bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycogen metabolism supports effector function and energy homeostasis of dendritic cells
Authors: Thwe Phyu M. , Beauchamp Saritha , Amiel Eyal
Published: January 2023

As professional antigen presenting cells of the immune system, dendritic cells (DCs) serve as a bridge between innate and adaptive immune responses. Activation of DCs by a stimulus through toll-like receptors (TLRs) is coupled with an increase in metabolic demand, which is fulfilled by a TLR-driven burst in glycolytic metabolism. Up-regulation of glycolysis in activated DCs provides metabolites and energy required for DC effector function, and inhibition of glycolysis impairs the survival and effector function of these cells. The prevailing model in the field proposes that TLR-driven glycolysis is sustained primarily by an increase in glucose uptake. While non-immune cells store glucose in the form of glycogen as an intracellular energy reserve, the role of glycogen metabolism in DCs has not been reported. Our data indicate that glycogen metabolism supports the activation of DCs, particularly before these cells increase the surface expression of glucose transporter-1 (GLUT1). We show that DCs express the enzymes essential for glycogen metabolism, that the glycogen metabolism is regulated upon TLR stimulation, and that inhibition of the glycogen metabolism in DCs impairs the expression of key maturation-associated molecules in these cells. Additionally, our data suggest that glycogen-derived glucose carbons feed into the tricarboxylic acid (TCA) cycle to support early maturation of DCs. These data indicate that the glycogen metabolism in DCs plays an important role in energy homeostasis in these cells to support their effector function. Understanding the role and regulatory mechanisms of glycogen metabolism in DCs has important implications for the future development of enhanced cellular based immunotherapy.
Downregulation of human IL4 and IL13 expression by CRISPR/Cas9-mediated deletion of DNase I hypersensitive site HS11/12 from a transgenic human Th2 cytokine locus
Authors: Pivniouk Vadim I , Rosenbaum David , Pivniouk Oksana , Vercelli Donata
Published: January 2023

Production of the Th2 cytokines IL4 and IL13 is frequently dysregulated in allergy and asthma. Multiple genetic elements regulating expression of murine Th2 cytokines have been characterized by gene targeting. To study the regulation of human Th2 cytokines, we created BAC transgenic mice (BAC5TG) carrying a 160 kb fragment of the human Th2 locus encompassing RAD50, IL13 and IL4. This transgene includes all the regulatory elements required for physiological expression of mouse Th2 cytokines.

BAC5TG mice appear normal, are born at the expected Mendelian ratio, and demonstrate tissue-specific and transgene copy number-dependent expression of human Th2 cytokines. DNA methylation profiles across the transgenic human Th2 locus are highly comparable if not identical to those observed in human cells.

A DNase I hypersensitive site (HS) in the human IL13-IL4 intergenic region, previously identified in our lab and designated HS11/12, colocalizes with an area of extensive and profound CpG hypomethylation both in human and mouse transgenic T cells. However, unlike most Th2 regulatory elements identified to date, HS11/12 is not conserved between humans and mice. Using the CRISPR/Cas9 approach, we deleted HS11/12 from the human Th2 transgene in BAC5TG mice. Notably, production of both human IL13 and IL4 by in vitro differentiated Th2 cells from BAC5DHS11/12 TG mice was reduced by ~50%.

Our results suggest that HS11/12 represents a novel cis-regulatory element required for optimal production of the human Th2 cytokines IL4 and IL13. The application of CRISPR/Cas9 technology to BAC transgenic mice may provide a powerful tool to study human gene regulation.
Activation of Interferon Regulatory Factor 3 (IRF3) is Required in Both T cells and APCs for Optimal Expression of IFN-γ, IL-17, and Granzyme B during T cell responses.
Authors: Petro Thomas M , Guinn Zachary , Lampe Anna
Published: January 2023

Recently we saw a link between activation of Interferon Regulatory Factor (IRF)-3 and expression of IFN-γ, as well as Granzyme B (GrB) from T cells during viral infections in C57Bl/6 (B6) and IRF3KO mice. To investigate this further, we stimulated splenic mononuclear cells (SMC) from B6 and IRF3KO mice in vitro with anti-CD3±anti-CD28± poly I:C, a TLR3/IRF3 agonist. We show during responses with anti-CD3/anti-CD28 that IRF3 is activated within 24 h in B6 SMC and expression of IFN-γ, IL-17 and GrB is impaired in IRF3KO SMC. Adding poly I:C with anti-CD3/anti-CD28 accelerated IRF3 activation and significantly increased IFN-γ, but not IL-17 or GrB, from IRF3KO SMC. Interestingly, Foxp3 expression and percentage of Foxp3+ T cells were significantly lower in unstimulated IRF3KO SMC compared with B6 SMC. To determine the contribution of IRF3 activation in T cells vs APCs, B6 and IRF3KO bone marrow dendritic cells (DCs; 3×104) were mixed in various combinations with WT or IRF3KO purified T cells (3×105), as well as anti-CD3±anti-CD28± poly I:C. After 48 h, IFN-γ, IL-17, and GrB expression was greatest in cultures where both B6 DCs and B6 T cells were mixed. IFN-γ, IL-17 and GrB decreased when either IRF3KO DCs or IRF3KO T cells were part of the mixture. Least production of IFN-γ, IL-17, and GrB occurred when DCs and T cells were from IRF3KO mice. These results suggest that IRF3 in both APCs and T cells is required to achieve optimum production of IFN-γ, IL-17, and GrB during T cell responses. Moreover, the data unexpectedly show that IRF3 is activated during T cell immune responses without a TLR3 agonist. The factor(s) responsible for IRF3 activation during APC/T cell responses is unclear but appears to be a soluble factor whose expression is stimulated through CD28.
The LFA-1 signal for cell migration influences Notch and TGF-β pathways with functional consequences for T-cells
Authors: Verma Navin Kumar , Fazil Mobashar Hussain Urf Turabe , Theng Ong Seow , Chalasani Madhavi Latha S. , Praseetha P , Kumar Sunil , Panda Aditya K , Freeley Michael , Smith Sinead , Boehm Bernhard Otto , Kelleher Dermot
Published: January 2023

