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M López-Botet

Publications and source records attributed to M López-Botet.

At least 55 records · Page 3Linked to original sources
PubMed1996

Functional analysis of alpha 1 beta 1 integrin in human natural killer cells.

Upon activation with interleukin (IL)-2 human natural killer (NK) cells acquire on their surface the alpha 1 beta 1 and alpha 2 beta 1 integrins and down-regulate the expression of alpha 6 beta 1. By employing alpha 1 beta 1-specific monoclonal antibody (mAb) HP-2B6, characterized in our laboratory, we examined the functional role of the alpha 1 beta 1 integrin in NK cells. Treatment with HP-2B6 mAb partially interfered with attachment of cultured NK cells to type I collagen, and combined with an anti-alpha 2 beta 1 (TEA 1/41) mAb, it completely abrogated cell adhesion to this extracelular matrix protein. In contrast, NK cell attachment to laminin was completely blocked by the anti-beta 1 LIA 1/2 mAb, but was unaffected by alpha 1 and alpha 2-specific mAb; as alpha 3 beta 1 and alpha 6 beta 1 were undetectable, the data indicate that the alpha 1 beta 1 integrin binding sites for type I collagen and laminin are different. Incubation with anti-alpha 1 HP-2B6 or its F(ab')2 fragments specifically induced a rapid homotypic aggregation of NK cells that was dependent on active metabolism, an intact cytoskeleton and the presence of divalent cations (Ca2+ and Mg2+); homotypic cell adhesion was selectively blocked by anti-CD18, CD11a or CD54 mAb. In addition, stimulation of cultured NK cells with the anti-alpha 1 HP-2B6 enhanced TNF-alpha production and induced tyrosine phosphorylation of a 110-kDa protein. Pretreatment with specific inhibitors of protein tyrosine kinase (PTK) activity (tyrphostin 25 and herbimycin A) completely abrogated the functional effects induced by the anti-alpha 1 HP-2B6 mAb. Our data show that ligation of the alpha 1 beta 1 integrin positively modulates IL-2-activated NK cell function via a PTK-dependent pathway.

Animals↗
PubMed1996

NK-cell receptors and recognition of MHC class I molecules.

Two families of natural killer (NK)-cell receptors exist that interact with major histocompatibility complex (MHC) molecules, thereby preventing cytotoxic cells from killing target cells. The expanding knowledge of these receptors provided the focus for a recent meeting.

Animals↗
PubMed1996

Expression and function of alpha 4/beta 7 integrin on human natural killer cells.

In this report we have analysed the expression and function of the alpha 4/beta 7 heterodimer in human natural killer (NK) cells. The expression of alpha 4 beta 7 is induced in NK cells upon activation as the anti alpha 4 beta 2 ACT-1 monoclonal antibody (mAb) family stained a minority of peripheral blood NK cells, whereas it strongly reacted with in vitro long-term interleukin-2 (IL-2)-activated NK cells. Incubation with ACT-1 on its F(ab) fragments induced a strong homotypic adhesion of NK cells, comparable to than stimulated by the anti-alpha 4 HPI 7 mAb. Cell cell interaction induced by the ACT-1 mAb was only prevented by another anti-alpha 4 mAb (HP2.1) that recognizes a different epitope. In alpha 4 beta 7-mediated cell aggregation the alpha 4 beta 7 heterodimer was redistributed to intercellular contact sites thus, suggesting a direct involvement of this integrin in the formation of cell clusters. In NK cells attached to Fibronectin (FN38) or vascular cell adhesion molecule-1 (VCAM-1), both alpha 4 beta 7 and alpha 4 beta 7 integrins were redistributed at the ventral cellular membrane forming discrete contact sites. The ACT-1 mAb only partially blocked NK cell binding to FN38, but in combination with the anti-beta 7 mAb LIAI 2, NK cell binding to FN38 was completely inhibited. In contrast. ACT-1 did not modify NK cell adhesion to VCAM-1 thus supporting the theory that the alpha 4 beta 7 binding sites for both ligands appear to be different. Our results indicate that upon IL-2-activation, expression of functional alpha 4 beta-integrin is induced on NK cells potentially participating in their interaction with both extracellular matrix and endothelial cells.

