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Bio Med Central Page 1 of 16(page number not for citation purposes) Journal of Hematology & Oncology Open Access ResearchPhenotype and functional evaluation of ex vivo generated antigen-specific immune effe ctor cells with potential for therapeutic applicationsShuhongHan1, YujuHuang2, YinLiang2, YuchinHo2, YichenWang2 and Lung-JiChang*1Address: 1Department of Molecular Genetics and Micr obiology, Co llege of Medici ne, University of Florida, Gainesville, FL 32610-0266 and 2Vectorite Biomedica, Inc., Taipei, Taiwan, Republic of China Email: ShuhongHan-hansh72@ufl.edu; YujuHuang-Yuju.huang@vectorite.com; YinLiang-Yin.liang@vectorite.com; YuchinHo-Malineho@gmail.com; YichenWang-yichen.wa ng@vectorite.com; Lung-JiChang*-lchang@mgm.ufl.edu Corresponding author AbstractEx vivo activation and expansion of lymphocytes for ad optive cell therapy ha s demonstrated great success. To improve safety and therapeutic efficacy, increased antigen specificity and reduced nonspecific response of the ex vivo generated immune cells are nece ssary. Here, using a complete protein-spanning pool of pent adecapeptides of the latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV), a weak viral antigen wh ich is associated with EBV lymphoproliferative diseases, we investigated the phe notype and function of immune effector cells generated based on IFNor CD137 activation marker selection an d dendritic cell (DC) activation. These ex vivo prepared immune cells exhibite d a donorand antigen-depend ent T cell response; the IFNselected immune cells displaye d a donor-related CD4or CD 8-dominant T cell phenotype; however, the CD137-enriched cell s showed an increased ratio of CD4 T cells. Importantly, the pentadecapeptide antigens access ed both class II and class I MHC antigen processing machineries and effectively activated EBV-specific CD4 an d CD8 T cells. Phenotyp e and kinetic analyses revealed that the IFNand the CD137 selections enriched more central memory T (Tcm) cells than did the DC-activation approa ch, and after expansion, the IFN-selected effecto r cells showed the highest level of antigen-specif icity and effector activi ties. While all three approaches generated immune cells with comparable anti gen-specific activities, the IFNselection followed by ex vivo expansion produced high quality and quantity of antigen-specific effecto r cells. Our studies presented the optimal approach for generating therapeutic imm une cells with potential for emergency and routine clinical applications.BackgroundThe key to the success of immune cell therapy is to increase specific and decrease non-specific immune response. Ex vivo expanded antigen-specific T cells targeting cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus have been successfully applied to treating hematopoietic stem cell or solid organ transplant patients who have developed post-transplant viral diseases. [1,2] The labor-intensive and time-consuming process of isolating and expanding antigen-specific immune cells, how-Published: 6 August 2009 Journal of Hematology & Oncology 2009, 2 :34doi:10.1186/1756-8722-2-34 Received: 30 June 2009 Accepted: 6 August 2009 This article is available from: h ttp://www.jhoonline.org/content/2/1/34 2009 Han et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons. org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the orig inal work is properly cited.

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Journal of Hematology & Oncology 2009, 2 :34http://www.jhoonline.org/content/2/1/34 Page 2 of 16(page number not for citation purposes)ever, has hindered common practice of this medical advent. Donor leukocyte infusion has attained a response rate of >50% in treating post-transplant infections or cancer relapse of hematopoietic stem cell transplant (HSCT) patients. [3-5] Non-specific leukocyte infusion, however, may cause severe graft-versus-host disease (GvHD) with high morbidity and mortality. Adoptive transfer of antigen-specific T cell clones combined with clinical regimens to increase immune cell homeostasis has illustrated high response rate (50%) in melanoma patients. [6-8] While the amplified T cell clones display increased antigen-specific effector functions, the ex vivo expansion process takes longer than 4–6 weeks, which often results in terminal differentiation of the T cells with reduced in vivo proliferation potential. [9] A few approaches have been developed to directly isolate antigen-specific immune cells, such as the use of MHCpeptide multimers and selection of IFN-secreting cells with affinity-magnetic beads. [10-14] Recently, CD137 (4-1BB), a member of the tumor necrosis factor receptor family, has been reported to be a suitable surface marker for antigen-specific T cell isolation. [15] Although both the IFNand the CD137 selection methods generate only a small number of immune effector cells, these cells may be further expanded in culture to obtain more functional cells. [12] Detailed phenotype and functional characterizations of these ex vivo prepared immune effector cells are necessary to facilitate their clinical applications. Here, we refined and compared three of the state-of-the-art immune effector cell preparation approaches, the IFNand the CD137 selection methods for emergency preparation of therapeutic cells, and a DC-immune cell coculture method for the expansion of antigen-specific immune cells. We targeted EBV using LMP2A pentadecapeptides as antigens because EBV-associated lymphoproliferative disorders represent one of the most severe problems in HIV/AIDS patients and transplantation patients. The IFN-selected cells showed an increased ratio of CD4 or CD8 effector cell population depending on the donor, whereas the CD137 selection method enriched a higher ratio of CD4 T cells regardless of the donor's T cell dominance response. Both of the rapid protocols yielded more central memory and effector memory T cells than did the DC-activation method. Our detailed side-by-side comparison concludes that IFNselection followed by ex vivo expansion represents the preferred method for the generation of antigenspecific immune effector cells with potential for clinical applications.MethodsPeripheral blood mononuclear cells (PBMC) and B lymphoblastoid cell lines (BLCL)Healthy donors' buffy coats were obtained from Civitan Blood Center (Gainesville, FL, USA). PBMC were prepared by gradient density centrifugation in Ficoll-Hypaque (GE Healthcare Bio-Sciences AB, NJ, USA) as previously described. [16] Viability was determined by trypan blue staining. Autologous B lymphoblastoid cell line (BLCL) was generated by transforming peripheral blood B lymphocytes with EBV as described previously. [17] The BLCL were continuously propagated in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 g/ml streptomycin, 100 IU/ml penicillin and 10% heat inactivated fetal bovine serum (FBS) at 37C with 5% CO2.PeptidesThe mixtures of 11 amino acid overlapping pentadecapeptides (122 peptides) spanning the entire 497 amino acids (NCBI Accession number P13285) of LMP2A of the EBV (human herpesvirus 4, strain B95-8) and the Wilms' tumor antigen (WT1, 449 amino acids, 110 peptides) were purchased from JPT Peptide Technologies GmbH (Berlin, Germany).Preparation of 2 day and 5 day dendritic cells (DC)PBMC were plated into 6-well plate at 1 107 cells/well and adhered for 2 hours in AIM-V (Gibco-BRL, CA, USA). The non-adherent cells were removed gently and frozen as source of lymphocytes for co-culture use. Adherent monocytes were cultured in AIM-V supplemented with 50 ng/ml of GM-CSF and 25 ng/ml IL-4 (eBiosource International, Inc. Camarillo, CA, USA). For the generation of 2 day DC, the adherent cells were cultured with GM-CSF and IL-4 for 24 h and incubated for another 24 h with TNF (50 ng/ ml), IL-1 (10 ng/ml), IL-6 (10 ng/ml, all from R&D systems, MN, USA) and PGE2 (1 uM, Sigma-Aldrich, MO, USA) to induce maturation. For the generation of 5 day DC, cells were cultured with GM-CSF and IL-4 for 5 days. On day 3, half of the medium was replaced with fresh medium containing GM-CSF and IL-4. On day 5, the immature DC were induced into maturation with TNF (50 ng/ml), LPS (1 g/ml, Sigma-Aldrich) and IFN(50 ng/ml, R&D systems).Isolation of antigen-specific IFNsecreting or CD137 positive cellsPBMC were resuspended in AIM-V plus 5% human AB serum at 1 107 cells/ml and mixed with pentadecapeptides for EBV-LMP2A (10 ug/ml), mouse anti-human CD28 antibody (Ab, 1 ug/ml, eBioscience, San Diego, USA) and human 2-microglobulin (1 ug/ml, Sigma) to enhance antigen presentation and costimulation. The cells were incubated in a 37C humidified incubator for 3–13 hours. IFNsecreting cells were enriched with the

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Journal of Hematology & Oncology 2009, 2 :34http://www.jhoonline.org/content/2/1/34 Page 3 of 16(page number not for citation purposes)IFNCatch Reagent and CD137 positive cells with PEconjugated monoclonal anti-CD137 Ab (clone 4B4-1), followed by affinity isolation using anti-PE microbeads according to the manufacturer's instruction (Miltenyi Biotech Inc. Auburn, CA, USA).Generation of DC-activated antigen-specific immune cellsDC-activated antigen-specific immune cells were generated as previously described. [16] In brief, mature 2 day DC were loaded with LMP2A peptides (2.5 ug/ml) for 2 h and irradiated (20 Gy, or 2,000 rads). The antigen-pulsed DC were cocultured with autologous non-adherent PBMC at a ratio of 1:20 in AIM-V with 5% human AB serum. On day 3, half of the medium was replaced with fresh medium supplemented with IL-2 (12.5 U/ml), IL-7 (5 ng/ ml) and IL-15 (20 ng/ml, all from Gentaur, Aachen, Germany). Half of the medium was replaced with fresh medium with cytokines every other day.Multi-color flow cytometryMature DC were analyzed using a four-color panel of monoclonal Ab including PE-anti-CD14, FITC-anti-HLADR, PE-anti-CD86, APC-anti-CD1a, PE-anti-CD 83, APCanti-CD40 and FITC-anti-HLA-I (BD Biosciences, San Jose, CA, USA), APC-anti-DC-SIGN and PE-cy7-antiCD11c (eBioscience), and incubated for 30 min at 4C. Isotype-matched antibodies were used for controls. The Ab-labeled cells were washed twice with PBS containing 1% FBS and analyzed with FACSCaliber or FACSAria using FACSDiva software (BD Biosciences) and Flowjo software (Tree Star, Inc., Ashland, OR, USA). For memory T cell analysis, the cells were stained with PE-anti-IFNor PE-anti-CD137, in combination with APC-anti-CD4 (clone RPA-T4), Pacific blue-anti-CD8 (clone RPA-T8), FITC-anti-CD45RA (clone HI100), PE-cy7-anti-CCR7 (clone 3D12), Percp-cy5.5-anti-CD28 (clone 293, all from BD Biosciences) and Alexa-fluo 750-anti-CD27 (clone O323, eBioscience) at 4C for 30 min and washed twice with PBS containing 1% FBS. The percentage of different T cell subsets was analyzed using FACSAria with FACSDiva and Flowjo softwares.Immune effector assays: CD107a degranulation and intracellular cytokine stainingThese assay were performed as described. [18] Briefly, 2 105 LMP2A-specific T cells were stimulated for 5 h in a 96well plate with irradiated (20 Gy) antigen-loaded autologous DC. Monensin A (Sigma-Aldrich) and FITC-conjugated Abs for CD107a or isotype matched Abs (BD Pharmingen, San Diego, CA, USA) were added 1 hour after stimulation and incubated for 5 hours. Cells were then stained with Abs against CD4 and CD8 and fixed, permeabilized with Cytofix/Cytoperm solution and stained with Ab against IFN(all from BD Pharmingen) at 4C for 20 min. Unrelated peptide group was included as a negative control for spontaneous CD107a expression and/or cytokine production.Detection of peptide-specif ic CD8+ T cells by MHC multimer analysisPeptide-major histocompatibility complex (MHC)-pentamer conjugate specific for EBV-LMP2A/TYGPVFMCL (HLA-A*2402 restricted) was purchased from Proimmune (Springfield, VA, USA). T cells were incubated with PE-labeled peptide MHC-pentamer at room temperature for 10 min, washed and stained with APC-anti-CD3 and FITC-anti-CD8 Ab (BD Pharmingen) on ice for 30 min, and analyzed using FACSAria. At least 1 105 events were collected for each sample.T cell proliferation assay with carboxy-fluorescein diacetate succinimidyl ester (CFSE) stainingThe CFSE-based proliferation assay was performed as previously described [19]. Briefly, LMP2A-specific T cells were washed and labeled with 1 uM CFSE (Molecular Probes, Inc., Eugene, OR, USA). The labeled cells were washed and plated into 96-well U-bottom wells at 1 105cells per well. Autologous DC were loaded with peptides (2.5 ug/ml) for 2 hours and irradiated (20 Gy). The irradiated DC were added to the CFSE-labeled T cells at a ratio of 1:20 and cultured in AIM-V with 5% human AB serum. After 4 days, cells were harvested and analyzed with flow cytometry.Antigen-specific cytotoxicity assay of immu ne effector cellsThe immune cell cytotoxicity assay was based on Jedema et al. with minor modifications. [20] The target cells were washed with PBS, and labeled with 1 uM CFSE (Molecular Probes) at 5 106 cells per ml at 37C for 15 minutes. The reaction was stopped with the addition of 10 volumes of complete RPMI containing 10% FCS, followed with a 30 min incubation at 37C. After two washes, the CFSElabeled target cells were resuspended in AIM-V containing 5% human AB serum. The cell concentration was adjusted to 1 105 cells/ml before plating into 96-well microtiter plates at 100 ul/per well. The effector cells were then mixed with target cells at a ratio of 1:1. The plates were incubated in a humidified atmosphere of 5% CO2 and 37C. The target cells included irradiated (20 Gy) autologous DC loaded with LMP2A peptides or control WT1 peptides, and irradiated (100 Gy) autologous BLCL. The effector cells included LMP2A peptide-stimulated IFNselected cells, LMP2A pulsed DC-expanded effector cells, LMP2A peptide-stimulated IFN-negative PBMC, and control PBMC. After 6 hour of incubation, the cells were mixed with 10,000 Flow-Count Fluorospheres (BD Pharmingen) and followed by flow cytometry analysis. To stain for dead cells, 7-AAD (10 ug/ml) was added and incubated for 30 min on ice. For each sample, 5,000

