Abstract
Peptide–MHC (pMHC) multimers have become the “gold standard” for the detection and isolation of antigen-specific T-cells but recent evidence shows that normal use of these reagents can miss fully functional T-cells that bear T-cell receptors (TCRs) with low affinity for cognate antigen. This issue is particularly pronounced for anticancer and autoimmune T-cells as self-reactive T-cell populations are enriched for low-affinity TCRs due to the removal of cells with higher affinity receptors by immune tolerance mechanisms. Here, we stained a wide variety of self-reactive human T-cells using regular pMHC staining and an optimized technique that included: (i) protein kinase inhibitor (PKI), to prevent TCR triggering and internalization, and (ii) anti-fluorochrome antibody, to reduce reagent dissociation during washing steps. Lymphocytes derived from the peripheral blood of type 1 diabetes patients were stained with pMHC multimers made with epitopes from preproinsulin (PPI), insulin-β chain, glutamic acid decarboxylase 65 (GAD65), or glucose-6-phospate catalytic subunit-related protein (IGRP) presented by disease-risk allelles HLA A*02:01 or HLA*24:02. Samples from ankylosing spondylitis patients were stained with a multimerized epitope from vasoactive intestinal polypeptide receptor 1 (VIPR1) presented by HLA B*27:05. Optimized procedures stained an average of 40.5-fold (p = 0.01, range between 1.4 and 198) more cells than could be detected without the inclusion of PKI and cross-linking anti-fluorochrome antibody. Higher order pMHC dextramers recovered more cells than pMHC tetramers in parallel assays, and standard staining protocols with pMHC tetramers routinely recovered less cells than functional assays. HLA A*02:01-restricted PPI-specific and HLA B*27:05-restricted VIPR1-specific T-cell clones generated using the optimized procedure could not be stained by standard pMHC tetramer staining. However, these clones responded well to exogenously supplied peptide and endogenously processed and presented epitopes. We also showed that anti-fluorochrome antibody-conjugated magnetic beads enhanced staining of self-reactive T-cells that could not be stained using standard protocols, thus enabling rapid ex vivo isolation of autoimmune T-cells. We, therefore, conclude that regular pMHC tetramer staining is generally unsuitable for recovering self-reactive T-cells from clinical samples and recommend the use of the optimized protocols described herein.
Highlights
Conventional T-cells orchestrate the immune response to pathogens by recognizing foreign protein antigens in the form of peptides presented at the cell surface bound to MHC molecules
The best anti-pathogen T-cell receptor (TCR) tend to bind with TCR affinities with dissociation constants (KDs) in the range of 0.1–10 μM by surface plasmon resonance (SPR) whereas self-reactive TCRs isolated from anticancer and autoimmune T-cells bind with much weaker affinities (KD 20–500 μM) [9,10,11]
Purified CD8 T-cells from an HLA A*02:01+ type I diabetes patient were stimulated with peptides from CMV, glutamic acid decarboxylase 65 (GAD65), Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), Islet Antigen-2 (IA-2), and the insulin-β chain (Table 2) to create T-cell lines for testing with standard and optimized tetramer staining protocols (Figure 2A)
Summary
Conventional T-cells orchestrate the immune response to pathogens by recognizing foreign protein antigens in the form of peptides presented at the cell surface bound to MHC molecules. TCR–pMHC binding parameters are selected in the thymus via recruitment of the signal-initiating kinase Lck, which is sequestered by the intracellular tails of CD4 and CD8 [4] These coreceptors bind to sites on MHC class I and class II, respectively, that are distinct from the TCR-docking platform thereby enabling formation of TCR–pMHCI-CD8 or TCR– pMHCII-CD4 quadripartite complexes [3, 4]. Cells bearing TCRs that bind strongly to self pMHC, and thereby have potential to react to self, are eliminated from the pool of developing T-cells [8] This central tolerance mechanism ensures that T-cells with TCRs that interact strongly with self-peptides do not enter the peripheral tissues and, in conjunction with peripheral tolerance mechanisms, explains why self-reactive TCRs bind with lower affinity, and with shorter dwell times than TCRs specific for foreign, pathogen-derived peptides [9, 10]. Anti-pathogen TCRs sit at the stronger end of this affinity window, followed by anticancer TCRs [1, 9, 10], whereas autoimmune TCRs reside at the other end of the spectrum and can have TCRs that bind very weakly (KD > 150 μM) [(10, 15) and unpublished data] (Figure 1)
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