Triggering via the alternative CD2 pathway induces apoptosis in activated human T lymphocytes

Activation of immature thymocytes or transformed (i.e. leukemic) T lymphocytes via CD3/T cell receptor (TcR) signaling can induce programmed cell death (apoptosis). Recent data indicate that anti-CD3/TcR monoclonal antibodies (mAb) also trigger apoptosis in activated (but not resting) mature peripheral LT cells. We now report that interleukin-2 (IL-2) dependent human polyclonal T cell lines as well as T cell clones undergo programmed cell death when triggered via the alternative CD2-dependent activation pathway. In the presence of exogenous IL-2, a pair of mitogenic anti-CD2 mAb suppressed the IL-2-driven proliferative response. Growth inhibition was associated with cell death and DNA fragmentation as revealed by propidium iodide staining and gel electrophoresis, respectively. Induction of apoptosis by anti-CD2 mAb was prevented by cyclosporine A and FK 506. We conclude that programmed cell death can be initiated in activated human T cells by signaling via the CD2 pathway.     

Cell suppression in PPD-induced blast specific response of human peripheral blood lymphocytes.

Secific stimulation of T-cells by PPD was inhibited by their autologous B cells. This inhibition was obtained with B cells separated either by depletion of E-RFC or by elution, with human IgG of lymphocytes bound to Sephadex beads coated with rabbit antibodies anti-human Fab fragments. The suppression was proportional to the number of B cells added to 10(6) T cells incubated with PPD and as previously reported was more marked in the case of B or T cells from BCG-vaccinated subjects with negative skin tests. The suppressive phenomenon required viable B cells and was inhibited by cycloheximide but was not altered by pretreatment of suppressor cells with actinomycin D or colchicine. It seems that B-suppressor cells interfere with recognition of PPD by T cells rather than with the proliferative phase of the specific blast response. Using various surface markers (i.e. Ig, C3 and Fc receptors) it was shown that the suppressor cells represent a subset of Ig-bearing B cells which do not carry Fc receptors.     

Ethanol-induced CD3 and CD2 hyporesponsiveness of peripheral blood T lymphocytes

Abstract

The functional relevance of a direct ethanol effect on the membrane structure of T lymphocytes and accessory cells (APC), as well as on signal transduction systems was studied in ten normal subjects. Ethanol incubation (80 mM for 24h) of highly purified T cells increased the number of CD4⁺/CD45RA⁺ lymphocytes. In contrast, ethanol exposure induced a drop in CD14⁺/LFA-3⁺ APC values. These changes were accompanied by faulty T-cell proliferation in response to anti-CD3 and anti-CD2 mAb and inhibition of CD3- and CD2-mediated rises in intracellular calcium and, to a lesser extent, inositol 1,4,5-triphosphate levels.

These data clearly indicate that a membrane-specific ethanol interaction both modifies surface glycoproteic and/or glycolipidic structures and alters transmembrane transduction of the activation signals.     

Ethanol-induced CD3 and CD2 hyporesponsiveness of peripheral blood T lymphocytes

The functional relevance of a direct ethanol effect on the membrane structure of T lymphocytes and accessory cells (APC), as well as on signal transduction systems was studied in ten normal subjects. Ethanol incubation (80 mM for 24h) of highly purified T cells increased the number of CD4⁺/CD45RA⁺ lymphocytes. In contrast, ethanol exposure induced a drop in CD14⁺/LFA-3⁺ APC values. These changes were accompanied by faulty T-cell proliferation in response to anti-CD3 and anti-CD2 mAb and inhibition of CD3- and CD2-mediated rises in intracellular calcium and, to a lesser extent, inositol 1,4,5-triphosphate levels.

These data clearly indicate that a membrane-specific ethanol interaction both modifies surface glycoproteic and/or glycolipidic structures and alters transmembrane transduction of the activation signals.     

Selective suppressive effects of Trypanosoma cruzi on activated human lymphocytes.

