Effects of Stimulating Interleukin-2/Anti-Interleukin-2 Antibody Complexes on Renal Cell Carcinoma

Kyu-Hyun Han; Ki Won Kim; Ji-Jing Yan; Jae-Ghi Lee; Eun Mi Lee; Miyeon Han; Eun Jin Cho; Seong Sik Kang; Hye Jin Lim; Tai Yeon Koo; Curie Ahn; Jaeseok Yang

Disclosures

BMC Urol. 2016;16(2) 

In This Article

Results

IL-2/Anti-IL-2 Antibody Complex Treatment Induces the Expansion of CD8+ Memory T and NK Cells in the Spleen

IL-2C were injected into healthy mice for 5 consecutive days to evaluate its immune-enhancing effects. The total numbers of splenocytes (Fig. 1a; P < 0.010) and CD8+ T cells (Fig. 1b; P < 0.010) were increased in the IL-2C group as compared to the PBS group. IL-2C treatment also increased the numbers of CD44+CD8+ memory T (Fig. 1c; P < 0.010) and CD49b+ NK (Fig. 1d; P < 0.010) cells. These results suggest that IL-2C treatment can enhance anti-tumor immunity.

Figure 1.

IL-2C treatment induces the expansion of CD8+ memory T and NK cells in the spleen. Mice were treated with IL-2C by intraperitoneal injection for 5 days. The total numbers of (a) splenocytes, (b) CD8+ T cells, (c) CD8+ memory T cells, (d) and NK cells were higher in IL-2C-treated than in PBS-treated mice (P < 0.010)

IL-2/Anti-IL-2 Antibody Complex Treatment Induces the Expansion of CD8+ Memory T and NK Cells in the Spleen of RCC Mice

Mice were subcutaneously injected with syngeneic RENCA cells, followed by IL-2C or PBS administration every other day for 4 weeks. A sham group received PBS without RENCA cell implantation. Among RCC mice, there was no difference in the number of CD4+ T cells between the IL-2C and PBS groups (Fig. 2a; P = 0.498). However, IL-2C treatment induced the expansion of CD8+ T, CD8+ memory T, and NK cells as well as macrophages (Fig. 2b–e; P < 0.010), and increased the number of splenic CD4+Foxp3+ regulatory T cells (Fig. 2f; P = 0.040), albeit to a lesser degree than for CD8+ memory T or NK cells. As a result, CD8+ memory T cell/regulatory T cell (Fig. 2g; P < 0.010) and NK cell/regulatory T cell (Fig. 2h; P < 0.010) ratios were increased in the IL-2C relative to the PBS group. These data indicate that IL-2C treatment enhances anti-tumor immunity against RCC.

Figure 2.

IL-2C treatment induces the expansion of immune cells in the spleen of mice with RCC. Syngeneic RENCA cells were implanted subcutaneously in mice, and IL-2C or PBS was administered every other day for 4 weeks. (a) The number of CD4+ T cells was similar between the two groups (P = 0.498), while the numbers of (b) CD8+ T cells, (c) CD8+ memory T cells, (d) NK cells, (e) macrophages, and (f) Tregs were higher in the IL-2C group than in the PBS group (P < 0.010). (g, h) CD8+ memory T cell/Treg and NK cell/Treg ratios were higher in the IL-2C group than the PBS group (P < 0.010 in both cases). Treg, regulatory T cell

IL-2/Anti-IL-2 Antibody Complex Treatment Increases IFN-γ+ and Decreases IL-10+ Splenocyte Populations

We analyzed Th1 and Th2 cytokine responses in the spleen of RCC mice (Fig. 3). The number of IFN-γ-producing splenocytes was lower in RCC mice treated with PBS than in the sham group. Meanwhile, IL-2C-treated mice had a higher number of IFN-γ+ splenocytes than those in the PBS group (Fig. 3a; P < 0.010). The number of IL-10-producing splenocytes was higher in RCC mice treated with PBS than in the sham group (P < 0.010), but this was decreased by IL-2C treatment (Fig. 3b; P < 0.01). These results indicate that IL-2C can shift the immune response from Th2 to Th1 in the RCC environment.

Figure 3.

IL-2C treatment increases the number of IFN-γ+ splenocytes, but decreased the number of IL-10+ splenocytes. After syngeneic RENCA cells were implanted in mice, splenocytes were harvested on day 28 and analyzed for IFN-γ and IL-10 production by ELISPOT. a, b The number of IFN-γ-producing splenocytes was higher (a) but the number of IL-10-producing splenocytes were lower (b) in IL-2C-treated mice than in PBS-treated mice (P < 0.010 in both cases)

IL-2/Anti-IL-2 Antibody Complex Treatment Increases Immune Cell Infiltration Into RCC Lesions

Given the immune-stimulating effects of IL-2C on the spleen in RCC, we investigated whether immune cell infiltration of immune cells into RCC lesions was induced by IL-2C treatment. Flow cytometric analysis showed higher numbers of infiltrating CD45+ (Fig. 4a; P < 0.010), CD4+ T (Fig. 4b; P < 0.010), CD8+ T (Fig. 4c; P = 0.037), and NK (Fig. 4d; P = 0.033) cells as well as macrophages (Fig. 4e; P < 0.010) in the IL-2C group than in the PBS group. In addition, an immunohistochemical analysis found that IL-2C treatment increased CD4+ T, CD8+ T, and NK cells as well as macrophages recruitment to RCC lesions (Fig. 5). However, there was no perigraft infiltration of regulatory T cells (data not shown). Taken together, these data demonstrate that IL-2C stimulates the infiltration of immune cells into RCC lesions.

