Biology subjects

Patel, R. K.

Publications and source records attributed to Patel, R. K..

14 recordsLinked to original sources

bioRxiv2026

Immune activation during broadly neutralizing antibody-mediated HIV suppression prior to post-intervention control

Broadly neutralizing antibodies (bNAbs) have been associated with enhancement of HIV-specific T or B cell responses and sustained partial control of HIV replication in some people with HIV (PWH). The mechanisms through which bNAbs may potentiate host immunity in this context are not known. We previously reported the outcomes of a clinical trial in which ten PWH on antiretroviral therapy (ART) received a combination of immunotherapies including two bNAbs administered immediately preceding an analytic treatment interruption (ATI). After bNAb levels waned, seven participants exhibited varying degrees of post-intervention control of HIV linked to a robust expansion of activated CD8+ T cells in response to rebounding virus. To investigate the role of the bNAbs in enhancing endogenous immune responses, we looked for evidence of HIV-specific or broader immune activation during the period after ART was paused and prior to rebound when bNAbs were controlling HIV replication. At a timepoint early post-ART interruption and at least one month before virus emerged in plasma, we detected an increase in levels of plasma inflammatory proteins as well as phenotypic and transcriptional activation of innate and adaptive immune cells. Compared to non-controllers, post-intervention controllers demonstrated unique transcriptional activation patterns as well as differential longitudinal plasma inflammatory protein trends. No enhancement of HIV-specific T cell or antibody responses was observed in this window. This study identifies activated cell types and inflammatory pathways that are recruited early during bNAb-mediated HIV suppression and that may play a role in potentiating long-lasting HIV immune control after bNAb therapy. One Sentence SummaryIn a combination immunotherapy trial with high rates of post-intervention control, bNAb-mediated HIV suppression was associated with increased immune activation compared to HIV suppression by ART.

immunology↗

bioRxiv2025

Congenital CMV infection drives oligoclonal expansion of cytotoxic γδ T cells from early fetal progenitors

Gamma delta T cells ({gamma}{delta} T cells) emerge early during human gestation and are uniquely equipped to protect the fetus and infant following in utero infection with viruses such as cytomegalovirus (CMV). Previous research showed that fetal {gamma}{delta} T cells in early gestation are transcriptionally pre-programmed for effector functions in the thymus. Infants with congenital CMV infection (cCMV) exhibit expansions of {gamma}{delta} T cells with CMV-reactive TCRs; however, the functional and transcriptional programming of these innate-like effector cells has not been characterized. Here, we analyzed cord blood mononuclear cells from cCMV+ and uninfected neonates in Uganda using flow cytometry and single-cell RNA and TCR sequencing. We find that {gamma}{delta} T cells in cCMV+ neonates are more differentiated, activated, cytotoxic, and proliferative. TCR repertoires of cCMV+ infants exhibit oligoclonal expansions with an enrichment of {gamma}{delta}TCRs that possess shorter CDR3 lengths and fewer N additions, which suggest they arise from early fetal progenitor cells. These expanded {gamma}{delta} T cell clonotypes in cCMV+ infants are more frequently public and exhibit cytotoxic transcriptional programming. These findings demonstrate that cCMV infection drives an oligoclonal expansion of highly cytotoxic effector {gamma}{delta} T cells with fetal-like TCR features, underscoring their specialized roles in early-life immunity.

immunology↗

bioRxiv2025

Clinical cure of chronic hepatitis B is dependent on activation and perpetuation of robust CD4+ T cell responses

