An optimized method for the functional analysis of human regulatory T cells

Naturally occurring regulatory T cells (Treg) suppress the activation of antigen-responsive T cells in a cell contact-dependent manner. In order to investigate the impact of soluble mediators and receptor-ligand interactions on the interplay between naive T cells and Treg, a reproducible suppressor cell assay which functions in the absence of additional feeder cells or antigen-presenting cells is mandatory. Here, we describe such a method which is suited to study the modulation of responder T cell/Treg interactions in vitro. Treg were isolated from negatively purified total human CD4+ T cells by positive selection using anti-CD25 monoclonal antibody (MoAb)-coated Dynabeads followed by a detachment step. The remaining CD4+ CD25- responder T cells were cocultured with CD4+ CD25+ Treg in the presence of T-cell Activation/Expansion Beads from Miltenyi Biotec pre-coated with anti-CD3 plus anti-CD28 monoclonal antibody (MoAb). The optimal concentration for coating was 5 microg/ml for both MoAb. At this concentration, strong proliferation of responder T cells was elicited which was almost completely suppressed by Treg at 1:1 cell ratios. When higher concentrations of anti-CD3/anti-CD28 MoAb were used for coating, Treg also showed some degree of proliferation. The optimized suppressor assay proved to be highly reproducible and was used here to confirm the partial or complete reversal of Treg-mediated T-cell suppression by some cytokines (IL-2, IL-15), soluble IL-6 receptor/IL-6 fusion protein and recombinant GITR-ligand. Furthermore, our data confirm that Treg do not need other cell types to suppress proliferation of CD4+ CD25- responder T cells.     

The Stimulation of T Cells with Anti-CD2 Monoclonal Antibodies Facilitates the Induction of Polyclonal B-Cell Responses but does not Enhance the Activation of Antigen-Specific B Cells

In this report we compare the effect of stimulation of peripheral mononuclear cells (PBMC) by using two monoclonal antibodies (MoAb) directed against the CD2 receptor on T cells or by using autologous erythrocytes (E) which express on their surface lymphocyte function-associated antigen 3 (LFA3), a natural ligand for CD2. The addition of autologous erythrocytes to pokeweed mitogen (PWM)-stimulated PBMC results in the enhancement of polyclonal immunoglobulin synthesis and of antigen-specific B-cell responses. Because B cells lack the CD2 molecule, it can be concluded that their enhanced activity is a consequence of the delivery of activating signals by activated T lymphocytes. When PBMC cultures were stimulated with a pair of anti-CD2 MoAb (Leu5b and VIT13) we were able to induce polyclonal immunoglobulin synthesis, particularly IgM, in cultures supplemented with interleukin 2(IL-2). Specific responses to tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH) were also enhanced by the addition of autologous E to PWM-stimulated PBMC. Significant anti-TT responses were observed in cultures stimulated with E + TT + IL-2. In contrast, stimulation of PBMC with VIT13 + Leu5b + IL-2 + antigen was not effective in inducing anti-TT antibody and only weakly effective in inducing anti-KLH antibodies. Replacing Leu5b by anti-CD3 had no effect on the induction of specific antibody responses; in contrast, replacement of Leu5b by E enhanced anti-TT antibody production while the effect on polyclonal production of IgM was minimal. Therefore, it appears that the signal delivered by the association of CD2 with LFA3 is a better potentiating signal for specific B-cell responses than the signal delivered by pairs of MoAb to different epitopes of CD2 or to CD2 and CD3 epitopes.     

Inhibition by glucocorticoid and staurosporine of IL-4-dependent CD23 production in B lymphocytes is reversed on engaging CD40.

