bluuebunny/crossref_metadata_2025 huggingface dataset:
TCR signal strength and antigen affinity independently modulate CD8+ memory T cell development
Authors: Solouki Sabrina , August Avery , Huang Weishan
Published: January 2023

CD8+ T cells play a critical role in adaptive immunity by maintaining the ability to differentiate into CD8+ memory T cells, which provide the basis of protective immunity. During an intracellular infection, CD8+ T cells pass through several characteristic phases before becoming mature memory cells. Initial antigen stimulation causes naïve CD8+ T cells to clonally expand and differentiate into short-lived effector cells (SLECs). Subsequently, SLECs undergo a contraction phase to give rise to memory precursor effector cells (MPECs); 5–10% of the MPECs survive the initial contraction phase and further develop into CD8+ memory T cells. We have shown that the non-receptor tyrosine kinase Itk, which regulates TCR signal strength, can significantly suppress CD8+ memory T cell development. We hypothesize that TCR signal strength, regulated by Itk, intersects with antigen affinity to modulate the development of SLECs and MPECs, leading to an increased proportion of memory CD8+ T cells. Utilizing Itk deficient OT-1 TCR transgenic mice, which carry CD8+ T cells specific for the Ovalbumin protein, an adoptive transfer model was established to exam the influence of TCR signal strength and antigen affinity in the development of CD8+ memory T cells. Our findings suggest that TCR signal strength and affinity independently contribute to CD8+ memory T cell development. Our data reveals that reducing both antigen affinity and TCR signal strength leads to enhanced and accelerated antigen-mediated proliferation and development of memory cells, and enhanced secondary expansion of these memory cells. In sum, our data suggests that TCR signal strength and antigen affinity are independent parameters that may be used to tune vaccine development.
Single cell multiomic analysis of chronically stimulated T cells displaying hallmarks of T-cell exhaustion
Authors: Nakamoto Margaret , Corselli Mirko , Taylor Ian , Saksena Suraj
Published: January 2023

T-cell exhaustion is a dysfunctional state caused by persistent antigen stimulation mediated by chronic infections or cancer. Current immunotherapy approaches focus on reinvigorating exhausted T cells through immune-receptor-blocking antibodies. A deeper understanding of the exhausted T-cell state is crucial for identification of new therapeutic targets. Single cell RNA-sequencing (scRNA-seq) is a powerful tool for resolving heterogeneity in complex systems like these; however the lack of protein information for the same cells can make it difficult to identify cells of interest or detect cellular responses. To resolve both mRNA and protein information in the same assay, we utilized DNA-conjugated BD™ AbSeq antibodies to profile protein and mRNA expression, using high-throughput sequencing analysis at the single cell level. Sorted T cells that had been transiently and chronically stimulated were multiplexed using the BD™ Single-Cell Multiplexing Kit and pooled before undergoing cell partitioning and cDNA capture on the BD Rhapsody™ system; this minimized batch effects and saved time and reagents. A panel of 400 mRNA targets alongside the 39-plex antibody panel was used for the study. We showed that cell-type resolution increased with higher protein plex, and the addition of mRNA information alongside protein markers revealed candidate protein markers for specific cell states. This study showcased the power of multiomics in analyzing heterogenous cell populations and cell states and answering complex biological questions.

Research Use Only. Not for use in diagnostic or therapeutic procedures.

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Lipid talk: Endocannabinoids regulate mucosal inflammation following helminth infection
Authors: Wiley Mark B , Batugedara Hashini , Bobardt Sarah , Argueta Donovan , Dillman Adler , DiPatrizio Nicholas V , Nair Meera G
Published: January 2023

More than 2 billion humans carry infectious parasites leading to chronic co-morbidities and growth retardation in children. Parasitic infections induce a T helper type 2 (Th2) immune response in the host to promote clearance which can lead to fibrosis if chronic. We recently showed that infection with the soil-transmitted nematode Nippostrongylus brasiliensis (Nb) induces overproduction of endocannabinoids (eCBs) in the host. Endocannabinoids are endogenous cannabis-like molecules that influence the development of obesity and are anti-inflammatory, however their function in infection is largely unknown. The overproduction of eCBs in Nb-infected mice was observed throughout infection in the infected lung and intestine. Pharmacological inhibition of cannabinoid receptor subtype 1 (CB-1R) with AM6545 (10 mg/Kg/2mL), a peripherally-restricted CB1R neutral antagonist, throughout Nb infection exacerbated weight loss of mice without altering food or water intake. This inhibition of CB1Rs led to a prolonged presence of eosinophils and neutrophils in the broncho-alveolar fluid and an accumulation of lymphocytes in the spleen. Furthermore, CB1R inhibition caused an increase in the expression of the mannose receptor on alveolar macrophages at day 8 post infection, indicating a sustained need for inflammatory resolution. Strikingly, we also found that Nb produces its own eCBs which vary in concentration based on stage of development, and that the eCB system is present in many parasitic nematodes including those that infect humans. These findings suggest that the eCB system is active in several hookworm species, and that host and helminth eCBs may influence immune and anti-inflammatory functions.
Type I Interferon confers susceptibility to Mycobacterium tuberculosis infection within granulomas of SIV co-infected non-human primates
Authors: Phuah Jia Yao , O’Connor Shelby , Scanga Charles A. , Mattila Joshua T.
Published: January 2023

