V gamma 1+ T cells suppress and V gamma 4+ T cells promote susceptibility to coxsackievirus B3-induced myocarditis in mice

Coxsackievirus B3 infections of C57BL/6 mice, which express the MHC class II IA but not IE Ag, results in virus replication in the heart but minimal myocarditis. In contrast, Bl.Tg.Ealpha mice, which are C57BL/6 mice transgenically induced to express IE Ag, develop significant myocarditis upon Coxsackievirus B3 infection. Despite this difference in inflammatory damage, cardiac virus titers are similar between C57BL/6 and Bl.Tg.Ealpha mice. Removing gammadelta T cells from either strain by genetic manipulation (gammadelta knockout(ko)) changes the disease phenotype. C57BL/6 gammadelta ko mice show increased myocarditis. In contrast, Bl.Tg.Ealpha gammadelta ko mice show decreased cardiac inflammation. Flow cytometry revealed a difference in the gammadelta cell subsets in the two strains, with Vgamma1 dominating in C57BL/6 mice, and Vgamma4 predominating Bl.Tg.Ealpha mice. This suggests that these two Vgamma-defined subsets might have different functions. To test this possibility, we used mAb injection to deplete each subset. Mice depleted of Vgamma1 cells showed enhanced myocarditis, whereas those depleted of Vgamma4 cells suppressed myocarditis. Adoptively transfusing enriched Vgamma4(+) cells to the C57BL/6 and Bl.Tg. Ealpha gammadelta ko strains confirmed that the Vgamma4 subset promoted myocarditis. Th subset analysis suggests that Vgamma1(+) cells biased the CD4(+) T cells to a dominant Th2 cell response, whereas Vgamma4(+) cells biased CD4(+) T cells toward a dominant Th1 cell response.     

Differential Th1 and Th2 cell responses in male and female BALB/c mice infected with coxsackievirus group B type 3.

Male and female BALB/c mice differ dramatically in susceptibility to myocarditis subsequent to coxsackievirus B3 (CVB3) infection. CVB3 infection of male mice results in substantial inflammatory cell infiltration of the myocardium, and virus-immune lymphocytes from these animals give predominantly a Th1 cell phenotypic response, as determined by predominant immunoglobulin G2a isotypic antibody production and elevated numbers of gamma interferon and interleukin-2 (IL-2)-producing CD4+ T lymphocytes. Females infected with the same virus give predominantly a Th2 cell phenotypic response, as determined by preferential immunoglobulin G1 antibody isotypic responses and increased precursor frequencies of IL-4- and IL-5-producing CD4+ T cells. Treatment of females with testosterone or males with estradiol prior to infection alters subsequent Th subset differentiation, suggesting that the sex-associated hormones have either a direct or indirect effect on CD4+ lymphocyte responses in this model. Treatment of females with 0.1 mg of monoclonal antibody to IL-4 reduces precursor frequencies of IL-4-producing CD4+ T cells and increases frequencies of gamma interferon-producing cells. This treatment also enhances myocardial inflammation, indicating a correlation between Th1-like cell responses and pathogenicity in CVB3 infection. The Th2-like cell may regulate Th1 cell activation. Adoptive transfer of T lymphocytes from CVB3-infected female mice into male animals suppresses the development of myocarditis in the recipients. Treatment of the female donors with monoclonal antibodies to either CD3, CD4, or IL-4 molecules abrogates suppression.     

Gammadelta T cells promote a Th1 response during coxsackievirus B3 infection in vivo: role of Fas and Fas ligand

Fas/Fas ligand (FasL) interactions regulate disease outcome in coxsackievirus B3 (CVB3)-induced myocarditis. MRL(+/+) mice infected with CVB3 develop severe myocarditis, a dominant CD4(+) Th1 (gamma interferon [IFN-gamma(+)]) response to the virus, and a predominance of gammadelta T cells in the myocardial infiltrates. MRL lpr/lpr and MRL gld/gld mice, which lack normal expression of Fas and express a mutated FasL, respectively, have minimal myocarditis and show a dominant CD4(+) Th2 (interleukin-4 [IL-4(+)]) phenotype to CVB3. Spleen cells from virus-infected wild-type, lpr, and gld animals proliferate equally to virus in vitro. Adoptive transfer of gammadelta T cells from hearts of CVB3-infected MRL(+/+) mice (FasL(+)) into infected MRL gld/gld recipients (FasL(-)/Fas(+)) restores both disease susceptibility and Th1 cell phenotype. However, transfer of these cells into MRL lpr/lpr recipients (FasL(+)/Fas(-)) did not promote myocarditis and the viral response remained Th2 biased. This paralleled the expression of very high surface levels of FasL by myocardial gammadelta T cells, as well as their propensity to selectively lyse Th2 virus-specific CD4(+) T cells. These results demonstrate that Fas/FasL interactions conferred by gammadelta T cells on lymphocyte subpopulations may regulate the cytokine response to CVB3 infection and pathogenicity.     

