Bidesh Mahata

Steroid hormones are well-known regulators of mast cell function, but whether mast cells themselves produce steroids de novo and influences their effector function has remained unknown. Here, we show that mast cells constitutively express the steroidogenic enzyme cytochrome P450 family 11 subfamily A member 1 (Cyp11a1) and produce pregnenolone. Cyp11a1 expression was detected across tissue-resident mast cells from multiple organs, was conserved in human mast cells, and was enhanced by interleukin-3 signaling and after activation. We identified Cyp11a1 as a Gata2-dependent, superenhancer-associated mast cell gene, placing steroidogenesis within the core mast cell transcriptional program. Loss of Cyp11a1 altered transcriptomic and proteomic programs in mast cells, with effects on pathways linked to cell growth, immune signaling, and effector function. Functionally, Cyp11a1-deficient mast cells showed increased degranulation in response to both immunoglobulin E (IgE)–dependent and IgE-independent stimulation. In vivo, loss of Cyp11a1 reduced mast cell abundance in several tissues but exacerbated passive cutaneous anaphylaxis. These findings identify cell-intrinsic steroidogenesis as a regulatory pathway that restrains mast cell activation and anaphylactic responses.

Glioblastoma (GBM) remains a devastating disease with few meaningful therapeutic advances over the past three decades. Dendritic cell (DC) vaccination is a promising immunotherapeutic strategy for GBM, but its efficacy is limited by the clinical use of dexamethasone to control cerebral oedema and associated symptoms. Here we show that steroid signalling is a central regulator of DC dysfunction in GBM. Through targeted metabolomics of primary GBM samples, we identified a steroid-rich tumour microenvironment in which dexamethasone is present at high levels. Across bulk and single-cell transcriptomic and epigenomic datasets, NR3C1 emerged as the dominant steroid receptor in GBM immune cells and was negatively associated with activated DC states. In patient-derived DCs, dexamethasone altered NR3C1 chromatin occupancy, induced broad transcriptional and chromatin remodelling, and suppressed co-stimulatory antigen-presentation and cytokine programmes. DC-specific deletion of Nr3c1 restricted syngeneic glioblastoma growth, enhanced DC activation, promoted cytotoxic CD8+ T cell responses and remodelled myeloid states in vivo. In GBM patient-derived DCs, pharmacological or non-viral CRISPR-mediated disruption of NR3C1 restored inflammatory, antigen presentation and T cell-stimulatory programmes, enhanced antigen-specific CD8+ T cell priming, and improved tumour lysate-loaded DC vaccination. Together, these findings identify glucocorticoid signalling as a key barrier to DC immunotherapy in GBM and establish NR3C1-targeted, steroid-resistant DCs as a potential therapeutic strategy.

Background: T helper 2 (Th2) lymphocytes orchestrate type-2 immunity and drive allergic diseases that disproportionately affect females. Sexual dimorphism in Th2 responses is well-documented, yet current models attribute sex differences exclusively to circulating gonadal hormones and sex chromosomes. Whether cell-intrinsic steroidogenesis, mediated by the enzyme Cyp11a1, contributes to female-biased Th2 differentiation and function remains unknown. Methods: Transcriptomes of in vitro generated Th2 cells from male and female T cell-specific Cyp11a1 -knockout ( Cyp11a1 ^fl/fl ; Cd4 ^Cre ) and control ( Cyp11a1 ^fl/fl ) mice were compared. Differential expression, hallmark pathway analysis, transcription factor activity scoring, and functional assays were performed across sexes and genotypes. Cyp11a1-dependent differentially expressed genes were integrated with sex-stratified human Th2 transcriptomes obtained from the type-2 inflammatory skin disease atopic dermatitis. Results: Cyp11a1 deletion markedly reduced the transcriptional signature distinguishing female from male Th2 cells. Female Cyp11a1 -knockout Th2 cells underwent extensive transcriptomic reprogramming converging toward the male profile, while male cells were largely unaffected. Female-specific pathway changes included reduced inflammatory signatures and enhanced cell-cycle programmes. Functionally, female Cyp11a1 -deficient Th2 cells exhibited significantly increased proliferation and elevated IL-13 production; male knockout cells showed no comparable changes. These effects were developmentally stage-specific, emerging during Th2 differentiation but not in naïve precursors. Cross-species analysis identified a conserved gene module shared between Cyp11a1 -deficient female mouse Th2 cells and female-biased human Th2 cells in atopic dermatitis. Conclusions: Cyp11a1-mediated steroidogenesis is a cell-intrinsic regulator of the female-biased Th2 transcriptional and functional state, identifying de novo steroidogenesis as a mechanism of immunological sexual dimorphism with direct relevance for female-predominant allergic disease.

