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Biomedical subjects

M C Mingari

Publications and source records attributed to M C Mingari.

At least 91 records · Page 5Linked to original sources
PubMed1986

Functional analysis of cloned germinal center CD4+ cells with natural killer cell-related features. Divergence from typical T helper cells.

Granular lymphocytes co-expressing the Leu-7 (NK-related) and CD4 (T helper cell) markers are selectively localized in the germinal centers of lymphoid tissues. Leu-7+ cells (greater than 98% of which co-expressed CD4) were isolated from inflammatory tonsils and were cloned by the limiting dilution technique. Clones were analyzed for their phenotypic and functional characteristics. CD4+-Leu-7+ cell-derived clones retained their CD3 and CD4 surface antigens, lost the Leu-7 marker, and acquired HLA-DR determinants. In comparison with clones derived from peripheral blood or tonsil CD4+ cells, CD4+-Leu-7+ tonsil cell-derived clones showed similar low frequencies of cytotoxic precursors. In contrast, the frequency of interleukin 2 (IL 2) and B cell growth factor producing clones was much lower for tonsil CD4+-Leu-7+ cells than for CD4+ blood or tonsil progenitors. We conclude that germinal center CD4+-Leu-7+ cells are a subset of T cells unable to produce IL 2 in response to phytohemagglutinin or anti-CD3 stimulation, which is effective on the majority of T helper cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1986

T cell nature of some lymphokine-activated killer (LAK) cells. Frequency analysis of LAK precursors within human T cell populations and clonal analysis of LAK effector cells.

The cell lineage of the lymphokine-activated killer (LAK) cells has been reinvestigated. Both T and non-T cells, isolated on the basis of rosette formation with sheep erythrocytes (E), generated LAK activity after 3-4 days of culture in recombinant interleukin 2 (rIL 2) in 8 different individuals tested. By applying a microculture technique which allows clonal expansion of virtually all E rosetting T cells, we further analyzed the frequency of clonogenic LAK precursors within T cell populations. Approximately 1 of 25 T cells was found to be a LAK precursor. Moreover, microcultures with LAK activity lysed both the natural killer-sensitive K562 cell line and the P815 target cells in the presence of phytohemagglutinin (PHA). Since cytolytic T lymphocytes capable of lysing P815 cells in the PHA-dependent assay were approximately 1/3, it is evident that only a minor subset of cytolytic T lymphocyte precursors can acquire LAK activity even in the presence of large amounts of IL 2. Several LAK clones obtained by limiting dilution were further expanded and analyzed for their phenotypic and functional properties. Twelve out of 14 clones analyzed expressed the T3+ T11+ phenotype whereas 2 were T3- T11+. All had maintained their original cytolytic pattern; moreover, the large majority of the T3+ clones produced IL 2 and interferon-gamma following PHA stimulation.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1986

Interleukin-2 production in response to phytohemagglutinin is not necessarily dependent upon the T3-mediated pathway of T-cell activation.

The present study was designed to investigate whether heterogeneity exists in the requirement for T3 molecules in the mechanism of T-cell activation and interleukin-2 (IL-2) release by IL-2-producing T-cell clones. Clones were derived from peripheral blood by a culture system which allows clonal expansion of essentially all T lymphocytes or from mixed lymphocyte culture (MLC) activated T-cell population. IL-2-producing clones were selected and cell aliquots treated with anti-T3 monoclonal antibodies (mAb) to induce modulation of T3 surface molecules. Although stimulation of modulated clones with different anti-T3 mAb did not lead to IL-2 production, 4/39 of these clones produced IL-2 after stimulation with phytohemagglutinin (PHA). The ability of these clones to be activated by PHA could not be explained by incomplete T3 modulation. In addition, two alloreactive clones were isolated from MLC population in which modulation of T3 antigens abrogated the IL-2 production induced by either anti-T2 mAb or allogeneic stimulation but had no effect on PHA-induced IL-2 release. These data further support the concept that PHA may trigger some T cells via surface molecules that are independent from the T3-Ti cell receptor molecular complex.

