CD28 activation promotes Th2 subset differentiation by human CD4+ cells

Ligation of CD28 provides a costimulatory signal to T cells necessary for their activation resulting in increased interleukin (IL)-2 production in vitro, but its role in IL-4 and other cytokine production and functional differentiation of T helper (Th) cells remains uncertain. We studied the pattern of cytokine production by highly purified human adult and neonatal CD4⁺ T cells activated with anti-CD3, phorbol 12-myristate 13-acetate (PMA) and ionomycin, or phytohemagglutinin (PHA) in the presence or absence of anti-CD28 in repetitive stimulation-rest cycles. Initial stimulation of CD4⁺ cells with anti-CD3 (or the mitogens PHA or PMA+ionomycin) and anti-CD28 monoclonal antibodies induced IL-4, IL-5 and interferon-γ (IFN-γ) production and augmented IL-2 production (6- to 11-fold) compared to cells stimulated with anti-CD3 or mitogen alone. The anti-CD28-induced cytokine production corresponded with augmented IL-4 and IL-5 mRNA levels suggesting increased gene expression and/or mRNA stabilization. Most striking, however, was the progressively enhanced IL-4 and IL-5 production and diminished IL-2 and IFN-γ production with repetitive consecutive cycles of CD28 stimulation. The enhanced Th2-like response correlated with an increased frequency of IL-4-secreting cells; up to 70% of the cells produced IL-4 on the third round of stimulation compared to only 5% after the first stimulation as determined by ELISPOT. CD28 activation also promoted a Th2 response in naive neonatal CD4⁺ cells, indicating that Th cells are induced to express a Th2 response rather than preferential expansion of already established Th2-type cells. This CD28-mediated response was IL-4 independent, since enhanced IL-5 production with repetitive stimulation cycles was not affected in the presence of neutralizing anti-IL-4 antibodies. These results indicate that CD28 activation may play an important role in the differentiation of the Th2 subset in humans.     

Accessory signaling by CD40 for T cell activation: induction of Th1 and Th2 cytokines and synergy with interleukin-12 for interferon-γ production

The interaction of CD40 ligand (CD40L) on activated T cells with CD40 on B cells, monocytes and dendritic cells is essential for humoral immunity and for up-regulation of antigen-presenting cell (APC) functions, as a result of signaling through CD40. There are also some indications that after interaction with CD40, CD40L can directly signal T cells. In this study we demonstrate that upon stimulation of human peripheral blood T cells through the T cell receptor (TCR)/CD3 complex, CD40/CD40L interaction strongly enhances the production of Th1 cytokines such as interleukin (IL)-2 and interferon (IFN)-γ and Th2 cytokines such as IL-4, IL-5 and IL-10 by a direct effect on T cells. Furthermore, CD40/CD40L interaction synergizes with IL-12 in selectively enhancing IFN-γ production by purified anti-CD3-stimulated T cells. These effects were observed at both the protein and the mRNA level. Both CD4⁺ and CD8⁺ T cells were able to produce IFN-γ in the presence of helper signals from IL-12 and CD40, although CD8⁺ T cells were less active. Since CD40/CD40L interaction also up-regulates IL-12 production and B7 expression by APC, our results suggest that CD40/CD40L interaction is bidirectional, and promotes activation of both APC and T cells.     

Interleukin-13 is produced by activated human CD45RA+ and CD45R0+ T cells: modulation by interleukin-4 and interleukin-12

