bluuebunny/crossref_metadata_2025 huggingface dataset:
HSV-2 increases replication of HIV in human T cells
Authors: Pierce Carl A , Preston-Hurlburt Paula , Loh Lip Nam , Steach Holly , Sidoli Simone , Zhang Fengrui , Philbrick William , Nassar Michel , Krishnaswamy Smita , Herold Kevan C , Herold Betsy C
Published: January 2023

Herpes simplex virus 2 (HSV-2) infection is a major factor driving the global HIV epidemic, leading to increased HIV plasma viral loads (PVLs) and transmission. Previous studies which focused on mucosal responses cannot explain the observed effects of HSV-2 on HIV PVLs. We sought to determine mechanisms by which HSV-2 increases HIV PVLs. CD4 T cells are recruited to sites of HSV-2 lesions and HSV-2 can infect these cells. Phenotyping of CD4 cells infected with HSV-2 in vitro suggested a preference for T follicular helper cells, an HIV latent reservoir. HSV infection of latently infected Jurkat-2D10 cells resulted in a 10-fold increase in HIV ltr expression. After exposing CD4 cells from HIV+ donors to HSV-2, HIV p24 was detected only in cells expressing HSV-2 glycoprotein B (gB), a marker of productive HSV-2 infection. Bulk and single cell RNAseq on FACS-purified, HSV-infected and uninfected CD4 cells demonstrated reduced expression of HIV restriction factors in HSV+ cells; HSV exposed but uninfected cells exhibited higher expression of interferon response genes, potentially explaining their resistance to HIV reactivation. HSV+ cells exhibited increased expression of the lncRNA MALAT1, which may promote HIV reactivation through epigenetic modification, and HSV infection of 2D10 and CD4 cells led to decreased histone deacetylase activity. Transfection of 2D10 cells with HSV protein VP16 resulted in significantly increased HIV ltr and MALAT1 gene expression, and reduced epigenetic silencing marks. HIV proteins were also increased by proteomic analyses. These findings identify several mechanisms by which HSV-2 directly triggers HIV reactivation, such as the up-regulation of the lncRNA MALAT1.

Supported by grants from the NIH (R01 AI134367, T32 AI007501, T32 GM007288)
IL-27 signaling contributes to pathogenic T cells in a mouse model of Sjögren’s
Authors: Debreceni Ivy L , Chen Yi-Guang , Lieberman Scott M
Published: January 2023

Sjögren’s is an autoimmune disease with limited therapies due to an incomplete understanding of the immunological mechanisms that contribute to disease. Non-obese diabetic (NOD) mice spontaneously develop lacrimal gland inflammation dominated by early T cell infiltration, mirroring human disease. IL-27 is elevated in serum and saliva of Sjögren’s patients and is required for disease development in NOD mice.

Here, we define the requirements of IL-27 signaling in effector T cell subsets in the context of lacrimal gland autoimmunity. We utilized IL-27Rα KO NOD mice and an adoptive transfer model to determine which T cell subsets require IL-27 signaling to mediate lacrimal gland inflammation in NOD-SCID recipients. Both CD4 and CD8 T cells require IL-27Rα to transfer disease. Through co-transferring different combinations of KO and WT effector T cells, we found significantly decreased disease development when CD4 effector T cells did not express IL-27Rα. The role of IL-27 signaling in CD4 T cells is further supported by co-transfers of bulk lymphocytes from WT and KO mice which implicate IL-27 in driving the imbalance of CD4 effector T cells to Tregs in lacrimal glands. Additionally, Nanostring analyses performed on whole lacrimal glands from WT and KO mice support a role for IL-27 in driving pathogenic T cell infiltration. Together, these data demonstrate a pathogenic role for IL-27 in the T cell dysregulation in Sjögren’s.

Supported by grants from NIH (2T32 AI007485, DK121747 and EY027731)
Investigating immune tolerance: characterization of immunoregulatory DN T cells
Authors: Pasquin Sarah , Pelletier Adam-Nicolas , Lombard-Vadnais Félix , Chabot-Roy Geneviève , Collin Roxanne , Coderre Lise , Lesage Sylvie
Published: January 2023

Autoimmunity results from defects in immune tolerance pathways that impair the capacity of immune cells to distinguish between self and non-self. One of these immune tolerance processes involves immunoregulatory TCRαβ+ CD4− CD8− double negative T cells (DN T). Indeed, DN T cells protect from autoimmune pathologies and graft rejection in mice. Similarly, high DN T cell numbers correlate with a low incidence of chronic graft-versus-host disease in humans, an autoimmune-like pathology. While the thymic differentiation process of DN T cells was recently described, the function of these unconventional T cells is still poorly documented. To characterize DN T cells, we performed bulk RNA sequencing on CD4, CD8, γδ and DN T cells isolated from mouse spleen. Comparison of the transcriptome profiles revealed that DN T cells express a unique signature. Moreover, following in vitro stimulation, we find that DN T cells also present a distinct cytokine secretion pattern. Interestingly, we observed a lower induction of CD69, LAG-3 and PD-1 on DN T cells in comparison to CD4 and CD8 T cells, whereas LCK phosphorylation is unabated. DN T cell proliferation in mixed lymphocyte reactions is also lower than that of CD4 and CD8 T cells. Overall, our transcriptomic and cytokine analyses clearly identify DN T cells as a unique T cell subset. Moreover, while DN T cells do not express an exhaustion profile, they are more refractory to in vitro T cell stimulation than conventional T cells. A better characterization of DN T cell function will help conceive novel immunotherapies to treat autoimmune diseases.

