bluuebunny/crossref_metadata_2025 huggingface dataset:
PD-L1 interacts specifically with B7-1 to regulate T cell function (88.24)
Authors: Butte Manish J , Keir Mary E , Phamduy Theresa B , Sharpe Arlene H , Freeman Gordon J
Published: January 2023

Pathways in the B7:CD28 family regulate T cell activation and tolerance. The demonstration of B7 dependent responses in CD28/CTLA-4−/− T cells together with studies pointing to stimulatory and inhibitory functions for PD-L1 and PD-L2, have suggested additional receptors for these B7 family members. We show that B7-1 and PD-L1 can bind to one another with an affinity greater than the affinity of B7-1 for CD28 but less than CTLA-4. The B7-1:PD-L1 interaction can be shown in both biophysical and cell-based assays, and is functionally significant. We demonstrate that this interaction is restricted to B7-1 and PD-L1. B7-2 does not interact with PD-L1, nor does PD-L2 interact with B7-1. We have identified anti-PD-L1 antibodies with either dual-specificity, which can block two interactions (PD-L1:B7-1 and PD-L1:PD-1), or mono-specificity, which can block only one (PD-L1:B7-1). The effects of this pathway on T cell proliferation and cytokine production will be discussed. Our findings give increased significance to B7-1 and PD-L1 on T cells, and add an additional dimension to immunoregulatory functions of the B7:CD28 family.
Regulation of CD28 costimulation versus anergy in T cells by RasGRP and DGK-α (88.27)
Authors: Gajewski Thomas F , Zha Yuan-yuan , Praveen Kesavannair , Janardhan Sujit
Published: January 2023

Although the functional attributes of CD28 costimulation and T cell anergy are well-defined, the biochemical basis of these processes have been incompletely understood. Details of Ras regulation in T cells have provided new insights into CD28 costimulation and anergy. Using confocal microscopy, we have observed that CD28 costimulation facilitates recruitment of the DAG-activatable guanine nucleotide exchange factor RasGRP to the plasma membrane. In addition, T cells from RasGRP-deficient mice show defective IL-2 production and ERK activation in response to TCR/CD28 ligation but not through the IL-2R. Moreover, introduction of CA Ras can bypass the need for CD28 costimulation for multiple functional outputs. In contrast, anergic T cells show upregulated expression of the DAG-metabolizing enzyme diacylglycerol kinase-α (DGK-α). Introduction of DGK-α can mimic the anergic state, and inhibition of endogenous DGK-α can recover function of anergic cells. Together, these results support a yin-yang model in which T cell activation is centrally influenced by DAG-dependent signaling, with CD28 costimulation promoting RasGRP-dependent Ras activation and DGK-α negating this process in the anergic state.
IL-10 secretion from BALB-neuT mice cells after stimulation with murine breast tumor cell lines (88.28)
Authors: Cabrera Rosalyn , Nava-Parada Pilar , Pease Larry R
Published: January 2023

Failures in tolerance mechanisms cause autoimmunity. Self reactive cells in the periphery are kept in check by peripheral tolerance. Mice expressing a neoself antigen, called BALB-neuT, were used as a model for delineating mechanisms of peripheral tolerance. Two questions were addressed: Are BALB-neuT tolerant to neoself antigen? Can BALB-neuT tolerance be broken? We first established that BALB-neuT are tolerant to Her2/neu peptide while parental BALB/c mounted a significant response after immunization with Her2/neu peptide in CFA and repeated CpG stimulations. Next we depleted CD25+ cells and used the immunomodulating antibody B7-DC XAb to study the mechanism involved. CD8+ T cells from treated BALB-neuT generated a potent response when compared to untreated BALB-neuT or BALB/c, as measured by IFNγ secretion. Interestingly, IL-10 secretion from BALB-neuT T cells was high after incubation with murine breast cell lines, irrespective of expression of BALB-neuT antigen. After depletion of CD4+CD25+ T cells, secretion of IL-10 was diminished, suggesting that the IL-10 is being secreted by T regulatory cells. In conclusion, BALB-neuT are tolerant to neoself antigen. Tolerance can be broken by depleting Tregs or using B7-DC Xab to modulate immunity. Future studies are aimed at identifying mechanisms regulating IL-10 secretion to unravel pathways important in regulation of autoimmunity.
Definition of a Qa-1-Qdm-dependent interaction between NK cells and activated T cells that inhibits expansion and memory development of pathogenic T cells (88.31)
Authors: Lu Linrong , Ikizawa Koichi , Werneck Miriam B.F. , Cantor Harvey
Published: January 2023

The ability of natural killer (NK) cells to recognize and lyse activated T cells in vitro may allow these cells to participate in regulating adaptive immune response through inhibition of clonal expansion of antigen-activated T cells. This interaction may be tightly constrained by an interaction between inhibitory NKG2A receptors on NK cells and Qa-1-Qdm ligands on activated T cells (Qa-1-NKG2A interaction).

