We have the advantage to be able to induce monophasic and relapsing uveitis in the same strain of rats by the same immunization method, the only difference being the antigen peptide. Thus, T cells from both disease courses can easily be compared. Since it is almost impossible to obtain rat T cell clones we used highly specific T cell lines after at least two restimulations in vitro with the respective peptides. These cell lines consist of more than 90% CD4+ T cells. mRNA from three different T cell lines of each antigen specificity were separately analyzed by microarrays, followed by quantitative real time polymerase chain reaction (qPCR) confirmation with another 6- 8 different T cell lines of each specificity.
Microarray and qPCR revealed upregulation of several genes in R14-specific T cells inducing relapsing disease. Only gene expression of at least twofold change compared to PDSAg-specific T cells was considered for the analysis. Surprisingly, there was no downregulation of genes detected in R14-specific T cells inducing relapsing EAU compared to PDSAg-specific T cells inducing monophasic EAU. Most of the upregulated genes are involved in mechanisms that lead to prolonged survival and activation of T cells. This indicates that T cells from monophasic and relapsing disease are basically equipped with the same features, but T cells inducing relapsing disease show a stronger expression of genes associated with regulatory pathways of antigen presentation and activation as well as the WNT/Hedgehog (HH) pathways. We could define 26 upregulated genes in R14-specific T cell lines, belonging to different signaling pathways that are connected with each other (Figure 7).
Figure 7 Signal transduction pathways of regulated genes in R14-specific T cells. Transcriptomic profiling of PDSAg- and R14-specific T cells resulted in the detection of upregulated genes in R14-specific T cells. These genes participate in different intracellular signaling pathways such as WNT, Hedgehog (HH), mitogen-activated protein kinase (MAPK) and antigen presentation pathway. * indicates significantly upregulated genes. (Courtesy of Gerhild Wildner).
WNT/HH signaling pathways
Many of the significantly upregulated genes contribute to the WNT and to the HH signaling pathway, which are as well interconnected. Both signaling pathways are evolutionarily conserved and use several related or even identical factors. They do not only play important roles in embryogenesis, but also contribute to proliferation, cell polarity, cell movement and survival of cells, which also explains their participation in cancer (Logan and Nusse, 2004; Lum and Beachy, 2004). WNT signaling is involved in induction of long-lived self-renewing populations of memory T cells (Zhao et al., 2010). In autoimmune responses, memory T cells play a central role. Among the significantly upregulated genes in R14-specific T cells E-cadherin, connector enhancer of kinase suppressor of ras 3 / membrane-associated guanylate
E C a d * WNT N o tc h Jn k 2/ Map k 9 Ip 10/ Cx cl 1 0 Jak 2 JAK/ STAT Wn t 5b MAPK Casp 1* Tg m 2 WNT-PCP AP1 Nq o1 IFN-γ Fo xp 3* Cd 74 MIF-Recept or Ct sh Rt 1.Da* Rt 1.Ba Rt 1.Db Hl a-d m b Cn k sr 3/ Mag i 1* HH Gl i 1* Ral g p s2 Casp 3* D e lt a -l ik e 1 Ja g 1 β- Cat en i n An t i g en p r esen t at i on Gzm g / CTLA-7* Cell membrane Cytoplasm Act g 2 Ral A Ral GTP Rab 27a* Il 4r Ct sc IFN-γ Recep t or IL-18 p 73 St at 1 E C a d * upregulated mRNA and protein upregulated tested, but not different not tested only protein expression tested
kinase-interacting protein-like 1, glioma-associated oncogene homolog 1 and caspase 3 (Ecad, Cnksr3/Magi1, Gli1 and Casp3) contribute to the WNT and HH pathways. E-cadherin is a transmembrane protein and is important for cell adhesion. Its cytoplasmic domain is linked via β-catenin to CNKRS3/MAGI1, which is also significantly increased in R14-specific T cells. The cleavage of E-cadherin or CNKRS3/MAGI1 by caspase 3, which is also upregulated in R14-specific T cells, leads to the release of β-catenin that can translocate to the nucleus and act as a transcription factor. Another transcription factor Gli1, which is significantly increased in R14-specific T cells, is part of the HH signaling pathway that suppresses apoptosis and induces proliferation of activated CD4+ T cells (Lowrey et al., 2002). The upregulation of these genes in T cells capable of inducing relapsing disease could be one explanation for their prolonged survival in the eye, even after resolution of the first attack of disease and their ability to become reactivated and induce relapses. By contrast, PDSAg-specific T cells that induce monophasic disease show no upregulation of genes involved in WNT and HH signaling pathways and could not be detected in high numbers in the eyes after resolution of EAU.
