bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-35 is essential for protection against collagen-induced arthritis (CIA) following intervention with Escherichia coli colonization factor antigen I (CFA/I) fimbriae (116.2)
Authors: Kochetkova Irina , Golden Sarah , Holderness Kathryn , Pascual David
Published: January 2023

Mucosal intervention with CFA/I fimbriae effectively diminishes CIA by stimulation of TGF-β+ and IL-10+ regulatory CD39+CD4+ T cells (Tregs), resulting in suppression of Th1 and Th17 cells and IL-27. CFA/I also stimulates Tregs expressing p35 and EBI3 subunits simultaneously, suggesting an essential role of IL-35 in CFA/I-mediated protection. We hypothesize CFA/I fimbriae stimulate endogenous IL-35 production to facilitate protection against autoimmune disease. In testing the relevance of endogenous IL-35 for protection, oral delivery of recombinant CFA/I into EBI3-/- mice on day 14 after CIA induction was found ineffective against CIA compared to similarly treated WT mice, as evidenced by the lack of suppression of IL-17 and IFN-γ (the latter increased 5-fold ). CFA/I-treated EBI3-/- mice lacked enhanced CREB phosphorylation that, in turn, failed to stimulate the transcription of CD39 (apyrase). In fact, surface apyrase activity by EBI3-/- CD4+ T cells was uninduced. Because of the lack of induced CD39 expression, IL-10 production was greatly diminished. Clinical improvement of CIA in CFA/I-treated EBI3-/- mice required further intervention with recombinant IL-35, resulting in the induction of CD39+CD4+ T cells, enhanced TGF-β production, and reduction in IFN-γ and IL-17. Thus, in the absence of endogenous IL-35, mucosal CFA/I fimbriae intervention is ineffective against autoimmune arthritis. Supported by AT-004986.
Neutrophil-derived IL-1β is required for abscess formation in immunity against Staphylococcus aureus (118.2)
Authors: Cho John , Guo Yi , Hebroni Frank , Ramos Romela , Plasier Seema , Matsushima Hironori , Takashima Akira , Iwakura Yoichiro , Cheung Ambrose , Cheng Genhong , Lee Delphine , Simon Scott , Miller Lloyd
Published: January 2023

Neutrophils have historically been viewed as phagocytes involved in direct killing of pathogens. Here, the mechanism of neutrophil recruitment and abscess formation during an infection was investigated by using a mouse model of Staphylococcus aureus cutaneous infection. Global gene expression analysis of S. aureus-infected skin of wild-type and IL-1R-deficient mice demonstrated that IL-1R activation was crucial for the upregulation of genes involved in neutrophil recruitment. IL-1β triggered IL-1R activation in this model and using IL-1β-reporter mice line, we surprisingly found that neutrophils were the predominant IL-1β-producing cells at the site of infection. S. aureus-induced IL-1β production by mouse and human neutrophils was mediated by the pattern recognition receptors, TLR2 and NOD2, and the ASC/NLRP3 inflammasome. Importantly, IL-1β-expressing neutrophils were essential for abscess formation and bacterial clearance since adoptive transfer of wild-type, but not IL-1β-deficient neutrophils, rescued the immune defects in IL-1β-deficient mice. Taken together, this study defines a new mechanism for abscess formation in which neutrophil-derived IL-1β, induced by TLR2, NOD2 and inflammasome activation, is required for effective neutrophil recruitment and host defense during a S. aureus skin infection.
Effects of anti-CD52 antibody treatment on immune cell depletion and progression of disease in murine systemic lupus erythematosus (119.13)
Authors: Boutin Paula , Severy Peter , Roberts Bruce , Kaplan Johanne , Rao Sambasiva
Published: January 2023

CD52 is expressed at high levels on lymphocytes and to a lesser extent on innate immune cells in humans and mice. Given this expression pattern, we investigated the effect of an anti-mouse CD52 antibody on immune cell depletion and potential therapeutic benefit in lupus prone NZB/NZW-F1 mice. 27 week old mice received either one or two courses of 25mg/kg anti CD52 antibody, three times a week for 2 weeks. Control mice received PBS or a control mouse IgG2a antibody. Anti-CD52 treatment resulted in a significant depletion of nearly 90% of blood lymphocytes. Repopulation kinetics in blood showed that B and T lymphocytes returned to baseline levels within two months post anti-CD52 treatment. Anti-nuclear antibodies decreased transiently but significantly one month post treatment. Proteinuria levels were significantly decreased in the anti CD52 treated group and remained low compared to the control group until 49 weeks of age suggesting a marked improvement in kidney function. Anti-CD52 treatment also provided a significant survival benefit with more than 80%of mice in the treated group surviving at 49 weeks of age compared to only 10% in the control group. These data provide evidence for a beneficial effect of anti-CD52 treatment on disease progression in NZB/NZW-F1 mice and suggest a potential application to human SLE.
Short-term helminth infection inhibits type 1 diabetes independently of the Th2-type response (119.5)
Authors: Mishra Pankaj , Patel Nirav , Wu Wenhui , Bleich David , Gause William
Published: January 2023

