Showing papers in "Journal of Experimental Medicine in 2000"
TL;DR: It is reported here that the ligand of PD-1 (PD-L1), an immunoinhibitory receptor expressed by activated T cells, B cells, and myeloid cells, is a member of the B7 gene family.
Abstract: PD-1 is an immunoinhibitory receptor expressed by activated T cells, B cells, and myeloid cells. Mice deficient in PD-1 exhibit a breakdown of peripheral tolerance and demonstrate multiple autoimmune features. We report here that the ligand of PD-1 (PD-L1) is a member of the B7 gene family. Engagement of PD-1 by PD-L1 leads to the inhibition of T cell receptor-mediated lymphocyte proliferation and cytokine secretion. In addition, PD-1 signaling can inhibit at least suboptimal levels of CD28-mediated costimulation. PD-L1 is expressed by antigen-presenting cells, including human peripheral blood monocytes stimulated with interferon gamma, and activated human and murine dendritic cells. In addition, PD-L1 is expressed in nonlymphoid tissues such as heart and lung. The relative levels of inhibitory PD-L1 and costimulatory B7-1/B7-2 signals on antigen-presenting cells may determine the extent of T cell activation and consequently the threshold between tolerance and autoimmunity. PD-L1 expression on nonlymphoid tissues and its potential interaction with PD-1 may subsequently determine the extent of immune responses at sites of inflammation.
4,633 citations
TL;DR: It is reported that the Treg cells that control intestinal inflammation express the same phenotype (CD25+CD45RBlowCD4+) as those that control autoimmunity, suggesting that Treg cell function contributes to the immune suppression characteristic of CTLA-4 signaling.
Abstract: It is now clear that functionally specialized regulatory T (Treg) cells exist as part of the normal immune repertoire, preventing the development of pathogenic responses to both self- and intestinal antigens. Here, we report that the Treg cells that control intestinal inflammation express the same phenotype (CD25+CD45RBlowCD4+) as those that control autoimmunity. Previous studies have failed to identify how CD25+ Treg cells function in vivo. Our studies reveal that the immune-suppressive function of these cells in vivo is dependent on signaling via the negative regulator of T cell activation cytotoxic T lymphocyte–associated antigen 4 (CTLA-4), as well as secretion of the immune-suppressive cytokine transforming growth factor β. Strikingly, constitutive expression of CTLA-4 among CD4+ cells was restricted primarily to Treg cells, suggesting that CTLA-4 expression by these cells is involved in their immune-suppressive function. These findings raise the possibility that Treg cell function contributes to the immune suppression characteristic of CTLA-4 signaling. Identification of costimulatory molecules involved in the function of Treg cells may facilitate further characterization of these cells and development of new therapeutic strategies for the treatment of inflammatory diseases.
2,238 citations
TL;DR: Interference with this role of CTLA-4 suffices to elicit autoimmune disease in otherwise normal animals, presumably through affecting CD25+CD4+ T cell–mediated control of self-reactive T cells.
Abstract: This report shows that cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) plays a key role in T cell–mediated dominant immunologic self-tolerance. In vivo blockade of CTLA-4 for a limited period in normal mice leads to spontaneous development of chronic organ-specific autoimmune diseases, which are immunopathologically similar to human counterparts. In normal naive mice, CTLA-4 is constitutively expressed on CD25+CD4+ T cells, which constitute 5–10% of peripheral CD4+ T cells. When the CD25+CD4+ T cells are stimulated via the T cell receptor in vitro, they potently suppress antigen-specific and polyclonal activation and proliferation of other T cells, including CTLA-4–deficient T cells, and blockade of CTLA-4 abrogates the suppression. CD28-deficient CD25+CD4+ T cells can also suppress normal T cells, indicating that CD28 is dispensable for activation of the regulatory T cells. Thus, the CD25+CD4+ regulatory T cell population engaged in dominant self-tolerance may require CTLA-4 but not CD28 as a costimulatory molecule for its functional activation. Furthermore, interference with this role of CTLA-4 suffices to elicit autoimmune disease in otherwise normal animals, presumably through affecting CD25+CD4+ T cell–mediated control of self-reactive T cells. This unique function of CTLA-4 could be exploited to potentiate T cell–mediated immunoregulation, and thereby to induce immunologic tolerance or to control autoimmunity.
2,215 citations
TL;DR: Investigation of the influence of the maturational state of dendritic cells on priming and differentiation of T cells shows that immature and mature DCs induce different types of T cell responses: inflammatory Th1 cells are induced by maturity DCs, and IL-10–producing T cell regulatory 1–like cells by immature DCs.
Abstract: The functional properties of dendritic cells (DCs) are strictly dependent on their maturational state. To analyze the influence of the maturational state of DCs on priming and differentiation of T cells, immature CD83− and mature CD83+ human DCs were used for stimulation of naive, allogeneic CD4+ T cells. Repetitive stimulation with mature DCs resulted in a strong expansion of alloreactive T cells and the exclusive development of T helper type 1 (Th1) cells. In contrast, after repetitive stimulation with immature DCs the alloreactive T cells showed an irreversibly inhibited proliferation that could not be restored by restimulation with mature DCs or peripheral blood mononuclear cells, or by the addition of interleukin (IL)-2. Only stimulation of T cells with mature DCs resulted in an upregulation of CD154, CD69, and CD70, whereas T cells activated with immature DCs showed an early upregulation of the negative regulator cytotoxic T lymphocyte–associated molecule 4 (CTLA-4). These T cells lost their ability to produce interferon γ, IL-2, or IL-4 after several stimulations with immature DCs and differentiated into nonproliferating, IL-10–producing T cells. Furthermore, in coculture experiments these T cells inhibited the antigen-driven proliferation of Th1 cells in a contact- and dose-dependent, but antigen-nonspecific manner. These data show that immature and mature DCs induce different types of T cell responses: inflammatory Th1 cells are induced by mature DCs, and IL-10–producing T cell regulatory 1–like cells by immature DCs.