Figure 4.

Flow cytometric analysis of immune cell infiltration into RCC lesions. Tumors were harvested on day 28 after mice were implanted with RENCA cells and immune cells were detected by flow cytometry. IL-2C treatment increased infiltration of (a) CD45+ cells (P < 0.010), (b) CD4+ T cells (P < 0.010), (c) CD8+ T cells (P = 0.037), (d) NK cells (P = 0.033) (e) and macrophages (P < 0.010) into tumors

Figure 5.

Immunohistochemical analysis of immune cell infiltration into RCC lesions. Tumors were harvested on day 28 after mice were implanted with RENCA cells and immune cells were detected by immunohistochemistry. The size of immune cell populations, including (a) CD4+ T cells, (b) CD8+ T cells, (c) NK cells and (d) macrophages along tumor margins was increased by IL-2C treatment. Images are shown at 200× magnification. Insets show immunoreactive cells at 1000× magnification

Anti-tumorigenic Effects of IL-2/Anti-IL-2 Antibody Complex Were not Sufficient to Suppress RCC Growth

The size of RCC lesions increased progressively over time in both PBS-treated and IL-2C-treated mice (Fig. 6a; P < 0.010); however, the rate of growth was higher in the former group (Fig. 6a; P = 0.036), although the difference was slight. However, tumor weights on day 28 did not differ significantly between the two groups (Fig. 6b; P = 0.176). These data suggest that the potentiation of anti-tumor immunity by IL-2C was not sufficient to suppress RCC growth significantly.

Figure 6.

IL-2C does not suppress growth of RCC significantly. a IL-2C slowed the growth of syngeneic RENCA cells implanted subcutaneously into mice (P = 0.036, linear mixed model). b Tumor weight on day 28 did not differ significantly between the IL-2C and the PBS groups (P = 0.176, Student's t-test)

IL-2/anti-IL-2 Antibody Complex Treatment Does not Induce Pulmonary Edema

Pulmonary edema is a manifestation of capillary leak syndrome and is the most serious side effect of high-dose of IL-2 therapy.[23] On day 28, there was no significant difference in lung weights between IL-2C- and PBS-treated mice (Fig. 7a; P = 0.184). A histologic examination revealed no evidence of increased pulmonary edema by IL-2C treatment (Fig. 7b). These results demonstrate that IL-2C is safe for use, as it does not carry a significant risk of pulmonary edema development.

Figure 7.

IL-2C does not exacerbate pulmonary edema in mice with RCC. Lung weight was measured by subtracting dry from wet weight immediately after harvesting on day 28. a Lung weight did not differ significantly between IL-2C-treated mice and PBS-treated mice (P = 0.184). b IL-2C treatment did not increase pulmonary edema, as visualized by hematoxylin and eosin staining. Images are shown at 400× magnification

Comparison Between IL-2/Anti-IL-2 Antibody Complex Treatment and High-dose IL-2 Therapy

When immune potentiating effects of IL-2C were compared with those of high-dose IL-2 therapy, the IL-2C therapy increased total leukocytes, CD8+ T cells, NK cells, and macrophages in both spleen (Fig. 8) and peritumor tissues (data not shown) to greater extent than the high-dose IL-2 therapy. The ratio of either splenic CD8+CD44+ T cells/Tregs or CD49b + NK cell/Tregs were not significantly increased in the high-dose IL-2 group (Fig. 8d-e). There was no difference in RCC weight between the IL-2C group and the high-dose IL-2 group (Fig. 8f). Pulmonary edema looked more severe in the high-dose IL-2 group than IL-2 complex group (Fig. 8g); however there was no significant difference in lung weight between the two groups (P > 0.05). Taken together, IL-2C induced more immune potentiating effects with lesser dose than high-dose IL-2 therapy; however IL-2C did not show significant benefits in either tumor reduction or pulmonary edema in the present dose.

Figure 8.

Comparison between IL-2C therapy and high-dose IL-2 therapy. IL-2C treatment induces more expansion of splenic immune cells than high-dose IL-2 therapy (a-e). a Both IL-2C (P = 0.004) and high-dose IL-2 (P = 0.008) increased the number of splenocytes; however, the effect of IL-2C was greater than that of high-dose IL-2 (P = 0.019). b CD8+ T cells were also increased more by IL-2C than high-dose IL-2 (P = 0.006). c Only IL-2C increased the number of NK cells (P = 0.002). d-e IL-2C increased both ratio of CD8+CD44+ T cells/Tregs (P = 0.002, d), and ratio of CD49b+ NK cells/Tregs (P = 0.001, e), whereas high-dose IL-2 did not. f Either IL-2C or high-dose IL-2 did not suppress growth of RCC significantly. Tumor weight on day 28 did not differ significantly between the IL-2C and the high-dose IL-2 groups (P = 0.353). g Pulmonary edema looked more severe in the high-dose IL-2 group than IL-2 complex group; however difference was not significant. Images are shown at 200× magnification. IL-2C, interleukin-2/anti-interleukin-2 antibody complex; HD, high dose; Treg, regulatory T cell

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