Bacterial carbohydrates have long been considered T cell-independent antigens that primarily induce humoral immune responses. Recently, it has been demonstrated that bacterial capsules that possess a zwitterionic charge motif can activate CD4(+) T cells after processing and presentation by antigen-presenting cells. Here we show that these zwitterionic polysaccharides can prevent T helper 1-mediated fibrosis by signaling for the release of IL-10 from CD4(+) T cells in vivo. IL-10 production by these T cells and their ability to prevent fibrosis is controlled by the inducible costimulator (ICOS)-ICOS ligand pathway. These data demonstrate that the interaction of the zwitterionic polysaccharides with T cells results in modulation of surgical fibrosis in vivo and suggest a previously undescribed approach to "harnessing" T cell function to prevent inflammatory tissue disorders in humans.
The small GTPase Rap1 is transiently activated during TCR ligation and regulates integrin-mediated adhesion. To understand the in vivo functions of Rap1 in regulating T cell immune responses, we generated transgenic (Tg) mice, which express the active GTP-bound mutant Rap1E63 in their T lymphocytes. Although Rap1E63-Tg T cells exhibited increased LFA-1-mediated adhesion, ERK1/2 activation and proliferation of Rap1E63-Tg CD4+ T cells were defective. Rap1E63-Tg T cells primed in vivo and restimulated with specific Ag in vitro, exhibited reduced proliferation and produced reduced levels of IL-2. Rap1E63-Tg mice had severely deficient T cell-dependent B cell responses, as determined by impaired Ig class switching. Rap1E63-Tg mice had an increased fraction of CD4+CD103+ regulatory T cells (Treg), which exhibited enhanced suppressive efficiency as compared with CD4+CD103+ Treg from normal littermate control mice. Depletion of CD103+ Treg significantly restored the impaired responses of Rap1E63-Tg CD4+ T cells. Thus Rap1-GTP is a negative regulator of Th cell responses and one mechanism responsible for this effect involves the increase of CD103+ Treg cell fraction. Our results show that Rap1-GTP promotes the generation of CD103+ Treg and may have significant implications in the development of strategies for in vitro generation of Treg for the purpose of novel immunotherapeutic approaches geared toward tolerance induction.
Positive selection during thymocyte development is driven by the affinity and avidity of the TCR for MHC-peptide complexes expressed in the thymus. In this study, we show that programmed death-1 (PD-1), a member of the B7/CD28 family of costimulatory receptors, inhibits TCR-mediated positive selection through PD-1 ligand 1 (PD-L1):PD-1 interactions. Transgenic mice that constitutively overexpress PD-1 on CD4+CD8+ thymocytes display defects in positive selection in vivo. Using an in vitro model system, we find that PD-1 is up-regulated following TCR engagement on CD4+CD8+ murine thymocytes. Coligation of TCR and PD-1 on CD4+CD8+ thymocytes with a novel PD-1 agonistic mAb inhibits the activation of ERK and up-regulation of bcl-2, both of which are downstream mediators essential for positive selection. Inhibitory signals through PD-1 can overcome the ability of positive costimulators, such as CD2 and CD28, to facilitate positive selection. Finally, defects in positive selection that result from PD-1 overexpression in thymocytes resolve upon elimination of PD-L1, but not PD-1 ligand 2, expression. PD-L1-deficient mice have increased numbers of CD4+CD8+ and CD4+ thymocytes, indicating that PD-L1 is involved in normal thymic selection. These data demonstrate that PD-1:PD-L1 interactions are critical to positive selection and play a role in shaping the T cell repertoire.
