Natural killer T cell activation protects mice against experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) serves as a prototypic model for T cell-mediated autoimmunity. V(alpha)14 natural killer T (NKT) cells are a subset of T lymphocytes that recognize glycolipid antigens presented by the nonpolymorphic major histocompatibility complex (MHC) class I-like protein CD1d. Here, we show that activation of V(alpha)14 NKT cells by the glycosphingolipid alpha-galactosylceramide (alpha-GalCer) protects susceptible mice against EAE. beta-GalCer, which binds CD1d but is not recognized by NKT cells, failed to protect mice against EAE. Furthermore, alpha-GalCer was unable to protect CD1d knockout (KO) mice against EAE, indicating the requirement for an intact CD1d antigen presentation pathway. Protection of disease conferred by alpha-GalCer correlated with its ability to suppress myelin antigen-specific Th1 responses and/or to promote myelin antigen-specific Th2 cell responses. alpha-GalCer was unable to protect IL-4 KO and IL-10 KO mice against EAE, indicating a critical role for both of these cytokines. Because recognition of alpha-GalCer by NKT cells is phylogenetically conserved, our findings have identified NKT cells as novel target cells for treatment of inflammatory diseases of the central nervous system.     

Natural killer T cell ligand alpha-galactosylceramide enhances protective immunity induced by malaria vaccines

The important role played by CD8(+) T lymphocytes in the control of parasitic and viral infections, as well as tumor development, has raised the need for the development of adjuvants capable of enhancing cell-mediated immunity. It is well established that protective immunity against liver stages of malaria parasites is primarily mediated by CD8(+) T cells in mice. Activation of natural killer T (NKT) cells by the glycolipid ligand, alpha-galactosylceramide (alpha-GalCer), causes bystander activation of NK, B, CD4(+), and CD8(+) T cells. Our study shows that coadministration of alpha-GalCer with suboptimal doses of irradiated sporozoites or recombinant viruses expressing a malaria antigen greatly enhances the level of protective anti-malaria immunity in mice. We also show that coadministration of alpha-GalCer with various different immunogens strongly enhances antigen-specific CD8(+) T cell responses, and to a lesser degree, Th1-type responses. The adjuvant effects of alpha-GalCer require CD1d molecules, Valpha14 NKT cells, and interferon gamma. As alpha-GalCer stimulates both human and murine NKT cells, these findings should contribute to the design of more effective vaccines against malaria and other intracellular pathogens, as well as tumors.     

The natural killer T (NKT) cell ligand alpha-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells

The natural killer T (NKT) cell ligand alpha-galactosylceramide (alpha-GalCer) exhibits profound antitumor activities in vivo that resemble interleukin (IL)-12-mediated antitumor activities. Because of these similarities between the activities of alpha-GalCer and IL-12, we investigated the involvement of IL-12 in the activation of NKT cells by alpha-GalCer. We first established, using purified subsets of various lymphocyte populations, that alpha-GalCer selectively activates NKT cells for production of interferon (IFN)-gamma. Production of IFN-gamma by NKT cells in response to alpha-GalCer required IL-12 produced by dendritic cells (DCs) and direct contact between NKT cells and DCs through CD40/CD40 ligand interactions. Moreover, alpha-GalCer strongly induced the expression of IL-12 receptor on NKT cells from wild-type but not CD1(-/-) or Valpha14(-/-) mice. This effect of alpha-GalCer required the production of IFN-gamma by NKT cells and production of IL-12 by DCs. Finally, we showed that treatment of mice with suboptimal doses of alpha-GalCer together with suboptimal doses of IL-12 resulted in strongly enhanced natural killing activity and IFN-gamma production. Collectively, these findings indicate an important role for DC-produced IL-12 in the activation of NKT cells by alpha-GalCer and suggest that NKT cells may be able to condition DCs for subsequent immune responses. Our results also suggest a novel approach for immunotherapy of cancer.     

