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Biomedical subjects

M F Mescher

Publications and source records attributed to M F Mescher.

At least 73 records · Page 4Linked to original sources
PubMed1985

Carbohydrate moieties of major histocompatibility complex class I alloantigens are not required for their recognition by T lymphocytes.

The ability to generate specific cytotoxic responses using purified major histocompatibility complex (MHC) antigen in liposomes has made it possible to directly assess the importance of class I carbohydrate moieties in T cell recognition of alloantigen. Deglycosylation of affinity-purified H-2Kk to yield a single glycan-free product did not alter the specificity, the magnitude, nor the dose range of the cytotoxic T lymphocyte (CTL) response to the class I antigen. It can be concluded that carbohydrate moieties are not required to maintain the necessary conformation of the MHC protein, nor to interact with either the antigen-specific receptor or accessory proteins on precursor CTL.

Animals↗
PubMed1985

The ability of Ia and H-2Kk-bearing membranes to replace the antigen-presenting cell in an H-2Kk allogeneic cytotoxic T cell response.

Induction of an allogeneic cytotoxic T lymphocyte (CTL) response is dependent, in part, on uptake and processing of the class I alloantigen by antigen-presenting cells and subsequent Ia-restricted recognition of the alloantigen by helper T cells, resulting in lymphokine production. The nature of the antigen-processing event has been investigated using reconstituted membranes to replace the antigen-presenting cells in the generation of a secondary allogeneic CTL response. Membranes were isolated from an Iad-positive antigen presenting B cell lymphoma (D2N), detergent solubilized and then reconstituted together with affinity-purified H-2Kk antigen in the presence of protease inhibitors. These reconstituted vesicles, containing both syngeneic Ia and alloantigen, were able to induce the helper T cell arm of the CTL response in cultures depleted of antigen-presenting cells. A variety of control experiments provided strong evidence that the helper T cells recognized the H-2Kk, probably in its native form, in an Ia-restricted manner on the vesicles, while the pre-CTL can directly recognize H-2Kk. Recognition was only effective if both the Ia and alloantigen were inserted into the same membrane bilayer. The results strongly suggest that the obligatory antigen processing event required for helper T cell recognition of alloantigen is simply the insertion of the alloantigen into the same membrane bilayer as the syngeneic Ia restricting element.

Animals↗
PubMed1985

Analysis of antigen presentation by metabolically inactive accessory cells and their isolated membranes.

Several amino acid copolymers are potent immunogens under the control of major histocompatibility complex (MHC)-encoded Ir genes. We have further characterized their accessory-cell-dependent, MHC-restricted presentation to T lymphocytes. We initially characterized their processing requirements by investigating the ability of paraformaldehyde-fixed antigen-presenting cells (APC) to present these copolymers. Fixed APC can present poly(Glu56Lys35Phe9) and poly(Glu60Ala30Tyr10) provided that they have been incubated with antigen prior to fixation. The inability of these same fixed preparations to present soluble antigen indicates a fixation-sensitive antigen-processing step. In contrast, the antigens poly(Glu55Lys35Leu10) and poly(Glu55Lys35Tyr10) can be presented by APC fixed before antigen exposure. This differential requirement for antigen processing was exploited to analyze the events of antigen presentation in two related systems. First, the ability of isolated APC membranes to process and present antigen was assessed. APC membranes can present the antigens poly(GluLysLeu) and poly(GluLysTyr) in a specific and MHC-restricted manner. However, the isolated membranes fail to present either poly(GluLysPhe) or poly(GluAlaTyr), suggesting that such preparations can present but not process antigen. Second, the distinct properties of the various copolymers were used with fixed APC to test the effects of antigen processing on the phenomenon of antigen competition. APC that had processed poly(GluLysPhe) or poly(GluAlaTyr) were subsequently fixed and used to present antigen in the presence or absence of various antagonists. Under these conditions, poly(GluLysLeu) and poly(Glu50Tyr50) could effect specific inhibition, clearly indicating that antigen competition occurs distal to and does not require antigen processing. In contrast, native antigen with an absolute processing requirement is not capable of competing with preprocessed antigen on fixed APC. Taken together, these results suggest that processing is important for the molecular interactions between the copolymer antigens and the APC cell surface that are relevant to both antigen presentation and competitive inhibition.

Antigen-Presenting Cells↗
PubMed1985

Triton X-100 extraction of P815 tumor cells: evidence for a plasma membrane skeleton structure.

