Deutsches Krebsforschungszentrum

Introduction

CD4⁺CD25^highFoxp3⁺ regulatory T cells (Treg) can suppress other immune cells and, thus, are critical mediators of peripheral self-tolerance. On the one hand Treg can prevent autoimmune disease and transplant rejection, while, on the other hand, Treg can avert immune reactions against tumors and pathogens. Thus, the understanding of Treg homeostasis and function may be exploited for the treatment of autoimmunity and cancer in the future. The main projects of our group focus on (1) suppression of conventional CD4⁺CD25⁻ T cells (Tcon) by Treg and (2) understanding CD95-mediated apoptosis and homeostasis of Treg.

Suppression of conventional T cell signaling by regulatory T cells

Proliferation and cytokine production of Tcon can be directly inhibited by Treg. Treg are also able to indirectly suppress Tcon activation via inhibition of antigen presenting cells. Direct suppression of Tcon by Treg can involve soluble factors and/or cell contact. The molecular mechanisms of contact-dependent Tcon suppression remain largely unknown and are analyzed in detail by our group. We demonstrated that human Treg suppress Tcon cytokine transcription very rapidly, within 1 to 3 hours after direct cell contact. Suppression is independent of known immunosuppressive soluble factors such as TGF-β and IL-10 (Oberle et al., 2007). More recently, we could show that the inhibited state of Tcon is sustained after removal of Treg after a pre-coculture of only 30 minutes. We found that pre-activated Treg rapidly suppress T cell receptor (TCR)-induced signaling in Tcon. In detail, TCR-induced calcium store depletion and, consequently, NFAT1 dephosphorylation and activity, are blocked in suppressed Tcon immediately after TCR stimulation. NF-κB activation, represented by phosphorylation of IKK, IκBα and p65, is also strongly and rapidly inhibited. Both NFAT and NF-κB suppression can be abrogated by artificially raising intracellular Ca²⁺ concentrations. In contrast, Ca²⁺-independent events such as TCR-proximal signaling and the AP-1 pathway are not affected in suppressed Tcon. PLCγ1- and IP₃-triggered Ca²⁺ store depletion, which is the main pathway to induce Ca²⁺ influx in T cells, are not inhibited by Treg. Thus, Treg inhibit Ca²⁺ store depletion in Tcon via so far unknown mechanisms independent of IP₃ generation. This inhibition of Tcon signaling elucidates a previously unrecognized and rapid mechanism of Treg-mediated suppression.

CD95-mediated apoptosis of Treg and signaling in Treg-suppressed Tcon. T cell receptor (TCR) stimulation activates several signaling pathways in Tcon, some of which are suppressed upon coculture with Treg (indicated in red), while others are not suppressed (indicated in green). Upon TCR restimulation, Tcon express the death receptor ligand CD95L, which triggers activation-induced cell death (AICD). Treg, in contrast, do not express CD95L due to repression of the CD95L gene by the transcription factor Foxp3. Thus, Treg do not undergo AICD, although they are sensitive to CD95-induced apoptosis in vitro and in vivo. | © Dr. Angelika Schmidt

CD95-mediated apoptosis of Treg in vitro and in vivo

Treg homeostasis has to be tightly controlled to ensure a balanced Treg-mediated suppression. One mechanism that regulates the CD4⁺ T cell pool is activation-induced cell death (AICD). This is mimicked in vitro by TCR restimulation-induced expression of the CD95 ligand (CD95L) in expanded Tcon. These cells express CD95 (Fas/APO-1) and binding of CD95L to CD95 results in AICD. In contrast, as shown previously by our group, Treg do not undergo AICD upon TCR (re)stimulation in vitro despite a functional CD95 cell death pathway (Fritzsching et al., 2005). More recently, we could demonstrate that human and murine Treg express low levels of CD95L upon stimulation. Knockdown of the Treg-specific transcription factor Foxp3 partially rescues CD95L expression and AICD in human Treg. Moreover, upon stimulation Foxp3-mutant Treg from Scurfy mice express CD95L similar to Tcon. We further addressed whether exogenous CD95 stimulation provides a mechanism of Treg homeostatic control in vivo in mice. Triggering of CD95 reduced Treg numbers systemically as reflected by in vivo imaging and decreased Treg numbers ex vivo. Our study reveals that Foxp3 negatively regulates CD95L expression in Treg and demonstrates that Treg are susceptible to homeostatic control by CD95 stimulation (Weiss et al., 2011).

