Biology subjects

Roychoudhuri, R.

Publications and source records attributed to Roychoudhuri, R..

10 recordsLinked to original sources

bioRxiv2026

Lysosomal adaptation to extracellular acidity compromises CD8+ T cell cytotoxicity

Extracellular acidosis is a hallmark of aggressive tumors and suppresses cytotoxic-T lymphocyte (CTL)-mediated killing, but how sustained acid exposure reshapes T cell cytotoxicity is poorly understood. Here we show that chronic acidosis drives lysosomal remodeling in human CD8+ T cells, converting the lysosome into a proton sink whose adaptation ultimately impairs killing by depleting the intracellular granzyme B reservoir through constitutive vesicle release. Quantitative proteomics of CD8+ T cells cultured at pH 6.5 identified lysosomes as the dominant target of acid-driven remodeling: CTLs upregulated the V1 sector of the vacuolar H+-ATPase and hyper-acidified the lysosomal lumen. The remodeled compartment accumulated lysosomal membrane proteins (LAMP1, LAMP2, CD63, CD68) post-transcriptionally, acquired higher cathepsin abundance and increased proteolytic activity. Chronic acidity also enlarged endoplasmic reticulum (ER) and lysosomal Ca2+ stores without altering cytosolic Ca2+, and drives constitutive, Ca2+-dependent release of LAMP1+ lysosome-derived vesicles, which drains the intracellular reservoir of granzyme B, contributing to reduced CTL activity. These findings establish lysosomal adaptation as a cost of surviving extracellular acidosis and identify lysosomal hydrolase activity as a possible target for improving CTL function in the acidic tumor environment.

immunology↗

bioRxiv2026

Targeting glucocorticoid signalling in dendritic cells for glioblastoma treatment

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.

immunology↗

bioRxiv2025

Local glucocorticoid signalling promotes pancreatic cancer

Pancreatic cancer (PC) represents one of the biggest challenges in terms of cancer treatment, mainly due to its continuously rising incidence, advanced stage at time of diagnosis, and dismal 5-year overall survival, which has not improved in recent decades despite the major advances made in oncological therapies. The limited progress in developing more effective therapies is, in part, attributable to the vast desmoplastic stroma present in PC. Additionally, immunosuppressive steroid-signalling has recently been shown to aid the development and metastasis of various tumour types. Therefore, we sought to explore whether local steroidogenesis and steroid signalling within the tumour microenvironment (TME) play a role in pancreatic cancer development. Reanalysis of publicly available datasets, including single cell RNA sequencing, as well as in vivo metastatic pancreatic ductal adenocarcinoma (PDAC) mouse models, allowed us to identify Hsd11b1 as the key enzyme responsible for locally elevated levels of the immunosuppressive glucocorticoid hormone, corticosterone. We identified fibroblasts as the major Hsd11b1-expressing populations in the pancreatic TME. Specifically, in mice, Hsd11b1 expression is primarily observed in iCAFs. Additionally, we found that patients with higher HSD11B1 expression present an increased mortality rate as well as an enriched fibrotic signature and inhibited immune activity. Collectively, these findings suggest that Hsd11b1 upregulation in iCAFs could be aiding PDAC development by promoting the activation of glucocorticoids directly in the TME. The presence of glucocorticoids inhibits inflammation and could also be enhancing local fibrosis by autocrine signalling in the fibroblast population. Given the urgent need for effective treatments in this fatal disease, targeting HSD11B1 represents a promising therapeutic strategy to overcome the immunosuppressive desmoplastic barrier and improve patient outcomes in pancreatic cancer.

cancer biology↗

bioRxiv2025

Drug repurposing reveals Posaconazole as a CYP11A1 inhibitor enhancing anti-tumour immunity

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 Posaconazoles 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.

immunology↗

bioRxiv2025

Primitive steroidogenesis in mast cells: A novel regulatory mechanism for mast cell function

