NK cell recovery after haploidentical HSCT with posttransplant cyclophosphamide: dynamics and clinical implications.

The use of posttransplant cyclophosphamide (PT-Cy) as graft-versus-host disease (GVHD) prophylaxis has revolutionized haploidentical hematopoietic stem cell transplantation (HSCT), allowing safe infusion of unmanipulated T cell-replete grafts. PT-Cy selectively eliminates proliferating alloreactive T cells, but whether and how it affects natural killer (NK) cells and their alloreactivity is largely unknown. Here we characterized NK cell dynamics in 17 patients who received unmanipulated haploidentical grafts, containing high numbers of mature NK cells, according to PT-Cy-based protocols in 2 independent centers. In both series, we documented robust proliferation of donor-derived NK cells immediately after HSCT. After infusion of Cy, a marked reduction of proliferating NK cells was evident, suggesting selective purging of dividing cells. Supporting this hypothesis, proliferating NK cells did not express aldehyde dehydrogenase and were killed by Cy in vitro. After ablation of mature NK cells, starting from day 15 after HSCT and favored by the high levels of interleukin-15 present in patients' sera, immature NK cells (CD62L+NKG2A+KIR-) became highly prevalent, possibly directly stemming from infused hematopoietic stem cells. Importantly, also putatively alloreactive single KIR+ NK cells were eliminated by PT-Cy and were thus decreased in numbers and antileukemic potential at day 30 after HSCT. As a consequence, in an extended series of 99 haplo-HSCT with PT-Cy, we found no significant difference in progression-free survival between patients with or without predicted NK alloreactivity (42% vs 52% at 1 year, P = NS). Our data suggest that the majority of mature NK cells infused with unmanipulated grafts are lost upon PT-Cy administration, blunting NK cell alloreactivity in this transplantation setting.

The impact of amino acid variability on alloreactivity defines a functional distance predictive of permissive HLA-DPB1 mismatches in hematopoietic stem cell transplantation.

A major challenge in unrelated hematopoietic stem cell transplantation (HSCT) is the prediction of permissive HLA mismatches, ie, those associated with lower clinical risks compared to their nonpermissive counterparts. For HLA-DPB1, a clinically prognostic model has been shown to be matching for T cell epitope (TCE) groups assigned by cross reactivity of T cells alloreactive to HLA-DPB1∗09:01; however, the molecular basis of this observation is not fully understood. Here, we have mutated amino acids (aa) in 10 positions of HLA-DPB1∗09:01 to other naturally occurring variants, expressed them by lentiviral vectors in B cell lines, and quantitatively measured allorecognition by 17 CD4(+) T cell effectors from 6 unrelated individuals. A significant impact on the median alloresponse was observed for peptide contact positions 9, 11, 35, 55, 69, 76, and 84, but not for positions 8, 56, and 57 pointing away from the groove. A score for the "functional distance" (FD) from HLA-DPB1∗09:01 was defined as the sum of the median impact of polymorphic aa in a given HLA-DPB1 allele on T cell alloreactivity. Established TCE group assignment of 23 alleles correlated with FD scores of ≤0.5, 0.6 to 1.9 and ≥2 for TCE groups 1, 2, and 3, respectively. Based on this, prediction of TCE group assignment will be possible for any given HLA-DPB1 allele, including currently 367 alleles encoding distinct proteins for which T cell cross reactivity patterns are unknown. Experimental confirmation of the in silico TCE group classification was successfully performed for 7 of 7 of these alleles. Our findings have practical implications for the applicability of TCE group matching in unrelated HSCT and provide new insights into the molecular mechanisms underlying this model. The innovative concept of FD opens new potential avenues for risk prediction in unrelated HSCT.

Integrated Multiomic Profiling Identifies the Epigenetic Regulator PRC2 as a Therapeutic Target to Counteract Leukemia Immune Escape and Relapse.

Immune escape represents a major driver of acute myeloid leukemia (AML) reemergence after allogeneic hematopoietic cell transplantation (allo-HCT), with up to 40% of relapses prompted by nongenomic loss of HLA class II expression in leukemia cells. By integrative analysis of gene expression, DNA methylation, and chromatin accessibility in paired diagnosis/relapse primary samples and in the respective patient-derived xenografts (PDX), we identify the polycomb repressive complex 2 (PRC2) as a key epigenetic driver of this immune escape modality. We report that loss of expression of HLA class II molecules is accompanied by a PRC2-dependent reduction in chromatin accessibility. Pharmacologic inhibition of PRC2 subunits rescues HLA class II expression in AML relapses in vitro and in vivo, with consequent recovery of leukemia recognition by CD4+ T cells. Our results uncover a novel link between epigenetics and leukemia immune escape, which may rapidly translate into innovative strategies to cure or prevent AML posttransplantation relapse. SIGNIFICANCE: Loss of HLA class II expression represents a frequent mechanism of leukemia posttransplantation relapse. Here we identify PRC2 as the main epigenetic driver of this immune escape modality and show that its chemical inhibition can reinstate a proficient graft-versus-leukemia effect, providing an innovative rationale for personalized epigenetic immunotherapies. See related commentary by Köhler and Zeiser, p. 1410. This article is highlighted in the In This Issue feature, p. 1397.

Immune signature drives leukemia escape and relapse after hematopoietic cell transplantation.

