bluuebunny/crossref_metadata_2025 huggingface dataset:
The production of macrophage inflammatory protein-1 alpha by human basophils.
Authors: Li H , Sim T C , Grant J A , Alam R
Published: December 2022

Macrophage inflammatory protein-1alpha (MIP-1alpha) has previously been shown to be produced by mononuclear cells, eosinophils, and neutrophils. Its production by basophils has not been investigated. The objective of this study was to investigate the production of MIP-1alpha by basophils. Peripheral blood basophils were separated by Percoll gradient centrifugation, cultured overnight, and processed for double immunocytochemistry using Abs against MIP-1alpha and FcepsilonRIalpha (alpha subunit of IgE receptor type 1). We demonstrated that basophils expressed immunoreactive MIP-1alpha upon stimulation with anti-IgE. Less than 5% of the basophils stained for MIP-1alpha without stimulation. The secretion of MIP-1alpha by basophils was studied by ELISA. In these experiments, basophils were further enriched to 65 to 99% (median, 86%) by a negative selection method. Basophils released MIP-1alpha when stimulated by Abs against IgE and FCepsilonRIalpha as well as IL-3 and the calcium ionophore, A23187. In parallel experiments, PBMC, eosinophils, and neutrophils did not produce MIP-1alpha in response to anti-IgE, but they did so in response to A23187. No MIP-1alpha release was detected in platelet preparations. Preincubation with IL-3 (15 min or 18 h) augmented anti-IgE-included basophil MIP-1alpha production. The secretion of MIP-1alpha by basophils was detectable shortly after stimulation and gradually increased over 24 h. Since MIP-1alpha has potent inflammatory and histamine-releasing activities, its production by basophils may indicate a positive feedback mechanism for allergic inflammation.
Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn's disease LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis LP cells manifest increased secretion of IL-5.
Authors: Fuss I J , Neurath M , Boirivant M , Klein J S , de la Motte C , Strong S A , Fiocchi C , Strober W
Published: December 2022

In this study, we investigate whether human inflammatory bowel disease (IBD) (ulcerative colitis and Crohn's disease) is associated with altered lymphokine secretion profiles, as recently found in various animal models of chronic intestinal inflammation. In initial studies, we determined the proliferative responses of purified lamina propria (LP) CD4+ T cells from patients with IBD under defined conditions of T cell stimulation. We found that IBD LP CD4+ T cells in comparison with control LP CD4+ T cells have diminished TCR/CD3 pathway proliferative responses, whereas CD2/CD28 accessory pathway proliferative responses are relatively preserved. In further studies centering on lymphokine production, we showed that LP T cells from inflamed Crohn's disease mucosa manifest increased IFN-gamma secretion compared with control LP T cells, particularly when stimulated via the CD2/CD28 pathway. Subsequent ELISPOT analysis indicated that this was due to an increased number of IFN-gamma-secreting CD4+ T cells. In contrast, IL-4 and IL-5 production by Crohn's disease LP T cells was decreased compared with that of control LP T cells. Of interest, IL-2 production by Crohn's disease LP T cells was also reduced, as was IL-2 production by peripheral blood T cells. In parallel studies, LP T cells from inflamed ulcerative colitis mucosa stimulated via either the TCR/CD3/CD28 or CD2/CD28 produced increased amounts of IL-5, again when measured either as secreted IL-5 or by ELISPOT analysis. Such increased IL-5 production was not associated with increased IL-4 secretion and, in contrast to Crohn's disease, ulcerative colitis LP T cell production of IL-2 and IFN-gamma was normal. Taken together, these studies provide strong evidence that the immunopathologic process characteristic of the two major forms of IBD is associated with very different cytokine secretion patterns. These different patterns may determine the type of inflammatory process present.
Costimulatory effects of T cell proliferation during infection with human T lymphotropic virus types I and II are mediated through CD80 and CD86 ligands.
Authors: Lal R B , Rudolph D L , Dezzutti C S , Linsley P S , Prince H E
Published: December 2022

The modulation of expression of CD80 and CD86 on T cells following infection with human T lymphotropic virus (HTLV)-I/II and its functional importance in T-T cell interactions was examined. Infection with HTLV-I/II leads to constitutive expression of CD80 and CD86, concomitant to down-modulation of CD28 on T cells. The CD80/CD86+ HTLV-infected T cells stimulated proliferation of allogeneic and autologous resting T cells, which could be specifically blocked by a soluble CTLA-4Ig chimeric protein, anti-CD80 or anti-CD86, but not by anti-CD54. It was necessary to inhibit interaction with both ligands (CD80 and CD86) to optimally block HTLV-mediated proliferation of allogeneic and autologous resting T cells. Simultaneous addition of anti-CD8O and anti-CD86 Abs also inhibited production of IFN-gamma, TNF-alpha, and IL-4, with no effect on IL-10 production, for both allo- and autologous T cell proliferation. Further, there was a direct correlation between the spontaneous proliferation of lymphocytes from patients infected with HTLV-II and expression of CD80, which could be blocked by simultaneous addition of anti-CD80 and anti-CD86. Taken together, these results suggest that HTLV-infected CD80/CD86+ T cells serve as APCs, leading to a sustained proliferation of T cells, and that both ligands participate in allostimulation, autologous proliferation, as well as spontaneous proliferation of HTLV-II-infected PBMC.
Identification through bioinformatics of cDNAs encoding human thymic shared Ag-1/stem cell Ag-2. A new member of the human Ly-6 family.
Authors: Capone M C , Gorman D M , Ching E P , Zlotnik A
Published: December 2022

