To help protect the body from disease, small immune cells called T lymphocytes move rapidly, searching for signs of infection. These signs are antigens – processed pieces of proteins from invading microbes – that are displayed on the surface of so-called antigen-presenting cells.Before it encounters its specific antigen, a T cell is called naive. After encountering its antigen, the naive T cell activates and then develops into a variety of immune cells, each with a specific activity. These immune cells include so-called peripherally induced regulatory T cells (or “pTreg cells” for short), which, as the name suggests, help to regulate the immune response.

In addition to foreign antigens from microbes, antigen-presenting cells display fragments of the body’s own proteins too. All naive T cells recognize some “Self-antigens”, but not as strongly as they recognize foreign antigens. As a naive T cell travels around the body, it repeatedly interacts with antigen-presenting cells that display Self-antigens, which triggers a low level of signaling in the T cell. While this background signaling was known to help the T cell survive, in 2013, researchers reported that: it also makes the T cell more responsive to foreign antigens; and it shapes how these cells will respond when activated. For example, the naive T cells that respond the most to Self-antigens were seen to be much more likely to become pTreg cells when activated than other T cells.

Guichard et al. – who include several of the researchers involved in the 2013 work – set out to understand why the most Self-reactive T cells show this bias toward becoming pTreg cells. The experiments used a range of approaches with T cells both in the laboratory and in mice. By looking at which genes were active in the most Self-reactive T cells, Guichard et al. narrowed in on a signaling pathway that involves calcium ions and an enzyme called Calcineurin. Blocking this pathway caused the most Self-reactive T cells to lose their bias, and instead develop in the same way as the least Self-reactive T cells.

Guichard et al. propose that the continuous interactions with Self-antigens trigger waves of calcium ions in a naive T cell that shapes its behavior and future development. In a related study, Dong, Othy et al. also conclude that contact with antigen-presenting cells causes calcium signals that shape how the T cells behave.

In addition to providing more detail about the inner workings of immune cells, these findings may also have implications in a clinical setting. Calcineurin inhibitors are often used to suppress the immune system in transplant patients to prevent rejection of the transplanted organ. However, it has proved difficult to safely interrupt these therapies even after many years. These new findings may provide a possible explanation for this, by suggesting that the inhibitors may also interfere with the generation of pTreg cells. Without these cells’ regulatory influence, the immune system is unlikely to ever become tolerant of the transplant.

T-cell precursors originate in the bone-marrow and are educated in the thymus through processes called positive and negative selections, which result in MHC-restriction and self-tolerance, respectively (Stritesky et al., 2012). Only those T cells that bear an αβT-cell receptor (TCR) recognizing self-MHC with a relatively low affinity will differentiate and exit into the systemic circulation as self-MHC restricted T cells. T cells carrying an αβ TCR that reacts with self-MHC with very low affinity die by neglect, whereas those recognizing self-MHC with high affinity are mostly deleted by apoptosis or differentiate into regulatory T cells called ‘natural’ or thymically derived (tTreg) in order to prevent autoimmunity (Bautista et al., 2009; Leung et al., 2009). Therefore, self-MHC and the associated self-reactivity of T cells influence both T-cell production and phenotype in the thymus.

In the periphery, the pre-immune repertoire of T cells is composed of almost 70% of naive T cells. The remaining 30% are divided between recent thymic emigrants with a comparable phenotype, regulatory T cells (Foxp3⁺) and cells with an activated/memory phenotype. Naive T cells are kept alive through continuous TCR interactions with MHC molecules complexed with various self-peptides. Such TCR/MHC interactions plus contacts with IL-7 cause low-level signaling, which promotes long-term survival of naive T cells in interphase through the synthesis of anti-apoptotic molecules such as Bcl-2 (Martin et al., 2006; Takada and Jameson, 2009).

