Showing papers in "International Immunology in 1996"
TL;DR: The results suggest that the expression of thePD-1 antigen is tightly regulated and induced by signal transduction through the antigen receptor and do not exclude the possibility that the PD- 1 antigen may play a role in clonal selection of lymphocytes although PD-1 expression is not required for the common pathway of apoptosis.
Abstract: A mAb J43 has been produced against the product of the mouse PD-1 gene, a member of the Ig gene superfamily, which was previously isolated from an apoptosis-induced T cell hybridoma (2B4.11) by using subtractive hybridization. Analyses by flow cytometry and immunoprecipitation using the J43 mAb revealed that the PD-1 gene product is a 50-55 kDa membrane protein expressed on the cell surface of several PD-1 cDNA transfectants and 2B4.11 cells. Since the molecular weight calculated from the amino acid sequence is 29, 310, the PD-1 protein appears to be heavily glycosylated. Normal murine lymphoid tissues such as thymus, spleen, lymph node and bone marrow contained very small numbers of PD-1(+) cells. However, a significant PD-1(+) population appeared in the thymocytes as well as T cells in spleen and lymph nodes by the in vivo anti-CD3 mAb treatment. Furthermore, the PD-1 antigen expression was strongly induced in distinct subsets of thymocytes and spleen T cells by in vitro stimulation with either anti-CD3 mAb or concanavalin A (Con A) which could lead T cells to both activation and cell death. Similarly, PD-1 expression was induced on spleen B cells by in vitro stimulation with anti-IgM antibody. By contrast, PD-1 was not significantly expressed on lymphocytes by treatment with growth factor deprivation, dexamethasone or lipopolysaccharide. These results suggest that the expression of the PD-1 antigen is tightly regulated and induced by signal transduction through the antigen receptor and do not exclude the possibility that the PD-1 antigen may play a role in clonal selection of lymphocytes although PD-1 expression is not required for the common pathway of apoptosis.
1,445 citations
TL;DR: Examination of expression of the PD-1 protein during differentiation of thymocytes in normal adult, fetal and RAG-2(-/-) mice with or without anti-CD3 mAb stimulation shows thatPD-1 expression is specifically induced at the stages preceding clonal selection.
Abstract: PD-1, a member of the Ig superfamily, was previously isolated from an apoptosis-induced T cell hybridoma 2B4.11 by subtractive hybridization. Expresson of the PD-1 mRNA is restricted to thymus in adult mice. Using an anti-PD-1 mAb (J43), we examined expression of the PD-1 protein during differentiation of thymocytes in normal adult, fetal and RAG-2(-/-) mice with or without anti-CD3 mAb stimulation. While PD-1 was expressed only on 3-5% of total normal thymocytes, approximately 34% of the CD4(-)CD8(-) double-negative (DN) fraction are PD-1(+) cells with two distinct expression levels (low and high). PD-1(high) thymocytes belonged to TCR gammadelta lineage cells. In the DN compartment of the TCR alphabeta lineage, PD-1 expression started at the low level from the CD44(+)CD25(+) stage and the majority of thymocytes expressed PD-1 at the CD44(-)CD25(-) stage in which the thymocytes express TCR beta chains. The anti-CD3epsilon antibody administration augmented the PD-1 expression as well as the differentiation of the CD44(-)CD25(+) DN cells into the CD44(-)CD25(-) DN stage, not only in normal mice but also in RAG-2-deficient mice. The fraction of the PD-1(low) cells in the CD4(+)CD8(+) double-positive (DP) compartment was very small (<5%) but increased by stimulation with the anti-CD3 antibody, although the total number of DP cells was drastically reduced. The results show that PD-1 expression is specifically induced at the stages preceding clonal selection.
265 citations
TL;DR: Increases in plasma Trx levels correlate with decreases in monochlorobimane staining and with changes in surface antigen expression that occur in the later stages of HIV infection, suggesting that elevation of plasma TrX levels may be an important component of advanced HIV disease.
Abstract: Thioredoxin (Trx), a ubiquitous protein intimately involved in redox and protein disulfide reductions, has been shown to be released from cells and to have cytokine-like activities. In addition, Trx has been implicated in the redox regulation of immunological responses and shown to be deficient in tissues from AIDS patients. In studies presented here, plasma Trx levels were measured by ELISA in plasma samples from HIV-infected individuals (n = 136) and HIV-negative controls (n = 47). To account for the release of Trx into plasma due to hemolysis, the Trx measurements were corrected according to the level of hemoglobin in the plasma sample. Data presented show that, in contrast to tissue Trx levels, corrected plasma Trx levels are significantly higher in HIV-infected individuals than in controls (P < 0.0001). Furthermore, approximately 25% of the HIV-infected individuals studied have plasma Trx levels greater than the highest levels found in controls (37 ng/ml). Detailed multiparameter FACS analysis of peripheral blood mononuclear cells (PBMC) from the infected individuals demonstrates that those with higher plasma Trx levels (37 ng/ml or greater) tend to have lower overall CD4 counts. In addition, increases in plasma Trx levels correlate with decreases in monochlorobimane staining (indicative of lower intracellular glutathione levels in PBMC) and with changes in surface antigen expression (CD62L, CD38 and CD20) that occur in the later stages of HIV infection. These correlations suggest that elevation of plasma Trx levels may be an important component of advanced HIV disease, perhaps related to the oxidative stress that often occurs at this stage.
210 citations
TL;DR: It is found that the extents of methylation and the amounts of germline transcripts do not necessarily correlate with the efficiency of recombination in induced CH12F3 cells, leading to the proposal that switch recombination can be separated into at least two phases.