Chronic infection with hepatitis B virus (HBV), a major global pathogen, often leads to immune-mediated progressive liver injury and liver cancer. While seroclearance of the surface antigen (HBsAg) defines clinical cure and reduces disease-associated risks, HBsAg clearance is rarely observed and remains therapeutically elusive. Here we overcome some of the challenges to studying immune mechanisms of HBsAg clearance in chronic hepatitis B (CHB) using our mouse model of age-dependent HBsAg clearance and persistence, and samples from our BeNEG-DO clinical trial that provided longitudinal PBMCs from patients who either cleared HBsAg or retained stable HBsAg levels after stopping nucleos(t)ide analog therapy. We show that young mice fail to clear HBsAg and have impaired ability to efficiently initiate and sustain HBV-specific CD4+ T cell responses. We also demonstrate a role for CD4+ T cells in hepatic leukocyte organization and cytotoxicity, and in HBV-specific CD8+ T cell cytotoxicity and HBsAg clearance. Upstream of the CD4+ T cell response, we reveal that hepatic dendritic cells, particularly cDC2s, direct effective CD4+ T cell activation and differentiation. Studies in CHB patients identified immune features of HBsAg clearance that overlap with the mouse model, including TH1 and cytotoxic CD4+ T cell activation and CD8+ T cell cytotoxic effector function. These findings identify an essential role for potent CD4+ T cell activation in the clinical cure of CHB and illuminate potential immunotherapeutic targets for enhancing CD4+ T cell responses to achieve greater HBsAg clearance rates. One Sentence SummaryUsing a mouse model of hepatitis B and longitudinal PBMCs from patients with chronic hepatitis B, we identify shared mechanisms of HBsAg seroclearance.

immunology↗

bioRxiv2025

Single-cell RNA sequencing of peripheral blood defines two immunological subtypes of Sjögren's disease

Sjogrens disease (SjD) is a heterogeneous autoimmune disorder with significant clinical and molecular diversity. While anti-SSA antibodies serve as a hallmark serological biomarker, nearly half of patients lack them, raising fundamental questions about distinct pathogenic mechanisms. To address this, we performed single-cell RNA sequencing with surface protein profiling on 1.5 million PBMCs from 333 participants in the Sjogrens International Collaborative Clinical Alliance cohort, comparing those with and without SjD and stratifying them by anti-SSA status. Our analysis identified two immunologically distinct subtypes of SjD, with SSA-positive patients exhibiting a dominant and persistent IFN-I signature, profoundly impacting immune cell maturation. Notably, transitional B cells were disproportionately affected, displaying signs of premature maturation, reduced BCR diversity, and shorter CDR3 regions, reinforced interactions with proinflammatory cells, thereby fostering an environment conducive to autoreactivity. By contrast, SSA-negative SjD participants lacked an upregulated IFN-I signature, challenging prevailing pathogenic models and suggesting alternative immune dysregulation pathways. These findings support a two-disease model of SjD and highlight transitional B cells as both a key biomarker and a potential therapeutic target. Targeting IFN-I signaling and transitional B cell maturation may represent a novel therapeutic avenue to modulate immune dysregulation and prevent autoreactivity in SjD.

genomics↗

bioRxiv2024

Coordinated variation in the immune systems and microbiomes of healthy humans is linked to tonic interferon states

Human immune systems are highly variable, with most variation attributable to non-genetic sources. The gut microbiome crucially shapes the immune system; however, its relationship with the baseline immune states of healthy humans remains incompletely understood. Therefore, we performed multi-omic profiling of 110 healthy participants through the ImmunoMicrobiome study. A factor-based integrative approach identified coordinated variation, revealing that the tonic interferon response was amongst the most variable immune features in healthy participants. Microbiome composition, pathways, and stool metabolites varied concomitantly with interferon response pathways. Distinct transcriptional programs involving inflammation and TGF-{beta} in SIGLEC-1high monocytes and CD69high activated MAIT and NK cells were representative of these programs. Our study provides extensive data to examine the relationship between the immune states and microbiomes of healthy individuals at steady state, which paves the way for delineating inter-individual differences relevant for disease susceptibility and responses to therapy.

immunology↗

bioRxiv2024

Unraveling SARS-CoV-2 Host-Response Heterogeneity through Longitudinal Molecular Subtyping

