Requirements for Phytohaemagglutinin Activation of Resting Pure CD4+ and CD8+ T Cells

We have utilized a new method for obtaining highly purified cells using positive selection by immunomagnetic separation to study the conditions required for phytohaemagglutinin (PHA) activation of pure T4 and T8 cells. In the presence of accessory cells (AC), a comparable proliferative response was obtained in the two subsets. In the absence of AC, PHA induced low levels of interleukin 2 (IL-2) receptor expression as well as responsiveness to IL-2 in both T4 and T8 cells. If AC or 12- O -tetradecanoyl-phorbol-13-acctate (TPA) were also present, IL-2 production and DNA synthesis were seen in both subsets. A short preincubation with PHA primed' T fells for subsequent responsiveness to IL-2 or TPA, while preincubation with TPA did not induce response to PHA. Thus, PHA alone is sufficient for the first step of T cell activation lending to IL-2 receptor expression. The second step leading to IL-2 production, is dependent on direct interaction with AC in the presence of PHA. While T8 cells are dependent on help by T4 cells for proliferation to occur during stimulation with antigen, in PHA stimulation the requirements for activation and proliferation seem to he identical for T4 and T8 cells.     

Activation of Human T Lymphocytes by Phorbol-12,13-Dibutyrate and Ionomycin

The calcium ionophore ionomycin and the phorbol ester phorbol-12,13-dibutyrate (PDBu) are shown to have a synergistic effect upon interleukin 2 (IL-2) production, interleukin 2 receptor expression, and T-lymphocyte proliferation. The proliferative response was inhibited by addition of a monoclonal antibody directed against the IL-2R (Tac antigen) demonstrating that PDBu and ionomycin induce T-cell growth through an IL-2-dependent autocrine pathway. Sequential stimulation with PDBu and ionomycin failed to induce IL-2 production, IL-2R expression, and consequently proliferation of the T cells, indicating that T-cell activation requires simultaneous activation of protein kinase C (PKC) and elevation of cytosolic calcium. Exposure of T cells to both agents for different times resulted in IL-2 production, IL-2R expression, and proliferation in proportion to the duration of incubation with at least 4 h required for maximal T-cell activation. Further, in the presence of PDBu maximal T-cell activation was found to require stimulation with ionomycin for 4 h, indicating that a sustained increase in free cytoplasmic calcium of several hours' duration is essential for T-cell activation. In contrast T cells incubated with ionomycin were induced to produce IL-2 and express IL-2Rs upon brief exposure to PDBu with a 2-h incubation period being sufficient for maximal T-cell activation. Thus transient activation of PKC seems to be sufficient for activation of the IL-2 gene and IL-2R gene. However, maximal T-cell activation requires activation of PKC for at least 2 h.     

Proliferation of highly purified T cells in response to signaling via surface receptors requires cell-cell contact

Lymphocyte proliferation is associated with cell-cell aggregation. In order to assess the importance of cell-cell contact in T-cell proliferation we examined the effect of disruption of cellular aggregation by anti LFA-1⁴ mAb on T-cell proliferation. Monocyte-dependent T-cell proliferation induced by anti-CD3 mAb, pairs of anti-CD2 mAbs, or PHA was inhibited by anti-LFA-1 mAb. Monocyte-independent proliferation of highly purified T cells to anti-CD3 mAb plus PMA or plus IL-2 and to PHA plus IL-2 was, surprisingly, also inhibited by anti-LFA-1 mAb. Anti-LFA-1 mAb caused the partial inhibition of both low-affinity and high-affinity IL-2 receptor and the complete inhibition of IL-2 synthesis. In contrast to the above, the proliferation of highly purified T cells to PMA plus ionomycin was not inhibited by anti-LFA-1 mAb. These results suggest that optimal activation of highly purified T cells via cell surface receptors requires LFA-1-dependent cell-to-cell contact between proliferating T cells as well as between T cells and accessory cells. Such contact appears to be crucial for initiating IL-2 production and for optimal action of IL-2 through its receptor.     

