Biology subjects

Nellore, A.

Publications and source records attributed to Nellore, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Tfh dysfunction underlies poor responses to HBV vaccination

The primary goal of vaccination is to induce durable protection through generation of antigen-specific humoral immunity and cellular immune memory. Yet the same vaccine may elicit durable protection in some and less durable responses in others. Antigen-specific antibody responses arise from germinal centers (GCs), where T follicular helper (Tfh) cells help cognate B cells undergo somatic hypermutation (SHM), affinity maturation to generate humoral protection and cellular immune memory. In humans, however, the mechanisms linking GC dynamics to downstream antibody output remain incompletely characterized. To identify the molecular and cellular events that underlie differential vaccine responsiveness, we leveraged the known, pronounced variability in vaccine antigen-specific antibody concentration and durability observed after Hepatitis B virus (HBV) immunization. We longitudinally profiled 101 healthy adults undergoing either de novo (n=59) or booster (n=42) immunization. We then stratified participants into High (>100 mIU/mL) and Low (<100 mIU/mL) responder groups based on vaccine antigen-specific antibody responses. From this cohort, we obtained core needle biopsies (CNBx) of vaccine-draining lymph nodes from 10 participants (High Responder n=6, Low Responder n=4), 10-21 days post-final vaccination and performed single-cell RNA sequencing with paired antigen receptor sequencing. Lymph node BCR repertoire analysis revealed convergent, semi-public clonotypes across unrelated participants with elevated SHM. Transcriptomic profiling of GC-B cells uncovered stark functional divergence between response groups, with High Responders showing robust activation across both dark zone (DZ) and light zone (LZ) compartments and enrichment of MYC and mTORC1 signaling. In contrast, Low Responders exhibited a contracted LZ compartment and a stalled GC B cell response. This divergence in B cell responses corresponded to changes in the T cell compartment. High Responders harbored an expanded Tfh subset marked by increased expression of CXCL13, ICOS, and GNG4, indicative of their GC localization and capacity to provide B cell help. Moreover, receptor-ligand analysis indicated that these GNG4+Tfh formed coordinated costimulatory networks capable of driving LZ GC-B cell licensing. Low Responders demonstrated a divergent program in which their GNG4+Tfh displayed aberrant inflammatory transcriptional activity. This inflammatory signaling was associated with reduced specificity of BCL6 regulon activity despite maintained expression of canonical Tfh genes. Together, these findings reveal that variable HBV vaccine responsiveness was associated with transcriptional network alterations in the GNG4+Tfh subset, offering a molecular framework for understanding poor vaccine responses. One Sentence SummaryLymph node Tfh dysfunction is associated with poor vaccine responses to Hepatitis B virus vaccination

immunology↗

bioRxiv2026

Terminally Differentiated Influenza-Specific Effector Memory B Cells Circulate after Live Attenuated Influenza Vaccination

Live attenuated influenza vaccination (LAIV) is the only FDA approved mucosal vaccine. Easily assayed, circulating correlates of protection after LAIV are lacking. Using fluorochrome labeled hemagglutinin (HA) antigen, we previously identified a subset of HA-specific (HA+) IgDneg memory B cells that circulate after inactivated intramuscular vaccination (IIV), express the master transcriptional regulator, T-bet, as well as effector memory genes and predict durable antibody (Ab) responses to IIV. Here we profile the circulating HA+ IgDneg memory B cell response in a cohort of immunized patients who seroconvert after LAIV to identify which, if any, circulating HA+ B cells predict antibody responses after LAIV. Although we report LAIV elicits circulating T-bet+ HA+ IgDneg B cells that are phenotypically similar to those we described after IIV, we find no correlation between the magnitude of these cells and systemic HA-IgG responses after LAIV. Supervised and unsupervised analyses demonstrate that unlike IIV, LAIV preferentially elicits circulating HA+ IgDneg B cells that co-express TBX21 and the terminal effector cell gene, Zeb2. Consistent with their terminal differentiation status, LAIV-elicited T-bet+ cells cannot be recalled as short-lived antibody-secreting cells (ASCs) after systemic or mucosal antigen re-challenge. We conclude that the transcriptional profiles and functions of HA+ IgDneg B cells vary by influenza vaccine platform.

