InVivoMAb anti-mouse CD2

Previous studies have reported an association of the cell surface adhesion molecule CD2 with the T cell receptor and with CD45 on mouse and human T lymphocytes. In this study the association of CD2 with cell surface molecules was investigated using cell surface biotinylation of T lymphocytes, coupled with immunoprecipitation using two CD2-specific monoclonal antibodies (mAb) (RM2-5 and 12-15) and analysis by SDS-PAGE. Although both CD2 mAb immunoprecipitated CD2 from lysates of murine lymphocytes, it was found that mAb 12-15, but not RM2-5, co-precipitated two other molecules of 95 and 180 kDa. Subsequent studies revealed that the 95- and 180-kDa molecules were associated with a subspecies of CD2 (approximately 5%) on thymocytes, the antigen-specific T cell line D10, and splenic T cells but not B cells. Two lines of evidence were obtained consistent with the 95- and 180-kDa molecules being the beta and alpha chains of LFA-1. Firstly, an analysis of 12-15 mAb immunoprecipitates on 4-12% gels under reducing and nonreducing conditions shows that the 95- and 180-kDa molecules have a molecular weight and migration pattern identical to LFA-1. Secondly, depletion of LFA-1 from lysates with LFA-1 mAb abolished the ability of CD2 mAb 12-15 to co-precipitate the 95- and 180-kDa molecules, thereby identifying these as the beta and alpha chains of mouse LFA-1, respectively. These results provide evidence for the first time for an association of LFA-1 and CD2 on mouse T lymphocytes, and suggest that the association occurs with an immunologically distinct subspecies of CD2 molecules.

We have examined the functional property of murine CD2 as an intercellular adhesion molecule by using five anti-murine CD2 mAb which were classified into two groups according to their mutual competition in binding to cell surface CD2. Hamster fibroblasts transfected with murine CD2 cDNA exhibited increased conjugate formation with a murine mastocytoma P815 which expresses the putative murine LFA-3 mRNA detected by cross-hybridization with human LFA-3 cDNA under conditions of low stringency. This increase in conjugate formation was abrogated by both groups of anti-CD2 mAb, although some differences in the extent of inhibition were observed at lower concentrations of the mAb. We then examined the involvement of CD2 in several murine T cell responses by using these mAb to abrogate CD2-mediated cellular interactions. Anti-CD2 mAb significantly inhibited mitogenic T cell responses induced by suboptimal doses of Con A and PHA. In the allogenic MLR response and in the Ag response of two KLH/I-Ak-specific Th cell clones, the inhibitory effect of anti-CD2 mAb was also greatest under suboptimal conditions, i.e., with lesser doses of the Ag. These results indicate that the contribution of CD2 as an accessory molecule is variable, depending on the Ag dose used for stimulation, and they suggest that CD2 is involved in the Ag response of murine T cells under the physiologic conditions where only a limited amount of Ag is available. We next examined the contribution of CD2 to MHC-restricted cytotoxicity by CTL and to MHC-unrestricted cytotoxicity by NK and lymphokine-activated killer cells. Only a marginal inhibition by anti-CD2 mAb alone was observed. Anti-lymphocyte function-associated Ag (LFA)-1 mAb alone exhibited greater inhibitory effects than anti-CD2 mAb in all of the cases tested. In most cases, however, substantial levels of cytotoxicity remained, even in the presence of both anti-CD2 and anti-LFA-1 mAb. These results indicate a minor contribution of CD2, as compared with LFA-1, to cytotoxicity by murine CTL, NK cells, and lymphokine-activated killer cells, and they reveal the presence of undefined cellular interaction pathways other than those mediated by CD2 and LFA-1.

CD2 is a T cell surface glycoprotein that mediates cellular adhesion and can participate in signal transduction. It is expressed early in thymocyte ontogeny and consequently has been proposed to participate in T cell development. To study the in vivo function of CD2, the murine gene was inactivated using the technique of homologous recombination in embryonic stem cells. Homozygous mutant mice are healthy and have an apparently normal complement of lymphocytes. They mount effective immune responses similar to those of wild type controls. In particular, the generation and function of cytotoxic T cells was found to be normal as was the production of antibodies following immunization. Selection of thymocytes expressing either MHC class I- or class II-restricted transgenic T cell receptors was also grossly normal in the absence of CD2. Thus, CD2 may be dispensable for the development and function of T cells. Within the context of other targetted mutations, these mice should be useful in defining the precise roles of various cell surface molecules involved in T cell responses.

CD2 is an intercellular adhesion molecule that has been implicated in T cell activation and differentiation both in humans and mice. Although the ligand for human CD2 has been defined as LFA-3, that for murine CD2 has not been identified yet. To identify the ligand for mouse CD2, we generated a chimeric molecule consisting of the extracellular domain of mouse CD2 and human immunoglobulin (Ig)G1 Fc (mCD2Rg). A hamster monoclonal antibody (mAb), HM48-1, was established by screening mAbs that could block the binding of mCD2Rg to T cell lines at the ligand site. The putative mouse CD2 ligand recognized by this mAb was a glycosyl phosphatidylinositol-anchored glycoprotein with an apparent molecular mass of 45 kD, which were shared characteristics with human LFA-3. However, its expression was predominantly restricted to hematopoietic cells, unlike human LFA-3. Protein microsequencing analysis for the NH2-terminal 18 amino acid residues of the affinity-purified HM48-1 antigen revealed that it is almost identical with mouse CD48. This identity was further confirmed by the reactivity of HM48-1 with a soluble recombinant CD48 (sCD48) protein and the molecule recognized by a rat mAb raised against sCD48. A rat anti-CD48 mAb blocked the mCD2Rg binding as well as HM48-1. Moreover, sCD48 also inhibited the mCD2Rg binding to the cellular ligand. Finally, like anti-CD2 mAb, HM48-1 inhibited the phytohemagglutinin response and, when crosslinked, augmented the anti-CD3 response of splenic T cells. These results indicate that CD48 is a ligand for mouse CD2 and is involved in regulating T cell activation.