CD4+ T lymphocytes expressing CD40 ligand help the IgM antibody response to soluble pneumococcal polysaccharides via an intermediate cell type

Streptococcus pneumoniae causes serious infections in children, the elderly, and immunocompromised patients. Protection against infections with S. pneumoniae is mediated through Abs against the capsular polysaccharides (caps-PS). We previously showed that the murine Ab response to caps-PS is dependent on CD40-CD40L interaction. In the present paper, we addressed the question of whether the CD40-CD40L-mediated modulation of the anti-caps-PS immune reaction is the result of a direct interaction between B lymphocytes and T lymphocytes or of an indirect interaction. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice did not mount anti-caps-PS Abs. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice and CD4+ T lymphocytes from wild-type mice but not CD4+ T lymphocytes from CD40L knockout mice stimulated the anti-caps-PS Ab response. This indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in a CD40L-dependent manner. SCID/SCID mice reconstituted with B lymphocytes from CD40 knockout mice and CD4+ T lymphocytes from wild-type mice generated an anti-caps-PS Ab response that could be inhibited by MR1, a blocking anti-CD40L Ab. These data indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in an indirect way. Finally, lethally irradiated CD40 knockout mice reconstituted with bone marrow from wild-type mice mounted an anti-caps-PS Ab response that was comparable to the Ab response in wild-type mice, revealing that the required CD40 was on hemopoietic cells. In conclusion, we provide evidence that CD4+ T lymphocytes expressing CD40L stimulate the Ab response to soluble caps-PS by interacting with CD40-expressing non-B cells.     

Essential role for CD40 ligand interactions in T lymphocyte-mediated modulation of the murine immune response to pneumococcal capsular polysaccharides

Protection against infection with pneumococci is provided by anti-capsular polysaccharide (caps-PS) Abs. We investigated whether CD40 ligand (CD40L) plays a role in T lymphocyte-mediated regulation of the immune response to caps-PS, which are considered thymus-independent Ags. Administration of MR1, an antagonist mAb against murine CD40L, in BALB/c mice immunized with Pneumovax resulted in an inhibition of the IgM and IgG Ab response for various caps-PS serotypes. Evidence for the involvement of CD4(+) T lymphocytes in the Ab response to caps-PS was obtained in SCID/SCID mice that, when reconstituted with B lymphocytes and CD4(+) T lymphocytes, mounted a higher specific IgM response compared with SCID/SCID mice reconstituted with only B lymphocytes. This helper effect of CD4(+) T lymphocytes was abrogated by MR1. Blocking CD40L in vitro decreased the IgM response to caps-PS and abolished the helper effect of CD4(+) T lymphocytes. CD8(+) T lymphocyte-depleted murine spleen cells mounted a higher in vivo immune response than total murine spleen cells, which provided evidence for a suppressive role of CD8(+) T lymphocytes on the anti-caps-PS immune response. CD4(+) T lymphocyte-depleted murine spleen cells, leaving a B and CD8(+) T lymphocyte fraction, elicited only a weak in vivo and in vitro Ab response, which was enhanced after MR1 administration. In summary, our data provide evidence that T lymphocytes contribute to the regulation of the anti-caps-PS immune response in a CD40L-dependent manner.     

Murine B1 B cells require IL-5 for optimal T cell-dependent activation

T helper cell-driven activation of murine B cells has been shown to depend upon CD40-CD40 ligand (CD40L) interactions and a defined set of cytokines. These observations are primarily based on the use of conventional B cells obtained from the spleen. Therefore, it is presently unclear whether all mature B cell subsets found in the mouse have an equal dependence upon CD40-CD40L interactions and use the same T cell-derived cytokines. The present study tested the response of splenic follicular and marginal zone as well as peritoneal B2 and B1 B cells to Th cell stimulation. Splenic and peritoneal B cell subsets were sort purified based on CD23 expression, and cultured with rCD40L and cytokines or Th2 cells. The results demonstrate that follicular, marginal zone, and peritoneal B2 B cells require CD40-CD40L interactions and preferentially use IL-4 for optimal proliferation, differentiation, and isotype switching. In contrast, peritoneal B1 B cells use IL-5 in conjunction with CD40-CD40L interactions for maximal Th cell-dependent responses. Furthermore, B1 B cells are capable of proliferating, differentiating, and isotype switching in the absence of CD40-CD40L interactions. B1 B cells are able to respond to Th2 clones in the presence of anti-CD40L mAb as well as to Th2 clones derived from CD40L(-/-) mice. The CD40-CD40L-independent response of B1 B cells is attributable to the presence of both IL-4 and IL-5, and may explain the residual Ab response to T cell-dependent Ags in CD40L- or CD40-deficient mice, and in X-linked hyper-IgM (X-HIM) patients.     

