bluuebunny/crossref_metadata_2025 huggingface dataset:
SIV-specific rhesus CD4 T cells expressing African green monkey TRIM5α exhibit prolonged persistence and enhanced antiviral function. (VIR1P.1153)
Authors: Jain Sumiti , Trivett Matthew , Ayala Victor , Coren Lori , Barsov Eugene , Ohlen Claes , Ott David
Published: January 2023

Infection of CD4 T cells by HIV/SIV inhibits the formation of robust antiviral responses. Furthermore, HIV-specific CD4 T cells are preferentially infected and eliminated in vivo. This highlights the importance of protecting virus-specific CD4 T cells to effectively study their antiviral mechanisms. In this study, we have shown that African green monkey (Agm) TRIM5α restricts SIVmac239 replication in primary rhesus macaque CD4 T cells and synergizes with an intrinsic resistance present in some CD4 T-cell clones. Importantly, AgmTRIM5α-transduced effector CD4 T cells exhibit an increased ability to suppress viral replication in infected target cells. This increase in antiviral effector function was strongly linked to decreased infection and prolonged persistence of the AgmTRIM5α-expressing effectors. Hence, AgmTRIM5α restriction in rhesus CD4 T cells offers an approach to evaluate their anti-SIV effector functions. Currently, we are pursuing studies to determine the effectiveness of SIV-specific CD8 and CD4 T cells in controlling infection in an adoptive transfer model, where the genetically modified CD4 T-cell clones express AgmTRIM5α to prolong their persistence in vivo.
Stress perception alters cellular and molecular immunity in the immune response in the brain to a neurotropic viral pathogen (VIR1P.1154)
Authors: Steel Christina , Machida Mayumi , Lundberg Patric , Wellman Laurie , Yu Maureen , Kennedy Krista , Yanev Petar , Vonu Peter , Sanford Larry , Ciavarra Richard
Published: January 2023

Vesicular stomatitis virus (VSV) applied intranasally produces a well-characterized encephalitis. However, mice that experienced controllable stress via an escapable foot-shock (ES) demonstrated an enhanced immune response relative to mice that experienced uncontrollable stress (inescapable foot shock, IS) or non-stressed control mice (NS). These differences began as early as 1 day post-infection, where IS mice show a more robust cytokine response than ES or NS mice. By 3 days post-infection, however, ES mice showed enhanced inflammatory cytokine responses in the olfactory bulb. We used the Ingenuity Pathways Analysis (IPA) platform to identify regulatory genes responsible for the differences. IPA identified miRNA146a as an upstream regulator of the antiviral response and qPCR found elevated levels (~5-fold) of miRNA146a in the olfactory bulb from ES versus IS mice. miRNA levels in the non-stressed group were below detectable limits. These results suggested increased inflammation in the brains of ES mice, which were supported by more aggressive weight loss in ES mice compared to IS or NS mice as the encephalitis progressed. By 28 days post-infection, surviving mice subjected to ES continue to show increased immune activation as evidenced by increased leukocyte infiltration and enhanced production or infiltration of CD103+ (memory phenotype) T cells relative to IS or NS mice.
Evaluation of bone marrow in cowpox virus infection in cynomolgus macaques (VIR1P.1127)
Authors: Josleyn Nicole , Yellayi Srikanth , Martinez Mark , Janosko Krisztina , Adams Ricky , Burdette Tracey , Cohen Melanie , Zhang Tengfei , Jahrling Peter , Johnson Reed
Published: January 2023

Evaluation of countermeasures against variola virus, the causative agent of smallpox, must be performed through extrapolation from suitable animal models of human smallpox. In previous studies, the gross and histopathological findings of nonhuman primates intravenously (IV) infected with Cowpox Brighton Red (CPXV) included the presence of necrotic areas of bone marrow that were orthopox-viral antigen positive by immunohistochemistry (IHC). Here, we describe changes in bone marrow in regards to cellular composition, orthopox-viral antigen positivity, viral load, and histological findings during a serial disease progression study of cynomolgus macaques following IV inoculation of CPXV. Our results demonstrate infectious CPXV as early as day 3 post inoculation in the bone marrow. Flow cytometry and histological analysis indicate changes in bone marrow composition. Furthermore, flow cytometry analysis indicates many cell populations including monocytes, CD34+ stem cells, and neutrophils were orthopox-viral antigen positive, while mesenchymal stromal cells were not. We have previously shown immunopositivity in the bone marrow by IHC. Flow cytometry revealed no significant differences in early or late apoptosis between CPXV- and mock-infected NHPs. These data indicate an important role for bone marrow function during the progression of disease in the CPXV- nonhuman primate model of smallpox.
IL-10 inhibits neuraminidase activated-TGF-β and facilitates Th1 phenotype during early phase of infection (VIR1P.1129)
Authors: Huang Ching-Tai , Dutta Avijit , Chen Tse-Ching , Lin Chun-Yen , Chiu Cheng-Hsun , Lin Yung-Chang , Chang Chia-Shiang , He Yueh-Chia
Published: January 2023

