Synergy of B cell growth factor and interleukin 2 in the proliferation of activated human B cells

The activity of purified interleukin 2 (IL2), obtained by the recombinant DNA technology, on the proliferative response of human B cells stimulated with low concentrations of anti-mu antibody was investigated. Recombinant IL2 was capable of augmenting the proliferative response of anti-mu-activated B cells and the T cell activation (Tac) antigen was expressed on a substantial proportion of normal B cells stimulated with anti-mu antibody. However, crude supernatants from protein A-stimulated peripheral blood mononuclear cells, which were found to possess both IL2 and B cell growth factor (BCGF) activities, maintained the ability to promote proliferation of anti-mu-activated B cells after depletion of IL2. In addition, supernatants from some T cell clones, apparently free of IL2 activity, displayed strong BCGF activity in the co-stimulation assay with anti-mu antibody. This BCGF activity was found in 25 kDa fractions by gel filtration and it was unaffected by addition to the cultures of anti-Tac antibody, which consistently inhibited the B cell proliferative response promoted by recombinant IL2. The proliferative response of anti-mu-activated B cells to clonal, IL2-free supernatants containing BCGF and recombinant IL2 present together from the beginning of culture was close to the sum of responses to the two stimulants, separately. In addition, the presence of clonal supernatant containing BCGF from the beginning of culture had a synergistic effect in the response of activated B cells to the subsequent addition of IL2, whereas the initial presence of IL2 had no such an effect on the reactivity of anti-mu-stimulated B cells to the late addition of clonal supernatant containing BCGF. The synergistic effect of BCGF in the IL2-promoted B cell proliferation was probably the result of the recruitment of a greater number of IL2-reactive B cells. In fact, the number of Tac-positive cells was significantly higher in 36-h cultures established in the presence of anti-mu antibody plus clonal supernatant containing BCGF than in cultures stimulated with anti-mu antibody alone. Taken together, these data indicate that anti-mu antibody promotes the expression by normal human B cells of distinct receptors for IL2 and a BCGF distinct from IL2. They also suggest that BCGF can exert a synergistic effect in the IL2-promoted proliferation of activated B cells.     

Analysis of the role of interferon-gamma, interleukin 2 and a third factor distinct from interferon-gamma and interleukin 2 in human B cell proliferation. Evidence that they act at different times after B cell activation

Recombinant interferon-gamma (rIFN-gamma) was able to induce proliferation of human tonsillar B cells activated with suboptimal concentrations of anti-mu antibody. The B cell growth factor (BCGF) activity of rIFN-gamma was not due to substances contaminating the IFN-gamma preparation, nor was it mediated by factors released by T cells or large granular lymphocytes following activation by rIFN-gamma. The response of B cells to rIFN-gamma peaked on day 3 of culture and rapidly declined thereafter, whereas the response of parallel anti-mu-activated B cell cultures to recombinant interleukin 2 (rIL2) appeared on day 3, but continued at least until day 5. In addition, B cells responsive to rIFN-gamma could be at least in part separated from those responsive to rIL2, the former being primarily contained in B cell fractions enriched for high-density small B lymphocytes. Finally, the addition to anti-mu-stimulated B cell cultures of very low concentrations of rIFN-gamma potentiated the B cell proliferation promoted by rIL2. The simultaneous addition of monoclonal antibodies against IFN-gamma and T cell activation antigen to anti-mu-stimulated B cell cultures strongly reduced the B cell proliferative response promoted by three different crude BCGF preparations obtained by polyclonal T cell activation in mixed lymphocyte culture. However, the supernatant from a T cell clone (DP5/11) apparently free of IL2, which manifested a BCGF activity similar to that of rIFN-gamma, still maintained its ability to promote proliferation of anti-mu-activated B cells after complete removal of IFN-gamma. Taken together, our data indicate that although some T cell clones are able to produce a BCGF distinct from both IFN-gamma and IL2, these lymphokines account for most of the BCGF activity of supernatants obtained from polyclonal T cell populations. They also suggest that IFN-gamma and the BCGF distinct from IFN-gamma and IL2 act primarily in the earlier phases of B cell activation and potentiate the proliferative response of activated B cells to IL2.     

Effects of anti-immunoglobulin antibodies, interleukin-4 and second messenger agonists on B cells from neonatal mice.

