Anti-Ig-mediated proliferation of human B cells in the absence of protein kinase C

Cross-linking of surface Ig has been shown to stimulate phosphatidylinositol hydrolysis in murine B cells, leading to increases in [Ca2+]i and activation of protein kinase C (PKC). Preliminary evidence suggests that a similar activation mechanism occurs in human B cells. We wished to examine whether anti-Ig antibody-stimulated human B cell proliferation is as dependent upon the presence of PKC as is anti-Ig-mediated murine B cell proliferation. Using highly purified, small, dense peripheral-blood B lymphocytes from healthy adult donors, we confirmed that PMA, a direct activator of PKC, is a potent mitogen for human B cells that synergizes with anti-mu antibody. Furthermore, we demonstrated that PMA treatment abolishes detectable cellular stores of immunoreactive PKC. However, after such depletion of cellular PKC, anti-mu antibody is still capable of delivering a proliferative signal to human B cells. It is unlikely that this signal occurs solely on the basis of increases in [Ca2+]i, because the calcium ionophore A23187 does not induce a proliferative response in PMA-treated B cells similar in magnitude to that seen with anti-mu. Additionally, the finding that pretreatment of B cells with PMA ablates the ability of anti-Ig antibody to mobilize intracellular and extracellular calcium also suggests that the ability of PMA to enhance anti-Ig mediated stimulation does not depend on elevations of [Ca2+]i induced by anti-Ig. Together, these observations suggest that anti-Ig signaling of human B cells may occur via other pathways in addition to the phosphatidylinositol system of calcium influx and PKC activation.     

Role of the CD22 human B cell antigen in B cell triggering by anti-immunoglobulin

As B cells mature during ontogeny the CD22 human differentiation Ag is exported from the cytoplasm onto the membrane. Surface expression is lost in terminal differentiation and after activation. In tonsils, CD22 is expressed on the surface of 60 to 80% of the dense B cells. Some IgM+ dense cells, however, and buoyant in vivo activated B cells are CD22-. This differential expression of CD22 and the finding that an anti-CD22 mAb augmented anti-Ig induced B cell proliferation suggested that CD22 may play a role in B cell activation. In this study we have found that CD22+ but not CD22- B cells could be triggered by anti-IgM or anti-IgD to have increased free intracellular calcium ([Ca2+]i). The presence of CD22 rather than of IgD seems to determine the ability of B cells to respond to anti-Ig with a [Ca2+]i flux. Also the proliferative response to anti-Ig or anti-Ig + B cell growth factor was restricted to the CD22+ population. Anti-CD22 mAb, although not inducing [Ca2+]i on their own after binding to B cells, did augment [Ca2+]i fluxes by anti-Ig when cross-linked. Together these results suggest that CD22 may regulate triggering of B cells through surface Ig perhaps by acting as a "bridge" to transmit an early signal into the cytoplasm.     

Antibody to a novel 95-kDa surface glycoprotein on human B cells induces calcium mobilization and B cell activation

These findings characterize a 95-kDa glycoprotein on the surface of B lymphocytes recognized by the mAb G28-8. This protein (designated Bgp95), previously classified as a CD39 molecule, is unique based on functional, cell distribution, and immunochemical criteria. Biochemical analyses revealed that Bgp95 is a 95-kDa glycoprotein with N-linked carbohydrate and is reduced to about 70-kDa after treatment with endoglycopeptidase F. In functional studies, stimulation by G28-8 mAb or its F(ab')2 fragments induced a G0 to G1 cell cycle transition and was synergistic with PMA, anti-mu, or anti-CDw40 in stimulating proliferation of resting B cells. G28-8 mAb also could induce increases of cytoplasmic free calcium concentration [Ca2+]i in a subpopulation of tonsillar or peripheral blood B cells. The G28-8 mAb alone induced a steady increase in [Ca2+]i detectable even 1 h after stimulation. Cross-linking the G28-8 mAb with a second mAb specific for murine kappa light chains induced a more rapid increase of [Ca2+]i which peaked at 10 to 20 min and then declined. At 1 h after stimulation, [Ca2+]i was higher in B cells stimulated with G28-8 alone than in B cells stimulated with G28-8 plus anti-kappa. The same conditions of cross-linking with the anti-kappa which increased the kinetics of the [Ca2+]i response decreased the proliferative response which otherwise followed co-incubation of the mAb with B cell growth factor or PMA. Thus, conditions leading to rapid but transient [Ca2+]i increase via Bgp95 may not be as effective at stimulating B cell proliferation as conditions favoring a slower prolonged [Ca2+]i response. Although the Bgp95 molecule is present on activated buoyant tonsillar B cells, mAb to Bgp95 did not trigger [Ca2+]i increases in these cells. These results suggest that the Bgp95 protein may function in early B cell activation and that its signal mechanisms are altered by the activation state of the cell.     

