Seeing is believing: Visualizing immune cells and calcium signals at different stages of neuroinflammation

Multiple sclerosis (MS) is a neuroinflammatory disease characterized by demyelination of neurons in the central nervous system (CNS) (1). While the ultimate cause of the disease is unknown, MS is commonly considered to be an autoimmune disorder in which autoreactive CD4⁺ T cells are activated by an (unknown) autoantigen presented by antigen-presenting cells (APCs) outside the CNS. Once in the CNS, T cells are activated by microglia-derived or CNS-infiltrating APC, resulting in inflammation and myelin damage. The main T cell subsets causing disease in MS are T helper (Th) 1 and Th17 cells, which produce the proinflammatory cytokines interferon gamma (IFN-γ), interleukin (IL)-17A, and granulocyte–macrophage colony-stimulating factor. Studies in MS patients have shown that IL-17A and IFN-γ levels are increased in T cells isolated from CNS lesions and the cerebrospinal fluid (2, 3). A similar role of Th1 and Th17 cells has been found in the experimental autoimmune encephalomyelitis (EAE) rodent model of MS. The proinflammatory function of Th1 and Th17 cells is kept in check by regulatory T (Treg) cells, a subset of CD4⁺ T cells that express the transcription factor Foxp3 by suppressing effector T cell functions and thus CNS inflammation. Treg cells attenuate the severity of EAE and contribute to recovery from CNS inflammation (4). A report by Othy et al. (5) sheds light on the spatiotemporal interaction of Th17 cells, Treg cells, and APCs and their migration at three different stages of EAE. It also shows how Treg cells modulate Ca²⁺ signals in Th17 cells in the spinal cord.

For their studies, Othy et al. (5) developed Foxp3^EGFP IL-17^TdT reporter mice to express enhanced green fluorescent protein (EGFP) in Treg cells and TdTomato in Th17 cells, as well as Foxp3^EGFP CD11c^EYFP reporter mice. Using this fate-mapping approach, the authors were able to simultaneously track encephalitogenic Th17 cells, Treg cells, and APCs in the CNS during the onset, peak, and chronic phase of EAE. During EAE onset when mice develop tail and hindlimb paralysis, Th17 cells are mainly localized in the lumbar region of the spinal cord with only very few Treg cells present (Fig. 1). At the peak of EAE, the numbers of Th17 cells markedly increase along the entire length of the spinal cord and exceed those of Treg cells. During the chronic phase of EAE when mice partially recover from paralysis, similar numbers of Th17 and Treg cells are found in the spinal cord. Using two-photon microscopy of spinal cords at the peak of EAE shows that Th17 cells and Treg cells display different mobility patterns. Treg cells turned frequently and showed a confined motility pattern, whereas Th17 cells migrated along relatively more straight tracks covering larger distances. Autoreactive Th17 cells adoptively transferred into mice displayed a more confined and slower motility pattern compared to endogenous Th17 cells, presumably because of interactions with autoantigen-presenting APCs. By contrast, the motility of Treg cells consistently showed a confined and rapid motility pattern suggesting that this is an intrinsic property of Treg cells in the spinal cord. Using Foxp3^EGFP CD11c^EYFP reporter mice, Othy et al. (5) demonstrate that the recurring “U-turn” behavior of Treg cells, which they term repetitive scanning motility (RSM), allows Treg cells to remain spatially confined and engage in prolonged contacts with APCs. During this process, Treg cells displace Th17 cells from APCs, thus presumably preventing Th17 cell reactivation in the leptomeningeal space. In agreement with a previous study (6), the authors furthermore show that deletion of Treg cells results in a significant increase in EAE severity and the numbers of APCs in the spinal cord of mice, suggesting an inhibitory role of Treg cells in the proliferation or survival of APCs in the CNS.

Fig. 1.

Treg cells suppress Th17 cell Ca²⁺ signaling in the spinal cord during EAE. During the onset of EAE, Th17 cells are located in the caudal region of the spinal cord and have robust, high-frequency Ca²⁺ signals (Left). At the peak of EAE, Th17 cells spread toward the brain and Treg cells infiltrate the spinal cord but are outnumbered by Th17 cells (Middle). During the chronic phase of EAE, the numbers of Th17 and Treg cells are similar; Treg cells displace Th17 cells from APCs and suppress Ca²⁺ signals in Th17 cells (Right). For details see text.

Othy et al. provide an elegant toolkit to image T cell responses in the CNS, which can be further expanded to address a number of follow-up questions, for instance to analyze the effects of Treg–APC interactions on Th17 cell function or to determine dynamic changes of Ca²⁺ signals in Treg cells over the course of disease using Foxp3^Salsa mice.

