After informed consent according to the Declaration of Helsinki, healthy donors (homozygously) expressing HLA-A^*01:01 and HLA-B^*08:01 or HLA-A^*02:01 and HLA-B^*07:02 were selected from the Sanquin database and the biobank of the department of Hematology, Leiden University Medical Center (LUMC). Two donors expressing HLA-A^*02:01/HLA-B^*07:02 were not homozygous. Peripheral blood mononuclear cells (PBMCs) were isolated by standard Ficoll-Isopaque separation and used directly or thawed after cryopreservation in the vapor phase of liquid nitrogen. Donor characteristics (HLA typing, CMV and EBV serostatus) are provided in Table 1 (Donors 1–24). Healthy donors expressing HLA-B^*08:01 and HLA-B^*13:02 or HLA-B^*35:01 (Table 1; donors 25–30) were selected from the biobank of the department of Hematology (LUMC).

Phycoerythrin (PE), allophycocyanin (APC), BV421, BV510 and/or peridinin-chlorophyll-protein (PerCP)-labeled pMHC-tetramer complexes were used for fluorescence-activated cell sorting (FACSorting). The pMHC-tetramers used (for generation see Supplementary Material and Methods) are shown in Supplementary Table 1. PeptideMHC-tetramer positive, CD8^pos/CD4^neg T cells were sorted and seeded at 10,000 cells per well in U-bottom microtiter plates for the generation of bulk T-cell populations. After 2 weeks of culture, pMHC-tetramer^pos T-cell populations were considered pure if they contained ≥97% pMHC-tetramer^pos cells. Polyclonality of the sorted virus-specific T cells was assessed by T-cell receptor-variable β (TCR-Vβ) family analysis using the TCR-Vβ kit (Beckman Coulter, Fullerton, USA). Sub-populations expressing a single TCR-Vβ family were sorted from the bulk using monoclonal antibodies from the TCR-Vβ kit. Sub-populations were then non-specifically expanded. Sub-populations using one specific TCR-Vβ family were considered pure if ≥95% of the population was positive for that TCR-Vβ family. Sorting was performed on a FACS ARIA (BD) and analyzed using Diva software (BD). All analyses were performed on a FACS Calibur (BD), and analyzed using Flowjo Software (TreeStar, Ashland, USA). Procedures to isolate and expand virus-specific T cells are described in the Supplementary Material and Methods.

EBV-transformed lymphoblastoid cell-lines (EBV-LCLs) were generated according to standard protocols (25). EBV-LCLs were selected to cover a total of 116 frequently occurring HLA molecules, as listed in Table 2. HLA-deficient K562 cells were transduced with 40 different single HLA-class-I molecules (Supplementary Table 2) including common and rare HLA-class-I molecules. EBV-LCLs and HLA-deficient K562 cell-lines were cultured in stimulator medium consisting of Iscove's Modified Dulbecco's Medium (IMDM; Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS; Invitrogen, Carlsbad, USA), 100 U/mL penicillin (Lonza), 100 μg/mL streptavidin (Lonza) and 2.7 mM L-glutamine (Lonza). Generation of various cell-lines is described in the Supplementary Material and Methods.

Interferon-γ (IFN-γ) production by virus-specific T cells was quantified using standard enzyme-linked immunosorbent assays (ELISA) according to the manufacturer's instructions (Sanquin Reagents, The Netherlands). Responder T cells were co-cultured with stimulator cells at a ratio of 1:10 (responder: stimulator) for 16 h at 37°C in T-cell medium used for expansion of T-cell populations as described in Supplementary Material using 25 IU/ml Interleukin-2 (IL-2) instead of 100 IU/ml IL-2. Recognition of HLA-mismatched EBV-LCLs, HLA-matched peptide-pulsed EBV-LCLs and K562 cells transduced with specific HLA molecules was defined as production of ≥200 pg/ml of IFN-γ.

Statistical analyses were only performed on quantitative data and were performed using non-parametric tests. The Fisher's-Exact-test was used to assess the differences in cross-reactivity (present or absent) of HLA-mismatched EBV-LCLs between groups (i.e., HLA-A^*01:01- and HLA-B^*08:01-restricted virus-specific T cells). Differences in the numbers of recognized HLA-mismatched EBV-LCLs was first assessed by the Kruskal-Wallis test. Differences between two groups were then further assessed with the non-parametric Mann-Whitney U test. p-values were adjusted by the Bonferroni correction for multiple testing. Statistical analyses were conducted using GraphPad Prism (GraphPad Software, version 8).