Development of functional B cells in a line of SCID mice with transgenes coding for anti-double-stranded DNA antibody

Deletion or inactivation of anti-self (DNA) B cells has been reported in non-autoimmune mice bearing Ig transgenes that code for Abs with specificity for dsDNA or ssDNA. However, we report a case in which anti-dsDNA B cells appear to escape both deletion and inactivation. We show that B cells (B220+IgM+) can develop in non-autoimmune SCID mice bearing two site-directed transgenes, 3H9(56R) and Vkappa8, that together code for an anti-dsDNA Ab. The B cells appear inactive, because the mice (56RVkappa8 SCID mice) generally lack serum Ig. However, 56RVkappa8 SCID mice are able to produce IgG Ab with specificity for dsDNA when they become "leaky" for T cells or are reconstituted with exogenous T cells from B cell-deficient JH-/- donors. Thus, anti-dsDNA B cells that escape deletion in 56RVkappa8 SCID mice appear fully functional and can differentiate, class switch, and give rise to IgG-producing cells in the presence of T cells and self-Ag.     

Prevention of experimental colitis in SCID mice reconstituted with CD45RBhigh CD4+ T cells by blocking the CD40-CD154 interactions

Increased expression of CD40 and CD40 ligand (CD40L or CD154) has been found in inflamed mucosa of human inflammatory bowel disease (IBD), and interactions between these molecules seem to be involved in local cytokine production by macrophages. However, the precise role of CD40 signaling in the pathogenesis of IBD is still poorly understood. The aim of the present study was to investigate the in vivo relevance of CD40 signaling in experimental colitis in SCID mice reconstituted with syngeneic CD45RBhighCD4+ T cells. The results demonstrated that CD40+ and CD40L+ cells as well as their mRNA levels were significantly increased in inflamed mucosa. Administration of anti-CD40L neutralizing mAb over an 8-wk period starting immediately after CD45RBhighCD4+ T cell reconstitution completely prevented symptoms of wasting disease. Intestinal mucosal inflammation was effectively prevented, as revealed by abrogated leukocyte infiltration and decreased CD54 expression and strongly diminished mRNA levels of the proinflammatory cytokines IFN-gamma, TNF, and IL-12. When colitic SCID mice were treated with anti-CD40L starting at 5 wk after T cell transfer up to 8 wk, this delayed treatment still led to significant clinical and histological improvement and down-regulated proinflammatory cytokine secretion. These data suggest that the CD40-CD40L interactions are essential for the Th1 inflammatory responses in the bowel in this experimental model of colitis. Blockade of CD40 signaling may be beneficial to human IBD.     

Costimulation requirements for antiviral CD8+ T cells differ for acute and persistent phases of polyoma virus infection

The requirement for costimulation in antiviral CD8+ T cell responses has been actively investigated for acutely resolved viral infections, but it is less defined for CD8+ T cell responses to persistent virus infection. Using mouse polyoma virus (PyV) as a model of low-level persistent virus infection, we asked whether blockade of the CD40 ligand (CD40L) and CD28 costimulatory pathways impacts the magnitude and function of the PyV-specific CD8+ T response, as well as the humoral response and viral control during acute and persistent phases of infection. Costimulation blockade or gene knockout of either CD28 or CD40L substantially dampened the magnitude of the acute CD8+ T cell response; simultaneous CD28 and CD40L blockade severely depressed the acute T cell response, altered the cell surface phenotype of PyV-specific CD8+ T cells, decreased PyV VP1-specific serum IgG titers, and resulted in an increase in viral DNA levels in multiple organs. CD28 and CD40L costimulation blockade during acute infection also diminished the memory PyV-specific CD8+ T cell response and serum IgG titer, but control of viral persistence varied between mouse strains and among organs. Interestingly, we found that CD28 and CD40L costimulation is dispensable for generating and/or maintaining PyV-specific CD8+ T cells during persistent infection; however, blockade of CD27 and CD28 costimulation in persistently infected mice caused a reduction in PyV-specific CD8+ T cells. Taken together, these data indicate that CD8+ T cells primed within the distinct microenvironments of acute vs persistent virus infection differ in their costimulation requirements.     

