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Biomedical subjects

M M Simon

Publications and source records attributed to M M Simon.

At least 127 records · Page 7Linked to original sources
PubMed1988

Release of biologically active fragments from human plasma-fibronectin by murine T cell-specific proteinase 1 (TSP-1).

We have studied the proteolytic activity of TSP-1, a tissue-specific serine proteinase expressed by activated murine T cells, on human plasma fibronectin and have investigated by affinity chromatography the biological activity of the polypeptides released after limited proteolysis. A Mr approximately 2.9 x 10(4) peptide bound to denatured collagen (gelatine) but not to heparin, a Mr approximately 3.0 x 10(4) fragment contained heparin-binding but not collagen-binding sites and a third Mr approximately 3.5 x 10(4) peptide did not express either of the two activities. All larger fragments - an array of five to six polypeptides with relative molecular masses between 15 x 10(4) and 19 x 10(4) - were bound to heparin-Sepharose but not to gelatine-Sepharose and could be eluted with high salt concentration. These data confirm recent results suggesting multiple, proteinase-resistant domains with discrete biological functions within fibronectins. The release of small, biologically active fibronectin fragments by TSP-1, an enzyme specifically released by activated T cells upon contact with antigen, suggests a role for this enzyme in the numerous cellular functions elicited by T lymphocytes in vivo.

Animals↗
PubMed1987

A secretable serine proteinase with highly restricted specificity from cytolytic T lymphocytes inactivates retrovirus-associated reverse transcriptase.

TSP-1, a murine T cell specific proteinase, is expressed in cytolytic T lymphocytes and secreted upon their interaction with antigen bearing target cells. In searching for possible extracellular substrates of the enzyme in the physiological environment of cytolytic effector cells, we have investigated the proteolytic activity of TSP-1 on retroviral proteins. It is shown that reverse transcriptase derived from the retrovirus Moloney murine leukemia virus is inactivated by TSP-1 via limited proteolysis. The data suggest the possibility that cytolytic T lymphocytes are able to interfere with retroviral replication by secreting a serine proteinase which degrades viral proteins.

Animals↗
PubMed1987

The T cell-specific serine proteinase TSP-1 is associated with cytoplasmic granules of cytolytic T lymphocytes.

This study describes the localization of the previously purified T cell-specific serine proteinase, termed TSP-1 (M. M. Simon et al., EMBO J. 1986. 5: 3267), within cytoplasmic granules of cytolytic T cell lines (CTLL). Subcellular fractionation of disintegrated CTLL (ruptured by nitrogen cavitation) was accomplished by Percoll density gradient centrifugation of cell lysates (postnuclear supernatant). Individual fractions were tested for proteinase activity on chromogenic peptide substrates and for the presence of TSP-1 by Western blot analysis. In addition, each fraction was assayed for cytolytic activity against sheep red blood cells (SRBC), for protein and for additional marker enzymes to assess the enrichment for cellular organells. All serine enzyme-type molecules including TSP-1 expressed by CTLL were identified by labeling cell lysates or gradient fractions with the serine proteinase-specific affinity ligand tritiated diisopropyl fluorophosphate [( 3H]DFP) in the presence or in the absence of class-specific or enzyme-specific proteinase inhibitors and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data demonstrate that the Percoll gradient fraction, which was shown by morphological examination in the electron microscope to be highly enriched for cytoplasmic granules, also contained greater than 80% of proteinase activity in addition to the granule-associated structures cytolysin and arylsulfatase. The identity of the granule-associated proteinase in two independent cell lines, CTLL HY3-Ag3 and CTLL 1.D.9, with the serine proteinase TSP-1 is indicated by its specificity for the chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide, its sensitivity to class-specific as well as TSP-1-specific enzyme inhibitors and by its reactivity with a polyvalent TSP-1-specific rabbit antiserum. Both CTLL contain a [3H]DFP-labeled protein that migrates with a molecular mass of 60 kDa under nonreducing conditions and with 30 kDa under reducing conditions and which can be inactivated by the TSP-1-specific inhibitor H-D-Pro-Phe-Arg-chloromethylketone. CTLL HY3-Ag3 (a long-term culture CTLL with natural killer-like activity) but not CTLL 1.D.9 (an antigen-specific short-term cultured CTLL) express in addition a further [3H]DFP-binding protein which migrates with 27 kDa under nonreducing or reducing conditions. No substrate specificity was found for this molecule. The possible function of the granule-associated serine proteinase TSP-1 is discussed.

