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The Role of CD25 in Interleukin-2 Signaling in Type 1 Diabetes

Permanent Link: http://ufdc.ufl.edu/UFE0043748/00001

Material Information

Title: The Role of CD25 in Interleukin-2 Signaling in Type 1 Diabetes
Physical Description: 1 online resource (125 p.)
Language: english
Creator: Hulme, Maigan A
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: cd25 -- diabetes -- il2 -- immunology -- interleukin -- regulatory -- t
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Through its diverse functional activity as part of the interleukin-2 (IL-2) receptor, CD25 is involved both in the progression to and prevention of autoimmunity at multiple levels. The high IL-2 affinity that CD25 confers allows for cellular proliferation during early conventional T cell (Tconv) activation, yet also predisposes these activated cells to activation induced cell death (AICD). Regulatory T cells (Treg) require high CD25 expression to maintain their phenotype. Proteolytic cleavage of CD25 from the cell surface therefore has opposite effects on Treg and Tconv by lessening their IL-2 response. In this work, we have demonstrated a role for the proteolytic release of soluble CD25 (sCD25) in providing additional fine-tuning of the IL-2 response. Production of sCD25 was identified to associate with both proliferation and cell death in T-cell activation cultures, suggesting that the loss of CD25 on the cell surface may be an attempt to avoid AICD in an activated cell. Additionally, we found that exogenous addition of sCD25 affected cell proliferation in a manner dependent on culture conditions. Through expression of mutant CD25 constructs in primary human T cells as well as human T cell lines, we identified that sCD25 appears to enact an IL-2 dependent 12 increase in cellular expansion in vitro. The in vivo effects, however, remain to be elucidated. Deficiencies or defects in several IL-2 pathway genes - including CD25 - associate with increased susceptibility to T1D and other autoimmune diseases. This is due, at least in part, to the disruption of an equilibrium that exists between Treg and pathogenic autoreactive T cells. Efforts to restore or alter this balance through provision of exogenous IL-2 to support Treg or blockade of CD25 to prevent autoreactive T cell expansion have had mixed success in autoimmune disease, suggesting the need for continued therapy development. We believe this can be accomplished through modulation directed at the high-affinity component of CD25, due to its unique role in directing cellular responsiveness to IL-2. Our efforts included the targeting of CD25 protein-protein interactions within the IL-2 receptor structure with small molecule compounds, and the screening of these compounds on human PBMC to view IL-2 response.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Maigan A Hulme.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Atkinson, Mark A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043748:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043748/00001

Material Information

Title: The Role of CD25 in Interleukin-2 Signaling in Type 1 Diabetes
Physical Description: 1 online resource (125 p.)
Language: english
Creator: Hulme, Maigan A
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: cd25 -- diabetes -- il2 -- immunology -- interleukin -- regulatory -- t
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Through its diverse functional activity as part of the interleukin-2 (IL-2) receptor, CD25 is involved both in the progression to and prevention of autoimmunity at multiple levels. The high IL-2 affinity that CD25 confers allows for cellular proliferation during early conventional T cell (Tconv) activation, yet also predisposes these activated cells to activation induced cell death (AICD). Regulatory T cells (Treg) require high CD25 expression to maintain their phenotype. Proteolytic cleavage of CD25 from the cell surface therefore has opposite effects on Treg and Tconv by lessening their IL-2 response. In this work, we have demonstrated a role for the proteolytic release of soluble CD25 (sCD25) in providing additional fine-tuning of the IL-2 response. Production of sCD25 was identified to associate with both proliferation and cell death in T-cell activation cultures, suggesting that the loss of CD25 on the cell surface may be an attempt to avoid AICD in an activated cell. Additionally, we found that exogenous addition of sCD25 affected cell proliferation in a manner dependent on culture conditions. Through expression of mutant CD25 constructs in primary human T cells as well as human T cell lines, we identified that sCD25 appears to enact an IL-2 dependent 12 increase in cellular expansion in vitro. The in vivo effects, however, remain to be elucidated. Deficiencies or defects in several IL-2 pathway genes - including CD25 - associate with increased susceptibility to T1D and other autoimmune diseases. This is due, at least in part, to the disruption of an equilibrium that exists between Treg and pathogenic autoreactive T cells. Efforts to restore or alter this balance through provision of exogenous IL-2 to support Treg or blockade of CD25 to prevent autoreactive T cell expansion have had mixed success in autoimmune disease, suggesting the need for continued therapy development. We believe this can be accomplished through modulation directed at the high-affinity component of CD25, due to its unique role in directing cellular responsiveness to IL-2. Our efforts included the targeting of CD25 protein-protein interactions within the IL-2 receptor structure with small molecule compounds, and the screening of these compounds on human PBMC to view IL-2 response.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Maigan A Hulme.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Atkinson, Mark A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043748:00001