Surface markers on human activated T lymphocytes. IV. Comparison of high-affinity E-rosette receptor expression with the expression of other activation markers (receptor for interleukin 2, MHC class II (antigens).

In order to re-examine the value of high-affinity E rosette receptor (Eh-R) as an activation marker of human T lymphocytes, its existence on resting and activated T cells was compared with the expression of such known activation markers as receptor for interleukin 2 (IL-2R; Tac antigen) and MHC class II antigens (DR/DP and DQ). To this aim expression of the above surface markers on lymphocytes of TEe subset, derived from early E rosettes (Eh-R+) and on lymphocytes of TEl subset, derived from late E rosettes (Eh-R-), was examined immediately after purification of the cells from peripheral blood as well as during cell activation with PHA. The phenotypic studies were done by using monoclonal antibodies and indirect immunofluorescence technique. We confirmed previous observation that in the course of PHA stimulation Eh-R like IL-2R marked currently activated T cells. However, it was also found that in the long-term cultures of lymphocytes activated with PHA, the expression of Eh-R was sustained on the cells which lost their IL-2R and DR/DP antigens. The above findings and the fact that TEe cell subset consisting of Eh-R+ lymphocytes was almost completely depleted from cells bearing IL-2R and MHC class II antigens allowed us to conclude that this subset of peripheral blood T lymphocytes represented not currently activated cells but the cells which had been previously activated in vivo.     

Mechanism of Calcium Ionophore and Phorbol Ester-Induced T-cell Activation

We examined the role of monocytes in T-cell activation induced by phorbol myristate acetate (PMA) and calcium ionophore ionomycin. Depletion of monocytes from peripheral blood mononuclear cells (PBMC) was associated with the loss of interleukin-2 (IL-2) production, IL-2 receptor (IL-2R) expression and proliferation, in response to either PMA or ionomycin. Addition of monocytes to highly purified T cells resulted in the complete reconstitution of IL-2 production, IL-2R expression and proliferation by PMA-stimulated lymphocytes. Exogenous IL-2, but not interleukin-1 (IL-1), could reconstitute the T-cell responsiveness. Addition of monocytes to highly purified T cells stimulated with ionomycin resulted in partial reconstitution of IL-2 production, IL-2R expression and proliferation. Similarly, the addition of exogenous IL-2 to ionomycin-stimulated T cells only partially reconstituted the response compared with PBMC. These results suggest that monocyte-T-cell interactions contribute to IL-2 production and IL-2R expression and are crucial events for PMA-induced T-cell proliferation. With ionomycin, monocytes play a role, in part, in inducing IL-2 production, IL-2R expression and proliferation. However, IL-2 is not a sufficient signal to induce T-cell proliferative response to ionomycin, suggesting that an IL-2-independent mechanism may exist in ionomycin-induced T-cell proliferation.     

Early events during primary activation of T cells: antigen receptor cross-linking and interleukin 1 initiate proliferative response of human T cells

To study early events during primary activation of human T cells, a simple method was developed which simultaneously allows positive selection of T cells from peripheral blood lymphocytes (PBL) and their polyclonal, antigen receptor-mediated stimulation with anti-T3 monoclonal antibodies. In the absence of accessory cells, T cells activated with matrix-bound OKT3 express high levels of the Tac antigen within 15 h and produce interleukin 2 (IL2). Tac expression was further enhanced by addition of exogenous IL2. However, under these conditions purified T cells were unable to mount a proliferative response, whereas unfractionated PBL proliferated already after 24 h of culture. This unresponsiveness of purified T cells could be overcome by either re-addition of low numbers of autologous accessory cells or semipurified human IL1. As IL1 had no significant effect on Tac expression of T3-stimulated T cells, we conclude from these data that IL1 exerts in addition to its influence on IL2 production an effect, which allows antigen receptor-triggered T cells to enter the cell cycle.     

Effect of phorbol myristate acetate on T cell colony formation, interleukin-2 (IL-2) receptor expression and IL-2 production by cells from patients at all stages of HIV infection.

