bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycolipid-loaded Nanoparticles Harness iNKT cells for Tumor Immunotherapy
Authors: Shute Travis J , Dudley Elizabeth , Naranjo Joshua , Griffith Wendell , Nash Kelly , Leadbetter Elizabeth
Published: February 2023

Invariant natural killer T cells (iNKT) have a well-documented role in anti-tumor immunity through their release of proinflammatory cytokines and cytotoxic compounds. As iNKT cells can have direct and indirect killing effects on tumor cells, we propose a novel strategy for activating iNKT cells, via a PLGA nanoparticle delivery platform, to promote anti-tumor immune responses. Poly-lactic-co-glycolic acid (PLGA) nanoparticles can be reproducibly loaded with an iNKT cell glycolipid agonist, alpha-galactosylceramide (alpha-GalCer), and a model tumor associated antigen, ovalbumin (OVA). These dual-loaded PLGA nanoparticles rapidly activate iNKT cells in vivo to produce IFNgamma. Furthermore, in an in vivo model of melanoma, using B16F10-OVA cells, both prophylactic and therapeutic administration of nanoparticles containing alpha-GalCer and OVA led to decreased tumor cell growth and increased survival. We also show our nanoparticle therapy has synergistic potential with two immune checkpoint blockade therapies, anti-PD-1 and anti-CTLA-4, currently used in the clinic for human cancer patients. This novel delivery system provides a platform with tremendous potential to harness iNKT cells for cancer immunotherapy purposes against many different cancer types.
The mRNA-LNP platform’s lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory
Authors: Ndeupen Sonia , Qin Zhen , Jacobsen Sonya , Igyarto Botond Z
Published: February 2023

Vaccines based on mRNA-containing lipid nanoparticles (LNPs) are a promising new platform used by two leading vaccines against coronavirus disease in 2019 (COVID-19). Clinical trials and ongoing vaccinations present with very high protection levels with varying degrees of side effects. However, the nature of the reported side effects remains poorly defined. Here we present evidence that LNPs used in many preclinical studies are highly inflammatory in mice. Intradermal injection of these LNPs led to rapid and robust inflammatory responses, characterized by massive neutrophil infiltration, activation of diverse inflammatory pathways, and production of various inflammatory cytokines and chemokines. The same dose of LNP delivered intranasally led to similar inflammatory responses in the lung and resulted in a high mortality rate.

In summary, here we show that the LNPs used for many preclinical studies are highly inflammatory. Thus, their potent adjuvant activity and reported superiority comparing to other adjuvants in supporting the induction of adaptive immune responses could stem from their inflammatory nature. Furthermore, the preclinical LNPs are similar to the ones used for human vaccines, which could also explain the observed side effects in humans using this platform.
Co-immunization with adenosine deaminase rescues age-associated impairment of SARS-COV-2 synDNA vaccine-induced responses
Authors: Gary Ebony Nicole , Connors Jennifer , Tursi Nicholas J. , Warner Bryce , Bell Matthew , Cuismano Gina , Taramangalam Bhavani , Zhang Shiyu , Canziani Gabriela , Chaiken Irwin C. , Wootton Sarah , Weiner David , Kutzler Michele A. , Haddad Elias , Kobasa Darwyn
Published: February 2023

SARS-CoV-2 is responsible for a global pandemic claiming over 2 million lives and infecting over 100 million people. Elderly patients display increased COVID-19 morbidity and mortality. Vaccine candidates have been studied and deployed in the clinic. However, age-associated immune deficits are known to cause sub-optimal responses, and the longevity of vaccine-induced responses is under investigation. Germinal center follicular helper T cells (TFH) promote affinity maturation of B cell receptors and memory B cell differentiation and are defined by expression of adenosine deaminase-1 (ADA-1). We investigated the adjuvant properties of plasmid-encoded adenosine deaminase (pADA) in the context of a SARS-CoV-2 spike synDNA antigens. Young and aged mice were immunized with plasmid-encoded spike (pS) alone or co-immunized with pS and pADA and cellular and humoral responses were evaluated. When immunized with pS alone, aged mice had decreased spike-binding IgG and neutralization titers. However, young and aged animals co-immunized with pADA had similar humoral responses. We observed similar trends in serum antibody affinity as measured by SPR. pADA co-immunization also rescued age-associated decreases in spike-specific IFNy secretion in the spleens and lungs of co-immunized aged mice as measured by ELISpot. Finally, preliminary data analysis indicates that co-immunization with pADA significantly impacts viral load in a model of SARS-CoV-2 infection. These data suggest that pADA enhances antigen-specific cellular and humoral immunity in aged mice and supports further study of this molecule as an immunoadjuvant for vaccines targeting elderly populations.
An adjuvant strategy enabled by modulation of the physical properties of fungal mannans elicits pan-coronavirus reactive anti-SARS-CoV-2 Spike antibodies
Authors: Borriello Francesco , Spreafico Roberto , Poli Valentina , Shrock Ellen , Chou Janet , Barrett Nora A. , Lacanfora Lucrezia , Franco Marcella E. , Marongiu Laura , Iwakura Yoichiro , Pucci Ferdinando , Kruppa Michael D. , Ma Zuchao , Lowman Douglas W. , Ensley Harry E. , Nanishi Etsuro , Saito Yoshine , O’Meara Timothy R. , Seo Hyuk-Soo , McGrath Marisa E. , Logue James , Haupt Robert E. , Dhe-Paganon Sirano , Dowling David J. , Frieman Matthew , Elledge Stephen J. , Levy Ofer , Irvine Darrell J. , Williams David L. , Zanoni Ivan
Published: February 2023

