The Abl-1 Kinase is Dispensable for NK Cell Inhibitory Signalling and is not Involved in Murine NK Cell Education.

Natural killer (NK) cell responsiveness in the mouse is determined in an education process guided by inhibitory Ly49 and NKG2A receptors binding to MHC class I molecules. It has been proposed that inhibitory signalling in human NK cells involves Abl-1 (c-Abl)-mediated phosphorylation of Crk, lowering NK cell function via disruption of a signalling complex including C3G and c-Cbl, suggesting that NK cell education might involve c-Abl. Mice deficient in c-Abl expression specifically in murine NK cells displayed normal inhibitory and activating receptor repertoires. Furthermore, c-Abl-deficient NK cells fluxed Ca2+ normally after triggering of ITAM receptors, killed YAC-1 tumour cells efficiently and showed normal, or even slightly elevated, capacity to produce IFN-γ after activating receptor stimulation. Consistent with these results, c-Abl deficiency in NK cells did not affect NK cell inhibition via the receptors Ly49G2, Ly49A and NKG2A. We conclude that signalling downstream of murine inhibitory receptors does not involve c-Abl and that c-Abl plays no major role in NK cell education in the mouse.

CD8 beta increases CD8 coreceptor function and participation in TCR-ligand binding.

To study the role of CD8 beta in T cell function, we derived a CD8 alpha/beta-(CD8-/-) T cell hybridoma of the H-2Kd-restricted N9 cytotoxic T lymphocyte clone specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260. This hybridoma was transfected either with CD8 alpha alone or together with CD8 beta. All three hybridomas released interleukin 2 upon incubation with L cells expressing Kd-peptide derivative complexes, though CD8 alpha/beta cells did so more efficiently than CD8 alpha/alpha and especially CD8-/- cells. More strikingly, only CD8 alpha/beta cells were able to recognize a weak agonist peptide derivative variant. This recognition was abolished by Fab' fragments of the anti-Kd alpha 3 monoclonal antibody SF1-1.1.1 or substitution of Kd D-227 with K, both conditions known to impair CD8 coreceptor function. T cell receptor (TCR) photoaffinity labeling indicated that TCR-ligand binding on CD8 alpha/beta cells was approximately 5- and 20-fold more avid than on CD8 alpha/a and CD8-/- cells, respectively. SF1-1.1.1 Fab' or Kd mutation D227K reduced the TCR photoaffinity labeling on CD8 alpha/beta cells to approximately the same low levels observed on CD8-/- cells. These results indicate that CD8 alpha/beta is a more efficient coreceptor than CD8alpha/alpha, because it more avidly strengthens TCR-ligand binding.

TCR/CD3 coupling to Fas-based cytotoxicity.

We studied the coupling of the TCR/CD3 complex to a T cell effector function, namely Fas-based T-cell-mediated cytotoxicity. Encounter or re-encounter with antigen was mimicked by treating 5 d mixed lymphocyte culture cells or T cell hybridomas with anti-CD3 antibody. This TCR/CD3 engagement induced swift expression of Fas-based cytotoxicity in these cells. Induction of Fas-based cytotoxicity was Ca(2+)-dependent, while its execution was not; induction was sensitive to macromolecular synthesis inhibitors, in line with a demonstrable increase of the Fas ligand (Fas-L) message. We also used T cell hybridomas transfected with various constructs to dissect the involvement of distinct components of the TCR/CD3 complex. The cytoplasmic domain of the CD3 zeta chain was able to transduce by itself a signal leading to Fas-L expression, unless there were mutations in its activation receptor homology sequence 1 (ARH-1) motifs. On the one hand, these findings are relevant to signal transduction pathways coupled to the TCR/CD3, and on the other hand, to the involvement of Fas-based T cell-mediated cytotoxicity in various physiological and possibly pathophysiological situations.

Developmental regulation of a mucinlike glycoprotein selectively expressed on natural killer cells.

