Acute Myeloid Leukemia Relapses after Allogenenic HSCT Display a Distinctive Immune-Related Signature, with Frequent and Functionally Relevant Alterations in HLA Class II Antigen Presentation and T Cell Costimulation
Authors: Toffalori Cristina , Riba Michela , Zito Laura , Barcella Matteo , Spinelli Orietta , Crucitti Lara , Cieri Nicoletta , Peccatori Jacopo , Bernardi Massimo , Bonini Chiara , Cittaro Davide , Lazarevic Dejan , Rambaldi Alessandro , Barlassina Cristina , Stupka Elia , Ciceri Fabio , Fleischhauer Katharina , Vago Luca 
Published: October 2019

Abstract
INTRODUCTION: Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) represents an effective form of adoptive immunotherapy for Acute Myeloid Leukemia (AML), thanks to the antitumor effect mediated by donor immune cells infused as part of the graft. Unfortunately, post-transplantation relapses remain a frequent observation, warranting further investigation on AML immunobiology. Our group demonstrated that relapses after partially HLA-incompatible HSCT are frequently due to the outgrowth of immune-resistant mutant AML clones characterized by genomic loss the mismatched histocompatibility determinants targeted by alloreactive donor T cells, and have thus gained a selective advantage upon pressure from the transplanted immune system, in a process called “leukemia immunoediting” (Vago et al., N Engl J Med, 2009). In the present study, we took advantage of high-throughput technologies to profile post-transplantation AML relapses with no genomic loss of HLA, to identify novel targets of the leukemia immunoediting process.
METHODS: Serial AML samples were collected and FACS-purified from 9 patients at diagnosis, relapse after sole chemotherapy and relapse after allo-HSCT, and employed for whole transcriptome profiling using Illumina HumanHT12 microarrays. Deregulated genes were identified by pair-wise LIMMA analysis, and unsupervised enrichment analysis was performed interrogating the Gene Ontology database. High-throughput results were confirmed in a validation cohort of 12 patients by ad hoc designed qPCR assays, immunophenotypic analyses and functional experiments. In particular, co-cultures were performed using as responders peripheral blood lymphocytes harvested from patients after allo-HSCT, and as stimulators and targets their respective AML blasts collected at diagnosis or at relapse: read-outs included antigen-specific T cell activation, cytotoxicity, and cytokine release.
RESULTS: Amongst all the genes expressed by purified AML blasts, 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. Alterations in costimulatory molecules were confirmed by immunophenotypic analyses in a validation cohort: AML blasts at both diagnosis and post-transplantation relapse expressed high levels of co-inhibitory molecules (PDL-1, B7-H3 and Vista) and low levels of costimulatory ligands (ICOS-L, TNFSF4). At relapse after allo-HSCT we documented a marked reduction in Vista surface expression on AML blasts, and very high levels of PD-1 on donor-derived T cells. 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 the 12 patients analyzed, occurring exclusively in patients transplanted from partially HLA-incompatible donors. In 2 of the 4 cases Class II expression was recovered upon culturing AML blasts in the presence of interferon-γ. SNP arrays documented no genomic alterations in Class II genes or their regulators, suggesting an epigenetic origin of the alteration. Importantly, loss of HLA Class II surface expression in AML blasts resulted in the complete abrogation of functional recognition and killing by donor-derived T cells, supporting the hypothesis that these changes might foster immune evasion and relapse.
CONCLUSIONS: Our results demonstrate that the deregulation of immune-related processes, and in particular of molecules and pathways involved in T cell-mediated allo-recognition, are pervasive and distinctive features of AML relapses after allo-HSCT. Identification of patient-specific mechanisms of immune evasion might be translated into personalized therapeutic approaches, tailored to restore or circumvent inefficient antigen presentation and T cell costimulation.

Disclosures
Bonini: MolMed S.p.A.: Consultancy.