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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

Background: Evasion from immune control represents one of the main drivers of acute myeloid leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (allo-HCT). In particular, up to 40% of AML relapses display complete loss of surface expression of HLA class II molecules without any genetic lesion explaining this phenotype (Christopher et al, N Engl J Med, 2018; Toffalori et al, Nat Med, 2019). This led us to investigate the links between epigenetic changes, immune evasion and post-transplantation relapse. Methods: Starting from primary AML samples pairwise collected from five patients at diagnosis and relapse with non-genomic loss of HLA class II expression, we generated Patients-Derived Xenografts (PDXs) into NOD-SCID γ-chain null mice. Leukemic cells expanded in the mice and their original human counterparts were characterized for changes in gene expression (by RNA-Seq), DNA methylation profile (by RRBS), and chromatin accessibility (by ATAC-Seq). The results obtained by all these approaches and in the different patients were integrated by Multi-Omics Factor Analysis (MOFA) and Gene Set Enrichment Analysis (GSEA). Finally, we tested the immunological effects of epigenetic drugs on AML cells and on their recognition by T cells in ex-vivo short-term cultures and in PDXs. Results: We verified that PDXs faithfully recapitulate immune-related differences between AML diagnosis and post-transplantation relapse, including loss of expression of HLA class II molecules. Differences between diagnosis and post-transplantation relapse samples were mostly explained by changes in chromatin accessibility, and largely unrelated to the DNA methylation profile. In particular, in all five patients analyzed, we documented genomewide chromatin compaction at time of relapse, that was particularly evident for HLA class II genes and their master regulator CIITA, and not detected in relapses after sole chemotherapy. Integration of all the high-throughput technologies by MOFA, and of results from different patients by GSEA, pointed to the Polycomb Repressive Complex 2 (PRC2) as the main candidate mediator of HLA class II silencing.Pharmacological inhibition of PRC2 subunits rescued HLA class II expression in AML relapses ex vivo and in vivo, with consequent recovery of leukemia recognition by CD4+ T cells. Conclusions: Our results uncover a novel link between epigenetics and leukemia immune escape, which may rapidly translate into innovative strategies to cure or prevent AML post-transplantation relapse. Citation Format: Valentina Gambacorta, Stefano Beretta, Martina Ciccimarra, Laura Zito, Kety Giannetti, Angela Andrisani, Daniela Gnani, Lucia Zanotti, Giacomo Oliveira, Matteo G. Carrabba, Davide Cittaro, Ivan Merelli, Fabio Ciceri, Raffaella Di Micco, Luca Vago. Integrated multiomic profiling identifies the epigenetic regulator PRC2 as a therapeutic target to counteract leukemia immune escape and relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB563.

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-gamma 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.

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.

