bluuebunny/crossref_metadata_2025_split huggingface dataset:
Using split-pool combinatorial barcoding to simultaneously characterize the transcriptome and TCR diversity of 1 million T cells
Authors: Papalexi Efthymia , Kim Grace , Hariadi Bryan , Schroeder Sarah , Matulich Peter , Tran Vuong , Diaz Daniel , Roco Charles , Rosenberg Alexander 
Published: August 2023

Abstract
Recent advances in single-cell sequencing allow for simultaneous profiling of T cell receptors (TCRs) and transcriptomes leading to the characterization of key T cell subsets with pathogen recognition and clearance capabilities. Despite their success, these methods rely on microfluidic devices or plate-based protocols with limited sensitivity and throughput (1,000s–10,000s of cells) making the study of disease-relevant T cells time consuming and costly.
To overcome these limitations, we have extended our split pool combinatorial barcoding technology to simultaneously characterize the TCRs alongside the full transcriptomes of up to 1 million T cells. As a proof of concept experiment, we applied our method to 920,000 primary T cells from 8 healthy human donors to characterize their TCR diversity. Applying our method, we recovered TCR sequences from at least one chain in 88% of T cells (807,000 cells). Furthermore, we detected hundreds of thousands of unique alpha and beta chains across all donors and found that most of these chains represent rare clonotypes detected in 1 or 2 cells. Lastly, we performed transcriptome-based clustering analyses to identify all major T cell subsets and discovered donor and subset-specific hyper-expanded clonotypes that could help us reconstruct each donor’s recent history of infection.
In summary, we report on the extension of our highly flexible and scalable combinatorial barcoding technology to allow researchers to profile up to 1 million T cells in a single experiment and investigate their functional responses during infection, cancer, autoimmunity, or therapeutic interventions.
Conserved epigenetic hallmarks of T-cell age
Authors: Zebley Caitlin C , Mi Tian , Soerens Andrew , Alli Shanta , Vasandan Anoop , Hines Christopher , Mueller April , Baylin Stephen , Jones Peter , Goldstein Harris , Mullighan Charles , Masopust David , Youngblood Ben A 
Published: August 2023

Abstract
As the immune system ages, the protective capacity from established immunological memory wanes over time and is often referred to as immune senescence. Yet the molecular hallmarks of lymphocyte age and replicative senescence remain largely undefined. Here we report our use of a multi-lifetime murine model of repeat vaccination to define a core epigenetic signature coupled to memory T cells that evade replicative senescence. Through adoptive transfer and boosting over a period of approximately four mouse lifetimes, memory T cells became enriched for repressive DNA methylation programs at cell cycle regulators including the canonical senescence gene P19Arf (CDKN2A/2B locus – human P14ARF). The senescence-resistance program was progressively acquired throughout phycological and non-physiological aging of murine T cells, leading to the development of a lymphocyte epigenetic clock that can be used to accurately measure a T cell’s age. Application of this clock in physiologically aged human polyclonal and CMV-specific T cells correlated with age-estimates using established epigenetic chronologic clocks. Moreover, T-cell clock-associated senescence-resistance programs were highly enriched in human malignancies of lymphoid origin, specifically delineating known T-cell acute lymphoblastic leukemia (T-ALL) subtypes and revealing non-physiological aging of HOXA9 and MLL pediatric leukemias (estimated to be ~100 years old). Discovery of the senescence-resistance epigenetic clock provides a novel molecular hallmark for describing physiological and non-physiological lymphocyte age.
Tissue-specific heterogeneity of Human T follicular helper cells marked by unique transcriptomes and regulatory architecture
Authors: Powell Michael Duane , Rose James R , Guopta Mansi , Rahmberg Andrew , Scharer Christopher D , Boss Jeremy M 
Published: August 2023