The acute phase of Chagas' disease is accompanied by immunosuppression. To explore the underlying mechanism(s), we used an in vitro culture system in which the capacities of activated human peripheral blood mononuclear cells to express interleukin-2 receptors (IL-2R) and proliferate are markedly inhibited in the presence of Trypanosoma cruzi, the etiologic agent. The present work was designed to define the earliest time at which T. cruzi-induced suppression is manifested in terms of IL-2R expression on the cell surface and establish whether expression of other lymphocyte activation markers is also suppressed by the parasite. We found that expression of IL-2R by human peripheral blood mononuclear cells cocultured with T. cruzi and stimulated with either phytohemagglutinin or anti-CD3 (a monoclonal antibody specific for an epitope of the T cell receptor complex T3-Ti) was significantly suppressed as early as 12 h after culture initiation. Both the percentage of IL-2R+ cells and the surface density of IL-2R, measured by flow cytometry, were affected. However, expression of EA1, a human lymphocyte activation antigen known to be expressed 4 to 6 h after stimulation, was not altered by T. cruzi whether phytohemagglutinin or anti-CD3 was used. On the other hand, expression of transferrin receptors (TfR), which first occurs between 20 and 24 h after lymphocyte activation, was markedly suppressed by T. cruzi. This effect was denoted by significant reductions in both the percentage of TfR+ cells and the cell surface density of TfR whether phytohemagglutinin or anti-CD3 was used as the mitogen and was observed at all test times, i.e., at 24, 48, 72, and 96 h. Because expression of IL-2R and TfR is required for lymphoproliferation but that of the EA1 lymphocyte activation marker is apparently not, these results are consistent with the possibility that T. cruzi, at a relatively early stage during lymphocyte activation, selectively affects certain key events on which clonal expansion is dependent. Inhibition of IL-2R and TfR expression by the parasite might play a role in causing the suppressive effects associated with acute Chagas' disease.     

Stimulation of B lymphocytes via CD72 (human Lyb-2).

A monoclonal antibody to CD72, the 45-kDa human homolog of murine Lyb-2, was found to augment selective activation pathways in tonsillar B lymphocytes. By itself, CD72 antibody provided a weak direct stimulus to resting B cells as assessed by [3H]dThd incorporation; this was modestly enhanced on the addition of interleukin 4 (IL4) or following ligation of surface CD40. CD72-delivered signals were more evident in co-stimulation assays with phorbol ester and with a synergistic combination of IL4 and CD40 antibody, but not with calcium ionophore or a CD23 antibody; rapidly cycling B cells were refractory to signaling via CD72 whether or not other co-stimuli were present. A unique feature of the CD72-delivered signal was its ability to enhance synergistically stimulations triggered with immobilized antibody to IgM, while failing to augment responses initiated by soluble anti-mu. On direct culture of resting B lymphocytes with CD72 antibody, an approximately twofold increase in the expression of major histocompatibility complex class II DQ antigen was observed, an augmentation similar to that achieved with IL 4; CD72 antibody also mimicked IL 4 in its ability to drive G0 cells into the early G1 phase of cell cycle. In contrast to IL 4-promoted stimulation, CD72 antibody failed to up-regulate the surface expression of either IgM or the CD23 antigen. CD72 expression itself was found to be weak on resting B lymphocytes and was modestly enhanced following culture with IL 4. The findings are discussed with reference to observations made on the triggering of murine B lymphocytes through Lyb-2 and within the context of previously defined human B cell activation pathways.     

Prednisone increases apoptosis in in vitro activated human peripheral blood T lymphocytes

Glucocorticoid hormones (GCH) regulate, through the apoptotic process, the negative selection of immature T cells in the thymus. Because apoptosis seems to occur also in the maintenance of peripheral tolerance, we have investigated whether GCH may induce apoptosis in human mature lymphocytes. Peripheral blood lymphocytes (PBL) or peripheral CD4⁺ and CD8⁺ T cell subsets were cultured in the presence of phytohaemaglutinin (PHA) or PHA and prednisone (PDN) at 10⁻³-10⁻¹²M concentrations for 72, 96 and 120h. Cell cycle and membrane antigen expression were evaluated by flow cytometry and DNA degradation was detected by agarose gel electrophoresis. PDN blocks PBL growth in the G₁ phase of cell cycle and inhibits both IL-2 receptor (IL-2R) expression and IL-2 secretion. Apoptosis is clearly increased by PDN in PHA-activated human PBL, and the apoptotic effect of PDN is stronger on CD8⁺ than on CD4⁺ T lymphocytes. All these effects are dose- and time-dependent. The addition of exogenous IL-2 did not rescue lymphocytes from PDN-increased apoptosis. These results show that PDN increases apoptosis in mature activated human peripheral blood lymphocytes, suggesting a possible role of GCH in the maintenance of immune tolerance at post-thymic level.     

