Functional activity of MIP-3α produced by HRS cells. (A) RT-PCR analysis of MIP-3α mRNA expression in various Hodgkin and non-Hodgkin cell lines, as indicated. Expression of GAPDH was analyzed as a control. (B) MIP-3α is regulated by IL-21. KM-H2 cells were left untreated, incubated with BSA (carrier for the :Fc constructs), or treated with 50 μg/mL of the IgG1:Fc control or the specific IL-21R:Fc construct, as indicated. After 6 hours, mRNA was prepared and analyzed by RT-PCR for expression of MIP-3α. Expression of GAPDH was analyzed as a control. (C) Measurement of secreted MIP-3α by ELISA. For the analysis of the secreted amount of MIP-3α by the various cell lines, supernatants were analyzed by a MIP-3α–specific ELISA. The amount of MIP-3α in the supernatants is shown in picograms per milliliter. As controls, standard medium and the reagent diluent for the standard (reagent D) were included. Error bars denote SD. (D) Chemotaxis assay of PMNCs toward SN of the HRS cell lines KM-H2 (□) and L591 (■) and the non-Hodgkin cell line Namalwa (□), as indicated. The migration of freshly isolated PMNCs toward SNs, each without (−) or with preincubation with a neutralizing MIP-3α antibody or the respective control antibody, is shown as fold migration relating to the spontaneous migration toward medium, which was set at 1.0. All assays were performed in triplicate. Error bars denote SD. (E) MIP-3α–induced migration of T_reg cells toward KM-H2 cells. Migrated cells and cells incubated in KM-H2 SN for the time of the assay (control) were analyzed by FACS for expression of CCR6, CD4, CD25, FoxP3, and CD127, as indicated.