bluuebunny/crossref_metadata_2025 huggingface dataset:
Impaired AHR Signaling Contributes To Intestinal ILC3/ILC1 Conversion In The Inflamed Terminal Ileum Of Crohn’s Disease Patients
Authors: Li Jian , Doty Andria , Glover Sarah
Published: January 2023

Introduction

Crohn’s disease (CD) is one form of inflammatory bowel disease (IBD). Traditionally, adaptive immunity has been presumed to play a major role in the pathogenesis of CD. Recently, increasing evidence indicates that innate immunity also plays an essential role in this process. The phenotype and function of newly discovered innate lymphoid cells (ILCs) are crucial for the maintenance of intestinal homeostasis. Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which is important for the regulation of ILC biology in the mouse gut. We wanted to explore the potential involvement of AHR in the regulation of intestinal ILC of CD patients.

Hypothesis

AHR signaling pathway is critical for the regulation of the phenotype and function of human intestinal ILC in the terminal ileum of CD patients.

Methods

Surgical terminal ileum samples were collected from CD patients. Histology, Real-time PCR analysis, flow cytometric analysis and immunohistochemistry staining were performed.

Results

The expression of AHR was correlated with CD117 expression on human intestinal ILC subsets. The IFN-γ-producing-ILC1s accumulated in the inflamed terminal ileum of CD patients at the expense of protective IL-22-producing NKp44+ILC3s. Also, the expression of both AHR and CD117 were downregulated in the ILCs from the inflamed tissue. Additionally, there was a disparity between AHR protein and mRNA expression in the inflamed gut of CD patients which suggested the involvement of a posttranscriptional mechanism.

Conclusions

The AHR signaling was impaired in the inflamed gut of CD patients. This transcriptional modification contributed to the ILC3/ILC1 conversion and promoted the process of intestinal inflammation.
Flow Cytometry Multiplexing and its Use in Detailed Characterization of Cell Health
Authors: Low Quentin , Yan Michelle , Wickett Marcy , O’Grady Michael , Montoya Leticia
Published: January 2023

High throughput screening (HTS) is extremely effective method for allowing researchers to identify putative compounds of interest which play a role in their area of cellular research. Recently, flow cytometry has emerged as a powerful HTS tool with the added benefit of cell-by-cell analysis. Flow cytometry not only allows a researcher to study drug effectiveness towards different cell types but also can be used to analyze protein-protein interactions, metabolic activities, as well as DNA/mRNA content in a single or multiplexed assay format.

In this study, we screened the MicroSource Discovery System Killer Plate compound library and compared HTS using a plate reader with flow cytometer multiplexing. Jurkat and Ramos cells, T and B lymphocyte cell types respectively, were used as cell models and cultured under standard conditions and screened either at hypoxic (1%) or hyperoxic (19%) oxygen levels at varied lengths of time (24, 48, or 72 hrs). To assess compound veracity, post-screening analysis was implemented to establish EC50s of “hits” from the compound library. Membrane integrity and metabolic activity were measured as an initial screening output for evaluating cellular viability. Using the multiplexed capabilities of flow cytometry, we performed secondary and tertiary assays to further characterize the possible mechanism of action by analysis of cell cycle, specific RNA expression levels, oxidative stress, and caspase activation across differing concentrations of the compounds. This method and analysis highlights the complicated nature of assessing toxicity in cellular screening assays and the advantage of Flow Cytometry in particular for characterizing cell health.
Tissue infiltration of plasma cells/plasmablasts in patients with active Systemic Lupus Erthematosus (SLE)
Authors: Blanco Isabella K , Heuer Sarah , Catalina Michelle , Robl Robert , Madamanchi Sushma , Lipsky Peter , Grammer Amrie
Published: January 2023

To understand the role of plasma cells/plasmablasts (PC/PB) in organ pathology in SLE, we used GSVA (Gene Set Variation Analysis) to interrogate multiple lupus gene expression datasets. Differentially expressed (DE) genes in active SLE peripheral blood, B cells, mononuclear cells and tissues [skin, synovium, glomerulus, kidney tubulointerstitum (TI)] were queried for the previously reported (Lugar et al, 2012) PC/PB signature. There was considerable overlap between the PC/PB signature (20–44%) with the tissue datasets, with skin overlapping the most and synovium the least. IGM heavy chain and light chains (kappa, IGKC and lambda, IGLJ3 and IGLDE) were upregulated in the blood samples and all four tissue datasets, whereas IGHG1 was upregulated in the blood (B cell, PBMC), synovium & TI and downregulated in the skin. The presence of both IgK and IgL and numerous VL genes implied the polyclonal nature of the PC/PB infiltration. XBP1 governs late events in PC differentiation and was increased in the datasets that also had increased IGHG1 (B cell, TI). PRDM1, required for PC/PB differentiation, was increased in the blood datasets as well as in the synovium and glomerulus. Transcripts of genes in the “ER-mediated translocation to the cytosol” (Tram1, Derl1, and Ssr1) were upregulated in the blood and all four tissue datasets. IPA upstream regulator analysis revealed IL4, IL5 and CD40L for both the reference PC/PB set and all blood and tissue datasets with the exception of TI. IPA also revealed IL-6 as an upstream regulator in the PC/PB signature as well for the blood and four tissues datasets. In summary, interrogation of blood and tissue datasets for a PC/PB signature implicated these Ig secreting cell populations in tissue and organ pathology in SLE.
PD-1 blockade alters fate commitment of myeloid progenitors during tumor-mediated “emergency” myelopoiesis
Authors: Strauss Laura , Weaver Jessica , Freeman Gordon J. , Boussiotis Vassiliki A.
Published: January 2023

