bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of the innate immune response by IL-36 mediated inflammation during active ticks mediated ehrlichial transmission in mice
Authors: Kempaiah Kempaiah Rayavara , Harrison Mack , Saito Tais B.
Published: January 2023

Tick borne ehrlichiosis is a life-threatening disease affecting humans and animals worldwide. Early host response to pathogen-tick interaction at the site of transmission has not been well studied due to difficulties associated with natural model of infection. To study the host immune interaction with Ehrlichia muris eauclarensis (EME), a human pathogen, we compare tick transmission of EME in C57Bl/6 mice with needle inoculation. Our objective was to analyze the local and systemic infection to identify the immune pathways involved in the pathogenesis of tick borne ehrlichiosis. As previously reported, our studies show inflammatory infiltration of neutrophils and eosinophils at the site of infection within 24–48h of tick infestation. Further evaluation of the site of tick transmission demonstrated increased expression of IL36α, IL36γ, IL1β and IL18 cytokines during initial ehrlichial infection. Our analysis of the peak of infection identified an increased epidermal thickening, massive inflammatory infiltration at the site of infection and systemically, and increased levels of proinflammatory cytokines in serum. The disease progression is associated with increased bacterial load in lungs, spleen, liver and other tissues tested, with increased liver ANG-2 and apoptosis, suggesting acute vascular dysfunction and liver injury. These results demonstrate that ehrlichial transmission by ticks causes severe inflammation with up-regulation of IL36 group of cytokines, potentially activating innate immune system to facilitate bacterial dissemination and severity of the disease. Understanding the key determinants of tick mediated ehrlichiosis will enable us to identify susceptibility markers and new therapeutic targets.
SIV and CD4 depletion distinctly reactivate latent Mycobacterium. tuberculosis infection
Authors: Diedrich Collin R , Rutledge Tara , Maiello Pauline , Baranowski Tonilynn , White Alex , Borish H. Jacob , Karell Paul , Hopkins Forrest , Brown Jessica , Fortune Sarah , Flynn JoAnne L , Ambrose Zandrea , Lin Philana Ling
Published: January 2023

Human immunodeficiency virus infection is the most common risk factor for severe forms of tuberculosis (TB) regardless of CD4 T cell count. Using a well-characterized cynomolgus macaque model, we compared reactivation of latent M. tuberculosis (Mtb) infection induced by simian immunodeficiency virus (SIV) or anti-CD4 antibody (αCD4). Reactivation, as defined by the appearance of a new lung lesion by PET CT, occurred in 5 of 7 αCD4 treated animals compared to 4 of 8 SIV infected animals within 2 months of treatment. αCD4 animals had significantly fewer CD4 T cells within granulomas compared to SIV/Mtb infected animals. However, compared to αCD4 groups, SIV infection caused more dissemination of lung granulomas and higher granuloma bacterial burdens. Within the granulomas, SIV replication was associated with greater Mtb growth and reduced Mtb killing. Granulomas from SIV/Mtb animals displayed a distinctly different profile of T cell cytokine and granzyme B expression compared to αCD4 animals and controls. PET CT imaging prior to treatment could predict reactivation in αCD4-treated animals, but not in SIV/Mtb animals. These data suggest that SIV infection disrupts protective immune responses against Mtb infection beyond the loss of CD4 T cells, and that synergy between SIV and Mtb occurs within granulomas.
Regulation of Id2 gene expression in plasmacytoid dendritic cells
Authors: Babcock Rachel Lauren , Li Haiyan S , Zhou Yifan , Chrisikos Taylor T , Watowich Stephanie S
Published: January 2023

Plasmacytoid dendritic cells (pDCs) are specialized type I interferon (IFN-I) producing cells that mediate anti-viral responses, anti-tumor immunity, and autoimmunity. When exposed to Toll-like receptor 7 (TLR7)- and TLR9-ligands, pDCs mature and produce IFN-Is; pDCs also acquire conventional DC (cDC)-like morphology and features, including the cell surface expression of antigen presentation and co-stimulatory molecules, secretion of additional cytokines, and ability to activate adaptive T cell responses. Yet, the transcriptional pathways regulating TLR-induced pDC maturation are poorly characterized. We found the mRNA and protein expression of the inhibitor of DNA binding protein 2 (Id2) was induced in TLR7/9-stimulated pDCs, and Id2 expression correlated with greater cDC-like features. Id2 normally blocks pDC development by antagonizing the pDC-master regulator, E2-2, while E2-2 suppresses Id2 transcription. Thus, we hypothesize TLR-activated signaling pathways in pDCs control the abundance of transcriptional regulators (e.g., E2-2) at the Id2 promoter as well as the promoter chromatin state, leading to induction of Id2 transcription during pDC maturation. Using genetic knockout mouse models, we found Id2 induction was independent of interferon alpha receptor and nuclear factor kappa beta signaling pathways. Chromatin-immunoprecipitation assays suggested E2-2 abundance at the Id2 promoter decreased following TLR stimulation, while activating histone modifications increased. These data are consistent with transcriptional induction of Id2 upon TLR stimulation. Future work will investigate the effect of additional regulators and TLR-regulated pathways on Id2 induction in TLR7/9-stimulated pDCs.
NOD2 promotes protective responses and recovery from Candida tropicalis infection in dextran sodium sulfate (DSS) colitic mice
Authors: Buela Kristine-Ann G. , De Salvo Carlo , Havran Hannah , Hager Christopher , Di Martino Luca , Cominelli Fabio , Ghannoum Mahmoud , Pizarro Theresa
Published: January 2023