Tumors foster an immunosuppressive microenvironment to evade the antitumor immune response. However, the influence of intratumoral immunosuppressive steroids on tumor-infiltrating natural killer (NK) cells and their implications for effective immunotherapy has remained largely unexplored. Here, we report that the functional enrichment of glucocorticoid cortisol signaling in the lung tumor microenvironment (TME) impairs NK cell anti-tumor cytotoxicity and exacerbates hypoxic stress. Cancer-associated fibroblasts (CAFs) and macrophages convert inactive cortisone to active cortisol, while T cells, fibroblasts, myeloid cells, macrophages, and cancer cells contribute to de novo steroid biosynthesis, collectively establishing a steroid-rich niche. Pharmacological inhibition of the glucocorticoid receptor (GR) in vivo alleviates cortisol-mediated immune suppression, resulting in reduced tumor growth and enhanced cytotoxicity of tumor-infiltrating NK cells. To overcome the cortisol-induced dysfunction of solid tumor targeting immunotherapy, we engineered chimeric antigen receptor (CAR) -NK cells specific to the C arcino e mbryonic a ntigen-related c ell a dhesion m olecule 5 (CEACAM5) (highly expressed in lung tumors) and rendered them cortisol-resistant by genetic deletion of the cortisol receptor gene NR3C1 . In cortisol-rich niches, cortisol-resistant CAR-NK cells sustained antitumor cytotoxicity. Mechanistically, NR3C1 deletion relieved cortisol-mediated suppression of PI3K-AKT-NF-κB signaling, restored anti-tumor activity, and markedly reduced hypoxic stress. In lung metastasis models, cortisol-resistant CAR-NK cells achieved superior tumor control and significantly reduced tumor burden compared with conventional CAR-NK cells. Together, these findings identify local cortisol signaling as a critical barrier to solid tumor immunotherapy and establish cortisol-resistant CAR-NK cells as a promising strategy for targeting steroidogenic solid tumors, which can be combined with therapeutic glucocorticoids.

Chimeric antigen receptor (CAR) T‐cell therapy has shown unprecedented success in haematological cancers but faces challenges in solid tumours. Although carcinoembryonic antigen‐related cell adhesion molecule 5 (CEACAM5) is differentially expressed in many solid tumours, anti‐CEACAM5 CAR T‐cells are ineffective. Here, we have studied the interaction of CEACAM5 targeting primary CAR T‐cells with colorectal cancer (CRC) cells using fluorescence microscopy. We found that CRC cells’ glycocalyx is much thicker than that of the CAR T cell causing delayed activation. Oscillating calcium fluxes, indicative of non‐sustained CAR T cell activation, are observed when CAR T cells interacted with CRC cells, which increased with increasing cell‐seeding time. Significant reduction in cytotoxicity is observed on going from early to longer‐seeded CRC monolayers. Imaging revealed that this effect correlated with a progressive loss of accessible CEACAM5 antigen on the CRC cell surface, possibly due to their sequestration in the intercellular junction, rendering CAR T cell engagement less effective. Local proteolytic treatment with trypsin to disrupt the CRC cell monolayer, using a micropipette, increased CEACAM5 availability, decreased glycocalyx thickness, and restored sustained CAR T cell calcium fluxes. Similar enhanced interaction is observed after treatment of CRC cell monolayer with hyaluronidase, approved for use in humans. Enzymatic treatment significantly enhanced CAR T cell‐mediated cytotoxicity and increased the percentage of TNF‐α–secreting CAR T cells. We observed limited availability of CEACAM5 on human colorectal cancer tissues, whereas treatment with trypsin or hyaluronidase increased accessibility. Our results reveal why CAR T cells targeting CEACAM5 are ineffective and suggest possible routes to improved therapy for CRC.

