A fusion of GMCSF and IL-21 initiates hypersignaling through the IL-21Ralpha chain with immune activating and tumoricidal effects in vivo

Abstract

We hypothesized that fusing granulocyte-macrophage colony-stimulation factor (GMCSF) and interleukin (IL)-21 as a single bifunctional cytokine (hereafter GIFT-21) would lead to synergistic anticancer immune effects because of their respective roles in mediating inflammation. Mechanistic analysis of GIFT-21 found that it leads to IL-21Ralpha-dependent STAT3 hyperactivation while also contemporaneously behaving as a dominant-negative inhibitor of GMCSF-driven STAT5 activation. GIFT-21's aberrant interactions with its cognate receptors on macrophages resulted in production of 30-fold greater amounts of IL-6, TNF-alpha, and MCP-1 when compared to controls. Furthermore, GIFT-21 treatment of primary B and T lymphocytes leads to STAT1-dependent apoptosis of IL-21Ralpha(+) lymphocytes. B16 melanoma cells gene-enhanced to produce GIFT-21 were immune rejected by syngeneic C57Bl/6 mice comparable to the effect of IL-21 alone. However, a significant GIFT-21-driven survival advantage was seen when NOD-SCID mice were implanted with GIFT-21-secreting B16 cells, consistent with a meaningful role of macrophages in tumor rejection. Because GIFT-21 leads to apoptosis of IL-21Ralpha(+) lymphocytes, we tested its cytolytic effect on IL-21Ralpha(+) EL-4 lymphoma tumors implanted in C57Bl/6 mice and could demonstrate a significant increase in survival. These data indicate that GIFT-21 is a novel IL-21Ralpha agonist that co-opts IL-21Ralpha-dependent signaling in a manner permissive for targeted cancer immunotherapy.

Figure 1

Characterization of GIFT-21. (a) Immunoblotting performed on the CM from B16 cells retrovirally transduced to express GIFT-21; rmGMCSF and CM from B16 expressing mIL-21 were used as controls. (b) Amino acid sequence of the GIFT-21 fusokine. CM, conditioned media; GMCSF, granulocyte-macrophage colony-stimulation factor; IL-21, interleukin-21.

Figure 2

Cellular biochemistry of GIFT-21. (a) STAT5 phosphorylation in RAW264.7 cells. Cells were stimulated for 20 minutes with rmGMCSF, rmIL-21, both cytokines, or with B16 GIFT-21 CM, and the cell lysates were probed for phosphorylated STAT5. Total STAT5 protein was used as a loading control. (b) STAT1/STAT3/STAT5 phosphorylation in EL-4 cells. Cells were stimulated for 20 minutes with a gradient of IL-21 and a gradient of B16 GIFT-21 CM, and the western blot of cell lysates was immunoblotted for phosphorylated STAT1 and STAT3. Total STAT1 or STAT3 protein was used as a loading control. EL-4 cells were stimulated for 20 minutes with B16 GIFT-21 CM and its controls, and the cell lysates were probed for phosphorylated STAT5. RAW264.7 cells were treated with rmGMCSF and used as a positive control for STAT5 phosphorylation in this experiment. CM, conditioned media; GMCSF, granulocyte-macrophage colony-stimulation factor; IL-21, interleukin-21.

Figure 3

GIFT-21 induces the secretion of pro-inflammatory cytokines by macrophages. (a) 5 × 10⁴ macrophages were treated for 24 or 48 hours with equimolar concentrations of GIFT-21 versus controls. Production of secreted IGF-1, IL-6, MCP-1, and TNF-α were measured by enzyme-linked immunosorbent assay (mean ± SEM, n = 3). Graphs represent the average of three independent experiments. (b) 5 × 10⁴ macrophages were treated with GIFT-21 with and without IL-21R blockade. GMCSF, granulocyte-macrophage colony-stimulation factor; IL-21, interleukin-21.

Figure 4

GIFT-21 induces macroscopic changes in macrophage morphology. (a) Giemsa staining of macrophages treated with GIFT-21 versus controls. Bar = 50 µm. (b) Intracellular immunofluorescent staining of IL-6 (versus isotype control) in peritoneal macrophages treated with B16 GIFT-21 conditioned media (left panels) versus no treatment (right panels). Bar = 10 µm. GMCSF, granulocyte-macrophage colony-stimulation factor; IL-21, interleukin-21.

Figure 5

Effect of GIFT-21 on IL-21Rα expressing lymphoid subsets. Unfractionated splenocytes from normal C57Bl/6 mice were treated with GIFT-21 versus controls for 24 hours; (a) apoptosis was measured by flow cytometry using annexin V and propidium iodide staining; and analysis of IL-21Rα expressing lymphocyte subsets analyzed by flow cytometry (mean ± SEM, n = 3); (b) CD19⁺, (c) CD3⁺CD4⁺, and (d) CD3⁺CD8⁺ cells were gated for IL-21Rα expression 24 hours following treatment with GIFT-21. Percentage of the IL-21Rα expressing fraction is indicated in top right of each flow histogram. (e) Splenocytes isolated from STAT1^−/− mice were treated with GIFT-21 versus controls, and the expression of the IL-21Rα was measured 24 hours later. The figures are representative of two independent experiments. GMCSF, granulocyte-macrophage colony-stimulation factor; IL-21, interleukin-21.

Figure 6

GIFT-21 elicits a robust immune response against cancer. Implantation of cytokine secreting B16 tumors in vivo. (a) B16 cells producing GIFT-21 versus all controls were implanted in WT C57Bl/6 mice. B16 IL-21 and B16 GIFT-21 were robustly rejected. Data are representative of three separate experiments. (b) B16 IL-21 and B16 GIFT-21 survivors were challenged with unmodified B16, and the challenge was rejected at equal rates in both groups. (c) Only NOD-SCID mice injected with B16 GIFT-21 survived significantly longer than the controls. Data are the pooling of two separate experiments. (d) B16 GIFT-21 was rejected from STAT1^−/− mice. GMCSF, granulocyte-macrophage colony-stimulation factor; IL-21, interleukin-21.

Figure 7

GIFT-21 can act as an IL-21R-specific chemotherapeutic drug. (a) EL-4 express the IL-21Rα and CTLL-2 do not. (b) GIFT-21 induces the apoptosis of EL-4, but not CTLL-2 (mean ± SEM, n = 3). (c) Only mice treated with MSC GIFT-21 survived significantly longer than the unmodified MSC control when challenged with a 10⁶ injection of EL-4 subcutaneously. IL-21, interleukin-21; MSC, mesenchymal stromal cell; NS, not significant.