Although it is well established that the gut microbiome plays an important role in GI health and disease, recent studies also underscore the complexity and contribution of intestinal fungal communities by, for example, promoting dysbiosis in mouse models of colitis and Crohn’s disease patients, which both have higher levels of Candida tropicalis (Ct) vs controls. Also, proteins encoded by IBD susceptibility loci, such as pattern recognition receptors (PRRs), not only recognize bacterial products, but in the case of NOD2, also detects the fungal cell wall component, chitin. Yet, how PRRs regulate intestinal fungal immunity and how fungi impact colitis is poorly understood. Herein, we show that while Ct infection does not exacerbate colitis in WT controls, Nod2−/− mice show a higher fungal burden and delayed recovery after DSS challenge, indicating NOD2’s protective role and ability to promote fungal clearance during experimental colitis. In the absence of NOD2, epithelial barrier function is compromised, with increased immune cell infiltration and muscle wall thickening. Nod2−/− mice specifically infected with Ct, and not other commensal species, express reduced colonic IL-6 (promotes Th17 and ILC3s), as well as IL-22 and IL-17 (cytokines that maintain epithelial barrier integrity), and show a decreased frequency of MLN-derived IL-17+ ILC3s vs WT, suggesting that delay in fungal clearance and recovery from colitis in Nod2−/− mice may be due to the inability to mount protective type 3 immune responses. Overall, these findings reveal that opportunistic fungal infections during colitis impede gut mucosal healing, and that NOD2 may not only impact the gut microbiome, but also the mycobiome, possibly through an IL-17-mediated mechanism.
Alveolar macrophages provide an early Mycobacterium tuberculosis niche and initiate dissemination
Authors: Cohen Sara Beth , Gern Benjamin H , Delahaye Jared L , Plumlee Courtney R , Gerner Michael Y , Urdahl Kevin B
Published: January 2023

Mycobacterium tuberculosis (Mtb) infection is initiated in the distal airways, a site patrolled by alveolar macrophages (AM), and infection outcomes are governed by early immune events that remain poorly understood. We show that Mtb replicates almost exclusively within airway-resident AM during the first week after aerosol exposure. Although AM are typically thought to reside within the alveolar lumen, confocal imaging demonstrated that Mtb-infected AM establish a novel niche within the lung interstitium, where they undergo proliferation and physically associate with recruited monocyte-derived cells (MC). Localization of AM to the interstitium precedes subsequent Mtb uptake by MC and neutrophils and is driven by non-hematopoietic MyD88/IL-1R inflammasome signaling and the Mtb ESX-1 secretion system. The interstitial localization of infected AM occurs in the absence of recruited monocytes and neutrophils, suggesting that this pulmonary niche may result from AM-intrinsic egress from the alveolar space rather than inflammation-driven alveolar consolidation. Comparisons of the transcriptomes of infected AM localized to the airway or interstitium by RNA-sequencing revealed unique transcriptional profiles in the two populations, suggesting that these two AM subsets may be functionally distinct. For example, Mtb-infected AM in the interstitium dramatically upregulated interferon-response genes, lending support to the idea that these cells are in unique lung environments. Thus, crosstalk between Mtb-infected AM and non-hematopoietic cells establishes pulmonary Mtb infection by promoting the translocation of infected cells from the alveoli to lung interstitium, facilitating dissemination to other myeloid subsets.
Addition of rexinoids during T cell activation enhances mucosal-homing receptor expression and migratory ability of effector cells in vitro
Authors: Manhas Kavita , Marshall Pamela , Wagner Carl E , Jurutka Peter W , Blattman Joseph N
Published: January 2023

Certain vitamins directly impact immune cell migration, indicating potential to function as a vaccine adjuvant. We previously showed that all-trans retinoic acid (ATRA), the biologically active form of vitamin A, modifies gut-homing receptor expression on effector T cells upon binding to the retinoic acid receptor (RAR), which joins with the retinoid X receptor (RXR) to function. Giving ATRA during vaccination also resulted in enhanced mucosal protection following viral challenge in murine models. While promising, certain factors limit adding ATRA to a vaccine, including its instability and poor solubility. Rexinoids, synthetic ligands for the RXR, may mimic or better ATRA immune effects by enhancing RAR-mediated transcription. Their improved stability and solubility also indicates a greater potential for clinical use. Here we explored the ability of different rexinoids to program effector T cell expression of gut-homing receptors CCR9, a4b7, and CD103. Naïve T cells from transgenic mice were activated with appropriate peptide and set concentration of each rexinoid. Flow cytometric analysis showed increased expression of gut-homing receptors in the presence of select rexinoids, indicating their ability to act as an ATRA mimic. A cooperative relationship was seen between other rexinoids and ATRA as co-culturing with suboptimal doses of each showed enhanced receptor expression, suggesting they could potentially act with ATRA normally found in serum. Some rexinoids also promoted better effector T cell migration compared to ATRA, following analysis of a transwell migration assay. Overall, our findings support the prospective use of rexinoids as adjuvants during vaccination.
Metabolic utilization of propanediol by adherent-invasive E. coli regulates intestinal tissue immunity
Authors: Viladomiu Monica , Metz Maeva L , Lima Svetlana F , Chou Lance , Guo Chun-Jun , Simpson Kenneth W , Scherl Ellen J , Longman Randy
Published: January 2023

