Contribution of interleukin-12 (IL-12) and the CD28/B7 and CD40/CD40 ligand pathways to the development of a pathological T-cell response in IL-10-deficient mice

The ability of interleukin-10 (IL-10) to suppress accessory cell functions required for optimal T-cell activation makes it an important inhibitor of cell-mediated immunity. Thus, after infection with the protozoan parasite Toxoplasma gondii, IL-10 knockout (KO) mice develop a CD4(+)-T-cell-dependent shock-like reaction with high levels of IL-12 and gamma interferon (IFN-gamma) in serum, leading to death of mice during the acute phase of infection. Previous studies from this laboratory have shown that simultaneous blockade of CD28 and CD40 can prevent this lethal reaction by inhibiting the production of IFN-gamma. However, the blockade of costimulation did not affect systemic levels of IL-12. To better understand the relationship between IL-12 and the CD28 and CD40 pathways in mediating immune hyperactivity, antagonists of these factors were used to determine their effects on the development of a pathological T-cell response in IL-10 KO mice. Blockade of IL-12 or the CD28/B7 interaction alone did not affect survival; however, the combined blockade of both pathways resulted in decreased production of IFN-gamma and the survival of IL-10 KO mice. To assess the role of the two ligands for CD28, B7.1 and B7.2, IL-10 KO mice were treated with alphaIL-12 plus alphaB7.1 or alphaB7.2 or the combination of all three antibodies. These studies revealed that blockade of both B7 molecules is required for decreased production of IFN-gamma and survival of infected IL-10 KO mice, suggesting that B7.1 and B7.2 can contribute to the lethal shock-like reaction in IL-10 KO mice. In contrast, neutralization of IL-12 and blockade of the CD40/CD40 ligand (CD40L) interaction in vivo did not alter the production of IFN-gamma and only resulted in a small delay in time to death of mice. Together, these data suggest that the CD28/B7 interaction has a central role in the development of a pathological T-cell response in IL-10 KO mice, which is distinct from the role of the CD40/CD40L and IL-12 pathways.     

Modulation of T-cell costimulation as immunotherapy or immunochemotherapy in experimental visceral leishmaniasis

CD40 ligand (CD40L)-deficient C57BL/6 mice failed to control intracellular Leishmania donovani visceral infection, indicating that acquired resistance involves CD40-CD40L signaling and costimulation. Conversely, in wild-type C57BL/6 and BALB/c mice with established visceral infection, injection of agonist anti-CD40 monoclonal antibody (MAb) induced killing of approximately 60% of parasites within liver macrophages, stimulated gamma interferon (IFN-gamma) secretion, and enhanced mononuclear cell recruitment and tissue granuloma formation. Comparable parasite killing was also induced by MAb blockade (inhibition) of cytotoxic T lymphocyte antigen-4 (CTLA-4) which downregulates separate CD28-B7 T-cell costimulation. Optimal killing triggered by both anti-CD40 and anti-CTLA-4 required endogenous IFN-gamma and involved interleukin 12. CD40L(-/-) mice also failed to respond to antileishmanial chemotherapy (antimony), while in normal animals, anti-CD40 and anti-CTLA-4 synergistically enhanced antimony-associated killing. CD40L-CD40 signaling regulates outcome and response to treatment of experimental visceral leishmaniasis, and MAb targeting of T-cell costimulatory pathways (CD40L-CD40 and CD28-B7) yields macrophage activation and immunotherapeutic and immunochemotherapeutic activity.     

Susceptibility of interleukin-2-deficient mice to Toxoplasma gondii is associated with a defect in the production of gamma interferon