Mycobacterium tuberculosis (Mtb) co-infection with human immunodeficiency virus (HIV) typically results in poor disease outcome for tuberculosis (TB). While the loss of CD4+ T cells due to HIV is known to cause increased TB disease susceptibility, the mechanisms which govern the loss of disease control in co-infected individuals are not well understood particularly within the lung granulomas that keep TB contained. This study aims to profile the transcriptional patterns of lung granulomas from non-human primates (NHP) co-infected with Mtb and simian immunodeficiency virus (SIV) as a model for TB-SIV co-infection in humans. We compared the transcriptional profiles of granulomas from four Mauritian cynomolgus macaques that were infected with SIV for six months and then co-infected with Mtb for eight weeks against animals that were infected with Mtb for eight weeks. Granulomas from co-infected animals were found to have generally elevated transcripts involved in Type I interferon pathways (IFIT1), anti-bacterial effectors (defensin A, cathepsin G) and evidence of increased neutrophilic inflammation compared to Mtb-only control lung granulomas. Pathways that were downregulated in co-infected animals were related to Type II interferon signaling (IRF8). These results suggest that SIV infection alters the host immune response in ways that may be advantageous to Mtb growth by lowering IFNγ responsiveness and promoting myeloid cell-associated inflammation. We hypothesize this leads to a more permissive and less antibacterial environment that promotes bacterial survival, dissemination, and ultimately leads to increased susceptibility of co-infected individuals towards TB disease.
Ruxolitinib, a selective JAK1/2 inhibitor, for the treatment of serious diseases caused by Staphylococcus aureus superantigens
Authors: Carnes Henry , Mehrkens Brittney , Stegman Molly , Rajagopalan Govindarajan
Published: January 2023

Staphylococcal superantigens (SSAgs), produced by Staphylococcus aureus, non-specifically activate 30–50% of all CD4+ and CD8+ T cells expressing certain T cell receptor (TCR) Vb gene families. SSAg-activated T cells rapidly produce large quantities of cytokines and chemokines resulting in a systemic inflammatory response syndrome (SIRS), which culminates in multi organ failure (MOF) and ultimately death. Through this process, SSAgs play a significant role in the immunopathogenesis of pneumonia, sepsis, and toxic shock syndrome. Because most cytokines produced during SIRS act through the Janus Kinase (JAK) signaling pathway, we hypothesized that therapeutics inhibiting JAK functions would attenuate SIRS, MOF, and lower mortality caused by SSAgs. We tested ruxolitinib (RUXO), an FDA-approved selective JAK 1/2 inhibitor. Several in vitro and in vivo studies were performed using HLA-DR3 transgenic mice because SSAgs bind to HLA class II molecules with higher affinity. In vitro, at 100, 10, and 1 mm concentrations, RUXO significantly inhibited SSAg-induced upregulation of CD25 and CD69 on both CD4+ and CD8+ T cell subsets as well as SSAg-induced production of IL-2, IL-17, and IFN-g in a dose-dependent manner at 24 and 48 hours. For in vivo studies, mice (5–6 per group) were gavaged with RUXO (100 mg/kg) or vehicle, twice daily on days −1, 0 and 1, challenged with SSAg (20 mg/mouse, i.p) on day 0 and euthanized on day 2. SSAg-induced SIRS, thymocyte deletion, upregulation of activation markers (CD69 and CD25), and immunopathology were significantly attenuated in RUXO-treated mice compared to vehicle-treated mice. Overall, JAK inhibitors such as ruxolitinib can be used as an adjunct to treat serious diseases caused by SSAg.
Pathogenic Mechanisms of Endothelial Damage in Severe Scrub Typhus
Authors: Soong Lynn , Trent Brandon , Liang Yuejin , Bouyer Donald W , Sun Jiaren
Published: January 2023

Our newly established lethal and sublethal Orientia tsutsugamushi infection mouse models, which mimic clinical/pathologic features of human scrub typhus, are valuable for mechanistic studies. A hallmark of lethal Orientia infection is Th1-skewed, but Th2-impared, immune responses, accompanied with severe endothelial damage and acute tissue injury. Importantly, while tissue bacteria peak at disease onset (D6) in mice, neutrophil and CD8 T cell influx/activation reach peaks in the lungs around D10 (prior to host death). We hypothesize a two-stage-dysfunction loop for lethal infection: 1) Orientia replication in phagocytes and endothelial cells (EC) triggers inflammatory responses and host defense machinery; and 2) infection-mediated release of DAMP molecules exacerbates vascular dysfunction. This hypothesis was supported by immunofluorescent co-staining of bacteria with leukocyte/EC/platelet-specific markers, multi-color FACS, Western blot, and qRT-PCR. The progressive loss of vasculature and junction proteins (VE-cad, occludin) and reduction in vascular function (angiopoietin 1, Tie2, pTie2) and CD41+ platelets in the lungs were positively linked to neutrophil and CD8 T cell influx and activation, rather than to body bacterial loads. The mechanisms of immune dysregulation were further examined by using infected human endothelial cells and mouse primary macrophage and neutrophil cultures, as well as knockout mice. While Orientia infection directly altered the Ang-Tie2 axis in EC, activated IL-33/IL-36-like DAMP molecules further contributed to vascular/tissue injury. Our studies, for the first time, have revealed immune dysregulation and prognostic biomarkers in the context of scrub typhus pathogenesis.
The increased permeability and inflammatory response of porcine intestinal epithelial cells caused by Escherichia coli and Salmonella enterica Typhimurium infections are mitigated by bovine colostrum
Authors: Blais Mylene , Bouchard Michael , Touchette Vincent , Asselin Claude , Lessard Martin
Published: January 2023

