MHC and Peptide Specificity Underpin CD8+ T cell Direct Alloresponse Open Access

Abstract

The direct alloresponse, pivotal in transplant rejection, occurs when recipient T cells recognize intact allogeneic peptide-MHC complexes. Despite extensive research, our understanding of alloreactive CD8+ T cells against an individual MHC allele in humans remains limited, especially their precursor frequency, MHC specificity, and peptide specificity. By utilizing K562 cell-based artificial antigen- presenting cells (aAPCs) expressing HLA-A*01:01, HLA-A*02:01, or HLA-A*03:01, we determined that the precursor frequency of alloreactive CD8+ T cells against a single MHC allele ranges from 0.1% to 0.5%. Further, these cells exhibited MHC-specificity regarding proliferation, activation, IFN-γ secretion, and cytolytic ability, with limited cross-reactivity towards non-targeted MHC alleles. Focusing on anti-A2 alloreactive CD8+ T cells, we developed a peptide-exchangeable aAPC that displays selected peptides on HLA-A*02:01. From a set of 95 computationally curated A2-restricted peptides most abundant in renal tubular cells, we identified two immunogenic kidney peptides across multiple donors. Overall, our findings significantly enhance the understanding of direct alloresponse and provide a toolkit for future mechanistic studies and reproducible patient monitoring. 

Table of Contents

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

1.1 Introduction to direct alloresponse.......................................................................................................1 1.1.1 Transplantation and rejection.........................................................................................................1 1.1.2 Transplant rejection is primarily mediated by T cells .................................................................4 1.1.3 Allorecognition pathways involved in transplant rejection........................................................5

1.2 Contribution of memory vs naive T cells toward direct allo-response ........................................... 8

1.3 The high precursor frequency of direct alloreactive T cells............................................................11

1.4 Binding correlates of direct allorecognition.......................................................................................15 1.4.1 Peptide-centric vs MHC-centric...................................................................................................15 1.4.2 Polyspecificity vs molecular mimicry...........................................................................................18

1.5 Immunogenicity of MHC molecules ..................................................................................................19

1.6 Advances in uncovering self-peptides recognized in direct alloresponse.....................................20

Chapter 2 Establish K562 cell-based aAPCs to assess alloreactive CD8+ T cells against a single HLA-A allele .....................................................................................................................................................24

2.1 Introduction............................................................................................................................................24

2.2 Method ....................................................................................................................................................25 2.2.1 Generation of KCo2, KA1, KA2, and KA3 cells......................................................................25 2.2.2 Lentivirus transduction..................................................................................................................25

2.3 Result .......................................................................................................................................................26

2.4 Discussion...............................................................................................................................................28

Chapter 3 Monoallelic MHC expressing aAPCs induced proliferation, activation, and TCR signaling of alloreactive CD8+ T cells against a single MHC allele..........................................................................29

3.1 Introduction............................................................................................................................................29

3.2 Method ....................................................................................................................................................29 3.2.1 Human CD8+ T cells preparation...............................................................................................29 3.2.2 In vitro allo-stimulation of human CD8+ T cells with aAPCs...............................................30 3.2.3 Statistical analysis............................................................................................................................31

3.3 Result .......................................................................................................................................................32 3.3.1 Monoallelic aAPCs induce proliferation and activation of alloreactive CD8+ T cells........32 3.3.2 Monoallelic aAPCs induced TCR signaling in alloreactive CD8+ T cells.............................38

3.4 Discussion...............................................................................................................................................40

Chapter 4 Estimate the precursor frequency of alloreactive CD8+ T cells against a single MHC allele ............................................................................................................................................................................. 42

4.1 Introduction............................................................................................................................................42 4.2 Method ....................................................................................................................................................42 4.3 Result .......................................................................................................................................................43 4.4 Discussion...............................................................................................................................................45

Chapter 5 Alloreactive CD8+ T cells exhibit MHC-specificity ................................................................46

5.1 Introduction............................................................................................................................................46

5.2 Method ....................................................................................................................................................47 5.2.1 Serial allo-stimulation with aAPCs to establish anti-A1, A2 and A3 human CD8+ T cells ..................................................................................................................................................................... 47 5.2.2 Cross-stimulation of anti-A1, A2 and A3 human CD8+ T cells ............................................47 5.2.3 Statistical analysis............................................................................................................................49

5.3 Result .......................................................................................................................................................50 5.3.1 Alloreactive CD8+ T cells exhibit MHC-specific proliferation and activation....................50 5.3.2 Alloreactive CD8+ T cells exhibit MHC-specific IFN-γ secretion........................................52 5.3.3 Alloreactive CD8+ T cells exhibit MHC-specific cytolytic activity........................................53

5.4 Discussion ...............................................................................................................................................57 Chapter 6 Establish and validate novel peptide exchangeable KA2 cellular system .............................59 6.1 Introduction............................................................................................................................................59

6.2 Method ....................................................................................................................................................60 6.2.1 Generation of PXKA2 cells..........................................................................................................60 6.2.2 qPCR analysis to confirm gene knock out..................................................................................60 6.2.3 3C protease-based linker cleavage and peptide exchange system...........................................61 6.2.4 Statistical analysis............................................................................................................................62

6.3 Result .......................................................................................................................................................62 6.3.1 Generation of PXKA2 cells..........................................................................................................62 6.3.2 Verify the 3C protease-based linker cleavage and peptide exchange system ........................65 6.3.3 Functional validation of PXKA2 cells.........................................................................................65

6.4 Discussion...............................................................................................................................................69 Chapter 7 Identifying immunogenic kidney peptides in anti-HLA-A*02:01 response .........................71

7.1 Introduction............................................................................................................................................71

7.2 Method ....................................................................................................................................................71 7.2.1 Computational approach to identify candidate immunogenic A2 restricted kidney peptides ..................................................................................................................................................................... 71 7.2.2 Synthesis of UV-cleavable Tetramer for 95 candidate kidney peptides.................................72 7.2.3 Expansion of A2- CD8+ T cells specific to kidney peptides..................................................73 7.2.4 Human ICCS assay coupled with Tetramer staining ................................................................74

7.3 Result .......................................................................................................................................................75 7.3.1 Selection of candidate HLA-A*02:01 restricted kidney peptides............................................75 7.3.2 Anti-A2 alloreactive CD8+ T cells showed exquisite peptide and MHC specificity...........79 7.3.3 Mapping public immunodominant A2-restricted kidney peptides .........................................80

7.4 Discussion...............................................................................................................................................87 Chapter 8 Conclusion ......................................................................................................................................90 8.1 Importance of direct alloreactive T cells............................................................................................90 8.2 Establishment of K562 cell based aAPCs..........................................................................................91 8.3 The precursor frequency of alloreactive CD8+ T cells ...................................................................91 8.4 MHC specificity of direct alloreactive CD8+ T cells.......................................................................93 8.5 Peptide specificity of direct alloreactive CD8+ T cells....................................................................94 8.6 Limitations of our study........................................................................................................................97 Chapter 9 Reference.......................................................................................................................................100 

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Cell
Keyword	Precursor Frequency Direct Allorecognition Cross-Reactivity Allopeptide CD8+ T cell
Committee Chair / Thesis Advisor	Christian Larsen, Emory University
Committee Members	Byron Au-Yeung, Emory University Haydn Kissick, Emory University Eliver Ghosn, Emory University Jyothi Rengarajan, Emory University

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