bluuebunny/crossref_metadata_2025 huggingface dataset:
The MHC-E Locus Is the Most Well Conserved of All Known Primate Class I Histocompatibility Genes
Authors: Knapp Leslie A. , Cadavid Luis F. , Watkins David I.
Published: January 2023

The HLA-E locus is characterized by limited polymorphism and low levels of cell surface expression. However, the function of the products of this nonclassical MHC class I gene remains unknown. To evaluate the conservation of the MHC-E locus throughout anthropoid primate evolution, we identified the homologue of the HLA-E locus in six different New World monkey species. Full-length sequencing of MHC-E cDNAs in four unrelated cotton-top tamarins (Saguinus oedipus) revealed no evidence for polymorphism. Using the PCR, denaturing gradient gel electrophoresis, and direct sequencing, we also identified MHC-E alleles in five other New World monkey species, representing all extant platyrrhine families. In contrast to all other classical and nonclassical MHC class I genes in primates, the rate of synonymous nucleotide substitution is much greater than the rate of nonsynonymous nucleotide substitution within exons 2 and 3 encoding the peptide binding region (PBR) in MHC-E genes. The PBR of the MHC-E molecule, therefore, has evolved under purifying selective pressures, and the very unusual evolutionary history of this ancient gene provides further evidence that the products of the HLA-E locus serve a critical immunological function. Given the remarkable conservation of the PBR during primate evolution, this critical immunological function is probably related to the peptide binding ability of the MHC-E protein.
Cutting Edge: Stimulation of CD28 with B7–2 Promotes Focal Adhesion-Like Cell Contacts Where Rho Family Small G Proteins Accumulate in T Cells
Authors: Kaga Shuji , Ragg Scott , Rogers Kem A. , Ochi Atsuo
Published: January 2023

Unless a costimulatory signal is provided, TCR recognition of Ag bound to the MHC is insufficient to induce optimal T cell proliferation or the production of IL-2. Here we show that the stimulation of CD28, a T cell costimulatory receptor, by a specific Ab increases F-actin contents in T cells. The interaction between T cells and B7–2-transfected Chinese hamster ovary cells expressing the CD28 ligand leads to the rearrangement of the actin cytoskelton in the region of cell-cell contact. Within the Rho family of G proteins, Rac1, but not Rho, translocates to the sites of cell-cell contact where Tailin also accumulates. These results indicate that the interaction between B7–2 and CD28 establishes a focal adhesion-like cell contact between T cell and APCs. The results also suggest that CD28 signaling is primarily transduced by a cytoskeletal rearrangment/signaling pathway mediated by the Rho family G proteins.
Cutting Edge: HIV-1 Infection Induces a Selective Reduction in STAT5 Protein Expression
Authors: Pericle Federica , Pinto Ligia A. , Hicks Stuart , Kirken Robert A. , Sconocchia Giuseppe , Rusnak Janice , Dolan Matthew J. , Shearer Gene M. , Segal David M.
Published: January 2023

HIV-1 infection is accompanied by qualitative and quantitative defects in CD4+ T lymphocytes. Loss of immune function in HIV patients is usually associated with a profound dysregulation of cytokine production. To investigate whether cytokine signaling defects occur during HIV infection, PHA blasts from healthy human donors were infected with two strains of HIV-1 and screened for the expression of STAT proteins used in cytokine signaling. A selective decrease in STAT5B was seen 8 days after infection with the BZ167 dual-tropic HIV isolate, but not with the Ba-L, M-tropic strain. Based on these findings, purified T cells from HIV-infected patients in different stages of disease were also tested for STAT expression; decreases in STAT5A, STAT5B, and STAT1α were observed in all patients. The reduction in STATs seen in vivo and in vitro after HIV infection may contribute to the loss of T cell function in HIV disease.
Accumulation of the p53 Protein Allows Recognition by Human CTL of a Wild-Type p53 Epitope Presented by Breast Carcinomas and Melanomas
Authors: Gnjatic Sacha , Cai Zhenzi , Viguier Mireille , Chouaib Salem , Guillet Jean-Gérard , Choppin Jeannine
Published: January 2023

The p53 protein is accumulated in tumor cells of many human cancers and can elicit in vivo humoral and proliferative responses. Rare reports about p53-mediated tumor recognition by CTLs have remained questioned. We therefore studied a panel of breast tumor and melanoma cell lines that we assayed for the presence of accumulated p53 and surface HLA-A2 and for the presentation of p53 epitopes. From PBMC of a healthy donor, we have generated a CTL line, D5/L9V, directed against HLA-A2-restricted peptide 264–272 from wild-type p53. It efficiently lysed breast adenocarcinomas MCF-7, MCF7/RA1, and MDA-MB-231, and melanoma M8, which all accumulate the p53 protein. Using competition assays, we made sure that tumor lysis by D5/L9V was due to recognition of endogenously produced p53 peptide 264–272 associated with the HLA-A2.1 molecule on the surface of these tumor cells. Cells with undetectable levels of wild-type p53, such as lymphoblastoid cells and melanoma M74, were not recognized by D5/L9V. Neither were breast tumor cell line MCF7/ADR nor melanoma line M44 because of HLA loss. This study therefore shows that it is possible to obtain in vitro CTL lines that specifically recognize a p53 epitope spontaneously presented by a variety of HLA-A2+ transformed cell lines provided they display abnormal patterns of p53 expression. This work points out that breast tumors and melanomas share a p53 epitope, and raises hopes for future immunotherapeutic approaches.
Normal Responsiveness of CTLA-4-Deficient Anti-Viral Cytotoxic T Cells
Authors: Bachmann Martin F. , Waterhouse Paul , Speiser Daniel E. , McKall-Faienza Kim , Mak Tak W. , Ohashi Pamela S.
Published: January 2023