Vgamma4(+) T cells promote autoimmune CD8(+) cytolytic T-lymphocyte activation in coxsackievirus B3-induced myocarditis in mice: role for CD4(+) Th1 cells

T cells expressing the Vgamma4 T-cell receptor (TCR) promote myocarditis in coxsackievirus B3 (CVB3)-infected BALB/c mice. CD1, a major histocompatibility complex (MHC) class I-like molecule, is required for activation of Vgamma4(+) cells. Once activated, Vgamma4(+) cells initiate myocarditis through gamma interferon (IFN-gamma)-mediated induction of CD4(+) T helper type 1 (Th1) cells in the infected animal. These CD4(+) Th1 cells are required for activation of an autoimmune CD8(+) alphabeta TCR(+) effector, which is the predominant pathogenic agent in this model of CVB3-induced myocarditis. Activated Vgamma4(+) cells can adoptively transfer myocarditis into BALB/c mice infected with a nonmyocarditic variant of CVB3 (H310A1) but cannot transfer myocarditis into either uninfected or CD1(-/-) recipients, demonstrating the need for both infection and CD1 expression for Vgamma4(+) cell function. In contrast, CD8(+) alphabeta TCR(+) cells transfer myocarditis into either infected CD1(-/-) or uninfected recipients, showing that once activated, the CD8(+) alphabeta TCR(+) effectors function independently of both virus and CD1. Vgamma4(+) cells given to mice lacking CD4(+) T cells minimally activate the CD8(+) alphabeta TCR(+) cells. These studies show that Vgamma4(+) cells determine CVB3 pathogenicity by their ability to influence both the CD4(+) and CD8(+) adaptive immune response. Vgamma4(+) cells enhance CD4(+) Th1 (IFN-gamma(+)) cell activation through IFN-gamma- and CD1-dependent mechanisms. CD4(+) Th1 cells promote activation of the autoimmune CD8(+) alphabeta TCR(+) effectors.     

V gamma 1 gammadelta T cells regulate type-1/type-2 immune responses and participate in the resistance to infection and development of heart inflammation in Trypanosoma cruzi-infected BALB/c mice

Many different cell populations or lineages participate in the resistance to Trypanosoma cruzi infection. gammadelta T cells may also take part in a network of interactions that lead to control of T. cruzi infection with minimal tissue damage by controlling alphabeta T cell activation, as was previously suggested. However, the gammadelta T cell population is not homogeneous and its functions might vary, depending on T cell receptor usage or distinct stimulatory conditions. In this study, we show that the in vivo depletion of V gamma 1-bearing gammadelta T cells, prior to the infection of BALB/c mice with the Y strain of T. cruzi, induces an increased susceptibility to the infection with lower amounts of IFN-gamma being produced by conventional CD4+ or CD8+ T cells. In addition, the production of IL-4 by spleen T cells in V gamma 1-depleted mice was increased and the production of IL-10 remained unchanged. Since V gamma 1(+) gammadelta T cell depletion diminished the conversion of naive to memory/activated CD4 T cells and the production of IFN-gamma during the acute infection, these cells appear to function as helper cells for conventional CD4+ Th1 cells. Depletion of V gamma 1(+) cells also reduced the infection-induced inflammatory infiltrate in the heart and skeletal muscle. More importantly, V gamma 1(+) cells were required for up-regulation of CD40L in CD4+ and CD8+ T cells during infection. These results show that a subset of gammadelta T cells (V gamma 1(+)), which is an important component of the innate immune response, up-regulates the type 1 arm of the adaptative immune response, during T. cruzi infection.     

