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 Carcinoembryonic antigen-related cell adhesion molecule 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 are potent immune regulators, yet how cortisol controls human CD8 T cell function remains poorly defined. Here, we show that cortisol reshapes the transcriptional landscape of human CD8 T cells through cooperation between the glucocorticoid receptor (GR) and RUNX transcription factors. Integrative RNA sequencing (RNA-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq) analyses identified genome-wide cortisol-responsive immunoregulatory genes, and NR3C1 deletion confirmed GR dependency. GR chromatin occupancy was enriched at RUNX motifs rather than canonical glucocorticoid response elements, and co-immunoprecipitation confirmed a ligand-dependent interaction between GR and RUNX3, requiring the N-terminal activation function-1 (AF1) domain of GR and the C-terminal region of RUNX3. Single-cell transcriptomic analyses across multiple solid tumors revealed consistent enrichment of GR-RUNX co-regulated genes in tumor-infiltrating CD8 T cells, predominantly within the predysfunctional state. These findings identify RUNX3 as a critical non-canonical GR partner and uncover a therapeutically actionable mechanism by which endogenous glucocorticoids drive CD8 T cell dysfunction in human cancer.

Abstract 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.

Diabetic retinopathy (DR) arises from intertwined inflammatory, metabolic, and hypoxia-driven angiogenic programs, yet upstream regulators coordinating these processes remain incompletely defined. Here, we used an integrative multi-omics and experimental framework to identify cathepsin H (CTSH) as a candidate causal driver of proliferative DR (PDR). By combining GWAS, eQTL, pQTL, and mQTL datasets with Mendelian randomization, summary-data-based Mendelian randomization, and Bayesian colocalization, CTSH emerged as the strongest genetically supported candidate across discovery and validation analyses. In the UK Biobank (UKB), circulating CTSH was elevated in diabetic retinopathy and independently predicted incident disease. Single-cell transcriptomic analyses localized CTSH predominantly to myeloid compartments within fibrovascular membranes and linked CTSH-high states to inflammatory, hypoxic, and angiogenic programs. In high-glucose-stimulated THP-1 monocytes, CTSH promoted reactive oxygen species accumulation, NF-κB activation, and increased IL-6, TNF-α, HIF-1α, and VEGF expression, whereas CTSH silencing reversed these effects. Structure-guided virtual screening identified Eriocitrin as a lead CTSH-binding candidate. In db/db mice, intravitreal Eriocitrin improved inner-retinal function, restored OCTA-derived vascular metrics, and partially rescued retinal structure, with efficacy comparable to anti-VEGF treatment across several endpoints. Molecular analyses further showed coordinated suppression of inflammatory, hypoxic, angiogenic, and NF-κB signaling. Together, these findings identify CTSH as an upstream immunometabolic regulator of DR-related inflammatory and angiogenic biology, with the strongest genetic support observed for PDR, and support CTSH targeting as a potential multi-pathway therapeutic strategy beyond VEGF inhibition.

ABSTRACT 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.

Abstract Introduction Regional lymph node (LN) metastasis critically influences distant metastatic progression, anti-tumour immunity, and patient prognosis. While tumour-induced immune modulation in tumour-draining LNs (TDLNs) has been extensively studied using murine models, the systematic reconstruction of the immune system from primary tumours through TDLNs and subsequent lymph nodes in human cancer progression remains understudied. Methods Here, we utilised integrated multi-omics approaches, including imaging mass cytometry, single-cell RNA sequencing, Visium and Xenium spatial transcriptomics, and multi-colour immunofluorescence to systematically characterise immune cell dynamics across 147 paired primary tumours, sentinel TDLNs (S-TDLNs), and secondary axillary LNs (ALNs) obtained from 50 treatment-naïve triple-negative breast cancer patients with different progression statuses. Results We observe depletion of cDC2 and naïve T cells, with expansion of immunosuppressive MARCO+ macrophages associated with survival, as identified by a newly developed single-cell transformer model. Spatial analysis and a unified cell—cell interaction framework reveal disrupted immune communication and impaired dendritic cell—T cell priming circuits within metastatic LNs and matched tumours. In an independent neoadjuvant immunotherapy cohort (36 patients, 52 LNs), preservation of CD1c+ cDC2 predicts pathological complete response and prolonged event-free survival, positioning cDC2 as a biomarker and rational therapeutic target in TNBC. Conclusion Collectively, this systematic mapping of immune landscape alterations during human sequential LN metastasis provides essential insights for understanding cancer metastasis mechanisms and paves the way for innovative immunotherapeutic strategies. Funding Source Royal Society Fund (IEC\NSFC\233577); CRUK Career Development Fellowship (RCCFEL\100095); National Natural Science Foundation of China (Grant No. 81672638, W2421095) Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

