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The challenge of eradicating leukemia in patients with acute myelogenous leukemia (AML) after initial cytoreduction has motivated modern efforts to combine synergistic active modalities including immunotherapy. Recently, the ETCTN/CTEP 10026 study tested the combination of the DNA methyltransferase inhibitor decitabine together with the immune checkpoint inhibitor ipilimumab for AML/myelodysplastic syndrome (MDS) either after allogeneic hematopoietic stem cell transplantation (HSCT) or in the HSCT-naïve setting. Integrative transcriptome-based analysis of 304 961 individual marrow-infiltrating cells for 18 of 48 subjects treated on study revealed the strong association of response with a high baseline ratio of T to AML cells. Clinical responses were predominantly driven by decitabine-induced cytoreduction. Evidence of immune activation was only apparent after ipilimumab exposure, which altered CD4+ T-cell gene expression, in line with ongoing T-cell differentiation and increased frequency of marrow-infiltrating regulatory T cells. For post-HSCT samples, relapse could be attributed to insufficient clearing of malignant clones in progenitor cell populations. In contrast to AML/MDS bone marrow, the transcriptomes of leukemia cutis samples from patients with durable remission after ipilimumab monotherapy showed evidence of increased infiltration with antigen-experienced resident memory T cells and higher expression of CTLA-4 and FOXP3. Altogether, activity of combined decitabine and ipilimumab is impacted by cellular expression states within the microenvironmental niche of leukemic cells. The inadequate elimination of leukemic progenitors mandates urgent development of novel approaches for targeting these cell populations to generate long-lasting responses. This trial was registered at www.clinicaltrials.gov as #NCT02890329.

Patient responses to autologous CD19 chimeric antigen receptor (CAR) T-cell therapies are limited by insufficient and inconsistent cellular functionality. Here, we show that controlling the precise level of stimulation during T-cell activation to accommodate individual differences in the donor cells will dictate the functional attributes of CAR-T cell products. The functionality of CAR-T cell products, consisting of a diverse set of blood samples derived from healthy donors, acute lymphoblastic leukemia (ALL), and chronic lymphocytic lymphoma (CLL) patient samples, representing a range of patient health status, is tested upon culturing on artificial antigen-presenting cell scaffolds to deliver T-cell stimulatory ligands (anti-CD3/anti-CD28) at highly defined densities. A clear relationship is observed between the dose of stimulation, the phenotype of the T-cell blood sample prior to T-cell activation, and the functionality of the resulting CAR-T cell products. We present a model, based on this dataset, that predicts the precise stimulation needed to manufacture a desired CAR-T cell product, given the input T-cell attributes in the initial blood sample. These findings demonstrate a simple approach to enhance CAR-T functionality by personalizing the level of stimulation during T-cell activation to enable flexible manufacturing of more consistent and potent CAR-T cells.

Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses1–3; however, the relationship between phenotypic characteristics and TCR properties is not well elucidated. Here we show, by linking the antigenic specificity of TCRs and the cellular phenotype of melanoma-infiltrating lymphocytes at single-cell resolution, that tumour specificity shapes the expression state of intratumoural CD8+ T cells. Non-tumour-reactive T cells were enriched for viral specificities and exhibited a non-exhausted memory phenotype, whereas melanoma-reactive lymphocytes predominantly displayed an exhausted state that encompassed diverse levels of differentiation but rarely acquired memory properties. These exhausted phenotypes were observed both among clonotypes specific for public overexpressed melanoma antigens (shared across different tumours) or personal neoantigens (specific for each tumour). The recognition of such tumour antigens was provided by TCRs with avidities inversely related to the abundance of cognate targets in melanoma cells and proportional to the binding affinity of peptide–human leukocyte antigen (HLA) complexes. The persistence of TCR clonotypes in peripheral blood was negatively affected by the level of intratumoural exhaustion, and increased in patients with a poor response to immune checkpoint blockade, consistent with chronic stimulation mediated by residual tumour antigens. By revealing how the quality and quantity of tumour antigens drive the features of T cell responses within the tumour microenvironment, we gain insights into the properties of the anti-melanoma TCR repertoire. The authors use single-cell profiling and T cell receptor specificity screening to show how tumour antigen recognition shapes the phenotypes of CD8+ T cells and antitumour immune responses.

The year 2020 marked the 30th anniversary of the Nobel Prize in Medicine awarded to E. Donnall Thomas for the development of allogeneic hematopoietic stem cell transplantation (allo-HSCT) to treat hematologic malignancies and other blood disorders. Dr. Thomas, “father of bone marrow transplantation,” first developed and reported this technique in 1957, and in the ensuing decades, this seminal study has impacted fundamental work in hematology and cancer research, including advances in hematopoiesis, stem cell biology, tumor immunology, and T-cell biology. As the first example of cancer immunotherapy, understanding the mechanisms of antitumor biology associated with allo-HSCT has given rise to many of the principles used today in the development and implementation of novel transformative immunotherapies. Here we review the historical basis underpinning the development of allo-HSCT as well as advances in knowledge obtained by defining mechanisms of allo-HSCT activity. We review how these principles have been translated to novel immunotherapies currently utilized in clinical practice and describe potential future applications for allo-HSCT in cancer research and development of novel therapeutic strategies.

Relapsed myeloid disease after allogeneic stem cell transplantation (HSCT) remains largely incurable. We previously demonstrated the potent activity of immune checkpoint blockade in this clinical setting with ipilimumab or nivolumab. To define the molecular and cellular pathways by which CTLA-4 blockade with ipilimumab can reinvigorate an effective graft-versus-leukemia (GVL) response, we integrated transcriptomic analysis of leukemic biopsies with immunophenotypic profiling of matched peripheral blood samples collected from patients treated with ipilimumab following HSCT on the Experimental Therapeutics Clinical Trials Network 9204 trial. Response to ipilimumab was associated with transcriptomic evidence of increased local CD8+ T-cell infiltration and activation. Systemically, ipilimumab decreased naïve and increased memory T-cell populations and increased expression of markers of T-cell activation and costimulation such as PD-1, HLA-DR, and ICOS, irrespective of response. However, responding patients were characterized by higher turnover of T-cell receptor sequences in peripheral blood and showed increased expression of proinflammatory chemokines in plasma that was further amplified by ipilimumab. Altogether, these data highlight the compositional T-cell shifts and inflammatory pathways induced by ipilimumab both locally and systemically that associate with successful GVL outcomes. This trial was registered at www.clinicaltrials.gov as #NCT01822509.

Within the tumour microenvironment, CD4(+) T cells can promote or suppress antitumour responses through the recognition of antigens presented by human leukocyte antigen (HLA) class II molecules(1,2), but how cancers co-opt these physiologic processes to achieve immune evasion remains incompletely understood. Here we performed in-depth analysis of the phenotype and tumour specificity of CD4(+) T cells infiltrating human melanoma specimens, finding that exhausted cytotoxic CD4(+) T cells could be directly induced by melanoma cells through recognition of HLA class II-restricted neoantigens, and also HLA class I-restricted tumour-associated antigens. CD4(+) T regulatory (T(Reg)) cells could be indirectly elicited through presentation of tumour antigens via antigen-presenting cells. Notably, numerous tumour-reactive CD4(+) T(Reg) clones were stimulated directly by HLA class II-positive melanoma and demonstrated specificity for melanoma neoantigens. This phenomenon was observed in the presence of an extremely high tumour neoantigen load, which we confirmed to be associated with HLA class II positivity through the analysis of 116 melanoma specimens. Our data reveal the landscape of infiltrating CD4(+) T cells in melanoma and point to the presentation of HLA class II-restricted neoantigens and direct engagement of immunosuppressive CD4(+) T(Reg) cells as a mechanism of immune evasion that is favoured in HLA class II-positive melanoma.

Neoantigens are prime targets for cancer immunotherapy, but their identification in low mutational burden malignancies remains challenging. In this issue of Immunity, Ehx et al. show that atypical transcripts, and particularly retained introns, expand the spectrum of leukemia immunotherapy targets.

Objective Memory stem T (Tscm) cells are long-lived, self-renewing T cells that play a relevant role in immunologic memory. This study was undertaken to investigate whether Tscm cells accumulate in rheumatoid arthritis (RA). Methods The polarization and differentiation profiles of circulating T cells were assessed by flow cytometry. Antigen-specific T cells were characterized by staining with major histocompatibility complex class II tetramers. The T cell receptor (TCR) repertoire was analyzed by high-throughput sequencing using an unbiased RNA-based approach in CD4+ T cell subpopulations sorted by fluorescence-activated cell sorting. Results We analyzed the dynamics of circulating Tscm cells (identified as CD45RA+CD62L+CD95+ T cells) by flow cytometry in 27 RA patients, 16 of whom were also studied during treatment with the anti-tumor necrosis factor (anti-TNF) agent etanercept. Age-matched healthy donors were used as controls. CD4+ Tscm cells were selectively and significantly expanded in RA patients in terms of frequency and absolute numbers, and significantly contracted upon anti-TNF treatment. Expanded CD4+ Tscm cells displayed a prevalent Th17 phenotype and a skewed TCR repertoire in RA patients, with the 10 most abundant clones representing up to 53.7% of the detected sequences. CD4+ lymphocytes specific for a citrullinated vimentin (Cit-vimentin) epitope were expanded in RA patients with active disease. Tscm cells accounted for a large fraction of Cit-vimentin-specific CD4+ cells. Conclusion Our results indicate that Tscm cells, including expanded clones specific for relevant autoantigens, accumulate in RA patients not exposed to biologic agents, and might be involved in the natural history of the disease. Further analysis of Tscm cell dynamics in autoimmune disorders may have implications for the design and efficacy assessment of innovative therapies.

Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses

Relapsed/refractory Peripheral T-cell Lymphomas are characterized by a poor prognosis, especially for patients who are not candidates for allogeneic hematopoietic stem-cell transplantation. We conducted a retrospective analysis on 73 consecutive patients affected by relapsed/refractory T-Cell lymphomas who were considered eligible for allogeneic transplant. All patients were referred at our center from 2001 to 2017. With a median follow-up of 40 months (range 9–192 months), 4-year second-line failure-free survival and overall survival were 14% (CI95%:7–24) and 34% (CI95%:22–46). Extranodal disease at relapse (HR 2.25, CI95%: 1.11–4.56, p = 0.02) and first-line failure-free survival

