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M C Mingari

Publications and source records attributed to M C Mingari.

At least 73 records · Page 4Linked to original sources
PubMed1990

Cytolytic function of clonable T cells after human bone marrow transplantation.

We evaluated T-cell mediated lymphokine activated killer (LAK) function during the late (greater than 5 months) reconstitution phase after T cell-depleted allogeneic bone marrow transplantation (BMT) for hematologic malignancy. Since LAK cells are sustained by interleukin-2 (IL-2), we also investigated the ability of post-BMT T cells to produce IL-2. These functions were investigated at the clonal level. More than 200 T-cell clones from six long-term BMT recipients were generated and compared with 60 T-cell clones derived from two normal controls. Almost all the CD8+ clonal cultures from BMT recipients expressed cytolytic activity in a lectin-dependent cellular cytoxicity assay. Interestingly, a higher proportion of BMT recipient-derived cytolytic clones were able to mediate LAK activity in comparison with control clones (28% versus 4%, P less than .05). However, T-cell clones from BMT recipients, as opposed to control clones, were largely incapable of producing IL-2. Given the high proportions of post-BMT circulating CD8+ T cells, it appears that, in long-term BMT recipients, the precursors of nonspecific LAK effectors are present at above normal levels. However, their function may be defective in vivo due to poor IL-2 production.

Bone Marrow Transplantation↗
PubMed1990

Human lymphocytes expressing a TCR gamma/delta.

A minor subset of T lymphocytes express a TCR composed of gamma and delta chains. This subset differs from conventional T cells for a number of phenotypic and functional characteristics. TCR gamma/delta + cells simultaneously lack both CD4 and CD8 antigens. Cloning of CD4-8-peripheral blood lymphocytes, under limiting dilution conditions, revealed that they are homogeneously composed of cytolytic cells which efficiently lyse tumor target cells. Formal proofs have been provided that TCR gamma/delta + cells are able to recognize antigens. For example, they proliferated in response to allogeneic mixed lymphocyte culture (MLC), in addition MLC-derived TCR gamma/delta + cells specifically lysed PHA-induced blast cells bearing the stimulating alloantigens. The selection of monoclonal antibodies (MoAbs) specific for TCR gamma/delta molecules allowed to identify two distinct subsets of TCR gamma/delta + cells. These MoAbs, termed BB3 and delta-TCS-1 (or the equivalent A13) respectively, induced specific activation of cloned cells expressing the corresponding antigenic determinants (as assessed by measurements of intracellular Ca2+ and/or lymphokine production of cytolytic activity). Analysis of the distribution of subsets expressing different forms of TCR gamma/delta, showed that the BB3-reactive form is prevalent in the peripheral blood. In contrast, delta-TCS-1-reactive cells are relatively infrequent in peripheral blood, but represent the majority of TCR gamma/delta + in the tissues.

Animals↗
PubMed1989

Phenotypic and functional characteristics of tumor-associated lymphocytes in patients with malignant ascites receiving intraperitoneal infusions of recombinant interleukin-2 (rIL-2).

In the course of a phase I trial, in which recombinant IL-2 (rIL-2) was infused intraperitoneally (i.p.) in patients with peritoneal carcinomatosis, we evaluated the effect on "tumor-associated lymphocytes" (TAL) isolated from the ascitic fluid. No major changes in the percentages of cells expressing the CD3, CD4, CD8, Leu-7, OKM1 and WT-31 antigens were detected either in TAL or in peripheral blood lymphocytes (PBL) after 7 days of rIL-2 infusion. In contrast the percentages of TAL (but not PBL) expressing surface IL-2 receptor (Tac), or LAK-1 antigen were sharply increased. Analysis of cytolytic functions showed a potentiation of the lytic activity against natural-killer (NK) sensitive K562 target cells and the de novo appearance of lytic activity against fresh melanoma cells. In one patient IFN-gamma was detected in the ascitic fluid following rIL-2 infusion. T-cell clones derived from the patient were analyzed for the IFN-gamma production. While only approximately 40% of PB-derived control clones produced medium to low amounts of IFN-gamma, all of the TAL-derived clones produced medium to high amounts of the lymphokine.

