Biological and clinical implications of interleukin-7 and lymphopoiesis.

Interleukin 7 (IL-7) is a stromal cell-derived cytokine that stands out as being the only cytokine identified to date on which development of B and T lymphocytes is absolutely dependent. IL-7 functions primarily as a growth and antiapoptosis factor for B- and T-cell (alphabeta and gammadelta TCR+ cells) precursors, and is essential for differentiation of gammadelta TCR+ cells. IL-7 can function as a cofactor during myelopoiesis, and is capable of activating monocytes/macrophages and natural killer (NK) cells. Its receptor (IL-7R) is a heterodimer of an alpha chain that specifically binds IL-7 and the common gamma chain gammac that is also a component of the receptors for IL-2, IL-4, IL-9 and IL-15. The functions of IL-7 in normal lymphocyte development and activation have led to the demonstration of the ability of IL-7 to stimulate lymphopoiesis in lymphopenic mice, suggesting a possible clinical application of IL-7 in accelerating lymphoid reconstitution in lymphopenic patients. There have also been a number of preclinical studies pointing to the possible utility of IL-7 in antitumor clinical applications, and clinical trials involving IL-7 gene therapy of metastatic disease are underway. IL-7 has also been shown to promote engraftment of stem cells in mice receiving bone marrow transplants, pointing to a possible use of IL-7 in patients receiving bone marrow or peripheral blood stem cell transplants. Areas of IL-7 biology that are essentially unexplored include the mechanisms of regulation of the expression of IL-7 and IL-7Ralpha, as well as the mechanisms by which IL-7 is a growth and differentiation factor for gammadelta T cells but a growth factor only for alphabeta T cells.

Characterization of the stage in natural killer cell development in 14.5-day mouse fetal liver using adult bone marrow stroma.

Nonstimulated fetal liver (FL) from 14.5-day gestation mice had no natural killer (NK) cell activity and <3% expressed NK1.1. Even after short-term (3-4 day) culture of FL with the late-acting cytokines, interleukin (IL)-15 or IL-2, little or no NK activity was detected. However, longer-term (13 day) culture with IL-2 plus stroma derived from bone marrow (BM) of adult mice, resulted in extensive proliferation and differentiation to mature NK cells. Cell numbers began to increase after 4 days, and by day 13, they had increased 40-fold and 69% of the cells were NK1.1+ with high NK activity and 5%-10% were NK1.1- B220+. With stroma, but no IL-2, equivalent proliferation occurred, but differentiated cells were predominantly NK1.1- B220+, not NK cells. Culture for 13 days without stroma, but with either IL-2, IL-15, FLTK3-ligand (L) or stroma-conditioned medium, resulted in less than fivefold expansion, and minimal NK activity. Culture with combinations of FLTK3-L or ckit-L plus IL-15 or IL-2 increased both cell number and NK activity, but the increase in cell number was less than that seen with stroma plus IL-2. By limiting dilution assay on stroma plus IL-2, the precursor frequency was 1/(2660+/-292) whole FL cells and the absolute number, but not the frequency, increased during culture on stroma without IL-2. The NK cell progenitors were found in sorted NK1.1- and Sca-1+ c-kit+ lineage- subpopulations at a frequency of 1/(156+/-52.5). Together, these data suggest that the NK lineage cells in FL are primarily in early stages of development. They are highly proliferative, respond to early acting cytokines and express stem cell markers.

Matrix metalloproteinases produced by rat IL-2-activated NK cells.

We have previously documented that adoptively transferred IL-2-activated NK (A-NK) cells can accumulate within cancer metastases. Electron microscopic studies of pulmonary metastases have revealed that adoptively transferred A-NK cells that accumulate within metastases bind to endothelial cells and are able to traverse basement membranes. We have now extended these morphologic studies. We report that rat A-NK cells produce two matrix metalloproteinases: MMP-2 and MMP-9, as determined by SDS-PAGE gelatin zymography. These activities are inhibited following incubation with BB-94 (batimastat), a specific inhibitor of matrix metalloproteinases but not with 3,4-dichloroisocoumarin, an inhibitor of neutral serine proteases. The identity of MMP-2 was confirmed by Western blots using a polyclonal Ab against human MMP-2, whereas reverse transcriptase-PCR analysis of mRNA extracts of A-NK cells has confirmed the presence of MMP-9. In addition, we report for the first time that A-NK cells can migrate through a model basement membrane-like extracellular matrix. Moreover, the ability of A-NK cells to migrate through this model basement membrane was partially inhibited by BB-94; however, BB-94 has no effect on A-NK cell-mediated cytotoxicity, suggesting that matrix metalloproteinases do not contribute to cytolytic function of A-NK cells. In sum, our studies show that A-NK cells employ BB-94-inhibitable matrix metalloproteinases to degrade extracellular matrices. This suggests that matrix metalloproteinases may play a role in the accumulation of A-NK cells within cancer metastases.

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.

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.

Therapy of murine tumors with p53 wild-type and mutant sequence peptide-based vaccines.

