bluuebunny/crossref_metadata_2025 huggingface dataset:
AID‐ and Ung‐dependent generation of staggered double‐strand DNA breaks in class switch DNA recombination: a post‐cleavage role for AID
Authors: Zan Hong , Casali Paolo
Published: June 2021

Double‐strand DNA breaks (DSBs) are critical for Ig class switch (S) DNA recombination (CSR). They would be generated through deamination of dC by AID, subsequent dU deglycosylation by uracil DNA glycosylase (Ung) and nicking by apurinic/apyrimidic endonuclease of nearby abasic sites on opposite DNA strands. We found that in human and mouse B cells, S region DSBs can be generated in an AID‐ and Ung‐independent fashion. These DSBs were blunt and 5′‐phosphorylated, and could be readily detected in human and mouse AID‐ or Ung‐deficient B cells. In B cells undergoing CSR, blunt DSBs were processed in an AID‐ and Ung‐dependent fashion to yield staggered DNA ends. Accordingly, we readily detected blunt‐DSB in B cells from AID‐ or Ung‐deficient patients and AID‐ or Ung‐deficient mice. These B cells were CSR defective, but showed evidence of intra‐S region recombination. Enforced expression of AID in AID‐negative B cells converted S region blunt DSBs to staggered DSBs. Conversely, enforced expression of dominant negative AID or inhibition of Ung by Ung inhibitor (Ugi) in switching B cells abrogated the emergence of staggered DSBs and concomitant CSR. Thus, AID and Ung generate staggered DSBs not only by cleaving intact double‐strand DNA, but also by processing blunt DSB ends, whose generation is AID‐ and Ung‐independent, thereby outlining a post‐cleavage role for AID in CSR.

Supported by NIH grants AI 45011, AI 60573 and AR 40908.
Characterizing an engineered chimeric anti‐ricin A chain antibody
Authors: Johnson Clorinda Channell , Song Kejing , Pincus Seth
Published: June 2021

Ricin toxin, derived from the castor bean, is a potent toxin that inhibits protein synthesis. It has been considered a possible agent of bioterrorism for many years. In experimental studies antibodies have been shown to be an effective treatment against ricin poisoning. Maddaloni et al, described a murine monoclonal anti‐ricin A chain antibody, mRAC 18, that was shown to neutralize in vitro and protect in vivo. This antibody has been found effective, even when administered 12 hours post exposure. The use of monoclonal antibodies in therapy has steadily increased since their development. A limitation to the utility of mAb therapy is their immunogenicity. In an effort to create an antibody for use in post‐exposure human therapy of ricin intoxication, we have created a chimeric ricin A chain antibody. We combined the antigen‐specific variable region genes of the mouse antibody RAC 18 to human gamma‐1 and kappa constant region genes. The resultant chimeric antibody retained the same specificity for ricin as the parental antibody, offered passive protection in vivo, and had enhanced in vitro neutralizing ability. A lectin binding assay showed the chimeric antibody had significantly less sialic acid than the parental antibody. We conclude that differences in sialic acid content may be responsible for the increased in vitro neutralizing ability, and that this effect is independent of Fc receptor binding.
The inositol phosphatase SHIP negatively regulates late stage B‐cell development and class switch recombination
Authors: Leung Wai‐Hang , Biesova Zuzana , Bolland Silvia
Published: June 2021

SHIP is a haemopoietic‐specific phosphatase that regulates antigen receptor, growth factor and cytokine‐mediated cell activation. Mice with germline deletion of SHIP develop a lethal pathology caused by myeloid cell dysregulation. To determine SHIP's role in B cells while avoiding the pheitropic phenotype of SHIP‐null mice, we have bred SHIP‐floxed mice to CD19cre mice. CD19cre SHIP‐floxed mice have less mature B cells in the bone marrow due to a faster rate of maturation of SHIP‐deficient B cells. We also observe that B cells that lack SHIP have activated phenotypes and generate switched antibodies, particularly IgG2a, in the periphery even in the absence of high affinity antigen. In contrast, mice with B cell specific deletion of SHIP exhibit hyporesponsiveness to both T‐cell dependent and T‐independent immunizations. Overall these results suggest that SHIP‐deficient B cells have lower thresholds of spontaneous activation and cytokine stimulation while at the same time they become more refractory to foreign antigen stimulation.
Ceramide Disrupts HLA Class II‐restricted Antigen Processing and Presentation
Authors: Haque Azizul , Hajiaghamohseni Laela M. , Harbeson Caroline , Amria Shereen , Xiang Liu , Bielawska Alicja , Hannun Yusuf , Norris James S.
Published: June 2021

Ceramide is a bioactive sphingolipid that mediates a variety of cell functions. However, the effect of ceramide on antigen processing and HLA class II‐mediated delivery to CD4+ T cells remains unclear. We have previously shown that intracellular ceramide levels can be regulated by ceramide anlogs that directly interfere with ceramide‐metabolizing enzymes such as acid ceramidases. In this study, we examined the actions of these cell permeable ceramide analogs on professional antigen presenting cells (APC) that constitutively express HLA class II proteins and functionally present antigens to CD4+ T cells. Our results show that the treatment of APC with ceramide analogs perturbs Ag processing and presentation in the context of HLA class II molecules. Cellular uptake of ceramide analogs into the endolysosomal compartments did not alter intracellulaer class II protein levels as confirmed by confocal microscopy and western blot analysis. Mass spectroscopic analysis showed that ceramide analogs decreased the activity of acid ceramidase elevating intracellular ceramide levels in the APC. Western blot and flow cytometric analysis confirmed that the ceramide analogs diminished acid ceramidase protein expression in APC, but not cell surface class II molecules. Data also showed that the elevation of intracellular ceramide modulates endolysosomal proteases as well as the components of the HLA class II pathway. These findings suggest that ceramide regulates multiple pathways in APC including antigen processing and presentation via HLA class II molecules.