Transplantation of hematopoietic cells from a healthy individual (allogeneic hematopoietic cell transplantation (allo-HCT)) demonstrates that adoptive immunotherapy can cure blood cancers: still, post-transplantation relapses remain frequent. To explain their drivers, we analyzed the genomic and gene expression profiles of acute myeloid leukemia (AML) blasts purified from patients at serial time-points during their disease history. We identified a transcriptional signature specific for post-transplantation relapses and highly enriched in immune-related processes, including T cell costimulation and antigen presentation. In two independent patient cohorts we confirmed the deregulation of multiple costimulatory ligands on AML blasts at post-transplantation relapse (PD-L1, B7-H3, CD80, PVRL2), mirrored by concomitant changes in circulating donor T cells. Likewise, we documented the frequent loss of surface expression of HLA-DR, -DQ and -DP on leukemia cells, due to downregulation of the HLA class II regulator CIITA. We show that loss of HLA class II expression and upregulation of inhibitory checkpoint molecules represent alternative modalities to abolish AML recognition from donor-derived T cells, and can be counteracted by interferon-γ or checkpoint blockade, respectively. Our results demonstrate that the deregulation of pathways involved in T cell-mediated allorecognition is a distinctive feature and driver of AML relapses after allo-HCT, which can be rapidly translated into personalized therapies.

Allorecognition of HLA-DP by CD4+ T cells is affected by polymorphism in its alpha chain.

Alloreactivity to HLA-DP molecules, class II heterodimers of an oligomorphic alpha and a polymorphic beta chain, is increasingly being studied due to its relevance in clinical transplantation. We hypothesized that not only polymorphisms in the peptide binding groove encoded by exon 2 of HLA-DPB1, but also in other regions of the molecule and the alpha chain, could play a role in CD4+ T cell allorecognition. To test this possibility, we comparatively investigated CD4+ T cell allorecognition, measured by upregulation of the activation marker CD137, against HLA-DPB1*13:01, *05:01, *03:01, *17:01 or their allele counter parts DPB1*107:01, *135:01, *104:01, *131:01, with identical exon 2 sequences but polymorphism in exons 1, 3 or 4, in the context of different HLA-DPA1 (DPA1) polymorphisms (DPA1*01:03 and *02:01). No significant differences in CD4+ T cell allorecognition levels could be demonstrated for any of the beyond exon 2 DPB1 variants studied. Interestingly, however, the mean fold change in CD4+ CD137+ cells was significantly higher when the target shared at least one DPA1 allele with the allogeneic stimulator, compared to a distinct DPA1 background (1.65 vs 0.23, P<0.005). Structural homology modeling suggested specific amino acid residues in the alpha chain, in particular position 31, to impact CD4+ T cell allorecognition of HLA-DP. Our data argue against a significant role of beyond exon 2 DPB1 polymorphisms for T cell alloreactivity, but show relevance of DPA1 polymorphism in this mechanism. These new findings impact HLA matching strategies in unrelated stem cell transplantation.

Effects of transmembrane region variability on cell surface expression and allorecognition of HLA-DP3.

The functional relevance of polymorphisms outside the peptide binding groove of HLA molecules is poorly understood. Here we have addressed this issue by studying HLA-DP3, a common antigen relevant for functional matching algorithms of unrelated hematopoietic stem cell transplantation (HSCT) encoded by two transmembrane (TM) region variants, DPB1(*)03:01 and DPB1(*)104:01. The two HLA-DP3 variants were found at a overall allelic frequency of 10.4% in 201 volunteer stem cell donors, at a ratio of 4.2:1. No significant differences were observed in cell surface expression levels of the two variants on B lymphoblastoid cell lines (BLCL), primary B cells or monocytes. Three different alloreactive T cell lines or clones showed similar levels of activation marker CD107a and/or CD137 upregulation in response to HLA-DP3 encoded by DPB1(*)03:01 and DPB1(*)104:01, either endogenously on BLCL or after lentiveral-vector mediated transfer into the same cellular background. These data provide, for the first time, direct evidence for a limited functional role of a TM region polymorphism on expression and allorecognition of HLA-DP3 and are compatible with the notion that the two variants can be considered as a single functional entity for unrelated stem cell donor selection.

Frequency and targeted detection of HLA-DPB1 T cell epitope disparities relevant in unrelated hematopoietic stem cell transplantation.

The majority of unrelated donor (UD) hematopoietic stem cell (HSC) transplants are performed across HLA-DP mismatches, which, if involving disparity in a host-versus-graft (HVG) direction for an alloreactive T cell epitope (TCE), have been shown by our group to be associated with poor clinical outcome in 2 cohorts of patients transplanted for hematopoietic malignancies and beta-thalassemia, respectively. Using site-directed mutagenesis of DPB1*0901, we show here that the TCE is abrogated by the presence of amino acids LFQG in positions 8-11 of the DP beta-chain. Based on this and on alloreactive T cell responsiveness, we have determined the presence or absence of the TCE for 72 DPB1 alleles reported in the ethnic groups representative of the worldwide UD registries, and predict that 67%-87% (mean 77%) of UD recipient pairs will not present a DPB1 TCE disparity in the HVG direction. We developed and validated in 112 healthy Italian blood donors an innovative approach of DPB1 epitope-specific typing (EST), based on 2 PCR reactions. Our data show that DPB1 TCE disparities may hamper the clinical success of a considerable number of transplants when DPB1 matching is not included into the donor selection criteria, and that a donor without DPB1 TCE disparities in the HVG direction can be found for the majority of patients. Moreover, the study describes the first protocol of targeted epitope-specific DPB1 donor-recipient matching for unrelated HSC transplantation. This protocol will facilitate large-scale retrospective clinical studies warranted to more precisely determine the clinical relevance of DPB1 TCE disparities in different transplant conditions.