Activation of human T cell clones and Jurkat cells by cross-linking class I MHC molecules

Cross-linking class I MHC molecules on human T cell clones by reacting them with various mAb directed at either monomorphic or polymorphic determinants on class I MHC molecules followed by cross-linking with GaMIg stimulated a rise in intracellular free calcium concentration ([Ca2+]i), and induced proliferation and IL-2 production. T cell clones varied in the mean density of class I MHC molecules and the capacity to respond to mAb to class I MHC molecules. However, the functional responses of the clones did not correlate with class I MHC density or the CD4/CD8 phenotype. mAb to polymorphic class I MHC determinants were less able to induce an increase in [Ca2+]i and a functional response in the T cell clones. Additive stimulatory effects were noted when mAb against both HLA-A and HLA-B determinants were employed. Cross-linking class I MHC molecules on Jurkat cells induced a rise by [Ca2+]i and induced IL-2 production upon co-stimulation with PMA. Cross-linking class I MHC molecules on mutant Jurkat cells that expressed diminished levels of CD3 and were unable to produce IL-2 in response to anti-CD3 stimulation triggered both a rise in [Ca2+]i and IL-2 production with PMA co-stimulation. In contrast, cross-linking class I MHC molecules on mutant Jurkat cells that were CD3- stimulated neither a rise in [Ca2+]i nor IL-2 production. The combination of mAb to CD28 or ionomycin and PMA, however, was able to induce IL-2 production by CD3- Jurkat cells. The data demonstrate that cross-linking class I MHC molecules delivers a functionally important signal to T cell clones and Jurkat cells and indicate that class I MHC molecules may function to transduce activation signals to T cells. In addition, the data demonstrate that transmission of an activation signal via class I MHC molecules requires CD3 expression. The data, therefore, support a central role for CD3 in the transduction of activation signals to T cells via class I MHC molecules.     

Phorbol myristate acetate inhibits increases in membrane fluidity induced by anti-IgM in B cells

Anti-IgM or anti-IgD stimulates B cells to induce increases in inositol phospholipid metabolism and intracellular free calcium concentration [( Ca2+]i). Anti-IgM also causes increases in membrane fluidity that occur more promptly than those in [Ca2+]i in resting B cells as well as BAL17 B lymphoma cells. However, other B cell activators such as LPS or PMA did not induce the membrane fluidity changes. Furthermore, sodium fluoride, which is considered to be an activator of the guanine nucleotide-binding protein, caused increases in membrane fluidity as well as increased [Ca2+]i or inositol phospholipid metabolism. Anti-IgM- or sodium fluoride-induced increases in membrane fluidity were inhibited by 20-min pretreatment of cells with PMA, but not by 24-h pretreatment. These results indicate that membrane fluidity changes are closely associated with increased [Ca2+]i after cross-linkage of membrane Ig and are regulated by protein kinase C in B cells.     

