Biomedical subjects

C C Stebbins

Publications and source records attributed to C C Stebbins.

9 recordsLinked to original sources

PubMed2001

Inhibition of natural killer cell activation signals by killer cell immunoglobulin-like receptors (CD158).

The killer cell immunoglobulin-like receptor (KIR) family includes receptors that bind to HLA class I molecules on target cells and inhibit natural killer (NK)-cell cytotoxicity, and receptors such as KIR3DL7 with no known ligand and function. Inhibitory KIR recruit the tyrosine phosphatase SHP-1 to block signals transduced by any one of a number of activation receptors. Inhibition of overall protein tyrosine phosphorylation by SHP-1 during binding of KIR to MHC class I on target cells is selective, suggesting that a limited number of substrates are dephosphorylated by SHP-1. We have chosen to study KIR inhibition as it occurs during binding of KIR to MHC class I on target cells, despite the technical limitations inherent to studies of processes regulated by cell contact. KIR binding to MHC class I on target cells inhibits tyrosine phosphorylation of the activation receptor 2B4 (CD244) and disrupts adhesion of NK cells to target cells. Inhibition of proximal events in NK activation may increase the availability of NK cells by liberating them from non-productive interactions with resistant target cells. As the receptors and the signaling pathways that induce NK cytotoxicity are not fully characterized, elucidation of the inhibitory mechanism employed by KIR may provide insight into NK activation.

Amino Acid Sequence↗

PubMed2000

NK cell inhibitory receptors prevent tyrosine phosphorylation of the activation receptor 2B4 (CD244).

2B4 is an NK cell activation receptor that can provide a co-stimulatory signal to other activation receptors and whose mode of signal transduction is still unknown. We show that cross-linking of 2B4 on NK cells results in its rapid tyrosine phosphorylation, implying that this initial step in 2B4 signaling does not require coligation of other receptors. Ligation of 2B4 in the context of an NK cell-target cell interaction leads to 2B4 tyrosine phosphorylation, target cell lysis, and IFN-gamma release. Coligation of 2B4 with the inhibitory receptors killer cell Ig-like receptor (KIR)2DL1 or CD94/NKG2 completely blocks NK cell activation. The rapid tyrosine phosphorylation of 2B4 observed upon contact of NK cells with sensitive target cells is abrogated when KIR2DL1 or CD94/NKG2 are engaged by their cognate MHC class I ligand on resistant target cells. These results demonstrate that NK inhibitory receptors can interfere with a step as proximal as phosphorylation of an activation receptor.

Antigens, CD↗

PubMed2000

Natural killer cells and mast cells from gp49B null mutant mice are functional.

Immune responses are controlled by a combination of positive and negative cellular signals. Effector cells in the immune system express inhibitory receptors that serve to limit effector cell expansion and to protect the host from autoreactivity. gp49B is a receptor of unknown function that is expressed on activated mast cells and natural killer (NK) cells and whose cytoplasmic tail endows it with inhibitory potential. To gain insight into the function of gp49B in mice, we disrupted the gp49B gene by homologous recombination. gp49B(0) mice were born at expected ratios, were healthy and fertile, and displayed normal long-term survival rates. gp49B(0) mice showed no defect in NK or mast cell development. Furthermore, NK and mast cells from the gp49B(0) mice showed activation properties in vitro similar to those of cells isolated from wild-type mice. Therefore, gp49B is not critical for the development, expansion, and maturation of mast cells and NK cells in vivo. The healthy status of gp49B(0) mice makes them suitable for testing the role of gp49B in immune responses to infectious agents.

Animals↗

PubMed1999

Essential role of LAT in T cell development.

The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement. Phosphorylated LAT binds many critical signaling molecules. The central role of this molecule in TCR-mediated signaling has been demonstrated by experiments in a LAT-deficient cell line. To probe the role of LAT in T cell development, the LAT gene was disrupted by targeting. LAT-deficient mice appeared healthy. Flow cytometric analysis revealed normal B cell populations but the absence of any mature peripheral T cells. Intrathymic development was blocked within the CD4- CD8- stage. No gross abnormality of NK or platelet function was observed. LAT is thus critical to both T cell activation and development.