PD-1 is an immune inhibitory receptor for which blocking agents have achieved significant success as anti-cancer therapeutics. The mechanism(s) of the how PD-1 compromises anti-tumor function and how such effect is reversed by PD-1 blockade remain poorly understood. The rapid change in hematopoietic output that occurs in response to immunologic stress is known as “emergency” myelopoiesis. This process is co-opted by tumors to enhance the generation of tumor-promoting myeloid cells, mostly undifferentiated myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). We analyzed the myeloid compartment of tumor-bearing mice and determined that myeloid cells express PD-1 and PD-L1. Using PD-1 KO mice or WT mice treated with PD-1 blocking antibody in cancer-driven myelopoiesis experiments we determined a cell-switch fate from MDSCs to effector myeloid cells. PD-1 deletion or blockade, which impaired tumor growth and metastases, stimulated the differentiation of hematopoietic progenitor cells and resulted in increase of Ly6Chimonocytes and monocyte-derived DC (mo-DC). Abrogation of PD-1 signaling enhanced the maturation of MDSCs by inducing the lineage commitment transcription factors IRF8, IRF4 and RORC1. IRF8 and IRF4 promote monocytes/macrophage and hinder granulocyte differentiation, whereas RORC1 is required for expansion of Ly6Chi monocytes and resolution of inflammation. These results reveal a role of the PD-1:PD-L1 pathway in the differentiation of lineage-committed myeloid progenitors to promote undifferentiated MDSCs and suppress effector monocytes/DCs. Switch of myeloid progenitor fate commitment might be a key mechanism by which PD-1 blockade mediates anti-tumor function.
Investigating the effect of anti-CTLA-4 on tumor-infiltrating effector T cells
Authors: Stone Erica Lyn , O’Brien Erin M
Published: January 2023

Despite the relative success of checkpoint inhibitors, a majority of patients fail to respond to these therapies. Thus it is necessary to understand mechanisms of resistance in order to extend the benefits of immunotherapy to more patients. Checkpoint inhibitors are designed to prevent the interaction of immune checkpoints with their ligands, thereby allowing the body’s anti-tumor immune response to be reactivated. Recently, it has been appreciated that anti-CTLA4 therapies, including Ipilimumab, have a second mode of action. They can mediate antibody-dependent cell-mediated cytotoxicity (ADCC). This leads to death of cells bound by anti-CTLA4. While CTLA4 is largely intracellular, it is expressed on the surface of tumor-infiltrating T regulatory cells (Tregs). Thus anti-CTLA4-mediated ADCC can deplete tumor-infiltrating Tregs. In mice, ADCC of tumor-infiltrating Tregs is required for anti-CTLA4 efficacy. Likewise, Ipilimumab can mediate Fc-receptor dependent depletion of human Tregs. We have found that a portion of effector T cells infiltrating mouse and human tumors express surface CTLA-4 at levels similar to tumor-infiltrating Tregs. We further find that this portion of tumor-infiltrating effector T cells is likely to include tumor-reactive T cells. We will discuss the possibility that anti-CTLA4 can lead to Fc-receptor-dependent depletion of effector T cells and the implications for therapy resistance.
The MLL1-H3K4me3 Axis-Mediated PD-L1 Expression and Pancreatic Cancer Immune Evasion
Authors: Lu Chunwan , Paschall Amy V , Shi Huidong , Savage Natasha , Liu Kebin
Published: January 2023

Background

Pancreatic cancer is one of the cancers where anti-PD-L1/PD-1 immunotherapy has been unsuccessful. The aim of this study is to elucidate the underlying mechanism of PD-L1 expression regulation in the context of pancreatic cancer immune evasion.

Methods

Pancreatic cancer mouse models and human specimens were used to determine PD-L1 and PD-1 expression and cancer immune evasion. Histone methyltransferase inhibitors, RNAi and overexpression were used to elucidate underlying molecular mechanism of PD-L1 expression regulation. All statistical tests were two-sided.

Results

PD-L1 is expressed in 60–90% of tumor cells in human pancreatic carcinomas and in 9 of 10 human pancreatic cancer cell lines. PD-1 is expressed in 51.2–52.1% of pancreatic tumor-infiltrating cytotoxic T lymphocytes (CTLs). Tumors grow statistically significantly faster in FasL-deficient mice than in wild type mice and when CTLs are neutralized. H3K4 trimethylation (H3K4me3) is enriched in the cd274 promoter in pancreatic tumor cells. MLL1 directly binds to the cd274 promoter to catalyze H3K4me3 to activate PD-L1 transcription in tumor cells. Inhibition or silencing of MLL1 decreases H3K4me3 level in the cd274 promoter and PD-L1 expression in tumor cells. Accordingly, inhibition of MLL1 in combination with anti-PD-L1 or anti-PD-1 antibody immunotherapy effectively suppresses pancreatic tumor growth in a FasL- and CTL-dependent manner.

Conclusions

The Fas-FasL/CTLs and the MLL1-H3K4me3-PD-L1 axis play contrasting roles in pancreatic cancer immune surveillance and evasion. Targeting the MLL1-H3K4me3 axis is an effective approach to enhance the efficacy of checkpoint immunotherapy against pancreatic cancer.
PD-1 blockade induces phosphorylated STAT3 and results in an increase of Tregs with reduced suppressive function
Authors: Woods David M , Ramakrishnan Ruap , Sodré Andressa L , Berglund Anders , Weber Jeffrey
Published: January 2023