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Journal of Hematology & Oncology 2009, 2 :34http://www.jhoonline.org/content/2/1/34 Page 4 of 16(page number not for citation purposes)microbeads were acquired, facilitating the calculation of absolute numbers of target cells. The percentage of survival was determined as the following: (R refers to different CTL ratio groups; R = 0 refers to the CTL = 0 control)Statistical analysisData were analyzed using GraphPad Prism 4 analysis software (GraphPad Software Inc. San Diego, CA) and Student's t-test A 2-sided P value of less than 0.05 was considered statistically significant.ResultsBoth IFNand CD137 are effective markers for the isolation of antigen-specific immune effector cellsThe small population of immune effector cells specific for a particular antigen may be isolated based on expression of antigen-specific activation markers. IFNand CD137 have been identified as antigen-specific activation markers. Upon antigen stimulation, the IFNsecreting cells can be captured with anti-IFNAb conjugated with an anti-surface marker Ab. Alternatively, CD137 positive cells can be directly isolated using anti-CD137 Ab. Specific T cell immune response to the peptide library spanning the entire LMP2A sequence has been previously documented. [21] We first established the expression kinetics of IFNand CD137 in PBMC after stimulation with a pool of EBV LMP2A pentadecapeptides, as reports by others, the expression peaked at 6–12 hr for IFNand 24 hr for CD137 (data not shown). [15,22] The IFNand CD137-expressing cells were isolated using Ab-conjugated magnetic bead affinity columns (Miltenyi Biotech). After examining a large number of donors, we found that the antigen-specific response of an individual could be either CD8 or CD4 T cell dominant, which can be donorand/or antigen-dependent. Representative results are illustrated in Fig. 1A (a CD4-dominant donor to the left, and a CD8-dominant donor to the right). The results indicated that the CD4/CD8 ratio of the IFN-selected cells correlated with the donor's T cell dominance phenotype. However, the CD137-enriched cells consistently showed an increased CD4 to CD8 ratio regardless of the donor's phenotype (Fig. 1A, bottom); the latter could be due to the increased proportion of CD137 positive CD4 cells in the total population of T cells before and after stimulation (Fig. 1B). However, quantitative analysis of the expression level of CD137 demonstrated that CD8 T cells expressed higher density of surface CD137 than did CD4 T cells in both CD4 and CD8 dominant individuals (see geometric means in Fig. 1C).The antigen-activated IFNand CD137 positive immune cells display different surface phenotypesThe phenotype of the immune cells of the two affinity isolation methods has not been characterized in the past due to the limited number of the total harvested cells. We subjected the IFNand CD137 affinity-purified cells to a multi-color flow cytometry analysis for central memory (Tcm, CCR7+, CD45RA-), effector memory (Tem, CCR7-, CD45RA-), terminal effector (Teff, CCR7-, CD45RA+), differentiation (CD27 and CD28) and migration (CCR7) markers of T cells in addition to CD4, CD8 and IFN(or CD137). We found that there was a trend of increased early-differentiated Tcm and Tem cells in the dominant populations (Fig. 2A and 2B, CD4and CD8-dominant donors, respectively).Ex vivo expansion of antigen-activated IFNand CD137 positive immune cellsTo see if the enriched immune effector cells could be further expanded, we cultured them on irradiated autologous feeder PBMC; the cells expanded approximately 600-fold in two weeks (not shown). Before expansion, the CD137 enriched cells contained a higher ratio of CD4 T cells (Fig. 1B, 37.6 vs. 13.1); however, the donor's dominant phenotype was restored after expansion in culture (Fig. 3A). Furthermore, we consistently observed an increased population of CD3-CD56+ cells in the CD137+ cell expansion culture compared with the IFNcell expansion culture (Fig. 3A top, 32.7% and 8.5% versus 2.3% and 2.6%, for a CD4-dominant and a CD8-dominant donors, respectively). Compared with the freshly isolated effector cells that contained more memory effector cells (Fig. 2), the cultured cells differentiated toward Tem and (Teff) cells after expansion (Fig. 3B and 3C). Intracellular staining for effector cytokines showed that the expanded IFNand CD137 cells displayed high antigen-specific activities, up to 59% and 34% for IFNand CD137 selected cells, respectively, when restimulated with DC-pulsed with the specific antigens (LMP2A petadecapeptides) or autologous EBV positive BLCL (Fig. 4A and 4B, control: WT-1 peptide-pulsed DC). To demonstrate antigen-specific cytolytic activity, we incubated these cells with different target cells as illustrated in Fig. 4C. The effector cells killed specific target cells with high specificity including autologous BLCL and LMP2A peptide-pulsed DC, but not control WT-1 peptide-pulsed DC (Fig. 4C, effector to target ratio 1:1).Ex vivo expansion of antigen-specific immune effector cells with dendritic cells (DC)Antigen-specific immune effector cells can also be generated through DC activation. The latter protocol includes a DC preparation step, followed by antigen exposure and lymphocyte coculture. For the preparation of DC, we compared the 2 day and the 5 day protocols. [23] We ana% #() Survival Absoluteof viable CFSEtarget cellsRx Absolu = += t teof viable CFSEtarget cellsR #() += 0 100

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Journal of Hematology & Oncology 2009, 2 :34http://www.jhoonline.org/content/2/1/34 Page 5 of 16(page number not for citation purposes)lyzed the surface markers for plastic-adherent monocytes and mature DC with flow cytometry (data shown in Additional file 1), and analyzed phenotypes of the 2 day and the 5 day mature DC (Fig. 5). The 2 day DC expressed higher levels of class I/II MHC (HLA-I and HLA-DR), costimulatory molecules (CD86 and CD40) and maturation marker CD83. We also found that the 2 day DC induced primary and secondary immune response against viral or cancer antigens at efficiencies equal to or better than the 5 day DC (manuscript in preparation). Therefore, the 2 day DC protocol was adopted for later experiments. The 2 day DC were pulsed with the pooled LMP2A pentadecapeptides, irradiated and cocultured with autologous lymphocytes at a ratio of 1:20. The cell number usually decreases around day 5 after coculture, followed by an increase of a few fold around day 17, suggesting a loss of non-specific cells followed by expansion of antigen-specific cells (see representative growth curves in Fig. 6A). Flow cytometry analysis of the DC-activated cells in culture from four different donors at day 0, 12 and 19 indicated that most donors generated CD3+CD56T cells, but some generated a large proportion of CD3-CD56+ NK cells and CD3+CD56+ cells (e.g. donor 3, Fig. 6B). The relative ratios of CD4+ T cells, CD8+ T cells, NK cells and CD3+CD56+ cells in the coculture appeared to be donordependent (Fig. 6B and 6C). Furthermore, phenotype analysis with multi-color flow cytometry demonstrated IFNor CD137-based enrichment of antige n-specific immune effector cells Figure 1 IFNor CD137-based enrichment of antigen-specific immune effector cells (A) CD4 and CD8 T cell distribution in IFNor CD137-positive cell populati on after antigen stimulation. The ratios of CD4 to CD8 T cells of five donors were presented. (B) and (C) CD137 expression in CD4 and CD8 T cells before and after Ab affinity column purification. PBMC were mixed with EBV LMP2 pentadecap eptides and 3…24 hr later, IFN-secreting cells or CD137-positi ve cells were isolated by using MACS magnetic bead affinity columns as described in Mate rials and Methods. The cells we re stained with PE-conjugated anti-IFNor anti-CD137 Ab and CD3, CD4 and CD8 sp ecific Ab and analyzed with flow cytometry.

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Journal of Hematology & Oncology 2009, 2 :34http://www.jhoonline.org/content/2/1/34 Page 6 of 16(page number not for citation purposes)Phenotype analysis of IFNor CD137-selecte d antigen-specific immune cells Figure 2 Phenotype analysis of IFNor CD137-selected anti gen-specific immune cells PBMC were stimulated with EBV LMP2A pentadecapeptides and IFNor CD137 positive cells were isolated for analysis. (A) & (B) Memory and effector phenotype analysis based on sevencolor flow cytometry with surface staining for CD27, CD28, CD45RA, CCR7, CD4, CD8, CD137 and intracellular staining for IFNimmediately after cell isolation without furthe r culture. The percentage of different populations (IFNor CD137 plus CD4 or CD8 gated) of T cells is illustrated for a CD4-do minant donor (A) and a CD8-dominant donor (B); Tcm, central memory T cell s (CD27+/-, CD28+, CCR7+, CD45RA-); Te m, effector memory T cells (CD27+/-, CD28+/-, CCR7-, CD45RA-); Teff, terminal effe ctor T cells (CD27-, CD28 -, CCR7+/-, CD45RA+).

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Journal of Hematology & Oncology 2009, 2 :34http://www.jhoonline.org/content/2/1/34 Page 7 of 16(page number not for citation purposes)Phenotype analyses of IFNor CD137-selected immune effector cells after ex vivo expansion Figure 3 Phenotype analyses of IFNor CD137-selected immu ne effector cells after ex vivo expansion (A) Phenotype analysis after ex vivo expansion. After the rapid se lection, the antigen-specific immune cell s were cultured for fifteen days with irradiated autologous PBMC as feeder cells. The distributio n of CD3, CD4, CD8 and CD56 ce ll populations was determined and representative FACS graphs are shown. (B) and (C) Memo ry and effector phenotype analysis based on seven-color flow cytometry with surface staining for CD27, CD28, CD45RA, CCR 7, CD4, CD8, CD137 and intracellular staining for IFN. Representative results of a CD4-dominant dono r (B) and a CD8-dominant donor (C) are shown.

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Journal of Hematology & Oncology 2009, 2 :34http://www.jhoonline.org/content/2/1/34 Page 8 of 16(page number not for citation purposes)Effector function analyses of IFNor CD137-selected immune effector cells after ex vivo expansion Figure 4 Effector function analyses of IFNor CD137-selected immu ne effector cells after ex vivo expansion (A) and (B) Flow cytometry analysis of IFNand CD137 expression after restimulation of the culture expanded IFNor CD137 effector cells. The cells were restimulated with di fferent cells as indicated and subjected to antibody staining and flow cytometry anal ysis; control, WT-1 peptide-pulsed DC; specific, LMP2A pept ide-pulsed DC; BLCL, autologous EBV-transformed B cells. (C) Analysis of antigen-specif ic cytolytic activity. The ex vivo expanded IFNor CD137-enriched cells were incubated with autologous EBV transformed B cells (BLCL), mature DCs loaded wi th either LMP2A pentadecapep tides (DC-LMP2A) or WT1 pentadecapeptides (DC-WT1, as cont rol) at 1:1 ratio in a cytotoxici ty assay based on CFSE labeling as described in Materials and Methods.

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Journal of Hematology & Oncology 2009, 2 :34http://www.jhoonline.org/content/2/1/34 Page 9 of 16(page number not for citation purposes)that the ex vivo expanded cells contained mostly Tem and Teff cells (Fig. 7A and 7B, representative of CD4and CD8-dominant donors).The DC-activated immune cells display antigen-specific effector functionsWe next evaluated the DC-activated immune cells for antigen-specific effector functions based on intracellular staining of IFN, IL-2, and the degranulation marker CD107a after restimulation with peptide-loaded DC (Fig. 8A). For direct comparison, the same donor whose cells were analyzed for the IFN-selected effector functions (Fig. 4B) was chosen. Analyses of the immune effector cells without further stimulation (IE cell alone), or stimulated with non-specific peptides (Non-specific) or with the LMP2A pentadecapeptides (LMP2A-specific) demonstrated LMP2A-specific expression of IFN, IL-2 and CD107a in the CD8 T cells, and to a lesser extent, the CD4 T cells (Fig. 8A top: IL-2 and IFN, and bottom: CD107a and IFN). The antigen-specific effector function was further confirmed with the CFSE-based proliferation assay, as well as MHC-peptide pentamer specific for the LMP2A epitope-specific T cell receptors of the CD8 T cells (data not shown). To demonstrate antigen-specific killing, we directly compared the DC-activated cells to the IFNcaptured cells (as shown in Fig. 4C) using an in vitro cytolytic assay (Fig. 8B). At an effector to target cell ratio of 1:1, the immune cells effectively killed autologous BLCL and LMP2A-pulsed DC (DC-LMP2A), but not DC pulsed with control WT1 pentadecapeptides (DC-WT1). Therefore, effector function Phenotype comparison of 5 day versus 2 day mature DC Figure 5 Phenotype comparison of 5 day versus 2 day mature DC Surface markers related to an tigen presentation function were stained with multi-color fl uorochrome-labeled Ab and analyzed with FACS Aria. Data represent MFI of mature DC generated from three healthy dono rs with p value calculated.

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Journal of Hematology & Oncology 2009, 2 :34http://www.jhoonline.org/content/2/1/34 Page 10 of 16(page number not for citation purposes)Ex vivo expansion and phenotype analyses of DC-activated immune effector cells Figure 6 Ex vivo expansion and phenotype analyses of DC-activated immune effector cells EBV LMP2A-specific effector T cells were generated by stimulation of non-adherent PBMC with DC pulsed with LMP2A pentadecapeptides. (A) Growth kinetics of DC-activated immune ef fector cells. The viable cells we re counted with trypan blue st aining at different time point s after coculture and the growth curves of 5 samples are shown. (B) and (C) CD3, CD56, CD4 and CD8 phenotype analysis. The DC-activated cells from day 0, 12 and 19 were stained with antibodies against CD4, CD8, CD 3 and CD56 and analyzed with flow cytometry. The percen tages of different lymphocyte s ubsets were analyzed and shown in bar graphs. Donor 1*, PBMC collected at a di fferent time poin t from donor 1.

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Journal of Hematology & Oncology 2009, 2 :34http://www.jhoonline.org/content/2/1/34 Page 11 of 16(page number not for citation purposes)Memory and effector T cell analyses of DC-activated immune effector cells Figure 7 Memory and effector T cell analyses of DC-activated immu ne effector cells (A) & (B) Memory and effector phenotype analysis of a CD4 dominant donor (A ) and a CD8 dominant donor (B). The DC-acti vated cells from day 0, 12, and 17 after coculture were analyzed for CD 3, CD4, CD8, CD27, CD28, CD 45RA, and CCR7 using a sevencolor panel of fluorochromelabeled Ab with FACSAria. The cells were gated for CD3 and CD 4 or CD8 as total cells for th e percentage analysis. One representative flow graph of 5 perf ormed experiments is presented.

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Journal of Hematology & Oncology 2009, 2 :34http://www.jhoonline.org/content/2/1/34 Page 12 of 16(page number not for citation purposes)Figure 8 (see legend on next page)

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Journal of Hematology & Oncology 2009, 2 :34http://www.jhoonline.org/content/2/1/34 Page 13 of 16(page number not for citation purposes)analyses including IFNrelease and cytotoxicity assays suggest that the DC-activated cells had lower activities than the expanded IFNeffector cells.DiscussionAdoptive immune cell therapy has shown great promise in treating viral diseases and melanoma. [2,6] Continued efforts are focused on the generation of sufficient amount of antigen-specific immune cells and optimal conditioning of immune homeostasis in patients in order to achieve a sustained in vivo immune surveillance. [24-26] Here, we compared three ex vivo immune cell preparation protocols and phenotypically and functionally characterized these cells. The rapid protocols based on IFNand CD137 selection generate a small number of antigen-specific effector cells with high percentage of central memory T cells in a very short period of time. The DC-activation protocol generates more immune cells, albeit, with more differentiated phenotype and reduced proportion of antigenspecific effector cells. Based on analyses of a large number of donors, we found that individual response to a given antigen could be either CD4or CD8-dominant, which is antigenand donordependent. Immune effector cells isolated based on IFNexpression displayed a CD4 or CD8 bias consistent with the donor's immune dominance. However, antigen-specific CD137 positive cells showed a higher ratio of CD4 effector cells regardless of the subject's immune phenotype; this is in contrast to previous reports that emphasize the induction of CD8 effector cells after CD137 enrichment. [15,22,27] We did, however, show that CD8 T cells displayed higher density of CD137 than did CD4 T cells. It is well documented that CD137 costimulation promotes both CD4 and CD8 T cell expansion and long term memory. [28-30] Our finding that more CD4 T cells than CD8 T cells are detected in the CD137-positive cell population suggests a rapid induction of CD137 in the memory T helper repertoire immediately after antigen stimulation. Although the enriched CD137 immune cells contained a higher CD4 T cell ratio, further expansion in culture restored the donor's original dominant phenotype, with a higher CD3-CD56+ NK cell population than those found in the expanded IFNenriched immune cells (Fig. 3A). This result suggests that IFNis a more restricted adoptive immune response marker and represent less of an innate immune marker as does CD137. The ex vivo DC-activation protocol generated different ratios of CD4, CD8 and NK cells in culture, which again, appeared to be donor-dependent. Whether the immune dominance has any effect on in vivo efficacy of the cultured immune effector cells awaits further investigation. As CD4 T cells are important for the maintenance of longterm anti-viral CD8 T cell memory [31], therapeutic immune cells should include polyclonal CD4 and CD8 T cells. Both IFNand CD137 selection approaches generated increased number of memory type of cells representative of polyclonal CD4 and CD8 T cells that may have increased proliferation potential after infusion. Although the antigen-specific memory T cells from PBMC may be low; for examples, the average yield of LMP2A-specific IFNpositive immune effector cells from healthy EBVseropositive donors is only 0.22 0.13% (n = 6, after two rounds of affinity column purification), they can be expanded to more than two orders of magnitude in culture in two weeks and maintain their high antigen specificity. It is evident that the LMP2A pentadecapeptides efficiently activate both CD4 and CD8 T cells in a short exposure period (3–13 hr). This was surprising since CD8 T cells are activated through class I MHC loaded with short 9–11 amino acid peptide epitopes, different from CD4 T cells, which are activated through class II MHC loaded with 12– 15 amino acid peptide epitopes. The pentadecapeptide antigens apparently activated CD8 T cells with high efficiency through cross-presentation. This has been confirmed with various pentadecapeptide antigens (unpublished). The processing of class II MHC peptides into class I epitopes for cross-presentation to CD8 T cells appears to be highly efficient with both the IFNand the CD137 protocols, as with the DC-activation method. The Functional analyses of DC-activated and ex vivo expanded immune effector cells Figure 8 (see previous page) Functional analyses of DC-activated and ex vivo expanded immune effector cells EBV LMP2A-specific effector cells were generated by stimulation of non-adhe rent PBMC with DC pulsed with LMP2A pent adecapeptides. (A) Analysis of antigenspecific effector cytokines and CD107a e xpression. The DC-activated immune cells from day 19 coculture were stimulated with autologous DC pulsed with LMP2A peptides, WT1 peptides (non -specific control) or no stim ulation (IE cell alone) for 6 hours. The cells were stained with Ab against CD4, CD8, CD107a and IFN. Flow cytometry analyses of IL-2, IFNand CD107a-positive cells in CD4-gated or CD8gated populations were illustrated as re presentative of five experiments. (B) Comparison of cytolytic function of IFN-selected (same donor as in Fig. 4C) versus DC-activa ted LMP2-specific immune effector cells. The LMP2A-specific effector cells were mixed wi th target cells including auto logous BLCL, DC-LMP2A, or control DC-WT1 at 1:1 ratio and analyzed fo r cytolytic activity based on the CFSE-labelin g method as describe d in Materials and Methods.

