Engagement of the T lymphocyte antigen receptor regulates association of son-of-sevenless homologues with the SH3 domain of phospholipase Cgamma1.

One mechanism for transducing signals downstream of lymphocyte receptor activation involves the stable association between signaling proteins. To identify protein ligands of the signal activator phospholipase Cgamma1 (PLCgamma1), we screened T cell cDNA libraries with the PLCgamma1-SH3 domain by the yeast two-hybrid assay. We observed association between the PLCgamma1-SH3 domain and the human Ras guanine nucleotide exchange factor son-of-sevenless-2 (hSos2) through a proline-rich domain interaction. Stable and abundant hSos2 / PLCgamma1 and hSos1 / PLCgamma1 complexes were observed in unstimulated T cells. The interaction between these enzymes was augmented following engagement of the T cell antigen receptor (TCR / CD3). The kinetics of protein complex enhancement correlated with TCR / CD3-induced tyrosine phosphorylation of PLCgamma1; however, those PLCgamma1 molecules in complex with hSos2 were non-phosphorylated after TCR / CD3 stimulation, in contrast to the phosphorylated PLCgamma1 associated with the linker for activation of T cells, LAT. The Grb2 adapter protein was detected in complex with hSos / PLCgamma1, suggesting a regulatory role for Grb2. SH3 domains from both Grb2 and PLCgamma1, but not RasGAP, bound directly to hSos homologues. The SH2 domain from Grb2 formed an association with the hSos / PLCgamma1 complex, which was enhanced following TCR / CD3 ligation. Together, the data suggest a mechanism for the son-of-sevenless and PLCgamma1 signal transducing enzymes in recruitment to protein complexes with potentially differential signaling consequences in T lymphocytes.

LeIF: a recombinant Leishmania protein that induces an IL-12-mediated Th1 cytokine profile.

We have evaluated the ability of the Leishmania protein LeIF to influence the Th1/Th2 cytokine responses and the generation of LeIF-specific T cell clones in the absence of adjuvant. We characterized LeIF-specific T cell responses in Leishmania major-infected and uninfected BALB/c mice. These mice develop a strong Th2 response during infection with L. major. When lymph node cells from infected BALB/c mice were stimulated in vitro with LeIF, only IFN-gamma (and no detectable IL-4) was found in the culture supernatant. In addition, LeIF down-regulated Leishmania Ag-specific IL-4 production by lymph node cells from infected BALB/c mice. Subsequently, Th responses were evaluated in naive BALB/c mice following immunization with LeIF. T cell clones derived from mice immunized with LeIF preferentially secreted IFN-gamma. Finally, to understand the basis for the preferential Th1 cytokine bias observed with LeIF, the ability of LeIF to influence the early cytokine profile was evaluated in splenocytes of SCID mice. We found that LeIF stimulated fresh spleen cells from naive SCID mice to secrete IFN-gamma by IL-12/IL-18-dependent mechanisms. The N-terminal half of the molecule (amino acid residues 1-226) maintained the ability to stimulate IFN-gamma from splenocytes of SCID mice. Finally, we also demonstrated that LeIF was able to provide partial protection of BALB/c mice against L. major. Thus, our results suggest the potential of LeIF as a Th1-type adjuvant and as a therapeutic and prophylactic vaccine Ag for leishmaniasis when used with other leishmanial Ags.

Mitogenic properties of a bispecific single-chain Fv-Ig fusion generated from CD2-specific mAb to distinct epitopes.

The combination of anti-CD2 mAb 9.6 and 9-1, specific for distinct epitopes, induces proliferation of resting human T cells. The mitogenic activity of this mAb mixture depends upon accessory cells and the 9-1 mAb Fc domain. To further study the functional properties of these mAb, their variable regions were cloned and expressed as monospecific single-chain Fv (scFv) proteins fused to the human IgG1 Fc domain (scFvIg). A novel bispecific scFvIg was constructed by cloning the two monospecific scFv binding sites in tandem, with the 9.6 scFv placed N-terminal to the 9-1 scFvIg. Monospecific scFvIg binding to CD2 was comparable to that of the corresponding parental mAb, while the bispecific scFvIg exhibited binding activity similar to that of the 9-1 scFvIg. The combination of 9.6 scFvIg and 9-1 mAb was mitogenic, whereas mixtures including the 9-1 scFvIg were non-stimulatory, confirming the unique properties of the 9-1 IgG3 Fc. Without the IgG3 tail, the bispecific 9.6/9-1 scFvIg was directly mitogenic and was a more potent mitogen than the mAb mixture, but was accessory cell dependent. Unlike the combination of mAb, the bispecific reagent did not directly mobilize calcium in T cells. In comparison to the mAb mixture, bispecific 9.6/9-1 scFvIg-mediated stimulation of a mixed lymphocyte reaction was significantly more resistant to inhibition of the CD28 co-stimulatory pathway by the inhibitor CTLA-4-Ig. These results show that expression of the 9.6 and 9-1 binding sites together on a bispecific scFvIg increased the mitogenic properties of the mAb and altered the degree of accessory cell signals required for T cell activation.