CTLA-4 has been proposed to negatively regulate immune responses, and mice deficient for CTLA-4 expression succumb to a lymphoproliferative disorder within a few weeks after birth. This study assessed the responsiveness of CTLA-4-deficient T cells expressing a class I-restricted TCR specific for lymphocytic choriomeningitis virus (LCMV). The kinetics of T cell proliferation were studied in vitro after stimulation of T cells with full and partial T cell agonists. No gross abnormalities in CTLA-4-deficient T cells could be detected. Using adoptive transfer experiments, T cell responses were also measured in vivo after infection with LCMV. Low dose infection with LCMV leads to strong expansion of specific T cells followed by a reduction in T cells that parallels the elimination of Ag. The kinetics of T cell expansion and elimination after low dose LCMV infection were not affected by the absence of CTLA-4. High dose infection of mice with LCMV leads to a transient expansion of T cells followed by T cell exhaustion, where all specific T cells are eliminated. T cell exhaustion also occurred in the absence of CTLA-4. Thus, surprisingly, the absence of CTLA-4 did not interfere with T cell activation, down-regulation of ongoing T cell responses after the elimination of Ag, or the exhaustion of T cell responses in the presence of excessive amounts of Ag.
Redundant Role of the Syk Protein Tyrosine Kinase in Mouse NK Cell Differentiation
Authors: Colucci Francesco , Turner Martin , Schweighoffer Edina , Guy-Grand Delphine , Di Bartolo Vincenzo , Salcedo Margarita , Tybulewicz Victor L. J. , Di Santo James P.
Published: January 2023

Syk and ZAP-70 subserve nonredundant functions in B and T lymphopoiesis. In the absence of Syk, B cell development is blocked, while T cell development is arrested in the absence of ZAP-70. The receptors and the signaling molecules required for differentiation of NK cells are poorly characterized. Here we investigate the role of the Syk protein tyrosine kinase in NK cell differentiation. Hemopoietic chimeras were generated by reconstituting alymphoid (B−, T−, NK−) recombinase-activating gene-2 × common cytokine receptor γ-chain double-mutant mice with Syk−/− fetal liver cells. The phenotypically mature Syk−/− NK cells that developed in this context were fully competent in natural cytotoxicity and in calibrating functional inhibitory receptors for MHC molecules. Syk-deficient NK cells demonstrated reduced levels of Ab-dependent cellular cytotoxicity. Nevertheless, Syk−/− NK cells could signal through NK1.1 and 2B4 activating receptors and expressed ZAP-70 protein. We conclude that the Syk protein tyrosine kinase is not essential for murine NK cell development, and that compensatory signaling pathways (including those mediated through ZAP-70) may sustain most NK cell functions in the absence of Syk.
Tolerance and Alloreactivity of the Ly49D Subset of Murine NK Cells
Authors: George Thaddeus C. , Ortaldo John R. , Lemieux Suzanne , Kumar Vinay , Bennett Michael
Published: January 2023

Class I-specific stimulatory and inhibitory receptors expressed by NK cell subsets contribute to the alloreactive potential of the self-tolerant murine NK cell repertoire. In this report, we have studied potential mechanisms of tolerance to the function of the positive signaling Ly49D receptor in mice that express one of its ligands, H2-Dd. Our results demonstrate that H2-Dd-expressing mice possess a large Ly49D+ subset of NK cells that is functionally capable of rejecting bone marrow cell (BMC) allografts in vivo and lysing allogeneic Con A lymphoblasts in vitro. Also, we show that the Ly49D receptor is responsible for the ability of H2b/d F1 hybrid mice to reject H2d/d parental BMC (hybrid resistance). Thus, deletion or anergy of Ly49D+ cells in H2-Dd+ hosts cannot explain self tolerance. Our functional studies revealed that coexpression of the Dd-specific Ly49A or Ly49G2 inhibitory receptors by Ly49D+ cells resulted in tolerance to Dd+ targets, while coexpression of Kb-specific inhibitory receptors Ly49C/I resulted in tolerance to Kb+ targets. Only in H2d/d cells did Ly49C/I dominantly inhibit Ly49D-Dd stimulation. This correlated with an increased mean fluorescence intensity of Ly49C expression, as well as an increased percentage of Ly49C+ cells in the Ly49D+A/G2− compartment. Therefore, we conclude that self tolerance of the Ly49D subset can be achieved through coexpression of a sufficient level of self-specific inhibitory receptors.
Cytolytic T Lymphocyte-Associated Antigen-4 and the TCRζ/CD3 Complex, But Not CD28, Interact with Clathrin Adaptor Complexes AP-1 and AP-2
Authors: Schneider Helga , Martin Margarita , Agarraberes Fernando A. , Yin Li , Rapoport Iris , Kirchhausen Tomas , Rudd Christopher E.
Published: January 2023

The negative signaling receptor cytolytic T lymphocyte-associated Ag-4 (CTLA-4) resides primarily in intracellular compartments such as the Golgi apparatus of T cells. However, little is known regarding the molecular mechanisms that influence this accumulation. In this study, we demonstrate binding of the clathrin adaptor complex AP-1 with the GVYVKM motif of the cytoplasmic domain of CTLA-4. Binding occurred primarily in the Golgi compartment of T cells, unlike with AP-2 binding that occurs mostly with cell surface CTLA-4. Although evidence was not found to implicate AP-1 binding in the retention of CTLA-4 in the Golgi, AP-1 appears to play a role in shuttling of excess receptor from the Golgi to the lysosomal compartments for degradation. In support of this, increased CTLA-4 synthesis resulted in an increase in CTLA-4/AP-1 binding and a concomitant increase in the appearance of CTLA-4 in the lysosomal compartment. At the same time, the level of intracellular receptor was maintained at a constant level, suggesting that CTLA-4/AP-1 binding represents one mechanism to ensure steady state levels of intracellular CTLA-4 in T cells. Finally, we demonstrate that the TCRζ/CD3 complex (but not CD28) also binds to AP-1 and AP-2 complexes, thus providing a possible link between these two receptors in the regulation of T cell function.
Anergy, IFN-γ Production, and Apoptosis in Terminal Infection of Mice with Mycobacterium avium
Authors: Gilbertson Brad , Zhong Jie , Cheers Christina
Published: January 2023