Although B7 on APCs has a well-recognized role in T cell costimulation, little is known about the functional significance of constitutive and activation-induced B7 expression that also occurs on T cells. To analyze the role of B7 on T cells, B7-1/B7-2-deficient mice (B7 double knockout) and mice overexpressing B7-2 exclusively on T cells (B7-2 transgenic) were used as T cell donors for allogeneic transplant recipients, and graft-vs-host disease (GVHD) was assessed. B7 double-knockout T cells resulted in significant GVHD acceleration compared with wild-type T cells. Conversely, B7-2 transgenic donor T cells mediated reduced GVHD mortality compared with wild-type T cells. Data indicated that B7 expression on T cells down-regulated alloresponses through CTLA-4 ligation. This study is the first to provide definitive in vivo data illustrating the importance of T cell-associated B7 as a negative regulator of immune responses in a clinically relevant murine model of GVHD. The up-regulation of B7 on T cells may be an important component of normal immune homeostasis.
BACKGROUND: Several lines of evidence indicate that T-cell responses influence the progression of atherosclerotic disease. Interferon-gamma (IFN-gamma)-producing T cells specific for lesional antigens, including oxidized LDLs and heat shock protein 60 (HSP60), may promote lesion development as well as plaque instability. B7-1 and B7-2 are closely related molecules expressed on antigen-presenting cells that provide costimulatory signals for T-cell activation. This study tested the hypothesis that the ability of T cells to influence atherosclerosis depends on B7-1/B7-2 costimulation.
METHODS AND RESULTS: B7-1/B7-2/LDL receptor (LDLR)-deficient mice and LDLR-deficient control mice were fed a 1.25% cholesterol or control diet for 8 and 20 weeks. Total serum cholesterol levels and extent and phenotype of atherosclerosis were analyzed. Splenic and lymph node CD4+ T cells from the animals were cultured with mouse recombinant HSP60 or media and antigen-presenting cells and analyzed for IFN-gamma and interleukin-4 production. The absence of B7-1 and B7-2 significantly reduced early cholesterol diet-induced atherosclerotic lesion development in LDLR-deficient mice compared with B7-1/B7-2-expressing control mice. Furthermore, CD4+ T cells from the cholesterol-fed B7-deficient mice secreted a significantly lower amount of IFN-gamma in response to mouse HSP60 in vitro than did T cells from B7-expressing control mice.
CONCLUSIONS: The data show that B7-1 and B7-2 regulated the development of atherosclerotic lesions and the priming of lesional antigen-specific T cells. This study highlights the B7-CD28 pathway as a potentially important target for immunomodulation of atherosclerosis.
Mice deficient in OX40 or 4-1BB costimulatory pathways show defects in T cell recall responses, with predominant effects on CD4 vs CD8 T cells, respectively. However, OX40L can also stimulate CD8 T cells and 4-1BBL can influence CD4 T cells, raising the possibility of redundancy between the two TNFR family costimulators. To test this possibility, we generated mice deficient in both 4-1BBL and OX40L. In an adoptive transfer model, CD4 T cells expressed 4-1BB and OX40 sequentially in response to immunization, with little or no overlap in the timing of their expression. Under the same conditions, CD8 T cells expressed 4-1BB, but no detectable OX40. Thus, in vivo expression of 4-1BB and OX40 can be temporally and spatially segregated. In the absence of OX40L, there were decreased CD4 T cells late in the primary response and no detectable secondary expansion of adoptively transferred CD4 T cells under conditions in which primary expansion was unaffected. The 4-1BBL had a minor effect on the primary response of CD4 T cells in this model, but showed larger effects on the secondary response, although 4-1BBL(-/-) mice show less impairment in CD4 secondary responses than OX40L(-/-) mice. The 4-1BBL(-/-) and double knockout mice were similarly impaired in the CD8 T cell response, whereas OX40L(-/-) and double knockout mice were similarly impaired in the CD4 T cell response to both protein Ag and influenza virus. Thus, 4-1BB and OX40 act independently and nonredundantly to facilitate robust CD4 and CD8 recall responses.
Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) plays a critical role in downregulating T cell responses. A number of autoimmune diseases have shown genetic linkage to the CTLA-4 locus. We have cloned and expressed an alternatively spliced form of CTLA-4 that has genetic linkage with type I diabetes in the NOD mice. This splice variant of CTLA-4, named ligand-independent CTLA-4 (liCTLA-4), lacks exon2 including the MYPPPY motif essential for binding to the costimulatory ligands B7-1 and B7-2. Here we show that liCTLA-4 is expressed as a protein in primary T cells and strongly inhibits T cell responses by binding and dephosphorylating the TcRzeta chain. Expression of liCTLA-4, but not full-length CTLA-4 (flCTLA-4), was higher in memory/regulatory T cells from diabetes-resistant NOD congenic mice compared to susceptible NOD mice. These data suggest that increased expression and negative signaling delivered by the liCTLA-4 may regulate development of T cell-mediated autoimmune diseases.
Signaling lymphocyte activation molecule (SLAM), a glycoprotein expressed on activated lymphocytes and antigen-presenting cells, has been shown to be a coregulator of antigen-driven T cell responses and is one of the two receptors for measles virus. Here we show that T cell receptor-induced interleukin (IL)-4 secretion by SLAM(-/-) CD4(+) cells is down-regulated, whereas interferon gamma production by CD4(+) T cells is only slightly up-regulated. Although SLAM controls production of IL-12, tumor necrosis factor, and nitric oxide in response to lipopolysaccharide (LPS) by macrophages, SLAM does not regulate phagocytosis and responses to peptidoglycan or CpG. Thus, SLAM acts as a coreceptor that regulates signals transduced by the major LPS receptor Toll-like receptor 4 on the surface of mouse macrophages. A defective macrophage function resulted in an inability of SLAM(-/-) C57Bl/6 mice to remove the parasite Leishmania major. We conclude that the coreceptor SLAM plays a central role at the interface of acquired and innate immune responses.
Helper T cell differentiation involves silencing as well as activation of gene expression. We have identified a conserved silencer of the gene encoding interleukin 4 (Il4) marked by DNase I hypersensitivity (HS IV) and permissive chromatin structure in all helper T cells. Deletion of HS IV increased Il4 and Il13 transcription by naive T cells and led to T helper type 2 skewing in vitro. HS IV controlled Il4 silencing during T helper type 1 differentiation, as HS IV-deficient T helper type 1 cells that expressed interferon-gamma also produced abundant interleukin 4 in vitro and in vivo. Despite mounting a vigorous interferon-gamma response, HS IV-deficient mice were more susceptible to Leishmania major infection than were wild-type littermate control mice, showing a critical function for Il4 silencing in T helper type 1-mediated immunity.
Kidney podocytes and their slit diaphragms form the final barrier to urinary protein loss. This explains why podocyte injury is typically associated with nephrotic syndrome. The present study uncovered an unanticipated novel role for costimulatory molecule B7-1 in podocytes as an inducible modifier of glomerular permselectivity. B7-1 in podocytes was found in genetic, drug-induced, immune-mediated, and bacterial toxin-induced experimental kidney diseases with nephrotic syndrome. The clinical significance of our results is underscored by the observation that podocyte expression of B7-1 correlated with the severity of human lupus nephritis. In vivo, exposure to low-dose LPS rapidly upregulates B7-1 in podocytes of WT and SCID mice, leading to nephrotic-range proteinuria. Mice lacking B7-1 are protected from LPS-induced nephrotic syndrome, suggesting a link between podocyte B7-1 expression and proteinuria. LPS signaling through toll-like receptor-4 reorganized the podocyte actin cytoskeleton in vitro, and activation of B7-1 in cultured podocytes led to reorganization of vital slit diaphragm proteins. In summary, upregulation of B7-1 in podocytes may contribute to the pathogenesis of proteinuria by disrupting the glomerular filter and provides a novel molecular target to tackle proteinuric kidney diseases. Our findings suggest a novel function for B7-1 in danger signaling by nonimmune cells.