Background Genomic loss of mismatched HLAs ("HLA loss") represents a frequent modality by which acute myeloid leukemia (AML) evades immune recognition from donor T cells after partially HLA-incompatible allogeneic hematopoietic cell transplantation (allo-HCT). One important consequence of this post-transplantation relapse mechanism is that infusions of lymphocytes from the original donor become ineffectual, prompting the search for alternative therapeutic options. Here, to circumvent the loss of physiological T cell receptor-HLA interactions in these patients, we tested the ability of an anti-CD3/CD33 bispecific antibody (BsAb) to re-target donor T cells towards HLA loss relapses. Methods For short-term in vitro experiments, T cells were co-cultured with the MOLM-13 AML cell line or with primary patient blasts for 96 hours in presence or absence of an anti-CD3/CD33 BsAb. As readouts, we measured T cell activation (as surface expression of CD25 and CD69) and the absolute counts and relative proportion of effectors and targets. For long-term in vitro experiments, we established mixed lymphocyte cultures (MLCs) of T cells purified from two patients after haploidentical HCT and primary AML blasts obtained from the same patients at the time of diagnosis. After sequential stimulations, the co-cultures were tested against targets of interest, with or without addition of the BsAb. Functional readouts were T cell degranulation (measured as CD107a expression), antigen-specific activation (as CD137/41-BB expression) and target-specific cytotoxicity (measured by time-lapse live cell imaging over a 48 hour time span). For in vivo experiments, human leukemic cells were infused intravenously into non-irradiated NSG mice, followed by intraperitoneal infusion of T cells and daily administration of the BiTE compound. Results First, we retrospectively analyzed immunophenotypic data of 36 AML patients who experienced HLA loss relapses at our Institution, documenting robust expression of CD33 on the surface of the relapsed leukemia in 35 of them (97%; Figure 1A). By short-term co-culture experiments we titrated the BsAb concentration to be used for subsequent in vitro assays to 100 ng/ml, and the most informative effector:target ratio to 1:3. Then, we established MLCs by stimulating T cells collected from two patients after partially HLA-incompatible allo-HCT with AML blasts collected from the same patients at the time of diagnosis. In both cases, donor-derived T cells robustly responded against the patient blasts both in term of degranulation (Figure 1B) and of antigen-specific activation (Figure 1C). As expected, when we tested the same T cells against the patient leukemia at time of HLA loss relapse, we detected no T cell-mediated responses. Noticeably, when the BsAb was added, in both cases we detected a strong response not only against the diagnosis but also against the HLA loss variants, indicating that T cells were effectively re-targeted towards leukemic cells. Similar results were obtained also by live cell imaging, measuring target cell apoptosis over 48 hours of recording: also in this assay, in fact, donor T cells recognized and killed leukemia at diagnosis (45% of detection area positive for apoptosis dye) and failed to recognize its HLA loss relapse counterpart (32% of area positive for apoptosis dye). Addition of the BsAb to the co-cultures had a minor effect on recognition of the original disease (45% of area positive for apoptosis dye) but drove dramatic cell death of HLA loss blasts (80% of area positive for apoptosis dye), demonstrating that the BsAb induced not only T cell activation but also and most importantly target cell killing (Figure 1D). Finally, we modeled the BsAb activity in vivo, showing that, whereas the sole infusion of human T cells is not able to prevent the outgrowth of leukemia in the bone marrow of NSG mice, addition of the bispecific antibody leads to effective disease clearance (Figure 1E). Conclusions Our results demonstrate that anti-CD3/CD33 BsAbs can effectively redirect donor T cells against HLA loss leukemia variants, resulting in their rapid and effective killing. Taken together, these promising findings strongly support translation of this approach to ad hoc designed early-phase clinical trials, to provide a rational therapy for this increasingly recognized but still treatment-orphan modality of post-transplantation relapse. Figure 1 Disclosures Subklewe: Janssen: Consultancy; Miltenyi: Research Funding; Pfizer: Consultancy, Honoraria; Oxford Biotherapeutics: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Morphosys: Research Funding; Roche: Consultancy, Research Funding; AMGEN: Consultancy, Honoraria, Research Funding. Vago:Moderna Therapeutics: Research Funding; GenDx: Research Funding.

