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. 2017 Jan 31;8(5):8604-8621.
doi: 10.18632/oncotarget.14365.

Bortezomib augments lymphocyte stimulatory cytokine signaling in the tumor microenvironment to sustain CD8+T cell antitumor function

Samuel T Pellom Jr  1   2   3 , Duafalia F Dudimah  1 , Menaka C Thounaojam  1 , Roman V Uzhachenko  1 , Ashutosh Singhal  1 , Ann Richmond  4   5   6   7   8   9 , Anil Shanker  1   3   7   8   9
Affiliations

Bortezomib augments lymphocyte stimulatory cytokine signaling in the tumor microenvironment to sustain CD8+T cell antitumor function

Samuel T Pellom Jr et al. Oncotarget. .

Abstract

Tumor-induced immune tolerance poses a major challenge for therapeutic interventions aimed to manage cancer. We explored approaches to overcome T-cell suppression in murine breast and kidney adenocarcinomas, and lung fibrosarcoma expressing immunogenic antigens. We observed that treatment with a reversible proteasome inhibitor bortezomib (1 mg/kg body weight) in tumor-bearing mice significantly enhanced the expression of lymphocyte-stimulatory cytokines IL-2, IL-12, and IL-15. Notably, bortezomib administration reduced pulmonary nodules of mammary adenocarcinoma 4T1.2 expressing hemagglutinin (HA) model antigen (4T1HA) in mice. Neutralization of IL-12 and IL-15 cytokines with a regimen of blocking antibodies pre- and post-adoptive transfer of low-avidity HA518-526-specific CD8+T-cells following intravenous injection of 4T1HA cells increased the number of pulmonary tumor nodules. This neutralization effect was counteracted by the tumor metastasis-suppressing action of bortezomib treatments. In bortezomib-treated 4T1HA tumor-bearing mice, CD4+T-cells showed increased IL-2 production, CD11c+ dendritic cells showed increased IL-12 and IL-15 production, and HA-specific activated CD8+T-cells showed enhanced expression of IFNγ, granzyme-B and transcription factor eomesodermin. We also noted a trend of increased expression of IL-2, IL-12 and IL-15 receptors as well as increased phosphorylation of STAT5 in tumor-infiltrating CD8+T-cells following bortezomib treatment. Furthermore, bortezomib-treated CD8+T-cells showed increased phosphorylation of mitogen-activated protein kinase p38, and Akt, which was abrogated by phosphatidylinositide 3-kinase (PI3K) inhibitor. These data support the therapeutic potential of bortezomib in conjunction with other immunotherapies to augment the strength of convergent signals from CD8+T-cell signaling molecules including TCR, cytokine receptors and downstream PI3K/Akt/STAT5 pathways to sustain CD8+T-cell effector function in the tumor microenvironment.