Costimulation through the B7-CD28 interaction is an important second signal for T-cell activation, and previous studies have shown that CD28(-/-) mice infected with Toxoplasma gondii generate suboptimal CD4(+) T-cell responses, associated with a defect in production of the T-cell growth factor interleukin-2 (IL-2). To address the role of IL-2 in the expansion of T cells during toxoplasmosis, IL-2(-/-) mice were infected with T. gondii and their ability to generate a protective T-cell response was assessed. Although IL-2(-/-) mice produced normal levels of IL-12p40, they had reduced levels of gamma interferon (IFN-gamma) in serum, had an increased parasite burden, and succumbed to infection with T. gondii within 20 days. Fluorescence-activated cell sorter analysis revealed that, although uninfected IL-2(-/-) mice had an increased number of activated T cells compared with uninfected IL-2(+/+) mice, following infection they were unable to further upregulate this population. Examination of the ability of splenocytes from uninfected and infected mice to produce IFN-gamma revealed that IL-2(-/-) mice were hyporesponsive to stimulation with anti-CD3 or parasite antigen compared with wild-type mice, and the addition of IL-2 alone or in combination with IL-12 or stimulation with phorbol myristate acetate and ionomycin did not restore the production of IFN-gamma. Together, these studies reveal that IL-2(-/-) mice are unable to generate a protective IFN-gamma response following infection with T. gondii and suggest that IL-2(-/-) mice have an intrinsic defect in their ability to activate and expand IFN-gamma-producing T cells required for resistance to T. gondii.     

The CD40/CD40 ligand interaction is required for resistance to toxoplasmic encephalitis

Since the CD40/CD40 ligand (CD40L) interaction is involved in the regulation of macrophage production of interleukin 12 (IL-12) and T-cell production of gamma interferon (IFN-gamma), effector cell functions associated with resistance to Toxoplasma gondii, the role of CD40L in immunity to this parasite was assessed. Infection of C57BL/6 mice with T. gondii results in an upregulation of CD40 expression on accessory cell populations at local sites of infection as well as in lymphoid tissues. Splenocytes from C57BL/6 mice infected with T. gondii for 5 days produced high levels of IL-12 and IFN-gamma when stimulated with toxoplasma lysate antigen, and blocking CD40L did not significantly alter the production of IFN-gamma or IL-12 by these cells. Similar results were observed with splenocytes and mononuclear cells isolated from the brains of chronically infected mice. Interestingly, although CD40L(-/-) mice infected with T. gondii produced less IL-12 than wild-type mice, they produced comparable levels of IFN-gamma but succumbed to toxoplasmic encephalitis 4 to 5 weeks after infection. The inability of CD40L(-/-) mice to control parasite replication in the brain correlated with the ability of soluble CD40L, in combination with IFN-gamma, to activate macrophages in vitro to control replication of T. gondii. Together, these results identify an important role for the CD40/CD40L interaction in resistance to T. gondii. However, this interaction may be more important in the control of parasite replication in the brain rather than the generation of protective T-cell responses during toxoplasmosis.     

CD4+-T-cell effector functions and costimulatory requirements essential for surviving mucosal infection with Citrobacter rodentium

Citrobacter rodentium causes an attaching and effacing infection of the mouse colon. Surprisingly, protective adaptive immunity against this mucosal pathogen requires a systemic T-cell-dependent antibody response. To define CD4+ T-cell effector functions promoting this systemic defense of infected epithelial surfaces, studies were undertaken in weaning-age mice lacking costimulatory molecules CD28 or CD40L or cytokines gamma interferon (IFN-gamma) or interleukin-4 (IL-4). Adoptive transfer of CD4+ T cells from wild-type, CD28(-/-), CD40L(-/-), or IFN-gamma(-/-) donors to CD4(-/-) recipients delineated functions of these CD4+ T-cell-expressed molecules on the outcome of infection. Wild-type and IL-4(-/-) mice successfully resolved infection, while 70% of IFN-gamma(-/-) mice survived. In contrast, all CD28(-/-) mice succumbed during acute infection. While fewer than half of CD40L(-/-) mice succumbed acutely, surviving mice failed to clear infection, resulting in progressive mucosal destruction, polymicrobial sepsis, and death 1 to 2 weeks later than in CD28(-/-) mice. Downstream of CD28-mediated effects, CD4+ T-cell-expressed CD40L proved essential for generating acute pathogen-specific immunoglobulin M (IgM) and early IgG, which reduced pathogen burdens. However, deficiency of CD4+ T-cell-expressed IFN-gamma did not adversely impact survival or development of protective antibody in adoptively transferred CD4(-/-) recipients, though it impacted Th1 antibody responses. These findings demonstrate that CD4+ T-cell-expressed CD40L promotes the rapid production of protective systemic antibody during acute infection, while deficiencies of IL-4 or of CD4+ T-cell-expressed IFN-gamma can be overcome. These findings have important implications for understanding the role of T-helper-cell responses during infections involving mucosal surfaces.     

