bluuebunny/crossref_metadata_2025 huggingface dataset:
Glutamate receptors provide costimulatory signals to improve T cell immune response.
Authors: Shanker Anil , de Aquino Maria Teresa P. , Hodo Thomas , Uzhachenko Roman
Published: January 2023

The interaction between the nervous system and immune system has sparked interest in recent years. Emerging evidence shows an intricate neuro-immune network. Studies revealed that neurotransmitters can trigger immune cell responses such as cytokine secretion, integrin expression, and chemotaxis. We were intrigued by the expression of various neurotransmitter receptors on mouse T lymphocytes. Specifically, following TCR stimulation, glutamate receptors (GluR) were significantly upregulated on both CD4+ and CD8+T cells, with a peak at 48 h. Concomitant with the upregulation of activation molecules CD69, CD25 and CD44, proliferating CD8+T cells presented higher levels of GluR3 and GluR1 when compared with non-proliferating cells, as well as a higher production of IFN-g and granzyme B. Based on the augmentation of GluR expression upon T cell activation, we evaluated the role of GluR signaling in modulating T cells in orthotopic mammary tumors that express a low avidity epitope of a model antigen hemagglutinin (4T1HA). We found that HA-reactive CD8+ T cells in the tumor-infiltrate had an upregulated expression of GluR3 and GluR1 when compared with control group. The blockade of GluR signaling down-regulated expression of CD69 and CD25, impaired T cell proliferation, and interfered with their cytolytic activity. Early TCR-activation pathways such as Lck/Akt as well as calcium signaling were affected by GluR antagonists, suggesting their signaling interaction. Overall, data suggest that glutamate receptors may have costimulatory effects on T cell activation and glutamate agonists may boost T cell response in an immunosuppressive setting such as cancer.
Role of the CD27/CD70 pathway in regulatory T cell function
Authors: Bowakim Natalia , Acolty Valérie , Dhainaut Maxime , Yagita Hideo , Oldenhove Guillaume , Leo Oberdan , Moser Muriel
Published: January 2023

There is evidence that the CD27/CD70 pathway plays a major role in the regulation of immunity versus tolerance in vivo. In particular, the transgenic expression of CD70 by dendritic cells breaks tolerance and induces a lethal autoimmune disorder.

Although CD27 engagement on conventional T cells (Tconv) has been shown to induce Th1-type responses, the role of CD27, expressed at higher levels on regulatory T cells (Treg), remains elusive. Recent data from our laboratory have shown that CD27 expressed on Tregs was involved in the inhibition of CD70, suggesting that CD27 may display opposite functions, i.e. pro- or anti-inflammatory when expressed on Tconv and Treg, respectively.

The objective of this work was therefore to decipher the role of CD27 on Treg function.

Our data show that (i) injection of agonistic anti-CD27 mAb increased the development and function of Tregs, as assessed by increased proliferation and upregulation of hallmarks of Treg activation such as ICOS, GATA-3, Helios, and inhibitory molecules such as CTLA-4, PD-1 ; (ii) similarly, Tregs were activated in mice expressing CD70 in a constitutive manner (WT x CD70tg mice); (iii) CD27+ Tregs from WT mice were more activated and displayed higher suppressive capacity than CD27− Tregs.

These observations suggest that the CD27/CD70 pathway potentiates the homeostasis and inhibitory function of Tregs. Experiments are under way to identify the transcriptomic profile induced by CD27 engagement in Tregs and to analyze their function in various settings in vivo, using a CD27fl/fl mouse to selectively delete CD27 in Tregs.
The dynein-binding protein DISC1 plays important roles in relocating organelles to the immunological synapse during T cell activation.
Authors: Poenie Martin F. , Maskalenko Nicholas A. , Nath Shubhankar
Published: January 2023

DISC1 (Disrupted in Schizophrenia-1) is a dynein-binding scaffold protein that interacts with many other proteins. In neurons, DISC1 is important for proliferation, migration, and signaling. We found that DISC1 is expressed in Jurkat cells and human NK cells and that it coimmunoprecipitates with dynein in Jurkat cells. Immunostaining for DISC1 in unstimulated Jurkat cells showed that it was generally located around the MTOC. When Jurkat cells were stimulated with SEE-coated Raji cells, depending on the antibody used, we found DISC1 either clustered around mitochondria or colocalized with dynein and NDE1 at the immunological synapse (IS). Biochemical studies showed that after Jurkat cells are activated (SEE-coated Raji cells or PMA), DISC1 coimmunoprecipitates with talin and paxillin. Sequence analysis of cloned DISC1 cDNAs from Jurkat cells revealed that at least two isoforms (L and Lv) are expressed. When GFP-DISC1 isoforms were individually expressed in Jurkat cells, we found that the Lv isoform concentrated around mitochondria whereas the L isoform colocalized with dynein at the synapse as seen in the immunostaining data. While RNAi was relatively ineffective at reducing DISC1 expression, we could abolish expression by using a DISC1-targeted CAS9/CRISPR construct. In these DISC1-disrupted cells, we found that mitochondria failed to accumulate at the immunological synapse and the MTOC did not appear to move all the way to the contact site (perhaps suggesting a defect in docking). When GFP-DISC1 Lv was expressed in the DISC1 CAS9/CRISPR cells mitochondria accumulation at the IS was restored. Similarly, expression of the GFP-DISC1 L restored the normal positioning of the MTOC upon T cell activation.
Transcriptional profiling of pathogen-specific CD4 T cells reveals key events that regulate Th17 cell priming and differentiation
Authors: Gao Yajing , Deason Krystin L. , Jain Aakanksha , Dozmorov Igor , Wakeland Edward K. , Pasare Chandrashekhar
Published: January 2023

