bluuebunny/crossref_metadata_2025 huggingface dataset:
Disease tolerance and immune response in bovine trypanosomiasis is mediated by CD14 promoter gene
Authors: Morenikeji Olanrewaju , Thomas Bolaji
Published: January 2023

Bovine trypanosomiasis is a major drawback for cattle production in sub-Saharan Africa. Immune response to infection is thought to be mediated by genetic diversity in pattern recognition receptors, leading to disease tolerance or susceptibility. We elucidated naturally occurring variations within the bovine CD14 gene promoter in trypanotolerant (N’dama) and trypanosusceptible (White Fulani) cattle, using genomic and transcriptomic methods. We compared our gene sequences with reference bovine genome UMD 3.1 assembly from the Ensembl database. In all, we identified a total of 54 and 41 SNPs at the CD14 promoter for Ndama and White Fulani respectively, including 10 (rs210109064, rs473185091, rs521360374, rs454858160, rs432371570, rs446947915, rs442402639 and rs456854916) common to both. Interestingly, we found 36 SNP’s in 9 SNP regions that were unique to Ndama that were either functional or transcript overlap variants. The significantly higher SNP variation and density at the CD14 promoter region in Ndama could be responsible for its trypanotolerance, potentially an evolutionary adaptation to disease. To further validate this, we examined the expression of CD14 in tissue samples, with our results showing a significant upregulation and higher expression in Ndama compared to White Fulani (p<0.05). These results confirm that trypanotolerant Ndama significantly expressed CD14 and the presence of a higher SNP variation at the CD14 gene promoter plays an important role in pathogen recognition and initiation of immune response, which is absent in White Fulani (trypanosusceptible). This is a major player in host defense mechanism against trypanosomiasis that could be harnessed for breeding and selection purposes.
Transcriptome analysis of distinct coelomocyte populations in Strongylocentrotus purpuratus
Authors: Fugmann Sebastian D , Cheng Jun Fang , Huang Hsiao Han
Published: January 2023

Coelomocytes are important cellular mediators of immunity in echinoderms. In the purple sea urchin (Strongylocentrotus purpuratus) these cells can readily be divided into phagocytes, vibratile cells, red spherule cells, and colorless spherule cells based on their morphology. While some information has been reported about genes that are expressed in specific types of coelomocytes, a comprehensive analysis of the gene expression pattern in each cell type was not available. Using a density gradient coelomocytes from individual adult urchins were separated into three populations (phagocytes, a mixture of vibratile and colorless spherule cells, and red spherule cells), RNA was isolated, and next generation sequencing was employed to determine the transcriptome of each cell population. All three cell populations showed clear differences in their gene expression patterns, and the differences in transcript levels for selected genes that were found to be uniquely expressed in one cell type were confirmed by qRT-PCR using cDNA obtained from additional sea urchins. This bulk population data was then complemented by single-cell RNAseq to obtain information about suitable population and sub-population markers for subsequent studies of coelomocyte biology.
Annotation of the WC1 gene family in Sus scrofa and evaluation of individual SRCR domain affinity for Mycobacterium bovis and Leptospira spp
Authors: Le Page Lauren Ann , Buck Jessica , Boisvert Natalie , Gillespie Alexandria , Yirsaw Alehegne , Hudgens Edward , Hsu Haoting , Baldwin Cynthia L , Telfer Janice C
Published: January 2023

γδ T cells respond to a variety of proteins and non-proteinaceous molecules independent of MHC presentation, making them less susceptible to immune evasion and thus; an attractive target for next-generation vaccines. Unfortunately, definitive methods to prime these cells remain elusive. WC1, a member of the group B Scavenger Receptor Cysteine-Rich (SRCR) superfamily, is expressed exclusively on the γδ T cells of swine and ruminants and functions as hybrid co-receptor and pattern recognition receptor for the γδ TCR. It is known that there are thirteen unique WC1 genes expressed in cattle, each containing up to eleven extracellular SRCR domains. Bovine WC1+ γδ T cells share restriction in their TCR gene usage, yet respond to different pathogens based on which WC1 molecule(s) they express. This can be attributed to the ability of the expressed WC1 molecule to directly bind whole pathogens via its SRCR domains. Because WC1 is expressed as a multigene array, we hypothesize that each WC1 gene has co-evolved with a different set of pathogens, and may be exploited to generate a protective immune response from WC1+ γδ T-cells. We sought to identify and characterize WC1 genes expressed in swine, and evaluate their pathogen binding potential. We have obtained cDNA evidence for ten swine WC1 genes, containing up to six extracellular SRCR domains. Through annotation with MAKER, we have confirmed the presence of seven of these genes in the current assembly, Sus scrofa 11.1, and identified four unique exon-intron gene structures. We have shown that SRCR domains from multiple WC1 genes differentially bind Leptospira spp, and Mycobacterium bovis BCG Danish and Pasteur strains.
Isolation and characterization of acute phase protein C-reactive protein in shark serum: a potential biomarker for assessing health in wild sharks
Authors: Orndorff Samantha Mae , Merly Liza
Published: January 2023

