Search PubMed⌕ Search

Biomedical subjects

E M Shevach

Publications and source records attributed to E M Shevach.

At least 127 records · Page 7Linked to original sources
PubMed1986

Role of Ly-6 in lymphocyte activation. II. Induction of T cell activation by monoclonal anti-Ly-6 antibodies.

The Ly-6 locus controls the expression and/or encodes for alloantigenic specificities found primarily on subpopulations of murine T and B lymphocytes. We have recently identified and characterized a new rat mAb, D7, that recognizes a nonpolymorphic Ly-6 specificity. After crosslinking by anti-Ig reagents or by Fc receptor-bearing accessory cells, mAb D7 could induce IL-2 production from T cell hybridomas, and in the presence of PMA could trigger a vigorous proliferative response in resting peripheral T cells. The addition of mAb D7 to cultures of antigen- and alloantigen-, but not mitogen-stimulated T cells resulted in a marked augmentation of the proliferative response. A number of other well-characterized mAbs to Ly-6 locus products could also stimulate a T cell proliferative response after crosslinking by anti-Ig and in the presence of PMA. These results strongly suggest that Ly-6 molecules may play a critical role in the T cell activation cascade, either as receptors for an unidentified soluble or cell-associated ligand or as transducing molecules that modulate signals initiated by antigen stimulation of the T3-Ti complex.

Animals↗
PubMed1986

Interleukin 2 receptors on cultured murine epidermal Langerhans cells.

Rat monoclonal antibodies 3C7 and 7D4 detect two distinct functional regions of the murine interleukin 2 (IL 2) receptor. When studying the emergence kinetics of IL 2 receptors in mixed epidermal cell (EC)-lymphocyte cultures by using 3C7 and 7D4 in an indirect immunofluorescence assay, we regularly encountered a distinctive membrane fluorescence not only on lymphocytes, but also on a subpopulation of cells exhibiting a dendritic morphology. Reasoning that these 3C7/7D4-reactive dendritic cells might represent a subpopulation of epidermal dendritic cells, we studied mouse EC for the presence of 3C7/7D4- reactive cells. Although 3C7/7D4 reactivity was never detected on freshly isolated EC or on epidermal sheets, a small number of 3C7/7D4+ cells was encountered after 24 to 48 hr of culture. These cells exhibited a dendritic shape, expressed Ia antigens, lacked Thy-1 antigens, and displayed the ultrastructural features of Langerhans cells (LC) with the notable exception of Birbeck granules. Although after 24 hr, only 20% of Ia+ EC were 3C7/7D4+, the vast majority of LC displayed 3C7/7D4 binding sites after 4 to 5 days of culture. Preincubation of cultured LC-enriched EC with recombinant human IL 2 prevented subsequent 3C7-but not 7D4-binding to these cells. Western blot analysis of 7D4-reactive material of detergent extracts from LC-enriched EC revealed three bands in the same m.w. range as reported for CTLL cells. These results demonstrate that cultured LC express IL 2 receptors and may bear important implications for a better understanding of growth regulation, differentiation, and immunologic functions of LC.

Animals↗
PubMed1986

Induction of T cell activation by monoclonal anti-Thy-1 antibodies.

We have analyzed the requirements for T cell activation by monoclonal anti-Thy-1 antibodies (MAb). A large panel of unselected anti-Thy-1 MAb was capable of inducing a strong proliferative response in resting peripheral T cells and a rise in cytoplasmic free calcium ([Ca2+]i) in both peripheral T cells and a T cell hybridoma. Both of these responses required the interaction of a MAb bound to Thy-1 with a second layer of anti-Ig antibody. Induction of T cell proliferation also required an additional signal, which could be provided by PMA. T cell activation in this system was specific for the Thy-1 molecule, independent of the epitope on Thy-1 recognized by a given MAb, with the anti-Ig reagent was also independent of the type of anti-Ig used, as both polyvalent rabbit anti-rat Ig sera and a mouse MAb to rat Ig functioned as effective cross-linkers. All signals provided by the interaction of anti-Thy-1 MAb with anti-Ig preparations could also be reproduced by the simultaneous binding of two MAb recognizing independent epitopes on Thy-1. Although the physiological role of Thy-1 remains unknown, the model system described here should prove to be very useful in further analysis of the steps involved in the polyclonal activation of murine T cells.

Animals↗
PubMed1986

Functional expression of the murine Thy-1.2 gene in transfected human T cells.

