Characterizing the role of mitochondrial Ca2+ overload and permeability transition during the BCR-dependent activation of murine B lymphocytes

Mitochondria have proved their role as major players for immunometabolism, regulating cell function by metabolic signals. Indeed, mitochondrial function during lymphocyte activation goes beyond ATP supply, as recent evidence shows that mitochondrial Ca2+ uptake and mitochondrial ROS (mROS) production regulate lymphocyte activation. Furthermore, the adaptive immune response depends on the differentiation of lymphocytes into various subsets. Whether metabolic signals determine lymphocyte differentiation has just began to be explored. Interestingly, recent evidence shows that mitochondrial signals determine B lymphocyte differentiation into memory cells. During lymphocyte activation, Ca2+ uptake into mitochondria has proven to be an important mechanism of regulation. However, if mitochondrial Ca2+ uptake capacity impacts B cell differentiation and activation is not yet determined. Here, we have generated an in vitro model of B lymphocyte mitochondrial Ca2+ overload to explore whether this mechanism is involved in B cell activation. Primary mice B lymphocytes were subjected to activation or thapsigargin treatment to induce Ca2+ overload. The effect on the expression of activation surface marker CD69 was explored using flow cytometry. Mitochondrial contribution was explored by Ca2+ uptake inhibition using Ru360 or treatment with the mitochondrial antioxidant MitoTEMPO. Mitochondrial membrane potential, Ca2+ uptake, and content were determined. Results show that our in vitro model effectively induces mitochondrial Ca2+ overload, as seen by higher Ca2+ content and mitochondrial membrane depolarization. Moreover, mitochondrial Ca2+ overload contributes to the expression of CD69 via mROS production. Lastly, results suggest that during BCR-dependent activation, mitochondria take up Ca2+ to increase mROS production that can alter the signaling cascade. The in vitro model generated herein, should allow for further exploration of whether mitochondrial Ca2+ overload impacts B cell differentiation.