Background It is becoming increasingly recognized that evasion from immune control represents one of the main drivers of acute myeloid leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (allo-HCT). In particular, alterations in the antigen processing and presentation machinery represent one of the most effective strategies enacted by tumor cells to avoid recognition from T cells. Whereas it is now well recognized that genomic loss of HLA is frequently at the basis of post-transplantation relapse, it was only recently reported that up to 40% of AML relapses display transcriptional downregulation and complete loss of surface expression of HLA class II molecules without any genetic lesion explaining this phenotype (Christopher et al, N Engl J Med, 2018; Toffalori et al, Nat Med, 2019). This led us to investigate the links between epigenetic changes, immune evasion and post-transplantation relapse. Methods Starting from primary AML samples pairwise collected from patients at diagnosis and relapse with non-genomic loss of HLA class II expression, we generated Patients-Derived Xenografts (PDXs) into NOD-SCID γ-chain null mice. Leukemic cells expanded in the mice and their original human counterparts were analyzed for surface expression of selected immune-related markers (HLA class I and II, PD-L1, B7-H3), and characterized for changes in gene expression (by RNA-Seq), DNA methylation profile (by RRBS), histone modifications associated with active promoters (H3K4me3) or regulatory elements (H3K27ac) (by ChIP-seq) and chromatin accessibility (by ATAC-Seq). The results obtained by all these approaches were integrated by Multi-Omics Factor Analysis (MOFA), followed by Gene Set Enrichment Analysis (GSEA). Finally, the immunological effects of epigenetic drugs and recombinant immune-modulatory cytokines on primary and PDX-derived AML samples were tested in ex-vivo short-term cultures on a layer of mesenchymal stromal cells. Results We verified that PDXs faithfully recapitulate immune-related differences between diagnosis and post-transplantation relapse, including loss of expression of HLA class II molecules (Figure 1A). Integration of all the high-throughput technologies by MOFA evidenced that the differences between diagnosis and post-transplantation relapse samples were mostly explained by changes in chromatin accessibility and histone marks, and largely unrelated to the DNA methylation profile (Figure 1B). We documented that the gene sets that emerged upon integrating epigenetic analyses by MOFA matched our previously published immune-related relapse signature (Toffalori et al, Nat Med, 2019) but also and most intriguingly lists of genes known to be targeted by EZH2, the enzymatic subunit of the PRC2 chromatin repressor complex (Figure 1C). These computational analyses were supported by the evidence of a relapse-specific closed chromatin status of HLA class II genes and their regulators (Figure 1D). To revert these epigenetic changes, we inhibited EZH2 with tazemetostat (EPZ-6438), an epigenetic drug currently being tested in early-phase clinical trials for lymphomas, in two AML relapses with non-genomic loss of HLA class II expression. EZH2 inhibition reduced the levels of the repressor mark H3K27me3 (Figure 1E), increased the surface expression of HLA class II molecules on leukemia cells (Figure 1F) and ultimately improved leukemia recognition by CD4+ T cells (Figure 1G). Notably, these effects were even more pronounced when EZH2 inhibition was combined with IFN-g treatment (Figure 1F,G), suggesting synergism between this epigenetic compound and cytokines released by immune cells upon target recognition. Conclusions Our results provide mechanistic insights into epigenetic regulation of HLA class II downregulation in leukemia and a strong therapeutic rationale to test EZH2 inhibition as an innovative strategy for the treatment of AML post-transplantation relapses. Figure 1 Disclosures Vago: GenDx: Research Funding; Moderna Therapeutics: Research Funding.

