CD25+CD4+ cells contribute to Th2 polarization during helminth infection by suppressing Th1 response development

Mice infected with Schistosoma mansoni develop polarized Th2 responses in which Th1 responses are prevented by IL-10-mediated suppression of IL-12 production. We show that dendritic cells from infected mice are primed to make IL-12 in response to CD40 ligation, and that IL-10 acts by inhibiting this process. In infected mice, two subpopulations of CD4(+) cells, separable by their expression of CD25, make IL-10. CD25(+)CD4(+) cells expressed forkhead box P3, inhibited proliferation of CD4(+) T cells, and made IL-10, but little IL-5. In contrast, CD25(-)CD4(+) cells failed to express forkhead box P3 or to inhibit proliferation and accounted for all the IL-5, IL-6, and IL-13 produced by unseparated splenic populations. Thus, CD25(+) and CD25(-) subpopulations could be characterized as regulatory T cells (Treg cells) and Th2 cells, respectively. Consistent with their ability to make IL-10, both CD25(+) and CD25(-)CD4(+) T cells from infected mice were able, when stimulated with egg Ag, to suppress IL-12 production by CD40 agonist-stimulated dendritic cells. Additionally, in adoptive transfer experiments, both CD4(+) subpopulations of cells were able to partially inhibit the development of Th1 responses in egg-immunized IL-10(-/-) mice. The relationship of Treg cells in infected mice to natural Treg cells was strongly suggested by the ability of CD25(+)CD4(+) cells from naive mice to inhibit Th1 response development when transferred into egg-immunized or infected IL-10(-/-) mice. The data suggest that natural Treg cells and, to a lesser extent, Th2 cells play roles in suppressing Th1 responses and ensuring Th2 polarization during schistosomiasis.     

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.     

CD8 T cells inhibit IgE via dendritic cell IL-12 induction that promotes Th1 T cell counter-regulation.

Th1 and Th2 cells are counterinhibitory; their balance determines allergic sensitization. We show here that CD8 T cell subsets break these rules as both T cytotoxic (Tc)1 and Tc2 cells promote Th1 over Th2 immunity. Using IL-12(-/-), IFN-gamma(-/-), and OVA(257-264)-specific Valpha2Vbeta5 TCR-transgenic mice, we have identified the key steps involved. OVA-specific IFN-gamma(-/-) CD8 T cells inhibited IgE responses equivalent to wild-type CD8 T cells (up to 98% suppression), indicating that CD8 T cell-derived IFN-gamma was not required. However, OVA-specific CD8 T cells could not inhibit IgE in IFN-gamma(-/-) recipients unless reconstituted with naive, wild-type CD4 T cells, suggesting that CD4 T cell-derived IFN-gamma did play a role. Transfer of either Tc1 or Tc2 Valpha2Vbeta5 TCR-transgenic CD8 T cells inhibited IgE and OVA-specific Th2 cells while promoting OVA-specific Th1 cell responses, suggesting a potential role for a type 1 inducing cytokine such as IL-12. CD8 T cells were shown to induce IL-12 in OVA(257-264)-pulsed dendritic cells (DC) in vitro. Furthermore, CD8 T cells were unable to inhibit IgE responses in IL-12(-/-) recipients without the addition of naive, wild-type DC, thus demonstrating a pivotal role for IL-12 in this mechanism. These data reveal a mechanism of IgE regulation in which CD8 T cells induce DC IL-12 by an IFN-gamma-independent process that subsequently induces Th1 and inhibits Th2 cells. Th1 cell IFN-gamma is the final step that inhibits B cell IgE class switching. This demonstrates a novel regulatory network through which CD8 T cells inhibit allergic sensitization.     