The acute phase protein, C-reactive protein (CRP), has become an established clinical biomarker in both animal and human health, where it is used to assess the degree of acute and/or chronic inflammation. CRP has also been proposed as a potential biomarker for measuring health in sharks by indicating when a shark is experiencing an inflammatory state or is under a chronic state of stress that may have significant physiological impact. Despite the promise in using increased levels of CRP as well as other acute phase proteins as indicators of compromised health, there has not to date been a developed shark-specific assay to accurately measure CRP. An initial step in the development of a specific bioassay for CRP is isolation and characterization of the protein. The purpose of the present study is to use both field and laboratory techniques to obtain serum samples and isolate CRP from nurse sharks, Ginglymostoma cirratum. A secondary aim is to characterize the molecule and produce shark CRP specific antisera. A two step isolation procedure is now being refined to positively identify the pentraxins present and characterize them further. Protein affinity chromatography was used to identify serum amyloid P component (SAP) in addition to CRP given that they are both pentraxin proteins displaying similar binding properties. Isolated protein bands resulting from concentrated chromatographic peaks were analyzed by LC-MS/MS. Results thus far include the isolation of a pentraxin domain-containing protein within shark serum.
Inflammatory changes and melanization in the Atlantic salmon (Salmo salar) targeted by In situ hybridization
Authors: Bjoergen Haavard , Hordvik Ivar , Koppang Erling Olaf
Published: January 2023

Research on the immune system of salmonid fish is attracting increased attention, however, progress within this field has been hampered by the general lack of cell markers targeting different immune molecules. Here, we circumvent these challenges by applying in situ hybridization (ISH) to reveal basic cellular responses in salmon immune reactions.

Using RNAscope® (ACD, Newark, CA), we have established ISH targeting immunoglobulin (Ig) M and T RNA. Further, we also target other immune genes where we previously had no markers, and tyrosinase, thought to be important for the function of the melano-macrophages which are special, melanin-producing immune cells found in ectothermic vertebrates. We provided target gene sequences to the manufacturer, which supplied us with probes, and we performed ISH according to the manufacturer’s instructions. Different inflammatory conditions in farmed salmon, including cancerous changes from the gut and muscle tissue with chronic myositis and melanization due to the presence of melano-macrophages, were screened by standard histology and subsequently subjected to ISH.

For the IgM results, we compared IHC and ISH results, as we have antisera targeting IgM. The results were highly similar. ISH thus seems as a good method that may be used in salmon when applicable and especially to target transcripts where no antibody recognizing the resulting protein is available. Further, we reveal hitherto non-described cellular compositions of inflammatory changes in salmon using probes detecting several different immune cell transcripts. Our results show that ISH using RNAscope® works in Atlantic salmon and is highly reproducible.
Evaluation of epitope specificity in the immune response to PR8 H1N1 influenza virus infection in neonatal and adult African Green monkeys
Authors: Clemens Elene A , Angeletti Davide , Holbrook Beth C , Aycock Scott Tyler , Yewdell Jonathan W. , Alexander-Miller Martha A
Published: January 2023

Generation of effective antibody responses following virus infection is challenging in neonates as a result of impairments in the innate and adaptive immune systems that characterize early life. This leaves them particularly susceptible to severe disease following virus infection, e.g. influenza A virus. A potential contributor to the efficacy of the immune response generated following infection is the epitope specificity of the elicited antibodies. Previous studies in adult mice have reported a defined and reproducible pattern of immunodominance among antibodies directed to the neutralizing epitopes on the head of the influenza hemagglutinin (HA) molecule. The impact of age on the immunodominance pattern of HA-specific antibodies has not been explored. To address this, epitope recognition was quantified in plasma collected from newborn and adult African Green monkeys on d14 following PR8 influenza virus infection. For these analyses we used HA molecules engineered to singly express each of the neutralizing epitopes identified on the HA head. In addition, we used a construct that restricts recognition to the stem region of PR8 HA. Our analyses revealed a similar pattern of recognition by HA-specific IgG between infants and adults in the 14 day period following infection. In contrast, the HA-specific IgM pools exhibited distinct binding patterns in these two groups. The most striking of these differences was in the production of antibodies capable of recognizing the stem portion of HA. These data suggest antibody immunodominance patterns may be modulated with age and sheds new light on the regulation of potentially broadly protective stem responses to influenza virus.
The immune responses to influenza vaccine in aged and metabolic disease occurred cynomolgus macaques
Authors: Urano Emiko , Murakata Yoshiko , Okamura Tomotaka , Yasutomi Yasuhiro
Published: January 2023

The analysis of age-related immune senescent is extremely meaningful to understand the vital homeostasis in individuals among the aging society. Influenza virus (FluV) infection is a major public health concern due to its high infectivity and ability to severe respiratory illness in elderly people. The reduced immune responses after Flu vaccination in humans are recognized in aging and also in lifestyle diseases such as diabetes mellitus (DM). Understanding the hypofunction in aging and lifestyle diseases is necessary to optimize vaccination resume and the comprehensive analysis of that will provide a great impact on public health burden. However, several factors affect the biofunctional analysis in human.