Polymorphisms in the 3' untranslated region (3'UTR) of HLA-G, an important player in immunological tolerance, could be involved in post-transcriptional expression control, and their association with different clinical immune-related conditions including autoimmunity and transplantation is of mounting interest. Most studies have focused on a 14 base pair (bp) insertion/deletion (ins/del), while additional single-nucleotide polymorphisms (SNPs) in the HLA-G 3'UTR have been described but not extensively investigated for their clinical relevance. Here we have comparatively studied the association between 3'UTR haplotypes of HLA-G, or the 14 bp ins/del, with clinical outcome of HLA-identical sibling hematopoietic stem cell transplantation (HSCT) in 147 Middle Eastern beta-thalassemia patients. Sequence based typing of 3'UTR HLA-G polymorphisms in the patients and in 102 healthy Italian blood donors showed strong linkage disequilibrium between the 14 bp ins/del and five 3'UTR SNPs, which together could be arranged into eight distinct haplotypes based on expectation-maximization studies, with four predominant haplotypes (UTRs1-4). After HSCT, we found a moderate though not significant association between the presence of UTR-2 in double dose and protection from acute graft versus host disease (hazard ratio (HR) 0.45, 95% confidence intervals (CI): 0.14-1.45; P = 0.18), an effect that was also seen when the corresponding 14 bp ins/ins genotype was considered alone (HR 0.42, 95% CI: 0.16-1.06; P = 0.07). No association was found with rejection or survival. Taken together, our data show that there is no apparent added value of considering entire 3'UTR HLA-G haplotypes for risk prediction after allogeneic HSCT for beta-thalassemia.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents an effective treatment for many hematological malignancies, but post-transplantation relapses remain frequent, and their biological bases poorly understood. Here we combined Whole Exome Sequencing (WES) and RNA-Seq to compare the features of 15 cases of Acute Myeloid Leukemia before allo-HSCT and at post-transplantation relapse. Leukemic blasts were collected and purified at the two timepoints, and compared between each other and with patient and donor germline controls. Three donors were HLA-identical siblings, 7 were matched unrelated volunteers (all of which were matched to the patient for 10/10 HLA loci) and 5 were HLA-haploidentical relatives. Median time from allo-HSCT to relapse was 144 days (range 33-574). The average coverage for WES, consistent between all cases, was 120x for leukemia samples and 60X for germline controls. Analysis of copy number alterations did not reveal major genomic alteration acquired at relapse, except for one patient who gained trisomy for chr. 6 and 21. Also the transitions/transversions ratio remained constant between the pre-Tx and post-Tx samples. We detected an average of 15 somatic variants (SNV and small InDel) per leukemia sample, and the overall mutational burden increased significantly between pre- and post-Tx samples (Wilcoxon test, pvalue = 0.0088). We evidenced 4 major patterns of clonal evolution. In pattern 1 (n=3), the pre-Tx clones persisted unchanged at relapse, in pattern 2 (n=6) and 3 (n=2), subclones were gained or lost, respectively, whereas in pattern 4 (n=4) we found a mixed scenario. Sixty somatic variants were present only in relapsed samples, encompassing known AML driver genes, including KRAS and WT1 (de novo mutated at relapse in 2 patients). Unexpectedly, WES analysis did not detect relapse-specific mutations in genes related to immune function, and even an ad hoc developed pipeline for the analysis of somatic mutations in HLA class I and class II genes did not detect any denovo acquired sequence abnormality. Conversely, a linear model analysis of RNA-seq showed ~800 genes significantly deregulated in AML blasts at post-transplantation relapse. The down-regulated genes were mainly immune-related, encompassing in particular those involved in HLA class II antigen presentation. Upregulated genes comprised genes relevant to DNA replication and cell cycle control (including several component of Minichromosome Maintanance Complex). Of interest, these two processes appeared also to cluster independently in our patient series, suggesting the presence of different transcriptional mechanisms of relapse. Of interest, we observed HLA class II downregulation also in several cases in which donor and patient were matched for those loci. Thus, to understand whether the decreased expression of antigen presentation molecules could in these cases be driven by an increase in the levels of presented antigens, we extracted from the RNA-seq dataset information regarding known leukemia associated antigens, finding several of them upregulated at post-transplantation relapse (MPO, TERT, PRTN3) Moreover, we combined WES and RNA-seq data to predict the number of patient-specific neoantigens and minor histocompatibility antigens (MiHAgs) presented on leukemia blast before and after allo-HSCT. In line with the low overall burden of mutations, we predicted a very low number of neoantigens per case (on average 3 per sample, ranging from 0 to 20), increasing at relapse in 5/15 patients. The number of predicted MiHAgs was sizably higher, and varied considerably in relation to donor-recipient matching (on average 481 in haploidentical HSCTs, 874 in HSCTs from HLA-identical siblings, and 1435 in unrelated donor HSCTs). However, the overall level of expression of MiHAgs did not vary between pre- and post-Tx samples Our results provide for the first time a detailed landscape of the many features that shape the interplay between immune system and leukemia in allo-HSCT. The resulting picture is composite, suggesting that mechanisms of relapse are highly patient-specific and combine genomic and non-genomic, immunological and non-immunological changes. For this reason we modeled our data in a comprehensive framework that we termed "relapsogram", that might help in elucidating the peculiarity of each case of relapse and in customizing the therapy. Disclosures Stoelzel: Neovii: Speakers Bureau. Bonini:Intellia Therapeutics: Research Funding. Vago:Moderna TX: Research Funding; GENDX: Research Funding.