Mast cells, ancient immune sentinels, are crucial in immune responses, tissue homeostasis and inflammatory pathologies. This study unveils a previously unknown regulatory mechanism in mast cell biology: de novo steroidogenesis. Through comprehensive multi-omics analysis and functional assays, we demonstrate that mast cells express Cyp11a1 and produce pregnenolone, representing a primitive form of steroidogenesis. This cell-intrinsic steroidogenic pathway is essential for mast cell development, survival, and functional regulation. Genetic abrogation of mast cell steroidogenesis leads to exaggerated inflammatory and anaphylactic responses in vivo. Our integrative approach reveals extensive transcriptional and proteomic remodelling during mast cell regranulation, with steroidogenesis playing a pivotal role in coordinating recovery and tissue repair processes. We uncover significant sexual dimorphism in mast cell proteomes and a global uncoupling of transcriptional and translational programmes. These findings advance our understanding of mast cell physiology and provide a foundation for developing targeted therapies for mast cell-associated pathologies.

immunology↗

bioRxiv2024

Hexa-acylated lipopolysaccharides from the gut microbiota enhance cancer immunotherapy responses

Immune checkpoint inhibitors (ICI), such as anti-PD-1, have revolutionized cancer treatment, but they are only effective for a minority of patients. The gut microbiome plays a crucial role in modulating immunotherapy treatment responses, and previous studies correlated lipopolysaccharide (LPS)-producing gut microbes with poorer prognosis. However, LPS from diverse bacterial species have activities ranging from immunostimulatory to inhibitory. By functionally analyzing fecal metagenomes from 112 melanoma patients prior to anti-PD-1 therapy, we found that a subset of LPS-producing bacteria encoding immunostimulatory hexa-acylated LPS was enriched in the microbiomes of clinical responders. We confirmed robust activation of the NF-kB pathway by hexa-acylated LPS in vitro, and this activation was significantly inhibited by penta-acylated LPS in a dose-dependent manner. Importantly, oral administration of hexa-acylated LPS augmented anti-PD-1-mediated anti-tumor immunity in an in vivo mouse model of cancer immunotherapy. Microbiome hexa-acylated LPS may therefore represent an accessible predictor and potential enhancer of clinical anti- PD-1 immunotherapy responses. Statement of significanceFunctional rather than taxonomic profiling of patient gut microbiomes reveals hexa-acylated LPS as a novel biomarker of responsiveness and a targetable pathway for enhancing responses to anti-PD-1, informing future studies and current patient treatment.

microbiology↗

bioRxiv2024

Lack of phosphatidylinositol 3-kinase VPS34 in regulatory T cells leads to a fatal lymphoproliferative disorder without affecting their development

Regulatory T (Treg) cells are essential for the maintenance of immunological tolerance, yet the molecular components required for their maintenance and effector functions remain incompletely defined. Inactivation of VPS34 in Treg cells led to an early, lethal phenotype, with massive effector T cell activation and inflammation, like mice lacking Treg cells completely. However, VPS34-deficient Treg cells developed normally, populated the peripheral lymphoid organs and effectively supressed conventional T cells in vitro. Our data suggest that VPS34 is required for the maturation of Treg cells or that mature Treg cells depend on VPS34 for survival. Functionally, we observed that lack of VPS34 activity impairs cargo processing upon transendocytosis, that defective autophagy contributes to, but is not sufficient to explain this lethal phenotype, and that loss of VPS34 activity induces a state of heightened metabolic activity that may interfere with metabolic networks required for maintenance or suppressive functions of Treg cells.