immunology↗

bioRxiv2025

Coordinated circulating and tissue-based T cell responses precede xenograft rejection

Despite the life-saving successes of solid organ transplantation, the number of individuals needing organ transplant far exceeds the number of organs available for use each year. Porcine xenotransplantation, or the use of pig organs for transplantation in people, holds substantial promise but xenograft rejection in humans is poorly understood. T cell rejection by the host immune system is a major challenge for human allografts and may limit the longevity of porcine xenografts. To study the xenograft rejection, we evaluated T cell responses and repertoire dynamics across tissues following porcine thymokidney transplantation in a decedent model over 61 days after bilateral native kidney nephrectomy. Despite induction with anti-thymocyte globulin and ongoing immune suppression consisting of rituximab, corticosteroids, calcineurin inhibition, and mycophenolate mofetil, human T cell infiltration of the xenograft was observed and was associated with xenograft dysfunction. Longitudinal analysis of T cell clonotypes in biopsies of thymokidney revealed accumulation of clonal human CD4 and CD8 T cell responses. Moreover, circulating activated T cells, including circulating T follicular helper (cTfh), were xeno-reactive and increased in frequency around rejection events. We confirmed clonal dominance of a single CD8 clonotype - identified as donor-reactive in a mixed lymphocyte reaction - in the circulation leading up to the acute cellular rejection event. Following re-treatment with anti-thymocyte globulin and intensification of corticosteroids, the T cell clonotypes were dramatically diminished in frequency in thymokidney and lymph nodes, though not eliminated. Over time of observation, the T cell clonotypes were shared across multiple compartments, including xenograft, circulation and lymph nodes and formed clonal families with known xeno-reactive clonotypes, suggesting a coordinated immune response against a limited pool of antigenic targets. Together, these data demonstrate T cell repertoire dynamics across tissues in the setting of xenograft rejection and highlight opportunities for early surveillance, prediction and potential intervention. One-sentence summaryAfter pig-to-human kidney xenotransplantation, xenoreactive T cells form clonotypic families across blood, graft, and lymphoid tissues, indicating a coordinated and targeted response.

immunology↗

bioRxiv2022

Discordant results among MHC binding affinity prediction tools

A large number of machine learning-based Major Histocompatibility Complex (MHC) binding affinity (BA) prediction tools have been developed and are widely used for both investigational and therapeutic applications, so it is important to explore differences in tool outputs. We examined predictions of four popular tools (netMHCpan, HLAthena, MHCflurry, and MHCnuggets) across a range of possible peptide sources (human, viral, and randomly generated) and MHC class I alleles. We uncovered inconsistencies in predictions of BA, allele promiscuity and the relationship between physical properties of peptides by source and BA predictions, as well as quality of training data. Our work raises fundamental questions about the fidelity of peptide-MHC binding prediction tools and their real-world implications.

bioinformatics↗

bioRxiv2022

Low Molecular Weight Hyaluronan Inhibits Lung Epithelial Ion Channels by Activating Calcium-Sensing Receptor

