Adoptive transfer of Lrch1 KO CD4⁺T cells accelerates EAE with enhanced T cell migration. (A) Generation of Lrch1 KO mice. The exon 1 of the Lrch1 gene was specifically targeted by TALEN, and DNA sequencing confirmed the nucleotide mutation in the Lrch1 locus adjacent to the FOKI cleavage site (arrow). (B) Numbers of CD4⁺ T cells in spleen and LNs from unimmunized mice. n = 3. (C) EAE incidence of WT and Lrch1 KO mice in response to MOG (35–55) treatment. n = 5. (D–F) Numbers of CD4⁺ T cells in spleen and draining LNs (D); TCR usage analyzed by anti-TCR Vα and anti-TCR Vβ antibodies (E); and percentages of BrdU⁺ CD4⁺ T cells, IL-2 secretion, and the surface expression of CD25 and CD44 in CD4⁺ T cells from the draining LNs (F) of WT and Lrch1 KO mice after EAE induction. n = 5. (G) The sublethally irradiated WT recipient mice were reconstituted with WT or Lrch1^−/− encephalitogenic CD4⁺ T cells to assess their clinical scores (left) and EAE incidence (right). n = 5. (H) The total numbers of WT or Lrch1^−/− encephalitogenic CD4⁺ T cells in the CNS from the sublethally irradiated WT recipient mice presented in G. The encephalitogenic CD4⁺ T cells were purified from the WT or Lrch1^−/− mice for a transwell assay in response to CCL5 (right). n = 5. (I) CD4⁺ T cells were reconstituted with LRCH1 or the vector control, for a transwell assay in response to CCL5 (left). The surface expression of CCR5 in the spleen CD4⁺ T cells from WT and Lrch1 KO mice after EAE induction (right). n = 3–4. NS, not significant (P > 0.05); *, P < 0.05; **, P < 0.01. Data are representative of three experiments (G, mean ± SEM), or two experiments (B–F and H­–I, mean ± SD). Statistical significance was determined using unpaired Student’s t test.