Biology subjects

Mathew, D.

Publications and source records attributed to Mathew, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Large-scale production of melanin nanoparticles from Pseudomonas stutzeri strain BTCZ109

Past few decades witnessed a boom in pharmaceutical and bioproduct industry with the help of bioprocess technology. Industrially important bioproducts can be produced in large scale for commercialization with the help of fermenters. Here in, pharmaceutically valuable bioproduct melanin, synthesized from Pseudomonas stutzeri strain BTC109 by using two different sized bioreactors. Under controlled conditions the bacteria were allowed to synthesis melanin nanoparticles. The important parameters to be monitored here are pH, dissolved oxygen, agitation, aeration, melanin production and cell biomass concentration. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/737634v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1cc9ca0org.highwire.dtl.DTLVardef@3eeb0borg.highwire.dtl.DTLVardef@b95bd3org.highwire.dtl.DTLVardef@13b8860_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPharmaceutical and bioproduct development industries witnessed a shoot up due to bioprocess technology. C_LIO_LIIndustrially important bioproduct like melanin can be produced in large scale with the help of industrial fermentation technology. C_LIO_LIThe product thus obtained was found to be nano sized and it can be commercialized. C_LI

microbiology↗

bioRxiv2026

Response surface methodology for melanin nanoparticle production optimization from producer strain Pseudomonas stutzeri BTCZ305 with invitro anti-inflammatory and wound healing potential

Culture conditions were optimized for the production of melanin nanoparticle by the bacterial strain Pseudomonas stutzeri BTCZ 305. Response surface methodology was employed for determining the most significant fermentation conditions using variables including, pH, temperature and L-tyrosine concentration identified through one-factor-at-a time approach. Box-behnken design consisting of 17 different combinations of all these factors were performed. Using this methodology, a quadratic regression model was built and the optimal combinations of media constituents for maximum melanin production 1192.27 g/mL were determined as temperature (32.5 {degrees}C), pH (8.5) and L-tyrosine concentration (7 g/L). Melanin productionwas obtained experimentally coincident with the predicted value and the model was proven to be adequate. The nanostructural distribution, its stability in colloidal suspension and particle size were also characterized with the help of TEM, particle size analysis and Zeta potential. The potent applicability of this molecule in anti-inflammation and wound healing was also elucidated. HighlightsO_LIMelanin nanoparticle synthesis by marine bacterial strain Pseudomonas stutzeri BTCZ 305 C_LIO_LIMedia optimization using one-factor-at-a-time method C_LIO_LIStatistical media optimization using Response surface methodology and Box-behnken design of experiment C_LIO_LINanoparticle characterization using TEM, particle size analysis and zeta potential. Its functional role in anti-inflammation and wound healing. C_LI

microbiology↗

bioRxiv2026

Optimization of process parameters for melanin nanoparticles synthesised from Pseudomonas stutzeri (BTCZ 109) using OFAT method and its anticancer property evaluation

Melanins are biological macromolecule with immense functionality synthesised by a wide spectrum of living organism. It is mainly synthesised by the oxidative polymerization of indolic and phenolic compounds through several enzymatic process. It has wide spread application in agriculture, cosmetic and therapeutic industry due to its various properties including antioxidation ability, UV protection efficiency and anticancer activity. Because of this wide range of application in different sectors, large scale production and commercialization attains enormous consideration. The present study deals with the effect of 12 different process parameters on melanin production viz., production media, incubation time, inoculum concentration, pH, temperature, agitation, carbon source, phosphate and magnesium source, CuSO4.5H2O, sodium chloride and L-tyrosine on melanin production by Pseudomonas stutzeri strain BTCZ 109 obtained from Arabian sea sediments was evaluated. After optimizing the important process parameters, the bacteria showed about [~] 4.65 fold increase in melanin production compared to unoptimized cultural conditions. The melanin optimized through this method was found to be nano sized. The Nano sized DOPA melanin in treating Skin cancer cell line SK ML28 which showed a dose-dependent activity with an IC50 value of 164 g/mL. All these results highlight the therapeutic efficiency of DOPA melanin Nano particle as promising bioactive molecule. HighlightsO_LIProcess parameter optimization for large scale production of Nano-melanin obtained from Pseudomonas stutzeri strain BTCZ 109 through one-factor-at-a-time method. C_LIO_LIOptimization of 12 different process parameters were carried out which led to an increase in yield of [~] 4.65 fold compared to unoptimized culture conditions. C_LIO_LIIts efficiency in treating skin cancer cell line SK ML28 was also detected. C_LI

microbiology↗

bioRxiv2025

A cytochrome P450 G subfamily member, CYP4G35, is highly expressed in antennae and modulates olfactory response in Aedes aegypti mosquitoes.

