Tor Lea

Depletion of T lymphocytes from human bone marrow. Use of magnetic monosized polymer microspheres coated with T-lymphocyte-specific monoclonal antibodies.

A new technique for depletion of T cells from bone marrow is presented. Bone marrow cells (BMC) were rosetted with magnetic monosized polystyrene micro-spheres coated with monoclonal antibodies (MAbs) specific for T cell CD2 and CD3 antigens. Rosetted T cells were subsequently removed from non-T cells with the aid of a magnet. This immunomagnetic separation procedure was carried out in less than 40 min and reproducibly removed T cells, leaving a maximum of 0.025% sheep-red-blood-cell (SRBC) rosette-forming cells and less than 0.02% T cells as detected by a T cell limiting dilution assay. The efficacy of the depletion procedure was further shown by flow cytometry data, by effective removal of cells from a T cell line added to the BMC prior to immunomagnetic separation, and by abrogation of interleukin 2 (IL-2)-producing capacity in T-cell-depleted BMC (BMC-T). The T cell depletion procedure provided a 43–74% recovery of non-T cells present in the Isopaque-Ficoll-isolated bone marrow mononuclear cell fraction and did not disturb the growth potential of stem cells, as assayed by hematopoietic stem cell assays.

Cutting Edge: T Cell-Specific Adapter Protein Inhibits T Cell Activation by Modulating Lck Activity

We previously reported the isolation of a cDNA encoding a T cell-specific adapter protein (TSAd). Its amino acid sequence contains an SH2 domain, tyrosines in protein binding motifs, and proline-rich regions. In this report we show that expression of TSAd is induced in normal peripheral blood T cells stimulated with anti-CD3 mAbs or anti-CD3 plus anti-CD28 mAbs. Overexpression of TSAd in Jurkat T cells interfered with TCR-mediated signaling by down-modulating anti-CD3/PMA-induced IL-2 promoter activity and anti-CD3 induced Ca2+ mobilization. The TCR-induced tyrosine phosphorylation of phospholipase C-γ1, SH2-domain-containing leukocyte-specific phosphoprotein of 76kDa, and linker for activation of T cells was also reduced. Furthermore, TSAd inhibited Zap-70 recruitment to the CD3ζ-chains in a dose-dependent manner. Consistent with this, Lck kinase activity was reduced 3- to 4-fold in COS-7 cells transfected with both TSAd and Lck, indicating a regulatory effect of TSAd on Lck. In conclusion, our data strongly suggest an inhibitory role for TSAd in proximal T cell activation.

Intrathecal complement activation in neurological diseases evaluated by analysis of the terminal complement complex

The terminal complement complex (TCC) was determined in plasma and cerebrospinal fluid (CSF) from 208 neurological patients. Elevated CSF TCC levels were observed in higher frequencies in patients with infectious diseases (80%), radiculoneuritis (62%), multiple sclerosis (30%), and miscellaneous autoimmune diseases (27%) than in patients with miscellaneous non-inflammatory diseases (2-13%). The plasma level of TCC was significantly increased only in the infectious group. No positive correlation was observed between the plasma and the CSF TCC concentration in the whole patient population nor in subgroups divided according to blood-brain barrier function. Furthermore, the CSF TCC concentration did not correlate with the serum/CSF albumin ratio or with CSF total protein concentration when this was below 1.0 g/l. It is concluded that an elevated TCC concentration in CSF reflects intrathecal complement activation and that quantification of TCC in CSF may be a valuable supplement in the examination of neurological diseases.