The Ly-6 family of cell surface molecules includes many members that have been characterized in the mouse. Until recently, very few Ly-6 family members had been described in the human. A significant development with important implications for novel gene discovery has been the growth of the public Expressed Sequence Tag (EST) database. Here we report that, through the application of bioinformatics analysis to the dbEST database, we obtained the sequence of human TSA-1/SCA-2, a new member of the human Ly-6 family. In addition, we identified full-length clones encoding this molecule as well as expression data in various tissues. Sequencing of the clones identified this way confirmed the sequence predicted through bioinformatics. This study constitutes an example of the application of bioinformatics to the analysis of the recently expanded databases for the identification of genes of potential importance in the immune system.
Ligation of MHC class I molecules on peripheral blood T lymphocytes induces new phenotypes and functions.
Authors: Bregenholt S , Röpke M , Skov S , Claesson M H
Published: December 2022

Microgram concentrations of immobilized anti-MHC class I (MHC-I) Ab induced proliferation of resting CD3+ T cells from peripheral blood. In contrast, soluble Ab did not activate T cells. Exposure of T cells to immobilized anti-MHC-I Ab for only 24 h was followed by proliferation and development of T cell-mediated cytotoxicity. Immediately following MHC-I ligation, the T cells responded with increased protein tyrosine phosphorylation, with new bands appearing in the SDS-PAGE. Exposure of T cells to immobilized anti-MHC-I Ab for 24 h induced an increased surface expression of the TCR/CD3 and CD28 molecules. MHC-I-induced proliferation of purified T cells was dependent on cellular interactions with non-T cells. Under certain conditions, in which MHC-I was ligated by picogram concentrations of immobilized anti-MHC-I Ab, anti-TCR/CD3 Ab-induced proliferation of T cells was strongly inhibited. These data clearly demonstrate that ligation of the MHC-I complex on T cells may induce both positive and negative signals. Since the physiologic ligands for MHC-I molecules are TCR and the CD8 molecules, our data may suggest that MHC-I molecules are instrumental in cellular interactions between T cells.
Murine T lymphocytes modulate activity of an ATP-activated P2Z-type purinoceptor during differentiation.
Authors: Chused T M , Apasov S , Sitkovsky M
Published: December 2022

Murine T, but not B, lymphocytes constitutively express a membrane receptor for adenosine nucleotides that opens a nonspecific pore that admits Ca2+ and ethidium (314 Da), but not propidium (415 Da) ions. ATP, ADP, and AMP show decreasing potency; UTP and adenosine are inactive. Nonhydrolyzable ATP analogues are completely ineffective. Oxidized ATP inhibits the response. Activity is detectable at ATP concentrations of 125 microM and peaks at 1 mM. The intracellular free Ca2+ ([Ca2+]i) rise is not reversed by removing ATP by centrifugation or apyrase. The kinetics, agonist and antagonist profiles, and the passage of ions as large as ethidium are the characteristics of a P2z-type purinoceptor. No expression of classical P2x-, P2u-, or P2Y-type purinoceptors can be detected. The [Ca2+]i elevating activity of the ATP receptor is modulated during T cell differentiation. CD4+8+ double-positive thymocytes are the least responsive. CD4-8+ single-positive thymocytes, CD8+ splenic T cells, CD4+8- single-positive thymocytes, and CD4+ splenic T cells show increasing reactivity. Measurement of P2Z expression by the rate of ethidium ion uptake correlates with the [Ca2+]i. The trimodal expression of P2Z by splenic CD4+ T cells correlates with the subsets defined by CD44 and CD45RB, differentiation Ags that distinguish memory cells: P2Zlow cells are CD44brightCD45RBbright; P2Zint are CD44dullCD45RBint; P2Zhigh are CD44brightCD45RBdull. It is suggested that P2Z receptor-mediated signaling could be involved in the regulation of differentiation and cell death in the thymus and peripheral T lymphocytes.
Activated B cells express CD28/B7-independent costimulatory activity.
Authors: Ding L , Shevach E M
Published: December 2022

Resting and activated B cells display distinct phenotypes and functional properties. Resting B cells are incompetent accessory cells whereas activated B cells are capable of triggering T cell activation. The up-regulation of expression of the B7 family of molecules has been considered to be the primary reason for this functional conversion of activated B cells. We report here that activation of B cells induces a novel costimulatory activity for induction of T cell proliferation, which is independent of the CD28/B7 costimulatory pathway. B cells activated by different stimuli expressed comparable levels of many of the known counter-receptors for costimulation and intercellular adhesion (B7-1, B7-2, HSA, ICAM-1), but differed markedly in their capacity to activate CD4+ T cells from CD28-deficient (-/-) mice. Activation of B cells via CD40, and to a lesser extent with LPS, induced potent B7/CD28-independent costimulatory activity that resulted in marked augmentation of IL-2-mediated proliferative responses of CD4+ T cells from CD28 -/- mice. The B7/CD28-independent costimulatory pathway was capable of triggering the activation of naive CD4+ T cells, as both sorted CD45RBhigh and isolated high density naive CD4+ T cells from CD28 -/- mice responded vigorously to the costimulation provided by CD40L-activated B cells.
Inhibition of transcription factor Stat1 activity in mononuclear cell cultures and T cells by the cyclic AMP signaling pathway.
Authors: Ivashkiv L B , Schmitt E M , Castro A
Published: December 2022