Hospitalized COVID-19 patients exhibit diverse immune responses during acute infection, which are associated with a wide range of clinical outcomes. However, understanding these immune heterogeneities and their links to various clinical complications, especially long COVID, remains a challenge. In this study, we performed unsupervised subtyping of longitudinal multi-omics immunophenotyping in over 1,000 hospitalized patients, identifying two critical subtypes linked to mortality or mechanical ventilation with prolonged hospital stay and three severe subtypes associated with timely acute recovery. We confirmed that unresolved systemic inflammation and T-cell dysfunctions were hallmarks of increased severity and further distinguished patients with similar acute respiratory severity by their distinct immune profiles, which correlated with differences in demographic and clinical complications. Notably, one critical subtype (SubF) was uniquely characterized by early excessive inflammation, insufficient anticoagulation, and fatty acid dysregulation, alongside higher incidences of hematologic, cardiac, and renal complications, and an elevated risk of long COVID. Among the severe subtypes, significant differences in viral clearance and early antiviral responses were observed, with one subtype (SubC) showing strong early T-cell cytotoxicity but a poor humoral response, slower viral clearance, and greater risks of chronic organ dysfunction and long COVID. These findings provide crucial insights into the complex and context-dependent nature of COVID-19 immune responses, highlighting the importance of personalized therapeutic strategies to improve both acute and long-term outcomes.

immunology↗

bioRxiv2024

Deciphering gene regulatory programs underlying functionally divergent naive T cell subsets

Naive CD8+ T cells are a heterogeneous population, with different subsets possessing distinct functions and kinetics upon activation. However, the gene regulatory circuits differentiating these naive subsets are not well studied. In this work, we analyzed a large collection of public and newly generated RNA-seq and ATAC-seq profiles of different subsets of naive CD8+ T cells, revealing significant differences in the gene regulatory landscapes between subsets. We leveraged these data by employing a network inference algorithm, Inferelator, to identify the transcriptional regulatory circuits active in each subset. The predicted transcriptional network of the naive CD8+ T cell pool was validated by multiple orthogonal approaches, including CUT&Tag and Micro-C. Interestingly, our network analysis revealed a novel role for Eomes in promoting effector cell differentiation in specific cell subsets. Moreover, we uncovered multiple novel regulators across a variety of subsets and discovered several modules of genes that were co-regulated by shared sets of transcription factors in distinct subsets. Collectively, our data defines the gene regulatory programs differentiating naive CD8+ T cells and facilitates the identification of novel transcription factors that may alter the propensity of naive CD8+ T cells to become effector or memory cells after infection.

systems biology↗

bioRxiv2023

Integrated longitudinal multi-omics study identifies immune programs associated with COVID-19 severity and mortality in 1152 hospitalized participants

Hospitalized COVID-19 patients exhibit diverse clinical outcomes, with some individuals diverging over time even though their initial disease severity appears similar. A systematic evaluation of molecular and cellular profiles over the full disease course can link immune programs and their coordination with progression heterogeneity. In this study, we carried out deep immunophenotyping and conducted longitudinal multi-omics modeling integrating ten distinct assays on a total of 1,152 IMPACC participants and identified several immune cascades that were significant drivers of differential clinical outcomes. Increasing disease severity was driven by a temporal pattern that began with the early upregulation of immunosuppressive metabolites and then elevated levels of inflammatory cytokines, signatures of coagulation, NETosis, and T-cell functional dysregulation. A second immune cascade, predictive of 28-day mortality among critically ill patients, was characterized by reduced total plasma immunoglobulins and B cells, as well as dysregulated IFN responsiveness. We demonstrated that the balance disruption between IFN-stimulated genes and IFN inhibitors is a crucial biomarker of COVID-19 mortality, potentially contributing to the failure of viral clearance in patients with fatal illness. Our longitudinal multi-omics profiling study revealed novel temporal coordination across diverse omics that potentially explain disease progression, providing insights that inform the targeted development of therapies for hospitalized COVID-19 patients, especially those critically ill.

immunology↗

bioRxiv2023

Cyclone: an accessible pipeline to analyze, evaluate and optimize multiparametric cytometry data

