bluuebunny/crossref_metadata_2025 huggingface dataset:
STAT1 expression controls CD4 T cell Th1 and Th17 effector functionality during influenza virus infection
Authors: Finn Caroline M , Prokop Emily , Dhume Kunal , McKinstry Kai
Published: August 2023

Most CD4 T cells primed by influenza A virus (IAV) express the transcription factor T-bet, the Th1 ‘master regulator’, and are marked by phenotypic and functional Th1 hallmarks. The extent to which STAT1 and STAT4 activation, both needed for maximal T-bet induction in vitro, are required to direct IAV-primed CD4 T cell responses is not clear. Here, we transferred STAT-deficient (STAT−/−) or wildtype CD4 T cells recognizing IAV to congenic wildtype mice, challenged them with IAV, and analyzed the donor cells in the infected lungs 7 days later. STAT1−/− cells were barely detected in host mice but were rescued when NK cells were depleted in host mice prior to infection. Functional analysis revealed compromised Th1 identity in STAT1−/− effectors mirroring that of T-bet−/− cells. However, while T-bet−/− cells develop Th17 functions, STAT1−/− cells do not. We show that this is due to alternative receipt of type I IFN signals by the STAT1−/− cells, as blocking type I IFN receptor in IAV-primed mice promotes robust Th17 identity in STAT1−/− but not in WT cells. In contrast, STAT4−/− donor cells closely resemble WT cells. To ask if IAV-primed CD4 T cells can respond to STAT4-activating signals we treated mice receiving STAT4−/− donor cells with IL-12 during IAV infection. IL-12 dramatically increased T-bet and Th1 cytokine production of wildtype, but not of STAT4−/− or T-bet−/− cells. Our findings thus indicate that STAT1 activation is sufficient to direct prototypical antiviral effector identity, but that this response is not fully Th1-polarized without therapeutic STAT4 activation. This work impacts vaccine strategies that aim to promote protective T cell immunity against IAV by shaping the priming environment with STAT-activating cytokine signals.
Kidney disease impairs B cells response against infection via inhibiting germinal center formation and antibody titers
Authors: Peroumal Doureradjou , Jawale Chetan V , Antos Dani , Li De-dong , Wang Shuxia , Alcorn John F , Biswas Partha Sarathi
Published: August 2023

Immunity to infections is crucial to healthy life. However, recent Covid-19 pandemic has shown that comorbidity such as kidney disease and associated uremia impairs immunity against infections. Uremic patients show high susceptible to infections and also exhibit poor antibody responses to vaccine. The mechanisms by which uremia negatively impacts antibody response is unknown. Using multiple mouse models of uremia, we assessed B cells response to model antigen NP-KLH in alum. We show that uremia inhibits both canonical germinal center (GC) and non-canonical extra-follicular B cells response following NP-KLH immunization. Immunized uremic mice demonstrated compromised affinity maturation, isotype switching, antigen-specific antibody secreting cells, antibody titer and T follicular helper cells response. Consequently, uremic mice exhibited diminished GC and antibody response following prime-boost immunization. B cells showed increased cell cycle arrest, apoptosis and impaired migration between light and dark zone in the uremic GCs. We identified uremic toxin hippuric acid as a major driver of loss of mitochondrial membrane potential leading to increased apoptosis in mouse and human B cells. Finally, we show that GC B cells, T follicular helper cells and antibody response were similarly diminished in uremic mice during influenza virus infection, a major cause of mortality in patients with kidney disease. These results shed light on how uremic toxin(s) suppress antimicrobial immunity and vaccine response in individuals with kidney disease. Knowledge gained from this study may pave the path for developing effective therapeutic and preventive strategies in uremic patients against infections including SARS-CoV-2.

R01AI142354, R01AI162616 and R21AI159058
CRISPR/Cas9 screen of B cell differentiation pathways
Authors: Mulpur Roy Neal , Patterson Dillon , Scharer Christopher D , Boss Jeremy M
Published: August 2023

To provide immunity to pathogens, naïve B cells undergo differentiation to several states such as plasma cells and memory B cells. The transcription factors and epigenetic modifiers that program a naïve B cell to adopt a cell fate have not been fully characterized. During differentiation, B cells undergo extensive changes in transcription factor networks and gene expression that are underpinned by changes in the epigenome. One such example is a transition from expression of glycolysis related genes to increased expression of genes related to oxidative phosphorylation metabolism pathways. We previously demonstrated that in vivo, naïve B cells take 8 cell divisions to reach the plasma cell terminal differentiation and that at each division, the B cells also go through a hierarchy of reprogramming before becoming plasma cells after exposure to T independent antigens. Our data also showed that at division 3–4 a second branch emerged that expressed genes associated with the memory B cell fate. In order to find genes that play a key role in committing a naïve B cell to the plasma cell or memory B cell fate, cells committed to the two branches after T independent antigen exposure were sorted and analyzed with RNA sequencing. Analysis of this data revealed 67 genes involved in transcriptional and epigenetic regulation that were significantly differentially expressed between the two branches. In order to determine the role these genes play in B cell lineage path commitment, retrovirus libraries containing gRNAs for each of the 67 genes were constructed and tested in a targeted CRISPR/Cas9 knock out screen. This study identifies mechanisms of early memory B cell fate commitment.

