Figure 4. Dual blockade of CCL2 and CCL5 sensitizes HCC tumors to the PD-1/PD-L1 inhibition therapy.

(a) Treatment scheme for HCC tumors established by hemi-spleen approach. (b) Average tumor weight of different treated groups. n = 12 biologically independent samples; one-way ANOVA and Tukey’s multiple comparisons test. (c) Kaplan-Meier survival curve in different treated groups. n = 10 biologically independent samples; Log-rank (Mantel-Cox) test. (d) Kaplan-Meier survival analysis of HCC tumor bearing-mice (for CD4⁺ or CD8⁺ T-cell depletion and BisCCL2/5i plus PD-Li treatments). n = 11 biologically independent samples; Log-rank (Mantel-Cox) test. (e-l) Change of the immunocellular composition in the HCC TME 4 days following Mock mRNA-LNPs and BisCCL2/5i mRNA-LNPs treatments (mRNA: 1 mg/kg), measured by flow cytometry (n = 4 biologically independent samples; one-way ANOVA and Tukey’s multiple comparisons test). (e-f) The counts of total CD8⁺ T (e) cells and CD4⁺FOXP3⁻ T cells (f) in the HCC tumor site. (g-h) Representative flow cytometry histograms of FOXP3 expression (g) and the percentage of Treg cells (h) in the CD4⁺ T cells. (i-j) The percentages of effector memory CD8⁺ T cells (T_EM) (i) and central memory CD8⁺ T cells (T_CM) (j) in the CD8⁺ T population in HCC TME. (k-l) The percentages of CD8⁺ T_EM cells (i) and CD8⁺ T_CM cells (j) in the CD8⁺ T population in peripheral blood. (m-n) Relative expressions of IFN-γ and TNF-α cytokines in HCC TME 4 days following indicated treatments, detected by quantitative RT-PCR (n = 8 biologically independent samples; one-way ANOVA and Tukey’s multiple comparisons test). CD8⁺ T (CD45⁺CD3⁺CD8⁺); CD4⁺ T (CD45⁺CD3⁺CD4⁺); Treg (CD45⁺CD3⁺CD4⁺CD25⁺FOXP3⁺), CD8⁺ T_EM (CD44⁺CD62L⁻ gated from CD3⁺CD8⁺ T cells), CD8⁺ T_CM (CD44⁺CD62L⁺gated from CD3⁺CD8⁺ T cells). Data are represented as the mean ± s.d.