PD-1 blockade has remarkable response rates in the treatment of melanoma; however, many patients fail to respond, and biomarkers and mechanisms of response remain unknown. We investigated the roles of Tregs, a population of immunosuppressive T-cells, in patient response to nivolumab. Tregs had a decrease in suppressive function in patients with positive outcomes (p=0.03), but not in patients with negative outcomes. Patients with no evidence of disease (NED) displayed increased percentages of Tregs post-nivolumab (p=0.04), but relapsing patients did not. RNA-seq analysis revealed genes significantly changed after treatment were distinct between Tcons and Tregs (~13% overlap) and between NED and relapsing patient Tregs (~2% overlap). Pathway analysis showed an increase in proliferation associated pathways in NED patient Tregs, but not relapsing patients. We found increased phosphoSTAT3 (pSTAT3) expression in Tregs from patients with positive outcomes (p=0.01), but not in patients with negative outcomes. Mechanistically, in vitro culturing of T-cells with αPD-1 resulted in increases in pSTAT3 expression (p=0.03) and increased percentages of Tregs (p=0.001). Culturing with αPD-1 also enhanced production of IL-10 (p=0.02), and the addition of a STAT3 inhibitor reduced the increases in IL-10 levels (p=0.01) and Treg percentages (p=0.01). The addition of an IL-10 neutralizing antibody also reduced the increased Tregs resulting from αPD-1 (p=0.01). These results support a model in which PD-1 blockade increases pSTAT3 expression leading to enhanced IL-10 production and Treg percentages, suggesting that pSTAT3 induction and reduced suppressive function are biomarkers of melanoma patient response to nivolumab.
Comparative analysis of conventional natural killer cell responses to acute infection with Toxoplasma gondii strains of different virulence
Authors: Gigley Jason P. , Ivanova Daria
Published: January 2023

Toxoplasma gondii is an obligate intracellular parasitic protozoan present in 30% of humans and a significant health concern in immunocompromised people. Conventional Natural Killer Cells (cNK), members of group 1 innate lymphoid cells, are critical for early immunity to T. gondii via IFNγ production. However, little is understood about how cNK cell functionality, subpopulations, maturation and proliferation are affected by parasite infection and parasite virulence. Here we comprehensively performed this analysis with Type I virulent RH, Type II avirulent ME49 and fully attenuated Type I cps1-1 strains. In response to these three parasite strains, murine cNK cells produce IFNγ and become cytotoxic and polyfunctional (IFNγ+CD107a+) at the site of infection. In contrast to virulent RH and avirulent ME49 T. gondii strains, attenuated cps1-1 strain induced only local cNK cell responses. RH and ME49 infection significantly decreased cNK cell frequency and numbers in spleen compared with cps1-1 parasites. Responding cNK cell subsets expressing activating receptors and inhibitory receptors were similar regardless of parasite infection strain. 5 days post infection cNK cells did not proliferate, however, cellular maturation (CD27, CD11b and KLRG1) was impacted by parasite strain. cNK cell maturation was reduced after RH or ME49 infection compared with cps1-1 infection. Interestingly, KLRG1 was highly expressed on immature not mature cNK cells after RH infection. Results suggest increased parasite virulence differentially impacts cNK cell maturation during acute T. gondii infection. Different cNK cell responses could impact early immunity and susceptibility to these strains.
Human exposure to Angiostrongylus cantonensis on east Hawaii Island
Authors: Jarvi Susan Irene , Quarta Stefano , Eamsobhana Praphathip , Howe Kathleen , Jacquier Steven , McHugh Robert , Kramer Kuikei , Meyers McKayla
Published: January 2023

A study was completed to quantify exposure to Angiostrongylus cantonensis among 435 human volunteers from east Hawaii Island. Volunteers completed a questionnaire and provided blood samples (at Puna Community Medical Center and Clinical Labs of Hawaii). Serum was screened by indirect ELISA using crude antigen isolated from adult A. cantonensis harvested from wild Hawaiian rats, and 28% tested positive. Of volunteers who defined themselves as definitively diagnosed by a clinician (n = 15), 67% were positive by ELISA and reported being infected in 2004 and later. Of those self-reported as previously infected with A. cantonensis (n=105), 31% were positive; those who self-reported as uninfected (n=315), 25% tested positive by ELISA. All ELISA positives were further tested by ELISA dot-blot using the more definitive 31 kDa protein isolated from Thailand adult female A. cantonensis as antigen. Of the 186 tested by ELISA dot-blot, 30% were positive. Of the 15 definitively diagnosed, 47% were positive and reported being infected in 2007 and later, however antibodies were also detected in a volunteer infected in 1981, which may have been due to a more recent re-exposure. Of those self-reported as previously infected (n=45), 29% were positive and those who self-reported as uninfected (n=126), 29% were positive. The sensitivity of the ELISA dot-blot as compared with ELISA was 100%, but the specificity was only 25% due to the high number of false positives by crude antigen ELISA. No samples were dot blot positive and ELISA negative, thus the ELISA test appears to be an effective initial screening method and the ELISA dot-blot appears to have good efficacy as a diagnostic. Human exposure to A. cantonensis in east Hawaii is higher than previously thought.
Longitudinal analysis of Plasmodium falciparum-specific atypical and classical memory B cell responses to natural malaria infection in children and adults
Authors: Hopp Christine , Krishnamurty Akshay T , Portugal Silvia , Thouvenel Chris , Doumbo Ogobara K , Traore Boubacar , Pierce Susan K , Rawlings David J , Pepper Marion , Crompton Peter D
Published: January 2023