Interleukin (IL)-13 is a cytokine originally identified as a product of activated T cells. Little is known, however, about IL-13 production by human T cells and its modulation by other cytokines. Here, we show that IL-13 is produced by activated human CD4⁺ and CD8⁺ CD45R0⁺ memory T cells and CD4⁺ and CD8⁺ CD45RA⁺ naive T cells. In contrast, IL-4, which shares many biological activities with IL-13, is only produced by CD45R0⁺ T cells following activation. Analysis of intracellular cytokine production by single CD45RA⁺ and CD45R0⁺ T cells indicated that IL-13 continued to be produced for more than 24 h after stimulation, whereas IL-4 could not be detected after 24 h. These data were confirmed by measurement of specific mRNA and suggest that IL-13, unlike IL-4, but like interferon-γ (IFN-γ), is a cytokine with long-lasting kinetics. The majority of human CD45R0⁺ T cells produced IL-4 and IL-13 simultaneously. In contrast, IFN-γ protein was generally not co-expressed with IL-4 or IL-13. IL-4 added to primary cultures of highly purified peripheral blood T cells activated by the combination of anti-CD3+anti-CD28 mAb enhanced IL-13 production by CD45RA⁺ and to a lesser extent by CD45R0⁺ T cells. Under these conditions, however, IL-12 inhibited IL-13 production by CD45RA⁺ T cells and to a lesser extent by CD45R0⁺ T cells in a dose-dependent fashion. These inhibiting effects were not related to enhanced IFN-γ production induced by IL-12, since IFN-γ by itself did not affect IL-13 production. Collectively, our data indicate that IL-13 is produced by peripheral blood T cells which also produce IL-4, but not IFN-γ, and by naive CD45RA⁺ T cells which, in contrast, fail to produce IL-4. These observations, together with the long-lasting production of IL-13, suggest that IL-13 may have IL-4-like functions in situations where T cell-derived IL-4 is still absent or where its production has already been down-regulated.     

Development in vitro of human CD4+ thymocytes into functionally mature Th2 cells. Exogenous interleukin-12 is required for priming thymocytes to produce both Th1 cytokines and interleukin-10

Fresh postnatal thymocyte cell suspensions were directly cloned under limiting dilution conditions with either phytohemagglutinin or toxic shock syndrome toxin-1 (TSST-1), a bacterial superantigen. Cultures contained allogenic irradiated feeder cells and interleukin (IL)-2, in the absence or presence of exogenous IL-4, interferon (IFN)-γ or IL-12. The resulting CD4⁺ T cell clones generated under these different experimental conditions were then analyzed for their ability to produce IL-2, IL-4, IL-5, IL-10, IFN-γ and tumor necrosis factor (TNF)-β in response to stimulation with phorbol 12-myristate 13-acetate (PMA)+anti-CD3 monoclonal antibody or PMA + ionomycin. Different from T cell clones generated from peripheral blood, virtually all CD4⁺ T cell clones generated from human thymocytes produced high concentrations of IL-2, IL-4 and IL-5, but no IFN-γ, TNF-β or IL-10. Moreover, after activation, these clones expressed on their surface membrane both CD30 and CD40 ligand, but not the product of lymphocyte activation gene (LAG)-3, and provided strong helper activity for IgE synthesis by allogeneic B cells. The Th2 cytokine pattern could not be modified by the addition of IFN-γ. However, upon addition of exogenous IL-12, the resulting CD4⁺ thymocyte clones produced TNF-β, IFN-γ, and IL-10 in addition to IL-4 and IL-5. These results suggest that CD4⁺ human thymocytes have the potential to develop into cells producing the Th2 cytokines IL-4 and IL-5, whereas the ability to produce both Th1 cytokines and IL-10 is acquired only after priming with IL-12.     

Schistosoma-specific helper T cell clones from subjects resistant to infection by Schistosoma mansoni are Th0/2

Although T helper cells play a critical role in human immunity against schistosomes, the properties of the T lymphocytes that govern resistance and pathogenesis in human schistosomiasis are still poorly defined. This work addresses the question as to whether human resistance to Schistosoma mansoni is associated with a particular T helper subset. Twenty-eight CD3⁺, CD4⁺, CD8^? parasite-specific T cell clones were isolated from three adults with high degree of resistance to infection by S. mansoni. The lymphokine secretion profiles of these clones were determined and compared to those of 21 CD3⁺, CD4⁺, CD8^? clones with unknown specificity, established from these same subjects in the same cloning experiment. Almost all parasite-specific clones produced interleukin (IL)-4 and interferon (IFN)-γ in large amounts. However, they generally produced more IL-4 than IFN-γ; variations in IL-4/IFN-γ ratios were accounted for by differences in IFN-γ production since IL-4 levels were comparable for the clones from the three subjects. T cell clones of unknown specificity produced significantly less IL-4 and more IFN-γ than parasite-specific T cell clones. Most clones produced IL-2, and IL-2 production did not differ between the two types of clones. Parasite-specific T cell clones from the resistant subjects were compared to specific T cell clones from a sensitized adult from a nonendemic area: T cell clones from this latter subject were the highest IFN-γ and the lowest IL-4 producers, compared to those of resistant subjects. Thus, parasite-specific T cell clones isolated from adults resistant to S. mansoni belong to the Th0 subset and produced more IL-4 than IFN-γ (Th0/2), whereas clones of a sensitized adult from a nonendemic area are also Th0, but produce more IFN-γ than IL-4 (Th0/1). These results support previous conclusions on the role of IgE in protection against schistosomes in humans, and may indicate that IFN-γ is required for full protection.     