The induction of T cell unresponsiveness by rapidly modulating CD3

The immunomodulatory effects of an IgM anti-CD3 mAb (38.1) were investigated. 38.1 was distinct from other anti-CD3 mAb, in that it was rapidly modulated from the cell surface in the absence of a secondary antibody. Although 38.1 induced an immediate increase in intracellular free calcium [Ca2+]i by highly purified T cells, it did not induce entry of the cells into the cell cycle in the absence of accessory cells (AC) or a protein kinase C-activating phorbol ester. Clearing of 38.1 from the surface of AC-depleted T cells, documented both by immunofluorescence and by functional activity, was rapid, with markedly reduced levels of initially bound mAb observed after a 1 to 2 h incubation at 37 degrees C and complete modulation noted after a 5-h incubation. Despite rapid modulation of 38.1, the T cells continued to express substantial amounts of surface CD3, suggesting there is a rapid rate of turnover of CD3 molecules on resting T cells. After modulation of 38.1 bound CD3, T cells were markedly inhibited in their capacity to respond to PHA. Inhibition could be overcome by culturing the cells with supplemental AC or IL-2. The inhibitory effects of 38.1 could be mimicked by briefly pulsing cells with the calcium ionophore, ionomycin, that had no effect on surface expression of CD3. 38.1- or ionomycin-pulsed cells were inhibited in their subsequent response to PHA even when exposures were carried out in the presence of EGTA to prevent increases in [Ca2+]i from extracellular sources. Inhibition could not be accounted for by an inability of the ionomycin-treated or 38.1-modulated T cells to increase [Ca2+]i in response to PHA. These studies demonstrate that a state of T cell nonresponsiveness can be induced by modulating CD3 with an anti-CD3 mAb in the absence of co-stimulatory signals. A brief increase in [Ca2+]i resulting from mobilization of internal calcium stores appears to be sufficient to induce this state of T cell nonresponsiveness.     

Intact antigen receptor-mediated generation of inositol phosphates and increased intracellular calcium in CD4 CD8 T lymphocytes from MRL lpr mice

The predominant T lymphocytes that accumulate in the peripheral lymphoid tissues of mice homozygous for the lpr gene bear the phenotype CD3+CD4-CD8-. By certain functional criteria these cells would appear to have impaired CD3-mediated signal transduction, in that they do not respond to alloantigen and produce little if any detectable IL-2 or other lymphokines. However, the signal pathway appears adequate for achieving other T cell functions, including induction of high affinity IL-2R, and thymic deletion. To clarify the basis of this seeming discrepancy, we examined transmembrane signal transduction in T cell subsets of lpr/lpr (lpr) and +/+ mice, as defined by increased [Ca2+]i and the generation of inositol phosphates (InsPs). Stimulation of lpr CD4-CD8- cells with anti-CD3 antibody produced prompt and sustained increases in the concentration of [C2+]i and in InsPs. Similar responses occurred in mature T cells from lpr and +/+ mice, except for the somewhat slower kinetics of their increased [Ca2+]i. In marked distinction to the anti-CD2-mediated response, Con A, even in high doses, could not stimulate any increase of [Ca2+]i in lpr CD4-CD8- cells, and only modest increases in InsPs. Mature T cells, whether of lpr or +/+ origin, yielded normal increased [Ca2+]i with Con A. The reason for the differences in signal transduction between anti-CD3 and Con A stimulation of lpr CD4-CD8- cells may relate to the absence of surface structures on these immature T cells that are required for activation by Con A but not by anti-CD3. The data demonstrate that the CD3 complex in lpr CD4-CD8- T cells can couple to phospholipase C to hydrolyze phosphoinositides. These activation properties of lpr CD4-CD8- T cells have interesting functional parallels to thymocytes at the time of thymic selection, as well as tolerance induction of mature T lymphocytes.     

Anti-immunoglobulin, cytoplasmic free calcium, and capping in B lymphocytes

This paper examines, in mouse spleen lymphocytes, the effect of anti-immunoglobulin (anti-Ig) on the cytoplasmic free calcium concentration, [Ca2+]i, measured with the fluorescent indicator quin2, and the relationship of [Ca2+]i to the capping of surface Ig. Anti-Ig causes a rapid rise of [Ca2+], which precedes capping. Assuming that only those 40-50% of the cells which can bind anti-Ig (the B cells) undergo a [Ca2+]i response, [Ca2+]i in those cells approaches 500 nM. It declines to resting levels over many minutes, roughly paralleling the formation of caps. Part of the [Ca2+]i signal is due to stimulated influx across the plasma membrane, since in Ca2+-free medium, anti-Ig gives a smaller and shorter [Ca2+]i rise. The amplitude of this reduced transient now varies inversely with quin2 content, as if some 0.25 mmol Ca per liter of cells was released into the cytoplasm from internal stores. These stores are probably sequestered in organelles since A23187 in Ca2+-free medium also causes a transient [Ca2+]i rise after which anti-Ig has no effect. These organelles seem not to be mitochondria because uncouplers have hardly any effect on [Ca2+]i. Though anti-Ig normally raises [Ca2+]i before causing capping, there seems to be no causal link between the two events. Cells in Ca2+-free medium whose stores have been emptied by A23187, still cap with anti-Ig even though there is no [Ca2+]i rise. Cells loaded with quin2 in the absence of external Ca2+ still cap anti-Ig normally even though their [Ca2+]i remains steady at below 30 nM, four times lower than normal resting [Ca2+]i.     