The degree of TCR self-reactivity of a given T-cell clone has been correlated with its expression of CD5 and Nur77 (Azzam et al., 1998; Moran et al., 2011). We have recently identified the cell surface GPI-anchored protein, Ly-6C, as an additional and complementary sensor of T-cell self-reactivity (Martin et al., 2013). Indeed, we have shown that, in contrast to CD5 and Nur77 which expression directly correlates with self-reactivity, the expression of Ly-6C by peripheral naive CD4 T cells (CD4 T_N cells) inversely correlates with their ability to interact with self-MHC (Martin et al., 2013). Ly-6C^- CD4 T_N cells were therefore identified as more self-reactive than their Ly-6C⁺-cell counterparts.

In the absence of foreign antigen, peripheral naive T cells continuously recirculate between lymphoid organs (Gowans, 1959), in which they migrate along the fibroblastic reticular cells network (Bajénoff et al., 2006) and interact frequently and briefly with dendritic cells (DC) (Bajénoff et al., 2006; Mempel et al., 2004). It is generally accepted that these frequent DC-T-cell interactions increase the probability of contacts between very rare antigen-specific naive T cells and the few DCs presenting their cognate antigen during the initial course of an infection. Experimental evidences indicate that self-MHC recognition in the periphery is also required to maintain T cells in a state of responsiveness toward foreign antigen (Persaud et al., 2014; Stefanová et al., 2002; Wülfing et al., 2002), suggesting a crucial role for self-MHC mediated ‘education’ and TCR self-reactivity in determining the intrinsic functional attributes of CD4 T_N cells. Altogether, this steady-state tonic TCR signaling was shown to influence CD4 T_N-cell effector fate by increasing the magnitude of their response toward their cognate antigens.

Following activation by antigen-presenting cells (APCs) in the periphery, the bulk of CD4 T_N cells can differentiate into a variety of well documented T-helper (T_H) cell subsets, such as T_H1, T_H2, T_H17 or peripherally induced regulatory T cells (pTreg cells), characterized by their cytokine production profiles, specific effector functions and lineage-specific transcription factors (T-bet for T_H1 cells, GATA-3 for T_H2 cells, RORγt/RORα for T_H17 cells and Foxp3 for pTreg cells) (Abbas et al., 1996; Bilate and Lafaille, 2012; Fontenot et al., 2003; Hori et al., 2003; Ivanov et al., 2006; Liang et al., 2006; Mosmann et al., 1986; Szabo et al., 2000; Ye et al., 2001; Zheng and Flavell, 1997). Among these effector CD4 T cells, pTreg cells produce TGF-β and share phenotypic and functional characteristics with tTreg cells (Bilate and Lafaille, 2012). The immunological context in which CD4 T_N cells are immersed at the time of their activation is known to drive lineage commitment. The strength of the activating TCR signals received by a CD4 T_N cell also influences its subsequent polarization toward particular differentiation pathways (Corse et al., 2011). Indeed, in weakly polarizing conditions, low TCR signals favor T_H2- and pTreg-cell differentiation, whereas T_H1- and T_FH-cell differentiation arises from stronger signals (Gottschalk et al., 2010; Rogers and Croft, 1999; Turner et al., 2009). Most of these data were obtained in vitro by modulating signal strength with graded dose of peptide-MHC ligands of varying potency. However, only relatively high-affinity TCR–MHC interactions were shown to facilitate the induction of persistent Foxp3⁺ T cells in vivo (Gottschalk et al., 2010).

Our recent work has reinforced the link between the tonic TCR signaling received by CD4 T_N cells in the steady-state and their fate in the effector phase. Indeed, we have demonstrated that TCR/self-MHC interactions not only increase quantitatively but also shape qualitatively the response of CD4 T_N cells to their cognate antigens in the effector phase (Martin et al., 2013). More precisely, by taking advantage of our data showing that Ly-6C expression can be considered as a new sensor of CD4 T_N-cell self-reactivity, we have demonstrated that CD4 T_N cells with the highest avidity for self-MHC (Ly-6C^-) have a biased commitment toward the iTreg/pTreg-cell lineage (Martin et al., 2013).