Abstract: We have developed an efficient in vitro class switching system using a subclone (CH12F3) of the IgM+ CH12.LX lymphoma cell line. CH12F3 cells switched from surface IgM+ cells to surface IgA+ cells at a high frequency (50%) after 72 h stimulation with IL-4, transforming growth factor (TGF)-beta and CD40L. No other class isotype-producing cells were detected, indicating that the CH12F3 clone is exclusively committed to IgA isotype switching. To understand the molecular basis of the isotype commitment, we studied the methylation profiles of I region promoters and I region transcription of CH12F3 cells. No germline transcripts other than those from the I alpha region were detected and only the I alpha promoter was demethylated in uninduced CH12F3 cells. TGF-beta, CD40L and IL-4 synergistically induced efficient switch recombination in CH12F3 cells, suggesting that the three stimulations up-regulate different steps of switch recombination in isotype-committed B cells such as CH12F3 cells. Stimulation of CH12F3 cells by IL-4 or TGF-beta, but not by CD40L, induced transient but complete methylation of the I alpha region. TGF-beta and CD40L, but not IL-4, increased the amounts of germline alpha transcripts. We found that the extents of methylation and the amounts of germline transcripts do not necessarily correlate with the efficiency of recombination in induced CH12F3 cells. These results led to the proposal that switch recombination can be separated into at least two phases, i.e. commitment and recombination. The roles of IL-4, TGF-beta and CD40L in the two phases are discussed.
203 citations
TL;DR: The combined inactivation of Tnf and Ltalpha confirms their essential role in the normal development and function of the immune system.
Abstract: To investigate the roles of tumor necrosis factor (TNF) and lymphotoxin (LT)-alpha in the development and function of the immune system, the Tnf and Ltalpha genes were simultaneously inactivated in mice by homologous recombination. These mutant mice are highly susceptible to Listeria monocytogenes infection and resistant to endotoxic shock induced by the combined administration of D-galactosamine (D-GaIN) and lipopolysaccharide (LPS). Their splenic microarchitecture is disorganized, characterized by the loss of the clearly defined marginal zone, ill defined T and B cell areas, and absence of MAdCAM-1 and reduced ICAM-1, VCAM-1 and Mac-1 expression. They are devoid of peripheral lymph nodes and Peyer's patches, and show a strong reduction of IgA+ plasma cells in the intestinal lamina propria. The alymphoplasia is accompanied by a marked B lymphocytosis and reduced basal lg levels. Ig depositions in the renal glomerulus and a strong up-regulation of MHC class I antigen expression on endothelial cells of different tissues are observed. The primary humoral immune response towards sheep red blood cells reveals a defective IgG isotype switch, while that against vesicular stomatitis virus is normal. The cytotoxic T cell responses are attenuated, although still effective, against vaccinia, lymphocytic choriomeningitis virus (LCMV-ARM) and LCMV-WE. In conclusion, the combined inactivation of Tnf and Ltalpha confirms their essential role in the normal development and function of the immune system.
196 citations
TL;DR: It is demonstrated that IL-4 up-regulates Fc epsilon RI in cultured human mast cells, which are grown from cord blood mononuclear cells in the presence of stem cell factor and IL-6, and usually lack Fc EpsilonRI expression.
Abstract: The high-affinity IgE receptor (Fc epsilon RI) is necessary for the induction of IgE-mediated allergic reactions. Cross-linking of Fc epsilon RI expressed on mast cells causes the release of various inflammatory mediators, which trigger allergic reactions. Recently, mast cells lacking Fc epsilon RI have been observed in vivo and in vitro, suggesting the presence of regulational mechanisms in the induction of Fc epsilon RI. In this report, we demonstrate that IL-4 up-regulates Fc epsilon RI in cultured human mast cells, which are grown from cord blood mononuclear cells in the presence of stem cell factor and IL-6, and usually lack Fc epsilon RI expression. At the protein level, the induction of Fc epsilon RI was observed by flow cytometric analysis and the induced Fc epsilon RI expression was stable for at least 23 days in the presence of IL-4. Consistently, Northern blot analysis demonstrated the increase of alpha chain mRNA of Fc epsilon RI and revealed that the up-regulation of Fc epsilon RI occurred at the transcriptional level. Fc epsilon RI induced by IL-4 was functional and caused histamine and beta-hexosaminidase release from mast cells upon cross-linking. Taken together, these results indicate a new role of IL-4 in allergic reactions, besides the stimulation of IgE production.
186 citations
TL;DR: The results establish the importance of both class I- and class II-restricted T cells in immune control of circulating blood stages and intracellular states of T. cruzi and reinforces the relationship between tissue parasite load and the severity of the inflammatory lesions in chronically infected animals.
Abstract: The role of T cell population in immune control of Trypanosoma cruzi infection and subsequent development of disease has been examined using gene knockout mice deficient in the expression of either or both class I and Class II MHC. Mice deficient in either class I- or class II-restricted T cell populations show a striking similarity in their mortality rate, parasite load and tissue inflammatory response following infection with the Brazil strain of T. cruzi. In both cases all animals died during the acute phase of the infection with high parasitemias and high parasite loads in the heart and skeletal muscle, but with reduced tissue inflammatory response. Mice deficient in both class I and class II MHC expression demonstrated even higher numbers of circulating and tissue parasites, essentially non-existent tissue inflammatory responses, and succumbed to infection earlier than single-deficient mice. MHC class I-deficient mice which survive into the chronic phase following infection with the M/78 or M/80 clones of T. cruzi have both relatively higher tissue parasite loads and more extensive and severe inflammatory responses than control immunocompetent mice. Immunologically, the acute infection in the double-deficient mice was accompanied by a marked increase in CD4(-)CD8(-)alphabetaTCR+ cells in the spleen. Surprisingly, both class I- and class II-deficient mice produce detectable but sub-normal levels of anti-parasite antibodies while double-deficient mice produced little to no detectable anti-parasite antibody. These results establish the importance of both class I- and class II-restricted T cells in immune control of circulating blood stages and intracellular states of T. cruzi. In addition, this work reinforces the relationship between tissue parasite load and the severity of the inflammatory lesions in chronically infected animals.
171 citations
TL;DR: The roles of CD28 and CTLA-4 in the response of murine T cells to the superantigen staphylococcal enterotoxin B are examined to conclude that the SEB response is significantly augmented by CD28-derived signaling and this in turn may be attenuated by signals through CTLA.