B cells and TCR avidity determine distinct functions of CD4+ T cells in retroviral infection

The T cell-dependent B cell response relies on cognate interaction between B cells and CD4(+) Th cells. However, the consequences of this interaction for CD4(+) T cells are not entirely known. B cells generally promote CD4(+) T cell responses to pathogens, albeit to a variable degree. In contrast, CD4(+) T cell responses to self- or tumor Ags are often suppressed by B cells. In this study, we demonstrated that interaction with B cells dramatically inhibited the function of virus-specific CD4(+) T cells in retroviral infection. We have used Friend virus infection of mice as a model for retroviral infection, in which the behavior of virus-specific CD4(+) T cells was monitored according to their TCR avidity. We report that avidity for Ag and interaction with B cells determine distinct aspects of the primary CD4(+) T cell response to Friend virus infection. Virus-specific CD4(+) T cells followed exclusive Th1 and T follicular helper (Tfh) differentiation. High avidity for Ag facilitated expansion during priming and enhanced the capacity for IFN-γ and IL-21 production. In contrast, Tfh differentiation was not affected by avidity for Ag. By reducing or preventing B cell interaction, we found that B cells promoted Tfh differentiation, induced programmed death 1 expression, and inhibited IFN-γ production by virus-specific CD4(+) T cells. Ultimately, B cells protected hosts from CD4(+) T cell-mediated immune pathology, at the detriment of CD4(+) T cell-mediated protective immunity. Our results suggest that B cell presentation of vaccine Ags could be manipulated to direct the appropriate CD4(+) T cell response.     

Identification of CD25+ gamma delta T cells as fetal thymus-derived naturally occurring IL-17 producers

We previously reported that resident gammadelta T cells in the peritoneal cavity rapidly produced IL-17 in response to Escherichia coli infection to mobilize neutrophils. We found in this study that the IL-17-producing gammadelta T cells did not produce IFN-gamma or IL-4, similar to Th17 cells. IL-17-producing gammadelta T cells specifically express CD25 but not CD122, whereas CD122(+) gammadelta T cells produced IFN-gamma. IL-17-producing gammadelta T cells were decreased but still present in IL-2- or CD25-deficient mice, suggesting a role of IL-2 for their maintenance. IFN-gamma-producing CD122(+) gammadelta T cells were selectively decreased in IL-15-deficient mice. Surprisingly, IL-17-producing gammadelta T cells were already detected in the thymus, although CD25 was not expressed on the intrathymic IL-17-producing gammadelta T cells. The number of thymic IL-17-producing gammadelta T cells was peaked at perinatal period and decreased thereafter, coincided with the developmental kinetics of Vgamma6(+) Vdelta1(+) gammadelta T cells. The number of IL-17-producing gammadelta T cells was decreased in fetal thymus of Vdelta1-deficient mice, whereas Vgamma5(+) fetal thymocytes in normal mice did not produce IL-17. Thus, it was revealed that the fetal thymus-derived Vgamma6(+) Vdelta1(+) T cells functionally differentiate to produce IL-17 within thymus and thereafter express CD25 to be maintained in the periphery.     

Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.     

PD-1 protects against inflammation and myocyte damage in T cell-mediated myocarditis

PD-1, a member of the CD28 family of immune regulatory molecules, is expressed on activated T cells, interacts with its ligands, PD-L1/B7-H1 and PD-L2/B7-DC, on other cells, and delivers inhibitory signals to the T cell. We studied the role of this pathway in modulating autoreactive T cell responses in two models of myocarditis. In a CD8(+) T cell-mediated adoptive transfer model, we found that compared with Pd1(+/+) CD8(+) T cells, Pd1(-/-) CD8(+) T cells cause enhanced disease, with increased inflammatory infiltrate, particularly rich in neutrophils. Additionally, we show enhanced proliferation in vivo and enhanced cytotoxic activity of PD-1-deficient T lymphocytes against myocardial endothelial cells in vitro. In experimental autoimmune myocarditis, a disease model dependent on CD4(+) T cells, we show that mice lacking PD-1 develop enhanced disease compared with wild-type mice. PD-1-deficient mice displayed increased inflammation, enhanced serum markers of myocardial damage, and an increased infiltration of inflammatory cells, including CD8(+) T cells. Together, these studies show that PD-1 plays an important role in limiting T cell responses in the heart.     