Helminth infection can control a number of autoimmune inflammatory diseases in experimental models including type 1 diabetes (T1D).The molecular mechanisms of heminth mediated control of T1D are not yet determined,but it is generally thought that the associated immunoregulation is caused by the potent and highly polarized Th2-type response driven by IL-4.In these studies, nonobese diabetic (NOD) IL-4-/- mice were infected with a strictly enteric nematode parasite.Remarkably,we showed that the helminth-induced control of T1D remained intact. Elevations in CD4+ T cell STAT6 phosphorylation were inhibited,while suppression of STAT1 phosphorylation was sustained,as were increases in FOXP3-,CD4+T cell IL-10 production.Furthermore, protection still occurred if parasites were expulsed with an anti-helminth Rx after only two weeks infection.Oral antibiotic treatment did not effect helminth-mediated protection indicating that intestinal microbiota did not play a major role.Blockade of IL-10 signaling in NOD-IL-4-/-,but not NOD mice,during this short interval abrogated protective effects resulting in pancreatic β-cell destruction and T1D development.These studies indicate that helminth infection triggers multiple immune regulatory components that are induced and can act independently.Their combined effects help explain the potency of helminth-mediated control of pathologic inflammation and suggest future strategies for helminth-mediated therapeutic treatment of this autoimmune disease.
Interleukin 4 increases the production of indduced regulatory T cells in C57BL/6 mice (120.12)
Authors: Haribhai Dipica , Schmitt Erica , Ziegelbauer Jennifer , Upchurch Kyle , Salzman Nita , Williams Calvin
Published: January 2023

Naïve peripheral T cells can express the transcription factor Foxp3 and develop regulatory function. These “induced” regulatory T (iTreg) cells are essential for tolerance and complement the function of other regulatory populations. In this study we used the lymphopenia-associated model of colitis to compare the kinetics of iTreg cell production in both C57BL/6 and BALB/c mice, the prototypical Th1- and Th2-type strains. Following the adoptive transfer of naïve CD4+ T cells into Rag-/- recipients, we tracked the development of iTreg cells over a period of 40 days. Whereas iTreg cell production in C57BL/6 was minimal, by 10 days there was a relative five-fold increase in the number of iTreg cells in BALB/c mice that grew to 35 fold by 40 days. This dramatic increase in iTreg cells correlated with a similar increase in IL-17A producing cells in the mesenteric lymph nodes and gut of BALB/c mice. In contrast, the proportion of CD4+ T cells that produced IFN-γ was comparable between the two strains. Pretreatment of C57BL/6 Rag-/- recipient mice with IL-4 resulted in a six-fold increase in the number of iTreg cells compared to untreated mice. Pretreatment with in vitro derived M2 macrophages also resulted in a five to fifteen fold increase in the number of iTreg cells compared to pretreated mice with M0 or M1 macrophages, respectively. These results indicate that a Th2 milieu supports iTreg cell induction.
The role of Ndfip1 in CD8+ T cell activation and effector function (121.25)
Authors: Kurzweil Vanessa , O'Leary Claire , Oliver Paula
Published: January 2023

Nedd4-family interacting protein 1 (Ndfip1) is an adapter protein for Nedd4-family E3 ubiquitin ligases. Mice lacking Ndfip1 in both CD4+ and CD8+ T cells develop a severe atopic inflammatory disease at sites of environmental exposure and die prematurely. In part, this is because Ndfip1 expression in CD4+ T cells is needed to inhibit IL-4 production and permit differentiation into non-Th2 lineages. However, Ndfip1-/- mice that lack MHC Class II-restricted CD4+ T cells still develop inflammation at a young age. This suggests that Ndfip1 might also be important in CD8+ T cells. We show that Ndfip1-/- CD8+ T cells are more sensitive to TCR stimulation than WT cells. After activation, Ndfip1-/- CD8+ T cells produce more IFN-γ than their WT counterparts. Consistent with this, Ndfip1-/- CD8+ T cells have higher levels of Eomesodermin than control cells. Future studies will attempt to identify E3 ligases and substrates that regulate these CD8+ T cell functions.
Mimicry epitope from Ehrlichia canis for interphotoreceptor retinoid-binding protein ameliorates autoimmune uveoretinitis by acting as altered peptide ligand in A/J mice (123.29)
Authors: Gangaplara Arunakumar , Massilamany Chandirasegaran , Madayiputhiya Nadakumar , Nandakumar Renu , Kuszynski Charles , Steffen David , Reddy Jay
Published: January 2023

Uveoretinitis is one of the sight-threatening inflammatory diseases of eyes, and autoimmune response to retinal antigens is suspected in the causation of disease. Although environmental triggers are suspected, their association remains tenuous and clinically not proven. We report here identification of novel mimicry epitopes for interphotoreceptor retinoid-binding protein (IRBP) 201-216 and they represent Ehrlichia canis (EHC), Pseudomonas putida, Trypanosoma cruzi, Chlamydia trachomatis, Clostridium novyi, Hepatitis C virus and Paracoccidoides brasiliensis. We demonstrate that one mimic from E. canis (EHC 44-59) although can generate cross-reactive T cells for IRBP 201-216, capable of producing T helper (Th)1 and Th17 cytokines, fail to induce autoimmune uveoretinitis by active immunization in A/J mice. Likewise, the animals previously immunized with IRBP 201-216 and subsequently challenged with EHC 44-59 fail to show the disease. The data suggest that exposure to microbial products containing the mimicry sequences for retinal antigens does not necessarily leads to the development of eye autoimmunity rather they can behave as natural altered peptide ligands.
Regulation of selection and autoimmunity by miR-181 family miRNAs (123.36)
Authors: Schaffert Steven , Wang Song , Axtell Robert , Newell Evan , Steinman Lawrence , Davis Mark , Chen Chang-Zheng
Published: January 2023