Activation of T cells results in a cascade of gene activation and subsequent proliferation and differentiation into effector phenotypes. The regulation of transcription factors belonging to the signal transducer and activator of transcription (STAT) family was analyzed in PHA-activated mononuclear cells and in purified T cells activated by cross-linking cell surface CD3. Cell activation resulted in a delayed induction of STAT DNA-binding activity, which was sustained for several days, was composed predominantly of Stat1 and Stat3, and was blocked by cycloheximide and actinomycin D. Increased Stat1 and Stat3 mRNA and protein levels were detected, respectively 4 and 24 h after activation. Stimulation of the cAMP signal transduction pathway, which skews cytokine production toward a Th2 pattern, resulted in the preferential suppression of Stat1 activity. cAMP inhibited the induction of expression of IL-2 receptor components, but did not inhibit IL-4 receptor alpha-chain and CD69 expression or the induction of activator protein 1 transcription factors. cAMP signaling inhibited Stat1 at several different levels, including suppression of DNA binding and down-regulation of Stat1 protein and mRNA levels. Our results demonstrate the regulation of STAT activity by a signaling pathway that regulates the T cell functional phenotype and is distinct from the cytokine-activated Janus kinase-STAT signaling pathway.
CD40 ligation results in protein kinase C-independent activation of ERK and JNK in resting murine splenic B cells.
Authors: Li Y Y , Baccam M , Waters S B , Pessin J E , Bishop G A , Koretzky G A
Published: December 2022

CD40 is a 45- to 50-kDa transmembrane glycoprotein that plays an important role in B cell proliferation, survival, memory, and Ig isotype switching. How CD40 engagement couples to these distal events in B cell activation remains poorly understood. In this study, we have examined signal transduction events mediated by CD40 cross-linking in resting murine splenic B cells. In comparison to signaling via the B cell Ag receptor (BCR), CD40 cross-linking was less effective at activating protein tyrosine kinases. Interestingly, however, CD40 engagement resulted in the phosphorylation of both extracellular signal-regulated protein kinase (ERK) and the Ras guanine nucleotide exchange factor, Son of sevenless. In addition, both ERK and c-Jun NH2-terminal kinase activities were increased after both CD40 and BCR ligation. Overnight treatment of cells with phorbol ester as well as pharmacologic inhibitors of protein kinase C abrogated these signaling events after BCR treatment; however, no effect was seen on CD40-mediated activation of ERK or c-Jun NH2-terminal kinase, suggesting that the BCR and CD40 differentially utilize protein kinase C to couple with these signaling pathways.
Somatic mutations in human Ig variable genes correlate with a partially functional CD40-ligand in the X-linked hyper-IgM syndrome.
Authors: Razanajaona D , van Kooten C , Lebecque S , Bridon J M , Ho S , Smith S , Callard R , Banchereau J , Brière F
Published: December 2022

X-linked hyper-IgM (HIGM-1) syndrome is a rare disorder resulting from mutations in the CD40-ligand (CD40L) gene. This defect is associated with normal or elevated serum levels of IgM, and with low to undetectable levels of serum IgG, IgA, and IgE. We analyzed the somatic mutation status in Ig V genes from three unrelated HIGM-1 patients by reverse-transcription PCR and sequence analysis. Two patients (B.S. and P.S.) expressed unmutated VH6 genes. In contrast, one patient (A.T.) was found to express mutated VH6 genes. Whether the presence of somatic mutations in this patient was related to a functional CD40L was assessed by deriving T cell clones from his peripheral blood cells. Upon activation, these T cell clones expressed weakly and transiently surface CD40L, and were able to induce limited isotype switch of normal native B cells, indicating residual CD40L function. Altogether, our results 1) confirm the central role played by CD40L in the generation of somatic mutation (patients B.S. and P.S.), 2) provide an unusual illustration of the relative dissociation between somatic mutation and isotype switching (patient A.T.), and 3) demonstrate a further complexity of the X-linked HIGM syndrome that may occur despite a partially functional CD40L.
The transcription factor B-cell-specific activator protein is not involved in the IL-4-induced activation of the human IgE germline promoter.
Authors: Albrecht B , Peiritsch S , Messner B , Woisetschläger M
Published: December 2022

Transcriptional activity of the human IgE germline gene is a prerequisite for a subsequent deletional rearrangement of the Ig heavy-chain locus, the hallmark of isotype switching to IgE. The B-cell-specific transcription factor B cell-specific activator protein (BSAP) was described for being critically involved in the IL-4 up-regulation of the murine IgE germline gene. Our study was initiated to evaluate the regulatory role of BSAP in the human IgE germline promoter. It is shown that BSAP binds to a DNA element located immediately upstream of the most 5' transcriptional start site. The authenticity of BSAP was determined by electrophoretic mobility shift assays in which oligonucleotides corresponding to published BSAP binding sites efficiently competed for binding to the novel identified sequence. In addition, recombinant purified BSAP protein bound this motif and comigrated with the band seen with nuclear extracts. Finally, a polyclonal anti-BSAP antiserum specifically prevented interaction of the protein with its DNA recognition sequence. The affinity of BSAP for its recognition sequence was low compared with the sites identified in the CD19, the blk gene, and an LR1 transcription factor binding sequence located in the Ig gamma 1 switch region. Reporter gene constructs in which binding of BSAP was abolished by site-directed mutagenesis responded to IL-4 stimulation better than the wild-type construct in both cell lines tested. In addition, the basal activity of the mutated promoter did not change significantly despite the close proximity of the BSAP motif to the transcriptional start site. It is concluded that BSAP plays no direct regulatory role in the cytokine-induced response of the human IgE germline promoter.
Stat1 alpha expression is involved in IFN-gamma induction of the class II transactivator and class II MHC genes.
Authors: Lee Y J , Benveniste E N
Published: December 2022