Regulation of T cell-dependent autoantibody production by a gammadelta T cell line derived from lupus-prone mice

Lupus-prone (MRLxC57BL/6) F(1) mice lacking gammadelta T cells show more severe lupus than their T cell-intact counterparts, suggesting that gammadelta T cells down-modulate murine lupus. To determine the mechanisms for this effect, we assessed the capacity of gammadelta T cell lines derived from spleens of alphabeta T cell-deficient MRL/Mp-Fas(lpr) (MRL/Fas(lpr)) mice to down-regulate anti-dsDNA production generated by CD4(+)alphabeta T helper cell lines and activated B cells from wild-type MRL/Fas(lpr) mice. One line, GD12 (gd TCR(+), CD4(-)CD8(-)), had the capacity to reduce anti-dsDNA production in a contact-dependent manner. GD12 also killed activated MRL/Fas(lpr) (H-2(k)) B cells, with less cytolysis of resting B cells than that generated by in comparison to cytokine-matched gammadelta T cell lines. In addition, GD12 also killed activated B cells derived from C57BL/6-Fas(lpr) (H-2(b)) or beta(2)-microglobulin (beta(2) M)-deficient MRL/Fas(lpr) mice, suggesting cytolysis was neither MHC- nor CD1-restricted. Killing by GD12 was inhibited by anti-TNFalpha and anti-TNF-R1, and partially blocked by anti-gd TCR Fab fragments, but not by anti-FasL, anti-TNF-R2 (p75) or concanamycin A. IL-10 produced by GD12 also partially inhibited alphabeta Th1-dependent but not alphabeta Th2-dependent autoantibody production. These findings prove that we have identtified a gammadelta T cell line that suppresses autoantibody synthesis by alphabeta T-B cell collaboration in vitro.     

IL-17 production is dominated by gammadelta T cells rather than CD4 T cells during Mycobacterium tuberculosis infection

IL-17 is a cytokine produced by T cells in response to IL-23. Recent data support a new subset of CD4 Th cells distinct from Th1 or Th2 cells that produce IL-17 and may contribute to inflammation. In this study, we demonstrate that, in naive mice, as well as during Mycobacterium tuberculosis infection, IL-17 production is primarily from gammadelta T cells and other non-CD4(+)CD8(+) cells, rather than CD4 T cells. The production of IL-17 by these cells is stimulated by IL-23 alone, and strongly induced by the cytokines, including IL-23, produced by M. tuberculosis-infected dendritic cells. IL-23 is present in the lungs early in infection and the IL-17-producing cells, such as gammadelta T cells, may represent a central innate protective response to pulmonary infection.     

Deficiency of gammadelta T lymphocytes contributes to mortality and immunosuppression in sepsis

Studies have indicated that gammadelta T lymphocytes play an important role in the regulation of immune function and the clearance of intracellular pathogens. We have recently reported that intraepithelial lymphocytes (IEL), which are rich in gammadelta T cells, within the small intestine illustrated a significant increase in apoptosis and immune dysfunction in mice subjected to sepsis. However, the contribution of gammadelta T cells to the host response to polymicrobial sepsis remains unclear. In this study, we initially observed that after sepsis induced by cecal ligation and puncture (CLP), there was an increase in small intestinal IEL CD8+gammadelta+ T cells in control gammadelta+/+ mice. Importantly, we subsequently found an increased early mortality in mice lacking gammadelta T cells (gammadelta-/- mice) after sepsis. This was associated with decreases in plasma TNF-alpha, IL-6, and IL-12 levels in gammadelta-/- mice compared with gammadelta+/+ mice after sepsis. In addition, even though in vitro LPS-stimulated peritoneal macrophages showed a reduction in IL-6 and IL-12 release after CLP, these cytokines were less suppressed in macrophages isolated from gammadelta-/- mice. Alternatively, IL-10 release was not different between septic gammadelta+/+ and gammadelta-/- mice. Whereas T helper (Th)1 cytokine release by anti-CD3-stimulated splenocytes was significantly depressed in septic gammadelta+/+ mice, there was no such depression in gammadelta-/- mice. However, gammadelta T cell deficiency had no effect on Th2 cytokine release. These findings suggest that gammadelta T cells may play a critical role in regulating the host immune response and survival to sepsis, in part by alteration of the level of IEL CD8+gammadelta+ T cells and through the development of the Th1 response.     

Distinct effects of STAT5 activation on CD4+ and CD8+ T cell homeostasis: development of CD4+CD25+ regulatory T cells versus CD8+ memory T cells

Using transgenic mice that express a constitutively active version of STAT5b, we demonstrate that STAT5 plays a key role in governing B cell development and T cell homeostasis. STAT5 activation leads to a 10-fold increase in pro-B, but not pro-T, cells. Conversely, STAT5 signaling promotes the expansion of mature alphabeta T cells (6-fold increase) and gammadelta and NK T cells (3- to 4-fold increase), but not of mature B cells. In addition, STAT5 activation has dramatically divergent effects on CD8(+) vs CD4(+) T cells, leading to the selective expansion of CD8(+) memory-like T cells and CD4(+)CD25(+) regulatory T cells. These results establish that activation of STAT5 is the primary mechanism underlying both IL-7/IL-15-dependent homeostatic proliferation of naive and memory CD8(+) T cells and IL-2-dependent development of CD4(+)CD25(+) regulatory T cells.     