1,638 citations
TL;DR: Critical roles for IL-15 in the development of specific lymphoid lineages are revealed and the ability to rescue lymphoid defects inIL-15−/− mice by IL- 15 administration represents a powerful means by which to further elucidate the biological roles of this cytokine.
Abstract: C57BL/6 mice genetically deficient in interleukin 15 (IL-15−/− mice) were generated by gene targeting. IL-15−/− mice displayed marked reductions in numbers of thymic and peripheral natural killer (NK) T cells, memory phenotype CD8+ T cells, and distinct subpopulations of intestinal intraepithelial lymphocytes (IELs). The reduction but not absence of these populations in IL-15−/− mice likely reflects an important role for IL-15 for expansion and/or survival of these cells. IL-15−/− mice lacked NK cells, as assessed by both immunophenotyping and functional criteria, indicating an obligate role for IL-15 in the development and functional maturation of NK cells. Specific defects associated with IL-15 deficiency were reversed by in vivo administration of exogenous IL-15. Despite their immunological defects, IL-15−/− mice remained healthy when maintained under specific pathogen-free conditions. However, IL-15−/− mice are likely to have compromised host defense responses to various pathogens, as they were unable to mount a protective response to challenge with vaccinia virus. These data reveal critical roles for IL-15 in the development of specific lymphoid lineages. Moreover, the ability to rescue lymphoid defects in IL-15−/− mice by IL-15 administration represents a powerful means by which to further elucidate the biological roles of this cytokine.
1,586 citations
TL;DR: In this article, the authors investigated whether exposure to apoptotic or necrotic cells affected dendritic cells' maturation and found that only exposure to the latter induces maturation.
Abstract: Cell death by necrosis is typically associated with inflammation, in contrast to apoptosis. We have identified additional distinctions between the two types of death that occur at the level of dendritic cells (DCs) and which influence the induction of immunity. DCs must undergo changes termed maturation to act as potent antigen-presenting cells. Here, we investigated whether exposure to apoptotic or necrotic cells affected DC maturation. We found that immature DCs efficiently phagocytose a variety of apoptotic and necrotic tumor cells. However, only exposure to the latter induces maturation. The mature DCs express high levels of the DC-restricted markers CD83 and lysosome-associated membrane glycoprotein (DC-LAMP) and the costimulatory molecules CD40 and CD86. Furthermore, they develop into powerful stimulators of both CD4(+) and CD8(+) T cells. Cross-presentation of antigens to CD8(+) T cells occurs after uptake of apoptotic cells. We demonstrate here that optimal cross-presentation of antigens from tumor cells requires two steps: phagocytosis of apoptotic cells by immature DCs, which provides antigenic peptides for major histocompatibility complex class I and class II presentation, and a maturation signal that is delivered by exposure to necrotic tumor cells, their supernatants, or standard maturation stimuli, e.g., monocyte-conditioned medium. Thus, DCs are able to distinguish two types of tumor cell death, with necrosis providing a control that is critical for the initiation of immunity.
1,461 citations
TL;DR: Results indicate that, like other cytokine mediators of endotoxin lethality (e.g., TNF and IL-1), extracellular HMG-1 is a regulator of monocyte proinflammatory cytokine synthesis.
Abstract: Lipopolysaccharide (LPS) is lethal to animals because it activates cytokine release, causing septic shock and tissue injury. Early proinflammatory cytokines (e.g., tumor necrosis factor [TNF] and interleukin [IL]-1) released within the first few hours of endotoxemia stimulate mediator cascades that persist for days and can lead to death. High mobility group 1 protein (HMG-1), a ubiquitous DNA-binding protein, was recently identified as a “late” mediator of endotoxin lethality. Anti–HMG-1 antibodies neutralized the delayed increase in serum HMG-1, and protected against endotoxin lethality, even when passive immunization was delayed until after the early cytokine response. Here we examined whether HMG-1 might stimulate cytokine synthesis in human peripheral blood mononuclear cell cultures. Addition of purified recombinant HMG-1 to human monocyte cultures significantly stimulated the release of TNF, IL-1α, IL-1β, IL-1RA, IL-6, IL-8, macrophage inflammatory protein (MIP)-1α, and MIP-1β; but not IL-10 or IL-12. HMG-1 concentrations that activated monocytes were within the pathological range previously observed in endotoxemic animals, and in serum obtained from septic patients. HMG-1 failed to stimulate cytokine release in lymphocytes, indicating that cellular stimulation was specific. Cytokine release after HMG-1 stimulation was delayed and biphasic compared with LPS stimulation. Computer-assisted image analysis demonstrated that peak intensity of HMG-1–induced cellular TNF staining was comparable to that observed after maximal stimulation with LPS. Administration of HMG-1 to Balb/c mice significantly increased serum TNF levels in vivo. Together, these results indicate that, like other cytokine mediators of endotoxin lethality (e.g., TNF and IL-1), extracellular HMG-1 is a regulator of monocyte proinflammatory cytokine synthesis.