To study the influence of HLA restriction and antigen specificity on the level of allo-HLA cross-reactivity mediated by virus-specific T cells, bulk virus-specific T-cell populations targeting single epitopes were isolated from total PBMCs of(homozygous) HLA-A^*01:01/HLA-B^*08:01^pos or HLA-A^*02:01/HLA-B^*07:02^pos healthy donors. Two donors did not homozygously express HLA-A^*02:01/HLA-B^*07:02. All donors were EBV seropositive and 5 out of 12 HLA-A^*01:01/HLA-B^*08:01^pos donors and 9 out of 12 HLA-A^*02:01/HLA-B^*07:02^pos donors were CMV seropositive (Table 1). The serostatus for AdV was unknown for all donors. Virus-specific T cells were isolated by FACS using pMHC-tetramers for various peptides (n = 21) from CMV, EBV, and AdV (Supplementary Table 1). In total, 45 CMV, 95 EBV and 24 AdV-specific T-cell populations were isolated (Table 3). CMV and EBV-specific T-cell populations could be isolated from all CMV^pos and EBV^pos donors, respectively. Although no AdV serostatus was known, AdV-specific T-cell populations could be isolated from 18 out of 24 donors. We isolated 22 different HLA-A^*01:01-restricted virus-specific T-cell populations, 69 HLA-A^*02:01-restricted virus-specific T-cell populations, 34 HLA-B^*07:02-restricted virus-specific T-cell populations and 39 HLA-B^*08:01-restricted virus-specific T-cell populations (Table 3). These T-cell populations were analyzed for allo-HLA cross-reactivity using a panel of HLA-mismatched EBV-LCLs, expressing the most frequent (>2%) HLA-class-I and class-II antigens in the Caucasian population (Table 2). EBV-specific T-cell populations were only tested against HLA-mismatched EBV-LCLs that did not express the specific restriction molecules to avoid recognition of EBV-derived peptides in self-HLA. In total, 65 out of 164 (39%) virus-specific T-cell populations produced interferon-γ in response to stimulation with at least one HLA-mismatched EBV-LCL (Supplementary Figure 1A).

Next, we investigated whether the T-cell populations that did not recognize any HLA-mismatched EBV-LCL contained smaller sub-population(s) of T cells that could recognize HLA-mismatched EBV-LCLs, but were missed in the initial bulk analysis. Sub-populations were sorted based on expression of single TCR-Vβ families. Twenty-four different TCR-Vβ families can be identified with the provided monoclonal antibodies in the kit that was used for flow cytometry, covering around 70% of the human TCR-Vβ repertoire (26). Sub-populations that could not be stained with the antibody kit could not be separated from the bulk populations using this strategy and were not analyzed for recognition of HLA-mismatched EBV-LCLs. In total, 165 sub-populations expressing a single TCR-Vβ family were isolated from the 99 bulk T-cell populations that initially did not show reactivity against the EBV-LCL panel. These sub-populations were subsequently analyzed for their capacity to recognize HLA-mismatched EBV-LCLs. We observed that 31 of these isolated sub-populations contained T cells that were capable of exerting allo-HLA cross-reactivity (Supplementary Figure 1B). Additionally, 193 sub-populations were sorted from bulk T-cell populations that did already demonstrate HLA-mismatched EBV-LCL recognition in the initial analysis (derived from 65 initial bulk populations). Eighty-six of these isolated sub-populations contained T cells that recognized HLA-mismatched EBV-LCLs. Recognition of additional HLA-mismatched EBV-LCLs could be observed that were not detected in the initial analysis of 25 different bulk T-cell populations (Supplementary Figure 2). In summary, a total of 83 bulk T-cell populations contained T cells that showed detectable cross-reactivity against one or more HLA-mismatched EBV-LCL(s).