Th1 cytokine-conditioned bone marrow-derived dendritic cells can bypass the requirement for Th functions during the generation of CD8+ CTL

Bone marrow-derived dendritic cell (BMDC) subsets have distinct immunoregulatory functions. Th1 cytokine-induced BMDC (BMDC1), compared with Th2 cytokine-induced BMDC2, have superior activities for the differentiation and expansion of CTL. To evaluate the cellular interactions between dendritic cells and CD8+ T cells for the induction of CTL, BALB/c-derived BMDC subsets were cocultured with purified CD8+ T cells from C57BL/6 mice. Our results demonstrate that BMDC1 support the generation of allogeneic CD8+ CTL in the absence of CD4+ Th cells. In contrast, BMDC0 (GM-CSF- plus IL-3-induced BMDC) and BMDC2 failed to promote the differentiation of CD8+ CTL. Using Ab-blocking experiments and studies with gene knockout mice, IL-2 and LFA-1 are demonstrated to be critical for BMDC1-induced CTL differentiation. Unexpectedly, BMDC1 were able to induce CTL from CD8+ T cells isolated from IFN-gamma-/- and IFN-gamma receptor-/- mice. However, BMDC1 produced higher levels of IFN-beta than other BMDC subsets, and anti-IFN-beta mAb blocked BMDC1-dependent CTL generation. These results indicated an indispensable role of IFN-beta, but not IFN-gamma, during BMDC1-induced CTL differentiation. We conclude that Th1-cytokine-conditioned BMDC1 can bypass Th cell function for the differentiation of naive CD8+ T cells into CTL.     

IFN-gamma production by Th1 cells generated from naive CD4+ T cells exposed to norepinephrine

During activation in vivo, naive CD4(+) T cells are exposed to various endogenous ligands, such as cytokines and the neurotransmitter norepinephrine (NE). To determine whether NE affects naive T cell differentiation, we used naive CD4(+) T cells sort-purified from either BALB/c or DO11.10 TCR-transgenic mouse spleens and activated these cells with either anti-CD3/anti-CD28 mAbs or APC and OVA(323-329) peptide, respectively, under Th1-promoting conditions. RT-PCR and functional assays using selective adrenergic receptor (AR) subtype antagonists showed that naive CD4(+) T cells expressed only the beta 2AR subtype to bind NE and that stimulation of this receptor generated Th1 cells that produced 2- to 4-fold more IFN-gamma. This increase was due to more IFN-gamma produced per cell upon restimulation instead of more IFN-gamma-secreting cells, as determined by IFN-gamma-specific immunofluorescence and enzyme-linked immunospot. In contrast, Th1 cell differentiation was unaffected when naive T cells were exposed to NE and activated either in the presence of a neutralizing anti-IL-12 mAb or by APC from IL-12-deficient mice. Moreover, the addition of IL-12 to the IL-12-deficient APC cultures restored the ability of NE to increase Th1 differentiation. Taken together, these results indicate that a possible link may exist between the signaling pathways used by NE and IL-12 to increase naive CD4(+) T cell differentiation to a Th1 cell.     

Cellular basis of the role of diesel exhaust particles in inducing Th2-dominant response

There is growing evidence that diesel exhaust particles (DEP) can induce allergic diseases with increased IgE production and preferential activation of Th2 cells. To clarify the cellular basis of the role of DEP in the induction of Th2-dominant responses, we examined the effects of DEP on the cytokine production by T cells stimulated with anti-CD3/CD28 Ab and on that by monocyte-derived dendritic cells (MoDCs) stimulated with CD40L and/or IFN-gamma. We examined IFN-gamma, IL-4, IL-5, IL-8, and IL-10 produced by T cells and TNF-alpha, IL-1beta, IL-10, and IL-12 produced by MoDCs using real-time PCR analysis or by ELISA. To highlight the effects of DEP, we compared the effects of DEP with those of dexamethasone (DEX) and cyclosporin A (CyA). DEP significantly suppressed IFN-gamma mRNA expression and protein production, while it did not affect IL-4 or IL-5 mRNA expression or protein production. The suppressive effect on IFN-gamma mRNA expression was more potent than that of DEX and comparable at 30 mug/ml with 10(-7) M CyA. The suppressive effect on IFN-gamma production was also more potent than that of either DEX or CyA. DEP suppressed IL-12p40 and IL-12p35 mRNA expression and IL-12p40 and IL-12p70 production by MoDCs, while it augmented IL-1beta mRNA expression. Finally, by using a thiol antioxidant, N-acetyl cysteine, we found that the suppression of IFN-gamma production by DEP-treated T cells was mediated by oxidative stress. These data revealed a unique characteristic of DEP, namely that they induce a Th2 cytokine milieu in both T cells and dendritic cells.     

