Molecular cloning of the human B cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains.

CD20 is an antigen expressed on normal and malignant human B cells that is thought to function as a receptor during B cell activation. Here we report the isolation of a CD20-specific cDNA clone from a lambda gt11 library using a polyclonal antiserum raised against purified CD20 antigen. Additional cDNA clones were then isolated from a lambda gt10 library. Alignment of the sequences of overlapping lambda clones reveal a single consensus sequence except for a divergence that preceded the first methionine within the open reading frame. Normal B cells and B cell lines contain a prominent 2.6 kb mRNA and a lower level of a 3.3 kb mRNA. An oligonucleotide derived from one of the divergent sequences hybridized to the 3.3 kb mRNA only, indicating that the two mRNA species are derived from an alternative splicing mechanism. The predicted amino acid sequence of CD20 reveals three major hydrophobic regions of approximately 53, 25 and 20 amino acids. CD20 lacks an NH2-terminal signal peptide and contains a highly charged COOH-terminal domain. Although CD20 is immunoprecipitated as a doublet of 33 and 35 kd proteins from B cells, in vitro translation of CD20 cDNA produced a single 33 kd protein that was specifically immunoprecipitated with monoclonal CD20 antibodies. CD20 was strongly phosphorylated on resting B cells after CDw40 stimulation, suggesting that CD20 may be functionally regulated by a protein kinase(s).     

Signal transduction via CD4, CD8, and CD28 in mature and immature thymocytes. Implications for thymic selection.

The positive and negative selection of immature thymocytes that shapes the mature T cell repertoire appears to occur at an intermediate stage of development when the cells express low levels of TCR/CD3. These cells are also CD4+CD8+ and CD28+ (dull), and signals delivered by these three accessory molecules have been implicated in the selection process. We have examined the regulatory function of these accessory molecules on responses of immature thymocytes stimulated through the TCR/CD3 complex. Cross-linking CD4 or CD8 with CD3 strongly enhanced signal transduction via CD3 as assessed by protein tyrosine phosphorylation and calcium mobilization. Subsequent cell proliferation could be induced by soluble anti-CD28 mAb, which was comitogenic for cells stimulated with CD3 x CD4 or CD3 x CD8 cross-linking, but was without effect on cells stimulated with CD3 x CD3 cross-linking. A potential role for CD28 signal transduction in thymic maturation is suggested by the demonstration that the BB-1 molecule, a natural ligand for CD28, is expressed on thymic stromal cells. Taken together, our data suggest a model of thymic development in which CD4 or CD8 may enhance TCR/CD3 signaling upon coligation by an MHC molecule. If the CD28 surface receptor is simultaneously stimulated by a BB-1 expressing stromal cell, this set of interactions could lead to proliferation and positive selection. In the absence of CD28 stimulation the enhanced TCR/CD3 signals might lead to apoptosis and negative selection.     

New chromosomal syndrome: Miller-Dieker syndrome and monosomy 17p13

Summary The Miller-Dieker Syndrome (MDS) consists of lissencephaly, characteristic facies, pre- and postnatal growth retardation, plus various other birth defects. Autosomal recessive inheritance has been presumed based on four reported families with two or more affected siblings. We present substantial evidence that monosomy 17p13.3 causes the MDS phenotype. This includes two patients with ring chromosome 17, one patient with a de novo 17p13 deletion, and one patient with monosomy 17p due to an unbalanced 7p; 17p translocation. We report the first prenatal diagnosis of MDS in a 20-week fetus from this latter family. Additionally, we report a balanced translocation between chromosome 17 and different autosomes (8, 12, and 15) in three of the four familial cases of lissencephaly. The finding of a chromosomal basis for this presumed autosomal recessive disorder significantly alters genetic counseling and makes prenatal diagnosis possible in some families.United States Air Force Medical Corps     

Molecular confirmation of Wolf-Hirschhorn syndrome with a subtle translocation of chromosome 4.

