Reduced IL-12 production by monocytes upon ultraviolet-B irradiation selectively limits activation of T helper-1 cells

The capacity of APC to stimulate the proliferation of human peripheral blood T cells decreases upon ultraviolet-B (UVB) irradiation. The aim of this study was to investigate whether all T cell subsets are equally sensitive to this reduced APC function. Established human Th1, Th2, and Th0 clones were stimulated with monocytes in a soluble CD3 mAb-mediated assay that is dependent on the presence of APC. Monocytes were exposed to low nonlethal doses of UVB radiation before coculture with T cells. UVB irradiation inhibited the capacity of monocytes to stimulate the proliferation and IFN-gamma production of Th1 cells in a dose-related fashion. In contrast, UVB-treated monocytes induced normal proliferation and IL-4 production in Th2 cells. Stimulation of Th0 cell proliferation by UVB-irradiated monocytes was normal, but a preferential suppression of IFN-gamma production was observed, thus leading to a more Th2-like cytokine response. The loss of Th1 proliferation upon stimulation with UVB-irradiated monocytes could be overcome by rIL-2; however, IFN-gamma production remained suppressed. IFN-gamma production could be completely restored by rIL-12, whereas the addition of IL-1 beta, TNF-alpha, or indomethacin had no such effect, nor did the addition of mAb to CD28, added to compensate for the reduced B7 expression of UVB-irradiated monocytes. Monocytes exposed to UVB radiation exhibited reduced expression of mRNA for the IL-1 2 subunits p35 and p40 and suppressed production of the IL-12 p70 protein. Our results thus indicate that UVB irradiation of APC selectively impairs Th1-like responses, a phenomenon caused by the UVB-induced suppression of monocyte IL-12 production.     

Use of intranasal IL-12 to target predominantly Th1 responses to nasal and Th2 responses to oral vaccines given with cholera toxin

We have investigated the effects of IL-12 and cholera toxin (CT) on the immune response to tetanus toxoid (TT) given by intranasal or oral routes. CT inhibited IL-12-induced IFN-gamma secretion both in vivo and in vitro. Intranasal administration of IL-12 to mice nasally immunized with the combined vaccine of TT and CT resulted in increased TT-specific IgG2a and IgG3 Abs, while IgG1 and IgE Ab responses were markedly reduced. This shift of the CT-induced immune response toward Th1 type was associated with TT-specific CD4+ T cells secreting IFN-gamma and reduced levels of Th2-type cytokines (i.e., IL-4, IL-5, IL-6, and IL-10). In contrast, intranasal IL-12 enhanced the CT-induced serum IgG1 and IgE Ab responses in mice given the combined vaccine orally. IFN-gamma secretion by TT-specific CD4+ T cells was also enhanced; however, Th2-type cytokine responses were predominant. Mucosal secretory IgA responses to oral or nasal vaccines were not affected by intranasal IL-12. Thus, intranasal IL-12 delivery influences Th cell subset development in mucosal inductive sites that are dependent on the route of vaccine delivery.     

Lymphotactin acts as an innate mucosal adjuvant

Lymphotactin (Lptn) is a C chemokine produced predominantly by NK and CD8-positive (CD8+) T cells including gammadelta TCR-positive (TCR+) intraepithelial lymphocytes. Lptn is chemotactic for NK and T cells and likely plays an important role in maintaining the integrity of the epithelium and in mucosal immune responses. In this study, we characterized the immune responses to OVA given intranasally with Lptn to mice. This regimen enhanced OVA-specific serum Ab responses and Ab titers in mucosal secretions. Lptn also enhanced OVA-specific Ab-forming cells in mucosal and systemic compartments. CD4-positive (CD4+) T cells isolated from mucosal compartments and spleens of mice intranasally immunized with OVA plus Lptn displayed higher OVA-specific proliferative responses and greater synthesis of IFN-gamma, IL-2, IL-4, IL-5, IL-6, and IL-10 than did CD4+ T cells from mice given OVA without Lptn. These studies indicate that Lptn has adjuvant properties and suggest that Lptn present in the mucosa has the potential to enhance mucosal and systemic Ab responses through help provided by Th1- and Th2-type cells to link the initial innate signals of the mucosa with the acquired immune system.     