This work was supported by NIH R01 AI123733, P01 AI125180 and T32 GM008490 to JMB
Thymus antibody-secreting cells possess an interferon-responsive gene signature and are comprised of a CD154(CD40L) dependent plasmablast population
Authors: Pioli Peter , Pioli KimAnh
Published: August 2023

Antibody-secreting cells (ASCs) consist of short-lived plasmablasts and post-mitotic mature plasma cells. While the major function of these cells is the production of antibodies, ASCs have been shown to regulate hematopoiesis and as well as the immune response in a context dependent manner. For years, it has been known that ASCs accumulate in the thymus of individuals with autoimmune disorders such as myasthenia gravis. In these instances, thymus ASCs persist long-term and generate pathogenic autoantibodies. However, it remains unclear how these cells are produced and regulated in the thymus under healthy conditions. Understanding these basic concepts may provide clues as to the emergence of select autoimmune diseases. As such, we have investigated the mechanisms of generation and the transcriptional landscape of thymus ASCs and compared these features to their counterparts in the bone marrow and spleen. Examination of ASC numbers demonstrated an organ-specific sex bias which manifested as increased numbers in the female thymus relative to males. Flow cytometric analysis for the proliferation marker Ki-67 demonstrated the presence of Ki-67+ plasmablasts. The administration of CD154(CD40L) blocking antibodies led to significant reduction in Ki-67+ ASCs as well as Ki-67+ B cells in the thymus. As CD40 expression was nearly exclusive to thymus B cells, the effect of CD154(CD40L) blockade was most likely mediated on upstream B cells. Finally, single cell RNA-sequencing revealed that thymus ASCs possessed an interferon-responsive gene signature which was validated at the protein level. Overall, these data provide insights as to the generation and regulation of thymus ASCs which can be leveraged for future studies.

This work was supported by the National Institute on Aging of the National Institutes of Health under Award Number R03AG071955.
Quantifying how TCR sequence variation affects T cell fate at single-cell resolution
Authors: Lagattuta Kaitlyn , Kwun Mujin , Raychaudhuri Soumya
Published: August 2023

Mucosal-Associated Invariant T (MAIT) and Natural Killer T (NKT) cells demonstrate that some T cell transcriptional fates are driven by the T cell antigen receptor (TCR). Though activation through the TCR is integral to T cell differentiation, the contribution of TCR sequence features to other T cell transcriptional fates remains yet to be defined. In this study, we identify how αβ V(D)J recombination affects T cell differentiation at single-cell resolution. By applying Canonical Correlation Analysis (CCA) to 340,557 TCR clones collected from 256 individuals, we define a set of TCR scoring functions that quantify transcriptional fate predispositions conferred by the TCR. Unsurprisingly, the strongest fate predispositions correspond to cognate peptide presentation molecules: MR1- or CD1d-restricted PLZFhigh innate-like (MAIT/NKT) transcriptional fate (72.6-fold increase in fate likelihood for top percentile compared to bottom percentile TCRs, AUC = 0.85) and MHC class I-restricted CD8 fate versus MHC class II-restricted CD4 fate (18.7-fold increase, AUC = 0.75). However, our results also uncover that hydrophobic CDR3 residues promote regulatory T cell transcriptional states in both the CD8 and CD4 lineages, and that TCR sequence features preferred by thymic positive selection continue to promote memory formation in the periphery. Applying our TCR scoring functions to 10× dextramer-labeled T cells reveals that not all antigen-specific TCRs are equally capable of mounting an effector response. Even among T cells that recognize the same antigen, biophysical variation in the TCR sequence directs which T cells undergo transcriptional reprogramming to form immunological memory.

Supported by grant from NIH (T32GM007753)
Characterization, functional validation, and analysis of the cross-reactive T cell repertoire in the context of heterosubtypic influenza A virus infection
Authors: Gaevert Jessica A , Duan Susu , Minervina Anastasia , Pogorelyy Mikhail , Mayer-Blackwell Koshlan , Kirk Allison , Crawford Jeremy , Thomas Paul
Published: August 2023

The T cell receptor (TCR) repertoire is extremely diverse and plays an instrumental role in fighting off pathogens individuals encounter in a lifetime. A key aspect of the immune system combatting the diverse array of pathogens an individual sees may be TCR cross-reactivity, where a TCR can recognize more than one peptide displayed on an MHC-I molecule. Specifically, cross-reactive TCRs may play a key role in allowing the immune system to adapt to rapidly evolving pathogens, such as influenza A virus (IAV). Utilizing 6 variant influenza viruses for priming and secondary challenge, our preliminary data has shown that cross-reactive TCRs seen in primary infection with one IAV can mount a robust and effective memory response to a different IAV, across a range of mutations. However, selection for cross-reactive receptors over multiple infections may create a narrowed repertoire consisting of a few clonotypes, covering a narrow range of antigenic space representing the mutant epitopes encountered. Our data shows that cross-reactive TCRs are more similar to each other than single epitope specific cells and that cross-reactivity can be replicated during peptide stimulation when cross-reactive TCRs are transduced into TCR null cell lines invitro. Given the narrowing nature of cross-reactive responses elicited over different infections, selection for cross-reactivity may provide a substantial escape opportunity with future antigenic variations. Ongoing work focuses on how repeated IAV challenges shape the repertoire, and how previous infections inform future responses by selected cross-reactive T cell pools.