1,412 citations
TL;DR: In this article, expression of CXC chemokine receptor 5 (CXCR5) defines a novel subpopulation of B helper T cells localizing to follicles, based on the characteristic localization within secondary lymphoid organs.
Abstract: Chemokines and their receptors have been identified as major regulators controlling the functional organization of secondary lymphoid organs. Here we show that expression of CXC chemokine receptor 5 (CXCR5), a chemokine receptor required for B cell homing to B cell follicles, defines a novel subpopulation of B helper T cells localizing to follicles. In peripheral blood these cells coexpress CD45RO and the T cell homing CC chemokine receptor 7 (CCR7). In secondary lymphoid organs, CD4(+)CXCR5(+) cells lose expression of CCR7, which allows them to localize to B cell follicles and germinal centers where they express high levels of CD40 ligand (CD40L), a costimulatory molecule required for B cell activation and inducible costimulator (ICOS), a recently identified costimulatory molecule of the CD28 family. Thus, when compared with CD4(+)CD45RO(+)CXCR5(-) cells, CD4(+)CD45RO(+)CXCR5(+) tonsillar T cells efficiently support the production of immunoglobulin (Ig)A and IgG. In contrast, analysis of the memory response revealed that long-lasting memory cells are found within the CD4(+)CD45RO(+)CXCR5(-) population, suggesting that CXCR5(+)CD4 cells represent recently activated effector cells. Based on the characteristic localization within secondary lymphoid organs, we suggest to term these cells "follicular B helper T cells" (T(FH)).
1,353 citations
TL;DR: Results indicate that CD8+ DCs play an important role in the generation of cytotoxic T lymphocyte responses specific for cell-associated antigens, suggesting an endosome to cytosol transport.
Abstract: Bone marrow–derived antigen-presenting cells (APCs) take up cell-associated antigens and present them in the context of major histocompatibility complex (MHC) class I molecules to CD8+ T cells in a process referred to as cross-priming. Cross-priming is essential for the induction of CD8+ T cell responses directed towards antigens not expressed in professional APCs. Although in vitro experiments have shown that dendritic cells (DCs) and macrophages are capable of presenting exogenous antigens in association with MHC class I, the cross-presenting cell in vivo has not been identified. We have isolated splenic DCs after in vivo priming with ovalbumin-loaded β2-microglobulin–deficient splenocytes and show that they indeed present cell-associated antigens in the context of MHC class I molecules. This process is transporter associated with antigen presentation (TAP) dependent, suggesting an endosome to cytosol transport. To determine whether a specific subset of splenic DCs is involved in this cross-presentation, we negatively and positively selected for CD8− and CD8+ DCs. Only the CD8+, and not the CD8−, DC subset demonstrates cross-priming ability. FACS® studies after injection of splenocytes loaded with fluorescent beads showed that 1 and 0.6% of the CD8+ and the CD8− DC subsets, respectively, had one or more associated beads. These results indicate that CD8+ DCs play an important role in the generation of cytotoxic T lymphocyte responses specific for cell-associated antigens.
1,306 citations
TL;DR: A new model enabling incremental ROS accumulation in individual mitochondria in isolated cardiac myocytes via photoactivation of tetramethylrhodamine derivatives, which also served to report the mitochondrial transmembrane potential is devised, which is termed mitochondrial “ROS-induced ROS release” (RIRR).
Abstract: We sought to understand the relationship between reactive oxygen species (ROS) and the mitochondrial permeability transition (MPT) in cardiac myocytes based on the observation of increased ROS production at sites of spontaneously deenergized mitochondria We devised a new model enabling incremental ROS accumulation in individual mitochondria in isolated cardiac myocytes via photoactivation of tetramethylrhodamine derivatives, which also served to report the mitochondrial transmembrane potential, ΔΨ This ROS accumulation reproducibly triggered abrupt (and sometimes reversible) mitochondrial depolarization This phenomenon was ascribed to MPT induction because (a) bongkrekic acid prevented it and (b) mitochondria became permeable for calcein (∼620 daltons) concurrently with depolarization These photodynamically produced “triggering” ROS caused the MPT induction, as the ROS scavenger Trolox prevented it The time required for triggering ROS to induce the MPT was dependent on intrinsic cellular ROS-scavenging redox mechanisms, particularly glutathione MPT induction caused by triggering ROS coincided with a burst of mitochondrial ROS generation, as measured by dichlorofluorescein fluorescence, which we have termed mitochondrial “ROS-induced ROS release” (RIRR) This MPT induction/RIRR phenomenon in cardiac myocytes often occurred synchronously and reversibly among long chains of adjacent mitochondria demonstrating apparent cooperativity The observed link between MPT and RIRR could be a fundamental phenomenon in mitochondrial and cell biology
1,303 citations
TL;DR: A strong and persistent CTL response in resolving acute HCV infection is demonstrated, and rationale to explore immune augmentation as a therapeutic intervention in chronic HCv infection is provided.