Reconstitution of donor hematopoiesis after allogeneic hematopoietic stem cell transplantation (HSCT) forms the basis for effective graft-versus-leukemia responses, but mixed chimerism is not an infrequent outcome. How the donor and host hematopoietic system interact under conditions of mixed chimerism remains incompletely understood. Multi-modal single cell sequencing platforms are increasingly available and provide information regarding cell identities and interactions at high resolution. However, the analysis of post-transplant immune reconstitution requires consistent distinguishing of donor- and recipient-derived cells, which for sparse single cell sequencing data until now has remained a challenge. Recently, mitochondrial DNA (mtDNA) mutations have been recognized for their potential as personal genetic barcodes that can be detected with mitochondrial single cell assay for transposase-accessible chromatin with sequencing (mtscATAC-seq). We hypothesized that individual-specific mtDNA mutations could provide a sensitive and robust approach for distinguishing donor- from recipient-derived cells, and therefore tested this approach on bone marrow (BM) samples from patients with relapsed acute myeloid leukemia (AML) post-HSCT. We employed ATAC with select cell surface antigen profiling by sequencing (ASAP-seq), which enables the detection of mtDNA mutations within distinct surface marker-defined cell populations alongside chromatin accessibility. We selected serial samples collected from the ETCTN 10026 study, which tested combined decitabine (days 1-5, every 4 weeks, start cycle 0) and ipilimumab (day 1, every 4 weeks, start cycle 1) in relapsed AML post-HSCT. We focused on 13 samples (study entry, on treatment and disease progression) from 3 patients: AML1012 (HSCT from a matched related donor [MRD]), and AML1010 and AML1026 (matched unrelated donor [MUD]-HSCT). In total, we obtained 33,943 ASAP-seq profiles, including 3,283 single T cells. While clustering using single cell chromatin profiles alone only allowed identification of either CD4 + or CD8 + T cells, integration with surface marker expression enabled more detailed annotations of 8 T cell subpopulations and NK cells. Further, phenotypically distinct subpopulations such as CD57 + CD4 + and CD8 + T cells shared highly similar chromatin profiles, and 11.1% of CD4 + and 33.7% of CD8 + T cells would have been mislabeled based on clustering of chromatin profiles alone. Thus, ASAP-seq identified T cell subsets with markedly improved accuracy and resolution than scATAC-seq alone. Upon evaluation of mtDNA mutations to discriminate donor- and recipient-derived single T cells, we found that this was unreliable for MRD-HSCT (AML1012), but highly robust in the setting of MUD-HSCT (AML1010, AML1026), consistent with maternal inheritance of mitochondrial genomes. For the latter two patients, we identified 48 donor- and 26 recipient-specific mtDNA mutations, all with high heteroplasmy (range 82 - 99%). Presence of donor- and recipient-derived mtDNA mutations was mutually exclusive, and recipient-specific mtDNA mutations were also detectable in AML cells. Clinical bulk and mtDNA mutation-based single T cell chimerisms were highly correlated (r = 0.97). AML1010 had sustained complete T cell chimerism (>97%) during study treatment. In AML1026, the mtDNA mutation-based T cell chimerism rose from 55% to 71% after 1 cycle of decitabine and then remained stable until disease progression 3 months later. This was associated with increased percentage of donor-derived CD4 + T cells (45% [study entry] vs. 71% [after 1 cycle of decitabine], p < 0.01), while donor-derived CD8 + T cells remained unchanged at 76%. Across all studied timepoints in AML1026, donor versus recipient skewing was also highest in CD4 + T cell subsets, with fewer naïve (20% vs. 31%, p < 0.01) but more donor-derived CD57 + CD4 + T cells (13% vs. 3%, p < 0.01). We demonstrate that mtDNA mutations can discriminate between donor- and recipient-derived single cells, enabling detection and in-depth characterization of chimeric immune cell dynamics after MUD HSCT. This approach will allow to systematically dissect conditions of mixed chimerism in the post-transplant setting with larger studies. Disclosures DeAngelo: Abbvie: Research Funding; Takeda: Consultancy; Servier: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding; Jazz: Consultancy; Incyte: Consultancy; Forty-Seven: Consultancy; Autolus: Consultancy; Amgen: Consultancy; Agios: Consultancy; Blueprint: Research Funding; Glycomimetrics: Research Funding. Neuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Sankaran: Cellarity: Consultancy; Forma: Consultancy; Novartis: Consultancy; Branch Biosciences: Consultancy; Ensoma: Consultancy. Soiffer: Jasper: Consultancy; Jazz Pharmaceuticals, USA: Consultancy; Precision Biosciences, USA: Consultancy; Juno Therapeutics, USA: Other: Data Safety Monitoring Board; Kiadis, Netherlands: Membership on an entity's Board of Directors or advisory committees; Rheos Therapeutics, USA: Consultancy; Gilead, USA: Other: Career Development Award Committee; NMPD - Be the Match, USA: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy. Garcia: Genentech: Research Funding; Prelude: Research Funding; Pfizer: Research Funding; AstraZeneca: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Wu: Pharmacyclics: Research Funding; BioNTech: Current equity holder in publicly-traded company. OffLabel Disclosure: ipilimumab to modulate anti-leukemia immunity in the post-transplant and transplant-naive context

BackgroundWithin the tumor microenvironment, distinct CD4+ T cell subsets can play different and even opposite roles either promoting or suppressing anti-tumor responses through the recognition of antigens presented by human leukocyte antigen (HLA) class II molecules. However, how cancers co-opt these processes to shape the intratumoral CD4+ landscape and achieve immune evasion remains incompletely understood.MethodsWe performed single-cell characterization of CD4+ tumor infiltrating lymphocytes (TILs) collected from four human melanoma with low or high HLA-class II expression and we utilized TCR reconstruction and antigen specificity screening to unambiguously discover the tumor reactivity of CD4+ TILs. By testing TCR-transduced T cells against autologous patient-derived melanoma cell lines or against autologous antigen presenting cells (APCs) loaded with tumor lysates, we assessed the capacity of CD4+ TCRs to directly or indirectly recognize tumor cells. We defined the antigen-specificity of antitumor CD4+ TCRs by assessing their reactivity towards personal neoantigens (NeoAg) or public melanoma associated antigens (MAAs). Finally, we correlated NeoAg burden and HLA-class II expression in a series of 116 melanoma specimens from 4 independent cohorts of patients.ResultsAnalysis of single-cell data showed that the cluster distribution of cells within each CD4+ TCR clonotype family was highly homogeneous and appeared to follow 3 distinct major phenotypes, corresponding to non-exhausted memory cells, exhausted cells and regulatory cells (TRegs). Strikingly, clonally expanded CD4+ TReg-TILs were highly abundant within the tumor microenvironment of HLA class IIpos melanomas. We found that TCRs from exhausted cytotoxic CD4+ T cells could be directly triggered by melanoma cells not only through recognition of HLA class II restricted antigens, but also through presentation of HLA class I restricted MAAs. TReg-TCRs could be indirectly elicited through presentation of tumor antigens via APCs. Notably, numerous tumor-reactive CD4+ TReg-TCRs were directly stimulated by HLA class IIpos melanoma and demonstrated specificity for melanoma NeoAgs. In HLA class IIpos melanomas, the clonal expansion of numerous tumor-reactive and NeoAg-specific TRegs-clones appeared to be favored by a dramatically high tumor NeoAg load. Analysis of 116 melanoma specimens confirmed the association of elevated HLA-class II expression with extremely high NeoAg burden.ConclusionsOur data elucidate the landscape of infiltrating CD4+ T cells in melanoma and point to presentation of HLA-class II restricted NeoAgs and direct engagement of immunosuppressive CD4+ TRegs as a novel mechanism of immune evasion favored in HLA class IIpos melanoma.

Summary To elucidate mechanisms by which T cells eliminate leukemia, we study donor lymphocyte infusion (DLI), an established immunotherapy for relapsed leukemia. We model T cell dynamics by integrating longitudinal, multimodal data from 94,517 bone marrow-derived single T cell transcriptomes in addition to chromatin accessibility and single T cell receptor sequencing from patients undergoing DLI. We find that responsive tumors are defined by enrichment of late-differentiated T cells before DLI and rapid, durable expansion of early differentiated T cells after treatment, highly similar to “terminal” and “precursor” exhausted subsets, respectively. Resistance, in contrast, is defined by heterogeneous T cell dysfunction. Surprisingly, early differentiated T cells in responders mainly originate from pre-existing and novel clonotypes recruited to the leukemic microenvironment, rather than the infusion. Our work provides a paradigm for analyzing longitudinal single-cell profiling of scenarios beyond adoptive cell therapy and introduces Symphony, a Bayesian approach to infer regulatory circuitry underlying T cell subsets, with broad relevance to exhaustion antagonists across cancers.

Relapse after allogeneic hematopoietic stem cell transplantation (HSCT) remains an unmet medical need. The ETCTN 9204 study evaluated in 71 study subjects if immune checkpoint blockade with anti-CTLA-4 (Ipilimumab (Ipi), n = 43) or anti-PD-1 (Nivolumab (Nivo), n = 28) antibodies could stimulate graft-versus-leukemia (GvL) responses in this setting. We focused mainly on patients (pts) with relapsed AML/MDS, which constituted the majority of pts (n = 38; 54%). Ipi induced both long-term complete remissions (CR; n = 3) and transient CRs (TR; n = 3), while Nivo did not generate any CRs, but 4 patients demonstrated partial remissions (PR). To define the molecular features associated with response to Ipi, we performed bulk transcriptomic analyses of formalin-fixed paraffin-embedded biopsies of sites of AML/MDS involvement (n = 35) collected before and after Ipi treatment from 13 pts. Our analysis of matched pre- and post-Ipi samples of patients with CRs identified 50 differentially expressed genes. By gene ontology analysis, these were enriched for signatures of 'lymphocyte activation' and 'antigen-receptor signaling'. Principal component analysis using these genes separated post-Ipi CR samples as a distinct cluster, transcriptionally apart from pre-Ipi CR samples and from non-responder (NR, n = 15) pre and post-Ipi samples. Of note, post-Ipi CR samples were similar to both pre- and post-Ipi TR samples as well as samples from GvHD/toxicity biopsies (n = 9). Consistent with these findings, we detected increased CD8+ T cell abundance post-Ipi in CR but not NR samples with CIBERSORTx (pre vs post, median score 0.043 vs. 0.56, p < 0.01). Likewise, reconstruction of T cell receptor (TCR) CDR3 sequences using the tool TRUST showed an increase in TCR clonotypes per million reads post-Ipi in CR samples (pre vs post, median 0.33 vs. 1.65, p < 0.05). In sum, response to CTLA-4 blockade is characterized by transcriptional evidence of T cell infiltration and activation within the tumor microenvironment, with similar gene programs observed in the setting of GvHD. To determine if the changes within tissue sites following Ipi are also observed in peripheral blood (PB), we analyzed the immunophenotype and TCR repertoire of T cells from serially collected PB samples from pts with AML/MDS (n = 14) or non-myeloid malignancy (n = 6). In responders and non-responders, exposure to Ipi was associated with decreased naïve, increased effector memory, and increased expression of HLA-DR on central memory T cells (p < 0.05), consistent with T cell differentiation and activation. From 9 of 14 AML/MDS pts (CR = 4, NR = 5), bulk TCR sequencing before and after 1 cycle of Ipi yielded 572,017 PB TCR sequences. Only 776 clonotypes demonstrated significant change in frequency, and these TCRs were detected in greater proportion among responders (613/776 versus NR 163/776, p < 0.01). Thus, Ipi alters the differentiation states of circulating T cells irrespective of response and leads to higher TCR repertoire turnover in CR patients. Of the AML patients achieving PR following Nivo, we also observed transcriptional evidence of CD8+ T cell infiltration in one patient. This signature, however, was not detected in a second such patient. In a separate instance, we had the opportunity to dissect the molecular features of a partial response in a patient with JAK2V617F+ secondary AML following Nivo through single-cell RNA and ATAC-sequencing (scRNA-seq and scATAC-seq, 10x Genomics) of PB mononuclear cells collected serially across 6 timepoints. In total, we analyzed the transcriptomes and chromatin accessibility data from of 27,777 and 28,713 individual cells, respectively. At time of response, non-exhausted CD4+ T cells expanded while both exhausted CD8+ T cells and a malignant subpopulation with increased expression of PD-L1 and features of megakaryocytic differentiation preferentially contracted. The subsequent expansion of a PD-L1- malignant population at progression suggests that decreased leukemic PD-L1 expression was associated with relapse. Altogether, these data highlight the molecular and cellular features of successful reinvigoration of GvL using CTLA-4 blockade, from increased local T cell infiltration and activation in the leukemic microenvironment to peripheral T cell turnover. In addition, the selection of therapy-resistant subclones after PD-1 blockade underscores the need for further high-resolution studies of GvL responses. Disclosures Zeiser: Novartis: Honoraria; Incyte: Honoraria; Malinckrodt: Honoraria. Ritz:Rheos Medicines: Consultancy; LifeVault Bio: Consultancy; Infinity Pharmaceuticals: Consultancy; Falcon Therapeutics: Consultancy; Avrobio: Consultancy; Kite Pharma: Research Funding; Talaris Therapeutics: Consultancy; Equillium: Research Funding; Amgen: Research Funding; TScan Therapeutics: Consultancy. Neuberg:Madrigak Pharmaceuticals: Current equity holder in publicly-traded company; Celgene: Research Funding; Pharmacyclics: Research Funding. Soiffer:alexion: Consultancy; Gilead: Consultancy; Be the Match/ National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Rheos Therapeutics: Consultancy; Juno: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Mana Therapeutics: Consultancy; Precision Bioscience: Consultancy; VOR Biopharma: Consultancy; Kiadis: Membership on an entity's Board of Directors or advisory committees; Cugene: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees. Liu:GV20 Oncotherapy: Consultancy, Membership on an entity's Board of Directors or advisory committees; 3DMed Care: Consultancy; Sanofi: Research Funding; Takeda: Research Funding. Davids:AbbVie: Consultancy; Gilead Sciences: Consultancy; Sunesis: Consultancy; BeiGene: Consultancy; AstraZeneca: Consultancy, Research Funding; Syros Pharmaceuticals: Consultancy; Research to Practice: Honoraria; Pharmacyclics: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; Janssen: Consultancy; Surface Oncology: Research Funding; Novartis: Consultancy, Research Funding; MEI Pharma: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Eli Lilly: Consultancy; Zentalis: Consultancy; Celgene: Consultancy; Bristol Myers Squibb: Research Funding; Merck: Consultancy; Ascentage Pharma: Consultancy, Research Funding. Wu:Pharmacyclics: Research Funding; BionTech: Current equity holder in publicly-traded company. OffLabel Disclosure: Ipilimumab and Nivolumab were tested in the setting of relapsed hematological malignancies after allogeneic stem 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-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 major cause of death after allogeneic Hematopoietic Stem Cell Transplantation (HSCT) for acute myeloid leukemia (AML) is disease relapse. We investigated the expression of Inhibitory Receptors (IR; PD-1/CTLA-4/TIM-3/LAG-3/2B4/KLRG1/GITR) on T cells infiltrating the bone marrow (BM) of 32 AML patients relapsing (median 251 days) or maintaining complete remission (CR; median 1 year) after HSCT. A higher proportion of early-differentiated Memory Stem (TSCM) and Central Memory BM-T cells express multiple IR in relapsing patients than in CR patients. Exhausted BM-T cells at relapse display a restricted TCR repertoire, impaired effector functions and leukemia-reactive specificities. In 57 patients, early detection of severely exhausted (PD-1+Eomes+T-bet−) BM-TSCM predicts relapse. Accordingly, leukemia-specific T cells in patients prone to relapse display exhaustion markers, absent in patients maintaining long-term CR. These results highlight a wide, though reversible, immunological dysfunction in the BM of AML patients relapsing after HSCT and suggest new therapeutic opportunities for the disease., Allogeneic hematopoietic cell transplantation is the standard treatment of acute myeloid leukemia, but many patients relapse. Here the authors show increased markers of exhaustion and cancer antigen specificity within bone marrow-residing memory T cells precede and potentially predict the relapse.