Supported by NIH Grant R01112404040 and the St. Jude Graduate School of Biomedical Sciences
Venous plexus-associated lymphoid hubs support meningeal humoral immunity
Authors: Zanluqui Nagela Ghabdan , Fitzpatrick Zachary , Rosenblum Jared S , Tuong Zewen Kelvin , Chandrashekhar Vikram , Negro-Demontel Maria Luciana , Allinson Kieren S J , Mastorakos Panagiotis , Chittiboina Prashant , Maric Dragan , Donahue Danielle , Ferdinand John R , Portet Anais , Penalver Ana , Gillman Eleanor , Zhuang Zhengping , Clatworthy Menna R , McGavern Dorian B
Published: August 2023

There is increasing interest in how immune cells in the meninges, the membranes that surround the brain and spinal cord, contribute to homeostasis and disease in the central nervous system. The outer layer of meninges, the dura mater, contains both innate and adaptive immune cells and functions as a site for B cell development. In this study, we identified previously undescribed lymphoid structures surrounding fenestrated venous plexi in the dura mater. We found the most elaborate immune organization, including lymphatic vessels, surrounding the rostral-rhinal confluence of sinuses. We termed this structure that interfaced with the skull bone marrow and a comparable venous plexus at the skull base, the rostral-rhinal venolymphatic hub. This hub emerged during development in mice at P8/9 before formation of the superior sagittal sinus. Single cell RNA sequencing demonstrated that rostral rhinal hub hosts a diverse array of resident innate and adaptive immune cells during steady state. Immune aggregates were also present in this structure during homeostasis and expanded with age or following challenge with systemic or nasal antigens. Following intranasal VSV infection, the rostral-rhinal hub supported local germinal center (GC) reactions consisting of T follicular helper cells as well as GC B cells that underwent proliferation, somatic hypermutation, class switch, and conversion into plasma cells locally. These data demonstrate lymphoid architecture around vascular plexi in the dura mater that can sample antigens and rapidly elaborate local humoral immune responses to protect the meninges and underlying brain parenchyma from pathogens.

This research was supported by the intramural program of the National Institute of Neurological Disorders and Stroke, NIH
Differential T Cell Responsiveness to Proliferative Stimuli Assessed with a Novel, Homogeneous Ki-67 Immunoassay
Authors: Lazar Dan F , Kupcho Kevin R , Niles Andrew L , Cali James J
Published: August 2023

Proliferative capacity is an important component of T cell fitness and a relevant parameter for characterization of CAR T cells and T cell activating agents. Ki-67 protein is a well-validated marker of cell proliferation. We used Lumit™ technology to develop a homogeneous, luminescent immunoassay for determination of intracellular Ki-67 levels. Purified CD8+ T cells from different human donors were plated in 96-well format, then treated with a panel of T cell activators +/− IL-2, including αCD3/αCD28, αCD3/αCD28/αCD2, phytohemagglutinin, concanavalin A, and a cocktail of PMA and ionomycin. An optimized lytic reagent was added to control and treated cells, followed by two anti-Ki-67 monoclonal antibodies labeled with SmBiT and LgBiT subunits, respectively. Upon coincident binding of these labeled antibodies to Ki-67 protein molecules, the resultant proximity of SmBiT and LgBiT reconstitutes NanoBiT luciferase. Addition of Lumit Detection Reagent produces light proportional to the level of Ki-67 expression, with a total assay time of 2 h. It was determined that stimulation of Ki-67 levels varied markedly between T cell activators tested, with the maximum responses observed exceeding 13-fold. Notably, T cell responses varied significantly between donors, including with respect to the degree of IL-2 dependence. This assay system was compatible with same-well fluorescent determination of cell death. In parallel treated wells, Lumit cytokine immunoassays were used to assess concomitant increases in cytokine levels. This simple, no-transfer, no-wash immunoassay for quantitative determination of Ki-67 levels provides an improved workflow for rapid and efficient comparative assessments of T cell proliferative responses.
Salivary gland response to type I interferons is independent of circulating cytokine levels: Implications for understanding Sjögren’s syndrome pathogenesis
Authors: Bagavant Harini , Pyclik Marcelina , Papinska Joanna , Durslewicz Justyna , Deshmukh Umesh S
Published: August 2023

Sjögren’s syndrome (SS) is a chronic autoimmune disorder affecting the salivary glands. Viral infection-mediated activation of innate immunity and elevated type I IFNs are considered risk factors for SS. However, the frequency of individuals developing SS is far lower than those infected with viruses. To address the role of genetic susceptibility for SS development, we treated 9 inbred strains of mice covering a diverse genetic repertoire with the TLR3 agonist poly (I:C). Serum cytokines were measured by bead-based assays, and salivary gland immune cell populations were studied by spectral flow cytometry. Primary salivary gland cell cultures were stimulated with IFNα, and the expression of IFN-responsive genes was analyzed. Poly (I:C) induced circulating cytokine levels were widely different between the mouse strains, and they correlated with the presence of a CD11b+ macrophage population in the peritoneum. All poly (I:C) treated mice showed significantly increased frequencies of salivary gland NKp46+ (NK and ILC1) innate immune cells compared to saline-treated controls. IFNα-treated primary salivary gland cultures showed strain-specific differences in IFN-responsive gene expression. Surprisingly, the IFN-responsive gene signature score and elevated frequency of NKp46+ cells in salivary glands did not correlate with the levels of circulating cytokines. These data show that the magnitude of salivary gland responses to type I IFNs are not concordant with the systemic innate immune response. Our study suggests that following a viral infection, genes dictating salivary gland response to type I IFNs might play a critical role in SS development. Identifying such genes will lead to a better understanding of SS etiopathogenesis.

