Hematopoietic switch from lymphoid to granulocytic development in 3LL tumor-bearing mice.

A significant splenomegaly and lymphadenopathy develops during the progressive growth of Lewis Lung (3LL) tumors in mice. Enlarged spleen and lymph nodes occur because of a pronounced increase in granulocytes in these organs. This granulocytosis in spleen and lymph node was not simply due to recruitment of granulocytes from peripheral blood to spleen and lymph nodes, but also a result of development and/or differentiation of granulocytes from the bone marrow. There was a marked increase in development of myeloid lineage cells, whereas lymphoid populations including T cells and B cells, were dramatically decreased in bone marrow and peripheral blood of 3LL tumor-bearing mice. These data demonstrate that host hematopoiesis shifts from lymphoid to granulocytic development in the 3LL tumor-bearing mice. Interestingly, a somatic mutation of N-Ras gene was found in 3LL tumor cells at codon 61, suggesting that mutated N-Ras may contribute to induction of granulocytosis in 3LL tumor-bearing mice.

IFN-gamma and Fas/FasL are required for the antitumor and antiangiogenic effects of IL-12/pulse IL-2 therapy.

Systemic administration of IL-12 and intermittent doses of IL-2 induce complete regression of metastatic murine renal carcinoma. Here, we show that overt tumor regression induced by IL-12/pulse IL-2 is preceded by recruitment of CD8(+) T cells, vascular injury, disrupted tumor neovascularization, and apoptosis of both endothelial and tumor cells. The IL-12/IL-2 combination synergistically enhances cell surface FasL expression on CD8(+) T lymphocytes in vitro and induces Fas and FasL expression within tumors via an IFN-gamma-dependent mechanism in vivo. This therapy also inhibits tumor neovascularization and induces tumor regression by mechanisms that depend critically on endogenous IFN-gamma production and an intact Fas/FasL pathway. The ability of IL-12/pulse IL-2 to induce rapid destruction of tumor-associated endothelial cells and regression of established metastatic tumors is ablated in mice with a dysregulated Fas/FasL pathway. The common, critical role for endogenous IFN-gamma and the Fas/FasL pathway in early antiangiogenic effects and in antitumor responses suggests that early, cytokine-driven innate immune mechanisms and CD8(+) T cell-mediated responses are interdependent. Definition of critical early molecular events engaged by IL-12/IL-2 may provide new perspective into optimal therapeutic engagement of a productive host-antitumor immune response.

Interleukin-7 restores immunity in athymic T-cell-depleted hosts.

Thymic-deficient hosts rely primarily on antigen-driven expansion to restore the peripheral T-cell compartment following T-cell depletion (TCD). The degree to which this thymic-independent pathway can restore immune competence remains poorly understood but has important implications for a number of clinical conditions including stem cell transplantation and human immunodeficiency virus (HIV) infection. A model of HY-mediated skin graft rejection by athymic, TCD mice was used to show that restoration of naive and recall responses via peripheral expansion requires transfer of only 25 x 10(6) lymph node (LN) cells representing approximately 10% of the T-cell repertoire. Constitutive expression of bcl-2 in the expanding inocula restored recall responses to HY at a substantially lower LN cell dose (1 x 10(6)), which is normally insufficient to induce HY-mediated graft rejection in athymic hosts. Interestingly, bcl-2 had no effect on primary responses. Interleukin-7 (IL-7) potently enhanced thymic-independent peripheral expansion and led to HY graft rejection using an LN cell dose of 1 x 10(6) in both primary and recall models. The restoration of immune competence by IL-7 appeared to be mediated through a combination of programmed cell death inhibition, improved costimulation, and modulation of antigen-presenting cell (APC) function. These results show that immune competence for even stringent antigens such as HY can be restored in the absence of thymic function and identify IL-7 as a potent modulator of thymic-independent T-cell regeneration.

Gastrointestinal cells of IL-7 receptor null mice exhibit increased sensitivity to irradiation.