Keywords: CD8+ T cells; adoptive cell therapy; cancer immunotherapy; immunosuppression; proteasome inhibition.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1. Modulation of cytokine/chemokine expression by bortezomib in 4T1HA tumor-bearing mice
A. Orthotopic tumors were established in mammary pads of Balb/c wild type mice following injection of 2 × 106 4T1HA tumor cells. On day 14 (after the tumor reached at least a size of 125 mm3), mice were injected intravenously with 1 mg/kg body weight of bortezomib (Bzb, ~20 nM by total blood volume in 8-wk mouse). After 4 h, the mice were euthanized and total cell lysates were made from the RBC-depleted splenocytes and 245 μg of protein from these lysates were analyzed for cytokine/chemokine concentration by MagPix array (Millipore). B. MagPix output data constitutes protein concentration (pg/mL) of cytokines/chemokines modulated in the spleen of 4T1HA tumor-bearing mice. Four experimental groups were compared: Saline control (gray bar), naïve mice treated with Bzb alone (black bar), mice with tumor alone (red bar), and tumor-bearing mice treated with Bzb (blue bar). Protein concentration of analytes compared among the 4 groups is shown as means ± SD from 4 independent experiments. *p values are representative as *p<0.05 (ANOVA, one-way) and used to compare tumor alone to tumor+Bzb group.
Figure 2
Figure 2. Effect of bortezomib administration on IL-2, IL-12, and IL-15 proteins and mRNA expression in vivo
Protein expression of cytokines IL-2 A. IL-12p40 B. IL-12p70 C. IL-15 D. in total cell lysates of RBC-depleted splenocytes from Balb/c WT mice were quantified 4 h after Bzb (1 mg/kg body weight) administration by MagPix multiplex array. Cytokine mRNA expression levels measured by RT-PCR are shown as the ratio of target gene expression to housekeeping gene, β-actin, for IL-2 E. IL-12 F. IL-15 G. The bar graph is shown as mean ± SD from 2 independent experiments. *p < 0.05; (ANOVA, one-way).
Figure 3
Figure 3. Time kinetics of splenic IL-2, IL-12 and IL-15 expression in vivo following bortezomib treatment
Balb/c WT mice were administered intravenously with Bzb (1 mg/kg body weight) at the indicated time points over a 72 h period. Total cell lysate samples of RBC-depleted splenocytes were prepared from a group of mice at different time points. Protein levels for IL-2, IL-12p40, IL-12p70, and IL-15 were quantified using the MagPix multiplex array. Values are presented as mean ± SEM; ANOVA, one-way; *p<0.05 compared to other time points.
Figure 4
Figure 4. Effect of bortezomib on lung metastases of 4T1HA mammary tumor cells
Balb/c wild type (WT) mice injected with 4T1HA tumor cells (0.5 × 106; i.v.; day 0) were administered intravenously with bortezomib (1 mg/kg body weight, ~20 nM by total blood volume) on days 4, 7, 11, and 15. Tumor metastases pulmonary nodules were counted on day 18. Nodule counts are presented as mean ± SEM; **p <0.01; two-tailed t-test.
Figure 5
Figure 5. Bortezomib counteracts IL-12 and IL-15 neutralization by enhancing CD8+T cell effector molecules and reducing pulmonary nodules of 4T1HA tumor cells
A. 0.5 × 106 4T1HA tumor cells were injected intravenously in Balb/c WT mice along with intravenous injections of IL-12 and IL-15 neutralizing antibodies on days 0, 3, 6, 9, and 12. On day 7, adoptive transfer of TCRHA CD8+T cells (2 × 106/mouse, i.v.) was performed. Bzb (1 mg/kg body weight) was administered on days 10 and 14. B. Lungs were harvested on day 14 to count tumor pulmonary nodules. Central lines depict values as means ± SEM. C. Bar graphs present data for IFNγ+Vb8.1/2+ and granzyme-B+Vβ8.1/2+ CD8+T cells. Values are depicted as means ± SD. *p<0.05, **p<0.01; (ANOVA, one-way).
Figure 6
Figure 6. Bortezomib-mediated modulation of IL-2 secretion in CD4+T cells and IL-12 and IL-15 cytokines in CD11c+ cells of tumor-bearing mice
0.5 × 106 4T1HA tumor cells were injected orthotopically under mammary pads to establish tumors approximately 125 mm3 in size. Bzb (1 mg/kg body weight) was administered intravenously in mice. Spleens were harvested 4 h later for analysis. Intracellular secretion of IL-2 from gated CD4+T cells A. or IL-12 and IL-15 from CD11c+ cells B. is shown by flow cytometry. Numbers inside plots depict % positive cells. Representative data are shown from 2 independent experiments.