Crohn’s Disease (CD) has been associated with alterations in the intestinal microbiota, but the microbial mechanisms that regulate host immunity are largely unknown. We have recently shown that Adherent-Invasive E. coli (AIEC), which are enriched in CD patients, are sufficient to induce intestinal Th17 cells. Although AIEC lack pathogenic factors including type III secretion systems, many CD-derived isolates express virulence-associated metabolic enzymes including propanediol dehydratase (PduC), which enables AIEC to use fucose-derived propanediol as an alternate carbon source. We found that pduC is enriched in the microbiome and among E. coli genomes in CD patients compared to healthy controls. To evaluate the physiologic contribution of propanediol utilization to AIEC Th17 induction, we generated a pduC-deficient mutant of a CD-derived, AIEC isolate. Deletion of pduC resulted in reduced, inflammatory Th17 cells and attenuated weight loss following T cell transfer colitis. Using genetic mouse models, we found that CX3CR1+ mononuclear phagocytes are required for AIEC-mediated Th17 induction and IL-10 is required to restrain pduC-dependent colitis. Using a catalytically-inactive mutant, we determined that PduC metabolic activity was required for this immune phenotype. Cell-free supernatants from WT AIEC (but not the isogenic, pduC-deficient clone) promoted ex vivo Th17 cell polarization and metabolomics analysis (LC-MS) of these supernatants defined PduC-dependent metabolites capable of promoting Th17 polarization. These studies reveal a link between AIEC metabolism and inflammatory Th17 cells with the potential to serve as a therapeutic target in the treatment of CD.
Lymphotoxin-beta receptor expression by neutrophils prevents metabolic activation and colitis pathogenesis
Authors: Riffelmacher Thomas , Giles Daniel , Zahner Sonja , Andreyev Alexander , McArdle Sara , Perez Tamara , van der Gracht E.T.I. , Morris Venetia , Tumanov Alexei , Kronenberg Mitchell
Published: January 2023

Inflammatory bowel disease, while multifactorial, is largely characterized by exacerbated intestinal immune activation. However, the critical mechanisms regulating immune activation remain to be fully defined. We have previously reported that a member of the TNF superfamily, TNFSF14 (LIGHT), and its interaction with the lymphotoxin beta receptor (LTβR), is required for preventing severe disease in a mouse model of dextran sulfate sodium (DSS) induced colitis. Thus, we aimed to determine the cell type and mechanism critical to LTβR mediated suppression of DSS-induced colitis. Employing the use of LTβRfl/fl mice and tissue-specific Cre-mediated deletion, we found that neutrophils were the critical LTβR expressing cell type. Deletion of LTβR expression in neutrophils (LtbrΔneut)resulted in exacerbated DSS-induced colitis and drastically increased colonic neutrophil accumulation. Mechanistically, RNA-Seq analysis of neutrophils revealed transcriptional alterations in genes associated with cellular metabolism, and mitochondrial function. Ex vivo studies of bone marrow neutrophils from LtbrΔneut, compared to Cre- controls, revealed increased mitochondrial mass and number, as well as elevated mitochondrial ROS production in neutrophils lacking the receptor. Subsequent metabolic studies demonstrated that LTβR deficient neutrophils exhibited elevated mitochondrial respiration and glycolysis. Interestingly, these increased levels of mitochondrial mass, ROS production and glycolytic activity persist in LtbrΔneut colonic neutrophils during DSS-induced colitis. In sum, our results demonstrate that neutrophil LTβR signaling plays a critical role in the immune activation associated with DSS-induced colitis.
Intestinal colonization with IL-10 inducing adherent E. coli protects from intestinal inflammation and tumor development
Authors: Diehl Gretchen , Ruiz Daniel Zegarra , Kim Dasom , Norwood Kendra , Hill Andrea A. , Morales Fatima Beatriz Saldana
Published: January 2023

Colorectal cancer (CRC) is the third most common and third most deadly cancer in the US. Chronic intestinal inflammation corresponds with advanced disease stage and decreased survival in CRC. Intestinal inflammation corresponds with changes in the microbiota, including expanded proteobacteria, such as E. coli. This altered microbiota is thought to support inflammation and disease progression in both CRC and inflammatory bowel disease (IBD). However, how individual microbes interact with host cells and the pathways they activate remains incompletely defined. We characterized immune effects after colonization with individual isolates from a panel of human mucosally associated E. coli. In contrast to our expectations, a subset induced intestinal macrophage production of IL-10, protecting from colitis. Further characterization of a single isolate revealed colonization with this isolate limited tumor incidence in a model of inflammation associated colorectal tumors. Intestinal macrophages, macrophage production of IL-10, and epithelial expression of IL-10 receptor were all required for anti-inflammatory and anti-tumor effects. Colonization with an isogenic mutant E. coli that lacked a single gene important for epithelial attachment did not induce IL-10 and amplified intestinal tumors with increased tumor infiltration of anti-inflammatory immune cells. While the inherent diversity of the microbiota makes it difficult to pinpoint singular pathways of tumorigenesis, we have identified a regulatory loop between adherent microbes, intestinal epithelium and intestinal immune cells which promotes barrier integrity and limits tumor development, identifying novel ways to therapeutically target inflammation and CRC.
B cell-specific S1P1 deletion reshapes both B and T cell activation profiles in hypersensitivity pneumonitis
Authors: Huppé Carole-Ann , Lecours Pascale Blais , Morissette Mathieu , Marsolais David
Published: January 2023