B7H1-Ig fusion protein activates the CD4+ IFN-gamma receptor+ type 1 T regulatory subset through IFN-gamma-secreting Th1 cells

It has been demonstrated in our previous work that, in the human skin-grafting model, the expression of costimulatory molecule B7H1 (PD-L1) by keratinocytes plays an essential role in inducing local tolerance via activation of IL-10-secreting T cells. This study further analyzes the role of B7H1 in differentiation of type 1 T regulatory (Tr1) cells and explores underlying mechanisms. Mouse fusion protein B7H1-Ig is used, together with immobilized anti-CD3 mAb, to costimulate the purified naive CD4+ T cells. B7H1-Ig-treated CD4+ T cells were found to activate a characteristic Tr1 population possessing a CD4+ CD25- Foxp3- CD45RBlow phenotype. These regulatory T cells strongly inhibited the Th1-dominated MLR by secretion of IL-10 and TGF-beta. Moreover, B7H1-treated Tr1 cells also resulted in suppressed clinical scores and demyelination when adoptively transferred into mice with experimental allergic encephalomyelitis. Furthermore, analysis of the cytokine profile indicated that there were two differential reaction patterns during the B7H1-Ig-induced Tr1 development. These two patterns were characterized by activation of IFN-gammaR+ IL-10R- Th1 and IFN-gammaR+ IL-10R+ Tr1 cells, respectively. Secretion of IFN-gamma by Th1 and the expression of IFN-gammaR on Tr1 were critical for further Tr1 differentiation, as demonstrated by mAb blocking and by analysis in IFN-gamma(-/-) mice. In conclusion, B7H1 is capable of inducing Tr1 differentiation from naive CD4+ T cells by coactivation in an IFN-gamma- or Th1-dependent manner. Our study may shed some light upon the clinical usage of B7H1 as a therapeutic reagent for induction of tolerance.     

Synergistic effect of IL-2, IL-12, and IL-18 on thymocyte apoptosis and Th1/Th2 cytokine expression

In the periphery, IL-18 synergistically induces the expression of the Th1 cytokine IFN-gamma in the presence of IL-12 and the Th2 cytokines IL-5 and IL-13 in the presence of IL-2. Although the expression of these cytokines has been described in the thymus, their role in thymic development and function remains uncertain. We report here that freshly isolated thymocytes from C57BL/6 and BALB/c mice stimulated in vitro with IL-2-plus-IL-18 or IL-12-plus-IL-18 produce large amounts of IFN-gamma and IL-13. Analysis of the thymic subsets, CD4(-)CD8(-) (DN), CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) revealed that IL-18 in combination with IL-2 or IL-12 induces IFN-gamma and IL-13 preferentially from DN cells. Moreover, DN2 and DN3 thymocytes contained more IFN-gamma(+) cells than cells in the later stage of maturation. Additionally, IL-18 in combination with IL-2 induces CCR4 (Th2-associated) and CCR5 (Th1-associated) gene expression. In contrast, IL-18-plus-IL-12 specifically induced CCR5 expression. The IL-2-plus-IL-18 or IL-12-plus-IL-18 effect on IFN-gamma and IL-13 expression is dependent on Stat4 and NF-kappaB but independent of Stat6, T-bet, or NFAT. Furthermore, IL-12-plus-IL-18 induces significant thymocyte apoptosis when expressed in vivo or in vitro, and this effect is exacerbated in the absence of IFN-gamma. IL-12-plus-IL-18-stimulated thymocytes can also induce IA-IE expression on cortical and medullary thymic epithelial cells in an IFN-gamma-dependent manner. Thus, the combination of IL-2, IL-12, and IL-18 can induce phenotypic and functional changes in thymocytes that may alter migration, differentiation, and cell death of immature T cells inside the thymus and potentially affect the Th1/Th2 bias in peripheral immune compartments.     

