B7-dependent Costimulation Research Articles

Role of the immune system in vascular function and blood pressure control induced by faecal microbiota transplantation in rats.

High blood pressure (BP) is associated with gut microbiota dysbiosis. The aim of this study was to investigate whether changes in gut microbiota induced by exchanging the gut microbiota between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) alter the gut-immune system interaction inducing changes in vascular function and BP. Twenty-week-old recipient WKY and SHR were orally gavaged with donor faecal contents from WKY or SHR. In additional experiments, we used a design to determine whether blockade of B7-dependent costimulation with CTLA4-Ig or blockade of IL-17 with IL-17-neutralizing antibody could prevent hypertension caused by faecal microbiota transplantation (FMT) from SHR to WKY. Correlation analyses identified the bacterial abundance of Turicibacter and S24-7_g that, respectively, positively and negatively correlated with systolic BP. FMT from WKY rats to SHR rats reduced basal systolic BP, restored the imbalance between Th17/Treg in mesenteric lymph nodes (MLNs) and aorta, and improved endothelial dysfunction and vascular oxidative status found in SHR transplanted with SHR faeces. FMT from SHR to WKY increased CD80 and CD86 mRNA levels and T cells activation in MLNs, circulating T cells, aortic T cell infiltration, impaired endothelial function and increased basal SBP. These effects were abolished by blockade of B7-dependent costimulation with CTLA4-Ig. IL-17a neutralizing antibody reduced SBP and improved endothelial dysfunction induced by FMT from SHR to WKY. Gut microbiota is an important factor involved in the control of BP, as a consequence of its effect in T-cell activation in gut immune system and vascular T-cells accumulation.

Accreditation Council for Pharmacy Education: Annual Report

In vivo anti-polysaccharide Ig responses to isolated polysaccharide (PS) are T cell independent, rapid, and fail to generate memory. However, little is known regarding PS-specific Ig responses to intact Gram-positive and Gram-negative extracellular bacteria. We previously demonstrated that intact heat-killed <i>Streptococcus pneumoniae</i>, a Gram-positive bacterium, elicited a rapid primary pneumococcal capsular PS (PPS) response in mice that was dependent on CD4⁺ T cells, B7-dependent costimulation, and CD40–CD40L interactions. However, this response was ICOS independent and failed to generate a boosted PPS-specific secondary IgG response. In the current study, we analyzed the murine meningococcal type C PS (MCPS)-specific Ig response to i.p.-injected intact, heat-killed <i>Neisseria meningitidis</i>, serogroup C (MenC), a Gram-negative bacterium. In contrast to <i>S. pneumoniae</i>, the IgG anti-MCPS response to MenC exhibited delayed primary kinetics and was highly boosted after secondary immunization, whereas the IgG anti-MCPS response to isolated MCPS was rapid, without secondary boosting, and consisted of only IgG1 and IgG3, as opposed to all four IgG isotypes in response to intact MenC. The secondary, but not primary, IgG anti-MCPS response to MenC was dependent on CD4⁺ T cells, CD40L, CD28, and ICOS. The primary and secondary IgG anti-MCPS responses were lower in TLR4-defective (C3H/HeJ) but not TLR2^−/− or MyD88^−/− mice, but secondary boosting was still observed. Of interest, coimmunization of <i>S. pneumoniae</i> and MenC resulted in a boosted secondary IgG anti-PPS response to <i>S. pneumoniae</i>. Our data demonstrate that the nature of the in vivo anti-PS response is markedly influenced by the composition and/or architecture of the bacterial subcapsular domain.

Structurally Identical Capsular Polysaccharide Expressed by Intact Group B Streptococcus versus Streptococcus pneumoniae Elicits Distinct Murine Polysaccharide-Specific IgG Responses In Vivo