Supported by grants from NIH (NIDCR DE031166)
Acquired Lipodystrophy is Mediated by a Treg Specific Deletion of Helios
Authors: Thornton Angela M , Penna Vinay , Gavrilova Oksana , Shevach Ethan M
Published: August 2023

The selective deletion of the transcription factor, Helios, in Regulatory T Cells (Treg) leads to systemic immune activation characterized by a Th1 phenotype, hypergammaglobulinemia, and enhanced germinal center formation. Strikingly, we also observe acquired lipodystrophy, hepatic steatosis, and insulin resistance. Further analysis of the lipodystrophy had revealed a significant lymphocytic infiltrate in both the inguinal and perigonadal adipose tissue, indicating autoimmune mediated destruction of the white adipose tissue (WAT). We show here that the destruction of adipocytes is dependent on CD8+ T cells, as Heliosfl/fl × Foxp3Cre mice crossed to B2m deficient mice (lacking Class I MHC) maintain normal adipose tissue volume. Flow cytometry analysis of the infiltrate demonstrates that the destruction of the adipose tissue is mediated by CD8+ T cells through both cytotoxic and Fas-L dependent mechanisms.

Preliminary analysis of the adipose tissue Tregs suggests that the Treg fail to acquire or maintain an effector state in the absence of Helios. Thus, Helios deficiency in Treg disrupts immune homeostasis in the adipose tissue, leading to the expansion and activation of CD8+ T cells, the destruction of the tissue and metabolic disease. This work was supported by the Intramural Research Program of NIAID, NIH.

This work was supported by the Intramural Research Program of NIAID, NIH.
Bacterial DNA Mediated Protection from Systemic Lupus Erythematosus
Authors: Oakland David N , Appiah Michael , Zhu Jing , Lipsky Peter , Luo Xin M
Published: August 2023

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with no known cure. Current therapies primarily rely on symptom control with corticosteroids, however these therapies carry many adverse effects. In recent decades, the gut microbiome has gained appreciation as a major immunomodulator of autoimmune disease. Toll-like receptor 9 (TLR9) is known to interact with microbial DNA readily provided by the gut microbiota. Polymorphisms of TLR9 have been implicated in increased susceptibility to SLE, and overexpression of the receptor has been shown to be protective in mice. Agonization of TLR9 with bacterial DNA (bDNA) could provide a passive immunity to lupus and other autoimmune conditions without the side effects of corticosteroids.

We previously showed that in vivo administration of bDNA was protective against lupus in mice, however the mechanism behind this protection was unknown. In vitro stimulation of lupus prone splenocytes showed increased production of IL-10, a potent anti-inflammatory cytokine, along with an increase in IL-6, a cytokine known to induce the proliferation of IL-10 producing B cells and antibody producing plasma cells. This phenomenon is dependent on TLR9 expression, but expression of TLR9 on either B cells or dendritic cells is sufficient to increase IL-10. However, TLR9 activation on B cells may cause an increase in IL-10 with a less dramatic increase in IL-6, which may lead to a more beneficial, anti-inflammatory profile as a potential therapeutic.

Our results suggest bDNA as a possible anti-inflammatory avenue of autoimmune management that avoids the use of corticosteroids. Future studies will focus on B cell targeted delivery of bDNA that promotes IL-10 without the side effect of increasing IL-6.
Human gut commensals support development of ocular autoimmunity in retina-specific T cell receptor transgenic mice
Authors: Zhang Amy , Horai Reiko , Jittayasothorn Yingyos , Badger Jonathan H , Nagarajan Vijayaraj , Murphy Caitlin Elizabeth , Yuan Wuxing , O’hUigin Colm , Honda Kenya , Caspi Rachel
Published: August 2023

Autoimmune uveitis is a major cause of blindness, in which intraocular inflammation is driven by T cells that target the neuroretina. In a mouse model of spontaneous autoimmune uveitis (R161H), retina-specific T cells are primed in the gut through their transgenic TCR, and trigger disease. To support the relevance to human disease, we examined the development of uveitis and its association with gut microbiota in gnotobiotic R161H mice colonized with gut flora from healthy human donors. We then performed fecal metagenomic sequencing, gut immunophenotyping by flow cytometry and disease monitoring. Our results indicate that human gut commensals support development of autoimmunity in the spontaneous uveitis model. Human flora mice compared to SPF mice harboring normal mouse flora displayed altered intestinal effector and regulatory CD4 T cell profiles, and had decreased fecal metabolites including microbe-derived short chain fatty acids. Microbiome analyses showed that human flora mice retained a distinct but simplified gut microbial community compared to their original donor sample. Mice with high disease scores appeared to harbor more diverse gut flora than those with low scores. Verrucomicrobia, Actinobacteria, and Fusobacteria were enriched in mice with high disease scores, whereas Firmicutes appeared enriched in mice with low disease scores. In-vivo association studies are underway to examine the ability of candidate microbes to modulate autoimmune uveitis. These findings suggest that some taxa of human commensals may have enhancing or suppressive effects on the development of ocular autoimmunity. These candidate microbes can be explored as targets or as probiotics for therapeutic use.
Benzo[a]pyrene exposure reduces cell-type diversity in lupus-prone mice
Authors: Zhu Runqi , Kennicott Kameron , Liang Yun
Published: August 2023

