October 2017 | Volume 8 | Article 1313 1 ORIGINAL R ESEARCH published: 26 October 2017 doi: 10.3389/mmu.2017.01313 Frontiers in Immunology | www.frontiersin.org Edited by: Herman Waldmann, University of Oxford, United Kingdom Reviewed by: Richard DiPaolo, Saint Louis University School of Medicine, United States Bruce Milne Hall, University of New South Wales, Australia *Correspondence: Todd M. Brusko email@example.com Specialty section: This article was submitted to Immunological Tolerance and Regulation, a section of the journal Frontiers in Immunology Received: 15June Accepted: 28September Published: 26October Citation: YehW-I, SeayHR, NewbyB, PosgaiAL, MonizFB, MichelsA, MathewsCE, BluestoneJA and BruskoTM (2017) Avidity and Bystander Suppressive Capacity of Human Regulatory T Cells Expressing De Novo Autoreactive T-Cell Receptors in Type 1 Diabetes. Front. Immunol. 8:1313. doi: 10.3389/mmu.2017.01313 Avidity and Bystander Suppressive Capacity of Human Regulatory T Cells Expressing De Novo Autoreactive T-Cell Receptors in Type 1 Diabetes Wen-I Yeh1, Howard R. Seay1, Brittney Newby1, Amanda L. Posgai1, Filipa Botelho Moniz1, Aaron Michels2, Clayton E. Mathews1, Jeffrey A. Bluestone3 and Todd M. Brusko1* 1 Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, FL, United States, 2 Barbara Davis Center for Diabetes, University of Colorado School of Medicine, Aurora, CO, United States, 3Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, United States The ability to alter antigen specicity by T-cell receptor (TCR) or chimeric antigen receptor (CAR) gene transfer has facilitated personalized cellular immune therapies in cancer. Inversely, this approach can be harnessed in autoimmune settings to attenuate inammation by redirecting the specicity of regulatory Tcells (Tregs). Herein, we demonstrate efcient protocols for lentiviral gene transfer of TCRs that recognize type 1 diabetes-related autoantigens with the goal of tissue-targeted induction of antigen-specic tolerance to halt -cell destruction. We generated human Tregs expressing a high-afnity GAD555-reactive TCR (clone R164), as well as the lower afnity clone 4.13 specic for the same peptide. We demonstrated that de novo Treg avatars potently suppress antigen-specic and bystander responder T-cell (Tresp) proliferation invitro in a process that requires Treg activation (P < 0.001 versus unactivated Tregs). When Tresp were also glutamic acid decarboxylase (GAD)-reactive, the high-afnity R164 Tregs exhibited increased suppression (P < 0.01) with lower Tresp-division index (P < 0.01) than the lower afnity 4.13 Tregs. These data demonstrate the feasibility of rapid expansion of antigen-specic Tregs for applications in attenuating -cell autoimmunity and emphasize further opportunities for engineering cellular specicities, afnities, and phenotypes to tailor Treg activity in adoptive cell therapies for the treatment of type 1 diabetes. Keywords: type 1 diabetes, regulatory Tcells, Tcell receptor, avidity, suppression mechanisms, adoptive cellular therapies, antigen-specic Tcells, glutamic acid decarboxylase 65 IN T R ODU C T I O N T-cell receptor (TCR) transgenic regulatory Tcells (Tregs) may represent a promising personalized treatment for T-cell-mediated autoimmune diseases such as type 1 diabetes. A curative therapy that targets the underlying immunological cause of disease to restore antigen-specic immunological tolerance represents an essential objective for the preservation of -cell mass and function in the treatment of type 1 diabetes ( 1 ). Non-antigen-specic therapies involving hematopoietic stem
2 Yeh et al. Frontiers in Immunology | www.frontiersin.org October 2017 | Volume 8 | Article 1313 cell transplantation combined with T-cell depletion, via high-dose anti-thymocyte globulin (ATG) or udarabine, plus immunomodulation with cyclosporine and granulocytecolony stimulating factor (G-CSF) have been shown to preserve -cell function (2 3 ), but the risks associated with these aggressive protocols preclude common clinical use. Comparatively, non-specic polyclonal immunotherapies, including immunoregulatory or depleting agents [e.g., alefacept (human LFA-3/IgG1-Fc fusion protein), teplizumab or otelixizumab (anti-CD3), and rituximab (anti-CD20)], have been better tolerated and oered some temporary ecacy but not long-term induction of tolerance (4 10). Until recently, most antigen-specic tolerance induction eorts have involved mucosal or peripheral administration of autoantigen(s), but thus far, such attempts have yielded limited ecacy in only a subset of patients, again with no indication for long-term toler ance induction (11, 12). Indeed, a safe treatment that controls persistent immune memory and induces long-term tolerance is needed. Islet cell antigen-reactive Tregs, isolated from BDC2.5 TCR transgenic mice, could be expanded invitro and following adoptive transfer, migrate to the pancreatic draining lymph node/nodes (13). ese Treg prevent and reverse autoimmune diabetes in non-obese diabetic (NOD) mice (14). In contrast, Tregs isolated and expanded from GAD286 TCR transgenic mice could suppress responder Tcells (Tresp) invitro but did not proliferate invivo aer transfer into recipient animals (14). Moreover, expression of cognate autoantigen is required for ecient tracking of Tregs to the target organ and suppression of diabetes in NOD mice (15). ese preclinical data support the notions that autoantigen-specic Tregs may oer an important therapy for type 1 diabetes, but also that intrinsic factors such as TCR specicity and/or avidity may play an important role in determining the capacity for immunomodulation and ecacy. e need for continued autoantigen expression by the host may render insulin-reactive TCRs less eective in patients with long-standing type 1 diabetes and support a need to investigate additional, potentially bystander, TCRs specic for additional/ alternative autoantigen targets such as glutamic acid decar boxylase (GAD). Moreover, antigen localization, density, and persistence in -cells along with risk of eector cell reprogramming support the use of alternative TCRs (16). Genetically modied Tcells with TCRs specic for tumor or viral antigens have become a valuable tool for the treatment of certain cancers or infections in humans (1719). We previously demonstrated successful HLA class I-restricted TCR gene transfer in human Tregs using a high-anity model receptor specic for the melanoma antigen tyrosinase presented by HLA-A*02:01 (20). We also generated a murine form of these tyrosinase-specic Tregs, and when transferred invivo the cells were capable of suppressing anti-tumor immunity in murine tumor models (20). is prompted us to ask whether candidate TCRs specic for type 1 diabetes-related autoantigens could be used to generate regulatory TCR avatars for human therapy. Two TCR clones (R164 and 4.13) specic for the same -cell peptide (GAD555) presented by HLA-DR4, but with dierent binding anities, have been identied from the peripheral blood of subjects with or at-risk for T1D (2123). Indeed, we recently identied Tcells expressing the TCR -chain complementarity determining region (CDR3) of the GAD 4.13 clone from tissues of seven organ donors with type 1 diabetes, including the pancreatic islets of one type 1 diabetes subject. Interestingly, for one donor with long-standing disease, the TCR CDR3 was highly enriched in the pLN (>25% of all productive sequences), representing the most prevalent clone in both the Treg and conventional CD4+ T-cell (Tconv) populations (24). Interestingly, 4.13 TCR transgenic HLA-DR4 mice were reported to contain a mixture of and Tr1 cells capable of producing IL-10 (21). Conversely, R164 TCR transgenic HLA-DR4 mice exhibited greater thymic negative selection, and the Tcells that escaped the thymus were skewed toward a phenotype (21). ese observations support the notion that TCR avidity may impart important functional distinctions. In a recent report by Ali etal., human CD4+ Tcells were engineered to express the R164 TCR clone, and importantly, when administered to NSG-Ab0 DRB*04:01 mice, these R164 cells established long-term engrament and islet inltration, up to 12weeks, without gra versus host disease (GvHD) (25). e creation of these autoreactive T-cell avatars presents the exciting possibility of autologous Treg therapy for type 1 diabetes with the benet of antigen specicity to potentially enhance Treg trafcking to the target organ and associated draining lymph nodes. ese antigen-specic Tregs would likely represent a signicant improvement upon autologous polyclonal Treg therapy, which has already been shown to be safe for use in human subjects (26, 27). Indeed, antigen-specic Tregs oer the potential for long-term tolerance to the target antigen and possibly, to other key -cell epitopes via bystander suppression and infectious tolerance (14, 28). To expand on these eorts, we generated primary human Tregs expressing the two GAD555-reactive TCR clones (R164 and 4.13), and investigated the pre-transfer conditions needed to optimize suppressive activity for potential use in adoptive cell therapy.RESEARCH DESIGN AND METHODS Design and Synthesis of Lentiviral ConstructsLentiviral vectors were generated to express TCR clones 4.13 and R164, both of which react to GAD555 (21, 25) (Table1 ). Equimolar expression of TCR and -chains was achieved by inclusion of a multicystronic P2A element, followed by a T2A element and the reporter, enhanced green uorescent protein (eGFP). e constructs were cloned into pCNFW lentiviral vectors with expression driven by a cytomegalovirus promoter as previously described (25) (Figure1A). Lentiviral vectors containing the Melan-A reactive TCR clone melanoma antigen recognized by Tcells 1 (MART-1) were generated as previously described (29) (Table).Lentivirus ProductionLentiviral vectors were generated as described (20). Briey, 55g of lentiviral vector and 18.3g of each helper plasmid
