bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual T cell receptor expression promotes agonist selection of regulatory T cells
Authors: Balakrishnan Amritha , Jama Burhan , Morris Gerald P.
Published: January 2023

Generation of CD4+FoxP3+ regulatory T cells (Treg) in the thymus is essential for maintenance of self-tolerance. Treg lineage commitment occurs via agonist selection, via TCR high-affinity interaction with self-peptide-MHC (pMHC) ligand. We have previously demonstrated that natural co-expression of dual TCRs, occurring in ~10% of T cells, results in increased ability to recognize self-pMHC ligands and promote positive selection. We hypothesized that dual TCR expression could similarly promote agonist Treg selection. Examination of Treg development in B6.TCRa+/− mice, heterozygous for disruptive mutation of TCRAC and unable to express dual TCRs, demonstrated decreased frequency of Tregs in the thymus (2.76+0.08% of CD4 single-positive thymocytes) compared to age-matched wild-type B6 mice (3.26+0.09%, P < 0.001). To more precisely compare Treg agonist selection, we co-transferred 1:1 ratios of T cell-depleted B6.TCRa+/−.Thy1.1 and B6.Ly5.1 bone marrow to lethally-irradiated B6 recipients and monitored thymic selection. TCRa+/− cells were deficient in Treg selection compared to co-transferred wild-type cells; TCRa+/− cells produced 37.57+4.16% of donor-derived thymic Tregs (P = 0.003). This defect was specific for Treg selection, as TCRa+/− cells comprised 44.68+6.33% of double-positive thymocytes and 45.50+5.77% of CD4 single-positive thymocytes. Tregs generated from TCRa+/− or wild-type cells were phenotypically similar, with no differences in expression of FoxP3 or CD5. We propose that the difference in agonist selection of dual TCR and single TCR Tregs could have important consequences in the TCR repertoire and antigenic specificity of peripheral Tregs, impacting immunologic tolerance.
Expression of Ly6C/6G defines a novel Aire-dependent subset of medullary thymic epithelial cells
Authors: Matsumoto Mitsuru , Matsumoto Minoru , Nishijima Hitoshi , Morimoto Junko
Published: January 2023

Ly6 family proteins are mainly expressed by myeloid-cell lineages such as granulocytes (expressing Ly6G) and monocytes (expressing Ly6C). However, exact function of Ly6 family protein in each cell-type remains unknown. For example, although expression of Ly6C has been used to define functionally distinct subset of monocytes (Ly6C+ monocytes vs. Ly6C− monocytes), molecular basis for the expression of Ly6C and functional difference between the two populations are largely unknown.

Medullary thymic epithelial cells (mTECs) are composed of heterogeneous populations in terms of the spectrum of tissue-restricted antigens (TRAs) expression for ensuring the elimination of autoreactive T-cell repertoire. We have recently identified mTEC subset(s) that express Ly6C and Ly6G both recognized by monoclonal antibody Gr-1 (i.e., Ly6C/6G+ mTECs). Approximately 10% of total mTECs expressed Ly6C/6G and Ly6C/6G+ mTECs were preferentially localized at the cortico-medullary junction. We found that Ly6C/6G+ mTECs and Ly6C/6G− mTECs do not form distinct lineage(s). Instead, Ly6C/6G− mTECs are able to convert into Ly6C/6G+ mTECs and vice versa in a reaggregated thymic organ culture (RTOC). Interestingly, Aire-deficient mice do not possess Ly6C/6G+ mTECs.

RNA-seq analyses have revealed that Ly6C/6G+ mTECs express higher levels of chemokines, integrins and extracellular matrix proteins compared with those from Ly6C/6G− mTECs, suggesting that Ly6C/6G+ mTECs might constitute a unique tolerogenic subset among heterogenous mTECs.
Critical roles for microbiota-mediated regulation of Th17 responses in a maternal immune activation model of autism
Authors: Lukens John R. , Lammert Catherine R. , Frost Elizabeth L. , Bellinger Calli E.
Published: January 2023

Recent studies suggest that autism is often associated with altered microbiota composition and gastrointestinal inflammation. This work has led to growing speculation of a potential role for the microbiome in autism spectrum disorder (ASD). However, how microbiota diversity mechanistically influences the development of autistic phenotypes has not been studied in great detail to date. To formally investigate this, we evaluated how environmentally induced changes in gut microbiota landscape influence the incidence and severity of MIA-induced neurodevelopmental disorders. Herein, we report critical roles for prenatal microbiota composition in the development of behavioral abnormalities in a MIA mouse model of autism. Mechanistically, we show that microflora-dependent calibration of the immune response underlies the effects of the microbiome on inflammation-induced ASD-like phenotypes. Specifically, we find that neutralization of IL-17a ameliorates the ability of the microbiome to affect the development of autism-related behaviors. Our results identify the immune system as a link between gut microbiota and the brain in neurodevelopmental disorders, and suggest that targeting the microbiome and maternal immune responses during gestation may offer strategies to limit autism development in at-risk pregnancies.
Cardiac endothelial cells produce complement C3 in response to hypoxia
Authors: Zhang Ming , Li Xiang , Kahyaoglu Seran , Sironi Marina , Mantovani Alberto
Published: January 2023

