Specific recognition by CD3- NK cells: a limiting dilution analysis of the frequency of alloreactive CD3- lymphocyte precursors.
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Antigens, Differentiation, T-Lymphocyte↗
Biomedical subjects
D Pende
Publications and source records attributed to D Pende.
Specific lysis of allogeneic cells after activation of CD3- lymphocytes in mixed lymphocyte culture.
Human CD3- lymphocyte populations were obtained by treating peripheral blood lymphocytes with mAbs directed to CD3, CD4, and CD8 surface antigens. The resulting populations were cultured with irradiated allogeneic cells; at day 4, 100 U/ml IL-2 were added and cultures continued for an additional 10 d. The resulting populations were CD3-CD2+CD7+ and displayed cytolytic activity against PHA-induced blast cells bearing the stimulating alloantigens but not against autologous or unrelated allogeneic blast cells. When CD3- populations were cultured with irradiated autologous cells, no cytolytic activity could be detected either against autologous or allogeneic blast cells. On the other hand, K562 target cells were lysed by both MLC-derived CD3- cell populations regardless of the origin (autologous or allogeneic) of the stimulating cells. CD3- clones were further derived from MLC-stimulated CD3- populations. These clones displayed a cytolytic pattern similar to the original MLC populations as only specific PHA blasts could be lysed. These clones did not express detectable surface TCR-alpha/beta or -gamma/delta molecules and lacked productive mRNA for TCR alpha and beta chains, while small amounts of TCR-gamma mRNA were detectable in one of four clones tested. Also mRNA for CD3 gamma and delta chains were undetectable in all clones, however, CD3 epsilon mRNA was consistently present.
Antigens, Differentiation, T-Lymphocyte↗
Antigen recognition by human T cell receptor gamma-positive lymphocytes. Specific lysis of allogeneic cells after activation in mixed lymphocyte culture.
These experiments were designed to define the ability of human TCR-gamma+ cells to recognize allogeneic cells. TCR-gamma+-enriched populations were obtained by treating peripheral blood E-rosetting cells with anti-CD4 and anti-CD8 mAbs. The resulting populations were CD2+4-8- expressed variable proportions of CD3+ cells (40-90%), and did not react with the WT31 mAb, which is specific for a framework determinant of the alpha/beta heterodimer that serves as receptor for antigen on most human T lymphocytes. After mixed lymphocyte culture with irradiated allogeneic cells for 7 d and 3 additional days in rIL-2 (100 U/ml), cells underwent proliferation in three of five individuals tested. In addition, MLC-derived cells lysed 51Cr-labeled PHA-induced blasts derived from the allogeneic cells used as stimulator, but not allogeneic unrelated or autologous blast cells. No cytotoxicity against autologous or allogeneic target cells could be induced by culturing CD3+4-8-WT31- lymphocytes in MLC with irradiated autologous cells. Surface iodination of allogeneic MLC-activated CD3+4-8-WT31- cells followed by lysis in 1% digitonin and immunoprecipitation with anti-CD3 mAb indicated that the CD3-associated molecules consisted of a major 45-kD band and a minor band of 43 kD. Northern blot analysis showed that mRNA for the gamma chain was expressed at high levels, whereas mRNAs for alpha and beta chains were missing. These data support the notion that TCR-gamma rather than TCR-alpha/beta is expressed in allospecific CD3-4-8-WT31- cell populations. Clones were further derived from MLC-stimulated CD3+4-8-WT31- populations. All the seven clones studied in detail maintained the surface phenotype as well as the cytolytic pattern of the original MLC populations, thus only specific allogeneic PHA-induced blasts were lysed. NK-sensitive as well as NK-resistant tumor targets were variably susceptible to lysis; therefore, specific cytolytic activity against allogeneic cells was not necessarily linked to the expression of MHC-nonrestricted cytotoxicity against tumor cells.
Clone Cells↗
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↗
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↗
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↗
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↗
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↗
In vitro enhancement of the proliferative response of human T cells to autologous non-T cells by hydralazine.
While high doses of hydralazine inhibit the proliferative response of T lymphocytes to mitogens and antigens, low doses (0.15 microgram/ml) selectively enhance the proliferative response of T cells to autologous non-T cells. The effect is especially pronounced on lymphocytes which express the HLA-DR4 allospecificity. These results suggest that the autologous mixed lymphocyte response with non-T cells may represent a useful in vitro model to analyse the mechanism(s) of the immunologic abnormalities induced by hydralazine.
Antibodies, Monoclonal↗
The effects of a new phthalazine derivative (MDL 899) on human lymphocyte functions.