Abstract: Co-stimulation via the CD28/CTLA-4 system appears critical for T cell proliferation to peptide antigens presented in association with MHC. In this study, we examine the roles of CD28 and CTLA-4 in the response of murine T cells to the superantigen staphylococcal enterotoxin B (SEB). In vitro, antibodies against B7-1/B7-2 or Fab fragments of anti-CD28 antibodies significantly inhibit the response of splenocytes to SEB. Conversely, Fab fragments of anti-CTLA-4 antibodies augment the proliferative response. Further, addition of blocking antibodies directed against B7-1/B7-2 augment proliferation co-stimulated by intact anti-CD28 antibodies. These data support the hypothesis that CD28 and CTLA-4 exert opposing effects upon early T cell activation. In vivo, intact anti-CD28 antibodies and non-stimulatory Fab fragments of anti-CD28 appear to have similar inhibitory effects upon the expansion of V beta 8+ T cells. In contrast, both intact and Fab fragments of anti-CTLA-4 appear to amplify this expansion. We conclude that the SEB response is significantly augmented by CD28-derived signaling and this in turn may be attenuated by signals through CTLA-4.
156 citations
TL;DR: It is reported that blocking Thp/B7-1 or B7-2 interactions in a primary response differentially affects the cytokine profile observed in a secondary stimulation, even in the absence of additional anti-B7 antibody.
Abstract: The CD28/CTLA-4 ligands, B7-1 (CD80) and B7-2 (CD86), provide a co-stimulatory signal necessary for optimal T cell activation. We have examined the effect of blocking B7-1 and B7-2 in an in vitro system using ovalbumin-specific T cells from alpha beta TCR-transgenic mice. This system allowed us to examine the interaction of B7 co-stimulators on physiologic antigen-presenting cells (APC) with antigen-specific T helper precursor (ThP) cells. We report that blocking Thp/B7-1 or B7-2 interactions in a primary response differentially affects the cytokine profile observed in a secondary stimulation, even in the absence of additional anti-B7 antibody. Engagement of B7-2 in the primary stimulation was found to be essential for production of the Th2 cytokine, IL-4, but not the Th1 cytokines, IL-2 and IFN-gamma, in a secondary stimulation. Conversely, inclusion of the anti-B7-1 mAb in cultures using highly purified naive T cells increased levels of IL-4 and significantly depressed levels of IFN-gamma, upon re-stimulation. The effect of the anti-B7-2 mAb in reducing IL-4 production could be overcome by the addition of recombinant IL-4 in the primary stimulation. The effects of the anti-B7-2 mAb appear to be due to blocking and not cross-linking, as F(ab) fragments mimicked the intact antibody. Taken together, our data demonstrate that the interaction between Thp and B7-2 favors the development of Th2 cells.
143 citations
TL;DR: These findings address the mechanisms underlying peripheral T cell tolerance following i.n. administration of a high dose of immunogenic peptide and have implications for understanding the consequences of peptide immunothearapy.
Abstract: We have previously demonstrated that intranasal (l.n.) administration of an immunodomlnant peptide (p1 111-139) derived from the house dust mite (HDM) allergen Der p 1 Inhibits antigenspecific CD4+ T cell responses in H-2b mice. Here we report that i.n. peptide induced a rapid but transient activation of MHC class II restricted CD4+ T cells that peaked 4 days after peptide treatment and was of similar magnitude to that induced by parenteral immunization with antigen In adjuvant. During the early phase of the response lymph node and splenic T cells secreted a range of lymphoklnes when re-stlmulated In vitro with p1 111-139; however, by day 14 IL-2 and IFN-y secretion by T cells were down-regulated. Mice deficient in CD8+ T cells became tolerant by l.n. treatment with peptide, suggesting that CD8+ T cells are not involved In down-regulating the CD4+ T cell response. Rechallenging mice with a single dose of p1 111-139 21 days after the initial treatment elicited a further transient T cell response, which was subsequently down-regulated over time. Although the i.n. peptide induced a strong transient CD4+ T cell response, only low levels of peptlde-speclfic antibodies were detected either after the initial or subsequent l.n. exposures to p1 111-139. Our findings address the mechanisms underlying peripheral T cell tolerance following l.n. administration of a high dose of Immunogenic peptide and have Implications for understanding the consequences of peptide immunotherapy.
138 citations
TL;DR: It is concluded that under the conditions tested, peptide immunotherapy did not reduce immediate- or late-phase skin reactivity to cat extract containing Fel d 1 or modify cat antigen-specific cytokine production significantly.
Abstract: We tested peptide immunotherapy in cat-allergic humans, using a formulation of two synthetic peptides, IPC-1 and IPC-2, each of which is 27 amino acids long and contains T cell-reactive regions of Fel d 1, the major cat allergen. In this exploratory, randomized, double-blind, parallel-group study, 42 subjects received s.c. injections of treatment peptides 250 |xg or placebo weekly for four consecutive weeks. Changes in immediate- and late-phase skin test reactivity, and in antigen-driven cytokine synthesis were assessed. Epicutaneous (end-point titration) and intradermal tests were performed with cat extract (ALK SQ Cat Hair) containing Fel d 1, before the first injection, then 2,6 and 24 weeks after the fourth and last injection of peptides or placebo. IL-4, IL-10 and IFN-y expression by circulating peripheral blood mononuclear cells (PBMC) in response to cat extract was measured using short-term bulk culture of PBMC and short-term limiting dilution analysis. Subjects who received peptide immunotherapy did not tolerate significantly more cat extract containing Fel d 1 in the skin tests 2,6 or 24 weeks after the last injection than they did at baseline, and their late-phase responses did not decrease significantly compared to baseline. Substantial IL-4, IL-10 and IFN-y responses were observed following primary culture of cat antigenstimulated PBMC; however, the intensity of cytokine synthesis and the IFN-y:IL-4 ratio were unchanged in peptide- and placebo-treated groups 6 and 24 weeks after the last injection. A few hours after the injections, subjects receiving peptides reported more allergic rhinitis and asthma symptoms and more pruritus than those receiving placebo. We conclude that under the conditions tested, peptide immunotherapy did not reduce immediate- or late-phase skin reactivity to cat extract containing Fel d 1 or modify cat antigen-specific cytokine production significantly.
TL;DR: Strategies that supplement the stromal environment may enhance B lymphopoiesis in aged animals and reduce the ability of pro-B cells to proliferate in the presence of Stromal cells was reduced by 24 months of age.
