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Biomedical subjects

L Moretta

Publications and source records attributed to L Moretta.

At least 235 records · Page 13Linked to original sources
PubMed1985

Involvement of T3 and T8 molecules in human T cell receptor function. Anti-T3 or anti-T8 antibodies do not block the antigen-dependent activation of a subset of alloreactive cytolytic T cell precursors.

In the present study, we have analyzed the effects of anti-T8 and anti-T3 monoclonal antibodies (mAb) on the generation of human allospecific cytolytic T lymphocytes (CTL). A sensitive limiting dilution microculture system, in which graded numbers of responder T cells were added to 10(5) irradiated allogeneic spleen cells and saturating amounts of exogeneous interleukin 2, has been employed to provide minimal estimates of the frequencies of alloreactive CTL precursors (CTL-P) present in T lymphocyte populations freshly isolated from peripheral blood. Addition of anti-T8 or anti-T3 mAb at the onset of micro mixed lymphocyte culture containing peripheral blood T lymphocytes as responding cells caused a 65-80% reduction in the frequency of specific CTL-P which proliferate under these conditions. Moreover, in most instances (greater than 80%) the cytolytic activity of those CTL-P which underwent clonal expansion in the presence of either anti-T8 or anti-T3 mAb was found to be resistant to inhibition by the corresponding mAb added during the cytolytic assay. In addition, a great degree of overlap existed between microcultures resistant to anti-T3 and those resistant to anti-T8 mAb. In contrast, neither antibody had any inhibitory effect when added later to the cultures, presumably because at this stage CTL-P had been already triggered by alloantigens. Taken together our data indicate that the heterogeneity in the susceptibility to inhibition by anti-T3 or anti-T8 mAb, previously observed at the level of CTL clones, is predetermined already at the level of peripheral blood CTL-P.

Antibodies, Monoclonal↗
PubMed1985

Involvement of T11 molecules in antigen receptor-mediated T lymphocyte functions: effect of anti-T11 monoclonal antibody on functional capabilities of alloreactive T cell clones.

As shown by previous studies, the sheep erythrocyte-binding T11 molecule is involved in T cell activation, as well as in mechanisms of specific allogeneic target cell lysis. In this study, we utilized two anti-T11 monoclonal antibodies (mAb) that inhibited the specific cytolytic activity of mixed lymphocyte culture (MLC)-activated T cells to analyze, at the clonal level, the involvement of T11 molecules in (a) antigen-specific vs. nonspecific mechanisms of target cell lysis, and (b) antigen-driven T cell proliferation and interleukin 2 (IL 2) production vs. IL 2-induced cell proliferation. In contrast to anti-T3 or anti-T8 mAb, antibodies to T11 molecules inhibited the cytolytic activity of MLC-derived allospecific clones in a uniform manner. In addition, anti-T11 antibodies inhibited the specific activity of cytotoxic T lymphocyte clones resistant to anti-T3 antibodies, even after antibody-induced modulation of T3 molecules (while anti-T3 mAb had no effect). Similarly, anti-T11 antibodies inhibited the alloantigen-induced proliferation and IL 2 release of alloreactive clones independent of their T4+ or T8+ phenotype. The inhibitory activity of anti-T11 antibodies appears to be confined to antigen-specific T cell functions since neither natural killer-like activity of cytotoxic T lymphocyte clones nor the IL 2-induced clonal proliferation was affected. Thus, our results indicate that T11 molecules are functionally involved in antigen recognition by T cell regardless of their function and T4/T8 phenotype. The possible mechanisms of anti-T11 antibody-mediated inhibition are discussed.

Antibodies, Monoclonal↗
PubMed1985

Proliferation of T8-positive cytolytic T lymphocytes in response to thyroglobulin in human autoimmune thyroiditis: analysis of cell interactions and culture requirements.

