Role of capsular polysaccharide in Group B Streptococccus interactions with dendritic cells

Group B Streptococcus (GBS) type III is an important agent of life-threatening invasive infections. Albeit the immune system plays a dual role in development and protection against disease, mechanisms leading to an efficient immune response against GBS remain obscure. Mouse bone marrow-derived dendritic cells (DCs) and primary spleen DCs were used to evaluate GBS capacity to modulate the functions of these important antigen-presenting cells. The role of capsular polysaccharide (CPS), one of the most important GBS virulence factors, in bacterial-DC interactions was evaluated by using a non-encapsulated mutant. Phagocytosis assays, confocal and electron microscopy showed that DCs efficiently internalize encapsulated GBS, but the latter possesses strong intracellular survival capacity. GBS devoid of CPS was internalized and killed at higher and faster rates than encapsulated GBS early after infection. Among several cytokines tested, GBS internalization was required for modulation of IL-12, IL-10 and CXCL10 pathways. In contrast, GBS induced DC expression of co-stimulatory molecules in a phagocytosis-independent manner. Finally, the production of pro-inflammatory and Th1 cytokines by GBS-stimulated DCs was differentially modulated by CPS expression, depending on DC origin. Our data suggest multiple mechanisms involved in GBS modulation of DC functions, which were selectively regulated by the presence of CPS.     

Manipulating dendritic cells to induce regulatory T cells

Dendritic cells (DCs) induce and regulate T-cell responses, and tolerogenic DCs can promote the development of regulatory T cells with suppressive activity. The possibility of manipulating DCs using different pharmacological or biological agents, enabling them to exert tolerogenic activities, could be exploited to better control a variety of chronic inflammatory conditions, from autoimmune diseases to allograft rejection.     

Identification of glycosylated regions in pneumococcal PspA conjugated to serotype 6B capsular polysaccharide

Conjugate vaccines are being widely used since their introduction. Nowadays the interest in these vaccines is still growing and new antigens and conjugate chemistry are being studied and developed. Pneumococcal surface protein A (PspA) is one of the most studied pneumococcal antigens and is an important vaccine candidate. One approach to broaden the conjugate vaccine coverage could be the conjugation of the polysaccharide to a pneumococcal protein such as PspA. Previous results have shown that conjugated recombinant fragment of PspA (rPspA) not only maintained but also in some conjugates improved the induction of protective antibodies raised against the protein carrier. We describe here a characterization study to identify the domains of Streptococcus pneumoniae recombinant PspA (rPspA), from family 1 clade 1 and family 2 clade 3, involved in the conjugation with serotype 6B capsular polysaccharide.     

Requirement for B cells for activation of contrasuppressor T cells by type III pneumococcal polysaccharide

Type III pneumococcal polysaccharide (S3) is unable to activate S3-specific contrasuppressor T cells (Tcs) in mice depleted of B cells by chronic anti-IgM treatment or in immune defective xid mice that lack the B cell subset required for anti-S3 antibody responses. The inability of S3 to activate Tcs in xid mice was shown to be due to a requirement of B cells for Tcs activation rather than to an absence of Tcs in xid mice. The B cells from normal mice that are required for Tcs activation apparently function to present the S3 Ag to Tcs. S3 physically coupled to spleen cells (S3-SC) prepared from normal BCF1 SC could activate Tcs in both xid and BCF1 mice whereas S3-SC prepared from xid SC or B cell-depleted BCF1 SC could not activate Tcs in either strain. B cell APC function was abrogated by 3000 R irradiation and by treatment of the B cells with either chloroquine or paraformaldehyde. Interestingly, B cells from mice previously immunized with S3 were unable to function in Tcs activation; preimmunization of B cell donors with an irrelevant Ag or with a T-dependent form of S3 had no effect on their ability to function as APC. These latter observations are discussed in terms of the in vivo persistence of polysaccharide Ag and their ability to induce B cell tolerance under the experimental conditions used for these experiments. The results of this study provide evidence that B cells play an important and apparently obligatory role in the activation of Tcs by S3; B cells apparently function to present Ag to Tcs, resulting in the activation of this regulatory T cell subset.     