We previously reported distinct differences in the murine in vivo Ig polysaccharide (PS)-specific responses to intact Streptococcus pneumoniae compared with responses to Neisseria meningitidis and that in each case, the bacterial subcapsular domain markedly influences the Ig response to the associated PS. In light of potentially unique contributions of biochemically distinct capsular PS and/or their characteristic attachments to the underlying bacterium, it remains unresolved whether different bacterial subcapsular domains can exert differential effects on PS-specific Ig responses to distinct bacterial pathogens. In this report, we used a mutant strain of group B Streptococcus (Streptococcus agalactiae) type III (GBS-III) that expresses desialylated capsular polysaccharide of GBS-III, biochemically identical to capsular pneumococcal polysaccharide type 14 (PPS14) of Streptococcus pneumoniae (intact inactivated Streptococcus pneumoniae, capsular type 14, Pn14), directly to compare the in vivo PPS14-specific IgG responses to two distinct gram-positive bacteria. Although both GBS-III and Pn14 elicited relatively rapid primary PPS14-specific IgG responses dependent on CD4(+) T cells, B7-dependent costimulation, and CD40-CD40L interactions, only GBS-III induced a highly boosted ICOS-dependent PPS14-specific IgG response after secondary immunization. Of note, priming with Pn14 and boosting with GBS-III, although not isolated PPS14, elicited a similar boosted PPS14-specific IgG response that was dependent on CD4(+) T cells during secondary immunization, indicating that Pn14 primes for memory but, unlike GBS-III, fails to elicit it. The inability of Pn14 to elicit a boosted PPS14-specific IgG response was overcome by coimmunization with unencapsulated GBS-III. Collectively, these data establish that structurally identical capsular PS expressed by two distinct gram-positive extracellular bacteria can indeed elicit distinct PS-specific IgG responses in vivo.

The Nature of an In Vivo Anti-Capsular Polysaccharide Response Is Markedly Influenced by the Composition and/or Architecture of the Bacterial Subcapsular Domain

In vivo anti-polysaccharide Ig responses to isolated polysaccharide (PS) are T cell independent, rapid, and fail to generate memory. However, little is known regarding PS-specific Ig responses to intact gram-positive and gram-negative extracellular bacteria. We previously demonstrated that intact heat-killed Streptococcus pneumoniae, a gram-positive bacterium, elicited a rapid primary pneumococcal capsular PS (PPS) response in mice that was dependent on CD4(+) T cells, B7-dependent costimulation, and CD40-CD40L interactions. However, this response was ICOS independent and failed to generate a boosted PPS-specific secondary IgG response. In the current study, we analyzed the murine meningococcal type C PS (MCPS)-specific Ig response to i.p.-injected intact, heat-killed Neisseria meningitidis, serogroup C (MenC), a gram-negative bacterium. In contrast to S. pneumoniae, the IgG anti-MCPS response to MenC exhibited delayed primary kinetics and was highly boosted after secondary immunization, whereas the IgG anti-MCPS response to isolated MCPS was rapid, without secondary boosting, and consisted of only IgG1 and IgG3, as opposed to all four IgG isotypes in response to intact MenC. The secondary, but not primary, IgG anti-MCPS response to MenC was dependent on CD4(+) T cells, CD40L, CD28, and ICOS. The primary and secondary IgG anti-MCPS responses were lower in TLR4-defective (C3H/HeJ) but not TLR2(-/-) or MyD88(-/-) mice, but secondary boosting was still observed. Of interest, coimmunization of S. pneumoniae and MenC resulted in a boosted secondary IgG anti-PPS response to S. pneumoniae. Our data demonstrate that the nature of the in vivo anti-PS response is markedly influenced by the composition and/or architecture of the bacterial subcapsular domain.

Found in translation: integrated approaches to drug development

The 44th Annual Scientific Meeting of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists; ‘Found in translation: integrated approaches to drug development’, was held in Melbourne, Australia, from 28 November to 1 December 2010. This was a wide-ranging conference that included some discussion of new drugs and new targets for drug development. The new drugs included dynamin inhibitors, Xen2174, an oxytocin receptor agonist and annexin-A12–26. The new targets included B7-dependent costimulation, chemoattractant receptors, granulocyte–macrophage colony-stimulating factor and glutathione peroxidases.

