bluuebunny/crossref_metadata_2025 huggingface dataset:
Bystander CD8+ T cells are Important Antiviral Effectors in the Brain
Authors: Berger Jennifer , Monogue Brendan , Heiden Dustin , Klarquist Jared , Beckham John David , Berg Leslie J
Published: August 2023

The central nervous system (CNS) presents a unique immunological environment with restricted infiltration of immune cells from the periphery. Since much is unknown about CNS-infiltrating immune cells during infection, we utilized the attenuated Venezuelan equine encephalitis virus vaccine strain TC83 (VEEV-TC83) to study T cell responses to this viral infection. VEEV-TC83 intracranial (IC) infection of wild-type mice produces high viral titers in the brain without lethality, enabling the study of the complete antiviral immune response in the CNS. Many T cells rapidly (5dpi) infiltrate the CNS, in a seemingly antigen-independent manner, upon IC VEEV-TC83 infection. During infection, CD8+ T cells (~40% of CNS-infiltrating T cells) in the brain express high levels of innate-memory markers. These bystander CD8+ T cells can produce IFNγ when treated with IL-12 and IL-18, cytokines known to induce innate signaling. IL-12, IL-18, and IL-15 (another innate-memory T cell supporter) are upregulated upon IC VEEV-TC83 infection. IC infection of IL-15 knockout (KO) mice results in decreased CNS infiltration by bystander CD8+ T cells. CD8+ T cells that do infiltrate the brains of IL-15 KO mice express fewer activation markers and cytokines, and are therefore likely not fully functional. Additionally, microglia express innate-memory cytokines during infection and likely support the recruitment and function of bystander CD8+ T cells in the CNS during infection. Data deposited in GEO indicate that infection with many neurotropic viruses induces Il15, Il12, and Il18 expression in CNS cells and tissues. Bystander CD8+ T cells are likely an important first line of defense during viral infection of the CNS.

Supported by grants from the NIH (Training grant T32 AI07405 and RO1 NS123431)
Oral and intranasal Ad5 influenza vaccination induces hemagglutinin-specific and influenza neutralizing antibodies in serum and milk of pregnant and lactating ferrets
Authors: Langel Stephanie N , Beverly Christopher , Byrne Jake , Sun Francis , Gurley Sarah Anne , Tomaras Georgia D , Tucker Sean N , Johnson Susan
Published: August 2023

Pregnant women and young children are at increased risk for severe influenza infection. While there are intramuscular (IM) inactivated influenza vaccines available to pregnant individuals, IM immunization may not be an ideal route to boost neutralizing antibodies in breastmilk (BM). Therefore, innovative vaccine strategies are needed to increase BM antibodies that promote neonatal protection against influenza. We hypothesize that maternal mucosal immunization stimulates a protective influenza-specific antibody response, by generating antibodies in both blood and BM. To test this, we immunized pregnant and lactating ferrets at two weeks prepartum and two weeks postpartum with an H1N1 hemagglutinin (HA) adenovirus type 5 (Ad5) vaccine via oral or intranasal routes. We detected HA-specific IgG antibodies in dam serum and HA-specific IgG and IgA antibodies in secreted milk after oral and intranasal immunization by ELISA and chemiluminescence assays. We demonstrated that serum and BM from oral and intranasally immunized dams neutralized influenza virus in vitro. Additionally, ferret kits born to oral and intanasally immunized dams had HA-specific IgG antibodies in circulation at 5–6 weeks of life. These preclinical data led to the announcement by Vaxart, Inc. that they will test their oral tablet vaccine in human lactating mothers to determine if vaccination induces BM antibodies. Future work includes using our ferret model to determine the relationship between BM antibody quantity and function and neonatal protection against influenza. An easy-to-deliver mucosal vaccine that boosts systemic and mucosal protective antibodies in the maternal-neonatal dyad is an ideal strategy for protection of these vulnerable populations.

Bill and Melinda Gates Foundation grant INV-22595D
Antiviral and autoimmune antibody levels for two years post SAR-CoV-2 infection show uniquely durable vaccination responses and partial waning of self-reactive IgG
Authors: McConney Christina , Ayuso Maria-Jose , Smith Erika , Volfson-Sedletsky Victoria , Sagar Manish , Olson Alex , Bhadelia Nahid , Snyder-Cappione Jennifer
Published: August 2023

As temporal distance from the start of the COVID-19 pandemic increases, there is mounting concern of the long-term immunological consequences of SARS-CoV-2 infection(s). Abnormally high levels of a panoply of autoantibodies are found during acute COVID-19, and to date it is still unclear if these antibodies are sustained for a year or longer post-infection. Additionally, if there is improved longevity of vaccine-induced antibodies when administered against the immune backdrop of previous SARS-CoV-2 infection remains to be elucidated. To address these questions, we measured a series of anti-viral and autoimmune antibodies from samples taken at intervals from seven subjects infected with SARS-CoV-2 in the spring of 2020 (five outpatient and two hospitalized). Blood draws were performed beginning at acute disease and for an additional 10–25 months. Plasma IgG and IgA antibodies reactive with SARS-CoV-2 spike, RBD, and nucleocapsid, as well as CMV Mosaic, EBV GP 350, and Flu H1 were measured using the BU ELISA protocol, autoantibodies to self-antigens associated with connective tissue disease were measured using a commercial kit. Anti-viral antibody responses induced by vaccination were sustained for more than one year post SARS-CoV-2 infection in multiple subjects. Autoantibody trajectories varied, with many positive ‘hits’ falling below baseline after 1 year, and others remaining high for all time points measured. Future work will include increasing subject numbers to a larger cohort with differing clinical presentations and outcomes to define connections between serological signatures, protection from re-infection, and PASC.