CD4 T cells play a central role in adaptive immunity through secretion of specific cytokines which exert effector functions against invading pathogens. They also drive B cell activation and coordinate CD8 T cell responses. Productive CD4 T cell responses are determined not only by the specificity of the T cell receptor, but also by innate immune cues provided by dendritic cells (DCs) which shape the nature of the effector lineage commitment. However, to what extent pathogen-recognizing DCs prime naïve T cells on a global transcriptional level remains elusive, because of the inability to track and identify pathogen-specific T cells during cognate DC-T cell interaction. Here, we analyzed transcriptional profiles of pathogen-specific mouse Th17 cells utilizing an in vitro approach, where naïve CD4 T cells were differentiated by mature DCs in a highly antigen-specific manner. These pathogen-specific Th17 cells exhibit enormous lineage heterogeneity and resemble Th17 cells found in vivo following infections. We further discovered that DC-mediated priming not only generates a distinct transcriptional landscape in pathogen-specific Th17 cells, but also warrants their acquisition of memory gene signature and metabolic switch from glycolysis to oxidative phosphorylation. These results indicate that pathogen-activated dendritic cells play a critical role in CD4 T cell transcription programming that extends beyond determining lineage commitment. By mining this dataset, we have identified a previously unappreciated gene in promoting Th17 lineage commitment. Conceptually, our study underlines the necessity of investigating physiologically differentiated CD4 T cells in order to reveal novel regulators of T cell biology.
Role of TNF-α reception in epidermal γδ T cell function during homeostasis and after TCR stimulation
Authors: Matthews Carolyn J. , Barba Cindy , Mendoza German , Jameson Julie M.
Published: January 2023

Inflammatory responses mediated by the cellular components of epithelial tissues are essential to the wound healing process. Epidermal γδ T cells, also known as dendritic epidermal T cells (DETC), play key roles in the wound healing process by producing growth factors, chemokines and cytokines necessary for the proliferation and migration of epithelial cells. DETC activation occurs via the γδ TCR, however, second and third signals are required to sustain the activation and production of factors needed in functions such as wound repair. While progress has been made on elucidating signal 2 costimulatory signals such as JAML and CAR, less is understood about what signals are required for signal 3 in DETC and what roles TNF-α plays in regulating and sustaining DETC function. TNF-α signals through two receptors, tumor necrosis factor receptor 1 and 2 (TNFR1 and TNFR2). These receptors elicit varied responses that include apoptosis, survival or inflammation. Our data indicate that DETC upregulate type 1 chemokines and their corresponding chemokine receptor expression during inflammation. We have also found that a subset of DETC express TNFR1 and TNFR2 on their cell surface constitutively suggesting a requirement for tonic TNFR signaling for particular DETC populations. We have quantified chemokine, cytokine and growth factor production from DETC stimulated in the presence or absence of TNF-α using Luminex technology. Additionally, we have investigated which TNFR is responsible for modulating DETC function by comparing DETC function in cells isolated from wild-type, TNFR1 and/or 2 knockout mice. Identifying which factors regulate DETC function and activation could provide potential targets for improving DETC function in wound repair.
IRF4 regulates the rate of cell cycle during B cell differentiation.
Authors: Patterson Dillon , Scharer Christopher D. , Boss Jeremy M.
Published: January 2023

Cell division is required for the initiation of B cell differentiation, the regulation of isotype class switching, and ultimately entry of cells into the plasma cell (PC) lineage. Division coupled changes in the expression of transcription factors, such as interferon regulatory factor-4 (IRF4), that coordinate the PC transcriptional program also occur; however, little is known regarding how these factors coordinate cell division and exit from the cell cycle after differentiation. To begin to address this gap in knowledge, we assessed the cell division kinetics of wildtype (WT) B cells responding to the T cell independent antigen lipopolysaccharide (LPS). Interestingly, we found that WT B cells undergo at least 8 divisions before differentiating into PCs. Computational modeling of division rates over time defined a proliferative burst between 48 and 60 hours after LPS injection that is characterized by a rapid increase in the rate of cell division. IRF4-deficient B cells divided but failed to undergo the proliferative burst resulting in fewer activated B cells. Indeed, IRF4 is expressed at low levels during these divisions before being upregulated in post-mitotic CD138+ PCs after 8 divisions. Notably, while more than 40% of WT B cells reached division 8+ by 72 hours, IRF4-null B cells appeared to be stalled at divisions 2–5. Taken together, these data suggest that IRF4 regulates the rate of cellular proliferation during B cell differentiation that occurs around divisions 2–5 and that this role may be concentration-dependent.
Small molecule inhibitors of Activation-Induced Deaminase
Authors: Alvarez-Gonzalez Juan Antonio , Maul Robert , Kohli Rahul M. , Gearhart Patricia J.
Published: January 2023