Adaptor Proteins, Signal Transducing↗

PubMed1996

DM-mediated release of a naturally occurring invariant chain degradation intermediate from MHC class II molecules.

The nonpolymorphic, nonclassical class II molecule DM has been shown to be important in the processing and presentation of antigenic epitopes by MHC class II molecules. The dependence of class II molecules on the coexpression of DM varies with the particular allele or epitope studied. In an effort to resolve disparities that exist for some alleles of class II in their requirements for DM, we have constructed a species-matched murine transfection model with which we can test both the functional and biochemical consequences of DM expression for different alleles of murine class II. When we evaluated the ability of class II molecules to form SDS-stable dimer and release CLIP, we find that while I-A(d) requires DM for the formation of SDS-stable dimers and the release of CLIP, I-A(k) is capable of both SDS-stable dimer formation and CLIP release in the absence of DM. Despite the apparent differences in the biochemical consequences of DM expression for I-A(d) and I-A(k), we find that Ag presentation by both alleles can be enhanced by the expression of DM. Interestingly, we find that DM can facilitate the removal of a natural intermediate in Ii processing (p12). The ability of DM to catalyze the dissociation of p12 and possibly larger Ii fragments from class II in vivo offers a possible mechanism to account for the observed DM enhancement of Ag presentation for alleles that have a low affinity for CLIP. Our findings indicate that DM may function in multiple compartments and on multiple class II/Ii substrates.

Alleles↗

PubMed1995

The requirement for DM in class II-restricted antigen presentation and SDS-stable dimer formation is allele and species dependent.

Recently several cell lines have been identified with mutations in a major histocompatibility complex (MHC)-linked protein that lead to defects in class II-restricted antigen presentation and a defect in the formation of class II SDS-stable dimers. The defect in these cells has recently been shown to result from the inability to express the MHC-encoded nonclassical class II molecule called DM. To further examine the role of DM in class II-restricted antigen presentation, we asked if this defect would equally affect different allelic and species variants of class II molecules. To investigate this, we transfected the parent cell lines T1 and 8.1.6 and their respective antigen presentation mutants T2 and 9.5.3 with the genes encoding I-Ad and examined the derived transfectants for their ability to present antigen, the conformation of I-Ad at the cell surface, association of I-Ad with invariant chain (Ii), and the ability to form I-Ad SDS-stable dimers. The lack of functional DM expression did not affect any of the anti-I-Ad monoclonal antibody (mAb) epitopes tested or the ability of I-Ad to associate and dissociate with Ii. Surprisingly, these studies also revealed that the antigen presentation defect observed for DR in the 9.5.3 cells did not compromise I-Ad-restricted antigen presentation. In addition, we found that the level of SDS-stable dimer formation did not correlate with antigen presentation capacity for I-Ad and that the amount of SDS-stable I-Ad dimer depends on the cellular context in which the class II molecule is expressed. Our results suggest that the ability to form SDS-stable dimer is not strictly correlated with class II-restricted antigen presentation. Finally, when two allelic forms of murine class II molecules were compared in the defective T2 cell line, it was found that I-Ak but not I-Ad forms SDS-stable dimers equivalent to that seen in the parental cell lines. Overall, our results suggest that DM may modulate rather than play a requisite role in I-Ad-restricted antigen presentation and SDS-stable dimer formation and that dependency on DM may be allele or species specific.

Animals↗

PubMed1991

Complementary roles for CD2 and LFA-1 adhesion pathways during T cell activation.