Crosslinking of surface Ig receptors on mature B cells with mitogenic anti-Ig antibodies stimulates phosphoinositide breakdown with subsequent activation of protein kinase C (PKC) and elevation of [Ca2+]i leading to B-cell activation. This response can be mimicked using second messenger agonists such as phorbol 12,13-dibutyrate (PDB) plus a Ca2+ ionophore. Furthermore, interleukin-4 (IL-4) synergizes with sub-mitogenic concentrations of anti-Ig (or with PDB) to activate adult B cells. In contrast anti-Ig does not activate neonatal B cells but rather desensitizes or kills them. The nature of the signals involved in these effects on neonatal B cells is poorly understood. Here we have investigated the proliferative responses of small, resting B cells from 1-, 4-, 8-, and 12-week-old mice stimulated with combinations of anti-Ig, PDB, ionomycin and IL-4. We find that B cells from 8- and 12-week-old mice show an adult pattern of reactivity. B cells from 4-week-old mice respond to high doses of anti-Ig, anti-Ig + IL-4 and also to PDB + ionomycin + IL-4, but not to PDB + ionomycin alone. Neonatal (1-week-old) B cells respond only to the combination of PDB, ionomycin and IL-4. Most strikingly, pre-incubation of neonatal cells with anti-Ig completely abrogates this response, whilst IL-4 renders them refractory to such anti-Ig mediated inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Down-regulation of protein kinase C activation in human lamina propria T lymphocytes: influence of intestinal mucosa on T cell reactivity.

Human lamina propria T lymphocytes (LPL-T) were shown to have lower proliferative responses to CD3 triggering than autologous peripheral blood T lymphocytes (PBL-T), yet preserved their responsiveness to CD2 stimulation. In order to elucidate the basis of these differences, freshly recovered human LPL-T and autologous PBL-T were stimulated with CD2 monoclonal antibodies anti-T11(2/3) plus sheep red blood cells and phorbol 12,13-dibutyrate (PBu2) plus ionomycin, respectively. LPL-T showed invariably lower responses to PBu2 plus ionomycin than PBL-T. In contrast, LPL-T still preserved proliferation to CD2 activation even when their responses to PBu2 plus ionomycin were decreased almost to background levels. Preincubation of PBL-T with intestinal mucosa supernatant led to a similar reactivity as observed in fresh LPL-T. Moreover, the protein kinase C (PKC) inhibitor sphinganine was able to inhibit DNA synthesis to stimulation with PBu2 plus ionomycin but not to CD2 triggering. This study suggests that CD2-induced proliferation is not dependent on PKC activation and that down-regulation of PKC activation may be one of the mechanisms for inhibition of the CD3-Ti-dependent activation pathway in LPL-T by intestinal mucosa-derived influences in vivo.     

Cross-talk between B cell surface immunoglobulin and interleukin 4 receptors: the role of protein kinase C and Ca2+-mediated signals

A well-known property of IL 4 is its capacity to synergize with low concentrations of anti-immunoglobulin (Ig) antibodies to induce B cells to synthesize DNA. Cross-linking of surface Ig receptors stimulates phosphoinositide hydrolysis, with consequent production of two signals: the elevation of intracellular Ca²⁺ levels and activation of protein kinase C (PKC). Little is known about the second messengers utilized by interleukin (IL)4 receptors. In this study we have investigated the relative contributions of the two signals emanating from the ligation of surface Ig receptors to the synergistic activation of B cells by IL 4. We show that IL 4 plus carefully titrated concentrations of PKC-activating phorbol esters [such as phorbol 12,13-dibutyrate (PBu₂)] induce cell cycle entry of virtually all murine B cells and substantial levels of DNA synthesis. Ca²⁺ ionophores, in contrast do not act as co-mitogens with IL 4. However, a critical concentration of ionomycin further enhanced DNA synthesis induced by PBu₂ plus IL 4. Taken together, these results suggest that PKC activation alone is sufficient to synergize with IL 4 in inducing B cells to enter cell cycle. However, the co-mitogenic effects of anti-Ig and IL 4 are evidently also dependent on Ca²⁺ signals. This interpretation is supported by the findings that cyclosporin, which abrogates the activation of lymphocytes by Ca²⁺-dependent stimuli, inhibits B cell proliferation induced by anti-Ig plus IL 4, but not the response to PBu₂ plus IL 4.     