IL-17A produced by gammadelta T cells plays a critical role in innate immunity against listeria monocytogenes infection in the liver

IL-17A is originally identified as a proinflammatory cytokine that induces neutrophils. Although IL-17A production by CD4(+) Th17 T cells is well documented, it is not clear whether IL-17A is produced and participates in the innate immune response against infections. In the present report, we demonstrate that IL-17A is expressed in the liver of mice infected with Listeria monocytogenes from an early stage of infection. IL-17A is important in protective immunity at an early stage of listerial infection in the liver because IL-17A-deficient mice showed aggravation of the protective response. The major IL-17A-producing cells at the early stage were TCR gammadelta T cells expressing TCR Vgamma4 or Vgamma6. Interestingly, TCR gammadelta T cells expressing both IFN-gamma and IL-17A were hardly detected, indicating that the IL-17A-producing TCR gammadelta T cells are distinct from IFN-gamma-producing gammadelta T cells, similar to the distinction between Th17 and Th1 in CD4(+) T cells. All the results suggest that IL-17A is a newly discovered effector molecule produced by TCR gammadelta T cells, which is important in innate immunity in the liver.     

Cytokine production by Vgamma(+)-T-cell subsets is an important factor determining CD4(+)-Th-cell phenotype and susceptibility of BALB/c mice to coxsackievirus B3-induced myocarditis

Two coxsackievirus B3 (CVB3) variants (H3 and H310A1) differ by a single amino acid mutation in the VP2 capsid protein. H3 induces severe myocarditis in BALB/c mice, but H310A1 is amyocarditic. Infection with H3, but not H310A1, preferentially activates Vgamma4 Vdelta4 cells, which are strongly positive for gamma interferon (IFN-gamma), whereas Vgamma1 Vdelta4 cells are increased in both H3 and H310A1 virus-infected animals. Depletion of Vgamma1(+) cells using monoclonal anti-Vgamma1 antibody enhanced myocarditis and CD4(+)-, IFN-gamma(+)-cell responses in both H3- and H310A1-infected mice yet decreased the CD4(+)-, IL-4(+)-cell response. Depleting Vgamma4(+) cells suppressed myocarditis and reduced CD4(+) IFN-gamma(+) cells but increased CD4(+) IL-4(+) T cells. The role of cytokine production by Vgamma1(+) and Vgamma4(+) T cells was investigated by adoptively transferring these cells isolated from H3-infected BALB/c Stat4 knockout (Stat4ko) (defective in IFN-gamma expression) or BALB/c Stat6ko (defective in IL-4 expression) mice into H3 virus-infected wild-type BALB/c recipients. Vgamma4 and Vgamma1(+) T cells from Stat4ko mice expressed IL-4 but no or minimal IFN-gamma, whereas these cell populations derived from Stat6ko mice expressed IFN-gamma but no IL-4. Stat4ko Vgamma1(+) cells (IL-4(+)) suppress myocarditis. Stat6ko Vgamma1(+) cells (IFN-gamma(+)) were not inhibitory. Stat6ko Vgamma4(+) cells (IFN-gamma(+)) significantly enhanced myocarditis. Stat4ko Vgamma4(+) cells (IL-4(+)) neither inhibited nor enhanced disease. These results show that distinct gammadelta-T-cell subsets control myocarditis susceptibility and bias the CD4(+)-Th-cell response. The cytokines produced by the Vgamma subpopulation have a significant influence on the CD4(+)-Th-cell phenotype.     

IL-12, but not IFN-alpha, promotes STAT4 activation and Th1 development in murine CD4+ T cells expressing a chimeric murine/human Stat2 gene