Cynomolgus macaques (CMs) show human-like characteristics such as higher brain functions, long life span, single pregnancy. In the present study, several groups of CMs, including DM and hyperlipidemia (HL) occurred CMs, were subcutaneously immunized with seasonal trivalent Flu vaccine and assessed immune responses. The reduced humoral and cellular immune responses to Flu vaccine were observed in the elderly CMs group compare to the young group and induced anti-HA antibody titers were negatively correlated with age. In particular, Flu-specific immune responses were considerably decreased in DM group despite their youth. However, the reduced humoral and cellular immune responses were not observed in HL group, unlike DM CMs. These results suggested that elderly or DM and HL occurred CMs should be a powerful tool to reveal the comprehensive mechanisms for hypofunction in aging and lifestyle diseases through a long-life span. This study will help to enhance the immune response to vaccine in metabolic disease and aged person.
Mechanism of Influenza Virus Induced Inflammatory Cell Death
Authors: Sannula Kesavardana , Kanneganti Thirumala-Devi
Published: January 2023

RNA viruses and host cell death responses are co-evolved which prompt the fate of immune responses. Inflammatory cell death is a form of programmed signaling cascade which lead to the loss of cell integrity and secretion of leaderless pro-inflammatory cytokines. Host innate immune system exerts inflammatory cell death to curtail virus replication and initiate protective inflammatory and adaptive responses. This also causes host tissue damage if uncontrolled. Elevated inflammatory responses and lung damage are primary causes for morbidity and mortality during pathogenic influenza infections. However, little is known about key mechanisms regulating influenza A virus (IAV) induced cell death and inflammation.

We identified Z-DNA Binding Protein 1 (ZBP1) as a sensor of IAV infection to trigger inflammatory cell death in in vitro and in vivo. ZBP1 induced inflammatory cell death is specific to nuclear replicating IAV but not the other RNA viruses those replicate in the cytosol. We found that the ZBP1 translocate to the nucleus prior to the cell death execution. Unlike other innate receptors, ZBP1 recognizes unique patterns of RNA in ribonucleoprotein complexes (vRNPs) of IAV. This nucleate RHIM domain driven oligomeric RIPK3 signaling scaffolds to facilitate RIPK3-Caspase-8 dependent inflammatory cell death (both necroptosis and pyroptosis). Our biochemical studies identified RIG-I dependent activation, ubiquitination of ZBP1 and loss of mitochondrial integrity precedes inflammatory cell death which facilitate the assembly of cell death signaling complexes proximal to the cellular membrane. Overall, our work demonstrates the mechanism of sensing of nuclear replicating IAV vRNPs to trigger programmed inflammatory cell death.
Fibroblasts from individuals with Trisomy 21 have increased Type-I Interferon responses to HSV-1 compared to typical adults
Authors: Royzman Kristina , Drews Caitlyn , Maxwell Tyler , Singh Gurkirat , Keefe Caitlin , James Stephanie
Published: January 2023

Down syndrome (Trisomy 21; T21) results from an extra copy of chromosome 21. This results in a myriad of abnormalities, including immune dysfunction. Previous research has focused only on children with T21. However, improved medical interventions and treatments have led to a rapid increase in the lifespan of people with T21. Thus, there is now a population of adults with T21 and a critical gap in our understanding of immunity in these adults. It is well known that adults with T21 are far more likely to die from respiratory infections compared to their disomic counterparts. This is despite recent published reports that individuals with T21 have an increased interferon response, and the Interferon-α receptor is located on q21. Our objective was to determine if a.) IFNα stimulated fibroblasts with trisomy 21 had greater interferon stimulated gene (ISG) and protein expression compared to disomic fibroblasts; and b.) If virally infected fibroblast cells with trisomy 21 had an increased type I IFN response after viral infection compared to disomic fibroblasts. Cells were treated with IFNα or virally infected with Herpes Simplex Virus-1 (HSV) for 6 or 24 hours. Gene expression and viral replication was measured by qPCR and protein expression measured by Western blot. Our results indicate that fibroblast cells with trisomy 21 have a much greater expression of the ISG15 and Mx1 proteins compared to disomic fibroblasts when stimulated with IFNα. During HSV infection, fibroblasts with trisomy 21 had a significantly greater gene expression of ISGs compared to disomic fibroblasts although viral replication rates were similar between the two groups. This data suggests that individuals with T21 have a greater type I IFN response compared to typical adults.
A novel proviral function for IL-17 during gammaherpesvirus infection
Authors: Jondle Christopher Ned , Johnson Kaitlin E , Aurubin Carlie A , Xin Gang , Cui Weiguo , Huppler Anna , Tarakanova Vera L
Published: January 2023

Gammaherpesviruses are ubiquitous pathogens that establish lifelong infection and are associated with B cell lymphomas. These viruses infect naïve B cells and induce a polyclonal germinal center (GC) response to establish long-term infection in memory B cells. We showed that Interferon Regulatory Factor 1 (IRF-1), an antiviral and tumor suppressor transcription factor, selectively restricts the GC reaction driven by murine gammaherpesvirus 68 (MHV68). Further, latent MHV68 infection was poorly controlled in IRF-1−/− mice, including in the peritoneal cavity where the latency is not regulated by the GC response. In our search for the mechanism underlying the observed phenotypes, we have unexpectedly found a significant increase in Th17 cells in MHV68 infected IRF-1−/− mice. Herpesvirus saimiri (HVS), a related simian gammaherpesvirus, encodes a viral IL-17, posing an intriguing possibility that IL-17 is proviral. MHV68 does not encode an IL-17 homologue, suggesting that MHV68 may rely on a host derived IL-17. Intriguingly, latent MHV68 infection was significantly attenuated in IL-17RA−/− mice establishing, for the first time, a proviral role of host IL-17 signaling during gammaherpesvirus infection. Further, there was a significant decrease in the GC B cell response in IL-17RA−/− mice following MHV68, but not LCMV infection, indicating that IL-17 signaling is selectively required to support the GC response during gammaherpesvirus infection. Altogether, our study establishes IRF-1 as a suppressor of Th17 differentiation and reveals a novel proviral role of IL-17 during gammaherpesvirus infection.
The innate signaling adaptor MAVS regulates the quality of the antibody response to West Nile Virus
Authors: Schenten Dominik , O’Ketch Marvin , Larson Cameron , Hall Brenna
Published: January 2023

