3. RESULTS
3.7 Chemokine receptor and integrin expression profile of T MBP-GFP and T βSyn-GFP cells during ptEAE
It has previously been shown that after transfer TMBP-GFP cells undergo in the peripheral organs a complex reprogramming that allow them to overcome the blood-brain barrier (BBB).
More specifically the effector T cells down-regulate the activation markers and up-regulate the expression of several integrins and chemokine receptors (Odoardi et al., 2012). Therefore, I investigated whether the expression of chemokine receptors and integrins on the surface of effector TMBP-GFP and TβSyn-GFP cells is crucial for the observed divergent CNS homing pattern (see chapter 3.3 and 3.4). TGFP cells of both specificities were purified via fluorescence activated cell sorting (FACS) from the blood of the TGFP cell-injected animals at the onset of the disease and analysed for the expression of their surface receptor profile by QRT-PCR.
TMBP-GFP and TβSyn-GFP cells expressed a similar pattern of the chemokine receptors CXCR4, CCR7, CCR6, CXCR3, CCR5 and of the integrins VLA-4 and LFA-1. The expression of CXCR3, CXCR4 and VLA-4 was more pronounced than the expression of the other surface receptors for both antigen-specific T cells (fig. 3.12 A). To flank the gene expression analysis with functional data a T cell migration assay was performed in which the chemotactic migration of the TMBP-GFP or TβSyn-GFP cell towards the chemokines CCL5, CCL19, CCL20, CXCL11 and CXCL12 was analysed. Both effector T cell types sorted from blood in the early phase of the disease showed a preferential migratory response to CCL20 and CXCL11 (fig.
3.12 B). According to these data the chemokine receptor/integrin expression profile in the periphery was not responsible for the observed different infiltration pattern of TMBP-GFP and TβSyn-GFP cells in the target tissues.
Next I investigated the chemokine receptor and integrin expression profile and the chemotactic behaviour of effector T cells of both specificities sorted from CNS tissue at the onset and peak of the disease. At both time points of investigation, auto-reactive TMBP-GFP and TβSyn-GFP cells show a similar expression pattern, as discovered previously for blood-purified effector T cells: Independently from the original CNS compartment, CXCR3 and CXCR4 were the most expressed chemokine receptors and in the integrin group VLA-4 was more expressed than LFA-1 (fig. 3.13 A and B). In addition, the same migratory response pattern of effector T cells to the inflammatory and the homeostatic or lymphoid chemokines was detected at the peak of disease. CCL20 and CXCL11 seemed to be chemo-attractive for both effector T cell types (fig. 3.14 A and B). In summary, there was no difference in the expression of surface receptors or the chemokine response pattern detectable in grey- or white
Results 78 matter-specific T cells. Therefore, it seems unlikely that these proteins are responsible for the differences in T cell homing behaviour between TMBP-GFP and TβSyn-GFP cells.
Figure 3.12 Surface expression of chemokine receptors and integrins and chemotactic response of blood-derived effector TMBP-GFP and TβSyn-GFP cells during ptEAE. EAE was induced by the transfer of TMBP-GFP or TβSyn-GFP cells to Lewis rats. Peripheral blood TGFP cells were collected and (A) purified by FACS sorting at the early disease onset or (B) enriched by an OptiPrep gradient at the same time point. (A) The expression of the chemokine receptors CXCR4, CCR7, CCR6, CXCR3, CCR5 and the integrins VLA-4 and LFA-1 was analysed by QRT-PCR. Specific copies in relation to β-actin copies are shown. Data of two independent experiments are shown. Average and SD are depicted. (B) The ability of effector T cells to migrate towards the chemokines CCL5, CCL19, CCL20, CXCL11 or CXCL12 was tested by a transwell migration assay. Migrated T cells were counted by flow cytometry and the migratory response of the T cells was calculated as cell migration index (ratio of transmigrated T cells in response to chemokines versus control without chemokines). One representative data set of two independent experiments is shown.
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Figure 3.13 The surface expression pattern of chemokine receptors and integrins of CNS-derived effector TMBP-GFP and TβSyn-GFP cells during ptEAE. EAE was induced by the transfer of TMBP-GFP or TβSyn-GFP cells to Lewis rats. TGFP cells in CNS tissue homogenate were enriched by a percoll gradient and further purified by FACS at disease onset (A) or at the peak of the disease (B). The expression of the chemokine receptors CXCR4, CCR7, CCR6, CXCR3, CCR5 and the integrins VLA-4 and LFA-1 was analysed by QRT-PCR. Specific copies in relation to β-actin copies are shown. Data of three independent experiments are shown. Average and SD are depicted. Men Brain, brain meninges; Men SC, SC meninges; Brain, brain parenchyma; SC, SC parenchyma.
Figure 3.14 The migratory response pattern of CNS-isolated effector TMBP-GFP and TβSyn-GFP cells in T MBP-GFP or TβSyn-GFP cell-induced ptEAE. EAE was induced by the transfer of TMBP-GFP or TβSyn-GFP cells to Lewis rats. Animals were sacrificed at the peak of the disease. TGFP cells in CNS tissue homogenate were enriched by an OptiPrep gradient. The ability of effector T cells to migrate towards the chemokines CCL5, CCL19, CCL20, CXCL11 or CXCL12 was analysed by a transwell migration assay. Migrated T cells were counted by flow
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cytometry and the migratory response was calculated as cell migration index (ratio of transmigrated T cells in response to chemokines versus control without chemokines). One representative data set of two independent experiments is shown. Men Brain, brain meninges; Men SC, SC meninges; Brain, brain parenchyma; SC, SC parenchyma.