Tyrosine phosphorylation is required for mast cell activation by Fc epsilon RI cross-linking.

We investigated the possible role of tyrosine phosphorylation in the activation process of mast cells by cross-linking of cell-bound IgE antibodies. Bone marrow-derived mouse mast cells (BMMC) were sensitized with mouse IgE antiDNP mAb and then challenged with multivalent Ag DNP conjugates of human serum albumin. Analysis of phosphotyrosine-containing proteins in their lysates by SDS-PAGE and immunoblotting revealed that cross-linking of cell-bound IgE antibodies induced a marked increase in tyrosine phosphorylation of several proteins. To obtain direct evidence for activation of protein-tyrosine kinases (PTK), phosphotyrosine-containing proteins in lysates of mast cells were affinity purified, and kinase activity of the immunoprecipitates was assessed by an in vitro kinase assay. The results clearly showed activation of PTK upon cross-linking of Fc epsilon RI. Activation of PTK was not detected by the same assay when the sensitized BMMC were challenged with monovalent DNP-lysine. Treatment of sensitized BMMC with either Ca2+ ionophore or PMA failed to induce the activation of PTK. A representative IgE-independent secretagogue, thrombin, induced histamine release from BMMC but failed to induce activation of PTK. The results excluded the possibility that PTK activation is the consequence of an increase in intracellular Ca2+ or activation of protein kinase C. Addition of genistein, a PTK inhibitor, to sensitized BMMC before Ag challenge inhibited not only Ag-induced PTK activation, but also inositol 1,4,5-trisphosphate production, and histamine release in a similar dose-response relationship. Other PTK inhibitors, such as lavendustin A and tyrphostin RG50864, also inhibited the Ag-induced activation of PTK and histamine release. The results collectively suggest that activation of PTK is an early event upstream of the activation of phospholipase C, and is involved in transduction of IgE-dependent triggering signals to mediator release.     

A therapeutic CD4 monoclonal antibody inhibits TCR-zeta chain phosphorylation,zeta-associated protein of 70-kDa Tyr319 phosphorylation,and TCR internalization in primary human T cells

The molecular mechanisms mediating the inhibitory effects of a humanized CD4 mAb YHB.46 on primary human CD4(+) T cells were investigated. Preincubation of T cells with soluble YHB.46 caused a general inhibition of TCR-stimulated protein tyrosine phosphorylation events, including a reduction in phosphorylation of p95(vav), linker for activation of T cells, and Src homology 2 domain-containing leukocyte protein of 76-kDa signaling molecules. A marked reduction in activation of the Ras/mitogen-activated protein kinase pathway was also observed. Examination of the earliest initiation events of TCR signal transduction showed that YHB.46 inhibited TCR-zeta chain phosphorylation together with recruitment and tyrosine phosphorylation of the zeta-associated protein of 70-kDa tyrosine kinase, particularly at Tyr(319), as well as reduced recruitment of p56(lck) to the TCR-zeta and zeta-associated protein of 70-kDa complex. These inhibitory events were associated with inhibition of TCR endocytosis. Our results show that the YHB.46 mAb is a powerful inhibitor of the early initiating events of TCR signal transduction.     

Triggering of co-mitogenic signals in T cell proliferation by anti-LFA-1 (CD18, CD11a), LFA-3, and CD7 monoclonal antibodies

Proliferative T cell responses were elicited in a comitogenic assay when purified mAb against CD 18, CD11a, LFA-3, and CD7 were immobilized onto solid plastic surfaces together with submitogenic doses of mAb against the CD3 complex. The proliferative response was associated to the production of IL-2 and to the expression of IL-2R. We explored the possibility that a second signal provided by either PMA or a Ca2+ ionofore could replace the anti-CD3 mAb in the comitogenic assay. Interestingly, our data clearly indicate that PMA but not the ionofore was capable of mediating the co-mitogenic effect in conjunction with solid-bound mAb (CDw18, CD11a, LFA-3, and CD7). We also demonstrate that the mAb (anti-CD4 and anti-CD2) which have been previously described as co-mitogenic in combination with anti-CD3 are capable of eliciting this activating signal in the presence of PMA. These data indicate that mAb to certain cell surface differentiation Ag that in soluble form inhibit T cell function such as LFA-1, LFA-3, and CD2 can under appropriate conditions induce co-mitogenic signals on T cells. Our results support the hypothesis that several cell surface differentiation Ag may participate in conjunction with the T3-Ti complex in the transmembrane signal transduction leading to T cell activation.     