Herein, we tested the hypothesis low molecular weight hyaluronan (LMW-HA) inhibits lung epithelial ion transport in-vivo, ex-vivo, and in-vitro by activating the calcium-sensing receptor (CaSR). Intranasal instillation of LMW-HA (150g/ml) to C57BL/6 mice inhibited their alveolar fluid clearance (AFC) by 75%, increased the epithelial lining fluid (ELF) thickness threefold, and lung wet/dry (W/D) ratio by 20% 24hrs later. Incubation of lung slices from mouse and human lungs with 150g/ml LMW-HA decreased the open probability (Po) of ENaC in ATII cell by more than 50% in 4hrs, inhibited amiloride sensitive short circuit current (SCC) 4hrs post exposure, and Cl- current through CFTR by more than 70%, and Na,K-ATPase current by 66% at 24hrs. In all cases the inhibitory effect of LMW-HA on lung epithelial ion transport in vivo, ex vivo, and in vitro preparations were reversed by the administration of 1M of NPS2143, a CaSR inhibitor, or 150g/ml HMW-HA. In HEK-293 cells co-transfected with CaSR and the calcium sensitive Cl- channel TMEM16-A, LMW-HA activated an inward Cl- current. These data are the first demonstration of the inhibitory effects of LMW-HA on lung epithelial ion and water transport, and are due to the activation of CaSR and its downstream signaling cascades.

physiology↗

bioRxiv2022

recountmethylation enables flexible analysis ofpublic blood DNA methylation array data

Thousands of DNA methylation (DNAm) array samples from human blood are publicly available on the Gene Expression Omnibus (GEO), but they remain underutilized for experiment planning, replication, and cross-study and cross-platform analyses. To facilitate these tasks, we augmented our recountmethylation R/Bioconductor package with 12,537 uniformly processed EPIC and HM450K blood samples on GEO as well as several new features. We subsequently used our updated package in several illustrative analyses, finding (1) study ID bias adjustment increased variation explained by biological and demographic variables, (2) most variation in autosomal DNAm was explained by genetic ancestry and CD4+ T-cell fractions, and (3) the dependence of power to detect differential methylation on sample size was similar for each of peripheral blood mononuclear cells (PBMC), whole blood, and umbilical cord blood. Finally, we used PBMC and whole blood to perform independent validations, and we recovered 40-46% of differentially methylated probes (DMPs) between sexes from two previously published epigenome-wide association studies (EWAS).

bioinformatics↗

bioRxiv2022

Retained introns in long RNA-seq reads are not reliably detected in sample-matched short reads

There is growing interest in retained introns in a variety of disease contexts including cancer and aging. Many software tools have been developed to detect retained introns from short RNA-seq reads, but reliable detection is complicated by overlapping genes and transcripts as well as the presence of unprocessed or partially processed RNAs. We compared introns detected by 5 tools using short RNA-seq reads with introns observed in long RNA-seq reads from the same biological specimens and found: (1) significant disagreement among tools (Fleiss{kappa} = 0.231) such that 52.4% of all detected intron retentions were not called by more than one tool; (2) that no tool achieved greater than 20% precision or 35% recall under generous conditions; and (3) that retained intron detectability was adversely affected by greater intron length and overlap with annotated exons.

genomics↗

bioRxiv2021

recount3: summaries and queries for large-scale RNA-seq expression and splicing

We present recount3, a resource consisting of over 750,000 publicly available human and mouse RNA sequencing (RNA-seq) samples uniformly processed by our new Monorail analysis pipeline. To facilitate access to the data, we provide the recount3 and snapcount R/Bioconductor packages as well as complementary web resources. Using these tools, data can be downloaded as study-level summaries or queried for specific exon-exon junctions, genes, samples, or other features. Monorail can be used to process local and/or private data, allowing results to be directly compared to any study in recount3. Taken together, our tools help biologists maximize the utility of publicly available RNA-seq data, especially to improve their understanding of newly collected data. recount3 is available from http://rna.recount.bio.

genomics↗

bioRxiv2021

Pepsickle rapidly and accurately predicts proteasomal cleavage sites for improved neoantigen identification

Proteasomal cleavage is a key component in protein turnover, as well as antigen presentation and subsequent immune response. Herein we present pepsickle, an open-source tool for proteasomal cleavage prediction with better in vivo prediction performance (AUC) and computational speed than current models available in the field, and with the ability to predict sites based on both constitutive and immunoproteasome profiles. Post-hoc filtering of predicted patient neoepitopes using pepsickle significantly enriches for immune-responsive epitopes and may represent a significant opportunity to improve current epitope prediction and vaccine development pipelines.

