Jesus Colino

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.

Two Distinct Mechanisms For Induction of Dendritic Cell Apoptosis in Response to Intact Streptococcus pneumoniae

Apoptotic dendritic cells (DCs) are ineffective at inducing immunity. Thus, parameters that regulate DC viability during a primary infection will help to determine the outcome of the subsequent immune response. In this regard, pathogens have developed strategies to promote DC apoptosis to counterbalance the nascent primary immune response. We demonstrate, using cultured bone marrow-derived DCs, that Streptococcus pneumoniae can induce DC apoptosis through two distinct mechanisms: 1) a rapid, caspase-independent mechanism of apoptosis induction, critically dependent on bacterial expression of pneumolysin, and 2) a delayed-onset, caspase-dependent mechanism of apoptosis induction associated with terminal DC maturation. Delayed-onset apoptosis does not require bacterial internalization, but rather is triggered by the interaction of bacterial subcapsular components and bone marrow-derived DC (likely Toll-like) receptors acting in a myeloid differentiation factor 88-dependent manner. In this regard, heavy polysaccharide encapsulation interferes with both DC maturation and apoptosis induction. In contrast, neither CD95/CD95 ligand interactions nor TNF-α appear to play a role in the delayed onset of apoptosis. These data are the first to define two mechanistically distinct pathways of DC apoptosis induction in response to an extracellular bacterium that likely have important consequences for the establishment of antibacterial immunity.

Exosomes from Bone Marrow Dendritic Cells Pulsed with Diphtheria Toxoid Preferentially Induce Type 1 Antigen-Specific IgG Responses in Naive Recipients in the Absence of Free Antigen

Exosomes derived from dendritic cells (DC) activate T cells in vivo, but whether exosomes are able to induce and/or modulate humoral immune responses is still unknown. We show that murine bone marrow DC pulsed in vitro with an intact protein (diphtheria toxoid (DT)) produce exosomes that induce, in the absence of free protein, in vivo Ig responses specific for DT in naive recipients. Furthermore, these exosomes stimulate secondary IgG anti-DT responses in mice primed with intact DT. Exosomes from mature, relative to immature, DC were more effective at inducing primary, although not secondary, IgG anti-DT responses. Whereas intact DT preferentially induced a type 2 (IgG1) anti-DT response, exosomes from DT-pulsed bone marrow DC favored induction of type 1 (IgG2b and IgG2a) DT-specific IgG. These results are the first to demonstrate the ability of exosomes derived from Ag-pulsed DC to induce and modulate Ag-specific humoral immunity in vivo.

Distinct Types of T-cell Help for the Induction of a Humoral Immune Response to Streptococcus pneumoniae

Studies have indicated that purified soluble polysaccharide antigens can elicit T cell-independent Ig responses in vivo, although these responses can be modulated by T cells in a noncognate manner. Relatively little is known, however, concerning the parameters that regulate polysaccharide-specific, as well as protein-specific, Ig isotype responses to an intact extracellular bacterium. Using the murine in vivo humoral response to intact Streptococcus pneumoniae as a model it can be shown that CD4+ T-cell receptor alphabeta+ T cells deliver help for both polysaccharide- and protein-specific Ig responses. However, these responses differ fundamentally in their mechanism of action.

Dendritic Cell-Derived Exosomes Express a Streptococcus pneumoniae Capsular Polysaccharide Type 14 Cross-Reactive Antigen That Induces Protective Immunoglobulin Responses against Pneumococcal Infection in Mice

ABSTRACT Exosomes activate T cells in vivo, but whether exosomes are able to induce humoral immune responses is still unknown. We found that dendritic cells, but not other immune cells, constitutively release an exosome-associated glycoconjugate that is cross-reactive with the capsular polysaccharide of Streptococcus pneumoniae type 14 (Cps14-CRA). Cps14-CRA was localized to the cholesterol-enriched microdomains or rafts of the exosomes and was mapped to the β1→6 branched N-acetyl-lactosamine derivatives of the Cps14-CRA. Injection of CFA-primed naive mice with purified dendritic cell exosomes induced immunoglobulin (Ig) anti-Cps14 responses composed predominantly of IgM, IgG3, and IgG1. These responses were associated with protection against a lethal challenge with live S. pneumoniae type 14, but not with type 3 bacteria, and was correlated with the titer of elicited IgM and IgG3 anti-Cps14. These data show, for the first time, that exosomes can induce a humoral immune response to an associated unprocessed, autologous antigen. Although anti-Cps14 Ig responses are specifically demonstrated, these could reflect a broader mechanism that modulates both natural immunity and autoimmunity to other glycotopes.

