BAD-LAMP is a novel biomarker of nonactivated human plasmacytoid dendritic cells by Axel Defays, Alexandre David, Aude de Gassart, Francesca De Angelis Rigotti, Till Wenger, Voahirana Camossetto, Pierre Brousset, Tony Petrella, Marc Dalod, Evelina Gatti, and Philippe Pierre Blood Volume 118(3):609-617 July 21, 2011 ©2011 by American Society of Hematology

BAD-LAMP mRNA expression profile in human leukocytes. BAD-LAMP mRNA expression profile in human leukocytes. (A) Gene microarray quantitation of BAD-LAMP mRNA expression in human leukocytes. Results are shown as the fluorescent signal intensity for Affymetrix Human Genome U133 PLUS 2.0 ProbeSet 219463_at (expressed in arbitrary units in log scale). Quality controls, data sources, and data normalization have been described previously.32 Neu indicates neutrophils; pMθ, PBMC-derived macrophages; Mo-Mθ, monocyte-derived macrophages; Mo-DC, monocyte-derived GM-CSF + IL-4 DCs; CD16 DC, blood Lin−HLA−DR+CD16+ DCs; BDCA1 DC, blood BDCA-1+ DCs; BDCA3 DC, blood BDCA-3+ DCs; BL, blood B lymphocytes; CD4 TL, blood CD4+ T lymphocytes; CD8 TL, blood CD8+ T lymphocytes; (B) Tissue expression of BAD-LAMP assessed by northern blot. A signal was detected only in the adult human brain. Actin mRNA levels are shown as a control. (C) Detection of BAD-LAMP transcript in human spleen RNA total extracts by nested RT-PCR. (D) Detection of BAD-LAMP transcript in total RNA extracts from human samples. Actin levels after the first PCR round are shown as a control. A plasmid containing BAD-LAMP cDNA was used as a positive control. Axel Defays et al. Blood 2011;118:609-617 ©2011 by American Society of Hematology

BAD-LAMP is detected specifically in pDCs. BAD-LAMP is detected specifically in pDCs. (A) Detection of BAD-LAMP in human lymphoid tissue. Paraffin human spleen sections were stained with mAbs against CD123 (red) and BAD-LAMP (green). Overlay shows that BAD-LAMP+ cells are also CD123+ (merge, yellow). Bar indicates 20μm. Paraffin-fixed human tonsil sections were stained for immunohistochemistry (bottom right). BAD-LAMP+ cells display a pDC morphology (arrows, ×800) next to HEV. (B) Intracellular FACS staining on human PBMCs. A rare cell population can be isolated based on BAD-LAMP expression (left). BAD-LAMP+ cells were identified as pDCs based on BDCA-4 expression (right). (C) BAD-LAMP staining on purified pDCs. Cells stained in intracellular FACS (left) are homogenously BAD-LAMP+ (solid line) compared with isotype control background (filled graph). BAD-LAMP is localized in intracellular membrane compartments of BDCA-4+ pDCs, as shown by confocal microscopy (green, right). Nucleus (Nu) staining is shown in blue. Bar indicates 20μm. Axel Defays et al. Blood 2011;118:609-617 ©2011 by American Society of Hematology

Regulation of BAD-LAMP during pDC activation. Regulation of BAD-LAMP during pDC activation. (A) BAD-LAMP detection by immunoblot. Cell lysates from different cell types were separated by SDS-PAGE and revealed using the 34.2 mAb against BAD-LAMP. A single specific band was detected in pDC extracts around 35 kDa and not in immature MoDCs (MoDCs i), lipopolysaccharide-activated MoDCs (MoDCs m), nor in total PBMCs. HeLa cells transfected with BAD-LAMP cDNA (HeLa BAD) and control (HeLa nt) were used as a positive control both for specificity and as a reference for the glycosylation pattern. Asterisk (*)–marked lanes were loaded with a lower amount of total proteins to compensate for the high BAD-LAMP expression levels in transfected cells. Actin levels are shown as loading controls. (B) BAD-LAMP mRNA levels are down-regulated upon IL-3 treatment and CpG activation. Purified pDCs were cultivated in presence of IL-3 for 6 or 24 hours and stimulated or not with A-, B- or C-type CpG ODNs. BAD-LAMP mRNA levels were determined using quantitative RT-PCR. Levels for CpG-treated cells were normalized relative to the IL-3–only condition. Results are from one representative experiment (n = 3). (C) BAD-LAMP is down-regulated at the protein level upon CpG activation. After 24 hours of culturing freshly isolated pDCs (filled graph) with IL-3 (solid black line) and A-type CpG ODN (dashed gray line), BAD-LAMP expression monitored by intracellular FACS staining was down-regulated in pDCs. IL-3 treatment was sufficient to decrease BAD-LAMP levels. (D) BAD-LAMP is no longer detectable by immunoblot in pDCs after 24 hours of A-type CpG ODN stimulation. Low amounts of HeLa BAD and HeLa nt (*) were used as a specificity control. Actin levels are shown as loading controls. Axel Defays et al. Blood 2011;118:609-617 ©2011 by American Society of Hematology