Ascites↗
PubMed1989

Human thymocytes expressing gamma/delta T-cell receptors.

Expression of T-cell receptors of gamma/delta type characterizes a small subset of peripheral T lymphocytes which is homogeneously composed of cytolytic cells and, in most instances, lack CD4 and CD8 differentiation antigens. By the use of anti-TCR gamma/delta MAbs it is possible to identify two distinct subsets of TCR gamma/delta+ cells that are characterized by a C gamma 1 or C gamma 2-encoded forms of gamma-chain, respectively. While the BB3 MAb-reactive (C gamma 1 encoded) cell subset is prevalent in peripheral blood (PB), these cells represent less than 10% in TCR gamma/delta+ thymocyte populations. In thymus, the majority of cells was found to react with delta-TCSI (or A13) MAbs. Culture of CD4-8- thymocytes (highly enriched in TCR gamma/delta+ cells) in IL-2 resulted in the de novo expression of CD8 surface antigen and of non MHC-restricted cytolytic activity. Cloning of CD4-8- thymocytes resulted, for the most part, in CD3+ TCR gamma/delta+ cells. Moreover, the majority of clones expressed the unusual delta-TCSI+ CD8+ phenotype and lysed the NK-sensitive K562 target cells. Analysis of the immunoprecipitated TCR molecules showed the existence of the (rare) heavy (55 kDa) form of gamma-chain. A redirected killing assay using murine P815 target cells and appropriate "stimulatory" antibodies was further employed for functional analysis of thymus-derived TCR gamma/delta+ clones. While anti-CD3 MAbs efficiently triggered the cytolytic activity of all clones irrespective of their phenotype, MAbs directed to TCR gamma/delta induced efficient lysis only of BB3+ or delta-TCSI+CD8 clones, but not of delta-TCSI+ CD8+ clones.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

Surface molecules involved in CD3-negative NK cell function. A novel molecule which regulates the activation of a subset of human NK cells.

Natural killer cells are characterized by the lack of CD3/TCR molecules and by the expression of CD16 and CD56 (NKHI or Leu19) surface antigens. In addition to their ability to lyse certain tumor target cells, they release lymphokines including tumor necrosis factor and interferon gamma. Another unexpected functional capability of at least some NK cells is the ability to specifically recognize and lyse certain normal allogeneic cells (PHA-induced blasts). MAbs directed to CD2 or to CD16 surface molecules induced triggering of NK cells leading to target cell (p815) lysis in a redirected killing assay. Importantly, different from induction of T cell activation, single anti-CD2 MAbs were sufficient to trigger NK cell function. Another MAb (GL183) inducing NK cell triggering recognized a novel surface molecules expressed on 20-50% of resting or cultured NK cells. Cloned GL183+ cells displayed a variable degree of cytolytic activity against a number of human target cells of different histotype; moreover, this activity was strongly enhanced by the addition of GL183 MAb. On the other hand, GL183 MAb inhibited lysis of murine lines (including P815). Thus on P815 target cells GL183 MAb has an effect antithetical to that of other stimuli including PHA, anti-CD2 or anti-CD16 MAbs. GL183 MAb, added simultaneously to one or another of the stimuli above, strongly inhibited the target cell lysis induced by these stimuli. Thus, GL183 may represent an important molecule in the process of activation/regulation of phenotypically-defined NK cell subsets.

Antibodies, Monoclonal↗
PubMed1989

Human T lymphocytes expressing gamma/delta T cell antigen receptor.