Class II MHC Ags are critical in the regulation of immune responses by presenting Ag to T lymphocytes, resulting in their activation and differentiation. Class II expression is rare in the normal central nervous system, but elevated expression on glial cells has been observed in several neurologic diseases. We have previously demonstrated that IFN-gamma-induced class II expression in glial cells involves activation of both tyrosine kinase and protein kinase C. IFN-gamma induces tyrosine phosphorylation of the tyrosine kinases Jak1 and Jak2 and of Stat1 alpha. In addition, IFN-gamma enhances expression of Stat1 alpha mRNA and protein. We utilized antisense oligonucleotides against Stat1 alpha to determine directly whether IFN-gamma-induced activation and/or enhancement of Stat1 alpha is involved in class II expression. Antisense oligonucleotides complementary to Stat1 alpha mRNA were introduced in CH235-MG astroglioma cells by transient transfection; such treatment inhibited both constitutive and IFN-gamma-enhanced expression of Stat1 alpha. IFN-gamma-induced class II MHC expression was also inhibited in cells exposed to Stat1 alpha antisense oligonucleotides. The fact that the class II promoter does not contain IFN-gamma-activated sequences for binding Stat1 alpha suggests that Stat1 alpha must activate another protein that is directly involved in class II expression. A likely candidate is the class II MHC transactivator (CIITA). IFN-gamma induction of CIITA mRNA was also inhibited in cells treated with antisense oligonucleotides against Stat1 alpha. These findings demonstrate that Stat1 alpha is involved in IFN-gamma induction of CIITA expression, resulting in class II MHC expression.
Induction of autoimmunity in the absence of CD28 costimulation.
Authors: Bachmaier K , Pummerer C , Shahinian A , Ionescu J , Neu N , Mak T W , Penninger J M
Published: December 2022

Ag-specific activation of T lymphocytes requires two signals, one by the TCR and a second by costimulatory molecules. In a CD4+ T helper cell-dependent experimental autoimmune myocarditis model, we provide genetic evidence that cardiac myosin-induced autoimmune myocarditis and the production of IgG auto-Abs is dependent on functional T cells and did not occur in mice lacking the tyrosine kinase p56lck or the tyrosine phosphatase CD45. By contrast, animals lacking the T cell-costimulatory molecule CD28 (CD28 -/-) developed autoimmune heart disease, although at significantly lower severity than in heterozygous littermates, and produced IgG auto-Abs depending on the concentration of the autoantigen administered. In addition, the isotypes of IgG auto-Abs specific for cardiac myosin differed between CD28 +/- and CD28 -/- mice. Whereas CD28 +/- mice predominantly produced Th2-mediated IgG1 auto-Abs, CD28 -/- mice produced predominantly IgG2a. These data suggest that CD28 costimulation plays a crucial role in induction and maintenance of autoimmune heart disease and that CD28 expression is required for predominant Th2-IgG1 responses in an autoimmune setting.
Pathogenesis of post-thymectomy autoimmune gastritis. Identification of anti-H/K adenosine triphosphatase-reactive T cells.
Authors: Suri-Payer E , Kehn P J , Cheever A W , Shevach E M
Published: December 2022

Autoimmune gastritis spontaneously develops following thymectomy of 3-day-old BALB/c mice (d3Tx). These mice develop autoantibodies to the gastric parietal cell proton pump, H/K ATPase, and aberrant expression of the H/K ATPase in the neonatal thymus prevents the induction of disease post-thymectomy. To characterize the effector cells mediating autoimmune gastritis, we isolated H/K ATPase-enriched preparations of parietal cell microsomes and further purified the enzyme by lectin affinity chromatography. Both preparations induced significant proliferative responses of gastric lymph node cells, which were mediated by CD4+, MHC class II-restricted T cells. Surprisingly, T cells reactive to the Ag could only be demonstrated in lymph nodes in the immediate proximity of the stomach; little or no response was seen when mesenteric or peripheral lymph nodes were tested. It is likely that the H/K ATPase-reactive T cells are actually the effector cells in this disease, as they could only be detected in mice that developed gastritis, as indicated by anti-parietal cell Ab, gastric inflammation, and the presence of cells capable of transferring disease into nu/nu mice. H/K ATPase-specific T cell proliferative responses could first be detected 5 wk post-thymectomy and were accompanied by high background responses at this time point. These latter responses may represent enhanced syngeneic MLRs, which we have previously shown to be elevated in d3Tx mice. Characterizations of the H/K ATPase-reactive and self-reactive T cell populations may reveal the factors that break peripheral T cell tolerance and lead to the development of organ-specific autoimmune disease.
Intraepithelial lymphocytes in human gut have lytic potential and a cytokine profile that suggest T helper 1 and cytotoxic functions.
Authors: Lundqvist C , Melgar S , Yeung M M , Hammarström S , Hammarström M L
Published: December 2022

