RNA aggregates harness the danger response for potent cancer immunotherapy.

Cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Here, we create "onion-like" multi-lamellar RNA lipid particle aggregates (LPAs) to substantially enhance the payload packaging and immunogenicity of tumor mRNA antigens. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for Toll-like receptor engagement in immune cells, systemically administered RNA-LPAs activate RIG-I in stromal cells, eliciting massive cytokine/chemokine response and dendritic cell/lymphocyte trafficking that provokes cancer immunogenicity and mediates rejection of both early- and late-stage murine tumor models. In client-owned canines with terminal gliomas, RNA-LPAs improved survivorship and reprogrammed the TME, which became "hot" within days of a single infusion. In a first-in-human trial, RNA-LPAs elicited rapid cytokine/chemokine release, immune activation/trafficking, tissue-confirmed pseudoprogression, and glioma-specific immune responses in glioblastoma patients. These data support RNA-LPAs as a new technology that simultaneously reprograms the TME while eliciting rapid and enduring cancer immunotherapy.

Expanded specific T cells to hypomutated regions of the SARS-CoV-2 using mRNA electroporated antigen-presenting cells.

The COVID-19 pandemic has caused about seven million deaths worldwide. Preventative vaccines have been developed including Spike gp mRNA-based vaccines that provide protection to immunocompetent patients. However, patients with primary immunodeficiencies, patients with cancer, or hematopoietic stem cell transplant recipients are not able to mount robust immune responses against current vaccine approaches. We propose to target structural SARS-CoV-2 antigens (i.e., Spike gp, Membrane, Nucleocapsid, and Envelope) using circulating human antigen-presenting cells electroporated with full length SARS-CoV-2 structural protein-encoding mRNAs to activate and expand specific T cells. Based on the Th1-type cytokine and cytolytic enzyme secretion upon antigen rechallenge, we were able to generate SARS-CoV-2 specific T cells in up to 70% of unexposed unvaccinated healthy donors (HDs) after 3 subsequent stimulations and in 100% of recovered patients (RPs) after 2 stimulations. By means of SARS-CoV-2 specific TCRß repertoire analysis, T cells specific to Spike gp-derived hypomutated regions were identified in HDs and RPs despite viral genomic evolution. Hence, we demonstrated that SARS-CoV-2 mRNA-loaded antigen-presenting cells are effective activating and expanding COVID19-specific T cells. This approach represents an alternative to patients who are not able to mount adaptive immune responses to current COVID-19 vaccines with potential protection across new variants that have conserved genetic regions.

Vaccines: a promising therapy for myelodysplastic syndrome.

Myelodysplastic neoplasms (MDS) define clonal hematopoietic malignancies characterized by heterogeneous mutational and clinical spectra typically seen in the elderly. Curative treatment entails allogeneic hematopoietic stem cell transplant, which is often not a feasible option due to older age and significant comorbidities. Immunotherapy has the cytotoxic capacity to elicit tumor-specific killing with long-term immunological memory. While a number of platforms have emerged, therapeutic vaccination presents as an appealing strategy for MDS given its promising safety profile and amenability for commercialization. Several preclinical and clinical trials have investigated the efficacy of vaccines in MDS; these include peptide vaccines targeting tumor antigens, whole cell-based vaccines and dendritic cell-based vaccines. These therapeutic vaccines have shown acceptable safety profiles, but consistent clinical responses remain elusive despite robust immunological reactions. Combining vaccines with immunotherapeutic agents holds promise and requires further investigation. Herein, we highlight therapeutic vaccine trials while reviewing challenges and future directions of successful vaccination strategies in MDS.

mRNA-based precision targeting of neoantigens and tumor-associated antigens in malignant brain tumors.