Peptide mimotopes of pneumococcal capsular polysaccharide of 6B serotype: a peptide mimotope can bind to two unrelated antibodies

Two groups of bacteriophage clones displaying the antigenic properties of serotype 6B pneumococcal capsular polysaccharide (PS) were obtained from different phage libraries expressing random heptameric peptides. One group, biopanned with a mouse mAb (Hyp6BM1), is comprised of 17 phage clones expressing 10 unique sequences of linear peptides. The other group, selected with another mAb (Hyp6BM8), contained six clones, all of which expressed the identical circular peptide. Phage clones expressing the linear peptides (e.g., PhaM1L3) bound only to Hyp6BM1, but not other 6B PS-specific mAb, and their binding could be inhibited with pneumococcal capsular type 6B PS only. In contrast, a phage clone expressing the circular peptide (PhaM8C1) cross-reacted with several other 6B PS-specific mAbs, and their binding could be inhibited with pneumococcal capsular PS of 6A and 6B serotypes. Two short peptides, PepM1L3 and PepM8C1, reflecting the peptide inserts of the corresponding phage clones, could inhibit the binding of the two clones to their respective mAb. Interestingly, the peptide insert in PhaM8C1 was identical to that in PhaB3C4, a previously reported mimotope of alpha(2-->8) polysialic acid, Neisseria meningitidis group B PS. Indeed, PhaM8C1 bound to HmenB3 (a meningococcal Ab), and their association could be inhibited with alpha(2-8) polysialic acid, but not with 6B PS. Conversely, alpha(2-8) polysialic acid could not inhibit the binding of PhaM8C1 to Hyp6BM8. The two-dimensional nuclear magnetic resonance studies indicate that PepM8C1 peptide can assume several conformations in solution. The ability of this peptide to assume multiple conformations might account for its ability to mimic more than one Ag type.     

Apoptotic cells induce immunosuppression through dendritic cells: critical roles of IFN-gamma and nitric oxide

Apoptotic cells induce immunosuppression through unknown mechanisms. To identify the underlying molecular mediators, we examined how apoptotic cells induce immunoregulation by dendritic cells (DC). We found that administration of DC exposed to apoptotic cells (DC(ap)) strongly inhibited the expansion of lymphocytes in draining lymph nodes in vivo and the subsequent Ag-specific activation of these lymphocytes ex vivo. Unexpectedly, DC(ap) supported T cell activation to a similar extent as normal DC in vitro, leading to proliferation and IL-2 production, except that DC(ap) did not support T cell production of IFN-gamma. Surprisingly, when DC(ap) were cocultured with normal DC, they completely lost their ability to support T cell activation, an effect reversed by anti-IFN-gamma or inhibitors of inducible NO synthase (iNOS). As expected, exposure to apoptotic cells rendered DC(ap) capable of producing much more NO in response to exogenous IFN-gamma than normal DC. Furthermore, DC(ap) from iNOS(-/-) or IFN-gammaR1(-/-) mice were not inhibitory in vitro or in vivo. Therefore, the IFN-gamma-induced production of NO by apoptotic cell-sensitized DC plays a key role in apoptotic cell-mediated immunosuppression.     