Abstract: Although hepatitis C virus (HCV) infection is very common, identification of patients during acute infection is rare. Consequently, little is known about the immune response during this critical stage of the disease. We analyzed the T lymphocyte response during and after acute resolving HCV infection in three persons, using interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) and human histocompatibility leukocyte antigen (HLA) peptide tetramer assays. Acute infection was associated with a broadly directed T helper and cytotoxic T lymphocyte (CTL) response, which persisted after resolution of clinical hepatitis and clearance of viremia. At the earliest time point studied, highly activated CTL populations were observed that temporarily failed to secrete IFN-γ, a “stunned” phenotype, from which they recovered as viremia declined. In long-term HCV-seropositive persons, CTL responses were more common in persons who had cleared viremia compared with those with persistent viremia, although the frequencies of HCV-specific CTLs were lower than those found in persons during and after resolution of acute HCV infection. These studies demonstrate a strong and persistent CTL response in resolving acute HCV infection, and provide rationale to explore immune augmentation as a therapeutic intervention in chronic HCV infection.
TL;DR: What makes a protein immunogenic, particularly for strong T cell–mediated immunity?
Abstract: What makes a protein immunogenic, particularly for strong T cell–mediated immunity? To a first approximation, this determination seems to be made by dendritic cells (DCs). Immature DCs, as in skin ([1][1])([2][2])([3][3])([4][4]), lung ([5][5]), blood ([6][6])([7][7]), and spleen ([7][7])([8][8
TL;DR: It is demonstrated that TNF-α stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL–RANK system.
Abstract: Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF–dependent bone marrow macrophages (M-BMMφ) appeared within 3 d. Tartrate-resistant acid phosphatase–positive osteoclasts were also formed when M-BMMφ were further cultured for 3 d with mouse tumor necrosis factor α (TNF-α) in the presence of M-CSF. Osteoclast formation induced by TNF-α was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti–RANK (ODF/RANKL receptor) antibody. Experiments using M-BMMφ prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-α. Osteoclasts induced by TNF-α formed resorption pits on dentine slices only in the presence of IL-1α. These results demonstrate that TNF-α stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL–RANK system. TNF-α together with IL-1α may play an important role in bone resorption of inflammatory bone diseases.
TL;DR: The results suggest that, in addition to its microbicidal activity, LL-37 may contribute to innate and adaptive immunity by recruiting neutrophils, monocytes, and T cells to sites of microbial invasion by interacting with FPRL1.
Abstract: We have previously shown that antimicrobial peptides like defensins have the capacity to mobilize leukocytes in host defense. LL-37 is the cleaved antimicrobial 37-residue, COOH-terminal peptide of hCAP18 (human cationic antimicrobial protein with a molecular size of 18 kD), the only identified member in humans of a family of proteins called cathelicidins. LL-37/hCAP18 is produced by neutrophils and various epithelial cells. Here we report that LL-37 is chemotactic for, and can induce Ca2+ mobilization in, human monocytes and formyl peptide receptor–like 1 (FPRL1)-transfected human embryonic kidney 293 cells. LL-37–induced Ca2+ mobilization in monocytes can also be cross-desensitized by an FPRL1-specific agonist. Furthermore, LL-37 is also chemotactic for human neutrophils and T lymphocytes that are known to express FPRL1. Our results suggest that, in addition to its microbicidal activity, LL-37 may contribute to innate and adaptive immunity by recruiting neutrophils, monocytes, and T cells to sites of microbial invasion by interacting with FPRL1.
TL;DR: These properties portray CXCR5+ T cells as a distinct memory T cell subset with B cell helper function, designated here as follicular B helper T cells (TFH).
Abstract: Leukocyte traffic through secondary lymphoid tissues is finely tuned by chemokines. We have studied the functional properties of a human T cell subset marked by the expression of CXC chemokine receptor 5 (CXCR5). Memory but not naive T cells from tonsils are CXCR5+ and migrate in response to the B cell–attracting chemokine 1 (BCA-1), which is selectively expressed by reticular cells and blood vessels within B cell follicles. Tonsillar CXCR5+ T cells do not respond to other chemokines present in secondary lymphoid tissues, including secondary lymphoid tissue chemokine (SLC), EBV-induced molecule 1 ligand chemokine (ELC), and stromal cell–derived factor 1 (SDF-1). The involvement of tonsillar CXCR5+ T cells in humoral immune responses is suggested by their localization in the mantle and light zone germinal centers of B cell follicles and by the concomitant expression of activation and costimulatory markers, including CD69, HLA-DR, and inducible costimulator (ICOS). Peripheral blood CXCR5+ T cells also belong to the CD4+ memory T cell subset but, in contrast to tonsillar cells, are in a resting state and migrate weakly to chemokines. CXCR5+ T cells are very inefficient in the production of cytokines but potently induce antibody production during coculture with B cells. These properties portray CXCR5+ T cells as a distinct memory T cell subset with B cell helper function, designated here as follicular B helper T cells (TFH).
TL;DR: It is reported that inflammatory exudates from mice treated with ω-3 polyunsaturated fatty acid and aspirin (ASA) generate a novel array of bioactive lipid signals, which provides a novel mechanism for the therapeutic benefits ofπ-3 dietary supplementation, which may be important in inflammation, neoplasia, and vascular diseases.