Th1 cells control their activity by producing regulatory IL-10. Here we report that Th1 cell-derived IL-10 facilitates their expansion and in addition, augments Th1 cell production of IFN-γ, TNF-α, and IL-2 during the early phase of influenza. In our antigen-specific mouse experimental system, influenza hemagglutinin-specific CD4+ T cells respond to infection with the induction of T-bet, and produce both IFN-γ and IL-10. In the early phase of infection, an abundance of viral neuraminidase causes TGF-β activation of hemagglutinin-specific CD4+ T cells. CD4+ T cell-derived IL-10 inhibits neuraminidase-driven TGF-β activation and counteracts the virus-mediated immune suppression. As the host eradicates the virus, neuraminidase activity wanes and IL-10 receptors are up-regulated on CD4+ T cells in the late phase of infection. IL-10 then suppresses immune activation and aids in recovery from infection and inflammation. These results reveal a previously unrecognized function of Th1 cell-derived IL-10 in vivo.
LCMV-specific CD4 T cells induce immunopathology and impair immune protection following chronic LCMV infection (VIR1P.1132)
Authors: Penaloza-MacMaster Pablo , Barber Daniel , Wherry E. , Provine Nicholas , Teigler Jeffrey E. , Parenteau Lily , Blackmore Stephen , Borducchi Erica , Larocca Rafael , Yates Kathleen , Shen Hao , Haining W. , Sommerstein Rami , Pinschewer Daniel , Ahmed Rafi , Barouch Dan
Published: January 2023

CD4 T cells promote memory immune responses, but it is unclear whether vaccine-induced CD4 T cells contribute to immune protection. We interrogated if virus-specific CD4 T cells would protect mice against infection with chronic lymphocytic choriomeningitis virus (LCMV). Immunization with vaccines that induced CD4 T cell responses resulted in mortality following infection with persistent strains of LCMV. Immunopathology was associated with high levels of virus-specific CD4 T cells, generalized inflammation and collapse of adaptive immunity. Interestingly, virus-specific CD8 T cells or antibodies abrogated these lethal effects of CD4 T cell vaccines. Our data demonstrate the importance of tightly regulating CD4 T cell responses following vaccination, and suggest that CD4 T cells in the absence of effective antiviral immune responses could induce lethal immunopathology.
Rapid recovery from severe H7N9 disease requires a diversity of response mechanisms driven by CD8+ T cells (VIR2P.1162)
Authors: Xu Jianqing
Published: January 2023

The avian origin A/H7N9 influenza virus causes high admissions rates (>99%) and mortality (>30%), with ultimately favorable outcomes ranging from rapid recovery to prolonged hospitalisation. Using a novel multi-colour assay for monitoring adaptive and innate immunity, we dissected the kinetic emergence of different effector mechanisms across the spectrum of H7N9 disease and recovery, to find that a diversity of response mechanisms contribute to resolution and survival. Patients discharged within 2-3 weeks had an early peak in H7N9-specific CD8+ T-cell responses, while individuals with prolonged hospital stays had late recruitement of CD8+/CD4+ T cells and antibodies simultaneously (recover by week 4), augmented even later by prominent NK cell responses (recovery >30 days). In contrast, those who succumbed had minimal influenza-specific immunity and little evidence of T-cell activation. Our study illustrates the importance of robust CD8+ T-cell memory for protection against severe influenza disease caused by newly-emerging influenza A viruses.
Bundibugyo ebolavirus replicates to lower viral titer in innate immune cells (VIR2P.1171)
Authors: Versteeg Krista , Mire Chad , Geisbert Thomas
Published: January 2023

The current Zaire ebolavirus (ZEBOV) epidemic in West Africa reinforces the need for effective vaccines and therapeutics that protect against all species of ebolavirus (EBOV). To achieve this, we characterized the effect of virus infection of macrophages and dendritic cells using the recently discovered Bundibugyo ebolavirus (BEBOV). BEBOV infection of humans and nonhuman primates has a lower case fatality rate, differing clinical presentations, and longer time to death compared to ZEBOV. More importantly, survivors and non-survivors of BEBOV have detectable antibodies against BEBOV, indicating that BEBOV interaction with immune cells may differ from ZEBOV. To better understand BEBOV pathogenicity and determine the effects of BEBOV on macrophages and dendritic cells, we performed growth curves and cell characterization in monocyte-derived dendritic cells and PMA-induced THP-1 macrophage-like cells. Over 96 hours, virus production was measured by virus titer, flow cytometry, and qRT-PCR. During monocyte derived dendritic cell experiments, cell surface markers were characterized over time to determine changes induced by EBOV infection. In all cell types tested, BEBOV grows to a lower viral titer. This may play an important role in the overall pathogenesis of BEBOV. Understanding the systemic immune response to BEBOV will aid in determining the mechanism of decreased pathogenesis and identify antiviral targets to be used in developing broad spectrum EBOV therapeutics.
Generation and evaluation of simian-human immunodeficiency virus subtype AE challenge stocks (VIR2P.1166)
Authors: Tartaglia Lawrence , Chang Hui-Wen , Lee Benjamin , Abbink Peter , Whitney James , Lim So-Yon , Sanisetty Srisomya , Lavine Christy , Seaman Michael , Barouch Dan
Published: January 2023

The majority of preclinical and clinical human immunodeficiency virus type 1 (HIV-1) vaccine development efforts have focused on HIV-1 subtype B strains from the western world. However, the overwhelming majority of HIV/AIDS-related deaths occur in the developing world and are from other HIV-1 subtype strains. Therefore, we sought to develop and generate the first mucosally transmissible simian-human immunodeficiency virus (SHIV) challenge stocks from HIV-1 subtype AE env sequences. The sequences were identified from acutely infected HIV-1 individuals from Southeast Asia. The challenge stocks efficiently infected rhesus monkeys, replicated to high levels during acute infection, and established chronic setpoint viremia. The SHIV challenge stocks should facilitate the evaluation of vaccines and other interventions aimed at controlling the spread of HIV-1 subtype AE infection.
T cell-mediated protection and epitope mapping with a trivalent alphavirus-like replicon particle vaccine (VIR2P.1167)
Authors: Cohen Courtney , Morazzani Elaine , Kalinyak Laura , Glass Pamela
Published: January 2023