Differential modulation of T helper type 1 (Th1) and T helper type 2 (Th2) cytokine secretion by prostaglandin E2 critically depends on interleukin-2

Prostaglandin E₂ (PGE₂) favors T helper type 2 (Th2)-like cytokine secretion profiles in murine and human CD4⁺ T cells by inhibiting the production of the Th1-associated cytokines interleukin-2 (IL-2) and interferon-γ (IFN-γ) and up-regulating the production of the Th2-associated cytokines IL-4 and IL-5 in a dose-dependent way. However, the potent inhibition of IL-2 production by PGE₂ seems to be in contrast with the simultaneous up-regulation of IL-4 and IL-5 production, because the induction of these cytokines requires IL-2. We, therefore, investigated to which extent the net modulatory effect of PGE₂ is determined by the availability of IL-2. To this aim, we examined the effects of PGE₂ on the cytokine secretion profiles of a panel of human Th0 clones upon stimulation via different activation pathways, resulting either in high or low IL-2 production. The differential modulation of Th1 and Th2 cytokines by PGE₂ was observed only upon modes of stimulation resulting in high IL-2 production. When IL-2 production was low, PGE₂ inhibited the secretion of all four cytokines. These different modulation patterns were directly related to the IL-2 availability, because (i) neutralizing antibody to IL-2 abrogated the up-regulatory effect of PGE₂ on IL-4 and IL-5 secretion in experiments with high endogenous IL-2 levels, (ii) lack of differential cytokine modulation by PGE₂ in conditions with low levels of endogenous IL-2 could be restored with exogenous IL-2, and (iii) cell viability was comparable in all conditions. These results demonstrate that the net modulatory effect of PGE₂ on the cytokine secretion profile of T cells critically depends on the availability of IL-2. Since this parameter varies with the experimental conditions and the T cell population studied, this finding may explain why certain immune responses may be either up- or down-regulated by PGE₂ under different conditions.     

Selective stimulation of T helper 2 cytokine responses by the anti-psoriasis agent monomethylfumarate

Type 2 cytokines are thought to have a protective role in psoriasis vulgaris by dampening the activity of T helper 1 (Th1) lymphocytes. The aim of the present study was to determine the effect of monomethylfumarate (MMF), the most active metabolite of the new anti-psoriatic drug Fumaderm®, on the production of cytokines and the development of Th subsets. MMF was found to enhance interleukin (IL)-4 and IL-5 production by CD2/CD8 monoclonal antibody-stimulated peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Maximal effects of MMF were found at a concentration of 200 μM and resulted in tenfold enhanced levels of IL-4 and IL-5 production. MMF did not affect the levels of IL-2 production, interferon (IFN)-γ production or proliferative T cell responses in these cultures. Similar effects of MMF were observed in cultures of purified peripheral blood T cells indicating that this compound can act directly on T cells. MMF did not influence cytokine production by purified CD4⁺CD45RA⁺ (unprimed) T cells, but greatly enhanced IL-4 and IL-5 production without affecting IFN-γ production by purified CD4⁺CD45R0⁺ (primed) T cells. Furthermore, MMF also augmented IL-4 and IL-5 production in established Th1/Th0 clones that were stimulated with CD2/CD28 monoclonal antibody. Finally, when PBMC were challenged with Mycobacterium tuberculosis that typically induces Th1 recall responses with strong IFN-γ secretion, MMF again appeared to induce high levels of IL-4 and IL-5 secretion while IFN-γ production was unaffected. These results may be relevant for the development of therapeutic regimens designed to correct inappropriate Th1 subset development in immunopathologic conditions.     