T cell sensitivity to antigens is regulated during development to ensure proper immunity and tolerance. We previously showed that miR-181a regulates the strength and threshold of T cell receptor signaling by targeting negative feedback molecules. Here we used miR-181 knockout mice to examine the role of miR-181 miRNAs in selection and T cell reactivity to antigens. We found that loss of mir-181ab1 caused an increase in the percent of positively selected CD8 single-positive (SP) cells in the thymi of female HY TCR transgenic mice. However, loss of miR-181 had no effect on the deletion CD8 SP cells in the thymi of male HY mice. Moreover, we showed that loss of mir-181ab1 resulted in two-fold more self-reactive T cells in the periphery after immunization with a self-peptide derived from myelin oligodendrocyte glycoprotein (MOG). Paradoxically, knockout mice exhibited a delayed disease onset in a MOG-dependent mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). In particular, the central nervous systems of knockout mice during disease had similar percentages of MOG-specific T cells, but an underrepresentation of TH17 and T H1-polarized CD4 T cells compared to the periphery. These findings demonstrate that mir-181ab1 also plays a role in modulating the differentiation and function of self-reactive T cells. Together, data from our study illustrate the diverse roles of mir-181ab1 in selection and autoimmunity.
Inhibition of p300 impairs Foxp3+ T-regulatory (Treg) function and promotes anti-tumor immunity (127.40)
Authors: Liu Yujie , Wang Liqing , Han Rongxiang , Predian Jarrod , Beier Ulf , Cole Philip , Albelda Steven , Wayne Wayne
Published: January 2023

Foxp3+ Treg cells limit autoimmunity and maintain immune homeostasis but also restrict immune responses to cancer. Thus, Tregs are a promising target for modulation of host anti-tumor immune responses. We have shown that targeting of Foxp3+ Tregs using histone/protein deacetylase inhibitors (HDACi) promotes Foxp3 acetylation and increases Treg production and suppression function. By contrast, there are no studies concerning the role of histone/protein acetyltransferases (HAT) targeting in Tregs. HATs are classified into 3 main families; GNAT, MYST and p300/CBP. Here, we found that conditional deletion of p300 in Tregs caused lymphoproliferation, weight loss, arthritis and dermatitis in mice. Likewise, deletion of p300 in Tregs, or its pharmacologic inhibition, decreased Foxp3+ Treg production and impaired Treg-mediated suppression of homeostatic proliferation, and blocked Treg-dependent donor-specific allograft tolerance. In contrast with these data, p300 targeting decreased TC1 lung tumor volume in immunocompetent C57BL/6 mice, but had no direct effect on TC1 tumor cell proliferation in vitro, or on the growth of TC1 implanted tumors in RAG1-/- immunodeficient mice. We conclude that in real world tumor models using immunocompetent mice, p300 targeting impaired Treg function without overt impairing effector T cell responses or inducing autoimmunity. We thereby identify p300 as a critical target for modulation of Foxp3+ Treg function in the context of anticancer therapy.
MARCKS protein plays a critical role in proinflammatory cytokine production (160.7)
Authors: Nordone Shila , Li Jingjing , D’Annibale Melissa , Birkenheuer Adam , Levy Mike , Yoder Jeff , Jones Sam , Adler Ken
Published: January 2023

Myristoylated alanine-rich C kinase substrate (MARCKS) is a ubiquitously expressed Protein Kinase C substrate that has emerged as a potential therapeutic target for amelioration of mucin secretion during chronic obstructive pulmonary disease. MARCKS also plays a key role in regulation of neutrophil adhesion, migration and degranulation. Given its biological role in immune cells, we hypothesized that MARCKS may play an integral role in cytokine secretion. MARCKS protein is highly conserved between the dog and human, and importantly, the N-terminal 24 amino acids are virtually identical between species, allowing us to use the well-characterized MANS peptide to inhibit canine MARCKS function through the N-terminus. Using isolated neutrophils, we evaluated the effect of MARCKS inhibition on LPS-induced cytokine production. We found that pre-treatment of canine neutrophils with MANS peptide significantly reduced the secretion of a broad range of LPS-induced cytokines, including IL-8, CXCL1 and TNFα, in comparison to untreated cells or those treated with a scrambled control peptide. This reduction in IL-8 was maintained when MANS was administered 2 hours after LPS-treatment of cells. Reduction in IL-8 secretion was found not to be due to protein retention or cell death. Our observations identify MARCKS protein as a promising therapeutic target in treatment of inflammatory disease, particularly those syndromes attributed to neutrophil influx and inflammatory cytokine production.
Anti-IL-20 monoclonal antibody suppresses breast cancer progression and bone osteolysis in murine models (161.10)
Authors: Hsu Yu-Hsiang , Chang Ming-Shi
Published: January 2023

IL-20 is a proinflammatory cytokine involved in rheumatoid arthritis and osteoporosis. However, little is known about the role of IL-20 in breast cancer. We explored the function of IL-20 in tumor growth and metastasis, and its clinical outcome. Tumor expression of IL-20 was assessed by IHC staining among 198 breast invasive ductal carcinoma (IDC) patients with available clinical and survival data. IL-20 expression was associated with advanced tumor stage, high tumor metastasis, and worse survival. RTQ-PCR showed that clinical breast tumor tissue expressed higher IL-20 and its receptors than did non-tumorous breast tissue. IL-20 was also highly expressed in breast cancer bone-metastasis tissue. In vitro, IL-20 upregulated matrix metalloproteinase-9, matrix metalloproteinase-12, cathepsin K, and cathepsin G, and enhanced proliferation and migration of breast cancer cells, which were inhibited by anti-IL-20 monoclonal antibody (mAb) 7E. In vivo, 7E reduced tumor growth, suppressed bone colonization, reduced tumor-mediated osteolysis, and lessened bone density decrement in mice injected with breast cancer cells. In conclusion, our results suggest that IL-20 plays pivotal roles in the tumor progression of breast cancer. IL-20 expression in breast cancer tissue is associated with a poor clinical outcome. Anti-IL-20 mAb 7E suppressed bone colonization, and decreased osteolytic bone lesions. Therefore, IL-20 may be a novel target in treating breast tumor-induced osteolysis.
Immune-mediated cure of established B16F10 melanoma with a nonvirulent strain of Toxoplasma gondii (162.12)
Authors: Fiering Steven , Baird Jay , Byrne Katelyn , Lizotte Patrick , Mullins David , Turk Mary Jo
Published: January 2023