Defective interleukin 2 receptor expression is associated with the T cell disfunction subsequent to bone marrow transplantation

In the present work we have used monoclonal antibodies (mAb) as probes to attempt a dissection of the mechanisms underlying the immunodeficiency subsequent to bone marrow transplantation (BMT). To this end we have studied 19 allogeneic BMT recipients, analyzing the proliferative response of peripheral blood mononuclear cells (PBMC) after activation with either phytohemagglutinin (PHA), anti-CD3 or anti-CD2 mAb. All patients presented normal proportions of CD2+ and CD3+ lymphocytes, as assessed by flow cytometry. Our results indicated that in most cases both CD2 and CD3-mediated activation pathways were inefficient to trigger normal T cell proliferation. The addition of exogenous interleukin 2 (IL2) did not restore in most cases the proliferative response, pointing out that additional defects contribute to the hyporesponsiveness. This was more evident in the group of patients studied during the first 6 months. To further dissect the T cell defect we analyzed the effect of a phorbol ester (phorbol myristate acetate, PMA), which activates protein kinase C, on the anti-CD3-induced response. Our data showed that PMA synergized with anti-CD3 similarly to exogenous IL2, and restored the proliferative response only in certain cases. The expression of IL2 receptors (CD25) as assessed by cytofluorimetry, after either PHA or anti-CD3 and PMA stimulation, was shown to be depressed, and the addition of IL2 did not restore it. Finally, we observed that the early increase of intracytoplasmic Ca2+ after anti-CD3 stimulation was comparable to that detected in normal PBMC. Altogether these results indicate that a diminished CD25 expression is associated with the T cell defect, and cannot apparently be attributed to an inability of the CD3 molecule to transduce early activation signals thus suggesting that either protein kinase C itself or an as yet undefined metabolic step preceding IL2 receptor expression is abnormal in variable proportions of T cells after BMT, and constitutes another manifestation of this complex immunodeficiency.     

Establishment of different T cell sublines using either interleukin 2 or interleukin 4 as growth factors

Purified protein derivative reactive T cell lines were established under identical conditions with the exception that different lymphokines, namely interleukin (IL) 2 and IL 4 were employed as growth factors. IL 2 favored the development of T cell lines (LNC.2) which upon activation by concanavalin A (Con A) secreted predominantly lymphokines characteristic of T_H1 cells. By contrast, T cell lines established with the aid of IL4 as growth factor (LNC.4) produced mainly lymphokines representative of T_H2 cells. Apart from their pattern of lymphokine secretion LNC.2 and LNC.4 T cells were found to differ in their proliferative response to lymphokines and Con A. LNC.2 T cells proliferated only marginally in the presence of IL 4, Con A or a combination of Con A and IL 1. Furthermore, the IL 2-dependent proliferation of LNC.2 T cells was slightly but significantly diminished by IL 4. In contrast, LNC.4 T cells showed a substantial IL 4-induced proliferative response which was on the one hand synergistically enhanced by minimal amounts of IL 2 and, on the other hand, strongly inhibited by interferon-γ. In addition, LNC.4 T cells displayed a strong proliferation when stimulated by low concentrations of Con A in the presence of IL 1 as co-stimulator.     

Cross-linking of membrane IgM on B CLL cells: dissociation between intracellular free Ca2+ mobilization and cell proliferation

It has been shown previously that chronic lymphocytic leukemia (CLL) B cells are frozen at different stages of activation with unique requirements for proliferation. Although most B CLL cells express surface IgM, anti-mu antibodies are able to trigger only some of them to proliferate and/or respond to B cell growth factor (BCGF) or interleukin 2 (IL2), as normal B cells. In this report we extend these observations using three different monoclonal antibodies (mAb) to human mu chain (one mitogenic in soluble form for normal B cells, the two others mitogenic only when coupled on Sepharose 4B beads). Cells from only 3 out of 11 B CLL patients proliferated in the presence of either mitogenic anti-mu. When the early events following surface Ig cross-linking, such as calcium mobilization (by flow cytometry on indo-1-labeled cells), were studied all three mAb in soluble form were able to induce a similar increase in the intracellular free calcium concentration ([Ca2+]i); analogous to [Ca2+]i rise after the mitogenic F(ab')2 anti-mu stimulation. This response was seen only in 8 out of the 12 CLL B cells studied. All B CLL cells, however, proliferate in response to a combination of phorbol ester 12,13-dibutyrate (PBt2) and ionomycin. Therefore, three patterns of response to sIg cross-linking by anti-mu could be distinguished: cells from 4 out of 12 cases proliferate and mobilize Ca2+ upon anti-mu triggering (behaving like resting B lymphocytes); in 4 other cases anti-mu lead to Ca2+ mobilization without cell proliferation; in the last 4 cases neither Ca2+ mobilization, IP3 generation (in the one case studied) nor cell proliferation are observed although these cells do proliferate directly in response to growth factors. Moreover, anti-mu stimulation in this group leads to increased proliferation in response to BCGF and IL2 suggesting an anti-Ig signaling independent of inositol phosphate metabolism. These results are interpreted in terms of differential anti-mu signaling on B cells at different stages of activation.     

