Atglistatin br Results br Discussion Analysis of EBI express
Discussion Analysis of EBI2 expression showed that EBI2 is highly expressed in central memory CD8+ T cells that is in line with previous data from human T cells in which these Atglistatin express higher levels of EBI2 than naive or effector memory CD8+ T cells (Hannedouche et al., 2011). Such cells were shown to have a specific role in fast viral defense in secondary infections (Wherry et al., 2003). Recently, it was suggested that EBI2 has a function in egress of T cells from LNs, because immunized CH25H-deficient animals contained enriched numbers of CD44+ effector T cells in draining LNs (Chalmin et al., 2015). This, together with our findings of differential expression of EBI2 in distinct stages of T cell differentiation, may suggest that EBI2 has an important role in T cell re-localization and possibly in LN egress reminiscent of the S1P-S1P receptor system, similar to its role in germinal center B cells. In two different types of co-transfer experiments, we analyzed where encephalitogenic T cells reside either in the active EAE model or in the transfer model, either before EAE or at onset of EAE. In active EAE, we found similar numbers of proliferating effector T cells irrespective of the EBI2 genotype in draining LNs but an accumulation of EBI2-deficient T cells in the blood. In the transfer model, we found a clear ratio change of EBI2-expressing T cells over EBI2-deficient T cells from periphery to the CNS in early EAE. Both experiments point to a role of EBI2 and its ligand in CNS extravasation. Therefore, our data do not support the hypothesis that EBI2 plays a major role in the regulation of T cell LN egress but also does not exclude that certain T cell subpopulations might be differentially released of draining lymph nodes (dLNs) after priming in absence of EBI2. In accordance with its original discovery, EBI2 may play its major role in response to viral infections or in a pathological context such as in autoimmunity. Although we found a significant delay of onset in the Th17 transfer EAE model, active EAE was unchanged in absence of EBI2. At the moment, we can only speculate about this difference. It might be that active EAE induction using CFA/MOG induces a stronger inflammatory array of immune cells, pro-inflammatory factors, cytokines, and chemokines, which might override the need for EBI2-mediated migration. Alternatively, there might be compensatory mechanisms for the lack of this GPCR, so that mice are susceptible to EAE also independently of EBI2 in knockout animals. We showed in transfer experiments with EBI2 mutant T cells that EAE was significantly delayed but after onset, disease proceeded normally. This might be due to the previously described plasticity of Th17 cells, which tend to change their expression profile toward the Th1 lineage together with high levels of GM-CSF (Hirota et al., 2011, Kurschus et al., 2010) (and herein). In absence of EBI2, EAE initiation may more depend on Th1 cells co-expressing GM-CSF than on Th17 cells. This hypothesis is supported by our data showing a major population of Th1 cells co-expressing GM-CSF in the CNS during disease. We found that Th17 cells, derived either from in vivo immunization or in vitro polarization under pathogenic conditions (in presence of IL-23 and/or IL-1β) expressed high levels of EBI2. In contrast, Th17 cells that were generated in absence of IL-23 or IL-1β lost EBI2 expression during differentiation. Addition of various cytokines revealed that IL-2 was the strongest in promoting down-modulation of EBI2 (data not shown). This result is in line with the inhibitory action of IL-2 on Th17 development (Laurence et al., 2007). Our data are also in line with a previous report showing that Gpr183 is specifically upregulated in Th17 differentiation under pathogenic conditions (using medium containing TGF-β3) (Lee et al., 2012). Hence, EBI2 expression seems to be part of a pathogenic T cell signature and, based on our results with the Th17-mediated EAE transfer model, we suggest that EBI2 confers pathogenicity to encephalitogenic T cells by enhancing migration into the inflamed CNS.