bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single-cell multiomics defines tolerogenic extrathymic Aire-expressing cells with unique homology to thymic epithelium
Authors: Wang Jiaxi , Lareau Caleb A , Bautista Jhoanne L , Sun Im Hong , Gupta Alexander R , Gardner James M 
Published: January 2023

Abstract
The autoimmune regulator (Aire) protein, a key transcriptional regulator expressed in medullary thymic epithelial cells (mTECs), is crucial for central tolerance. Aire is also found in extrathymic Aire-expressing cells (eTACs) in secondary lymphoid organs. eTACs are hematopoietic antigen-presenting cells that are capable of inducing immune tolerance, but the precise identity of eTACs has remained unclear. To define eTACs, we utilized a combination of single-cell multiomics, transgenic murine models, and functional approaches. We found that eTACs consist of two similar cell types: CCR7+ Aire-expressing migratory dendritic cells and an Aire-high population co-expressing Aire and retinoic acid receptor–related orphan receptor γt (RORγt) that we termed Janus cells (JCs). eTACs, particularly JCs, have highly accessible chromatin and share remarkable homology with mTECs. Additionally, transgenic self-antigen expression by eTACs is sufficient to induce negative selection of cognate autoreactive T cells and prevent autoimmune diabetes. To better define the function of extrathymic Aire in eTACs and its contribution to peripheral immune tolerance, we have generated a Peripheral Aire KnockOut (PAKO) mouse (Vav1-Cre x Aire-fl/fl). Using intracellular Aire staining by flow cytometry, we validated that PAKO mice lack all eTACs in secondary lymphoid organs while maintaining normal thymic Aire expression in mTECs. Studying the biology of eTACs and peripheral Aire will help further elucidate the function of Aire and define basic peripheral tolerance mechanisms, which may have significance for a range of clinical applications from autoimmunity to tumor immunity to maternal-fetal tolerance.
Supported by ARCS Foundation
Sterile neutrophilia induced via CXCR4 antagonism ameliorates colonic inflammation by increasing immunosuppressive regulatory T cells
Authors: Saha Piu , Yeoh Beng San , Golonka Rachel , Abokor Ahmed , Vijay-Kumar Matam 
Published: January 2023

Abstract
Sterile neutrophilia (SN) is commonly associated with chronic disease, but its role in inflammatory bowel diseases (IBD) is largely unknown. AMD3100 (plerixafor; AMD) is a CXCR4 antagonist that promotes neutrophil egress from bone marrow resulting in SN. To study the role of SN in IBD, we induced chronic intestinal inflammation in C57BL6 mice via 4 weekly injections of IL-10R neutralizing antibody and administered AMD (10 mg/kg bw, i.p., twice weekly) or vehicle. As expected, AMD-treated control and colitic mice had increased peripheral and splenic neutrophils. AMD treatment remarkably reduced epithelial damage and hyperplasia, immune cell infiltration, and disease activity index. To elucidate the protective mechanism, we analyzed immune cells in peripheral circulation, spleen and lamina propria. In circulation, colitic mice with AMD treatment had elevated % CD3+ CD4+ helper T cells (Th) but reduced % CD3+ CD8+ cytotoxic T (Tc) cells. Yet, the % CD3+ CD4+ FoxP3+ cells (Tregs) and % CD3+ CD4+ RORγt+ cells (Th17) remained unchanged. In spleen, AMD treatment significantly increased Tregs, but decreased Th and Tc cells. In lamina propria, AMD treatment markedly elevated Tregs, including RORγt+ Tregs and tolerogenic IL-10+ Tregs. Conversely, the inflammatory IL-17+ Th cells were decreased in AMD-treated colitic mice. In addition to the immunosuppressive milieu, AMD-treated colitic mice had increased ileal expression of the antimicrobial peptide angiogenin 4, which further suggests SN to be important in maintaining gut microbiota homeostasis. Collectively, our results demonstrate that SN could be an adaptive and beneficial immune response during chronic intestinal inflammation.
This research was funded by R01 grant from the National Institutes of Health (NIH) [grant number CA219144] and Crohn’s and Colitis Foundation (CCF) [grant number 855256].
Triggering receptor expressed on myeloid cells (Trem)-1 plays important roles in UVB induced immune suppression and cutaneous carcinogenesis
Authors: Aguilar Carlos Alberto Mier , Sherwani Mohammad Asif , Tsuruta Yuko , Rashid Harunur , Crossman David Keith , Yusuf Nabiha , Xu Hui 
Published: January 2023

