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Biomedical subjects

R Galandrini

Publications and source records attributed to R Galandrini.

At least 19 recordsLinked to original sources
PubMed2001

The adaptor protein shc is involved in the negative regulation of NK cell-mediated cytotoxicity.

The activation of protein tyrosine kinase(s) (PTK) is a critical event required for the development of NK cell-mediated cytotoxicity. Here we demonstrate that the adaptor protein shc undergoes tyrosine phosphorylation during the generation of antibody-dependent cellular cytotoxicity (ADCC) and natural killing. In addition, we report that, upon direct or antibody-dependent target cell interaction, shc coprecipitates with the Src homology 2 (SH2)-containing inositol phosphatase, SHIP. To gain information on the functional role of shc in NK cytotoxicity, we overexpressed wild-type or dominant negative shc constructs in the human NKL cell line. Our findings show a consistent shc-mediated down-regulation of ADCC and natural killing. Such functional effect correlates with a perturbation of the phosphoinositide (PI) metabolism by means of a shc-mediated negative regulation of inositol 1,4,5 triphosphate (IP3) generation and intracellular calcium flux upon CD16 ligation. Furthermore, our data show that dominant-negative shc-mediated perturbation of shc/SHIP interaction leads to inhibition of ligand-dependent SHIP recruitment to CD16 zeta chain. We suggest that shc plays a role of negative adaptor by modulating SHIP recruitment to activation receptors involved in the generation of NK cytotoxic function.

Adaptor Proteins, Signal Transducing↗
PubMed2000

The GTPase rho controls a p53-dependent survival checkpoint during thymopoiesis.

During the early stages of thymopoiesis, cell survival is controlled by cytokines that regulate the expression of antiapoptotic proteins such as Bcl-2. At the pre-T cell stage, a critical checkpoint for beta chain selection is monitored by the tumor suppressor p53: pre-T cells can survive and differentiate when p53 is removed genetically or when its proapoptotic function is inactivated physiologically as a consequence of signaling through the pre-T cell receptor complex. Previous work has shown that the guanine nucleotide binding protein Rho controls cell survival in T cell progenitors. Here we define the survival pathways controlled by Rho in pre-T cells and show that this GTPase is a pivotal regulator of the p53-mediated checkpoint operating at the time of beta selection: loss of Rho function results in apoptosis in pre-T cells, but this cell death is prevented by loss of p53. The prevention of cell death by loss of p53 restored numbers of early T cell progenitors but did not fully restore thymic cellularity. Further analysis revealed that loss of Rho function caused survival defects in CD4/8 double-positive thymocytes that is independent of p53 but can be prevented by ectopic expression of Bcl-2. These studies highlight that the GTPase Rho is a crucial component of survival signaling pathways in at least two different thymocyte subpopulations: Rho controls the p53 survival checkpoint in pre-T cells and is also crucial for a p53 independent survival signaling pathway in CD4/8 double positives.

ADP Ribose Transferases↗
PubMed1999

Role for the Rac1 exchange factor Vav in the signaling pathways leading to NK cell cytotoxicity.

Here we investigate the activation of and a possible role for the hematopoietic Rac1 exchange factor, Vav, in the signaling mechanisms leading to NK cell-mediated cytotoxicity. Our data show that direct contact of NK cells with a panel of sensitive tumor targets leads to a rapid and transient tyrosine phosphorylation of Vav and to its association with tyrosine-phosphorylated Syk. Vav tyrosine phosphorylation is also observed following the activation of NK cells through the low-affinity Fc receptor for IgG (Fc gamma RIII). In addition, we demonstrate that both direct and Ab-mediated NK cell binding to target cells result in the activation of nucleotide exchange on endogenous Rac1. Furthermore, Vav antisense oligodeoxynucleotide treatment leads to an impairment of NK cytotoxicity, with Fc gamma RIII-mediated killing being more sensitive to the abrogation of Vav expression. These results provide new insight into the signaling pathways leading to cytotoxic effector function and define a role for Vav in the activation of NK cell-mediated killing.