We have followed the course of experimental infection of mice with Mycobacterium avium over an extended period, assessing bacterial numbers and T cell responsiveness. When mice were infected intranasally, bacteria spread to the spleen and liver, but remained in highest numbers in the lungs. Both CD4+ and CD8+ T cells, assayed at any time from 6–28 wk after infection, produced IFN-γ. After initial rapid growth, bacterial numbers slowly increased from ∼107 at 6 wk to more than 5 × 108 at 28 wk, indicating that the resistance mechanisms so generated were not adequate to contain the infection. During infection, apoptosis of both CD4+ and CD8+ T cells, measured immediately ex vivo by staining with Annexin V, increased steadily. With some individual exceptions, there was a close correlation between apoptosis of CD4+ cells and level of IFN-γ production by cultured spleen cells. By 34 wk postinfection, there was an abrupt cessation of IFN-γ production. No IL-4 was detected, ruling out a switch to Th2 profile. Subsequently, bacterial numbers increased still further to >5 × 109 per lung, and the mice lost body weight and would have died if not killed for experimental or humane reasons. The possibility that T cells exposed over this prolonged period to extremely high doses of Ag may become tolerant by a process of terminal differentiation is discussed.
Differential Monocyte Chemoattractant Protein-1 and Chemokine Receptor 2 Expression by Murine Lung Fibroblasts Derived from Th1- and Th2-Type Pulmonary Granuloma Models
Authors: Hogaboam Cory M. , Bone-Larson Cynthia L. , Lipinski Scott , Lukacs Nicholas W. , Chensue Stephen W. , Strieter Robert M. , Kunkel Steven L.
Published: January 2023

Recent studies suggest that monocyte chemoattractant protein-1 (MCP-1) is involved in fibrosis through the regulation of profibrotic cytokine generation and matrix deposition. Changes in MCP-1, C-C chemokine receptor 2 (CCR2), procollagen I and III, and TGF β were examined in fibroblasts cultured from normal lung and from nonfibrotic (i.e., Th1-type) and fibrotic (i.e., Th2-type) pulmonary granulomas. Th2-type fibroblasts generated 2-fold more MCP-1 than similar numbers of Th1-type or normal fibroblasts after 24 h in culture. Unlike normal and Th1-type fibroblasts, Th2-type fibroblasts displayed CCR2 mRNA at 24 h after IL-4 treatment. By flow cytometry, CCR2 was present on 40% of untreated Th2-type fibroblasts, whereas CCR2 was present on <20% of normal and Th1-type fibroblasts after similar treatment. IL-4 increased the number of normal fibroblasts with cell-surface CCR2 but IFN-γ-treatment of normal and Th2-type fibroblasts significantly decreased the numbers of CCR2-positive cells in both populations. Western blot analysis showed that total CCR2 protein expression was markedly increased in untreated Th2-type fibroblasts compared with normal and Th1-type fibroblasts. IL-4 treatment enhanced CCR2 protein in Th1- and Th2-type fibroblasts whereas IFN-γ treatment augmented CCR2 protein in normal and Th1-type fibroblasts. All three fibroblast populations exhibited MCP-1-dependent TGF-β synthesis, but only normal and Th2-type fibroblasts showed a MCP-1 requirement for procollagen mRNA expression. Taken together, these findings suggest that lung fibroblasts are altered in their expression of MCP-1, TGF-β, CCR2, and procollagen following their participation in pulmonary inflammatory processes, and these changes may be important during fibrosis.
B7.2 (CD86) But Not B7.1 (CD80) Costimulation Is Required for the Induction of Low Dose Oral Tolerance
Authors: Liu Liming , Kuchroo Vijay K. , Weiner Howard L.
Published: January 2023

Oral administration of Ag leads to systemic unresponsiveness (oral tolerance) to the fed Ag. Oral tolerance is mediated through active suppression by Th2 or TGF-β-secreting cells or clonal anergy/deletion, depending on the Ag dose used, with low dose favoring active suppression and high dose favoring anergy/deletion. The nature of APC and inductive events leading to the generation of oral tolerance have not been well defined. To determine the role of costimulatory molecules in the induction of oral tolerance, we have tested the effect of anti-B7.1 or anti-B7.2 mAb on the induction of tolerance by both high and low dose Ag feeding regimens. Our results show that the B7.2 molecule is critical for the induction of low-dose oral tolerance. Injection of anti-B7.2 but not anti-B7.1 intact Ab or Fab fragments inhibited the oral tolerance induced by low-dose (0.5 mg) but not high-dose OVA (25 mg) feeding. In addition, anti-B7.2, but not anti-B7.1, inhibited secretion of TGF-β, one of the primary cytokines that mediates low-dose oral tolerance. Finally, in the in vivo model of experimental allergic encephalomyelitis, anti-B7.2 mAb treatment abrogated protection offered against disease by low-dose myelin basic protein feeding, while anti-B7.1 had no effect. Anti B7.2 had no effect on disease suppression by high-dose oral Ag. These data demonstrate that B7.2 costimulatory molecules play an essential role in the induction of low-dose oral tolerance.
B Cells Are Required for Lupus Nephritis in the Polygenic, Fas-Intact MRL Model of Systemic Autoimmunity
Authors: Chan Owen T. M. , Madaio Michael P. , Shlomchik Mark J.
Published: January 2023