It has been shown that a Triton X-100-insoluble protein matrix can be isolated from the plasma membranes of P815 tumor cells and murine lymphoid cells (Mescher, M. F., M. J. L. Jose and S. P. Balk, 1981, Nature (Lond.), 289:139-144). The properties of the matrix suggested that this set of proteins might form a membrane skeletal structure, stable in the absence of the lipid bilayer. Since purification of plasma membrane results in yields of only 20 to 40%, it was not clear whether the matrix was associated with the entire plasma membrane. To determine if a detergent-insoluble structure was present over the entire cell periphery and stable in the absence of the membrane bilayer or cytoskeletal components, we have examined extraction of whole cells with Triton X-100. Using the same conditions as those used for isolation of the matrix from membranes, we found that extraction of intact cells resulted in structures consisting of a continuous layer of protein at the periphery, a largely empty cytoplasmic space, and a nuclear remnant. Little or no lipid bilayer structure was evident in association with the peripheral layer, and no filamentous cytoskeletal structures could be seen in the cytoplasmic space by thin-section electron microscopy. Analysis of these Triton shells showed them to retain approximately 15% of the total cell protein, most of which was accounted for by low molecular weight nuclear proteins. 5'-Nucleotidase, a cell surface enzyme that remains associated with the plasma membrane matrix, was quantitatively recovered with the shells. Included among the polypeptides present in the shells was a set with mobilities identical to those of the set that makes up the plasma membrane matrix. The polypeptide composition of the shells further confirmed that cytoskeletal proteins were present to a very low extent, if at all, after the extraction. The results demonstrate that a detergent-insoluble protein matrix associated with the periphery of these cells forms a continuous, intact macrostructure whose stability is independent of the membrane bilayer or filamentous cytoskeletal elements, and thus has the properties of a membrane skeletal structure. Although not yet directly demonstrated, the results also strongly suggest that this peripheral layer is composed of the previously described set of plasma membrane matrix proteins. This article discusses possible roles for this proposed membrane skeletal structure in stabilizing the membrane bilayer and affecting the dynamics of other membrane proteins.

Animals↗
PubMed1984

Hairpin configuration of H-2Kk in liposomes formed by detergent dialysis.

H-2Kk is a transmembrane glycoprotein having the N-terminal region of the heavy chain exposed at the cell surface and the C-terminal region exposed at the cytoplasmic face in its native configuration in the plasma membrane. The configuration of H-2Kk in liposomes formed by detergent dialysis was investigated by using fluorescently labeled H-2 and Co2+ ions to quench fluorescence. H-2Kk was incorporated into sealed lipid vesicles when deoxycholate was removed by dialysis from a mixture of protein and lipid. Including 20 mM carboxyfluorescein (CF) in the mixture prior to dialysis resulted in CF trapped inside the vesicles at concentrations where self-quenching occurred. Vesicles with CF trapped inside were shown to be osmotically active and impermeable to Na+ and Co2+ ions. In order to examine the configuration of H-2Kk in these liposomes, the heavy chain was covalently labeled by using the sulfhydryl reactive fluorescent reagents fluorescein-5-ylmaleimide (NFM) or 5-[[2-[(iodoacetyl)amino]-ethyl]amino]naphthalene-1-sulfonic acid (IAEDANS). In both cases, approximately equal amounts of fluorescent label were incorporated into the N- and C-terminal regions of the protein. Incorporation of the labeled H-2 into liposomes and examination of the effect of Co2+ on the fluorescence showed that all of the label was accessible to quenching by Co2+ and thus exposed on the outside of the liposome. The results demonstrate that the H-2Kk is incorporated into these liposomes in a hairpin configuration, not in the transmembrane configuration found in native membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Inhibition of normal lymphocyte responses by cell membranes.

Cell membranes bearing the appropriate antigen are known to stimulate a variety of cell-mediated immune responses. This report confirms that tumor cell membranes at doses of 2-5 micrograms protein/ml will stimulate in vitro generation of allogeneic cytotoxic T lymphocytes (CTL). However, higher doses (50-100 micrograms protein/ml) of the same membranes completely abrogate the generation of lytic activity. Responding lymphocytes are inhibited by membranes from either syngeneic or allogeneic cells. The inhibition appears to act at a proliferative or differentiation step in the generation of the CTL response, since membranes are known to have little direct effect on the lytic phase of CTL activity. Similar doses of membranes also inhibit LPS-induced B-cell proliferation. B-Cell proliferation is inhibited equally well by allogeneic and syngeneic membranes, and membranes from normal spleen cells are as inhibitory as tumor cell membranes. The inhibitory activity copurifies with the plasma membrane. The results raise important considerations regarding the use of subcellular forms of antigen in studies of lymphocyte recognition. In addition, these data suggest that cell-cell contacts might provide signals regulating the proliferation of lymphocytes.

