Elevations in cytosolic calcium (Ca²⁺) drive a wide array of immune cell functions, including cytokine production, gene expression, and cell motility. Live-cell imaging of cells loaded with ratiometric chemical Ca²⁺ indicators remains the gold standard for visualization and quantification of intracellular Ca²⁺ signals; ratiometric imaging can be accomplished with dyes such as Fura-2, the combination of Fluo-4 and Fura-Red, or, alternatively, by expressing genetically-encoded Ca²⁺ indicators (GECI) such as GCaMPs. Here, we describe a detailed protocol for Ca²⁺ imaging of T cells in vitro using genetically encoded or chemical indicators that can also be applied to a wide variety of cell types. The protocol addresses the challenge of facilitating T cell attachment on various substrates prepared on glass-bottom dishes to enable T cell imaging on an inverted microscope. The protocol also emphasizes cell preparation steps that ensure optimal cell viability - an essential requirement for recording dynamic changes in cytosolic Ca²⁺ levels - and that ensure reproducibility between multiple samples. Finally, we describe a simple algorithm to analyze single-cell Ca²⁺ signals over time using Fiji (ImageJ) software.

Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Here, we describe development and characterization of 'Salsa6f', a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca²⁺, and a transgenic Ca²⁺ reporter mouse with Salsa6f targeted to the Rosa26 locus for Cre-dependent expression in specific cell types. The development and function of T cells was unaffected in Cd4-Salsa6f mice. We describe Ca²⁺ signals reported by Salsa6f during T cell receptor activation in naive T cells, helper Th17 T cells and regulatory T cells, and Ca²⁺ signals mediated in T cells by an activator of mechanosensitive Piezo1 channels. Transgenic expression of Salsa6f enables ratiometric imaging of Ca²⁺ signals in complex tissue environments found in vivo. Two-photon imaging of migrating T cells in the steady-state lymph node revealed both cell-wide and localized sub-cellular Ca²⁺ transients ('sparkles') as cells migrate.

T lymphocyte motility and interaction dynamics with other immune cells are vital determinants of immune responses. Regulatory T (Treg) cells prevent autoimmune disorders by suppressing excessive lymphocyte activity, but how interstitial motility patterns of Treg cells limit neuroinflammation is not well understood. We used two-photon microscopy to elucidate the spatial organization, motility characteristics, and interactions of endogenous Treg and Th17 cells together with antigen-presenting cells (APCs) within the spinal cord leptomeninges in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Th17 cells arrive before the onset of clinical symptoms, distribute uniformly during the peak, and decline in numbers during later stages of EAE. In contrast, Treg cells arrive after Th17 cells and persist during the chronic phase. Th17 cells meander widely, interact with APCs, and exhibit cytosolic Ca²⁺ transients and elevated basal Ca²⁺ levels before the arrival of Treg cells. In contrast, Treg cells adopt a confined, repetitive-scanning motility while contacting APCs. These locally confined but highly motile Treg cells limit Th17 cells from accessing APCs and suppress Th17 cell Ca²⁺ signaling by a mechanism that is upstream of store-operated Ca²⁺ entry. Finally, Treg cell depletion increases APC numbers in the spinal cord and exaggerates ongoing neuroinflammation. Our results point to fundamental differences in motility characteristics between Th17 and Treg cells in the inflamed spinal cord and reveal three potential cellular mechanisms by which Treg cells regulate Th17 cell effector functions: reduction of APC density, limiting access of Th17 cells to APCs, and suppression of Th17 Ca²⁺ signaling.

Ca²⁺ influx through Orai1 channels is crucial for several T cell functions, but a role in regulating basal cellular motility has not been described. Here, we show that inhibition of Orai1 channel activity increases average cell velocities by reducing the frequency of pauses in human T cells migrating through confined spaces, even in the absence of extrinsic cell contacts or antigen recognition. Utilizing a novel ratiometric genetically encoded cytosolic Ca²⁺ indicator, Salsa6f, which permits real-time monitoring of cytosolic Ca²⁺ along with cell motility, we show that spontaneous pauses during T cell motility in vitro and in vivo coincide with episodes of cytosolic Ca²⁺ signaling. Furthermore, lymph node T cells exhibited two types of spontaneous Ca²⁺ transients: short-duration 'sparkles' and longer duration global signals. Our results demonstrate that spontaneous and self-peptide MHC-dependent activation of Orai1 ensures random walk behavior in T cells to optimize immune surveillance.