We tested the hypothesis that Qa-1-NKG2A interaction protects activated T cells from NK lysis and is essential for clonal expansion and development of immunologic memory. We found that Qa-1-deficient CD4 cells fail to undergo antigen-induced or homeostatic expansion and develop autoimmune EAE disease due to increased susceptibility to NK cells lysis. Re-expression of Qa-1-Qdm on autoreactive CD4 cells rescues expansion of self-reactive T cell clones. These findings were confirmed using Qa-1 knock-in mice which express an amino acid exchange mutation (R72A) that eliminates Qa-1 binding to NKG2A but spares binding to the TCR. Dissection of the NK cell population revealed that the NKG2A+ but not NKG2A– subset of NK cells mediate lysis of Qa-1-deficient CD4 cells. Blockage of the Qa-1-NKG2A interaction that constrains NK lysis of activated T cells resulted in potent NK-dependent elimination of activated autoreactive T cells and amelioration of autoimmune EAE. These findings extend the functional reach of the NK system to include regulation of adaptive T cell responses and demonstrate that antibody-mediated blockade of NKG2A-Qdm interactions as a new clinical approach to eliminate antigen-activated T cells in the context of autoimmune disease or transplantation.
Role of Epitope Valency in inducing Tolerance or Immunity (88.34)
Authors: Shah Shivanee , Qiao Liang
Published: January 2023

Different antigens result in different immune responses. Soluble antigens, such as food, are tolerogenic; whereas microbes, which generally have repeating eptitopes, are immunogenic. Thus, we hypothesize that epitope valency of the antigen plays a role in deciding the immune outcome. B cells are antigen presenting cells that specifically recognize antigenic epitopes and become activated in the presence of multi-valent epitopes. Thus, we studied the effect of different epitope valencies on B cell activation and consequently T cell activation. Using monovalent and divalent fragments of the same antigen, we determined B cell activation markers by flow cytometry; and T cell activation by proliferation assays and cytokine analysis (ELISA).

We found that like unstimulated B cells, univalent epitopes cannot activate B cells but can expand a population of CD4+CD25+Foxp3+T cells (Tregs); whereas divalent epitopes activate B cells and consequently help T cell activation, but do not induce T regs. Interestingly, the mechanism of expansion of this Treg population is via TGFβ3 and not via known cytokines TGFβ1 or IL-10. This implies not only that structure of the antigen plays a critical role in setting off a competitive immune response, but also that resting B cells can maintain a population of regulatory T cells that can prevent the activation of self-reactive T cells and thus prevent autoimmunity.
Complement-bearing immune complexes induce IL-10 and granzyme B production by human CD4+ T cells (88.35)
Authors: Price Jeffrey D. , Kemper Claudia , Atkinson John P.
Published: January 2023

Induction of complement-induced regulatory T cells (cTregs) occurs through concurrent cross-linking of the TCR and the complement regulatory protein CD46 on naive human CD4+ T cells. The phenotype of cTregs includes the secretion of large amounts of IL-10 as well as production of granzyme B, providing cell-contact independent and cell-contact dependent mechanisms for immunosuppression. Previous work in our lab has demonstrated cTreg induction by both natural and pathogenic ligands for CD46: C3b dimers and the M protein of group A streptococci, respectively. The current studies examine whether a physiological presentation of complement components on immune complexes can stimulate CD46 in a similar fashion. BSA:α-BSA complexes and antibodies to CD3 were used to stimulate human CD4+ T cells. Immune complexes that were opsonized with complement C3b and C4b fragments through treatment with normal human serum induce IL-10 secretion by T cells and production of intracellular granzyme B. Immune complexes that lack opsonizing complement components due to heat-inactivation or EDTA treatment of serum do not induce IL-10 or granzyme B production. As immune responses mature towards many pathogens, immune complexes are formed. We propose that these complexes, when presented to T cells by antigen-presenting cells bearing Fc or complement receptors, generate a regulatory phenotype and thereby quench the immune response.

Funding provided by NIH grant AI070489 AADCRC, and the Sandler Program for Asthma Research.
Inhibition of glycogen synthase kinase-3 promotes human CD4+ T regulatory type 1 activity (88.48)
Authors: Garcia Carlos Armando , Rehani Kunal , Martin Michael
Published: January 2023

T regulatory type 1 cells (Tr1) are a distinct subset of a heterogeneous population of regulatory CD4+ T cells present in humans whose main function is to prevent chronic immune activation and maintain normal immune homeostasis via the production of interleukin-10 (IL-10). Although several studies have identified particular in vitro conditions that promote Tr1 differentiation, none to date have identified a putative molecule when pharmacologically targeted promotes Tr1 differentiation. Identifying such a target has proven difficult due to the inherent complexity and lack of information regarding IL-10 gene regulation in T cells. In the present study, we examined the influence of the serine/threonine kinase, glycogen synthase kinase-3 (GSK3) to regulate IL-10 production by CD4+ T cells. CD3 stimulated peripheral blood mononuclear cells (PBMCs) or CD4+ T cells treated with GSK3 inhibitors promotes IL-2 and IL-10 production while suppressing interferon-gamma (IFN-?) levels as compared to cells treated with anti-CD3 alone. The ability of GSK3 inhibition to promote IL-10 and suppress IFN-? production in CD4+ T cells was restricted to the memory (CD45RA− CD45RO+) population of CD4+ T cells, whereas naïve (CD45RA+ CD45RO-) CD4+ T cells solely increased IL-2 production. Memory CD4+ T cells activated in the presence of GSK3 inhibition acquired the capacity to suppress the bystander activation of CD4+ T cells; an effect dependent on the presence of IL-10. Our findings illustrate a dichotomy present in the role of GSK3 in naïve and memory CD4+ T cells and identify GSK3 as a key kinase in Tr1 differentiation.
CD28 Regulates de novo Generation of Tregs through IL-2 in the Periphery (88.49)
Authors: Yu Xue-Zhong , Liang Yaming , Anasetti Claudio , Guo Fei
Published: January 2023