from left to right: Eva-Maria Weiß (former PhD student), Ursula Matiba, Diana Vobis, Angelika Schmidt, Nina Oberle | © dkfz.de

Key references

Oberle, N., Eberhardt, N., Falk, C.S., Krammer, P.H., and Suri-Payer, E. (2007). Rapid suppression of cytokine transcription in human CD4+CD25- T cells by CD4+Foxp3+ regulatory T cells: independence of IL-2 consumption, TGF-beta, and various inhibitors of TCR signaling. J Immunol 179(6), 3578-3587.
Haas, J., Fritzsching, B., Trübswetter, P., Korporal, M., Milkova, L., Fritz, B., Vobis, D., Krammer, P.H., Suri-Payer, E., Wildemann, B. (2007). Prevalence of newly generated naive regulatory T cells (Treg) is critical for Treg suppressive function and determines Treg dysfunction in multiple sclerosis. J Immunol 179(2), 1322-30.
Franz, B., Fritzsching, B., Riehl, A., Oberle, N., Klemke, C.D., Sykora, J., Quick, S., Stumpf, C., Hartmann, M., Enk, A., Ruzicka, T., Krammer, P.H., Suri-Payer, E., Kuhn, A. (2007). Low number of regulatory T cells in skin lesions of patients with cutaneous lupus erythematosus. Arthritis Rheum 56(6), 1910-20.
Wendel, M., Galani, I.E., Suri-Payer, E., Cerwenka, A. (2008). Natural killer cell accumulation in tumors is dependent on IFN-gamma and CXCR3 ligands. Cancer Res 68(20), 8437-45.
Heid, J.B., Schmidt, A., Oberle, N., Goerdt, S., Krammer, P.H., *Suri-Payer, E., *Klemke, C.D. *shared senior authors (2009). FOXP3+CD25- tumor cells with regulatory function in Sézary syndrome. J Invest Dermatol 129(12), 2875-85.
Liesz, A., Suri-Payer, E., Veltkamp, C., Doerr, H., Sommer, C., Rivest, S., Giese, T., Veltkamp, R. (2009). Regulatory T cells are key cerebroprotective immunomodulators in acute experimental stroke. Nat Med 15(2), 192-9.
Galani, I.E., Wendel, M., Stojanovic, A., Jesiak, M., Müller, M.M., Schellack, C., Suri-Payer, E., Cerwenka, A. (2010). Regulatory T cells control macrophage accumulation and activation in lymphoma. Int J Cancer 127(5), 1131-40.
Klein, S., Kretz, C.C., Ruland, V., Stumpf, C., Haust, M., Hartschuh, W., Hartmann, M., Enk, A., Suri-Payer, E., Oberle, N., Krammer, P.H., Kuhn, A. (2011). Reduction of regulatory T cells in skin lesions but not in peripheral blood of patients with systemic scleroderma. Ann Rheum Dis 70(8): 1475-81. Epub Nov. 19, 2010
Fritzsching, B., Haas, J., König, F., Kunz, P., Fritzsching, E., Pöschl, J., Krammer, P.H., Brück, W., Suri-Payer, E., Wildemann, B. (2011). Intracerebral Human Regulatory T Cells: Analysis of CD4CD25FOXP3 T Cells in Brain Lesions and Cerebrospinal Fluid of Multiple Sclerosis Patients. PLoS One. 6(3), e17988.
Weiss, E.M., Schmidt, A., Vobis, D., Garbi, N., Lahl, K., Mayer, C.T., Sparwasser, T., Ludwig, A., Suri-Payer, E., *Oberle, N., *Krammer, P.H. *shared senior authors (2011). Foxp3-mediated suppression of CD95L expression confers resistance to activation-induced cell death in regulatory T cells. J Immunol 187(4):1684-91.
Schmidt, A., Oberle, N., Weiss, E.M., Frischbutter, S., Baumgrass, R., Falk, C.S., Haag, M., Brügger, B., Lin, H., Mayr, G.W., Reichardt, P., Gunzer, M., Suri-Payer, E., Krammer, P.H. (2011). Human Regulatory T Cells Rapidly Suppress T Cell Receptor-Induced Ca2+, NK-kB, and NFAT Signaling in Conventional T Cells. Sci Signal 4(204):ra90