The FASEB Journal express article 10.1096/fj.99-0874fje. Published online July 24, 2000.
A molecular basis for T cell suppression by IL-10: CD28associated IL-10 receptor inhibits CD28 tyrosine phosphorylation and phosphatidylinositol 3-kinase binding Cezmi A. Akdis, Andrea Joss, Mübeccel Akdis, Alexander Faith, and Kurt Blaser Swiss Institute of Allergy and Asthma Research, Davos, Switzerland Corresponding author: Cezmi A. Akdis, Swiss Institute of Allergy and Asthma Research, Obere Strasse 22, CH-7270 Davos, Switzerland. E-mail:
[email protected] ABSTRACT 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 antiCD28 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. Key Words: anergy • tolerance • costimulation • antigen-specific responses • natural antigen exposure
T
he immune system has the potential to recognize any foreign protein or microorganism. Suppression of specific immune response to antigenic load of dietary and airborne antigens and induction of tolerance are normal regulatory events in the homeostasis of the immune system (1-3). Specific activation of T cells requires stimulation through the T cell receptor (TCR) and a costimulatory signal generated by the engagement of multiple cell surface receptors with their ligands (2,3). A major costimulatory signal is delivered to the T cells by the interaction of CD28 with
molecules of the B7 family (CD80, CD86) displayed by antigen-presenting cells (APC) (4,5). TCR stimulation without costimulatory signals induces anergy in T cells (6-9). Anergy is a state of immune suppression characterized by abolished proliferative and cytokine responses. It is actively generated by a number of molecular events and can be reversed by certain cytokines (3,8-12). Although the molecular mechanisms have not been elucidated so far, specific T cell anergy induced by autocrine action of IL-10 has been demonstrated during natural exposure to antigens and during specific immunotherapy and various diseases in human and mice (12-17). The costimulatory signal induced by complexing CD28 with specific mAb or by interaction with B7 counter-receptors enhances the antigen-dependent T cell proliferation and cytokine production (5-7). In contrast, anergy was elicited in T cells by blocking the CD28/B7mediated cellular interaction, as shown in vitro and in vivo (6,7). Investigation of the immunological mechanisms of specific immunotherapy (SIT) in allergy to bee venom (BV) and after bee stings in healthy individuals provides a human model well-suited to study the molecular basis of antigen-specific T cell tolerance (1214). The present study demonstrates the mechanism of IL-10-induced T cell suppression. This plays a role in T cell inactivation during specific allergen immunotherapy and antigenic challenge by exposure to a high number of bee stings. IL-10 inhibits CD28 signaling pathway in T cells by inhibiting CD28 tyrosine phosphorylation. Consequently, it blocks the binding of the downstream signaling molecule phosphatidylinositol 3-kinase (PI3-K) to CD28. Coprecipitation experiments revealed a physical association between the IL-10 receptor (R) and CD28 in T cells upon activation that elucidates the mechanism as to why IL-10-mediated T cell suppression specifically operates through the CD28 signal transduction. MATERIALS AND METHODS Study population Nine BV allergic individuals (mean age 32 years) with a history of severe systemic anaphylactic reactions of grade III-IV (18) after a bee sting were studied. All patients demonstrated positive intracutaneous reactions to honey BV (ALK, Horsholm, Denmark) at a concentration of 2 kU/l as estimated by CAP immunoassay (Pharmacia Diagnostics AG, Uppsala, Sweden). Under intensive care conditions 0.1, 1, 10 and 20 µg of BV (ALK) were administered subcutaneously in the upper arms at 30 min intervals and then 30 and 50 µg at 60 min intervals, reaching a cumulative dose of 111.1 µg. On day 7, two booster injections of 50 µg were administered, followed by 100 µg boosters given at 4 wk intervals (19). Blood samples were taken before BV-SIT and 1, 7, and 28 days after starting BV-SIT. Four healthy nonallergic BV hyperimmune subjects (beekeepers, mean age 42 years) who were stung >20 times by bees 1 wk before were included in the study. Blood samples from 32 healthy individuals were used for signal transduction analysis. T cell cultures
PBMC were isolated by Ficoll (Biochrom, Berlin, Germany) density gradient centrifugation of peripheral venous blood. Cells were washed three times and resuspended in RPMI 1640 medium supplemented with 1 mM sodium pyruvate, 1% MEM nonessential amino acids and vitamins, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-ME (all from Life Technologies), and 10% heatinactivated FCS (12,13). Human T cell clones and CD45RO+ T cells were generated or purified as described (8,20). PBMC of sensitized individuals (105 cells/well in a 96-well flat-bottom plates, Costar, Cambridge, U.K.) were stimulated with the antigens: 10 µg/ml bee venom phospholipase A2 (Boehringer Mannheim, Mannheim, Germany), 10 µg/ml Derp1 (Allergopharma KG, Reinbeck, Germany), 1 µg/ml purified protein derivative of M. tuberculosis (PPD), and 0.1 U/ml tetanus toxoid (TT) (both from the