Animals↗
PubMed1997

Selective binding of shc-SH2 domain to tyrosine-phosphorylated zeta but not gamma-chain upon CD16 ligation on human NK cells.

Fc gamma RIII (CD16) is a hetero-oligomeric receptor composed of a ligand-binding alpha subunit associated with homo- or heterodimers of the TCR zeta- and Fc epsilon RI gamma-chains. We have previously demonstrated that CD16 ligation promotes complex formation between tyrosine-phosphorylated shc and Grb2, leading to activation of ras signaling pathway in human NK cells. Here we report that CD16 engagement induces rapid shc association with the tyrosine-phosphorylated receptor complex in human NK cells. In vitro binding studies demonstrate that this interaction is mediated by the shc-SH2 domain, and immunodepletion experiments indicate that the zeta- but not the gamma-chain has the capability to mediate this association. Jurkat cell clones expressing CD16-zeta or -gamma homodimers have been used to gain more information about the mechanism of shc/CD16 association. Our data show that, while engagement of both receptors induces tyrosine phosphorylation of shc and Grb2 recruitment, shc-SH2/receptor complex association is evident only in CD16-zeta but not in CD16-gamma transfectants. Overall, our data demonstrate that the adaptor protein shc can be recruited to the activated CD16 complex by interaction with tyrosine-phosphorylated zeta-chain in a SH2-dependent manner. These results also provide further support to the notion that zeta- and gamma-chains might couple to different biochemical pathways.

Cells, Cultured↗
PubMed1997

The GTPase Rho has a critical regulatory role in thymus development.

The present study employs a genetic approach to explore the role of Rho GTPases in murine thymic development. Inactivation of Rho function in the thymus was achieved by thymic targeting of a transgene encoding C3 transferase from Clostridium botulinum which selectively ADP-ribosylates Rho within its effector domain and thereby abolishes its biological function. Thymi lacking functional Rho isolated from C3 transgenic mice were strikingly smaller and showed a marked (90%) decrease in cellularity compared with their normal litter mates. We also observed a similar decrease in levels of peripheral T cells in C3 transgenic mice. Analysis of the maturation status of thymocytes indicated that differentiation of progenitor cells to mature T cells can occur in the absence of Rho function, and both positive and negative selection of T cells appear to be intact. However, transgenic mice that lack Rho function in the thymus show maturational, proliferative and cell survival defects during T-cell development that severely impair the generation of normal numbers of thymocytes and mature peripheral T cells. The present study thus identifies a role for Rho-dependent signalling pathways in thymocyte development. The data show that the function of Rho GTPases is critical for the proliferative expansion of thymocytes. This defines a selective role for the GTPase Rho in early thymic development as a critical integrator of proliferation and cell survival signals.

ADP Ribose Transferases↗
PubMed1997

Different functions of the GTPase Rho in prothymocytes and late pre-T cells.

Mice lacking thymic function of the GTPase Rho show severe defects in fetal and adult thymopoiesis. Rho thymi are deficient in CD44+ CD25+ pro-T cells and CD44- CD25+ early pre-T cells because Rho function is required for survival but not G1/S phase cell cycle progression in these populations. The selective apoptosis defect in Rho prothymocytes can be rescued by expression of a bcl-2 transgene. A second function for Rho is seen in CD44- CD25- late pre-T cells: Rho regulates cell cycle progression but not survival of this population. These studies show that the critical processes of proliferation and survival are independently regulated during thymopoiesis and establish two different functions for Rho in the development of early thymic progenitors.

Aging↗
PubMed1996

CD16-mediated p21ras activation is associated with Shc and p36 tyrosine phosphorylation and their binding with Grb2 in human natural killer cells.