CD28 plays a critical role in Treg development in the thymus and also contributes to Treg survival and homeostasis in the periphery. However, the role of CD28 in the differentiation of Tregs (CD4+Foxp3+) from naïve T cells (CD4+Foxp3-) in the periphery is unclear. To address this question, we stimulated naïve T cells (CD4+CD25-) with APCs plus anti-CD3 mAb in the presence of TGF-beta. We found that differentiation of Tregs was essentially blocked when CD28 costimulation was absent either by using CD28−/− T cells or B7−/− APCs in the culture. The requirement of CD28 for Treg differentiation was further confirmed in vivo, where WT or CD28−/− naïve T cells were adoptively transferred into irradiated syngeneic or allogeneic recipient. Using CFSE labeling, we found that differentiation of Tregs from WT naïve T cells was irrelevant to the number of cell division, and even T cells without division could also be “converted” into Tregs. Naïve T cells without CD28 divided much more slowly and did not differentiate into Tregs regardless of cell division. The requirement of CD28 for Treg differentiation was mediated through IL-2, in that neutralization of IL-2 with its specific antibody blocked Treg differentiation from WT naïve T cells and additional IL-2 restored Treg differentiation from CD28−/− naïve T cells. We further asked whether other common gamma-chain cytokines have a similar role as IL-2, and found that IL-7, but not IL-4 or IL-15, could partially restored Treg differentiation in the replacement of IL-2. In conclusion, our current study indicates that CD28 is required for Treg differentiation from naïve T cells through IL-2 production in the periphery.
Polymorphic alleles of murine CD244 exhibit divergent function in NK cells (89.1)
Authors: westcott jill m , mooney jill m , wakeland edward , schatzle john d
Published: January 2023

CD244 (2B4) is a member of the signaling lymphocyte activation molecule (SLAM) family of receptors and is expressed on all murine NK cells and a subset of CD8+ T cells. The SLAM family of receptors exists as one of two stable haplotypes (b and z haplotype) in common laboratory strains of mice. The b haplotype is associated with C57-derived mouse strains while the z haplotype is found in most other inbred strains. Previous studies have shown that CD244 exhibits inhibitory function in NK cells from C57BL/6 (B6) mice. Here we employ a congenic mouse strain (B6.Sle1b) that differs from the parental solely at a region of chromosome 1 containing the SLAM family of genes to characterize the function of the z haplotype of CD244. We show that the more common haplotype of CD244 exhibits activating function in an SH2D1a dependent manner as evidenced by enhanced lytic activity towards targets expressing the ligand for CD244, CD48. The activating function of the z version of CD244 is due to polymorphic differences that appear to be clustered in the ligand-binding region of the receptor. In addition, we observed differential baseline phosphorylation and adaptor association for each of the CD244 haplotypes in cultured NK cells accounting for their functional differences.
Characterization of the inhibitory receptor CD300a in human neutrophils (89.10)
Authors: Borrego Francisco , Alvarez Yelina , Tang Xiaobin , Coligan John E.
Published: January 2023

To achieve an adequate response, cells of the immune system must be tightly regulated to avoid hypo- or hyper-responsiveness. One of the mechanisms used by the immune system to avoid excessive inflammation is the ligation of inhibitory receptors containing immunoreceptor tyrosine based inhibitory motifs (ITIM). In this report, we show that human neutrophils express the ITIM containing CD300a (IRp60) receptor and that its expression is developmentally regulated. Stimulation of neutrophils with LPS and GM-CSF increased the cell surface expression of CD300a as a result of the rapid translocation of an intracellular pool of the receptor to the cell surface. Cross-linking experiments showed that CD300a inhibited FcãRIIa (CD32a) receptor immunoreceptor tyrosine based activating motif (ITAM) mediated signaling, but not TLR-4 mediated reactive oxygen species (ROS) production. Therefore, in human neutrophils, the ITIM containing CD300a receptor is able to specifically down-modulate ITAM derived signals.
Lithium-induced upregulation of the granzyme B inhibitor proteinase inhibitor 9 (PI-9) in monocytes: Role of glycogen synthase kinase-3? (89.13)
Authors: Classen Carl Friedrich
Published: January 2023

Proteinase inhibitor 9 (PI-9) - the only known natural antagonist of the lymphocyte protease granzyme B (GrB) - is an intracellular serpin expressed in lymphocytes and monocyte-derived cells. By intracellular flow cytometry, we have previously shown that ex-vivo stimulation by lipopolysaccharides leads to upregulation of PI-9 within 24 hours in the monocyte, but not the lymphocyte fraction; this can be inhibited by the NF-kappaB inhibitor pyrrolidin dithiocarbamate (PTDC). Glycogen synthase kinase 3 (GSK) is a lithium-inhibitable, constutitively active serine/threonine kinase that controls a number of intracellular functions, especially those involving NF-kappaB regulated genes. We now asked whether lithium-induced inhibition of GSK might influence expression of PI-9 in monocytes and lymphocytes. A 24 hours incubation assay was done using heparinized full blood of healthy volonteers, and PI-9 expression was analysed by intracellular flow cytometry. We found that lithium chloride leads to PI-9 upregulation in monocytes at a medium concentration (1.5 μM), which could not further be enhanced by higher concentrations (10 or 20 μM lithium chloride). In lymphocytes, no alteration of PI-9 expression was induced. When cells were stimulated by LPS in the presence of lithium, only marginal additional upregulation of PI-9 could be induced. Our data show that a lithium-regulated factor, probably GSK, is involved in PI-9 expression in monocytes, down-regulating PI-9 in resting cells. Since it has been shown that PI-9 over-expression in antigen presenting cells, e.g. dendritic cells, leads to an enhanced immune response, our finding may be relevant for specific immune regulation. Pharmacological manipulation of PI-9 expression may represent a useful tool to study immune regulatory mechanisms as well as for therapy .
A novel role of Janus Kinase 3 and IL-6 in M-CSF-mediated CCL2 production in monocytes and macrophages (89.17)
Authors: Kim Hee-Jung , Wang Yijie , Hunter Melissa , Mo Xiaokui , Marsh Clay B
Published: January 2023

