Detection of Antigen Specific T Lymphocytes by Determination of IntracellularCalcium Concentration Using Flow Cytometry

摘要

We present a method for the detection of lymphocytes with specific reactivity toantigens on stimulator cells using flow cytometry. Cultured human T lymphocytes were loaded with the intracellular fluorochrome indo-1 and were mixed with stimulator cells. Using flow cytometry we could detect ii specific increase in intracellular calcium in the T lymphocytes as well as conjugation between the T cells and the stimulator cells. Examination of antigen-specific CD4 -+ and CD8 + T cell clones demonstrated that the vast majority of T cells which were conjugated to antigen-bearing stimulator cells manifested a rapid increase in intracellular calcium. In contrast T cells conjugated to stimulator cells which did not bear specific antigen demonstrated no such increase in calcium. A similar finding was observed when examining polyclonal tumor infiltrating lymphocytes obtained from patients with melanoma. Tumor infiltrating lymphocytes with specific antitumor reactivity demonstrated an increase in intracellular calcium when conjugated to autologous tumor but not to allogeneic melanoma. In contrast to the T cell clones, only a small subpopulation of tumor infiltrating lymphocytes manifested this specific signal upon conjugation with autologous tumor. Calcium; T lymphocyte; Activation: Flow cytometry(Human).