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.     

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.     

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.     

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.     

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 /+ clones, and V1 or V2 TCR Y/+ 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 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.