bioinformatics↗

bioRxiv2020

Human methylome variation across Infinium 450K data on the Gene Expression Omnibus

While DNA methylation (DNAm) is the most-studied epigenetic mark, few recent studies probe the breadth of publicly available DNAm array samples. We collectively analyzed 35,360 Illumina Infinium HumanMethylation450K DNAm array samples published on the Gene Expression Omnibus (GEO). We learned a controlled vocabulary of sample labels by applying regular expressions to metadata and used existing models to predict various sample properties including epigenetic age. We found approximately two-thirds of samples were from blood, one-quarter were from brain, and one-third were from cancer patients. 19% of samples failed at least one of Illuminas 17 prescribed quality assessments; signal distributions across samples suggest modifying manufacturer-recommended thresholds for failure would make these assessments more informative. We further analyzed DNAm variances in seven tissues (adipose, nasal, blood, brain, buccal, sperm, and liver) and characterized specific probes distinguishing them. Finally, we compiled DNAm array data and metadata, including our learned and predicted sample labels, into database files accessible via the recountmethylation R/Bioconductor companion package. Its vignettes walk the user through some analyses contained in this paper.

bioinformatics↗

bioRxiv2019

Putatively cancer-specific alternative splicing is shared across patients and present in developmental and other non-cancer cells

We compared cancer and non-cancer RNA sequencing (RNA-seq) data from The Cancer Genome Atlas (TCGA), the Genotype-Tissue Expression (GTEx) Project, and the Sequence Read Archive (SRA). We found that: 1) averaging across cancer types, 80.6% of exon-exon junctions thought to be cancer-specific based on comparison with tissue-matched samples are in fact present in other adult non-cancer tissues throughout the body; 2) 30.8% of junctions not present in any GTEx or TCGA normal tissues are shared by multiple samples within at least one cancer type cohort, and 87.4% of these distinguish between different cancer types; and 3) many of these junctions not found in GTEx or TCGA normal tissues (15.4% on average) are also found in embryological and other developmentally associated cells. This study probes the distribution of putatively cancer-specific junctions across a broad set of publicly available non-cancer human RNA-seq datasets. Overall, we identify a subset of shared cancer-specific junctions that could represent novel sources of cancer neoantigens. We further describe a framework for characterizing possible origins of these junctions, including potential developmental and embryological sources, as well as cell type-specific markers particularly related to cell types of cancer origin. These findings refine the meaning of RNA splicing event novelty, particularly with respect to the human neoepitope repertoire. Ultimately, cancer-specific exon-exon junctions may affect the anti-cancer immune response and may have a substantial causal relationship with the biology of disease.

cancer biology↗

bioRxiv2019

Alternative Splicing of MR1 regulates antigen presentation to MAIT cells

Mucosal Associated Invariant T (MAIT) cells can sense intracellular infection by a broad array of pathogens. These cells are activated upon encountering microbial antigen(s) displayed by MR1 on the surface of an infected cell. Human MR1 undergoes alternative splicing. The full length isoform, MR1A, can activate MAIT cells, while the function of the isoforms, MR1B and MR1C, are not well characterized.\n\nIn this report, we sought to characterize these splice variants. Using a transcriptomic analysis in conjunction with qPCR, we find that that MR1A and MR1B transcripts are widely expressed. Despite the widespread expression of MR1A and MR1B, only MR1A can present mycobacterial antigen to MAIT cells. Coexpression of MR1B with MR1A serves to decrease MAIT cell activation following bacterial infection. However, expression of MR1B prior to MR1A lowers total MR1A abundance, suggesting competition between MR1A and MR1B for either ligands or chaperones required for folding and/or trafficking. Finally, we evaluated CD4/CD8 double positive thymocytes expressing surface MR1. Relative MR1A/MR1B expression in MR1-expressing thymocytes is associated with their prevalence.\n\nOur results suggest alternative splicing of MR1 represents a means of regulating MAIT activation in response to microbial ligand.\n\nFundingThis work was supported by NIH T32HL083808 (EK, GAN, EM), VA Merit Award I01CX001562 (MJH), NIH R01AI29976 (MJH), NIH R01AI048090 (DML), NIH R21AI124225-01A1 (FT) and VA Merit Award I01BX000533 (DML). The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