Supported by the Canadian Institutes of Health Research - PJT 159603. S.P. was supported by a postdoctoral CIHR fellowship
The X-linked gene for the helicase DDX3X is required for lymphoid differentiation and MYC-driven lymphomagenesis
Authors: Lacroix Marion , Beauchemin Hugues , Ross Julie , Fraszczak Jennifer , Shooshtarizadeh Peiman , Chen Riyan , Moroy Tarik
Published: January 2023

The X-linked gene DDX3X encodes an RNA helicase and is mutated at high frequencies in several types of human B-cell lymphoma. Females have two active DDX3X alleles and males carry a DDX3Y homolog on the Y chromosome. We show here that pan-hematopoietic, homozygous deletion of Ddx3x in female mice perturbs erythropoiesis causing early developmental arrest. However, both hemizygous male and heterozygous female embryos develop normally, suggesting that one Ddx3x allele is sufficient for fetal hematopoietic development in females and that the Ddx3y allele can compensate for the loss of Ddx3x in males. In adult mice, loss of DDX3X affects hematopoietic progenitors, early lymphoid development, marginal zone and germinal center B-cells and lymphomagenesis driven by an Em-Myc transgene in a sex-dependent manner. Loss of one Ddx3x allele slightly accelerated MYC-driven lymphomagenesis in female mice, while male Em-Myc mice lacking Ddx3x remained almost lymphoma-free. However, the few tumors that appeared in male mice showed upregulated expression of DDX3Y indicating a minimal requirement for DDX3 activity for lymphomagenesis. Our data reveal sex specific roles of DDX3X in erythro- and lymphopoiesis as well as in MYC-driven lymphomagenesis, which are important when considering inhibition of DDX3 as a treatment of B-cell lymphoma.

Tarik Möröy holds a Canada Research Chair (Tier 1), a Foundation grant from the CIHR (FDN-148372) and support from the Cancer Research Society (CRS). Marion Lacroix is supported by fellowships from the Cole Foundation and the IRCM.
Epithelial mesenchymal transition of epithelial cells induced by TGF-β signaling in lymphedema
Authors: Park Hyeung Ju , Kataru Raghu P , Shin Jinyeon P , Mehrara Babak J
Published: January 2023

TGF-β driven fibrosis of the skin is a key pathologic feature of secondary lymphedema. However, the cellular mechanisms that regulate this process remain unknown. Epithelial mesenchymal transition (EMT) is a mechanism by which TGF-β induces fibrosis in other skin diseases. This process is characterized by loss of epithelial cell markers and polarity and increased migratory capacity and invasiveness. Therefore, the purpose of this study was to determine if EMT of skin keratinocytes is activated in lymphedema and if this process is mediated by TGF-β.

Using clinical lymphedema biopsy specimens and mouse models of lymphedema, we found that lymphedema results in fibrosis in the papillary dermis and a greater number of rete ridges in the basal layer of epithelium. Keratinocytes in the basal layer of lymphedematous skin express EMT markers (vimentin, fsp1, itgb4, slug, mmp9, twist) and fail to express epithelial markers (keratin14, E-cadherin). Interestingly, we found psamd3 expression in these EMT-like epithelial cells with accumulated TGF-β in papillary dermis by immunohistochemistry. Inhibition of TGF-β signaling markedly decreased EMT and prevented development of fibrosis in a mouse model. To determine if EMT of epithelial cells is induced by lymphedema fluid in skin, we treated keratinocytes with lymph fluid obtained from patients with lymphedema and analyzed in cultures with or without TGF-β inhibitors. We found that keratinocytes cultured with arm fluid only rapidly underwent EMT. In contrast, inhibition of TGF-β signaling prevented this response. Taken together, our findings show for the first time that EMT is activated by TGF-β signaling in lymphedema and that this process plays an important role in the pathology of this disease.

Supported by grants from NIH (R01HL111130).
The contribution of fetal-derived tissue resident macrophages to cytomegalovirus-associated sensorineural hearing loss
Authors: Otsuka Kelly , Nielson Chris , Suarez Daniel , Park Albert , Beaudin Anna E
Published: January 2023

Cytomegalovirus (CMV) is the most common congenital viral infection in the developed world and the leading cause of non-genetic sensorineural hearing loss (SNHL) in children. Congenital CMV causes progressive SNHL long after acute infection has resolved, suggesting a lasting effect on the developing host immune response within the cochlea and surrounding temporal bone marrow (TB). Using fate-mapping models, we have investigated the contribution of fetal-derived tissue resident macrophages (TRMs) to normal cochlear development and function, and their role in mediating CMV-associated SNHL. Our analysis defined two distinct myeloid population within the inner ear: 1) classical tissue resident macrophages (F4/80hi CD11blo) that highly express CD64 and are found exclusively in cochlear tissue; and 2) transitional monocytes (F4/80mid CD11bhi) that express both CD64 and Ly6C and are found in cochlea and TB. Fate mapping of these populations uncovered dual contribution of fetal-derived cochlear TRMs from yolk sac (YS) and fetal liver (FL) hematopoiesis. Transitional monocytes increase during acute CMV infection, acting as drivers of expanded lymphoid populations after CMV resolves. Importantly, analysis of Flt3-Cre labeling within myeloid populations suggests that TRMs are replaced by BM-derived populations at later postnatal stages following CMV infection, coinciding with SNHL. Ongoing work examines spatial distribution of distinct fetal-derived TRMs along cochlear architecture, defining transcriptional regulation differences between YS-, FL-, and adult-derived TRMs in response to CMV, and examining how replacement of fetal-derived TRMs with adult-derived TRMs affects tissue function and response to infection.

