Self-mediated shaping of naïve CD4 T cell effector fate – Self-shaped-CD4_TN

This project is divided into four distinct parts:
• The first aim will focus on the interactions of CD4 TN cells with their cellular partners in secondary lymphoid organs. We will address whether they differ depending on the Self-reactivity capacity of CD4 TN cells. To achieve this task, we will first document the localization of Ly-6C- and Ly-6C+ CD4 TN cell subsets within lymph nodes by multi-color immunofluorescence microscopy. Productive interactions will be visualized and quantified thanks to the appearance of intracellular Ca2+ fluxes or to the nuclear translocation of NFAT within CD4 TN cells.
• The second aim relies on new preliminary data demonstrating that a sustained intracellular Ca2+ increase is sufficient to induce the phenotypic conversion of Ly-6C+ CD4 TN cells into Ly-6C- CD4 TN cells, through a Calcineurin (CN) dependent pathway. The transcriptional signature of Ly-6C- and Ly-6C+ CD4 TN cells will be obtained by deep RNA sequencing. The Ca2+-dependent and CN-dependent components of this signature will also be determined. This analysis will give us key information about the genes regulated by tonic TCR signaling in CD4 TN cells.
• The third aim is to identify the molecular mechanisms involved in the greater iTreg-cell potential of Ly-6C- CD4 TN cells when compared to Ly-6C+ CD4 TN cells. We will first document the activation of a comprehensive set of signaling pathways by analyzing the phosphorylation status of key molecules at different time points of our robust in vitro iTreg-cell differentiation assay. In parallel, the candidate genes identified as dependent on both Self-reactivity and Ca2+ / CN signaling (Aim #2) will be tested for their influence on iTreg-cell generation.
Finally, the fourth aim will point to validate in vivo the results of the three first tasks. We will investigate the possibility to modulate iTreg-cell generation in vivo in three experimental models.

We have focused on the molecular mechanisms involved in the tonic TCR signaling mediated shaping of the CD4 TN-cell compartment. We have first compared the transcriptomic profile of the 2 CD4 TN-cell subsets that can be defined on the basis of Ly-6C expression. In-depth analysis of these profiles confirms the role of the TCR signaling pathway in the molecular signature of the most Self-reactive CD4 TN cells (Ly-6C-). We have then identified the calcium-Calcineurin signaling cascade as key for the acquisition of both the phenotype of Ly-6C- CD4 TN cells and their enhanced cell-intrinsic ability to commit into iTreg cells upon activation in vitro and in vivo.
The enhanced ability of the most autoreactive CD4 TN cells to commit to the iTreg-cell lineage may represent an efficient mechanism of Self-tolerance. Indeed, this would diminish the risk for an autoreactive CD4 TN-cell clone to differentiate into a deleterious effector cell in the course of an immune response. The crucial role of the calcium-Calcineurin signaling cascade in this education process should be taken into account. Indeed, we show that chronic treatment with a Calcineurin inhibitor leads to the disappearance of Ly-6C- CD4 TN cells. These inhibitors, widely used in transplant patients, could thus interfere with the neoconversion of CD4 TN cells into iTreg cells. This could potentially limit the installation of an efficient tolerance to the graft and 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 further studied to better assess and adapt their use.

The phenotype of the most Self-reactive CD4 TN cells is maintained over time through chronic interactions with Self-MHC molecules. We will focus on the identification of the tonic TCR signaling partners of (Ly-6C-) CD4 TN cells. To achieve this aim, we will first analyze the distribution of Ly-6C- and Ly-6C+ CD4 TN cells within secondary lymphoid organs in the steady state. As bone marrow chimeras experiments indicate that the MHC-II expressing cells required to maintain the Ly-6C- CD4 TN cell phenotype are of hematopoietic origin, we will focus our efforts on these cells.
Lymph nodes are encapsulated bean-shaped lymphoid organs that are subdivided into three main regions: the cortex, the paracortex and the medulla. Naive (T and B) lymphocytes enter the lymph node via high endothelial venules (HEVs) or afferent lymphatic vessels, and exit through cortical sinuses, medullary sinuses and the efferent lymphatic vessel in the medulla. Among putative tonic TCR signaling partners for CD4 TN cells, DCs represent the most obvious candidates. However, extrafollicular localization of B cells within the T cell zone has been described as well as their persistence around HEVs in close contact with transmigrated T cells. It was also shown that CD4 TN cells accumulate in LN egress zones (medullary cord and medullary sinuses) in the vicinity of macrophages. These three MHC-II expressing cell types, DCs, B cells and macrophages, are thus localized within zones where they could frequently interact with CD4 TN cells in the steady state.
Our recently published data strongly suggest that the ability of a CD4 TN cell to commit into the iTreg-cell lineage pathway upon appropriate stimulation is shaped by its capacity to interact with Self-MHC in its original environment. We will first compare the cellular response of both Ly-6C- and Ly-6C+ CD4 TN cells to iTreg cell promoting conditions. Secondly, candidate molecules will be targeted to modulate iTreg cell generation.

An article describing our recent results is currently submitted for publication.

T cell precursors originate in the BM and are educated in the thymus through processes called positive and negative selections, which result in MHC-restriction and self-tolerance, respectively. Only those T cells that bear an aß T 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 aß TCR that reacts with self-MHC with very low affinity die by neglect, whereas those that recognize self-MHC with high affinity are mostly deleted by apoptosis or differentiate into regulatory T cells called "natural" (nTreg) in order to prevent autoimmunity.

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 naïve T cells in interphase through synthesis of anti-apoptotic molecules such as Bcl-2.

In the absence of foreign antigen, peripheral naive T cells continuously recirculate between lymphoid organs, in which they interact frequently and shortly with antigen presenting cells (APC). The resulting tonic TCR signaling received by CD4 T cells influence their effector fate by increasing quantitatively their responsiveness towards their cognate antigens.

Following activation by antigen presenting cells (APCs) in the periphery, the bulk of naïve CD4 T cells (CD4 TN cells) can differentiate into a variety of well documented T-helper (TH) cell subsets, such as TH1, TH2, TH17 or induced regulatory T cells (iTreg), characterized by their cytokine production profiles and specific effector functions. The immunological context in which CD4 TN cells are immersed at the time of their activation is known to drive this lineage commitment. Among these effector CD4 T-cell subsets, iTreg cells, whose generation is governed by TGFß, are of particular interest. Indeed, iTreg cells share phenotypic and functional characteristics with nTreg cells that play a crucial role in maintaining peripheral self-tolerance.

Our recent works reinforce the link between the tonic TCR signaling that CD4 TN cells receive in the steady state and their fate in the effector phase. We have demonstrated that TCR/self-MHC interactions not only increase quantitatively but also shape qualitatively the response of CD4 TN cells to their cognate antigens in the effector phase. Indeed, we have demonstrated that CD4 TN cells with the highest avidity for self-MHC have a biased commitment toward the iTreg cell lineage.

Altogether, our recent results suggest that continuous / chronic interactions with self-MHC shape both the phenotype of CD4 TN cells and their behavior in the effector phase by favoring their differentiation into iTreg cells. We now plan to extend this study by:
1- Identifying the tonic TCR signaling partners
2- Deciphering the molecular pathways initiated by tonic TCR signaling.
3- Identifying molecules involved in the higher sensitivity of the most self-reactive CD4 TN cells to iTreg cell polarization signals.
4- Identifying targets to modulate iTreg cell polarization potential in vivo in different pathological conditions.