Biology subjects

Verma, A.

Publications and source records attributed to Verma, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Scalable assessment of genome editing off-targets associated with genetic variants

Genome editing with RNA-guided DNA binding factors carries risk of off-target editing at homologous sequences. Genetic variants may introduce sequence changes that increase homology to a genome editing target, thereby increasing risk of off-target editing. Conventional methods to verify candidate off-targets rely on access to cells with genomic DNA carrying these sequences. However, for candidate off-targets associated with genetic variants, appropriate cells for experimental verification may not be available. Here we develop a method, Assessment By Stand-in Off-target LentiViral Ensemble with sequencing (ABSOLVE-seq), to integrate a set of candidate off-target sequences along with unique molecular identifiers (UMIs) in genomes of primary cells followed by clinically relevant gene editor delivery. Gene editing of dozens of candidate off-target sequences may be evaluated in a single experiment with high sensitivity, precision, and power. We provide an open-source pipeline to analyze sequencing data. This approach enables experimental assessment of the influence of human genetic diversity on specificity evaluation during gene editing therapy development.

genomics↗

bioRxiv2024

A novel repressor-activator-competitor module comprising C2H2 zinc finger and NAC transcription factors regulates rice grain development

Grain size and quality are crucial agronomic traits. We have characterized a seed-preferential C2H2 zinc finger transcriptional repressor, ZOS1-15. Its overexpression, knock-down and knock-out plants indicated a negative control over grain size due to altered cell expansion. ZOS1-15 homodimerized and directly interacted with co-repressor TOPLESS and histone deacetylases to form a repression complex. ZOS1-15 also interacted with Mediator subunit MED14_1 and a seed-preferential transcriptional activator, ONAC024, with three alternatively spliced isoforms. The ectopic expression of ONAC024 negatively affected plant growth and development. Seed-preferential overexpression and knock-down plants showed ONAC024 as a positive regulator of grain length due to increased cell proliferation and expansion. CRES-T generated transgenic rice plants indicated a functional divergence amongst ONAC024 isoforms. Tandem interactions were observed between ONAC024-ONAC023-ONAC026-ONAC020. ZOS1-15 and ONAC024 functioned antagonistically to regulate grain amylose and SSP accumulation while ONAC023 affected only amylose. ZOS1-15 and ONAC024 directly regulated the expression of two SSP encoding genes. Binding of ONAC024 was competed by ONAC025-MADS29 complex. The seed-preferential overexpression of SS1/ ONAC025 resulted in decreased grain size and amylose content, but higher yield. This study proposes a repressor-activator-competitor module, wherein ZOS1-15, ONAC024, ONAC023, ONAC025 along with their interactors synergistically and antagonistically regulate multiple aspects of rice grain development.

plant biology↗

bioRxiv2024

APV9WR: An Integrated Web Resource of Alpha papillomavirus 9 for Genomics, Proteomics, Phylogenetic and Therapeutic Analysis

PurposeAlphapapillomavirus 9 is a virus belonging to the Papillomaviridae family. It has a close genetic relationship with high-risk HPV-16 and other HPV strains such as HPV-31, HPV-52, HPV-35, HPV-58, HPV-67, and HPV-35. This virus is responsible for causing warts and malignant tumors and is responsible for about 75% of cervical malignancies and pre-cancerous lesions worldwide. As a result, it requires specialized research and attention. Our goal is to create a comprehensive resource that can assist researchers and scientific groups in their work. Material and MethodologyA total of 1230 full genome sequences and 9140 protein sequences were obtained from GenBank and NCBI Virus, respectively. Further Phylogenetic Analysis, Codon usage and context analysis, CpG islands analysis, Glycosylation sites, Diagnostic Primers, B cell Epitopes and MHC I and MHC II binders were identified and analyzed using relevant Bioinformatics tools and Python program ResultsAPV9WR is a web resource that was developed after analysis. Our data indicate that HPV 35 and HPV 38 have the most genomic diversity. From codon usage analysis, it was observed that AAA, AUU, UAU, UGU, and UUU are the most used codons, while ACG, CCG, CGA, CGG, CGU, GCG, and UCG are some of the unusual codons in APV9 nucleotide sequence with accession id - LC626346.1. We found 4714 CpG island locations in 1230 complete nucleotide sequences of APV9, and only 663 CpG island locations were unique. Further N-linked glycosylation, O-linked glycosylation, diagnostic primers, Potential B-cell epitopes and MHC I and MHC II binders were also analyzed and tabulated. ConclusionWe have consolidated basic information about the virus, such as entire genomic sequences and proteins. It primarily comprises a wide range of studies and outcomes, including genome alignment, phylogenetic inferences, codon context and usage bias, and important CpG island statistics. Furthermore, primers for molecular diagnostics were identified, and glycosylation sites were located and investigated. Most significantly, potential therapeutic elements such as vaccine epitopes and obtaining potential information about them were investigated. Our collective effort on this tool is meant to serve the greater good of the research community for therapeutic intervention for Alphapapillomavirus 9. Using this link https://apv9nsut.web.app will take you to the web app.

