Fig. 6: MARCH5 knockdown improves anti-tumor immunity and suppresses tumor growth.
a Effects of MARCH5 knockdown on the level of γc in lymphocytes from spleen. Splenocytes from sex- and age-matched March5+/f or March5+/f:Vav1-Cre mice were stained with the indicated antibodies and analyzed by flow cytometry. Graph shows mean ± SEM, n = 6 independent samples. Data were analyzed using Student’s unpaired t-test with GraphPad Prism 8. MFI, median fluorescence intensities. b March5 knockdown increases the percentage of CD8+ single-positive (CD8SP) cells in thymocytes. Thymocytes from sex- and age-matched March5+/f or March5+/f:Vav1-Cre mice were analyzed by flow cytometry for the percentage of CD4-CD8- double-negative (DN), CD4+CD8+ double-positive (DP), CD4+ single-positive (CD4SP) and CD8+ single-positive (CD8SP). The CD4-CD8- double-negative (DN) cells were analyzed by flow cytometry for the percentage of CD44+ single-positive (DN1), CD44+CD25+ double-positive (DN2), CD25+ single-positive (DN3) and CD44-CD25- double-negative (DN4).Graph shows mean ± SEM, n = 6 independent samples. Data were analyzed using Student’s unpaired t-test with GraphPad Prism 8. c Effects of MARCH5 knockdown on the percentages of CD8+ T and NK cells from spleen and the peripheral blood. Splenocytes or peripheral blood leukocytes from sex- and age-matched March5+/f or March5+/f:Vav1-Cre mice were analyzed by flow cytometry for the percentage of CD8+ T (CD3+CD8+), NK (CD3-NKp46+), native CD8+ T (CD44lowCD62Lhigh CD8+ T cells), central memory CD8+ T (CD44highCD62Lhigh CD8+ T cells, CM) and effector/effector memory CD8+ T (CD44highCD62Llow CD8+ T cells, Effector/EM) cells. Graph shows mean ± SEM, n = 6 independent samples. Data were analyzed using Student’s unpaired t-test with GraphPad Prism 8. d MARCH5 knockdown inhibits tumor growth. Sex- and age-matched March5+/f or March5+/f:Vav1-Cre mice were subcutaneously injected with MC38 cells (5 × 105). On day 3 after tumor cell inoculation, tumor sizes were measured every two days by caliper. Tumor-bearing mice were euthanized on day 13, and then tumor tissues were separated from the mice. Tumor weights were measured by Analytical Balance. Graph shows mean ± SEM, n = 6. Data were analyzed using Student’s unpaired t-test with GraphPad Prism 8. e MARCH5 knockdown increases the percentages of CD8+ T and NK cells in TILs. TILs were isolated from the MC38 tumor tissues in d. TILs were stained with the indicated antibodies and analyzed by flow cytometry. Graph shows mean ± SEM, n = 6 independent samples. Data were analyzed using Student’s unpaired t-test with GraphPad Prism 8. f Combination of IL-2 and PD-1 blockade has increased anti-tumor efficacy in March5+/f:Vav1-Cre mice. March5+/f and March5+/f:Vav1-Cre mice were subcutaneously injected with MC38 cells (5 × 105). On day 5 after tumor cell inoculation, mice were intraperitoneally injected with control, IL-2 (50,000 IU per mouse) or anti-PD-1 (100 μg per mouse) (Supplementary information, Fig. S7d). Tumor sizes were measured every two days by caliper from day 5. WT: March5+/f, CHZ: March5+/f:Vav1-Cre. Graph shows mean ± SEM, n = 8. Data were analyzed using two-way ANOVA with GraphPad Prism 8. g Combination of IL-2 and PD-1 blockade increases the survival rate in March5+/f:Vav1-Cre mice. March5+/f and March5+/f:Vav1-Cre mice were subcutaneously injected with MC38 cells (5 × 105). On day 5 after tumor cell inoculation, mice were intraperitoneally injected with control, IL-2 (50,000 IU per mouse) or anti-PD-1 (100 μg per mouse) (Supplementary information, Fig. S7d). Mice were sacrificed when the tumor size was bigger than 15 mm of the mean tumor diameter, tumor volume exceeded 2000 mm3, or tumor had ulcers with diameter reached 10 mm. Statistical analysis was performed using the GraphPad Prism 8 software, n = 8. Kaplan–Meier survival curves and corresponding log-rank (Mantel-Cox) tests were used to evaluate the statistical differences between groups in survival studies. There is a significant difference when the P < 0.05.