Inhibition and Genetic Ablation of the B7/CD28 T-Cell Costimulation Axis Prevents Experimental Hypertension

The pathogenesis of hypertension remains poorly understood, and treatment is often unsuccessful. Recent evidence suggests that the adaptive immune response plays an important role in this disease. Various hypertensive stimuli cause T-cell activation and infiltration into target organs such as the vessel and the kidney, which promotes vascular dysfunction and blood pressure elevation. Classically, T-cell activation requires T-cell receptor ligation and costimulation. The latter often involves interaction between B7 ligands (CD80 and CD86) on antigen-presenting cells with the T-cell coreceptor CD28. This study was therefore performed to examine the role of this pathway in hypertension. Angiotensin II-induced hypertension increased the presence of activated (CD86(+)) dendritic cells in secondary lymphatic tissues. Blockade of B7-dependent costimulation with CTLA4-Ig reduced both angiotensin II- and deoxycorticosterone acetate (DOCA)-salt-induced hypertension. Activation of circulating T cells, T-cell cytokine production, and vascular T-cell accumulation caused by these hypertensive stimuli were abrogated by CTLA4-Ig. Furthermore, in mice lacking B7 ligands, angiotensin II caused minimal blood pressure elevation and vascular inflammation, and these effects were restored by transplantation with wild-type bone marrow. T-cell costimulation via B7 ligands is essential for development of experimental hypertension, and inhibition of this process could have therapeutic benefit in the treatment of this disease.

Inhibition of T cell Costimulation Prevents the Development of Hypertension

Classically, T cell activation requires antigen recognition by the T cell receptor and costimulation. The latter often involves interaction between B7 ligands (CD80 and CD86) on antigen presenting cells (APCs) with the T cell co receptor CD28. We hypothesized that inhibition of costimulation would reduce hypertension and its associated vascular inflammation. Blockade of B7 dependent costimulation with the human recombinant fusion protein, CTLA4Ig, prevented both Ang II (490 ng/kg/min) and DOCA salt induced hypertension in mice (Ang II: 168 ± 1.5 Vs 139 ± 2.8 mmHg, P&lt;0.001; DOCA: 153 ± 3.2 Vs 135 ± 2.7 mmHg, P&lt;0.001). FACS revealed that circulating T cell activation and vascular T cell infiltration caused by these hypertensive stimuli was completely abrogated by CTLA4Ig. Additionally, B7KO mice were protected from Ang II induced hypertension (154 ± 2.5 Vs 124 ± 3.3 mmHg, P&lt;0.001) and vascular T cell infiltration. Engrafting B7 mice with WT bone marrow restored Ang II induced hypertension. Finally, we found that angiotensin II infusion increased the percent of CD11c+CD86+ cells in lymph nodes, suggesting APC activation in hypertension. These findings indicate that T cells are activated classically in hypertension, suggestive of a possible neoantigen produced by or in response to hypertensive stimuli. This study suggests a novel approach to hypertension treatment. Funded by NIH R01 HL39006 and PPG HL58000.

Parameters Underlying Distinct T Cell-Dependent Polysaccharide-Specific IgG Responses to an Intact Gram-Positive Bacterium versus a Soluble Conjugate Vaccine

IgG anti-polysaccharide (PS) responses to both intact Streptococcus pneumoniae (Pn) and PS conjugate vaccines are dependent on CD4(+) T cells, B7-dependent costimulation, and CD40-CD40-ligand interactions. Nevertheless, the former response, in contrast to the latter, is mediated by an ICOS-independent, apoptosis-prone, extrafollicular pathway that fails to generate PS-specific memory. We show that pre-existing PS-specific Igs, the bacterial surface or particulation, selective recruitment of B cell subsets, or activation and recruitment of Pn protein-specific CD4(+) T cells do not account for the failure of Pn to generate PS-specific IgG memory. Rather, the data suggest that the critical factor may be the lack of covalent attachment of PS to protein in intact Pn, highlighting the potential importance of the physicochemical relationship of PS capsule with the underlying bacterial structure for in vivo induction of PS-specific Igs.

