Virus-specific T cells have been shown to cross-react with allogeneic HLA (allo-HLA) at a clonal level. However, the impact of a single virus on the allorepertoire has never been investigated at... Show moreVirus-specific T cells have been shown to cross-react with allogeneic HLA (allo-HLA) at a clonal level. However, the impact of a single virus on the allorepertoire has never been investigated at the polyclonal level.We made an inventory of the incidence and specificity of allo-HLA-cross-reactive-virus-specific CD8(+) T cells in 24 healthy individuals. T cells were stained for 25 virus-specific tetramers, and mixed-lymphocyte reactions were performed against a panel of HLA-typed allostimulators. Allospecificity was confirmed by IFN gamma-ELISA using T-cell clones against a panel of HLA-typed cell-lines.The polyclonal immune repertoire directed against CMV alone was associated with a memory response against six allo-HLA molecules. Besides, a single allostimulator activated memory T-cell responses with multiple viral specificities.Concluding, a single virus can substantially broaden the allo-HLA memory T-cell repertoire. This study only looked at CMV- and EBV-specific T cells, whereas the immune repertoire consists of T cells directed against many different viruses. Hence, transplant patients receiving an HLA-mismatched graft may already express a poly clonal repertoire of anti-donor-memory T cells before transplantation. Show less
Zwan, A. van der; Meer-Prins, E.M.W. van der; Miert, P.P.M.C. van; Heuvel, H. van den; Anholts, J.D.H.; Roelen, D.L.; ... ; Heidt, S. 2018
Virus-specific T cells can recognize allogeneic HLA (allo-HLA) through TCR cross-reactivity. The allospecificity often differs by individual (private cross-reactivity) but also can be shared by... Show moreVirus-specific T cells can recognize allogeneic HLA (allo-HLA) through TCR cross-reactivity. The allospecificity often differs by individual (private cross-reactivity) but also can be shared by multiple individuals (public cross-reactivity); however, only a few examples of the latter have been described. Because these could facilitate alloreactivity prediction in transplantation, we aimed to identify novel public cross-reactivities of human virus-specific CD8(+) T cells directed against allo-HLA by assessing their reactivity in mixed-lymphocyte reactions. Further characterization was done by studying TCR usage with primer-based DNA sequencing, cytokine production with ELISAs, and cytotoxicity with 51 chromium-release assays. We identified three novel public allo-HLA cross-reactivities of human virus-specific CD8(+) T cells. CMV B35/IPS CD8(+) T cells cross-reacted with HLA-B51 and/or HLA-B58/B57 (23% of tetramer-positive individuals), FLU A2/GIL (influenza IMP[58-66] HLA-A* 02: 01/GILGFVFTL) CD8(+) T cells with HLA-B38 (90% of tetramer-positive individuals), and VZV A2/ALW (varicella zoster virus IE62[593-601] HLA-A*02:01/ALWALPHAA) CD8(+) T cells with HLA-B55 (two unrelated individuals). Cross-reactivity was tested against different cell types including endothelial and epithelial cells. All cross-reactive T cells expressed a memory phenotype, emphasizing the importance for transplantation. We conclude that public allo-HLA cross-reactivity of virus-specific memory T cells is not uncommon and may create novel opportunities for alloreactivity prediction and risk estimation in transplantation. Show less
Heuvel, H. van den; Meer-Prins, E.M.W. van der; Heutinck, K.M.; Berge, I.J.M. ten; Claas, F.H.J. 2014
Viral infection is a major cause of morbidity and mortality, and there are few therapeutic options available to augment a virus-specific T cell response. Although allo-HLA cross-reactivity from... Show moreViral infection is a major cause of morbidity and mortality, and there are few therapeutic options available to augment a virus-specific T cell response. Although allo-HLA cross-reactivity from virus-specific memory T cells is common, it is unclear whether priming with specific allogeneic cells could conversely elicit a viral peptide/self-HLA restricted cytotoxic T cell response in humans. First, we used the previously described allo-HLA-B*44:02 cross-reactivity of EBV peptide/HLA-B8 restricted T cells, to determine whether allogeneic HLA stimulation can elicit a cytolytic immune response against EBV. HLA-B8(+) HLA-B44(-) EBV-seropositive PBMCs were stimulated with either HLA-B*44:02(+) or HLA-B*44:03(+) mismatched irradiated PBMCs in a 7-10 d MLR. The allo-HLA stimulated responder cells were then evaluated for cytotoxicity using EBV peptide loaded autologous target cells and unloaded HLA-B8(+) EBV LCL target cells. PBMCs from EBV-seropositive donors gained EBV-specific cytolytic effector function following specific allo-HLA stimulation. Finally, we also elicited cytolytic CMV-specific responses using specific allogeneic cell stimulation, to confirm that this technique can be used to elicit viral peptide/self-HLA restricted responses even from nonpublic TCR responses. Allogeneic cell stimulation used as a cell therapy may be a potential tool to augment an antiviral T cell response in patients with EBV or CMV infection. Show less