IL-7 is a critical cytokine in the development of T and B cells but little is known about its activity on nonhematopoietic cells. An unexpected finding was noted in allogeneic bone marrow transplant studies using IL-7 receptor null (IL-7R alpha(-/-)) mice as recipients. These mice exhibited a significantly greater weight loss after total body irradiation compared with wild type, IL-7R alpha(+/+), mice. Pathological assessment indicated greater intestinal crypt damage in IL-7R alpha(-/-) recipients, suggesting these mice may be predisposed to gut destruction. Therefore, we determined the effect of the conditioning itself on the intestinal tract of these mice. IL-7R alpha(-/-) mice and IL-7R alpha(+/+) mice were irradiated and examined for lesions and apoptosis within the small intestine. In moribund animals, IL-7R alpha(-/-) mice had extensive damage in the small intestine, including marked ablation of the crypts and extreme shortening of villi following 1500 cGy total body irradiation. In contrast, by 8 days after irradiation, the small intestines of IL-7R alpha(+/+) mice had regenerated as distinguished by normal villus length and hyperplastic crypts. Following 750 cGy irradiation, IL-7R alpha(-/-) mice had a higher proportion of apoptotic cells in the crypts and an accompanying increase in the pro-apoptotic protein Bak was expressed in intestinal epithelial cells. These results demonstrate the increased radiosensitivity of intestinal stem cells within the crypts in IL-7R alpha(-/-) mice and a role for IL-7 in the protection of radiation-induced apoptosis in these same cells. This study describes a novel role of IL-7 in nonhematopoietic tissues.

IL-7 administration alters the CD4:CD8 ratio, increases T cell numbers, and increases T cell function in the absence of activation.

IL-7 is vital for the development of the immune system and profoundly enhances the function of mature T cells. Chronic administration of IL-7 to mice markedly increases T cell numbers, especially CD8(+) T cells, and enhances T cell functional potential. However, the mechanism by which these effects occur remains unclear. This report demonstrates that only 2 days of IL-7 treatment is needed for maximal enhancement of T cell function, as measured by proliferation, with a 6- to 12-fold increase in the proportion of CD4(+) and CD8(+) T cells in cell cycle by 18 h of ex vivo stimulation. Moreover, a 2-day administration of IL-7 in vivo increases basal proliferation by 4- and 14-fold in CD4(+) and CD8(+) T cells, respectively. These effects occur in the absence of cytokine production, increases in most activation markers, and changes in memory markers. This enhanced basal proliferation is the basis for the increase in T cell numbers in that IL-7 induces an additional 60% and 85% of resting CD4(+) and CD8(+) T cells, respectively, to enter cell cycle in mice given IL-7 for 7 days. These results demonstrate that in vivo administration of IL-7 increases T cell numbers and functional potential via a homeostatic, nonactivating process. These findings may suggest a unique clinical niche for IL-7 in that IL-7 therapy may increase T cell numbers and enhance responses to specific antigenic targets while avoiding a general, nonspecific activation of the T cell population.

Pathogen-specific loss of host resistance in mice lacking the IFN-gamma-inducible gene IGTP.

Interferon-gamma (IFN-gamma) is critical for defense against pathogens, but the molecules that mediate its antimicrobial responses are largely unknown. IGTP is the prototype for a family of IFN-gamma-regulated genes that encode 48-kDa GTP-binding proteins that localize to the endoplasmic reticulum. We have generated IGTP-deficient mice and found that, despite normal immune cell development and normal clearance of Listeria monocytogenes and cytomegalovirus infections, the mice displayed a profound loss of host resistance to acute infections of the protozoan parasite Toxoplasma gondii. By contrast, IFN-gamma receptor-deficient mice have increased susceptibility to all three pathogens. Thus, IGTP defines an IFN-gamma-regulated pathway with a specialized role in antimicrobial resistance.

IFN-gamma-dependent delay of in vivo tumor progression by Fas overexpression on murine renal cancer cells.