Abstract: Although it is reported that B lymphopoiesis declines with age, the precursor stage(s) affected and the age of onset are ambiguous. Each progressive phase of B cell differentiation has distinct requirements; therefore, precise identification of the stage(s) that decline would yield insight into the age-related mechanisms affecting humoral immunity. We analysed the composition of B lineage cells of mice 1, 4, 12 and 24 months of age using flow cytometry. Numbers of prepro-B and pro-B cells were unchanged, and a profound decrease occurred only in the numbers of pre-B cells. This decrease occurred in two distinct phases: between 1 and 4 months and between 12 and 24 months. Notably, the numbers of newly formed B cells did not decline in parallel, suggesting that mechanisms are established to overcome the deficiency of pre-B cells. Since the age-related changes are limited to the pre-B cell stage, we hypothesized that the impairment acts at the pro-B to pre-B transition. We therefore evaluated whether the pro-B cells or the supporting stromal cells, which are necessary for normal progression of this stage, changed with age. The ability of pro-B cells to proliferate in the presence of stromal cells was reduced by 24 months of age, as was the ability of the stromal cells to support pro-B cell proliferation. In contrast, the ability to mature into IgM+ cells was unchanged. Thus, strategies that supplement the stromal environment may enhance B lymphopoiesis in aged animals.
TL;DR: The data clearly show that IL-4 is not necessary for the hepatic granuloma formation which occurs during experimental schistosomiasis.
Abstract: Immunopathology and immune responses to Schistosoma mansoni were examined in IL-4 -/- mice. IL-5 and IL-10 production by lymphoid cells stimulated with soluble egg antigen (SEA), peripheral eosinophilia and serum levels of soluble IL-4 receptor but not IgE were all significantly elevated over background normal levels in IL-4 -/- mice as a result of infection. Additionally, IL-10 and IL-5 in addition to IL-2 and IFN-gamma transcripts were equally evident in diseased liver tissue from infected IL-4 -/- and wild-type mice. Nevertheless, analysis of antigen-stimulated IL-2, IL-4, IL-5, IL-10 and IFN-gamma production by lymphoid organ cells from infected or egg-injected IL-4 -/- mice revealed a more Th1-like pattern of cytokine production (IFN-gamma > IL-5) than in (wild-type) mice in which a stronger type 2 response to SEA was detectable (IL-4, IL-5 > IFN-gamma). Despite this, at 8 and 16 weeks after infection, liver pathology, as indicated by the size, cellularity, cellular composition and collagen content of granulomas, was similar in IL-4 -/- and wild-type animals. As in wild-type animals, granuloma size at week 16 was smaller than at week 8, indicating that modulation had occurred in the absence of IL-4. Differences in pathology were seen only when eggs were experimentally embolized to the lungs, in which case IL-4 -/- mice made smaller granulomatous responses than did wild-type animals. These data clearly show that IL-4 is not necessary for the hepatic granuloma formation which occurs during experimental schistosomiasis.
TL;DR: The role of transcription factor NF-kappa B in the induction of cytokines and ICAM-1 upon stimulation with proinflammatory cytokines, IL-1 and tumor necrosis factor (TNF)-alpha was investigated in primary synovial fibroblasts obtained from patients with rheumatoid arthritis, highlighting the possibility of a novel therapeutic strategy.
Abstract: The role of transcription factor NF-kappa B in the induction of cytokines and ICAM-1 upon stimulation with proinflammatory cytokines, IL-1 and tumor necrosis factor (TNF)-alpha was investigated in primary synovial fibroblasts obtained from patients with rheumatoid arthritis (RA). Nuclear translocation of NF-kappa B was demonstrated after 30 min of treatment with IL-1 or TNF-alpha. Thereafter, the production of several cytokines including granulocyte macrophage colony stimulating factor, IL-6 and IL-8, that are known to be abundantly produced in the synovial cavity of RA patients, was greatly augmented. Similarly, cell surface expression of ICAM-1 was induced by the IL-1 or TNF-alpha treatment. Since expression of these genes is induced in rheumatoid synovial tissue, this experimental system is considered to represent the in vivo situation of RA pathophysiology. Using this cell culture system we attempted to modulate the intracellular signaling cascade for NF-kappa B activation and examined the effects of N-acetyl-L-cysteine (NAC) and acetylsalicylic acid (aspirin), which were previously reported to inhibit NF-kappa B activation. Pretreatment of the primary synovial fibroblasts with NAC inhibited nuclear translocation of NF-kappa B. Subsequently, the induction of these cytokines and ICAM-1 was considerably suppressed. On the other hand, pretreatment with aspirin blocked these phenomena only partially. These observations indicate the pivotal role of NF-kappa B in RA pathogenesis thus highlighting the possibility of a novel therapeutic strategy.
TL;DR: It is concluded that CsA and FK506 block activation-induced apoptosis in T cell hybridomas predominantly by interfering with activation signals leading to FasL expression and that the regulation of the expression of Fas and FasL on activated T cells is differentially controlled.
Abstract: We have previously reported that activation of murine T cell hybridomas leads to expression of Fas (CD95) and Its ligand (FasL) which subsequently interact, even on the same cell, leading to apoptotlc cell death. Since the immunosuppressive drugs cyclosporin A (CsA) and FK506 block activation-Induced apoptosis in T cell hybridomas, we examined whether such compounds affect cell death by interfering with expression of Fas, FasL or both, or whether they block Fas signal transduction. We have found that CsA- and FK5O6-treated cells did not exhibit transcription of FasL mRNA after activation and were lacking functional FasL protein on their surface as determined by staining and the ability to induce apoptosis In Fas+ target cells. In contrast, no inhibition of the elevated Fas mRNA expression was observed in cells activated in the presence of CsA or FK506. Surprisingly, however, cell surface Fas levels were consistently lower on cells activated in the presence of Immunosuppres sive drugs than on activated cells, suggesting that Fas expression is regulated at several levels. Nevertheless, cells activated In the presence of CsA or FK5O6 underwent apoptosis upon treatment with anti-Fas antibody, while unactivated cells did not Furthermore, CsA and FK506 do not interfere with Fas signaling since anti-Fas induced apoptosis in Fas+ target cells was unaffected by these drugs. We therefore conclude that CsA and FK506 block activation-induced apoptosis in T cell hybridomas predominantly by interfering with activation signals leading to FasL expression and, further, that the regulation of the expression of Fas and FasL on activated T cells is differentially controlled.