Abstract: Aspirin therapy inhibits prostaglandin biosynthesis without directly acting on lipoxygenases, yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins (LXs) epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered LX [ATL]). Here, we report that inflammatory exudates from mice treated with ω-3 polyunsaturated fatty acid and aspirin (ASA) generate a novel array of bioactive lipid signals. Human endothelial cells with upregulated COX-2 treated with ASA converted C20:5 ω-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Each was used by polymorphonuclear leukocytes to generate separate classes of novel trihydroxy-containing mediators, including 5-series 15R-LX5 and 5,12,18R-triHEPE. These new compounds proved to be potent inhibitors of human polymorphonuclear leukocyte transendothelial migration and infiltration in vivo (ATL analogue > 5,12,18R-triHEPE > 18R-HEPE). Acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant COX-2 as well as ω-5 and ω-9 oxygenations of other fatty acids that act on hematologic cells. These findings establish new transcellular routes for producing arrays of bioactive lipid mediators via COX-2–nonsteroidal antiinflammatory drug–dependent oxygenations and cell–cell interactions that impact microinflammation. The generation of these and related compounds provides a novel mechanism(s) for the therapeutic benefits of ω-3 dietary supplementation, which may be important in inflammation, neoplasia, and vascular diseases.
TL;DR: The data demonstrate that the antiapoptotic effect of HO-1 in ECs is mediated by CO and more specifically via the activation of p38 MAPK by CO.
Abstract: Heme oxygenase 1 (HO-1) inhibits apoptosis by regulating cellular prooxidant iron. We now show that there is an additional mechanism by which HO-1 inhibits apoptosis, namely by generating the gaseous molecule carbon monoxide (CO). Overexpression of HO-1, or induction of HO-1 expression by heme, protects endothelial cells (ECs) from apoptosis. When HO-1 enzymatic activity is blocked by tin protoporphyrin (SnPPIX) or the action of CO is inhibited by hemoglobin (Hb), HO-1 no longer prevents EC apoptosis while these reagents do not affect the antiapoptotic action of bcl-2. Exposure of ECs to exogenous CO, under inhibition of HO-1 activity by SnPPIX, substitutes HO-1 in preventing EC apoptosis. The mechanism of action of HO-1/CO is dependent on the activation of the p38 mitogen-activated protein kinase (MAPK) signaling transduction pathway. Expression of HO-1 or exposure of ECs to exogenous CO enhanced p38 MAPK activation by TNF-alpha. Specific inhibition of p38 MAPK activation by the pyridinyl imidazol SB203580 or through overexpression of a p38 MAPK dominant negative mutant abrogated the antiapoptotic effect of HO-1. Taken together, these data demonstrate that the antiapoptotic effect of HO-1 in ECs is mediated by CO and more specifically via the activation of p38 MAPK by CO.
TL;DR: Results show that a distinct DC subset constitutively endocytoses and transports apoptotic cells to T cell areas and suggest a role for these DCs in inducing and maintaining peripheral self-tolerance.
Abstract: This study identifies a dendritic cell (DC) subset that constitutively transports apoptotic intestinal epithelial cell remnants to T cell areas of mesenteric lymph nodes in vivo. Rat intestinal lymph contains two DC populations. Both populations have typical DC morphology, are major histocompatibility complex class IIhi, and express OX62, CD11c, and B7. CD4+/OX41+ DCs are strong antigen-presenting cells (APCs). CD4−/OX41− DCs are weak APCs and contain cytoplasmic apoptotic DNA, epithelial cell–restricted cytokeratins, and nonspecific esterase (NSE)+ inclusions, not seen in OX41+ DCs. Identical patterns of NSE electrophoretic variants exist in CD4−/OX41− DCs, intestinal epithelial cells, and mesenteric node DCs but not in other DC populations, macrophages, or tissues. Terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick-end labeling (TUNEL)-positive DCs and strongly NSE+ DCs are present in intestinal lamina propria. Peyer's patches and mesenteric but not other lymph nodes contain many strongly NSE+ DCs in interfollicular and T cell areas. Similar DCs are seen in the ileum and in T cell areas of mesenteric nodes in gnotobiotic rats. These results show that a distinct DC subset constitutively endocytoses and transports apoptotic cells to T cell areas and suggest a role for these DCs in inducing and maintaining peripheral self-tolerance.
TL;DR: Clonal composition and T cell receptor (TCR) repertoire of CD4+ and CD8+ T cells infiltrating actively demyelinating multiple sclerosis (MS) lesions were determined with unprecedented resolution at the level of single cells.
Abstract: Clonal composition and T cell receptor (TCR) repertoire of CD4+ and CD8+ T cells infiltrating actively demyelinating multiple sclerosis (MS) lesions were determined with unprecedented resolution at the level of single cells. Individual CD4+ or CD8+ T cells were isolated from frozen sections of lesional tissue by micromanipulation and subjected to single target amplification of TCR-β gene rearrangements. This strategy allows the assignment of a TCR variable region (V region) sequence to the particular T cell from which it was amplified. Sequence analysis revealed that in both cases investigated, the majority of CD8+ T cells belonged to few clones. One of these clones accounted for 35% of CD8+ T cells in case 1. V region sequence comparison revealed signs of selection for common peptide specificities for some of the CD8+ T cells in case 1. In both cases, the CD4+ T cell population was more heterogeneous. Most CD4+ and CD8+ clones were represented in perivascular infiltrates as well as among parenchymal T cells. In case 2, two of the CD8+ clones identified in brain tissue were also detected in peripheral blood. Investigation of the antigenic specificities of expanded clones may help to elucidate their functional properties.