IL-4 synergizes with signals delivered through CD40 both for the induction of CD23/Fc epsilon RII expression and for IgE synthesis. Moreover, engagement of CD40 on the B cell surface by MoAb overcomes the ability of interferons, transforming growth factor-beta, or anti-CD19 to inhibit IL-4-dependent change. We now report that occupancy of CD40 relieves potent suppression of IL-4-induced CD23 production by glucocorticoid or the relatively broad-acting kinase inhibitor staurosporine. Interruption of the IL-4 signal was observed with concentrations of staurosporine considered to be selective for protein kinase C (PKC) inhibition (IC50 = 10 nM) but not with genistein or tyrphostins, effective inhibitors of tyrosine kinase activity. On ligation of CD40, staurosporine no longer inhibited the IL-4 signal: at concentrations of between 1 and 20 nM, staurosporine actually increased by as much as 100% the rate of CD23 production stimulated on simultaneous activation through CD40 and IL-4R. Such augmentation was not observed when the more specific PKC inhibitor RO-31-8220 was used; indeed, CD40 engagement was unable to overcome the ability of this inhibitor to block IL-4-promoted CD23 induction (IC50 = 10 microM). Occupancy of CD40 did, however, thwart completely the usual ability of prednisolone to inhibit the IL-4 signal leading to CD23 induction. Activation through CD40 left inhibition of phorbol ester-induced CD23 expression by staurosporine, RO-31-8220, or glucocorticoid unchecked. These findings further highlight the intimate level of cross-talk existing between CD40 and IL-4R on resting B lymphocytes to promote CD23 expression, a phenotypic change which preludes IgE synthesis.     

CD3-Mediated T-Cell Activation is Inhibited by Anti-CD44 Monoclonal Antibodies Directed to the Hyaluronan-Binding Region

The CD44 molecule has been shown to play a role in T cell adhesion and activation. We have investigated the ability of five anti-CD44 monoclonal antibodies (MoAb) including 15C6, 18A3, BU75 (Ancell), J173 (Immunotech), and L178 (Becton Dickinson) to regulate T cell activation. Three MoAb: 15C6, BU75, and J173 were found to selectively inhibit DNA synthesis, interleukin-2 (IL-2) receptor expression, and G1→S transition of the cell cycle in T cells stimulated with anti-CD3 MoAb. None of anti-CD44 MoAb had influence on T cell proliferation induced by IL-2 or phorbol 12-myristate 13-acetate plus ionomycin. Inhibition of the CD3 pathway by anti-CD44 MoAb occurred by binding of MoAb directly to T cells without the involvement of monocytes or Fc receptors. In addition, the inhibitory anti-CD44 MoAb clearly suppressed intracellular calcium mobilization in T cells stimulated with anti-CD3 MoAb. Interestingly, the ability of anti-CD44 MoAb to inhibit T cell activation was well correlated with their capability to block the binding of hyaluronan (HA) to CD44 molecules. These results suggest that anti-CD44 MoAb directed to HA-binding site could selectively inhibit CD3-mediated T cell activation. Furthermore, CD44-mediated inhibitory signals would be linked to the blocking of early CD3-mediated signal transduction.     

CD3-mediated T cell activation is inhibited by anti-CD44 monoclonal antibodies directed to the hyaluronan-binding region.

The CD44 molecule has been shown to play a role in T cell adhesion and activation. We have investigated the ability of five anti-CD44 monoclonal antibodies (MoAb) including 15C6, 18A3, BU75 (Ancell), J173 (Immunotech), and L178 (Becton Dickinson) to regulate T cell activation. Three MoAb: 15C6, BU75, and J173 were found to selectively inhibit DNA synthesis, interleukin-2 (IL-2) receptor expression, and G1-->S transition of the cell cycle in T cells stimulated with anti-CD3 MoAb. None of anti-CD44 MoAb had influence on T cell proliferation induced by IL-2 or phorbol 12-myristate 13-acetate plus ionomycin. Inhibition of the CD3 pathway by anti-CD44 MoAb occurred by binding of MoAb directly to T cells without the involvement of monocytes or Fc receptors. In addition, the inhibitory anti-CD44 MoAb clearly suppressed intracellular calcium mobilization in T cells stimulated with anti-CD3 MoAb. Interestingly, the ability of anti-CD44 MoAb to inhibit T cell activation was well correlated with their capability to block the binding of hyaluronan (HA) to CD44 molecules. These results suggest that anti-CD44 MoAb directed to HA-binding site could selectively inhibit CD3-mediated T cell activation. Furthermore, CD44-mediated inhibitory signals would be linked to the blocking of early CD3-mediated signal transduction.     

