Biology subjects

Mazumder, T.

Publications and source records attributed to Mazumder, T..

8 recordsLinked to original sources

bioRxiv2026

A genome-wide CRISPR activation map of surface protein expression in human CD4 T cells

Surface proteins define T cell identity and function, but the abundance of each protein is not determined by transcription alone. Existing genome-wide CRISPR screens in primary human T cells either profile the transcriptome or isolate cells based on a single functional or protein phenotype. Here we present SCITO-Perturb-seq, a novel platform that couples combinatorial-indexed single-cell cytometry sequencing with pooled CRISPR activation (CRISPRa) to map the causal regulation of 201 surface proteins across 3.6 million human CD4 T cells. We find that 16% of activated genes significantly alter the expression of at least one surface protein. By applying semi-nonnegative matrix factorization to the perturbation effect matrix, we identified five modules corresponding to known CD4 T cell states. Notably, these modules group surface proteins by their shared response to perturbation, revealing coordinated regulation of proteins that are not co-expressed in unperturbed cells. SCITO-Perturb-seq represents the first genome-wide CRISPRa screen paired with direct, high-dimensional surface protein profiling, providing a comprehensive regulatory map of the CD4 T cell surface proteome.

genomics↗

bioRxiv2026

Allergen-responsive T helper type 2 cells revealed by high-dimensional profiling in allergen challenged human airways

Asthma is a chronic inflammatory disease affecting over 300 million people worldwide. This disease has multiple underlying etiologies, and a major endotype of asthma is characterized by cellular and molecular signatures of type 2 (allergic) inflammation. In this study we conducted bronchoscopies with airway segmental allergen challenge in allergic asthmatics to dissect airway responses to allergen. Using mass cytometry and single-cell RNA sequencing, we characterized with high resolution the airway immune landscape before and after allergen challenge and the heterogeneity present between subjects. This heterogeneity generally falls along a type 1/ type 2 axis. In type 2 high individuals, we identified allergen-reactive Th2 cells by using TCR sequences to barcode clonal T cell populations in single-cell genomic and activation-induced marker expression assays. These potentially pathogenic Th2 cell clones were present systemically and expanded following allergen challenge, connecting local lung inflammation to systemic clonal Th2 cell dynamics. Th2 cell airway ingress was coordinated with myeloid cell expression of T cell chemoattractants including CCL17 and CCL22. This study provides insight into the molecular and cellular components of allergen-induced tissue inflammation in asthma. Deeper resolution of the T cell response to aeroallergens may inform novel diagnostic and therapeutic strategies for asthma and other allergic airway diseases.

immunology↗

bioRxiv2026

DPP9-mediated inflammasome repression protects against checkpoint inhibitor lung toxicity

Over one million patients receive cancer immunotherapy annually, yet the mechanisms underlying life-threatening immune-mediated toxicities remain poorly understood. Checkpoint inhibitor pneumonitis (CIP) is the leading cause of immunotherapy-related mortality, with a case fatality rate approaching 10%, and no genetic risk factors have been described to date. We identified Dipeptidyl-peptidase 9 (DPP9) as the first genetic susceptibility gene for CIP in a clinico-genomics cohort of 4,397 patients treated with immune checkpoint inhibitors. Mechanistically, DPP9 suppresses CARD8 inflammasome activation and IL-18 secretion in human monocytes, a pathway which is engaged prior to CIP onset, with IL-18 selectively elevated in the plasma of patients who subsequently develop CIP. Myeloid-restricted ablation of Dpp8 and Dpp9 in mice recapitulated the pulmonary histopathological and immunological hallmarks of CIP, including granuloma formation, accumulation of IFN{gamma}-producing T cells and monocyte-derived macrophages. Each of these phenotypes were driven by excessive IL-18 secretion. Together, these findings establish DPP9 as a genetic determinant of CIP and nominate IL-18 blockade as a mechanistically rational therapeutic strategy.

immunology↗

bioRxiv2025

Programmable microparticles rewire CAR signaling to enable super-physiological expansion of human T cells in vitro

T cell proliferative capacity and persistence critically determine the therapeutic success of chimeric antigen receptor (CAR) T cells. However, it remains unknown if and how human CAR-T cells can be externally programmed to reach maximal proliferative capacity. Here, we use programmable PLGA microparticles functionalized with CAR-antigens and CD28-costimulatory antibodies (CAREp) to repeatedly stimulate human CD8+ CAR-T cells in vitro. CAREp-stimulated CAR-T cells expanded continuously for over 100 days--versus [~]30 days with tumor cell stimulation--and achieved up to 1018-fold cumulative expansion, greatly surpassing CD3/28-Dynabeads. Early-phase transcriptomic responses-- upregulation of DNA repair, cell cycle, telomere maintenance, and mitochondrial pathways--aligned with long-term outcomes: massive proliferation, telomere stability, robust respiration, and preserved progenitor phenotype by single-cell sequencing. Differentiation and exhaustion signals were broadly suppressed. Transient telomerase activity further supported physiologic expansion. These findings demonstrate that nanoscale-controlled extracellular cues can rewire intracellular signaling to drive durable, super-physiological expansion of functional CAR-T cells.

