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In this study we reported that on T cell receptor (TcR) gamma/delta+ cells from three cell lines Peer, MOLT-13 and ICRF-1, the T cell antigen CD7 (gp40) can be directly involved in the activation process. This is shown by a rapid increase in cytoplasmic free calcium after stimulation of these cells with an anti-CD7 monoclonal antibody (mAb). Activation through CD7 was further confirmed by measuring the production of interleukin 2 in ICRF-1 cells stimulated with anti-CD7 mAb. In addition induction of mRNA for tumor necrosis factor (TNF)-alpha and TNF-beta in Peer and for granulocyte-macrophage-colony-stimulating factor in MOLT-13 was observed in these anti-CD7-stimulated cells. The same anti-CD7 antibody was unable to activate TcR alpha/beta+ Jurkat cells or normal resting peripheral blood T lymphocytes. We further showed that normal resting TcR gamma/delta+ cells were likewise activated via the CD7 molecule. TcR gamma/delta+ cells obtained from a patient with acute lymphoblastic leukemia 3 months after autologous bone marrow transplantation were induced to proliferate, as measured by [3H]thymidine incorporation after stimulation with anti-CD7 mAb but not with anti-CD3 mAb. Interestingly TcR alpha/beta+ cells from the same donor tested in parallel were not stimulated by anti-CD7 but by anti-CD3 mAb. In essence these findings contribute to the idea that on TcR gamma/delta+ cell, the CD7 antigen could play an important role during T cell differentiation.

The expression on several established human glioma cell lines of two well-defined differentiation antigens, HLA-DR and the common acute lymphoblastic leukemia antigen (CALLA) has been demonstrated. Rabbit anti-CALLA antiserum and monoclonal anti-Ia antibodies specifically lysed glioma cells in the presence of complement. Absorption of anti-CALLA antiserum and anti-Ia antibodies by glioma cells abolished their cytotoxicity against blasts isolated from a common acute lymphoblastic leukemia. Immunoprecipitation of solubilized glioma cells by monoclonal anti-Ia antibodies revealed two polypeptide chains of 28 and 33 kDa, whereas the anti-CALLA antiserum precipitated a single polypeptide chain of 100 kDa.

Two monoclonal antibodies (mAb) directed against idiotypic determinants of the T cell receptor (anti-Ti) from HPB-ALL cells induce interleukin 2 (IL2) production in Jurkat T cells without evidence of binding to these cells as judged by fluorescence-activated cell sorter (FACS) analysis, indirect antibody-binding radioimmunoassay and direct binding studies with 125I-labeled mAb. The IL2 response induced by these mAb observed both in the presence and absence of phorbol myristate acetate was in the range of that obtained when Jurkat cells were stimulated with phytohemagglutinin or anti-T3 mAb (Leu 4). The idiotypic specificity of the two anti-HPB-ALL Ti mAb was demonstrated by several criteria. Both mAb bound specifically to HPB-ALL cells as determined by radioimmunoassay or FACS analysis but not with 8 other T cell lines. The anti-HPB-ALL Ti mAb precipitated a disulfide-linked heterodimer of 85 kDa only from 125I-labeled HPB-ALL cells and not from other cell lines tested. Incubation of HPB-ALL cells with anti-T3 abrogated the expression of T3 and induced co-modulation of the idiotypic structures detected by the two anti-HPB-ALL Ti mAb. Conversely, incubation of HPB-ALL cells with either one of the anti-Ti mAb abrogated the expression of T3 and of the idiotypic structures. Our results suggest that mAb with an apparent unique specificity for the receptor of the immunizing T cell line HPB-ALL can activate Jurkat cells by a very weak cross-reaction with these cells, which is not detectable by conventional binding tests.