The integrin LFA-1 plays a key role in T-cell motility, which is critical for host defense and is also main driver of autoimmune diseases. However, whether and how LFA-1 signalling can modulate T-cell effector functions remain to be understood. We performed gene expression analysis of LFA-1-stimulated human T-cells and identified genomic signatures associated with Notch and TGF-β pathways, both involved in T-cell differentiation. The activation of Notch pathway in motile T-cells was confirmed by the nuclear translocation of cleaved Notch intracellular domain and up-regulation of target genes Hey1 and Hes1. LFA-1 stimulation of naïve peripheral blood mononuclear cells was found to promote T-cell Th1 polarization through a GSK3β signalling-dependent Notch pathway. We further detected that LFA-1 signalling up-regulated Stat3 and/or JNK activation-dependent expression of Smad7, Smurf2 and Ski causing T-cell TGF-β unresponsiveness. LFA-1-stimulated naïve T-cells were refractory to TGF-β-mediated induction of Foxp3+ iTreg or RORγt+ Th17 differentiation. Of note, peripheral or splenic T-cells isolated from patients with rheumatoid arthritis, type1 diabetes or thyroiditis showed constitutively high expression of Tbet. Pretreatment of cells with blocking anti-LFA-1 antibody, specific inhibitors or siRNA against identified molecules restored LFA-1-mediated modulation of T-cell functional phenotypes. Thus, LFA-1-mediated signalling is crucial for T-cell immunoregulation concurrent with T-cell motility, involving both Notch and TGF-β pathways. These findings have implications for normal immunologic functions and also have therapeutic relevance for inflammatory diseases.
Function of protein kinase CK2 in CD4+ T cell polarization and EAE
Authors: Gibson Sara , Qin Hongwei , Liu Yudong , Rowse Amber , Benveniste Etty
Published: January 2023

Auto-reactive CD4+ T cells drive many autoimmune diseases including multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS). CK2 is a constitutively expressed and highly conserved serine/threonine kinase that promotes a number of pro-survival and pro-inflammatory signaling pathways including PI3K/Akt/mTOR, NF-kB and JAK/STAT. These pathways are essential for CD4+T cell activation and polarization, but little is known about how CK2 functions in T cells. Here we demonstrate that protein levels of the major catalytic subunit of CK2, CK2α, are induced in murine CD4+ T cells upon activation, correlating with an increase in overall kinase activity. In addition, CK2α expression is elevated in CD4+ T cells that are activated and infiltrate the CNS during experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Targeting the activity of CK2α using the small molecule inhibitor CX-4945 in vitro significantly inhibited Th17 cell polarization and promoted FOXP3 expression in both Th17 and Treg polarizing conditions with no appreciable effect on the generation of Th1 cells. In vivo treatment with CX-4945 during EAE resulted in a dampened Th17 response in the periphery and a decrease in IL-17A+ and IL-17A+IFN-γ+ CD4+ T cells present in the spinal cord. Continued CX-4945 treatment significantly reduced the severity of EAE clinical symptoms and increased the percentage of FOXP3+ Tregs in the spinal cord during disease resolution. Our data implicate CK2 as a novel regulator of the Th17/Treg cell axis, and suggest that CK2 could be targeted for the treatment of CD4+ T cell-driven autoimmune disorders.
Image Based Assessment of NFkB Nuclear Translocation in Murine Cells with ImageStream Cytometry
Authors: Kong Raymond K , Pugsley Haley , Friend Sherree , Morrissey Phil
Published: January 2023

Nuclear Factor kappa B (NFkB) transcription factor plays a central role in regulating many key processes in mammalian cells, including differentiation, cell growth, inflammation, immunity, and stress responses. The activation of NFkB and its translocation from the cytoplasm to the nucleus has become a focal point for studying cellular function, signal transduction pathways, disease mechanisms and drug discovery efforts. The nuclear translocation of NFkB in human cells has been extensively studied with the ImageStream technology during the last decade. However, similar studies in murine cells has not been done. In this project, NFkB nuclear translocation in a murine cell line is measured using ImageStream cytometry. Images of activated and non-activated cells labeled with anti-NFkB antibody and a nuclear dye are acquired on the ImageStream cytometer. Images analysis is performed using IDEAS image analysis software. A Similarity score is determined for every cell based on a pixel by pixel correlation of the nuclear image to the NFkB image. As a result the mean similarity values in the non-activated and activated cell groups are determined. The increased mean similarity value is correlated with higher level of nuclear translocation. The project result will provide a robust method for measuring the NFkB nuclear translocation in the murine cells using ImageStream technology.
A diverse repertoire of memory CD4 T cells is maintained in the absence of the common gamma chain
Authors: Matson Courtney Alexandra , Johnson Lakesha , Warner Ross , Singh Nevil J
Published: January 2023