The interaction of certain mAbs with the Thy-1 molecules of murine T lymphocytes leads to cell activation and proliferation. To examine the signal transduction mechanism underlying this process and to determine what, if any, relationship exists between Thy-1-dependent triggering and T cell activation mediated through the T3-antigen receptor (T3-Ti) complex, a genomic clone of murine Thy-1.2 was isolated and transfected into the human T cell tumor, Jurkat. The transfected gene was actively transcribed in these human cells and high levels of Thy-1.2 glycoprotein were found on the cell membrane. Although certain mAbs to Thy-1.2 failed to bind to the Thy-1 transfected Jurkat cells, several known mitogenic anti-Thy-1 mAbs did react, and in the presence of phorbol ester, induced IL-2 secretion. One Thy-1+ transfectant out of five failed to produce IL-2 in response to anti-T3/Ti antibodies even though it retained the ability to increase intracytoplasmic calcium concentration [( Ca2+]i) in response to these ligands. A Thy-1 negative revertant of this cell regained anti-T3/Ti reactivity, suggesting a regulatory defect in signal transmission via T3/Ti in the original transfectant. These data confirm the ability of Thy-1 to act as an activation receptor for T cells. They reveal a potential role for changes in [Ca2+]i in this process, in common with other pathways of T cell activation, but also indicate a more complex series of events is involved.

Animals↗
PubMed1986

Dendritic cells with antigen-presenting capability reside in airway epithelium, lung parenchyma, and visceral pleura.

In this study, we identified a population of dendritic cells (DC) that exists throughout human and mouse pulmonary tissues, including the trachea, bronchi, alveoli, and visceral pleura. In human tissue, these DC were shown to be positive for HLA-DR and T200 antigens. In the mouse, the DC expressed not only Ia and the T200 antigen, but also Fc-IgG and C3bi receptors. Unlike alveolar macrophages, the DC were negative for nonspecific esterase staining and shared ultrastructural similarities with the DC described by Steinman (1), and with Langerhans' cells, even though they did not contain Birbeck granules. We were able to demonstrate that mouse pulmonary DC function in antigen presentation, as observed with the other DC. Thus, the respiratory tract contains DC that are capable of functioning in antigen presentation and that may be important in pulmonary immune responses.

Animals↗
PubMed1986

Photoaffinity labeling demonstrates binding between Ia molecules and nominal antigen on antigen-presenting cells.

We have used radioiodinated photoreactive bovine insulin as antigen to examine the molecular nature of immunogenic complexes that form on antigen-presenting cells. The probe was allowed to bind to either insulin-presenting B-hybridoma cells, lipopolysaccharide-stimulated blasts, or bovine insulin-specific helper-T-hybridoma cells in the dark. Samples were then exposed to light to induce crosslinkage, solubilized, and analyzed by gel electrophoresis. Two protein bands at about 36 kDa and 27 kDa were specifically labeled on antigen-presenting cells but not on helper T cells. Treatment of these bands with dithiothreitol or endo-beta-N-acetylglucosaminidase F showed that each is composed of a single glycoprotein. These proteins are immunoprecipitable with haplotype-specific but not control anti-Ia antibodies. This identifies the labeled bands as the alpha and beta subunits of class II major histocompatibility antigens. We conclude that a molecular complex may form between Ia and antigen on antigen-presenting cells and that formation of this complex does not require the presence of a helper-T-cell antigen receptor.

Affinity Labels↗
PubMed1986

Alpha 1-microglobulin is mitogenic for guinea pig lymphocytes.

Guinea pig alpha 1-microglobulin (alpha 1-m) was found to exert mitogenic effects on guinea pig lymphocytes, i.e. peritoneal exudate lymphocytes (PEL) or T lymphocytes from regional or mesenteric lymph nodes (LNL). On the other hand, spleen T lymphocytes did not respond to alpha 1-m. Stimulation of LNL required the presence of accessory cells. The stimulatory effect was strain-specific: alpha 1-m clearly induced proliferation of lymphocytes from Strain 2 guinea pigs, whereas Strain 13 lymphocytes responded poorly or not at all. Moreover, alpha 1-m inhibited antigen-driven proliferation of guinea pig lymphocytes. None of the effects described were species-specific, e.g. human alpha 1-m exerted similar effects on the guinea pig lymphocytes. These data indicate that alpha 1-m may be involved in the regulation of lymphocyte activation.

Alpha-Globulins↗
PubMed1985

The Thy-1-bearing cell of murine epidermis. A distinctive leukocyte perhaps related to natural killer cells.

