Insights into Gene Expression of Activated Pathogenic Autoimmune T Cells-Studies in Experimental Multiple Sclerosis-like Model

Multiple Sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS) and the leading cause of paralysis in young adults [1]. The cause of MS is not well defined but it is acknowledged that pro-inflammatory, self-myelin-reactive clones of CD4+ T cells play a critical role in MS pathology and progression [2,3]. These myelin-specific T cells are primed by antigen presenting cells (APC) to target the subject own myelin. Indeed, an immunization with myelin peptides or myelin containing particles induces T cell-driven MS-like pathology in animals, defined as experimental autoimmune encephalomyelitis (EAE), a common model of MS-like condition [4,5]. Memory T cells, despite their low quantity, exhibit enhanced proliferation, high cytotoxic effector functions, prolonged lifespan and re-activation in response to self-antigens. These distinct features are controlled by unique pattern of signaling pathways and reprogrammed gene expression [6]. It was observed that autoreactive memory T cells secrete interleukin(IL)-17 cytokine via retinoic acid receptorrelated orphan receptor γt (RORγt)/signal transducer and activator of transcription 3 (STAT3)-dependent manner and were therefore defined as Th17 phenotype [7-9]. Adoptive transfer of such autoreactive encephalitogenic Th17 cells to healthy animals results in rapid and severe EAE defined as passive EAE [4,5]. Indeed, T lymphocytes of the Th17 phenotype were found to be present in MS patients as well as in *Corresponding author: Ewa Kozela, The Dr Miriam and Sheldon G. Adelson Center for the Biology of Addictive Diseases, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel, Tel: +972 584543248; E-mail: kozelae@post.tau.ac.il or ewa.kozela@weizmann.ac.il


Introduction
Multiple Sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS) and the leading cause of paralysis in young adults [1]. The cause of MS is not well defined but it is acknowledged that pro-inflammatory, self-myelin-reactive clones of CD4 + T cells play a critical role in MS pathology and progression [2,3]. These myelin-specific T cells are primed by antigen presenting cells (APC) to target the subject own myelin. Indeed, an immunization with myelin peptides or myelin containing particles induces T cell-driven MS-like pathology in animals, defined as experimental autoimmune encephalomyelitis (EAE), a common model of MS-like condition [4,5]. Memory T cells, despite their low quantity, exhibit enhanced proliferation, high cytotoxic effector functions, prolonged lifespan and re-activation in response to self-antigens. These distinct features are controlled by unique pattern of signaling pathways and reprogrammed gene expression [6]. It was observed that autoreactive memory T cells secrete interleukin(IL)-17 cytokine via retinoic acid receptorrelated orphan receptor γt (RORγt)/signal transducer and activator of transcription 3 (STAT3)-dependent manner and were therefore defined as Th17 phenotype [7][8][9]. Adoptive transfer of such autoreactive encephalitogenic Th17 cells to healthy animals results in rapid and severe EAE defined as passive EAE [4,5]. Indeed, T lymphocytes of the Th17 phenotype were found to be present in MS patients as well as in several other T cell driven autoimmune conditions in humans [10].
Antigen re-activation of quiescent, circulating memory T cells may contribute to MS relapses [11]. Targeting these activated memory T cells appears to be an efficient strategy in MS treatment [12]. However, this approach is still challenging due to the difficulty in distinguishing autoreactive T cells from their non-pathogenic counterparts. Thus, further mapping of T cell specific pathways and co-regulated genes are of high importance.
In our current study, we analyzed the changes in gene expression following antigen-specific re-activation of autoreactive T cells. For this purpose, we used a mouse memory myelin oligodendrocyte glycoprotein (MOG)35-55-specific CD4 + T cell line (T MOG ) isolated from MOG35-55-immunized EAE C57BL/6 mice [13][14][15]. Previously, we showed that these T MOG cells produce and secrete IL-17 cytokine in response to MOG35-55 stimulation [16]. IL-17 production was accompanied by increased STAT3 phosphorylation [17] confirming self-antigen-induced Th17-like activity of these T MOG cells. Here we performed whole gene expression analysis of the activated T MOG and Volume  identified the main pathways and gene networks that are driving their acute activation and effector functions. Mapping these pathways and affected genes could lead to better understanding of the processes taking place in autoimmune diseases and eventually to better treatment.

