CXCR4-expressing T cells are increased in LNs during the development of CIA. DBA/1J mice were immunized with IIC and CFA, or CFA only, on day 0 and day 21. At day 30, arthritis development was determined, and LN cells were collected. (a) CXCR4 expression was analyzed with flow cytometry in CD4+ T cells from IIC and CFA-immunized mice (arthritic and nonarthritic), CFA-immunized mice (nonarthritic), and normal nonimmunized DBA/1J mice. Representative data from two similar experiments are presented. (b) Proportion of CXCR4+ cells in CD4+ cells from normal DBA/1J mice without immunization (n = 3), IIC and CFA-immunized nonarthritic (n = 4) and arthritic (n = 7) and CFA-immunized nonarthritic (n = 7) mice are shown. Data from two similar experiments were combined. *P < 0.05 by Student's t test. (c) The expression of CXCR4 was analyzed with immunohistochemistry. CXCR4 was labeled with green fluorescence, and CD3 was labeled with red fluorescence. An increased number of CXCR4-expressing T cells is found in LNs from arthritic mice than those from normal mice. Representative figures among three sections from normal mice and three sections from arthritic mice are presented. White bar represents 20 μm. (d) CXCR4 and CD62L coexpression was analyzed in CD4+ cells. Representative plots gated on CD4+ cells are presented, and all the data from 10 normal nonimmunized mice and 11 IIC/CFA-immunized arthritic mice are summarized on the right. Averages and SDs are shown. *P < 0.05 by Student's t test. (e) LN cells from two nonimmunized normal or IIC/CFA-immunized arthritic mice were pooled and cultured in the absence or presence of 1 μg/ml of anti-CD3 antibody for 72 hours, and CXCR4 and CD44 expression was analyzed in CD4+ cells. Representative plots gated on CD4+ cells from two independent experiments are shown.