HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 14/12/1666 99-0874fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by AKDIS, C. A. Articles by BLASER, K. Search for Related Content PubMed PubMed Citation Articles by AKDIS, C. A. Articles by BLASER, K.

A molecular basis for T cell suppression by IL-10: CD28-associated IL-10 receptor inhibits CD28 tyrosine phosphorylation and phosphatidylinositol 3-kinase binding

Swiss Institute of Allergy and Asthma Research (SIAF), CH-7270 Davos, Switzerland

¹Correspondence: Swiss Institute of Allergy and Asthma Research (SIAF), Obere Strasse 22, CH-7270 Davos, Switzerland. E-mail: akdisac{at}siaf.unizh.ch

SPECIFIC AIM

THE PRESENT STUDY demonstrates that interleukin 10 (IL-10) induces T cell tolerance in natural high-dose antigen exposure and specific immunotherapy by blocking the CD28 pathway, and thus inhibits T cells stimulated by low numbers of triggered T cell receptors that require costimulation.

PRINCIPAL FINDINGS

1. IL-10 induces T cell tolerance in natural high-dose antigen exposure and specific immunotherapy
PeripheralT cell tolerance is induced in subjects naturally receiving high antigen loads as well as in specific immunotherapy of allergy. During the course of specific immunotherapy, the T cell proliferative response to major antigen is significantly suppressed in parallel to IL-4, IL-5, IL-13, and interferon (IFN-) production. Simultaneously with the specific suppression of the T cells, the antigen-induced secretion of IL-10 increased within 7 days. The same features of T cell suppression with increased IL-10 production were detected in individuals who received high doses of antigens (i.e., multiple bee stings in healthy individuals). By neutralization of IL-10, the abrogated specific T cell proliferation and production of both Th1 (IFN-) and Th2 (IL-4, IL-5, IL-13) cytokines were fully reestablished, indicating that IL-10 is actively involved in the induction of specific T cell tolerance. Together, these data demonstrate the pivotal role of IL-10 in the elicitation of an unresponsive state in T lymphocytes, thereby representing an essential regulatory event in tolerating high doses of exogenously administered antigen.

2. IL-10 inhibits T cells only if costimulation by CD28 is required
The molecular mechanisms of T cell suppression by IL-10 was further investigated in antigen-specific PBMC cultures and purified CD45RO⁺ T cells and T cell clones. IL-10 inhibited the proliferative T cell response in PBMC to various antigens and the superantigen staphylococcal enterotoxin B. However, IL-10 did not affect the proliferative responses of T cells that were stimulated by anti-CD3. In contrast, IL-10 significantly inhibited the anti-CD28-stimulated proliferation. An analysis of T cell receptor (TCR) numbers on T cells demonstrated the essential requirement for costimulation in T cell activation and its relation to the number of triggered TCRs (Fig. 1A ). IL-10 inhibited T cell proliferation within a certain range of triggered TCRs that require costimulation. T cells that were stimulated by different concentrations of anti-CD3 and a constant amount of anti-CD28 showed that low numbers of triggered TCRs required CD28 costimulation. Thus, IL-10 suppressed only those T cells that had low numbers of TCRs triggered and required CD28 for proliferation.

View larger version (32K): [in this window] [in a new window]   Figure 1. IL-10 inhibits CD28 signaling in T cells. A) Human T cell clones were stimulated by different concentrations of soluble anti-CD3, ranging from 1 to 0.007 µg/ml and a constant amount of 2 µg/ml anti-CD28, in the presence or absence of IL-10. TCR surface expression and triggered TCR numbers were determined after 8 h. T cell proliferation was measured by 3H-thymidine incorporation on day 3. B) IL-10 inhibits tyrosine phosphorylation of CD28. PBMC or purified T cells were stimulated for 10 min with immobilized anti-CD28 mAb in the presence or absence of IL-10. Cell lysates were immunoprecipitated (IP) with anti-CD28 mAb, immunoblotted, and detected with anti-phosphotyrosine (ptyr) mAb. C) IL-10 inhibits CD28 binding to PI3-K. Cell lysates were immunoprecipitated by anti-CD28 immunoblotted and detected with anti-PI3-K-p85. The blocking effect of IL-10 on CD28–PI3-K association was hindered by anti-IL-10R mAb, which blocks and does not stimulate the IL-10R. D) IL-10R shows cytoplasmic association to CD28 in activated T cells. The immunoprecipitation of the T cell lysates by anti-CD28 and immunoblotting by anti-IL-10R stained the coprecipitated IL-10R 48 h after PHA stimulation. An EBV transformed B cell line that expresses IL-10R (but not CD28) and the HuT 78 T cell line that does not express any of the IL-10R or CD28 molecules did not show any staining in comparison to PBMCs.