Activation-Induced Deaminase (AID) is a cytosine deaminase that converts cytosine into uracil in DNA, which initiates a cascade of mutagenic DNA repair to introduce point mutations and double-strand breaks. Specific targeting of AID to the immunoglobulin heavy chain locus promotes somatic hypermutation in antibody variable genes for affinity maturation, and breaks in switch regions for class switch recombination (CSR). However, mis-targeting of AID to other loci could initiate tumor development and lead to greater drug resistance among cancer cells when continually expressed. To identify a small molecule inhibitor of AID, we screened ~400,000 compounds in conjunction with the NCATS core facility at NIH. Using FRET based analysis of cytosine deamination, we identified 150 potential inhibitors of AID catalytic activity. To confirm biological function, we examined their effects on CSR in an in vitro murine B-cell activation assay using CH12 cells, wherein 30 were confirmed inhibitor candidates. We then selected the top seven molecules to proceed with further characterization in wild type primary splenocytes, and found two near-identical compounds that had inhibitory activity. From these structures, we tested commercially available analogues and identified two molecules with inhibitory efficacy in the nanomolar range. Using these approaches, we hope to identify a small molecule with great efficacy and low toxicity for use as a molecular probe to further characterize AID’s intricacies, or even as a therapeutic agent.
IRF4 and IRF5 transcription factors exhibit shared and distinct roles in regulating human B cell differentiation and function
Authors: Shih Tiffany , De Saurav , Zhong Baohong , Barnes Betsy J.
Published: January 2023

Upon recognition of antigen, B cells undergo rapid proliferation followed by differentiation to specialized antibody secreting cells (ASCs). Increased levels of ASCs are seen in autoimmune diseases, such as systemic lupus erythematosus (SLE), and some B cell-associated malignancies. Studies suggest that altered transcription factor (TF) expression and/or activation play a role in the imbalance of B cell subsets in these diseases. Interferon regulatory factor 5 (IRF5) is one such TF, as polymorphisms in IRF5 associate with risk of numerous autoimmune diseases and correlate with elevated IRF5 expression in SLE patients. Recent findings from IRF5 knockdown (KD) in purified naïve B cells identified IRF5 as an early intrinsic regulator of B cell activation, proliferation and plasma cell differentiation in response to TLR9 activation. In this study, IRF4 was identified as a new IRF5 target gene by ChIP-Seq and RNA-Seq analysis. IRF4 expression is high in ASCs and murine studies show that it plays an essential, cell-intrinsic role in the generation of germinal center B cells. To date, little is known of IRF4 function in human ASC differentiation or whether these two factors (IRF4 and 5) cooperate. Activation of TLR9 and B cell receptor (BCR) revealed distinct kinetic changes in IRF4 and IRF5 during the early transition of naïve B cells to ASCs. Moreover, IRF4 KD of human primary naïve B cells resulted in significant IgD retention and reduced plasmablast (PB) differentiation without loss of early B cell activation (CD86) or key regulatory factors of class switch recombination in response to TLR9/BCR stimulation. Together, these results show that IRF4 and IRF5 are kinetically distinct TFs that have non-redundant roles in human ASC differentiation.
Immune regulation by glucocorticoids can be linked to cell-lineage-specific transcriptional responses
Authors: Franco Luis Miguel , Gadkari Manasi , Howe Katherine , Sun Jing , Kumar Parag , Kardava Lela , Biancotto Angélique , Hu Zonghui , Fraser Iain D.C. , Moir Susan , Germain Ronald N. , Tsang John S.
Published: January 2023

Glucocorticoids remain the most widely used class of immunosuppressive and anti-inflammatory drugs, yet significant gaps exist in our understanding of glucocorticoid-mediated immunoregulation. We have studied the transcriptional response to glucocorticoids, by total RNA sequencing and small-RNA sequencing, in nine human primary cell populations: B cells, CD4+ T cells, monocytes, neutrophils, endothelial cells, myoblasts, osteoblasts, fibroblasts, and preadipocytes. Our results suggest that the human response to glucocorticoids displays cell-lineage dependency, in terms of the individual genes and pathways that are differentially affected, as well as the magnitude and direction of transcriptional regulation. While decreased expression of genes that function in critical immune pathways is widespread, increased expression of negative regulators of immune activation is also common. Despite the critical role of B cells in many diseases for which glucocorticoids are used, glucocorticoid effects on human B cells are particularly poorly understood. We have identified significant transcriptional and functional changes in key molecules involved in B cell receptor signaling, intracellular Toll-like receptor signaling, B cell activation, differentiation, and antibody production. We have validated these results at the RNA and protein levels in vivo, by assaying B cells isolated from healthy human volunteers given a single intravenous dose of a glucocorticoid. Overall, our results illustrate the ability of an unbiased, genome-wide approach to uncover previously undescribed, cell-type-dependent transcriptional responses that can be linked to the immunoregulatory actions of glucocorticoids in humans.
Hepatic Myeloid Cell Diversity During Fibrosis and Nonalcoholic Steatohepatitis
Authors: Troutman Ty Dale , Seidman Jason S. , Link Verena M. , Hosseini Mojgan , Sakai Mashito , Bruni Cassi , Ouyang Zhengyu , Pasillas Martina , Glass Christopher K.
Published: January 2023