TABLE 1 | T-cell receptor (TCR) clone information. TCR (IMGT) TRA gene TRB gene pMHC restriction Source S. no. Clone V J CDR3 AA sequence V D J CDR3 AA sequence HLA Antigen 1 5 TRAV21 TRAJ6 CAVKRTGGSYIPTF TRBV11-2 TRBD1 TRBJ2-2 CASSSFWGSDTGELFF DQ8 InsB (9) Roep, personal communication 2 GSE.20D11aTRAV12-3 TRAJ4 CAILSGGYNKLIF TRBV02-01*01 TRBD02-01 TRBJ02-05*01 CASSAETQYF DQ8 InsB (9) (30) 3 GSE.6H9aTRAV26-1 TRAJ40 CIVRVDSGTYKYIF TRBV7-2 TRBD2 TRBJ2-1 CASSLTAGLASTYNEQFF DQ8/ DQ8-trans InsB (9) (30) 4 T1D#3 C8 TRAV17 TRAJ23 CATDAGYNQGGKLIF TRBV5-1 TRBD2 TRBJ1-3 CASSAGNTIYF DQ8 InsB (9) (31) 5 T1D#10 C8 TRAV12-3 TRAJ26 CATAYGQNFVF TRBV4-1 TRBD2 TRBJ2-2 CASSRGGGNTGELFF DQ8 InsB (9) (31) 6 PM1#11 TRAV35*02 TRAJ54*01 CAGHSIIQGAQKLVF TRBV5-1*01 TRBD2*02 TRBJ2-1*01 CASGRSSYNEQFF DRB1*03:01 GAD (339) (32) 7 MHB10.3 TRAV4*01 TRAJ27*01 CLVGDSLNTNAGKSTF TRBV29-1*01 TRBD2*01 TRBJ2-2*01 CSVEDRNTGELFF DRB1*03:01 InsB (11) (33) 8 SD32.5 TRAV26-1*01 TRAJ23*01 CIVRVSSAYYNQGGKLIF TRBV27*01 TRBD2*01 TRBJ2-3*01 CASSPRANTDTQYF DRB1*04:01 InsA (5) (34) 9 SD52.c1 TRAV4*01 TRAJ27*01 CLVGDSLNTNAGKSTF TRBV27*01 TRBD1*01 TRBJ1-5*01 CASSWSSIGNQPQHF DRB1*04:01 PPI (C18A1) (34) 10 R164 TRAV19*01 TRAJ56*01 CALSEEGGGANSKLTF TRBV05-01*01 TRBD02-01*01 TRBJ01-06*01 CASSLAGGANSPLHF DRB1*04:01 GAD (555) (23) 11 4.13 TRAV19*01 TRAJ44*01 CALSENRGGTASKLTF TRBV05-01*01 TRBD01-01*01 TRBJ01-01*01 CASSLVGGPSSEAFF DRB1*04:01 GAD (555) (21) 12 1E6 TRAV12-3 TRAJ12 CAMRGDSSYKLIF TRBV12-4 TRBD2 TRBJ2-4 CASSLWEKLAKNIQYF A*02-01 PPI (15) (35) 13 D222D TRAV17*01 TRAJ36*01 CAVTGANNLFF TRBV19*01 TRBD1*01 TRBJ2-2*01 CASSIEGPTGELFF A*02-01 ZnT8 (186) Patent WO2017046335 A1 14 32 TRAV12-1 TRAJ48*01 CVVNILSNFGNEKLTF TRBV20 TRBD01-01*01 TRBJ2-01*01 CSASRQGWVNEQFF A*02-01 IGRP (265) (36) 15 MART-1 TRAV12-2 TRAJ23 CAVNFGGGKLIF TRBV6-4 TRBD2 TRBJ1-1 CASSLSFGTEAFF A*02-01 Melan-A (27) (37)For the experiments described herein, Tcells were transduced to express TCR clones 4.13 or R164, which were rst identied from the peripheral blood or pancreas of a type 1 diabetes patient or an autoantibody positive subject at risk for T1D. CD8+ Tcells were transduced to express melanoma antigen recognized by Tcells 1 (MART-1) TCR. Remaining TCR clones [sourced from the international ImMunoGeneTics information system, IMGT.org (IMGT)] listed are those with known reactivities to type 1 diabetes-related autoantigen peptides with which we can generate lentivirus constructs to create additional Treg avatars. TCR (TRA) and TCR (TRB) V, D, and J genes as well as complementarity determining region 3 (CDR3) amino acid (AA) sequence, HLA restriction, and antigen target are listed for each clone.aIntra-islet source material. 3 Yeh et al. Frontiers in Immunology | www.frontiersin.org October 2017 | Volume 8 | Article 1313
A C B FIGURE 1 | Verifying transfection and activation of Jurkat cells expressing T-cell receptor (TCR) clones. (A) Lentiviral constructs were designed containing the TCR and TCR -chain genes (TRA and TRB, respectively) for known glutamic acid decarboxylase (GAD)-reactive clones (R164 and 4.13, additional clone information is listed in Table). The TRA and TRB coding regions were joined by a multicystronic P2A element, and TRB was linked by a multicystronic T2A element to an enhanced green uorescent protein (eGFP) reporter. Amino-acid sequences for the complementarity determining region 3 (CDR3) are shown for both clones. (B) Jurkat Tcells were untransduced (Mock; top), transduced with lentivirus expressing the R164 TCR (middle), and lentivirus expressing the 4.13 TCR (bottom), and expression was conrmed by ow cytometry. Double positivity for TCR Va12.1 and V5.1, which is comparable between both clones, and eGFP indicates successful transduction. Untransduced cells were negative for both markers. (C) Mock (top), 4.13 (middle), and R164 (bottom) TCR-transduced cells were unstimulated (black), stimulated with plate bound anti-CD3 (p.b. -CD3, blue), irrelevant antigen inuenza hemagglutinin (HA306, orange), or cognate antigen (GAD555, green). TCR expression (left panels) was comparable across all unstimulated, HA-stimulated, and GAD-stimulated cells, and p.b. -CD3 stimulation induced TCR downregulation. p.b. -CD3 stimulation also induced the highest level of CD69 expression (right panels), and unstimulated cells exhibited low CD69 expression. These observations were comparable across mock, R164, or 4.13 transduced cells. 4 Yeh et al. Frontiers in Immunology | www.frontiersin.org October 2017 | Volume 8 | Article 1313 were co-transfected in 293Tcells using TransIT-2020 transfection reagent (Mirus, Madison, WI, USA). Supernatants were collected 72h aer transfection, ltered through a 0.45-m lter, and concentrated by ultracentrifugation at 198,000 g for 1.5h.Subject Enrollment and T-Cell IsolationHealthy control blood donors provided written informed consent prior to inclusion in the study in accordance with the Declaration of Helsinki and according to Institutional Review Board-approved protocols at the University of Florida (Protocol no. IRB201600092) and the University of Colorado Denver (Protocol no. COMIRB92-292). Tcells where enriched by negative selection from whole blood by Ficoll-Paque density gradient in combination with a total T-cell enrichment cocktail by following manufacturers instructions (Catalog no. 15061, STEMCELL Technologies, Cambridge, MA, USA). Cells were stained with uorescently labeled antibodies [CD4-PB (clone RPA-T4), CD8-APC.H7 (SK1), CD25-APC (BC96), CD127-PE (A019D5), and CD45RA-PE-Cy7 (HI100)]. CD4+CD25+CD127lo/ Tregs, CD4+CD25CD127+CD45RA+ nave Tconv cells, and CD8+CD45RA+ nave CD8+ Tcells were puried by uorescenceactivated cell sorting (FACS) using a BD FACSAria III (BD Biosciences, San Jose, CA, USA).