T-Cell-Independent Immunoglobulin G Responses In Vivo Are Elicited by Live-Virus Infection but Not by Immunization with Viral Proteins or Virus-Like Particles

Immunoglobulin G (IgG) responses to viruses are generally assumed to be T-cell dependent (TD). Recently, however, polyomavirus (PyV) infection of T-cell-deficient (T-cell receptor β chain [TCR-β] −/− or TCR-β×δ −/−) mice was shown to elicit a protective, T-cell-independent (TI) antiviral IgM and IgG response. A repetitive, highly organized antigenic structure common to many TI antigens is postulated to be important in the induction of antibody responses in the absence of helper T cells. To test whether the repetitive structure of viral antigens is essential and/or sufficient for the induction of TI antibodies, we compared the abilities of three forms of PyV antigens to induce IgM and IgG responses in T-cell-deficient mice: soluble capsid antigens (VP1), repetitive virus-like particles (VLPs), and live PyV. Immunization with each of the viral antigens resulted in IgM production. VLPs and PyV elicited 10-fold-higher IgM titers than VP1, indicating that the highly organized, repetitive antigens are more efficient in IgM induction. Antigen-specific TI IgG responses, however, were detected only in mice infected with live PyV, not in VP1- or VLP-immunized mice. These results suggest that the highly organized, repetitive nature of the viral antigens is insufficient to account for their ability to elicit TI IgG response and that signals generated by live-virus infection may be essential for the switch to IgG production in the absence of T cells. Germinal centers were not observed in T-cell-deficient PyV-infected mice, indicating that the germinal center pathway of B-cell differentiation is TD even in the context of a virus infection.     

Contributions of antinucleoprotein IgG to heterosubtypic immunity against influenza virus

Influenza A virus causes recurring seasonal epidemics and occasional influenza pandemics. Because of changes in envelope glycoprotein Ags, neutralizing Abs induced by inactivated vaccines provide limited cross-protection against new viral serotypes. However, prior influenza infection induces heterosubtypic immunity that accelerates viral clearance of a second strain, even if the external proteins are distinct. In mice, cross-protection can also be elicited by systemic immunization with the highly conserved internal nucleoprotein (NP). Both T lymphocytes and Ab contribute to such cross-protection. In this paper, we demonstrate that anti-NP IgG specifically promoted influenza virus clearance in mice by using a mechanism involving both FcRs and CD8(+) cells. Furthermore, anti-NP IgG rescued poor heterosubtypic immunity in B cell-deficient mice, correlating with enhanced NP-specific CD8 T cell responses. Thus, Ab against this conserved Ag has potent antiviral activity both in naive and in influenza-immune subjects. Such antiviral activity was not seen when mice were vaccinated with another internal influenza protein, nonstructural 1. The high conservation of NP Ag and the known longevity of Ab responses suggest that anti-NP IgG may provide a critically needed component of a universal influenza vaccine.     

Role of CD4 T cell help and costimulation in CD8 T cell responses during Listeria monocytogenes infection

