Preferential HLA-B27 Allorecognition Displayed by Multiple Cross-Reactive Antiviral CD8+ T Cell Receptors

Abstract

T cells provide essential immunosurveillance to combat and eliminate infection from pathogens, yet these cells can also induce unwanted immune responses via T cell receptor (TCR) cross-reactivity, also known as heterologous immunity. Indeed, pathogen-induced TCR cross-reactivity has shown to be a common, robust, and functionally potent mechanism that can trigger a spectrum of human immunopathologies associated with either transplant rejection, drug allergy, and autoimmunity. Here, we report that several virus-specific CD8⁺ T cells directed against peptides derived from chronic viruses (EBV, CMV, and HIV-1) presented by high frequency HLA-A and -B allomorphs differentially cross-react toward HLA-B27 allotypes in a highly focused and hierarchical manner. Given the commonality of cross-reactive T cells and their potential contribution to adverse outcomes in allogeneic transplants, our study demonstrates that multiple antiviral T cells recognizing the same HLA allomorph could pose an extra layer of complexity for organ matching.

Keywords: CMV; EBV; HIV-1; HLA; T cells; TCR; cross-reactivity.

Figure 1

Characterization of virus-specific CD8⁺ T cells. Virus-specific CD8⁺ T cell lines and clones for (A) EBV, (B) CMV, and (C) HIV-1 were examined for specificity following either re-stimulation with HLA-restricted APCs pulsed with cognate viral peptide or bulk PBMC sorting, using both anti-CD8 and specific tetramer. The functionality of virus-specific CD8⁺ tetramer⁺ T cells was assessed either using IFNγ production for T cell lines or via the CD137 activation marker for HIV-1 T cell clones. Cells were gated on FSC vs. SSC, single cells, CD8⁺, CD8⁺tetramer⁺, CD8⁺IFNγ⁺ cells. Representative plots are shown.

Figure 2

Activation of SKW3.A2_NLV TCR cells by HLA-B27–expressing APCs. SKW3.TCR activation was measured using cell surface CD69 upregulation after 16–20 h stimulation with C1R.A*02:01 ± cognate NLV peptide and a panel of C1R.B27 transfectants. CD69 MFI values were calculated after gating on FSC vs. SSC, single cells, GFP⁺ cells, live cells, CD3⁺CD8⁺ cells then CD69⁺ cells. Mean ± SEM are shown (a single experiment with triplicate data is shown from independent biological replicates performed at least twice). Statistical significance denoted by *p < 0.05 and **p < 0. 01 was determined by repeated measures non-parametric ANOVA (Kruskal–Wallis test) with post-hoc Dunn's multiple comparison test.

Figure 3

αβTCR expression of SKW3 reporter cells. Retrovirally transduced SKW3 cells expressing cross-reactive virus-specific αβTCRs for (A) EBV, (B) CMV, and (C) HIV-1 were monitored for stable cell surface TCR expression. Cells were gated on FSC vs. SSC, single cells, GFP⁺CD3⁺ cells. Representative plots are shown.

Figure 4

EBV B7_RPP allorecognition of HLA-B27 molecules. (A) Day 13 in vitro expanded B7_RPP-specific CD8⁺ T cells were stimulated with C1R.B*07:02 ± cognate RPP peptide and a panel of C1R.B27 transfectants before performing a 6 h ICS, with T cell responses measured by the production of Th1 cytokines (i.e., TNFα⁺ or IFNγ⁺ alone or dual TNFα⁺IFNγ⁺) after gating on CD8⁺tetramer⁺ T cells. (B) SKW3.TCR activation was measured using cell surface CD69 upregulation after 16–20 h stimulation with C1R.B*07:02 ± cognate RPP peptide and a panel of C1R.B27 transfectants. CD69 MFI values were calculated after gating on FSC vs. SSC, single cells, GFP⁺ cells, live cells, CD3⁺CD8⁺ cells then CD69⁺ cells. Mean ± SEM are shown (single experiments with duplicate data for ICS assay and CD69 assay are shown from independent biological replicates each performed at least twice).

Figure 5

HIV-1 B57_TW10 allorecognition of HLA-B27 molecules. TCR activation was measured using cell surface CD69 upregulation after 16–20 h stimulation with C1R.B*57:01 ± cognate TW10 peptide and a panel of C1R.B27 transfectants. CD69 MFI values were calculated after gating on FSC vs. SSC, single cells, GFP⁺ cells, live cells, CD3⁺CD8⁺ cells then CD69⁺ cells. Mean ± SEM are shown (a single experiment with duplicate data is shown from independent biological replicates performed at least twice).

Figure 6

HLA-B27 presentation of cognate viral peptide does not confer additional immunogenicity. (A) SKW.HC5 (i.e., CMV A2_NLV), (B) SKW3.LTR54.1 (i.e., EBV B7_RPP), and (C) SKW3.457 (i.e., HIV-1 B57_TW10) TCR activation was measured using cell surface CD69 upregulation after 16–20 h stimulation with HLA-restricted C1R transfectants ± cognate peptide and a panel of C1R.B27 transfectants. CD69 MFI values were calculated after gating on FSC vs. SSC, single cells, GFP⁺ cells, live cells, CD3⁺CD8⁺ cells then CD69⁺ cells. Mean ± SEM are shown (single experiment with triplicate data). Statistical significance using unpaired Student's t-test is denoted by ****p < 0.0001.

Figure 7

HLA-B27 polymorphisms and amino acid chemical properties. B27:01-B*27:04, B*27:06-B*27:10 amino acid polymorphisms were compared to the consensus sequence for B*27:05. The chemical properties of substituted residues and their influence on HLA-I peptide-binding pockets are detailed.