Interleukin-7 activates human naive CD4+ cells and primes for interleukin-4 production

Interleukin (IL)-4 is considered to be essential for T helper (Th)2 cell development, yet in areas of primary T cell activation, CD4⁺ cells are its only source. This implies that other signals must drive the initial expression of IL-4 production. The role of CD28 co-stimulation in Th2 subset development has been described. However, in mice deficient for CD28, Th2 responses are diminished, but not abrogated. Cytokines produced within the lymphoid tissue, e.g. IL-7, may be important in the primary activation of naive CD4⁺ cells. We have found that human naive CD4⁺ cells purified from umbilical cord blood express the IL-7 receptor and respond vigorously to IL-7 during primary stimulation. Naive CD4⁺ cells grown in IL-4, in the presence or absence of IL-2, fail to produce Th2 cytokines upon restimulation. In contrast, IL-7 induces development of a population of T cells that produce large amounts of IL-4. Growth in IL-7 also increases IL-2-induced production of interferon (IFN)-γ and IL-10 production. IL-7-induced IL-4 production is not inhibited by neutralizing antibodies to IL-4 on its receptor. This implies that IL-7 acts directly to induce Th2 subset development and not by up-regulating either production of IL-4 during culture or expression of the IL-4 receptor. Moreover, IL-7 potentiates the effects of CD28 co-stimulation on both naive CD4⁺ cell proliferation and subsequent IL-4 production. Following primary stimulation, CD4⁺ cells lose expression of the IL-7 receptor, resulting in IL-7 unresponsiveness. This work reveals a novel role for IL-7 in the primary activation of CD4⁺ cells. We propose that in conjunction with CD28 co-stimulation, IL-7 induces the initial expression of IL-4 production and that IL-4 acts subsequently to expand Th2 cytokine-producing cells at the appropriate anatomical site.     

Human recombinant interferon-β influences T helper subset differentiation by regulating cytokine secretion pattern and expression of homing receptors

Type I interferons (IFN) are important regulators of both innate and acquired immunity. We have used an in vitro system of human CD4⁺ T cell differentiation to determine how IFN-β influences development of T helper (Th) subsets and homing receptor expression. IFN-β promoted differentiation of CD4⁺ T cells that produce low levels of both IFN-γ and lymphotoxin compared to interleukin (IL)-12-derived Th1 CD4⁺ T cells. IFN-β inhibited production of Th2 cytokines (IL-5 and IL-13) and augmented IL-12-mediated IL-10 secretion. In addition, IFN-β significantly enhanced L-selectin expression on CD4⁺ T cells and synergized with IL-12 to induce expression of cutaneous lymphocyte-associated antigen (CLA). This Th1 L-selectin⁺, CLA⁺ phenotype is characteristic of T cells found in normal human skin and suggests a role for type I IFN in the regulation of Th subset differentiation and tissue-specific homing receptors.     

Bacille Calmette Guérin and interleukin-12 down-modulate interleukin-4-producing CD4+ NK1+ T lymphocytes

Early production of interleukin-12 (IL-12) by macrophages and of IL-4 from CD4⁺ NK1⁺ T cells influence development of the acquired immune response against infectious agents, namely differentiation of interferon-γ-secreting T helper 1 (Th1) cells against intracellular pathogens and of IL-4-producing Th2 cells against helminths. Evidence has been presented for transient convertibility of Th1 and Th2 cells in the presence of the polarizing cytokines IL-4 or IL-12, respectively. Moreover, it is likely that IL-4 dominates over IL-12, suggesting that Th2 cell development is preferred in the presence of both cytokines. Mycobacterium bovis Bacille Calmette Guérin (BCG) and IL-12 are potent inducers of Th1 responses. Here we show that BCG and IL-12 down-modulate IL-4-producing CD4⁺ NK1⁺ TCRα/β^intermediate liver lymphocytes. Our data provide further insights into the mechanisms by which BCG and IL-12 may promote unrestricted development of Th1 responses in vivo: BCG and IL-12 not only provide the positive stimuli for Th1 cell differentiation, but also interfere with antagonizing signals.     