Glycolipid antigen induces long-term natural killer T cell anergy in mice

Natural killer T (NKT) cells recognize glycolipid antigens presented by the MHC class I-related glycoprotein CD1d. The in vivo dynamics of the NKT cell population in response to glycolipid activation remain poorly understood. Here, we show that a single administration of the synthetic glycolipid alpha-galactosylceramide (alpha-GalCer) induces long-term NKT cell unresponsiveness in mice. NKT cells failed to proliferate and produce IFN-gamma upon alpha-GalCer restimulation but retained the capacity to produce IL-4. Consequently, we found that activation of anergic NKT cells with alpha-GalCer exacerbated, rather than prevented, B16 metastasis formation, but that these cells retained their capacity to protect mice against experimental autoimmune encephalomyelitis. NKT cell anergy was induced in a thymus-independent manner and maintained in an NKT cell-autonomous manner. The anergic state could be broken by IL-2 and by stimuli that bypass proximal TCR signaling events. Collectively, the kinetics of initial NKT cell activation, expansion, and induction of anergy in response to alpha-GalCer administration resemble the responses of conventional T cells to strong stimuli such as superantigens. Our findings have important implications for the development of NKT cell-based vaccines and immunotherapies.     

Efficient ex vivo expansion of Valpha24+ NKT cells derived from G-CSF-mobilized blood cells

Natural killer T (NKT) cells are involved in the function of innate immune systems and also play an important role in regulating acquired immune responses. In previous reports, we showed that Valpha24+ NKT cells proliferated more efficiently from granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (PBMC) than from non-mobilized PBMC. However, the mechanism of this enhanced NKT cell expansion is not yet clear. The goal of this research was to develop culture conditions for the more efficient ex vivo expansion of NKT cells. G-CSF-mobilized PBMC was cultured in AIM-V medium supplemented with 10% auto-plasma, 100 ng/mL alpha-galactosylceramide (alpha-GalCer) and 100 IU/mL recombinant human (rh) interleukin (IL)-2. The efficiency of the expansion of Valpha24+ NKT cells was evaluated on day 12. The expansion-fold of Valpha24+ NKT cells was augmented depending on the proportion of CD14+ cells at the beginning of culture. The depletion of Valpha24+ NKT cells abrogated the expansion of Valpha24+ NKT cells. Depletion of CD56+ NK cells from mobilized PBMC enhanced, and add-back of purified CD56+ NK cells suppressed the expansion of Valpha24+ NKT cells. Experiments with different timings for the addition of cells, IL-2 and alpha-GalCer suggested that follow-up supplementation with IL-2 or CD14+ cells should be avoided for the efficient expansion of Valpha24+ NKT cells. These results should be useful for the development of an efficient and practical expansion protocol for adoptive immunotherapy with Valpha24+ NKT cells.     

IL-21-induced Bepsilon cell apoptosis mediated by natural killer T cells suppresses IgE responses

Epidemiological studies have suggested that the recent increase in the incidence and severity of immunoglobulin (Ig)E-mediated allergic disorders is inversely correlated with Mycobacterium bovis bacillus Calmette Guerin (BCG) vaccination; however, the underlying mechanisms remain uncertain. Here, we demonstrate that natural killer T (NKT) cells in mice and humans play a crucial role in the BCG-induced suppression of IgE responses. BCG-activated murine Valpha14 NKT cells, but not conventional CD4 T cells, selectively express high levels of interleukin (IL)-21, which preferentially induces apoptosis in Bepsilon cells. Signaling from the IL-21 receptor increases the formation of a complex between Bcl-2 and the proapoptotic molecule Bcl-2-modifying factor, resulting in Bepsilon cell apoptosis. Similarly, BCG vaccination induces IL-21 expression by human peripheral blood mononuclear cells (PBMCs) in a partially NKT cell-dependent fashion. BCG-activated PBMCs significantly reduce IgE production by human B cells. These findings provide new insight into the therapeutic effect of BCG in allergic diseases.     