The human p167 gene encodes a unique structural protein that contains centrosomin A homology and associates with a multicomponent complex.

The characterization of novel cytoplasmic, structural, and enzymatic proteins has been enhanced by a panel of monoclonal antibodies specific for protein substrates of transforming and nontransforming c-Src mutants. These protein substrates have included the focal adhesion kinase (FAK), cortactin, AFAP-110, p120CAS, and p130CAS. The monoclonal antibody 4G8 was generated as part of this panel of antibodies and was used to isolate the human gene for a 167-kD polypeptide. The cDNA sequence is 5,238 nucleotides in length with a predicted open reading frame consisting of 1,382 amino acids. The polypeptide is largely hydrophilic and highly charged. The central region of p167 has 88% identity with the entire 278-amino-acid encoded sequence of the murine centrosomin A gene. The carboxyl third of p167 contains a unique cluster of 10 amino acid repeats with the consensus sequence (A/M)DDDRGPRRG. The p167 protein was found primarily in the cytoplasm of lymphocytes and is part of a multicomponent protein complex with prominent members of 167, 120, 64, 45, 40, 38, and 25 kD. Finally, we illustrate the conservation of p167 and its associated complex, and demonstrate its expression in different human tissues and cell types. The data suggest that p167 is novel and has an important cellular function as a cytoplasmic structural protein.

Facile preparation of nuclease resistant 3' modified oligodeoxynucleotides.

An efficient chemical procedure for the immobilization of carboxylate containing conjugate groups onto controlled pore glass (CPG) is described. The derivatized supports were used in the automated synthesis of an oligodeoxynucleotide (20-mer ODN) containing a 3' phosphodiester linked hexanol, aminohexyl, acridine, or cholesterol group. The stability of the oligomer in a hepatoma cell culture was found to be prolonged two to three fold by the presence of any of the 3' tails. By contrast, an aminohexyl group appended to the 5' terminus of the ODN only marginally improved its nuclease resistance. These data support the notion that antisense ODNs are primarily degraded by 3' exonucleases. Introduction of simple 3' tails which incorporate a normal phosphodiester linkage can increase ODN stability by interfering with these enzymes.

A novel telomeric gene conversion in Trypanosoma brucei.

Gene conversion is one mechanism of antigenic variation in Trypanosoma brucei. Variant surface glycoprotein (VSG) genes are duplicated by this process to telomeric locations from which they may be expressed. We examined four independent antigenic switches in which the IsTaR 1.1 minichromosomal VSG gene is duplicated to a large chromosome where it is expressed. An unusual feature of three of these telomeric gene conversions is that the distance between the VSG gene and the end of the chromosome is identical for both the basic and duplicated copies following the antigenic switch. This suggests that the gene conversion is initiated 5' to the VSG gene and extends to the end of the telomere. The data also suggest that events other than simple nucleotide addition account for telomeric growth.

Transcripts from the co-transposed segment of variant surface glycoprotein genes are in Trypanosoma brucei polyribosomes.

In Trypanosoma brucei the 5' proximal flanking sequences of a variant surface glycoprotein (VSG) gene, the co-transposed segment, are transcribed in a variant antigenic type- and stage-specific fashion along with the VSG gene. The precursor transcripts are subsequently processed to yield smaller transcripts from the co-transposed segment as well as the VSG mRNA. These co-transposed segment transcripts are quite abundant, polyadenylated and contain the spliced leader sequence, all characteristics of trypanosome mRNAs. We have found that all of the co-transposed segment transcripts from two VSG genes are present in polyribosomes. The nucleotide sequence of much of the co-transposed segment of one of these VSG genes, however, has no open reading frames coding for proteins longer than 49 amino acids. These results suggest that co-transposed segment transcripts do not encode essential proteins even though they are present in polyribosomes and may be translated.

Multiple events associated with antigenic switching in Trypanosoma brucei.

The genomic environments of six unrelated variant surface glycoprotein (VSG) gene families in the IsTaR 1 serodeme of Trypanosoma brucei were examined by Southern blot analysis of DNA resolved by conventional and pulse field gradient electrophoresis. The genomic locations of these gene family members were characterized in variants arising from 19 independent antigenic switches of which 13 represent single relapses. We have identified 9 distinct telomeric sites containing VSG genes on 7 different chromosomes ranging in size from approximately 65 kilobase pairs to greater than 3 megabase pairs. VSG genes are expressed in at least 6 of these telomeric sites. All of the observed antigenic switches can be explained by two types of processes: gene conversion and telomeric activation, with almost half (9/19) the result of combinations of these processes. The implications of these observations are discussed with particular reference to the frequencies of occurrence of each process.