Hypoxia takes places in life threatening conditions, such as heart attack. Endothelial cells are on the first line being affected when hypoxia occurs. Complements as innate immune factors have been reported to play an important role in re-oxygenation after hypoxia being corrected, but little is known the complement response in endothelia cells during hypoxia. Using a murine cardiac endothelial line H5V, we investigated this. Under hypoxia condition without serum (thus no exogenous complements provided), these cardiac endothelial cells produced complement C3 for a period time and died. However, when serum was supplemented during hypoxia, these cells did not produce endogenous C3 and maintained a reasonable survival rate. The results indicate that endogenous production of complement C3 is promoted by the hypoxia stress and linked to cell death of cardiac endothelial cells.
Early Inhibition of Fatty Acid Synthesis Reduces Generation of Memory Precursor Effector T cells in Chronic Infection
Authors: Ibitokou Samad , Dillon Brian E. , Sinha Mala , Szczesny Bartosz , Delgadillo Añahi , Abdelrahman Doaa Reda , Szabo Csaba , Abu-Elheiga Lutfi , Porter Craig , Tuvdendorj Demidmaa , Stephens Robin
Published: January 2023

Understanding the mechanisms of CD4 memory T cell (Tmem) differentiation in malaria is critical for vaccine development. However, the metabolic regulation of CD4 Tmem differentiation is not clear, particularly in persistent infections. In this study, we investigated the role of fatty acid synthesis (FAS) in Tmem development in Plasmodium chabaudi chronic mouse malaria infection. We show that T cell-specific deletion, and early pharmaceutical inhibition of acetyl coA carboxylase 1, the rate limiting step of FAS, inhibit generation of early memory precursor effector T cells (MPEC). To compare the role of FAS during early differentiation or survival of Tmem in chronic infection, a specific inhibitor of acetyl coA carboxylase 1, 5-(Tetradecyloxy)-2-furoic acid, was administered at different times postinfection. Strikingly, the number of Tmem was only reduced when FAS was inhibited during T cell priming, but not during the Tmem survival phase. FAS inhibition during priming increased effector T cells (Teff) proliferation, and strongly decreased peak parasitemia, which is consistent with improved Teff function. Conversely, MPEC were decreased, in a T cell-intrinsic manner, upon early FAS inhibition in chronic, but not acute, infection. Early cure of infection also increased mitochondrial volume in Tmem compared to Teff, supporting previous reports in acute infection. We demonstrate that the MPEC-specific effect was due to the higher fatty acid content and synthesis in MPEC compared to terminally differentiated Teff. In conclusion, FAS in CD4 T cells regulates the early divergence of Tmem from Teff in chronic infection.
Correlative Recurrent Expression of Predicted Elements (CREPE): A Novel Computational Approach to Predict LncRNA Function
Authors: Pyfrom Sarah , Koues Olivia , Kowalewski Rodney , Oltz Eugene M. , Payton Jacqueline
Published: January 2023

Long non-coding RNAs (lncRNAs) act as transcriptional regulators, scaffolds, and signaling modulators in development, immune response, and oncogenesis. Our -omics study of >100 human Non-Hodgkin Lymphoma (NHL) and normal B cell samples revealed altered expression of lncRNAs in NHL. LncRNAs have not been characterized in NHL or B cells, and there are few guidelines for functional prediction. To address this gap, we developed a novel computational approach: Correlative Recurrent Expression of Predicted Elements (CREPE). This method calculates and tracks the following for each lncRNA: expression, cell-type specificity, subcellular localization, differential expression (eg, tumor/normal), correlation with neighboring gene transcripts (RNAseq); local enhancer and 3D genome interaction landscapes (ChIPseq, Hi-C, ChIA-PET). From this, CREPE calculates a rankable score to predict the likelihood of potential functions, including 1) transcriptional regulation via lncRNA transcript, 2) enhancer-associated regulation, and 3) scaffold/modulator cytoplasmic function. The likelihood score formula incorporates an if-then logic, correlative regression analysis, and statistical significance. Finally, CREPE enables heuristic ranking of lncRNAs by pathway analysis. CREPE identified lncRNAs that may regulate NHL oncogenes and modulate B-cell receptor signaling. CREPE analysis of data from The Cancer Genome Atlas and a CRISPRi lncRNA growth screen validate its ability to identify lncRNAs with oncogenic or growth-promoting potential in diverse studies. In summary, we developed a novel approach that fills an unmet need for predictive modeling of lncRNA function and identified lncRNAs likely to promote lymphomagenesis.
Type 2 diabetes mellitus induces TNFR1 mediated necroptotic cell death of mice alveolar macrophages infected with Mycobacterium tuberculosis
Authors: Radhakrishnan Rajesh Kumar , Tripathi Deepak , Thandi Ramya Sivangala , Paidipally Padmaja , Vankayalapati Ramakrishna
Published: January 2023

Previously, we developed a mouse model of type 2 diabetes mellitus (T2DM) using streptozotocin and nicotinamide and found that T2DM mice are susceptible to Mycobacterium tuberculosis (Mtb) infection. We also found that alveolar macrophages from T2DM mice were more permissive to Mtb growth ex vivo compared to non-diabetic controls. In the current study, we determined the defective mechanisms that make T2DM mice alveolar macrophages more susceptible to Mtb infection. Mtb infected alveolar macrophages from T2DM mice produced more TNF-α (973.8 ± 13.3 pg/ml vs. 614.6 ± 27.2 pg/ml, p<0.003) and less apoptotic (4.7 ± 1.8% vs. 28.6 ± 2.3%, p<0.001) compared to Mtb infected non-diabetic control mice. Mtb infected alveolar macrophages from T2DM mice expressed higher levels of TNFR1 (12.8 ± 0.5 vs. 2.09 ± 0.01, p<0.001) and markers of necroptosis RIPK1 (11.5 ± 0.57 vs. 2.94 ± 0.20, p<0.002), RIPK3 (16.1 ± 0.58 vs. 2.89 ± 0.2, p<0.001) and MLKL (9.2 ± 0.56 vs. 1.58 ± 0.72, p<0.02) compared to Mtb infected alveolar macrophages from non-diabetic control mice as determined by real-time PCR. This finding was again proved by Western blot and confocal microscopy. Anti-TNFR1 antibody treatment of alveolar macrophages from T2DM mice before or after Mtb infection reduced RIPK1, RIPK3 and MLKL expression as determined by RT-PCR. Our findings demonstrate that T2DM induces necroptosis of alveolar macrophages upon Mtb infection. Enhanced TNFR1 signaling in T2DM mice alveolar macrophages is responsible for enhanced necroptosis. We are also determining the subpopulations of T2DM mice alveolar macrophages that express higher levels of TNFR1 upon Mtb infection. Studies are underway to determine the in vivo relevance of our current findings to Mtb growth in T2DM mice.
Kupffer cells restricts Mycobacterium tuberculosis growth better than alveolar macrophages
Authors: Thandi Ramya Sivangala , Tripathi Deepak , Radhakrishnan Rajesh Kumar , Paidipally Padmaja , Vankayalapati Ramakrishna
Published: January 2023

