Journal: eLife

Article Title: Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction

doi: 10.7554/eLife.62927

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-human LAIR-1 (rabbit polyclonal) , ATLAS antibodies , Cat number: HPA011155 , (Immunohistochemistry: 1:500).

Techniques: In Vitro, In Vivo, Transduction, Flow Cytometry, Immunohistochemistry, Immunofluorescence, Recombinant, Cell Stimulation, Luminex, Selection, Lysis, Plasmid Preparation, Software

Journal: Nature cell biology

Article Title: The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development

doi: 10.1038/ncb3158

Figure Lengend Snippet: A, Effects of shRNA-mediated silencing of expression of indicated ITIM receptors on growth of MV4-11 and NB4 AML cells. The cells were counted on day 6 post-infection, and normalized to cells treated with scrambled shRNAs (mean ± s.e.m., Student's t-test; n=3 samples, sh-LAIR1 *** p < 0.0001; sh-KIR3DL1 ** p = 0.0051; sh-PECAM1 ** p = 0.0061; sh-SIGLEC11 ** p = 0.0089; sh-SIGLEC6 * p = 0.0326; sh-LILRB1 * p = 0.0412; sh-LILRB2 ** p = 00059; sh-LILRB3 *** p < 0.0001; sh-LILRB4 *** p < 0.0001; sh-KIR2DL2 * p = 0.0182; sh-KIRG1 ** p = 0.0096). B, Flow cytometry analysis showing that LAIR1 is highly expressed on the cell surfaces of RCH-ACV, 697, Kasumi2, MV4-11, U937, and THP1 human leukemia cells but not on K562 cells. X-axis indicates LAIR1 expression, and Y-axis indicates cell numbers. The bar graph summarized MFIs of cell surface LAIR1 (relative fold changes to isotype control). C, Endogenous LAIR1 expression was inhibited by lentivirus vector-based expression of different shRNAs (226, 277, 593) in MV4-11 cells as determined by western blotting at 48 hours after lentiviral infection. D, Cell-surface LAIR1 expression was inhibited by shRNAs in MV4-11 cells as determined by flow cytometry at 48 hours after lentiviral infection. MFIs (obtained after subtraction of the background MFI of the unstained control) indicate LAIR1 expression levels; GFP expression indicates infection. Data are from a single experiment, representative of 3 independent experiments. E, Treatment with shRNA targeting lair1 inhibited the growth of MV4-11 cells. F, Expression from Flag- lair1 -6m is not silenced by shRNA 226 as determined by western blotting. G, Rescue of lair1 -knockdown phenotype. RFP-tagged mutant lair1 (6m) infected MV4-11 cells are resistant to the shRNA 226-induced growth inhibition. H, Apoptosis is increased in MV4-11 cells after treatment with shRNA 226 to inhibit lair1 expression (mean ± s.e.m., Student's t-test; n=3 samples, Day 2 * p = 0.0112; Day 4 *** p < 0.0001; Day 6 *** p < 0.0001; Day 9 *** p < 0.0001). I, Treatment with each shRNA targeting lair1 inhibited the growth of 697 cells. J, Lair1 knockdown inhibited the growth of THP1, U937, RCH-ACV, and Kasumi2 cells as measured on day 6. In Figs 1B, E, G, I, and J, data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results.

Article Snippet: For analysis of human hematopoietic engraftment in NSG mice, we followed our previously published protocol , and used anti-human LAIR1 (clone 342219, R&D Systems) and anti-human CD45-PE (mouse, HI30, BD Pharmingen, 1:50 dilution) to quantify the total human AML engraftment.

Techniques: shRNA, Expressing, Infection, Flow Cytometry, Control, Plasmid Preparation, Western Blot, Knockdown, Mutagenesis, Inhibition

Journal: Nature cell biology

Article Title: The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development

doi: 10.1038/ncb3158

Figure Lengend Snippet: A, MV4-11 cells (1×10 6 cells) were infected with virus designed to express GFP and either scrambled shRNA or shRNA 226. GFP + cells were collected and transplanted into NSG mice (n = 7 mice) one day post-infection. Left panels show representative flow cytometry plots indicating decreased BM engraftment of MV4-11 cells treated with shRNA targeting lair1 . Staining with anti-human CD45 and anti-LAIR1 antibodies confirmed engraftment was from transplanted human leukemia cells. The percentages of each population are indicated in red numbers, and the median fluorescent intensity of LAIR1 expression is indicated in black numbers. The panel on the right plots percentages of GFP + cells in BM, spleen, liver, and PB at 1 month after transplantation (mean ± s.e.m., Student's t-test; n=7 samples, BM *** p < 0.0001; Spleen ** p = 0.0004; Liver * p = 0.0233; PB * p = 0.01471). B, Comparison of the sizes of spleens of the mice transplanted with control MV4-11 leukemia cells or cells expressing shRNA targeting lair1 (mean ± s.e.m., Student's t-test; n=7 samples, ** p = 0.0028). C, GFP + 697 cells infected by scrambled shRNA or shRNA 226 virus were collected, and 5×10 5 cells were transplanted into NSG mice (n = 5 mice for Scramble, n = 7 mice for lair1 null). Left are representative flow cytometry plots showing the decreased BM engraftment of lair1- knockdown 697 cells. Human CD45 and LAIR1 antibody staining confirmed engraftment was from transplanted human leukemia cells. The percentages of each population are indicated in red numbers, and the median fluorescent intensity of LAIR1 expression is indicated in black numbers. Shown on the right are percentages of GFP + cells in BM, spleen, liver, and PB at 1 month after transplantation (mean ± s.e.m., Student's t-test; n = 5 mice for Scramble, n = 7 mice for lair1 null, BM ** p = 0.0004; Spleen ** p = 0.0002; Liver *** p <0.0001; PB * p = 0.0057). D, Comparison of the sizes of livers of the mice transplanted with control or lair1 -knockdown 697 leukemia cells (mean ± s.e.m., Student's t-test; n = 5 mice for Scramble, n = 7 mice for lair1 null, * p = 0.0048). E, Summary of the effects of lair1 silencing in the indicated human leukemia cell lines on inhibition of cell growth in vitro and xenograftment in NSG mice.

Article Snippet: For analysis of human hematopoietic engraftment in NSG mice, we followed our previously published protocol , and used anti-human LAIR1 (clone 342219, R&D Systems) and anti-human CD45-PE (mouse, HI30, BD Pharmingen, 1:50 dilution) to quantify the total human AML engraftment.

