T helper type 1 development of naive CD4+ T cells requires the coordinate action of interleukin-12 and interferon-γ and is inhibited by transforming growth factor-β

It was observed in vitro and in vivo that both interferon (IFN)-γ and interleukin (IL)-12 can promote the development of T helper type 1 (T_H1) cells. Since IL-12 was shown to be a costimulator for the production of IFN-γ by T or natural killer (NK) cells, IL-12 might play only an indirect role in T_H1 differentiation by providing IFN-γ which represents the essential differentiation factor. Using anti-CD3 monoclonal antibody (mAb) for activation of naive CD4⁺ T cells in the absence of accessory cells we could demonstrate that costimulation by IFN-γ alone results only in marginal T_H1 development. Similarly, IL-12 in the absence of IFN-γ is only a poor costimulator for inducing differentiation towards the T_H1 phenotype. Our data indicate that both cytokines are required to allow optimal T_H1 development and that IL-12 has a dual role, it promotes differentiation by direct costimulation of the T cells and also enhances the production of IFN-γ which serves as a second costimulator by an autocrine mechanism. Another cytokine that was reported to favor T_H1 differentiation in certain experimental systems is transforming growth factor (TGF)-β. With naive CD4⁺ T cells employed in this study TGF-β strongly inhibited the production of IFN-γ triggered by IL-12 as well as the IL-12-induced T_H1 development. When TGF-β was combined with anti-IFN-γ mAb for neutralization of endogenous IFN-γ the T_H1-inducing capacity of IL-12 was completetly suppressed.     

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.     

The interleukin-12 subunit p40 specifically inhibits effects of the interleukin-12 heterodimer

The recently discovered cytokine interleukin (IL)-12 is a heterodimeric protein of two disulfide-bonded subunits of 35 and 40 kDa. IL-12 has multiple effects on T cells and natural killer (NK) cells. In particular it appears to be a major factor for the development of cellular immunity. So far activity of the single subunits alone has not been described, however their expression is regulated independently. In this report we demonstrate for the first time that the mouse IL-12 subunit p40 (IL-12p40) specifically antagonizes the effects of the IL-12 heterodimer in different assay systems. The proliferation of mouse splenocytes activated by phorbol ester and IL-12 was inhibited by IL-12p40, whereas the proliferation induced by phorbol ester and IL-2 was not affected. Furthermore, the synthesis of interferon (IFN)-γ by mouse splenocytes activated with IL-2 and IL-12 was suppressed by IL-12p40. Purified mouse splenic CD4⁺ T cells produced IFN-γ upon activation with plate-bound anti-CD3 monoclonal antibody which was enhanced more than tenfold in the presence of IL-12. In this system IL-12p40 inhibited only the enhancement caused by IL-12 but not IFN-γ synthesis of CD4⁺ T cells stimulated with anti-CD3 alone. Moreover, IL-12p40 inhibited the effects of IL-12 on differentiated T helper type 1 (T_h1) cells. IFN-γ production by T_h1 cells induced in a T cell receptor-independent way by macrophages and IL-2 or macrophages and IL-12 was greatly reduced by IL-12p40 providing evidence for the endogenous synthesis of IL-12 in the Th1 cell, macrophage and IL-2 co-cultures. The specificity of inhibition was clearly demonstrated in the homotypic aggregation assay of Th1 cells. Incubation of Th1 cells with either IL-2 and IL-12 or IL-2 and tumor necrosis factor induces LFA-1/ICAM-1-dependent aggregation. Only IL-2 + IL-12 but not IL-2 + tumor necrosis factor-induced aggregation was inhibited in a dose-dependent manner by IL-12p40. Thus, the IL-12 subunit p40 appears to be a specific inhibitor for the IL-12 heterodimer.     

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.     