immunology↗

bioRxiv2019

Burden of tumor mutations, neoepitopes, and other variants are cautionary predictors of cancer immunotherapy response and overall survival

BackgroundTumor mutational burden (TMB, the quantity of aberrant nucleotide sequences a given tumor may harbor) has been associated with response to immune checkpoint inhibitor therapy and is gaining broad acceptance as a result. However, TMB harbors intrinsic variability across cancer types, and its assessment and interpretation are poorly standardized. MethodsUsing a standardized approach, we quantify the robustness of TMB as a metric and its potential as a predictor of immunotherapy response and survival among a diverse cohort of cancer patients. We also explore the additive predictive potential of RNA-derived variants and neoepitope burden, incorporating several novel metrics of immunogenic potential. ResultsWe find that TMB is a partial predictor of immunotherapy response in melanoma and non-small cell lung cancer, but not renal cell carcinoma. We find that TMB is predictive of overall survival in melanoma patients receiving immunotherapy, but not in an immunotherapy-naive population. We also find that it is an unstable metric with potentially problematic repercussions for clinical cohort classification. We finally note minimal additional predictive benefit to assessing neoepitope burden or its bulk derivatives, including RNA-derived sources of neoepitopes. ConclusionsWe find sufficient cause to suggest that the predictive clinical value of TMB should not be overstated or oversimplified. While it is readily quantified, TMB is at best a limited surrogate biomarker of immunotherapy response. The data do not support isolated use of TMB in renal cell carcinoma.

cancer biology↗

bioRxiv2019

Fcrl5 and T-bet define influenza-specific memory B cells that predict long-lived antibody responses

Seasonal influenza vaccination elicits hemagglutinin (HA)-specific CD27+ memory B cells (Bmem) that differ in expression of T-bet, BACH2 and TCF7. T-bethiBACH2loTCF7lo Bmem are transcriptionally similar to effector-like memory cells while T-betloBACH2+TCF7+ Bmem exhibit stem-like central memory properties. T-bethi Bmem do not express plasma cell-specific transcription factors but do exhibit transcriptional, epigenetic, metabolic and functional changes that poise the cells for antibody production. Consistent with these changes, D7 HA+ T-bethi Bmem express intracellular immunoglobulin and T-bethi Bmem differentiate more rapidly into ASCs in vitro. The T-bethi Bmem response positively correlates with long-lived humoral immunity and clonotypes from T-bethi Bmem are represented in the early secondary ASC response to repeat vaccination, suggesting that this effector-like population can be used to predict vaccine durability and recall potential.

immunology↗

bioRxiv2019

IFNγ induces epigenetic programming of human T-bethi B cells and promotes TLR7/8 and IL-21 induced differentiation