After successfully combating a pathogen, a small number of antigen-experienced T cells are retained in the body to offer better protection against future infections by the same agent. In the case of CD8 T cells, the stable maintenance of these memory phenotype T cells critically requires homeostatic turnover driven by cytokines such as IL-7 and IL-15. Intriguingly, CD4 T cells that are deficient in the shared receptor for IL-2, IL-7 and IL-15 – i.e. the cytokine common gamma chain (γc), can still generate memory phenotype (MP) T cells in vivo. Here we show that such naturally generated γc-deficient CD4 MP T cells have a diverse T-cell receptor repertoire usage, despite a profound deficiency in the naïve CD4 T cell compartment in such mice. Furthermore, adoptive transfer experiments show that γc-deficient CD4 MP T cells can be stably maintained even in a competitive environment (of a γc+ host) where other CD4 MP T cells can utilize IL-2, IL-7 and IL-15. CD4 MP T cells from γc-deficient animals also differentiated to IFN-γ producers. Therefore, unlike CD8 memory T cells, the maintenance of a diverse repertoire of CD4 memory T cells is independent of trophic cytokines such as IL-7 and IL-15.
Fast and easy isolation of CD27-positive human memory B cells using EasySep&[trade] Releasable RapidSpheres&[trade]
Authors: Maeshima Nina M , Jovanovic Vida K , Ewen Catherine L , Clarke Samuel J , Kokaji Andy I , Woodside Steven M , Thomas Terry E , Eaves Allen C
Published: January 2023

Over the course of a lifetime, memory B cells are key to maintaining antibody-mediated protection from viral and bacterial pathogens and they are one outcome of an effective vaccine. Since memory B cells are typically found at frequencies of less than 5% of normal human peripheral blood mononuclear cells (PBMC), research into their function would be aided by a fast and easy isolation method. To meet this need, we have developed an improved EasySep™ kit for the isolation of CD27-positive (CD27+) human memory B cells from fresh PBMC.

First, CD27+ cells are labeled using an antibody cocktail and EasySep™ Releasable RapidSpheres™ and are positively selected using a hand-held EasySep™ magnet. Next, magnetic particles are released from the positively-selected cells, and then non-B cells within the CD27+ fraction are targeted for depletion using a second antibody cocktail and EasySep™ Dextran RapidSpheres™. Following an additional magnetic separation step, the particle-free, CD27+ memory B cells are poured off into a new tube and ready for use. With one additional step, CD27− naive B cells can be isolated from the same starting sample. The entire isolation protocol is completed in under 40 minutes without any centrifugation steps.

Using this method, CD19+CD27+ memory B cells can be isolated to 97±2% purity and 37±14% recovery (average of n=19 ± S.D.). The CD19+CD27− naive B cells can be isolated to 93±5% purity and 28±11% recovery (n=9 ± S.D.). When stimulated with CpG and IL-15, the isolated memory and naive B cells are functional, as assessed by both proliferative responses and the secretion of IgG.
Experimental food-induced anaphylaxis is driven by C5aR1 actvivation on mast cells
Authors: Koehl Joerg , Kordowski Anna , Wu David , Hogan Simon P
Published: January 2023

Food-induced anaphylaxis is a serious allergic reaction mainly caused by antigen cross-linking of IgE-loaded Fce-receptors on mast cells (MCs). Such MC activation leads to the release of pro-inflammatory mediators which results in the manifestation of the disease. Here, we demonstrate that the complement fragment C5a plays an important and until now not recognized role in the development and severity of food allergy. We subjected C5aR1−/− and wild-type (wt) Balb/c mice to oral ovalbumin (OVA)-induced anaphylaxis. We observed that 78% of wt but only 9% of C5aR1−/− mice suffered from diarrhea and hypothermia after the 7th oral OVA challenge. Importantly, peritoneal MCs strongly expressed C5aR1 after the 7th oral OVA treatment. OVA-specific serum IgE and MCPT-1 levels were significantly decreased in C5aR1-deficient as compared with wt mice. To directly examine the impact of the C5a/C5aR1 axis on MC function, we generated bone marrow-derived MCs (BMMCs) and tested their activation and degranulation potency in response to FceR1 cross-linking. Degranulation of C5aR1−/− BMMCs was reduced by 50% and IL-6 production was decreased by 35% as compared with wt cells. Also, FceR1 cross-linking markedly upregulated C5aR1 MC expression. Further, in contrast to wt mice, C5aR1−/− mice were largely protected from histamine-induced temperature drop in vivo. In line with this finding, C5aR targeting protected human endothelial cells from histamine-induced barrier dysfunction and disruption of tight junction proteins in vitro. Our findings identify a critical role of the C5a/C5aR1 axis in IgE-mediated oral antigen-induced anaphylaxis. C5aR1 may serve as a novel therapeutic target to suppress the inflammatory response in food-induced anaphylaxis.
Peptide-deficient forms of HLA-B: Prevalence and Function
Authors: Geng Jie , Altman John , Krishnakumar Sujatha , Raghavan Malini
Published: January 2023

Human leukocyte antigen class I (HLA-I) molecules comprise a highly polymorphic heavy chain complexed to a conserved light chain, b2-microglobulin (b2m), and peptide. When bound to antigenic peptides, HLA-I molecules mediate CD8+ T cell responses against cancers and intracellular pathogens. The transporter associated with antigen processing (TAP) is the major source of cellular peptides for the assembly of HLA-I molecules in the endoplasmic reticulum (ER). Consistent with previous findings of varying stabilities of peptide-deficient forms of HLA-B, we show allotype-dependent variations in the cell surface expression of HLA-B molecules in TAP-deficient and TAP-inhibited cells. HLA-B allotypes that are resistant to inhibition of TAP (RIT-HLA-B) exist on the cell surface in empty or suboptimally loaded forms under TAP-deficiency conditions, even at physiological temperatures. Furthermore peptide-deficient forms of RIT-HLA-B allotypes bind to primary CD8+ T cells via a mode of binding that is CD8-dependent. Together, these findings indicate allotype-dependent variations in the prevalence of peptide-deficient HLA-B molecules and demonstrate previously unrecognized functions for “empty” HLA-I.
Indispensable role of IL-10 in the development of antigen cross-presentation capacity of the cDC1 subset
Authors: Hanakawa Sho , Kitoh Akihiko , Kabashima Kenji
Published: January 2023

The conventional type 1 dendritic cell (cDC1) is a distinct DC subset with specialized functions including cross-presentation of antigens to CD8 T cells and CD103 expression. cDC1s require GM-CSF signal for gaining their signature functions; however, the involvement of other cytokines remains unknown.

