bluuebunny/crossref_metadata_2025 huggingface dataset:
Mast cell-driven clearance in N. brasiliensis is masked by IL-25-dependent B1 cell IgE.
Authors: Martin Rebecca , Valentine Yolander , Davis Elijah , Lownik Joseph Cornelius , Luker Andrea , Zellner Matthew , Urban Joseph F. , Finkelman Fred D. , Conrad Daniel
Published: January 2023

A large amount of poly-specific IgE is generated during helminth infection. We have previously shown that innate-like B1 cells are responsible for this IgE production during infection with the nematode parasite Nippostrongylus brasiliensis. In vitro analysis demonstrated a novel role for IL-25-enhancement of IgE in B1 cells from helminth infected mice. In T cell-reconstituted RAG1−/− mice, N. brasiliensis clearance was enhanced with the addition of B2 cells in an IgE-dependent manner. This enhanced clearance was impeded by reconstitution with IgE sufficient B1 cells. IL-25 receptor-deficient B1 cells did not impede this clearance. Mucosal mast cells mediated the B2 cell-enhancement of clearance only in the absence of B1 cells. This was shown using mast cell depletion with anti-ckit (ACK.2) in a helminth-infected RAG1−/− reconstitution model. Additionally, gene expression analysis of the jejunum on day 7 post-inoculation found increased mast cell protease genes and mucus related genes in RAG1−/− mice reconstituted with B2 cells over T cells alone. This revealed a potential mechanism for B2 cell-enhanced clearance. Finally, R1E4, an antibody that prevents IgE from binding to FcɛRI, was administered in a model of RAG1−/− reconstitution infected with helminth and results confirmed that the B2 cell IgE/mast cell clearance enhancement was mediated through FcɛRI. Overall, these data shine new light on B2 cell derived IgE and mast cells and their role in helminth clearance that is impeded by IL-25-driven B1 cell IgE.
Selective Serotonin Reuptake Inhibitors Suppress Mast Cell Function
Authors: Haque Tamara , Ryan John J.
Published: January 2023

There is considerable evidence of links between depression and inflammation. Selective serotonin reuptake inhibitors (SSRIs) are effective anti-depressants with well documented off-target effects. Because SSRI also has anti-inflammatory effects, we tested the hypothesis that SSRIs can attenuate mast cell function. We show that SSRIs reduce IgE-mediated degranulation and cytokine production in mouse bone marrow derived and peritoneal mast cells in vitro, and suppress IgE-mediated anaphylaxis in vivo. Since exogenous serotonin had no effect on IgE-induced mast cell activation, we suspected an SSRI mechanism independent of serotonin inhibition. We show that inhibiting the purinergic receptors P2X3 and P2X7 mimic SSRI effects, and combining these inhibitors and SSRIs has no additive effects. SSRIs blocked ATP-induced activation and ATP uptake in mouse mast cells, further suggesting SSRIs are targeting P2X signaling. Because IgE induces ATP release, we postulate that SSRIs suppress an IgE-ATP positive feedback response by limiting P2X signaling. These data indicate that SSRIs may be effective therapeutics for mast cell-associated diseases.
The sins of first exposure: simultaneous allergen- and influenza-mediated signaling results in mast cell hyperactivity
Authors: Temple Rachel Mary , Obar Joshua J.
Published: January 2023

Each year hundreds of thousands of individuals in United States are hospitalized due to influenza A virus infection (IAV). Asthma is one of the leading co-morbidities among these hospitalized patients. Given the detrimental role mast cells can play during both asthma and IAV infection, we hypothesized simultaneous stimulation of mast cells by allergens and IAV may contribute to the asthmatic exacerbations often accompanying IAV infection. Here we show that signaling by both FcɛRI cross-linking and IAV infection (A/WSN/33 or A/PR/8/34) results in a heightened release of proinflammatory cytokines by these cells in vitro. At the same time, the type I interferon response is suppressed. As early as 1.5 hours post infection Ptgs2 mRNA is upregulated. Furthermore, chemical inhibition of COX-2 or mPGES-1 reduces this heightened proinflammatory response, suggesting a role for prostaglandin E2 in the mast cell hyperactivity. The addition of exogenous prostaglandin E2 can partially mimic this phenotype, though not as drastically as cells infected with IAV, suggesting there is an as yet unidentified viral component contributing to the mast cell hyperactivity. Surprisingly active viral replication is not necessary, as UV-inactivated IAV is capable of inducing mast cell hyperactivity as strongly as untreated virus. Interestingly, within 24–48 hours, infected mast cells die irrespective of FcɛRI stimulation. Taken together, our data suggest that IAV may incite asthmatic exacerbation through enhanced cytokine and prostaglandin production from mast cells receiving two distinct stimuli simultaneously, but ultimately mast cell death induced by IAV infection limits the duration of the asthmatic exacerbation.
TNFα Inhibits the Expression of Germinal Center Phenotype on T-bet+ CD11c+ IgM Memory B Cells without Altering Clonal Diversity
Authors: Popescu Maria , Winslow Gary
Published: January 2023

Our studies have identified a population of T-bet+ CD11c+ IgM memory B cells that arise within 30 days following infection with the intracellular bacterium, E. muris. The memory cells require for their generation T cell help and IL-21; however, they are generated in a GC-independent fashion, because GCs are suppressed during infection. Previous studies have shown that TNFα is necessary for proper GC formation. Given that we detected an increase in serum TNFα following infection, we addressed a role for this cytokine in IgM memory cell development. Relative to wild-type, we observed a substantial increase in the frequency of developing IgM memory. These B cells were positive for GL7+ CD38lo CD95+ on day 16 post-infection. Imaging studies corroborated these findings by demonstrating the appearance of GC-like structures in infected TNFα-deficient, but not in wild-type, mice.