Stimulation via CD3-Ti but not CD2 induces rapid tyrosine phosphorylation of a 68-kDa protein in the human Jurkat T cell line.

Tyrosine phosphorylation is an early biochemical event associated with surface receptor triggering in many cellular systems. In T lymphocytes, Ag receptor (CD3-Ti) stimulation results in tyrosine phosphorylation of the CD3 zeta subunit. The tyrosine kinase responsible for this modification after CD3-Ti triggering has not been identified. Here we reported that a 68-kDa T cell membrane-associated protein (pp68) in human Jurkat T cells is phosphorylated on tyrosine residues within 1 min after anti-CD3 mAb addition. This induced tyrosine phosphorylation is detected either by in vivo [32P]orthophosphate labeling of the Jurkat T cells or by in vitro [32P]ATP labeling after immunoprecipitation by antiphosphotyrosine antibody. In contrast, mAb stimulation via CD2 and CD4 structures does not induce phosphorylation of pp68. These data are among the first to provide evidence that CD3-Ti and CD2 activation pathways are distinct. Furthermore, they imply that pp68 is itself a tyrosine kinase and/or is a rapidly phosphorylated substrate of a tyrosine kinase.     

Intracellular events involved in CD5-induced human T cell activation and proliferation.

In this report we describe a novel pathway of human T cell activation and proliferation involving the CD5 surface Ag. The CD5-specific Cris1 mAb induces by itself monocyte-dependent proliferation of PBMC. Among a panel of CD5-specific mAb (Leu1, OKT1, LO-CD5a, F101-1C5, and F145GF3), only the F145GF3 mAb shared this property with Cris1. The analysis of the biochemical pathway involved in this activation showed the lack of detectable hydrolysis of inositol phosphates or early increments in the intracellular cytosolic calcium concentration, after triggering cells with the mitogenic CD5 mAb. However, stimulation with CD5 induces activation of protein kinase C, as measured by phosphorylation of a specific peptide substrate (peptide GS), which can be inhibited by a pseudosubstrate peptide inhibitor. Stimulation with CD5 mAb induces also tyrosine kinase activity, with a substrate pattern that differs from that induced after triggering lymphocytes through the TCR-CD3 complex. On the other hand the IL-2/IL-2R pathway seems involved in the CD5-mediated proliferation of PBMC because anti-IL-2R-specific mAb inhibits CD5-induced proliferation, and stimulation with mitogenic CD5 mAb induces production of IL-2 and expression of IL-2R alpha and beta chains. Therefore, the triggering of the CD5 Ag can induce IL-2- and monocyte-dependent human T cell proliferation by a biochemical pathway that differs, at least in the first stages, from the one that mediates TCR-CD3 complex-induced T cell activation.     

CD7 augments T cell proliferation via the interleukin-2 autocrine pathway.

The role of CD7, a T cell differentiation antigen, in T cell function is not known at present; this study evaluates the effect of anti-CD7 mAb in PMBC cultures activated with suboptimal concentrations of lectins, antigens, and anti-CD3 mAb. We found that the inclusion of anti-CD7 resulted in increased IL-2 production and IL-2R-alpha expression in these cultures. H-7, a protein kinase C (PKC) inhibitor, and genistein, a protein tyrosine kinase (PTK) inhibitor, significantly suppressed the proliferation of T cells in comitogenic assays. This suggested that the comitogenic effect mediated by CD7 molecule involved both the PKC and the PTK pathways of T cell activation. These drugs appeared to affect the CD7-mediated effects by inhibiting the IL-2 autocrine pathway, especially the up-regulation of IL-2R-alpha since inhibition was not relieved with exogenous rIL-2. Taken together, our results suggest that CD7 augments T cell function by up-regulating IL-2R-alpha expression and IL-2 production via multiple pathways of protein phosphorylation.     