Differentiation of human peripheral blood B lymphocytes.

Human B-lymphocyte differentiation was studied by measuring the capacity of such cells, isolated from peripheral blood, to synthesize and secrete Ig after pokeweed stimulation. Results show that a maximum incorporation of [3H]-thymidine took place 2 days before the appearance of detectable Ig-secreting cells. On the 7th day after pokeweed stimulation, when Ig synthesis and secretion are at a maximum, [3H]-thymidine uptake was low. Since inhibition of DNA synthesis 3 days after pokeweed stimulation completely prevents the generation of Ig-secreting plasma cells, initial DNA synthesis is apparently essential before Ig-secreting plasma cells can develop in response to pokeweed stimulation.     

Normal development of lymphokine activated killing (LAK) in peripheral blood lymphocytes from hyperprolactinemic patients.

The effect of prolactin on the interleukin 2 (IL2)-driven development of Lymphokine Activated Killing (LAK) by normal PBL and by PBL from hyperprolactinemic patients was investigated. Concentrations of PRL corresponding to the physiological serum levels of the hormone and to the Kd of the PRL receptors on NK cells (6-20 ng/ml, 0.3-1 nM) had no effect on the generation of LAK activity by normal PBL, whereas 100-200 ng/ml were slightly, although significantly, inhibitory. By contrast, PBL from 16 hyperprolactinemic patients developed levels of LAK activity comparable with those generated by PBL from age- and sex-matched normoprolactinemic donors.     

ICAM-3 (CD50) cross-linking augments signaling in CD3-activated peripheral human T lymphocytes.

ICAM-3 is a pan-hematopoietic, constitutive adhesion molecule. ICAM-3 binds to LFA-1 on antigen-presenting cells (APC) stabilizing the T cell-APC interaction, facilitating signaling through the CD3/TCR complex. However, recent evidence using cultured and transformed T cells suggests ICAM-3 may also function in signaling. Because ICAM-3 is constitutively expressed on resting T cells, we postulated that signaling through ICAM-3 in resting T cells represents an important costimulatory mechanism in these cells. In purified resting human T cells, cross-linking both ICAM-3 and CD3 with plate-bound antibodies resulted in a marked increase in cell size (consistent with blastogenesis), synergistically increased surface expression of CD25 and CD69, and increased T cell metabolism. Similarly, concomitant ICAM-3 and CD3 stimulation significantly (P < 0.001) increased resting human T cell phosphatidylinositol hydrolysis and phospholipase C-gamma1 phosphorylation. These results indicate that ICAM-3 augments signaling through CD3, functioning as a costimulatory molecule for resting T cells in the initial activation step.     

Activation of swine peripheral blood lymphocytes with human recombinant interleukin-2.

These studies examined the effect of the lymphokine interleukin-2 (IL-2) on porcine peripheral blood lymphocytes (PBL). Cultures of porcine (Yorkshire) PBL in human recombinant (r) IL-2 had a time-dependent increase in activation of killer cells. Previous reports of porcine killer cells derived from PBL indicated maximal lysis 18 hr after killer and target cells were mixed, with very little lytic activity at 4 hr. After culturing for 2 days in rIL-2, the cells acquire enhanced lytic capabilities resulting in substantial target lysis in the 4-hr assay. Enhanced lytic activity in the 18 hr assay occurs as early as 30 min after exposure to rIL-2, with a more significant increase after 2 days' exposure. The IL-2-treated cells have an increase in the incorporation of tritiated thymidine and an increase in percentage of granular cells. Tumour targets, such as Daudi, which are resistant to lysis, and L929, Mdt4 and P815, which are moderately susceptible to lysis by untreated swine PBL, had a substantial increase in lysis in IL-2-treated swine PBL. These results indicate human rIL-2 induces functional and morphological changes in swine peripheral blood lymphocytes.     

