Human pDCs present KLH to PBLs after DCIR-mediated uptake. (A) Anti–DCIR-KLH binds specifically to DCIR-expressing CHO cells. The CHO cell line stably expressing human DCIR was incubated on ice with anti–DCIR-KLH, anti–DC-SIGN-KLH, rabbit polyclonal anti-KLH, and fluorochrome-conjugated secondary antibodies. FACS analysis showed that anti–DCIR-KLH binds well to the CHO-DCIR cell line, whereas binding of the anti–DC-SIGN conjugate cannot be detected (left). Binding of anti–DCIR-KLH (gray shaded area) can be inhibited by preincubation with anti-DCIR antibodies (thick line). Preincubation with a matching isotype control (dashed line) does not block anti–DCIR-KLH binding (right). Anti–DCIR-KLH does not bind to untransfected CHO cells (data not shown). (B) PDCs were freshly isolated and loaded with anti–DC-SIGN-KLH and anti–DCIR-KLH conjugates, KLH alone, or KLH together with patient-derived serum containing anti-KLH antibodies (positive control). To block DCIR-mediated antigen uptake, pDCs were also preincubated with anti-DCIR mAb before antigen targeting. PDCs were cultured with IL-3 and matured with 5 μg/mL of CpG-C. Fresh PBLs were added and thymidine incorporation was measured after 4 days of coculture. DCIR targeting induces PBL proliferation clearly above background of pDCs loaded with KLH alone or anti–DC-SIGN-KLH (*P ≤ .012 and **P ≤ .019, respectively). Proliferation can be blocked by preincubation with anti-DCIR antibody (***P ≤ .051). Similar data were obtained with pDCs incubated without CpG-C (data represent mean ± SEM of experiments performed in triplicate). Similar data were obtained from 2 independent patients.