Differential association of CD45 isoforms with CD4 and CD8 regulates the actions of specific pools of p56lck tyrosine kinase in T cell antigen receptor signal transduction.

An investigation into the role of CD45 isoforms in T cell antigen receptor signal transduction was carried out by transfecting CD45-negative CD4(+)CD8(+) HPB-ALL T cells with the CD45R0, CD45RBC, and CD45RABC isoforms. Fluorescence resonance energy transfer analysis showed that the CD45R0 isoform, but not the CD45RBC or CD45RABC isoforms, was found as homodimers and also preferentially associated with CD4 and CD8 at the cell-surface. A comparison was therefore made of T cell antigen receptor signaling between sub-clones expressing either CD45R0 or CD45RBC. Under basal conditions CD4-associated p56(lck) tyrosine kinase activity and cellular protein tyrosine phosphorylation levels were higher in the CD45R0(+) than in the CD45RBC(+) sub-clones. Upon CD3-CD4 ligation, TCR-zeta phosphorylation, ZAP-70 recruitment to the p21/p23 TCR-zeta phosphoisomers, ZAP-70 phosphorylation, as well as p56(lck), c-Cbl and Slp-76 phosphorylation, were all markedly increased in CD45R0(+) compared with CD45RBC(+) cells. T cell antigen receptor (TCR) stimulation alone also promoted c-Cbl phosphorylation in CD45R0(+) but not in CD45RBC(+) cells. Our results are consistent with a model in which association of CD45R0 with CD4 generates a more active pool of CD4-associated p56(lck) kinase molecules. Upon CD3-CD4 co-ligation, the active p56(lck) increases the intensity of T cell antigen receptor signal transduction coupling by promoting TCR-zeta chain phosphorylation and ZAP-70 recruitment.     

Differential requirement of protein tyrosine kinase and protein kinase C in the generation of IL-2-induced LAK cell and αCD3-induced CD3-AK cell responses

This study examined the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the signal transduction pathways for lymphocyte activation through IL-2R to generate LAK cells and through TCR—CD3 to generate CD3-AK cells. Two PTK inhibitors [herbimycin A and genistein (PTK-I)] and two PKC inhibitors [calphositin C and staurosporine (PKC-I)] were used in the experiments. It was found that the primary activation pathway through IL-2R was PTK-dependent; that is, generation of both the IL-2-induced proliferative and the cytotoxic responses was completely abrogated by PTK-I and not by PKC-I. Quite different results were obtained with the αCD3-induced CD3-AK cell response. First, the αCD3-induced proliferation was only partially inhibited by PTK-I or PKC-I alone. Second, generation of CD3-AK cytotoxic response was primarily PKC-dependent; that is, only PKC-I induced significant inhibition. Genistein was found to reduce protein tyrosine phosphorylation in both LAK cells and CD3-AK cells, indicating that CD3-AK cells were also susceptible to PTK-I treatment. Further studies showed that PTK-I and not PKC-I suppressed perforin mRNA expression and N-2-benzyoxycarbonyl-l-lysine thiobeneylester esterase production in LAK cells, and the opposite was true for CD3-AK cells. These results indicate that different pathways were employed in lymphocyte activation through IL-2R and TCR—CD3. The former pathway is primarily PTK-dependent. Activation through TCR—CD3 is a more complex event. Induction of a proliferative response can employ either a PTK- or a PKC-dependent pathway, whereas induction of a cytotoxic response is primarily PKC-dependent. Furthermore, it appears that a PTK-independent pathway exists for the induction of a CD3-AK response and thus suggests that activation of the second messenger PKC may not necessarily be preceded by PTK activation.     