B cells are required for both the expression of lupus nephritis and spontaneous T cell activation/memory cell accumulation in MRL-Faslpr mice (MRL/lpr). Autoimmunity in the MRL/lpr strain is the result of Fas-deficiency and multiple background genes; however, the precise roles of background genes vs Fas-deficiency have not been fully defined. Fas-deficiency (i.e., the lpr defect) is required in B cells for optimal autoantibody expression, raising the possibility that the central role for B cells in MRL/lpr mice may not extend to MRL/+ mice and, thus, to lupus models that do not depend on Fas-deficiency (“polygenic lupus”). To address this issue, B cell-deficient, Fas-intact MRL/+ mice (JHd-MRL/+) were created; and disease was evaluated in aged animals (>9 mo). The JHd-MRL/+ animals did not develop nephritis or vasculitis at a time when the B cell-intact littermates had severe disease. In addition, while activated/memory CD4+ and CD8+ T cells accumulated in B cell-intact mice, such accumulation was substantially inhibited in the absence of B cells. This effect appeared to be restricted to the MRL strain because it was not seen in B cell-deficient BALB/c mice (JHd-BALB) of similar ages. The results indicate that B cells are essential in promoting systemic autoimmunity in a Fas-independent model. Therefore, B cells have an important role in pathogenesis, generalizable to lupus models that depend on multiple genes even when Fas expression is intact. The results provide further rationale for B cell suppression as therapy for systemic lupus erythematosus.
Oligodeoxynucleotides Containing Palindrome Sequences with Internal 5′-CpG-3′ Act Directly on Human NK and Activated T Cells to Induce IFN-γ Production In Vitro
Authors: Iho Sumiko , Yamamoto Toshiko , Takahashi Takayuki , Yamamoto Saburo
Published: January 2023

Previous studies have shown that the action of bacterial or synthetic oligodeoxynucleotide (oligo-DNA) on mouse NK cells to produce IFN-γ is mediated mostly by monocytes/macrophages activated by olig-DNA. However, its action on human IFN-γ-producing cells has not been well investigated. In the present study, we examined the effect of oligo-DNAs on highly purified human NK and T cells. Bacillus Calmette-Guérin-derived or synthetic oligo-DNAs induced NK cells to produce IFN-γ with an increased CD69 expression, and the autocrine IFN-γ enhanced their cytotoxicity. The response of NK cells to oligo-DNAs was enhanced when the cells were activated with IL-2, IL-12, or anti-CD16 Ab. T cells did not produce IFN-γ in response to oligo-DNAs but did respond independently of IL-2 when they were stimulated with anti-CD3 Ab. In the action of oligo-DNAs, the palindrome sequence containing unmethylated 5′-CpG-3′ motif(s) appeared to play an important role in the IFN-γ-producing ability of NK cells. The changes of base composition inside or outside the palindrome sequence altered its activity: The homooligo-G-flanked GACGATCGTC was the most potent IFN-γ inducer for NK cells. The CG palindrome was also important for activated NK and T cells in their IFN-γ production, although certain nonpalindromes acted on them. Among the sequences tested, cell activation- or cell lineage-specific sequences were likely; i.e., palindrome ACCGGT and nonpalindrome AACGAT were favored by activated NK cells but not by unactivated NK cells or activated T cells. These results indicate that oligo-DNAs containing CG palindrome act directly on human NK cells and activated T cells to induce IFN-γ production.
Expression of the Transcription Factor Lung Krüppel-Like Factor Is Regulated by Cytokines and Correlates with Survival of Memory T Cells In Vitro and In Vivo
Authors: Schober Sonya L. , Kuo Chay T. , Schluns Kimberly S. , Lefrancois Leo , Leiden Jeffrey M. , Jameson Stephen C.
Published: January 2023

The transcription factor lung Krüppel-like factor (LKLF) is involved in naive T cell survival. Expression of LKLF is rapidly down-regulated upon T cell stimulation, raising the question of whether LKLF is reexpressed after activation, and what factors are required for such reexpression. Furthermore, the expression of LKLF in resting memory cells has not been determined. Here, we use the OT-I TCR transgenic mouse system to address these issues. LKLF was found to be reexpressed following culture of activated CD8 T cells in certain cytokines (IL-2, IL-7) but not others (IL-12) known to influence CTL development. Interestingly, induction of LKLF reexpression corresponded with long-term T cell survival and development of memory T cell phenotype. Furthermore, using OT-I cells stimulated in vivo, we demonstrated that Ag induced rapid LKLF down-regulation and that the factor is expressed by in vivo-derived memory T cells.
Induction of CD4+ T Cell Alloantigen-Specific Hyporesponsiveness by IL-10 and TGF-β
Authors: Zeller Jay C. , Panoskaltsis-Mortari Angela , Murphy William J. , Ruscetti Francis W. , Narula Satwant , Roncarolo Maria G. , Blazar Bruce R.
Published: January 2023

Induction and maintenance of Ag-specific tolerance are pivotal for immune homeostasis, prevention of autoimmune disorders, and the goal of transplantation. Recent studies suggest that certain cytokines, notably IL-10 and TGF-β, may play a role in down-regulating immune functions. To further examine the role of cytokines in Ag-specific hyporesponsiveness, murine CD4+ T cells were exposed ex vivo to alloantigen-bearing stimulators in the presence of exogenous IL-10 and/or TGF-β. Primary but not secondary alloantigen proliferative responses were inhibited by IL-10 alone. However, the combined addition of IL-10 + TGF-β markedly induced alloantigen hyporesponsiveness in both primary and secondary MLR cultures. Alloantigen-specific hyporesponsiveness was observed also under conditions in which nominal Ag responses were intact. In adoptive transfer experiments, IL-10 + TGF-β-treated CD4+ T cells, but not T cells treated with either cytokine alone, were markedly impaired in inducing graft-vs-host disease alloresponses to MHC class II disparate recipients. These data provide the first formal evidence that IL-10 and TGF-β have at least an additive effect in inducing alloantigen-specific tolerance, and that in vitro cytokines can be exploited to suppress CD4+ T cell-mediated Ag-specific responses in vivo.
Restricted Receptor Segregation into Membrane Microdomains Occurs on Human T Cells During Apoptosis Induced by Galectin-1
Authors: Pace Karen E. , Lee Christina , Stewart Phoebe L. , Baum Linda G.
Published: January 2023