IL-4 is a mediator of IL-12p70 induction by human Th2 cells: reversal of polarized Th2 phenotype by dendritic cells

IL-12 is a key inducer of Th1-associated inflammatory responses, protective against intracellular infections and cancer, but also involved in autoimmune tissue destruction. We report that human Th2 cells interacting with monocyte-derived dendritic cells (DC) effectively induce bioactive IL-12p70 and revert to Th0/Th1 phenotype. In contrast, the interaction with B cells preserves polarized Th2 phenotype. The induction of IL-12p70 in Th2 cell-DC cocultures is prevented by IL-4-neutralizing mAb, indicating that IL-4 acts as a Th2 cell-specific cofactor of IL-12p70 induction. Like IFN-gamma, IL-4 strongly enhances the production of bioactive IL-12p70 heterodimer in CD40 ligand-stimulated DC and macrophages and synergizes with IFN-gamma at low concentrations of both cytokines. However, in contrast to IFN-gamma, IL-4 inhibits the CD40 ligand-induced production of inactive IL-12p40 and the production of either form of IL-12 induced by LPS, which may explain the view of IL-4 as an IL-12 inhibitor. The presently described ability of IL-4 to act as a cofactor of Th cell-mediated IL-12p70 induction may allow Th2 cells to support cell-mediated immunity in chronic inflammatory states, including cancer, autoimmunity, and atopic dermatitis.     

Conventional, naive CD4+ T cells provide an initial source of IL-4 during Th2 differentiation

IL-4 is known to promote the differentiation of CD4+ T cells into IL-4-secreting Th2 cells. However, the cellular source of the early burst of IL-4 that drives Th2 responses in vivo has not been conclusively identified. Mice deficient for the IL-4 receptor alpha-chain (IL-4Ralpha-/-) retain the capacity to secrete IL-4 and can be used to identify those cell types that produce IL-4 without a requirement for prior IL-4-mediated stimulation. To address whether naive, conventional CD4+ T cells may act as initial producers of IL-4 in Ag-specific responses, we crossed the BALB/c IL-4Ralpha-/-mice to DO11.10/scid TCR transgenic mice. Lymph node cells from wild-type and IL-4Ralpha-/- DO11.10/scid mice secreted approximately 50 pg of IL-4 per10(6) cells within 48 h after peptide stimulation. This small amount of IL-4 was sufficient to cause the differentiation of wild-type CD4+ T cells into Th2 cells, particularly if IFN-gamma and IL-12 were neutralized during the priming cultures. CD4+ cells from the IL-4Ralpha-/- mice gave rise to a minor proportion (approximately 2%) of IL-4-producing cells upon stimulation in the presence of anti-IFN-gamma and anti-IL-12. These data show that conventional, naive CD4+ T cells may be considered as initial sources of IL-4 and, in the absence of IFN-gamma and IL-12, this IL-4 can induce Th2 polarization.     

CCR4 participation in Th type 1 (mycobacterial) and Th type 2 (schistosomal) anamnestic pulmonary granulomatous responses

CCR4 is purported to be a Th type 2 (Th2) cell-biased receptor but its functional role is unclear. Recent studies suggest that chemokine receptor expression and function are more complex in vivo and raise doubts regarding restricted CCR4 expression by Th2 cells. To address these issues, we analyzed the role of CCR4 in highly polarized models of Th type 1 (Th1) and Th2 cell-mediated pulmonary granulomas, respectively, elicited by i.v. challenge of primed mice with either mycobacterial purified protein derivative or schistosomal egg Ag-coated beads. CCR4 agonists were expressed during both responses, correlating with a shift of CCR4+ CD4+ T cells from blood to lungs. CCL22 dominated in draining nodes during the Th1 response. Analysis of CD4+ effector T cells revealed CCR4 expression and CCR4-mediated chemotaxis by both IFN-gamma and IL-4 producers. Studies of CCR4 knockout (CCR4(-/-)) mice showed partial impairment of the local type-2 cytokine response and surprisingly strong impairment of the Th1 response with abrogated IFN-gamma production during secondary but not primary challenge. Adoptive transfer indicated CCR4(-/-)CD4+ Th1 cell function was defective but this could not be reconstituted with wild-type (CCR4(+/+)) CD4+ T cells indicating involvement of another CCR4+ population. Coculture of CCR4(+/+)CD4+ T cells and CCR4(-/-) dendritic cells revealed intact IL-2 but impaired IFN-gamma production, pointing to a role for CCR4+ dendritic cells in effector cell expression. Therefore, CCR4 is not Th2-restricted and was required for sustenance and expression of the Th1 effector/memory response to mycobacterial Ags.     