CD4 T cells are known to assist the CD8 T cell response by activating APC via CD40-CD40 ligand (L) interactions. However, recent data have shown that bacterial products can directly activate APC through Toll-like receptors, resulting in up-regulation of costimulatory molecules necessary for the efficient priming of naive T cells. It remains unclear what role CD4 T cell help and various costimulation pathways play in the development of CD8 T cell responses during bacterial infection. In this study, we examined these questions using an intracellular bacterium, Listeria monocytogenes, as a model of infection. In CD4 T cell-depleted, CD4(-/-), and MHC class II(-/-) mice, L. monocytogenes infection induced CD8 T cell activation and primed epitope-specific CD8 T cells to levels commensurate with those in normal C57BL/6 mice. Furthermore, these epitope-specific CD8 T cells established long-term memory in CD4(-/-) mice that was capable of mounting a protective recall response. In vitro analysis showed that L. monocytogenes directly stimulated the activation and maturation of murine dendritic cells. The CD8 T cell response to L. monocytogenes was normal in CD40L(-/-) mice but defective in CD28(-/-) and CD137L(-/-) mice. These data show that in situations where infectious agents or immunogens can directly activate APC, CD8 T cell responses are less dependent on CD4 T cell help via the CD40-CD40L pathway but involve costimulation through CD137-CD137L and B7-CD28 interactions.     

CD4 T cell-independent antibody response promotes resolution of primary influenza infection and helps to prevent reinfection

It is generally believed that the production of influenza-specific IgG in response to viral infection is dependent on CD4 T cells. However, we previously observed that CD40-deficient mice generate influenza-specific IgG during a primary infection, suggesting that influenza infection may elicit IgG responses independently of CD4 T cell help. In the present study, we tested this hypothesis and show that mice lacking CD40 or CD4 T cells produce detectable titers of influenza-specific IgG and recover from influenza infection in a manner similar to that of normal mice. In contrast, mice completely lacking B cells succumb to influenza infection, despite the presence of large numbers of functional influenza-specific CD8 effector cells in the lungs. Consistent with the characteristics of a T-independent Ab response, long-lived influenza-specific plasma cells are not found in the bone marrow of CD40-/- and class II-/- mice, and influenza-specific IgG titers wane within 60 days postinfection. However, despite the short-lived IgG response, CD40-/- and class II-/- mice are completely protected from challenge infection with the same virus administered within 30 days. This protection is mediated primarily by B cells and Ab, as influenza-immune CD40-/- and class II-/- mice were still resistant to challenge infection when T cells were depleted. These data demonstrate that T cell-independent influenza-specific Ab promotes the resolution of primary influenza infection and helps to prevent reinfection.     

CD40-CD40 ligand costimulation is required for generating antiviral CD4 T cell responses but is dispensable for CD8 T cell responses.

This study documents a striking dichotomy between CD4 and CD8 T cells in terms of their requirements for CD40-CD40 ligand (CD40L) costimulation. CD40L-deficient (-/-) mice made potent virus-specific CD8 T cell responses to dominant as well as subdominant epitopes following infection with lymphocytic choriomeningitis virus. In contrast, in the very same mice, virus-specific CD4 T cell responses were severely compromised. There were 10-fold fewer virus-specific CD4 T cells in CD40L-/- mice compared with those in CD40L+/+ mice, and this inhibition was seen for both Th1 (IFN-gamma, IL-2) and Th2 (IL-4) responses. An in vivo functional consequence of this Th cell defect was the inability of CD40L-/- mice to control a chronic lymphocytic choriomeningitis virus infection. This study highlights the importance of CD40-CD40L interactions in generating virus-specific CD4 T cell responses and in resolving chronic viral infection.     

Differential requirements for expression of CD80/86 and CD40 on B cells for T-dependent antibody responses in vivo

The CD80/86-CD28 and CD40-CD40 ligand costimulatory pathways are essential for Th cell-dependent B cell responses that generate high-affinity, class-switched Ab in vivo. Disruption of either costimulatory pathway results in defective in vivo humoral immune responses, but it remains unclear to what extent this is due to deficient activation of Th cells and/or of B cells. To address this issue, we generated mixed chimeras in which CD80/86- or CD40-deficient bone marrow-derived cells coexist with wild-type (WT) cells, thereby providing the functional T cell help and accessory cell functions required for fully competent B cell responses. We were then able to assess the requirement for CD80/86 or CD40 expression on B cells producing class-switched Ig in response to a T-dependent Ag. In CD80/86 WT plus CD80/86 double-knockout mixed chimeras, both WT- and CD80/86-deficient B cells produced IgG1 and IgE responses, indicating that direct signaling by CD80/86 is not essential for efficient B cell activation. In marked contrast, only WT IgG1 and IgE responses were detected in the chimeras containing CD40-deficient cells, demonstrating that CD40 expression on B cells is essential for class switching by those B cells. Thus, while disrupting either the CD80/86-CD28 or the CD40-CD40 ligand costimulatory pathway abrogates T-dependent B cell immune responses, the two pathways are nonredundant and mediated by distinct mechanisms.     