Hypersensitivity pneumonitis (HP) is a TH1/TH17-polarized chronic disease caused by repetitive exposure to airborne agents. Efficient acquired B cell responses are thought to sustain pulmonary inflammation in HP. We found that an S1P1 pharmacological ligand prevented the reactivation of hypersensitivity upon airway antigenic exposure, which translated into a decrease of antigen-specific antibodies. Given that mice with B cell-specific S1P1 deletion (B-S1P1KO) feature altered bone marrow egress, lower peripheral B cell levels, as well as a defect in antigen uptake, we hypothesized that S1P1 deletion on B cell could mitigate HP progression. B-S1P1KO mice were exposed three times a week for three weeks to an HP-causing agent, Methanosphaera stadtmanae (MSS), and were euthanized 24 hours or 4 days after the last exposure for flow cytometry analyses. Lymphocyte populations in the lung of B-S1P1KO mice were more activated in response to MSS compared to the control strain. Indeed, we observed an increase surface expression of CD80, CD86 on B cells and surprisingly, an increase of surface levels of CD69, CD44 and CD25 on T cells. MHCII levels at the B cell surface were reduced in B-S1P1KO mice, compared to the control strain, while there were no differences in MHCII expression on dendritic cells or macrophages. B cell-specific S1P1 deletion increased the frequency of B cells with the ability to produce pro-inflammatory cytokines (IFNg+, IL-4+ and IL-6+). Concomitantly, the frequency of both B cells and T cells with the ability to produce IL-10 was reduced. Thus, B cell-specific S1P1 deletion induces a proinflammatory B cell phenotype in the HP lung and reduces regulatory populations.
Heterogeneity of regulatory T cells during lung inflammation
Authors: Huang Weishan , Ye Kaixiong , Nidetz Natalie F , McGee Michael C , Islam Rezwanul , Limper Candice B , August Avery
Published: January 2023

Pulmonary inflammation is the leading cause of respiratory illness worldwide. Although strong T cell responses are desired for defending the hosts against infections, the cytotoxic effector function of the innate and adaptive immune responses can lead to the development of pulmonary immunopathology, which may lead to death. While T cells produce inflammatory cytokines during infections and allergies, they can also produce the immunomodulatory cytokine IL-10, which is critical for limiting the immunopathology caused by the excessive effector immune responses. However, the composition of IL-10-producing lymphocyte population and their molecular signatures are unclear. Using mouse models that report the production of IL-10 by GFP and expression of T regulatory cell marker Foxp3 by RFP, mouse models of lung inflammation (including allergic asthma, hypersensitivity pneumonitis, and flu), transcriptomic analyses at the population and single cell levels, and transgenic mouse models that are impaired in T cell-derived IL-10 production, we found that, in the disease models used, regulatory T cells in the mouse airway are mainly comprised of CD4+ Foxp3+, CD4+ Foxp3− and CD8+ subsets, and they differ under different disease conditions. Furthermore, within each subset, IL-10-producing T cells exhibit significant molecular heterogeneity. Information gained from this dataset provides insights into the T cell subset heterogeneity and signature markers, and shed light for future strategic design for therapeutic development utilizing the immunomodulatory features of T cells for the treatment of pulmonary immunopathology.
Distinct memory γδ T cell subsets are fine-tuned by TCR engagement and soluble signals to shape IFNγ and IL-17A responses
Authors: Khairallah Camille F , Imperato Jessica N , Yang Daniella , Qiu Zhijuan , Sheridan Brian S
Published: January 2023

Unconventional T cells are essential for tissue homeostasis and protection against invading pathogens at barrier sites. γδ T cells are enriched at these interfaces where they can form memory populations that can be either beneficial or detrimental depending on the functional response elicited. However, our understanding of the factors regulating memory γδ T cell generation, activation and function remains limited. Foodborne Listeria monocytogenes (Lm) infection of Batf3−/− mice revealed a critical role for Batf3-dependent dendritic cells in the generation and function of adaptive γδ T cells. In line with this observation, IFNγ and IL-17A production and IFNγ/IL-17A co-production by memory γδ T cells were induced by heat-killed Lm after 24 hours in culture, and these responses were inhibited after MHC-II or CD11c depletion. The induction of cytokine production was tightly associated with Zap70/Syk phosphorylation, indicative of TCR activation. Although IL-12 is usually associated with type 1 responses, neutralization of IL-12/23p40 dramatically impaired IL-17A production whereas IFNγ response was only marginally affected. Surprisingly, classical TH17 polarizing cytokines had little impact on IL-17A production. Despite this conventional-like response, memory γδ T cells remained able to quickly produce IL-17A in response to supernatant or live Lm independently of TCR signaling. Thus, adaptive γδ T cells are a functionally diverse population that encompasses innate- and adaptive-like responses that are fine-tuned by the γδTCR and soluble signals. These insights may facilitate the development of new strategies eliciting specific functional subsets of memory γδ T cells tailored to various intestinal disease settings.
ST2-expressing B1 B cells acquire an anti-inflammatory capacity during pregnancy in mice
Authors: Valeff Natalin Jimena , Ventimiglia Maria Silvia , Quadrana Florencia , Jensen Federico
Published: January 2023

In the context of pregnancy, it is known that IL-33 induces the production of anti-inflammatory molecules by decidual B1 cells, protecting against preterm birth (PTB) in human and mouse. We have previously showed that expression of IL-33 receptor, Il1rl1 (ST2) is significantly upregulated in splenic B cells during mid pregnancy, predominantly in B1 cell subset. Furthermore, using an LPS induced PTB mouse model, we observed increased numbers of splenic and decidual ST2-expressing B1 cells in the acute phase of PTB as compared to term pregnant females.

We aimed to investigate here the anti-inflammatory properties of ST2-expressing B1 cells during pregnancy. Total splenocytes from pregnant (P) and non-pregnant (NP) mice were isolated and cultured for 24h with/without LPS (10 μgr/ml), ST2 expression and cytokine production by ST2+ B1 cells was evaluated by flow cytometry. B1 cells from P mice stimulated with LPS showed significantly higher levels of ST2 expression. ST2+ B1 cells produced significantly higher levels of IL-10 and significantly lower levels of TNF-α and IL-17 as compared to ST2+ B1 cells from NP mice.

