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.     

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.     

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.     

The central nervous system environment controls effector CD4+ T cell cytokine profile in experimental allergic encephalomyelitis

In experimental allergic encephalomyelitis (EAE), CD4⁺ T cells infiltrate the central nervous system (CNS). We derived CD4⁺ T cell lines from SJL/J mice that were specific for encephalitogenic myelin basic protein (MBP) peptides and produced both Th1 and Th2 cytokines. These lines transferred EAE to naive mice. Peptide-specific cells re-isolated from the CNS only produced Th1 cytokines, whereas T cells in the lymph nodes produced both Th1 and Th2 cytokines. Mononuclear cells isolated from the CNS, the majority of which were microglia, presented antigen to and stimulated MBP-specific T cell lines in vitro. Although CNS antigen-presenting cells (APC) supported increased production of interferon (IFN)-γ mRNA by these T cells, there was no increase in the interleukin (IL)-4 signal, whereas splenic APC induced increases in both IFN-γ and IL-4. mRNA for IL-12 (p40 subunit) was up-regulated in both infiltrating macrophages and resident microglia from mice with EAE. We have thus shown that a Th1 cytokine bias within the CNS can be induced by CNS APC, and that IL-12 is up-regulated in microglial cells within the CNS of mice with EAE. Microglia may therefore control Th1 cytokine responses within the CNS.     

Differential regulation of Th1 responses and CD154 expression in human CD4+ T cells by IFN-alpha

Like interleukin (IL)-12, interferon (IFN)-alpha has been shown to play an important role in inducing human Th1 responses. Recent studies have shown that human Th1 responses driven by IL-12 are associated with enhanced expression of CD154. The present study examined the effects of IFN-alpha on CD154 expression in human CD4+ T cells, with special attention to the relationship with Th1 responses. Highly purified CD4+ T cells from healthy donors were stimulated with immobilized anti-CD3 with or without IFN-alpha and IL-12 in the complete absence of accessory cells. IFN-alpha suppressed CD154 protein and mRNA expression in CD4+ T cells at the initial phase of activation with immobilized anti-CD3, but enhanced it in the subsequent maturation phase irrespective of the presence of IL-12. By contrast, IFN-alpha by itself did not enhance IFN-gamma production or mRNA expression in CD4+ T cells in the absence of IL-12 even in the presence of stimulation with anti-CD28, but enhanced it in the presence of IL-12. Accordingly, IFN-alpha enhanced IL-12Rbeta2 mRNA expression in anti-CD3-stimulated CD4+ T cells. Neither IFN-alpha nor IL-12 influenced the stability of CD154 mRNA in anti-CD3-activated CD4+ T cells. These results indicate that IFN-alpha by itself enhances CD154 expression in CD4+ T cells independently of the induction of IFN-gamma mRNA expression. The data also suggest that the optimal induction of human Th1 responses by IFN-alpha might require the presence of IL-12 and that the induction of Th1 responses and CD154 expression in human CD4+ T cells might be regulated through different mechanisms.     

Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response

Mice with homologous disruption of the gene coding for either the p35 subunit or the p40 subunit of interleukin-12 (IL-12) and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in resistance to infection and the differentiation of functional CD4⁺ T cell subsets in vivo. Wild-type 129/Sv/Ev mice are resistant to infection with L. major showing only small lesions which resolve spontaneously within a few weeks and develop a type 1 CD4⁺ T cell response. In contrast, mice lacking bioactive IL-12 (IL-12p35^?/? and IL-12p40^?/?) developed large, progressing lesions. Whereas resistant mice were able to mount a delayed-type hypersensitivity (DTH) response to Leishmania antigen, susceptible BALB/c mice as well as IL-12-deficient 129/Sv/Ev mice did not show any DTH reaction. To characterize the functional phenotype of CD4⁺ T cells triggered in infected wild-type mice and IL-12-deficient mice, the expression of mRNA for interferon-γ (IFN-γ) and interleukin-4 (IL-4) in purified CD4⁺ lymph node cells was analyzed. Wild-type 129/Sv/Ev mice showed high levels of mRNA for IFN-γ and low levels of mRNA for IL-4 which is indicative of a Th1 response. In contrast, IL-12- deficient mice and susceptible BALB/c mice developed a strong Th2 response with high levels of IL-4 mRNA and low levels of IFN-γ mRNA in CD4⁺ T cells. Similarly, lymph node cells from infected wild-type 129 mice produced predominantly IFN-γ in response to stimulation with Leishmania antigen in vitro whereas lymph node cells from IL-12-deficient mice and susceptible BALB/c mice produced preferentially IL-4. Taken together, these results confirm in vivo the importance of IL-12 in induction of Th1 responses and protective immunity against L. major.     