A Novel ICOS-Independent, but CD28- and SAP-Dependent, Pathway of T Cell-Dependent, Polysaccharide-Specific Humoral Immunity in Response to IntactStreptococcus pneumoniaeversus Pneumococcal Conjugate Vaccine

Polysaccharide (PS)- and protein-specific murine IgG responses to intact Streptococcus pneumoniae (Pn) are both dependent on CD4(+) T cell help, B7-dependent costimulation, and CD40/CD40 ligand interactions. However, the primary PS-specific, relative to protein-specific, IgG response terminates more rapidly, requires a shorter period of T cell help and B7-dependent costimulation, and fails to generate memory. In light of the critical role for ICOS/ICOS ligand interactions in sustaining T cell-dependent Ig responses and promoting germinal center reactions, we hypothesized that this interaction was nonessential for PS-specific IgG responses to Pn. We now demonstrate that ICOS(-/-), relative to wild-type, mice elicit a normal PS-specific IgG isotype response to Pn, despite marked inhibition of both the primary and secondary IgG anti-protein (i.e., PspA, PspC, and PsaA) response. A blocking anti-ICOS ligand mAb injected during primary Pn immunization inhibits both the primary anti-protein response and the generation of protein-specific memory, but has no effect when injected during secondary immunization. In contrast to Pn, both PS- and protein-specific IgG responses to a pneumococcal conjugate vaccine are inhibited in ICOS(-/-) mice. ICOS(-/-) mice immunized with intact Pn or conjugate exhibit nearly complete abrogation in germinal center formation. Finally, although mice that lack the adaptor molecule SAP (SLAM-associated protein) resemble ICOS(-/-) mice (and can exhibit decreased ICOS expression), we observe that the PS-specific, as well as protein-specific, IgG responses to both Pn and conjugate are markedly defective in SAP(-/-) mice. These data define a novel T cell-, SAP-, and B7-dependent, but ICOS-independent, extrafollicular pathway of Ig induction.

Accreditation Council for Pharmacy Education: Annual Report

In vivo anti-polysaccharide Ig responses to isolated polysaccharide (PS) are T cell independent, rapid, and fail to generate memory. However, little is known regarding PS-specific Ig responses to intact Gram-positive and Gram-negative extracellular bacteria. We previously demonstrated that intact heat-killed <i>Streptococcus pneumoniae</i>, a Gram-positive bacterium, elicited a rapid primary pneumococcal capsular PS (PPS) response in mice that was dependent on CD4⁺ T cells, B7-dependent costimulation, and CD40–CD40L interactions. However, this response was ICOS independent and failed to generate a boosted PPS-specific secondary IgG response. In the current study, we analyzed the murine meningococcal type C PS (MCPS)-specific Ig response to i.p.-injected intact, heat-killed <i>Neisseria meningitidis</i>, serogroup C (MenC), a Gram-negative bacterium. In contrast to <i>S. pneumoniae</i>, the IgG anti-MCPS response to MenC exhibited delayed primary kinetics and was highly boosted after secondary immunization, whereas the IgG anti-MCPS response to isolated MCPS was rapid, without secondary boosting, and consisted of only IgG1 and IgG3, as opposed to all four IgG isotypes in response to intact MenC. The secondary, but not primary, IgG anti-MCPS response to MenC was dependent on CD4⁺ T cells, CD40L, CD28, and ICOS. The primary and secondary IgG anti-MCPS responses were lower in TLR4-defective (C3H/HeJ) but not TLR2^−/− or MyD88^−/− mice, but secondary boosting was still observed. Of interest, coimmunization of <i>S. pneumoniae</i> and MenC resulted in a boosted secondary IgG anti-PPS response to <i>S. pneumoniae</i>. Our data demonstrate that the nature of the in vivo anti-PS response is markedly influenced by the composition and/or architecture of the bacterial subcapsular domain.

TCR subunit specificity of CTLA-4-mediated signaling.

Cytotoxic T-lymphocyte-associated antigen (CTLA)-4 is an activation-induced receptor that down-regulates T cell responses by antagonizing B7-dependent costimulation and/or by transducing a negative signal. The mechanism of CTLA-4-mediated negative signaling is unknown. Recently, it has been postulated that CTLA-4 inhibits T cell activation by causing specific dephosphorylation of the T cell receptor (TCR)-zeta chain of the antigen-receptor complex through an lck-dependent recruitment of the Src homology-2-containing tyrosine phosphatase-2. To test this hypothesis, we generated stably transfected T cell clones expressing doxycycline-inducible CTLA-4 with CD25:TCR-zeta (CD25-zeta) or CD25:CD3-epsilon (CD25-epsilon) fusion proteins. In these clones, ligation of CD25-zeta or of CD25-epsilon with antibodies against CD25 induced full T cell activation, as illustrated by extracellular signal-regulated kinase (ERK) activation and interleukin (IL)-2 production. More importantly, coligation of CTLA-4 with CD25-zeta or of CTLA-4 with CD25-epsilon in the respectively transfected clones inhibited ERK activation and IL-2 production, demonstrating that CTLA-4 does not specifically inhibit signals from TCR-zeta but can also inhibit signals from CD3-epsilon. Our results suggest that the target specificity of CTLA-4 is determined by its coligation with any given transmembrane receptor rather than by its intracellular mediators.