Purpose of review The most substantial advancement in the treatment of indolent B-cell non-Hodgkin lymphoma (NHL), since the advent of combination chemotherapy, has been the introduction of the monoclonal anti-CD20 antibody rituximab. However, the optimal schedule, timing, and duration of rituximab therapy remain controversial. Recent findings Since its initially reported single-agent activity in 1997, the role of rituximab has greatly expanded and it is now ubiquitously integrated in all treatment phases of indolent NHL. Yet, several questions remain to be addressed: should asymptomatic patients be treated at diagnosis with single-agent rituximab or still kept in watchful waiting, what are the optimal first-line treatments to combine with rituximab, what is the role of maintenance therapy, and is there a benefit in incorporating rituximab in autologous and allogeneic stem cell transplantation schemes for these diseases? Recent and ongoing clinical trials tackling these relevant issues will be presented and critically discussed in this article. Summary Excellent outcomes are reported with rituximab therapy in indolent NHL, both early and late in the disease course. Continued study of this most valuable therapeutic agent is warranted to set the optimal treatment approach leading to cure the majority of patients.

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 effects of graft or donor characteristics in haploidentical hematopoietic cell transplantation (HCT) using post-transplant cyclophosphamide (PT-Cy) are largely unknown. In this multicenter retrospective study we analyzed the correlations between graft cell composition (CD34+, CD3+) and donor features on transplant outcomes in 234 patients who underwent HCT between 2010 and 2016. On multivariate analysis, the use of peripheral blood stem cells (PBSC) was associated with an increased incidence of grade 2–4 acute GVHD [HR 1.94, 95% confidence Interval (CI) = 1.01–3.98, p = 0.05]. An elevated CD3+ graft content was associated with an increased incidence of all-grade chronic GVHD [HR 1.36 (95% CI = 1.06–1.74), p = 0.01]. This effect was confirmed only for the PBSC graft group. A higher CD34+ graft content had a protective role on non-relapse mortality [HR 0.78 (95% CI = 0.62–0.96), p = 0.02] but this was confirmed only for the bone marrow (BM)-derived graft cohort. Donor characteristics did not influence any outcomes. GVHD prophylaxis should be modulated accordingly to CD3+ graft content, especially when a PBSC graft is used. These results need further validation in prospective trials.

Background. Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the only cure for high-risk acute myeloid leukemia (AML); nonetheless, relapse remains the major cause of death after such therapeutic option. Patients and Methods . We investigated the expression of Inhibitory Receptors (IR; i.e. PD-1, CTLA-4, TIM-3, LAG-3 and KLRG1) on different T-cell subsets infiltrating the bone marrow (BM) of 8 healthy donors (HD) and 32 allogeneic HSCT recipients diagnosed with Acute Myeloid Leukemia, collected at relapse (median 251 days) or at complete remission (CR) 1 year after HSCT. Inclusion criteria were: a diagnosis of acute myeloid leukemia or myelodysplastic syndrome, a relapse-free survival of at least 4 months after allogenein HSCT, absence of active GvHD, CMV infections or other complications at the time of sampling. Samples were analysed by multi-parametric flow cytometry for the expression of inhibitory receptors on T-cell subsets and the results were validated with BH-SNE, an unbiased dimensionality reduction algorithm. We exploited HLA-mimicking fluorescent molecules loaded with a specific epitope to screen anti-tumour and anti-viral T cells whereas the T-cell receptor repertoire was assessed by TRAC and TRBC RNA sequencing and the relative frequency of each T-cell receptor calculated. To evaluate T-cell function and specificity, CD107a expression, cytokine profiles and killing of autologous blasts were quantified. Results. After Haploidentical-HSCT PD-1, CTLA-4, 2B4 and Tim-3 were expressed at higher percentage when compared to HD, independently from the clinical outcome. In contrast, after HLA-matched HSCT, patients who relapsed displayed a higher frequency of BM-infiltrating T cells expressing PD-1, CTLA-4 and Tim-3 than CR pts (p To gain insights on the inhibited T-cell subpopulation identified in the BM of relapsing patients, we separately profiled the different T-cell memory subsets: in both HD and CR patients the IR expression was confined to effector memory and effectors whereas at relapse PD-1, 2B4, KLRG1 and Tim-3 were also expressed in BM-infiltrating central memory (TCM) and memory stem T cells (TSCM, p Interestingly, the TCR repertoire of BM-infiltrating T cells at relapse displayed a restricted clonality, suggesting that immune inhibitory signals are active on discrete and specific T-cell clones. To gain further insights on such clones, we assessed the IR expression profile on CD8 T cells specific for viral (CMV) and tumor-associated antigens (including peptides from WT1, EZH2 and PRAME). We observed a higher IR expression and co-expression on tumor-specific T cells when compared to viral-specific CD8 cells, particularly in case of patients who experienced post-transplant relapse. In accordance, IRpos sorted T cells harvested from relapsing patients showed a restricted TCR repertoire and, when challenged with autologous leukemic blasts, proved enriched in leukemic specificities as shown by higher expression of the activation marker HLA-DR (p Conclusion. These results highlight a wide, yet reversible, immunological dysfunction likely mediated by AML blasts in the BM of patients relapsing after allogeneic HSCT, that is particularly evident on memory T cells specific for tumor antigens. This suggest and open new therapeutic opportunities for AML. Figure. Figure. Disclosures Bondanza: Novartis: Employment. Vago:GENDX: Research Funding; Moderna TX: Research Funding. Bonini:Intellia Therapeutics: Research Funding.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only cure for several patients with high-risk hematological malignancies. Recent advances in supportive therapies have significantly reduced the treatment-related mortality over the last decades. Nevertheless graft-versus-host disease (GvHD) still represents a major complication, and research is needed to achieve further progress in this area of transplantation. Moreover, although initial studies suggested relative equivalence, recent and large prospective studies showed higher rates of acute and chronic GvHD, and worse survival in allo-HSCT from unrelated donors (URD) compared with HLA-identical sibling donors. Following our encouraging results in haplodentical setting with the more recently post-transplantation cyclophosphamide (PTCy) and sirolimus as GvHD prophylaxis (Cieri et al, 2015), in this study we investigated whether the incorporation of low dose anti-thymocyte globulin (ATG) to PTCy and sirolimus GvHD prophylactic approach, may alleviate some of the increased alloreactivity associated with URD transplants. We retrospectively evaluated a cohort of 21 haematological patients receiving URD allo-HSCT in the San Raffaele Haematology and BMT Unit, from 2013 to 2015. All subjects had high-risk hematologic malignancies, with the most common diagnosis being myeloid diseases (52%). A total of 11 patients had active disease at HSCT, while 9 were in hematologic remission. Nine patients scored high according to disease risk index defined by Armand et al, 10 scored intermediate, while only 2 had a low disease score. Myeloablative conditioning consisted of treosulfan (14 g/m2/day) on days -6 to -4, fludarabine (30 mg/m2/day) on days -6 to -2, and melphalan (70 mg/m2/day) on days -2 and -1, followed by T-replete G-CSF-mobilized PBSCs (n=19) or BM grafts (n=2). Whether 12 patients received stem cells from HLA- matched URD (fully 10/10 HLA matched), the remaining 9 had one-locus HLA-mismatched from their donors. During conditioning patients received ATG-Fresenius 5 mg/kg per day IV on day -4 to -2. In vivo B-cell depletion was obtained by rituximab (i.v. 375mg/m2 given as a single dose on day -1). Postgrafting immunosuppression consisted of PTCy (50 mg/kg/day) on days 3 and 4. Sirolimus was given orally from day 5, and monitored to maintain a target therapeutic plasma level of 5-14 ng/ml. According to chimeric status and evidence of GVHD, the dosage of sirolimus was tapered during the second month post-transplantation, ending in complete withdrawal within the sixth month after HSCT. Mycophenolate mofetil (MMF) was initiated orally at 10 mg/kg t.i.d. on day 5 after HSCT, and withdrawn on day 30. Median follow up was 247 days (range 29-486) from HSCT. All patients were engrafted in a median time of 17 days (range, 14-51 days), with full donor chimerism achieved by day 30. Poor graft function was observed in 11 patients. Post-HSCT recovery of lymphocyte subsets was lengthy as compared to other unmanipulated HSCT, with a median time to CD3>100/ml of 41 days (range, 22-389 days) by flow-cytometry, while 5 patients never reached immune reconstitution. All except one developed febrile neutropenia during aplasia. We observed a high rate of serious infections: severe pneumonia in 7 cases (5 required NIV, 4 were admitted to ICU), CMV reactivation in 13 (3 CMV disease), HHV6 reactivation in 10, BK virus haemorrhagic cystitis in 2 patients. Although the overall mortality associated with these complications was limited, as evidenced by a favourable NRM, it caused significant morbidity in patients and often required aggressive therapies, potentially contributing to poor graft function. The incidences of acute and chronic GVHD were low. A grade II-IV acute GvHD was reported in 10% patients, while five patients developed chronic GvHD (mild in 4 cases, only one moderate). Non-relapse mortality (NRM) at 100 days and 1 year was 4.8% and 20.7%, respectively. The estimated 1-year overall survival (OS) and relapse incidence were 51% and 22.5% respectively. Although limited by a small number of patients with relatively short follow-up, our study suggests that URD allo-HSCT with ATG, PTCy and sirolimus-based GvHD prophylaxis, can be very effective in preventing GvHD, but at the expense of delayed immune reconstitution and high rate of infectious complications, while warranting acceptable NRM and relapse incidence. Disclosures Bonini: MolMed S.p.A: Consultancy.

