Biology subjects

Seshasayee, D.

Publications and source records attributed to Seshasayee, D..

3 recordsLinked to original sources

bioRxiv2026

A Co-culture Cell-Based Reporter Assay for Quantitative Measurement of Integrin αvβ8-Mediated Activation of Latent TGF-β1

Integrin v{beta}8 is a major activator of latent transforming growth factor-{beta} (TGF-{beta}) and an emerging therapeutic target in cancer and immune regulation. However, functional assays that directly measure v{beta}8-mediated activation of latent TGF-{beta} in a physiologically relevant context remain limited. Here, we report a co-culture cell-based reporter assay for quantitative measurement of v{beta}8-mediated activation of latent TGF-{beta}1. NIH/3T3 reporter cells were engineered to express a SMAD-responsive NanoLuc reporter, constitutive firefly luciferase for internal normalization, and cell-surface GARP-latent TGF-{beta}1. When co-cultured with v{beta}8-expressing LN-229 cells, reporter cells produced a robust signal that directly reflected localized latent TGF-{beta}1 activation. The assay demonstrated stable expression of the required biological components, reproducible signal-to-background performance, and sensitivity to benchmark v{beta}8-blocking antibodies. Inhibition studies showed potent dose-dependent blockade by an anti-v{beta}8 antibody. In contrast, pan-TGF-{beta} neutralizing antibody displayed markedly weaker apparent potency, suggesting that targeting localized v{beta}8-mediated activation is more effective than neutralizing released TGF-{beta} in this assay context. The assay also enabled screening and ranking of anti-v{beta}8 antibodies, identifying several high-potency clones, and detected v{beta}8-mediated activation of a non-cleavable latent TGF-{beta}1 mutant. This platform provides a sensitive, internally normalized, and scalable approach for mechanistic studies and therapeutic discovery targeting the v{beta}8-TGF-{beta} axis.

immunology↗

bioRxiv2025

A single MHCII neoepitope mRNA vaccine elicits CD4 T- and B- cell responses promoting endogenous CD8 anti-tumor immunity

Recent progress in therapeutic cancer vaccines has shown promising clinical activity, especially when targeting MHC class I (MHCI) neoantigen-specific CD8+ T cell responses in post-surgical patients. To explore the role of CD4+ T cells in vaccine-dependent tumor rejection, we constructed an mRNA lipoplex vaccine encoding a single MHCII-restricted neoantigen. The vaccine elicited Tfh and Th1 cell responses while decreasing Tregs, leading to rejection of established tumors in mice. IL-21 and IFN-{gamma}, crucial for Tfh and Th1 function respectively, contributed to anti-tumor activity. B cells and neoantigen-specific antibodies were also shown to participate in vaccine efficacy. Additionally, conventional type 1 dendritic cells (cDC1s) were essential for eliciting vaccine-induced CD4+ T cells, and both cDC1s and CD4+ T cells were required to enhance endogenous CD8+ responses, which were crucial for tumor control. Our results suggest that immunizing against MHCII neoantigens alone is sufficient to orchestrate a potent and cooperative immune response against cancer.

immunology↗

bioRxiv2019

Massively parallel single-cell B-cell receptor sequencing enables rapid discovery of diverse antigen-reactive antibodies

Using high-throughput single-cell B-cell receptor sequencing (scBCR-seq) we obtained accurately paired full-length heavy- and light-chain variable domains from thousands of individual B cells in a massively parallel fashion. We sequenced more than 250,000 B cells from rat, mouse and human repertoires to characterize their lineages and expansion. In addition, we immunized rats with chicken ovalbumin and profiled antigen-reactive B cells from lymph nodes of immunized animals. The scBCR-seq data recovered 81% (n = 56/69) of B-cell lineages identified from hybridomas generated from the same set of B cells that were subjected to scBCR-seq. Importantly, scBCR-seq identified an additional 710 candidate lineages that were not recovered as hybridomas. We synthesized, expressed and tested 93 clones from the identified lineages and found that 99% (n = 92/93) of the clones were antigen-reactive. Our results establish scBCR-seq as a powerful tool for antibody discovery.

immunology↗