Cytokines regulate the capacity of CD8alpha(+) and CD8alpha(-) dendritic cells to prime Th1/Th2 cells in vivo

Prior studies have shown that subclasses of dendritic cells (DC) direct the development of distinct Th populations in rodents and in humans. In the mouse, we have recently shown that administration of Ag-pulsed CD8alpha(-) DC induces a Th2-type response, whereas injection of CD8alpha(+) DC leads to Th1 differentiation. To define the DC-derived factors involved in the polarization of Th responses, we injected either subset purified from mice genetically deficient for IFN-gamma, IL-4, IL-12, or IL-10 into wild-type animals. In this work, we report that DC-derived IL-12 and IFN-gamma are required for Th1 priming by CD8alpha(+) DC, whereas IL-10 is required for optimal development of Th2 cells by CD8alpha(-) DC. The level of IL-12 produced by the DC appears to determine the Th1/Th2 balance in vivo. We further show that the function of DC subsets displays some flexibility. Treatment of DC with IL-10 in vitro induces a selective decrease in the viability of CD8alpha(+) DC. Conversely, incubation with IFN-gamma down-regulates the Th2-promoting capacities of CD8alpha(-) DC and increases the Th1-skewing properties of both subsets.     

Either IL-2 or IL-12 is sufficient to direct Th1 differentiation by nonobese diabetic T cells

Th cell differentiation from naive precursors is a tightly controlled process; the most critical differentiation factor is the action of the driving cytokine: IL-12 for Th1 development, IL-4 for Th2 development. We found that CD4(+) T cells from nonobese diabetic mice spontaneously differentiate into IFN-gamma-producing Th1 cells in response to polyclonal TCR stimulation in the absence of IL-12 and IFN-gamma. Instead, IL-2 was necessary and sufficient to direct T cell differentiation to the Th1 lineage by nonobese diabetic CD4(+) T cells. Its ability to direct Th1 differentiation of both naive and memory CD4(+) T cells was clearly uncoupled from its ability to stimulate cell division. Autocrine IL-2-driven Th1 differentiation of nonobese diabetic T cells may represent a genetic liability that favors development of IFN-gamma-producing autoreactive T cells.     

The role of B cells in the development of CD4 effector T cells during a polarized Th2 immune response

Previous studies have suggested that B cells promote Th2 cell development by inhibiting Th1 cell differentiation. To examine whether B cells are directly required for the development of IL-4-producing T cells in the lymph node during a highly polarized Th2 response, B cell-deficient and wild-type mice were inoculated with the nematode parasite, Nippostrongylus brasiliensis. On day 7, in the absence of increased IFN-gamma, IL-4 protein and gene expression from CD4 T cells in the draining lymph nodes were markedly reduced in B cell-deficient mice and could not be restored by multiple immunizations. Using a DO11.10 T cell adoptive transfer system, OVA-specific T cell IL-4 production and cell cycle progression, but not cell surface expression of early activation markers, were impaired in B cell-deficient recipient mice following immunization with N. brasiliensis plus OVA. Laser capture microdissection and immunofluorescent staining showed that pronounced IL-4 mRNA and protein secretion by donor DO11.10 T cells first occurred in the T cell:B cell zone of the lymph node shortly after inoculation of IL-4-/- recipients, suggesting that this microenvironment is critical for initial Th2 cell development. Reconstitution of B cell-deficient mice with wild-type naive B cells, or IL-4-/- B cells, substantially restored Ag-specific T cell IL-4 production. However, reconstitution with B7-1/B7-2-deficient B cells failed to rescue the IL-4-producing DO11.10 T cells. These results suggest that B cells, expressing B7 costimulatory molecules, are required in the absence of an underlying IFN-gamma-mediated response for the development of a polarized primary Ag-specific Th2 response in vivo.     