TL;DR: The possibility that infection with filarial nematodes causes a reduction in the co-stimulatory or antigen-presenting capacity of macrophages resulting in a failure to activate specific T cells is tested and may provide a murine model for the immune suppression seen in lymphatic filariasis.
Abstract: Specific T cell hyporesponsiveness and depressed antibody production is a key feature of human infection with the filarial nematodes, Brugia malayi and Wuchereria bancrofti. Despite this immune suppression, responses indicative of Th2 subset activation are present, including unusually high levels of specific IgG4. We tested the possibility that infection with filarial nematodes causes a reduction in the co-stimulatory or antigen-presenting capacity of macrophages resulting in a failure to activate specific T cells. Adherent peritoneal exudate cells (PEC) from mice implanted with adult B. Malayi were used to present antigen to the conalbumin-specific T cell clone, D10.G4. Proliferation of the D10 cells at even background levels was completely blocked by the presence of implant-derived adherent PEC. However, cytokine production by these cells in response to antigen was intact, and thus PEC from implanted mice are capable of functionally processing and presenting antigen. The elicitation of a suppressive cell population was specific for live adults as cells from mice implanted with dead adult parasites effectively stimulated D10 proliferation. The block in cellular proliferation is not due to the production of factors typically associated with macrophage suppression such as nitric oxide, prostaglandins or catalase. These observations are consistent with the T cell hyporesponsiveness seen in human cases of patent Brugia infection and may provide a murine model for the immune suppression seen in lymphatic filariasis.
TL;DR: A major role for SCF is demonstrated in the generation of intestinal mastocytosis and the host protective immune response following parasitic infection and the reduction in the mast cell response and worm expulsion observed after SCF neutralization were reversible following cessation of monoclonal treatment.
Abstract: In common with many intestinal nematode infections, Trichinella spiralis infections in mice are associated with a pronounced intestinal mast cell hyperplasia. The expulsion of the parasite from the gut is temporally associated with intestinal mastocytosis and mast cell function reflected by the secretion of mast cell protease into tissue and serum. In vivo, mucosal mast cell production is highly dependent upon T cell-derived cytokines including IL-3 and IL-4. We present data here to show that intestinal mast cell hyperplasia induced by helminth infection is also dependent upon the production of stem cell factor (SCF). Neutralization of SCF by anti-SCF or anti-SCF receptor mAb completely abrogated the mast cell hyperplasia generated by T. spiralis infection. Moreover, worm expulsion was dramatically delayed in treated mice and a reduced intestinal eosinophilia was observed. These effects did not appear to be mediated through alteration of Th cell responses and the parasite-specific serum antibody response was not affected. The reduction in the mast cell response and worm expulsion observed after SCF neutralization were reversible following cessation of monoclonal treatment. The data presented here clearly demonstrate a major role for SCF in the generation of intestinal mastocytosis and the host protective immune response following parasitic infection.
TL;DR: Results strongly indicated that endogenous IL-12, which stimulates Th1-dominant cellular immunity and IFN-gamma production, may be an essential cytokine on the course of T cell-dependent liver injury.
Abstract: Intravenous injection of Propionibacterium acnes and lipopolysaccharide (LPS) with a 7 day interval caused CD4+ T cell-dependent severe liver injury in the C57BL/6 (H-2b) mouse strain. In contrast, BALB/c (H-2d) mice were resistant to P. acnes and LPS-induced liver injury. The different susceptibilities of the two mouse strains to liver injury appeared to be closely correlated with their different abilities to produce IFN-gamma after P. acnes priming. Namely, the sensitive C57BL/6 mouse strain produced a significant level of IFN-gamma 7-10 days after P. acnes injection, whereas no significant amount of serum IFN-gamma was detected in the resistant BALB/c mouse strain. The important role of IFN-gamma in liver injury was demonstrated from the finding that in vivo administration of anti-IFN-gamma mAb abrogated P. acnes and LPS-induced liver injury in C57BL/6 mice. Moreover, it was demonstrated that in vivo administration of recombinant IL-12, a key cytokine for the induction of IFN-gamma, into mice induced P. acnes and LPS-induced liver injury in the resistant BALB/c mouse strain. Conversely, in vivo administration of anti-IL-12 mAb blocked the development of liver injury in the sensitive C57BL/6 mouse strain. Moreover, it was demonstrated that the failure of the induction of liver injury in BALB/c mice appeared to be derived from the lack of expression of IL-12 at the local site of liver in P. acnes-primed mice. These results strongly indicated that endogenous IL-12, which stimulates Th1-dominant cellular immunity and IFN-gamma production, may be an essential cytokine on the course of T cell-dependent liver injury.
TL;DR: The results indicate that peptides binding to DQ2 have anchor residues in relative positions 4, 7 and (P4, P7 and P9).
Abstract: To understand the rules determining peptide binding to the celiac disease and type 1 diabetes mellitus associated HLA-DQ2 molecule, we have studies in detail the binding of a peptide OVA 258-276Y (IINFEKLTEWTSSNVMEERY) which exhibits strong binding to DQ2. First we tested a set of N- and C-terminal truncated variants, and found the core binding region to comprise residues 267-276Y. Single alanine substitution analysis of the OVA 267-276Y peptide revealed that replacements of V272, E275 and the C-terminal Y had negative effects whereas the substitution of N271 had a positive effect. A polyalanine analogue of the OVA 267-276Y peptide with V272, E275 and a C-terminal Y bound at least as well as the original peptide. A variant peptide with a deletion of R276 displayed decreased binding, suggesting that the anchor residues were out of frame in this analogue. To further characterize the residues playing a role in the binding of the OVA 267-276Y peptide to DQ2 we tested the binding of several analogues with substitutions for V272, E275 and the C-terminal Y residue. Our results indicate that peptides binding to DQ2 have anchor residues in relative positions 4, 7 and (P4, P7 and P9). Residues with negatively charged or hydrophobic aliphatic but not positively charged side chains are preferred in P4 and P7, whereas residues with bulky hydrophobic side chains are preferred in P9.