Immune recognition and elimination of tumors protects us from cancer. Tumors frequently manifest immunosuppressive mechanisms to avoid anti-tumor immunity and thereby become clinical challenges. The immune system is quite sensitive to microbial pathogens and microbes or microbial extracts are very effective adjuvants to stimulate an immune response. Here we present the results of treatment of established cutaneous B16F10 murine melanoma by intratumoral injection of a fully attenuated uracil auxotroph strain of Toxoplasma gondii (ΔCPS) that cannot replicate in vivo. ΔCPS exposure breaks immunosuppression and stimulates a strong Th1 antitumor immune response in vivo that cures established primary melanoma and develops immune memory that confers protection against subsequent rechallenges. This treatment modifies the tumor microenvironment, increases MHC Class I expression on tumor cells and reverses immunosuppression, permitting a tumor-antigen specific CD8 T cell response to eliminate the tumor. The approach has efficacy against multiple tumor types and has potential for clinical utility.
Myeloid derived suppressor cells(MDSCs) emergence from distinct splenic precursors (162.28)
Authors: Park Young-Jun , Song Boyeong , Kim Yun-Sun , Kim Eun-Kyung , Lee Jung-Mi , Lee Ga-Eun , Kim Jae-Ouk , Kim Yeon-Jeong , Chang Woo-Sung , Kang Chang-Yuil
Published: January 2023

Myeloid derived suppressor cells(MDSCs) are a heterogenous population of immature myeloid cells, which accumulate in various pathological conditions, especially in tumor. In mice, MDSCs are characterized by the co-expression of Gr1 and CD11b molecules, and these cells could be classified into two subsets, CD11b+Ly6G+Ly6Clow polymorphonuclear neutrophil-like(PMN) MDSCs and CD11b+Ly6G-Ly6Chigh monocytic(Mo) MDSCs, by their expression of Ly6C and Ly6G molecules. Functional characteristics and fate of MDSCs are defined quite well. On the other hands, accumulation mechanism and origins of MDSC need to be more elucidated, and have not been fully understood whether MDSC could be generated in the periphery from distinct precursors in tumor microenvironment. For the first time, we identified the presence of splenic MDSC precursors in tumor bearing mice, characterized by CD11b+Ly6G-Ly6Cneg/low. We demonstrated that cytokines related to MDSC accumulation convert CD11b+Ly6G-Ly6Cneg/low cells into CD11b+Ly6G+Ly6Clow PMN-MDSC and/or CD11b+Ly6G-Ly6Chigh Mo-MDSC. These converted cells exerted strong suppressive activity on DO11.10 Tg splenocytes proliferation. Thus, these data suggest that there are peripheral distinct precursors, CD11b+Ly6Cneg/low cells, from which immunosuppressive MDSC could be generated.
Avidity maturation of γ9δ2T-cell receptor engineered T-cells by CDR3 modulation (162.37)
Authors: Kuball Jürgen , Gründer Cordula , van Dorp Suzanne , Drent Esther , Hol Samantha , Scholten Kirsten , Heijhuurs Sabine , Strong Roland
Published: January 2023

Immunotherapy with innate immune cells has recently evoked a broad interest as a novel treatment option for cancer patients. γ9δ2T-cells are an emerging innate cell population with strong anti-tumor-reactivity and a promising candidate for immune interventions. Especially γ9δ2T-cell receptors (TCR) which recognize a broad panel of tumor cells with high avidity are clinically attractive since αβT-cells can be efficiently redirected against a variety of tumor cells by introducing a γ9δ2TCR. Here we demonstrate that distinct γ9δ2TCRs mediate different functional avidities and present the concept of combinatorial-γδTCR-chain exchange (CTE) as an efficient method to create γ9δ2TCRs that mediate strong anti-tumor-responses. Thereby γ9- and δ2-chains derived from individual γ9δ2T-cell clones are newly combined allowing the design of γ9δ2TCRs that mediate a higher functional avidity against a broad tumor cell panel in vitro and in vivo when compared to a reference γ9δ2TCR. We furthermore demonstrate that this phenomenon is selectively caused by differences in the CDR3-domains of γ9- and δ2-chain. Accordingly, alanine-scanning-mutations were performed and elucidated important residues within the CDR3 sequence and stress the impact of the CDR3 length for optimal γ9δ2TCR function. This knowledge allowed improving tumor-control by engineered T-cells not only in vitro but also in vivo in a humanized mouse model.
Novel functions of cFLIP in lymphocyte death signaling (163.22)
Authors: Zhang Haibing , Zhang Jianke
Published: January 2023

Fas/Apo-1 signals through the FADD (Fas-associated death domain) adaptor protein, which recruits and activates the apical caspase 8 and leads to apoptosis. cFLIP is a homologue of caspase 8 but lacks caspase activities, and presumably inhibits apoptosis induced by the death receptors by competing with caspase 8 for binding to FADD. FADD, Caspase 8, and cFLIP form a signaling complex during death receptor-induced apoptosis. In addition to the cell death function, deletion of FADD, Caspase 8, and cFLIP results in embryonic lethality and lymphocytes proliferation defects upon TCR-induced or Toll-like receptor stimulation. Previous studies have shown that death receptors also induce a particular type of necrotic death in certain cell types, termed necroptosis, which is dependent on RIP1 and RIP3. Recently, our group and other groups showed that RIP1 or RIP3 deficiency could rescue the embryonic lethality and T cell proliferation defects caused by FADD or Caspase 8 deficiency. These studies identified a critical function of FADD and caspase-8 in vivo in suppressing RIP1/RIP3-dependent necrosis that is essential for embryonic development and T cell proliferation. Using a conditional cFLIP knockout mouse model and various in vitro systems, our recent studies reveal novel functions for cFLIP in regulating apoptosis and necrosis in lymphocytes.
Multiparameter flow cytometric analysis of RORγt expression in mouse models of T cell development and differentiation (163.25)
Authors: Wu Xiaowei , Le Hai , Nobida Naomi , Boyce Christopher , Sepulveda Homero , Rohrer Jurg , Ernst David
Published: January 2023