Anti-Ig-stimulated B lymphoblasts can be restimulated via their surface Ig

Engaging AgR (surface Ig) on B lymphocytes leads to rapid inositol phosphate turnover and elevation of intracellular [Ca2+]. Continuous receptor occupancy (greater than 18 h) by anti-Ig leads to transit of most B lymphocytes from G0 to G1 stage of the cell cycle (blast transformation); a fraction of cells continue into S phase but do not proliferate continuously in the absence of growth factors. Prolonged exposure to ligand can induce receptor desensitization of some receptors. We therefore investigated whether such desensitization occurs in B cells activated by insolubilized anti-Ig. Resting B cells and anti-Ig-activated blasts were examined for their potential to elevate [Ca2+]i, maintain viability, and synthesize DNA in response to reexposure to anti-Ig. B cells and anti-Ig blasts had similar basal [Ca2+]i levels. Anti-Ig blasts retained the capacity to increase [Ca2+]i in response to anti-Ig; the magnitude of the increase was equal to or greater than that observed with resting B cells and occurred in more than 90% of cells. Isolated anti-Ig blasts subcultured in the presence of T cell-derived growth factors for 3 to 5 days responded to restimulation by anti-Ig with an increase in [Ca2+]i similar to that observed in freshly isolated blasts. The B cell and B lymphoblast ion channels were found to be permeable to Ca2+ but impermeable to Mn2+. Finally, blasts restimulated by anti-Ig retained viability and incorporated low levels of [3H]thymidine for 24 h. These results suggest that AgR on activated B lymphocytes can remain functionally coupled to intracellular signaling pathways and can participate in immune responses subsequent to initial activation.     

Patterns of costimulation of T cell clones by cross-linking CD3, CD4/CD8, and class I MHC molecules

The ability of mAb to class I MHC molecules, CD3, or CD4/CD8 to stimulate human T cell clones alone or in combination was examined. Cross-linking each of these surface Ag with appropriate mAb and goat anti-mouse Ig (GaMIg) resulted in a unique pattern of increase in intracellular free calcium ([Ca2+]i) and different degrees of functional activation. Cross-linking class I MHC molecules provided the most effective stimulus of IL-2 production and proliferation. Cross-linking more than one surface Ag induced a compound calcium signal with characteristics of each individual response. Cross-linking CD3 + HLA-A,B,C caused a rapid and prolonged increase in [Ca2+]i and synergistically increased IL-2 production and proliferation of all clones. Cross-linking CD3 + CD4/CD8 also generated a compound calcium signal and increased IL-2 production and DNA synthesis. Purposeful inclusion of CD3 was not required for costimulation as cross-linking HLA-A,B,C + CD4/CD8 also increased [Ca2+]i, IL-2 production, and proliferation. Cross-linking three surface Ag, CD3 + HLA-A,B,C + CD4/CD8, resulted in the greatest initial and sustained [Ca2+]i, IL-2 production, and DNA synthesis. Although there was a tendency for the various stimuli to increase both [Ca2+]i and functional responsiveness, neither the magnitude nor duration of the increased [Ca2+]i correlated with the amount of IL-2 produced or the ultimate proliferative response. To determine whether costimulation required that the various surface molecules were cross-linked together, experiments were carried out using isotype specific secondary antibodies. Augmentation of [Ca2+]i and costimulation of functional responses were noted when class I MHC molecules were cross-linked and CD3 was bound, but not cross-linked. Similarly, costimulation through CD3 and CD4/CD8 was observed when CD4/CD8 was cross-linked and the CD3 complex was engaged by an anti-CD3 mAb which was not further cross-linked. In contrast, costimulation by class I MHC molecules and CD4/CD8 was only observed when these molecules were cross-linked together. These data demonstrate that cross-linking class I MHC determinants or CD4/CD8 provides a direct signal to T cell clones that can be enhanced when CD3 is independently engaged. The results also indicate that T cell clones can be stimulated without engaging CD3 by the combination of signals delivered via class I MHC molecules and CD4/CD8, but only when these determinants were cross-linked together. These studies have demonstrated that these cell surface molecules differ in their capacity to deliver activation signals to T cell clones and also exhibit unique patterns of positive cooperativity in signaling potential.     