These experiments were designed to analyze the involvement of T-lymphocyte subpopulations in autoimmune thyroid disorders such as Graves' Disease (GD) and Hashimoto's Disease (HD). In a first set of experiments, lymphocytes isolated from thyroid infiltrates or from peripheral blood of GD and HD patients were analyzed for the expression of various surface antigens. While HLA-DR + T cells were numerous among thyroid infiltrating T lymphocytes in both groups of patients, the proportions of T8 + cells (as defined by their reactivity with the B 9.4 monoclonal antibody specific for T8 surface molecule) were strikingly different in HD and GD. In the latter group of patients only 19% of infiltrating T cells were T8 +, whereas these cells represented approximately 50% in four HD patients. Given the previous demonstration that all T cells expressing T8 antigen are cytolytic T lymphocytes (CTL) or their precursors (CTL-P) in conjunction with the fact that lymphocytes from HD or GD patients are known to proliferate in vitro in response to human tg (Htg), we further analyzed the T-cell subset(s) responsible for in vitro proliferation to Htg. In these experiments, peripheral blood T lymphocytes purified from patients with GD or HD were cultured with 1 microgram/ml Htg and irradiated autologous T-depleted mononuclear cells as the source of antigen presenting cells (APC). The proportions of T8 + cells declined considerably during culture in GD patients, but at Days 6 to 9, T8 + cells represented as much as 51% of cultured T lymphocytes from patients with HD. Moreover, the majority of T8 + cells were medium-large size lymphoblasts. Removal of Htg at Day 6 resulted in both abrogation of proliferative responsiveness and in decreases of T8 + percentages. Further analysis of the cell interactions leading to T8 + cell proliferation in response to Htg showed that helper/inducer T cells, as defined by 5/9 antigen expression, were strictly required. Collectively, these features are reminiscent of the T-cell involvement in experimental autoimmune thyroiditis of mice and stress for the first time the potential role of CTL in tissue damage occurring in Hashimoto's thyroiditis.

Adult↗
PubMed1985

Phenotypic and functional heterogeneity of human T lymphocytes producing B-cell growth factor(s): a clonal analysis.

Human T cells active on B-cell proliferation are phenotypically and functionally heterogeneous. A series of 43 human T-cell clones, selected according to their ability to release factors active on B-cell proliferation, were analyzed. B-Cell proliferation was evaluated by two different assays, namely, a costimulation assay with anti-mu antibody or by an assay based on B-cell preactivation with Staphylococcus aureus. Eight of these clones expressed the T4-/T8+ phenotype while the remaining were T4+/T8-. The large majority had T-cell growth factor activity as well. However, some clones appeared to have B-cell growth factor activity only. Fourteen clones (eight T8+ and six T4+) also displayed cytolytic activity in a phytohemagglutinin-dependent cytolytic assay.

Antibodies, Anti-Idiotypic↗
PubMed1985

Analysis of the expression of the 4F2 surface antigen in normal and neoplastic fibroblastic human cells of embryonic and adult origin.

4F2 monoclonal antibody recognizes a 120-kD glycoprotein on the surface of human spread fibroblastic cells of embryonic and neoplastic origin, but it does not bind to normal spread adult fibroblasts. Flow cytometric analysis reveals that human adult fibroblasts become 4F2-positive when they are analyzed as round-shaped cells; this means that, in normal adult cells, 4F2 antigen behaves as a cryptic molecule. Thus, the basic difference between embryonic, neoplastic and normal adult cells consists in a different organization in the architecture of the cell membrane, since in embryonic and neoplastic cells there is a continuous expression of the 4F2 antigen independently of the cell shape and cell cycle phase. Quantitative flow cytometry shows that the mean surface density (MSD) of the 4F2 antigen 1, does not vary as a function of the cell cycle; 2, is inversely related to cell size and "metabolic time". This suggests that at the plateau phase the surface organization of G1 resting cells changes as a function of the number of days spent in culture; and 3, sarcoma and SV40-transformed cells show significantly increased MSD levels of the 4F2 antigen in comparison with normal cells of similar size. Electrophoretic analysis under reducing conditions confirms the quantitative differences in the expression of the 4F2 antigen described with the cell sorter. It also reveals, in a way different from that previously found with lymphoid cells, the coexistence of two molecules (85 and 73 kD) in the heavy chain regions. The 73 kD is, however, much more strongly expressed in the fibrosarcoma than in the embryonic cells. Finally, it shows that 4F2 antigen is a very useful tool for studying the organization and the structure of the cell membrane of human fibroblasts and can provide new insights to understand better the developmental and transformation processes.