BACKGROUND: Despite advancements in the successful use of immunotherapy in treating a variety of solid tumors, applications in treating brain tumors have lagged considerably. This is due, at least in part, to the lack of well-characterized antigens expressed within brain tumors that can mediate tumor rejection; the low mutational burden of these tumors that limits the abundance of targetable neoantigens; and the immunologically "cold" tumor microenvironment that hampers the generation of sustained and productive immunologic responses. The field of mRNA-based therapeutics has experienced a boon following the universal approval of COVID-19 mRNA vaccines. mRNA-based immunotherapeutics have also garnered widespread interest for their potential to revolutionize cancer treatment. In this study, we developed a novel and scalable approach for the production of personalized mRNA-based therapeutics that target multiple tumor rejection antigens in a single therapy for the treatment of refractory brain tumors. METHODS: Tumor-specific neoantigens and aberrantly overexpressed tumor-associated antigens were identified for glioblastoma and medulloblastoma tumors using our cancer immunogenomics pipeline called Open Reading Frame Antigen Network (O.R.A.N). Personalized tumor antigen-specific mRNA vaccine was developed for each individual tumor model using selective gene capture and enrichment strategy. The immunogenicity and efficacy of the personalized mRNA vaccines was evaluated in combination with anti-PD-1 immune checkpoint blockade therapy or adoptive cellular therapy with ex vivo expanded tumor antigen-specific lymphocytes in highly aggressive murine GBM models. RESULTS: Our results demonstrate the effectiveness of the antigen-specific mRNA vaccines in eliciting robust anti-tumor immune responses in GBM hosts. Our findings substantiate an increase in tumor-infiltrating lymphocytes characterized by enhanced effector function, both intratumorally and systemically, after antigen-specific mRNA-directed immunotherapy, resulting in a favorable shift in the tumor microenvironment from immunologically cold to hot. Capacity to generate personalized mRNA vaccines targeting human GBM antigens was also demonstrated. CONCLUSIONS: We have established a personalized and customizable mRNA-therapeutic approach that effectively targets a plurality of tumor antigens and demonstrated potent anti-tumor response in preclinical brain tumor models. This platform mRNA technology uniquely addresses the challenge of tumor heterogeneity and low antigen burden, two key deficiencies in targeting the classically immunotherapy-resistant CNS malignancies, and possibly other cold tumor types.

Pulmonary microbiome and transcriptome signatures reveal distinct pathobiologic states associated with mortality in two cohorts of pediatric stem cell transplant patients.

Lung injury is a major determinant of survival after pediatric hematopoietic cell transplantation (HCT). A deeper understanding of the relationship between pulmonary microbes, immunity, and the lung epithelium is needed to improve outcomes. In this multicenter study, we collected 278 bronchoalveolar lavage (BAL) samples from 229 patients treated at 32 children's hospitals between 2014-2022. Using paired metatranscriptomes and human gene expression data, we identified 4 patient clusters with varying BAL composition. Among those requiring respiratory support prior to sampling, in-hospital mortality varied from 22-60% depending on the cluster (p=0.007). The most common patient subtype, Cluster 1, showed a moderate quantity and high diversity of commensal microbes with robust metabolic activity, low rates of infection, gene expression indicating alveolar macrophage predominance, and low mortality. The second most common cluster showed a very high burden of airway microbes, gene expression enriched for neutrophil signaling, frequent bacterial infections, and moderate mortality. Cluster 3 showed significant depletion of commensal microbes, a loss of biodiversity, gene expression indicative of fibroproliferative pathways, increased viral and fungal pathogens, and high mortality. Finally, Cluster 4 showed profound microbiome depletion with enrichment of Staphylococci and viruses, gene expression driven by lymphocyte activation and cellular injury, and the highest mortality. BAL clusters were modeled with a random forest classifier and reproduced in a geographically distinct validation cohort of 57 patients from The Netherlands, recapitulating similar cluster-based mortality differences (p=0.022). Degree of antibiotic exposure was strongly associated with depletion of BAL microbes and enrichment of fungi. Potential pathogens were parsed from all detected microbes by analyzing each BAL microbe relative to the overall microbiome composition, which yielded increased sensitivity for numerous previously occult pathogens. These findings support personalized interpretation of the pulmonary microenvironment in pediatric HCT, which may facilitate biology-targeted interventions to improve outcomes.

Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable success as an immunotherapy for hematological malignancies, and its potential for treating solid tumors is an active area of research. However, limited trafficking and mobility of T cells within the tumor microenvironment (TME) present challenges for CAR T cell therapy in solid tumors. To gain a better understanding of CAR T cell function in solid tumors, we subjected CD70-specific CAR T cells to a challenge by evaluating their immune trafficking and infiltration through a confined 3D microchannel network in a bio-conjugated liquid-like solid (LLS) medium. Our results demonstrated successful CAR T cell migration and anti-tumor activity against CD70-expressing glioblastoma and osteosarcoma tumors. Through comprehensive analysis of cytokines and chemokines, combined with in situ imaging, we elucidated that immune recruitment occurred via chemotaxis, and the effector-to-target ratio plays an important role in overall antitumor function. Furthermore, through single-cell collection and transcriptomic profiling, we identified differential gene expression among the immune subpopulations. Our findings provide valuable insights into the complex dynamics of CAR T cell function in solid tumors, informing future research and development in this promising cancer treatment approach. STATEMENT OF SIGNIFICANCE: The use of specialized immune cells named CAR T cells to combat cancers has demonstrated remarkable success against blood cancers. However, this success is not replicated in solid tumors, such as brain or bone cancers, mainly due to the physical barriers of these solid tumors. Currently, preclinical technologies do not allow for reliable evaluation of tumor-immune cell interactions. To better study these specialized CAR T cells, we have developed an innovative in vitro three-dimensional model that promises to dissect the interactions between tumors and CAR T cells at the single-cell level. Our findings provide valuable insights into the complex dynamics of CAR T cell function in solid tumors, informing future research and development in this promising cancer treatment approach.

Allogeneic hematopoietic cell transplantation for blastic plasmacytoid dendritic cell neoplasm: a CIBMTR analysis.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematological malignancy with a poor prognosis and considered incurable with conventional chemotherapy. Small observational studies reported allogeneic hematopoietic cell transplantation (allo-HCT) offers durable remissions in patients with BPDCN. We report an analysis of patients with BPDCN who received an allo-HCT, using data reported to the Center for International Blood and Marrow Transplant Research (CIBMTR). We identified 164 patients with BPDCN from 78 centers who underwent allo-HCT between 2007 and 2018. The 5-year overall survival (OS), disease-free survival (DFS), relapse, and nonrelapse mortality (NRM) rates were 51.2% (95% confidence interval [CI], 42.5-59.8), 44.4% (95% CI, 36.2-52.8), 32.2% (95% CI, 24.7-40.3), and 23.3% (95% CI, 16.9-30.4), respectively. Disease relapse was the most common cause of death. On multivariate analyses, age of ≥60 years was predictive for inferior OS (hazard ratio [HR], 2.16; 95% CI, 1.35-3.46; P = .001), and higher NRM (HR, 2.19; 95% CI, 1.13-4.22; P = .02). Remission status at time of allo-HCT (CR2/primary induction failure/relapse vs CR1) was predictive of inferior OS (HR, 1.87; 95% CI, 1.14-3.06; P = .01) and DFS (HR, 1.75; 95% CI, 1.11-2.76; P = .02). Use of myeloablative conditioning with total body irradiation (MAC-TBI) was predictive of improved DFS and reduced relapse risk. Allo-HCT is effective in providing durable remissions and long-term survival in BPDCN. Younger age and allo-HCT in CR1 predicted for improved survival, whereas MAC-TBI predicted for less relapse and improved DFS. Novel strategies incorporating allo-HCT are needed to further improve outcomes.

Feasibility study of busulfan, fludarabine, and thiotepa conditioning regimen for allogeneic hematopoietic stemcell transplantationfor children and young adults with nonmalignant disorders.