A filarial nematode-secreted product signals dendritic cells to acquire a phenotype that drives development of Th2 cells

Although exogeneous "danger" signals such as LPS can activate APC to produce a Th1 response, the nature of events initiating a Th2 response is controversial. We now show that pathogen-derived products have the capacity to induce bone marrow-derived dendritic cell cultures to acquire a phenotype that promotes the differentiation of naive CD4+ T cells toward either a Th1 or Th2 phenotype. Thus, LPS-matured dendritic cells (DC1) promote a Th1 response (increased generation of IFN-gamma and reduced production of IL-4) by Ag-stimulated CD4+ T cells from the DO.11.10 transgenic mouse expressing a TCR specific for an OVA peptide (OVA323-339). In contrast, a phosphorylcholine-containing glycoprotein, ES-62, secreted by the filarial nematode, Acanthocheilonema viteae, which generates a Th2 Ab response in vivo, is found to induce the maturation of dendritic cells (DC2) with the capacity to induce Th2 responses (increased IL-4 and decreased IFN-gamma). In addition, we show that the switch to either Th1 or Th2 responses is not effected by differential regulation through CD80 or CD86 and that a Th2 response is achieved in the presence of IL-12.     

Activated STAT4 has an essential role in Th1 differentiation and proliferation that is independent of its role in the maintenance of IL-12R beta 2 chain expression and signaling

In this study we demonstrated that CD4(+) T cells from STAT4(-/-) mice exhibit reduced IL-12R expression and poor IL-12R signaling function. This raised the question of whether activated STAT4 participates in Th1 cell development mainly through its effects on IL-12 signaling. In a first approach to this question we determined the capacity of CD4(+) T cells from STAT4(-/-) bearing an IL-12Rbeta2 chain transgene (and thus capable of normal IL-12R expression and signaling) to undergo Th1 differentiation when stimulated by Con A and APCs. We found that such cells were still unable to exhibit IL-12-mediated IFN-gamma production. In a second approach to this question, we created Th2 cell lines (D10 cells) transfected with STAT4-expressing plasmids with various tyrosine-->phenylalanine mutations and CD4(+) T cell lines from IL-12beta2(-/-) mice infected with retroviruses expressing similarly STAT4 mutations that nevertheless express surface IL-12Rbeta2 chains. We then showed that constructs that were unable to support STAT4 tyrosine phosphorylation (in D10 cells) as a result of mutation were also incapable of supporting IL-12-induced IFN-gamma production (in IL-12Rbeta2(-/-) cells). Thus, by two complementary approaches we demonstrated that activated STAT4 has an essential downstream role in Th1 cell differentiation that is independent of its role in the support of IL-12Rbeta2 chain signaling. This implies that STAT4 is an essential element in the early events of Th1 differentiation.     

The source of early IFN-gamma that plays a role in Th1 priming

When naive CD4 T cells are primed, they rapidly differentiate into polarized Th1 and/or Th2 phenotypes. A major factor in producing such polarization is the early production of cytokines (IL-12 and IFN-gamma in the case of Th1 cells and IL-4 in the case of Th2 cells). One issue that remains unresolved is the source of the early IFN-gamma that synergizes with IL-12 to fully polarize CD4 T cells into Th1 cells. We have examined this question by injecting mice with anti-CD3 and examining cells from normal and various MHC-knockout mice. We found that IFN-gamma is induced rapidly in a small subset of CD8 T cells. This subset is absent in mice that lack beta2-microglobulin, but not in K(b)D(b)-double-knockout mice, indicating that these CD8 T cells are dependent on nonclassical MHC class Ib molecules. The early burst of IFN-gamma polarizes CD4 T cells toward Th1 cells, in part by stimulating the release of IL-12 from APC. We also use TAP- and CD1-knockout mice to show that such cells are not CD1-restricted NK T cells, nor are they dependent on TAP-1 transport for surface expression of the relevant MHC class Ib molecule. Therefore, they arise on MHC class Ib molecules that do not depend on TAP-1 transporters.     

