Biomedical subjects

E M Shevach

Publications and source records attributed to E M Shevach.

At least 109 records · Page 6Linked to original sources

PubMed1988

Characterization of a cell surface-expressed disulfide-linked dimer involved in murine T cell activation.

We have produced a hamster mAb, H1.2F3, which was derived by immunization with a murine TCR-gamma delta + epidermal T cell line. H1.2F3 immunoprecipitates a cell surface-expressed disulfide-linked dimer that has a m.w. of 85,000 under non-reducing conditions and consists of subunits of 35,000 to 39,000 m.w. This dimer is distinct from the CD3-associated TCR-gamma delta complex (CD3/TCR), inasmuch as H1.2F3 does not co-precipitate or co-modulate with the CD3/TCR complex and recognizes an Ag with a single-peptide backbone of 22,000 m.w. after N-Glycanase treatment. H1.2F3 is weakly reactive with a small percentage of cells from unfractionated thymus, spleen, or lymph node, but reactivity with both T and B lymphocytes is markedly enhanced by a brief period of stimulation with Con A or PMA in vitro. This enhancement requires de novo protein synthesis. Enhanced expression of the H1.2F3 Ag can also be induced in vivo by injection of Con A or anti-CD3. H1.2F3 is a potent stimulator of T, but not B, cell proliferation in the presence of PMA and FcR-bearing accessory cells. These functional and biochemical studies strongly suggest that the Ag recognized by H1.2F3 is the murine homologue of the human CD28 Ag recognized by mAb 9.3.

Aging↗

PubMed1988

Thy-1- and Ly-6-mediated lymphokine production and growth inhibition of a T cell hybridoma require co-expression of the T cell antigen receptor complex.

Repetitive subcloning of an Ag-specific T cell hybridoma yielded a variant that lacked functional mRNA for the Ag receptor (Ti, T cell Ag receptor alpha/beta heterodimer) beta-chains and failed to express CD3/Ti on the cell surface. Transfection with the original Ti alpha- and beta-chain genes restored CD3/Ti expression to normal levels. Whereas the parental T cell hybridoma produced IL-2 when stimulated with mAb against CD3, Thy-1, and Ly-6, the CD3/Ti negative cell failed to do so. Reconstitution of CD3/Ti expression restored normal IL-2 production in response to these mAb. A separate response to activation, the inhibition of transformed growth, was also dependent upon co-expression of CD3/Ti. These data demonstrate that cell surface expression of CD3/Ti is required for IL-2 production and growth inhibition initiated by two distinct activating molecules, and suggest that CD3/Ti may be a final common pathway for many transmembrane activation signals.

Animals↗

PubMed1988

T cell receptor gamma/delta chain diversity.

The TCR-gamma and -delta chains of six murine hybridomas were compared by one-dimensional SDS-PAGE and two-dimensional NEPHGE/SDS-PAGE analysis. This allowed the identification of three distinct gamma chains (gamma a, gamma b, and gamma c) and three distinct delta chains (delta a, delta b, and delta c). Four gamma/delta chain combinations (gamma a delta a, gamma b delta b, gamma b delta c, and gamma c delta a) were observed. These results indicate that multiple forms of the delta chain are expressed and suggest that the delta chains are encoded for by an Ig-like rearranging gene. This delta chain polymorphism significantly enhances the potential diversity of TCR-gamma/delta, which may be of importance for a better understanding of the putative ligand(s) recognized by this receptor.

Animals↗

PubMed1988

Characterization of T cell receptors on resident murine dendritic epidermal T cells.

Cell lines derived from Thy-1+ dendritic epidermal cells (Thy-1+DEC) display a marked heterogeneity in T cell receptor (TcR) expression including CD3-associated alpha/beta, C gamma 1/delta or C gamma 2/delta and C gamma 4/delta TcR. In order to investigate whether this heterogeneity is primarily imposed by in vitro culture conditions or, alternatively, is already present within the epidermis, we studied TcR expression by Thy-1+DEC in situ and on freshly isolated epidermal cell suspensions greatly enriched for Thy-1+DEC. Immunolabeling experiments showed that resident Thy-1+DEC are CD45+, Thy-1+, CD3+, TcR V beta 8-, CD5-, CD4- CD8-, CD25- lymphocytes. Immunoprecipitation of lysates from 125I-surface-labeled Thy-1+DEC-enriched epidermal cells with anti-CD3 epsilon, anti-C gamma 1,2,3 and anti-C gamma 4 antibodies, respectively, and subsequent analysis of the precipitates by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed only CD3-associated 35 kDa/45 kDa gamma/delta heterodimers. The demonstration of TcR heterodimers on resident Thy-1+DEC strongly implies that these cells are functional T cells. The selective expression of C gamma 1/delta (C57BL/6) and C gamma 1/delta or C gamma 2/delta (C3H/He) TcR makes these cells useful for the study of gamma/delta TcR function.