Techniques: Infection, Virus, shRNA, Flow Cytometry, Staining, Expressing, Transplantation Assay, Comparison, Control, Knockdown, Inhibition, In Vitro

Journal: Nature cell biology

Article Title: The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development

doi: 10.1038/ncb3158

Figure Lengend Snippet: A, Survival curves of mice receiving 3,000 pooled YFP + BM cells that were collected from primary recipients transplanted with WT or lair1 -null MLL-AF9 AML cells (n = 10 mice; p = 0.00002, log-rank test). B, Comparison of the spleen sizes of mice that were transplanted with WT or lair1 -null MLL-AF9 AML cells collected from secondary recipients at 18 days or 28 days after transplantation (mean ± s.e.m., Student's t-test; n = 5 mice, 18 days * p = 0.0245; 28 days ** p = 0.0006). C, Summary of the percentages of YFP + WT and lair1 -null MLL-AF9 leukemia cells in PB (open squares or open diamonds) or BM (red squares or red diamonds) of secondarily transplanted mice over time. Mice that were moribund and euthanized are indicated by red spots, in which the percentages of YFP + cells in BM (instead of PB) were used in this analysis (n = 10 mice). D, Summary of percentages of YFP + AML cells in PB, BM, and spleen of secondary recipient mice transplanted with the WT or lair1 -null MLL-AF9 AML cells at day 18 (upper panel) and at day 28 (lower panel) post-transplant (mean ± s.e.m., Student's t-test; n = 5 mice, 18 days PB * p = 0.0124; BM * p = 0.0243; Spleen * p = 0.0332; 28 days PB ** p = 0.00035; BM *** p < 0.0001; Spleen ** p =0.0014). E, Histological analysis of AML infiltration in the livers, spleens, and PB of mice after secondary transplant of WT and lair1 -null AML cells at 4 weeks and 20 weeks (hematoxylin/eosin staining for livers and spleens, HEMA 3 staining for cytospin-prepared PB samples). Shown are representative images from at least three similar images. Scale bar is 100 μM. F, Survival curves of mice transplanted with 1×10 5 pooled GFP + BM cells that were collected from primary recipients transplanted with WT or lair1 -null AML1-ETO9a cells (n = 10 mice; p = 0.00012, log-rank test). G, Percentages of AML1-ETO9a GFP + leukemia cells in BM at 1 month after secondary transplantation (mean ± s.e.m., Student's t-test; n = 10 mice; *** p < 0.0001).

Article Snippet: For analysis of human hematopoietic engraftment in NSG mice, we followed our previously published protocol , and used anti-human LAIR1 (clone 342219, R&D Systems) and anti-human CD45-PE (mouse, HI30, BD Pharmingen, 1:50 dilution) to quantify the total human AML engraftment.

Techniques: Comparison, Transplantation Assay, Staining

Journal: Nature cell biology

Article Title: The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development

doi: 10.1038/ncb3158

Figure Lengend Snippet: A, Representative flow cytometry plots showing that secondarily transplanted mice receiving lair1 -null AML cells had markedly decreased percentages of Mac1 + Kit + cells and increased differentiated B220 + and CD3 + cells in YFP + compartments compared to mice receiving WT AML cells. Three pairs of mice were used for the comparison. B-D, Percentages of YFP + Mac-1 + Kit + , YFP + Mac-1 + Gr-1 + , YFP + B220 + , and YFP + CD3 + cells in (B) BM, (C) PB, and (D) spleen of secondary recipient mice transplanted with the WT or lair1 -null MLL-AF9 AML cells at day 18 post-transplant (mean ± s.e.m., Student's t-test; n = 5 mice; (B) Mac1+/Gr1+ * p = 0.0195; Mac1+/kit+ ***p <0.0001; B220+ ** p = 0.0025; CD3+ * p = 0.0256; (C) Mac1+/Gr1+ * p = 0.0211; Mac1+/kit+ ***p <0.0001; CD3+ ***p <0.0001; (D) Mac1+/Gr1+ * p = 0.0342; Mac1+/kit+ **p =0.0015; B220+ *** p <0.0001; CD3+ ** p = 0.0027). E, Comparison of colony-forming activity of WT and lair1 -null MLL-AF9 + BM cells during serial replating. Data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. F, Left panel shows typical morphology of WT and lair1 -null colonies formed by YFP + Mac-1 + Kit + cells collected from secondarily transplanted mice at 28 days post-transplant. The right panel is a comparison of colony-forming activity of Mac1 + Kit + cells from MLL-AF9 + bone marrow cells harvested at 28 days after secondary transplantation. Scale bar is 10 μM. Data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. G, Survival curve of tertiary recipient mice transplanted with 1,000 YFP + Mac1 + Kit + cells from MLL-AF9 + WT or lair1 -null BM cells harvested at 28 days after secondary transplantation (n = 10 mice; p = 0.000022, log-rank test). H, Flow cytometry analysis of apoptosis in mouse MLL-AF9 YFP + (AML) cells and YFP - (normal) cells. Early apoptosis was detected as Annexin V positive/PE positive/7-AAD negative staining and late apoptosis was detected as Annexin V positive/PE positive/7-AAD positive staining (mean ± s.e.m., Student's t-test; n =3 samples, *** p < 0.0001). I, Limiting dilution assays comparing the frequencies of AML stem cells in WT and lair1 -null MLL-AF9 + AML. The indicated YFP + WT and lair1 -null MLL-AF9 + BM cells that were collected from primary recipients were co-transplanted with 2×10 5 bone marrow competitor cells into lethally irradiated recipients. The CRUs were calculated by L-Calc software.

Article Snippet: For analysis of human hematopoietic engraftment in NSG mice, we followed our previously published protocol , and used anti-human LAIR1 (clone 342219, R&D Systems) and anti-human CD45-PE (mouse, HI30, BD Pharmingen, 1:50 dilution) to quantify the total human AML engraftment.

Techniques: Flow Cytometry, Comparison, Activity Assay, Transplantation Assay, Negative Staining, Staining, Irradiation, Software

Journal: Nature cell biology

Article Title: The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development

doi: 10.1038/ncb3158

Figure Lengend Snippet: A, Phospho-SHP-1 and total SHP-1 levels in lair1 -null and control MLL-AF9 + BM cells from both primarily and secondarily transplanted mice were determined by western blotting. The experiment was repeated for 5 times, which gave similar results. B, Retrovirally-expressed SHP-1 increased CFU numbers of lair1 -null AML cells compared to cells that were infected with control or SHP-2-expressing viruses in colony-forming assays. Retrovirally-expressed SHP-1 and SHP-2 had similar levels as endogenous proteins in WT controls. C-D, Retrovirally-expressed SHP-1 rescued lair1 -null phenotype upon tertiary transplantation. MLL-AF9 + WT or lair1 -null BM cells in secondarily transplanted mice were harvested at 28 days and were infected by SHP-1 encoding or control virus. (C) Survival curves of mice transplanted with 3,000 of these ectopically SHP-1-expressing, SHP-2-expressing, or control cells (n= 5 mice; p < 0.0001, log-rank test). (D) Percentages of retrovirus-infected (GFP + ) AML cells in PB of tertiary recipient mice after 28 days of transplantation (mean ± s.e.m., Student's t-test; n =3 samples, *** p < 0.0001). E-G, Bone marrow cells isolated from 5-fluorouracil pretreated SHP-1 fl/fl mice were transformed with retroviral MLL-AF9 and then transduced by infection with empty vector or Cre-expressing vector to generate control (SHP-1 fl/fl + Con) or SHP-1-deficient (SHP-1 fl/fl + Cre) MLL-AF9 AML cells. (E) Survival curves of mice receiving 3,000 GFP + SHP-1 fl/fl + Con or SHP-1 fl/fl + Cre MLL-AF9 AML cells (n = 10; p < 0.0001, log-rank test). (F) Comparison of percentages of GFP + AML cells in the PB from SHP-1 fl/fl + Con and SHP-1 fl/fl + Cre MLL-AF9 AML cells injected mice (mean ± s.e.m., Student's t-test; n = 10 mice; ** p = 0.0067). (G) Comparison of colony-forming abilities of SHP-1 fl/fl + Con and SHP-1 fl/fl + Cre MLL-AF9 AML cells. H, Bone marrow cells isolated from 5-fluorouracil pretreated SHP-1 fl/fl mice were transduced by infection with empty vector or Cre expression vector to generate control (SHP-1 fl/fl + Con) or SHP-1-deficient (SHP-1 fl/fl + Cre) cells without the MLL-AF9 transformation. Shown is the comparison of colony-forming abilities between control and SHP-1-deficient bone marrow cells. I, Total SHP-1 and CAMK1 levels in WT and lair1 -null BM cells were determined by western blotting. In Fig. 5B, G, and H, data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results.