The majority of mature T lymphocytes express CD3-associated antigen receptor molecules (TCR) formed by alpha and beta chains. Recently, a minor subset has been identified that expresses a different CD3-associated heterodimer composed of gamma and delta chains. The TCR gamma/delta+ cell subset differs from conventional T cells for a number of phenotypic and functional characteristics. The simultaneous lack of both CD4 and CD8 antigens allows to greatly enrich TCR gamma/delta+ cells (by monoclonal antibodies and complement). Cloning of CD4-8- peripheral blood lymphocytes, under limiting dilution conditions, revealed that they are homogeneously composed of cytolytic cells which, in most instances, lyse tumor target cells. The formal proof has been provided that TCR gamma/delta+ cells are able to recognize antigens. Indeed they proliferated in response to allogeneic cells in mixed lymphocyte culture (MLC) and MLC-derived TCR gamma/delta+ cells specifically lysed PHA-induced blast cells bearing the stimulating alloantigens. The use of different monoclonal antibodies specific for TCR gamma/delta molecules allowed to identify two distinct subsets which bound BB3 and delta-TCS-1 mAbs, respectively. The BB3-reactive TCR molecules were represented by C gamma 1-encoded disulfide-linked heterodimers, whereas delta-TCS-1 reacted with C gamma 2-encoded nondisulfide-linked molecules. Both BB3 and delta-TCS-1 mAb induced activation of cloned cells expressing the corresponding antigenic determinants (as assessed by measurements of intracellular Ca2+ and lymphokine production or cytolytic activity). Analysis of the unfrequent delta-TCS-1+ clones which express surface CD8 molecules revealed that the "heavy" 55 kDa form of (C gamma 2-encoded) gamma chain is selectively expressed by this cell type. Analysis of the distribution of subsets expressing different TCR gamma/delta isotypes showed that the C gamma 1-encoded, BB3-reactive form is prevalent in the peripheral blood, but virtually absent in the thymus. In contrast, cells expressing the C gamma 2-encoded, delta-TCS-1 reactive form are relatively unfrequent in peripheral blood, but represent the majority of TCR gamma/delta+ thymocytes. In addition, upon culture in rIL-2, approximately half of the delta-TCS-1+ thymocytes expressed CD8 antigen, thus providing further evidence that major differences exist in the distribution of TCR gamma/delta+ subsets in thymus and in peripheral blood.

Antibodies, Monoclonal↗
PubMed1989

Human T cells expressing the gamma/delta T-cell receptor (TcR-1): C gamma 1- and C gamma 2-encoded forms of the receptor correlate with distinctive morphology, cytoskeletal organization, and growth characteristics.

BB3 and delta-TCS1 monoclonal antibodies identify two distinct nonoverlapping populations of T-cell receptor (TcR) gamma/delta (TcR-1)-positive cells, which express a disulfide-linked and a nondisulfide-linked form of TcR, respectively. BB3+ cells represented the majority of circulating TcR-1+ cells, but they were virtually undetectable in the thymus. On the other hand, delta-TCS1+ cells were largely predominant among TcR-1+ thymocytes but represented a minority in peripheral blood (PB). Similar distributions were observed by clonal analysis of thymocytes or PB TcR-1+ populations. The use of joining region (J)-specific probes indicated that BB3+ and delta-TCS1+ clones displayed different patterns of J rearrangement. Thus, the disulfide-linked form of TcR-1 (BB3+ clones) was associated with the expression of J segments upstream to the C gamma 1 gene segment, whereas the nondisulfide-linked form (delta-TCS1+ clones) was associated with the expression of J segments upstream to C gamma 2. delta-TCS1+ clones, in most instances, exhibited a growth pattern different from that of BB3+ or conventional TcR alpha/beta+ clones as they adhered promptly to surfaces, spread, and emitted long filopodia ending with adhesion plaques. Ultrastructural analyses showed, exclusively in delta-TCS1+ cells, nuclear deformations, uropod formation, and abundant cytoskeletal structures. In addition, immunofluorescence studies of this subset of TcR-1+ cells revealed the presence of abundant microtubules, intermediate filaments, and submembranous microfilaments. Thus, our findings suggest that delta-TCS1+ cells are capable of active motility.

Blotting, Southern↗
PubMed1989

Surface molecules involved in the activation and regulation of T or natural killer lymphocytes in humans.

