Costimulatory Blockade-Resistant Transplant Rejection: Mechanistic Characterization and Evaluation of Novel Immunomodulatory Regimens Pubblico

Abstract

The success of belatacept in late-stage clinical trials inaugurates the arrival of a new class of immunosuppressants based on costimulatory blockade (CoB), an immunomodulatory strategy that disrupts signals required for alloreactive T cell activation. Despite having improved renal function, kidney transplant recipients treated with belatacept experienced increased rates of acute rejection. This finding has renewed focus on costimulatory blockade-resistant rejection, a process likely mediated by subsets of T cells with diminished requirements for costimulation, such as memory and Th17 T cells. To study the contribution of memory T cells to CoB-resistant rejection, we developed a murine transplant system that models a donor-specific memory CD8⁺ T cell response. After confirming that alloreactive memory T cells can mediate CoB-resistant rejection, we then demonstrated that these donor-specific memory T cells require intact VLA-4 and LFA-1 integrin pathways to mediate rejection. Indeed, the resistance of memory T cells to CoB was abrogated when costimulatory blockade was coupled with either anti-VLA-4 or anti-LFA-1. Mechanistic studies revealed that in the presence of CoB, anti-VLA-4 impaired T cell trafficking to the graft, whereas anti-LFA-1 predominantly attenuated memory T cell effector responses. We extended our findings to both a murine transplant model of polyclonal heterologous memory alloresponses, as well as to a non-human primate kidney transplant system. Next, we evaluated whether Th17 T cells participate in CoB-resistant rejection, exploiting the requirement of IL-23 for Th17 T cell proliferation. We showed that combined CoB and anti-IL-12/23 can significantly prolong survival of murine skin and vascularized cardiac allografts. Further work with selective IL-12 and IL-23 blockade indicated that IL-23 blockade was responsible for the enhanced efficacy of CoB. Combined costimulatory and IL-12/23 blockade inhibited alloreactive T cell proliferation and promoted immunodeviation away from Th1 and Th17 alloresponses. Finally, we extended our findings in a non-human primate kidney transplant system using rhesus macaques treated with belatacept and ustekinumab (humanized anti-IL-12/23), demonstrating prolonged graft survival compared to recipients treated with belatacept monotherapy. Given that integrin antagonists and ustekinumab are in clinical use, these findings have significant translational potential for future clinical transplant trials with belatacept.

Table of Contents

Chapter 1: Introduction........... 1

Costimulatory blockade: a novel immunosuppression strategy with clinical potential

Memory T cells as a barrier to costimulatory blockade

Integrin antagonists as a means to target alloreactive memory T cells

Integrin antagonists in transplantation

Th17 T cells: a newly defined T cell subset

Th17 cells and transplantation

Th17 T cells and costimulatory blockade

Chapter 2: Methods and Materials........... 17

Chapter 3: Integrin Antagonists prevent costimulatory blockade-resistant transplant rejection by CD8⁺ memory T cells... 25

A novel murine transplant system models rejection by donor-specific memory T cells

Combined integrin and costimulatory blockade prolongs graft survival against a donor-specific memory response

Combined integrin and costimulatory blockade does not curtail recall accumulation of donor-specific memory T cells

LFA-1 blockade attenuates donor-specific memory T cell effector responses

Both VLA-4 and LFA-1 blockade inhibit donor-specific memory T cell trafficking to graft

Combined LFA-1 and costimulatory blockade prolongs skin graft survival against a heterologous immune alloresponse

Combined blockade surmounts barrier posed by heterologous immunity to allogeneic bone marrow engraftment

Combined blockade inhibits alloreactive T cell proliferation and effector responses

Combined blockade promotes retention of Tregs in draining LNs

Combined blockade suppresses cytokine responses of human memory CD8⁺ T cells

Integrin and costimulatory blockade prolongs kidney allograft survival in non-human primates

Chapter 4: Th17 cells as a barrier to costimulatory blockade........... 35

Anti-IL-12/23 synergizes with costimulatory blockade to prolong murine skin graft and heterotopic cardiac allograft survival

Prolongation of transplant survival with adjunct anti-IL-12/23 is mediated predominantly by IL-23 blockade

Combined costimulatory and IL-12/23 blockade inhibits alloreactive T cell proliferation

Treatment with combined costimulatory and IL-12/23 blockade causes no additional expansion of alloreactive Tregs compared to costimulatory blockade alone

Combined costimulatory and IL-12/23 blockade induces immunodeviation away from Th1 and Th17 alloresponses

Cellular sources of alloreactive IL-17A production

Characterization of belatacept-resistant kidney allograft rejection in non-human primates

Belatacept synergizes with ustekinumab to prolong kidney allograft survival in non-human primates

Chapter 5: Discussion........... 43

Figures........... 56

References........... 98

Figure Index

Fig. 1: Schematic of mOVA transplant system to model graft-specific memory responses ......... 56

Fig. 2: Kinetics of memory OT-I cell generation after LM-OVA infection.......... 57

Fig. 3: Memory OT-I cells are resistant to CoB and reject mOVA skin grafts.......... 58