Regulatory role of CD4/L3T4 molecules in IL-2 production by affecting intracellular Ca2+ concentration of T cell clone stimulated with soluble anti-CD3

To elucidate the role of CD4 molecule in T cell activation, the effect of anti-CD4 on T cell IL-2 production was examined by using an alloreactive Th clone. The alloreactive T cell used in the present experiments produced IL-2 in response to soluble anti-CD3 epsilon-chain (anti-CD3) without accessory cell or insoluble antibody carrier. The IL-2 production was suppressed by the addition of anti-CD4 in cultures. An intracellular free Ca2+ concentration ([Ca2+]i) of the T cell clone was elevated by anti-CD3 stimulation, but the elevation was suppressed in the presence of anti-CD4. When the clone was stimulated in Ca2(+)-free medium, the elevation of [Ca2+]i was not observed. When Ca2+ influx was induced by calcium ionophore A23187 or ionomycin, the clone produced IL-2 in response to anti-CD3 in the presence of anti-CD4. When polyclonal T cell line or several other alloreactive T cell clones were examined for their anti-CD3 response, essentially the same results as mentioned above were obtained. Taken together, these results suggest that the slow and sustained elevation of [Ca2+]i is an essential signal for IL-2 production of T cells, and that anti-CD4 suppresses the IL-2 production by interfering the [Ca2+]i elevation. The significance of CD4 molecules in murine T cell activation was discussed.     

Molecular signals in B cell activation. I. Differential refractory effects of incomplete signaling by ionomycin or PMA relate to autocrine IL-2 production and IL-2R expression

The molecular signals required by resting (G0) B cells for the induction of cell cycle entry, IL-2 production, and high-affinity IL-2 receptor (IL-2R) expression were defined and the effects of incomplete activation signals on the subsequent response to complete signals were examined. Highly enriched rabbit peripheral blood B cells were activated with a calcium ionophore, ionomycin, and a protein kinase C (PKC) activating phorbol ester, phorbol myristate acetate (PMA). It was observed that cell cycle entry to early G1 was induced by either reagent acting alone, but both reagents were required to stimulate IL-2 production, IL-2R expression, and DNA synthesis. These effects of ionomycin and PMA were shown to be mediated by increased intracellular calcium ion concentration [Ca2+]i and PKC activation, respectively. Although, increased [Ca2+]i or PKC activation each led to cell cycle entry, the subsequent response of these preactivated cells to complete activation with both signals was different: Cells pretreated with PMA alone for up to 24 hr could progress further to DNA synthesis after the addition of ionomycin. In contrast, cells activated with ionomycin alone, or those cultured without any stimulus, progressively lost the ability to show DNA synthesis after complete activation. The failure to progress to DNA synthesis in these two cases was, however, differentially regulated by the ability of these cells to produce IL-2 and to express IL-2R. Ionomycin-pretreated cells retained the ability to produce IL-2 but showed about 70% reduction in the numbers of IL-2R; whereas cells cultured without any stimulus lost the ability to produce IL-2 after subsequent complete activation, but showed lesser reduction in IL-2R expression.     

