Humoral and cellular immune responses against IIC are normal in Cxcr4flox/flox / Lck-Cre mice. (a) IIC-specific IgG and IgM levels in the serum were determined with ELISA 60 days after immunization with IIC/CFA. Each circle represents an individual mouse, and averages and SDs are shown. Cxcr4+/+/Lck-Cre mice: open columns (n = 9); Cxcr4flox/flox/Lck-Cre mice: closed columns (n = 11); Cxcr4flox/flox mice: light gray columns (n = 6); and WT DBA/1J mice: dark gray columns (n = 6). Data are representative of two independent experiments. (b) T-cell recall proliferative response toward IIC was assessed 1 week after immunization with IIC/CFA. LN cells from Cxcr4+/+/Lck-Cre or Cxcr4flox/flox/Lck-Cre mice were harvested and cultured in the presence or absence of 50, and 100, 200 μg/ml of denatured IIC at 37°C for 72 hours, and T-cell proliferation was assessed with [3H]-thymidine incorporation by using three wells for each mouse. The average and SD of four mice are shown for each column. Representative data from three independent experiments are shown. IL-17 (c) and IFN-γ (d) levels in the culture supernatants were measured with ELISA. Triplicate (proliferation, IFN-γ) or duplicate (IL-17) wells were used for each mouse, and averages and SDs of three mice are shown. Data are representative of at least two independent experiments. In the right columns, IL-17 (C) and IFN-γ (D) levels in cultures of LN cells from two immunized mice after stimulation with 1 μg/ml of anti-CD3 antibody at 37°C for 72 hours are shown. Averages and SDs of triplicate wells are shown, and data are representative of two independent experiments. (e) LN cells from immunized mice were pooled and stimulated with or without 100 μg/ml of IIC for 72 hours. The proportion of CD25+, CD62L-, and CCR6+ cells in CD4+ cells was analyzed with FACS. Data from three independent experiments were combined, and averages and SDs are shown.