Amides↗
PubMed1987

The immune response of the mouse to lymphocytic choriomeningitis virus. V. High numbers of cytolytic T lymphocytes are generated in the spleen during acute infection.

In the spleens of C57BL/6J (B6) and CBA/J (CBA) mice undergoing acute infection with lymphocytic choriomeningitis (LCM) virus, lymphocytes with the ability to develop in vitro into LCM virus-specific cytolytic clones were enumerated by use of the limiting dilution method. At intervals after virus inoculation, defined numbers of cells were cultivated with virus-infected syngeneic stimulator cells and T cell growth factor in multiple wells of microculture plates. After 7 days, individual cell cultures were tested for their ability to cause release of 51Cr from infected and uninfected syngeneic target cells. In cultures seeded with spleen cells from uninfected mice or from mice infected 3 days previously, no cytolytic activity was observed. On day 5, cells developing into LCM virus-specific cytolytic effector cells were detected. They rose in numbers, and on days 8 to 9 after infection, values of approximately 1/10 and 1/200 in B6 and CBA mice, respectively, were calculated. A low proportion of microcultures proved cytolytic also for noninfected syngeneic target cells, but the counts thus released were consistently much lower than the counts set free from infected targets, and no regular dose-response relationships existed between seeded cells and positive cultures. Determination of cell surface antigens of responder cells by negative and positive selection procedures disclosed that they were predominantly T lymphocytes and expressed Lyt-2 but not L3T4 surface markers. Lysis by the great majority of LCM virus-specific clones was restricted by products of the major histocompatibility gene complex (MHC), but a few lysed, in addition, allogeneic infected or uninfected targets; however, a consistent pattern of alloreactivity was not observed. Furthermore, cells of a proportion of the cultures also lysed uninfected YAC cells. Probably this natural killer-like activity was acquired by T lymphocytes during prolonged cultivation. We conclude that most spleen cells that during acute infection with LCM virus attained the ability to develop in vitro into LCM virus-specific cytolytic clones were derived from MHC-restricted Lyt-2+, L3T4- antigen-specific cytolytic T lymphocytes and their activated precursors.

Acute Disease↗
PubMed1987

A novel serine proteinase (HuTSP) isolated from a cloned human CD8+ cytolytic T cell line is expressed and secreted by activated CD4+ and CD8+ lymphocytes.

A novel proteinase, termed human T cell-associated serine proteinase (HuTSP), has been highly purified from a human CD8+ T lymphocyte clone. By using a panel of chromogenic model peptide substrates the enzyme was found to specifically recognize L-arginine and to cleave Tos-Gly-Pro-Arg-nitroanilide with high efficiency at a pH optimum of 10.5-11. Exposure to class-specific proteinase inhibitors revealed that HuTSP is a serine endopeptidase. The enzyme has a mol. mass of approximately 50 kDa (non-reduced) and of approximately 25-30 kDa (reduced) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting HuTSP to be a disulfide-linked dimer. The enzyme is shown to be inducible by lectin in both CD4+ and CD8+ lymphocytes. Moreover, HuTSP was detected in a number of independent CD4+ and CD8+ T cell clones and was found to be released from effector cells upon ligand binding to the CD3-T cell receptor complex.

Clone Cells↗
PubMed1987

Cloned cytolytic T-effector cells and their malignant variants produce an extracellular matrix degrading trypsin-like serine proteinase.