The antiviral CD8+ T cell response is differentially dependent on CD4+ T cell help over the course of persistent infection

Although many studies have investigated the requirement for CD4(+) T cell help for CD8(+) T cell responses to acute viral infections that are fully resolved, less is known about the role of CD4(+) T cells in maintaining ongoing CD8(+) T cell responses to persistently infecting viruses. Using mouse polyoma virus (PyV), we asked whether CD4(+) T cell help is required to maintain antiviral CD8(+) T cell and humoral responses during acute and persistent phases of infection. Though fully intact during acute infection, the PyV-specific CD8(+) T cell response declined numerically during persistent infection in MHC class II-deficient mice, leaving a small antiviral CD8(+) T cell population that was maintained long term. These unhelped PyV-specific CD8(+) T cells were functionally unimpaired; they retained the potential for robust expansion and cytokine production in response to Ag rechallenge. In addition, although a strong antiviral IgG response was initially elicited by MHC class II-deficient mice, these Ab titers fell, and long-lived PyV-specific Ab-secreting cells were not detected in the bone marrow. Finally, using a minimally myeloablative mixed bone marrow chimerism approach, we demonstrate that recruitment and/or maintenance of new virus-specific CD8(+) T cells during persistent infection is impaired in the absence of MHC class II-restricted T cells. In summary, these studies show that CD4(+) T cells differentially affect CD8(+) T cell responses over the course of a persistent virus infection.     

Characterization of the CD154-positive and CD40-positive cellular subsets required for pathogenesis in retrovirus-induced murine immunodeficiency

Genetically susceptible C57BL/6 (B6) mice that are infected with the LP-BM5 isolate of murine retroviruses develop profound splenomegaly, lymphadenopathy, hypergammaglobulinemia, terminal B-cell lymphomas, and an immunodeficiency state bearing many similarities to the pathologies seen in AIDS. Because of these similarities, this syndrome has been called murine AIDS (MAIDS). We have previously shown that CD154 (CD40 ligand)-CD40 molecular interactions are required both for the initiation and progression of MAIDS. Thus, in vivo anti-CD154 monoclonal antibody (MAb) treatment inhibited MAIDS symptoms in LP-BM5-infected wild-type mice when either a short course of anti-CD154 MAb treatment was started on the day of infection or a course was initiated 3 to 4 weeks after LP-BM5 administration, after disease was established. Here, we further characterize this required CD154-CD40 interaction by a series of adoptive transfer experiments designed to elucidate which cellular subsets must express CD154 or CD40 for LP-BM5 to induce MAIDS. Specifically with regard to CD154 expression, MAIDS-insusceptible B6 nude mice reconstituted with highly purified CD4+ T cells from wild-type, but not from CD154 knockout, B6 donors displayed clear MAIDS after LP-BM5 infection. In contrast, nude B6 recipients that received CD8+ T cells from wild-type B6 donors did not develop MAIDS after LP-BM5 infection. B6 CD40 knockout mice, which are also relatively resistant to LP-BM5-induced MAIDS, became susceptible to LP-BM5-induced disease after reconstitution with highly purified wild-type B cells but not after receiving purified wild-type dendritic cells (DC) or a combined CD40+ population composed of DC and macrophages obtained from B6 SCID mouse donors. Based on these and other experiments, we thus conclude that the cellular basis for the requirement for CD154-CD40 interactions for MAIDS induction and progression can be accounted for by CD154 expression on CD4+ T cells and CD40 expression on B cells.     

Mechanisms of central nervous system viral persistence: the critical role of antibody and B cells.

