MYO1F deficiency enhances the immunosuppressive function of TANs. (A) Volcano plot of mRNA sequencing data of sorted tumor-infiltrating neutrophils from WT and Myo1f−/− B16F10 model. (B) Gene mRNA level was detected by RT-qPCR from sorted tumor-infiltrating neutrophils from WT and Myo1f−/− B16F10 model (N = 5). (C) FACS analyses of PD-1hi and TIM3+ of CFSE-labeled CD8+ T cell co-culture with sorted tumor neutrophils from WT and Myo1f−/− B16F10 tumor models of day 21 (N = 5). (D) FACS analyses of recipient intratumoral CD45.1+ neutrophils in the BM neutrophil transfer tumor model as described in Fig. 2 O (N = 5). (E) FACS analyses of apoptosis in intratumoral CD45.1+ neutrophils by staining with annexin V and PI in the BM neutrophil transfer tumor model as described in Fig. 2 O (N = 5). (F) FACS analyses of PD-L1 in intratumoral CD45.1+ neutrophils in recipient (N = 5). (G) Cultured neutrophils from WT mice were transfected with siCtrl, siMyo1f, and PD-L1 on neutrophils were analyzed 48 h after transfection by FACS (N = 5). (H) Cultured neutrophils from WT mice were transfected with siCtrl and siCd274; after 24 h, cells were treated with cisplatin at 1 µM, and annexin V and PI staining were analyzed at 48 h after transfection by FACS (N = 3). (I) Cultured neutrophils from WT and Myo1f−/− were incubated with anti–PD-L1 neutralizing antibody at 10 µg/ml, and annexin V and PI staining were analyzed at 48 h after incubation by FACS (N = 5). (J) FACS analyses of CXCL9 on intratumoral neutrophils from WT and Myo1f−/− B16F10 model (N = 5). (K)Cxcl9 mRNA levels were detected by RT-qPCR from cultured neutrophils treated with siCtrl or siMyo1f (N = 5). (L) Chemotaxis assay of CD8+ T cell induced by cytokine and cell lysate. CD8+ T cell in the lower chamber counts by FACS with anti-CD8 flow staining with different treatment (N = 5). (M) Chemotaxis assay of CD8+ T cell induced by neutrophils. Cultured neutrophils from day 4 were transfected with siRNA or siCxcl9 for 24 h and set into the lower chamber with 3 × 106 cells. Sorted CD8+ T cells were placed into the upper chamber of transwell set with 1 × 106 cells. After 6 h, CD8+ T cell in the lower chamber counts by FACS with anti-CD8 flow staining with different treatment (N = 5). (N) FACS analyses of DCFH-DA–labeled tumor neutrophils from WT and Myo1f−/− B16F10 model of day 21 (N = 5). (O) FACS analyses of DCFH-DA–labeled cultured neutrophils from WT mice with transfections of siRNA of siCtrl or siMyo1f (N = 5). (P)Cybb mRNA levels were detected by RT-qPCR from cultured WT neutrophils treated with siCtrl or siMyo1f (N = 5). (Q) FACS analyses of DCFH-DA in cultured neutrophils from Myo1f−/− mice with treatment of NOX2i for 24 h (N = 5). (R) Chemotaxis assay of CD8+ T cell induced by cytokine and small molecular compound. CD8+ T cell in the lower chamber counts by FACS with anti-CD8 flow staining (N = 5). (S) FACS analyses of CFSE–labeled CD8+ T cell from BM neutrophil and CD8+ T cell co-culture assay (N = 3). (T) ELISA detection of IFN-γ level from the supernatant of BM neutrophil and CD8+ T cell co-culture assay (N = 3). (U) Cultured neutrophil transfer effect on tumor growth curve over time. Cultured neutrophils at day 5 treated with stattic (5 µM) for 24 h before transfer (N = 5). (V) FACS analyses of intratumoral CD8+ (GZMB+) T cells described in U. Data in B–V represent one experiment of three independent repeats; A represents one experiment of two independent repeats. Data are presented as mean ± SD. P values were analyzed by one-way ANOVA test (H, I, L, M, O, and R–V); two-tailed unpaired Student’s t test (B–G, J, K, N, P, and Q); *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 3.