Gastroenteric infections caused by Escherichia coli and Salmonellaenterica Typhimurium are the source of important losses in porcineindustry. The use of antibiotics in piglets’ food to improve post-weaning performances and prevent enteric infections is raising many concerns, leading the governments to call for finding alternatives that contribute to enhance gut health and resistance to infections. Bovine colostrum is loaded with bioactive pro-immune components that could mediate anti-microbial activities. Therefore, we tested the effect of complete defatted bovine colostrum (BC), as well as bovineserocolostrum and casein fractions (SC and CAS respectively) on porcineintestinal epithelial cell (IPEC J2) in response to the two enteric pathogens. Bothpathogens increased the cell monolayer permeability 4h following the infection, as determined by TEER assays. BC treatment prevented the monolayer disruption caused by both pathogens, while SC only prevented the one caused by Salmonella. After 2h of incubation, the expression of IL8, IL6, TNFA, CCL20, CXCL2 and CXCL10 was increased by bothpathogens, while IL1B, CCL5 and SAA2 were only induced by Salmonella, as measured by Q-PCR. BC and SC reduced the induction of IL8, CCL20 and CXCL2 genes by E. coli(p<0,05). Both BC and SC reduced the expression of IL6 and CCL20 genes by Salmonella (p<0,05), while only BC reduced the induction of IL8, IL6, CCL20, IL1B, SAA2 (p<0,05) and TNFA (p<0,1). Altogether, these results show that the complete form of bovine colostrum can reduce both E. coli and Salmonella infections of porcineintestinal epithelial cells and could be a good alternative to antibiotics in order to control these infections.
Trehalose-6-phosphate synthase-deletion in Cryptococcus neoformans elicits rapid innate fungal clearance from the lungs with early neutrophil accumulation
Authors: Xu Jintao , Reynolds Mack , He Xiumiao , Gao Xueli , Ivey Michael , Kolbe Jess , Lopez Raphael , Mechler Claire , Perfect John , Olszewski Michal A
Published: January 2023

Clearance of the invasive fungus Cryptococcus neoformans (Cn) infection requires T-cell immune responses and individuals with impaired adaptive immunity are highly vulnerable to Cn infection. The innate immune response thus may be the only functional arm against the expansion of pathogens in patients with severe T cell deficiencies. Accordingly, targeting pathways responsible for Cn-evasion of early innate defenses could rapidly resolve Cn-disease in immunocompromised patients.

Trehalose-6-phosphate synthase (TPS1), a fungal enzyme for trehalose sugar synthesis absent in mammal, was recently identified as a key virulence gene. Using a mouse model of pulmonary Cn infection, we found that TPS1-deficient Cn (tps1Δ) elicits rapid clearance by the host. Within 72 hours, tps1Δ was either completely cleared or reduced by 5 orders of magnitude, while WT-Cn expanded in the lungs of infected mice. Mechanistically, we found that rapid tps1Δ clearance was associated with early neutrophils recruitment and induction of neutrophils - recruiting CXCL1 and CXCL2. Notably, this occurred without improved Cn-killing by macrophages. Tps1Δ stains were rapidly cleared from CD4+ T cell depleted mice that receive anti-CD4 antibody treatment, suggesting that Tps1Δ clearance is independent of host adaptive immunity. Furthermore, our in vitro killing assay showed that tps1Δ was more vulnerable to neutrophils killing compared to WT strain. Together, our data revealed that cnTPS1 contributes to early innate immune evasion by inhibiting neutrophil recruitment and killing. We identify TPS1-interception as an important therapeutic strategy to both compromise the microbe fitness and to enhance efficiency of the innate defenses in C.n elimination.
Bacteria in human intestine contribute to mucosal immune function in utero
Authors: Rackaityte Elze , Halkias Joanna , Fukui Elle M , Mendoza Ventura , Crawford Emily D. , Fujimura Kei , Burt Trevor , Lynch Susan V
Published: January 2023

Mucosal immunity influences host-microbial interactions and is evident in the human fetal intestine by 11–14 weeks of gestation; the developing intestine is populated by lymphoid aggregates, memory T cells and dendritic cells capable of responding to microbial stimuli. However, whether intestinal encounters with viable microbes occur in utero and shape immune maturation has not been investigated. Here, we identified subsets of fetal meconium relatively enriched in Lactobacillus or Micrococcaceae, using culture-independent methods, which related to divergent epithelial cell layer transcriptomes and proportions of lamina propria PLZF+ CD161+ CD4+ T cells in paired intestinal samples. Mimicking conditions in the fetal intestine permitted isolation of viable Lactobacillus and Micrococcus strains from fetal meconium. In contrast with phylogenetically related reference strains, fetal isolates utilized placental hormones for growth, remained viable within antigen presenting cells, and exhibited species-specific capacity to promote immune homeostasis. Fetal Lactobacillus isolates reduced activation of antigen presenting cells, while the Micrococcus isolate induced IL-10 production in antigen presenting cells and inhibited IFNγ production by memory PLZF+T cells. Fetal isolates were identified as strains of Lactobacillus jensenii or Micrococcus luteus by whole genome sequencing, with the latter encoding strain-specific genes for intracellular survival, immune modulation, and steroid uptake. These data suggest that pre-natal immunity is influenced by bacterial exposure in the fetal intestine, identifying a previously unknown component of human mucosal immune development.
NKT cells induce IgG1 class switch against C. difficile carbohydrate antigens and enhance immunity against a live pathogen challenge
Authors: Lang Mark L , Lang Gillian A , Amani Souwelimatou Amadou , Shrestha Binu , Shadid Tyler , Gillaspy Allison F , Ballard Jimmy D
Published: January 2023