Natural killer (NK) cells are CD3:TCR-, CD16+, CD56+ large granular lymphocytes capable of recognizing and eliminating a variety of virus-infected, malignant, and antibody-coated target cells. Two functionally distinct populations of peripheral blood NK cells can be differentiated by their surface expression of an isoform of the neural cell adhesion molecule (CD56). CD56bright NK cells have the attributes of an undifferentiated cell, in that they proliferate in response to exogenous cytokines, but exert poor cytolytic activity. CD56dim NK cells have the attributes of a more differentiated cell, in that they proliferate poorly in response to exogenous cytokines, but are potent cytolytic effector cells. Here we describe the molecular characterization of a NK cell restricted epitope (PEN5) that is selectively expressed on the functionally differentiated CD56dim NK cells. PEN5+ NK cells proliferate poorly in response to interleukin 2 (IL-2), but are potent cytolytic effectors, whereas PEN5- NK cells proliferate in response to IL-2, but are poor cytolytic effectors. Biochemical and immunochemical analyses reveal the PEN5 epitope to be an unusual sulfated poly-N-lactosamine carbohydrate related to keratan sulfate glycosaminoglycans. Immunoprecipitates prepared using a monoclonal antibody reactive with PEN5 include two polydisperse membrane-bound glycoproteins, PEN5 alpha (120-170 kD) and PEN5 beta (210-245 kD). Enzymatic deglycosylation reduces the apparent molecular weight of both PEN5 isoforms by 80-90%, and classifies PEN5 beta as a mucinlike glycoprotein. The surface expression of the PEN5 epitope is downmodulated by stimuli that induce NK cell proliferation, and it is absent from leukemic NK cells of patients with granular lymphocyte proliferative disorder. Taken together, these results indicate that PEN5 is a developmentally regulated poly-N-lactosamine epitope associated with a mucin-type glycoprotein, whose expression is restricted to the population of nonproliferative NK cells fully committed to cytolytic effector function.

Different roles for the Fc epsilon RI gamma chain as a function of the receptor context.

The high affinity immunoglobulin E receptor (Fc epsilon RI) and the B and T cell antigen receptors (TCR) are multimeric complexes containing subunits with cytoplasmic antigen recognition activation motifs (ARAMs). The presence of multiple motifs may be a way to amplify a single signal or provide independent activation modules. Here we have compared the signaling capacity of the same Fc epsilon RI gamma motif in the context of two different receptors, Fc epsilon RI and TCR/CD3, simultaneously reconstituted on the surface of the same zeta-deficient T cell line. Both reconstituted receptors mediate early (phosphorylation) and late (interleukin [IL]-2 release) signals. Mutation of the two tyrosine residues of ARAM gamma alters early signaling by both receptors, but the set of substrates phosphorylated via ARAM gamma is different for each receptor and is thus dependent on the receptor context. Furthermore, the mutations prevent Fc epsilon RI- but not TCR/CD3-mediated IL-2 release. These data demonstrate that ARAM gamma is necessary for allowing both receptors to phosphorylate the complete set of substrates, and that the CD3 complex, unlike the Fc epsilon RI beta chain, contains activation modules capable of compensating for the absence of a functional ARAM gamma in generating late signals such as IL-2 release.

[The outer membrane complex and its development at the time of the formation of daughter cells in Toxoplasma gondii]. / Le complexe membranaire superficiel et son évolution lors de l'élaboration des individus-fils chez Toxoplasma gondii.

The parasitic protozoan Toxoplasma gondii has been examined with the electron microscope in order to study the fine structure and the formation of the membranes surrounding the cell. The study of the ultrastructure of the membranes covering the parasite shows the existence of a three-membraned complex. Only the outer membrane is considered to be the plasma membrane; the two membranes below it form an inseparable whole of changeable molecular architecture (modifications in appearance depending on the methods of fixation, local differentiation). During reproduction, which takes place by fission or more often by endogeny, the membranes of the daughter individuals are formed from the membranes of the parent. At first the middle and inner membranes of the parent extend, separating the cytoplasm of the daughter cells from that of the parent. The three-membrane complex of the endozoites is completed at the time of their liberation; the external membrane of the parent covers the leaving endozoites; thus, the plasma membrane of the daughter cells derives also from that of the parent. These findings on the origin and role of limiting membranes during reproduction differ entirely from those described so far for other cells.