Supported by grants from University of Utah Molecular Medicine and Immunology, Inflammation and Infectious Disease (3i) Initiative and NIAID (T32 5T32AI138945)
Identification of a Novel Protein Interactions that Elucidates the Mechanism of Hydatidiform Molar Pregnancies in Women with NLRP2 and NLRP7 Mutations
Authors: Son Nayeon , So Matthew , Lupfer Christopher R
Published: January 2023

Mutations in NLRP2 and NLRP7 genes result in genetic maternal imprinting disorders. These genes have been identified to be maternal effect genes regulating early embryo development in idiopathic recurrent miscarriage. Previous research suggests that NLRP2 and NLRP7 regulate DNA methylation and immune signaling through inflammasome formation. However, the exact mechanisms underlying recurrent miscarriages are not known. In this study, we report a novel protein interaction of Human proliferation-associated 2G4 (PA2G4, aka: EBP1) with NLRP2 and NLRP7 through an unbiased yeast 2-hybrid screen of a human HeLa cell cDNA library. Protein interactions were confirmed by co-immunoprecipitation and confocal microscopy. Furthermore, global DNA methylation decreased in cells that overexpressed NLRP2/7 and EBP1. Our results further support the role for NLRP2/7 in regulating DNA methylation as a mechanism for recurrent miscarriages. Since EBP1 is implicated in apoptosis, cell proliferation, and differentiation, our discovery significantly advances our understanding of NLR biology and helps to explain the cellular pathways involved in idiopathic recurrent miscarriages.

Supported by grants from Beta Beta Beta Biological Honor Society
Epithelial C15ORF48/miR-147 is an essential regulator of gut inflammation and microbiome homeostasis
Authors: Xiong Min , Liu Ze , Graham Natalie Marie , Sokolich Thomas , Boldin Mark P
Published: January 2023

Aberrant inflammation plays an important role in the pathogenesis of several gastrointestinal diseases, including inflammatory bowel disease (IBD) and colorectal cancer (CRC). Insufficient understanding of the molecular mechanisms that control gut inflammation is one of the critical barriers to effective IBD and CRC therapies. To this end, we have uncovered C15ORF48/miR-147 as a novel negative regulator of gut inflammation. The C15ORF48/miR-147 gene encodes a small protein (C15ORF48) and a microRNA (miR-147-3p). Both molecular products are expressed predominantly in the large intestine, and their genetic ablation significantly exacerbates dextran sodium sulfate (DSS)-induced colitis in mice. The fulminant DSS-induced colitis phenotype in C15ORF48/miR-147−/− mice is driven by a cell-autonomous defect in gut epithelial cells and is mediated by gut dysbiosis. miR-147-3p and C15ORF48 synergize to silence the expression of NDUFA4, an accessory subunit of complex IV (CIV) in the mitochondrial electron transport chain. Our findings suggest that NDUFA4 plays an essential role in the activation of NF-kB signaling and the downstream inflammatory responses. Furthermore, C15ORF48, being an NDUFA4 structural paralog, can replace NDUFA4 in CIV and thus attenuate CIV activity and mitochondrial respiration, which may subsequently impact gut microbiome homeostasis. Collectively, our findings have established the C15ORF48/miR-147-NDUFA4 molecular axis as a novel, indispensable regulator of gut inflammation and a critical mediator between gut epithelial cells and microbiota.
CD300e Acts As A Ligand To Inhibit T Cell Immunity
Authors: Wang Jinghua , Manick Brian , Li Jun , Bi Ming , Kalabokis Vassilios , Person Anthony , Wu Guoping
Published: January 2023

CD300e has been reported as either an activating or inhibitory receptor and is involved in the tuning of immune responses. In this study, we demonstrate that CD300e acts as a ligand through an unknown receptor to inhibit T cell immunity. In PBMCs, CD300e protein is expressed on the cell surface of CD14+ monocytes, but not on T and B cells. CD300e-Fc fusion protein significantly reduced the expression of CD69 and CD25 on T cells, which suggest that CD300e inhibits the activation of T cells. Furthermore, CD300e-Fc fusion protein significantly inhibited TCR-mediated T cell proliferation and cytokine production including IL-2, IFN-gamma, IL-17, TNF-alpha, IL-8 and IP-10/CXCL10. Florescent conjugated CD300e-Fc fusion protein significantly bound to activated T cells and weakly to inactive T cells, suggesting that the putative CD300e counter-receptor is expressed on the cell surface of T cells, and the expression levels are upregulated upon T cell activation. CD300e-Fc fusion protein inhibited TCR-mediated p38 MAPK phosphorylation which indicates that CD300e negatively regulates T cell activation by impairing the p38 MAPK dependent antigen presentation. Moreover, our results support the notion that CD300e might be a new player in the regulation of the expansion of T cell-mediated responses. This CD300e novel T cell inhibitory pathway may provide a new strategy to modulate T cell-mediated immunity to treat immune-related diseases.
Metabolic fuel choices control MAIT cell functions at homeostasis and after infection
Authors: Riffelmacher Thomas , Paynich Mallory Murray , Chandra Shilpi , Wientjens Chantal , Seumois Gregory , Vijayanand Pandurangan , Kronenberg Mitchell
Published: January 2023