5 Yeh et al. Frontiers in Immunology | www.frontiersin.org October 2017 | Volume 8 | Article 1313Lentiviral Transduction (L V TD) of Human T CellsJurkat CellsHuman Jurkat Tcells were plated at 2 105cells/well in a 24-well plate and transduced in the presence of protamine sulfate (8g/mL; Sigma-Aldrich, St. Louis, MO, USA). Transgene expression was assessed 72h post-transduction by ow cytometry (Figure).Primary Human T CellsPrimary human Tcells were transduced as previously described ( 3 ). Briey, FACS-puried CD4+ Tcells (total), Tregs, nave Tconv cells, and nave CD8+ Tcells were plated at 2.5 05 cells/ well in a 24-well plate. Total CD4+ Tcells, nave Tconv, and CD8+ Tcells were activated with anti-CD3 and anti-CD28 dynabeads (Catalog no. 11161D, ermoFisher Scientic, Waltham, MA, USA), while Tregs were expanded with anti-CD3 and anti-CD28 conjugated microbeads (Catalog no. 130-091-441, Miltenyi Biotec, San Diego, CA, USA) according to the manufacturers instructions. Aer 48h of activation, cells were supplied with protamine sulfate (8g/mL) and transduced with 3TU/cell of lentivirus for TCR expression followed by spinoculation. Total CD4+ Tcells were supplied IL-2 (30IU/mL) every 2days and restimulated with the HLA-DR4 (DRB1*04:01) expressing K562 articial antigen-presenting cell (aAPC) line and GAD555 peptide on day 9 and day 16 for serial activation (Figure2). For T-cell subsets, IL-2 (300IU/mL for Treg; 20IU/mL for Tconv; 100IU/mL for CD8+ Tcells) was supplied every 1days during expansion (Figure3). K562 aAPCs were kindly provided by Drs. James Riley and Bruce Levine (University of Pennsylvania).Flow CytometryCells were rst stained with live/dead near-IR (Invitrogen) followed by uorescently labeled antibodies specic for the following surface markers: CD4-PB (clone RPA-T4), CD69-BV711 (FN50), TCR V 12.1-Alexa Fluor 647 (6D6.6), TCR V 5.1-PE (IMMU 157), OX40-APC (ACT35), and CD25-PE (BC96). e TCR V 12.1 monoclonal antibody was labeled with Alexa Fluor 647 using Zenon labeling kit (ermoFisher Scientic, Waltham, MA, USA) before staining. Intracellular FOXP3 was stained using a FOXP3-Alexa Fluor 488 (206D) antibody with a FOXP3/transcription factor staining kit (Catalog no. 00-552300, ermoFisher Scientic, Waltham, MA, USA) according to the manufacturers instructions. Flow cytometry data were collected using an LSRFortessa (BD Biosciences) and analyzed with FlowJo soware (Tree Star, Ashland, OR, USA).Treg Suppression AssayT-cell-receptor-redirected Tregs were FACS-puried based on their eGFP expression and tested for the ability to suppress polyclonal or TCR-transduced Tresp proliferation, as described previously (38). For suppression assays involving polyclonal Tresp, cells were stimulated with 2g/mL soluble anti-CD3 (clone Hit3a) and 1g/mL soluble anti-CD28 (clone 28.2, BD PharMingen). Proliferation was determined by the incorporation of 3H-thymidine by pulsing cultures with 1mCi of 3H-thymidine for the nal 12h of culture. Plates were har vested on a Packard FilterMate harvester and read on a Packard TopCount Scintillation & Luminescence Counter (Perkin Elmer; Waltham, MA, USA). Interferon-gamma (IFN) was measured from the supernatant by ELISA. For suppression assays involving TCR-redirected Tresp, Tregs expressing the 4.13 TCR were stained with cell proliferation dye eFluor670 (5M; Catalog no. 65-0840-85, ermoFisher Scientic, Waltham, MA, USA), whereas Tresp expressing the 4.13 TCR or a Melan-A27 reac tive MART-1 TCR were labeled with CellTrace Violet (5M; Catalog no. C34571, ermoFisher Scientic, Waltham, MA, USA) following the manufacturers instructions. Tregs were plated in two-fold serial dilution, co-cultured with Tresp, and activated with the indicated peptide presented by irradiated CD3-depleted peripheral blood mononuclear cells (PBMCs) (HLA-DRB1*04:01 and A*02:01) for 3days. Triplicate cultures were pooled, harvested, stained with live/dead dye and for the surface markers, CD4 and CD8, and then analyzed by ow cytometry as described above. Proliferation was calculated by division and replication index of Tresp cells. Assay conditions are detailed in Table S1 in Supplementary Material.Statistical AnalysisData were analyzed by two-way analysis of variance (ANOVA) and graphs prepared using GraphPad Prism version 6 soware (La Jolla, CA, USA).RESUL TS Validation of TCR Expression and Activation in Human Jurkat CellsTwo lentiviral constructs with identical backbone each contained the TCR and TCR -chain genes (TRA and TRB, respectively) for the GAD555-reactive clones R164 or 4.13 followed by an eGFP reporter sequence as shown in Figure1A. Multi-cystronic and equal molar expression of TCR and -chains is achieved by including P2A and T2A elements between TRA, TRB, and the eGFP reporter. We used lentivirus carrying these constructs to transduce human Jurkat cells and express one of the two de