BACKGROUND: Hematopoietic stem cell transplant (HSCT) is the only curative treatment for several pediatric non-malignant disorders. A widely used conditioning backbone is busulfan, fludarabine, and rabbit anti-thymocyte globulin (rATG). Thiotepa has improved engraftment when added to this regimen, however the minimum effective dose (MED) of thiotepa to achieve engraftment while minimizing toxicities has not been well established. OBJECTIVES: The primary objective of this prospective feasibility study was to determine the MED of thiotepa (5mg/kg) in combination with reduced-dose busulfan, fludarabine or cyclophosphamide, and rATG required to achieve engraftment in >90% of HSCT recipients for non-malignant disorders with acceptable toxicity. RESULTS: Six patients who received fully matched HSCT were enrolled. Patient diagnoses included Wiskott-Aldrich syndrome (n = 1), CD40L deficiency (n = 1), sickle cell disease (n = 2), autoinflammatory syndrome (n = 1), and paroxysmal nocturnal hemoglobinuria (n = 1). All six patients achieved engraftment prior to Day +42 and five patients had stable full donor engraftment. Two of the six patients (33%) developed acute GVHD and/or chronic GHVD, both of whom had sickle cell disease. At a median follow-up of 2.25 years post-transplant, all patients were alive without evidence of disease recurrence. None of the patients experienced grade 4 or 5 toxicities. Three out of six patients (50%) developed grade 3 adverse events. Neurocognitive functioning of children under 10 years of age was not adversely affected by this regimen. CONCLUSION: This approach shows acceptable toxicity and reliable engraftment in children with non-malignant disorders receiving related or unrelated HLA-matched transplants.

Three-Dimensional Bioconjugated Liquid-Like Solid (LLS) Enhance Characterization of Solid Tumor - Chimeric Antigen Receptor T cell interactions.

Cancer immunotherapy offers lifesaving treatments for cancers, but the lack of reliable preclinical models that could enable the mechanistic studies of tumor-immune interactions hampers the identification of new therapeutic strategies. We hypothesized 3D confined microchannels, formed by interstitial space between bio-conjugated liquid-like solids (LLS), enable CAR T dynamic locomotion within an immunosuppressive TME to carry out anti-tumor function. Murine CD70-specific CAR T cells cocultured with the CD70-expressing glioblastoma and osteosarcoma demonstrated efficient trafficking, infiltration, and killing of cancer cells. The anti-tumor activity was clearly captured via longterm in situ imaging and supported by upregulation of cytokines and chemokines including IFNg, CXCL9, CXCL10, CCL2, CCL3, and CCL4. Interestingly, target cancer cells, upon an immune attack, initiated an "immune escape" response by frantically invading the surrounding microenvironment. This phenomenon however was not observed for the wild-type tumor samples which remained intact and produced no relevant cytokine response. Single cells collection and transcriptomic profiling of CAR T cells at regions of interest revealed feasibility of identifying differential gene expression amongst the immune subpopulations. Complimentary 3D in vitro platforms are necessary to uncover cancer immune biology mechanisms, as emphasized by the significant roles of the TME and its heterogeneity.

mRNA challenge predicts brain cancer immunogenicity and response to checkpoint inhibitors.

To prospectively determine whether brain tumors will respond to immune checkpoint inhibitors (ICIs), we developed a novel mRNA vaccine as a viral mimic to elucidate cytokine release from brain cancer cells in vitro. Our results indicate that cytokine signatures following mRNA challenge differ substantially from ICI responsive versus non-responsive murine tumors. These findings allow for creation of a diagnostic assay to quickly assess brain tumor immunogenicity, allowing for informed treatment with ICI or lack thereof in poorly immunogenic settings.

mRNA aggregates harness danger response for potent cancer immunotherapy.