The surface molecules that mediate activation of different subsets of T or NK cells have been reviewed. A suitable approach to the study of different lymphocyte activation pathways is provided by mAbs specific for these molecules. MAbs directed to the CD3 surface molecules mediate a polyclonal T-cell activation, whereas mAbs to "clonotypic" structures of TCR only trigger cells bearing the corresponding clonotypic determinant (thus mimicking the effect of antigen/MHC). MAbs directed to appropriate epitopes of CD2 molecules or to CD28 molecules mediate polyclonal T-cell activation, leading to triggering of the functional program of the cell (i.e. proliferation, lymphokine production or activation of the cytolytic machinery). Interaction of specific mAbs with CD3/TCR molecules leads to surface modulation of these molecules which lasts for 48-72 h. During this interval the cell is refractory to any further activation stimulus. No such refractoriness occurs following mAb-induced modulation of CD2 or CD28 surface molecules. The mechanisms by which CD3/TCR modulation results in the inactivation of T-cell function appears to involve the early metabolic steps of T-cell activation, as neither Ca++ mobilization nor IP3 formation could be further induced by any stimulus. The surface molecules and mechanisms involved in the activation of TCR gamma/delta cells are similar to those of TCR alpha/beta + cells. TCR gamma/delta molecules are heterogeneous in size and charge mobility. MAbs directed to one or another form of TCR gamma/delta trigger the functional program of the cell (primarily cytolytic function). However, a receptor form composed of a heavy form (55 kD) of the gamma chain appears to be relatively inefficient in signal transduction upon binding with anti-TCR mAbs. Evidence has also been provided that TCR gamma/delta + cells are capable of (allo)antigen responses and that polymorphic determinants of class I can be recognized (specific lysis of P815 cells transfected with HLA-A24 allele). Although the mechanisms and the surface receptor molecules involved in (CD3-, CD16+) NK cell activation are still poorly understood, several surface molecules have been identified that mediate NK-cell triggering. These include CD2 and CD16 and the novel GL183 molecule which is selectively expressed by a fraction of NK cells and thus identifies a well-defined NK subsets. Under appropriate conditions, mAbs to CD16 or GL183 mediate an inhibitory effect on the NK cell activation. These data suggest that also NK cells are characterized by surface molecules capable of initiating distinct pathways of cell activation and that, similarly to T lymphocytes, mechanisms exist which regulate NK cell function.

Antibodies, Monoclonal↗
PubMed1989

Lysozyme-induced inhibition of the lymphocyte response to mitogenic lectins.

Both human lysozyme (HL) and hen egg white lysozyme (HEWL) inhibited the proliferative response of peripheral blood lymphocytes to T cell mitogens such as the lectins phytohemagglutinin and concanavalin A. This inhibition was observed both when HL or HEWL was added to the lymphocyte cultures in combination with phytohemagglutinin or concanavalin A and when lymphocytes were pretreated with either lysozyme and extensively washed prior to culture with mitogens. Under both conditions, the effects were strictly dose dependent; the lysozyme concentrations yielding maximal inhibitory effect were 5 micrograms/ml for HL and 1 microgram/ml for HEWL, while both lower and higher concentrations were less effective. Specific antilysozyme rabbit sera completely prevented the inhibitory effects of both HL and HEWL on the proliferative response of lymphocytes to phytohemagglutin or concanavalin A. Chitotriose (a lysozyme inhibitor) caused a strong reduction in the inhibitory effects of the two lysozymes on the lymphocyte response to either lectin. HL and HEWL also were found to markedly inhibit the polyclonal B cell proliferation and differentiation induced by pokeweed mitogen and T cells. A less marked inhibition was also obtained when T cells, but not B cells, were pretreated with HL or HEWL. Again, as in the experiments with T cell mitogens, the effects were dose dependent and 5 micrograms/ml HL and 1 microgram/ml HEWL proved to be the most effective concentrations. The possible mechanisms by which lysozyme inhibits the lymphocyte response to mitogenic lectins are considered and discussed. The enzymatic activity seemed to perform an essential function, as shown by the loss of effect when the heat- or trypsin-inactivated lysozymes were used and by the fact that only the enzymatically active compound, among certain semisynthetic derivatives of HEWL, inhibited the lymphocyte response to the mitogens. However, the cationic properties of the lysozyme molecule appeared to be essential too, since enzymes with a similar specificity of action showed effects similar to those observed with HL or HEWL only when they carried a strong positive charge. It is suggested that lysozyme, which is naturally secreted by monocytes and macrophages, might interact with lymphocyte surface receptor sites and participate in the complex mononuclear phagocyte-lymphocyte interactions and in the modulation of lymphocyte activation.

