In hematopoietic cell transplantation (HCT), permissive HLA-DPB1 mismatches between patients and their unrelated donors (UD) are associated with improved outcomes compared to non-permissive mismatches, but the underlying mechanism is incompletely understood. Here we used mass spectrometry, T-cell receptor-beta (TCRb) deep sequencing, and cellular in vitro models of alloreactivity to interrogate the HLA-DP immunopeptidome and its role in alloreactive T cell responses. We find that permissive HLA-DPB1 mismatches display significantly higher peptide repertoire overlaps compared to their non-permissive counterparts, resulting in lower frequency and diversity of alloreactive TCRb clonotypes in healthy individuals and transplanted patients. Permissiveness can be reversed by the absence of the peptide editor HLA-DM, or the presence of its antagonist HLA-DO, through significant broadening of the peptide repertoire. Our data establish the degree of immunopeptidome divergence between donor and recipient as the mechanistic basis for the clinically relevant permissive HLA-DPB1 mismatches in HCT, and show that permissiveness is dependent on HLA-DM-mediated peptide editing. Its key role for harnessing T-cell alloreactivity to HLA-DP highlights HLA-DM as a potential novel target for cellular and immunotherapy of leukemia.

HLA expression levels have been suggested to be genetically controlled by single nucleotide polymorphisms (SNP) in the untranslated regions (UTR), and expression variants have been associated with the outcome of chronic viral infection and hematopoietic stem cell transplantation (HSCT). In particular, the 3′UTR rs9277534-G/A SNP in HLA-DPB1 has been associated with graft-versus-host-disease after HSCT (Expression model); however its relevance in different immune cells and its mode of action have not been systematically addressed. In addition, there is a strong though not complete overlap between the rs9277534-G/A SNP and structural HLA-DPB1 T cell epitope (TCE) groups which have also been associated with HSCT outcome (TCE Structural model). Here we confirm and extend previous findings of significantly higher HLA-DPB1 expression in B cell lines, unstimulated primary B cells, and monocytes homozygous for rs9277534-G compared to those homozygous for rs9277534-A. However, these differences were abrogated by interferon-γ stimulation or differentiation into dendritic cells. We identify at least seven 3′UTR rs9277534-G/A haplotypes differing by a total of 37 SNP, also characterized by linkage to length variants of a short tandem repeat (STR) in intron 2 and TCE group assignment. 3′UTR mapping did not show any significant differences in post-transcriptional regulation assessed by luciferase assays between two representative rs9277534-G/A haplotypes for any of eight overlapping fragments. Moreover, no evidence for alternative splicing associated with the intron 2 STR was obtained by RT-PCR. In an exemplary cohort of 379 HLA-DPB1 mismatched donor-recipient pairs, risk prediction by the Expression model and the Structural TCE model was 36.7% concordant, with the majority of discordances due to non-applicability of the Expression model. HLA-DPB1 from different TCE groups expressed in the absence of the 3′UTR at similar levels by transfected HeLa cells elicited significantly different mean alloreactive CD4+ T-cell responses, as assessed by CD137 upregulation assays in 178 independent cultures. Taken together, our data provide new insights into the cell type-specific and mechanistic basis of the association between the rs9277534-G/A SNP and HLA-DPB1 expression, and show that, despite partial overlap between both models in HSCT risk-prediction, differential alloreactivity determined by the TCE structural model occurs independently from HLA-DPB1 differential expression.

T cell alloreactivity is mediated by a self-human leukocyte antigen (HLA)-restricted T cell receptor (TCR) repertoire able to recognize both structurally similar and dissimilar allogeneic HLA molecules (i.e., differing by a single or several amino acids in their peptide-binding groove). We hypothesized that thymic selection on self-HLA molecules could have an indirect impact on the size and diversity of the alloreactive response. To test this possibility, we used TCR Vβ immunophenotyping and immunosequencing technology in a model of alloreactivity between self-HLA selected T cells and allogeneic HLA-DPB1 (DPB1) differing from self-DPB1*04:02 by a single (DPB1*02:01) or several (DPB1*09:01) amino acids in the peptide-binding groove. CD4+ T cells from three different self-DPB1*04:01,*04:02 individuals were stimulated with HeLa cells stably transduced with the relevant peptide processing machinery, co-stimulatory molecules, and HLA-DP. Flow cytometric quantification of the DPB1-specific T cell response measured as upregulation of the activation marker CD137 revealed significantly lower levels of alloreactivity against DPB1*02:01 compared with DPB1*09:01 (mean CD4+CD137+ frequency 35.2 ± 9.9 vs. 61.5 ± 7.7%, respectively, p < 0.0001). These quantitative differences were, however, not reflected by differences in the breadth of the alloreactive response at the Vβ level, with both alloantigens eliciting specific responses from all TCR-Vβ specificities tested by flow cytometry, albeit with higher levels of reactivity from most Vβ specificities against DPB1*09:01. In line with these observations, TCRB-CDR3 immunosequencing showed no significant differences in mean clonality of sorted CD137+CD4+ cells alloreactive against DPB1*02:01 or DPB1*09:01 [0.39 (0.36–0.45) and 0.39 (0.30–0.46), respectively], or in the cumulative frequencies of the 10 most frequent responding clones (55–67 and 58–62%, respectively). Most of the clones alloreactive against DPB1*02:01 (68.3%) or DPB1*09:01 (75.3%) were characterized by low-abundance (i.e., they were not appreciable among the pre-culture T cells). Interestingly, however, their cumulative frequency was lower against DPB1*02:01 compared with DPB1*09:01 (mean cumulative frequency 35.3 vs. 50.6%, respectively). Our data show that, despite lower levels of alloreactivity, a similar clonal diversity can be elicited by structurally similar compared with structurally dissimilar HLA-DPB1 alloantigens and demonstrate the power of TCRB immunosequencing in unraveling subtle qualitative changes not appreciable by conventional methods.

