The combination of common viral hits and heterologous immunity strongly influences clinical outcomes in ‘at risk’ lungs

The intimate interaction of a T cell receptor (TCR) with a specific peptide/human leukocyte antigen (HLA) complex is a cornerstone of cell-mediated immunity, although some T cells can exhibit promiscuity by recognising more than one peptide/HLA complex. This mechanism of dual recognition frames our investigations of the role of cross-reactive T cells in transplantation. Initial investigations focused on the identification of T cell cross-reactivity across three common heterologous viruses (Epstein-Barr virus [EBV], Cytomegalovirus [CMV] and Influenza A virus [IAV]), which have been reported to contribute to significant episodes of allograft dysfunction in transplant recipients, particularly in the respiratory setting. Immunodominant EBV epitopes restricted to high frequency HLA allotypes (HLA-A*02:01, B*07:02 and B*08:01) were screened for T cell cross-reactivity towards immunodominant epitopes derived from CMV or IAV, restricted to the same HLA allotype. Despite extensive investigations, EBV-specific CD8+ T cells failed to demonstrate cross-reactivity towards any of the CMV or IAV epitopes examined. Furthermore, analyses of the viral peptide/HLA structures revealed distinct molecular structures consistent with the lack of T cell cross-reactivity. Thus, heterologous viral T cell cross-reactivity was found not to be as common as predicted despite the ubiquitous nature of these viruses and the immunodominance of the selected epitopes. Following solid organ transplantation, virus-specific memory T cells have been shown to cross-recognise allogenic HLA molecules expressed on the donor allograft. Our laboratory identified HLA-A*02:01-restricted CMVNLV-specific CD8+ T cells that cross-recognise a subset of HLA-B27 allomorphs in a hierarchical manner, HLA-B*27:07>B*27:09>B*27:05>B*27:03/B*27:10. In two unrelated individuals (healthy donor and lung transplant recipient [LTR5]), the cross-reactive TCR signature expressed by the CMVNLV-specific CD8+ T cells was almost identical (coined OTN5 TCR). Strikingly, in the absence of post-transplantation CMV reactivation the cross-reactive CMVNLV-specific CD8+ T cells were found to be stable, however upon reactivation these cells significantly increased and demonstrated clonality at the height of the infection. Fortunately, no allograft damage was reported in this case study as LTR5 received a non-cross-reactive allotype (HLA-B*27:04). In addition, we explored an alternate model of allo-HLA cross-reactivity first identified by collaborators at Leiden University Medical Centre, with HLA-B*07:02-restricted EBV¬RPP-specific CD8+ T cells demonstrating cross-reactivity towards HLA-B*40:02. We also observed allo-HLA cross-reactivity in five individuals (two healthy donors and three LTR) and have identified two different TCRs expressed by two unrelated individuals both capable of the same allo-HLA cross-recognition. Finally, studies that have been able to identify peptide(s) presented by allo-HLA capable of facilitating ligation to the cross-reactive TCR have been limited. We undertook an immunopeptidomics approach that used mass spectrometry (MS) to identify HLA-B27-restricted naturally presented peptides that exhibit cross-reactive potential in the HLA-A*02:01/CMVNLV → HLA-B27 model. Whilst preliminary investigations have yet to confirm a specific allopeptide identity, we demonstrated that a subset of peptides eluted from both cross-reactive (B*27:07 and B*27:09) and non-cross-reactive (B*27:01 and B*27:02) allomorphs stimulated the cross-reactive OTN5 TCR, but only when presented by a cross-reactive allomorph (HLA-B*27:09). This has important ramifications for models of alloreactivity and suggests that common peptides can be presented in a differential immunogenic manner by closely related allo-HLA. Overall, this thesis combined cellular, proteomic and biophysical technologies to investigate the functional and structural elements of cross-reactive virus-specific CD8+ T cells and their clinical importance in the setting of lung transplantation