The binding of antigen/MHC complexes to the TCR triggers the recruitment of a series of signaling molecules and adaptors to the TCR/CD3 complex that ultimately results in the phosphorylation and activation of phospholipase C-γ (PLCγ). PLCγ then cleaves the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂) in the plasma membrane to generate diacylglycerol, which activates protein kinase C (PKC) and Ras-dependent pathways, as well as 1,4,5-inositol trisphosphate (IP₃), which induces the release of calcium (Ca²⁺) from intracellular stores (the endoplasmic reticulum (ER)). This Ca²⁺ store release only transiently elevates intracellular Ca²⁺ concentrations but this transient rise induces in turn a massive and sustained Ca²⁺ entry from the extracellular space (Hogan et al., 2010).

With the aim of deciphering the molecular mechanisms involved in the tonic TCR-signaling-mediated shaping of the CD4 T_N-cell compartment, we have focused on the TCR signaling cascade. By using transcriptomic and phenotypic approaches as well as in vitro and in vivo assays, we have identified the Ca²⁺ signaling pathway as key for the acquisition of both the phenotype of the most self-reactive CD4 T_N cells and their enhanced cell-intrinsic ability to commit into regulatory T cells upon activation in vitro (iTreg) and in vivo (pTreg).

In the steady-state, naive T cells continually recirculate between the blood, lymph and secondary lymphoid organs, scanning dendritic cells (DCs) for the presence of foreign antigens. In the course of their journey, naive T cells also make weak, but functional, interactions with self-peptides presented by self-MHC molecules (self-MHC). Such contacts with self-MHC are required for the long-term survival of peripheral naive T cells (Martin et al., 2006, 2003; Stritesky et al., 2012; Tanchot et al., 1997). The signals derived from the recognition of self-MHC by TCRs also allow maintaining naive T cells in a state of greater sensitivity for responses to foreign antigens (Dorfman et al., 2000; Stefanová et al., 2002). The seminal work of Štefanová et al. showed a rapid decline in the ability of CD4 T_N cells to respond to their cognate antigen once contacts with self-MHC were disrupted (Stefanová et al., 2002). These findings were confirmed by several groups using various elegant experimental models (Hochweller et al., 2010; Lo et al., 2009; Mandl et al., 2013; Persaud et al., 2014). Beside these works, we have recently demonstrated that CD4 T_N-cell self-reactivity not only increases quantitatively but also shapes qualitatively their response toward their cognate antigens in the effector phase by increasing their ability to commit toward the iTreg/pTreg-cell lineage (Martin et al., 2013). In the present paper, we first wondered whether the enhanced ability of the most self-reactive CD4 T_N cells to convert into iTreg/pTreg cells upon appropriate stimulation was a cell-intrinsic property. The unchanged ability of both Ly-6C^- and Ly-6C⁺ CD4 T_N cells to polarize into Foxp3-expressing iTreg cells in vitro whether they were cultured together or separately demonstrate that the biased commitment of the most self-reactive CD4 T_N cells toward the iTreg-cell lineage is cell-intrinsic.