Reagents
Lyophilized MOG35-55 peptide [MEVGWYRSPFSRVVH-LYRNGK] purchased from GenScript (Piscataway, NJ, USA) was reconstituted in sterile PBS and the stock solution stored in aliquots at -20°C. The working MOG35-55 solutions were prepared fresh just before the experiments. Fetal calf serum (FCS) and other tissue culture reagents were obtained from Biological Industries (Kibbutz Beit HaEmek, Israel).

T MOG stimulation and CD4+ microbead purification from APC/T MOG co-cultures
The MOG35-55-specific T MOG cell line was isolated and maintained as described earlier [14][15][16]. T MOG were stimulated with MOG35-55 in the presence of mouse spleen-derived APC as described before [16,17] with few modifications. Freshly isolated APC (50 × 10 6 cells/10 cm plate) were pre-adhered for 2 h at 37°C, washed with PBS containing Ca ++ and Mg ++ to remove floating cells and then co-cultured with 2.5 × 10 6 T MOG cells. The APC/T MOG co-cultures were stimulated with 5 µg/ml of MOG35-55. After 8 h of incubation, the media containing mostly T MOG cells (but not the adherent APC cells) were carefully collected and the T MOG cells further purified using anti-CD4 (L3T4) magnetic beads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) as described earlier [17]. As shown by us before, CD4 + column purification allows the separation of the T MOG cells from any residual APC cells while preserving the antigen specificity and antigen-induced Th17-function of the T MOG cells [16]. mRNA isolated from these purified T MOG cells was subjected to global mRNA expression analysis using Illumina microarray as described below.
The MOG35-55 incubation time of 8 h was chosen based on previous time-and dose-response experiments, including MOG35-55induced T MOG proliferation, IL-17 cytokine production and release as well as intracellular pathways' activation [16][17][18].

RNA extraction and microarray transcript analysis
Purified T MOG cells were lysed and subjected to RNA extraction (5Prime, Darmstadt, Germany) as described earlier [17,19]. For comparative microarray analysis, 200 ng samples of total RNA were amplified, labelled and hybridized onto Illumina MouseRef-8 v 2.0 Expression Bead-Chip, querying the expression of 25,600 RefSeqcurated gene targets and 796 random sequences used for the assessment of background noise. Arrays were processed and scanned with Illumina BeadStation platform according to the manufacturer's protocol. Raw data were log2 transformed and normalized using quantile normalization.
Statistical microarray analysis and gene expression analysis of the raw data were performed at the Informatics Center for Neurogenetics and Neurogenomics core at UCLA using R scripts (www.r-project. com) and Bioconductor packages (http://www.bioconductor.org; [20]) as described before [21].
Pathway and global functional analyses were performed using QIAGEN's Ingenuity Pathway Analysis (IPA®, QIAGEN Redwood City CA, USA www.qiagen.com/ingenuity). A data set containing gene identifiers and corresponding expression values was uploaded into the application to identify the biological functions as well as the pathways, gene networks and upstream regulators from the IPA library that were most significantly affected by the treatment with MOG35-55 as compared to control, purified, not stimulated T MOG cells. Genes that met the p-value cutoff of 0.05 (using Fisher's exact test) were used to build the network plots using the IPA tool.

qPCR analysis
Selected gene products found by microarray analysis to be affected by MOG35-55 stimulation (as compared to unstimulated, purified T MOG ) were validated by qPCR as described [17,19] using β2microglobulin (β2MG) gene product for normalization. Supplementary Table S1 presents the list of specific primers used. The qPCR runnings were repeated 3-4 times using mRNA preparations from independent experiments and analyzed statistically using t test.

Study approval
Mice used in this study were maintained according to the guidelines of the Institutional Animal Care and Use Committee (IACUC). Animal experiments were approved by the Weizmann Institute of Science and the Tel Aviv University IACUCs.