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.     

CD40 ligand-positive CD8+ T cell clones allow B cell growth and differentiation

A fraction of activated CD8⁺ T cells expresses CD40 ligand (CD40L), a molecule that plays a key role in T cell-dependent B cell stimulation. CD8⁺ T cell clones were examined for CD40L expression and for their capacity to allow the growth and differentiation of B cells, upon activation with immobilized anti-CD3. According to CD40L expression, CD8⁺ clones could be grouped into three subsets. CD8⁺ T cell clones expressing high levels of CD40L (≥80% CD40L⁺ cells) were equivalent to CD4⁺ T cell clones with regard to induction of tonsil B cell proliferation and immunoglobulin (Ig) production, provided the combination of interleukin (IL)-2 and IL-10 was added to cultures. CD8⁺ T cell clones, with intermediate levels of CD40L expression (10 to 30% CD40L⁺ cells), also stimulated B cell proliferation and Ig secretion with IL-2 and IL-10. B cell responses induced by these CD8⁺ T cell clones were neutralized by blocking monoclonal antibodies specific for either CD40L or CD40. By contrast, CD40L^?? T cell clones (?5 % CD40L⁺ cells), only induced marginal B cell responses even with IL-2 and IL-10. All three clone types were able to activate B cells as shown by up-regulation of CD25, CD80 and CD86 expression. A neutralizing anti-CD40L antibody indicated that T cell-dependent B cell activation was only partly dependent on CD40-CD40L interaction. These CD40L^?? clones had no inhibitory effects on B cell proliferation induced by CD40L-expressing CD8⁺ T cell clones. Taken together, these results indicate that CD8⁺ T cells can induce B cell growth and differentiation in a CD40L-CD40-dependent fashion.     

Enhanced HIV expression during Th2-oriented responses explained by the opposite regulatory effect of IL-4 and IFN-γ on fusin/CXCR4

The human α-chemokine receptor fusin/CXCR4 is an important cofactor for entry of T lymphocyte-tropic HIV-1 strains. We investigated the possible regulatory role of T cell cytokine patterns on CXCR4 as well as HIV expression by using in vitro models of both secondary and primary immune responses. Antigen-specific memory CD4⁺ T cells infected with a T-tropic HIV-1 strain showed significantly higher CXCR4 and HIV-1 expression in Th0/2-oriented responses in comparison with Th1-oriented responses. Similarly, in naive CD4⁺ T cells activated in the presence of IL-4 or IL-12 and infected with the same T-tropic strain, IL-4 up-regulated whereas IL-12 down-regulated both CXCR4 and HIV-1 expression. The down-regulatory effect of IL-12 on CXCR4 expression was found to be dependent on its capacity to induce IFN-γ production. These observations can account for the higher risk of progression in HIV-1-infected individuals undergoing Th0/2-oriented immune responses.     

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.     

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.     

Diversion of CD4+ T cell development from regulatory T helper to effector T helper cells alters the contact hypersensitivity response

