IL‐10 induces T cell anergy in numerous mouse models and specific immunotherapy of allergy in humans. Here, we demonstrate that IL‐10 directly acts on T cells which are stimulated via CD28 by efficiently blocking proliferation and cytokine production. T cells tolerized by IL‐10 showed high viability and the unresponsive state was reversed by anti‐CD3 monoclonal antibody (mAb) stimulation and IL‐2, but not by anti‐CD28 mAb stimulation. Signal transduction via CD28 requires CD28 tyrosine phosphorylation and binding of phosphatidylinositol 3‐kinase. IL‐10 inhibited tyrosine phosphorylation of CD28; thus, the phosphatidylinositol 3‐kinase binding to CD28 was blocked. Consequently, IL‐10 inhibited the antigen‐induced secretion of both Th1 and Th2 cytokines, including IL‐2, IFN‐γ, IL‐4, IL‐5 and IL‐13. Furthermore, neutralization of endogenously produced IL‐10 significantly increased T cell proliferation and both Th1 and Th2 cytokine production in vitro. Using superantigen stimuli, T cell suppression by IL‐10 was merely induced at low doses when co‐stimulation by CD28 was essential. Together, these data demonstrate that IL‐10 directly acts on the CD28 signaling pathway and this represents an important T cell suppression mechanism leading to anergy.

Specific immune suppression and induction of anergy in T cells are essential processes in regulation and circumvention of immune defense. IL‐10, a suppressor cytokine of T cell proliferative and cytokine responses, plays a key regulatory role in tolerizing exogenous antigens during specific immunotherapy and natural exposure. The present study demonstrates that IL‐10 induces T cell suppression by blocking the CD28 costimulatory signal. T cell receptor counting and T cell proliferation studies by anti‐CD3 and anti‐CD28 stimulation in the presence or absence of IL‐10 revealed that IL‐10 only inhibits T cells stimulated by low numbers of triggered T cell receptors and that depend on CD28 costimulation. T cells receiving a strong signal by the T cell receptor alone and that do not require CD28 costimulation are therefore not affected by IL‐10. Coprecipitation experiments demonstrated that CD28 and the IL‐10 receptor are associated in activated T cells. IL‐10 inhibited CD28 tyrosine phosphorylation, the initial step of the CD28 signaling pathway. In consequence, phosphatidylinositol 3‐kinase p85 binding to CD28 was inhibited. Thus, IL‐10‐induced selective inhibition of the CD28 costimulatory pathway demonstrates a decisive mechanism in determining whether a T cell will contribute to an immune response or become anergic.

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Background: Induction of specific unresponsiveness (tolerance/anergy) in peripheral T cells and recovery by cytokines from the tissue microenvironment represent two key steps in specific immunotherapy (SIT) with whole allergen or antigenic T cell peptides. Methods: Antigen-specific T cell responses and molecular mechanisms of T cell inactivation were investigated during conventional SIT, T cell epitope peptide immunotherapy and natural exposure to bee venom in allergic and hyperimmune individuals. Results: T cell unresponsiveness, initiated by autocrine action of IL-10, is characterized by suppressed proliferative and cytokine responses. The unresponsive T cells can be reactivated by different cytokines that may mimic the microenvironmental cytokine influence. IL-10 initiates peripheral tolerance by blocking the CD28 costimulatory signal in T cells. Coprecipitation experiments reveal that upon stimulation CD28 and IL-10 receptor are physically associated in T cells. Accordingly, IL-10 binding to its receptor inhibits CD28 tyrosine phosphorylation, the initial step of the CD28 signaling pathway. This leads to inhibition of phosphatidylinositol 3-kinase p85 binding to CD28. IL-10 only affects T cells that receive a stimulation with low numbers of triggered T cell receptors and that require costimulatory signals by CD28. Conclusion: These data demonstrate the pivotal role of autocrine IL-10 and the interaction of its receptor with CD28 in the induction of T cell tolerance as an immunoregulatory mechanism controlling antigen-specific T cell responses.

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