T cell receptor stimulation initiates a cascade of reactions that cause an increase in intracellular calcium (Ca²⁺) concentration mediated through inositol 1,4,5-trisphosphate (IP₃). To understand the basic mechanisms by which the immune response in T cells is activated, it is useful to understand the signaling pathways that contain important targets for drugs in a quantitative fashion. A computational model helps us to understand how the selected elements in the pathways interact with each other, and which component plays the crucial role in systems. We have developed a mathematical model to explore the mechanism for controlling transcription factor activity, which regulates gene expression, by the modulation of calcium signaling triggered during T cell activation. The model simulates the activation and modulation of Ca²⁺ release-activated Ca²⁺ (CRAC) channels by mitochondrial dynamics and depletion of endoplasmic reticulum (ER) store, and also includes membrane potential in T-cells. The model simulates the experimental finding that increases in Ca²⁺ current enhances the activation of transcription factors and the Ca²⁺ influx through CRAC is also essential for the NFAT and NFκB activation. The model also suggests that plasma membrane Ca²⁺-ATPase (PMCA) controls a majority of the extrusion of Ca²⁺ and modulates the activation of CRAC channels. Furthermore, the model simulations explain how the complex interaction of the endoplasmic reticulum, membrane potential, mitochondria, and ion channels such as CRAC channels control T cell activation.

Gain-of-function RyR1-p.R163C mutation in ryanodine receptors type 1 (RyR1) deregulates Ca²⁺ signaling and mitochondrial function in skeletal muscle and causes malignant hyperthermia in humans and mice under triggering conditions. We investigated whether T lymphocytes from heterozygous RyR1-p.R163C knock-in mutant mice (HET T cells) display measurable aberrations in resting cytosolic Ca²⁺ concentration ([Ca²⁺]_i), Ca²⁺ release from the store, store-operated Ca²⁺ entry (SOCE), and mitochondrial inner membrane potential (ΔΨ_m) compared with T lymphocytes from wild-type mice (WT T cells). We explored whether these variables can be used to distinguish between T cells with normal and altered RyR1 genotype. HET and WT T cells were isolated from spleen and lymph nodes and activated in vitro using phytohemagglutinin P. [Ca²⁺]_i and ΔΨ_m dynamics were examined using Fura 2 and tetramethylrhodamine methyl ester fluorescent dyes, respectively. Activated HET T cells displayed elevated resting [Ca²⁺]_i, diminished responses to Ca²⁺ mobilization with thapsigargin, and decreased rate of [Ca²⁺]_i elevation in response to SOCE compared with WT T cells. Pretreatment of HET T cells with ryanodine or dantrolene sodium reduced disparities in the resting [Ca²⁺]_i and ability of thapsigargin to mobilize Ca²⁺ between HET and WT T cells. While SOCE elicited dissipation of the ΔΨ_m in WT T cells, it produced ΔΨ_m hyperpolarization in HET T cells. When used as the classification variable, the amplitude of thapsigargin-induced Ca²⁺ transient showed the best promise in predicting the presence of RyR1-p.R163C mutation. Other significant variables identified by machine learning analysis were the ratio of resting cytosolic Ca²⁺ level to the amplitude of thapsigargin-induced Ca²⁺ transient and an integral of changes in ΔΨ_m in response to SOCE. Our study demonstrated that gain-of-function mutation in RyR1 significantly affects Ca²⁺ signaling and mitochondrial fiction in T lymphocytes, which suggests that this mutation may cause altered immune responses in its carrier. Our data link the RyR1-p.R163C mutation, which causes inherited skeletal muscle diseases, to deregulation of Ca²⁺ signaling and mitochondrial function in immune T cells and establish proof-of-principle for in vitro T cell-based diagnostic assay for hereditary RyR1 hyperfunction.