Antibodies, Monoclonal↗
PubMed1985

Clonal analysis of the involvement of T11 molecules in antigen receptor-mediated T lymphocyte functions.

In this study, we utilized two anti-T11 monoclonal antibodies (mAbs) that inhibited the specific cytolytic activity of mixed lymphocyte culture (MLC) activated T cells to analyze, at the clonal level, the involvement of T11 molecules in 1) antigen specific versus non-specific mechanisms of target cell lysis 2) antigen-driven T cell proliferation and IL-2 production versus IL-2-induced cell proliferation. In contrast to anti-T3 or anti-T8 mAbs, antibodies to T11 molecules inhibited the cytolytic activity of MLC-derived allospecific clones in a uniform manner. In addition, anti-T11 antibodies inhibited the specific activity of CTL clones resistant to anti-T3 antibodies, even after antibody-induced modulation of T3 molecules (while anti-T3 mAbs had no effect). Similarly, anti-T11 antibodies inhibited the alloantigen induced proliferation and IL-2 release of alloreactive clones independent on their T4+ or T8+ phenotype. The inhibitory activity of anti-T11 antibodies appears to be confined to antigen-specific T cell functions since neither natural killer-like activity of CTL clones nor the IL-2 induced clonal proliferation was affected. Thus, our results indicate that T11 molecules are functionally involved in antigen recognition by T cell regardless of their function and T4/T8 phenotype.

Antibodies, Monoclonal↗
PubMed1985

Phenotypic and functional heterogeneity of human peripheral blood T lymphocytes producing colony stimulating factor. A clonal and precursor frequency analysis.

In this report we describe the precursor frequency and the subset distribution of peripheral blood human T cells producing lymphokine(s) acting on the proliferation and differentiation of bone marrow precursors of the granulocyte/macrophage series, as assessed in a liquid microculture assay. Because the sensitivity of this system was similar to that of the classic colony formation assay in semi-solid (methylcellulose) medium, it is likely that the lymphokine activity measured in this assay corresponds to the colony-stimulating factor (CSF) activity. Single human T lymphocytes, isolated from peripheral blood by E rosetting and Ficoll-Hypaque gradients, were seeded into microculture wells by limiting dilution or micromanipulation techniques and were incubated under culture conditions that allow clonal expansion of essentially all T cells. After 15 to 20 days, microcultures were stimulated with PHA and CSF activity was assayed in culture supernatants 24 hr thereafter. About 45% (1/2.3) of peripheral blood T cells were found to give rise to CSF-producing progenies. Moreover, when fluorescence-activated cell sorter-purified T4+ and T4- (or T8- and T8+) were analyzed, the frequency of the precursors of CSF-producing cells was 1/1.5 in the T4+ subset, whereas approximately one-third of the T8+ cells had this functional potential. To additionally characterize T cells responsible for CSF production, unfractionated T cells as well as T4+ and T4- cells were cloned by single cell micromanipulation. The resulting clones were analyzed simultaneously for the production of IL 2, production of CSF and for cytolytic activity in a lectin-dependent assay. It was found that 25/48 clones obtained from unfractionated T cells produced CSF, whereas 23/48 and 19/48 produced IL 2 or had cytolytic activity respectively. Six of the 25 CSF-producing clones had only this functional capability, whereas the remaining clones in addition displayed cytolytic activity (4/25), IL 2 production (10/25), or both (5/25). A similar functional heterogeneity was observed among T4+ and T4- clones, thus indicating that T cells producing CSF are functionally heterogeneous within both the T4+ and T8+ subsets.

Cell Differentiation↗
PubMed1985

Mutant EL-4 thymoma cells polyclonally activate murine and human B cells via direct cell interaction.