Mucosal-associated-invariant-T (MAIT) cells are an innate-like T cell subset that recognizes microbial-derived vitamin B metabolites. In contrast to conventional T cells, MAIT cells have an antigen-experienced phenotype and express memory markers by default. When activated, MAIT cells rapidly produce copious amounts of cytokines, resembling IFNg+ Th1- or IL-17+ Th17 effectors cells. While memory-like vs effector-like states in conventional CD4 and CD8 T cells are controlled by mutually exclusive metabolic states, the question remains as to which metabolic programs MAIT cells adopt at steady-state and after infection. Here we integrate scRNA-seq analysis with single-cell metabolic characterization of MAIT cells from humans and mice. We discovered a memory-like metabolic program that is acquired in the thymus and at steady state in the periphery. We identify a novel cluster of circulatory MAIT cells which is metabolically similar to IFNg+ MAIT1-like cells and preferentially consumes glucose. MAIT17 clusters, most prevalent in mice, uniquely engage in fatty acid uptake and mitochondrial metabolism. Following exposure to bacteria, mouse MAIT cells expand as CD127− KLRG1+ and CD127+ KLRG1− populations that adopt divergent transcriptomic and metabolic profiles with enhanced functionality. They remain altered long-term. CD127+, but not KLRG1+ MAIT cells engage in MAIT17-like metabolic and effector pathways and protect mice from lung infection with Streptococcus pneumoniae. In contrast, KLRG1+ MAIT cells depend on Hif1a-driven glycolysis and remain metabolically dormant but ready to respond, more rapidly engaging multiple metabolic programs to protect from viral infection.

Supported by R01AI137230 (MK), R01AI71922 (MK), Wellcome Trust 210842_Z_18_Z (TR)
Eos regulates IL-7 signaling and central memory T cell differentiation
Authors: Pokhrel Srijana , Read Kaitlin A , Jones Devin M , Powell Michael D , Warren Robert T , Oestreich Kenneth J
Published: January 2023

CD4+ central memory T cells (TCM) are key players in recall immune responses and thus are critical to long-term immunity. Despite their importance, much remains to be defined regarding the mechanisms that promote their differentiation and survival. Ikaros zinc finger (IkZF) transcription factors are known regulators of T and B cell differentiation programs. To date, the IkZF factor Eos has been mainly linked to regulatory T cell suppressive functions. Now, we have found that Eos is expressed at elevated levels in CD4+ TCM populations. Comparison between WT and Eos-deficient T cells in a murine model of influenza infection revealed a decrease in the percentage of CD4+ TCM cells in the absence of Eos, suggesting that Eos promotes TCM differentiation and/or survival. Using an established in vitro model of CD4+ TCM-like cell differentiation, we found that loss of Eos correlated with reduced expression of genes encoding key TCM transcription factors and cell surface markers. Further, Eos deficiency resulted in decreased STAT5 activation downstream of IL-7 signaling, a known positive regulator of TCM populations. In WT cells, IL-7 treatment increased Eos expression, suggesting a positive feed-forward relationship exists between Eos expression and IL-7/STAT5 signaling. Overall, our findings demonstrate that Eos is an important regulator of TCM populations. Understanding the mechanisms by which Eos regulates TCM function and survival may therefore be of interest in exploiting TCM protection against re-infection for therapeutic benefit.

Supported by a grant from NIAID (NIH- R01 AI134972) and funds through The Ohio State University College of Medicine
The strength of antigenic and IL-2 signals epigenetically programs functional memory CD8+ T cells
Authors: Guillen Erik , Chin Shu Shien , Chorro Laurent , Achar Sooraj , Ng Karina , Oberle Susanen , Alfei Francesca , Zehn Dietmar , Altan-Bonnet Gregoire , Delahaye Fabien , Lauvau Gregoire
Published: January 2023

Priming of naïve CD8+ T cells requires three signals: 1) cognate antigen, 2) co-stimulation, and 3) cytokines. These signals orchestrate CD8+ T cell differentiation. Particularly, strong T cell receptor (TCR) signals induce a more robust effector response, while weak TCR signals preferentially favor memory CD8+ T cells. However, memory cells induced upon varied TCR signaling strengths have been reported to be functionally similar. In this work, we show that, in contrast to this assumption, the strength of T cell receptor (TCR) signaling determines the requirement for interleukin-2 (IL-2) signals to form a pool of memory CD8+ T cells that are functionally different. We found that upon secondary antigen encounter, memory CD8+ T cells induced upon priming with lower TCR and IL-2 signals exhibited delayed cell cycle entry, impaired expansion and intracellular calcium accumulation compared to those primed with strong TCR and IL-2 signals. Furthermore, we also establish that the combination of both robust TCR and IL-2 signals, but not either one alone, are necessary to induce genome-wide changes in chromatin accessibility in regions targeting a wide range of biological processes consistent with their greater functional fitness. These notably included greater chromatin accessibility in promoters of genes encoding for stem cell, cell cycle and calcium-related proteins. Taken together, these findings demonstrate that modulating TCR strength and IL-2 signaling at the time of CD8+ T cell priming, lead to the differentiation of functionally distinct pools of memory CD8+ T cells.

Supported by T32GM7288
IL-23 signaling limits ferroptosis-driven immunopathology during systemic fungal infection
Authors: Millet Nicolas Olive Jean , Solis Norma V , Aguilar Diane , Lionakis Michail S , Wheeler Robert T , Jendzjowsky Nicholas , Swidergall Marc
Published: January 2023

Pattern recognition receptors initiate antifungal immune responses during infection. Recently, we identified that the ephrin type-A receptor 2 (EphA2) is a fungal β-glucan receptor which orchestrates innate immune responses and prevents oral Candida infection. However, the role of EphA2 during invasive candidiasis (IC) remains unknown. Here we found that EphA2 induces renal inflammation and injury; thus, promoting immunopathology during IC. In Epha2−/− mice, dendritic cells migrated more efficient in infected tissues, while secreting more IL-23. Furthermore, Epha2−/− mice had reduced lipid peroxidation in kidneys, a marker of ferroptotic cell death. Pharmacological inhibtion of ferroptosis increased macrophage fungal killing, reduced inflammation, and increased disease outcomes during IC. Previously, IL-23 signaling has been associated with myeloid cell survival during candidiasis. Treatment of macrophages with IL-23 reduced ferroptotic macrophage cell death and excessive inflammation, while increasing killing capacity. Furthermore, IL-23 treatment of infected mice increased survival and reduced ferroptosis during IC. Collectively, our study demonstrates that ferroptotic host cell death is linked to immunopathology and can be targeted by recombinant cytokine therapy during fungal infection.