Animals↗

PubMed1988

A single cDNA encodes multiple Ly-6 antigenic specificities.

To determine the relationship between the structure and function of proteins coded for by the Ly-6 gene complex, we have transfected a cDNA for a Ly-6.2 specificity into COS-7 cells. A number of monoclonal antibodies which have been shown to be capable of inducing T cell activation and which have been previously considered to recognize distinct proteins all reacted with the same transfected gene product. The approach used in these studies should be useful to further elucidate the complexities of the Ly-6 alloantigen system.

Animals↗

PubMed1988

Induction of IL-2 receptor expression in vivo: response to concanavalin A.

The requirements for the induction of IL-2 receptor expression following lymphocyte stimulation in vitro are well understood but little is known about the role of IL-2 and the IL-2 receptor in lymphocyte activation in vivo. Following the subcutaneous injection of concanavalin A, cells from popliteal and lumbar lymph nodes draining the footpad became enlarged and expressed the IL-2 receptor. At the peak of the response, 9-15 hr after injection, more than 70% of all cells were IL-2 receptor positive; the majority were T cells of both Lyt-2+ and L3T4+ subsets. IL-2 receptor expression was closely associated with both spontaneous and IL-2-driven proliferation. Analysis of the biodistribution of 125I-labeled concanavalin A revealed that much of the injectate remains in the footpad injection site but the mitogen also accumulates in the draining lymph nodes and eventually in the major organs. This model of lymphocyte activation was used to demonstrate that in vivo cyclosporin A inhibits both IL-2 receptor expression and the induction of spontaneous proliferation.

Animals↗

PubMed1988

Mitogenic activation of EL-4 cells does not require surface Thy-1 expression.

The ability of monoclonal anti-Thy-1 antibodies to stimulate IL-2 production and T-cell proliferation has raised the possibility that Thy-1 may play an important role in T-cell activation. To examine this postulated role we have produced Thy-1-negative variants of the murine T lymphoma EL-4 by mutagenesis with ethyl methanesulfonate (EMS) and subsequent negative selection with anti-Thy-1 monoclonal antibodies (mAbs) and complement. Although the parental EL-4 cell line produced interleukin-2 (IL-2) in response to concanavalin A (Con A), phytohemagglutinin, anti-Thy-1 mAbs, and an anti-T3 mAb, as well as after exposure to phorbol myristate acetate (PMA), only PMA was capable of inducing IL-2 production by several Thy-1-negative cell lines. The loss of responsiveness to cell surface stimulatory ligands appeared to be correlated with loss of Thy-1 expression because mutagenized cells selected for high levels of Thy-1 expression all responded normally to Con A. However, when Thy-1 expression was reconstituted in the "nonresponder" (Thy-1-negative) cell lines either by transfection of a Thy-1.2 gene or by 5-azadeoxycytidine treatment, the revertant cell lines were still unable to produce IL-2 when stimulated with Con A, anti-Thy-1, or anti-T3. Furthermore, several other independently derived Thy-1-negative EL-4 cell lines responded normally to mitogens and mitogenic mAbs. Taken together, these results suggest that Thy-1 expression is not required for the T-cell activation process and that the EMS mutagenesis procedure resulted in an additional mutation(s) responsible for the inability of certain Thy-1-negative cell lines to be triggered by mitogens and mitogenic mAbs. These cell lines may prove to be valuable tools for further biochemical and molecular studies of the sequence of events associated with T-cell activation.

Animals↗

PubMed1988

Comparative aspects of T cell activation in vivo following stimulation with anti-CD3 MAB, allogeneic cells and Trypanosoma cruzi.