Antibodies, Monoclonal↗
PubMed1985

Anticlonotypic monoclonal antibodies induce proliferation of clonotype-positive T cells in peripheral blood human T lymphocytes. Evidence for a phenotypic (T4/T8) heterogeneity of the clonotype-positive proliferating cells.

Three previously selected monoclonal antibodies (mAb) directed against the clonotypic structure of a variant (termed JA3) of the interleukin 2 (IL-2)-producing Jurkat leukemia cell line (anti-JTi1-3 mAb) were found to induce an adherent cell-dependent proliferation of peripheral blood T cells in 20 different donors. Unlike the early cell proliferation induced by anti-T3 mAb, anti-JTi mAb-induced proliferation was detectable at day 5-6 of culture and reached peak levels at day 7-9. Less than 1% JTi+ cells were consistently detected in the starting peripheral blood lymphocytes or in control cultures in which cells were stimulated with anti-T3, phytohemagglutinin, or allogeneic cells. However, JTi+ cells were found in increasing proportions after culture with anti-JTi mAb and they were mostly represented by large blast cells expressing either the T4 or the T8 antigen, together with typical activation antigens including HLA-DR, IL-2 receptor, and 4F2. Immunoprecipitation experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that anti-JTi-reactive molecules present on antibody-stimulated lymphocytes or on JA3 cells were similar, disulphide-linked heterodimeric structures.

Animals↗
PubMed1985

B cell growth factor activity of immunoaffinity-purified and recombinant human interleukin 2.

We investigated the effect of recombinant and affinity-purified human interleukin 2 (IL2) on human B cell proliferation. Five X 10(4) nonadherent spleen cells that had been depleted twice of T cells were activated by 3-day culture with formaldehyde-killed Staphylococcus aureus Cowan strain I (SAC) prior to addition of tested growth factors. Cultures were harvested 72 h later. It was found that both IL2 preparations led to optimal cell proliferation compared with a control supernatant obtained by 36 h phytohemagglutinin stimulation of spleen mononuclear cells. Moreover, the effect of such spleen supernatant on B cell proliferation correlated with the IL2 activity since its B cell growth factor activity (BCGF) was not greater than that of purified IL2 and no residual BCGF activity could be detected after absorption of all IL2 activity by the IL2-dependent cytotoxic T lymphocyte line cells. T cells, enumerated as E-rosetting cells as well as T3+ cells, represented 0.2 to 2% of the cells recovered at termination of the cultures (day 6) and there were less than 1% E-rosetting cells in freshly purified or SAC-activated (day 3) B cell populations. Therefore, we conclude that IL2 is a growth factor not only for activated T cells but also for activated human B cells.

B-Lymphocytes↗
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

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↗
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

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↗
PubMed1983

Direct demonstration of the clonogenic potential of every human peripheral blood T cell. Clonal analysis of HLA-DR expression and cytolytic activity.

In an attempt to determine the clonogenic properties of human peripheral blood T cells, we have developed a limiting dilution microculture system using phytohemagglutinin (PHA) as T cell activator and supernatant from PHA-stimulated spleen cultures as a source of T cell growth factors. The frequencies of cells capable of extensive proliferation under these culture conditions were 0.52-0.73, 0.98-1.11, and less than 0.02 in peripheral blood mononuclear, E-rosette-positive, and E-rosette-negative cell populations, respectively. The clonogenic potential of virtually all T cells was confirmed in experiments using single cells isolated by micromanipulation. Clone size ranged between 5 and 30 X 10(4) cells on day 14 of culture. The same microculture system was used to determine the precursor frequency of all cytolytic T lymphocytes (CTL-P). As assessed by a lectin-dependent 51Cr release assay, the CTL-P frequency in purified T cell populations ranged between 0.30 and 0.34. In comparison, the precursor frequency of T cells capable of lysing K562 target cells was ranging between 0.14 and 0.16. Parallel analysis of individual clonal cultures for both lytic activities showed that 50% of the clones exhibiting lectin-dependent lysis were also active against K562 target cells. All of the proliferating clones expressed HLA-DR antigens, although to a varying degree as assessed by flow cytofluorometry. Given the high cloning efficiency of this culture system, it appears now possible to determine the precursor frequencies of the various classes of functional cells in T cell populations.