Glucocorticoids (GCs) are potent modulators of immune responses; however, the mechanisms by which GCs regulate gene expression in human CD8 T cells remain incompletely defined. Here, we delineate how physiological cortisol signalling shapes the transcriptional and chromatin landscapes of primary human CD8 T cells. We identify a substantial cohort of GC-responsive genes that are co-regulated through the cooperative activity of the glucocorticoid receptor (GR) and RUNX transcription factors. Integrative RNA sequencing and ChIP sequencing analyses identified genome-wide cortisol-responsive immunoregulatory genes. Genetic deletion of the GR (encoded by NR3C1 ) abolished cortisol-induced gene expression changes, confirming GR-dependency. Notably, GR chromatin occupancy in cortisol-treated CD8 T cells was strongly enriched at RUNX transcription factor (TF) motif rather than canonical GC response elements (GREs). Co-immunoprecipitation assays validated a ligand-dependent physical interaction between GR and RUNX and revealed the interacting domains. Single-cell transcriptomic analyses of tumour infiltrating CD8 T cells revealed significant enrichment of cortisol-responsive genes, indicating an active GC signalling (response) within the tumour microenvironment. GR-RUNX dual controlled genes were enriched in tumour-infiltrating CD8 T cells across multiple cancer types, including lung adenocarcinoma, head and neck squamous carcinoma, pancreatic cancer, and breast cancer. We found GR-RUNX co-regulated genes are predominantly expressed in the predysfunctional state of CD8 T cell population of different solid tumours. These results suggest that local cortisol signalling within tumour microenvironments drives CD8 T cell dysfunction through GR-RUNX TF cooperation. Collectively, our findings identify RUNX3 TF as a critical mediator of GR signalling in human CD8 T cells and reveal a novel mechanism by which endogenous GCs influence antitumour immunity, which could be therapeutically targetable.

Breast cancer, particularly triple-negative breast cancer (TNBC), evades the body’s immune defences, in part by cultivating an immunosuppressive tumour microenvironment. Here, we show that suppressing local steroidogenesis can augment anti-tumour immunity against TNBC. Through targeted metabolomics of steroids coupled with immunohistochemistry, we profiled the existence of immunosuppressive steroids in TNBC patient tumours and discerned the steroidogenic activity in immune-infiltrating regions. In mouse, genetic inhibition of immune cell steroidogenesis restricted TNBC tumour progression with a significant reduction in immunosuppressive components such as tumour associated macrophages. Steroidogenesis inhibition appears to bolster anti-tumour immune responses in dendritic and T cells by impeding glucocorticoid signalling. Undertaking metabolic modelling of the single-cell transcriptomics and targeted tumour-steroidomics, we pinpointed the predominant steroidogenic cells. Inhibiting steroidogenesis pharmacologically using a identified drug, posaconazole, curtailed tumour expansion in a humanised TNBC mouse model. This investigation paves the way for targeting steroidogenesis and its signalling pathways in breast cancer affected by immune-steroid maladaptation.

Summary: Steroid hormones regulate cell physiology and immune function, with dysregulated steroidogenesis promoting cancer progression by supporting tumour growth and suppressing anti-tumour immunity. Targeting CYP11A1, the first and rate-limiting enzyme in steroid biosynthesis, has shown promise in cancer therapy, but safe and effective inhibitors remain an unmet need. Undertaking in silico structure-based drug repurposing approach, we found Posaconazole as an inhibitor of CYP11A1. The docking pose analysis showed that Posaconazole can form multiple hydrogen bonds and hydrophobic interactions with the key residues at the binding site and the cofactor, stabilising the protein-ligand complex. We validated its inhibition efficiency in cell-based assays. In a mouse model of lung metastasis, we demonstrated that Posaconazole restricts metastatic cancer growth by stimulating anti-tumour immunity. These findings highlight Posaconazole’s potential as a research tool to study steroidogenesis and as a candidate for further preclinical and clinical evaluation in pathological conditions associated with local steroid production, such as steroidogenic tumours.