bioinformatics↗

bioRxiv2024

Differences in Methanotrophic Community Structure in Two Methane-Rich Habitats: Oil Natural Gas Field & Paddy Field

Methanotrophic bacterial isolates were identified in this study using the molecular detection method, isolated using microbiological techniques, and studied their cellular shape using atomic force microscopy. Two methanotrophic bacterial species belonging to the Methylocaldum and Methylomonas genera were provisionally designated as Isolate 1 and Isolate 5, thus isolated from the Oil-Natural Gas Field and Paddy Field, respectively. The Oil-Natural Gas Field Isolate 1 showed 91.82-97.25% sequence homology to the reference Methanotrophic species, whereas Paddy Field Isolate 5 showed 79.72-84.99% sequence homology to the reference Methylomonas species in the NCBI database. As per the phylogenetic analysis, Oil-Natural Gas Field Isolate 1 and Paddy Field Isolate 5 are possibly new species of Methylocaldum and Methylomonas genus, respectively. In addition, the microscopic study also supported the molecular identification and phylogenetic analysis of isolated species by showing the cocci and rod shapes for the Oil-Natural Gas Field Isolate 1 and Paddy Field Isolate 5, respectively.

microbiology↗

bioRxiv2024

Accelerating Genome- and Phenome-Wide Association Studies using GPUs - A case study using data from the Million Veteran Program

The expansion of biobanks has significantly propelled genomic discoveries yet the sheer scale of data within these repositories poses formidable computational hurdles, particularly in handling extensive matrix operations required by prevailing statistical frameworks. In this work, we introduce computational optimizations to the SAIGE (Scalable and Accurate Implementation of Generalized Mixed Model) algorithm, notably employing a GPU-based distributed computing approach to tackle these challenges. We applied these optimizations to conduct a large-scale genome-wide association study (GWAS) across 2,068 phenotypes derived from electronic health records of 635,969 diverse participants from the Veterans Affairs (VA) Million Veteran Program (MVP). Our strategies enabled scaling up the analysis to over 6,000 nodes on the Department of Energy (DOE) Oak Ridge Leadership Computing Facility (OLCF) Summit High-Performance Computer (HPC), resulting in a 20-fold acceleration compared to the baseline model. We also provide a Docker container with our optimizations that was successfully used on multiple cloud infrastructures on UK Biobank and All of Us datasets where we showed significant time and cost benefits over the baseline SAIGE model.

