The Confluence of Protective Immunity and Alloreactivity: Analysis of Memory CD4+ Th17 and CD8+ T cells During Heterologous Immunity Open Access

Krummey, Scott Michael (2014)

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Effective immunodulation following solid organ transplantation presents a tremendous clinical challenge. Microbe-elicited T cells that cross-react with allogeneic antigen and mediate graft rejection, a process termed allogeneic heterologous immunity, are a strong barrier to transplantation acceptance and tolerance. Although this phenomenon has been described in both murine models and human studies, the specific parameters that control allogeneic heterologous immunity are poorly understood. While the susceptibility of CD8+ memory T cells to CD28/CTLA-4 blockade with belatacept (a CTLA-4 Ig derivative) are well defined, the role of Th17 memory cells in costimulation blockade resistant rejection is not known. We found that the frequency of Th17 memory cells was specifically associated with acute cellular rejection in patients treated with belatacept. Murine and human Th17 cells were resistant to costimulation blockade with CTLA-4 Ig both in vitro and in vivo, due at least in part to higher expression of the coinhibitory receptor CTLA-4. An additional undefined facet of allogeneic heterologous T cell responses is the role of T cell priming affinity. As heterologous immunity requires a T cell to recognize two distinct p:MHC complexes, T cell priming affinity is hypothesized to be a critical feature of heterologous immunity. Here we report that compared to high affinity memory CD8+ T cells, memory T cells primed with low affinity antigen are capable of potent heterologous rechallenge responses. We found that large CD45 isoforms, represented as CD45RBhi, predominated on low affinity primed memory cells, and that CD45RBhi expression conferred a proliferative advantage during secondary rechallenge characterized by distinct IL-2/IL-2Rα production. Low affinity primed secondary effectors also upregulated the TNF receptor 2 (TNFR2) and were more dependent on TNF:TNFR2 signals during graft rejection than high affinity primed secondary effectors. Together, these data provide important phenotypic and functional description of pathogen elicited T cell subsets in the context of transplantation. As the successful modulation of pathogenic T cell responses depends on a more granular understanding of T cell functionality, this work provides important description of the role of T cell subsets in the context of heterologous immunity that may generate novel therapeutic strategies.

Table of Contents

Chapter 1. Introduction 1

Chapter 2. High CTLA-4 Expression on Th17 Cells Results in Increased Sensitivity to CTLA-4 Coinhibition and Resistance to Belatacept 31

Introduction 31

Materials & Methods 33

Results 36

Alloreactive Th17 memory cells are resistant to belatacept

Th17 memory cells significantly upregulate the coinhibitor CTLA-4

CD45RA- Th17 cells are uniquely sensitive to CTLA-4 coinhibition

Renal transplant recipient-derived lymph node CD45RA- Th17 cells are resistant to belatacept

An elevated Th17 memory cell frequency correlates to rejection during belatacept therapy in renal transplant recipients

Discussion 41

Figures 44

Chapter 3. Candida-Elicited Murine Th17 Cells Express High CTLA-4 Compared to Th1 Cells and Are Resistant to Costimulation Blockade 50

Introduction 50

Materials & Methods 53

Results 57

Candida antigen immunization yields costimulation blockade resistant graft rejection

Candida immunization elicits a Th17 skewed phenotype compared to M.Tb.

M.Tb and Candida elicit similar frequencies of effector memory cells

Candida elicited Th17 cells have a more pathogenic phenotype compared to M.Tb elicited Th17 cells.

Th17 cells express greater amounts of CTLA-4 than Th1 cells.

Th17 cells are less susceptible to CTLA-4 Ig and more sensitive to CTLA-4 coinhibition than Th1 cells.

Pathogen elicited Th1 and Th17 cells similarly regulate CD154 expression

Neutrophils are recruited to skin grafts in a costimulation blockade independent manner by Candida-elicited T cell responses.

Discussion 65

Figures 68

Chapter 4. High CTLA-4 Expression Corresponds with Diminished FOXO3 Expression on Human Th17 Memory Cells 81

Introduction 81

Results & Discussion 82

Human Th17 cells are resistant to selective CD28 blockade

Th1 and Th17 cells are similarly activated by CD28 signals

FOXO3 expression correlates inversely with CTLA-4 expression

FOXO3 expression is diminished in Th17 cells

Materials & Methods 85

Figures 88

Chapter 5. Low Affinity Memory CD8+ T Cells Mediate Robust

Heterologous Immunity 92

Introduction 92

Results 94

Low affinity CD8+ T cell priming efficiently generates memory cells

Low affinity memory CD8+ T cells have a distinct central memory phenotype

Low affinity memory CD8+ T cells are tuned to generate robust secondary recall responses

CD45RB is a stable marker of the affinity experience of memory CD8+ T cells

High CD45RB expression tunes CD8+ T cells to respond to heterologous rechallenge antigen

Low affinity primed secondary effector cells have a distinct phenotype

Low affinity primed secondary effector CD8+ T cell produce high levels of IL-2 and upregulate IL-2Rα

Low affinity polyclonal CD8+ T cells express high levels of CD45RB

Discussion 102

Materials & Methods 105

Figures 109

Chapter 6. Enhanced Requirement for TNF Signaling on Low Affinity Memory CD8+ T Cells During Heterologous Immunity 120

Introduction 120

Materials & Methods 122

Results 124

Low affinity primed memory CD8+ T cells produce high levels of TNF

Low affinity primed memory CD8+ T cells produce high levels of TNF

Low affinity primed secondary effectors upregulate TNFR2 expression

Low affinity primed memory CD8+ T cells are dependent on TNFR2 signaling in order to optimally mediate graft rejection during heterologous rechallenge

Discussion 128

Figures 130

Chapter 7. Discussion 134

References 149

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