The 77C->G mutation in the human CD45 (PTPRC) gene leads to increased intensity of TCR signaling in T cell lines from healthy individuals and patients with multiple sclerosis

The 77C-->G mutation in exon A of the human CD45 gene occurs with low frequency in healthy individuals. An enhanced frequency of 77C-->G individuals has been reported in cohorts of patients suffering from multiple sclerosis, systemic sclerosis, autoimmune hepatitis, and HIV-1. To investigate the mechanisms by which the variant allele may contribute to disease susceptibility, we compared T cell reactivity in heterozygous carriers of the mutation (healthy individuals and multiple sclerosis patients) and wild-type controls. In vitro-generated T cell lines and freshly isolated CD4+CD45R0+ primed/memory T cells from 77C-->G individuals aberrantly expressed CD45RA isoforms and showed enhanced proliferation and IL-2 production when stimulated with anti-TCR/CD3 mAb or Ag. Mutant T cell lines contained a more active pool of p56lck tyrosine kinase and responded with increased phosphorylation of Zap70 and TCR-zeta and an enhanced Ca2+ flux to TCR/CD3 stimulation. These data suggest that 77C-->G may act as a risk factor for certain diseases by increasing the intensity of TCR signaling.     

Phosphorylation of two cytosolic proteins. An early event of T-cell activation.

T lymphocytes can be activated to proliferate by triggering the T-cell antigen-receptor complex (CD3-Ti) with anti-CD3 (Cluster of Differentiation 3) monoclonal antibody (mAb) or with the mitogenic lectin phytohaemagglutinin A (PHA). We have investigated the relationship between lymphocyte activation and protein phosphorylation in the human leukaemic T-cell line Jurkat. Incubation of 32P-labelled Jurkat cells with anti-CD3 mAb or PHA induced the phosphorylation of two cytosolic proteins that migrate with apparent Mr values of 21,000 (pp21) and 23,000 (pp23) and pI values of 5.1 and 5.0 respectively. Peptide mapping of the two proteins produced the same phosphopeptides pattern, suggesting that pp21 and pp23 are closely related. The phosphorylation of pp21 and pp23 induced by anti-CD3 mAb appeared to be transient, since it was already detected 2 min after the addition of the mAb, reached a maximum at 10 min and recovered its basal level after 1 h. Phosphorylation of pp21 and pp23 could also be elicited by the Ca2+ ionophore A23187 and sodium orthovanadate (Na3VO4), two agents that bypass the T-cell-receptor complex and produced an increase in cytosolic Ca2+ concentration. In addition, we found that vanadate, like the Ca2+ ionophore, induced the secretion of interleukin-2 (IL-2) when used in combination with a submitogenic concentration of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. These results show that the Ca2+-dependent phosphorylation of pp21 and pp23 represents an early event in the process of signal transduction through the CD3-Ti receptor complex.     

Differential in vitro activation of CD8-CD4+ and CD4-CD8+ T lymphocytes by combinations of anti-CD2 and anti-CD3 antibodies

Purified peripheral blood T lymphocytes and the CD8-CD4+ and CD4-CD8+ T cell subsets, exhaustively depleted of APC have been studied for their capacity to respond to mAb directed against the CD3-Ti Ag-specific TCR complex and against the CD2 SRBCR. It is demonstrated that high affinity IL-2R can be readily induced by either anti-CD3 and/or anti-CD2 stimulation. However, IL-2 production can be observed only in the CD4+CD8- T cell subset. These results clearly contrast data obtained with purified CD4-CD8+ T cells, which are able to proliferate when the CD3-Ti complex is activated in the presence of APC. The data presented in the present study demonstrate that a simplified model for T cell activation and clonal expansion of the two major T cell subsets involve only the CD3-Ti complex and the CD2 Ag. Under conditions where the activation signals for the T cells are restricted only to the activation of CD3-Ti and CD2, the CD4+CD8- T cells respond with IL-2 production and expression of high affinity IL-2R, whereas the CD4-CD8+ T cell subset depends on exogenous IL-2 provided by the CD4+CD8- cells. These data do not, however, exclude an involvement of other cell-surface signals for regulation and control of T cell activation and T cell effector functions.     