Defective Th1 and Th2 cytokine synthesis in the T-T cell presentation model for lack of CD40/CD40 ligand interaction

In this study, T or NK cell clones used as antigen-presenting cells (T- or NK-APC) were shown to be significantly less efficient than professional APC in inducing Th1 and Th2 cytokines by antigen-specific T cell clones. This phenomenon was not related to a limited engagement of TCR by T-APC, since comparable thresholds of TCR down-regulation were shown when antigen was presented by either T-APC or professional APC. Rather, the stimulatory T-APC weakness was due to their inability, because they are CD40-, to provide the appropriate co-stimuli to responder T cells both indirectly via IL-12, and partially via direct CD40L triggering on T cells. Indeed, the simultaneous addition of IL-12 and reagents directly engaging CD40L on responder T cells restored T cell cytokine synthesis when antigen was presented by T-APC. In addition, either IL-12 production or blocking of T cell cytokine synthesis by anti-IL-12 p75 antibodies was evident only when professional APC were used in our antigen-specific system. The down-regulation of cytokine synthesis in the system of T-T cell presentation could represent a novel mechanism of immune regulation, which may intervene to switch off detrimental Th1- or Th2-mediated responses induced by antigen presentation among activated T cells infiltrating inflamed tissues.     

Crossregulation between T helper cell (Th)1 and Th2: inhibition of Th2 proliferation by IFN-gamma involves interference with IL-1

The Th1-derived cytokine IFN-gamma inhibits the proliferation of Th2 lymphocytes, but the mechanism of inhibition is not known. Under certain disease conditions, an established Th2-mediated immune response is undesirable and a Th1-mediated response is beneficial. However, established Th2 cells appear to be phenotypically stable. Thus, learning more about cytokine-mediated regulation of established Th2 cells is important if deleterious immune responses are to be altered. We studied the effects of IFN-gamma on a panel of recently derived Th2 lines and clones, as well as a previously established Th2 clone, 13.26. Inhibition by IFN-gamma was observed only when there was a concomitant response to IL-1, a known costimulator of Th2. Clone 13.26 was particularly sensitive to both IL-1 and IFN-gamma, so it was studied in greater detail. We examined cytokine responses using stimulation by anti-TCR mAb-coated plates, or Ag presented by APC populations that do or do not produce IL-1. All IL-1-mediated proliferative responses of 13.26 were inhibited by IFN-gamma, whereas IL-1-independent (IL-4-associated) responses were unaffected. Our data suggest that IFN-gamma inhibits Th2 proliferation through an IL-1-dependent mechanism, and furthermore, that the costimulatory pathways used by APCs may be critical for subsequent Th cell responses to cytokines.     

Differential regulation of human T cell cytokine patterns and IgE and IgG4 responses by conformational antigen variants

Bee venom phospholipase A2 (PLA) represents the major allergen and antigen in allergic and non-allergic individuals sensitized to bee sting. We have studied specific activation of peripheral T cells by different structural and conformational variants of PLA and secretion of cytokines regulating IgE and IgG4 antibody (Ab) formation. PLA molecules expressing the correctly folded tertiary structure, which show high affinity to membrane phospholipids and were recognized by Ab from bee sting allergic patients, induced high IL-4, IL-5 and IL-13 production in peripheral blood mononuclear cell cultures. In contrast, non-refolded recombinant PLA (rPLA) and reduced and alkylated native PLA (nPLA) induced more IFN-gamma and IL-2 and higher proliferative responses. Differences in proliferation and cytokine patterns among correctly folded and non-refolded PLA resulted from conformation-dependent involvement of different antigen-presenting cell (APC) types. Antigen (Ag)-presenting B cells recognized PLA only in its natural conformation, stimulated Th2 type cytokines and induced IgE Ab. Non-refolded PLA was recognized, processed and presented exclusively by monocytes and induced a Th1 dominant cytokine profile leading to IgG4 production by B cells. The possibility that production of particular cytokine patterns and Ig isotype was influenced by the enzymatic activity of PLA was excluded by using enzymatically inactive H34Q point-mutated, refolded rPLA. These findings demonstrate the decisive role of specific Ag recognition by different APC, depending on structural features, membrane phospholipid binding property and the existence of conformational B cell epitopes, in the differential regulation of memory IgE and IgG4 Ab. Furthermore, they show that a change from IgE-mediated allergy to normal immunity against a major allergen can be induced by rPLA variants that are not recognized by specific Ab and B cells but still carry the T cell epitopes. These features may enable new applications for safer immunotherapy.     