Figure 7
Figure 7. Cytokine expression in lymphoid and myeloid cell lines following bortezomib treatment
Expression of target cytokine mRNA was analyzed by RT-PCR in cell lines: Jurkat (CD4+T cell), T1 (CD8+T cell), and RAW264.7 (macrophage) 4 h after 10 nM Bzb treatment. Data are presented as the ratio of target gene expression to housekeeping gene β-actin from two independent experiments. Values are means ± SD; *p <0.05; two-tailed t-test.
Figure 8
Figure 8. Bortezomib-mediated modulation of IL-2 receptor chains, IL-12Rβ, and IL-15Rα on tumor-infiltrating CD8+T cells
Orthotopic tumors were established under mammary pads in Balb/c WT mice following injection of 2 × 106 4T1HA tumor cells. On day 14 (after the tumor reached an approximate size of 125 mm3), mice were injected intravenously with 1 mg/kg body weight of bortezomib. After 4 h, the mice were euthanized and single cell suspensions were obtained from the tumor mass harvested. The expression of cytokine receptors IL-2Rα A. IL-2Rβ B. IL-2Rγ C. IL-12Rβ D. and IL-15Rα E. is shown on tumor-infiltrating CD8+T cells by flow cytometry, with corresponding bar graphs presenting values as means ± SD. Numbers inside plots depict % positive cells. Representative data are shown from 2 independent experiments with 3 mice per group. *p<0.05, **p<0.01; two-tailed t-test.
Figure 9
Figure 9. Bortezomib modulates the expression of phosphorylated p38, Akt, and STAT5 in CD8+T cells
Levels of phosphorylated and total proteins were analyzed by Western blot in CD8+T cells following treatment in vitro with Bzb (20 nM) for the indicated duration of time. A. Protein levels of phosphorylated and total p38 and Akt. B. Protein levels of phosphorylated and total Akt following treatment with a combination of Bzb and PI3K inhibitor Ly294002 (50 μM). C. Protein levels of phosphorylated and total STAT5. The values on the Western blots represent fold change in expression analyzed by the densitometry with GAPDH used as a loading control. D. Orthotopic tumors were established in mammary pads of Balb/c WT mice following the injection of 2 × 106 4T1HA tumor cells. On day 14 (after the tumor reached an approximate size of 125 mm3), mice were injected intravenously with 1 mg/kg body weight of bortezomib. After 4 h, the mice were euthanized and single cell suspensions were obtained from the tumor mass harvested. Intracellular expression of phosphorylated STAT5 on tumor-infiltrating CD8+T cells was analyzed by flow cytometry. Numbers inside plots depict % positive cells. Representative data are from 2 individual experiments.
Figure 10
Figure 10. Effect of bortezomib treatment on the expression of transcription factors eomesodermin, T-bet and FoxP3 in T cells
A. Purified CD8+T cells from spleens of naïve Balb/c mice were stimulated in vitro with soluble anti-mouse CD3 and CD28 antibodies (1 μg/ml each) for 48 h with or without treatment with Bzb (10 nM) for another 4 h. Intracellular expression of transcription factors Eomes and T-bet were analyzed by flow cytometry. Numbers inside plots depict % positive cells. Bar graphs present data as means ± SEM for Eomes+CD8+T cells and T-bet+CD8+T cells from 2 independent experiments. **p<0.01; two-tailed t-test. B. Orthotopic (4T1HA) or subcutaneous (RencaHA) tumors were established in Balb/c WT mice following the injection of 2 × 106 tumor cells. On day 14, mice were injected intravenously with 1 mg/kg body weight of bortezomib. After 4 h, the mice were euthanized and single cell suspensions were obtained from the tumor mass harvested. Intracellular expression of FoxP3 on tumor-infiltrating CD8+T cells was analyzed by flow cytometry. Numbers inside plots depict % positive cells. Representative data are from 2 individual experiments.
Figure 11
Figure 11. Bortezomib enhances cytokine receptor signaling to enhance the expression of CD8+T cell effector molecules
Bortezomib treatment increases lymphocyte-stimulatory signaling through cytokines, IL-2, IL-12, and IL-15 via p38/PI3K/Akt/STAT5 pathways. This increases the transcription of effector molecules eomesodermin, granzyme-B and IFNγ in CD8+T lymphocytes.
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