Bone marrow-derived leukocytes of murine epidermis can express two phenotypes: typical Langerhans cells, which are Ia+ and Thy-1-, and a recently discovered second population that is Thy-1+ and Ia-. To verify that these phenotypes are expressed by two different cell types, and to help understand their lineage and function, we have studied morphology and reactivity with a large panel of antibodies. Dual antibody immunofluorescence combined with electron microscopy showed that Thy-1+ and Ia+ cells were each distributed in a regular fashion and formed adjacent dendritic systems in or close to the basal layer. Double-labeling studies with anti-Ia and a second monoclonal antibody revealed that all Langerhans cells expressed F4/80 (macrophage), Mac-1 (C3bi receptor), and 2.4G2 (Fc receptor), as well as the thymus leukemia (TL) and heat-stable (M1.69/16) antigens. A large fraction expressed S100 and all exhibited membrane ATPase and nonspecific esterase. In contrast, Thy-1+ cells lacked all these features of Langerhans cells, except that a minority were strongly reactive with 2.4G2. Thy-1+ cells also lacked differentiation antigens of most other types of leukocytes, except they were rich in asialo GM1. By electron microscopy, Thy-1+ cells had cytoplasmic granules that were similar in structure and in their aryl sulfatase content to those previously described in natural killer cells. The granules were enlarged in beige mice, suggesting a lysosomal origin, and were present in mast cell-deficient W/Wv mice, indicating no relation to mast cells. We conclude that Thy-1+ epidermal cells are thoroughly distinct from Langerhans cells. On the basis of morphology and phenotype, they may represent a type of tissue natural killer cell. Thy-1+ natural killer cells are now being identified in several nonlymphoid sites, such as gut epithelium and the livers of mice given adjuvants. If Thy-1+ epidermal cells prove to be natural killer cells, it is noteworthy that they represent a resident population regularly distributed in the basal layer of all mouse strains. The notion that Thy-1+ epidermal cells are immature natural killer cells is intriguing in light of recent evidence that Ia+ Langerhans cells are also immature with respect to accessory cell function. The epidermis may not have the functional capacities of a lymphoid organ, but it could contribute immature cells important for both natural and acquired resistance.

Animals↗
PubMed1985

Rapid production of monoclonal antibodies to T lymphocyte functional antigens.

Brief immunization of rats with mouse lymphoid cells was combined with the rat/mouse hybridoma technology and functional hybridoma screening to yield a rapid method for the production of monoclonal antibodies (MAb) against functionally important T lymphocyte cell surface antigens. Two protocols were used. In one, rats were immunized once with mouse thymocytes followed by fusion and screening of the hybridomas for interference with the thymocyte co-stimulator (interleukin 1) assay. The resultant hybridomas included producers of MAbs against the L3T4-antigen (inhibitory), the Ly-1-antigen (stimulatory), and the Thy-1-antigen (inhibitory?). In the second protocol, rats were immunized twice with a T cell hybridoma. The resultant hybridomas were screened for inhibition of polyclonal T cell activation, induced by an anti-Thy-1 (MAb G7). A panel of MAbs against the Thy-1 antigen with different reactivity profiles was generated by this procedure. Most of the MAbs were of the IgM class. Short-term immunization may lead to less selection of response to highly immunogenic determinants than a protocol involving several boosters. Thus, this alternative may be useful for producing MAbs against rare or weakly immunogenic cell surface molecules, as suggested by the ease with which we were able to make MAbs against the L3T4-molecule.

Animals↗
PubMed1985

Expression of interleukin 2 receptor on murine fetal thymocytes.

Rat monoclonal antibodies AMT-13, 3C7 and 7D4 which react to the mouse interleukin 2 (IL 2) receptors were used to define cell populations with the putative IL 2 receptors in the mouse thymus as a part of series of investigations to elucidate the mechanism of intrathymic cell proliferation and differentiation. With freshly dissociated cells from the thymus of 15 gestational days, the anti-IL 2 receptor monoclonal antibodies (anti-IL 2 receptor) reacted with about half of them. The proportion of the reactive cells decreased rapidly thereafter till birth as the numbers of thymus cells expanded. The antibodies reacted with only two to three percent of thymic cells from newborn mice and less than one percent of cells from adult thymus. Through the gestational period, the fetal liver cells did not react with the antibodies. When the thymus cells at early gestational days were subjected to a two-color analysis, one for the anti-IL 2 receptor and the other for anti-Thy-1 or anti-asialo GM1, by a fluorescence-activated cell sorter, it was found that the majority (up to 80%) of anti-IL 2 receptor-reactive cells (IL 2 receptor-positive cells) was also reactive with anti-Thy-1. Some of the IL 2 receptor-positive cells but Thy-1-negative cells reacted with anti-asialo GM1, a marker of the immature thymocytes. Immunohistochemically, the IL 2 receptor-positive cells were found mainly in the subcapsular area and in the border region of cortex and medulla. Collectively, these results suggest that the pre-T lymphocytes are stimulated immediately after their arrival to the thymus from the stem cell source such as fetal liver and bone marrow and are driven into the proliferation via the IL 2 receptor system.