Donor-specific transfusion (DST) can synergize with T cell co-stimulatory blockade in inducing tolerance in several transplant models, but the mechanism of action of DST is poorly characterized. This study used genetically altered mice in an established model of cardiac transplantation to study the role of MHC and co-stimulatory molecule expression on DST cells in mediating the immunomodulatory effects of DST. In addition, to examine the role of indirect antigen presentation in the effect of DST, experiments used recipient mice that do not express MHC class II molecules on peripheral antigen-presenting cells, but do have functional CD4(+) T cells (II(-)4(+)). As previously reported, treatment with DST from wild-type donors in combination with CD154 blockade induced tolerance in wild-type recipients of cardiac allografts. Tolerance in this model is also induced despite the absence of MHC class I and II, CD40, or B7 molecules on transfused cells. In contrast, eliminating the indirect pathway using II(-)4(+) recipients blocked the induction of long-term cardiac allograft survival by DST. These results indicate that the indirect antigen recognition pathway mediates the immunomodulatory effect of DST in inducing transplantation tolerance in vivo.
Both positive and negative regulatory roles have been suggested for the B7 family member PD-L1(B7-H1). PD-L1 is expressed on antigen-presenting cells (APCs), activated T cells, and a variety of tissues, but the functional significance of PD-L1 on each cell type is not yet clear. To dissect the functions of PD-L1 in vivo, we generated PD-L1-deficient (PD-L1(-/-)) mice. CD4(+) and CD8(+) T cell responses were markedly enhanced in PD-L1(-/-) mice compared with wild-type mice in vitro and in vivo. PD-L1(-/-) dendritic cells stimulated greater wild-type CD4(+) T cell responses than wild-type dendritic cells, and PD-L1(-/-) CD4(+) T cells produced more cytokines than wild-type CD4(+) T cells in vitro, demonstrating an inhibitory role for PD-L1 on APCs and T cells. In vivo CD8(+) T cell responses also were significantly enhanced, indicating that PD-L1 has a role in limiting the expansion or survival of CD8(+) T cells. Studies using the myelin oligodendrocyte model of experimental autoimmune encephalomyelitis showed that PD-L1 on T cells and in host tissues limits responses of self-reactive CD4(+) T cells in vivo. PD-L1 deficiency converted the 129S4/SvJae strain from a resistant to experimental autoimmune encephalomyelitis-susceptible strain. Transfer of encephalitogenic T cells from wild-type mice into PD-L1(-/-) recipients led to exacerbated disease. Disease was even more severe in PD-L1(-/-) recipients of PD-L1(-/-) T cells. These results demonstrate that PD-L1 on T cells, APCs, and host tissue inhibits naïve and effector T cell responses and plays a critical role in T cell tolerance.
In these studies, we examined the effects of OX40 ligand (OX40L) deficiency on the development of Th2 cells during the Th2 immune response to the intestinal nematode parasite Heligmosomoides polygyrus. Elevations in IL-4 production and total and Ag-specific serum IgE levels were partially inhibited during both the primary and memory immune responses to H. polygyrus in OX40L(-/-) mice. The host-protective memory response was compromised in OX40L(-/-) mice, as decreased worm expulsion and increased egg production were observed compared with H. polygyrus-inoculated OX40L(+/+) mice. To further examine the nature of the IL-4 defect during priming, adoptively transferred DO11.10 T cells were analyzed in the context of the H. polygyrus response. Although Ag-specific T cell IL-4 production was reduced in the OX40L(-/-) mice following immunization with OVA peptide plus H. polygyrus, Ag-specific T cell expansion, cell cycle progression, CXCR5 expression, and migration were comparable between OX40L(+/+) and OX40L(-/-) mice inoculated with OVA and H. polygyrus. These studies suggest an important role for OX40/OX40L interactions in specifically promoting IL-4 production, as well as associated IgE elevations, in Th2 responses to H. polygyrus. However, OX40L interactions were not required for serum IgG1 elevations, increases in germinal center formation, and Ag-specific Th2 cell expansion and migration to the B cell zone.