The cytochrome P450 enzymes of the CYP4G subfamily are some of the most enigmatic insect P450s. The dipterans with sequenced genomes have two CYP4G paralogs. In Drosophila melanogaster, CYP4G1 is highly expressed in the oenocytes and catalyzes the last enzymatic step in the biosynthesis of cuticular hydrocarbons. In contrast, CYP4G15 is expressed in the brain glial cells, but its function is unknown. The Aedes aegypti genome encodes two CYP4Gs: CYP4G36 (ortholog of DmCYP4G1) and CYP4G35 (ortholog of DmCYP4G15). Here, we show that CYP4G35 is highly expressed in mosquito antennae, and the RNAi knockdown of CYP4G35 results in delayed host-seeking. Ae. aegypti CYP4G knockout lines confirmed delayed host-seeking behavior in CYP4G35 knockout females. Proteomics analysis of CYP4G35 KO females also corroborates the physiological findings and shows upregulation of proteins related to olfaction and other CYP4Gs to compensate for the lack of CYP4G35. Immunohistochemistry and in situ hybridization were used to localize CYP4G35 and demonstrated its expression in the sensilla lymph of the antennae and the tip of the proboscis. CYP4G35 and CYP4G36 fusion proteins with cytochrome P450 reductase demonstrated that, unlike CYP4G36, CYP4G35 lacks an oxidative decarbonylase function. Together, our data support a novel function of CYP4G35 in modulating olfactory response.

molecular biology↗

bioRxiv2025

Geometric Tuning of Cytokine Receptor Association Modulates Synthetic Agonist Signaling

Cytokines signal by bringing receptor subunits together, but the role of receptor geometry in shaping signaling remains unclear because natural ligands enforce fixed assemblies. Here, we present a de novo protein design platform that rigidly scaffolds receptor-binding domains into defined spatial arrangements. Applying this across IL-7, type I and III interferons, IL-10, gp130, {beta} common, and synthetic receptor pairs, we show that by varying geometry, we can bias pSTAT pathway usage and tune functional outcomes. Geometric control allowed us to decouple pSTAT1 from pSTAT5 in IL-7, separate antigen presentation (MHC-I) from checkpoint induction (PD-L1) in type I interferons, and suppress pro-inflammatory IFN{gamma} secretion while retaining anti-inflammatory activity in IL-10. We further created minimal IL-6 and IL-3 agonists and strengthened synthetic receptor pairings inaccessible with present cytokines. These results establish receptor geometry as a central determinant of cytokine activity and provide a platform for programmable immune modulation.

synthetic biology↗

bioRxiv2025

High-Throughput De Novo Protein Design Yields Novel Immunomodulatory Agonists

Cytokines regulate cell behavior by bringing together specific receptor subunits to trigger downstream signaling. Designed molecules that bring together non-natural receptor pairs could have novel signaling responses and cell specificities. We present a high-throughput de-novo design approach to create novel cytokines by generating and fusing pairs of computationally designed binders. By combining 33 designed receptor-binding domains, we generated over a thousand potential de novo designed "Novokines", of which 75 activated pSTAT signaling in peripheral blood mononuclear cells. We characterized 18 of these, including new pairings of established common receptors, cross-family pairings such as TrkA-{gamma}common, and a series of pairings with interferon receptor-1 (IFNAR1), revealing that IFNAR1 can function as a versatile common receptor similar to {gamma}common or {beta}common. We identify novokines that drive monocyte proliferation, T cell survival and CD4+ T cell-specific proliferation. Our framework provides a blueprint for expanding the understanding of cytokine signaling and generating novel therapeutic proteins.

synthetic biology↗

bioRxiv2025

Downregulation of Transducin Delays Photoreceptor Degeneration in P23H Rhodopsin Retinitis Pigmentosa

