Chronic alcohol consumption enhances myeloid-derived suppressor cells in B16BL6 melanoma-bearing mice

Abstract

We previously found that chronic alcohol consumption decreases the survival of mice bearing subcutaneous B16BL6 melanoma. The underlying mechanism is still not completely understood. Antitumor T cell immune responses are important to inhibiting tumor progression and extending survival. Therefore, we examined the effects of chronic alcohol consumption on the functionality and regulation of these cells in C57BL/6 mice that chronically consumed 20% (w/v) alcohol and subsequently were inoculated subcutaneously with B16BL6 melanoma cells. Chronic alcohol consumption inhibited melanoma-induced memory T cell expansion and accelerated the decay of interferon (IFN)-gamma producing T cells in the tumor-bearing mice. Foxp3(+)CD4(+)CD25(+) regulatory T cells were not affected; however, the percentage of myeloid-derived suppressor cells (MDSC) was significantly increased in the peripheral blood and spleen. T cell proliferation as determined by carboxyfluorescein succinimidyl ester labeling experiments in vitro was inhibited by alcohol consumption relative to control water-drinking melanoma-bearing mice. Collectively, these data show that chronic alcohol consumption inhibits proliferation of memory T cells, accelerates the decay of IFN-gamma producing CD8(+) T cells, and increases MDSC, all of which could be associated with melanoma progression and reduced survival.

Fig. 1

Effects of chronic alcohol consumption on CD44^hiCD8⁺ T cells. a Dot plot showing the gated CD8⁺ T cells in splenocytes. b Histogram showing the CD44^hi cells in the gated splenic CD8⁺ T cells of melanoma-bearing mice. c Percentage of CD8⁺CD44^hi cells in CD8⁺ splenocytes from non-tumor injected mice (Cont) and melanoma-bearing mice at the indicated time points after tumor inoculation. Circle alcohol-consuming mice. Square water-drinking mice. Alcohol group different from water group, *P < 0.05, **P < 0.001. Each group contained ten mice and the results are representative to two separate experiments

Fig. 2

Chronic alcohol consumption decreases B16BL6 melanoma-associated gp100-specific CD8⁺ T cells. a Dot plot of the gp100/H-2D^b tetramer (3700) positive CD8⁺ cells in the gated splenic CD8⁺ T cell population from melanoma-bearing mice after 3 weeks. b Percentage of gp100-specific T cells in the splenic CD8⁺ T cell population of melanoma-bearing mice at the indicated time points after tumor inoculation. Circle water-drinking mice. Square alcohol-consuming mice. ETOH group different from water group, *P < 0.05. c Number of gp100-specific CD8⁺ T cells in the spleen of melanoma-bearing mice 3 weeks after tumor inoculation. ETOH group different from water group, **P < 0.001. Each group contained ten mice and the results are representative to two separate experiments. ETOH Alcohol-consuming group, Water water-drinking group

Fig. 3

Chronic alcohol consumption accelerates the decay of IFN-γ producing cells within the splenic CD8⁺ T cell population in the melanoma-bearing mice. Closed diamonds water-drinking mice. Closed squares alcohol-consuming mice. Alcohol-consuming group different from water consuming group, *P < 0.05, **P < 0.001. Each group contained ten mice and the results are representative to two separate experiments

Fig. 4

Chronic alcohol consumption increases the percentages of MDSC in the PBL and of CD124⁺ cells in MDSC and CD11b⁺ cells. MDSC were determined 1 week after tumor inoculation. a Dot plot of CD11b⁺Gr-1^int MDSC in the PBL of water-drinking mice. b Dot plot of CD11b⁺Gr-1^int MDSC in the PBL of alcohol-consuming mice, respectively. c Percentage of CD11b⁺Gr-1^int MDSC in the PBL. d Percentage of CD124⁺ cells in CD11b⁺Gr-1^int MDSC cells. ETOH group significantly different from water group, *P < 0.05, **P < 0.001. ETOH Alcohol-consuming group, Water water-drinking group. Each group contained ten mice and the results are representative to two separate experiments

Fig. 5

Alcohol consumption does not alter Foxp3⁺CD4⁺CD25⁺ Treg cells. Treg cells were determined 2 weeks after tumor inoculation. a Dot plot of Treg cells (upper right quadrant) in cells from the inguinal lymph nodes (LN). b Percentage of Foxp3⁺CD4⁺CD25⁺ Treg cells in the spleen, PBL and LN of melanoma-bearing mice

Fig. 6

Effects of chronic alcohol consumption on T cell proliferation in vitro. Splenocytes were isolated from mice consuming alcohol for 3 months and inoculated with 2 × 10⁵ B16BL6 sc for 2 weeks, labeled with CFSE and stimulated with anti-CD3 and anti-CD28 monoclonal antibodies for 90 h. a, b Representative histograms indicating the MFI of CFSE in CD8⁺ T cells from water-drinking tumor-bearing mice and alcohol-consuming tumor-bearing mice, respectively. The marker (bar region) M1 stands for proliferating cells. c Histogram of the MFI of CFSE in CD8⁺ T cells, ETOH different from water, *P < 0.05. d Histogram of the percentage of proliferating cells in CD8⁺ T cells, ETOH different from water, **P < 0.001. Each group contained seven mice. ETOH Alcohol-consuming group, Water water-drinking group. The results are representative of two separate experiments