Cell Adhesion↗
PubMed1995

Functional ambivalence of the Kp43 (CD94) NK cell-associated surface antigen.

We originally characterized the Kp43 (CD94) surface Ag, whose expression is restricted to human NK cells and a minor T lymphocyte subset. As shown in the present study, anti-Kp43 mAbs but not their F(ab')2 fragments markedly inhibited the cytolytic activity of polyclonal-activated NK cells in a redirected lysis assay against the murine P815 cell line. Furthermore, anti-Kp43 mAbs also down-regulated the CD16-dependent redirected killing and PHA-induced lectin-dependent cellular cytotoxicity. However, the intensity of the inhibitory effect was variable and no significant down-regulation of cytotoxicity was substantiated in NK cell populations from some individuals. A similar variability in the responsiveness to anti-Kp43 mAb was noticed when fresh CD3- lymphocyte populations were tested: in some donors we observed the induction of redirected lysis, whereas in other samples the Kp43-specific mAb inhibited cytotoxicity. The analysis of single cell-derived microcultures provided a clue to interpret the variable responsiveness of polyclonal cell populations; remarkably, the cytolytic activity of some NK clones was inhibited, whereas that of others was either stimulated or unaffected. The pattern of responsiveness in the cytolytic assay correlated with TNF production upon stimulation with solid phase-bound anti-Kp43 mAbs. The different types of clones could be derived from the same individual, although their relative proportions varied from donor to donor. Moreover, Kp43 appeared to be coupled differently to signal transduction pathways, because (Ca2+)i mobilization in the presence of the Kp43-specific mAbs was substantiated only in clones that were activated in the redirected lysis assay.

Antibodies, Monoclonal↗
PubMed1995

The NKB1 and HP-3E4 NK cells receptors are structurally distinct glycoproteins and independently recognize polymorphic HLA-B and HLA-C molecules.

NK cells lyse hematopoietic cells that lack expression of MHC class I molecules on the cell surface. Transfection of certain MHC class I negative cell lines with MHC class I genes renders these cells resistant to NK cell-mediated cytotoxicity. Recently, we described an NK cell receptor, NKB1, that inhibits NK cells from killing target cells expressing Bw4-reactive HLA-B molecules (-B*2705, -B*5101, -B*5801). In this study, we have demonstrated that another structurally distinct NK cell membrane glycoprotein, HP-3E4, is involved in the recognition of certain polymorphic HLA-C molecules (-Cw*0401 and -Cw*1503). NK cell clones co-expressing both the NKB1 and HP-3E4 receptors fail to lyse targets expressing HLA-Cw*0401 and -B*5801, but are able to kill the transfectants in the presence of mAbs against both receptors. These studies demonstrate that a single NK cell clone may possess multiple structurally distinct receptors for different polymorphic HLA class I molecules that function independently.

Adult↗
PubMed1995

Molecular characterization of human CD94: a type II membrane glycoprotein related to the C-type lectin superfamily.

Natural killer (NK) cells preferentially express several genes of the C-type lectin superfamily which have been implicated in the regulation of NK cell function. We demonstrate that CD94 is a type II membrane protein encoded by a unique gene of the C-type lectin superfamily. While homology of CD94 with the NK cell-associated NKR-P1 and NKG2 C-type lectin genes is limited to the structural motifs conserved in the carbohydrate recognition domain, all of these genes are on human chromosome 12, the syntenic of mouse chromosome 6, where genes of the NK complex (NKR-P1 and Ly-49) are located. An unexpected feature of CD94 is the essential absence of a cytoplasmic domain, implying that association with other receptors may be necessary for the function of this molecule.

