Role of macrophage migration inhibitory factor in the regulatory T cell response of tumor-bearing mice

Abstract

Macrophage migration inhibitory factor (MIF) is involved in tumorigenesis by facilitating tumor proliferation and evasion of apoptosis; however, its role in tumor immunity is unclear. In this study, we investigated the effect of MIF on the progression of the syngenic, CT26 colon carcinoma and the generation of tumor regulatory T cells (Tregs). The results showed that the tumor growth rate was significantly lower in MIF knockout (MIF(-/-)) mice than in wild-type (MIF(+/+)) mice. Flow cytometric analysis of both spleen and tumor cells revealed that MIF(-/-) mice had significantly lower levels of tumor-associated CD4(+)Tregs than MIF(+/+) mice. The splenic cells of MIF(-/-) mice also showed a decrease in CD8(+)Tregs, which was accompanied by an increase in CD8-induced tumor cytotoxicity. Interestingly, the inducible Treg response in spleen cells to anti-CD3/CD28 plus IL-2 plus TGF-β was greater in MIF(-/-) mice than in MIF(+/+) mice. Spleen cells of MIF(-/-) mice, stimulated with anti-CD3/CD28, produced lower levels of IL-2, but not TGF-β, than those of MIF(+/+) mice, which was recovered by the addition of recombinant MIF. Conversely, a neutralizing anti-MIF Ab blocked anti-CD3-induced IL-2 production by splenocytes of MIF(+/+) mice and suppressed the inducible Treg generation. Moreover, the administration of IL-2 into tumor-bearing MIF(-/-) mice restored the generation of Tregs and tumor growth. Taken together, our data suggest that MIF promotes tumor growth by increasing Treg generation through the modulation of IL-2 production. Thus, anti-MIF treatment might be useful in enhancing the adaptive immune response to colon cancers.

Figure 1. Reduced growth of the CT26 colon carcinoma in syngeneic MIF−/− mice

(A and B) Decrease in tumor size and weight in MIF−/− mice. CT26 colon cancer cells (1×10⁶/mouse) were administered subcutaneously into the right flank of MIF−/− mice and wild type (MIF +/+) mice. The tumor volume was calculated using the formula of width²×length×0.5 (length > width). Four weeks after the tumor inoculation, tumor mass was excised and its weight was measured. *, P < 0.001. Representative photographs are shown inside the figure; WT: wild type, KO: knock-out. (C) Changes in body weight of MIF−/− mice versus MIF +/+ mice after the tumor injection. *, P < 0.05. (D) Comparison of spleen weight between MIF−/− and MIF +/+ mice, which was assessed at 4 weeks after the tumor injection. *, P < 0.05. (E) Immuno-histochemical staining of tumor mass of MIF−/− mice (n=6) and MIF+/+ mice (n=6) using anti-CD3 antibody. The number of T cells was manually counted and presented as a ratio of T cells over non-T cells, including tumor cells. Data are the mean ± SD of CD3 ratios. (F) MIF mRNA expression in CT-26 tumor cells. Total RNA was isolated from CT26 colon cancer cells (lane 1), spleen cells of MIF+/+ mouse (lane 2), and spleen cells of MIF−/− mouse (lane 3). The mRNA expressions in these cells were determined by reverse transcription PCR analysis. Expression of GAPDH mRNA is presented as a control. (G) MIF concentrations in the sera of tumor-bearing MIF+/+ mice (n=14) and MIF−/− mice (n=14). Four weeks after the tumor inoculation, the sera were collected from the two groups of mice, and circulating MIF levels were measured by ELISA. *, P < 0.05.

