T cell deletion induced by chronic infection with mouse mammary tumor virus spares a CD25-positive, IL-10-producing T cell population with infectious capacity.

We found that T cells recognizing viral superantigen (vSAG) can be subdivided into two distinct functional subsets based on IL-2R alpha (CD25) expression. CD4+Vbeta6+CD25- and CD4+Vbeta6+CD25+ T cells were sensitive to vSAG activation. When obtained from BALB/c(SW) mice, both subsets were infected and capable to induce the tolerance process when transferred into noninfected recipients. However, in contrast to CD4+Vbeta6+CD25- cells, which were gradually deleted in MMTV(SW)-infected mice, the pool of CD4+Vbeta6+CD25+ lymphocytes was constant even at the end of the deletion process, and maintained a limited reactivity to vSAG-induced activation. The constant number of Vbeta6+CD25+ observed in infected mice could not be explained by their rapid turnover (deletion and renewal), as their proliferative rate measured by BrdU incorporation was similar in infected and naive mice, as well as in virus-nonspecific (Vbeta8.2+) cells. Neither was the Vbeta6+CD25+ subset dependent on vSAG activation since it was also present in MMTV-free mice and was not generated from Vbeta6+CD25- cells upon in vivo vSAG stimulation. Vbeta6+CD25+ T cells constitutively expressed IL-4 and IL-10 mRNA. IL-10 has been shown to be associated with viral, bacterial, and parasitic infections. This permanent CD25+ subpopulation may play a role in the control of viral infection and tolerance induction via vSAG recognition and IL-10 production.

Persistence of V beta 6+ T cells in Mls-1a mice. A role for the third complementarity-determining region (CDR3) of the T cell receptor beta chain in superantigen recognition.

We have studied V alpha 2 and J beta usage by V beta 6+CD4+ peripheral T cells isolated from the congenic mice strains BALB/c (Mls-1b) and BALB.D2 (Mls-1a). We found that the TCR beta-chain of V beta 6+CD4+ T cells present in adult Mls-1a mice differed from those in Mls-1b mice; the fraction of V beta 6+CD4+T cells using the J beta 2.7 segment was reduced, while the number of V beta 6+CD4+ T cells using J beta 1.2 was augmented. These results indicate that the CDR3 region of the TCR beta-chain participates in recognition of the Mls superantigen. We also found that in Mls-1a mice an increased fraction of V beta 6+CD4+ T cells expressed the V alpha 2 chain. The study of J beta usage by V beta 6+CD4+V alpha 2+ and V beta 6+CD4+V alpha 2- T cells indicates that both J beta segment and TCR V alpha 2 chain expression confer complementary protection against deletion by Mls-1a superantigen. These results suggest a novel view of Mls-1a-driven selection, where the CDR3 region of the V beta chain modulates superantigen recognition, and the affinity/avidity of the TCR-MHC-superantigen complex determine the fate of the T cell.

An antisense interferon-beta RNA abolishes repression of c-fos gene expression.

To determine the cellular functions which are modified when interferon-beta (IFN-beta) gene expression is inhibited, a plasmid allowing the constitutive expression of RNA complementary to IFN-beta mRNA was constructed and stably introduced into L929 cells. Some of the selected clones expressing this antisense IFN-beta mRNA, named L-ASI, were unable to produce IFN-beta and lost the ability to arrest in the G0 phase of the cell cycle. Indeed, the usual transrepression of the c-fos gene observed in quiescent cells was blocked in IFN-beta antisense L-ASI clones and the c-fos gene was permanently stimulated. This overexpression of c-fos was not modified in response to protein kinase C agonists such as phorbol esters, but increased in response to the adenylate cyclase activator forskolin. In addition, the ability to induce major histocompatibility class I genes following recombinant IFN-beta treatment was impaired in antisense IFN-beta L-ASI clones, suggesting an important alteration of this cell with regard to the interferon system. Unexpectedly, the tumorigenicity of the clones was significantly diminished. We postulate that IFN-beta antisense RNA blocks the repression of the c-fos gene and thus prevents the arrest of cells in the G0 phase of the cycle.

A common cellular pathway for v-mos and v-Ki-ras is not required for v-Ki-ras-induced tumorigenicity in a nonmalignant, v-mos-expressing revertant cell.

