CD4~+T-cell Epitope Based Heterologous Prime-boost Vaccination Potentiates Anti-tumor Immunity And PD-1/PD-L1 Immunotherapy

Tumor-infiltrating CD8⁺T cells become exhaustion in tumor microenvironment（TME）due to the prolonged antigen stimulation and the suppressive microenvironment.Exhaustion CD8⁺T cells express inhibitory immune checkpoint receptors companied with poor function,such as PD-1,CTLA-4,TIM-3,and 2B4.Immune checkpoint blockade（ICB）has been reported to re-invigorate the exhaustion CD8⁺T cells.However,only about 20-30%patient can respond to it.The combination of ICB treatment with other strategis can improve the anti-tumor efficacy of ICB,such as anti-tumor therapeutic vaccines.Anti-tumor therapeutic vaccines are generally based on the MHC-I restricted CD8⁺T-cell epitopes to induce tumor-specific CD8⁺T cells.However,the high-load MHC-I restricted CD8⁺T-cell epitopes can accelerate T cell exhaustion.CD4⁺T cells also play an important role in tumor control.Nonetheless,MHC-II-restricted CD4⁺T-cell epitopes based anti-tumor therapeutic vaccines receive less attention.Here,we construct a CD4⁺T-cell epitope based heterologous prime-boost vaccination by expressing lymphocytic choriomeningitis virus（LCMV）glycoprotein-specific I-A^b-restricted CD4⁺T cell epitope(GP_61–80)or ovalbumin-specific CD4⁺T cell epitope(OVA_323-339)on Listeria monocytogenes vector and influenza A virus（PR8 strain）vector.We also develop a tumor model-melanoma or colorectal adenocarcinoma expressing LCMV-GP or ovalbumin.To analyze the mechanism of anti-tumor efficacy,we perform flow-cytometer staining,single-cell RNA sequencing and single-cell TCR sequencing.We observe CD4⁺T cell epitope-based heterologous prime-boost vaccination inhibits melanoma and colorectal adenocarcinoma.This prime-boost vaccination strategy induces potent tumor-specific T_H1 response and tumor-specific CD8⁺T cells response.Tumor-specific CD8⁺T cells induced by prime-boost vaccination have better effector function,clonal breadth and expansion.Moreover,this prime-boost vaccination strategy synergized PD-L1 blockade promote the efficacy of suppressing tumor growth.Furthermore,CD4⁺T-cell epitope based heterologous prime-boost vaccination can prevent re-invigorated CD8⁺T cells induced by PD-1/PD-L1 treatment interruption from re-exhaustion.In conclusion,CD4⁺T-cell epitope based heterologous prime-boost vaccination potentiates anti-tumor immunity and PD-1/PD-L1 immunotherapy.