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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

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 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.

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.

Background: Patient-derived xenografts (PDXs) are key models for interrogating the biology of tumor cells that poorly survive in vitro. In particular, over the last decade, immunodeficient mouse models have been extensively used to assess the in vivo growth potential of human leukemia, to provide insights into its biology, and to perform preclinical validation of therapies. Still, only a fraction of the cases of acute myeloid leukemia (AML) are able to engraft into mice, and the biological and clinical correlates of the ability to generate PDXs are unknown. Methods: Primary AML harvested from 52 patients at diagnosis (n=37, 71%), at relapse after treatments (n=15, 29%), or both (n=6) were purified and infused into non-irradiated NOD-SCID γ-chain null (NSG) mice. Upon leukemia engraftment, assessed by multiparametric flow cytometry, mice were sacrificed and leukemic cells were isolated, characterized, and reinfused in serial recipients, in up to four serial passages. Gene expression profile was analyzed using Illumina microarray, and deregulated genes and processes identified by pairwise LIMMA analysis and classified using Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) curated databases. The mutational asset of infused AML was assessed through targeted resequencing, using a custom panel comprising 192 targets and based on the Agilent Haloplex HS technology. Results: Twenty-six out of 52 primary AML samples (50%) generated xenografts. Engraftment and growth kinetics of the human leukemic cells were highly consistent among littermates, and specific for each tested leukemia. Circulating leukemic cells were firstly detected in the peripheral blood of animals at a median time of 22.5 days (range 14 - 150). In vivo growth allowed expansion of infused AMLs in bone marrows and spleens of the animal, with a median fold increase of 3.5 (range 0.1 - 351.4). The gene expression profile of xenografts was reproducible amongst littermates and recapitulated the features of parental AML: genes deregulated in xenografts accounted for 9.1% of the transcript assessed, with substantial overlap in the genes and processes deregulated in each of the studied cases. GO and GSEA demonstrated the selective deregulation of genes involved in cell proliferation (CDC20, AURKA), syster chromatyde organization (CENPF CEP170) and myeloid differentiation (AZU1, MPO, MYADM, CTSG). Of note, the ability to generate xenografts was conserved when AML cells were challenged at different time-points during the clinical history of the patients, with leukemia harvested at relapse after transplantation displaying a more aggressive behavior. Similarly, upon serial transfer AML exhibited an accelerated growth kinetic. Engraftment in mice significantly correlated with poor patient prognosis: AML engrafters had dramatically lower leukemia free-survival rates compared to non-engrafters (median 5.9 vs. 21.8 months after induction chemotherapy, p=0.0022, Fig. 1A), confirmed also by multivariate analysis (p=0.002). Also the mutational profile differed greatly between engrafters and non-engrafters, as summarized in Fig. 1B. In particular, while the presence of an aberrant karyotype was not associated with PDX generation, FLT3 internal tandem duplication, DNMT3A and NPM1 mutation were all significantly associated to engraftment (p=0.0244, p=0.009 and p=0.0437 respectively). In particular the co-occurrence of mutations in these three genes, recently reported to confer very poor prognosis to AML patients (Papaemmanuil et al, NEJM 2016), markedly enhanced the ability to generate PDXs (Fig.1C). Conclusion: These data show that engraftment into immunodeficient mice mirrors the biology of primary human leukemia, providing a proxy to select cases with a higher chance to generate PDXs. Further comparisons between AML capable or not to generate PDXs might provide novel markers of leukemia aggressiveness and rationales for targeted therapies. Figure 1 Figure 1. Disclosures Bonini: TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy. Ciceri:MolMed SpA: Consultancy.