Venezuelan (VEEV), eastern (EEEV) and western (WEEV) equine encephalitis viruses cause debilitating and sometimes fatal illness. These pathogens are highly infectious when aerosolized, and thus favorable candidates for weaponization, yet no licensed vaccines exist. In a subcutaneous model of infection protection correlates with neutralizing antibody response, but studies suggest that protection from aerosol challenge is partially mediated by cellular immunity. We have previously shown that a trivalent (VEEV/EEEV/WEEV) virus-like replicon particle (triVRP) vaccine protected mice against lethal aerosol challenge. To investigate the cell-mediated response, splenocytes from triVRP- and mock-vaccinated mice were cultured for 6 days ex vivo in the presence of VEEV, EEEV or WEEV peptides. Fifteen-mer peptide pool libraries against E1 and E2 glycoproteins were used for T cell epitope mapping. Splenocytes were analyzed via flow cytometry for T cell-specific markers and cytokine production. Restimulation with specific regions of the VEEV E2 protein induced a memory CD8+CD44+ T cell response, as well as an increase in proliferating, non-cytokine producing CD8+CD44+ T cells. Thus, triVRP-vaccinated mice developed a cell-mediated, peptide-specific memory response. Studies are ongoing to precisely map T cell epitopes within VEEV, EEEV and WEEV glycoproteins. Future studies will evaluate the importance of these responses to protection from lethal aerosol alphavirus challenge.
Comparative analysis of CTL-repertoires induced by different vaccine vectors
Authors: Bernhard Caroline Andrée
Published: January 2023

Dendritic cells (DCs) are considered the most potent antigen-presenting cells (APCs), which directly prime or cross-prime MHCI-restricted cytotoxic T cells (CTLs). Although we know much about the molecular mechanism of cross-presentation, its actual contribution to cytotoxic T cell (CTL) immunity under physiological conditions in vivo is still unclear. Cross-presentation is based on the idea that DCs are the only professional antigen-presenting cells able to prime naïve T cells. However, recent evidence suggests the existence of other, as-yet unidentified APCs also able to prime T cells. To identify those APCs, we used adenoviral (rAd) vectors, which do not infect DCs but selectively accumulate in CD169+ macrophages (MPs). In mice that lack DCs, infection of CD169+ MPs was sufficient to prime CTLs specific for all epitopes tested. In contrast, CTL responses relying exclusively on cross-presenting DCs were biased to selected strong MHC I-binding peptides only. When both DCs and MPs were absent, no CTL responses could be elicited. Therefore CD169+ MPs can be considered APCs that significantly contribute to CTL responses. However, other vaccine vectors, such as Modified Vaccinia Ankara (MVA), seem to have different APC-requirements to be efficient. Side-by-side comparison of CTL responses initiated by different APC types and vaccines might greatly advance our knowledge for vaccine development.
Inhibition of vasoactive intestinal polypeptide signaling augments T cell activation by plasmacytoid dendritic cells: implications for cellular immunotherapy
Authors: Li Jingxia , Li Jian-Ming , Waller Edmund K
Published: January 2023

Introduction

Murine models of allogeneic hematopoietic stem cell transplant (allo-HSCT) have shown that adding donor plasmacytoid dendritic cell (pDC) to allogeneic HSC and T cell grafts can: 1) augment the graft-versus-leukemia (GvL) activity of T cells by inducing donor Th1 polarization; and 2) limit the subsequent development of graft-versus-host disease (GvHD) through interferon gamma-licensed IDO production by pDC. Vasoactive intestinal polypeptide (VIP) is an immunosuppressive neuropeptide, which may regulate activation of allo-specific T cells, making it a potential target for regulating GvHD and GvL in allo-HSCT.

Methods and results

We used a model system of indirect presentation by pDC of a I-Ed derived allo-peptide (EaP 52–68) to transgenic T cells expressing the TEa TCR. The effect of blocking VIP signaling from pDC was assessed with VIP-knockout (KO) pDC or by adding a VIP antagonist, VIPhyb. Peptide-primed pDCs from VIP-KO mice increased expression of activation markers (CD69 and CD71) and stimulated TEa T cell proliferation compared with pDCs from WT mice. Similar results were observed in T cells cultured with VIPhyb treated pDCs. T cells proliferated more when cultured with BM VIP-KO pDCs than splenic VIP-KO pDCs.

Conclusions

These data indicate a novel role for VIP signaling in T cell activation and proliferation in response to indirect antigen presentation. These data support published data on the role of donor pDC in modulating the GvHD activity of T cells and indicate important functional differences in pDC activity according to their source. Ongoing experiments support the immune regulatory effect of VIP on GvHD and GvL in murine allo-HSCT.
Are the extracellular IgV and IgC domains of butyrophilin 3A1 required for prenyl pyrophosphate stimulation of human Vγ2Vδ2 T cells?
Authors: Morita Craig T , Wang Hong
Published: January 2023