Supported by grants from CTSI Pilot grant (NIH/NCATS 5UL1TR001430-05), BU Internal Funding, MassCPR PASC Subaward #FP-0034311, and NIH R01 AG065050-01A1
Subcapsular sinus macrophage infection after viral dissemination to distal lymph nodes maximizes germinal center responses
Authors: Kalia Anurag , Aguilar Cynthia C , Hickman Heather D
Published: August 2023

Neutralizing antibodies are a hallmark of protective humoral response against many viral infections. Microanatomical structures called germinal centers (GCs) within the secondary lymphoid organs are the sites where B cells get differentiated into high affinity antibody secreting, class switched and long-lasting plasma and memory B cells. Most studies of antigen movement leading to B cell activation have been performed using subcutaneous injection, mimicking the natural route of infection. Under this condition, virus infects sinusoidal lymph node macrophages which relay the antigen to B cells to drive the GC responses. However, not much is known about antigen movement during the generation of GCs in the non-draining (or distal lymph nodes) after systemic viral dissemination. Using ZIKV infection of mice, we have mapped virus dissemination through two separate lymph node chains. Surprisingly, GCs efficiently formed in distal LNs, although with delayed kinetics compared to those directly draining the infection site. Remarkably, virus in the blood alone was not sufficient to produce GCs. Instead, virus still reached the LNs through the lymphatics and infected LN sinusoidal macrophages. This coincided with virus-induced vascular permeability in the tissue. Thus, GC formation still proceeds through sinusoidal LN macrophages even when virus is spread through the blood. Our findings reveal a conserved mechanism for antiviral B cell development in LNs distal from the site of infection.
Coxsackievirus B3 elicits a protective, sex-specific CD8+ T cell response in female mice
Authors: Robinson Christopher M , Dhalech Adeeba H , Condotta Stephanie A , Corn Caleb , Richer Martin J
Published: August 2023

Sex is a significant contributor to the outcome of human infections. Males are frequently more susceptible to viral, bacterial, and fungal infections, often attributed to weaker immune responses. In contrast, a heightened immune response in females enables better pathogen elimination but leaves females more predisposed to autoimmune diseases. Unfortunately, the underlying basis for sex-specific immune responses remains poorly understood. Here, we show a sex difference in the CD8+ T cell response to an enteric virus, Coxsackievirus B3 (CVB3). We found that CVB3 induced expansion of CD8+ T cells in female Ifnar−/−mice but not in male Ifnar−/−mice. Using a recombinant CVB3 virus expressing a model CD8+ T cell epitope, we found that the expansion of CD8+ T cells is viral-specific and not due to bystander activation. CVB3 also increased the proportion and number of CD11ahiCD62Llo CD8+ T cells in female mice, indicative of T cell activation. However, CD8+ T cell activation is dampened by the primary male sex hormone, testosterone, in male mice. Finally, using antibodies to deplete CD8+ T cells, we found that CD8+ T cells are required to limit CVB3-induced disease in female mice. These data demonstrate that CVB3 induces a sex-dependent CD8+ T cell response that is important for viral clearance in female mice and highlights the importance of sex differences in the immune response to viral pathogens.

This work was funded by a K01 DK110216, R03 DK124749, and a Biomedical Research Grant from the Indiana Clinical and Translational Sciences Institute to CMR.
B cells regulate early lung macrophage activation during influenza virus infection via production of acetylcholine
Authors: Prieto Antonio Cembellin , Murray Kaitlin , Luo Zheng , Reardon Colin , Baumgarth Nicole
Published: August 2023

The neurotransmitter Acetylcholine (ACh) is produced by the enzyme choline acetyltransferase (ChAT). Recently, ChAT expression was found in leukocytes, however, the function of leukocyte-derived ACh is incompletely understood. Flow cytometry showed that most ChAT-expressing cells in the spleen, lymph nodes, lung, and pleural cavity of ChAT-GFP reporter mice are B cells. B-1 cells contain the highest frequencies of ChAT-expressors. B cell-specific Mb1-Cre+ ChATfl/fl knockouts (BChat-KO) showed comparable bone marrow B cell development and steady-state serum IgG and IgM production as wild-type littermate controls. Given earlier studies showing TLR-mediated induction of ChAT in B cells, we studied infection of ChAT-GFP mice with Influenza A/Puerto Rico/8/34. Infection resulted in an accumulation of ChAT-expressing B cells in the lungs by 1- and 3-days post infection (dpi). Moreover, BChat-KO mice showed significant reductions in viral loads at 1 dpi, suggesting that B cell-derived ACh regulates early innate antiviral immunity. Indeed, BChat-KO showed increased TNFa-expressing and CD86hi lung macrophages at 1dpi. Consistent with this, acetylcholine reduced TNFa-expression in LPS-stimulated macrophages in vitro. In contrast, Lck-Cre+ ChATfl/fl that lack ChAT in T cells showed no difference in lung viral loads at 1dpi compared to controls, and the numbers of ChAT-expressing T cells in the lungs and draining lymph nodes did not increase until 7dpi. Together these data suggest a new regulatory function for B cells in early virus-induced lung inflammation via production of ACh.