The BALB/c Meth A sarcoma carries a p53 missense mutation at codon 234, which occurs in a peptide, termed 234CM, capable of being presented to cytotoxic T lymphocytes (CTL) by H-2Kd molecules (Noguchi, Y., E.C. Richards, Y.-T. Chen, and L.J. Old. 1994. Proc. Natl. Acad. Sci. USA. 91:3171-3175). Immunization of BALB/c mice with bone marrow-derived dendritic cells (DC), generated in the presence of granulocyte macrophage colony-stimulating factor and interleukin 4, and prepulsed with the Meth A p53 mutant peptide, induced CTL that specifically recognized peptide-pulsed P815 cells, as well as Meth A cells naturally expressing this epitope. Immunization with this vaccine also protected naive mice from a subsequent tumor challenge, and it inhibited tumor growth in mice bearing day 7 subcutaneous Meth A tumors. We additionally determined that immunization of BALB/c mice with DC pulsed with the p53 peptide containing the wild-type residue at position 234, 234CW, induced peptide-specific CTL that reacted against several methylcholanthrene-induced BALB/c sarcomas, including CMS4 sarcoma, and rejection of CMS4 sarcoma in vaccination and therapy (day 7) protocols. These results support the efficacy of DC-based, p53-derived peptide vaccines for the immunotherapy of cancer. The translational potential of this strategy is enhanced by previous reports showing that DC can readily be generated from human peripheral blood lymphocytes.

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.

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.

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.

Characterization and comparison of the lytic function of NKR-P1+ and NKR-P1-rat natural killer cell clones established from NKR-P1bright/TCR alpha beta-cell lines.

From sorted rat NKR-P1bright/T cell receptor (TCR) alpha beta-cells, we established interleukin (IL)-2-dependent cell lines with the morphology, phenotype and function of natural killer (NK) cells. The cell lines NKbr11.3 and NKbr1.28 had large-granular-lymphocyte morphology, were capable of lysing NK-and lymphokine-activated-killer-susceptible target cells, and had the phenotype NKR-P1bright/CD3-/CD4-/CD5-/CD25-/gp42+/TCR alpha beta-. Both cell lines mediated reverse antibody-dependent cellular cytotoxicity via NKR-P1. NKR-P1-subpopulations, identical in all other aspects of phenotype, spontaneously developed in both cell lines. Cloning of NKbr11.3 and NKbr1.28 by limiting dilution resulted in two NKR-P1+ clones, 11.3(6B) and 1.28(3D), and three NKR-P1- clones, 11.3(8A), 11.3(10B), and 1.28(9F). The NKR-P1- clones were lytic and their target preference resembled that of the parental lines, except that C1498 and P815 appeared to be poor targets for 11.3(8A) and 11.3(10B). These cells represent the first reported rat IL-2-dependent NK cell lines and clones. They will be useful for the study of NK cell function as well as the function and expression of NKR-P1.

Immunocytochemical demonstration of prostaglandin synthase (cyclooxygenase) in thymic macrophages of normal and cyclosporin-treated rats.

As revealed with ED1 and ED2 monoclonal antibodies, macrophages are scattered throughout the thymic tissue. However, in contrast to the cortex and medulla, in the cortico-medullary zone macrophages are large and show strong reactivity with rabbit polyclonal antisera to cyclooxygenase. Only few smaller cortical macrophages also show weaker presence of prostaglandin synthase. After cyclosporin treatment cortical macrophages become strikingly similar to the macrophages of the cortico-medullary zone of the normal thymus. Cortical macrophages become enlarged and develop the strong expression of prostaglandin synthase. Our results show that a specific type of macrophages (with distinct histochemical characteristics, enzyme profile and ultrastructural organization, which is strategically positioned within the thymic tissue--as we demonstrated earlier) possesses the enzyme capacity required for prostaglandin synthesis. After cyclosporin treatment, which interferes with the maturation of thymocytes, cortical macrophages thoroughly change and develop the strong prostaglandin synthase expression, similar to that of normal cortico-medullary zone macrophages.

Promoter activity of the proliferating-cell nuclear antigen gene is associated with inducible CRE-binding proteins in interleukin 2-stimulated T lymphocytes.

The proliferating-cell nuclear antigen (PCNA) gene encodes an auxiliary factor of DNA polymerase delta and functions in DNA replication during S phase. It is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the regulatory role of the 5'-flanking sequence of the murine PCNA gene in interleukin 2 (IL-2)-responsive cloned T cells (L2). Analysis of a set of deletion constructs in transient transfection assays measuring heterologous reporter gene (luciferase) activity demonstrated that the 182-bp 5'-flanking region provides full promoter activity in IL-2-stimulated L2 cells. While many elements contribute to PCNA promoter strength in IL-2-stimulated cells, the largest decrease in activity occurred with deletion of the tandem CRE (cyclic AMP response element) binding sites located at nucleotides -37 to -52. With a gel mobility shift assay, several IL-2-inducible DNA-protein complexes were detected, including CREB (CRE-binding) and ATF1 (activating transcription factor) proteins that are specific for the PCNA-CRE sequence. Methylation interference analysis confirmed specific binding of these proteins to the CRE sites. Mutation at the PCNA-CRE motif abolishes IL-2-inducible binding and reduces substantially PCNA promoter activity. These results indicate that IL-2-stimulated PCNA transcription may be partially mediated by these CRE-binding proteins.

