Faculty Bibliography

T cells bearing T cell receptor (TCR) gamma and delta chain heterodimers are first generated early in ontogeny. They form distinct subsets that differ in their TCR repertoires and tissue distribution. Disruption of the mouse TCR C delta gene segment by a gene targeting method caused the complete loss of T cells bearing TCR gamma delta chains, but had little or no effect on the development of T cells bearing TCR alpha beta chains. The analyses of TCR gamma and delta genes in the mutant mice suggest that intracellular mechanisms acting at the level of DNA rearrangement play key roles in the differential gamma and delta gene rearrangements and in the generation of the highly restricted junctional sequences during fetal thymic development

Analysis of mice carrying mutant T-cell antigen receptor (TCR) genes indicates that TCR-beta gene rearrangement or expression is critical for the differentiation of CD4-CD8- thymocytes to CD4+CD8+ thymocytes, as well as for the expansion of the pool of CD4+CD8+ cells. TCR-alpha is irrelevant in these developmental processes. The development of gamma delta T cells does not depend on either TCR-alpha or TCR-beta

We constructed a recombinant baculovirus that expressed part of a Trypanosoma cruzi flagellar repetitive antigen (FRA). Both cell- associated and secreted forms of recombinant FRA were detected in cultures of virus-infected Spodoptera frugiperda (Sf9) cells. These forms show a complex pattern after polyacrylamide gel electrophoresis and Western blot analysis using either an anti-FRA rabbit serum or human Chagasic sera. Competitive Western-blot experiments revealed that all bands react with the same antibodies as a bacterially-derived FRA. Polymerase chain reaction and Southern blots of the recombinant viral DNA also showed a complex pattern, suggesting the presence of more than one repeat unit in the viral genome. When tested against a panel of human sera from an endemic area for Chagas' disease, FRA recombinant-Sf9 culture supernatant showed the same reactivity as purified FRA produced in bacteria

We tested two Trypanosoma cruzi recombinant antigens in a diagnostic test for Chagas' disease. These antigens were a cytoplasmic repetitive antigen (CRA) and a flagellar repetitive antigen (FRA). The results indicate that the recombinant antigens give better results when used in combination than when used separately, and that the removal of the beta-galactosidase portion of the recombinant fusion proteins increases the specificity of the diagnostic test for Chagas' disease. In addition, a direct enzyme-linked immunosorbent assay (ELISA), which involves the use of peroxidase-labeled antigens to detect the immune-complexes, was developed and compared with a conventional ELISA. The results indicate that the recombinant (CRA+FRA) ELISA is better than the conventional ELISA in the diagnosis of Chagas' disease, providing 100% specificity and sensitivity in all sera tested to date. The recombinant ELISA was compared with conventional serologic tests (hemagglutination and immunofluorescence) for Chagas' disease diagnosis, and the results show that the recombinant ELISA does not give rise to false-positive results that are observed with the two other tests. The use of the recombinant ELISA should be useful in the prevention of transmission of Chagas' disease by blood transfusions

The issue of T-cell repertoire selection has been addressed recently by several laboratories. While evidence has been provided for both negative and positive selection of CD4+ and CD8+ alpha beta T cells, the molecular basis of positive selection remains unclear. In this article Juan Lafaille and colleagues describe molecular features of gamma delta T-cell selection in the fetal thymus. These features were deduced from extensive junctional sequence data of gamma delta T-cell receptor genes in fetal thymocytes. Their data suggest the active participation of a self peptide in the positive selection of gamma delta T cells

In mice gamma delta T-cell populations with distinct T-cell receptor (TCR) repertoires and homing properties have been identified. Diversified populations are found in lymphoid organs and intestinal epithelia. By contrast, the gamma delta T-cells that have been found in the murine skin are homogeneous. They express a TCR consisting of one particular V gamma 5 and one particular V delta 1 chain and seem to originate from early fetal thymocytes. We have now systematically analysed many tissues by immunohistochemistry and TCR gene sequencing aided by the polymerase chain reaction. These studies revealed a second homogeneous gamma delta T-cell subset in epithelia not of the intestine and skin, but of the vagina, uterus and tongue. The TCR expressed by this gamma delta T-cell subset consists of the same V delta 1 chain. Cells that express this particular TCR have previously been shown to be positively selected in the late fetal thymus

Nucleotide sequences of a large number of V-(D)-J junctions of T cell receptor (TCR) gamma and delta genes show that most fetal thymocytes express on their surface one of just two gamma delta TCRs known to be expressed by epidermal gamma delta T cells (s-IEL) or intraepithelial gamma delta T cells associated with female reproductive organs (r-IEL). In contrast, gamma delta TCRs expressed on adult thymocytes are highly diverse as a result of multiple combinations of gene segments as well as junctional deletions and insertions, indicating that developmental time-and cell lineage-dependent mechanisms exist that control the extent of gamma delta TCR diversity. In addition, this study revealed a new type of junctional insertion (P nucleotides), which led to a new model of V-(D)-J joining generally applicable to immunoglobulin and TCR genes

Trypanosoma cruzi genes were cloned in lambda gt11 and screened with an anti-trypomastigote antiserum. Two out of twelve clones were selected in view of their reactivity with human chagasic sera. One clone encodes a flagellar antigen (FRA) of more than 300 kDa, whereas the other corresponds to a roughly 225-kDa cytoplasmic antigen (CRA). The flagellar antigen is present in both epimastigotes and trypomastigotes, but the cytoplasmic antigen is not found in trypomastigotes. The CRA clone is entirely composed of at least 23 copies of a 42-bp repeat and the FRA gene contains at least 14 copies of a 204-bp motif. The FRA gene hybridizes to a RNA of about 10 kb, while the CRA gene detects a transcript of 5.2 kb

Swiss albino, C57BL/10J, and B10.A mice previously inoculated with an LD50 dose of T. cruzi Y strain were protected against subsequent challenge with the homologous strain, but had a parasitemia comparable to that of control mice when challenged with F strain trypomastigotes, although they were protected against mortality. In contrast, animals previously inoculated with an LD50 of T. cruzi F strain were protected both against a subsequent challenge with the homologous strain and the Y strain