Macrophage Colony-Stimulating Factor (M-CSF) enhances differentiation and survival of monocytes and macrophages. Elevated levels of M-CSF and newly infiltrated monocyte/macrophages are detected in inflammatory diseases and cancer. However, the mechanism regulating M-CSF-mediated mononuclear phagocyte activation remains unclear. Since Janus kinase 3 (JAK3) regulates myeloid development and is a target to regulate the immune system in transplantation and cancer therapy, we determined function of JAK3 in M-CSF-mediated inflammatory responses. JAK3 in monocytes was induced by M-CSF in dose- and time-dependent manner and was higher in macrophages compared to monocytes. A specific JAK3 inhibitor (JANEX-1) suppressed M-CSF-induced CCL2 and IL-10 production in monocytes, but did not reduce M-CSF-induced VEGF production or caspase-3 activation. These results correlated with the observation that bone marrow-derived macrophages (BDMs) from JAK3 KO mice had less M-CSF-induced CCL2 production than WT BDMs, although both BDMs were not different in differentiation based on CD11b and F4/80 staining. Interestingly, exogenous IL-6 augmented M-CSF-induced CCL2 and IL-10 production and enhanced JAK3 expression. Since c-Jun binds the promoter regions of CCL2 and IL-10, we also utilized SP600125 (a specific Jun kinase inhibitor). Both JANEX-1 and SP600125 suppressed M-CSF- and M-CSF/IL-6-induced CCL2 and IL-10 production in a dose-dependent manner, linking JAK3 and JNK signaling pathways. Increased CCL2 may recruit additional mononuclear phagocytes and prolong the inflammatory response, while IL-10 may counter this inflammatory cascade. These data suggest that JAK3 regulation is an important target in chronic inflammatory disease and provides a mechanism by which IL-6 affects these events.
IFN-gamma Limits Peripheral T cell Expansion of Extrathymic T cells. (89.18)
Authors: Blais Marie-Eve , Brochu Sylvie , Perreault Claude
Published: January 2023

When overexpressed under the p56Lck proximal promotor, Oncostatin M induces extrathymic T cell development in the lymph nodes. During LCMV infection, extrathymic (ET) T cells produce copious amounts of IFN-γ and their expansion is precocious but of limited amplitude because of early apoptosis. Moreover, ET T cells show significant apoptosis in uninfected mice. Hence, we sought to understand why ET T cells are more susceptible to apoptosis than thymic T cells. We found that the mitochondrial apoptotic pathway is likely involved in apoptosis of CD4+ ET T cells because of lower Il-7Rα and Bcl-2 levels compared to thymic T cells. In contrast, this deficit was not seen in CD8+ ET T cells. However, CD8+ ET T cells displayed higher Fas and active caspase 8 expression indicating apoptosis via the death receptor pathway. Moreover, we found that ET T cells and thymic T cells in homeostatic peripheral expansion (HPE) shared many similarities: memory phenotype, accelerated turnover and increased susceptibility to apoptosis. Moreover, spontaneous IFN-γ secretion by CD8+ ET T cells leading to massive accumulation in sera was observed. Finally, we found that CD8+ T cell survival was restored during HPE induced with ifngR−/−T cells as well as in ifng−/−ET T cells compared to IFN-γ-responsive T cells. Taken together, these results show that IFN-γ regulates apoptosis of ET T cells T cells in HPE and thereby limits their accumulation in vivo.
A novel role for Ku70 in differential regulation of pro-inflammatory cytokine response to CpG-DNA (89.22)
Authors: Wang Xin , Dragoi Ana-Maria , Riolo Matthew , Peng Michelle (Xiao) , Chu Wen-Ming
Published: January 2023

CpG-DNA or synthetic oligodeoxynucleotides containing CpG motif (CpG-ODNs) strongly activate dendritic cells (DCs) and macrophages to produce proinflammatory cytokines including IL-6, IL-12 and TNF?. It has been suggested that CpG-DNA activates TLR9, which in turn recruits MyD88, IRAK4, IRAK1 and TRAF6, leading to activation of AP-1 and NF-?B, which are critical for immune gene expression. However, whether DNA binding proteins are involved in activation of the CpG-DNA pathway remains elusive. Here we demonstrate that Ku70 is involved in this process. Administration of CpG-ODN into Ku70-deficient mice and in vitro stimulation of bone marrow-derived DCs (BMDCs) as well as bone marrow-derived macrophages (BMDMs) from these mice resulted in defective induction of IL-6 and TNF?. Loss of Ku70 impaired activation of its downstream kinases, the I?B kinase (IKK) and the mitogen-activated protein kinases (MAPKs), by CpG-ODN. Intriguingly, in macrophages ablation of Ku70 largely abrogated IRF1 expression and subsequent IL-12 response to CpG-ODN; in BMDCs Ku70 deficiency only reduced IL-12 response to CpG-ODN at a low dose within early treatments, and had no apparent effects on this response in late treatments. Thus, our results suggest that Ku70 is important for differential regulation of inflammatory cytokine response to CpG-DNA. In addition, we have identified a mechanism for involvement of Ku70 in activation of the CpG-DNA pathway.
CD303 (BDCA-2) mediated inhibition of interferon type I production in plasmacytoid dendritic cells is linked to SYK, BLNK and PKC delta signaling (89.25)
Authors: Röck Jürgen , Schmitz Jürgen , Winkels Gregor
Published: January 2023