Animals↗
PubMed1985

Analysis of autoreactive I region-restricted T cell colonies isolated from the guinea pig syngeneic mixed leukocyte reaction and from immune responses to conventional foreign antigens.

Guinea pig proliferating T cell colonies were isolated from T cell populations stimulated during the syngeneic mixed leukocyte reaction (SMLR) or following positive selection of immune T lymphocytes specific for pork insulin (PI) or the copolymer of L-glutamic acid, L-lysine (GL). SMLR-responding T cell colonies could be isolated in the absence of any extrinsic antigen and were strictly restricted to the recognition of Ia molecules on stimulator peritoneal exudate cells (PEC) and required both stimulator cells and interleukin 2-enriched fluids for optimal proliferative responses. Blocking of T cell colony proliferation with a panel of monoclonal anti-Ia antibodies showed that SMLR T cell colonies were restricted by discrete and distinct self-Ia epitopes. Analysis of individual T cell colonies generated against PI and GL revealed three types of colonies: (a) antigen specific, I region-restricted; (b) autoreactive, I region-restricted; and (c) antigen specific, but also autoreactive. These doubly reactive colonies were restricted to the same Ia epitope when stimulated with self-PEC alone or when stimulated with self-PEC in the presence of the relevant exogenous antigen. These results substantiate the hypothesis that both the SMLR and antigen-specific responses are mediated by a common set of precursor T lymphocytes and that the guinea pig SMLR is at least in part the result of the polyclonal proliferative responses of several distinct antigen-reactive T cell clones in the absence of exogenous antigen.

Animals↗
PubMed1985

Role of the Ly 1 antigen in interleukin 1-induced thymocyte activation.

Interleukin 1 (IL 1), but not IL 2-induced thymocyte proliferation was augmented by monoclonal antibodies (mAb) against the Ly 1 antigen. The anti-Ly 1 mAb could also co-stimulate thymocytes with phytohemagglutinin (PHA) alone, mimicking the effect of IL 1. Moreover, overnight culture with PHA of thymocytes, but not of spleen T lymphocytes led to selectively enhanced expression of the Ly 1 antigen on the cell surface. Similar data were generated at the clonal level. Thus, a thymocyte hybridoma (22A6) could be stimulated to release IL 2 in response to PHA plus IL 1, but also to PHA plus anti-Ly 1 mAb. In addition, 22A6 exhibited enhanced cell surface expression of the Ly 1 antigen after overnight culture with PHA. These data suggest a critical role for the Ly 1 antigen in thymocyte proliferation, perhaps by serving as an IL 1-receptor.

Animals↗
PubMed1985

Role of the L3T4 antigen in T-cell activation. I. Description of a monoclonal IgM antibody to a distinct epitope (L3T4b) of the L3T4 antigen and its effect on interleukin 1-induced thymocyte proliferation.

This report describes a new rat monoclonal IgM/k antibody, monoclonal antibody (MAb) 2B6, which reacts with a cell surface antigen present on a subpopulation of both thymocytes (85%) and peripheral T lymphocytes (55-60%). The antigen recognized by MAb 2B6 has multiple properties in common with the L3T4 antigen, as defined by the recently described MAb GK1.5. Thus, MAb 2B6 and MAb GK1.5 give very similar flow cytometry staining patterns on thymocytes, purified spleen T cells and all tested T-cell hybridomas. Depletion of MAb 2B6-positive cells with antibody and complement led to simultaneous depletion of MAb GK1.5-positive cells, and vice versa. Depletion of Lyt 2-positive cells led to enrichment of both MAb 2B6- and MAb GK1.5-positive cells. Both MAb 2B6 and MAb GK1.5 immunoprecipitate the same pattern of cell surface molecules from detergent extracts of radiolabeled thymocytes, the main components being a 55-kDa and a 115-kDa band. We therefore conclude that MAb 2B6 reacts with the L3T4 antigen. Interestingly, MAb 2B6 and MAb GK1.5 do not cross-block and therefore most probably react with distinct epitopes on the L3T4 molecule. The determinant recognized by MAb GK1.5 is called L3T4a. We suggest that the determinant recognized by MAb 2B6 be named L3T4b. As MAb 2B6 was selected for its ability to inhibit the action of interleukin 1 (IL-1) in the thymocyte costimulator assay, it is likely that the L3T4 molecule is functionally involved in the events taking place during IL-1 induction of thymocyte proliferation.