Protein kinase C plays a role in the induction of tyrosine phosphorylation of lymphoid microtubule-associated protein-2 kinase. Evidence for a CD3-associated cascade that includes pp56lck and that is defective in HPB-ALL.

Ligation of the CD3 receptor induces multiple signal transduction events that modify the activation state of the T cell. We have compared two lines that express biologically active CD3 receptors but differ in their biochemical activation pathways during ligation of this receptor. Jurkat cells respond to anti-CD3 with Ca2+ mobilization, PKC activation, induction of protein tyrosine phosphorylation, and activation of newly characterized lymphoid microtubule associated protein-2 kinase (MAP-2K). MAP-2K itself is a 43-kDa phosphoprotein that requires tyrosine phosphorylation for activation. Although ligation of the CD3 receptor in HPB-ALL could stimulate tyrosine phosphorylation of a 59- kDa substrate, there was no associated induction of [Ca2+]i flux, PKC, or MAP-2K activation. A specific PKC agonist, PMA, which bypasses the CD3 receptor, could, however, activate MAP-2K in HPB-ALL cells. This implies that defective stimulation of PKC by the CD3 receptor is responsible for its failure to activate MAP-2K in HPB-ALL. The defect in PKC activation is likely distal to the CD3 receptor as A1F14- failed to activate MAP-2K in HPB-ALL but was effective in Jurkat cells. The stimulatory effect of PMA on MAP-2K activity in HPB-ALL was accompanied by tyrosine phosphorylation of this kinase which implies that PKC may, in some way, regulate tyrosine phosphorylation of MAP-2K. A candidate for this role is pp56lck which underwent posttranslational modification (seen as mobility change on SDS-PAGE) during anti-CD3 and PMA stimulation in Jurkat or PMA treatment in HPB-ALL. There was, in fact, exact coincidence between induction of PKC activity, posttranslational modification of lck and tyrosine phosphorylation/activation of MAP-2K. Lck kinase activity in an immune complex kinase assay was unchanged during PMA treatment. An alternative explanation is that modification of lck may alter its substrate profile. We therefore looked at the previously documented ability of PKC to dissociate lck from the CD4 receptor and found that PMA could reduce the stoichiometry of the lck interaction with CD4 in HPB-ALL and to a lesser extent in Jurkat cells. These results imply the existence of a kinase cascade that is initiated by PKC and, in the course of which, lck and MAP-2K may interact.     

Regulation of CD4 and CD8 surface expression on human thymocyte subpopulations by triggering through CD2 and the CD3-T cell receptor

Human thymocytes bearing the CD4 and/or CD8 antigens can be fractionated into cells with an immature and more mature phenotype based on their quantitative expression of the CD3 Ag (J. Immunol. 138:3108; J. Immunol. 139:1065). We show that the expression of CD4 and CD8 on thymocyte subpopulations with low CD3 (CD3L) and high CD3 (CD3H) is regulated by activation through the CD2 molecule and perturbation of the CD3-T cell receptor complex (CD3-Ti). Similar to its previously reported effects on peripheral T cells, PMA was able to induce the down-regulation of surface CD4, but not CD8, on thymocyte subpopulations. PMA could induce CD4 and CD8 phosphorylation in both CD3L and CD3H fractions. These results suggest that if changes in phosphorylation represent the mechanism by which CD4 and CD8 are able to transmit signals, this mechanism is operative in both CD3L and CD3H subpopulations. Treatment with anti-T11(2) and anti-T11(3) antibodies (CD2 activation pathway) resulted in partial down-regulation of CD4 but not CD8 surface expression on both CD3L and CD3H thymocytes. Similar treatment had no detectable effect on peripheral T cells. The down-regulation of surface CD4 induced by activation via CD2 could be inhibited by treatment of thymocytes with anti-CD3 antibodies. Treatment of thymocytes with anti-CD3 alone or following CD2 activation induced the selective down-regulation of surface CD8 within 15 minutes. These results suggest that CD2 and CD3-Ti triggering may regulate CD4 and CD8 surface expression on thymocytes. Furthermore, these results suggest that "cross-talk" between the CD2 and CD3-Ti pathway of activation may involve CD4 and CD8 molecules.     