CD3-Mediated T-Cell Activation is Inhibited by Anti-CD44 Monoclonal Antibodies Directed to the Hyaluronan-Binding Region

The CD44 molecule has been shown to play a role in T cell adhesion and activation. We have investigated the ability of five anti-CD44 monoclonal antibodies (MoAb) including 15C6, 18A3, BU75 (Ancell), J173 (Immunotech), and L178 (Becton Dickinson) to regulate T cell activation. Three MoAb: 15C6, BU75, and J173 were found to selectively inhibit DNA synthesis, interleukin-2 (IL-2) receptor expression, and G1→S transition of the cell cycle in T cells stimulated with anti-CD3 MoAb. None of anti-CD44 MoAb had influence on T cell proliferation induced by IL-2 or phorbol 12-myristate 13-acetate plus ionomycin. Inhibition of the CD3 pathway by anti-CD44 MoAb occurred by binding of MoAb directly to T cells without the involvement of monocytes or Fc receptors. In addition, the inhibitory anti-CD44 MoAb clearly suppressed intracellular calcium mobilization in T cells stimulated with anti-CD3 MoAb. Interestingly, the ability of anti-CD44 MoAb to inhibit T cell activation was well correlated with their capability to block the binding of hyaluronan (HA) to CD44 molecules. These results suggest that anti-CD44 MoAb directed to HA-binding site could selectively inhibit CD3-mediated T cell activation. Furthermore, CD44-mediated inhibitory signals would be linked to the blocking of early CD3-mediated signal transduction.     

Accessory Signalling by B7-1 for T Cell Activation Induced by Anti-CD2: Evidence for IL-2-Independent CTL Generation and CsA-Rcsistant Cytokine Production

Resting T cells can be activated by selected pairs of anti-CD2 MoAb. Activation is dependent on the presence of accessory cells, which can be replaced by either anti-CD28, or by the combination of IL-1β and IL-6. The present study was undertaken to investigate accessory signalling by B7-1, the natural ligandof CD28, in this pathway of T cell activation. 3T6 mouse fibrobiasts were transfected with human B7-1 and used as accessory cells in cultures of purified resting human T cells. In the presence of a stimulating pair of anti-CD2 MoAb, T cell proliferation, production of cytokines (IL-2, IL-4, IL-10, GM-CSF, IFN-α and TNF-α), and generation of cytotoxic T lymphocytes were all supported by B7-l(+) 3T6 cells but not by control 3T6 cells. Blocking studies with anti-IL-2 + anti-IL-2R MoAb revealed both IL-2-dependent and IL-2-independent CTL generation after B7-1 -mediated costimulation. Moreover, a partial or complete resistance to inhibition with CsA was observed for IL-2 production and CTL generation respectively in the presence of the costimulatory signal derived from B7-1 - CD28 interaction. Anti-CD2 MoAb with B7-1 costimulation could directly induce proliferation, IL-2 production and generation of CTL activity in highly purified CD8⁺ T cells without the heip of CD4⁺ T cells. We conclude that CD28 ligation with the natural ligand B7-1 provides a strong accessory signal for CD4 and CD8 cell activation through CD2.     

Study of induction of activation of human peripheral blood mononuclear cells with a non-activating form of anti-CD3 MoAb in autoimmune thyroid disease (AITD).

Anti-CD3 (OKT3) MoAb is a mitogenic agent which activates lymphocytes. We have studied the effects of murine anti-human OKT3 MoAb (IgG1) alone or in combination with IL-2, human thyroglobulin (Tg) and thyroperoxidase (TPO) antigens on the proliferation of whole peripheral blood mononuclear cells (PBMC) (including monocytes) or subtypes (T, CD4+, CD8+, B) as measured by tritiated thymidine (3H-TdR) incorporation. B cell differentiation was studied by measuring numbers of IgG-secreting cells and specific anti-TPO/anti-Tg-secreting cells by SPOT ELISA. PBMC or lymphocyte subtypes, obtained from 45 patients with Hashimoto's thyroiditis (HT), 40 Graves' disease (GD) and 51 normal controls were cultured in 96 microtitre plates for 6 days in the presence of OKT3 MoAb at final concentrations 25-250 ng/ml, IL-2 15 U/ml, Tg and TPO (1 micrograms/ml). Then cultures were pulsed with 0.2 microCi 3H-TdR/well and incorporation was measured after 18 h. IgG and anti-TPO/Tg-secreting cells were detected at 7 days. Higher proliferative responses from whole PBMC preparations in response to any of the combinations including OKT3 MoAb were observed in the HT preparations, while the basal values were the lowest. IL-2 alone increased these responses markedly, but equally in all groups. IL-2 in combination with OKT3 had an additive effect on proliferation, with higher responses in HT. Tg and TPO antigens did not change these responses. Most HT preparations responded with their maximum proliferation to the lowest concentration of OKT3 MoAb (25 ng/ml), whereas in GD and control preparations of PBMC these responses were shifted to higher concentrations (250 ng/ml); even with those, proliferation was not so enhanced in controls when compared with HT and GD preparations. In contrast, the proliferative responses of T cells alone and subpopulations of CD8+ suppressor/cytotoxic cells were decreased in HT preparations compared with controls. Monocytes were necessary for proliferation. In the subpopulation of B cells (> 95% pure) and CD4+ helper/inducer cells, differences did not reach significance. In spite of the effect on proliferation, OKT3 MoAb only mildly but significantly increased the numbers of IgG-secreting cells in HT and GD preparations and did not stimulate synthesis of specific antibodies. Our data suggest that the increased proliferative responses of whole PBMC to OKT3 MoAb in HT preparations might be due to insufficient activation of T suppressor/cytotoxic cells.     