In inherited blinding diseases, such as Retinitis Pigmentosa (RP), photoreceptors progressively degenerate, eventually leading to blindness. Unfortunately, effective treatments to prevent or delay vision loss do not exist for most RPs. Dark rearing is known to delay retinal degeneration in preclinical models of RP. Therefore, in this study we evaluated the impact of reducing photoreceptor light signaling on RP progression. This was done by genetically ablating or downregulating transducin in rods or cones in a preclinical RP model carrying a single P23H mutant rhodopsin allele (P23H mice). Ablating rod transducin significantly improved photoreceptor survival in the P23H retina. Additionally, downregulating rod transducin promoted photoreceptor survival and improved rod light response in P23H mice. Remarkably, male P23H mice retained robust cone function until old age in the absence of rod transducin whereas female P23H carriers experienced significantly faster loss of cone function. In these females, reducing cone transducin improved cone function whereas the same treatment was not effective in male P23H carriers. Our data demonstrate that reducing rod or cone transducin expression in P23H mice improves the survival and function of rods and cones, and suggest transducin downregulation as an effective therapeutic strategy to delay photoreceptor degeneration in RP.

physiology↗

bioRxiv2025

Deciphering Cell Fate and Clonal Dynamics via Integrative Single-Cell Lineage Modeling

Through natural or synthetic lineage barcodes, single-cell technologies now enable the joint measurement of molecular states and clonal identities, providing an unprecedented opportunity to study cell fate and dynamics. Yet, most computational methods for inferring cell development and differentiation rely exclusively on transcriptional similarity, overlooking the lineage information encoded by lineage barcodes. This limitation is exemplified by T cells, where subtle transcriptional differences mark divergent fates with distinct biological activity. Single-cell RNA and matched TCR sequencing is now ubiquitous in the analysis of clinical samples, where the TCR sequence provides an endogenous clonal barcode and could reveal clonal T cell responses. We present Clonotrace, a computational framework that jointly models gene expression and clonotype information to infer cell state transitions and fate biases with higher fidelity. While motivated by challenges in analyzing T cell populations, especially in the tumor microenvironment and immunotherapy settings, Clonotrace is broadly applicable to any lineage-barcoded single-cell dataset. Across diverse systems including T cells, hematopoietic differentiation, and cancer therapy resistance models, Clonotrace reveals differentiation hierarchies, distinguishes unipotent from multipotent states, and identifies candidate fate-determining genes driving lineage commitment.

bioinformatics↗

bioRxiv2025

Divergent ontogeny of Tissue Resident Memory and Tissue Resident Exhausted CD8+ T cells underlies distinct functional potential

Persistent antigen stimulation promotes differentiation of exhausted CD8+ T (TEX) cells. TEX cells are distinct from circulating memory T (TCIRCM) cells but share many features with tissue-resident memory (TRM) cells established following infection resolution. CD8+ T cells co-expressing residency- and exhaustion-associated molecules in chronic diseases often correlate with clinical outcomes. However, the relationship between these cells and conventional TRM or TEX cells remains unclear. Here, we show that chronic antigen stimulation drives development of tissue-resident TEX (TR-TEX) cells that are ontologically and functionally distinct from TRM cells generated after antigen clearance. TR-TEX phenotypically resembled TRM cells but were regulated by distinct transcriptional networks and were uniquely dependent on Tox for residency programming. Although TEX progenitor cells acquired residency features upon entering chronically infected tissues, they failed to generate conventional TRM cells after antigen withdrawal. Conversely, TRM cells were able to differentiate into TEX cells during chronic antigen stimulation. Deriving cell-state specific transcriptional signatures revealed a selective association of TR-TEX cells with patient responses to immune checkpoint blockade, and only TR-TEX but not TRM cells responded to PD-1 pathway inhibition in vivo. These data suggest that TR-TEX and TRM cells are developmentally distinct cell types that share a tissue-residency program but have distinct roles in disease control.

immunology↗

bioRxiv2025

YPEL controls synapse development through p62-Nrf2 antioxidant pathway in Drosophila neuromuscular junction

The Yippee-like (YPEL) genes are highly conserved among all eukaryotic species, yet the molecular and cellular pathways that YPEL regulate are poorly understood. Human and animal studies suggest that YPEL is involved in nervous system functions. Here we report that YPEL is necessary for synapse development in neuromuscular junction and motor functions in Drosophila. YPEL interacts with the Drosophila p62, Refractory to sigma P, which forms cytoplasmic proteinaceous bodies for selective autophagy and signaling. YPEL overexpression decreased p62 bodies, while increased p62 bodies were observed in YPEL mutant neurons. Suppressing p62 bodies by reducing p62 gene dosage significantly alleviated both synaptic and locomotion defects in YPEL mutants, suggesting that YPEL acts through p62 for synapse development. On the other hand, suppressing p62 bodies via autophagy did not restore synapse development in YPEL mutants. Interestingly, reduced levels of reactive oxygen species were found in YPEL mutants, which is consistent with the role of p62 in promoting the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant pathway. Overexpressing Drosophila Nrf2, Cap n collar (Cnc), phenocopied the synaptic and locomotion deficits in YPEL mutants. Importantly, both synaptic and locomotion defects were completely rescued by knocking down Cnc in YPEL mutant motor neurons. Taken together, our study demonstrates that YPEL negatively controls p62 - Nrf2 antioxidant pathway for neuromuscular synapse development and locomotion.