Streptococcal preparation OK-432 is a potent inducer of IL-12 and a T helper cell 1 dominant state

Streptococcal preparation OK-432 is a bacterial immunopotentiator extensively used in Japan for adjuvant cancer therapy. Using a C57BL/6 mouse model, OK-432 was found to induce multiple cytokines including the Th1 polarizing cytokine IL-12. Expression of IL-12 protein by murine splenocytes was restricted to macrophages and B cells and led to high levels of IFN-gamma production from both CD4+ and CD8+ T cells. Of the Th2 cytokines IL-4 and IL-10, only IL-10 protein was detected and originated primarily from the adherent cell population. Its expression was delayed relative to IL-12. A similar pattern of cytokine induction was observed from human PBMCs. OK-432-driven IFN-gamma production was inhibited by anti-IL-12 Ab, anti-IL-2 Ab, anti-TNF-alpha Ab, and anti-IL-2R alpha Ab, suggesting that IFN-gamma production from Th1 cells is induced by the cooperation action of these cytokines through the IL-2R alpha pathway. When compared with another widely used immunopotentiator bacillus Calmette-Guérin (BCG), OK-432 was a stronger IL-12 and IFN-gamma inducer. Furthermore, the mechanism of IFN-gamma induction by OK-432 differed from BCG in that coincident granulocyte-macrophage CSF and IL-1 expression played little to no role. These results suggest that OK-432 is a potent multicytokine inducer, specifically a strong inducer of IL-12, and that OK-432 may exert its antitumor effect by promoting a Th1-dominant state.     

Failure of P strain mice to respond to vaccination against schistosomiasis correlates with impaired production of IL-12 and up-regulation of Th2 cytokines that inhibit macrophage activation

In contrast to most inbred strains, P mice fail to develop significant resistance to Schistosoma mansoni infection as a result of vaccination with either radiation-attenuated cercariae or schistosome antigens plus Bacillus Calmette Guérin, and this failure correlates with defects in macrophage larvicidal activity. Supernatant fluids from antigen-treated in vitro cultures of splenocytes from vaccinated P mice demonstrate less macrophage stimulatory activity than do supernatants from cells of vaccine-responsive strains such as C57BL/6. This is not due either to diminished production of the macrophage-activating cytokine IFN-gamma by P mice, or to a lesser responsiveness of macrophages from P mice to activation by IFN-gamma. Rather, P splenocytes produce two-to threefold higher amounts of IL-4 and IL-10, cytokines which down-regulate the cytotoxic potential of IFN-gamma-treated macrophages. Thus, the macrophage-activating potential of cytokine preparations from vaccinated P mice can be completely recovered by in vitro treatment with antibodies to IL-4 or IL-10. Moreover, lower levels of IL-12, a cytokine involved in promoting development of Th1 responses, are produced by splenocytes from P mice as compared to C57BL/6 counterparts. These studies indicate that a genetic predisposition toward an impaired production of IL-12 and an increased production of down-regulatory Th2 cytokines correlate with low response to vaccination against S. mansoni.     