Detection of Fc-receptor-bearing human lymphocytes. The majority of Tμ cells carry HLA-DR antigens

Abstract The Fc receptors of peripheral blood mononuclear cells were studied. The cells detected with anti-Rh Ripley-coated erythrocytes (EAHu) were the same as those detected with rabbit IgG-coated ox erythrocytes (EARa), since depletion of EA-RFC with one system caused an almost complete removal of EA-RFC as detected with both systems. For the isolation of Fc-receptor-bearing T lymphocytes (Tγ and Tμ cells) untreated, AET-treated, and neuraminidase-treated sheep red blood cells were equally good. The effect of anti-HLA-DR antibodies and anti-β2 microglobulin (anti-β2m) antibodies on EA-rosette formation was also studied. Anti-HLA-DR antibodies completely inhibited EAIgM-rosette formation of T lymphocytes, whereas no inhibition of EAIgG-rosette formation was observed in any mononuclear cell population. Anti-β2m antibodies completely inhibited both EAIgM- as well as EAIgG-rosette formation. Less than 5% of freshly isolated T lymphocytes formed EAIgM rosettes and were HLA-DR positive. In contrast approximately 50% of T lymphocytes formed EAIgM rosettes and were HLA-DR positive after overnight incubation. More than 90% of the Tμ cells were HLA-DR positive. Thus, overnight cultivation of T lymphocytes induce expression of HLA-DR antigens along with the Fcμ receptors. This is first report of HLA-DR antigens detected on the majority of nonstimulated human T cells.

The C terminus of T cell-specific adapter protein (TSAd) is necessary for TSAd-mediated inhibition of Lck activity.

T cell‐specific adapter protein (TSAd), encoded by the SH2D2A gene, is expressed in activated T cells. The function of TSAd is as yet unknown. We previously showed that TSAd may modulate T cell receptor‐triggered signaling events. TSAd contains a Src homology (SH)2 domain, ten tyrosines and a C‐terminal proline‐rich region. Here, we show that human TSAd interacts with Lck through the Lck SH2 and SH3 domains and is a substrate for Lck. The TSAd C terminus, including the proline‐rich region and five tyrosines, is both necessary and sufficient for TSAd interaction with and phosphorylation by Lck. Expression of TSAd in Jurkat TAg cells results in hyperphosphorylation of endogenous Lck on Y394 and to an even larger extent on Y505, resulting in a reduced Y394/Y505 phosphorylation ratio in these cells. Furthermore, full‐length TSAd, but not TSAd lacking the C terminus, inhibits the hyperactive Lck Y505F mutant when both are expressed in Jurkat T cells. In contrast, expression of the TSAd C terminus alone is sufficient to inhibit Lck Y505F in phosphorylating its substrates in Jurkat T cells. Our results indicate that the TSAd C terminus is essential for inhibition of Lck activity by TSAd, and suggest a mechanism for how TSAd may inhibit early T cell activation events.

Modulation of Lck Function through Multisite Docking to T Cell-specific Adapter Protein

T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, interacts with Lck through its C terminus and thus modulates Lck activity. Here we mapped Lck phosphorylation and interaction sites on TSAd and evaluated their functional importance. The three C-terminal TSAd tyrosines Tyr280, Tyr290, and Tyr305 were phosphorylated by Lck and functioned as docking sites for the Lck Src homology 2 (SH2) domain. Binding affinities of the TSAd Tyr(P)280 and Tyr(P)290 phosphopeptides to the isolated Lck SH2 domain were similar to that observed for the Lck Tyr(P)505 phosphopeptide, whereas the TSAd Tyr(P)305 peptide displayed a 10-fold higher affinity. The proline-rich Lck SH3-binding site on TSAd as well as the Lck SH2 domain were required for efficient tyrosine phosphorylation of TSAd by Lck. Interaction sites on TSAd for both Lck SH2 and Lck SH3 were necessary for TSAd-mediated modulation of proximal TCR signaling events. We found that 20–30% of TSAd molecules are phosphorylated in activated T cells and that the proportion of TSAd to Lck molecules in such cells is ∼1:1. Therefore, in activated T cells, a considerable number of Lck molecules may potentially be engaged by TSAd. In conclusion, Lck binds to TSAd prolines and phosphorylates and interacts with the three C-terminal TSAd tyrosines. We propose that through multivalent interactions with Lck, TSAd diverts Lck from phosphorylating other substrates, thus modulating its functional activity through substrate competition.