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Journal of Hematology & Oncology 2009, 2 :34http://www.jhoonline.org/content/2/1/34 Page 14 of 16(page number not for citation purposes)detailed molecular mechanism of the efficient cross-presentation requires further investigation. To assess differentiation and maturation status of the ex vivo generated T cells, we applied multi-color flow cytometry to detect differentiation and homeostatic marker CD45RA, trafficking marker CCR7, and costimulatory marker CD27 and CD28. [32,33] It is not surprising that both the IFNand the CD137-enriched antigen-specific effector cells displayed more memory markers than did the DC coculture-expanded cells. While preserving Tcm cells is critical to in vivo therapeutic efficacy,[7,34,35] clinical studies have proven that ex vivo expanded effector cells can persist many years after infusion. [36] Clinical benefits of these different protocols will require detailed evaluation in a large cohort of patients. The differentiation status of the ex vivo generated immune cells may contribute to their in vivo therapeutic efficacy. Homeostasis of antigen-specific memory cells can vary depending on antigen source, the immune milieu and individual donor. It is known that Tcm cells are mainly located in lymphoid tissues and Tem cells are distributed in diverse non-lymphoid sites including lung, liver and intestine. [37] In addition, bone marrow has been shown to embrace increased number of anti-cancer or anti-virus memory T cells. [38-40] After ex vivo expansion, however, wherever the T cells come from, they tend to bestow exhausted proliferation and replicative senescence associated with down-regulation of anti-apoptotic protein Bcl-2 and Bcl-xL, and decreased telomere length. [33,34,41] Modification of antigen presentation protocol and culture condition may help overcome the immune cell exhaustion problem. [9,42] For patients with acute infections or illness, direct isolation of antigen-specific immune cells from partly HLAmatched healthy donors represents an attractive emergency approach to obtain therapeutic cells. [43,44] This approach offers several advantages including a shortened handling time and increased proliferation potential in vivo Although the number of immune cells is limited with the direct isolation approach, clinical evidence supports that only a small number of such immune cells, in the range of 103-104/kg body weight, is sufficient to attain therapeutic efficacy in transplant patients. [13,45,46] Ex vivo expansion of immune cells, nevertheless, may be necessary for patients with a compromised immunity. [47,48]ConclusionThe two rapid immune cell isolation methods generate functional effector cells in less than 24–48 hr suitable for emergency immune cell preparation. On the other hand, the DC-activation method expands antigen-specific immune effector cells while effectively reduce the number of non-specific cells. Depending on clinical needs, for examples, the urgency for treatment, patient's body weight (e.g. less cells are needed for pediatric patients), or patient's immune cell proliferative potential in vivo the method of immune cell preparation may differ. Our data indicate that IFNselection followed by ex vivo expansion represents the best approach for the generation of high amount of antigen-specific immune effector cells. Further efforts to overcome immune tolerance and expand antigen-specific immune cells with prolonged in vivo persistence are critical to the success of immune cell therapy.List of abbreviationsIFN: interferon-gamma; IL: interleukine; DC: dendritic cell; CTL: cytotoxic T lymphocyte; MHC: major histocompatibility complex; Ab: antibody; Ag: antigen; TCR: T cell receptor; TNF: tumor necrosis factor; BLCL: B lymphoblastoid cell line; EBV: Epstein-Barr virus; LMP2A: late membrane protein 2A; CMV: cytomegalovirus; ICCS: intracellular cytokine staining; CFSE: carboxy-fluorescein diacetate succinimidyl ester.Competing interestsYH, YL, YH and YW are employees of Vectorite Biomedica Inc. LJC is consultant to a biotech company.Authors' contributionsAll authors are accountable for the integrity of the research results; Chang is responsible for the conception of the research and Han, Huang, Liang, Ho and Wang are responsible for the execution and for data collection; Chang is responsible for initial drafting and revisions of the manuscript.Additional material AcknowledgementsWe thank the technical assistance of Liheng Guo, Lily Lien, Fuhung Yang, Yinchieh Fu and Meifang Lin. The study was funded by Vectorite Biomedica Inc. and Yongling Foundation.Additional file 1Phenotype analysis of monocytes and 2 day and 5 day mature DC Surface markers related to antigen presentation function were analyzed using fluorochrome-labeled Ab. The light-co lored lines in the FACS graphs represent control Ab and the numbers represent geometric means with percentages shown in parentheses. Representatives of two monocyte experiments and three DC experiments are illustrated. Click here for file [http://www.biomedcentral.co m/content/supplementary/17568722-2-34-S1.tiff]

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Publish with Bio Med Central and every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.asp Bio Med central Journal of Hematology & Oncology 2009, 2 :34http://www.jhoonline.org/content/2/1/34 Page 16 of 16(page number not for citation purposes)37.Masopust D, Vezys V, Marzo AL, Lefrancois L: Preferential localization of effector memory ce lls in nonlymphoid tissue. Science 2001, 291: 2413-2417. 38.Letsch A, Keilholz U, Assfalg G, Mailander V, Thiel E, Scheibenbogen C: Bone marrow contains melanom a-reactive CD8+ effector T cells and, compared with peripheral blood, enriched numbers of melanoma-reactive CD8+ memory T cells. Cancer Res 2003, 63: 5582-5586. 39.Slifka MK, Whitmire JK, Ahmed R: Bone marrow contains virusspecific cytotoxic T lymphocytes. Blood 1997, 90: 2103-2108. 40.Wood AH, Zhang X, Fa rber DL, Strome SE: CD8+ memory T lymphocytes from bone marrow … immune function and therapeutic potential. Crit Rev Immunol 2007, 27: 527-537. 41.Soares MV, Borthwick NJ, Maini MK, Janossy G, Salmon M, Akbar AN: IL-7-dependent extrathymic expansion of CD45RA+ T cells enables preservation of a naive repertoire. J Immunol 1998, 161: 5909-5917. 42.van Stipdonk MJ, Sluijter M, Han WG, Offringa R: Development of CTL memory despite arrested clonal expansion. Eur J Immunol 2008, 38: 1839-1846. 43.Haque T, Wilkie GM, Taylor C, Amlot PL, Murad P, Iley A, Dombagoda D, Britton KM, Swerdlow AJ, Crawford DH: Treatment of Epstein-Barr-virus-positive po st-transplantation lymphoproliferative disease with partly HLA-matched allogeneic cytotoxic T cells. Lancet 2002, 360: 436-442. 44.Lucas KG, Salzman D, Garcia A, Sun Q: Adoptive immunotherapy with allogeneic Epstein-Barr virus (EBV)-specific cytotoxic T-lymphocytes for recurrent, EBV-positive Hodgkin disease. Cancer 2004, 100: 1892-1901. 45.Cobbold M, Khan N, Pourgheysari B, Tauro S, McDonald D, Osman H, Assenmacher M, Billingham L, St eward C, Crawley C, Olavarria E, Goldman J, Chakraverty R, Mahe ndra P, Craddock C, Moss PA: Adoptive transfer of cytomeg alovirus-specific CTL to stem cell transplant patients after selection by HLA-peptide tetramers. J Exp Med 2005, 202: 379-386. 46.Amrolia PJ, Muccioli-Casadei G, Hu ls H, Adams S, Durett A, Gee A, Yvon E, Weiss H, Cobbold M, Gaspar HB, Rooney C, Kuehnle L, Ghetie V, Schindler J, Krance R, Heslop HE, Veys P, Vitetta E, Brenner MK: Adoptive immunotherapy with allodepleted donor T-cellsimproves immune reconstituti on after haploidentical stem cell transplantation. Blood 2006, 108: 1797-1808. 47.Overwijk WW: Breaking tolerance in cancer immunotherapy: time to ACT. Curr Opin Immunol 2005, 17: 187-194. 48.Rabinovich GA, Gabrilovich D, Sotomayor EM: Immunosuppressive strategies that are mediated by tumor cells. Annu Rev Immunol 2007, 25: 267-296.