TL;DR: The majority of circulating CD8+ T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-γ and MIP-1β but not TNF-α.
Abstract: The use of peptide–human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8+ T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8+ T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1β, and perforin is analyzed by FACS® within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8+ T cells compared with cytomegalovirus (CMV)-specific CD8+ T cells in HIV chronic infection. We show that the majority of circulating CD8+ T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-γ and MIP-1β but not TNF-α. However, a striking finding is that HIV-specific CD8+ T cells express significantly lower levels of perforin than CMV-specific CD8+ T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8+ T cells are impaired in cytolytic activity.
TL;DR: It is shown that tetramers of mouse CD1d loaded with α-GalCer are a sensitive and highly specific reagent for identifying Vα14+ NK T cells and that α- GalCer–specific T lymphocytes are more widely distributed than was previously appreciated.
Abstract: A major group of natural killer (NK) T cells express an invariant Vα14+ T cell receptor (TCR) specific for the lipoglycan α-galactosylceramide (α-GalCer), which is presented by CD1d. These cells may have an important immune regulatory function, but an understanding of their biology has been hampered by the lack of suitable reagents for tracking them in vivo. Here we show that tetramers of mouse CD1d loaded with α-GalCer are a sensitive and highly specific reagent for identifying Vα14+ NK T cells. Using these tetramers, we find that α-GalCer–specific T lymphocytes are more widely distributed than was previously appreciated, with populations of largely NK1.1− but tetramer-binding T cells present in the lymph nodes and the intestine. Injection of α-GalCer leads to the production of both interferon γ and interleukin 4 by nearly all NK T cells in the liver and the majority of the spleen within 2 h. These cells mostly disappear by 5 h, and they do not reappear after 1 wk. Curiously, tetramer-positive thymocytes do not rapidly synthesize cytokines, nor do they undergo decreases in cell number after lipid antigen stimulation, although they express equivalent TCR levels. In summary, the data presented here demonstrate that α-GalCer–specific NK T cells undergo a unique and highly compartmentalized response to antigenic stimulation.
TL;DR: Virus-activated IPCs may play two master roles in antiviral immune responses: directly inhibiting viral replication by producing large amounts of IFN-α/β, and subsequently triggering adaptive T cell–mediated immunity by differentiating into DCs.
Abstract: Innate immune responses to pathogens critically impact the development of adaptive immune responses. However, it is not completely understood how innate immunity controls the initiation of adaptive immunities or how it determines which type of adaptive immunity will be induced to eliminate a given pathogen. Here we show that viral stimulation not only triggers natural interferon (IFN)-α/β–producing cells (IPCs) to produce vast amounts of antiviral IFN-α/β but also induces these cells to differentiate into dendritic cells (DCs). IFN-α/β and tumor necrosis factor α produced by virus-activated IPCs act as autocrine survival and DC differentiation factors, respectively. The virus-induced DCs stimulate naive CD4+ T cells to produce IFN-γ and interleukin (IL)-10, in contrast to IL-3–induced DCs, which stimulate naive CD4+ T cells to produce T helper type 2 cytokines IL-4, IL-5, and IL-10. Thus, IPCs may play two master roles in antiviral immune responses: directly inhibiting viral replication by producing large amounts of IFN-α/β, and subsequently triggering adaptive T cell–mediated immunity by differentiating into DCs. IPCs constitute a critical link between innate and adaptive immunity.
TL;DR: T cell receptor Jα281 gene-targeted mice confirmed a critical function for NKT cells in protection from spontaneous tumors initiated by the chemical carcinogen, methylcholanthrene, which is the first description of an antitumor function in the absence of exogenously administered potent stimulators such as IL-12 or α-galactosylceramide.
Abstract: Natural tumor surveillance capabilities of the host were investigated in six different mouse tumor models where endogenous interleukin (IL)-12 does or does not dictate the efficiency of the innate immune response. Gene-targeted and lymphocyte subset–depleted mice were used to establish the relative importance of natural killer (NK) and NK1.1+ T (NKT) cells in protection from tumor initiation and metastasis. In the models examined, CD3− NK cells were responsible for tumor rejection and protection from metastasis in models where control of major histocompatibility complex class I–deficient tumors was independent of IL-12. A protective role for NKT cells was only observed when tumor rejection required endogenous IL-12 activity. In particular, T cell receptor Jα281 gene-targeted mice confirmed a critical function for NKT cells in protection from spontaneous tumors initiated by the chemical carcinogen, methylcholanthrene. This is the first description of an antitumor function for NKT cells in the absence of exogenously administered potent stimulators such as IL-12 or α-galactosylceramide.
TL;DR: In vivo findings strongly suggest that uNK cell–derived IFN-γ modifies the expression of genes in the uterine vasculature and stroma, which initiates vessel instability and facilitates pregnancy-induced remodeling of decidual arteries.