Cutaneous sensitization to reactive haptens and subsequent challenge results in a T cell-mediated response, contact hypersensitivity (CHS). Recent results from this laboratory have indicated that hapten sensitization induces two populations of reactive T cells: CD8⁺ T cells producing interferon (IFN)-γ which mediate the response and CD4⁺ T cells producing interleukin (IL)-4 and IL-10 which negatively regulate the magnitude and duration of the response. Since CD4⁺ T cell development to either IFN-γ- (Th1) or IL-4/IL-10- (Th2)-producing cells is dependent upon the cytokine environment during antigen priming, we hypothesized that CD4⁺ T cell induction in a Th1-promoting environment would not only alter the CD4⁺ T cell cytokine-producing phenotype but also the course of the CHS response. Administration of the Th1-promoting cytokine IL-12 during hapten sensitization resulted in a CHS response of greater magnitude following challenge and extended the duration of the response. In hapten-sensitized mice depleted of CD8⁺ T cells, treatment with IL-12 induced effector CD4⁺ T cells. Histological examination of challenged ear tissue from these mice indicated minimal edema and an acute mononuclear cell infiltration more typical of classical delayed-type hypersensitivity than CHS. Hapten-primed CD4⁺ T cells from IL-12 treated, sensitized mice produced IFN-γ, but not IL-4 in response to T cell receptor-mediated stimulation. Use of neutralizing anti-IFN-γ antibody indicated that IL-12 not only directly promoted Th1 development but also indirectly inhibited Th2 development through stimulation of IFN-γ production at the time of hapten sensitization. Overall, these results demonstrate that diversion of CD4⁺ T cell development to Th1 effector cells rather than to Th2 cells alters the efferent nature of CHS and removes a primary regulatory mechanism of the immune response.     

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.     

Role of CD40 antigen and interleukin-2 in T cell-dependent human B lymphocyte growth

In the present study, we examined the participation of CD40 ligand (L)-CD40 interaction in T cell-dependent B cell responses. To this end, purified B lymphocytes were cultured over irradiated CD4⁺ cloned T cells activated with immobilized anti-CD3 antibody. The anti-CD40 mAb 89 strongly blocked, in a specific fashion, both proliferation and Ig secretion of tonsil B cells. Interestingly, proliferation of surface (s)IgD⁺ B cell was significantly less inhibited by anti-CD40 than that of sIgD^? cells. Preactivated T cells induced B cells to grow and secrete immunoglobulins preferentially in response to IL-2. This contrasts with the CD40 system where B cells are essentially responsive to IL-4 and IL-10 but not to IL-2 alone. Collectively, these data indicate that CD40L-CD40 interaction plays an important role in IL-2-mediated T cell-dependent B cell responses. However, the activation of a subset of sIgD⁺ cells may be independent of this interaction.     

The induction of a protective response in Leishmania major-infected BALB/c mice with anti-CD40 mAb

A protective immune response to the intracellular parasite Leishmania major requires the development of a Th1 CD4⁺ T cell phenotype. We demonstrate herein that BALB/c mice, which normally develop a susceptible Th2 response to L. major infection, are protected when co-injected with an agonistic anti-murine CD40 mAb. Anti-CD40 mAb-mediated protection in this system was found to be T cell dependent, since it was not observed in C57BL/ 6 × 129 mice that were rendered T cell deficient (TCR β^–/– × TCR δ^–/–) and L. major susceptible. Anti-CD40 mAb stimulation of L. major-infected BALB/c mice was accompanied by increased IL-12 and IFN-γ production in draining lymphnodes, analyzed either by direct expression, or in an antigen-specific in vitro recall assay. The protective role of these cytokines was indicated by the finding that anti-CD40 mAb-mediated protection of L. major-infected BALB/c mice could be reversed by co-treating the animals with neutralizing anti-IL-12 and/or anti-IFN-γ mAb. Collectively, these data suggest that BALB/c mice develop a protective Th1 CD4⁺ T cell response to L. major infection when co-injected with anti-CD40 mAb. While the CD40-CD40L interaction has been previously shown to be vital in the control of murine Leishmaniasis, the current study establishes in vivo that anti-CD40 mAb treatment alone is sufficient to protect BALB/c mice from L. majorinfection and raises the possibility of utilizing this approach for vaccination strategies.     

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.     

Suppressive influences of IFN-α on IL-17 expression in human CD4+ T cells

We examined the direct effects of IFN-α on the development of Th17 with a system using immobilized anti-CD3, which permits activation of CD4+ T cells in the complete absence of accessory cells. Highly purified CD4+ T cells obtained from healthy donors were stimulated with immobilized anti-CD3 with or without IFN-α. IFN-α suppressed the production of IL-17 of immobilized anti-CD3-stimulated CD4+ T cells in a dose–response manner. Accordingly, IFN-α inhibited IL-17 mRNA expression in immobilized anti-CD3-stimulated CD4+ T cells. IFN-α did not affect the production of TGF-β or IL-6, but inhibited RORC mRNA expression of anti-CD3-stimulated CD4+ T cells. These results indicate that IFN-α suppresses IL-17 expression and Th17 differentiation through down-regulation of RORC mRNA expression. It is therefore suggested that these effects might play a role in the mode of action of IFN-α in the treatment of various inflammatory diseases.     