Here we found that cDC1s were normal in number but lacked CD103 expression in IL-10-deficient (Il10−/−) mice, suggesting that IL-10 has some effects on cDC1 function in vivo. Thus, next we assessed the CD8 T-cell priming capacity of cDC1s from Il10+/+ and Il10−/− mice by coculture with OT-I CD8+ T cells in the presence of ovalbumin in vitro. In contrast to Il10+/+ cDC1s, Il10−/− cDC1s failed to induce the expansion and IFN-γ production of OT-I cells. In addition, Il10−/− mice had a significantly lower number of memory CD8 T cells in the lymph nodes in the steady state. These results indicate that cDC1s in Il10−/− mice were defective in antigen cross-presentation. We also found that, whereas Flt3-ligand-induced DCs derived from Il10+/+ bone marrow cells upregulated CD103 expression by and CD8 T-cell cross-priming capacity of the cDC1-equivalent population in response to GM-CSF stimulation, Il10−/− bone marrow-derived DCs did not, suggesting that dysfunction of cDC1s in Il10−/− mice is likely due to unresponsiveness to GM-CSF. Finally, we found that mRNA expression of Csf2ra, which encodes GM-CSF receptor α-chain, was completely deficient in both cDC1s in Il10−/− mice and Il10−/− cDC1-equivalent bone marrow-derived DCs. Collectively, our data suggest that IL-10 critically controls the development of antigen cross-presentation capacity of cDC1s under homeostatic conditions, likely through regulating GM-CSF receptor expression.
An allosteric site in the T cell receptor β-chain constant domain plays a critical role in T cell signaling
Authors: Sgourakis Nikolaos , Natarajan Kannan , McShan Andrew , Jiang Jiansheng , Zhao Huaying , Schuck Peter , Boyd Lisa F , Tilahun Mulualem E , Ying Jinfa , Bax Ad , Margulies David H
Published: January 2023

The molecular mechanism through which the interaction of a clonotypic αβ TCR with peptide/MHC (p/MHC) complexes leads to T cell activation is not yet fully understood. Here we exploit a high affinity TCR (B4.2.3) to examine the structural changes that accompany binding to its p/MHC ligand (P18-I10/H2-Dd) by combining crystallographic, NMR, and functional data. In addition to conformational changes in complementarity determining regions (CDR) of the TCR seen in comparison of unliganded and bound X-ray structures, NMR characterization of the TCR β chain dynamics reveals significant chemical shift effects in sites removed from the MHC binding site. In particular, a remodeling of electrostatic interactions near the Cβ H3 helix at the membrane-proximal face of the TCR, a region implicated in interactions with the CD3 co-receptor, suggests an allosteric mechanism for TCR signaling. The contribution of these TCR residues to signal transduction is supported by mutagenesis and T cell functional assays.
Blockade of Interleukin-10 signaling during BCG vaccination enhances protection against Mycobacterium tuberculosis infection in mice
Authors: Dwivedi Varun , Headley Colwyn , Ault Russell , Torrelles Jordi , Turner Joanne
Published: January 2023

Interleukin-10 (IL-10) is a major immune regulator in Mycobacterium tuberculosis (M.tb) infection. We have shown that the absence of IL-10 signaling during early phases of M.tb infection results in enhanced protection in mice. We further showed that BCG immunization alone induces IL-10 production in the lungs as early as day 7. We therefore hypothesized that blocking IL-10 signaling during BCG vaccination would increase the magnitude of protection against M.tb challenge. Mice were subcutaneously co-immunized with a single dose of anti-IL-10R1 or respective IgG1 isotype antibody with BCG followed by low dose aerosol M.tb infection 6 weeks later. Protective efficacy was determined at day 30 and 120 post challenge. BCG/anti-IL-10R1 immunized mice showed a significant reduction (~1.5 log) in M.tb CFU in the lungs at day 120, compared to control mice. CD4+ and CD8+ T cell populations in BCG/anti-IL10R1 immunized mice (in the absence of M.tb infection) showed significant increases in central memory markers in the lung at day 7, which reduced at day 28 post immunization. Effector memory markers were reduced in CD4+ T cells at day 7 and increased at day 28 in BCG/anti-IL-10R1 immunized mice but were unchanged in CD8+ T cells. Our findings demonstrate that transient IL-10 signaling blockade during BCG vaccination alters memory T cell populations and results in long-term protection against M.tb challenge. Our findings are relevant to vaccine development in humans as they demonstrate that modulation of IL-10 at the time of vaccination can generate long-term protective efficacy against M.tb.
Infection-permissive immunity against influenza virus provided by vaccination prevents loss of alveolar macrophages and modulates virus-induced cross-reactive cellular immune responses during subsequent influenza infections.
Authors: Schotsaert Michael , Choi Angela , Spitaels Jan , Mina Andrea Joselin Morales , Garcia-Sastre Adolfo
Published: January 2023

Conventional influenza vaccines aim at the induction of virus-neutralizing antibodies. Influenza viruses can escape neutralization by antibodies through antigenic drift or shift or because antibody levels diminish. We wondered to what extent infection-permissive immunity provided by a trivalent inactivated influenza virus vaccine (TIV) could modulate disease and virus-induced host immune responses after homologous H1N1 infection. We first focused on alveolar macrophages, cells that are transiently lost during influenza infection in the mouse-influenza challenge model. Later we focused on how vaccine-induced immunity modulates induction of cross-reactive CD8+ T cells by virus infection. TIV vaccination did not result in detectable HI titers but correlated with lower viral lung titers and faster recovery after challlenge. Alveolar macrophages were abolished at 7dpi in negative control mice, but not in TIV-vaccinated mice. TIV vaccination allowed the induction but also affected levels of cross-reactive NP-specific CD8+ T cell responses, which correlated with protection against heterosubtypic H3N2 virus. The effect of TIV vaccination on TCR Vbeta-region bias of CD8+ T cells after secondary challenge with H3N2 virus was also investigated.