Article Snippet: For analysis of human hematopoietic engraftment in NSG mice, we followed our previously published protocol , and used anti-human LAIR1 (clone 342219, R&D Systems) and anti-human CD45-PE (mouse, HI30, BD Pharmingen, 1:50 dilution) to quantify the total human AML engraftment.

Techniques: Control, Western Blot, Infection, Expressing, Transplantation Assay, Virus, Isolation, Transformation Assay, Retroviral, Plasmid Preparation, Comparison, Injection

Journal: Nature cell biology

Article Title: The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development

doi: 10.1038/ncb3158

Figure Lengend Snippet: A, Retrovirally-expressed WT SHP-1 or mutants C 453 S or 4YF (278, 303, 538, 566), but not the SHP-1 PTP domain (PTPc), increased CFU numbers of lair1 -null AML cells. Data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. B-C, Retrovirally-expressed SHP-1 WT, C 453 S, and 4YF, but not SHP-1 PTPc, rescued lair1 -null phenotype upon transplantation. The green, blue, and purple dotted lines representing SHP-1 WT, C 453 S, and 4YF virus infected leukemia BM cells reversed lair1 -null MLL-AF9 + leukemia development. The red dotted line representing control virus infected leukemia BM cells and black dotted line representing SHP-1 PTP domain virus infected leukemia BM cells were unable to rescue (n= 5 mice; (B) p < 0.0001, log-rank test; (C) *** p < 0.0001, mean ± s.e.m., Student's t-test). D, Total CAMK1 and CAMK5 levels in lair1 -null and control MLL-AF9 + BM cells from both primarily and secondarily transplanted mice were determined by western blotting. E, Retrovirally-expressed CAMK1 increased CFU numbers of lair1 -null AML cells. Data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. F-G, Retrovirally-expressed CAMK1 rescued lair1 -null phenotype upon transplantation. MLL-AF9 + WT or lair1 -null BM cells in secondarily transplanted mice were harvested at 28 days and were infected by CAMK1-encoding or control virus (n= 5 mice, (F) p < 0.0001, log-rank test; (G) *** p < 0.0001, mean ± s.e.m., Student's t-test). H, Endogenous CAMK1 and SHP-1 interact with each other as determined by bi-directional pull down assays. WT MLL-AF9 + BM cells (1×10 7 cells) were lysed by RIPA buffer, and indicated antibodies were used for immunoprecipition and western blot. I, The C-terminal phosphatase-active domain of SHP-1 did not bind to CAMK1 in transfected 293T cells. The indicated Flag-tagged WT or mutant SHP-1 proteins were overexpressed in 293 cells. Flag antibody was used to precipitate SHP-1 proteins, and the Flag or CAMK1 antibodies were used in western blots.

Article Snippet: For analysis of human hematopoietic engraftment in NSG mice, we followed our previously published protocol , and used anti-human LAIR1 (clone 342219, R&D Systems) and anti-human CD45-PE (mouse, HI30, BD Pharmingen, 1:50 dilution) to quantify the total human AML engraftment.

Techniques: Transplantation Assay, Virus, Infection, Control, Western Blot, Transfection, Mutagenesis

Journal: Nature cell biology

Article Title: The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development

doi: 10.1038/ncb3158

Figure Lengend Snippet: A, Phospho-CREB and total CREB levels in lair1 -null and control MLL-AF9 + BM cells from both primarily and secondarily transplanted mice were determined by western blotting. B, Retrovirally-expressed CREB WT, but not CREB S129A, S133A, or S129/S133A, increased CFU numbers of lair1 -null AML cells. Data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. C-D, Retrovirally-expressed CREB WT, but not CREB mutants, rescued lair1 -null phenotype upon transplantation. While the red dotted line representing CREB WT virus infected leukemia BM cells reversed the lair1 -null MLL-AF9 + leukemia development, the black, blue, brown, and green dotted lines representing control, CREB S129A, CREB S133A, CREB S129/133A virus each infected leukemia BM cells were unable to do so (n= 5 mice; (C) p < 0.0001, log-rank test; (D) *** p < 0.0001, mean ± s.e.m., Student's t- test). E, Treatment with a CREB inhibitor XX15 reduced CREB phosphorylation and the colony forming ability of WT AML cells. The molecule structure of the CREB inhibitor XX15 is shown. Data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results.

Article Snippet: For analysis of human hematopoietic engraftment in NSG mice, we followed our previously published protocol , and used anti-human LAIR1 (clone 342219, R&D Systems) and anti-human CD45-PE (mouse, HI30, BD Pharmingen, 1:50 dilution) to quantify the total human AML engraftment.

Techniques: Control, Western Blot, Transplantation Assay, Virus, Infection, Phospho-proteomics