The effects of B7-dependent costimulation on T cell division and survival in vivo and in vitro are dependent on antigen concentration.

We used 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester labeled TCR-transgenic CD4(+) T cells to investigate the contribution of B7 costimulation to T cell activation and clonal expansion. B7 costimulation was blocked with the fusion protein cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)-Ig, which prevents the interaction of B7 with its receptor CD28 on T cells. CTLA4-Ig had different effects depending on the density of antigen (Ag)/MHC ligands available by T cells. In the presence of CTLA4-Ig, tenfold higher concentrations of Ag were required for T cells to undergo cell division in vitro. At high Ag concentrations, T cell division occurred at comparable rates whether in the presence or absence of CTLA4-Ig; however, T cell survival and clonal expansion were strongly inhibited. Addition of IL-2 restored T cell survival but not responsiveness to low doses of Ag. In vivo, B7 costimulation was similarly required for the survival of Ag-specific T cells but not for cell division in response to high amounts of Ag. Thus, B7 costimulation regulates CD4(+) T cell responses by promoting cell division in the presence of limiting amounts of Ag, and by protecting T cells from the onset of apoptosis.

4-1BB (CD137) differentially regulates murine in vivo protein- and polysaccharide-specific immunoglobulin isotype responses to Streptococcus pneumoniae.

4-1BB (CD137) is induced on activated CD4(+) and CD8(+) T cells and delivers a costimulatory signal upon binding the 4-1BB ligand (4-1BBL) expressed on antigen-presenting cells. Induction of 4-1BB is dependent on activation via the T-cell receptor (TCR) and possibly CD28. It was previously demonstrated that both an in vivo protein (pneumococcal surface protein A [PspA])- and polysaccharide (phosphorylcholine [PC] determinant of teichoic acid)-specific immunoglobulin (Ig) isotype response to Streptococcus pneumoniae was dependent on CD4(+) TCRalphabeta(+) T cells and B7-dependent costimulation through CD28. We thus postulated that 4-1BB costimulation would also play a role in regulating the in vivo anti-PspA and anti-PC response to S. pneumoniae. We demonstrate that mice genetically deficient in 4-1BBL elicit a markedly reduced IgM and IgG anti-PC but normal primary and secondary IgG anti-PspA responses to S. pneumoniae relative to those for wild-type mice. However, injection of an agonistic anti-4-1BB monoclonal antibody (MAb), while having no significant effect on the anti-PC response, strongly inhibits the primary anti-PspA response, the generation of PspA-specific memory, and germinal center formation but does not induce a lasting state of tolerance. In contrast, anti-4-1BB MAb has no effect on the anti-PspA response when injected only at the time of secondary immunization. Delay of the addition of anti-4-1BB leads to progressively less inhibition of the primary response up to day 8. This inhibition is independent of CD8(+) T cells and is associated with the expansion of CD4(+) T cells with an activated phenotype, which is partly dependent on B7-dependent costimulation. These data are the first to suggest a stimulatory role for endogenous 4-1BB-4-1BBL interactions during a humoral immune response to a pathogen and further underscore significant differences in costimulation requirements for an in vivo protein- versus polysaccharide-specific Ig isotype response to an extracellular bacterium.

The mechanism underlying T cell help for induction of an antigen-specific in vivo humoral immune response to intact Streptococcus pneumoniae is dependent on the type of antigen.