Galectin-1 induces apoptosis of human thymocytes and activated T cells by an unknown mechanism. Apoptosis is a novel function for a mammalian lectin; moreover, given the ubiquitous distribution of the oligosaccharide ligand recognized by galectin-1, it is not clear how susceptibility to and signaling by galectin-1 is regulated. We have determined that galectin-1 binds to a restricted set of T cell surface glycoproteins, and that only CD45, CD43, and CD7 appear to directly participate in galectin-1-induced apoptosis. To determine whether these specific glycoproteins interact cooperatively or independently to deliver the galectin-1 death signal, we examined the cell surface localization of CD45, CD43, CD7, and CD3 after galectin-1 binding to human T cell lines and human thymocytes. We found that galectin-1 binding resulted in a dramatic redistribution of these glycoproteins into segregated membrane microdomains on the cell surface. CD45 and CD3 colocalized on large islands on apoptotic blebs protruding from the cell surface. These islands also included externalized phosphatidylserine. In addition, the exposure of phosphatidylserine on the surface of galectin-1-treated cells occurred very rapidly. CD7 and CD43 colocalized in small patches away from the membrane blebs, which excluded externalized phosphatidylserine. Receptor segregation was not seen on cells that did not die in response to galectin-1, including mature thymocytes, suggesting that spatial redistribution of receptors into specific microdomains is required for triggering apoptosis.
Chemokine Amplification in Mesangial Cells
Authors: Luo Yi , Lloyd Clare , Gutierrez-Ramos Jose-Carlos , Dorf Martin E.
Published: January 2023

Mesangial cells are specialized cells of the renal glomerulus that share some properties of vascular smooth muscle cells and macrophages. They are implicated in the pathogenesis of many forms of nephritis. The murine CXC-chemokines macrophage inflammatory protein-2 (MIP-2) and KC induce migration of mouse mesangial cells. Mesangial cells also exhibit a unique chemokine feedback mechanism. Treatment with nanomolar concentrations of MIP-2 or KC markedly up-regulates monocyte chemoattractant protein-1 and RANTES expression in mesangial cells. Autoinduction of MIP-2 and KC mRNA was also noted. Low levels of MIP-1α, MIP-1β, and IFN-γ-inducible protein-10 were induced following treatment with higher doses of MIP-2 or KC. These effects are specific to mesangial cells, as MIP-2 or KC treatment of renal cortical epithelial cells or peritoneal macrophages failed to induce chemokine production. This cascade of chemokine interactions may contribute to renal infiltration and leukocyte activation. The abilities of MIP-2 or KC to stimulate their own synthesis may also contribute to the maintenance and chronic course of glomerular inflammation. The mesangial cell receptor for MIP-2 and/or KC is unknown but is not CXC-chemokine receptor-2.
Regulation of IL-1beta secretion by defensins (100.11)
Authors: Shi Jishu , Aono Shelly , Lu Wuyuan , Ouellette Andre J , Wang Lei , Lenz Stephen , Elson Charles O
Published: January 2023

Impaired expression of antimicrobial peptides α defensins and overproduction of proinflammatory cytokine IL-1β have been associated with inflammatory bowel disease (IBD). In this study, we examine the interactions between defensins and IL-1β and the role of defensin-deficiency in the pathogenesis of IBD. It was found that matrix metalloproteinase-7 deficient (MMP-7−/−) mice, which produce procryptdins but not mature cryptdins (α-defensins) in the intestine, were more susceptible to dextran sulfate sodium (DSS)-induced colitis. Furthermore, the baseline and DSS-induced IL-1β production in the intestine are significantly up-regulated in MMP-7−/− mice compared with that in WT mice. To elucidate the molecular mechanism for the increased IL-1β production in defensin-deficiency in vivo, we evaluated the effect of defensins on IL-1β posttranslational processing and release. It was found that α defensins, including human neutrophil defensin HNP-1, human Paneth cell defensin HD-5, and mouse Paneth cell defensins cryptdin-3 and cryptdin-4, can block the release of IL-1β from LPS-activated monocytes. However, TNF α expression and release from LPS-activated monocytes are not affected by defensins. Unlike α defensins, human β defensins and mouse procryptdins do not have any effect on IL-1β processing and release. Thus, α-defensins may play an important role in intestinal homeostasis by controlling the production of IL-1β.
Through melanocortin 1 receptor (MC1r), alpha-melanocyte stimulating hormone (α-MSH) regulates macrophage function (100.2)
Authors: Taylor Andrew W , Li Dayu
Published: January 2023

The neuropeptide α-MSH suppresses macrophage innate inflammatory activity, their drive to activate Th1 cells, and induce anti-inflammatory activity by the macrophages. This anti-inflammatory activity has led to evaluating the use of α-MSH peptides in therapy to suppress inflammation. Three melanocortin receptors (MCr) for α-MSH are expressed by macrophages. Although macrophages express mRNA for MC1r, MC3r, and MC5r, it is assumed that the anti-inflammatory activity of α-MSH is through MC1r or MC3r. To understand the mechanisms of α-MSH signaling in macrophages, we synthesized double stranded small interfering RNA (siRNA) to target mouse MC1r and MC3r mRNA. We knocked-down MC1r and MC3r mRNA and protein in siRNA transfected mouse macrophage cell line RAW264.7. The MC1r siRNA transfection neutralized α-MSH suppression of LPS-induced production of nitric oxide, IL-1β, TNF-α, and LPS-induced p38 and NF-κB activation. Although MC3r siRNA effectively knocked-down MC3r, it had no effect on α-MSH suppression of LPS-induced NF-κB activation. Therefore, of the three naturally expressed MCr on macrophages, it is through at least MC1r that α-MSH suppresses the prototypical LPS-induced activity in macrophages. These results with our previous reports of α-MSH regulation of T cell activity imply an important role for melanocortin proteins and receptors in immune regulation.