Modulation of experimental blood stage malaria through blockade of the B7/CD28 T-cell costimulatory pathway

Recent studies have implicated cytokines associated with CD4+ T lymphocytes of both T helper (Th)1 and Th2 subsets in resistance to experimental blood stage malaria. As the B7/CD28 costimulatory pathway has been shown to influence the differentiation of Th cell subsets, we investigated the contribution of the B7 molecules CD80 and CD86 to Th1/Th2 cytokine and immunoglobulin isotype profiles and to the development of a protective immune response to malaria in NIH mice infected with Plasmodium chabaudi. Effective blockade of CD86/CD28 interaction was demonstrated by elimination of interleukin (IL)-4 and up-regulation of interferon (IFN)-gamma responses by P. chabaudi-specific T cells and by reduction of P. chabaudi-specific immunoglobulin G1 (IgG1). The shift towards a Th1 cytokine pattern corresponded with efficient control of acute parasitaemia but an inability to resolve chronic infection. Moreover, combined CD80/CD86 blockade by using anti-CD80 and anti-CD86 monoclonal antibodies raised IFN-gamma production over that seen with CD86 blockade alone, with augmentation of this Th1-associated cytokine reducing levels of peak primary parasitaemia. These results demonstrate that IL-4 production by T cells in P. chabaudi-infected NIH mice is dependent upon CD86/CD28 interaction and that IL-4 and IFN-gamma contribute significantly, at different times of infection, to host resistance to blood stage malaria. In addition, combined CD80/CD86 blockade resulted in preferential expansion of IFN-gamma-producing T cells during P. chabaudi infection, suggesting that costimulatory pathways other than B7/CD28 may contribute to T-cell activation during continuous antigen stimulation. This study indicates a role for B7/CD28 costimulation in modulating the CD4+ T-cell response during malaria, and further suggests involvement of this pathway in other infectious and autoimmune diseases in which the Th cell immune response is also skewed.     

Both IL-12 and IL-18 contribute to small intestinal Th1-type immunopathology following oral infection with Toxoplasma gondii, but IL-12 is dominant over IL-18 in parasite control

Oral infection of C57BL/6 mice with Toxoplasma gondii results in small intestinal Th1-type immunopathology mediated by local production of IFN-gamma, TNF-alpha, and NO. To analyze whether the proinflammatory cytokines IL-12 and IL-18 play a role in the induction of immunopathology, IL-12p35/p40(-/-) and IL-18(-/-) mice were orally infected with T. gondii. Wild-type mice developed massive necrosis in their small intestines and died 7-10 days post infection. Even though IL-12p35/40(-/-) mice did not develop the necrosis they all died between day 9 and 11 after infection. In contrast, 50% of IL-18(-/-) mice died during the acute phase of infection. Compared to wild-type mice, IL-12p35/p40(-/-) but not IL-18(-/-) mice showed significantly higher parasite numbers in their small intestines and significantly higher numbers of parasite-associated inflammatory foci in their livers. IFN-gamma production was similar in infected wild-type and IL-18(-/-) mice but significantly decreased in IL-12p35/p40(-/-) mice. Treatment of mice with anti-IL-12- or anti-IL-18 antibodies after infection prevented the development of intestinal necrosis. These results reveal that both IL-12 and IL-18 play an important role in the development of intestinal immunopathology following oral infection with T. gondii. However, IL-12 is dominant over IL-18 in the host defense against parasite replication. Therefore, neutralization of IL-18 (rather than TNF-alpha, IL-12, and IFN-gamma) may be a safe strategy for the treatment of Th1-associated diseases.     

Role of interleukin-10 in regulation of T-cell-dependent and T-cell-independent mechanisms of resistance to Toxoplasma gondii.