Role of monocytes in anti-CD3-induced T-cell DNA synthesis: Effect of chloroquine and monensin on anti-CD3-induced human T-cell activation

Anti-CD3 monoclonal antibody (MoAb) induces proliferation of freshly isolated peripheral blood T cells only in the presence of monocytes/macrophages and requires binding of the Fc portion of antibody to monocytes/macrophages. In this investigation, we examined whether monocytes process anti-CD3 similar to any soluble antigen and present to T cells in context with HLA-DR to induce maximal DNA synthesis. Adherent monocytes were pulsed with anti-CD3 MoAb in the presence or absence of the lysozomotropic agents chloroquine and monensin, which are known to inhibit processing of soluble antigens, washed extensively, and then incubated with autologous T cells in the absence of soluble anti-CD3, and³H-thymidine incorporation and CD25 expression were measured. Both monensin and chloroquine inhibited anti-CD3-pulsed monocyte-induced T-cell DNA synthesis and CD25 expression in a dose-dependent manner. This inhibitory effect was not due to any loss in cell viability or the effect on the expression of HLA-DR on monocytes. Paraformaldehyde-fixed monocytes pulsed with anti-CD3 MoAb induced significantly less DNA synthesis, HLA-DR expression, and CD25 antigen expression on autologous T cells as compared to responses induced by unfixed anti-CD3-pulsed monocytes. The treatment of anti-CD3-pulsed monocytes with frame-work-specific anti-HLA-DR MoAb inhibited their capacity to induce T-cell DNA synthesis. These data suggest that monocytes, in addition to serving as the matrix for cross-linking, also process anti-CD3 MoAb and present to the T cells in the context of HLA-DR antigens to induce optimal DNA synthesis.     

Somatropin promotes dermatophagoides farinae-specific IgE generation independently of IL-4 and IL-10

Interleukin (IL)-10 accelerates the IgE production of anti-CD40- and IL-4-stimulated PBMC by enhancing the IL-6 production of T lymphocytes or antigen-primed spleen cells, in addition to its role as a regulator of the inflammatory responses. To further investigate the mechanisms enhancing IgE synthesis, we determined the effect of somatropin as well as IL-10 on the secretion of Dermatophagoides farinae (Df)-specific IgE by K7 cells, which originate from an EBV-immortalized cell line. Df-pulsed autologous T cells, as well as the supernatants of these cultures, increased the synthesis of Df-specific IgE. Antigen-specific IgE was also enhanced when K7 cells were treated with anti-CD40 antibody and with both IL-4 and IL-10, or with IL-4 and IL-10 without anti-CD40 antibody. The treatment of K7 cells with anti-CD40 antibody and IL-4, or anti-CD40 antibody and IL-10 did not increase IgE production. The Df-specific IgE activity of the supernatants of K7 cells treated with somatropin alone was increased significantly although somatropin did not show any additive effect on the IgE production of anti-CD40 antibody-treated cells. The results indicate that IL-10, a Th2-type cytokine, directly affects the mature B cells that produce IgE, and that the secretion of IgE is increased by treatment with IL-10 in cells that are stimulated with anti-CD40 and IL-4 at the level of the EBV-immortalized cell line, which has already switched to IgE production. Somatropin similarly stimulates activated mature B cells to enhance their production of antigen-specific IgE without class switching, independently of IL-4 and IL-10.     

T lymphocyte anergy during acute infectious mononucleosis is restricted to the clonotypic receptor activation pathway.