Supported by the Leukemia and Lymphoma Society (3024), American Lung Association (RG‐10435‐N), MUSC HCC Funds and NC1 PO1 CA97132
Estrogen and the environmental estrogen bisphenol A differentially regulate cathepsin activity in a mouse model for Systemic Lupus Erythematosus
Authors: Roberts Rebecca A. , Heayn Michelle , Leichner Theresa , Mercadante Emily , Patel Priya , Seczech Laura , Stanert William , Stasulli Nikolas
Published: June 2021

The autoimmune disease Systemic Lupus Erythematosus (SLE) is linked to estrogen (E2) and may be affected by the environmental estrogen bisphenol A (BPA) that is used in plastics production. E2 and BPA bind to estrogen receptors (ER), altering cellular pathways. We investigated the effect of E2 and BPA on the regulation of cathepsins, central enzymes in major histocompatibility class II (MHC‐II) antigen presentation. A bioinformatic analysis showed that many cathepsins have putative estrogen response elements (ERE) making them candidates for regulation by compounds that activate ER. Splenocytes isolated from C57/Bl6 and lupus‐prone NZB/WF1 female mice were treated with varying concentrations of hormone. B cells were also purified using magnetic activated cell sorting and populations were routinely enriched to >90% as verified by fluorescence activated cell scanning. Following treatment, cathepsin expression (via Western blotting) and activity (by fluorescence enzyme activity assays) were monitored. In both cell populations we found that cathepsins B and L were increased in NZB/WF1 but not control mice when exposed to E2, while the opposite effect was seen with BPA. These results indicate that MHC‐II antigen processing and presentation is differentially regulated in SLE animals and that environmental estrogens can affect immune function. Funded by NIH grant 1R15ES013947‐01, Merck/AAAS USRP, and Van Sant.
B Cells Induce Tolerance by Presenting Endogenous Peptide‐IgG on MHC Class II Molecules via a GILT‐dependent Pathway
Authors: Su Yan , maric Maja , Scott David W
Published: June 2021

Classically, MHC class I and II molecules present endogenous and exogenous antigens, respectively. MHC class II can also present endogenously derived antigens, even though the mechanisms largely remain unclear. In this study, we addressed on the mechanisms of in vivo tolerance induction by peptide‐IgG fusion protein producing B cells, particularly on the processing and presentation of peptide‐IgG by tolerogenic B cells. We show that endogenously produced antigen (peptide‐IgG fusion protein) was processed and presented on MHC class II molecules on tolerogenic antigen presenting B cells. More importantly, the peptide‐IgG is processed in the lysosomes/endosomes in a gamma‐interferon‐inducible lysosomal thiol reductase (GILT) dependent manner. The lysosomal processing and MHC class II presentation for endogenous peptide‐IgG are critical for tolerance induction by B cells. In addition, our data suggest that chaperone‐mediated autophagy might be involved in the intracellular delivery of peptide‐IgG to the lysosomes/endosomes. Together, we conclude that, in our model, tolerogenic peptide:MHC class II complexes on B cells are derived from lysosomal/endosomal processing of endogenously expressed antigen. (Supported by NIH, RO1 AI035622)
Variability in HLA class I viral peptide presentation during infection with two different Influenza A H1N1 strains
Authors: Wahl Angela , Schafer Fredda , Bardet Wilfried , Buchli Rico , Eckerd Annette , Air Gillian , Hildebrand William
Published: June 2021

The influenza virus infects up to 20% of the US population yearly eliciting a mild to severe respiratory illness. During infection, the HLA (Human Leukocyte Antigen) class I molecule presents viral‐derived peptides to surveying CTL (cytotoxic T lymphocytes) allowing CTL to recognize and kill influenza infected cells. A better understanding of the influenza CTL response and the class I ligands involved may aid the development of anti‐influenza vaccines and therapeutics. To identify class I ligands that mark infected cells, we collect soluble HLA A*0201 and B*0702 molecules from influenza infected/uninfected cells. The uninfected/infected peptide ligands are eluted from class I heavy and light chains by an acid boil, size separated and fractionated by RP‐HPLC and comparatively mapped via mass spectrometry. Thus far, we have identified one A*0201 and five B*0702 viral‐derived ligands during infection with two different Influenza A H1N1 strains. While infection with both H1N1 strains resulted in presentation of previously described B*0702 nucleoprotein418–426 ligand, they varied in presentation of one A*0201 and three B*0702 influenza‐derived ligands whose amino acid sequence is shared between strains. To investigate the cause of varied presentation we have performed synthetic peptide binding assays and are analyzing infection time course samples with real‐time PCR, Western blot and TCR mimics.
Plasmacytoid Dendritic cells prime naïve CD4+ and CD8+ T cells after antigen uptake via mPDCA‐1
Authors: Fischer Jens A. A. , Janz Monika , Voelkel Tobias , Schmitz Jürgen , Dzionek Andrzej
Published: June 2021

Plasmacytoid Dendritic cells (PDC) produce large amounts of IFN upon microbial stimulation and are believed to link innate and adaptive immune responses. However, it remains controversial whether PDC are in fact able to prime naïve T cells. Here we investigated the function of the recently described PDC‐specific receptor mPDCA‐1. Ligation of mPDCA‐1 with specific mAb resulted in a rapid internalization of the complex. Targeting of PDC with OVA‐conjugated anti‐mPDCA‐1 mAb, but not with an equivalent amount of soluble OVA or OVA conjugated to isotype control antibody, resulted in strong proliferation of OVA‐specific naïve CD4+ T cells. The same was also observed for OVA‐specific naïve CD8+ T cells. Blocking the receptor with excess of unconjugated anti‐mPDCA‐1 mAb inhibited priming of CD4+ and CD8+ T cells. These results indicate that mPDCA‐1 may serve as an antigen uptake receptor delivering its ligands for MHC‐I and MHC‐II presentation. Interestingly, processing and presentation of antigens taken up via mPDCA‐1 was strongly dependent on stimulation, since only activated but not immature PDC were able to prime naïve antigen‐specific T cells. In contrast, antigen uptake was independent on activation as unstimulated PDC also internalized the mAb‐receptor complex. Our results demonstrate that PDC take up and process antigen for efficient priming of CD4+ and CD8+ T cells and thus combine innate and adaptive function.
IRF4 regulates the expression of specific Th2 cytokines
Authors: Ahyi Ayele‐Nati Nathalie , Chang Hua‐Chen , Kaplan Mark H
Published: June 2021