These results suggest that suboptimal vaccination with conventional influenza vaccines may still positively modulate disease outcome, thereby allowing induction of heterosubtypic immunity by virus infection.
Lung extracellular matrix changes mediated by ADAMTS-4 modulate immune responses to influenza A infection
Authors: Boyd David F , Guo Xizhi , Allen Emma Kaitlynn , Thomas Paul Glyndwr
Published: January 2023

The lung extracellular matrix (ECM) undergoes dramatic changes during and after respiratory viral infection. Matrix proteases, including members of matrix metalloproteinases (MMPs) and A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) gene families, play a critical role in remodeling the ECM following tissue damage. Alterations in the ECM feed into immunological pathways, which subsequently regulate repair and regeneration. The mechanisms that regulate matrix proteases and the effect of lung ECM changes on immune responses to viral infection have not been well characterized. We previously found that ADAMTS-4 was upregulated in the lung within 3 days after influenza infection and that genetic knockout of ADAMTS-4 resulted in improved survival following infection in mice. Using quantitative PCR, we determined the transcriptional levels of 96 ECM-related genes in lung homogenates from ADAMTS-4 −/− and +/+ mice during a 15-day infection time course. At early time points, lungs from ADAMTS-4 −/− mice exhibited lower expression of inflammatory cytokines, including IL-6 and IL-1β, but higher expression of several ECM remodeling enzymes, including neutrophil elastase and MMP-13. Flow cytometric analysis of immune cell populations from lungs revealed altered neutrophil and T cell kinetics between ADAMTS-4 −/− and +/+ mice over the course of influenza infection. Lungs from ADAMTS-4 −/− mice exhibited higher numbers of neutrophils at 3 dpi and a higher percentage of IFNγ-producing CD4+ and CD8+ T cells at 6 dpi, but lower numbers of these cells by 12 dpi. These results suggest that ADAMTS-4-mediated alterations of the lung ECM modulate immune responses to influenza infection and may contribute to immunopathology.
PD-1 expression on Foxp3+ Treg cells modulates CD8+ T cell function in prostatic tumor microenvironment
Authors: Kumar Sanjay , Malik Shalie , Singh Udai P. , Ponnazhagan Selvarangan , Scissum-Gunn Karyn , Manne Upender , Mishra Manoj K.
Published: January 2023

Regulatory T (Treg) cells act as terminators of T cell immune response during prostate cancer progression and development. However, their role and immunosuppressive mechanism(s) in context to programmed death 1 (PD-1) is not completely understood. The present investigation is aimed to determine the role of PD-1 on Treg cells and their impact on CD8+ T cell function in prostatic tumor microenvironment using transgenic adenocarcinoma of the mouse prostate (TRAMP) cells (TRAMP C1, C2 and C3) as a model system. To execute the above aim, tumor induction studies were performed. Briefly, the C57BL/6 mice were administered with serial log concentrations of TRAMP (C1-3) cells. Interestingly, the TRAMP-C3 cells do not form tumor, however, TRAMP-C1 and TRAMP-C2 cells do form tumor. After tumor development, mice were sacrificed by cervical dislocation; tumor, lung, spleen, and draining lymph nodes were harvested when the tumor size reached approximately 20mm. Single cell suspensions were prepared from different organs, cells were then stained with specific antibodies, and flow analyzed for the expression of different immune markers. Our preliminary findings demonstrated that PD-1 expression on Foxp3+ Treg cells displayed greater suppressive capacity against CD8+ T cell function in tumor, lung, spleen, and draining lymph node when compared to the control. More importantly, Foxp3+ Treg(high) PD-1(high) interaction with PDL-1 induced immunosuppression by blocking CD8+ T cells function in prostatic tumor microenvironment. In conclusion, Foxp3+ Treg(high), PD-1(high), and CD8+(low) T cells may enhance tumor progression; thus, targeting the PD-1 on Treg cells may be a possible therapy to treat prostate cancer.
Secondary iTreg generation has a major impact on the tumor-infiltrating Treg population in cancer patients.
Authors: Xydia Maria , Ruggiero Eliana , Domschke Christof , Mastitskaya Svetlana , Pritsch Maria , Schmidt Manfred , von Kalle Christof , Schütz Florian , Beckhove Philipp
Published: January 2023

CD4+ regulatory T cells (Treg) have a detrimental effect in cancer-immunity, as they suppress tumor-specific effector T cells (Teff) hindering tumor eradication. Indeed, increased Treg numbers in various cancer types, including breast cancer, correlate with poor patient prognosis. Based on animal studies, Treg enrichment may result from the expansion of thymic natural Treg but also through the conversion of conventional T cells (Tconv) into induced Treg (iTreg) under tumor-suppressive conditions. However, little is known about tumor-specific iTreg generation in humans and its impact on the TCR repertoire of peripheral Treg.

Therefore, we compared the TCRβ repertoire of total Treg and tumor-reactive Teff or total Tconv from peripheral blood of breast cancer patients or healthy individuals, as negative control, for the identification of overlapping TCRs, representing iTreg. In addition, using high-throughput single-cell transcriptome sequencing combined with TCRαβ characterization we analyze the functional profile of individual Treg and Tconv clones, in order to elucidate their specific role in anti-tumor immunity in humans.