- Chan Zuckerberg Initiative to Colin Reardon and Nicole Baumgarth - NIH/NIAID R01AI148652 Nicole Baumgarth
TGF-β promotes the resolution of acute hepatitis B virus infection
Authors: Cimino Jesse L , Moshkani Safiehkhatoon , Bailey Jacob T , Rexhouse Catherine , Robek Michael D
Published: August 2023

An estimated 296 million people live with a chronic hepatitis B virus (HBV) infection, resulting in approximately 800,000 annual deaths. Thus, despite the availability of an effective vaccine, HBV remains a global health concern. Most children and a fraction of adults infected with HBV develop a persistent liver infection that yields lifelong complications. It is believed that the immune tolerogenic environment of the liver and the dysfunction of T and B cells contribute to HBV persistence in these individuals. Here, an adeno-associated virus (AAV)-based mouse model of HBV infection was utilized to investigate the underlying mechanisms of immune dysfunction that impede viral clearance. HBV infection was initiated in mice by AAV-HBV transduction, and TGF-β was depleted to determine the role of this cytokine in HBV persistence. As TGF-β is often associated with negative regulation of the immune response, we hypothesized that its inhibition might improve HBV-specific T and B cell responses. However, the lack of TGF-β resulted in impaired HBV surface antigen (HBsAg) clearance and diminished anti-HBsAg antibody levels, revealing a positive role for TGF-β in the antibody response to HBV. This effect of TGF-β on the antibody response to HBsAg was unique to AAV-HBV transduction, as a similar increase in anti-HBsAg was not observed by TGF-β depletion following infection with recombinant vesicular stomatitis virus expressing HBsAg. We also observed differences in B cell activation marker expression during HBsAg clearance in mice transduced with AAV-HBV and depleted of TGF-β. Together, these findings highlight the impact of TGF-β on the B cell response to HBV and have begun to define a unique role for this cytokine in promoting HBV clearance.

Supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R01AI148354. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Terminally exhausted CD8+ T cells contribute to age-dependent severity of human metapneumovirus
Authors: Parks Olivia Bartlett , Eddens Taylor John , Sojati Jorna , Lan Jie , Zhang Yu , Ramsey Manda , Erickson John , Byersdorfer Craig A , Williams John V
Published: August 2023

Objective

Human metapneumovirus (HMPV) is a leading cause of acute respiratory infection with severe morbidity and mortality among older adults. HMPV seropositivity is universal due to childhood exposure, yet antibodies do not fully protect. We hypothesized that severe HMPV disease in the elderly was due to deficient CD8+T cell responses.

Methods

B6 aged 70–71wk mice and young 6–7wk mice were infected with 2×106 PFU HMPV. Seven days post infection, mice were euthanized and lung, bronchoalveolar lavage (BAL), spleen, and draining lymph nodes were collected for flow cytometry. Lung and BAL supernatant was collected for viral titer, histology, and cytokine measurement.

Results

Aged 70–71wk C57BL/6 mice exhibited worsened weight loss, disease, and lung pathology with delayed viral clearance compared to young adult mice. Aged mice developed fewer HMPV tetramer+ CD8+ T cells, which demonstrated higher expression of PD-1 and other inhibitory receptors and were less polyfunctional with decreased granzyme B. Transplant of aged T cells into young mice and vice versa, as well as adoptive transfer of young or aged CD8+ T cells into Rag1−/− mice, recapitulated the HMPV aged phenotype, suggesting a cell-intrinsic age-associated defect. HMPV-specific aged CD8+ T cells exhibited a terminally exhausted TCF1− TOX+ EOMES+ phenotype. PD-1 blockade or 4-1BB agonist failed to improve the aged CD8+ T cell response.

Conclusion

Aged mice exhibit more severe respiratory disease when infected with HMPV. There is a cell-intrinsic age-associated defect in CD8+ T cells as shown through the transplant models. This study also identifies terminal CD8+ T cell exhaustion as a mechanism of severe HMPV disease in the elderly.

NIH AI085062 (JVW)

1F30HL159915-01A1 (OBP)

T32 GM-008208 (OBP, JS)

Henry L. Hillman Foundation (JVW)

T32 AI089443 (YZ)

K12 HD000850 (TE)
Determination of autoantibody repertoire in the mouse model of viral myocarditis and in patients with non-ischemic cardiomyopathy by PhIP-Seq
Authors: Rasquinha Mahima T , Jayaraman Sahana , Lasrado Ninaad , Duryee Michael J , Qiu Haowen , Riethoven Jean-Jack , Mone Kiruthiga , Sur Meghna , Anderson Daniel , Larman H Benjamin , Reddy Jay
Published: August 2023

Various infectious and non-infectious agents can cause myocarditis that may lead to cardiomyopathy with viruses being the most common suspects. Mechanistically, autoantibodies have been demonstrated for various antigens, in animal models and dilated cardiomyopathy patients, but their role continues to be enigmatic. First, by using the Coxsackievirus B3 (CVB3) infection model in A/J mice, we applied PhIP-Seq, a high-throughput platform to evaluate antibodies for various antigens. The data revealed the identification of antibodies to 32 antigens that were not previously reported. By investigating the biological significance of a few select antigens, we noted that cytochrome c oxidase assembly factor 4 homolog (COA4)-reactive antibodies were generated only in mice infected with CVB3 but not in influenza-infected animals proving virus specificity for antibody reactivity to COA4. Further immunogenicity studies revealed that animals immunized with COA4 did not develop myocarditis, nor was the T cell response generated, implying that COA4 is unlikely to act as a bonafide autoantigen. Alternatively, the COA4-reactive antibodies may represent biomarkers of viral myocarditis. Second, using the PhIP-Seq platform, we investigated the antibody profiles of human sera obtained from 25 age-matched non-ischemic cardiomyopathy patients and healthy controls. The analysis revealed significantly elevated levels of antibodies to 33 antigens, and the detection of the major candidates is being analyzed by ELISA as a secondary assay. Overall, our data suggest that the PhIP-Seq allows us to comprehensively analyze antibody repertoires that may lead to the identification of novel autoantibodies or their use as biomarkers.