BAD-LAMP is a marker of blastic pDC neoplasms. BAD-LAMP is a marker of blastic pDC neoplasms. (A) Immunohistochemistry on paraffin sections of skin lesions from patients with BPDCNs reveal a massive infiltration of BAD-LAMP+ cells (arrows, ×400). Negative staining with mouse IgG isotype control is shown on the right. (B) A larger-scale analysis by tissue arrays revealed that > 78% of biopsies were BAD-LAMP+ among 33 patients diagnosed with a CD4+/CD56+ malignancy (left). An example of a BAD-LAMP+ biopsy from the tissue array is also shown (right). Axel Defays et al. Blood 2011;118:609-617 ©2011 by American Society of Hematology

BAD-LAMP is localized in the ERGIC BAD-LAMP is localized in the ERGIC. (A) Immunofluorescence staining for BAD-LAMP in purified pDCs. BAD-LAMP is localized in the ERGIC. (A) Immunofluorescence staining for BAD-LAMP in purified pDCs. BAD-LAMP (green, top panels) costaining with early endosomal marker transferrin receptor (TfR, red) and the lysosomal marker LAMP1 (blue) show no overlap. The BAD-LAMP (green, bottom panels) and the ERGIC marker ERGIC53 (red) display a strong colocalization (arrow) confirmed by Pearson coefficient calculation (0.6). Bar indicates 10μm. (B) Analysis of BAD-LAMP glycosylation by enzymatic treatments. Immunoprecipitation from pDC lysate and subsequent endoglycosidase H (EndoH) treatment reveals that BAD-LAMP glycosylation remains endo H sensitive. Total lysate and antibodies alone (Ab) are shown as controls. (C) Immunofluorescence staining for BAD-LAMP in pDCs cultured with IL-3 and A-type CpG ODN for 6 hours. BAD-LAMP (green) was lost whereas ERGIC53 distribution (blue) was not affected by activation. Bar indicates 10μm. (D) Confocal microscopy of BAD-LAMP heterologous expression in human monocyte-derived DCs. Six hours after transfection, BAD-LAMP (green) and ER-resident PDI (red) displayed extensive colocalization. Bar indicates 10μm. Pearson coefficient values are shown as R. Axel Defays et al. Blood 2011;118:609-617 ©2011 by American Society of Hematology

BAD-LAMP colocalizes with UNC93B1 in transfected HeLa cells. BAD-LAMP colocalizes with UNC93B1 in transfected HeLa cells. (A) BAD-LAMP and UNC93B1 have different cellular localization when overexpressed together in HeLa cells. BAD-LAMP (green, top) is mainly targeted to the plasma membrane with a small portion is found in endocytic compartments. UNC93B1 (red, bottom) is localized in the ER. Bar indicates 20μm. (B) When coexpressed, BAD-LAMP (green) and UNC93B1 (red) colocalize in large endosomal intracellular vesicles (top panels, arrows). Conversely, upon expression of the structurally related endosomal resident DC-LAMP (green), intracellular trafficking UNC93B1 (red) remains unchanged (bottom panels). (C) Flag-tagged BAD-LAMP mutants have different sorting behaviors. Flag-BAD-LAMP (wt) is targeted to the cell surface and partially to endosomes (green, left panels), whereas the Flag-BAD-LAMP Y276A mutant is almost exclusively localized at the plasma membrane. Flag-BAD-LAMP ΔCt mutant is retained in the ER. Upon cotransfection with His-UNC93B1 (red, right panels), all of the different flag-tagged forms of BAD-LAMP (green, right panels) are sorted together with His-UNC93B1 (red) in the same intracellular endosomal compartments (arrows). Pearson coefficient values are shown as R. Axel Defays et al. Blood 2011;118:609-617 ©2011 by American Society of Hematology

BAD-LAMP–dependent sorting of UNC93B1 to the endosomes. BAD-LAMP–dependent sorting of UNC93B1 to the endosomes. Immunofluorescence confocal microscopy of HeLa cells transfected with an eGFP-tagged BAD-LAMP fusion. BAD-LAMP-GFP (green) is sorted to intracellular compartments that are mostly LAMP1+ (blue, arrow, top panels). In cells cotransfected with BAD-LAMP-GFP and UNC93B1 (red, bottom panels), the 2 molecules are sorted together in LAMP1+ intracellular compartments. Pearson coefficient values are shown as R. Axel Defays et al. Blood 2011;118:609-617 ©2011 by American Society of Hematology