No major TCR overlap was observed between Treg and tumor-reactive Teff or total Tconv in peripheral blood of patients and healthy individuals, arguing against the existence of tumor-specific iTreg in the circulation. Nevertheless, within the tumor Treg and Tconv shared dominant highly-expanded clones, representing 10–65% of the tumor-infiltrating Treg population but with no effect in peripheral blood.

Taken together, our data suggest that intratumoral secondary Treg conversion from dominant Tconv clones may have a major impact on the tumor-infiltrating but not on the circulating Treg population.
Regulation of Myeloid Derived Suppressor Cell (MDSC) dynamics by T regulatory cells
Authors: Tanwar Shalini , Shevach Ethan M
Published: January 2023

Multiple sclerosis is an inflammatory demyelinating autoimmune disorder affecting the central nervous system whose severity is reduced using immune suppressive drugs. Therapeutic intervention with interferon-b reduces disease exacerbations and delays relapses. The receptor for type 1 interferon, IFNAR, is present on virtually all cell types making it difficult to dissect the mechanisms involved. We generated mice with a conditional deletion (cKO) of IFNAR in Treg cells (IFNARfl/flFoxp3cre). cKO mice developed severe EAE with an earlier onset than control mice. The activation status and effector cytokine production of CD4+ and CD8+ T cells in the draining lymph nodes (dLN) was similar in WT and cKO mice during the priming phase. Treg activation and expansion were also similar in WT and cKO mice. However, we noted a substantial reduction of myeloid derived suppressor cells (MDSCs) in the dLN of cKO mice while equivalent numbers of MDSCs were present in bone marrow and spleen of WT and cKO mice. IFNARfl/flLysMcre mice do not show a similar reduction in MDSC number indicating that the enhanced disease severity in the cKO mice is dependent on IFNAR signaling on Treg cells. CD4+ T cells from cKO mice were found to be defective in chemokine secretion suggesting that IFNAR signaling on Treg modulates the capacity of CD4+ T cells to secrete MDSC recruiting chemokines during the priming phase. While modulation of Treg number and function by MDSCs has been documented, this study is one of the first to demonstrate that Treg may modulate MDSC homing and raises the possibility of a novel role for Treg cells in regulating the kinetics of MDSC recruitment during inflammatory conditions.

Supported by the Division of Intramural Research NIAID.
Identification of a suppressor of autoimmune phenotype that affects deoxynucleotide synthesis in T cells
Authors: Bolland Silvia , Sule Gautam , Scott Bethany , Tarasenko Tatyana
Published: January 2023

FcγRIIB-deficient mice represent a well-characterized animal model of systemic lupus erythematosus. They develop spontaneous anti-nuclear antibodies (ANA) and fatal glomerulonephritis when on the C57BL/6 (B6) background while the same mutation on the BALB/c background is phenotypically benign, indicating differences in lupus susceptibility between the BALB/c and B6 strains. We mapped the BALB/c derived protection to a 151KB genomic fragment on chromosome 12. Insertion of this fragment in transgenic form (A12Tg) results in protection from lupus disease. Mixed bone marrow reconstitution experiments pointed to a T cell intrinsic effect of the protective gene in the BALB/c A12 interval. This region contains a single gene, Rrm1, which encodes for a subunit of the ribonucleotide reductase responsible for the synthesis of deoxiribonucleotides. We hypothesize that reduced levels of this enzyme affect the ability of T cell to expand upon activation and that is why allelic differences in Rrm2 expression can alter tolerance in mouse models of lupus.
‘Interleukin-7 production by dendritics cells is critical for negative selection of CD4+ lymphocytes in the thymus’
Authors: Moutuou Moutuaata Mohamed , Thiant Stéphanie , Leboeuf Dominique , Guimond Martin
Published: January 2023

Introduction

IL-7 plays a crucial role in thymopoiesis, this cytokine is mainly expressed by thymic epithelial stroma cells and dendritics cells (DCs). During thymopoiesis, thymocytes undergo positive and negative selection which ensures the generation of a functional T-cell repertoire. In the thymus, Antigen presenting cells including medullary thymic epithelial cells (mTEC) and DCs, express a variety of self-peptides that contribute to purge the repertoire of self-reactive thymocytes. Studies have indicated that co-stimulation through cell surface molecules and cytokine mediated by DCs is important during negative selection. Given that DCs produce IL-7 and IL-7 can modulate TCR signaling, we hypothesized that IL-7 production by DCs might increases TCR sensitivity and modulates negative selection.

Objective

We limited IL-7 production to thymic epithelial cells by engineering chimeric Rag−/− mice transplanted with hematopoietic stem cells from IL-7−/− mice. Following bone marrow (BM) transplantation, control mice (C57BL/6 BM in Rag−/−) remains healthy whereas recipients of IL-7−/− BM developed signs of illness and dies within 5–6 weeks. Adoptive transfer of CD4+ but not CD8+ T cells recapitulated the disease host. We confirmed a defect in negative selection by observing the production of autoreactive anti-HY T cells TCR transgenic Marilyn male mice receiving IL-7−/− BM.

Conclusion

Our data identified an unsuspected role of DCs-derived IL-7 in negative selection of CD4+ T cells.
T regulatory cells follow CXCL10 to suppress T effector cells through a contact-dependent mechanism in the skin during vitiligo
Authors: Essien Kingsley Imeh , Richmond Jillian M , Strassner James P , Harris John E
Published: January 2023