Regulation by interleukin 2 of CD23 expression of leukemic and normal B cells: comparison with interleukin 4

Recombinant interleukin (IL) 4 has been shown to be able to up-regulate low-affinity Fc receptor for IgE (Fc epsilon RII)-CD23 expression on B cells as well as on other human mononuclear cells. We demonstrate here that, in opposition with previous reports, recombinant IL2 also can up-regulate CD23 expression on B cells. This was first observed on CLL cells which represent a monoclonal proliferation of B cells arrested at an intermediate stage of activation. Cells of 5 out of 12 B-CLL studied display such an increase and all 5 proliferated, in vitro, directly in response to IL2. Similarly, upon triggering with anti-mu and IL2, normal tonsillar B lymphocytes also demonstrate an increase of CD23 expression but this was observed only on day 3. Interferon-gamma was able to inhibit this IL2-mediated up-regulation of CD23 on normal B cells but not on CLL-B cells; on those cells interferon-gamma was similarly unable to inhibit the IL4-mediated CD23 up-regulation. These results suggest that CD23 regulation is complex and related to the stage of activation and the cell type.     

Proliferative responses induced by the activation of protein kinase C during the development of human T lymphocytes

We have studied the effects of phorbol-dibutyrate (PBu2), a protein kinase C (PKC) activator, on the proliferation of peripheral human T cells and thymocyte subpopulations selected by treatment with monoclonal antibodies and complement: pre-thymocytes (CD1a-CD3-CD4-CD8-), cortical thymocytes (CD3-, class I- antigens) and medullary thymocytes (enriched as CD1a- cells). PBu2 induces a dose-dependent proliferative response in human peripheral blood T cells at concentrations greater than 6 ng/ml, this proliferation being mediated by the autocrine interleukin 2 (IL2)/IL2 receptor (IL2R) pathway. Pre-thymocytes respond to PBu2 in a way similar to T cells, being able to secrete IL2 in significant amounts and express the p55 chain of IL2R. On the other hand, cortical thymocytes are not induced to proliferate after PKC activation and neither expression of the p55 chain of IL2R nor IL2 secretion is observed. Human medullary thymocytes, phenotypically identical to peripheral blood T cells, show no proliferation in response to PBu2 at any concentration tested unless IL2 is supplied to the cultures. The activation of PKC induces the expression of IL2R in these cells, but not IL2 secretion. The implications of PKC activation in thymic maturation, the role of IL2 and the relevance of the differences between medullary thymocytes and peripheral blood T cells are discussed.     

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.     

Monoclonal antibodies to epitope of CD45R (B220) inhibit interleukin 4-mediated B cell proliferation and differentiation.

A mAb, ALP-2, produced by immunizing rats with proliferating lymph node cells from MRL/Mp-lpr/lpr mice, had an inhibitory effect on recombinant interleukin 4 (rIL-4)-mediated proliferation and differentiation of murine B cells. Kinetic analysis revealed that the ALP-2 exerted these inhibitory effects at an early phase of B cell proliferation and differentiation. Nevertheless, the proliferative response of thymocytes to rIL-4 was not inhibited by ALP-2. In addition, ALP-2 inhibited neither the B cell proliferation induced by interleukin 2 nor the B cell differentiation by interleukin 5. Cross-inhibition experiments, together with immunoblotting analysis, revealed that the LP-2 recognized by ALP-2 appears to be an epitope of CD45R (B220) molecule(s), the isoform(s) of the Ly-5 antigen system. Epitope mapping analysis using CD45 gene transfectants showed that Lp-2 epitope is dependent upon the expression of the first alternatively spliced exon of CD45 gene. Functional studies showed that the mAb to an epitope of CD45R, RA3-2C2, but not RA3-6B2, had similar inhibitory effects on the IL-4-mediated proliferative response of B cells. These findings suggest that the CD45R molecules associated with Lp-2 and RA3-2C2 epitopes are probably related to a signal transduction provided by IL-4 in B cells. The possibility that different pathways are operative for IL-4-mediated signaling between B and T cells has to be considered.     