Th2 cells can be subdivided into subpopulations depending on the level of a cytokine and the subsets of cytokines they produce. We have recently identified the ETS family transcription factor PU.1 as regulating heterogeneity in Th2 populations. To define additional factors that might contribute to Th2 heterogeneity, we examined the PU.1 interacting protein IFN‐regulatory factor (IRF)‐4, a transcription factor expressed in lymphocytes and macrophages. When Th2 cells are separated based on levels of IL‐10 secretion, IRF4 expression segregates into the subset of Th2 cells expressing high levels of IL‐10. Th2 cells infected with retrovirus expressing IRF4 secreted significantly higher levels of IL‐10 compared to cells infected with a control vector while other Th2 cytokines were not affected. IRF4 directly activated expression from Il10 regulatory elements in a reporter assay, and using ChIP assay we show that IRF4 directly binds the Il10 locus. Our data also showed that IRF4 negatively regulates IL‐9. Thus, IRF4 is important for establishing specific cytokine expression patterns in subpopulations of Th2 cells.
Comparison in the effects of IL‐2, IL‐12, IL‐15 and IFN[alpha] on gene regulation of granzymes of human NK cell line NK‐92
Authors: Zhang Jian , Zhang Bin , Tian Zhigang
Published: June 2021

Serine protease granzymes (GZM) are key effector molecules of granule‐mediated cytotoxicity of NK cell, but the requirement for their expression regulation remains largely undefined. In this study, it was showed that IL‐2, IL‐12, IL‐15 and IFNα differently regulated the expression of granzymes in NK cells. Although IL‐2, IL‐12, IL‐15 and IFNα exerted overlapping positive effects on transcripts of GZM‐A and GZM‐B, the argument extent and the peaked time course were different for each GZM member after stimulation with these cytokines. Additionally, only IFNα up‐regulated the transcription of GZM‐K, and IL‐15 dramatically down‐regulated the GZM‐H mRNA level. Together, these findings illustrated that IL‐2, IL‐12, IL‐15 and IFNα exerted differential regulating effects on granzyme genes of NK cells.
Differential effects of cigarette smoke extract on cytokine production from IL‐1beta‐activated mast cells
Authors: Chi David S. , Lin Ta‐Chang , Hall Kenton , Wu Zong Dao , Moorman Jonathan , Krishnaswamy Guha
Published: June 2021

Human mast cells are multifunctional cells capable of a wide variety of inflammatory responses and associated with allergy, asthma, and atherosclerosis. Our previous studies have shown that IL‐1β activates human mast cells to produce selected inflammatory cytokines. We examined effects of cigarette smoke extract (CSE) on the production of inflammatory cytokines from IL‐1β‐activated human mast cells. Mainstream (Ms) and Sidestream (Ss) cigarette smoke were collected onto fiber filters and extracted in RPMI‐1640 medium. Two ml of HMC‐1 at 1 × 106 cells/mL were cultured with Ms and Ss CSE at various concentrations in the presence or absence of IL‐1β (10 ng/mL) for 24 hrs. The supernatants were harvested and assayed for IL‐6, IL‐8, and MCP‐1 by ELISA. The Ms and Ss CSE (32.5 to 500 μg/mL) alone induced either no or trace amounts of cytokines from HMC‐1. However, both Ms and Ss CSE significantly increased IL‐6 and IL‐8 production (all p < 0.001) from IL‐1β‐activated HMC‐1 in a dose dependent fashion, except Ms CSE at 500 μg/mL, which significantly decreased IL‐6 production (p < 0.0001). Contrarily, both Ms and Ss CSE at concentrations from 62.5 to 500 μg/mL, but not 32.5 μg/mL, significantly inhibited MCP‐1 production (all p < 0.05) by IL‐1β‐activated HMC‐1 in a dose dependent fashion. The effects of Ms CSE appeared to be more potent than that of Ss CSE. The results show that CSE had differential effects on the production of inflammatory cytokines from IL‐1β‐activated mast cells, which may partially explain why cigarette smoke contributes to lung and cardiovascular diseases. (Supported by the Ruth R. Harris Endowment and RDC of ETSU)
Interleukin‐1 facilitates recruitment of leukocytes into the skin through a complex assortment of chemokines
Authors: Jensen Liselotte E
Published: June 2021

The skin constitutes an important barrier against the environment. Keratinocytes in the skin are often the first cells to encounter an intruding potential pathogen. Keratinocytes express the cytokine interleukin‐1 (IL‐1) which is a potent pro‐inflammatory cytokine. However, the specific function of IL‐1 in the skin and skin pathologies such as eczema and psoriasis remains unclear. Using pathway‐focused microarrays interrogating expression of 113 mRNAs encoding cytokines and receptors, the mRNAs for CCL5, CCL20, CXCL1, CXCL2, CXCL5, CXCL11 and IL‐8 were found to be regulated by IL‐1 in primary keratinocytes. IL‐1 dependent differential expression was confirmed using real‐time PCR and ELISA in primary and HaCaT keratinocytes. Synergy between IL‐1 and tumor necrosis factor‐alpha and interferon‐gamma was observed. These results imply that IL‐1 plays an important role in recruiting leukocytes into the skin. Further studies established that IL‐1 facilitates chemoattraction of T cells and neutrophils in the transwell system. In conclusion, IL‐1 plays a fundamental role in the dynamic interplay between keratinocytes and leukocytes aimed at establishing an effective barrier against microorganisms. This work was supported in part by the National Institutes of Health Grant AR053672, the Transdisciplinary Program in Translational Medicine and Therapeutics, and a grant from the American Heart Association.
Validation of antibody specificity using RNA Interference
Authors: Sullivan Yvonne B. , Narahari Janaki , Hughes Douglas E. , Webb Brian L.
Published: June 2021