genetics↗

bioRxiv2024

Oxysterol accumulation in aging cells alters GPCR signalling

Organismal aging is accompanied by the accumulation of senescent cells in the body, which drives tissue dysfunction. Senescent cells have a distinctive profile, including proliferation arrest, resistance to apoptosis, altered gene expression, and high inflammation. Despite global signalling and metabolic dysregulation during senescence, the underlying reasons for changes in signalling remain unclear. GPCRs are pivotal in cellular signalling, dynamically mediating the complex interplay between cells and their surrounding environment to maintain cellular homeostasis. The chemokine receptor CXCR4 plays a crucial role in modulating immune responses and inflammation. It has been shown that expression of CXCR4 increases in cells undergoing senescence, which enhances inflammation post-activation. Here we examine CXCR4 signalling in deeply senescent cells, where cholesterol and its oxidized derivatives, oxysterols, affect receptor function. We report elevated oxysterol levels in senescent cells, which altered classical CXCL12-mediated CXCR4 signalling. Tail-oxidized sterols disrupted signalling more than ring-oxidized counterparts. Molecular dynamics simulations revealed that 27-hydroxycholesterol displaces cholesterol and binds strongly to alter the conformation of critical signalling residues to modify the sterol-CXCR4 interaction landscape. Our study provides a molecular view of the observed mitigated GPCR signalling in the presence of oxysterols, which switched G-protein signalling from Gi/o to Gs class. Overall, we present an altered paradigm of GPCR signalling where cholesterol oxidation alters the signalling outcome in aged cells. Significance StatementOur study brings to light a novel and significant discovery in aged cells: the accumulation of oxysterols, oxidized forms of cholesterol, critically impairs CXCR4-dependent signalling and alters G-protein coupling specificity. This effect of oxysterols is demonstrated for the first time in aged cellular models, providing a molecular basis for a multitude of observed alterations in senescence, such as compromised immune functions and a decline in cellular responsiveness with age. Our research not only fills a crucial gap in understanding the aging process at the molecular level but also identifies potential targets for therapeutic interventions aimed at mitigating age-related cellular dysfunctions and diseases.

cell biology↗

bioRxiv2024

Experimental type 1 diabetes metabolically rejuvenates CD8+ T cells for improved control of tumor growth through an IGF1-IGF1R axis

AbstractEpidemiological studies suggest that patients with pre-existing type 1 diabetes (T1D) have a decreased risk of developing melanoma, prostate cancer, and breast cancer, although the underlying mechanism remains to be elucidated. In translational modelling, we observed that streptozotocin (STZ) induced T1D mice exhibited restricted melanoma and carcinoma (mammary, lung and colon) growth in association with extended overall survival. Tumor-infiltrating CD8+ T cells were found to be responsible for tumor growth restriction. Tumor infiltrating CD8+ T cells but not tumor cells themselves exhibited higher glycolytic and cytotoxic activities in T1D hosts. Such improved anti-tumor T cell function was linked to selective upregulated expression of insulin-like growth factor 1, insulin-like growth factor 1 receptor, and phospho-mTOR in CD8+ T cells in the TME. T1D patient derived CD8+ T cells displayed superior activation in vitro after tumor antigen stimulation vs. non-diabetic CD8+ T cells. Activation of T1D patient derived CD8+ T cells was sensitive to targeted antagonism of IGF1R and mTOR, supporting the operational involvement of the IGF1R-mTOR signaling axis. Our results suggest that selective activation of the intrinsic IGF1R-mTOR signaling axis in CD8+ T cells represents a preferred endpoint to achieving more effective immunotherapy outcomes and improved cancer patient management. SignificanceExperimental type 1 diabetes decelerates tumor growth through metabolic activation of cytotoxic T cells dependent on an IGF1R-mTOR signaling pathway. CD8+IGF1R+IGF1+ T cells play a crucial role in T1D dependent tumor control.

cancer biology↗

bioRxiv2024

Development and tissue specific expression of RAPGEF1 (C3G) transcripts having exons encoding disordered segments with predicted regulatory function.

The ubiquitously expressed RAPGEF1(C3G), regulates differentiation, and is essential for development of mouse embryos. While multiple transcripts have been predicted, evidence of their expression and function is scarce. We demonstrate tissue and development specific expression of novel transcripts with exons 12-14 in various combinations, in the mouse. These exons encode an intrinsically disordered serine-rich polypeptide, that undergoes phosphorylation. Isoform switching occurred during differentiation of myoblasts and mouse embryonic stem cells. In silico structure and docking studies indicated that the additional exons alter intra-molecular interactions keeping it in a closed confirmation, and interaction with its target, RAP1A. Our results demonstrate the expression of novel RAPGEF1 isoforms, and suggest cassette exon inclusion as an additional means of regulating RAPGEF1 activity during differentiation.

molecular biology↗

bioRxiv2024

Broad de-regulated U2AF1 splicing is prognostic and augments leukemic transformation via protein arginine methyltransferase activation

