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Published on: October 28, 2014
Detection of antigen specific T lymphocytes by determination of intracellular calcium concentration using flow
R B Alexander1, E S Bolton, S Koenig
1Surgery Branch, National Cancer Institute, Bethesda, MD 20892.
Insights
We developed a flow cytometry method to detect antigen-specific T cells by measuring intracellular calcium changes upon cell conjugation. This technique identifies reactive lymphocytes in real-time for potential sorting and further analysis.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- T lymphocytes play a crucial role in adaptive immunity by recognizing specific antigens.
- Detecting antigen-specific T cell responses is vital for understanding immune function and developing immunotherapies.
- Current methods for assessing T cell reactivity can be complex and may not provide real-time, single-cell resolution.
Purpose of the Study:
- To establish a novel flow cytometry-based method for detecting antigen-specific T lymphocytes.
- To characterize the intracellular calcium flux in T cells upon recognition of specific antigens on stimulator cells.
- To evaluate the utility of this method for analyzing T cell clones and tumor-infiltrating lymphocytes.
Main Methods:
- Human T lymphocytes were loaded with indo-1, a calcium-sensitive fluorochrome.
- T lymphocytes were co-cultured with antigen-bearing or non-antigen-bearing stimulator cells.
- Flow cytometry was used to measure intracellular calcium increases and cell conjugation in real-time.
Main Results:
- A rapid increase in intracellular calcium was observed in T lymphocytes conjugated to antigen-specific stimulator cells.
- This calcium response was specific, as T cells conjugated to non-antigen-bearing cells showed no significant increase.
- The method successfully identified antigen-specific T cell clones and a subpopulation of tumor-infiltrating lymphocytes with antitumor reactivity.
Conclusions:
- The developed flow cytometry method enables real-time, single-cell detection of antigen-specific T cell responses.
- This technique allows for the identification and potential isolation of functionally active T cells.
- The method holds promise for immunological research, diagnostics, and therapeutic cell sorting.
Abstract:
We present a method for the detection of lymphocytes with specific reactivity to antigens 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 a 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. This suggests that tumor infiltrating lymphocyte cultures contain T cells with varying reactivities to tumor or may also imply heterogeneity in the stimulating tumor cell lines. The method allows for the detection of specific T cells on an individual cell basis in real time. The procedure is not lethal to the cell and sorting and subculturing of reactive T cell populations can be readily performed. The method could also be used to sort stimulator cells based on their ability to elicit an increase in intracellular calcium in selected T cells.