Increased GL7 expression on IgM memory cells in absence of TNFα was not correlated with changes in clonal diversity on day 30 post-infection. This suggests that TNFα does not affect B cell selection.

To address mechanisms whereby the absence of TNFα may facilitate formation of pseudo-GC structures, we quantitated CXCL13, a chemokine known for its role as a chemoattractant during GC morphogenesis. CXCL13 expression was 10-fold lower in TNFα-deficient mice, indicating its requirement, in part, for splenic disorganization in wild-type mice during infection.

Our findings suggest that although GCs are suppressed during ehrlichial and other bacterial infections, GC structures can, when present, magnify IgM memory cell generation. Ongoing studies will address whether changes in lymphoid architecture impact other properties and functions of T-bet+ CD11c+ IgM memory cells.
Dissecting the contribution of antigen-dependent B cell receptor engagement and T cell help to Germinal Center B cell selection and differentiation.
Authors: Grigorova Irina , Turner Jackson S.
Published: January 2023

Proper selection of somatically hyper mutated B cells in the Germinal Centers (GCs) and their differentiation into long-lived plasma cells is key for the development of high-affinity long-term humoral immunity. However, relative contribution towards GC B cell selection by the two key factors: antigen-driven B cell receptor (BCR) signaling and T cell help, remains an open question. We have addressed this long-standing question in the field using an experimental model that enables independent manipulation of both antigen-driven BCR engagement and antigenic peptide presentation in GC B cells. Our studies suggest that in the absence of antigen-driven BCR engagement, T cell help is sufficient for robust selection of GC B cells and their differentiation into plasmablasts. In contrast, neither is induced by antigen-dependent BCR crosslinking in the absence of T cell help. Interestingly, when the levels of MHCII/peptide presentation by GC B cells are suboptimal, antigen-dependent BCR engagement can synergize with T cell help to promote selection of GC B cells and their differentiation into plasmablasts. Our findings reconcile somewhat contradictory data reported in previous studies and point towards the importance of the two signal model in the GC response.
Dendritic cell expressed H-2Db is required for generating antiviral CD8+ T cell responses and blood brain barrier disruption during Theiler’s virus infection
Authors: Tritz Zachariah Peter , Malo Courtney Sarah , Yokanovich Lila , Jin Fang , Hansen Michael J. , Johnson Aaron J.
Published: January 2023

Blood brain barrier (BBB) disruption is a feature of multiple neurological disorders for which there is an increasingly recognized role for CD8+ T cells. Theiler’s murine encephalomyelitis virus (TMEV) infection serves as an excellent model to study CD8+ T cell mediated BBB disruption because of the well-characterized immune response to the immunodominant VP2121–130 peptide presented in the context of the H-2Db class I molecule. Intravenous injection of VP2121–130 peptide seven days post TMEV infection results in severe BBB disruption. BBB disruption is dependent on CD8+ T cell interaction with the Db: VP2121–130 epitope, but the antigen presenting cell required for BBB disruption is undefined. We hypothesized that dendritic cells are responsible for presentation of the immunodominant VP2121–130 peptide. We tested this hypothesis using a novel H-2Db Cre-LoxP conditional knockout mouse bred to MHC class I deficient CD11c-Cre expressing mice. We then evaluated these animals at 7 days post infection for induction of BBB disruption, following VP2121–130 peptide administration, using gadolinium enhanced MRI, fluorescent albumin leakage into the brain, and confocal microscopy for the tight junction protein occludin. Control CD11c-Cre(−) mice generated robust Db: VP2121–130 epitope specific CD8+ T cell responses and displayed extensive BBB disruption. In contrast, CD11c-Cre(+) littermates generated no brain infiltrating antiviral CD8+ T cell responses and were protected from BBB disruption. We conclude that CD11c+ dendritic cells are required for priming of CD8+ T cell responses and are necessary for BBB disruption during neuroinflammation in this model.
Sodium Pyruvate Alters the Immune Response to Influenza A Virus Infection in Macrophages
Authors: Abysalamah Hazzar M.
Published: January 2023

Pyruvate is the end product of glycolysis. It can either be transported into the mitochondria for use in the TCA cycle or be used to regenerate NAD+ during aerobic glycolysis. We recently discovered that addition of sodium pyruvate to the culture medium during infection of macrophages with influenza A virus affects the production of cytokines involved in immune signaling. The purpose of the present study was to determine whether sodium pyruvate’s role in energy production in the macrophages may alter the immune response to the infection. While infection of macrophages with influenza A virus resulted in high levels of cytokines (IL-6, IL-1β, and TNF-α) in the absence of sodium pyruvate, the addition of sodium pyruvate significantly impaired cytokine production. Furthermore, sodium pyruvate did not affect virus growth, suggesting the effect of sodium pyruvate is on the immune response produced by the macrophages and not the viability of the virus.
Ssu72 phosphatase is involved in immunometabolism
Authors: Lee Si-Yeon , Park Eun-Ji , Kim Sumin , Lee Chang-Woo
Published: January 2023