RNA-binding protein conserved in both microtubule- and microfilament-based RNA localization

IL-12 is a key cytokine in the development of Th1 responses. IL-12 production by antigen-presenting cells (APC) can be induced by the interaction between CD40 on the APC and CD40 ligand (CD40L) expressed on T cells after activation. Our previous study indicated that in dendritic cells (DC), the only APC that can activate naive T(h) cells efficiently, the mere CD40 engagement is insufficient to induce IL-12 production. The aim of the present study was to dissect the conditions for efficient IL-12 production by DC further. Using populations of naive and memory Th cells, recombinant CD40L, neutralizing and blocking antibodies, and by determining IFN-gamma production and CD40L expression levels, we here show that T cell-induced IL-12 production by DC results from the action of two signals, mediated by CD40L and IFN-gamma, and that the inability of naive T(h) cells to induce IL-12 production resides in their inability to produce IFN-(gamma). Other factors than CD40L and IFN-gamma can provide the required signals for IL-12 production by DC, as either factor could be replaced by lipopolysaccharide (LPS). The two-signal requirement proved unique for the production of IL-12, since either CD40 engagement or LPS was sufficient for the efficient production of tumor necrosis factor-alpha, IL-8 and the p40 subunit of IL-12, and may be considered as a safety mechanism for optimal control of potentially harmful T(h)1 responses.     

Augmentation of naive, Th1 and Th2 effector CD4 responses by IL-6, IL-1 and TNF

The role of antigen-presenting cell (APC)-derived cytokines in T cell activation is still controversial. Highly purified CD4 T cell populations of the naive and short-term Th1 and Th2 effector subsets were examined. Stimulation from anti-CD3 in the absence of APC was used to analyze directly T occurring cell-mediated effects, and the requirement for co-signaling was addressed using anti-CD28. Exogenous IL-6, IL-1 and TNF each enhanced proliferation and IL-2 secretion from naive cells, although IL-6 was most active in this regard. Peak responses, however, were obtained with IL-1 or TNF in combination with IL-6 resulting in up to 11-fold increases in IL-2 secretion. Enhanced naive T cell responses were only observed with anti-CD3 and anti-CD28, suggesting that co-signaling through surface-bound receptors was required to initiate IL-2 production. Although the cytokines enhanced naive activation, little effect was seen on differentiation into effector populations. IL-6 alone, or in combination, partially suppressed effectors secreting IFN-gamma, but did not promote generation of effectors secreting IL-4. In contrast to reports on cloned cell lines, IL-6, TNF and IL-1 had enhancing activities on all cytokines elicited from already generated Th1 and Th2 effector populations. Again combinations of IL-6, TNF and IL-1 were most effective and generally required CD28 signaling. Induced responses with preexisting effector cells were far less than with naive cells and predominantly directed at augmenting IFN-gamma and IL-5 secretion rather than IL-2 and IL-4. These studies show that APC-derived cytokines can promote T cell responses directly but largely after co-stimulation from accessory molecule co-receptors, that the effect is not specific for one T cell subset or cytokine, and that the naive T cell is the main target of action.     

Mechanisms for induction of acquired host immunity by neutrophil peptide defensins

Human neutrophil peptide (HNP) defensins were studied to determine their potential effects on adaptive mucosal immunity. Intranasal delivery of HNPs plus ovalbumin (OVA) enhanced OVA-specific serum IgG antibody (Ab) responses. However, OVA-specific IgA Abs were not induced in mucosal secretions or in serum. CD4(+) T cells of intranasally immunized mice displayed higher OVA-specific proliferative responses and elevated production of interferon gamma, interleukin (IL) 5, IL-6, and IL-10 when compared with control groups receiving OVA alone. In vitro, HNPs also enhanced both proliferative responses and T helper (Th) cytokine secretion profiles of CD3epsilon-stimulated spleen- and Peyer's patch-derived naive CD4(+) T cells. HNPs modulated the expression of costimulatory molecules by lipopolysaccharide- or CD3epsilon-stimulated splenic and Peyer's patch B or T cell populations, respectively. These studies show that defensins enhance systemic IgG, but not IgA, Ab responses through help provided by CD4(+) Th1- and Th2-type cytokines and foster B and T cell interactions to link innate immunity with the adaptive immune system.     

Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene

Replication-deficient adenovirus (Ad) vectors are effective to specifically target the respiratory epithelium for either corrective gene therapy such as cystic fibrosis or for mucosal immunization. As a consequence of transducing the lower respiratory tract with an E1/E3 deleted Ad5 vector, host responses have been characterized by the duration of transgene expression and by the induction of CTL responses. However, limited emphasis has been devoted to understanding the contribution of CD4+ T cell responses to the Ad vector. Both CD4+ and CD8+ T cells migrate into the lung following sequential intratracheal Ad5 transgene instillations. Isolated CD3+ T lymphocytes from the lungs were predominantly of the Th2 type, and after cell sorting, the IL-4-producing T cells were largely CD4+, while IFN-gamma expression was associated with both CD4+ and CD8+ T cells. Ab responses to the Ad5 vector and to the expressed transgene beta-galactosidase (beta gal) revealed elevated bronchial and serum IgA and IgG Abs with low neutralization titers. Analysis of serum IgG subclass responses showed IgG1 and IgG2b with lower IgG2a Abs to Ad5 and IgG2a and IgG2b Ab responses to beta gal. Ad5-specifc CD4+ T cells produced both Th1 (IFN-gamma and IL-2)- and Th2 (IL-4, IL-5, IL-6)-type cytokines, while beta gal-specific CD4+ T cells secreted IFN-gamma and IL-6. This study provides direct evidence for the concomitant induction of Th2- with Th1-type responses in both the pulmonary systemic and mucosal immune compartments to the Ad5 vector as well as a Th1-dominant response to the transgene.     

Regulation of T helper cell differentiation in vivo by soluble and membrane proteins provided by antigen-presenting cells

The aim of this study was to test whether the nature of the antigen-presenting cell (APC) can influence the Th1/Th2 balance in vivo. Our data show that dendritic cells (DC), pulsed extracorporeally with antigen, induced the development of cells secreting IL-2, IFN-gamma and IL-4 upon antigen rechallenge in vitro. Priming with peritoneal macrophages sensitized cells that produced IL-4 but not IFN-gamma. To identify the factors involved in T helper development, mice were primed with APC with or without treatment with neutralizing antibodies to costimulatory molecules or cytokines. Our results indicate that priming with DC or macrophages is strictly dependent on the CD28-CTLA4/B7 interaction. Of note, CD86 provides the initial signal to induce naive T cells to become IL-4 producers, whereas CD80 is a more neutral differentiation signal. IL-12, released by the DC, appears as a potent and obligatory inducer of differentiation for IFN-gamma-producing cells. IL-6, although produced by both APC populations, is necessary to direct activation of the Th2-type response by macrophages but not by DC.     

Decreased drug accumulation in a mitoxantrone-resistant gastric carcinoma cell line in the absence of P-glycoprotein

The developmental commitment to a T helper 1 (Th1)- or Th2-type response can significantly influence host immunity to pathogens. Extinction of the IL-12 signaling pathway during early Th2 development provides a mechanism that allows stable phenotype commitment. In this report we demonstrate that extinction of IL-12 signaling in early Th2 cells results from a selective loss of IL-12 receptor (IL-12R) beta 2 subunit expression. To determine the basis for this selective loss, we examined IL-12R beta 2 subunit expression during Th cell development in response to T cell treatment with different cytokines. IL-12R beta 2 is not expressed by naive resting CD4+ T cells, but is induced upon antigen activation through the T cell receptor. Importantly, IL-4 and IFN-gamma were found to significantly modify IL-12 receptor beta 2 expression after T cell activation. IL-4 inhibited IL-12R beta 2 expression leading to the loss of IL-12 signaling, providing an important point of regulation to promote commitment to the Th2 pathway. IFN-gamma treatment of early developing Th2 cells maintained IL-12R beta 2 expression and restored the ability of these cells to functionally respond to IL-12, but did not directly inhibit IL-4 or induce IFN-gamma production. Thus, IFN-gamma may prevent early Th cells from premature commitment to the Th2 pathway. Controlling the expression of the IL-12R beta 2 subunit could be an important therapeutic target for the redirection of ongoing Th cell responses.     