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title
p Phenotype and functional evaluation of it ex vivo generated antigen-specific immune effector cells with potential for therapeutic applications
aug
au id A1
snm Han
fnm Shuhong
insr iid I1
email hansh72@ufl.edu
A2
Huang
Yuju
I2
Yuju.huang@vectorite.com
A3
Liang
Yin
Yin.liang@vectorite.com
A4
Ho
Yuchin
Malineho@gmail.com
A5
Wang
Yichen
yichen.wang@vectorite.com
ca yes A6
Chang
Lung-Ji
lchang@mgm.ufl.edu
insg
ins
Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL 32610-0266
Vectorite Biomedica, Inc., Taipei, Taiwan, Republic of China
source Journal of Hematology & Oncology
issn 1756-8722
pubdate 2009
volume 2
issue 1
fpage 34
url http://www.jhoonline.org/content/2/1/34
xrefbib
pubidlist
pubid idtype doi 10.1186/1756-8722-2-34
pmpid 19660111
history
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date
day 30
month 6
year 2009
acc
6
8
2009
pub
6
8
2009
cpyrt
2009
collab Han et al; licensee BioMed Central Ltd.
note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
abs
sec
st
Abstract
Ex vivo activation and expansion of lymphocytes for adoptive cell therapy has demonstrated great success. To improve safety and therapeutic efficacy, increased antigen specificity and reduced non-specific response of the ex vivo generated immune cells are necessary. Here, using a complete protein-spanning pool of pentadecapeptides of the latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV), a weak viral antigen which is associated with EBV lymphoproliferative diseases, we investigated the phenotype and function of immune effector cells generated based on IFN-γ or CD137 activation marker selection and dendritic cell (DC) activation. These ex vivo prepared immune cells exhibited a donor- and antigen-dependent T cell response; the IFN-γ-selected immune cells displayed a donor-related CD4- or CD8-dominant T cell phenotype; however, the CD137-enriched cells showed an increased ratio of CD4 T cells. Importantly, the pentadecapeptide antigens accessed both class II and class I MHC antigen processing machineries and effectively activated EBV-specific CD4 and CD8 T cells. Phenotype and kinetic analyses revealed that the IFN-γ and the CD137 selections enriched more central memory T (Tcm) cells than did the DC-activation approach, and after expansion, the IFN-γ-selected effector cells showed the highest level of antigen-specificity and effector activities. While all three approaches generated immune cells with comparable antigen-specific activities, the IFN-γ selection followed by ex vivo expansion produced high quality and quantity of antigen-specific effector cells. Our studies presented the optimal approach for generating therapeutic immune cells with potential for emergency and routine clinical applications.
meta
classifications
classification endnote subtype user_supplied_xml type bmc
bdy
Background
The key to the success of immune cell therapy is to increase specific and decrease non-specific immune response. Ex vivo expanded antigen-specific T cells targeting cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus have been successfully applied to treating hematopoietic stem cell or solid organ transplant patients who have developed post-transplant viral diseases. abbrgrp abbr bid B1 1B2 2 The labor-intensive and time-consuming process of isolating and expanding antigen-specific immune cells, however, has hindered common practice of this medical advent.
Donor leukocyte infusion has attained a response rate of 50% in treating post-transplant infections or cancer relapse of hematopoietic stem cell transplant (HSCT) patients. abbrgrpabbr bid="B3"3/abbrabbr bid="B4"4/abbrabbr bid="B5"5/abbr/abbrgrp Non-specific leukocyte infusion, however, may cause severe graft-versus-host disease (GvHD) with high morbidity and mortality. Adoptive transfer of antigen-specific T cell clones combined with clinical regimens to increase immune cell homeostasis has illustrated high response rate (50%) in melanoma patients. abbrgrpabbr bid="B6"6/abbrabbr bid="B7"7/abbrabbr bid="B8"8/abbr/abbrgrp While the amplified T cell clones display increased antigen-specific effector functions, the itex vivo /itexpansion process takes longer than 4–6 weeks, which often results in terminal differentiation of the T cells with reduced itin vivo /itproliferation potential. abbrgrpabbr bid="B9"9/abbr/abbrgrp/p
pA few approaches have been developed to directly isolate antigen-specific immune cells, such as the use of MHC-peptide multimers and selection of IFN-γ-secreting cells with affinity-magnetic beads. abbrgrpabbr bid="B10"10/abbrabbr bid="B11"11/abbrabbr bid="B12"12/abbrabbr bid="B13"13/abbrabbr bid="B14"14/abbr/abbrgrp Recently, CD137 (4-1BB), a member of the tumor necrosis factor receptor family, has been reported to be a suitable surface marker for antigen-specific T cell isolation. abbrgrpabbr bid="B15"15/abbr/abbrgrp Although both the IFN-γ and the CD137 selection methods generate only a small number of immune effector cells, these cells may be further expanded in culture to obtain more functional cells. abbrgrpabbr bid="B12"12/abbr/abbrgrp/p
pDetailed phenotype and functional characterizations of these itex vivo /itprepared immune effector cells are necessary to facilitate their clinical applications. Here, we refined and compared three of the state-of-the-art immune effector cell preparation approaches, the IFN-γ and the CD137 selection methods for emergency preparation of therapeutic cells, and a DC-immune cell coculture method for the expansion of antigen-specific immune cells. We targeted EBV using LMP2A pentadecapeptides as antigens because EBV-associated lymphoproliferative disorders represent one of the most severe problems in HIVAIDS patients and transplantation patients. The IFN-γ-selected cells showed an increased ratio of CD4 or CD8 effector cell population depending on the donor, whereas the CD137 selection method enriched a higher ratio of CD4 T cells regardless of the donor's T cell dominance response. Both of the rapid protocols yielded more central memory and effector memory T cells than did the DC-activation method. Our detailed side-by-side comparison concludes that IFN-γ selection followed by itex vivo /itexpansion represents the preferred method for the generation of antigen-specific immune effector cells with potential for clinical applications./p
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pMethods/p
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pPeripheral blood mononuclear cells (PBMC) and B lymphoblastoid cell lines (BLCL)/p
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pHealthy donors' buffy coats were obtained from Civitan Blood Center (Gainesville, FL, USA). PBMC were prepared by gradient density centrifugation in Ficoll-Hypaque (GE Healthcare Bio-Sciences AB, NJ, USA) as previously described. abbrgrpabbr bid="B16"16/abbr/abbrgrp Viability was determined by trypan blue staining. Autologous B lymphoblastoid cell line (BLCL) was generated by transforming peripheral blood B lymphocytes with EBV as described previously. abbrgrpabbr bid="B17"17/abbr/abbrgrp The BLCL were continuously propagated in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 μgml streptomycin, 100 IUml penicillin and 10% heat inactivated fetal bovine serum (FBS) at 37°C with 5% COsub2/sub./p
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pPeptides/p
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pThe mixtures of 11 amino acid overlapping pentadecapeptides (122 peptides) spanning the entire 497 amino acids (NCBI Accession number P13285) of LMP2A of the EBV (human herpesvirus 4, strain B95-8) and the Wilms' tumor antigen (WT1, 449 amino acids, 110 peptides) were purchased from JPT Peptide Technologies GmbH (Berlin, Germany)./p
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pPreparation of 2 day and 5 day dendritic cells (DC)/p
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pPBMC were plated into 6-well plate at 1 × 10sup7 /supcellswell and adhered for 2 hours in AIM-V (Gibco-BRL, CA, USA). The non-adherent cells were removed gently and frozen as source of lymphocytes for co-culture use. Adherent monocytes were cultured in AIM-V supplemented with 50 ngml of GM-CSF and 25 ngml IL-4 (eBiosource International, Inc. Camarillo, CA, USA). For the generation of 2 day DC, the adherent cells were cultured with GM-CSF and IL-4 for 24 h and incubated for another 24 h with TNFα (50 ngml), IL-1β (10 ngml), IL-6 (10 ngml, all from R&D systems, MN, USA) and PGE2 (1 uM, Sigma-Aldrich, MO, USA) to induce maturation. For the generation of 5 day DC, cells were cultured with GM-CSF and IL-4 for 5 days. On day 3, half of the medium was replaced with fresh medium containing GM-CSF and IL-4. On day 5, the immature DC were induced into maturation with TNFα (50 ngml), LPS (1 μgml, Sigma-Aldrich) and IFN-γ (50 ngml, R&D systems)./p
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pIsolation of antigen-specific IFN-γ secreting or CD137 positive cells/p
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pPBMC were resuspended in AIM-V plus 5% human AB serum at 1 × 10sup7 /supcellsml and mixed with pentadecapeptides for EBV-LMP2A (10 ugml), mouse anti-human CD28 antibody (Ab, 1 ugml, eBioscience, San Diego, USA) and human β2-microglobulin (1 ugml, Sigma) to enhance antigen presentation and costimulation. The cells were incubated in a 37°C humidified incubator for 3–13 hours. IFN-γ secreting cells were enriched with the IFN-γ Catch Reagent and CD137 positive cells with PE-conjugated monoclonal anti-CD137 Ab (clone 4B4-1), followed by affinity isolation using anti-PE microbeads according to the manufacturer's instruction (Miltenyi Biotech Inc. Auburn, CA, USA)./p
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pGeneration of DC-activated antigen-specific immune cells/p
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pDC-activated antigen-specific immune cells were generated as previously described. abbrgrpabbr bid="B16"16/abbr/abbrgrp In brief, mature 2 day DC were loaded with LMP2A peptides (2.5 ugml) for 2 h and irradiated (20 Gy, or 2,000 rads). The antigen-pulsed DC were cocultured with autologous non-adherent PBMC at a ratio of 1:20 in AIM-V with 5% human AB serum. On day 3, half of the medium was replaced with fresh medium supplemented with IL-2 (12.5 Uml), IL-7 (5 ngml) and IL-15 (20 ngml, all from Gentaur, Aachen, Germany). Half of the medium was replaced with fresh medium with cytokines every other day./p
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pMulti-color flow cytometry/p
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pMature DC were analyzed using a four-color panel of monoclonal Ab including PE-anti-CD14, FITC-anti-HLA-DR, PE-anti-CD86, APC-anti-CD1a, PE-anti-CD 83, APC-anti-CD40 and FITC-anti-HLA-I (BD Biosciences, San Jose, CA, USA), APC-anti-DC-SIGN and PE-cy7-anti-CD11c (eBioscience), and incubated for 30 min at 4°C. Isotype-matched antibodies were used for controls. The Ab-labeled cells were washed twice with PBS containing 1% FBS and analyzed with FACSCaliber or FACSAria using FACSDiva software (BD Biosciences) and Flowjo software (Tree Star, Inc., Ashland, OR, USA). For memory T cell analysis, the cells were stained with PE-anti-IFN-γ or PE-anti-CD137, in combination with APC-anti-CD4 (clone RPA-T4), Pacific blue-anti-CD8 (clone RPA-T8), FITC-anti-CD45RA (clone HI100), PE-cy7-anti-CCR7 (clone 3D12), Percp-cy5.5-anti-CD28 (clone 293, all from BD Biosciences) and Alexa-fluo 750-anti-CD27 (clone O323, eBioscience) at 4°C for 30 min and washed twice with PBS containing 1% FBS. The percentage of different T cell subsets was analyzed using FACSAria with FACSDiva and Flowjo softwares./p
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pImmune effector assays: CD107a degranulation and intracellular cytokine staining/p
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pThese assay were performed as described. abbrgrpabbr bid="B18"18/abbr/abbrgrp Briefly, 2 × 10sup5 /supLMP2A-specific T cells were stimulated for 5 h in a 96-well plate with irradiated (20 Gy) antigen-loaded autologous DC. Monensin A (Sigma-Aldrich) and FITC-conjugated Abs for CD107a or isotype matched Abs (BD Pharmingen, San Diego, CA, USA) were added 1 hour after stimulation and incubated for 5 hours. Cells were then stained with Abs against CD4 and CD8 and fixed, permeabilized with CytofixCytoperm solution and stained with Ab against IFN-γ (all from BD Pharmingen) at 4°C for 20 min. Unrelated peptide group was included as a negative control for spontaneous CD107a expression andor cytokine production./p
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pDetection of peptide-specific CD8+ T cells by MHC multimer analysis/p
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pPeptide-major histocompatibility complex (MHC)-pentamer conjugate specific for EBV-LMP2ATYGPVFMCL (HLA-A*2402 restricted) was purchased from Proimmune (Springfield, VA, USA). T cells were incubated with PE-labeled peptide MHC-pentamer at room temperature for 10 min, washed and stained with APC-anti-CD3 and FITC-anti-CD8 Ab (BD Pharmingen) on ice for 30 min, and analyzed using FACSAria. At least 1 × 10sup5 /supevents were collected for each sample./p
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pT cell proliferation assay with carboxy-fluorescein diacetate succinimidyl ester (CFSE) staining/p
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pThe CFSE-based proliferation assay was performed as previously described abbrgrpabbr bid="B19"19/abbr/abbrgrp. Briefly, LMP2A-specific T cells were washed and labeled with 1 uM CFSE (Molecular Probes, Inc., Eugene, OR, USA). The labeled cells were washed and plated into 96-well U-bottom wells at 1 × 10sup5 /supcells per well. Autologous DC were loaded with peptides (2.5 ugml) for 2 hours and irradiated (20 Gy). The irradiated DC were added to the CFSE-labeled T cells at a ratio of 1:20 and cultured in AIM-V with 5% human AB serum. After 4 days, cells were harvested and analyzed with flow cytometry./p
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pAntigen-specific cytotoxicity assay of immune effector cells/p
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pThe immune cell cytotoxicity assay was based on Jedema et al. with minor modifications. abbrgrpabbr bid="B20"20/abbr/abbrgrp The target cells were washed with PBS, and labeled with 1 uM CFSE (Molecular Probes) at 5 × 10sup6 /supcells per ml at 37°C for 15 minutes. The reaction was stopped with the addition of 10 volumes of complete RPMI containing 10% FCS, followed with a 30 min incubation at 37°C. After two washes, the CFSE-labeled target cells were resuspended in AIM-V containing 5% human AB serum. The cell concentration was adjusted to 1 × 10sup5 /supcellsml before plating into 96-well microtiter plates at 100 ulper well. The effector cells were then mixed with target cells at a ratio of 1:1. The plates were incubated in a humidified atmosphere of 5% COsub2 /suband 37°C. The target cells included irradiated (20 Gy) autologous DC loaded with LMP2A peptides or control WT1 peptides, and irradiated (100 Gy) autologous BLCL. The effector cells included LMP2A peptide-stimulated IFN-γ-selected cells, LMP2A pulsed DC-expanded effector cells, LMP2A peptide-stimulated IFN-γ-negative PBMC, and control PBMC. After 6 hour of incubation, the cells were mixed with 10,000 Flow-Count Fluorospheres (BD Pharmingen) and followed by flow cytometry analysis. To stain for dead cells, 7-AAD (10 ugml) was added and incubated for 30 min on ice. For each sample, 5,000 microbeads were acquired, facilitating the calculation of absolute numbers of target cells. The percentage of survival was determined as the following:/p
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p(R refers to different CTL ratio groups; R = 0 refers to the CTL = 0 control)/p
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pStatistical analysis/p
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pData were analyzed using GraphPad Prism 4 analysis software (GraphPad Software Inc. San Diego, CA) and Student's itt-test/it. A 2-sided P value of less than 0.05 was considered statistically significant./p
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pResults/p
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pBoth IFN-γ and CD137 are effective markers for the isolation of antigen-specific immune effector cells/p
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pThe small population of immune effector cells specific for a particular antigen may be isolated based on expression of antigen-specific activation markers. IFN-γ and CD137 have been identified as antigen-specific activation markers. Upon antigen stimulation, the IFN-γ secreting cells can be captured with anti-IFN-γ Ab conjugated with an anti-surface marker Ab. Alternatively, CD137 positive cells can be directly isolated using anti-CD137 Ab. Specific T cell immune response to the peptide library spanning the entire LMP2A sequence has been previously documented. abbrgrpabbr bid="B21"21/abbr/abbrgrp We first established the expression kinetics of IFN-γ and CD137 in PBMC after stimulation with a pool of EBV LMP2A pentadecapeptides, as reports by others, the expression peaked at 6–12 hr for IFN-γ and 24 hr for CD137 (data not shown). abbrgrpabbr bid="B15"15/abbrabbr bid="B22"22/abbr/abbrgrp The IFN-γ- and CD137-expressing cells were isolated using Ab-conjugated magnetic bead affinity columns (Miltenyi Biotech). After examining a large number of donors, we found that the antigen-specific response of an individual could be either CD8 or CD4 T cell dominant, which can be donor- andor antigen-dependent. Representative results are illustrated in Fig. figr fid="F1"1A/figr (a CD4-dominant donor to the left, and a CD8-dominant donor to the right). The results indicated that the CD4CD8 ratio of the IFN-γ-selected cells correlated with the donor's T cell dominance phenotype. However, the CD137-enriched cells consistently showed an increased CD4 to CD8 ratio regardless of the donor's phenotype (Fig. figr fid="F1"1A/figr, bottom); the latter could be due to the increased proportion of CD137 positive CD4 cells in the total population of T cells before and after stimulation (Fig. figr fid="F1"1B/figr). However, quantitative analysis of the expression level of CD137 demonstrated that CD8 T cells expressed higher density of surface CD137 than did CD4 T cells in both CD4 and CD8 dominant individuals (see geometric means in Fig. figr fid="F1"1C/figr)./p
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pFigure 1/p
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pIFN-γ- or CD137-based enrichment of antigen-specific immune effector cells/p
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pbIFN-γ- or CD137-based enrichment of antigen-specific immune effector cells/b. (A) CD4 and CD8 T cell distribution in IFN-γ- or CD137-positive cell population after antigen stimulation. The ratios of CD4 to CD8 T cells of five donors were presented. (B) and (C) CD137 expression in CD4 and CD8 T cells before and after Ab affinity column purification. PBMC were mixed with EBV LMP2 pentadecapeptides and 3–24 hr later, IFN-γ-secreting cells or CD137-positive cells were isolated by using MACS magnetic bead affinity columns as described in Materials and Methods. The cells were stained with PE-conjugated anti-IFN-γ or anti-CD137 Ab and CD3, CD4 and CD8 specific Ab and analyzed with flow cytometry./p
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graphic file="1756-8722-2-34-1"/
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pThe antigen-activated IFN-γ and CD137 positive immune cells display different surface phenotypes/p
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pThe phenotype of the immune cells of the two affinity isolation methods has not been characterized in the past due to the limited number of the total harvested cells. We subjected the IFN-γ- and CD137 affinity-purified cells to a multi-color flow cytometry analysis for central memory (Tcm, CCR7+, CD45RA-), effector memory (Tem, CCR7-, CD45RA-), terminal effector (Teff, CCR7-, CD45RA+), differentiation (CD27 and CD28) and migration (CCR7) markers of T cells in addition to CD4, CD8 and IFN-γ (or CD137). We found that there was a trend of increased early-differentiated Tcm and Tem cells in the dominant populations (Fig. figr fid="F2"2A/figr and figr fid="F2"2B/figr, CD4- and CD8-dominant donors, respectively)./p
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pFigure 2/p
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pPhenotype analysis of IFN-γ- or CD137-selected antigen-specific immune cells/p
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pbPhenotype analysis of IFN-γ- or CD137-selected antigen-specific immune cells/b. PBMC were stimulated with EBV LMP2A pentadecapeptides and IFN-γ or CD137 positive cells were isolated for analysis. (A) & (B) Memory and effector phenotype analysis based on seven-color flow cytometry with surface staining for CD27, CD28, CD45RA, CCR7, CD4, CD8, CD137 and intracellular staining for IFN-γ immediately after cell isolation without further culture. The percentage of different populations (IFN-γ or CD137 plus CD4 or CD8 gated) of T cells is illustrated for a CD4-dominant donor (A) and a CD8-dominant donor (B); Tcm, central memory T cells (CD27+-, CD28+, CCR7+, CD45RA-); Tem, effector memory T cells (CD27+-, CD28+-, CCR7-, CD45RA-); Teff, terminal effector T cells (CD27-, CD28-, CCR7+-, CD45RA+)./p
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graphic file="1756-8722-2-34-2"/
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pitEx vivo /itexpansion of antigen-activated IFN-γ and CD137 positive immune cells/p
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pTo see if the enriched immune effector cells could be further expanded, we cultured them on irradiated autologous feeder PBMC; the cells expanded approximately 600-fold in two weeks (not shown). Before expansion, the CD137 enriched cells contained a higher ratio of CD4 T cells (Fig. figr fid="F1"1B/figr, 37.6 vs. 13.1); however, the donor's dominant phenotype was restored after expansion in culture (Fig. figr fid="F3"3A/figr). Furthermore, we consistently observed an increased population of CD3sup-/supCD56sup+ /supcells in the CD137+ cell expansion culture compared with the IFN-γ cell expansion culture (Fig. figr fid="F3"3A/figr top, 32.7% and 8.5% versus 2.3% and 2.6%, for a CD4-dominant and a CD8-dominant donors, respectively). Compared with the freshly isolated effector cells that contained more memory effector cells (Fig. figr fid="F2"2/figr), the cultured cells differentiated toward Tem and (Teff) cells after expansion (Fig. figr fid="F3"3B/figr and figr fid="F3"3C/figr). Intracellular staining for effector cytokines showed that the expanded IFN-γ and CD137 cells displayed high antigen-specific activities, up to 59% and 34% for IFN-γ and CD137 selected cells, respectively, when restimulated with DC-pulsed with the specific antigens (LMP2A petadecapeptides) or autologous EBV positive BLCL (Fig. figr fid="F4"4A/figr and figr fid="F4"4B/figr, control: WT-1 peptide-pulsed DC). To demonstrate antigen-specific cytolytic activity, we incubated these cells with different target cells as illustrated in Fig. figr fid="F4"4C/figr. The effector cells killed specific target cells with high specificity including autologous BLCL and LMP2A peptide-pulsed DC, but not control WT-1 peptide-pulsed DC (Fig. figr fid="F4"4C/figr, effector to target ratio 1:1)./p