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate long-lasting adaptive immunity. While it is known that PRRs detect unique chemical patterns associated with invading microorganism, if and how the physical properties of PRR ligands influence development of the immune response is largely overlooked. Through the study of fungal mannans we present data that put the physical form of PRR ligands at the center of the process that determines the outcome of the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both periphery and dLN. When combined with SARS-CoV-2 Spike, this formulation elicits neutralizing anti-SARS-CoV-2 antibodies that cross-react with pathogenic coronaviruses. Thus, the physical properties of fungal ligands can be harnessed for rational adjuvant design and vaccine development.
Optimizing immunoassay-based selection of donors for COVID-19 convalescent plasma
Authors: Van Ert Hanora A , Rogers Kai , Bohan Dana , Han Chanze , Dempewolf Spencer , Sevcik Kristina , Ruggio Natalie , Rojas-Chavez Anthony , Boyt Devlin , Pentella Michael , Jackson Brooks , Merrill Anna , Knudson Michael , Maury Wendy , Haim Hillel
Published: February 2023

COVID-19 convalescent plasma (CCP) is currently administered to hospitalized COVID-19 patients under an EAU by the FDA. CCP units are selected by commercially available ELISA quantification of anti-SARS-CoV-2 (SARS2) antibodies under the rationale that higher levels of anti-SARS2 antibodies correlate with higher neutralizing capabilities and thus greater efficacy. The validity of this selection process has yet to be investigated. We analyzed serum samples from 60 convalesced donors with PCR-confirmed COVID-19. Samples were analyzed using commercial SARS2 ELISAs (DiaSorin, Euroimmun, Ortho, and Roche) and a cell-based enzyme-linked immunosorbent assay (cbELISA) that detects antibodies against the native conformation of the SARS2 spike glycoprotein (GP). Sera were also tested for neutralizing activity against wild-type SARS2 and a pseudovirus expressing the SARS2 GP. Immunoassay and neutralization values were compared to determine the precision of each immunoassay to predict differing levels of neutralizing activity, and ability of each immunoassay to identify high neutralizing activity. Results of immunoassays that apply SARS2 GP as the antigen (DiaSorin, Euroimmun, Ortho, and cbELISA) correlated well with neutralization titers measured by wild-type SARS2 or SARS2 GP-pseudotyped virus as compared with assays using other SARS2 antigens (Roche; N protein). Differences in precision of these assays were evident when samples within the top quintile or decile of immunoassay values were analyzed, with DiaSorin and Ortho assays showing superior performance in predicting neutralizing capability. We hope this approach will assist in the selection of highly neutralizing CCP units that confer greater clinical efficacy.
EGR2 and EGR3 function in a cell-intrinsic and partially redundant manner to suppress the accumulation of B1a cells and CD21low CD23low age-associated B cells
Authors: Masle-Farquhar Etienne , Peters Timothy , Oakes Christopher , Reed Joanne , Goodnow Christopher C
Published: February 2023

Egr2 and Egr3 genes encode early growth response (EGR) transcription factors, act as immediate early genes following B cell receptor (BCR) signalling and are highly up-regulated in anergic mouse B cells. Anergy preserves microbial epitope responsiveness from a finite pool of pre-immune B cells, but is also reversible – creating a risk of autoimmune disease. Pathological proliferation of self-reactive B cells can also cause chronic lymphocytic leukemia (CLL), 3.8% of which harbour somatic missense EGR2 mutations resulting in loss- or change-of-function and correlated with poor prognosis. We analysed B cells in mice lacking one or both alleles of Egr2 and/or Egr3 and show that Egr2 and Egr3 deletion cause the cell-intrinsic accumulation in spleen, blood and bone marrow of populations enriched for self-reactive BCRs: B1a and CD21low CD23low age-associated B cells. Global single-cell RNA profiling of these expanded populations in vivo demonstrated their differential expression of genes involved in their survival and maintenance. We used chromatin immunoprecipitation sequencing (ChIP-Seq) to show that several of these genes are direct EGR2 transcriptional targets in human CLL. This is the first report on the roles of Egr2/3 in B cells and we demonstrate herein that Egr2 and Egr3 transcription factors are crucial in suppressing the accumulation of B1a and age-associated or atypical memory B cells.
The role of berberine, a clinically relevant plant-derived alkaloid, in T-cell mediated immunosuppression
Authors: Vita Alexandra Adorno , Pullen Nicholas A.
Published: February 2023

Preliminary research revealed a novel function of berberine (BBR), a clinically relevant plant-derived alkaloid, as a suppressor of follicular T helper (Tfh) cell proliferation in secondary lymphoid organs of BBR-treated mice that underwent immunization for arthritis induction in a collagen induced arthritis (CIA) mouse model, a common murine model for rheumatoid arthritis. As the interaction between Tfh cells and B cells is crucial for thymus-dependent humoral responses, this raises concern that the prolonged use of berberine could suppress the generation of such responses. In our current research we explored the effects of BBR in vitro on the differentiation of Tfh cells from naïve CD4+T cells (>95% pure) by examining the expression of the key Tfh cell surface molecules CXCR5, ICOS, and PD1 following BBR treatment. Such molecules are crucial for Tfh cell effector function. The treatment of BBR at 0.25 mg/mL, 0.50 μg/mL, and 1 mg/mL significantly reduced the expression of both CXCR5 (p <0.01) and ICOS (p < 0.01), but not PD1. It is important to note that in preliminary dose-response experiments, 0.50 mg/mL was the highest dose of BBR that did not significantly lead to cell death, indicating that the decreased expression of CXCR5 and ICOS at 0.25 mg/mL and 0.50 mg/mL are likely not due to cytotoxic effects. In the future, we plan to elucidate the mechanism by which BBR inhibits the expression of CXCR5 and ICOS by examining key cell signaling pathways that lead to Tfh activation and differentiation, and ultimately to CXCR5 and ICOS expression, following BBR treatment.
Expansion of T follicular regulatory cells by OX40L-JAG1 co-signaling ameliorates experimental lupus nephritis
Authors: Kumar Prabhakaran , Setty Suman , Epstein Alan L , Chiba Shigeru , Prabhakar Bellur S
Published: February 2023