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. ### Competing Interest Statement The authors have declared no competing interest. CRUK, RCCFEL\100095 NSF-BIO/UKRI-BBSRC, BB/V006126/1 MRC, MR/V028995/1

Tumour microenvironments (TME) accumulate immunosuppressive steroids, impairing NK cell anti-tumour immunity. We found glucocorticoid cortisol signalling enrichment in lung TME exacerbates hypoxic stress and impairs NK cell function. Single-cell transcriptomics revealed cancer-associated fibroblasts and macrophages convert inactive cortisone to active cortisol, while T cells, mast cells, and macrophages induce de novo steroid biosynthesis. Inhibiting the glucocorticoid receptor in mice reduced tumour growth and improved NK cell cytotoxicity.To overcome steroid-mediated immunosuppression, we engineered CEACAM5-specific CAR-NK cells with CRISPR-mediated deletion of the glucocorticoid receptor (NR3C1). These cortisol-resistant CAR-NK cells showed enhanced tumour cell killing efficacy, even in the presence of glucocorticoids. This approach offers promising applications against steroidogenic solid tumours and potential use alongside therapeutic glucocorticoids. Our study addresses a critical challenge in CAR-NK cell therapy for lung cancer - the immunosuppressive TME. By targeting CEACAM5, highly expressed in lung tumours, and making CAR-NK cells cortisol-resistant, we’ve developed a novel strategy to enhance the efficacy of immunotherapy in steroid-rich environments. This advancement could significantly improve outcomes for lung cancer patients, addressing limitations of current CAR-NK therapies in solid tumours. The work is supported by CRUK Career Development Fellowship (RCCFEL\100095), NSF-BIO/UKRI-BBSRC project grant (BB/V006126/1), MRC project grant (MR/V028995/1), CRUK Cambridge Centre Cancer Immunology Programme Pump Priming award, and CRUK CC MRes/PhD Studentship. Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Steroid hormones regulate cell physiology and immune function, with dysregulated steroidogenesis promoting cancer progression by supporting tumor growth and suppressing anti-tumor 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, stabilizing 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 metastasis by stimulating anti-tumor 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 pathologies associated with local steroidogenesis, such as steroidogenic tumors.

The immunosuppressive tumour microenvironment dampens anti-tumour immunity and reduces the efficacy of immunotherapy in aggressive malignancies like triple-negative breast cancer (TNBC) and glioblastoma (GBM). Recent research highlights the immunosuppressive effects of steroid hormones. However, the extent to which these hormones inhibit the anti-tumour immune response remains largely unknown. Through targeted metabolomics of steroids coupled with immunohistochemistry techniques, we comprehensively profiled the existence of different types of steroids in TNBC and GBM patient tumours and discerned steroidogenic activity in immune infiltrating regions. In mouse models, genetic inhibition of immune cell steroidogenesis restricted tumour progression, significantly reducing tumour-associated macrophages. Using metabolic modelling of single-cell transcriptomics, we identified mast cells as the predominant steroidogenic agent. Notably, pharmacologically inhibiting steroidogenesis curtailed tumour expansion in a humanised mouse model of TNBC. In addition to endogenous source, considering the frequent use of exogenous steroids in GBM treatments, we explored impeding glucocorticoid signalling appears to bolster the ability of dendritic cells to prime T cells. Altogether, these studies will shed light on the role of steroid hormones in promoting anti-tumour immunosuppression. The new knowledge is anticipated to create new therapeutic avenues for TNBC and GBM patients. Cancer Research UK (CRUK) - RCCFEL\100095 RCUK | Biotechnology and Biological Sciences Research Council (BBSRC) - BB/V006126/1 RCUK | MRC | Medical Research Foundation - MR/V028995/1 Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

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. ### Competing Interest Statement The authors have declared no competing interest.

Effective resolution of inflammation following acute lung infection or injury is critical for restoring immune and tissue homeostasis to ensure functional recovery. Prolonged or unresolved inflammation can impair lung repair, promote fibrosis, and contribute to pulmonary dysfunction. While systemic steroid signalling is known to modulate general immune responses, the specific role of immune cell-mediated steroidogenesis in regulating lung inflammation and repair remains unknown. Here, we show that immune cell de novo steroidogenesis is essential for resolving inflammation and promoting recovery in a murine model of acute lung injury. During the resolution phase, steroid-synthesizing immune cells, predominantly basophils, are enriched in the lung. Mice with immune cell-specific ablation of de novo steroidogenesis exhibit exacerbated lung injury, impaired resolution of inflammation, and defective tissue repair. These findings reveal a previously unrecognized immunoregulatory function of immune cell-derived steroids and identify immune cell steroidogenesis as a potential therapeutic target for promoting resolution and recovery in inflammatory lung diseases. ### Competing Interest Statement The authors have declared no competing interest. This work is supported by CRUK Career Development Fellowship (RCCFEL\100095), NSF-BIO/UKRI-BBSRC project grant (BB/V006126/1), and MRC project grant (MR/V028995/1).