Activation of human T4 cells by cross-linking class I MHC molecules

These studies examined whether cross-linking class I MHC molecules results in functional or biochemical responses in human T4 cells. The initial studies demonstrated that cross-linking class I MHC molecules either by culturing highly purified T4 cells with immobilized mAb to class I MHC Ag or reacting the T4 cells with mAb to class I MHC Ag and then cross-linking the mAb with goat antimouse Ig (GaMIg) enhanced T4 cell proliferation induced by an immobilized mAb to CD3, OKT3. More-over, immobilized but not soluble mAb to class I MHC Ag enhanced T4 cell proliferation induced by the combination of two mAb to CD2, OKT11, and D66.2. Finally, T4 cells reacted with mAb to CD3 and class I MHC Ag proliferated in the presence of IL-2 when cross-linked with GaMIg more vigorously than T4 cells reacted with either mAb alone. Cross-linking class I MHC molecules was also found to stimulate T4 cells directly. T4 cells reacted with mAb to class I MHC Ag or beta 2 microglobulin and cross-linked with GaMIg proliferated vigorously in the presence of IL-2 or PMA. In addition, it was demonstrated that cross-linking class I MHC molecules by culturing T4 cells with immobilized mAb to class I MHC Ag induced T4 cell proliferation in the presence of IL-2. T4 cell proliferation in the presence of IL-2 and PMA could also be induced by reacting the cells with specific mAb to polymorphic determinants on class I MHC molecules and cross-linking with GaMIg. Cross-linking mAb to CD4 or CD11a did not have a similar functional effect on T4 cells. Finally it was demonstrated that adding GaMIg to T4 cells reacted with mAb to class I MHC Ag but not CD11a resulted in an increase in intracellular calcium concentration. The data demonstrate that cross-linking class I MHC molecules results in the generation of at least one activation signal, a rise in intracellular calcium concentration, and, thereby, stimulates human T4 cells.     

Engagement of CD14 on human monocytes terminates T cell proliferation by delivering a negative signal to T cells.

We have recently shown that engagement of the human monocytic Ag CD14 by murine mAb induces lymphocyte function-associated antigen-1/intercellular adhesion molecule-1-dependent homotypic adhesion. To determine whether CD14 plays a role in monocyte-T cell interactions, we tested the effect of anti-CD14 mAb on the proliferation of human T cells. Our results show that anti-CD14 mAb strongly inhibited T cell proliferation induced by Ag, anti-CD3 mAb, and mitogenic lectins. Inhibition by anti-CD14 mAb was epitope-dependent and required physical contact between monocytes and T cells. CD14 engagement did not affect IL-2R expression or IL-2 synthesis but induced a state of unresponsiveness that was not IL-2 specific; proliferation of anti-CD3-activated T cell blasts in response to both IL-2 and IL-4 was abrogated by addition of monocytes preincubated with anti-CD14 mAb. Inhibition of T cell proliferation after engagement of CD14 on monocytes was likely to result from delivery of a negative signal to T cells, rather than from disruption of a costimulatory monocyte-derived signal, because incubation of monocytes with anti-CD14 mAb also inhibited monocyte-independent T cell proliferation induced by PMA and ionophore. These results, together, point to a role of CD14 in the monocyte-dependent regulation of T cell proliferation.     

