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Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) can cure Acute Myeloid Leukemia (AML) thanks to the synergistic combination of chemotherapy and antitumor immunity. Still, relapse after transplantation remains an unsolved issue for a large proportion of patients, warranting further investigation on the immunobiology of AML. Our group demonstrated that, after partially HLA-compatible HSCT, AML relapse is frequently due to the genomic loss of the HLA determinants targeted by alloreactive donor T cells (Vago, N Engl J Med, 2009; Crucitti, Leukemia, 2014), suggesting that relapse might represent an expression of immune veasion from the donor-derived immune system.

To identify and characterize novel mechanisms of post-transplantation leukemia relapse.

Serial AML samples purified from 9 patients at diagnosis, relapse after chemotherapy and relapse after allo-HSCT were employed for whole transcriptome profiling using Illumina microarrays. Deregulated genes were identified by pair-wise LIMMA analysis and used in an unsupervised gene ontology enrichment analysis. Results obtained by gene expression microarrays were confirmed in a validation cohort of 21 patients by ad hoc optimized molecular and cellular assays, including qPCR, immunophenotypic analysis, and functional ex vivo and in vivo experiments.

A 110-gene signature (p<0.1) was able to discriminate between AML collected at disease diagnosis and at relapse after allo-HSCT. Most of the transcripts deregulated at post-transplantation relapse were involved in immune-related processes, and in particular in T cell costimulation and in the antigen presentation machinery. Conversely, no significant enrichment in immune-related processes was documented when comparing diagnosis with relapses after sole chemotherapy. We validated the significant upregulation of the PDL1 and B7H3 coinhibitory ligands on leukemic cells, accompanied by high levels of PD1 on the respective donor-derived T cells. Blocking this inhibitory axis by the usage of an anti-PDL1 monoclonal antibody, we rescued the ability of donor T cells to proliferate in response to the patient AML blasts. Even more evident were the de novo changes observed in the antigen presentation machinery at post-transplantation relapse: selective loss of surface expression of all HLA class II molecules and downregulation of their master regulator CIITA were detected in 4 out of 14 patients analyzed, occurring exclusively in patients transplanted from partially HLA-incompatible donors. SNP arrays documented no genomic rearrangement in HLA Class II genes or their regulators, suggesting an epigenetic origin of the alteration: accordingly, surface expression could be recovered upon culturing AML blasts in the presence of interferon-γ. Notably, loss of HLA-II expression on AML blasts abolished their recognition and killing by donor-derived T cells both in vitro and in vivo, which was recovered upon exposure of AML blasts to γ-interferon.

Our results demonstrate that the deregulation of immune-related processes, and in particular of the pathways involved in T cell-mediated allorecognition, is a distinctive feature of AML relapses after allo-HSCT. Identification of patient-specific mechanisms of immune evasion and relapse might be rapidly translated into personalized therapeutic approaches.