A revertant cell line was selected from Moloney sarcoma virus-transformed BALB/c cells after long-term treatment with type I interferon. Despite an actively transcribed and transfectable v-mos gene, these revertant cells were nontumorigenic in nude mice. The functionality of the mos protein was investigated, focusing on the alpha 2(1) collagen promoter regulation, which is known to be affected by mos-induced trans-acting factors. Both in transient expression assays and after stable integration into the cellular genome, the transfected alpha 2(1) collagen promoter fused to the cat reporter gene was activated in the revertant while being downregulated in the original transformed cells. In retransformation assays of the revertant by Moloney sarcoma virus strains homologous to the original transforming virus, no detectable change was noted in the in vitro phenotype or in tumorigenicity. These results reveal that the mos-directed factors were no longer effective on their specific targets. Thus, the R.MSVIF cell could be either an oncoprotein-deficient or a target-related revertant. Attempts at retransformation with unrelated sarcoma viruses bearing v-sis, v-fms, or v-fos oncogenes were also negative. In contrast, tumorigenicity was obtained with the unrelated Kirsten sarcoma virus without any change in the revertant morphology or collagen expression. These findings showed that the common pathway blocked by the reversion and shared by v-mos and v-ras was not required for ras-induced tumorigenicity.

Increased proto-oncogene expression in peripheral blood T lymphocytes from patients with systemic sclerosis.

The expression of c-myc, c-myb, and c-ras proto-oncogenes, determined using RNA hybridization techniques (slot-blot), was significantly increased in peripheral blood T lymphocytes, but not in B cells, from 17 patients with systemic sclerosis with diffuse scleroderma, compared with the expression in normal control subjects. The magnitude of expression of c-myc and c-myb tended to be higher in patients with early, active disease. These results demonstrate an in vivo activation of T cells from systemic sclerosis patients, which may play an important role in the pathogenesis of the disease.

Presence of a constitutive paracrine beta-interferon in v-mos-bearing nonmalignant reverted cells.

A stable nonmalignant revertant cell line was derived from Moloney murine sarcoma virus-transformed BALB/c cells after long-term cultivation in the presence of murine type I interferon (IFN). These cells gradually established resistance to exogenous IFN and were also seen to contain IFN-dependent proteins. The presence of an endogenous IFN was confirmed by the results of Northern blot analysis and in situ hybridization with an IFN-beta probe, showing that only mRNA specific for IFN-beta- could be found in the uninduced reverted cells. The latter synthesized only a small amount of IFN-beta protein and exhibited few IFN-specific membrane receptors, which bound recombinant IFN-beta with a high affinity. After treatment with IFN antibody, the overexpression of H-2 major histocompatibility antigen genes was significantly down-regulated. These findings strongly suggest the existence in this reverted cell line of a constitutive IFN which, acting through an autocrine and/or paracrine mechanism, might play a role in maintaining the reverted state.

Persistent expression of v-mos oncogene in transformed cells that revert to nonmalignancy after prolonged treatment with interferon.

BALB/c embryonic fibroblasts productively transformed by Moloney sarcoma virus and cultivated for over 600 generations in the presence of mouse alpha/beta interferon reverted to an apparently normal phenotype and were unable to produce tumors in nude mice. Nevertheless, the presence of an integrated Moloney sarcoma virus genome in the nonmalignant Moloney sarcoma virus-transformed interferon-treated cell DNA could be shown by focus formation upon transfection and by hybridization with a v-mos probe. After digestion with various restriction endonucleases, similar hybridization patterns of v-mos sequences were obtained with DNAs from both reverted and transformed cells. However, additional integration sites and at least twice as many copies of the oncogene were found in the nonmalignant Moloney sarcoma virus-transformed interferon-treated cell DNA. Polyadenylylated RNA extracted from reverted and control cells contained two mos-specific transcripts. Interestingly, the nonmalignant Moloney sarcoma virus-transformed interferon-treated cells produced helper virus, but no detectable mos-containing virions, suggesting that a posttranscriptional block in the v-mos gene expression had occurred in these cells. It should be stressed that, after up to 100 additional passages, cells cultured in the absence of interferon maintained their nontumorigenic character in spite of the persistent transcription of the mos oncogene.