Interactions between CD8+ T cells and endothelial cells are important in both protective and pathologic immune responses. Endothelial cells regulate the recruitment of CD8+ T cells into tissues, and the activation of CD8+ T cells by antigen presentation and costimulatory signals. PD-L1 and PD-L2 are recently described B7-family molecules which bind to PD-1 on activated lymphocytes and down-regulate T cell activation. We found that PD-L1 is expressed on interferon-gamma stimulated cultured human and mouse endothelial cells, while PD-L2 was found on stimulated human but not mouse endothelial cells. Expression was further up-regulated by TNF-alpha. Antibody blockade of endothelial cell PD-L1 and PD-L2 enhanced endothelial cell costimulation of PHA-activated human CD8+ T cells. Antibody blockade of mouse endothelial cell PD-L1 enhanced both IFN-gamma secretion and cytolytic activity of CD8+ T cells in response to endothelial cell antigen presentation. These results show that IFN-gamma activated endothelial cells can inhibit T cell activation via expression of the immunoinhibitory PD-L1 and PD-L2 molecules. Endothelial expression of PD-ligands would allow activation and extravasation of T cells without excessive vessel damage. Our findings highlight a potentially important pathway by which endothelial cells down-regulate CD8+ T cell-mediated immune responses.
When antigen-presenting cells (APCs) encounter inflammatory stimuli, they up-regulate their expression of B7. A small amount of B7 is also constitutively expressed on resting APCs, but its function is unclear. Here we show that initiation of T cell responses requires the expression of B7 on immunizing APCs, but the responses are much greater in the absence of basal B7 expression. Transfer of antigen-specific CD4+CD25+ cells reverses the increased responsiveness, and tolerance to a self-protein is broken by immunization in the absence of basal B7, thereby inducing autoimmunity. Similar loss of self-tolerance is seen on depletion of CD25+ cells. Thus, constitutively expressed B7 costimulators function to suppress T cell activation and maintain self-tolerance, principally by sustaining a population of regulatory T cells.
OX40 (CD134) is expressed on activated T cells; its ligand, OX40 ligand (OX40L) is expressed on dendritic cells, B cells, and activated endothelial cells. To determine how OX40-OX40L interaction affects graft-versus-host disease (GVHD), we used antagonistic anti-OX40L monoclonal antibody (mAb) or OX40(-/-) donor or OX40L(-/-) recipient mice. Similar degrees of GVHD reduction were observed with each approach. Despite the fact that OX40 is up-regulated on both CD4(+) and CD8(+) T cells isolated during GVHD, the major effects of OX40 ligation were on CD4(+) and not CD8(+) T-cell-mediated alloresponses as assessed in both GVHD and engraftment model systems. GVHD inhibition by blockade of the OX40/OX40L pathway did not require CD28 signaling. Some studies have indicated OX40 is essential for inducing T-helper type 2 (Th2) responses. However, in vivo blockade of OX40-OX40L interactions reduced GVHD mortality induced by either signal transducer and activator of transcription-6(-/-) (Stat-6(-/-)) (Th2-defective) or Stat-4(-/-) (Th1-defective) major histocompatibility complex (MHC)-disparate splenocytes, indicating that the GVHD-ameliorating effects did not require Stat-4 or Stat-6 signaling. Although OX40L has been reported to be expressed on activated T cells, no effects on GVHD were observed when OX40L(-/-) versus OX40L(+/+) T cells were infused in different models. These data provide insights as to the mechanisms responsible for OX40/OX40L regulation of GVHD.
Adoptive transfer experiments using C57BL/6 mice lacking B7-1 and B7-2 as recipients of wt (wt) encephalitogenic T cells demonstrate a key role for B7 costimulation during the effector phase of experimental autoimmune encephalomyelitis (EAE). Following transfer of encephalitogenic T cells, B7-1/B7-2-deficient (-/-) recipients develop a transient and mild disease as compared to wt recipients. To understand the mechanism by which B7-1/B7-2 may influence the effector phase of EAE, we analyzed T cells, pro-inflammatory cytokines and chemokines within the CNS of wt and B7-1/B7-2-/- recipients at different times after adoptive transfer of activated myelin specific T cells. There was a marked decline in T cells and inflammatory mediators in the CNS of B7-1/B7-2-/- recipients by day 30 post transfer. B7-1/B7-2-/- mice developed more TUNEL+ apoptotic cells in the parenchyma and greater ratios of TUNEL+ cells/parenchymal foci than wt mice resulting in virtual disappearance of parenchymal foci. Therefore, without B7-1 and B7-2 costimulation in the target organ, there is increased T cell apoptosis and attenuation of inflammation. These results indicate that B7-1 and B7-2 provide critical costimulatory signals for sustaining survival of pathogenic T cells within the central nervous system parenchyma during the effector phase of EAE and suggest novel treatment approaches in the effector phase of autoimmune diseases.
