Differential expression of cytokine receptors accounts for an important regulatory mechanism in differentiation of Th1/Th2 subsets. Here, we report that human Th0 and Th2 clones constitutively express transcripts for the IFN-gammaR beta-chain, whereas mRNA for this signaling component of the IFN-gamma receptor is absent in Th1 clones. Activation of T cell clones, however, resulted in a transient induction or enhancement of IFN-gammaR beta-chain mRNA expression in Th1 clones and Th0/Th2 clones, respectively. IL-12-mediated Th1 cell differentiation of naive CD4+, CD45RA+ cord blood T cells, which constitutively express IFN-gammaR beta-chain mRNA, resulted in a loss of expression of this cytokine receptor chain after 6 to 12 days of culture. In contrast, Th2 populations, differentiated from CD4+, CD45RA+ cord blood T cells in the presence of IL-4, continued to express high levels of IFN-gammaR beta-chain transcripts. The loss of IFN-gammaR beta-chain expression in Th1 populations was accompanied by a failure of IFN-gamma to induce the expression of the IFN-gamma-inducible gene, IFN response factor-1, whereas IFN-gamma was effective in inducing IFN response factor-1 mRNA expression in Th0 and Th2 cells. These results indicate that down-regulation of the IFN-gammaR beta-chain correlates with impaired IFN-gamma-induced signaling in Th1 cells. Finally, Th2 populations, generated in the presence of both IL-4 and IFN-gamma, expressed levels of IFN-gammaR beta-chain transcripts similar to those produced by cells differentiated in the presence of IL-4 only, demonstrating that IFN-gamma does not modulate the expression of its receptor. Together, these data indicate that human Th0/Th2 and Th1 subsets, respectively, can be distinguished based on the expression of the IFN-gammaR beta-chain.

In this study it is shown that both membrane-bound and soluble forms of signaling lymphocytic activation molecule (SLAM) induce proliferation and Ig synthesis by activated human B cells. Activated B cells express the membrane-bound form of SLAM (mSLAM), the soluble (s) and the cytoplasmic (c) isoforms of SLAM, and the expression levels of mSLAM on B cells are rapidly upregulated after activation in vitro. Importantly, recombinant sSLAM and L cells transfected with mSLAM efficiently enhance B cell proliferation induced by anti-μ mAbs, anti-CD40 mAbs or Staphylococcus aureus Cowan I (SAC) in the presence or absence of IL-2, IL-4, IL-10, IL-12, or IL-15. sSLAM strongly enhances proliferation of both freshly isolated B cells and B cells derived from long-term in vitro cultures, indicating that SLAM acts not only during the initial phase of B cell activation but also during the expansion of preactivated B cells. In addition, sSLAM enhances production of IgM, IgG, and IgA by B cells activated by antiCD40 mAbs. SLAM has recently been shown to be a high affinity self-ligand, and the present data suggest that signaling through homophilic SLAM–SLAM binding during B–B and B–T cell interactions enhances the expansion and differentiation of activated B cells.

The expression and function of the novel signaling lymphocytic activation molecule (SLAM) are described. SLAM is present on immature thymocytes, CD45R0(high) memory T cells, T cell clones, CD56(+) T cells, EBV-transformed B-cell lines and on a proportion of B cells. SLAM is rapidly induced on naive T cells, TCR gamma delta(+) T cells, and B cells following activation. Engagement of SLAM by the agonistic anti-SLAM mAb A12 resulted in proliferation of T cell clones and Ag- or PHA-activated peripheral blood T cells, mAb A12-induced T cell proliferation is independent of IL-2 and sensitive to inhibition by cyclosporin A. The extent of the anti-SLAM-induced proliferation was related to the activation state of the T cells, since fully rested T cell clones failed to proliferate in response to mAb A12 stimulation, despite unchanged SLAM expression on their surface. Ligation of SLAM on activated peripheral T cells or T cell clones by mAb A12 induced IFN-gamma production in the absence of other exogenous stimuli, even in allergen-specific Th2 clones. These data indicate that stimulation via SLAM reverses the cytokine production profile of Th2 clones to a Th0 phenotype, whereas it further polarizes cytokine production by Th1 clones. Thus, SLAM is a novel receptor that mediates IL-2-independent expansion of activated T cells during immune responses, while concomitantly directing these proliferating cells to a Th0/Th1 pathway.

