Article Figures & Data
Figures
- Figure 1.
TGFβ1 prolongs PD-1 mRNA half-life in an SRSF3/5-dependent manner in T cells. A, qRT-PCR analysis of PD-1 mRNAs in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) stimulated with TGFβ1 (10 ng/mL) for the indicated time points. B, CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) were stimulated with TGFβ1 (10 ng/mL) prior to incubation with ActD (5 μg/mL), and the remaining PD-1 mRNA or control TNF, IL10, β-actin, and α-tubulin mRNAs were quantified. The half-lives were determined from three independent experiments. Data were analyzed using Student t test. C, Schematic indicating the construction of the Tet-off system. PDCD1, gene for PD-1; tTA, Tet transactivator. D, HEK293 Tet-off cells were transfected with inducible Tet-off PD-1 plasmids and stimulated with TGFβ1 for 24 hours. Doxycycline (1 μg/mL) was added, the remaining PD-1 mRNA was quantified, and the half-life was determined from three independent experiments. Data were analyzed using Student t test. E, A heatmap revealing potential protein binding sites in the PD-1 mRNA 3′-UTR. F, Western blot analysis of HEK293 Tet-off cells transfected with inducible Tet-off PD-1 plasmids and then transfected with pcDNA3, CUG-BP, SRSF1, HNRNPA1, SRSF5, SRSF9, MBNL1, YBX1, or SRSF3 plasmids. Doxycycline (1 μg/mL) was added, the remaining PD-1 mRNA was quantified, and the half-life was determined from three independent experiments. Data were analyzed using Student t test. G, Western blot of the indicated proteins in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) stimulated with TGFβ1 (10 ng/mL) for 24 hours. H, CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) were transfected with siSRSF3 and/or siSRSF5 prior to incubation with ActD (5 μg/mL) for the indicated times, and the remaining PD-1 mRNA or control TNF, IL10, β-actin, and α-tubulin mRNAs were quantified. The half-lives were determined from three independent experiments. Data were analyzed using Student t test. All data are presented as the mean ± SD and are representative of three independent experiments. Significant results are presented as ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Dox, doxycycline.
- Figure 2.
The P38-SRSF3/5 axis activated by ccRCC-derived TGFβ1 prolongs the PD-1 mRNA half-life in T cells. A, Western blot analysis of the indicated proteins in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) stimulated with TGFβ1 (10 ng/mL) for 24 hours. B, CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) were transfected with MKK6EE for 24 hours prior to incubation with ActD (5 μg/mL) for the indicated times, and the remaining PD-1 mRNA or control TNF, IL10, β-actin, and α-tubulin mRNAs were quantified. The half-lives were determined from three independent experiments. Data were analyzed using Student t test. C, CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) were transfected with MKK6EE and treated with SB203580 (10 μmol/L) for 24 hours prior to incubation with ActD (5 μg/mL) for the indicated times, and the remaining PD-1 mRNA or control TNF, IL10, β-actin, and α-tubulin mRNAs were quantified. The half-lives were determined from three independent experiments. Data were analyzed using Student t test. D and E, CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) with P38 knockdown (KD) or treated with an inhibitor were stimulated with TGFβ1 (10 ng/mL) for 24 hours prior to incubation with ActD (5 μg/mL) for the indicated times, and the remaining PD-1 mRNA or control TNF, IL10, β-actin, and α-tubulin mRNAs were quantified. The half-lives were determined from three independent experiments. Data were analyzed using Student t test. F, Western blot analysis of the indicated proteins in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) transfected with MKK6EE or treated with SB203580 (10 μmol/L) for 24 hours. All data are presented as the mean ± SD and are representative of three independent experiments. Significant results are presented as ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
- Figure 3.