In a separate project in our laboratory we demonstrated that prophylactic treatment with probiotic Lactobacillus kefiri prevented LPS-induced PTB in mice. Interestingly, we observed that numbers of splenic and decidual ST2-expressing B1 cells were increased in L. kefiri treated mice that were protected against LPS-induced PTB.

Our data strongly suggest an anti-inflammatory capacity of ST2-expressing B1 cells during gestation, presumably to ensure pregnancy wellbeing and prevent inflammation induced PTB.
Antigen recognition by CD8+ T cells in non-lymphoid tissues initiates a transcriptional program of tissue-resident memory differentiation
Authors: Nolz Jeffrey C , Hobbs Samuel J
Published: January 2023

Tissue-resident memory (TRM) CD8+ T cells permanently reside within non-lymphoid tissues to provide a first-line of defense against invading pathogens. TRM T cells are largely derived from recently activated effector T cells, but the mechanisms that control the extent of TRM differentiation once they are recruited into non-lymphoid tissue microenvironments are largely undefined. To determine how T cell receptor (TCR) engagement promotes TRM formation, we utilized interferon-gamma (IFNγ)-YFP reporter T cells and found that only ~25% of the antigen-specific CD8+ T cells in the skin produced IFNγ during the course of a Vaccinia virus (VacV) infection. Strikingly, expression of IFNγ was strictly antigen-dependent and genome-wide transcriptional profiling revealed that IFNγ+ CD8+ T cells in the VacV-infected skin were becoming tissue-residents, suggesting that local antigen recognition identifies the dominant TRM precursor population. Mechanistically, TCR engagement caused effector CD8+ T cells to express Blimp1 and repress expression of the tissue egress receptor S1PR1 and the transcription factor KLF2, collectively preventing S1P-dependent migration. Interestingly, the S1PR1 antagonist CD69 was not transcriptionally upregulated by TCR stimulation. However, CD69 surface expression was Blimp1 dependent, suggesting that Blimp1-mediated repression of S1PR1 causes increased availability of CD69 protein to remain on the cell surface, a hallmark of TRM differentiation. Thus, antigen recognition by effector CD8+ T cells in non-lymphoid tissues is a critical factor that promotes Blimp1 expression, ultimately causing stable expression of CD69 and retention of TRM CD8+ T cells in the skin following viral infection.
Fli-1 inhibitors reduce the production of inflammatory cytokines in human renal glomerular endothelial cells and ameliorate lupus nephritis in NZB/W F1 mice
Authors: Wang Xuan , Gilkeson Gary S , Zhang Xian
Published: January 2023

Expression of transcription factor Fli-1 impacts lupus nephritis (LN) development in both human patients and murine models of lupus. We studied the effects of camptothecin and topotecan, Fli-1 inhibitors, on LN in NZB/W F1 mice and renal endothelial cells. Human Renal Glomerular Endothelial Cells (HRGEC) were treated with Fli-1 inhibitors, and inflammatory cytokines were measured after stimulation with IFN-α and IFN-γ. NZB/W F1 female mice were treated with camptothecin, topotecan, cyclophosphamide, or vehicle control. Blood and urine were collected, and autoantibodies and proteinuria were monitored.

The expression of Fli-1 in HRGEC, when treated with 0.1 μM or higher concentrations of camptothecin, was significantly reduced. The production of MCP-1 and IP-10, inflammatory cytokines associated with LN development, were significantly lower in the HRGEC treated with camptothecin compared to the HRGEC treated with DMSO at 4 and 24 hours following stimulation with IFN-α and IFN-γ at the concentration of 100 ng/ml. NZB/W F1 mice were treated with camptothecin, topotecan, cyclophosphamide, or PBS control when more than 40% of mice had proteinuria higher than 30 mg/dL. At the age of 37 weeks, 70% of NZB/W F1 mice treated with vehicle had proteinuria higher than 2,000 mg/dL. None of the NZB/W F1 mice treated with camptothecin or topotecan had proteinuria higher than 100 mg/dL. One of the NZB/W F1 mice treated with cyclophosphamide had proteinuria higher than 100 mg/dL. Blood urea nitrogen (BUN) was lower in the sera from NZB/W F1 mice treated with camptothecin, topotecan, or cyclophosphamide compared to that of the mice treated with vehicle. Together, these results indicate that Fli-1 inhibitors are novel therapeutic approaches in treating LN.
Impact of curcumin and its metabolites on the immune response of type 1 diabetes-prone mice
Authors: Alard Pascale , Kosiewicz Michele M , Ma Jing
Published: January 2023

Recent studies have shown that diferuloylmethane (curcumin) ameliorates autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, lupus and diabetes in animal and human models. We examined the effects of curcumin and its metabolites on the immune response in NOD mice. We assessed their ability to regulate pro-inflammatory cytokines and the differentiation of IFNγ-producing T cells. We also determined whether curcumin could affect Treg development and production of the anti-inflammatory cytokine, IL-10. NOD dendritic cells (DC) treated with curcumin in vitro produce decreased pro-inflammatory cytokines, including IL-12. Treatment of DC with ferulic acid and vanillin, two metabolites of curcumin, also downregulated production of IL-12. This effect was not due to an decrease in DC viability or increased production of IL-10. More importantly, NOD DC treated with curcumin and its metabolites induce fewer IFNγ-producing T cells, shown to be responsible for beta islet destruction. Mice treated with curcumin were protected from disease and this protection was associated with a decrease in IFNγ production in the spleen and pancreatic LN. Although CD103+ DC harvested from the mesenteric LN of curcumin-treated NOD mice were better at inducing in vitro Treg conversion, the levels of CD4+Foxp3+ and CD4+Foxp3+Neuropilin- cells in the pancreatic LN were actually impaired in those mice. Overall, our in vitro and in vivo data show that curcumin and its metabolites suppress the differentiation of IFNg-producing cells involved in type 1 diabetes development. Curcumin or its metabolites may have therapeutic potential for the prevention of type 1 diabetes.
Antigen targeted to the apoptotic pathway via dying erythrocytes leads to profound T cell exhaustion and tolerance
Authors: Watkins Elyse A , Roberts Jaeda , Dunaif Anya A , De Loera Robert , Bailey Luke , Hubbell Jeffrey
Published: January 2023

