The combination therapy augments immune cells infiltration and activation. Mice with established MC38 tumors were treated with nano-PTX and PD-1 antibody as described in Fig 7A, Tumors were isolated on day 23 for analysis. A Immunohistochemistry staining of CD8 within MC38 tumor after treatment (scale bar, 100 µm). An enlargement of the squared area was showed below (Scale bar, 50 µm). B The numbers of tumor-infiltrating CD3+, CD4+ and CD8+ T cells. C Tumor growth with CD4+ T cells or CD8+ T cells depletion, n = 9 ~ 10 mice per group. E CD69 expression on CD8+ T cells. F PD-1 expression on CD8+ T cells. G The percentages of PD-1+ LAG3+, PD-1+ TIM-3+ and PD-1+ TIGIT+ double positive CD8+ T cells. H-I Spleen cells were stimulated with leukocyte activation cocktail (BD Bioscience) containing brefeldin A for 4 h, and the expression of IFN-γ (H) and GzB (I) was determined by flow cytometry, gated on CD8+ T cells. Mean ± SEM was shown. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns (no statistical significance).