Abstract
American Cancer Society acknowledges that most skin cancers result from exposure to ultraviolet (UV) rays in sunlight. UVB radiation induces mutant cells as well as immune suppression. Tumors only occur when mutant cells are in an immune suppressive environment. The mechanisms for UVB induced immune suppression are yet to be fully elucidated. The goal of this work is to explore new mechanisms for UVB induced immune suppression and skin carcinogenesis by using advanced technology. Analysis of UVB exposed mouse (Nanostring) and human (RNAseq) skin shows that triggering receptor expressed on myeloid cells (TREM)-1 signal pathway is the top canonic pathway increased by UVB. TREM-1 has roles in inflammatory diseases and cancers. However, its role in UVB induced immune suppression and skin cancers is not understood. We have found by using multi-color flow cytometry that UVB induces TREM-1 expression in a subset of conventional dendritic cell type 2 (cDC2) in the draining lymph nodes (LN). TREM-1+ cDC2 cells are barely detected in normal LN and express a high level of the co-inhibitory molecule PD-L1. In functional assays, treatment of mice with a specific TREM-1 blocking peptide LP-17 significantly diminishes UVB-induced suppression of contact hypersensitivity responses that are mediated by T cells. Further, mice treated with this peptide show decreased incidence of photocarcinogenesis and reduced number of tumors. In summary, our findings demonstrate that UVB induces the development of a novel TREM-1+ cDC2 cell subset and that TREM-1 has an important role in UVB induced immune suppression and skin carcinogenesis. Importantly, our study implies new strategies for targeting TREM-1 in the prevention and treatment of UVB induced skin cancers.
Supported by grants from NIH (R01AR072213 and R01AI154842)
A role for a DEAD box RNA helicase in natural killer cells
Authors: Mukhopadhyay Eshana , Krishnamurthy Durga , Tuazon Jasmine , Ohayon David , Ali Ayad , Stevens Joseph , Waggoner Stephen N 
Published: January 2023

Abstract
The X chromosome linked RNA helicase DDX3X plays numerous roles in regulation of cellular RNA metabolism and immune responses. Loss of function mutations in this helicase are linked to neurological disease and malignancies, including NK/T-cell lymphoma. To investigate how DDX3X contributes to development of NK cells, we generated mice with a conditional deletion Ddx3x in Ncr1-expressing cells. Female mice with homozygous deletion of NK cells exhibit a complete loss of NK cells. By comparison, the frequency of NK cells was significantly reduced in hemizygous male mice. The reduction in NK cells is associated with increased apoptosis in this lineage early during differentiation in the bone marrow. Our ongoing work is focused on elucidation of the mechanisms by which DDX3X regulates NK cell survival, differentiation, and malignant transformation, as well as on the capacity of DDX3Y to compensate for loss of DDX3X in hemizygous male mice. Our data identify a novel role for DDX3 RNA helicases in the development and survival of NK cells in mice.
ITK signaling regulates IL-10 production by CD8+ T cells and lung immunopathology during influenza infection
Authors: McGee Michael C , Solouki Sabrina , Limper Candice B , Zhang Tianyi , Magazine Nicholas , Ye Kaixiong , Nidetz Natalie , August Avery , Huang Weishan 
Published: January 2023

Abstract
Influenza (flu) infections cause 250,000 deaths and 3–5 million cases of severe illness during the average flu season. Severe influenza infections are associated with a combination of strong pro-inflammatory and weak anti-inflammatory immune responses. Production of the immunomodulatory cytokine IL-10 by T cells restricts immunopathology during flu infections, however knowledge of the signaling pathways regulating IL-10 induction during flu is limited. Using IL-10GFP reporter mouse models, we found Interleukin-2 inducible T cell kinase (ITK), a critical component in T cell receptor (TCR) signaling, regulates the development of IL-10-producing CD8+ T cells during influenza A infection. Compared to wild type (WT) mice, Itk−/− mice displayed increased morbidity and mortality after influenza infection, accompanied by a significant reduction of IL-10-producing CD8+ T cells in the airways. The absence of ITK and chemical inhibition of the PI3K/AKT/mTOR signaling pathway impaired both BLIMP1 expression and IL-10 production in CD8+ T cells in vitro. Introduction of Blimp-1 or a constitutively active AKT mutant via retroviral transduction rescued IL-10 production and Blimp-1 expression in Itk−/− CD8+ T cells. Utilizing a transgenic allele sensitive Itk (ITKas) mouse model to specifically inhibit ITK kinase activity in coculture, we found ITK kinase activity is not required for the suppressive function of IL-10 producing CD8+ T cells. Together, our data suggests that ITK is a critical regulator of the development and suppressive function of IL-10 producing CD8+ T cells and flu-induced pulmonary immunopathology. Modulating ITK signaling may be a strategy for regulating immunopathology due to respiratory viral infections.
This work was supported in part by grants from the National Institutes of Health (R01 AI151139, R56 AI146226 and P20 GM130555). W. Huang also received research support from MegaRobo Technologies Corporation.
Androgen receptor regulation of CD8 T cell immune responses
Authors: Hawkins Reed M , Polesso Fanny , Moran Amy E 
Published: January 2023