Recombinant interferon-gamma (IFN-gamma) induced the expression of HLA-DR when added to the culture medium of HLA-DR- melanoma cell lines. In addition, IFN-gamma induced the expression of another class II antigen, HLA-DC, on a HLA-DR+ and -DC-melanoma cell line and to a lower level on a -DR- and -DC-melanoma line. IFN-gamma also enhanced the expression of HLA-ABC and beta 2-microglobulin, as well as HLA-DR on DR+ melanoma cells. In contrast, IFN-alpha gave no induction of expression of HLA-DR and DC on two DR- melanoma lines, while it did enhance the expression of HLA-ABC and of beta 2-microglobulin. The expression of 3 out of 6 melanoma-associated differentiation antigens was enhanced by IFN-gamma treatment. The modulation of antigens by IFN-gamma was both dose and time dependent. A minimum incubation time of 48 h was necessary for the appearance of HLA-DR on the two HLA-DR- melanoma lines, whereas HLA-ABC and beta 2-microglobulin were already increased after 24 h. A dose of 20 U/ml IFN-gamma started to induce the expression of HLA-DR and DC on melanoma cells GLL-19 and Me-43 and a plateau of maximum antigen expression was reached with 100 U/ml. Analyses of IFN-gamma-treated cells by flow microfluorometry showed a homogeneous distribution of increased staining intensity rather than the appearance of two cell populations. Immunoprecipitation experiments using detergent-solubilized 125I-labeled membrane proteins of IFN-gamma-treated melanoma cells and a monoclonal anti-HLA-DR antibody confirmed the presence of HLA-DR antigens. When IFN-gamma-treated cells were cultured without IFN the induced or enhanced expression of HLA antigens was reversible. Eight days after removal of IFN, the HLA-DR level was reduced by more than 90% and the level of HLA-ABC and beta 2-microglobulin by more than 50%. The demonstration of the ability of HLA-DR- melanoma cells to express HLA-DR after IFN-gamma treatment was extended to cells from other types of tumor such as gliomas, colon carcinomas and one cervical carcinoma cell line.

The expression at the cell surface level of two previously defined human Ia antigen subsets, NG1 and NG2, was studied. Two monoclonal hybridoma antibodies, D1-12 (anti-NG1) and D4-22 (anti-NG2), labeled with distinct fluorochromes, were used as probes. The results indicate that NG1 and NG2 molecules are simultaneously expressed on the same cell, and moreover, that they are identifiable as spatially separate structures. In addition, when a series of 6 other anti-Ia monoclonal antibodies previously described were analyzed in a checkerboard test of binding inhibition at the cell surface level, they were found to closely mimic the reactivity pattern of either D1-12 or D4-22 antibody, suggesting that NG1 and NG2 Ia subsets express independent clusters of highly immunogenic epitopes.

The T3 complex is known to be expressed on the cell surface of mature T cells together with either the alpha-beta heterodimeric T cell receptor (TCR) or the TCR gamma protein. In a number of immature T cell malignancies, however, T3 has been described exclusively in the cytoplasm. We have investigated five such T cell lines with cytoplasmic T3 and could demonstrate by biosynthetic labeling the presence of the alpha and beta chains of the TCR in the cytoplasm of two of them, CEM and Ichikawa. No surface TCR alpha-beta protein could be detected by staining with the WT31 antibody. These observations, therefore, argue against the concept that expression of the TCR alpha chain controls the surface expression of the T3/TCR complex. Interestingly, phorbol 12-myristate 13-acetate (PMA) induced cell surface expression of T3 protein in these two cell lines only. Moreover, on surface-iodinated CEM cells no association of T3 and TCR molecules could be demonstrated after treatment with PMA, and expression of TCR alpha and beta chains was limited to the cytoplasm. In Ichikawa cells, however, PMA induced surface expression of a mature T3/TCR complex. Our findings indicate that separate regulatory mechanisms may exist for the surface expression of the T3 proteins and for the assembly of the T3/TCR complex.