Antibodies play a key role in naturally acquired immunity to malaria; however, protective antibodies are only acquired after years of repeated Plasmodium falciparum (Pf) infections. We have shown in Mali that this inefficiency in humoral immunity to malaria is associated with an expansion of CD21loCD27− ‘atypical’ MBCs that are isotype-switched and somatically hypermutated, but that exhibit markedly reduced effector functions. A similar subset of ‘exhausted’ MBCs has been observed in patients with chronic viral infections. However, in the context of malaria, it remains unclear whether atypical MBC expansion is Pf-specific or represents a global expansion as a result of chronic immune activation from repeated Pf infections and/or co-infections common in malaria-endemic areas. Using PfMSP1/PfAMA1 B cell tetramer staining we recently identified an expanded population of Pf-specific MBCs in malaria-exposed individuals that were 60 fold more frequent as compared to healthy U.S. adults. Preliminary findings show that monoclonal antibodies expressed from PfMSP1-specific MBCs are indeed PfMSP1-specific by ELISA. Using this B cell tetramer approach, we are tracking Pf-specific classical and atypical MBCs in a longitudinal cohort study of children and adults in Mali. With tetramers specific for tetanus and influenza we will determine the relative role of Pf in driving atypical MBC expansion by comparing the Pf-, tetanus-and influenza-specific MBC responses from age 3 months to 25 years before, during and after an acute Pf infection. This analysis should provide new insights into the mechanisms underlying the delayed acquisition of immunity to malaria in children, and may aid in the development of an urgently needed malaria vaccine.
Viral, bacterial and host determinants converge on impairment of alveolar macrophage function to promote influenza and S. pneumoniae coinfection
Authors: Bansal Shruti , Yajjala Vijaya Kumar , Bauer Christopher , Sun Keer
Published: January 2023

Secondary bacterial pneumonia is a major cause of influenza-associated morbidity and mortality. The mechanism for this influenza-induced susceptibility to bacterial superinfection is incompletely understood. In this study, we examined the relative contribution of three central components in the virus-host-pneumococcus interaction, i.e., viral virulence, bacterial virulence, and host resistance to disease severity. Specifically, wild-type and interleukin-1 receptor type 1 deficient (Il1r1−/−) mice were infected with influenza virus X31 (H3N2) or highly virulent PR8 (H1N1), and then super-challenged with noninvasive pneumococcal serotype 19A or 14. The coinfections were followed by comparative analyses of animal survival, viral and bacterial loads, and inflammatory responses in the lungs. Several lines of our data indicate that lethal influenza and Streptococcus pneumoniae coinfection is primarily due to impairment of alveolar macrophage (AM)-mediated bacterial control. First, from a bacterial perspective, the requirement for AMs for bacterial killing increases the virulence of S. pneumoniae serotype 14 compared with 19A; from a viral perspective, the ability of PR8 to induce prominent IFN-γ production contributes to its enhanced virulence compared with X31; and finally from a host perspective, while IL-1 cytokines primarily enhance host resistance to coinfection by preventing AM depletion, IFN-γ impairs antibacterial defense by disrupting AM antibacterial function. Collectively, our data establish that dysregulation of alveolar macrophage function is the central pathogenic mechanism of influenza-induced susceptibility to secondary pneumococcal infection.
Murine models of Pneumocystis infection recapitulate human primary immune disorders
Authors: Elsegeiny Waleed Abdel Wahab , Zheng Mingquan , Eddens Taylor John , Alcorn John F , Chen Kong , Kolls Jay
Published: January 2023

Despite the discovery of key pattern recognition receptors and CD4+ T-cell subsets in laboratory mice, there is ongoing discussion of the value of murine models to reflect human disease. We recognize a strong parallel between human and mouse CD4+ T-cell immunity using the hallmark example of CD4+ T-cell dependent immunity, Pneumocystis pneumonia. Pneumocystis jirovecii infection was the first AIDS-defining illness and most closely tied with peripheral CD4+ T-cell counts. Due to successful control of HIV, the epidemiology of Pneumocystis infection in children is now primarily due to primary human immunodeficiencies and immunosuppressive therapies. To this end, we found that every human genetic immunodeficiency associated with Pneumocystis infection, recapitulated susceptibility in Pneumocystis infection in mice bearing similar mutations. Similar to human IL-21R deficiency, we found that IL-21R and CD4+ T-cell STAT3 signaling were required for both protective anti-fungal class-switched antibody responses and CD4+ effector T-cell mediated protection. Furthermore, we discerned that CD4+ T-cell intrinsic IL-21R/STAT3 signaling was required for inducing a Pneumocystis-specific IL-22 response, and that IL-22 inhibited trophozoite binding to alveolar epithelial cells. Moreover, recombinant IL-22 administration to Il21r−/− mice induced the expression of a fungicidal peptide, cathelicidin anti-microbial peptide which showed in vitro fungicidal activity. In conclusion, SPF laboratory mice faithfully replicate many aspects of human primary immunodeficiency and provide useful tools to understand the generation and nature of effector CD4+ T-cell immunity.
2,3,7,8-tetrachlorodibenzo-p-dioxin induced gut dysbiosis modulates delayed type hypersensitivity reaction
Authors: Abdulla Osama Azeldeen , Nagarkatti Prakash S , Nagarkatti Mitzi
Published: January 2023