This work was supported in part by the institutional funds from the University of Nebraska-Lincoln (J.R.), the Transformational Grant 18TPA34170206 from the American Heart Association (J.R.), and the National Institutes of Health grants R01 HL114669 (J.R), R01 GM136724 (H.B.L.).
Steady-state Langerhans cells induce Tfh and B cell responses through a type I interferon and IL-6 independent mechanism
Authors: Bouteau Aurélie , Zurawski Sandra M , Zurawski Gerard , Igyártó Botond Z
Published: August 2023

Langerhans cells (LCs) can support the induction of antibody responses against foreign antigens without a danger signal. These findings contradict the danger model proposed to define how the adaptive immune responses are mounted. Deciphering how LCs, and some other dendritic cell subsets, support adaptive immune responses in steady-state could lead to a better understanding of how immune responses are induced and regulated and, ultimately, to developing more efficient immunotherapeutics for autoimmune- and infectious diseases. Here we used a well-established steady-state antigen targeting model to dissect the mechanism by which LCs support T follicular helper (Tfh) cells and antibody responses. Using bone marrow chimeras, Cre-lox system, and blocking antibodies, we found that IL-6, which is critical in the induction of Tfh cells and antibody responses in inflammatory conditions, played no role in LC-induced adaptive immune responses at steady-state. Type I interferon signaling was also dispensable in this regard. However, our preliminary data support that induction of humoral immune responses by steady-state LCs depends on membrane-bound co-stimulatory molecules, such as ICOS/ICOS-L. Thus, these data suggest that adaptive immune responses against foreign antigens in the absence of inflammation are generated through a distinct mechanism that likely does not involve inflammatory cytokines.

Supported by grants from NIH R01AI146420 and Thomas Jefferson University startup funds.
Intrinsic B cell TLR-BCR linked coengagement induces class-switched, hypermutated, neutralizing antibody responses in absence of T cells
Authors: Rivera Carlos Eduardo , Zhou Yulai , Chupp Daniel P , Yan Hui , Fisher Amanda D , Simon Raphael , Zan Hong , Xu Zhenming , Casali Paolo
Published: August 2023

Maturation of an antibody response entails somatic hypermutation (SHM), class-switch DNA recombination (CSR), plasma cell differentiation and generation of memory B cells, and is thought to be generally dependent on T cell help. We show here that B cell TLR4-BCR (B cell receptor for antigen) coengagement by NP-LPS (from E. coli) or TLR5-BCR coengagement by Salmonella flagellin induced specific, mature antibody responses in Tcrβ−/− Tcrδ−/− mice and in NSG/B mice (B cells only). Such coengagement requires physical linkage of TLR and BCR ligands, “linked coengagement”, which induced germinal center-like differentiation, clonal expansion, CSR/SHM, intraclonal diversification, plasma cell differentiation, TLR expression, production of specific antibodies and generation of memory B cells supporting anamnestic antibody responses, overall indicating an affinity maturation process. In Tcrβ−/− Tcrδ−/− mice, linked coengagement of TLR4 and cognate BCR by LPS (Lipid A and polysaccharidic O-antigen) or TLR5 and cognate BCR by flagellin induced neutralizing and protective antibodies to E. coli or S. Typhimurium, limiting infection and pathology. Intrinsic B cell TLR-BCR linked coengagement likely plays an important role in inducting mature and neutralizing antibody responses in the absence of T cells. It would also play an important role in early stages of T-dependent anti-microbial responses when T cell help is not yet available, as well as potentiate the late-stage response when T help is available. Finally, it may inform the development of B cell TLR-based vaccines to microbial pathogens, particularly for subjects with an immature T cell compartment, such as the infant, or declining T cell function and clonality, such as the elderly.

Supported by NIH grants AI 079705, AI 105813/105813S1, AI138944, AI 167416 and Lupus Research Alliance grant LRA 641363 to PC and PHS grant K12 GM111726.
Diminished γδ T Cells during allergic skin inflammation is mediated by IL-4 signaling in keratinocytes
Authors: Zhang Wenwu , Pajulas Abigail , Akhtar Nahid , Zhao Jennifer , Kaplan Mark H
Published: August 2023

Atopic dermatitis results in profound changes in the function of the skin that include diminished barrier function and altered production of antimicrobial peptides. Dendritic epidermal γδ T cells (DETC) are a subset of γδ T cells that play an important role in the wound repair and inflammation process. Our previous work identified a loss of DETC in Stat6VT mice and in MC903 induced skin inflammation mouse model that was dependent on IL-4. However, the mechanisms through which IL-4 mediates this loss of DETCs are still not clear. In this study, we show that IL-4Rα germline knockout mice (Il4ra−/−) have an increased DETC population in skin that correlates with a higher rate of wound closure and increased EDC gene and fibronectin 1 expression. In Il4ra−/− mice, MC903 induced loss of DETCs was alleviated. Bone marrow chimeras of Il4ra−/− and WT bone marrow into Il4ra−/− mice retained the increased DETCs in the skin of Il4ra−/− host mice. Importantly, IL-15 secretion from skin keratinocytes was decreased dramatically in the MC903 induced skin inflammation model and injection of IL-15 rescued the loss of DETCs. Furthermore, conditional knock out of IL-4Rα from Keratinocytes of Il4ra fl/fl K14cre mice also showed an increase of this DETC populations in skin, increased IL-15 expression and less skin inflammation. Together, these results identify an IL-4-dependent circuit that regulates DETCs in the skin by repressing IL-15 expression in keratinocytes. This provides further insights into the ability of IL-4 to promote the development of atopic lesions and impair wound healing.