The influence of T cell receptor (TcR) triggering on T cell adhesion function has been systematically investigated in the present studies; we show that the adhesion function of LFA-1 is minimal in non-activated T cells but is augmented within minutes following TcR-mediated activation. In contrast, CD2 function is essentially optimal in non-activated T cells and undergoes no detectable modification within 12 h of TcR stimulation. Protein kinase C activation augments LFA-1 but not CD2 adhesion function and cyclic AMP reduces LFA-1 adhesion without affecting CD2-LFA-3 interactions. Up-regulation of the LFA-1 pathway occurs in the absence of any detectable surface redistribution of this molecule, suggesting an activation dependent modification leading to a high-affinity ICAM-1 binding state. The TcR independence of CD2 adhesion function implies a critical role of the CD2 pathway in initiating cell-cell interactions prior to TcR engagement and LFA-1-ICAM-1 binding and underscores the complementary nature of the CD2 and LFA-1 adhesion pathways during the immune response.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1990

Human natural killer cells and mature T lymphocytes express identical CD3 zeta subunits as defined by cDNA cloning and sequence analysis.

In order to characterize the CD3 zeta-related protein found in human natural killer (NK) cells and compare it with CD3 zeta expressed in T lymphocytes, the present study was performed. A polyclonal CD3-CD16+NK population displaying a strong non-major histocompatibility complex-restricted cytotoxic activity against the NK target K-562 was isolated and a product corresponding to CD3 zeta amplified using the polymerase chain reaction method. This 0.6-kb product was present in similar amounts in NK cells and T cells. In contrast, a product corresponding to CD3 delta was amplified from T lymphocytes exclusively. Thus, the CD3 zeta product detected in NK cells did not originate from contaminating T cells. DNA sequence analysis of two independent polymerase chain reaction products from the NK cells demonstrates that human NK cells and mature T cells share a CD3 zeta subunit with an identical primary amino acid sequence. The nucleotide sequence of a third NK-derived cDNA revealed an insertion of a CAG triplet encoding an additional glutamine residue in the cytoplasmic domain. Since this residue is encoded by nucleotides at a putative RNA splice junction, it possibly results from a difference in pre-mRNA splicing. Taken together, these data show that CD3 zeta is not structurally distinct in NK cells and in T lymphocytes.

Amino Acid Sequence↗

PubMed1990

Characterization of functional GTP binding proteins in Jurkat T cell mutants lacking either CD3-Ti or CD2 surface receptors.

G proteins are membrane-bound molecules involved in coupling of surface receptors with signal transduction effector systems in multiple cell types including T lymphocytes. Given that mature T cells which lack antigen receptors (CDl-Ti) are refractory to stimulation through CD2 or other accessory molecules, T cell receptor components likely play a critical role in coupling surface receptors with signal transduction effectors. It has recently been proposed that modulation of T cell receptor components with MAbs results in a physical loss or functional inactivation of G protein(s). In view of the importance of the T cell activation process, we herein examined G proteins in untreated or antibody-modulated Jurkat T cells as well as in genetic variants lacking either CD3-Ti or CD2 surface receptors. 43- and 41-kDa G protein alpha chains are ADP ribosylated with cholera (CTX) and pertussis (PTX) toxins, respectively, in wild type and receptor minus cell populations. In the wild type Jurkat cell line as well as in CD3- and CD2- variants, AlF4- can activate the G protein(s) presumably associated with phospholipase C to generate polyphosphoinositide turnover as well as an increase in cytoplasmic free calcium ions. Furthermore, G protein(s) linked to adenylylcyclase, a pathway which inhibits T lymphocyte activation, can be directly activated with CTX in the absence of CD3-Ti or CD2 on the membrane. Importantly, AlF4- can also induce polyphosphoinositide turnover in Jurkat cells whose T cell receptor proteins have been modulated with anti-CD3 MAb. These data provide functional and biochemical evidence that at least certain G proteins are intact in the absence of surface expression of CD3-Ti or CD2 molecules and imply that CD3-Ti desensitization is not singularly due to G protein loss.

Adenosine Diphosphate Ribose↗