Fig 2: Proportion of CD4+/CD25+ T-cells was significantly increased following treatment with 4-1BB mAb and MEP. (A) The proportion of CD4+1/CD25+ T-cells was increased following 4-1BB mAb treatment. 4-1BB mAb was injected into the mouse tail vein at either 2 h prior to or 2 h following Con A injection. Data are presented as the mean ± standard deviation *P<0.01 vs. the Con A + control IgG group; #P<0.05 vs. the control group. (B) The combination of MEP and 4-1BB mAb treatment further increased the CD4+/CD25+ T-cell levels. Mice were intraperitoneally injected with MEP (3 mg/kg) at 2 h post-Con A injection. Blood samples were collected 8 h after Con A injection and incubated with CD4-phycoerythrin and CD25-fluorescein isothiocyanate antibodies and then analyzed by flow cytometry. Data are presented as the mean ± standard deviation (n=10). *P<0.05 vs. the control group. CD, cluster of differentiation; Con A, concanavalin A; mAb, monoclonal antibody; MEP, methylprednisolone.

Fig 3: AdIL-10-transduced MSCs reduce the number of CD4+ T cells in the popliteal and inguinal lymph nodes 6 weeks after injection. CD4 and CD25 expression by lymphocytes isolated from the popliteal and inguinal lymph nodes, 6 weeks post treatment. a, b AdIL-10-transduced MSCs reduce CD4+ T-cell levels in the popliteal lymph nodes compared with AdIL-10, with no significant difference in the number of activated (CD25+) CD4 T cells between any experimental groups. c, d No significant difference was observed between any of the treatment groups in the amount of CD4+ T cells present in the inguinal lymph nodes. However, AdIL-10 only and AdIL-10-transduced MSCs significantly decreased the amount of activated CD4+ cells compared with vehicle-treated animals. Data points represent n = 4, pooled from eight animals. Data points in the AdIL-10 group represent n = 5, with three samples pooled from two animals and two single samples. Statistical significance was determined using a Kruskal–Wallis test, followed by Dunn’s multiple comparisons test. *p < 0.05, **p < 0.005, ***p < 0.001. Lines indicate significant difference between the individual groups. ILN inguinal lymph node, MSC mesenchymal stem cell, PLN popliteal lymph node

Fig 4: 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.

Fig 5: AdIL-10-transduced MSCs reduce the number of CD8 T cells in the popliteal and inguinal lymph nodes 6 weeks after injection. CD8 and CD25 expression by lymphocytes isolated from the popliteal and inguinal lymph nodes, 6 weeks post treatment. a, b AdIL-10-transduced MSCs reduced the amount of CD8+ T cells compared with AdNull-transduced MSCs in the popliteal lymph nodes, whereas no significant difference in the amount of activated CD8+ T cells was observed. c, d AdIL-10-transduced MSCs significantly decreased the amount of CD8+ cells in the inguinal lymph nodes and reduced the amount of activated CD8+ T cells compared with MSCs alone. Data points represent n = 4, pooled from eight animals. Data points in the AdIL-10 group represent n = 5, with three samples pooled from two animals and two single samples. Statistical significance was determined using a Kruskal–Wallis test, followed by Dunn’s multiple comparisons test. *p < 0.05, **p < 0.005, ***p < 0.001. Lines indicate significant difference between the two groups. ILN inguinal lymph node, MSC mesenchymal stem cell, PLN popliteal lymph node