Both gut microbiota and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been shown to have an impact on immune response and disease development. The present study investigates how TCDD alters gut microbiota, thereby inducing immunosuppression in delayed type hypersensitivity reaction (DTHR). C57BL/6 mice were sensitized with methylated bovine serum albumen (mBSA) and rechallenged after six days with mBSA. On day 5, one group received intraperitoneally TCDD (10μg/kg) and the other group received vehicle. On day 8, all groups were sacrificed. TCDD treated mice showed significant reduction in the DTHR in comparison with the vehicle group. Furthermore, we found an increase in the T regulatory cells (Tregs) in draining lymph nodes of TCDD-treated mice, and in the expression of TGFβ. Moreover, cecal flushes were collected and 16S rRNA sequencing was performed. We determined alpha and beta diversity. We found that at the level of phylum, there was a significant decrease in Bacteroidetes and a significant increase in Firmicutes in TCDD-treated mice in comparison with vehicle group. Interestingly, TCDD-treated group showed significant increase in members of the class Clostridia, which is known to maintain the immune homeostasis by inducing T-regs. Furthermore, we found a significant decrease in the species, Prevotella in TCDD-treated group in comparison with vehicle-treated group. It is known that decreased abundance of Prevotella in the gut correlates with reduction in inflammation in the joints. Together, TCDD treatment alters gut microbiota which may be responsible for amelioration from inflammatory diseases.
Inhaled silica induces autoimmunity in a strain-dependent manner
Authors: Clark Amy , Zhao Emma J. , Birukova Anastasiya , Buckley Elizabeth S. , Ord Jeffrey R. , Asfaw Yohannes G. , Tighe Robert M. , Foster Mary H.
Published: January 2023

Background

Respiratory exposure to crystalline silica in man is associated with autoimmunity that includes a prominent humoral component. We hypothesize that autoimmune B cell recruitment and regulation are altered in silica-exposed lungs in genetically susceptible individuals.

Methods

Wildtype and autoAb transgenic (Tg+) mice of B6 and lupus-prone NZB, MRL, and BXSB backgrounds were exposed to silica dust or vehicle by oropharyngeal aspiration. After 1–3 months, lung pathology and lymphocytic infiltrates were scored. Lupus and vasculitis autoAb levels were measured in bronchoalveolar lavage fluid (BALF), serum, and supernatants from cultured lung and spleen cells.

Results

All silica-exposed strains showed extensive lung pathology and focal B and T cell infiltrates consistent with ectopic lymphoid structures (ELS). In wildtype mice, % lung area containing ELS after silica exposure varied by strain (BXSB>MRL>B6>NZB, p=0.03). Significant increases in autoAb production with silica exposure were observed in BALF, lung cell supernatants, and serum, and varied by genetic background. Among Tg+ mice, Tg+ Ig levels in BALF were higher in silica- vs vehicle-exposed mice in B6 and BXSB strains (p<0.05).

Conclusions

Respiratory exposure to silica leads to pulmonary B and T cell accumulation with ELS formation and enhanced autoAb production. This occurs in a strain-dependent manner, highlighting the impact of genetic influences on silica-induced autoimmunity. Evidence of local autoAb secretion implicates dysregulated control of autoreactive B cells at the environment/lung interface, possibly through the intermediary of silica-induced ELS.
Exposure to bacterial Curli triggers antibody production and type I interferon activation in lupus patients
Authors: Pachucki Ryan J , Corradetti Chelsea , Kohler Lynne , Tursi Sarah , Gallucci Stefania , Kilpatrick Laurie , Tukel Cagla , Caricchio Roberto
Published: January 2023

Infections are a major contributor to lupus disease. We have previously demonstrated that bacterial amyloid curli, produced by E.coli, can accelerate disease in mouse models of lupus. Interestingly curli incorporates extracellular DNA, which in turn can be both adjuvant and a self-antigen in lupus. Finally, uropathogenic E. coli (UPEC) is responsible for the majority of urinary tract infections in SLE.

Based on our previous results, we hypothesize that exposure to UPEC triggers anti-curli antibodies, and that curli can also trigger the innate immune system.

We investigated 34 lupus patients who met at least 4 SLICC criteria. Results were compared to 17 age, sex and race matched healthy controls. We tested the production of anti-curli/DNA complex for both IgG and IgA subclasses. We than correlated the levels of anti-curli/DNA antibodies with clinical parameters, including anti-dsDNA antibodies and bacteriuria. Finally, we used curli/DNA complexes to stimulate PBMCs from lupus patients and controls and determined the levels by rt-PCR of IL-6 (bacterial response) and MX-1 (type I interferon (IFN I) activation). We found that lupus patients and healthy controls generated anti-curli antibodies of IgG and IgA subclasses. The levels of anti-curli antibodies significantly correlated with the levels of anti-dsDNA antibodies (p=0.029) and persistent bacteriuria (p=0.035) in lupus patients. Finally we found that curli/DNA complexes triggered production of IL-6 and MX-1 in both healthy and lupus PBMCs. Nevertheless, the expression of MX-1 was significantly higher in PBMCs from lupus patients (p=0.012). Our results show that bacterial curli/DNA is a trigger of systemic autoimmunity via activation of IFN I and production of autoantibodies.
Prostaglandin I2 -orchestrates regulatory and effector T cell–mediated autoimmunity
Authors: Bloodworth Melissa Harintho , Boyd Kelli L. , Rogers Lisa M. , Aronoff David M , Peebles R. Stokes
Published: January 2023

Tregs maintain tolerance and prevent autoimmune disease by inhibiting T cell activation. There is extensive interest in the application of Tregs in transplantation, autoimmune diseases, and allergy. Our group and others have shown that prostacyclin I2 (PGI2) signaling through the I prostanoid (IP) receptor inhibits allergic airway inflammation. However, it remains unknown whether PGI2 regulates Tregs and Treg-mediated inflammation. We hypothesized that PGI2 skews the immunological balance towards tolerance and away from inflammation. To determine whether PGI2 mediates systemic tolerance, we performed whole animal histopathology on aged WT and IP KO mice. PGI2 protected against autoimmune manifestations including splenomegaly, perivascular-bronchiolar lymphocytic cuffing, and epicarditis that developed in aged IP KO mice. Endogenous PGI2 also promoted Treg Foxp3 expression, suppressive function, and polarization. PGI2 concurrently decreased Teff proliferation and increased Teff susceptibility to Treg-mediated suppression. These studies are the first to show that PGI2 protects against autoimmunity and may represent a novel treatment strategy for Treg-mediated diseases.
Induction of antigen-specific tolerance with Hybrid Insulin Peptides in the NOD mouse model of autoimmune diabetes
Authors: Jamison Braxton , Neef Tobias , Miller Stephen D , Haskins Kathryn
Published: January 2023