Wolf-Hirschhorn syndrome is a clinically recognizable, multiple congenital anomaly syndrome usually associated with terminal deletion of the short arm of chromosome 4. A girl with clinical features of Wolf-Hirschhorn syndrome did not show an obvious deletion of chromosome 4, and a molecular defect was suspected. RFLPs of genomic DNA from the proband and her parents were studied using DNA probes from the distal region of chromosome 4p. Fluorescence in situ hybridization using a cosmid p847.351 containing the fragment 847 E-C was performed to investigate the possibility of a subtle translocation. Cytogenetic analyses done on the child and on both parents did not conclusively reveal abnormalities of chromosome 4. Molecular studies using two probes mapped to distal 4p showed the absence of the maternal haplotype in the child. These findings are thus consistent with a molecular deletion of 4p and confirm the diagnosis of Wolf-Hirschhorn syndrome. Cytogenetic experiments involving fluorescence in situ hybridization showed that the mother carried a subtle translocation between chromosomes 4 and 19, 46,XX,t(4,19)(p16.3; p13.3), which resulted in an unbalanced form in the child. Chorionic villus sampling for prenatal diagnosis in a subsequent pregnancy showed the fetus to be unaffected. This provides the first evidence, in chromosome 4p, of a molecular deletion due to a subtle, inherited translocation leading to the Wolf-Hirschhorn phenotype. Such subtle translocations may become an important mechanism for some recurrent genetic defects.     

Chromosomal assignment of a large tRNA gene cluster (tRNALeu,tRNAGln, tRNALys,tRNAArg, tRNAGly) to 17p13.1

Summary A cluster of tRNA genes (tRNA _UAG ^Leu , tRNA _CUG ^Gln , tRNA _UUU ^Lys , tRNA _UCU ^Arg ) and an adjacent tRNA _GCC ^Gly have been assigned to human chromosome 17p12–p13.1 by in situ hybridization using a 4.2 kb human DNA fragment for tRNA^Leu, tRNA^Gln, tRNA^Lys, tRNA^Arg, and, for tRNA^Gly, 1.3 kb and 0.58 kb human DNA fragments containing these genes as probes. This localization was confirmed and refined to 17p13.100–p13.105 using a somatic cell hybrid mapping panel. Preliminary experiments with the biotiny lated tRNA Leu, Gln, Lys, Arg probe and metaphase spreads from other great apes suggest the presence of a hybridization site on the long arm of gorilla (Gorilla gorilla) chromosome 19 and the short arm of orangutan (Pongo pygmaeus) chromosome 19 providing further support for homology between HSA17, GGO19 and PPY19.     

Evidence for the involvement of three distinct signals in the induction of IL-2 gene expression in human T lymphocytes

The regulation of IL-2 gene expression during T cell activation and proliferation has been investigated in primary cultures of purified human peripheral blood T cells. Prior results indicated that stimulation of T cells by anti-CD28 mAb plus PMA could induce IL-2 expression and T cell proliferation that was entirely resistant to cyclosporine. The present studies examined whether CD28 augments IL-2 expression by a unique pathway or merely acts at a point common to CD3-induced proliferation but distal to the effects of cyclosporine. The induction of maximal IL-2 gene expression required three signals provided by phorbol ester, calcium ionophore, and anti-CD28 mAb. Stimulation of cells by optimal amounts of calcium ionophore and PMA induced IL-2 mRNA that was completely suppressed by cyclosporine. The addition of anti-CD28 to T cells stimulated with PMA plus calcium ionophore induced a 5- to 100-fold increase in IL-2 gene expression and secretion that was resistant to cyclosporine. The CD28 signal was able to increase steady state IL-2 mRNA levels even in cells treated with maximally tolerated amounts of calcium ionophore and PMA. The three-signal requirement did not reflect differential regulation of lymphokine gene expression between the CD4 and CD8 T cell subsets or differences in the kinetics of IL-2 mRNA expression. The signal provided by CD28 is distinct from that of CD3 because although anti-CD28 plus PMA-induced proliferation is resistant to cyclosporine, anti-CD3 or anti-CD3 plus PMA-induced IL-2 expression is sensitive. Thus, these studies show that three biochemically distinct signals are required for maximal IL-2 gene expression. Furthermore, these studies suggest that lymphokine production in T cells is not controlled by an "on/off" switch, but rather, that CD28 regulates a distinct intracellular pathway which modulates the level of IL-2 production on a per cell basis. The observation that CD28 stimulation results in IL-2 concentrations that exceed 1000 U/m1 in tissue culture supernatants suggests that a role in vivo for CD28 might be to amplify immune responses initiated by the CD3/T cell receptor complex. Finally, the observation that CD28 interacts with the signals provided by PMA and calcium ionophore shows that the function of CD28 is not merely to act as a scaffold to stabilize or enhance signalling through the CD3/TCR complex.     

