Fig. 9.

Blockade of CCL2 or addition of Arg1 suppress CD8⁺ T cell activation and migration in co-culture with LILRB4-KD microglia. (A, B) Differential expression of LILRB4 in BV2 microglia transfected by knockdown and negative control lentiviral vectors. The expression of LILRB4 in BV2 was detected by PCR (A) and Flow cytometry assay (B) (n = 3; **p = 0.0025/0.0065/0.0057). (C) Experimental procedure. Transwell-placed, Control, and LILRB4-KD microglia (without or with CCL2 inhibitor or IgG) were cultured for 4 h under OGD conditions. During reoxygenation, the t-cell-containing Transwell device was placed on a 24-well plate and exposed to medium on its lower surface for 24 h, and the levels of t-cell migration to the lower layer were measured by Flow cytometry. In another experiment, microglia cells were co-cultured with CD8⁺ T cells. Control and LILRB4-KD microglia were collected and cultured under OGD conditions for 4 h, and CD8⁺ T cells were added directly to the medium during reoxygenation. One group was added recombinant Arg-1 and another group was not. After 24 h, CD69 and IFN-γ expression in CD8⁺ T cells were detected by flow cytometry. (D) T cell migration after exposure to OGD/R, measured by the number and type of T cells microglia into 24-well plates, with or without CCL2 inhibition. Flow cytometry tests for T cell migration and ratio of CD8⁺ T cell. (n = 4; *p = 0.0187/0.0383/0.0104, **p = 0.0029). (E) Control or LILRB4-KD microglia were exposed to OGD/R and co-cultured with CD8⁺ T cells with or without the addition of recombinant Arg-1. CD8⁺ T cells were collected for flow cytometry detection of CD69 and IFN-γ expression. (F) Quantitation and statistical evaluation of data in (E). (n = 6; *p = 0.0106/0.0427, ***p = 0.0006, **p = 0.0024/0.0024/0.0029). (G) Control or LILRB4-KD microglia were exposed to OGD/R and co-cultured with CD8⁺ T cells with or without the addition of recombinant Arg-1. Flow cytometry tests for T cell proliferation. (n = 5; *p = 0.0348/0.0487/0.0153)