The Src homology (SH) 2/SH3 domain-containing protein Grb2 and the oncoprotein Shc have been implicated in a highly conserved mechanism that regulates p21ras activation. We investigated the involvement of these adaptor proteins in the signaling pathway induced by CD16 or interleukin (IL) 2R triggering in human natural killer (NK) cells. Both p46 and p52 forms of Shc were rapidly and transiently tyrosine phosphorylated upon CD16 or IL-2 stimulation with different kinetics. Shc immunoprecipitates from lysates of CD16- or IL-2-stimulated NK cells contained Grb2 and an unidentified 145-kD tyrosine phosphoprotein. Grb2 immunoprecipitates from anti-CD16-stimulated NK cells contained not only Shc, but also a 36-kD tyrosine phosphoprotein (p36). The interaction between Grb2 and Shc or p36 occurred via the Grb2SH2 domain as indicated by in vitro binding assays using a bacteriologically synthesized glutathione S-transferase-Grb2SH2 fusion protein. We also present evidence that p21ras is activated by CD16 and IL-2R cross-linking. Accumulation of guanosine triphosphate-bound Ras was detected within 1 minute and occurred with kinetics similar to inductive protein tyrosine phosphorylation and Grb2 association of Shc and p36 adaptor proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Tyrosine kinase-dependent activation of human NK cell functions upon triggering through CD44 receptor.

We recently showed evidence of CD44-mediated enhancement of natural killer (NK) cell cytotoxic activity and induction of intracellular Ca2+ flux. In this study, we evaluated whether CD44 plays a stimulatory role in NK cell functions, such as cytokine production and activation antigen expression. Our results indicate that ligation of the CD44 receptor results in the induction of expression of the CD69 surface activation antigen as well as in the enhancement of phorbol ester-induced TNF-alpha secretion. We report also evidence for the coupling of CD44 receptor to a protein tyrosine kinase(s) pathway. CD44 engagement rapidly stimulates the tyrosine phosphorylation of several cellular substrates. Pretreatment of NK cells with the tyrosine kinase inhibitor herbimycin A resulted in marked decrease of CD44-stimulated phosphorylation, indicating that it activates tyrosine kinase(s). Furthermore, the drug also prevents CD44-mediated TNF-alpha production and CD69 expression. These findings indicate that protein tyrosine phosphorylation is an early and critical event in CD44-mediated activation of NK cell functions.

Antigens, CD↗
PubMed1994

CD44 triggering enhances human NK cell cytotoxic functions.

CD44, a major hyaluronate receptor, is involved in a variety of lymphocyte functions including lympho-hemopoiesis, adhesion to high endothelial venules or the extracellular matrix, and T cell activation. Here we investigated the ability of CD44 to affect the cytotoxic functions of human NK cells. Ligation of CD44 by selected mAb (J173 and F10442) resulted in a rapid, dose-response-dependent enhancement of NK cytotoxic activity against a panel of tumor target cells that varied in their sensitivity to NK killing. Neither enhanced killing against NK-resistant target cells nor CD44 mAb-mediated redirected lysis was not observed. CD44 cross-linking also was found to up-regulate CD16-mediated lysis. In an attempt to investigate the early biochemical events that occur after CD44 ligation, we found that optimal cross-linking conditions induce a rapid increase of intracellular free calcium levels, which is abrogated by extracellular Ca2+ chelation. Moreover, enhanced and more sustained Ca2+ rise resulted from CD16 and CD44 coengagement. In contrast, no inositol 1,4,5-trisphosphate generation was found after optimal CD44 cross-linking. These results suggest that although CD44 is not capable of delivering a lytic signal in human NK cells, it coactivates spontaneous or CD16-mediated NK cytotoxicity. The variation in intracellular free calcium may be one of the signals that account for the costimulation of the lytic activity.

Antibodies, Monoclonal↗
PubMed1994

Hyaluronate is costimulatory for human T cell effector functions and binds to CD44 on activated T cells.