immunology↗

bioRxiv2023

Intracellular K+ limits T cell exhaustion and preserves antitumor function

The cancer-killing activity of T cells is often compromised within tumors, allowing disease progression. We previously found that intratumoral elevations in extracellular K+ related to ongoing cell death constrained CD8+ T cell Akt-mTOR signaling and effector function (1,2). To alleviate K+ mediated T cell suppression, we pursued genetic means to lower intracellular K+. Transcriptomic analysis of CD8+ T cells demonstrated the Na+/K+ ATPase to be robustly and dynamically expressed. CRISPR-Cas9 mediated deletion of the catalytic alpha subunit of the Na+/K+ ATPase lowered intracellular K+ but produced tonic hyperactivity in multiple signal transduction cascades along with the acquisition of co-inhibitory receptors and terminal differentiation in mouse and human CD8+ T cells. Mechanistically, Na+/K+ ATPase disruption led to ROS accumulation due to depletion of intracellular K+ in T cells. Antioxidant treatment or high K+ media prevented Atp1a1 deficient T cells from exhausted T (TEx) cell formation. Consistent with transcriptional and proteomic data suggesting a TEx cell phenotype, T cells lacking Atp1a1 had compromised persistence and antitumor activity in a syngeneic model of orthotopic murine melanoma. Translational application of these findings will include efforts to lower intracellular K+ while limiting ROS accumulation within tumor specific T cells. SynopsisHigh extracellular K+ ({uparrow}[K+]e) is found within tumors and suppresses T cell effector function. Collier et al. find that deletion of the Na+/K+ ATPase in T cells lowers intracellular K+ and promotes ROS accumulation, tonic signal transduction and T cell exhaustion owing to ROS accumulation. Engineering T cell ion transport is an important consideration for cancer immunotherapy.

immunology↗

bioRxiv2023

Time-, tissue- and treatment-associated heterogeneity in tumour-residing migratory DCs

Tumour dendritic cells (DCs) internalise antigen and upregulate CCR7, which directs their migration to tumour-draining lymph nodes (dLN). CCR7 expression is coupled to a maturation programme enriched in regulatory molecule expression, including PD-L1, termed mRegDC. However, the spatio- temporal dynamics and role of mRegDCs in anti-tumour immune responses remain unclear. Using photoconvertible mice to precisely track DC migration, we found that mRegDCs were the dominant DC population arriving in the dLN, but a subset remained tumour-resident despite CCR7 expression. These tumour-retained mRegDCs were phenotypically and transcriptionally distinct from their dLN counterparts and were heterogeneous. Specifically, they demonstrated a progressive reduction in the expression of antigen presentation and pro-inflammatory transcripts with more prolonged tumour dwell-time. Tumour mRegDCs spatially co-localised with PD-1+CD8+ T cells in human and murine solid tumours. Following anti-PD-L1 treatment, tumour-residing mRegDCs adopted a state enriched in lymphocyte stimulatory molecules, including OX40L, which was capable of augmenting anti- tumour cytolytic activity. Altogether, these data uncover previously unappreciated heterogeneity in mRegDCs that may underpin a variable capacity to support intratumoural cytotoxic T cells, and provide insights into their role in cancer immunotherapy.

immunology↗

bioRxiv2022

Multimodal single-cell profiling of intrahepatic cholangiocarcinoma defineshyperactivated Tregs as a potential therapeutic target

The quality of the immune infiltrate of intrahepatic cholangiocarcinoma (iCCA), a rare, yet aggressive tumor of the biliary tract, remains poorly characterized, limiting development of successful immunotherapies. We used high-dimensional flow cytometry to characterise the T cell and myeloid compartments of iCCA comparing these with their tumor-free peritumoral and circulating counterparts. We found poor infiltration of putative tumor-specific CD39+ CD8+ T cells accompanied by abundant infiltration of hyperactivated CD4+ regulatory T cells (Tregs), whose frequency in relation to that of CD4+ CD69+ T cells and conventional type 2 dendritic cells was associated with poor prognosis. Single-cell RNA-sequencing identified an altered network of transcription factors in iCCA-infiltrating compared to peritumoral T cells, suggesting reduced effector functions by tumor-infiltrating CD8+ T cells and enhanced immunosuppression by CD4+ Tregs. Specifically, we found that expression of mesenchyme homeobox 1 (MEOX1) was highly enriched in tumor-infiltrating Tregs, and demonstrated that MEOX1 overexpression is sufficient to reprogram circulating precursors to acquire the transcriptional and epigenetic landscape of tumorinfiltrating Tregs. Interfering with hyperactivated Tregs should be thus explored to enhance antitumor immunity in iCCA.

immunology↗