Vγ2Vδ2 T cells play important roles in human immunity to pathogens and in cancer immunotherapy by responding to isoprenoid metabolites such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) and isopentenyl pyrophosphate (IPP). The immunoglobulin superfamily protein, butyrophilin 3A1 (BTN3A1), is required for prenyl pyrophosphate stimulation. Our mutational studies and the work of others has shown that HMBPP and IPP compounds are not antigens but instead are sensed molecules that bind to the intracellular B30.2 domain of BTN3A1. How this binding is detected by the Vγ2Vδ2 TCR is unclear. One possibility is that HMBPP binding alters the conformation and/or distribution of the extracellular domains (ECD) of the BTN3A1 dimer allowing direct binding to the Vγ2Vδ2 TCR or to another molecule that binds the Vγ2Vδ2 TCR. Alternatively, the Vγ2Vδ2 TCR could bind to a molecule that is recruited by the intracellular B30.2 domain upon HMBPP binding. To distinguish between these mechanisms, we mutagenized 32 residues on the surfaces of the IgV and IgC domains of BTN3A1 to alanine and tested the mutant proteins for their ability to mediate prenyl pyrophosphate stimulation of Vγ2Vδ2 T cells. Mutagenesis of residues in the IgV and IgC domains had no effect whereas mutagenesis of the B30.2 binding site abrogated Vγ2Vδ2 T cell responses to HMBPP. Therefore, these findings do not support direct binding to the Vγ2Vδ2 TCR (or to another protein) by the ECD of BTN3A1. Instead, Vγ2Vδ2 TCR recognition may be to a protein recruited to the B30.2 domain upon HMBPP binding. Identification of this second protein will help clarify the puzzling process of human Vγ2Vδ2 T cell receptor recognition.
Transporter associated with antigen processing (TAP) dependencies and cell surface peptide occupancies of HLA-B allotypes
Authors: Geng Jie , Raghavan Malini
Published: January 2023

Human leukocyte antigens (HLA) class I molecules comprise a highly polymorphic heavy chain complexed to beta2-microglobulin (β2m). The heterodimers bind and display short peptides on the cell surface for intracellular surveillance by cytotoxic T cells. Peptides that assemble with HLA class I molecules are typically derived from the cytosol of cells, whereas the assembly of HLA class I occurs within the ER lumen. The transporter associated with antigen processing (TAP) is essential for peptide transport from the cytosol into the ER. In this study we showed that frequently occurring HLA-B variants display distinct patterns of TAP dependencies. Some HLA-B allotypes are detectable at significant levels in TAP-deficient and TAP-inhibited cells. Such allotypes are predominantly peptide-occupied in TAP-sufficient cells, but display higher percentages of empty protein in cells with inhibited or dysfunctional forms of TAP. TAP activity is known to be blocked by many viral proteins and in some cancers. Our findings indicate TAP dependency-influenced variations in cell surface expression and peptide occupancies of HLA-B allotypes under pathoglocical conditions.
Association of functionally distinct ERAP1 variants with prognosis in HPV positive cancer.
Authors: Reeves Emma , King Emma , Elliott Tim , Thomas Gareth , James Edward N
Published: January 2023

Human papillomavirus (HPV) infection accounts for 5% cancers worldwide, with cervical carcinoma and squamous cell carcinoma of the head and neck (SCCHN) being most common. HPV can enable immune evasion, with 50% of HPV+SCCHN having reduced pMHC I expression, correlating with fewer tumor infiltrating lymphocytes (TIL) and a worse clinical outcome. Polymorphisms in the endoplasmic reticulum aminopeptidase1 (ERAP1), a key component of antigen processing, trimming N-terminally extended peptides for MHC I, are associated with prognosis and outcome in cervical carcinoma. We have previously demonstrated ERAP1 to be highly polymorphic, forming distinct allotypes with functional differences. Here we investigate the contribution of ERAP1 in the pathogenesis of SCCHN.

We identified a number of novel ERAP1 allotype pairs (both chromosomal copies) from HPV+ SCCHN. Assessment of trimming function revealed a range of abilities to generate the model epitope SIINFEHL from individual amino acid or AIVMK precursors. Furthermore, the ability to generate HPV-16 E6/E7 specific epitopes also demonstrated varying trimming phenotypes. Interestingly, the overall ability to generate the final epitopes (model and HPV) in TILLOW patients was poor, whereas TILHIGH patients efficiently generated them. This difference revealed ERAP1 gene sequence and function stratifies with TIL levels and prognosis. These results demonstrate that the successful presentation of HPV-16 E6/E7 epitopes at the cell surface depends on the ERAP1 allotype pairs expressed within an individual. Furthermore, the allotype pairs correlate with levels of tumor infiltrate, suggesting ERAP1 activity as an indicator of prognosis in HPV+ SCCHN patients.
ERAP1 deficiency protects HLA-B27 transgenic rats from arthritis
Authors: Tran Tri M , Gill Tejpal , Hong Sohee , Bennett Joshua , Colbert Robert A
Published: January 2023

HLA-B27 (B27) confers a strong predisposition to spondyloarthritis (SpA). Rats transgenic for B27 (B27-TG) develop SpA-like disease with gut inflammation and arthritis. Common variants in ERAP1, which trims peptides for HLA class I in the ER, have been associated with SpA. We sought to determine how ERAP1 loss-of-function affects SpA-like disease in rats. Part of the first exon of ERAP1 was deleted using a TALEN, resulting in complete loss of ERAP1 expression in cells from ERAP1−/− animals. Strains were crossed to generate cohorts of B27-TG with ERAP1+/+, ERAP1+/−, and ERAP1−/− genotypes. B7-TG rats and WT rats with ERAP1+/+, +/− or −/− were used as controls. B27+ males with either ERAP1+/+ or ERAP1+/− genotype developed arthritis with a frequency of 28% (5/18), and 35% (8/23), respectively, with comparable arthritis severity scores (2.0) and age of onset (4.1 months), whereas only 13% (2/15) of rats with an ERAP1−/− genotype developed arthritis with a lower arthritis score (0.75). Only 13% of B27+ERAP1+/− females developed arthritis (arthritis score of 0.75), while ERAP1+/+ and ERAP1−/− females remained healthy. Furthermore, B7-TG and WT rats were healthy regardless of the ERAP1 genotype. All B27+ rats developed gut inflammation with no differences in clinical colitis, although histology scores were slightly higher in the complete absence of ERAP1 (7.0) compared to ERAP1+/+ (5.0) with p<0.05. Taken together, complete absence of ERAP1 protected B27-TG rats from arthritis, no appreciable difference in gut inflammation, suggesting different effects on pathogenic mechanisms involved in arthritis and colitis. This novel model provides an opportunity to better understand the mechanism(s) by which HLA-B27 contributes to SpA pathogenesis
Participation of plasmacytoid dendritic cells and NKT cells in the mouse model of lupus induced by non-bilayer phospholipid arrangements
Authors: Saldivar Carla Elizabeth Landa , Reséndiz Albany , Zárate Luz Angela , Hernández Teresa , Sánchez Sandra , Flores-Romo Leopoldo , Wong-Ramírez Carlos , Baeza-Ramírez Isabel , Wong-Baeza Carlos
Published: January 2023