Lymphohematopoiesis, cell matrix adhesion, homing of leukocytes, T cell activation, and tumor metastasis are mediated through the CD44 family of cell surface receptors. We have recently shown that anti-CD44 mAb trigger protein tyrosine kinase-dependent activation of T cell effector functions. Here, we show that hyaluronate (HA), a CD44 ligand, in conjunction with CD3/TCR-mediated stimuli, is costimulatory for human peripheral blood T cell proliferation, for IL-2 production by Th clones, and for release of trypsin-like esterase by cytolytic T cell clones. A human T cell line, HUT-78, was found to bind HA and on HA coating it was used as a target for cytolytic T cell clones. After anti-CD3 stimulation, CD3+/CD8+ clones acquire the ability of lysing HA-coated HUT-78 cells more efficiently than the same HA-uncoated targets. Resting peripheral blood T cells and T cell clones do not adhere to HA-coated plates. However, 24-h anti-CD3 mAb stimulation gives them the transient ability to bind HA. HA adhesion of activated T cells and T cell clones, as well as that of T cell lines, is blocked by one anti-CD44 mAb (J-173). Two other anti-CD44 mAbs induce a 10-fold increase in HA adhesiveness of anti-CD3-stimulated peripheral blood T cells. This impressive HA adhesiveness is also readily blocked by J-173 anti-CD44 mAb. These data indicate that 1) HA is costimulatory for human T cell effector functions in conjunction with CD3/TCR-mediated stimuli, 2) the capacity to bind HA is acquired by resting T cells and T cell clones after anti-CD3 stimulation, and 3) HA binding occurs via specific interaction with CD44 molecules expressed on activated T cells.

CD4-Positive T-Lymphocytes↗
PubMed1993

Antibodies to CD44 trigger effector functions of human T cell clones.

mAb against the lymphocyte homing receptor CD44/Hermes up-regulate the proliferation of human T PBL induced by anti-CD3 or anti-CD2 mAb. Moreover, certain anti-CD44 mAb can activate human resting T cells and mouse cytotoxic T cells in the absence of anti-CD3 or anti-CD2 mAb. Here, we show that anti-CD44 mAb trigger proliferation of human CD3+/CD4+ T cell clones in a fashion similar to that observed with mAb to CD3. Such an effect is IL-2-dependent, as shown by IL-2 production induced by anti-CD44 mAb and by complete inhibition of cell proliferation in the presence of anti-IL-2 antibodies or cyclosporin A. Moreover, anti-CD44 mAb trigger human cytolytic T cell clones to lyse Fc gamma-R+ P815 cells in the absence of additional stimuli. The magnitude of the cytolytic response induced by anti-CD44 mAb is comparable to that observed in the presence of anti-CD3 mAb for both CD4+ and CD8+ TCR-alpha/beta+ clones, and for V delta 1 or V delta 2 TCR-gamma/delta+ clones. By contrast, in CD3-/CD16+ NK cell clones, no cytolytic responses to anti-CD44 mAb could be observed. Granule trypsin-like esterase enzyme (granzyme) release by cytolytic T cell clones is induced by plastic-immobilized anti-CD44 mAb. Anti-CD44 mAb-triggered proliferation ([3H]thymidine incorporation) and cytotoxicity are blocked by the protein tyrosine kinase inhibitor, genestein. In addition, ligation of the CD44 molecule induces tyrosine phosphorylation of proteins identical, by molecular mass, to those phosphorylated after anti-CD3 mAb stimulation. Notably, anti-CD44 mAb does not induce tyrosine phosphorylation of a 21-kDa protein (the phosphorylated zeta-chain of the TCR molecular complex) typically observed upon anti-CD3 mAb stimulation. In conclusion, this study shows that the ligated CD44 molecule provides the necessary stimuli for a variety of T cell-mediated functions triggered via protein tyrosine kinase-dependent signal transduction pathways at least in part similar to those that follow stimulation of the CD3/TCR complex.

CD4-Positive T-Lymphocytes↗
PubMed1993

Ligation of the lymphocyte homing receptor CD44 triggers T-helper and cytolytic functions of human T cells.