Supported by grants from NIH (R01AI095282)
DJ-X-013 ameliorates inflammation by reducing TNF-α/NF-κB expression and experimental colitis by inducing myeloid-derived suppressor cells
Authors: Mandal Mousumi , Rakib Ahmed , Kiran Sonia , Hwang Dong-Jin , Li Wei , Miller Duane D , Singh Udai P
Published: August 2023

Inflammation is a body’s defense response for a short duration however if persists for long periods, it causes tissue damage and pathological consequences. Moreover, it paves the way for several diseases including inflammatory bowel disease (IBD). Therefore, there is an urgent need for a natural and safe anti-inflammatory therapeutic compound. To address this issue, we aimed to determine the anti-inflammatory effects of DJ-X-013, using lipopolysaccharide (LPS) activated RAW 264.7 macrophage in vitro and dextran sodium sulfate (DSS) induced experimental model of colitis in vivo. DJ-X-013 drastically reduced tumor necrosis factor-alpha and inducible nitric oxide synthase expression in LPS-activated RAW 264.7 macrophages. Further, DJ-X-013 also impeded the expression of NF-κB and other inflammatory markers and pathways like IL-6, IL-1β, STAT3, ERK, and JNK as compared to LPS-activated cells. Additionally, DJ-X-013 also modulates the migratory activity of RAW 264.7 macrophages during wound healing. Next, using DSS induced colitis model, we observed that the reduction of body weight and colon length were slightly improved in the DJ-X-013 group compared to the control. Intriguingly, myeloid-derived suppressor cells, neutrophils, and CD8+ T cells increased, while Th17 cells frequency decreased in the spleen, mesenteric lymph nodes, and colon lamina propria in DJ-X-013 treated group as compared to DSS. However, natural killer and natural killer T cells were marginally enhanced in the DJ-X-013 group as compared to the DSS alone. Hence, the DJ-X-013 exhibits an immunomodulatory effect and might be a promising drug for IBD and other inflammatory diseases, however, further investigation is required for prudent conclusions.

This research is supported by NIH grant R01 AI140405
Host metabolic reprogramming ameliorates disease severity and colitis during Clostridioides difficile infection
Authors: Tubau-Juni Nuria , Bassaganya-Riera Josep , Leber Andrew J , Alva Sameeksha S , Baker Ryan , Hontecillas Raquel
Published: August 2023

Clostridioides difficile is an anaerobic, opportunistic bacterium that can cause severe infections in the gastrointestinal tract upon disruption of gut microbiome. C. difficile infection (CDI) affects 450,000 people yearly only in the U.S. The high recurrence rates associated to antibiotic regimen, the current standard of care, highlight the need to investigate novel antimicrobial free therapeutic approaches for the treatment of CDI. Here, we have conducted a global transcriptomics analysis and metabolic computational modeling, using our developed M2 pipeline, of an in vivo murine 10-day time course study of CDI to identify novel host metabolic mechanisms with therapeutic potential. Our analysis has reported a biphasic modulation of colonic metabolism, characterized by increased glycolysis during the induction of effector responses, while mitochondrial metabolism is favored in the recovery phase of the infection, when regulatory and healing processes are predominant. Inhibition of glycolysis through administration of 2-Deoxy-D-glucose (2-DG) ameliorates diseases severity, protects from mortality, and reduces colitis in a mouse model of CDI. In vitro treatment with 2-DG, an inhibitor of glycolysis, in T-84 cells exposed to C. difficile toxins A and B protects epithelial barrier integrity and decreases secretion of inflammatory mediators. In conclusion, host immunometabolic reprogramming represents a promising novel antimicrobial-free therapeutic approach for CDI.

Supported by a grant from DTRA (HDTRA1-20-1-0021)
Skin immunity during pregnancy
Authors: Sun Lillian , Corral Dan , Chi Liang , Enamorado Michel , Bouladoux Nicolas , Belkaid Yasmine
Published: August 2023

Pregnancy presents a challenging immunological dilemma. The maternal immune system must tolerate fetal antigens, which implies pregnancy may induce specific mechanisms of immunoregulation. Yet within this regulatory milieu, protection against pathogens must be maintained. How does the maternal immune system navigate these opposing demands? Successfully navigating this immunological dilemma is essential for supporting the reproductive fitness of the organism, and generally, the survival of any mammalian species.

Of particular importance in this context are barrier tissues, like the skin, which represent the primary portal of entry for pathogens. These barrier tissues are an especially important first line of defense during pregnancy, since adaptive immunity must be regulated to tolerate the fetal antigens which distribute throughout maternal tissues. However, little is known about how immunity in barrier tissues is altered during pregnancy.

Our preliminary studies have identified an increase in ceramide levels in the skin during pregnancy. Ceramides have been shown to be critical mediators of skin barrier function, and may prevent pathogens from breaching the skin. Additionally, in the setting of S. aureus infection, pregnant skin demonstrated increased infiltration of innate immune cells, as well as an increase in type I immunity among adaptive cells. Together, these data suggest that the protective mechanisms of the skin are altered during pregnancy. Further insights in this area may elucidate an especially critical role for barrier tissue immunity in defending against infection during pregancy, and may eventually help guide improved clinical strategies to prevent and treat infections in pregnant mothers.