Kupffer cells protect liver from bacterial infections. In the current study, we compared Mycobacterium tuberculosis (M.tb) growth and cytokine production by mice kupffer cells and alveolar macrophages. M.tb H37Rv infected kupffer cells and alveolar macrophages produced equal amounts of TNF-α, IL-6, IL-10 & IL-1β. In contrast, kupffer cells restricted M.tb growth better than alveolar macrophages (1 ± 0.346×106 CFU vs. 4 ± 0.916×106 CFU, p<0.03). There is no significant difference in the apoptosis of M.tb infected kupffer cells and alveolar macrophages. In contrast, M.tb infected kupffer cells expressed significant higher amounts of autophagy molecules LC-3B, ATG-7, ATG-5 and Beclin-1 (p<0.005, p<0.002, p<0.0032 & p<0.004 respectively) compared to M.tb infected alveolar macrophages as determined by real-time PCR. This was confirmed by Western blot and confocal microscopy. Our results suggest autophagy is involved in better restriction of M.tb growth by kupffer cells. Prime PCR analysis for 35 intracelluar signaling molecules those are involved in autophagy as well as in cytoskeleton indicated that M.tb infected kupffer cells significantly express higher levels of VASP (Vasodilator-stimulated phosphoprotein), RhoA (Ras homolog gene family member A) and Arp3 (Actin–related protein 3) genes (two fold, p<0.01, p<0.02&p<0.02 respectively) compared to M.tb infected alveolar macrophages. Studies are underway 1. To confirm whether autophagy is involved in better restriction of M.tb growth in kupffer cells. 2. Determine the role of cytoskeleton proteins those are involved in enhanced autophagy and M.tb growth inhibition in kupffer cells. 3. Determining in vivo relevance to our current findings using mouse model of M.tb infection.
Lung tissue resident memory T cells coordinate effector T cell dynamics during the protective recall response to influenza
Authors: Paik Daniel H. , Farber Donna L.
Published: January 2023

Influenza remains a serious public health challenge as the current vaccination strategy cannot protect against new strains that emerge every season. Tissue resident memory T cells (TRM) are a non-circulating memory subset that are generated in the lung after influenza infection or intranasal live vaccination, and these lung TRM have been shown to confer broad cross-strain protection. However, the mechanisms by which both CD4+ and CD8+ TRM mediate their protective response are not well defined. We investigated the tissue specific events during an active murine influenza response, including TRM-mediated protection and effector T cell trafficking. To specifically determine the role of TRM, we treated mice with the drug Fingolimod (FTY720), which sequesters circulating lymphocytes into secondary lymphoid organs, enriching the lung for tissue resident memory cells. We found that TRM immediately confer protection, with reduced viral titers evident as early as day 3 after infection. At later time points beginning at days 4–5 post infection, we determined that CD4+ and CD8+ TRM enhance the recruitment of influenza-specific effector T cells into the lung resident niche as well as CD4+ and CD8+ lymphocyte in situ proliferation as measured by BrdU incorporation. Furthermore, total lung RNA sequencing during active infection and FTY720 treatment reveal candidate TRM-associated chemokine pathways for tissue lymphocyte trafficking. These findings indicate that TRM uniquely influence the cellular dynamics of the lung resident niche to mediate cross-strain protection against influenza.
Fast and easy immunomagnetic isolation of untouched human naïve T cells in less than 15 minutes
Authors: Ewen Catherine , MacDonald Neil , de Jong Susan , Kokaji Andy I. , Woodside Steven M. , Eaves Allen C. , Thomas Terry E.
Published: January 2023

Peripheral blood circulating T cells can be subdivided into either antigen experienced effector and memory T cells or naïve T cells that have not yet encountered their cognate antigen. Naïve T cells are valuable tools when studying mechanisms of T cell activation and are required for foundational research into infectious diseases, cancer and transplantation.

We describe here a simple immunomagnetic cell isolation protocol for the isolation of untouched human naïve T cells from fresh or previously frozen PBMC or leukapheresis samples. The column-free, negative selection procedure involves labelling and removing unwanted cells using bispecific antibody complexes that crosslink cell surface antigens to magnetic particles. The labelling cocktail is composed of antibodies that target non-T cells, memory T cells, and an optional cocktail for depleting gamma delta T cells. Briefly, the procedure involves a five minute incubation with the antibody cocktail followed by the addition of magnetic particles and two three minute magnetic separations in a hand-held EasySep™ magnet. Following the second magnetic separation, the untouched naïve T cells are simply poured off into a new tube and are ready for use.