BACKGROUND: Long-term T-cell survival is pivotal for the development of effective therapeutic approaches against pathogens and cancer, since the success of immunotherapy requires the generation of a robust, safe but also durable immune response. Even if it is established that memory cells can survive and persist for years, little is known about the requirements for their long-term persistence. Suicide gene therapy after T-cell depleted haploidentical hematopoietic stem cell transplantation (haplo-HSCT) provides a unique model to study memory T cells. In this setting, patients receive the post-transplant infusion of donor-derived gene-modified memory T lymphocytes retrovirally transduced to express the Herpes Simples Virus Thymidine Kinase (TK) suicide gene and the DLNGFR selection marker. The presence of a safety switch allows the infusion into patients of a broad T-cell repertoire in the absence of immune suppression, while the surface marker enables unambiguous detection and close monitoring of gene-modified cells circulating in treated patients. In the present work we characterize the immunological profile of a cohort of long-term survivors after suicide gene therapy and we studied the fate of persisting TK cells to shed light on memory T cell dynamics in vivo and to unravel the requirements for long-term persistence directly in humans. RESULTS: We studied 10 adult patients who underwent haplo-HSCT and infusion of suicide-gene modified donor T cells (median dose: 1.9x107 cells/kg, range:1-39.5x106) for high-risk hematologic malignancies between 1995 and 2012. Three out of 10 patients (33%) experienced GvHD early after HSCT; in all cases, ganciclovir (GCV) administration proved effective in abrogating the adverse reaction. At a median follow-up of 7 years (range 2-14), all patients were in complete remission and free of GvHD, and displayed a complete and broad donor-derived immune system characterized by physiological counts of NK cells, B lymphocytes, γδ T cells and naïve and memory CD4+ or CD8+ T cells. TK cells were detected in all patients, at low levels (median=4cells/uL), even in patients treated with GCV. Ex vivo selection of pure TK-cells confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory T cells to persist for years. Importantly, GCV sensitivity was preserved in long-term persisting TK cells, independently from their differentiation phenotype. Longitudinal follow up revealed that TK cells circulated in patients at stable levels and displayed a conserved phenotype comprising effector memory (TEM), central memory (TCM) and stem memory (TSCM) T cells. The low level of Ki-67 positivity suggested the maintenance of a pool of gene-modified memory cells through homeostatic proliferation. Polyclonality was demonstrated by sequencing among TK cells of thousands of diverse TCRs with a broad usage of V and J alpha and beta genes. The number of TK cells persisting at the longest follow-up did not correlate with the amount of infused cells, but instead with the peak of TK cells measured within the first months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory T cells. Accordingly, we documented the persistence of CMV and Flu-specific TK cells only after post-transplant CMV reactivation or after Flu infection. Characterization of TK cell products infused to patients showed that the amount of infused TSCM cells positively correlates with early expansion and long-term persistence of gene-marked cells. By combining sorting of memory T-cell subsets with sequencing of integration sites, TCRα and TCRβ clonal markers, we longitudinally traced T-cell clones from infused products to late follow-up time-points. We showed that although T cells retrieved long-term are enriched in clones originally shared in different memory T-cell subsets, dominant long-term clonotypes preferentially originate from infused TSCM clones, suggesting that TSCM might play a privileged role in the generation of a long-lasting immunological memory. CONCLUSION: In a completely restored immune system, suicide gene-modified donor T cells persist for up to 14 years in treated patients. Long-term persistence of memory T cells is determined by antigen exposure, and by the original phenotype of infused cells, according to a hierarchical model in which TSCM are superior to TCM and TEM/EFF. Disclosures Lambiase: MolMed S.p.A: Employment. Traversari:MolMed S.p.A: Employment. Bordignon:MolMed S.p.A: Membership on an entity's Board of Directors or advisory committees. Bonini:MolMed S.p.A: Consultancy.

Haploidentical hematopoietic stem cell transplantation (HSCT) performed using bone marrow (BM) grafts and post-transplantation cyclophosphamide (PTCy) has gained much interest for the excellent toxicity profile after both reduced-intensity and myeloablative conditioning. We investigated, in a cohort of 40 high-risk hematological patients, the feasibility of peripheral blood stem cells grafts after a treosulfan-melphalan myeloablative conditioning, followed by a PTCy and sirolimus-based graft-versus-host disease (GVHD) prophylaxis (Sir-PTCy). Donor engraftment occurred in all patients, with full donor chimerism achieved by day 30. Post-HSCT recovery of lymphocyte subsets was broad and fast, with a median time to CD4 > 200/mu L of 41 days. Cumulative incidences of grade II to IV and III-IV acute GVHD were 15% and 7.5%, respectively, and were associated with a significant early increase in circulating regulatory T cells at day 15 after HSCT, with values < 5% being predictive of subsequent GVHD occurrence. The 1-year cumulative incidence of chronic GVHD was 20%. Nonrelapse mortality (NRM) at 100 days and 1 year were 12% and 17%, respectively. With a median follow-up for living patients of 15 months, the estimated 1-year overall and disease-free survival (DFS) was 56% and 48%, respectively. Outcomes were more favorable in patients who underwent transplantation in complete remission (1-year DFS 71%) versus patients who underwent transplantation with active disease (DFS, 34%; P = .01). Overall, myeloablative haploidentical HSCT with peripheral blood stem cells (PBSC) and Sir-PTCy is a feasible treatment option: the low rates of GVHD and NRM as well as the favorable immune reconstitution profile pave the way for a prospective comparative trial comparing BM and PBSC in this specific transplantation setting. (C) 2015 American Society for Blood and Marrow Transplantation.

Memory stem T cells (TSCM) have been proposed as key determinants of immunologic memory. However, their exact contribution to a mounting immune response, as well as the mechanisms and timing of their in vivo generation, are poorly understood. We longitudinally tracked TSCM dynamics in patients undergoing haploidentical hematopoietic stem cell transplantation (HSCT), thereby providing novel hints on the contribution of this subset to posttransplant immune reconstitution in humans. We found that donor-derived TSCM are highly enriched early after HSCT. We showed at the antigen-specific and clonal level that TSCM lymphocytes can differentiate directly from naive precursors infused within the graft and that the extent of TSCM generation might correlate with interleukin 7 serum levels. In vivo fate mapping through T-cell receptor sequencing allowed defining the in vivo differentiation landscapes of human naive T cells, supporting the notion that progenies of single naive cells embrace disparate fates in vivo and highlighting TSCM as relevant novel players in the diversification of immunological memory after allogeneic HSCT. Memory stem T cells (TSCM) have been proposed as key determinants of immunologic memory. However, their exact contribution to a mounting immune response, as well as the mechanisms and timing of their in vivo generation, are poorly understood. We longitudinally tracked TSCM dynamics in patients undergoing haploidentical hematopoietic stem cell transplantation (HSCT), thereby providing novel hints on the contribution of this subset to post transplant immune reconstitution in humans. We found that donor-derived TSCM are highly enriched early after HSCT. We showed at the antigen specific and clonal level that TSCM lymphocytes can differentiate directly from naïve precursors infused within the graft and that the extent of TSCM generation might correlate with interleukin 7 serum levels. In vivo fate mapping through T-cell receptor sequencing allowed defining the in vivo differentiation landscapes of human naïve T cells, supporting the notion that progenies of single naive cells embrace disparate fates in vivo and highlighting TSCM as relevant novel players in the diversification of immunological memory after allogeneic HSCT.

Hematopoietic SCT (HSCT) from HLA haploidentical family donors is a promising therapy for high-risk hematological malignancies. In the past 15 years at San Raffaele Scientific Institute, we investigated several transplant platforms and post transplant cellular-based interventions. We showed that T cell-depleted haploidentical transplantation followed by the infusion of genetically modified donor T cells (TK007 study, Eudract-2005-003587-34) promotes fast and wide immune reconstitution and GvHD control. This approach is currently tested in a phase III multicenter randomized trial (TK008 study, NCT00914628). We targeted patients with advanced leukemia with a sirolimus-based, calcineurin inhibitor-free prophylaxis of GvHD to allow the safe infusion of unmanipulated PBSCs from haploidentical family donors (TrRaMM study, Eudract 2007-5477-54). Results of these approaches are summarized and discussed. Hematopoietic SCT (HSCT) from HLA haploidentical family donors is a promising therapy for high-risk hematological malignancies. In the past 15 years at San Raffaele Scientific Institute, we investigated several transplant platforms and post transplant cellular based interventions. We showed that T cell-depleted haploidentical transplantation followed by the infusion of genetically modified donor T cells (TK007 study, Eudract-2005-003587-34) promotes fast and wide immune reconstitution and GvHD control. This approach is currently tested in a phase III multicenter randomized trial (TK008 study, NCT00914628). We targeted patients with advanced leukemia with a sirolimus-based, calcineurin inhibitor-free prophylaxis of GvHD to allow the safe infusion of unmanipulated PBSCs from haploidentical family donors (TrRaMM study, Eudract 2007-5477-54). Results of these approaches are summarized and discussed.

Hematopoietic stem cell transplantation from a healthy donor (allo-HSCT) represents the most potent form of cellular adoptive immunotherapy to treat malignancies. In allo-HSCT, donor T cells are double edge-swords: highly potent against residual tumor cells, but potentially highly toxic, and responsible for graft versus host disease (GVHD), a major clinical complication of transplantation. Gene transfer technologies coupled with current knowledge on cancer immunology have generated a wide range of approaches aimed at fostering the immunological response to cancer cells, while avoiding or controlling GVHD. In this review, we discuss cell and gene therapy approaches currently tested in preclinical models and in clinical trials.

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.