In this report we show that three mutagenized sublines of (murine) EL-4 thymoma cells can constitutively activate human and/or murine B cells via an MHC-nonrestricted cell-cell interaction. The activation signal is not by itself mitogenic but renders B cells capable of proliferating in response to interleukin 2 (IL 2). In addition, one of the mutant EL-4 sublines can constitutively respond by release of IL 2 in the presence of IL 1-containing macrophage (P388D1) supernatant. The exact relationships between these functional properties of the mutant EL-4 thymoma cells and those associated with activated normal T helper-cells remain to be established. However, the EL-4 cells provide a unique system to study in parallel murine and human B cell responses. In particular, the following observations were made during the present study. First, anti-Ig antibodies (anti-Ig) were required for B cell activation in conjunction with two EL-4 sublines acting only on murine B cells, whereas with a third subline acting on both murine and human B cells, anti-Ig was not required. Anti-Ig by itself did not lead to significant B cell activation in the absence of mutant EL-4 (or normal T) cells. Second, the growth factor-stimulated proliferation of EL-4-activated B cells, following separation of the B cells from the EL-4 cells, lasted only 2 days. These results, thus, indicate that the requirement for a surface Ig-mediated B cell activation signal depends on the quality/intensity of a direct T cell signal and that cell-cell interactions may exert a more stringent control over the growth factor responsiveness of B cells as compared with T cells.

Animals↗
PubMed1985

Functional characterization of human thymocytes: a limiting dilution analysis of precursors with proliferative and cytolytic activities.

In this report we have analyzed the pool size of human thymocytes capable of proliferating or mediating cytolytic activity. Furthermore, the relationship between these functional capabilities and cell surface antigen expression was studied. Graded numbers of human thymocytes were plated under limiting dilution conditions with irradiated human spleen cells (as a source of feeder cells) and phytohemagglutinin (PHA) in the presence of a saturating concentration of interleukin 2. Cell proliferation, which was evaluated after 20 days of culture, was usually compared with the proliferation of peripheral blood T cell populations cultured under identical conditions. Although essentially all peripheral blood T cells were clonogenic, only 3 to 8% of thymocytes proliferated. Of proliferating microcultures, 48 to 86% showed cytolytic activity in a PHA-dependent assay, whereas 26 to 83% killed the NK-sensitive target cell K-562 in the absence of added lectin. Similar frequency analysis of functional precursors was performed on thymocyte subsets selected according to their expression of T3, T6, T4, and T8 antigens. All precursors of proliferating cells (PTL-P) were found in the T3+ subset. From the comparison of the percentages of total thymocytes capable of proliferation and the proportion of cells brightly stained with anti-T3 antibody, it was evident that only a fraction of T3+ cells was clonogenic. Although the large majority of PTL-P was found in the T6- subpopulation, a small fraction of functional precursors was detected in the T6+ subset. When thymocytes were fractionated according to T4 or T8 antigen expression, it was found that 80 to 90% of the recovered PTL-P were confined to the T4+ fraction, whereas only 20 to 28% of the recovered PTL-P were present in the T8+ subset. Analysis of the precursors of cytolytic T lymphocytes (CTL-P) in thymocyte populations fractionated according to T4 or T8 antigen expression showed that 70 to 90% of the recovered CTL-P were found in the T4+ fraction and 17 to 30% were in the T8+ subset. Because approximately 80% of proliferating T4+ thymocytes had CTL activity (as compared with less than 5% in peripheral blood T4+ lymphocytes), it appears that the subset distribution of thymic CTL-P differs from that of peripheral blood T cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1984

Clonal heterogeneity in the requirement for T3, T4, and T8 molecules in human cytolytic T lymphocyte function.