A key difference that distinguishes viral infections from protein immunizations is the recognition of viral nucleic acids by cytosolic pattern recognition receptors (PRRs) during the course of infections. Cytosolic PRRs play a crucial role in the control of innate immunity to many viral infections. In contrast to transmembrane PRRs such as Toll-like receptors, however, the functions of cytosolic PRRs in the regulation of protective adaptive immunity have remained poorly understood. Rig-I-like Receptors (RLRs) are a family of PRRs that recognize microbial RNA in the cytosol and induce cytokine and interferon responses via the essential signaling adaptor MAVS. Infection of MAVS-deficient mice with West Nile Virus (WNV) results in a defective adaptive immune response. While this finding suggests a role for RLRs in the regulation of adaptive immunity to WNV, it is difficult to interpret due to the high WNV viremia and associated antigen loads in the absence of a MAVS-dependent innate immune response. In order to overcome these limitations, we have infected MAVS-deficient mice with a single-round-of-infection mutant of WNV. MAVS-deficient mice failed to produce an effective neutralizing antibody response to WNV despite normal titers of antibodies targeting the neutralizing lateral ridge epitope in the DIII domain of WNV-E protein. Instead, the antibodies of MAVS-deficient mice bound the neutralizing epitope with lower affinity. Our findings suggest that RLR-dependent signals are important for the quality of the humoral immune response to WNV.
Diets rich in omega-3s decrease CD8 T cell 2-dimensional affinity and short lived effector cell development and function during viral infection
Authors: Kolawole Elizabeth Motunrayo , Evavold Brian D
Published: January 2023

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), which are key components of fish oil, are increasingly consumed for their health benefits and have been shown to dramatically influence chronic disease. Although omega-3s can lead to the production of less inflammatory metabolites, we focused on the direct effects of a high omega-3 diet on T cell activation during viral infection. Specifically, we analyzed T cell recognition of antigen and the resulting functional responses during lymphocytic choriomeningitis (LCMV) infection. The omega-3 based diet manipulated structural lipid content in plasma membranes leading to reduced T cell frequency and changes in the prevalence of immunodominant epitopes. We further found that a high omega-3 diet decreased 2D affinity and number of tetramer positive polyclonal as well as CD8 T cell receptor transgenic cells at peak anti-viral immunity. In addition to affinity, the bond lifetime of antigen recognition was also decreased. This resulted in decreased numbers of SLEC and effector cytokines without impeding viral clearance. Our work indicate that a major effect of dietary omega-3 fatty acids on T cells is the modification of the cell membrane to dampen the initial recognition of antigen leading to decreased but effective immunity.
Patients with Natural Killer (NK) Cell Chronic Active Epstein-Barr Virus Have a Unique Phenotype, With Increased Activation of the PI3K/Akt/mTOR and STAT1 Pathways and Immature NK Cells
Authors: Dowdell K C , Howe Matthew , Kuehn Hyesun , Hart Geoffrey T , Hsu Amy , Su Hua , Niemela Julie , Uzel Gulbu , Shereck Evan , Schulz Laura , Feldman Tatyana , Stoddard Jennifer , Rosenzweig Sergio , Long Eric O , Dropulic Lesia , Cohen Jeffrey
Published: January 2023

Epstein-Barr virus (EBV) infection is usually asymptomatic and latency occurs in B cells. We studied three patients with NK cell chronic active EBV (CAEBV) disease and EBV hepatitis. All patients had an increased number of NK cells, and high levels of EBV in the blood, with EBV predominantly in NK cells; two patients who were tested had an NK cell receptor repertoire consistent with immature cells. All three patients had increased phosphorylation of Akt and ribosomal protein S6 in NK cells and a marked increase in STAT1; two of the patients had increased phosphorylation of STAT1 in their T cells, NK cells, and monocytes. Treatment of one of these patients with sirolimus, an mTOR inhibitor, reduced phosphorylation of S6 in the patient’s T and B cells, but not in her NK cells and did not reduce the level of NK cells or EBV DNA in the peripheral blood. The increased STAT1 activity observed in two of the patients was reduced to normal when the cells were treated with JAK inhibitors in vitro. Unlike other patients with STAT1 gain-of-function (GOF) mutations, no mutations were identified in STAT1 or STAT1 regulatory proteins. Patients with T or B cell CAEBV also had increased phosphorylation of Akt and S6, but not STAT1. In summary, the increase in Akt and S6 phosphorylation, and the increase in STAT1 protein, as well as immature NK cells in our NK cell patients describe a novel phenotype in NK cell CAEBV disease.
Accumulation of CXCR5+ NK cells during chronic SHIV infection is associated with enhanced viral control
Authors: Velu Vijayakumar , Rahman Sheikh abdul , Billingsley James M , Ibegbu Chris C , Shiferaw Ayalnesh , Villinger Francois J , Bosinger Steve , Johnson Paul , Amara Rama Rao , Velu Vijayakumar
Published: January 2023