neuroscience↗

bioRxiv2024

Automated Cytometric Gating with Human-Level Performance Using Bivariate Segmentation

Recent advances in cytometry technology have enabled high-throughput data collection with multiple single-cell protein expression measurements. The significant biological and technical variance between samples in cytometry has long posed a formidable challenge during the gating process, especially for the initial gates which deal with unpredictable events, such as debris and technical artifacts. Even with the same experimental machine and protocol, the target population, as well as the cell population that needs to be excluded, may vary across different measurements. To address this challenge and mitigate the labor-intensive manual gating process, we propose a deep learning framework UNITO to rigorously identify the hierarchical cytometric subpopulations. The UNITO framework transformed a cell-level classification task into an image-based semantic segmentation problem. For reproducibility purposes, the framework was applied to three independent cohorts and successfully detected initial gates that were required to identify single cellular events as well as subsequent cell gates. We validated the UNITO framework by comparing its results with previous automated methods and the consensus of at least four experienced immunologists. UNITO outperformed existing automated methods and differed from human consensus by no more than each individual human. Most critically, UNITO framework functions as a fully automated pipeline after training and does not require human hints or prior knowledge. Unlike existing multi-channel classification or clustering pipelines, UNITO can reproduce a similar contour compared to manual gating for each intermediate gating to achieve better interpretability and provide post hoc visual inspection. Beyond acting as a pioneering framework that uses image segmentation to do auto-gating, UNITO gives a fast and interpretable way to assign the cell subtype membership, and the speed of UNITO will not be impacted by the number of cells from each sample. The pre-gating and gating inference takes approximately 2 minutes for each sample using our pre-defined 9 gates system, and it can also adapt to any sequential prediction with different configurations.

immunology↗

bioRxiv2024

Single-cell Masked Autoencoder: An Accurate and Interpretable Automated Immunophenotyper

High-throughput single-cell cytometry data are crucial for understanding involvement of immune system in diseases and responses to treatment. Traditional methods for annotating cytometry data, specifically manual gating and clustering, face challenges in scalability, robustness, and accuracy. In this study, we propose a cytometry masked autoencoder (cyMAE), which offers an automated solution for immunophenotyping tasks including cell type annotation. The cyMAE model is designed to uphold user-defined cell type definitions, thereby facilitating easier interpretation and cross-study comparisons. The cyMAE model operates on a pre-train and fine-tune approach. In the pre-training phase, cyMAE employs Masked Cytometry Modelling (MCM) to learn relationships between protein markers in immune cells solely based on protein expression, without relying on prior information such as cell identity and cell type-specific marker proteins. Subsequently, the pre-trained cyMAE is fine-tuned on multiple specialized tasks via task-specific supervised learning. The pre-trained cyMAE addresses the shortcomings of manual gating and clustering methods by providing accurate and interpretable predictions. Through validation across multiple cohorts, we demonstrate that cyMAE effectively identifies co-occurrence patterns of bound labeled antibodies, delivers accurate and interpretable cellular immunophenotyping, and improves the prediction of subject metadata status. Specifically, we evaluated cyMAE for cell type annotation and imputation at the cellular-level and SARS-CoV-2 infection prediction, secondary immune response prediction against COVID-19, and prediction of the infection stage in COVID-19 progression at the subject-level. The introduction of cyMAE marks a significant step forward in immunology research, particularly in large-scale and high-throughput human immune profiling. This approach offers new possibilities for predicting and interpreting cellular-level and subject-level phenotypes in both health and disease.

bioinformatics↗

bioRxiv2023

Breeding and identification of promising Mauritius x Kew pineapple hybrids with high heterosis for fruit and plant traits