INTRODUCTION The use of high-dose cyclophosphamide as post-transplant Graft versus Host Disease (GvHD) prophylaxis has revolutionized haploidentical hematopoietic stem cell transplantation (haplo-HSCT), allowing the safe infusion of T cell replete grafts. The efficacy of post-transplant cyclophosphamide (PT-Cy) has its basis in its capacity to selectively eliminate proliferating cells, including alloreactive T cells. It is however to date unknown whether PT-Cy affects the reconstitution of Natural Killer (NK) cells, whose alloreactivity is known to play a major role in T cell-depleted haplo-HSCT. PATIENTS AND METHODS We analyzed the grafts and serial peripheral blood (PB) and bone marrow (BM) samples from 14 patients who received T cell replete haplo-HSCT followed by PT-Cy at the San Raffaele Scientific Institute, Milan (n=10, OSR) or the Johns Hopkins University, Baltimore (n=4, JHU). OSR patient received a myeloablative conditioning, PB stem cell grafts, and sirolimus and mycophenolate as pharmacological GvHD prophylaxis. JHU patients received the "classical" Baltimore nonmyeloablative conditioning, unmanipulated BM grafts, and tacrolimus and mycophenolate as pharmacological GvHD prophylaxis. To characterize NK cells reconstitution, we monitored absolute counts and employed a 27-marker flow cytometry panel with high dimensional single-cell analysis using the bh-SNE algorithm. We used intracellular staining to determine the frequency of Ki67+ proliferating cells and expression of Aldehyde Dehydrogenase (ALDH), known to confer resistance to PT-Cy. Interleukin-15 (IL-15) serum concentration was quantified using the Bio-Plex Pro Human Cytokine 4-plex assay. To directly assess the effect of PT-Cy on proliferating NK cells we exposed graft NK cells to IL-15 and mafosfamide, a cyclophosphamide analogue active in vitro. Functional assays against leukemic cell lines and primary AML blasts were performed measuring CD107A degranulation on NK cells and Annexin V on targets. RESULTS All patients received high numbers of mature donor NK cells as part of the graft (OSR median 17x106/kg, JHU median 7.25x106/kg ), and donor-derived NK cells were detectable as early as day 3 after HSCT and throughout the entire follow-up. At day 3 after HSCT, all subsets of NK cells, including single KIR+ alloreactive cells, were actively proliferating (mean 61.23% of Ki-67+ cells for OSR patients, and 58% for JHU patients), possibly driven by the high levels of IL-15 detected in patient serum after conditioning (Fig.1A). After PT-Cy, a marked reduction in the frequency and counts of proliferating NK cells was evident irrespectively of the transplantation platform, suggesting selective killing of dividing cells by PT-Cy. In line with this hypothesis, NK cells from the graft and from patient PB at day 3 after HSCT showed no detectable ALDH expression, and NK cells prompted to proliferate in vitro were killed in a dose-dependent manner by mafosfamide (Fig.1B). The phenotype of NK cells also changed upon PT-Cy: whereas before the infusion they resembled their mature counterparts from the graft, after PT-Cy an immature phenotype, CD62L+NKG2A+KIR-, became prevalent, suggesting derivation from donor HSCs rather than from infused NK cells (Fig.1C). Accordingly, bhSNE maps demonstrated differential clustering of NK cells from the graft and analyzed 30 days after HSCT (Fig.1D). In line with these features, we detected very low numbers of putatively alloreactive single KIR+ NK cells both in the PB and in the BM of patients at day 30 after HSCT, and these NK cells displayed impaired cytotoxic potential against leukemic targets. Finally, consistent with these observations, when we analyzed the impact of predicted NK alloreactivity in an extended series of 99 patients who received myeloablative haplo-HSCT with PT-Cy, we detected no significant difference in progression-free survival (Fig.1E). CONCLUSION Our data suggest that the majority of mature NK cells infused with unmanipulated grafts are eliminated upon PT-Cy administration and that in this transplantation platform NK cell alloreactivity might be blunted by the elimination of donor single KIR+ NK cells and by the competition between reconstituting NK and T cells. Still, the high levels of IL-15 detected in patients' sera at early time-points might provide a biological rationale for the infusion of mature donor NK cells early after PT-Cy administration. Figure 1 Figure 1. Disclosures Bonini: TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy. Ciceri:MolMed SpA: Consultancy.