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pFigure 3/p
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pPhenotype analyses of IFN-γ- or CD137-selected immune effector cells after itex vivo /itexpansion/p
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pbPhenotype analyses of IFN-γ- or CD137-selected immune effector cells after itex vivo /itexpansion/b. (A) Phenotype analysis after itex vivo /itexpansion. After the rapid selection, the antigen-specific immune cells were cultured for fifteen days with irradiated autologous PBMC as feeder cells. The distribution of CD3, CD4, CD8 and CD56 cell populations was determined and representative FACS graphs are shown. (B) and (C) Memory and effector phenotype analysis based on seven-color flow cytometry with surface staining for CD27, CD28, CD45RA, CCR7, CD4, CD8, CD137 and intracellular staining for IFN-γ. Representative results of a CD4-dominant donor (B) and a CD8-dominant donor (C) are shown./p
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graphic file="1756-8722-2-34-3"/
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pFigure 4/p
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pEffector function analyses of IFN-γ- or CD137-selected immune effector cells after itex vivo /itexpansion/p
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pbEffector function analyses of IFN-γ- or CD137-selected immune effector cells after itex vivo /itexpansion/b. (A) and (B) Flow cytometry analysis of IFN-γ and CD137 expression after restimulation of the culture expanded IFN-γ or CD137 effector cells. The cells were restimulated with different cells as indicated and subjected to antibody staining and flow cytometry analysis; control, WT-1 peptide-pulsed DC; specific, LMP2A peptide-pulsed DC; BLCL, autologous EBV-transformed B cells. (C) Analysis of antigen-specific cytolytic activity. The itex vivo /itexpanded IFN-γ- or CD137-enriched cells were incubated with autologous EBV transformed B cells (BLCL), mature DCs loaded with either LMP2A pentadecapeptides (DC-LMP2A) or WT1 pentadecapeptides (DC-WT1, as control) at 1:1 ratio in a cytotoxicity assay based on CFSE labeling as described in Materials and Methods./p
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graphic file="1756-8722-2-34-4"/
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pitEx vivo /itexpansion of antigen-specific immune effector cells with dendritic cells (DC)/p
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pAntigen-specific immune effector cells can also be generated through DC activation. The latter protocol includes a DC preparation step, followed by antigen exposure and lymphocyte coculture. For the preparation of DC, we compared the 2 day and the 5 day protocols. abbrgrpabbr bid="B23"23/abbr/abbrgrp We analyzed the surface markers for plastic-adherent monocytes and mature DC with flow cytometry (data shown in Additional file supplr sid="S1"1/supplr), and analyzed phenotypes of the 2 day and the 5 day mature DC (Fig. figr fid="F5"5/figr). The 2 day DC expressed higher levels of class III MHC (HLA-I and HLA-DR), costimulatory molecules (CD86 and CD40) and maturation marker CD83. We also found that the 2 day DC induced primary and secondary immune response against viral or cancer antigens at efficiencies equal to or better than the 5 day DC (manuscript in preparation). Therefore, the 2 day DC protocol was adopted for later experiments./p
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pAdditional file 1/p
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pbPhenotype analysis of monocytes and 2 day and 5 day mature DC/b. Surface markers related to antigen presentation function were analyzed using fluorochrome-labeled Ab. The light-colored lines in the FACS graphs represent control Ab and the numbers represent geometric means with percentages shown in parentheses. Representatives of two monocyte experiments and three DC experiments are illustrated./p
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pClick here for file/p
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pFigure 5/p
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pPhenotype comparison of 5 day versus 2 day mature DC/p
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pbPhenotype comparison of 5 day versus 2 day mature DC/b. Surface markers related to antigen presentation function were stained with multi-color fluorochrome-labeled Ab and analyzed with FACSAria. Data represent MFI of mature DC generated from three healthy donors with p value calculated./p
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graphic file="1756-8722-2-34-5"/
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pThe 2 day DC were pulsed with the pooled LMP2A pentadecapeptides, irradiated and cocultured with autologous lymphocytes at a ratio of 1:20. The cell number usually decreases around day 5 after coculture, followed by an increase of a few fold around day 17, suggesting a loss of non-specific cells followed by expansion of antigen-specific cells (see representative growth curves in Fig. figr fid="F6"6A/figr). Flow cytometry analysis of the DC-activated cells in culture from four different donors at day 0, 12 and 19 indicated that most donors generated CD3+CD56- T cells, but some generated a large proportion of CD3-CD56+ NK cells and CD3+CD56+ cells (e.g. donor 3, Fig. figr fid="F6"6B/figr). The relative ratios of CD4+ T cells, CD8+ T cells, NK cells and CD3+CD56+ cells in the coculture appeared to be donor-dependent (Fig. figr fid="F6"6B/figr and figr fid="F6"6C/figr). Furthermore, phenotype analysis with multi-color flow cytometry demonstrated that the itex vivo /itexpanded cells contained mostly Tem and Teff cells (Fig. figr fid="F7"7A/figr and figr fid="F7"7B/figr, representative of CD4- and CD8-dominant donors)./p
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pFigure 6/p
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pitEx vivo /itexpansion and phenotype analyses of DC-activated immune effector cells/p
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pbitEx vivo /itexpansion and phenotype analyses of DC-activated immune effector cells/b. EBV LMP2A-specific effector T cells were generated by stimulation of non-adherent PBMC with DC pulsed with LMP2A pentadecapeptides. (A) Growth kinetics of DC-activated immune effector cells. The viable cells were counted with trypan blue staining at different time points after coculture and the growth curves of 5 samples are shown. (B) and (C) CD3, CD56, CD4 and CD8 phenotype analysis. The DC-activated cells from day 0, 12 and 19 were stained with antibodies against CD4, CD8, CD3 and CD56 and analyzed with flow cytometry. The percentages of different lymphocyte subsets were analyzed and shown in bar graphs. Donor 1*, PBMC collected at a different time point from donor 1./p
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graphic file="1756-8722-2-34-6"/
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pFigure 7/p
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pMemory and effector T cell analyses of DC-activated immune effector cells/p
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pbMemory and effector T cell analyses of DC-activated immune effector cells/b. (A) & (B) Memory and effector phenotype analysis of a CD4 dominant donor (A) and a CD8 dominant donor (B). The DC-activated cells from day 0, 12, and 17 after coculture were analyzed for CD3, CD4, CD8, CD27, CD28, CD45RA, and CCR7 using a seven-color panel of fluorochrome-labeled Ab with FACSAria. The cells were gated for CD3 and CD4 or CD8 as total cells for the percentage analysis. One representative flow graph of 5 performed experiments is presented./p
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graphic file="1756-8722-2-34-7"/
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pThe DC-activated immune cells display antigen-specific effector functions/p
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pWe next evaluated the DC-activated immune cells for antigen-specific effector functions based on intracellular staining of IFN-γ, IL-2, and the degranulation marker CD107a after restimulation with peptide-loaded DC (Fig. figr fid="F8"8A/figr). For direct comparison, the same donor whose cells were analyzed for the IFN-γ-selected effector functions (Fig. figr fid="F4"4B/figr) was chosen. Analyses of the immune effector cells without further stimulation (IE cell alone), or stimulated with non-specific peptides (Non-specific) or with the LMP2A pentadecapeptides (LMP2A-specific) demonstrated LMP2A-specific expression of IFN-γ, IL-2 and CD107a in the CD8 T cells, and to a lesser extent, the CD4 T cells (Fig. figr fid="F8"8A/figr top: IL-2 and IFN-γ, and bottom: CD107a and IFN-γ). The antigen-specific effector function was further confirmed with the CFSE-based proliferation assay, as well as MHC-peptide pentamer specific for the LMP2A epitope-specific T cell receptors of the CD8 T cells (data not shown)./p
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pFigure 8/p
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pFunctional analyses of DC-activated and itex vivo /itexpanded immune effector cells/p
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pbFunctional analyses of DC-activated and itex vivo /itexpanded immune effector cells/b. EBV LMP2A-specific effector cells were generated by stimulation of non-adherent PBMC with DC pulsed with LMP2A pentadecapeptides. (A) Analysis of antigen-specific effector cytokines and CD107a expression. The DC-activated immune cells from day 19 coculture were stimulated with autologous DC pulsed with LMP2A peptides, WT1 peptides (non-specific control) or no stimulation (IE cell alone) for 6 hours. The cells were stained with Ab against CD4, CD8, CD107a and IFN-γ. Flow cytometry analyses of IL-2, IFN-γ and CD107a-positive cells in CD4-gated or CD8-gated populations were illustrated as representative of five experiments. (B) Comparison of cytolytic function of IFN-γ-selected (same donor as in Fig. 4C) versus DC-activated LMP2-specific immune effector cells. The LMP2A-specific effector cells were mixed with target cells including autologous BLCL, DC-LMP2A, or control DC-WT1 at 1:1 ratio and analyzed for cytolytic activity based on the CFSE-labeling method as described in Materials and Methods./p
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graphic file="1756-8722-2-34-8"/
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pTo demonstrate antigen-specific killing, we directly compared the DC-activated cells to the IFN-γ captured cells (as shown in Fig. figr fid="F4"4C/figr) using an itin vitro /itcytolytic assay (Fig. figr fid="F8"8B/figr). At an effector to target cell ratio of 1:1, the immune cells effectively killed autologous BLCL and LMP2A-pulsed DC (DC-LMP2A), but not DC pulsed with control WT1 pentadecapeptides (DC-WT1). Therefore, effector function analyses including IFN-γ release and cytotoxicity assays suggest that the DC-activated cells had lower activities than the expanded IFN-γ effector cells./p
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pDiscussion/p
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pAdoptive immune cell therapy has shown great promise in treating viral diseases and melanoma. abbrgrpabbr bid="B2"2/abbrabbr bid="B6"6/abbr/abbrgrp Continued efforts are focused on the generation of sufficient amount of antigen-specific immune cells and optimal conditioning of immune homeostasis in patients in order to achieve a sustained itin vivo /itimmune surveillance. abbrgrpabbr bid="B24"24/abbrabbr bid="B25"25/abbrabbr bid="B26"26/abbr/abbrgrp Here, we compared three itex vivo /itimmune cell preparation protocols and phenotypically and functionally characterized these cells. The rapid protocols based on IFN-γ and CD137 selection generate a small number of antigen-specific effector cells with high percentage of central memory T cells in a very short period of time. The DC-activation protocol generates more immune cells, albeit, with more differentiated phenotype and reduced proportion of antigen-specific effector cells./p
pBased on analyses of a large number of donors, we found that individual response to a given antigen could be either CD4- or CD8-dominant, which is antigen- and donor-dependent. Immune effector cells isolated based on IFN-γ expression displayed a CD4 or CD8 bias consistent with the donor's immune dominance. However, antigen-specific CD137 positive cells showed a higher ratio of CD4 effector cells regardless of the subject's immune phenotype; this is in contrast to previous reports that emphasize the induction of CD8 effector cells after CD137 enrichment. abbrgrpabbr bid="B15"15/abbrabbr bid="B22"22/abbrabbr bid="B27"27/abbr/abbrgrp We did, however, show that CD8 T cells displayed higher density of CD137 than did CD4 T cells. It is well documented that CD137 costimulation promotes both CD4 and CD8 T cell expansion and long term memory. abbrgrpabbr bid="B28"28/abbrabbr bid="B29"29/abbrabbr bid="B30"30/abbr/abbrgrp Our finding that more CD4 T cells than CD8 T cells are detected in the CD137-positive cell population suggests a rapid induction of CD137 in the memory T helper repertoire immediately after antigen stimulation. Although the enriched CD137 immune cells contained a higher CD4 T cell ratio, further expansion in culture restored the donor's original dominant phenotype, with a higher CD3-CD56+ NK cell population than those found in the expanded IFN-γ enriched immune cells (Fig. figr fid="F3"3A/figr). This result suggests that IFN-γ is a more restricted adoptive immune response marker and represent less of an innate immune marker as does CD137./p
pThe itex vivo /itDC-activation protocol generated different ratios of CD4, CD8 and NK cells in culture, which again, appeared to be donor-dependent. Whether the immune dominance has any effect on itin vivo /itefficacy of the cultured immune effector cells awaits further investigation. As CD4 T cells are important for the maintenance of long-term anti-viral CD8 T cell memory abbrgrpabbr bid="B31"31/abbr/abbrgrp, therapeutic immune cells should include polyclonal CD4 and CD8 T cells. Both IFN-γ and CD137 selection approaches generated increased number of memory type of cells representative of polyclonal CD4 and CD8 T cells that may have increased proliferation potential after infusion. Although the antigen-specific memory T cells from PBMC may be low; for examples, the average yield of LMP2A-specific IFN-γ positive immune effector cells from healthy EBV-seropositive donors is only 0.22 ± 0.13% (n = 6, after two rounds of affinity column purification), they can be expanded to more than two orders of magnitude in culture in two weeks and maintain their high antigen specificity./p
pIt is evident that the LMP2A pentadecapeptides efficiently activate both CD4 and CD8 T cells in a short exposure period (3–13 hr). This was surprising since CD8 T cells are activated through class I MHC loaded with short 9–11 amino acid peptide epitopes, different from CD4 T cells, which are activated through class II MHC loaded with 12–15 amino acid peptide epitopes. The pentadecapeptide antigens apparently activated CD8 T cells with high efficiency through cross-presentation. This has been confirmed with various pentadecapeptide antigens (unpublished). The processing of class II MHC peptides into class I epitopes for cross-presentation to CD8 T cells appears to be highly efficient with both the IFN-γ and the CD137 protocols, as with the DC-activation method. The detailed molecular mechanism of the efficient cross-presentation requires further investigation./p
pTo assess differentiation and maturation status of the itex vivo /itgenerated T cells, we applied multi-color flow cytometry to detect differentiation and homeostatic marker CD45RA, trafficking marker CCR7, and costimulatory marker CD27 and CD28. abbrgrpabbr bid="B32"32/abbrabbr bid="B33"33/abbr/abbrgrp It is not surprising that both the IFN-γ- and the CD137-enriched antigen-specific effector cells displayed more memory markers than did the DC coculture-expanded cells. While preserving Tcm cells is critical to itin vivo /ittherapeutic efficacy,abbrgrpabbr bid="B7"7/abbrabbr bid="B34"34/abbrabbr bid="B35"35/abbr/abbrgrp clinical studies have proven that itex vivo /itexpanded effector cells can persist many years after infusion. abbrgrpabbr bid="B36"36/abbr/abbrgrp Clinical benefits of these different protocols will require detailed evaluation in a large cohort of patients./p
pThe differentiation status of the itex vivo /itgenerated immune cells may contribute to their itin vivo /ittherapeutic efficacy. Homeostasis of antigen-specific memory cells can vary depending on antigen source, the immune milieu and individual donor. It is known that Tcm cells are mainly located in lymphoid tissues and Tem cells are distributed in diverse non-lymphoid sites including lung, liver and intestine. abbrgrpabbr bid="B37"37/abbr/abbrgrp In addition, bone marrow has been shown to embrace increased number of anti-cancer or anti-virus memory T cells. abbrgrpabbr bid="B38"38/abbrabbr bid="B39"39/abbrabbr bid="B40"40/abbr/abbrgrp After itex vivo /itexpansion, however, wherever the T cells come from, they tend to bestow exhausted proliferation and replicative senescence associated with down-regulation of anti-apoptotic protein Bcl-2 and Bcl-xL, and decreased telomere length. abbrgrpabbr bid="B33"33/abbrabbr bid="B34"34/abbrabbr bid="B41"41/abbr/abbrgrp Modification of antigen presentation protocol and culture condition may help overcome the immune cell exhaustion problem. abbrgrpabbr bid="B9"9/abbrabbr bid="B42"42/abbr/abbrgrp/p
pFor patients with acute infections or illness, direct isolation of antigen-specific immune cells from partly HLA-matched healthy donors represents an attractive emergency approach to obtain therapeutic cells. abbrgrpabbr bid="B43"43/abbrabbr bid="B44"44/abbr/abbrgrp This approach offers several advantages including a shortened handling time and increased proliferation potential itin vivo/it. Although the number of immune cells is limited with the direct isolation approach, clinical evidence supports that only a small number of such immune cells, in the range of 10sup3/sup-10sup4/supkg body weight, is sufficient to attain therapeutic efficacy in transplant patients. abbrgrpabbr bid="B13"13/abbrabbr bid="B45"45/abbrabbr bid="B46"46/abbr/abbrgrpitEx vivo /itexpansion of immune cells, nevertheless, may be necessary for patients with a compromised immunity. abbrgrpabbr bid="B47"47/abbrabbr bid="B48"48/abbr/abbrgrp/p
/sec
sec
st
pConclusion/p
/st
pThe two rapid immune cell isolation methods generate functional effector cells in less than 24–48 hr suitable for emergency immune cell preparation. On the other hand, the DC-activation method expands antigen-specific immune effector cells while effectively reduce the number of non-specific cells. Depending on clinical needs, for examples, the urgency for treatment, patient's body weight (e.g. less cells are needed for pediatric patients), or patient's immune cell proliferative potential itin vivo/it, the method of immune cell preparation may differ. Our data indicate that IFN-γ selection followed by itex vivo /itexpansion represents the best approach for the generation of high amount of antigen-specific immune effector cells. Further efforts to overcome immune tolerance and expand antigen-specific immune cells with prolonged itin vivo /itpersistence are critical to the success of immune cell therapy./p
/sec
sec
st
pList of abbreviations/p
/st
pIFN-γ: interferon-gamma; IL: interleukine; DC: dendritic cell; CTL: cytotoxic T lymphocyte; MHC: major histocompatibility complex; Ab: antibody; Ag: antigen; TCR: T cell receptor; TNF: tumor necrosis factor; BLCL: B lymphoblastoid cell line; EBV: Epstein-Barr virus; LMP2A: late membrane protein 2A; CMV: cytomegalovirus; ICCS: intracellular cytokine staining; CFSE: carboxy-fluorescein diacetate succinimidyl ester./p
/sec
sec
st
pCompeting interests/p
/st
pYH, YL, YH and YW are employees of Vectorite Biomedica Inc. LJC is consultant to a biotech company./p
/sec
sec
st
pAuthors' contributions/p
/st
pAll authors are accountable for the integrity of the research results; Chang is responsible for the conception of the research and Han, Huang, Liang, Ho and Wang are responsible for the execution and for data collection; Chang is responsible for initial drafting and revisions of the manuscript./p
/sec
/bdy
bm
ack
sec
st
pAcknowledgements/p
/st
pWe thank the technical assistance of Liheng Guo, Lily Lien, Fuhung Yang, Yinchieh Fu and Meifang Lin. The study was funded by Vectorite Biomedica Inc. and Yongling Foundation./p
/sec
/ack
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au
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au
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au
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au
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Abstract
Ex vivo activation and expansion of lymphocytes for adoptive cell therapy has demonstrated great success. To improve safety and therapeutic efficacy, increased antigen specificity and reduced non-specific response of the ex vivo generated immune cells are necessary. Here, using a complete protein-spanning pool of pentadecapeptides of the latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV), a weak viral antigen which is associated with EBV lymphoproliferative diseases, we investigated the phenotype and function of immune effector cells generated based on IFN-γ or CD137 activation marker selection and dendritic cell (DC) activation. These ex vivo prepared immune cells exhibited a donor- and antigen-dependent T cell response; the IFN-γ-selected immune cells displayed a donor-related CD4- or CD8-dominant T cell phenotype; however, the CD137-enriched cells showed an increased ratio of CD4 T cells. Importantly, the pentadecapeptide antigens accessed both class II and class I MHC antigen processing machineries and effectively activated EBV-specific CD4 and CD8 T cells. Phenotype and kinetic analyses revealed that the IFN-γ and the CD137 selections enriched more central memory T (Tcm) cells than did the DC-activation approach, and after expansion, the IFN-γ-selected effector cells showed the highest level of antigen-specificity and effector activities. While all three approaches generated immune cells with comparable antigen-specific activities, the IFN-γ selection followed by ex vivo expansion produced high quality and quantity of antigen-specific effector cells. Our studies presented the optimal approach for generating therapeutic immune cells with potential for emergency and routine clinical applications.
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Han, Shuhong
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Shuhong Han et al.; licensee BioMed Central Ltd.
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Journal of Hematology & Oncology. 2009 Aug 06;2(1):34
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Journal of Hematology & Oncology 0
BioMed Central