3. IL-10 inhibits tyrosine phosphorylation of CD28 and phosphatidylinositol 3-kinase (PI3-K) binding
Stimulation of CD28 by B7 surface molecules leads to tyrosine phosphorylation of CD28. Ligation of IL-10 receptor (IL-10R) at the time of CD28 stimulation inhibits tyrosine phosphorylation of CD28 as detected after 10 min (Fig. 1B ). The inhibitory effect of IL-10 on CD28 appeared to be specific for the CD28 pathway, because IL-10 did not affect ZAP-70 tyrosine phosphorylation stimulated by CD3 cross-linking. As a consecutive event for signal transduction, PI3-K should bind to CD28 with the p85 subunit. The association of CD28 with the PI3-K p85 molecule was inhibited by IL-10. This inhibition was specifically blocked by preventing IL-10 binding to its receptor with an anti-IL-10R monoclonal antibody (mAb) (Fig. 1C ).

4. IL-10 R shows association to CD28 in activated T cells
Resting human T cells contain very low or undetectable levels of IL-10Rs. These are increased after stimulation of T cells by anti-CD28, anti-CD3, and mitogens. Immunoprecipitation of CD28 and coprecipitation of the 110 kDa IL-10R in lysates of activated T cells revealed an association of the two receptors. HuT 78 T cells that did not express any of these molecules and EBV-transformed B cells (BuB1) that expressed only the IL-10R (but not CD28) did not show the association of IL-10R with CD28 in control experiments (Fig. 1D ).

5. Regulation of T cell IL-10R expression by CD28 and CD3 stimulation
T cells were stimulated with anti-CD28, anti-CD3, and IL-10. Surface expression of IL-10R chain, IL-10Rß chain, and CD28 was determined by flow cytometric analysis. Freshly purified T cells expressed few IL-10R. CD28 stimulation significantly enhanced IL-10R and partially up-regulated IL-10Rß expression after 24 h. With the same stimulation, CD28 itself was fully down-regulated by receptor internalization. Anti-CD3 stimulation strongly enhanced both IL-10R chains and CD28 expression. Surface CD28 expression and IL-10R and ß chain expression did not change by IL-10 stimulation in T cells. These results demonstrate a relationship between IL-10R and CD28 in which CD28 stimulation up-regulates IL-10R expression, rendering the cells more susceptible to IL-10-mediated suppression. Moreover, they demonstrate that the specific effect of IL-10 on inhibition of CD28 costimulation does not involve sterical interactions of the two receptors and antibodies at the cell surface.

CONCLUSION

IL-10-induced peripheral T cell tolerance during specific immunotherapy as well as natural antigen exposure represents a key event in the control of specific immune responses to high antigen doses. The inhibition of the CD28 costimulatory pathway by IL-10 delineates a major mechanism in peripheral T cell tolerance. IL-10 inhibits T cell responses even at high antigen doses, and the number of TCR triggered by peptide-MHC complex on the surface of APC appears to be in the range of the requirement for costimulation. IL-10 efficiently increases the threshold for T cell activation, rendering optimal conditions provided by professional APC and CD28/B7 interaction. In consequence, IL-10 induces an immunological unresponsive, anergic state in T cells.

Numerous studies demonstrated that IL-10 acts as a general inhibitor of proliferative and cytokine responses of both Th1 and Th2 cells in vitro and in vivo. The inhibitory effect of IL-10 in T cell clones was observed exclusively in APC-dependent culture systems, but not in T cells stimulated by solid-phase bound anti-CD3. This can be explained by the present study showing that IL-10 blocks the CD28 stimulation and subsequent signaling pathways in T cells. IL-10 specifically inhibits the tyrosine phosphorylation of CD28 and its binding to PI3-K (Fig. 2 ). PI3-K is a heterodimer that comprises a 85 kDa regulatory and a 110 kDa catalytic subunit possessing both protein serine-kinase and lipid-kinase activity. The p85 subunit contains a p110 binding site as well as two src-homology-2 (SH2) domains. Binding of PI3-K to CD28 occurs by direct interaction between SH2 domain motifs of p85 PI3-K and a (p)YXXM motif in the cytoplasmic part of CD28. This requires CD28 tyrosine phosphorylation. Recently, CTLA-4, a negative regulator of T cell function, was reported to be associated with the TCR complex chain in T cells. The IL-10R/CD28 association reported here appears to be similar to CTLA-4/TCR association. Both mechanisms have broad implications for the negative regulation of T cell function and T cell anergy.