Retinoic acid-related Orphan Receptor gamma t (RORγt) is a DNA-binding transcription factor that is selectively expressed by lymphoid cells including CD4+CD8+ thymocytes, CD4+ Th17 and CD8+ Tc17 cells, NKT cells and lymphoid tissue inducer (LTi) cells. RORγt plays essential roles in thymopoiesis, T cell homeostasis, effector T cell differentiation and the development of secondary lymphoid tissues, eg, lymph nodes and Peyer’s patches. A new mouse mAb, Q31-378, that recognizes mouse RORγt was developed to provide a sensitive probe for analyzing the cellular and molecular mechanisms that underlie the development and differentiation of T cells and lymphoid tissues. With a newly developed transcription factor buffer, Q31-378 was successfully used in multicolor flow cytometric analyses to analyze RORγt expression in thymocyte subsets defined by CD4 and CD8 expression. Among all thymocytes, CD4+CD8+ cells comprised the main cell subset that expressed RORγt. Analyses of splenocytes cultured under Th17-inducing conditions revealed that most activated CD4+IL-17A+IFN-γ± T cells expressed RORγt when compared with other T cell subsets. These new reagents, along with additional fluorescent antibodies to other cytokines, transcription factors and phenotypic markers are being applied to analyze RORγt expression patterns in the development and differentiation of T cells and LTi cells.
Signals via TCR and common-γ-chain receptors concurrently coordinate the generation of antigen-specific human Th17 (163.3)
Authors: Ni Ling , Joo HyeMee , Duluc Dorothee , Kluca Peter , Xue Yaming , Zurawski Sandy , Zurawski Gerard , Banchereau Jacques , Oh SangKon
Published: January 2023

IL-17-producing CD4+ T cells (Th17) can confer protective and therapeutic immunity against microbial infections and cancers. However, mechanisms for the induction of antigen-derived peptide-specific human Th17 remain unknown. To study this, we employed an in vitro system in which influenza viral antigen HA1 was delivered to dendritic cells (DCs) via Dectin-1. Notably, MHC II/peptide complexes presented by DCs expanded memory Th17, but could not induce HA1-specific Th17 even with Th17-promoting cytokines. We found that signals via TCR and common-γ-chain receptors, which utilize Stat1/3, first induce naïve CD4+ T cells to express IL-1R1 followed by programming naïve CD4+ T cells to express Rorc in response to IL-1β in a MyD88 dependent manner. IL-6-mediated Stat3 activation simply amplifies IL-1β-MyD88-mediated Rorc expression. A failure in any of those signaling components, Stat1, Stat3, or MyD88, significantly diminished HA1-specific Th17 induction. Thus, the differentiation of naïve CD4+ T cells into pathogen-specific human Th17 cells requires interplays between innate and adaptive immune signals.
Secondary CD4+ T cell effectors retain heterogeneity during recall responses but are functionally superior to primary effectors (163.8)
Authors: Strutt Tara , McKinstry Karl , Kuang Yi , Bradley Linda , Swain Susan
Published: January 2023

It is not known whether or how distinctions between secondary (2°) CD4+ T cell effectors, generated from memory precursors, and primary (1°) effectors, arising from naïve cells, contribute to protective recall responses. Here, we compare these two populations in vivo following influenza A virus infection (IAV). We show that 2° effectors are functionally superior to 1° effectors, and that their generation is critical to memory CD4+ T cell mediated protection against IAV. 2° effectors develop and migrate to infected lungs with similar kinetics, but 2° effectors accumulate in greater number and demonstrate enhanced multifunctional capabilities. The 2° effector response in the lung is a critical component of protection against influenza mediated by memory CD4+ T cells and upon transfer to unprimed hosts, 2° effectors are superior to 1° effectors at mediating protection. Transcriptome analysis indicates that 1° and 2° effectors responding in different organs share key genetic pattterns, however, 2° effectors possess unique gene signatures. Finally, by targeting differentially expressed surface molecules, we demonstrate specific regulation of 1° and 2° effector functions in vivo. Our approach provides novel insight into the regulation of effector CD4+ T cells during pathogen challenge and may reveal ideal candidates for manipulation with the goal of directing the generation of larger populations of multifunctional effectors in a variety of settings apart from IAV infection.
Anaplasma marginale infection results in an impaired pathogen-specific CD4 T-cell response (164.16)
Authors: Brown Wendy , Turse Joshua
Published: January 2023

Anaplasma marginale infection of cattle previously immunized with a non-protective outer membrane (OM) protein resulted in complete loss of functional CD4 T cell responses to the specific protein and rapid deletion of tetramer+ CD4 T cells in peripheral blood. Furthermore, lack of A. marginale-specific responses during persistent infection suggested a failure of infection to prime for normal effector and memory T-cell responses. Dysfunctional memory T-cell responses were confirmed following infection of naïve cattle, as only transient and sporadic A. marginale-specific responses were detected in blood and spleen. We hypothesize that high antigen load during acute and persistent infection results in apoptosis and/or suppression of antigen-specific T cells. The objective of this study was to determine if immunization with a protective whole OM vaccine, which prevents disease, and in some cases, infection, would similarly result in loss of memory T cells primed by OM immunization. Upon infection of OM vaccinees with A. marginale, OM-specific T cell responses disappeared in all cattle, even though infection was blunted compared with a naïve control. To determine if Anaplasma-specific T cell responses recover once pathogen load is removed, infection was cleared with long-acting oxytetracycline and oral Aureomycin. The response did recover in all animals by 11 weeks. The induction of suppressive Treg cells by A. marginale infection is currently under investigation.
Chronic infection with Toxoplasma gondii potentiates the immune response against polymicrobial infection in an IFN-gamma and TNF - mediated pathway, which can be harmful to the host (164.18)
Authors: Souza Maria , Fonseca Denise , Kanashiro Alexandre , Bonfá Giuliano , Benevides Luciana , Alves-Filho José Carlos , Cunha Fernando , Silva João
Published: January 2023

