Present difficulties and future promise of MHC multimers in autoimmune exploration.

Class I tetramers have been used to track cytotoxic T cells during bacterial and viral infections. During the past year, the use of such molecules has revealed important information about the role of CD8(+) T cells in autoimmune diabetes. Furthermore, class II multimers have been produced and successfully used to stain autoreactive CD4(+) T cells from patients with rheumatoid arthritis or Borrelia-burgdorferi-induced Lyme arthritis.

Listeria monocytogenes as a short-lived delivery system for the induction of type 1 cell-mediated immunity against the p36/LACK antigen of Leishmania major.

Listeria monocytogenes has been used as an experimental live vector for the induction of CD8-mediated immune responses in various viral and tumoral experimental models. Susceptibility of BALB/c mice to Leishmania major infection has been correlated to the preferential development of Th2 CD4 T cells through an early production of interleukin 4 (IL-4) by a restricted population of CD4 T cells which react to a single parasite antigen, LACK (stands for Leishmania homologue of receptors for activated C kinase). Experimental vaccination with LACK can redirect the differentiation of CD4(+) T cells towards the Th1 pathway if LACK is coadministrated with IL-12. As IL-12 is known to be induced by L. monocytogenes, we have tested the ability of a recombinant attenuated actA mutant L. monocytogenes strain expressing LACK to induce the development of LACK-specific Th1 cells in both B10.D2 and BALB/c mice, which are resistant and susceptible to L. major, respectively. After a single injection of LACK-expressing L. monocytogenes, IL-12/p40 transcripts showed a rapid burst, and peaks of gamma interferon (IFN-gamma)-secreting LACK-specific Th1 cells were detected around day 5 in the spleens and livers of mice of both strains. These primed IFN-gamma-secreting LACK-reactive T cells were not detected ex vivo after day 7 of immunization but could be recruited and detected 15 days later in the draining lymph node after an L. major footpad challenge. Although immunization of BALB/c mice with LACK-expressing L. monocytogenes did not change the course of the infection with L. major, immunized B10.D2 mice exhibited significantly smaller lesions than nonimmunized controls. Thus, our results demonstrate that, in addition of its recognized use for the induction of effector CD8 T cells, L. monocytogenes can also be used as a live recombinant vector to favor the development of potentially protective IFN-gamma-secreting Th1 CD4 T lymphocytes.

Presentation of the Leishmania antigen LACK by infected macrophages is dependent upon the virulence of the phagocytosed parasites.

We have previously demonstrated that murine macrophages (Mphi) infected with Leishmania promastigotes, in contrast to Mphi infected with the amastigote stage of these parasites, are able to present the Leishmania antigen LACK (Leishmania homologue of receptors for activated C kinase) to specific, I-Ad-restricted T cell hybrids and to the T cell clone 9.1-2. These T cells react with the LACK (158-173) peptide, which is immunodominant in BALB/c mice. Here, we show that the level of stimulation of the LACK-specific T cell hybridoma OD12 by promastigote-infected Mphi is clearly dependent upon the differentiation state of the internalized parasites. Thus, shortly after infection with log-phase or stationary-phase promastigotes of L. major or of L. amazonensis, Mphi strongly activated OD12. The activity was transient and rapidly lost. However, under the same conditions, activation of OD12 by Mphi infected with metacyclic promastigotes of L. major or of L. amazonensis was barely detectable. At the extreme, Mphi infected with amastigotes were incapable to stimulate OD12. Thus, the presentation of LACK by infected Mphi correlates with the degree of virulence of the phagocytosed parasites, the less virulent being the best for the generation/expression of LACK (158-173)-I-Ad complexes. While the intracellular killing of the parasites appears to be an important condition for the presentation of LACK, it is not the only requisite. The partial or total destruction of intracellular L. amazonensis amastigotes does not allow the presentation of LACK to OD12. A preferential interaction of LACK (158-173) with recycling rather than newly synthesized MHC class II molecules does not explain the transient presentation of LACK by Mphi infected with log-phase or stationary-phase promastigotes because brefeldin A strongly inhibited the presentation of LACK to OD12. Taken together, these results suggest that virulent stages of Leishmania, namely metacyclics and amastigotes, have evolved strategies to avoid or minimize their recognition by CD4+ T lymphocytes.

Expression cloning of a protective Leishmania antigen.

