Memory CD4+ T Cells Recognize Divergent HLA-DP Immunopeptidomes and Contribute to Leukemia Control after Allogeneic HCT
Authors: Kittisares Kulvara , Crivello Pietro , Walther Thorben , Mohamaddokht Maryam , Bolaños Thuja Arrieta , Betke Luisa , Lux Dominik , Bracht Thilo , Bayer Malte , Flossdorf Sarah , Petersdorf Effie Wang , Turki Amin T. , Horn Peter , Köster Johannes , Westendorf Astrid M. , Falkenburg J.H. Frederik , Griffioen Marieke , Sitek Barbara , Schroeder Thomas , Bleakley Marie , Fleischhauer Katharina , Arrieta-Bolanos Esteban 
Published: December 2024

While naïve T cells are considered the main mediators of alloreactivity after hematopoietic cell transplantation (HCT), the role of memory T cells in these allo-immune responses and their clinical consequences is more debated. Differences between naïve and memory subsets are at the basis of clinical trials using naïve cell-depleted grafts in allogeneic HCT (Bleakley, J Clin Invest 2015 and JCO 2020). In unrelated HCT, patient and donor HLA-DP allotypes present immunopeptidomes with different degrees of divergence regulated by the peptide editor HLA-DM, which determine the strength and diversity of clinically relevant CD4+ T-cell alloresponses (Meurer, Blood 2021). In particular, HLA-DPB1 allele matches and core permissive mismatches involve immunopeptidomes with low divergence, the former composed mainly of minor histocompatibility antigen (mHAg) peptides, while non-core permissive and non-permissive mismatches present immunopeptidomes with high divergence (Arrieta-Bolaños, Blood 2022 and 2024). It is currently unknown how these immunopeptidome differences influence the alloreactive capacity of naïve vs memory CD4+ cells. Here, we hypothesized that the relative contribution of naive vs memory CD4+ T cells to alloreactivity against HLA-DP and its clinical consequences in HCT could vary according to the degree of immunopeptidome divergence between the allotypes from patient and donor. In CD137 upregulation assays, memory cell-mediated alloresponses from healthy donors were significantly weaker against HeLa cells expressing HLA-DP with low (n=48) compared to high (n=34) immunopeptidome divergence in the presence of HLA-DM (mean ± standard deviation 5.9% ± 7.6% vs 33.4 ± 22.3%, p&lt;0.01). Mass spectrometry-based analysis of the HLA-DP immunopeptidome coupled with whole-exome sequencing of responder and target cells showed that its de-regulation by the absence of HLA-DM increased the presentation of mHAg peptides. This in turn recruited memory T cells into the alloresponse against HLA-DP with low immunopeptidome divergence (7.9 ± 9.9% vs 33.9 ± 14.6%, p&lt;0.0001), and also significantly increased their alloreactive T-cell receptor-αβ diversity. Both naïve and memory cells produced high amounts of pro-inflammatory cytokines interferon-γ and tumor necrosis factor-α when exposed to highly divergent HLA-DP immunopeptidomes, while in this context interleukin-17 was produced exclusively by memory cells. Healthy donor alloreactive memory CD4+ T cells readily recognized and killed leukemic and non-leukemic cell lines expressing HLA-DP allotypes with high immunopeptidome divergence. In samples collected from patients one month after standard unmanipulated unrelated HCT, donor-derived peripheral blood CD4+ T cells were predominantly of memory phenotype and also showed significantly better recognition of HLA-DP with high (N=9) compared to low (N=10) immunopeptidome divergence in CD137 upregulation (4.62 ± 3.61% vs 0.82 ± 1.12%, p&lt;0.01) and cytotoxicity assays against both HeLa and leukemic cell lines expressing the relevant patient HLA-DP allotype. In a prospective clinical trial with naïve T cell-depleted allografts (Bleakley, JCO 2022; NCT02220985, NCT01858740, NCT03970096), the probability of relapse-free survival differed among patients with HLA-DP disparities with high (n=12) compared to low (n=47) immunopeptidome divergence (p=0.02), with the 5-year probabilities of relapse-free survival being 100% versus 62%, respectively. This was correlated with a lower incidence disease relapse in highly divergent patient-donor pairs, but similarly low cumulative incidences of severe acute and chronic graft-versus-host disease regardless of HLA-DP immunopeptidome divergence. There were no statistically significant differences in overall survival. Overall, these findings support an important participation of memory CD4+ T cells in alloresponses against mismatched HLA-DP with high immunopeptidome divergence that is associated with a beneficial effect for malignant disease control after unrelated HCT. This bears implications for harnessing and exploiting memory T-cell alloreactivity in HCT and cellular therapies.