Introduction: The majority of studies regarding host-pathogen interactions have focused on a single pathogen. However, host organisms in a natural environment are commonly exposed to multiple pathogens, such as T. gondii infection, which induces a strong immune response by Th1 pathway, that can persists throughout the life of the host. Therefore, we hypothesized that chronic infection with T. gondii could alter the course of host response against bacterial infection. Methodology: C57BL/6 mice were orally infected with 5 cysts of ME-49 strain of T. gondii and 40 days post infection, subjected infection by cecal ligation and puncture (CLP) sepsis model. Results: Our data showed that, 12 and 24 hours after of CLP, the group of mice infected with T. gondii exhibited decreased bacterial count, increased inflammation characterized by an increase in myeloperoxidase content in lung and increase in IFN-γ and TNF cytokines levels, as compared mice subjected to the same stimulus infectious without previous T. gondii infection. Moreover, after CLP, these animals showed a significantly increase in mortality rate, which was reduced in mice TLR-2 knockout or treated with low concentrations of anti-IFN-γ or anti-TNF antibodies. Conclusion: This study demonstrates that T. gondii infection potentiates the host response against polymicrobial infection, which can be harmful to the host. The mechanism by which this happens seems to be mediated via the overproduction of IFN-γ and TNF.
Identification and characterization of the recently evolved littoral cell as the chief source of SIRPα in human spleen (169.1)
Authors: Ogembo Javier , Milner Dan , Fingeroth Joyce
Published: January 2023

Asplenic individuals are compromised not only in their ability to destroy infectious agents, but are at increased risk of death from autoimmune disease, certain tumors, and ischemic heart disease. Enhanced mortality is attributed to lack of phagocytes sequestered in spleen that efficiently engulf and destroy appropriate targets, though related cells are found elsewhere in man. To investigate whether a unique population(s) regulates RBC and pathogen clearance and possibly filtration of altered self, we focused on a little-characterized cell that dominates the splenic red pulp of man and closely related primates, the venous sinus lining or littoral cell (LC). High expression of the formin FHOD1 outlines the LC population. Though endothelial-like in distribution LCs express several macrophage-associated proteins, the RBC antigen DARC and T-cell co-receptor CD8α/β though they lack lineage markers CD34 and CD45. Strikingly, SIRPα (CD172a) expression in human spleen concentrates on the LC, consistent with a key role in RBC turnover and elimination versus release of infected or altered self. Human LCs (SIRPα+, FHOD1+, CD8α+, CD34-, CD45-) comprise a highly specialized barrier cell population that emerged late in the course of evolution upon CD8 expression. Unique to higher primates (Hominidae), LCs may provide the ultimate filtration function(s) of human spleen.
Systemic administration of metallic nanoparticles initiates an acute, NALP3-mediated, inflammatory response (169.8)
Authors: Fernandez-Cabezudo Maria , Sarawathiamma Dhanya , Nemmar Abderrahim , Haik Yousef , Al-Ramadi Basel
Published: January 2023

Nanoparticles are increasingly being commercialized for use in biomedicine. Given their inorganic metal makeup, nanoparticles have been thought to be biologically inert. However, there is accumulating evidence demonstrating that, due to their exceedingly small size, inorganic particles may have deleterious effects on pulmonary function. In this study, we investigated the potential immunotoxicity associated with systemic administration of composite inorganic nanoparticles. Using a murine model, the acute effect of intraperitoneal inoculation of metallic nanoparticles was studied by monitoring changes in hematological and inflammatory parameters as well as serum levels of organ toxicity indicators, including AST, ALT, LDH, BUN and creatinine. Our findings indicate that high dose systemic exposure is associated with acute liver injury due to preferential accumulation of nanoparticles in this organ. Moreover, injection of nanoparticles resulted in the activation of phagocytic cells and the secretion of proinflammatory cytokines. Utilizing several lines of gene-deficient animals, we demonstrate that the nanoparticle-triggered response is dependent on the NALP3 inflammasome. These findings suggest that nanoparticles may be subject to recognition by the innate immune system.
Coxsackie virus entry and spread in HeLa cells is aided by microvesicle release (170.22)
Authors: Jorfi Samireh , Ansa-Addo Ephraim , Inal Jameel
Published: January 2023

Microvesicles(MVs) released from plasma membrane expressing surface phosphatidylserine and ranging from 0.2-≤1 μm in diameter are reported to carry various membrane proteins, lipids and cytoplasmic components characteristic of the parental cell. Coxsackie virus B (CVB), a member of the enterovirus family is the main cause of meningitis and encephalitis in infants which may result in neurodevelopmental defects. Calpains are calcium-dependant cysteine proteases that degrade cytoplasmic and cytoskeletal proteins. They regulate a variety of actin-dependant cellular processes such as microvesiculation. CVB1 requires calpain activation for both entry and virus replication. Here, we show that knocking down calpain, using approaches such as small interfering RNA (siRNA), culminates in reduction of MV release, which then abrogates CVB1 entry and spread in HeLa cells. The calpain inhibitor calpeptin also caused similar reduction in CVB1 entry and spread to healthy target cells. Together, our findings provide evidence that CVB1 infected HeLa cells enhance MV production, and these MVs aid the spread of infection. Furthermore, inhibition of MV release using siRNA results in inhibition of CVB1 entry and spread.
Reduced OPP susceptibility in naturally exposed lambs with homozygous TMEM154 K35 genotypes. (170.30)
Authors: Chitko-McKown Carol , Leymaster Kreg , Heaton Michael , Clawson Michael , Harhay Gregory
Published: January 2023