The transient T cell anergy associated with acute infectious mononucleosis (IM) caused by the Epstein-Barr virus has been analysed in a sample of 14 IM children. Peripheral blood mononuclear cells (PBMC) obtained from IM patients showed a significant specific impairment in their proliferative response to both phytohaemagglutinin (PHA; P less than 0.05) and to an anti-CD3 MoAb (P less than 0.001), although both responses reached normal control levels by addition of a submitogenic dose of either phorbol myristate acetate (PMA) or recombinant IL-2 (rIL-2). In contrast, activation signals delivered through other surface molecules (CD2, CD28) or other transmembrane pathways (PMA plus a calcium ionophore) elicited normal or high proliferative responses in most IM PBMC. In a group of five patients tested, the synthesis of IL-2 by IM PBMC in the presence of PMA was impaired when PHA or anti-CD3 was used as stimulus, but it reached normal levels with anti-CD2 or ionophore. Lastly, PHA failed to induce IL-2 alpha receptor (IL-2R alpha) expression in IM PBMC from four tested patients, but the presence of PMA completely corrected this defect. Taken together, these results strongly suggest that the T cell anergy associated with acute IM is due to a T cell receptor (TCR)-specific impairment in the induction of genes involved in T cell proliferation (including those coding for IL-2 and IL-2R alpha) upon membrane signalling to otherwise normal T lymphocytes, since CD2, CD28 and certain transmembrane activation pathways are uncoupled from CD3 in these particular pathological conditions (and perhaps in most in vivo situations). This and other similar experimental approaches to transient secondary immunodeficiencies may help to unravel the physiopathological role of different surface molecules in T cell activation.     

Xanthine derivatives: comparison between suppression of tumour necrosis factor-alpha production and inhibition of cAMP phosphodiesterase activity.

Several in vitro and in vivo studies have demonstrated suppression of tumour necrosis factor-alpha (TNF-alpha) synthesis by pentoxifylline. In the present study we compared the effect of pentoxifylline with that of five other xanthine derivatives. We addressed two questions. First, what is the relative potency of those chemically related compounds in suppressing the lipopolysaccharide (LPS)-induced production of TNF-alpha in human mononuclear cells? Second, does suppression of TNF-alpha production by these xanthine derivatives correlate with their capacity to inhibit 3',5'-cAMP phosphodiesterase (PDE) activity? The experimental drug A 80 2715 [1-(5-hydroxy-5-methylhexyl)-3-methyl-7-propylxanthine] was identified as the most potent agent with an IC50 (concentration exerting 50% suppression of LPS-induced TNF-alpha production) of 41 microM (mean of 13 individuals). The IC50 values of the other substances ranged between 106 microM for HWA 138 and 419 microM for theophylline. The LPS-induced interleukin-1 beta (IL-1 beta) production was not influenced by all substances tested at comparable concentrations. Inhibition of PDE activity was determined in a cell-free system using PDE isolated from bovine heart. All xanthine derivatives dose-dependently inhibited PDE activity. Furthermore, with the exception of theophylline, there was a high degree of correlation between the potency to suppress TNF-alpha production in the cell culture system and the potency to inhibit PDE activity in the cell-free enzymatic assay. This argues for a crucial role of PDE inhibition in the suppression of TNF-alpha synthesis by xanthine derivatives.     

Selective T cell subset depletion with anti-CD4 and anti-CD8 intact ricin immunotoxins.

In the present study we have comparatively analysed the specific activity of a panel of immunotoxins (ITs) prepared by coupling Ricin to several monoclonal antibodies (MoAbs) directed against the CD3, CD4 and CD8 T cell membrane molecules. Peripheral blood and bone marrow mononuclear cells (PBMC and BMMC) were incubated with the different ITs for 2 h, in the presence of 0.1 M lactose, washed and subsequently stimulated with either phytohemagglutinin (PHA) or an anti-CD3 MoAb. Our results indicate that the proliferative response of PBMC to both stimuli was specifically inhibited (greater than 95%) by either anti-CD3 IT or a combination of anti-CD4 and CD8 ITs, at concentrations comparable to those previously used for ex vivo T cell depletion (300 ng/ml). When used individually at the same dose, anti-CD8 and anti-CD4 ITs inhibited the PHA-induced PBMC proliferative response 40 and 70% respectively. When either anti-CD4 or anti-CD8 IT-treated cells were activated with PHA and cultured for 14 days in the presence of IL-2, less than 2% of the blasts expressed the corresponding antigens as assessed by flow cytometry analysis. Similar results were observed when BMMC were treated with the different ITs. In contrast, the growth of CFU-GM was minimally affected (0-25% inhibition). Our data indicate that ITs directed against T cell subsets are highly active and specific reagents that may be potentially useful for pre-transplant bone marrow purging.     