Amino Acid Sequence↗
PubMed1994

Tyrosine kinase-dependent activation of human NK cell functions upon stimulation through a 58-kDa surface antigen selectively expressed on discrete subsets of NK cells and T lymphocytes.

We have raised a mAb, termed HP-3E4 (IgM), by immunizing BALB/c mice against human IL2-activated NK cells. The HP-3E4 mAb recognized in different donors variable proportions (< 2-70%) of either fresh or activated NK cells. A small population of T cells (alpha/beta and gamma/delta) appeared HP-3E4+ in PBL. No reactivity was detected on other leukocytes and a panel of cell lines from different lineages. By immunohistochemical staining of different tissues, few HP-3E4+ cells were detected only in lymphoid organs. Analysis of CD56+CD16+CD3- clones (n = 167) from unrelated donors (n = 6), showed that the Ag was stably expressed on 8 to 70%, moreover it was detected on some gamma/delta + T cell clones, whereas all CD3+ alpha/beta +(CD4+ and CD8+) clones analyzed (n = 90) were HP-3E4-. As assessed by SDS-PAGE analysis, the HP-3E4 mAb immunoprecipitated a 58-kDa surface structure. When compared with two mAbs (GL183 and EB6) previously reported to bind also a clonally distributed 54- to 58-kDa Ag, the HP-3E4 mAb appeared to recognize a distinct epitope, thus allowing to further define NK cell subsets. Stimulation of IL2-activated NK cells with the mAb triggered TNF-alpha and IFN-gamma production, which was enhanced by using the mAb attached to plastic or in the presence of suboptimal concentrations of phorbol esters. Although the HP-3E4 mAb did not significantly modify NK cell-mediated cytotoxicity against different targets, with the exception of the Hmy-C1R cell line, it activated BLT esterase secretion. Remarkably, the HP-3E4 mAb triggered phosphoinositide turnover and an early increase of [Ca2+]i, as well as tyrosine phosphorylation of several cellular substrates including CD3 zeta; inhibition of tyrosine kinase activity with genistein hampered the HP-3E4-mediated stimulation of cytokine production. Our data provide further support for the structural diversity of a 58-kDa surface Ag, whose expression is restricted to discrete NK and T cell subsets. Moreover, the results support the fact that the molecule plays an active role in regulating NK cell functions through signal transduction mechanisms comparable to those triggered via Fc gamma RIII.

Antibodies, Monoclonal↗
PubMed1993

Stimulation of IL-2-activated natural killer cells through the Kp43 surface antigen up-regulates TNF-alpha production involving the LFA-1 integrin.

We have previously described that a mAb directed against a surface dimer (Kp43) expressed by NK cells was able to regulate cell proliferation, enhance the cytotoxicity of IL-2-activated NK cells, and activate phospholipase D. In this work we have analyzed the ability of the anti-Kp43 mAb to regulate the production of TNF-alpha. Our results show that the stimulation of IL-2-activated NK cells with soluble anti-Kp43 mAb or its F(ab')2 activated the secretion of the cytokine, inasmuch as this effect is associated with the induction of cellular aggregation. The intensity of the Kp43-mediated stimulation varied among different IL-2-activated NK cell samples. Even in those instances where the anti-Kp43 mAb alone could not detectably enhance TNF-alpha production, it displayed a synergistic effect combined to a soluble anti-CD16 mAb, which on its own did not efficiently activate cytokine production. The anti-Kp43 mAb also cooperated with a phorbol ester, although it did not modify the TNF-alpha production triggered by a Ca2+ ionophore. The anti-Kp43-mediated effect, which required the preactivation of cells with IL-2, was inhibited by cycloheximide and actinomycin D and was associated with an increase in the levels of TNF-alpha-specific mRNA. It is noteworthy that the Kp43-mediated production of TNF-alpha was partially inhibited by anti-CD18, anti-CD11a and anti-ICAM-1 mAb that blocked LFA-1-dependent cellular interactions, which impaired NK cell aggregation and, moreover, was dependent on the presence of extracellular Mg2+, thus suggesting that the leukocyte integrin is involved in the activation process triggered through Kp43.