Th1 cells induce and Th2 inhibit antigen-dependent IL-12 secretion by dendritic cells

Dendritic cells are the most relevant antigen-presenting cells (APC) for presentation of antigens administered in adjuvant to CD4+ T cells. Upon interaction with antigen-specific T cells, dendritic cells (DC) expressing appropriate peptide-MHC class II complexes secrete IL-12, a cytokine that drives Th1 cell development. To analyze the T cell-mediated regulation of IL-12 secretion by DC, we have examined their capacity to secrete IL-12 in response to stimulation by antigen-specific Th1 and Th2 DO11.10 TCR-transgenic cells. These cells do not differ either in TCR clonotype or CD40 ligand (CD40L) expression. Interaction with antigen-specific Th1, but not Th2 cells, induces IL-12 p40 and p75 secretion by DC. The induction of IL-12 production by Th1 cells does not depend on their IFN-gamma secretion, but requires direct cell-cell contact mediated by peptide/MHC class II-TCR and CD40-CD40L interactions. Th2 cells not only fail to induce IL-12 secretion, but they inhibit its induction by Th1 cells. Unlike stimulation by Th1, inhibition of IL-12 production by Th2 cells is mediated by soluble molecules, as demonstrated by transwell cultures. Among Th2-derived cytokines, IL-10, but not IL-4 inhibit Th1-driven IL-12 secretion. IL-10 produced by Th2 cells appears to be solely responsible for the inhibition of Th1 -induced IL-12 secretion, but it does not account for the failure of Th2 cells to induce IL-12 production by DC. Collectively, these results demonstrate that Th1 cells up-regulate IL-12 production by DC via IFN-gamma-independent cognate interaction, whereas this is inhibited by Th2-derived IL-10. The inhibition of Th1 -induced IL-12 production by Th2 cells with the same antigen specificity represents a novel mechanism driving the polarization of CD4+ T cell responses.     

Down-regulating effects of IL-4 and IL-10 on the IFN-gamma response in atopic dermatitis

Atopic dermatitis (AD) is a chronic allergic disease associated with toxin (superantigen)-producing Staphylococcus aureus skin infections, impaired delayed hypersensitivity responses, and the expansion of IL-4-secreting Th2 cells, as well as diminished IFN-gamma synthesis. IL-12 is known to induce IFN-gamma synthesis and to augment Th1 responses. In this study, therefore, we examined the potential role of IL-12 in the immunopathogenesis of AD. We show that, after stimulation with staphylococcal toxic shock syndrome toxin-1 (TSST-1) or IL-12, PBMC from patients with AD are deficient in their ability to produce IFN-gamma. PBMC from AD patients, however, produced normal quantities of IL-12 and expressed normal levels of IL-12R. Induction of IFN-gamma by TSST-1 was decreased by neutralizing anti-IL-12 Ab in normal donors, but not in AD patients. The latter observation is consistent with a defective response to IL-12 in AD PBMC. Because AD is associated with increased production of IL-4 and IL-10, we examined the effect of IL-4 on IL-12- or TSST-1-induced IFN-gamma production in normal donors. IL-4 inhibited IL-12-induced IFN-gamma production. Furthermore, Ab neutralization of IL-4 caused increased production of IFN-gamma in AD PBMC. However, neutralization of IL-10 activity caused an even greater augmentation of IFN-gamma production. Our data suggest that despite normal levels of IL-12 production and IL-12R expression, PBMC from AD patients are unable to generate normal IL-12-induced IFN-gamma responses. This defective response may be due to the excess production of IL-4 and IL-10 in this common allergic condition.     