Cross-linkage of Ly-6A/E induces Ca2+ translocation in the absence of phosphatidylinositol turnover and mediates proliferation of normal murine B lymphocytes

Ly-6A/E is a phosphatidylinositol (PI)-linked membrane protein whose expression is induced or upregulated on normal murine T and B cells by IFN-gamma. Cross-linkage of Ly-6A/E expressed on normal murine T cells stimulates Ca2+ translocation, and in the presence of a protein kinase C (PKC) activator, lymphokine secretion, and cellular proliferation. Utilizing an anti-Ly-6A/E mAb, we studied the effect of cross-linking Ly-6A/E on IFN-gamma-treated resting B cells, for Ca2+ translocation, PI turnover, and cellular proliferation. Since these events are known to be stimulated by cross-linkage of B cell membrane (m)Ig, we compared the changes mediated through these respective membrane proteins. We show that cross-linkage of B cell Ly-6A/E stimulates a large, rapid, and sustained increase in the concentration of intracellular free calcium ([Ca2+]i) comparable in magnitude, though somewhat delayed, relative to that observed after cross-linking of mIg. Cross-linkage of B cell Ly-6A/E does not, however, stimulate detectable PI turnover, in contrast to PI turnover induced by ligation of mIg. Both the Ly-6A/E- and mIg-mediated increase in [Ca2+]i occur through mobilization of internal Ca2+ stores as well as entry of Ca2+ into the cell from the extracellular compartment. Ly-6A/E-mediated Ca2+ translocation appears to be under the regulation of PKC in that short term pretreatment of B cells with the PKC activator, PMA, inhibits the Ly-6A/E- as well as the mIg-mediated increase in [Ca2+]i, whereas prolonged exposure to PMA, under conditions that lead to depletion of PKC, results in an augmentation in Ca2+ translocation after ligation of either Ly-6A/E or mIg. Co-capping studies indicate that Ly-6A/E and mIg cap independently in the B cell membrane, thus suggesting that the Ly-6A/E-induced effects on Ca2+ translocation are not mediated through simultaneous modulation of mIg. Anti-Ly6A/E, by itself, does not stimulate an increase in [3H]thymidine incorporation by IFN-gamma-treated resting B cells, but induces a striking increase in the presence of PMA. By contrast, anti-Ig by itself stimulates significant increases in [3H]thymidine incorporation that is inhibited by PMA. Thus, Ly-6A/E is a potent mediator of B cell activation that may use a signal transduction system in quiescent B cells that is distinct from that of the Ag receptor.     

Modeling of T cell contact-dependent B cell activation. IL-4 and antigen receptor ligation primes quiescent B cells to mobilize calcium in response to Ia cross-linking.

The generation of antibody secretory cells from resting B lymphocytes after immunization with most protein Ag requires B cell signaling by Ag, direct Th cell contact and lymphokines. Previous studies suggest that cell contact-mediated signals may be transduced by Ia after Ia binding by alpha beta TCR and/or CD4. Seemingly inconsistent with this concept are findings that cross-linking of Ia molecules on quiescent B cells leads to cAMP generation that is antagonistic for B cell mitogenesis. Here we show that ligand binding to IL-4 and Ag receptors on quiescent B cells induce transition of these cells into a competent state in which Ia molecules transduce signals via a distinct mechanism. This mechanism involves the tyrosine kinase-dependent activation of phospholipase C leading to Ca2+ mobilization from intracellular stores and the extracellular space. This competence, which is seen within 4 h of priming, is not simply a function of increased Ia expression by the B cell because the response can be induced by cross-linking of less than 5% of cell surface Ia molecules on primed cells. Finally, cross-linking of Ia molecules leads to more than fivefold greater increase in [Ca2+]i than is induced by membrane Ig ligation. These findings are consistent with alpha beta TCR/CD4 delivery via Ia of proliferative signals mediated by tyrosine kinase activation, phosphoinositide hydrolysis and Ca2+ mobilization.     