We define naïve T cells phenotypically as CD3+CD45RA+CD45RO-CD197+. Starting from fresh PBMC, we were able to obtain purities of 96.1 ± 2.3% (mean ± SD, n=14) and recoveries of 62.6% ± 24.5% (mean ± SD, n=14). Additionally, isolated naïve T cells are functional and respond by upregulating the expression of CD25 and CD69 upon stimulation. Our EasySep™ Human Naïve Pan T Cell Isolation Kit offers a fast and easy isolation method for naïve T cells that is suitable for downstream applications such as flow cytometry, cell culture or DNA/RNA extraction.
Depletion of Dead Cells from Primary Tissue in 6 Minutes
Authors: MacDonald Neil , Barr Christine , McQuenn Karina , Woodside Steven M. , Thomas Terry E. , Eaves Allen C.
Published: January 2023

Tissue specific research often requires mechanical and/or enzymatic digestion to isolate and study certain cell types. The digestion can be a harsh process resulting in a significant number of dead cells in the final cell suspension. Subsequent analysis by flow cytometry is difficult to interpret due to non-specific binding of antibodies to dead cells and dead cell auto fluorescence. Factors released by dead cells can also interfere with downstream assays, complicating the study of primary tissues.

During apoptosis, the cell membrane loses its phospholipid asymmetry resulting in exposure of negatively charged phospholipids on the cell surface. Relocation of phosphatidylserine (PS) to the outer leaflet of the cell membrane is a well-established marker of apoptosis. By targeting exposed PS with Annexin V we have developed a rapid method (EasySep™) to immunomagnetically remove dead cells from primary tissue samples.

Performance of this kit was examined on various mouse and human tissue types. Using this method, we were able to improve viability of a single cell suspension of mouse lungs digested with collagenase/hyaluronidase from an initial viability of 39.6 ± 12.3% AnxV−/PI− to 70.9 ± 11.8% AnxV−/PI. From 1×10^8 total start cells, 1.43 ± 0.68 ×10^7 live cells were recovered (n=10). From human polymorphonuclear leukocytes cultured overnight, viability was improved from 23.7 ± 9.8% AnxV−/PI− to 67.7 ± 12.1% AnxV−/PI− with recovery of 1.27 ± 0.52 ×10^7 live cells from 1×10^8 total start cells (n=7). This equates to removal of 88.9 ± 8.3% and 91.8 ± 5.3% dead cells respectively. Since live cells are untouched, subsequent isolation of desired cell types can be performed, resulting in a more viable population of cells for downstream applications.
Integrative analysis reveals CD38 as a therapeutic target for plasma cell-rich rheumatoid arthritis, pre-rheumatoid arthritis and systemic lupus erythematosus
Authors: Cole Suzanne , Walsh Alice , Yin Xuefeng , Wechalekar Mihir , Smith Malcolm , Proudman Susanna M. , Veale Douglas J. , Fearon Ursula , Pitzalis Constantino , Chiu Christopher , Sharp Michael , Alvarez John , Anderson Ian , Madakamutil Loui , Nagpal Sunil , Guo Yanxia
Published: January 2023

Autoantibodies play a significant role in the progression and pathogenesis of many autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). No studies have analyzed the expression of plasma cell/plasmablast genes during RA disease progression and the potential of an anti-CD38 antibody in depleting plasma cells and plasmablasts for efficacy in autoimmunity. Therefore, we interrogated the rationale of daratumumab, an anti-CD38 monoclonal antibody, as a potential therapeutic in RA and SLE. RNA-Seq analysis of synovial biopsies from various stages of RA disease development shows that plasma cell/plasmablast-related genes CD38, XBP1, IRF4, PRDM1, IGJ and TNFSF13B are significantly up-regulated in synovial biopsies from arthralgia, undifferentiated arthritis, early RA and established RA compared to healthy and osteoarthritis controls. In addition, flow cytometry analysis reveals highest CD38 expression on plasma cells and plasmablasts compared to natural killer cells, classical dendritic cell (DC), plasmacytoid DC, and T cells, in peripheral blood of healthy control, SLE and RA donors. We show that IGJ expression mRNA strongly correlates with the number of plasma cell/plasmablast in SLE patient PBMCs. Most importantly, IGJ mRNA down-regulation correlates with daratumumab-mediated depletion of these cells ex vivo. The data indicates a potential use of IGJ mRNA as a surrogate pharmacodynamic biomarker in clinical testing of daratumumab. Taken together, our data provides rationale for daratumumab in the treatment of RA and SLE.
Novel targeted therapies for African-American and European-American SLE patients identified from E-Genes elucidated by transancestral SNP mapping
Authors: Aidukaitis Bryce N. , Labonte Adam C. , Catalina Michelle D. , Bachali Prathyusha , Rouffa Sean , Ainsworth Hannah C. , Marion Miranda C. , Howard Timothy D. , Langefeld Carl D. , Lipsky Peter E. , Grammer Amrie C.
Published: January 2023

SLE is more prevalent and severe in African-American (AA) compared with European-American (EA) populations but standard-of-care drugs vary in effectiveness between these groups. To gain insight, drugs specific for E-gene containing pathways were investigated using the Immunochip transancestral SNP mapping and a curated database of SLE DE genes. SNPs proxy to SLE-associated SNPs were compared with known eQTLs contained in the GTEx database. Since GTEx contains genotype data as well as gene expression data from cell lines, tissues and whole blood from healthy individuals, an independent query of GTEx for expression of RNAs abnormally expressed in SLE patients compared to healthy individuals was carried out. The results of the eQTL and DE gene queries of GTEx were combined and eQTLs and associated E-Genes were pooled by ancestry. Drugs targeting E-genes/pathways shared by EA and AA include ibrutinib, ruxolitinib and ustekinumab. Predicted EA-specific drugs include antimalarials and cyclosporine, as well as drugs-in-development targeting CD40, CXCR1 and CXCR2; AA-specific drugs include retinoids and HDAC inhibitors. This analysis of the distinct genetic profiles of EA and AA populations indicates that unique sets of drugs may be particularly effective at treating lupus in each ancestral group.
A Low molecular weight BAFF receptor antagonist inhibits differentiation of human peripheral B cells into plasma blasts/plasma cells in vitro.
Authors: Yoshimoto Keiko , Seki Noriyasu , Suzuki Katsuya , Sugahara Kunio , Takeuchi Tsutomu
Published: January 2023