Supported by Perelman School of Medicine’s Medical Scientist Training Program and intramural research funding from NIAID.
Discovery of TIGIT inhibitors by phage display
Authors: Huang Xueting , Hsiao Chia-Hung Christine , Wiemer Andrew J
Published: August 2023

TIGIT is an emerging immune checkpoint receptor expressed higher than PD-1 in some tumors especially anti-PD1 resistant tumors. By competing with the co-stimulatory receptor CD226/DNAM-1 for binding to CD155/PVR, TIGIT transduces inhibitory signals and suppresses the immune response. Therefore, we sought to discover TIGIT inhibitors for improving tumor immunotherapy. Through directional cloning, bacterial transformation and phage amplification techniques, we generated a novel genetically encoded, phage-displayed bicyclic peptide library comprised of more than a million peptide sequences. Depending on the two combinations of different disulfide bonds, each peptide backbone is predicted to be folded into either endo-bicyclic or exo-bicyclic structure. We conducted three rounds of phage display panning against recombinant TIGIT IgV domain that enriched high-affinity peptides and identified promising TIGIT binding peptides. Phage ELISA showed binding of phage-presented peptides to recombinant TIGIT IgV and competitive ELISA evaluated the ability of the peptides to disrupt interactions between TIGIT and PVR. This work validated the efficiency of the phage-displayed bicyclic peptide library for discovery of ligands targeting protein receptors and led to identification of novel TIGIT inhibitors.

AAI Careers in Immunology Fellowship
Systems approaches to characterize the local heterogeneities in NK cell antitumor immunity in MHC I-disparate human non-small cell lung cancer
Authors: Dolatshahi Sepideh , Erdogan Remziye , Ageeb Nardin , Obeid Youssef , Mauldin Ileana S , Goundry Kate , Hossain Mahdin , Slingluff Craig , Bullock Timothy N , Brown Michael G
Published: August 2023

Checkpoint immunotherapy has shown encouraging yet limited success in non-small cell lung cancer (NSCLC) patients. We combined multiplexed fluorescence imaging with quantitative spatial analysis to investigate antitumoral immunity in NSCLC. As seen before, we observed frequent MHC class I (MHC I) loss and development of CD8 T cell refractory tumors. MHC I-deficiency suggested NK cells may confer protection in NSCLC. Indeed, we found higher CD3−CD56+ NK cell numbers were associated with both disease-free and overall survival. Using partial least squares regression, we found that both T cells and NK cells were significantly enriched in MHC I-bearing tumors. Quantitative spatial characterization of tumor cell MHC I expression revealed vast intra- and inter-tumoral heterogeneity, which was highly associated with the local lymphocyte landscape. To infer functional differences in local cell-cell communication, we built computational models based on spatial single cell neighborhood profiles to discriminate between IFNg-disparate lymphocytes. We found IFNg+ NK cells more frequently associated with other IFNg+ NK cells or T cells in comparison to IFNg− NK cells, or IFNg+ T cells which more frequently had IFNg− lymphocyte neighbors. Moreover, IFNg+ NK cells were unique in their capacity to also associate with MHC I− tumor cells. Together these data implicate a key role for NK cells in recruiting and activating other lymphocytes in MHC I-bearing tumors, and further suggest NK cells may be key to developing effective therapies even in CD8 T cell refractory tumors.

Supported by UVA Carter Immunology Center Collaborative Research Award
Single cell RNA sequencing reveals heterogenous immune landscape of tumor infiltrated immune cell population against glioblastoma
Authors: Kang ByeongHoon
Published: August 2023

Glioblastoma (GBM) is the most aggressive glioma. Recently, immunotherapy using immune-checkpoint blockage (ICB) has provided therapeutic efficacy to various tumors. Despite an attractive ICB therapy, there have not been beneficial outcomes about GBM patients due to the characteristic of immunosuppressive environment. Thus, it is essential to understand the features of GBM immune microenvironment. Here, we profile immune cells of GBM microenvironment via single cell RNA sequencing (scRNAseq) according to GBM progression in orthotopic glioma model. We identify that kinetics of immune cell population throughout the GBM : macrophages are recruited first and T cells are recruited later. In addition, we analyze early and late stage of microglia and macrophages. Especially, GBM progression down-regulates pro-inflammatory genes and interferon responsive genes in microglia and induces M2 phenotype of recruited macrophages. We further identify that at later stage, overall T cells are increased, but regulatory T cells (Tregs) are significantly increased. In particular, CD8T cells express Nur77 but they also increase multiple inhibitory molecules and show reduction of effector molecules. These findings reveal overall profiling and features of immune cells in GBM microenvironment and provide comprehensive landscape of immune cells throughout GBM progression.
IL-17A-mediated JMJD3 regulation of integrin alpha 3 gene expression alters migration of diabetic keratinocytes and impairs wound repair
Authors: Moon Jadie Yoonjoo , Wolf Sonya , Audu Christopher , Melvin William , Joshi Amrita , Mangum Kevin , Barrett Emily , Davis Frank , Mazumder Purba , Rocco Sabrina , Erturk Ilkim , Gallagher Katherine
Published: August 2023

Keratinocytes are key structural cells in cell migration during wound repair. Keratinocyte migration is dysfunctional in diabetes and results in non-healing, however, the reason for this is unclear. Using bulk and single cell sequencing, we identified that Th17 CD4+ T-cells predominate as does an IL-17A signature in human diabetic wound tissue compared to non-diabetic controls. The effects of increased IL-17A in diabetic skin on keratinocyte function is unknown. To examine this, we subjected isolated keratinocytes to scratch injury and a 24hr rIL-17A (20ng) stimulation. We observed a significant decrease in migration rate in IL-17A stimulated keratinocytes compared to their controls. Next, we examined migration related genes in keratinocytes from mice fed with a normal (ND) or a high fat (HFD) diet. Integrin alpha 3 subunit, Itga3, (a protein known to delay keratinocyte migration during wound re-epithelialization) was higher in diabetic (HFD) keratinocytes compared to ND controls at baseline and with rIL-17A. We analyzed epigenetic enzymes known to increase gene expression and identified that JMJD3, a histone demethylase that relaxes chromatin, was increased both in diabetic keratinocytes from our human scRNA-seq and in murine diabetic keratinocytes with rIL17A. Further, treatment with a JMJD3-specific inhibitor (GSK-J4) decreased Itga3 in diabetic keratinocytes, suggesting that JMJD3 may be relevant to IL-17A-mediated regulation of integrin alpha 3 and other keratinocyte migration genes. Continued investigation into upstream IL-17A signaling in normal and diabetic keratinocytes that alters downstream migration is crucial for our understanding of impaired keratinocyte functions associated with diabetic wound repair.