novo TCRs. As expected, untransduced cells did not express eGFP, TCR -chain V gene family 12.1 (TCRV12.1), and TCR -chain V gene family 5.1 (TCRV5.1), which are common to both R164 and 4.13 clones (Figure1B). Over 94% of Jurkat cells transduced with either the R164 or 4.13 TCR lentiviral construct were double positive for both TCRV12.1 and V5.1 with comparable mean uorescence intensity (MFI) (Figure1B). To verify stable transfection and antigen-specic activation of Jurkat cell lines, we stimulated mock (eGFP), R164, and 4.13 transduced cells with K562 aAPCs loaded with cognate antigen (GAD555) and evaluated for TCR and CD69 expression levels. Positive and negative controls included stimulation of transduced cells with plate bound anti-CD3 or K562 aAPCs loaded with irrelevant antigen inuenza hemagglutinin (HA306), respectively. Compared with unstimulated cells, anti-CD3 induced TCR downregulation concurrent with high expression of the activation marker CD69
FIGURE 2 | Serial activation increases transduction efciency. (A) Primary CD4+ Tcells remain untransduced (Mock) or transduced with lentivirus (LV TD) expressing T-cell receptor (TCR) clones 4.13 or R164 were activated with -CD3/-CD28 coated beads on day 0 (D0). Cells were restimulated with articial APC (aAPCs; K562 cell line expressing HLA-DR4) and GAD555 peptide for an additional two rounds on day 9 (D9) and day 16 (D16). IL-2 (30IU/mL) was given every 2days. Transduction efciency was detected by uorescence-activated cell sorting (FACS) every 4days after stimulation (D4, D13, and D20). (B) TCR V5.1 and enhanced green uorescent protein (eGFP) reporter were assessed by ow cytometry on day 4 (D4, top), day 13 (D13, middle), and day 20 (D20, bottom). At each time point, a portion of untransduced cells were positive for TCR V5.1, but no eGFP was observed. TCR V5.1 and eGFP positivity was observed for 4.13 and R164 transduced cells at each time point, and the proportion of dual positive cells increased with time. 6 Yeh et al. Frontiers in Immunology | www.frontiersin.org October 2017 | Volume 8 | Article 1313 in each of the three cell lines (Figure1C). GAD555 stimulation of both R164 and 4.13 cell lines resulted in high CD69 expression without TCR downregulation, whereas irrelevant antigen resulted in only modest upregulation of CD69, likely due to interaction with the costimulatory molecule, CD80 constituently expressed by the aAPCs (Figure1C). ese data support both surface receptor expression and activation in the presence of the cognate peptide presented by HLA-DR4.
FIGURE 3 | Verication of T-cell receptor (TCR) overexpression and stability of transfected regulatory Tcells (Tregs). (A) CD25+CD127lo/ Tregs (top and middle) and CD25lo/CD127+CD4+CD45RA+ nave conventional Tcells (Tconv, bottom) were puried from adult peripheral blood by uorescent-activated cell sorting (FACS). (B) Tregs were activated with -CD3/-CD28 coated beads on day 0 (D0). Lentiviral transduction (LV TD) was performed on day 2 (D2). Successfully transduced cells expressing enhanced green uorescent protein (eGFP) were FACS-puried on day 19 (D19) for further analysis. (C) Treg (top) and Tconv cells (bottom) were untransduced (Mock; black), transduced with lentivirus containing the expression vector for the GAD-reactive TCR clone 4.13 (blue) or R164 (red), and eGFP expression among TCR transduced cells was conrmed by ow cytometry. (D) Mock (left), 4.13 (middle), and R164 (right) transduced Tregs (top) and Tconv cells (bottom) were stained for the TCRV12.1 and TCRV5.1 chains that comprise the TCR clones 4.13 and R164, and overexpression was conrmed for TCR transduced cells. (E) Cell activation markers OX40 and CD25 were measured after co-culturing Tcells with HLA-DR4 expressing K562 articial antigen-presenting cells (aAPCs) loaded with GAD GAD555 peptide for 1 day. (F) The majority of Tregs in all conditions (non-transduced, 4.13, or R164) maintain FOXP3 expression (top left). Low frequency of FOXP3 expression was observed in Tconv (bottom left). 7 Yeh et al. Frontiers in Immunology | www.frontiersin.org October 2017 | Volume 8 | Article 1313Optimizing TCR Expression in Primary Human CD4+ T CellsWe next transduced primary human peripheral blood CD4+ Tcells to express the GAD-reactive 4.13 and R164 TCRs and assessed transduction eciency. Cells were stimulated with anti-CD3/anti-CD28 coated beads on day 0, transduced on day 2, and restimulated on days 9 and 16 with K562-DR4 aAPCs loaded with GAD555 peptide (Figure2A). As expected, a por tion of untransduced cells expressed TCRV 5.1 but not eGFP ( Figure2B). In addition, 35% of GAD 4.13 and 13% of GAD R164 cells were TCRV 5.1+eGFP+ on day 4, and by day 20, 85 and 71% of 4.13 and R164 cells, respectively, were double positive for TCRV 5.1 and eGFP (Figure2B) suggesting that serial activation resulted in enriched T-cell avatars.