CD303 is a calcium dependent type II lectin also known as Blood-Dendritic-Cell-Antigen-2 (BDCA-2) specifically expressed by human plasmacytoid dendritic cells (PDC). We have previously shown that monoclonal antibody (mAb) ligation of CD303 induces mAb endocytosis, calcium mobilization, protein tyrosine phosphorylation and inhibition of type I interferon (IFN I) production in stimulated PDC. Here we show that CD303 signaling and internalization in many aspects resembles B cell receptor (BCR) signaling and internalization. Instead of CD79a and CD79b, CD303 appears to use the Fc-receptor-common-gamma-chain as transmembrane adaptor protein. Like BCR signaling, CD303 triggering leads to SYK and BLNK phosphorylation. Signal transduction most likely involves activation of the phospholipase C-gamma 2, the phosphoinositide-3 kinase and the protein kinase C delta. Therefore, inhibition of IFN I production in stimulated PDC can be mimicked by PMA. Western blotting and peptide mass fingerprinting show that tyrosine phosphorylation occurs at cytoskeletal proteins (actin, alpha/beta-tubulin, profilin, alpha-actinin), proteasome activator subunit and the clathrin heavy chain, indicating clathrin-mediated endocytosis and vesicle trafficking. Finally, CD303 triggering inhibits TNF-alpha and CpG ODN-induced NF-kappa B activation in PDC.
Interplay of Activating and Inhibitory Receptors in NK cells (89.28)
Authors: Das Asmita , Long Eric
Published: January 2023

Natural Killer (NK) cells are lymphocytes capable of killing tumor cells and virus-infected cells without prior sensitization. Each NK cell expresses several different activating and inhibitory receptors. A balance of activating and inhibitory signals delivered by these receptors determines the outcome of NK–target cell interactions. However, it is often difficult to identify the individual contribution of each activating receptor and its susceptibility to inhibition. To avoid this complexity, SC2 insect cells have been transfected with ligands for several activating and inhibitory receptors. CD16 is the only receptor tested that is capable of inducing degranulation and cytokine production. Other receptors, such as NKG2D, LFA-1 enhance CD16-induced responses. Target cell lysis by NK cells requires polarized release of cytotoxic granule. The insect cell system is now being used to test if co-engagement of inhibitory receptors for MHC class I blocks granule polarization, degranulation or both.
Knockout of Mkp-1 enhances the host inflammatory responses to Gram-positive bacteria (89.3)
Authors: Liu Yusen , Wang Xianxi , Meng Xiaomei , Kuhlman Joshua R. , Nelin Leif D. , English B. Keith
Published: January 2023

MAPK phosphatase (MKP)-1 is an archetypal member of the dual specificity protein phosphatase family that dephosphorylates MAPK. We have previously demonstrated that MKP-1 acts as a negative regulator of p38 and JNK in immortalized macrophages after stimulation with peptidoglycan isolated from Gram-positive bacteria. To define the physiological function of MKP-1 in host defense during Gram-positive bacterial infection, we studied the innate immune responses to Gram-positive bacteria using Mkp-1 knockout mice. Here we show that Mkp-1−/− macrophages exhibited prolonged activation of p38 and JNK, but not of ERK, following exposure to either peptidoglycan or lipoteichoic acid. Compared to wild type macrophages, Mkp-1−/− macrophages produced more pro-inflammatory cytokines such as TNF-α and IL-6. Moreover, after challenge with peptidoglycan, lipoteichoic acid, live or heat-killed Staphylococcus aureus bacteria, Mkp-1 knockout mice also mounted a more robust production of cytokines and chemokines, including TNF-α, IL-6, IL-10, and MIP-1α, than did wild type mice. Accordingly, Mkp-1 knockout mice also exhibited greater NO production, more robust neutrophil infiltration, and more severe organ damage than did wild type mice. In response to challenge with heat-killed S. aureus, Mkp-1 knockout mice exhibited a substantially higher mortality rate compared to wild type mice. Our studies indicate that MKP-1 plays a critical role in the inflammatory response to Gram-positive bacterial infection. MKP-1 serves to limit the inflammatory reaction by inactivating JNK and p38, and in the absence of MKP-1 the exaggerated inflammatory response results in devastating consequences to the host.
Natural killer cells from mice deficient in protein kinase C-theta have reduced production of interferon-gamma after stimulation with interleukin-12 (89.32)
Authors: Bourque Karen M. , Kasaian Marion T.
Published: January 2023

NK cells play a key role in innate immune responses. PKC𝛉 is important for T cell activation, but is also expressed in NK cells. NK cells from PKC𝛉 −/− and WT mice were enriched from splenocyte cultures by incubation with IL-15 for one week and then treated for 24 hours with IL-12 or IL-18 to induce IFNγ production. Cytotoxicity of NK cells from PKC𝛉 −/− and WT mice was activated in vivo with poly I:C or in vitro with cytokines, and target cell specific killing measured by 51chromium release assay. NK cell-enriched splenocytes from PKC𝛉 −/− animals produced significantly less IFNγ in response to IL-12, but not in response to IL-18. In contrast, NK cell mediated cytotoxicity was unaffected by the absence of PKC𝛉. T cells, which can also make IFNγ, were not a source of IFNγ in these experiments, nor did they affect the ability of NK cells to produce it. No differences in spleen NK cell numbers or viability were detected. NK cells from PKC𝛉 −/− mice also exhibited normal expression of IL-12Rβ1, and STAT4 proteins and normal IL-12-induced phosporylation of STAT4. Phosphorylation of T538 within the catalytic domain of PKC𝛉 was detectable in NK cells from WT mice, but was not enhanced by IL-12. IFNγ mRNA increased to a similar extent in NK cells from WT and PKC𝛉 −/− mice in response to IL-12. IFNγ mRNA stability was unaffected by the absence of PKC𝛉. These findings indicate a role for PKC𝛉 in the post-transcriptional regulation of IL-12-induced IFNγ.
KIR2DL4 signals downstream functions in human NK cells through two distinct structural elements (89.34)
Authors: Miah Shah Md. Shahjahan , Hughes Tracey L. , Campbell Kerry S.
Published: January 2023

