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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.

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.

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

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