Background The new generation of adoptive T cell therapy utilizing high-precision neoantigen targeting is gaining increasing interest, especially in solid tumors, where the unmet medical need is high. Personalized tumor trained lymphocytes (pTTL) is a novel autologous adoptive T cell therapy targeting patient-specific neoantigens. A phase I/IIa First in Human (FIH) clinical trial of pTTL in Stage IV colorectal cancer (CRC) patients has started 2023. Methods pTTL is produced through in vitro expansion of T cells derived from tumor-adjacent regional lymph nodes (RLN). The T cells are stimulated with a patient-specific array of neoantigens utilizing the proprietary EpiTCer® technology. Tumor-specific mutations are identified from next generation sequencing data obtained from tumor and normal tissue samples, and personal neoantigen epitopes are selected and ranked using the in-house bioinformatic software PIOR®. Polypeptides containing the selected neoantigens are designed, produced and linked to paramagnetic micro-particles to form EpiTCer® beads, a tumor-selective T cell expansion stimulus used for pTTL manufacturing. Results pTTL can be manufactured with a high rate of success despite the product’s personalized nature. pTTL consists mainly of T cells, with small proportions of NK and B cells. The CD4/CD8 T cell ratio varies between products. pTTL contains a significant proportion of memory T cells expressing markers indicating functionality, with limited levels of late-stage T cells. TCR sequencing has demonstrated increased T cell clonality in pTTL compared to in the RLN starting material, indicating antigen-specific expansion. Conclusions The ongoing FIH Phase I/II trial will include up to 16 patients with Stage IV CRC. pTTL is administered as a single-dose monotherapy after chemotherapy-based preconditioning with cyclophosphamide and fludarabine. A dose-escalation design is applied. The trial is divided into three parts. Part I: Collection of materials for sequencing and pTTL production (tumour biopsy, surgical collection of RLNs), and manufacturing of EpiTCer® beads and pTTL. Part II: pre-conditioning, administration of pTTL and follow-up for 6 months. Part III: Long-term follow-up to 5 years after pTTL administration. The primary endpoint is safety. Secondary outcomes include response, overall survival, and progression-free survival. Biomarkers for pTTL persistence, pTTL characteristics, and response will be evaluated. Trial Registration Trial Registration EUDRA CT #2022–000394-96. Clinicaltrials.gov Identifier #NCT05908643 Ethics Approval The trial is approved by the Swedish MPA (5.1–2022-89272) and the Swedish Ethical Review Authority (2022–01842-01). All patients must give their informed consent to participation before inclusion in the trial.

Background Adoptive T cell therapy as a treatment for solid tumours is gaining increasing interest. Cancer neoantigens as targets for such therapy is also gaining recognition. Personalised tumour trained lymphocytes (pTTL) is a novel autologous T cell therapy targeting patient-specific neoantigens. A phase I/II First in Human (FIH) clinical trial of pTTL in Stage IV colorectal cancer (CRC) patients will be initiated in the near future. Methods pTTL is produced through in vitro expansion of T cells derived from regional lymph nodes (RLNs). The T cells derived from RLNs, nodes in anatomical proximity of the tumour, contain a pool of naive and antigen-experienced T cells enriched for tumour-antigen specificity. This enriched population is stimulated during pTTL production with an array of neoantigen epitopes individually designed using PIOR®, an in house-developed software for neoantigen detection and selection. Selected neoantigens are linked to paramagnetic particles using EpiTCer® technology. The resulting EpiTCer® particles are used to stimulate the RLN T cells via phagocytosis and presentation of the neoantigen epitopes by antigen-presenting cells. This process is HLA-independent. Each pTTL product is unique due to the personalised nature of cellular and molecular players (cancer characteristics, immune cell properties and neoantigens are specific to one single individual). In the planned FIH trial patients with Stage IV CRC which have either no remaining relevant standard care therapies or which are in a scheduled break in such therapy can be included. pTTL will be administered as a single dose monotherapy after chemotherapy-based preconditioning with cyclophosphamide and fludarabine. The primary endpoint of the trial is safety of pTTL. Biomarker analysis of pTTL persistence and characteristics will also be central in trial assessments, and clinical outcome and response will be evaluated. Results Clinical results are not yet available. We here present central pTTL product characteristics. We have found that pTTL can be manufactured with a high rate of success despite the patient-specific nature of each product. The majority of the cells in pTTL are T cells, with small proportions of remaining NK and B cells originating from the RLN. The CD4 and CD8 T cell ratio are variable. Main pTTL characteristics include a significant proportion of memory T cells and phenotypic markers indicating maintained functionality such as limited levels of Temra (late stage memory cells re-expressing CD45RA) and CD57+ T cells, and maintained expression of CD28. TCRseq data have shown increased clonality in pTTL compared to RLNs, indicating antigen-specific expansion. Trial Registration EUDRA CT #2022-000394-96 Ethics Approval The trial have been approved by the Swedish Ethical Review Authority, Approval # 2022-01842-01 Consent Written informed consent will be requested from study participants in future, but no study subjects have yet been included.