Development of murine pre-T cells into gamma delta T-cell receptor bearing cells.

Murine T cells bearing the gamma delta T-cell receptor (gamma delta TCR) are the major lymphocyte subset in the thymus early in fetal development, and postnatally they are the major population of T cells in the epithelia of nonlymphoid tissues including the intestine, skin, tongue, lung, and reproductive organs. The site of origin of gamma delta T-cell precursors (pre-T cells) changes during fetal development, reflecting the sites of active hematopoiesis. In addition, the pattern of expression of specific gamma delta TCR variable (V) region genes changes during fetal and neonatal development, and is unique in different epithelial tissues postnatally. We herein review the literature describing these developmental changes and provide a model for the developmental pathways of murine gamma delta T cells.

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.

IL 7-induced T cell receptor-gamma gene expression by pre-T cells in murine fetal liver cultures.

IL-7 has previously been shown to stimulate proliferation of immature CD4-CD8- thymocytes from fetal and adult mice. We show that IL-7 mRNA levels are much higher in the day-14 fetal thymus than in the adult thymus, i.e., when the thymus is primarily composed of CD4-CD8- cells. Inasmuch as IL-7 may, therefore, be one of the first cytokines to which pre-T cells from the fetal liver are exposed when they migrate to the thymus during development, we hypothesized that IL-7 may promote differentiation of pre-T cells from the fetal liver. We found that human rIL-7 promotes growth of cells obtained from the liver of fetal mice at day 14 of gestation. Moreover, we found that IL-7 stimulated expression of mRNA encoding TCR-gamma, alpha-, and beta-genes. The size of the transcripts induced by IL-7 suggests that TCR-gamma was expressed in a rearranged form in response to IL-7, whereas TCR-alpha and -beta genes appeared to be expressed from nonrearranged loci. Culture of fetal liver cells for 7 days with IL-7 also caused the appearance of a population of cells expressing Thy-1 and Pgp-1 Ag, a phenotype similar to that seen in the early (day-14) fetal thymus. The Thy-1+ Pgp-1+ population was enriched in TCR-gamma mRNA, as determined by flow cytometric sorting followed by Northern blot analysis. These data support the hypothesis that IL-7 can stimulate some of the early events associated with thymocyte differentiation in the fetus.

Expression of prostaglandin G/H synthase (cyclooxygenase) during murine fetal thymic development.

Fetal thymic lobes in organ culture have been shown to have the capacity to metabolize [14C]arachidonic acid (AA) to prostaglandins (PGs), including 6-ketoPGF1 alpha, PGF2 alpha, PGE2, and PGA2. Inhibition of AA metabolism results in inhibition of growth and Thy 1 expression during thymic organ culture. We report herein that freshly-isolated fetal thymic lobes also have the capacity to metabolize [14C]AA to PGs and HETEs at Days 14 and 16 of prenatal murine development. RNA encoding phospholipase A2, which liberates arachidonic acid from membrane phospholipids, and cyclooxygenase (prostaglandin G/H synthase), the first enzyme involved in the conversion of AA to PGs, are expressed during thymic development. We have localized the cyclooxygenase protein to stromal cells in the fetal and adult thymus. Exogenous AA or an analogue of PGI2 (iloprost) stimulated growth of fetal thymocytes in organ culture. These findings, together with our studies of the morphology of thymic lobes cultured with inhibitors of arachidonate metabolism, support the hypothesis that PGs are required for thymocyte proliferation during thymic development.

Nucleotide sequence of murine PCNA: interspecies comparison of the cDNA and the 5' flanking region of the gene.

Proliferating cell nuclear antigen (PCNA) RNA levels are regulated by transcription as well as changes in stability, in growing cells. We have cloned the murine PCNA cDNA and a fragment of the murine PCNA gene flanking the transcription initiation site. Comparison of the murine deduced amino acid sequence with the PCNA sequence from rat, human, Drosophila, Saccharomyces cerevisiae, and higher plants, reveals extensive homology between species. The homology is likely to be related to the fundamental role of PCNA as an auxiliary protein for DNA replication. Consensus sequences for transcriptional regulatory factors identified within 520 bp 5' of the cap site of the murine PCNA gene include: an inverted CCAAT site, an enhancer core element (EBP-1), three cAMP-response elements (CRE-BP), one AP-2 site, three Sp1 sites, and two octamer sequences. The first 20 bp of the transcriptional unit are homologous to an initiator element, which may direct transcription from RNA polymerase II in the absence of a TATAA box. The consensus elements in the murine PCNA gene are similar in sequence and/or location to elements identified in the genes for human, Drosophilia, and yeast PCNA.