INTRODUCTION: Although allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) can accomplish apparent leukemia eradication in most patients, residual tumor cells can persist over time and eventually outgrow, resulting in clinical relapse. The genetic landscape of relapsing leukemia is often markedly different from its counterpart at diagnosis, due to clonal evolution (Ding et al, Nature, 2012) and selection of treatment-resistant variants (Vago et al, N Engl J Med, 2009). A potential solution to treat, or even prevent, relapse is to identify and specifically target mutations occurring very early in the leukemic transformation process, and thus putative hallmarks of cancer progenitors. Next-Generation Sequencing (NGS) provides the opportunity to track disease subclones during the clinical history of patients, pinpoint founder alterations and identify the mechanisms by which leukemia evades elimination. METHODS: In the present study, we combined immunogenetic analyses and NGS to detail the complex clinical history of a patient with therapy-related myelodysplasia (t-MDS), diagnosed years after sequential intensive chemotherapy for B cell Acute Lymphoblastic Leukemia (B-ALL). Leukemic cells collected at serial time-points during the patient disease history (initial diagnosis of B-ALL, first presentation of t-MDS, relapse after first allo-HSCT, relapse after second allo-HSCT) were characterized by means of genomic HLA typing, HLA allele-specific quantitative PCR and whole exome sequencing, with a minimum depth of coverage of 70x. Patient fibroblasts and peripheral blood mononuclear cells (PBMCs) collected before the occurrence of t-MDS, as well as PBMCs from the two allogeneic HSC donors, served as controls and reference exomes. Targeted resequencing of mutations of interest was performed using the Fluidigm Access Array system. Gene segments encompassing newly-identified mutations in TP53, NHEJ1 and BTNL8and their respective wild-type counterparts were cloned into plasmids, and used to design and validate specific droplet digital PCR assays, using the Bio-Rad QX100 instrument. RESULTS: A 54-year-old patient with high-risk t-MDS received two subsequent allo-HSCTs from partially-incompatible family donors, and after each transplant experienced disease recurrences. Genomic HLA typing and HLA allele-specific qPCR demonstrated that both relapses were due to mutant variants of the original leukemia that had evaded immune pressure through genomic loss of the HLA haplotype targeted by the respective donor’s T cells. Immunogenetic studies ruled out any linear relationship between the two relapses, suggesting that both might have derived from a common HLA-heterozygous progenitor. Accordingly, whole exome sequencing demonstrated direct clonal evolution from the initial presentation of t-MDS to first relapse, whereas the large majority of mutations present at second relapse were unique to the sample. Only five non-synonymous coding mutations were present in all three t-MDS samples, comprising disrupting mutations in TP53, in NHEJ1 (a key factor in DNA double strand-break repair), and in the T cell costimulatory receptor BTNL8. Of notice secondary mutations, detected only in one or two of the three disease presentations, were predicted to result in partially overlapping functional effects, and could be modeled in a conjoint DNA damage repair network, centered around the putative founder mutation in TP53. By ultra-sensitive droplet digital PCR, the same TP53 mutation was backtracked throughout the whole nine years of patient clinical history, including the phases of apparent complete remission, up to a preleukemic progenitor present in the patient bone marrow at the time of B-ALL initial diagnosis. CONCLUSIONS: Our results demonstrate that therapies, and the immune pressure of allo-HSCT in particular, can have a dramatic effect in shaping leukemia clonal evolution. Importantly, by identifying leukemia founder mutations, we might further our insights into leukemogenesis, and identify novel targets for post-transplantation diagnostics and leukemia-eradicating therapies. Disclosures Bonini: MolMed S.p.A.: Consultancy.

Long-living memory stem T cells (T-SCM) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here, we show that it is possible to differentiate in vitro, expand, and gene modify in clinically compliant conditions CD8(+) T-SCM lymphocytes starting from naive precursors. Requirements for the generation of this T-cell subset, described as CD62L(+)CCR7(+)CD45RA(+)CD45R0(+)IL-7R alpha(+)CD95(+), are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly, T-SCM accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T-lymphocyte subset, intermediate between naive and central memory cells. When transplanted in immunodeficient mice, gene-modified naive-derived T-SCM prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GVHD. Furthermore, gene-modified T-SCM are the only T-cell subset able to expand and mediate GVHD on serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for T-SCM generation and pave the way for their clinical rapid exploitation in adoptive cell therapy. (Blood. 2013; 121(4): 573-584) Long-living memory stem T cells (TSCM) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here we show that it is possible to differentiate in vitro, expand and gene modify in clinically compliant conditions CD8+ TSCM lymphocytes starting from naïve precursors. Requirements for the generation of this T-cell subset, described as CD62L+CCR7+CD45RA+CD45R0+IL-7Rα+CD95+, are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly TSCM accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T lymphocyte subset, intermediate between naïve and central memory cells. When transplanted in immunodeficient mice, gene-modified naïve-derived TSCM prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GvHD. Furthermore, gene-modified TSCM are the only T-cell subset able to expand and mediate GvHD upon serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for TSCM generation and pave the way for their clinical rapid exploitation in adoptive cell therapy. "Long-living memory stem T cells (T(SCM)) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here, we show that it is possible to differentiate in vitro, expand, and gene modify in clinically compliant conditions CD8(+) T(SCM) lymphocytes starting from naive precursors. Requirements for the generation of this T-cell subset, described as CD62L(+)CCR7(+)CD45RA(+)CD45R0(+)IL-7Rα(+)CD95(+), are CD3\/CD28 engagement and culture with IL-7 and IL-15. Accordingly, T(SCM) accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T-lymphocyte subset, intermediate between naive and central memory cells. When transplanted in immunodeficient mice, gene-modified naive-derived T(SCM) prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GVHD. Furthermore, gene-modified T(SCM) are the only T-cell subset able to expand and mediate GVHD on serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for T(SCM) generation and pave the way for their clinical rapid exploitation in adoptive cell therapy."

Background: Post transplant immune reconstitution plays a major role in determining the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT), and is currently monitored with different techniques in different Centers, with the aim of identifying clinically relevant immunological biomarkers. However, it is unclear which and how many of these immunological tests are currently performed on a routine basis, and which ones have the potential to predict patient outcome, and possibly guide patient care after allo-HSCT. Methods: The EBMT Cellular Therapy & Immunobiology Working Party (CTIWP) conducted a survey to identify current policies to monitor immune reconstitution in patients undergoing allo-HSCT and possibly reach a general consensus. This study followed the EBMT study guidelines. All EBMT Centers were invited to participate. Each participating Center received a questionnaire on the availability of specific immunomonitoring assays, specifying the use in clinical practice and/or within investigational trials. Assays were based on relatively simple and readily available parameters such as absolute lymphocyte counts (ALC) to more complex cellular and molecular tests. Moreover, the Centers were asked to define the transplant platform (HLA-identical sibling, matched unrelated donor, haploidentical and/or cord blood) on which each test is generally performed. Results: Policies for post-transplant immunomonitoring have been reported by 35 participating EBMT Centers active in 14 Countries and performing allo-HSCT from HLA identical related (35 centers), matched unrelated (33), haploidentical (34), unrelated cord blood (27). Complete blood counts and immunoglobulins are routinely tested for patients' care by all centers. Relative proportions of T cell subsets are currently tested by flow-cytometry as "standard of care" or "investigational" by 82% and 17% of centers respectively. B cell and NK cell counts are quantified routinely by 46% and 23% of Centers, and investigationally by 40% of Centers. The availability of molecular tests (STR, qPCR, Fish) to measure post-transplant engraftment are reported by all Centers, except two, as a standard of care measure. T cell receptor-expressing circles (TRECs) and/or K-deleting recombination excision circles (KRECs) are quantified within selected clinical trials by 37% of Centers. Interestingly, 60% of Centers evaluate, mostly as an investigational measure, antigen specific T cell responses by: proliferation assays (49%), interferon-gamma enzyme-linked immunospot-Elispot (49%), intracellular cytokine staining (46%) and tetramer/dextramer staining (37%). Most of these Centers test responses to Cytomegalovirus and Epstein Barr Virus, and 5 Centers use at least one of these assays on a routine basis. About half of the participating Centers (43%) commonly test antigen-specific antibodies, mainly as responses to vaccines, and not routinely. T-cell receptors (TCR) and B-cell receptors (BCR) repertoires are measured by spectratyping in 14 out of 35 Centers (4 as clinical practice and 10 in selected trials), or, in selected trials, by next generation sequencing (in 11 out of 35 the participating Centers). Conclusions: Results of this survey indicate that country- and center expertise are associated with heterogeneous and distinct protocols, and underline the clinical need to harmonize methods and to provide practical recommendations for monitoring post-transplant immune reconstitution, both for routine purposes and investigational studies. Adequate reporting and connection between individual Centers exploiting these data will foster collaborative and comparative research studies, with the ultimate goals of improving patient care and refining our understanding of the immunological correlates to clinical outcome. Acknowledgments: R. Ram, M. A. Diaz, G. McQuaker, D. Russo, E. Faber, P. Chiusolo, C. Rössig, S. M. Martin, A. Anagnostopoulos, M. Stelljes, K. Orchard, P. Jindra, A. Sampol, K. Patrick, M. A. Bekadja, J. Gayoso, A. Olivieri, J. Passweg, E. Jost, H Labussiere-Wallet, Y Koc, A. Lange, I. Garcia Cadenas, N. Kröger, A. Biondi, N. Milpied, D. Olive, E. Lanino, G. Stuhler, J.H. Dalle, J.R. Cabrera Marín, F. Ciceri, D. Uckan-Cetinkaya, R. Parody Porras, G. Kriván. Disclosures Ciceri: MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.

Background:Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the only cure for high-risk acute myeloid leukemia (AML). Unfortunately, relapse still remains the major cause of death after HSCT. We investigated if T-cell dysfunction is associated to post-transplant relapse. Patients and Methods: To this,we longitudinally analyzed the T-cell dynamics in bone marrow (BM) and peripheral blood (PB) of 32 AML patients receiving HSCT from HLA identical (HLAid, 20 pts) or HLA haploidentical (haplo, 12 pts) donors. Samples were analysed by multi-parametric flow cytometry to investigate the expression of inhibitory receptors (IRs) on CD4 and CD8 T-cell subsets defined by CD45RA, CD62L and CD95 expression, and to assess the proportion of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Results were also analyzed with the BH-SNE algorithm, an unbiased computational method for the analysis of FACS data. To evaluate T-cell effector functions, the CD107a degranulation assay was performed and the production of cytokines (IL-2, IFNg and TNFa) was measured by intracellular staining. BM and PB were collected 60 days after HSCT and at relapse (median 237 days; 16 pts) or, when complete remission was maintained (CR; 16 pts), at 1 year. Samples from 8 healthy donors (HD) were used as controls. Results:After transplant, BM and PB T cells showed a lower CD4/CD8 ratio (p We next investigated the expression of several IRs as T-cell exhaustion markers. After haplo-HSCT, PD-1, CTLA-4, 2B4 and Tim-3 were significantly upregulated in BM and PB T cells at all time-points, compared to HD and independently from the clinical outcome. Conversely, after HLAid-HSCT, at the late time-point, patients who relapsed, displayed a higher frequency of BM infiltrating T cells expressing PD-1, CTLA-4 and Tim-3 than CR pts (p To verify whether the T-cell exhaustion phenotypic profile at relapse associates with functional impairment, we evaluated T-cell effector functions upon polyclonal stimulation. Strikingly, we observed a lower degranulation ability of CD8 cells at relapse when compared to CR (p Conclusions: After HSCT, the molecular signature of exhausted CD8 cells in relapsing pts includes PD-1, CTLA-4, 2B4 and Tim-3. The expression of IRs on early differentiated central memory and memory stem T cells at relapse suggests a wide, though reversible, immunological dysfunction mediated by AML relapsing blasts. Disclosures Bondanza: TxCell: Research Funding; MolMed SpA: Research Funding; Formula Pharmaceuticals: Honoraria. Ciceri:MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.