Abstract: The dominant lymphocytes in human and murine implantation sites are transient, pregnancy-associated uterine natural killer (uNK) cells. These cells are a major source of interferon (IFN)-γ. Implantation sites in mice lacking uNK cells (alymphoid recombinase activating gene [RAG]-2−/− common cytokine receptor chain γ [γc]−/−) or IFN-γ signaling (IFN-γ−/− or IFN-γRα−/−) fail to initiate normal pregnancy-induced modification of decidual arteries and display hypocellularity or necrosis of decidua. To investigate the functions of uNK cell–derived IFN-γ during pregnancy, RAG-2−/−γc−/− females were engrafted with bone marrow from IFN-γ−/− mice, IFN-γ signal-disrupted mice (IFN-γRα−/− or signal transducer and activator of transcription [Stat]-1−/−), or from mice able to establish normal uNK cells (severe combined immunodeficient [SCID] or C57BL/6). Mated recipients were analyzed at midgestation. All grafts established uNK cells. Grafts from IFN-γ−/− mice did not reverse host vascular or decidual pathology. Grafts from all other donors promoted modification of decidual arteries and decidual cellularity. Grafts from IFN-γRα−/− or Stat-1−/− mice overproduced uNK cells, all of which were immature. Grafts from IFN-γ−/−, SCID, or C57BL/6 mice produced normal, mature uNK cells. Administration of murine recombinant IFN-γ to pregnant RAG-2−/−γc−/− mice initiated decidual vessel modification and promoted decidual cellularity in the absence of uNK cells. These in vivo findings strongly suggest that uNK cell–derived IFN-γ modifies the expression of genes in the uterine vasculature and stroma, which initiates vessel instability and facilitates pregnancy-induced remodeling of decidual arteries.
TL;DR: Two redundant parallel pathways may lead to chromatin processing during apoptosis, including one which involves Apaf-1 and caspases, as well as CAD, and leads to oligonucleosomal DNA fragmentation and advanced chromatin condensation and the other which is caspase-independent.
Abstract: Apaf-1−/− or caspase-3−/− cells treated with a variety of apoptosis inducers manifest apoptosis-associated alterations including the translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, large scale DNA fragmentation, and initial chromatin condensation (stage I). However, when compared with normal control cells, Apaf-1−/− or caspase-3−/− cells fail to exhibit oligonucleosomal chromatin digestion and a more advanced pattern of chromatin condensation (stage II). Microinjection of such cells with recombinant AIF only causes peripheral chromatin condensation (stage I), whereas microinjection with activated caspase-3 or its downstream target caspase-activated DNAse (CAD) causes a more pronounced type of chromatin condensation (stage II). Similarly, when added to purified HeLa nuclei, AIF causes stage I chromatin condensation and large-scale DNA fragmentation, whereas CAD induces stage II chromatin condensation and oligonucleosomal DNA degradation. Furthermore, in a cell-free system, concomitant neutralization of AIF and CAD is required to suppress the nuclear DNA loss caused by cytoplasmic extracts from apoptotic wild-type cells. In contrast, AIF depletion alone suffices to suppress the nuclear DNA loss contained in extracts from apoptotic Apaf-1−/− or caspase-3−/− cells. As a result, at least two redundant parallel pathways may lead to chromatin processing during apoptosis. One of these pathways involves Apaf-1 and caspases, as well as CAD, and leads to oligonucleosomal DNA fragmentation and advanced chromatin condensation. The other pathway, which is caspase-independent, involves AIF and leads to large-scale DNA fragmentation and peripheral chromatin condensation.
TL;DR: The results provide a rationale for using type I IFNs as vaccine adjuvants and support the concept that a natural alliance between these cytokines and monocytes/DCs represents an important early mechanism for connecting innate and adaptive immunity.
Abstract: Type I interferons (IFNs) are cytokines exhibiting antiviral and antitumor effects, including multiple activities on immune cells. However, the importance of these cytokines in the early events leading to the generation of an immune response is still unclear. Here, we have investigated the effects of type I IFNs on freshly isolated granulocyte/macrophage colony-stimulating factor (GM-CSF)–treated human monocytes in terms of dendritic cell (DC) differentiation and activity in vitro and in severe combined immunodeficiency mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID) mice. Type I IFNs induced a surprisingly rapid maturation of monocytes into short-lived tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)–expressing DCs endowed with potent functional activities, superior with respect to the interleukin (IL)-4/GM-CSF treatment, as shown by FACS® analyses, mixed leukocyte reaction assays with allogeneic PBLs, and lymphocyte proliferation responses to HIV-1–pulsed autologous DCs. Type I IFN induced IL-15 production and strongly promoted a T helper cell type 1 response. Notably, injection of IFN-treated HIV-1–pulsed DCs in SCID mice reconstituted with autologous PBLs resulted in the generation of a potent primary immune response, as evaluated by the detection of human antibodies to various HIV-1 antigens. These results provide a rationale for using type I IFNs as vaccine adjuvants and support the concept that a natural alliance between these cytokines and monocytes/DCs represents an important early mechanism for connecting innate and adaptive immunity.
TL;DR: Results show that in the presence of an effective HBV-specific CD8 response, inhibition of virus replication can be independent of liver damage and may contribute to liver pathology not only directly but by causing the recruitment of nonvirus-specific T cells.