Swiss Serum and Vaccine Institute Bern, Switzerland), and 1 ng/ml recombinant SEB (a kind gift from J. Lamb, Edinburg, U.K.). The proliferative responses were measured by 3H-thymidine incorporation after 5 days as described (12,13). Parallel cultures were expanded in medium supplemented with a mixture of IL-2 (25 U/ml) and IL-4 (25 ng/ml) (both from Novartis, Basel, Switzerland). After 12 days, cells were washed three times with PBS and 106 cells were restimulated with the same antigen at the same concentrations as before, in the presence of 106 autologous, 3000 Rad irradiated PBMC in 24-well tissue culture plates, in duplicates (12). T cell clones, CD45RO+ T cells and PBMC were stimulated with 10 µg/ml plate bound anti-CD28 (15E8, Red Cross, Amsterdam, The Netherlands) and anti-CD3 (OKT3, Ortho Diagnostic Systems Inc., Raritan, N.J.). 3H-Thymidine incorporation was measured after 3 days. IL-10 (PeproTech Inc., Rocky Hill, N.J.) was used at 50 ng/ml and neutralizing anti-IL-10 and blocking anti-IL-10R mAbs (both from DNAX Research Institute, Palo Alto, Calif.) were used at 10 µg/ml (21). Quantification of cytokines IL-4, IL-5, IL-10, IL-13, and IFN-γ were determined by sandwich ELISAs as described (8,12,13,20). The sensitivity of the IFN-γ ELISA was 10 pg/ml (mAbs and IFN-γ standard were gifts from Dr. S. S. Alkan, Novartis, Basel, Switzerland). The sensitivity of IL-4 ELISA was 20 pg/ml (mAbs and the IL-4 standard were provided by Dr. C.H. Heusser, Novartis, Basel). The detection limit of the IL-5 ELISA was 50 pg/ml (mAbs and IL-5 standard were from PharMingen, St. Louis, Mo.). The sensitivity of the IL-10 ELISA was 50 pg/ml (mAbs and IL-10 standard were from PharMingen). The detection limit of IL-13 ELISA was 100 pg/ml of IL-13 (mAbs and IL-13 standard were from PharMingen). Flow cytometric analysis TCR number counting on T cell surface was done by indirect immune fluorescence staining with 50 µg/ml anti-CD3 mAb, followed by FITC-conjugated anti-mouse IgG (Dako A/S, Glostrup, Denmark) (22). Beads coated by known numbers of mouse mAbs were used as standards (Qifikit, Dako A/S). Stained cells were determined by an Epics
XL (Coulter Corp., Hialeah, Fla.). Triggered TCRs were calculated by subtracting TCR numbers of stimulated cells from TCR numbers of unstimulated cells, measured after 8 h. For analysis of IL-10R and CD28 expression, 5 × 104 cells were stained with 50 µg/ml of anti-IL-10Rα mAb, IL-10Rβ Ab (a kind gift from Sydney Pestka, Robert Wood Johnson Medical School, N.J.) or anti-CD28 mAb for 30 min and washed with 2% FCS containing PBS. FITC-conjugated anti-mouse Ig's and FITC-conjugated anti-rabbit Ig’s were the second Abs used for 30 min. Stained cells were fixed in 2% paraformaldehyde. The controls were mouse IgG2a, mouse IgG1 and rabbit IgG. Immunoprecipitation and Western blotting analysis Cells were lysed in 0.5% Triton X-100 containing leupeptin, pepstatin A, and aprotinin (each at 10 µg/ml), sodium orthovanadate (100 µM), EDTA (5 mM), and iodoacetamide (50 mM) (all from Sigma, St. Louis, Mo.). Lysates were incubated 45 min on ice, centrifuged for 10 min and supernatants were collected (8). Postnuclear lysates were precleared by 40 µg/ml isotype control mAb for 1 h, followed by Sepharose-protein G (Pharmacia, Uppsala, Sweden) for 1 h at 4oC. Immunoprecipitations were performed in precleared supernatants for 2 h at 4oC with anti-CD28 mAb, anti-IL-10R α chain mAb (Serotec, Oxford, U.K.), anti-p85 PI3-K (a kind gift from M. Thelen, Theodor Kocher Inst. Bern, Switzerland), anti-phosphotyrosine mAb (4G10, Upstate Biotechnology Inc. Lake Placid, NY) and anti-ZAP-70 mAb (Transduction Laboratories, Lexington, Ky.). Sepharose-protein G was added for 2 h at 4oC (Sigma). Immunoprecipitates were washed with cold lysis buffer, boiled in Laemmli sample buffer, then subjected to electrophoresis on Tris-glycine gels (Novex AG, Frankfurt, Germany). Immunoblotting was performed on nitrocellulose membranes (Amersham Life Science, Buckinghamshire, U.K.) by antiphosphotyrosine, anti-PI3-K p85, anti-IL-10R and visualized by chemiluminescence detection system (ECL, Amersham Pharmacia Biotech Limited, Buckinghamshire, U.K.). Experiments were repeated with different anti-CD28 mAb from PharMingen (Hamburg, Germany) and anti-IL-10R α chain mAb from DNAX Research Institute (Palo Alto, Calif.) for specificity control. PHA (Sigma) was used at 3 µg/ml to activate T cells for IL-10R-CD28 association experiments. Statistics Results are shown as mean ± standard deviation. Stimulation index was calculated by dividing measurements of PLA-stimulated cultures by unstimulated cultures. Student's t test for paired samples was used for statistical analysis to compare results at different time points of immunotherapy. RESULTS IL-10-induced T cell tolerance in specific immunotherapy and natural high-dose antigen exposure
In both BV-SIT and healthy subjects who received high numbers of bee stings, high doses of antigen induce peripheral tolerance in T cells against the bee venom major antigen, the phospholipase A2 (PLA). As shown in Fig. 1A, the T cell proliferative response to PLA was significantly suppressed in parallel to IL-4, IL-5, IL-13, and IFN-γ production after 7 days (P