Antigen presentation
A further group of upregulated genes including RT1 class II locus DA, CD74, cathepsin H and MHC class II DM beta (Rt1.Da, Cd74, Ctsh and Hla-dmb) could be grouped together as they are related to antigen presentation. Rt1.Da is a rat MHC class II molecule, equivalent to human HLA-DR and mouse I-E, and is additionally the restriction element of R14-specific T cells. CD74, CTSH and HLA-DMB are involved in peptide loading onto MHC class II molecules. Initially, it seems uncommon that T cells upregulate genes important for antigen presentation. However, this phenomenon was already observed in humans as well as in rats and is associated with T cell activation (Gansbacher and Zier, 1989; Taams et al., 1999). In addition, it could be demonstrated that T cells expressing MHC class II molecules acquire B7 costimulatory molecules from APCs and can therefore act as antigen presenting cells (Sabzevari et al., 2001). This mechanism may be important for the amplification of the specific immune response. In case of the R14-specific T cell line, this could be a mechanism allowing long-lasting immune responses and could probably be important for reactivation of T cells during relapses. However, memory T
cells, which acquire B7 costimulatory molecules, die by apoptosis (Sabzevari et al., 2001). Moreover, expression of MHC class II molecules on T cells is also associated with T cell anergy. If T cells are activated by antigen presenting T cells, they cannot be restimulated by professional APCs and become anergic (Taams et al., 1999). This could lead to the termination of the immune response and to the resolution of disease. In case of MHC class II expressing R14-specific T cells one could speculate that they may present antigen to regulatory T cells and subsequently anergize them, thus escaping from regulation. On the other hand, it is also possible that anergic effector T cells become reactivated and induce relapses.
CD74 has dual roles in the immune response. Besides its role as invariant chain (Ii), which blocks the peptide binding groove of MHC class II molecules to prevent peptide binding in the ER, CD74 can also be expressed on the surface of T cells and act as a receptor for MIF. In the latter function it induces the expression of pro- inflammatory genes and is involved in proliferation and inhibition of apoptosis (Leng et al., 2003). This emphasizes the pro-inflammatory role of R14-specific cells in relapsing disease as well.
IFN-γ signaling pathway
Another gene possibly contributing to the pro-inflammatory status of R14-specific T cells is Casp1. The protease Caspase-1 processes IL-1β and IL-18 from their inactive pro-cytokines. IL-18 was originally discovered as an IFN-γ inducing factor (Okamura et al., 1995); however, it has pleiotropic effects and can also induce TH2
(Nakanishi et al., 2001) and TH17 responses (Lalor et al., 2011).
IFN-γ, which is upregulated in R14-specific T cells on the mRNA as well as protein level, is the signature cytokine of TH1 cells. It induces interferon gamma-induced
protein 10 / interferon-inducible cytokine 10 (IP-10/CXCL10) that is also increased in R14-specific T cells and functions as a chemoattractant for monocytes/macrophages, T cells and NK cells. It also promotes T cell adhesion to endothelial cells and thus may facilitate the traffic through the blood-retina-barrier, potentially explaining the earlier onset of R14-induced as compared to PDSAg-mediated EAU. IP10/CXCL10 acts via Janus Kinase 2 (JAK2), which is upregulated in R14-specific T cells and is an important component of the JAK/STAT (signal transducer and activator of
transcription) pathway. JAK2 is critical for IL-12 signal transduction and TH1 cell
differentiation (O'Shea and Plenge, 2012).
Foxp3 expression
Most of the upregulated genes are closely connected with inflammation or proliferation. An exception is Foxp3, which is the master transcription factor of regulatory T cells (Fontenot et al., 2003). However, in addition to its expression in regulatory T cells, Foxp3 is also expressed in activated T effector cells in humans, but only transiently and at a lower level than in regulatory T cells (Allan et al., 2007). The expression of Foxp3 in R14-specific T cells lines could thus rather represent the activated status of the T cells and thus do not necessarily indicate a regulatory phenotype.
Regulated genes with unexplained functions
Some of the upregulated genes are involved in the release of cytotoxic granules, such as granzyme G, cathepsine C and ras-related protein 27A (Gzmg, Ctsc and Rab27a). However, their exact functions in T helper cells have not been completely elucidated.
In principle, all of the upregulated genes support the pro-inflammatory role of R14- specific T cells and are probably the determining factors for induction of relapses in R14-induced EAU, in contrast to the monophasic EAU-inducing PDSAg-specific T cells, where these genes were not upregulated.