Studies indicate that genetic factors only account for approximately thirty percent of all autoimmune diseases, while the rest of autoimmune pathogenesis is attributed to environmental factors including toxic chemicals, dietary components, and infections. To understand if and how toxic chemicals trigger autoimmunity, we investigated the effect of benzo[a]pyrene (BaP) exposure on the development of autoimmune phenotypes in the lupus-prone MRL/MpJ strain. Since BaP is a polycyclic aromatic hydrocarbon commonly found in byproducts of industrial incineration, motor vehicle emissions and cigarette smoke, we subjected mice to skin plus lung exposure to BaP over the course of 8 weeks before lupus onset. BaP treatment resulted in total body weight loss in male mice, while marginal changes in anti-dsDNA levels occurred. Multi-organ analyses of BaP-treated and control MRL mice suggested that kidney is a major organ directly affected by the metabolism of benzene-containing compounds, with increased expression of BaP-target genes including Cyp4b1 and Hao2. Intriguingly, spatial transcriptomic data showed that BaP caused drastic reduction in cell type diversity in both kidney and spleen of MRL mice. In addition, BaP exposure triggered C3 deposit in a more severe manner in the kidney of male MRL mice compared to female. Collectively, our results show that BaP exposure reduces cell type diversity in lupus-prone mice before disease onset, and stimulates sex-biased molecular pathways which may help explain the increased likelihood of end organ damage in males with lupus.
Clonally Expanded Cytotoxic CD8+ T cells Target Citrullinated Antigens In ACPA+ Rheumatoid Arthritis
Authors: Moon Jae Seung , Younis Shady , Sharpe Orr , Rao Navin L , Carman Julie A , James Eddie A , Buckner Jane H , Deane Kevin D , Holers V Michael , Donlin Laura T , Davis Mark M , Robinson William H
Published: August 2023

The immune mechanisms that mediate synovitis and joint destruction in rheumatoid arthritis (RA) remain poorly defined. Although increased levels of CD8+ T cells have been described in RA, their role in pathogenesis remains unclear. Here we perform single cell transcriptome and T cell receptor (TCR) sequencing of CD8+ T cells derived from anti-citrullinated protein antibodies (ACPA)+ RA blood. We identify GZMB+CD8+ subpopulations containing large clonal lineage expansions that express cytotoxic and tissue homing transcriptional programs, while a GZMK+CD8+ memory subpopulation comprises of smaller clonal expansions that express effector T cell transcriptional programs. We demonstrate RA citrullinated autoantigens presented by MHC class I activate RA blood-derived GZMB+CD8+ T cells to expand, express cytotoxic mediators, and mediate killing of target cells. We also demonstrate that these clonally expanded GZMB+CD8+ cells are present in RA synovium. These findings suggest that cytotoxic CD8+ T cells targeting citrullinated antigens have a role in contributing to synovitis and joint tissue destruction in ACPA+ RA.

The study was supported by following grants: National Institutes of Health grant R01 AR063676, U19 AI110491, R01 AR078268, and Janssen Biotech, Inc.
Distinct phenotypes of rare autoreactive and virus-reactive CD8+ T cells revealed through novel clustering algorithm
Authors: Kus Anna , Wiedeman Alice , Long Alice
Published: August 2023

Antigen-specific cells are implicated in the development and progression of disease in autoimmunity, infectious disease, and cancer, and are an attractive target for therapies. While robust detection of antigen-specific cells is possible with tetramer and activation-induced marker assays, extensive characterization of these cells remains challenging due to their rarity. Here we present a novel computational tool, DISCOV-R, that enables deep phenotyping of rare antigen-specific cells through unbiased clustering of a parent population into subsets defined by expression of phenotyping markers and overlaying the antigen-specific population. Using this tool with 35-parameter mass cytometry (CyTOF) along with class I MHC-tetramer assays, we identified phenotypes enriched among rare islet autoantigen-reactive and chronic virus-reactive CD8+ T cells when compared to total CD8+ T cells from 46 type 1 diabetics. CD8+ T cells were clustered based on expression of 24 phenotypic markers, revealing 12 CD8+ T cell phenotypes shared across subjects. Overlaying antigen-specific tetramer-positive cells on this phenotypic landscape revealed that both islet- and virus-reactive cells were enriched for an exhausted-like memory phenotype (p=1.7E-07 and 1.6E-05, respectively) consistent with chronic antigen exposure, while islet-reactive cells were enriched for two transitional memory phenotypes (p=0.01 and 0.03). The characterization of these rare antigen-specific cells using DISCOV-R reveals unique phenotypes associated with their specificity that may indicate different function, and enables comparison over time and between individuals to identify biomarkers and potential pathways to disease development and progression.
Tuning peptide specificity for T cell tolerance in Type 1 diabetes
Authors: Smith Jennifer Anne , Balolong Jared , Jin Nijun , Whitty Caroline , Kappler John W , Anderson Mark S
Published: August 2023

Type 1 Diabetes (T1D) is a classical T-cell mediated autoimmune disease, and fundamental data have implicated insulin as the dominant autoantigen in T1D disease. In the NOD mouse model of T1D, notable studies have found that mice lacking native insulin expression but with an altered insulin sequence to maintain blood glucose levels are protected from insulitis and diabetes. Growing evidence also indicates how the insulin peptide is bound within MHC Class II (peptide register) is essential in determining the strength of interactions and recognition of autoreactive T-cells. We have uncovered a unique peptide binding characterized by the dominant insulin epitope InsB:9–23. The majority of InsB:9–23-specific CD4 T-cells in the periphery recognize insulin bound in the unusual register 3, and knocking in this single amino acid variation into just one copy of the insulin gene in NOD mice, confers protection against T1D. In addition, these mice were protected from T1D, even when treated with checkpoint inhibitor blockade, unlike their wild-type littermates. Introducing this superagonist epitope into the NOD mouse has allowed us to address several key questions surrounding pathways of peptide generation, presentation by MHC molecules, and recognition by auto-reactive pathogenic T cells. In addition, we have been able to model how central and peripheral tolerance mechanisms can be altered when major epitopes in the Insulin gene are mutated, allowing us to explore means of dominant tolerance to understand the potential for translation into treatments for T1D.

