Sepsis arises from diverse and incompletely understood dysregulated host response processes following infection that leads to life-threatening organ dysfunction. Here we showed that neutrophils and ...emergency granulopoiesis drove a maladaptive response during sepsis. We generated a whole-blood single-cell multiomic atlas (272,993 cells, n = 39 individuals) of the sepsis immune response that identified populations of immunosuppressive mature and immature neutrophils. In co-culture, CD66b
sepsis neutrophils inhibited proliferation and activation of CD4
T cells. Single-cell multiomic mapping of circulating hematopoietic stem and progenitor cells (HSPCs) (29,366 cells, n = 27) indicated altered granulopoiesis in patients with sepsis. These features were enriched in a patient subset with poor outcome and a specific sepsis response signature that displayed higher frequencies of IL1R2
immature neutrophils, epigenetic and transcriptomic signatures of emergency granulopoiesis in HSPCs and STAT3-mediated gene regulation across different infectious etiologies and syndromes. Our findings offer potential therapeutic targets and opportunities for stratified medicine in severe infection.
Mucosal-associated invariant T (MAIT) cells are T cells that recognise vitamin-B derivative Ag presented by the MHC-related-protein 1 (MR1) antigen-presenting molecule. While MAIT cells are highly ...abundant in humans, their role in tumour immunity remains unknown. Here we have analysed the frequency and function of MAIT cells in multiple myeloma (MM) patients. We show that MAIT cell frequency in blood is reduced compared to healthy adult donors, but comparable to elderly healthy control donors. Furthermore, there was no evidence that MAIT cells accumulated at the disease site (bone marrow) of these patients. Newly diagnosed MM patient MAIT cells had reduced IFNγ production and CD27 expression, suggesting an exhausted phenotype, although IFNγ-producing capacity is restored in relapsed/refractory patient samples. Moreover, immunomodulatory drugs Lenalidomide and Pomalidomide, indirectly inhibited MAIT cell activation. We further show that cell lines can be pulsed with vitamin-B derivative Ags and that these can be presented via MR1 to MAIT cells in vitro, to induce cytotoxic activity comparable to that of natural killer (NK) cells. Thus, MAIT cells are reduced in MM patients, which may contribute to disease in these individuals, and moreover, MAIT cells may represent new immunotherapeutic targets for treatment of MM and other malignancies.
Chimeric antigen receptor (CAR) T cells have been highly successful in treating hematological malignancies, including acute and chronic lymphoblastic leukemia. However, treatment of solid tumors ...using CAR T cells has been largely unsuccessful to date, partly because of tumor-induced immunosuppressive mechanisms, including adenosine production. Previous studies have shown that adenosine generated by tumor cells potently inhibits endogenous antitumor T cell responses through activation of adenosine 2A receptors (A2ARs). Herein, we have observed that CAR activation resulted in increased A2AR expression and suppression of both murine and human CAR T cells. This was reversible using either A2AR antagonists or genetic targeting of A2AR using shRNA. In 2 syngeneic HER2+ self-antigen tumor models, we found that either genetic or pharmacological targeting of the A2AR profoundly increased CAR T cell efficacy, particularly when combined with PD-1 blockade. Mechanistically, this was associated with increased cytokine production of CD8+ CAR T cells and increased activation of both CD8+ and CD4+ CAR T cells. Given the known clinical relevance of the CD73/adenosine pathway in several solid tumor types, and the initiation of phase I trials for A2AR antagonists in oncology, this approach has high translational potential to enhance CAR T cell efficacy in several cancer types.
While adoptive transfer of T cells bearing a chimeric antigen receptor (CAR) can eliminate substantial burdens of some leukemias, the ultimate challenge remains the eradication of large solid tumors ...for most cancers. We aimed to develop an immunotherapy approach effective against large tumors in an immunocompetent, self-antigen preclinical mouse model.
In this study, we generated dual-specific T cells expressing both a CAR specific for Her2 and a TCR specific for the melanocyte protein (gp100). We used a regimen of adoptive cell transfer incorporating vaccination (ACTIV), with recombinant vaccinia virus expressing gp100, to treat a range of tumors including orthotopic breast tumors and large liver tumors.
ACTIV therapy induced durable complete remission of a variety of Her2
tumors, some in excess of 150 mm
, in immunocompetent mice expressing Her2 in normal tissues, including the breast and brain. Vaccinia virus induced extensive proliferation of T cells, leading to massive infiltration of T cells into tumors. Durable tumor responses required the chemokine receptor CXCR3 and exogenous IL2, but were independent of IFNγ. Mice were resistant to tumor rechallenge, indicating immune memory involving epitope spreading. Evidence of limited neurologic toxicity was observed, associated with infiltration of cerebellum by T cells, but was only transient.
