Multiple actins in drosophila melanogaster

The tissue and developmental specificities of the three Drosophila isoactins, originally identified in primary myogenic cultures and in the permanent Schneider L-2 cell line, have been investigated. Of these three isoactins (I, II, and III), actins I and II are stable and actin III is unstable. Two-dimensional polyacrylamide gel electrophoretic analyses of total cellular extracts after 1-h [(35)S]methionine pulses were performed on a large variety of embryonic, larval, and adult muscle and nonmuscle tissues. The results suggest that isoactins II and III are generalized cellular actins found in all drosophila cell types. Actin I, on the other hand, is muscle-associated and is found exclusively in supercontractile muscle (such as larval body wall and larval and adult viscera) including primary myogenic cell cultures. Although actin I synthesis is not detectable during very early embryogenesis, it is detectable by 25 h and actin I is a major stable actin in all larval muscle tissues. Actin I is synthesized in reduced amounts relative to the other actins in late third instar larvae but is again a major product of actin synthesis in the adult abdomen. A stable actin species with the same pI as actin III has been identified in the adult thorax and appears to be unique to flight muscle tissue. This new stable form of thoracic actin may be the result of a stabilization of the actin III found in other tissues or may be an entirely separate gene product.     

CD43 regulates the threshold for T cell activation by targeting Cbl functions

T cell (TC) activation requires the coordinated signaling of the T cell receptor (TCR) and coreceptor molecules, allowing TCs to respond to lower degrees of TCR occupancy. Coreceptor molecules set the threshold for TC activation by controlling different regulatory signaling loops. The Cbl family members prevent undesired activation of T cells by regulating TCR signals. In this report, we show that TC prestimulation by the CD43 coreceptor molecule before TCR engagement inhibits TCR-dependent c-Cbl tyrosine phosphorylation, c-Cbl interaction with the adapter molecule Crk-L and promotes Cbl-b degradation in a PKCθ-dependent manner. Consequently, the prolonged tyrosine phosphorylation and delayed degradation of ZAP-70 and of the ζ chain lead to enhanced mitogen-activated protein kinase activation and robust TC response. These data indicates that CD43-mediated signals lower the threshold for TC activation by restricting the c-Cbl and Cbl-b inhibitory effects on TCR signaling. In addition to the strength and duration of intracellular signals, our data underscore temporality with which certain molecules are engaged as yet another mechanism to fine tune TC signal quality, and ultimately immune function.     

Expression and ontogeny of CD2 on murine B cells.

Expression of CD2 on developing and mature murine B cells was examined by using an antipeptide antiserum (L50). Most Ig-bearing splenic B cells were found to express CD2. Anti-CD5 and anti-B220 mAb divided the peritoneal B cells into two populations expressing high and low levels of these proteins; both populations were found to express uniform levels of CD2. Abelson murine leukemia virus-transformed pre-B cell lines derived from fetal liver and adult bone marrow were analyzed to delineate the ontogeny of CD2 in the B cell lineage. The results show that onset of CD2 expression correlates with the presence of cytoplasmic mu-chain. Therefore, the earliest CD2+ pre-B cell in the developing B cell population appears to be the classical pre-B cell.     

Human lymphocyte function associated antigen-1 (LFA-1): identification of multiple antigenic epitopes and their relationship to CTL-mediated cytotoxicity

Human lymphocyte function-associated antigen (LFA)-1, a heterodimeric lymphocyte surface glycoprotein of 177,000 and 95,000 relative molecular weight has been implicated to function in the cytotoxic T lymphocyte (CTL) effector mechanism. Seven mouse hybridoma lines producing monoclonal antibodies (MAb) reactive with this structure were studied. Three unique and 3 partially over-lapping epitopes on human LFA-1 were defined by competitive cross inhibition binding assays using biosynthetically labeled anti-LFA-1 MAb. In contrast, of five rat antimouse LFA-1 MAb, all five recognized a common or shared epitope. An HLA-B7 specific human CTL line expressed 1.1 X 10(5) LFA-1 sites per cell with a direct saturation binding assay. Human CTL expressed two to four times more LFA-1 than peripheral blood lymphocytes or B and T lymphoblastoid cell lines. Titration of each of the anti-LFA-1 MAb in a 51chromium release cytolytic assay revealed quantitative differences in the ability of the different anti-LFA-1 MAb to block cytolysis indicating distinct functional and antigenic epitopes exist on the human LFA-1 molecule. Anti-LFA-1 MAb reversibly inhibited the CTL reaction by slowing the initial rate of cytolysis. These results suggest anti-LFA-1 MAb inhibit CTL function by specific blockade of a functionally relevant molecule.     