Antibodies, Monoclonal↗
PubMed1993

A novel functional cell surface dimer (kp43) serves as accessory molecule for the activation of a subset of human gamma delta T cells.

Kp43 is a cell surface molecule expressed by NK cells, gamma delta T lymphocytes, and a small subset of CD56+ alpha beta T cells. In the present study the distribution and function of Kp43 on gamma delta T cells is described. The expression of Kp43 on freshly isolated gamma delta T-cell populations derived from fetal and adult tissues was biased toward memory V gamma 2+/V delta 2+ T lymphocytes and appeared to be up-regulated after activation. Several gamma delta T-cell clones were derived from different sources. Adult V gamma 2+/V delta 2+ T-cell clones expressed the highest levels of Kp43, V delta 1 T-cell clones derived from periphery expressed moderate levels, whereas some thymus V delta 1 T cells and fetal gamma delta T-cell clones displayed low or undetectable levels of Kp43 molecule. Based on the effect of a specific mAb, this dimer appears to be fully functional as previously shown for NK cells. The stimulation of Kp43+ gamma delta T-cell clones with a combination of anti-CD3 and anti-Kp43 co-immobilized on a solid substratum enhanced both the expression of cell surface CD25 and the secretion of TNF-alpha. The restricted expression and functional activity of the Kp43 dimer suggests that it may play an important regulatory role in activation, selection, Ag recognition, and responsiveness of a subset of gamma delta T cells.

Antigen-Presenting Cells↗
PubMed1993

Signaling through the LFA-1 leucocyte integrin actively regulates intercellular adhesion and tumor necrosis factor-alpha production in natural killer cells.

The LFA-1 leucocyte integrin is known to participate in natural killer (NK) cytolytic activity, mediating effector target interactions. The possibility that LFA-1 may also play an active regulatory role in NK cells has been explored. To this end, we have employed a monoclonal antibody (HP1N) raised against recombinant interleukin-2 (rIL-2)-activated NK cells, which recognizes the alpha chain of the LFA-1 heterodimer (CD11a). In contrast to other anti-CD11a mAb the HP1N and its F(ab')2 fragment did not affect NK cell-mediated cytotoxicity and triggered a strong homotypic adhesion of NK cells and other LFA-1+ cells. Cellular aggregation was inhibited by anti-CD18 mAb, anti-ICAM-1 mAb, and other anti-CD11a mAb. Remarkably, the HP1N mAb was also shown to induce tumor necrosis factor-alpha (TNF-alpha) production from NK cells upon costimulation with anti-CD16 mAb. Such an effect appeared to be independent from homotypic adhesion since it took place in Mg(2+)-free medium, where NK cell aggregation was inhibited. Moreover, incubation with the HP1N mAb triggered a Ca2+ influx into the cytosol; this effect was clearly observed upon cross-linking of cell bound HP1N and was also substantiated with other anti LFA-1 (CD11a and CD18) mAb. Taken together these results indicate that the LFA-1 molecule is capable of transducing signals in NK cells, which regulate the intercellular interaction with its ligand, and enhance the activation via Fc gamma receptor type III.

Antibodies, Monoclonal↗
PubMed1993

Costimulation of cAMP and protein kinase C pathways inhibits the CD3-dependent T cell activation and leads to a persistent expression of the AP-1 transcription factor.