Parasite-specific CD4+ T cells have been shown to transfer protection against Leishmania major in susceptible BALB/c mice. An epitope-tagged expression library was used to identify the antigen recognized by a protective CD4+ T cell clone. The expression library allowed recombinant proteins made in bacteria to be captured by macrophages for presentation to T cells restricted to major histocompatibility complex class II. A conserved 36-kilodalton member of the tryptophan-aspartic acid repeat family of proteins was identified that was expressed in both stages of the parasite life cycle. A 24-kilodalton portion of this antigen protected susceptible mice when administered as a vaccine with interleukin-12 before infection.

Coordinated posttranscriptional control of gene expression by modular elements including Alu-like repetitive sequences.

We previously reported that in rat fibroblasts, accumulation of a set of mRNAs ("pIL genes") was modulated as a function of cell growth and transformation, at a posttranscriptional stage, and by a mechanism that depends on a short nucleotide sequence containing an ID repetitive element. In mouse fibroblasts, hybridization with rat pIL probes identified mRNAs with the same pattern of expression, which did not contain ID sequences but contained a different regulatory element, encompassing a repetitive sequence of the B1 family. Expression in mouse cells of a reporter beta-globin gene carrying this element inserted in its 3' noncoding region was growth- and transformation-dependent. The nucleotide sequences of two murine and of three rat pIL cDNAs showed clear similarities in the region immediately adjacent to the ID and B1 repeats. Both the repeat and the flanking sequence were required to confer on beta-globin constructs the pattern of expression characteristic of the pIL genes. The hypothesis is presented that repetitive sequences in the eukaryotic genome might be modular parts of complex regulatory elements ensuring the coordinated expression of various mRNA species.

A transcriptional switch between the Pig-1 and Sgs-4 genes of Drosophila melanogaster.

Pig-1 and Sgs-4 are a pair of closely linked and divergently transcribed Drosophila melanogaster genes, which are both expressed in larval salivary glands but at different times during development. While Sgs-4 is expressed at high levels only at the end of the third instar, Pig-1 exhibits a major peak of expression during late second and early third instar. Thus, Pig-1 expression declines as Sgs-4 expression is induced. In this paper, we show that three adjacent elements located within the short region between these genes can account for the switch from Pig-1 to Sgs-4 expression. A 170-bp segment acts as an enhancer to direct Sgs-4 expression in late-third-instar salivary glands. A 64-bp sequence located just upstream from the enhancer can modify its temporal specificity so that it works throughout the third instar. Expression induced at mid-third instar by a combination of these two elements can be repressed by a negative regulatory sequence located still further upstream. We present evidence suggesting that the changing interactions between these regulatory elements and the Sgs-4 and Pig-1 promoters lead to the correct pattern of expression of the two genes.

Tumorigenic transformation of rat FR3T3 fibroblasts carrying an activated myc oncogene requires subsequent mutational events.

Early passage FR3T3 rat cells were co-transfected with a neo resistance gene and pSVc-myc-1, an SV40-driven expression vector carrying an activated murine myc oncogene. Selection in G418 medium produced clones expressing the exogenous myc gene at various levels, with a concomitant loss of expression of the normal c-myc allele. These clones were phenotypically normal, but, in fluctuation tests performed according to Luria and Delbrück (1943) on subcultures independently derived from the same clone, transformed foci appeared as stochastic events with a wide range of fluctuation. These results indicate that expression of the oncogene was not sufficient to induce the appearance of transformed growth properties, and that secondary genetic changes are required, most likely mutations in cellular proto-oncogenes. Within a single clone, independent transformants exhibited different tumorigenic potentials, spanning from high efficiency to no detectable tumor induction, without any clear correlation with their degree of in vitro transformation. Tumors and cell lines established from independent tumors, while maintaining the exogenous myc gene without gross rearrangement of its structure, no longer expressed the oncogene and resumed the expression of the normal allele.

Transfer of 'immortalizing' oncogenes into rat fibroblasts induces both high rates of sister chromatid exchange and appearance of abnormal karyotypes.

Cell lines established after transfer into FR3T3 rat fibroblast cells of 'immortalizing' oncogenes (plt gene (large T protein) of polyoma virus, v-myc gene of MC29 virus, rearranged forms of c-myc) exhibited increased rates of sister chromatid exchange (SCE). This was observed neither in cells which expressed one of the oncogenes responsible for the terminal stages of tumorigenic transformation (polyoma virus pmt (middle T protein), mutated ras genes), nor in cell lines carrying oncogenes of both types. Abnormal chromosome numbers were observed in cell lines expressing plt or myc, but not after transformation by pmt or ras oncogenes.