Isotype-specific cross-linking of select human Fc gamma R isoforms triggers release of IL-6.

Anti-CD3 MoAbs are widely used in T cell activation studies, and are effective in immunosuppressive therapy. We used a panel of mouse (m) anti-CD3 switch variant MoAbs of five different isotypes to study IL-6 release from accessory cells. Incubation of human (h) mononuclear cells with anti-CD3 MoAbs resulted in increased IL-6 levels with MoAbs of mIgG1 and mIgG2a isotypes, with no effect of mIgG2b or mIgA. This suggested involvement of IgG Fc receptors (Fc gamma R) in triggering IL-6 production. To evaluate the role of different Fc gamma R molecules individually we used a panel of hFc gamma R-transfected mouse fibroblasts, and Jurkat T cells as a model. IL-6 secretion by CD32 transfectants expressing the hFc gamma RIIa high-responder (HR) allelic form was triggered by mIgG1 anti-CD3 MoAb, with no effect of four other isotypes. None of the anti-CD3 MoAbs induced IL-6 secretion by CD32 transfectants expressing either a variant of this receptor, containing only a single intracellular amino acid (CT-), the hFc gamma RIIa low-responder (LR) allelic form, or hFc gamma RIIb1. hFc gamma RI (CD64) transfectants exhibited IL-6 production after incubation with mIgG2a anti-CD3 MoAb, and to a lesser extent with mIgG2b, and mIgG1 MoAb. Indirect involvement of T cells in triggering IL-6 secretion could be excluded by experiments in which transfectants were cultured with immobilized anti-CD3 MoAb. These data indicate that cross-linking of either hFc gamma RI, or hFc gamma RIIaHR by appropriate anti-CD3 MoAbs triggers IL-6 production of accessory cells, and not T cells. This may also take place in vivo during immunosuppressive therapy with anti-CD3 MoAbs, and related antibody-mediated immune responses.     

Selective impairment of T lymphocyte activation through the T cell receptor/CD3 complex after cytomegalovirus infection.

Cytomegalovirus (CMV) infection is reported to cause transient immunosuppression in man. In this study we have analysed the effect of CMV on T lymphocyte function in 29 children diagnosed for acute CMV infection. Peripheral blood mononuclear cells (PBMC) obtained from the patients showed a significant specific impairment in their proliferative response to enterotoxins A and C1, to concanavalin A and to the anti-CD3 MoAb OKT3. The impaired responses were corrected with exogenous IL-2. In contrast, stimulation using phytohaemagglutinin, as well as activation signals delivered through the surface molecules CD26 or CD28, elicited normal proliferative responses in CMV PBMC. The results indicate that the T cell anergy associated with CMV infection is restricted to the T cell receptor/CD3 activation pathway.     

水仙环素抑制T细胞增殖的作用机制

目的 探究水仙环素抑制T细胞增殖与功能的作用机制.方法 密度梯度离心法及免疫磁珠法纯化人外周血T细胞,anti-CD3/anti-CD28抗体活化T细胞.使用流式细胞仪检测细胞增殖、CD25的表达、细胞毒性及细胞周期;酶联免疫吸附试验检测细胞因子IL-2、IL-6、IL-17及IFN-γ的分泌水平.结果 水仙环素抑制a...     

Interleukin 2 receptor expression by human blood lymphocytes after vaccination with pneumococcal polysaccharides.