Journal: Nature cell biology

Article Title: The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development

doi: 10.1038/ncb3158

Figure Lengend Snippet: A, Expression of lair1 mRNA differs significantly in BM and PB of AML patients (n = 7 BM or 19 PB respectively) from those in normal samples (n = 10 BM or 9 PB; mean ± s.e.m., Student's t-test; BM * p = 0.0329; PB * p = 0.0421). B, Representative flow cytometry plots showing different LAIR1 expression patterns in normal mononuclear cells (Cord blood) and primary AML cells (type 1 and type 2). A representative plot of a flow cytometry analysis (from at least 3 similar images) of an type 1 sample shows that cells are present that express both high and low levels of LAIR1. Type 2 samples mainly contain LAIR1 high cells. Data on these primary AML samples are listed in sTable 3. C, Summary of percentages of hCD45 + cells in PB of NSG mice transplanted with LAIR1 low or LAIR1 high primary human AML cells from 2 patient samples at 4 months after transplantation (mean ± s.e.m., Student's t-test, n= 5 mice, *** p < 0.0001). D&G, Expression of shp-1 or camk1 mRNA negatively correlates with the overall survival of AML patients. n = 82 samples for high and n = 83 samples for low (TCGA database); (D) p =0.0205; (G) p =0.0037, log-rank test). E&H, Treatment with shRNAs targeting shp-1 or camk1 inhibited the growth of MV4-11 cells. F&I, Increased apoptosis in shp-1 -silenced (by shRNA 698) or camk1 -silenced MV4-11 cells (mean ± s.e.m., Student's t-test; n =3 samples, (F) Day 3 *p =0.0324; Day 9 ** p = 0.0019; (I) Day 3 *p =0.0302; Day 9 ** p = 0.0031). J, Endogenous SHP-1 and CAMK1 expression was silenced with shRNAs (226, 277) in MV4-11 cells as determined by western blotting at 72 hours after shRNA-encoding lentiviral infection. K, Expression of lair1 , camk1 , and shp-1 in 165 AML patients (TCGA database) showed highly significant positive correlations. These correlations were also supported by clustering analysis. L, CREB inhibitor XX15 treatment inhibited the growth of MV4-11 cells. M, Flow cytometry analysis of apoptosis of MV4-11, U937, and 697 cells after one day of treatment with XX15 (mean ± s.e.m., Student's t-test; n =3 samples, MV4-11 *p =0.0411; U937 ** p = 0.0216; 697 *p =0.0294). In Fig. 8E, H, and L, data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results.

Article Snippet: For analysis of human hematopoietic engraftment in NSG mice, we followed our previously published protocol , and used anti-human LAIR1 (clone 342219, R&D Systems) and anti-human CD45-PE (mouse, HI30, BD Pharmingen, 1:50 dilution) to quantify the total human AML engraftment.

Techniques: Expressing, Flow Cytometry, Transplantation Assay, shRNA, Western Blot, Infection

Journal: eLife

Article Title: Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction

doi: 10.7554/eLife.62927

Figure Lengend Snippet: ( A ) LAIR-2 overexpression is associated with improved overall survival in some tumors: head and neck squamous cell carcinoma (HNSC), thyroid carcinoma (THCA), thymoma (THYM) and skin cutaneous melanoma (SKCM). Patients were grouped in low 25% quantile (black) and high 25% quantile (red) of LAIR-2 mRNA expression for overall survival analysis. Hazards ratio indicating if high expression is associated with poor survival (HR(High)), and p-value (p(High)) indicating the significance of association was determined using Wald test as indicated. ( B ) NC410 is a biologic fusing LAIR-2 with a functional IgG1 to generate a dimeric fusion protein. ( C ) Avidity characterization of NC410 to human, mouse and rat collagen I and III as measured by Octet analysis. ( D ) Indicated amounts of collagen I were plate coated, and the binding of soluble LAIR-1 was inhibited by NC410. Asterisks indicate statistical significance (****p<0.0001, two-way ANOVA). ( E, F ) The human LAIR-1 (hLAIR-1) extracellular domain was fused with CD3z and stably expressed in a cell line containing an NFAT-GFP pathway reporter. LAIR-1 ligation and CD3 ligation induce NFAT-GFP signaling. A parental cell line containing the CD3 NFAT-GFP reporter without LAIR-1 was used as control (WT). NC410 protein was added at increasing concentrations and inhibited human collagen I (5 µg/mL)-mediated NFAT-GFP signaling through LAIR-1 binding by ( E ) FACS analysis and ( F ) Incucyte microscopy. Total green integrated intensity of WT and hLAIR-1 reporter cells is shown over time. Points represent the median of n = 3 (with experimental triplicates in each independently performed experiment), and the whiskers indicate the 95% confidence interval (CI). Isotype control was used at the highest concentration (100 µg/mL) and showed no inhibition of NFAT-GFP signaling. Anti-human LAIR (8A8 clone) and anti-mouse CD3 were used as positive controls. Closed circles in ( F ) indicate NC410 treatment, and open circles indicate control treatment. Significant differences between different treatment groups of hLAIR-1 reporter cells are indicated (and tested using a two-way ANOVA with Dunnett’s correction). In all plots: *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Article Snippet: Peptide, recombinant protein , LAIR-2 FES , This paper/NextCure , , See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation.

Techniques: Over Expression, Expressing, Functional Assay, Binding Assay, Stable Transfection, Ligation, Control, Microscopy, Concentration Assay, Inhibition

Journal: eLife

Article Title: Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction

doi: 10.7554/eLife.62927

Figure Lengend Snippet: NC410 dose-response measured by Incucyte imaging during 24 hr. Representative microscopic images (10×) of WT and hLAIR-1 reporter cells over time. NC410 protein was added at increasing concentrations and inhibited collagen I (5 µg/mL)-mediated NFAT-GFP signaling through LAIR-1 binding. Isotype control was used at the highest concentration (100 µg/mL) and showed no inhibition of NFAT-GFP signaling. Pictures were taken every 1 hr for 24 hr. Representative images from one out of three independently performed experiments (each with experimental triplicates).

Article Snippet: Peptide, recombinant protein , LAIR-2 FES , This paper/NextCure , , See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation.

Techniques: Imaging, Binding Assay, Control, Concentration Assay, Inhibition

Journal: eLife

Article Title: Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction

doi: 10.7554/eLife.62927

Figure Lengend Snippet: In a non-tumor model, 1 × 10 7 total human peripheral blood mononuclear cells were adoptively transferred intravenously to NSG mice (N = 6/group) on day 0. Mice were treated with indicated doses of NC410 by intravenous injection on days 0 and 2. On day 6, mice were euthanized and spleens were analyzed for naïve (CD45RA + CCR7 + ), central memory (CM, CD45RA - CCR7 + ), effector memory (EM, CD45RA - CCR7 - ) and effector (CD45RA + CCR7 - ) CD4 + ( A ) and CD8 + ( B ) T cell populations. The graph shows the percentage of T cell subpopulations as a percentage of total human T cells. ( C–E ) Cell counts of ( C ) CD4 + EM, ( D ) CD8 + EM and ( E ) CD8 + effector T cells in the spleen. The graphs show the means ± SD (error bars). Asterisks indicate statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA followed by Tukey’s multiple comparisons.

Article Snippet: Peptide, recombinant protein , LAIR-2 FES , This paper/NextCure , , See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation.