D'Orsogna, L.J.; Heuvel, H. van den; Meer-Prins, E.M.W. van der; Roelen, D.L.; Doxiadis, I.I.N.; Claas, F.H.J. 2012
BACKGROUND.: The crossreactivity of Epstein-Barr virus (EBV Epstein-Barr virus nuclear antigen 3A [EBNA3A])-specific CD8 T cells against allogeneic human leukocyte antigen (HLA)-B*44:02 has been... Show moreBACKGROUND.: The crossreactivity of Epstein-Barr virus (EBV Epstein-Barr virus nuclear antigen 3A [EBNA3A])-specific CD8 T cells against allogeneic human leukocyte antigen (HLA)-B*44:02 has been shown to be dependent on presentation of self-peptide EEYLQAFTY by the target antigen. In this study, we report that allogeneic HLA-B*44:02 proximal tubular epithelial cells (PTECs) and human umbilical vein endothelial cells (HUVECs) are poor targets for EBV EBNA3A-specific T cells. METHODS.: The EEY peptide was exogenously loaded onto HLA-B*44:02 and HLA-B*44:03-expressing PTECs and HUVECs. EEY-peptide-loaded, and unloaded, PTECs and HUVECs were then incubated with serial dilutions of our EBNA3A T-cell clone, in a cytotoxicity assay. RESULTS.: Although HLA-B*44:02-expressing PTECs were specifically lysed in proportion to the effector/target ratio by the EBNA3A T-cell clone, without peptide loading, lysis was greatly increased by exogenous EEY peptide loading (15% vs. 75%; P<0.0001). HLA-B*44:02-expressing HUVECs were only lysed when loaded with exogenous EEY peptide (0% vs. 64%; P<0.0001). Lack of HLA expression and lack of ABCD3 gene expression were excluded as a cause for these results. PTECs and HUVECs were specifically targeted by another alloreactive T-cell clone without exogenous peptide loading, suggesting that the lack of recognition of HLA-B*44:02 epithelial and endothelial cells by the EBV EBNA3A T-cell clone was due to lack of EEYLQAFTY peptide presentation. CONCLUSIONS.: Tissue-specific (peptide dependent) alloreactivity may have important implications for transplantation monitoring and rejection. Show less
D'Orsogna, L.J.A.; Roelen, D.L.; Meer-Prins, E.M.W. van der; Pol, P. van der; Franke-van Dijk, M.E.; Eikmans, M.; ... ; Claas, F.H.J. 2011
BACKGROUND.: Allo-human leukocyte antigen (HLA) reactivity by naturally acquired viral-specific memory T cells is common. However, the effect of successful vaccination on the alloreactive memory T... Show moreBACKGROUND.: Allo-human leukocyte antigen (HLA) reactivity by naturally acquired viral-specific memory T cells is common. However, the effect of successful vaccination on the alloreactive memory T-cell repertoire is unclear. We hypothesized that vaccination could specifically induce allo-HLA-reactive memory T cells. METHODS.: A varicella-zoster virus (VZV) immediate early 62 (IE62)-specific CD8 memory T-cell clone was single cell sorted from a VZV seronegative renal transplant candidate after response to live attenuated varicella vaccination. To analyze the allo-HLA reactivity, the VZV IE62-specific T-cell clone was tested against HLA-typed target cells and target cells transfected with HLA molecules, in both cytokine production and cytotoxicity assays. RESULTS.: The varicella vaccine-induced VZV IE62-specific T-cell clone specifically produced interferon-γ when stimulated with HLA-B*55:01-expressing Epstein-Barr virus-transformed B cells and HLA-B*55:01-transfected K562 cells (single HLA antigen expressing cell line [SALs]) only. The clone also demonstrated specific cytolytic effector function against HLA-B*55:01 SALs and phytohemagglutinin blasts. Cytotoxicity assays using proximal tubular epithelial cell and human umbilical vein endothelial cell targets confirmed the kidney tissue specificity of the allo-HLA-B*55:01 reactivity, and the relevance of the cross-reactivity to clinical kidney transplantation. The results also suggest that molecular mimicry, and not bystander proliferation, is the mechanism underlying vaccine-induced alloreactivity. CONCLUSIONS.: Varicella vaccination generated a de novo alloreactive kidney cell-specific cytolytic effector memory T cell in a patient awaiting renal transplantation. Vaccination-induced alloreactivity may have important clinical implications, especially for vaccine timing and recipient monitoring. Show less