TL;DR: It is demonstrated that the Mart-1 peptide-specific cytotoxic T lymphocytes lyse melanocytes in the eye of patients with VKH disease or SO, suggesting that these diseases are autoimmune diseases directed toward the MART-1 antigen in HLA-A2(+) patients.
Abstract: The MART-1/Melan-A melanoma antigen recognized by the majority of HLA-A2-restricted tumor-infiltrating lymphocytes is a self antigen expressed on melanocytes and the retina. We have investigated whether Vogt-Koyanagi-Harada (VKH) disease and sympathetic ophthalmia (SO), systemic inflammatory disorders affecting various organs containing melanocytes, are autoimmune diseases directed toward the MART-1 antigen. In two of three patients with VKH disease and one patient with SO, CD8(+) T cell clones (TCC) form intraocular fluid of HLA-A2(+) patients lysed T2 cells when pulsed with a HLA-A2-binding MART-1 peptide, but not a HLA-A2-binding pMel-17 or tyrosinase peptide, in a HLA-A2-restricted manner. In addition, Th, TCC recognizing a HLA-A2-binding MART-1 peptide were also established from peripheral blood mononuclear cells of a patient with VKH disease. In contrast, either CD4(+) TCC from these patients or CD8(+) TCC from the intraocular fluid of HLA-A2(+) patients with uveitis associated with Behcet's disease or HTLV-1 uveitis did not show this cytotoxicity. The results demonstrate that the MART-1 peptide-specific cytotoxic T lymphocytes lyse melanocytes in the eye of patients with VKH disease or SO, suggesting that these diseases are autoimmune diseases directed toward the MART-1 antigen in HLA-A2(+) patients.
TL;DR: The role of HLA and non-HLA genes in the antibody response to two merozoite surface antigens and a sexual stage antigen of Plasmodium falciparum are investigated, and it is concluded that host genotype is not a major determinant of responsiveness.
Abstract: Malaria Infection Induces the production of serum antibodies to a variety of malaria antigens but the prevalence of antibodies to any particular antigen Is typically much less than 100%. It has been assumed that non-responsiveness to defined antigens In malaria Immune subjects is due to HLAmedlated restriction of the immune response. In this study we have investigated the role of HLA and non-HLA genes in the antibody response to two merozolte surface antigens (MSP1 and MSP2) and a sexual stage antigen (Pfs260/230) of Plasmodium falclparum, and conclude that host genotype is not a major determinant of responsivenes s. Although antibody levels vary in . accordance with seasonal variations In malaria transmission In semi-immune children, antibody levels remain stable In clinically immune adults. Antigen recognition Is selective with Individual donors showing consistent high tttre responses to some antigens/epitopes whilst consistently falling to recognize adjacent reglons/epltopes from the same protein. An alternative explanation, consistent with the data presented here, Is that selective antibody responses to malaria antigens in immune individuals result from a process akin to clonal Imprinting (original antigenlc sin).
TL;DR: It is demonstrated for the first time in a protozoan parasite that activation induced CD4(+) T cell unresponsiveness occurs during acute T. gondii infection in mice, and may be important in immune down-regulation and parasite persistence in the infected host.
Abstract: Infection of mice with Toxoplasma gondii has been shown to induce a transient state of immune down-regulation. Earlier reports have demonstrated the role of cytokines, in particular IL-10, in this host response. Here evidence is presented that T. gondii, a major opportunistic pathogen of the newborn and those with AIDS, is able to induce CD4(+) T cell apoptosis in the infected murine host. We have examined the changes in the CD4(+) T cell population that occur during acute infection in an experimental mouse model. Seventy-six percent of the CD4(+) T cell population increased in volume by day 7 post-infection and expressed T cell maturation markers (CD44(hi), IL-2Rhi, Mel-14(lo)). Further noted was a clonal activation of several CD4(+) T cell to mitogen or parasite antigen stimulation was observed, in particular Vbeta5 T cells. Addition of rIL-2 partially restored the CD4(+) T cell proliferative response in vitro. The T cell activation marker CTLA-4 could not be detected and the co-stimulatory molecule, CD28, was down-regulated. Electrophoretic and morphologic analysis of these cells post-culture demonstrated a DNA fragmentation pattern and cell death consistent with apoptosis. These studies demonstrate for the first time in a protozoan parasite that activation induced CD4(+) T cell unresponsiveness occurs during acute T. gondii infection in mice, and may be important in immune down-regulation and parasite persistence in the infected host.
TL;DR: In this paper, the anti-tumor effects of IL-12 were associated with enhanced induction of IFN-gamma because these effects were abrogated by pretreatment of hosts with anti-IFNgamma antibody.
Abstract: The present study investigates the molecular mechanisms by which IFN-gamma produced as a result of in vivo IL-12 administration exerts its anti-tumor effects. rIL-12 was administered three or five times into mice bearing CSA1M fibrosarcoma, OV-HM ovarian carcinoma or MCH-1-A1 fibrosarcoma. This regimen induced complete regression of CSA1M and OV-HM tumors but only transient growth inhibition of MCH-1-A1 tumors. The anti-tumor effects of IL-12 were associated with enhanced induction of IFN-gamma because these effects were abrogated by pretreatment of hosts with anti-IFN-gamma antibody. Exposure in vitro of the three types of tumor cells to rRFN-gamma resulted in moderate to potent inhibition of tumor cell growth. IFN-gamma stimulated the expression of mRNAs for an inducible type of NO synthase (iNOS) in CSA1M cells and indoleamine 2,3-dioxygenase (IDO), an enzyme capable of degrading tryptophan, in OH-HM cells, but induced only marginal levels of these mRNAs in MCH-1-A1 cells. In association with iNOS gene expression, IFN-gamma-stimulated CSA1M cells produced a large amount of NO which functioned to inhibit their own growth in vitro. Although OV-HM and MCH-1A1 cells did not produce NO, they also exhibited NO susceptibility. Whereas the tumor masses from IL-12-treated CSA1M-bearing or OV-HM-bearing mice induced higher levels of iNOS (for CSA1M) or IDO and iNOS (for OV-HM) mRNAs, the MCH-1-A1 tumor mass expressed lower levels of iNOS mRNA alone. Moreover, massive infiltration of CD4(+) and CD8(+) T cells and Mac-1(+) cells was seen only in the CSA1M and OV-HM tumors. Thus, these results indicate that IFN-gamma produced after IL-12 treatment induces the expression of various genes with potential to modulate tumor cell growth by acting directly on tumor cells or stimulating tumor-infiltrating lymphoid cells and that the effectiveness of IL-12 therapy is associated with the operation of these mechanisms.
