François Denizot

A Differential Molecular Biology Search for Genes Preferentially Expressed in Functional T Lymphocytes: The CTLA Genes

One approach to the isolation of molecules involved in T cell-mediated cytolysis stems from the postulate of a possible correlation between molecular phenotype and molecular functional involvement. Accordingly, CTL-specific molecules have been looked for, using a strategy based on the differential screening of a subtracted cDNA library. This led to the isolation and characterization of the following structures, expressed mostly (but no exclusively) in CTLs and inducible upon lymphocyte activation: CTLA-1 and CTLA-3 (serine-proteases), CTLA-4 (a member of the Ig superfamily) and CTLA-2 alpha and beta (homologues to the proregion of cysteine-proteases). The theoretical and practical limitations and the prospects of this type of approach are discussed.

The gene encoding L1, a neural adhesion molecule of the immunoglobulin family, is located on the X chromosome in mouse and man.

The murine and human genes for the L1 neural adhesion molecule were shown to lie on conserved regions of the X chromosome to which genes responsible for several neuromuscular diseases have been mapped and which are adjacent to the fragile site (FRAXA) associated with mental retardation. By pulsed-field gel mapping we have demonstrated physical linkage between the L1 gene and other genes located in Xq28: L1 lies between the eye pigment RCP, GCP locus and the glucose-6-phosphate dehydrogenase (G6PD) gene. This location is compatible with the implication of the L1 molecule in one of the X-linked neuromuscular diseases mapped to this region.

YAC-assisted cloning of a putative G-protein mapping to the MHC class I region

We report the successful use of whole yeast artificial chromosomes (YACs) as probes for direct positional cloning of novel expressed sequences in a given genomic fragment. The class I region of the human major histocompatibility complex, in particular the chromosomal fragment spanning the HLA-E locus, was investigated. The screening of a cDNA library with a 210-kb-long YAC clone led to the identification of a new gene, positionally conserved in the major histocompatibility complex of the mouse genome and encoding a putative GTP binding protein. Although its precise function remains unknown, the interspecies conservation of both sequence and map position suggests a regulatory or functional link with the histocompatibility cluster.

Control of T- and B-lymphocyte differentiation: Preliminary characterization of lymphocyte promoter factor(s) made by FCS-induced T-cell line and clones

Abstract A T-cell line and some of its clones, induced by fetal calf serum, and their supernates have the ability to trigger the differentiation of normal spleen cells into cytotoxic T cells and plaque-forming cells. The cytolysis-promoting factors in these supernates were investigated. From a functional point of view, they triggered the differentiation of normal rather than primed cells. From a biochemical point of view, the activity was found in two major (of 45K- 30K-dalton apparent molecular weight) and two minor (of 90K and 12K dalton) gel chromatography peaks. This pattern was found using supernates from a line and also from cloned cells. Rechromatography of each of the major peaks gave a similar multipeak pattern. The supernates of some of the investigated clones had either T- or B-cell promoting activity. These results are briefly discussed in the light of published work on other interleukins. They provide a preliminary biochemical characterization of promoter factors and pave the way for their purification and further use to study T- and B-cell differentiation.

Specific cooperative induction by KLH or invertebrate hemolymphs of mouse polyclonal T-cell-mediated cytolysis☆

Abstract We previously showed that cells from mice primed in vivo with xenogeneic vertebrate serum can generate cytolytic T cells in vitro after boosting with the same serum. We investigated whether this would occur using any antigenic stimulus. We found in fact that (1) a number of conventional antigens were relatively inefficient at inducing cytolysis, (2) in contrast, KLH (a partially purified preparation of keyhole limpet hemocyanin) was efficient at inducing cytolysis, (3) induction in this case was KLH specific while cytolysis once induced had a polyclonal specificity for syngeneic and allogeneic tumor target cells, (4) mixtures of irradiated KLH-primed cells and normal spleen cells led to the generation of cytolytic cells, which was consistent with the existence of a first population of KLH-specific “promoter” cells triggering a second population of cells to polyclonally differentiate into cytolytic cells, and (5) not only vertebrate xenosera and KLH (a component of an invertebrate hemolymph) but also invertebrate hemolymphs themselves could specifically induce T-cell-mediated cytolysis. The question is raised in the Discussion section as to the nature and possible homology of those components present in both vertebrate sera and invertebrate hemolymphs, which are especially efficient at stimulating promoter cells. Also, from these and other results it is clear to us that indirectly KLH may do much more to the T-cell immune system than just stimulate KLH-specific cells.

P815 mastocytoma cells, classical targets for cytotoxic T lymphocytes, exert natural cytotoxicity

While investigating the tissue distribution of cytotoxic-T-lymphocyte-associated (CTLA) gene transcripts, we found that some of these could be detected in mast cells. This led us to test the cytolysis exerted by a number of mast cell populations. We briefly report here that P815 cells, classically known as excellent target cells for cytotoxic T cells, exert natural cytotoxicity toward WEHI-164 target cells.

A molecular biology approach to the mechanism of T-cell-mediated cytotoxicity.

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