Regulation of 4-1BB expression by cell-cell interactions and the cytokines,interleukin-2 and interleukin-4

4-1BB expression increased gradually following T cell activation, and by day 3 post-stimulation with immobilized anti-CD3 (anti-CD3i) or concanavalin A (Con A), splenic T cells were routinely 35–45% 4-1BB⁺ by flow cytometric analysis. 4-1BB was expressed on activated CD8⁺, CD4⁺, CD28⁺ and CD45RB⁺ T cells. Optimal 4-1BB expression was seen by day 6 post-stimulation and was cell density dependent. When T cells were cultured for 6 days at 1 × 10⁶/well in a 24-well plate with anti-CD3i, 82% of the cells were 4-1BB⁺. In contrast, at lower cell densities (4 × 10⁵, 2 × 10⁵ and 1 × 10⁵), optimal 4-1BB expression was observed only if the cultures were supplemented with recombinant interleukin-2 (IL-2) or recombinant IL-4 (IL-4). In agreement, with these results, modes of inducing endogenous IL-2 production such as cross-linking the costimulatory molecule, CD28, or the addition of syngeneic accessory cells to T cells activated with anti-CD3i, resulted in high levels of 4-1BB expression. The addition of interleukin-1α(IL-1α) or interferon-γ (IFN-γ) did not increase 4-1BB expression on anti-CD3i-activated T cells. In addition, if T cells were incubated with IL-2, IL-4, IL-1α, IFN-γ or anti-CD28 alone, no 4-1BB expression was induced. T cells activated with soluble anti-CD3 (anti-CD3s) in the presence of IL-2, IL-4, or accessory cells, did not express higher levels of 4-1BB on their cell surface. These data suggest that initial events crucial for efficient T cell activation, such as signals delivered through the T cell receptor/CD3 complex and the CD28 molecule, are instrumental in regulating subsequent 4-1BB expression.     

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.     

Normal B cells fail to secrete interleukin-12

Interleukin-12 is a key regulatory cytokine produced by antigen-presenting cells (APC) which drives the development of interferon-γ (IFN-γ)-producing cells and promotes cell-mediated immunity. Following subcutaneous immunization with protein antigen in adjuvant, dendritic cells (DC) but not small nor large B cells in immune lymph nodes express antigenic complexes and secrete substantial amounts of bioactive IL-12 p75 upon antigen-specific interaction with T cells. We have analyzed secretion of IL-12 p40 and p75 by cell populations enriched in DC, macrophages or B cells in response to nonspecific stimulation or to interaction with antigen-specific CD4⁺ cells. These APC populations do not produce IL-12 constitutively but, upon stimulation with heat-fixed Staphylococcus aureus and IFN-γ, IL-12 p40 and p75 are secreted by DC and macrophages, whereas B cells fail to produce IL-12. B cells also fail to secrete IL-12 in response to stimulation with LPS and IFN-γ. Co-culture with CD4⁺ T hybridoma cells and antigen induces IL-12 secretion by DC. Up-regulation of IL-12 secretion by interaction with antigen-specific CD4⁺ T cells is abrogated by anti-class II monoclonal antibodies (mAb), by soluble CD40 molecules and by anti-CD40 ligand mAb, demonstrating a positive feedback between T cells and DC mediated by TCR-peptide/class II and by CD40-CD40 ligand interactions. Expression of class II and CD40 molecules is comparable in B cells and DC, and both APC types activate CD4⁺ T cells. Yet, even upon interaction with antigen-specific T cells, B cells fail to secrete IL-12. The capacity of B cells to present antigen but not to secrete IL-12 may explain their propensity to selectively drive T helper type 2 cell development.     

Regulation of IL-10 and IL-17 mediated experimental autoimmune encephalomyelitis by S-nitrosoglutathione