Role of the CD40-CD40 ligand interaction in CD4+ T cell contact-dependent activation of monocyte interleukin-1 synthesis

Most studies of the induction of cytokine synthesis in monocytes have employed an exogenous triggering agent such as lipopolysaccharide. However, in nonseptic inflammatory responses (e.g. rheumatoid arthritis) monocyte activation occurs as a result of T cell-generated signals. In previous reports, we and others have demonstrated that contact-dependent T cell-generated signals are capable of contributing to macrophage activation. We have shown that plasma membranes from anti-CD3 activated purified peripheral CD4⁺ T cells (Tm^A) but not from resting CD4⁺ cells (Tm^R) induce monocytes to synthesize interleukin (IL)-1 in the absence of co-stimulatory cytokines. Studies to determine the expression kinetics of the molecule(s) unique to activated CD4⁺ T cells which interact with monocytes to induce IL-1 revealed that optimal expression occurred at 6 h post activation. This matched the previously reported kinetics of expression of CD40 ligand (CD40L) on activated peripheral T cells, implicating the CD40-CD40L interaction as a candidate for the initiator of the IL-1 signaling event. The ability of Tm^A to induce IL-1 synthesis in resting monocytes could be markedly reduced by addition of a monoclonal anti-CD40L antibody, 5c8. In addition, a monoclonal anti-CD40 IgM (BL-C4) proved dramatic in its ability to induce resting monocytes to synthesize IL-1. In summary, these results demonstrate that the CD40-CD40L interaction provides a critical component of CD4⁺ T cell contact-dependent activation of monocyte IL-1 synthesis.     

Differential effects of interleukin-12 on the development of naive mouse CD4+ T cells

The influence of interleukin (IL)-12 and IL-4 on the differentiation of naive CD4⁺ T cells was studied in an accessory cell-free in vitro system. Dense CD4⁺ T cells were purified from unimmunized mice and activated using immobilized anti-CD3 monoclonal antibodies (mAb) in the presence of IL-4, IL-12, or a combination of both cytokines, and restimulated after 6 days by re-exposure to anti-CD3-coated culture wells. T cells initially activated in the presence of IL-4 produced substantial amounts of IL-4 and trace amounts of interferon (IFN)-γ after restimulation at day 6 with plate-bound anti-CD3 mAb. By contrast, T cells primed in the presence of IL-12 produced high levels of IFN-γ and only minimal amounts of IL-4, thus indicating that IL-12 and IL-4 by acting directly on stimulated naive CD4⁺ T cells support the development of T_H1 and T_H2 cells, respectively. When naive CD4⁺ T cells were stimulated in the presence of IL-12 together with IL-4 in comparable concentrations, the effect of IL-12 on T_H1 differentiation was largely inhibited by IL-4. On the other hand, IL-12 exerted no inhibitory effect on IL-4-induced T_H2 differentiation but rather enhanced the production of IL-4 after restimulation of the respective T cells. Decreasing amounts of IL-4 in combination with a high level of IL-12 led to an increasing production of IFN-γ by the emerging T cells and, simultaneously, to a relatively high production of IL-4. These data were confirmed by time-course experiments which revealed that the delayed addition of IL-4 to IL-12-primed T cell cultures resulted in a gradual restoration of IFN-γ production whereas in parallel the secretion of IL-4 was not reduced over a wide period of delay (6–72 h). These results, therefore, demonstrate that (a) IL-4 dominates the effect of IL-12, (b) IL-12 promotes the development of T_H1 cells; however, in the presence of IL-12 and relatively high levels of IL-4 also the development of T_H2-like cells is slightly but significantly enhanced by IL-12, and (c) high amounts of IL-12 in combination with relatively low levels of IL-4 give rise to a T cell population that upon rechallenge exhibited a cytokine profile resembling that of T_H0 cells.