Identification of functional networks in activated T MOG
MOG35-55 significantly affected genes were uploaded onto the IPA analysis program to identify the main gene networks associated with diseases, functional subsets, pathways and upstream regulators. Figure   1 shows the IPA-identified functional categories that were the most significantly (p<0.005) enriched following MOG35-55 stimulation. Supplementary Figure S1 complements this list with functional categories falling into lower significance values (between p>0.005  Figures 1 and S1 links the MOG35-55 affected genes to several known autoimmune pathologies such as rheumatoid arthritis and type I diabetes (p<0.005, Figure 1) as well as systemic lupus erythematosus and multiple sclerosis (p<0.05, Figure S1) suggesting prevailing autoimmune type of inflammatory activation. Furthermore, among the IPA-identified categories we find recurring annotations related to Th17 activity. These annotations include "IL-17A Signaling in Fibroblasts", "Role of IL17F in Allergic Inflammatory Airway Diseases" and "IL-17A Signaling in Gastric Cells" (at p<0.005) as well as "IL-17 Signaling", "Role of IL-17A in Arthritis", "Differential Regulation of Cytokine Production in Macrophages and T Helper cells by IL-17A and IL-17F" and "IL-17A Signaling in Airway Cells" (at p>0.005-p<0.05) suggesting a dominant Th17 transcriptional mode of the activated T MOG cells. In parallel, several pathways related to other cytokines were also recognized as significantly enriched in activated T MOG . These include: IL-1, IL-6, IL-8, IL-10, IL-12, IL-15 and their cognate receptors such as Lymphotoxin β Receptor, TNFR2, (p<0.005) followed by IL-2, IL-9 and IGF1 signaling (p<0.05). IPA analysis highlighted the role of iNOS, MAPK, JAK/STAT, PKCθ, ILK and NFκβ signaling, pathways that are known to be downstream of 4-1BB (Tnfrsf9), CD28, April (Tnfsf13), CD27, CD40, TREM1 and RIG1-like receptors, in MOG35-55 stimulated T cells (p<0.005). Several pathways related to antigen presentation were recognized by IPA to be enriched following MOG35-55, emphasizing the role of dendritic cells (the most competent APC) and B cells (the major type of APC in the periphery) in the process of T MOG stimulation. This includes "Cross Talk Between Dendritic and Natural Killer Cells", "Altered T Cell and B Cell Signaling in Rheumatoid Arthritis", "B Cell Activating Factor Signaling" and "Dendritic Cell Maturation". Interestingly, the IPA identified a substantial number of pathways that relate to a crosstalk between innate and adaptive immunity as being involved in MOG35-55 stimulation. These include "Activation of IRF by Cytosolic Pattern Recognition Receptors", "Role of PKR in Interferon Induction and Antiviral Response", "Role of RIG1-like Receptors in Antiviral Innate Immunity", "Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses", "Toll-like receptor signaling" and "LPS Stimulated MAPK Signaling".

