Cytotoxic T lymphocyte antigen-4 ( CTLA-4 ) regulates primary and secondary peptide-specific CD 4 1 T cell responses ( CD 152 y inhibitory costimulation y homeostasis y tolerance )

CTLA-4-deficient mice develop a fatal lymphoproliferative disorder, characterized by polyclonal expansion of peripheral lymphocytes. To examine the effect of restricting the CD41 TCR repertoire on the phenotype of CTLA-4-deficient mice and to assess the inf luence of CTLA-4 on peptide-specific CD41 T cell responses in vitro, an MHC class II-restricted T cell receptor (AND TCR) transgene was introduced into the CTLA-42y2 animals. The expression of the AND TCR transgene by CD41 T cells delays but does not prevent the lymphoproliferation in the CTLA-42y2 mice. The CD41 T cells become preferentially activated and expand. Interestingly, young AND TCR1 CTLA-42y2 mice carrying a null mutation in the rag-1 gene remain healthy and the T cells maintain a naive phenotype until later in life. We demonstrate that CTLA-4 regulates the peptide-specific proliferative response generated by naive and previously activated AND TCR1 RAG2y2 T cells in vitro. The absence of CTLA-4 also augments the responder frequency of cytokine-secreting AND TCR1 RAG2y2 T cells. These results demonstrate that CTLA-4 is a key regulator of peptide-specific CD41 T cell responses and support the model that CTLA-4 plays a differential role in maintaining T cell homeostasis of CD41 vs. CD81 T cells. Optimal T cell activation requires two signals, one via the T cell receptor (TCR) and the second via costimulatory molecules. The primary positive costimulatory molecule for T cell activation is CD28 (1). CD28-mediated costimulation functions to facilitate and sustain T cell-proliferative responses (1–3). Conversely, recent studies indicate that CD28 homologue CTLA-4 inhibits T cell responses (4). CTLA-4-mediated costimulation inhibits T cell-proliferative and cytokine responses in vitro, and blockade of CTLA-4yligand interaction enhances T cell responses in vivo (4). Although CD28 and CTLA-4 share common ligands, namely CD80 (B7.1) and CD86 (B7.2), it appears that CTLA-4 does not function simply to counteract CD28-mediated costimulatory signals; rather, accumulating evidence indicates that CTLA-4 may directly modulate proximal TCR signaling (5–11). CTLA-4-deficient mice die at 3–4 weeks of age as a result of a lymphoproliferative disorder caused by polyclonal T cell expansion (12–14). Peripheral CD41 T cells are primarily responsible for the lymphoproliferative disorder in CTLA42y2 mice expressing an unmanipulated TCR repertoire (15). Mice depleted of CD81 T cells from birth by antibody treatment develop a lymphoproliferative disorder similar in onset and severity to unmanipulated CTLA-42y2 mice, whereas lymphoproliferation does not occur in CTLA-42y2 mice depleted of CD41 T cells from birth. Moreover, the CD81 T cells in these animals maintain a naive phenotype. These results suggest that CTLA-4 has differential effects on the CD41 vs. the CD81 T cells in vivo. The nature of TCR–ligand interaction that initiates the lymphoproliferation and the extent to which individual naive CD41 T cells become dysregulated by the absence of CTLA-4 in vivo is unknown. Based on the phenotype of the CTLA-42y2 mice, we proposed (15, 16) that CTLA-4 may provide an inhibitory signal that prevents CD41 T cell activation during the continuous interaction of TCR and MHC that is shown to be required for T cell survival (17–21). Further, we have proposed a two-signal model for T cell activation in which T cell responses are regulated by the dynamic integration of TCR, CD28, and CTLA-4 signals (14). To test these models and to determine antigenic peptide reactivity of naive T cells in the absence of CTLA-4, we have initiated studies using MHC class I or class II-restricted TCR transgenic CTLA-42y2 mice. The role of CTLA-4 in regulating homeostasis and antigen reactivity of CD81 T cells has been examined in three different MHC class I-restricted TCR transgenic mouse strains (21–23). CTLA-42y2 mice expressing MHC class I-restricted TCR transgenes remain healthy for several months but eventually develop a lymphoproliferative disorder, apparently initiated by activation of the normally small population of CD41 T cells present in these animals (22, 23). The CD81 TCR transgenic CTLA-42y2 T cells maintain a naive phenotype in these animals (23, 24). The magnitude and the kinetics of the peptide-specific proliferative and cytokine responses by naive CD81 peptide-specific responses are equivalent between the CTLA-42y2 and littermate control T cells (23, 24), but there is a dramatic increase in the magnitude of secondary peptidespecific responses by 2C TCR1 CD81 CTLA-42y2 compared with wild-type T cells (23). In this report we examine the effects of the CTLA-4 null mutation on the development and function of T cells expressing an MHC class II-restricted TCR. The CTLA-41y2 mice were crossed to an MHC class II-restricted TCR (AND TCR) (Vb3Va11) transgenic mouse strain that is selected on MHC class II Ab molecules and specific for pigeon cytochrome c (PCC88–104) in the context of I-Ek (25), to generate CTLA-4deficient AND TCR transgenic mice (AND TCR1 CTLA42y2). Further, we introduced a RAG null mutation in these mice to generate AND TCR1RAG2y2 CTLA-42y2 mice that express only the transgenic TCR. We demonstrate that although there is a delay in the onset, AND TCR1 CTLA-42y2 mice develop a fatal lymphoproliferative disorder mediated primarily by activated CD41 T cells whereas the minor CD81 population present in these mice maintain a naive phenotype. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS is available online at www.pnas. org. Abbreviations: AND TCR, MHC class II-restricted T cell receptor; APC, antigen-presenting cell; ELISPOT, enzyme-linked immunospot assay; CTLA-4, cytotoxic T lymphocyte antigen-4. *To whom reprint requests should be addressed at: 447 LSA, University of California, Berkeley, CA 94720. e-mail: jallison@ uclink4.berkeley. edu.