The role of splicing dysregulation in cancer is underscored by splicing factor mutations; however, its impact in the absence of such rare mutations is poorly understood. To reveal complex patient subtypes and putative regulators of pathogenic splicing in Acute Myeloid Leukemia (AML), we developed a new approach called OncoSplice. Among diverse new subtypes, OncoSplice identified a biphasic poor prognosis signature that partially phenocopies U2AF1-mutant splicing, impacting thousands of genes in over 40% of adult and pediatric AML cases. U2AF1-like splicing co-opted a healthy circadian splicing program, was stable over time and induced a leukemia stem cell (LSC) program. Pharmacological inhibition of the implicated U2AF1-like splicing regulator, PRMT5, rescued leukemia mis-splicing and inhibited leukemic cell growth. Genetic deletion of IRAK4, a common target of U2AF1-like and PRMT5 treated cells, blocked leukemia development in xenograft models and induced differentiation. These analyses reveal a new prognostic alternative-splicing mechanism in malignancy, independent of splicing-factor mutations. Statement of significanceUsing a new in silico strategy we reveal counteracting determinants of patient survival in Acute Myeloid Leukemia that co-opt well-defined mutation-dependent splicing programs. Broad poor-prognosis splicing and leukemia stem cell survival could be rescued through pharmacological inhibition (PRMT5) or target deletion (IRAK4), opening the door for new precision therapies. Competing InterestsConflict-of-interest disclosure: DTS. serves on the scientific advisory board at Kurome Therapeutics; is a consultant for and/or received funding from Kurome Therapeutics, Captor Therapeutics, Treeline Biosciences, and Tolero Therapeutics; and has equity in Kurome Therapeutics. AV has received research funding from GlaxoSmithKline, BMS, Jannsen, Incyte, MedPacto, Celgene, Novartis, Curis, Prelude and Eli Lilly and Company, has received compensation as a scientific advisor to Novartis, Stelexis Therapeutics, Acceleron Pharma, and Celgene, and has equity ownership in Throws Exception and Stelexis Therapeutics.

cancer biology↗

bioRxiv2024

Fluorescence Lifetime Imaging for quantification of targeted drug delivery in varying tumor microenvironments

RationaleTrastuzumab (TZM) is a monoclonal antibody that targets the human epidermal growth factor receptor (HER2) and is clinically used for the treatment of HER2-positive breast tumors. However, the tumor microenvironment can limit the access of TZM to the HER2 targets across the whole tumor and thereby compromise TZMs therapeutic efficacy. An imaging methodology that can non-invasively quantify the binding of TZM-HER2, which is required for therapeutic action, and distribution within tumors with varying tumor microenvironments is much needed. MethodsWe performed near-infrared (NIR) fluorescence lifetime (FLI) Forster Resonance Energy Transfer (FRET) to measure TZM-HER2 binding, using in vitro microscopy and in vivo widefield macroscopy, in HER2 overexpressing breast and ovarian cancer cells and tumor xenografts, respectively. Immunohistochemistry was used to validate in vivo imaging results. ResultsNIR FLI FRET in vitro microscopy data show variations in intracellular distribution of bound TZM in HER2-positive breast AU565 and AU565 tumor-passaged XTM cell lines in comparison to SKOV-3 ovarian cancer cells. Macroscopy FLI (MFLI) FRET in vivo imaging data show that SKOV-3 tumors display reduced TZM binding compared to AU565 and XTM tumors, as validated by ex vivo immunohistochemistry. Moreover, AU565/XTM and SKOV-3 tumor xenografts display different amounts and distributions of TME components, such as collagen and vascularity. Therefore, these results suggest that SKOV-3 tumors are refractory to TZM delivery due to their disrupted vasculature and increased collagen content. ConclusionOur study demonstrates that FLI is a powerful analytical tool to monitor the delivery of antibody drug tumor both in cell cultures and in vivo live systems. Especially, MFLI FRET is a unique imaging modality that can directly quantify target engagement with potential to elucidate the role of the TME in drug delivery efficacy in intact live tumor xenografts.