Reversible protein phosphorylation is the primary type of various post-translational modifications in multiple cellular signaling pathways of eukaryotic cell. Dynamic alterations of phosphorylation state of many cellular signaling-mediated proteins including adipogenesis and thermogenesis regulate their molecular and cellular fates. Ssu72 is dual protein phosphatase that can act upon tyrosine or serine/threonine residues and transcription/RNA-processing factor. Recently, we found that Ssu72 expressed in adipose tissue, especially strongly in BAT, indicating that the potential involvement of Ssu72 in regulating BAT function. In this aspect, we generated conditional knock out mice which Ssu72 is deleted specifically in adipose tissue and found that the deficiency of Ssu72 leads to BAT dysfunction and abnormal thermoregulation compared to wild type mice. Moreover, we observed not only dramatically reduction of macrophage population but also defective M2 macrophage generation in Ssu72-deficient BAT. Notably, it has been reported that IL-4-induced M2 activation of macrophages is required for adaptive thermogenesis in mice. Thus, we further generated Ssu72-deleted macrophage mouse model. This study includes the physiological relevance of Ssu72 loss-of-function in macrophage during metabolic and thermogenic responses.
Roles for MS4A4 in FcɛRI signal transduction and store-operated calcium entry in human mast cells.
Authors: Arthur Greer K. , Cruse Glenn
Published: January 2023

The membrane spanning 4A (MS4A) gene family have similar topology to tetraspanins and are clustered around chromosome 11q12-13, a region linked to allergy and asthma susceptibility. However, other than the well-established roles of FcɛRIβ and CD20 in mast cell and B cell signaling, respectively, functional studies for the remaining genes in the MS4A family are lacking. We have demonstrated that MS4A4 promotes recruitment of KIT into caveolin-1-enriched microdomains and signaling through PLCγ1 in mast cells. PLCγ1 signaling and caveolin-1 are important regulators of store-operated Ca2+entry (SOCE) and caveolin-1 promotes expression of the store-operated Ca2+ channel pore-forming unit, Orai1. We thus hypothesized that MS4A4 regulates SOCE in response to FcɛRI-mediated stimulation through PLCγ1 and Orai1-dependent pathways. We began by identifying that human mast cells express a full length and truncated splice variant of MS4A4. Transfection of MS4A4 variants into HEK293 cells demonstrates that the full length variant of MS4A4 traffics to the plasma membrane, but the truncated MS4A4 variant does not. Knockdown of the full length MS4A4 variant with shRNA inhibits IgE-mediated degranulation of human mast cells, signaling through PLCγ1 and SOCE, likely through Orai1. Compound 48/80 and ionomycin stimulation were less affected by knockdown of MS4A4 or Orai1. These data suggest that MS4A4 plays an important role in FcɛRI signaling and suggest a possible novel link between KIT and FcɛRI signaling pathways.
Comparison of the immunomodulatory and epigenetic regulatory effects of natural estrogen 17-β estradiol and its synthetic analog 17α-ethinyl estradiol in NZB/WF1 mice
Authors: Dai Rujuan , Edwards Michael , Heid Bettina , Ahmed S. Ansar
Published: January 2023

Concern is increasing regarding the effects of environmental exposures to different Estrogenic Endocrine Disrupting Chemicals (EEDCs) on immune system function. 17α-ethinyl estradiol (EE), a synthetic analog of natural estrogen 17-β estradiol (E2), is a primary component in oral contraceptive pills (OCP) and has been found in sewage effluents. In this study, to compare the immune and epigenetic regulatory effects of environmental E2 and EE exposure in NZB/WF1 mice, we implanted gonadal intact female NZB/WF1 mice with E2 or EE. While the effect of EE was more profound, both E2 and EE exposures caused splenomegaly, altered splenic and thymic T cell composition and activation, increased splenic neutrophil percentage, and promoted inflammatory molecules iNOS/NO and the chemokine MCP-1 in ex vivo-activated splenocytes. Impressively, we found that E2 and EE had differential effects on the induction of different inflammatory cytokines in ex vivo activated splenocytes (e.g. IFNβ and IL-1β were increased in LPS-activated splenocytes from EE-treated NZB/WF1 mice, but were decreased in LPS-activated splenocytes from E2-treated mice when compared to placebo controls). Also, in vivo EE exposure promoted, and E2 reduced, TLR9 agonist-induced IFNα production in splenocytes. We further demonstrated that E2 and EE regulated the expression of numerous immune-related genes and miRNAs in a similar manner, while EE had a stronger effect than E2 on the regulation of miRNAs expression. Moreover, we found that EE, but not E2 treatment, significantly reduced the global DNA methylation level in NZB/WF1 splenocytes. Together, our data revealed similar and also differential regulatory effects of E2 and EE on the different aspects of immune function.
Effects of TLR 7 and 8 agonists on human monocyte activation and differentiation
Authors: Kayraklioglu Neslihan , Klinman Dennis M.
Published: January 2023

TLR7 and TLR8 are located on endosomal membranes and function to detect ssRNA released by viral or bacterial pathogens. Differences in the response characteristics of these two receptors, in terms of cell type preference and cytokine profile of human PBMC was being revealed. However, the effect of these two agonists on the same cell still remains as a question. Human monocytes express both TLR7 and TLR8 are highly plastic cells which is an important feature for immunotherapies. This study investigates the effects of TLR7 and 8 agonists on human classical monocytes in terms of activation and differentiation into macrophages. CD14+ HLA DR+ monocytes from healthy donors were cultured with the TLR7 specific agonist 3M-055, TLR8 specific agonist CL-075, and TLR7/8 dual agonist R848. All three agonists induced human monocytes to mature into macrophage significantly when compared to the unstimulated monocytes. A majority of those macrophages were 25F9+/CD200R−/CD206+ with an increased CD163 expression observed only with TLR8 agonists. Although CD163 and CD206 are commonly expressed on M2-like macrophage, functional analysis revealed that the macrophages differentiated by TLR7 or TLR8 agonists overall does not have significant endocytic ability (a prominent M2-macrophage function). TLR8 stimulation resulted in prominent pro-inflammatory cytokine increase whereas TLR7 agonist stimulation did not. Furthermore, TLR7 agonists decreased the cytokine induction by TLR8 agonists and this decrease was more prominent when TLR7 agonists were given prior to the TLR8 agonist. This study reveals differences in TLR7 vs TLR8 response of the monocytes and regulatory role of TLR7 on TLR8.
NF-kB c-Rel Dictates the Inflammatory Threshold by Acting as a Transcriptional Repressor
Authors: Ramakrishnan Parameswaran , de Jesus Tristan , Shukla Sudhanshu
Published: January 2023