All clinical Clostridioides difficile strains identified to date express a surface capsule-like polysaccharide structure known as polysaccharide II (PSII). The PSII antigen is immunogenic and when conjugated to a protein carrier and induces a protective antibody response in animal models. Given that CD1d-restricted Natural Killer T (NKT) cells promote antibody responses, including those against carbohydrates, we tested the hypothesis that immunization with PSII and a CD1d-binding glycolipid adjuvant could lead to enhanced protection against a live C. difficile challenge. We purified PSII from a clinical isolate of C. difficile and immunized B6 mice with PSII alone or PSII plus the CD1d-binding glycolipid α-galactosylceramide (α-GC). PSII-specific IgM and IgG3 titers were evident in sera from immunized mice, but the inclusion of α-GC led to additional isotype switch and production of IgG1. Enhanced protection against C. difficile disease was achieved by inclusion of the α-GC ligand and was associated with reduced bacterial numbers in fecal pellets. In contrast, NKT-deficient Traj18−/− mice were not protected by the PSII/α-GC immunization modality. These results indicate that α-GC-driven NKT cells stimulate a protective IgG1 response directed at the C. difficile PSII antigen. This study suggests that NKT activation represents a pathway for additional B cell help that could be used to supplement existing efforts to develop a C. difficile vaccine.
Alveolar macrophage dysfunction and increased PD-1 expression during chronic SIV infection of rhesus macaques
Authors: Hunegnaw Ruth , Mushtaq Zuena , Enyindah-Asonye Gospel , Hoang Tanya , Robert-Guroff Marjorie
Published: January 2023

HIV infected individuals have been shown to be predisposed to pulmonary infections even while receiving anti-retroviral therapy. Alveolar macrophages (AMs) play a critical role in innate immunity of the lung, but their functional and immunological attributes after infection remain contradictory. Here, we use flow cytometry and phagocytosis assays to document phenotypic and functional responses of SIV infected rhesus macaque AMs. We assessed AMs by sampling bronchoalveolar lavage (BAL) from acute through chronic phases of SIV infection and compared these to BAL samples taken from naïve macaques. We found that expression of pro-inflammatory cytokines TNF-□, IL-6, IL-1β and the chemokine RANTES by AMs drastically increased at 2 weeks post-infection compared to naïve animals (p<0.0001 for all) and was quickly followed by a significant drop as infection progressed into the chronic phase of infection. We further show that AMs from chronically infected macaques have diminished capacity to perform SIV-specific antibody-dependent phagocytosis (ADP). Moreover, SIV infection was associated with reduced AM FcγRIII expression, which negatively correlated with viral load (r=−0.6819; p=0.0013). Importantly, PD-1 was shown to be expressed on AMs and showed a strong trend toward correlation with plasma viral load (r=0.8266; p=0.0583), indicating that similar to T-cells, PD-1 expression on AMs may also be associated with disease progression. These findings provide new insight into the dynamics of SIV infection leading to AM dysfunction and alteration of pulmonary innate immunity. Our results suggest new pathways to exploit in developing therapies targeting pulmonary disease susceptibility in HIV-infected individuals.
In vivoeffectsof α4β7 blockade on size of viral reservoirs in SIV-infected rhesus macaques
Authors: XU HUANBIN
Published: January 2023

Cellular viral reservoirs are rapidly established upon HIV/SIV infection, which persist throughout viral infection, even under long-term antiretroviral therapy (ART). Recent studies report α4β7 blockade in vivo could preserve mucosal CD4+ T cells, and control persistent viral infection in SIV-infected macaques. In this study, we investigated the effects of early administration with α4β7 antagonist (antibody against α4β7 or small-molecule dual inhibitor TR-14035) for the prevention of infection, and for reducing cellular reservoirs in systemic and lymphoid tissues post SIV infection. Rhesus macaques (Macaca mulatta, RMs) were intravenously (i.v.) inoculated with 100 TCID50 SIVmac251, then administrated a single i.v. dose of anti-α4β7mAb one week prior to SIV inoculation and another 3 weeks post SIV inoculation, or orally treated with TR-14035 for two weeks beginning on the day of SIV inoculation. Our results indicated that early α4β7 blockade did not result in viral containment or reduction of the reservoir size post SIV infection. Plasma viral loads and cell-associated SIV DNA/RNA levels in blood and lymphoid tissues in treated animals were similar to untreated cohorts. Interestingly, TR-14035, a dual inhibitor for α4β7 and α4β1, resulted in persistently higher viremia throughout infection, and more rapid disease progression when administrated in the acute stage of SIV infection.

Conclusions

These findings suggest that α4β7 blockade alone may not effectively control viral infection, replication, and dissemination and reservoir establishment in HIV/SIV infection. Its usage for potential preventive or/and therapeutic strategies in combined with ART requires further investigation.
Uncharted Territory: The CD4+ T cell response to human metapneumovirus
Authors: Lamens Kristina D , Rogers Meredith C , Tometich Justin T , Hand Timothy W , Williams John V
Published: January 2023

