The murine homolog of the HIN 200 family, Ifi 204, is constitutively expressed in myeloid cells and selectively induced in the monocyte/macrophage lineage.

To examine the expression of Ifi 200 genes in vivo and add new information about their function, polyclonal monospecific rabbit antibodies, designated N-term or C-term, were raised against both the N-terminus and C-terminus of the 204 protein (p204) respectively. Western blotting analysis demonstrated that p204 and D3, another member of the Ifi 200 gene family, are constitutively expressed, though at different degrees, in bone marrow, thymus and lymph nodes, and barely detectable in the spleen. Poly rI:rC treatment did not modulate their expression. Peritoneal resident macrophages (Mphi) from untreated mice were negative, but displayed high levels of both p204 and D3 on poly rI:rC treatment. A significant increase of these proteins is also observed when Mphi are cultured overnight in vitro with IFNs or LPS. Lung, kidney and brain were negative for p204 and D3 expression. These results, together with immunohistochemical analysis, demonstrate that the 204 gene has an expression pattern restricted to cells of the myelomonocytic lineage similar to that observed for the human homolog, the myeloid nuclear differentiation antigen (MNDA) suggesting its potential involvement in the differentiation and maturation of this cell lineage.

Inhibition of cell proliferation by the interferon-inducible 204 gene, a member of the Ifi 200 cluster.

The role of the IFN-inducible p204 as growth regulator was investigated by transfecting an expression vector constitutively expressing p204 into several cell lines. Like pRB and p107, p204 is a potent growth inhibitor in sensitive cells, as demonstrated by the cell focus assay. Since stable transfectants of sensitive lines constitutively overexpressing p204 could not be established in vitro, we inserted the 204 cDNA into a vector bearing an heavy-metal-inducible promoter. Here we show that proliferation of B6MEF fibroblasts lacking endogenous p204 is strongly inhibited by transient p204 expression in the nucleus. p204 delays G1 progression into the S-phase and cells accumulate with a DNA content equivalent to cells arrested in late G1. Moreover, the role of p204 in the control of cell growth in vivo was investigated by generating transgenic mice in which the Ifi 204 gene was constitutively expressed in all tissues. To this end, expression vectors bearing the 204 cDNA under the control of the SV40 viral promoter were constructed. The overexpression of the p204 transgene achieved by injecting fertilized mouse eggs with these vectors was compatible with embryo development up to the four-cell stage in an in vitro follow-up of 4.5 days. However, no viable animals with an intact copy of the transgene were obtained, suggesting that high and constitutive levels of p204 expression can impair normal embryo development. These findings indicate that p204 plays a negative role in growth regulation and provide new information about the molecular mechanisms exploited by IFNs to inhibit cell proliferation.

Restriction of the T-cell receptor V delta gene repertoire is due to preferential rearrangement and is independent of antigen selection.

To determine whether the limited V gene usage by the T-cell receptor delta (TCRD) chain is dictated by preferential rearrangement or by antigen selection, we characterized and compared the TCRDV gene repertoire of the productive with that of the unproductive allele in 80 human TCRG/TCRD clones. Six different V genes were found on the expressed allele; two of them, provisionally named DV7 and DV8, have not been described before on the surface of TCRG/TCRD T cells. Overall, six V genes and six non-V elements were isolated from the unproductive allele. Interestingly, the same set of genes was rearranged both in the productive and in the unproductive chromosome. These findings seem to suggest that antigen-independent mechanisms play a major role in the restriction of the TCRDV gene repertoire.