In this study, we investigated IL-10 and IL-17 specific immunomodulatory potential of S-nitrosoglutathione (GSNO), a physiological nitric oxide carrier molecule, in experimental autoimmune encephalomyelitis (EAE). In active EAE model, GSNO treatment attenuated EAE severity and splenic CD4⁺ T cells isolated from these mice exhibited decreased IL-17 expression without affecting the IFN-γ expression compared to the cells from untreated EAE mice. Similarly, adoptive transfer of these cells to nave mice resulted in reduction in IL-17 expression in the spinal cords of recipient mice with milder EAE severity. CD4⁺ T cells isolated from GSNO treated EAE mice, as compared to untreated EAE mice, still expressed lower levels of IL-17 under T_H17 skewing conditions, but expressed similar levels of IFN-γ under T_H1 skewing condition. Interestingly, under both T_H17 and T_H1 skewing condition, CD4⁺ T cells isolated from GSNO treated EAE mice, as compared to untreated EAE mice, expressed higher levels of IL-10 and adoptive transfer of these T_H17 and T_H1 skewed cells seemingly exhibited milder EAE disease. In addition, adoptive transfer of CD4⁺ T cells from GSNO treated EAE mice to active EAE mice also ameliorated EAE disease with induction of spinal cord expression of IL-10 and reduction in of IL-17, thus suggesting the participation of IL-10 mechanism in GSNO mediated immunomodulation. GSNO treatment of mice passively immunized with CD4⁺ T cells either from GSNO treated EAE mice or untreated mice further ameliorated EAE disease, supporting efficacy of GSNO for prophylaxis and therapy in EAE. Overall, these data document a modulatory role of GSNO in IL-17/IL-10 axis of EAE and other autoimmune diseases.     

Opposite role for interleukin-4 and interferon-γ on CD30 and lymphocyte activation gene-3 (LAG-3) expression by activated naive T cells

Polarized human type 1 and type 2 T helper cells not only produce different sets of cytokines, but they also preferentially express certain activation markers, such as lymphocyte activation gene-3 (LAG-3) and CD30, respectively. In this study we have examined the LAG-3 and CD30 expression in relation to the lineage commitment of human naive CD4⁺ T cells, as assessed at the single-cell level of committed T cells. Purified CD45RA⁺ umbilical cord blood T lymphocytes were activated with phytohemagglutinin and interleukin (IL)-2 in the absence or presence of interleukin IL-4 or IL-12 and assessed for CD30 and LAG-3 expression, as well as for intracellular cytokine synthesis. Significant numbers of CD30⁺ cells were only found in CD4⁺ and CD8⁺ T lymphocytes of cultures primed with IL-4, which developed into cells able to produce IL-4 and IL-13 in addition to interferon (IFN)-γ. By contrast, LAG-3 expression was strongly up-regulated in CD4⁺ and CD8⁺ T cells from cultures primed with IL-12, which developed into high numbers of IFN-γ producers. The addition of a neutralizing anti-IFN-γ antibody to IL-12-primed CD4⁺ T cell cultures virtually abolished the development of LAG-3-expressing CD4⁺ T cells. Taken together, these data suggest that CD30 expression is dependent on the presence of IL-4, whereas LAG-3 expression is dependent on the production of IFN-γ during the lineage commitment of human naive T cells.     

IL-4-producing NK T cells are biased towards IFN-γ production by IL-12. Influence of the microenvironment on the functional capacities of NK T cells

NK T cells are an unusual T lymphocyte subset capable of promptly producing several cytokines after stimulation, in particular IL-4, thus suggesting their influence in Th2 lineage commitment. In this study we demonstrate that, according to the cytokines present in the micro environment, NK T lymphocytes can preferentially produce either IL-4 or IFN-γ. In agreement with our previous reports showing that their IL-4-producing capacity is strikingly dependent on IL-7, CD4⁻ CD8⁻ TCRα β⁺ NK T lymphocytes, obtained after expansion with IL-1 plus granulocyte-macrophage colony-stimulating factor, produced almost undetectable amounts of IL-4 or IFN-γ in response to TCR/CD3 cross-linking. However, the capacity of these T cells to produce IFN-γ is strikingly enhanced when IL-12 is added either during their expansion or the anti-CD3 stimulation, while IL-4 secretion is always absent. A similar effect of IL-12 on IFN-γ production was observed when NK T lymphocytes were obtained after expansion with IL-7. It is noteworthy that whatever cytokines are used for their expansion, IL-12 stimulation, in the absence of TCR/CD3 cross-linking, promotes consistent IFN-γ secretion by NK T cells without detectable IL-4 production. Experiments in vivo demonstrated a significant up-regulation of the capacity of NK T cells to produce IFN-γ after anti-CD3 mAb injection when mice were previously treated with IL-12. In conclusion, we provide evidence that the functional capacities of NK T cells, which ultimately will determine their physiological roles, are strikingly dependent on the cytokines present in their microenvironment.     