The role of Fas in the regulation of solid tumor growth was investigated. Murine renal carcinoma (Renca) cells were constitutively resistant to Fas-mediated killing in vitro, but exhibited increased expression of Fas and sensitivity to Fas-mediated killing after exposure to IFN-gamma and TNF. Transfected Renca cells overexpressing Fas were efficiently killed in vitro upon exposure to anti-Fas Ab (Jo2). When Fas-overexpressing Renca cells were injected into syngenic BALB/c mice, there was a consistent and significant delay in tumor progression, reduced metastasis, and prolonged survival that was not observed for Renca cells that overexpressed a truncated nonfunctional Fas receptor. The delay of in vivo tumor growth induced by Fas overexpression was not observed in IFN-gamma-/- mice, indicating that IFN-gamma is required for the delay of in vivo tumor growth. However, there was a significant increase of infiltrated T cells and in vivo apoptosis in Fas-overexpressing Renca tumors, and Fas-overexpressing Renca cells were also efficiently killed in vitro by T cells. In addition, a strong therapeutic effect was observed on Fas-overexpressing tumor cells by in vivo administration of anti-Fas Ab, confirming that overexpressed Fas provides a functional target in vivo for Fas-specific ligands. Therefore, our findings demonstrate that Fas overexpression on solid tumor cells can delay tumor growth and provides a rationale for therapeutic manipulation of Fas expression as a means of inducing tumor regression in vivo.

Molecular mechanisms of immune-mediated lysis of murine renal cancer: differential contributions of perforin-dependent versus Fas-mediated pathways in lysis by NK and T cells.

Mice bearing the experimental murine renal cancer Renca can be successfully treated with some forms of immunotherapy. In the present study, we have investigated the molecular pathways used by NK and T cells to lyse Renca cells. Renca cells normally express low levels of Fas that can be substantially enhanced by either IFN-gamma or TNF-alpha, and the combination of IFN-gamma + TNF-alpha synergistically enhances cell-surface Fas expression. In addition, cells pretreated with IFN-gamma and TNF-alpha are sensitive to lysis mediated by Fas ligand (FasL)-expressing hybridomas (dllS), cross-linking of anti-Fas Abs or soluble Fas (FasL). Lysis via Fas occurs by apoptosis, since Renca shows all the typical characteristics of apoptosis. No changes in levels of bcl-2 were observed after cytokine treatments. We also examined cell-mediated cytotoxic effects using activated NK cells and T cells from gld FasL-deficient mice, and perforin-deficient mice, as well as wild-type C57BL/6 and BALB/c mice. Interestingly, the granule-mediated pathway predominated in killing of Renca by activated NK cells, while the Fas/FasL pathway contributed significantly to cell-mediated killing of Renca by activated T cells. These results suggest that killing of Renca tumor cells by immune effector cells can occur by both granule and Fas-mediated cytotoxicity. However, for the Fas-mediated pathway to function, cell surface levels of Fas need to be increased beyond a critical threshold level by proinflammatory cytokines such as IFN-gamma and TNF-alpha.

Development and characterization of a mouse prostate adenocarcinoma cell line: ductal formation determined by extracellular matrix.

BACKGROUND: Tumor vaccines show promise as a new approach for treating cancer. We have developed a murine prostate cancer cell line which can be used to study growth factor and extracellular matrix regulation of prostate differentiation and will be useful for generating tumor vaccines using the C3(1)/TAG transgenic model of prostate cancer. METHODS: Pr-14 cells were established in defined growth media (GM) and grown in GM, GM + 2% fetal bovine serum (FBS) or DMEM + 10% FBS on plastic, collagen, or Matrigel. Immunofluorescence and Western blot analyses were performed using antibodies to cytokeratin, vimentin, SV40 large T-antigen, and androgen receptor (AR). RESULTS: Pr-14 cells are cytokeratin-positive, vimentin-negative, and express SV40 large T-antigen. These cells are tumorigenic when injected into athymic nude mice and appear to be androgen-independent. Pr-14 cell lines are nontumorigenic when injected into syngeneic FVB/N mice, but form tumors in transgenic TAG-expressing FVB/N mice. Cell growth and morphology are dependent on media composition which determines whether ductal or acinar structures form when grown on Matrigel. CONCLUSIONS: The mouse prostate adenocarcinoma cell line, Pr-14, undergoes alterations in the state of differentiation dependent upon serum concentration when grown on Matrigel. The Pr-14 cell line is a useful reagent to study prostate cell/extracellular matrix interactions, and for immunotherapy and cancer vaccine studies in C3(1)/TAG transgenic mice.

Bone marrow and thymus expression of interferon-gamma results in severe B-cell lineage reduction, T-cell lineage alterations, and hematopoietic progenitor deficiencies.