Supported By EY010752.
TNF-like weak inducer of apoptosis (TWEAK) in pathogenesis of autoimmune renal disease (101.7)
Authors: Gao Hua-Xin , Schwartz Noa , Campbell Sean , Burkly Linda , Jarchum Irene , Banas Bernard , Saleem Moin , Mathieson Peter , Michaelson Jennifer , Putterman Chaim
Published: January 2023

Lupus nephritis (LN), which manifests histologically by glomerulonephritis, tubulointerstitial inflammation and mesangial cell proliferation, is a major cause of mortality and morbidity in systemic lupus (SLE). TNF ligands and receptors are important in the pathogenesis of LN; furthermore, we recently found that TWEAK, a member of the TNF ligand superfamily, was significantly increased in urine from patients with active LN as compared to patients with inactive renal disease. We studied the effects of TWEAK in human kidney cells including mesangial cells (MC), podocytes and tubular cells, following our demonstration of the presence of the TWEAK receptor Fn14 on these cells. TWEAK induces kidney cells to secret multiple inflammatory mediators, including RANTES, MCP-1, IP-10, MIP-1a, GM-CSF, ICAM-1, and VCAM-1. Cytokine production is primarily mediated through NF-kB activation, and could be inhibited by novel anti-TWEAK mAb. TWEAK-stimulated chemokines induced PBMC migration, especially monocyte/macrophages, which may then infiltrate into lupus kidneys in vivo. Furthermore, we found that TWEAK promotes MC proliferation. In conclusion, TWEAK may play a pathogenic role in the development of LN by enhancing renal inflammation and MC proliferation. Furthermore, blocking TWEAK/Fn14 interactions may be a promising therapeutic target in LN and perhaps other immune-mediated renal diseases.
Characterization of the interaction between human LAIR-1 and collagens. (101.8)
Authors: Tang Xiaobin , Narayanan Sriram , Coligan John E. , Borrego Francisco
Published: January 2023

Human leukocyte-associated Ig-like receptor-1 (LAIR-1) is an immunoreceptor tyrosine based inhibitory motif (ITIM) containing inhibitory receptor that is expressed on the majority of PBMCs and thymocytes. The ligand for LAIR-1 has recently been identified as collagens. Since collagens represent the most abundant type of proteins in vertebrates it is reasonable to think that LAIR-1 could have a very important role in controlling many aspects of the immune response. To study the interaction of LAIR-1 with collagens, we use a reporter cell system expressing a chimeric receptor composed of the extracellular domain of LAIR-1 and the transmembrane and cytoplasmic domains of the CD3æ chain. Using this reporter system we have found that both purified collagens type I and type IV are ligands for LAIR-1. Moreover, we have also found that LAIR-1 can interact with collagen type IV that is present in the extracellular matrix and basement membranes. In addition, we have identified a pivotal residue in the extracellular domain of LAIR-1 that could be required for a functional interaction with collagens type I and type IV. Then, by confocal microscopy, we have observed the accumulation and polarization of LAIR-1-EGFP at the binding sites with collagens type I and IV and matrigel. This polarization is very fast and dynamic in nature. An analysis of the role of ITIM motifs in the interaction of LAIR-1 with collagens will be discussed.
Surprising Kinetics of the T cell Response to HY (102.25)
Authors: Tyznik Aaron Jacob , Bevan Michael J
Published: January 2023

Cooperation of CD4+ and CD8+ T cells is required for the development of primary effector and memory CD8+ T cells following immunization with non-inflammatory immunogens. In this study, we characterized endogenous CD4+ and CD8+ T cell responses to male-specific minor histocompatibility (HY) antigens. We find that male cells are rejected 10–12 days after transfer into female mice of the same strain, and this rejection coincides with the expansion and effector function of CD8+ CTLs to two H-2Db-restricted epitopes. While anti-male CD4+ T cell responses are readily detectable day 5 post-transfer, CD8+ T cell response are not detectable until day 10. The early CD4+ T cell response is not dependent on direct presentation of antigen by donor male cells, but depends on processing and presentation of the male cells by female APC. The primary and recall CD8+ T cell responses and rejection of HY incompatible cells are dependent on a sustained CD4+ T cell response and the absence of this response is not replaced by inflammation or treatment with anti-CD40 agonist Ab. Unexpectedly, HY-specific CD4+ T cells are also capable of efficiently lysing target cells in vivo. The delay in the CD8 effector response can be largely abrogated by depleting T cells from the male inoculum, and we further demonstrate that donor CD8+ T cells in particular suppress or veto the host anti-HY CD8+ T cell responses.
Treatment of autoimmune diseases in MRL/lpr mice by allogenic bone marrow transplantation plus adult thymus transplantation (102.4)
Authors: Hosaka Naoki , Miyake Takashi , Cui Wenhao , Nishida Teruhisa , Takaki Takashi , Inaba Muneo , Ikehara Susumu
Published: January 2023