Background: T cells are key players in immunotherapy but our understanding of their role and function in AML is limited. We have previously shown that following induction chemotherapy, the phenotypic and transcriptional profiles of peripheral blood (PB) CD8+ T cells in AML diverge between complete responders (CR) and non-responders (NR) to treatment (Knaus et al, JCI insight 2018). Further defining AML-induced T cell dysfunction is critical for immune monitoring and developing novel immunotherapeutic strategies. Aims: 1. To characterize transcriptional differences between PB vs BM T cells of AML patients, similar to concept of circulating vs tumor infiltrating lymphocytes (TILs), and compare them to established immunologic signatures of solid tumors. 2. To examine the cumulative expression of multiple inhibitory receptors (IRs) on AML CD8+ T cells and their dynamics in PB vs BM at diagnosis (PRE-treatment) and POST-induction chemotherapy. Methods: To study transcriptional signatures, we FACS-purified CD8+ T cells from BM of 6 AML patients (3 CR and 3 NR) PRE- and POST-induction chemotherapy whose PB T cells we analyzed and published previously (Knaus et al, JCI insight 2018). Samples were hybridized to the Human Prime View Gene Expression Array (Affymetrix). Expression fold change (FC), p-values and FDR were calculated. We compared our data set to those of dysfunctional T-cell signatures from non-small cell lung cancer (NSCLC: Guo et al, Nature 2018), colorectal cancer (CRC; Zhang et al, Nature 2018) and hepatocellular carcinoma (HCC; Zheng et al, Cell 2017) using gene set enrichment analyses (GSEA) and considered a normalized enrichment score NES>2 and FDR<0.1 as enriched. Flow cytometry data on paired PB and BM samples from AML patients (n=32) PRE- and POST-treatment and healthy controls (HC; n=21) were used to calculate the IR-score (Thommen et al, Cancer Immunol Res 2015). The IR-score summarizes the cumulative relative amount of expression of 7 different IRs on CD8+ T cells. To determine the predictive ability of the IR score to differentiate CR from NR, Receiver Operating Characteristic (ROC) analysis was conducted using logistic regression, and the area under the ROC curve (AUC) was calculated. Results: Immune signatures of AML CD8+ T cells at diagnosis, particularly in the BM, overlapped with transcriptomic exhaustion signatures of TILs from other malignancies (Figure 1-Overlap CRC, HCC, NSCLC), in particular that of CRC. Further, AML BM but not PB T cells showed a high enrichment for a common immune exhaustion signature shared by all 3 solid tumor entities and included IRs like HAVCR2, PDCD1, CTLA4, LAG3 and TIGIT, and other genes (e.g. TNFRSF9, CD27, IFNG, FASLG, CD39/ENTPD1) (Figure 1). Interestingly, this signature seemed to discriminate CR from NR patients only in the BM compartment, and at both PRE- and POST-treatment, thus providing a rationale for future exploration as a predictive biomarker of response. We found that the flow-cytometry-based IR-score strongly discriminated between CR and NR in both the PB and the BM compartment POST-treatment with an AUC of 0.70 and 0.84, respectively. While IR-score including all 7 markers provided the best discriminatory value in the PB, the combination of only two senescence markers, CD57 and KLRG1, discriminated CR from NR patients in both PB and BM with an AUC of 0.69 and 0.79, respectively. The combination of 5 exhaustion markers (2B4, BTLA, Tim3, PD-1, and CD160) discriminated CR from NR patients only in the BM (AUC 0.79). Conclusion: We found that in AML, T cells display a signature of immune exhaustion which is more prominent in the tumor milieu (e.g BM), similar to exhaustion signatures of solid cancers, can discriminate CR from NR, and persists in CR. Multiple genes, such as TNFRSF9, CD27, IFNG, FASLG, and CD39/ENTPD1, were higher expressed in CR patients both PRE and POST-treatment, suggesting that this signature also includes genes that could be useful for discriminating less dysfunctional T cell states. The phenotypic IR score was increased only in NR POST-treatment, indicating that assessment of a limited number of phenotypic markers may not be sufficient to address overall changes within T cells. Further exploration of AML-imposed T cell dysfunction is ongoing to inform better integration of immune-based therapies to augment anti-leukemia immunity. Disclosures Zeidner: Celgene: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Tolero: Honoraria, Research Funding; Pfizer: Honoraria; AsystBio Laboratories: Consultancy; Merck: Research Funding; Takeda: Research Funding; AbbVie: Honoraria; Agios: Honoraria. Gojo:Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Merck: Research Funding; Juno: Research Funding; Amgen Inc: Consultancy, Honoraria, Research Funding; Amphivena: Research Funding. Luznik:Merck: Research Funding, Speakers Bureau; Genentech: Research Funding; AbbVie: Consultancy; WindMiL Therapeutics: Patents & Royalties: Patent holder.