Trypanosoma cruzi-induced immunosuppression: selective triggering of CD4+ T-cell death by the T-cell receptor-CD3 pathway and not by the CD69 or Ly-6 activation pathway.

In a model of experimental Chagas' disease induced with metacyclic forms of Trypanosoma cruzi, CD4+ but not CD8+ T cells undergo T-cell receptor (TCR)-CD3-mediated activation-induced cell death (AICD) in vitro. CD4+ T cells from T. cruzi-infected mice also developed unresponsiveness in proliferative responses to TCR-CD3-mediated stimulation. A linear correlation was found between extent of proliferative unresponsiveness and loss of CD4+ T-cell viability. CD4+ T-cell activation through the CD69 or Ly-6 A/E pathway, on the other hand, did not result in proliferative unresponsiveness compared with controls. Lack of suppression in proliferation assays correlated with lack of AICD by cells stimulated through the CD69 or Ly-6 A/E pathway. Concomitant stimulation through CD69, however, did not rescue CD4+ T cells from CD3-induced death. Flow cytometry study of cells stimulated in vitro showed no defect in interleukin-2 receptor expression by CD4+ T cells from infected donors, which escaped TCR-mediated AICD. In vivo injection of anti-CD3 into acutely infected mice, but not into control mice, led to splenocyte DNA fragmentation and failed to increase splenic CD4+ T-cell numbers. These results show that TCR-CD3-mediated AICD is involved in CD4+ T-cell unresponsiveness in vitro following infection with T. cruzi. In addition, successful activation of these cells through the CD69 and Ly-6 pathways is due to differences in the inability of these stimuli to trigger AICD. Since TCR-CD3-mediated AICD can be induced in vivo in infected mice, these findings may be relevant for the onset of immunological disturbances in the host.     

Trypanosoma cruzi immunosuppressive factor decreases the interleukin-2 mRNA level in cultured normal activated human lymphocytes.

We show here that the marked inhibition of interleukin-2 production seen in hosts acutely infected with Trypanosoma cruzi can be reproduced in vitro by adding a T. cruzi suspension filtrate to cultures of activated human peripheral blood mononuclear cells. This effect was not due to an accelerated decay of interleukin-2 mRNA and appeared to be selective since the levels of other mRNA molecules relevant to lymphocyte activation were not noticeably decreased. The use of normal lymphoid cells in this in vitro system makes it possible to examine the mechanisms used by T. cruzi to induce abnormalities in lymphocyte functions that also occur in infected hosts.     

Binding of C-reactive protein (CRP) by human peripheral blood lymphocytes in vivo.

Antigenicity of C-reactive protein (CRP) on the surface of human lymphocytes was investigated by use of indirect immunofluorescence technique with anti-CRP antibodies. CRP on the lymphocyte surface (sd-CRP) belongs to two different categories: i) CRP produced by lymphocytes and inserted into cell membrane (s-CRP), ii) CRP produced primarily by the liver and bound by the lymphocytes (sb-CRP) in calcium-dependent manner. In human peripheral blood of healthy donors approximately 2.5% of lymphocytes expressed membrane CRP (s-CRP) and 1.5% of lymphocytes bound CRP in calcium-dependent manner (sb-CRP). Percentage of s-CRP lymphocytes increased in patients with rheumatoid arthritis, while population of sb-CRP lymphocytes did not change significantly, except cases where serum CRP concentration reached more than 50 micrograms/ml. Thus, it can be concluded that CRP is bound to the distinct population of lymphocytes, bearing specific membrane receptors.     

Suppression by Trypanosoma cruzi of T-cell receptor expression by activated human lymphocytes.