Augmentation of Valpha14 NKT cell-mediated cytotoxicity by interleukin 4 in an autocrine mechanism resulting in the development of concanavalin A-induced hepatitis

The administration of concanavalin A (Con A) induces a rapid severe injury of hepatocytes in mice. Although the Con A-induced hepatitis is considered to be an experimental model of human autoimmune hepatitis, the precise cellular and molecular mechanisms that induce hepatocyte injury remain unclear. Here, we demonstrate that Valpha14 NKT cells are required and sufficient for induction of this hepatitis. Moreover, interleukin (IL)-4 produced by Con A-activated Valpha14 NKT cells is found to play a crucial role in disease development by augmenting the cytotoxic activity of Valpha14 NKT cells in an autocrine fashion. Indeed, short-term treatment with IL-4 induces an increase in the expression of granzyme B and Fas ligand (L) in Valpha14 NKT cells. Moreover, Valpha14 NKT cells from either perforin knock-out mice or FasL-mutant gld/gld mice fail to induce hepatitis, and hence perforin-granzyme B and FasL appear to be effector molecules in Con A-induced Valpha14 NKT cell-mediated hepatocyte injury.     

Cutaneous immunization rapidly activates liver invariant Valpha14 NKT cells stimulating B-1 B cells to initiate T cell recruitment for elicitation of contact sensitivity

T cell recruitment to elicit contact sensitivity (CS) requires a CS-initiating process mediated by B-1 cells that produce IgM, which activates complement to promote T cell passage into the tissues. We now show that Valpha14i NKT cells induce B-1 cell activation likely by releasing IL-4 early postimmunization. The CS initiation process is absent in Jalpha18-/- and CD1d-/- NKT cell-deficient mice and is reconstituted by populations enriched for Valpha14i NKT cells. Transfers are not effective if cells are derived from IL-4-/- mice. Staining with specific tetramers directly showed that hepatic Valpha14i NKT cells increase by 30 min and nearly double by 2 h postimmunization. Transfer of immune B-1 cells also reconstitutes CS responses in NKT cell-deficient mice. The B-1 cells act downstream of the Valpha14i NKT cells to restore CS initiation. In addition, IL-4 given systemically to Jalpha18-/- or CD1d-/- NKT cell-deficient mice reconstitutes elicitation of CS. Further, splenocytes from immune Jalpha18-/- mice produce less antigen (Ag)-specific IgM antibodies compared with sensitized WT mice. Together these findings indicate that very early after skin immunization Valpha14i NKT cells are stimulated to produce IL-4, which activates B-1 cells to produce Ag-specific IgM, subsequently needed to recruit effector T cells for elicitation of CS responses.     

CD4(+) Valpha14 natural killer T cells are essential for acceptance of rat islet xenografts in mice

Pancreatic islet transplantation represents a potential treatment for insulin-dependent diabetes mellitus. However, the precise cellular and molecular mechanisms of the immune reactions against allogeneic and xenogeneic transplanted islets remain unclear. Here, we demonstrate that CD4(+) Valpha14 natural killer T (NKT) cells, a recently identified lymphoid cell lineage, are required for the acceptance of intrahepatic rat islet xenografts. An anti-CD4 mAb, administrated after transplantation, allowed islet xenografts to be accepted by C57BL/6 mice, with no need for immunosuppressive drugs. The dose of anti-CD4 mAb was critical, and the beneficial effect appeared to be associated with the reappearance of CD4(+) NKT cells at around 14 days after transplantation. Interestingly, rat islet xenografts were rejected, despite the anti-CD4 mAb treatment, in Valpha14 NKT cell-deficient mice, which exhibit the normal complement of conventional lymphoid cells; adoptive transfer of Valpha14 NKT cells into Valpha14 NKT cell-deficient mice restored the acceptance of rat islet xenografts. In addition, rat islet xenografts were accepted by Valpha14 NKT mice having only Valpha14 NKT cells and no other lymphoid cells. These results indicate that Valpha14 NKT cells play a crucial role in the acceptance of rat islet xenografts in mice treated with anti-CD4 antibody, probably by serving as immunosuppressive regulatory cells.     