This work was supported in part by NIH grant R00DE026856, an American Association of Immunologists Careers in Immunology Fellowship, and a TLI Seed grant to MS
Developing a transgenic mouse model to better understand the role of CD8 and granulysin against Mycobacterium tuberculosis infection.
Authors: Thakur Preeti , Sutiwisesak Rujapak , Lu Yu-Jung , Behar Samuel
Published: January 2023

Human cytotoxic lymphocytes kill infected cells through release of lytic proteins contained in cytotoxic granules. The cytotoxic granules of human NK and CD8 T cells contain perforin, granzyme and granulysin (GNLY), and GNLY has direct antimicrobial activity against Mycobacterium tuberculosis (Mtb). Murine studies suggest that CD8 T cells have only a modest role in immunity to tuberculosis (TB). We hypothesize that mouse CD8 T cells are ineffective at containing Mycobacterium tuberculosis (Mtb) as they don’t express GNLY. To test this hypothesis, GNLY-transgenic mice that express human granulysin protein under the control of human regulatory elements were infected with Mtb. GNLY-Tg and non-tg control mice control Mtb similarly. There was no difference in the ability of GNY-Tg and non-tg CD8 T cells to transfer protection to T cell deficient mice. Anti-CD3 stimulation led to expression of granulysin in human CD8 T cells, but not in murine CD8 T cells. However, after Mtb infection, GNLY was expressed in NK cells but not CD8 T cells. We conclude that GNLY-expressing NK cells do not alter resistance to Mtb. Importantly, GNLY-Tg mice are inadequate model to study CD8 function during TB. We are developing a new strategy to express granulysin in CD8 T cells. We expressed GNLY (isoform 2) in primary murine CD8 T cells by retroviral gene transfer and detected GNLY expression in 60% of CD8 T cells.

Future directions

We are assessing the cytolytic activity of GNLY-expressing CD8 T cells and their ability to kill intracellular pathogens in vitro and in vivo. We expect to develop a new GNLY-Tg model that can be used to study the function of CD8 T cells and granulysin in many different diseases. This work is supported by NIH AI159374.

This work is supported by NIH grant R21 AI159374.
Sphingolipids: A double-edged sword in the defense against Mycobacterium tuberculosis.
Authors: Guzman Gaelen D , Leier Hans , Niekamp Patrick , Holthuis Joost , Tafesse Fikadu G
Published: January 2023

Mycobacterium tuberculosis (Mtb) is a pathogen with a massive global health impact, and yet fundamental questions remain regarding the underlying mechanisms of its entry and subversion of innate immune mechanisms – particularly the manners in which host lipids influence the course of an infection. This pathogen gains entry to host alveolar macrophages via phagocytosis and, within the phagosome, Mtb perturbs host lipid metabolism and distribution. Here, we’ve shown that Mtb relies on an intact sphingolipid biosynthesis pathway to gain entry to host cells but that sphingolipids restrict the intracellular growth of this pathogen. We used genetic ablation and small molecule inhibition to perturb sphingolipid biosynthesis, resulting in reduced Mtb uptake across multiple phagocytic cell types without affecting receptor mediated endocytosis. Mechanistically, we visualized the dynamics of several signaling events during phagocytosis to demonstrate that sphingolipid deficiency results in poor initiation of phagocytic signaling. Intriguingly, sphingolipid inhibition at later stages of infection may result in enhanced intracellular Mtb growth and accelerated lysosomal damage and subsequent phagolysosomal escape. This study has revealed that the sphingolipid family may constitute a double-edged sword which plays multiple roles during Mtb infection. In understanding the interplay between Mycobacterium tuberculosis and host factors such as sphingolipids, we hope to identify novel methods of treatment for this ancient and global pathogen.

Supported by grants from NIH (R21 3R21AI124225-01A1 and R01 1R01AI14154 9-01A1)
A GABA-producing probiotic for the protection of CNS demyelinating inflammation
Authors: Ochoa-Reparaz Javier , Hoffman Kristina , Long Tyrel , Doyle William J , Kohl Hannah M , Staben Kendall , Sargent Alivia , Linton Rachel , Ristig Molly , Harris Rylee , Shi Xutong , Gibson K Michael , Roullet Jean-Baptiste , Castillo Andrea R
Published: January 2023

The gut-microbiota-brain axis has emerged as a critical pathway in the regulation of neuroinflammation. The gut microbiome regulates the severity of many experimental models of autoimmune central nervous system (CNS) demyelinating inflammatory diseases. Our most recent findings demonstrate that the microbiota of mice from different sources affects the severity of CNS inflammatory demyelination. Neuroinflammation triggered in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, modified the gut microbiota composition. The disease progression resulted in a significant reduction in the relative abundances of members of lactic acid bacteria when compared to healthy control mice. Among the altered taxa, bacteria producing gamma-aminobutyric acid (GABA) were significantly reduced. We hypothesized that modifying the microbiota with a probiotic while increasing intestinal GABA levels would reduce EAE’s severity. We genetically engineered a Lactococcus lactis probiotic that produces excessed levels of GABA. Real-time quantitative PCR analysis demonstrated an elevated expression of glutamic acid decarboxylase (GAD). GABA-specific ELISA showed a significant increase in neurotransmitter production when exposed to increasing concentrations of glutamic acid and time. In vivo, five times/week oral gavages with 5 × 108 CFU/mouse of GAD L. lactis but not with empty-plasmid carrier L. lactis protects against EAE in mice compared with sham-treated mice and prevents weight loss of animals while modulating the microbiome’s composition. Our results show that the increase of GABA at the intestinal level with the oral treatment with a probiotic strain protects against neuroinflammation in the CNS.