This report describes the distribution of a trypsin-like proteinase in defined homogeneous cytolytic T-cell lines (CTLL) and their in vitro and in vivo derived malignant T-lymphoma variants. By means of chromogenic peptide substrates, we found the enzyme to attack preferentially at the carboxy terminus of arginine, in particular when non-polar amino acids were present in the amino terminal neighbouring position. The enzyme was identified by means of various inhibitors as a serine type proteinase having a pH optimum around 8 X 5. Affinity chromatography in connection with molecular sieving resulted in a 200-fold purification and indicated a molecular weight (MW) of about 50,000 for the proteinase. The enzyme was found to be highly expressed in antigen-specific CTLL as well as in their tumorigenic variants. Both intact lymphocytes of all CTLL tested and Triton X-100 lysates or enriched proteinase preparations thereof were able to degrade a high molecular weight protein (casein) and to release high molecular weight split products from the sulphated proteoglycans in subendothelial extracellular matrix. The results are discussed with respect to the invasiveness of normal and malignant T lymphocytes and the proteinase is suggested to be crucially involved in the process of cellular migration in vivo.

Animals↗
PubMed1986

Purification and characterization of a T cell specific serine proteinase (TSP-1) from cloned cytolytic T lymphocytes.

We describe the purification of a T cell specific serine proteinase derived from a cloned murine cytolytic T lymphocyte line. Analysis of the enzyme by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a mol. wt of approximately 60 kd under non-reducing conditions and of approximately 30 kd under reducing conditions. The proteinase cleaves the model peptide substrate H-D-Pro-Phe-Arg-NA, at the 4 nitroanilide (NA) group with high efficiency. Much lower or no activity of the enzyme is found against synthetic peptide substrates carrying other amino acid (AA) sequences at position P2, P3 adjacent to L-arginine or against substrates in which AA other than L-arginine are bound to the NA group. Optimal pH for the cleavage of H-D-Pro-Phe-Arg-NA is in the range of 8.0-8.5. The enzyme is strongly inhibited by inhibitors of serine proteinases, diisopropylfluorophosphate, phenylmethane-sulfonyl fluoride, m-aminobenzamidine, aprotinin, and leupeptin but not by inhibitors of either thiol-, metallo- or carboxyl-proteinases. We propose the designation TSP-1 (T-cell derived serine proteinase 1) for this enzyme. TSP-1 has the capacity to stimulate B lymphocytes for proliferation in the absence of antigen.

Animals↗
PubMed1986

Characterization and isolation of a trypsin-like serine protease from a long-term culture cytolytic T cell line and its expression by functionally distinct T cells.

We describe the characterization and purification of a trypsin-like serine protease isolated from cloned long-term culture cytolytic T cell line (CTLL AK). High amounts of proteolytic activity were isolated from extracts of CTLL AK after either nitrogen cavitation or detergent lysis. Trypsin-like protease was detected by using either the ester compound N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester or a panel of low molecular amide substrates. The latter compounds were preferentially cleaved at the carboxyl termini of lysine and arginine residues. The enzyme activity was completely inhibited by two serine esterase inhibitors, diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and by aprotinin and meta-aminobenzamidine, which are known to block trypsin-like proteases. The pH optimum for CTLL AK-derived protease activity is 8 to 9. Analysis of the enzyme by gel filtration revealed that the cell-bound proteolytic activity was associated with a complex that could not be dissociated by treatment with Triton X-100. The CTLL AK-derived protease activity was found to reside in two proteins with relative molecular masses (Mr) of 32,000 and 40,000 daltons as determined by affinity labeling with [3H]diisopropylfluorophosphate and sodium dodecyl sulfate gel electrophoresis. High levels of enzyme activity were found in a panel of H-Y-specific cloned T cell lines with either cytolytic/suppressor (CTLL) or helper potential (THL), indicating a lack of correlation between trypsin-like protease activity and a particular T cell function. High enzyme activity was also detected in tumorigenic variants of CTLL. Furthermore, it was excluded that the trypsin-like activity detected was attributable to plasminogen activator activity. In contrast to cloned T effector cells and their in vitro or in vivo derived variants, considerably less activity was found in normal nonactivated or activated lymphocyte populations. The possible role of the trypsin-like serine protease in the function of T effector cells is discussed.