Animals↗
PubMed1989

Lymphokine production by T-cell clones after human bone marrow transplantation.

T cells recovering after bone marrow transplantation (BMT) were analyzed for their phenotypic and functional features by two-color immunofluorescence and a high efficiency cloning technique. A predominance of cells co-expressing natural killer (NK)-related surface antigens, such as Leu 7 (CD57) and CD11b, was detected within both the CD4+ and CD8+ subsets from 5 months postgrafting onward. Such cells are virtually absent among normal circulating CD4+ cells and account for a minority (approximately 30%) of normal CD8+ cells. Postgrafting T cells representative of the whole range of NK-related antigen co-expression were selected from six patients for clonal analyses. In control subjects, 63% and 41% of the CD4+ and CD8+ clones, respectively, produced interleukin-2 (IL-2) whereas approximately 30% of either CD4+ or CD8+ control clones produced interferon (IFN)-gamma. At variance, and irrespective of their CD4+/CD8+ phenotype, lower proportions of BMT recipient-derived clones produced IL-2 (20% and 12%, respectively), whereas the majority of both CD4+ and CD8+ clones (75% and 71%, respectively) released high amounts of IFN-gamma. Purified populations of CD57+/CD11b+ v negative cells from two BMT recipients and two control subjects were cloned and subsequently evaluated for IL-2 and IFN-gamma production. CD57+/CD11b+ cell-derived clones were poor IL-2 producers in both normal subjects and BMT patients. In contrast, IL-2-producing clones were frequent (62% to 79%) among those derived from CD57-/CD11b- cells from normal subjects, whereas they were still represented at lower than normal proportions, ie, 25% to 41%, among clones generated from BMT recipients. CD57+/CD11b+ cells gave rise to comparably high proportions of IFN-gamma producing clones in both normal subjects and BMT recipients (approximately 80%). In contrast, IFN-gamma producing clones were approximately 25% to 50% of CD57-/CD11b- cell-derived clones in both normal subjects and BMT patients. Therefore, while the predominance of NK-related antigen-positive T cells may be predictive of poor IL-2 and high IFN-gamma production, the immune derangement in long-term BMT recipients is further enhanced by the finding that all T cells may be poor IL-2 producers. It is also suggested that IL-2 production is a preferential function of T cells that do not express CD57 and CD11b, whereas IFN-gamma production is attributable to T cells that express CD57 and CD11b.

Antigens, CD↗
PubMed1988

Human peripheral blood lymphocytes bearing T cell receptor gamma/delta. Expression of CD8 differentiation antigen correlates with the expression of the 55-kD, C gamma 2-encoded gamma chain.

We analyzed the CD3-associated molecules present on peripheral blood-derived TCR-gamma/delta+ clones that express CD8 surface antigens. Clones were derived by limiting dilution from CD3+WT31- FACS-purified populations derived from several donors. Eight of greater than 300 TCR-gamma/delta+ clones analyzed expressed CD8 and reacted with delta-TCS-1 mAb. Cell numbers suitable for more detailed analyses could be obtained from four clones, including one derived from thymus. Analysis of CD3-associated TCR molecules immunoprecipitated by anti-Leu-4 (anti-CD3) mAb under conditions that preserve the CD3/TCR association (1% digitonin) showed a predominant 55-60-kD molecule both under reducing and nonreducing conditions. On the other hand, the delta-TCS-1-reactive molecules immunoprecipitated from 25 CD3+ delta-TCS-1+ CD8- clones, in all instances, displayed a 40-44-kD mol mass. In two-dimensional PAGE, TCR-gamma molecules precipitated from delta-TCS-1+ CD8+ clones appeared more acidic than those of BB3+ or delta-TCS-1+ CD8+ clones. Southern analysis confirmed that this type of non-disulphide-linked TCR-gamma/delta is also coded for by the C gamma 2 gene segment.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

Two subsets of human T lymphocytes expressing gamma/delta antigen receptor are identifiable by monoclonal antibodies directed to two distinct molecular forms of the receptor.