Techniques: Injection

Journal: eLife

Article Title: Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction

doi: 10.7554/eLife.62927

Figure Lengend Snippet: ( A ) Histogram plot showing mouse P815 tumor cells stained with NC410-biotin or control Fc-biotin. ( B ) Mouse P815 cells and human HT-29 cells were stained with titrated concentrations of NC410-biotin. ( C ) 1.5 × 10 7 total human peripheral blood mononuclear cells were adoptively transferred by intravenous injection to NSG mouse on day 0, and 2 × 10 5 P815 tumor cells in Matrigel were injected subcutaneously on day 1. Mice were treated on days 1 and 3 with indicated dose of NC410 or control IgG1 (10 mg/kg) by intraperitoneal injection (N = 6/group). P815 tumor was measured every 2–3 days with a caliper, and tumor volume was calculated. Asterisks indicate statistical significance: **p<0.01, ***p<0.001, two-way ANOVA with Sidak multiple comparisons correction. ( D–G ) CD8 + ( D, E ) and CD4 + ( F, G ) T cell analysis of cell numbers in blood on days 6 ( D, F ) and 13 ( E, G ). The graphs show the means ± SD (error bars). Asterisks indicate statistical significance: *p<0.05 **p<0.01 ***p<0.001, two-way ANOVA with Sidak multiple comparisons correction.

Article Snippet: Peptide, recombinant protein , LAIR-2 FES , This paper/NextCure , , See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation.

Techniques: Staining, Control, Injection, Cell Analysis

Journal: eLife

Article Title: Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction

doi: 10.7554/eLife.62927

Figure Lengend Snippet: ( A ) Humanized tumor model of HT-29 tumor cells injected subcutaneously in the presence of human peripheral blood mononuclear cells (PBMCs). 2 × 10 7 total human PBMCs were adoptively transferred intravenously to NSG mice (N = 6/group) on day 0. 1 × 10 6 HT-29 tumor cells were injected subcutaneously with Matrigel on day 1. Mice were treated with 10 mg/kg NC410 or control by intraperitoneal injection, Q4D × 4 doses followed by Q7D until endpoint. Tumor growth was monitored 1–3 times a week. Asterisks indicate statistical significance: ****p<0.0001, two-way ANOVA with Sidak multiple comparisons correction. ( B ) Dose-dependent effect of NC410 (N = 6/group). Experimental conditions are the same as ( A ), with different doses of NC410. Asterisks indicate statistical significance: *p<0.05, ***p<0.001, two-way ANOVA with Tukey's multiple comparisons correction. ( C–G ) On day 27 after treatment with 10 mg/mL NC410, tumor and spleen tissues were isolated for tumor-infiltrating T cells (TILs) and cytokine analysis. ( C ) CD4 + and ( D ) CD8 + TIL cell numbers in the tumor. The cell number was counted by flow cytometry and normalized to weight (gram) of tumor tissue. Asterisks indicate statistical significance: *p<0.05, two-tailed t-test. ( E ) Cytokine production by TILs following ex vivo restimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 5 hr. Cells were intracellularly stained for IFN-γ and TNF-α, and the indicated cell populations were counted by flow cytometry and normalized to weight (gram) of tumor tissue. Asterisks indicate statistical significance: *p<0.05, two-tailed t-test. ( F–I ) Tissue lysate protein was extracted from tumor and spleen tissues. Analysis of tumor ( F, H ) and spleen ( G, I ) for cytokines ( F, G ) and chemokines ( H, I ) for analysis of local and systemic effects, respectively. Cytokines and chemokines were analyzed by Luminex and presented as the relative levels normalized to weight (gram) of tissue. ( J ) CXCL10, CXCL11 and CXCL12 correlation with tumor weight. The graphs show the means ± SD (error bars). Asterisks indicate statistical significance: *p<0.05, **p<0.01, two-tailed t-test.

Article Snippet: Peptide, recombinant protein , LAIR-2 FES , This paper/NextCure , , See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation.

Techniques: Injection, Control, Isolation, Flow Cytometry, Two Tailed Test, Ex Vivo, Staining, Luminex

Journal: eLife

Article Title: Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction

doi: 10.7554/eLife.62927

Figure Lengend Snippet: Immunofluorescence analysis of HT-29 cells stained with ( A ) isotype control, ( B ) NC410 or ( C ) pan-collagen antibody. 40× magnification ( D ) HT-29 cells were removed from culture flasks using increasing concentrations of EDTA for 10 min. Mean fluorescence intensity (MFI) of NC410 staining is shown, demonstrating that EDTA-treated HT-29 cells keep surface collagen expression. ( E ) HT-29 cells treated with 0.1 mM collagenase lose NC410 binding. Data from three independently performed experiments. ( F ) In vitro chromium release assay after 24 hr using HT-29 and peripheral blood mononuclear cells at three different effector to target ratios. 20:1 and 50:1 n = 16 and 100:1 n = 25 in 17 independently performed experiments. Closed circles indicate NC410 treatment, and open circles indicate control treatment. The graphs show the means ± SD (error bars). ***p<0.001, two-way ANOVA with Dunnett’s correction.

Article Snippet: Peptide, recombinant protein , LAIR-2 FES , This paper/NextCure , , See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation.

Techniques: Immunofluorescence, Staining, Control, Fluorescence, Expressing, Binding Assay, In Vitro, Release Assay

Journal: eLife

Article Title: Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction

doi: 10.7554/eLife.62927

Figure Lengend Snippet: ( A ) Humanized tumor model ( A ) of HT-29 tumor injected subcutaneously in the presence of human peripheral blood mononuclear cells (PBMCs). 1 × 10 7 total human PBMCs with or without T cell depletion were adoptively transferred intravenously to NSG mice (N = 5 or 6/group) on day 0. 1 × 10 6 HT-29 tumor cells were injected subcutaneously with Matrigel on day 1. Mice were treated with NC410, LAIR-2-FES (Fc dead FES mutant NC410) or controls by intraperitoneal injection, Q4D × 4 doses followed by Q7D until endpoint. Tumor growth was monitored two times a week. ( B ) Analysis of tumor growth. Asterisks indicate statistical significance compared to control. The graphs show the means ± SD (error bars). ****p<0.0001, two-way ANOVA followed by Tukey’s multiple comparisons. ( C ) The images of tumors obtained from PBMC-transferred mice on day 53.

Article Snippet: Peptide, recombinant protein , LAIR-2 FES , This paper/NextCure , , See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation.

Techniques: Injection, Mutagenesis, Control

Journal: eLife

Article Title: Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction

doi: 10.7554/eLife.62927

Figure Lengend Snippet: ( A ) Schematic representation of the humanized murine tumor model used. HT-29 tumor was injected subcutaneously in the presence of human peripheral blood mononuclear cells. Mice were treated with NC410 or control by intraperitoneal injection, Q4D × 4 doses followed by Q7D until endpoint. Mice were bled prior to start of experiment and weekly for 4 weeks. ( B ) Tumor growth kinetics with NC410. Asterisks indicate statistical significance: *p<0.05, ****p<0.0001, two-way ANOVA with Sidak multiple comparisons. ( C ) Analysis of collagen degradation products in serum at baseline, at weeks 1, 2, 3 and 4. reC1M: neo-epitope of MMP-2,9,13-mediated degradation of type I collagen; C3M: type III collagen degradation by MMP; C4M: type IV collagen degradation by MMP; C6M: neo-epitope of MMP-2-mediated degradation of type VI collagen; PRO-C3: pro-peptide of type III collagen/ECM formation/fibroblast activity; PRO-C6: pro-peptide of type VI collagen; VICM: neo-epitope of MMP-2,8, trypsin-mediated degradation of citrullinated vimentin; C4G: type IV collagen degraded by granzyme B (T cell activity/infiltration); closed circles indicate NC410 treatment, and open circles indicate control treatment. The graphs show the means ± SD (error bars). Asterisks indicate statistical significance: ****p<0.0001, two-way ANOVA followed with Sidak multiple comparisons.

