J E de Vries

The X-linked lymphoproliferative-disease gene product SAP regulates signals induced through the co-receptor SLAM

In addition to triggering the activation of B- or T-cell antigen receptors, the binding of a ligand to its receptor at the cell surface can sometimes determine the physiological outcome of interactions between antigen-presenting cells, T and B lymphocytes. The protein SLAM (also known as CDw150), which is present on the surface of B and T cells, forms such a receptor–ligand pair as it is a self-ligand. We now show that a T-cell-specific, SLAM-associated protein (SAP), which contains an SH2 domain and a short tail, acts as an inhibitor by blocking recruitment of the SH2-domain-containing signal-transduction molecule SHP-2 to a docking site in the SLAM cytoplasmic region. The gene encoding SAP maps to the same area of the X chromosome as the locus for X-linked lymphoproliferative disease (XLP) and we found mutations in the SAP gene in three XLP patients. Absence of the inhibitor SAP in XLP patients affects T/B-cell interactions induced by SLAM, leading to an inability to control B-cell proliferation caused by Epstein–Barr virus infections.

Theory and practice of centrifugal elutriation (Ce): factors influencing the separation of human blood cells

Centrifugal elutriation (CE) is currently a widely used preparative cell separation technique. In order to optimize the separation of cells that show only small differences in sedimentation velocity, several conditions that might influence the resolution capacity, such as rotor speed, counterflow, jetstream, cell load, density, and viscosity of the elutriation medium, were analyzed.Experiments carried out with human red blood cells (rbc) indicated that aselective losses of rbc from the rotor caused by the jetstream, could be prevented if the separations were carried out at high rotor speeds, as predicted by the theory. In addition, high cell loads (5×108 rbc) resulted in better separations than low cell loads (5×107 rbc).Human monocytes were separated into subpopulations that differed only about 0.003 g/mL in density, but have virtually the same size. The separation was carried out either by increasing the density or viscosity of the elutriation medium or by decreasing the rotor speed. In all cases similar results were obtained.These results indicated that under optimal conditions CE can be applied for the separation of cells that differ only slightly in sedimentation velocity.

Epitope mapping of recombinant human gamma interferon using monoclonal antibodies

Five monoclonal antibodies (MAbs B22, B27, 3-6, 32 and 35) specific for human recombinant IFN-gamma were characterized. These MAbs were used to set up quantitative sandwich ELISAs which allowed the detection of 1.25 ng/ml of IFN-gamma when diluted in normal human serum. Epitope mapping of the IFN-gamma molecule using these MAbs demonstrated that antibodies 3-6 and 32 which did not inhibit the biological activity of IFN-gamma recognized an epitope localized on the 15 C-terminal amino acids, suggesting that this portion of the molecule was not implicated in the biological activity of IFN-gamma. Sandwich ELISAs were performed using various pairs of MAbs. The level of reactivity obtained when antibodies B22 and B27 were used simultaneously as catcher and tracer was similar to the result obtained with two antibodies recognizing different epitopes. These results confirm that the IFN-gamma molecule is a dimer in solution and indicate that the two sites of the IFN-gamma dimeric molecule which are associated with the biological activity (epitope B22/B27) are fully exposed. In contrast, the C-terminus is only partially accessible to the antibodies 3-6/32, suggesting that the dimerization of IFN-gamma molecule results in the interaction of regions of the monomers that are homologous and adjacent to the C-terminus.

Cord blood B cells are mature in their capacity to switch to IgE-producing cells in response to interleukin-4 in vitro.