Vitiligo is an autoimmune disease of the skin driven by CD8+ T effector cells (Teffs) that infiltrate the epidermis to kill melanocytes, resulting in visible white spots. To investigate the role of T regulatory cells (Tregs) during vitiligo we depleted Tregs in our mouse model with CD4 depleting antibody or diphtheria toxin-treated Foxp3-DTR mice and saw increased disease (p<0.0001 and p<0.02, respectively) and increased Teff skin infiltration, suggesting that Tregs suppress Teffs during vitiligo. We investigated the kinetics of Treg and Teff migration to the skin in our mouse model and saw that both cell types accumulate in a similar pattern, suggesting that Tregs may follow the same signals that recruit Teffs to the skin. Previous studies revealed that CXCL10, which is upregulated in human patients as well as in our mouse model of disease, is primarily expressed in the epidermis and necessary for the migration of CXCR3+Teffs to the epidermis. Using flow cytometry we saw that both mouse and human Tregs in the skin express CXCR3 during vitiligo and confocal microscopy revealed that mouse Tregs co-localize with CXCL10 expression in the skin during vitiligo. Using confocal microscopy, we observed that Tregs directly contact Teffs in mouse skin, suggesting that Tregs may use contact-dependent mechanisms of suppression in the skin. We found similar interactions in human skin and hypothesize that Tregs follow CXCL10 to localize with Teffs in the epidermis where they suppress Teffs through a contact-dependent mechanism.
A novel method to classify subpopulations in murine spleen and blood using multiparametric imaging flow cytometry.
Authors: Kong Raymond K , Zhou Weiyang , Venkatachalam Vidya , McCann Kelly , Pugsley Haley Renee , Friend Sherree , Morrissey Philip
Published: January 2023

Being able to identify the lymphocytes, monocytes, and neutrophils (granuolcytes) subpopulations in murine spleen and peripheral blood is an important step of the analysis of the immune system. Conventionally, this task is accomplished by performing immunofluorescence staining of specific cell surface markers. However, given the distinct morphology of each of these subpopulations as well as the cell imageries obtained using the Image Stream technology, we attempt to identify 3cell types in the spleen and peripheral blood samples of C57BL/6 and BALB/c mice based solely on the Bright Field (BF), Side Scatter (SSC) and nuclear images without using any specific markers for the cell types. We will then compare the results obtained using this novel method with that obtained using immunofluorescence. We hope that the results will serve to both validate our methodology and highlight the value offered by this objective, automated, and quantitative approach towards other murine immune cell analysis, particularly in those situations where immunofluorescence based subpopulation detections are either unfeasible(due to fluorochrome conflicts) or impractical (due to high sample sizes).
Prime and Target Immunization Protects Against Liver-Stage Malaria
Authors: Gola Anita , Walters Adam A , Uderhardt Stefan , Salman Ahmed M , Halbroth Benedict R , Khan Shahid M , Janse Chris J , Germain Ronald N , Spencer Alexandra J , Hill Adrian VS
Published: January 2023

A highly effective vaccine against malaria is urgently needed, with leading vaccination strategies targeting primarily the pre-erythrocytic liver stages of the Plasmodium falciparum lifecycle. Malaria antigen specific CD8+ T-cells to are known to be protective, with heterologous prime-boost viral vector immunization being an effective method of induction. Non-replicating viral vectored vaccines have shown a remarkable capacity to induce systemic CD8+ T-cell responses in animals and humans. To date, the greatest immunogenicity has been obtained through a heterologous prime boost regimen, where vaccination with an adenoviral vector is followed 8 weeks later by a Modified Vaccinia Ankara virus (MVA) boost. A novel vaccine strategy aimed at priming CD8+ T-cells in the periphery and subsequently targeting them to hepatic tissue with protein loaded poly(lactic-co-glycolic acid) nanoparticles or recombinant viral vectors administered by specific immunization routes was developed. Durable Ag-specific CD8+ T-cells exhibiting a phenotype of tissue-resident memory (TRM) T-cells were generated in the liver, with a ten-fold increase over the conventional heterologous vector regime. Importantly, in a P. berghei sporozoite challenge model of liver-stage malaria, this strategy was found to result in unprecedented sterile protection across several clinically relevant antigens and mouse strains. This prime and target immunization strategy for malaria may provide a general approach for prevention or immunotherapy against pathogens that infect the liver.
Suppression of vaccination-induced antigen-specific regulatory T cells enhances the antiviral immunity against influenza virus infection
Authors: Lin Pin-Hung , Yang Hung-Chih
Published: January 2023

T cell immunity targeting the conserved epitopes of influenza virus has the potential to provide cross-protection against distinctly-related strains. Peptide vaccines are an ideal strategy to induce specific antiviral T cell immunity, but subimmunogenic stimulation by peptide vaccines tend to induce regulatory T (Treg) cells. However, it remains unclear about the roles of vaccine-induced antigen-specific Treg cells in vivo. Here, we aimed to investigate how vaccine-induced antigen-specific Treg cells respond during acute influenza virus infection, and the effects of adjuvants on them. By adoptive transfer experiments with OT-II TCR transgenic T cells, we found that the OVA OT-II peptide vaccine induced Treg cells and secondary vaccination further expanded them. Infection with influenza virus containing the OVA OT-II peptide also drove the expansion of pre-existing vaccine-induced Treg cells. Importantly, specific depletion of vaccine-induced antigen-specific Treg cells enhanced the antiviral immunity. Vaccination combined with adjuvants, especially with CpG, suppressed the development of vaccine-induced antigen-specific Treg cells. Finally, CpG-adjuvanted OVA protein or whole inactivated influenza vaccine by the subcutaneous-prime-intranasal-boost strategy reduced the ratio of antigen-specific Treg cells in lung and protected mice from the heterosubtypic influenza infection. In conclusion, unadjuvanted peptide vaccines promoted antigen-specific Treg cells, which were further expanded upon acute influenza infection. The CpG adjuvant restricted the development of vaccine-induced antigen-specific Treg cells, and enhanced the antiviral T cell immunity against heterosubtypic influenza infection.
Model specific role of B cell-intrinsic IFNαR signaling in promoting spontaneous germinal centers and autoimmunity
Authors: Domeier Phillip P , Chodisetti Sathi Babu , Soni Chetna , Schell Stephanie L , Fasnacht Melinda J , Imamura Yuka , Salzberg Anna , Rahman Ziaur
Published: January 2023