The effects mediated by a combined stimulation of cAMP- and protein kinase C (PKC)-dependent pathways have been investigated in different cellular systems, and it has been shown that they may complement each other in activating cell proliferation and differentiation. In this report, we show that upon the stimulation of both pathways T lymphocytes became refractory to activation via the CD3/T cell receptor (TcR) complex. T cells preincubated with phorbol 12-myristate 13-acetate (PMA) and dibutyryl cAMP (Bt2cAMP) displayed a deficient proliferative ability in response to anti-CD3 mAb stimulation, whereas lymphocytes treated individually with either Bt2cAMP or PMA responded comparably to untreated samples. We detected an association between the reduced mitogenic response and low expression of both interleukin-2 (IL-2) and the alpha chain (CD25) of the IL-2 receptor (IL-2R). Analysis of intracellular Ca2+ mobilization suggested that the CD3/TcR-dependent signal transduction was impaired in PMA/Bt2cAMP-treated cells. Remarkably, we observed that these samples displayed a persistent expression of the c-fos protooncogene, associated to an increased AP-1 DNA-binding activity, whereas no variations of CREB or NF-kB were detected. Neither Bt2cAMP nor PMA individually mediated these sustained effects, which therefore appear as a consequence of the interplay between both metabolic stimuli. Altogether, the data provide the evidence that both pathways complement each other in regulating gene expression and, conversely, downregulate the TcR transduction mechanisms.

Base Sequence↗
PubMed1993

Expression of lymphocyte activation surface antigens in bronchoalveolar lavage and peripheral blood cells from young healthy subjects.

Cell surface phenotypes of bronchoalveolar lavage (BAL) and peripheral blood lymphocytes (PBL) obtained from a group of young healthy volunteers, including smokers and nonsmokers were studied. Results indicate that the proportions of T (CD3+) and B (CD19+) lymphocytes were comparable in PBL and BAL lymphocytes, whereas the numbers of cells bearing natural killer cell-associated markers (CD16, CD11b, CD56, and CD57) were significantly higher in PBL; in BAL lymphocytes of smokers, the CD4+/CD8+ ratio was < 1. The surface density of CD3 in every case was lower in BAL T cells. The expression of different surface antigens considered to reflect lymphocyte activation was variable; the human lymphocyte antigen (HLA)-DR, CD25 (alpha chain of the interleukin-2 receptor), and CD71 (transferrin receptor) were displayed at low levels in both types of samples. In contrast, the expression of 4F2, CD49a, and particularly, the early activation antigen CD69 were significantly higher in BAL cells compared with PBL; BAL samples from smokers included significantly lower proportions of lymphocytes bearing the CD69, HLA-DR, and 4F2 activation antigens.

Adult↗
PubMed1993

Variability in the expression of a beta 2-microglobulin epitope on hepatocytes in chronic type C hepatitis on treatment with interferon.

Cytotoxic CD8+ T lymphocytes recognize viral antigens in the context of human leukocyte antigen class I molecule coexpression by target cells. Analysis of beta 2-microglobulin reactivity is useful in evaluating changes in human leukocyte antigen class I antigen distribution. In this study we analyzed liver biopsy specimens obtained from 15 patients with chronic active hepatitis type C who underwent a clinical trial with recombinant interferon-alpha 2b. We comparatively studied by immunohistochemical analysis the expression of human leukocyte antigen class I antigens in frozen liver samples obtained before entry in the protocol and in specimens taken 8 mo after initiation of treatment. Six normal liver samples were used as controls. For immunohistochemical analysis, a panel of several human leukocyte antigen class I monoclonal antibodies, specific for beta 2-microglobulin or different heavy-chain determinants, was used. In addition, we included a novel monoclonal antibody (HP-1H8), characterized in this report, which is specific for a distinct beta 2-microglobulin epitope. On entry, mean serum ALT was 240 +/- 89 IU/L and mean Knodell's index was 9.9 +/- 2.4, whereas at the time of the second biopsy mean values had diminished to 45 +/- 22 IU/L and 4.7 +/- 3.0, respectively. Liver sections from controls and patients expressed human leukocyte antigen class I light- and heavy-chain determinants in hepatocytes, biliary duct epithelium, sinusoidal lining cells and lymphocytes. Remarkably, the beta 2-microglobulin epitope recognized by the HP-1H8 monoclonal antibody was undetectable on hepatocytes from normal livers but clearly evident on hepatocytes from patients with chronic active hepatitis C before interferon treatment. Positive staining was more intense in areas of piecemeal and lobular necrosis. Double immunostaining with a CD2 monoclonal antibody demonstrated that labeling with HP-1H8 was predominantly associated with T-cell infiltration. Interestingly, the reactivity of HP-1H8 with hepatocytes was diminished or disappeared in specimens obtained during interferon treatment; the pattern of reactivity then resembled that of samples from normal controls. Our data indirectly suggest that, in addition to the increased expression of human leukocyte antigen class I molecules on hepatocytes in viral infections, conformational changes may take place in these antigens. These changes can be revealed by immunostaining with the HP-1H8 monoclonal antibody. Interferon therapy could down-regulate this expression through its effect in reducing the histological activity resulting from the lysis of virus-infected hepatocytes by cytotoxic T cells.