Serum amyloid A inhibits dendritic cell apoptosis to induce glucocorticoid resistance in CD4+ T cells

Mediators produced by the airway epithelium control the activation, recruitment, and survival of pulmonary dendritic cells (DC) that present antigen to CD4⁺ T cells during the genesis and exacerbation of allergic asthma. The epithelial-derived acute phase protein, serum amyloid A (SAA), induces DC maturation and T_H17 polarization. T_H17 responses are associated with severe forms of allergic asthma that are poorly controlled by corticosteroids. We sought to determine whether SAA would enhance the survival of DC during serum starvation and could then contribute to the development of a glucocorticoid-resistant phenotype in CD4⁺ T cells. Bone marrow-derived dendritic cells (BMDC) that were serum starved in the presence of SAA were protected from activation of caspase-3 and released less lactate dehydrogenase. In comparison with untreated serum-starved BMDC, treatment with SAA downregulated mRNA expression of the pro-apoptotic molecule Bim, increased production of the pro-survival heat shock protein 70 (HSP70), and induced secretion of pro-inflammatory cytokines. SAA-treated BMDC that were serum starved for 48 h remained capable of presenting antigen and induced OTII CD4⁺ T cells to secrete IL-17A, IL-17F, IL-21, IL-22, and IFNγ in the presence of ovalbumin. IL-17A, IL-17F, IL-21, and IFNγ production occurred even when the CD4⁺ T cells were treated with dexamethasone (Dex), whereas glucocorticoid treatment abolished cytokine secretion by T cells cocultured with untreated serum-starved BMDC. Measurement of Dex-responsive gene expression demonstrated CD4⁺ T cells as the target of glucocorticoid hyperresponsiveness manifest as a consequence of BMDC stimulation by SAA. Finally, allergic airway disease induced by SAA and antigen inhalation was unresponsive to Dex treatment. Our results indicate that apo-SAA affects DC to both prolong their viability and increase their inflammatory potential under apoptosis-inducing conditions. These findings reveal mechanisms through which SAA enhances the CD4⁺ T-cell-stimulating capacity of antigen-presenting cells that may actively participate in the pathogenicity of glucocorticoid-resistant lung disease.     

A strain-independent method to induce progressive and lethal pneumococcal pneumonia in neutropenic mice

Background

Experimental models of pneumonia with penicillin non-susceptible Streptococcus pneumoniae (PNSSP) are hard to reproduce because the majority of strains with clinical relevance (like serotypes 6B, 9 V and 19 F) have low murine virulence. By optimization of culture and inoculum conditions of PNSSP (using porcine mucin), our aim was to develop a suitable, reliable and reproducible pneumonia mouse model for anti-infective pharmacology research.

Results

Seven PNSSP strains, including serotypes 6B, 9 V, 14 and 19 F were included. Strain INS-E611 displayed the highest murine virulence and was chosen to validate the lung model. Nose-instilled pneumococci grew between 2.1 and 2.5 log₁₀ CFU/g of lung in 24 hours when an optimized culture of bacterial cells was used, but animals were all alive and recovered of infection after 36 h. In contrast, inoculum supplementation with mucin led to 100% mortality related to a successful lung infection confirmed by histopathology. These findings were reproduced with all seven PNSSP strains in neutropenic mice. Immunocompetent animals cleared all strains spontaneously.

Conclusions

This pneumonia model produces a progressive and uniformly fatal lung infection with diverse serotypes of PNSSP independently of their intrinsic murine virulence.     

Characterization and activity of contrasuppressor T cells induced by type III pneumococcal polysaccharide

Optimally immunogenic amounts of type III pneumococcal polysaccharide (S3) activate a population of contrasuppressor T cells (Tcs), which have been shown to play an important role in the induction of anti-S3 antibody responses. These Tcs belong to a unique T cell subset that has the surface phenotype Lyt 1+2- L3T4- I-J+ I-A+. These Tcs are also cyclophosphamide (Cy)-sensitive and sensitive to antilymphocyte serum (ALS) and mitomycin C. Tcs have antigen-binding receptors, indicating that any interactions of Tcs with B cells or T suppressor cells (Ts) (both of which also have antigen-binding receptors) must be via an antigen bridge rather than an idiotype-anti-idiotype interaction. Tcs are also Igh restricted in their action. Contrasuppression is manifest only when the Tcs are Igh compatible with both the Ts and the responding B cells. Tcs apparently mediate their effects by releasing a soluble factor, since a soluble factor extracted from Tcs is able to abrogate the effects of S3-specific Ts.     