8 Yeh et al. Frontiers in Immunology | www.frontiersin.org October 2017 | Volume 8 | Article 1313 TCR Expression in Primary Human Regulatory and Conventional T-Cell SubsetsCD4+CD25+CD127lo/ Tregs and CD4+CD25CD127+CD45RA+ nave Tconv were FACS-puried from peripheral blood (Figure3A). We then generated primary human Tregs and Tconv expressing the GAD 4.13 and GAD R164 TCRs and expanded them with anti-CD3/CD28-coated beads for 19days (Figure3B). Again, compared with untransduced cells, 4.13 and R164 cells were conrmed to express high levels of eGFP (Figure3C) as well as the V12.1 and V5.1 chains of the GAD-reactive TCRs as measured by ow cytometry (Figure3D). e activation markers OX40 and CD25 were upregulated on 4.13 and R164 transduced Tregs compared with mock transduced Tregs 1 day post co-culture with HLA-DRB1*04:01 expressing K562 aAPC loaded with cognate peptide (Figure3E). Similarly, OX40 was slightly upregulated on the surface of 4.13 and R164 transduced Tconv following aAPC-antigen activation, while CD25 upregulation was more pronounced for R164 Tconv compared with 4.13 Tconv (Figure3E) (39). Aer transduction and anti-CD3/28 stimulation, Tregs maintained FOXP3 positivity whereas Tconv cells showed low/intermediate expression of FOXP3 (Figure3F) indicating transduction aected neither Treg dierentiation nor development.Suppressive Capacity of R164 and 4.13 Treg A vatarse capacity to impact type 1 diabetes progression prior to symptomatic onset (i.e., in the context of multiple autoantibody positive high-risk individuals) or at the time of symptomatic disease will likely require the capacity to control a polyclonal memory T-cell response. Depletion of these cells is one potential approach but would require broad targeting resulting in a period of immunosuppression. We hypothesize that tissue targeting and dominant suppression of a broad repertoire by TCR-redirected Tregs may confer persistent tolerance. erefore, we sought to understand if Treg avatars functionally suppress Tresp in an antigen-specic and/or bystander manner. We rst demonstrated that LV TD does not aect Treg capacity to suppress polyclonal Tresp using well-described invitro suppression assays (38). Both proliferation and IFNproduction by polyclonal stimulated Tresp were comparable between R164, 4.13, and mock transduced Treg groups (Figure S1 in Supplementary Material). en, we assessed Treg suppressive capacity in both antigen-specic and bystander mechanisms with or without Treg activation (Figure4A). At physiological ratios, Tregs showed excellent suppression of Tresp against cognate antigen by culturing both CD4+ Tresp and Tregs engineered to express a GAD-reactive TCR clone 4.13 and activated with cognate GAD555 peptide (Figures4B,C, Ag-specic; Table S1 in Supplementary Material). Specically, Tresp division was signicantly blunted, and Treg percent suppression was signicantly greater than in settings of bystander suppression (FigureC). Importantly, CD8+ Tcells are thought to drive type 1 diabetes pathogenesis invivo through the direct killing of -cells (40). We therefore sought to understand whether Treg avatars are capable of suppressing CD8+ Tcells in a bystander manner in the islets or periphery. We tested the capacity of GAD-specic Tregs to suppress MART-1 CD8+ Tcells recognizing the tumor antigen Melan-A, with or without Treg activation. While unactivated 4.13 Tregs exhibited limited suppression of MART-1 CD8+ Tresp proliferation, GAD-activation of 4.13 Tregs resulted in signicantly reduced Tresp proliferation and increased suppression of MART-1 CD8+ Tresp (Figures4B,C). is supports two notions: rst, that Treg activation is required for functional suppression and second, that TCR transgenic Treg avatars are capable of both antigen-specic and bystander suppression. Finally, we examined if TCR avidity aects Treg suppressive ability with the advantage of using two GAD555-reactive TCR clones, R164 and 4.13, where R164 exhibits higher avidity. Either GAD R164 or 4.13 Tregs were cultured with R164 CD4+ Tresp in the presence of peptide presented by CD3-depleted APCs from HLA-DRB1*04:01/A*02:01 individuals. We normalized the Treg suppression capacity against reporter eGFP MFI allowing us control for potential variation in TCR expression levels. Indeed, cells expressing the high-avidity R164 TCR were signicantly more suppressive than cells expressing the lower avidity 4.13 TCR (Figure5). ese data support the notion that Treg avatars engineered to receive a higher anity signal through the TCR are more ecient suppressors of bystander T-cell responses. It remains to be investigated how costimulatory signals will impact suppressive activity, a notion that may be particularly important for assessing signaling through CAR-T vectors if expressed by Tregs.DISCUSSIONFor tolerogenic adoptive cell therapy, autologous polyclonal Tregs can be expanded from peripheral blood which provides an attractive Treg source given the abundant cell numbers, allowing for repeat dosing if needed, and the safety associated with autologous cell therapy (26, 27). Concerns remain, however, regarding the lack of antigen specicity by administrating polyclonal Tregs. Indeed, Tregs have a highly diverse repertoire (24), which indicates the precursor frequency of autoreactive Tregs will likely be quite low in peripheral blood, especially considering that Tregs do not enrich to the extent that is observed for Tconv during conversion to eector Tcells and expansion. Hence, we expect polyclonal Treg therapy to confer potentially limited ecacy and tracking to the pancreas or PLN to induce immunological tolerance for -cell antigens. To address this, we utilized LV TD to generate primary human T-cell avatars expressing two GAD555-reactive TCRs (R164 and 4.13) originally identied from the peripheral blood of subjects with or at risk for type 1 diabetes (2123). ese clones, which dier by only 10 amino acids in TRAV and TRBV genes and only three amino acid charge dierences in the CDR3 region (Table1 ), exhibit dierent binding anities for their cognate antigen peptide (21). Regulatory Tcell avatars maintained FOXP3 positivity, indicating that LV TD did not impair Treg stability. Functionally, 4.13 Treg avatars eectively suppressed antigen-specic 4.13 CD4+ Tresp. Beyond this, 4.13 Treg avatars exhibited a moderate ability to suppress MART-1 CD8+ Tresp in a bystander suppressive