Abstract
T follicular helper (TFH) cells represent a distinct lineage of CD4 T cells that engage with cognate B cells, facilitating the production of high affinity vaccine- and pathogen-specific antibodies. In humans, TFH cells can be detected in several tissues throughout the body, each representing a unique cellular environment complete with distinct signaling molecules. Despite TFH cell importance to human health, it remains unclear if and how anatomical location results in diversity among TFH cells. Here, we detail tissue-specific heterogeneity of human TFH cells reflected in both transcriptome and chromatin architecture. TFH cells sourced from circulating peripheral blood (cTFH), lung lymph nodes (lnTFH), tonsils (tTFH), and an in vitro differentiation model (exTFH), were subjected to RNA-sequencing and ATAC-sequencing. When comparing expression of conical TFH genes, including BCL6 and PDCD1, a spectrum in magnitude emerged: cTFH &lt; exTFH &lt; lnTFH &lt; tTFH. Increased chromatin accessibility at proximal cis-regulatory elements occurred within the loci of TFH genes in a similar tissue-dependent fashion and correlated with transcript levels. Additionally, unique gene signatures were detected in each TFH population, including the presence of distinct transcription factor networks, implying tissue specific TFH functionality. Integrating PageRank and scRNA-seq of tTFH cells revealed BCL-6, ASCL2, TOX, TOX2, and AIOLOS as potential regulators of the TFH gene program. Collectively, these findings detail tissue specific diversity amongst human TFH cells, while highlighting key developmental networks which could influence the design of future immunological therapies targeting TFH cells.
Supported by grants from NIH: (RO1 AI113021, F32 AI161857)
Interferon-beta mediated STAT4 up-regulation are distinct in the ex vivo human CD4+ T-cells priming dendritic cells
Authors: Tangtanatakul Pattarin , Makarathut Pannaphan , Phonboon Terdkiat , Amornsupak Kamolporn , Makchareon Jiradej , Ritparchak Patcharee , Hirankarn Nattiya , Sodsai Pimpayao 
Published: August 2023

Abstract
STAT4 (Signal Transducer Activator of Transcription factor 4) polymorphisms are risk factors contributed to autoimmune disease. While STAT4 has a dominant role in promoting interferon-gamma (IFN-g) production in T and NK cells, its existence and function exert in human dendritic cells (DCs) are still unknown. To characterize the STAT4 expression in human peripheral DCs, we isolated DCs from buffy coat of health blood donors (n = 4). The ex vivo DCs were enriched and sorted into 3 subtypes including plasmacytoid DCs (pDCs, CD123+), CD8+ T-cells priming DC (cDC1, CLEC9A+), and CD4+ T-cells priming DC (cDC2/3, CD1c+) through negative selection bead and flow cytometry. The presence of STAT4 as well as its potential target genes (interleukin-12, IL-12 and interleukin-23, IL-23) expression were examined in IFN-beta induced DCs maturation compared to immature DCs, using Real-Time PCR. We found the proportion of DCs subsets in peripheral mononuclear cells which are 0.05%, 0.02% and 0.3% for pDC, cDC1 and cDC2/3, respectively. The IFN-beta enhanced DC maturation markers (HLA-DR+ CD86+), especially in cDC2/3. Interestingly, up-regulated STAT4 expression (2.5-fold) were significantly prominent in mature cDC2/3 compared to immature (p &lt; 0.01), while IFN-beta showed no influence on STAT4 expression in the cDC1 and pDC (p &gt; 0.05). Interestingly, elevated IL-12 and IL-23 expression were significantly observed in activated cDC2/3 (p &lt; 0.05). Further study to investigate the role of STAT4 in human cDC2/3 are needed. Our result firstly discover the existing of STAT4 expression in the ex vivo peripheral human DC.
Supported by Chulalongkorn University (Fundamental Fund, CU_FRB65_hea (84)_179_37_09).
The role of interleukin-12-PRMT5 axis in generating suppressive Th1-like induced regulatory T cells
Authors: Jadon Nidhi 
Published: August 2023