KIR2DL4 is a member of the killer cell Ig-like receptor (KIR) family expressed on human natural killer (NK) cells. A transmembrane Arginine (R) in KIR2DL4 binds to the transmembrane adaptor FcεRI-γ and stimulates potent IFN-γ production and weak cytolytic activity. In this study, we have found that the mutation of transmembrane R to Glycine (G) (RG mutant) in KIR2DL4 abrogates FcεRI-γ association, IFN-γ production, cytolytic activity and calcium responses. We further found that crosslinking of KIR2DL4 activates the MAP kinases JNK and ERK, as well as the NF-κB pathway in KHYG-1, a human NK-like cell line. Engagement of KIR2DL4 also activates the transcription of a variety of cytokine and chemokine genes including TNF-α, IFN-γ, MIP1α, MIP1β, IL-8 and RANTES. Pharmacological inhibitors of JNK and MEK1/2 blocked IFN-γ and IL-8 production, suggesting that these MAP kinases are regulating KIR2DL4 mediated cytokine production. Surprisingly, we have found that the RG mutant of KIR2DL4 still activates MAP kinases and the NF-κB pathway and differentially regulates chemokine production. In conclusion, our data suggests that the activating functions of KIR2DL4 are mediated by two distinct pathways in NK cells:through transmembrane association with FcεRI-γ andthrough its cytoplasmic tail, which is independent of the transmembrane R.

This work was supported by National Institutes of Health Grant CA-100226 to K.S.C.
Disparity in mycobacterium tuberculosis-induced IL-12 release in dendritic cells and macrophages is due to utilization of distinct Toll-like receptors (89.35)
Authors: Pompei Luca , Jang Sihyug , Zamlynny Beata , McBride Amanda
Published: January 2023

In the pursuit of understanding the molecular basis for a differential release of interleukin (IL)-12 between dendritic cells (DCs) and macrophages in response to Mycobacterium tuberculosis (Mtb), we first evaluated the expression of IL-12p35 and IL-12p40. The two cell types showed similar transcription levels of IL-12p35, while IL-12p40 mRNA was expressed in a rapid and enhanced manner only in murine bone-marrow DCs. An analysis of the remodeling at the nucleosome 1 of the IL-12p40 promoter also provided evidence of a rapid and extensive accessibility to the transcription factor NF-kB in DCs. Despite restoring the impaired Mtb-dependent release of IL-12, addition of interferon (IFN) 2 □ or removal of IL-10 did not affect the extent of remodeling in the IL-12 promoter of macrophages. By evaluating the IL-12p40 remodeling and release in knock-out mice models we demonstrate that DCs mainly rely on signaling from toll-like receptor (TLR)9 for their extensive Mtb induced IL-12 expression. Moreover the TLR9 ligand CpG induced extensive remodeling and release of IL-12p40 in comparison to a TLR2 agonist. Our work suggests a different handling of Mtb from the two cell types where TLR9 engagement occurs prevalently in DCs and leads to high expression of IL-12 and supports the rising opinion, which favors a role for TLR2 in Th2 type responses.
Acute alcohol induces SOCS3 and SOCS1 via STAT3 activation in human monocytes (89.36)
Authors: Norkina Oxana , Dolganiuc Angela , Kodys Karen , Mandrekar Pranoti , Szabo Gyongyi
Published: January 2023

Consumption of moderate amounts of alcohol inhibits the inflammatory responses in human monocytes. Our study reports the effect of in vitro and vivo alcohol treatment on pathways leading to expression of SOCS3 and SOCS1, potent inhibitors of cytokine signaling. Human monocytes treated in vitro with 25–50mM of alcohol for 3–24h had significantly increased SOCS1 and SOCS3 mRNA compared to controls, similar to in vivo alcohol-exposed cells. Activation of STAT3, a regulator of SOCS3 and SOCS1, was induced by alcohol, as indicated by STAT3 tyrosine and serine phosphorylation and DNA binding. We found that STAT3 activation by alcohol can be directly regulated by Src tyrosine kinases and through a crosstalk with MAPKs. Acute alcohol treatment activated Src kinases in monocytes as detected by increased Src Tyr 416 phosphorylation. Consistent with Src kinases activation, alcohol decreased the levels of the inactive form of Lyn kinase, represented by phosphorylation at the Tyr 507. Using specific inhibitors, we identified that STAT3 activation by Src kinases occurred directly at Tyr 705 or indirectly at Ser 727 via JNK and p38, but not ERK MAP kinases. Our finding that acute alcohol activates Src-STAT3 pathway and induces SOCS3 and SOCS1 indicates that the immunosuppressive effects of acute alcohol may influence multiple signaling pathways employed by inflammatory cytokines and regulated by SOCS3 and SOCS1.
TLR5 is not critical for humoral immune responses to purified flagellin or co-administered antigens (89.37)
Authors: Sanders Catherine J. , Akira Shizuo , Gewirtz Andrew T.
Published: January 2023