Unresolved inflammation is an underlying reason for several immune and inflammatory diseases as well as cancer. Both initiation and resolution of inflammatory response rely largely on the genes induced by NF-kB/Rel family of transcription factors. Previous studies have categorized NF-kB family proteins as transcriptional activators (homo or hetero dimers containing RelA, RelB or c-Rel) and transcriptional inhibitors (homodimers of p50 or p52). Here, we report the function of the subunit c-Rel with a transactivation domain, as a transcriptional repressor of selected proinflammatory genes. Genetic deletion of c-Rel enhances the expression of a subset of proinflammatory genes and also potentiates the expression of TNF-induced RelA-dependent mediators of inflammation. Interestingly, v-Rel, the viral homologue of c-Rel, also possesses this repressive function, indicating that the repressive function of c-Rel is evolutionarily conserved. We also found that c-Rel’s ability to repress transcription depends on it competency to bind consensus NF-kB-binding sites in the DNA. Our findings reveal a novel role of c-Rel as a transcriptional repressor in the maintenance of inflammatory homeostasis.
Pioneer transcription factor BATF controls chromatin accessibility and CTCF-mediated chromatin architecture in CD4+ T cells
Authors: Pham Duy , Moseley Carson E. , Savic Daniel , Myers Richard , Kee Barbara L. , Hatton Robin D. , Weaver Casey T.
Published: January 2023

Pioneer transcription factors are a unique class of proteins that access heterochromatin, facilitate chromatin accessibility, and recruit other transcription factors to regulate gene transcription. The basic leucine zipper transcription factor ATF-like (BATF) is a critical factor for the development of T helper cell lineages. While BATF has been proposed as a pioneer factor in both T and B cells, its molecular functions are incompletely understood. In this study, we have identified a novel aspect of the pioneer function of BATF in controlling the chromatin landscape in developing effector T cells. We find that BATF is indispensable for the recruitment of chromatin-organizing complexes containing the DNA binding factor CTCF and other co-factors such as Oct1 and Ets1 to a subset of enhancer and chromatin-looping nodes during effector T cell lineage specification. Accordingly, BATF has a unique function in modulating genomic spatial organization in a CTCF-dependent manner. This study establishes BATF as a bona fide pioneer factor and provides novel insight into its role as a spatial genome organizer that modulates chromatin accessibility and initiates chromatin looping for the coordinate and differential gene expression in CD4+ T cells.
IL-6 counteracts IL-2-dependent suppression of T follicular helper cell responses
Authors: Papillion Amber M. , Bachus Holly , Fuller Michael , León Beatriz , Ballesteros-Tato Andre
Published: January 2023

T follicular helper (Tfh) cells are a specialized subset of CD4+ T cells that aide in the production of high-affinity antibody (Ab) responses. In the absence of Tfh cells, there is impairment in the generation of protective Ab responses to pathogens. In autoimmune patients, however, the expansion of unwanted self-reactive Tfh cells favor auto-Ab responses and subsequent Ab-mediated pathology. Thus, understanding the factors that regulate the generation of Tfh cells is critical for developing more efficient vaccination strategies and for designing new therapeutic approaches to prevent Ab-mediated autoimmune diseases. In this regard, it is generally acknowledged that IL-2 inhibits CD4 T cells from differentiating into Tfh cells. Conversely, previous studies suggest that IL-6 signaling is required for normal Tfh cell responses. However, Tfh cells normally differentiate in the absence of IL-6 in some experimental models, suggesting that IL-6 is not absolutely necessary for Tfh cell formation. Thus, the exact role played by IL-6 in controlling Tfh cell responses remains elusive. Using an influenza infection model, we found that IL-6 is dispensable for the priming of influenza-specific Tfh cell precursors, but essential for the maintenance of the Tfh cells at the peak of the influenza immune response. Mechanistically, our results demonstrate that already differentiated Tfh cells produce large amounts of IL-2 in the GCs and that intrinsic IL-6 signaling is needed to antagonize the negative impact of IL-2 in the ongoing Tfh cell response. Collectively, our data demonstrate that IL-6 induces a program that allows for Tfh cells to produce IL-2 without succumbing to the deleterious effects of IL-2 on Tfh cell differentiation.
Regulation of Notch activation in CD4+ T cells
Authors: Mitra Ankita , Torres Joe Angel , Shanthalingam Sudarvili , Vijayaraghavan Jyothi , Lawlor Rebecca , Osborne Barbara Anne
Published: January 2023

