Costimulatory receptors in a teleost fish: Typical CD28, elusive CTLA4
T cell activation requires both specific recognition of the peptide-MHC complex by the TCR and additional signals delivered by costimulatory receptors. We have identified rainbow trout sequences similar to CD28 (rbtCD28) and CTLA4 (rbtCTLA4). rbtCD28 and rbtCTLA4 are composed of an extracellular Ig-superfamily V domain, a transmembrane region, and a cytoplasmic tail. The presence of a conserved ligand binding site within the V domain of both molecules suggests that these receptors likely recognize the fish homologues of the B7 family. The mRNA expression pattern of rbtCD28 and rbtCTLA4 in naive trout is reminiscent to that reported in humans and mice, because rbtCTLA4 expression within trout leukocytes was quickly up-regulated following PHA stimulation and virus infection. The cytoplasmic tail of rbtCD28 possesses a typical motif that is conserved in mammalian costimulatory receptors for signaling purposes. A chimeric receptor made of the extracellular domain of human CD28 fused to the cytoplasmic tail of rbtCD28 promoted TCR-induced IL-2 production in a human T cell line, indicating that rbtCD28 is indeed a positive costimulator. The cytoplasmic tail of rbtCTLA4 lacked obvious signaling motifs and accordingly failed to signal when fused to the huCD28 extracellular domain. Interestingly, rbtCTLA4 and rbtCD28 are not positioned on the same chromosome and thus do not belong to a unique costimulatory cluster as in mammals. Finally, our results raise questions about the origin and evolution of positive and negative costimulation in vertebrate immune systems. T cell activation is initiated through complex cell-to-cell interactions. TCRs expressed on the surface of T cells first recognize antigenic peptides presented by MHC molecules on the surface of APCs. TCRs and other surface receptors and ligands stabilize the contact between T cells and APCs, which triggers signal transduction pathways resulting in T cell activation. According to the two-signal model ( 1 , 2 ), T cell activation by Ag requires both specific recognition of the peptide by the TCR (signal 1) and additional signals delivered by other costimulatory receptors (signal 2). Among the known costimulatory receptors, CD28 and CTLA4 (also known as CD152), which are expressed on T cells and bind their corresponding ligands B7-1/B7-2 (CD80/CD86) on APCs, represent a well-studied system in mammals ( 3 ). CD28 is a glycosylated homodimeric protein expressed on the surface of double-positive thymocytes, mature CD4 + T cells, CD11b − CD8 + T cells, and γδ CD3 + T cells ( 4 , 5 ). CD28 is expressed at higher levels on activated T cells than on resting cells ( 6 ). In mammals, CD28 binds to B7-1 and B7-2, which are expressed on the surface of APCs, and delivers a critical costimulatory signal to T lymphocytes. In the absence of CD28 ligation, TCR binding either induces apoptosis or anergy in T cells ( 5 ). Engagement of CD28 alone cannot activate T cells, even if synergetic effects are provided via stimulation by T cell mitogens or anti-CD3 treatment ( 7 ). A tyrosine-based motif in cytoplasmic tail of CD28 functions as a binding site for the p85 PI3K subunit when it is phosphorylated ( 8 , 9 ). Other CD28 intracytoplasmic motifs, which associates with IL-2-inducible tyrosine kinase, lymphocyte-specific tyrosine kinase, and the adaptor growth-factor receptor bound protein-2 are responsible for signal transduction ( 10 , 11 , 12 ). The binding of growth-factor receptor bound protein-2 to CD28 also activates the GTPase RAS ( 13 ). CD28 signaling is also thought to contribute to the mobilization of lipid rafts to the immunological synapse ( 14 ), the region of contact between T cell and APC, which lowers the overall threshold of TCR engagement required for effective cytokine production and proliferation ( 15 ). In contrast, CTLA4 is a powerful negative regulator of T cell activation and was first cloned via differential screening of a cytotoxic T cell cDNA library ( 16 ). CD28 and CTLA4 belong to the same family and share high sequence similarity to each other ( 16 , 17 ). Both receptors interact with B7-1 and/or B7-2 but induce different signals: CD28 amplifies signaling triggered by the TCR-CD3 complex, whereas CTLA4 generates negative signals that inhibit T cell activation ( 18 ). CTLA4 has higher affinity for B7-1 and B7-2 compared with CD28, and its expression is also induced by TCR engagement ( 19 ). The mechanisms of CTLA4-mediated suppression likely involve both competition with CD28 for B7-1/B7-2 binding and potent inhibitory signals delivered by CTLA4 ( 20 ). The inhibitory signaling mechanisms triggered by CTLA4 are not clear. The CTLA4 cytoplasmic tail lacks typical ITIM motifs and shares with CD28 a p85 binding site ( 21 ). Thus, the extent of an immune response is likely controlled through the finely tuned expression of costimulatory receptors from the CD28/CTLA4 family on the surface of activated T cells ( 22 , 23 ). Very little is known about T cell responses and activation in bony fish. TCR-αβ cDNAs ( 24 , 25 , 26 , 27 ) and polymorphic class IA and class IIA/B MHC molecules ( 28 , 29 , 30 ) have been reported in several species, suggesting that the fish TCR recognizes antigenic peptides presented by MHC molecules similar to that of mammals. T cell-mediated responses in fish are supported by several lines of evidence. Allograft rejection provided the first experimental indications suggesting that bony fish possess a functional T cell-mediated immune response ( 31 , 32 ). In vitro assays for allospecific cytotoxicity have also been established using clonal and nonclonal catfish cell lines ( 33 , 34 ) or clonal rainbow trout ( 35 ). Autologous cell-mediated specific lysis of virus-infected syngenic target cells has also been reported in cloned goldfish ( 36 , 37 ). Finally, both public and private T cell-specific expansions have been observed in rainbow trout during secondary immune responses to viral hemorrhagic septicemia virus (VHSV), 3 a fish rhabdovirus, using TCR-β CDR3-length spectratyping ( 38 ). TCR-MHC-peptide interactions induce signaling events through CD3 in mammals and most likely in lower vertebrates because a complete set of CD3 genes, including TCR ζ, CD3 ε, and CD3 γδ, has been found in Pufferfish and Xenopus ( 39 , 40 ). CD8α has been identified in rainbow trout ( 41 ); however, the cytoplasmic tail lacks the consensus p56 lck motif, suggesting that TCR/CD3/CD8-mediated signaling events may be different in teleosts. In this study, we have identified and initially characterized two members of the CD28 family in rainbow trout, rbtCD28 and rbtCTLA4. Their sequence features and expression patterns suggest that they are the likely homologues of mammalian CD28 and CTLA4. The potential costimulatory capacities of these fish receptors were investigated using a human T cell line expressing chimeric receptors composed of the extracellular domain of human CD28 (hCD28) fused to the cytoplasmic tail of rbtCD28 or rbtCTLA4. The chimeric hCD28-rbtCD28 receptor mediated enhanced TCR-induced IL-2 production, suggesting a costimulatory function for rbtCD28. In contrast, the divergent cytoplasmic tail from rbtCTLA4 did not mediate similar signaling activities. This study therefore provides the first characterization of costimulatory receptors in lower vertebrates.