1. Introduction [1]
T cells are a diverse and important class of lymphocytes that undergo positive and negative selection to mature in the thymus. It plays an important role in both components of active immunity, namely cell-mediated immunity and to some extent humoral immunity. There are various types of T cells, with the most common being CD4+T cells (helper T cells) and CD8+T cells (cytotoxic T cells or killer T cells). T cells cannot recognize soluble free antigens. T cells can only recognize antigens based on proteins that bind to receptors. This recognition is achieved through the use of MHC (also known as HLA) 1 and 2 receptors, which combine with TCR (T cell receptor) to form a complex that allows T cells to recognize antigens. CD4+T cells can recognize antigens that bind to MHC 2, while CD8+T cells can recognize antigens that bind to MHC 1.
2. Technical process
2.1 Isolation of peripheral blood mononuclear cells (PBMCs)
(1) Collect 10ml of human venous blood using a sterile collection tube containing heparin sodium, and gently invert the tube several times to mix evenly.
(2) Take 10ml Ficoll in a 50ml centrifuge tube.
(3) Gently stratify the blood at the top of Ficoll using an automatic pipette. The stratification should be very slow, and blood and Ficoll should be kept in two different layers.
(4) At 4 ℃, increase and decrease by 1900g, centrifuge for 30 minutes.
(5) After centrifugation, extract the cells from the PBMC cell layer and transfer them to a 50ml centrifuge tube. Add an equal volume of 1640 medium to the centrifuge tube, gently blow the cell suspension 3-5 times with a pipette, and resuspend the cells.
(6) At 4 ℃, 400g, centrifuge for 5 minutes, collect PBMC cell pellet for later use.
2.2 Anti-CD3 antibody coating
(1) Dilute anti-CD3 antibody with sterile PBS (concentration of 1 µ g/ml), then coat anti-CD3 antibody in a 6-well plate, add 2ml of coating solution to each well, place the plate in a 5% CO2 incubator, and incubate at 37 ° C for 2 hours.
(2) After 2 hours, remove the 6-well plate from the incubator. Suck out the anti-CD3 solution and discard it. Do not clean the culture plate for future use.
2.3 Activation culture of peripheral blood T lymphocytes
(1) Take PBMC cell pellet and resuspend in 1640 medium (containing 10% inactivated FBS+2 mM L-glutamine+5 µ g/ml anti-CD28+1% double antibody) until 5x105 cells/ml.
(2) Connect to a pre packaged 6-hole plate at a rate of 3m per hole.
(3) Incubate in a 37 ℃, 5% CO2 incubator.
(4) Administer fluid once every 3 days, observe the activation and culture of peripheral blood T lymphocytes, and draw a growth curve.
3. Research progress of T lymphocytes in tumors [2]
CD4+T lymphocytes participate in regulating the innate and adaptive immunity of tumors through a series of mechanisms. According to their different functions, they can be divided into multiple subgroups, mainly including Th1, Th2, Th17, etc. [3]. CD8+T lymphocytes account for 24% to 30% of peripheral blood lymphocytes, and their main function is to directly kill target antigens (such as viruses and tumor cells), hence they are called cytotoxic T lymphocytes (CTLs).
At present, it is widely believed that CD8+T cells are the main driving factor of anti-tumor immunity, and CD4+T cells also play a prominent role in tumor control, which can promote or inhibit anti-tumor responses [4-5]. Most cancer cells do not express MHC class II molecules (MHC II), and therefore cannot be directly recognized by CD4T cells. Instead, they recognize tumor antigens through cross presentation by tumor stromal cells [6]. The abundance of tumor infiltrating CD8+T lymphocytes and the characteristics of elevated T expression are significantly related to the disease-free survival or overall survival of many patients with different tumor types, including breast cancer, lung cancer, colorectal cancer, melanoma, head and neck cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, ovarian cancer, cervical cancer, endometrial adenocarcinoma, and bladder cancer. Tumor specific CD8+T lymphocytes can directly kill tumor cells in an MHC-I restricted manner by producing cytotoxic molecules [7].
4. References
【1】Sauls RS, McCausland C, Taylor BN. Histology, T-Cell Lymphocyte. [Updated 2023 May 1]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls
【2】Chen Fei, Yan Dong Research progress of T lymphocytes in tumors [J] Clinical Medical Progress, 2024, 14 (4): 3012-3020
【3】Basu, A., Ramamoorthi, G., Albert, G., et al. (2021) Differentiation and Regulation of TH Cells: A Balancing Act for Cancer Immunotherapy. Frontiers in Immunology, 12, Article 669474.
【4】Poncette, L., Bluhm, J. and Blankenstein, T. (2022) The Role of CD4 T Cells in Rejection of Solid Tumors. Current Opinion in Immunology, 74, 18-24.
【5】Kim, H.-J. and Cantor, H. (2014) CD4 T-Cell Subsets and Tumor Immunity: The Helpful and the Not-So-Helpful. Cancer Immunology Research, 2, 91-98.
【6】Ohshima, K. and Morii, E. (2021) Metabolic Reprogramming of Cancer Cells during Tumor Progression and Metastasis. Metabolites, 11, Article 28.
【7】Martinez-Usatorre, A., Carmona, S.J., Godfroid, C., et al. (2020) Enhanced Phenotype Definition for Precision Isolation of Precursor Exhausted Tumor-Infiltrating CD8 T Cells. Frontiers in Immunology, 11, Article 340.