Kristin L. Komschlies

IFN-γ 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.

Antitumor effects of interleukin-7 and adoptive immunotherapy on human colon carcinoma xenografts.

The antitumor properties of recombinant human IL-7 (rhIL-7) on a human tumor was evaluated by engrafting a human colon carcinoma into immunodeficient mice and then treating the mice with rhIL-7 and adoptively transferred human peripheral blood T cells. It was found that rhIL-7 alone had no effect on the survival of the tumor-bearing recipients. However, the combination of rhIL-7 and human T cells significantly promoted the survival of the recipients compared with mice receiving either treatment by itself. When the surviving mice were analyzed 6 mo later for the degree of human cell engraftment, the recipients receiving both rhIL-7 and human T cells had greater numbers of human CD8+ T cells in the spleens. However, the human T cells recovered from the surviving mice showed low lytic activity against the tumor in vitro. Supernatants from human T cells cultured with the tumor and rhIL-7 in vitro were found to inhibit tumor growth and were demonstrated to contain high levels of IFN-gamma. Antibodies to IFN-gamma neutralized the growth inhibition of the tumor both in vitro and in vivo demonstrating that the in vivo mechanism underlying the antitumor effects of this regimen was partly dependent on the production of IFN-gamma by the T cells and not their cytolytic capability. Interestingly, systemic administration of rhIFN-gamma to tumor-bearing mice yielded little antitumor effect suggesting that adoptive immunotherapy with rhIL-7 was superior possibly because of the continuous local release of the cytokines. Therefore, rhIL-7 may be of clinical use as an antineoplastic agent and the human/mouse model is a potentially important preclinical model for in vivo evaluation of the efficacy of this and other immunotherapies.

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 Igμ‐, B220+, CD2‐ and Igμ‐, 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. J. Leukoc. Biol. 58: 151–158; 1995.

Substrate-specific proteases (BLT-esterase)are localized predominantly in the natural killer cells of unprimed mice

In leukocytes isolated from unprimed mice, the levels of extractable Nα‐Cbz‐Lys‐thiobenzylester‐ esterase (BLT‐esterase) closely correlated with the number of natural killer (NK) cells. The spleens of mice that exhibit severe combined immunodeficiency (SCID) contained much higher levels of this enzyme than other mouse strains. Treatments that resulted in a local accumulation of NK cells (as assessed by lytic activity) produced a concomitant increase in BLT‐esterase activity. However, short‐term in vitro treatment of spleen cells with interferon (IFN)‐α/β indicated that BLT‐esterase levels correlated more closely with absolute numbers of NK cells than with their lytic capacity. There was a very good correlation between the numbers of cells bearing the NK phenotype (NK‐1. 1+) and BLT‐esterase levels. Cells positively sorted using the NK‐specific antibodies NK‐1. 1 and LGL‐1 had high enzymatic activity. The BLT‐ esterase levels were high in both the NK‐1. 1+LGL‐1” and NK‐l. r/LGL‐r subsets. Highly purified CD4+ and CD8+ T cells and slg+ B cells demonstrated negligible enzyme, as did populations of cells highly enriched for macrophages or neutrophils. However, it should be stressed that the inbred mice used on this study have been maintained in a pathogen‐free facility. It would be anticipated that mice maintained under less stringent conditions could exhibit appreciable levels of BLT‐esterase activity in their T cells. Nonetheless, BLT‐esterase is present at high levels in NK cells and cannot be regarded as a T cell‐specific enzyme.

Cellular and Molecular Considerations for the Treatment of Murine Renal Cancer

Approximately 25,000 new cases of renal cell cancer are diagnosed each year in the United States (1). Renal cell carcinoma is devastating because many patients have metastatic disease at the time of presentation, and treatment with chemotherapeutic drugs is largely ineffective. Some BRMs, such as α-interferon (IFNα) and Interleukin-2 (IL-2), have shown therapeutic efficacy as good as or better than chemotherapy (2), raising the possibility that this disease is capable of inducing an immune response under some circumstances.

Cytokine-Based Combined Modality Approaches to the Treatment of Murine Renal Cancer

Biological response modifiers (BRM) are being developed as a potential fourth modality for cancer treatment to supplement the three currently accepted modalities, chemotherapy, radiotherapy, and surgery (1). There are several possible approaches to the use of immunoactive cytokines as part of combined modalities for cancer treatment. First, cytokines can be used in combination with one or more of the traditional modalities for cancer treatment. The rationale for such an approach is that either the mechanisms for the antitumor effects of the cytokines versus those of the other modality will differ leading to enhanced antitumor effects, or that the effects of one will enhance the activity of the other. In either case the efficacy of the combination should be greater than either modality alone. Second, different combinations of cytokines should be complementary based on their interaction as part of the cytokine cascade that regulates immune responses. We are studying different preclinical models that fall into both types of combined modality approaches. It has been reported that interleukin 1 (IL-1) can protect mice from the acute toxicity of some chemotherapeutic drugs (2–4) and that this ability to dose escalate chemotherapy translates into enhanced antitumor efficacy (4), although some late toxicity becomes evident (4). In addition, we are studying the hematoimmunological effects of various T cell-stimulating cytokines (5–7) under the hypothesis that the enhancement of the appropriate T cell functions will lead to increased antitumor efficacy.