Interleukin-10 (IL-10) is a cytokine which can inhibit T-cell and natural killer (NK) cell functions associated with cell-mediated immunity to intracellular infections. The production of IL-10 by mice infected with Toxoplasma gondii has been implicated in the suppression of lymphocyte proliferation observed during acute toxoplasmosis, as well as susceptibility to infection with this parasite. We have used C57BL/6 mice which lack a functional IL-10 gene (IL-10(-/-) mice) to investigate the role of IL-10 in acute toxoplasmosis. Intraperitoneal infection of IL-10(-/-) mice with T. gondii resulted in 100% mortality by day 13, whereas wild-type C57BL/6 (WT) mice survived acute infection. IL-10(-/-) mice infected with T. gondii had significantly higher serum levels of IL-12 and gamma interferon (IFN-gamma) than WT mice. Early mortality of infected IL-10(-/-) mice was prevented by treatment with IL-10 and significantly delayed by neutralizing antibodies to IL-12 and IFN-gamma. Further studies revealed that SCID/IL-10(-/-) mice infected with T. gondii had delayed time to death compared to IL-10(-/-) mice, indicating that lymphocytes contributed to death of IL-10(-/-) mice. In addition, infected SCID/IL-10(-/-) mice survived longer than infected SCID mice. These latter data indicate that in mice lacking lymphocytes, endogenous IL-10 is associated with increased susceptibility to T. gondii. However, the lack of IL-10 does not alter the infection-induced suppression of T cell and NK cell functions. Our experiments reveal that IL-10 is associated with protection or increased susceptibility to infection with T. gondii, depending on whether mice possess lymphocytes, and demonstrate the important roles of IL-12 and IFN-gamma in the early infection-induced mortality observed in the IL-10(-/-) mice.     

Critical contribution of CD28-CD80/CD86 costimulatory pathway to protection from Trypanosoma cruzi infection

The CD28-CD80/CD86-mediated T-cell costimulatory pathway has been variably implicated in infectious immunity. In this study, we investigated the role of this costimulatory pathway in resistance to Trypanosoma cruzi infection by using CD28-deficient mice and blocking antibodies against CD80 and CD86. CD28-deficient mice exhibited markedly exacerbated T. cruzi infection, as evidenced by unrelenting parasitemia and 100% mortality after infection with doses that are nonlethal in wild-type mice. The blockade of both CD80 and CD86 by administering specific monoclonal antibodies also exacerbated T. cruzi infection in wild-type mice. Splenocytes from T. cruzi-infected, CD28-deficient mice exhibited greatly impaired gamma interferon production in response to T. cruzi antigen stimulation in vitro compared to those from infected wild-type mice. The induction of T. cruzi antigen-specific CD8(+) T cells was also impaired in T. cruzi-infected, CD28-deficient mice. In addition to these defects in natural protection against T. cruzi infection, CD28-deficient mice were also defective in the induction of CD8(+)-T-cell-mediated protective immunity against T. cruzi infection by DNA vaccination. These results demonstrate, for the first time, a critical contribution of the CD28-CD80/CD86 costimulatory pathway not only to natural protection against primary T. cruzi infection but also to DNA vaccine-induced protective immunity to Chagas' disease.     

Influence of costimulatory molecules on immune response to Leishmania major by human cells in vitro

The importance of CD40, CD80, and CD86 costimulatory molecules in anti-Leishmania immune responses has been established in murine models. A role for these costimulatory molecules in human anti-Leishmania immune responses was investigated in this study. Autologous macrophages and peripheral blood leukocytes (PBL) were prepared from peripheral blood mononuclear cells of Leishmania-naive donors and cultured with or without Leishmania major in various combinations. After 7 days of culture, high levels of CD40 and CD86 were expressed on macrophages in the presence or absence of L. major. When macrophages were cultured for an additional 7 days with PBL, expression of all three costimulatory molecules was detected. When L. major was present in these cultures, the expression of CD80, and to a lesser extent CD40, on macrophages was enhanced. Blockade of CD80, CD86, or both molecules (in the order of greatest effect) in cultures containing macrophages, PBL, and L. major significantly inhibited the production of gamma interferon, interleukin-5 (IL-5), and IL-12. Blockade of CD40-CD154 interactions also significantly inhibited production of these cytokines in response to L. major. Production of IL-10 was unaltered by the blockade of these costimulatory molecules. Thus, these data suggest that CD40, CD80, and CD86 expression and regulation may significantly impact anti-Leishmania immune responses in humans.     

