Journal: Cancer Biology & Medicine
Article Title: Neoepitope BTLA P267L -specific TCR-T cell immunotherapy unlocks precision treatment for hepatocellular carcinoma
doi: 10.20892/j.issn.2095-3941.2024.0434
Figure Lengend Snippet: Schematic diagram of the anti-tumor activity of BTLA P267L -specific CD8 + TCR-T cells. Tumor tissues and paired peripheral blood were collected from 7 patients with HLA-A*02:01-restricted HCC for WES and bioinformatics analyses, followed by identification of neoepitope BTLA P267L and corresponding TCR sequences (85-3, 126-5, or 52-3) and preparation of BTLA P267L -specific CD8 + TCR-T cells. TAP is located on the endoplasmic reticulum and is essential for epitope presentation. Epitopes cannot be transported and presented to the membrane in T2 cells lacking TAP, and therefore cannot bind to TCR on T cells to further initiate T cell cytotoxicity. BTLA P267L -pulsed T2 cells were used as target cells to exclude the interference of endogenous epitopes. While BTLA P267L -specific CD8 + TCR-T cells were co-cultured with exogenous BTLA P267L -pulsed T2 cells or endogenous BTLA P267L -overexpressed HCC cell lines, the expression of markers on activated CD8 + TCR-T cells changed with the stimulation of neoepitope BTLA P267L . Activation markers (CD25, CD69, 4-1BB, and OX40), inhibitory markers (PD-1 and CTLA-4) indicating feedback mechanisms in response to persistent antigen stimulation, and the cytotoxic marker (CD107a) leading to the release of cytotoxic granules (e.g., perforin and granzyme B) on the surface of CD8 + TCR-T cells co-cultured with BTLA P267L -pulsed T2 cells, along with the intracellular pro-inflammatory cytokine (IFN-γ) were significantly upregulated. The levels of CD69, 4-1BB, OX40, and CD107a expression were also markedly increased on the surface of CD8 + TCR-T cells co-cultured with BTLA P267L -overexpressed HCC cell lines. CD8 + TCR-T cells activated by T2 or HCC cell lines released a large amount of pro-inflammatory cytokines (e.g., IFN-γ and TNF-α) to synergistically promote the anti-tumor effect. The murine experiments subsequently validated the results of the in vitro studies. SK-HEP-1-neo-luc cells were subcutaneously injected in the mid-backs of NCG mice on day -12, while BTLA P267L -specific CD8 + TCR-T cells (85-3, 126-5, or 52-3) were injected via the tail vein on days 0 and 12. CD8 + TCR-T cells had a lifespan of 2–3 weeks in the peripheral circulation and mediated robust elimination of HCC by releasing perforin. The scalable workflow provides new insights for personalized precision treatment on HCC. BTLA P267L , B and T lymphocyte attenuator P267L ; TCR, T cell receptor; HLA, human leukocyte antigen; pHLA, peptide-HLA complex; HCC, hepatocellular carcinoma; WES, whole exome sequencing; TAP, transporter associated with antigen processing; T2, human hybrid B/T lymphoblastic cell line that expresses HLA-A*02:01 but lacks TAP; ER, endoplasmic reticulum; CD, cluster of differentiation; PD-1, programmed cell death protein 1; CTLA-4, cytotoxic T-lymphocyte associated protein 4; IFN-γ, interferon-gamma; TNF-α, tumor necrosis factor-alpha; SK-HEP-1-neo-luc, hepatocellular carcinoma cell line that endogenously expresses HLA-A*02:01 and overexpresses neoepitope BTLA P267L and luciferase through lentiviral infection; NCG, NOD/ShiLtJGpt- Prkdc em26Cd52 Il2rg em26Cd22 /Gpt.
Article Snippet: All antibodies were purchased from BioLegend and diluted at a 1:200 ratio. (2) Cytotoxicity of BTLA P267L -specific CD8 + TCR-T cells at different effector-to-target ratios (E:T) was determined with the LDH release assay (G1780; Promega, Madison, WI, USA) or the luciferase reporter assay [for suspension cells (DD1201-02; Vazyme, Nanjing, China); for adherent cells (11401ES60; Yeasen, Shanghai, China)]. (3) IFN-γ spots produced by BTLA P267L -specific CD8 + TCR-T cells were detected using an ELISPOT assay (2110005; Dakewe, Shenzhen, China) and an ImmunoSPOT S6 Micro Analyzer (CTL, Shaker Heights, OH, USA). (4) IFN-γ (88-7316-88; Thermo Fisher Scientific), TNF-α (88-7346-88; Thermo Fisher Scientific), perforin (3465-1HP-2; Mabtech, Nacka Strand, STO, Sweden), or granzyme B (3486-1HP-2; Mabtech) were detected using an ELISA assay and a microplate reader (BioTek, Winooski, VT, USA).
Techniques: Activity Assay, Membrane, Cell Culture, Expressing, Activation Assay, Marker, In Vitro, Injection, Sequencing, Luciferase, Infection
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