FIGURE 4 | Regulatory T-cell (Treg) suppression is optimal with activation. (A) Antigen-specic suppression by 4.13 Tregs was tested on 4.13 T-cell receptor (TCR) transduced conventional Tcells (Tconv) invitro (left). Bystander suppression by 4.13 Tregs was assessed on CD8+ Tcells expressing the melanoma antigen recognized by Tcells 1 (MART-1) TCR, with (middle) or without (right) Treg activation. (B) Tregs were isolated from adult peripheral blood and transduced to express glutamic acid decarboxylase (GAD) 4.13 TCR. Transduced Tregs were sorted, labeled with cell proliferation dye (CPD) eFluor670, and plated in decreasing proportions with GAD 4.13 TCR transduced CD4+ responder Tcells (Tresp) (Ag-specic) or MART-1 transduced CD8+ Tresp (Bystander) stained with cell trace violet (CTV) dye. For Ag-specic suppression, GAD 4.13 Tresp and Treg were activated with cognate GAD555 peptide presented by CD3-depleted peripheral blood mononuclear cell (PBMC) from an HLA-DR4 individual. For bystander suppression, MART-1 CD8+ Tresp and GAD 4.13 Tregs were activated with Melan-A27 with or without GAD555 peptide, again presented by CD3-depleted PBMC from an HLA-DR4 individual. Cell proliferation was evaluated via dye dilution for Tresp (top) and Tregs (bottom). Tresp proliferation decreased as the Treg to Tresp ratio increased only when Tregs were activated, and suppression was most effective when Treg activation was antigen-specic. Unactivated Tregs exhibited little to no proliferation. (C) Suppression was evaluated by Tresp division index (left) and percent (%) suppression (right). Tresp division index was signicantly lower and percent suppression of Tresp proliferation was signicantly greater in antigen-specic settings (Ag-specic, black) followed by bystander suppression when Tregs were activated (red). Two-way analysis of variance (ANOVA) (*P<.05, ***P<.001, ****P<.0001). 9 Yeh et al. Frontiers in Immunology | www.frontiersin.org October 2017 | Volume 8 | Article 1313
AB FIGURE 5 | High-avidity T-cell receptor (TCR) activation augments regulatory T-cell (Treg) suppression. (A) Tregs expressing the high-avidity GAD555-reactive R164 TCR (left) or the low-avidity GAD555-reactive 4.13 TCR (right) were activated with their cognate antigen. Activated Tregs were plated in decreasing proportions with Tresp expressing the R164 TCR and stained with cell trace violet (CTV) dye as indicated in the gure. Tresp proliferation was evaluated via dye dilution. Both Treg clones were able to suppress Tresp proliferation at 1:1 cell ratio, but R164 Tregs were more effective in suppressing Tresp proliferation at lower ratios (1/2:1/16:1). (B) Both Tresp proliferation and Treg suppression were normalized to the reporter enhanced green uorescent protein (eGFP) mean uorescence intensity (MFI) to control for potential variation in TCR expression levels. The Tresp division index (left) was signicantly lower and percent (%) suppression (right) based on replication index was signicantly greater for suppression assays using high-avidity R164 Tregs (green) compared with 4.13 Tregs (blue). 10 Yeh et al. Frontiers in Immunology | www.frontiersin.org October 2017 | Volume 8 | Article 1313 mechanism that required Treg activation. Interestingly, when the high-avidity R164 or lower avidity 4.13 Treg avatars were cultured with R164 CD4+ Tresp in the presence of GAD555 peptide presented by CD3-depleted HLA-DRB1*04:01/HLA-A*02:01 APCs, R164 TCR were signicantly more suppressive. is suggests that Treg TCR avidity aects suppressive ability and importantly, that the optimal avidity of TCR or CAR signals may be required for eective Treg avatar cellular therapy. Importantly, however, there is the potential for heterologous TCR chain pairing with the endogenous receptors, and further experiments are needed to empirically determine this for each receptor. Recent developments in gene editing technologies could be used to correct for this potential caveat. Specically, knockout of endogenous TCR and -chains via CRISPR/Cas9, silencing of endogenous TCR via shRNA with expression of a codon optimized de novo TCR, or the domain-swap approach described by Bethune etal. (41) could be applied. Although preproinsulin (PPI) and alternative forms of this antigen (e.g., hybrid insulin peptides, alternative mRNA transcripts) (42, 43) are considered key type 1 diabetes autoantigens, we anticipate continued expression of cognate antigen will be imperative for Treg survival and tracking to the target organ (15, 44). Hence, we focused our eorts on the development of Tregs against GAD65, which exhibits a high autoantigen density in T1D (45) and is the target of persistent autoimmunity, as evidenced by maintenance of autoantibodies (46). We anticipate that adoptive cell therapy with GAD-specic Tregs will lead to bystander suppression and infectious tolerance (47) with the hope for inducing long-term antigen-specic tolerance to GAD as well as other -cell antigens. A recent report by Hull etal. described the generation and invitro characterization of peripheral blood-derived human Tregs expressing TCRs specic for insulinoma-associated protein-2 (IA-2) and insulin (48). Although the authors did not conduct functional comparisons of TCR avidity, invivo investigations of these and the GAD-specic clones generated herein are certainly warranted to determine the optimal clone(s) for tolerogenic cell therapy as we move forward toward clinical testing. Chimeric antigen receptor (CAR) Treg therapy should also be considered given the promising outcomes observed from CAR eector Tcells in cancer immunotherapy (49, 50). CAR Treg therapy could be particularly advantageous given that CAR Tcells are not constrained by HLA restriction, hence, oering the opportunity for o-the-shelf clinical utility. However, the need for surface expression of the target antigen on islets or -cells represents a clear limitation compared with TCR gene transfer, which allows for recognition of intracellular antigens in the context of class II HLA. An