Identification of CD4+ T cells targeting DM-sensitive antigens presented on mismatched HLA-DP alleles, as mediators of a selective graft-versus-leukemia effect.

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Abstract Introduction: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) enables hematopoietic reconstitution through donor stem cells matched at essential HLA loci. While it offers potential for a graft-versus-leukemia (GvL) effect through elimination of residual malignant cells, it also carries the risk of graft-versus-host disease (GvHD) due to the recognition of healthy tissues. We identified two groups of HLA-II restricted antigens with distinct behavior towards HLA-DM. DM-resistant antigens are presented when HLA-DM is expressed. In contrast, DM-sensitive antigens require inhibition of HLA-DM by HLA-DO. Because HLA-DO expression is confined to hematopoietic antigen-presenting cells, DM-sensitive antigens cannot be presented on non-hematopoietic tissues, even under inflammatory conditions. Since HLA-DP is frequently mismatched in unrelated donor transplants, we hypothesized that CD4+ T cells targeting DM-sensitive antigens in a mismatched HLA-DP allele could be an ideal way to achieve GvL effect without inducing GvHD. Methods: To identify T cells recognizing DM-sensitive antigens, isolated CD4⁺ T cells from an HLA-DP–mismatched donor were co-cultured with HeLa cells expressing invariant chain (li) and one of the five most common HLA-DP molecules in the Caucasian population (DPB1*01:01, *02:01, *03:01, 04:01, 04:02). Activated T cells were isolated based on expression of CD137 and clonally expanded. Reactivity of T-cell clones against HeLa + Ii ± HLA-DP ± HLA-DM, malignant hematopoietic and non-hematopoietic cell lines, EBV-infected B cells, and primary AML blasts was characterized, along with cytotoxicity and cytokine profiles. T-cell receptors (TCR) of T-cell clones with the most favorable characteristics were sequenced in order to generate TCR-engineered T cells. TCR reexpression was achieved by orthotopic T-cell receptor replacement (OTR) using CRISPR. Results: Of 105 T-cell clones from nine donors, 79 targeted DM-sensitive and 26 DM-resistant antigens, distinguished by their reactivity in presence of HLA-DM. T-cell clones directed against DM-sensitive antigens did not recognize non-hematopoietic cells, even under inflammatory conditions. In contrast, HLA-DO positive malignant hematopoietic cell lines and primary AML blasts were recognized by T-cell clones targeting DM-sensitive antigens, indicating potential leukemia-specificity. Testing against cell lines from various donors suggests that antigen recognition by T cells was independent of donor-specificity, but rather directed against the mismatched HLA-DP possibly complexed with a monomorphic peptide. Across all experimental conditions, the recognition profile (sensitive or resistant) remained consistent regardless of target cell concentration. Donor-dependent cytotoxicity for isolated T-cell clones was observed and mainly mediated by Granzyme A and B. Extended cytokine analysis revealed secretion of INF-γ, IL-5, IL-13, and IL-6, accompanied by lower levels of IL-4. After reexpression, TCR-engineered T cells demonstrated high functionality as shown by IFN-γ ELISA and activation marker analysis via flow cytometry. Conclusion: We show feasibility to identify T cells directed against DM-sensitive antigens presented in mismatched HLA-DP alleles recognizing malignant hematopoietic cells, which in future may contribute to the development of GvL directed T-cell therapies with reduced risk of GvHD.

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