Cell, Vol. 60, 929-939, March 23, 1990, Copyright 0 1990 by Cell Press Chimeric lmmunoglobulin-T Cell Receptor Proteins Form Functional Receptors: Implications for T Cell Receptor Complex Formation and Activatiin Joan Goverman,’ Stephen M. Gomez,t Kathleen D. Segesman: Tim Hunkapiller: Walter E. Laug,t and Leroy Hood’ l Division of Biology California Institute of Technology Pasadena, California 91125 t Department of Hematology-Oncology Childrens Hospital of Los Angeles Los Angeles, California 90027 Summary We constructed chimeric receptor chains in which an immunoglobulin heavy chain variable region (V,) from a phosphorylcholine-specific antibody is sub- stituted for T cell receptor (Tcr) a and 3 V regions. We demonstrate that the VH region joined to either the C, or the CB region can form stable chime& proteins in EL4 T cells. Both chimeric receptor chains associate with CD3 polypeptides in functional receptor com- plexes and respond to phosphorylcholine coupled to Sepharose beads. The V&Z, chimeric chain associ- ates with the EL4 f3 chain, while the Vu-C, chimeric protein appears to form either a homodimer or a het- erodimer with the native EL4 p chain. Thus, functional receptor complexes can be formed using two C, regions, and the C, region may not be required for CD3 association and surface expression of Tcr com- plexes. Introduction Two types of lymphocytes are responsible for the recogni- tion of foreign antigen in the immune response. The cell surface receptors of T cells recognize antigenic peptides only when they are complexed with polypeptides encoded in the major histocompatibility complex (MHC) and ex- pressed on the surface of antigen-presenting cells. Thus, engaging a Tcr requires the formation of a trimolecular complex in which all components are displayed on a cell surface (Schwartz, 1984). B cells, in contrast, use cell sur- face immunoglobulin to recognize the three-dimensional conformation of antigens independent of a cellular or MHC-associated context (Davies et al., 1988). Other inter- actions between T cells and target cells also contribute toward limiting T cell responses to cellularly presented antigen. The T cell accessory molecules CD4 and CD8 appear to stabilize cell-cell interactions by interacting with nonpolymorphic determinants on MHC molecules and may also participate in transmitting regulatory signals (Parnes, 1989). Also, activation of resting T cells appears to require two signals (Chiller et al., 1982; Mizel, 1982). One signal consists of engaging the Tcr, and the other is transmitted by lymphokines produced by the antigen- presenting cell. Thus, to receive a signal required for proliferation, T cells must be proximal to another cell that is producing that signal. Both B and T cell receptors are disulfide-linked hetero- dimers consisting of light (L) and heavy (H) chains for im- munoglobulin and a and 6 chains for the predominant Tcrs. Each chain is divided into variable (V) and constant (C) regions. The V regions of both chains associate to form the antigen-binding domain, and the C regions link the receptor to the cell surface and, in the case of Tcr, to other cell surface accessory molecules (Davies and Metzger, 1983; Davis, 1985; Kronenberg et al., 1988). The immuno- globulin C regions are associated with the effector func- tion of the molecule. For cell surface expression, the Tcr must be associated with a complex of polypeptides called CD3 (Clevers et al., 1988). These interactions appear to involve the C regions and mediate signal transduction be- tween the receptor and cell (Weiss et al., 1986). A second type of Tcr associated with CD3 is composed of y and S chains and is expressed on immature thymocytes and 10/o-2% of peripheral T cells (Saito et al., 1984; Chien et al., 1987; Brenner et al., 1988). While S and y chains ex- hibit sequence similarity to a and 8 chains, respectively, they express a more limited V region repertoire and do not appear to play a major role in the general MHC-restricted response to antigens (Strominger, 1989). The V regions of the H and 8 chains are formed from separate V, diversity (D), and joining (J) gene segments that rearrange together to form a functional V gene. The L and a V regions are formed by rearranging only V and J gene segments. Predicted secondary and tertiary struc- tures based on sequence comparisons of these V regions indicate a fundamental similarity between the antigen- binding domains of the two receptors (Goverman et al., 1986; Novotny et al., 1986). Thus, no structural features are apparent in the V, and VP regions of the Tcr that could account for its unique properties of antigen recogni- tion in relation to those of immunoglobulin. We are interested both in defining the molecular interac- tions between T cells and target cells, which signal activa- tion, and in identifying features of Tcr chains that mediate their interaction with one another and CD3 components. Our approach is to construct chimeric immunoglobu- lin-Tcr genes in which the V region genes of the Tcr are replaced with those of an immunoglobulin of known speci- ficity. Transfecting these chimeric receptor genes into T cells should confer a specificity for an antigen that can be easily manipulated and is independent of MHC deter- minants. The first step in setting up this system is to deter- mine whether there is a unique character to Tcr V regions not shared by immunoglobulin V regions. To examine this question directly, we determined the ability of individual chimeric receptor chains to pair with native Tcr chains and form receptor complexes. We report here the characterization of chimeric receptor genes encoding an immunoglobulin Vu region that is joined to either the C, or CB regions of a Tcr. The VH re-