Bacterial lipoproteins differentially regulate human primary and memory CD4+ T and B cell responses to pneumococcal protein antigens through Toll-like receptor 2.

BACKGROUND: Bacterial lipoproteins (BLPs) are expressed across a range of bacteria and are able to activate Toll-like receptor 2 (TLR-2). BLPs enhance immune responses in naive individuals and have therefore been tested as candidate vaccine adjuvants. It is not known whether BLPs affect any preexisting immunity (eg, memory cell response in primed individuals). Colonization with pneumococcus (PNC), which primes for memory cell response, is common in young children. METHODS: We studied effects of BLPs on memory and primary B and CD4+ T cell responses to pneumococcal proteins using adenoidal cells from children. RESULTS: Although BLPs enhanced the primary antibody responses seen in some children with no detectable nasal PNC, BLPs unexpectedly reduced the memory antibody responses in children with positive nasal culture results. Likewise, BLPs augmented the naive but inhibited the memory antigen-driven CD4+ T cell response. The downregulation of the memory responses was associated with increases in interleukin 10 and inducible costimulatory molecule expression, as well as a decrease in CD28 expression in memory CD4+ T cells; all were blocked by anti-TLR2 and anti-B7h antibodies. Augmentation of naive CD4+ T cell proliferation was blocked by anti-B7.2. CONCLUSION: Differential regulation of primary and memory responses by BLPs through TLR2 may have important implications for therapeutic and vaccination strategies against bacterial infection.

Interleukin-17A mediates acquired immunity to pneumococcal colonization.

Although anticapsular antibodies confer serotype-specific immunity to pneumococci, children increase their ability to clear colonization before these antibodies appear, suggesting involvement of other mechanisms. We previously reported that intranasal immunization of mice with pneumococci confers CD4+ T cell-dependent, antibody- and serotype-independent protection against colonization. Here we show that this immunity, rather than preventing initiation of carriage, accelerates clearance over several days, accompanied by neutrophilic infiltration of the nasopharyngeal mucosa. Adoptive transfer of immune CD4+ T cells was sufficient to confer immunity to naïve RAG1(-/-) mice. A critical role of interleukin (IL)-17A was demonstrated: mice lacking interferon-gamma or IL-4 were protected, but not mice lacking IL-17A receptor or mice with neutrophil depletion. In vitro expression of IL-17A in response to pneumococci was assayed: lymphoid tissue from vaccinated mice expressed significantly more IL-17A than controls, and IL-17A expression from peripheral blood samples from immunized mice predicted protection in vivo. IL-17A was elicited by pneumococcal stimulation of tonsillar cells of children or adult blood but not cord blood. IL-17A increased pneumococcal killing by human neutrophils both in the absence and in the presence of antibodies and complement. We conclude that IL-17A mediates pneumococcal immunity in mice and probably in humans; its elicitation in vitro could help in the development of candidate pneumococcal vaccines.

Low CD4 T cell immunity to pneumolysin is associated with nasopharyngeal carriage of pneumococci in children.

BACKGROUND: Recent studies in mice have suggested that T cell immunity may be protective against pneumococcal infection. METHODS: CD4 T cell proliferative responses to the pneumococcal proteins pneumolysin (Ply), Ply toxoid (F433), and choline-binding protein A were investigated in peripheral blood mononuclear cells (PBMCs) and adenoidal mononuclear cells (MNCs) obtained from children undergoing adenoidectomy. RESULTS: Ply and F433 induce significant proliferation of CD4 T cells in both PBMCs and adenoidal MNCs, and both memory and naive phenotypes of CD4 T cells proliferated after stimulation. In PBMCs, CD4 T cell proliferation induced by Ply and F433, which was associated with increased production of interferon (IFN)- gamma and tumor necrosis factor (TNF)- alpha , was significantly lower in children who were culture positive for pneumococcus than in those who were culture negative for pneumococcus (P<.05). Between groups, no such difference was observed in adenoidal MNC CD4 T cell proliferation, which was associated with production of IFN- gamma and interleukin (IL)-10. The CD4 T cell proliferation induced by Ply and F433 was inhibited by antibodies to Toll-like receptor 4. CONCLUSION: These data suggest that Ply induces CD4 T cell proliferative responses with production of IFN- gamma and TNF- alpha in PBMCs or of IFN- gamma and IL-10 in adenoidal MNCs, which may be important in modulating pneumococcal carriage in children.

Regulation of production of mucosal antibody to pneumococcal protein antigens by T-cell-derived gamma interferon and interleukin-10 in children.

Nasopharyngeal tonsils (adenoids) are part of human nasopharynx-associated lymphoid tissue, which may play an important role in local defense against pneumococci. Recent studies with animals have suggested that several pneumococcal proteins, including CbpA and pneumolysin (Ply), may be vaccine candidates. Our recent data obtained with children suggest that antibodies to these proteins may protect against carriage. This study was performed to investigate the regulation of the T-cell-dependent antibody responses to CbpA and pneumolysin by cytokines in adenoidal immune cells from children. Adenoidal mononuclear cells (MNC) were cultured with pneumococcal concentrated culture supernatants (CCS) or recombinant proteins. Cytokine expression profiles in adenoidal MNC after antigen stimulation were analyzed by reverse transcription-PCR, protein array analysis, and an immunoassay, along with an antibody production analysis. The roles, interactions, and cellular sources of the main cytokines identified were evaluated further. Pneumococcal CCS induced production of CbpA- and Ply-specific antibodies in association with several chemokines and cytokines, including gamma interferon (IFN-gamma) and interleukin-10 (IL-10) in MNC. The antibody production correlated well with the concentrations of these two cytokines. Addition of recombinant IFN-gamma or IL-10 enhanced antibody production, and monoclonal antibodies to these two cytokines and T-cell depletion significantly reduced antibody production. Intracellular cytokine staining showed that T cells are a major source of IFN-gamma and IL-10. Recombinant Ply and, to a lesser extent, recombinant CbpA induced significant production of IFN-gamma and IL-10 in MNC. T-cell-derived IFN-gamma and IL-10 may be key regulators of production of mucosal antibody to pneumococcal protein antigens in the nasopharynx and may play an important role in local protection against pneumococcal infection in children.

Serum and mucosal antibody responses to pneumococcal protein antigens in children: relationships with carriage status.

Streptococcus pneumoniae causes significant morbidity and mortality especially in children. Some pneumococcal protein antigens can protect mice against infection. Little information is available concerning the nature of naturally acquired protective immunity to pneumococci in humans induced by these antigens. This study investigates the relationships between systemic and local antibody production and carriage in children. Children undergoing adenoidectomy (n=112, ages 2-12 years) were studied. Nasopharyngeal swabs were collected for pneumococcal culture. Serum and saliva were assayed for antibodies to several pneumococcal proteins: choline binding protein A (CbpA), pneumolysin (Ply), pneumococcal surface adhesin A (PsaA) and pneumococcal surface protein A (PspA). Adenoidal mononuclear cells (MNC) were cultured with pneumococcal culture supernatants or recombinant proteins. Cell culture supernatants were analyzed for antigen-specific antibodies. Carriage rates fell with age and serum levels of anti-CbpA, Ply and PspA rose. Anti-CbpA and -Ply serum and salivary IgG antibody levels were higher in children who were culture negative than those who were colonized. Antigen stimulation increased respective antigen-specific IgG production by adenoidal MNC and these responses were greater in those who were colonized than in culture-negative children. Antibodies to CbpA and Ply may protect children aged 2 years and older against pneumococcal colonization. Adenoids may be important local induction and effector sites for both mucosal and systemic antibody production to pneumococcal proteins in children.