cancer biology↗

bioRxiv2024

Contrast-enhanced photon-counting micro-CT of tumor xenograft models

Photon-counting micro computed tomography (micro-CT) offers new potential in preclinical imaging, particularly in distinguishing materials. It becomes especially helpful when combined with contrast agents, enabling the differentiation of tumors from surrounding tissues. There are mainly two types of contrast agents in the market for micro-CT: small molecule-based and nanoparticle-based. However, despite their widespread use in liver tumor studies, there is a notable gap in research on the application of these commercially available agents for photon-counting micro-CT in breast and ovarian tumors. Herein, we explored the effectiveness of these agents in differentiating tumor xenografts from various origins (AU565, MDA-MB-231, and SKOV-3) in nude mice, using photon-counting micro-CT. Specifically, ISOVUE-370 (a small molecule-based agent) and Exitrone Nano 12000 (a nanoparticle-based agent) were investigated in this context. To improve tumor visualization, we proposed a novel color visualization method for photon-counting micro-CT, which changes color tones to highlight contrast media distribution, offering a robust alternative to traditional material decomposition methods with less computational demand. Our in vivo experiments confirm its effectiveness, showing distinct enhancement characteristics for each contrast agent. Qualitative and quantitative analyses suggested that Exitrone Nano 12000 provides superior vasculature enhancement and better quantitative consistency across scans, while ISOVUE-370 gives more comprehensive tumor enhancement but with a significant variance between scans due to its short blood half-time. This variability leads to high sensitivity to timing and individual differences among mice. Further, a paired t-test on mean and standard deviation values within tumor volumes showed significant differences between the AU565 and SKOV-3 tumor models with the nanoparticle-based (p-values < 0.02), attributable to their distinct vascularity, as confirmed by immunohistochemistry. These findings underscore the utility of photon-counting micro-CT in non-invasively assessing the morphology and anatomy of different tumor xenografts, which is crucial for tumor characterization and longitudinal monitoring of tumor development and response to treatments.

bioengineering↗

bioRxiv2023

E-proteins set the threshold for optimal TCF1 expression during αβ T cell development.

Expression of T Cell Factor-1 (TCF1), encoded by Tcf7, regulates lineage fate decisions during T cell development. Here we demonstrate that E-proteins control the threshold of TCF1 expression required for development of T cells. E-proteins bind to five elements (EPEs) in the Tcf7 locus. The third element, EPE3, interacts directly with Tcf7 promoter in Hi-ChIP analyses, suggesting it is an active enhancer. CRISPR-ablation of EPE3 reduces TCF1 protein expression in precursor thymocytes by 2-fold and dramatically impairs development of {beta} and {gamma}{delta} T cells. Single cell gene expression analysis identified differentiation blocks at multiple CD4-CD8- stages and subsequent transition to CD4+CD8+ stage. These data identify E-proteins and EPE3 as critical for the optimal TCF1 expression required for T cell development.

immunology↗

bioRxiv2023

Nucleotide metabolism constrains prime editing in hematopoietic stem and progenitor cells

Therapeutic prime editing of hematopoietic stem and progenitor cells (HSPCs) holds great potential to remedy blood disorders. Since quiescent cells have low nucleotide levels and resist retroviral infection, we hypothesized that nucleotide metabolism could limit reverse transcription mediated prime editing in HSPCs. We demonstrate that deoxynucleoside supplementation and Vpx-mediated degradation of SAMHD1 improve prime editing efficiency in HSPCs, especially when coupled with editing approaches that evade mismatch repair.

genetics↗

bioRxiv2023

ACSL4 activity drives TNBC metastasis by positively regulating Histone H3 Acetylation mediated SNAIL expression