Fatty liver diseases account for rapidly growing morbidity in the United States where it is estimated that 80 to 100 million individuals have non-alcoholic fatty liver disease (NAFLD), and 6 to 16 million have the more severe variant nonalcoholic steatohepatitis (NASH). The mechanisms underlying development of NASH and progression to cirrhosis are physiologically complex. Using scRNA-Seq, we profiled CD45+ cells from the livers of mice subjected to a dietary NASH model. This approach revealed that the NASH model diet induced transcriptional diversification of Kupffer cells, and resulted in expansion of myeloid cell types. Kupffer cells, defined as F4/80High CD11bIntermediate, play a key role in the development of liver disease through their role as sentinel cells. They respond to noxious stimuli by producing chemokines and cytokines, which promote both recruitment of inflammatory leukocytes and pro-fibrogenic activation of hepatic stellate cells. Through flow cytometry, we confirmed scRNA-Seq suggestions of hepatic macrophage diversification beyond the previously reported F4/80Low CD11bHigh Ly6CHigh and F4/80Low CD11bHigh Ly6CLow recruited macrophages. Further profiling of these hepatic macrophages by RNA-Seq suggested the differential involvement of these cell subsets in the disease process. These results were corroborated using an alternative mouse model of liver fibrosis. Ongoing experiments are targeted towards assessment of the developmental origin of the distinct Kupffer cell subsets, with preliminary data suggesting a monocytic and not embryonic origin. Further efforts are focused on comparisons of myeloid populations in humans and mouse towards informing the design of future experiments.
Indispensable role of protein kinase D1 in inflammatory responses and host defense in Group B streptococci infection
Authors: Yoon Taewon , Halder Priyanka , Yi Ae-Kyung
Published: January 2023

Group B streptococcus (GBS) causes life-threatening diseases in neonates and immune compromised individuals. We previously found that GBS induce activation of protein kinase D1 (PKD1) in mouse and human. This PKD1 activation by GBS is dependent on the TLR signaling adaptor MyD88 and its downstream IRAK4 and IRAK1. GBS-mediated proinflammatory cytokine production is substantially suppressed by pretreatment with a PKC/PKD inhibitor Gö6976. However, it is currently unknown whether PKD1 plays a regulatory role in GBS-mediated proinflammatory responses and host defense. In the present study, to further understand contribution of PKD1 in inflammatory responses and host defense in GBS infection, tamoxifen-inducible PKD1-deficient mice were generated. We found that GBS-mediated activation of MAPKs and NF-kB and expression of cytokines/chemokines are significantly inhibited in PKD1-deficient mice. Furthermore, increased susceptibility to GBS infection was observed in PKD1-deficient mice. However, PKD1-deficient mice sensitized with D-galactosamine were more resistant to the shock-mediated death caused by antibiotic-killed GBS compared to wild-type. Our findings imply that PKD1 is one of the critical factors that plays a regulatory role in GBS-induced proinflammatory reactions and is indispensable for host defense against GBS infection and that inhibition of PKD1 activation together with antibiotic treatment in GBS-infected neonates might be an effective way to control GBS diseases.
A potential for crosstalk between inflammation, coagulation and oxidative stress host responses in malaria induced mid-gestational pregnancy loss in a rodent model
Authors: Andrew Alicer Keendalyn , Moore Julie M. , Sarr Demba , Shastri Sachi , Bracken Tara
Published: January 2023

Despite an overall reduction in the global malaria burden, placental malaria (PM) is a major complication of Plasmodium falciparum malaria that impacts hundreds of thousands of pregnancies annually. Primigravid women living in Sub-Saharan Africa are especially prone to PM and often experience poor maternal-fetal health outcomes though pregnancy loss, preterm delivery and infant low birth weight. Mouse models for malaria infection during pregnancy have been instrumental in our current understanding of PM, however our understanding remains incomplete. In the laboratory of Dr. Julie Moore, we have developed a mouse model of infection during pregnancy using the murine-infective species P. chaubaudi chaubaudi AS (PccAS). Infection initiated at gestation day 0 (GD 0) in mice deficient in tumor necrosis factor (TNF) function or signaling and intact C57BL/6J control mice treated with antioxidant and anticoagulant drugs reveals distinct differences in pregnancy outcome at mid-gestation (GD 10). While control mice lose their pregnancies, TNF deficient and drug-treated mice experience rescue of mid-gestational pregnancy and display improved offspring viability at sacrifice. These preliminary findings suggest that by disrupting the function of key host mediators of severe malaria, it may be possible to mitigate negative pregnancy outcomes associated with malaria. Through strategic use of TNF modified mice, combined with studies of gene expression and tissue localization, this work aims to investigate crosstalk between three major host responses to infection - inflammation, coagulation and oxidative stress – and characterize how their interplay may contribute to PM pathogenesis and associated negative pregnancy outcomes.
The host ubiquitin system in innate immunity and virus replication: proviral and antiviral functions of the host E3-ubiquitin ligase TRIM family
Authors: Rajsbaum Ricardo , Giraldo Maria Isabel , Bharaj Preeti , Atkins Colm , Xia Hongjie , Rossi Shannan L. , Lee Benhur , Shi Pei-Yong , Freiberg Alexander N.
Published: January 2023