NIH T32 AI 007413, Research Training in Experimental Immunology Rackham Pre-Candidate Graduate Student Research Grant, University of Michigan
High-dimensional analysis of the T cell response in multiple myeloma and the potential efficacy of harnessing tumor-reactive CD8 T cells
Authors: Prazich Jack , Jiang Jenny , Dhodapkar Madhav
Published: August 2023

Multiple myeloma is an incurable malignancy of plasma cells in the bone marrow increasingly being treated by immunotherapies with improved response and survival over traditional chemotherapy, however an unexplored potential immunotherapy is the adoptive transfer of tumor-specific CD8+ T cells. Here, we investigated the tumor-specific CD8+ T cell response in highly progressed multiple myeloma patients. We predicted neoantigens and tumor-associated antigens for each patient and grew out tumor-specific CD8 T cells from patient TIL’s using autologous B cells loaded with the predicted epitopes. We then utilized TetTCR-SeqHD, a high-throughput tetramer-based workflow, to isolate paired tumor-specific T cell receptor (TCR) sequence and antigen specificities. We subsequently transduced and functionally validated these TCR specificities and their cytotoxicity in vitro. In addition, we performed single-cell transcriptomic and proteomic analyses on TIL’s and peripheral blood T cells before and after immunomodulatory treatment to capture high-dimensional data on the cellular states of the T cell response and their response to stimulation. We observed T cells within the tumor were largely exhausted, but that patients had neoantigen-specific CD8+ T cell populations present responding to a broad array of antigens suggesting adoptive transfer of tumor-specific CD8 T cells is potentially efficacious. Last, we measured TCR repertoire changes and identified T cell clones that had expanded. We also found a number of peripheral blood T cells markers that correlated with treatment response in our patients. Next, we aim to transfer transduced tumor-specific T cells into PDX mouse models to measure preclinical therapeutic potential.

Supported by grants from CRI-STAR and NIH R01
Impaired pathogen-specific CD4+ T cell functionality in patients with CLL
Authors: Wu Jinghua , Ribeiro Oriana Jesus , Österborg Anders , Gao Yu , Buggert Marcus
Published: August 2023

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in adults and remains an incurable disease. Many patients with CLL are affected by non-malignant complications, including infectious diseases, as witnessed during the past years, with very high COVID-19 mortality rates. Accumulating data indicate that T cells might become exhausted in patients with CLL, which might be a contributing factor to poor control of pathogenic infections. Here we developed flow cytometry protocols to combine activation-induced molecule (AIM) assessment and functional read-outs to understand if bacteria- and virus-specific CD4+ T cell responses are impaired in patients with CLL. PBMCs were collected from indolent CLL patients (n=23) and age and gender-matched blood donors (n=17) to assess CD4+ T cell dysfunction against pathogenic bacteria (S. Pneumonia, H. Influenzae, M. Catarrhalis, K. Pneumoniae) and viruses (CMV, EBV, IAV). We show that the frequency of CD4+ T cell responses against most assessed bacterial species was of lower frequencies in CLL patients compared to matched control groups. Both bacteria- and virus-specific CD4+ T cells experienced, in general, signs of functional exhaustion, including diminished capacity to produce IFN-γ, TNF, IL-2, IL-17, and/or IL-21, during indolent CLL. Functional exhaustion was not coupled to increased expression of inhibitory receptors but instead reduced stem-like phenotypes of CD4+ T cells. These data indicate that untreated CLL is coupled to impaired functional capacity of bacteria and virus-specific CD4+ T cells, which might lead to poor pathogen control.

Swedish Cancer Foundation(Cancerfonden)
UCP2 Deficiency Renders Th1/2 Pathogenic Phenotype to CD4+ T cells
Authors: Choi Seungho , Mehrotra Shikhar
Published: August 2023

Uncoupling protein 2 (UCP2) is an anion transporter in the mitochondrial matrix that controls ROS production, calcium influx, and C4 metabolites. It is regarded as a metabolic regulator. A lack of UCP2 has led to an extreme clinical score with higher oxidative stress and inflammation in autoimmune illness, even though elevated UCP2 has been found in many tumors and is known to be a potential target for tumor therapy. Uncertainty still exists regarding UCP2’s function in T cells. A significant rise in the Th2/Th1 ratio was seen in this work using CD4 cells from UCP2−/− mice. These cells exhibited an improved Th2 phenotype with decreased calcium influx, elevated iNOS expression, and elevated urea metabolism. CD4 cells were treated with NOC-18, a source of nitric oxide, or Genipin to demonstrate nitric oxide-mediated Th2 polarization (inhibitor for UCP2). There were more cytotoxic markers and Th2/Th1 double positive cells. The B16/F10 tumor cells were controlled in vitro in a tumor killing assay utilizing genipin-treated CD4 cells, according to the findings. However, in UCP2−/− mice, the severity of EAE worsened, and heterogenic Th2 cells were infiltrated into central nerves system. Our findings imply that the loss of UCP2 in CD4 increases Th2 polarization with a cytotoxic phenotype and may be relevant to various illnesses.