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.     

Interleukin-12/T cell stimulating factor,a cytokine with multiple effects on T helper type 1 (Th1) but not on Th2 cells

At least two subsets of CD4⁺ T helper cell lymphocytes termed T_h1 and T _h, 2 exist in the mouse and probably in humans. They are characterized by the secretion of different lymphokines and by their functional behavior. Dysregulated expansion of one or the other subset may be one reason for the development of certain diseases. Thus, it is of importance to define the signals involved in the differentiation and activation of the two T_h cell subsets. It is known and has been confirmed in this report that the cytokine interleukin (IL)-1 acts onT_h2 cells but not on T_h1 cells. We now report that a previously identified cytokine which was provisionally termed T cell stimulating factor is identical with IL-12 and exhibits a reciprocal behaviour to IL-1. IL-12 has several effects on T_h1 cells. It can induce the proliferation of certain T_h1 cells in combination with IL-2. Synthesis of interferon (IFN)-γ by T_h1 cells can be triggered by IL-2 plus IL-12. In contrast to the IFN-γ production observed after T cell receptor (TcR) CD3 stimulation of T_h1 cells with lectin Concanavalin A the IFN-γ production induced by IL-12+IL-2 is insensitive to the immunosuppressive drug cyclosporin A. Furthermore, IL-12 enhances the TcR/CD3-induced synthesis of IFN-γ of several T_h1 clones. Finally, IL-12 (+ IL-2) induces homotypic cell aggregation of T_h1 clones. This type of cell aggregation depends on the participation of LFA-1 and ICAM-1 molecules. In all activation systems with T_h1 cells no effect of IL-1 was demonstrable. In contrast, only IL-1 but not IL-12 served as a co-stimulatory signal for several T_h2 cell lines activated via the TcR/CD3 complex.     

Human Th1 responses driven by IL-12 are associated with enhanced expression of CD40 ligand

IL- 12 is the prominent inducer of Th1 responses in humans and in the mouse. CD40 ligand (CD40L) plays important roles in regulation of immune responses, including T cell-dependent activation of B cells and cytokine production by monocytes and dendritic cells. The present study examined the influences of IL-12 on the CD40L expression of activated human CD4⁺ T cells. IL-12 enhanced CD40L expression on CD4⁺ T cells stimulated with immobilized anti-CD3 in the complete absence of accessory cells, whereas IL-4 and IL-10 decreased it. Exogenous interferon-gamma (IFN-γ) did not increase CD40L expression on immobilized anti-CD3 stimulated CD4⁺ T cells at any time up to 168 h of culture. The IL-12-induced enhancement of CD40L expression on anti-CD3 activated CD4⁺ T cells was not influenced in the presence of a metalloproteinase inhibitor KB8301, which up-regulated CD40L expression by preventing the processing of membrane-bound CD40L, or B cells, which down-regulated CD40L expression by receptor-mediated endocytosis. These results indicate that IL-12 enhances the CD40L expression of activated CD4⁺ T cells independently of the IFN-γ production. The data thus suggest that Th1 responses induced by IL-12 might play an important role in the regulation of humoral immune responses through up-regulated CD40L expression.     

T cells made deficient in interleukin-2 production by exposure to staphylococcal enterotoxin B in vivo are primed for interferon-γ and interleukin-10 secretion