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.

Transgenic expression of Bcl-xL or Bcl-2 by murine B cells enhances the in vivo antipolysaccharide, but not antiprotein, response to intact Streptococcus pneumoniae.

IgG antipolysaccharide (PS) and antiprotein responses to Streptococcus pneumoniae (Pn) are both CD4+ T cell dependent. However, the primary IgG anti-PS response terminates more quickly, uses a shorter period of T cell help, fails to generate memory, and is more dependent on membrane Ig (mIg) signaling. We thus determined whether this limited anti-PS response to Pn reflected a greater propensity of PS-specific B cells to undergo apoptosis. We used mice that constitutively expressed the antiapoptotic protein Bcl-xL or Bcl-2 as a B cell-specific transgene. Both transgenic (Tg) mice exhibited increased absolute numbers of splenic B-1 and peritoneal B-1b and B-2 cells, subsets implicated in anti-PS responses, but not in marginal zone B (MZB) cells. Both Tg mouse strains elicited, in an apparently Fas-independent manner, a more prolonged and higher peak primary IgM and IgG anti-PS, but not antiprotein, response to Pn, but without PS-specific memory. A similar effect was not observed using purified PS or pneumococcal conjugate vaccine. In vitro, both splenic MZB and follicular Tg B cells synthesized DNA at markedly higher levels than their wild-type counterparts, following mIg cross-linking. This was associated with increased clonal expansion and decreased apoptosis. Using Lsc−/− mice, the Pn-induced IgG response specific for the capsular PS was found to be almost entirely dependent on MZB cells. Collectively, these data suggest that apoptosis may limit mIg-dependent clonal expansion of PS-specific B cells during a primary immune response to an intact bacterium, as well as decrease the pool of PS-responding B cell subsets.

Dendritic Cells, New Tools for Vaccination

Our rapidly expanding knowledge of the biology of the dendritic cell (DC), a major antigen-presenting cell connecting innate and adaptive immunity, suggests new possibilities for the development of vaccines and therapeutic strategies against pathogens, through the manipulation of their function in vivo, or the injection of the DC itself, once properly instructed ex 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.

B and CD4 + T-cell expression of TLR2 is critical for optimal induction of a T-cell-dependent humoral immune response to intact Streptococcus pneumoniae

TLR2−/− mice immunized with Streptococcus pneumoniae (Pn) elicit normal IgM, but defective CD4+ T‐cell‐dependent type 1 IgG isotype production, associated with a largely intact innate immune response. We studied the T‐cell‐dependent phosphorylcholine (PC)‐specific IgG3 versus the T‐cell‐independent IgM response to Pn to determine whether TLR2 signals directly via the adaptive immune system. Pn‐activated TLR2−/− BMDC have only a modest defect in cytokine secretion, undergo normal maturation, and when transferred into naïve WT mice elicit a normal IgM and IgG3 anti‐PC response, relative to WT BMDC. Pn synergizes with BCR and TCR signaling for DNA synthesis in purified WT B and CD4+T cells, respectively, but is defective in cells lacking TLR2. Pn primes TLR2−/− mice for a normal CD4+ T‐cell IFN‐γ recall response. Notably, TLR2−/− B cells transferred into RAG‐2−/− mice with WT CD4+T cells, or TLR2−/− CD4+T cells transferred into athymic nude mice, each elicit a defective IgG3, in contrast to normal IgM, anti‐PC response relative to WT cells. These data are the first to demonstrate a major role for B‐cell and CD4+ T‐cell expression of TLR2 for eliciting an anti‐bacterial humoral immune response.