Differential CD28 and inducible costimulatory molecule signaling requirements for protective CD4+ T-cell-mediated immunity against genital tract Chlamydia trachomatis infection

Th1 cells and gamma interferon (IFN-gamma) production play critical roles in protective immunity against genital tract infections by Chlamydia trachomatis. Here we show that inducible costimulatory molecule (ICOS)(-/-) mice develop greatly augmented host resistance against chlamydial infection. Protection following a primary infection was characterized by strong Th1 immunity with enhanced CD4(+) T-cell-mediated IFN-gamma production in the genital tract and high expression of T-bet in the draining para-aortic lymph node. This Th1 dominance was associated with low expression of interleukin 10 (IL-10) mRNA in the uteruses of protected ICOS(-/-) mice. By contrast, CD28(-/-) mice were severely impaired in their adaptive immune response, demonstrating a lack of CD4(+) T cells and IFN-gamma in the genital tract, with a substantial delay in bacterial elimination compared to that seen in wild-type (WT) mice. Upon reinfection, WT mice exhibited a transient local infection with evidence of regulatory T-cell (Treg)/Foxp3 mRNA and a more balanced Th1 and Th2 response in the genital tract than ICOS(-/-) mice, whereas 90% of the latter mice developed sterile immunity, poor expression of local Treg/Foxp3 mRNA, and macroscopic signs of enhanced local immunopathology. Therefore, different requirements for CD28 signaling and ICOS signaling clearly apply to host protection against a genital tract infection by C. trachomatis. Whereas, CD28 signaling is critical, ICOS appears to be dispensable and can have a dampening effect on Th1 development by driving Th2 immunity and anti-inflammation through IL-10 production and promotion of the Foxp3(+) Treg populations in the genital tract. Both the CD28-deficient and the ICOS-deficient mice demonstrated poor specific antibody production, supporting the fact that antibodies are not needed for protection against genital tract chlamydial infections.     

Targeting OX40 and OX40L for the treatment of autoimmunity and cancer

The optimal activation of na?ve T cells requires TCR-mediated recognition of cognate peptide-MHC complexes on antigen presenting cells in the presence of costimulatory signals. Although signals provided via CD28-B7 interactions are important for enhancing the initial T-cell response, other costimulatory signals are required for sustaining the response and promoting both T-cell differentiation and survival. In particular, engagement of OX40 (CD134) by its natural ligand OX40L (CD134L) or OX40 agonists has been shown to provide key signals that can augment CD4 and CD8 T-cell responses. Importantly, numerous studies have highlighted the ability of OX40-specific agonists or antagonists to enhance antitumor immunity or ameliorate autoimmune disease, respectively. On the basis of these studies, the development of OX40- and OX40L-specific reagents has been pursued for clinical use. Given the emerging role of OX40 and OX40L as novel therapeutic targets, this review will focus on the cellular and molecular mechanisms of OX40-mediated T-cell costimulation with a special emphasis on the role of OX40-OX40L interactions in the etiology and treatment of autoimmunity and cancer.     

CD28 and IL-4: two heavyweights controlling the balance between immunity and inflammation

The costimulatory receptor CD28 and IL-4Rα-containing cytokine receptors play key roles in controlling the size and quality of pathogen-specific immune responses. Thus, CD28-mediated costimulation is needed for effective primary T-cell expansion and for the generation and activation of regulatory T-cells (Treg cells), which protect from immunopathology. Similarly, IL-4Rα signals are required for alternative activation of macrophages, which counteract inflammation by type 1 responses. Furthermore, immune modulation by CD28 and IL-4 is interconnected through the promotion of IL-4 producing T-helper 2 cells by CD28 signals. Using conditionally IL-4Rα and CD28 deleting mice, as well as monoclonal antibodies, which block or stimulate CD28, or mAb that deplete Treg cells, we have studied the roles of CD28 and IL-4Rα in experimental mouse models of virus (influenza), intracellular bacteria (L. monocytogenes, M. tuberculosis), and parasite infections (T. congolense, L. major). We observed that in some, but not all settings, Treg cells and type 2 immune deviation, including activation of alternative macrophages can be manipulated to protect the host either from infection or from immunopathology with an overall beneficial outcome. Furthermore, we provide direct evidence that secondary CD8 T-cell responses to i.c. bacteria are dependent on CD28-mediated costimulation.     