Notch signaling provides an important cue in the mammalian developmental process. It is a key player in T cell development and function. Mature T cells express Notch receptors 1, 2 and 3. Data from our lab and others indicate that T cell receptor (TCR) signaling activates Notch. Conventional ligand dependent Notch signaling involves, interaction of the Notch receptor with ligands such as Delta like ligand (DLL) 1,3, 4 or Jagged (JAG) 1, 2. However, in T cells it is unclear whether TCR induced activation of Notch is dependent upon engagement of Notch receptor with ligand. In-vitro stimulation of purified CD4+T cells, using antibodies against CD3 and costimulatory molecule CD28, results in activation of Notch. This occurs in absence of APC or any apparent ligand presenting cell. Thus, the mechanism that drives Notch activation during TCR stimulation is unclear. When naïve CD4+ T cells were cultured with bone marrow derived dendritic cells pulsed with antigen, we observed activation of Notch in the apparent absence of ligand on APC or activated T cells. However, when we examined ligand expression on naïve CD4+ T cell, activated by anti-CD3/CD28, we found resting T cells express constitutively low levels of DLL1 which increases with activation and is maintained through activated state. Additionally, DLL4 and JAG1 are expressed only after T cell activation and maintained only for 24hrs after activation. This upregulation is completely dependent upon signaling through CD28. However, whether the expression of ligand is sufficient to activate Notch remains unclear. Our preliminary data thus suggests that TCR mediated signaling in the absence of ligand may be is sufficient to activate Notch.
Critical role of ASK signaling in promoting inflammatory disease in Ptpn6spin mice
Authors: Tartey Sarang , Kanneganti Thirumala-Devi
Published: January 2023

Mice homozygous for the Tyr208Asn amino acid substitution in the carboxy terminus of SHP-1 (referred to as Ptpn6spin mice) spontaneously develop a severe inflammatory disease resembling neutrophilic dermatosis in humans. Disease in Ptpn6spin mice is characterized by persistent footpad swelling and suppurative inflammation. Recently, in addition to IL-1α and IL-1R signaling, we demonstrated pivotal role for several kinases, such as SYK, RIPK1, and TAK1 in promoting inflammatory disease in Ptpn6spin mice. In order to identify new kinases involved in SHP1 mediated inflammation, we undertook genetic approach and discovered Apoptosis signal-regulating kinases (ASK1 and ASK2) as novel kinases regulating the Ptpn6 mediated footpad inflammation. Double deletion of ASK1 and ASK2 abrogated cutaneous inflammatory disease in Ptpn6spin mice. This double deletion further rescued the splenomegaly and lymphomegaly caused by excessive neutrophil infiltration in Ptpn6spin mice. Mechanistically, ASK regulates Ptpn6spin mediated disease by controlling pro-inflammatory signaling in the neutrophils. Collectively, this study identifies SHP1 and ASK signaling crosstalk as a critical regulator of IL-1α driven inflammation and open future avenues for finding novel drug targets to treat neutrophilic dermatosis in humans.
Bhlhe40 maintains CD8+ T cell fitness and functionality in non-lymphoid tissues and tumors
Authors: Li Chaofan , Taneja Reshma , Sun Jie
Published: January 2023

Recent evidence has suggested that tissue-resident and tumor-infiltrating CD8 T cells share common molecular signatures for their maintenance in the non-lymphoid environment. Currently, the molecular mechanisms regulating the residency, health and function of effector and memory CD8 T cells in non-lymphoid tissue remain largely elusive. Here we have identified that the transcription factor Bhlhe40 is critical for the fitness and function of effector & memory CD8 T cells in the non-lymphoid tissue and tumor. Bhlhe40 and its associated gene signature are highly expressed in CD8 T cells, which reside in non-lymphoid tissues and tumor, compared with their counterparts in the spleen following viral infection and tumor transplantation. We find that Bhlhe40 deficiency results in the progressive loss of effector and memory CD8 T cells in non-lymphoid tissues following influenza infection and tumor transplantation. As such, Bhlhe40 abolition leads to defective anti-cancer immunity and impaired heterologous anti-viral immunity mediated by the tissue-resident effector and memory CD8 T cells. Mechanistically, we show that Bhlhe40 is dispensable for the activation and metabolic programming of CD8 T cells, but is vital for them maintaining metabolic and mitochondrial fitness. As such, Bhelhe40 deficiency results in energy failure of CD8 T cells and impaired maintenance of active histone marks in the loci of CD8 T cell effector molecules. Therefore, we have identified Bhlhe40 as a key TF for the maintenance of CD8 T cell fitness and functionality in non-lymphoid tissues. Furthermore, our data suggest that the manipulation of Bhlhe40 expression and function may serve as a potential strategy to boost the efficacy of vaccination and immunotherapy.
TRAF3-mediated regulation of the T cell receptor complex
Authors: Arkee Tina , Wallis Alicia M. , Bishop Gail A.
Published: January 2023

TRAF3 is an adaptor protein with diverse cell type-specific functions that plays an important role in T cell biology. T cell conditional TRAF3−/− mice, despite normal numbers of T cells, have poor responses to immunization and infection. Further investigation revealed that early events in T cell receptor (TCR) signaling are defective in TRAF3-deficient T cells. Examining the basis of this defect, we recently reported that TRAF3 normally inhibits localization of the negative regulators Csk and PTPN22 to the TCR membrane complex. We now wish to understand how TRAF3 is recruited to this complex. TRAF3 associates with the TCR-CD28 complex upon stimulation; engagement of both the TCR and CD28 is required for TRAF3 recruitment. We hypothesize that TRAF3 associates with the TCR complex either indirectly, by binding to other CD28-associated adaptor proteins, or directly, by binding to motifs in the cytoplasmic tail of CD28. Results of preliminary experiments suggest that the TRAF3 TRAF-C domain associates with Linker of Activated T cells (LAT), but not with the LAT-binding adaptor protein Gads. We are currently investigating the structural requirements for the association of TRAF3 with CD28, the adaptor protein Grb2, and other TCR signaling proteins, to determine how TRAF3 is recruited to the TCR complex. Based upon our previous finding that TRAF3 enhances TCR signaling by inhibiting the localization of negative regulatory proteins associated with the TCR, as well as our preliminary data indicating that TRAF3 interacts with members of the LAT complex, we are also investigating the interactions between TRAF3 and negative regulatory proteins associated with the LAT complex.
The transcription factor T-bet is regulated by microRNA-155 in murine anti-viral CD8+ T cells via SHIP-1.
Authors: Hope Jennifer L. , Stairiker Christopher J. , Spantidea Panagiota I. , Gracias Donald T. , Carey Alison J. , Fike Adam J. , van Meurs Marjan , Brouwers-Haspels Inge , Rijsbergen Laurine C. , Fraietta Joseph A. , Mueller Yvonne , Klop Rosemarieke C. , Stelekati Erietta , Wherry E. John , Erkeland Stefan , Katsikis Peter D.
Published: January 2023