The in vivo administration of the immunosuppressive drug, Cyclosporin A (CSA), has allowed us to define IL-2 dependent and IL-2 independent pathways of T cell activation in vivo. Thus, CSA inhibited T cell activation and the production of IL-2 mRNA in the draining lymph node (LN) population following footpad injection of anti-CD3 mAb. In contrast, even though CSA completely inhibited the induction of IL-2 mRNA in the draining LN following the injection of allogeneic cells, T cell activation proceeded normally. In the present study, we have analyzed the effects of CSA on the T cell activation induced in vivo by T. cruzi. BALB/c and C57BL/6 mice were injected subcutaneously in the footpad with irradiated, cultured T. cruzi trypomastigotes (CMTs, clone sylvio-X10/4). CSA was delivered to the mice via an osmotic pump, Alzet 2001 at a concentration of 35mg/Kg/day. The injection of CMTs resulted in a dose dependent activation of the draining LN population including an increase in the number of cells, an increase in cell size, induction of expression of the IL-2 receptor and other T cell activation antigens (Ly-6, CD28), induction of responsiveness to IL-2, and a vigorous proliferative response when the freshly explanted node was cultured for 18 h in vitro in the presence of 3H-TdR. CSA markedly inhibited all of these parameters of T cell activation. Thus, the early T cell activation response observed after injection of irradiated T. cruzi CMTs appears to be mediated by an IL-2 dependent, CSA sensitive T cell activation pathway.

Animals↗

PubMed1987

Mechanism of action of cyclosporin A in vivo. I. Cyclosporin A fails to inhibit T lymphocyte activation in response to alloantigens.

Although cyclosporin A (Cy A) has been widely used clinically as a potent suppressor of organ allograft rejection and has been shown to block T lymphocyte activation in vitro by inhibiting the generation of interleukin 2 (IL-2) and other lymphokines, little direct evidence is available to support the view that the immunosuppressive effects of Cy A in vivo are mediated by a similar inhibition of the autocrine lymphokine cascade. We have used a quantitative assay for the assessment of the role of the IL-2/IL-2 receptor system in the activation of the draining popliteal lymph node population after the injection of allogeneic cells in the footpad to define the effects of Cy A on the early events of lymphocyte activation in vivo and to compare them with the effects of Cy A on lymphocyte activation in vitro. The administration of Cy A in vivo had no effect on alloantigen-induced increases in cell size, percentage of cells expressing the IL-2 receptor, the spontaneous or IL-2-driven proliferation of freshly explanted cells, or the induction of cytotoxic T lymphocyte activity. These findings raise major questions about the mechanism of action of Cy A in vivo and suggest that more experimentation is required to probe the mechanisms of Cy A-induced suppression of the response to allografts.

Animals↗

PubMed1987

Cell surface T3 expression requires the presence of both alpha- and beta-chains of the T cell receptor.

We have identified a surface T3- Jurkat variant which has a defective alpha-chain but which possesses an intact beta-chain. The transfection of a functional mouse alpha-chain into this human T cell induces the expression of surface T3 molecules associated with mouse alpha-human beta-heterodimers detected by anticlonotypic antibodies. Treatment of the transfectant with anti-T3, anti-mouse Ti-alpha, or anti-human Ti-beta antibodies clears all Ti-T3 complexes from the surface. These results demonstrate that functional alpha- and beta-chains are both required for expression of T3 on the cell membrane, and that the Ti heterodimers present and associated with T3 on Jurkat cells involve only alpha- and beta-chains.

Antibodies, Monoclonal↗

PubMed1987

Production of a T cell hybridoma that expresses the T cell receptor gamma/delta heterodimer.

We have produced a T cell hybridoma line by fusion of an IL-2-dependent, long-term T cell receptor (TCR) gamma/delta+ Thy-1+, bone marrow-derived, dendritic epidermal cell line to the BW5147 tumor line. The resultant hybridoma was rapidly growing, lymphokine independent, and expressed T3 in association with the TCR gamma/delta heterodimer. Several subclones of the hybridoma line produced easily detectable levels of IL-2 after stimulation by anti-T3 or Con A. The availability of these cloned cell lines should greatly facilitate further functional, biochemical, and molecular studies of the TCR delta chain.

Animals↗

PubMed1987

Receptor diversity of insulin-specific T cell lines from C57BL (H-2b) mice.

To characterize the T cell receptor repertoire in an immune response in which the Ia and nominal antigenic determinants are defined and limited, we have cloned and sequenced the expressed receptors from four independent, beef insulin-specific T cell lines from C57BL mice. Each of these lines responded to beef but not to the pork insulin, thus defining the nominal antigenic determinant recognized. Furthermore, each of these lines could only be presented antigen by B6 but not mutant B6.C-H-2bm12 antigen-presenting cells, thus defining the requisite Ia recognition or antigen-association site. In spite of this functional similarity in ligand specificity, each of these T cell lines was found to use different V alpha and V beta gene segments. Moreover, structural comparisons of implied protein sequences of each of these receptors showed no stretches of conserved amino acid residues that could be implicated in ligand interaction. However, the V alpha genes used by these four clones appeared considerably more homologous to each other than were their V beta genes.