Natural Killer (NK) cells play an essential role in antiviral immunity, however knowledge pertaining to their function in lymphoid organs during chronic HIV infection is not clearly elucidated. Lymph node (LN) follicles constitute major reservoir sites for HIV persistence. Cure strategies could benefit from the LN CXCR5+ NK cells able to access/eliminate viral reservoirs. Here we studied the phenotype, distribution and function of CXCR5+ NK cells in the LN of SHIV naive and chronic SHIV-infected (>14 weeks PI) rhesus macaques (RM) and its association with plasma viral RNA levels. We found that prior to infection, a significant proportion of NK cells (~15%) expressed CXCR5. Following infection, the frequency of CXCR5+ NK cells was significantly higher in chronic SHIV-infected RM. Phenotypically CXCR5+ NK cells express higher levels of CD32a and CD16 compared to CXCR5− cells, which might be important for ADCC function. RNA expression profiling of sorted CXCR5+ and CXCR5− NK cells from LNs of chronic SHIV-infected RM revealed that CXCR5+ cells are activated and express increased levels of cytolytic markers (perforin, granzyme-B, granulysin and CD107a), suggesting that these cells have a higher capacity to kill. Gene set enrichment analysis showed elevated expression in CXCR5+ cells of transcripts associated with cell activation, TNF-alpha, Interferon signaling and apoptosis. Importantly, the higher frequency of CXCR5+ NK cells was associated with lower SHIV plasma viral RNA levels and lower germinal center Tfh cells. Thus these data suggest that CXCR5+ NK cells could play an important role in controlling chronic SHIV infection. HIV cure strategies should focus on inducing CXCR5+ NK cells that may contribute to sustained viral remission.
Identification of dendritic cell precursors in the culture of bone marrow with hematopoietic cytokines
Authors: In Hyunju , Na Hye Young , Ryu Seul Hye , Sohn Moah , Choi Wanho , Shin Hyun Soo , Park Ji Soo , Park Sejung , Park Chae Gyu
Published: January 2023

Dendritic cells (DCs) play crucial parts in a variety of immune responses by capturing and presenting antigens to naïve T cells and by producing the subsequent T-cell activation or tolerance. In vitro culture systems of DCs have been widely used. The culture of bone marrow (BM) in the presence of a hematopoietic cytokine, Fms-like tyrosine kinase 3 ligand (FLT3L), produces plasmacytoid DCs (pDCs) and classical DCs (cDCs), whereas the culture of BM with granulocyte-macrophage colony-stimulating factor (GM-CSF) generates DCs and macrophages considered to possess inflammatory phenotypes. In the present study, we examined the culture of BM with both cytokines, i.e., FLT3L and GM-CSF in combination and identified 4 different subsets in CD11c⁺ MHCII+ cells. Meanwhile, in the culture of BM with FLT3L, we discovered a novel population of CD11c⁺ MHCIIhi cells that possess a limited capacity of antigen-presenting activity, suggesting precursors for DCs. This novel population of DC precursors from the BM culture with FLT3L was isolated and evaluated for their potential to differentiate to DCs in the BM culture containing both FLT3L and GM-CSF. Consequently, we demonstrated that the presence of a novel population of DC precursors in the in vitro culture system of DCs.
Activation of Dendritic Cells Alters the Mechanism of MHC Class II Antigen Presentation to CD4 T Cells
Authors: Roche Paul A , Ishido Satoshi , Eisenlohr Laurence C , Cho Kyung-Jin
Published: January 2023

Both immature and mature DCs can process and present foreign antigens to CD4 T cells, however the mechanism by which MHC-II in mature DCs acquires antigenic peptides remains unknown. To address this we have studied antigen processing and presentation of two distinct CD4 T cells epitopes of the influenza virus haemagglutinin coat protein by both immature and mature DCs. We find that immature DCs almost exclusively use newly-synthesized MHC-II targeted to DM+ late endosomes for presentation to influenza virus-specific CD4 T cells. By contrast, mature DCs exclusively use recycling MHC-II that traffics to both early and late endosomes for antigenic peptide binding. Knock-down of the small GTPase Rab11a partially inhibits recycling of MHC-II in mature DCs and inhibits presentation of an influenza virus hemagglutinin CD4 T cell epitope generated in early endosomes. By contrast, knock-down of Rab11a does not affect presentation of an influenza virus hemagglutinin CD4 T cell epitope that is DM-dependent and is generated in late endosomes in both immature and mature DCs. These studies highlight a “division of labor” in MHC-II peptide binding in which immature DCs preferentially present antigens acquired in Rab11a− DM+ late endosomes whereas mature DCs use recycling MHC-II to present antigenic peptides acquired in both Rab11a+ early endosomes and Rab11a− endosomes for CD4 T cell activation.
Activation of virtual memory CD8+ T cells specific for self antigens requires antigen presentation by CD11c+ cells
Authors: Gillig Marc A , Karandikar Sukrut H , Srivastava Pramod K
Published: January 2023