Increased CD45RO CD62L CD4 T-cell subpopulation responsible for Th2 response in Kimura’s disease

Kimura’s disease is characterized by subcutaneous masses, eosinophilia, and markedly elevated serum immunoglobulin E, suggesting that T helper (Th)2 cells may play a role in the pathogenesis. We investigated Th2 cytokine synthesis by mononuclear cells and possible Th1/Th2 subpopulations in Kimura’s disease. Peripheral blood samples were obtained from seven patients with Kimura’s disease and CD4⁺ T-cell subpopulations separated by CD45RO and CD62L were isolated. Purified cells were stimulated with PHA or anti-CD3 mAb, and the cytokine levels were measured by Cytometric Bead Array kit. Peripheral blood mononuclear cells in the majority of the patients produced Th2 cytokines such as interleukin (IL)-3, IL-4, IL-5, IL-13 or GM-CSF higher than those of controls. The ratio of CD45RO⁺ CD62L⁺ cells in CD4⁺ T cells was increased in six out of seven patients compared to age-matched controls. Especially, patient 1 had remarkably increased levels of CD45RO⁺ CD62L⁺ population in CD4⁺ T cells. In addition, IL-4 production levels by CD45RO⁺ CD62L⁺ CD4⁺ T cells of patients 1 and 2 were higher than those of their CD45RO⁺ CD62L⁻ CD4⁺ T cells, in the same manner as those by a normal control. Taken together, the synthesis of Th2 cytokines and CD62L-positive subpopulation in CD45RO⁺ CD4⁺ T cells, which may represent characteristics of Th2, are increased in patients with Kimura’s disease, suggesting that deviation to Th2 may involve in pathogenesis of the disease.     

T helper type 2-like cells and therapeutic effects of interferon-γ in combined immunodeficiency with hypereosinophilia (Omenn's syndrome)

We characterized the defects of CD4+ cells in a 17-month-old girl suffering from combined immunodeficiency with hypereosinophilia (Omenn's syndrome). Because the vast majority of peripheral blood CD4⁺ cells expressed the CD45R0 isoform, we purified circulating CD4⁺ CD45R0⁺ cells from the patient and healthy individuals in order to compare their production of cytokines. The patient's CD4⁺ CD45R0⁺ cells spontaneously produced high levels of interleukin-5 (IL-5) in vitro (1600 pg/ml after 24 h of culture) and this was associated with the presence of IL-5 in serum (323 pg/ml). After stimulation with phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187, they produced higher levels of IL-4 (306 vs. 55 ± 4 pg/ml) and IL-5 (2900 vs. 213 ± 72 pg/ml) and lower levels of IL-2 (17 vs. 63 ± 17 IU/ml) and interferon-γ (IFN-γ) (16 vs. 299 ± 70 IU/ml) than controls CD4⁺ CD45R0⁺ cells. This T helper type 2 (Th2) pattern was confirmed by the detection using reverse polymerase chain reaction of IL-4, IL-5 and IL-10 mRNA within peripheral blood mononuclear cells. During a therapeutic trial with human IFN-γ (40 μg/day) which ameliorated the clinical status of the patient, we observed a down-regulation of the in vivo expression of IL-5 and IL-10, a normalization of the eosinophil count and an improvement of the Tcell response to phytohemagglutinin. This observation indicates for the first time that Th2-like cells might be involved in certain forms of congenital immunodeficiency and that IFN-γ might down-regulate their activities in vivo.     

CD8+ T cells and not CD4+ T cells are hyporesponsive to CD28- and CD40L-mediated activation in HIV-infected subjects