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 die 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 DPB I 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. (c) 2007 American Society for Blood and Marrow 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 die 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 DPB I 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. (c) 2007 American Society for Blood and Marrow Transplantation Inflammation and immune reaction, or pre-existing immunity towards commonly used viral vectors for gene therapy severely impair long-term gene expression in the central nervous system (CNS), impeding the possibility to repeat the therapeutic intervention. Here, we show that injection of a helper-dependent adenoviral (HD-Ad) vector by lumbar puncture into the cerebrospinal fluid (CSF) of non-human primates allows long-term (three months) infection of neuroepithelial cells, also in monkeys bearing a pre-existing anti-adenoviral immunity. Intrathecal injection of the HD-Ad vector was not associated with any sign of systemic or local toxicity, nor by signs of a CNS-specific immune reaction towards the HD-Ad vector. Injection of HD-Ad vectors into the CSF circulation may thus represent a valuable approach for CNS gene therapy allowing for long-term expression and re-administration.

Background Despite the constant improvement in the outcome of allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) for Acute Myeloid Leukemia (AML), relapses remain frequent, warranting investigation on their biological bases. After haploidentical HSCT up to one third of AML relapses feature selective genomic loss of the HLA haplotype targeted by alloreactive donor T cells (Vago, N Engl J Med, 2009; Crucitti, Leukemia, 2015), evading their control and gaining the ability to outgrow. Yet, Natural Killer (NK) cells mediate alloreactivity in response to loss of specific HLA allotypes from target cells: thus, in theory, HLA loss relapses should represent excellent targets for donor NK cell recognition. Here we investigated the dynamics of NK cells in this unique immunogenetic context, to understand the biological bases of their failure in preventing the emergence of HLA loss variants. Methods We took into consideration 23 patients who after T cell replete haploidentical HSCT experienced HLA loss relapses. NK cell alloreactivity was predicted according to the Perugia algorithm (Ruggeri, Science, 1999). Killer Cell Immunoglobulin-like Receptor (KIR) typing was performed using a commercially-available kit, and KIR B-content estimated using the EMBL-EBI calculator. The phenotypic features of peripheral blood NK cells were assessed by multiparametric flow cytometry, and high dimensional single-cell analysis was performed using the viSNE bioinformatic tool. Results Based on donor-recipient HLA typing, at the time of HSCT NK cell alloreactivity in the graft-versus-leukemia direction was predicted in 10/23 patients who experienced HLA loss relapses (43.5%). In 7/23 additional patients (30.4%), conditions for predicted NK cell alloreactivity were fulfilled at time of relapse, upon genomic loss of the mismatched HLA haplotype from AML blasts. In all cases KIR genotyping confirmed the presence in the donor repertoire of the necessary KIR genes. Only 3/17 HSC donors were homozygous for KIR A haplotypes, encoding preferentially inhibitory KIR genes, and most carried equal or higher numbers of activating KIR genes than expected (Cooley, Blood, 2010). Thus, the absence of NK cell-mediated control of HLA loss variants can not be explained by an unfavorable immunogenetic asset of HSC donors. Therefore we characterized the phenotypic features of NK cells circulating in the peripheral blood of 7 patients at the time of HLA loss relapse (median time after HSCT 307 days, range 147-703), and compared them to their counterparts in healthy individuals (n=6), and matched-paired transplanted patients in remission (n=6), or at the time of non HLA loss ("classical") relapse (n=7). We analyzed a total of 27 markers involved in NK cell target recognition (KIRs, NKG2A, NKG2C, SIGLEC7, SIGLEC9), activation (NKp30, NKp44, NKp46, NKG2D, 2B4, DNAM1), maturation (CD57, CD16, CD62L), and exhaustion (PD1, TIM3, KLRG1). At the time of HLA loss relapse, NK cells had recovered a mature phenotype, although with a slightly higher frequency of CD56bright cells. In all cases in which NK cell alloreactivity had been predicted we detected the single-KIR+ NK cells of interest, without significant differences between patients and controls. However NK cells from transplanted patients expressed lower levels of the SIGLEC9 (p Conclusions Even at late timepoints after partially-incompatible allo-HSCT, when HLA loss relapses typically occur, the reconstituted NK cell repertoire displays profound differences from its counterpart in healthy subjects, hinting for defective immunosurveillance. Therapeutic protocols employing freshly isolated mature donor NK cells should thus be further investigated for the prevention and treatment of HLA loss relapses. Figure 1. The same viSNE map, obtained by analysis of the entire dataset, is differentially colored to evidence the spatial distribution, and thus phenotypic similarity, of NK cells from each cohort (upper row) or the intensity of expression of the indicated markers (lower row). Figure 1. The same viSNE map, obtained by analysis of the entire dataset, is differentially colored to evidence the spatial distribution, and thus phenotypic similarity, of NK cells from each cohort (upper row) or the intensity of expression of the indicated markers (lower row). Disclosures No relevant conflicts of interest to declare.

