Junctional sequences influence the specificity of gamma/delta T cell receptors.

T lymphocytes bearing the gamma/delta T cell receptor (TCR-gamma/delta) express a limited number of germline variable gene segments, generating receptor sequence diversity primarily through junctional mechanisms. To examine the role of V(D)J junctional sequences in antigen recognition by TCR-gamma/delta, we derived an alloreactive murine TCR-gamma/delta+ T cell line, LKD1, specific for the I-Ad class II major histocompatibility complex (MHC) molecule, and compared its receptor with that expressed by a previously characterized class II MHC alloreactive T cell line, LBK5, specific for I-Ek,b,s Ia molecules. Both LKD1 and LBK5 express receptors encoded by rearranged V gamma 1.2J gamma 2 and V delta 5D delta 2J delta 1 gene elements, differing in sequence only in the V(D)J junctional regions of the gamma and delta genes. These results demonstrate that junctionally encoded sequences corresponding to the putative third complementarity determining region can influence the antigen specificity of TCR-gamma/delta.

Signal transduction through class I MHC by a monoclonal antibody that detects multiple murine and human class I molecules.

We have generated a hamster anti-mouse class I reactive mAb that is capable of activating T cells in the presence of the cofactor PMA, as assayed by both IFN-gamma production and cellular proliferation. This mAb detects an epitope present on the majority of murine class I molecules, with the known exceptions of H-2Kk and H-2Kq, and is therefore not beta 2-microglobulin-specific. It also recognizes multiple human class I molecules. The epitope recognized by this antibody maps to the class I alpha 1 domain. The activation properties of this mAb are not mediated exclusively through the glycosylphosphatidylinositol-linked Qa-2 molecule, as the antibody activates spleen cells from Qa-2 negative strains. Although class I molecules are not usually considered as activation Ag, these data demonstrate their potential for involvement in signal transduction.

Cross-linking of Ly 6-linked alloantigens: association between ThB and Ly 5.

The bifunctional cross-linking reagent dithiobis(succinimidyl propionate) (DSP) was used to cross-link 125I surface-labelled glycoproteins from viable thymocytes. The cells were solubilized, and the cross-linked material immunoprecipitated and analysed by SDS-PAGE. When DSP cross-linked thymocyte material was immunoprecipitated with either anti-ThB or anti-Ly 5 monoclonal antibodies, and then cleaved, molecules with masses identical to Ly 5 (Mr 180 kD) and ThB (Mr 16-18 kD) were obtained. However, if the cross-linker was not cleaved, the intact product had a molecular mass of greater than 200 kD. The identity of these co-precipitated, cross-linked moieties was formally proved by limited proteolysis peptide map analysis. The data indicated that the ThB and Ly 5 antigens were associated on the thymocyte cell surface but no such association could be found on peripheral lymphocytes. The ThB-Ly 5 interaction may indicate an association relevant to the differentiation of thymocytes.

Structure and specificity of T cell receptor gamma/delta on major histocompatibility complex antigen-specific CD3+, CD4-, CD8- T lymphocytes.

Analyses of TCR-bearing murine and human T cells have defined a unique subpopulation of T cells that express the TCR-gamma/delta proteins. The specificity of TCR-gamma/delta T cells and their role in the immune response have not yet been elucidated. Here we examine alloreactive TCR-gamma/delta T cell lines and clones that recognize MHC-encoded antigens. A BALB/c nu/nu (H-2d)-derived H-2k specific T cell line and derived clones were both cytolytic and released lymphokines after recognition of a non-classical H-2 antigen encoded in the TL region of the MHC. These cells expressed the V gamma 2/C gamma 1 protein in association with a TCR-delta gene product encoded by a Va gene segment rearranged to two D delta and one J delta variable elements. A second MHC-specific B10 nu/nu (H-2b) TCR-gamma/delta T cell line appeared to recognize a classical H-2D-encoded MHC molecule and expressed a distinct V gamma/C gamma 4-encoded protein. These data suggest that many TCR-gamma/delta-expressing T cells may recognize MHC-linked antigens encoded within distinct subregions of the MHC. The role of MHC-specific TCR-gamma/delta cells in immune responses and their immunological significance are discussed.

The 33,000 protein precipitated by Ly-6A.2-specific antibodies is not associated with the Ly-6 polymorphism.

In this study, the relative mass of the Ly-6A.2 antigen was shown to be 12,000-14,000, in contrast to initial studies which showed the relative mass to be 33,000. Using polymorphic Ly-6-specific antibodies, the 33,000 molecules could be immunoprecipitated from surface-iodinated thymocytes of Ly-6A.2+, Ly-6A.2- strains and a Ly-6A.2- mutant cell line BW(Thy-1-e). This clearly demonstrated that 33,000 molecules were not associated with the Ly-6 polymorphism. By contrast, when biosynthetically labeled Ly-6A.2+ spleen cell lysates were analyzed, the major species immunoprecipitated by the polymorphic Ly-6A.2-specific antibody was 12,000-14,000, although a minor 33,000 species were also evident. The Ly-6A-specific antibody D7 which detects a monomorphic epitope on the Ly-6A molecule could immunoprecipitate the 12,000-14,000 molecules from surface-labeled cells. By contrast, the Ly-6A.2-specific antibodies detecting the polymorphic Ly-6A.2 determinant could not, though the reasons for this difference are not clear. Thus 12,000-14,000 molecules were only immunoprecipitated from Ly-6A.2+ cells, whereas 33,000 molecules were precipitated from both Ly-6A.2+ cells and Ly-6A.2- cells. These findings suggest that the 33,000 molecules immunoprecipitated by 5041-24.2 are most likely to be an unrelated protein, possibly cross-reactive with some Ly-6A.2 antibodies.

Interrelationships of the "Ly-6 complex" antigens.

Competitive binding studies and immunoprecipitation experiments define at least five distinct epitopes encoded by Ly-6-linked genes--Ly-6A.2, Ly-6B.2, Ly-6C.2, Ly-6D.2, and ThB. Ly-6A.2, a 33 kd protein, and Ly-6D.2 are closely overlapping epitopes that can be distinguished by their unique thymus reactions of 10-20% or greater than 90%, respectively. Similarly, the Ly-6C.2 antigen present on a 14 kd moiety loosely overlaps the Ly-6B.2 antigen. Ly-6C.2 and Ly-6B.2 antigens are distinct from Ly-6A.2 and Ly-6D.2, however. ThB is a 16-18 kd antigen which is not associated on the cell surface with any other "Ly-6" antigens. In addition, independently derived antibodies made to the Ly-6C.2 antigen detect an identical epitope, as do antibodies to Ly-6A.2 and Ly-6B.2. These results imply the existence of a single antigenic site on each of these molecules.