Antinuclear autoantibodies (Nabs) promote tissue inflammation involving multiple organs including the kidneys in Systemic Lupus Erythmatosus (SLE). T-follicular helper (TFH) cell-dependent B-cell response plays an essential role in lupus nephritis (LN). TFH and B-cells receive mutual help in the germinal centers (GCs) to enable high-affinity autoantibody production. T follicular regulatory cells (TFR) cells, a specialized subset of CD4+Foxp3+Tregs, suppress GC formation and TFH-dependent B-cell response. Previously, we have shown that the expansion of functional Tregs using soluble OX40L and JAG1 proteins suppressed experimental autoimmune diabetes and thyroiditis. However, unlike other autoimmune diseases, the role of Tregs in lupus remains unresolved. Here, we show that NZBWF1/j mice with severe proteinuria and higher anti-dsDNA IgG levels had increased GC-TFH, GC-B cells, and GC-TFH/GC-TFR ratio in their renal lymph nodes compared to mice with no proteinuria. While total Tregs were significantly higher in mice with proteinuria, GC-TFRs were significantly reduced. Intriguingly, OX40L-JAG1 treatment significantly increased Tregs, GC-TFRs, and reduced GC-TFH/GC-TFR ratio. Periodic OX40L-JAG1 treatment resulted in a significant reduction in GC-B-cells, anti-dsDNA IgG level, proteinuria and kidney pathology, and increased survival. Mechanistic studies showed increased PD1 expression in Tconv cells, Tregs, TFH, and TFR cells, and PD-L1 expression in GC-B-cells suggesting a key role for PD1 signaling in the observed protective effect. Our results show that OX40L-JAG1 treatment expanded TFRs which inhibited GC-B cell activation and NAb production, proteinuria and LN.
T-cell receptor repertoire of mice with organ-specific autoimmunity resulting from a partial defect in T cell negative selection and dendritic cell enhancement
Authors: Yin Mianmian , Smith Jennifer A , Chou Marissa , Chan Jackie , Hu Yongmei , Lowell Clifford A , Gould Douglas B , Anderson Mark S , DeFranco Anthony L
Published: February 2023

Previously, we reported a new genetic model of spontaneous autoimmune disease in which mice with a hypomorphic mutation of Aire (AireGW/+) combined with deletion of Lyn in dendritic cells develop autoimmune uveitis with a 50% penetrance. Here we demonstrate that a key autoantigen in this model is interphotoreceptor retinoid-binding protein (IRBP) as AireGW/+ Lyn−/− IRBP−/− mice failed to develop uveitis. The expansion of CD4+ T cells recognizing amino acids 271–290 (“P2”) of IRBP correlated strongly with autoimmune disease in mice with intact IRBP. To study the role of these T cells, we did single-cell RNA-seq with TCR sequencing for P2-specific CD4+ T cells isolated from eye-draining lymph nodes (LN) of AireGW/+ Lyn−/− mice with and without uveitis. Both mice showed an expansion of these T cells in eye-draining LNs, but it was greater in mice with disease. A remarkable clonal diversity of TCRs was seen, with some sharing of TCR Vα or Vβ sequences. Expansions of particular T cell clonotypes were more evident in LN of mice with disease, and even greater in the retinas of mice with disease. We reconstituted several P2–specific TCR clonotypes in a T cell hybridoma line and verified their specificity. One of the TCR clonotypes was expressed in developing thymocytes by retroviral transduction of bone marrow stem cells from Rag2−/− mice and transplanted into WT or AireGW/+ recipient mice. Remarkably, the small residual expression of IRBP in the thymus of AireGW/+ mice was sufficient to induce substantial negative selection of thymocytes with this TCR clonotype. Thus, deficiency of negative selection of P2 tetramer–specific TCR promotes the development of uveitis in AireGW/+ Lyn−/− mice, even if negative selection is only partly compromised.
ICOS is critical for T follicular regulatory cell differentiation
Authors: Panneton Vincent , Mindt Barbara , Bouklouch Yasser , Chang Jinsam , Witalis Mariko , Li Joanna , Stancescu Albert , Bouchard Antoine , Bradley John , Randall Troy D , Fritz Jörg H , Suh Woong-Kyung
Published: February 2023

