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Introduction Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in hematopoietic stem cell transplantation (HSCT) (1-6). Current dogma holds that donor T cells, either transferred with or [...], Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in hematopoietic stem cell transplantation (HSCT). Donor T cells are key mediators in pathogenesis, but a contribution from host T cells has not been explored, as conditioning regimens are believed to deplete host T cells. To evaluate a potential role for host T cells in GVHD, the origin of skin and blood T cells was assessed prospectively in patients after HSCT in the absence of GVHD. While blood contained primarily donor-derived T cells, most T cells in the skin were host derived. We next examined patient skin, colon, and blood during acute GVHD. Host T cells were present in all skin and colon acute GVHD specimens studied, yet were largely absent in blood. We observed acute skin GVHD in the presence of 100% host T cells. Analysis demonstrated that a subset of host T cells in peripheral tissues were proliferating ([Ki67.sup.+]) and producing the proinflammatory cytokines IFN-[gamma] and IL-17 in situ. Comparatively, the majority of antigen-presenting cells (APCs) in tissue in acute GVHD were donor derived, and donor-derived APCs were observed directly adjacent to host T cells. A humanized mouse model demonstrated that host skin-resident T cells could be activated by donor monocytes to generate a GVHD-like dermatitis. Thus, host tissue-resident T cells may play a previously unappreciated pathogenic role in acute GVHD.

Chronic graft-versus-host disease (cGVHD) is a life-threatening impediment to allogeneic hematopoietic stem cell transplantation, and current therapies do not completely prevent and/or treat cGVHD. CD4+ T cells and B cells mediate cGVHD; therefore, targeting these populations may inhibit cGVHD pathogenesis. Ibrutinib is an FDA-approved irreversible inhibitor of Bruton's tyrosine kinase (BTK) and IL-2 inducible T cell kinase (ITK) that targets Th2 cells and B cells and produces durable remissions in B cell malignancies with minimal toxicity. Here, we evaluated whether ibrutinib could reverse established cGVHD in 2 complementary murine models, a model interrogating T cell-driven sclerodermatous cGVHD and an alloantibody-driven multiorgan system cGVHD model that induces bronchiolar obliterans (BO). In the T cell-mediated sclerodermatous cGVHD model, ibrutinib treatment delayed progression, improved survival, and ameliorated clinical and pathological manifestations. In the alloantibody-driven cGVHD model, ibrutinib treatment restored pulmonary function and reduced germinal center reactions and tissue immunoglobulin deposition. Animals lacking BTK and ITK did not develop cGVHD, indicating that these molecules are critical to cGVHD development. Furthermore, ibrutinib treatment reduced activation of T and B cells from patients with active cGVHD. Our data demonstrate that B cells and T cells drive cGVHD and suggest that ibrutinib has potential as a therapeutic agent, warranting consideration for cGVHD clinical trials.

Introduction Relapse of acute myeloid leukemia (AML) occurs in up to 50% of patients after allogeneic hematopoietic stem cell transplant (HCT) and heralds an extremely poor prognosis (1). In several [...], BACKGROUND. Responses to conventional donor lymphocyte infusion for postallogeneic hematopoietic cell transplantation (HCT) relapse are typically poor. Natural killer (NK) cell-based therapy is a promising modality to treat post-HCT relapse. METHODS. We initiated this ongoing phase I trial of adoptively transferred cytokine-induced memory-like (CIML) NK cells in patients with myeloid malignancies who relapsed after haploidentical HCT. All patients received a donor-derived NK cell dose of 5 to 10 million cells/kg after lymphodepleting chemotherapy, followed by systemic IL-2 for 7 doses. Highresolution profiling with mass cytometry and single-cell RNA sequencing characterized the expanding and persistent NK cell subpopulations in a longitudinal manner after infusion. RESULTS. In the first 6 enrolled patients on the trial, infusion of CIML NK cells led to a rapid 10- to 50-fold in vivo expansion that was sustained over months. The infusion was well tolerated, with fever and pancytopenia as the most common adverse events. Expansion of NK cells was distinct from IL-2 effects on endogenous post-HCT NK cells, and not dependent on CMV viremia. Immunophenotypic and transcriptional profiling revealed a dynamic evolution of the activated CIML NK cell phenotype, superimposed on the natural variation in donor NK cell repertoires. CONCLUSION. Given their rapid expansion and long-term persistence in an immune-compatible environment, CIML NK cells serve as a promising platform for the treatment of posttransplant relapse of myeloid disease. Further characterization of their unique in vivo biology and interaction with both T cells and tumor targets will lead to improvements in cell-based immunotherapies. TRIAL REGISTRATION. ClinicalTrials.gov NCT04024761. FUNDING. Dunkin' Donuts, NIH/National Cancer Institute, and the Leukemia and Lymphoma Society.