Introduction : Cell source and graft composition are known to have a prognostic role in allogeneic hematopoietic cell transplant (HCT). Currently, there are no data in the setting of haploidentical-HCT with post-transplant cyclophosphamide (PT-Cy). Patients and methods : One-hundred patients undergoing haplo-HCT with PT-Cy from Sept 2011 to Nov 2015 were included in this multicenter retrospective analysis. Bone marrow (BM) was used as graft source in 25 patients and peripheral blood stem cell (PBSC) in 75. The two groups (BM vs PBSC) were similar in terms of age, Disease Risk Index (DRI) and HCT-CI. The only differences were on disease type (lymphoid malignancies 76% vs 57%, p=0.02) and conditioning regimen (myeloablative: 100% versus 30%, p Graft cellular subsets analysis was performed by means of flow cytometry (CD34, CD3, CD4, CD8, CD56, CD19, CD38, CD31, CD25, CD45RA, CD95, CD127, CCR4, CCR6, CCR7). Overall Survival (OS) and Progression Free Survival (PFS) were performed with Kaplan-Meier analysis. Acute GVHD, chronic GVHD, Non-Relapse Mortality (NRM) and Relapse Incidence/Progression of disease (RI/POD) were obtained with competing risk analysis. Results : For the whole cohort, neutrophil and platelet engraftment cumulative incidences at day +30 were 97% (95% CI: 91-99) and 64% (95% CI: 53-73), with no differences between BM or PBSC. Grade II-IV and grade III-IV aGVHD cumulative incidences at day +100 were 36% (95% CI: 26-48) and 10% (95%CI: 5-18), respectively. Acute GVHD cumulative incidence was significantly lower for BM grafts [16% (95% CI: 5-33) vs 43% (95% CI: 31-54), p=0.02], but no differences were observed regarding grade III-IV acute GVHD [8% (CI 95%: 1-24) vs 11% (CI 95%: 5-20), p=0.73]. Chronic GVHD cumulative incidence at 18 months was 23% (95%CI: 15-32) with no difference between BM or PBSC. With a median follow up of 16.5 months (range 0.8 - 40.6), the 18-months PFS and OS were 43% (95%CI: 32-54) and 63% (95% CI: 52-73), respectively. NRM and RI/POD at 18 months were 18% (95%CI: 11-26) and 38% (95%CI: 27-49). Multivariable analysis was performed using patient (age, gender, HCT-CI, DRI) and transplant characteristics (donor gender, donor-recipient relation, graft source). A DRI>1 was the only factor associated with higher RI/POD (HR 3.45, 95% CI: 1.4-8.2, p Graft cell composition analysis was performed separately for BM and PBSC cohorts. For the BM group, univariable analysis showed that CD4 graft count ≥20x10^6/kg was associated with a prolonged PFS [60% (95%CI: 28-81) vs 0%, p Conclusions : In our retrospective analysis, we did not observe significant differences in survival outcomes based on graft source. Patients receiving BM grafts developed fewer grade II-IV aGVHD, but grade III-IV aGVHD incidence was similar between the two groups. For the BM group, a higher CD4 count was predictive of better PFS, TRM and OS, as reported with standard GVHD prophylaxis and matched donors (Waller EK, J Clin Oncol 2014. 32: 2365-2372). Interestingly, of the CD4 population, only naive T cells and RTEs were associated with an improved PFS and OS. A protective effect of CD8 cell count was observed in the PBSC group. This is similar to the non PT-Cy setting where higher CD8 cells are associated with better survival outcomes (Reshef R, J Clin Oncol 2015). These data should be validated in a prospective analysis to guide the choice of cell source in the haplo PT-Cy setting. Figure 1 Kaplan-Meier estimates of progression free survival from time of transplant associated to the CD4 graft content. Figure 1. Kaplan-Meier estimates of progression free survival from time of transplant associated to the CD4 graft content. Disclosures Corradini: Celgene: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Roche: Honoraria; Takeda: Honoraria; Sanofi: Honoraria; Servier: Honoraria; Gilead: Honoraria; Takeda: 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.

Background. Patients who experienced leukemia relapse after allogeneic hematopoietic stem cell transplantation (HSCT) have a dismal prognosis. Strategies to prevent or overcome post-transplant disease recurrence are limited and their efficacy still remains unclear. Recently, HSCT using haploidentical T-replete source has found increasing feasibility and acceptance thanks to new transplant platforms (Luznik L. et al., Biol Blood Marrow Transplant, 2008; Peccatori J. et al., Leukemia, 2014), allowing lower rates of graft-versus-host disease (GvHD) and non-relapse mortality (NRM). We evaluated the outcome of haploidentical transplant approach performed as second transplant (HSCT2) for patients who suffered of acute leukemia (AL) relapse after a first allogeneic HSCT (HSCT1). Methods. We analyzed 42 consecutive patients (median age 48 years) who underwent HSCT2 from a haploidentical donor, different from the previous one, between 2007 and 2013 in our Institute. Patients suffered of acute myeloid leukemia (n=31), acute lymphoblastic leukemia (n=9) or biphenotypic AL (n=2) relapsing after HSCT1 (matched related: 21; unrelated: 17; mismatched related: 4; umbilical cord blood: 4). Thirty-seven patients (88%) received re-induction therapy before HSCT2: 13 of them achieved complete remission (CR), 16 did not respond and 8 patients were transplanted directly in aplasia. In 5 cases HSCT2 was performed without re-induction therapy, in active disease (AD). Median Hematopoietic Cell Transplant-Co-morbidity Index was 2 (range 0-6) at time of HSCT2. All patients received treosulfan (14 g/m2 for 3 days) and fludarabine (30 mg/m2 for 5 days) as part of preparative regimen for HSCT2; conditioning was intensified with total body irradiation (4 Gray) in 18 (43%) or with melphalan (70 mg/m2 for 2 days) in 6 patients (14%). GvHD prophylaxis consisted of mycophenolate mofetile and sirolimus combined with pre-transplant anty-thymocyte globuline (n=36) or with post-transplant cyclophosphamide (n=6). Unmanipulated peripheral blood stem cells were used as graft source in all cases. Results. Three out of 42 patients (7%) experienced early death in aplasia, while all the others achieved neutrophil engraftment (n=39, 93%), with no primary graft rejection. Complete remission of disease at day +30 was reached in 37 out of 39 evaluated cases (95%). Estimated overall survival (OS) and leukemia-free survival (LFS) (± SE) rates at 1 year from HSCT2 were 37% (± 8%) and 35% (± 7%), respectively (Figure 1A). One-year relapse incidence (± SE) amounted to 32% (± 7%) and NRM (± SE) to 31% (± 7%) (Figure 1A) (7 patients died for infectious, 5 for GvHD and 1 for central nervous system toxicity). Sixteen patients (38%) experienced of acute GvHD grade II-IV and 20 (47%) of chronic GvHD. Disease status at HSCT2 (AD vs CR) had no impact on OS (HR:1.26, 95% CI 0.58-2.76, p=0.564) and relapse (HR:1.35, CI 95% 0.46-3.76, p=0.61). Conversely, time of remission from HSCT1>6 months provided the better outcome in terms of estimated OS (±SE) at 1 year (50% ± 10%) compared to patients with shorter duration of remission (18% ± 9%) (HR:0.49, 95% CI 0.24-1.02, p=0.05) and reduced risk of relapse (HR:0.28, 95% CI 0.11-0.75, p=0.011) (Figure 1B). After a median follow-up time of 22 months (range 6-68), 11 patients (26%) were alive with sustained leukemia remission. Conclusions. These results demonstrate the feasibility of HSCT2 from a haploidentical donor as treatment for AL relapse after initial allogeneic transplant, showing rates of OS, LFS and NRM comparable to the reported in literature for HSCT2 from matched related or unrelated graft, using the same or different donor (Christopeit M. et al., J Clin Oncol, 2013). Interestingly, disease status at HSCT2 did not influence the outcome of transplant. While, in accordance with previous reports, duration of remission after HSCT1 is the major factor involved in predicting outcome of a second transplant due to an increased relapse incidence after HSCT2 in patients with shorter HSCT1 remission, reflecting diseases biologically different. If confirmed in a larger cohort, these data could be useful in decision making after HSCT1 failure for disease recurrence. Disclosures Bonini: MolMed S.p.A.: Consultancy.

Introduction: The T cell memory compartment is multi-faceted and encompasses multiple subsets with divergent properties. In addition to central memory (TCM) and effector memory (TEM) cells, the spectrum of immunological memory has been recently extended with the identification of memory stem T cells (TSCM). Gene expression profiling, corroborated by in vitro and in vivo experimental results, posits TSCM upstream TCM and TEM in T-cell ontogeny. However, TSCM role in immune reconstitution (IR) following allogeneic HSCT remains largely unknown. Here, we tracked TSCM dynamics in patients undergoing haploidentical HSCT. By this process, we unconventionally exploited HSCT as model system to provide novel insights into the contribution of TSCM to a mounting immune response. Patients and Methods: We characterized T cell dynamics during the first month post HSCT in 20 patients receiving myeloablative conditioning, T-replete haploidentical peripheral blood stem cell graft, and GvHD prophylaxis consisting of cyclophosphamide (PT-Cy day 3,4), MMF and sirolimus from day 5. Results: Upon infusion, naïve T cells (TN) cells gradually disappeared from circulation, and by day 8 post HSCT the peripheral compartment was composed only by memory lymphocytes. At day 8, TSCM cells were the most represented circulating subset, and their frequency was significantly higher than that of the infused graft (LK). To investigate whether such high TSCM frequency was due to expansion of TSCM cells infused or to their direct in vivo generation, T cell subset proliferation was analyzed. At day 3 upon infusion (in the absence of immunosuppression), all memory subsets robustly proliferated, with TSCM cells displaying significantly higher frequencies of Ki-67+ cells compared to all other subsets. In sharp contrast, TN cells were scantly Ki-67+, in line with the longer timeframe required for their priming. PT-Cy abrogated proliferation in all subsets. T cell counts however did not drop between day 5 and 8 post HSCT, and TSCM cells increased in percentages and absolute numbers. We thus explored whether TSCM cells were resistant to PT-Cy, but failed to record ALDH activity, the major mechanism of Cy inactivation, in CD3+ cells. neither within LK nor upon infusion. Accordingly, we detected high percentages of apoptotic cells within all memory subsets at day 5 post HSCT. Nonetheless, at day 8 significantly lower percentages of TSCM cells were Annexin V+ compared to TCM/TEM, while by day 15 post-HSCT all memory subsets displayed very low levels of apoptosis. Thus, we reasoned that direct conversion of TN into TSCM could be the preferential mechanism underlying TSCM expansion. To prove this, we exploited the TCR sequences harbored by each individual T cell as surrogate clonal markers. Purified T cell subsets from LK and harvested 30 days post HSCT in 3 consecutive patients were subjected to TCR sequencing. Sequences harbored by LK-TN were retrieved in all memory subsets at day 30 after HSCT, showing that TN were able to generate the complete spectrum of immunological memory, including TSCM. Quantitative analysis revealed that only clonotypes originally harbored by LK-TN were significantly increased in counts at day 30 post-HSCT. In contrast, sequences unique to LK-TSCM, LK-TCM or LK-TEM were similar or reduced in counts at day 30 post-HSCT compared to LK, indicating that either PT-Cy efficiently dampened their expansion and/or that such memory lymphocytes persisted unchallenged upon in vivo infusion. Overall, in vivo fate mapping through TCR sequencing allowed defining the in vivo differentiation landscapes of human naïve T cells upon HSCT, highlighting TSCM as privileged players in the diversification of immunological memory. We next validated these results at the antigen-specific level. In suitable 5 patients, we recorded the presence of WT1 or PRAME-specific TN cells within donor LK. Upon HSCT, tumor-specific T cells increased in frequency and displayed a memory phenotype, comprising TSCM. Finally, by quantifying patient serum cytokines, we found that the degree of IL-7 availability at day 1 post HSCT correlated with the extent of TSCM expansion at day 8. Conclusions: These data provide novel insights into TSCM biology and ongoing analyses will define correlations with clinical events. Longer immunological follow-up will advance our understanding of the contribution of early TSCM generation to the overall success of post HSCT IR. Disclosures Bordignon: MolMed: Employment. Bonini:MolMed S.p.A.: Consultancy.