Motile flagellum with a "3 + 0" ultrastructure.

The male gamete of the parasitic protozoan Diplauxis hatti has a flagellum consisting of three doublet microtubules. This flagellum exhibits a helicoidal waveform in which bends propagate toward the tip with a frequency of about 1.5 hertz. It is the simplest motile eukaryotic flagellum yet described.

Inhibition of IgE-mediated mast cell activation by the paired Ig-like receptor PIR-B.

The potential of the paired Ig-like receptors of activating (PIR-A) and inhibitory (PIR-B) types for modifying an IgE antibody-mediated allergic response was evaluated in mouse bone marrow-derived mast cells. Although mast cells produced both PIR-A and PIR-B, PIR-B was found to be preferentially expressed on the cell surface, where it was constitutively tyrosine phosphorylated and associated with intracellular SHP-1 protein tyrosine phosphatase. PIR-B coligation with the IgE receptor (FcepsilonRI) inhibited IgE-mediated mast cell activation and release of serotonin. Surprisingly, the inhibitory activity of PIR-B was unimpaired in SHP-1-deficient mast cells. A third functional tyrosine-based inhibitory motif, one that fails to bind the SHP-1, SHP-2, and SHIP phosphatases, was identified in parallel studies of FcepsilonRI-bearing rat basophilic leukemia (RBL) cells transfected with constructs having mutations in the PIR-B cytoplasmic region. These results define the preferential expression of the PIR-B molecules on mast cells and an inhibitory potential that can be mediated via a SHP-1-independent pathway.

Regulation of T cell function by NK cell receptors for classical MHC class I molecules.

Inhibitory receptors for MHC class I molecules were initially characterised on NK cells. Human and mouse NK cell receptors (NKRs) are also expressed on T cells, predominantly on a subset of memory-phenotype CD8(+) T cells. This review focuses on the precise determination of interactions between NKRs and MHC class I, as well as on the unexpected in vivo function of NKRs on T cells.

Strategies of natural killer cell recognition and signaling.

The participation of natural killer (NK) cells in multiple aspects of innate and adaptive immune responses is supported by the wide array of stimulatory and inhibitory receptors they bear. Here we review the receptor-ligand interactions and subsequent signaling events that culminate in NK effector responses. Whereas some receptor-ligand interactions result in activation of both NK cytotoxicity and cytokine production, others have more subtle effects, selectively activating only one pathway or having distinct context-dependent effects. Recent approaches offer ways to unravel how the integration of complex signaling networks directs the NK response.

[NK cells: new insights on physiology and clinical implication in diseases]. / Les cellules natural killer: acquisitions récentes et implication en pathologie humaine.

INTRODUCTION: Natural killer cells are cytotoxic lymphocytes of innate immunity. These last ten years our knowledge about the mechanisms that regulates NK cell function has greatly improved. Our purpose is to present a review of these new acquisitions and their potential implications in human disease. CURRENT KNOWLEDGE AND KEY POINTS: NK cell function is regulated by a repertoire of NK cell receptors and is diversified by recognition of MHC class I by a multigenic and multi-allelic family of NK receptors. Analysis of NK cell repertoire has been used to investigate features that characterize NK cells in pathological situations. Apart from their direct cytotoxic potential to eliminate target cells, recently identification of mechanisms that control NK cell mediated cytokine production and cross talk with dendritic cells emphasize the role of NK cells in the regulation of acquired immune response. FUTURE PROSPECTS AND PROJECTS: These findings have lead to a better knowledge of the importance of the NK cells in several human diseases. It has been shown that NK cells are actors of the immunosurveillance of tumoral and infectious challenges. Allo or auto reactivity of the NK cell compartment have also been suggested in autoimmune diseases, infertility or foetal loss and transplantation. Ongoing research on NK cells in the fields of human diseases is increasing and will clarify the utility of the evaluation of the NK cell compartment and their receptors in clinical practice.

Decreased expression of the signal-transducing zeta chains in tumor-infiltrating T-cells and NK cells of patients with colorectal carcinoma.