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease where animal models are used to study its pathogenesis. We have developed a mouse model of an autoimmune disease that resembles human lupus by the injection of liposomes bearing non-bilayer phospholipid arrangements (NPA). We detected the presence of IgG antibodies against NPA in the serum of mice with lupus and patients with SLE. In this work, we determined by citofluorometry the presence of plasmacytoid dendritic cells (pDC) the main producer of type I interferons, and of NKT cells in the secondary lymphoid organs of mice 30 and 60 days after the injection of liposomes with or without NPA induced with 8 mM promazine. In both groups of mice injected with liposomes bearing NPA a significant increase of pDC cells (5-fold) was found which correlates with the high concentration of type I interferon previously detected in this mouse model of lupus and in patients with SLE. A significant increase of NKT (3-fold) was detected at 30 days, specifically in the NKT subpopulation CD4+ that is known to cooperate with B cells in response of lipid antigens; this increase suggests its probable involvement in adaptive immune responses, which lead to the production of anti-NPA IgG antibodies. The increase of pDC and NKT cells found by cytometry in secondary lymphoid organs in this work, suggests their involvement in the formation of anti-NPA IgG antibodies and the development of the disease resembling human lupus.
Neutrophils modulate the progression of B cell auto-reactivity in systemic lupus erythematosus
Authors: Bird Anna Kathleen , Rangel-Moreno Javier , Meednu Nida , Hossler Jennifer , Anolik Jennifer
Published: January 2023

The central feature of systemic lupus erythematosus (SLE) pathogenesis is loss of immunologic self-tolerance. Here, we address a current controversy regarding whether neutrophils influence the development of B cell auto-reactivity SLE. While both human and murine lupus neutrophils produce factors known to fuel adaptive immune dysregulation, including type I interferon, B cell survival factors, and extracellular traps (a putative source of self-antigen), some studies have contended that neutrophils in peripheral lymphoid organs (PLO) suppress B cell activation and differentiation. In order to ask whether neutrophils exert regulatory or deleterious effects on loss of B cell self-tolerance and immune activation, we compare changes in SLE progression following neutrophil depletion in the NZB/W lupus model, either during or after establishment of disease. Following depletion early in disease, we observe pronounced acceleration of proteinuria, anti-dsDNA titers, and germinal center formation. However, neutrophil depletion after onset of overt disease pathology (including proteinuria) does not strongly alter SLE progression, implicating neutrophils as playing a protective role only apparent during disease onset. To elucidate the specific mechanisms underlying neutrophil effects on B cell auto-reactivity, we examine the cytokine profile of neutrophils in direct proximity to B cells in PLO during several stages of disease. With progression of disease, we observe an influx of interferon-alpha-producing neutrophils in direct proximity to B cells in PLO. These data delineate a shifting balance of both regulatory and activating roles for neutrophils in auto-reactive B cell activation/proliferation during SLE progression.
TNF receptor type 2 (TNFR2) in the maintenance of gut microbiota and autoimmune disease susceptibility
Authors: McKarns Susan C , Miller Patrick G. , Franklin Craig , Ericsson Aaron C , Bonn Michael B
Published: January 2023

Tumor necrosis factor (TNF) antagonism is therapeutic for some inflammatory autoimmune disorders such as Crohn’s disease and rheumatoid arthritis. However, TNF blockade inexplicably exacerbates CNS autoimmunity, including multiple sclerosis. We now demonstrate that selective TNFR2 deficiency augments spontaneous autoimmunity in female, but not male, myelin oligodendrocyte glycoprotein (MOG)–specific 2D2 TCR transgenic mice. Disease in TNFR2−/− 2D2 mice correlates with CNS lymphocyte infiltration and increased myelin oligodendrocyte glycoprotein–induced IL-17, IFN-γ, and IgG2b production. Attenuated disease in TNF−/− 2D2 mice relative to TNFR2−/− 2D2 mice identified distinctive roles for TNFR1 and TNFR2. Antibiotic treatment eliminated spontaneous autoimmunity in TNFR2−/− 2D2 mice to suggest a role for gut microbiota. Illumina sequencing of fecal 16S rRNA identified a distinct microbiota profile in male TNFR2−/− 2D2 mice that associated with disease protection. While Akkermansia muciniphila, Sutterella sp., Oscillospira sp., Bacteroides acidifaciens, and Anaeroplasma sp. were selectively more abundant in male TNFR2−/− 2D2 mice, Bacteroides sp., Bacteroides uniformis, and Parabacteroides sp. were more abundant in affected female TNFR2−/− 2D2 mice. Overall, selective TNFR2 blockade appears to target commensal bacteria–host immune dysbiosis to potentiate autoimmunity in genetically susceptible female mice. Under this paradigm, microbes likely contribute to an individual’s response to anti-TNF therapy. This model provides a foundation for host immune–microbiota-directed measures for the prevention and treatment of CNS-demyelinating autoimmune disorders.
TLR2 tolerance in EAE
Authors: Anstadt Emily J , Fujiwara Mai , Nichols Frank , Clark Robert B
Published: January 2023

TLR2 expression is enhanced in MS and EAE, but its role in these diseases is controversial. In adoptive transfer EAE, exogenous TLR ligands are not administered yet TLR2-deficient mice have been reported to develop attenuated disease. Despite this evidence for a disease-promoting function of TLR2, others have shown the opposite, i.e. that administration of TLR2 ligands can inhibit EAE in WT mice. To understand this paradox we tested the postulate that although TLR2 signaling may be EAE-promoting, repeated TLR2 ligand administration can lead to diminished TLR2 responsiveness (tolerance) and attenuated disease.