The superantigen staphylococcal enterotoxin B (SEB) induces a defect in interleukin (IL)-2 production by T cells expressing specific T cell receptor Vβ domains. The present study was undertaken to determine the capacity of T cells, made deficient in IL-2 production by exposure to SEB in vivo, to secrete interferon (IFN)-γ and IL-10 and to induce pathology upon SEB rechallenge. For this purpose, BALB/c mice received two intraperitoneal injections of 100 μg SEB with a 48-h interval. First, we compared peak serum levels of IL-2, IFN-γ and IL-10 after SEB rechallenge with those measured after a single SEB injection in control mice. The expected defect in IL-2 production in SEB-pretreated mice was associated with a major increase in IL-10 and IFN-γ levels which were about fivefold higher than in controls. Experiments in mice depleted of CD4⁺ or CD8⁺ cells as well as studies in which purified T cell populations were rechallenged with SEB in vitro indicated that both CD4⁺ and CD8⁺ cells from SEB-pretreated mice were primed for IL-10 and IFN-γ production. Furthermore, SEB-pretreated mice were sensitized to the toxic effects of the superantigen as indicated by a 30-70% lethality rate (vs. 0% in naive mice) within 48 h after SEB rechallenge. IFN-γ was involved in the lethal syndrome as it could be prevented by injection of neutralizing anti-IFN-γ monoclonal antibody. We conclude that SEB-reactive T cells made deficient for the production of IL-2 by exposure to SEB in vivo are primed for IFN-γ and IL-10 production, and that IFN-γ up-regulation is involved in the shock syndrome occurring upon SEB rechallenge.     

Identification of the association of CD28+CD244+ Tc17/IFN-γ cells with chronic hepatitis C virus infection

CD8⁺ T cells play multiple and complex immunological roles including antiviral, regulatory, and exhaustive effects in hepatitis C virus (HCV) infected patients. Some CD8⁺ T-cell subsets were confirmed to be closely related to HCV infection such as T_CM, T_EM, T_EMRA, Tc17, and CD8⁺ Treg. Herein, we report a new subset of interleukin (IL)-17/interferon (IFN)-γ producing CD8⁺ T (Tc17/IFN-γ) cells that markedly correlate with CD28⁺CD244⁺ cells, IL-17 levels, and HCV RNA in HCV patients. During early treatment with peg-IFN-a2a plus ribavirin, the imbalance of these Tc17/IFN-γ cells could be partially restored, together with normalized serum alanine aminotransferase but not aspartate transaminase. Also, we analyzed the dynamic change of the percentage of this T cells subset in patients with different outcome after 4-week course of treatment with peg-IFN-a2a plus ribavirin and found that the percentage of CD8⁺CD28⁺CD244⁺ T cells significantly decreased in recovered patients but not in nonrecovered patients. In vitro, CD28⁺CD244⁺ T cells were the only CD8⁺ T-cell group that secreted both IL-17 and IFN-γ in this axis and blockade with anti-CD244 antibodies significantly reduced cytokine production. Taken together, this study demonstrates that the frequency and regulatory functions of CD28⁺CD244⁺ Tc17/IFN-γ cells may play an important role in persistent HCV infection.     

Interleukin-12 but not interferon-γ production correlates with induction of T helper type-1 phenotype in murine candidiasis

By means of polymerase chain reaction-assisted mRNA amplification, we have monitored message levels of interleukin (IL)-12 in splenic macrophages and of interferon-γ (IFN-γ), IL-4, and IL-10 in CD4⁺ and CD8⁺ T cells using Candida albicans/host combinations that result either in a T helper type-1 (Th1)-associated self-limiting infection (“healer mice”) or in a Th2-associated progressive disease (“nonhealer mice”). The timing and pattern of message detection did not differ qualitatively by the expression of IFN-γ or IL-10 mRNA in CD4⁺ and CD8⁺ cells from healer (i.e. PCA-2 into CD2F1) vs. nonhealer (i.e. CA-6 into CD2F1 or PCA-2 into DBA/2) mice. In contrast, IL-4 mRNA was uniquely expressed by CD4⁺ cells from nonhealer animals. IL-12p40 was readily detected in macrophages from healer mice but was detected only early in infection in mice with progressive disease. Cytokine levels were measured in sera, and antigen-driven cytokine production by CD4⁺ and CD8⁺ cells was assessed in vitro, while IFN-γ-producing cells were enumerated in CD4^? CD8^? cell fractions. Overall, our results showed that (i) antigen-specific secretion of IFN-γ protein in vitro by CD4⁺ cells occurred only in healing infection; (ii) IL-4- and IL-10-producing CD4⁺ cells would expand in nonhealer mice in the face of high levels of circulating IFN-γ, likely released by CD4^? CD8^? lymphocytes; (iii) a finely regulated IFN-γ production correlated in the healer mice with IL-12 mRNA detection, and IL-12 was required in vitro for yeast-induced development of IFN-γ-producing CD4⁺ cells. Although the mutually exclusive production of IL-4/IL-10 and IFN-γ by early CD4⁺ cells may be the major discriminative factor of cure and noncure responses in candidiasis, IL-12 rather than IFN-γ production may be an indicator of Th1 differentiation.     