Research


Open Access I


Phenotype and functional evaluation of ex vivo generated
antigen-specific immune effector cells with potential for
therapeutic applications
Shuhong Han', Yuju Huang2, Yin Liang2, Yuchin Ho2, Yichen Wang2 and
Lung-Ji Chang*


Address: 'Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL 32610-0266 and
2Vectorite Biomedica, Inc., Taipei, Taiwan, Republic of China
Email: Shuhong Han hansh72@ufl.edu; Yuju Huang Yuju.huang@vectorite.com; Yin Liang Yin.liang@vectorite.com;
Yuchin Ho Malineho@gmail.com; Yichen Wang yichen.wang@vectorite.com; Lung-Ji Chang* lchang@mgm.ufl.edu
* Corresponding author



Published: 6 August 2009 Received: 30June 2009
journal ofHematology & Oncology 2009, 2:34 doi:10.1 186/1756-8722-2-34 Accepted: 6 August 2009
This article is available from: http://www.jhoonline.org/content/2/1/34
2009 Han et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Ex vivo activation and expansion of lymphocytes for adoptive cell therapy has demonstrated great
success. To improve safety and therapeutic efficacy, increased antigen specificity and reduced non-
specific response of the ex vivo generated immune cells are necessary. Here, using a complete
protein-spanning pool of pentadecapeptides of the latent membrane protein 2A (LMP2A) of
Epstein-Barr virus (EBV), a weak viral antigen which is associated with EBV lymphoproliferative
diseases, we investigated the phenotype and function of immune effector cells generated based on
IFN-y or CD137 activation marker selection and dendritic cell (DC) activation. These ex vivo
prepared immune cells exhibited a donor- and antigen-dependent T cell response; the IFN-y-
selected immune cells displayed a donor-related CD4- or CD8-dominant T cell phenotype;
however, the CD137-enriched cells showed an increased ratio of CD4 T cells. Importantly, the
pentadecapeptide antigens accessed both class II and class I MHC antigen processing machineries
and effectively activated EBV-specific CD4 and CD8 T cells. Phenotype and kinetic analyses
revealed that the IFN-y and the CD137 selections enriched more central memory T (Tcm) cells
than did the DC-activation approach, and after expansion, the IFN-y-selected effector cells showed
the highest level of antigen-specificity and effector activities. While all three approaches generated
immune cells with comparable antigen-specific activities, the IFN-y selection followed by ex vivo
expansion produced high quality and quantity of antigen-specific effector cells. Our studies
presented the optimal approach for generating therapeutic immune cells with potential for
emergency and routine clinical applications.



Background adenovirus have been successfully applied to treating
The key to the success of immune cell therapy is to hematopoietic stem cell or solid organ transplant patients
increase specific and decrease non-specific immune who have developed post-transplant viral diseases. [1,2]
response. Ex vivo expanded antigen-specific T cells target- The labor-intensive and time-consuming process of isolat-
ing cytomegalovirus (CMV), Epstein-Barr virus (EBV) and ing and expanding antigen-specific immune cells, how-


Page 1 of 16
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Journal of Hematology & Oncology 2009, 2:34


ever, has hindered common practice of this medical
advent.

Donor leukocyte infusion has attained a response rate of
>50% in treating post-transplant infections or cancer
relapse of hematopoietic stem cell transplant (HSCT)
patients. [3-5] Non-specific leukocyte infusion, however,
may cause severe graft-versus-host disease (GvHD) with
high morbidity and mortality. Adoptive transfer of anti-
gen-specific T cell clones combined with clinical regimens
to increase immune cell homeostasis has illustrated high
response rate (50%) in melanoma patients. [6-8] While
the amplified T cell clones display increased antigen-spe-
cific effector functions, the ex vivo expansion process takes
longer than 4-6 weeks, which often results in terminal
differentiation of the T cells with reduced in vivo prolifer-
ation potential. [9]

A few approaches have been developed to directly isolate
antigen-specific immune cells, such as the use of MHC-
peptide multimers and selection of IFN-y-secreting cells
with affinity-magnetic beads. [10-14] Recently, CD137
(4-1BB), a member of the tumor necrosis factor receptor
family, has been reported to be a suitable surface marker
for antigen-specific T cell isolation. [15] Although both
the IFN-y and the CD 137 selection methods generate only
a small number of immune effector cells, these cells may
be further expanded in culture to obtain more functional
cells. [12]

Detailed phenotype and functional characterizations of
these ex vivo prepared immune effector cells are necessary
to facilitate their clinical applications. Here, we refined
and compared three of the state-of-the-art immune effec-
tor cell preparation approaches, the IFN-y and the CD 137
selection methods for emergency preparation of therapeu-
tic cells, and a DC-immune cell coculture method for the
expansion of antigen-specific immune cells. We targeted
EBV using LMP2A pentadecapeptides as antigens because
EBV-associated lymphoproliferative disorders represent
one of the most severe problems in HIV/AIDS patients
and transplantation patients. The IFN-y-selected cells
showed an increased ratio of CD4 or CD8 effector cell
population depending on the donor, whereas the CD 137
selection method enriched a higher ratio of CD4 T cells
regardless of the donor's T cell dominance response. Both
of the rapid protocols yielded more central memory and
effector memory T cells than did the DC-activation
method. Our detailed side-by-side comparison concludes
that IFN-y selection followed by ex vivo expansion repre-
sents the preferred method for the generation of antigen-
specific immune effector cells with potential for clinical
applications.


Methods
Peripheral blood mononuclear cells (PBMC) and B
lymphoblastoid cell lines (BLCL)
Healthy donors' buffy coats were obtained from Civitan
Blood Center (Gainesville, FL, USA). PBMC were prepared
by gradient density centrifugation in Ficoll-Hypaque (GE
Healthcare Bio-Sciences AB, NJ, USA) as previously
described. [16] Viability was determined by trypan blue
staining. Autologous B lymphoblastoid cell line (BLCL)
was generated by transforming peripheral blood B lym-
phocytes with EBV as described previously. [17] The BLCL
were continuously propagated in RPMI 1640 medium
supplemented with 2 mM L-glutamine, 100 lg/ml strep-
tomycin, 100 IU/ml penicillin and 10% heat inactivated
fetal bovine serum (FBS) at 37C with 5% CO2.

Peptides
The mixtures of 11 amino acid overlapping pentade-
capeptides (122 peptides) spanning the entire 497 amino
acids (NCBI Accession number P13285) of LMP2A of the
EBV (human herpesvirus 4, strain B95-8) and the Wilms'
tumor antigen (WT1, 449 amino acids, 110 peptides)
were purchased from JPT Peptide Technologies GmbH
(Berlin, Germany).

Preparation of 2 day and 5 day dendritic cells (DC)
PBMC were plated into 6-well plate at 1 x 107 cells/well
and adhered for 2 hours in AIM-V (Gibco-BRL, CA, USA).
The non-adherent cells were removed gently and frozen as
source oflymphocytes for co-culture use. Adherent mono-
cytes were cultured in AIM-V supplemented with 50 ng/ml
of GM-CSF and 25 ng/ml IL-4 (eBiosource International,
Inc. Camarillo, CA, USA). For the generation of 2 day DC,
the adherent cells were cultured with GM-CSF and IL-4 for
24 h and incubated for another 24 h with TNFa (50 ng/
ml), IL-13 (10 ng/ml), IL-6 (10 ng/ml, all from R&D sys-
tems, MN, USA) and PGE2 (1 uM, Sigma-Aldrich, MO,
USA) to induce maturation. For the generation of 5 day
DC, cells were cultured with GM-CSF and IL-4 for 5 days.
On day 3, half of the medium was replaced with fresh
medium containing GM-CSF and IL-4. On day 5, the
immature DC were induced into maturation with TNFa
(50 ng/ml), LPS (1 gg/ml, Sigma-Aldrich) and IFN-y (50
ng/ml, R&D systems).

Isolation of antigen-specific IFN-y secreting or CD 137
positive cells
PBMC were resuspended in AIM-V plus 5% human AB
serum at 1 x 107 cells/ml and mixed with pentadecapep-
tides for EBV-LMP2A (10 ug/ml), mouse anti-human
CD28 antibody (Ab, 1 ug/ml, eBioscience, San Diego,
USA) and human p2-microglobulin (1 ug/ml, Sigma) to
enhance antigen presentation and costimulation. The
cells were incubated in a 37C humidified incubator for
3-13 hours. IFN-y secreting cells were enriched with the


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Journal of Hematology & Oncology 2009, 2:34


IFN-y Catch Reagent and CD137 positive cells with PE-
conjugated monoclonal anti-CD137 Ab (clone 4B4-1),
followed by affinity isolation using anti-PE microbeads
according to the manufacturer's instruction (Miltenyi Bio-
tech Inc. Auburn, CA, USA).

Generation of DC-activated antigen-specific immune cells
DC-activated antigen-specific immune cells were gener-
ated as previously described. [16] In brief, mature 2 day
DC were loaded with LMP2A peptides (2.5 ug/ml) for 2 h
and irradiated (20 Gy, or 2,000 rads). The antigen-pulsed
DC were cocultured with autologous non-adherent PBMC
at a ratio of 1:20 in AIM-V with 5% human AB serum. On
day 3, half of the medium was replaced with fresh
medium supplemented with IL-2 (12.5 U/ml), IL-7 (5 ng/
ml) and IL-15 (20 ng/ml, all from Gentaur, Aachen, Ger-
many). Half of the medium was replaced with fresh
medium with cytokines every other day.

Multi-color flow cytometry
Mature DC were analyzed using a four-color panel of
monoclonal Ab including PE-anti-CD14, FITC-anti-HLA-
DR, PE-anti-CD86, APC-anti-CD1a, PE-anti-CD 83, APC-
anti-CD40 and FITC-anti-HLA-I (BD Biosciences, San
Jose, CA, USA), APC-anti-DC-SIGN and PE-cy7-anti-
CDllc (eBioscience), and incubated for 30 min at 40C.
Isotype-matched antibodies were used for controls. The
Ab-labeled cells were washed twice with PBS containing
1% FBS and analyzed with FACSCaliber or FACSAria
using FACSDiva software (BD Biosciences) and Flowjo
software (Tree Star, Inc., Ashland, OR, USA). For memory
T cell analysis, the cells were stained with PE-anti-IFN-y or
PE-anti-CD137, in combination with APC-anti-CD4
(clone RPA-T4), Pacific blue-anti-CD8 (clone RPA-T8),
FITC-anti-CD45RA (clone HI100), PE-cy7-anti-CCR7
(clone 3D12), Percp-cy5.5-anti-CD28 (clone 293, all
from BD Biosciences) and Alexa-fluo 750-anti-CD27
(clone 0323, eBioscience) at 4 C for 30 min and washed
twice with PBS containing 1% FBS. The percentage of dif-
ferent T cell subsets was analyzed using FACSAria with
FACSDiva and Flowjo software.

Immune effector assays: CD107a degranulation and
intracellular cytokine staining
These assay were performed as described. [18] Briefly, 2 x
105 LMP2A-specific T cells were stimulated for 5 h in a 96-
well plate with irradiated (20 Gy) antigen-loaded autolo-
gous DC. Monensin A (Sigma-Aldrich) and FITC-conju-
gated Abs for CD107a or isotype matched Abs (BD
Pharmingen, San Diego, CA, USA) were added 1 hour
after stimulation and incubated for 5 hours. Cells were
then stained with Abs against CD4 and CD8 and fixed,
permeabilized with Cytofix/Cytoperm solution and
stained with Ab against IFN-y (all from BD Pharmingen)
at 4 C for 20 min. Unrelated peptide group was included


as a negative control for spontaneous CD107a expression
and/or cytokine production.

Detection of peptide-specific CD8+ T cells by MHC
multimer analysis
Peptide-major histocompatibility complex (MHC)-pen-
tamer conjugate specific for EBV-LMP2A/TYGPVFMCL
(HLA-A*2402 restricted) was purchased from Proim-
mune (Springfield, VA, USA). T cells were incubated with
PE-labeled peptide MHC-pentamer at room temperature
for 10 min, washed and stained with APC-anti-CD3 and
FITC-anti-CD8 Ab (BD Pharmingen) on ice for 30 min,
and analyzed using FACSAria. At least 1 x 105 events were
collected for each sample.

T cell proliferation assay with carboxy-fluorescein
diacetate succinimidyl ester (CFSE) staining
The CFSE-based proliferation assay was performed as pre-
viously described [19]. Briefly, LMP2A-specific T cells
were washed and labeled with 1 uM CFSE (Molecular
Probes, Inc., Eugene, OR, USA). The labeled cells were
washed and plated into 96-well U-bottom wells at 1 x 105
cells per well. Autologous DC were loaded with peptides
(2.5 ug/ml) for 2 hours and irradiated (20 Gy). The irradi-
ated DC were added to the CFSE-labeled T cells at a ratio
of 1:20 and cultured in AIM-V with 5% human AB serum.
After 4 days, cells were harvested and analyzed with flow
cytometry.