Backgrounds and Purpose

We have demonstrated that the elevated expression of BAFF receptor (BR3) on monocytes is involved in the overproduction of IgG by B cells in patients with primary Sjögren’s syndrome (pSS) which is often accompanied with hypergammaglobulinemia. We discovered a low molecular weight compound, BIK13, which inhibits binding of BAFF to BR3, by our original high-throughput screening system. We found that BIK-13 suppressed IL-6 production by BAFF-stimulated pSS monocytes. In this study, we investigated inhibitory effects of BIK-13 on B cell functions.

Methods

PBMCs were cultured with a B cell-stimulants cocktail, such as anti-IgM and CD40 antibodies, human IL-21 and human BAFF, in the presence of BIK-13. B cell-differentiation was monitored through analysis of the expression levels of CD19/CD38/IgD and activation-induced cytidine deaminase (AID) by FACS and qPCR, respectively. The amounts of IL-6 and IgG produced in vitro by the cells were measured by ELISA.

Results

Increased production of IgG and IL-6 by PBMC upon stimulation with B cell-stimulants was suppressed by BIK13 in a dose dependent manner. FACS analysis indicated that differentiation of B cells into plasma blasts and/or plasma cells was inhibited by BIK-13. In addition, the expression level of AID was also suppressed by the compound, suggesting that the IgG class switching was impaired.

Conclusion

These data collectively suggest that BIK-13 suppresses the B cell functions and may provide a novel therapeutic possibility to treat autoimmune diseases such as pSS.
Neutrophils contribute to T cell activation via reactive oxygen species production in HIV infection
Authors: Dunsmore Garett John , Shahbaz Shima , Houston Stan , Elahi Shokrollah
Published: January 2023

Neutrophils are the most abundant leukocytes in the human immune system contributing to 50–80% of all the white blood cells in the peripheral blood. Interestingly, the role of neutrophils in HIV infection has been poorly investigated. Previous studies have demonstrated that during HIV infection neutrophils are capable of both activating T cells and suppressing T cells. Our preliminary findings suggest that T cell activation could be modulated by neutrophils through various mechanisms. Galectin-9 has been demonstrated to play an integral role in HIV pathogenesis by suppressing CD8+ T cell function through the engagement with TIM-3. Interestingly, we have observed neutrophils have high expression of galectin-9, which is reduced after prolonged activation. In confirmation with previous studies demonstrating that HIV infected patients have hyperimmune activation, galectin-9 expression is decreased on neutrophils from HIV infected individuals, while the expression of the neutrophil activation marker CD32 is increased. Additionally, galectin-9 expression on neutrophils could be involved in suppressing T cell responses via. galectin-9:TIM-3 engagement.

We found that neutrophils from HIV patients enhance T cells activation when co-cultured, whereas this was not the case for healthy controls. Interestingly, these effects could be reversed by inhibiting neutrophil reactive oxygen species (ROS) production. These findings suggest that HIV infection results in hyperimmune activation of neutrophils contributing to T cell activation.
The surprising role of Indoleamine 2,3-dioxygenase (IDO) in supporting the survival of bone marrow resident long lived plasma cells (LLPC)
Authors: Lightman Shivana M , Carlson Louise M , Lee Kelvin P
Published: January 2023

Long lived plasma cells (LLPC) are a key component of protective humoral immunity. The ability of these cells to maintain longevity and a durable antibody response is largely dependent on the complex nature of the bone marrow (BM) microenvironment in which these cells reside. Our lab has demonstrated that the co-stimulatory receptor, CD28, is essential for the survival and function of LLPC. Furthermore, when CD28 on LLPC ligates with CD80/CD86 expressed on DC, it induces upregulation of the enzyme indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan (Trp) into kynurenine (kyn), a well-known ligand of the aryl hydrocarbon receptor (AhR). AhR is a nuclear transcription factor that can modulate immune responses and is highly expressed in plasma cells (PC). Our preliminary data reveals that IDO knock-out (KO) mice in comparison to wild type (Wt) mice have a decrease in bone marrow resident PCs and a decrease in overall Ig levels. Additionally, 15 weeks post vaccination IDO KO mice have significantly less antigen-specific long lived antibody titers compared to Wt. It was also observed that interaction with DC both in vitro and in vivo is needed for LLPC survival and function. Moreover, AhR activation by kyn causes an increase in CD28 mRNA in LLPC. This leads us to propose a model where CD28 interaction with CD80/CD86 induces IDO production, which through its metabolite kyn activates AhR which can then regulate CD28 expression- thus creating a mechanism that promotes LLPC survival and function. These findings challenge the traditional idea of IDO as an inhibitory molecule and provide the rationale for investigating its essential role in maintaining LLPC survival and sustained antibody responses.
Infant T cells exhibit increased TCR signaling and proliferation to respiratory infection
Authors: Thapa Puspa , Guyer Rebecca S , Farber Donna L
Published: January 2023