Two mAbs directed to the TCR-gamma/delta were analyzed for their pattern of reactivity with CD3+WT31- cell populations or clones. In normal individuals, the BB3 mAb reacted with approximately 2/3 of peripheral blood CD3+WT31- lymphocytes, whereas delta-TCS-1 stained approximately 1/3 of such cells. In addition, the sum of the percentages of BB3+ and delta-TCS-1+ cells approximated the percentages of peripheral blood CD3+WT31- lymphocytes in seven normal donors tested. Also, in peripheral blood-derived polyclonal CD3+WT31- populations, cultured in IL-2, cells reacting with one or another mAb accounted for the whole cell population. On the other hand, only delta-TCS-1-reactive cells, but not BB3+ cells, could be detected in unfractionated as well as in CD4-8-thymocyte populations. Analysis of peripheral blood-derived CD3+WT31- clones showed that 70% of 72 clones analyzed reacted with BB3 mAb, but not with delta-TCS-1 mAb. On the other hand, delta-TCS-1 mAb stained the remaining BB3- clones. Five clones expressing medium-low amounts of CD8 antigen were BB3- delta-TCS-1+. Both types of clones lysed the Fc gamma receptor-bearing P815 target cell in the presence of anti-CD3 mAb (but not of mAb directed against HLA-DR, CD7 molecules, or TCR-alpha/beta). In this cytolytic assay, BB3 mAb induced target cell lysis only by BB3+ clones, whereas delta-TCS-1 mAb was effective only with delta-TCS-1+ clones. The CD3-associated surface molecules expressed by BB3+ or delta-TCS-1+ clones were analyzed after cell surface iodination and immunoprecipitation with the corresponding anti-TCR mAb or with anti-CD3 mAb (in digitonin-containing buffer). In SDS-PAGE, molecules immunoprecipitated from 13 BB3+ clones displayed, under nonreducing conditions, a molecular weight of 80 kD (in some cases, a minor 38-kD band could be detected). Under reducing conditions, two major components of 44 and 41 kD (and a minor component of 38 kD) were detected. On the other hand, TCR molecules immunoprecipitated from 11 different delta-TCS-1+ clones appeared as a diffuse band of 41-44 kD, both under reducing and nonreducing conditions (under non-reducing condition, an additional 38-kD band was present). Therefore, BB3+ cells express a disulphide-linked form of TCR-gamma/delta whereas delta-TCS-1+ cells express a non-disulphide-linked form.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed1988

Clonal analysis of CD4-CD8- human thymocytes expressing a T cell receptor gamma/delta chain. Direct evidence for the de novo expression of CD8 surface antigen and of cytolytic activity against tumor targets.

CD4-CD8- human thymocytes were obtained by treating total thymocyte suspensions with anti-CD4 and anti-CD8 monoclonal antibodies (mAb) and complement. The resulting cell populations contained virtually no CD4+, CD8+ or WT31+ cells and 17-65% CD3+ cells. In addition, analysis of cell reactivity with delta-TCS-1 mAb (specific for the C gamma 2-encoded, nondisulfide-linked form of TcR gamma/delta), revealed the presence of a variable proportion of delta-TCS-1+ cells (the % of delta-TCS-1+ cells were lower than the percentage of CD3+ cells). Upon culture in recombinant interleukin 2 (IL2, in the presence of irradiated mononuclear cells), CD4-CD8- thymocytes underwent extensive proliferation. In addition, a progressive increase of CD8+ cells (but not of CD4+ or WT31+ cells) could be detected. Cells also progressively acquired cytolytic activity against K-562 or fresh melanoma cells. Fresh CD4-CD8- thymocytes were cloned under limiting dilution conditions. The cloning efficiencies were relatively high (1/3 cells); in addition, virtually all the clonal progenies obtained displayed cytolytic activity and expressed the CD3+WT31-delta-TCS-1+ surface phenotype. About half of the clones analyzed were CD8+, whereas none expressed CD4 antigens. We conclude that (a) only delta-TCS-1-reactive, TcR gamma/delta+ cells can be isolated from CD4-CD8- thymocytes cultured in IL2, and (b) the expression of CD8 antigen and of cytolytic activity reflects a true in vitro phenotypic change of CD8-, noncytolytic precursors (and not the preferential growth of few contaminating cells).