The Inducible Costimulator (ICOS) is a T cell costimulatory receptor critical for humoral immunity. ICOS-deficient patients suffer from recurrent infections due to lack of protective antibodies. However, some patients also display signs of antibody-mediated autoimmunity. These findings may reflect a dual role of ICOS: facilitating the differentiation and function of T follicular helper (Tfh) and T follicular regulatory (Tfr) cells. While Tfh cells are known to provide help to B cells to produce high affinity antibodies, the main role of Tfr cells seems to be preventing autoantibody generation. Using Foxp3-cre-mediated ICOS knockout (ICOS FC) mice, we show that T regulatory (Treg)-specific ICOS deletion drastically reduces the number of Tfr cells without altering Treg cell numbers. Single cell RNA sequencing further revealed shifts in transitory Tfr precursor populations in immunized ICOS FC mice. Importantly, we observed a lowered ratio of antigen-specific germinal center B (GCB) cells and increased anti-nuclear antibodies in ICOS FC mice, suggesting a rise in autoreactive GCB cells. We also noted variations in isotype composition of total and virus-specific antibodies in infected ICOS FC mice. Mechanistically, our data suggests that ICOS could promote the Treg-to-Tfr transition by regulating CXCR5 expression. Thus, our study demonstrates that ICOS is critical for Tfr cell generation and supports the role of Tfr cells in preventing generation of autoantibodies during germinal center reactions.
AIM2 in regulatory T cells restrains autoimmune diseases
Authors: Chou Wei-Chun , Guo Zengli , Wan Yisong , Ting Jenny P-Y
Published: February 2023

The inflammasome comprised of an innate immune receptor/sensor, pro-caspase-1, and a common adaptor molecule, ASC (apoptotic speck-containing protein with a CARD) initiates innate defense and inflammatory response by activating caspase-1 and pyroptotic cell death in myeloid cells. Consistent with their pro-inflammatory function, caspase-1, ASC and NLRP3 were known to exacerbate autoimmunity during experimental autoimmune encephalomyelitis (EAE) by enhancing IL-1b and IL-18 secretion in myeloid cells. Here we reveal an unexpected function of a DNA-binding inflammasome receptor, AIM2 (Absent in Melanoma 2), in T regulatory cells (Tregs) to restrain two models of autoimmune disease, including experimental autoimmune encephalomyelitis and T cell-mediated colitis, by studying whole-body and Treg-specific Aim2-deficient mice. AIM2 is highly expressed by human and mouse Tregs, with its expression induced by TGF-b and its promoter occupied by transcription factors associated with Tregs, including Runx1, Ets1, Bcl11b and CREB. Moreover, we demonstrate that AIM2 promotes the stability of Tregs during inflammation in lineage tracing mice. RNA-seq, biochemical and metabolic analyses revealed that AIM2 attenuates Akt-phosphorylation, mTOR, Myc and glycolysis, but promotes lipid oxidative phosphorylation in Tregs. Mechanistically, AIM2 interacts with the RACK1/PP2A-phosphatase complex to restrain Akt-phosphorylation. While AIM2 is generally accepted as an inflammasome effector in myeloid cells, this report unveils a T cell-intrinsic role of AIM2 in restraining autoimmunity by diminishing Akt-mTOR signaling and altering immune-metabolism to enhance Treg stability.
Helios expression in CD4+Foxp3− T cells
Authors: Tison Kole , Penna Vinay , Skadow Mathias , Oguz Cihan , Lack Justin , Thornton Angela M , Shevach Ethan M
Published: February 2023

The transcription factor Helios is expressed in the majority of Foxp3+ Treg cells, where it is necessary for the maintenance of self-tolerance. Mice with a Foxp3-specific deficiency in Helios develop systemic immune activation with increased T follicular helper (Tfh) and T helper 1 (Th1) effector responses. Notably, mice with a CD4-specific deletion of Helios do not exhibit autoimmunity, pointing to an additional role for Helios in CD4+ T cells. In fact, Helios is expressed in a minor population of conventional CD4+ T cells and its expression is upregulated upon activation of CD4+ T cells, yet the role of Helios in these cells remains unknown. We generated TCR Tg mice with a CD4-specific Helios deletion and have shown that Helios deficient naive CD4+ T cells are fully able to differentiate into Th1, Th2 and Tfh effector cells in vivo. Helios deficient CD4+T cells show no defect in expansion or the production of cytokines. However, Helios deficiency increased the propensity of naive T cells to differentiate into antigen-specific pTregs under inflammatory as well as tolerizing conditions and attenuated the induction of a memory response. To further characterize CD4+Foxp3−Helios+ cells, Next-generation sequencing (NGS) analysis was performed. This analysis has yielded evidence for a heterogenous population that is comprised of Tfh, Tmem, iNKT and a population of cells that have Treg-like features. In vitro activation assays and qPCR have validated differentially expressed cytokines between Helios+ and Helios− CD4+ T cell populations, in agreement with the NGS data. Together, these findings suggest that Helios may play a role in stabilizing effector function.
Kidney macrophages in human lupus nephritis and mouse models express conserved inflammatory and remodeling gene programs and localize to the same anatomic sub-compartments.
Authors: Hoover Paul , Peters Michael , Lieb David , Jones Tony , Mishra Rakesh , Hacohen Nir , Davidson Anne
Published: February 2023