Amelioration of acute graft vs host disease due to minor histocompatibility antigens by in vivo administration of anti-interleukin 2 receptor antibody

Lethal acute graft vs host disease (GVHD) elicited by minor histocompatibility antigens was studied in a murine model of bone marrow transplantation (B10.BR----CBA). The severity of GVHD was reduced by both clinical and histologic parameters when transplant recipients received injections of a monoclonal antibody directed against the interleukin 2 receptor. This study suggests that anti-interleukin 2 receptor antibodies may be useful in clinical marrow transplantation and provides additional evidence that monoclonal antibodies that block T cell function in vitro may be of therapeutic value in vivo.     

The alpha-3 domain of class I MHC proteins influences cytotoxic T lymphocyte recognition of antigenic determinants located within the alpha-1 and alpha-2 domains

An interspecies class I MHC molecule, Kb1+2/A2 (in which the alpha-1 and alpha-2 domains of the H-2Kb molecule have been linked to the alpha-3, transmembrane and intracytoplasmic domains of the HLA-A2 molecule) has been expressed on both human and mouse target cells by gene transfer. Maintenance of serologic determinants has been demonstrated. However, decreased lysis by allospecific CTL populations of cell lines that expressed a hybrid interspecies class I molecule, Kb1+2/A2, as compared with lines that expressed the native Ag, H-2Kb, has been described. An analysis with a limited panel of H-2Kb allospecific clones demonstrated that not all H-2Kb-specific CTL can lyse cells that express Kb1+2/A2 Ag. This suggested that the reduction of lysis by CTL populations was due to the loss of specific alloreactive clones in the population. Each clone used in this study was then defined as having high or low affinity characteristics. No correlation between the affinity of the CTL and the ability to recognize the interspecies hybrid molecule could be shown. Rather, these data suggest that antigenic determinants that are located within the polymorphic domains, alpha-1 and alpha-2, may be conformationally influenced by the alpha-3 domain.     

The induction of cytolytic T lymphocytes with syngeneic trinitrophenyl-coupled membranes.

Evidence is presented that trinitrophenyl-coupled tumor membranes are able to induce cytolytic T lymphocytes (CTL) when co-cultured with syngeneic spleen cells. These haptenated membranes stimulate spleen cells from naive and immune mice. The specificity of these CTL is determined by the H-2 antigens of the membranes used for stimulation.     

p56lck association with CD4 is required for the interaction between CD4 and the TCR/CD3 complex and for optimal antigen stimulation.

By fluorescence resonance energy transfer, we have previously demonstrated that upon anti-CD3 mAb-mediated activation of a murine T cell hybridoma expressing human CD4, CD4 moves into close association with the TCR/CD3 complex. It was shown that this association between CD4 and the TCR/CD3 complex was dependent upon the presence of an intact CD4 cytoplasmic domain. We have now expressed, in a murine T cell hybridoma, mutated forms of CD4 containing cysteine to serine point mutations at positions 420, 422, or 430. The mutations at positions 420 and 422, but not 430, abolish association with p56lck. By using fluorescence resonance energy transfer, we demonstrate that mutations of CD4 which fail to interact with p56lck are unable to associate with the TCR/CD3 complex under conditions in which wild-type CD4 and the 430 mutant CD4 do associate with the TCR/CD3 complex. In addition, these mutants have a diminished response to CD4-dependent stimuli. We conclude that the association between CD4 and the TCR/CD3 complex during T cell activation plays an important role in CD4-dependent responsiveness and this association requires the interaction of CD4 with p56lck. These results also suggest that a substrate for p56lck may be expressed in the TCR/CD3 complex.     

Specificity of rat xenogeneic cell-mediated cytolysis for the products of the K and D loci of the mouse H-2 complex.

Lewis rat lymph node (LN) cells, when cultured with irradiated mouse spleen cells, develop cytotoxic effectors that were shown to be specific for the sensitizing mouse strain. Alloantisera directed to the products of the K and D loci of the target inhibited cytolysis whereas an antiserum to the I region gene products did not inhibit cytolysis even though LPS-induced spleen blasts were used as targets. Thus, it appears that even when effector cells from another species are generated to mouse spleen cells, the gene products of the K and D loci continue to play a predominant role.