MicroRNAs (miRNAs) are small, single-stranded non-coding RNAs that play essential roles in regulating key cellular processes, and are highly conserved across species. However, the regulatory role of microRNAs in generating effective CD8+ T cell (CTL) responses to viral infection and tumors is only now being elucidated. Microarray miRNA expression profiling identified unique expression patterns of miRNAs in naïve and effector anti-viral CTL. In particular, microRNA-155 was significantly upregulated in effector CTL. Previously, we have demonstrated that miR-155 is essential to CD8+ T cell responses to viral and intracellular bacterial infections. In the present study, we show that miR-155 overexpression in CTL augments anti-viral effector CTL and skews memory CD8+ T cells towards an effector memory phenotype. MiR-155 overexpression resulted in enhanced T-bet expression, which is known to be required for effector CTL and to promote effector memory cell formation. Importantly, we show that the proliferative effect of miR-155 on CD8+ T cells is mediated by T-bet. In CTL, T-bet levels were controlled in vivo by miR-155 via SH2 (Src homology 2)-containing inositol phosphatase-1 (SHIP-1), a known direct target of miR-155. Our studies have revealed an important and unexpected link between miR-155, T-bet transcription factor and SHIP-1 in the context of CTL responses to an in vivo viral infection.
The IRF4 gene regulatory module functions as a read-write integrator to dynamically control T helper cell fate
Authors: Sciammas Roger , Krishnamoorthy Veena , Kannanganat Sunil , Maienschein-Cline Mark , Cook Sarah , Chen Jianjun , Bahroos Neil , Corse Emily , Chong Anita S. F.
Published: January 2023

Transcriptional regulation during CD4+ T cell fate decisions enables their differentiation into distinct states, guiding immune responses towards antibody production via Tfh cells or inflammation by Teff cells. Tfh–Teff fate commitment is regulated by mutual antagonism between the transcription factors Bcl6 and Blimp-1. Here we examined how T cell receptor (TCR) signals establish and arbitrate Bcl6–Blimp-1 counter-antagonism. We found that the TCR-signal induced transcription factor IRF4 is essential for the differentiation of Bcl6-expressing Tfh and Blimp-1-expressing Teff cells. Increased TCR signaling raised IRF4 amounts and promoted Teff fates at the expense of Tfh ones. Importantly, orthogonal induction of IRF4 expression redirected Tfh fate trajectories towards those of Teff and this occurred independently of IL-2 signals. Mechanistically, we linked greater IRF4 abundance with its recruitment towards low affinity binding sites within Teff cis-regulatory elements, including those of Prdm1. We propose that the Irf4 locus functions as the “reader” of TCR signal strength, in turn, concentration-dependent activity of IRF4 “writes” T helper fate choice.
Hands on, Minds on: How to create effective, student-driven learning without becoming overwhelmed
Authors: Lukin Kara R. , Aviszus Katja
Published: January 2023

When done effectively, student-driven approaches to learning increase student satisfaction and learning outcomes. In addition, encouraging students to be more actively involved in their learning better prepares them for graduate school and for joining the professional workforce. For those used to a more traditional lecturing role in the classroom, the prospect of developing and integrating approaches that require a higher degree of engagement of students can be intimidating. Here, we share our experiences and provide examples of simple strategies and approaches we adopted to implement a classroom environment that requires students to be more actively involved in the learning process. We demonstrate how to create and implement the following methods slowly into courses with any size enrollment:

1) content organizers and hierarchy maps that scaffold concepts and drive students to integrate material, 2) distributive recall practices that increase neural networking, 3) online resources for content mastery so class time can be for application and group activities, 4) IF-AT (immediate feedback) scratch cards and problem sets that drive discussion and learning better than clickers, 5) activities that explain immunologic concepts with which students often struggle, and 6) mini and full case studies that are woven through the term.

All resources will be available for immediate use.
The prevalence of digital note-taking trends in professional pharmacy students
Authors: Hussein Mohamed , Lana Angelica , Reda Betlhem , Bizuayehu-Mekonnen Menorwork , Nassan Motaz , Fazal Nadeem
Published: January 2023