Animals↗
PubMed1986

Monoclonal antibodies to interferon-gamma inhibit interleukin 2-dependent induction of growth and maturation in lectin/antigen-reactive cytolytic T lymphocyte precursors.

In this study we tested the effect of monoclonal antibodies (moAb) AN-18 to murine IFN-gamma on the generation of cytolytic T cells (CTL) from a homogeneous population of precursor cells (CTL-P). As responder cells, highly purified Lyt-2+ C57BL/6 lymph node T cells were used that had been positively selected by flow cytofluorometry on a cell sorter. Lyt-2+ cells were set up in bulk culture or in limiting dilution (LD) either with Con A or with P815 tumor cells as antigen and recombinant human interleukin 2 (rec.hIL 2) in the presence or absence of moAb AN-18 and tested for growth and development of CTL. The results show that moAb AN-18 but not the unrelated moAb AN-37 diminished or abrogated proliferative and cytolytic responses of Lyt-2+ lymphocytes to lectin and rec.hIL 2 in a dose-dependent manner. The inhibitory activity of the antibodies could be abolished by neutralizing moAb AN-18 with recombinant murine IFN-gamma (rec.mIFN-gamma) before their addition to culture. Kinetic analysis shows that the inhibitory effect of moAb AN-18 is only optimal when added at the beginning of culture or up to 48 hr after initiation. The frequencies of CTL-P responding either to Con A or to P815 tumor cells and rec.hIL 2 were reduced up to 10-fold in the presence of moAb AN-18. The inhibitory capacity of moAb AN-18 was also operative in cultures containing on the average one antigen-specific CTL-P. Together with the finding that activated CTL-P secrete IFN-gamma in response to rec.hIL 2 in a dose-dependent manner, the data suggest that endogenous IFN-gamma collaborates with exogenous IL 2 in the induction of CTL-P. The generation of CTL may therefore represent a case of autocrine growth regulation of normal lymphocytes, in which the same cell synthesizes and responds to its own factor.

Animals↗
PubMed1986

Leukemic cells arise from cloned cytotoxic lymphocytes during cell culture.

In spite of many promising attempts to apply T cell clones to questions of in vitro and in vivo function of T cells it is still unclear to what extent continuous propagation of T lymphocytes in vitro effects their original properties. This study describes the appearance of malignant cells from long-term cultured C57BL/6 (B6) cytotoxic T lymphocytes (CTL). Four out of five T cell lines (CTLL.1,3,4,5) representing distinct stages of development of T effector cells in vitro were repeatedly cloned and all five CTLL were tested for various cellular parameters. It is shown that transformation of H-Y-specific CTLL into malignant cells in vitro was accompanied by alterations in growth characteristics, successive loss of specificity and cytolytic function and by quantitative changes in the expression of cell surface markers. Whereas growth of the H-Y-specific CTLL (CTLL.1) was dependent on antigen and concanavalin A (Con A) supernatant (Con ASN) the CTLL variants could be either maintained in Con ASN alone (CTLL.3) or in the absence of both antigen and lymphokine sources (CTLL.4,5). CTLL.1 was cytolytic for male B6 target cells and lysed P815 tumor targets in the presence but not the absence of lectin. In contrast, CTLL.3 lost its original specificity but lysed P815 cells in the absence or presence of lectin. CTLL.2 representing an intermediary stage showed cytolytic activity on both male B6 and P815 target cells. In contrast, CTLL.4 and CTLL.5 lost the ability to lyse any of the indicated target cells. Although all CTLL expressed the surface markers Thy-1, Lyt-2, Kb, Db and interleukin 2 receptor (IL 2 R), Thy-1 and Lyt-2 markers were drastically reduced and Kb/Db and IL 2 R structures significantly increased on CTLL.4 and CTLL.5 compared to CTLL.1,2,3. In addition, multiple karyotypic alterations including the appearance of metacentric chromosomes were observed in long-term cultured CTLL. Investigations on the expression of the alpha-, beta-, and gamma-chains of the T cell antigen receptor in CTLL.1-5 indicate that all three chains were expressed as mRNA irrespective of whether the lymphocytes expressed their original specificity and/or function. However, distinct beta variable chain genes were used by H-Y-specific CTLL and its long-term culture variants CTLL.2 and CTLL.3 suggesting that the expression of the new specificity was accompanied by the rearrangement of a new beta-chain gene in T effector cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

A specific serine proteinase is inducible in Lyt-2+,L3T4- and Lyt-2-,L3T4+ T cells in vitro but is mainly associated with Lyt-2+,L3T4- effector cells in vivo.