Changes in telomere length associated with antigenic variation in Trypanosoma brucei.

In the IsTaR 1 serodeme, we have identified variant surface glycoprotein (VSG) genes in nine different telomeric sites. We have measured the distance from the 3' end of these VSG genes to the end of the chromosome (the 'telomere length') in 20 variant antigen types (VATs) of the serodeme. Analyses of the changes in telomere length during 19 antigenic switches involving eight telomeric sites indicate a median increase in telomere length of 0.6 kilobase pairs during each switch. This may be accounted for by the 6-10 bp increase in telomere length per generation associated with DNA replication described by others. The changes in telomere lengths do not form a normal distribution since a substantial fraction show unusually large increases in telomere length or decreases in telomere length during an antigenic switch. These changes are probably caused by recombinations 3' to the VSG gene. No significant differences were detected in the behavior of telomeres at each of the eight different telomeric sites, nor were changes in telomere lengths significantly different between different antigenic switches. However, it was found that those telomeres where transcription was activated during the antigenic switch showed a significantly greater increase in telomere length than those telomeres not involved in regulation of VSG gene expression. Conversely, there was a strong correlation between transcriptional inactivation of a telomeric expression site and a decrease in telomere length. These findings suggest that processes (possibly genomic recombinations) 3' to the VSG gene coding region may be associated with a change in the transcriptional status of the VSG gene.

Variant specific transcripts from the co-transposed segments of variant surface glycoprotein genes in Trypanosoma brucei.

We examined steady-state transcripts from the sequences immediately upstream of the 1 and 11 variant surface glycoprotein (VSG) genes in Trypanosoma brucei of the IsTaR 1 serodeme. These sequences, the co-transposed segment, differ in size and sequence, and produce a variety of variant-specific polyadenylated transcripts. Most, if not all, of these transcripts, some of which may be precursors, contain the spliced leader (SL). Variant specific transcription extends from 5' of the co-transposed segment to 100-400 nucleotides downstream of the major VSG mRNA polyadenylation site. The 1 VSG mRNA has at least 2, and possibly 3, SL addition sites; the major site is 120 nucleotides downstream of the polyadenylation site identified by sequencing a cDNA from the co-transposed segment. Two general models for the production of the co-transposed segment transcripts are discussed.

Molecular karyotype of species and subspecies of Leishmania.

The DNA karyotypes of three species and several subspecies of New World Leishmania were found to be distinct. The karyotypes were more similar among closely related isolates than among more distantly related groups. Two classes of chromosomal DNA differences were detected among stocks; +/- 50 kb size differences among DNAs, some of which were shown to contain homologous sequences, and DNAs having no obvious corresponding chromosomal DNA in other isolates. A total of 14-24 chromosomal DNA bands were resolved, depending on the isolate, but densitometric analyses suggest that these isolates contain 26-33 distinct DNA molecules. These molecules total about 2.5 X 10(7) bp, a substantial fraction of the genomic DNA. The chromosomal DNA locations of gene sequences homologous to alpha- and beta-tubulin, ribosomal RNA, thymidylate synthetase-dihydrofolate reductase, and the H-region sequence were determined. The homologous sequences were located on chromosomal DNAs of similar, but not identical sizes among different stocks. We also found species- and some subspecies-specific beta-tubulin chromosomal loci. We conclude that the DNA karyotype is useful for stock identification, taxonomy, and gene localization in Leishmania. Its potential for identifying the species and subspecies in natural infections appears less useful unless applied in conjunction with specific hybridization probes.

Preferential activation of telomeric variant surface glycoprotein genes in Trypanosoma brucei.

During an infection, Trypanosoma brucei expresses diverse variant surface glycoprotein (VSG) genes in a quasi-sequential order. Numerous VSG genes have intrachromosomal locations but many are located adjacent to telomeres. We have tested whether telomeric VSG genes are preferentially activated compared to intrachromosomal VSG genes during an antigenic switch. The frequency with which the IsTat 11 VSG gene is expressed in first relapse populations has been compared for variant antigenic types (VATs) A3 and A11. These VATs express the same A VSG gene from the same chromosome but VAT A11 contains an inactive telomeric 11 VSG gene which is absent in VAT A3. The 11 gene is activated at a much higher frequency in first relapse populations from VAT A11 than from VAT A3. A resultant VAT 11 clone was examined in detail and shown to have reactivated the telomeric 11 VSG gene. These results suggest that a telomeric location can result in a greater frequency of activation of a VSG gene. This preferential activation may explain, in part, the order of expression of VSG genes.