Even in experimentally naïve mice, a significant fraction of CD8+ T cells express CD44, a marker associated with previous exposure to cognate antigen. Many of these cells express additional hallmarks of the memory phenotype, leading to questions about their specificity, development, and functional relevance. It has been suggested that lymphopenic conditions experienced by early T cells in prenatal/neonatal mice can give rise to these unexpected “virtual” memory T cells. However, their antigen specificity remains largely unknown. Here, we report several peptide sequences derived from self antigens, which are capable of eliciting robust interferon gamma (IFNγ) secretion in CD8+ T cells isolated from naïve mice. Only CD44+ (memory phenotype), and not CD44− (naïve phenotype) T cells show this unexpected self-reactivity, and the antigens are cross presented by professional antigen-presenting cells (pAPCs) but are not presented to T cells by non-pAPCs expressing these antigens. Interestingly, antigen donor cells pretreated with IFNγ also failed to stimulate these T cells. The implications of having such self-reactive memory-phenotype T cells in circulation remain unclear, although their role in autoimmunity and self-tolerance can be imagined. There is also the possibility that such responses are a manifestation of immunological nihilism.
TGF-β downregulates MHC I surface expression through a Smad3-dependent mechanism
Authors: Berglund Alix K , Long Julie M , Schnabel Lauren V
Published: January 2023

Major histocompatibility complex (MHC) expression is tightly regulated in nucleated cells to prevent unnecessary and damaging immune responses. Transforming growth factor-β (TGF-β) has previously been shown to downregulate MHC I surface expression, although this mechanism is unknown. We hypothesized that TGF-β downregulates MHC I gene expression thereby inhibiting MHC I surface expression. TGF-β1, TGF-β2, and TGF-β3 were all similarly effective at downregulating constitutive MHC I expression. Inhibition of TGF-βRII (ALK5) or Smad3 prevented downregulation of MHC I surface expression by TGF-β indicating this mechanism is through canonical TGF-β signaling. RNA-sequencing revealed that TGF-β significantly downregulated gene expression of accessory proteins necessary for stable MHC I protein expression including β2-microglobulin (B2M). qPCR confirmed that B2M expression was significantly lower in TGF-β-treated MSCs compared to untreated, while MHC I gene expression was not significantly different. However, downregulation of B2M gene expression did not occur until at least 24 hours after TGF-β treatment suggesting an intermediate step may be involved between Smad3 activation and downregulation of B2M. These results support that TGF-β regulates MHC I surface expression by downregulating gene expression of proteins associated with antigen processing and presentation through a Smad3-dependent pathway. Further investigation is needed to analyze which antigen processing and presentation genes regulated by TGF-β are the limiting factor for MHC I surface expression and how Smad3 influences expression of these genes.
Liposomes represent a perfect tool to enhance the cross-presentation effectivity of viral HIV-1 p24 protein fused with heat shock protein 70
Authors: Zachova Katerina , masek Josef , Bartheldyova Eliska , Krupka Michal , Kulich Pavel , Turanek Jaroslav , Raska Milan
Published: January 2023

Current approaches focusing on anti-viral and anti-tumor vaccines developement tend to induce strong CD4 T cell response leading in antibodies production and simultaneously, efficient CD8 T cell activation responsible for cytotoxic elimination of infected cells. The p24 protein from HIV-1 virus is a relatively conserved but poorly immunogenic protein. Its fusion with host heat shock protein 70 (hsp70) enhanced the immune response to p24 including cross-presentation on MHC I molecules, despite the recognition by antigen presenting cells as an exogenous antigen. Cross-presentation efficacy and immune response was supported by binding this construct to liposomes containing aminooxy lipids allowing further modification by click chemistry. Hsp70-p24 was bound to liposome surface and used in cDC1 cell line (DCs from murine spleen) and mice vaccination model. Activation markers, MHCI expression, apoptosis assay and proliferation efficacy was analyzed. Expression of DC cell-surface activation markers CD40, CD86 and also MHC I was increased in hsp70-p24 pulsed cells as much as LPS, but when antigens coupled to liposome expression was significantly higher. None of the constructs activated CD86 expression as much as the LPS. Studies on hepatocytes transfected by hsp70-p24 encoding plasmid DNA showed increased death of hepatocytes co-cultivated with splenocytes from mice vaccinated by hsp70-p24-liposome in comparison to hsp70-p24 alone. Activation markers on CD8 T cells from vaccinated mice were enhanced after co-cultivation with hsp70-p24-liposome pulsed cDC1 cells or bone marrow derived dendritic cells. All these results clearly show that coupling of antigen with liposomes leads to effective CD4 and also CD8 T cell activation.
Unusual distribution of a non-classical MHC I restricted CD8+ T population
Authors: Guan Jian , Shastri Nilabh
Published: January 2023

The normal peptide repertoire presented by MHC class I molecules on the cell surface is essential for immune surveillance of intracellular pathogens. The generation of this peptide repertoire is critically dependent upon the ER aminopeptidase associated with Ag processing (ERAAP). Interestingly, ERAAP-deficient cells are recognized by a unique population of cytotoxic T cells, called QFL-T cells, that are specific for the ligand presented by the non-classical MHC molecule, Qa-1b. The QFL-T cells are similar to other non-classical MHC restricted NKT or MAIT cells in their αβ TCRs, but their physiological function(s) are unknown. To identify the potential function of QFL-T cells, we assessed the expression of the Qa-1b restricted ligand as well as the distribution of QFL-T cells in various tissues. We found the ERAAP-dependent Qa-1b restricted ligand as well as the QFL-T cells were highly expressed in the intestine. Remarkably, relative to wild-type mice, the overall number and frequency of QFL-T cells among the intraepithelial lymphocytes(IELs) decreased significantly in the germ-free mice whereas the splenic QFL-T population remained unaffected. These findings suggest a role for gut-microbiota in the homing/maintenance of the QFL-T cells in the intestine.
The (tremendous) gap between predicted epitopes and those actually recognized by CD8 T cells
Authors: Kirchenbaum Greg A , Lehmann Alexander , Lehmann Paul V
Published: January 2023