Animals↗

PubMed1987

Identification of a T3-associated gamma delta T cell receptor on Thy-1+ dendritic epidermal Cell lines.

The murine epidermis contains a subpopulation of bone marrow-derived lymphocytes that have a dendritic morphology and that express Thy-1 and T3 cell-surface antigens but not other markers (L3T4 or Lyt-2) characteristic of mature peripheral T lymphocytes. An alternative type of T cell receptor was earlier identified on a subpopulation of murine thymocytes with a similar phenotype (T3+, L3T4-, Lyt-2-), but not on peripheral murine T lymphocytes. Two independently derived Thy-1+, L3T4-, and Lyt-2- dendritic cell lines of epidermal origin that express a T3-associated disulfide-linked heterodimer composed of a 34-kilodalton gamma-chain and 46-kilodalton partner (the delta chain) have now been identified. Analysis of N-linked glycosylation revealed that this receptor is similar to that detected on thymocytes. These results demonstrate that Thy-1+ dendritic epidermal cell lines can express gamma delta T cell receptors in vitro and suggest that Thy-1+ dendritic epidermal cells express such receptors in vivo. The localization of these gamma delta T cell receptor-expressing cells in the epidermis may be of importance for understanding the function of these receptors.

Animals↗

PubMed1987

B cell stimulatory factor 1 (interleukin 4) is a potent costimulant for normal resting T lymphocytes.

Resting T cells proliferate in response to B cell stimulatory factor 1 (BSF-1; interleukin 4) plus phorbol myristate acetate (PMA). This response is obtained with highly purified T cells and is density independent, suggesting that accessory cells are not required. Both L3T4+ and Lyt-2+ T cells respond to BSF-1 plus PMA. Although BSF-1 alone does not cause T cell proliferation, it maintains the viability of small, dense T cells, indicating that it acts on resting T cells. Furthermore, BSF-1 is required early in the proliferative response of resting T cells to BSF-1 plus PMA, further supporting the concept that it acts on G0 or early G1 cells. However, BSF-1 is also needed late in the first round of division of T cells stimulated with BSF-1 plus PMA. Removing BSF-1 at 24 h of stimulation prevents entry into S phase. These results indicate that BSF-1 is involved in both the induction of competence and in the progression phases of T cell division.

Animals↗

PubMed1987

Thy-1+ dendritic epidermal cells belong to the T-cell lineage.

The murine epidermis contains a population of dendritic, Thy-1+ cells (Thy-1+ DEC). Although it is now clear that these cells are of bone marrow origin, extensive morphological, histochemical, and cell-membrane marker studies have not definitively placed them in any known hematopoietic differentiation pathway. Based on the observation that Thy-1+ DEC can be propagated in vitro with Con A and interleukin 2, we have established three cell lines (Tehy 184, Tehy 245, and Yety 245) that can be continuously grown in medium with lectin-lymphokine-rich culture supernatants of rat spleen cells. With the exception of the loss of reactivity with anti-asialo-GM1 antibodies (Tehy 184 and Tehy 245) and the gain of interleukin 2 receptor expression, the cultured cell lines bear the same surface characteristics as freshly isolated Thy-1+ DEC: Thy-1+, Ly-5+, Lyt-1-, Lyt-2-, L3T4-, Ia-, sIg-. Using Southern and RNA gel blot analysis, we now demonstrate that these Thy-1+ DEC-derived cell lines exhibit various patterns of rearrangements in the gene complexes encoding the T-cell receptor (related) beta and gamma chains and contain mature and/or incomplete transcripts from the T-cell receptor alpha- and beta-chain genes, as well as transcripts from the receptor-related gamma-chain genes. Tehy 184 cells, the only cells containing both mature alpha- and beta-chain transcripts, react positively with the F23.1 monoclonal antibody, which recognizes the product of a subset of T-cell receptor beta-chain variable region gene segments. This antibody precipitates a surface protein of 84-88 kDa from these cells that after reduction separates into two 40- to 44-kDa chains, characteristic of Ti alpha/beta heterodimers. These data strongly suggest that Thy-1+ DEC belong to the T-cell lineage and point to the epidermis as a site either of immature thymocyte migration or of extrathymic T-cell differentiation.

