PMC

DCAL-2 ligation induces CCR7 expression on iDCs and TLR-stimulated DCs. iDCs were stimulated with or without LPS (1 μg/mL) or zymosan (100 μg/mL) in the presence or absence of anti-DCAL-2 (10 μg/mL) or IgM control (10 μg/mL). After 24 hours, cells were harvested and washed, then double stained with (A) anti-CD86 and anti-CCR7 or (B) anti-CD83 and anti-DC-LAMP as described in “Materials and methods.” Results are representative of 4 independent experiments.

The presence of DCAL-2 signaling during DC maturation modulates DC-directed T-cell differentiation. iDCs were stimulated with LPS, Zymosan, and CD40L with or without anti-DCAL-2 for 24 hours, then mixed with allogeneic CD4⁺CD45RA⁺ T cells labeled with CFSE for 4 days to stimulate T-cell proliferation (A), or T cells were recovered and restimulated with PMA and ionomycin for 1 hour before adding brefeldin A and cultured for a further 5 hours (B). Cells were then fixed and stained for IL-4 and IFN-γ. Results are representative of 4 independent experiments.

Human DCAL2 gene structure and sequence. (A) The nucleotide and protein sequence of hDCAL-2. The ITIM in the cytoplasmic tail is outlined and indicated in bold; the putative transmembrane region is underlined. The 6 cysteine residues that create 3 disulphide bonds in the CRD are circled and shaded, and 4 potential glycosylation sites are boxed. (B) Alignment of the hDCAL-2 CRD protein sequence with related CLRs. ^*Conserved cysteine residues; two dots, a conserved charge residues. The WIGL domain is shown in bold. (C) A phylogenetic tree of the CLRs most closely related to hDCAL-2.

DCAL-2 signals synergize with CD40 signals. iDCs were stimulated with CD40L fusion protein or CD40L-transfected L cells alone or combined with anti-DCAL-2 or IgM control for 24 hours. DC maturation was analyzed by monitoring the expression of maturation markers using FACS (A), and culture supernatants were collected and analyzed for cytokine or chemokine expression by ELISA (B). Results are representative of 3 independent experiments using different donors. Mean of triplicate wells ± SEM. ^*P < .05; ^**P < .01, paired Student t test.

The mRNA and protein expression of hDCAL-2 is restricted to myeloid cells. (A) RT-PCR analysis of hDCAL-2 in a panel of primary cells shows that hDCAL-2 is expressed in human CD14⁺ monocytes, human iDCs, CD1a⁺ DCs, and macrophages but not in NK cells, PDCs, B cells, activated B cells (activated by IgM and anti-CD40 mAb for 24 hours), T cells, or activated T cells (activated by CD3 mAb). Results are representative of 3 different donors. (B) Fluorescence-activated cell sorter (FACS) analysis of hDCAL-2 in a panel of PBMCs shows that hDCAL-2 is expressed on human CD1a⁺ DCs but not on B cells, T cells, NK cells, or pDCs. Results are representative of 3 different donors. (C) FACS analysis of human primary cells and cell lines shows hDCAL-2 is expressed strongly on CD14⁺ monocytes and iDCs but only weakly on the HL-60, U937, and Nalm-6 cell lines. Results are representative of 3 independent experiments.

Regulation of DC cytokine expression in the presence of a DCAL-2 signal. iDCs were stimulated with or without indicated doses of LPS or Zymosan in the presence or absence of anti-DCAL-2 (10 μg/mL) or IgM control (10 μg/mL). After 24 hours, supernatants were harvested and the levels of cytokines or chemokines analyzed by ELISA. (A) Induction of cytokines and chemokines by anti-DCAL-2 stimulation. (B) DCAL-2 signal modulated LPS- or zymosan-induced IL-12p40 and p70 expression. Results are representative of 4 independent experiments. Mean of triplicate wells ± SEM. ^*P < .05; ^**P < .01, paired Student t test.

Ligation of hDCAL-2 on iDCs induces activation of kinases and receptor internalization. iDCs were stimulated with anti-DCAL-2 mAb for the indicated time periods before being lysed. Total cell lysates were used for Western blot and probed with (A) antiphosphorylated tyrosine Ab (4G10), (B) anti-phospho-p38 MAPK, anti-phospho-ERK antibody, and anti-total p38MAPK, and anti-ERK as loading control. (C) iDCs were stimulated with anti-DCAL-2 mAb or IgM control for 15 minutes. After washing with cold PBS, cells were fixed and then permeabilized in 90% methanol. Cells were then washed twice and stained with anti-phospho-ERK1/2-PE and anti-phospho-p38 MAPK-Alexa fluor 488. The cells were analyzed by FACS. (D) The internalization of DCAL-2 after ligand binding was measured after coating iDCs with anti-DCAL-2 using GAM Ig-FITC and flow cytometry as described in “Materials and methods.” Results are representative of 3 independent experiments.