In order to monitor CD8 cell immunity it is essential to know the peptide determinant(s) of the antigen that these T cells recognize. Relevant peptide determinants of antigens were initially identified through characterization of T cell lines or hybridomas, but more recently they are increasingly predicted based on MHC-peptide binding algorithms. Systematic, brute force testing of the actually engaged CD8 T cell repertoire ex vivo is an alternative approach, but has been challenging due to (a) the large number of candidate peptides for even simple antigens, (b) the volume of blood required for testing these peptides, and (c) the scope of effort being at the border of feasibility. To highlight the utility of ELISPOT to perform such epitope mapping, we performed a feasibility study in which PBMC from HCMV infected, HLA-A*02:01 subjects were assessed for reactivity against the 553 individual nonamer peptides that encompass the entire HCMV pp65 protein. Reactivity against a single peptide of the HCMV pp65 protein, pp65495–503, was of particular interest because numerous reports have indicated that this peptide is immunodominant in HLA-A*02:01 subjects. Strikingly, our epitope mapping showed that pp65495–503 was immunodominant in only a fraction of our HLA-A*02:01, HCMV infected test subjects. Instead, responses to pp65495–503 were either subdominant or absent in other HLA-A*02:01, HCMV infected test subjects. In the latter subjects, other peptides of pp65 elicited robust responses, providing further confirmation of HCMV status. Collectively, our findings suggest that systematic testing against all potential epitopes of the antigen of interest is both feasible and is needed for reliably monitoring of T cell immunity.
Structural Insights into the Mechanism(s) of Peptide Loading in MHC-I dependent Antigen Presentation
Authors: Jiang Jiansheng , Natarajan Kannan , Kim Ellen , Dhobi Javeed A. , Mage Michael G. , Boyd Lisa F. , Margulies David H.
Published: January 2023

Class I MHC molecules (MHC-I) provide crucial cell surface elements that signal health or status of cells for recognition by T cells via their T cell receptor or natural killer (NK) cells via various NK receptors. MHC-I dependent antigen presentation thus influences TCR and NK recognition in immunity to infection, in cancer, and in autoimmunity. Antigen presentation in the MHC-I-dependent pathway depends, in part, on the loading of peptides, derived from proteolysis, aberrant translation, or protein splicing onto MHC-I molecules in the endoplasmic reticulum (ER). The classical pathway of peptide loading and exchange involves components of the peptide loading complex (PLC): the transporter TAP1/2; a lectin, calreticulin; an oxidoreductase, ERp57; a chaperone, tapasin; as well as the peptide-receptive MHC-I molecule and its light chain, b2-microglobulin (b2m). To understand the mechanism of peptide loading and exchange, several laboratories have explored functional and structural aspects of MHC-I interactions with a tapasin homolog, TAP binding protein, related (TAPBPR). We previously reported the structure of a TAPBPR/MHC-I complex at 3.4 Å resolution. However, details of the direct interaction of tapasin with MHC-I are more desirable. Here we report the X-ray crystal structure of tapasin/MHC-I complexes at higher resolution. We discuss similarities and differences between the tapasin/MHC-I and TAPBPR/MHC-I interactions and their implications for MHC-I dependent T cell and NK cell recognition.
Maturation of dendritic cells derived from type 1 diabetes-susceptible NOD mice is differentially regulated by two strains of generally regarded as safe (GRAS) Lactobacillus
Authors: Larkin Joseph , Mohamed Zaynab Sidi , Ardissone Alexandria , Bedoya Simone , Collins Erin , Wasserfall Clive , Lorca Graciela , Triplett Eric W. , Atkinson Mark
Published: January 2023

Although commensal bacteria have been implicated in type 1 diabetes (T1D), specific mechanistic insight is limited. We hypothesized that specific strains of commensal bacteria can modulate the diabetogenic peripheral T cell effector repertoire (PTER) through differential dendritic cell (DC) maturation. To test this, we assessed the presence of an IL17+ PTER and compared the microbial profiles present within female non-obese diabetic (NOD) mice, which spontaneously develop TID, and non-obese resistant (NOR) mice, which are resistant to T1D. We also assessed changes in DC maturation mediated by generally regarded as safe (GRAS) Lactobacillus johnsonii N6.2 (LjN6.2) versus Lactobacillus reuteri TD1 (LrTD1), which were previously shown to inhibit and accelerate T1D, respectively, in bio-breeding diabetes-prone (BBDP) rats. We found that NOR mice possessed a distinct Th17 bias and higher CD11c+MHCII+ DCs frequencies within lymph nodes and pancreas when compared to NOD mice. In vitro, LjN6.2 induced production of IL-6, IL-10, and TGF-b, previously shown to prime protective IL17+ T lymphocytes. Additionally, LjN6.2 promoted dendritic cell expression of the tolerogenic signaling molecules SOCS1, B7-H4, and PD-L1. In contrast, LrTD1 promoted production of proinflammatory cytokines, IL-12 and IL-23, which share a common p40 subunit. Together these data suggest that certain bacterial strains may enhance tolerance mechanisms and, thereby, contribute toward an environment that might protect against T1D, whereas bacterial strains such as LrTD1 may act as an “inducing” environmental factor by enhancing IL-12 production. Hence, these results emphasize a need to elucidate immunological mechanisms mediated by GRAS bacteria.
TLR9 stimulation by the gut microbiota promotes tissue-specific immunosuppression in autoimmune-prone MRL/lpr mice
Authors: Edwards Michael Richard , Mu Qinghui , Swartwout Brianna , Cabana-Puig Xavier , Mao Jiangdi , Reilly Christopher M. , Luo Xin M.
Published: January 2023