To establish our approach, we administered Pam2Cys to SJL/J mice for 5 days and induced TLR2 tolerance that persisted for 4–5 days, reflected in decreased serum TNFα in response to a second TLR2 ligand (p<0.0001 day 1; p<0.01 day 3; p=0.069 day 5). Next, we used this approach to induce TLR2 tolerance in the context of EAE. SJL/J mice received EAE-causing, PLP-reactive LNCs on day 0 and tolerance was induced at various time points. Surprisingly, initiating the TLR2 tolerance protocol at day −2 or day +8 showed no effect on disease. In contrast, initiating the protocol between day +4 and +6 lead to significant inhibition of EAE onset and severity that lasted at least 5 days (p=0.025) and was associated with TLR2 tolerance (p<0.0001).

In sum, we have used TLR tolerance as a novel probe to identify a narrow kinetic window in which TLR2 is required for EAE. Additionally, we have demonstrated the potential use of TLR tolerance as a new therapeutic approach in EAE and MS. Ongoing studies will characterize the relevant TLR2-expressing cells in EAE and the potential role of the microbiome in inducing homeostatic TLR tolerance that regulates autoimmunity.
Gut microbiota dysbiosis in patients with Multiple Sclerosis
Authors: Mangalam Ashutosh K , Chia Nicholas , Chen Jun , Kalari Krishna R , Yao Janet Z , Novotna Martina , Soldan Mateo Paz , Luckey David , Marietta Eric V , Jeraldo Patricio R , Chen Xianfeng , Weinshenker Brian G , Rodriguez Moses , Kantarci Orhun H , Kantarci Orhun H , Nelson Heidi , Murray Joseph A
Published: January 2023

Multiple sclerosis (MS) is an inflammatory and demyelinating autoimmune disease that is known to involve environmental predisposition—an important component of which may be influenced by the GI microbiota. This study was designed to investigate whether gut microbiota may play a role in the etiopathogenesis of MS, by comparing the composition of fecal microbiota in MS patients to that of age- and gender-matched healthy controls. Phylotype profiles of the microbiome populations were generated using deep sequencing of the hyper variable V3–V5 region of the 16S ribosomal RNA (rRNA) gene and high quality sequences were then analyzed for taxonomic composition and clustering (OTU) using QIIME (Quantitative Insights Into Microbial Ecology) and LEfSe (LDA Effect Size). High-throughput multiplexed MiSeq sequencing yielded over 50,000 reads/sample, ensuring detection of both dominant and rare members of the microbiome. Detailed fecal microbiome analyses revealed that patients with MS had a distinct microbial community profile compared to healthy controls. We observed a decreased abundance of taxa involved in metabolism of phytoestrogen and bile acid, both of which play important roles in the maintenance of gut homeostasis and the induction of anti-inflammatory pathways. This study suggests that microbial dysbiosis, particularly a decrease in anti-inflammatory microbes, may lead to an increased pro-inflammatory immune response and subsequent predisposition to the development of MS. In particular, our study consistent with the hypothesis that microbial dysbiosis is a contributing environmental factor involved in the etiopathogenesis of MS.
Influenza infection modulate the extent and duration of inflammation in the central nervous system of EAE pathogenesis
Authors: Chen Qingyun , Liu Yinping , Ni Ke , Lv Aizhen , Xiang Zheng , Zheng Jian , Tu Wenwei
Published: January 2023

Multiple sclerosis (MS) starts with increased migration of auto-reactive lymphocytes across the blood-brain barrier, resulting in persistent neurodegeneration. Evidence from clinical and epidemiological studies indicated that viral infections are important in the induction of autoimmunity including MS. Using the experimental autoimmune encephalomyelitis (EAE) as a model for MS, we demonstrated that influenza infection can modulate the development of the central nervous system (CNS) inflammation. It was shown that mice with influenza infection were unable to recover from EAE, exhibiting a long-term chronic disease course. We found that the exacerbated disease after influenza virus infection was due to elevated Th1 cells infiltration into CNS, and this enhancement in the early stage of EAE disease is accompanied by increased CCR5+CD4+, CXCR3+CD4+ T cell and MOG35–55 specific CD4+ T cells localized in the lung and its draining lymph node in influenza virus-infected mice. We also observed much more CD45highCD4+CD44high effector T cell and CCR5, CXCR3, IFNγ expressing Th1 type cells entering the CNS at both acute phase and later stage of EAE disease in influenza virus-infected mice. Finally, the administration of a CCR5 and CXCR3 antagonist inhitibs the Th1 cells infiltration and significantly decreases the difference of clinical disease course between mock and influenza virus-infected mice. Taken together, our data suggested that prior influenza infection may affect subsequent EAE pathogenesis by selectively recruit CCR5 and CXCR3 bearing CD4 T cells into CNS.
The effects of Estrogen on the IFN signature in Lupus
Authors: Lee Michael Hweemoon , Sriram Uma , Chakhtoura Marita , Chain Robert W , Caricchio Roberto , Gallucci Stefania
Published: January 2023