In an attempt to define the requirement of T8, T4, and T3 surface molecules in functional interactions occurring between human cytolytic T lymphocytes (CTL) and specific target cells, we have analyzed a large number of CTL clones derived from primary mixed lymphocyte culture (MLC) T cell populations for their susceptibility to inhibition by monoclonal antibodies (mAb) directed against these surface antigens. In most experiments, MLC T cells were stained with B9.4 (anti-T8) or OKT4 (anti-T4) mAb, separated into positive and negative cells using a fluorescence-activated cell sorter (FACS) and cloned under limiting conditions. While the lytic activity of the majority of T8+ CTL clones was inhibited by B9.4 mAb, approximately 15% of these clones were unaffected even in the presence of excess antibody. Flow cytofluorometric analysis of T8 antigen in individual clones did not show any correlation between the amount of T8 antigen expressed, the magnitude of cytolytic activity and the susceptibility (or lack thereof) to inhibition by B9.4 mAb. Of the 16 T4+ CTL clones analyzed, 7 were resistant to inhibition by OKT4 mAb even at doses 10-fold higher than that sufficient for complete inhibition of susceptible clones. Again, no correlation was found between the amount of T4 antigen expressed and the susceptibility to inhibition by the corresponding antibody. The same sets of T8+ and T4+ CTL clones were also analyzed for their susceptibility to inhibition by OKT3 mAb. Although all of the clones expressed the T3 surface antigen, only 15/23 T8+ clones and 9/14 T4+ clones were inhibited by anti-T3 mAb. To further document this clonal heterogeneity, we selected two T3+ T4- T8+ CTL clones that had no concomitant NK-like activity. One clone was resistant to inhibition by OKT3 mAb, whereas the other was highly susceptible. Incubation with OKT3 mAb resulted in modulation of the T3 molecules in both clones. Following modulation, however, the cytolytic activity of the resistant clones was unaffected, whereas the lytic activity of the susceptible clone was abrogated. These results thus indicate extensive clonal heterogeneity in the requirement for T3, T4, and T8 molecules in CTL function. Moreover, it appears that T3 molecules are not always physically and functionally linked to CTL receptor structures.

Antibodies, Monoclonal↗
PubMed1984

Assignment of human natural killer (NK)-like cells to the T cell lineage. Single allospecific T cell clones lyse specific or NK-sensitive target cells via distinct recognition structures.

The aim of the present study was to define the cell lineage of mixed lymphocyte culture (MLC)-induced natural killer (NK) effector cells. Human MLC cells were plated under limiting microculture conditions in the presence of irradiated spleen cells and interleukin 2-containing supernatant. After 18 days, microcultures were scored for proliferation and for cytolytic activity against specific lymphoblasts and NK-sensitive K562 target cells. About 1 in 7 and 1 in 5 proliferating microcultures had specific or NK-like cytolytic activity, respectively. Moreover, several microcultures exhibited dual (specific and NK-like) cytolytic activity, even when they had been established at relatively low numbers of responding cells/well (0.5-0.25) to ensure a high probability of monoclonality. Direct evidence for the existence of cytolytic effector cells with dual activity was achieved by using clones derived from single MLC T cells by micromanipulation. Out of 26 cytolytic clones so derived, 16 exhibited specific cytolytic activity, whereas 22 lysed K562 target cells. More interestingly, 12 of these 26 clones were active against both types of target cells. Only one of these clones was able to lyse autologous or unrelated target cells. In contrast, all such clones lysed the NK-sensitive cell lines G11, MOLT-4, Raji, Daudi, Chang and T-24. Addition of saturating amounts of B9-4 monoclonal antibody in the lytic assays resulted in the inhibition of the specific cytolysis, but not the NK-like activity of clones with dual cytolytic activity. It thus appears that (a) alloreactive cytotoxic T lymphocytes can mediate both specific and NK-like cytolysis and (b) two independent recognition structures are involved in this dual activity.

Cell Differentiation↗
PubMed1984

Frequent coexpression of cytolytic activity and lymphokine production among human T lymphocytes. Production of B cell growth factor and interleukin 2 by T8+ and T4+ cytolytic clones.

These studies were designed to determine the degree of overlapping between cytolytic function and lymphokine production among peripheral blood human T lymphocytes. T8+ and T4+ cells were obtained by sorting purified T cells using the fluorescence-activated cell sorter (FACS) and cloned at 0.25 cells/well in a microculture system that allows clonal growth of every T cell. 49/49 T8+ clones but only 5/102 T4+ clones displayed cytolytic activity, as assessed by a lectin-dependent assay. Cytolytic clones were further analyzed for their ability to release interleukin 2 (IL2) and B cell growth factor(s) (BCGF) upon 24 h stimulation with phytohemagglutinin. Fifteen percent and 18% of T8+ cytolytic clones were producing IL2 and BCGF, respectively; in addition 3 out of 5 T4+ cytolytic clones released both lymphokines. Although the majority of cytolytic clones producing IL2 were also producing BCGF, 5 clones released either BCGF or IL2. The present studies indicate that the coexpression of cytolytic and helper/inducer functions is relatively common among peripheral blood T lymphocytes, irrespective of their T8+ or T4+ phenotype.