In the present study the expression and function of signaling lymphocytic activation molecule (SLAM) in lymphocytes from patients with rheumatoid arthritis (RA) were investigated. The expression levels of SLAM were significantly up-regulated on synovial fluid and synovial tissue T cells from patients with RA compared with peripheral blood T cells from the same patients or from healthy volunteers. In addition, the expression of SLAM on peripheral blood B cells from patients with RA was elevated compared with that in healthy volunteers. SLAM+ T cells in synovial fluid coexpressed CD45RO and demonstrated decreased expression of CD27, indicative of a primed phenotype. In addition, the activation state of SLAM+ T cells was enhanced, as judged by increased expression of CD25, CD28, CD69, and CD95 on these cells. Interestingly, SLAM expression on activated CD4+ and CD8+ T cells from both patients and healthy individuals could be down-regulated by IL-10, which has been previously shown to function as an anti-inflammatory molecule in rheumatoid synovium. Furthermore, anti-SLAM mAbs increased the production of IL-10, IFN-gamma, and TNF-alpha by in vitro activated synovial fluid mononuclear cells, supporting the idea that signaling through SLAM may play a role in the regulation of synovial inflammation in patients with RA. Given the fact that SLAM was recently shown to be a high affinity self ligand, our data suggest that synovial T cells may stimulate their own cytokine production through homophilic SLAM-SLAM interactions.

Following the initial events of T cell activation, triggered by binding of specific peptide-MHC complex to the TCR for antigen and engagement of costimulatory molecules, a number of activation molecules are expressed on the cell surface. Many of these molecules regulate T cell function, T-T cell interactions and the interaction of T cells with other cells. One such molecule is SLAM, a multifunctional 70 kDa glycoprotein member of the Ig superfamily with multiple isoforms. SLAM is rapidly induced on naive T cells and B cells following activation. Engagement of SLAM by a specific antibody (mAb A12) results in IL-2-independent T cell expansion and induction/up-regulation of IFN-gamma by activated T cells, including Th2 cells. SLAM was found to be a high-affinity self-ligand mediating molecular and cellular homophilic interactions. In this review we discuss SLAM as a receptor involved in T cell expansion and in directing immune responses to a Th0-Th1 pathway.

The effects of a novel cytokine FLK2/FLT3 ligand (FL) on human fetal bone marrow-derived CD34+CD19+ pro-B cells were analyzed in a stromal- cell-independent, serum-deprived culture system. FL, like interleukin-3 (IL-3), synergized with IL-7 in promoting pro-B cell growth, and differentiation of these cells into CD34-CD19+clgM+slgM- pre-B cells, whereas a small proportion of these cells even differentiate into more mature slgM+ B cells. In contrast, KIT ligand (KL) and granulocyte- macrophage colony-stimulating factor (GM-CSF) were ineffective in promoting IL-7-dependent pro-B cell growth and differentiation. Maximal levels of pro-B cell expansion, generally resulting in 15- to 30-fold increases in cellularity, were obtained in cultures supplemented with optimal doses of FL + IL-7 + IL-3. The addition of mouse bone marrow stromal cells further enhanced the proliferation and differentiation of pro-B cells obtained in the presence of these three cytokines. Under these conditions, cultures could be maintained for more than 4 weeks, and in general 40- to 50-fold increases in cell numbers were observed by 3 weeks of culture. The percentages of clgM+ and slgM+ B cells increased 1.5- to 3-fold and 2-fold, respectively, suggesting that stromal cells may provide additional costimulatory signals for human B- cell growth and differentiation that are different from IL-7, IL-3, and FL. Collectively, our results indicate that FL, in contrast to KL, strongly promotes long-term expansion and differentiation of human pro- B cells in the presence of IL-7 or in combination of IL-7 and IL-3, which is a novel property of this hematopoietic growth factor.

Human CD4+ T cells, activated by allogeneic monocytes in a primary mixed lymphocyte reaction in the presence of exogenous interleukin (IL) 10, specifically failed to proliferate after restimulation with the same alloantigens. A comparable state of T cell unresponsiveness could be induced by activation of CD4+ T cells by cross-linked anti-CD3 monoclonal antibodies (mAbs) in the presence of exogenous IL-10. The anergic T cells failed to produce IL-2, IL-5, IL-10, interferon gamma, tumor necrosis factor alpha, and granulocyte/macrophage colony-stimulating factor. The IL-10-induced anergic state was long-lasting. T cell anergy could not be reversed after restimulation of the cells with anti-CD3 and anti-CD28 mAbs, although CD3 and CD28 expression was normal. In addition, restimulation of anergized T cells with anti-CD3 mAbs induced normal Ca2+ fluxes and resulted in increased CD3, CD28, and class II major histocompatibility complex expression, indicating that calcineurin-mediated signaling occurs in these anergic cells. However, the expression of the IL-2 receptor alpha chain was not upregulated, which may account for the failure of exogenous IL-2 to reverse the anergic state. Interestingly, anergic T cells and their nonanergic counterparts showed comparable levels of proliferation and cytokine production after activation with phorbol myristate acetate and Ca2+ ionophore, indicating that a direct activation of a protein kinase C-dependent pathway can overcome the tolerizing effect of IL-10. Taken together, these data demonstrate that IL-10 induces T cell anergy and therefore may play an important role in the induction and maintenance of antigen-specific T cell tolerance.