Figure 2. Decrease in CD4+Tregs in MIF−/− mice

(A) Decrease in CD4+Tregs in MIF−/− mouse as compared to MIF+/+ mouse. Representative data of similar results in more than five mice are shown. Using flow cytometry analysis, the frequency of CD4+CD25+Foxp3+ T cells, CD4+GITR+ T cells, and CD4+CTLA4+ T cells was determined in single cell suspensions of tumor mass of MIF−/− versus MIF+/+ mouse. (B) Representative flow cytometry analysis of CD4+Tregs in the spleen of MIF−/− mouse versus MIF+/+ mouse. (C) Comparison of the percentage of CD4+Tregs in the spleen and tumor tissue between tumor-bearing MIF−/− and MIF+/+ mice. The frequency of Tregs was calculated as the percentage of CD4+CD25+Foxp3+ cells in the CD4+ T-cell population. Mice without tumor inoculation were used as a control. **, P < 0.01. (D) Tumor rejection in MIF−/− mice. Left panel represents a photograph of tumor-bearing and tumor-rejected MIF−/− mouse taken 4 weeks after tumor inoculation. Right panel shows changes in tumor volume in tumor-rejected versus tumor-bearing MIF−/− mice. Representative data are shown and the arrow indicates the injection site. (E and F) Recovery of CD4+ and CD8+ T cells in the spleen of tumor-rejected MIF−/− mice. The frequency of CD4+ or CD8+ T cells was determined by flow cytometry and calculated as the percentage of CD4+ or CD8+ cells in the splenocytes from mice. Tumor-naïve MIF−/− and MIF +/+ mice were used as a control. *, P < 0.05. (G) Decrease in CD4+ Tregs in the spleen of tumor-rejected MIF−/− mice, as compared to that of tumor-bearing MIF−/− and MIF +/+ mice. The frequency of Treg was calculated as the percentage of CD4+CD25+Foxp3+ cells in the CD4+ T-cell population. *, P < 0.05, **, P < 0.01. (H) Correlation of the frequency of CD4+Tregs with tumor size determined at 4 weeks after tumor injection, in all of MIF−/− and MIF+/+ mice tested.

Figure 3. Inducible CD4+Treg response and cytokine production in spleen cells of MIF−/− versus MIF+/+ mice

(A and B) Representative data and quantification of inducible CD4+Tregs. Sorted CD4+ T cells of the spleen of tumor-naïve mice were stimulated with or without anti-CD3/CD28 Ab+IL-2 (1 ng/ml) +TGF-β (3 ng/ml). After 72 hours of stimulation, the frequency of CD4+CD25+Foxp3+ in CD4+ cells was determined by flow cytometry. Overlay histogram for Foxp3 is also shown in Fig. 3B. (C) Comparison of the percentage of inducible CD4+Tregs between tumor-naive MIF−/− and MIF +/+ mice. *, P < 0.05. (D to F) Cytokine production by splenic T cells of tumor-bearing MIF−/− versus MIF+/+ mice. The spleen cells (1×10⁶) of the two groups of mice were stimulated with anti-CD3/CD28 Ab for 48 or 72 hours. Levels of IL-2, IFN-γ, IL-10 and TGF-β in the culture supernatants were measured by ELISA. *, P < 0.05.