Animals↗
PubMed1985

A comparison of the effects of cyclosporin A, dexamethasone, and ouabain on the interleukin-2 cascade.

We have studied the mechanism by which cyclosporin A (CsA), dexamethasone (DEX), and ouabain (OUA) inhibit T cell proliferation by measuring the effects of these agents on 1) interleukin-2 (IL-2) production, 2) acquisition of IL-2 responsiveness, 3) the induction of IL-2 receptor expression, and 4) the specific interaction of IL-2 with its receptor. DEX primarily inhibited IL-2 production and did not block acquisition of responsiveness to IL-2 or interaction of IL-2 with its receptor. OUA mainly interfered with the mitogenic activity of IL-2 on IL-2 dependent cells and showed a modest inhibitory effect on IL-2 production. In contrast, CsA blocked acquisition of responsiveness of resting T cells to IL-2, inhibited IL-2 production, and also inhibited IL-2 receptor expression at 48 hrs but not at 24 hrs following mitogen stimulation. The protocol described in these studies should prove to be useful in dissecting the mechanisms responsible for the depressed lymphoproliferative responses in different autoimmune and immunodeficiency disease states.

Animals↗
PubMed1985

Guinea pig T lymphocyte development analyzed by enzyme histocytochemistry, monoclonal antibodies, and flow cytometry.

Studies of T (thymus-derived) lymphocyte ontogeny in the guinea pig have been hampered by the lack of suitable antigenic or other markers for various T cell subpopulations in this species. Monoclonal antibodies that recognize three distinct surface proteins of guinea pig T cells and react with all peripheral T cells have been used in combination with membrane alkaline phosphatase (AP) to characterize stages of guinea pig T cell development and to determine anatomical localization of different T cell subpopulations. Flow cytofluorographic analysis of thymus, spleen, and lymph node lymphocytes was used to characterize monoclonal antibody specificity. Cortical thymocytes in tissue sections expressed membrane AP activity and contained nuclear terminal deoxynucleotidyl transferase; medullary thymocytes reacted strongly with one of the monoclonal antibodies (8BE6), minimally with a second (5CD2), and not at all with a third (11AE3). In contrast, polyclonal rabbit antiguinea pig T cell antiserum reacted with both cortical and medullary thymocytes. Staining of tissue sections of lymph node and spleen revealed AP+ lymphocytes to be present peripheral to the mantle region of lymph node follicles and to be randomly scattered throughout the splenic red pulp. T cells reactive with monoclonal antibodies were located primarily in paracortical regions of lymph node and the central region around the periarteriolar regions of the spleen. Dual staining of frozen sections and cell suspension of guinea pig lymphoid tissues for AP activity and surface proteins unique to T cells showed that AP+ cells lacked T cell markers. Dual staining for AP activity and surface immunoglobulins or esterase activity showed that AP+ cells are not likely to be derived from either B cell or monocyte-macrophage lineages. AP+ cells in guinea pig secondary lymphoid tissue may represent a unique subset of lymphocytes of unknown function.

Animals↗
PubMed1985

Role of the L3T4-antigen in T cell activation. II. Inhibition of T cell activation by monoclonal anti-L3T4 antibodies in the absence of accessory cells.

We have used two monoclonal antibodies (Mab) to the L3T4 antigen to reexplore the role of this molecule in the process of T cell activation. Both Mab (Gk1.5 and 2B6) were capable of inhibiting Con A-induced IL 2 production by a number of antigen-specific T cell hybridomas in an assay system that was free of major histocompatibility complex (MHC) class II antigen-bearing cells. The inhibition produced by the anti-L3T4 Mab was specific, because other Mab to cell surface antigens expressed on the hybridomas were without inhibitory effects. These studies rule out the possibility that the mechanism of inhibition by anti-L3T4 in this model is mediated by blocking interaction of L3T4 with MHC class II products. Taken together, these results and those of other groups of investigators, are most compatible with a dual function for L3T4 in T cell activation. L3T4 might first interact with MHC class II molecules or other molecules on target or accessory cells; L3T4 would subsequently transmit a signal that would regulate the activation process. Mab to L3T4 might exert inhibitory effects at one or both of these steps.

Animals↗