The immunosuppression that develops during Chagas' disease and African sleeping sickness is thought to facilitate survival of the causative agents in their mammalian hosts. Whereas a number of manifestations of immunosuppression manifested during the course of these diseases has been reported in patients and animals, the mechanisms by which they are induced remain obscure. An in vitro system in which phytohaemagglutinin (PHA)-stimulated human peripheral blood mononuclear (PBMC) were co-cultured with purified Trypanosoma cruzi or T. brucei rhodesiense was used in the present work to establish whether these organisms were able to alter the capacity of activated helper/inducer (CD4+) or cytotoxic/suppressor (CD8+) cells to express T-cell receptor (TcR). Suppressed interleukin-2 receptor (IL-2R), known to be caused by both the trypanosomes and supernatants containing their secretion products, was the independent parameter used to demonstrate the occurrence of immunosuppression in all experiments. We found marked reductions in the percentage of TcR+ cells in T. cruzi-containing cultures as early as 18 hr after PHA stimulation. This alteration was still readily demonstrable after 72 hr of culture, i.e. when last tested for. Suppressed TcR expression occurred concomitantly with reduced levels of CD4 or CD8 molecules on the surface of helper/inducer and cytotoxic/suppressor T lymphocytes, respectively, indicating that the parasite had induced more than one alteration in the same cells. These effects were reproduced when the trypanosomes were separated from the PBMC by a 0.45 micron pore size filter or when filtrates from T. cruzi suspensions substituted for the parasite in the cultures, indicating that TcR suppression was mediated by a parasite secretion product(s). Interestingly, neither T. b. rhodesiense nor filtrates of suspensions of this organism altered significantly the level of TcR expression in cultures in which suppressed IL-2R expression by activated human T cells took place. Thus despite sharing the ability to impair IL-2R expression, T. cruzi and T.b. rhodesiense appear to differ in other mechanisms by which they affect human T-cell function. If occurring in infected hosts, the alterations that T. cruzi causes in the expression of TcR, CD4, CD8 and IL-2R--all molecules playing important roles in lymphocyte activation--could contribute to the development of the immunosuppression observed during the acute phase of Chagas' disease.     

Sodium tungstate (Na2WO4) exposure increases apoptosis in human peripheral blood lymphocytes

The potential for adverse health effects of using tungsten and its alloys in military munitions are an important concern to both civilians and the US military. The toxicological implications of exposure to tungsten, its alloys, and the soluble tungstate (Na₂WO₄) are currently under investigation. To examine tungstate toxicity, a series of experiments to determine its in vitro effects on cells of the immune system were performed. We identified alterations in isolated human peripheral blood lymphocytes (PBL) treated in vitro with sodium tungstate (0.01, 0.1, 1.0, and 10?mM). Analyses of apoptosis with annexin V and propidium iodide revealed a dose- and time-dependent increase in the quantity of cells in early apoptosis after tungstate exposure. Reductions in the number of cells entering into the cell cycle were also noted. Exposure of PBL to tungstate (1?mM) and Concanavalin A (ConA) for 72?h reduced the number of cells in S and G₂/M phases of the cell cycle. There were alterations in the numbers of cells in G₀/G₁, S, and G₂/M phases of the cell cycle in long-term THP-1 (acute leukemic monocytes) cultures treated with tungstate (0.01, 0.1, 1.0, and 10?mM). Gel electrophoresis, silver staining, and LC-MS/MS showed the cytoplasmic presence of histone H1b and H1d after 72?h of tungstate exposure. The addition of tungstate to cultures resulted in significant reductions in the quantity of interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and IL-6 produced by stimulated [CD3/CD28, ConA, or lipopolysaccharide (LPS)] and tungstate-treated lymphocytes. Taken together, these data indicate that tungstate increases apoptosis of PBL, alters cell cycle progression, reduces cytokine production, and therefore warrants further investigation.     

Restriction of the alternative pathway of human complement by intact Trypanosoma brucei subsp. gambiense.