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.

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, CEACAM5 CAR T-cells are ineffective. Here, we have studied the interaction of CEACAM5 targeting CAR primary T-cells with colorectal cancer (CRC) cells using fluorescence microscopy. We find that CRC cells glycocalyx is much thicker than the CAR T-cell and likely contributes to immune-escape. Oscillating calcium flux, a signature of non-sustained triggering and decreased killing, was observed when CAR T-cells interacted with CRC, which increased with increasing cell-seeding time. This was because CEACAM5 became increasingly unavailable on the CRC cell monolayer, as revealed by fluorescence imaging. 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, increasing the killing of CRC cells. Our results reveal why CAR T-cells targeting CEACAM5 are ineffective and suggest possible routes for improved therapy. ### Competing Interest Statement The authors have declared no competing interest.

Osteoclasts play a central role in cancer -cell -induced osteolysis, but the molecular mechanisms of osteoclast activation during bone metastasis formation are incompletely understood. By performing RNA sequencing on a mouse breast carcinoma cell line with higher bone -metastatic potential, here we identify the enzyme CYP11A1 strongly upregulated in osteotropic tumor cells. Genetic deletion of Cyp11a1 in tumor cells leads to a decreased number of bone metastases but does not alter primary tumor growth and lung metastasis formation in mice. The product of CYP11A1 activity, pregnenolone, increases the number and function of mouse and human osteoclasts in vitro but does not alter osteoclast-specific gene expression. Instead, tumor -derived pregnenolone strongly enhances the fusion of pre-osteoclasts via prolyl 4-hydroxylase subunit beta (P4HB), identified as a potential interaction partner of pregnenolone. Taken together, our results demonstrate that Cyp11a1-expressing tumor cells produce pregnenolone, which is capable of promoting bone metastasis formation and osteoclast development via P4HB.

Age-related macular degeneration (AMD) is a significant cause of visual impairment in the aging population, with the pathophysiology driven by a complex interplay of genetics, environmental influences and immunometabolic factors. These immunometabolic mechanisms, in particular, those distinguishing between the dry and wet forms of AMD, remain incompletely understood. Utilizing an integrated multiomic approach, incorporating Mendelian Randomization (MR) and single-cell RNA sequencing (scRNA-seq), we have effectively delineated distinct immunometabolic pathways implicated in the development of AMD. Our comprehensive analysis indicates that the androgen-IL10RA-CD16+ monocyte axis could protect against wet AMD. We have also identified several immune and metabolic signatures unique to each AMD subtype, with TNFα and Notch signaling pathways being central to disease progression. Furthermore, our analysis, leveraging expression Quantitative Trait Loci (eQTLs) from the Genotype-Tissue Expression (GTEx) project coupled with MR, have highlighted genes such as MTOR , PLA2G7 , MAPKAPK3 , ANGPTL1 , and ARNT as prospective therapeutic targets. The therapeutic potential of these candidate genes was validated with observations from existing drug trial databases. Our robust genetic and transcriptomic approach has identified promising directions for novel AMD interventions, emphasizing the significance of an integrated multiomic approach in tackling this important cause of visual impairment.

Pregnenolone (P5) is synthesized as the first bioactive steroid in the mitochondria from cholesterol. Clusters of differentiation 4 (CD4+) and Clusters of differentiation 8 (CD8+) immune cells synthesize P5 de novo; P5, in turn, play important role in immune homeostasis and regulation. However, P5’s biochemical mode of action in immune cells is still emerging. We envisage that revealing the complete spectrum of P5 target proteins in immune cells would have multifold applications, not only in basic understanding of steroids biochemistry in immune cells but also in developing new therapeutic applications. We employed a CLICK-enabled probe to capture P5-binding proteins in live T helper cell type 2 (Th2) cells. Subsequently, using high-throughput quantitative proteomics, we identified the P5 interactome in CD4+ Th2 cells. Our study revealed P5’s mode of action in CD4+ immune cells. We identified novel proteins from mitochondrial and endoplasmic reticulum membranes to be the primary mediators of P5’s biochemistry in CD4+ and to concur with our earlier finding in CD8+ immune cells. Applying advanced computational algorithms and molecular simulations, we were able to generate near-native maps of P5–protein key molecular interactions. We showed bonds and interactions between key amino acids and P5, which revealed the importance of ionic bond, hydrophobic interactions, and water channels. We point out that our results can lead to designing of novel molecular therapeutics strategies.

EDITORIAL article Front. Endocrinol., 28 November 2023Sec. Cellular Endocrinology Volume 14 - 2023 | https://doi.org/10.3389/fendo.2023.1323185

EDITORIAL article Front. Immunol., 20 December 2021Sec. Cytokines and Soluble Mediators in Immunity Volume 12 - 2021 | https://doi.org/10.3389/fimmu.2021.825577