TL;DR: Cross-linking experiments indicate that the purified soluble molecule binds a 120 kDa molecule expressed by monocytoid cells and identified as a candidate ligand for human mCD38, which displays a M(r) of 39 kDa.
Abstract: Human CD38 is a transmembrane glycoprotein involved in lymphocyte activation and adhesion to endothelium. The ectocellular domain of the molecule possesses properties of a bifunctional enzyme catalyzing both the synthesis from NAD+ and the hydrolysis of the calcium-releasing metabolite cyclic ADP-ribose (cADPR). Surface expression of CD38 (mCD38) is rapidly and almost completely down-modulated upon ligation by specific mAb in cells from different lineages. The data presented here also show that, in addition to the existence of a mCD38, a soluble form of CD38 (sCD38) is detectable in the cell culture supernatant of allo-activated T lymphocytes and of several tumor cell lines. sCD38 is also present in vivo and is assayable in normal (fetal serum and amniotic fluid) and pathological (serum and ascites from patients with multiple myeloma, and serum from patients with AIDS) biological fluids. Immunoaffinity chromatography, SDS-PAGE and Western blot analyses with mAb and polyclonal antibodies, along with metabolic labeling, yield a body of data concerning the structure of sCD38, which displays a M(r) of 39 kDa. Native sCD38 maintains the ability to inhibit the binding activity of different anti-CD38 mAb and still catalyzes the synthesis and the hydrolysis of cADPR at the same ratio observed with mCD38. Furthermore, cross-linking experiments indicate that the purified soluble molecule binds a 120 kDa molecule expressed by monocytoid cells and identified as a candidate ligand for human mCD38.
TL;DR: It is suggested that efficient expression of CD40L on naive CD4 cells does require accessory molecules on APC, and the role of these molecules forCD40L induction is not one of co-stimulation but one of adhesion, presumably allowing stronger or more prolonged signals to be generated through the TCR.
Abstract: We have investigated the roles of TCR and accessory co-stimulatory signals in the induction of CD40 ligand (CD40L) on CD4 cells. Using naive T cells form TCR transgenic mice, specific for a peptide of pigeon cytochrome c, we show that in contrast to IL-2 secretion, CD40L expression is regulated primarily by signalling through the TCR, is enhanced by accessory molecule interactions, but co-stimulatory signals play little if any role. CD40L was induced at high levels on naive T cells, peaking at 5 h, by class II MHC+ fibroblast antigen-presenting cells (APC) which expressed either ICAM-1, B7-1 or both molecules, whereas only low levels were induced by fibroblasts which did not express any accessory molecules. Differences in intensity and duration of expression were seen following stimulation with ICAM- and B7-expressing APC, with the presence of ICAM resulting in greater and longer expression, although both molecules together were most efficient. The involvement of co-stimulatory signals delivered from accessory molecules was investigated in systems where there was no effect on TCR signalling from adhesive interactions. Anti-CD3, or antigen-pulsed APC lacking accessory molecules, were used to provide the TCR signal, with co-stimulus from either anti-CD28 or accessory molecule-expressing fibroblasts not presenting antigen. Anti-CD3 in the absence of co-stimuli induced high CD40L expression but no IL-2 production and provision of co-stimulatory signals, although inducing large quantities of IL-2, did not increase CD40L expression. In addition, low CD40L expression induced by antigen presented in the absence of accessory molecules was not enhanced by co-stimulation, although IL-2 was strongly up-regulated. These studies suggest that efficient expression of CD40L on naive CD4 cells does require accessory molecules on APC. However, the role of these molecules for CD40L induction, as opposed to IL-2 secretion, is not one of co-stimulation but one of adhesion, presumably allowing stronger or more prolonged signals to be generated through the TCR. The synergistic role of ICAM and B7 during naive CD4 activation was confirmed using dendritic cells as APC, with nearly complete inhibition of CD40L expression as well as IL-2 secretion being seen when both CTLA-4-Ig and anti-LFA-1 were used to block these molecules.
TL;DR: It is suggested that CD86 plays a critical role in the initiation of primary immune responses in the skin, while CD80 and CD86 are not essential in the effector phase of CH reactions.
Abstract: Contact hypersensitivity (CH) has served as a useful model for investigating the allergen-specific immune responses of T cells and skin-associated antigen-presenting cells. We examined the distinct role between CD80 and CD86 on hapten-induced CH in both an induction and an effector phase. Intraperitoneal injection of mAb against CD86, but not CD80, 2 h before sensitization with epicutaneous application of dinitrofluorobenzene led to an almost complete inhibition of ear swelling, and histologically a marked reduction of edema, inflammatory polymorphonuclear cells and lymphocyte infiltration in the dermis. In contrast, the administration with either anti-CD80 or CD86 mAb 2 h before challenge partially inhibited CH reactions and a combination of both mAb did not improve the inhibitory effect. Although Langerhans cells (LC) expressing MHC class II were observed in both the epidermis and dermis 24 h after primary sensitization, CD86(+) LC were observed only in the subepidermal regions and CD80-bearing cells were not detected. Dendritic cells (DC) expressing both CD80 and CD86 were preferentially observed in the T cell areas of draining lymph nodes 24 h after challenge. The administration of anti-CD86 mAb in the induction phase prominently reduced the up-regulation of CD80 and CD86 on DC in the lymph nodes. The predominant role of CD86 was further supported by a marked reduction in proliferation of lymph node T cells against the sensitized hapten after the anti-CD86 treatment. These results suggest that CD86 plays a critical role in the initiation of primary immune responses in the skin, while CD80 and CD86 are not essential in the effector phase of CH reactions.