Introduction:Haploidentical hematopoietic stem cell transplantation (HSCT) performed using T–replete BM grafts and post-transplantation cyclophosphamide (PT-Cy) has gained much interest in the transplantation community for the excellent toxicity profile, achieving outcomes equivalent to those of HSCT performed using matched related or unrelated donors (Bashey, JCO 2013). Nonetheless, relapse remains a major challenge with the use of this non-myeloablative regimen (Luznik, BBMT 2008). Thus, we investigated whether the use of PBSC graft, an intensified myeloablative conditioning and sirolimus instead of tacrolimus might help increasing the therapeutic index of this procedure. Patients and Methods:Here we report the results on 39 patients treated between Nov 2012 and June 2014. The median age was 56 years (range, 21–78). Diagnoses included AML (21), ALL (5), CML-BC (1), MDS (1), NHL (5), and HD (6). The majority (62%) of patients were not in disease remission at time of HSCT. Eight patients (20%) received a prior allo-HSCT. For the remaining patients who underwent haplo-PTCy as a first allogeneic transplant, 19 patients (61%) scored high/very high according to the CIBMTR disease score, 12 (39%) scored intermediate, while no patient had a low disease score. The median Sorror Comorbidity Index was 3 (range: 0-9). Conditioning consisted of treosulfan (14 g/m2/day) on days –6 to –4, fludarabine (30 mg/m2/day) on days –6 to –2, and melphalan (70 mg/m2/day) on days –2 and –1, followed by T-replete G-CSF-mobilized PBSC. Postgrafting immunosuppression consisted of PT-Cy (50 mg/kg/day) on days 3 and 4, followed by mycophenolate mofetil for 30 days, and sirolimus for at least 3 months. Overall survival (OS) and disease-free survival (DFS) were estimated using the Kaplan-Meier method. Cumulative incidence (CI) of non-relapse mortality (NRM) was calculated using relapse as a competing risk, for GvHD the competing risk was death. Results:PBSC grafts contained an infused median CD34/kg of 6.03 e6 (4.15–8.02 e7), CD3/kg 21.12 e7 (10.5–48.26 e7). All patients but 1 engrafted (97.5%), with a median time to neutrophil and platelet recovery of 18 days (range 13–45) and 19 days (9–65), respectively. Notably, the only patient with primary graft failure had a HLA-DP mismatch in HvG direction. Post-HSCT recovery of lymphocyte subsets was broad and fast: median time to CD3>100/ml was 28 days (range: 8-97), to CD4>200/ml 35 days (21-137) and to CD19>0/ml 34 days (24-137). Circulating T cells comprised naïve and memory subsets, with a recovery of CD31+ recent thymic emigrants (RTEs) starting from day 30. All patients had a significantly higher proportion of circulating RTEs at day 30, 90 and 180 compared to their pre-HSCT levels, suggesting an improvement in their thymic function after HSCT. We observed a significant early increase in circulating Tregs at day 15 post HSCT. With a median follow-up for living patients of 327 days, the 1-year cumulative CI of NRM and relapse were 15% and 32%, respectively. Two of the 9 relapses were HLA-loss variants (Vago, NEJM 2009) occurring at a median time of 277 days post-HSCT. Classical relapses had instead an earlier occurrence (median time to relapse: 92 days, range 37-134). CI of acute GvHD grade II-IV and III-IV at 6 months were 18% and 8%, while CI of chronic GvHD at 12 months was 19%. Estimated 1-year probability of OS and DFS were 58% and 51%. Outcomes were particularly favorable in patients transplanted in CR (1-year OS 93%, DFS 95%) as compared to those of patients transplanted with active disease (1-year OS 37%, DFS 29%; p=0.005 and p=0.001, respectively). Importantly, stratification according to CIBMTR score held similar results: in patients with intermediate score, 1-year OS was 91% vs. 33% in patients with high/very high score (p=0.015); similarly, 1-year DFS was 92% vs. 34% (p=0.009). Conclusions: PT-Cy coupled to sirolimus allow the use of haploidentical PBSC grafts with low rates of GvHD, and intensified myeloablative regimen is a valid option for patients with aggressive/advanced hematologic malignancies. The acceptable rates of GvHD and NRM as well as the favorable immune reconstitution profile open the way for combining it with novel immunomodulatory or cellular therapies to improve DFS in patients at high risk for relapse who would benefit from further treatment intensification early after transplant. Disclosures Bonini: MolMed S.p.A.: Consultancy.

BACKGROUND: Suicide gene therapy applied to haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is one of the widest clinical applications of gene therapy. By the infusion of donor lymphocytes transduced to express the Herpes Simplex Virus Thymidine Kinase (TK) suicide gene, patients achieve a rapid immune reconstitution and substantial protection against tumor recurrence. TK-cells are promptly eliminated in case of graft versus host disease (GvHD), with complete resolution of the adverse reaction. In previous studies, we showed that TK-cell infusions are necessary and sufficient to promote the generation of a fast, polyclonal and full competent T cell repertoire. In the present work we characterize the immunological profile of a cohort of long-term survivors after suicide gene therapy and we studied the long-term fate of TK-cells to shed light on memory T cell dynamics after transplantation. RESULTS: We studied 9 adult patients who underwent haplo-HSCT and infusion of purified suicide-gene modified donor T cells (median dose: 1.9x107 cells/kg, range:0.9x106-39.5x106) for high-risk hematologic malignancies between 1995 and 2010 (TK patients). At a median follow-up of 7,4 years (range 3.2-12.3), all patients are in complete remission. Two out of 9 patients (22%) experienced GvHD in the early phase post immune reconstitution; in all cases, ganciclovir (GCV) administration proved effective in abrogating the adverse reaction. No symptoms or complications related to GvHD were observed during the long-term follow up, and none of the patient is receiving immunosuppressive drugs. A complete recovery of NK cells, B lymphocytes and αβ or γδ T cells was observed. The CD8+ and CD4+ T cell compartment of TK patients were characterized by level of naïve and memory cell comparable to age and sex matched healthy controls. The quantification of CD4+ CD31+ CD62L+ CD45RA+ CD95- recent thymic emigrants and measure of single joint T-cell receptor excision circles demonstrated that the normalization of the T cell compartment was supported by a completely recovered thymic output. TK-cells were detected in all patients (100%), at low levels (median=4cells/uL). Ex vivo selection of pure TK-cells after polyclonal stimulation and LNGFR-purification confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory T cells to persist for years. Of notice TK-cells could be retrieved also in patients successfully treated with GCV for GvHD, thus confirming the selective action of GCV only on proliferating TK-cells. Accordingly, GCV sensitivity was preserved in long-term persisting TK-cells, independently from their differentiation phenotype. TK-cells circulating in patients displayed a memory phenotype comprising effector memory (TEM), central memory (TCM) and stem memory (TSCM) T cells and exhibited a low level of Ki-67 positivity, thus suggesting the maintenance of a pool of gene modified memory cells through homeostatic proliferation. The number of TK-cells circulating at the longest follow-up did not correlate with the number of infused cells, nor patients or donors’ age, but instead with the peak of TK-cells observed within the first months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory T cells. We evaluated whether the phenotype of infused TK-cells was able to affect the long-term fate of gene-modified memory T cells. We observed that the number of infused TSCM cells positively correlated with early TK-cell expansion and with their long-term persistence, suggesting that TSCMmight play a privileged role in the generation of a long-lasting immunological memory. CONCLUSION: These data show that a complete and physiological donor-derived immune system is restored in adult surviving long-term after suicide gene therapy. After infusion, gene modified cells persist for up to 12 years in treated patients. This setting can be exploited to investigate the requirements at the basis of the generation of a long-lasting immunological memory in vivo. Further studies on TK-cell TCR repertoire and vector integrations are currently being performed to elucidate the in vivo dynamics of infused memory T cells. Disclosures Lambiase: MolMed S.p.A: Employment. Traversari:MolMed S.p.A: Employment. Bordignon:MolMed S.p.A: Chairman and CEO Other. Bonini:MolMed S.p.A: Consultancy.

Introduction Achieving a cure for relapsed leukemia remains a challenge. Hematopoietic stem cell transplantation (HSCT) from an HLA-haploidentical relative provides a potentially curative treatment for such patients in urgent need. Based on previous studies on the feasibility and low toxicity profile of haploidentical HSCT with high-dose post-transplant Cyclophosphamide (PT-Cy), we planned to intensify this protocol for patients with active disease by the use of: 1) myeloablative conditioning, 2) peripheral blood stem cells (PBSCs) graft instead of bone marrow (BM), and 3) sirolimus instead of tacrolimus, for its direct anti-leukemic effect. Patients and Methods Here we report the results on 18 patients treated between November 2012 and July 2013. The median age was 59 years (range, 30–78). Diagnoses included AML (10 refractory, 3 CR1, and 2 CR>1), ALL (2 refractory), and 1 CML in lymphoid blast crisis. The majority (78%) of patients were not in remission at the time of HSCT. Consistently, 13 (72%) patients scored high/very high accordingly to the CIBMTR disease score (Todisco et al., Leukemia 2013). Fourteen (78%) patients had a Sorror comorbidity score ≥ 3. Conditioning consisted of treosulfan (14 g/m2/day) on days –6 to –4, fludarabine (30 mg/m2/day) on days –6 to –2, and melphalan (70 mg/m2/day) on days –2 and –1, followed by T-repleted peripheral blood stem cells (PBSCs). Postgrafting immunosuppression consisted of PT-Cy (50 mg/kg/day) on days 3 and 4, followed by mycophenolate mofetil for 30 days, and sirolimus for 3 months. Results PBSC grafts contained an infused median TNC/kg of 10.5 e8 (range 5.5–20.7 e8), CD3/kg 18.4 e7 (range 10.5–40.9 e7) and a CD34/kg of 7.01 e6 (range 4.3–8.02 e6). All patients but 1 engrafted (94%), with a median time to neutrophil and platelet recovery of 18 days (range 14–26) and 19 days (range 9–51) respectively. Notably, the patient with primary graft failure had a HLA-DP mismatch in HvG direction. No patient experienced secondary graft failure. Post-HSCT recovery of lymphocyte subsets was broad and fast: the median CD3 count at day 30 after HSCT was 642/µl and exceeded 1000/µl by day 60, with a median value of CD4 T cell counts of 329/µl at day 30; NK cells reached a median value of 192/µl at day 30 and remained stable thereafter, while B cells steadily increased from a median value of 2/µl at day 30 to 48/µl at day 180. Circulating T cells comprised both naïve and memory subsets, with a recovery of CD31+ recent thymic emigrants (RTEs) starting from day 30. All patients had a significantly higher proportion of circulating RTEs at day 30, 90 and 180 compared to their pre-HSCT levels, suggesting an improvement in their thymic function after HSCT. Peri-engraftment syndrome (PES) occurred in 4 patients with a median time to onset of 15 days and was promptly resolved with short-course steroids. Interestingly, the percentage of circulating Ki-67+, dividing, effector memory CD8 T cells at day 8 post HSCT was able to accurately predict the occurrence of PES. Grade I/II skin acute GvHD occurred in 6 patients, while 4 patients experienced grade III/IV acute GvHD (22%). Acute GvHD were accompanied by a rise in circulating Ki-67+ CD8 T cells, while response to first line therapy resulted in a drop in Ki-67 expression. Chronic GVHD occurred in 2 of the 7 patients at risk, but only one patient required systemic therapy. Of the 16 patients at high risk, 7 had CMV reactivations. No patient had EBV-related PTLD. With a median follow-up of 100 days (range 15–260), 14 (78%) out of the 18 patients are alive. Only two patients died of non-relapse mortality (NRM, 11%). All patients in CR at time of transplant are alive and disease-free, while 8 (57%) out of 14 patients with active disease at transplant are alive and leukemia-free. In 4 patients tumor-specific T cells against PRAME or WT1 were detectable both in the peripheral blood and BM up to 180 days after HSCT, suggesting that PT-Cy did not hamper the GvL effect. Conclusions Myeloablative haploidentical HSCT with PBSCs, PT-Cy and sirolimus is feasible and might be a valid option for patients with refractory/active leukemia. If confirmed in a larger cohort of patients and with a longer follow-up, these results suggest that the acceptable rates of GvHD and NRM as well as the favorable immune reconstitution profile open the way for combining it with novel immunomodulatory or cellular therapies to improve leukemia-free survival. Disclosures: Bonini: MolMed SpA: Consultancy.