Leading cultivars of pineapple Mauritius and Kew were hybridized and 25 hybrids were evaluated under open field conditions, using randomized block design with two replications. Performance of the female parent cum check variety Mauritius, male parent Kew and check variety in Kerala state, India, Amritha, were also evaluated and compared. Based on the performance, heterobeltiosis, average heterosis and standard heterosis over two check varieties, in each hybrid, for 10 plant growth traits and 24 fruit traits, were calculated. For fruit weight, hybrid H35 had the highest heterobeltiosis and standard heterosis over Amritha whereas H62 had highest standard heterosis over Mauritius and average heterosis. For pulp weight, hybrid H17 had the highest values for all heterosis parameters. For TSS, hybrid H62 had the highest heterobeltiosis and other parameters were highest in H43. For days to attain physiological maturity, crown weight, peel weight and acidity, H27, H30, H77 and H43, respectively were lowest in all heterosis parameters. Based on the selection criterion [{sum}average heterosis (fruit weight, TSS, pulp weight) - {sum}average heterosis (crown weight, peel weight, eye profile, eye relative surface, time taken for physiological maturity, acidity)] developed using the average heterosis values for desirable and undesirable fruit traits, six hybrids H66, H17, H59, H43, H70 and H35 were identified for further evaluation. The identified hybrids also satisfied the requirements in fruit weight ([≥]1.0 kg), pulp weight ([≥]750.58 g), TSS ([≥]14.49 {circ}Brix), days to attain physiological maturity ([≤]185.70 days), crown weight ([≤]305.50 g), peel weight ([≤]159.27 g) and acidity ([≤]1.05).

plant biology↗

bioRxiv2023

Deep Phenotyping of the Lipidomic Response in COVID and non-COVID Sepsis

Lipids may influence cellular penetrance by pathogens and the immune response that they evoke. Here we find a broad based lipidomic storm driven predominantly by secretory (s) phospholipase A2 (sPLA2) dependent eicosanoid production occurs in patients with sepsis of viral and bacterial origin and relates to disease severity in COVID-19. Elevations in the cyclooxygenase (COX) products of arachidonic acid (AA), PGD2 and PGI2, and the AA lipoxygenase (LOX) product, 12-HETE, and a reduction in the high abundance lipids, ChoE 18:3, LPC-O-16:0 and PC-O-30:0 exhibit relative specificity for COVID-19 amongst such patients, correlate with the inflammatory response and link to disease severity. Linoleic acid (LA) binds directly to SARS-CoV-2 and both LA and its di-HOME products reflect disease severity in COVID-19. AA and LA metabolites and LPC-O-16:0 linked variably to the immune response. These studies yield prognostic biomarkers and therapeutic targets for patients with sepsis, including COVID-19. An interactive purpose built interactive network analysis tool was developed, allowing the community to interrogate connections across these multiomic data and generate novel hypotheses.

systems biology↗

bioRxiv2023

Signal recovery in single cell batch integration

Data integration to align cells across batches has become a cornerstone of single cell data analysis, critically affecting downstream results. Yet, how much biological signal is erased during integration? Currently, there are no guidelines for when the biological differences between samples are separable from batch effects, and thus, data integration usually involve a lot of guesswork: Cells across batches should be aligned to be "appropriately" mixed, while preserving "main cell type clusters". We show evidence that current paradigms for single cell data integration are unnecessarily aggressive, removing biologically meaningful variation. To remedy this, we present a novel statistical model and computationally scalable algorithm, CellANOVA, to recover biological signal that is lost during single cell data integration. CellANOVA utilizes a "pool-of-controls" design concept, applicable across diverse settings, to separate unwanted variation from biological variation of interest. When applied with existing integration methods, CellANOVA allows the recovery of subtle biological signals and corrects, to a large extent, the data distortion introduced by integration. Further, CellANOVA explicitly estimates cell- and gene-specific batch effect terms which can be used to identify the cell types and pathways exhibiting the largest batch variations, providing clarity as to which biological signals can be recovered. These concepts are illustrated on studies of diverse designs, where the biological signals that are recovered by CellANOVA are shown to be validated by orthogonal assays. In particular, we show that CellANOVA is effective in the challenging case of single-cell and single-nuclei data integration, where the recovered biological signals are replicated in an independent study.

bioinformatics↗

bioRxiv2023

Prior vaccination enhances immune responses during SARS-CoV-2 breakthrough infection with early activation of memory T cells followed by production of potent neutralizing antibodies