In Type 1 Diabetes (T1D), autoreactive CD4 T cells are important contributors to the inflammatory infiltrate in the pancreatic islets which leads to the destruction of insulin-producing β-cells. We recently found that CD4 T cell lines and clones isolated from non-obese diabetic (NOD) mice and T1D patients are strongly activated by novel peptide ligands which are hybrid insulin peptides (HIPs) consisting of proinsulin fragments fused to other peptides present in β-cell secretory granules. Our data suggest that HIPs play a central role in the pathogenesis of T1D and we hypothesize that loss of tolerance to the pancreatic islets involves HIP-reactive T cells. In this project we have investigated whether HIPs can be used for the induction of antigen-specific tolerance in NOD mice. The specific objective is to determine whether HIP-loaded biodegradable nanoparticles can prevent disease transfer by diabetogenic T cells into NOD mice. Our experimental system involves HIPs coupled to or encapsulated within poly(lactide-co-glycolide) (PLG) nanoparticles which are administered to NOD mice following injection of BDC-2.5 TCR transgenic T cells to trigger disease. Preliminary data from these experiments indicates that a hybrid between an insulin C-peptide sequence and a peptide from ChgA can completely block transfer of BDC-2.5 diabetogenic T cells. Long-term goals of this project are (1) to test the ability of nanoparticles containing other HIPs in preventing transfer of diabetogenic T cells and (2) to determine whether HIP-loaded nanoparticles are capable of reversing spontaneous disease in NOD mice. Results from these studies could have significant implications for the development of antigen-specific therapy in T1D.
HYPERACTIVATION OF PI3 KINASE CAUSES DEFECTS IN HUMAN B-CELL DEVELOPMENT AND DIFFERENTIATION
Authors: Tangye Stuart
Published: January 2023

Gain of function (GOF) mutations in PIK3CD, encoding the p110δ subunit of PI3-kinase, cause immune dysregulation characterized by recurrent respiratory infections, increased susceptibility to herpesvirus infection, lymphoma, organomegaly and autoimmunity. Affected individuals display poor Ab responses to protein and polysaccharide Ags suggesting defective humoral immune responses. To better understand humoral immune defects due to hyperactive PI3K, we analysed B cells in these individuals. In peripheral blood, transitional B cells were markedly increased while memory B cells were reduced. The phenotype of naïve B cells with PIK3CD GOF mutations resembled that of normal transitional B cells, indicating an aberrant arrest of developing B cells. Analysis of bone marrow revealed increased proportions of pro/pre-B cells and reduced recirculating mature B cells patients with PIK3CD GOF mutations. Within the memory B cell population there was a reduction in IgG+ cells. Consistent with this, there was a selective impairment in IgG production by transitional of naive B cells following in vitro stimulation. To extend these studies in vivo, we generated a mouse model using CRISPR/Cas9 to introduce the common disease-causing mutation (E1021K) into Pik3cd. Analysis of bone marrow from these mice revealed disrupted B-cell development. Investigation of Ag-specific T-dependent B cell responses using mice expressing a transgenic HEL-specific BCR combined with Pik3cd GOF allowed us to dissect defects in specific Ab generation that are seen in human PIK3CD GOF mutations. These results indicate that intrinsic defects in B-cell development and differentiation underlie poor humoral immune responses in individuals with PIK3CD GOF mutations.
Phenotypic Characterization of ILC2s in an EoE Mouse Model
Authors: Kinard Jessica L. , Laky Karen , Guerrerio Anthony , Frischmeyer-Guerrerio Pamela
Published: January 2023

Loeys-Dietz Syndrome (LDS) is a genetic disorder caused by mutations in genes essential to TGFβ signaling including TGFBRI, which encodes a TGFβ receptor subunit. LDS patients exhibit a high prevalence of allergic disease. A mouse model of LDS Type I (TGFBR1M318R+/−) has been developed to further study this disease and its associated allergic conditions including eosinophilic esophagitis (EoE). EoE is characterized by the abnormal influx of eosinophils into esophageal tissue, basal cell hyperplasia, and smooth muscle dysfunction. The molecular mechanisms underlying this disease are not well understood, but Th2 cytokines are thought to play an essential role. TGFBR1M318R+/− mice spontaneously develop EoE with 100% penetrance, thereby providing an ideal model to study this allergic condition. Remarkably, TGFBR1M318R+/− mice continue to develop EoE in the absence of lymphocytes, suggesting that Th2 effector cells are not essential for driving EoE development. Further investigation has revealed increased numbers of type two innate lymphoid cells (ILC2s) in the esophagus of TGFBR1M318R+/− mice compared to wild type (WT) littermates. These cells are lineage negative, ST2+, GATA3+, and produce large amounts of IL-5 and IL-13, and to a lesser extent IL-4, following in vitro stimulation. The differences in ILC2 frequency between TGFBR1M318R+/− and WT mice appears around 10 days of age, while striking differences start to occur around 19 days, concurrently when ILC2 numbers peak in the normal esophagus. These large differences are sustained throughout the mouse’s lifespan, indicating that these cells may play a critical role in perpetuation of this disease. These findings support a novel role for ILC2s in driving EoE pathogenesis.
Signature Gene Expression Patterns Revealed in Hypoplastic Thymii from Mouse Models of DiGeorge-22q11.2 Deletion Syndrome
Authors: van Oers Nicolai S , Du Qiumei , de la Morena M Teresa , Dozmorov Igor , Khan Shaheen , Cleaver Ondine B
Published: January 2023