TL;DR: It is suggested that continuous oral exposure to a tolerogen may be a biologically relevant strategy to tolerize both Th1- and Th-dependent responses, and extend the potential clinical use of oral tolerance to ailments mediated by Th2 lymphocytes.
Abstract: We established conditions for inducing antigen-specific tolerance in Th2 lymphocytes by means of oral tolerance. Mice were continuously exposed to ovalbumin in their drinking water for a minimal period of 20 days and then immunized against antigen in either complete Freund's adjuvant or Al(OH)3. This feeding regimen tolerized both Th2 and Th1 responses as shown by diminished proliferation, cytokine secretion (IL-4, IL-2 and IFN-gamma) and specific cytokine mRNA expression (IL-4, IL-2 and IFN-gamma) in vitro, as well as by absence of specific antibody production (IgG1, IgG2a, IgG2b and IgE) in vivo. Conditions for generating Th2 lymphocyte tolerance were different from those required to generate tolerance in Th1 lymphocytes: these included extended, continuous exposure to high dosages of antigen, rather than a single or intermittent feeding regimen which was sufficient to induce tolerance in Th1 lymphocytes. These findings suggest that continuous oral exposure to a tolerogen may be a biologically relevant strategy to tolerize both Th1- and Th-dependent responses, and extend the potential clinical use of oral tolerance to ailments mediated by Th2 lymphocytes.
TL;DR: The results strongly suggest that the cytokines IL-4 and IL-13 are both important in modulating endothelial cell function, and may act through a single receptor complex on human endothelial cells that includes the IL- 4R alpha chain but not the gamma c chain.
Abstract: The cytokine IL-4 has unique effects on human endothelial cells, It specifically increases expression of vascular cell adhesion molecule (VCAM)-1 promoting adhesion of lymphocytes but not neutrophils, and causes profound effects on the morphology of endothelial monolayers characterized by formation of cell clusters and the appearance of holes in the cultured monolayer, In this study we show that the effects of IL-13 on human umbilical vein endothelial cells (HUVEC) are indistinguishable from those of IL-4, Both cytokines induce the same morphological changes in cultured HUVEC monolayers which are distinct from any other cytokine, In addition, IL-13 and IL-4 stimulate comparable levels of VCAM-1 expression with similar time kinetics, but at doses 10-fold less than those required for B cell activation and proliferation. Using a combination of mutant IL-4 antagonists and mAb to the IL-4R alpha chain (CD124), we show that expression of IL-4R alpha is essential for HUVEC responses to both IL-4 and IL-13, consistent with this receptor subunit being a component of the receptors for both cytokines. In contrast, the common gamma chain (gamma c), which is a component of the classical IL-4 receptor, was not detected on endothelial cells by flow cytometry or immunogold histochemistry. In addition, RT-PCR showed extremely low or absent gamma c mRNA, consistent with the absence of detectable surface protein. These results strongly suggest that the cytokines IL-4 and IL-13 are both important in modulating endothelial cell function, and may act through a single receptor complex on human endothelial cells that includes the IL-4R alpha chain but not the gamma c chain.
TL;DR: The identified 31 candidate epitopes in the HCV that have the potential to be recognized by either HLA-A1, A2.1-, A3, A11- or A24-restricted cytotoxic T lymphocytes (CTL) illustrate the value of this approach for the development of virus-specific, peptide-based vaccines.
Abstract: We have focused on conserved regions of the hepatitis C Virus (HCV) genome to identify viral peptides that contain HLA class I binding motifs and bind with high affinity to the corresponding purified HLA molecules. Accordingly, we have identified 31 candidate epitopes in the HCV that have the potential to be recognized by either HLA-A1, A2.1-, A3, A11- or A24-restricted cytotoxic T lymphocytes (CTL). Twelve conserved peptides that bind HLA-A2.1 with high or intermediate affinity were tested for immunogenicity in vitro in human primary CTL cultures and in vivo by direct immunization of HLA-A2.1/Kb transgenic mice. Six HLA-A2.1-restricted CTL epitopes were immunogenic in both systems. At least three of these peptide epitopes were endogenously processed and presented for CTL recognition. Overall, these data illustrate the value of this approach for the development of virus-specific, peptide-based vaccines.
TL;DR: It is shown that cross-linking GM1 by EtxB causes a differential effect on mature CD4(+) and CD8(+) T cells from lymph node cultures proliferating in response to an unrelated antigen, ovalbumin, which provides an insight into the potent immunogenicity and immunomodulatory properties of E. coli enterotoxins.
Abstract: Gangliosides are glycosphingolipids found ubiquitously on the surface of mammalian cells. They contain a ceramide tail that is inserted into the membrane and exposed carbohydrate and sialic acid moieties. The non-toxic B subunit oligomer (EtxB) of Escherichia coli heat-labile enterotoxin (Etx) is a potent immunogen in vivo and has profound modulatory effects on EtxB-primed lymphocytes in vitro, properties which are dependent on its ability to bind to GM1 ganglioside receptors. Here, it is shown that cross-linking GM1 by EtxB causes a differential effect on mature CD4(+) and CD8(+) T cells from lymph node cultures proliferating in response to an unrelated antigen, ovalbumin. Addition of EtxB to such cultures led to the complete depletion of CD8(+) T cells compared with enhanced activation of CD4(+) cells [as measured by expression of CD25 (IL-2Ralpha)]. By contrast, addition of a mutant EtxB, EtxB(G33D), which does not bind to GM1, failed to trigger CD8(+) T cell depletion. When EtxB was added to isolated non-immune CD8(+) lymphocytes rapid (12-18 h) alterations in nuclear morphology and the appearance of sub-G0/G1 levels of DNA were induced; properties which are characteristic of cells undergoing apoptosis. EtxB(G33D) failed to trigger apoptosis, indicating that the induction of the apoptotic signal was dependent on the binding of GM1. These findings provide an insight into the potent immunogenicity and immunomodulatory properties of E. coli enterotoxins as well as heralding a novel method for the selective induction of apoptosis in mature CD8(+) T lymphocytes.