Tissue macrophages promote kidney homeostasis through tightly regulated initiation, maintenance, and resolution of tissue repair. In lupus nephritis (LN), kidney macrophages may drive damage in glomeruli and other kidney sub-compartments leading to proteinuria and fibrosis. Recent single-cell RNA sequencing work uncovered 4 novel kidney macrophage subsets in LN patient biopsies: M2-like “reparative”, “inflammatory”, “phagocytic”, and “resident” subsets. But how macrophage gene programs translate into cellular functions driving proximal tissue damage has been difficult to study because we do not fully understand how mouse models recapitulate human LN. Here, we used single cell RNA sequencing to identify kidney macrophage subsets conserved in human and mouse models of LN. We compared the transcriptomes of macrophages collected from human LN patients at peak clinical disease to those from 4 mouse strains at early and peak clinical disease. We discovered that human “reparative”, “inflammatory”, “phagocytic” subsets were conserved across species. In particular, human “reparative” and mouse resident macrophages shared inflammatory and remodeling programs, expressed interferon stimulated genes, and localized to the same kidney sub-compartments. In nephritic mice, this subset converted to a mixed M1-inflammatory/M2-repair phenotype also observed in humans, thus identifying potential conserved programs driving tissue damage in mice and humans. These findings strongly support shared roles for conserved human and mouse macrophages. Critically, they open a new path utilizing mice to study how conserved programs translate into cellular functions driving proximal tissue damage.
Disease-associated dominant negative CARD11 mutations perturb the CARD11 Opening step during T cell receptor-induced activation of NF-κB and JNK2
Authors: Bedsaul Jacquelyn Renee , Pomerantz Joel
Published: February 2023

CARD11 is an oligomeric, multidomain scaffold that mediates lymphocyte activation. Upon antigen receptor engagement, CARD11 becomes active and signals through NF-κB, JNK, and mTOR to elicit the adaptive immune response. The CARD11 signaling cycle consists of at least four stages beginning with the Opening step, followed by Cofactor Recruitment, Enzyme Activation, and Complex Disassembly. Primary Immunodeficiency Diseases (PIDDs) have been attributed to both autosomal dominant and recessive CARD11 variants. Combined Immunodeficiency with atopy is caused by the inheritance of a single dominant negative CARD11 allele, but the mechanistic basis for the dominant negative effects is not well understood. In this study, we investigated the mechanistic basis for dominant negative CARD11 mutations in T cells by assessing the activity of loss-of-function variants in the presence and absence of co-expressed wild-type CARD11. In the absence of wild-type CARD11, the variants perturbed the Opening step and downstream signaling steps, including recruitment of Bcl10, HOIP, and MALT1 to CARD11, Bcl10 polyubiquitinylation, MALT1 protease activity, NF-κB and JNK2 activation, and IL-2 production. When the variants were co-expressed with wild-type CARD11, a range of dominant negative effects on NF-κB activation was observed, which correlated with the ability of the CARD11 oligomer to open, recruit cofactors, and mediate IL-2 production. We conclude that dominant negative mutations interfere with the CARD11 Opening step and provide insights into the manifestation of PIDDs caused by the inheritance of dominant and recessive CARD11 alleles.
The deficiency of Cbl family in human elicits heightened T-dependent immune activation with augmentation of TFH responses
Authors: Lee Kyungwoo , Kim Sun jung
Published: February 2023

T follicular helper (TFH) cells are functional subset CD4+ T cells in providing help to B cells and aberrant expansion of TFH cells is observed in systemic lupus erythematosus (SLE), which suggests a global defect in the maintenance of T and B cell tolerance. The mechanisms underlying uncontrolled TFH cells development and differentiation in SLE pathogenesis is fragmentary understand. Casitas B-lineage lymphoma (Cbl) family is an E3 ubiquitin ligase and has been implicated in SLE pathogenesis as a potent regulator in activating the immune cells and maintaining peripheral tolerance. The deficiency of cbl-b mice spontaneously develops autoimmunity and is highly susceptible to develop pathogenic auto-antibodies of autoimmune diseases. Through CRISPR KO system, we identified the deficiency of cbl/cbl-b in human naïve CD4+ T cells induces the TFH expansion in T cell stimulation with Activin A & IL12. Indeed, higher levels of chemokine (C-X-C motif) ligand 13 (CXCL13) which is constitutively produced in germinal center (GC) TFH cells were detected. Functionally, cbl/cbl-b KO TFH cells are more potent B cell activators compared to cbl/cbl-b competent TFH cells. There was increased plasmablast differentiation which secrete IgG. These data suggest that the deficiency of Cbl family in human elicits heightened T-dependent immune activation with augmentation of TFH responses, thus regulating humoral immune responses and the susceptibility to SLE in human. This project will advance our understanding of the mechanism of activation and maintenance of TFH cells in lupus and dissect possible avenues of therapeutic intervention in disease.
Lymphocyte phenotype in a mouse model of 22q11.2 deletion syndrome
Authors: Crockett Alexis Mary , Benallegue Nail , Adelman Philippa , Gur Raquel , Sullivan Kathleen E , Anderson Stewart A , Alvarez Jorge Ivan
Published: February 2023

Mice modeling the hemizygous deletion of chromosome 22q11.2 (22qMc) have been utilized to address various clinical phenotypes associated with the disease, including cardiac malformations, altered neural circuitry and behavioral deficits. Yet, the status of the immune system, an important clinical concern among 22q11.2 deletion syndrome (22qDS) patients, has not yet been addressed in this model despite the well-known effects of the immune system in regulating body homeostasis. While infancy and early childhood in 22qDS are associated with deficient T cell numbers due to thymic hypoplasia, which can be severe in a small subset of patients, clinical studies suggest a normalization of the T cell counts by adulthood. For the first time, we have studied the immune system in 22qMc and find that adult 22qMc do not exhibit thymic hypoplasia, immunodeficiencies of lymphoid organs, or altered T cell activation. Furthermore, deletion of the 22q11.2 homologous region does not affect T cell differentiation. Thus, this model accurately reflects the normalization of the T cell compartment in adulthood, as has been reported in human 22qDS. Altogether, our data suggest that 22qMc may serve as novel model to address experimental and translational aspects of immunity in adult 22qDS.
Borrelia burgdorferi peptidoglycan triggers inflammatory responses in Lyme arthritis
Authors: Rouse Joe , Wahhab Amanda , Danner Rebecca , Jutras Brandon , Lochhead Robert
Published: February 2023

Background:

Lyme arthritis (LA) is the predominant late-stage manifestation of Lyme disease, caused by the tick-borne spirochete Borrelia burgdorferi (Bb). Bacterial peptidoglycan (PG) is abundant in inflamed joints of LA patients, even months to years after antibiotic therapy, but the role of PG in LA pathogenesis is unclear.