Cells, Cultured↗
PubMed1983

Ultrastructural characteristics of human T cell clones with various cytolytic activities.

Fifteen T cell clones with different (specific, antibody-dependent or natural killer-like) cytolytic activity were derived from mixed lymphocyte culture activated T cells and analyzed for their morphological characteristics and, in some instances, for their surface markers. All of the five cytolytic clones analyzed by electron microscopy possessed numerous electron-dense granules and in some instances multivesicular bodies, with or without an electron-dense matrix, that are the putative precursors of the granules. In addition, light microscopy examination of semithin sections of other ten cytolytic clones showed that the large majority of the cells in each clone had numerous toluidine blue-stained cytoplasmic granules. It is of note that nine clones without detectable cytolytic activity analyzed by electron microscopy did not possess granules and presented the features of large agranular blasts. Ten cytolytic clones were analyzed for different surface markers including rosette formation with sheep erythrocytes (E rosettes), receptors for the Fc portion of IgG or IgM ((Fc gamma R and Fc mu R) and a group of antigens recognized by monoclonal antibodies including Ia, 4F2, OKT8 or OKT4. All the clones were E-rosette+, Ia+ and 4F2+. Expression of Fc gamma R was restricted to the clones active in antibody-dependent cell-mediated cytotoxicity. Expression of OKT8 and OKT4 antigens was variable; in particular, five clones were OKT8+/OKT4-, whereas the other five expressed the OKT8-/OKT4+ phenotype. It is noteworthy that the ultrastructural features of cytolytic T cell clones are similar to those of large granular lymphocytes, known to be the only lymphoid cells in normal peripheral blood which possess cytolytic activity. Thus, it is possible that the presence of electron-dense granules may represent a morphological marker for all human cytolytic lymphocytes.

Antigens, Surface↗
PubMed1983

Relationship between expression of Fc gamma receptors or Ia antigens and cytolytic activities of alloactivated human T cells.

The relationship between expression of Ia antigens or Fc gamma receptors (Fc gamma R) and different cytolytic activities of mixed leukocyte culture (MLC)-activated T-cell populations was studied. Lymphocytes mediating specific lysis of target cells bearing the stimulating alloantigens (CTL) could clearly be distinguished from cells mediating antibody-dependent cellular cytotoxicity (ADCC) in that CTL were Fc gamma R- while cells mediating ADCC were Fc gamma R+. In contrast, MLC-generated natural killer (NK) cells were distributed in both the Fc gamma R- and Fc gamma R+ cell fractions. The presence or absence of Ia antigens did not correlate with any of the cytolytic activities studied. In addition, Fc gamma R- and not Fc gamma R+ cells responded in the secondary MLC. Thus, although Fc gamma R+ cells are generated in large numbers in MLC, they do not appear to play a direct role in specific cytotoxicity nor do they proliferate in response to secondary stimulation. However, their ability, following MLC generation, to mediate NK activity and ADCC, both of which may contribute to in vitro and in vivo lysis of allogeneic cells, might explain their appearance following allogenic stimulation.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1983

Generation of CFUC suppressor T cells in vitro: VII. T derived colony inhibitory activity (Td/CIA) has no suppressor effect on in vitro immunoglobulin production.

T derived colony inhibitory activity (Td/CIA) was obtained from unstimulated T cells from aplastic anemia patients (SAA), or from PWM primed normal T cells. Td/CIA suppressed CFUC growth of normal allogeneic marrow to less than 30% of expected growth. Td/CIA was then added to normal peripheral blood T and B cells, primed with PWM, to test whether it would interfere with in vitro immunoglobulin (Ig) production. When Td/CIA from normal T cells was added to cultures of T + B cells + PWM there was a 2-2.1-fold increase in Ig production. Similarly the addition of Td/CIA from SAA patients also resulted in a 1.4 up to 166-fold increase in Ig production. These results indicate that either (a) the targets for Td/CIA are expressed on hemopoietic but not on T and B cells, or (b) that Td/CIA inactivates an accessory cell which is essential for CFUC growth but not for the PWM driven in vitro B cell differentiation system.

Anemia, Aplastic↗