Inhibition of B cell proliferation with anti-CD19 monoclonal antibodies: anti-CD19 antibodies do not interfere with early signaling events triggered by anti-IgM or interleukin 4.

The 95-kDa antigen recognized by the anti-CD19 panel of monoclonal antibodies is found on the surface of most cells of the B cell lineage. Anti-CD19 antibodies inhibit B cell proliferation in response to anti-Ig plus interleukin 4 (IL4), but enhance the response to mitogenic concentrations of either phorbol 12-myristate 13-acetate (PMA) or Epstein-Barr virus. This dichotomy in the effect of anti-CD19 antibodies suggested that the inhibitory action may be directed at the transmembrane signaling pathways utilized by anti-IgM and IL4. To investigate this hypothesis, an attempt was made to determine the mechanism of signal transduction utilized by the CD19 antigen, and elucidate its effect on transmembrane signaling invoked by anti-immunoglobulin and IL4. Binding of anti-CD19 antibody to B cells did not promote activation of either the phosphoinositide or cAMP signaling pathways. In addition, anti-CD19 antibody did not inhibit phosphatidylinositol bisphosphate (PIP2) hydrolysis induced by anti-IgM or IL4, nor did it interfere with cAMP induction by IL4. We also found that anti-CD19 antibody inhibited PMA plus calcium ionophore-induced B cell proliferation. This evidence indicates that anti-CD19 mAb interrupts the signaling cascade at a point distal to receptor-mediated breakdown of PIP2 and/or activation of adenyl cyclase. This conclusion was fully consistent with experiments in which anti-CD19 antibody was shown to inhibit DNA but not RNA synthesis, and the observation that anti-CD19 antibody must be present between 6 h and 20 h after the initiation of the culture suggesting that anti-CD19 mAb exerts its inhibitory effect in late G0 or G1, after the initial signaling events.     

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.     

Selective effects of cholera toxin on the activation of mouse B cells by different polyclonal activators

Murine B cells were stimulated in vitro with anti-immunoglobulin (Ig) antibodies, lipopolysaccharide, or with various combinations of phorbol dibutyrate and ionomycin. Very low concentrations (ca. 10(-14) M) of cholera toxin inhibited anti-Ig-stimulated DNA synthesis, while the response to LPS was only abrogated by 2 X 10(4)-10(5)-fold greater concentrations of the toxin. Earlier responses in anti-Ig-stimulated B cells, such as increases in Ia antigen levels, were not affected by the toxin. Protein kinase C-activating phorbol esters, together with Ca2+ ionophores, are believed to stimulate DNA synthesis in lymphocytes by mimicking the two second messengers resulting from ligation of the antigen receptors. However, concentrations of cholera toxin which totally abolish anti-Ig-induced B cell proliferation significantly enhanced DNA and RNA synthesis induced by phorbol dibutyrate plus ionomycin. The results are discussed in terms of possible effects of cholera toxin on guanine nucleotide-binding (G) proteins controlling receptor coupling to second messenger-generating systems in B cells.     

Immobilized anti-CD5 together with prolonged activation of protein kinase C induce interleukin 2-dependent T cell growth: evidence for signal transduction through CD5.

Monoclonal antibodies (mAb) identifying the CD5 antigen were used to stimulate human peripheral blood T lymphocytes. Three out of three anti-CD5 mAb, 10.2, OKT1 and anti-Leu-1 induced vigorous proliferation of purified T cells in the presence of 1.6 nM phorbol 12-myristate 13-acetate (PMA). Immobilization of anti-CD5 mAb on a solid support was necessary for the induction of a proliferative response. Neither 1.6 nM PMA, nor immobilized anti-CD5 mAb were mitogenic as a sole stimulus. mAb identifying CD4, CD7, CD11a, CD18, and major histocompatibility complex class I molecules were not comitogenic with PMA. Anti-CD5/PMA-induced cell proliferation proceeded by an interleukin 2 (IL 2)-dependent mechanism, as was demonstrated by the cell surface expression of the p55 chain of the IL 2 receptor (IL 2R), the production of IL 2 and the inhibition of the proliferative response by anti-IL 2R mAb anti-Tac. There was no strict requirement for detectable numbers of monocytes, although cell proliferation could be enhanced by the monocyte-derived cytokines IL 1 and IL 6. Phorbol 12,13-dibutyrate and mezerein could substitute for PMA in this activation pathway, but synthetic diacylglycerols and phorbol esters that do not activate protein kinase C (PKC) could not, indicating a need for prolonged activation of PKC. T cells activated by anti-CD5/PMA are sensitive to inhibition by cyclosporin A (CsA) and by prostaglandin E2 (PGE2). This contrasts with anti-CD28/PMA-induced T cell proliferation, which is resistant to CsA and PGE2. Cell surface expression of CD5 was strongly up-regulated by PMA, whereas CD3 expression was down-regulated. We conclude that T cell activation can be triggered by engagement of CD5 by immobilized anti-CD5 mAb, combined with prolonged activation of PKC. These data support a role for CD5 as an independent signal transducing molecule.     