This study supports a view that it is possible to design a highly effective combination immunotherapy for solid cancers, with acceptable transient toxicity, even when the target antigen is also expressed in vital tissues.
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Adoptive immunotherapy utilizing chimeric antigen receptor (CAR) T cells has demonstrated high success rates in hematologic cancers, but results against solid malignancies have been limited to date, ...due in part to the immunosuppressive tumor microenvironment. Activation of the 4-1BB (CD137) pathway using an agonistic α-4-1BB antibody is known to provide strong costimulatory signals for augmenting and diversifying T-cell responses. We therefore hypothesized that a combination of α-4-1BB and CAR T-cell therapy would result in improved antitumor responses. Using a human-Her2 self-antigen mouse model, we report here that α-4-1BB significantly enhanced CAR T-cell efficacy directed against the Her2 antigen in two different established solid tumor settings. Treatment also increased the expression of IFNγ and the proliferation marker Ki67 in tumor-infiltrating CAR T cells when combined with α-4-1BB. Strikingly, α-4-1BB significantly reduced host immunosuppressive cells at the tumor site, including regulatory T cells and myeloid-derived suppressor cells, correlating with an increased therapeutic response. We conclude that α-4-1BB has a multifunctional role for enhancing CAR T-cell responses and that this combination therapy has high translational potential, given current phase I/II clinical trials with α-4-1BB against various types of cancer.
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Bach2 codes for a transcriptional regulator exerting major influences on T cell‐mediated immune regulation. Effector CTLs derived from in vitro activation of murine CD8+ T cells showed increased ...proliferative and cytolytic capacity in the absence of BACH2. Before activation, BACH2‐deficient splenic CD8+ T cells had a higher abundance of memory and reduced abundance of naïve cells compared to wild‐type. CTLs derived from central memory T cells were more potently cytotoxic than those derived from naïve T cells, but even within separated subsets, BACH2‐deficiency conferred a cytotoxic advantage. Immunofluorescence and electron microscopy revealed larger granules in BACH2‐deficient compared to wild‐type CTLs, and proteomic analysis showed an increase in granule content, including perforin and granzymes. Thus, the enhanced cytotoxicity observed in effector CTLs lacking BACH2 arises not only from differences in their initial differentiation state but also inherent production of enlarged cytolytic granules. These results demonstrate how a single gene deletion can produce a CTL super‐killer.
CTLs generated from mice lacking BACH2 show increased target cell killing. This arises both from increased numbers of memory CD8+ (CD8+mem) relative to naïve (CD8+n) splenocytes as well as the increased expression of cytolytic proteins localised in enlarged granules. This study shows that loss of BACH2 generates “super‐killer” CTLs.
BackgroundHigh-grade gliomas including glioblastoma (GBM) and diffuse midline gliomas (DMG) represent the most lethal and aggressive brain cancers where current treatment modalities offer limited ...efficacy. Chimeric antigen receptor (CAR) T cell therapies have emerged as a promising strategy, boasting tumor-specific targeting and the unique ability to penetrate the blood-brain barrier. However, the effective clinical application hinges on the optimal choice of antigen, with a limited number, currently under investigation.MethodsWe employed cell surface proteomic analysis of primary human high-grade glioma samples from both adult and pediatric patients. This led to the identification of Ephrin type-A receptor 3 (EphA3) as a prevalently expressed target. We engineered a second-generation EphA3-targeted CAR T cell and assessed function using in vitro and in vivo models of GBM and DMG.ResultsEphA3-targeted CAR T cells demonstrated robust antigen-specific killing of human GBM and DMG cell lines in vitro. In an orthotopic xenograft NSG mouse model, EphA3-targeted CAR T cells not only effectively eradicated tumors but also established a functional T cell population protective on rechallenge. Remarkably, mice rechallenged with a second contralateral orthotopic tumor implantation achieved complete tumor clearance and maintained a sustained complete response 6 months following initial treatment.ConclusionBuilding on the proven safety profile of EphA3 antibodies in clinical settings, our study provides compelling preclinical evidence supporting the efficacy of EphA3-targeted CAR T cells against high-grade gliomas. These findings underscore the potential for transitioning this innovative therapy into clinical trials, aiming to revolutionize the treatment landscape for patients afflicted with these formidable brain cancers.