Article Snippet: Peptide, recombinant protein , LAIR-2 FES , This paper/NextCure , , See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation.

Techniques: Injection, Control, Activity Assay

Journal: eLife

Article Title: Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction

doi: 10.7554/eLife.62927

Figure Lengend Snippet: ( A ) Schematic representation of the immunohistochemical stainings performed. ( B ) Representative hematoxylin and eosin (H&E), Masson Trichrome, NC410 and LAIR-1 staining in a stomach tumor specimen. ( C ) Representative NC410 and LAIR-1 analysis. The percentage of NC410 binding to tissue within a tumor was calculated by dividing the NC410 + stained area by the total tumor area. The number of LAIR-1 + cells was calculated by dividing the total number of positive cells within five regions of interest (ROIs) by the total surface in mm 2 of these ROIs. ( D ) Quantification of LAIR-1 and NC410 staining of 9–10 patients per tumor type across six different tumor types (head and neck squamous cell carcinoma [HNSC], skin cutaneous melanoma [SKCM], non-small cell lung carcinoma [NSCLC], high-grade serous ovarian carcinoma [HGSC], pancreatic adenocarcinoma [PDAC] and stomach adenocarcinoma [STAD]). ( E ) Higher magnification pictures of stomach cancer specimens show LAIR-1 + cells (depicted in red, right side) co-localizing with NC410-positive areas (depicted in red, left side).

Article Snippet: Peptide, recombinant protein , LAIR-2 FES , This paper/NextCure , , See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation.

Techniques: Immunohistochemical staining, Staining, Binding Assay

Journal: eLife

Article Title: Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction

doi: 10.7554/eLife.62927

Figure Lengend Snippet: ( A ) Representative NC410, LAIR-1, CD45, CD3, CD68 and CD163 staining in a stomach tumor specimen. For quantification of the immune cell counts, regions of interest (ROIs) were annotated on the tissue slides by drawing circles with a diameter of 600 µm at five random spots within the NC410-positive part of the tumor. Positive cells were quantified using the Positive Cell Detection tool. ( B ) Number of positive LAIR-1 mAb, CD45, CD3, CD68 and CD163 cells is shown across seven different tumor types (glioblastoma [GBM], head and neck squamous cell carcinoma [HNSC], skin cutaneous melanoma [SKCM], non-small cell lung carcinoma [NSCLC], high-grade serous ovarian carcinoma [HGSC], pancreatic adenocarcinoma [PDAC] and stomach adenocarcinoma [STAD]).

Article Snippet: Peptide, recombinant protein , LAIR-2 FES , This paper/NextCure , , See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation.

Techniques: Staining

Journal: eLife

Article Title: Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction

doi: 10.7554/eLife.62927

Figure Lengend Snippet: Representative hematoxylin and eosin (H&E), Masson Trichrome, NC410 and LAIR-1 staining for seven different tumor types (glioblastoma [GBM], head and neck squamous cell carcinoma [HNSC], skin cutaneous melanoma [SKCM], non-small cell lung carcinoma [NSCLC], high-grade serous ovarian carcinoma [HGSC], pancreatic adenocarcinoma [PDAC] and stomach adenocarcinoma [STAD]).

Article Snippet: Peptide, recombinant protein , LAIR-2 FES , This paper/NextCure , , See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation.

Techniques: Staining

Journal: eLife

Article Title: Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction

doi: 10.7554/eLife.62927

Figure Lengend Snippet: Representative hematoxylin and eosin (H&E), Masson Trichrome, NC410 and LAIR-1 staining for healthy tissue matching the tumors used in .

Article Snippet: Peptide, recombinant protein , LAIR-2 FES , This paper/NextCure , , See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation.

Techniques: Staining

Journal: eLife

Article Title: Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction

doi: 10.7554/eLife.62927

Figure Lengend Snippet: ( A ) Representative image of the three different immune phenotypes, immune-desert, immune-excluded and immune-inflamed, in a high-grade serous ovarian carcinoma (HGSC) specimen. ( B ) Patients within each tumor type as shown were characterized into immune-desert, immune-excluded and immune-inflamed based on CD3 + T cell presence and localization. ( C ) Quantification of NC410 staining across the different immune phenotypes in the seven different tumor types analyzed.

Article Snippet: Peptide, recombinant protein , LAIR-2 FES , This paper/NextCure , , See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation.

Techniques: Staining

Journal: eLife

Article Title: Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction

doi: 10.7554/eLife.62927

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , LAIR-2 FES , This paper/NextCure , , See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation.

Techniques: In Vitro, In Vivo, Transduction, Flow Cytometry, Immunohistochemistry, Immunofluorescence, Recombinant, Cell Stimulation, Luminex, Selection, Lysis, Plasmid Preparation, Software

Journal: eLife

Article Title: Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction

doi: 10.7554/eLife.62927

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , LAIR-2 FES , This paper/NextCure , , See Materials and methods LAIR-1- Fc, LAIR-2-Fc (NC410) and LAIR-2-Fc (NC410) FES generation.

Techniques:

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: C1q binds to CD33M and CD33m isoforms on the cell surface. ( A ) A representative flow cytometry plot showing binding of biotin C1q (20 ug/ml) to HEK293T cells transfected with plasmids encoding CD33m and CD33M and control mock plasmid. Cells were incubated with C1q as outlined in Materials and Methods. Numbers in graphs = % positive/MFI. Debris/dying cells was excluded on the basis of forward and side scatter. ( B ) Pooled flow cytometry data illustrating binding of C1q (at comparable levels) on the surface of CD33m/M transfected HEK293T cells and a lack of C1q binding on mock transfected cells. For A and B, N = 3. ( C ) Representative slot blot showing that C1q binds to cell-associated CD33m, CD33M or LAIR-1 in a dose dependent manner. Protein prepared from whole cell lysates (HEK293T cells transfected with plasmid encoding CD33m, CD33M or LAIR-1) was immobilized on membranes at increasing doses; biotin-whole C1q was in solution at 10 ug/ml. HSA = human serum albumin control. Protein concentration was calculated by the BCA method. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). ( D ) Pooled data analyses illustrating dose related interactions between biotin-C1q and cell-associated CD33m or CD33M represented in ( C) . ( B , D ), the mean ± SE of pooled data is shown; for all proteins and doses, N = 3–5. *P = < 0.05, **P = <0.01, ***P < 0.001. One-way ANOVA followed by Tukey’s pairwise multiple comparison was used.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Flow Cytometry, Binding Assay, Transfection, Plasmid Preparation, Incubation, Dot Blot, Protein Concentration