Supported by grants from NIH (R01 DK133443-01A)
Single cell characterisation of tissue homing CD4+ and CD8+ T cell clones in immune-mediated refractory arthritis
Authors: Bhattacharya Dipabarna , Theodoropoulos Jason , Kelkka Tiina , Nurmi Katariina , Juutilainen Timo , Eklund Kari , Koivuniemi Riitta , Mustjoki Satu , Lönnberg Tapio
Published: August 2023

Background

Immune-mediated arthritis (IMA) is a group of autoimmune diseases where chronic inflammation primarily targets different joints in the body. Rheumatoid Arthritis (RA) is the most common subtype. In childhood, RA mostly manifests as juvenile idiopathic arthritis (JIA). In this study, we aimed to characterize the tissue homing inflammatory pathogenic T cells in patients with IMA.

Methods

We collected paired synovial tissue (ST) and peripheral blood (PB) samples from three patients with IMA and analyzed CD45+, CD4+ and CD8+ sorted cells with paired single-cell RNA and TCRα/β sequencing and validated the findings with published datasets.

Results

Despite being disparate diseases, CD4+ T cells in IMA represented uniform phenotype across patients with no major patient specific clusters. The ST cells were more cytotoxic (GZMB), highly activated (LAG3, PDCD1) and proliferative (NME1, FABP5, G0S2 and MKI67). A modest clonal expansion within the Treg cluster was found in the ST compartment as compared to PB (p<0.05). Conversely, the CD8+ T cell clusters showed strong patient specific characteristics. All T cell clones enriched to the ST upregulated genes belonging to TNF receptor superfamilies, inflammation markers (LMNA, RGCC, CREM), and transcription factor NR4A2. Pathway enrichment analysis showed enrichment of the TNFA signalling via NFKB and apoptosis pathways.

Conclusions

Our data suggests that in patients with IMA, the synovial tissue is dominated with cytotoxic, highly activated CD4+ T cells with proliferation-associated gene expression signature. Shared CD4+ and CD8+ T cell clones can be found between PB and ST, but they have a more proliferative and activated phenotype when trafficking from PB to ST.

Supported by grants from University of Helsinki, Finland
Neutrophil peptidylarginine deiminase 4 plays a systemic role in obesity-induced chronic inflammation in mice
Authors: Van Bruggen Stijn , Sheehy Casey E , Kraisin Sirima , Wagner Denisa D , Martinod Kimberly
Published: August 2023

Obesity is an increasing problem in our current society. In morbidly obese patients, the level of circulating neutrophil extracellular traps (NETs) was found to be higher than in lean control subjects. Essential in this process of NET formation is the activation of the enzyme peptidylarginine deiminase 4 (PAD4).

We aimed to understand how NETs contribute to the detrimental effects associated with a prolonged state of (morbid) obesity. Therefore, an animal model was adopted in which wild type (WT) and neutrophil specific PAD4 deficient (Ne-PAD4−/−) mice on a C57BL6/J background were fed a high fat containing diet (HFD – 60kcal% fat) for a period of 10 weeks.

Neutrophil activation status, evaluated through the formation of NETs in response to calcium ionophore ionomycin (4 μM) stimulation, was initially reduced after 3 days of HFD feeding. At later timepoints (5- and 10-weeks), neutrophils isolated from HFD-fed mice showed increased levels of NET formation in response to ionomycin stimulation in comparison to neutrophils from control diet-fed mice. Ne-PAD4−/− mice on the HFD gained less weight than WT mice, which had a constant gain of body weight over the 10-week experimental period. Finally, after 10-weeks of high fat intake, cardiac function was evaluated in WT and Ne-PAD4−/− mice by echocardiography. Interestingly, in WT mice diastolic function (early passive vs late active filling, E/A ratio) decreased as compared to baseline values, while Ne-PAD4−/− mice kept E/A ratios comparable to baseline levels.

Here, we demonstrate the importance of systemic NET release under metabolic challenge, and how this can contribute to cardiac deterioration at a young age due to obesity-induced alteration in the innate immune system.
Potent Anti-inflammatory Effects of the Adiponectin Receptor Agonist, ALY688
Authors: Sater Ali Abdul , Tang Jialing , Lone Abdul Hadee , Pignalosa Anglica , Crawford Kathryn , Sweeney Gary
Published: August 2023

Low circulating levels of adiponectin are associated with increased inflammation in a wide range of disease states. In particular, this applies to obesity-related diseases such as type 2 diabetes, heart failure and non-alcoholic fatty liver disease. Numerous studies have shown that adiponectin exerts anti-inflammatory and beneficial metabolic effects. Hence there has been tremendous interest in developing adiponectin-based therapeutics. Here we examined the effect of ALY688, a small peptide adiponectin receptor agonist, on lipopolysaccharide (LPS)-driven inflammation in vivo and in vitro. We show that ALY688 administration significantly diminished LPS-driven production of pro-inflammatory cytokines and chemokines (i.e., IL-1β, TNF-α, IL-12, IFN-β, MCP-1, KC, RANTES) in mice. Moreover, mice injected with ALY688 exhibited a rapid increase in the anti-inflammatory cytokine, TGF-β. Apart from an increase in total CD3+ T cells, there were no marked changes in the circulating immune cells following ALY688 injection. No alterations in cytokine profile was seen in normal mice given ALY688, suggesting that the effects of ALY688 are context-dependent. Mechanistically, we employed phospho-flow cytometry to demonstrate that circulating immune cells responded rapidly to ALY688 injection by upregulating p-AMPK and p-P38, two well established adiponectin downstream signaling events. These results were confirmed in human peripheral blood mononuclear cells (PBMCs), where ALY688 treatment led to a robust increase in phospho-AMPK and phospho-P38. Altogether, these results show that the adiponectin agonist, ALY688, has strong anti-inflammatory effects by triggering AMPK and P38 activation in vivo and in vitro.
Overexpression of miR-10a-3p mediates TGF-β1/Smad Signaling Pathway to suppress adipogenesis and AT inflammation
Authors: Singh Udai P , Kiran Sonia , Mandal Mousumi , Rakib Ahmed
Published: August 2023