Abstract
After activation, naïve CD4 T cells can differentiate into T helper cell subsets (Th1, Th2, Th17, and Follicular helper T cells) or into regulatory T cells (Tregs), depending on the type of antigen-presenting cells (APC) and cytokine milieu encountered during differentiation. Recent studies suggest that CD4+Foxp3+ Tregs can produce the pro-inflammatory cytokine IFNg when stimulated in a Th1 cytokine environment, such as in the presence of IL-12, and stimulation under these conditions makes iTregs sensors for inflammatory cytokines.
The stability and suppressive functions of the iTregs depend on multiple factors. The goal of this study is to explore an epigenetic enzyme protein arginine methyltransferase 5 (PRMT5) and to understand the significance of this protein in the development of Th1-like iTregs. PRMT5 can either represses or activate transcription of target genes via symmetric di-methylation of arginine residues on histones. However, the role of PRMT5 in the differentiation of Th1-like iTregs has not been thoroughly investigated.
We demonstrate that differentiating iTregs in a Th1 cytokine environment generated a specific population of Th1-like iTregs with increased suppressive function. In Th1-like iTregs, we observed symmetric arginine di-methylation on histone (H3R2me2s) mediated by PRMT5, while this epigenetic marker is absent in iTregs. We also demonstrate that si-RNA mediated knockdown of PRMT5 reduced the suppressive capacity of Th1-like iTregs. Therefore, we hypothesize that the IL12-PRMT5 axis is crucial for developing suppressive Th1-like iTregs. Goals of further research are to generate stable immunosuppressive iTregs for therapeutic use.
Rab4A controls the overexpression of CD38 and depletion of IL-2 in CD4+ T cells: Potential involvement in proinflammatory lineage development in systemic lupus erythematosus
Authors: Park Joy Seonghee , Patel Akshay , Krakko Daniel , Nolan Jessica , Perl Andras 
Published: August 2023

Abstract
Rab4A is a GTPase that is overexpressed in systemic lupus erythematosus (SLE) patients’ T cells and enhances cell surface receptor recycling. Increased expression of CD38 and loss of IL-2 production have independently been associated with proinflammatory T cell development in SLE. We investigated the impact of Rab4A on the expression of CD38 and the production of IL-2.
Our lab created Rab4A-mutant Jurkat cell lines, containing GFP-expressing vector alone (control), doxycycline-inducible vectors that overexpress Rab4A (Rab4Ahi) or the dominant-negative mutant Rab4AS27N (Rab4ADN). They were co-stimulated with anti-CD3 antibody and PMA. After 24 hours, cell surface markers and cytokines were analyzed by flow cytometry and protein levels by western blot. NAD+ levels were measured by LC-MS/MS.
In the Rab4Ahi cells compared to the control, CD38 expression was 2-fold upregulated (p=2.49×10−13), intracellular production and secretion of IL-2 were significantly decreased (fold change=−0.566, p=1.16×10−7 and fold change= −0.481, p=0.0401, respectively), NAD+ concentration was decreased (fold change=0.647, p=0.0039), while pSTAT3 levels were 2-fold increased (p=0.0318).
CD38 is an NAD+ hydrolase, which regulates Sirtuin-1 activity, a NAD+-dependent histone deacetylase that suppresses STAT3 activity. Elevated pSTAT3 levels may underlie diminished IL-2 production by binding to the promoter of FoxO1, which inhibits IL-2 production. The overexpression of Rab4A and CD38, increased pSTAT3, and diminished IL-2 production reflect changes observed in SLE patients. Our results suggest that increased expression of Rab4A may underlie the overexpression of CD38 and diminished secretion of IL-2 in SLE.
Human OTULIN mutations can cause distinct inflammatory disease phenotypes depending on the protein domain that is mutated
Authors: Gil Deb , Liu Qi , Dang Elizabeth , Bhattad Sagar , Markle Janet 
Published: August 2023

Abstract
Linear ubiquitination is a post-translational modification that controls many immune signaling pathways. Linear ubiquitin chains are added to targets by LUBAC and removed by OTULIN, thus the opposing actions of LUBAC and OTULIN are critical for immune homeostasis.
To understand mechanisms of how ubiquitination controls inflammation, we leveraged the use of biospecimens from patients with a novel bi-allelic OTULIN mutation. These patients have pyoderma gangrenosum (PG), an extremely rare inflammatory skin disease. OTULIN mutations have been previously described to cause a systemic autoinflammatory disease called ORAS, yet these patients define a new monogenic disease. We sought to understand this discrepancy through functional analyses of OTULIN mutations that culminate in PG vs ORAS.
ORAS-causing OTULIN mutations impair the catalytic domain. However, the mutation in PG patients affects the domain responsible for binding LUBAC. Further, OTULIN’s ability to downregulate linear ubiquitin or NF-kB activity was unaffected. Patients’ transcriptional signatures showed elevated expression of genes involved in neutrophil activation and antigen presentation, and reduced expression of genes involved in Th17 differentiation, TLR signaling, and NOD-like receptor signaling. Deep immunophenotyping by CyTOF revealed that PG patients have higher frequencies of T cells and subtle defects of B cells and myeloid cells. These patients’ unique phenotype suggests an unrecognized function for OTULIN in skin inflammation. This discovery adds to the emerging spectrum of human immune-mediated diseases caused by defects in the ubiquitin pathway.
Impacts of the A20 protein polymorphism, rs2230926, in human B cells
Authors: Sutton Rachel Elizabeth , Gupta Mansi , Scharer Christopher D 
Published: August 2023