Adult↗
PubMed1992

Cyclic AMP and calcium regulate at a transcriptional level the expression of the CD7 leukocyte differentiation antigen.

CD7 is a 40-kDa cell surface glycoprotein expressed on T-cell precursors before their entry into the thymus during fetal development and whose functional role remains uncertain. T-cell activation has been shown to increase the expression of this surface molecule. In this report we describe the intracellular signals and the mechanisms involved in the regulation of CD7 antigen expression on human T lymphocytes. The elevation of intracellular calcium by using the A23187 ionophore increased the cell surface expression of CD7, whereas protein kinase C activation caused its down-regulation. Interestingly, the increase of intracellular cAMP with Bt2cAMP stimulated CD7 expression as well. Upregulation of CD7 on the cell surface following either Bt2cAMP or calcium ionophore stimulation of T lymphocytes correlated with a raise of the steady-state levels of CD7-specific mRNA, without de novo protein synthesis requirements. No differences between the half-life of basal CD7 mRNA and that induced by either Bt2cAMP or calcium ionophore were detected. Run-on experiments showed that both stimuli enhanced the transcriptional rate of the CD7 gene. Our results provide the evidence for a positive regulatory effect mediated by cAMP on the expression of a leucocyte differentiation antigen.

Antigens, CD↗
PubMed1992

Phospholipase D activation in human natural killer cells through the Kp43 and CD16 surface antigens takes place by different mechanisms. Involvement of the phospholipase D pathway in tumor necrosis factor alpha synthesis.

We have recently described a novel glycoprotein, Kp43, expressed on the surface of human natural killer (NK) cells that appears to regulate their functional activity. In this report, signaling mechanisms through the Kp43 surface antigen have been studied. Incubation of interleukin 2 (IL-2)-treated NK cells with anti-Kp43 monoclonal antibody F(ab')2 fragments resulted in the time- and dose-dependent stimulation of NK cell phospholipase D. Phospholipase D activation through the Kp43 surface antigen was found to take place in the absence of polyphosphoinositide turnover and appeared not to depend on the presence of Ca2+ in the extracellular medium. On the other hand, signaling mechanisms through the CD16 receptor (FcR-III) on NK cells were comparatively studied. Stimulation of IL-2-treated NK cells with anti-CD16 monoclonal antibody F(ab')2 fragment also resulted in time- and dose-dependent activation of phospholipase D. However, CD16-triggered phospholipase D activation took place concomitant to phospholipase C-mediated polyphosphoinositide breakdown and showed a strong dependence on extracellular Ca2+. These results provide, to our knowledge, the first evidence for the presence of activatable phospholipase D in NK cells, as well as the first indication that distinct receptor-modulated pathways exist for activation of phospholipase D within the same cell type. On the other hand, phosphatidic acid, the physiologic product of phospholipase D action on phospholipids, was found to mimic the effect of anti-Kp43 monoclonal antibody regarding tumor necrosis factor alpha (TNF-alpha) biosynthesis and secretion by NK cells. Addition of phosphatidic acid vesicles to IL-2-treated NK cell cultures stimulated a TNF-alpha production that was abolished when the cells were previously treated with actinomycin D. Other phospholipids, including lysophosphatidic acid, were ineffective. However, phosphatidic acid-induced TNF-alpha production was strongly inhibited by the presence of propranolol, an inhibitor of phosphatidic acid phosphohydrolase. Moreover, in cells responding to phorbol myristate acetate, a compound that triggers activation of phospholipase D, TNF-alpha synthesis was also inhibited by propranolol. Thus, these data suggest a second messenger role for phosphatidic acid-derived diradylglycerol in the induction of TNF-alpha gene expression.