Methods:

Primary murine macrophages and synovial fibroblasts, two cell types abundant in LA synovial tissue, were stimulated for 24–72 hours with IFNγ, PG, or both. Inflammatory responses were determined by flow cytometric analysis of CD80, CD86, MHC-II, CD206, ICAM1, and IL-15R.

Results:

Macrophage stimulation with PG+IFNγ for 24 hours led to up-regulation of costimulatory molecules CD80/CD86, compared with PG or IFNγ alone, consistent with classical (M1) activation. In contrast, stimulation for 72 hours resulted in marked suppression of MHC-II and upregulation of CD206, consistent with M2 macrophage polarization. When stimulated with PG+IFNγ for 24 hours, synovial fibroblasts upregulated activation markers ICAM1 and IL-15R, whereas these activation markers were only partially up-regulated when stimulated with PG or IFNγ alone. Current experiments include analyzing cytokine secretion by these cells at different time points.

Conclusion:

Both innate immune and stromal cells abundant in joint tissue are responsive to stimulation with PG, but these responses may be either pro-inflammatory or anti-inflammatory, depending on cell type and duration of stimulation. This highlights the complex relationship between PG and the cellular environment of infected joints. Future experiments will identify relevant immune response pathways, such as TLR2 and NOD2 signaling, in shaping these responses.
PI3Kd coordinates transcriptional, epigenetic and metabolic changes to promote effector CD8 T cells at the expense of memory
Authors: Cannons Jennifer Leigh , Villarino Alejandro V , Kapnick Senta M , Preite Silvia , Shih Han-Yu , Gomez-Rodriguez Julio Washington , Reilley Julie , Huang Bonnie , McBain Ian , Wu Tuoqi , Su Helen C , McGavern Dorian B , O’Shea John J , McGuire Peter J , Uzel Gulbu , Schwartzberg Pamela L
Published: February 2023

Patients with Activated-PI3Kd Syndrome (APDS) present with sinopulmonary infections, lymphadenopathy and CMV and/or EBV viremia, yet why patients fail to clear certain viral infections remains poorly understood. Using APDS patient samples and a mouse model (Pik3cdE1020K/+ mice), we demonstrate that, upon activation, Pik3cdE1020K/+ CD8+ T cells exhibit exaggerated features of short-lived effectors both in vitro and post-viral infection, associated with increased Fas-mediated apoptosis due to sustained phosphorylation of FoxO1 and derepression of FasL. In addition, Pik3cdE1020K/+ CD8+ T cells exhibit enhanced mTORC1 and c-Myc signatures; metabolic perturbations; and reorientation of their chromatin landscape. Conversely, Pik3cdE1020K/+ CD8+ T cells failed to sustain expression of proteins critical for maintenance of long-lived memory cells, including TCF1. Strikingly, activated Pik3cdE1020K/+ CD8+ T cells exhibit altered transcriptional and epigenetic circuits characterized by a pronounced IL-2/STAT5 signature associated with heightened IL-2 responses that prevented differentiation to memory-like cells in the presence of IL-15. Our data position PI3Kd as a central driver integrating multiple signaling circuits that promote terminal CD8+ T cell effector differentiation at the expense of memory and long-lived T cell responses.

This work was funded in part by the Intramural Research Program of NIAID, NIH.
Dendritic Cells Supply CD4+ T Cells With L-arginine
Authors: Crowther Rebecca R , Schmidt Stephanie M , Zhao Junfang , McKell Melanie C , Setchell Kenneth D , Qualls Joseph E
Published: February 2023

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is responsible for over 1 million deaths each year. Mycobacteria-infected dendritic cells (DCs) migrate to the lymph node to initiate adaptive immune priming, which is vital to antimycobacterial immunity. This response is intimately tied to nutrient availability – especially the amino acid L-arginine (L-ARG), metabolism of which is altered in TB patients. We have characterized a pathway utilized by immune cells to synthesize L-ARG. Loss of L-ARG synthesis in CD11c+ cells, which includes DCs, results in increased mycobacterial burden following infection in mice. To characterize the role of this pathway in DCs, we developed a co-culture system with mycobacterial-specific CD4+ T cells and bone marrow derived DCs. Using CD4+ T cells and DCs with differing capabilities of L-ARG synthesis, we found 1) DC L-ARG synthesis supports CD4+ T cell proliferation and 2) activated T cells contain DC-derived L-ARG. We hypothesize DCs “share” synthesized L-ARG to support CD4+ T cell activation when L-ARG is limiting. Our data suggest nutrient availability as a 4th signal – following antigen presentation, co-stimulation, and cytokine receptor ligation – required for T cell activation. This work expands the current model of DC-T cell interactions and provides insight into the effects of nutrient availability in immune cells.
CD40-CD40L interaction is critical in mounting host immunity against m-CoV Mouse Hepatitis Virus induced neuroinflammatory demyelination
Authors: Sarma Jayasri Das , Saadi Fareeha , Chakravarty Debanjana , Kumar Saurav , Kamble Mithila , Khan Reas , Shindler Kenneth S
Published: February 2023