additional approach to potentially address this challenge could involve the use of a CD8-restricted TCR that functions independently of the CD8 co-receptor. In fact, this type of activity has been demonstrated previously with a high-anity melanoma antigen tyrosinase-reactive TCR expressed by CD4+ Tcells (20). Yet an additional approach could involve the identication of a CAR capable of recognizing an islet epitope in the context of HLA-A2, given the observation that beta cells hyperexpress class I HLA in settings of type 1 diabetes (51). RNA TCR or CAR gene transfer has been demonstrated as one potential approach to confer T-cell antigen-specicity (52), and could be further explored in the context of tolerogenic adoptive Treg therapy for type 1 diabetes. Specically, mRNA encoding the TCR or a CAR of choice can be introduced to Tcells via electroporation, thereby eliminating the need for LV TD and associated safety requirements. is approach would, of course, be transient with transgene expression lasting only a few days (53), but might be accomplished with multiple autologous dosings. Temporary transgene expression presents lower risk of o-target eects such as bystander suppression of anti-tumor or anti-infection immunity. However, lentivirus transduced Treg avatars likely oer greater potential for long-term ecacy in
11 Yeh et al. Frontiers in Immunology | www.frontiersin.org October 2017 | Volume 8 | Article 1313clearing islet inltration/inammation and leading to persistent engrament. Importantly, adoptive cell therapy with polyclonal autologous peripheral blood Tregs has been demonstrated to be safe in Phase I clinical trials (26, 27). While we anticipate a similar safety prole with TCR transgenic Treg therapy, tolerogenic cell therapies always carry with them possible associations with increased risk of infection or cancer due to bystander suppression and infectious tolerance mechanisms. us, there is a need to perform Phase I safety studies and simultaneously, investigate co-transfection of suicide genes for inducible apoptosis of TCR transgenic Tregsa biological o-switch (54). We recently demonstrated that cryopreserved umbilical cord blood Tregs (cryoCB Tregs) could be isolated and expanded eciently while retaining their nave phenotype as well as suppressive capacity (55). Beyond the possibility for polyclonal autologous cryoCB Treg therapy, these cells oer the potential to generate antigen-specic Treg avatars from precursors with an optimal nave phenotype and without the need for a large-volume peripheral blood draw and leukapher esis, which is generally contraindicated in pediatric patients. is is a goal currently being pursued by our lab and others. Additional optimization, such as further genetic manipulation of TCR transgenic Tregs, could be implemented to correct intrinsic single-nucleotide polymorphisms (SNPs) with putative implications for Treg function and known associations with type 1 diabetes as identied by genome-wide association studies (GWAS) (56). Beyond this, there is potential for delivery of tissue repair factors directly to the pancreas via production by antigen-specic Tregs or via conjugation to the Treg sur face using poly lactic-co-glycolic acid (PLGA) nanoparticles ( 57 59). For these approaches to be successful, we expect that Treg survival invivo and tracking to the target organ will depend largely upon TCR specicity. Hence, we anticipate the functional eects of TCR avidity on human Treg phenotype and function, as demonstrated herein, will be extremely important as we rene adoptive cell therapies to reverse autoimmunity in type 1 diabetes.ETHICS ST A TEMENTHealthy control blood donors provided written informed consent prior to inclusion in the study in accordance with the Declaration of Helsinki and according to Institutional Review Board-approved protocols at the University of Florida (Protocol no. IRB201500059) and the University of Colorado Denver (Protocol no. COMIRB92-292).GUARANTOR ST A TEMENTAs the guarantor of this work, Todd Brusko assumes responsibility for ethical completion of the study, integrity of the data, and accuracy of the data analysis reported herein.AUTHOR CONTRIBUTIONSW-IY researched and analyzed the data and wrote the manuscript. HS and BN researched the data and reviewed/edited the manuscript. AP and FM contributed to discussion and wrote the manuscript. AM and CM contributed to discussion and reviewed/ edited the manuscript. JB conceived of the study and reviewed/ edited the manuscript. TB conceived of the study, researched the data, and wrote the manuscript.ACKNOWLEDGMENTSWe would like to thank Rita Bortelli and Dale L. Greiner (University of Massachusetts, Worcester, MA, USA) who gener ously provided 4.13 and R164 TCR constructs. We also thank Drs. James Riley and Bruce Levine (University of Pennsylvania) for kindly providing the K562 articial APC cell line.FUNDINGis project was supported with funding provided by the JDRF Career Development Award (grant number 2-2012-280 to TB), the National Institutes of Health (grant number R01 DK106191 and P01 AI42288), and the NIH Human Islet Research Network (HIRN) (grant number UC4 DK104194 and U01 DK104162). Publication of this article was funded in part by the University of Florida Open Access Publishing Fund.SUPPLEMENT AR Y MA TERIALe Supplementary Material for this article can be found online at http://www.frontiersin.org/article/10.3389/mmu.2017.01313/ full#supplementary-material.FIGURE S1 | Comparable regulatory T-cell (Treg) avatar suppression of polyclonal stimulated responder Tcells (Tresp). (A) Tregs were transduced with GAD-reactive TCR clones (R164 or 4.13) or remained untransduced (Mock) and cultured with autologous polyclonal Tresp cells in decreasing proportions for 4days with soluble anti-CD3 (2g/mL) and anti-CD28 (1g/mL) stimulation. Tresp proliferation was assessed by 3H-thymidine incorporation (left), and the percent suppression was determined by upon Tresp division index relative to the proliferation of Tresp when no Tregs were present (right). (B) IFNproduction by polyclonal Tcells was inhibited by Treg cells with or without TCR transduction. 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