The functional properties of intraepithelial lymphocytes (IEL) in normal human jejunum, ileum, and colon were investigated. Cytokine mRNA expression in IEL and enterocytes was determined by reverse transcriptase-PCR and IFN-gamma+ IEL by immunohistochemistry. Polyclonal activators were used to study proliferation and IFN-gamma secretion of IEL, and an anti-CD3-mediated redirected cytotoxicity assay was used to determine the lytic potential of IEL. Freshly isolated IEL at all three gut levels expressed mRNA for IL-1 beta, IL-2, IL-8, IFN-gamma, and TNF-alpha. Approximately 10% of IEL produced IFN-gamma, suggesting that IEL are immunologically active in vivo, performing similar functions along the intestine. IEL could be stimulated further in vitro to express IL-10, TNF-beta, and TGF-beta 1, while no Th2-type cytokines were induced, suggesting suppressive and cytolytic functions for IEL. All three jejunal IEL subpopulations (CD4-CD8-TCR-gamma delta+, CD4+TCR-alpha beta+, CD8+TCR-alpha beta+) expressed the same four cytokines, IL-2, IL-8, IFN-gamma, and TNF-alpha, indicating that CD4+TCR-alpha beta+ IEL are Th1 cells and that TCR-gamma delta+ IEL and CD8+TCR-alpha beta+ IEL include cytotoxic effector cells. Indeed, freshly isolated jejunal IEL displayed cytolytic activity. IEL were induced to proliferation by anti-CD3/TCR complex mAbs and leukoagglutinin, but not by Con A. There was no correlation between the magnitude of the proliferative response and the amounts of secreted IFN-gamma. Enterocytes expressed IL-1 beta and IL-8, and sometimes TNF-alpha. Although jejunal enterocytes express HLA-DR and hsp60, Ag presentation by these cells may induce anergy since their cytokine profile is different from that of classical APCs.
Switching on of the proliferation or apoptosis of activated human T lymphocytes by IFN-gamma is correlated with the differential expression of the alpha- and beta-chains of its receptor.
Authors: Novelli F , Bernabei P , Ozmen L , Rigamonti L , Allione A , Pestka S , Garotta G , Forni G
Published: December 2022

To find out how physiologically secreted IFN-gamma controls either the proliferation or the apoptosis of human T lymphocytes, the kinetics of expression of the alpha- and beta-chains of its receptor (IFN-gamma R) were sequentially followed on T lymphocytes first activated with PHA and then cultured in the presence of IL-2, and related to the kinetics of expression of Fas, Bcl-2, and IL-2R p55 chain. Both IFN-gamma R chains were poorly expressed on the membrane of resting T lymphocytes. Following their stimulation with PHA, IFN-gamma R alpha but not IFN gamma R beta-chain up-modulated before T lymphocyte entry into the S phase, and then IFN-gamma R alpha down-modulated when they passed through the S and G2/M. The ensuing proliferative response was inhibited by an anti-IFN-gamma R alpha mAb that impeded the binding of IFN-gamma. When PHA-activated T lymphoblasts were cultured for 16 days with IL-2, IFN-gamma R alpha expression increased, whereas that of the beta-chain remained barely detectable. Fas and Bcl-2 were both highly expressed. When these T lymphoblasts were restimulated by PHA, OKT3, or Staphylococcus enterotoxin beta-pokeweed mitogen, both chains up-modulated and most cells underwent apoptosis in a way apparently independent of Bcl-2, but not of Fas. This apoptosis, too, was prevented by the anti-IFN-gamma R alpha mAb. Physiologically secreted IFN-gamma is thus involved in the activation of resting T lymphocytes and in the apoptosis of reactivated lymphoblasts. However, high expression of IFN-gamma R beta took place when IFN-gamma induced apoptosis, but not when it induced proliferation. In conclusion, a correlation exists between differential expression of the IFN-gamma R beta-chain and the delivery by IFN-gamma of proliferative or apoptotic signals.
Induction of "infectious" tolerance to MHC-incompatible cardiac allografts in CD4 monoclonal antibody-treated sensitized rat recipients.
Authors: Onodera K , Lehmann M , Akalin E , Volk H D , Sayegh M H , Kupiec-Weglinski J W
Published: December 2022

LBNF1 heart grafts are rejected in an accelerated manner within 36 h by LEW rats that have been sensitized with Brown Norway rat skin grafts on day -7. Treatment with RIB-5/2, a CD4-nondepleting mAb (10 doses of 5 mg/rat/day, i.v., from day -7 to day +21) abrogated rejection at <36 h and produced indefinite (>200 days) cardiac allograft survival. Transplantation tolerance in this model developed within several weeks, and during the maintenance phase (>100 days) it was associated with diminished host circulating allo-Ab responses and induction of peripheral allospecific T cell unresponsiveness both in vitro and in vivo. Tolerant cells in mAb-treated hosts could disable naive or alloimmune cells, so that they failed to trigger graft rejection. Moreover, donor-specific and organ-nonspecific tolerance could be adoptively transferred by spleen cells alone into new sets of primary (100%) and secondary (>40%) test recipients. CD4+ T cells were instrumental for the induction of such readily transferable tolerance. The first and second generation suppressive regulatory cells were also critical for the inhibition of allograft recognition by normal or even alloimmune cells. Hence, the features of an "infectious" tolerance pathway to minor histocompatibility-mismatched skin grafts, originally described in thymectomized mice, may be applied to the euthymic primed rats rendered tolerant to fully MHC-incompatible vascularized organ allografts. Such reprogramming of host cell-mediated regulatory mechanisms following CD4-targeted therapy adds to our appreciation of the potential utility and applicability of infectious tolerance in transplant recipients treated with a perioperative course of CD4-targeted monotherapy.
TNF receptors in murine Candida albicans infection: evidence for an important role of TNF receptor p55 in antifungal defense.
Authors: Steinshamn S , Bemelmans M H , van Tits L J , Bergh K , Buurman W A , Waage A
Published: December 2022

