Lymphopenia is mainly independent of GPCRs. Splenocytes were cultured for 3 hours in medium with or without 20 ng/mL PTX (Sigma), labeled with CFSE or TAMRA, and adoptively transferred into WT mice. (A) Mice were killed after 20 hours, and spleens and LNs were analyzed. Note that in contrast to untreated cells (green), PTX-treated cells (purple) did not enter splenic white pulp and LNs, indicating quantitative blockade of GPCR signaling. Images were visualized and acquired using a Zeiss LSM 510 Meta laser scan microscope equipped with a 10×/0.3 NA Plan Neofluor objective. Moviol (Calbiochem, Darmstadt, Germany) was used as an imaging medium. (B) After treatment with poly(I:C) or PBS, transferred control cells (left) and PTX-treated lymphocytes (right) were counted in blood at the indicated time points. Data are expressed as mean ± SD (n = 3) and are representative of 3 similar experiments. (C) BL/6 splenocytes cultured for 3 hours in medium with or without PTX or (D) BL/6 and CCR7^–/– cells were differentially labeled (CFSE^hi or CFSE^lo) and transferred to BL/6 mice. After treatment with R-848, blood samples were taken at the indicated time points. Labeled cells are depicted as percentages of pretreatment values. Data are expressed as mean ± SD (n = 3).