We and others have shown that several T cell responses induced by the mitogen phytohaemagglutinin (PHA), including T cell colony formation, IL-2 receptor (IL-2R) expression, and IL-2 production are impaired in patients with AIDS and lymphadenopathy syndrome (LAS). We investigated whether phorbol myristate acetate (PMA) could act in synergy with PHA (as it does in healthy subjects) to enhance in vitro T cell responses of patients at all stages of infection by HIV. In AIDS patients with opportunistic infections (AIDS/OI), PHA + IL-2 + PMA led to a total disappearance of T cell colonies in 10/11 patients, among whom six already displayed very low numbers of colonies induced by PHA + IL-2 (less than 50 colonies/5 x 10(4) cells). In contrast, T cell colony formation induced by PHA + IL-2 + PMA was maintained or increased, compared with that induced by PHA + IL-2, in five out of six AIDS patients with Kaposi's sarcoma (AIDS/KS), 10/14 LAS and six out of seven HIV-seropositive asymptomatic (HIV+/AS) homosexuals. In these three groups of patients, a low percentage of colony cells induced by PHA + IL-2 + PMA expressed CD3 and CD4 molecules, but 50-89% of cells were IL-2R (Tac) positive, as in healthy controls. Studies on T cell activation and IL-2 production were performed on a selected group of 12 HIV-infected patients for whom sufficient numbers of lymphocytes could be obtained. PMA induced CD4 down-modulation in controls and in HIV-infected patients. However, CD3 down-modulation and induction of the Tac chain of IL-2R by PMA were significantly impaired in patients, compared with controls, and these two parameters were correlated. Although PHA alone induced virtually normal levels of Tac antigen on patients' cells, Tac induction by PHA + PMA was significantly decreased in patients versus controls. Cells from five out of 10 patients tested failed to produce detectable amounts of IL-2 after PHA stimulation, whereas IL-2 production increased significantly in all patients tested (n = 9) after PHA + PMA, with a level of IL-2 activity significantly higher than in controls. No correlation was found in this group of patients between the effects of PMA + PHA on T cell colony formation, Tac expression, or IL-2 production, as compared with PHA alone. Taken together, our results indicate that in vitro T cell functional studies with PMA may be useful to evaluate better the defects of T cell activation in HIV-infected patients.     

Role of Ia antigens and interleukin 1 in T-cell proliferation to phytohemagglutinin

Highly purified human T cells were obtained by a four-step purification procedure which included: removal of plastic adherent cells, rosetting with sheep red cells, passage over nylon-wool columns, and treatment with mouse monoclonal antibodies to human Ia antigens and complement. The resulting T cells did not proliferate to phytohemagglutinin (PHA). Purified human interleukin 1 (IL-1) could not substitute for accessory cells in supporting a PHA response. Reconstitution with as little as 0.03% adherent cells resulted in a proliferative response to PHA. T-Cell proliferation to PHA was supported by monocytes, by Ia+ Epstein-Barr virus-transformed lymphoblastoid B-cells lines, and by Ia- cultured human dermal fibroblasts but not by Ia-containing liposomes. Addition of anti-Ia antibodies to monocyte-containing cultures did not inhibit the T-cell proliferative response to PHA. These results suggest that Ia antigen expression by accessory cells is neither necessary nor sufficient to support T-cell proliferation to PHA and that IL-1 is not sufficient to support the proliferation of T cells to PHA.     

Cellular responses to human chondrocytes: absence of allogeneic responses in the presence of HLA-DR and ICAM-1.

To assess the accessory cell function of human articular chondrocytes, we assessed the ability of human chondrocytes to stimulate allogeneic peripheral blood mononuclear cells (PBMC) and to support phytohaemagglutinin (PHA)-induced proliferation of highly purified T cells. We also examined the surface expression of HLA-DR and ICAM-1 on the chondrocytes both unstimulated and stimulated with cytokines in vitro. Chondrocytes failed to stimulate allogeneic PBMC despite the constitutive expression of MHC class I molecules and the cytokine-induced expression of class II molecules but were able to support T cell proliferation to PHA, IFN-gamma and to a limited extent, IL-1 beta, induced class II expression on chondrocytes. ICAM-1 was present on 94-99% of freshly isolated cells; this declined with culture (17-59%; P < 0.005) but was readily induced by IFN-gamma, IL-1 beta, and tumour necrosis factor-alpha. Alloreactivity and, presumably, autoreactivity to chondrocytes requires factors in addition to the surface expression of DR and ICAM-1. However the presence of these molecules suggests a capacity for cell-cell interactions in inflammatory sites such as the cartilage pannus junction.     