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Type 1 Diabetes Development and Complications

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Treatment of Type 1 Diabetes

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Increasing Incidence of Type 1 Diabetes and Healthcare Burden Natural History and Etiology of Type 1 Diabetes

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Defined Genetic Susceptibility

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Environmental I nfluences

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,

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Autoimmune P athogenesis

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Regulatory imbalance

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IL 2 and the IL2 Receptor: P hysiological R ole and F unction. Functional I mpact of IL -2 S ignaling

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Receptor c lustering and s ignal t hresholds dictates cellular activity a role for membrane CD25

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IL -2 signaling in nonT cells Genetic D efects in the IL -2 S ignaling P athway Predispose to Autoimmunity

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CD25 CD25 IL2 Escape from AICD

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Imbalance in Treg and effector T cell activity in vitro

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Lineage plasticity in Thelper and Treg cells

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IL -2 P athway -T argeted T herapy for Immunomodulation IL -2 therapy in animal models

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Clinical usage of IL -2 for the treatment of autoimmunity

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Modified versions of IL -2 allow for lower dose and optimal delivery. in vivo

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IL -2 signaling blockade differentially affects Treg and effector populations.

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Alternative approaches to therapeutically target the IL -2 signaling pathway Personalized medicine and pathway targeted interventions. Summary

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Background

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Methods Participants

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Staining and Flow Cytometry Cell Purification and FluorescenceActivated Cell Sorting

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Isolation of Purified Treg and Teff Cell Populations by FACS Cell Culture

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Suppression Assay Coculture System PBMC Proliferation Assays

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Supernatant Cytokine, sCD25 and Nuclear Matrix Protein Detection Reagents Statistical Methods

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Results Production of sCD25 Duri ng Suppression Assay

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The Production of sCD25 Correlates with Cellular Proliferation in the Suppression Assay

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Exogenous sCD25 Does Not Affect Suppression, but Alters Teff P roliferation sCD25 has CultureDependent Effects on PBMC Proliferation

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Exogenous sCD25 May Increase Survival in PBMC Cultures

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Production of sCD25 by PBMC Correlates with Cell Death Interpretation

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Summary

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Background

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Methods Participants Mouse S erum Samples

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sCD25 Protein Detection by ELISA Cell C ulture of C ell L ines and P rimary C ells Preparation of L entiviral V ectors for CD25 P rotein E xpression

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Lentiviral V ector P roduction

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Lentiviral Transduction of C ell L ines and P rimary C ells Cell lines Primary cells Flow C ytometry

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Western B lotting Statistical Methods Results sCD25 P roduction in K nockout M ouse S erum

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Lentiviral C onstruct F ormation

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Lentiviral Transduction of CD25 in Jurkat Cells

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Lentiviral Transduction of P rimary CD4+ T cells

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Interpretation

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+

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Background

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Methods Molecular Docking

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Compound Receipt and Storage Participants Cell C ulture

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Preliminary Compound Screening on PBMCs Functional A nalysis of CD4 T cells Flow C ytometry

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Supernatant and Cell Lysate Protein Detection by ELISA Statistical Analysis Results Preliminary Screening on Human PBMC and CD4 T cells

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Functional Analysis of IL-2 S pecific E ffects of Candidate C ompounds

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Interpretation

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A PBMC response B CD4+ response

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