The utility of an antibody as a research tool is dependant on the antibody recognizing the proper protein target. Most often antibody function is determined by Western blot analysis, with recognition of a band of the appropriate molecular weight denoting proper function. In this study we used siRNA‐mediated protein knockdown to evaluate antibody specificity. Lysates from siRNA‐transfected cells were probed using antibodies from Thermo Scientific SuperSignal® siRNA/Antibody kits as well as other commercially available antibodies. Interestingly, we observed for several targets that some commercially available antibodies recognized a band of the appropriate molecular weight that was not the correct protein, as judged by lack of siRNA‐mediated protein knockdown. Other commercially available antibodies required considerable Western blot optimization. The antibodies in the Thermo Scientific SuperSignal® siRNA/Antibody Modules all detected a protein band that was specifically reduced with corresponding siRNA treatment. These studies highlight the value of siRNA‐validation of antibodies for Western blotting and other protein detection applications.
Induction of granulysin in T cells by common gamma‐chain cytokines IL‐2, IL‐15 and IL‐21
Authors: Hogg Alison E. , Bowick Gavin A. , Herzog Norbert K. , Endsley Janice J.
Published: June 2021

Granulysin displays potent anti‐microbial activity against many prevalent pathogens including multi‐drug resistant M. tuberculosis. Granulysin is a constituent of cytotoxic granules within T cells and NK cells and its bactericidal activity is dependent on its co‐expression with perforin. The regulation of granulysin expression in T cells is poorly defined due to the lack of a murine gene homologue. The aim of this project is to define the signals required by human T cells to express granulysin. We found that the common gamma‐chain cytokines, IL‐2, IL‐15 and IL‐21 induced the highest levels of mRNA and protein, of perforin and granulysin in T cells compared to TCR stimulation or mitogen. In response to these cytokines, the kinetics of protein expression differed, with perforin levels peaking at 3 days and granulysin at 5 days. Preliminary results indicate that activation of T cells by both IL‐15 and IL‐21 resulted in the degradation of IkB and a concomitant increase in NF‐kB p65 expression. However, dramatic increases in total STAT5 and p‐STAT5 expression were observed in response to IL‐15, but not IL‐21, and coincided with granulysin and perforin expression. Our data suggest that the induction of granulysin and perforin expression by IL‐15 and IL‐21 may be mediated by activation of different signaling pathways. This research is supported by the Dept. of Microbiology and Immunology, UTMB.
Herpesvirus Entry Mediator and Cytomegalovirus ORF UL144 bind a common region of B and T Lymphocyte Attenuator
Authors: Sedy John Richard , Cheung Tim C , Nelson Christopher A , Norris Paula S , Benedict Chris A , Murphy Kenneth M , Fremont Daved H , Ware Carl F
Published: June 2021

Herpesvirus entry mediator (HVEM) interacts with the TNF ligands LIGHT and Lymphotoxin, and with the immunoglobulin (Ig) domains of B and T lymphocyte attenuator (BTLA) and herpes simplex virus‐1 glycoprotein D (gD), demonstrating complex ligand interactions. The crystal structures of mouse BTLA and of human BTLA bound to human HVEM show that BTLA folds as an I‐set Ig domain, and that BTLA binds HVEM similar to gD. BTLA also binds the Cytomegalovirus (CMV) mimic of HVEM, ORF UL144, with lower affinity but causing greater T cell inhibition. We sought to determine how BTLA interacts with both HVEM and ORF UL144 and to clarify how BTLA may interact with multiple ligands to induce different activities. Here we show by single point mutation of human BTLA that key solvent exposed residues clustered at one face of the molecule are critical for binding both human HVEM and ORF UL144. In addition, a monoclonal antibody to human BTLA that blocks binding to human HVEM recognizes one of the residues in this region. Furthermore, we show that ORF UL144 from rhesus monkey CMV also binds human BTLA. The requirement of the same residues for BTLA binding to its ligands indicates a conserved binding mode between mammalian and viral proteins, providing insight to how BTLA is activated by diverse ligands. This research is supported by a fellowship from the Diabetes & Immune Disease Natioinal Research Institute.
Recombinant human IL‐7 (rhIL‐7) preferentially expands CD4+ and CD8+ naïve T cells and enhances T cell receptor (TCR) repertoire diversity
Authors: Sportes Claude , Hakim Frances T , Memon Sarfraz A , Zhang Hua , Brown Margaret R , Fleisher Thomas A , Krumlauf Michael C , Babb Rebecca R , Chow Catherine K , Fry Terry J , Engels Julie , Buffet Renaud , Morre Michel , Venzon David J , Korngold Robert , Pecora Andrew , Mackall Crystal L , Gress Ronald E
Published: June 2021

Aging is associated with a decline in T cell generation and TCR repertoire diversity and a progressive accumulation of oligoclonal, terminally differentiated and functionally limited CD8+ effector T cells which may impede new responses. A phase I study of rhIL‐7 (subcutaneously every other day for 2 weeks) evaluated toxicity and biologic activity in 16 adults (age 20–71). RhIL‐7 was well tolerated. It resulted in a dose but not age dependent preferential expansion of naïve CD8+ T cells and of CD4+ RTEs, naïve and central memory T cells. (4 to 5‐fold at the higher doses of 30 and 60 μg/Kg/dose). T cells remained functional in vitro and persisted for 6–8 weeks. The naïve T cell expansions resulted in increased TCR diversity assessed before and one week after treatment. We scored divergence between each Vβ spectratype and normal standard comparing all the pre and post scores by Wilcoxon paired test. The increase in diversity was statistically significant in 4 of 6 subjects in CD4+, CD8+ or both.