A new heterodimeric structure, Tp135-145, which can mediate interleukin 2 (IL2) production and Ca2+ mobilization by Jurkat cells is described. This structure was identified by a monoclonal antibody, MX24, on the surface of either T3/TcR+ or T3/TcR- human T cell lines as well as on B cell lines. Biochemical studies showed that antibody MX24 precipitated two polypeptide chains of 135 and 145 kDa, respectively, in lysates from 125I-labeled T cells. After reduction the 135-kDa polypeptide chain shifted to 140 kDa, whereas the molecular mass of the other polypeptide remained unchanged. The apparent molecular masses of the desialylated polypeptides differed by 5 kDa. No common peptide fragments between the two polypeptide chains were found after limited proteolysis by Staphylococcus aureus V8 protease. The expression of Tp135-145 was independent of the expression of the T3/TcR molecular complex. Incubation of Jurkat cells with anti-TcR or anti-T3 monoclonal antibody induced complete modulation only of the T3/TcR complex but not of Tp135-145. Conversely complete modulation of Tp135-145 was observed after incubation of these cells with MX24 antibody. Functional studies showed that anti-Tp135-145 antibody MX24 induced high levels of IL2 production in Jurkat cells. In addition, incubation of these cells with MX24 resulted in Ca2+ mobilization from internal stores. In peripheral blood, Tp135-145 was found to be expressed by 39%-76% of resting T cells in individual donors. Two-color flow microfluorimetry showed that the Tp135-145+ cells were equally distributed on the CD4+ and CD8+ subsets. Incubation of peripheral blood T cells with antibody MX24 resulted in IL2 production and cell proliferation. Taken together these results suggest that Tp135-145 is a novel surface molecule involved in antigen-independent pathway of T cell activation.

The expression on a significant number of thymocytes of idiotypic structures (Ti) restricted to HPB-ALL or Jurkat cells is demonstrated. As many as 2-4% of thymocytes were stained with anti-Ti HPB-ALL or anti-Ti Jurkat monoclonal antibodies, when analyzed by flow microfluorometry. Immunohistochemical localization studies performed on frozen thymus specimens of either fetal or pediatric origin indicated a scattered distribution of Ti-positive cells in both the cortex and the medulla. From lysates of 125I-labeled pediatric thymocytes, anti-Ti HPB-ALL and anti-Ti Jurkat monoclonal antibodies precipitated disulfide-linked heterodimers comparable to those precipitated from 125I-labeled HPB-ALL or Jurkat cells as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.

A novel surface molecule, Tp90, is described which appears to be involved in an antigen-independent pathway of human T lymphocyte activation. The Tp90 molecule was identified by a monoclonal antibody (mAb), MX20, obtained from a fusion using spleen cells of a mouse immunized with cells from two T cell leukemia lines, Jurkat and HPB-ALL. Biochemical data show that Tp90 is distinct and physically independent from the structures already known to be involved in T cell activation, namely T11, T44 or T3/TCR. These results were confirmed by antibody-induced antigen modulation experiments. Modulation of Tp90 had no effect on the expression of T3 and of the T cell receptor. Conversely, the expression of Tp90 was not affected by modulation of the T3/TCR molecular complex by either anti-T3 or anti-TCR antibody. Functional studies showed that anti-Tp90 mAb MX20 induced high levels of interleukin 2 production in Jurkat cells. Modulation of the T3/TCR complex significantly decreased the response of Jurkat cells to stimulation by antibody MX20, suggesting that the T3/TCR complex regulates the ability of the Tp90 molecule to induce IL 2 synthesis. In addition to its effect on Jurkat cells, anti-Tp90 mAb was found to be mitogenic for peripheral blood T cells. As the magnitude of the proliferative response elicited by anti-Tp90 mAb was lower than that induced by anti-T3 mAb, the possibility was considered that only a subpopulation of T cells is reactive with anti-Tp90. Indeed as determined by FACS analyses, only 3-14% of E-rosette-positive cells were stained with mAb MX20. In addition, multicolor flow cytometry analysis showed that the Tp90+ cells belong preferentially to the CD8 subset.