Natural killer T cell activation inhibits hepatitis B virus replication in vivo

We have previously reported that hepatitis B virus (HBV)-specific CD8(+) cytotoxic T lymphocytes and CD4(+) helper T lymphocytes can inhibit HBV replication in the liver of HBV transgenic mice by secreting interferon (IFN)-gamma when they recognize viral antigen. To determine whether an activated innate immune system can also inhibit HBV replication, in this study we activated natural killer T (NKT) cells in the liver of HBV transgenic mice by a single injection of alpha-galactosylceramide (alpha-GalCer), a glycolipid antigen presented to Valpha14(+)NK1.1(+) T cells by the nonclassical major histocompatibility complex class I-like molecule CD1d. Within 24 h of alpha-GalCer injection, IFN-gamma and IFN-alpha/beta were detected in the liver of HBV transgenic mice and HBV replication was abolished. Both of these events were temporally associated with the rapid disappearance of NKT cells from the liver, presumably reflecting activation-induced cell death, and by the recruitment of activated NK cells into the organ. In addition, prior antibody-mediated depletion of CD4(+) and CD8(+) T cells from the mice did not diminish the ability of alpha-GalCer to trigger the disappearance of HBV from the liver, indicating that conventional T cells were not downstream mediators of this effect. Finally, the antiviral effect of alpha-GalCer was inhibited in mice that are genetically deficient for either IFN-gamma or the IFN-alpha/beta receptor, indicating that most of the antiviral activity of alpha-GalCer is mediated by these cytokines. Based on these results, we conclude that alpha-GalCer inhibits HBV replication by directly activating NKT cells and by secondarily activating NK cells to secrete antiviral cytokines in the liver. In view of these findings, we suggest that, if activated, the innate immune response, like the adaptive immune response, has the potential to control viral replication during natural HBV infection. In addition, the data suggest that therapeutic activation of NKT cells may represent a new strategy for the treatment of chronic HBV infection.     

Mechanisms imposing the Vbeta bias of Valpha14 natural killer T cells and consequences for microbial glycolipid recognition

Mouse and human natural killer T (NKT) cells recognize a restricted set of glycosphingolipids presented by CD1d molecules, including self iGb3 and microbial alpha-glycuronosylceramides. The importance of the canonical Valpha14-Jalpha18 TCR alpha chain for antigen recognition by NKT cells is well recognized, but the mechanisms underlying the Vbeta8, Vbeta7, and Vbeta2 bias in mouse have not been explored. To study the influences of thymic selection and the constraints of pairing with Valpha14-Jalpha18, we have created a population of mature T cells expressing Valpha14-Jalpha18 TCR alpha chain in CD1d-deficient mice and studied its recognition properties in vitro and in vivo. Transgenic cells expressed a diverse Vbeta repertoire but their recognition of endogenous ligands and synthetic iGb3 was restricted to the same biased Vbeta repertoire as expressed in natural NKT cells. In contrast, alpha-GalCer, a synthetic homologue of microbial alpha-glycuronosylceramides, was recognized by a broader set of Vbeta chains, including the biased NKT set but also Vbeta6, Vbeta9, Vbeta10, and Vbeta14. These surprising findings demonstrate that, whereas Vbeta8, Vbeta7, and Vbeta2 represent the optimal solution for recognition of endogenous ligand, many Vbeta chains that are potentially useful for the recognition of foreign lipids fail to be selected in the NKT cell repertoire.     