Background Suicide gene therapy applied to allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the widest clinical applications of gene therapy. By the infusion of donor lymphocytes transduced to express the Herpes Simplex Virus Thymidine Kinase (TK) suicide gene, patients achieve a rapid immune reconstitution and substantial protection against tumor recurrence. TK-cells are promptly eliminated in case of graft versus host disease (GvHD), with complete resolution of the adverse reaction. In previous studies, we showed that TK-cell infusions are necessary and sufficient to promote the generation of a fast, polyclonal and full competent T cell repertoire. In the present work we characterize the immunological profile of a cohort of long-term survivors after suicide gene therapy and we studied the long-term fate of TK-cells to shed light on memory T cell dynamics after transplantation. Results We studied 14 adult patients who underwent allo-HSCT (haploidentical HSCT: n=11; HLA-identical HSCT n=3) and infusion of purified suicide-gene modified donor T cells (median dose: 1.9x107 cells/kg, range:0.9x106-2.8x108) for high-risk hematologic malignancies between 1995 and 2010. At a median follow-up of 8,7 years (range 3-17), all patients are in complete remission. Five out of 14 patients experienced GvHD in the early phase post immune reconstitution; in all cases, ganciclovir administration proved effective in abrogating the adverse reaction. No symptoms or complications related to GvHD were observed during the long-term follow up, and none of the patient is receiving immunosuppressive drugs. We observed a complete recovery of NK cells, comprising of mature (CD56+CD16+) and immature (CD56+CD16-) NK cells. Interestingly the proportion of B cells circulating long-term in patients was significantly higher than that observed in age-related healthy controls (p TK-cells were detected in the majority of analyzed patients (90%), at low levels (median=0,43%±6,9%). Ex vivo selection of pure TK-cells after polyclonal stimulation and NGFR-purification confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory T cells to persist for years. The proportion of TK-cells detectable at the longest follow-up did not correlate with the number of infused cells, nor patients or donors’ age, but instead with the peak of TK-cells observed within the first 3 months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory T cells. Of notice TK-cells could be retrieved also in patients successfully treated with ganciclovir for GvHD, thus confirming the selective action of ganciclovir only on proliferating TK-cells. Accordingly, ganciclovir sensitivity was preserved in long-term persisting TK-cells, independently from their differentiation phenotype. While infused TK-cells displayed a predominant effector memory phenotype, gene modified T cells persisting long-term were enriched for central memory (CD45RA-CD62L+) and stem memory (CD45RA+CD62L+CD95+) phenotypes, suggesting the higher ability of these T cell subsets to persist and shape the immunological profile long-term in treated patients. Conclusion These data show that a complete donor-derived immune system is restored in adult surviving long-term after suicide gene therapy. After infusion, gene modified cells persist for up to 14 years in treated patients. Further studies on TK-cell TCR repertoire and vector integrations are currently being performed to elucidate the in vivo dynamics of infused memory T cells. Disclosures: Valtolina: MolMed S.p.A: Employment. Traversari:MolMed S.p.A: Employment. Bordignon:MolMed S.p.A: Employment. Bonini:MolMed S.p.A: Consultancy.

Haploidentical hematopoietic stem cell transplantation (HSCT) with T–replete grafts and post-transplant cyclophosphamide (PT-Cy) has gained much interest in the transplantation community for the low rates of GvHD, non-relapse mortality and opportunistic infections. This platform, devoid of anti-thymocyte globulins, allows a thorough analysis of circulating cells in the early phase post-HSCT. Indeed, several biological events that play a critical role for transplant outcome occur within the first month after HSCT; while engraftment and hematological reconstitution are carefully monitored, the shape of T cell dynamics within this timeframe remains largely unknown. We characterized immune reconstitution (IR) during the first month post HSCT in 18 high-risk leukemia patients receiving myeloablative conditioning, T-replete haploidentical peripheral blood stem cell graft (PBSCs), and GvHD prophylaxis consisting of PT-Cy (day 3, 4), followed by mycophenolate mofetil and sirolimus from day 5. Infused PBSCs and blood samples harvested at day 1, 3, 5, 8, 15 and 30 post HSCT were analyzed by multiparametric flow cytometry. T cells were infused in the absence of immunosuppressive agents, and by day 3, prior to PT-Cy, a large fraction of memory lymphocytes, possibly enriched for allo-specificities, proliferated (assessed by Ki-67 staining). Conversely, naïve T cells (TN) were scantly Ki-67+ (P< 0.001). A high CD4:CD8 ratio was observed at this time-point (10). PT-Cy efficiently abated T cell proliferation and appeared to affect CD4 more than CD8 T cells. Nevertheless, T cell numbers progressively increased (mean CD3 counts, day 5: 19 cells/µL; day 8: 27 cells/µL; day 15: 97 cells/µL), suggesting that residual proliferation in extravascular sites was likely to fuel the surge in circulating T cells. Consistently, we observed an expansion of antigen-experienced T cells including central memory (TCM), effector memory (TEM), effectors (TEFF) and the recently described stem memory T cells (TSCM). TSCM are a subset of memory cells hierarchically superior to TCM and TEM, for self-renewal, long-term persistence and functional capacity. Similarly to TN, TSCM coexpress CD45RA and CD62L but differently from TN, TSCM express CD95, a marker of memory cells. As early as day 8 post HSCT, the T cell compartment was predominantly composed by TSCM cells (P < 0.01 compared to all other subsets). Such enrichment in TSCM was not due to a selective resistance to PT-Cy, as suggested by the lack of activity of the ALDH enzyme, which converts Cy to a non-toxic metabolite, in TSCM infused with the graft. Rather, we hypothesized that TSCM expansion came directly from the differentiation of TN infused within the graft, which escaped the purging effect of PT-Cy thanks to a delayed activation kinetics compared to alloreactive memory T cells. We demonstrated the in vivo differentiation of WT1 and PRAME specific TN cells, present in the graft, into memory lymphocytes, comprising TSCM cells, in 4/7 patients suitable for dextramer tracking. Such tumor specific T cells were detected in the peripheral blood and bone marrow of treated patients, suggesting that PT-Cy did not hamper GvL players. Of note in the remaining patients for whom tumor-specific TN were not detectable in the graft, no tumor response could be documented in vivo. From day 15 post HSCT, TSCM were outnumbered by other memory subsets, suggesting their differentiation into more committed TCM TEM and TEFF. The quality of IR correlated with clinical events. The percentage of circulating Ki-67+ CD8 TEMcells at day 8 post HSCT accurately predicted the occurrence of periengraftment syndrome, observed in 4 patients with a median time to onset of 15 days. Acute GvHD (Grade I/II in 6 patients, grade III/IV in 4) was accompanied by a rise in circulating Ki-67+ CD8 cells, and response to therapy resulted in a drop in Ki-67 expression. No immunological parameter correlated with chronic GvHD, observed in 2 patients. In all patients with a CMV-seropositive donor, CMV-specific T cells were tracked in the graft, at early time-points and up to 180 days post HSCT, indicating that virus-specific T cells escaped PT-Cy. These results suggest that PT-Cy acts mainly on alloreactive memory T cells infused within the graft, while sparing infused virus-specific, non cross-reactive, memory cells and TN, which can differentiate predominantly in TSCM, but also in TCM TEM and TEFF, thus promoting a rapid and broad IR. Disclosures: Bonini: MolMed SpA: Consultancy.

The genetic transfer of tumor-specific T cell receptors (TCRs) into mature T lymphocytes enables T cell specificity to be redirected towards cancer cells, however the transfer of novel TCRs into polyclonal T cells, while overcoming tolerance barriers, may be limited by factors intrinsic to TCR biology. Specifically, the tumor-specific a and b TCR chains are expressed in lymphocytes that already bear an endogenous TCR on their cell surfaces. Gene-modified cells thus express at least two different TCRs that compete for binding to the CD3 complex, resulting in mutual TCR dilution and reduced avidity. Furthermore, since TCRs are heterodimers, the a and b chains of the endogenous TCR have the potential to mispair with the respective α and β chains of the transgenic TCR to produce a new hybrid TCR, with unpredictable and potentially harmful specificity. This represents a major concern in TCR transfer adoptive immunotherapy, both in autologous and allogeneic settings. To permanently eliminate the expression of the endogenous TCR and the risk of mispairing, our group recently developed a TCR gene editing approach. This technique is based on the transient transfer of zinc-finger nucleases (ZFN) to induce DNA double strand breaks in the constant regions of the endogenous TCR a and/or b chain genes, leading to permanent gene disruption. Upon lentiviral transfer of a tumor-specific TCR, such fully TCR-edited T cells express only the exogenous tumor-specific TCR transgenes at high levels (Provasi, Genovese et al., Nature Medicine 2012). While the complete editing procedure (both a and b TCR chains) currently requires multiple manipulation steps, ‘single TCR editing’, based on the ZFN-mediated knock-down of a single endogenous TCR chain (a or b) followed by the introduction of the tumor-specific TCR, enables the generation of redirected T cells devoid of their natural TCR repertoire during a single round of T cell activation, improving the feasibility of the clinical translation of this approach. This might be particularly useful to reduce the risk of GvHD after allogeneic hematopoietic stem cell transplantation. We exploited a HLA-A2 restricted TCR specific for NY-ESO-1, a cancer testis antigen expressed by solid tumors and hematological malignancies, to directly compare the safety and efficacy profile of unedited TCR transferred T cells (TR), single TCR edited (SE) lymphocytes and completely TCR edited (CE) T cells. We observed that gene editing does not detectably affect the phenotype, function or proliferative potential of engineered lymphocytes. Our protocols ensured the maintenance of the early differentiated memory phenotype, with enrichment in central memory and CD45RA+/CD62L+/CD95+ memory stem T (TSCM) cells. Upon lentiviral transfer of the NY-ESO-1-specific TCR, we observed significantly higher levels of the tumor-specific TCR expression, evaluated as NY-ESO-1 specific dextramer binding, in edited versus transferred T cells (relative fluorescence intensity to untransduced cells: CE: 37; SE: 31; TR: 19). Edited T cells were more efficient than unedited-TCR transferred T cells in killing NY-ESO-1-pulsed cell lines (half maximal effective peptide concentration in a 51Cr release assay: 310, 210, 186 nM for TR, SE and CE T cells respectively) and NY-ESO-1+ myeloma cell lines naturally processing the antigen. Importantly our SE and CE T cells displayed no activity against NY-ESO-1- targets. Importantly, in NSG mice, NY-ESO-1 redirected single edited and complete edited T cells completely eliminated an NY-ESO1+ HLA-A2+, WT1- myeloma cell line, that, on the contrary, expanded in bone marrow in the presence of WT1-redirected CE T cells. Our results demonstrate that the TCR single editing approach is effective in redirecting T cell specificity as evidenced by the potent anti-tumor effect observed while potentially eliminating the risk of GvHD associated with the infusion of donor-derived lymphocytes. Moreover, the relative speed and simplicity of the TCR single editing protocol should facilitate its clinical application to patients with hematological malignancies. Disclosures: Reik: Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment.