Stat3 inhibition augments the immunogenicity of B-cell lymphoma cells, leading to effective antitumor immunity

Abstract

Mantle cell lymphoma (MCL) is an aggressive and incurable subtype of B-cell non-Hodgkin lymphomas. Although patients often respond initially to first-line treatment with chemotherapy plus monoclonal antibodies, relapse and decreased response to further lines of treatment eventually occurs. Harnessing the immune system to elicit its exquisite specificity and long-lasting protection might provide sustained MCL immunity that could potentially eradicate residual malignant cells responsible for disease relapse. Here, we show that genetic or pharmacologic disruption of Stat3 in malignant B cells augments their immunogenicity leading to better activation of antigen-specific CD4(+) T cells and restoration of responsiveness of tolerized T cells. In addition, treatment of MCL-bearing mice with a specific Stat3 inhibitor resulted in decreased Stat3 phosphorylation in malignant B cells and anti-lymphoma immunity in vivo. Our findings therefore indicate that Stat3 inhibition may represent a therapeutic strategy to overcome tolerance to tumor antigens and elicit a strong immunity against MCL and other B-cell malignancies.

Figure 1. Disruption of Stat3 in malignant B-cells augments their antigen-presenting function

A20 B-cells were left untransfected, mock transfected or transiently transfected with GFP vector or Stat3β GFP expression vector. Then, 1×10⁵ transfected cells as well as non-transfected or mock transfected cells were incubated with either 5×10⁴ naïve anti-HA CD4⁺ T-cells (left panel) or with 5×10⁴ tolerized anti-HA CD4⁺ T-cells isolated from the spleen of A20HA-bearing mice (right panel), in the presence of 12.5 mcg of HA peptide_110-120 SFERFEIFPKE. After 48 hours, supernatants were collected and IL-2 (A, C) and IFN-γ (B, D) production by antigen-specific CD4⁺ T-cells were determined by ELISA. Values represent mean ± S.E of triplicate cultures and are representative of three independent experiments (*p statistically significant for the difference in cytokine production between treatment with STAT3β and GFP).

Figure 2. Increased Stat3 activity in malignant B-cells inhibits antigen-specific CD4⁺ T-cell responses

A20 B-cells were transiently transfected with either pcDNA3 empty vector or Stat3c expression vector. Then, 1×10⁵ transfected cells as well as non-transfected cells were incubated with 5×10⁴ naïve anti-HA CD4⁺ T-cells in the presence or not of 12.5 mcg of HA peptide. After 48 hours, supernatants were collected and IL-2 (A) and IFN-γ (B) production were determined by ELISA. Values represent mean ± S.E of triplicate cultures and are representative of three independent experiments (*p statistically significant for the difference in cytokine production between none and Stat3C).

Figure 3. Comparison of the effects of CPA-7 treatment and Stat3 siRNA inhibition upon human MCL cells

JEKO cells were transiently transfected with Stat3-specific siRNA (Stat3 siRNA) or non-targeting control (Control). In parallel, JEKO cells were treated or not with CPA-7 (30μM) for 24 hours. Cells were harvested and protein extracts were obtained and subjected to western blot using antibodies against p-Stat3, Stat3, p-MAPK, MAPK, p-Akt and Akt. Shown is a representative experiment of two with similar results.

Figure 4. Phenotypic and functional characteristics of FC-muMCL1 cells

(A) Expression of Cyclin D1, CD19 and CD5 by FC-muMCL1 cells (open histogram). Gray histogram: Isotype control. (B) In vivo growth of FC-muMCL1 tumors. C57BL/6 mice were injected either sc in the right leg (closed circles) or ip (open circles) with 5×10⁶ FC-muMCL1 cells. Five mice were included in each group and they were inspected three times a week for the development of tumor nodules (sc model) or abdominal girth (ip model). Shown is a representative experiment of two with similar results. (C–D). Antigen-presenting function of MCL cells. FC-muMCL1 cells (1×10⁵ cells/well) were treated with LPS (2 mcg/ml), LPS + increasing concentrations of CPA-7, or left untreated (Media) for 24 hours. Then, cells were washed and plated with either 5×10⁴ naïve anti-OVA CD4⁺ T-cells/well or tolerized anti-OVA CD4⁺ T-cells isolated from mice bearing an OVA-expressing tumor together with 3mcg/ml of OVA peptide_323-339. Forty-eight hours later supernatants were collected and the production of IL-2 and IFN-γ by naïve T-cells (C) and the production of IFN-γ by tolerized T-cells (D) were determined by ELISA. Shown is a representative experiment of three independent experiments with similar results (*p statistically significant for the difference in cytokine production between treatment).

Figure 5. In vivo treatment with CPA-7 results in decreased Stat3 phosphorylation in malignant B-cells and an anti-lymphoma effect

(A) 5×10⁶ FC-muMCL1 cells were injected ip into C57BL/6 mice. Twenty-one days later animals were treated or not with 5 mg/kg of CPA-7 given iv every three days (days +21, +24 and +27). On day +29 animals were sacrificed and tumor nodules were carefully dissected from their livers. Malignant B-cells were then isolated and the expression of phopho-Stat3 was determined by western blot using an anti-p-Stat3 (Tyr705) antibody. Shown is a representative experiment of two independent experiments with similar results. (B) C57BL/6 mice (n=10) were challenged with 5×10⁶ FC-muMCL1 cells given sc in the right leg. Half the mice were then treated with CPA-7 (5mg/kg) given IV every three days, starting on day +5 after tumor challenge (dashed line). The other half of the mice received vehicle control (solid line). Tumor volumes were calculated (LengthxWidthxWidthX1/2) from measurements made at times indicated. Two independent experiments were performed with similar results. (C) BALB/c mice (n=10) were challenged with 1×10⁶ A20 lymphoma cells given sc in the right leg. Half the mice were treated with CPA-7 (dash line), and the other half with vehicle control (solid line) as indicated in B. Animals were monitored for the development of tumor nodules and tumor volumes measured on days indicated. Two independent experiments were performed with similar results.

Figure 6. Similar kinetics of lymphoma growth in CPA-7 treated SCID mice

(A) C57BL/6 SCID and BALB/c SCID mice were treated as indicated in (B) and (C). Mice were sacrificed after 19 days of tumor challenge and tumor nodules were collected. Malignant B-cells were then isolated and the expression of phopho-Stat3 was determined by western blot using an anti-p-Stat3 (Tyr705) antibody. (B) C57BL/6 SCID mice (n=10) were challenged with 5×10⁶ Fc-muMCL1 cells given sc. Half the mice were then treated with CPA-7 (dash line) (5mg/kg/iv given every three days, starting on day +5 after tumor challenge) and the other half received vehicle control (solid line). (C) BALB/c SCID mice (n=10) were challenged with 1×10⁶ A20 cells given sc. Mice were then treated with CPA-7 (dash line) or received vehicle control (solid line) as indicated in A. Tumors were measured at times indicated. Two independent experiments were performed with similar results.