The activation of CD4⁺ T lymphocytes requires assistance of antigen-presenting cells (APCs) such as dendritic cells and macrophages. APCs harvest antigens from the environment, process them to peptides and present them on the cell surface in a complex with MHC class II molecules. Binding of peptide-MHC complex to the T-cell receptor (TCR) triggers a phosphorylation cascade (e.g. Lck, Zap70, LAT) and activates phospholipase C (PLCg). This eventually leads to the release of intracellular Ca²⁺ from the endoplasmic reticulum (ER) and Ca²⁺ influx into the cytosol of T cells through CRAC channels in the plasma membrane. Raising the calcium level in the cytosol of T cells activates the phosphatase calcineurin which dephosphorylates the nuclear factor of activated T cells (NFAT) protein. This translocates NFAT to the cell nucleus, where it binds to specific promoters and activates the transcription of target genes (e.g. IL-2, IFNg, etc.), which is essential for differentiation of T cells and execution of their effector functions.

The N-terminal portion of NFAT fused to a fluorescent protein can be expressed in effector T cells by means of retroviral transduction. This enables live monitoring of the activation process using time laps epifluorescence microscopy (animation 1). The video shows dendritic cells (red), derived from bone marrow of RFP-transgenic host, presenting myelin basic protein to MBP-specific T_{NFAT-YFP/CherryH2B} cells. The contact of a T cell with an antigen-presenting cell (APC) induces rapid nuclear translocation of NFAT into the nucleus (yellow).

Meningeal and perivascular macrophages are the first APCs encountered by T cells crossing the blood brain barrier and entering the perivascular/meningeal space. These cells are of myeloid lineage and can be visualized in bone marrow-chimeric animals reconstituted with GFP-expressing hematopoietic stem cells (animations 2 & 3). In contrast to microglia, meningeal macrophages express a high level of MHC class II molecules on their surface. Meningeal macrophages, regarded also as phagocytes, efficiently take up macromolecules (e.g. fluorescent dextran (animations 3 and 4)) and soluble antigens. This property can be exploited to label meningeal APCs in vivo and to visualize their interaction with T cells by intravital 2-photon imaging of the spinal cord.

The transfer of MBP-specific T cells results in an induction of EAE disease in Lewis rats. If these T-cells express the double fluorescent NFAT/H2B reporter, they can be visualized in the meninges of the spinal cord shortly before the onset of clinical symptoms (animation 4), where they arrive from the blood circulation. Extravasated T cells crawl along pial vessels and meet meningeal phagocytes there. Upon contact of non-activated T cells (gray arrow) to dextran-TxR-labelled phagocytes, NFAT translocates to the nucleus (marked by yellow circle). This allows real time quantification of the frequency of T cell activation events in situ.

Antigenic receptors expressed on the surface of T cells are encoded by specialized sets of genomic segments that are assembled as functional units via V(D)J recombination during T cell development.

In a normal immune repertoire only few T lymphocytes are specific for a given antigen. Genetic engineering has been used to generate mice with monospecific T cell repertoires. These T cell receptor transgenic mouse strains have become extremely useful tools in immunological research. We are working on engineering transgenic Lewis rats expressing T-cell receptors (TCRs) of different specificities including autoantigens expressed in nervous tissue. To identify coding sequences for TCR alpha and beta chains, clonal population are generated using T cell culture techniques. These sequences are then integrated into lentiviral expression vectors and tested in TCR-negative hybridoma cell lines for their responsiveness to antigens. Viruses with functional TCR are used to perform transgenesis in preimplantation rat embryos to  generate transgenic strains.

Recently, using these techniques, we were able to generate Lewis rats which express a TCR specific for rat beta-synuclein protein (Lodygin, Hermann et al., 2019). With this rat model we could show that T cells which recognize a neuronal autoantigen (b-Synuclein) trigger inflammation in the grey matter of the CNS. In contrast, myelin-specific T cells trigger inflammation in the white matter of the CNS. Additionally, we could show in our transgenic animals that repeated attacks from CD4 T-effector cells evoke permanent gliosis, neuronal damage and cortical brain atrophy. This new animal model is therefore suitable for studying mechanisms in CNS autoimmune diseases that involve progressive tissue damage.

Engagement of immunoreceptors, such as TCR, results in an increase in the concentration of Ca²⁺ ions in the cytosol. This elevation in Ca²⁺ in turn activates several transcription factors including NFAT, CREB and NF-kappaB, which regulate the expression of genes essential for T‑cell function. The increase in intracellular Ca²⁺ levels in lymphocytes is mediated mainly through store-operated calcium entry (SOCE) and calcium-release-activated calcium channels (CRAC).

The endoplasmic reticulum (ER) and endosomes/lysosomes are intracellular stores of Ca²⁺. The ER in lymphocytes is relatively small, so that the release of Ca²⁺ from the ER to the cytosol results in a moderate and transient increase of cytoplasmic Ca²⁺ concentration. The concomitant depletion of the calcium stores activates CRAC channels in the plasma membrane and leads to a massive influx of extracellular Ca²⁺.

The depletion of Ca²⁺ stores can be triggered by several means, e.g. by second messengers, such as inositol-1,4,5-triphosphate (InsP3), cyclic ADP ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). These second messenger molecules are generated upon activation of the T-cell receptor and signal transduction by tyrosine kinases. Recently, we have identified BZ194 as an NAADP inhibitor which interferes with Ca²⁺-mediated signaling during the early phase of T-cell activation (Dammermann et al., PNAS 2009). Both active and passive EAE in Lewis rats were ameliorated by preventive and therapeutic treatments with BZ194, indicating that the NAADP/Ca²⁺ pathway may be a novel target for the treatment of autoimmune disorders (Cordiglieri et al., 2010).