Newer members of the B7-CD28 superfamily include the receptor PD-1 and its two ligands, PD-L1 and PD-L2. Here, we characterize the expression of PD-1, PD-L1, and PD-L2 in tissues of naive miceand in target organs from two models of autoimmunity, the pancreas from non-obese diabetic (NOD) mice and brain from mice with experimental autoimmune encephalomyelitis (EAE). In naive mice, proteiexpression of PD-1, PD-L1, and PD-L2 was detected in the thymus, while PD-1 and PD-L1 were detected in the spleen. PD-L1, but not PD-L2, was also detected at low levels on cardiac endothelium, pancreatic islets, and syncyciotrophoblasts in the placenta. In pre-diabetic NOD mice, PD-1 and PD-L1 were expressed on infiltrating cells in the pancreatic islets. Furthermore, PD-L1 was markedly up-regulated on islet cells. In brains from mice with EAE, PD-1, PD-L1, and PD-L2 were expressed on infiltrating inflammatory cells, and PD-L1 was up-regulated on endothelium within EAE brain. The distinct expression patterns of PD-L1 and PD-L2 led us to compare their transcriptional regulation in STAT4(-/-), STAT6(-/-), or NF-kappaB p50(-/-)p65(+/-) dendritic cells (DC).PD-L2, but not PD-L1, expression was dramatically reduced in p50(-/-)p65(+/-) DC. Thus, PD-L1 and PD-L2 exhibit distinct expression patterns and are differentially regulated on the transcriptional level.
Although costimulation plays an important role in activating naive T cells, its role in negative selection is controversial. By following thymocyte deletion induced by endogenous superantigens in mice lacking B7-1 and/or B7-2, we have identified a role for both B7-1 and B7-2 in negative selection. Studies using CD28-deficient and CD28/CTLA-4-double-deficient mice have revealed that either CD28 or another as yet undefined coreceptor can mediate these B7-dependent signals that promote negative selection. Finally, CTLA-4 delivers signals that inhibit selection, suggesting that CTLA-4 and CD28 have opposing functions in thymic development. Combined, the data demonstrate that B7-1/B7-2-dependent signals help shape the T cell repertoire.
Inducible costimulatory molecule (ICOS) plays a pivotal role in T cell activation and Th1/Th2 differentiation. ICOS blockade has disparate effects on immune responses depending on the timing of blockade. Its role in transplantation immunity, however, remains incompletely defined. We used a vascularized mouse cardiac allograft model to explore the role of ICOS signaling at different time points after transplantation, targeting immune initiation (early blockade) or the immune effector phase (delayed blockade). In major histocompatibility-mismatched recipients, ICOS blockade prolonged allograft survival using both protocols but did so more effectively in the delayed-treatment group. By contrast, in minor histocompatibility-mismatched recipients, early blockade accelerated rejection and delayed blockade prolonged graft survival. Alloreactive CD4+ T cell expansion and alloantibody production were suppressed in both treatment groups, whereas only delayed blockade resulted in suppression of effector CD8+ T cell generation. After delayed ICOS blockade, there was a diminished frequency of allospecific IL-10-producing cells and an increased frequency of both IFN-gamma- and IL-4-producing cells. The beneficial effects of ICOS blockade in regulating allograft rejection were seen in the absence of CD28 costimulation but required CD8+ cells, cytotoxic T lymphocyte antigen-4, and an intact signal transducer and activator of transcription-6 pathway. These data define the complex functions of the ICOS-B7h pathway in regulating alloimmune responses in vivo.