Increased cyclic AMP levels block interleukin 2-induced protein kinase C substrate phosphorylation but not the mitogenic response

Protein kinase C (PKC) has been implicated in the signaling of a number of cellular responses including activation of T cells. In the present report we have evaluated the effect of increased cAMP levels on PKC activation after stimulation of two distinct receptor systems on normal human T cells. PKC substrate phosphorylation can be induced via either the CD3 complex or, to a limited extent, the high affinity interleukin 2 (IL 2) receptor. Substrate phosphorylation via both pathways is shown to be blocked by increased intracellular levels of cAMP. In accordance with previous reports, the CD3-dependent autocrine proliferative response could also be blocked by a cAMP-dependent mechanism. Since direct activation of PKC with a phorbol ester reversed this inhibition, a causal relationship between cAMP-dependent PKC blockage and inhibition of the CD3 response is suggested. In contrast, however, initiation of IL 2-induced proliferation was essentially unaltered by cAMP and could progress in the apparent absence of PKC activity. Thus, this study indicates that IL 2-induced proliferation can under such conditions be completely uncoupled from IL 2-induced PKC activation in normal T cells.     

Enhancement of Human B-Cell Proliferation by a Monoclonal Antibody to CD43

Monoclonal antibodies (MoAb) to human leucocyte sialoglycoprotein, CD43, have been shown to deliver mitogenic signals to human T cells or to enhance T-cell proliferation induced by concanavalin A, anti-CD3 antibodies or phorbol ester. In this paper, we studied the effects of anti-CD43 MoAb B1B6 on the activation of human B cells. Anti-CD43 MoAb B1B6 was not mitogenic by itself for human B cells. However, when added together with TPA, both resting and in vivo activated tonsillar B cells, containing 5-10% and about 35% CD43+ respectively, responded with three- to fivefold higher proliferation compared to that obtained with TPA alone. A peak in the proliferative response was reached on day 3. Optimal proliferation was obtained when the antibody was present from the start of culturing. Addition of MoAb B1B6 together with a calcium ionophore, ionomycin, did not induce B-cell proliferation. Neither did mAb B1B6 sustain the growth of B cells that were already in the cell cycle, i.e. precultured with phorbol ester (PDB) and ionomycin for 3 days. The results are similar to those obtained with antibodies to CD22 and CD23 and show that early progression signals are delivered to resting B cells through CD43 in the presence of primary activators of protein kinase C.     

Involvement of the CD4 molecule in a post-activation event on T cell proliferation

A monoclonal antibody (mAb) directed to a human leukocyte 55-kDa cell surface molecule with identical cellular distribution and biochemical properties to the CD4 was able to inhibit T cell proliferation induced either in a mixed lymphocyte culture or by activation with mAb anti-CD3, anti-CD2 or phytohemagglutinin. The inhibitory effect of anti-CD4 was observed in the absence of monocytes and was directly exerted on T4+ cells. This effect on cellular proliferation appears to be due to an inhibition of a postactivation event since the rise of cytoplasmic Ca2+ after activation with anti-CD3 mAb is not affected by the presence of anti-CD4 and the proliferation that occurs after an activation pulse of 3 h with ionophore and phorbol myristate acetate can be inhibited when the anti-CD4 is added after the pulse period. Kinetic studies demonstrated that the inhibition of cellular proliferation by anti-CD4 mAb was observed even if the antibody was added as late as 18-24 h after the initiation of the culture. The effect of this blocking anti-CD4 mAb on the interleukin (IL) 2/IL 2 receptor signalling pathways was also examined. The presence of anti-CD4 slightly affected the production of IL2. In fact, addition of exogenous recombinant IL2 at the initiation of the cultures did not restore the proliferative response. However, anti-CD4 had a strong inhibitory effect on the expression of IL2 receptors as analyzed by direct immunofluorescence cytometry. Taken together, these results indicate that the binding of the anti-CD4 mAb to T cells interferes with a late metabolic step being capable of abolishing the proliferative activity of fully activated cells.