SARS-CoV-2 infection of vaccinated individuals is increasingly common but rarely results in severe disease, likely due to the enhanced potency and accelerated kinetics of memory immune responses. However, there have been few opportunities to rigorously study early recall responses during human viral infection. To better understand human immune memory and identify potential mediators of lasting vaccine efficacy, we used high-dimensional flow cytometry and SARS-CoV-2 antigen probes to examine immune responses in longitudinal samples from vaccinated individuals infected during the Omicron wave. These studies revealed heightened Spike-specific responses during infection of vaccinated compared to unvaccinated individuals. Spike-specific CD4 T cells and plasmablasts expanded and CD8 T cells were robustly activated during the first week. In contrast, memory B cell activation, neutralizing antibody production, and primary responses to non-Spike antigens occurred during the second week. Collectively, these data demonstrate the functionality of vaccine-primed immune memory and highlight memory T cells as rapid responders during SARS-CoV-2 infection.

immunology↗

bioRxiv2022

Enhanced STAT5a activation rewires exhausted CD8 T cells during chronic stimulation to acquire a hybrid durable effector like state

Rewiring exhausted CD8 T cells (TEX) towards more functional states is a major goal of cancer immunotherapy but has proven challenging due to the epigenetic stability of TEX. Indeed, TEX are epigenetically programmed by the transcription factor Tox. However, epigenetic changes continue to occur as TEX transition from progenitor (TEXprog), to intermediate (TEXint) and terminal (TEXterm) subsets, suggesting potential developmental flexibility in mature TEX subsets. By examining the transition of TEXprog into TEXint cells, we discovered a reciprocally antagonistic circuit between Stat5a and Tox in TEX cells. Stat5-activity controlled TEXint development, antagonized Tox, and instigated partial effector biology. Stat5 was also essential for TEX reinvigoration by PD-1 blockade. Indeed, temporal induction of Stat5-activity in TEX using an orthogonal IL-2/IL2R{beta}-pair fostered TEXint cell accumulation and synergized with PD-L1 blockade. Constitutive Stat5a activity (STAT5CA) antagonized Tox-dependent TEX epigenetic programming to generate a durable hybrid effector/NK-like population with enhanced tumor control. Finally, enforcing Stat5-signals in established TEXprog partially rewired the TEX epigenetic landscape towards the effector/memory lineage. Together, these data highlight therapeutic opportunities of manipulating Stat5 to rewire TEX towards a durably protective hybrid program.

immunology↗

bioRxiv2022

Induction of a CD8 T cell intrinsic DNA damage and repair response is associated with clinical response to PD-1 blockade in uterine cancer.

Despite the success of immune checkpoint blockade (ICB), many patients fail to achieve durable clinical benefit, and the underlying immunological mechanisms remain poorly understood. Here, we investigated immune reinvigoration by ICB in advanced or recurrent hypermutated or microsatellite instability-high, mismatch repair deficient (MSI-H/MMRd) uterine cancer patients treated with anti-PD-1 (nivolumab). CD8 T cells underwent rapid pharmacodynamic proliferation 2-4 weeks after initiating PD-1 blockade. This immunological response, however, did not correlate with clinical response. We hypothesized that the T cell-intrinsic response to proliferative and genotoxic stress might contribute to the disparity between immunological and clinical response. We developed a high-dimensional single cell cytometric platform to simultaneously analyze T cell differentiation with changes in DNA damage and repair (DDR) pathways. This DDR-Immune platform revealed T cell subset-specific patterns of DDR, and distinct DDR pathways induced by different types of DNA damage. Applying this platform to MSI-H/MMRd or hypermutated uterine cancer patients revealed a signature of DDR exemplified by rapid increase in phosphorylated-ATM (pATM) intrinsic to CD8 T cells proliferating in response to PD-1 blockade that distinguished clinical responders and non-responders. ATM regulated transcriptional circuits in T cells were associated with better clinical response to PD-1 blockade. These findings highlight a previously unrecognized role for CD8 T cell-intrinsic DDR as a potential determinant of immune fitness and clinical outcome of PD-1 blockade therapy. STATEMENT OF SIGNIFICANCEUsing high-dimensional immune and DNA damage response (DDR) profiling in T cell subsets we identified distinct signatures in anti-PD-1 treated MSI-H/hypermutated uterine cancer patients associated with clinical response versus non-response. Thus, T cell-intrinsic DDR is a potential determinant of immune responsiveness and clinical outcome to PD-1 blockade therapy in cancer.

immunology↗