Human metapneumovirus (HMPV) is a leading cause of lower respiratory tract infection in pediatric and immunocompromised populations. Clearance of respiratory viruses including HMPV relies primarily on the destruction of infected cells by cytotoxic CD8+ T cells. However, signals provided by CD4+ helper T cells significantly impact the magnitude and effectiveness of CD8+ T cells. Using a mouse model of acute infection, we performed an in-depth analysis of CD4+ helper T cells in the immune response to HMPV. We identified and validated an immunodominant CD4+ T cell epitope in C57BL/6 mice and constructed the first MHC-II tetramer for HMPV. Kinetic analysis showed that the percentage of CD44+tetramer+ cells in the lung peaked at day 10 post-infection. Ex vivo peptide stimulation of pulmonary T cells revealed that most virus-specific CD4+ T cells produced IFNγ, followed by a small but consistent population of IL-17a producing cells. To determine the contribution of CD4+ T cells in the primary immune response to HMPV, CD4+ T cells were antibody-depleted prior to infection. Depletion of CD4+ T cells exacerbated infection-induced CD8+ T cell impairment, led to enhanced PD-1 expression on virus-specific CD8+ T cells, and delayed viral clearance. Next, global PD-1−/− mice were infected with HMPV to further explore the role of PD-1. Both CD4+ and CD8+ T cells displayed improved functionality in HMPV-infected PD-1−/− mice, suggesting that PD-1-mediated impairment following respiratory virus infection affects CD4+ as well as CD8+ T cells. Further characterization of virus-specific CD4+ helper T cells, their regulation by PD-1, and their role in CD8+ T cell impairment will provide new insights that aid in the design of effective vaccines against HMPV.
Eosinophils undergo temporally regulated phenotypic and physiologic changes during influenza A virus infection
Authors: Samarasinghe Amali , Tiwary Meenakshi , LeMessurier Kim
Published: January 2023

Eosinophils are traditionally known for their antiparasitic responses and as biomarkers of allergic diseases, including asthma. Using our model of asthma and influenza (AF) co-morbidity wherein disease outcomes varied by the presence of eosinophils in the airways, we determined that eosinophils played a dynamic antiviral role during influenza A virus (IAV) infections mainly by regulating CD8+ T cell immunity. We have also found that eosinophils in different niches including bone marrow, lymphoid organs, and lungs, undergo distinct immunophenotypic changes depending on the disease state in mice with asthma, influenza, or AF co-morbidity. Therefore, we hypothesized that eosinophil activation and migratory capacity are temporally regulated during IAV infection and that these changes in eosinophils are sensitive to the environment. Virus-exposure resulted in the upregulation of ICAM-1, CD69, and CD11b and downregulation of CD62L on eosinophils within 20 minutes. This expression pattern was temporally regulated suggestive of their ability sense IAV and alter functional responses such as migratory potential. Furthermore, mice infected with eosinophil pre-exposed IAV had a significantly reduced viral burden in the lungs. Eosinophils prevented IAV-induced cytopathology on lung epithelial cells when co-cultured directly or separated by transwell. In addition, eosinophil recipient IAV-infected mice showed no damage to the bronchial airway epithelium. Cumulatively, these data suggest a direct antiviral role for eosinophils through self-activation, reducing virus infectivity, and protecting the respiratory barrier from virus-induced damage further establishing an antiviral role for eosinophils in allergic airways.
Unmasking a killer: discovery of a gene that controls the pathogenic activation of CD8 T cells in Type 1 diabetes
Authors: Ye Cheng , Serreze David V , Chen Yi-Guang , Driver John P
Published: January 2023

CD4 T cell help is critical for the activation of autoreactive CD8 T cells that cause most β-cell destruction in type-1 diabetes (T1D). We previously identified a 10 Mb C57BL/6 (B6)-origin recessive Idd susceptibility locus on chromosome 11, designated Idd32 that controls whether CD4 T cells cause pathogenic CD8 T cells to undergo activation or become tolerized. To identify the underlying gene, we adoptively transferred CD8 T cells transgenically expressing the diabetogenic AI4 T cell receptor into B6 mice expressing the NOD-derived H2g7 MHC haplotype (B6.H2g7) and one of 9 different NOD-derived segments of the Idd32 interval. This reduced the interval to a <520 kb region containing 18 polymorphic genes. To test these candidate genes (Δ) we used the CRISPR/Cas9 system to generate two sets of F1 strains: (B6.H2g7.Idd32NOD/NOD.Δ−/− x B6.H2g7)F1 and (B6.H2g7.Idd32NOD/NODx B6.H2g7)F1 that were monitored for T1D development after infusing them with AI4 T cells. We found that F1 mice lacking the NOD allele of the sialic acid transferase gene St6galnac1 (B6.H2g7.Idd32NOD/NOD. St6galnac1−/− x B6.H2g7)F1, but not other candidates, developed T1D. Furthermore, CD4 T cells from these mice strongly promoted diabetogenic AI4 T cell activation when co-transferred into NOD.Rag1−/− mice. In contrast, CD4 T cells from St6galnac1-intact (B6.H2g7.Idd32NOD/NODx B6.H2g7)F1 mice suppressed AI4 activation and T1D development. In summary, we have identified a novel, recessively-acting T1D susceptibility gene hidden in the normally T1D-resistant B6 mouse strain, with striking effects on pathogenic CD8 T cell activation. Our results may aid the screening of humans at future risk for T1D as well as provide a target for possible disease interventions.
Intranodal dendritic cell relocalization during inflammation impacts T cell immunity
Authors: Leal Joseph M. , Olin Brandy , Gerner Michael Y
Published: January 2023