Interferon-gamma (IFN-gamma) is an immunoregulatory lymphokine that is primarily produced by T cells and natural killer cells. It has effects on T-cell, B-cell, and macrophage differentiation and maturation. We have developed transgenic mice that express elevated levels of IFN-gamma mRNA and protein by inserting multiple copies of murine IFN-gamma genomic DNA containing an Ig lambda-chain enhancer in the first intron. The founder line carrying eight copies of this transgene has eightfold to 15-fold more IFN-gamma-producing cells in the bone marrow and spleen than do nontransgenic littermates. Transgenic mice show a pronounced reduction in B-lineage cells in the bone marrow, spleen, and lymph nodes. In addition, single positive (CD4+,CD8- and CD4-,CD8+) thymocyte numbers are increased twofold, yet the number of splenic T cells is reduced by 50%. There is also a twofold to threefold decrease in the frequency and total number of myeloid progenitors in the bone marrow. Granulomatous lesions and residual degenerating cartilaginous masses are also present in the bones of these mice. Overall, our data show that the abnormal expression of IFN-gamma in these transgenic mice results in multiple alterations in the immune system. These animals provide an important model to examine the role of IFN-gamma expression on lymphoid and myeloid differentiation and function.

Quantitative and cell-cycle differences in progenitor cells mobilized by recombinant human interleukin-7 and recombinant human granulocyte colony-stimulating factor.

Administration of recombinant human interleukin-7 (rhIL-7) to mice increases the exportation of myeloid progenitors (colony-forming unit [CFU]-c and CFU-granulocyte erythroid megakaryocyte macrophage [CFU-GEMM]) from the bone marrow (BM) to peripheral organs, including blood, and also increases the number of primitive progenitor and stem cells in the peripheral blood (PB). We now report that combined treatment of mice with rhIL-7 and recombinant human granulocyte-colony stimulating factor (rhG-CSF) stimulates a twofold to 10-fold increase in the total number of PB CFU-c, and a twofold to fivefold increase in the total number of PB CFU-spleen at day 8 (CFU-S8) over the increase stimulated by rhIL-7 or rhG-CSF alone. In addition, the quality of mobilized cells with trilineage, long-term marrow-repopulating activity is maintained or increased in mice treated with rhIL-7 and rhG-CSF compared with rhIL-7 or rhG-CSF alone. These differences in mobilizing efficiency suggest qualitative differences in the mechanisms by which rhIL-7 and rhG-CSF mobilize progenitor cells, in fact, the functional status of progenitor cells mobilized by rhIL-7 differs from that of cells mobilized by rhG-CSF in that the incidence of actively cycling (S-phase) progenitors obtained from the PB is about 20-fold higher for rhIL-7-treated mice than for mice treated with rhG-CSF. These results suggest the use of rhIL-7-mobilized progenitor/stem cells for gene-modification and tracking studies, and highlight different functions and rates of repopulation after reconstitution with PB leukocytes obtained from mice treated with rhIL-7 versus rhG-CSF.

Administration of interleukin 12 with pulse interleukin 2 and the rapid and complete eradication of murine renal carcinoma.

