Since multiple different T-cell receptor (TCR) sequences can bind to the same peptide-MHC combination and the number of TCR-sequences that can theoretically be generated even exceeds the number of T cells in a human body, the likelihood that many public identical (PUB-I) TCR-sequences frequently contribute to immune responses has been estimated to be low. Here, we quantitatively analyzed the TCR-repertoires of 190 purified virus-specific memory T-cell populations, directed against 21 epitopes of Cytomegalovirus, Epstein-Barr virus and Adenovirus isolated from 29 healthy individuals, and determined the magnitude, defined as prevalence within the population and frequencies within individuals, of PUB-I TCR and of TCR-sequences that are highly-similar (PUB-HS) to these PUB-I TCR-sequences. We found that almost one third of all TCR nucleotide-sequences represented PUB-I TCR amino-acid (AA) sequences and found an additional 12% of PUB-HS TCRs differing by maximally 3 AAs. We illustrate that these PUB-I and PUB-HS TCRs were structurally related and contained shared core-sequences in their TCR-sequences. We found a prevalence of PUB-I and PUB-HS TCRs of up to 50% among individuals and showed frequencies of virus-specific PUB-I and PUB-HS TCRs making up more than 10% of each virus-specific T-cell population. These findings were confirmed by using an independent TCR-database of virus-specific TCRs. We therefore conclude that the magnitude of the contribution of PUB-I and PUB-HS TCRs to these virus-specific T-cell responses is high. Because the T cells from these virus-specific memory TCR-repertoires were the result of successful control of the virus in these healthy individuals, these PUB-HS TCRs and PUB-I TCRs may be attractive candidates for immunotherapy in immunocompromised patients that lack virus-specific T cells to control viral reactivation.
T-cell products derived from third-party donors are clinically applied, but harbor the risk of off-target toxicity via induction of allo-HLA cross-reactivity directed against mismatched alleles. We used third-party donor-derived virus-specific T cells as model to investigate whether virus-specificity, HLA restriction and/or HLA background can predict the risk of allo-HLA cross-reactivity. Virus-specific CD8pos T cells were isolated from HLA-A*01:01/B*08:01 or HLA-A*02:01/B*07:02 positive donors. Allo-HLA cross-reactivity was tested using an EBV-LCL panel covering 116 allogeneic HLA molecules and confirmed using K562 cells retrovirally transduced with single HLA-class-I alleles of interest. HLA-B*08:01-restricted T cells showed the highest frequency and diversity of allo-HLA cross-reactivity, regardless of virus-specificity, which was skewed toward multiple recurrent allogeneic HLA-B molecules. Thymic selection for other HLA-B alleles significantly influenced the level of allo-HLA cross-reactivity mediated by HLA-B*08:01-restricted T cells. These results suggest that the degree and specificity of allo-HLA cross-reactivity by T cells follow rules. The risk of off-target toxicity after infusion of incompletely matched third-party donor-derived virus-specific T cells may be reduced by selection of T cells with a specific HLA restriction and background.
Reactivations of cytomegalovirus (CMV), Epstein Barr virus (EBV) and adenovirus (AdV) occur frequently in immune compromised patients after allogeneic stem cell transplantation (alloSCT) and cause high morbidity and mortality. T-cell immunity is essential for anti-viral protection, but a fully competent T-cell repertoire generally does not develop until 3-6 months after transplantation. Especially patients transplanted with a graft from a virus non-experienced donor are at risk. Adoptive transfer of partially HLA-matched virus-specific T cells from healthy third party donors is a potential strategy to temporarily provide anti-viral immunity to these patients. However, such T cells harbor a risk of mediating off-target toxicity due to allo-HLA cross-reactivity. It is not currently known whether the degree of allo-HLA cross-reactivity is random or whether rules exist that might allow prediction of specific T-cell populations. Here, we investigated whether virus specificity, HLA type of the donor or HLA restriction of the virus-specific T cells influence the risk of allo-HLA cross-reactivity. Through cell sorting using tetramers for various peptides from CMV, EBV and AdV, 164 CD8 T-cell populations (21 specificities) were isolated from peripheral blood of 24 healthy donors, homozygous for HLA-A*01:01/B*08:01 and HLA-A*02:01/B*07:02. Allo-HLA cross-reactivity was tested using an allogeneic EBV-LCL panel covering 116 different HLA molecules and confirmed using K562 cells retrovirally transduced with single HLA alleles of interest. Forty percent of all virus-specific T-cell populations exerted allo-HLA cross-reactivity. Similar frequencies were found for the various viral specificities showing 33% of the CMV, 43% of the EBV and 38% of the AdV-specific T-cell populations to be allo-HLA cross-reactive. Surprisingly, a much larger fraction of the HLA-B*08:01-restricted virus-specific T-cell populations exhibited allo-HLA cross-reactivity (77%) than from those restricted by the other HLAs (32% of HLA-A*01:01, 38% of HLA-A*02:01 and 26% of HLA-B*07:02-restricted virus-specific T-cell populations). HLA-B*08:01-restricted virus-specific T cells also exhibited the broadest allo-HLA reactivity, reacting to a median of 5 different allogeneic EBV-LCLs (range 1-17). In contrast, HLA-A*01:01, HLA-A*02:01 and HLA-B*07:02-restricted virus-specific T cells reacted to a median of 1, 2 and 3 (range 1-7) different allogeneic EBV-LCLs, respectively. Dissection of the diversity/specificity of the allo-HLA reactivities using a panel of 40 different single HLA-A, B, or C-transduced K562 cells further illustrated recurrent recognition of a restricted group of allogeneic HLA-B molecules by HLA-B*08:01-restricted T-cell populations, mediated by single T-cell clones. Heterozygosity for recurrently recognized allo-HLA-B molecules led to a significant decrease in the broadness of allo-HLA cross-reactivity by HLA-B*08:01-restricted T-cell populations, presumably due to negative thymic selection. In contrast, heterozygosity HLA-B molecules that were not part of the restricted group of cross-recognized alleles did not significantly decrease allo-HLA cross-reactivity. These data show that allo-HLA cross-reactivity by virus-specific T cells is highly influenced by their HLA restriction and the HLA background of the donors, but not by their virus specificity. Of the HLA-A*01, A*02, B*07 and B*08-restricted virus-specific T-cell populations isolated from homozygous donors, HLA-B*08:01-restricted virus-specific T cells showed the highest frequency and diversity of allo-HLA cross-reactivity with recurrent recognition of groups of specific mismatched allogeneic HLA-B alleles. Our results indicate that selection of virus-specific T cells with specific HLA restrictions and HLA backgrounds may decrease the risk of off-target toxicity after infusion of third-party virus-specific T cells to patients with uncontrolled viral reactivation after alloSCT. Disclosures No relevant conflicts of interest to declare.