An impaired immune response is frequently observed in cancer patients and tumor-bearing mice. T-cells from mice with an experimental colon carcinoma were recently shown to express T-cell receptors that completely lacked the signal-transducing molecule CD3 zeta. Here, we have investigated the expression of the signal-transducing molecule zeta on lymphocytes from 14 patients with colorectal carcinomas using flow cytometric analysis of permeabilized cells with a monoclonal antibody (TIA-2; IgG1) specific for the cytoplasmic domain of the zeta chain as well as with immunoprecipitation and analysis on diagonal gel electrophoresis. We demonstrate that T-cells isolated from the tumors of the patients express significantly less CD3 zeta than T-cells in the peripheral blood of the same patients and that the peripheral blood of the patients express decreased levels of zeta chains, as compared to the levels found in lymphocytes from healthy controls. This decreased expression was also observed on zeta chains associated with the low affinity Fc receptor for IgG found in tumor-infiltrating NK cells (Fc gamma RIIIA alpha; CD16).

Analysis of donor NK and T cells infused in patients undergoing MHC-matched allogeneic hematopoietic transplantation.

We retrospectively analyzed the percentages and absolute numbers of T cells, natural killer (NK) cells and NK cell subsets in cryopreserved samples of either bone marrow or blood non-T cell-depleted allogeneic MHC-matched hematopoietic grafts. Using flow cytometry, we found higher numbers of NK cells in aphereses than in bone marrow collections. We further investigated the distribution of NK cell subsets, defined by the cell surface expression of MHC class I-specific receptors, in these allogeneic grafts. The distribution of NK cell subsets from the two different origins were similar, with the exception of the CD158a/h(+) NK cell subset, whose size appeared to be smaller in bone marrow. The search for relations between the numbers of infused cells and post-transplantation events demonstrated that increasing numbers of infused T cells but not NK cells are related with decreased overall survival. Our study highlights the toxicity of infused T cells but not NK cells in allogeneic MHC-matched hematopoietic grafts. These data pave the way for further trials to investigate the effect of NK cell infusion in MHC-matched allogeneic transplantation, and in particular whether ex vivo NK cell expansion and activation may enhance the anti-tumoral effect of the procedure and decrease its morbidity.

CD antigens 2001.

This paper reviews the Seventh Human Leucocyte Differentiation Antigen (HLDA7) workshop. Due to the limitations of "blind" antibody screening, which had been evident at the previous meeting in 1996, participants at HLDA7 adopted a more selective approach to the choice of antibodies by identifying new CD specificities. This resulted in the addition of more than 80 new CD specificities. Plans for the eighth and subsequent workshops are also previewed.

Function of killer cell inhibitory receptors for MHC class I molecules.

NK- and T-cells express at their surface, members of a multigenic family of killer-cell inhibitory receptors (KIR) for MHC Class I molecules. KIR engagement leads to the inhibition of NK- and T-cell activation programs. These receptors recruit the protein tyrosine phosphatases (PTPase), SHP-1 and SHP-2, upon tyrosine phosphorylation of immunoreceptor tyrosine-based inhibition motif (ITIM) expressed in both human and mouse KIR. We further define the ITIM amino acids sequence required in that recognition and demonstrate the critical role of the phosphoY-2 amino acid residue in this V/IxYxxL/V motif. In addition, using RBL-2H3 cells expressing endogenous Fc epsilonRI receptors as well as transfected CD25/CD3zeta chimera and p58.183 human KIR, we show that KIR inhibitory function requires co-engagement of KIR and ITAM-containing receptors. These results document the pathway used by KIR to down-regulate NK- and T-cell activation programs.

Early signaling via inhibitory and activating NK receptors.

This review focuses on recent findings on the structural features of inhibitory NK cell receptors containing immunoreceptor tyrosine-based inhibition motif (ITIM) and of NK cell activating receptors, both in human and mouse. First, the study of the inhibitory killer cell immunoglobulin-like receptors (KIR) unveiled the presence of intracytoplasmic ITIM and their capacity to recruit protein tyrosine phosphatases such as SHP-1 in vivo. A brief summary of the known SHP-1 targets may help us to understand the inhibition mediated by the KIR. The characterization of ITIM thus allowed the definition of a large group of inhibitory cell surface receptors. The second part of the review describes the known NK cell activating receptors. Most of them require association with ITAM-containing polypeptides in order to mediate cell activation.