Neonatal B cells have been considered immature because of their impaired capacity to produce immunoglobulins in response to polyclonal activators in vitro. Here we demonstrate that cord blood mononuclear cells (MNC) produce normal levels of IgE in vitro when cultured in the presence of interleukin‐4 (IL‐4), indicating that the B cells are mature in their capacity to switch to IgE‐producing cells. However, in contrast to adult peripheral blood T cells, cord blood T cells failed to produce detectable levels of IL‐4 upon activation by phytohaemagglutinin (PHA) concanavalin A (Con A) or combinations of PHA and the phorbol ester TPA. Interferon‐gamma (IFN‐γ) production by cord blood T cells following activation by Con A or PHA was also strongly reduced. However, high levels of IFN‐γ, significantly higher than those produced by adult T cells, were synthesized in response to combinations of PHA and TPA, indicating that IFN‐γ production by cord blood T cells is not intrinsically defective. In contrast, cord blood T cells produced levels of IL‐2 that were significantly higher than those obtained by adult T cells tested in parallel. Collectively, our data indicate that the minimal levels of IgE production measured in cord blood (< 1 U/ml) are not due to immaturity of the cord blood B cells, but may be associated with the failure of cord blood T cells to produce detectable levels of IL‐4, which has been shown to be responsible for induction of IgE synthesis both in vitro and in vivo.

Chimerism and tolerance to host and donor in severe combined immunodeficiencies transplanted with fetal liver stem cells.

We have studied the peripheral T cell repertoire of two patients with severe combined immunodeficiency who were successfully treated with human histocompatibility leukocyte antigen (HLA)-mismatched fetal liver stem cell transplantation. The patients presented a split chimerism. T cells were of donor origin, whereas the B cells/monocytes were of the host phenotype. Interestingly, the natural killer (NK) cells in one patient were donor derived and in the other patient of host origin. The NK cells were functional but did not have antihost or donor reactivity. Despite the HLA mismatch between donor and host cells, complete tolerance was achieved in vivo, and a specific unresponsiveness of peripheral blood mononuclear cells from both patients toward the host cells was demonstrated in vitro. Nevertheless, we could isolate T cell receptor (TCR)alpha beta, CD4+ or CD8+, T cell clones specifically reacting with HLA class I and II molecules of the host. The CD4+ host-reactive T cell clones from both patients produced interleukins 2 and 5, interferon-gamma, granulocyte/macrophage colony-stimulating factor but are specifically defective in interleukin 4 production. The frequencies of CD8+ host-reactive T cells were high, and were in the same range as those observed for CD8+ alloreactive T cells. In contrast, no donor-reactive CD8+ T cells or host or donor-reactive TCR gamma delta + T cells were detected. These data indicate that, after fetal stem cell transplantation, donor-reactive, but not host-reactive cells, are deleted from the T cell repertoire. Therefore, a peripheral mechanism of suppression or clonal anergy, rather than clonal deletion, is involved in maintaining in vivo tolerance toward the host.

Presence of Host-Reactive and MHC-Restricted T Cells in a Transplanted Severe Combined Immunodeficient (SCID) Patient Suggest Positive Selection and Absence of Clonal Deletion

Mature T cells in the periphery are derived from progenitor cells that originate in the fetal liver or bone marrow and migrate to the thymus during ontogeny. These pre-T cells undergo a series of complex maturational events that result in the expression of clonotypic TCRs on these cells. During these maturation processes the T cells learn to distinguish self from non-self. The mechanisms of this selection that occurs in the thymus are incompletely understood. It is believed that T cells carrying aP T-cell receptors with potential to recognize foreign peptide sequences associated vnth self MHC are selected by interactions with self MHC antigens expressed on thymic cortical epithelial cells (Zinkernagel et al. 1978, Bevan & Fink 1978, Kruisbeek et al. 1985, Farr et al. 1986), while those able to recognize self peptide are eliminated when they interact with MHC on bone marrow-derived cells present in the thymic medulla (Jenkinson et al. 1985, Lo et al. 1986, von Boehmer & Schubiger 1984, Marrack et al. 1988). Besides clonal deletion, two other possible mechanisms for T-cell tolerance have been proposed: clonal anergy involving specific inactivation of self-reactive T cells (Nossal & Pike 1983, Rammensee et al. 1990), and suppression of self-reactive T cells by other T cells (Gershon & Kondo 1971). Elegant studies using transgenic mice

The Replacement of Monocytes and Interleukin-1 by Phorbol Ester in Lectin-Induced Proliferation of Human Thymocytes and T Cells