Requirements for activation of contrasuppressor T cells by type III pneumococcal polysaccharide

Either S3-coupled spleen cells (S3-SC) or soluble S3 activates two populations of regulatory T cells, T suppressor cells (Ts) and contrasuppressor T cells (Tcs). The latter cells function to mask the activity of Ts in unfractionated T cell populations, so that Ts can be detected only after removal of Tcs. Activation of Tcs by S3 may be required for induction of an antibody response to S3. This is suggested by the findings that Tcs are activated only by immunogenic doses of S3, that Tcs are not detectable in the spleens of mice tolerant to S3, and that (CBA/N X BALB/c)F1 male (xid) mice, which are genetically unresponsive to S3, do not develop Tcs after immunization with S3. Moreover, the kinetics of activation of Tcs by S3 closely parallels the kinetics of the antibody response to S3. Tcs have no detectable activity in the absence of Ts, indicating that these cells do not function as amplifier or helper T cells.     

Regulation of IgG responses by helper and suppressor T cells activated by pneumococcal capsular polysaccharides

Type 2 antigens are usually unable to prime the helper T cells (TH) required for secondary IgG antibody responses. However, previous results from this laboratory indicated that low doses of the type 2 antigen polyvinylpyrrolidone (PVP) could activate T cells which provided help to PVP-primed B cells for the production of PVP-specific IgG antibody. Therefore, it was of interest to determine if other type 2 antigens may also be able to activate TH. Low doses of S19 or S3 (subimmunogenic for a primary IgM response) activated TH capable of providing help to S19- or S3-CRBC-primed B cells for a secondary IgG response. Higher doses of these antigens (optimally immunogenic for a primary IgM response) activated suppressor T cells (TS). Removal of these TS prior to transfer of T cells to recipient mice resulted in expression of TH function. Therefore, the preferential activation of TH versus TS was dependent on the dose of antigen used for priming. TH activated by low doses of S19 expressed Thy 1 and L3T4 and were antigen specific. In contrast to the ability of low doses of PVP to prime B cells for secondary IgG responses, low doses of S3 and S19 did not prime capsular polysaccharide-specific IgG memory B cells. High doses of S3 were able to prime B cells if TS precursors were first removed by treatment of mice with cyclophosphamide (Cy), whereas high doses of S19 did not prime B cells for secondary IgG responses in either Cy-treated or control mice. These results are discussed in relation to the general observations that type 2 antigens may not activate antigen-specific TH.     

Distinct roles of dendritic cells and B cells in Va14Ja18 natural T cell activation in vivo

Va14Ja18 natural T (iNKT) cells are innate, immunoregulatory lymphocytes that recognize CD1d-restricted lipid Ags such as alpha-galactosylceramide (alpha GalCer). The immunoregulatory functions of iNKT cells are dependent upon either IFN-gamma or IL-4 production by these cells. We hypothesized that alpha GalCer presentation by different CD1d-positive cell types elicits distinct iNKT cell functions. In this study we report that dendritic cells (DC) play a critical role in alpha GalCer-mediated activation of iNKT cells and subsequent transactivation of NK cells. Remarkably, B lymphocytes suppress DC-mediated iNKT and NK cell activation. Nevertheless, alpha GalCer presentation by B cells elicits low IL-4 responses from iNKT cells. This finding is particularly interesting because we demonstrate that NOD DC are defective in eliciting iNKT cell function, but their B cells preferentially activate this T cell subset to secrete low levels of IL-4. Thus, the differential immune outcome based on the type of APC that displays glycolipid Ags in vivo has implications for the design of therapies that harness the immunoregulatory functions of iNKT cells.     