To investigate the ability of gut microbiota to modify immune regulation in autoimmune disease, we treated lupus-prone mice with oral vancomycin prior to disease development, and subsequently administered bacterial DNA isolated from commensal bacteria. Vancomycin given prior to disease initiation promotes exacerbation of autoimmune disease, likely due to a decrease in the frequency of B regulatory (Breg) cells, whereas vancomycin given after autoimmune disease onset has suppressive effects on disease phenotype in MRL/lpr mice. Bacterial DNA administration during the pre-disease stage promoted Breg cell production and suppressed autoimmune disease parameters in MRL/lpr mice. Further, we showed that activation of toll-like receptor 9 (TLR9) on B cells was involved in the suppressive effects of bacterial DNA seen in the MRL/lpr mouse model. We found that during the pre-disease period, joint treatment with vancomycin and oral administration of a TLR9 specific agonist, ODN 1826, promoted Breg cells in the lamina propria, increased total CD8+ T cells and effector memory CD44 expressingCD8+ T cells in the spleen, and suppressed Th17 and IFNγ producing CD4+ T cells in the mesenteric lymph nodes. Oral ODN 1826 administration also increased the Treg/Th17 ratio in the mesenteric lymph nodes. The alterations in suppressive vs. proinflammatory cell phenotypes by oral exposure to ODN 1826 suggests that the suppressive effects of bacterial DNA from the gut microbiota are modulated through TLR9 activation in a tissue-specific manner.
Efficacy of cannabidiol treatment in experimental MS is due to immunosuppressive activity of myeloid cells in CNS downregulating recruitment of CD4+ T cells, proinflammatory chemokines and gasdermin D expression
Authors: Dopkins Nicholas , Wilson Kiesha , Miranda Kathryn , Nagarkatti Prakash S , Nagarkatti Mitzi
Published: January 2023

Cannabidiol (CBD) is a nonpsychoactive ingredient from Cannabis that has garnered attention in the medical community due to its anti-inflammatory properties and therapeutic potential. CBD has in particular become a popular alternative medicine among individuals with autoimmune disorders due to CBD lacking the side effects and costs associated with immunosuppressants. To better define how CBD inhibits inflammation, we studied the effects of orally administering CBD (20mg/kg) in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. Using single cell RNA sequencing on cells isolated from the central nervous system (CNS) of EAE mice, we demonstrated that CBD treatment inhibits neuroinflammation by regulating gene expression and infiltration of myeloid cells. Within the resident and infiltrating myeloid cells of the CNS, there was decreased expression of inflammatory cytokines and chemokines (CXCL9, CXCL10, IL-18) associated with neuroinflammation and CD4+ T cell recruitment, pyroptosis initiators (gasdermin D; Gsdmd), oxide synthesizers (Arg1) and antigen presentation mediators (CD40) in CBD-treatment group. Additionally, CBD yielded an increase in the polymorphonuclear myeloid derived suppressor cells in the CNS, signifying immunotolerance induction at the site of inflammation. In the VEH treated mice, we found CD4+ T cells that express the receptor for IL-18 (IL-18r1) that are absent in CBD treated mice, suggesting CBD affects T cell activity via inhibition of myeloid cell activity. This data shows that CBD treatment ameliorates EAE by inhibiting infiltration, pro-inflammatory cytokine production, antigen presentation, T cell recruitment and pyroptosis of myeloid cells in the CNS.
Exhausted CD8 T cells exhibit phenotypic and functional heterogeneity in type I diabetes (T1D)
Authors: Kus Anna , Wiedeman Alice , Wall Valerie , Muir Virginia , Speake Cate , Long S. Alice
Published: January 2023

CD8 T Cell exhaustion is associated with poor clinical outcome in cancer and chronic infection, but positive outcome in autoimmunity. Recently, phenotypic and functional heterogeneity within exhausted CD8 T cells suggests lineage subsets. We observed similar heterogeneity in the phenotype of exhausted cells in the context of T1D. Here we further explore the phenotypic and functional features of exhausted subsets, and ask about correlation with features of T1D. Using a CyTOF panel, we identified 12 major phenotypes (clusters) among total CD8 T cells in T1D subjects. Four of these clusters were characterized by features of exhaustion, namely PD-1, EOMES, KLRG-1, and TIGIT expression. Of these four, a CD127 high, but Granzyme B and TBET low cluster was enriched for islet-specific autoreactive CD8 T cells, and was associated with slower disease progression. As compared with other exhausted clusters, CD127-high exhausted cells proliferated more and produced increased IFNγ and TNFα upon in vitro anti-CD3 stimulation, measured by flow cytometry. This phenotype and functionality is consistent with a stem-like exhausted population. In comparison, the other three other clusters expressed higher TBET and Granzyme B but lower CD127, two of which were enriched for insulin-specific cells. This is consistent with a transitory exhausted phenotype driven by repeated antigen exposure as occurs with insulin treatment of T1D. Thus, varying stages of exhaustion are present in T1D and the stem-like exhausted phenotype may be beneficial in slowing disease progression in T1D. Understanding exhausted CD8 T cell heterogeneity and the role these subsets play in T1D will lead to improved therapies.