The Response Pattern of Human T Cells to Stimulation Following Expansion in IL-2

The ability to stimulate and induce responses of T cells is influenced by their age and state of differentiation. We have examined the response upon restimulation of T cells expanded in IL-2. Our results demonstrate that large numbers of T cells, which after activation react by secreting IL-2, can be obtained from small numbers of PBL. In this study T cells were grown in IL-2 and, after 12–14 days, IL-2 was withdrawn in order to deprive them of growth factors. Our findings showed that after resting for 48 hours without IL-2, IL-2- dependent cells reverted to small lymphocytes, ceased to incorporate ³H-TdR, and had no mRNA for activation antigens such as Tac or IL-2. The cells could be reactivated to proliferate by stimulation with a calcium ionophore ionomycin and phorbol dibutyrate (PdB). Cells from insulin dependent diabetes mellitus patients with a defined immunoregulatory defect were then studied. Our results demonstrated that the IL-2 expanded cells evidenced the immunoregulatory defect for IL-2 synthesis that we had initially defined using virgin T cells from peripheral blood. These results demonstrate that studies of immune function can be undertaken in donors from whom limited numbers of peripheral blood lymphocytes are available using primed cells which have been allowed to dedifferentiate.     

The Response Pattern of Human T Cells to Stimulation Following Expansion in IL-2

The ability to stimulate and induce responses of T cells is influenced by their age and state of differentiation. We have examined the response upon restimulation of T cells expanded in IL-2. Our results demonstrate that large numbers of T cells, which after activation react by secreting IL-2, can be obtained from small numbers of PBL. In this study T cells were grown in IL-2 and, after 12-14 days, IL-2 was withdrawn in order to deprive them of growth factors. Our findings showed that after resting for 48 hours without IL-2, IL-2- dependent cells reverted to small lymphocytes, ceased to incorporate ³H-TdR, and had no mRNA for activation antigens such as Tac or IL-2. The cells could be reactivated to proliferate by stimulation with a calcium ionophore ionomycin and phorbol dibutyrate (PdB). Cells from insulin dependent diabetes mellitus patients with a defined immunoregulatory defect were then studied. Our results demonstrated that the IL-2 expanded cells evidenced the immunoregulatory defect for IL-2 synthesis that we had initially defined using virgin T cells from peripheral blood. These results demonstrate that studies of immune function can be undertaken in donors from whom limited numbers of peripheral blood lymphocytes are available using primed cells which have been allowed to dedifferentiate.     

Cell-mediated immune functions in a patient with MHC class II deficiency

This report focuses on cell-mediated immune functions in a patient with MHC class II deficiency. The patient described presented with a case of "classical" MHC class II deficiency (T and B cells within the normal range, normal lymphocyte proliferation in response to stimulation with mitogens, gene encoding for MHC class II present, no expression of MHC class II). The absence of MHC class II expression resulted in an incapability of the patient's antigen-presenting cells to function as accessory cells in the presentation of soluble protein antigens, while accessory functions required for the induction of alloantigen-induced lymphocyte proliferation or for the generation of cytotoxic T cells in response to an allostimulus were normal. The patient's T cells responded normally to alloantigenic stimulation and also had the capacity to develop antigen-specific cytotoxic functions. However, the T cells were completely naive with respect to activation by soluble protein antigens, even after presentation by accessory cells derived from the patient's healthy histoidentical brother. In this context it was interesting to note that the patient's CD4-positive cells showed a normal pattern of expression of the 4B4 marker, a marker generally present on memory T cells. These data make it tempting to speculate that in the absence of MHC class II, other cell surface structures may at least partially take over immune functions normally under the control of the MHC class II complex.     