Supported by grant from NIH (R15 NS107743)
Aging impairs regulatory T cell (Treg) cells to affect late-onset (aged) multiple sclerosis (MS) – with the model of experimental autoimmune encephalomyelitis (EAE)
Authors: Wang Weikan , Thomas Rachel , Oh Jiyoung , Su Dong-Ming
Published: January 2023

A master immunoregulator FoxP3-expressing CD4+ Treg cellsplay an ameliorative role in the severity of MS/EAE disease. However, it is contradictory that the aged T cell immune system possesses relatively enhanced thymic CD4+ Treg generation and accumulated peripheral CD4+ Treg cells, but coupled with severe MS/EAE symptoms and pathology. Using single-cell (sc)-RNA-Seq assay, we found that CNS-infiltrating CD4+ Treg cells in aged EAE mouse model had increased pathogenic properties, showing co-expression of infg and il17a with foxp3, and reduced suppressive effect, exhibiting increased clonal expansion of pathogenic CD4+ T cells. These indicate changed Treg quality in aged EAE mice. Transient inhibition of aged peripheral FoxP3+ Treg cells mitigated the disease severity in the aged mice. The ameliorative effect was more significant when partially inhibiting FoxP3+ Treg cells with a drug P300i than completely depleting FoxP3+ Treg cells in FoxP3DTR transgenic mice. The mitigation is probably attributed to the correction of Treg cell distribution outside and inside the CNS. By inhibiting accumulated FoxP3+ Treg cells adhering to the brain’s choroid plexus (outside the CNS), the IFN-g-producing cells can be restored, thereby, the impediment of immune cell trafficking into the inflamed CNS is released in aged EAE mice. As a result, the proportion of myelin-specific CD4+ Treg cells inside the CNS was increased for repairing neuroinflammatory damage. Together, the underlying mechanism of severe MS symptoms in elderly patients is associated with the accumulation of Treg cells outside the CNS, which prevents the reparative antigen-specific Treg cells from entering the CNS during the disease.

Supported by NIH/NIAID R01AI121147
Estradiol Effects on Polymorphonuclear Cell Production and Actions Contribute to Estrogen-Mediated Lymphangioleiomyomatosis (LAM) Progression
Authors: Minor Briaunna M N , Koudouvoh Jules , Gibbons Erin , Seger Christina , Hammes Stephen
Published: January 2023

The estrogen sensitivity of Lymphangioleiomyomatosis (LAM)—a rare lung disease involving the slow growth of smooth muscle-like adenomas that result in cystic change in the lung parenchyma—is traditionally examined to assess the direct effects of estradiol on the tumor cells. However, we have observed that the estrogen sensitivity of in vitro models is markedly less pronounced than that of in vivo models, suggesting that estradiol may act elsewhere to promote tumor progression. We have previously reported that polymorphonuclear cells (PMNs) are preferentially increased in the immune microenvironment and regulate growth of LAM-like TSC2-null uterine tumors. We hypothesized that, in addition to direct effects of estrogen on tumor cells, estrogen might also stimulate tumor growth by promoting PMN production in the bone marrow and actions in the tumor microenvironment. Here, we show that estrogen availability influences the levels of PMNs in naïve and tumor-burdened mice. Using bone marrow cultures and CFC assay, we demonstrate that estradiol is a potent inducer of PMN production in chronic inflammatory conditions. Employing both pharmacologic agents and estrogen receptor (ER)-null bone marrow, we showed that ER□ is necessary for promoting a PMN fate for myeloid progenitors. While we see that the migratory, invasive, and proliferative capacities of TSC2-null cell lines are not augmented when stimulated with E2 directly, evidence shows estrogen promotes the pro-tumorigenic function of PMNs co-cultured with TSC2-null cell lines. These data provide insight into the robust effect of in vivoestrogen stimulation as estradiol may be a dual effector in LAM tumor progression and great target for anti-LAM therapeutic strategy.

Supported by grants from NIH (R01CA193583, F31 CA254132-01).
Assembly and localization of extracellular matrix protein fibronectin modulates Natural killer cell infiltration in tumor spheroids.
Authors: Rajasekaran Narendiran , House Trenton , Magdaleno Carina Crystal , Varadaraj Archana
Published: January 2023

Natural Killer cells (NK), the major innate effector cells, with their broad cytotoxicity against tumors are ideal candidates for immunotherapy. Once having entered the malignant site, NK cells encounter a complex environment composed of tumor cells, non-tumor cells along with the extra cellular matrix (ECM). As one of the integral components of the tumor microenvironment (TME), the normal regulation of the extra cellular matrix (ECM) is necessary to prevent tumor progression and metastasis. Nevertheless, the impact of the matrix architecture found in solid tumors on immune cells and especially NK cells is not well characterized. Here we investigated the role of Fibronectin, a fibrous ECM protein, and its assembly and localization on NK cell infiltration into clear cell renal cell carcinoma (ccRCC) spheroids. 3D tumor spheroids established from the ccRCC cell line 786-O (RCC-) that lacks the Von Hippel-Lindau (VHL) gene are incapable of fibronectin assembly in their ECM. However, addition of the VHL gene into the 786-O cells (RCC+) reversed this phenotype facilitating fibronectin assembly. Fibronectin in RCC-spheroids exhibited prominent peripheral localization in contrast to RCC+ spheroids where fibronectin was detected towards the center of the spheroid. Coincident with fibronectin localization, PKH26 labeled Natural killer cell line, NKL, showed significantly increased infiltration into RCC+ spheroids compared to RCC-spheroids that lacked the tumor suppressor VHL. Thus, assembly and localization of fibronectin modulates NK cell infiltration in RCC tumors.