Infants are highly susceptible to respiratory infections and frequently experience recurrent episodes. Our previous study demonstrated that tissue resident memory T cells (TRM), which are critical in mediating long-term protection against pathogens, are reduced in infant mice compared to adult mice after influenza infection. However, the underlying mechanisms for reduced TRM generation in infants is still unknown. Our preliminary data show that infant T cells have enhanced TCR sensitivity leading to increased activation compared to adult T cells and thus are biased towards effector differentiation at the expense of memory formation. We show that infant OVA-specific (OTII) T cells exhibit lower T cell activation threshold for proliferation and enhanced sensitivity for low does of antigen. Infant T cells express higher levels of CD69 and CD25 with a greater induction of Nur77, a direct indicator of TCR signal strength, at peptide doses that fail to fully activate adult T cells. Co-transfer of infant and adult OTII T cells into a congenic host resulted in infant T cells dominating the effector response in the lungs (at a ratio of 3:1) after primary influenza challenge. At the peak of infection, infant effector T cells also expressed lower levels of TCF1 and Bcl-2, indicating transcriptional bias for terminal effector differentiation with reduced survival. Moreover, infant derived effector and memory T cells express lower levels of CD5, suggesting enhanced survival of lower affinity T cells after influenza infection. Taken together, these results show that enhanced sensitivity for antigenic stimulation promotes cell intrinsic mechanisms that govern decreased potential of infants to form durable and functional lung TRM.
Tumor necrosis factor receptor associated factor 3 (TRAF3) regulates B cell antigen receptor (BCR) signaling
Authors: Whillock Amy Louise , Bishop Gail A
Published: January 2023

Tumor necrosis factor receptor associated factor 3 (TRAF3) is an intracellular adaptor protein that plays multiple cell type- and context-dependent roles. In B cells, TRAF3 negatively regulates signaling by several receptors important to survival and activation, including the B cell activating factor receptor (BAFF-R), CD40, toll-like receptors and the IL-6 receptor. Deletions or inactivating mutations of TRAF3 are common in the human B cell malignancies multiple myeloma and B cell lymphoma (BCL). Enhanced homeostatic B cell survival in B cell-specific TRAF3-deficient mice (B-Traf3−/−) causes enlarged secondary lymphoid organs, spontaneous germinal centers, and autoantibody production. Highlighting the role TRAF3 plays as a B cell tumor suppressor, B-Traf3−/− mice develop BCL at an increased rate as they age.

The B cell antigen receptor (BCR) is crucial for B cell antigen recognition, survival, proliferation, and antibody production. Defects in BCR signaling can lead to malignancy, and many BCLs are dependent upon BCR signaling for survival. TRAF3 regulates the signaling of several pathways that interact with the BCR pathway, but the role of TRAF3 in regulating BCR signals is unknown, a knowledge gap we are currently addressing. Our initial studies show that TRAF3 associates with CD79b of the BCR complex. Phosphorylation of Syk, a proximal BCR signaling target, is increased after BCR stimulation in the absence of TRAF3, with increased downstream phosphorylation and activation of Btk, PLCγ2, Akt, Erk, and canonical NF-κB. This suggests that TRAF3 plays an important role as a regulator of BCR signaling. Ongoing studies are examining the mechanisms of this regulation.
A screening platform for human plasma cells (PCs) reveals a selective response to SYK inhibition
Authors: Campbell Michelle , Stephenson Sophie J , Whale Kevin , Rapecki Stephen , Finney Helene , Doody Gina M , Tooze Reuben M
Published: January 2023

Therapeutic targeting of PC populations has potential value in various auto-immune, allergic and neoplastic cell states, but at present few options exist for selective targeting. However, the unique physiology and niche dependence of PCs suggests that such targeting may be achievable. To this end we have developed a sensitive screening platform capable of detecting changes in PC biology. Starting from our published methods for in vitro PC generation we optimised conditions to allow maintenance of PCs in polarised niche environments reflective of homeostatic and inflammatory conditions in microtitre plates. Using this assay format we probed PC biology via small molecule inhibitors (SMIs) targeting BCL2 family members, proteasome, and kinase signalling pathways. We were able to identify SMIs with broad and selective toxicity, as well as SMIs with no effect on PCs. Under all conditions analysed we found consistent divergence in the impact of targeting aspects of B cell receptor (BCR)-associated signalling pathways. Targeting SYK via two distinct SMIs consistently resulted in selective killing of subsets of PCs. By contrast BTK inhibition had no significant effect on PC viability. Whilst the importance of SYK signalling has been well documented at the B cell stage its importance to PCs has to our knowledge not been extensively documented. Retention of functional BCRs has been recently described in BM IgA+ and IgM+ PCs, and examination of expression data indicates selective retention of components of the BCR signalling complex in our assay system. The data presented here demonstrate the potential utility of an in vitro screening platform to evaluate the selective dependency of subsets of PCs on therapeutically targetable pathways.
CD209 activation promotes survival of lymphoblastic human B cells
Authors: Valentin Anibal J , Díaz Glorianne Y
Published: January 2023