In the past decade, high-dimensional single cell technologies have revolutionized basic and translational immunology research and are now a key element of the toolbox used by scientists to study the immune system. However, analysis of the data generated by these approaches often requires clustering algorithms and dimensionality reduction representation which are computationally intense and difficult to evaluate and optimize. Here we present Cyclone, an analysis pipeline integrating dimensionality reduction, clustering, evaluation and optimization of clustering resolution, and downstream visualization tools facilitating the analysis of a wide range of cytometry data. We benchmarked and validated Cyclone on mass cytometry (CyTOF), full spectrum fluorescence-based cytometry, and multiplexed immunofluorescence (IF) in a variety of biological contexts, including infectious diseases and cancer. In each instance, Cyclone not only recapitulates gold standard immune cell identification, but also enables the unsupervised identification of lymphocytes and mononuclear phagocytes subsets that are associated with distinct biological features. Altogether, the Cyclone pipeline is a versatile and accessible pipeline for performing, optimizing, and evaluating clustering on variety of cytometry datasets which will further power immunology research and provide a scaffold for biological discovery.

immunology↗

bioRxiv2022

Magnetic Levitation and Sorting of Neoplastic Circulating Cell Hybrids

Circulating hybrid cells (CHCs) are a novel, rare cell population that harbor tumor and immune cell phenotypes and genotypes and are detectible in peripheral blood. Several recent reports implicated CHCs in the metastatic cascade and found their enumeration to provide better prognostic value than conventionally-defined circulating tumor cells (CTCs). However, methods for isolation and enrichment of CHCs are not well-studied or established. Here, we developed an ultrasensitive, antigen-independent platform leveraging the principles of magnetic levitation for the detection and isolation of disseminated neoplastic CHCs. For the first time, we demonstrate that CHCs can be magnetically focused to different levitation heights, under various paramagnetic conditions using a static levitation system, and we quantified the biophysical properties of CHCs (i.e., levitation heights). In addition, we investigated whether magnetic levitation approach can be combined with the affinity-based strategies to enrich CHCs under the magnetic field. Using clinical samples from breast and colorectal cancer patients, we demonstrated that neoplastic cells can be sorted with a magnetic levitation-based sorting device, without relying on any surface markers. Overall, we demonstrated the feasibility of the magnetic levitation method for unbiased enrichment of rare neoplastic-immune hybrid cells from peripheral blood specimens from cancer patients. This approach can be expanded to more clinical samples and cancer types to unprecedentedly explore the biology of rare neoplastic cells and develop metastasis-tailored therapies broadly impacting personalized and precision clinical treatments.

bioengineering↗

bioRxiv2020

RNA-Seq data analysis of a rodent model of adolescent binge drinking reveals pathways and candidate genes involved in neuronal remodeling and neuroimmune activation

IntroductionBinge drinking is common among adolescents and young adults and is associated with an increased risk of developing alcohol use disorder (AUD) and long-term cognitive deficits. We analyzed RNA-seq data from male Sprague Dawley rats to identify candidate genes that may play a role in the acute and chronic changes in cognitive function during binge-like adolescent alcohol/EtOH exposure and after a period of abstinence. MethodsAt postnatal day (PND) 30, male rats received chronic intermittent EtOH across 16 days. RNA was extracted from hippocampal tissue and sequenced at two acute timepoints, PND 35 and PND 46, and after 24 days forced abstinence (PND 70). We processed RNA-seq data, compiled gene counts, and performed normalization and differential expression analysis (DESeq2). Gene set enrichment analysis was performed through the R package fgsea. Gene sets of the Molecular Signatures Database (MSigDB) collections were used to identify gene pathways that were dysregulated following EtOH exposure. We also evaluated overlapping gene pathways that were affected across all timepoints. ResultsMultiple gene pathway analyses revealed that EtOH has robust effects on neuroinflammation, cellular remodeling, sleep, and bioenergetics. Changes were heavily dependent on whether gene expression was assessed during acute EtOH exposure or after abstinence. Genes involved in sleep regulation were selectively impacted during the acute timepoints, whereas dysregulation of genes involved in bioenergetics were only impacted after abstinence. The most striking changes occurred in genes that regulate neuroinflammatory processes and cellular remodeling. ConclusionThese data reveal acute and chronic effects of EtOH on multiple gene pathways that persist across analytic approaches and identify genes that have increased sensitivity to EtOH. These findings contribute to our understanding of the temporal effects of adolescent EtOH exposure and how gene pathway dysregulation contributes to the protracted emergence of neuronal remodeling in the hippocampus during a critical period of brain maturation.