Introduction
Diffuse intrinsic pontine glioma (DIPG), recently reclassified as a subtype of diffuse midline glioma, is a highly aggressive brainstem tumor affecting children and young adults, with no ...cure and a median survival of only 9 months. Conventional treatments are ineffective, highlighting the need for alternative therapeutic strategies such as cellular immunotherapy. However, identifying unique and tumor-specific cell surface antigens to target with chimeric antigen receptor (CAR) or T-cell receptor (TCR) therapies is challenging.
Methods
In this study, a multi-omics approach was used to interrogate patient-derived DIPG cell lines and to identify potential targets for immunotherapy.
Results
Through immunopeptidomics, a range of targetable peptide antigens from cancer testis and tumor-associated antigens as well as peptides derived from human endogenous retroviral elements were identified. Proteomics analysis also revealed upregulation of potential drug targets and cell surface proteins such as Cluster of differentiation 27 (CD276) B7 homolog 3 protein (B7H3), Interleukin 13 alpha receptor 2 (IL-13Rα2), Human Epidermal Growth Factor Receptor 3 (HER2), Ephrin Type-A Receptor 2 (EphA2), and Ephrin Type-A Receptor 3 (EphA3).
Discussion
The results of this study provide a valuable resource for the scientific community to accelerate immunotherapeutic approaches for DIPG. Identifying potential targets for CAR and TCR therapies could open up new avenues for treating this devastating disease.
Immunotherapy is widely accepted as a powerful new treatment modality for the treatment of cancer. The most successful form of immunotherapy to date has been the blockade of the immune checkpoints ...PD-1 and CTLA-4. Combining inhibitors of both PD-1 and CTLA-4 increases the proportion of patients who respond to immunotherapy. However, most patients still do not respond to checkpoint inhibitors, and prognostic biomarkers are currently lacking. Therefore, a better understanding of the mechanism by which these checkpoint inhibitors enhance antitumor immune responses is required to more accurately predict which patients are likely to respond and further enhance this treatment modality. Our current study of two mouse tumor models revealed that CD4
Foxp3
cells activated by dual PD-1/CTLA-4 blockade modulated the myeloid compartment, including activation of conventional CD103
dendritic cells (DC) and expansion of a myeloid subset that produces TNFα and iNOS (TIP-DCs). CD4
Foxp3
T cell-mediated activation of CD103
DCs resulted in enhanced IL12 production by these cells and IL12 enhanced the therapeutic effect of dual PD-1/CTLA-4 blockade. Given the importance of these myeloid subsets in the antitumor immune response, our data point to a previously underappreciated role of CD4
Foxp3
cells in modulating this arm of the antitumor immune response.
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BMP9, originating from the liver, and BMP10 are circulating BMPs that preserve vascular endothelial integrity. We assessed BMP9, BMP10 and soluble endoglin (sEng) levels and their relationships to ...liver disease severity and associated pulmonary vascular syndromes in a cohort of well-characterised liver disease patients.
Plasma samples from patients with liver disease (n = 83) and non-disease controls (n = 21) were assayed for BMP9, BMP10 and sEng. Levels were also assessed in a separate cohort of controls (n = 27) and PoPH patients (n = 8). Expression of mRNA and immunohistochemical staining was undertaken in liver biopsy specimens. Plasma BMP activity was assessed using an endothelial cell bioassay.
Plasma BMP9 and BMP10 levels were normal in patients with compensated cirrhosis or fibrosis without cirrhosis, but markedly reduced in patients with decompensated cirrhosis, including those with hepatopulmonary syndrome (HPS) or portopulmonary hypertension (PoPH). Liver biopsy specimens revealed reduced mRNA expression and immunostaining for these ligands. Patient plasma samples with reduced BMP9 and BMP10 levels exhibited low BMP activity that was restored with exogenous BMP9. Endoglin mRNA expression was increased in cirrhotic livers and elevated circulating sEng levels in PoPH and HPS patients suggested increased endothelial sEng shedding in these syndromes.
Plasma BMP9 and BMP10 levels are reduced in decompensated cirrhosis, leading to reduced circulating BMP activity on the vascular endothelium. The pulmonary complications of cirrhosis, PoPH and HPS, are associated with markedly reduced BMP9 and BMP10 and increased sEng levels, suggesting that supplementation with exogenous ligands might be a therapeutic approach for PoPH and HPS.
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