The ability of Th17 cells to induce CNS inflammation is critically dependent on Ca²⁺ influx in response to T cell receptor (TCR) stimulation. The most important source of Ca²⁺ influx in T cells is the Ca²⁺ release-activated Ca²⁺ (CRAC) channel in the plasma membrane. Pharmacological inhibition of CRAC channel function or genetic deletion of ORAI and STIM family proteins that constitute the channel was shown to prevent EAE onset and progression, which was associated with a reduced ability of encephalitogenic T cells to expand in the CNS, produce Th1 and Th17 cytokines, and migrate down a chemokine gradient (7–10). Treg cells have been shown to attenuate Ca²⁺ signaling in effector T cells in vitro (11, 12), providing a potential mechanism for their ability to suppress Th17 cell function in EAE. Several studies have explored the role of Ca²⁺ signals in T cell migration in the CNS during EAE using two-photon microscopy and genetically encoded Ca²⁺ indicators (GECIs) introduced ex vivo into myelin-specific T cells (13, 14). After having crossed the blood–brain barrier, T cells were shown to be motile in the CNS parenchyma and exhibit Ca²⁺ signals that were dependent on TCR–major histocompatibility complex (MHC) interactions between T cells and APCs (13, 14). For their study, Othy et al. (5) generated IL-17^Salsa mice, which express the GECI Salsa6f (15) in Th17 cells, and crossed them to Foxp3^EGFP mice. This approach allowed them to simultaneously measure Ca²⁺ signaling in Th17 cells and the dynamics of Th17–Treg cell interactions during EAE. The authors find high frequencies and amplitudes of Ca²⁺ signals in Th17 cells at the onset of EAE, which are significantly reduced at the peak of disease (Fig. 1). These findings correlate with the number of Treg cells in the spinal cord. In addition, Th17 cells isolated from the CNS at EAE onset had large, high-frequency Ca²⁺ signals when stimulated by TCR cross-linking in vitro, whereas signals in Th17 cells isolated during the established phase of EAE showed smaller amplitudes and lower frequencies. Together these data support a model in which Treg cells suppress Ca²⁺ signals in encephalitogenic Th17 cells during the resolution of EAE.

Before the study by Othy et al. (5), in situ two-photon imaging of T cells in the context of EAE had been conducted by adoptive transfer of fluorescently tagged, myelin-specific CD4⁺ T cells to mice and rats (13, 14, 16). While elegant and ground-breaking in their own right, these studies were limited by the fact that they monitored the behavior of ex vivo-activated effector T cells in the CNS, did not differentiate between T cell subsets, and were focused on T cell motility immediately after adoptive transfer. Othy et al. (5) use a number of elegant transgenic tools to overcome these limitations. By generating transgenic IL-17^TdT Foxp3^EGFP and CD11c^EYFP reporter mice, the authors are able to analyze endogenous T cells and APCs. Furthermore, they can distinguish between the location and motility of proinflammatory Th17 and immunomodulatory Treg cells and determine their numbers, location, and motility in the spinal cord in relation to APCs. These approaches provide a greater level of resolution at which T cell responses in the inflamed CNS during EAE can be visualized. Adding to the toolkit, the authors generated IL-17^Salsa mice to measure Ca²⁺ signals in endogenous Th17 cells during EAE. Unlike other GECIs Salsa6f is ratiometric, which is critical for in vivo imaging to correct for changes in GECI expression levels and cell movement. By using Salsa6f, the authors show that Ca²⁺ signals in encephalitogenic Th17 cells are attenuated during the chronic phase of EAE when disease activity tapers off, providing compelling evidence that one of the mechanisms by which Treg cells in the spinal cord decrease EAE activity is suppression of Ca²⁺ influx in Th17 cells (Fig. 1). This is a reasonable conclusion if one considers that CRAC channels are absolutely essential for the ability of Th17 cells to cause EAE and that even small reductions in Ca²⁺ influx attenuate disease onset and progression (7–10). A particularly intriguing insight of this study is that Th17 cells cover larger distances in the CNS until they become restrained by contacts with APCs, whereas Treg cells are more spatially confined and exhibit an RSM. The authors interpret the RSM behavior as a means by which Treg cells restrict the interaction of Th17 cells with APCs, thus preventing Th17 cell activation. This is an attractive model consistent with earlier in vitro studies which showed that Treg cells outcompete effector T cells by forming aggregates around DCs, thus physically blocking the access of effector T cells to DCs (17). Future imaging studies will need to show that deletion of Treg cells affects the interaction of APC and Th17 cells, for instance by using IL-17^TdT CD11c^EYFP mice in which Treg cells are deleted.

Othy et al. (5) provide an elegant toolkit to image T cell responses in the CNS, which can be further expanded to address a number of follow-up questions, for instance to analyze the effects of Treg–APC interactions on Th17 cell function or to determine dynamic changes of Ca²⁺ signals in Treg cells over the course of disease using Foxp3^Salsa mice. This is relevant because Ca²⁺ influx is required for Treg cell function, as demonstrated by the impaired differentiation and immunosuppressive function of Treg cells in mice with T cell-specific deletion of Stim1 and Stim2 genes (18). Another follow-up question pertains to the mechanisms by which Treg cells suppress encephalitogenic Th17 cells in the inflamed CNS. The suppression of Ca²⁺ signaling appears to be long-lasting but is not due to down-regulation of the CRAC channel and may instead involve more proximal TCR signaling steps. The study by Othy et al. (5) emphasizes the importance of Treg cells in the resolution of EAE and provides support for the idea that enhancing Treg cell numbers or function may be a useful approach for the treatment of MS (4).