Administration of interleukin 13 to simian immunodeficiency virus-infected macaques: induction of intestinal epithelial atrophy.

Increase Th2 cytokine production may contribute to some clinical manifestations of HIV infection, and studies have suggested that IL-13 rather than IL-4 is involved in these conditions. We directly tested this hypothesis by administrating IL-13 to SIV-infected macaques. SIV-infected rhesus macaques received a daily subcutaneous injection for 21 days of either IL-13 (10 microg/kg/day) or a placebo. The four macaques treated with IL-13 experienced body weight loss (9.95 +/- 0.71%) related to intestinal tract damage: they all suffered from a complete atrophy of duodenal villi. This was presumably due to premature epithelial cell death: proliferating Ki67+ cells in glandular crypts were as numerous as in control animals, but many epithelial cells developed apoptosis. The duodenal mucosa was infiltrated with cells expressing CD56 and PEN5, two markers of NK cells, and there was a deregulation of local cytokine and chemokine production characterized by a decrease in IL-10 gene expression (25% of controls) and an increase in gene expression for IFN-gamma (4-fold control), MIP-1alpha (8-fold control), and MIP-1beta (13-fold control). Thus, IL-13 can induce digestive epithelial cell injury in vivo in primates infected with a retrovirus. Therefore, its role should be considered in digestive manifestations of HIV infection as well as in other disorders associated with intestinal epithelial atrophy.

A freeze-fracture study of microsporidia (Protozoa: Microspora): II. The extrusion apparatus: Polar filament, polaroplast, posterior vacuole.

Developmental stages (sporoblasts and spores) of five species of microsporidia (Amblyospora bracteata, A. varians, A. weiseri, Nosema apis, Tuzetia sp.), fixed by conventional EM fixatives or by glutaraldehyde supplemented with potassium chloride, were studied by freeze-fracture using ultrathin sections as controls. It was found that the polar filament is formed by coalescence of cytoplasmic vesicles of the Golgi zone. The filament is enveloped by a single cytoplasmic membrane ("filament ensheathing membrane" = FEM) showing more intramembrane particles (IP) on the EF face (in the order of 1000 IP/µm(2)) and less IP (in the order of 500 IP/µm(2)) on the PF face. During maturation of the spore the IP on the FEM progressively disappear. The polaroplast is a stack of cytoplasmic membranes revealing more IP (in the order of 1500 IP/µm(2)) on the PF face and less particles (in the order of 400 IP/µm(2)) on the EF face. When KCl is added to the fixative the polaroplast remains unswollen and its membranes are smooth. In swollen polaroplasts the membranes are modified, probably by mechanical stretching. The polaroplast membranes are not direct outgrowths of the FEM. The posterior vacuole is limited by a single cytoplasmic membrane. No evidence was found supporting the idea that the filament ends in the posterior vacuole.

Immunoregulatory functions of paf-acether. VI. Dual effect on human B cell proliferation.

The role of paf-acether (paf), a phospholipid cytokine, in the modulation of human B cell function was investigated. Paf, from 1 x 10(-5) M to 10(-6) M, decreased B cell proliferation induced by both phorbol myristate acetate (PMA) and anti-IgM antibodies (anti-IgM Ab). By contrast, 1 x 10(-7) M to 1 x 10(-9) M paf enhanced PMA triggered, but not anti-IgM triggered B cell proliferation. B cell proliferation was modulated between 24 and 72 hr of culture indicating that the effect of paf did not merely reflect a shift in proliferation kinetics. Interestingly, paf also enhanced the spontaneous proliferation of a Burkitt lymphoma-derived B cell line, Raji, which suggests that paf can directly act on B cells. The modulatory effect of paf on peripheral blood B cells was independent of PMA concentration, yet the effect on Raji cells was dependent upon cell density. The data suggest that paf is a potent modulator of B cell function, and may be involved in the control of humoral immune response.