The use of technology in the classroom may have positive and negative effects on student learning. We determined the use of note taking technologies to precept students learning. The research in question is to find out the use of integration of modern technology into didactic courses of pharmacy education. We surveyed students’ perceptions of note taking regarding the type, frequency, and appropriateness of use of note-taking technology in the classroom. We carefully prepared 10 questions for an anonymous survey pertaining to students’ perception of habits and ways of taking notes in the class and how it affected their learning capability in the school. A questionnaire survey was delivered to 59 P1 students in the classroom. 100% of the students who responded owned electronic device and use it while in school. The data determined the following trends. When students were asked about which operating system they are using, 39% used MacBook, 34% used windows and 21% reported use of Ipad for their notes-taking needs. 60% of the students either used a digital product to take notes or used windows. 71% of students who responded said that they take 75-100% of their notes on electronic devices in the P1 courses. The majority of those students used a laptop to take notes during class, 60% of students reported using Notability for note taking while in classrooms. Among other digital products PDF writer, Evernote, and OneNote were also used. 23% of the students still prefer taking notes the old traditional way by printing the lectures and writing notes on paper. This study demonstrated how modern digital technology helps and enhances note-taking ability among pharmacy students. Price and easy use of technological gadgets influences the final selection for students.
The Role of Food Allergy Education in Schools: Measuring Attitudes, Beliefs, and Knowledge
Authors: Canon Nicole , Gharfeh Maya , Anvari Sara , Davis Carla
Published: January 2023

Introduction

Almost 6 million children suffer from food allergies with roughly 2 affected per classroom. However, deficiencies in knowledge and discrepancy in attitudes within school staff when addressing food allergies are associated with barriers to care. In this study, school personnels’ knowledge, beliefs, and attitudes were measured before and after a food allergy educational workshop.

Methods

Six schools in Houston Independent School District were randomly assigned into an interventional (n=4) and control group (n=2). Seven-hundred and thirty-three personnel matched for age, socioeconomic status, ethnicity, and educational level completed the Chicago Food Allergy Research survey before and after a one-hour didactic session. Responses were measured using frequencies and percentages. The group score differences and survey question comparisons were measured with a linear mixed effects model.

Results

Post-test, the intervention group had knowledge scores 19.58% higher than control (95% CI=16.62, 22.53; p<0.001) with no differences pre-test. Odds of agreeing that injectable epinephrine is important was 874 times higher in the interventional schools post lecture. Within the interventional group, personnel were 207 times more likely to agree to an epi-pen for children post-education.

Conclusion

A didactic session improved knowledge and attitudes in personnel in the intervention schools. Given the growing prevalence of food allergy, the emphasis on food allergy education is crucial to allow for familiarization of the condition, early recognition of anaphylaxis, and promotion of injectable epinephrine use.
IL-18 from Batf3-dependent cells licenses natural killer cell IL-10 production during Listeria monocytogenes infection
Authors: Clark Sarah E. , Schmidt Rebecca L. , McDermott Daniel S. , Lenz Laurel L.
Published: January 2023

Natural killer (NK) cells are innate lymphoid cells that regulate the immune response to infection through secretion of both pro- and anti-inflammatory cytokines. The pathogen Listeria monocytogenes (Lm) capitalizes on NK cell production of the anti-inflammatory cytokine interleukin (IL)-10 during establishment of severe infection. Lm-stimulated NK cell IL-10 limits immune cell recruitment and activation, allowing for bacterial expansion. Here, we report that IL-18 from dendritic cells (DCs) promotes this IL-10 production. IL-18 acts directly on NK cells to license IL-10 secretion that is independent of IL-12 and STAT4, which co-stimulate IFNγ secretion during Lm infection and can induce NK cell IL-10 in other contexts. DC release of IL-18 is driven by the Lm p60 virulence protein and requires DC expression of nlrp3 and batf3. Mice lacking nlrp3, il18, il18R, or batf3 fail to accumulate serum IL-10 and are highly resistant to systemic Lm infection. Our data thus show that during systemic infection Lm selectively targets Baft3-dependent cells to drive IL-18 release that licenses NK cell IL-10 production. Exploiting this previously-unappreciated pathway to promote IL-10 production enables Lm to dampen inflammatory and anti-microbial host responses.
Innate-like activation of mucosal-associated invariant T cells in mycobacterial infection
Authors: Huang Shouxiong , Sharma Manju , Zhang Shuangmin , Niu Liang , Zhang Xiang
Published: January 2023

Unlike conventional T cells, mucosal-associated invariant T (MAIT) T cells respond to microbial riboflavin precursor metabolites presented by major histocompatibility complex (MHC)-like molecule MHC-related protein 1 (MR1). Recently, a high percentage of CD8+ T cells reactive to Mycobacterium tuberculosis in humans were identified as MAIT cells instead of conventional MHC class I-restricted T cells. To understand the activation mechanism and effector functions of MAIT cells in mycobacterial infections, we used mycobacterial-incubated human dendritic cells and MR1-overexpressed cells to stimulate primary and clonal human MAIT cells. As a result, the enzyme-linked immunospot assay showed a quick activation kinetics of MAIT cells within hours of mycobacterial stimulation in an MR1-dependent manner. Flow cytometry analysis demonstrated that mycobacterial-incubated antigen presenting cells quickly stimulated primary human MAIT cells to develop a dominant CD69+CD26+phenotype. Activated MAIT cells displayed an enhanced expression of cytokines TNFa, IFNg, IL-17, and granulysin, demonstrating a potential to induce pro-inflammatory and cytolytic responses. Further transcriptomic analysis of the CD69+CD26+subset revealed an integrated activation pathway from conventional CD8+ T cells and natural killer (NK) cells. Especially, activated MAIT cells showed an enhanced gene expression of mitogen-activated protein kinase (MAPK) signaling molecules, NF-kB, T-bet, RORg, and PLZF transcription factors, and pro-inflammatory cytokines and cytolytic molecules. Taken together, our data support that MAIT cells are quickly activated through an innate-like activation mechanism to induce anti-mycobacterial responses.
CHARACTERIZATION OF INTERLEUKIN-17 PRODUCING CELLS IN THE PERIPHERAL BLOOD OF PATIENTS WITH TUBERCULOSIS
Authors: Ruiz-Sánchez Bibiana Patricia , Zarate David Cruz , García-Paredes Victor Gabriel , Hernández-Solis Alejandro , Sabido Raúl Cisero , Serafín-López Jeanete , Estrada-Garcia Iris , Baeza Isabel
Published: January 2023