Type-I Interferons (IFN-I), which are innate immune cytokines with broad-spectrum antiviral effects, are regulated by the host Ubiquitin (Ub) system. Evidence has shown that new forms of poly-Ub chains, which are not covalently attached to any protein (termed unanchored poly-Ub), are important activators of kinase activity in vitro. We recently reported that unanchored K48-linked poly-Ub synthesized by the E3-Ub ligase TRIM6 promote IFN-mediated antiviral innate immunity in vivo by activating the IKKɛ kinase. Intriguingly, we found that two different highly pathogenic viruses, Nipah (NiV; Paramyxoviridae family) and Ebola (EBOV; Filoviridae family), hijack TRIM6 to promote virus replication through mechanisms that involve both covalent and unanchored Ub. This indicates that host antiviral factors can have direct pro-viral functions. We also found that Zika virus (ZIKV; Flaviviridae family), which can cause congenital microcephaly in newborns, requires ubiquitination of viral proteins for efficient replication. Importantly, ZIKV ubiquitination occurs in vivo in a tissue-specific manner. Recombinant infectious ZIKV mutants that lack ubiquitination on specific sites are attenuated in mice and appear to differentially replicate in tissues. As compared to other flaviviruses, ZIKV acquired an additional ubiquitination site, which may be responsible for providing ZIKV an advantage to replicate in brain and reproductive tissue. This would be the first example of a flavivirus utilizing the Ub system for tissue tropism. Taken together, our work suggests that divergent viruses hijack similar innate immune host factors to enhance their replication. This information could help design novel broad-spectrum antiviral strategies
The role of Indoleamine 2,3-dioxygenase (IDO) in the survival of bone marrow resident long lived Plasma cells
Authors: Lightman Shivana M. , Carlson Louise M. , Lee Kelvin P.
Published: January 2023

Long lived plasma cells (LLPCs) are essential for sustained antibody responses and protective humoral immunity. How these cells maintain longevity and a durable antibody response is largely dependent on the complex nature of the bone marrow microenvironment in which these cells reside, and the pro survival factors produced in this niche. Previous work in our lab has shown that CD28 is required for LLPC survival, and we know that CD28 back signaling to CD80/86 induces IDO production from dendritic cells (DCs) within the bone marrow microenvironment. IDO is classically known to contribute to an immunosuppressive environment (specifically with respect to T cell activity). However, upon investigation of IDO in the normal setting, we observed fewer plasma cells present in the bone marrow of IDO knockout mice in comparison to wild type mice, as well as fewer antigen-specific plasma cells in the bone marrow following vaccination. Additionally, knowing the role of DCs in IDO production in the bone marrow, we employed the use of CD11c-DTR mice to observe the effects of PC survival upon DC elimination. We found that depletion of DCs significantly decreases the amount of antigen-specific plasma cells in the bone marrow. This leads us to propose a model where CD28, through back signaling to CD80/86 induces IDO production in DCs, a mechanism to maintain the bone marrow niche for long lived plasma cell survival and sustained antibody production.
Varying pMHC-TCR affinity at the memory checkpoint alters the number and phenotype of memory CD4 T cells.
Authors: Jones Michael C. , Devarajan Priyadharshini , Nanaware Padma Pandurang , Stern Lawrence J. , Huseby Eric S. , Swain Susan L.
Published: January 2023

We have defined a memory checkpoint, 5–7 days post infection (dpi) with influenza A virus (IAV), during which responding CD4 effector cells need to re-encounter their cognate antigen (Ag) to effectively transition to memory. Ag encounter during this checkpoint promotes CD4 effector and memory survival, cytokine production potential, and leads to enhanced protection against lethal IAV challenge. Activated APC, pulsed with peptide, are sufficient to drive effectors to memory in vivo. Therefore, we can determine what influence Ag dose and affinity, at the effector stage, have on memory generation. We have developed an IAV-specific TCR transgenic mouse with CD4 T cells specific for an immunodominant, highly conserved, influenza nucleoprotein determinant (NP311–325). Using several altered peptide ligands with varying affinities that activate the transgenic CD4 T cells to varying degrees, we can analyze how varying pMHC-TCR affinity at the memory checkpoint influences the generation of the CD4 memory population. Our preliminary data indicate that when CD4 T cells encounter high, as compared to the medium and low affinity peptides during the memory checkpoint, they more efficiently develop into memory cells. If high affinity and/or dose of peptide is needed, it would imply continuing infection at the checkpoint is optimum for generating CD4 memory and should inform the kind of vaccine strategies that can successfully mimic live infection.
Defining the role of CD69 in the formation of resident memory CD8+ T cells
Authors: Walsh Daniel , da Silva Henrique Borges , Beura Lalit , Breed Elise , Ruscher Roland , Peng Changwei , Hamilton Sara E. , Masopust David , Jameson Stephen
Published: January 2023

Resident memory CD8+ T cells (TRM) reside in nonlymphoid tissues. There, they play a key role in preventing reinfection by exerting cytotoxic and inflammatory functions upon exposure to previously encountered pathogens. CD69 is often used as a definitive marker of TRM cells. CD69’s interaction with the G-protein-coupled receptor S1PR1 has been identified as one mechanism by which CD69 can regulate tissue residency. However, the functional requirement for CD69 in promoting the generation and maintenance of CD8+ TRM under a wide variety of circumstances remains unclear. We explored the role of CD69 in tissue residency using co-transfer of antigen specific CD69 sufficient and deficient CD8+ T cells in the context of acute LCMV, Influenza, and VSV infections. Strikingly, we found that CD69 was not necessary for TRM establishment in most tissues, although it can promote TRM localization under some circumstances. This seems to be influenced by the focal point of infection. Interestingly, the kidney appears to rely on CD69 for tissue residency with every model pathogen examined. We propose that the requirement for CD69 is context dependent rather than absolute, and that a combination of factors, including tissue microenvironment and infectious agent, dictate CD69’s influence on development of CD8+ resident memory.
Antigen recognition by CD8+ T cells in non-lymphoid tissues initiates a transcriptional program of tissue-resident memory differentiation
Authors: Hobbs Samuel J. , Nolz Jeffrey C.
Published: January 2023