Abstract
Patients with autoimmune diseases, such as systemic lupus erythematosus (SLE), often have skewed B cell population ratios, accumulation of autoantibodies, and an increased amount of proinflammatory cytokines aiding in disease development. Genome-wide association studies (GWAS) have strongly linked polymorphisms in A20, encoded by TNFAIP3, to autoimmune diseases including SLE. A20 functions to inhibit the activation of nuclear factor kappa B (NF-κB) through ubiquitination and deubiquitylation of key signaling proteins and has been extensively studied in epithelial cells in response to TNF. Given the role of autoantibodies in SLE and the importance of NF-κB in regulating B cell fate, the role of SLE linked polymorphisms in A20 and their effects on both the canonical and noncanonical NF-κB pathways in human B cells is poorly understood. Our lab has identified an A20 polymorphism, rs2230926 (F127C), that is enriched in a cohort of African American females with SLE. Herein, we investigate the impact of rs2230926 on B cell function. We employed CRISPR/Cas9 genome-editing and generated a TNFAIP3 knockout lymphoblastoid Raji human B cell line. To determine how the polymorphism rs2230926 affects the NF-kB pathway, we re-introduced WT A20, A20-rs2230926, and a truncated version of A20 into the TNFAIP3-KO cell line. Using a combination of qRT-PCR, western blotting, and flow cytometry, we determined how rs2230926 influences the expression of proinflammatory cytokines, response to various B cell stimuli, and ultimately NF-κB activity. Dissecting A20’s role in NF-kB signaling will help to understand the dysfunction and regulation of B cells in autoimmunity.
Supported by grants from NIH to CDS (R01 AI148471)
Commensal Bacteria Maintain a Qa-1b-restricted Unconventional CD8+ T Population in Gut Epithelium
Authors: Guan Jian , Valerio Michael Manoharan , Peske J David , Park Chansu , Robey Ellen A , Sadegh-Nasseri Scheherazade , Shastri Nilabh 
Published: August 2023

Abstract
Intestinal intraepithelial lymphocytes (IELs) are characterized by an unusual phenotype and developmental pathway, yet their specific ligands and functions remain largely unknown. Here by analysis of QFL T cells, a population of CD8+ T cells critical for monitoring the MHC I antigen processing pathway, we established that unconventional Qa-1b-restricted CD8+ T cells are abundant in intestinal epithelium. We found that QFL T cells showed a Qa-1b-dependent antigen-experienced phenotype in the spleen and small intestine of naïve wild-type mice. The splenic QFL T cells showed innate-like functionality exemplified by rapid response to cytokines or antigen, while the gut population was refractory to stimuli. Microbiota was required for the maintenance, but not the initial gut homing of QFL T cells. Interestingly, monocolonization with Pediococcus pentosaceus, which expresses a peptide that cross-activated QFL T cells, was sufficient to maintain QFL T cells in the intestine. Thus, microbiota is critical for shaping the Qa-1b-restricted IEL landscape.
RO1AI130210, R37AI060040
Protective colonic iNKT cells are expanded by Goblet cell-associated antigen passages in CD1d-dependent manner in adults
Authors: John Vini , Barrios Bibiana E , Udayan Sreeram , Floyd Alexandria N , Joyce Elisabeth S , Schill Ellen M , Knoop Kathryn A , Blumberg Richard S , McDonald Keely G , Newberry Rodney D 
Published: August 2023