Tuberculosis (TB), an infection caused by Mycobacterium tuberculosis, is one of the ten main causes of death by infectious disease. When the mycobacteria are inhaled, they interact with immune cells in the respiratory tract in a specialized structure known as granuloma. Interleukin 17 (IL-17) contributes to formation and size of the granuloma, and it correlates with the recruitment of neutrophils. It has been demonstrated that other innate lymphocytes, such as Tγδ cells, contribute to the production of IL-17 during TB. Innate lymphoid cells (ILCs) are other innate lymphocytes that are classified into groups (ILC1, ILC2 and ILC3) according to their cytokine patterns. ILC3 produce IL-17, but it is not known if these cells participate in the response to TB infection. We evaluated, by flow cytometry, the frequency and IL-17 production of Th, Tgd, NK and ILCs (ILC1, ILC2 and ILC3) in the peripheral blood of healthy individuals (negative and positive for the Quantiferon [QTF] test, which detects latent TB), and of patients with active pulmonary TB. The numbers of Th cells, NK cells and ILCs (ILC1, ILC2 and ILC3) were decreased in TB patients, compared to healthy individuals. Peripheral blood was stimulated with M. tuberculosis soluble extract (MTSE) for 5 h in the presence of brefeldin A. After stimulation, Th cells from QTF-positive healthy individuals and TB patients produced IL-17. Tγδ cells also produced IL-17, although the percentage of these cells did not change after activation with MTSE. In some cases, NK, ILC2 and ILC3 from TB patients produced IL-17, but this production did not change after MTSE activation. These results indicate that Th and Tγδ cells contribute to IL-17 production during TB.

This project was funded by SIP-IPN-México (20180254).
Neutrophil extracellular traps deficiency aggravates Citrobacter rodentium infection and the associated intestinal inflammation
Authors: Saha Piu , Yeoh Beng San , Wang Yanming , Vijay-Kumar Matam
Published: January 2023

Peptidyl arginine deiminase-4 (PAD4) is required for the formation of neutrophil extracellular traps (NETs), which is part of the host antimicrobial innate immune response. However, the role of NETs in the pathogenesis and clearance of Citrobacter rodentium (a well-characterized mouse enteropathogen) infection remains unclear. In this study, we challenged the PAD4-deficient (Pad4KO; NETs-deficient) and WT littermates with C. rodentium. Luminal colonization of C. rodentium in Pad4KO mice peaked between 11-14 days post-infection (pi), whereas WT mice cleared the infection with no detectable level of the pathogen in fecal shedding after 7-10 days. Bacterial dissemination to spleen and mesenteric lymph node was significantly higher in Pad4KO mice at day 8 and 28 pi compared to WT. Additionally; Pad4KO mice displayed splenomegaly, colomegaly and elevated colonic and systemic pro-inflammatory markers (lipocalin 2 and serum amyloid A). Histological analysis demonstrated that transmissible colonic hyperplasia and goblet cell depletion were more pronounced in Pad4KO mice. Pad4KO mice also exhibited epithelial barrier dysfunction as indicated by their heightened serum immunoreactivity to bacterial proteins. Flow cytometric analysis revealed a substantial reduction in IL-10-producing macrophages and Th17 cells in the lamina propria and spleens of Pad4KO mice compared to WT mice. However, colonic expression of IL-17A and IL-22 were highly elevated in Pad4KO mice on day 8 pi, but were attenuated on day 28 both in Pad4KO and WT mice. Taken together, our findings highlight that NETs are indispensable in promoting clearance of C. rodentium and resolving the associated intestinal inflammation.
Key role for CD103+ DCs in activating lung iNKT cells during Streptococcus pneumoniae infection
Authors: Crosby Catherine Marie , Mikulski Zbigniew , Kronenberg Mitchell
Published: January 2023

Invariant natural killer T cells (iNKT) are profoundly important for protection against Streptococcus pneumoniae. Prior work has shown iNKT cells are rapidly activated in a CD1d-dependent manner, however the critical antigen presenting cells remain unknown. We have begun to uncover a dominant role for a more recently described iNKT cell subset: NKT17 cells. Within 14 hours of infection, approximately 60% of NKT17 cells have received a TCR signal, and 45% produce IL-17A. Notably, only a minor percentage of NKT1 cells are TCR-activated or produce IFN-γ. Here, by generating conditional CD1d knockout mice, our goal was to determine which cell types directly activate NKT17 cells during infection. In mice with a CD11c+ cell-specific deletion, there was a 50% reduction of IL-17A-producing NKT17 cells at 14 hours, and a significant increase in bacteria loads at 48 hours. While these mice lack CD1d on several cell types, we focused on CD103+ DCs. If CD103+ DCs are absent, mice are highly susceptible to S. pneumoniae infection, iNKT cells do not expand, and IL-17A is diminished. Using confocal and two-photon microscopy of live tissue sections and flow cytometry we investigated their role as APCs. CD103+ DCs engulf a GFP-expressing S. pneumoniae strain within 2 hours and are primarily located within the interstitial lung tissue, notably where most NKT17 cells localize. We next generated a mouse with a CD103+ DC-specific deletion of CD1d. These mice also showed a 50% reduction of IL-17A-producing NKT17 cells at 14 hours and an increase in bacteria loads at 48 hours. Continuing studies include microscopy of live lung sections to further investigate the direct interaction between CD103+ DCs and iNKT cells during S. pneumoniae infection.