Theresa A. Wiltrout

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.

Synergistic Engagement of an Ineffective Endogenous Anti-Tumor Immune Response and Induction of IFN-γ and Fas-Ligand-Dependent Tumor Eradication by Combined Administration of IL-18 and IL-2

IFN-γ is a critical component of the endogenous and many cytokine-induced antitumor immune responses. In this study we have shown that the combination of IL-18 and IL-2 (IL-18/IL-2) synergistically enhances IFN-γ production both in vitro and in vivo, and synergizes in vivo to induce complete durable regression of well-established 3LL tumors in >80% of treated mice. We have observed a nascent, but ineffective, host immune response against 3LL that depends on endogenous IFN-γ and IL-12 production and the Fas/Fas ligand (Fas-L) pathway. The combined administration of IL-18/IL-2 engages this endogenous response to induce tumor regression via a mechanism that is independent of NK and NKT cells or IL-12, but is critically dependent on CD8+ T cells, IFN-γ, and the Fas/Fas-L pathway. These studies demonstrate the importance of IFN-γ as well as the Fas/Fas-L pathway in both endogenous and cytokine-driven antitumor immune responses engaged by IL-18/IL-2 and provide preclinical impetus for clinical investigation of this potent anti-tumor combination.

Cloning and expression of a second human natural killer cell granule tryptase, HNK-Tryp-2/granzyme 3.

Cytotoxic lymphocytes possess a number of serine proteases (granzymes) usually localised in cytoplasmic granules. To date, the DNA sequences of four human granzymes have been reported. A fifth human granzyme (granzyme 3) has been biochemically purified and its N‐terminal amino acid sequence has been reported. This enzyme was described as possessing tryptase activity, cleaving synthetic substrates after arginine or lysine. We recently cloned a rat granzyme tryptase (RNK‐Tryp‐2), and used this cDNA to screen human cDNA libraries. Isolation of cDNA fragments of a human gene could be overlapped to provide a complete cDNA sequence, which we designated HNK‐Tryp‐2. The N‐terminal amino acid sequence deduced from HNK‐Tryp‐2 was identical to that reported for granzyme 3. This gene appears to be a single copy gene that is expressed in isolated natural killer cells and T cells as well as in tissues containing these cells.

Trafficking and activation of murine natural killer cells: Differing roles for IFN-γ and IL-2

The repeated ip injection of highly purified recombinant IFN-γ or IL-2 resulted in a local increase in peritoneal NK activity. This increase in lytic activity was paralleled by increases in the number of peritoneal leukocytes reacting with a rat monoclonal antibody directed against the NK cell-associated surface antigen LGL-1. LGL-1 reacts specifically with the majority of murine NK cells in BALB/c and C57BL/6 mice. A single injection of IFN-γ induced more peritoneal NK activity at 24 hr than IL-2 on a protein basis. Both cytokines induced increases in the number of LGL-1+ peritoneal cells by 24 hr after injection. Simultaneous injection of suboptimal amounts of IFN-γ (100 U) and IL-2 (10,000 U) resulted in a significant augmentation of peritoneal NK activity over that observed with either cytokine alone. Also, the peritoneal NK activity generated in response to ip injection of high doses of IL-2 (100,000 U) could be dramatically reduced by simultaneous injection of a neutralizing monoclonal antibody to IFN-γ. Administration of IFN-γ 1 day prior to IL-2 resulted in a significant augmentation of the NK activity above that observed with the individual cytokines. In contrast, injection of IL-2 prior to IFN-γ did not enhance NK activity over that observed with the individual cytokines. Both cytokines must be injected ip for the complementary effects of IFN-γ and IL-2 on peritoneal NK activity to occur. In contrast, in vitro incubation of peritoneal leukocytes with IFN-γ resulted in neither a significant enhancement of NK lytic activity nor an increase in the number of LGL-1+ cells. In vitro treatment of peritoneal leukocytes with IL-2 always resulted in significant augmentation of NK lytic activity in the absence of any increase in the number of LGL-1+ cells. These data are consistent with the hypothesis that the local release of IFN-γ increases peritoneal NK activity by promoting the influx of blood-borne LGL-1+ NK cells from other sites. In contrast, low doses of IL-2 augment the lytic activity of local resident NK cells, whereas high doses of this cytokine induce both an activation of local NK cells and emigration of LGL-1+ NK cells from other sites due to the endogenous generation of IFN-γ within the peritoneal cavity. Therefore, the local release of IFN-γ may play an important role in regulating NK cell infiltration in vivo.

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.

Characterization of a non-granule associated pore-forming protein in agranular lymphocytes.

Recently, two populations of small lymphocytes (SL) have exhibited non‐major histocompatibility complex (MHC) restricted lysis. Recent studies by numerous laboratories have demonstrated that resting T cells triggered through CD3 and CD28 costimulations can result in immediate, non‐MHC restricted killing. Our recent studies with CD3‐, CD56+ SL demonstrated that although these cells contained no cytoplasmic granules detected with electron microscopy, they mediated potent NK and ADCC activity. In the present study, we have used a Western blotting technique that allows for the detection and quantitation of total cellular levels of pore‐forming protein (PFP). We have found that freshly isolated peripheral non‐granulated lymphocytes (both CD3+ and CD3‐) contain PFP. In addition, CD3‐ CD56+ SL contain levels of PFP similar to those of the highly granular CD3‐ LGL. In search of non‐granule PFP, we exploited the rat NK (RNK) cell lines as a source of other potential cytotoxic factors. A membrane associated PFP, based on Western blotting, was isolated from rat RNK cells. Unlike PFP isolated from granules, this PFP was active after culture in Ca2+‐containing medium. However, the lytic activity isolated from the non‐granule PFP of RNK cells was inhibited by monoclonal antibodies to PFP. Collectively, these studies indicate that PFP is present in small agranular lymphocytes (both NK and T cells) and that it is not stored in large cytoplasmic granules. The implication of our results for the acquisition and activation of lytic ability in NK and T cells will be discussed. J. Leukoc. Biol. 57: 897–903; 1995.