Molecular signals in B cell activation. II. IL-2-mediated signals are required in late G1 for transition to S phase after ionomycin and PMA treatment

We report that sustained increase of intracellular calcium ion concentration and protein kinase C (PKC) activation maintained throughout the G1 phase of cell cycle do not provide sufficient signals to cause S-phase entry in rabbit B cells, and that additional signals transduced by IL-2 and IL-2 receptor interaction are essential for G1 to S transition. We have shown earlier that rabbit B cells can be activated to produce IL-2 and express functional IL-2 receptors after treatment with ionomycin and PMA. Herein we have compared the response of rabbit PBLs, which contain about 50% T cells, with those of purified B cells. After activation with ionomycin or PMA, comparable numbers of PBLs and B cells entered the cell cycle; but DNA synthesis by the PBL cultures was three to four times higher than that of cultures of purified B cells. Interestingly, IL-2 production by the PBL cultures was also three to four times higher than in B cell cultures, suggesting an involvement of IL-2 in inducing DNA synthesis in these cells. The hypothesis that IL-2, which is produced in early G1, acts in late G1 and is required for G1 to S transition in B cells was supported by the following observations: (i) IL-2 production by B cells was detected as early as 6 hr after activation and preceded DNA synthesis by at least 24 hr. (ii) B cell blasts in G1 (produced by treatment of resting B cells with ionomycin and PMA) showed DNA synthesis in response to IL-2, but showed very little DNA synthesis in response to restimulation with ionomycin and PMA. (iii) A polyclonal rabbit anti-human IL-2 antibody caused nearly complete inhibition of DNA synthesis by B cells activated by ionomycin and PMA. (iv) A PKC inhibitor, K252b, inhibited DNA synthesis in ionomycin and PMA-stimulated cells if added at the beginning of culture but was not inhibitory if added 16 hr later. We conclude that increased [Ca2+]i and PKC activation are not sufficient signals for G1 to S transition in B cells; entry into S is signaled by IL-2, and IL-2-mediated signal transduction probably does not involve increased [Ca2+]i or PKC activation.     

Human resting B lymphocytes can serve as accessory cells for anti-CD2-induced T cell activation.

Although resting B cells are poor accessory cells for signals transmitted through the TCR/CD3 complex, we report that these B cells can support T cell proliferation when T cell activating signals are delivered through CD2. This was first suggested when leucine methyl ester treatment of PBMC abolished proliferation induced by anti-CD3, but not by the accessory cell-dependent anti-CD2 mAb combination, GT2 and OKT11. Then we demonstrated that unstimulated, resting B cells could support the proliferation of both CD4+ and CD8+ T cells. Aggregated IgG inhibited proliferation, suggesting that anti-CD2 mAb bound to T cells were cross-linked by attachment to B cell FcR. Two lines of evidence suggested that lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interaction was crucial for anti-CD2-induced proliferation. First, proliferation was blocked by mAb against these adhesion molecules. Second, intercellular adhesion molecule-1 expression rapidly increased on resting B cells after the addition of anti-CD2, but not anti-CD3. This was of interest because fixed monocytes, but not fixed B cells, were able to support the proliferative response. In contrast to lymphocyte function-associated Ag-1/intercellular adhesion molecule-1, CD28/B7 interaction was not required for anti-CD2-induced proliferation, although ligation of these molecules provided important costimulatory signals for stimulation by anti-CD3. Finally, neutralizing antibodies against IL-1 alpha, IL-1 beta, and IL-6 showed only modest inhibitory effects on T cell proliferation. The addition of IL-1 and/or IL-6 to T cells failed to substitute for accessory cells and were only partially effective with fixed B cells. Further evidence of a linkage between CD2 and CD45 isoforms was obtained. Anti-CD45RA, but not anti-CD45RO, potentiated anti-CD2-induced T cell proliferation. These studies have revealed a novel role for resting B cells as accessory cells and have documented costimulatory signals that are important for this effect. Because Ag-presentation by resting B cells to T cells generally leads to T cell nonresponsiveness, it is possible that this tolerogenic signal may be converted to an activation signal if there is concurrent perturbation of CD2 on T cells.     

T cell receptor aggregation, but not dimerization, induces increased cytosolic calcium concentrations and reveals a lack of stable association between CD4 and the T cell receptor.