Antigen-specific cytotoxicity assay of immune effector
cells
The immune cell cytotoxicity assay was based on Jedema
et al. with minor modifications. [20] The target cells were
washed with PBS, and labeled with 1 uM CFSE (Molecular
Probes) at 5 x 106 cells per ml at 37 C for 15 minutes. The
reaction was stopped with the addition of 10 volumes of
complete RPMI containing 10% FCS, followed with a 30
min incubation at 370C. After two washes, the CFSE-
labeled target cells were resuspended in AIM-V containing
5% human AB serum. The cell concentration was adjusted
to 1 x 105 cells/ml before plating into 96-well microtiter
plates at 100 ul/per well. The effector cells were then
mixed with target cells at a ratio of 1:1. The plates were
incubated in a humidified atmosphere of 5% CO2 and
37 C. The target cells included irradiated (20 Gy) autolo-
gous DC loaded with LMP2A peptides or control WT1
peptides, and irradiated (100 Gy) autologous BLCL. The
effector cells included LMP2A peptide-stimulated IFN-y-
selected cells, LMP2A pulsed DC-expanded effector cells,
LMP2A peptide-stimulated IFN-y-negative PBMC, and
control PBMC. After 6 hour of incubation, the cells were
mixed with 10,000 Flow-Count Fluorospheres (BD
Pharmingen) and followed by flow cytometry analysis. To
stain for dead cells, 7-AAD (10 ug/ml) was added and
incubated for 30 min on ice. For each sample, 5,000


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Journal of Hematology & Oncology 2009, 2:34


microbeads were acquired, facilitating the calculation of
absolute numbers of target cells. The percentage of sur-
vival was determined as the following:

% Survival = Absolute # of viable CFSE+target cells(R=x) 100
% Survival = ------------ -x 100
Absolute # of viable CFSE+target cells(R=0)

(R refers to different CTL ratio groups; R = 0 refers to the
CTL = 0 control)

Statistical analysis
Data were analyzed using GraphPad Prism 4 analysis soft-
ware (GraphPad Software Inc. San Diego, CA) and Stu-
dent's t-test. A 2-sided P value of less than 0.05 was
considered statistically significant.

Results
Both IFN-yand CD 137 are effective markers for the
isolation of antigen-specific immune effector cells
The small population of immune effector cells specific for
a particular antigen may be isolated based on expression
of antigen-specific activation markers. IFN-y and CD137
have been identified as antigen-specific activation mark-
ers. Upon antigen stimulation, the IFN-y secreting cells
can be captured with anti-IFN-y Ab conjugated with an
anti-surface marker Ab. Alternatively, CD137 positive
cells can be directly isolated using anti-CD137 Ab. Spe-
cific T cell immune response to the peptide library span-
ning the entire LMP2A sequence has been previously
documented. [21] We first established the expression
kinetics of IFN-y and CD137 in PBMC after stimulation
with a pool of EBV LMP2A pentadecapeptides, as reports
by others, the expression peaked at 6-12 hr for IFN-y and
24 hr for CD 137 (data not shown). [15,22] The IFN-y- and
CD137-expressing cells were isolated using Ab-conju-
gated magnetic bead affinity columns (Miltenyi Biotech).
After examining a large number of donors, we found that
the antigen-specific response of an individual could be
either CD8 or CD4 T cell dominant, which can be donor-
and/or antigen-dependent. Representative results are
illustrated in Fig. 1A (a CD4-dominant donor to the left,
and a CD 8-dominant donor to the right). The results indi-
cated that the CD4/CD8 ratio of the IFN-y-selected cells
correlated with the donor's T cell dominance phenotype.
However, the CD137-enriched cells consistently showed
an increased CD4 to CD8 ratio regardless of the donor's
phenotype (Fig. 1A, bottom); the latter could be due to
the increased proportion of CD137 positive CD4 cells in
the total population ofT cells before and after stimulation
(Fig. 1B). However, quantitative analysis of the expression
level of CD137 demonstrated that CD8 T cells expressed
higher density of surface CD137 than did CD4 T cells in
both CD4 and CD8 dominant individuals (see geometric
means in Fig. 1C).


The antigen-activated IFN-y and CD 137 positive immune
cells display different surface phenotypes
The phenotype of the immune cells of the two affinity iso-
lation methods has not been characterized in the past due
to the limited number of the total harvested cells. We sub-
jected the IFN-y- and CD137 affinity-purified cells to a
multi-color flow cytometry analysis for central memory
(Tcm, CCR7+, CD45RA-), effector memory (Tem, CCR7-,
CD45RA-), terminal effector (Teff, CCR7-, CD45RA+),
differentiation (CD27 and CD28) and migration (CCR7)
markers ofT cells in addition to CD4, CD8 and IFN-y (or
CD137). We found that there was a trend of increased
early-differentiated Tcm and Tem cells in the dominant
populations (Fig. 2A and 2B, CD4- and CD8-dominant
donors, respectively).

Ex vivo expansion of antigen-activated IFN-yand CD 137
positive immune cells
To see if the enriched immune effector cells could be fur-
ther expanded, we cultured them on irradiated autologous
feeder PBMC; the cells expanded approximately 600-fold
in two weeks (not shown). Before expansion, the CD137
enriched cells contained a higher ratio of CD4 T cells (Fig.
1B, 37.6 vs. 13.1); however, the donor's dominant pheno-
type was restored after expansion in culture (Fig. 3A). Fur-
thermore, we consistently observed an increased
population of CD3-CD56+ cells in the CD137+ cell expan-
sion culture compared with the IFN-y cell expansion cul-
ture (Fig. 3A top, 32.7% and 8.5% versus 2.3% and 2.6%,
for a CD4-dominant and a CD8-dominant donors,
respectively). Compared with the freshly isolated effector
cells that contained more memory effector cells (Fig. 2),
the cultured cells differentiated toward Tem and (Teff)
cells after expansion (Fig. 3B and 3C). Intracellular stain-
ing for effector cytokines showed that the expanded IFN-y
and CD137 cells displayed high antigen-specific activities,
up to 59% and 34% for IFN-y and CD137 selected cells,
respectively, when restimulated with DC-pulsed with the
specific antigens (LMP2A petadecapeptides) or autolo-
gous EBV positive BLCL (Fig. 4A and 4B, control: WT-1
peptide-pulsed DC). To demonstrate antigen-specific
cytolytic activity, we incubated these cells with different
target cells as illustrated in Fig. 4C. The effector cells killed
specific target cells with high specificity including autolo-
gous BLCL and LMP2A peptide-pulsed DC, but not con-
trol WT-1 peptide-pulsed DC (Fig. 4C, effector to target
ratio 1:1).

Ex vivo expansion of antigen-specific immune effector
cells with dendritic cells (DC)
Antigen-specific immune effector cells can also be gener-
ated through DC activation. The latter protocol includes a
DC preparation step, followed by antigen exposure and
lymphocyte coculture. For the preparation of DC, we
compared the 2 day and the 5 day protocols. [23] We ana-


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Journal of Hematology & Oncology 2009, 2:34


A CD4-dominant donor
IFN-Y+ cells CD137+ cells
74.9 96.4

83
IFN-y CD137

23 09 14.6 0.4
P ,
9 52 ;2..
-CD4



U10-
S20




S IF
IFN-y


CD8-dominant donor
IFN-f+ cells CD137+ cells
36,7 ,90.A



IFN-y CD137

61.6 0.6 52 0.5

16.2 21.6 AU I8.3

SCD4



-I-.


Before selection CD137+ cells


r, 75

0


A 78

/


Flow through


I.5


5.2


|


4.6 2.9 42.9 37.6 35 1.9


2 il6 8.4 M 2 4m

CD4
5.5 1.2 62.8 13.1 4,1 1.0


7 ,4,E. ?0 7 3.5 l0,4 I 6


CD137


S---. isotype
----- CD4+ T cells
CD8+ T cells
CD4-dominant donor CD8-dominant donor

162 278 219 A
278 319





CD137

Figure I
IFN-y- or CDI37-based enrichment of antigen-specific immune effector cells. (A) CD4 and CD8 T cell distribution
in IFN-y- or CD I 37-positive cell population after antigen stimulation. The ratios of CD4 to CD8 T cells of five donors were
presented. (B) and (C) CD 137 expression in CD4 and CD8 T cells before and after Ab affinity column purification. PBMC were
mixed with EBV LMP2 pentadecapeptides and 3-24 hr later, IFN-y-secreting cells or CD I37-positive cells were isolated by
using MACS magnetic bead affinity columns as described in Materials and Methods. The cells were stained with PE-conjugated
anti-IFN-y or anti-CD I37 Ab and CD3, CD4 and CD8 specific Ab and analyzed with flow cytometry.


lyzed the surface markers for plastic-adherent monocytes
and mature DC with flow cytometry (data shown in Addi-
tional file 1), and analyzed phenotypes of the 2 day and
the 5 day mature DC (Fig. 5). The 2 day DC expressed
higher levels of class I/II MHC (HLA-I and HLA-DR), cos-
timulatory molecules (CD86 and CD40) and maturation
marker CD83. We also found that the 2 day DC induced
primary and secondary immune response against viral or
cancer antigens at efficiencies equal to or better than the 5
day DC (manuscript in preparation). Therefore, the 2 day
DC protocol was adopted for later experiments.

The 2 day DC were pulsed with the pooled LMP2A penta-
decapeptides, irradiated and cocultured with autologous


lymphocytes at a ratio of 1:20. The cell number usually
decreases around day 5 after coculture, followed by an
increase of a few fold around day 17, suggesting a loss of
non-specific cells followed by expansion of antigen-spe-
cific cells (see representative growth curves in Fig. 6A).
Flow cytometry analysis of the DC-activated cells in cul-
ture from four different donors at day 0, 12 and 19 indi-
cated that most donors generated CD3+CD56- T cells, but
some generated a large proportion of CD3-CD56+ NK
cells and CD3+CD56+ cells (e.g. donor 3, Fig. 6B). The
relative ratios of CD4+ T cells, CD8+ T cells, NK cells and
CD3+CD56+ cells in the coculture appeared to be donor-
dependent (Fig. 6B and 6C). Furthermore, phenotype
analysis with multi-color flow cytometry demonstrated



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Journal of Hematology & Oncology 2009, 2:34


IFN-I+ cells
4,j (Tcm Tern Teff)


2:'




1 2 3


4 5 6 7


CD4-dominant donor


CD137+ cells
iTcm -> Tern Teffi


8 9 10


10
0 2 3 4 5
12345


6 7 8 9 10


1 2 3 4 5 6 7 8 9 10
CD28+ CD28+ CD28- CD28- CD28+ CD28+ CD28- CD28-
CD27+ CD27- CD27+ CD27- CD27+ CD27- CD27+ CD27- CCR7+ CCR7-
CCR7+ CCR7- CD45RA+
CD45RA-


CD8-dominant donor


IFN-y+ cells
(Tcm -- Tern Teff)







2 3 15 6 7 9 10


4 5 6 7 8


I 1 i
(9 10


CD137+ cells
80 ITem Tem Teffl

6(1

40

20

S2 3 4 5 6 10
1 2 3 4 5 6 7 8 9 10


80

60

40

20


1 2 3 4 5


6 7 8 9 10


1 2 3 4 5 6 7 8 9 10
CD28+ C028+ CD28- CD28- CD28+ CD28+ CD28- CD28-
CD27+ CD27- CD27+ CD27- CD27+ CD27- CD27+ C027- CCR7+ CCR7-
CCR7+ CCR7- CD45RA+
CD45RA-


Figure 2
Phenotype analysis of IFN-y- or CDI37-selected antigen-specific immune cells. PBMC were stimulated with EBV
LMP2A pentadecapeptides and IFN-y or CDI 37 positive cells were isolated for analysis. (A) & (B) Memory and effector pheno-
type analysis based on seven-color flow cytometry with surface staining for CD27, CD28, CD45RA, CCR7, CD4, CD8, CDI 37
and intracellular staining for IFN-y immediately after cell isolation without further culture. The percentage of different popula-
tions (IFN-y or CD 137 plus CD4 or CD8 gated) of T cells is illustrated for a CD4-dominant donor (A) and a CD8-dominant
donor (B); Tcm, central memory T cells (CD27+/-, CD28+, CCR7+, CD45RA-); Tem, effector memory T cells (CD27+/-,
CD28+/-, CCR7-, CD45RA-); Teff, terminal effector T cells (CD27-, CD28-, CCR7+/-, CD45RA+).





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3 4 5 F 7 8 9 10


1 2


S 6 7


9 1'i


2


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Journal of Hematology & Oncology 2009, 2:34


CD4-dominant donor
expanded expanded
IFN-f* cells CD137+ cells
23 32.7




SC03

953 61.9


3

D3


C
o
.3 a


06 949
... CDO4


3t 04

31 777


http://www.jhoonline.org/content/2/1/34


CD8-dominant donor
expanded expanded
IFN-f+ cells CD137+ cells
26 85

- Ie


..... CD3 -

956 87.4


C03





39 28 264
... C 4 ----


16


0s


CD4-domi'Mtna dnr
Expanded IFN.+ cells Expanded CDi37-* cll
T c ( rn Tern I T. ri ** T', T.:n. i..


1 3 J 4 ..,

W0


1 2 3 4 5 8 9 10
CD28+ CDl* 2CD28- CD28- CD2 C02D28 t 2B- CD28
CD207 CPD. CD27O CD227 C0~2l CD.7 C 27. C0D27 CCR?+ CCR-7
CCR7+ CCR7 CD45RA*
CD45RA


CD8-dominarnt donor
Expanded IFNi* cells Expanded CD137. cell
Il.n,i. r r rr T- .. 1,i ...r >. ruf


S 2 3 4 5 6 7 8 9 10
C028* CD28* C02- CD2B- CD28+ CD28 C028- CO28-
C027 CD27- CD27- CO27- CD27. CD27- C027- CD27- CCR7* CCR7.
CCR7* CCR7- CD45RA I
CD45RA.


Figure 3
Phenotype analyses of IFN-y- or CDI37-selected immune effector cells after ex vivo expansion. (A) Phenotype
analysis after ex vivo expansion. After the rapid selection, the antigen-specific immune cells were cultured for fifteen days with
irradiated autologous PBMC as feeder cells. The distribution of CD3, CD4, CD8 and CD56 cell populations was determined
and representative FACS graphs are shown. (B) and (C) Memory and effector phenotype analysis based on seven-color flow
cytometry with surface staining for CD27, CD28, CD45RA, CCR7, CD4, CD8, CDI 37 and intracellular staining for IFN-y.
Representative results of a CD4-dominant donor (B) and a CD8-dominant donor (C) are shown.






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Y[
0 1


C












80
I-


i


I i


I .








Journal of Hematology & Oncology 2009, 2:34


CD4-dominant donor


expanded IFN-y-selected cells
control specific BLCL
0A4 10 4 1.9 7 8 0,6 7.0


3.1 1 6.4 54 10 82.5
-- CD4
06 2.8 1.7 14.2 0.7 5.3


expanded CD137-selected cells
control specific BLCL
0,3 4.4 0.7 1 3 A 0.4 3,2


16 ;8 9 19.7 63.4 .9 9-5
CD4 -
2.0 5.7 3.4 17.6 1,6 7,0


i941 1111410 9 47P 36 ) 4 dr *'E-
CD4 CD4

B CDB-dominant donor


expanded IFN-y-selected cells


control specific BLCL control
01 02 05 ii 0.1 16.6 01 0


3.9 5 8.3 31.9 42 .I1 69.1
-CD8

02 1-0 09 582 0-4 9.7 0.6 1.9


4-4 D944 .5 1 .
COB


expanded CD137-selected cells


specific BLCL
05 30.6 02 22 3


32,5 _36.5 32 45.6
- C08

3-8 33-5 1.4 16.3



CD8


Antigen-seofic cytol~lic assaV


IFN-y+ cells -





CD137+ cells


L BLCL
SDC-LMP2A
I--- DC-WT1
rol'


0 10 20 30 40 50
% cell lysis

Figure 4
Effector function analyses of IFN-y- or CD 137-selected immune effector cells after ex vivo expansion. (A) and (B)
Flow cytometry analysis of IFN-y and CDI 37 expression after restimulation of the culture expanded IFN-y or CD 137 effector
cells. The cells were restimulated with different cells as indicated and subjected to antibody staining and flow cytometry analy-
sis; control, WT- I peptide-pulsed DC; specific, LMP2A peptide-pulsed DC; BLCL, autologous EBV-transformed B cells. (C)
Analysis of antigen-specific cytolytic activity. The ex vivo expanded IFN-y- or CDI 37-enriched cells were incubated with autol-
ogous EBV transformed B cells (BLCL), mature DCs loaded with either LMP2A pentadecapeptides (DC-LMP2A) or WTI pen-
tadecapeptides (DC-WTI, as control) at I:1 ratio in a cytotoxicity assay based on CFSE labeling as described in Materials and
Methods.