Recently, we and others reported on the expression of a serine proteinase in long-term cultured murine T lymphocyte cell lines. In an attempt to explore the distribution and possible regulation of this enzyme in T lymphocyte subsets, we performed the presented detailed study. We found that the proteinase is not expressed by thymocytes and resting T cells but can be induced by lectin or antigen in combination with lymphokine sources in vitro in macrophage-depleted unselected T cells as well as in both T cell subsets (Lyt-2+,L3T4- and Lyt-2-,L3T4+) separated by flow cytofluorometry. Furthermore, it appears that cell-associated proteinase activity is increasing with prolonged culture period of sensitized T lymphocytes and that it is higher in antigen-activated as compared to lectin-activated T cells. When tested for substrate specificity the T cell-associated proteinase was shown to preferentially cleave model peptide substrates carrying L-arginine at position P1 in combination with nonpolar amino acids at position P2 and P3. As concluded from its sensitivity to proteinase inhibitors the enzyme can be classified as a serine proteinase and by molecular sieving at high ionic strength it was shown to have a mol. mass of approximately 50-60 kDa. Analysis of in vivo activated T cells revealed that this particular proteinase was expressed in flow cytofluorometry sorted lymphocytic choriomeningitis virus-specific Lyt-2+,L3T4- cytolytic T lymphocytes but not in Lyt-2-,L3T4+ T cells presensitized with either Listeria monocytogenes or I-A alloantigens. The data demonstrate that the two T cell subsets (Lyt-2+,L3T4-; Lyt-2-,L3T4+) have distinct in vitro induction requirements for the expression of proteinase and that after activation of T cells in vivo the enzyme is preferentially associated with Lyt-2+,L3T4- effector cells.

Animals↗
PubMed1986

Interleukin 2 induces both, growth and maturation of lectin reactive Lyt-2+ but not Lyt-2-precursor cells and regulates the cytolytic potential of effector cells.

This study investigated the requirements for lymphokines derived by recombinant (rec.) DNA technology for the induction of growth and maturation in highly purified lectin reactive T cell subsets. Nylon purified C57BL/6 lymph node T cells were treated with monoclonal anti-Lyt-2.2 or anti-L3T4 antibodies and fluorescence labeled (FITC) anti-immunoglobulin antibodies and were positively selected into Lyt-2+ (L3T4-) and Lyt-2- (L3T4+) lymphocyte subsets using a fluorescence-activated cell sorter. Sorted T lymphocytes, which were devoid of accessory cells were incubated either in bulk culture (2 X 10(2) - 3 X 10(4) cells/microculture) or under limiting dilution conditions (2.5-1,000 cells/well) with lectin (Concanavalin A, Leukoagglutinin) and rec. human Interleukin 2 (rec. hIL-2) and/or rec. mouse Interferon gamma (rec. mIFN-gamma). The data show that Lyt-2+ lymphocytes respond to lectin and rec. hIL-2 with growth and development of cytolytic activity in the absence of other exogenous factor(s) or accessory cells. The presence of monoclonal antibodies to the Interleukin 2 receptor during the sensitization phase ablated the induction of Con A reactive precursor cells of cytolytic lymphocytes (CTL-P) by either rec. hIL-2 or conventional IL-2 containing lymphokine sources, indicating the essential role of IL-2 during activation of Lyt-2+ T lymphocytes. In contrast, Lyt-2- lymphocytes could not be induced by lectin and rec. hIL-2 alone for proliferation and always required the presence of accessory cells for significant growth. Exogenous rec. m IFN gamma was unable to induce growth and cytolytic activity in Con A reactive Lyt-2+ cells and did not significantly effect their response to rec. hIL-2. Limiting dilution experiments revealed that 10-16% of the Lyt-2+ lymphocytes responded to Con A and rec. hIL-2 with growth (GTL-P). The frequencies of CTL-P, determined under similar conditions, were always lower compared to GTL-P. However the results suggest that the differences observed between both precursor populations is due to differential sensitivity of the detection system rather than to the recruitment of distinct T cell subsets. Furthermore, it was shown that at least 50% of lectin reactive CTL-P were induced by rec. hIL-2 to secrete IFN-gamma under optimal conditions. The finding that some of the conventional lymphokine sources were superior to rec. hIL-2 in the induction of growth and cytolytic activity suggests the existence of mediators distinct from IL-2 that regulate the expansion of CTL-P.(ABSTRACT TRUNCATED AT 400 WORDS)