RhIL‐7 resulted in circulating T‐cell profiles resembling what is seen early in life with abundant naïve T‐cells and wide TCR diversity. We propose that rhIL‐7 has the potential of becoming a clinically usable T‐cell growth factor for rejuvenation of peripheral T‐cell populations in hosts with impaired immunity due to physiologic (age), iatrogenic (chemotherapy) or pathologic (HIV) lymphodepletion.
IL‐23 inhibits innate IFN‐γ secretion
Authors: Sieve Amy N. , Meeks Karen D. , Lee Suheung , Balch Joshua B. , Berg Rance E.
Published: June 2021

Most studies have concluded that interleukin‐23 (IL‐23) promotes IL‐17 secreting T cells. While some reports have also characterized IL‐23 as having redundant pro‐inflammatory effects with IL‐12, we have instead found that IL‐23 can regulate the innate immune response in a novel manner, by antagonizing IL‐12 induced secretion of interferon‐γ (IFN‐γ). Our recent data documents the rapid, innate production of IFN‐γ from memory CD8 T cells, as well as NK cells, stimulated with IL‐12 and IL‐18. When splenocytes are cultured with IL‐23, IFN‐γ secretion in response to IL‐12 is dramatically reduced, as measured by ELISA and intracellular cytokine staining. The impact of IL‐23 is most prominent in CD8 T cells, but can also be seen in NK, NK‐T, and CD4 T cells. We also show that IL‐23 can regulate the induction of IFN‐γ by endogenously produced IL‐12 from Listeria monocytogenes (LM) infected macrophages. In vivo, LM infection induces higher serum IFN‐γ levels and a greater percentage of IFN‐γ + CD8 T cells in IL‐23p19 deficient mice as compared to wild‐type mice. We are currently investigating the mechanism by which IL‐23 inhibits IFN‐γ secretion, and if IL‐23 can inhibit other effector functions. Our data suggest that IL‐23 may be a key player in determining the responsiveness of lymphocytes to IL‐12 and their subsequent secretion of IFN‐γ.
Regulation of P‐selectin Ligand Expression by Activated Cutaneous Lymphocyte‐Associated Antigen (CLA) T Cells
Authors: Ni Zhenya , Walcheck Bruce
Published: June 2021

T cells utilize E‐ and P‐selectin to enter inflamed skin; however, these C‐type lectins require different glycan modifications on T cells for binding. Sialyl‐Lewis x is sufficient for E‐selectin binding, whereas a core‐2 O‐glycan terminated with sLex (C2‐O‐sLex) is required for P‐selectin binding. We have characterized a mAb CHO‐131 specific to C2‐O‐sLex, which detects a subset of CLA+ T cells. A direct comparison of the selectin binding of CHO‐131+ and CHO131−CLA+ T cells from peripheral blood revealed a significantly greater P‐selectin binding activity by the former subset. Interestingly, we observed that the in vitro activation of CLA+ T cells transiently upregulated the CHO‐131 epitope and the mRNA level of the core‐2 O‐glycan branching enzyme C2GnT‐I. Moreover, the activated CLA+ T cells uniformly were reactive with P‐selectin/Fc in contrast to resting cells. PSGL‐1 isolated from activated CLA+ T cells revealed increased reactivity with CHO‐131, indicating an increased decoration of PSGL‐1 by P‐selectin glycan ligands following activation. Our findings suggest that P‐selectin ligand expression is upregulated in a transient manner by recently activated CLA+ T cells.
Ethanol alters neutrophil membrane mechanics and adhesion molecule expression
Authors: Oh Hana , Diamond Scott L.
Published: June 2021

Ethanol (EtOH) has been linked to both protective and damaging effects in inflammatory and immune responses of leukocytes. To characterize EtOH effect on neutrophil mechanics, we studied neutrophil rolling, tethering, and firm arrest behavior at physiologically relevant EtOH concentrations achieved in humans by alcohol consumption (<0.5 v/v%). Neutrophils were perfused over parallel‐plate flow chambers with P‐selectin‐coated beads or confluent HAEC (human aortic endothelial cells) monolayer at a venous shear rate of 100 s−1. EtOH decreased the adhesion efficiency of neutrophils from 0.085 to 0.031, increased the average tether length and tether growth velocity fourfold, and decreased bond forces, suggesting a lower degree of membrane‐cytoskeleton association. When perfused over IL‐1‐activated HAEC, the fraction of neutrophils converting to firm arrest decreased from 36% to 24% with EtOH, and average rolling length and rolling velocity both increased with EtOH (24.8 to 33.7 μm and 2.5 to 3.4 m/sec, respectively). EtOH resulted in ~20% increase in L‐selectin and PSGL‐1 expression in quiescent neutrophils as measured by flow cytometry, and significantly inhibited the upregulation of CD11b and shedding of L‐selectin in fMLP‐activated neutrophils. Our results indicate that EtOH affects bond mechanics and surface molecules to change neutrophil response. This work was supported by NIH R01 HL56621.
Innate cytokines and natural receptor agonist arrest natural killer T cell patrolling of liver sinusoids
Authors: Velazquez Peter , Cameron Thomas O. , Kinjo Yuki , Nagarajan Niranjana , Kronenberg Mitchell , Dustin Michael L
Published: June 2021