Abstract
Sex differences in disease susceptibility, including infection, cancer, and autoimmunity, have been characterized, with males generally more susceptible to infections and malignancies, and females to autoimmune diseases. While many factors underlie such differences, sex differences in T cell differentiation and function likely lead to disparate adaptive immune responses and disease control. Indeed, sex hormones, including androgens, are strong regulators of gene expression, and have been implicated as drivers of inflammation and regulators of T cell differentiation, but the mechanisms of such control remain unknown. We reveal that androgen receptor (AR) activity in CD8 T cells suppresses activation and reduces proliferation and effector function. We show that anti-CD3- and anti-CD28-activated or peptide-pulsed CD8 T cells exhibit enhanced proliferation, effector cytokine production, activation marker expression, and altered histone modifications upon treatment with AR inhibitors or genetic AR deletion. Further, we demonstrate ligand-dependent AR binding to genetic loci of effector and differentiation genes in CD8 T cells via chromatin immunoprecipitation and quantitative PCR. Finally, in vivo murine models of lymphocytic choriomeningitis virus reveal an increase in AR mRNA expression in virus-specific CD8 T cells following infection, which persists in memory cells and during chronic infection. These data suggest that AR regulates epigenetic and transcriptional changes induced during T cell activation and differentiation, thereby dampening the T cell response. Therefore, disruption of AR activity in T cells may be a viable therapeutic approach for altering the T cell response in the context of human diseases.
Supported by grants from NIH (NRSA T32 5T32GM71338-15, NRSA T32 1T32GM142619-01), Knight Cancer Institute (Knight Pilot Award), and OHSU Foundation
Blimp1 regulates the growth and function of human regulatory T cells
Authors: Ding Ying , Vujanac Milos , Nivelo Luis , Villarino Alejandro , Malek Thomas R 
Published: January 2023

Abstract
Blimp1 is a multifunctional transcriptional regulator that has established roles in mouse T cells. However, its function in human Tregs are not well-understood. To directly evaluate the role of Blimp1 in human Tregs, Blimp1α, the active and dominant isoform of Blimp1, was knocked out in human Tregs in vitro by the Crispr-Cas9 system using dual-targeting sgRNAs. High knockout efficiency was verified at the DNA, RNA, and protein levels. Transcriptome studies identified 356 Blimp1-repressed genes and 399 Blimp1-activated genes in Tregs. The significantly enriched gene sets are associated with cell cycle, apoptosis, cytokine-related signaling, and cytokine production. Consistent with normally restraining Treg growth, functional studies showed that knockout of Blimp1 increased Treg numbers, proliferative responses, and Ki67 expression, but decreased early apoptotic cells. Correspondingly, IL2-induced tyrosine phosphorylation of STAT5 (pSTAT5) was enhanced in Blimp1KO Tregs. Blimp-1 is also positively associated with some aspects of Treg function as loss of Blimp1 downregulated the expression of cytokines, including IL10, IL4, IL5, IL9, IL13, IFNγ, CCL3 and CCL4, and immune checkpoints, including LAG3, PD1, and CTLA4. In the absence of Blimp-1, Foxp3 and Helios levels and IL-6-induced pSTAT3 increased, suggesting a role for Blimp1 in regulating Treg stability. Treg suppressive function, in vitro, however, was not affected by the loss of Blimp1. Collectively, our results suggest that Blimp1 is a pivotal regulator of human Treg growth, function, and stability and is consistent with a model where Blimp1 contributes to Treg homeostasis in vivo. (Supported by R01AI131648).
Supported by grant from NIH (R01AI131648).
Batf Stabilizes the Th17 Cell Developmental Program through Impairment of Stat5-dependent Recruitment of Ets1-Runx1 Complexes
Authors: Pham Duy , Silberger Daniel J , Nguyen Kim , Gao Min , Hatton Robin D , Weaver Casey Todd 
Published: January 2023