In vivo role of interleukin 4 in T cell tolerance induced by aqueous protein antigen

High doses of aqueous protein antigens induce a form of immunological tolerance in which interleukin 2 (IL-2)- and interferon gamma (IFN- gamma)-secreting T helper type 1 (Th1) cells are inhibited, but IL-4- secreting (Th2) cells are not. This is manifested by reduced proliferation of antigen-specific T cells upon in vitro restimulation, and marked suppression of specific antibody responses of the immunoglobulin (Ig)G2a, IgG2b, and IgG3 isotypes, but not of IgG1 and IgE. The role of the immunomodulatory cytokine IL-4 in this model of unresponsiveness to protein antigens has been examined. Administration of tolerizing antigen itself primes splenic CD4+ T cells for secretion of lymphokines, both IL-2 and IL-4. Neutralization of IL-4 in vivo with the anti-IL-4 antibody 11B11 during tolerance induction augments IFN- gamma production by T cells of tolerant mice, and reverses the suppression of IgG2a, IgG2b, and IgG3. This blockade of IL-4 function does not, however, restore the proliferative responses of T cells, suggesting that reduced T cell proliferation is due to direct T cell inactivation or anergy. Inhibiting the activity of IL-4 in vivo also inhibits the expansion of antigen-specific Th2-like cells, which are resistant to the induction of unresponsiveness. Thus, the immunologic consequences of high-dose tolerance are due to a combination of clonal T cell anergy and IL-4-mediated immune regulation.     

Prevention of injury-induced suppression of T-cell immunity by the CD1d/NKT cell-specific ligand alpha-galactosylceramide

Infection, sepsis, and multiple organ failure continue to be significant factors leading to morbidity and mortality after severe injury. Studies by our laboratory and others have identified injury-induced defects in both innate and adaptive components of host defense. We previously reported that CD1d-restricted natural killer T (NKT) cells actively suppress effector T-cell immunity after burn injury via production of excess IL-4 and failure to produce IFN-gamma. alpha-Galactosylceramide (alpha-GalCer) is a synthetic NKT cell-specific ligand presented exclusively to invariant NKT cells and is known to improve immunity against tumors and infection by promoting IFN-gamma production. Here, we confirmed the role of Valpha14-Jalpha281 invariant NKT cells in mouse model of burn injury-induced suppression of T-cell immunity and further asked whether alpha-GalCer can improve immunity after injury via similar mechanisms. We observed that systemic treatment with alpha-GalCer prevented the injury-induced suppression of Ag-specific T-cell responsiveness both in vitro and in vivo and restored the ability of splenic lymphocytes to produce both IL-2 and IFN-gamma. Moreover, burn injury was associated with diminished expression of major histocompatibility complex II and CD40 on antigen presenting cells that were both restored by alpha-GalCer treatment to levels seen in sham-treated mice. Collectively, these data suggest that, via manipulation of the NKT cell population, we may be able to maintain T-cell function and improve host defense after burn injury.     

CD1d-restricted human natural killer T cells are highly susceptible to human immunodeficiency virus 1 infection

Human natural killer (NK) T cells are unique T lymphocytes that express an invariant T cell receptor (TCR) Valpha24-Vbeta11 and have been implicated to play a role in various diseases. A subset of NKT cells express CD4 and hence are potential targets for human immunodeficiency virus (HIV)-1 infection. We demonstrate that both resting and activated human Valpha24(+) T cells express high levels of the HIV-1 coreceptors CCR5 and Bonzo (CXCR6), but low levels of CCR7, as compared with conventional T cells. Remarkably NKT cells activated with alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells were profoundly more susceptible to infection with R5-tropic, but not X4-tropic, strains of HIV-1, compared with conventional CD4(+) T cells. Furthermore, resting CD4(+) NKT cells were also more susceptible to infection. After initial infection, HIV-1 rapidly replicated and depleted the CD4(+) subset of NKT cells. In addition, peripheral blood NKT cells were markedly and selectively depleted in HIV-1 infected individuals. Although the mechanisms of this decline are not clear, low numbers or absence of NKT cells may affect the course of HIV-1 infection. Taken together, our findings indicate that CD4(+) NKT cells are directly targeted by HIV-1 and may have a potential role during viral transmission and spread in vivo.     

