Homeostatic chemokines function as costimulatory factors during Ab- and peptide-induced T-cell activation. (A) Flow cytometry histogram of CFSE-labeled T-cell division after 48 and 72 hours of activation with anti-CD3ϵ mAb in the presence (bold line) or absence (gray fill) of 100 nM CCL21. Data are representative from 1 of 6 independent experiments. (B) Proliferation in the presence and absence of homeostatic chemokines. CFSE-labeled T cells were stimulated with plate-bound anti-CD3ϵ mAb with or without anti-CD28 mAb in the presence or absence of CCL21, CCL19, or CXCL12 (100 nM). Proliferation was determined after 48 hours by fluorescence-activated cell sorter and normalized to fold increase compared with cells cultured with anti-CD3ϵ mAb (proliferation index). The presence of chemokines increases the percentage of cells having undergone cell divisions within 48 hours. Data are pooled from 4 to 6 independent experiments. Statistical significance was determined using analysis of variance comparison of anti-CD3 or anti-CD3/CD28–stimulated T-cell proliferation with or without chemokines. *P < .05 compared with “no chemokine.” (C) DO11.10 T cells were cocultured with chicken or turkey OVA_323-339-loaded congenic splenocytes in the presence or absence of CCL21 (100 nM). T-cell activation was determined by ³H-thymidine-incorporation after 48 hours. Numbers indicate fold increase of proliferation in the presence of CCL21. *P < .05 compared with “no chemokine.” (D) Prior activation as in panel C; DO11.10 T cells were incubated with PTX. Data are pooled from 2 independent experiments.