Dendritic cells (DCs) are pivotal immune regulators and are important in the pathogenesis of Systemic Lupus Erythematosus (SLE). We have previously found that DCs from female lupus-prone mice constitutively over-express Type I Interferon (IFN) responsive genes resembling the IFN Signature found in SLE patients. Since this Signature is present in pre-diseased mice, it may contribute to disease pathogenesis. Nucleic acids, major auto-antigens in lupus, are potent stimulators of innate immunity and may contribute to the IFN signature by stimulating DCs through TLR7 and 9. As SLE is ~9 times more likely to affect women than men and the onset of SLE is most often in the reproductive years of women, estrogens have been suggested to affect the IFN response. To investigate the effects of estrogen on the IFN signature, we studied conventional DCs (cDCs), from male and female Sle1,2,3 lupus prone mice or from wild type C57BL/6 mice, generated in media void of sterols with supplemental titrations of 17-beta-estradiol (E2). We have found that DCs from male pre-diseased lupus-prone mice express the IFN Signature found previously in female DCs. Furthermore, E2 stimulation, regardless of the sex of the DCs, modulates the activation of cDCs in response to TLR stimulation. Interestingly, while high levels of E2 in hormone replete conditions were capable of exacerbating the IFN signature, the difference seen between wild-type and Sle DCs in standard conditions indicates both an E2 and E2-independent exacerbation in the activation of Sle DCs.
Chemotaxis to islets based on cellular insulin receptor expression
Authors: Esakov Emily L , Al-Dieri Ali , McInerney Marcia F
Published: January 2023

In the non-obese diabetic (NOD) mouse model, T1D is an autoimmune disease characterized by insulitis and T cell-mediated destruction of pancreatic islet beta cells. Insulin receptor (IR) is a chemotactic receptor capable of driving T cell movement in response to insulin. In published work, purified T cells, obtained from diabetic mice, and sorted into IR positive and negative cells were passaged into irradiated nondiabetic NOD mice. Only recipients that received IR positive T cells had insulitis and diabetes. If the IR expressing cells are moving into the pancreas based on an insulin gradient, then artificially making T cells with high IR expression should allow for movement into the pancreas in mice that are not susceptible to T1D.

A novel transgenic mouse model was made that has a FLAG tagged mouse insulin receptor behind a CD3 promoter for expression on T cells in a mouse not susceptible to T1D development. Transgenic BL/6-CD3FLAGmIR mice were successfully made, and show evidence for T cell trafficking into the pancreas. Although insulitis occurs, no diabetes has developed.

Characterizing IR expression on T cells may be of key importance for understanding the pathogenesis of diabetes. If IR expression is established as a mechanism to move T cells into islets, then down regulation of IR expression specifically in T cells will provide a new therapeutic target to block cell movement into the pancreas, thus preventing T1D.
Disease stage-dependent response of lupus-prone mice to antibiotics
Authors: Mu Qinghui , Zhang Husen , Luo Xin M
Published: January 2023

Little is known on the role of commensal bacteria in lupus. We previously showed that a higher abundance of Lactobacillaceae and a lower abundance of Lachnospiraceae are correlated with improved lupus symptoms in lupus-prone MRL/lpr mice. In the same study, it was noted that Lactobacillaceae (“good” bacteria) was more abundant in the gut of pre-disease lupus mice, while Lachnospiraceae (“bad” bacteria) was relatively more abundant after disease onset. We thus hypothesized that antibiotic use after disease onset might have a greater impact on “bad” than “good” bacteria, thus attenuating the disease. To test this hypothesis, we treated MRL/lpr mice with antibiotics starting from 3 weeks (pre-disease, Abx-3w) or 9 weeks of age (after disease onset, Abx-9w). Consistent with the results obtained from germ-free MRL/lpr mice, Abx-3w did not change disease course or severity. Treatment with Abx-9w, however, was able to significantly reduce spleen and lymph node weight, serum level of anti-dsDNA IgG, proteinuria, and renal pathology. Bacterial profiling analysis revealed that >99% of bacteria were removed by Abx-3w, while 20% bacteria remained with Abx-9w treatment, where Lactobacillales and Enterobacteriales were enriched. Further studies are underway to investigate the immunological mechanisms of how Abx-9w attenuated lupus in these mice. The results of this study will increase our understanding of how environmental triggers, such as antibiotic use, are associated with lupus.
Characterization of the microbiome in association with reduced diabetes incidence in AhR deficient NOD mice
Authors: Ehrlich Allison , Pennington Jamie , Greer Renee , Shulzhenko Natalia , Kerkvliet Nancy
Published: January 2023

AhR activation by the ligands TCDD and Cl-BBQ leads to potent immunosuppression and prevents hyperglycemia in NOD mice. In addition to exogenous treatment, AhR can be activated by endogenous ligands that control inflammation. AhR−/− mice on the C57Bl6 background have enhanced inflammatory responses following infectious or allergenic exposure in comparison to AhR wild type mice. We generated AhR−/− mice on the NOD background and hypothesized they would display an increased incidence of diabetes. Surprisingly, the opposite finding was observed; in the absence of AhR, female NOD mice have a significantly reduced onset of diabetes in comparison to wild type mice. A similar trend was observed between knockout and wild type male mice. While immune cell composition was unchanged in the pancreatic LN, an increase in the percentage of F4/80+ macrophages and CD25+Foxp3+ T cells was observed in mesenteric LN of NOD.AhR−/− mice. Changes in gene expression in the ileum of NOD.AhR−/− mice included reduced Gzmb, Il10 and Lcn2. Commensal bacteria (including SFB and Akkermansia) have been identified that influence diabetes incidence in the NOD mouse model. It has been shown that microbial biproducts can activate AhR, and in turn, AhR activation (or lack thereof) can impact the microbiome. Thus, we hypothesize that NOD.AhR−/− mice have altered microbiota and that these changes contribute to the reduction in diabetes incidence. Initial observations demonstrated changes in fecal consistency and an increase in SFB associated with AhR deficiency. Current studies are focused on determining if shifts in the microbiome are directly associated with the reduction of diabetes incidence in NOD.AhR−/− mice.
Metabolic inhibitors elimiante the expansion of autoimmune follicular helper T cells but not that induced by T-dependent-immunization in lupus mice
Authors: Choi Seung-Chul , Yin Yiming , Morel Laurence
Published: January 2023