Agglutinins↗
PubMed1986

Change in antigen specificity of cytotoxic T lymphocytes is associated with the rearrangement and expression of a T-cell receptor beta-chain gene.

Cloned H-Y-specific murine cytotoxic T lymphocytes, which alter antigen specificity in vitro ("aging"), simultaneously exhibit changes in the T-cell antigen receptor beta-chain rearrangements and respective mRNAs expressed. beta-chain cDNA clones were isolated from a library prepared from mRNA of aged killer T cells. The sequence of the beta-chain variable region element (VAK) was found to be identical with germ-line DNA. Four bases at the beta-chain diversity-joining region (D beta--J beta) junction cannot be explained by known germ-line D beta and J beta elements. These results illustrate that in T-cell clones altered antigen specificity correlates with a switch in productive beta-chain rearrangements of the T-cell receptor. When tested for its expression under physiological conditions, significant levels of VAK mRNA were found in normal lymphocyte populations.

Amino Acid Sequence↗
PubMed1985

Immunoregulation by mouse T cell clones. III. Cloned H-Y-specific cytotoxic T cells secrete a soluble mediator(s) that inhibits cytotoxic responses by acting on both Lyt-2- and L3T4- lymphocytes.

In this study we report that cloned Thy-1+, L3T4-, Lyt-1-, Lyt-2+, H-Y-specific and H-2Db-restricted cytotoxic T cell lines (CTLL) when induced by lectin or antigen secrete a soluble mediator(s) (SF) that inhibits proliferation and generation of cytotoxic lymphocytes (CTL) in mixed lymphocyte cultures (MLC). The biological activity was separable by gel filtration and appeared as a broad peak in the molecular mass range between 10 000 and 50 000 kDa. It was found that the suppressive activity released by CTLL neither strictly correlates with their cytotoxic potential nor with their ability to produce immune interferon or lymphotoxin. SF was shown to elicit its activity in an antigen-nonspecific manner in that it suppressed the maturation of T lymphocytes responding to both, the appropriate H-Y antigen as well as to unrelated H-2d alloantigens or to the hapten 2,4,6-trinitrophenyl (TNP). The effect of SF on CTL responses was most pronounced in early phases of primary or secondary MLC. When analyzed for its inhibitory activity on precursor cells in populations selected for either Lyt-2- or L3T4- lymphocytes, it was found that SF interfered with the maturation of both subsets. The inhibition of CTL responses elicited by SF could not be reversed by the addition of exogenous interleukin 2. The finding that SF also inhibited the proliferation of some but not all antigen-dependent cloned T cells with helper or cytotoxic potential provides evidence that the factor also may regulate effector lymphocytes. In addition, the results support the assumption that SF exerts its effect directly on the responder rather than the stimulator population, and demonstrate that the development of CTL from their precursor cells is controlled at least in part by the cytotoxic effector cells themselves via a soluble factor(s) that interferes with distinct stages of T cell maturation. These findings again emphasize the expression of multiple functions by CTL and indicate their possible role during the course of an immune response by their capability to eliminate target cells and to secrete a soluble product(s) that mediates feedback control.

Animals↗