The role of B cells in host protection and antibody production depends in part on their ability to recognize antigens and pathogen associated molecular patterns (e.g. carbohydrates and nucleic acids) mostly through membrane bound receptors. CD209 (i.e. DC-SIGN) is a C-type lectin receptor expressed on dendritic cells, macrophages and B cells which binds to different oligosaccharides and plays a role in antigen capture and cell adhesion. CD209 also functions as a receptor for many bacterial, fungal and viral pathogens. In B cells, CD209 expression enhances infections by HHV8 and H5N1 viruses and in HIV positive patients, promotes trans-infection of CD4+ T cells. Besides its role in cell adhesion and pathogen recognition, the role of CD209 in B cell physiology remains elusive. Our goal is to identify and characterize CD209-induced signaling pathways in human B cells and determine how these pathways relate to normal and pathologic B cell responses. Our data shows that CD209+ lymphoblastic B cells have a higher level of basal cell death suggesting that expression sensitizes B cells to apoptosis. Stimulating CD209+ B cells using an anti-CD209 antibody or Mannan, promotes cell survival. Moreover, CD209 stimulation inhibits BCR-mediated programmed cell death and modifies the expression of several genes involved in cell survival and apoptosis (e.g. Bim, BclXL, Xiap). Elucidating the role of CD209 in B cell physiology will have important implications towards understanding the development of the humoral immune response to pathogens such as HHV8, Zika, Dengue and HIV. Moreover, our results will set the groundwork for future studies on vaccine development and biomarker discovery. Supported by NIGMS-INBRE P20 GM103475-14
IL-33 induces a metabolic programming supporting dendritic cells tolerogenicity
Authors: Velayutham Murugesan , Roessing Anna S , Shiva Sruti , Turnquist Heth R
Published: January 2023

Objective

Delivery of the stromal cell-derived cytokine IL-33 protects allografts from rejection by acting on CD11c+ DC that facilitate the expansion of regulatory T cells (Treg). IL-33 administration also permits tolerance induction at doses of co-stimulatory blockade that fails on their own. Based on these data we hypothesized that IL-33-stimulated DC represent a tolerogenic DC subset and their precise examination will define biochemical pathways that contribute to tolerogenic DC functions.

Methods

Mouse bone marrow-derived CD11c+ DCs were treated with lipopolysaccharide (LPS) or IL-33 before nitric oxide (NO) generation, glucose and fatty acid uptake, and mitochondrial function and potential were assessed by EPR spectroscopy, flow cytometry, and Seahorse analysis.

Results

DC responded to the TLR4 ligand LPS by generating high levels of NO and shifting their metabolic activity to aerobic glycolysis. This was in stark contrast to IL-33, which caused a metabolic reprogramming in DC that involved augmented mitochondrial basal respiration, ATP production, and maximum respiration. IL-33-stimulated DC also displayed increase mitochondrial potential and a profound increase in the uptake of fatty acids.

Conclusions

It has emerged that immunogenic DCs undergo metabolic changes that enables their pro-inflammatory functions. The metabolic underpinnings of tolerogenic DCs is less understood. Here we show that IL-33-stimulated DC, which potently expand Treg, rely on oxidative phosphorylation and uptake fatty acids. These data suggest IL-33 initiates metabolic reprogramming matching those found in regulatory myeloid populations, including tumor associated DC and alternatively activated macrophages.
Investigation on the role of temperature in macrophage responses
Authors: Hassan Intisar Abdullah , Durdik Jeannine M , Satyajit Rath , Renfro Zachary , Blake Harrison
Published: January 2023

This study investigated temperature-associated changes in macrophage activation and metabolism across species. Macrophages were isolated from C57BL/6J mice, broiler chicken spleens, and rainbow trout head kidneys, treated with lipopolysaccharide (LPS) and nitric oxide (NO) responses measured. Mouse macrophage NO production was similar at 37°C (normal body temp.) and 35°C. However at 33°C and 28°C responses dropped notably. At 39°C, Mouse bone marrow-derived macrophages (BMDM) and mouse cell line, RAW264.7, showed higher NO production versus 37°C. In chicken splenic macrophages (CSM) LPS-induced NO responses were similar at 41°C (normal for avian) 37°C, and 39°C. A mild fever of 42°C had a large stimulatory effect on NO production compared to 41°C. During infection, trout induce fever by swimming to warmer water. We found NO responses in LPS-treated head kidney macrophages to be higher at 15, 28, and 37°C than at 4°C. Total protein synthesis was evaluated via fluorescence. Without LPS stimulation, peritoneal resident mouse macrophages (PRM) from young mice showed similar protein synthesis at 37°C and 39°C. In both young and aged LPS-stimulated PRM, significantly higher protein synthesis was observed at 39°C compared to 37°C, though the increase was smaller in aged PRM. However, this effect was not uniform across all tissues. LPS stimulated young (BMDM) showed an increased protein synthesis at 37°C but cells from aged did not. A further increase in LPS stimulation was observed at 39°C. Consistent with fever increasing protein synthesis, LPS-stimulated CSM and chicken cell line HTC, showed higher protein synthesis at 42°C compared with 41°C.
Activation of cannabinoid receptor type-1 (CB1) and type-2 (CB2) inhibits the release of vascular endothelial growth factor (VEGF-A) from human neutrophils
Authors: Braile Mariantonia , Scala Sara , Modestino Luca , Cristinziano Leonardo , Marcella Simone , Ferraro Anne Lise , Galdiero Maria Rosaria , Loffredo Stefania
Published: January 2023

Background

Neutrophils (PMNs) are innate immune cells with primary roles in the acute phase of inflammation and resistance against invading pathogens. Activation of cannabinoid receptor type-1 (CB1) and −2 (CB2) has been suggested as a new strategy to modulate angiogenesis in vitro and in vivo. Angiogenesis, the formation of new blood vessels is complex processes that require the coordinated action of several factors, called vascular endothelial growth factors (VEGFs) and angiopoietins (Ang). These factors are produced by several cells, included PMNs.

Objective

We sought to investigate whether endocannabinoids and their receptors modulate PMNs production of VEGF-A, Ang1, CXCL8 and reactive oxygen species (ROS) production.

Methods

PMNs were isolated from buffy coats of healthy donors. Cells were stimulated with CB1 and CB2 agonists/antagonists alone and in association with LPS. Mediators concentrations in cell-free supernatants were measured using ELISA, whereas ROS production was evaluated by fluorescence assay.