We have recently described the cell surface GPI-anchored protein, Ly-6C, as an additional and complementary sensor of T-cell self-reactivity (Martin et al., 2013). However, significant differences may be noticed between CD5 and Ly-6C. First, whereas CD5 surface levels directly correlate with self-reactivity, Ly-6C expression by peripheral CD4 T_N cells inversely correlates with their ability to interact with self-MHC. Second, in contrast to CD5, Ly-6C expression at the cell surface of CD4 T_N cells is stable over time in homeostatic conditions as its up-regulation after self-MHC deprivation takes several days (Martin et al., 2013). Notwithstanding these differences, Ly-6C^- CD4 T_N cells express higher protein and mRNA levels of CD5 than their Ly-6C⁺-cell counterparts. Two recent papers by the group of Daniel Hawiger have highlighted a crucial role of CD5 in promoting the conversion of CD4 T_N cells into iTreg/pTreg cells (Henderson et al., 2015; Jones et al., 2016). CD5 would block the activation of the mammalian target of rapamycin (mTOR) and would allow activated CD4 T_N cells to resist to the inhibition of iTreg-cell induction induced by T_H1- and T_H2-cell-derived cytokines. Accordingly, in the absence of effector-differentiating cytokines, CD5^hi and CD5^lo CD4 T_N cells were shown to differentiate with a similar efficiency into iTreg/pTreg cells (Henderson et al., 2015). Such a phenomenon is unlikely to account for the greater ability of Ly-6C^- CD4 T_N cells to commit to the iTreg/pTreg-cell lineage. Indeed, Ly-6C^- and Ly-6C⁺ CD4 T_N cells produced similar amounts of these cytokines after stimulation and Ly-6C^- CD4 T_N cells still differentiated more efficiently than their Ly-6C⁺-cell counterparts into iTreg cells in vitro in the presence of anti-cytokine (IL-4 and IFN-γ) blocking antibodies (Figure 1—figure supplement 1C–E). Moreover, whereas rapamycin drastically diminished the difference in the ability of CD5^hi and CD5^lo CD4 T_N cells to convert to iTreg/pTreg cells in the presence of cytokines known as restraining this effector fate (Henderson et al., 2015), this mTOR inhibitor similarly enhanced the generation of iTreg cells from both Ly-6C^- and Ly-6C⁺ CD4 T_N cells and thus preserved the difference between these two cell subsets (data not shown).

In the present study, we have identified the Ca²⁺ signaling pathway as sufficient to induce Ly-6C down-regulation at the cell surface of CD4 T_N cells in vitro. Indeed, incubation of Ly-6C⁺ CD4 T_N cells with the sarco/endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, led to multiple phenotypic changes including not only Ly-6C down-regulation but also variations in the expression of many other genes of the 6CSign (such as CD5, CD73, CD122, CD200 and Izumo1r). This phenotypic conversion of Ly-6C⁺ CD4 T_N cells into Ly-6C^- CD4 T_N cells takes four days to occur in vitro and relies on the activity of Calcineurin, as shown by its complete blocking in the presence of Cyclosporin A or FK506. Interestingly, calcium- and PKC/Ras-dependent signaling pathways had divergent effects on the expression of Ly-6C. Indeed, whereas TG induced Ly-6C down-regulation, PMA led to its upregulation (Figure 4A). In line with this observation, the 6CSign does not correlate with the changes in gene expression induced by PMA (Figure 3B). These opposite effects of TG and PMA may reflect the well-documented and complex interplay between the PKC and Ca²⁺ signaling pathways. For example, PKC translocation to the plasma membrane is strictly Ca²⁺ dependent (Reither et al., 2006) and calcineurin is phosphorylated and inhibited by PKC (Hashimoto and Soderling, 1989). Altogether, our results suggest that interactions with self-MHC in the steady-state result in a dominant Ca²⁺ signaling (when compared to PKC and Ras-dependent pathways) leading to down-regulation of Ly-6C expression. This hypothesis is consistent with our results showing that in vivo Calcineurin inhibition leads to an increase in Ly-6C expression at even higher levels than those observed at the cell surface of Ly-6C⁺ CD4 T_N cells from untreated mice. This hypothesis is reinforced by the work of Dong et al. (Dong TX et al., co-published with the present article) showing that Ca²⁺ fluxes can be measured in mouse total lymph node T cells in the steady-state and that anti-MHC blocking antibodies significantly reduced their frequency. Continuous interactions with self-MHC in the steady-state may thus induce calcium waves that shape both the phenotype of CD4 T_N cells and their behavior in the effector phase by favoring their differentiation into pTreg cells.

