nature immunology • volume 4 no 2 • february 2003 • www.nature.com/natureimmunology A RTICLES 182 Hélène Bour-Jordan 1,4 , Jane L. Grogan 2,3 , Qizhi Tang 1,4 , Julie A. Auger 5 , Richard M. Locksley 2,3 and Jeffrey A. Bluestone 1,4 Published online 13 January 2003; doi:10.1038/ni884 The relative importance of the cytokine milieu versus cytolytic T lymphocyte-associated antigen 4 (CTLA-4) and T cell receptor signal strength on T cell differentiation remains unclear. Here we have generated mice deficient for signal transducer and activator of transcription 6 (STAT6) and CTLA-4 to determine the role of CTLA-4 in cytokine-driven T cell differentiation. CTLA-4–deficient T cells bypass the need for STAT6 in the differentiation of T helper type 2 (T H 2) cells.T H 2 differentiation of cells deficient for both STAT6 and CTLA-4 is accompanied by induction of GATA-3 and the migration of T H 2 cells to peripheral tissues. CTLA-4 deficiency also affects the balance of the nuclear factors NFATc1 and NFATc2, and enhances activation of NF- κB. These results suggest that CTLA-4 has a critical role in T cell differentiation and that STAT6-dependent T H 2 lineage commitment and stabilization can be bypassed by increasing the strength of signaling through the T cell receptor. 1 UCSF Diabetes Center and Department of Medicine, 2 Howard Hughes Medical Institute, and 3 Departments of Medicine and Microbiology-Immunology, University of California San Francisco, San Francisco, CA 94143, USA. 4 The Committee on Immunology, 5 Immunology Applications Core Facility, University of Chicago, Chicago, IL 60637, USA. Correspondence should be addressed to J.A.B. (jbluest@diabetes.ucsf.edu). CTLA-4 regulates the requirement for cytokine-induced signals in T H 2 lineage commitment Naive TH cells can differentiate into two functionally distinct subsets defined by their cytokine profiles. TH1 cells produce interleukin-2 (IL- 2), interferon-γ (IFN-γ29 and tumor necrosis factor-α (TNF-α29 , whereas TH2 cells produce IL-4, IL-5 and IL-13. The strength of the T cell receptor (TCR) signal, the cytokine milieu and the cell cycle all influ- ence T cell differentiation; however, the relative importance of these different factors in the complex molecular process leading to stable TH1 or TH2 phenotypes is not fully understood. Binding of IL-4 to the IL-4 receptor activates STAT6 1,2 , which in turn induces expression of the tissue-specific transcription factor GATA-3 3,4 . The importance of STAT6 in TH2 cell differentiation was initially estab- lished by showing defective TH2 responses in STAT6-deficient mice 5,6 . Several studies suggest that STAT6 is essential for initiating TH2 cell differentiation, whereas other reports support the crucial importance of STAT6 in controlling the expansion of fully committed TH2 cells. The strength and duration of signaling during T cell activation also has a primary role in TH cell differentiation. Both antigen dose and co- stimulatory molecules determine the overall T cell receptor (TCR) sig- nal strength and influence T cell differentiation towards a TH1 or a TH2 phenotype. Indeed, strong TCR signals and CD28 costimulation induce TH2 differentiation 7–9 , whereas ligation of the immune attenuator CTLA-4 promotes TH1 differentiation 10 . The influence of CTLA-4 on T cell differentiation is further underscored by a skewing towards TH2 dif- ferentiation in CTLA-4–deficient mice during uncontrolled T cell expansion 11,12 . To evaluate the relative contribution of TCR signals versus cytokine signals in T cell differentiation, we have examined the effects of CTLA-4 deficiency on T cell differentiation in the absence of upstream signals characteristic of the TH2 lineage by analyzing the production of T cell cytokines in mice deficient for STAT6 and CTLA- 4. Unexpectedly, we found that, unlike STAT6-deficient T cells, STAT6– CTLA-4 doubly deficient (DKO) T cells efficiently differen- tiated towards a TH2 phenotype in vitro and in vivo. The TH2 phenotype in DKO mice was associated with induction of GATA-3 in vitro and with migration of CD4 + TH2 cells to peripheral tissues in vivo. In addi- tion, TCR crosslinking induced a relative increase of NFATc1 versus NFATc2 nuclear translocation and enhanced NF-κB activation in DKO T cells as compared with STAT6 –/– T cells. Thus, CTLA-4–deficient T cells bypass STAT6 to stably differentiate into mature TH2 cells. We propose that CTLA-4 regulates T cell differentiation by controlling the overall strength of the T cell activation signal, bypassing the cytokine dependency of TH2 differentiation. Results DKO T cells are skewed towards T H 2 differentiation To examine the influence of CTLA-4 on cytokine-driven CD4 + T cell differentiation pathways, we bred CTLA-4 –/– mice with STAT6 –/– mice to produce mice deficient in both proteins. Similar to CTLA-4 –/– mice 13,14 , the DKO mice died at 2–4 weeks from a lymphoproliferative disease (data not shown). To assess how CTLA-4 deficiency would affect TH2 cell differentiation in STAT6-deficient mice, we measured cytokine pro- duction by DKO cells upon in vitro stimulation with monoclonal anti- bodies (mAbs) to CD3 and to CD28. STAT6 –/– cells produced the TH1 cytokines IL-2 and IFN-γ, but the TH2 cytokine IL-4 was undetectable © 2003 Nature Publishing Group http://www.nature.com/natureimmunology