We show that antibodies to the CD44 molecule trigger proliferation of human CD3+/CD4+ T-cell clones. Such effect is IL2-dependent, as shown by IL2 production induced by anti-CD44 mAb and by inhibition of cell proliferation in the presence of anti-IL2 antibodies or cyclosporin A (CsA). Moreover, anti-CD44 mAb triggered human cytolytic CD4+ and CD8+ TCR alpha/beta+ clones, and V delta 1 or V delta 2 TCR Y/delta+ clones to lyse Fc-gamma-R+ P815 cells and to release granule trypsin-like esterase enzymes. Anti-CD44 mAb-triggered proliferation and cytotoxicity were blocked by the PTK-inhibitor, genestein. In addition, ligation of the CD44 molecule induced tyrosine phosphorylation of proteins identical, by molecular weight, to those phosphorylated following anti-CD3 mAb-stimulation. Notably, anti-CD44 mAb does not induce tyrosine phosphorylation of a 21 kD protein (the phosphorylated zeta chain of the TcR molecular complex) typically observed upon anti-CD3 mAb stimulation.

Antibodies, Monoclonal↗
PubMed1993

Residual clonable host cell detection for predicting engraftment of T cell depleted BMTs.

Rejection of T-depleted BMTs is predominantly mediated by alloreactive host T cells. A low but significant number of radiochemoresistant clonable T cells can be detected following a conventional cytoreductive protocol given prior to T-depleted BMT. Elimination of these cells increases the engraftment rate. We found no clonable T cells at the end of the conditioning regimen in 100 ml of peripheral blood from 47 patients who received an HLA-identical T-depleted BMT. None rejected the graft and none displayed mixed chimerism. In addition, although no clonable T cells were detected in nine patients who received a mismatched BMT, two rejected their graft. However, in three mismatched patients, who for clinical reasons received a modified pre-BMT schedule, the presence of host clonable T cells was associated with immunological rejection. These findings suggest that the detection of clonable T cells should prove a valuable indicator for optimising immunosuppression prior to T-depleted BMT.

Adolescent↗
PubMed1992

Adhesion molecule-mediated signals regulate major histocompatibility complex-unrestricted and CD3/T cell receptor-triggered cytotoxicity.

Appropriate experimental conditions were devised to demonstrate that CD58 (LFA-3), CD54 (ICAM-1) and CD11a/CD18 (LFA-1) adhesion molecules are the source of signals that regulate nonspecific major histocompatibility complex-unrestricted and CD3/T cell receptor (TcR)-triggered cytotoxicity. Using anti-LFA-3 monoclonal antibody (mAb)-treated, interleukin-2 (IL-2)-cultured peripheral blood lymphocytes (PBL) or cloned CD3+/CD8+ cells as lymphocyte-activated killer (LAK) effectors, and ligand (CD2)-negative tumor cell lines as targets, a down-regulation of CD3- and CD3+ cell-mediated LAK activity was consistently observed. Anti-LFA-3 mAb also down-regulated tumor cell lysis when T cell clones were triggered to kill P815 cells through stimulation of the CD3/TcR complex by an anti-CD3 mAb. The inhibitory effect of anti-LFA-3 mAb was not prevented by stimulatory anti-CD2 mAb. Anti-ICAM-1 mAb treatment of IL-2-cultured PBL consistently up-regulated LAK cytotoxicity against tumor target cells. However, this effect was only exerted on CD3- LAK effectors. Anti-LFA-1 mAb blocked conjugate formation between effector cells and tumor target cells, thus rendering this model unsuitable to evaluate the regulatory role of LFA-1. Therefore, a cytotoxicity model system was applied in which a hybrid anti-CD3/anti-human red blood cell (HuRBC) mAb triggers cytolytic T cells to lyse HuRBC. In these experiments, anti-LFA-1 mAb markedly up-regulated the lytic ability of IL-2-cultured PBL. We conclude that mAb against LFA-3, ICAM-1 and LFA-1 molecules deliver regulatory signals for LAK cells and cytotoxic T lymphocytes. As these stimuli may be delivered by ligands expressed on tumor targets as well as on other immune competent and inflammatory cells, the present observations are relevant in the context of both the host's immune response against tumors and the general functioning of the immune system.

Antibodies, Monoclonal↗
PubMed1992

Antibodies to adhesion molecules inhibit the lytic function of MHC-unrestricted cytotoxic cells by preventing their activation.