Reduced interleukin-2 (IL-2) production in common variable immunodeficiency is due to a primary abnormality of CD4+ T cell differentiation

Common variable immunodeficiency (CVI) is a condition characterized by hypogammaglobulinemia and impaired antibody responses, resulting in recurrent bacterial infections in untreated patients. In addition, affected individuals exhibit an increased incidence of autoimmunity, malignancy, and certain viral infections, suggesting the presence of an underlying generalized immune dysregulation. We have previously described a subgroup of CVI patients in whom T cells within PBMC populations exhibit a selective defect in lymphokine production. IL-2, IL-4, and IL-5 mRNA production was impaired in these patients, while proliferation, IL-2R expression, and c-myc mRNA production were normal. In the present series of experiments, using highly purified CD4+ T cells prepared by negative selection, we show that this lymphokine production defect is a primary abnormality of CVI CD4+ T cells: whereas CD4+ T cells from CVI patients proliferate normally in response to stimulation by PHA, staphylococcal enterotoxin B (SEB), or anti-CD2 antibodies, these stimuli induce significantly less IL-2 production than observed with CD4+ T cells from normal individuals. Furthermore, we show that this IL-2 production defect is not due to an accessory cell abnormality, since it was seen in the presence of normal (allogeneic) accessory cells, and patient accessory cells supported normal amounts of IL-2 production by PHA-stimulated CD4+ T cells obtained from normal individuals. Of interest, we also found that while IL-2 production by CD4+ T cells from CVI patients induced by stimulation with immobilized anti-CD3 antibody was reduced compared to CD4+ T cells from normal control individuals, this reduction was not statistically significant. Furthermore, stimulation of both CVI patient and normal CD4+ T cells with either ionomycin + phorbol myristate acetate or a combination of immobilized anti-CD3 antibody plus anti-CD28 antibody resulted in a 50-fold increase in IL-2 production compared to stimulation with immobilized anti-CD3 antibody alone, and, under these conditions, CVI and normal CD4+ T cells produced equivalent amounts of IL-2. Finally, minor defects in interferon- production by CD4+ T cells from CVI donors were observed, but these were less severe than the IL-2 production defects and were not statistically significant. We conclude that a primary abnormality of lymphokine production exists in the CD4+ T cells of a subset of patients with CVI. The fact that the IL-2 production defect is more severe upon stimulation with superantigen as opposed to anti-CD3 antibody, and could be overcome by stimulation with ionomycin + phorbol myristate acetate or by costimulation with immobilized anti-CD3+ anti-CD28 antibodies, implies that this defect is due to impairment of a specific signaling pathway.     

Oxidized phospholipids induce anergy in human peripheral blood T cells

Lipids are key regulators of immune responses. In this study we investigated the direct impact of oxidized phospholipids (ox-PL) on T cell activation and function. We could demonstrate that ox-PL strongly inhibit proliferation of purified human T cells induced with anti-CD3/CD28 or anti-CD3/CD63 mAb, whereas proliferation of naive T cells from human cord blood was not affected by ox-PL. Unoxidized phospholipids showed no such effect. Inhibition of T cell proliferation by ox-PL was not due to cell death. Moreover, T cell proliferation triggered by PMA/ionomycin activation was not diminished by ox-PL. T cells activated in the presence of ox-PL produced and released low amounts of IFN-gamma and IL-2, whereas IL-4 was only slightly diminished. Ox-PL prevented the expression of de novo synthesized activation markers (CD25, MHC class II) but not expression of CD63 or CD69. We further observed that T cells stimulated in the presence of ox-PL are poorly cytotoxic T cells. Most importantly, T cells activated in the presence of ox-PL failed to proliferate in response to restimulation. This hypo-proliferative state was accompanied with an up-regulation of early growth response gene 3 and Casitas B-lineage lymphoma protein B. Taken together, our results demonstrate that ox-PL are potent and specific regulators of T cell activation and function.     

