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Calcium response of helper T lymphocytes to antigen-presenting cells in a single-cell assay
1Department of Chemical Engineering, University of Michigan, Ann Arbor 48109, USA.
Insights
A novel assay reveals helper T lymphocyte (Th cell) activation is an all-or-none calcium response, independent of antigen concentration. However, more antigen increases the number of Th cell-APC interactions that trigger this response.
Area of Science:
- Immunology
- Cell Biology
- Biophysics
Background:
- Helper T lymphocytes (Th cells) are crucial for adaptive immunity.
- Th cell activation involves physical interaction with antigen-presenting cells (APCs).
- Intracellular calcium signaling is an early indicator of Th cell activation.
Purpose of the Study:
- To develop a dynamic, single-cell assay to visualize Th cell-APC interactions and monitor Th cell calcium responses.
- To investigate the role of Th cell motility in initiating contacts.
- To determine the quantitative requirements for Th cell activation.
Main Methods:
- Developed a dynamic, single-cell assay using microscopy and digital imaging.
- Observed live Th cell-APC interactions and intracellular calcium changes.
- Utilized micromanipulation to control cell proximity.
- Applied mathematical modeling to estimate activation thresholds.
Main Results:
- Th cell motility initiates but does not dictate the calcium response.
- Individual Th cell calcium responses are heterogeneous and all-or-none, irrespective of antigen concentration.
- The proportion of responding Th cell-APC conjugates increases with antigen concentration.
- Estimated 1-20 MHC class II-antigen complexes are sufficient for Th cell calcium response.
Conclusions:
- Th cell activation is a stochastic, all-or-none event at the single-cell level.
- Antigen concentration modulates the frequency of Th cell activation, not the response threshold.
- Quantified the minimal antigenic stimulus required for Th cell activation.
Abstract:
We developed a dynamic, single-cell assay involving alternating differential interference contrast and fluorescence microscopy, together with digital imaging, for both viewing the physical interaction of live helper T lymphocytes (Th cells) with antigen-presenting cells (APCs) and monitoring the increases in the intracellular free calcium concentration of the Th cell, an early event in Th cell activation. We obtained Th-APC conjugates by allowing the Th cells to migrate toward and interact with APCs that either settled nearby or had been micromanipulated in close proximity to the Th cells. Th cell motility played an important role in initiating Th-APC contacts but not in determining the Th cell calcium response. We found that the intracellular calcium responses of individual Th cells are heterogeneous and an all-or-none phenomenon, independent of antigen concentration. However, the fraction of Th-APC conjugates involving responding Th cells is an increasing function of the antigen concentration. Finally, we measured some characteristics of the developing Th-APC contact area. We used all of these data together with previously developed mathematical models to estimate that only 1 to 20 major histocompatibility class II-antigen complexes are required in the initial Th-APC contact area to elicit a Th cell calcium response.