Abstract
The emergence of alternative CD4 T cell fates is controlled by transcriptional networks activated downstream of TCR and cytokine signaling inputs. Th17 cells develop from antigen-activated naive CD4 T cells in the presence of IL-6 and TGF-b. An important facet of the Th17 cell program is its intrinsic instability; early development of Th17 cells shares overlapping features with that of Treg cells and Th17 cells are prone to transdifferentiation into Th1-like cells. The basic leucine zipper transcription factor ATF-like (Batf) contributes to the transcriptional programming of multiple effector T cells but is indispensable for Th17 cell development. Although Batf is required for development of Th17 cells, its mechanisms of action to underpin the Th17 program are incompletely understood. Here, we find that in addition to activating core genes of the Th17 program, Batf also acts to restrict expression of genes of the Treg and Th1 programs through its actions to restrain IL-2-induced Stat5 activation, thereby limiting Stat5-dependent recruitment of Ets1-Runx1 factors to Th1 and Treg cell-specific gene loci. Accordingly, Batf acts both to pioneer regulatory elements in Th17-specific genes and limit assembly of a Stat5-Ets1-Runx1 enhanceosome that appears to be required for optimal expression of Th1 and Treg cell-specific genes. Our findings define a new role for Batf in T cell differentiation and unveil an important role for Stat5-Ets1-Runx1 interactions in transcriptional networks that define alternative T cell developmental fates.
Supported by NIH R01 DK115172 and T32 AI007051-37
Cell-intrinsic expression of the hemichannel Pannexin-1 promotes effector and memory CD8+ T cells via distinct metabolic pathways
Authors: da Silva Henrique Borges , Vardam-Kaur Trupti , Zhou Maggie , de Gois Macedo Bruna , Van Dijk Sarah , Jameson Stephen C 
Published: January 2023

Abstract
Tissue-derived extracellular ATP (eATP) is a “danger signal” sensed by immune cells via purinergic receptors (e.g. P2RX7). In addition, healthy cells can export eATP through transmembrane channels, such as the hemichannel Pannexin-1 (Panx1). CD8+ T cells express Panx1 constitutively throughout developmental and immune response stages. However, the biological role of Panx1 in CD8+ T cells is undefined. Here, we used genetic and pharmacological tools to address this question. T-cell specific Panx1 deletion did not affect CD8+ T cell thymic development. In contrast, CD8+ T cell responses to viral infection (LCMV) are impaired by Panx1 inhibition or deletion. This is true for effector expansion of CD8+ T cells and for the survival of memory CD8+ T cells. Using adoptive transfer of tamoxifen-conditional Panx1 knockout LCMV-specific (P14) cells into LCMV-infected mice, we showed that memory CD8+ T cell survival requires sustained Panx1 – except for small intestinal-resident cells. Mechanistically, Panx1 promotes memory CD8+ T cell survival via P2RX7, which also favors memory CD8+ T cells - as suggested by our data with Panx1/P2RX7-double knockout P14 cells. The role of Panx1 for effector CD8+ T cell expansion, however, is unlikely to be via P2RX7 since this receptor does not have a role in effector CD8+ T cells. Our transcriptional and metabolic data suggests that, while memory CD8+ T cells require Panx1 for optimal mitochondrial function and expression of pro-memory genes, effector CD8+ T cells lacking Panx1 had decreased activation of the glycolytic pathway. Collectively, these results suggest that, in response to acute virus, Panx1 induces CD8+ T cell effector expansion and memory survival through distinct metabolic pathways.
Supported by grants from NIH (R00 AI139381, NIAID).
Defining the extra-thymic role of HEB in the development of CD8+ T stem cell-like immunological memory
Authors: Leung Joanne , Campbell Kieran , Anderson Michele K 
Published: January 2023

