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. 2020 Jun;19(6):3827-3838.
doi: 10.3892/ol.2020.11477. Epub 2020 Mar 27.

Gene silencing of indoleamine 2,3-dioxygenase 1 inhibits lung cancer growth by suppressing T-cell exhaustion

Ke Shang  1 , Zhigang Wang  2 , Yinying Hu  2 , Yanqin Huang  2 , Keng Yuan  2 , Yanrong Yu  2
Affiliations

Gene silencing of indoleamine 2,3-dioxygenase 1 inhibits lung cancer growth by suppressing T-cell exhaustion

Ke Shang et al. Oncol Lett. 2020 Jun.

Abstract

Indoleamine 2,3-dioxygenase 1 (IDO1), which degrades the essential amino acid tryptophan, exerts immunosuppressive functions and serves a crucial role in multiple types tumor progression, including non-small-cell lung cancer (NSCLC) and melanoma. Recent studies have reported that T-cell exhaustion is increased during tumor progression, which impairs the antitumor immune response. However, the association between IDO1 and T-cell exhaustion during tumor progression remains unknown. The present study evaluated the effect of IDO1 on T-cell exhaustion in lung cancer mice. The present study demonstrated that IDO1 knockdown by small interfering RNA in the LLC cell line inhibited T-cell exhaustion. Furthermore, the role of IDO1 in T-cell exhaustion during lung cancer progression was determined in an in vivo mouse model using IDO1 short hairpin RNA (shRNA). The results demonstrated that inhibition of IDO1 activity by shRNA administration in vivo significantly delayed the onset and growth of tumors. In addition, the expression levels of the inhibitory receptors programmed death-1 (PD-1) and B and T lymphocyte attenuator (BTLA) were increased in T-cells from the lung tumor-bearing mice, whereas interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) levels in serum were decreased compared with the control mice. However, no difference in the absolute number of T cells was observed, including CD4+ and CD8+ T cells. In addition, IDO1 knockdown by shRNA inhibited T-cell exhaustion in lung tumor-bearing mice, which was characterized by decreased expression of PD-1 and BTLA on T cells. By contrast, IL-2 and TNF-α levels in serum were increased in IDO1-shRNA-treated mice. By using a shRNA approach, the present study demonstrated that IDO1 activity may be involved in tumor growth, and that IDO1 silencing may inhibit tumor progression by impeding the process of T-cell exhaustion.

Keywords: T-cell exhaustion; immunotherapy; indoleamine 2,3 dioxygenase 1; lung cancer.

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Figures

Figure 1.
Figure 1.
IDO1-siRNA silences IDO1 expression in vitro. (A) IDO1 mRNA expression. LLC cells were transfected with IDO1-siRNA or GL2-siRNA (negative control). Untransfected cells were used as a blank control. IDO1 mRNA expression level was detected by reverse transcription-quantitative PCR (n=3). (B) IDO1 protein expression. Silencing of IDO1 was confirmed by western blotting (n=3) in LLC cells after transfection with IDO1-siRNA, GL2-siRNA or blank control. *P<0.05 and **P<0.01. GL2, glabra 2; IDO1, indoleamine 2,3-dioxygenase 1; siRNA, small interfering RNA.
Figure 2.
Figure 2.
IDO1-siRNA in LLC cells inhibits T-cell exhaustion in vitro. (A and B) LLC cells were transfected with IDO1-siRNA or GL2-siRNA and co-cultured with lymphocytes isolated from spleen. Lymphocytes were collected and (A) PD-1 and (B) BTLA were detected by flow cytometry. n=3. *P<0.05 and **P<0.01. BTLA, B and T lymphocyte attenuator; GL2, glabra 2; IDO1, indoleamine 2,3-dioxygenase 1; PD-1, programmed death-1; siRNA, small interfering RNA; control, untransfected cells.
Figure 3.
Figure 3.
Treatment with IDO1-shRNA inhibits lung cancer growth in vivo. Treatment with IDO1 or scrambled expression vectors was performed by hydrodynamic intravenous tail injection. (A) Tumor tissues were collected from lung cancer-bearing mice after 21 days. Expression of IDO1 in tumors was detected by immunohistochemistry (magnification, ×40). (B) IDO1 expression scores. (C) Time of tumor onset was recorded when the tumor diameter reached 5 mm. (D) Tumor growth curve. (E) Images of tumors. At day 21 after LLC cell inoculation, the mice were sacrificed, and the tumors were measured with a caliper. (F) Tumor weights were measured after 21 days. *P<0.05, **P<0.01 and ***P<0.001. IDO1, indoleamine 2,3-dioxygenase 1; shRNA, short hairpin RNA; control, untreated mice,
Figure 4.
Figure 4.
Tumors increase inhibitory receptor expression in both CD4+ and CD8+ T cells in vivo. Tumor-bearing mice were inoculated subcutaneously with LLC cells, and lymphocytes were collected for analysis after 21 days. Mice without tumors were used as the control. (A) Flow cytometry analysis of CD4+/CD8+ lymphocytes from lymph nodes of CD3+ T cells in mice without tumors and mice with tumors. (B) Flow cytometry analysis of CD4+/CD8+ lymphocytes from the spleen of CD3+ T cells in mice without tumors and mice with tumors. (C and D) Percentage of PD1 and BTLA in CD4+/CD8+ T cells from the lymph nodes and the spleen in mice without tumors vs. mice with tumors. *P<0.05 and **P<0.01. BTLA, B and T lymphocyte attenuator; LN, lymph node; PD-1, programmed death-1; SP, spleen.
Figure 5.
Figure 5.
IDO1-shRNA treatment decreases the expression of inhibitory receptors of CD4+/CD8+ T cells in vivo. Lymphocytes from lymph nodes and spleen of LLC-bearing tumor mice were collected on day 21 injection of LLC cells. CD4+ and CD8+ T cells were stained with anti-PD-1 and anti-BTLA and analyzed by flow cytometry. Expression of PD-1 and BTLA on CD4+ T cells in the (A) lymph nodes and (B) spleen of tumor-bearing mice without treatment, or treated with scrambled-shRNA or IDO1-shRNA. (C) Expression of PD-1 and BTLA on CD8+ T cells from the lymph nodes of tumor-bearing mice without treatment or treated with scrambled-shRNA or IDO1-shRNA. Statistical analysis of the expression of PD1 and BTLA on CD4+ (right panel in A and B) and CD8+ (right panels in C) was performed from three independent experiments. *P<0.05 and **P<0.01. BTLA, B and T lymphocyte attenuator; IDO1, indoleamine 2,3-dioxygenase 1; LN, lymph node; PD-1, programmed death-1; sh, short hairpin; SP, spleen.
Figure 6.
Figure 6.
Treatment with IDO1-shRNA in tumor-bearing mice in vivo recovers cytokine secretion. C57BL/6 mice were treated with IDO1-shRNA or scrambled-shRNA by hydrodynamic intravenous tail injection. At day 21, blood from tumor-bearing control and treated mice was collected to detect the serum levels of TNF-α and IL-2 by ELISA. (A and B) Serum TNF-α and IL-2 levels in mice with and without tumors. (C and D) Serum TNF-α and IL-2 levels in the control mice, IDO1-shRNA- and scrambled-shRNA-treated mice. n=3. *P<0.05 and **P<0.01. IL-2, interleukin-2; IDO1, indoleamine 2,3-dioxygenase; shRNA, short hairpin RNA; TNF-α, tumor necrosis factor-α.
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