T cell dysfunction in HIV-infected subjects could be the consequence of altered sensitivity of CD4⁺ or CD8⁺ T cells to various costimulatory signals. Therefore, we studied proliferation and cytokine production in highly purified CD8⁺ and CD4⁺ T cells from HIV-infected and HIV⁻ subjects, induced by co-activation via cell-bound CD80, CD86 and CD40 or by allo-activation. Regardless of the nature of the first and the costimulatory signal, CD8⁺ T cells from patients proliferated consistently less than controls, while responses from CD4⁺ T cells were similar in patients and controls. This phenomenon was observed after ligation of CD28 combined with anti-CD3 or phorbol myristate acetate (PMA), but also after allogeneic stimulation and after activation by CD40 and anti-CD3. Anti-CD3 combined with CD80 or CD86 induced a mixed Th1/Th2-type cytokine profile in both CD4⁺ and CD8⁺ T cells from controls, whereas anti-CD3 plus CD40 induced only low levels of Th2-type cytokines and no interferon-gamma (IFN-γ) in CD4⁺ T cells. Compared with controls, CD4⁺ T cells from patients produced slightly lower levels of IL-10 but equal amounts of IFN-γ, IL-4 and IL-5, while CD8⁺ T cells from patients produced less of all cytokines tested. In conclusion, responses of purified CD4⁺ T cells from HIV⁺ subjects to various costimulatory pathways are relatively intact, whereas CD8⁺ T cells are hyporesponsive at the level of proliferation and cytokine production. A generalized intrinsic CD8⁺ T cell failure might contribute to viral and neoplastic complications of HIV infection.     

Role of transforming growth factor-βthe preferential induction of T helper cells of type 1 by staphylococcal enterotoxin B

Stimulation of murine CD4⁺ T cells with staphylococcal enterotoxin B (SEB) results in the preferential development of T helper (Th) 1 cells [i.e. high interferon (IFN)-γ and low interleukin (IL)-4, IL-5 and IL-10]; whereas in response to plate-bound anti-CD3 or anti-T cell receptor-αβ, Th1 as well as Th2 cells develop. In the present study, we examined the mechanism which is responsible for the selective Th1 development in the SEB system. The addition of IL-4 resulted in a strong development of Th2 cells showing that SEB stimulation can result inTh2 differentiation. Co-stimulation with anti-CD28 was insufficient in this regard. Lack of Th2 development in the SEB system was in part due to the inhibitory effect of endogenously produced transforming growth factor-β (TGF-β), because anti-TGF-β allowed the development of Th2 cells. Similarly, TGF-β inhibited Th2 development and stimulated Th1 development in the anti-CD3 system. This shift was only partially prevented by also including IL-4 in the cultures. The effects of TGF-β could only partially be explained by stimulation of IFN-γ or inhibition of IL-4 as intermediatory cytokines: (1) TGF-β stimulated Th1 development even in the presence of anti-IL-4 and anti-IFN-γ, and (2) a strong inhibitory effect of anti-TGF-β on Th1 development was still observed when anti-IL-4 and IFN-γ were simultaneously added to the cultures. It is concluded that SEB favors Th1 development by stimulation of TGF-β production. Inhibition of Th2 development by TGF-β is due, in part, to inhibition of IL-4 and stimulation of IFN-γ, and, in part, to a direct effect of TGF-β on the responding T cells.     

Bach2 in CD4+ T cells from SLE patients modulates B-cell differentiation and IgG production

T and B cells participate in the development of systemic lupus erythematosus (SLE). BTB and CNC homology 2 (Bach2) is an irreplaceable regulator in the T and B lineages that helps to maintain immune homeostasis. However, the function of Bach2 in the pathogenesis of SLE has not been studied in depth. Flow cytometry and qRT‒PCR were used to assess Bach2 levels, bisulfite sequencing PCR was used to measure the methylation level, and silencing by electroporation and stimulation with a cytokine concentration gradient were used to investigate the effect of Bach2 on T cells. Bach2 expression was elevated in the helper T-cell subsets (T follicular helper, Th1, Th2, Th17, and Treg cells) of SLE patients and negatively correlated with disease severity and autoantibody levels. CD4⁺ T cells from SLE patients had decreased methylation levels in the Bach2 promoter region. Silencing Bach2 in CD4⁺ T cells induced increases in the CD19⁺ B-cell count, plasmablasts, and secretion of IgG by prompting the secretion of cytokines. The activation signals CD3/CD28, IL-6, and IL-21 upregulated Bach2 expression in CD4⁺ T cells. The regulation of Bach2 by cytokines and T-cell activation signals in CD4⁺ T cells was shown to act on B cells and play a protective role against SLE.     

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-γ 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-γ-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.