Natural killer T (NKT) cells are innate‐like lymphocytes that rapidly secrete large amounts of effector cytokines upon activation. Recognition of α‐linked glycolipids presented by CD1d leads to the production of IL‐4, IFNγ, or both, while direct activation by the synergistic action of innate cytokines, IL‐12 and IL‐18, leads to IFNγ production only. Using intravital fluorescence laser scanning microscopy, we previously demonstrated that NKT cells patrol within the hepatic sinusoids. Here we examine the relationship between hepatic sinusoidal NKT cell patrolling and activation. We report that NKT cell arrest results from activation driven by limiting doses of a bacterial derived agonist, GalA‐GSL, and a synthetic agonist, αGalCer. Interestingly, NKT cell arrest also results from IL‐12 and IL‐18 synergistic activation. Thus, both innate cytokines and natural NKT cell TCR agonist trigger arrest and an effector response by sinusoidal NKT cells.
Expression of TNF‐α, IFN‐γ, TGF‐β and IL‐7 on cells isolated from the Bronchoalveolar Fluid in a model of secondary immunodeficiency.
Authors: Luczak Erica Natalia , Calabrese Graciela Cristina , Roux Maria Estela
Published: June 2021

We have previously reported the early appearance (4 days post partum) of γδT cells that colocalize with TNF‐α in Bronchus‐Associated Lymphoid Tissue (BALT), indicating the important role of this cytokine for BALT development. In our experimental model of secondary immunodeficiency due to severe protein deprivation at weaning followed by administration of a 20% casein diet during 21 days (R21), we also demonstrated an increase of TCR γδ+ cells accompanied by an increase in TNF‐α+ cells number in R21. The objective of this work is to analyze and compare cells isolated from the bronchoalveolar fluid (BAL) in R21 vs control rats: 1) the expression of TNF‐α and IFN‐γ, as indicator of an inflammatory process and its regulation by TGF‐β and 2) the cellular regulation by IL‐7. Western Blot analysis showed an increase of all cytokines in cells from BAL of R21 animals when compared to control rats (Figure 1). Our results indicate the existence of an inflammatory process (due to TNF‐α and IFN‐γ) modulated by TGF‐β. The increment of IL‐7 correlates with its capacity for the proliferation and differentiation of γδ T cells, which are increased in BAL from R21 as previously reported. Moreover, we suggest a protective role of IL‐7 in the inflammatory focus.

Supported by B010 grant.
Interferon gamma selectively protects the cerebellum and brain stem from inflammation during EAE pathogenesis
Authors: Lees Jason R , Golumbek Paul T , Sim Julia , Russell John H
Published: June 2021

In multiple sclerosis (MS) the localization of inflammatory foci within the central nervous system (CNS) is associated with prognosis, with lesions of the cerebellum correlated to a severe clinical outcome. Previous studies of experimental autoimmune encephalomyelitis (EAE), a model of MS, revealed distinct clinical outcomes correlated with the capacity to produce IFN‐γ. Outcomes were linked to localization of inflammatory cells in either the spinal cord (wild type (WT)) or the cerebellum and brain stem (IFN‐γ deficient).

We demonstrate using an adoptive transfer system that the ability of the host to respond to IFN‐γduring EAE initiation determines the site(s) of CNS pathogenesis. Transfer of WT Th1 cells into IFNγR deficient mice results in invasion of the brain stem and cerebellum with attendant clinical symptoms, identical to the pathogenesis observed following transfer of IFN‐γ deficient T‐cells to WT hosts. Inflammation of the spinal cord associated with EAE in WT mice is abrogated in both IFN‐γdeficient models. These data demonstrate that interaction between cytokine(s) and host cells during the initiation of EAE determines localization of neuroinflammation. Further, these data reveal that IFN‐γ signaling can selectively protect CNS regions from T‐cell pathogenesis.

Supported by a National Multiple Sclerosis Society Fellowship, NMSS research grants RG3314, RG 3732, and NIH grant # 5 T32 NS07205‐22.
CD73‐generated adenosine regulation of colonic epithelial barrier permeability during inflammatory bowel disease
Authors: Mills Jeffrey H , Waickman Adam T , Mueller Cynthia , Bynoe Margaret S
Published: June 2021

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal (GI) tract that is characterized by weight loss, bloody diarrhea, and increased activated immune cell infiltration in the GI tract. Since extracellular adenosine signaling has been shown to modulate inflammation during IBD, we wanted to determine if CD73, the cell surface enzyme that catalyzes the dephosphorylation of extracellular AMP to adenosine, also has a role in IBD. Therefore, we induced IBD with dextran sodium sulfate (DSS) in drinking water in cd.73−/− and wild type mice. As expected, cd73−/−mice exhibited dramatically more severe IBD than wild type mice (based on weight loss and histology). However, in attempting to verify that T cells were responsible for mediating this severe IBD (by adoptively transferring naïve CD45RBHi T cells from wild type or cd73−/− mice into RAG‐1−/− recipients, which has been proven to induce passive IBD), surprisingly, we did not see any difference between the IBD severity in mice that received wild type or cd73−/− derived cells. These results suggest that other factors (besides T cells) are regulated by CD73/adenosine during IBD. Furthermore, our data indicates that CD73 generated adenosine is involved in the regulation of colonic epithelial barrier permeability, as decreased expression of junctional adhesion molecules (JAMs) is observed in cd.73−/− DSS‐treated mice. AI 57854 (MSB)
Macrophage‐dependent TGFBI‐mediated regulation of collagen levels following ingestion of apoptotic cells
Authors: Nacu Natalia , Luzina Irina G , Todd Nevins W , Atamas Sergei P
Published: June 2021