Animals↗
PubMed1984

Growth-inhibitory activity of lymphoid cell plasma membranes. I. Inhibition of lymphocyte and lymphoid tumor cell growth.

Membranes isolated from normal spleen cells or lymphoid tumor cells were found to inhibit in vitro growth of several murine tumor cell lines including a B cell hybridoma, a thymoma, and a mastocytoma. 50% inhibition occurred at membrane protein concentrations of 60-100 micrograms/ml. A similar concentration dependence was found for inhibition of [3H]-thymidine incorporation by tumor cells and for the lipopolysaccharide-induced mitogenic response of normal spleen cells. The inhibitory activity co-purified with the plasma membrane upon fractionation of crude membranes. Membrane solubilization with deoxycholate followed by dialysis to remove the detergent gave good recovery of inhibitory activity in the resulting reconstituted membranes. Membrane-mediated growth inhibition resulted from a decreased rate of proliferation and not from increased cell death. A toxic effect of the membranes was further ruled out by the finding that increasing the fetal calf serum content of the medium could substantially reverse the growth inhibition. Thus, the plasma membrane of lymphoid cells contains a component that can slow or stop the growth of cells in culture. This membrane component may have a role in cell contact-mediated regulation of growth.

Animals↗
PubMed1984

Growth-inhibitory activity of lymphoid cell plasma membranes. II. Partial characterization of the inhibitor.

We have shown that plasma membranes from lymphoid cells have inhibitory activity for the growth of normal lymphocytes and lymphoid tumor cells (Stallcup, K. C., A. Dawson, and M. F. Mescher, J. Cell Biol. 99:1221-1226). This growth-inhibitory activity has been found to co-purify with major histocompatibility complex class I antigens (H-2K and D) when these cell surface glycoproteins are isolated from detergent lysates of cells by affinity chromatography on monoclonal antibody columns. When incorporated into liposomes, the affinity-purified H-2 antigens inhibited the growth of both normal lymphocytes and tumor cells at concentrations of 1-3 micrograms/ml. Inhibition was readily reversed upon removal of the liposomes from the cell cultures, even after several days of exposure of cells to the inhibitor. Inhibitory activity was insensitive to protease digestion or heat treatment, indicating that it was not due to the H-2 glycoproteins. This was confirmed by the demonstration that inhibitory activity could be separated from the H-2 protein by gel filtration in the presence of deoxycholate and could be extracted from membranes or H-2 antigen preparations with organic solvents. The results demonstrate that the growth-inhibitory component(s) of the plasma membrane is a minor lipid or lipid-like molecule which retains activity in the absence of other membrane components. The findings reported here and in the preceding article suggest that this novel membrane component may have a role in control of lymphoid cell growth, possibly mediated by cell contacts.

Animals↗
PubMed1983

Large-scale purification of murine I-Ak and I-Ek antigens and characterization of the purified proteins.

Detailed analysis of the role of the structural characteristics of these molecules will require isolation of relatively large amounts of these antigens in serologically active form. We have purified murine Ia antigens on a large scale by affinity chromatography using monoclonal antibodies coupled to Sepharose 4B. Both I-Ak and I-Ek were isolated by sequential passage of cell lysate over columns prepared using specific monoclonal antibodies. Elution of the bound antigens required high pH (11-12) but, nonetheless, the purified material was 50-75% serologically active. Using LPS-stimulated spleen cells or B-lymphocyte tumor cells as starting material, 0.5 mg of each antigen can be readily purified. Based on antigen yields, it can be estimated that normal B-cells have about the same surface density of Class I and Class II MHC antigens. LPS blasts, in contrast, have normal levels of Class I antigen but 3-5 times higher levels of Class II antigens. We have now purified I-Ak and I-Ek from a number of different cell sources and have noted differences in both the mol. wts of the alpha- and beta-chains and in their apparent associations with cytoskeletal components. Proteins having the same apparent mol. wts as actin and myosin co-purify with both I-Ak and I-Ek antigens from various sources. These proteins do not co-purify with H-2K and D molecules obtained by similar methods, suggesting that Ia antigens may specifically interact with cytoskeletal elements.

Actins↗
PubMed1983

Stimulation of secondary anti-MSV cytolytic T lymphocytes with MBL-2 reconstituted membranes.