Mechanisms of immune memory. T cell activation and CD3 phosphorylation correlates with Ta1 (CDw26) expression

The Ta1 (CDw26) Ag distinguishes a subset of circulating T lymphocytes that is the major population proliferating to recall Ag challenge. Unlike receptors for growth factors such as IL-2 and transferrin, the Ta1 Ag is present on T cell lines and clones irrespective of cell cycle. The appearance of Ta1 on T cells that respond to recall Ag allowed us to investigate activation requirements that may be associated with T cell immune memory. Ta1+ peripheral blood T cells were induced to proliferate by mAb recognizing either the invariant chains of the TCR, or by pairs of mitogenic antibodies directed to the CD2 molecule. In contrast, Ta1- cells were not stimulated by these antibodies. In addition, Ta1-cells did not proliferate maximally after addition of the phorbol ester PMA in combination with the calcium ionophore Ionomycin, suggesting that the intracellular targets of these agents may not be fully active. Anti-CD3-induced elevation of intracellular calcium levels was equivalent in the two subpopulations, suggesting that calcium mobilization mechanisms were intact. In contrast, PMA-induced phosphorylation of TCR CD3 chains was significantly greater in Ta1+ cells as compared to Ta1- T cells. Taken together, our results indicate that Ta1 expression, which is associated with T cell activation and memory, may be causally related to TCR and CD2-mediated activation mechanisms. The PMA inducible TCR phosphorylation in Ta1+ memory cells associated with their increased ability to proliferate after CD3/TCR or CD2 stimulation suggests that intracellular phosphorylation events may be causally associated with T cell immune memory.     

The 1A4 molecule (CD27) is involved in T cell activation.

We developed a new mAb, anti-1A4, which recognizes an epitope on the CD27 molecule distinct from those recognized by several known anti-CD27 mAb. Although it has been suggested that the CD27 molecule is a T cell activation Ag, there was little direct evidence that the structure was involved in the T cell activation process. In this study, we showed that anti-1A4 inhibited anti-CD2, anti-CD3, mitogens, or soluble Ag-induced T cell proliferation as well as PWM-driven B cell IgG synthesis. Interestingly, anti-1A4 inhibited IL-2 secretion without affecting IL-2R expression. In addition, pretreatment of T cells with anti-1A4 inhibited the normally sustained intracellular calcium mobilization seen after triggering of T cells via the CD2 or CD3 pathways. Thus, binding of anti-1A4 to the CD27 molecule appears to induce a negative effect on T cell activation. This may be due to either a direct signal to T cells or the blocking of an interaction between T cells and accessory cells or both. These findings support the notion that the CD27 molecule plays an integral role in the process of T cell activation.     

A monoclonal antibody that detects a specific human neutrophil antigen involved in phorbol myristate acetate- and formyl-methionyl-leucyl-phenylalanine-triggered respiratory burst.

A mouse IgM mAb termed P1E3 was raised against resting human peripheral blood neutrophils and has been shown to recognize a cell-surface Ag with an apparent molecular mass of 155 kDa, as assessed by immunoprecipitation analysis. In addition to the main 155-kDa protein, an additional band of about 210 kDa was also recognized by P1E3 in Western blot analysis. Sequential immunoprecipitation assays showed that the Ag recognized by P1E3 differed from the CD29 and CD45 Ag. However, sequential immunoprecipitation assays carried out with two distinct anti-CD15 mAb and P1E3 showed that P1E3 reacted with CD15 or with a CD15-like Ag. P1E3 stained strongly resting human peripheral blood neutrophils, hardly reacted with peripheral blood monocytes and did not react with PBL and platelets, as assessed by immunofluorescence flow cytometry. P1E3 inhibited the respiratory burst induced by PMA or FMLP, but not the oxidative response induced by Con A or the calcium ionophores A23187 or ionomycin. Furthermore, P1E3 inhibited the activation of the Na+/H+ antiporter in response to PMA or FMLP and the phosphorylation of a protein of about 50 kDa in response to PMA. However, preincubation of neutrophils with P1E3 did not affect the increase in cytosolic free calcium concentration induced by FMLP. These data suggest that the Ag recognized by P1E3 may play a role in modulating the activation of the respiratory burst induced by PMA or FMLP, and that P1E3 seems to affect protein kinase C-mediated signal transduction mechanisms coupled to the induction of the respiratory burst.