Cyclosporin A increases IFN-γ production by T cells when co-stimulated through CD28

Despite its calcineurin-inhibiting properties, cyclosporin A (CsA) can not inhibit IL-2 production when T cells are co-stimulated by CD80/CD86 on the antigen-presenting cells. We studied the in vitro effect of CsA on IFN-γ production. Anti-CD3 monoclonal antibody (mAb) was used as the primary stimulus for activation of purified human T cells. A stimulating anti-CD28 mAb, or CD80 or CD86 on stably transfected P815 cells, provided the co-stimulatory signal. IL-2 production was hardly affected by CsA under these stimulating conditions, while IFN-γ (at the protein and mRNA level) was markedly stimulated by CsA. The use of anti-CD3 or phorbol 12-myristate 13-acetate with ionomycin as the primary stimulus, together with co-stimulation through either CD28 or CD2 using transfectants with the appropriate ligands, allowed us to demonstrate that the resistance of IFN-γ production to inhibition by CsA required both CD3 and CD28 triggering. Inhibition of IL-10 production, and to a lesser degree of IL-4 production, by CD4⁺ cells was responsible for the enhancement of IFN-γ production in the presence of CsA. In conclusion, IFN-γ production by CD28-co-stimulated CD4⁺ T cells is resistant to inhibition by CsA and can even be facilitated by CsA as a result of removing a negative regulatory signal which is mainly IL-10 mediated. This finding might have implications for immunosuppressive strategies based upon the use of CsA.     

Is TGFβ as an anti-inflammatory cytokine required for differentiation of inflammatory TH17 cells?

T-Helper 17 (T_H17) cells are a CD4⁺ T_H subset that plays a critical role in the pathophysiology of inflammatory disorders, especially chronic forms. It seems that the derivation of T_H17 cells from their precursors take place in inflammatory microenvironment. The role of transforming growth factor (TGF)-β as an anti-inflammatory cytokine in T_H17 cell differentiation is controversial. To address some of the discrepancies that exist among different studies, this study was undertaken to more clarify the TGFβ role in human T_H17 cell differentiation. Here, CD4⁺ T-cells were isolated from peripheral blood samples and cultured in X-VIVO 15 serum-free medium. Purified cells were then treated with different combinations of polarizing cytokines (interleukin [IL]1-β, -6, and -23, with or without TGFβ), neutralizing anti-interferon (IFN)-γ and anti-IL-4 antibodies and polyclonal stimulators anti-CD3 and -CD28 antibodies, and then analyzed for IL-17, IFNγ, Foxp3, and CD25 expression by flow cytometry and for release of IL-17, -21, -22, and -10 into culture media by ELISA. The effects of selective inhibition of TGFβ signaling pathway on T_H17 cell polarization were also determined by using small molecules SB-431542 and A83-01. The current study found that a combination of pro-inflammatory cytokines, including IL-1β, -6, and -23, but not TGFβ, could be used as a cytokine combination to induce development of human T_H17 cells. It was also shown that TGFβ acted as a negative regulator in this regard and also led to reduced IL-17 and IL-22 production while inducing Foxp3 expression. Indeed, blocking of TGFβ signaling pathways by selective inhibitors up-regulated T_H17 cell differentiation. From the data here, we concluded that TGFβ down-regulates human T_H17 cell differentiation and that a presence of pro-inflammatory cytokines (along with IFNγ and IL-4 neutralizing antibodies) is sufficient for optimal differentiation of human T_H17 cells.     

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.