View larger version (39K): [in this window] [in a new window]   Figure 2. The molecular basis of T cell suppression by IL-10: CD28-associated IL-10R inhibits CD28 tyrosine phosphorylation and phosphatidylinositol 3-kinase binding. IL-10 specifically inhibits the tyrosine phosphorylation of CD28 and its binding to PI3-K. IL-10 exerts its biological functions through the activation of Jak1 and Tyk2, members of the receptor associated Janus tyrosine kinases family, Stat1 and Stat3, and in certain cells, Stat5. In monocytes, IL-10 was shown to induce the expression of the suppressor of the cytokine signaling 3 (SOCS3) gene, which may play a role on inhibition of IFN-induced tyrosine phosphorylation of STAT-1. However, the effect of IL-10 on the inhibition of CD28 tyrosine phosphorylation is within minutes. Obviously, this rapid activity does not require new protein synthesis, and the mechanism of inhibition of CD28 tyrosine phosphorylation remains to be elucidated. Moreover, identification of the second chain of the IL-10R, termed IL-10R2 or IL-10Rß, as an essential molecule for the action of IL-10 receptor complex will further help to characterize IL-10 signaling. Both chains are significantly up-regulated by CD28 stimulation as a negative feedback mechanism for providing T cells sensitive to IL-10-mediated suppression.

Including various molecular mechanisms that generate T cell tolerance, an increase in IL-10 production and suppression of CD28 costimulation-mediated T cell proliferative and cytokine responses demonstrate important steps in tolerating exposure to high doses of exogenous antigen. The knowledge of this molecular basis is pivotal in understanding the equilibrated regulation of immune response and anergy to immunogenic agents and their possible therapeutic applications.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.99-0874fje

Received for publication January 11, 2000. Revision received March 27, 2000.

This article has been cited by other articles:

				P.-h. Park, H. Huang, M. R. McMullen, K. Bryan, and L. E. Nagy Activation of cyclic-AMP response element binding protein contributes to adiponectin-stimulated interleukin-10 expression in raw 264.7 macrophages J. Leukoc. Biol., May 1, 2008; 83(5): 1258 - 1266. [Abstract] [Full Text] [PDF]

				C. B. Schmidt-Weber, M. Letarte, S. Kunzmann, B. Ruckert, C. Bernabeu, and K. Blaser TGF-{beta} signaling of human T cells is modulated by the ancillary TGF-{beta} receptor endoglin Int. Immunol., July 1, 2005; 17(7): 921 - 930. [Abstract] [Full Text] [PDF]

				G. Grutz New insights into the molecular mechanism of interleukin-10-mediated immunosuppression J. Leukoc. Biol., January 1, 2005; 77(1): 3 - 15. [Abstract] [Full Text] [PDF]

				K. Ohkusu-Tsukada, N. Tominaga, H. Udono, and K. Yui Regulation of the Maintenance of Peripheral T-Cell Anergy by TAB1-Mediated p38{alpha} Activation Mol. Cell. Biol., August 15, 2004; 24(16): 6957 - 6966. [Abstract] [Full Text] [PDF]

				M. Akdis, J. Verhagen, A. Taylor, F. Karamloo, C. Karagiannidis, R. Crameri, S. Thunberg, G. Deniz, R. Valenta, H. Fiebig, et al. Immune Responses in Healthy and Allergic Individuals Are Characterized by a Fine Balance between Allergen-specific T Regulatory 1 and T Helper 2 Cells J. Exp. Med., June 7, 2004; 199(11): 1567 - 1575. [Abstract] [Full Text] [PDF]

				K. T. Nouri-Aria, P. A. Wachholz, J. N. Francis, M. R. Jacobson, S. M. Walker, L. K. Wilcock, S. Q. Staple, R. C. Aalberse, S. J. Till, and S. R. Durham Grass Pollen Immunotherapy Induces Mucosal and Peripheral IL-10 Responses and Blocking IgG Activity J. Immunol., March 1, 2004; 172(5): 3252 - 3259. [Abstract] [Full Text] [PDF]

				T. C. Tung, K. Oshima, G. Cui, H. Laks, and L. Sen Dual upregulation of Fas and Bax promotes alloreactive T cell apoptosis in IL-10 gene targeting of cardiac allografts Am J Physiol Heart Circ Physiol, August 7, 2003; 285(3): H964 - H973. [Abstract] [Full Text] [PDF]

				M. AKDIS, A. TRAUTMANN, S. KLUNKER, I. DAIGLE, U. C. KUCUKSEZER, W. DEGLMANN, R. DISCH, K. BLASER, and C. A. AKDIS T helper (Th) 2 predominance in atopic diseases is due to preferential apoptosis of circulating memory/effector Th1 cells FASEB J, June 1, 2003; 17(9): 1026 - 1035. [Abstract] [Full Text] [PDF]

				M. Schroder, C. Meisel, K. Buhl, N. Profanter, N. Sievert, H.-D. Volk, and G. Grutz Different Modes of IL-10 and TGF-{beta} to Inhibit Cytokine-Dependent IFN-{gamma} Production: Consequences for Reversal of Lipopolysaccharide Desensitization J. Immunol., May 15, 2003; 170(10): 5260 - 5267. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 14/12/1666 99-0874fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by AKDIS, C. A. Articles by BLASER, K. Search for Related Content PubMed PubMed Citation Articles by AKDIS, C. A. Articles by BLASER, K.