bioinformatics↗

bioRxiv2020

Robust partitioning of microRNA targets from downstream regulatory changes

The biological impact of microRNAs (miRNAs) is determined by their targets, and robustly identifying direct miRNA targets remains challenging. Existing methods suffer from high falsepositive rates and are unable to effectively differentiate direct miRNA targets from downstream regulatory changes. Here, we present an experimental and computational framework to deconvolute post-transcriptional and transcriptional changes using a combination of RNA-seq and PRO-seq. This novel approach allows us to systematically profile the regulatory impact of a miRNA. We refer to this approach as CARP: Combined Analysis of RNA-seq and PRO-seq. We apply CARP to multiple miRNAs and show that it robustly distinguishes direct targets from downstream changes, while greatly reducing false positives. We validate our approach using Argonaute eCLIP-seq and ribosome profiling, demonstrating that CARP defines a comprehensive repertoire of targets. Using this approach, we identify miRNA-specific activity of target sites within the open reading frame. Additionally, we show that CARP facilitates the dissection of complex changes in gene regulatory networks triggered by miRNAs and identification of transcription factors that mediate downstream regulatory changes. Given the robustness of the approach, CARP would be particularly suitable for dissecting miRNA regulatory networks in vivo.

genomics↗

bioRxiv2018

MiR-146a wild-type 3’ sequence identity is dispensable for proper innate immune function in vivo

The prevailing model of microRNA function is that the \"seed\" region (nucleotides 2-8) is typically sufficient to mediate target recognition and repression. However, numerous recent studies have challenged this model, either by demonstrating extensive 3 pairing between physically defined miRNA-mRNA pairs or by showing in C. elegans that disrupted 3 pairing can result in impaired function in vivo. To test the importance of miRNA 3 pairing in a mammalian system in vivo, we engineered a mutant murine mir-146a allele in which the 5 half of the mature microRNA retains its wild-type sequence, but the 3 half has been altered to be anti-complementary. Mice homozygous or hemizygous for this mutant allele are phenotypically indistinguishable from wild-type controls and do not recapitulate any of the immunopathology previously described for mir-146a-null mice. Our results indicate that 3 pairing is dispensable for the established myeloid function of this key mammalian microRNA.\n\nSummary Blurb3 sequence identity is dispensable for the established function of a mammalian miRNA in vivo.

genetics↗

bioRxiv2018

Analysis of novel domain-specific mutations in the zebrafish ndr2/cyclops gene induced by CRISPR-Cas9 RNPs

Nodal-related protein (ndr2) is a member of the transforming growth factor type {beta} superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in the ndr2 gene in the zebrafish (Danio rerio) lead to similar phenotypes, including loss of the medial floor plate, severe deficits in ventral forebrain development, and cyclopia. Alleles of the ndr2 gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different ndr2 alleles have been reported in zebrafish, of which eight were generated using chemical mutagenesis, four were radiation-induced, and the remaining alleles were obtained via random insertion, gene targeting (TALEN), or unknown methods. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of zebrafish ndr2 and observed cyclopia in the injected (G0) embryos. We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G0) embryos. Targeted PCR amplicon analysis using Sanger sequencing showed that most of the alleles had small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an in vitro nuclease assay and in vivo analysis using embryos. We describe one specific mutant allele resulting in loss of conserved terminal cysteine-coding sequences. This study is another demonstration of the utility of the CRISPR-Cas9 system in generating domain- specific mutations and provides further insights into the structure-function of the ndr2 gene.

genetics↗