Abstract Small human thymocytes isolated by counterflow centrifugal elutriation (CCE) failed to respond to lectins, and therefore they were considered to represent the immature thymocyte (IT) population. In addition, these IT also failed to respond to the tumor promotor 12-0-tetradecanoyl-13 acetate (TPA). Proliferation of the IT was induced if in addition to lectins irradiated allogeneic monocytes, crude interleukin-1 (IL-1) preparations or TPA at concentrations of 1–10 −3 µg/ml were added. Allogeneic monocytes, IL-1 or TPA also acted similarly in their synergistic effects with lectins (at concentrations that induced optimal proliferation) in the proliferative responses of unfractionated thymocytes (UT) and the medium sized to large thymocytes (MLT) which represent the mature thymocyte compartment. Irradiated autologous monocytes or TPA were also found to act in a similar synergistic way with lectins at suboptimal concentrations in the proliferation of mature T cells. These results indicate that TPA mimics the signal provided by monocytes or IL-1 in the mitogenesis of human thymocytes and mature T cells. However, at optimal lectin concentrations autologous monocytes were found to restore the reduced mature T-cell responses, whereas in contrast TPA caused strong reductions in the 3 H-thymidine ( 3 H-TdR) uptake of these cells. The mechanism of this depressed 3 H-TdR uptake remains unclear, but our data so far indicate that metabolites of TPA are toxic for those T cells that have relatively large quantities of lectins bound to their membranes, since the same metabolites had no toxic effects on mature T cells activated with suboptimal lectin concentrations.

Regulation of IgE synthesis by T cells and cytokines

Human IgE synthesis is tightly regulated by cytokines. IgE production by normal B cells is specifically induced by IL-4, but requires additional, yet to be defined, signals that are provided by CD4+ T cells. Single surface IgM+ B cells can be induced to proliferate and switch to IgG4 and IgE producing cells, indicating that the induction of IgE synthesis by IL-4 and CD4+ T cells reflects direct isotype switching. Although IL-4 is the sole inducing cytokine of IgE synthesis known thus far, multiple cytokines modulate IL-4 induced IgE synthesis in vitro. IFN-alpha, IFN-gamma TGF-beta and IL-10 are inhibitory, whereas IL-5, IL-6 and TNF-alpha act synergistically with IL-4. Results obtained with animal models, as well as from clinical studies in the human have indicated that IL-4, IFN-alpha and IFN-gamma are operational in vitro. Cocultivation of B cells with allergen-specific CD4+ T cell clones producing high levels of IL-4 and IL-5, but normal to undetectable levels of IL-2 and IFN-gamma, following activation resulted in the synthesis of IgE, in the absence of exogenously added IL-4. These results indicate that aberrant ratios of IL-4 and IFN-gamma production are sufficient for induction of IgE synthesis in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

The capacity of interleukin-4 to induce in vitro IgE synthesis by B cells of patients with common variable immunodeficiency

Interleukin‐4 (IL‐4) has been shown to induce IgE synthesis by peripheral blood mononuclear cells (PBMC) of normal donors in vitro. However, induction of PBMC of patients with common variable immunodeficiency (CVI) with IL‐4 resulted in IgE production in only two out of eight cases tested. PBMC of the first patient that produced IgE in response to IL‐4 also secreted normal levels of IL‐4 upon activation. PBMC of the second patient secreted very low levels of IL‐4 in vitro which may account for the very low serum IgE levels in this patient. Of the other six patients who had very low serum IgE levels and whose PBMC failed to produce IgE in response to IL‐4 in vitro, five did not secrete IL‐4 upon in vitro activation. The capacity of the T cells to produce IL‐4 was intact in the sixth patient. Collectively our data indicate the PBMC of the majority of patients with CVI are defective since they failed to respond appropriately to IL‐4 and they failed to produce IL‐4, contributing to the view that CVI is a heterogeneous disorder in which a variety of T and B cell defects occur.

Modulation of phenotypic and functional properties of human peripheral blood monocytes by interleukin-4 (IL-4)

IL-4, which was first described as B-cell stimulating factor-1 (BSF-1), has been shown to have important pleiotropic biologic effects in the mouse as well as in the human system [1]. In the present study we investigated the effect of rIL-4 on human peripheral blood monocytes. These cells were isolated by means of centrifugal elutriation, which prevented activation of the cells, and thus provided an excellent source of highly purified monocytes for functional and phenotypical studies upon culture with IL-4 [2].