Biology subjects

Kanagasabesan, N.

Publications and source records attributed to Kanagasabesan, N..

5 recordsLinked to original sources

bioRxiv2026

Proteomics of human cancer-associated T cells identifies regulators of T cell functionality

CD8+ T cells in solid cancers progressively lose anti-tumor activity, yet the cell-intrinsic mechanisms driving this loss of function remain incompletely defined. Here, we performed matched proteomic and transcriptomic profiling of dysfunctional and bystander CD8+ tumor-infiltrating T cells isolated from primary tumors of treatment-naive non-small cell lung cancer patients. Proteomic analysis revealed widespread discordance with mRNA expression, with 8% of all quantified proteins displaying differential expression exclusively at the protein level. Genetic perturbation of such differentially expressed proteins identified the chromatin remodeler CHD4 and fatty acid synthase (FASN) as cell-intrinsic regulators of T cell function. CHD4 deletion resulted in altered gene-regulatory networks that promoted effector differentiation and enhanced cytokine production. In contrast, FASN deletion preserved mitochondrial fitness and sustained T cell functionality under chronic T cell receptor stimulation. Together, these findings demonstrate that proteomic profiling uncovers regulators of T cell functionality that are not apparent from transcriptomic analyses alone, highlighting an additional layer of regulatory control.

immunology↗

bioRxiv2026

Decoding translational control by cis-regulatory elements and RNA binding proteins in human effector T cells

T cells are central players in killing virally infected and malignant cells. To achieve this, T cells depend on dynamic, tightly regulated alterations of the proteome. To decipher the rules instructing translation in T cells, we performed transcriptome and proteome analysis of polysome fractions from human effector CD8+ T cells. Transcriptome analysis informed on ribosomal occupancy of RNAs and uncovered the rapid and RNA-specific redistribution upon T cell activation. With machine learning, we identified RNA binding motifs that predict the RNA (re)distribution among the polysome fractions. Using matched proteome analysis, we identified polysome-associated RBPs and their swift shuttling across polysome fractions upon T cell activation. Integrating sequence feature analysis with polysome RBP localization uncovered PTBP1 as a positive translation regulator, through its interactions with cis-regulatory elements in 3'UTRs of target mRNAs. In conclusion, the multi-level analysis presented here identifies rules of selective translation control which shape the T cell proteome.

immunology↗

bioRxiv2025

Mapping the dynamic RNA binding proteome in human effector T 1 cells identifies differentiation and cytotoxicity regulators

RNA-binding proteins (RBPs) are key regulators of T cell function by controlling (m)RNA fate and fine-tuning protein expression dynamics. Dysregulated RBPs can drive immune diseases and malignancies, highlighting their potential as therapeutic targets. To achieve this, a systematic analysis of the dynamic RBP-RNA interactions is required. Here, we mapped the RNA-binding proteome in human T cells and measured its alterations upon T cell activation using orthogonal organic phase separation (OOPS), analysed with PROMOGEB, a Bayesian linear regression model. This approach uncovered the intricate RNA-binding dynamics of the RBProteome. Gene-editing of such dynamic RNA binders revealed that TUT1 (Star-PAP) maintains the integrity of the T cell differentiation program, and that mutating SF3A1 enhanced the cytotoxic molecule expression and thus target cell killing. Our work provides the most comprehensive analysis of the effector T cell RBProteome to date and shows the potential of identifying RBPs and their binding dynamics as therapeutic agents. TeaserOOPS analysed with PROMOGEB maps RBP dynamics in human Teff cells, identifying TUT1 and SF3A1 as regulators of T cell fidelity.

immunology↗

bioRxiv2024

Single molecule imaging of transcription dynamics, RNA localization and fate in T cells

T cells are critical effector cells against infections and malignancies. To achieve this, they produce pro-inflammatory cytokines, including IFN-{gamma} and TNF. Cytokine production is a tightly regulated process. The relative contribution of transcriptional and post-transcriptional regulation to mRNA expression is, however, unknown. We therefore optimized single-molecule FISH for primary human T cells (T-cell smFISH) to simultaneously quantify nascent RNA, mature mRNA levels and its localization with single-cell resolution. T-cell smFISH uncovered heterogeneous cytokine mRNA levels, with high cytokine producers displaying biallelic IFNG/TNF RNA transcription activity. Throughout activation, nuclear cytokine mRNAs accumulated, whereas cytoplasmic cytokine mRNA was degraded through translation-dependent decay. Lastly, T-cell smFISH uncovered cytokine-specific regulation by the RNA-binding protein HuR. Thus, T-cell smFISH provides novel insights in the intricate (post)-transcriptional processes in T cells.

immunology↗

bioRxiv2024

Tertiary lymphoid structure-related immune infiltrates in NSCLC tumor lesions correlate with low tumor-reactivity of TIL products

Adoptive transfer of tumor infiltrating lymphocytes (TIL therapy) has shown great potential for the treatment of solid cancers, including non-small cell lung cancer (NSCLC). However, not all patients benefit from this therapy, and the parameters that define the likelihood of TIL products to be tumor reactive are to date unknown. Defining prognostic markers that correlate with high level of tumor-reactivity is key for achieving better tailored immunotherapies. To determine whether the composition of immune cell infiltrates correlates with the tumor-reactivity of expanded TIL products, we employed multi-parameter flow cytometry to characterize the immune cell infiltrates from 26 early-stage, and 20 late-stage NSCLC tumor lesions. Unbiased flow cytometry analysis with Cytotree and Spearmans Rank Correlation was used to correlate immune infiltrates with the expansion rate, immune cell activation and T cell differentiation state, and the anti-tumor response of TIL products generated from the same lesions. The composition of tumor immune infiltrates was highly variable between patients, irrespective of the disease stage. High percentages of B cell infiltrates positively correlated with the presence of conventional CD4+ T cells, and an overall increase of naive T cell infiltrates. In contrast, high B cell infiltrates negatively correlated with the tumor-reactivity of expanded TIL products, as defined by cytokine production upon exposure to autologous tumor digest. Tumors with high B cell infiltrates contained IgD+BCL6+ B cells and CXCR5+BLC6+ CD4+ T cell infiltrates and an increased percentage of naive CD8+ T cells, indicative of the presence of tertiary lymphoid structures (TLS) in tumors with high B cell infiltrates. This study reveals that the composition of immune cell infiltrates in NSCLC tumors associates with the functionality of expanded TIL products from NSCLC tumor lesions. Importantly, the tumor-responsiveness of TIL products negatively correlated with the presence of TLS-associated immune infiltrates in tumors. Our finding may thus help improve patient selection for TIL therapy.

immunology↗