MDL-899 is new phthalazine derivative which has been developed as a substitute for hydralazine, which has several undesirable effects, in the treatment of hypertension. The effects of MDL-899 on human lymphocyte functions are analyzed. This drug inhibited in a dose-related fashion the blastogenesis of lymphocytes upon PHA and Con A activation and down-regulated the activation of allogeneic mixed lymphocyte reactions (MLR). On the contrary the drug enhanced the activation of autologous MLR of non T/T type. This effect was five times higher on cells which carried the HLA DR 4 phenotype. The above reported observations suggest that MDL-899, as well as hydralazine, affects the in vitro responsiveness of human lymphocytes mostly in subjects with HLA-DR 4 phenotype. Whether the impact of MDL-899 on immune function gives rise to a lupus like syndrome is not known. For this reason further studies are warranted to assess its long-term in vivo effects.
Antibodies, Monoclonal↗
Enhancement of natural killer cytotoxicity by cis-diamminedichloroplatinum (II) in vivo and in vitro.
To investigate the immunomodulating properties of cis-diamminedichloroplatinum (II) (CDDP), we studied the drug's effects on natural killer (NK) lymphocyte cytotoxicity. i.p. injections of CDDP (2-6 mg/kg) into adult mice significantly enhanced cytolysis of YAC-1 and K562 targets mediated by peritoneal and spleen cells. Lysis of the NK-resistant targets P815, EL-4, MOT, and RAJI was not increased, nor was the lysis of a YAC variant which had been specifically rendered resistant to NK lysis. Activated cytotoxicity was first noted 24 h after injection and returned to baseline by 7 days. Although i.p. injection enhanced peritoneal and spleen cell lysis, i.v. injection only activated spleen cells. Two analogues of CDDP, carbo- and iproplatin, effectively enhanced NK activity, but transplatin had no effect. Activated effector cells were non-adherent to nylon wool and serum-coated plates; they co-separated with lymphocytes on Percoll gradients, and they expressed asialo GM1 determinants. Incubation of targets with CDDP for 1 or 18 h significantly increased their sensitivity to lysis by normal murine spleen cells. These data indicate that CDDP has potent effects on NK cytotoxicity.
Animals↗
Analysis of the role of xenogeneic antigens in the proliferation of human T cells stimulated with autologous non-T cells and phytohemagglutinin-activated T cells.
Since conflicting results have been reported about the role of xenoantigens in the proliferation of T cells stimulated with autologous non-T cells, the effect of the exposure of cells to xenogeneic proteins during the isolation procedure and/or the culture period on autologous mixed lymphocyte reactions (AMLR) with non-T cells and phytohemagglutinin-activated T cells as stimulators was investigated. T and non-T cells were isolated by rosetting with 2-aminoethylisothiuronium bromide-treated sheep red blood cells (AET-SRBC), by nylon-wool filtration, and by positive or negative selection with anti-class II HLA antigens and anti-T-cell monoclonal antibodies. Isolation and cultures were performed in presence of fetal calf serum (FCS) or of autologous serum. In both types of AMLR, proliferation of responding cells did not require exposure to xenoantigens. However xenoantigens enhanced the proliferation of cells from some, although not all, the donors tested. There were differences in the degree of proliferation of the cells from the donors tested, but without correlation with the two types of AMLR. These results suggest that both types of AMLR reflect a self-recognition event and not a response to xenoantigens. However the potential interference of xenoantigens, as well as the individual variability, should be taken into account when interpreting the significance of abnormalities of AMLR in immunopathologic processes.
Antibodies, Monoclonal↗
Human T cells in cord blood: abnormalities in IA antigens induction by phytohemagglutinin and in autologous mixed lymphocyte reactions.
About 25% of human T cells isolated from cord blood acquired la antigens, following stimulation with phytohemagglutinin (PHA) for 72 hr. This percentage is markedly lower than that found in PHA-activated T-cell populations (PHA-T cells) isolated from peripheral blood of adults. The low expression of la antigens by human T cells from cord blood does not reflect abnormalities in the sensitivity to PHA stimulation and/or in the kinetics of induction of la antigens. PHA-T cells from cord blood display a low stimulatory activity in autologous mixed lymphocyte reactions (MLR). The defect does not reflect a nonspecific abnormality in the stimulatory activity of PHA-T cells from cord blood, since the latter do not differ from PHA-T cells from adults in their ability to stimulate allogeneic T cells from adults. Furthermore the defect does not reflect a nonspecific abnormality in the proliferative response of T cells from cord blood, since the latter display a normal proliferative response to PHA-T cells from adults. The defect in the proliferative response is not restricted to the autologous MLR with PHA-T cells, since it was found also in autologous MLR with non-T cells as stimulators. Correlation of the temporal evolution of the abnormalities of human T cells with the maturation of the immune system may contribute to our understanding of the role of la antigens in cell-cell interactions and of the biological significance of abnormalities of autologous MLR.