Introduction Graft-versus-leukemia (GvL) activity following allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents one of the most striking examples of effective human antitumor immunity and is the basis of curative responses [...], Background. Patients with advanced hematologic malignancies remain at risk for relapse following reduced-intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (allo-HSCT). We conducted a prospective clinical trial to test whether vaccination with whole leukemia cells early after transplantation facilitates the expansion of leukemia-reactive T cells and thereby enhances antitumor immunity. Methods. We enrolled 22 patients with advanced chronic lymphocytic leukemia (CLL), 18 of whom received up to 6 vaccines initiated between days 30 and 45 after transplantation. Each vaccine consisted of irradiated autol-ogous tumor cells admixed with GM-CSF-secreting bystander cells. Serial patient PBMC samples following transplantation were collected, and the impact of vaccination on T cell activity was evaluated. Results. At a median follow-up of 2.9 (range, 1-4) years, the estimated 2-year progression-free and overall survival rates of vaccinated subjects were 82% (95% CI, 54%-94%) and 88% (95% CI, 59%-97%), respectively. Although vaccination only had a modest impact on recovering T cell numbers, [CD8.sup.+] T cells from vaccinated patients consistently reacted against autologous tumor, but not alloantigen-bearing recipient cells with increased secretion of the effector cytokine IFN-γ, unlike T cells from nonvaccinated CLL patients undergoing allo-HSCT. Further analysis confirmed that 17% (range, 13%-33%) of CD8+ T cell clones isolated from 4 vaccinated patients by limiting dilution of bulk tumor-reactive T cells solely reacted against CLL-associated antigens. Conclusion. Our studies suggest that autologous tumor cell vaccination is an effective strategy to advance long-term leukemia control following allo-HSCT. Trial registration. Clinicaltrials.gov NCT00442130. Funding. NCI (5R21CA115043-2), NHLBI (5R01HL103532-03), and Leukemia and Lymphoma Society Trans-lational Research Program.

Introduction Natural killer (NK) cells are a critical component of the innate immune response against infectious pathogens and malignant transformation (1, 2). NK cells mediate this activity through the elaboration [...], Natural killer (NK) cells are primary effectors of innate immunity directed against transformed tumor cells. In response, tumor cells have developed mechanisms to evade NK cell-mediated lysis through molecular mechanisms that are not well understood. In the present study, we used a lentiviral shRNA library targeting more than 1,000 human genes to identify 83 genes that promote target cell resistance to human NK cell-mediated killing. Many of the genes identified in this genetic screen belong to common signaling pathways; however, none of them have previously been known to modulate susceptibility of human tumor cells to immunologic destruction. Gene silencing of two members of the JAK family (JAK1 and JAK2) increased the susceptibility of a variety of tumor cell types to NK-mediated lysis and induced increased secretion of IFN- γ by NK cells. Treatment of tumor cells with JAK inhibitors also increased susceptibility to NK cell activity. These findings may have important clinical implications and suggest that small molecule inhibitors of tyrosine kinases being developed as therapeutic antitumor agents may also have significant immunologic effects in vivo.