Conventional dendritic cells (cDCs) are the critical antigen-presenting cells that initiate adaptive immunity in response to external inflammatory stimuli. We have previously shown that LN-resident cDC2s, a DC subset critical for generating CD4 T cell responses, reside in regions highly proximal to the lymphatic sinuses, enabling superior acquisition of draining soluble and particulate antigens. However, given that this peripheral positioning is distal from the deep LN paracortex and the T cell zone, we hypothesized that for optimal generation of adaptive immunity, LN-resident cDC2s must migrate intranodally from peripheral sites into the deep T zone in order to engage in cognate DC-T cell interactions during priming. Here, we show that s.c immunization with TLR-agonists, as well as infection with West Nile Virus, induces rapid relocalization of cDC2s into the deep T zone of the LN. This cDC2 relocalization requires Type 1 interferon in some, but not all, adjuvant immunizations, and is predominantly driven by increased CCR7 expression on cDCs after maturation. When lacking CCR7, LN-resident cDC2s failed to reposition to the T cell zone across multiple adjuvant comparisons, and this in turn led to a marked reduction in the activation and proliferation of CD4+ T cells. A decrease in the differentiation of Tfh cells was also observed, suggesting a possible impact on humoral immunity. Collectively, this work identifies a novel CCR7-mediated spatial reorganization of LN-resident cDC2s during inflammation, independent of peripheral migratory DCs, and its role in the generation of adaptive immunity.
CXCL9/10 producing cell clusters optimize Th1 cell positioning for interaction with antigen presenting cells
Authors: Negatu Seble G , Prizant Hen , Fowell Deborah J
Published: January 2023

Intracellular pathogen clearance is dependent on successful T cell mediated immunity. Th1 cells must localize to the site of infection to be activated into IFN-γ producing effector T cells. How Th1 cells find and interact with antigen presenting cells (APC) in infected tissues is poorly understood. Preliminary data using intravital imaging and the REX3 mouse, in which CXCL9 and CXCL10 producing cells express red and blue fluorescent proteins (RFP/BFP) respectively, suggest that CXCL9/10-producing cells form dense perivascular clusters that attract and arrest CXCR3+ Th1 cells. We hypothesize that these CXCL10+ cell clusters are enriched for APC that optimize Th1 cell activation and IFN-γ production. To determine the phenotype of the immune cells within the chemokine-producing clusters we crossed the REX3 mouse with a photoactivatable green fluorescent protein (PA-GFP) mouse. This will enable selective labeling of cells within chemokine-rich clusters prior to flow cytometry. Using precise multiphoton delivery of 830 nm light, we have successfully photoactivated the CXCL10+ clustered regions in the inflamed ear and have developed a flow cytometry panel to distinguish between macrophages, cDCs, and monocyte-derived cells that may be positioned within or outside these chemokine-clusters. Additionally, we will determine if these cell clusters enhance Th1 function by sorting photoactivated (in clusters) and non-photoactivated (outside clusters) Th1 cells from the inflamed ear and analyzing IFN-γ expression. Understanding how Th1 cells are positioned and activated at sites of inflammation will allow the field to boost responses to infectious agents and therapeutically attenuate Th1 responses in autoimmunity.
Multi-parametric Cell-Based Inflammation Assays for Cytokines and Cell Surface Antigens
Authors: Cromwell Evan , Cho Byungrae , Hoxha Ori , Bristol Neil , Olsen Cathy , Sirenko Oksana
Published: January 2023

Macrophages and vascular cells upregulate expression of cytokines and adhesion molecules in response to inflammation stimuli. Monitoring these molecules can provide a physiological read-out for inflammation. We present results from a multiparametric assays that used a combination of low-volume ELISA for secreted cytokines and automated cell imaging for cell surface markers to evaluate the effect of different mediators on inflammatory response with two cell models. First, human monocytic THP-1 cells were differentiated to macrophages and activated by LPS. Phenotypic changes in cell morphology were quantified by cell area and number of adherent cells using TL imaging. Secretion of IL-8, IL-1β and TNF-α was quantified by an automated low-volume ELISA (PuMA System). Second, primary human umbilical vein endothelial cells (HUVEC) were stimulated with inflammation cytokines (TNF-α, IFN-γ, and IL-1β), then PuMA System was used to quantify amount of MCP-1, IL-8, and IL-6 in cell supernatants. Expression of adhesion molecules VCAM and HLADR was quantified by measurement of total fluorescence from antibody-stained cells using an ImageXpress Pico system. Concentration dependent inhibition of inflammation by the compounds AG126 and MG132, SB202190 and PDTC were measured and EC50s calculated. Clear differences in cytokine expression were seen between the compounds consistent with reported mechanism of actions of the kinase inhibitors. The combination of imaging and microfluidic-based assays provides an efficient multiparametric assay system that can test the efficacy of anti-inflammatory compounds versus toxicity and provide insight into their MoA by selective inhibition of markers triggered by different signaling pathways.
Comprehensive characterization of the mast cell surface proteome
Authors: Shubin Nicholas J , Niino Kerri , Kasprzak Victoria , James Richard , Piliponsky Adrian Martin
Published: January 2023

Mast cells are known for their involvement in many pathological conditions, suggesting that mast cell responsiveness to various stimuli requires the expression of a rich cell surface protein repertoire. Mast cell surface proteomes have not been comprehensively characterized; therefore, our aim in this study was to identify unknown mast cell surface proteins to obtain a better understanding of how these cells function in health and disease. For this purpose, we enriched for glycosylated surface proteins expressed in murine bone marrow-derived mast cells (BMCMCs) and we identified the enriched proteins by using mass spectrometry analysis. The use of this approach resulted in the identification of 435 glycoproteins in BMCMCs. Based on the UniProtKB database, we identified 60 plasma membrane proteins among the enriched glycoproteins, including cell surface receptors, ligands, proteases, and adhesion molecules. The usefulness of our approach was validated by the presence of surface molecules known to be expressed on BMCMCs, including c-Kit, IL2rα, FcɛrIα, Csf2rb, and Tgfβr1. Notably, we identified 22 proteins that have not been previously reported to be expressed in BMCMCs. The presence of these proteins in mast cells was validated by cross-referencing them against the ImmGen database and/or qPCR and western blot analyses. Importantly, variations or deletions in genes that encode for these surface proteins are associated with disorders in which mast cells play an active role, such as infections, asthma, and sepsis. Taken together, our study suggests that we can identify surface molecules that may influence mast cell biology in health and disease.
Profile of Dendritic Cells Surface Biomarkers in Nasal and Oral Mucosa of Human Subjects Reactive to Grass Pollen and House Dust Mites
Authors: Rivera-Mariani Felix Emanuel , Srour Hayat , Fomenko Ruslan , Bellinger Shandra , Stateman Ariel , Baguley Joshua
Published: January 2023