Abstract
The transcription factor HEB is involved in multiple stages of intrathymic T cell development, complexing with stage-specific partners to regulate different genes in a context-specific manner. Remarkably, little is known about how HEB influences the differentiation of peripheral T cells, and even less so of how the transcriptional circuitry in early T cell development may be re-invoked to generate long-lasting immunity. While T cells are detected in the periphery of HEB-deficient mice (HEB cKO), the contribution of HEB to the function of T cells upon pathogen encounter is obscure. Thus, HEB cKO mice were challenged with LCMV to determine the impact of HEB in the activation and differentiation of CD8+ T cells into effector and memory lineages. Throughout the course of infection, HEB cKO mice exhibited critical differences in their antigen-specific CD8+ response and had enhanced levels of stem cell-like memory T cells (Tscm). This implicated a role for HEB downstream of T cell activation, which we further confirmed using well-established in vitro cultures supplemented with or without Tscm-inducing cytokines. Consistently, HEB transcripts were detected in publicly available scRNA datasets from P14 transgenic mice infected with LCMV. To greater define the transcriptional pattern of HEB, and of others that orchestrate thymic T cell development in the context of peripheral T cell immunity, we will conduct a scRNA-seq comparative bioinformatic analysis of developing thymocytes to splenic LCMV-specific CD8+ T cells. This will advance our understanding of how early transcriptional cascades, like those initiated by HEB, are propagated in downstream immunological reactions, which may lead to therapies that can revitalize T cell immunity.
Supported by a grant from NIH (1P01AI102853-06) and the The AAI Intersect Fellowship Program for Computational Scientists and Immunologists
Normal microbial experiences accelerate memory T cell compartment maturation in infants
Authors: Stenger Terran D , Burger Sarah , Pierson Mark , Huggins Matt , Hamilton Sara , Schuldt Nathan J 
Published: January 2023

Abstract
Memory T cells cause faster and more effective elimination of pathogens during reinfection by the rapid production of effector T cells. Worldwide, the leading cause of death among children and infants is infection, partly due to under-developed T cell memory populations commonly seen in infants. In the context of biomedical research, the specific pathogen-free (SPF) facilities research animals are housed in further the deficit of memory T cells, as it is known that adult mice housed in SPF facilities have few memory T cells, but that exposure to normal microbial experiences (NME) replenishes this population of cells. This phenomenon, though, has not been investigated in infants. Therefore, we aimed to investigate how NME influences infant memory T cell commitment to reveal novel insights into natural mammalian immune development. At various time points, the trajectory of infant immune maturation of age-matched SPF and NME (pups birthed and raised in an environment exposed to NME) mice were examined using flow cytometry. We observed significant increases in both the number and frequency of memory T cell subsets (CD8+ and CD4+ Tcm and Tem cells) at all time points in pups exposed to NME. We also observed increased serum concentrations of cytokines required for memory T cell differentiation and survival (IL-7 and IL-15) in NME pups. These data support the hypothesis that NME accelerates the maturation of the memory T cell compartment in infant mice. We predict this more mature memory T cell compartment may contribute to improved immunity to novel infections. Our ongoing experiments test this hypothesis using a Listeria monocytogenes challenge model.
Dendritic cells instruct differentiation of tissue resident memory T cells in the skin to promote durable tumor immunity
Authors: Mohamed Asmaa , Vella Jennifer , Turk Mary Jo , Huang Yina H 
Published: January 2023

Abstract
A subset of melanoma patients treated with immune checkpoint inhibitors develops vitiligo, a CD8 T cell-mediated autoimmune disease associated with improved patient survival. Using a melanoma-associated vitiligo (MAV) mouse model, CD8 tissue resident memory (TRM) T cells in vitiligo skin were found to be necessary and sufficient for durable tumor immunity. Using immunofluorescence microscopy, we found that skin TRM cells formed large aggregates with CD11c myeloid cells proximal to hair follicles. CD11c depletion resulted in TRM cell loss, revealing an unexpected requirement for continued T cell-dendritic cell (DC) interactions in TRM cell maintenance. We hypothesize that DCs provide instructive signals that are required for continued in situ maturation of CD8 TRM cells. To identify these signals, we disrupted Toll Like Receptor (TLR) signaling in DCs by ablating the MyD88 adapter protein and observed a reduction in skin TRM cell accumulation. This prompted us to explore a role for the microbiota in CD8 TRM formation. Treatment with broad spectrum antibiotics resulted in a 50% reduction in vitiligo incidence. B6 mice from vendors with microbiota differences, exhibited divergent MAV incidence, but upon cohousing or fecal transplantation, mice from both sources exhibited high MAV incidence. Collectively, these findings indicates that the gut-skin microbiota axis plays a critical role in generating tumor protective TRM cells. Future studies seek to identify microbial antigens responsible for promoting TRM differentiation.
Supported by T32-AI007363 P30-CA023108