The current standard of care for autoimmune disease is administration of immunosuppressive agents that require a lifetime of treatment and leave patients susceptible to opportunistic infections. Thus, there is a great need for therapies that induce lasting and antigen-specific tolerance, which would require fewer treatments and allow the immune system to fully respond to pathogens. A promising target for the induction of antigen-specific tolerance is the apoptotic pathway. Antigen-presenting cells create a tolerogenic milieu upon recognition of apoptotic debris, and antigen presentation by APCs to T cells in the absence of inflammatory cues typically leads to anergy and deletion.

A large source of endogenous apoptotic cells is erythrocytes, or red blood cells. The purpose of this study is to discover antibody fragments (Fabs) that bind erythrocytes, and use these Fabs to deliver antigen to erythrocytes for processing via the apoptotic pathway and presentation to T cells for tolerance. Phage display on mouse erythrocytes yielded high affinity Fabs that lacked binding to peripheral blood mononuclear cells and induced no hematological changes. Erythrocyte-binding Fabs were recombinantly expressed with the model antigen ovalbumin and administered to mice. Targeting ovalbumin to erythrocytes led to robust abortive proliferation of antigen-specific T cells. Remaining cells appeared phenotypically exhausted, and had reduced capacity to produce inflammatory cytokines upon restimulation. Antigen presentation of erythrocyte-targeted antigen occurred in the spleen, because T cell exhaustion was completely reversed in splenectomized mice. Thus, targeting erythrocytes may induce lasting antigen-specific tolerance.
Novel compound JOAB-40 decreases inflammation in vitro and colitis severity in vivo by using the murine Dextran Sulfate Sodium (DSS) model
Authors: Szollosi Doreen E , Khajah Maitham , Hawai Sanaa , Ghoneim Ola , Bill Ashley , Daniels-Monk Mallory , Edafiogho Ivan O.
Published: January 2023

Crohn’s disease and ulcerative colitis are two disorders categorized as inflammatory bowel diseases (IBD). Patients with IBD may have inflammation extending the full thickness of the gut and non-gut organs mediated in part by Th-induced cytokines IL-1β, -2, -12, -13, -17, TNF-α, and IFN-Υ. To mimic this in vivo, DSS given in drinking water (3% w/v) can induce colitis in mice. Our previous work with novel anti-inflammatory enaminone E121 has shown that treating mice with E121 along with DSS-induced colitis (prophylactic approach) reduced colitis severity at gross and histological levels at the 30–60 mg/kg doses. Modifications made to E121 have resulted in new compounds (JOAB series) that include a piperazino ring which can not only decrease levels of pro-inflammatory cytokines but also inhibit chemokine signaling through CCR2. In vitro data suggest that JOAB-40 can reduce levels of TNF, IL-1, IL-6, and IL-12 in LPS-stimulated macrophages. Since JOAB-40 was successful in decreasing inflammation in a mouse model of hemarthrosis without toxicity, we assessed its efficacy in decreasing inflammation associated with colitis. Prophylactically, mice treated with JOAB-40 had a more profound reduction in severity of colitis at lower doses (1–10 mg/kg) than E121. At higher doses (30–60 mg/kg), both E121 and JOAB were equivalent in their potency in reducing colitis severity. When both agents were given after colitis induction (treatment approach), JOAB-40 reduced colitis severity at 10, 30, and 60 mg/kg doses (by 50% compared to vehicle), but E121 did not reduce colitis severity at histological level when used at similar dose range. These data suggest JOAB-40 is more effective in reducing colitis severity in mice compared to the parental E121 compound.
Contact-dependent suppression of FVIII-specific memory B cells by BAR-Tregs
Authors: Zhang Ai-Hong , De Paula Alves Sousa Alessandra , Venkatesha Shivaprasad H , Scott David W
Published: January 2023

Anti-drug antibody formation poses tremendous obstacles for optimal treatment of hemophilia A (HA). In this study, we sought to utilize chimeric receptor-modified natural regulatory T cells (Tregs) to target FVIII-specific memory B cells, which are responsible for persistent anti-FVIII neutralizing antibodies (inhibitors) in HA patients. Thus, CD4+CD25hiCD304+ natural Tregs were FACS sorted from naïve C57BL/6 mice and retrovirally transduced to express a chimeric B-cell antibody receptor (BAR) containing the immunodominant A2 domain of FVIII. Plasmablast-depleted (CD138neg) splenocytes from FVIII/IFA immunized FVIII-knockout HA mice served as the source for FVIII-specific memory B cells, which were then specifically stimulated in vitro with FVIII and enumerated in B-cell ELISPOT assays. Adding A2-BAR Tregs (1 per 150 splenocytes), but not conventional T cells, to the CD138− splenocytes significantly suppressed the formation of anti-FVIII producing cells, compared to the non-relevant OVA-BAR Tregs control group. Transwell experiments confirmed that the suppression was contact-dependent. Interestingly, even in the presence of antibody to FVIII (so-called inhibitors), similarly prepared CD4+CD25hiCD127low A2-BAR human natural Tregs completely suppressed polyclonal anti-FVIII Elispots formation, indicating potential by-stander suppression by A2-BAR Tregs on all FVIII-specific B cells in the local milieu. In conclusion, we demonstrated in vitro that FVIII domain-expressing BAR Tregs could efficiently target and suppress FVIII-specific memory B cells. Supported by a Bayer Hemophilia Grant [AHZ] and NIH HL126727 [DWS].
Mechanisms of “Peptide Alarm Therapy”: Can antiviral memory T cells directly kill cancer cells in an MHC Class I-dependent way?
Authors: Gavil Noah Veis , Joag Vineet , Rosato Pamela C. , Masopust David
Published: January 2023