Abstract: Hepatitis B virus (HBV) is a noncytopathic virus, and the recognition of infected hepatocytes by HBV-specific CD8 cells has been assumed to be the central mechanism causing both liver damage and virus control. To understand the role of cytotoxic T cells in the pathogenesis of HBV infection, we used functional assays that require T cell expansion in vitro and human histocompatibility leukocyte antigen (HLA)-peptide tetramers that allow direct ex vivo quantification of circulating and liver-infiltrating HBV-specific CD8 cells. Two groups of patients with persistent HBV infection were studied: one without liver inflammation and HBV replication, the other with liver inflammation and a high level of HBV replication. Contrary to expectation, a high frequency of intrahepatic HBV-specific CD8 cells was found in the absence of hepatic immunopathology. In contrast, virus-specific T cells were more diluted among liver infiltrates in viremic patients, but their absolute number was similar because of the massive cellular infiltration. Furthermore, inhibition of HBV replication was associated with the presence of a circulating reservoir of CD8(+) cells able to expand after specific virus recognition that was not detectable in highly viremic patients with liver inflammation. These results show that in the presence of an effective HBV-specific CD8 response, inhibition of virus replication can be independent of liver damage. When the HBV-specific CD8 response is unable to control virus replication, it may contribute to liver pathology not only directly but by causing the recruitment of nonvirus-specific T cells.
TL;DR: A model in which excessive BAFF-mediated survival of peripheral immature B cells contributes to the emergence and maturation of autoreactive B cells, skewed towards the MZ compartment is supported, providing new clues on mechanisms regulating B cell maturation and tolerance.
Abstract: B cell maturation is a very selective process that requires finely tuned differentiation and survival signals. B cell activation factor from the TNF family (BAFF) is a TNF family member that binds to B cells and potentiates B cell receptor (BCR)-mediated proliferation. A role for BAFF in B cell survival was suggested by the observation of reduced peripheral B cell numbers in mice treated with reagents blocking BAFF, and high Bcl-2 levels detected in B cells from BAFF transgenic (Tg) mice. We tested in vitro the survival effect of BAFF on lymphocytes derived from primary and secondary lymphoid organs. BAFF induced survival of a subset of splenic immature B cells, referred to as transitional type 2 (T2) B cells. BAFF treatment allowed T2 B cells to survive and differentiate into mature B cells in response to signals through the BCR. The T2 and the marginal zone (MZ) B cell compartments were particularly enlarged in BAFF Tg mice. Immature transitional B cells are targets for negative selection, a feature thought to promote self-tolerance. These findings support a model in which excessive BAFF-mediated survival of peripheral immature B cells contributes to the emergence and maturation of autoreactive B cells, skewed towards the MZ compartment. This work provides new clues on mechanisms regulating B cell maturation and tolerance.
TL;DR: It is shown that IL-1ra gene deficiency causes autoimmunity and joint-specific inflammation and suggested that IL -1ra is important in maintaining homeostasis of the immune system.
Abstract: Interleukin (IL)-1 is a proinflammatory cytokine that plays important roles in inflammation, host defense, and the neuro-immuno-endocrine network. IL-1 receptor antagonist (ra) is an endogenous inhibitor of IL-1 and is supposed to regulate IL-1 activity. However, its pathophysiological roles in a body remain largely unknown. To elucidate the roles of IL-1ra, IL-1ra–deficient mice were produced by gene targeting, and pathology was analyzed on different genetic backgrounds. We found that all of the mice on a BALB/cA background, but not those on a C57BL/6J background, spontaneously developed chronic inflammatory polyarthropathy. Histopathology showed marked synovial and periarticular inflammation, with articular erosion caused by invasion of granulation tissues closely resembling that of rheumatoid arthritis in humans. Moreover, elevated levels of antibodies against immunoglobulins, type II collagen, and double-stranded DNA were detected in these mice, suggesting development of autoimmunity. Proinflammatory cytokines such as IL-1β, IL-6, and tumor necrosis factor α were overexpressed in the joints, indicating regulatory roles of IL-1ra in the cytokine network. We thus show that IL-1ra gene deficiency causes autoimmunity and joint-specific inflammation and suggest that IL-1ra is important in maintaining homeostasis of the immune system. Possible involvement of IL-1ra gene deficiency in RA will be discussed.
TL;DR: Modifications of anchor residues that lead to an improved affinity for major histocompatibility complex (MHC), and altered conformation of the peptide–MHC complex may be a critical factor leading to T cell responses to gliadin and the oral intolerance of gluten found in CD.
Abstract: The great majority of patients that are intolerant of wheat gluten protein due to celiac disease (CD) are human histocompatibility leukocyte antigen (HLA)-DQ2(+), and the remaining few normally express HLA-DQ8. These two class II molecules are chiefly responsible for the presentation of gluten peptides to the gluten-specific T cells that are found only in the gut of CD patients but not of controls. Interestingly, tissue transglutaminase (tTG)-mediated deamidation of gliadin plays an important role in recognition of this food antigen by intestinal T cells. Here we have used recombinant antigens to demonstrate that the intestinal T cell response to alpha-gliadin in adult CD is focused on two immunodominant, DQ2-restricted peptides that overlap by a seven-residue fragment of gliadin. We show that tTG converts a glutamine residue within this fragment into glutamic acid and that this process is critical for T cell recognition. Gluten-specific T cell lines from 16 different adult patients all responded to one or both of these deamidated peptides, indicating that these epitopes are highly relevant to disease pathology. Binding studies showed that the deamidated peptides displayed an increased affinity for DQ2, a molecule known to preferentially bind peptides containing negatively charged residues. Interestingly, the modified glutamine is accommodated in different pockets of DQ2 for the different epitopes. These results suggest modifications of anchor residues that lead to an improved affinity for major histocompatibility complex (MHC), and altered conformation of the peptide-MHC complex may be a critical factor leading to T cell responses to gliadin and the oral intolerance of gluten found in CD.