Introduction Allogeneic hematopoietic stem cell transplantation can lead to several immunological outcomes, including graft-versus-host disease (GvHD) and graft-versus-leukemia effects (GvL) (1). The typical locations of GvHD reactions suggest that induction [...], Donor lymphocyte infusion (DLI), whereby donor mononuclear cells are infused into patients, is one of the few effective immunotherapeutic strategies that generate long-lasting tumor remissions.We previously demonstrated that chronic myelogenous leukemia (CML) patients treated with DLI develop high-titer plasma antibodies specific for CML-associated antigens, the majority of which have been reported to bind nucleic acids These observations led us to predict that circulating antibody-antigen complexes in DLI-responsive patients carry nucleic acids that can engage innate immune sensors. Consistent with this, we report here that post-DLI plasma from 5CMLpatients that responded to DLI treatment induced massive upregulation of MIP-1α, IP-10, and IFN-α in normal blood mononuclear cells. Importantly, this was not observed with plasma obtained before DLI and from DLI nonresponders and imatinib-treated patients. This endogenous immunostimulatory activity required nucleic acid and protein for its adjuvant effect and activated antigen-presenting cells through the RNA and DNA sensors TLR8 and TLR9. Presence of the immunoglobulin Fc receptor CD32 enhanced cellular responses, suggesting that immunoglobulins associate with this activity. Finally, a TLR-induced expression signature was detectable in post-DLI but not pre-DLI blood, consistent with an active circulating TLR8/9-stimulating factor.We have therefore demonstrated that effective tumor immunity correlates with the presence of endogenous nucleic acid-immunoglobulin complexes in patient plasma, thus providing a putative mechanism for the induction of potent antigen-specific immunity against malignant cells.

Introduction Allogeneic HSC transplantation (HSCT) provides curative therapy for patients with various hematologic malignancies, bone marrow failure syndromes, and congenital immune deficiencies. With improvements in immune suppressive therapy and supportive [...], [CD4.sup.+][CD25.sup.+][Foxp3.sup.+] Tregs have an indispensable role in the maintenance of tolerance after allogeneic HSC transplantation (HSCT). Patients with chronic graft-versus-host disease (GVHD) have fewer circulating Tregs, but the mechanisms that lead to this deficiency of Tregs after HSCT are not known. Here, we analyzed reconstitution of Tregs and conventional [CD4.sup.+] T cells (Tcons) in patients who underwent allogeneic HSCT after myeloablative conditioning. Following transplant, thymic generation of naive Tregs was markedly impaired, and reconstituting Tregs had a predominantly activated/memory phenotype. In response to [CD4.sup.+] lymphopenia after HSCT, Tregs underwent higher levels of proliferation than Tcons, but Tregs undergoing homeostatic proliferation also showed increased susceptibility to Fas-mediated apoptosis. Prospective monitoring of [CD4.sup.+] T cell subsets revealed that Tregs rapidly expanded and achieved normal levels by 9 months after HSCT, but Treg levels subsequently declined in patients with prolonged [CD4.sup.+] lymphopenia. This resulted in a relative deficiency of Tregs, which was associated with a high incidence of extensive chronic GVHD. These studies indicate that [CD4.sup.+] lymphopenia is a critical factor in Treg homeostasis and that prolonged imbalance of Treg homeostasis after HSCT can result in loss of tolerance and significant clinical disease manifestations.

Leukemia relapse is a major cause of death after allogeneic hematopoietic cell transplantation (allo-HCT). We tested the potential of targeting T cell (Tc) immunoglobulin and mucin-containing molecule 3 (TIM-3) for improving graft-versus-leukemia (GVL) effects. We observed differential expression of TIM-3 ligands when hematopoietic stem cells overexpressed certain oncogenic-driver mutations. Anti-TIM-3 Ab treatment improved survival of mice bearing leukemia with oncogene-induced TIM-3 ligand expression. Conversely, leukemia cells with low ligand expression were anti-TIM-3 treatment resistant. In vitro, TIM-3 blockade or genetic deletion in CD8+ Tc enhanced Tc activation, proliferation, and IFN-γ production while enhancing GVL effects, preventing Tc exhaustion, and improving Tc cytotoxicity and glycolysis in vivo. Conversely, TIM-3 deletion in myeloid cells did not affect allogeneic Tc proliferation and activation in vitro, suggesting that anti-TIM-3 treatment-mediated GVL effects are Tc induced. In contrast to anti-programmed cell death protein 1 (anti-PD-1) and anti-cytotoxic T lymphocyte-associated protein 4 (anti-CTLA-4) treatment, anti-TIM-3-treatment did not enhance acute graft-versus-host disease (aGVHD). TIM-3 and its ligands were frequently expressed in acute myeloid leukemia (AML) cells of patients with post-allo-HCT relapse. We decipher the connections between oncogenic mutations found in AML and TIM-3 ligand expression and identify anti-TIM-3 treatment as a strategy for enhancing GVL effects via metabolic and transcriptional Tc reprogramming without exacerbation of aGVHD. Our findings support clinical testing of anti-TIM-3 Ab in patients with AML relapse after allo-HCT.