The development of CD4+ T helper (Th) type 1 and 2 cells is essential for the eradication of pathogens, but can also be responsible for various pathological disorders. Therefore, modulation of Th cell differentiation may have clinical utility in the treatment of human disease. Here, we show that interleukin (IL) 12 and IL-4 directly induce human neonatal CD4- T cells, activated via CD3 and CD28, to differentiate into Th1 and Th2 subsets. In contrast, IL-13, which shares many biological activities with IL-4, failed to induce T cell differentiation, consistent with the observation that human T cells do not express IL-13 receptors. Both the IL-12-induced Th1 subset and the IL-4-induced Th2 subset produce large quantities of IL-10, confirming that human IL-10 is not a typical human Th2 cytokine. Interestingly, IL-4-driven Th2 cell differentiation was completely prevented by an IL-4 mutant protein (IL-4.Y124D), indicating that this molecule acts as a strong IL-4 receptor antagonist. Analysis of single T cells producing interferon gamma or IL-4 revealed that induction of Th1 cell differentiation occurred rapidly and required only 4 d of priming of the neonatal CD4+ T cells in the presence of IL-12. The IL-12-induced Th1 cell phenotype was stable and was not significantly affected when repeatedly stimulated in the presence of recombinant IL-4. In contrast, the differentiation of Th2 cells occurred slowly and required not only 6 d of priming, but also additional restimulation of the primed CD4+ T cells in the presence of IL-4. Moreover, IL-4-induced Th2 cell phenotypes were not stable and could rapidly be reverted into a population predominantly containing Th0 and Th1 cells, after a single restimulation in the presence of IL-12. The observed differences in stability of IL-12- and IL-4-induced human Th1 and Th2 subsets, respectively, may have implications for cytokine-based therapies of chronic disease.

In the present study, it is shown that a human interleukin (IL)-4 mutant protein (IL-4.Y124D) acts as a potent IL-4 and IL-13 receptor antagonist. Human (h) IL-4.Y124D efficiently inhibits both IL-4- and IL-13-induced IgE production in vitro. In addition, hIL-4.Y124D strongly inhibits ongoing human IgE synthesis in SCID-hu mice. These inhibitory effects are specific, since human IgG levels were not significantly affected. These results confirm the notion that the IL-4 and IL-13 receptor share a common component, which is required for signal transduction. In addition, they show that relatively large antagonistic polypeptides, such as hIL-4.Y124D have potential clinical utility in reducing IgE-mediated allergic diseases.

The induction of anergy in T cells is believed to be the result of triggering of the TCR in the absence of adequate costimulation mediated through the interaction of CD28 and its ligands, CD80 and CD86. Here, we demonstrate that stimulation of human group I allergen in Dermatophagoides pteronyssinus extract (Der p 1)-specific CD4+ Th2-like T cell clones with Der p 1-derived peptides in the absence of professional APC results in a state of nonresponsiveness. The induction of anergy occurred despite the expression of high levels of CD28, CD80, and CD86 on the surface of the T cell clones and was not prevented by the addition of anti-CD28 mAb. The anergic, Der p 1-specific, Th2 cells failed to mobilize calcium from intracellular stores, to proliferate, and to produce IL-2, IL-4, IL-13, GM-CSF, and TNF-alpha following optimal stimulation with Der p 1-derived peptide and autologous APC. However, they mobilized intracellular calcium following stimulation with Ca(2+)-ionophore and produced all of the above cytokines, including IFN-gamma, when stimulated with phorbol ester and Ca2+ ionophore. These results indicate that the anergic T cell clones are capable of responding to signals circumventing the TCR/CD3 complex activation pathway. In contrast to T cell clones optimally activated with peptide and APC, anergic T cells failed to induce IgG4 and IgE synthesis when cocultured with B cells, even in the presence of exogenous IL-4 or IL-13. Anergic T cells expressed normal levels of CD40L, suggesting that their inability to help in Ig production by B cells is due to conditions other than a lack of expression of this molecule. Finally, exogenous IL-2 restored the helper function of anergic Th2 T cells for IgE production by B cells, which was greatly enhanced by the addition of IL-4 or IL-13. These data suggest that induction of anergy in allergen-specific Th2 T cells by allergen-derived peptides may play an important role in the successful desensitization of allergic patients.