Contributions of humoral and cellular immunity in controlling neurotropic mouse hepatitis virus persistence within the CNS were determined in B cell-deficient J(H)D and syngeneic H-2(d) B cell+ Ab-deficient mice. Virus clearance followed similar kinetics in all mice, confirming initial control of virus replication by cellular immunity. Nevertheless, virus reemerged within the CNS of all Ab-deficient mice. In contrast to diminished T cell responses in H-2(b) B cell-deficient muMT mice, the absence of B cells or Ab in the H-2(d) mice did not compromise expansion, recruitment into the CNS, or function of virus-specific CD4+ and CD8+ T cells. The lack of B cells and lymphoid architecture thus appears to manifest itself on T cell responses in a genetically biased manner. Increasing viral load did not enhance frequencies or effector function of virus-specific T cells within the CNS, indicating down-regulation of T cell responses. Although an Ab-independent antiviral function of B cells was not evident during acute infection, the presence of B cells altered CNS cellular tropism during viral recrudescence. Reemerging virus localized almost exclusively to oligodendroglia in B cell+ Ab-deficient mice, whereas it also replicated in astrocytes in B cell-deficient mice. Altered tropism coincided with distinct regulation of CNS virus-specific CD4+ T cells. These data conclusively demonstrate that the Ab component of humoral immunity is critical in preventing virus reactivation within CNS glial cells. B cells themselves may also play a subtle role in modulating pathogenesis by influencing tropism.     

SAP is required for Th cell function and for immunity to influenza

Ab is a crucial component of protective immunity to infection, but Ab responses do not proceed normally when defects occur in a protein called signaling lymphocytic activation molecule-associated protein (SAP). To explain this Ab defect, we analyzed B cell and plasma cell responses under conditions of SAP deficiency. Our results demonstrate that SAP-deficient (SAP knockout (KO)) mice have a profound CD4 T cell-intrinsic defect in generating Ag-specific plasma cells following challenge with model Ags or influenza virus, resulting in low Ag-specific Ab titers. We also show that SAP is required in CD4 T cells for normal division and expansion of B cells. These B cell and plasma cell defects were observed during the expansion phase of the primary immune response, indicating early defects in Th cell activity. In fact, additional experiments revealed a nearly complete lack of T cell help for B cells in SAP KO mice. Our work suggests that the ability of SAP to promote T-dependent humoral immune responses is important for antiviral immunity because mice lacking SAP are unable to prevent high dose secondary influenza infection, and because passive transfer of IgG in immune serum from wild-type, but not SAP KO mice can protect mice from an otherwise lethal influenza infection. Overall, our results demonstrate that SAP is required in CD4 T cells for their ability to help B cell responses and promote influenza-specific immunity.     

CD40-CD40 ligand interactions promote trafficking of CD8+ T cells into the brain and protection against West Nile virus encephalitis

Recent studies have established a protective role for T cells during primary West Nile virus (WNV) infection. Binding of CD40 by CD40 ligand (CD40L) on activated CD4+ T cells provides an important costimulatory signal for immunoglobulin class switching, antibody affinity maturation, and priming of CD8+ T-cell responses. We examined here the function of CD40-dependent interactions in limiting primary WNV infection. Compared to congenic wild-type mice, CD40(-/-) mice uniformly succumbed to WNV infection. Although CD40(-/-) mice produced low levels of WNV-specific immunoglobulin M (IgM) and IgG, viral clearance from the spleen and serum was not altered, and CD8+ T-cell priming in peripheral lymphoid tissues was normal. Unexpectedly, CD8+ T-cell trafficking to the central nervous system (CNS) was markedly impaired in CD40(-/-) mice, and this correlated with elevated WNV titers in the CNS and death. In the brains of CD40(-/-) mice, T cells were retained in the perivascular space and did not migrate into the parenchyma, the predominant site of WNV infection. In contrast, in wild-type mice, T cells trafficked to the site of infection in neurons. Beside its role in maturation of antibody responses, our experiments suggest a novel function of CD40-CD40L interactions: to facilitate T-cell migration across the blood-brain barrier to control WNV infection.     