TGFβ1 activated the p38/p-Ser10 H3 axis to increase SRSF3/5 levels by recruiting p65 in T cells. A and B, Western blot analysis of the indicated proteins in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) stimulated with TGFβ1 (10 ng/mL) for the indicated times. C, Western blot of the indicated proteins in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) stimulated with TGFβ1 (10 ng/mL) for 24 hours and then treated with SB203580 (10 μmol/L) for the indicated times.D, ChIP-PCR analysis of the enrichment of p-Ser10 H3 in the SRSF3 and SRSF5 promoter regions in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) stimulated with TGFβ1 (10 ng/mL) for 24 hours. E, ChIP-PCR analysis of the enrichment of p-Ser10 H3 in the SRSF3 and SRSF5 promoter regions in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) stimulated with TGFβ1 for 24 hours and then treated with SB203580 (10 μmol/L) for 24 hours. F, Immunofluorescence of SRSF3 and SRSF5 in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) stimulated with TGFβ1 (10 ng/mL) for 24 hours. Scale bar, 25 μm. G, Immunofluorescence of SRSF3 and SRSF5 in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) stimulated with TGFβ1 (10 ng/mL) for 24 hours and then treated with SB203580 (10 μmol/L) for 24 hours. Scale bar, 25 μm. H, CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) with p65 overexpression (OE) were followed by ActD (5 μg/mL) for indicated times, and the remaining PD-1 mRNA or control TNF, IL10, β-actin, and α-tubulin mRNAs were quantified. The half-lives were determined from three independent experiments. Data were analyzed using Student t test. I, CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) were transfected with MKK6EE and then treated with QNZ (11 nmol/L) for 24 hours prior to incubation with ActD (5 μg/mL) for the indicated times. The remaining PD-1 mRNA or control TNF, IL10, β-actin, and α-tubulin mRNAs were quantified, and the half-lives were determined from three independent experiments. Data were analyzed using Student t test. J, Predicted p65 binding sites in the promoter region of SRSF3 and SRSF5 in the JASPAR database. K and L, ChIP-PCR analysis of the enrichment of p65 in the SRSF3 and SRSF5 promoter regions in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28-activated) transfected with MKK6EE or scramble for 24 hours. M, Luciferase activity of GV272-SRSF3 and GV272-SRSF5 in CD8+ T cells isolated from PBMCs (anti-CD3/anti-CD28 activated) transfected with pcDNA3-p65, pcDNA3-mut, or pcDNA3 plasmids (n = 3). Data were analyzed using Student t test. N, Western blot analysis of the indicated proteins in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) overexpressing p65 or treated with QNZ (11 nmol/L) for 24 hours. O, Top, EMSA of TGFβ1-induced NF-κB activation in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated). Bottom, ChIP-PCR assay of the enrichment of p65 in the SRSF3 and SRSF5 promoter regions in CD8+ T cells (anti-CD3/anti-CD28 activated) stimulated with TGFβ1 (10 ng/mL) for the indicated times. P, ChIP-PCR analysis of the enrichment of p65 in the SRSF3 and SRSF5 promoter regions in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) treated with SB203580 (10 μmol/L) for 24 hours. All data are presented as the mean ± SD and are representative of three independent experiments. Significant results are presented as ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scra, scramble.
- Figure 4.
SRSF3 binding with NXF1 promotes extranuclear transport of PD-1 mRNA in T cells. A, Schematic diagram of the nuclear and cytoplasmic RNA extraction process. qRT-PCR analysis of the nuclear and cytoplasmic fractions of PD-1 mRNA after knocking down or overexpressing SRSF3 in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28-activated). Data were analyzed using Student t test. B, qRT-PCR analysis of the nascent PD-1 mRNA level in the nucleus or cytoplasm after knockdown of SRSF3 expression in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated). Data were analyzed using Student t test. C, RIP-PCR analysis of the enrichment of SRSF3 on PD-1 mRNA after knocking down P38 expression in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated). Data were analyzed using Student t test. D, RIP-PCR analysis of the enrichment of SRSF3 on PD-1 mRNA in CD8+ T cells (anti-CD3/anti-CD28 activated) stimulated with TGFβ1 (10 ng/mL) for 24 hours. Data were analyzed using Student t test. E, RIP-PCR analysis of the enrichment of SRSF3 on the 3′-UTR of PD-1 mRNA after overexpression of SRSF3 in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated). Data were analyzed using Student t test. F, qRT-PCR analysis of the nuclear and cytoplasmic fractions of WT-PD-1 and ΔSRSF3-PD-1 mRNAs after knocking down SRSF3 expression in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated). Data were analyzed using Student t test. G, Top, co-IP analysis of CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) with an antibody against SRSF3, followed by labeling with an anti-NXF1. Bottom, co-IP with an antibody against NXF1, followed by labeling with an anti-SRSF3. H, Co-IP analysis of SRSF3-knockdown CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) with an antibody against SRSF3, followed by labeling with an anti-NXF1. I, Co-IP analysis of CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated) stimulated with TGFβ1 for 24 hours and immunoprecipitated with an antibody against SRSF3, followed by labeling with an anti-NXF1. J, RIP-PCR analysis of the enrichment of NXF1 or SRSF3 on WT-PD-1 and ΔSRSF3-PD-1 mRNAs in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated). Data were analyzed using Student t test. K, RIP-PCR analysis of the enrichment of SRSF3 on the 3′-UTR of PD-1 mRNA after overexpression of NFX1 in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated). Data were analyzed using Student t test. L, qRT-PCR analysis of the nuclear and cytoplasmic fractions of WT-PD-1 and ΔSRSF3-PD-1 mRNAs after knocking down NFX1 expression in CD8+ T cells isolated from PBMCs of healthy donors (n = 3, anti-CD3/anti-CD28 activated). Data were analyzed using Student t test. All data are presented as the mean ± SD and are representative of three independent experiments. Significant results are presented as ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. KD, knockdown; Scra, scramble.