CD40-CD40 ligand interactions are critical in T-B cooperation but not for other anti-viral CD4+ T cell functions

CD40-CD40 ligand (CD40L) interaction is required for the generation of antibody responses to T-dependent antigens as well as for the development of germinal centers and memory B cells. The role of the CD40-CD40L interaction in the induction of antigen-specific. Th cells and in mediating Th cell effector functions other than cognate help for B cells is less well understood. Using CD40- and CD40L-deficient mice together with lymphocytic choriomeningitis virus and vesicular stomatitis virus as viral model antigens, this study corroborates earlier findings that no lg isotype switching of virus-specific antibodies was measurable upon infection of CD40- or CD40L-deficient mice. In contrast, in vivo induction of virus-specific CD4+ T cells measured by proliferation and cytokine secretion of primed virus- specific Th cells in vitro was not crucially dependent on the CD40- CD40L interaction. In addition, virus-specific Th cells primed in a CD40-deficient environment, adoptively transferred into CD40-competent recipients, were able to mediate lg isotype switch. Th-mediated effector functions distinct from and in addition to T-B collaboration were analyzed in CD40- and CD40L-deficient and normal mice: (a) local inflammatory reactions upon LCMV infection mediated by LCMV-specific Th cells were not dependent on a functional CD40-CD40L interaction, (b) cytokine-mediated protection by CD4+ T cells primed by vesicular stomatitis virus against a challenge infection with recombinant vaccinia virus expressing the glycoprotein of vesicular stomatitis virus was found to be equivalent in CD40L-deficient and normal mice. Thus, CD40-CD40L interaction plays a crucial role in T-B interactions for Th-dependent activation of B cells but not, or to a much lesser extent, in T cell activation, antigen-specific Th cell responses in vitro, and for interleukin-mediated Th cell effector functions in vivo.     

Activation of natural killer T cells in NZB/W mice induces Th1-type immune responses exacerbating lupus

In vivo treatment of mice with the natural killer T (NKT) cell ligand, alpha-galactosylceramide (alphaGalCer), ameliorates autoimmune diabetes and experimental autoimmune encephalomyelitis (EAE) by shifting pathogenic Th1-type immune responses to nonpathogenic Th2-type responses. In the current study, in vivo activation of NKT cells in adult NZB/W mice by multiple injections of alphaGalCer induced an abnormal Th1-type immune response as compared with the Th2-type response observed in nonautoimmune C57BL/6 mice. This resulted in decreased serum levels of IgE, increased levels of IgG2a and IgG2a anti-double-stranded DNA (anti-dsDNA) Ab's, and exacerbated lupus. Conversely, treatment of NZB/W mice with blocking anti-CD1d mAb augmented Th2-type responses, increased serum levels of IgE, decreased levels of IgG2a and IgG2a anti-dsDNA Ab's, and ameliorated lupus. While total CD4+ T cells markedly augmented in vitro IgM anti-dsDNA Ab secretion by splenic B cells, the non-CD1d-reactive (CD1d-alphaGalCer tetramer-negative) CD4+ T cells (accounting for 95% of all CD4+ T cells) failed to augment Ab secretion. The CD1d-reactive tetramer-positive CD4+ T cells augmented anti-dsDNA Ab secretion about tenfold. In conclusion, activation of NKT cells augments Th1-type immune responses and autoantibody secretion that contribute to lupus development in adult NZB/W mice, and anti-CD1d mAb might be useful for treating lupus.     