Tissue-resident memory (TRM) CD8+ T cells permanently reside in non-lymphoid tissues where they are important mediators of both host defense and inflammatory disease. TRM cells are derived from effector cells after they have entered non-lymphoid tissues, but the mechanisms that control TRM differentiation are largely undefined. We have recently used a model of localized epicutaneous infection with Vaccinia viruses (VacV) expressing unique model antigens to demonstrate that antigen recognition in the skin microenvironment enhances TRM formation by ~50–100 fold. To understand how T cell receptor (TCR) engagement may promote TRM differentiation, we sought to identify CD8+ T cells that were actively recognizing cognate peptide-MHC in vivo. Using an endogenous interferon-gamma (IFNγ)-YFP reporter, we found that only ~20% of the antigen-specific CD8+ T cells in the skin microenvironment produce IFNγ during a VacV skin infection. However, genome-wide transcriptional profiling revealed that the T cells producing IFNγ in the skin were also undergoing changes in gene expression that resemble mature TRM cells. In fact, IFNγ+ CD8+ T cells also expressed the transcription factor Blimp1, which has been shown to be critical for the acquisition of the core TRM transcriptional profile. TCR stimulation induced Blimp1 expression in effector, but not naïve, CD8+ T cells, suggesting that a secondary antigen encounter promotes unique transcriptional programming when recently activated CD8+ T cells exhibit their effector functions. Thus, antigen recognition by effector CD8+ T cells in non-lymphoid tissues is a critical factor impacting gene expression, which ultimately promotes the differentiation of TRM cells during viral skin infection.
Interleukin-1 and IL-23 induce innate-like immune responses by bystander-activated memory CD4+ T cells contributing to the autoimmune pathogenesis
Authors: Lee Hong-Gyun , Lee Jae-Ung , Kang Insoo , Choi Je-Min
Published: January 2023

Antigen-nonspecific bystander activation of CD4+ T lymphocytes have been recently discovered. However, the immunopathological role of non-specific bystander activated CD4+ T cells responding to pro-inflammatory cytokines remains unknown. Here, we show that IL-1β acted directly on polyclonal memory, but not naïve, CD4+ T cells in synergy with IL-23 to promote IL-17A and IFN-γ expression in both mice and human. This bystander CD4+ T cell activation is dependent on IL-1 but not T cell antigen receptors (TCRs). In vivo, co-transfer of MOG-specific TCR transgenic (2D2) naïve T cells with MOG-nonspecific TCR transgenic (OT-II) memory-like Th17 cells increased susceptibility to EAE. IL-1 dependent MOG-nonspecific memory-like Th17 cells infiltrated to the CNS producing IL-17A, IFN-γ, and GM-CSF, which made important contributions to the development of autoimmune neuroinflammation. Our findings demonstrate that IL-1β and IL-23 activates TCR-independent innate-like immune responses by bystander activated memory CD4+ T cells contributing to the autoimmune disease pathogenesis.
TLR2-dependent bystander activation of T cells results in exacerbation of Lyme arthritis
Authors: Whiteside Sarah , Snook Jeremy P. , Ma Ying , Sonderegger F. Lynn , Fisher Colleen , Petersen Charisse , Zachary James F. , Round June L. , Williams Matthew A. , Weis Janis J.
Published: January 2023

Infection with the bacteria Borrelia burgdorferi results in a multidimensional disease including, fever, headache, arthritis, and a characteristic skin rash. When left untreated, bacteria can disseminate to multiple parts of the body including joints, heart, and nervous system. Approximately 10–20% of patients treated with antibiotic therapy for Lyme disease will continue to have persistent symptoms for months or years. T cells predominate the immune response in the synovial fluid of these patients; however, their role in Lyme disease remains poorly defined. Using a murine model of persistent Lyme arthritis, we observed that bystander activation of CD4+ and CD8+ T cells leads to the secretion of arthritis-promoting IFN-γ, similar to the inflammatory environment seen in the synovial tissue of patients with post-treatment Lyme disease (PTLD). T cell receptor (TCR) transgenic mice containing monoclonal specificity towards non-Borrelia epitopes confirmed that bystander T cell activation was responsible for disease development. RNA-sequencing of bystander activated T cells provided insight into the molecular characteristics of non-classical T cell activation. The microbial pattern recognition receptor Toll-like receptor 2 (TLR2) was upregulated on T cells following infection, implicating it as marker of bystander T cell activation. In fact, T cell intrinsic expression of TLR2 contributed to IFN-γ production and arthritis, providing a mechanism for microbial-induced bystander T cell activation during infection. These results reveal a novel TLR2-intrinsic role for T cells in Lyme arthritis, with potentially broader application to bystander T cell activation and immune pathogenesis.
Mda5/MAVS signaling is essential for resistance against Aspergillus fumigatus
Authors: Obar Joshua J. , Caffrey Alayna Katherine , Wang Xi , Espinosa Vanessa , Tang Yi-Wei , Hohl Tobias , Rivera Amariliz , Cramer Robert
Published: January 2023