TNF mediates multiple biologic activities through two distinct cell surface receptors, TNFR-p55 and TNFR-p75. TNF plays an important role in nonspecific resistance against the fungus Candida albicans. We used transgenic mice deficient for TNFR-p55 or TNFR-p75 to investigate the role of the TNFR in antifungal defense. Mice deficient for TNFR-p55 have highly impaired ability to clear infection with C. albicans and readily succumb to the infection. Also mice deficient for TNFR-p75 had a significant reduction in their ability to clear the fungus although lethality was not increased. These data demonstrate that TNFR-p55 in particular, but also TNFR-p75, plays a definite role in defense against infection with C. albicans. In NMRI mice, infection with C. albicans resulted in a significant systemic release of soluble (s)TNFR-p75. Cyclophosphamide-induced granulocytopenia led to a reduction of sTNFR-p75 release, whereas levels of bioactive TNF in response to fungal infection were increased. Release of sTNFR-p55 was not affected by induction of granulocytopenia. These observations suggest that granulocytes are a source of sTNFR-p75, possibly contributing to regulation of TNF activity during infection with C. albicans.
Delayed tyrosine phosphorylation and nuclear expression of STAT1 following antigen receptor stimulation of B lymphocytes.
Authors: Karras J G , Huo L , Wang Z , Frank D A , Zimmet J M , Rothstein T L
Published: December 2022

The regulation of the STAT1 alpha transcription factor was assessed during B cell activation induced by cross-linking the surface IgM Ag receptor. Surface Ig ligation or pharmacologic stimulation with PMA and ionomycin resulted in the delayed (2-3 h after stimulation) nuclear appearance of tyrosine-phosphorylated STAT1 alpha, in contrast to the rapid induction that follows cytokine treatment. Nuclear expression of phosphorylated STAT1 alpha was abrogated by co-incubation of anti-Ig-treated B cells with the protein synthesis inhibitor cycloheximide (CHX), with the protein kinase inhibitor H-7, or with the immunosuppressive drug rapamycin. Tyrosine-phosphorylated STAT1 alpha was found to be recruited to the STAT binding site of the IFN regulatory factor-1 (IRF-1) gene promoter only after 2 to 3 h, and this association was also inhibitable by CHX and rapamycin. The arrival of STAT1 alpha coincided with attenuation of anti-Ig-induced STAT-binding activity specific for the IRF-1 promoter site, and both rapamycin and CHX treatment counteracted the loss of this activity. Furthermore, basal transcription of the endogenous IRF-1 gene was decreased as a result of anti-Ig treatment, and this effect of anti-Ig was blocked by co-incubation with rapamycin. Thus, STAT1 alpha plays a dynamic role in the composition of IRF-1 promoter-specific DNA binding complexes stimulated by B cell Ag receptor ligation, and nuclear expression of phosphorylated STAT1 alpha is regulated in a unique fashion by Ag receptor engagement. In addition, surface Ig cross-linking imparts negative regulatory control of IRF-1 gene expression, possibly through activation of STAT1 alpha.
Inhibition of TC-1 cytokine production, effector cytotoxic T lymphocyte development and alloantibody production by 2,3,7,8-tetrachlorodibenzo-p-dioxin.
Authors: Kerkvliet N I , Baecher-Steppan L , Shepherd D M , Oughton J A , Vorderstrasse B A , DeKrey G K
Published: December 2022

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a widespread environmental contaminant and prototypic ligand for the aryl hydrocarbon receptor, is a potent immunotoxicant. To understand the underlying mechanisms of TCDD immunotoxicity, we have characterized the time course of changes in CTL, alloantibody, and cytokine responses to the P815 tumor allograft in C57B1/6 mice treated with 0 or 15 microg TCDD/kg. Suppression of CTL activity by TCDD directly correlated with reduced numbers of splenic CTL effector cells identified by their CD8+CD44 high CD45RB low phenotype, while suppression of the alloantibody response correlated with a lack of expansion of the B220+ splenocyte population. Cytokine production was differentially modulated following TCDD treatment. Although type 1 cytokine production (IFN-gamma, IL-2, and TNF) was initially induced in TCDD-treated mice, production failed to increase normally after day 5. In contrast, the production of IL-1 beta, IL-4, and IL-6 was mostly unaffected by TCDD exposure. This differential effect of TCDD on cytokine production was reflected in the degree of suppression of specific alloantibody isotypes. TCDD abrogated the production of IgG2a (promoted by IFN-gamma), but had much less effect on the level of IgG1 (promoted by IL-4). IgM Ab titers were also highly suppressed. CD8+ cells were the exclusive producers of IFN-gamma and IL-2 when spleen cells from P815-injected mice were cultured in vitro on days 4 to 7 after P815 injection. However, CD4+ cells were shown to play a crucial role in the generation of both CTL and alloantibody responses, since their depletion in vivo abolished both responses. Based on similar temporal effects produced by TCDD and anti-CD4 Ab on alloimmune responses, we postulate that TCDD interferes with the initial activation of CD4+ T cells, which leads to downstream inhibition of the activation and/or differentiation of CD8+ T cells and B cells. In addition, since delayed treatment with either anti-CD4 Ab or TCDD suppressed the alloantibody but not the CTL response, TCDD may also affect later CD4+ T helper-B cell interactions.
TCR-gamma delta cells in CD3 zeta-deficient mice contain Fc epsilon RI gamma in the receptor complex but are specifically unresponsive to antigen.
Authors: Khattri R , Sperling A I , Qian D , Fitch F W , Shores E W , Love P E , Bluestone J A
Published: December 2022