Fig. 4: CD8⁺ memory T cells are sufficient to mediate costimulatory blockade-resistant transplant rejection.......... 59

Fig. 5: Integrin upregulation on memory T cells.......... 61

Fig. 6: Dual costimulatory/integrin blockade prolongs graft survival.......... 62

Table 1: Combined costimulatory/integrin blockade prolongs graft survival even against a donor-specific CD8⁺ T cell response......... 63

Fig. 7: High memory OT-I frequency after serial LM-OVA infection prevents graft survival prolongation with combined integrin/costimulatory blockade.......... 64

Fig. 8: Dual blockade does not inhibit donor-reactive memory CD8+ T cell expansion and accumulation compared to CoB alone.......... 65

Fig. 9: LFA-1 blockade attenuates CD8⁺ memory T cell cytokine response.......... 66

Fig. 10: LFA-1 blockade attenuates CD8⁺ memory T cell degranulation.......... 67

Fig. 11: LFA-1 blockade attenuates CD8⁺ memory T cell cytotoxicity.......... 68

Fig. 12: VLA-4 blockade inhibits donor-specific CD8⁺ memory T cell trafficking to the graft.......... 69

Fig. 13: VLA-4 blockade inhibits donor-specific CD8⁺ memory T cell trafficking to the graft.......... 70

Fig. 14: VLA-4 blockade inhibits donor-specific CD8⁺ memory T cell trafficking to the graft.......... 71

Fig. 15: Combined costimulatory and LFA-1 blockade prolongs graft survival against a heterologous immune response.......... 72

Fig. 16: Short-course induction anti-LFA-1 fails to prolong skin graft survival.......... 73

Fig. 17: Combined costimulatory and LFA-1 blockade prevents graft infiltration.......... 74

Fig. 18: Combined costimulatory and LFA-1 blockade enables long-term donor chimerism despite an anti-donor heterologous immune response.......... 75

Fig. 19: Combined costimulatory and VLA-4 blockade did not prolong graft survival against a robust anti-donor heterologous immune response.......... 76

Fig. 20: Combined costimulatory and LFA-1 blockade suppresses alloreactive T cell proliferation in recipients with a robust anti-donor heterologous immune response.......... 77

Fig. 21: Combined costimulatory and LFA-1 blockade suppresses alloreactive T cell cytokine production in recipients with a robust anti-donor heterologous immune response.......... 78

Fig. 22: Combined costimulatory and LFA-1 blockade promotes allospecific T cell accumulation in dLN.......... 79

Fig. 23: LFA-1 blockade (but not costimulation blockade) inhibits human CD8⁺ memory T cell effector responses.......... 80

Fig. 24: Combined belatacept and natalizumab significantly prolong kidney allograft survival in rhesus macaques.......... 81

Fig. 25: CoB + anti-IL-12/23 significantly prolongs skin and cardiac allograft survival......... 82

Fig. 26: Prolonged graft survival with CoB + anti-IL-12/23 is mediated predominantly by IL-23 blockade......... 83

Fig. 27: Combined costimulatory and anti-IL-17A fails to prolong allograft survival......... 84

Fig. 28: Combined costimulatory and anti-IL-12/23 blockade potently suppresses alloreactive T cell proliferation......... 85

Fig. 29: Treatment with CoB or CoB+anti-IL-12/23 enables accumulation of allospecific FoxP3⁺ Tregs......... 86

Fig. 30: Treatment with costimulatory blockade induces higher levels of IL-17 by alloreactive dLN cells compared to untreated skin graft recipients......... 87

Fig. 31: Treatment with costimulatory blockade induces higher levels of IL-17 transcripts by alloreactive dLN cells compared to untreated skin graft recipients......... 88

Fig. 32: Anti-IL-12/23 suppresses the allospecific production of GM-CSF......... 89

Fig. 33: Allostimulation-induced IL-17A is produced predominantly by CD4⁺ T cells......... 90

Fig. 34: Characterization of kidney allograft rejection in belatacept-treated non-human primates......... 91

Fig. 35: Chemokine receptor profile of graft-infiltrating lymphocytes from primate kidney allografts undergoing belatacept-resistant rejection is consistent with Th17 cells......... 92

Fig. 36: Combined belatacept and ustekinumab significantly prolongs non-human primate kidney allograft survival......... 93

Fig. 37: Combined belatacept and ustekinumab prevents graft-infiltration of kidney allografts in non-human primates......... 94

Fig. 38: Combined belatacept and ustekinumab does not deplete B cell or T cell populations......... 95

Fig. 39: Combined belatacept and ustekinumab treatment does not cause reactivation of CMV viremia......... 96

Fig. 40: Integrated model of the effects of combined costimulatory and IL-12/23 blockade on alloresponses......... 97

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Health Sciences, Immunology
Parola chiave	memory T cells LFA-1 transplantation heterologous immunity costimulatory blockade Th17 T cells non-human primates VLA-4 IL-23 integrin antagonists
Committee Chair / Thesis Advisor	Ford, Mandy L, Emory University
Committee Members	Larsen, Christian P, Emory University Selvaraj, Periasamy, Emory University Williams, Ifor, Emory University Kirk, Allan D, Emory University

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