Obesity, an epidemic of our time is a multifactorial disease characterized by an enhanced amount of fat and energy storage in adipose tissue (AT). Growing evidence suggests that obesity promotes activation in resident T cells and macrophages to maintain low-grade inflammation. MicroRNAs (miRs) are shown to maintain immune response in the AT and are associated with adipocyte differentiation, and function during obesity. Microarray analysis shows differential expression of miRs in the AT immune cells in mice fed with a high-fat diet (HFD) for 12 weeks. Pathways analysis demonstrated that miR-10a-3p (miR-10a) expression is downregulated in AT and its expression has been correlated with obesity progression. Thus, we aimed to evaluate miR-10a overexpression effects on AT inflammation, metabolic markers, and signaling pathways during obesity. Our ex vivo analysis shows that mimicking miR-10a reduces the expression of inflammatory M1 macrophage, TGF-b1, KLF4, IL-17F, and inflammatory cytokine while inducing FoxP3 expression in immune cells derived from AT of HFD-fed mice as compared to ND. Correspondingly, we noticed that mimicking miR-10a in NIH3T3-L1 adipocytes reduces proinflammatory genes, adipocyte size, cytokines, and lipids accumulation as compared to the control. Further, overexpression of miR-10a also reduces TGF-b1, Smad3, CHOP10, and C/EBPa expression in NIH3T3-L1 as compared to the control. Thus, our findings suggest that miR-10a mimics mediate the TGF-β1/Smad3 signaling pathway, which suppresses cytokine, and metabolic markers, to reduce adipogenesis and AT inflammation. Our studies strongly suggest miR-10a as a new target to therapeutically cope with obesity and its associated metabolic disorders.

Supported by NIH grant R01 AI140405).
VEGFR-3 Stimulation on LECs Boosts Type 2 Immunity After Vaccination
Authors: Kurtanich Trevin Robert , Medellin Jorge Emiliano Gomez , Swartz Melody A
Published: August 2023

Lymphatic vessels are the main transport routes for fluid, antigens, exosomes, and immune cells from peripheral tissues to the lymph nodes (LNs). In addition to being pervasive through tissue, lymphatic vessels are also abundant within the LN, where they route lymph and cells around the B cell follicles and throughout the paracortical zone; thus, lymphatic endothelial cells (LECs) have unique access to lymph-borne molecules and interactions with immune cells. Upon acute inflammation or vaccination, LN LECs undergo expansion along with lymphocyte proliferation, a process dependent on the VEGF-C/VEGFR-3 signaling axis; however, it is not well understood how LN lymphatics and their expansion contribute to acute adaptive immune responses. Here, we asked how altering VEGFR-3 signaling LN LECs alters the adaptive immune response, using OVA + CpG (a TLR9 agonist) as a model for vaccination. We found that delivering VEGF-C156S, a VEGFR-3 specific agonist, multiple times prior to and concurrent with the inflammatory stimulus led to increased Th2 responses in terms of higher plasma levels of OVA-specific IgG1, more IgG1+ germinal center B cells, and increased production of Th2 cytokines (IL-4, IL-5, and IL-13) by CD4+ T cells upon restimulation. In loss-of-function experiments using a tamoxifen-inducible LEC-specific deletion of VEGFR-3, vaccination led to reduced plasma levels of OVA-specific IgG1 compared to tamoxifen-treated control littermates. Together, these findings suggest that in response to vaccination, VEGFR-3 signaling in LN LECs bias adaptive immune responses towards type 2 immunity. This highlights the role of lymphatics not just as passive conduits for fluid transport, but as having direct immunomodulatory function.
CD99’s highly conserved region III facilitates the homodimerization and function in cell-cell adhesion
Authors: Chang Hanna , Nam Giri , Choi Eun Young
Published: August 2023

CD99 is a type I transmembrane protein and its function has been known to be implicated in various cellular processes including cell-cell adhesion and leukocyte diapedesis. These functions were all related to actin polymerization and/or reorganization. Nonetheless, the molecular mechanism has remained unclear. In previous studies, we showed that CD99 is capable of forming a homodimer and the homodimerization requires the extracellular domain. This trait was confirmed to be conserved in mouse CD99. In this study, we investigated the molecular basis and functional role of homodimer formation. In sequence comparison analysis, CD99 amino acid sequences from different species have three conserved subdomains in the extracellular domain, named highly conserved region (HCR) I, II, and III. Based on this information, we generated various subdomain-deleted mutants. In the bimolecular fluorescence complementation assay, the HCRIII-deleted mutant (named HCRIIIΔ ext-mut), significantly reduced the ability to form a homodimer, compared with other mutants. Additionally, HCRI and II-deleted mutant, that is, a mutant expressing only HCRIII-subdomain on the cell surface (named HCR3ext-mut), was enough to form the homodimer. Overexpression of HCRIIIΔext-mut in T cells prevented T cells from forming stable interaction with the antigen-presenting cells (APCs), and polarization of polymerized actin to the T-APC interface. However, overexpressing HCRIIIext-mut enhanced the T-APC stable interaction. These results identify the HCRIII of CD99 as the key subdomain required for dimer formation and indicate that the homodimer would contribute to the formation of stable interaction with APCs of T cells via actin cytoskeleton reorganization.
VEGFR1 blockade reduces Mycobacterium tuberculosis-induced pathology in immunocompromised mice
Authors: Herbath Melinda , Priyathilaka Thanthrige Thiunuwan , Fabry Zsuzsanna , Sandor Matyas
Published: August 2023

Mycobacterium tuberculosis (Mtb) is a major public health concern that caused 1.6 million deaths in 2021. New treatments are needed, especially in immunocompromised individuals, such as patients with HIV coinfection, who are severely impacted by the disease.