Although B cells expressing the IFN{gamma}R or the IFN{gamma}-inducible transcription factor T-bet drive autoimmunity in Systemic Lupus Erythematosus (SLE)-prone mouse models, the role for IFN{gamma} signaling in human antibody responses is unknown. We show that elevated levels of IFN{gamma} in SLE patients correlate with expansion of the T-bet expressing IgDnegCD27negCD11c+CXCR5neg (DN2) pre-antibody secreting cell (pre-ASC) subset. We demonstrate that naive B cells form T-bethi pre-ASCs following stimulation with either Th1 cells or with IFN{gamma}, IL-2, anti-Ig and TLR7/8 ligand and that IL-21 dependent ASC formation is significantly enhanced by IFN{gamma} or IFN{gamma}-producing T cells. IFN{gamma} promotes ASC development by synergizing with IL-2 and TLR7/8 ligands to induce genome-wide epigenetic reprogramming of B cells, which results in increased chromatin accessibility surrounding IRF4 and BLIMP1 binding motifs and epigenetic remodeling of IL21R and PRDM1 loci. Finally, we show that IFN{gamma} signals poise B cells to differentiate by increasing their responsiveness to IL-21.

immunology↗

bioRxiv2018

ASCOT identifies key regulators of neuronal subtype-specific splicing

Public archives of next-generation sequencing data are growing exponentially, but the difficulty of marshaling this data has led to its underutilization by scientists. Here we present ASCOT, a resource that allows researchers to summarize, visualize, and query alternative splicing patterns in public RNA-Seq data. ASCOT enables rapid identification of splice-variants across tens of thousands of bulk and single-cell RNA-Seq datasets in human and mouse. To demonstrate the utility of ASCOT, we first focused on the nervous system and identified many alternative exons used only by a single neuronal subtype. We then leveraged datasets from the ENCODE and GTEx consortiums to study the unique splicing patterns of rod photoreceptors and found that PTBP1 knockdown combined with overexpression of MSI1 and PCBP2 activates rod-specific exons in HepG2 liver cancer cells. Furthermore, we observed that MSI1 targets intronic UAG motifs proximal to the 5 splice site and interacts synergistically with PTBP1 downregulation. Finally, we show that knockdown of MSI1 in the retina abolishes rod-specific splicing. This work demonstrates how large-scale analysis of public RNA-Seq datasets can yield key insights into cell type-specific control of RNA splicing and underscores the importance of considering both annotated and unannotated splicing events. ASCOT splicing and gene expression data tables, software, and interactive browser are available at http://ascot.cs.jhu.edu.

neuroscience↗

bioRxiv2018

neoepiscope Improves Neoepitope Prediction with Multi-variant Phasing

The vast majority of tools for neoepitope prediction from DNA sequencing of complementary tumor and normal patient samples do not consider germline context or the potential for co-occurrence of two or more somatic variants on the same mRNA transcript. Without consideration of these phenomena, existing approaches are likely to produce both false positive and false negative results, resulting in an inaccurate and incomplete picture of the cancer neoepitope landscape. We developed neoepiscope chiefly to address this issue for single nucleotide variants (SNVs) and insertions/deletions (indels), and herein illustrate how germline and somatic variant phasing affects neoepitope prediction across multiple datasets. We estimate that up to [~]5% of neoepitopes arising from SNVs and indels may require variant phasing for their accurate assessment. neoepiscope is performant, flexible, and supports several major histocompatibility complex binding affinity prediction tools. We have released neoepiscope as open-source software (MIT license, https://github.com/pdxgx/neoepiscope) for broad use.\n\nKEY POINTSO_LIGermline context and somatic variant phasing are important for neoepitope prediction\nC_LIO_LIMany popular neoepitope prediction tools have issues of performance and reproducibility\nC_LIO_LIWe describe and provide performant software for accurate neoepitope prediction from DNA-seq data\nC_LI

bioinformatics↗

bioRxiv2018

RNA-seq transcript quantification from reduced-representation data in recount2

More than 70,000 short-read RNA-sequencing samples are publicly available through the recount2 project, a curated database of summary coverage data. However, no current methods can be directly applied to the reduced-representation information stored in this database to estimate transcript-level abundances. Here we present a linear model taking as input summary coverage of junctions and subdivided exons to output estimated abundances and associated uncertainty. We evaluate the performance of our model on simulated and real data, and provide a procedure to construct confidence intervals for estimates.

genomics↗