A problem in developing vaccine adjuvants is that their effectiveness may be inherently tied to TLR activation and consequently “cytokine storms”. To examine whether this applied to the TLR5 ligand flagellin, a MyD88-dependent adjuvant, we compared immune responses of TLR5KO mice and WT littermates to purified flagellin or flagellin+OVA. Flagellin-treated TLR5KO mice lacked induction of serum IL-12(p40), TNFα, RANTES, IL-17, Eotaxin, or MIP-1β measured over a 24 hour period compared to WT, yet had reduced though significant serum cytokine levels of KC (9%), IL-1α (53%), IL-6 (24%), MCP-1 (22%), and G-CSF (16%) (% KO of WT). TLR5KO mice also lacked detectable activation of splenic DC and had modestly diminished Ab titers to flagellin (26% of WT) or OVA (40% of WT). These data indicate flagellin’s adjuvanticity does not require TLR5, detectable DC activation, or the relatively high serum cytokine levels found in WT mice. In contrast, MyD88KO mice exhibited complete absence of flagellin-induced cytokines and Abs. Such data suggest the positive antibody response in TLR5KO mice may have resulted from a TLR5-independent pathway of flagellin recognition, whose activation and subsequent immune events promote adaptive immunity. These data support and expand the concept that TLR ligands can promote antibody responses by means not requiring the TLRs thought necessary for their innate immune recognition.
Quantitative analysis of T Cell Homeostatic Proliferation Reveals Genetic Influence (89.4)
Authors: Li Cheng-Rui (Michael) , Lo David D
Published: January 2023

Conditions of lymphopenia can drive proliferation of residual T cells, a process known as T cell homeostatic proliferation. This situation can be reproduced experimentally by transferring T cells into lymphopenic recipient animals; transferred T cells undergo several rounds of proliferation without antigen stimulation. However, detailed mechanisms have not been fully elucidated. In this study, we developed a mathematical model of the proliferating T cells, considering factors such as apoptosis and proliferation. The modeling results show differential behaviors of T cells between CD4+ and CD8+ subsets. This model predicts that, at least for CD8+ T cells, both the proportion of cells entering cell cycle and the proportion of cells undergoing apoptosis increase with each successive division. These predictions can be confirmed by short-term BrdU labeling and AnnexinV staining. Using same modeling method, we analyzed T cell homeostatic proliferation from different mouse strains, including C57BL/6, BALB/c, CB6F1 and NOD. The results show that homeostatic proliferation is also subject to genetic influence. As homeostatic T cell proliferation has been implicated in precipitating autoimmune diseases, better understanding of the genetic influences over the parameters in this modeling system may yield insights into the behavior of T cells not only in lymphopenic conditions, but also in the context of antigen responses and autoimmune disease susceptibility.
Role of the SH2 domain-containing Phosphatase-1, SHP-1 in Dendritic Cell Activation (89.6)
Authors: Ramachandran Indu Rekha , Levitt Jonathan Michael
Published: January 2023

SHP-1 is an SH2 domain containing phosphatase activated upon recruitment to inhibitory receptors through their immunotyrosine inhibitory motif domains. Once activated, SHP-1 can dephosphorylate a wide range of receptors (e.g. antigen, chemokine and cytokine receptors), as well as their downstream signaling molecules, dampening cellular signals. Although SHP-1 is highly expressed in dendritic cells (DCs), its role in DC signaling and function is not well defined. By blocking SHP-1 activity with shRNA or a phosphatase dead dominant negative mutant (dn), our studies in the murine DC line, D2SC/1 show that SHP-1 inhibited AP-1 activity, upon LPS stimulation but did not affect NFκB activity. This is in contrast to SHP-1 function in RAW264.7 macrophages where it inhibits both AP-1 and NFκB activity after LPS and/or IFNγ treatment. Further, phenotypic characterization of primary bone-marrow derived DCs from SHP-1 deficient motheaten viable (MeV) mice shows, surprisingly, that the MeV DCs have drastically lower and uninducible levels of MHC class II surface expression compared to wild-type DCs. We are currently investigating the mechanisms by which SHP-1 affects class II surface expression in DCs. Taken together, these studies elucidate differences between SHP-1 signaling in DCs and macrophages which may have bearing on their differential ability to modulate antigen presentation and subsequent T cell activation.

Supported by DOD New Investigator Award PC061027 to JL.
High concentrations of CpG-B ODNs induce a novel IFNαβ-specific inhibitory pathway in dendritic cells (89.9)
Authors: Gray Reginald Courtney , Kuchtey John , Harding Clifford V
Published: January 2023

CpG oligodeoxynucleotides (ODNs) signal through TLR9 to induce type-I IFN (IFNαβ) and other immunomodulatory responses (i.e., TNF, IL-12p40, and MHC-I, etc.) by antigen presenting cells. CpG ODNs are currently in preclinical and clinical studies as vaccine adjuvants and therapies for allergies and cancers. Caveats to CpG ODN therapies are the possible provocation of autoimmune diseases such as systemic lupus erythematosis, which may be exacerbated by the induction of CpG-induced IFNαβ. Thus, further studies are needed to examine CpG induction of IFNαβ and to develop agents to inhibit the induction of IFNαβ by CpG ODNs. These studies address a mechanism for inhibiting induction of IFNαβ that could potentially offer an approach for therapeutic inhibition of IFNαβ expression in vivo. Our data suggest that high concentrations of CpG-B ODNs (H/CpG-B) induce an IFNαβ specific inhibitory mechanism as early as 3 h and sustained inhibition as long as 48 h, without similarly affecting TNF and IL-12p40 levels. Additionally, H/CpG-B inhibits MyD88-dependent (CpG-A ODNs) and -independent (Sendai virus) induction of IFNαβ. However, H/CpG-B fails to inhibit IFNαβ expression following induction with recombinant IFNαA protein, suggesting that H/CpG-B IFNαβ inhibition is upstream of the IFNαβ receptor and possibly due to specific obstruction of early type-I IFN induction (IFNα4/β). These data demonstrate a novel IFNαβ specific inhibitory mechanism induced by stimulation of DCs in vitro with H/CpG-B ODNs.
The DNA degradation product uric acid directly promotes T cell autoreactivity (90.1)
Authors: Webb Ryan , Jeffries Matlock , Sawalha Amr H
Published: January 2023

Purpose: Abnormally high serum uric acid levels have been associated with several disease conditions including gout and kidney stone disease. More recently, it was shown that uric acid crystals stimulate dendritic cell maturation, activate the NALP3 inflammasome, and enhance antigen-specific immune response. We hypothesize that uric acid can also stimulate T cells directly and in the absence of antigen presentation.