Triple-Negative Breast Cancer (TNBC) has profound unmet medical need globally for its devastating clinical outcome associated with rapid metastasis and lack of targeted therapies. Recently, lipid metabolic reprogramming has emerged as a major driver of breast cancer metastasis. Here, we unveil a strong association between the heightened expression of fatty acid metabolic enzyme, acyl-CoA synthetase 4 (ACSL4) and TNBC, which is primarily attributed by the selective absence of progesterone receptor (PR). Loss of ACSL4 function, either through genetic ablation or pharmacological inhibition significantly reduces metastatic potential of TNBC. Global transcriptome analysis reveals that ACSL4 activity markedly influences the gene expression pattern associated with TNBC migration. Mechanistically, ACSL4 alters fatty acid oxidation (FAO) and cellular acetyl-CoA levels, leading to the hyper-acetylation of particularly H3K27Ac and H3K9Ac marks resulting in overexpression of SNAIL during the course of TNBC metastatic spread to lymph node and lungs. Further, human TNBC metastasis exhibits positive correlation between ACSL4 and SNAIL expression. Altogether, our findings provide new molecular insights regarding the intricate interplay between metabolic alterations and epigenetic modifications, intertwined to orchestrate TNBC metastasis and posit a rational understanding for the development of ACSL4 inhibitors as a targeted therapy against TNBC.

cancer biology↗

bioRxiv2023

p38-MAPK is prerequisite for the synthesis of SARS-CoV-2 protein

The inhibition of p38 mitogen-activated protein kinase (p38-MAPK) by small molecule chemical inhibitors was previously shown to impair severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) replication, however, mechanisms underlying antiviral activity remains unexplored. In this study, reduced growth of SARS-CoV-2 in p38- knockout Vero cells, together with enhanced viral yield in cells transfected with construct expressing p38, suggested that p38-MAPK is essential for the propagation of SARS-CoV-2. The SARS-CoV-2 was also shown to induce phosphorylation (activation) of p38, at time when transcription/translational activities are considered to be at the peak levels. Further, we demonstrated that p38 supports viral RNA/protein synthesis without affecting viral attachment, entry, and budding in the target cells. In addition, we demonstrated that long-term culture of SARS-CoV-2 in the presence of p38 inhibitor SB203580 does not easily select resistant viral mutants. In conclusion, we provide mechanistic insights on the regulation of SARS-CoV-2 replication by p38 MAPK.

microbiology↗

bioRxiv2023

Apolipoprotein-E transforms intracellular Amyloid-β oligomers to a more toxic state

It is poorly understood why ApoE variants are major genetic risk factors in Alzheimers disease (AD), which is associated with the aggregation of amyloid beta (A{beta}). Here we directly image specific changes in small A{beta} oligomers in rat brain cells that correlate with the cellular ApoE content. An inhibitor of A{beta}-ApoE interaction suppresses this change and concomitantly reduces A{beta} toxicity in a dose-dependent manner. Single-molecule techniques show changes both in the conformation and the stoichiometry of the oligomers. hiPSC-derived neural stem cells from Alzheimers patients also show similar changes. Interaction with ApoE therefore changes the oligomeric state, membrane affinity, and toxicity of A{beta} oligomers, and can be directly read out in live cells. Our findings suggest a rapid and quantitative assay for AD drug discovery. One-sentence summaryApoE causes specific toxicogenic modifications of A{beta} oligomers, and these changes can be directly imaged in live cells.

biochemistry↗

bioRxiv2023

CCR7+ CD4 T Cell Immunosurveillance Disrupted in Chronic SIV-Induced Neuroinflammation in Rhesus Brain.