Ovine progressive pneumonia virus (OPPV) is a small ruminant lentivirus of the Retroviridae family that infects sheep worldwide. Our research group recently identified a mutation (K35) within ovine gene TMEM154 that associated with reduced susceptibility to OPPV. The ancestral state for TMEM154 is E35. The mechanism responsible for reduced OPPV susceptibility is unknown. Lambs that were homozygous K35 (n = 64), homozygous E35 (n = 58), or heterozygous E35K (n = 80) and born to OPPV sero-positive ewes were produced to test the association of E35K with OPPV susceptibility. Lambs were bled one week post-weaning and every 5 weeks thereafter to determine serological status. At 7 months of age, lambs were co-mingled with a flock of sero-positive mature ewes during a 35-day breeding season to re-expose lambs to OPPV as naturally occurs during breeding in infected flocks. Mature ewes were removed after breeding and lambs were again bled at 5-week intervals. At 7 months of age, 11% of homozygous K35 lambs were sero-positive for OPPV, compared to about 25% of lambs with one or two copies of the susceptible E35 ancestral allele. Two months after re-exposure to the infected ewes, 25% of homozygous K35 lambs were sero-positive, compared to 42% of heterozygous E35K lambs and 38% of homozygous E35 lambs. This study confirms that homozygous K35 lambs are less susceptible to OPPV infection and provides the first evidence of dominance of the E35 allele relative to K35.
Interaction of PB1 domain-containing signaling molecules in the regulation of Th2 response (174.16)
Authors: Yang Jun-Qi , Liu Hongzhu , Moscat Jorge , Diaz-Meco Maria
Published: January 2023

Phox and Bem1 (PB1) domains are protein interaction modules which contain approximately 80 amino acids and are found in a number of cytoplasmic signaling proteins. PB1 domain-containing molecules include signaling adapters p62 and Par-6, kinases such as MEK5α, MEKK3, atypical PKCs (aPKCs) PKCζ and PKCλ/ι, as well as the newly discovered NBR1 (neighbor of BRCA1 gene 1), etc. They participate in diverse biological processes. We have recently reported that PKCλ/ι and NBR1 can regulate Th2 differentiation and function both in vivo and in vitro. Here, we demonstrated that PKCλ/ι NBR1 and p62, the three important PB1 domain-containing signaling molecules, all were recruited to immunological synapses between activated CD4+ T cells and anti-CD3/CD28-coated latex beads, or between Jurkat cells and Raj B cells loaded with SEE super antigen. These three signaling molecules were found co-localized with lipid rafts of cell membranes, which is believed important for signaling transduction. Furthermore, PKCλ/ι, NBR1 and p62 were able to directly interact each other as confirmed by immunoprecipitation and confocal staining. The three signaling molecules can regulate nuclear factor of activated T cells (NFAT) c1 activity, which is actively involved in Th2 differentiation and function. Therefore, we identify a novel mechanism by which PB1 domain-containing signaling molecules control Th2 response, at least in part through NFATc1.
B cells as master regulators of a pro-inflammatory T cell balance in obesity and glucose intolerance (176.17)
Authors: Belkina Anna , Snyder-Cappione Jennifer , Jagannathan-Bogdan Madhumita , Carr Jordan , DeFuria Jason , Markham Douglas , Allen Jessica , Bouchard Jacqueline , Nersesova Yanina , Watkins Amanda , McDonnell Marie , Apovian Caroline , Denis Gerald , Nikolajczyk Barbara
Published: January 2023

Lymphocytes play key roles in the chronic inflammation critical for T2D pathogenesis. T2D patients have an elevated ratio of pro- to anti-inflammatory T cells, and B cells that fail to produce anti-inflammatory IL-10. New data show that B cells from the diet-induced obesity (DIO) mouse model of T2D secrete a pro-inflammatory balance of cytokines, including relatively low IL-10 production, which mirrors our findings in T2D patients. DIO B cells do not secrete auto-antibodies, as evidenced by anti-nuclear antigen staining. Complementary metabolic studies show that B cell-null μMT mice resist the development of glucose intolerance (but not obesity) in response to DIO. Taken together, these data support the conclusion that a pro-inflammatory B cell cytokine balance, rather than antibodies, promotes T2D. To further define roles for B cells in T2D, we immunophenotyped DIO μMT splenocytes. Regulatory T cells (Tregs) expand in DIO μMT, while Tregs fail to expand in WT mice. These data are consistent with new human studies showing B cells from T2D (but not healthy) subjects support pro-inflammatory T cells, and indicate clinical relevance of published studies showing decreased Th1 function in DIO μMT vs. WT mice. We conclude B cells are critical regulators of inflammation in T2D due to 1. Secretion of a pro-inflammatory cytokine balance; and 2. An ability to promote pro-inflammatory T cell function. Thus B cell depletion may represent a valuable tool in the T2D clinic.
Mast cells activate MDSC to promote inflammation (177.2)
Authors: Morales Johanna , Faber Travis , Saunders Bryan , Graham Laura , Norton Sarah , Bear Harry , Lantz Chris , Conrad Daniel , Ryan John
Published: January 2023