To the editor: Increasing evidence suggests that donor-recipient disparities for human leukocyte antigen (HLA)–DPB1 can be of clinical importance in unrelated hematopoietic stem cell transplantation (HSCT).[1][1] Two overlapping algorithms for functional T-cell epitope (TCE) matching involving 3

In the developed world, the survival and quality of life of patients with beta thalassemia (Bthal) has dramatically improved with optimization of blood transfusions and iron chelation. By contrast, in countries with limited resources most affected children die before the age of 20 because of the unavailability of safe blood products, expensive iron chelating drugs and inadequate management of co-morbidities. For these patients allogeneic stem cell transplantation (SCT) from matched donors offers a cure with low morbidity and mortality. Between June 2005 and May 2008, 47 consecutive Bthal patients underwent SCT from an HLA identical sibling in our center, among these, 3 patients underwent 2 SCTs. Median age was 8 years (2–15), country of origin: Lebanon (9), Iraq (19), Palestine (3), Syria (16). One pt was classified as Lucarelli class I, 24 as class II and 22 as class III. Most patients had severe iron overload evidenced by irregular iron chelation (83%), median ferritin 2973 (956–14280), median liver iron concentration 22 mg Fe/g (6.9–95.7). Most patients had liver toxicity due to iron overload and hepatitis evidenced by median ALT 71 (12–545), AST 59 (18–371), liver fibrosis Ishak 3 (0–5), HepC pos 16/47 (34%). Patients had inadequate transfusional management evidence by a median pre-transfusion Hb of 8 g/dL and anti HLA antibodies in 81% pts. Class I-II patients were conditioned with a regimen based on iv busulphan (iv Bu, Busilvex®, dosage according to weight, adjusted from the 5th dose to a target AUC 1200 umol/min) and cyclophosphamide (Cy) 200mg/kg (n19) with the addition of thiotepa (TT 10mg/kg) if

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 = 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. (C) 2015 American Society for Blood and Marrow Transplantation. OI Maiers, Martin/0000-0002-0198-2064