Type I and III interferons act as important activators of antifungal neutrophil response in the lungs. The RIG-I like receptor (RLR) family, including RIG-I and Mda5, are cytosolic RNA sensors that signal through the MAVS adaptor in order to activate interferon responses against viruses. Whether the RLR family has broader effects on host immunity against other pathogen families remains to be fully explored. Herein we demonstrate that Mda5/MAVS signaling was essential for host resistance against pulmonary Aspergillus fumigatus challenge through the regulation of antifungal leukocyte responses. Aspergillus fumigatus induction of type I interferons was partially dependent on Mda5/MAVS signaling, while type III interferon expression was entirely dependent on Mda5/MAVS signaling. Activation of the interferon pathway was driven by prolonged exposure to fungal spores, rather than germ tubes. Ultimately, interferon signaling drove the expression of CXCL9 and CXCL10, SNPs in the latter are associated with invasive aspergillosis in human patients. Our data suggest a broader role of the RLR family in the regulation of innate immunity to include invasive fungal infections.
The influence of gammaherpesvirus co-infection on malaria immunopathogenesis and parasite control
Authors: Netongo Palmer Masumbe , Mimche Patrice Nsangou , Matar Caline G. , Speck Samuel H. , Lamb Tracey J.
Published: January 2023

Malaria still kills up to 500,000 people annually. Risk factors contributing to severe disease remain poorly understood. There is compelling evidence that acute gammaherpesvirus such as Epstein-Barr Virus (EBV) coinfections are responsible for suppressing the development of humoral immunity during Plasmodium infection in children and may constitute a salient risk factor for disease severity. However, the actual mechanisms by which protective humoral immune responses to Plasmodium infection are suppressed by gammaherpesviruses are incompletely understood. It has previously been shown that acute MHV68 co-infection resulted in the transformation of a non-lethal Plasmodium yoelii XNL infection into a lethal one in C57BL/6 mice, an outcome linked to a defect in the maintenance of germinal center B cells and T follicular helper cells (Tfh) cells in the spleen and a defect in antibody production. Since the suppressive effect requires the M2 latency associated protein of MHV68, and M2 induces IL-10 secretion from the B cells it infects, we tested the hypothesis that the induction of IL-10 by M2 in MHV68-infected B cells impairs the ability of T follicular helper cells (Tfh) to provide B cell help for effective, broad spectrum antibody production to incoming Plasmodium infection. We tested our hypothesis by co-infecting mice that cannot produce IL-10 from B cells (IL-10Flox_CD19 Cre+), and measured the effect that this defect has on Tfh maintenance, plasma cell formation and antibody production in response to Plasmodium infection. Our findings suggest B cell derived IL-10 is an important component of MHV68-mediated disruption in germinal center formation during gammaherpesvirus/ Plasmodium co-infections.
Coinfection: influenza A virus and Aspergillus fumigatus
Authors: Rippee-Brooks Meagan D. , Lupfer Christopher R.
Published: January 2023

Bacterial coinfections with influenza A virus (IVA) are extremely serious and life-threatening. However, there exists limited data gathered about the importance of fungal infections with IAV. Clinical case reports indicate that fungal coinfections do happen and suggest the pandemic 2009 IAV has a propensity to predispose patients to secondary fungal infections more than previous IAV strains. However, the factors involved remain to be determined. We have developed an in vitro model using primary mouse bone marrow derived macrophages infected with IVA and coinfected with the opportunistic fungal pathogen Aspergillus fumigatus. Our results indicate that IVA and fungal coinfections synergistically enhance cell signaling and cytokine production. We propose this exacerbated immune response during IVA and A. fumigatus predisposes clinical patients to more severe pneumonia, and we seek to identify the pathways responsible for the heightened cytokine responses and their significance in vivo.
Investigating local and systemic immune changes following sub-chronic inhalation of Aspergillus versicolor spores
Authors: Barnes Mark A. , Croston Tara L. , Lemons Angela R. , Beezhold Donald H. , Green Brett J.
Published: January 2023

Fungi are environmentally ubiquitous, and can be particularly abundant in damp indoor locations which may lead to pulmonary fungal exposure via inhalation of bioaerosols. Some of the adverse pulmonary health effects associated with fungal challenges include asthma, allergy, hypersensitivity pneumonitis, and local or disseminated infection. Here, we study the innate immune effects following 13 weeks sub-chronic inhalation exposures to spores derived from a common indoor fungal contaminant, Aspergillus versicolor. Compared to air-only control mice, A. versicolor-exposed B6C3F1 mice exhibited increased recruitment of leukocytes to the lung, particularly eosinophils and type 2 innate lymphoid cells (ILC2s), but also alveolar macrophages, inflammatory monocytes, and neutrophils following 13 weeks of exposure in nose-only inhalation chambers. Local expression of ccl2, ccl7, eotaxin, and il13 mRNA was increased in lung tissue. In addition, IL-4, CCL3, CCL4, CXCL1, and CXCL5 were increased in bronchoaveolar lavage fluid of A. versicolor-exposed mice compared to controls. Systemically, circulating CCL7, CXCL1, CXCL5, and CXCL10 were increased in serum of mice following inhalation of spores. Taken together, these data indicate A. versicolor elicits a Th2-biased immune response that is driven by multiple innate immune cell populations, including monocytes, eosinophils, and ILC2s.
The role of transcription factor T-bet in B cell mediated responses to Plasmodium infection
Authors: Cimperman Clare K. , Sheehan Patrick W. , Theall Brandon P. , Pena Mirna , Pierce Susan K. , Akkaya Munir
Published: January 2023