Background and objectives Mucositis is a common complication after allogeneic hematopoietic stem cell transplantation (HSCT), and is caused by a combination of conditioning-induced mucosal damage and severe neutropenia. The symptoms include oral and abdominal pain, inability to swallow food and fluids, and severe diarrhoea. Severe mucositis is associated with increased risk of Graft-versus-Host disease and infection. Granulocyte transfusions (GCX) could be a treatment option, and our objective was to study its feasibility and potential benefits. Material and methods This retrospective, single-centre study included 30 patients receiving GCX because of severe oral mucositis after HSCT during 2005-2017. Clinical outcome, response to GCX, change in opiate administration and adverse events were studied. Results Twenty-seven patients received GCX from donors pre-treated with steroids and G-CSF, and three from donors pre-treated with steroids only. Overall response was 83% (24/29 evaluable patients). Fifteen patients reached a complete response. In 14 of 24 responders, a reduction of the administration of opiate pain relief was seen. In eight patients this reduction was >= 50% of the dose. Adverse events (AEs) were reported in 14 cases, and were mild to moderate, and well manageable with symptomatic treatment. No life-threatening or fatal AEs were recorded. Conclusions These results indicate that GCX could be a safe and effective treatment for oral mucositis after HSCT with the potential to reduce the necessity of opiate analgesic treatment in this disorder. No severe AEs were seen in this study, but the risk for severe pulmonary AEs after GCX needs to be considered.

Decreased T-lymphocyte response to T-cell-dependent vaccines after neurotrauma or neurosurgery

INTRODUCTION:In recent years, immunotherapy for the treatment of solid cancer has emerged as a promising therapeutic alternative. Adoptive cell therapy (ACT), especially T cell-based, has been found to cause tumor regression and even cure in a percentage of treated patients. Checkpoint inhibitors further underscore the potential of the T cell compartment in the treatment of cancer. Not all patients respond to these treatments; however, many challenges remain.AREAS COVERED:This review covers the challenges and progress in tumor antigen target identification and selection, and cell product manufacturing for T cell ACT. Tumor immune escape mechanisms and strategies to overcome those in the context of T cell ACT are also discussed.EXPERT OPINION:The immunotherapy toolbox is rapidly expanding and improving, and the future promises further breakthroughs in the T cell ACT field. The heterogeneity of the tumor microenvironment and the multiplicity of tumor immune escape mechanisms pose formidable challenges to successful T cell immunotherapy in solid tumors, however. Individualized approaches and strategies combining treatments targeting different immunotherapeutic aspects will be needed in order to expand the applicability and improve the response rates in future.

Gammadelta (γδ) T cells are found in both blood and tissues and have antiviral and antitumor properties. The frequency of γδ T cells in umbilical cord blood (UCB) is low, and the majority express δ1, in contrast to blood, whereas the main subset is δ2γ9 T cells. UCB γδ T cells are functionally immature, which together with their scarcity complicates the development of UCB γδ T cell therapies. We aimed to develop an effective expansion protocol for UCB γδ T cells based on zoledronate and IL-2. We found that culture with 5 μM zoledronate and 200 IU IL-2/ml medium for 14 days promoted extensive proliferation. The majority of the cultured cells were γ9δ2 T cells. The fold expansion of this, originally infrequent, subset was impressive (median and maximum fold change 253 and 1085, resp.). After culture, the cells had a polyclonal γδ T cell repertoire and the main memory subset was central memory (CD45RO+ CD27+). The cells produced cytokines such as IL-1B, IL-2, and IL-8 and displayed significant tumor-killing capacity. These results show that development of in vitro expanded UCB γδ T cell therapies is feasible. It could prove a valuable treatment modality for patients after umbilical cord blood transplantation.