tTreg and pTreg cells have complementary roles in immune-mediated tolerance (Haribhai et al., 2011). An attractive hypothesis would be that tTreg cells would be responsible for tolerance to self-antigens, whereas pTreg cells would be in charge of restraining deleterious immune responses to non-self-antigens. In particular, pTreg cells are involved in the control of the responses to non-self-antigens leading to allergy and asthma (Josefowicz et al., 2012) as well as to commensal organism- (Lathrop et al., 2011) and food-derived antigens (Mucida et al., 2005) in the gut. Foetus-derived and allograft-derived antigens represent other obvious examples of acute exposure to non-self-antigens arising in the adults and requiring the establishment of a tolerance. In both cases, pTreg cells are generated against non-self antigens (either conceptus-male-derived [Samstein et al., 2012] or allograft-derived [Francis et al., 2011; Wood et al., 2012]). These cells are needed to establish an efficient tolerance toward the foetus (Samstein et al., 2012). However, there is still a lack of evidence to definitely implicate pTreg cells in the induction of an efficient tolerance toward allograft, in part because of the difficulties to achieve such a state. We have previously demonstrated that self-reactivity in the steady-state increases the ability of CD4 T_N cells to differentiate into iTreg/pTreg cells (Martin et al., 2013). Accordingly, the most self-reactive CD4 T_N cells (i.e. Ly-6C^- CD4 T_N cells) should contribute predominantly to the pTreg-cell pool generated under physiologic and pathologic conditions. In the present study, our data suggest strongly that this tonic TCR-signaling-mediated shaping of the CD4 T_N-cell compartment is calcineurin-dependent. In particular, chronic treatment with a calcineurin inhibitor leads to the disappearance of Ly-6C^- CD4 T_N cells. Cyclosporin A and Tacrolimus treatments could thus interfere with the neoconversion of CD4 T_N cells into pTreg cells and limit the development of tolerance in transplant patients. This may explain the difficulty to safely interrupt these immunosuppressive therapies even after years. Thus, besides their obvious clinical utility, calcineurin inhibitors may have potentially harmful side effects that should be studied to better assess and adapt their use.

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Author details

1. Vincent Guichard

   1. Institut Cochin, Paris Descartes Université, CNRS UMR8104, INSERM U1016, Paris, France
   2. Paris Diderot Université, Paris, France

   Contribution

   Conceptualization, Formal analysis, Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0206-4924

2. Nelly Bonilla

   Institut Cochin, Paris Descartes Université, CNRS UMR8104, INSERM U1016, Paris, France

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

3. Aurélie Durand

   Institut Cochin, Paris Descartes Université, CNRS UMR8104, INSERM U1016, Paris, France

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

4. Alexandra Audemard-Verger

   Institut Cochin, Paris Descartes Université, CNRS UMR8104, INSERM U1016, Paris, France

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

5. Thomas Guilbert

   Institut Cochin, Paris Descartes Université, CNRS UMR8104, INSERM U1016, Paris, France

   Contribution

   Software, Investigation

   Competing interests

   No competing interests declared

6. Bruno Martin

   Institut Cochin, Paris Descartes Université, CNRS UMR8104, INSERM U1016, Paris, France

   Contribution

   Funding acquisition, Validation, Investigation

   Competing interests

   No competing interests declared

7. Bruno Lucas

   Institut Cochin, Paris Descartes Université, CNRS UMR8104, INSERM U1016, Paris, France

   Contribution

   Conceptualization, Data curation, Supervision, Funding acquisition, Project administration, Writing—review and editing

   Contributed equally with

   Cédric Auffray

   Competing interests

   No competing interests declared

8. Cédric Auffray

   Institut Cochin, Paris Descartes Université, CNRS UMR8104, INSERM U1016, Paris, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   Contributed equally with

   Bruno Lucas

   For correspondence

   cedric.auffray@inserm.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1012-0132

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