This report describes a surface molecule, Tp45, which appears to be involved in interleukin 2 production and Ca2+ mobilization by Jurkat cells. The Tp45 molecule was identified by a monoclonal antibody, MX13, on the surface of either T3/TCR+ or T3/TCR- human T cell lines. Biochemical data showed that mAb MX13 precipitated a single polypeptide chain of 45 kDa both under reduced and nonreduced conditions from lysates of 125I-surface-labeled cells. Sequential immunodepletion experiments using lysates of 125I-labeled T3/TCR+ cells showed that Tp45 was distinct from the alpha chain of the TCR complex. However, incubation of such cells with either anti-T3 or anti-TCR monoclonal antibody induced complete modulation of both the T3/TCR complex and Tp45. Conversely, complete modulation of both Tp45 and the T3/TCR complex was observed after incubation with anti-Tp45 antibody. Functional studies showed that anti-Tp45 antibody induced high levels of interleukin 2 production in Jurkat cells. In addition, incubation of these cells with the antibody resulted in Ca2+ mobilization from internal stores. Anti-Tp45 antibody reacted with 3-19% peripheral blood (E-rosette-positive) T cells in individual donors. The magnitude of the proliferative response elicited by anti-Tp45 antibody for peripheral blood T cells was lower than that induced by an anti-T3 antibody. This observation is compatible with the idea that only a subpopulation of T cells is reactive with anti-Tp45. Multicolor flow cytometry analysis showed that the Tp45+ cells belong preferentially to the T8 subset.

As shown by previous studies, the sheep erythrocyte-binding T11 molecule is involved in T cell activation, as well as in mechanisms of specific allogeneic target cell lysis. In this study, we utilized two anti-T11 monoclonal antibodies (mAb) that inhibited the specific cytolytic activity of mixed lymphocyte culture (MLC)-activated T cells to analyze, at the clonal level, the involvement of T11 molecules in (a) antigen-specific vs. nonspecific mechanisms of target cell lysis, and (b) antigen-driven T cell proliferation and interleukin 2 (IL 2) production vs. IL 2-induced cell proliferation. In contrast to anti-T3 or anti-T8 mAb, antibodies to T11 molecules inhibited the cytolytic activity of MLC-derived allospecific clones in a uniform manner. In addition, anti-T11 antibodies inhibited the specific activity of cytotoxic T lymphocyte clones resistant to anti-T3 antibodies, even after antibody-induced modulation of T3 molecules (while anti-T3 mAb had no effect). Similarly, anti-T11 antibodies inhibited the alloantigen-induced proliferation and IL 2 release of alloreactive clones independent of their T4+ or T8+ phenotype. The inhibitory activity of anti-T11 antibodies appears to be confined to antigen-specific T cell functions since neither natural killer-like activity of cytotoxic T lymphocyte clones nor the IL 2-induced clonal proliferation was affected. Thus, our results indicate that T11 molecules are functionally involved in antigen recognition by T cell regardless of their function and T4/T8 phenotype. The possible mechanisms of anti-T11 antibody-mediated inhibition are discussed.

There is experimental evidence that the CD4 molecule participates in the antigen-driven activation of T cells expressing this surface glycoprotein. Whether CD4, a member of the immunoglobulin supergene family, acts as a ligand-binding molecule and/or is directly involved in the activation pathway has yet to be established. In this study, we show that human CD4+ lymphocytes can be activated by exposure to the anti-CD4 monoclonal antibody (mAb) B66. Normal peripheral blood CD4+ cells were induced to proliferate and to synthesize interleukin 2 (IL 2) by the antibody. The specificity of the antibody stimulatory activity was tested by using IL 2-producing clones bearing either CD4 or CD8 on their surface. IL 2 production was induced by mAb B66 in CD4+, but not CD8+, clones, whereas both types of clones responded to stimulation by the anti-CD3 mAb Leu-4. Despite its unique stimulatory activity, mAb B66 shared with other anti-CD4 antibodies the ability to inhibit the specific cytolytic activity of CD4+ effector cells. These results clearly indicate that cross-linking of surface CD4 molecules with appropriate antibodies can fully activate CD4+ lymphocytes. Whether the natural ligand for CD4 can trigger this activation pathway remains to be defined.