Due to the central role in immune surveillance, the antigen peptide hold tremendous promise for treatment modalities in cancer, infectious disease, autoimmunity, and allergy. Almost all that T-lymphocytes immune response relies on the recognition of specific antigen presented by major histocompatibility complexes (MHC) on target cells. The cytotoxic T cells target cells via recognition of endogenous antigen-MHC class I complexes by T cell receptors (TCRs). The interaction of antigen peptide-MHC complexs (pMHCs) with TCR results in T cell activation and proliferation, which subsequently exert cytotoxic functions by killing virus-infected target cells or tumor cells [1]. Theoretically, the key to the body's effective immune response to tumors is the generation and presenting of tumor neoantigens, and then be-recognized by TCRs of tumor-specific T cells, which can recognize human leukocyte antigen (HLA) complexes and mediate corresponding responses. Due to the randomness of HLA typing and tumor mutations, there are great individual differences in tumor neoantigens, and tumor-specific T cells also vary from person to person, making it difficult to accurately recognize immunogenic antigen peptides in vitro [2], [3].

Despite considerable improvements in pMHC predictions in recent years, empirical observations continually reveal gaps between theoretical and naturally presented antigens. Successful activation of corresponding T cells and expansion to clinically required numbers for transfusion has been reported. This “precision targeting” mechanism of pMHC-TCR poses a great challenge to the screening of antigen-specific T cells. Studies had indicated that the effectiveness of personalized adoptive cellular immunotherapy on cancer depends on the percentage of tumor-associated antigens (TAAs)-specific T cells, particularly tumor neoantigens-specific T cells, which are able to effective target and kill tumor cells. The general strategy is to screen tumor antigen-specific T cells with TCR that recognize tumor antigenic peptide-MHC class I complexes [4], [5]. This screening process first requires the prediction of TAAs of tumors and identification of human leukocyte antigen (HLA) haplotypes of patients. Usually, the combination of whole-exome sequencing, RNA sequencing, and HLA class I molecular binding algorithms is used to predict antigenic peptide-MHC class I complexes [6], [7]. Then the predicted and synthesized antigenic peptides combined with the tetramer technique are used for T cell screening. It also reported the use of a tandem minigene library to encodes tumor mutations to identify antigen-specific T cells [8], [9]. However, both the identification and screen are expensive and time-consuming, limiting the clinical application [10], [11], [12], [13], [14].

The formation of tumor antigen recognition memory by T cells in vivo depends on the antigen peptide-MHC complexes presented on the surface of antigen-presenting cells (APCs). In study on tumor immunology, artificial APC (aAPCs) can serve as an alternative to APCs of innate immunity in vitro. As synthetic substances, aAPCs theoretically do not respond to environmental condition changes, thus maintaining their antigen-presenting function [15], [16]. However, the application of aAPCs is also limited by the antigenic-peptide identification and HLA typing, because the synthesis of aAPCs requires the pre-identification of immunogenic antigens and in vitro synthesis of the corresponding patient-specific MHC I haplotypes. The identification of immunogenic cancer neoantigens is a very expensive, cumbersome, and labor-intensive process with many caveats along the pipeline that further limit its success rate, [17] restricting the application of aAPCs in personalized adoptive cell therapy.

To simplify the previously mentioned process and overcome the limitations of the existing approaches, we propose an alternative method that does not require pre-identification or generation of antigenic pMHCs in vitro. This one-step shotgun approach focuses on the direct capture of pMHCs from cell lysates, avoiding the costly and time-consuming process of identifying TAAs as well as eliminating the APC process that results in TAA delivery to T cells. Our results demonstrated that the pMHCs thus obtained containing antigenic peptide-MHC class I complexes can effectively enrich CD8+ T cells in tumor infiltrating lymphocytes (TILs) and stimulate T cells activation and proliferation, especially the memory T cells..