Results

CB1 and CB2 agonists did not induce VEGF-A, Ang1 and CXCL8 release from PMNs but reduced spontaneous release of VEGF-A. LPS activated PMNs causing VEGF-A and CXCL8 release. Interestingly, preincubation of PMNs with agonists inhibited in concentration dependent manner VEGF-A release induced by LPS but did not affected CXCL8 production. The effect of CB agonists of LPS induced VEGF-A was reverted by the treatment with CB antagonists. Finally, CB agonists and antagonists promoted ROS production.

Conclusions

Activation of CB1 and CB2 agonists on PMNs might be a novel strategy to modulate immune cell-assisted angiogenesis.
Irgm1-deficiency leads to myeloid dysfunction in colon lamina propria and susceptibility to the intestinal pathogen Citrobacter rodentium
Authors: Hammer Gianna , Huang Hsin-I , Taylor Gregory
Published: January 2023

IRGM and its mouse orthologue Irgm1 are dynamin-like proteins that regulate vesicular remodeling, intracellular microbial killing and pathogen immunity. IRGM dysfunction is also linked to inflammatory bowel disease (IBD), and while it is thought that defective intracellular killing of microbes underscores IBD susceptibility, studies have yet to address how IRGM/Irgm1 regulates immunity to microbes relevant to intestinal inflammation. Here we find that loss of Irgm1 confers marked susceptibility to Citrobacter rodentium, a noninvasive intestinal pathogen that models inflammatory responses to intestinal bacteria similar to that of IBD. Irgm1-deficient mice fail to control C. rodentium outgrowth in the intestine, leading to systemic pathogen spread and host mortality. Surprisingly, susceptibility due to loss of Irgm1 function was not linked to defective intracellular killing of C. rodentium or exaggerated inflammation, but was instead linked to failure to remodel specific colon lamina propria (C-LP) myeloid cells that expand in response to C. rodentium infection and are essential for C. rodentium immunity. Defective immune remodeling was most striking in C-LP monocytes, which were successfully recruited to the infected C-LP, but subsequently underwent apoptosis. Apoptotic susceptibility was induced by C. rodentium infection and was specific to this setting of pathogen infection, and was not apparent in other settings of intestinal inflammation. These studies reveal a novel role for Irgm1 in host defense and suggest that deficiencies in survival and remodeling of C-LP myeloid cells that control inflammatory intestinal bacteria may underpin IBD pathogenesis linked to IRGM dysfunction.
TLR5 and TLR7 amplify different stage of myeloid cells
Authors: Jung Minho , Choi Eun Young
Published: January 2023

Balance between immune activation and immune suppression is important for immune homeostasis. Toll-like receptors (TLRs) play key roles in the innate immune system, initiating inflammatory responses against pathogen infections and internal danger signals. Different TLRs recognize different kinds of ligands, but not all the TLRs do have same effect: some TLRs are immune stimulatory, while the others are immune suppressive. For instance, TLR5 ligand has been known to be immune suppressive, while TLR7 ligand has been used for immune activation. To understand how these TLR ligands lead to opposite immune outcomes, we investigated the effects of the ligands on myeloid cell differentiation. Treatment of TLR5 ligand flagellin resulted in the increase of CD11b+ Ly6C+Ly6G+ myeloid-derived suppressor cells (MDSCs) on days 2 and 3 after culture initiation. However, TLR7 ligand gardiquimod led to enhancement of dendritic cell differentiation on day 1, and then increase of CD11c− MHCII− CD11b− Ly6C− Ly6G− progenitor population on day 2, which differentiate into CD11c− MHCIIow CD11b+ Ly6C+ cells with DC differentiation potential afterwards. Based on these results, we suggest that different immune outcomes of TLR ligands would depend on the stages of myeloid cells at which corresponding TLRs act, contributing to overall immune homeostasis.
Repeated malaria exposures associate with skewing of monocytes/macrophages toward a regulatory phenotype
Authors: Guha Rajan , Arora Gunjan , Li Shanping , Doumtabe Didier , Doumbo Ogobara k , Doumbo Safiatou , Kayentao Kassoum , Ongoiba Aissata , Traore Boubacar , Crompton Peter D
Published: January 2023

In malaria-naïve individuals, P. falciparum (Pf) infection results in numerous Pf-infected red blood cells (iRBCs) that trigger systemic inflammation and fever. Conversely, repeatedly infected individuals in endemic areas are often asymptomatic and have low levels of iRBCs, even children who have yet to acquire reliably protective antibodies. The molecular mechanisms underlying these clinical observations are unclear. PBMCs collected from Malian children before the malaria season responded to iRBCs by producing pyrogenic, pro-inflammatory mediators such as IL-1β, IL-6 and IL-8. However, following febrile malaria there was a marked shift in the response to iRBCs with the same children’s PBMCs producing lower levels of those cytokines. These data suggest that malaria-induced epigenetic reprogramming of innate immune cells may play a role in immunity to malaria. Accordingly, age-stratified analysis of monocytes collected before the malaria season showed an inverse relationship between age and pro-inflammatory cytokine production capacity. Monocytes of Malian adults expressed higher levels of CD163 and arginase1, associated with a regulatory phenotype. These observations were recapitulated with an in vitro system of monocyte-macrophage differentiation whereby re-exposure to iRBCs was associated with diminished expression of pro-inflammatory mediators and a corresponding decrease in epigenetic markers of active gene transcription (i.e. H3K4me3) at the TSS of the same pro-inflammatory mediators. Together these data support the hypothesis that epigenetic reprogramming of monocytes/macrophages toward a regulatory phenotype contributes to clinical immunity to malaria.