“Peptide alarm therapy” is a novel cancer immunotherapy that repurposes antiviral memory CD8+ T cells within tumors to drive antitumor immunity. After a primary infection, antigen-specific CD8+ T cells establish a surveillance program that positions T cells throughout the entire body. In particular, resident-memory CD8+ T cells (Trm) permanently reside in all tissues after primary antigen exposure and survey their immediate surroundings for possible re-exposure. In addition to normal tissue, antiviral Trm populate solid tumors. In contrast to tumor-specific T cells, intratumoral antiviral Trm are not exhausted and demonstrate potent reactivation with cognate peptide. This re-activation of Trm in tumors leads to upregulation of effector molecules and chemokines, the local recruitment of circulating immune cells, and the increased migration of dendritic cells to the tumor-draining lymph node. The intratumoral injection of peptide, and the subsequent reactivation of antiviral Trm, induces tumor growth suppression and exhibits synergy with immune checkpoint blockade, specifically PD-L1. The mechanisms driving this antitumor immune response remain uncharacterized. In this study, we have tested whether viral peptide presentation by cancer cells is necessary for the efficacy of peptide alarm therapy. A targeted knock-out of the MHC Class I allele, H-2Kb, in the B16.F10 melanoma cell line has been generated and employed in combination with the OT-I Tg CD8+ T cells, which bind the SIINFEKL:H-2Kb peptide:MHCI complex. Our data suggest that peptide presentation by cancer cells is not necessary for the reactivation of intratumoral antiviral Trm, and does not reduce tumor growth suppression in the context of peptide alarm therapy.
Inhibition of TET (Ten-Eleven Translocation) enzymes enhances systemic anti-tumor immune responses in T cells
Authors: Seo Hyungseok , Yang Chao , Zhang Wade , Hogan Patrick , Rao Anjana
Published: January 2023

TET enzymes facilitate DNA demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine and other oxidation products in DNA. Loss-of-function mutations in TET enzymes are associated with increased proliferation and increased stem cell function in many cellular lineages. Moreover, there are several hints that inhibition of TET enzymes in tumor-infiltrating T cells might enhance anti tumor immune responses in solid tumors. For instance, TET2 deficiency in mouse T cells increased the number of CD8+ central memory T cells, and TET deficiency human CD8 chimeric antigen receptor (CAR) T cells increased their antigen-specific proliferation and capacity to reject tumor cells in a patient with chronic lymphocytic leukemia. Furthermore, we previously showed that TET deficiency in regulatory T cells decreased the stability of Foxp3 expression; hence TET deficiency would be expected to counter the ability of regulatory T cells to suppress anti-tumor responses. In this study, we found that TET loss of function in CD8 TILs indeed slowed tumor growth and promoted anti tumor responses in a mouse CAR T cell model. The function of TET proteins and other Fe(II) and a-ketoglutarate dependent dioxygenases is inhibited by L-2-Hydroxyglutarate (L2HG), which is normally kept at low levels by the enzyme L-2HG dehydrogenase (L2HGDH). We found that when CAR T cells were rendered low in TET activity in any one of three ways: (i) deficiency of the TET enzymes, (ii) depletion of L2HGDH and (iii) pretreatment of L2HG, they showed decreased expression of inhibitory receptors as well as increased secretion of inflammatory cytokines. Based on these results, we suggest that TET inhibition in T cells might be a promising general approach in cancer immunotherapy.
Antigen-specificity/protein/mRNA single-cell sequencing profiling of bystander CD8+tumor-infiltrating lymphocytes (TILs) in renal cell carcinoma (RCC) reveals their transcriptomic similarities with tumor-specific T cells
Authors: Li Shamin , Simoni Yannick , Zhuang Summer , Newell Evan
Published: January 2023

Recently, using human colorectal and lung cancer, we showed that CD8+ T cells infiltrating tumor tissue (TILs) are not only specific for tumor antigens, but are also composed of CD8+TILs specific for cancer unrelated epitopes – called bystander – such as HCMV, EBV or flu epitopes. We also showed that the surface marker CD39 can be useful for discriminating bystander (CD39−) from tumor-specific (CD39+) CD8+TILs (Simoni et al, Nature, 2018). Here, our aim was to test these findings in human Renal cell Carcinoma (RCC) and to better understand the biology of these bystander CD8+TILs. Surprisingly, our primary CYTOF analyses showed heterogeneity within bystander CD8+TILs that possess various phenotypes including effector (CD45RO+), memory (CD45RO+ CCR7+), Trm (CD69+ CD103+/–), and senescent (CD57+ KLRG1+) cell features. Using targeted mRNA single-cell sequencing combined with BD AbSeq assay, we analyzed sorted CD8+TILs from RCC patients and identified bystander CD8+TILs using oligo-tagged tetramers. Gene signature analysis of these different subsets revealed transcriptomic similarities with the tumor-specific CD8+TILs. Our data provide a comprehensive perspective of cancer unrelated CD8+TILs in RCC and suggest functional roles for these cells in tumor immune responses, which could lead to new diagnostic and therapeutic strategies.
Amplification of neoantigen-specific antitumor responses using a long-lasting IL-2 fusion protein
Authors: Hernandez Rosmely , Toomer Kevin H , Põder Janika , LaPorte Kathryn M , Malek Thomas R
Published: January 2023