Main gene networks affected by MOG35-55 stimulation
The genes significantly affected by MOG35-55 stimulation were used by IPA analysis to build gene networks that are involved in MOG35-55 activation of encephalitogenic T cells (see Figures 2-9 and supplementary Figures S2-S5). These IPA-constructed networks include the genes that were significantly affected (p<0.05) by MOG35-   Figure S3), between IFNγ, IL-2 and TLR3 signaling pathways ( Figure S4) and between STAT4, TNFα and IFNγ ( Figure S5). It is worth noting that the genes comprising the interactomes presented in Figure  S3, and Figure S4 (TLRs and TLR-dependent Myd88 pathway) support a role of the innate immunity processes in T cell driven autoimmunity.
Several genes attributed by IPA into these interactomes include transcripts of genes known to exert anti-inflammatory functions that were found to be downregulated following MOG35-55 activation. These genes include: signaling threshold-regulating transmembrane adapter 1 (Sit1, Table 2), 11β-hydroxysteroid dehydrogenase type 1 (Hsd11b1, Table 2, Figure 7), Pias3 ( Figure  8) and peroxisome proliferator-activated receptor gamma (Pparg,  Figure S3). Figure 9 shows other anti-inflammatory genes that were suppressed following MOG35-55 activation, i.e. legumain (Lgmn), kallikrein 3 (Klk3) and tumor necrosis factor (TNF)-alpha-induced protein 8-like 2 (Tnfaip8l2) and links these effects via CBFβ, a known transcriptional co-regulator of FoxP3 to IL-4-dependent genes ( Figure 7) and to CTLA4, both known for their potent regulatory role in inflammatory processes, including autoimmunity. These findings suggest that intrinsic regulatory mechanisms may be suppressed in activated memory T cells to facilitate efficient activation. On the other hand, several other gene products known to exert anti-inflammatory effects in Th17 cells like Stat5a (Figure 3), Foxp3 ( Figure 4) and Egr2 ( Figure 5) were found to be upregulated, suggesting that feedback homeostatic processes may be initiated in parallel with T cell activation.

Validation by qPCR
Several genes that were identified by microarray analysis as differentially regulated were subjected to validation by qPCR using β2microglobulin as a reference gene. Table 3