MYO1F deficiency enhances the immunosuppressive function of TANs. (A) Volcano plot of mRNA sequencing data of sorted tumor-infiltrating neutrophils from WT and Myo1f−/− B16F10 model. (B) Gene mRNA level was detected by RT-qPCR from sorted tumor-infiltrating neutrophils from WT and Myo1f−/− B16F10 model (N = 5). (C) FACS analyses of PD-1hi and TIM3+ of CFSE-labeled CD8+ T cell co-culture with sorted tumor neutrophils from WT and Myo1f−/− B16F10 tumor models of day 21 (N = 5). (D) FACS analyses of recipient intratumoral CD45.1+ neutrophils in the BM neutrophil transfer tumor model as described in Fig. 2 O (N = 5). (E) FACS analyses of apoptosis in intratumoral CD45.1+ neutrophils by staining with annexin V and PI in the BM neutrophil transfer tumor model as described in Fig. 2 O (N = 5). (F) FACS analyses of PD-L1 in intratumoral CD45.1+ neutrophils in recipient (N = 5). (G) Cultured neutrophils from WT mice were transfected with siCtrl, siMyo1f, and PD-L1 on neutrophils were analyzed 48 h after transfection by FACS (N = 5). (H) Cultured neutrophils from WT mice were transfected with siCtrl and siCd274; after 24 h, cells were treated with cisplatin at 1 µM, and annexin V and PI staining were analyzed at 48 h after transfection by FACS (N = 3). (I) Cultured neutrophils from WT and Myo1f−/− were incubated with anti–PD-L1 neutralizing antibody at 10 µg/ml, and annexin V and PI staining were analyzed at 48 h after incubation by FACS (N = 5). (J) FACS analyses of CXCL9 on intratumoral neutrophils from WT and Myo1f−/− B16F10 model (N = 5). (K)Cxcl9 mRNA levels were detected by RT-qPCR from cultured neutrophils treated with siCtrl or siMyo1f (N = 5). (L) Chemotaxis assay of CD8+ T cell induced by cytokine and cell lysate. CD8+ T cell in the lower chamber counts by FACS with anti-CD8 flow staining with different treatment (N = 5). (M) Chemotaxis assay of CD8+ T cell induced by neutrophils. Cultured neutrophils from day 4 were transfected with siRNA or siCxcl9 for 24 h and set into the lower chamber with 3 × 106 cells. Sorted CD8+ T cells were placed into the upper chamber of transwell set with 1 × 106 cells. After 6 h, CD8+ T cell in the lower chamber counts by FACS with anti-CD8 flow staining with different treatment (N = 5). (N) FACS analyses of DCFH-DA–labeled tumor neutrophils from WT and Myo1f−/− B16F10 model of day 21 (N = 5). (O) FACS analyses of DCFH-DA–labeled cultured neutrophils from WT mice with transfections of siRNA of siCtrl or siMyo1f (N = 5). (P)Cybb mRNA levels were detected by RT-qPCR from cultured WT neutrophils treated with siCtrl or siMyo1f (N = 5). (Q) FACS analyses of DCFH-DA in cultured neutrophils from Myo1f−/− mice with treatment of NOX2i for 24 h (N = 5). (R) Chemotaxis assay of CD8+ T cell induced by cytokine and small molecular compound. CD8+ T cell in the lower chamber counts by FACS with anti-CD8 flow staining (N = 5). (S) FACS analyses of CFSE–labeled CD8+ T cell from BM neutrophil and CD8+ T cell co-culture assay (N = 3). (T) ELISA detection of IFN-γ level from the supernatant of BM neutrophil and CD8+ T cell co-culture assay (N = 3). (U) Cultured neutrophil transfer effect on tumor growth curve over time. Cultured neutrophils at day 5 treated with stattic (5 µM) for 24 h before transfer (N = 5). (V) FACS analyses of intratumoral CD8+ (GZMB+) T cells described in U. Data in B–V represent one experiment of three independent repeats; A represents one experiment of two independent repeats. Data are presented as mean ± SD. P values were analyzed by one-way ANOVA test (H, I, L, M, O, and R–V); two-tailed unpaired Student’s t test (B–G, J, K, N, P, and Q); *P < 0.05, **P < 0.01, and ***P < 0.001.

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