Dendritic cells (DCs) play key roles in human allergic pathophysiology at different anatomic sites, such as the respiratory tract. To identify relationships between DCs subpopulations in the upper respiratory tract and atopic status, a public-available dataset (http://doi.org/10.5281/zenodo.50228) on nasal and oral DCs cell-surface biomarkers expression following allergen challenge (through skin prick test) was re-analyzed, with regression and principal component analysis (PCA), to identify profiles of DC cell-surface biomarkers based on the allergen reactivity. The dataset included immunohistochemical measures on CD207 (langerin), CD1a, BDCA-1 to -4 nasal and oral epithelium and lamina propia, as well as demographic information (age, gender), history of respiratory disease, and allergen skin provocation (grass pollen or house dust mite). Subjects with reactivity to grass pollen had different DCs cell-surface biomarker profiles than subjects reactive to house dust mite in both nasal and oral mucosal epitheium and lamina propia. Except for BDCA-3 and 4, reactivity to grass pollen yielded higher expression of DC cell-surface biomarkers in nasal epithelium. In nasal lamina propia, subject reactive to grass pollen had higher expression of BDCA-1 and -2. In the oral epithelium and lamina propia, subjects reactive to house dust mite had higher expression to only CD1a. Graphical evaluation with PCA of both nasal and oral mucosa of DC cell-surface biomarkers yielded principal components explaining three clusters in oral mucosa; two in nasal mucosa. These findings suggest that different allergen activate differently specific innate immune cells, including different DCs subpopulations at different anatomical sites.
Lymph node conduits transport virions for rapid T cell activation
Authors: Reynoso Glennys V , Weisberg Andrea S. , Shannon John P. , McManus Daniel T , Shores Lucas , Americo Jeffrey L , Stan Radu V , Yewdell Jonathan W. , Hickman Heather D.
Published: January 2023

Despite intense interest in antiviral T cell priming, the routes of virion movement in lymph nodes (LNs) are imperfectly understood. Current models fail to explain how virus-infected cells rapidly appear within the LN interior after viral infection. To better understand virion trafficking in the LN, we determined virion and infected-cell locations after pox- and zika-virus administration. Surprisingly, many rapidly infected cells in the LN interior were adjacent to LN conduits. Using confocal and electron microscopy, we clearly visualized virions within conduits. Functionally, CD8+ T cells rapidly and preferentially associated with vaccinia-infected cells deeper in the LN, leading to T cell activation in the LN interior. These results reveal that, contrary to dogma, even large virions can flow through lymph node conduits to infect T cell zone DCs that prime CD8 + T cells.
Anti-viral CD8+ T cell responses are impaired by endogenous n-3 polyunsaturated fatty acids
Authors: Seo Young-Jin , Kim Seyoung , Cho Yong-Bin , Kang Kyung-Won , Lee Sang-Myeong
Published: January 2023

Omega-3 (n-3) polyunsaturated fatty acids (PUFAs) have been known to exert anti-inflammatory effects on various inflammatory diseases. However, its role in CD8+ T cell responses against an acute viral infection has not been well elucidated yet. To determine the role of n-3 PUFAs in anti-viral CD8+ T cell responses, we used FAT-1 transgenic mice that are able to convert n-6 PUFAs to n-3 PUFAs. The FAT-1 mice or mice orally administrated with n-3 PFUAs exhibited a significant reduction of anti-viral CD8+ T cell responses against an acute strain of lymphocytic choriomeningitis virus (LCMV). When LCMV-specific P14 CD8+ T cells carrying fat-1 gene (P14/FAT-1) were adoptively transferred into mice that were subsequently infected with LCMV, expansion of the cells was substantially suppressed. Similarly, when P14/FAT-1 cells were stimulated with LCMV gp33 peptide in vitro, their expansion was significantly reduced as compared to P14 cells. Collectively, our results indicate that n-3 PUFAs attenuate anti-viral CD8+ T cell responses against acute viral infection.
TSC1-mTOR-controlled metabolic-epigenetic crosstalk underlies DC control of CD8+ T cell homeostasis
Authors: Xiao Hui , Shi Lei , Chen Xia , Zang Aiping , Li Tiantian , Yang Jianbo
Published: January 2023