The expansion of follicular helper CD4 T (Tfh) cells is tightly associated with disease severity in lupus patients, following multiple studies in various mouse models showing that they are necessary for the production of class-switched high-affinity pathogenic autoAbs. We have recently shown that both glycolysis and mitochondrial oxidative metabolism are elevated in CD4 T cells from lupus prone B6.Sle1.Sle2.Sle3 (TC) mice as compared to B6 controls, and that a treatment combining 2-deoxy-D-glucose (2-DG), which inhibits glucose metabolism, and metformin, which inhibits oxygen consumption, normalized lupus T cell functions in vitro and reverted disease. We have expanded these results to B6.lpr, BXSB.Yaa and NZB/WF1 models of lupus. Remarkably, the number and the frequency of Tfh cells was normalized in these 4 models of lupus treated with a combination of 2DG and metformin, corresponding to a drastic reduction of autoantibodies. Treatment of normal or lupus-prone mice with the same inhibitors had however little effect on the production of TD antibodies or the expansion of Tfh cells following immunization with a nominal antigen. This suggests that Tfh cells supporting the production of autoAbs are quantitatively or qualitatively different from Tfh cells providing protective humoral immunity against pathogens. Moreover, it suggests that autoimmune Tfh cells have higher metabolic requirements than the metabolically quiescent Tfh cells that have been recently described in TD-responses. Studies to characterize the specific metabolic requirements of autoimmune Tfh cells are ongoing. Overall, our results predict that targeting Tfh cellular metabolism provides an effective and safe therapeutic approach for systemic autoimmunity.
CD4 Expression on Splenic DCs Coordinates their Positioning in a Cell-Intrinsic Manner and is Necessary for Optimal CD4+ T cell Activation
Authors: Stolley J. Michael , Campbell Daniel J.
Published: January 2023

The immunological dogma of MHC-class restriction states helper T cells expressing the co-receptor CD4 perceive antigen in the context of MHC-class II while cytotoxic CD8+ T cells recognize antigen presented on MHC-class I. Paradoxically, dendritic cells (DCs) which express high levels of MHC molecules (yet lack recombined antigen receptors) differentially express CD4 and CD8α, which have classically been used to subset DC populations within secondary lymphoid tissues. Why DCs concurrently express MHC molecules and T cell co-receptors has puzzled immunologists since their discovery on DCs in the mid 1990’s. However, studying the function of these molecules on DCs is confounded by the fact that their T cell counterparts are absent in CD4 or CD8α-deficient mice. Here using a mixed chimera system in which CD4 deficiency can be restricted specifically within the DC compartment, we show CD4-expression on DCs is necessary to drive optimal antigen-specific CD4+ T cell proliferation in vivo. Impaired T cell proliferation which occurs when DCs lack CD4 appears to result from their inability to efficiently access the T cell follicle. Therefore, DC-intrinsic CD4 expression coordinates the micro-anatomical positioning of DCs within the spleen and is necessary for optimal CD4+ T cell activation.
Trafficking of IL-17-producing CD4+, but not CD8+, T cells to the skin requires CCR6 expression
Authors: Kish Danielle , Fairchild Robert L
Published: January 2023

Contact hypersensitivity (CHS) is a CD8-mediated response to hapten sensitization and challenge of skin. During elicitation of CHS, hapten-primed CD8 T cells producing IFN-g and IL-17 are recruited to the challenge site by hapten-presenting endothelial cell (EC)-derived chemokines. Our previous results have shown that CXCL9 directs localization of IFN-g producing CD8 T cells to the challenge site, but chemokines directing localization of IL-17-producing CD8 T cells to the site remain unknown. DNFB hapten application to the skin of sensitized mice or to EC in culture induced TNFa-dependent expression of CCL20 within 1 h. Hapten-stimulated ELISPOT assay of flow-sorted CD8 T cells from draining lymph nodes of sensitized mice indicated many CCR6+CXCR3+ and CCR6+CXCR3− producing IL-17, but low numbers of CCR6−CXCR3+ cells producing IL-17. Hapten-induced IFN-g production was restricted to CXCR3+ CD8 T cells. Anti-CCL20 mAb did not inhibit CHS responses when given to sensitized WT mice during challenge and normal CHS responses were observed in sensitized and challenged CCR6−/− mice. Sensitized CCR6−/− mice had increased hapten-specific IFN-g and IL-17 producing CD8 T cells in the skin-draining lymph nodes when compared to sensitized WT mice. In contrast to sensitized WT mice, sensitization of CCR6−/− mice also induced high numbers of hapten-primed CD4+T cells producing IFN-g and IL-17. However, depletion of CD8, but not CD4, T cells from sensitized CCR6−/− mice abrogated the CHS response to hapten challenge. These results indicate that the CD8 T cell mediated CHS response can occur independently of CCR6 expression whereas trafficking of hapten-primed CD4 T cells to the skin to mediate the response requires CCR6.