Characterization of a membrane surface glycoprotein associated with T-cell activation

A cell surface antigen (gp140) was previously shown to exist on T cell subsets as well as on monocytes and macrophages in normal peripheral blood. Elevated expression of this antigen was associated with immune system disorders, acute lymphocytic leukemias, and in vitro activation of T cells. The antigen could be identified with monoclonal antibody (MAb) T305. Gp140 was a biosynthetic product of T cells because it could be labeled with [3H]leucine or [3H] glucosamine. Biochemical studies of gp140 used high performance liquid chromatography with nitrocellulose blotting to isolate aliquots suitable for 125I radiolabeling and immunoprecipitation to demonstrate: a) a reduction in m.w. of gp140 KD to 90 KD after deglycosylation by trifluoromethanesulfonic acid, b) alteration of isoelectric point from 4.1 to 5.7 after neuraminidase treatments, c) absence of N-linked sugars based on resistance to endoglycosidase F, d) resistance to trypsin and chymotrypsin digestion but susceptibility to pronase, and e) presence of sialic acid and lactosaminoglycan as O-linked sugars. Gp140 could be labeled with the periodate/NaB[3H]4 technique, indicating its similarity to a class of sialoglycoproteins previously described on activated T-cells in mouse and man. The antigenic epitope recognized by MAb T305 contains sialic acid linked (2----3) to galactose; however, periodate oxidation of the exocyclic ring of sialic acid did not affect binding by MAb T305. In an attempt to determine the functional role of gp140, we tested the ability of MAb T305 to block: a) proliferation of peripheral blood lymphocytes to mitogens, b) response to interleukin 2 (IL 2) of an IL 2 dependent T cell line, and c) growth of a T-ALL derived cell line. No inhibition of proliferation or growth was noted. Although the function of gp140 remains unknown, its association with lymphocyte activation and certain disease states suggests that it may provide a target for modulation of the immune response. These studies characterize the structural features of gp140 and further define the epitope recognized by MAb T305.     

Activation of human thymocytes via the 50KD T11 sheep erythrocyte binding protein induces the expression of interleukin 2 receptors on both T3+ and T3- populations

Recent studies have demonstrated that the 50KD T11 molecule is a surface component of a macrophage-independent alternative pathway of human T cell activation that is unrelated to the T3/Ti antigen-MHC receptor complex. Given the expression of T11 on all human thymocytes, it was of interest to determine whether they could be activated via this pathway. The triggering of T11 by monoclonal antibodies anti-T112 and anti-T113, directed at two unique epitopes on the molecule, induced IL 2 receptor expression on both T3+ and T3- thymocytes but did not induce IL 2 production. Consequently, in contrast to peripheral blood T cells, thymocytes did not proliferate in response to anti-T112 and anti-T113 in the absence of exogenous IL 2. These studies suggest that IL 2 receptor gene activation precedes IL 2 gene activation in T cell development. The ability of the alternative pathway of T cell activation to induce IL 2 receptor expression on T3- thymocytes implies that the T11 molecule may have an important role in early thymocyte ontogeny.     

Production of interleukin 2 (IL 2) by salivary gland lymphocytes in Sj?gren's syndrome. Detection of reactive cells by using antibody directed to synthetic peptides of IL 2

Because abnormalities in interleukin 2 (IL 2) production have been reported in the blood of patients with certain autoimmune diseases, we have examined the lymphocytes from patients with Sj?gren's syndrome (SS) in which it is possible to obtain simultaneous samples of inflammatory site (i.e., salivary gland) lymphocytes and blood lymphocytes. We found that IL 2 production by peripheral blood lymphocytes (PBL) after mitogen stimulation was markedly diminished (4 +/- 2 U/ml) in 8/32 SS patients. However, salivary gland lymphocytes (SGL) from six out of six SS patients (including three patients with low IL 2 production by their PBL) had a high level of IL 2 production (97 +/- 32 U/ml), suggesting that IL 2 production by inflammatory site lymphocytes may differ from blood lymphocytes in the same patients. Low IL 2 production by a patient's PBL was not correlated with the patient's age, duration of disease, immunoglobulin level, or presence of antinuclear antibodies. Low IL 2 production was associated with a decreased ratio of Leu-3a/Leu-2a positive cells (p less than 0.05) and with an increased proportion of "activated" T cells expressing HLA-DR and gp140 (p less than 0.05). To determine the proportion of PBL and SGL containing cytoplasmic IL 2-like material, we used affinity-purified rabbit antibodies prepared against chemically synthesized peptides of human IL 2. Before mitogen stimulation, PBL were not stained by these antibodies (less than 1% reactive cells), whereas SGL T cells eluted from the salivary gland of SS patients contained a small (3.4% +/- 1.8) proportion of reactive cells. A similar proportion (2.4% +/- 1.2) of reactive cells was noted when frozen tissue sections of salivary gland biopsies were examined with these antibodies. After mitogen stimulation, 35% +/- 17 of PBL and 56% +/- 18 of SS SGL were specifically stained with anti-IL 2 peptide antibodies. In summary, these studies demonstrate a significant difference in IL 2 production between PBL and SGL of the same patients. Furthermore, antibodies against IL 2 peptides provide a powerful tool for detection of T cells producing IL 2 in vitro and in situ, and for understanding the role of this lymphokine in pathogenesis.     