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: C1q triggers CD33 ITIM phosphorylation and CD33-LAIR-1 physical associations in human monocytes. Monocytes were either untreated (utx), treated with whole C1q, gC1q, C1q CLR or pervanadate (PV) as detailed in Materials and Methods. ( A , B ) Immunoblot analyses showing increased phosphorylated (p) CD33 after treatment with C1q and gC1q, but not C1q CLR. CD33 was immunoprecipitated with anti-CD33M antibody; tyrosine phosphorylation of CD33 (pCD33M) was detected with anti-phosphotyrosine (4G10) antibody. The membrane was stripped and re-probed with anti-CD33 WM53 antibody (CD33M). Arrows denote molecular weight of 67kD. Bar graphs denote pooled data expressed as fold change relative to total CD33 and corresponding statistical analyses. One-way ANOVA with post-hoc Tukey multiple comparisons was used to determine significance. N = 4 for A; N = 3 for B. ( C ) The addition of whole C1q (20 ug/ml) prompts concurrent increases in pLAIR-1 and pCD33 in a phospho-immunoreceptor array. ( D ) C1q CLR “tails” (20 ug/ml) do not elicit increases in pCD33M in the phospho-immunoreceptor array. Bar graphs represent fold change (plus/minus C1q), calculated from the mean pixel density of duplicates, as determined by densitometry analysis. Corresponding C1q minus/plus array data is located below each bar graph in C and D. Data from a typical array are shown. N = 3 for C and D. ( E ) Proximity ligation assay, performed on freshly isolated human monocytes as detailed in materials and methods, showing that whole C1q is required for CD33-LAIR-1 crosslinking. Red fluorescent dots represent molecular associations between CD33 and LAIR-1; blue represents nuclear staining with DAPI. CLR and gC1q represent the C1q collagen tail and globular heads of C1q, respectively. Original magnification = 60X. One of three representative experiments is shown.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Western Blot, Immunoprecipitation, Molecular Weight, Proximity Ligation Assay, Isolation, Staining

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Masking of CD33C2 domains impedes C1q binding on the cell surface. ( A , B ) Increased detection of the CD33C2, but not the CD33V domain or LAIR-1, occurs after sialidase treatment of THP-1 cells. Cells were treated with sialidase (20 mU/10 6 cells) for 60 min at 37 °C in a humidified incubator, resuspended twice in PBS, then in staining buffer for detection of CD33C2 domains (HIM3-4), CD33V domains (WM53) and LAIR-1. A = Pooled data, N ≥ 4. B = Representative experiment from A; gates were set according to isotype Ab controls. ( C ) Unmasking of CD33C2 domains with sialidase correlates with increased binding of C1q on THP-1 cells; N = 3–4. ( D ) Reduced levels of sialic acid on the THP-1 surface after sialidase treatment as revealed by decreased binding to Sambucus nigra lectin (SNL) (5 ug/ml). N = 5. ( E ) Pooled flow cytometry data showing highly expressed CD33C2 and CD33V domains and LAIR-1 on freshly isolated normal monocytes. Results represent mean +/− SEM. N = 3–4. For ( A , C , D ) solid bars = no sialidase; open bars = sialidase. Two-tailed Student’s t test was used to determine significance.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Binding Assay, Staining, Flow Cytometry, Isolation, Two Tailed Test

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Maturing DCs exhibit decreases in LAIR-1 and CD14 along with a reduced ability to detect CD33C2 epitopes. ( A , B ) Decreased expression of LAIR-1 and CD14 on DCs. ( C , D ) Pooled flow cytometry data showing CD33C2 domains (detected with mAb HIM3-4) and CD33V domains (detected with mAb WM53) on freshly isolated monocytes and DCs obtained after 5–6 days of culture with 50 U/ml rGM-CSF and 50 ng/ml rIL-4. ( E ) Pooled data showing co-detection of CD33C2 and CD33V domains on fresh monocytes and day 6 DCs. ( F ) Representative dot plot comparing co-detection of CD33C2/V epitopes on freshly isolated monocytes and day 6 DCs; gates were set based on isotype controls. (G ) Increased levels of sialic acid on immature DCs detected with Sambucus nigra lectin (SNL) linked to PECy7. For ( A – E ), N = 3–5; for (G), N = 2. Two-tailed Student’s t test was used to determine significance. Solid bars represent day 0; hatched bars represent day 6.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Expressing, Flow Cytometry, Isolation, Two Tailed Test

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Altered patterns of CD33C2/V and LAIR-1 expression on freshly isolated SLE versus normal blood monocytes. ( A , B ) Decreased CD33C2/CD33V, LAIR-1, CD14, on SLE blood myelomonocytes vs healthy monocytes as revealed by percent positive ( C ) Decreased LAIR-1, CD33C2/CD33V domains on SLE blood monocytes vs healthy monocytes as revealed by Mean Fluorescence Intensity (MFI). In ( A – C ) open bars represent normal (NL) monocytes; solid bars represent SLE myelomonocytes. Results represent the mean +/− SEM of pooled flow cytometry data; two-tailed Student’s t test ( A–C ) was used to determine significance. For ( A , B ); N = 9 for CD33C2 and CD33V; N = 8 for LAIR-1 and CD14 for both NL and SLE. For ( C ); N = 3 for both normal and SLE blood. Gate settings for flow analysis excluded debris and dead cells and are described in Supplementary Fig. .

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Expressing, Isolation, Fluorescence, Flow Cytometry, Two Tailed Test

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Myeloid cytokines distinctly control the coupled expression of CD33C2 and LAIR-1 in healthy and SLE cells. Freshly isolated adherent mononuclear cells from healthy subjects ( A , B ) and SLE patients ( C , D ) were cultured in parallel with either GM-CSF/IL-4 (G4) or M-CSF for 5 days, stained with anti-LAIR-1, CD33C2, CD33V antibodies and then analyzed by flow cytometry. ( A , C ): dot plots represent fluorescence versus forward light scatter patterns for a typical experiment; ( B , D ) represent the mean +/− SEM of pooled flow cytometry data. Two-tailed Student’s t test was used to determine significance. For normals, G4 vs M-CSF; N = 5 for CD33C2, N = 6 for CD33V, N = 4 for LAIR-1. For SLE, G4 vs M-CSF; N = 4 for CD33C2, LAIR-1; N = 3 for CD14.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Expressing, Isolation, Cell Culture, Staining, Flow Cytometry, Fluorescence, Two Tailed Test

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Evidence for domain-specific and concentration dependent associations between C1q and CD33 using purified proteins in slot blot assays. Unlabeled proteins were immobilized on membranes; biotin labeled proteins were in solution. ( A–C ) Dose related binding of immobilized whole C1q and gC1q, but not CLR, to biotin-CD33M (5 ug/ml). ( D ) Biotin-whole C1q (10 ug/ml) binds to immobilized CD33m (lacking the extracellular V-like domain and expressing the C2-like domain) in a dose specific manner. (HSA = human serum albumin control showing no reactivity. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). Pixel density was determined by densitometry. (C , D) Bar graphs represent the mean ± SE of pooled data; one-way ANOVA followed by Tukey’s pairwise multiple comparison was used to determine significance. N ≥ 3.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Concentration Assay, Purification, Dot Blot, Labeling, Binding Assay, Expressing