Animals↗

PubMed1987

Induction of IL-2 receptor expression in vivo. Response to allogeneic cells.

Although the requirements for the induction of IL-2 receptor expression following lymphocyte stimulation in vitro have been well characterized, little information is available on the role of IL-2 and the IL-2 receptor in lymphocyte activation in vivo. We have established a quantitative assay for the measurement of IL-2-receptor-positive cells in the draining popliteal lymph node following the injection of allogeneic cells in the footpad. At the peak of the response more than 20% of the lymph node cells were IL-2-receptor-positive, and the majority of the IL-2-receptor-positive cells were Ly-2+ T cells and B cells. IL-2 receptor expression in vivo was closely associated with an increase in cell size and spontaneous and IL-2-driven proliferation, as well as with the induction of CTL activity specific for the donor strain. The model system described here should be useful in the further characterization of the mechanism of action in vivo of immunosuppressive drugs, antibodies, or lymphokines.

Animals↗

PubMed1986

Role of Ly-6 in lymphocyte activation. I. Characterization of a monoclonal antibody to a nonpolymorphic Ly-6 specificity.

In studies with alloantisera and monoclonal antibodies (mAb) a number of antigenic determinants have been defined that are the products of the Ly-6 locus on murine chromosome 2 and that are expressed primarily on B and T lymphoid cells. It remains controversial whether these antigenic determinants are encoded by a single gene or a multigene complex. We have characterized a new rat mAb, D7, which recognizes a cell surface antigen whose expression on nonactivated peripheral lymphocytes varies from strain to strain. The phenotype of the staining profile, i.e., high or low percentage of D7-positive cells, mapped to the Ly-6 locus as assayed by strain distribution studies, RI lines, and Ly-6 congenic strains. The binding of D7 to Ly-6.1-positive strains could be inhibited by mAb directed to the Ly-6E.1 specificity, whereas D7 could inhibit the binding of mAb specific for Ly-6A.2 to cells from Ly-6.2-positive strains. Coprecipitation studies followed by Western blot analysis confirmed that D7 reacts with both Ly-6E.1- and Ly-6A.2-bearing molecules. The most likely explanation for these findings is that Ly-6A.2 and Ly-6E.1 represent allelic specificities. Further dissection of the complexity of the Ly-6 antigen system and determination of its possible functional importance in lymphocyte activation should be greatly facilitated by the availability of xenogeneic mAb that recognize framework determinants on multiple Ly-6 products.

Alleles↗

PubMed1986

In vivo and in vitro expression of an interleukin 2 receptor by murine B and T lymphocytes.

Interleukin 2 (IL 2), which is well established to be a T cell growth factor, has more recently been shown to stimulate B lymphocyte growth and differentiation in vitro. Responsiveness of B and T cells to IL 2 has been associated with expression of a cell membrane IL 2 receptor (IL 2R). To investigate the role of IL 2 in B cell growth and differentiation in vivo, a system was used in which the injection of mice with a goat antibody to mouse IgD (GaM delta) induces polyclonal T-independent B cell proliferation first, and later induces polyclonal T-dependent B cell proliferation and IgG secretion. IL 2R expression by splenic B and T lymphocytes from GaM delta injected mice was studied by a dual label immunofluorescence technique. Although GaM delta was found to be a strong inducer of B cell IL 2R expression in vitro, even in serum-free medium, and stimulated up to 50% of splenic T cells to express considerable quantities of IL 2R in vivo, it failed to induce more than minimal B cell IL 2R expression in vivo. Concanavalin A and bacterial lipid A also induced B cells to express IL 2R to a much greater extent in vitro than in vivo. Although these agents and GaM delta acted synergistically to stimulate B cell IL 2R expression both in vitro and in vivo, a single agent induced B cell IL 2R expression to a considerably greater extent in vitro than did all three agents acting together in vivo. In vitro GaM delta-induced B cell IL 2R expression was not suppressed by inclusion of IL 2 in the culture medium but was suppressed by the presence of 10% normal mouse serum or plasma. These observations suggest that polyclonal T-dependent B cell proliferation and antibody secretion may not require an interaction between B cells and IL 2; the in vivo environment may downregulate IL 2R expression by B cells: and in vivo B cell IL 2R expression and consequently, induction of B cell responsiveness to IL 2, may require stimuli beyond those sufficient to induce B cell IL 2R expression and IL 2 responsiveness in vitro.

Animals↗