Supported by grants from NIH U54MD012388 (NR and AV) and NIH U54CA143925 (NACP-NAU) to NR.
LTi-like type 3 innate lymphoid cells are associated with the transient induction of tertiary lymphoid structures in inflamed pulmonary tissue
Authors: Riffard Clemence , Teillaud Jean-Luc , Dieu-Nosjean Marie-Caroline
Published: January 2023

Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates observed in inflamed tissues. Their presence in pulmonary tissue has been associated with good prognosis in patients with lung cancer. Although TLS formation shares common features with secondary lymphoid organogenesis, the mechanisms underlying this process are yet to be fully elucidated. Presence of ILC3 in the vicinity of tumor-associated TLS correlates with the density of lymphoid structures. We hypothesized that RORγt+ ILC3 play a major role in TLS induction in inflamed lungs, as do RORγt+ fetal lymphoid tissue inducer cells (LTi) during lymph node formation. We developed a murine model of pulmonary inflammation induced by LPS intranasal instillation that triggers TLS formation. ILC3 presence in lungs was observed with a specific flow cytometry antibody panel. Three RORgt+ ILC3 subsets (NCR− CCR6−, NCR− CCR6+, NCR+ CCR6−) were detected. In particular, NCR− CCR6+ LTi-like cells represented about 25% of CD117+ CD127+ ILC (ILC2/ILC3) and less than 0.04% among CD45+ cells. Analyses of ILC subsets at different time points following LPS instillation showed an increase in the number and % (up to 0.59%) of ILC3 among CD45+ cells, as well as an increase in the % of LTi-like ILC3 among CD45+ cells, that peaked around day 10 (from 0.02% up to 0.15%). Quantification of TLS by histochemistry showed that they are induced in lung tissue in an LPS dose-dependent manner. TLS density increases between days 3 and 10 and decreases thereafter. They were no longer detected at day 25, while LTi-like ILC3 % returned to baseline. Gene expression analysis and functional assays of sorted LTi-like ILC3 are essential to determine if these cells are key players in TLS induction.

Supported by grants from La Ligue Nationale contre le cancer (TAHG21042) and Fondation ARC (R19232DD).
Resident memory T cells in antitumor immunity and cancer immunotherapy
Authors: Mami-Chouaib Fathia , Tihy Isabelle , Corgnac Stephanie
Published: January 2023

Tumor-resident memory T (TRM) cells play an important role in cancer diseases. Accumulating evidence indicates that subpopulations of CD3+ CD8+ tumor-infiltrating lymphocytes (TIL) are TRM cells, and are emerging as activated tumor-specific T cells. These TRM are characterized by expression of CD103 integrin and identify tumor-reactive cytotoxic T lymphocytes. Human lung tumor CD103+ CD8+ TRM co-express CD49a and CD69, and are associated with a favorable prognosis. They also express a panel of T-cell inhibitory receptors, including PD-1, and are able to kill autologous tumor cells upon blockade of PD-1 with neutralizing antibodies. This CD103+ CD8+ TRM subset also emerges as a predictive biomarker of response to PD-1 blockade and expands during anti-PD-1 treatment in responder, but not in non-responder patients. It is now widely admitted that expression of CD103 on activated CD8+ T lymphocytes and persistence of TRM in epithelial tissues requires TGF-β. This cytokine is secreted in the tumor microenvironment in its inactive form bound to latency-associated protein, and is activated by αV integrins on tumor cells and immune cells. Using multiplex immunofluorescence staining on cohorts of anti-PD-(L)1-treated lung cancer patients, we recently showed that decreased expression of tumor αV is associated with improved immunotherapy-related-progression-free survival and correlates with an increased density of CD8+ CD103+ TIL. Cancer cell αV activates autocrine TGF-β and participates in inducing CD103 on activated CD8+ T cells. Our more recent studies in mouse tumor models and human cancers demonstrate that different CD8+ TRM subsets participate to response different immune checkpoint inhibitors and therapeutic cancer vaccines.

Supported by ARC, INCa, Ligue

Supported by ARC, INCa, Ligue
Mucosal vaccination provides protection from HSV-2 infection and disease
Authors: Tucker Kiersten , Dave Veronica , Lund Jennifer M
Published: January 2023

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted pathogen that is estimated to infect around 23 million people per year. Despite high global prevalence, there are not any approved vaccines that are therapeutic or preventative. Most vaccines that we use today rely on injecting antigens intramuscularly in order to elicit an adaptive immune response. However, given that most pathogens gain entry to the host across barrier surfaces, a focus on eliciting mucosal immunity may enhance protection; vaccine-induced local immunity at the site of first pathogen exposure may have the best chance at preventing the spread of infection beyond the pathogen portal of entry. We hypothesized that a mucosal immunization would prime memory T cells to reside in vaginal tissues and provide better protection against vaginal HSV-2 exposure than other routes of immunization. Our initial data suggests that intranasal immunization is effective in protecting mice from vaginal HSV-2 infection. Ongoing work focuses on characterizing the role of vaccine-elicited mucosal CD8+ T cells in preventing infection to provide insight into the mechanisms of protection induced by mucosal immunization for HSV-2. These findings contribute to the efforts to generate an effective vaccine to prevent HSV-2 infection and disease.