B-Lymphocytes↗
PubMed1984

Interleukin 2 and interferon-gamma are not sufficient to induce natural killer-like activity in human T cell clones.

The role of interleukin 2 (IL 2) and interferon (IFN)-gamma in the maintenance of natural killer (NK)-like activity mediated by human cytolytic T cell clones was investigated. When cultured in standard supernatant (SN) obtained by phytohemagglutinin stimulation of human mononuclear cells (containing both IL2 and IFN activities), several mixed lymphocyte culture-derived clones displayed cytolytic activity directed against the K562 cell line; in addition, some of these clones also lysed specific target cells bearing the sensitizing alloantigens. When cultured in IL2-containing SN obtained from the Jurkat cell line, the NK-like activity was selectively abrogated within 4-5 days. However, by culturing cells again in standard SN the cytolytic activity was completely restored. The possibility that inhibitory molecules in Jurkat SN could be responsible for the loss of NK-like activity was ruled out by experiments showing that cytotoxicity could be induced by mixtures of Jurkat and standard SN. In addition, a source of affinity chromatography-purified IL2 also failed to maintain NK-like activity. Moreover, addition of recombinant IFN-gamma in combination with purified IL2 did not prevent the loss of NK-like activity. The present results indicate that neither IL2 nor IFN-gamma are sufficient to maintain NK-like activity and suggest that other molecule(s) are required.

Cells, Cultured↗
PubMed1984

4F2 monoclonal antibody recognizes a surface antigen on spread human fibroblasts of embryonic but not of adult origin.

The 4F2 monoclonal antibody (mAb) has been shown to recognize a 120-kilodalton glycoprotein expressed on the cell surface of human peripheral blood monocytes, activated (but not resting) T or B cells, and T and B lymphoblastoid cell lines. In this report we show that 4F2 mAb specifically binds to the surface of adherent human embryonic fibroblasts but fails to bind to normal adult fibroblasts. Moreover, 4F2 antigen was expressed on sarcoma-derived or SV40-transformed adult fibroblastic cells. Finally, addition of 4F2 mAb inhibited the growth of cultured HT-1080 fibrosarcoma cell line, but had no inhibitory effect on various embryonic and adult normal or transformed fibroblasts.

Animals↗
PubMed1983

Quantitative assessment of the pool size and subset distribution of cytolytic T lymphocytes within human resting or alloactivated peripheral blood T cell populations.

In order to directly assess the distribution of cytolytic T lymphocytes (CTL) and their precursors (CTL-P) in the two major subsets of human T cells, we have used limiting dilution microculture systems to determine their frequencies. The two subsets were defined according to their reactivity (or lack thereof) with B9.4 monoclonal antibody (the specificity of which is similar, if not identical, to that of Leu 2b monoclonal antibody). Both B9+ and B9- cells obtained by sorting peripheral blood resting T cells using the fluorescence-activated cell sorter (FACS) were assayed for total CTL-P frequencies in a microculture system that allows clonal growth of every T cell. As assessed by a lectin-dependent assay, approximately 30% of peripheral blood T cells were CTP-P. In the B9+ subset (which represents 20-30% of all T cells), the CTL-P frequency was close to 100%, whereas the B9- subset had a 25-fold lower CTL-P frequency. It is thus evident that 90% and 10% of the total CTL-P in peripheral blood are confined to the B9+ or B9- T cell subsets, respectively. Analysis of the subset distribution of CTL-P directed against a given set of alloantigens confirmed these findings. CTL-P frequencies were also determined in B9+ and B9- subsets derived from T cells that had been activated in allogenic mixed leucocyte cultures (MLC). Approximately 10% of MLC T cells were CTL-P. This frequency was increased 3.5-fold in the B9+ subset, whereas the B9- subset contained only a small, although detectable number of CTL-P. Moreover, the great majority of the (operationally defined) CTL-P in MLC T cell population were found to be directed against the stimulating alloantigens, thus indicating a dramatic increase in specific CTL-P frequencies following in vitro stimulation in bulk cultures.

Antibodies, Monoclonal↗