Abstract
Invariant natural killer T (iNKT) cells are unique innate immune cell which secrete cytokines and immune mediators. These cells recognize self or microbial glycolipids presented in the context of CD1d. iNKT cells can contribute to host protection or pathogenesis during intestinal inflammation. Colonic iNKT cells are established under the influence of the microbiota in early life and the current understanding is that colonic iNKT cells are fixed and not manipulable in later life. Goblet cells (GCs) or/and goblet cell associated passages (GAPs) deliver luminal antigens and maintain peripheral T regulatory cells in the steady state. However, GAPs in the colon are absent in adult mice due to GCs microbial sensing. We found that the glycolipids can be delivered through GAPs and that inducing GAPs in the colon in adult mice resulted in significant iNKT cell expansion. Deletion of GCs or deletion of CD1d on GCs prevented iNKT cell expansion suggesting a role for colonic GCs in presenting glycolipids to iNKT cells. Single cell RNA sequencing of colonic-iNKT cells showed significantly increased iNKT2 and iNKT1 subsets after colonic GAP induction. Proteomic analysis of these iNKT cells further confirmed the characteristics of expanded iNKT subsets. Moreover, iNKT cells expanding after colonic GAP induction persisted and were protective in DSS-induced colitis. Adoptive transfer of the expanded colonic iNKT cells protected in the DSS-induced colitis model, indicating that expanded colonic iNKT cells were sufficient for protection in this model. These findings indicate that colonic iNKT cells are not fixed during early life but can be expanded in a GAP dependent and GC CD1d dependent manner and can be protective in some models of colitis.
Crohn’s and Colitis Foundation
Low IL-13Ra1 Expression on Mast Cells Tunes Them Unresponsive to IL-13
Authors: Salomaa Tanja , González-Rodríguez Martín Ignacio , Kummola Laura , Hiihtola Lotta , Junttila Ilkka 
Published: August 2023

Abstract
Cytokine-mediated mast cell regulation enables precise optimization of their own pro-inflammatory cytokine production. During allergic inflammation, Interleukin(IL)-4 regulates mast cell functions, tissue-homing and proliferation, but the direct role of closely related IL-13 for mast cell activation remains unclear. Previous work has shown that mast cells are potent IL-13 producers, but here we show that mouse mast cells do not directly respond to IL-13 by Stat6 activation, as they do not express measurable amount of IL-13Ra1 mRNA. Consequently, IL-4 responses are mediated via type I IL-4R (IL-4/IL4Ra/gC) and IL-4-induced Stat6 activation is abolished in gC deficient mast cells. Type II IL-4R deficiency (IL-13Ra1 KO) has no effect on IL-4-induced Stat6 activation. In basophils, both IL-4 and IL-13 induce Stat6 activation in WT and gC deficient cells, while in type II IL-4R deficient basophils IL-4 signaling is impaired at low ligand concentration. Thus, mast cell and basophil sensitivity to IL-4/IL-13 is different and in mast cells, lack of IL-13Ra1 expression likely explains their unresponsiveness to IL-13.
Supported by grants from Academy of Finland (grants: 25013080481 and 25013142041), The Competitive State Research Financing of the Expert Responsibility Area of Fimlab Laboratories (grant: X51409), Tampere University Hospital Support Foundation (grant: MK247), Tampere Tuberculosis Foundation, Nordlab Laboratories (grant: X3710-KT0011) and Orion Research Foundation.
P2X3 Potentiates IgE Mediated Mast Cell Function
Authors: Burchett Jason , Ryan John J 
Published: August 2023

Abstract
The rising incidence of allergic disease is a public health challenge needing novel interventions. Mast cell activation by IgE is an established feature of allergy, but fundamental aspects of IgE-induced signaling remain unclear. Prior data from our lab testing the effects of fluoxetine on IgE-driven mast cell function indicated a role for the purinergic receptor P2X3. The P2X3 receptor is an ATP-activated cationic channel most often associated with pain that is expressed on the surface of mast cells. IgE cross-linkage elicits a rapid release of ATP from mast cells, suggesting ATP-P2X3 receptor signaling may potentiate IgE-driven responses. We show that diminishing P2X3 function pharmacologically and genetically reduces IgE-mediated cytokine production. P2X3 knockout bone marrow-derived mast cells showed a diminished capability to produce cytokine in vitro when stimulated by IgE cross-linkage. The P2X3 inhibitors BLU-5937 and Gefapixant administered intraperitoneally suppressed cytokine levels measured in the plasma of mice subjected to the mast cell-dependent passive systemic anaphylaxis model (PSA). Furthermore, P2X3 knock-out mice showed reduced IL-6 compared to wild-type mice in the PSA model. Interestingly, inhibition or deletion of the P2X3 receptor showed no effect on mast cell degranulation in vitro and did not reduce hypothermia during PSA. We propose that an ATP-P2X3 autocrine or paracrine loop augments mast cell-mediated inflammation, thus offering a potential new target for the treatment of allergic disease.
RO1 AI164710-01, TL1 DK 132771-1