Fig 2: LMWH treatment improved the infiltration of proliferative and cytotoxic CD8+ T cells mediated by anti-PD-1 therapy. (A) mIHC staining of CT26 tumors, CD8 (green), Ki67 (red), DAPI (blue). (B) mIHC staining of CT26 tumors, CD8 (red), GZMB (green), DAPI (blue). (C) Quantitative analysis of CD8+Ki67+ T-cell infiltration. (D) Quantitative analysis of CD8+GZMB+ T-cell infiltration. GZMB, granzyme-B; mIHC, multiplex immunohistochemistry; LMWH, low molecular weight heparin; PD-1, programmed cell death protein 1.

Fig 3: NsPEF activated inflammatory pathways and promoted the activation of immune cells. (A) Inflammatory factor micro-array of tumors in the nsPEF group and control group on the third day after treatment; heatmap showing the changes in cytokines (n = 5, p < 0.05). (B) KEGG pathway enrichment analysis of cytokines. (C) Macrophages and (D) memory activated CD4+ T cells in tumor on day 7 according to the results of CIBERSORTx based on the RNA-seq data. (E) Changes in the proportion of CD8+ T cells in CD45+ cells in the control group and the nsPEF group (n = 4). The flow plot showed the proportion of CD8+ T cells in the CD3+ T cell gate. (F) Immuno-histochemistry (IHC) of CD8 and GZMB (Granzyme B) in tumors of the two groups on day 7. Scale bar: 50 μm. Data are presented as the mean ± SEM; * p < 0.05, ** p < 0.01, according to Student’s t-test.

Fig 4: NIR-715-induced immune responses in mice bearing mEC01-3 tumors.A Schematic illustration of the mEC01-3 tumor model experimental design. B The tumor images obtained from tumor-bearing C57BL/6 mice on day 27 after NIR-715 with or without light treatment. C, D After establishment of subcutaneous mEC01-3 syngeneic mouse tumor model, tumor volumes were measured (C), along with tumor weights (D). Data are shown as mean ± SEM, n = 5, significance was determined by two-way ANOVA with Bonferroni’s multiple comparisons test. For tumor weight, significance was determined by unpaired two-tail Student’s t-tests. E Survival of mEC01-3-bearing mice (n = 7 independent samples). F, G Proportions of CD8+ T cells and GZMB+ CD8+ T cells in the TME were quantified by flow cytometry. Data represent mean ± SEM. H Representative multiplex immunofluorescent staining image showing distribution of CD8, PD-1, Tim-3 and GZMB expression cells in mEC01-3 syngeneic mouse tumor model. Scale bar: 500 μm. I–K Proportions of GZMB+ CD8+ T cells, Tim3+ CD8+ T cells and PD-1+ CD8+ T cells in mEC01-3 syngeneic mouse tumor model were examined on day 27 after NIR-715 treatment. Data represent mean ± SEM (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: CXCL9/10-DC potentiates ICB immunotherapy in murine NSCLC models(A) CXCL9/10-DC enhances the efficacy of anti-PD-1. On D6 post-tumor inoculation (1.25 × 105 KPL-3M delivered s.c.), FVB mice bearing ∼50-mm3 tumors were randomized and treated with PBS control; CXCL9/10-DC (IT 1 × 106 CXCL9-DC and CXCL10-DC each per injection on D6, D8, and D11); anti-PD-1 (i.p. injections at 200 μg/injection on D6, D8, D11, and D14); or the combination of CXCL9/10-DC and anti-PD-1 (n = 6–8 mice per group). Tumor growth curves and tumor weights at the time of euthanasia are presented.(B) Same as in (A), except that mice were s.c. inoculated with 1 × 105 1940A-KPL tumor cells.(C) In-depth scRNA-seq analysis identifies distinct CD8+ T cell subtypes (cells pooled from 5 tumors per group). Lineage trajectory interference (TI) analysis of CD8+ T cell changes following various treatments.(D) Spatial quantification of CD8+Ki67+ and CD8+GzmB+ T cells were performed on D23 per Figure S6 (n = 6–8 mice per group).(E) Therapy-induced changes in CD4+ T cells revealed by scRNA-seq. Same as in (C).(F) Spatial quantification of CD4+Ki67+ and FOXP3+ Treg cells. Same as in (D).Error bars represent SEM. p values were determined by one-way ANOVA, adjusting for multiple comparisons for (A) and (B) and two-tailed t test for (D) and (F).∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005; ∗∗∗∗p < 0.00005.