- Figure 5.
TGFβ1 induced SRSF3 to restrict the antitumor capability of T cells in ccRCC. A, Schematic diagram of the orthotopic ccRCC model and treatment. FCM, flow cytometry. B, Bioluminescence images of BALB/c mice at day 24 in the knockdown scramble plus IgG (KD_Scra + IgG), knockdown scramble plus anti–PD-L1 (KD_Scra + anti–PD-L1), knockdown TGFβ1 plus IgG (KD_TGFβ1 + IgG), and knockdown TGFβ1 plus anti–PD-L1 (KD_TGFβ1 + anti–PD-L1) groups (n = 12/group). C, Quantification of bioluminescence imaging signal intensities in BALB/c mice from B (n = 12/group). Data were analyzed using Student t test. D, Kaplan–Meier survival curves for the BALB/c mice from B. Data were analyzed using log-rank test. E, Density plots of Pd-1 and Srsf3 among tumor-infiltrating CD8+ T cells in the mice from B. Data represent three independent experiments. Data were analyzed using Student t test. F, Schematic diagram of adoptive cell transfer therapy for ccRCC in mice (n = 8/group). i.p., intraperitoneal; i.v., intravenous; s.c., subcutaneous. G, Transferred OTI CD8+ T cells overexpressing Srsf3 were analyzed in the dLNs and non-dLNs at day 25. Data were analyzed using Student t test. H, The Ki-67 and PD-1 expression of transferred CD8+ T cells overexpressing Srsf3 was analyzed in the dLNs at day 25. Data were analyzed using Student t test. I, Tumor volume of BALB/c mice in the PBS, overexpression scramble plus IgG (OE_Scra + IgG), overexpression Srsf3 plus IgG (OE_Srsf3 + IgG), overexpression scramble plus anti–PD-1 (OE_Scra + anti–PD-1), and overexpression Srsf3 plus anti–PD-1 (OE_Srsf3 + anti–PD-1) groups (n = 8/group). Data were analyzed using Student t test. J, Kaplan–Meier survival curves for the BALB/c mice from I. Data were analyzed using log-rank test. All data are presented as the mean ± SD and are representative of three independent experiments. Significant results are presented as ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. KD, knockdown; MFI, mean fluorescence intensity; Scra, scramble.
- Figure 6.
Mechanism by which ccRCC-derived TGFβ1 extends the half-life and mediates the nuclear export of PD-1 mRNA in T cells. Left, TGFβ1 attenuates the antitumor immune activity by prolonging the half-life of PD-1 mRNA and promoting PD-1 mRNA nuclear export in CD8+ T cells. Right, inhibition of TGFβ1 in ccRCC reduces the stability of PD-1 mRNA and decreases extranuclear transport in CD8+ T cells. Blue arrows indicate the increase (up) or decrease (down) of RNA/protein expression, molecular activity, or extranuclear transport. Black arrows indicate the direction and steps of regulation. Red “p” indicates “phosphorylation.”
Additional Files
Supplementary Data
- Supplementary Tables and Figures: Table S1, Table S2, Table S3, Table S4, Table S5, Table S6, Figure S1, Figure S2, Figure S3, Figure S4, Figure S5, Figure S6 - Supplementary Tables and Figures: Table S1, Table S2, Table S3, Table S4, Table S5, Table S6, Figure S1, Figure S2, Figure S3, Figure S4, Figure S5, Figure S6
Volume 8, Issue 12