Role of Accessory Cells in the Activation of Pure T Cells via the T Cell Receptor-CD3 Complex or with Phytohaemagglutinin

The ability of different subpopulations of blood mononuclear cells to serve as accessory cells in the activation of CD4+ and CD8+ T cells via Ti-CD3 or with phytohaemagglutinin (PHA) was studied. Pure CD4+ or CD8+ T cells did not respond to particle-bound anti-CD3 monoclonal antibodies (MoAb) or PHA, whereas responses were seen when non-T cells served as accessory cells. Removal of class II-positive cells from peripheral blood mononuclear cells (PBMC) or from non-T cells diminished, but did not completely abolish, the responses in both T cell subsets, indicating that the accessory cells are mainly found among the class II-positive cells. However, the class II molecules themselves were not involved, as demonstrated in antibody-blocking experiments. Removal of monocytes decreased the ability of non-T cells to serve as accessory cells for both CD4+ and CD8+ cells in PHA activation. In contrast, the removal of monocytes resulted in an enhanced activation by anti-CD3 MoAb in CD4+ T cells, while the activation of CD8+ T cells was less affected. Positively selected B cells were effective accessory cells in anti-CD3 and PHA activation. Furthermore, Epstein-Barr virus (EBV)-transformed B cell lines were very potent accessory cells both in anti-CD3 and PHA activation of T cells, and showed the strongest accessory cell function observed in this system on a per cell basis.     

CD28 activation promotes Th2 subset differentiation by human CD4+ cells

Ligation of CD28 provides a costimulatory signal to T cells necessary for their activation resulting in increased interleukin (IL)-2 production in vitro, but its role in IL-4 and other cytokine production and functional differentiation of T helper (Th) cells remains uncertain. We studied the pattern of cytokine production by highly purified human adult and neonatal CD4⁺ T cells activated with anti-CD3, phorbol 12-myristate 13-acetate (PMA) and ionomycin, or phytohemagglutinin (PHA) in the presence or absence of anti-CD28 in repetitive stimulation-rest cycles. Initial stimulation of CD4⁺ cells with anti-CD3 (or the mitogens PHA or PMA+ionomycin) and anti-CD28 monoclonal antibodies induced IL-4, IL-5 and interferon-γ (IFN-γ) production and augmented IL-2 production (6- to 11-fold) compared to cells stimulated with anti-CD3 or mitogen alone. The anti-CD28-induced cytokine production corresponded with augmented IL-4 and IL-5 mRNA levels suggesting increased gene expression and/or mRNA stabilization. Most striking, however, was the progressively enhanced IL-4 and IL-5 production and diminished IL-2 and IFN-γ production with repetitive consecutive cycles of CD28 stimulation. The enhanced Th2-like response correlated with an increased frequency of IL-4-secreting cells; up to 70% of the cells produced IL-4 on the third round of stimulation compared to only 5% after the first stimulation as determined by ELISPOT. CD28 activation also promoted a Th2 response in naive neonatal CD4⁺ cells, indicating that Th cells are induced to express a Th2 response rather than preferential expansion of already established Th2-type cells. This CD28-mediated response was IL-4 independent, since enhanced IL-5 production with repetitive stimulation cycles was not affected in the presence of neutralizing anti-IL-4 antibodies. These results indicate that CD28 activation may play an important role in the differentiation of the Th2 subset in humans.     

In Situ Hybridization of Interferon-Gamma Producing Peripheral Blood Mononuclear Cells

Individual interferon-gamma (IFN-gamma) producing cells in activated human peripheral blood mononuclear cells (PBMC) were characterized by in situ hybridization using [35S]-labelled antisense RNA probes. The proportion of positive cells expressing IFN-gamma mRNA varied according to the substances used for stimulation. IFN-gamma mRNA expressed a relatively low percentage of 1-8% PBMC after a single stimulus with mitogens or OKT-3 antibody and 20-30% of the cells were identified to synthesize IFN-gamma mRNA after stimulation with PHA + P-MA + OKT-3 antibody. The expression of IFN-gamma mRNA and production of the lymphokine was dependent on accessory cells. If accessory cells were replaced by recombinant interleukin-1 (IL-1) plus interleukin-6 (IL-6), then T-cell proliferation to phytohaemagglutinin (PHA) could be partially restored and measurable amounts of IFN-gamma were detected. The addition of interleukin-2 (IL-2) or phorbol-12-myristate-13-acetate to T cells stimulated with PHA, IL-1 and IL-6 did not restore the production of IFN-gamma to an extent comparable to that produced by T cells stimulated in the presence of accessory cells. In further studies, depletion of T-cell subsets showed that CD3+, CD4+, CD8+, CD29+ and CD45RA+ cells were involved in IFN-gamma production after mitogenic stimulation. In conclusion, our data demonstrate that IFN-gamma production is dependent on signals from accessory cells and IFN-gamma is synthesized by only a small proportion of T cells, that did not belong to a unique population, characterized by conventional cellular surface antigens.     