Mast cells play an important role in promoting inflammation, controlling parasitic infection, and potentiating allergic disease. Conversely, myeloid-derived suppressor cells (MDSC) are found in tumor-bearing individuals and in models of septic shock, where they are known to have regulatory effect. We aimed to determine a possible role for interactions between MDSC and mast cells and the effect of these interactions in a mouse model of parasitic infection. Surprisingly, co-culture of these cells resulted in a drastic increase in pro-inflammatory mediator release, which was attributed entirely to MDSC. Depletion of MDSC from mice infected with Nippostrongylus brasiliensis resulted in delayed worm expulsion and a drastically increased intestinal worm burden. Conversely, this inflammatory role of MDSC seems detrimental when inflammation is chronic, as MDSC depletion in an airway hyperresponsiveness model decreased the pathology of this disease. Interestingly, we have found that mast cell conditioned-media activates both the STAT3 and ERK pathways in MDSC, which could represent a novel means of pro-inflammatory cytokine induction in MDSC. We therefore conclude that mast cells can reprogram MDSC to a pro-inflammatory phenotype, suggesting that the evolutionary purpose of these suppressive cells may be to potentiate inflammatory Th2 responses necessary for parasite expulsion.
Lantrunculin B-induced filamentous actin disassembly enables human mast cell degranulation by SCF alone (177.7)
Authors: Smrž Daniel , Beaven Michael , Metcalfe Dean , Gilfillan Alasdair
Published: January 2023

The release of inflammatory mediators from mast cells following antigen/IgE-mediated FcϵRI aggregation induces allergic reactions including anaphylaxis. Stem cell factor (SCF), the ligand for the mast cell growth factor receptor KIT, potentiates such release. However, despite inducing similar early signaling events as antigen, SCF alone induces minimal, if any, degranulation in the absence of other stimuli. We therefore investigated whether the process underlying these differences may lie in receptor-induced distal events involving the reorganization of the actin cytoskeleton. To explore this issue, we utilized latrunculin B, an inhibitor of actin polymerization which is known to potentiate FcϵRI-mediated mast cell degranulation. As expected, latrunculin B moderately potentiated IgE/antigen-induced human mast cell degranulation, This response was, however, markedly enhanced in the presence of SCF (100 ng/ml), especially at lower antigen concentrations, suggesting that the SCF dependent component was more sensitive to latrunculin B than the antigen component. Furthermore, although SCF alone failed to induce degranulation, in the presence of latrunculin B, it induced degranulation in a concentration-dependent manner to a similar extent as that produced by IgE/antigen. These data thus suggest that SCF provides inadequate signals for the necessary remodeling of the actin filament network for degranulation which is rectified by latrunculin B.
Regulation of the ribosomal protein S6 by CARMA1 and MALT1 (178.1)
Authors: Hamilton Kristia , Kane Lawrence
Published: January 2023

The ribosomal protein S6 is a downstream target of the PI3K/Akt/mTOR pathway, which promotes cellular growth and proliferation. Hyper-activation of the mTOR pathway has been implicated in cancer progression as well as resistance to therapies. Activation of S6 occurs through engagement of growth factor receptors and through the T cell receptor and co-stimulatory molecules in T cells. Here we show that activating S6 upon T cell stimulation is mediated through the proteins CARMA1 and MALT1. Using a mutant T cell line deficient in CARMA1 (JPM50.6), we observed decreased S6 phosphorylation upon TCR/CD28 stimulation compared with wild type T cells. Knockdown of CARMA1 in Jurkats (CARMAshRNA) also followed this trend. Notably, we also found decreased S6 phosphorylation in Jurkats expressing little MALT1 (MALT1shRNA) with T cell stimulation. Investigation into the mechanism of CARMA1 and MALT1 activation of S6 revealed a prominent role for the kinase p70S6K, a downstream target of mTOR. We noted impaired induction of S6K phosphorylation in JPM 50.6 and MALT1shRNA cells, compared with Jurkats, after TCR/CD28 stimulation. Additionally, use of the MALT1 caspase inhibitor, z-VRPR-fmk, was shown to inhibit TCR/CD28 mediated S6 phosphorylation in Jurkats and primary CD4+ T cells. These findings reveal a novel pathway for S6 activation in T cells through CARMA1, MALT1 and p70S6K.
Protective action of regulatory T cells in ischemic kidney injury requires adenosine 2A receptor signaling and PD-1 (178.15)
Authors: Kinsey Gilbert , Huang Liping , Khutsishvili Konstantine , Ye Hong , Lobo Peter , Okusa Mark
Published: January 2023

Regulatory T cells (Tregs) suppress innate inflammation and injury associated with kidney ischemia-reperfusion injury (IRI). Tregs express CD73, which is the final enzyme in production of extracellular adenosine and previous studies have demonstrated that adenosine 2A receptor (A2AR) activation on immune cells protects against IRI. We hypothesized that the production of adenosine by adoptively-transferred Tregs is required to block innate immune responses in kidney IRI and that the Treg-generated adenosine would signal through A2ARs on inflammatory cells and in an autocrine manner on Tregs. Adoptively-transferred WT Tregs protected WT mice from kidney IRI, but in the absence of adenosine generation (CD73KO Tregs) or A2ARs (A2ARKO Tregs), Treg function was completely inhibited. Pharmacologic A2AR stimulation in CD73KO Tregs, prior to adoptive transfer, restored their protective ability, and augmented the ability of WT Tregs to suppress kidney IRI. Surface expression of the negative co-stimulatory molecule PD-1 is enhanced in Tregs by A2AR signaling. PD-1 blocking antibody treatment of Tregs, prior to adoptive transfer, reversed their protective effects in vivo, even if they had been pre-treated with an A2AR agonist. Taken together these findings demonstrate that the simultaneous ability to generate and respond to adenosine is required for Tregs to suppress innate immune responses in ischemia-reperfusion injury through a PD-1-dependent mechanism.