Methods: Purified primary human T cells were incubated with and without uric acid at concentrations of 50, 100, 150 and 200 μg/ml. This range of uric acid concentrations covers the normal physiologic serum uric acid concentration as well as a spectrum of hyperuricemic values in human. The expression of T cell activation markers CD25 and CD70 was assessed by flow cytometry. Purified human T cell proliferation response to the various uric acid concentrations was quantified using a colorimetric proliferation assay.

Results: Uric acid treatment resulted in antigen-independent T cell activation as determined by increased expression of CD25 in both CD4+ as well as CD8+ T cells. CD4+ T cells also overexpressed CD70, a co-stimulation molecule important in T cell-dependent B cell activation. In addition, we demonstrate a dose response relationship between T cell proliferation and uric acid concentration in vitro.

Conclusion: The finding that uric acid directly promotes T cell proliferation and activation in an antigen-independent system is novel and might play a mechanistic role in the inflammatory response observed in gout and other uric acid associated diseases.

This work is supported by NIH Grant Number P20-RR015577 and the University of Oklahoma Health Sciences Center.
Temporal development of B and T cell areas in the neonatal rabbit appendix. (90.14)
Authors: Lanning Dennis Keith , Hanson Nicholas B.
Published: January 2023

The rabbit appendix is organized structurally into multiple B cell follicles separated, at their sub-apical regions, by well-defined T cell areas. We found, by RNA in situ hybridization, that the B cell chemoattractant CXCL13 (B-lymphocyte chemoattractant, or BLC) is highly expressed in the B cell follicles. In addition, we found that the T cell chemoattractant CCL21 (secondary lymphoid tissue chemokine, or SLC) is highly expressed in the T cell areas. The localized expression of these chemoattractants likely organizes the B and T cell areas of the appendix. Surprisingly, however, we found that the localized expression of CXCL13 and CCL21 differs dramatically in early neonatal appendix. At two days after birth, CXCL13 expression was confined to the appendix domes and CCL21 expression was localized in a continuous layer significantly beneath the domes. Over subsequent days, as B cells accumulated in the domes and began proliferating, developing follicles gradually distended downward through the layer of CCL21 expression, ultimately generating the characteristic organization of B and T cell areas seen in the adult appendix. This developmental process might be important in establishing an environment supportive of B cell proliferation and antibody repertoire diversification, which occur in the rabbit appendix between 3 and 6 weeks of age.
IgG1, IgG2a, complement and FcR in immune complex disease (90.16)
Authors: Strait Richard , Meyer Cathy , Lambris John , Magotti Paola , Finkelman Fred
Published: January 2023

Immune complexes (IC) contribute to tissue damage in some diseases but prevent it in others. We hypothesize that this contrast occurs because different immunoglobulin (Ig) isotypes involved in the IC interact differently with both stimulatory and inhibitory FcγR and complement (C). To study this we compared IgG1 (which poorly activates C and stimulatory FcγR) to other Ig isotype-mediated IC processing using a model in which wild-type (WT) mice immunized with goat anti-mouse IgD antiserum (GaMD), produce a large, predominantly IgG1 Ab response, produce large amounts of IC, and eliminate them without developing renal disease. In GaMD-immunized IgG1-deficient mice, IgG2a (which activates C and interacts with stimulatory FcγR) predominates and mice develop severe, lethal IC-mediated renal disease that can be prevented by immunization with antigen-specific IgG1. Renal disease also develops in C3-deficient, FcγRIIb-deficient and anti-FcγRII/III mAb treated WT but not in FcRγ-deficient mice immunized with GaMD. Surprisingly, GaMD-immunized IgG1-deficient mice that are also lacking both FcRγ and C3 or lacking FcRγ and treated with a C5aR antagonist still develop renal disease. Together these findings suggest that IgG1 may protect against IC disease through a C- and FcγRIIb-dependent mechanism, while non-IgG1, presumably IgG2a, may mediate IC injury through a FcRγ- and either C3 or C5aR-independent mechanism.
Testosterone attenuates TLR4 expression on murine macrophages (90.5)
Authors: Rettew Jennifer , Marriott Ian
Published: January 2023

Gender has long been known to be a contributory factor in immune responses following infection, with females generally exhibiting greater immune responses than males. These differences have generally been assumed to be a consequence of the actions of reproductive hormones and such a hypothesis is supported by the presence of receptors for these hormones in a variety of immune cell types. Androgens such as testosterone have been shown to decrease immune functions including cytokine production. However, the mechanisms by which testosterone limits such responses remain undefined. In this study we have investigated the acute effects of testosterone on the level of expression of a key microbial pattern recognition receptor, Toll-like receptor 4 (TLR4), on isolated murine macrophages. We show that in vitro testosterone treatment of macrophages, generated in the absence of androgen, elicits a modest but significant decrease in cell surface TLR4 expression within 24 hours. In addition, we have studied the effect of in vivo removal of endogenous testosterone on TLR4 expression. We report that macrophages isolated from orchidectomized animals have significantly less (~30%) TLR4 cell surface expression than those derived from sham gonadectomized mice. Importantly, this effect was not apparent in orchidectomized animals that received exogenous testosterone treatment. As such, these data represent a novel means by which reproductive hormones can modulate immune responsiveness to infectious organisms.