BACKGROUND: Interleukin 2 (IL-2) and interleukin 12 (IL-12) are potent immunoregulatory cytokines that exhibit antitumor activity. Preliminary evidence suggests that combined administration of IL-2 and IL-12 may yield greater antitumor activity than that observed with either agent alone. PURPOSE: We evaluated the ability of combination regimens of IL-2 and IL-12 to induce regression of established primary and metastatic murine renal carcinoma (Renca) tumors. METHODS: BALB/c mice were given either subcutaneous or intrarenal injections of 10(5) Renca cells; tumor cell injections were given to 10-12 mice for each treatment group. Mice bearing subcutaneous primary tumors were treated with chronic IL-2 (300,000 IU given on a daily basis) or pulse IL-2 (300,000 IU given twice daily one day per week) alone, IL-12 alone (0.5 micrograms given on a daily basis), or IL-12 in combination with either chronic or pulse IL-2. Mice with metastatic tumors (arising from intrarenal implants; animals were nephrectomized to remove the primary tumors) were treated with IL-12 plus or minus pulse IL-2; in these experiments, IL-12 was given at doses of either 0.5 or 1.0 micrograms. In most experiments, treatment was continued for at least 3 weeks. Two-sided statistical tests were used to evaluate the data. RESULTS: Most mice with subcutaneous Renca tumors treated with the combination of IL-12 and chronic IL-2 died of treatment-related toxic effects within 7-14 days. In contrast, treatment with IL-12 plus pulse IL-2 was well tolerated, and six of 10 mice experienced complete tumor regression; none of the mice treated with either IL-12 alone or pulse Il-2 alone experienced a curative response. Seven of eight and nine of nine mice with metastatic tumors experienced complete tumor regression after treatment with 0.5 micrograms IL-12 plus pulse IL-2 or 1.0 microgram IL-12 plus pulse IL-2, respectively; two of 12 mice treated with pulse IL-2 alone and 10% or less of mice treated with IL-12 alone were cured of metastatic tumors (with 0.5 micrograms IL-12, none of 10 mice; with 1.0 micrograms IL-12, one of 10 mice). Five of 10 mice with metastatic tumors treated with a short-course regimen of IL-12 and pulse IL-2 (two pulses of IL-2 flanking 5 days of 0.5 micrograms IL-12) experienced complete tumor regression, while only one of the 12 mice treated with IL-2 alone and none of the mice treated with IL-12 alone experienced complete tumor regression. Virtually all curative response frequencies obtained with IL-12 and pulse IL-2 combination regimens differed significantly (P < .05) from those obtained with corresponding single-agent treatments. CONCLUSIONS: IL-12 administered in combination with pulse IL-2 induced rapid and complete regression of primary and metastatic Renca tumors and displayed greater antitumor activity than that observed with either IL-12 or IL-2 alone.

Diverse immunological and hematological effects of interleukin 7: implications for clinical application.

Interleukin-7 (IL-7) was originally discovered to be a pre-B cell growth factor. Soon thereafter, a broader role for IL-7 in leukocyte development and function began to be identified. IL-7 now has been shown to be a critical cytokine for normal T and B lymphopoiesis and a mobilizer of pluripotent stem cells and myeloid progenitors. IL-7 has been demonstrated to enhance T cell function and induce cytokine expression in monocytes. Preclinical studies have already found that IL-7 could accelerate murine lymphocyte regeneration following chemotherapy and bone marrow transplantation, induce antitumor effects in mice, and expand anti-HIV-specific human T cells. Thus it is essential that further preclinical and clinical research be performed to evaluate IL-7 as a potential therapy for leukopenia, bone marrow/stem cell transplantation, cancer, and HIV/AIDS.

Gradual loss of T-helper 1 populations in spleen of mice during progressive tumor growth.

BACKGROUND: The carefully orchestrated events that result in a protective immune response are coordinated to a large extent by cytokines produced by T helper 1 (Th1) and T helper 2 (Th2) T-cell subsets, which are two arms of the immune system. Th1 cells preferentially produce interleukin 2 (IL-2), interferon gamma (IFN gamma), and tumor necrosis factor (TNF), resulting in a cellular response that helps to eliminate infected cells. In contrast, Th2 cells produce IL-4, IL-5, IL-6, and IL-10 and stimulate an antibody response that helps to prevent the cells from becoming infected. The clinical progression of several infectious diseases, including human immunodeficiency virus, some types of parasitoses, and tuberculosis, is thought to be associated with the predominance of a Th2-type T-cell response. Recent reports have demonstrated the presence of T cells producing Th2 lymphokines (IL-4, IL-6, and IL-10) in tumor-infiltrating lymphocytes of renal cell carcinoma. PURPOSE: The purpose of this study was to investigate at the molecular level whether there was any change in the splenic T cells of mice with progressively growing tumors from a Th1 to a Th2 DNA-binding pattern or phenotype. METHODS: Splenic T cells from mice bearing renal cell carcinoma or MCA-38 colon carcinoma were tested for cytokine production after in vitro activation. Nuclear extracts of splenic T cells were used for the DNA-binding assay using IFN-gamma core promoter region, the kappa B (kappa B) site from immunoglobulin gene, and the nuclear factor of activated T-cell (NFAT) site from IL-2 gene. RESULTS: Splenic T cells from mice bearing renal cell carcinoma or MCA-38 colon carcinoma preferentially produced Th2 cytokines (i.e., IL-4) upon activation and showed a marked decrease in Th1 cytokine (particularly IFN gamma) production compared with the production observed in normal splenic T cells. The DNA-binding assay with the IFN-gamma core promoter region confirmed the gradual decline in the nuclear transcription factors associated with the Th1 phenotype during tumor progression in both tumor models. Renal cell carcinoma-bearing mice, successfully treated with flavone-8-acetic acid and recombinant human IL-2, showed a reversion to a Th1-like pattern. In addition, nuclear extracts of T cells from tumor-bearing animals showed a Th2-type kappa B-binding pattern. Moreover, the NFAT complex present in the normal splenic T cells was lost at the later stages of tumor progression; instead, a new complex was present in mice bearing long-term tumors. CONCLUSION: T cells from tumor-bearing mice lose the Th1 phenotype with progressive tumor growth.