Interleukin-12 inhibits hepatitis B virus replication in transgenic mice

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by antigen-presenting cells that has the ability to induce gamma interferon (IFN-gamma) secretion by T and natural killer cells and to generate normal Th1 responses. These properties suggest that IL-12 may play an important role in the immune response to many viruses, including hepatitis B virus (HBV). Recently, we have shown that HBV-specific cytotoxic T lymphocytes inhibit HBV replication in the livers of transgenic mice by a noncytolytic process that is mediated in part by IFN-gamma. In the current study, we demonstrated that the same antiviral response can be initiated by recombinant murine IL-12 and we showed that the antiviral effect of IL-12 extends to extrahepatic sites such as the kidney. Southern blot analyses revealed the complete disappearance of HBV replicative intermediates from liver and kidney tissues at IL-12 doses that induce little or no inflammation in these tissues. In addition, immunohistochemical analysis demonstrated the disappearance of cytoplasmic hepatitis B core antigen from both tissues after IL-12 treatment, suggesting that IL-12 either prevents the assembly or triggers the degradation of the nucleocapsid particles within which HBV replication occurs. Importantly, we demonstrated that although IFN-gamma, tumor necrosis factor alpha, and IFN-alpha/beta mRNA are induced in the liver and kidney after IL-12 administration, the antiviral effect of IL-12 is mediated principally by its ability to induce IFN-gamma production in this model. These results suggest that IL-12, through its ability to induce IFN-gamma, probably plays an important role in the antiviral immune response to HBV during natural infection. Further, since relatively nontoxic doses of recombinant IL-12 profoundly inhibit HBV replication in the liver and extrahepatic sites in this model, IL-12 may have therapeutic value as an antiviral agent for the treatment of chronic HBV infection.     

An in vitro model for infection with Leishmania major that mimics the immune response in mice

By using a primary in vitro response specific for Leishmania major, normal T cells from resistant CBA/CaH-T6J and susceptible BALB/c mice commit to a Th1 and a Th2 response, respectively. Since commitment occurred, we measured the production of gamma interferon (IFN-gamma), interleukin-1 (IL-1), IL-2, IL-4, IL-5, IL-10, and IL-12, prostaglandin E2 (PGE2), transforming growth factor beta (TGF-beta), and nitric oxide in the first 7 days of the response to identify factors that are critical for Th1 and Th2 development. While cells from resistant CBA mice produced more IFN-gamma, IL-10, and nitric oxide, cells from susceptible BALB/c mice produced more IL-1alpha, IL-5, PGE2, and TGF-beta. Although substantial amounts of IL-12 were detected, IL-12 did not associate with either Th1 or Th2 development. We did not anticipate that cells from resistant CBA mice would make more IL-10 in vitro. However, this also occurred in vivo since CBA mice produced substantial amounts of IL-10 following infection with L. major. Moreover, adding anti-IL-10 to primary in vitro responses enhanced production of IFN-gamma and nitric oxide by cells from CBA and BALB/c mice. Therefore, IL-10 cannot be regarded as a cytokine that associates with susceptibility to infection with L. major. Finally, the data presented here suggest that a collection of factors that can be produced by accessory cells influence Th commitment (e.g., IL-1, PGE2, and TGF-beta favor Th2 development).     

Interferon gamma derived from CD4(+) T cells is sufficient to mediate T helper cell type 1 development

Interferon gamma (IFN-gamma) has been implicated in T helper type 1 (Th1) cell development through its ability to optimize interleukin 12 (IL-12) production from macrophages and IL-12 receptor expression on activated T cells. Various systems have suggested a role for IFN-gamma derived from the innate immune system, particularly natural killer (NK) cells, in mediating Th1 differentiation in vivo. We tested this requirement by reconstituting T cell and IFN-gamma doubly deficient mice with wild-type CD4(+) T cells and challenging the mice with pathogens that elicited either minimal or robust IL-12 in vivo (Leishmania major or Listeria monocytogenes, respectively). Th1 cells developed under both conditions, and this was unaffected by the presence or absence of IFN-gamma in non-T cells. Reconstitution with IFN-gamma-deficient CD4(+) T cells could not reestablish control over L. major, even in the presence of IFN-gamma from the NK compartment. These data demonstrate that activated T cells can maintain responsiveness to IL-12 through elaboration of endogenous IFN-gamma without requirement for an exogenous source of this cytokine.