Sister chromatid exchange in FR3T3 rat fibroblasts transformed by Simian virus 40.

The frequency of Sister Chromatid Exchange (SCE) was determined at low (33 degrees C) and high (40.5 degrees C) temperatures in cell lines derived from FR3T3 rat fibroblast cells after transformation either with Wild-Type Simian Virus 40 (SV40-WT), with an origin-defective SV40 (SV40-ori-), or with the early temperature-sensitive mutant tsA30. Of these cell lines, SV40-WT-, SV40-ori--, and one class of tsA30-transformants (A-type) express the transformed phenotype both at 33 and 40.5 degrees C. The other tsA30-transformants (N-type) revert to a normal phenotype at high temperature. As compared with normal FR3T3 cells, all transformants exhibited, at 33 degrees C, increased numbers of metaphases with high SCE rates. At 40.5 degrees C, all cell lines which expressed a transformed phenotype (SV40-WT, tsA30 type A, SV40-ori-) exhibited substantially increased SCE rates. That this increase was not related to a possible induction of viral replication by BrdU, was proven by Southern blot analysis and by SCE data on SV40-ori--transformed cells. By contrast, no such temperature-induced increase of SCE rates was observed in tsA30-transformants of type N.

Biological activities of v-myc and rearranged c-myc oncogenes in rat fibroblast cells in culture.

Two distinct forms of the myc oncogene were assayed for their ability to induce, in cultured rat fibroblast cells, the alterations of cellular growth controls observed upon transfer of the gene of polyoma virus encoding only the large T protein (plt). Both of these rearranged myc genes and the plt gene had been previously shown to cooperate with ras oncogenes for transformation of rat embryo fibroblasts (REF) and were thought to induce the same early step ("immortalization") of the tumoral transformation pathway. We now report that these two different oncogenes elicite the same response in the following biological assays: (i) reduction of the requirements in serum factors for growth in culture of cells of the established FR3T3 line; (ii) expression of transformed properties in low serum medium after transfer into FR3T3 cells expressing only the middle T protein of polyoma virus (MTT lines); (iii) conferring on REF cells the ability to grow as clonal colonies after seeding at low cell density; (iv) conferring on REF cells the ability to grow continuously in cell culture. These congruent phenotypes suggest that the activities of the large T and myc proteins result in the induction of the same molecular events. These results also provide simple biological assays and selective systems for oncogenes of the myc class.

Simian virus 40 illegitimate recombination occurs near short direct repeats.

We have analysed nucleotide sequences at the junction between simian virus 40 (SV40) and cellular DNA in the Fisher rat transformed line tsA30-N2. This line contains a single insertion of one complete SV40 genome with a terminal duplication of 267 nucleotides, the recombination sites being located at nucleotides 439 and 705 in the late region of SV40. These two positions are located within short direct repeats in the virus genome. In order to test the significance of such repeats with respect to illegitimate recombination events, we analysed two series of published sequences of SV40 recombination sites: the first one consists of eight SV40 insertion endpoints derived from four SV40-transformed cell lines; the second one consists of 18 junction points from SV40 evolutionary variants. Our analysis demonstrates that in both cases, recombination preferentially takes place near short direct repeats in the virus genome. A model involving a "slipped mispairing" mechanism is proposed in order to account for this finding.

Integration sites and sequence arrangement of SV40 DNA in a homogeneous series of transformed rat fibroblast lines.

The state and organization of viral DNA sequences present in independently isolated rat cell lines transformed with SV40 were investigated using restriction-enzyme cleavage of the cellular DNA and blot hybridization with a viral probe. The transformed lines were established under conditions as identical as possible, except for a limited number of variables (multiplicity of infection, physiological state of the cells after infection and procedures used for selecting the transformed derivatives). They were characterized after a limited number of generations in culture. Two distinct types of organization were found: covalently integrated viral genomes were present either as single inserts or as head-to-tail oligomeric structures. The latter was observed among transformants derived from cells maintained after infection under growth-inhibiting conditions (suspension in agarose medium, confluency on a solid substrate). Single inserts were observed only among cell lines isolated after an initial period of active growth. Recurrent patterns of hybridizaton were observed in independently isolated lines, indicating that the sites of the integrative recombinations were close enough, both in the viral and the cellular sequences, not to be distinguished at the level of sensitivity of the technique (more than +/- 100 bp). Among cell lines with multiple integration sites, only part of the inserts were found in several instances to be identical to inserts observed in other transformed lines.