(a) APRIL expression in activated, but not in naive, T cells. Activated T cells have increased APRIL mRNA expression levels. DO11.10 spleen CD4⁺ T cells were isolated and activated in vitro toward a Th1 or Th2 phenotype. RNA was prepared from naive, Th1, and Th2 cells and used to analyze the expression of APRIL, BLyS, and GAPDH by RT-PCR. Generation and characterization of APRIL Tg mice. (b) Schematic diagram of constructs used for peripheral T cell–specific expression of the human APRIL transgene. (c) mRNA expression of APRIL in T cells of four different founders. RNA was prepared from purified T cells derived from spleen and lymph nodes of four Tg mice and analyzed by RT-PCR for APRIL RNA expression levels. To ensure that the amplified human APRIL signal was not due to contaminating genomic DNA, cDNA probes were amplified for Thy1 using primers that amplify a 300-bp fragment of cDNA and a 700-bp fragment of genomic DNA (genomic mouse DNA was used as control). (d) Protein expression of APRIL in Tg mouse T cells. Cell lysates were prepared from purified T cells derived from spleen and lymph nodes of four Tg mice, a control littermate, and a C57BL/6 mouse and analyzed in Western blot using anti-human APRIL antibodies (8). (e) Secreted APRIL circulates in serum of APRIL Tg mice. Sera (1 μl) of control and Tg mice were resolved under nonreducing conditions and immunoblots developed with anti-human APRIL antibodies. Recombinant (Rec.) APRIL protein (0.1–5 ng) added to control serum was used as standard.