Fig 2: Cyclin D3/CDK6 depletion or PTP1B/TCPTP inhibition increases cytokine production in T cells(A–D) Bar graphs show a comparison of IFN-? (A and C) and GzmB (B and D) production of T cells treated with 1 µM PTPIB-IN2, 1 µM Palbo, or 10 µM BSJ-03–123 for 24 h. In (D), 2 µM PTPIB-IN2 was applied for 16 h. CD8+ T cells were isolated from OT-I mice (A and B) or human T cells (C and D). n = 4 (A), n = 4 (B), n = 5 (C), n = 3 (D).(E and F) Normalized IFN-? (E) or GzmB (F) production in human T cells in which cyclin D3 or CDK6 was knocked down.(G) The population of memory T cells differentiated from T cells upon treatment with PTP1B-IN2 (0.5 µM), Palbo (0.5 µM), or BSJ-03–123 (5 µM) for 2 days in the presence of IL-15.(H–J) Bar graphs show a comparison of phospho-Tyr142 of CD3? (H), TNF-a (I), and GzmB (J) of T cells treated with inhibitors for LCK kinase (10 µM PP2) or PTPs (1 µM PTPIB-IN2) for 4 h(K–P) Bar graphs show a comparison of IFN-? (K and N) and GzmB (L and O) production and phospho-Tyr142 of CD3? levels (M and P) of PTP knockdown (K–M) or A-mutant expressing (N–P) T cells treated with 1 µM Palbo or 5 µM BSJ-03–123 for 24 hn = 2 (E–P). Mean ± SEM, 1-way ANOVA (Dunnett’s test). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3: CDK6 inhibition increases CD3? tyrosine phosphorylation mediated by PTP1B and TCPTP(A–C) Immunoprecipitation using Abs specific to CD3e, CD3?, or CD3?, followed by immunoblotting using pY99 phosphotyrosine Abs to detect phosphorylation of CD3 subunits in Jurkat cells treated with palbociclib (24 h).(D) Comparative CD3? phosphorylation measured with flow cytometry by phospho-Tyr142 Abs in Jurkat cells with and without palbociclib treatment (24 h). n = 2.(E–G) In vitro phosphatase assay showed that CD3e, CD3?, and CD3? were the substrates of PTP1B and/or TCPTP.(H) In vitro phosphatase assay to detect the phosphatase activity of PTP1B or TCPTP immunoprecipitated from Jurkat cells treated with palbociclib (8 h). The phosphorylation level of PTP1B/TCPTP was detected using p-S/TP Abs.(I) IB using pY99 to detect phosphorylated CD3? immunoprecipitated from Jurkat cells treated with PTP1B-IN2 (1 µM, 24 h).(J) Phosphorylation levels of CD3? at Tyr142 in CD3/CD28/IL-2-activated splenic CD4+ or CD8+ T cells from CDK6 KO mice or WT littermates. n = 4.(K) IB of tyrosine phosphorylation of CD3? in CD8+ T cells from CDK6 KO mice or WT littermates or in WT CD8+ T cells treated with a CDK6 degrader for 48 h (L and M) Comparative levels of tyrosine phosphorylated CD3? (phospho-CD3? [Tyr142] Abs) in OVA peptide-activated CD8+ T cells isolated from OT-I mice (L) or primary human T cells derived from peripheral blood mononuclear cells (M) upon treatment with PTP1B-IN2 (1 µM), palbociclib (1 µM), or BSJ-03–123 (5 µM) for 24 h n = 4 (L). Con, n = 6; PTPIB-IN2, n = 7; palbociclib (Palbo), n = 7; CDK6 degrader, n = 6 (M).(N) Phosphorylation levels of CD3? at Tyr142 in human T cells upon genetic deletion of CDK6 (shCDK6) or cyclin D3 (shcyclinD3). n = 3.(D), (J), and (L)–(N) represent mean ± SEM; two-tailed Student’s t-test (D and J) and 1-way ANOVA (Dunnett’s test) (L–N); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: Targeting the CDK6-PTPs axis enhances the efficacy of T cell killing and adoptive T cell immunotherapy.(A) In vitro tumor killing assays showed that targeting the CDK6-PTPs axis increased splenic CD8+ T cell killing capacity against OVA-expressing B16F10 cells. The control groups were without OT-I CD8+ T cells. n = 3.(B) CAR T tumor killing assay was applied to examine the cytotoxicity of human T cells targeting Raji cells expressing GFP upon compound treatments. n = 3. For (A) and (B), 1 µM PTP1B-IN2, 1 µM Palbo, or 10 µM BSJ-03–123 was applied.(C) Graph showing the mean of B16F10-OVA tumor volumes in C57BL/6-Ly5.1 mice receiving CD8+ T cells from OT-I mice. n = 5.(D) Activities of tumor-infiltrating OT-I CD8+ T cells (top) and analysis of memory T cells among tumor-infiltrating OT-I T cells from C57BL/6-Ly5.1 mice (bottom). Ly5.2+ T cells were quantified in this experiment. n = 5.(E and F) Bioluminescence images (E) and overall survival graph (F) of tumor progression in Raji tumor-bearing mice with/without receiving CAR T cells under treatment with BSJ-03–123 (50 mg/kg) or PTP1B-IN2 (5 mg/kg). Shown are representative bioluminescence images (E, top) and quantification of the regions of interest (ROIs) (E, bottom). n = 11/group.(G) Memory T cell analysis of CAR T cells from Raji tumor-bearing mice receiving treatment with PTP1B-IN2 or BSJ-03–123 for 2 weeks. n = 4/group.(H) Overall survival (left) and tumor-free survival (right) of mice challenged with B16F10 melanoma cells with/without receiving pmel CD8+ T cells.(I) Schematic depicting the presented study model. Created with BioRender.(A), (B), (D), and (G) represent mean ± SEM, 1-way ANOVA (Dunnett’s test); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.