We studied the interaction of African trypanosomes with human complement. Bloodstream forms of Trypanosoma brucei subsp. gambiense isolated from mice activated the alternative pathway of complement during a 30-min incubation in vitro. In human serum, all cells remained intact and motile during this period. C3 was detected on the surface by a direct binding assay with a monoclonal antibody which recognizes C3b and iC3b. C3 deposition could also be detected by this radioimmunoassay when parasites were incubated with purified C3. Such C3 binding was enhanced by factor B, factor D, and magnesium. Surface deposition of factor B was demonstrated both by flow immunofluorescence analysis and binding of radiolabeled factor B. C3 binding and factor B binding were inhibitable by EDTA but not by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N' -tetraacetic acid (EGTA). The inhibited binding could be restored by addition of magnesium. No human immunoglobulin G or mouse immunoglobulin was detected on the trypanosome surface. By flow cytometry, neither human C5 nor polymerized C9 was detected on trypanosomes incubated in serum, although this assay was able to detect C5 and C9 on the surface of complement-treated human erythrocytes. Using a radioimmunoassay which measures C5b-9 in serum, we found that there was no generation of SC5b-9 in serum which had been incubated with trypanosomes. We concluded that, although trypanosomes activate the alternative pathway of complement, they are not lysed, because the cascade does not continue beyond the establishment of C3 convertase.     

Interleukin-4 regulates the interleukin-2 receptors on human peripheral blood B lymphocytes.

The high-affinity interleukin-2 (IL-2) receptor (IL-2R) consists of the non-covalent association of at least two subunits, p55 and p70-75, capable of binding IL-2 with low and intermediate affinity, respectively. We studied the effects of cytokines on the IL-2R expressed on human peripheral blood B lymphocytes using monoclonal antibodies specific for IL-2R p55 and IL-2R p70-75, by means of two-colour flow cytometric analysis. In freshly isolated peripheral blood B lymphocytes, the p55 subunit was expressed only in a small population (7.0% of CD20+ cells), whereas the p70-75 subunit was expressed in a large population (89.0% of CD20+ cells). Of the cytokines studied, interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) were involved in the regulation of IL-2R on B cells. After a 2-day incubation with IL-4, expression of IL-2R p55 was markedly induced, but expression of IL2-R p70-75 was profoundly suppressed in a dose-dependent manner. These abilities of IL-4 to promote IL-2R p55 expression and suppress IL-2R p70-75 expression were inhibited by the presence of IFN-gamma. Other cytokines, including IL-1, IL-2, IL-5, and IL-6, had little effect on the expression of these two subunits. These findings suggest that IL-4 is a cytokine modulating B cell response through the regulation of IL-2R.     

Interactions between peripheral blood CD8 T lymphocytes and intestinal epithelial cells (iEC)

Intestinal intraepithelial lymphocytes (iIEL) are primarily CD8 cells and most of them have a CD28^? phenotype, the phenotype of effector cytotoxic T cells. We asked whether the predominance of CD8⁺ CD28^? T cells in the gut may result from peripheral blood T cells preferentially migrating to the iIEL compartment and adhering to iEC. Compared with CD4 cells, adhesion of resting CD8⁺ T cells to iEC cell lines was significantly higher. Adhesion could be blocked with a MoAb to gp180, a molecule expressed on iEC which is known to interact with CD8/lck. No significant difference in the level of adhesion was observed between CD8⁺ CD28⁺ and CD8⁺ CD28^? T cells. Thus CD8 cells may preferentially migrate to the iIEL compartment, but loss of CD28 expression could occur in situ after migration. Consistent with this hypothesis, the CD8⁺ CD28^? cells became enriched after co-culturing T cells with iEC cell lines and primary iEC. Induction of the CD8⁺ CD28^? phenotype in cord blood and adult T cells was observed in co-cultures with iEC and also with mitogens and superantigens. In the latter case, CD28 down-modulation was seen specifically in the Vβ subset targeted by the superantigen, indicating that loss of CD28 expression is a direct result of T cell receptor (TCR)-mediated stimulation. The combined results suggest that CD8⁺ CD28^? T cells are antigen experienced T cells, and that they may have a survival advantage in the presence of gut epithelial cells in vitro. This may contribute to the predominance of CD8⁺ CD28^? T cells in the iIEL compartment.