BACKGROUNDOur understanding of phenotypic and functional signatures of CD8+ T cell dysfunction in acute myeloid leukemia (AML) is limited. Deciphering these deranged T cell functional states and how they are impacted by induction chemotherapy is essential for incorporation of novel immune-based strategies to restore and maintain antileukemia immunity.METHODSWe utilized high-dimensional immunophenotyping, gene expression, and functional studies to characterize peripheral blood and bone marrow CD8+ T cells in 72 AML patients at diagnosis and after induction chemotherapy.RESULTSOur data suggest that multiple aspects of deranged T cell function are operative in AML at diagnosis, with exhaustion and senescence being the dominant processes. Following treatment, the phenotypic and transcriptional profile of CD8+ T cells diverged between responders and nonresponders. Response to therapy correlated with upregulation of costimulatory, and downregulation of apoptotic and inhibitory, T cell signaling pathways, indicative of restoration of T cell function. In functional studies, AML blasts directly altered CD8+ T cell viability, expansion, co-signaling and senescence marker expression. This CD8+ T cell dysfunction was in part reversible upon PD-1 blockade or OX40 costimulation in vitro.CONCLUSIONOur findings highlight the uniqueness of AML in sculpting CD8+ T cell responses and the plasticity of their signatures upon chemotherapy response, providing a compelling rationale for integration of novel immunotherapies to augment antileukemia immunity.FUNDINGThis work was supported by the Leukemia & Lymphoma Society grant no. 6449-13; NIH grants UM1-CA186691 and R01-HL110907-01; the American Society for Blood and Marrow Transplantation New Investigator Award/Gabrielle's Angel Foundation; the Vienna Fund for Innovative Cancer Research; and by fellowships from the Wenner-Gren Foundation and the Swedish Society for Medical Research.

Allogeneic stem cell transplantation (allo-SCT) can be a curative treatment for patients with a hematologic malignancy due to alloreactive T cell responses recognizing minor histocompatibility antigens (MiHA). Yet tumor immune escape mechanisms can cause failure of T cell immunity, leading to relapse. Tumor cells display low expression of costimulatory molecules and can up-regulate coinhibitory molecules that inhibit T cell functionality on ligation with their counter-receptors on the tumor-reactive T cells. The aim of this explorative study was to evaluate immune checkpoint expression profiles on T cell subsets and on cytomegalovirus (CMV)- and/or MiHA-reactive CD8+ T cells of allo-SCT recipients using a 13-color flow cytometry panel, and to correlate these expression patterns to clinical outcomes. MiHA-reactive CD8+ T cells exhibited an early differentiated CD27++/CD28++ phenotype with low KLRG-1 and CD57 expression. These T cells also displayed increased expression of PD-1, TIM-3, and TIGIT compared with total effector memory T cells and CMV-specific CD8+ T cells in healthy donors and allo-SCT recipients. Remarkably, high coexpression of PD-1, TIGIT, and KLRG-1 on MiHA-reactive CD8+ T cells was associated with relapse after allo-SCT. Taken together, these findings indicate that MiHA-specific CD8+ T cells of relapsed patients have a distinctive coinhibitory expression signature compared with patients who stay in remission. This phenotype may serve as a potential monitoring tool in patients. Moreover, these findings suggest that PD-1 and TIGIT play important roles in regulating T cell-mediated tumor control, providing a rationale for immunotherapy with blocking antibodies to treat relapse after allo-SCT.

BACKGROUND:Urinary bladder cancer is a common malignancy worldwide. Environmental factors and chronic inflammation are correlated with the disease risk. Diagnosis is performed by transurethral resection of the bladder, and patients with muscle invasive disease preferably proceed to radical cystectomy, with or without neoadjuvant chemotherapy. The anti-tumour immune responses, known to be initiated in the tumour and draining lymph nodes, may play a major role in future treatment strategies. Thus, increasing the knowledge of tumour-associated immunological processes is important. Activated CD4+ T cells differentiate into four main separate lineages: Th1, Th2, Th17 and Treg, and they are recognized by their effector molecules IFN-γ, IL-13, IL-17A, and the transcription factor Foxp3, respectively. We have previously demonstrated signature CpG sites predictive for lineage commitment of these four major CD4+ T cell lineages. Here, we investigate the lineage commitment specifically in tumour, lymph nodes and blood and relate them to the disease stage and response to neoadjuvant chemotherapy. RESULTS:Blood, tumour and regional lymph nodes were obtained from patients at time of transurethral resection of the bladder and at radical cystectomy. Tumour-infiltrating CD4+ lymphocytes were significantly hypomethylated in all four investigated lineage loci compared to CD4+ lymphocytes in lymph nodes and blood (lymph nodes vs tumour-infiltrating lymphocytes: IFNG -4229 bp p < 0.0001, IL13 -11 bp p < 0.05, IL17A -122 bp p < 0.01 and FOXP3 -77 bp p > 0.05). Examination of individual lymph nodes displayed different methylation signatures, suggesting possible correlation with future survival. More advanced post-cystectomy tumour stages correlated significantly with increased methylation at the IFNG -4229 bp locus. Patients with complete response to neoadjuvant chemotherapy displayed significant hypomethylation in CD4+ T cells for all four investigated loci, most prominently in IFNG p < 0.0001. Neoadjuvant chemotherapy seemed to result in a relocation of Th1-committed CD4+ T cells from blood, presumably to the tumour, indicated by shifts in the methylation patterns, whereas no such shifts were seen for lineages corresponding to IL13, IL17A and FOXP3. CONCLUSION:Increased lineage commitment in CD4+ T cells, as determined by demethylation in predictive CpG sites, is associated with lower post-cystectomy tumour stage, complete response to neoadjuvant chemotherapy and overall better outcome, suggesting epigenetic profiling of CD4+ T cell lineages as a useful readout for clinical staging.