Figure 4. MIF controls IL-2-mediated Treg generation

(A) Suppression of inducible CD4+ Tregs by anti-IL-2 Ab. Splenic CD4+ cells of MIF+/+ mice were stimulated with anti-CD3/CD28 Ab (CD3/CD28) plus TGF-β (3 ng/ml) in the presence of anti-IL-2 Ab (0.1 µg/ml) or isotype control IgG (IgG). After 72 hours of incubation, the frequency of CD4+CD25+Foxp3+ T cells over CD4+ cells was determined by flow cytometry analysis. Data shows mean±SD. *, P < 0.05 versus isotype control Ab. (B) Restoration of IL-2 production by recombinant MIF. Splenic T cells (2×10⁵) of MIF−/− mice were stimulated with anti-CD3/CD28 Ab in the presence of recombinant MIF (500 ng/ml) for 48 hours. IL-2 levels the culture supernatants were measured by ELISA. *, P < 0.05 versus IL-2 production by splenic cells of MIF+/+ mice treated with anti-CD3/CD28 Ab alone. (C and D) Inhibition of IL-2 production by neutralizing anti-MIF Ab (C) or MIF receptor antagonist (D). Splenic cells (2×10⁵) of MIF+/+ mice were stimulated with anti-CD3/CD28 Ab in the absence or presence of anti-MIF Ab (100 µg/ml) for 48 hours or D1036 (a MIF receptor antagonist: 1, 5, and 50 µM) for 24 hours. Isotype control IgG (100 µg/ml) was used as a control. IL-2 levels in the culture supernatants were measured by ELISA. *, P < 0.05 versus isotype control Ab or anti-CD3/CD28 Ab alone. (E) Effect of anti-MIF Ab on the inducible CD4+Treg production. Splenic CD4+ cells of MIF+/+ mice were cultured in RPMI1640 supplemented with 10% FBS, and stimulated with anti-CD3/CD28 Ab (CD3/CD28) plus TGF-β (3 ng/ml) in the presence of anti-MIF Ab (50 µg/ml) or isotype control IgG (50 µg/ml). After 72 hours of incubation, the frequency of CD4+Tregs was determined by flow cytometry analysis. A representative is shown on the left. Bar graph on the right shows the mean±SD of six independent experiments. *, P < 0.05 versus isotype control Ab. **, P < 0.01 versus anti-MIF Ab alone.

Figure 5. Changes in tumor-specific cytotoxicity and CD8+Tregs in MIF−/− mice

(A) Increase in tumor cell death induced by CD8+ T cells of MIF−/− mice. The CT26 carcinoma cells were subcutaneously injected in MIF−/− mice and MIF+/+ mice, and CD8+ T cells in the spleens of the two groups of mice then were isolated using anti-CD8 micro-beads. The CD8+ effector cells were co-cultured with CT26 target cells with E:T ratios of 40:1 for 4 hours and 8 hours. Cytotoxicity was measured by lactate dehydrogenase (LDH) release. *, P < 0.05. (B and C) Decrease in CD8+Tregs in tumor-bearing MIF−/− mice. Four weeks after the tumor injection, spleen cells and tumor tissues were harvested from MIF−/− mice and MIF+/+ mice. The frequency of CD8+CD25+Foxp3+ T cells was assessed by flow cytometry and calculated as the percentage of CD8+CD25+Foxp3+ cells in the CD8+ T-cell population. The representative data are shown in the left panel. *, P < 0.05. (D and E) Inducible CD8+ Tregs in tumor-naïve MIF−/− mice (n=3) and MIF+/+ mice (n=3). The splenic cells were stimulated by anti-CD3/CD28 Ab+IL-2+TGF-β as described in Materials and Methods. The number of CD8+CD25+Foxp3+ T cells was determined by flow cytometry. *, P < 0.05 versus MIF+/+ mice. A representative is shown in the left panel.

Figure 6. Recombinant IL-2 reverses tumor growth and Treg generation in MIF−/− mice

(A) Tumor-bearing MIF−/− mice were injected with recombinant 2,500 IU IL-2 (IL-2, n=3), or 12,500IU IL-2 (5×IL-2, n=9) 14 times every other day for 30 days every day after the tumor cell inoculation. MIF+/+ (n=5) and MIF−/− mice (n=9) injected with vehicle alone were used as a control. Tumor growth rate was monitored every day. *, P < 0.05. (B) Appearance of vehicle-treated MIF+/+ mice, vehicle-treated MIF−/− mice, and IL-2-injected MIF−/− mice at 4 weeks after tumor cell inoculation. (C and D) The percentage of CD4+ and CD8+Tregs in the spleens and tumor tissues of IL-2-treated MIF−/− mice (n=3 for IL-2, n=9 for 5×IL-2), vehicle-treated MIF−/− (n=9), and vehicle-treated MIF+/+ mice (n=5), determined by flow cytometry analysis.