Introduction: In 8/8 matched unrelated donor (UD) hematopoietic cell transplantation (HCT), permissive HLA-DPB1 (DP) mismatches within the same functional T Cell Epitope (TCE) group are associated with better outcomes compared to non-permissive mismatches across different TCE groups (Fleischhauer, Blood 2017). This clinical advantage has been shown to be associated with limited in vitro T cell alloreactivity (Meurer, Front Immunol 2019), which in turn is dependent on polymorphic peptide contact amino acids in the DP molecule (Crivello, Biol Blood Marrow Transplant 2015). The HLA class II immunopeptidome is shaped by the peptide editor HLA-DM (DM), and its natural antagonist HLA-DO (DO). Here we investigated the effect of DM/DO activity on the DP immunopeptidome, the breadth of the overall alloresponse to and immunogenicity of permissive and non-permissive DP mismatches, in healthy individuals and in patients after UD-HCT. Methods: HeLa cells expressing single DP alleles in the presence or absence of DM, or in the presence of DM and DO (Rutten, BBMT 2008), were generated for HLA-DPB1*04:02 (DP4) and *10:01 (DP10) as prototypes for 2 distinct TCE groups. The DP immunopeptidomes were analyzed by mass spectrometry. Alloresponses against DP were quantified by CD137 up-regulation assays after co-culture of irradiated HeLa cells with CD4+ responder T cells from 14 healthy blood donors permissive to DP4 and non-permissive to DP10, or from 2 patients referring to the University Hospital Essen, Germany, the latter alive and well >9 months after 8/8 matched UD-HCT with a permissive DP4 or a non-permissive DP10 mismatch, respectively. The breadth of the responding T cell receptor beta (TCRb) repertoire was determined by immunosequencing (Adaptive Biotechnologies, Seattle, USA). The study was performed under informed consent according to the declaration of Helsinki. Results: Reflecting their association with different TCE groups, DP4 and DP10 presented peptidomes with limited (<4%) overlap and different peptide motifs. These features were not changed by the presence or absence of DM. In contrast, the presence of DM resulted in a significant (>50%) shrinking of the peptide repertoire displayed by the same DP antigen in the absence of DM, with approximately 30% peptides shared by the same allele in the two conditions, both for DP4 and for DP10 (Figure 1A). In the presence of DM, the magnitude of the T cell alloresponse to non-permissive DP10 was significantly higher than to permissive DP4, both in healthy individuals (40.7% vs 16.3%, respectively, p<0.0001) and in the informative transplanted patients (Figure 1B). Neither the absence of DM (40.7% vs 45.3%, p=ns) nor the presence of DM with DO (71.6% vs 77.4%, p=ns) altered the magnitude of the non-permissive alloresponse to DP10. Compellingly, both the absence of DM (16.3% vs 39.0%, p<0.001) and the co-expression of DM and DO (21.6% vs 59.5%, p<0.001) significantly increased the response to permissive DP4, again both in healthy individuals and in the informative transplanted patients. The strength of the overall alloresponse was associated with the breadth of the corresponding TCRb repertoire, with significantly higher diversity (1-clonality) in response to non-permissive DP10 (mean 0.68) compared to permissive DP4 (mean 0.48) in the presence of DM, and similar high diversity against both DP antigens in its absence (mean 0.74 vs 0.75 against DP4 and DP10, respectively) in healthy individuals. In the transplanted patients, the permissive alloresponse to DP4 was dominated by a single TCRb that could be retrieved at high frequency also in ex-vivo follow-up samples from the same patient from day +195 and +363, while the non-permissive alloresponse to DP10 was polyclonal (mean 0.62 and 0.61 in the presence and absence of DM, respectively) (Figure 1C). Conclusion: Permissiveness of HLA-DPB1 TCE mismatches is dependent on the peptide editing by DM, and converted into non-permissiveness in its absence or in the presence of its antagonist DO. Permissiveness is associated with the immunopeptidomes of mismatched HLA-DP alloantigens on the MHC side, and with TCRb diversity on the alloreactive T cell side, both in healthy individuals and in patients after UD-HCT. These new mechanistic insights suggest that expression of DM and DO by leukemia or healthy tissues might modulate graft-versus-leukemia and graft-versus-host disease after permissively DP mismatched UD HCT. Disclosures No relevant conflicts of interest to declare.