Macrophages perform diverse functions within tissues during immune responses to pathogens and injury, but molecular mechanisms by which physical properties of the tissue regulate macrophage behavior are less well understood. Here, we examine the role of the mechanically activated cation channel Piezo1 in macrophage polarization and sensing of microenvironmental stiffness. We show that macrophages lacking Piezo1 exhibit reduced inflammation and enhanced wound healing responses. Additionally, macrophages expressing the transgenic Ca²⁺ reporter, Salsa6f, reveal that Ca²⁺ influx is dependent on Piezo1, modulated by soluble signals, and enhanced on stiff substrates. Furthermore, stiffness-dependent changes in macrophage function, both in vitro and in response to subcutaneous implantation of biomaterials in vivo, require Piezo1. Finally, we show that positive feedback between Piezo1 and actin drives macrophage activation. Together, our studies reveal that Piezo1 is a mechanosensor of stiffness in macrophages, and that its activity modulates polarization responses.

T lymphocytes encounter complex mechanical cues during an immune response. The mechanosensitive ion channel, Piezo1, drives inflammatory responses to bacterial infections, wound healing, and cancer; however, its role in helper T cell function remains unclear. In an animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we found that mice with genetic deletion of Piezo1 in T cells showed diminished disease severity. Unexpectedly, Piezo1 was not essential for lymph node homing, interstitial motility, Ca²⁺ signaling, T cell proliferation, or differentiation into proinflammatory T helper 1 (T_H1) and T_H17 subsets. However, Piezo1 deletion in T cells resulted in enhanced transforming growth factor-β (TGFβ) signaling and an expanded pool of regulatory T (T_reg) cells. Moreover, mice with deletion of Piezo1 specifically in T_reg cells showed significant attenuation of EAE. Our results indicate that Piezo1 selectively restrains T_reg cells, without influencing activation events or effector T cell functions.

The membrane protein TREM2 (Triggering Receptor Expressed on Myeloid cells 2) regulates key microglial functions including phagocytosis and chemotaxis. Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer's disease (AD). Because abnormalities in Ca²⁺ signaling have been observed in several AD models, we investigated TREM2 regulation of Ca²⁺ signaling in human induced pluripotent stem cell-derived microglia (iPSC-microglia) with genetic deletion of TREM2. We found that iPSC-microglia lacking TREM2 (TREM2 KO) show exaggerated Ca²⁺ signals in response to purinergic agonists, such as ADP, that shape microglial injury responses. This ADP hypersensitivity, driven by increased expression of P2Y₁₂ and P2Y₁₃ receptors, results in greater release of Ca²⁺ from the endoplasmic reticulum stores, which triggers sustained Ca²⁺ influx through Orai channels and alters cell motility in TREM2 KO microglia. Using iPSC-microglia expressing the genetically encoded Ca²⁺ probe, Salsa6f, we found that cytosolic Ca²⁺ tunes motility to a greater extent in TREM2 KO microglia. Despite showing greater overall displacement, TREM2 KO microglia exhibit reduced directional chemotaxis along ADP gradients. Accordingly, the chemotactic defect in TREM2 KO microglia was rescued by reducing cytosolic Ca²⁺ using a P2Y₁₂ receptor antagonist. Our results show that loss of TREM2 confers a defect in microglial Ca²⁺ response to purinergic signals, suggesting a window of Ca²⁺ signaling for optimal microglial motility.

The P522R variant of PLCG2, expressed by microglia, is associated with reduced risk of Alzheimer's disease (AD). Yet, the impact of this protective mutation on microglial responses to AD pathology remains unknown. Chimeric AD and wild-type mice were generated by transplanting PLCG2-P522R or isogenic wild-type human induced pluripotent stem cell microglia. At 7 months of age, single-cell and bulk RNA sequencing, and histological analyses were performed. The PLCG2-P522R variant induced a significant increase in microglial human leukocyte antigen (HLA) expression and the induction of antigen presentation, chemokine signaling, and T cell proliferation pathways. Examination of immune-intact AD mice further demonstrated that the PLCG2-P522R variant promotes the recruitment of CD8⁺ T cells to the brain. These data provide the first evidence that the PLCG2-P522R variant increases the capacity of microglia to recruit T cells and present antigens, promoting a microglial transcriptional state that has recently been shown to be reduced in AD patient brains.

The discovery of TREM2 as a myeloid-specific Alzheimer's disease (AD) risk gene has accelerated research into the role of microglia in AD. While TREM2 mouse models have provided critical insight, the normal and disease-associated functions of TREM2 in human microglia remain unclear. To examine this question, we profile microglia differentiated from isogenic, CRISPR-modified TREM2-knockout induced pluripotent stem cell (iPSC) lines. By combining transcriptomic and functional analyses with a chimeric AD mouse model, we find that TREM2 deletion reduces microglial survival, impairs phagocytosis of key substrates including APOE, and inhibits SDF-1α/CXCR4-mediated chemotaxis, culminating in an impaired response to beta-amyloid plaques in vivo. Single-cell sequencing of xenotransplanted human microglia further highlights a loss of disease-associated microglial (DAM) responses in human TREM2 knockout microglia that we validate by flow cytometry and immunohistochemistry. Taken together, these studies reveal both conserved and novel aspects of human TREM2 biology that likely play critical roles in the development and progression of AD.