Dendritic cells (DCs) play pivotal roles in T cell homeostasis and activation, and metabolic programing has been linked to DC development and function recently. However, the metabolic underpinnings corresponding to distinct DC functions remain largely unresolved. Here we demonstrate a special metabolic-epigenetic coupling mechanism orchestrated by TSC1-mTOR for the homeostatic DC function. Specific ablation of TSC1 in the DC compartment (TSC1DC-KO) largely preserved DC development, but led to pronounced reduction in naïve and memory-phenotype CD8+ T cells, a defect fully rescued by the simultaneous deletion of mTOR or Raptor in DCs. Moreover, TSC1DC-KO mice were unable to launch efficient antigen-specific CD8+ T effector responses required for containing Listeria monocytogenes and B16 melanomas. Mechanistically, our data suggest that steady-state DCs tune down de novo fatty acid synthesis through TSC1 and divert acetyl-CoA for histone acetylation. Correspondingly, TSC1-deficiency elevated ACC1 expression and fatty acid synthesis in the steady-state DCs, leading to impaired epigenetic imprinting on selective genes such as MHC-I and IL-7. Remarkably, tempering ACC1 activity was able to avert cytosolic acetyl-CoA for histone acetylation and restoration of the gene expression program compromised by TSC1-deficiency. Taken together, our results uncover a crucial role for TSC1-mTOR in metabolic programing the homeostatic DCs for T cell homeostasis, and implicate metabolic-coupled epigenetic imprinting as a paradigm for DC specification.
Commensal polysaccharide-induced T cell communication and peripheral immune regulation
Authors: Zhou Julie Y , Jones Mark B , Alvarez Carlos A , Johnson Jenny L , Cobb Brian A
Published: January 2023

The increasing prevalence of inflammatory and autoimmune diseases across the world has been attributed in part to a shift in microbial exposure both in the exogenous environment and the endogenous microbiome. Here we identify and adopt a strategy used by a common commensal gut microbe, Bacteroides fragilis, to suppress inappropriate immune activation. Bacteroides fragilis produces a protective capsule containing polysaccharide A (PSA), which has been shown to suppress a host of inflammatory diseases in an IL-10-dependent manner. We have discovered that PSA is endocytosed, processed, and presented by APCs through MHCII and αβTCR engagement, and clonally expands CD4+FoxP3−CD25−CD45Rblo cells of an effector memory subset (RbloTEM). RbloTEM are capable of suppressing peripheral inflammation in way dependent on IL-10, however, they themselves do not produce the IL-10 necessary for suppression of disease. We show here that RbloTEM cells communicate directly with FoxP3+ Regulatory T cells, resulting in enhanced IL-10 production. Using novel FoxP3RFP/IL-10GFP dual reporter mice, we further demonstrate that this communication induces synergistic IL-10 expression specifically in Tregs, enhances Treg proliferation, and drives Treg-mediated immune inhibition by preferentially favoring the expansion of IL-10+ Tregs. Through promoting a robust and rapid Treg response, we show that the cytokine signature of PSA-responding RbloTEM cells and coordinating FoxP3+ Tregs can suppress mouse models of inflammatory disease, including HDM-induced asthma and EAE.
Visualising interaction of monocytes with red blood cells (RBC) sensitised with “clinically significant” antibodies
Authors: Dean Melinda M , Ji Ju , Gu YuanTong , Sauret Emilie , Flower Robert L
Published: January 2023

Background

Anti-RBC antibodies in plasma are regarded as clinically significant when they promote clearance of transfused RBC by monocytes/macrophages. To date, processes involved in the attachment or phagocytosis of sensitised RBC by monocytes have been investigated using light or fluorescent microscopy. We used scanning electron microscopy (SEM) to facilitate a closer examination of the interaction between monocytes and RBCs sensitised with antibodies of specificities classified as clinically significant.

Methods

Peripheral blood mononuclear cells (PBMC) were seeded onto glass coverslips, and non-adhered cells were removed after 1hr. Antigen-positive RBCs were opsonised with clinically significant antibodies (anti-A, anti-B, anti-D and anti-K) before addition to adhered monocytes. Anti-M was used as an example of an antibody regarded as non-clinically significant. Coverslips were then prepared for SEM which included glutaraldehyde fixation followed by post-fixation with osmium tetroxide. Cells were further dehydrated with ethanol and hexamethyldisilazane before coated with gold nanoparticles

Results

We observed the interaction between monocytes and sensitised RBCs using SEM. Clinically significant antibodies induced monocyte activation, evidenced by multiple morphological changes including cell elongation, extension of membrane ruffles and the projection of pseudopodia to capture sensitised RBCs.

Conclusions

Detailed characterisation of interaction between monocytes and sensitised RBCs provides insight into the processes associated with RBC removal. The model provides a basis for in-depth analysis of the mechanisms associated with antibody-induced RBC destruction in vivo.
CD31 acts as a checkpoint molecule and is modulated by FcγR-mediated signaling in monocytes
Authors: Butchar Jonathan P , Merchand-Reyes Giovanna , Robledo-Avila Frank , Buteyn Nathaniel , Gautam Shalini , Santhanam Ramasamy , Fatehchand Kavin , Partida-Sanchez Santiago , Tridandapani Susheela
Published: January 2023

Monocytes and macrophages express Fc-gamma Receptors (FcγR) that engage IgG immune complexes such as antibody-opsonized pathogens or cancer cells to destroy them by various mechanisms including phagocytosis. FcγR-mediated phagocytosis is regulated by the concerted actions of activating FcγR and inhibitory receptors such as FcγRIIb and SIRPα. Here, we report that another ITIM-containing receptor, Platelet Endothelial Adhesion Molecule-1 (PECAM1/CD31), regulates FcγR function, and is itself regulated by FcγR activation. First, qRT-PCR and flow cytometry analyses revealed that monocyte FcγR activation leads to significant downregulation of CD31 expression, both at the message level and at surface expression. Interestingly, the kinetics of downregulation between the two varied, with surface expression reducing earlier than the message. Experiments to analyze the mechanism behind this discrepancy revealed that loss of surface expression was due to internalization, which depended predominantly on the PI3 Kinase pathway. Finally, functional analyses showed that downregulation of CD31 expression in monocytes by siRNA enhanced FcγR-mediated phagocytic ability. Together, these results suggest that CD31 functions as a checkpoint receptor that could be targeted to enhance FcγR functions in antibody-mediated therapies.