CD154 is essential for protective immunity in experimental salmonella infection: evidence for a dual role in innate and adaptive immune responses

CD40-CD154 interactions are of central importance in the induction of humoral and cellular immune responses. In the present study, CD154-deficient (CD154-/-) mice were used to assess the role of CD40-CD154 interactions in regulating the immune response to a systemic Salmonella infection. Compared with C57BL/6 (CD154+/+) controls, CD154-/- mice were hypersusceptible to infection by an attenuated strain of Salmonella enterica serovar Typhimurium (S. typhimurium), as evidenced by decreased survival rate and mean time to death, which correlated with increased bacterial burden and persistence in target organs. CD154-/- mice exhibited a defect both in the production of IL-12, IFN-gamma, and NO during the acute phase of the disease and in the generation of Salmonella-specific Ab responses and Ig isotype switching. Furthermore, when CD154-/- animals were administered a sublethal dose of attenuated S. typhimurium and subsequently challenged with a virulent homologous strain, all mice succumbed to an overwhelming infection. Similar treatment of CD154+/+ mice consistently resulted in > or =90% protection. The lack of protective immunity in CD154-/- mice correlated with a decreased T cell recall response to Salmonella Ags. Significant protection against virulent challenge was conferred to presensitized CD154-/- mice by transfer of serum or T cells from immunized CD154+/+ mice. For best protection, however, a combination of immune serum and T cells was required. We conclude that intercellular communications via the CD40-CD154 pathway play a critical role in the induction of type 1 cytokine responses, memory T cell generation, Ab formation, and protection against primary as well as secondary Salmonella infections.     

B cells are required for generation of protective effector and memory CD4 cells in response to Pneumocystis lung infection

B cell-deficient mice are susceptible to infection by Pneumocystis carinii f. sp. muris (PC). To determine whether this susceptibility is due to a requirement for B cells to prime T cells, we compared CD4 T cell responses to PC in bone marrow chimeric mice that express MHC class II (MHCII) on all APCs (wild-type (WT) chimeras) and in bone marrow chimeric mice that express MHCII on all APCs except B cells (MHCII(-/-) chimeras). Although PC was rapidly cleared by WT chimeric mice, PC levels remained high in chimeric mice that lacked MHCII on B cells. In addition, although T cells were primed in the draining lymph nodes of MHCII(-/-) chimeric mice, the number of activated CD4 T cells infiltrating the lungs of these mice was reduced relative to the number in the lungs of WT chimeras. We also adoptively transferred purified CD4 T cells from the draining lymph nodes of PC-infected normal or B cell-deficient mice into SCID mice. Mice that received CD4 cells from normal mice were able to mount a response to infection in the lungs and clear PC. However, mice that received CD4 cells from B cell-deficient mice had a delayed T cell response in the lungs and failed to control the infection. These data indicate that B cells play a vital role in generation of CD4(+) memory T cells in response to PC infection in the lungs.     

TLR signaling fine-tunes anti-influenza B cell responses without regulating effector T cell responses

Influenza is a ssRNA virus that has been responsible for widespread morbidity and mortality; however, the innate immunological mechanisms that drive the adaptive anti-influenza immune response in vivo are yet to be fully elucidated. TLRs are pattern recognition receptors that bind evolutionarily conserved pathogen-associated molecular patterns, induce dendritic cell maturation, and consequently aid the development of effective immune responses. We have examined the role of TLRs in driving effective T and B cell responses against influenza virus. We found TLR3 and its associated adapter molecule, Toll/IL-R domain-containing adaptor-inducing IFN-beta, did not play a role in the development of CD4(+) or CD8(+) T cell responses against influenza virus, nor did they influence influenza-specific B cell responses. Surprisingly, TLR7 and MyD88 also played negligible roles in T cell activation and effector function upon infection with influenza virus; however, their signaling was critical for regulating anti-influenza B cell Ab isotype switching. The induction of appropriate anti-influenza humoral responses involved stimulation of TLRs on B cells directly and TLR-induced production of IFN-alpha, which acted to reduce IgG1 and increase IgG2a/c class switching. Notably, direct TLR signaling on B cells or T cell help through the CD40-CD40L interaction was sufficient to support B cell proliferation and IgG1 production, whereas IFN-alpha was critical for fine-tuning the nature of the isotype switch. Taken together, these data reveal that TLR signaling is not required for anti-influenza T cell responses, but through both direct and indirect means orchestrates appropriate anti-influenza B cell responses.