Transport of Streptococcus pneumoniae Capsular Polysaccharide in MHC Class II Tubules

Bacterial capsular polysaccharides are virulence factors and are considered T cell–independent antigens. However, the capsular polysaccharide Sp1 from Streptococcus pneumoniae serotype 1 has been shown to activate CD4(+) T cells in a major histocompatibility complex (MHC) class II–dependent manner. The mechanism of carbohydrate presentation to CD4(+) T cells is unknown. We show in live murine dendritic cells (DCs) that Sp1 translocates from lysosomal compartments to the plasma membrane in MHCII-positive tubules. Sp1 cell surface presentation results in reduction of self-peptide presentation without alteration of the MHCII self peptide repertoire. In DM-deficient mice, retrograde transport of Sp1/MHCII complexes resulting in T cell–dependent immune responses to the polysaccharide in vitro and in vivo is significantly reduced. The results demonstrate the capacity of a bacterial capsular polysaccharide antigen to use DC tubules as a vehicle for its transport as an MHCII/saccharide complex to the cell surface for the induction of T cell activation. Furthermore, retrograde transport requires the functional role of DM in self peptide–carbohydrate exchange. These observations open new opportunities for the design of vaccines against microbial encapsulated pathogens

Streptococcus pneumoniae Serotype 1 Capsular Polysaccharide Induces CD8+CD28− Regulatory T Lymphocytes by TCR Crosslinking

Zwitterionic capsular polysaccharides (ZPS) of commensal bacteria are characterized by having both positive and negative charged substituents on each repeating unit of a highly repetitive structure that has an α-helix configuration. In this paper we look at the immune response of CD8+ T cells to ZPSs. Intraperitoneal application of the ZPS Sp1 from Streptococcus pneumoniae serotype 1 induces CD8+CD28− T cells in the spleen and peritoneal cavity of WT mice. However, chemically modified Sp1 (mSp1) without the positive charge and resembling common negatively charged polysaccharides fails to induce CD8+CD28− T lymphocytes. The Sp1-induced CD8+CD28− T lymphocytes are CD122lowCTLA-4+CD39+. They synthesize IL-10 and TGF-β. The Sp1-induced CD8+CD28− T cells exhibit immunosuppressive properties on CD4+ T cells in vivo and in vitro. Experimental approaches to elucidate the mechanism of CD8+ T cell activation by Sp1 demonstrate in a dimeric MHC class I-Ig model that Sp1 induces CD8+ T cell activation by enhancing crosslinking of TCR. The expansion of CD8+CD28− T cells is independent, of direct antigen-presenting cell/T cell contact and, to the specificity of the T cell receptor (TCR). In CD8+CD28− T cells, Sp1 enhances Zap-70 phosphorylation and increasingly involves NF-κB which ultimately results in protection versus apoptosis and cell death and promotes survival and accumulation of the CD8+CD28− population. This is the first description of a naturally occurring bacterial antigen that is able to induce suppressive CD8+CD28− T lymphocytes in vivo and in vitro. The underlying mechanism of CD8+ T cell activation appears to rely on enhanced TCR crosslinking. The data provides evidence that ZPS of commensal bacteria play an important role in peripheral tolerance mechanisms and the maintenance of the homeostasis of the immune system

Oligoclonal CD4+ T Cells Promote Host Memory Immune Responses to Zwitterionic Polysaccharide of Streptococcus pneumoniae▿

Zwitterionic polysaccharides of the normal flora bacteria represent a novel class of antigens in that they correct systemic CD4+ T-cell deficiencies and direct lymphoid organogenesis during colonization of the host. Presentation of these polysaccharides to CD4+ T cells depends on major histocompatibility complex class II- and DM-dependent retrograde transport from lysosomes to the cell surface. Yet the phenotype and clonality of the immune response to the polysaccharide in the mature host immune system have not been studied. Using the zwitterionic capsular polysaccharide Sp1 of Streptococcus pneumoniae, a transient member of the bacterial flora, in an experimental mouse model of cellular immunity, we demonstrated the accumulation of TH1- and TH17-polarized CD4+ CD44high CD62low CD25− memory T cells. Subcutaneous immunization with Sp1 resulted in an increase of serum immunoglobulin G (IgG), predominantly of the IgG1 subclass, and suggested the presence of a humoral memory response to the polysaccharide. CD4+ T cells stimulated with polysaccharide in vitro and in vivo showed a nonrestricted pattern for the T-cell receptor (TCR) β-chain variable region, as demonstrated by semiquantitative reverse transcription-PCR and flow cytometry. Clonotype mapping of in vivo and in vitro polysaccharide-activated CD4+ T cells revealed clonotypic TCR transcripts. Taken together, the data show the induction of clonal expansion of CD4+ T cells by polysaccharides of commensal bacteria. Cellular and humoral memory host responses imply the ability of these polysaccharides to mediate the expansion of T cells via recognition within the CDR3 region of the TCR

DM Dependency of Sp1 Retrograde Transport in MHCII-Positive Tubules

<div><p>(A) DM^−/− iDCs transfected with MHCII-GFP were treated for 30 min with Sp1-Alexa 594 (500 μg/ml) before live cell imaging by confocal microscopy. Merged fluorescence images of DM^−/− iDCs reveal co-localization of Sp1 with MHCII-GFP in lysosomes. No MHCII is found on the cell surface.</p><p>(B) DM^−/− iDCs transfected with MHCII-GFP were pre-incubated with Sp1-Alexa 594 (500 μg/ml) for 30 min and then treated with LPS for 4 h. Merged fluorescence live cell images of confocal microscopy demonstrate that DCs form MHCII-GFP–positive tubules by 4 h of stimulation with LPS that extend from the perinuclear area to the cell surface (arrows) while Sp1-Alexa 594 remains lysosomal (right panel). Scale bar, 10 μm.</p><p>(C) DCs from WT or DM^−/− mice expressing MHCII-GFP were stimulated with Sp1-Alexa 594 (500 μg/ml) for 30 min and LPS for various time intervals. Immune interactions with CFSE-labeled WT T cells were analyzed by live cell fluorescent microscopy imaging (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030032#ppat-0030032-g002" target="_blank">Figure 2</a>E and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030032#ppat-0030032-g002" target="_blank">2</a>F).</p></div