Proliferative responses of unseparated peripheral blood mononuclear cells (PBMC) and blood T cells to recombinant interleukin 2 (rIL-2) were significantly increased 7-21 days after the vaccination with pneumococcal polysaccharides (PPS). In contrast, non-T cells expressed increased responsiveness to rIL-2 only on post-vaccination day 7. Analysis of the proliferative response to rIL-2 among lymphocyte subsets (CD4+Leu8+, CD4+Leu8-, CD8+Leu8+, CD8+Leu8-, CD20+) in cultures of unseparated PBMC revealed that the CD8+Leu8- T cells expressed increased responsiveness 7-14 days after vaccination, whereas neither CD4+ (Leu8+ and Leu8-) nor CD8+Leu8+ T cells showed significantly increased responsiveness after vaccination. The CD20+ B cells, like non-T cells, expressed increased responsiveness to rIL-27 days after the vaccination only. Expression of the 55 kD low-affinity interleukin 2 receptor (IL-2R, CD 25) on freshly isolated PBMC, as judged by direct fluorescence staining with a MoAb anti-55 kD chain, was low (less than 3%) and an increased expression of this receptor was not detected following vaccination. In contrast, binding of 125I-labelled IL-2 to freshly isolated PBMC increased following vaccination (day 7). Scatchard plot analysis revealed a modest increase in the expression of high-affinity IL-2R (Kd = 1-2 pM), whereas the increase in expression of the 75-kD, intermediate-affinity IL-2R (Kd = 300 pM) was more pronounced (from 195 to 295 (means) receptors per PBMC). It is concluded that, following vaccination with PPS increased IL-2R expression is induced on blood lymphocytes. These investigations suggest a role for T cells in the human immune response against PPS.     

Activation of human T cells with NK cell markers by staphylococcal enterotoxin A via IL-12 but not via IL-18

We have reported recently that mouse liver NK cells and NK1 x 1+ T cells were activated by bacterial superantigens via the IL-12 production from Kupffer cells. In the present study, we examined the effect of staphyloccoccal enterotoxin A (SEA) on human T cells with NK cell markers, CD56 or CD57 (NK-type T cells). After stimulating peripheral blood mononuclear cells (PBMC) with SEA, PBMC produced a large amount of IFN- and acquired a potent antitumour cytotoxicity. The in vitro depletion of either CD56+ TCR NK cells, CD56+ T cells or 57+ T cells from PBMC significantly inhibited the IFN- production from PBMC. When purified NK-type T cells, NK cells and regular T cells were cultured with monocytes and SEA they all produced IFN-, while the IFN- amounts produced by both NK-type T cells were greater than those produced by NK cells. NK cells as well as CD56+ T cells showed cytotoxicity against NK-sensitive K562 cells, whereas both NK-type T cells showed a more potent cytotoxicity against NK-resistant Raji cells than did NK cells. The IFN- production from each population as well as from whole PBMC was greatly inhibited by anti-IL-12 antibody but not by anti-IL-18 antibody. The antitumour cytotoxicity of whole PBMC was also significantly inhibited by anti-IL-12 antibody while the SEA-induced proliferation of PBMC was not affected by anti-IL-12 antibody. Furthermore, SEA-activated NK-type T cells as well as NK cells showed cytotoxicities against vascular endothelial cells. Our findings suggest that human NK-type T cells are thus involved in bacterial superantigen-induced immune response.     

Selective type IV phosphodiesterase inhibitors prevent IL-4-induced IgE production by human peripheral blood mononuclear cells

Background Selective type IV phosphodiesterase (PDE) inhibitors elicit anti-inflammatory and bronchodilatory activities in vitro and in vivo which suggest that these drugs could provide a new therapeutic approach for asthma treatment. Objective Regarding the role of IgE production in allergic and inflammatory reactions of the airways, we investigated the effect of selective PDE inhibitors on IL-4-driven IgE production by peripheral blood mononuclear cells (PBMC) or by purified B lymphocytes. Methods PBMC or purified B lymphocytes from non-allergic donors were stimulated for 13 days with IL-4 (100U/mL) in the presence or in the absence of selective PDE inhibitors. IgE production is evaluated by an ELISA technique. Results The selective PDE IV inhibitors, rolipram and Ro 20–1724 (10μM), inhibit lL-4-induced IgE production by PBMC. but not by purified B lymphocytes. No modification of the IgE production was noted with the selective PDE III inhibitors, milrinone and SK&F94-836, or the selective PDE V inhibitor, SK&F 96–231 (10 μM). Flow cytometry experiments showed that the effect of Rolipram could not be explained by the inhibition of the cell surface expression of the IL-4 receptor. Similarly, no significant effect of PDE IV inhibitors was observed on PHA-induced cell proliferation. The incubation of monocytes only with rolipram was sufficient to achieve a significant reduction of IgE production induced by IL-4. Conclusion Taken together, these results indicate that PDE IV inhibitors reduce lL-4-induced IgE production by PBMC and suggest that the inhibition of IgE production could be explained by a failure of monocytes to provide the necessary costimulatory signals.