Activation of natural killer T cells by alpha-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a coadministered protein

The maturation of dendritic cells (DCs) allows these antigen-presenting cells to initiate immunity. We pursued this concept in situ by studying the adjuvant action of alpha-galactosylceramide (alphaGalCer) in mice. A single i.v. injection of glycolipid induced the full maturation of splenic DCs, beginning within 4 h. Maturation was manifest by marked increases in costimulator and major histocompatibility complex class II expression, interferon (IFN)-gamma production, and stimulation of the mixed leukocyte reaction. These changes were not induced directly by alphaGalCer but required natural killer T (NKT) cells acting independently of the MyD88 adaptor protein. To establish that DC maturation was responsible for the adjuvant role of alphaGalCer, mice were given alphaGalCer together with soluble or cell-associated ovalbumin antigen. Th1 type CD4+ and CD8+ T cell responses developed, and the mice became resistant to challenge with ovalbumin-expressing tumor. DCs from mice given ovalbumin plus adjuvant, but not the non-DCs, stimulated ovalbumin-specific proliferative responses and importantly, induced antigen-specific, IFN-gamma producing, CD4+ and CD8+ T cells upon transfer into naive animals. In the latter instance, immune priming did not require further exposure to ovalbumin, alphaGalCer, NKT, or NK cells. Therefore a single dose of alphaGalCer i.v. rapidly stimulates the full maturation of DCs in situ, and this accounts for the induction of combined Th1 CD4+ and CD8+ T cell immunity to a coadministered protein.     

Low dose Leishmania major promotes a transient T helper cell type 2 response that is down-regulated by interferon gamma-producing CD8+ T cells

An unresolved issue in the field of T helper (Th) cell development relates to the findings that low doses of antigen promote Th2 cell development in vitro, whereas several classic in vivo studies suggest the opposite. Here we resolve this paradox by studying the early immune response in mice after infection with different doses of Leishmania major. We found that low parasite doses induced a Th2 response in C57BL/6 (B6) mice, whereas high doses induced a Th1 response. However, the Th2 response in low dose-infected mice was transient and the animals healed. The appearance of a Th1 response after low dose infection was dependent upon the concomitant activation of interferon gamma-producing CD8+ T cells. In the absence of CD8+ T cells, the Th2 response was maintained. However, either neutralization of interleukin (IL)-4 or administration of IL-12 promoted a Th1 response after low dose infection of CD8-deficient mice, indicating that the required role for CD8+ T cells was limited to modulation of CD4+ T cell responses. Thus, the discrepant results seen between in vivo and in vitro studies on the effects of antigen dose on Th cell differentiation may depend upon whether CD8+ T cells participate in the immune response.     

Protein kinase C theta is critical for the development of in vivo T helper (Th)2 cell but not Th1 cell responses

The serine/threonine-specific protein kinase C (PKC)-theta is predominantly expressed in T cells and localizes to the center of the immunological synapse upon T cell receptor (TCR) and CD28 signaling. T cells deficient in PKC-theta exhibit reduced interleukin (IL)-2 production and proliferative responses in vitro, however, its significance in vivo remains unclear. We found that pkc-theta(-/-) mice were protected from pulmonary allergic hypersensitivity responses such as airway hyperresponsiveness, eosinophilia, and immunoglobulin E production to inhaled allergen. Furthermore, T helper (Th)2 cell immune responses against Nippostrongylus brasiliensis were severely impaired in pkc-theta(-/-) mice. In striking contrast, pkc-theta(-/-) mice on both the C57BL/6 background and the normally susceptible BALB/c background mounted protective Th1 immune responses and were resistant against infection with Leishmania major. Using in vitro TCR transgenic T cell-dendritic cell coculture systems and antigen concentration-dependent Th polarization, PKC-theta-deficient T cells were found to differentiate into Th1 cells after activation with high concentrations of specific peptide, but to have compromised Th2 development at low antigen concentration. The addition of IL-2 partially reconstituted Th2 development in pkc-theta(-/-) T cells, consistent with an important role for this cytokine in Th2 polarization. Taken together, our results reveal a central role for PKC-theta signaling during Th2 responses.