Mouse monoclonal antibodies to chicken VH idiotypic determinants reactivity with B and T cells

We have prepared mouse monoclonal antibodies against idiotypic (Id) determinants on chicken antibodies to N-acetylglucosamine (NAGA) and p-aminobenzoic acid (PABA) made by inbred line EL 6(3) birds. The monoclonal anti-NAGA Id antibody, termed CId-1, reacted with affinity purified antibodies to NAGA, but not with antibodies specific for PABA, arsanilic acid (Ars), phosphorylcholine (PC), or with normal chicken IgG and IgM. The monoclonal anti-PABA ID antibody, termed CId-2, reacted with anti-PABA antibodies and to a lesser extent with anti-Ars antibodies, but not with anti-NAGA, anti-PC, and normal IgG and IgM. The Id determinants were found among antibodies to NAGA and PABA made by outbred and inbred lines of White Leghorn chickens. The binding of the CId-1 and CId-2 antibodies to intact homologous anti-NAGA and anti-PABA antibodies, respectively, was not hapten-inhibitable in either case. Both anti-Id antibodies reacted specifically with isolated homologous heavy chains, suggesting VH Id specificities. The monoclonal CId-1 and CId-2 antibodies were reactive by immunofluorescence with approximately 0.9 and 0.2%, respectively, of the circulating lymphocytes and with approximately 0.4 and 0.15 of plasma cells. CId-1+ and CId-2+ bursal cells were first detected on the 16th and 14th days of incubation, respectively; both reached maximal frequencies by the 17th day of incubation. The CId-2 antibody reacted exclusively with immunoglobulin-positive cells. The CId-1 antibody also reacted with a subpopulation (0.4%) of immunoglobulin-negative lymphocytes from normal and agammaglobulinemic chickens, and thus would appear to recognize an idiotypic determinant expressed by certain clones of B and T cells.     

肺炎球菌1型荚膜多糖多克隆抗体制备与夹心ELISA法建立及其在多糖浓度测定中的应用

目的：利用肺炎球菌1型全菌体制备多克隆抗体,并且利用该抗体建立肺炎1型荚膜多糖夹心酶联免疫吸附分析法（ Enzyme-linked immunosorbent assay ,ELISA）,用于检测发酵和纯化过程中的多糖浓度。方法用灭活的1型肺炎链球菌免疫家兔6周,获得高滴度的抗多糖血清,经过亲和层析纯化,获得高纯度的兔抗肺炎1型多糖抗体IgG。以纯化IgG作为包被抗体,加入多糖样品,再以生物素化的抗体作为检测抗体,建立夹心ELISA法检测肺炎1型多糖浓度。确定标准曲线的最佳线性范围,并对该方法进行特异性、准确性和精密度验证。结果兔免疫血清经过双向免疫扩散检测抗体滴度可达1∶32;该方法的线性检测范围为1．56～50 ng/mL;最低检测限为3．13 ng/mL。在标准品中混入其他型别多糖或培养基,回收率分别为102％和108％;该方法批内精密度和批间精密度分别为6．08％和7．01％。结论建立的夹心ELISA方法,其特异性、准确性和精密度均良好,可以特异地检测肺炎球菌1型多糖浓度。     

In vivo priming of natural killer T cells by dendritic cells pulsed with hepatoma-derived acid-eluted substances