Cells, Cultured↗
Role of normal adherent cells in the regulation of the autologous mixed lymphocyte reactions in humans.
The effect of normal adherent suppressor cells on the blastogenesis of human T lymphocytes in the mixed lymphocyte reaction (MLR) was studied in both allogeneic and autologous combinations. Non-T cells and Ia+ T lymphocytes were used as stimulator cells in both allogeneic and autologous MLR. The addition of adherent cells to the stimulators inhibited blastogenesis of T lymphocytes in both types of MLR when the stimulator population was made up of non-T lymphocytes but did not interfere with blastogenesis when Ia+ T lymphocytes were used as stimulator cells. The present data indicate that the T lymphocytes able to respond to Ia+ T cells (in the MLR, autologous or allogeneic) may be different from those which respond to non-T lymphocytes or may be less sensitive to the regulatory function of normal adherent cells.
Cell Adhesion↗
Decreased sensitivity of T lymphocytes to normal adherent suppressor cells in patients with head and neck cancer.
The sensitivity of T Lymphocytes to the inhibitory action of normal adherent cells in the mixed lymphocyte reaction (MLR) was studied in 20 subjects with head and neck cancer. T lymphocytes from cancer patients proliferated in the MLR both in the absence and in the presence of increasing numbers of autologous as well as allogeneic adherent cells, while the blastogenesis of T lymphocytes from controls was inhibited up to 70% by the addition of adherent cells to the culture. Such a lack of sensitivity to adherent cells in cancer patients occurred both in allogeneic and in autologous MLR. These observations indicate that the immunocompetence of patients with head and neck cancer may be related to a defect of macrophage-T lymphocyte interaction similar to the one described in patients with common varied immunodeficiency.
Adult↗
Effect of single oral doses of prednisone and deflazacort on human lymphocyte distribution and functions. Analysis with monoclonal antibodies.
Two corticosteroids, prednisone and deflazacort, have been compared with respect to their capacity of inducing redistribution of T lymphocyte subsets, modification of expression of Ia antigens by PHA primed T cells, and inhibition of blastogenesis of T cells in autologous mixed lymphocyte reactions. Both corticosteroids were able to induce T lymphocytes depletion, increase of suppressor T cells, inhibit Ia expression upon mitogenic activation of T lymphocytes, and inhibit autologous MLRs either when non T cells or when PHA T lymphocytes were used as stimulators. We conclude that deflazacort may constitute a better glucocorticoid than prednisone for correcting the imbalance among T cells subsets, the helper T cells activity and the processes of cell-to-cell cooperation in immune diseases.
Adult↗
Inhibitory effect of a low dose of prednisone on PHA-induced Ia antigen expression by human T cells and on proliferation of T cells stimulated with autologous PHA-T cells.
Administration of a small dose of prednisone markedly reduced (1) the PHA-induced expression of Ia antigens by T cells, (2) the stimulatory activity of Ia antigen-bearing T cells in autologous and allogeneic mixed lymphocyte reactions (MLRs), and (3) the proliferative response of T cells stimulated with autologous PHA-activated T cells or autologous or allogeneic non-T cells. The inhibitory effects of prednisone are reversible and are not detectable on T cells isolated from blood drawn 24 hr following prednisone administration. The kinetics of the prednisone-mediated inhibition of MLRs with autologous PHA-T cells is different from that of MLRs with autologous non-T cells. These data in conjunction with the information available in the literature suggest that the mechanisms underlying these two types of autologous MLRs are different.
Adult↗
Human T lymphocytes in aging and malignancy: abnormalities in PHA-induced Ia antigen expression and in functional activity in autologous and allogeneic MLR.
T lymphocytes from patients with solid tumors and from aged donors are abnormal in their expression of Ia antigens following in vitro stimulation with phytohemagglutinin (PHA). Ia antigens were not detected on PHA-activated T lymphocytes from 15 of 27 patients with solid tumors. The abnormality in T lymphocytes from 25 donors older than 60 years was evidenced by a reduction in the percentage of T cells acquiring Ia antigens following stimulation with suboptimal amounts of PHA and by a delayed appearance of those antigens. In both groups of donors the defect in Ia antigen expression by PHA-activated T cells did not correlate with the reduced [3H]thymidine uptake. PHA-activated T cells from aged donors and from patients with solid tumors were poorly stimulatory in autologous and allogenic mixed lymphocyte reactions. Furthermore, T lymphocytes from these two groups of donors displayed a reduced proliferative response to autologous non-T cells, but a normal proliferative response to allogeneic PHA-activated T cells and to non-T cells from control subjects.
Adult↗