CD8alpha+ and CD11b+ dendritic cell-restricted MHC class II controls Th1 CD4+ T cell immunity

The activation, proliferation, differentiation, and trafficking of CD4 T cells is central to the development of type I immune responses. MHC class II (MHCII)-bearing dendritic cells (DCs) initiate CD4(+) T cell priming, but the relative contributions of other MHCII(+) APCs to the complete Th1 immune response is less clear. To address this question, we examined Th1 immunity in a mouse model in which I-A(beta)(b) expression was targeted specifically to the DCs of I-A(beta)b-/- mice. MHCII expression is reconstituted in CD11b(+) and CD8alpha(+) DCs, but other DC subtypes, macrophages, B cells, and parenchymal cells lack of expression of the I-A(beta)(b) chain. Presentation of both peptide and protein Ags by these DC subsets is sufficient for Th1 differentiation of Ag-specific CD4(+) T cells in vivo. Thus, Ag-specific CD4(+) T cells are primed to produce Th1 cytokines IL-2 and IFN-gamma. Additionally, proliferation, migration out of lymphoid organs, and the number of effector CD4(+) T cells are appropriately regulated. However, class II-negative B cells cannot receive help and Ag-specific IgG is not produced, confirming the critical MHCII requirement at this stage. These findings indicate that DCs are not only key initiators of the primary response, but provide all of the necessary cognate interactions to control CD4(+) T cell fate during the primary immune response.     

In vivo generation of pathogen-specific Th1 cells in the absence of the IFN-gamma receptor

The precise mechanisms that govern the commitment of CD4 T cells to become Th1 or Th2 cells in vivo are incompletely understood. Recent experiments demonstrate colocalization of the IFN-gammaR chains with the TCR during activation of naive CD4 T cells, suggesting that association of these molecules may be involved in determining lineage commitment. To test the role of IFN-gamma and its receptor in the generation of Th1 Ag-specific CD4 T cells, we analyzed mice after infection with Listeria monocytogenes or lymphocytic choriomeningitis virus. In the absence of IFN-gamma, Ag-specific CD4 T cells were generated in response to both these infections. In addition, IFN-gamma-producing (Th1) Ag-specific CD4 T cells were generated in mice lacking the ligand-binding chain of the IFN-gammaR (IFN-gammaR1-/-) or the signaling chain (IFN-gammaR2-/-). There was no increase in the number of IL-4-producing Ag-specific CD4 T cells, nor was there a decrease in the expression of T-bet in the absence of functional IFN-gamma signaling, indicating that the cells were committed Th1 cells. Thus, both chains of the IFN-gammaR are dispensable for the generation of Th1 Ag-specific CD4 T cells after infection in vivo.     

IL-12 administration accelerates autoimmune diabetes in both wild-type and IFN-gamma-deficient nonobese diabetic mice,revealing pathogenic and protective effects of IL-12-induced IFN-gamma

IL-12 administration to nonobese diabetic (NOD) mice induces IFN-gamma-secreting type 1 T cells and high circulating IFN-gamma levels and accelerates insulin-dependent diabetes mellitus (IDDM). Here we show that IL-12-induced IFN-gamma production is dispensable for diabetes acceleration, because exogenous IL-12 could enhance IDDM development in IFN-gamma-deficient as well as in IFN-gamma-sufficient NOD mice. Both in IFN-gamma(+/-) and IFN-gamma(-/-) NOD mice, IL-12 administration generates a massive and destructive insulitis characterized by T cells, macrophages, and CD11c(+) dendritic cells, and increases the number of pancreatic CD4(+) cells secreting IL-2 and TNF-alpha. Surprisingly, IL-12-induced IFN-gamma hinders pancreatic B cell infiltration and inhibits the capacity of APCs to activate T cells. Although pancreatic CD4(+) T cells from IL-12-treated IFN-gamma(-/-) mice fail to up-regulate the P-selectin ligand, suggesting that their entry into the pancreas may be impaired, T cell expansion is favored in these mice compared with IL-12-treated IFN-gamma(+/-) mice because IL-12 administration in the absence of IFN-gamma leads to enhanced cell proliferation and reduced T cell apoptosis. NO, an effector molecule in beta cell destruction, is produced ex vivo in high quantity by pancreas-infiltrating cells through a mechanism involving IL-12-induced IFN-gamma. Conversely, in IL-12-treated IFN-gamma-deficient mice, other pathways of beta cell death appear to be increased, as indicated by the up-regulated expression of Fas ligand on Th1 cells in the absence of IFN-gamma. These data demonstrate that IFN-gamma has a dual role, pathogenic and protective, in IDDM development, and its deletion allows IL-12 to establish alternative pathways leading to diabetes acceleration.