Many murine tumor cells express not only individual haplotype-matched class I MHC molecules, but also species-specific CD1d molecules. The former class I MHC molecules generally present internally synthesized tumor-derived peptide antigens to highly specific CD8⁺ cytotoxic T lymphocytes (CTLs) in acquired immunity. In contrast, the latter CD1d molecules may present tumor-associated glycolipid antigens to broadly crossreactive natural killer T (NKT) cells, which might correlate with controlling tumor metastasis. Here, we showed that murine hepatoma cell line Hepa1-6-derived acid-eluted substances might contain both D^b class I MHC-restricted antigens and CD1d-restriced substances, which could sensitize not only syngeneic bone marrow-derived DCs (BM-DCs), but also allogeneic BM-DCs expressing haplotype-mismatched class I MHC and species-specific CD1d molecules. To our surprise, intravenous (i.v.) immunization of C57BL/6 mice with the former syngeneic BM-DCs carrying acid-eluted materials primed both CD4^–CD8^– and CD8⁺ NKT cells in the spleen, whereas immunization with the latter allogeneic BM-DCs loaded the tumor-derived substances primed CD4^–CD8^–, but not CD8⁺ NKT cells. The findings shown in the present study will open a new area for cancer immunotherapy using allogeneic DCs and tumor-derived acid-eluted substances.Abbreviations CTLs cytotoxic T lymphocytes - NKT natural killer T - BM-DCs bone marrow-derived dendritic cells - CTM complete T-cell medium - FCS fetal calf serum - MMC mitomycin C - TCRs T cell receptors     

Cell surface antigens and other characteristics of T cells regulating the antibody response to type III pneumococcal polysaccharide

The administration of a subimmunogenic dose of type III pneumococcal polysaccharide (SSS-III) produces an antigen-specific T cell-dependent phenomenon termed low-dose paralysis (immunologic unresponsiveness). This form of unresponsiveness can be transferred by spleen cells obtained 5 to 24 hr after priming, and the suppressive activity of the transferred cells is abolished by prior treatment with monoclonal anti-Lyt-2 and anti-I-J antibody in the presence of complement, indicating that suppression is mediated by a distinct subset of T cells (suppressor T cells). If primed spleen cells are transferred 24 to 72 hr after immunization with SSS-III, however, the resulting antibody response of immunized recipients is enhanced. Greater enhancement is noted when transferred cells, pretreated with monoclonal anti-Lyt-2 antibody plus complement to remove suppressor T cells, are used; such enhancement is attributed to amplifier T cells. These findings indicate suppressor T cells regulate the antibody response to SSS-III by influencing the expansion of SSS-III-specific clones of B cells as well as the expression of amplifier T cell activity; the latter causes B cells to proliferate further in response to SSS-III.     

肺炎链球菌18C型糖蛋白结合物的制备及其免疫原性

制备肺炎链球菌18C型荚膜多糖－破伤风类毒素结合物（CPS－TT）,测定结合物的理化性质,抗原特异性及其在动物中的免疫原性。结果显示,结合物能与相应的多糖和破伤风抗血清形成明显的沉淀线,蛋白／多糖比率为1．86,结合物分子大小（Kd值）为0．058。注射小鼠后可诱导明显的抗体应答,而且随着注射针次的增加,抗体反应水平明显增高,显示加强效应。结果表明,制备的肺炎链球菌糖蛋白结合物抗原性良好,具有胸腺依赖性的特性,在小鼠中显示较好的免疫原性。     

Contribution of capsular and clonal types and beta-lactam resistance to the severity of experimental pneumococcal meningitis

We used a rabbit model to assess the effects of capsular serotype, genetic background and beta-lactam resistance on the course and severity of experimental meningitis. Meningitis was induced by five pneumococcal strains belonging to five different clones with known invasive potential: two serotype 3 strains (ST260(3) and Netherlands(3)-31 clones) and three serotype 23F strains with different beta-lactam susceptibility patterns (Spain(23F)-1 clone, Tennessee(23F)-4 clone and a double locus variant of the Tennessee(23F)-4 clone). Major differences in secondary bacteremia and mortality rates were observed between serotypes 3 and 23F, as were divergences in the CSF lactate, protein and lipoteichoic-teichoic acid concentrations. Minor differences in the CSF-induced inflammatory response were found among strains belonging to the same serotype. Our results suggest that capsular serotype might be the main factor determining the course and severity of pneumococcal meningitis and genetic background contributes to a lesser extent. The acquisition of beta-lactam resistance does not reduce the virulence of the invasive clones. Since five strains belonging to two serotypes were studied, our findings have to be confirmed with other pneumococcal serotypes.