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Phenotypic and functional analysis of human CD3+ and CD3- clones with "lymphokine-activated killer" (LAK) activity. Frequent occurrence of CD3+ LAK clones which produce interleukin-2.

Clones capable of lysing fresh, uncultured tumor cells ("lymphokine-activated killer": "LAK" activity) were selected from microcultures derived from either E-rosette-positive or E-rosette-negative cell populations. All the selected clones displayed a strong cytolytic activity against the NK-sensitive K562 cell line. Two major phenotypic groups of clones could be identified: a first group expressed the CD3 differentiation antigen, present exclusively on mature T lymphocytes; however, in contrast to typical cytolytic T lymphocytes, the majority of these clones expressed the unusual CD4- CD8- phenotype, whereas the remainder were CD4- CD8+. A second group was represented by CD3- clones which, in most instances, expressed the T-cell-lineage-specific CD2 antigen. Following stimulation with phytohemagglutinin (PHA), most of the CD3+ LAK clones produced Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) whereas those expressing the CD3- phenotype did not. Since previous studies indicated that PHA may be inefficient in inducing lymphokine production by T-cell variants lacking the CD3/T cell receptor complex (TCR), CD3- clones were further stimulated with the calcium ionophore A23187 plus phorbol 12-myristate 13-acetate (PMA). Only 2/11 CD3- LAK clones produced small amounts of IL-2, whereas the majority released IFN-gamma. Given the peculiar phenotypic and functional properties of many CD3 + LAK clones, we suggest that they may belong to a T-cell subset distinct from typical CTLs.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Glycoproteic nature of surface molecules of effector cells with lymphokine-activated killer (LAK) activity. Evidence that T11, T8 or T3 molecules are not involved in tumor-cell lysis by LAK effector T cells.

Human peripheral blood mononuclear cells cultured in the presence of interleukin-2 (IL-2) acquire the capability of lysing NK-resistant fresh tumor target cells. In an attempt to delineate the surface structure(s) present on the effector cells, the latter were first treated with different amounts of pronase and neuraminidase. The effect of the enzymes on cytolytic activity against fresh melanoma cells was evaluated and compared with the NK-like activity against K562 target cells of the same effector population. At a pronase concentration of 0.01 mg/ml, no inhibition of NK-like activity was detected, whereas LAK activity was inhibited by more than 75%. In addition, neuraminidase had no effect on NK-like activity, even at 1 U/ml, whereas as little as 0.03 U/ml inhibited LAK activity by more than 75%. Metabolic inhibition of N-linked glycosylation with Tunicamycin prevented the generation of LAK activity, even when added late (18 hr before termination of the culture). Tunicamycin, on the other hand, had no effect on the boost of NK activity induced by IL-2. Provided that LAK activity can also be generated in T-cell (E-rosetting) populations, in the presence of adherent cells, we analyzed the inhibitory activity of monoclonal antibodies (MAbs) to T11, T3 and T8 molecules. While all these MAbs strongly inhibited the specific target cell lysis by alloreactive CTLs, they had no effect on the LAK activity.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Human CD3+4-8-WT31- T lymphocyte populations expressing the putative T cell receptor gamma-gene product. A limiting dilution and clonal analysis.