Chronic progressive neuroinflammatory disease multiple sclerosis (MS) is characterized by loss of neuronal functions resulting from demyelination with or without axonal degeneration. Infiltration of T lymphocytes and activation of microglia and their interplay are the major pathophysiological events leading to neurodegeneration in MS. Our studies in Mouse Hepatitis Virus (MHV) induced neuroinflammatory model demonstrated a protective role of CNS infiltrating CD4+ T cells. In the absence of CD4+ T cells, microglial activation fails to resolve, and mice are more susceptible to acute poliomyelitis and chronic demyelination with axonal bulbar vacuolation. Our studies also revealed that CD40L (expressed on CD4+ T cells) upregulates upon MHV infection but is downregulated in CD4−/− mice CNS. This led to a further delineation of the CD4-microglia nexus at the molecular level using CD40L−/− mice. Results showed that the absence of CD40L renders mice highly susceptible to MHV infection due to reduced microglia/macrophage activation and significantly dampened effector CD4+ T recruitment to the CNS at the acute-adaptive bridging phase (day 7–10 p.i.) of inflammation. Moreover, CD40L−/− mice exhibited severe demyelination, axonal loss, and persistent poliomyelitis at the chronic phase of infection, highlighting the protective role of CD40-CD40L in MHV induced neuroinflammatory demyelination. Together, these studies highlight that migration of peripheral T cells and their interaction with microglia via CD40-CD40L is essential to eliminate the virus and provide long-term neuroprotection. These findings can lead to designing potential therapeutic interventions against MS, targeting CD40-CD40L interaction.
Eos differentially regulates Th1 versus Tfh differentiation through feed-forward propagation of the IL-2/STAT5 signaling pathway
Authors: Read Kaitlin , Sreekumar Bharath , Powell Michael Duane , Jones Devin M , Pokhrel Srijana , Saadey Abbey , Collins Patrick , Ghoneim Hazem , Oestreich Kenneth J
Published: February 2023

The Ikaros zinc finger (IkZF) protein Eos is a transcription factor with known roles in driving regulatory T cell (Treg) suppressive function. However, whether it regulates the differentiation of effector CD4+ T cell subsets is currently unknown. Here, we find that Eos deficiency compromises both expression of the T helper 1 (Th1) differentiation program and effector function in vitro and in vivo, including production of IFN-gamma and TNF-alpha. Despite historical associations between IkZF factors and changes in gene expression via the alteration of chromatin structure, our ATAC-seq analysis indicated that there were minimal changes in chromatin accessibility between wildtype and Eos-deficient cells cultured under Th1-polarizing conditions. Rather, we find that IL-2 signaling promotes the STAT5-dependent induction of Eos expression, and that Eos both interacts with STAT5 and supports tyrosine phosphorylation-mediated STAT5 activation. Thus, STAT5 enrichment is reduced at Th1 target genes in the absence of Eos, including Prdm1, which encodes Blimp-1, an antagonist of the T follicular helper (Tfh) cell transcriptional program. Consequently, we find that Eos-deficient T cells also exhibit increased expression of signature Tfh genes. Together, our findings indicate that modulation of IL-2/STAT5 signaling represents a novel mechanism by which Eos regulates the Th1 and Tfh gene programs. Given the important role for IL-2/STAT5 signaling in both the positive and negative regulation of other T helper cell subsets, it will be of interest to determine whether this mechanism is conserved across additional CD4+ T cell populations.
TET-mediated regulation of effector CD4 T cells
Authors: Johnson Jordan T , Issuree Priya , Butler Noah
Published: February 2023

Functionally distinct subsets of pathogen-specific CD4 helper T cells, including T follicular helper (Tfh) and T helper type 1 (Th1) subsets, are critical for orchestrating the induction and maturation of innate and adaptive immune responses. Proper genetic programming of Tfh and Th1 differentiation is required for regulating immune responses to clear primary infections and promote long-lived memory CD4 T cell-mediated protection against pathogen re-exposures. Genetic approaches to study CD4 T cell differentiation have been largely focused on transcriptional programming, but gene regulation is also critically tied to epigenetic modifications, which represents a major knowledge gap in our understanding of CD4 T cell biology. Ten-eleven-translocation (TET) family enzymes facilitate DNA demethylation to influence chromatin accessibility and promote gene expression. Prior work has shown that TET2 activity has been linked to optimal Th1 responses in vitro. TET1 and TET3 proteins mediate Cd4 locus demethylation and are required for stable CD4 expression during thymocyte development. However, the precise role of either TET1 or TET3 activity to mediate CD4 T cell fate and function in vivo has not been directly investigated. Thus, we hypothesized that TET1 and TET3 activity in peripheral, mature CD4 T cells is required for regulating T helper cell differentiation and function. We used conditional genetic and chimeric approaches, coupled with robust systems of infectious disease immunology, to directly test our hypothesis. Our data show substantial CD4 T cell developmental skewing towards Tfh fates in the absence of specific TET family members. This suggests that TET1 and TET3 enzymes play a role in governing Tfh and Th1 CD4 cell fate.
Inhibition of GATA3 and Th2 cytokines expression in type 2 high asthmatics by a small molecule targeting RNA-binding protein HuR
Authors: Fattahi Fatemeh , Ellis Jason S , Sylvester Michael , Bahelda Kristin , Hietanen Sam , Correa Luis O , Lugogo Njira , Atasoy Ulus
Published: February 2023