MRL/lpr mice (H-2k) with Fas gene mutation develop severe autoimmune diseases, and their hematolymphoid cells such as bone marrow and spleen cells showed a low apoptotic activity by irradiation. Therefore, conventional bone marrow transplantation (BMT) cannot be used to treat autoimmune diseases in these mice (chimeric resistance). In the present study, we examine the effects of additional adult thymus transplantation (TT) from the same donor on successful BMT. When the MRL/lpr mice were lethally irradiated (9.5Gy) and reconstituted with 3 X 107 of C57BL/6 mouse (H-2b) bone marrow cells (BMCs) in conjunction with TT, the mice significantly survived long-term and showed a high donor-derived chimerism in comparison with those treated with BMT alone. Interestingly, the numbers of not only donor-derived T cells but also B cells significantly increased in the mice treated with BMT plus TT even at the early phase of BMT. The number of aberrant CD3+B220+ cells significantly decreased, and the numbers of lymphocyte subsets were also normalized 4 weeks after the treatment. Finally, the autoimmune diseases in MRL/lpr mice could be cured by BMT with TT. These results indicate that the combination of BMT plus TT can overcome the chimeric resistance and treat the autoimmune diseases in MRL/lpr mice.
Physiological levels of insulin induce receptor editing in anti-insulin B cells to maintain tolerance (128.18)
Authors: Henry Rachel A. , Thomas James W.
Published: January 2023

Receptor editing (RE) is now recognized to play a major role in removing newly formed, autoreactive B cells from the repertoire. Although tolerance induction by monovalent BCR interactions may include clonal ignorance or anergy, it is not known if they can induce RE. Anti-insulin B cells harboring fixed Ig transgenes show evidence of attempted RE, including increased intracellular light chains and insulin-induced elevation of RAG-2 transcripts in vivo and in vitro. To formally test whether insulin could induce RE, we generated mice in which a targeted Ig Vκ encodes an anti-insulin BCR when paired with a fixed Ig VH125 transgene. This model allows the study of tolerance induced by Vκ RE when the BCR interacts with the non-cross-linking hormone, insulin. Anti-insulin B cells show strong evidence for RE in vivo; these findings suggest that BCR engagement with physiological levels of insulin trigger RE. This outcome was confirmed using bone marrow culture to demonstrate low levels of insulin effectively drive RE in vitro. These data extend the role of RE in tolerance to include regulation of BCRs directed at autoantigens such as protein hormones present at low concentrations. Accordingly, physiologically-regulated antigens may escape tolerance during reduced physiological demand or when emerging autoimmune disease reduces self antigen availability.

Support provided by 5T32 HL069765, 5T32 GM08554, AI051448, and JDRF.
Igf2 expression is required for complete tolerance to insulin (128.19)
Authors: Hansenne Isabelle Sylvie , Renard Chantal , Geenen Vincent
Published: January 2023

All members of insulin gene family are transcribed in human thymus according a hierarchy whereby IGF2 expression (thymic epithelial cells/TEC) exceeds IGF1 (macrophages), which exceeds INS (medullary TEC). Type 1 and type 2 IGF receptors are expressed by thymic T-cell populations.

To further study the expression of IGF-2 in thymus, the dominance of this factor was compared to insulin, while ontogenesis of Igf2, insulin1 and insulin2 transcription was studied in Balb/c pancreas and thymus. In 4-wk old thymi, IGF-2 concentration is higher than insulin content. Ontogenesis of Igf2, insulin1 and insulin2 transcription from E13 to post-natal day 2 does not differ in Balb/c thymus and pancreas.

In a second step, tolerance to IGF-2 and insulin was assessed by immunization of Igf2−/− mice. The profile of B-cell response in Igf2−/− mice immunized with IGF-2 evidenced a T-dependent profile of anti-IGF-2 antibodies that was absent in Igf2+/+ mice. This T-dependent isotype switch indicates the presence of specific anti-IGF-2 CD4+ T cells. Cloning of these T cells failed because Igf2−/− CD4+ T cells exhibit a low rate of proliferation in presence of IGF-2. After immunization with insulin, Igf2−/− mice developed a significantly higher humoral response against insulin, indicating that Igf2 expression is necessary for complete tolerance to insulin.
Prevention of diabetes development in NOD mice is associated with the increased ability of APC to activate CD4+CD25+ regulatory T cells. (128.2)
Authors: Manirarora Jean N. , Kosiewicz Michele M. , Parnell Sarah A. , Alard Pascale
Published: January 2023

CD4+CD25+regulatory T cells control the development of various autoimmune diseases. We have found previously that NOD APC are defective in their ability to activate regulatory cell function, and that appropriate APC stimulation could overcome this defect. In the present study, we examined whether NOD APC were defective in their ability to induce CD4+CD25+ regulatory T cells to express Foxp3 and Granzyme B, two molecules associated with the development and function of these cells. We found that regulatory cells from NOD mice expressed lower levels of Foxp3 mRNA and protein than regulatory cells from age-matched B6 mice and exhibit lower percentages of CD4+CD25+ regulatory T cells expressing Granzyme B. Since prevention of diabetes in NOD mice injected with complete Freund’s adjuvant (CFA) is due to activation of CD4+CD25+ regulatory T cells in the pancreatic LN, we tested the effect of CFA on the expression of these two molecules. Interestingly, the expression of Foxp3 on CD4+CD25+ regulatory T cells was increased in CFA-injected NOD mice. Moreover, APC from CFA-injected NOD mice enhanced Foxp3 expression and the percent of CD4+CD25+ regulatory T cells expressing Granzyme B in in vitro culture, suggesting that NOD APC were responsible for the impaired expression of these two molecules. Together, these data suggest that stimulation of NOD APC restores their ability to activate CD4+CD25+ regulatory T cells.
CpG ODN-induced NF-kB activation / IL-6 secretion through CD21-TLR9 dependent B cell signaling? (128.34)
Authors: Asokan Rengasamy , Chen Xiaojiang S , Holers V. Michael
Published: January 2023