Interleukin-12 regulates the production of Bacille Calmette-Guérin-induced interferon-gamma from human cells in a CD40-dependent manner.

Interferon (IFN)-gamma is a cytokine which plays a critical role in the host defence against human tuberculosis infection. There is evidence that interleukin (IL)-12 can exert a potent effect in stimulating the production of IFN-gamma, and it is well known that a costimulatory signal provided by CD40 may enhance IL-12 production by monocytes/macrophages. However, it is unclear whether CD40-CD40L stimulation is able to regulate the production of mycobacterial-induced IFN-gamma through an IL-12-dependent pathway. In this study, we investigated the capacity of the Bacille Calmette-Guérin (BCG) strain of Mycobacterium bovis to induce the production of interferon-gamma through IL-12 and/or CD40 costimulation from human cells. Our data demonstrate that anti-IL-12 antibodies markedly reduced the levels of IFN-gamma produced by the BCG-stimulated human cells, while exogenous recombinant (r)IL-12 up-regulated the production of IFN-gamma. In addition, the stimulatory effect of IL-12 on BCG-induced IFN-gamma secretion was specific, as it was significantly abolished in the presence of anti-IL-12 antibodies. We also observed that the presence of an anti-CD40L monoclonal antibody significantly inhibited the production of IL-12 and IFN-gamma by human cells activated with BCG. In contrast, an isotype control antibody showed no effect on cytokine production. Furthermore, the presence of a trimeric soluble CD40L agonist (CD40T) in cultures increased the production of IL-12 and IFN-gamma. Importantly, the stimulatory capacity of CD40T on BCG-induced IFN-gamma secretion was blocked by a monoclonal antibody against IL-12, indicating that the effect of CD40T on T cells was mediated through IL-12. Together, these studies are the first to demonstrate that BCG-induced IFN-gamma production by human cells appears to be mediated by IL-12 in a CD40-dependent manner and suggest that CD40-CD40L activation may be a critical mediator in regulating the immune response to stimulation with BCG.     

Cognate stimulatory B-cell-T-cell interactions are critical for T-cell help recruited by glycoconjugate vaccines

Covalent linkage of a bacterial polysaccharide to an immunogenic protein greatly enhances the carbohydrate's immunogenicity and induces polysaccharide-specific B-cell memory in vivo. These findings have spurred the development of glycoconjugate vaccines for serious bacterial infections. The specific B-cell-T-cell interactions responsible for recruitment of T-cell help by glycoconjugate vaccines are not well defined. We used mice deficient in molecules critical for stimulatory, cognate B-cell-T-cell interactions to study how T cells improve the immunogenicity of a glycoconjugate vaccine against group B streptococcal disease. Isotype switching to immunoglobulin G (IgG) was abrogated in mice deficient in major histocompatibility complex (MHC) class II antigen (Ag)-T-cell receptor (TCR), B7-CD28, or CD40-CD40L interactions. However, expression of either the B7-1 or the B7-2 molecule on antigen-presenting cells was sufficient for optimal T-cell costimulation. T cells activated by the vaccine also played a pivotal role in determining the magnitude of the IgM response to the polysaccharide. Comparable results were obtained with pathway antagonists. These data suggest that MHC class II Ag-TCR, B7-CD28, and CD40-CD40L interactions are critical for immune responses to glycoconjugate vaccines in vivo.     