RNA-binding protein human antigen R (HuR) is a master regulator of gene expression in human pathophysiology. Our animal studies in the past showed multiple critical roles for HuR in regulating proinflammatory markers in CD4+ T cells. We hypothesized HuR plays an important role in expression of GATA3 and Th2 cytokines in type 2 high asthma and its inhibition impairs these Th2 inflammatory responses. Peripheral CD4+ T cells were isolated from 26 healthy controls and 32 type 2 high asthmatics (blood eosinophil ≥300 cells/μl or FeNO levels ≥25ppb) and stimulated with anti-CD3/CD28 for 4 days, followed by determination of GATA3 and cytokines expression by flow cytometry and LEGENDplex. HuR levels were quantitated by Western blot, flow cytometry, and qPCR. Inhibition of inflammatory responses was evaluated using a cumarin-derived small molecule named CMLD-2 (a HuR-specific inhibitor). Actinomycin D treatment was used to measure mRNA decay rates of targeted HuR mRNAs. HuR protein levels in CD4+ T cells was significantly higher in type 2 high asthmatics compared to healthy controls. The expression and secretion of Th2 cytokines were significantly higher in asthmatics compared to controls. Our further mRNA and protein studies in type 2 high asthmatics showed reduced expression of GATA3 and Th2 cytokines after CMLD-2 treatment. There was also a significant reduction in gata3 mRNA stability with CMLD-2 treatment, compared with vehicle in the asthmatics. Our study is the first to demonstrate the role of RNA-binding protein HuR in type 2 high asthmatics and that interfering with HuR action using CMLD-2 ameliorates GATA3 and Th2 cytokines production. This data suggests HuR may serve as a novel and powerful therapeutic target to treat type 2 high asthmatics.
Student Application of Immunology Concepts and Mechanisms Through Test Based Learning on Canvas
Authors: Swanson-Mungerson Michelle Anne , Laddaga Richard , Szul Martin J. , Volin Michael V
Published: February 2023

Application of immunological concepts is a major challenge for students, especially during a pandemic when most students are learning remotely. Many students memorize the details, but often fail to critically process the information to form a comprehensive understanding of how the immune system works. To address this concern, we developed a “test-based learning” approach to engage students and reinforce important concepts. We converted our immunology lecture-based cases to a quiz format that was presented through our Learning Management Software (Canvas). This approach provides students the opportunity to remotely apply the information presented in lecture either independently or in a small group format through Teams. These cases engaged students to discuss the important aspects of the case and the immunological mechanisms involved. If students struggled, Faculty Members were available via Teams video chat or email to answer questions. We realized that this approach allowed for increased student participation, more active learning, and increased questions to faculty members in comparison to asking questions during lecture. A final advantage of this method is that faculty were able to quickly assess if students mastered key concepts by analyzing students’ scores. If faculty identified deficiencies, there was ample time to address misconceptions before an exam. This approach can be easily modified and used in any course that uses Learning Management Software (e.g. Canvas or Blackboard). Furthermore, this approach is not only applicable to medical education, but could be used at both the undergraduate and graduate levels.
Using the H5P digital platform as an active learning tool to build content-based critical thinking skills in an undergraduate immunology course
Authors: Carr William H
Published: February 2023

Recent studies have demonstrated that active learning or learning by doing has a more significant positive impact on learning outcomes than reading or watching videos. However the availability of free and open source tools for creating these active learning exercises has been limited by the requirement of computer programing skills. With the development of H5P technology, an open source, on-line tool, faculty and even undergraduate students can create interactive exercises that facilitate the learning process and promote formative learning. H5P is a plugin for existing publishing systems that enables the system to create interactive content, such as multiple-choice quizzes that provide feedback in real-time on quiz responses. Here are provided scaffolded assignments to enable implementation of this approach in an undergraduate immunology course that is taught online asynchronously. This activity was implemented at Medgar Evers College, City University of New York (Brooklyn, NY) in 2020–2021. These assignments enabled students to design interactive formative assessments, i.e., multiple choice questions, using the H5P digital platform (www.h5p.org). Results of undergraduate student surveys on their subjective impressions of using this tool are presented. Specifically, the learning outcome that this activity addressed was to increase understanding of the regulation of V(D)J recombination, which is often a difficult learning concept in undergraduate immunology courses. This is teaching innovation will also be presented as a workshop in the Education Session of Immunology 2021.
Engagement with In-class Activities Contributes to Student Success in an Accelerated, Flipped Classroom, Medical School Immunology Course
Authors: Steinel Natalie C , Corliss Stephanie , Lee Michael
Published: February 2023

In an era of decreasing basic science curriculum at medical schools, we sought to re-imagine how to optimally deliver three core basic science disciplines (immunology, microbiology, and pharmacology) together with infectious disease in a 5-week course. This course was developed as part of a new 1-year pre-clinical basic science curriculum at the recently established Dell Medical School (DMS) at the University of Texas at Austin. In line with the goals and missions of the DMS, course material was covered using primarily self-directed and flipped classroom pedagogies that incorporated approaches from the educational and cognitive psychology literature. Attendance and participation in flipped classroom activities was optional, and the materials and activities from these sessions were made available to all students. Analysis of student data of our integrated course shows a correlation between attendance in flipped classroom sessions and summative exam performance. We also observed a strong correlation between student performance on no-stakes formative assessments (weekly competency quizzes that did not count towards their course grade) and the summative exam as well. This suggests that, despite the abbreviated course time, engaging with the course as designed impacts achievement and may lead to superior outcomes. This offers valuable information that can be used to advise students on how to be successful in this type of high-impact course. It also may afford us the ability to predict which students may be at academic risk prior to the final summative exam.