Human CR2, a receptor for complement C3d, EBV-gp350, CD23 and IFN-ƒÑ is a B lymphocyte membrane glycoprotein that plays a central role in immunity and autoimmunity. Dysfunction or absence of CR2 promotes development of anti-DNA antibodies in models of systemic lupus erythematosus. In this study, we show that CpG ODN and TLR9 bind to CR2 using ELISA and surface plasmon resonance (SPR). Using SPR, in which carboxy-terminal biotin-tagged CR2 SCR1-4 on a streptavidin chip and CpG ODN and TLR9 as solution phase analyte were studied, the calculated equilibrium dissociation constant (kd/ka) for this binding, reveal 3.1¡Ñ10-2/5.4¡Ñ10-5 s-1 and /1.1¡Ñ10-2/2.4¡Ñ106 s-1 M-1 with a maximal KD of 571 nM and 42 nM respectively. Monoclonal antibodies developed against the first 2 short consensus repeats (SCR) blocked full-length CR2 binding to CpG ODN and TLR9 by ELISA, identically to their effect on C3d binding, indicating the first two SCR involved in this binding. Further more, treatment with chloroquine, a known inhibitor of TLR9 signaling blocks the CR2-TLR9 interaction by ELISA. We are currently determining whether, by using activation of Namalwa B cells with CpG ODN, blockade of CR2 inhibits the CpG-induced NF-kB activation and induction of IL-6 release. These data suggest that CR2 may functionally interact with TLR9 on B cells.
Serum levels of estradiol correlate with disability in men with multiple sclerosis (MS) (129.20)
Authors: Pelfrey Clara M. , Butler Robert , Cotleur Anne
Published: January 2023

MS occurs more frequently in women compared to men, however men are thought to deteriorate more rapidly. The underlying reasons for a sex bias in MS may relate to sex hormone effects. To determine whether hormone levels correlate with disease disability, we tested 51 untreated relapsing-remitting MS patients (31F, 20M) and 71 controls (38 F, 33M). Serum was tested for hormones (estrogen, testosterone, progesterone) and disability was assessed by the Multiple Sclerosis Functional Composite (MSFC). Testosterone (T) levels were significantly elevated in women with MS compared to female controls (p = 0.0005). 17-beta-estradiol (E2) levels strongly trended towards lower levels in male MS patients compared to male controls (p = 0.06). E2 was significantly positively correlated with T levels in all subjects except the MS males. In males with MS, E2 levels correlated with the MSFC (p = 0.011). T levels did not correlate with disability in men or women. Thus, E2 levels appear decreased in males with MS, which strongly correlates with greater disease disability. Interestingly, we observed that T levels were significantly increased in MS females and did not correlate with disability. Our findings suggest interference in hormone metabolism in patients with MS, which may contribute to altered clinical responses depending on the sex of the patient.

Supported by: NIH, NMSS, and CCF GCRC.
A distinct role for PI3Kgamma in suppressing the development and progression of experimental autoimmune encephalomyelitis (EAE) (129.30)
Authors: Gatson NaTosha N , Ji H , Gienapp I E , Song F , Papenfuss T L , Camps M , Ruckle T , Zimmerer J L , Shawler T M , Lu B , Gerard C , Kithcart A P , Powell N , Fischer A J , Satoskar A , Rommel C , Whitacre C C
Published: January 2023

Phosphoinositide 3-kinases (PI3K) are intracellular signaling proteins involved in cellular responses such as chemotaxis, proliferation and apoptosis. Selective inhibitors of the PI3Kγ-isoform have recently become available. This study explores the role of PI3Kγ in the development and progression of EAE. PI3Kγ +/+ (wt) and PI3Kγ −/− (ko) mice were immunized for EAE using myelin oligodendrocyte glycoprotein p35-55 (MOG) and assessed for clinical signs, CNS histopathology and T cell activation. WT mice showed a progressive disease course with elevations in inflammatory cytokines increased CNS mononuclear infiltrates. In contrast, ko mice exhibited a delayed onset and dramatically less severe EAE course characterized by decreased T cell activation and inflammatory cytokines (TNF-α, IFN-γ, IL-2, IL-12p40, IL-6, IL-17 and MCP-1). Male ko mice were significantly more protected than ko females. Adoptive transfer of MOG-activated T cells into ko recipients show that ko mice are resistant to passive disease induction. Interestingly, wt recipients of ko donors immunized for EAE were also protected. Furthermore, adoptive transfer of activated GFP+ T cells show marked decreased infiltration of GFP+ T cells into the CNS. Taken together, these findingssuggest that depletion of PI3Kγ results in a down-regulation of the inflammatoryresponse caused by impaired trafficking of cells critical to generation of the immune response.

(Supported by NIH grant AI 064320 and National MS Society Grant RG 3272).
Cytokines regulate miRNA expression in islet beta cells (129.41)
Authors: Mi Qing-Sheng , He Hong-Zhi , Kook-Heon Seo , Zhou Li
Published: January 2023

Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-secreting beta cells. The immune system utilizes diverse mediators, including the cytokines, to kill pancreatic beta cells. Accumulating evidence suggests that apoptosis is themain form of beta cell death in spontaneousT1D and islet transplantation, and that the cytokines are key contributors in triggering beta cell apoptosis.MicroRNAs (miRNAs) are a recently discovered class of evolutionarily conserved small noncoding RNAs that lead to post-transcriptional repression, which is involved in the regulation of cell proliferation and cell death. To test the role of miRNAs in cytokine-mediated apoptosis, we compared the miRNA expression profile in rat insulinoma cell line INS-1 (834/40) with or without cytokine treatment, and between cytokine-resistant (INS-1res 833/15) and cytokine-sensitive (INS-1 834/40) cells, using miRNA array. We found that miRNAs were differentially regulated in cytokine-treated INS-1 834/40 compared to untreated cells, and between INS-1 834/40 and INS-1res 833/15 before or after cytokine treatments. Some of these changed miRNAs were recently reported to be linked to control cell growth and apoptosis. These results are the first evidence, to our knowledge, that cytokines can regulate miRNA expression, which suggests that these cytokine-regulated miRNAs could be involved in cytokine-mediated beta cell apoptosis. (JDRF, 5-2006-386)