Helicobacter hepaticus-induced colitis in interleukin-10-deficient mice: cytokine requirements for the induction and maintenance of intestinal inflammation

We have previously shown that specific-pathogen-free interleukin-10 (IL-10)-deficient (IL-10 KO) mice reconstituted with Helicobacter hepaticus develop severe colitis associated with a Th1-type cytokine response. In the present study, we formally demonstrate that IL-12 is crucial for disease induction, because mice deficient for both IL-10 and IL-12 p40 show no intestinal pathology following H. hepaticus infection. By using monoclonal antibodies (MAbs) to IL-12, gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha), we have further analyzed the role of these cytokines in the maintenance of the Th1 response and inflammation in IL-10 KO mice with established H. hepaticus-induced colitis. Treatment of infected colitic IL-10 KO mice with anti-IL-12 p40 resulted in markedly reduced intestinal inflammation, colonic IFN-gamma, TNF-alpha, and inducible nitric oxide synthase (iNOS) mRNA levels, and H. hepaticus-specific IFN-gamma secretion by mesenteric lymph node (MLN) cells compared to the findings in control MAb-treated mice. Moreover, the diminished pathology was associated with decreased numbers of colonic CD3(+) T cells and significantly reduced frequencies of Helicobacter-reactive CD4(+) Th1 cells in MLN. In contrast, anti-IFN-gamma and/or anti-TNF-alpha had no effect on intestinal inflammation in IL-10 KO mice with established colitis. Using IL-10/IFN-gamma double-deficient mice, we further show that IFN-gamma is not required for the development of colitis following H. hepaticus infection. MLN cells from infected IL-10/IFN-gamma KO animals secreted elevated amounts of IL-12 and TNF-alpha following bacterial antigen stimulation, indicating alternative pathways of disease induction. Taken together, our results demonstrate a crucial role for IL-12 in both inducing and sustaining intestinal inflammation through recruitment and maintenance of a pool of pathogenic Th1 cells.     

Enhanced IL-10 production by CD4+ T cells primed in IL-15Rα-deficient mice

In this study, we investigated the functional outcomes of CD4(+) T cells primed in the absence of IL-15 transpresentation. Compared with their WT counterparts primed in WT mice, IL-15Rα KO CD4(+) T cells primed in KO mice were found to exclusively overproduce IL-10 upon in vitro restimulation(.) The comparable expression of IL-4 and Foxp3 in CD4(+) T cells primed in the WT and IL-15Rα KO mice indicated that this was neither due to T(H) 2- nor Treg cell-differentiation. IL-10 overproduction was also observed when OVA-specific TCR transgenic CD4(+) T (OT-II) cells were primed in KO mice, excluding an intrinsic deficiency of KO CD4(+) T cells. To investigate the WT and KO microenvironment, DCs from both WT and IL-15Rα KO mice were compared. DCs from both backgrounds were indistinguishable in their steady-state survival and in their expression of MHC class II and costimulatory molecules CD80, CD86, and CD40. However, IL-15Rα KO DCs primed OT-II cells in vitro to produce higher levels of IL-10 upon their restimulation. Additionally, IL-15Rα KO DCs produced significantly more IL-10 upon activation, and IL-10 neutralization during DC-mediated in vitro priming abolished IL-10 overproduction by CD4(+) T cells. Thus, IL-15Rα KO DCs provide an IL-10-enriched environment that preferentially primes CD4(+) T cells for more IL-10 production, highlighting a regulatory role for IL-15 transpresentation in CD4(+) T-cell priming.     

Importance of CD80/CD86-CD28 interactions in the recognition of target cells by CD8+CD122+ regulatory T cells

CD8+CD122+ regulatory T cells are a newly identified, naturally occurring type of regulatory T cell that produce interleukin-10 (IL-10) and effectively suppress interferon-gamma (IFN-gamma) production from both CD8+ and CD4+ target cells. Molecular mechanisms responsible for the recognition of target cells by CD8+CD122+ regulatory T cells were investigated in this study by using an in vitro culture system that reconstitutes the regulatory action of these cells. CD8+CD122( regulatory T cells did not produce IL-10 and did not suppress the IFN-gamma production of allogeneic target T cells when they were stimulated by immobilized anti-CD3 antibody alone, but they clearly produced IL-10 and suppressed the IFN-gamma production of target cells when stimulated by anti-CD3 plus anti-CD28-coated beads. IFN-gamma production by major histocompatibility complex-class I-deficient T cells was also suppressed by CD8+CD122+ regulatory T cells stimulated with anti-CD3 plus anti-CD28 antibody but was not suppressed by cells stimulated by anti-CD3 alone. Experiments examining the blockade of cell surface molecules expressed on either the regulatory cells or the target cells by adding specific neutralizing antibodies in the culture indicated that CD80, CD86, and CD28 molecules were involved in the regulatory action, but cytotoxic T lymphocyte antigen-4, inducible costimulatory molecule (ICOS) and programmed death-1 (PD-1) molecules were not. Finally, CD8+CD122+ cells isolated from CD28-knockout (CD28-/-) mice showed no regulatory activity. These results indicate that CD8+CD122(+) regulatory T cells recognize target T cells via the interaction of CD80/CD86-CD28 molecules to become active regulatory cells that produce suppressive factors such as IL-10.     