Chromosome 22q11.2 deletion syndrome (22q11.2 ΔS) is the most common microdeletion disorder reported (1/4000). Individuals with this deletion have variable, multi-system disorders including thymic hypoplasia, cardiac anomalies, hypoparathyroidism, and/or dysmorphic facial features. Over 90% of patients have a deletion of 2.4 Mb, which comprises 90 genes, 50% protein coding and the remainder microRNAs, long noncoding RNAs and pseudogenes. The principal cause of the development defects is a haploinsufficiency of the T-box 1 transcription factor (Tbx1). Between 40–60% of patients have some degree of thymic hypoplasia, resulting in systemic T cell lymphopenia. Defects in the thymic stromal tissue is the underlying cause of the hypoplasia. We analyzed the thymic tissue in the mouse models of 22q11.2 ΔS (Df1/+). Comparative transcriptome analyses of hypoplastic and normal-sized lobes derived from the same Df1/+ embryo revealed a signature mRNA expression pattern unique to hypoplastic lobes. Ingenuity pathway analysis uncovered selective pathways compromised in the hypoplastic lobes. Fetal thymic organ culture and reaggregate cultures are currently being used to identify the genes essential for the specification and expansion of the thymic stroma. In addition, normal and hypoplastic thymii, including some from 22q11.2 ΔS patients, are being characterized for the expression of the over- and under-represented genes identified in the mouse model. Findings from these studies may lead to better strategies for improving human thymopoiesis in patients with conditions including 22q11.2 and 10p syndromes, those undergoing chemoablative treatments, and other conditions leading to a thymic hypoplasia and ensuing T cell lymphopenia.
A delicate balance of PI3K activation is required for CD8 T cell survival and generation of memory cells
Authors: Cannons Jennifer Leigh , Kapnick Senta , Preite Silvia , Gomez-Rodriguez Julio , Reilley Julie , Handon Robin , Wu Tuoqi , Uzel Gulbu , Schwartzberg Pamela L
Published: January 2023

The p110d subunit of phosphatidylinositol-3-OH kinase (PI(3)K) is selectively expressed in leukocytes and is critical for lymphocyte biology. We previously reported patients heterozygous for three different germline, gain-of-function mutations in PIK3CD (encoding p110d). Patients presented with sinopulmonary infections, lymphadenopathy, nodular lymphoid hyperplasia and CMV and/or EBV viremia, suggestive of immune dysfunction. We established a mouse model (PIK3cd E1024K) that recapitulates features of this disease including lymphopenia, yet enlarged secondary lymphoid organs, as well as increased Akt phosphorylation and hyperactivation of the metabolic checkpoint kinase mTOR. In response to TCR stimulation, mutant T cells proliferate, but rapidly die via a Fas/FasL-mediated apoptosis. The few surviving mutant CD8 T cells exhibit features of short-lived effectors, expressing abundant cytokines and elevated granzymes, and kill target cells more efficiently than WT counterparts. This phenotype is mirrored at early time points following viral infection. However, at late time-points post-viral infection, mutant CD8 T cells fail to sustain expression of proteins critical for maintenance of long-lived memory cells. Thus, our data suggests that activated p110d limits effective CD8 T cell memory. Our mouse model therefore recapitulates facets of the human primary immunodeficiency, revealing insight into the pathogenesis of this disease.
Patients with Compound Heterozygous Mutations in Forkhead Box N1 have a Severe Immunodeficiency while Maintaining Normal Skin and Hair Development
Authors: van Oers Nicolai S , Khan Shaheen , Padron Grace T , Du Qiumei , Dozmorov Igor , Markert M. Louise , de la Morena M Teresa
Published: January 2023

Forkhead Box N1 (FoxN1) is an epithelial-specific transcription factor essential for the development of the thymus. Patients with mutations in Foxn1 (OMIM # 600838) are born with a severe T-cell lymphopenia that presents in combination with alopecia and nail dystrophy. The nude mouse, which developed from a spontaneous genetic mutation in Foxn1, phenocopies the human disease. We report on 3 independently identified patients that presented with low to absent circulating T cells. Genetic workup of these patients revealed mutations in Foxn1. Each patient had distinct compound heterozygous mutations that were localized in the distal exons of Foxn1. These were predicted to maintain Foxn1 expression while adversely affect its function. Of significance, each patient had normal hair and skin, without any evidence of nail dystrophy, distinct from previously reported phenotypes. To better define the molecular mechanisms leading to this novel clinical presentation, we used CRISPR/Cas techniques to introduce the corresponding mutations in the mouse Foxn1 sequence. We will present data on the phenotypes of these mice, using intercrosses between individual mutant mice. Comparative transcriptome analyses of fetal thymii from these mice will reveal how the Foxn1 mutations impacts thymic epithelial gene expression and function compared to normal Foxn1. Our findings may lead to better understanding of the role of Foxn1 epithelial cell development and function in both the thymus and skin.
A Novel Mendelian Disease of Autoimmunity caused by Mutations in a small GTPase
Authors: Leney-Greene Michael Alexander , Park Ann , Chao Brittany , Schwefel David , Balasubramaniyam Arasu , Ozen Ahmet , Vilarinho Silvia , Morawski Aaron , Daumke Oliver , Su Helen , Lenardo Michael J
Published: January 2023

In this study, we harness Whole Exome Sequencing (WES) to interrogate the underlying genetic cause in a patient cohort suffering from a Mendelian disease of immune dysregulation characterized by severe lymphopenia, splenomegaly, anemia, thrombocytopenia and liver failure. Bioinformatic analysis revealed novel mutations in a small GTPase that led to a near complete loss of mature protein in patient cells. Animal models lacking these genes develop a disease remarkably similar to that observed in the human patients, however, the molecular role of this GTPase in the immune system remains a fundamental gap in our knowledge. Due to a remarkable similarity in phenotypes between mice deficient for this small GTPase and T cell specific deletions of regulators of mTORC1, we hypothesized that this gene may be involved in the mTORC1 signaling pathway. We observe a robust association between this GTPase and members of the ragulator complex which is required for recruitment of mTORC1 to the lysosome and subsequent signalling. Furthermore, in human T cells deficient for this GTPase we observe increased phosphorylation of targets downstream of mTORC1. Overall, our data suggest that this GTPase is a novel T cell specific negative regulator of mTORC1 signalling.