These 83 T-cell populations were used to investigate whether the virus specificity (CMV, EBV or AdV) or HLA restriction (HLA-A^*01:01, HLA-A^*02:01, HLA-B^*07:02 or HLA-B^*08:01) of the virus-specific reactivity influences the occurrence and frequency of HLA-mismatched EBV-LCL recognition. A similar proportion of the virus-specific T-cell populations targeting CMV, EBV or AdV exerted reactivity against at least one HLA-mismatched EBV-LCL (Figure 1A). In contrast, a significantly larger fraction of the HLA-B^*08:01-restricted virus-specific T-cell populations showed recognition of HLA-mismatched EBV-LCLs, as compared to the HLA-A^*01:01, HLA-A^*02:01, and HLA-B^*07:02-restricted virus-specific T-cell populations (Figure 1B). To assess the broadness of the cross-reactivity patterns, we counted how many different HLA-mismatched EBV-LCLs were recognized by the individual T-cell populations (65 bulk T-cell populations including 86 additional sub-populations derived from these T-cell populations, and 31 sub-populations derived from the 18 bulk T-cell populations that did not recognize any HLA-mismatched EBV-LCLs in the initial analysis). In these analyses, HLA-B^*08:01-restricted virus-specific T cells exhibited a significantly broader cross-reactivity pattern, illustrated by reactivity against a median of 6 different HLA-mismatched EBV-LCLs, whereas HLA-A^*01:01, HLA-A^*02:01 and HLA-B^*07:02-restricted virus-specific T-cell populations showed reactivity against a median of only 2, 2 and 3 HLA-mismatched EBV-LCLs, respectively (Figure 1C). Within the different HLA-B^*08:01-restricted T-cell populations, similar high frequencies of T cells capable of exerting cross-reactivity against HLA-mismatched EBV-LCLs were observed, regardless of viral antigen-specificity (Figure 1D). These results show that the occurrence and frequency of cross-reactivity against HLA-mismatched EBV-LCLs is highly affected by HLA-restriction and not by virus-specificity (CMV, EBV or AdV).

To investigate if the recognition of HLA-mismatched EBV-LCLs was indeed caused by recognition of allogeneic HLA molecules, HLA-deficient EBV^neg K562 cell-lines transduced with single HLA-class-I molecules were used as stimulator cells (12, 27). T-cell populations exhibiting a clear pattern of EBV-LCL recognition, corresponding with the expression of a single HLA allele, were tested against K562 cells transduced with the respective HLA-molecule. For example, a population of EBV-EBNA3A^QAK-specific T cells recognized HLA-mismatched EBV-LCL ABF, which uniquely expressed HLA-A^*30:04 and HLA-B^*55:01 (Figure 2A). Recognition of K562 cells that were transduced with HLA-B^*55:01 confirmed part of this respective cross-reactivity pattern (Figure 2B). In another example, a population of EBV-BRLF1^YVL-specific T cells recognized HLA-mismatched EBV-LCLs ACD, WKD, AVZ and UBM that all expressed HLA-A^*24:02 (Figure 2A) and this was confirmed by recognition of K562 cells transduced with HLA-A^*24:02 (Figure 2B). Some virus-specific T-cell populations (especially HLA-B^*08:01-restricted T cells) showed more complex reactivity patterns when tested against the EBV-LCL panel, that could not be (fully) attributed to recognition of a single allogeneic HLA-molecule. The first representative example shows CMV-pp65^RPH-specific T cells that recognized multiple different EBV-LCLs, not allowing direct complete elucidation of the HLA allele(s) being recognized (Figure 2C). Only part of the reactivity could be explained by the unique shared expression of HLA-B^*40:01 in EBV-LCLs WKD and RHP, that were both recognized. EBV-LCL UCE was the only EBV-LCL expressing HLA-B^*27:05. However, the HLA alleles underlying the recognition of EBV-LCLs GML, GMS, and MWX could not be deduced. Similarly, EBV-LMP2^CLG-specific T cells recognized 4 EBV-LCLs with unique shared expression of HLA-B^*35:01 or HLA-B^*35:03, while the recognition of EBV-LCL GMK could not be traced back to a specific HLA allele (Figure 2C and Supplementary Figure 3). Recognition of the HLA molecules that were anticipated to partly underlie the cross-reactivity patterns was confirmed using K562 cells transduced with the respective HLA molecules (Figure 2D). No recognition was observed of K562 cells transduced with irrelevant HLA molecules, whereas recognition of K562 cells transduced with the HLA restriction molecule of the respective virus-specific T-cell population only occurred upon exogenous peptide loading (Figures 2B,D). Allo-HLA cross-reactive virus-specific T cells also showed to be able to lyse HLA-mismatched target cells (Supplementary Figure 4), in line with previous studies (11, 14). These results show that recognition of HLA-mismatched EBV-LCLs can be mediated by recognition of single or multiple allogeneic HLA-molecules.

The cross-reactivity patterns against the EBV-LCL panel of more than half of the HLA-B^*08:01-restricted T-cell populations were rather complex and extensive (observed in 11 out of the 12 HLA-B^*08:01^pos donors), even when the complexity of the T-cell populations was reduced by selecting for cells expressing a single TCR-Vβ family (Representative examples; Figure 3A). No correlation could be observed for recognition of EBV-LCLs that show shared expression of specific HLA-class-II molecules. To investigate whether the reactivity patterns of these HLA-B^*08:01-restricted T-cell populations could be (fully) attributed to recognition of a limited number of allogeneic HLA-class-I alleles, a panel of 40 different single HLA-class-I-transduced K562 cell-lines was used as stimulator cells (Supplementary Table 2). With this panel we covered 63% of the HLA-A, 73% of the HLA-B and 37% of the HLA-C alleles present in our EBV-LCL panel. Testing the HLA-B^*08:01-restricted T-cell populations against this K562 panel revealed recognition of multiple specific groups of allogeneic HLA alleles by single T-cell populations, which could in part explain the cross-reactivity patterns observed when tested against the EBV-LCL panel (Figures 3B,C).