Reactivations of cytomegalovirus (CMV), Epstein Bar virus (EBV) and adenovirus (AdV) are frequently seen in immune compromised patients after allogeneic stem cell transplantation (alloSCT), and are associated with high morbidity and mortality. T cell immunity is essential for anti-viral protection, but a fully competent T cell repertoire generally does not develop until 3-6 months after transplantation. Especially patients transplanted with a virus non- experienced donor are at risk of developing severe complications. Adoptive transfer of partially HLA-matched virus specific T cells from healthy third party donors is a potential strategy to temporarily provide anti-viral immunity to these patients. However, these partially HLA-matched T cells harbor a risk of mediating allo-HLA cross-reactivity. Here, we investigated whether virus specificity and HLA restriction of the virus specific T cells influence the risk of allo-HLA cross-reactivity, and thus the development of GVHD. To determine the occurrence and diversity of allo-HLA cross-reactivity, virus specific CD8 T cells from homozygous HLA-A*01:01/B*08:01 and HLA-A*02:01/B*07:02 donors were isolated by cell sorting using tetramers for various peptides from CMV, EBV and AdV. Allo-HLA cross-reactivity was tested using an allogeneic EBV-LCL panel covering 116 different HLA molecules and confirmed using K562 cells retrovirally transduced with single HLA alleles of interest. A significant proportion of the virus specific T cell populations (n=174; 20 specificities) isolated from 27 healthy donors exerted allo-HLA cross-reactivity, as measured by recognition of 1 or more HLA mismatched EBV-LCLs from the panel. Similar frequencies were found for the various viral specificities showing 30% of the CMV, 46% of the EBV and 36% of the AdV-specific T cell populations to be allo-HLA cross-reactive. However, for some specificities (e.g. HLA-A*0201-restricted EBV-LMP2-FLY) allo-HLA cross-reactivity was infrequent (n=1/11), whereas for other specificities (e.g. HLA-B*08:01-restricted EBV-BZLF1-RAK) the majority of the T cell populations (n=9/13) was allo-HLA reactive. Surprisingly, a much larger fraction of HLA-B*08:01 restricted virus specific T cell populations showed allo-HLA cross-reactivity (72%, 36 out of 50 T cell lines), compared to the other HLA restricted virus specific T cell populations (29% of HLA-A*01:01, 30% of HLA-A*02:01 and 26% of HLA-B*07:02 restricted virus specific T cell lines). HLA-B*08:01 restricted virus specific T cells also exhibited the broadest allo-HLA reactivity, reacting to a median of 5 allo EBV-LCLs (range 1-17). In contrast, HLA-A*01:01, HLA-A*02:01 and HLA-B*07:02 restricted virus specific T cells reacted to a median of 1, 2 and 3 (ranges 1-7) allo EBV-LCLs, respectively. Dissection of the diversity/specificity of the allo-HLA reactivity using the panel of 40 different single HLA transduced K562 cells further illustrated the extensive allo-HLA cross-reactivity for HLA-B*08:01 restricted T cells isolated from homozygous HLA-A*01/B*08 donors compared to virus specific T cells restricted by other HLA alleles. These data show that allo-HLA cross-reactivity by virus specific T cells is highly influenced by the HLA restriction and not by the viral specificity of the T cell populations. Of the HLA-A*01, A*02, B*07 and B*08-restricted virus specific T cell populations isolated from homozygous donors, HLA-B*08:01 restricted virus specific T cells showed the highest frequency and diversity of allo-HLA cross-reactivity. Our results indicate that selection of virus specific T cells with specific HLA restrictions may decrease the risk of developing GVHD after infusion of third-party virus specific T cells to patients with uncontrolled viral reactivation after alloSCT. Disclosures No relevant conflicts of interest to declare.
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