Humans and mice have evolved distinct pathways for Th1 cell development. Although IL-12 promotes CD4(+) Th1 development in both murine and human T cells, IFN-alphabeta drives Th1 development only in human cells. This IFN-alphabeta-dependent pathway is not conserved in the mouse species due in part to a specific mutation within murine Stat2. Restoration of this pathway in murine T cells would provide the opportunity to more closely model specific human disease states that rely on CD4(+) T cell responses to IFN-alphabeta. To this end, the C terminus of murine Stat2, harboring the mutation, was replaced with the corresponding human Stat2 sequence by a knockin targeting strategy within murine embryonic stem cells. Chimeric m/h Stat2 knockin mice were healthy, bred normally, and exhibited a normal lymphoid compartment. Furthermore, the murine/human STAT2 protein was expressed in murine CD4(+) T cells and was activated by murine IFN-alpha signaling. However, the murine/human STAT2 protein was insufficient to restore full IFN-alpha-driven Th1 development as defined by IFN-gamma expression. Furthermore, IL-12, but not IFN-alpha, promoted acute IFN-gamma secretion in collaboration with IL-18 stimulation in both CD4(+) and CD8(+) T cells. The inability of T cells to commit to Th1 development correlated with the lack of STAT4 phosphorylation in response to IFN-alpha. This finding suggests that, although the C terminus of human STAT2 is required for STAT4 recruitment and activation by the human type I IFNAR (IFN-alphabetaR), it is not sufficient to restore this process through the murine IFNAR complex.     

Induction of CTL and nonpolarized Th cell responses by CD8alpha(+) and CD8alpha(-) dendritic cells

Two distinct dendritic cell (DC) subpopulations have been evidenced in mice on the basis of their differential CD8alpha expression and their localization in lymphoid organs. Several reports suggest that CD8alpha(+) and CD8alpha(-) DC subsets could be functionally different. In this study, using a panel of MHC class I- and/or class II-restricted peptides, we analyzed CD4(+) and CD8(+) T cell responses obtained after i.v. injection of freshly purified peptide-pulsed DC subsets. First, we showed that both DC subsets efficiently induce specific CTL responses and Th1 cytokine production in the absence of CD4(+) T cell priming. Second, we showed that in vivo activation of CD4(+) T cells by CD8alpha(+) or CD8alpha(-) DC, injected i.v., leads to a nonpolarized Th response with production of both Th1 and Th2 cytokines. The CD8alpha(-) subset induced a higher production of Th2 cytokines such as IL-4 and IL-10 than the CD8alpha(+) subset. However, IL-5 was produced by CD4(+) T cells activated by both DC subsets. When both CD4(+) and CD8(+) T cells were primed by DC injected i.v., a similar pattern of cytokines was observed, but, under these conditions, Th1 cytokines were mainly produced by CD8(+) T cells, while Th2 cytokines were produced by CD4(+) T cells. Thus, this study clearly shows that CD4(+) T cell responses do not influence the development of specific CD8(+) T cell cytotoxic responses induced either by CD8alpha(+) or CD8alpha(-) DC subsets.     

The role of CTLA-4 in regulating Th2 differentiation.

To examine the role of CTLA-4 in Th cell differentiation, we used two newly generated CTLA-4-deficient (CTLA-4-/-) mouse strains: DO11. 10 CTLA-4-/- mice carrying a class II restricted transgenic TCR specific for OVA, and mice lacking CTLA-4, B7.1 and B7.2 (CTLA-4-/- B7.1/B7.2-/- ). When purified naive CD4+ DO11.10 T cells from CTLA-4-/- and wild-type mice were primed and restimulated in vitro with peptide Ag, CTLA-4-/- DO11.10 T cells developed into Th2 cells, whereas wild-type DO11.10 T cells developed into Th1 cells. Similarly, when CTLA-4-/- CD4+ T cells from mice lacking CTLA-4, B7. 1, and B7.2 were stimulated in vitro with anti-CD3 Ab and wild-type APC, these CTLA-4-/- CD4+ T cells produced IL-4 even during the primary stimulation, whereas CD4+ cells from B7.1/B7.2-/- mice did not produce IL-4. Upon secondary stimulation, CD4+ T cells from CTLA-4-/- B7.1/B7.2-/- mice secreted high levels of IL-4, whereas CD4+ T cells from B7.1/B7.2-/- mice produced IFN-gamma. In contrast to the effects on CD4+ Th differentiation, the absence of CTLA-4 resulted in only a modest effect on T cell proliferation, and increased proliferation of CTLA-4-/- CD4+ T cells was seen only during secondary stimulation in vitro. Administration of a stimulatory anti-CD28 Ab in vivo induced IL-4 production in CTLA-4-/- B7.1/B7.2-/- but not wild-type mice. These studies demonstrate that CTLA-4 is a critical and potent inhibitor of Th2 differentiation. Thus, the B7-CD28/CTLA-4 pathway plays a critical role in regulating Th2 differentiation in two ways: CD28 promotes Th2 differentiation while CTLA-4 limits Th2 differentiation.