In systemic lupus erythematosus (SLE), germinal centers can form spontaneously (Spt-GCs) in the absence of foreign antigen and drive high-affinity autoantibody (autoAb) production. Overexpression of Type 1 interferon (IFNα) accelerates autoimmunity and alters B cell selection in certain SLE mouse models, but in another model, B cell intrinsic IFNαR signaling is shown to be dispensable for autoimmune GC response and autoimmunity. Using B6.Sle1b autoimmune mice derived from the NZM2410 SLE mouse model, we show here that IFNαR signaling is not required for Spt-GC formation but is important for elevated Spt-GC response and autoreactive B cell selection into AFCs. IFNα receptor1 deficient B6.Sle1b mice (B6.Sle1b.IFNαR1−/−) have lower Spt-GC and Tfh responses, reduced CD86 expression on B cells and diminished autoAb titers as compared to B6.Sle1b mice. We also observe lower Ca2+ flux in B cells deficient in IFNα receptor compared to control B cells upon BCR cross-linking in vitro. Consistently, we find that B cell-intrinsic IFNαR signaling promotes differentiation of Sle1b-expressing DNA-reactive B cells into GC B cells. RNA sequencing studies show that B6.Sle1b and B6.Sle1b.IFNαR1−/− GC B cells have distinct transcriptional profiles that are associated with proliferation, cell survival, and antigen presentation pathways. Compared to B6.Sle1b control, B6.Sle1b.IFNαR1−/− B cells fail to form stable conjugates with T cells in vitro and GC B cells express reduced levels of ICOS-ligand in vivo, suggesting B6.Sle1b.IFNαR1−/− B cells may not acquire adequate T cell help. These data reveal novel information about the model specific role of B cell-intrinsic IFNαR signaling in the regulation of Spt-GCs and autoimmunity.
TCR signaling strength dependent regulation of T cell proliferation, survival and Th differentiation by TGF-βR3 (betaglycn)
Authors: Raman Chander , Ren Clark , Boas Stephanie , Sestero Christine M. , Chellappan Rajeshwari , Kesterson Robert A , De Sarno Patrizia , Soldevila Gloria
Published: January 2023

TGF-β receptor 3 (TGFβ-R3) or betaglycan, promotes high-affinity binding of TGF-β (1, 2 and 3) to TGFβ-R1 and TGFβ-R2. Additionally, it is also a receptor for bone morphogenic proteins (BMPs) and inhibins. Although TGFβ-R3 is widely expressed on cells of both the innate and adaptive immune system, embryonic lethality of Tgfbr3 knock-outs has been a limitation to obtain a mechanistic understanding of its function in immune response. We therefore developed a Tgfbr3f/fmouse and bred with the CD4Cre mouse. Remarkably, thymic inactivation of TGFβ-R3 had no significant effect on T cell development nor did the mice develop spontaneous autoimmunity. This led us infer that TGFβ-R3 is unlikely to have a necessary function in enhancing TGF-β signaling from TGFβ-R1/R2. To interrogate the function of TGFβ-R3 in mature T cells, we generated OT2.Tgfbr3f/f.dLckCre mice and control littermates. We observed that TGFβR-R3 deficient T cells exhibited greater early proliferation than controls, however, their ability to persist was compromised. This effect of TGFβ-R3 was more pronounced with increasing concentrations of OVA323–339 peptide. The differentiation of TGFβ-R3 deficient to Th1 cells under polarizing conditions was significantly reduced in proportion and absolute numbers compared to Tgfbr3f/f T cells and this correlated with lower Tbet expression. Encephalitogenic Th1 cells generated from Tgfbr3f/f.dLckCre mice induced significantly less severe EAE in naïve mice than that from Tgfbr3f/f littermates. Overall or data reveal a novel role for TGFβR3 in promoting of Th1 differentiation and regulation of regulation T-cell activation and survival.
A novel, intergenic enhancer contributes to Il2 gene expression via chromosomal looping.
Authors: Thomas Rajan M , Mehra Parul , Johnson Matthew , Grant Struan , Wells Andrew
Published: January 2023

IL-2 is a key immunoregulatory cytokine with pleotropic effects on many immune cell types. Its expression is highly regulated in T cells through multiple signaling pathways, chromatin remodeling, DNA methylation, and cooperative transcription factor binding. Recently, we identified an evolutionarily conserved intergenic enhancer 83 kb upstream of the Il2 gene that augments transcription from the Il2 promoter and upstream regulatory region (URR) by >50-fold in recombinant reporter assays. Upon TCR/CD28 co-stimulation of primary CD4+ T cells, a long-range chromosomal loop is established between the −83 kb CNS and the endogenous Il2 promoter, and both regions become epigenetically marked by histone acetylation and methylation. To determine the relative contribution of the URR vs. this new distal element to inducible transcription of the Il2 gene, we deleted the URR or the −83 kb CNS in mice using CRISPR/CAS genome editing. Deletion of the URR (~500 bp beyond the TATA element) completely abolished the Il2 transcription by CD4+ T cells, while a ~500 bp deletion of the −83 kb CNS resulted in a 2- to 4-fold decrease in IL-2 production. Young mice deficient for the −83 kb enhancer exhibited no major defects in lymphoid development, however, mutant CD4+ T cells showed reduced proliferative capacity in vitro. Furthermore, TGFB-induced iTreg differentiation from mutant conventional CD4+ precursors was strongly impaired, but could be fully restored by addition of exogenous IL-2. These results show that induction of Il2 depends upon cooperation between the promoter-URR and at least one intergenic enhancer, and suggest mechanisms by which graded expression of Il2 might be achieved through ‘modular’ recruitment of distinct enhancer elements.