The small peripheral blood CD3+ T cell population lacking both CD4 and CD8 surface antigens has been analyzed in the present study. Enriched CD3+4-8- populations were obtained by depletion with anti-CD4 or anti-CD8 monoclonal antibodies (mAb) and complement. The resulting populations contained greater than 99% CD2+ cells, whereas CD3+ represented approximately 50%. Virtually all of the cells were CD4-8- and did not react with the WT31 mAb, specific for a framework determinant of the alpha/beta T cell receptor (TCR). In order to analyze the molecular nature of CD3-associated molecules in CD3+WT31- populations, cells were stimulated with 0.5% phytohemagglutinin (PHA) for 24 h and expanded for an additional 7-14 days in interleukin 2 (IL 2). The resulting cells were greater than 95% CD3+ and expressed neither CD4/CD8 nor WT31 antigen. Cell surface iodination followed by cross-linking and immunoprecipitation with anti-CD3 mAb showed that CD3-associated molecules consisted of a major 45-kDa band and a minor band of 43 kDa. Thus, whereas CD3-associated molecules isolated from polyclonal CD3+WT31+ populations (expanded in IL 2 under the same culture conditions) appeared as diffuse bands, CD3-associated molecules isolated from CD3+WT31- populations displayed a homogeneous molecular mass. Northern blot analysis revealed the presence of mRNA for the TCR gamma chain whereas the mRNA for the alpha chain was mostly represented by a truncated (1.2 kb) form. Also small amounts of a nonproductive mRNA for the beta chain were detected. Freshly isolated CD3+WT31--enriched populations proliferated in response to PHA and concanavalin A, moreover, IL 2 was detected in the culture supernatants after cell stimulation. By applying culture conditions which allow virtually all T cells to undergo clonal expansion, approximately 1/3 CD3+WT31- were clonogenic. In addition, the large majority of proliferating microcultures lysed the K562 cell line and about half the natural killer (NK)-resistant fresh melanoma target cells. A large number of clones derived from CD3+WT31- enriched populations by limiting dilution has been further analyzed. More than 95% of the clones were CD3+4-8-WT31-; 12/15 clones analyzed in more detail displayed NK activity and 6/15 lysed melanoma cells; in addition, all lysed P815 target cells in the presence of PHA, thus indicating that all the clonogenic CD3+WT31- cells have a cytolytic potential.

Antibodies, Monoclonal↗
PubMed1987

CD3+4-8-WT31-(T cell receptor gamma+) cells and other unusual phenotypes are frequently detected among spontaneously interleukin 2-responsive T lymphocytes present in the joint fluid in juvenile rheumatoid arthritis. A clonal analysis.

T lymphocytes (E rosetting cells) isolated from the joint fluid of four patients with juvenile rheumatoid arthritis (JRA) were first analyzed for surface antigen expression. Approximately 15% of cells were CD25+ (interleukin, IL, 2 receptor positive), in addition, a remarkable proportion of cells expressed the CD2+3- phenotype. CD3+ cells outnumbered the sum of CD4+ and CD8+ cells as well as the cells reactive with the WT31 monoclonal antibody (which recognizes a framework determinant of the alpha/beta T cell receptor). Purified T cells were cloned under culture conditions (1% phytohemagglutinin, PHA plus IL2) which allow clonal expansion of most peripheral blood T lymphocytes. Under these conditions proliferating cells ranged from 25 to 65%; clones (derived from microcultures containing 0.5 or 0.25 cells/well) were tested for cytolytic activity against P815 cells (in the presence of PHA) or against the natural killer (NK)-sensitive K562 target cells. Fifty-four percent and 73% of clones obtained from the two patients with the polyarticular form of the disease displayed cytolytic activity in the lectin-dependent assay. Cytolytic clones were 22 and 29% in the two patients with single joint involvement. About half of all cytolytic clones displayed NK-like activity. Surface antigen analysis revealed that, in addition to conventional CD3+4+8- and CD3+4-8+, a noticeable fraction of clones (50/202) displayed unusual surface phenotypes. In particular, 33/50 coexpressed CD4 and CD8 antigens; 7/50 were CD2+3-4-8- and displayed NK-like activity; 10/50 expressed CD3 but lacked both CD4 and CD8 antigen and did not react with the WT31 monoclonal antibody. In order to allow selective growth of IL2-responsive cells, T lymphocytes were also cloned directly in IL2. As much as 57% of all clones thus obtained (48/84) displayed cytolytic activity. Moreover, about half expressed unusual surface phenotypes including CD2+3-4-8-, CD3+4+8+ and CD3+4-8-WT31-. Given the accumulation at the site of the joint involvement of unusual T cells, most of which displayed cytolytic activity and were likely to represent cells activated in vivo (IL2 responsive), one may speculate that these cells may be involved in the injury process.

Antigens, Differentiation, T-Lymphocyte↗