CD4 T cells survey and maintain immune homeostasis in the brain, yet their differentiation states and functional capabilities remain unclear. Our approach, combining single-cell transcriptomic analysis, ATAC-seq, spatial transcriptomics, and flow cytometry, revealed a distinct subset of CCR7+ CD4 T cells resembling lymph node central memory (TCM) cells. We observed chromatin accessibility at the CCR7, CD28, and BCL-6 loci, defining molecular features of TCM. Brain CCR7+ CD4 T cells exhibited recall proliferation and interleukin-2 production ex vivo, showcasing their functional competence. We identified the skull bone marrow as a local niche for these cells alongside other CNS border tissues. Sequestering TCM cells in lymph nodes using FTY720 led to reduced CCR7+ CD4 T cell frequencies in the cerebrospinal fluid, accompanied by increased monocyte levels and soluble markers indicating immune activation. In macaques chronically infected with SIVCL57 and experiencing viral rebound due to cessation of antiretroviral therapy, a decrease in brain CCR7+ CD4 T cells was observed, along with increased microglial activation and initiation of neurodegenerative pathways. Our findings highlight a role for CCR7+ CD4 T cells in CNS immune surveillance and their decline during chronic SIV-induced neuroinflammation highlights their responsiveness to neuroinflammatory processes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/555037v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@191f9caorg.highwire.dtl.DTLVardef@a67d2dorg.highwire.dtl.DTLVardef@645443org.highwire.dtl.DTLVardef@d81fd6_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefUtilizing single-cell and spatial transcriptomics on adult rhesus brain, we uncover a unique CCR7+ CD4 T cell subset resembling central memory T cells (TCM) within brain and border tissues, including skull bone marrow. Our findings show decreased frequencies of this subset during SIV- induced chronic neuroinflammation, emphasizing responsiveness of CCR7+ CD4 T cells to CNS disruptions. HighlightsO_LICCR7+ CD4 T cells survey border and parenchymal CNS compartments during homeostasis; reduced presence of CCR7+ CD4 T cells in cerebrospinal fluid leads to immune activation, implying a role in neuroimmune homeostasis. C_LIO_LICNS CCR7+ CD4 T cells exhibit phenotypic and functional features of central memory T cells (TCM) including production of interleukin 2 and the capacity for rapid recall proliferation. Furthermore, CCR7+ CD4 T cells reside in the skull bone marrow. C_LIO_LICCR7+ CD4 T cells are markedly decreased within the brain parenchyma during chronic viral neuroinflammation. C_LI

neuroscience↗

bioRxiv2023

CD4 T cell Responses in the CNS during SIV infection

Virologic suppression with antiretroviral therapy (ART) has significantly improved health outcomes for people living with HIV, yet challenges related to chronic inflammation in the central nervous system (CNS) - known as Neuro-HIV-persist. As primary targets for HIV-1 with the ability to survey and populate the CNS and interact with myeloid cells to co-ordinate neuroinflammation, CD4 T cells are pivotal in Neuro-HIV. Despite their importance, our understanding of CD4 T cell distribution in virus-targeted CNS tissues, their response to infection, and potential recovery following initiation of ART remain limited. To address these gaps, we studied ten SIVmac251-infected rhesus macaques using an ART regimen simulating suboptimal adherence. We evaluated four macaques during the acute phase pre-ART and six during the chronic phase. Our data revealed that HIV target CCR5+ CD4 T cells inhabit both the brain parenchyma and adjacent CNS tissues, encompassing choroid plexus stroma, dura mater, and the skull bone marrow. Aligning with the known susceptibility of CCR5+ CD4 T cells to viral infection and their presence within the CNS, high levels of viral RNA were detected in the brain parenchyma and its border tissues during acute SIV infection. Single-cell RNA sequencing of CD45+ cells from the brain revealed colocalization of viral transcripts within CD4 clusters and significant activation of antiviral molecules and specific effector programs within T cells, indicating CNS CD4 T cell engagement during infection. Despite viral suppression with ART, acute infection led to significant depletion of CNS CD4 T cells, persisting into the chronic phase. These findings underscore the functional involvement of CD4 T cells within the CNS during SIV infection, enhancing our understanding of their role in establishing CNS viral presence. Our results offer insights for potential T cell-focused interventions while also underscoring the challenges in eradicating HIV from the CNS, even with effective ART. AUTHOR SUMMARYAntiretroviral therapy (ART) has improved health outcomes of people living with HIV. However, there are still challenges, especially in the central nervous system (CNS), where ongoing inflammation can lead to neurological disorders. Our study focused on understanding the role of CD4 T cells in the brain during HIV infection and treatment. We used a model with SIV-infected rhesus monkeys to study the AIDS virus in the brain and surrounding tissues. We discovered that a subset of CD4 T cells, which are vulnerable to HIV, are present throughout the CNS. During the early stages of infection, we noticed high levels of the virus in both the brain and nearby tissues. By examining these CD4 T cells at a single-cell level, we found that they actively respond to the virus by activating specific antiviral effector functions to fight it. Although ART controlled virus replication in the brain, CD4 T cells were continuously depleted, indicative of immune perturbations. Overall, our study helps us understand the role of CD4 T cells within the CNS during both acute and chronic HIV infection. This knowledge could help us develop new ways to target the virus in the CNS and devise treatments for complications related to Neuro-HIV.

neuroscience↗