Inhibition of virus production in peripheral blood mononuclear cells from human immunodeficiency virus (HIV) type 1-seropositive donors by treatment with recombinant HIV-like particles.

We have previously reported on the assembly of recombinant human immunodeficiency virus (HIV)-like particles that contain gag structural proteins and present env glycoproteins gp120 and gp41 on their surfaces (O. Haffar,. J. Garriques, B. Travis, P. Moran, J. Zarling, and S.-L. Hu, J. Virol. 64:2653-2659, 1990). On the basis of their structures, we hypothesized that the recombinant particles would interfere with virus infection and tested our hypothesis in vitro by using peripheral blood mononuclear cells (PBMC) from HIV type 1-seropositive donors. Addition of the recombinant particles to PBMC concomitant with stimulation by anti-CD3 inhibited virus production, as determined by reduced levels of p24 in the culture supernatants. This inhibition of p24 production correlated with lower levels of cell-associated viral DNA. Several lines of evidence suggested that the recombinant particles exerted their antiviral effects primarily by inhibiting virus production from latently infected cells and not by inhibiting subsequent virus spread. Importantly, CD4+ T-cell stimulation by specific antigen or by anti-CD3 was not inhibited by treatment with the recombinant particles. This apparent selective inhibition of virus replication in infected PBMC represents a novel property of the recombinant HIV-like particles.     

Identification of the precursor protein to basement membrane heparan sulfate proteoglycans

The precursor protein of a basement membrane specific heparan sulfate proteoglycan has been identified as a 400,000 Mr polypeptide. Antibodies against large and small forms of this proteoglycan, isolated from a basement membrane (Engelbreth-Holm-Swarm, EHS) tumor, immunoprecipitated the same 400,000 protein from pulse-labeled EHS cells. The proteoglycan precursor protein was not recognized by antibodies against other basement membrane components or by antibodies to the cartilage proteoglycan. Furthermore, heparan sulfate proteoglycan purified from the EHS tumor blocked the immunoprecipitation of the precursor protein. Pulse-chase studies with [35S]methionine showed the precursor protein was converted to a proteoglycan. Pulse-chase studies with 35SO4 showed the large, low density proteoglycan appeared first and was degraded to a smaller, high density proteoglycan. We propose that the precursor protein is used after very little or no modification in the assembly of a large, low density heparan sulfate proteoglycan and that a portion of the population of these macromolecules are subsequently degraded to a smaller form.     

Antibodies to Tp67 and Tp44 augment and sustain proliferative responses of activated T cells

We have shown previously that binding of a monoclonal antibody (MAb) to Tp44 molecules increased the proliferation of anti-CD3-activated T cells by causing enhanced IL 2 receptor expression and IL 2 release. We now show that anti-CD5 (Tp67) antibodies have a similar effect under conditions in which monocytes are suboptimally activated or where monocytes are not present. The activity did not depend on antibody isotype or on the precise CD5 epitope recognized. Functional experiments indicated that both IL 2 production and IL 2 receptor expression were enhanced by antibody binding. Anti-Tp67 and anti-Tp44 appear to augment proliferation through distinct mechanisms, because both antibodies together had greater activity than either antibody alone. In neither system is the Fc portion of the antibody required, because F(ab')2 fragments had activity equivalent to that of the intact antibody and were effective at concentrations as low as 10 ng/ml. Fab fragments of anti-Tp67 were active, but Fab fragments of anti-Tp44 had no effect. Anti-Tp67 and anti-Tp44 were able to sustain continuous proliferation of anti-CD3-Sepharose-stimulated T cells for up to 2.5 wk without exogenous IL 2 or feeder cells. These experiments suggest that Tp67 and Tp44 are receptors that play a critical regulatory role in the control of T cell proliferation.