Messenger RNA (mRNA) has emerged as a remarkable tool for COVID-19 prevention but its use for induction of therapeutic cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Herein, we develop a facile approach for substantially enhancing immunogenicity of tumor-derived mRNA in lipid-particle (LP) delivery systems. By using mRNA as a molecular bridge with ultrapure liposomes and foregoing helper lipids, we promote the formation of 'onion-like' multi-lamellar RNA-LP aggregates (LPA). Intravenous administration of RNA-LPAs mimics infectious emboli and elicits massive DC/T cell mobilization into lymphoid tissues provoking cancer immunogenicity and mediating rejection of both early and late-stage murine tumor models. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for toll-like receptor engagement, RNA-LPAs stimulate intracellular pathogen recognition receptors (RIG-I) and reprogram the TME thus enabling therapeutic T cell activity. RNA-LPAs were safe in acute/chronic murine GLP toxicology studies and immunologically active in client-owned canines with terminal gliomas. In an early phase first-in-human trial for patients with glioblastoma, we show that RNA-LPAs encoding for tumor-associated antigens elicit rapid induction of pro-inflammatory cytokines, mobilization/activation of monocytes and lymphocytes, and expansion of antigen-specific T cell immunity. These data support the use of RNA-LPAs as novel tools to elicit and sustain immune responses against poorly immunogenic tumors.

Impact of pre-transplant induction and consolidation cycles on AML allogeneic transplant outcomes: a CIBMTR analysis in 3113 AML patients.

We investigated the impact of the number of induction/consolidation cycles on outcomes of 3113 adult AML patients who received allogeneic hematopoietic cell transplantation (allo-HCT) between 2008 and 2019. Patients received allo-HCT using myeloablative (MAC) or reduced-intensity (RIC) conditioning in first complete remission (CR) or with primary induction failure (PIF). Patients who received MAC allo-HCT in CR after 1 induction cycle had 1.3-fold better overall survival (OS) than 2 cycles to CR and 1.47-fold better than ≥3 cycles. OS after CR in 2 or ≥3 cycles was similar. Relapse risk was 1.65-fold greater in patients receiving ≥3 cycles to achieve CR. After RIC allo-HCT, the number of induction cycles to CR did not affect OS. Compared to CR in 1 cycle, relapse risk was 1.24-1.41-fold greater in patients receiving 2 or ≥3 cycles. For patients receiving only 1 cycle to CR, consolidation therapy prior to MAC allo-HCT was associated with improved OS vs. no consolidation therapy. Detectable MRD at the time of MAC allo-HCT did not impact outcomes while detectable MRD preceding RIC allo-HCT was associated with an increased risk of relapse. For allo-HCT in PIF, OS was significantly worse than allo-HCT in CR after 1-3 cycles.

Vitamin D Supplementation: Association With Serum Cytokines in Pediatric Hematopoietic Stem Cell Transplantation.

Vitamin D deficiency is prevalent in pediatric patients presenting for hematopoietic stem cell transplantation (HSCT) and has been linked to poor clinical outcomes. Using the data from a randomized control trial, in this paper we explore the effects of vitamin D supplementation on circulating cytokine levels during pediatric HSCT (www.clinicaltrials.gov as NCT03176849). A total of 41 children, 20 received Stoss therapy and 21 children received standard of care vitamin D supplementation. Levels of 25(OH)D and 20 cytokines were assessed at baseline and day +30. Significantly (P < 0.05) higher levels of mostly proinflammatory cytokines, FGF, GCSF, TNFα, IL-2, IL-6, IP10 were detected pre-transplant for patients with low compared to those with normal vitamin D levels. In sex stratified models that compare changes in cytokines between Stoss vs. standard of care, females in the Stoss group show greater changes in mostly pro -inflammatory cytokines- IP-10 (P = 0.0047), MIG (P = 0.009), and RANTES (P = 0.0047), IL-2R (P = 0.07) and IL-6(P = 0.069). Despite a small sample size, these findings suggest vitamin D deficiency affects the pre-transplant cytokine milieu and higher doses of vitamin D (Stoss therapy) appears to influence proinflammatory cytokine responses in a sex specific manner during pediatric HSCT. Larger clinical trials are warranted to validate these results.

CAR T Cell Locomotion in Solid Tumor Microenvironment.