The cell cycle dependence of thermotolerance. III. HeLa cells heated at 45.0 degrees C

We have extended our studies on the cell cycle dependence of thermotolerance to include HeLa cells heated at 45.0 degrees C to compare the results to Chinese hamster ovary (CHO) cells. We found that asynchronous HeLa cells were more resistant to heat than CHO cells but showed a similar development and decay of thermotolerance. Flow cytometry (FCM) was used to study redistributions in the cell cycle after an initial heat dose. Cells heated for 35 min at 45.0 degrees C were delayed in G1 by about 7 h compared to controls, with delays in late S and G2/M phase also. The heat sensitivity varied through the cell cycle; G1 cells were the most resistant to heat, while S-phase cells were uniformly sensitive throughout S phase, and G2 cells were resistant. Thermotolerance could be induced and expressed in early or late S-phase cells, but to a lesser extent than for G1 cells. The results were similar in many respects to CHO cells, but there were significant differences.     

The localization and rate of disappearance of a synaptic vesicle antigen following denervation

Summary A proteoglycan-specific antiserum has been used to monitor the effects of denervation in the electric organ of Torpedo marmorata. The antiserum was produced by injecting a highly purified synaptic vesicle fraction prepared from the electric organs of Torpedo marmorata. Following absorption the serum appears to be specific towards synaptic vesicles. The ultrastructural localization of the antigen determined by immuno-electron microscopy confirmed the specificity of the antiserum and showed that it did not crossreact with the proteoglycans of the basal lamina. The rate of disappearance of the vesicle proteoglycans following denervation was evaluated by means of the antiserum and was compared to the rate of disappearance of other vesicular and nerve terminal-associated markers. The results suggest that degeneration affects the vesicular constituents at varying rates resulting in a progressive disappearance of the entire functional capacity of the synaptic vesicles.     

Investigations into the sequence-selective binding of mithramycin and related ligands to DNA.

The preferred binding sites for mithramycin on four different DNA fragments have been investigated by DNAase I footprinting. Sites containing at least two contiguous GC base pairs are protected by the antibiotic, the preferred binding site consisting of the dinucleotide step GpG (or CpC). Related antibiotics chromomycin and olivomycin produce similar, but not identical footprinting patterns suggesting that they can recognize other sequences as well. All three antibiotics induce enhanced rates of enzyme cleavage at regions flanking some of their binding sites. These effects are generally observed in runs of A and T and are attributed to DNA structural variations induced in the vicinity of the ligand binding site. The reaction of dimethylsulphate with N7 of guanine was modified by the presence of mithramycin so that we cannot exclude the possibility that these antibiotics bind to DNA via the major groove.     

DNA repair, DNA synthesis and cell cycle delay in human lymphoblastoid cells differentially sensitive to the cytotoxic effects of nitrogen mustard

