Fig 2: Recombinant IFNß promotes apoptosis, activation, inhibitory receptor expression, and dysregulated expression of the CAR.a Annexin V expression on CD8 CAR T cells or untransduced (UTD) T cells cultured in IL2 (50 U/mL) in the presence or absence of recombinant murine IFNß and 1µM Ruxolitinib (JAK1/2 inhibitor) for 48 h. b Annexin V expression on CD8 CAR T cells cocultured with B16EGFRvIII cells at an E:T ratio of 1:5 in the presence or absence of recombinant murine IFNß and/or 1µM Ruxolitinib. Fas (c) or cleaved caspase (d) expression on CD8 CAR T cells or UTD cells cultured in IL2 with IFNß. e Median fluorescence intensity (MFI) of CAR expression measured using Biotin-Protein L and streptavidin- PE (Protein L + SA PE) compared to Annexin V on CAR T cells cultured in IL2 with or without IFNß (10 ng/mL). CAR expression on CD8 CAR T cells cultured in IFNß for 48 h in the absence (f) or presence of target tumor cells (g). Representative (h) and mean (i) expression of CD25 and CD69 for CAR T cells cultured in IL2 with IFNß. Statistical comparisons are indicated for CD25- CD69- populations in (i). j Co-expression of the CAR and activation markers on CD8 CAR T cells. k Mean expression of PD1, LAG3, TIM3 on CD8 CAR T or UTD cells cultured in IL2 or in the presence of B16EGFRvIII cells (E:T 1:5). l Fold change in inhibitory receptor and CAR expression is shown for cells grown in IL2 with additional recombinant IFNß relative to culture in IL2 alone. m WT or transgenic IFNAR1 KO CAR T cells were cocultured with B16EGFRvIII (E:T ratio of 1:5) which were mock infected or infected with VSVmIFNß 6 h prior to coculture. Surface CAR expression is represented as fold change relative to coculture with mock infected tumor cells. n Inhibitory receptor expression was quantified on WT or IFNAR1 KO CD8 CAR T cultured as in (m). Data are representative of two independent experiments (panels a–l). Experiments in panels (m, n) were performed once. Means ± SD of n = 4 (panel a) or n = 3 (panels b–d, f, g, i, k, m, n) technical replicates are shown. P-values were determined using a two-way ANOVA with a Tukey multiple comparisons post-test (a–d, f, g, i). Statistical significance set at p < 0.05, ns > 0.05. Source data are provided in the Source Data File.

Fig 3: Characterization of transient externalization of phosphatidylserine in activated T cells. (A, B) Antigen inexperienced or antigen experienced CD8+ cells were cultured with anti-CD3 and anti- CD28 (antigen inexperienced cells only) and stained with CD69 and AnnV at the indicated time points. Live and activated CD8+ cells were gated on forward scatter and side scatter profiles. Representative dot plots from one sample are shown. (C) Frequency of CD69+ AnnV+ cells at the indicated time points for antigen inexperienced CD8+ T cells stimulated with the indicated concentrations of anti-CD3. (D) Schematic of experiment workflow. (E) Frequency of viable cells after 3 days of re-culture was quantified by flow cytometry. (F) ELISA for IFN? in culture supernatant. Data is represented by mean ± SEM. P values were calculated by ordinary one-way ANOVA with the Tukey-Kramer multiple comparison test, and P < 0.05 is considered statistically significant. ** = < 0.01,*** = < 0.001. AnnV, Annexin V. NS, not significant. Schematic in 1D was generated using BioRender.com.

Fig 4: RAD21 suppresses antitumor immunity in vivo.(A) Tumor volume and tumor weight over time in C57BL/6 mice implanted with Rad21-KO and control B16-OVA mouse cells. Data are shown as mean ± SEM (2-way ANOVA) for tumor volume and as mean ± SD (2-tailed t test) for tumor weight (n = 6 mice per group). (B and C) Tumor volume in nude mice (n = 8 mice per group) (B) and C57BL/6 mice (n = 6 mice per group) (C) implanted with Rad21-KO and control B16-OVA mouse cells. Mice were pretreated with CD8-depleting antibodies at –1, 2, and 5 days. Data are shown as mean ± SEM (2-way ANOVA). (D and E) Flow cytometry analysis showing the numbers of tumor-infiltrating CD8+ T cells (D) and expression of activation marker CD69 and effector molecules IFN-? and GZMB (E) in CD8+ T cells. Data are shown as mean ± SD (n = 5, 2-tailed t test). (F) Mice with established Rad21-KO and control B16-OVA tumors were treated with anti–PD-1 at indicated time points. Tumor volume and survival rates are shown. Data are shown as mean ± SEM (2-way ANOVA). (G) Representative bioluminescence images of mice with established Rad21-KO and control ID8 tumors treated with anti–PD-1 formed by intraperitoneal injection at day 9 and day 12. (H) The bar graph shows the change in bioluminescence in mice. Data are shown as mean ± SEM (1-way ANOVA). (I and J) Flow cytometry analysis showing the numbers of tumor-infiltrating CD8+ T cells (I) and expression of CD69 and effector molecules IFN-? and GZMB (J). Data are shown as mean ± SD (2-tailed t test). **P < 0.01; ***P < 0.001.

Fig 5: PmTriTNE@CDA showed potent antitumor efficacy. A–D) CD8+ T cells or MDA-MB-231 cells were incubated with cy5-labeled a-Isotype-PAMAM@CDA pretreated with pH 6.5 buffer (i), cy5-labeled PmTiTNE@CDA pretreated with pH 7.4 buffer (ii) or cy5-labeled PmTiTNE@CDA pretreated with pH 6.5 buffer (iii) for 20 min at 4 °C. Then, Analyzed the specific binding of NPs to CD8+ T cells (A,B) or MDA-MB-231 cells (C,D) by flow cytometry. E) Schematic illustration of the co-culture system used to evaluate the in vitro antitumor efficacy of TriTNE. F–I) CD8+ T cells were sorted from hPMBCs and then cocultured with MDA-MB-231 cells in the presence of a-Isotype+a-Isotype-PAMAM (control), a-CD3-PAMAM + a-PD-L1-PAMAM + HA-PAMAM (combinational NPs) or TriTNE for 24 h. Then, CD69 (F,G) and CD25 (H,I) positive cells were analyzed by flow cytometry. J) Target cell lysis was evaluated at a 10:1 E:T ratio in the presence of TriTNE. K) Schematic illustration of the method used to evaluate the antitumor efficacy of PmTriTNE@CDA in the CDX model. MDA-MB-231 cells were inoculated into female human HSC-engrafted NPG mice (HSC-NPG) and treated with a-Isotype+a-Isotype-PAMAM (control), a-CD3 + a-PD-L1+ mPEG-HA-PAMAM@CDA (PHM@CDA) or PmTriTNE@CDA. L) Tumor volume and M) mouse body weight were monitored during the treatment period. N) Schematic illustration of the method used to evaluate the antitumor efficacy of PmTriTNE@CDA in the PDX model. PDX was inoculated into female HSC-NPG mice carrying PDX and treated with a-Isotype+a-Isotype-PAMAM (control), a-CD3 + a-PD-L1+ mPEG-HA-PAMAM@CDA (PHM@CDA) or PmTriTNE@CDA (O,P). O) Tumor volume and P) mouse body weight were observed during the treatment period. Data are presented as the mean ± SD (For in vitro assays (A–J), n = 3. For in vivo assays (K–P), n = 5). p-values are calculated using one-way ANOVA with Tukey's multiple comparison. *** p < 0.001.