Analysis of phenotypic and functional changes during ganglioside-induced inhibition of human T cell proliferation.

Glycosphingolipids added to the cell culture medium can be incorporated into the plasma membrane and interfere with the growth of certain cell types. In the past years, previous reports have shown that gangliosides, a class of glycosphingolipids bearing sialic acid can inhibit antigen or mitogen induced T cell proliferative responses in vitro. We report here that the inhibition of PHA induced proliferation by the trisialoganglioside GT1b was not reversed by addition of exogenous IL-1, IL-2, TPA and calcium ionophore. Furthermore, GT1b did not affect IL-2 production by activated T cells. In addition, GT1b ganglioside could also decrease strongly the expression of the T cell antigens CD3, CD2, CD4, CD8 and the alpha/beta T cell receptor antigenic complex whereas it did not affect HLA-class I antigens. By contrast, GT1b modulated only partially membrane expression of activation antigens such as CD25 (Tac) and transferrin receptor and increased the expression of HLA-class II antigens. Moreover CD25 messenger RNA induction was not affected by GT1b treatment of PHA-stimulated T cells. Our results demonstrate that gangliosides, in spite of their anti-proliferative capacity and their modulation effect on T cell antigen membrane expression, do not prevent the progression of T cells into early stages of the activation process.     

Effects of phorbol myristate and ionomycin on in vitro growth of aged Peyer's patch T and B cells.

The proliferative responses of Peyer's patch (PP) T cells from aged BALB/c mice to concanavalin A (Con A) are considerably reduced, as compared to those of the young (P < 0.001). This reduced reactivity of aged T cells could be partly, but not entirely, corrected by interleukin 2 (IL-2) (P < 0.001). PP T cells from aged mice responded synergistically to a protein kinase C (PKC) activator, phorbol myristate acetate (PHA), plus a calcium ionophore, ionomycin, at much lower concentrations than to Con A (P < 0.001); however, the maximal proliferative response still remained nearly at 8/10th of the young (P < 0.01) and higher levels of PMA (but not of ionomycin) were required (P < 0.001). Addition of IL-2 restored the diminished response to the levels of the young T cells (P < 0.05), but that of Con A did not (P > 0.05). The proliferative responses of PP B cells to lipopolysaccharide (LPS) do not differ from those of the young (P > 0.05), but the spontaneous proliferation of aged (unstimulated) B cells is enhanced nearly twofold versus that of the young (P < 0.001). Like the PP T cells, PP B cells from aged mice also responded synergistically to PMA plus ionomycin but to a lesser degree than those of the young under the same stimulation (P < 0.01). Their maximal proliferation required higher levels of PMA, but not of ionomycin and was also diminished (P < 0.01), compared to that of the young. B cell stimulatory co-factors, IL-4 and IL-6, failed to affect the response of aged and young B cells to PMA plus ionomycin (P > 0.05), whereas LPS remediates the reduced response of aged B cells to PMA plus ionomycin. Thus, T and B cells from senescent PP demonstrate an impaired proliferative responsiveness via the Ca-dependent PKC pathway. A T cell mitogen and B cell stimulatory cytokines did not alter this activation pathway, once optimally stimulated. Whereas, T cell stimulatory cytokine IL-2 and B cell mitogen LPS could restore the age-associated decline of the corresponding lymphocyte subsets, T and B cells, in activation of the Ca-dependent pathway. The altered transmembrane signal transduction appears to be intrinsically defective in these aged PP T and B cells.