We evaluated the effect of the antibodies to adhesion molecules CD2, CD11a/CD18 (LFA-1), and CD56 (N-CAM) on MHC-unrestricted cytotoxicity mediated by polyclonal NK cells and LAK cells or by CD3+ or CD3- cytolytic cell clones against a panel of tumor cell targets selected according to expression or absence of the corresponding ligands. We show that (i) antibodies to CD11a/CD18 and, to a lesser extent, antibodies to CD2 inhibit target cell lysis, whereas anti-CD56 antibodies exert little if any effect; (ii) in a model system using polyclonal NK/LAK cells as effectors and K562 or HL60-R (NK-resistant) cells as targets, inhibition of cytotoxicity occurs without a significant impairment of effector to target cell binding; (iii) the cytotoxic function of CD3+ or CD3- cytotoxic cell clones is inhibited differentially by antibodies to adhesion molecules; (iv) conjugates formed in the presence of antibodies which inhibit target cell lysis display a significant reduction of target to effector cell contact surface; and (v) this may lead to defective activation of effector cells, as indicated by lack of redistribution of the microtubular apparatus. We conclude that (i) MHC-unrestricted cytotoxicity is regulated by a number of molecular interactions that span far beyond our present knowledge and that it is strictly dependent on the surface phenotype of the effector cell and of the target cell; (ii) in certain types of effector/target cell interactions, antibodies to adhesion molecules do not prevent conjugate formation but reduce the extent of cell-to-cell surface contact which, in turn, leads to defective activation of the effector cell and, therefore, to inhibition of target cell lysis.

Actins↗
PubMed1992

Human T-helper clones induce IgG production in a subclass-specific fashion.

The mechanisms governing the induction of IgG subclasses by T-helper cells in humans were investigated. As preliminary bulk-culture experiments had indicated that a direct B cell contact with viable T cells was an essential requirement for optimal IgG subclass production, 256 CD4+ human T cell clones were preactivated with PHA and cultured in direct contact with autologous B cells. These clones induced IgG production in a strikingly subclass-specific fashion. Moreover, the distribution of subclass-specific helper clones was very similar to the IgG subclass profile observed in serum and peripheral lymphoid tissue plasma cells (IgG1 approximately 60%, IgG2 approximately 30%, IgG3 approximately 5-10%, IgG4 less than or equal to 5%) and unlike that observed in resting B cells (which is IgG1 approximately 40% and IgG2 approximately 50%). It would, therefore, seem that a predominance of T cells capable of delivering IgG1-specific, as opposed to IgG2-specific, help is an essential factor for the preferential induction of IgG1 antibodies during B cell proliferation and differentiation. There was no relationship between IL2, IL4, IL6, and IFN-gamma secreted by the T-helper clones and their IgG subclass induction patterns. In addition, only a few supernatants were able to reproduce the helper effects of the clones themselves. Therefore, direct contact of B cells with helper clones is crucial for IgG-subclass production in humans.

B-Lymphocytes↗
PubMed1991

Interleukin-6 is constitutively produced by human CTL clones and is required to maintain their cytolytic function.

Maturation of cytolytic T lymphocytes from nonlytic precursors requires cytokines in addition to IL2. Interleukin-6 is the principal cytokine that cooperates with IL2 in the induction of CTL differentiation from murine and human thymocyte precursors. However, a cytotoxic differentiation factor (CDF) role of IL6 for mature T cells is challenged by data indicating that IL2 alone is sufficient for CTL generation. The aim of this study was to identify a model system in which IL6 acted as a CDF for human peripheral T cells. We noted that IL6 was endogenously produced by CTL clones in the course of their expansion with APC, lectin, and IL2. The majority of several hundred T-cell clones, both CD4+ and CD8+, produced IL6 in response to relatively high doses of IL2. Other experiments that compared the cytolytic function of CTL clones cultured in the presence of IL6 with that of the same clones cultured in the absence of IL6 demonstrated that IL6 contributes to the cytolytic ability of the majority of human CTL clones. Our data suggest that IL6 acts in an autocrine fashion to support CTL differentiation in human T-cell clones.

CD4 Antigens↗