The Role of Signal 3 Cytokines in Costimulation Independent Rejection Open Access

Abstract

Solid organ transplantation has become the primary treatment for end-stage organ failure, driven by the advent of potent immunosuppressants. Non-specific immunosuppression with calcineurin inhibitors (CNIs) were instrumental in reducing the incidence of early graft failure due to acute rejection. Despite these advances, long-term transplant outcomes have remained largely unchanged over the past thirty years. While this is likely multi-factorial, the non-immune effects of CNIs play a significant role, leading to heart-attack, stroke, diabetes, and allograft vasculopathy which is the leading cause of transplant failure. In 2011, a high affinity variant of the CTLA4-Ig fusion protein, belatacept, was approved as the first alternative to CNIs. Belatacept specifically interrupts CD28-CD80/CD86 mediated T cell costimulation. Compared to patients treated with CNIs, patients treated with belatacept live longer and retain superior renal function, which amounts to the first improvement in long-term outcomes for transplant patients in over thirty years. However, a subset of belatacept treated patients experience increased rates of acute rejection. In order to understand and address costimulation independent rejection, we studied belatacept resistance in a pre-clinical model of non-human primate (NHP) renal transplantation. We found that animals resistant to belatacept had increased frequencies of CD28⁺CD8⁺ memory T cells prior to transplant. These cells leverage a proliferative advantage in order to prosecute costimulation independent rejection, characterized by a unique more terminally differentiated CD8⁺ T cell graft infiltrate. We hypothesized that signaling through the IL-2/IL-15Rb (CD122) and/or the IL-7Ra (CD127) may provide alternative pathways for T cell activation in the setting of costimulatory blockade. We found CD122 signaling was critical for costimulation independent memory CD8 T cell mediated graft rejection, and combination therapy improved graft survival in mice and NHPs. We also investigated the role of CD127 in costimulation independent rejection. Combined Costimulation blockade and CD127 blockade gave rise to increased frequencies of graft specific iTregs while controlling the expansion and effector function of graft reactive CD8 T cells. These data provide basic insights into the signaling requirements of T cells and outline a new strategy: targeting unique Signal 3 cytokines for the optimization of clinical costimulatory blockade in transplantation.

Table of Contents

Chapter 1. Introduction…………………………………………………………………1

           The Immunological Barrier to Graft Survival.............................................................................1

           Pharmacological Management of Acute Allograft Rejection.........................................................2

           Life After Year One: A Failure to Maximize Graft Half-life...................................................5           

Costimulatory Blockade as a Targeted Approach to Transplant Immunosuppression...................6

           Costimulation Independence is a Barrier to Better Outcomes in Transplantation..........................11

           Memory as Barrier to Transplant Tolerance................................................................................13

Signal 3 Cytokines.....................................................................................................................19

                      The Shared Biology of Interleukin-2 and Interleukin-15...............................................20

                      Interleukin-7 in T cell Responses..................................................................................27

           Costimulation Independence – A Pressing Clinical Need with Potential Solutions.......................31

Chapter 2. Belatacept Resistant Rejection is Associated with CD28+ Memory CD8 T cells…………33

           Introduction...........................................................................................................................33

           Materials and Methods.........................................................................................................36

           Results.....................................................................................................................................40

           Discussion..............................................................................................................................48

           Figures....................................................................................................................................52

Chapter 3. CD122 Signaling in CD8+ Memory T cells Drives Costimulation Independent Rejection……72

Introduction...........................................................................................................................72

           Materials and Methods.........................................................................................................75

           Results.....................................................................................................................................80

           Discussion..............................................................................................................................87

           Figures....................................................................................................................................92

Chapter 4. The role of IL-7Ra in Costimulation Independent Rejection…………..107

Introduction.........................................................................................................................107

           Materials and Methods.......................................................................................................109

           Results...................................................................................................................................112

           Discussion............................................................................................................................115

           Figures...................................................................................................................................118

Chapter 5. Discussion………………………………………………………………….127

References………………………………………………………………………………133

Figure Index

Chapter 2

           Figure 2.1 Kidney Transplant Treatment Schema

           Figure 2.2 Survival and Therapeutic Resistance

           Figure 2.3 Pre-Transplant Immune Phenotyping with CD28 and CD95

           Figure 2.4 Pre-transplant Multi-parametric Immune Phenotyping of CD8 T cells

           Figure 2.5 Kinetics of CD8+ TEMRA

           Figure 2.6 In-Vitro Functional Assessment of alloreactive CD28+ CD8+ TEMRA

           Figure 2.7 Increased Adhesion Molecule Expression in Rejection

Figure 2.8 Belatacept Resistance is Marked by Terminally Differntiated T cells in the Graft Infiltrate

           Figure 2.9 Distinct Intragraft Transcriptome Modules Define Belatacept Resistant Rejection

           Supplemental Figure 2.1 Graft Function and Survival

           Supplemental Figure 2.2 Pre-transplant immunophenotype Segregates Belatacept Resistance

           Supplemental Figure 2.3 Kinetics of CD8+ TEMRA in Belatacept Therapy

           Supplemental Figure 2.4 Mean Prograf Levels         

           Supplemental Figure 2.5 CMV Reactivation

Supplemental Figure 2.6 Gating Strategy for pre-transplant Immunophenotyping

Chapter 3

Figure 3.1 Kinetics of CD122 Expression on CD8 T cells in Acute Viral Infection and Allograft Rejection

Figure 3.2 CD122 Signaling Underlies Costimulation Independent Rejection

Figure 3.3 CD122 Signaling Supports Costimulation Independent Recall Responses

Figure 3.4 The High Affinity IL-2R is Dispensable for Recall Responses

Figure 3.5 CD122 Phenotype and function on Rhesus macaque CD8 T cells

Figure 3.6 Humanized αCD122 Synergizes with Belatacept to inhibit Alloreactivity and Prolong NHP Survival

Figure 3.7 Belatacept + αCD122 Combination Therapy does not impact overall T cell frequencies including Tregs

Supplemental Figure 3.1 CMV Reactivation

 Chapter 4

           Figure 4.1 NHP CD8 T cell Phenotype and Function

           Figure 4.2 Blocking IL-7Ra Prevents Costimulation Independent Rejection

Figure 4.3 Blocking IL-7Ra Prevents Costimulation Independent Proliferation

Figure 4.4 Addition of Anti-IL7Ra Synergistically Prevents Costimulation Independent Rejection

Figure 4.5 Anti-IL7Ra + CoB Results in Decreased Expansion and Effector Function of Graft Reactive T cells

Figure 4.6 Anti-IL7Ra + CoB Augments the Frequency of Graft Specific iTregs

Figure 4.7 Phenotypic Changes in Graft Specific CD8 T cells

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Health Sciences, Immunology
Keyword	Signal 3 Cytokines Transplantation Costimulation Blockade
Committee Chair / Thesis Advisor	Adams, Andrew B., Emory University
Committee Members	Araki, Koichi, Emory University Stowell, Sean, Emory University Boise, Lawrence, Emory University Ford, Mandy , Emory University Sanz, Inaki, Emory University

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