Structure function analysis of SH2D2A isoforms expressed in T cells reveals a crucial role for the proline rich region encoded by SH2D2A exon 7

BackgroundThe activation induced T cell specific adapter protein (TSAd), encoded by SH2D2A, interacts with and modulates Lck activity. Several transcript variants of TSAd mRNA exist, but their biological significance remains unknown. Here we examined expression of SH2D2A transcripts in activated CD4+ T cells and used the SH2D2A variants as tools to identify functionally important regions of TSAd.ResultsTSAd was found to interact with Lck in human CD4+ T cells ex vivo. Three interaction modes of TSAd with Lck were identified. TSAd aa239–256 conferred binding to the Lck-SH3 domain, whereas one or more of the four tyrosines within aa239–334 encoded by SH2D2A exon 7 was found to confer interaction with the Lck-SH2-domain. Finally the TSAd-SH2 domain was found to interact with Lck. The SH2D2A exon 7 encoding TSAd aa 239–334 was found to harbour information essential not only for TSAd interaction with Lck, but also for TSAd modulation of Lck activity and translocation of TSAd to the nucleus. All five SH2D2A transcripts were found to be expressed in CD3 stimulated CD4+ T cells.ConclusionThese data show that TSAd and Lck may interact through several different domains and that Lck TSAd interaction occurs in CD4+ T cells ex vivo. Alternative splicing of exon 7 encoding aa239–334 results in loss of the majority of protein interaction motives of TSAd and yields truncated TSAd molecules with altered ability to modulate Lck activity. Whether TSAd is regulated through differential alternative splicing of the SH2D2A transcript remains to be determined.

Activation-Dependent Epitopes in the Terminal Complement Pathway

Assembly of the terminal complement complex (TCC) during complement activation is associated with considerable antigenic changes in the individual C5, C6, C7, C8 and C9 components. Numerous neoepitopes specific for the TCC are exposed. Similarly, native-restricted epitopes specific for the nonactivated components are concealed in the TCC. The present paper reviews these antigenic changes with special reference to available monoclonal antibodies and their utilization in assays to detect and quantify the TCC. Application of such assays in clinical medicine is discussed. Finally, a recommended terminology for the terminal complement pathway is put forward.

Immunofluorescence staining of agarose-embedded cells. A new technique developed for immunological characterization of markers on a small number of cells.

A simple new method for the immunofluorescence staining of small numbers of cells is described: cell suspensions are mixed with low-temperature-gelling agarose at 37 degrees C and 2 microliter samples of agarose containing cells are dispensed onto multitest microslides precoated with agarose. The cells are subsequently stained by immunofluorescence techniques. Alternatively, the cell slides can be stored in liquid nitrogen until immunofluorescence staining is carried out. Since cells are entrapped within the agarose matrix, cell loss is prevented during staining and washing procedures. The method permits staining of as few as 250 cells for each marker, thus enabling simultaneous characterization of several separate cell markers in cerebrospinal fluid or other body compartments from which comparatively few cells are obtainable.

Streptolysin O neutralizing capacity and idiotypic properties of fragments, subunits and reassociated H and L chains from three human IgG monoclonal proteins.

Abstract Three IgG M-components with streptolysin O (SLO)∗ neutralizing activity were studied using anti-idiotype specific antisera. The heavy and light chains of the M-components were also studied and compared with the various recombinant molecules made by reassociation with normal human chains or chains from the other M-components. The results indicated that heavy and light chains of the M-components bound co-operatively to antigenic sites on SLO and that the three M-components were probably directed against different and independent antigenic determinants on the SLO molecules (no cross-idiotypy). Heavy and light chains had equal but low SLO neutralizing activity. The anti-idiotype specific antisera inhibited completely the SLO neutralizing activity of the M-components, but there was no cross-idiotypic inhibition. The SLO, on the other hand, could not completely inhibit the reaction between the idiotypic antisera and the M-components, indicating the presence of idiotypic sites not involved in antigen binding.