This research was supported by the Intramural Research Program of NIAID, NIH.
EBI2 contributes to central tolerance by promoting rapid motility of medullary thymocytes
Authors: Thyagarajan Hiran Maye , Ki Sanghee , Hu Zicheng , Lancaster Jessica , Ehrlich Lauren
Published: January 2023

Maturing thymocytes enter the thymic medulla, where they encounter numerous self-antigens presented by antigen presenting cells (APC). Those thymocytes that are strongly self-reactive undergo either negative selection or diversion into the regulatory T cell lineage. Although the majority of the proteome is expressed in the medulla, many self-antigens are expressed by only a minor fraction of medullary APC; thus, thymocytes must efficiently enter the medulla and scan APC to ensure central tolerance. Chemokine receptors promote lymphocyte migration, organization within tissues, and interactions with APC in lymphoid organs. The chemokine receptor EBI2 governs the localization of T cells, B cells, and dendritic cells (DC) during immune responses in secondary lymphoid organs. However, the role of EBI2 in thymocyte development has not been elucidated. Here, we demonstrate that EBI2 is expressed by murine CD4+ single positive (CD4SP) thymocytes and thymic DC. EBI2 deficiency did not impact thymocyte cellularity or subset distribution, but did alter the TCR repertoire. Furthermore, EBI2 deficiency impaired negative selection of OT-II TCR transgenic thymocytes responding to an endogenous self-antigen. Two-photon imaging revealed that EBI2 deficiency resulted in reduced migration and impaired medullary accumulation of CD4SP thymocytes. These data identify a role for EBI2 in promoting efficient central tolerance.
Deletion of the Dense Core Vesicle Proteins IA-2 and IA-2β Causes Profound Leukopenia in Mice
Authors: Gunti Sreenivasulu , Gottumukkala Raju VSRK , Carmona Gilberto N , Notkins Abner Louis
Published: January 2023

Previous studies showed that deletion of the genes encoding the dense core vesicle (DCV)-associated proteins IA-2 and/or IA-2β resulted in a decrease in the secretion of a number of hormones and neurotransmitters and that this, in turn, led to a variety of pathophysiologic and behavioral abnormalities. The effect of the deletion of these genes on the immune system has never been studied. In the present experiments, we found that the knockout of the IA-2 and IA-2β genes (double knockout) resulted in a profound leukopenia characterized by a 78% decrease in WBCs and an 84% decrease in absolute number of lymphocytes. The leukopenia was detected within 1.5 months after birth and persisted for at least 12 months. Both B and T cells were decreased by about 40–50% in bone marrow and thymus and by about 75% in the spleen. LPS-induced cytokine secretion (e.g., IL-6, MCP-1, INF-γ and TNF-α) also was severely impaired. The most likely explanation for these profound immunological abnormalities in single and double knockout mice is that they represent secondary events resulting from impaired secretion of hormones and neurotransmitters that are involved in immunological homeostasis.
On the mechanism of leukocytosis in patient WHIM-09, cured of WHIM syndrome by chromothripsis
Authors: Liu Qian , Li Zhanzhuo , Yang Alexander , Velez Daniel , Stregevsky Elina , Cho Elena , Holmes Kevin , McDermott David H. , Murphy Philip M.
Published: January 2023

Patient WHIM-09 was cured of WHIM syndrome, an autosomal dominant immunodeficiency disorder caused by gain-of-function CXCR4 mutations, by chromothripsis (chromosome shattering) in a single hematopoietic stem cell (HSC). Chromothripsis fortuitously deleted CXCR4WHIM, and one copy of 163 other genes, resulting in hematopoietic chimerism (100% chromothriptic myeloid cells and 100% WHIM lymphoid cells) and overcorrection of leukopenia in neutrophil and monocyte, but not lymphocyte, lineages to levels stably ~2-fold above normal. Earlier, splenectomy had increased the absolute lymphocyte count stably by ~2-fold. To address leukocytosis mechanisms, we analyzed animal models and patient samples. The total WBC count was increased by splenectomy in wild-type mice, and by Cxcr4 haploinsufficiency in sham or splenectomized mice, due to increases in lymphocytes and monocytes, not neutrophils. Cxcr4 haploinsufficiency also increased mouse bone marrow LSK cell proliferation and reduced neutrophil homing from blood to bone marrow. In the patient, the frequency, absolute number and myeloid colony-forming capacity of blood CD34+ cells were normal; however, bone marrow GMPs generated increased G/M/GM-CFU colonies in vitro. WHIM-09 serum contained increased levels of Groa, MIP-1d, MIG, IL-8 and IL-15, and decreased levels of CXCR4’s ligand SDF-1, which may regulate leukocyte levels. Thus, in WHIM-09 splenectomy and CXCR4 haploinsufficiency may have contributed to monocytosis; whereas HSC hyperproliferation, enhanced differentiation capacity of GMPs, impaired neutrophil homing to bone marrow and skewed serum cytokine levels may all contribute to aspects of leukocytosis.