R01 CA250458, R01 CA236379
Tet2 deficient conventional dendritic cells contribute to clonal hematopoiesis inflammation and alter immune response
Authors: Franceski Alana Marie , Luca Francesca , Goel Paran , Crown Brittany , Kuznetsova Valeriya , Costa Amanda , Welner Robert
Published: August 2023

Clonal mutations accrue with age and 20% of the elderly have dominant clonal hematopoiesis. Mutations to demethylating enzymes (including Tet methylcytosine dioxygenase 2, TET2) are common in these patients and loss of functional TET2 in mature myeloid cells increases pro-inflammatory cytokine secretion (such as interleukin 6, IL-6) leading to immune cell dysfunction.

Our group investigated the impact of TET2 mutations in immune cells and effects of altered hematopoiesis on non-mutated cells in chimeric mouse models. We utilized an MxCre x Tet2fl/fl mouse model on a CD45.1 background. After polyinosinic:polycytidylic acid excision, bone marrow was collected and transplanted retro-orbitally into CD45.1/CD45.2 hosts. Peripheral blood was analyzed at 8-, 14-, 18-, and 24-weeks using flow cytometry to test for engraftment of Tet2−/− cells. Mice were taken down and marrow, spleen, and mediastinal lymph nodes (medLN) were analyzed at 18- and 24-weeks of age.

We saw increases by frequency and counts of TET2−/− cDCs in the spleen and medLNs along with reduced production of host cDCs. Specifically, there was a slight increase in TET2−/− cDC1s and frequency of mutant cDC2s was three times greater than host, indicating host cDCs are being outcompeted. Loss of Hdac2 acylation in TET2−/− cDCs causes sustained production of IL-6, influencing Th17 and Tfh cell production and inflammatory cytokine secretion. This suggests cDCs play a role in disease progression and altering immune response. Mutant and disease-exposed cDCs are being explored using scRNAseq and ATACseq in combination with functional assays to determine gene regulatory networks governing mutant pro-inflammatory mechanisms and consequences in the education of adaptive immunity.
Live bacterial ligands universally regulate mTOR activity but are not essential for homeostatic innate immunity
Authors: Song Baobao , Zhang Xuan , Steele Hope , Chou Chih-Hsing , Ferchen Kyle , Salomonis Nathan , Herro Rana , Grimes H Leighton
Published: August 2023

Multiple studies have reported significant impact of gut microbiota on hematopoiesis, including granulocyte development and functional defects. Germ-Free (GF) mice show developmental defects due to lack of live bacteria; underscored by the development of Altered Schaedler Flora (ASF), a community of 8 bacterial species sufficient to rescue developmental immune and gastrointestinal defects. Mimicking gnotobiosis by using antibiotics perturbs the microbiome systemically. Therefore, results from such studies may represent sequelae of the drug perturbation rather than simply modifying microbiota. We studied the impact of gut microbiota signals on murine hematopoiesis without antibiotics or fecal transfer. Namely, by using mice (from Taconic) generated by embryonic transfer of C57/Bl6 fertilized eggs into females with controlled microbiota, totaling 4 groups of mice: 1. GF; 2. ASF (no Gammaproteobacteria); 3. Excluded Flora (EF, includes Gammaproteobacteria); 4. WildR7 (wild mouse microbiome). We found no significant differences in either number or effector function of bone-marrow neutrophil granulocytes, myeloid progenitors, or stem cells. The single-cell genomic landscape of CD117+ bone-marrow progenitors confirmed flow analyses but revealed a profound defect in gnotobiotic hematopoietic signatures. Importantly, standard pathogen free C57/BL6 mice were inappropriate controls. While gnotobiotic mice likely receive inert TLR stimulation via bedding and food, the addition of any group of live bacteria was sufficient to rescue gene expression downstream of mTOR signaling. Such robust signatures could be considered so essential to life that they do not depend on any single bacterial clade.

This project is supported by NIH R01HL122661.
The DEAD box RNA helicase DDX3X is required in natural killer cells
Authors: Mukhopadhyay Eshana , Krishnamurthy Durga , Ohayon David , Tuazon Jasmine , Ali Ayad , Waggoner Stephen N
Published: August 2023

The X-chromosome linked RNA helicase DDX3X plays multiple roles in RNA metabolism and antiviral interferon responses. Some viruses, including SARS-CoV-2, usurp the function of DDX3X to support viral genome replication and evasion of antiviral interferon. These findings have led to development of DDX3X inhibitors as potential antiviral agents. However, somatic loss of function mutations in this helicase are linked to neurological disease and malignancies, including a form of non-Hodgkin’s lymphoma initiated in the natural killer (NK) cell lineage. Given the critical role of NK cells in antiviral and antitumor immunity, we sought to determine how inhibition of DDX3X could affect NK-cell biology. Constitutive (Ncr1-iCre) or inducible (Ncr1-iCreERT2) deletion of Ddx3x in Ncr1-expressing cells (i.e. NK cells) in mice resulted in a complete loss of peripheral NK cells. Addition of the DDX3X inhibitor RK-33 to in vitro cultures of NK cells resulted in a similar loss of cellularity, while deletion of Ddx3x blocked development of NK cells from progenitor cells in an in vitro culture on stromal cells. Preliminary analyses suggest that loss of DDX3X results in rapid cell death of committed NK cells. Partial preservation of early stages of NK-cell development in the bone marrow of male but not female Ncr1DDdx3x suggests that the Y-chromosome DDX3Y may compensate for loss of DDX3X in supporting NK-cell survival at the earliest stages of differentiation. We identify a crucial role for DDX3 helicases in the development and survival of NK cells that may have implications for the clinical use of DDX3 inhibitors as antiviral agents.

R01 AI148080

R01 AR073228