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Journal of Hematology & Oncology 2009, 2:34


2000 -


1500



1000


500

400

300

200

100

0


I -


I--i 5 day DC
2 day DC


ii


Surface Markers of Mature DC

Figure 5
Phenotype comparison of 5 day versus 2 day mature DC. Surface markers related to antigen presentation function
were stained with multi-color fluorochrome-labeled Ab and analyzed with FACSAria. Data represent MFI of mature DC gener-
ated from three healthy donors with p value calculated.


that the ex vivo expanded cells contained mostly Tem and
Teff cells (Fig. 7A and 7B, representative of CD4- and
CD8-dominant donors).

The DC-activated immune cells display antigen-specific
effector functions
We next evaluated the DC-activated immune cells for anti-
gen-specific effector functions based on intracellular
staining of IFN-y, IL-2, and the degranulation marker
CD107a after restimulation with peptide-loaded DC (Fig.
8A). For direct comparison, the same donor whose cells
were analyzed for the IFN-y-selected effector functions
(Fig. 4B) was chosen. Analyses of the immune effector
cells without further stimulation (IE cell alone), or stimu-
lated with non-specific peptides (Non-specific) or with
the LMP2A pentadecapeptides (LMP2A-specific) demon-


strated LMP2A-specific expression of IFN-y, IL-2 and
CD 107a in the CD8 T cells, and to a lesser extent, the CD4
T cells (Fig. 8A top: IL-2 and IFN-y, and bottom: CD107a
and IFN-y). The antigen-specific effector function was fur-
ther confirmed with the CFSE-based proliferation assay, as
well as MHC-peptide pentamer specific for the LMP2A
epitope-specific T cell receptors of the CD8 T cells (data
not shown).

To demonstrate antigen-specific killing, we directly com-
pared the DC-activated cells to the IFN-y captured cells (as
shown in Fig. 4C) using an in vitro cytolytic assay (Fig. 8B).
At an effector to target cell ratio of 1:1, the immune cells
effectively killed autologous BLCL and LMP2A-pulsed DC
(DC-LMP2A), but not DC pulsed with control WT1 pen-
tadecapeptides (DC-WT1). Therefore, effector function


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G b eb N",s
0) ocije


ce)








Journal of Hematology & Oncology 2009, 2:34


c














B

100


80


60
CL










40
*







(C



B
100


80


]j 60
0
0.


http://www.jhoonline.org/content/2/1/34


0 2 4 6 8 10 12 14 16 18 20
Days


0
(Day) 01219
Donor 1


01219 01219 01219 01219
Donor 1 Donor2 Donor 3 Donor 4


60
c
_o
-m

o 40

02
s0


0
(Day) QJ2J1
Donor 1


01219 QJ2-I 01219
Donor 1* Donor 2 Donor 3


Figure 6
Ex vivo expansion and phenotype analyses of DC-activated immune effector cells. EBV LMP2A-specific effector T
cells were generated by stimulation of non-adherent PBMC with DC pulsed with LMP2A pentadecapeptides. (A) Growth
kinetics of DC-activated immune effector cells. The viable cells were counted with trypan blue staining at different time points
after coculture and the growth curves of 5 samples are shown. (B) and (C) CD3, CD56, CD4 and CD8 phenotype analysis. The
DC-activated cells from day 0, 12 and 19 were stained with antibodies against CD4, CD8, CD3 and CD56 and analyzed with
flow cytometry. The percentages of different lymphocyte subsets were analyzed and shown in bar graphs. Donor 1*, PBMC
collected at a different time point from donor I.





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01219
Donor 4








Journal of Hematology & Oncology 2009, 2:34


A

day 0
80 (Tcm --- Tern -- Teff)

I-
o
+ 40

20T

0


CD4-dominant donor
day 12 day 19
(Tem -- Tem -- Teff) (Tem --- Tm ---- Teff)






_s __


3 4 5 6 7 8 910


34567 8


10 1 2 3 4 5 6 7 8 9 10


1 2 3 4 5 6 7 8 9 10
CD28+ CD28+ CD28- CD28- CD28+ CD28+ CD28- CD28-
CD27+ C027- CD27+ C27- CD27+ CD27- C027+ CD27- CCR7+ CCR7
CCR7+ CCR7- CD45RA+
CD45RA-


day 0
80 (Tnm -- Tem -- Teff)

Z 60-
I-
+ 40'

S20

0 -


CD8-dominant donor
day 12
(Tcm -- Tern -- Teff)


day 19
(Tcm -- Tem -- Teff)


50
.T 40
30
S20,
0 10"
0-


12 34
1 2 3 4 5


6 7 8910 12 34 5 6 7 8 9 10 12 3 4 5 67


8 9 10


1 2 3 4 5 6 7 8 9 10
CD28+ CD28+ CD28- CD28- CD28+ CD28+ CD28- CD28-
CD27+ CD27- CD27+ CD27- CD27+ CD27- C027+ CD27- CCR7+ CCR7-
CCR7+ CCR7- CD45RA+
CD45RA-


Figure 7
Memory and effector T cell analyses of DC-activated immune effector cells. (A) & (B) Memory and effector pheno-
type analysis of a CD4 dominant donor (A) and a CD8 dominant donor (B). The DC-activated cells from day 0, 12, and 17 after
coculture were analyzed for CD3, CD4, CD8, CD27, CD28, CD45RA, and CCR7 using a seven-color panel of fluorochrome-
labeled Ab with FACSAria. The cells were gated for CD3 and CD4 or CD8 as total cells for the percentage analysis. One rep-
resentative flow graph of 5 performed experiments is presented.


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30

* 20
20
Co
o 10-

0 -
1 2


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Journal of Hematology & Oncology 2009, 2:34


IE cell al
0 9[





'0.4


I


one


Non-specific LMP2A-specific


0.1 3.8





0.1 1.41



08


0.6 J1.8I



1.4
0.3 0.8




08


S02
0

O


--I IFN-y


0.1 0.3



1.2

01 02



02 p


0.1 0.2 0.6



1.4 3.1

01 08 3.6



02 14.


Comparison of cytolytic activity of IFN-y-selected versus DC-
activated EBV LMP2A-specific immune effector cells


IFN-y+ cells -





DC-activated cells -


I
I I


^^ h1


0 10 20 30 40 50

% cell lysis


BLCL
DC-LMP2A
DC-WT1


Figure 8 (see legend on next page)


Page 12 of 16
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CD4
gated




CD8
gated


CD4
gated




CD8
gated


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Journal of Hematology & Oncology 2009, 2:34


Figure 8 (see previous page)
Functional analyses of DC-activated and ex vivo expanded immune effector cells. EBV LMP2A-specific effector cells
were generated by stimulation of non-adherent PBMC with DC pulsed with LMP2A pentadecapeptides. (A) Analysis of antigen-
specific effector cytokines and CDI07a expression. The DC-activated immune cells from day 19 coculture were stimulated
with autologous DC pulsed with LMP2A peptides, WTI peptides (non-specific control) or no stimulation (IE cell alone) for 6
hours. The cells were stained with Ab against CD4, CD8, CDI07a and IFN-y. Flow cytometry analyses of IL-2, IFN-y and
CD I07a-positive cells in CD4-gated or CD8-gated populations were illustrated as representative of five experiments. (B)
Comparison of cytolytic function of IFN-y-selected (same donor as in Fig. 4C) versus DC-activated LMP2-specific immune
effector cells. The LMP2A-specific effector cells were mixed with target cells including autologous BLCL, DC-LMP2A, or con-
trol DC-WTI at 1:1 ratio and analyzed for cytolytic activity based on the CFSE-labeling method as described in Materials and
Methods.


analyses including IFN-y release and cytotoxicity assays
suggest that the DC-activated cells had lower activities
than the expanded IFN-y effector cells.

Discussion
Adoptive immune cell therapy has shown great promise
in treating viral diseases and melanoma. [2,6] Continued
efforts are focused on the generation of sufficient amount
of antigen-specific immune cells and optimal condition-
ing of immune homeostasis in patients in order to achieve
a sustained in vivo immune surveillance. [24-26] Here, we
compared three ex vivo immune cell preparation protocols
and phenotypically and functionally characterized these
cells. The rapid protocols based on IFN-y and CD137
selection generate a small number of antigen-specific
effector cells with high percentage of central memory T
cells in a very short period of time. The DC-activation pro-
tocol generates more immune cells, albeit, with more dif-
ferentiated phenotype and reduced proportion of antigen-
specific effector cells.

Based on analyses of a large number of donors, we found
that individual response to a given antigen could be either
CD4- or CD8-dominant, which is antigen- and donor-
dependent. Immune effector cells isolated based on IFN-y
expression displayed a CD4 or CD8 bias consistent with
the donor's immune dominance. However, antigen-spe-
cific CD137 positive cells showed a higher ratio of CD4
effector cells regardless of the subject's immune pheno-
type; this is in contrast to previous reports that emphasize
the induction of CD8 effector cells after CD137 enrich-
ment. [15,22,27] We did, however, show that CD8 T cells
displayed higher density of CD137 than did CD4 T cells.
It is well documented that CD137 costimulation pro-
motes both CD4 and CD8 T cell expansion and long term
memory. [28-30] Our finding that more CD4 T cells than
CD8 T cells are detected in the CD137-positive cell popu-
lation suggests a rapid induction of CD137 in the memory
T helper repertoire immediately after antigen stimulation.
Although the enriched CD 137 immune cells contained a
higher CD4 T cell ratio, further expansion in culture
restored the donor's original dominant phenotype, with a


higher CD3-CD56+ NK cell population than those found
in the expanded IFN-y enriched immune cells (Fig. 3A).
This result suggests that IFN-y is a more restricted adoptive
immune response marker and represent less of an innate
immune marker as does CD 137.

The ex vivo DC-activation protocol generated different
ratios of CD4, CD8 and NK cells in culture, which again,
appeared to be donor-dependent. Whether the immune
dominance has any effect on in vivo efficacy of the cul-
tured immune effector cells awaits further investigation.
As CD4 T cells are important for the maintenance of long-
term anti-viral CD8 T cell memory [31], therapeutic
immune cells should include polyclonal CD4 and CD8 T
cells. Both IFN-y and CD137 selection approaches gener-
ated increased number of memory type of cells represent-
ative of polyclonal CD4 and CD8 T cells that may have
increased proliferation potential after infusion. Although
the antigen-specific memory T cells from PBMC may be
low; for examples, the average yield of LMP2A-specific
IFN-y positive immune effector cells from healthy EBV-
seropositive donors is only 0.22 0.13% (n = 6, after two
rounds of affinity column purification), they can be
expanded to more than two orders of magnitude in cul-
ture in two weeks and maintain their high antigen specif-
icity.

It is evident that the LMP2A pentadecapeptides efficiently
activate both CD4 and CD8 T cells in a short exposure
period (3-13 hr). This was surprising since CD8 T cells are
activated through class I MHC loaded with short 9-11
amino acid peptide epitopes, different from CD4 T cells,
which are activated through class II MHC loaded with 12-
15 amino acid peptide epitopes. The pentadecapeptide
antigens apparently activated CD8 T cells with high effi-
ciency through cross-presentation. This has been con-
firmed with various pentadecapeptide antigens
(unpublished). The processing of class II MHC peptides
into class I epitopes for cross-presentation to CD8 T cells
appears to be highly efficient with both the IFN-y and the
CD137 protocols, as with the DC-activation method. The



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Journal of Hematology & Oncology 2009, 2:34


detailed molecular mechanism of the efficient cross-pres-
entation requires further investigation.

To assess differentiation and maturation status of the ex
vivo generated T cells, we applied multi-color flow cytom-
etry to detect differentiation and homeostatic marker
CD45RA, trafficking marker CCR7, and costimulatory
marker CD27 and CD28. [32,33] It is not surprising that
both the IFN-y- and the CD137-enriched antigen-specific
effector cells displayed more memory markers than did
the DC coculture-expanded cells. While preserving Tcm
cells is critical to in vivo therapeutic efficacy,[7,34,35] clin-
ical studies have proven that ex vivo expanded effector cells
can persist many years after infusion. [36] Clinical bene-
fits of these different protocols will require detailed evalu-
ation in a large cohort of patients.

The differentiation status of the ex vivo generated immune
cells may contribute to their in vivo therapeutic efficacy.
Homeostasis of antigen-specific memory cells can vary
depending on antigen source, the immune milieu and
individual donor. It is known that Tcm cells are mainly
located in lymphoid tissues and Tem cells are distributed
in diverse non-lymphoid sites including lung, liver and
intestine. [37] In addition, bone marrow has been shown
to embrace increased number of anti-cancer or anti-virus
memory T cells. [38-40] After ex vivo expansion, however,
wherever the T cells come from, they tend to bestow
exhausted proliferation and replicative senescence associ-
ated with down-regulation of anti-apoptotic protein Bcl-2
and Bcl-xL, and decreased telomere length. [33,34,41]
Modification of antigen presentation protocol and culture
condition may help overcome the immune cell exhaus-
tion problem. [9,42]

For patients with acute infections or illness, direct isola-
tion of antigen-specific immune cells from partly HLA-
matched healthy donors represents an attractive emer-
gency approach to obtain therapeutic cells. [43,44] This
approach offers several advantages including a shortened
handling time and increased proliferation potential in
vivo. Although the number of immune cells is limited with
the direct isolation approach, clinical evidence supports
that only a small number of such immune cells, in the
range of 103-104/kg body weight, is sufficient to attain
therapeutic efficacy in transplant patients. [13,45,46]Ex
vivo expansion of immune cells, nevertheless, may be nec-
essary for patients with a compromised immunity.
[47,48]

Conclusion
The two rapid immune cell isolation methods generate
functional effector cells in less than 24-48 hr suitable for
emergency immune cell preparation. On the other hand,
the DC-activation method expands antigen-specific


immune effector cells while effectively reduce the number
of non-specific cells. Depending on clinical needs, for
examples, the urgency for treatment, patient's body
weight (e.g. less cells are needed for pediatric patients), or
patient's immune cell proliferative potential in vivo, the
method of immune cell preparation may differ. Our data
indicate that IFN-y selection followed by ex vivo expansion
represents the best approach for the generation of high
amount of antigen-specific immune effector cells. Further
efforts to overcome immune tolerance and expand anti-
gen-specific immune cells with prolonged in vivo persist-
ence are critical to the success of immune cell therapy.

List of abbreviations
IFN-y: interferon-gamma; IL: interleukine; DC: dendritic
cell; CTL: cytotoxic T lymphocyte; MHC: major histocom-
patibility complex; Ab: antibody; Ag: antigen; TCR: T cell
receptor; TNF: tumor necrosis factor; BLCL: B lymphoblas-
toid cell line; EBV: Epstein-Barr virus; LMP2A: late mem-
brane protein 2A; CMV: cytomegalovirus; ICCS:
intracellular cytokine staining; CFSE: carboxy-fluorescein
diacetate succinimidyl ester.

Competing interests
YH, YL, YH and YW are employees ofVectorite Biomedica
Inc. LJC is consultant to a biotech company.

Authors' contributions
All authors are accountable for the integrity of the research
results; Chang is responsible for the conception of the
research and Han, Huang, Liang, Ho and Wang are
responsible for the execution and for data collection;
Chang is responsible for initial drafting and revisions of
the manuscript.

Additional material


Additional file 1
Phenotype analysis of monocytes and 2 day and 5 day mature DC. Sur-
face markers related to antigen presentation function were analyzed using
fluorochrome-labeled Ab. The light-colored lines in the FACS graphs rep-
resent control Ab and the numbers represent geometric means with per-
centages shown in parentheses. Representatives of two monocyte
experiments and three DC experiments are illustrated.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1756-
8722-2-34-S1.tiff]


Acknowledgements
We thank the technical assistance of Liheng Guo, Lily Lien, Fuhung Yang,
Yinchieh Fu and Meifang Lin. The study was funded by Vectorite Biomedica
Inc. and Yongling Foundation.





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Title: Phenotype and functional evaluation of ex vivo generated antigen-specific immune effector cells with potential for therapeutic applications
Series Title: Journal of Hematology & Oncology 2009, 2:34
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