Inflammation resolution involves phagocytic clearance of apoptotic cells coinciding with activation of repair that includes accumulation of extracellular matrix. The regulation of collagen levels by macrophages during this process has not been studied. Collagen levels were increased in co‐cultures of primary fibroblasts with monocyte‐derived but not with alveolar macrophages following ingestion of apoptotic cells; direct contact between macrophages and fibroblasts was not required for collagen upregulation. Macrophages produced TGF‐β at the levels that were lower than those required for a significant upregulation of collagen, but the levels of TGF‐β‐induced (TGFBI), mRNA and protein were increased in monocyte‐derived but not alveolar macrophages following ingestion of apoptotic cells. Fibroblast activation with recombinant TGFBI led to decreased DNA binding by p53, increased DNA binding by PU.1, downregulation of matrix metalloproteinase 14 (MMP14) levels, and upregulation of collagen protein but not mRNA levels. Overexpression of p53 or MMP14, or siRNA‐mediated inhibition of PU.1 increased MMP14 and decreased collagen levels. In conclusion, following ingestion of apoptotic cells, monocyte‐derived but not alveolar macrophages produce TGFBI that employs p53 and PU.1 to downregulate MMP14, leading to a subsequent collagen accumulation. Support: NIH NHLBI 1R01HL074067 and VA Merit (SPA).
RasGRP1‐dependent Signals Are Required For Regulatory T cell Survival and Function.
Authors: Buhlmann Janet E. , Barrington Robert A. , Lin Michael L. , Lim Bing
Published: June 2021

Regulatory cells have been shown to have a critical role in controlling immune responses and preventing autoimmunity. In the absence of RasGRP1, mice develop a spontaneous lupus‐like disease, the reasons for which are poorly defined. T cell development is dramatically impaired by the loss of RasGRP1 and the diminished T cell population may be one factor for disease development. Given the important role of regulatory T cells, we undertook studies to monitor regulatory cell development and function in the absence of RasGRP1. Regulatory T cells develop normally in the absence of RasGRP1 and make up a larger portion of the CD4+ T cell pool than in wild type mice. Surprisingly, regulatory T cells from RasGRP1 mutant mice exhibit impaired function with increased susceptibility to apoptosis in vitro. We are currently testing in vivo regulatory cell function and investigating the reasons for the increased susceptibility to apoptosis. Together these data suggest that RasGRP1 is an important mediator of signals leading to both regulatory cell survival and function.
Adaptive regulatory T cells (TR) reverse diabetes and persist as memory cells
Authors: Summers‐Torres Daphne Mahriah , Godebu Elana , Bradley Linda M.
Published: June 2021

In a previous study we used the type 1 non‐obese diabetic mouse model (NOD) to show that (TR) can be generated ex vivo from monoclonal islet‐specific CD4 cells by the combination of TCR signaling and TGFB1. These T cells express FoxP3, produce IL‐10 and TGFB1 and prevent the spontaneous induction of diabetes after adoptive transfer to NOD mice. Using allelically marked donor cells we now show that these TR cells can reverse overt diabetes if transferred early following the onset. Moreover, TR cells can also be induced from polyclonal CD4 cells from NOD mice. Following the adoptive transfer these TR cells can persist for more that one‐year and are able to maintain FoxP3 expression as well as production of IL‐10 and TGFB1. However they do not express CD25, which distinguishes them from the innate TR cells. We can identify adaptive TR cells in the lymphoid compartment and in pancreatic infiltrates of the protected mice. As memory cells these adaptive TR cells undergo homeostatic turnover and retain the capacity to respond to antigen challenge. Following re‐isolation as memory cells they transfer protection against diabetes to NOD recipients of diabetogenic effectors. The results indicate that adaptive regulatory CD4 cells can be homeostatically maintained as memory cells that can contribute to the long‐term control of diabetes, making them possible candidates for use as immunotherapeutic agents. NIH RO1 DK 59438; JDRF
Endogenous IL‐2 production governs the in vitro induction of FoxP3‐expressing adaptive Treg in the NOD mouse
Authors: Goudy Kevin , Wang Bo , Tisch Roland
Published: June 2021

Lack of self‐tolerance to [beta] cells in Type 1 diabetes (T1D) is in part due to defects in the development and function of FoxP3‐expressing immunoregulatory CD4+ T cells (Treg). IL‐2 has been shown to be involved in Treg differentiation and maintenance. In addition, the IL‐2 gene located in the IDD3 locus is a candidate for diabetes susceptibility. To explore the role of IL‐2 in the development of adaptive Treg and T1D, NOD mice congenic for the C57BL/6 IDD3 interval(NOD.IDD3) and which have a reduced incidence of diabetes were studied. Stimulated CD25‐CD4+ T cells from NOD.IDD3 versus NOD mice produced ~2‐fold more IL‐2. To test the respective T cells to differentiate into adaptive Treg, CD25‐CD4+ T cells were stimulated with anti‐CD3 & CD28 antibodies and varying concentrations of TGF‐β1. A 3‐fold increase in FoxP3‐expressing cells was detected in NOD.IDD3 versus NOD cultures. Notably, the addition of anti‐IL‐2 ab blocked the induction of FoxP3‐expressing T cells in both sets of cultures. Furthermore, an equivalent frequency of FoxP3‐expressing T cells was detected in NOD.IDD3 and NOD cultures supplemented with IL‐2. These results demonstrate that elevated IL‐2 production by stimulated NOD.IDD3 CD25‐CD4+ T cells increases the efficiency of adaptive Treg differentiation. Furthermore, reduced production of IL‐2 by CD25‐CD4+ T cells in NOD mice may limit the induction of adaptive Treg and contribute to T1D.
CII‐pulsed immature dendritic cells suppress T cell proliferation and inflammatory cytokines in the HLA‐DQ6αβ8αβ tg mouse model of spontaneous polyarthritis
Authors: Sidhu Maninder , Bradley David S
Published: June 2021