Malaria is a global health concern which affects over 200 million individuals worldwide. Although the immune system rapidly responds to Plasmodium infection with specific antibodies, natural antibodies fail to establish long term protection, often leading to repetitive infections and chronicity. Recent studies showed that in a chronic malaria setting both B cells and T cells induce expression of T-bet transcription factor. It is not completely understood whether this is a part of the specific host defense or a result of disease pathogenesis. Here using transgenic mouse models, we show that knockout of T-bet or IFN-g similarly reduces survival of mice compared to WT upon infection with Plasmodium chabaudi, therefore suggesting a protective role for T-bet. To determine the specific role T-bet plays in B cell responses to Plasmodium, we first identified that T-bet expression in mouse B cells are triggered by dual stimulation of IFN-g and B cell receptor. Similarly using Tbet-zsgreen transgenic mice we showed that conditions specific to Plasmodium chabaudi infection drive T-bet expression in multiple B cell subsets in a time dependent fashion. However, no such induction is shown when mice are immunized with T-dependent protein antigens adjuvanted with Alum. On the other hand, bone marrow chimeric animals reconstituted with equal amounts of congenically labeled WT and T-bet KO bone marrow cells showed that for most B cell subsets the lack of T-bet does not change the ratio of WT and KO cells upon Plasmodium infection indicating that T-bet expression may not be equally critical for all B cell responses to infection. These findings suggest a complex role of T-bet in the development of humoral responses.
Acute high fat diet disrupts intestinal barrier repair
Authors: McAlester Andrea Hill , Kim Myunghoo , Song Rachel (Hyo) , Wu Wan-jun , Diehl Gretchen
Published: January 2023

Over-consumption of high fat diets (HFD) corresponds to increased incidence and severity of many chronic inflammatory diseases, including those of the intestinal tract. The impact of HFD feeding on bodily tissues has largely been studied in the context of obesity. Long-term exposure to HFD has been shown to decrease barrier function, allowing for bacterial penetration into the body resulting in chronic inflammation. However, this effect is secondary to adipose tissue dysfunction making it difficult to determine the direct impact of HFD on intestinal barrier function.

The intestine acts as a protective barrier and our immune system supports the proper functioning of this barrier, aiding in protection from pathogens. Sustained damage or loss of proper barrier function can result in chronic inflammation and diseases such as inflammatory bowel disease (IBD). We hypothesized that intestinal exposure to HFD would directly alter the intestinal barrier resulting in enhanced inflammation.

To test this, we utilized short-term HFD feeding in the context of mouse models of colitis. Our data demonstrates that HFD increases susceptibility to colitis with increased weightloss, aberrant epithelial cell proliferation, loss of goblet cells and the mucus layer necessary to prevent microbial penetration into the body. We also identified dysregulated immune responses resulting in an inability of intestinal immune cells to properly support barrier function and repair. Together, we find that HFD feeding directly promotes altered functions of intestinal epithelial and immune cells leading to a barrier repair defect resulting in an inability to repair epithelial damage and increasing pathology in models of IBD.
Interactions between microbiota and the teleost immune system in health and disease
Authors: Salinas Irene , Casadei Elisa , Takizawa Fumio , Shibasaki Yasuhiro , Sunyer Oriol J.
Published: January 2023

The symbiosis between metazoans and prokaryotes is one of the most ancient and successful relationships found in nature. Mucosal surfaces are the main interface between animal hosts and their environment, and these surfaces are colonized by unique microbial communities that shape every physiological system of the host. Teleost fish have both an innate and adaptive immune system and are the most ancient vertebrates with a dedicated mucosal immunoglobulin (Ig), known as IgT/IgZ. IgT is secreted via the polymeric Ig receptor across teleost mucosal epithelia, including the gills, skin, nose and gut. Recent studies have shed light on the interactions between mucosal Igs and microbiota in teleosts both during health and disease. Teleost fish symbiotic bacteria are coated by both secretory Igs (sIgT and sIgM) as well as pIgR. Using two different models, a stress model and an IgT depletion model, we provide evidence that any alterations in the levels of mucosal Igs result in different states of dysbiosis in rainbow trout (Oncorhynchus mykiss). Dysbiosis in trout is characterized by losses of certain taxa and subsequent invasion of new species in several mucosal sites. Unlike in mammalian models of IgA deficiency, where IgM compensatory responses occur, no compensatory IgM or IgD responses were observed in IgT-depleted rainbow trout. These results demonstrate that teleost fish, similar to mammals, utilize specialized mucosal Igs to maintain harmonic relationships with symbiotic bacteria.
Commensal gut fungi regulate susceptibility to colitis and colorectal cancer.
Authors: Malik Ankit , Sharma Deepika , Vogel Peter , Kanneganti Thirumala-Devi
Published: January 2023

The cross-talk between innate immune sensors and commensal microbes in the gut is crucial for maintaining intestinal barrier homeostasis. Fungi represent a major component of the resident gut flora, and fungal diversity narrows during colitis and colorectal cancer pathogenesis. Mutations in the genes that encode molecules involved in the fungal sensing pathways are associated with an increased risk of developing colitis and colorectal cancer. However, the precise roles of gut fungi and associated immune recognition cascade in colitis and colorectal tumorigenesis are not well established. We show here the role of commensal fungal sensing by CLR signaling pathway in the regulation of inflammation and tumorigenesis in the colon.