Unlike TCR-alpha beta cells, TCR-gamma delta cells express a distinct member of the zeta family, the gamma-chain of Fc epsilon RI (Fc epsilon RI gamma) within the TCR complex. To study the role of the Fc epsilon RI gamma-chain in TCR-gamma delta cells, a TCR-gamma delta transgenic mouse (G8) has been crossed with CD3 zeta-chain-deficient mice (G8.zeta-/-). Thy-1+ spleen and lymph node cells of these animals expressed low levels of CD3/TCR. These results suggested that the zeta-chain is required for effective TCR transport to the cell surface. In contrast, intraepithelial TCR-gamma delta cells of G8.zeta-/- mice expressed high levels of TCR. Immunoprecipitation with anti-CD3 showed that Fc epsilon RI gamma-chains were associated with the TCR complex in T cells isolated from zeta-deficient mice. Although the Fc epsilon RI gamma-expressing T cells proliferated in response to stimulation by TCR-specific Abs including anti-CD3 epsilon, anti-pan gamma delta, and anti-V gamma 2 mAb, the G8.zeta-/- T cells did not respond to the G8-specific Ag (T10b), anti-Thy-1 mAb, or Con A. The unresponsiveness to the Ag was not due to the reduced TCR expression, because intraepithelial TCR-gamma delta cells from the zeta-deficient mice did not respond to Ag. The inability of the G8.zeta-/- T cells to respond to Ag could not be overcome by providing an anti-CD28 costimulatory signal or by adding exogenous rIL-2. Taken together, our data suggest that the Fc epsilon RI gamma-chain associates with the TCR-gamma delta complex in the absence of the zeta-chain, but it is not able to substitute for the zeta-chain for effective transport of TCR to the cell surface or functional responses to Ag.
High dose oral tolerance in ovalbumin TCR-transgenic mice: systemic neutralization of IL-12 augments TGF-beta secretion and T cell apoptosis.
Authors: Marth T , Strober W , Kelsall B L
Published: December 2022

The immune response to oral Ag administration, including the development of oral tolerance, was explored with the use of OVA TCR-transgenic mice. Feeding high doses of OVA enhanced IFN-gamma production in the Peyer's patches, but induced tolerance in the peripheral lymphoid tissues marked by suppressed proliferative and cytokine responses. Systemic administration of Abs to IL-12 (anti-IL-12) simultaneous with Ag feeding modestly enhanced the degree of tolerance in the peripheral lymphoid tissues, as shown by increased suppression of proliferative responses after in vitro restimulation, and secondary responses in the popliteal lymph nodes following in vivo challenge and in vitro restimulation. Systemic anti-IL-12 treatment was associated with augmented TGF-beta production and T cell apoptosis in both Peyer's patches and peripheral lymphoid tissues. Cell mixing studies and proliferation assays in the presence of anti-TGF-beta provided evidence that the increased suppression of responses induced by anti-IL-12 was due primarily to the secretion of TGF-beta. These findings suggest that IL-12 negatively regulates two of the main mechanisms of oral tolerance, TGF-beta production and clonal deletion via apoptosis. in addition, they suggest that the combination of oral Ag feeding and systemic anti-IL-12 administration may be of benefit in the treatment of autoimmune diseases.
Mucosal or targeted lymph node immunization of macaques with a particulate SIVp27 protein elicits virus-specific CTL in the genito-rectal mucosa and draining lymph nodes.
Authors: Klavinskis L S , Bergmeier L A , Gao L , Mitchell E , Ward R G , Layton G , Brookes R , Meyers N J , Lehner T
Published: December 2022

The major routes of HIV transmission are through the rectal and cervico-vaginal mucosa. To prevent dissemination of HIV to the regional lymph nodes (LNs), an effective vaccine may need to stimulate CTL in the rectal or genital tract and the draining LNs. We report that mucosal immunization by the recto-oral and vagino-oral route or s.c. immunization targeting the iliac LNs with a particulate SIVp27:Ty-VLP vaccine elicits SIVgag-specific CTL in the regional LNs as well as in the spleen and PBMC. Targeted LN immunization with this vaccine elicited MHC class I-restricted CD8+ CTL responses, and the highest frequency of CTL was found in the iliac LNs. Moreover, SIVgag-specific CTL activity was detected in short term T cell lines established in mononuclear cells eluted from the rectal and cervico-vaginal mucosa. The relative frequency of CTL in short term cell lines prepared from the rectal mucosa (21/113 or 18.6%) was similar to that obtained from the cervico-vaginal mucosa (16/79 or 20.3%). Examination of the relative frequency of CTL to the T cell epitopes residing within SIVp27 showed a higher frequency in iliac LN cells to peptide aa 41-70 than in that to peptide aa 121-150, and this was significant after both recto-oral (chi-squared 6.500, p < 0.02) and vagino-oral (chi-squared = 10.391, p < 0.01) immunization. In contrast, the relative frequency of CTL in PBMC to peptide aa 41-70 (15.5%) was comparable to that elicited by peptide aa 121-150 (17.6%). This study provides novel evidence that mucosal or targeted LN immunization can generate anti-SIV CTL in the rectal and genital mucosa, in the draining LNs, and in the central lymphoid system.
Complement C4 protein expression by rat hepatic stellate cells.
Authors: Fimmel C J , Brown K E , O'Neill R , Kladney R D
Published: December 2022