Dendritic cell function during chronic hepatitis C virus and human immunodeficiency virus type 1 infection

Hepatitis C virus (HCV) infection can persist despite HCV-specific T-cell immunity and can have a more aggressive course in persons coinfected with human immunodeficiency virus type 1 (HIV-1). Defects in antigen-presenting, myeloid dendritic cells (DCs) could underlie this T-cell dysfunction. Here we show that monocyte-derived DCs from persons with chronic HCV infection, with or without HIV-1 coinfection, being treated with combination antiretroviral therapy produced lower levels of interleukin 12 (IL-12) p70 in response to CD40 ligand (CD40L), whereas the expression of DC surface activation and costimulatory molecules was unimpaired. The deficiency in IL-12 production could be overcome by addition of gamma interferon (IFN-gamma) with CD40L, resulting in very high, comparable levels of IL-12 production by DCs from HCV- and HIV-1-infected subjects. Smaller amounts of IL-12 p70 were produced by DCs treated with the immune modulators tumor necrosis factor alpha and IL-1beta, with or without IFN-gamma, and the amounts did not differ among the uninfected and infected subjects. Blocking of IL-10 with an anti-IL-10 monoclonal antibody in the CD40L-stimulated DC cultures from HCV-infected persons increased the level of IL-12 p70 production. The ability of DCs from HCV-infected persons to stimulate allogeneic CD4+ T cells or induce IL-2, IL-5, or IL-10 in a mixed lymphocyte reaction was not impaired. Thus, myeloid DCs derived from persons with chronic HCV infection or with both HCV and HIV-1 infections have defects in IL-12 p70 production related to IL-10 activity that can be overcome by treatment of the DCs with CD40L and IFN-gamma. DCs from these infected subjects have a normal capacity to stimulate CD4+ T cells. The functional effectiveness of DCs derived from HCV-infected individuals provides a rationale for the DC-based immunotherapy of chronic HCV infection.     

Disruption of the ICOS-B7RP-1 costimulatory pathway leads to enhanced hepatic immunopathology and increased gamma interferon production by CD4 T cells in murine schistosomiasis

Morbidity and mortality in schistosomiasis are largely due to an immune response mediated by CD4 T lymphocytes. Since lymphocyte activation is shaped by costimulatory signals, the specific functions of different costimulatory pathways are of increasing interest. We now examined the role of the inducible costimulatory molecule (ICOS) and its ligand B7-related protein 1 (B7RP-1) in the experimental murine schistosome infection by blocking this costimulatory pathway with monoclonal antibody against ICOS, administered daily by intraperitoneal injection during the patent phase of the disease. The treated mice exhibited enhanced hepatic immunopathology characterized by enlarged egg granulomas and pronounced parenchymal inflammation with hepatocellular necrosis, resulting in elevated liver enzyme levels in serum. Most strikingly, there was a sharp increase in gamma interferon (IFN-gamma) production by schistosome egg antigen-stimulated granuloma cells, bulk mesenteric lymph node (MLN) cells, and purified MLN CD4 T cells, which contrasted with a more discreet change in the Th2-type cytokines interleukin 4 (IL-4) and IL-10. These findings suggest that the ICOS-B7RP-1 costimulatory pathway serves primarily to control IFN-gamma production, thereby promoting a cytokine environment conducive to limited hepatic damage.