Based on: Paper published in The Journal of Immunology by researchers at the Institute of Blood and Marrow Transplantation under the guidance of Dr. Haian Liu and authored by Ziqi Jin.
Hepatocellular carcinoma (HCC) is the sixth most prevalent form of cancer and is the third most common cause of cancer-related deaths in the world (Forner et al., 2012). Unfortunately, the mechanisms that explain tumor progression and metastasis in HCC continue to be poorly understood. This paper examines the role that the cytokine IL-33 has on HCC tumor growth and investigates the underlying mechanism. IL-33 is found in a wide variety of cells, including liver, lung and CNS cells, and typically functions as an alarmin, a molecule that signals damage, when the cell is stressed or dying (Cayrol and Girard, 2009). Previous research showed that HCC patients with tumor-infiltrating cells that produced IL-33 survived for longer periods of time, indicating to the authors that IL-33 may have an important antitumor effect (Brunner et al., 2015).
The first experiment examined the effect of IL-33 on HCC development. Jin et al (2018) developed a mouse orthotopic HCC model by releasing IL-33 into the liver through an injection of minicircle (MC) IL-33 plasmids. Afterwards, they implanted hepa1-6 cells into the mice and monitored the growth of the tumors for two weeks. After two weeks, the size of the tumors and the weight of the livers both decreased in the presence of IL-33. Jin et al (2018) then injected hepa1-6 cells that expressed either IL-33 or a vector control and measured the weight of the tumors and the number of tumor masses after three weeks. In tumor cells that expressed IL-33, tumor growth was significantly reduced compared to the control. To further understand the role of IL-33 in HCC tumor development, the authors injected MC IL-33 or control plasmids into DEN-induced HCC mouse models every other month for eight months. The mice injected with IL-33 plasmids showed a decrease in the number of tumor masses and tumor volumes compared to those injected with the control. Collectively, these results indicated to the authors that releasing IL-33 in the liver, through injection or from tumoral cells, can hinder the development of HCC tumors (Jin et al., 2018). Typically, IL-33 binds the receptor ST2 in order to function normally so the authors looked to see if the ST2 receptor was needed for IL-33 to have the previously observed effects on tumor development. They injected H22 cells and MC IL-33 plasmids into wild type (WT) mice that expressed the ST2 receptor or ST2-/- mice that did not express the ST2 receptors. Tumor growth was inhibited in the WT mice however the ST2-/- mice showed a diminished antitumor effect which indicated that IL-33 needs the ST2 receptor to inhibit HCC tumor development (Jin et al., 2018).
After establishing that releasing IL-33 inhibits the growth of HCC tumors, the next step was to determine the mechanism through which IL-33 does this. Jin et al., (2018) tackled this by injecting MC IL-33 plasmids or control vector plasmids in the hepa1-6 orthotopic HCC model and used FACS analysis to look at the immune cells present in the spleen and liver. They found that the percentage and number of activated and effector CD4+ & CD8+ T cells in the IL-33 group increased while the percentage of naive CD4+ & CD8+ T cells decreased (Jin et al., 2018). All together, this indicates that IL-33 promotes the activation of CD4+ and CD8+ T cells in vivo (inside an organism).
After demonstrating that IL-33 promotes the activation of CD4+ and CD8+ T cells, the authors’ next step was to see if IL-33 plays a role in the T cell response after they have been activated. To answer this question, Jin et al (2018) injected mice with orthotopic HCC model tumors that expressed IL-33 or a vector control (no IL-33 expression) then collected splenocytes and intrahepatic leukocytes seven days later. The authors found that the number of TNF-⍺- producing CD4+ and CD8+ T cells increased in the liver of the IL-33 group compared to the controls. Furthermore, both the percentage and number of IFN-γ-producing CD4+ and CD8+ T cells increased in the spleen and liver of the IL-33 group compared to the controls (Jin et al., 2018). In addition, splenocytes in the IL-33 group were more effective in killing hepa1-6 cells than the control group (Jin et al., 2018). Collectively, these results indicate that IL-33 upregulates antitumor T cell responses via increased cytokine production and elimination of HCC tumor cells.
The authors showed in the first experiment that IL-33 promotes the upregulation of activated and effector CD4+ and CD8+ T cells and sought to explore whether IL-33 can directly promote T cell activation. To investigate this, naive T cells from the spleen were collected from tumor-containing mice and grown in vitro (outside an organism) in the presence of 100 ng/mL rIL-33 or PBS in cell culture plates coated with anti-CD3 and anti-CD28 antibodies. They found that rIL-33 treatment significantly increases the percentage of CD8+ T cells as well as activated and effector CD4+ and CD8+ T cells (Jin et al., 2018). Meanwhile, the percentage of naive CD4+ and CD8+ T cells decreased after they were treated with rIL-33 (Jin et al., 2018). These results confirmed that IL-33 can directly enhance T cell activation in vitro.
Finally, the authors sought to examine if CD4+ and CD8+ T cells are required for IL-33 to have the antitumor effect they demonstrated earlier. In order to investigate this, the authors depleted CD8+ or CD4+ in tumor-bearing mice expressing IL-33 or a vector control by injecting the mice with anti-CD8⍺ or anti-CD4 antibodies respectively. Depletion of either CD8+ or CD4+ T cells had no effect on the antitumor activity of IL-33 (Jin et al., 2018). However, when both CD8+ and CD4+ T cells were depleted, the antitumor effect of IL-33 was reduced (Jin et al., 2018). This result indicates that the IL-33 antitumor effect is mediated by activation of both CD8+ and CD4+ T cells.
This paper demonstrates several key conclusions regarding the effects of IL-33 on HCC tumors. The authors show that IL-33 released in mouse HCC models inhibited the growth of tumors and promoted anti-tumor T cell responses in vivo. In addition, they also showed that IL-33 can directly activate a T cell response in vitro. The demonstrated antitumor effect was mediated by both CD8+ and CD4+ T cells. Future directions could involve an investigation into whether the antitumor effect of IL-33 is organ-specific and the effect releasing IL-33 has in other cancers it’s been described in.
References
Forner, A., Llovet, J. M., Bruix, J. (2012). Hepatocellular carcinoma. The Lancet https://www.sciencedirect.com/science/article/pii/S0140673611613470?via%3Dihub
Cayrol, C., & Girard, J. P. (2009). The IL-1-like cytokine IL-33 is inactivated after maturation by caspase-1. Proceedings of the National Academy of Sciences, 106(22), 9021-9026.
Brunner, S. M., Rubner, C., Kesselring, R., Martin, M., Griesshammer, E., Ruemmele, P., ... & Fichtner‐Feigl, S. (2015). Tumor‐infiltrating, interleukin‐33–producing effector‐memory CD8+ T cells in resected hepatocellular carcinoma prolong patient survival. Hepatology, 61(6), 1957-1967.
Jin, Z., Lei, L., Lin, D., Liu, Y., Song, Y., Gong, H., ... & Zhang, G. (2018). IL-33 Released in the Liver Inhibits Tumor Growth via Promotion of CD4+ and CD8+ T Cell Responses in Hepatocellular Carcinoma. The Journal of Immunology, ji1800627.
Schmitz, J., Owyang, A., Oldham, E., Song, Y., Murphy, E., McClanahan, T. K., ... & Gorman, D. M. (2005). IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity, 23(5), 479-490.
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