Lrch1 transgenic mice are resistant to EAE with reduced T cell migration. (A) T8.1 cells were transfected with the vector control, LRCH1, or its fragments (LRR_1-9, L305-763) for a transwell assay in response to SDF-1α (n = 3). FACS assay was performed to check the cell surface expression of CXCR4. The transfected exogenous human LRCH1 was detected at 130 kD and the endogenous murine LRCH1 in T8.1 cells was detected at 95 kD by Western blot. (B) The expression of FLAG-Lrch1 in thymus from Lrch1 transgenic mice was assessed by immunoblotting. (C) The clinical scores (left) and EAE incidence (right) of WT and Lrch1 transgenic mice induced by the MOG (35–55) peptide. n = 5. (D and E) The total numbers of CD4⁺ T cells in the CNS, blood (D), spleen, and draining LNs (E; middle right and right) of the WT or Lrch1 transgenic mice; percentages of BrdU⁺ or Annexin V⁺ CD4⁺ T cells in spleen were checked at the peak stage of EAE (E; left and middle left). n = 4–5. (F) The number of lymphocytes in spleen and draining LNs were counted from WT and Lrch1 Tg mice after EAE induction. n = 5. (G) The percentage of Foxp3⁺ CD4⁺ T reg cells in draining LNs from the WT or Lrch1 transgenic mice at the peak stage of EAE. n = 4. (H and I) The encephalitogenic CD4⁺ T cells were purified from the WT or Lrch1 transgenic mice for a transwell assay in response to SDF-1α (H), or for a FACS assay to check the surface expression of CXCR4 (I). n = 4–5. NS, not significant (P > 0.05); *, P < 0.05; **, P < 0.01. Data are representative of four experiments (A, mean ± SD), three experiments (C, mean ± SEM), or two experiments (D–I, mean ± SD). Statistical significance was determined using unpaired Student’s t test.