We have previously shown that immunization with type II collagen (CII)‐loaded immature dendritic cells (iDCs) significantly delays the onset (> 90 days) and reduces the severity of spontaneous polyarthritis, compared to sham immunizations, in the HLA‐DQ6ab8ab transgenic (tg) model of spontaneous polyarthritis. Vaccinated mice received bone marrow‐derived DCs, cultured in vitro with GM‐CSF, and pulsed with CII, or control preparations of unpulsed DCs, or PBS. Flow cytometric analysis of the cellular phenotype of the DC populations indicated they were both maturation‐resistant. The presence of CII‐loaded iDCs blocked or greatly decreased the proliferation of anti‐CD3 activated T cells in vitro. The presence of CII‐loaded iDCs in vivo corresponded with a significant decrease in the levels of pro‐inflammatory cytokines, e.g. IFN‐g and TNF‐a, compared to mice receiving either unloaded iDCs or PBS. Interestingly mice receiving CII‐iDCs also had significantly reduced levels of IL‐10, suggesting a possible inflammatory role for IL‐10 in this model. Immunostaining of arthritic paws also indicated an absence of IFN‐g and TNF‐a associated only with the CII‐loaded‐iDC treated mice. This study emphasizes the potential of antigen‐pulsed dendritic cells as an immunotherapeutic vehicle for inducing tolerance in autoimmune diseases.
Development of a novel immunoliposomal delivery system for regulation of inflammation in target organs in murine models of Systemic Lupus Erythematosus
Authors: Bagavant Harini , Scindia Yogesh , Thimmalapura Puspha‐Rekha , Deshmukh Umesh
Published: June 2021

Murine models of Systemic Lupus Erythematosus have established the importance of local inflammatory responses and the end organ in the pathogenesis of nephritis. The mesangial cells of the renal glomerulus are key players in this process. However, there are no unique cell surface markers identified for murine and human mesangial cells. Our study shows that α8 is expressed in mouse glomeruli and increases with renal disease making it a potential candidate for immunotargetting of therapeutic agents to the glomerular mesangium. We used a novel strategy of sized liposomes composed of specific lipids conjugated to rabbit anti‐α8 intergrin antibody to target the glomerular mesangium. The anti‐α8 immunoliposomes (α8‐IL) were loaded with a fluorescent dye (DiI) to study trafficking of the immunoliposomes following tail vein injection. DiI was rapidly detected by fluorescence microscopy in glomeruli of mice injected with α8‐IL indicating intracellular uptake. Although expression of α8 intergin has been shown in the lung, brain and interstitial myofibroblasts, the sized liposomal preparation could not access these tissues. A small (<5%) of CD11b+ cells carrying DiI were detected in spleen, liver and lung. Control DiI loaded rabbit IgG ‐IL did not traffic to the glomeruli. This study establishes a novel therapeutic modality for the treatment of glomerular disease and targeted drug therapy in lupus nephritis.
The ability of dendritic cells to prevent diabetes development in NOD mice depends on their production of high levels of IL‐10 versus low levels of IL‐12
Authors: Manirarora Jean N , Parnell Sarah A , Hu Yoon‐Hyeon , Kosiewicz Michele M , Alard Pascale
Published: June 2021

NOD mice, similar to diabetic patients, exhibit various defects in different types of immune cells, including dendritic cells (DC) that produce pro‐inflammatory cytokines, and regulatory T cells. Recognition of TLR2 can lead to immune evasion of various microorganisms via induction of tolerogenic APC and regulatory cells. The goal of this study was to induce tolerogenic NOD DC able to prevent diabetes. DC were stimulated for 24 hrs with two TLR2 agonists, lipoteichoic acid (LTA) from S. aureus and Lactobacillus. DC stimulated with LTA produced 50 times more IL‐12 than IL‐10, and mediated acceleration of diabetes development when transferred into NOD mice. In contrast, high doses of Lactobacilli (LC) favored the production of IL‐10 versus IL‐12. Transfer of DC treated with high‐dose LC, not low‐dose LC, into NOD mice delayed diabetes onset and decreased diabetes incidence. Interestingly, DC treated with high‐dose LC produced 5 times more IL‐10 than those treated with low‐dose LC. Finally, significant increases in IL‐10 levels were detected in most of the samples from LC DC‐injected NOD mice. These data indicate that the levels of IL‐10 versus IL‐12 produced by DC cultured with various stimuli correlates with the ability of those cells to prevent diabetes in NOD mice. IL‐10 may play a significant role in diabetes prevention either directly, by suppressing pro‐inflammatory responses, and/or by inducing regulatory cells.
Identification of prohibitin as a target of IVIg on human B cells
Authors: Proulx Dominic Paquin , Aubin Éric , Lemieux Réal , Bazin Renée
Published: June 2021

IVIg are therapeutic preparations of human IgG prepared from the plasma of thousands of donors and are currently used to treat more than one hundred autoimmune or inflammatory diseases, although their mechanism of action is not precisely known in any of these diseases. Previous work from our laboratory showed that IVIg could directly influence the fate of human B cells by inducing their differentiation (Blood, 2003; 101:3065). The goal of the present study was to identify the membrane target of IVIg on human B cells. We used two different methods to isolate the B cell membrane proteins recognized by IVIg (classical immunoprecipitation following incubation with IVIg and cell solubilization, and affinity chromatography of soluble membrane proteins on IVIg‐Sepharose). The recovered proteins were identified by LC‐MS. Surprisingly, most of them were not membrane receptors but intracellular proteins such as actin, prohibitin 1 and 2, heat shock cognate 71KDa protein and RPL13. Since prohibitin is known to be associated with the BCR, at least in IgM‐expressing B cells (EMBO J, 1994; 13:3782), and to regulate events such as ERK phosphorylation and progression in the cell cycle, we have examined more closely the association between IVIg, BCR and prohibitin. Preliminary results suggest that IVIg interact with prohibitin in a BCR‐independent way. The role of prohibitin in the effects of IVIg on human B cells is currently under study.