Membranes and solubilized, reconstituted membranes from Moloney-infected tumors MBL-2 have been used to stimulate in vitro secondary cytolytic T lymphocytes (CTL) in C57BL/6 mice primed and Moloney murine sarcoma virus. Membranes are shown to stimulate the generation of Moloney-specific and H-2 restricted CTL. Stimulation with solubilized, reconstituted membranes required the presence of rat lymphocyte Con A supernatant (containing interleukin 2) during the culture. Reconstituted membranes made in the presence of the detergent-insoluble fraction from the plasma membrane were able to stimulate a response in the absence of Con A supernatant.

Animals↗
PubMed1982

Proteolytic modifications of the carboxyl-terminal region of H-2Kk.

Conditions were established for the generation of limited proteolysis products from purified H-2Kk in high yield (greater than 70%). Chymotrypsin, trypsin, or papain treatment in buffer containing Nonidet P-40 resulted in removal of discrete segments from the H-2 heavy chain without detectable alteration of the beta 2-microglobulin. The Mr = 47,400 heavy chain was converted to products with Mr = 44,200, 42,800, or 40,600 by treatment with chymotrypsin, trypsin, or papain, respectively. Papain digestion removed both the hydrophilic carboxyl terminus and the hydrophobic regions. The size, detergent binding properties, and products resulting from subsequent papain treatment demonstrated that chymotrypsin or trypsin removed segments of the hydrophilic carboxyl-terminal region of the heavy chain while leaving the hydrophobic (membrane-spanning) and glycosylated NH2-terminal regions intact. Chymotrypsin and trypsin caused rapid and extensive degradation of the H-2Kk heavy chain when treatment was done in buffer containing deoxycholate, suggesting that the protein undergoes partial, but readily reversible, denaturation in this detergent. This may account for the elution of H-2K and D antigens from monoclonal antibody affinity columns by deoxycholate-containing buffers.

Animals↗
PubMed1982

Structural heterogeneity of murine IgD during ontogeny.

We have studied the expression of the two membrane delta heavy chains (delta 1 and delta 2) and the two native IgD structures (IgDI and IgDII) in neonatal mice. Both delta-chains appear simultaneously during development and neonatal mice, like adults, express equal amounts of delta 1 and delta 2. IgDI and IgDII also appear simultaneously during ontogeny and in the same ratio as expressed by adult mice of the same strain. Thus, when IgD first appears during ontogeny it shows the same structural heterogeneity as observed in adult mice.

Aging↗
PubMed1982

Role of I-region gene products in T cell activation. I. Stimulation of T lymphocyte proliferative responses by subcellular membrane preparations containing Ia alloantigens.

A model system has been developed for exploring the requirements for activation of T cells by subcellular forms of Ia alloantigen. Lymph node cells from mice recently primed subcutaneously with viable allogeneic cells show strong proliferative responses in vitro to membrane preparations derived from cells bearing the appropriate I-region-encoded glycoproteins. This stimulation shows kinetics characteristic of a secondary response, with a peak at 24 to 48 hr. Primary responses to alloantigen-bearing membranes are weak or absent under these conditions. The predominant cell type involved in the secondary response is the Lyt-1+ T lymphocyte, and the major antigenic stimulus is the I-A subregion-encoded Ia glycoprotein. Syngeneic Ia+ accessory cells do not appear necessary for activation to occur. Detergent solubilized reconstituted membrane vesicles also will stimulate primed T lymphocytes to respond by proliferation. The applications of this approach to the study of T cell recognition of antigen and the role of nonspecific lymphokines in T cell triggering are discussed.

Animals↗
PubMed1982

Analysis of the two-signal requirement for precursor cytolytic T lymphocyte activation using H-2Kk in liposomes.

Activation of primed pre-cytolytic T lymphocytes (pCTL) requires two signals: recognition of antigen (signal 1) and interaction with a nonspecific helper factor (signal 2). The two signals necessary for generation of a secondary allogeneic CTL response have been analyzed using H-2Kk in liposomes as the stimulating antigen. Use of the liposomes allows the alloantigen to be separated from responder cells after a brief exposure. Thus, the requirements for effective delivery of each signal could be studied independently. A 12-hr exposure of pCTL to alloantigen was sufficient for optimum signal 1 delivery. pCTL recognition of the antigen occurs during this time, and no requirement for adherent cells could be demonstrated. The structure of the antigen-containing liposomes affects the efficiency of pCTL triggering. Factor(s) necessary for signal 2 could be provided by supernatants from mitogen-stimulated lymphocytes. Alternatively, it could be generated with alloantigen, providing that adherent cells were present. Optimum interaction of factor(s) with pCTL, i.e., optimum delivery of signal 2, occurred only if factor(s) was present at 12 to 24 hr after interaction of pCTL with alloantigen. The results suggest that alloantigen recognition triggers pCTL to synthesize and/or express receptors for the factor(s).

Animals↗