Discussion
We show here for the first time the transcript profiling of encephalitogenic T cells following activation by exposure to their antigen. The present report identifies the main pathways and gene networking mediating the MOG35-55-induced proinflammatory functions of memory T MOG cells as well as the interactions between them.
MOG is a CNS specific myelin glycoprotein that is expressed  preferentially on the outermost surface of the myelin sheath. Although being a minor component of CNS myelin, MOG is highly immunogenic, and its targeting by encephalitogenic memory T cells induces severe EAE in various rodents and primates [22][23][24]. Autoimmunity to MOG was recognized as one of the primary demyelinating triggers in MS (For review see [25]). These observations substantiate the use of T MOG memory cells as a tool to identify the molecular mechanisms mediating memory T cell pathological activity and especially the processes that can lead to the demyelination. In the present study, we applied gene array analysis to profile the changes in mRNA expression following MOG35-55 activation of memory T MOG as well as to identify the main mechanisms driving the T cells' unique properties including Th17 function, antigen-specificity and cytotoxicity.
In recent years, few large-scale genetic approaches have been used to study the signature gene networks controlling autoimmune processes and Th17 functions. In many cases, this research was carried out on purified, naïve spleen-derived T cells differentiated in vitro (e.g., using IL-6+TGFβ or IL-6+TGFβ+IL-23 cytokines) into IL-17 releasing T helper cells [26,27]. Due to the absence of APC cells in these in vitro systems, the newly differentiated Th17 cells lack antigen specificity. Consequently, antigen-activated pathways and correlated gene networks in such T cells are usually not identified by the subsequent bioinformatics analyzes. In other cases, antigen specific molecular  patterns, including gene expression, were addressed using tissues isolated from EAE mice [28]. However, in these later cases the presence of other cell types in addition to the memory T cells in the whole tissue homogenates could have influenced the accuracy of the results obtained regarding the gene expression in the T cells of interest. We overcame above obstacles by utilizing pure antigen specific T cells which were incubated with the antigen in the presence of APC but were carefully purified afterwards before subjecting them to the mRNA preparation and gene transcription analysis.
We observed that 8 h of stimulation with MOG35-55 led to a dramatic pro-inflammatory activation of T MOG cells manifested by increased transcription of cytokines, chemokines, growth factors and elements of pro-inflammatory signaling cascades. This result corresponds with the known profile of memory T cells defined by highly coordinated expression of array of cytokines, chemokines and transcription factors [29]. IPA analysis identified IFNγ, TNFα, IL-1, IL-2, IL-6, IL-12, IL-10, IL-15 signaling pathways as involved in MOG35-55 activation of T MOG . This led to the identification of MAPK, NFkB, JAK/STAT and Jun pathways as the leading co-activated intracellular cascades in T MOG activation in agreement with previous reports (for review see [30]).
Several "IL-17 signaling" pathways were recognized to be enriched in activated T MOG , clearly indicating powerful Th17-like activation of T MOG . Indeed, we previously showed that MOG35-55 stimulation leads to increased IL-17 mRNA production and to the release of IL-17 protein from T MOG co-cultured with APC [16]. It is known that the generation of Th17 is mainly dependent on IL-6 and TGFβ activation and that Th17 encephalitogenicity is determined by co-expressed IL-1 and IL-23 [31]. Moreover, GM-CSF and IL-3 were recently shown to drive the activation of autoreactive T cells [32,33]. Indeed, all these cytokines, the relevant transcription factors and intracellular cascades (Tbx21, Stat4, Irf4) were recognized by our analysis to be highly activated following MOG35-55 stimulation. Annotations related to efficient antigen presentation and processing, including pathways activated in the T cells  by dendritic cells and B cells (both present in the APC population) were also recognized to be enriched (e.g., "Crosstalk between Dendritic and NK cells", "Altered T and B cell Signaling in Rheumatoid Arthritis", "B Cell Activating Factor Signaling", "Dendritic Cell Maturation"). These findings highlight the fact that MOG35-55-induced stimulation of T MOG indeed comprises activation of both, (i) Th17 functional phenotype and (ii) antigen specific responses.
As described in Table 2 and the interactomes presented in Figures  8, 9 and S3, several transcripts of proteins with anti-inflammatory roles were down-regulated following MOG35-55 stimulation. These genes include Sit1, previously shown to negatively regulate TCR-mediated signaling in T-cells [34]; Hsd11b1, an enzyme participating in steroid biosynthetic pathways and shown to have restraining effects in MS-like demyelinations [35,36]. Pparg, well known for its anti-inflammatory role, including in MS-like pathologies [37]; Pias3, shown to serve as a potent suppressor of Th17 function [38]; Lgmn, a cysteine protease and a promoter of regulatory T cell function [39]; Klk3, exerting protective role in human systemic lupus erythematosus and lupus nephritis [40] and Tnfaip8l2 (TIPE2), a negative regulator of TLRs and T-cell receptor function, inhibiting JUN/AP1 and NFκB activation and promoting Fas-induced apoptosis [41]. Moreover, Tnfaip8l2, Lgmn and Klk3 gene products were shown by IPA to be directly regulated by CBFβ protein, a co-factor that (together with Runx1 or Runx3) binds to FoxP3 promoter and activates its regulatory function. Indeed, Cbfb-deleted regulatory T cells exhibit impaired suppressive function in vitro and in vivo with altered gene expression profiles including reduced expression of Tnfaip8l2, attenuated expression and activity of FoxP3 and high expression of IL-4, altogether resulting in autoimmunity [42,43]. These results imply that antigen-induced silencing of intrinsic inhibitory mechanisms may accompany memory T cell activation.