We have recently reported that blocking VEGFR1, a receptor that is present on monocytes and contributes to their recruitment to the sites of infection, limits Mtb-induced pathology in immunocompetent mice of both Mtb-resistant (C57BL/6J) and Mtb-supersusceptible (C3HeB/FeJ) strains (Harding et al., 2019). These results offer a way to reduce lung pathology without impeding host defense. Here, we extend this finding by showing that VEGFR1 blockade has similar effects in immunocompromised (RAG1KO) mice as well, measured by a reduction in the area of inflammation and without a change in bacterial burden. Following treatment with the VEGFR1 blocker SU5416, we have found an elevated ratio and an increase in the absolute number of neutrophil granulocytes in the Mtb-infected lungs in both immunosufficient and immunocompromised mice. Surprisingly however, this did not result in exacerbated pathology as the majority of the recruited neutrophils remained in the lung vasculature.

Our results indicate that further evaluation of VEGFR1 blockers as an adjunctive treatment to antitubercular drug therapy for immunocompromised populations could be worthwhile.

Supported by grants from NIH (R01 HL128778 and R01 NS123449)
Transcription factor Bhlhe40 plays a protective role during intravaginal Chlamydia muridarum infection in mice
Authors: Mercado Miguel Antonio Bernabe , Quick Charles Matthew , Li Qiang , Palmer Rachel , Kim Yejin , Huang Lu , Li Lin-Xi
Published: August 2023

The protein basic helix-loop-helix family member e40 (BHLHE40) is a transcription factor involved in cell cycle regulation and differentiation. It has recently emerged as a key player in immunity regulating T cell responses and cytokine production during infections.

To address the role of Bhlhe40 in Chlamydia infections, we infected WT and Bhlhe40−/− mice intravaginally with Chlamydia muridarum and monitored infection kinetics. Bhlhe40−/− mice exhibited a significant defect in their ability to clear infection compared to their WT counterparts. Single-cell RNA sequencing revealed distinct heterogeneity in the CD4+ T cell population in the female reproductive tract in WT and Bhlhe40−/− mice following C. muridarum infection. Accordingly, CD4+ T cell population in Bhlhe40−/− mice showed a marked increase in IL-10-producing T regulatory type 1 (Tr1) cells and decreased IFN-γ and GM-CSF-producing cells as assessed by flow cytometry. The overall CD4+ T cell cytokine response was diminished in the absence of Bhlhe40, as indicated by decreased levels of IFN-γ, GM-CSF and IL-17A measured by a multiplex cytokine assay. In contrast, Bhlhe40 did not appear to play an essential role in antibody responses, development of pathology, or metabolic fitness of CD4 T cells during C. muridarum infection. Lastly, IL-10 deficiency restored optimal bacterial clearance in Bhlhe40−/− mice. Altogether, our findings identified Bhlhe40 as a novel player in protective immune response against C. muridarum infection in mice.

NIH/NIAID R01 (AI139124)

NIH/NIGMS COBRE (GM103625)The American Association of Immunologists through a Careers in Immunology Fellowship
Cytokine production profiles of immune cells of stressed beta2-adrenergic receptor knockout mouse model during Chlamydia muridarum genital infection
Authors: Belay Tesfaye , Ray Alexis , Cook Kaitlyn
Published: August 2023

Chlamydia genital infection by Chlamydia trachomatis is the most common bacterial sexually transmitted disease worldwide. The relationships between stress and chlamydia genital infection remain unknown. Beta2-adrenergic receptor(β2-AR), the major receptor of the stress hormone norepinephrine (NE), is known to impair the function of immune cells. Stress was induced by immersing mice in cold water for five minutes daily for 21 days before infection. This study determined profiles of immune cells of stressed β2-AR KO and stressed WT C57BL/6J after 48 h of Chlamydia muridarum genital infection. Splenic T cells and differentiated bone marrow-derived macrophages and dendric cells (DCs) were purified by negative selection and stimulated by seeding T cells on anti-CD3/CD28 antibodies coated wells and by treating macrophages and DCs with LPS. After 48 h proliferation, cytokine levels in culture supernatants of the cells were determined using ELISA. Results show increased IFN-γ and IL-1β secretions but deceased IL-4 secretion by CD4+ T cells of β2-AR KO compared to WT (p<0.05). The secretion of IL-13, IL-5, and IL-10 but not IL-23 in CD4 T cells of stressed β2-AR KO mice was two-fold lower than that of stressed WT. TNF-α and IL-1β secretions in macrophages and DCs or IL-12 secretion in DCs was high in stressed β2-AR KO compared to WT (p<0.05). Overall, this study provides insights into the β2-AR signaling pathway’s role in suppressing the secretion of protective cytokines suggesting that inhibiting β2-AR signaling pathway may restore the function of immune cells during chlamydia genital infection. However, further study is warranted.

This work was funded by NIH grant # 1R15AI124156-01 awarded to Bluefield State University