Two cloned human lymphoblastoid cell lines, Raji and TK6, differ in their sensitivity to the cytotoxic effects of nitrogen mustard (HN2). Raji cells exhibit a biphasic response with an initial D value of 0.06 microgram/ml and a final slope of 0.25 microgram/ml. TK6 cells were considerably more sensitive, D0 value 0.02 microgram/ml. Dose-response relationships for delay in cell cycle progression were measured using flow cytometry. Delay in S-phase traverse was concentration-dependent in both cell lines, and at a given concentration was 2-fold greater in TK6 than in Raji. Numbers of crosslinks (determined by alkaline elution) increased linearly with increasing HN2 concentration and were approximately 2-fold higher in TK6 than in Raji. At equal levels of DNA crosslinks, rates of removal were similar in both cell lines. Inhibition of [3H]TdR uptake was concentration-dependent and the extent of inhibition was similar in both cell lines. Recovery from HN2-induced inhibition of cell cycle progression markedly preceded recovery from inhibition of [3H]TdR incorporation suggesting that nucleotide pools are markedly perturbed in HN2-treated cells. The difference in sensitivity of these two cell lines cannot be adequately explained by differences in amounts of initial DNA damage, rates of repair, differential S-phase delay or rate of loss of DNA crosslinks.     

Two new monoclonal antibodies (LN-1, LN-2) reactive in B5 formalin-fixed, paraffin-embedded tissues with follicular center and mantle zone human B lymphocytes and derived tumors

Two monoclonal antibodies (LN-1, LN-2) reactive with B lymphocytes in B5 formalin-fixed, paraffin-embedded tissue sections have been produced by utilizing cell extracts from pokeweed mitogen-stimulated peripheral blood lymphocytes and diffuse histiocytic lymphoma SU-DHL-4 cells, respectively. Both monoclonal antibodies were initially identified by indirect immunofluorescence screening techniques on paraformaldehyde-acetone-fixed cell preparations. Specificity screens with 36 well-characterized human lymphoma and leukemia cell lines showed that both LN-1 and LN-2 stained cell lines of B cell lineage but were unreactive with those of T cell or, with one exception, myeloid derivation. Null cell acute lymphoblastic leukemia cell lines were found to be LN-2+ but LN-1-. The B cell specificity of these reagents was confirmed on 15 lymphoma and 17 leukemia biopsy specimens by using indirect immunofluorescence techniques. Immunoperoxidase staining of sections from B5-fixed, paraffin-embedded human lymphoid tissues showed that LN-1 bound to the cell membrane and cytoplasm of germinal center cells whereas LN-2 stained the nuclear membrane and cytoplasm of germinal center and mantle zone B lymphocytes as well as interfollicular histiocytes and thymic medullary dendritic cells. Both monoclonal antibodies failed to stain cortical thymocytes, lymph node T cells, and peripheral blood T and myeloid cells. Immunoperoxidase staining of 20 nonlymphoid human organs and tissues revealed that LN-1 reacted positively with red blood cell precursors of the bone marrow, ciliated epithelial cells of the bronchus, distal tubular cells of the kidney, and ductal cells from several organs including the breast and prostate. In contrast, LN-2 was unreactive with all human nonlymphoid organs and tissues including the bone marrow. Indirect immunofluorescence staining of a panel of 26 solid tumor cells lines showed that LN-1 was reactive with the majority of epithelium-derived cell lines, glioblastomas, and astrocytomas but was unreactive with neuroblastomas, small cell carcinoma of the lung, and sarcomas. LN-2 was unreactive with 25 of 26 of the solid tumor cell lines by these techniques. Immunobiochemical studies have shown that LN-1 recognizes a cell surface sialoantigen whereas LN-2 is directed against a 35,000 dalton nuclear membrane protein. Because of their high specificity for B cell tumors and their ability to stain B5-fixed, paraffin-embedded tissues, LN-1 and LN-2 are useful reagents for the diagnosis and classification of the human lymphomas and leukemias.     

Ta1, a novel 105 KD human T cell activation antigen defined by a monoclonal antibody

By using a murine monoclonal antibody produced against an IL 2-dependent human T cell line, we defined a T lineage-specific molecule, termed Ta1, that is expressed strongly on activated T lymphocytes of both the T4 and T8 subsets, as well as on T cell lines and clones, but only weakly on a fraction of resting T cells. SDS-PAGE analysis of immunoprecipitates from 125I-labeled, activated T cells demonstrates a single major band of apparent m.w. 105 KD under both reducing and nonreducing conditions. Unlike anti-IL 2 receptor antibodies, anti-Ta1 does not inhibit T cell proliferative responses to mitogen, antigen, or IL 2-containing medium. Moreover, anti-Ta1 has no effect on T cell-mediated cytotoxicity. Ta1 appears to be a novel human T cell-specific activation antigen that may serve as a useful marker of T cell activation in human disease.