Low frequency of tumor-specific T cells represents one major reason for failed immunotherapy. IL-2 can boost tumor-reactive T cells but its benefit is limited due to toxicity and potential off-target effects. We developed a novel IL-2/CD25 fusion protein which exhibits more favorable pharmacology when compared to IL-2. At low doses, IL-2/CD25 targets the high affinity IL-2R and selectively boosts Tregs. Here we show that a single administration of high dose mouse IL-2/CD25 in conjunction with cognate peptide and LPS amplified tumor-specific Pmel-1 CD8+ and TRP-1 CD4+ Teff cells and Pmel-1 memory cells while only transiently increasing Tregs. This approach led to effective anti-tumor responses against B16.F10 melanoma. This scheme was modified to amplify endogenous polyclonal melanoma-reactive T cells, which is translationally more relevant. Development of tumor-specific T cells required priming and boosting with a mixture of B16.F10 neoantigens and poly (I:C) followed by application of IL-2/CD25 after the boost. This resulted in largely a CD4+ anti-neoantigen specific T cell response. This immunization scheme delayed B16.F10 growth and extend survival time not only when immunized mice were challenged with B16.F10 but also for mice with pre-established B16.F10. The immune response within the tumor microenvironment was accompanied by increased frequencies of granzyme B-expressing CD4+ and CD8+ T cells. Our data show that limited application of high-dose IL-2/CD25 in the context of neoantigen vaccines offers potential advantages over IL-2 for melanoma treatment by avoiding toxicities seen with IL-2 therapy and minimizing Treg-associated tolerance.
Immunometabolism regulation by PSGL-1 signaling in tumor-specific CD8 T cells
Authors: Hope Jennifer L , Otero Dennis C , Bae Eun-ah , Stairiker Christopher J , Palete Ashley B , Faso Hannah A , de Jong Petrus , Powis Garth , Bradley Linda M
Published: January 2023

We previously identified that the adhesion molecule P-selectin glycoprotein ligand-1 (PSGL-1) regulates T cell function and exhaustion in response to chronic viral infection and tumors. Subsequent studies have focused on investigating the role of PSGL-1 signaling in T cell responses, with an emphasis on understanding the mechanisms by which PSGL-1 regulates T cell exhaustion. Single-cell RNA-sequencing of tumor infiltrating PSGL-1−/− CD8+ T cells identified the upregulation and differential modulation of several genes associated with T cell metabolism and enhanced intratumoral responses, including Mtor, Hif1a, and Mki67 in Gzmb/Ifng double-positive cells from tumors and Tcf7 in both tumor draining and non-draining inguinal lymph nodes. These data suggest an important role for PSGL-1 signaling in the development and maintenance of effective anti-tumor T cell responses to melanoma. Using the Seahorse glycolysis stress test, we identified that both CD4+ and CD8+ PSGL-1−/− T cells demonstrate increased glycolysis after 72 hours of in vitro activation compared to wild-type T cells. In situ activation of PSGL-1−/− CD8+ T cells demonstrated that PSGL-1−/− CD8+ T cells have increased glycolysis and increased glycolytic capacity at both sub-optimal and optimal levels of α-CD3 stimulation, and 2-NBDG glucose uptake assays confirmed increased glucose uptake by PSGL-1−/− CD8+ T cells within two hours of stimulation. Importantly, this increased glycolytic phenotype does not come at the cost of CD8+ T cell stemness, as determined by TCF-1 staining. Taken together, these data show that PSGL-1 signaling has an intrinsic and immediate role in the development of T cell responses and their metabolic profile in response to melanoma tumors.
Metformin corrects immune dysfunction and modulates tumor gene expression in overweight individuals to yield context-specific improvement of lung cancer outcomes
Authors: Barbi Joseph J , Zollo Robert , Sass Stephanie , Srinivasan Aravind , Kannisto Eric , Patnaik Santosh , Yendamuri Sai
Published: January 2023

Non-small cell lung cancer (NSCLC) is the foremost cause of cancer-related mortality in the U.S. While early detection followed by surgery allows for the best possible NSCLC outcome, recurrence still occurs in 30% of these cases, and existing adjuvant therapies have largely failed to improve this statistic. Therefore, pressing need exists for more effective early stage NSCLC treatments. Metformin, a widely used diabetes drug with minimal side effects, is also known to show anti-cancer activity. A retrospective study of early and multistage NSCLC patients treated at Roswell Park revealed that the survival benefit of metformin is surprisingly restricted to overweight or obese patients with a body mass index (BMI)>25kg/m2. In line with this, murine lung cancer models showed that metformin treatment markedly slowed tumor growth in obese mice while minimally affecting normal controls. Flow cytometric analysis of tumor-infiltrating cells from obese mice revealed indications of immune dysfunction including elevated frequencies of suppressor cells and high levels of the inhibitory checkpoint molecules PD-1 and CTLA-4. Strikingly, metformin reversed these obesity-driven changes while promoting an abundance of activated, memory T cells in tumor-associated tissues. Transcriptome analysis of tumor gene expression in both mice and patients found that metformin counteracts discrete metabolic pressures brought on by obesity capable of dampening anti-tumor immune responses including altered expression of solute transporters and biosynthetic pathways. Our findings suggest that metformin has the potential to improve NSCLC treatment of high-BMI patients by reshaping obesity’s impact on tumor gene expression and anti-tumor immunity.