Next, we determined if the cross-reactivity of HLA-B^*08:01-restricted T-cell populations was skewed toward HLA-A, B, or C molecules. In total, 22 HLA-B^*08:01-restricted bulk or sub-populations (derived from the 11 HLA-B^*08:01^pos donors that contained complex and extensive cross-reactive virus-specific T-cell populations) were tested against the K562 panel expressing a selection of HLA-A, B, and C alleles. Twenty-one out of 22 HLA-B^*08:01-restricted T-cell populations recognized at least one allogeneic HLA-B molecule and 1 HLA-B^*08:01-restricted T-cell population (CMV-IE1^QIK from donor 7) only recognized multiple HLA-A molecules in this panel (Figure 4A and Supplementary Figure 5). Twelve out of 21 allo-HLA-B-reactive HLA-B^*08:01-restricted T-cell populations recognized only allogeneic HLA-B molecules and 9 T-cell populations additionally recognized allogeneic HLA-A and/or HLA-C molecules (Supplementary Figure 5). The number of allogeneic HLA-class-I molecules in the K562 panel recognized by the 22 HLA-B^*08:01-restricted virus-specific T-cell populations ranged from 1 to 10 per T-cell population (median of 3; Figure 4B). HLA-B^*35:01, B^*44:02 and B^*44:03 were most frequently recognized, whereas HLA-B^*13:02, HLA-B^*14:02 and HLA-B^*41:01 were never recognized by HLA-B^*08:01-restricted T cells (Figure 4C).

To investigate whether the complex and extensive recognition of allogeneic HLA molecules could be mediated by one T-cell clone, we generated T-cell clones from three cross-reactive EBV-EBNA3A^QAK-specific T-cell populations (donor #4; donor #8 and #12; Supplementary Figure 5). Indeed, all T-cell clones recognized multiple HLA-B alleles in the K562 panel, in the same pattern as the initial EBV-EBNA3A^QAK-specific T-cell populations (1 representative example per donor; Figure 5), demonstrating that single T-cell clones can exert complex and extensive cross-reactivity against allogeneic HLA molecules. Although these T-cell clones expressed different TCR-Vβ families, all T-cell clones showed a recurrent pattern of recognition of both HLA-B^*15:01, HLA-B^*35:01, HLA-B^*35:03, HLA-B^*40:01, and HLA-B^*44:03.

Virus-specific T cells from HLA-B^*08:01 homozygous donors frequently recognized HLA-B^*35:01. We therefore reasoned that heterozygosity for HLA-B^*35:01 would purge much of the cross-reactivity from the HLA-B^*08:01-restricted TCR-repertoire through thymic negative selection. For these reasons, HLA-B^*08:01-restricted EBV-EBNA3A^QAK and EBV-BZLF1^RAK-specific T-cell populations (n = 35) were isolated from 3 HLA-B^*08:01/HLA-B^*35:01^pos heterozygous donors (Table 1). Contrary, HLA-B^*13:02 was never recognized by T cells from HLA-B^*08:01^pos donors. Therefore, we also isolated T-cell populations (n = 10) with the same specificities from 3 HLA-B^*08:01^pos donors, heterozygous for HLA-B^*13:02 (Table 1). Strikingly, only 33% of the T-cell populations isolated from HLA-B^*08:01/B^*35:01^pos heterozygous donors recognized one or more HLA-mismatched EBV-LCLs, while 90% of the corresponding T-cell populations from HLA-B^*08:01^pos homozygous donors demonstrated recognition of HLA-mismatched EBV-LCLs (Figure 6A). In contrast, 80% of the T-cell populations isolated from HLA-B^*08:01/HLA-B^*13:02 heterozygous donors recognized one or more HLA-mismatched EBV-LCLs (Figure 6A). HLA-B^*08:01-restricted T cells isolated from HLA-B^*08:01/HLA-B^*35:01 donors also recognized significantly fewer HLA-mismatched EBV-LCLs than the corresponding T-cell populations isolated from HLA-B^*08:01/HLA-B^*13:02 heterozygous or HLA-B^*08:02 homozygous donors (Figure 6B). These results show that the occurrence and broadness of allo-HLA cross-reactivity by virus-specific-specific T cells is influenced by the HLA background of the donors.