Interleukin 7-induced expression of specific T cell receptor gamma variable region genes in murine fetal liver cultures.

We previously reported that culture of murine fetal liver (FL) cells with interleukin 7 (IL-7) results in expression of high levels of T cell receptor (TCR) gamma transcripts by a population of cells expressing Thy-1 and Pgp-1, suggesting that IL-7 promotes the growth and/or differentiation of pre-T cells. We demonstrate herein that culture of FL cells for 7 d with IL-7 caused the rearrangement and expression of TCR gamma variable (V) region genes V gamma 4 and V gamma 6, but not V gamma 5 or V gamma 7. Since this effect was not blocked by hydroxyurea, it appeared to represent induction of expression of these genes by IL-7 rather than expansion of a preexisting positive population. We also show that IL-7 induced RAG-1 and RAG-2 mRNA expression by FL cells. These data provide evidence that specific TCR gamma/delta V region genes can be rearranged and expressed by T lineage cells before their migration to the thymus, in response to IL-7.

Expression of diverse and functional TCR gamma and Ig heavy chain transcripts in fetal liver cells cultured with interleukin-7.

We have previously shown that specific T cell receptor (TCR) gamma V regions genes (V gamma 4 and V gamma 6) are rearranged and expressed by murine fetal liver (FL) cells cultured with IL-7. The present studies determined that the sequences of the TCR V region gene transcripts expressed in response to IL-7 included diverse and functional sequences expressed by thymocyte and peripheral V gamma 4+ and V gamma 6+ T cells, indicating that the IL-7-induced expression of these genes is functionally relevant and mimics normal in vivo developmental events of gamma delta T cells. We found that more than 50% of these TCR transcripts had N region diversity. The presence of N region diversity indicates that these TCR rearrangements took place in vitro, presumably in response to IL-7, because fresh (uncultured) FL cells do not produce detectable terminal deoxynucleotidyl transferase (TdT) mRNA or protein. We also found that 100% of immunoglobulin (Ig) VH7183-JH4 transcripts from FL cells cultured with IL-7 had N region diversity at the V-DJ region, while only 40% of Ig VH7183-JH4 transcripts from FL cells cultured in the absence of IL-7 had N region diversity at this region. FL cell cultures supplemented for 7 days with IL-7 had increased TdT mRNA and protein levels. However, since 1-day culture of FL cells with or without IL-7 resulted in induction of expression of TdT, IL-7 probably does not directly stimulate TdT expression, but increases the development and expansion of TdT+ lymphoid cells. These findings implicate IL-7 as a regulator of the molecular signals involved in controlling TCR gamma rearrangement and diversity, and provide an in vitro system for studying the regulation of TdT and N region diversity in B and T lymphoid progenitors by environmental signals.

Requirement for surface aminopeptidase activities during development of CD8+ fetal thymocytes.

The role of surface aminopeptidases (APs), enzymes that cleave amino-terminal residues from polypeptide chains, in the development of fetal thymocytes was studied using a murine fetal thymic organ culture (FTOC) model. FTOC AP activity was demonstrable for various amino acid-p-nitroanilide substrates, and specific inhibitors of AP (amastatin and bestatin) inhibited enzymatic activity. AP activity decreased from Day 4 to Day 7 in FTOC. Inhibition of AP activity during thymic development by FTOC in the presence of bestatin caused a significant selective decrease in the percentage of CD8+ cells (both CD4+CD8+ and CD4-CD8+). Bestatin did not downregulate expression of CD8 by a mature CD8+ T cell clone. These data suggest that APs are involved in the development of thymocytes expressing CD8.

NKR-P1dim/TCR alpha beta + T cells and natural killer cells share expression of NKR-P1A and NKR-P1D.

Natural killer (NK) cells and a T cell subset express NKR-P1s; however, it is not known if NKR-P1s on these cells are homologous. By molecular and biochemical means, we determined that NKR-P1s on NK cells and T cells were similar. Also, sequencing of polymerase chain reaction products derived from these populations indicated expression of a novel form, termed NKR-P1D, with approximately 60% nucleotide homology to NKR-P1A. NKR-P1D shares approximately 50% amino acid homology, overall and in the carbohydrate recognition domain, with rat NKR-P1A and mouse NKR-P1A -B, and -C. Transcripts for NKR-P1A (1.1 kb) and NKR-P1D (2.0 kb) were produced by NK cells and NKR-P1dim/TCR alpha beta + T cells. Some NK cell clones coexpressed NKR-P1A and NKR-P1D. However, other clones lacking NKR-P1A produced message for NKR-P1D.

Prolactin receptor expression by rat NK cells.

We have recently begun investigating the effects of prolactin (PRL) on the function, or growth and differentiation of rat NK cells. Initially, we sought to determine the expression of a receptor for PRL (PRLr) on rat NK cells and to determine the effects of interleukin 2 (IL-2) on its expression. By a combination of reverse transcription/polymerase chain reaction (RT/PCR) and Southern blotting, we were able to identify a PRLr specific transcript using RNA isolated from highly purified, IL-2-activated NK (A-NK) cells. Using primers specific for unique portions of the major forms of PRLr transcripts in liver and ovary, it was determined that the NK cell PRLr has some homology with the major form of transcript for PRLr found in rat liver. By flow cytometric and 251-PRL binding analyses, we determined that A-NK cells expressed detectable levels of PRLr internally. Surface PRLr was not detectable by flow cytometry on freshly isolated NK cells from normal rats, and incubation of NK cells with rIL-2 did not induce detectable surface PRLr. Similarly, incubation of NK cells with PRL was not found to induce expression of the alpha chain of the IL-2 receptor (IL-2ra; CD25) on NK cells or other lymphoid cells. However, we were able to detect surface expression of PRLr on NK cells from bromocriptine-treated rats. Taken together, these data indicate that both cultured and freshly isolated populations of NK cell express PRLr.

TCR-gamma genes are rearranged but not transcribed in IL-7R alpha-deficient mice.

IL-7, a cytokine produced by bone marrow and thymic stroma, is a growth factor for B and T lymphocytes very early in their development. The IL-7R is a heterodimer of an alpha-chain that specifically binds IL-7 and the common gamma-chain, gamma(c), which is also a component of the receptors for IL-2, IL-4, IL-9, and IL-15. IL-7 has also been hypothesized to play a role in the differentiation of gammadelta T cells, which is supported by the recent findings that mice deficient in the alpha-chain of the IL-7R (IL-7R alpha -/-) or IL-7 (IL-7 -/-) have a complete absence of gammadelta T cells, but not alphabeta T cells. We show in this work that Vgamma4 and Vgamma6 TCR genes are rearranged, and sterile Vgamma4 and Vgamma6 TCR-gamma transcripts are expressed in IL-7R alpha -/- thymocytes, but these TCR-gamma genes, and Vgamma5, are not transcribed in thymocytes from IL-7R alpha -/- mice. RAG-1 and RAG-2 genes are transcriptionally active in fetal and adult IL-7R alpha -/- thymocytes. The IL-7-inducible transcription factor, STAT5, is not active in the fetal thymus of IL-7R alpha -/- compared with IL-7R alpha +/+ mice. These data point to a specific role for IL-7/IL-7R signaling in regulating the transcriptional activity, possibly mediated by STAT5, of the rearranged TCR-gamma complex during development of gammadelta T cells, and point to mechanistic differences in the regulation of rearrangement of Vgamma4 and Vgamma6 genes vs Vgamma5.