immunology↗

bioRxiv2025

Ethanolic leaf extract of Cleome spinosa induces apoptosis in Ehrlich ascites carcinoma cells through upregulation of p53 and down-regulation of Bcl-xL in vitro and in vivo

Cleome genus is commonly known as spider flower plant or cats whiskers, and is the largest genus of the family Cleomaceae, with around 200 species containing a range of remedial applications. Several species of Cleome genus show anti-cancer properties, however, nothing has been reported with genus spinosa, an anti-inflammatory and immunomodulatory plant. Thus, the present study examined the likely anti-cancer property of ethanolic leaf extract of Cleome spinosa (CSE) in Ehrlich ascites carcinoma (EAC) cells in vitro and in vivo. CSE treatment in vitro significantly caused higher percentage of death in EAC in a dose dependent manner compared to control groups (untreated and ethanol treated). Bright field, fluorescence and electron microscopies revealed apoptotic changes such as surface blebbings, nuclear fragmentation, chromatin condensation and apoptotic body formation, which probably could be the cause of death in EAC cells with CSE treatment. Apoptosis of CSE-treated EAC cells appeared to be mediated through upregulation of pro-apoptotic protein p53 and downregulation of anti-apoptotic protein, Bcl-xL expression, as ascertained through western blot both in vitro and in vivo. CSE was protective toward murine splenocytes by scavenging free radicals; it also reduced the ascitic tumor burden and increased the survival of the tumor-bearing mice compared to the control. Gas Chromatography-Mass Spectrometry (GCMS) analysis identified 43 phytocompounds in CSE. In silico molecular docking analysis indicated that a number of bioactive compounds, such as cycloartenol, stigmasterol, clionasterol, beta-sitosterol, lupenone, and sitostenone that were abundant in the CSE extract, exhibited strong binding affinities for the key apoptosis-regulating proteins, p53 and Bcl-xL. Thus, CSE derived-compounds appear to act as natural antioxidants, and have anti-cancer properties via induction of apoptosis in EAC possibly through upregulation of p53 and downregulation of Bcl-xL proteins in murine model. Hence, CSE could be considered as a promising natural compound for future cancer therapies.

cancer biology↗

bioRxiv2024

Learning antibody sequence constraints from allelic inclusion

Antibodies and B-cell receptors (BCRs) are produced by B cells, and are built of a heavy chain and a light chain. Although each B cell could express two different heavy chains and four different light chains, usually only a unique pair of heavy chain and light chain is expressed--a phenomenon known as allelic exclusion. However, a small fraction of naive-B cells violate allelic exclusion by expressing two productive light chains, one of which has impaired function; this has been called allelic inclusion. We demonstrate that these B cells can be used to learn constraints on antibody sequence. Using large-scale single-cell sequencing data from humans, we find examples of light chain allelic inclusion in thousands of naive-B cells, which is an order of magnitude larger than existing datasets. We train machine learning models to identify the abnormal sequences in these cells. The resulting models correlate with antibody properties that they were not trained on, including polyreactivity, surface expression, and mutation usage in affinity maturation. These correlations are larger than what is achieved by existing antibody modeling approaches, indicating that allelic inclusion data contains useful new information. We also investigate the impact of similar selection forces on the heavy chain in mouse, and observe that pairing with the surrogate light chain significantly restricts heavy chain diversity.

immunology↗

bioRxiv2023

Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the chromosome, including in pre-clinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells,1 dramatically reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

cell biology↗

bioRxiv2023

In vitro and in vivo evidences propound therapeutic potential of Lipocalin 2 in cervical carcinoma

Cervical cancer (CC), the second most common in developing countries and the third most common in developed nations, is the fourth most common type of cancer in women overall. The HPV16 high-risk genotype of the virus, which is responsible for about 61% of cervical cancer incidences, was found to have higher LCN2 levels in advanced clinical CC stages. In this study, we assessed the impact of suppressing LCN2 activity after treatment with an anti-LCN2 monoclonal antibody (MAb) in both in vitro and in vivo settings. Anti-LCN2 antibody was found to reduce proliferation and invasion of HeLa cells, the first immortal cells from a HPV positive aggressive adenocarcinoma of the cervix. LCN2 and its ligand MMP9 was found to be highly expressed in the cells and abrogated on treatment with anti-LCN2. The five receptors of LCN2 - SLC22A17, MC1R, MC2R, MC4R and LRP2 were barely detected with or without treatment. Anti-LCN2 Mab caused tumors to regress and soften in vivo, in a xenograft mouse model. Analysis of histology images of the treated and untreated tumor established the necrotic capability of the therapeutic molecule explaining the regression and softening of the tumor. Differential gene expression analysis between untreated and treated tumor proved that LCN2 inhibition abolished the migratory, invasive, and hypoxic pathways while significantly increasing the necrosis and cell death pathways in tumor after treatment with the monoclonal antibody. LCN2 inhibition was shown molecularly to lead to tumor regression via a negative feedback loop of LCN2 through the TNF-IL17 axis exponentially increasing the effect of the anti-LCN2 monoclonal antibody. In conclusion, LCN2 appears to be a viable therapeutic target, and the monoclonal antibody used in this study can be further developed for clinical usage in cervical cancer.

cancer biology↗