Little is known concerning the role of T cells in regulating an anti-polysaccharide Ig response to an intact pathogen. We previously reported that the in vivo Ig responses to Streptococcus pneumoniae (strain R36A), specific for pneumococcal surface protein A (PspA) and for the phosphorylcholine (PC) determinant of C-polysaccharide, were both dependent on TCR-alphabeta(+) T cells and B7-dependent costimulation, although only PspA-specific memory was generated. In this report, we show that the T cell help underlying these two Ag-specific Ig responses is distinct. Using H-Y-specific T cell transgenic mice made "nonleaky" by crossing with mice genetically deficient for TCR-alpha, we demonstrate that the T cell help for the anti-PC, in contrast to the anti-PspA, response is TCR-nonspecific and occurs normally in the absence of germinal center formation, although it is still dependent on B7-dependent costimulation. Consistent with these data, we demonstrate, using cathepsin S(-/-) mice, that although the anti-PC response is largely dependent on CD4(+) T cells, there is a reduced (or lack of) dependence, relative to the anti-PspA response, on the generation of new peptide-MHC class II complexes. In this regard, the T cell help for an optimal anti-PC response is delivered more rapidly than that required for an optimal anti-PspA response. Collectively, these data demonstrate a novel accelerated TCR-nonspecific B7-dependent form of T cell help for augmenting a polysaccharide-specific Ig response to an intact bacterium without the generation of memory.

Dendritic cells pulsed with intact Streptococcus pneumoniae elicit both protein- and polysaccharide-specific immunoglobulin isotype responses in vivo through distinct mechanisms.

Immature bone marrow–derived myeloid dendritic cells (BMDCs) are induced to undergo phenotypic maturation and secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-12, and IL-10 when pulsed in vitro with intact Streptococcus pneumoniae. After transfer to naive mice, pulsed BMDCs induce immunoglobulin (Ig) isotype responses specific for both protein and polysaccharide pneumococcal antigens, having in common the requirement for viable BMDCs, T cells, and B7-dependent costimulation in the recipient mice. Whereas primary Ig isotype responses to bacterial proteins uniformly require BMDC expression of major histocompatibility complex class II, CD40, and B7, and the secretion of IL-6, but not IL-12, similar requirements for antipolysaccharide Ig responses were only observed for the IgG1 isotype.

B7-dependent T-cell costimulation in mice lacking CD28 and CTLA4.

To examine whether B7 costimulation can be mediated by a molecule on T cells that is neither CD28 nor CTLA4, we generated mice lacking both of these receptors. CD28/CTLA4(-/-) mice resemble CD28(-/-) mice in having decreased expression of T-cell activation markers in vivo and decreased T-cell proliferation in vitro, as compared with wild-type mice. Using multiple approaches, we find B7-dependent costimulation in CD28/CTLA4(-/-) mice. The proliferation of CD28/CTLA4(-/-) T cells is inhibited by CTLA4-Ig and by the use of antigen-presenting cells lacking both B7-1 and B7-2. CD28/CTLA4(-/-) T-cell proliferation is increased by exposure to Chinese hamster ovary cells transfected with B7-1 or B7-2. Finally, administration of CTLA4-Ig to CD28/CTLA4(-/-) cardiac allograft recipients significantly prolongs graft survival. These data support the existence of an additional receptor for B7 molecules that is neither CD28 nor CTLA4.

In vivo blockade of B7-dependent costimulation does not affect IL-12 signaling

In vivo blockade of B7-dependent costimulation does not affect IL-12 signaling

Absence of B7-dependent responses in CD28-deficient mice

Costimulation of T cell proliferation can occur through the CD28 signal transduction pathway. In addition, other cell surface receptors, including the CD28 homolog CTLA-4, have been proposed to be capable of providing costimulatory signals. We have examined the response of CD28-deficient T cells to activation by a variety of agonists. We demonstrate that proliferation of CD28-deficient T cells in the presence of antigen-presenting cells or B7-1 transfectants is markedly reduced. Although CTLA-4 can be expressed on CD28-deficient T cells, we observed no B7-dependent costimulation in the absence of CD28. This data demonstrates that CD28 is the major B7-binding costimulatory ligand on T cells. Furthermore, our data suggest that CD28 is the primary, and perhaps exclusive, costimulatory receptor used by traditional antigen-presenting cells to augment the proliferation of antigenactivated T cells.