Exposure of T94, a CD4+ V beta 8-expressing murine Th cell clone, or immediately ex vivo CD4+ T cells to deaggregated, bivalent antibodies specific for either the TCR or CD3 failed to induce an increase in [Ca2+]i, or activation of phosphatidylinositol hydrolysis unless cross-linked with a secondary anti-Ig antibody. In contrast, we show that a combination of two mAb directed against different components of the TCR/CD3 complex (145.2C11, anti-CD3 epsilon and F23.1, anti-V beta 8) successfully induce second messenger formation, that is, without any requirement for a secondary antibody. This requirement for either a secondary antibody or two independent bivalent antibodies to activate second messenger production in T cells suggested that the signal transduction apparatus may be activated by multiple TCR/CD3 complexes being brought together on the T cell surface. This was supported by the observation that conditions inducing increased T cell [Ca2+]i through the TCR/CD3 complex also resulted in aggregation of the TCR/CD3 complex on the T cell surface. Conversely, binding of anti-TCR/CD3 antibodies to the T cell under conditions that did not induce increased [Ca2+]i also failed to induce surface TCR/CD3 redistribution. Cross-linking of the CD4 accessory molecule on T94 also resulted in increased [Ca2+]i, with kinetics similar to those observed after TCR/CD3 oligomerization. CD4 is involved in the recognition of invariant regions of MHC class II during Ag presentation and has been proposed to be associated with TCR/CD3 in the absence of Ag. Aggregation of TCR/CD3 and subsequent second messenger formation was achieved by combinations of mAb to distinct determinants within the complex due to the stable association of these determinants within the T cell membrane. We therefore assessed the functional association of CD4 with the TCR/CD3 complex by examining whether a combination of mAb directed against CD4 and CD3 or TCR induced second messenger formation. We found that anti-CD4 in combination with F23.1 or with 145.2C11 failed to induce increases in [Ca2+]i. Furthermore, mAb to CD4 failed to inhibit the increase in [Ca2+]i observed with the combination of 145.2C11 and F23.1. We therefore conclude that CD4 is not stably associated with TCR or CD3 in the absence of Ag/MHC class II composites.     

CD28 delivers a costimulatory signal involved in antigen-specific IL-2 production by human T cells.

CD4+ T cells require two signals to produce maximal amounts of IL-2, i.e., TCR occupancy and an unidentified APC-derived costimulus. Here we show that this costimulatory signal can be delivered by the T cell molecule CD28. An agonistic anti-CD28 mAb, but not IL-1 and/or IL-6, stimulated T cell proliferation by tetanus toxoid-specific T cells cultured with Ag-pulsed, costimulation-deficient APC. Furthermore, the ability of B cell tumor lines to provide costimulatory signals to purified T cells correlated well with expression of the CD28 ligand B7/BB-1. Finally, like anti-CD28 mAb, autologous human APC appeared to stimulate a cyclosporine A-resistant pathway of T cell activation. Together, these results suggest that the two signals required for IL-2 production by CD4+ T cells can be transduced by the TCR and CD28.     

Murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore.

The calcium ionophore, A23187, when used alone was found to induce proliferation of murine T cells, at concentrations of 0.5-1 mM. This response required the presence of syngeneic splenic adherant cells (SAC) as a source of accessory cells. Interestingly, only CD4+ T cells but not CD8+ T cells or B cells responded to the calcium ionophore by proliferation. The inability of CD8+ T cells or B cells to respond was not related to decreased elevation in the intracellular ionized calcium [Ca2+]i concentration induced by the ionophore, because activated CD4+ T, CD8+ T and B cells all exhibited similar elevation in [Ca2+]i. The inability of CD8+ T cells to respond to calcium ionophore was probably due to insufficient production of autocrine growth factors, such as IL-2, inasmuch as the addition of exogenous IL-2 could completely restore the CD8+ T cell responsiveness. Also, exogenous rIL-1 could partially restore purified T cell response to calcium ionophore, whereas, rIL-6 failed to do so. IL-2, but not IL-4, acted as an autocrine growth factor for T cells responding to the calcium ionophore in the presence of SAC, since, antibodies against IL-2 or IL-2 receptor (IL-2R) but not against IL-4, could inhibit the T cell proliferation. Furthermore, exogenous rIL-2 but not rIL-4 supported the proliferation of T cells to calcium ionophore in the absence of accessory cells. Our results suggest that murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore and that this may not depend on the early activation signal such as the elevation in [Ca2+]i) induced by the ionophore but may depend on subsequent signals which regulate endogenous growth factor production.