Tissue-resident memory T (TRM ) cells are CD8+ T lymphocytes that reside in the tissues, including tumours. This T cell subset possesses a magnitude of cytotoxicity, but its epigenetic regulation has not been studied. Here, we investigate the impact of perforin DNA methylation in TRM cells and correlate it with their functional potential. Fifty-three urothelial urinary bladder cancer (UBC) patients were recruited prospectively. The DNA methylation status of the perforin gene (PRF1) locus in TRM cells was investigated by pyrosequencing. Flow cytometry with ViSNE analysis and in-vitro stimulation were used to evaluate TRM cell phenotypes. We discovered that tumour TRM cells have low DNA methylation in the PRF1 locus (32·9% methylation), which corresponds to increased numbers of perforin-expressing TRM cells. Surprisingly, programmed cell death 1 (PD-1) expression is high in tumour TRM cells, suggesting exhaustion. Following interleukin-15 and T cell receptor stimulation, perforin and T-bet expressions are enhanced, indicating that TRM cells from tumours are not terminally exhausted. Moreover, a high number of TRM cells infiltrating the tumours corresponds to lower tumour stage in patients. In conclusion, TRM cells from UBC tumours are epigenetically cytotoxic with signs of exhaustion. This finding identifies TRM cells as potential new targets for cancer immunotherapy.

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.

ABSTRACT Introduction: Umbilical cord blood (UCB), previously seen as medical waste, is increasingly recognized as a valuable source of cells for therapeutic use. The best-known application is in hematopoietic stem cell transplantation (HSCT), where UCB has become an increasingly important graft source in the 28 years since the first umbilical cord blood transplantation (UCBT) was performed. Recently, UCB has been increasingly investigated as a putative source for adoptive cell therapy. Areas covered: This review covers the advances in umbilical cord blood transplantation (UCBT) to overcome the limitation regarding cellular dose, immunological naivety and additional cell doses such as DLI. It also provides an overview regarding the progress in adoptive cellular therapy using UCB. Expert opinion: UCB has been established as an important source of stem cells for HSCT. Successful strategies to overcome the limitations of UCBT, such as the limited cell numbers and naivety of the cells, are being developed, including novel methods to perform in vitro expansion of progenitor cells, and to improve their homing to the bone marrow. Promising early clinical trials of adoptive therapies with UCB cells, including non-immunological cells, are currently performed for viral infections, malignant diseases and in regenerative medicine.

Abstract 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.

Introduction: AML pts have a poor prognosis with conventional chemotherapy regimens. Early lymphocyte recovery (ELR) following intensive timed sequential therapy (TST) induction is characterized by a dysfunctional immunosuppressive state. Pomalidomide (Pom), a small molecule immunomodulatory agent (IMiD), has direct effects on T cell co-stimulation by promoting the ubiquitination of Aiolos, an IL-2 transcriptional repressor. We hypothesized that the administration of Pom at the time of ELR after induction TST may influence T cell differentiation and enhance an anti-leukemia immune effect.

Background: T cell dysfunction in AML remains poorly understood. Our previous studies of AML-associated T cell dysfunction (Knaus, ASH 2015) have focused on expression of multiple inhibitory receptors by T cells in AML patients. Transcriptional signatures, however, remain relatively unexplored, as does the role of Blast/T cell interactions on T cell function. Deciphering those could be crucial for integration of future immunotherapies into clinical practice. Therefore, we aimed to characterize CD8+ T cell gene expression signatures in newly diagnosed AML patients before and after treatment, and to decipher the effects of AML blasts on the expression of co-signaling molecules by CD8+ T cells in co-culture experiments.