Partial degradation of T-cell signal transduction molecules by contaminating granulocytes during protein extraction of splenic T cells from tumor-bearing mice.

Flavone-8-acetic acid plus recombinant human interleukin 2 is a successful antitumor therapy in mice bearing the Renca murine renal cell carcinoma. This report demonstrates that T cells, particularly CD8+ T cells, are critical for the generation of this response. Initial experiments examining T-cell signal transduction proteins demonstrated that T cells from Renca-bearing mice had undetectable levels of p56lck and zeta-chain of the T-cell receptor and that flavone-8-acetic acid and recombinant human interleukin 2 therapy could be used as a model for reversal of these alterations. However, further experimentation showed that the majority of the reduction in zeta-chain and part of the reduction in p56lck was due to degradation of these molecules during protein extraction caused by mature granulocytes contaminating the enriched T-cell population. This was not the case for nuclear c-Rel or NF kappa B p65, which remained at undetectable/reduced levels in the absence of granulocytes, confirming our previous data that transcription factor alterations exist in tumor-bearing mice. Thus, most of the reduction in zeta-chain in T cells from Renca-bearing mice is due to granulocyte contamination and emphasizes the need to use pure T-cell populations and/or sufficient amounts and types of protease inhibitors when quantitating proteins in T cells from tumor-bearing mice.

Recombinant human IL-7 administration in mice affects colony-forming units-spleen and lymphoid precursor cell localization and accelerates engraftment of bone marrow transplants.

Murine reconstitution assays were used to investigate the effects of recombinant human interleukin-7 (rhIL-7) on myeloid and lymphoid precursors and on bone marrow engraftment. Reconstitution with bone marrow from rhIL-7-treated mice results in a 3.4-fold decrease in total colony-forming unit-spleen (CFU-S) activity (day 9) and an 18.1- and 11.9-fold decrease in its ability to generate thymocytes and splenic B lineage cells, respectively. In contrast, after reconstitution with splenocytes from rhIL-7-treated mice, CFU-S activity increased 23.6-fold (day 9) and the thymocyte and splenic B lineage cell regenerative capacity increased by 4.0- and 3.2-fold, respectively. In addition, CD43low+, B220low+ cells that contain pre-pro-B cells and pro-B cells were expanded two- to threefold and Ig mu-, B220+, CD2- and Ig mu-, B220+, CD2+ B lineage cells were expanded approximately 10-fold and 10- to 45-fold (depending on the tissue examined), respectively, after rhIL-7 treatment. Administration of rhIL-7 to irradiated mice transplanted with bone marrow resulted in accelerated T cell and B cell reconstitution by up to 2-4 weeks. Thus, rhIL-7 administration affects the distribution of myeloid and lymphoid precursors. Moreover, rhIL-7 administration accelerates murine bone marrow cell engraftment and therefore may be useful in reducing the engraftment time in bone marrow transplant patients.

Cellular and molecular studies in the treatment of murine renal cancer.

Antigen-nonspecific approaches to the use of BRMs for cancer treatment have resulted in only limited success to date. In particular, the use of large numbers of adoptively transferred, broadly cytotoxic LAK cells in combination with IL-2 has been effective for only small subsets of cancer patients. Recent demonstrations of T-lymphocyte-mediated antigen-specific responses against some human tumors, and the more potent effects of these cells in preclinical models, have refocused much of the dialogue for biological therapy to potentiation of T-lymphocyte-mediated antitumor effects. Our studies are using the well-characterized Renca murine renal cancer model to study the induction of antitumor T-lymphocyte-mediated responses, the mechanisms by which positive effects are achieved, and the reasons why T lymphocytes in tumor-bearing mice may not respond as predicted. One possible reason why T-lymphocyte responses may not be triggered easily by tumors could be an impairment of critical nuclear transcription factors. We also are studying two approaches for stimulating T-cells in tumor-conditioned hosts. (1) We have shown that IL-7 has potent costimulatory effects on T cells as well as some antitumor effects. (2) We are developing a comprehensive vaccine-type gene therapy approach whereby T cells and antigen-presenting dendritic cells are recruited through the use of antigen, chemokines and GM-CSF. Studies are in progress to determine whether the activity of these recruited cells can then be potentiated by Renca or fibroblast transfectants that express T-cell costimulatory cytokines (IL-2, IL-4, IL-7, or IL-12). This approach should optimize both MHC class I- and class II-dependent pathways for induction of T-lymphocyte-mediated responses to cancer, and perhaps overcome tumor-induced impairments in the T lymphocyte function.

Mobilization of long-term reconstituting hematopoietic stem cells in mice by recombinant human interleukin 7.

Administration of recombinant human interleukin 7 (rh)IL-7 to mice has been reported by our group to increase the exportation of myeloid progenitors (colony-forming unit [CFU]-c and CFU-granulocyte erythroid megakarocyte macrophage) from the bone marrow to peripheral organs (blood, spleen[s], and liver). We now report that IL-7 also stimulates a sixfold increase in the number of more primitive CFU-S day 8 (CFU-S8) and day 12 (CFU-S12) in the peripheral blood leukocytes (PBL) of mice treated with rhIL-7 for 7 d. Moreover, > 90% of lethally irradiated recipient mice that received PBL from rhIL-7-treated donor mice have survived for > 6 mo whereas none of the recipient mice that received an equal number of PBL from diluent-treated donors survived. Flow cytometry analysis at 3 and 6 mo after transplantation revealed complete trilineage (T, B, and myelomonocytic cell) repopulation of bone marrow, thymus, and spleen by blood-borne stem/progenitor cells obtained from rhIL-7-treated donor mice. Thus, IL-7 may prove valuable for mobilizing pluripotent stem cells with long-term repopulating activity from the bone marrow to the peripheral blood for the purpose of gene modification and/or autologous or allogeneic stem cell transplantation.

Administration of recombinant human IL-7 to mice alters the composition of B-lineage cells and T cell subsets, enhances T cell function, and induces regression of established metastases.

These studies investigate the effects of exogenously administered recombinant human IL-7 (rhIL-7) on mouse leukocyte subsets in vivo in normal and tumor-bearing mice. The administration of rhIL-7 to normal mice caused a pronounced leukocytosis (three- to fivefold increase over background) in the spleen and lymph nodes, with B-lineage and T cells, NK cells, and macrophages all being increased. CD8+ T cells increased disproportionately, such that the CD4 to CD8 ratio decreased dramatically. The rhIL-7-induced effects were dose-dependent, increased with duration of treatment, and were reversible after cessation of rhIL-7 administration. T cell number increases after rhIL-7 treatment were primarily a result of an expansion of the peripheral T cell population. Importantly, splenocytes from rhIL-7-treated mice have enhanced proliferative responses to various T cell stimuli in vitro and were able to potentiate an allogeneic CTL response in vivo. The rhIL-7-induced changes in T cell number and the CD4 to CD8 ratio also were observed in mice bearing early Renca renal adenocarcinoma pulmonary metastases, and these changes coincided with up to a 75% reduction in pulmonary metastases. Overall, these results demonstrate that the administration of rhIL-7 to mice profoundly increases the number of B and T cells, and reduces the number of pulmonary metastases. The results also suggest that IL-7 may be useful for restoring lymphoid subsets in immunosuppressed hosts and in enhancing T cell-mediated immune responses. Such effects may be useful in the treatment of microbial diseases and cancer.