Stellate cells play an important role in the production and turnover of the normal extracellular matrix of the liver and are key effector cells in the hepatic fibrogenesis that occurs in response to liver injury. In the present study, we used a rat model of long term dietary iron supplementation to identify stellate cell genes that are expressed during chronic hepatic iron overload. Using a subtraction cloning strategy, we identified a rat isoform of the complement C4 protein gene whose expression was strongly induced in stellate cells after iron overload. Highly purified, cultured stellate cells synthesized the C4 precursor protein and released its subunits into the culture medium. The C4 protein secreted in vitro was biologically active in a C4-specific hemolytic assay. C4 mRNA expression was minimal in freshly isolated stellate cells and increased between days 3 and 7 of primary culture, coincident with the expression of smooth muscle alpha-actin (alpha-SMA), a marker of cellular activation. C4 expression was absent in strongly alpha-SMA-positive, passaged cells, but was induced by IFN-gamma, which simultaneously inhibited alpha-SMA expression. Our studies establish hepatic stellate cells as a previously unrecognized source of C4 and raise the possibility that complement protein expression by the cells plays a role in the hepatic injury response and in fibrogenesis. Our in vitro data point to the presence of two distinct stimulatory pathways for C4 expression in stellate cells that differ with regard to their sensitivity to IFN-gamma and their relationship to cellular activation.
Regulation of superantigen-induced T cell activation in the absence and the presence of MHC class II.
Authors: Lando P A , Olsson C , Kalland T , Newton D , Kotb M , Dohlsten M
Published: December 2022

To study MHC class II-dependent and -independent SAg2 activation and the relative importance of CD80/CD28 costimulation, staphylococcal enterotoxin A (SEA) was presented to T cells as a fusion protein containing the Fab fragment of an mAb directed against the CA215 glycoprotein. Chinese hamster ovary (CHO) cells transfected with HLA-DR4, CA215, and CD80, individually or in combinations, were used as presenting cells. A strong T cell proliferation was obtained when C215Fab-SEA fusion proteins were presented by CHO-DR/CD80 or CHO-CA215/CD80 double transfectants, whereas only low levels of proliferation were seen in the absence of CD80. Large amounts of IL-2, IFN-gamma, and TNF were produced in addition to an increase in IL-2 mRNA as a result of CD80 costimulation. Only approximately 50% of the SEA-reactive T cells responded by expression of IL-2 receptor chains and by blast formation when activated with SEA in the absence of MHC class II. Reverse transcription-PCR-assisted repertoire analysis of SEA-reactive TCR V beta families showed that the CA215-dependent activation involved an expansion of fewer TCR V beta families compared with MHC class II-dependent activation. One-half of the six analyzed TCR V beta families were expanded independently of class II. This indicates that MHC class II has only a partial influence on the TCR V beta repertoire imprinted by SAg. This finding redefines the role of MHC class II in SAg presentation. It is suggested that MHC class II molecules are selected as SAg-binding molecules mainly as a suitable targeting receptor for professional APC expressing costimulatory molecules such as CD80 and CD86.
Extrathymic origin of human gamma delta T cells during fetal development.
Authors: McVay L D , Carding S R
Published: December 2022

We have identified the first TCRs to be generated in vivo during normal human fetal development. Before thymic formation, the liver is a site of generation for a subset of V gamma 9/V delta 2+ gamma delta T cells. Analysis of the expression of the male-specific gene, SRY, by V gamma 9/V delta 2+ gamma delta T cells isolated from the fetal liver of male donors has shown that these cells are generated de novo in the liver. Examination of TCR-V gamma and -V delta gene expression demonstrated that although multiple receptor rearrangements could be detected, V gamma 9-JP- and V delta 2-D delta 3-J delta 1/3-encoded receptors were preferentially expressed in each of five individual liver samples. Structural analysis of these receptor chains and those expressed by a panel of V gamma 9/V delta 2+ fetal T cell clones showed that the V gamma 9-JP receptors were invariant or canonical and that the delta-chains contained non-germ line-encoded structural motifs. gamma delta T cells expressing these structurally limited receptor chains were shown to be functional and capable of responding to mycobacterial Ags. Together with the observation that V gamma 9/V delta 2+ cells represented the majority of T cells present in the fetal liver between 7 and 11 wk of development, our findings demonstrate that this subset of gamma delta T cells is a major, and presumably important, component of the human fetal immune system.
Requirements for induction of vitamin D-mediated gene regulation in normal human B lymphocytes.
Authors: Morgan J W , Morgan D M , Lasky S R , Ford D , Kouttab N , Maizel A L
Published: December 2022

Mature human lymphocytes are unique targets of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) in that vitamin D receptors (VDR) are not constitutively expressed, and specific cellular activation signals are required for both the up-regulation of VDR and establishment of reactivity to the lipophilic ligand. Treatment of B lymphocytes with the cytokine IL-4 (IL-4), in the absence of prior activation, induces a weak up-regulation of VDR expression but fails to generate vitamin D-responsive element (VDRE)-reactive nuclear protein complexes or to initiate the genomic transcription of 25-hydroxyvitamin D3 24-hydroxylase. Stimulation of B lymphocytes by either ligation of CD40 Ag or cross-linking the Ig receptor is also insufficient to render B lymphocytes responsive to 1 alpha,25(OH)2D3. However, this apparent lack of response to the secosterol can be overcome by stimulation of B lymphocytes with a combination of these cellular activation signals, which are sufficient to lead to G1 cell cycle progression. In the presence of 1 alpha,25(OH)2D3, cellular activation associated with stimulation of such a progression appears to be sufficient for the up-regulation of VDR message and protein and necessary for the establishment of VDRE binding complexes and the induction of 24-hydroxylase message. Furthermore, biologic functions are modulated, in that the hormone inhibits proliferation in a subset of the activated B cells. These observations suggest that reactivity to 1 alpha,25(OH)2D3 is tightly regulated in B lymphocytes, requiring specific signals for its initiation.