The promising outcomes of chimeric antigen receptor (CAR) T cell therapy in hematologic malignancies potentiates its capability in the fight against many cancers. Nevertheless, this immunotherapy modality needs significant improvements for the treatment of solid tumors. Researchers have incrementally identified limitations and constantly pursued better CAR designs. However, even if CAR T cells are armed with optimal killer functions, they must overcome and survive suppressive barriers imposed by the tumor microenvironment (TME). In this review, we will discuss in detail the important role of TME in CAR T cell trafficking and how the intrinsic barriers contribute to an immunosuppressive phenotype and cancer progression. It is of critical importance that preclinical models can closely recapitulate the in vivo TME to better predict CAR T activity. Animal models have contributed immensely to our understanding of human diseases, but the intensive care for the animals and unreliable representation of human biology suggest in vivo models cannot be the sole approach to CAR T cell therapy. On the other hand, in vitro models for CAR T cytotoxic assessment offer valuable insights to mechanistic studies at the single cell level, but they often lack in vivo complexities, inter-individual heterogeneity, or physiologically relevant spatial dimension. Understanding the advantages and limitations of preclinical models and their applications would enable more reliable prediction of better clinical outcomes.

Impact of Adequate Day 30 Post-Pediatric Hematopoietic Stem Cell Transplantation Vitamin D Level on Clinical Outcome: An Observational Cohort Study.

This prospective observational study evaluated the impact of adequate vitamin D levels by day +30 after vitamin D supplementation on early post-HSCT outcomes, including acute graft-versus-host disease (aGVHD), immune recovery, infection rates, and overall survival. Forty children (age 2 to 16 years) undergoing hematopoietic stem cell transplantation (HSCT) were given vitamin D supplementation, were followed prospectively from day +30 post-transplantation, and had day +30 vitamin D levels measured. Thirty patients with normal vitamin D levels (≥30 ng/mL) were compared with 10 patients with low day +30 vitamin D levels (<30 ng/mL). The times to neutrophil and platelet engraftment was similar in both day +30 vitamin D groups (P = .13 and .32, respectively). At day +100, slower immune recovery in CD4+ cells (P = .027), CD19+ cells (P = .024), and natural killer cells (P = .042) was observed in the patients with a low vitamin D level (<30 ng/mL), and no between-group differences were detected in the incidence of infection (P = .72) or grade II-IV aGVHD (P = .46). Our findings show that patients with adequate vitamin D levels during transplantation had faster immune recovery and better overall survival. Vitamin D deficiency does not appear to impact engraftment or the risk of aGVHD and infection in pediatric HSCT.

Natural Killer Cell Alloreactivity Predicted By Killer Cell Immunoglobulin-Like Receptor Ligand Mismatch Does Not Impact Engraftment in Umbilical Cord Blood and Haploidentical Stem Cell Transplantation.

Natural killer cell alloreactivity is determined by killer cell immunoglobulin-like receptor (KIR) ligands in donor and recipient pairs. A small, single institution study suggested that the risk of primary graft failure after cord blood hematopoietic cell transplantation (CBT) can be predicted by host-versus-graft (HvG)-directed natural killer cell alloreactivity. In the haploidentical transplantation (Haplo HCT) cohort, graft failures were observed only in graft-versus-host (GvH) KIR ligand mismatched pairs. A subsequent study was designed to explore the association between HvG and GvH KIR ligand mismatching and engraftment in both CBT and Haplo HCT using the large, multicenter transplant population of the Center for International Blood and Transplant Research database. Nine hundred single CBT (sCBT), 954 double CBT (dCBT), and 671 Haplo HCT performed between 2008 and 2017 for acute leukemias and myelodysplastic syndrome were examined. Several models of KIR-L interactions were analyzed by multiple regression analyses for their association with engraftment, overall survival (OS), and transplant-related mortality (TRM). In sCBT, although HvG or bidirectional KIR ligand mismatch (KIR-L-MM) was initially associated with higher TRM in the first 6 months after transplantation, this effect was nullified after 6 months such that long-term survival was not different compared to GvH KIR-L-MM or KIR-L matched (KIR-L-M) pairs. There was no significant difference in neutrophil and platelet engraftment. In dCBT, no significant differences were seen in engraftment, OS and TRM. In the Haplo cohort there was faster platelet recovery in the GvH KIR-L-MM/KIR-L-M pairs versus HvG KIR-L-MM or bidirectional mismatch (HR 1.23, P= .0116). There was no significant association with OS, TRM, or neutrophil engraftment. In this large registry study, KIR-L mismatching did not significantly impact engraftment, TRM, or survival in CBT and Haplo HCT, although an association with platelet engraftment in Haplo HCT was demonstrated.