Antigens, Differentiation↗
PubMed1992

Functional analysis of peripheral blood lymphocytes isolated from patients with chronic hepatitis type B.

Cell-mediated immunity, evaluated by lymphocyte proliferation and expression of the activation antigen interleukin-2 receptor in response to mitogens such as phytohemagglutinin and concanavalin-A, has been reported to be defective in chronic hepatitis B virus carriers. However, no definite conclusion on the functional state of T cells from these patients can be drawn. In the present study, we have investigated the expression of a wide set of lymphoid activation molecules as well as the proliferative response of peripheral blood lymphocytes isolated from patients with chronic hepatitis type B after in vitro stimulation with monoclonal antibodies to both the T-cell receptor-CD3 complex and the CD2 molecule, which are the two main T-cell activation pathways. Our findings show that peripheral T lymphocytes from patients with chronic hepatitis type B express the activation antigens 4F2 molecule, interleukin-2 receptor, and activation inducer molecule (AIM) antigen, and proliferate normally after specific stimulation through either the T-cell receptor-CD3 complex or the CD2 molecule. These results suggest that the peripheral blood T cells of patients with chronic hepatitis B are fully operative and functionally competent in vitro.

Adult↗
PubMed1992

Analysis of different protein kinase C-dependent events in T cells from allogeneic bone marrow transplantation recipients.

In order to understand the mechanisms underlying the T lymphocyte dysfunction associated to allogeneic bone marrow transplantation (BMT), we assessed two different protein kinase C (PKC) dependent events in T cells from BMT recipients: the PKC-dependent membrane expression and function of the CD69 early activation antigen; and the rapid phorbol ester-induced phosphorylation of PKC protein substrates in lysates from T cells permeabilized with digitonin, in the presence of (gamma-32P)ATP. Most BMT recipient T cells detectably expressed the CD69 surface antigen after 24 h of stimulation with either phorbol 12-myristate 13-acetate (PMA) or anti-CD3 MoAb and PMA, thus indicating that PKC activity is sufficient to induce de novo gene expression. Nevertheless, it is noteworthy that the fluorescent staining intensity with anti-CD69 MoAbs was significantly lower in BMT recipient T cells than in normal T lymphocytes, although no clear-cut correlation was found between the expression of CD69 and the proliferative capacity. However, the pattern of PMA-induced phosphoproteins analysed as early as 1 min after PKC activation in T cells from BMT recipients displaying a low response to mitogenic stimuli, was undistinguishable from that detected in T cells from healthy subjects. In all cases a major 110-kD phosphoprotein was observed, which was inducible with PMA, phorbol 12,13-dibutyrate (PDBu), 1-oleoyl-2-acetylglycerol (OAG) and a phorbol-ester-related activator of PKC (mezerein); moreover, its phosphorylation was blocked by pretreating cells with the PKC inhibitor H-7. Altogether our results suggest that the depressed mitogenic responses, which were also observed in the present study when T cells were stimulated via CD69, cannot be simply attributed to a defective PKC activity.

Antigens, CD↗