Supported by the Mary Gates Research Scholarship and the Art Levinson Award through the University of Washington.
Genetic analysis of differential responses to adjuvanted influenza vaccination
Authors: Cruz Cisneros Marta C , Parotti Breantié , Noll Kelsey , Bell Timothy A , Hock Pablo , Heise Mark T , Ferris Martin T
Published: January 2023

Each year the seasonal influenza vaccine is administered to protect individuals from infection, but vaccination often shows variable efficacy against influenza infections. While the inclusion of adjuvants can boost vaccine efficacy, individuals also vary in their response to adjuvants, and understanding the basis of this variation would provide insights into adjuvant mechanisms and advance vaccine development. Host genetics contribute to variation in vaccine response, but the specific genetic factors that impact immune responses to adjuvanted vaccination are not well understood. To better understand how the genetic background of individuals can modify vaccine and adjuvant-induced immune responses, we screened a set of Collaborative Cross (CC) mouse strains with immunization via inactivated influenza with or without alum. Across these genetically diverse CC strains, we observed wide variation in levels of influenza specific antibodies and isotypes (e.g., IgG1, IgG2). We further focused on two strains, CC002 and CC072, that had contrasting antibody responses to vaccine + alum. To understand the genetic factors driving these responses, we generated an F2 population to map quantitative trait loci (qtl) driving the differences between CC002 and CC072 in vaccine responses. Among the F2 population, variation across antibody levels were observed in mice vaccinated with the vaccine with and without alum that reflected the variation in the parent strains. QTL were mapped for variation in antibody levels for mice vaccinated without alum, further supporting a role for host genetics in regulating vaccine responses. Future analysis will focus on analyzing specific host factors modulating antibody responses to adjuvanted vaccination.

Supported by grants from NIH (U19 AI100625, 5T32 GM007092, 1T32 GM135128, 5R25 GM055336-19) and Burroughs Wellcome Fund GDEP to MCC.
A live-attenuated mutant CVB3 vaccine virus protects against multiple coxsackievirus B infections
Authors: Lasrado Ninaad , Rasquinha Mahima T , Sur Meghna , Gangaplara Arunakumar , Massilamany Chandirasegaran , Arumugam Rajkumar , Steffen David , Reddy Jay
Published: January 2023

Group B Coxsackieviruses (CVB) contains six serotypes, namely CVB1 through CVB6, affecting various organs. But, no vaccines are currently available to prevent these infections. We recently derived a live attenuated vaccine virus termed mutant (Mt) 10 encoding a single amino acid substitution H790A within the viral protein 1 (VP1) of the CVB3 viral canyon region. This virus could prevent myocarditis and pancreatitis caused by both homologous (CVB3) and heterologous (CVB4) serotypes in A/J mice. Mechanistically, we noted that the vaccine virus induces cross-reactive neutralizing antibodies that skewed towards mainly IgG isotypes. Similarly, by using major histocompatibility complex class II dextramers and tetramers for various VP1 epitopes, we demonstrated that the vaccine recipients develop antigen-specific T cell responses producing preferentially interferon-γ responses. Furthermore, our preliminary studies revealed that the vaccine virus could prevent the progression of diabetes-induced by CVB4 in the non-obese diabetic mice. Together, our data suggest that the Mt10 vaccine can prevent infections caused by multiple CVB serotypes, paving the way for developing monovalent CVB vaccines to prevent heart and pancreatic diseases of enteroviral origin.

This work was supported by The Transformational Grant from the American Heart Association (18TPA34170206)
Control of established, CNS-resident lyssavirus infection by an adaptive immune response stimulated by single-dose monoclonal antibody therapy
Authors: Huaman Celeste , Mastraccio Kate E , Coggins Si’Ana A , Hussain Imran , Yan Lianying , Ahmed Anwar E , Ho Trung , Smith Ina L , Markotter Wanda , Weir Dawn , Laing Eric D , Broder Christopher C , Schaefer Brian C
Published: January 2023

Rabies virus and related lyssaviruses cause uniformly fatal disease, once the infection progresses to the central nervous system. Current lyssavirus immunotherapies are directed toward peripheral neutralization of virus to prevent CNS infection during the pre-symptomatic stage of disease. In this study, using an anti-lyssavirus human monoclonal antibody (mAb), F11, we evaluated the efficacy of immunotherapy on established lyssavirus infections in mice. Remarkably, using luminescence-based longitudinal tracing of virus infection in a mouse model of lethal disease, we found that a single dose of F11 reverses clinical signs of disease and protects animals from lethality following lyssavirus infection, even when administered after initiation of virus replication in the CNS. Investigation of the mechanisms of F11 efficacy revealed that F11-dependent neutralization is insufficient to protect animals from mortality. Control of infection requires an intact adaptive immune response, particularly CD4 T cells. Additionally, in vivo analysis of the F11-N297G mutant, which has defective FcRγ binding, showed a transiently increased viral load and increased morbidity when compared to F11 treated mice. Despite long-term survival and absence of clinical signs of disease in F11-treated animals, we found that lyssavirus infection persists chronically, concomitant with elevated expression of cellular immune response genes. These findings demonstrate that single-dose mAb therapy can stimulate an adaptive, T cell-dependent response that is durable and highly effective against an established CNS infection by a lethal neurotropic virus.

Supported by grants from the National Institute of Health (U01GM109887, R01AI125552), and a USU Program Project Grant (MIC-732515) and a USU Center for Global Health Engagement grant (HU00011920118)