Effects of immune checkpoint blockade on antigen-specific CD8+ T cells for use in adoptive cellular therapy.

Adoptive T-cell therapies have been successfully used as prophylaxis or treatment for immunocompromised patients at risk of viral infections or advanced cancers. Unfortunately, for some refractory cancers, they have failed. To overcome this, checkpoint inhibitors are used to rescue immune antitumor responses. We hypothesized that in vitro checkpoint blockade during T-cell stimulation and expansion with messenger RNA (mRNA)-pulsed DCs may enhance the activity of antigen-specific T cells and improve the efficacy of adoptive cellular therapy platforms. Human peripheral blood mononuclear cells were isolated from cytomegalovirus (CMV)-seropositive donors to generate DCs. These were pulsed with CMV matrix phosphoprotein 65 (CMVpp65)-mRNA to educate T cells in coculture for 15 days. Three checkpoint blockade conditions were evaluated (anti-PD1, anti-Tim3, and anti-PD1 + Tim3). IL-2 and antibodies blockades were added every 3 days. Immunophenotyping was performed on Day 0 and Day 15. Polyfunctional antigen-specific responses were evaluated upon rechallenge with CMVpp65 peptides. CMVpp65-activated CD8+ T cells upregulate Lag3 and Tim3 (P ≤ 0.0001). Tim3 antibody blockade alone or in combination led to a significant upregulation of Lag3 expression on CD8+ pp65Tetramer+ central memory, effector memory, and terminal effector memory cells re-expressing RA (TEMRA) T cells. This latter T-cell subset uniquely maintains double-positive Tim3/Lag3 expression after checkpoint blockade. By contrast, PD1 blockade had minimal effects on Tim3 or Lag3 expression. In addition, IFN-γ secretion was reduced in T cells treated with Tim3 blockade in a dose-dependent manner (P = 0.004). In this study, we have identified a potential activating component of Tim3 and linkage between Tim3 and Lag3 signaling upon blocking the Tim3 axis during T-cell-antigen-presenting cell interactions that should be considered when targeting immune checkpoints for clinical use.

Risk classification at diagnosis predicts post-HCT outcomes in intermediate-, adverse-risk, and KMT2A-rearranged AML.

Little is known about whether risk classification at diagnosis predicts post-hematopoietic cell transplantation (HCT) outcomes in patients with acute myeloid leukemia (AML). We evaluated 8709 patients with AML from the CIBMTR database, and after selection and manual curation of the cytogenetics data, 3779 patients in first complete remission were included in the final analysis: 2384 with intermediate-risk, 969 with adverse-risk, and 426 with KMT2A-rearranged disease. An adjusted multivariable analysis detected an increased risk of relapse for patients with KMT2A-rearranged or adverse-risk AML as compared to those with intermediate-risk disease (hazards ratio [HR], 1.27; P = .01; HR, 1.71; P < .001, respectively). Leukemia-free survival was similar for patients with KMT2A rearrangement or adverse risk (HR, 1.26; P = .002, and HR, 1.47; P < .001), as was overall survival (HR, 1.32; P < .001, and HR, 1.45; P < .001). No differences in outcome were detected when patients were stratified by KMT2A fusion partner. This study is the largest conducted to date on post-HCT outcomes in AML, with manually curated cytogenetics used for risk stratification. Our work demonstrates that risk classification at diagnosis remains predictive of post-HCT outcomes in AML. It also highlights the critical need to develop novel treatment strategies for patients with KMT2A-rearranged and adverse-risk disease.