Interestingly, various IL-17 receptor transcripts (including Il17re, v1 and v2 and Il17rc) belong to the group of down-regulated genes following the stimulation with MOG35-55. IL-17RA and IL-17RC form a heteromeric complex in which IL-17RC is required for IL-17A and IL-17F mediated signaling [44]. IL-17RC expression is low in haematopoetic tissues and is high in non-immune cells (e.g. prostate, liver, kidney, thyroid and joints). We hypothesize, that such downregulation of IL-17R on T MOG memory cells may protect these IL-17releasing cells from their own cytotoxic activity induced by this cytokine, a mechanism that was suggested by Gaffen et al., [44]. We observed enhanced levels of the transcript of FoxP3, known to serve as a master regulator of suppressory transcription in autoimmune pathologies [45]. This is in agreement with a previous observation reporting that moderately increased FoxP3 activity transiently accompanies the activation of pathogenic T cells [46,47] while the classic regulatory T cell phenotypes are usually characterized by stable high levels of FoxP3 [48].
STAT5 was found by us to be slightly upregulated following MOG35-55 stimulation. STAT5 was recently described as a negative regulator of STAT3-dependent Th17 function [49]. Moreover, STAT5 seems to play diverse roles in T cell activation as it can also induce proproliferative IL-2 signals and is indispensable for the encephalitogenicity of autoreactive CD4 + T cells via induction of pro-inflammatory GMCSF production [33]. Indeed, the levels of Csf2 mRNA (the gene transcript encoding GMCSF) were elevated in activated T MOG , indicating that STAT5 may play a role in promoting the activation of T MOG .
EGR2 is known as a main anergy regulator [50]. We found that Egr2 mRNA is up-regulated following MOG35-55 stimulation (see also [17]. This result suggests that feedback homeostatic processes may be initiated in parallel with T cell activation, tuning proper antigen activation of T MOG as suggested by Miao et al. [51]. Interestingly, IPA identified certain cancer-related pathways to be enriched in activated TMOG. This may imply the induction of mechanisms regulating cell cycle and proliferation. Indeed, memory T cells respond to activating antigens by increased proliferation [13,52], a finding that was confirmed by us using T MOG cells [15,18]. Accordingly, transcripts known for their pro-proliferative and anti-apoptotic roles in activated T cells e.g. regulator of cell cycle (Rgcc) and BCL2-Like 1 (Bcl2l1) [53] were upregulated and those with pro-apoptotic role were down-regulated in response to MOG35-55 stimulation, e.g. BCL2 binding component 3 (Bbc3). In agreement with this increase in antiapoptotic gene products another study showed anti-apoptotic gene reprogramming (increase in anti-apoptotic Bcl2 activity) in quiescent MOG35-55 selected T cell line derived from MS patients long time after their last activation, thus suggesting that memory T cells possess reprogrammed gene function that contributes to their resiliency to apoptosis and prolonged survival [54].
Various cytokines involved in autoimmune processes, including TNFα, IFNγ and IL-4 found to be activated in our hands, play important roles in innate immunity as well, linking both types of immune responses. Our analysis revealed a significant role of innate immunity processes in the antigen activation of encephalitogenic T MOG cells. This involved regulation of TLRs mRNAs (Tlr2, Tlr3, Tlr4, Tlr7) as well as activation of MyD88, a membrane-bound molecule initiating TLR-dependent signaling, mRNA. IPA analysis demonstrated substantial activation of IFNγ mRNA and of IFN-dependent pathways known to play important roles in innate immunity. Indeed, TLR-mediated activation of dendritic cells was shown to be critical for CD4+ and CD8+ T cells' effector functions [55]. Moreover, initiation of EAE requires functional MyD88-dependent signaling since MyD88-deficient mice are resistant to MOG-induced EAE [56,57]; for review see [58]). Indeed, Th17 cells are primarily activated in response to pathogen infections [59]). Our results indicate that these pathways are of high relevance not only at the initial phase of memory T cell differentiation but also during the re-activation of established antigen-specific T cell clones such as T MOG . Thus, parallel targeting of both innate and adaptive processes may be of high relevance for restraining the pathogenicity of autoreactive T cells as suggested by Waldner et al. [58] and Mills et al. [60].
The present study reveals that the various MOG35-55-enriched pathways and gene networks are similar to the processes driving other T cell-dependent autoimmune pathologies including psoriasis, type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis and inflammatory bowel disease. Indeed, memory Th17 were shown to be involved in several autoimmune disorders including MS, psoriasis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus and asthma [59]. This similarity includes as well other cytokines, co-receptors, signaling pathways and transcriptional regulators accompanying the Th17 activity [59] and described in the present report. This strongly suggests that despite the high epitope-specificity of the T MOG the mechanisms leading to autoimmune pathogenicity are shared by other types of autoreactive T cell lineages. Thus, using T MOG cells in vitro should be useful for studying the mechanisms and pathways involved in other autoimmune diseases including those listed above. In addition, it should have significant predictive values in screening for therapeutic potentials of various materials not only for MS-like pathologies but for other T cell driven autoimmune diseases as well. We therefore suggest that the T MOG system may serve as a reliable model for deciphering the pathways affected by potential therapeutics to diminish activity of autoimmune T cells.