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: C1q binds to CD33M and CD33m isoforms on the cell surface. ( A ) A representative flow cytometry plot showing binding of biotin C1q (20 ug/ml) to HEK293T cells transfected with plasmids encoding CD33m and CD33M and control mock plasmid. Cells were incubated with C1q as outlined in Materials and Methods. Numbers in graphs = % positive/MFI. Debris/dying cells was excluded on the basis of forward and side scatter. ( B ) Pooled flow cytometry data illustrating binding of C1q (at comparable levels) on the surface of CD33m/M transfected HEK293T cells and a lack of C1q binding on mock transfected cells. For A and B, N = 3. ( C ) Representative slot blot showing that C1q binds to cell-associated CD33m, CD33M or LAIR-1 in a dose dependent manner. Protein prepared from whole cell lysates (HEK293T cells transfected with plasmid encoding CD33m, CD33M or LAIR-1) was immobilized on membranes at increasing doses; biotin-whole C1q was in solution at 10 ug/ml. HSA = human serum albumin control. Protein concentration was calculated by the BCA method. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). ( D ) Pooled data analyses illustrating dose related interactions between biotin-C1q and cell-associated CD33m or CD33M represented in ( C) . ( B , D ), the mean ± SE of pooled data is shown; for all proteins and doses, N = 3–5. *P = < 0.05, **P = <0.01, ***P < 0.001. One-way ANOVA followed by Tukey’s pairwise multiple comparison was used.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Flow Cytometry, Binding Assay, Transfection, Plasmid Preparation, Incubation, Dot Blot, Protein Concentration

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: C1q triggers CD33 ITIM phosphorylation and CD33-LAIR-1 physical associations in human monocytes. Monocytes were either untreated (utx), treated with whole C1q, gC1q, C1q CLR or pervanadate (PV) as detailed in Materials and Methods. ( A , B ) Immunoblot analyses showing increased phosphorylated (p) CD33 after treatment with C1q and gC1q, but not C1q CLR. CD33 was immunoprecipitated with anti-CD33M antibody; tyrosine phosphorylation of CD33 (pCD33M) was detected with anti-phosphotyrosine (4G10) antibody. The membrane was stripped and re-probed with anti-CD33 WM53 antibody (CD33M). Arrows denote molecular weight of 67kD. Bar graphs denote pooled data expressed as fold change relative to total CD33 and corresponding statistical analyses. One-way ANOVA with post-hoc Tukey multiple comparisons was used to determine significance. N = 4 for A; N = 3 for B. ( C ) The addition of whole C1q (20 ug/ml) prompts concurrent increases in pLAIR-1 and pCD33 in a phospho-immunoreceptor array. ( D ) C1q CLR “tails” (20 ug/ml) do not elicit increases in pCD33M in the phospho-immunoreceptor array. Bar graphs represent fold change (plus/minus C1q), calculated from the mean pixel density of duplicates, as determined by densitometry analysis. Corresponding C1q minus/plus array data is located below each bar graph in C and D. Data from a typical array are shown. N = 3 for C and D. ( E ) Proximity ligation assay, performed on freshly isolated human monocytes as detailed in materials and methods, showing that whole C1q is required for CD33-LAIR-1 crosslinking. Red fluorescent dots represent molecular associations between CD33 and LAIR-1; blue represents nuclear staining with DAPI. CLR and gC1q represent the C1q collagen tail and globular heads of C1q, respectively. Original magnification = 60X. One of three representative experiments is shown.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Western Blot, Immunoprecipitation, Molecular Weight, Proximity Ligation Assay, Isolation, Staining

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Masking of CD33C2 domains impedes C1q binding on the cell surface. ( A , B ) Increased detection of the CD33C2, but not the CD33V domain or LAIR-1, occurs after sialidase treatment of THP-1 cells. Cells were treated with sialidase (20 mU/10 6 cells) for 60 min at 37 °C in a humidified incubator, resuspended twice in PBS, then in staining buffer for detection of CD33C2 domains (HIM3-4), CD33V domains (WM53) and LAIR-1. A = Pooled data, N ≥ 4. B = Representative experiment from A; gates were set according to isotype Ab controls. ( C ) Unmasking of CD33C2 domains with sialidase correlates with increased binding of C1q on THP-1 cells; N = 3–4. ( D ) Reduced levels of sialic acid on the THP-1 surface after sialidase treatment as revealed by decreased binding to Sambucus nigra lectin (SNL) (5 ug/ml). N = 5. ( E ) Pooled flow cytometry data showing highly expressed CD33C2 and CD33V domains and LAIR-1 on freshly isolated normal monocytes. Results represent mean +/− SEM. N = 3–4. For ( A , C , D ) solid bars = no sialidase; open bars = sialidase. Two-tailed Student’s t test was used to determine significance.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Binding Assay, Staining, Flow Cytometry, Isolation, Two Tailed Test

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Maturing DCs exhibit decreases in LAIR-1 and CD14 along with a reduced ability to detect CD33C2 epitopes. ( A , B ) Decreased expression of LAIR-1 and CD14 on DCs. ( C , D ) Pooled flow cytometry data showing CD33C2 domains (detected with mAb HIM3-4) and CD33V domains (detected with mAb WM53) on freshly isolated monocytes and DCs obtained after 5–6 days of culture with 50 U/ml rGM-CSF and 50 ng/ml rIL-4. ( E ) Pooled data showing co-detection of CD33C2 and CD33V domains on fresh monocytes and day 6 DCs. ( F ) Representative dot plot comparing co-detection of CD33C2/V epitopes on freshly isolated monocytes and day 6 DCs; gates were set based on isotype controls. (G ) Increased levels of sialic acid on immature DCs detected with Sambucus nigra lectin (SNL) linked to PECy7. For ( A – E ), N = 3–5; for (G), N = 2. Two-tailed Student’s t test was used to determine significance. Solid bars represent day 0; hatched bars represent day 6.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Expressing, Flow Cytometry, Isolation, Two Tailed Test

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Evidence for domain-specific and concentration dependent associations between C1q and CD33 using purified proteins in slot blot assays. Unlabeled proteins were immobilized on membranes; biotin labeled proteins were in solution. ( A–C ) Dose related binding of immobilized whole C1q and gC1q, but not CLR, to biotin-CD33M (5 ug/ml). ( D ) Biotin-whole C1q (10 ug/ml) binds to immobilized CD33m (lacking the extracellular V-like domain and expressing the C2-like domain) in a dose specific manner. (HSA = human serum albumin control showing no reactivity. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). Pixel density was determined by densitometry. (C , D) Bar graphs represent the mean ± SE of pooled data; one-way ANOVA followed by Tukey’s pairwise multiple comparison was used to determine significance. N ≥ 3.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Concentration Assay, Purification, Dot Blot, Labeling, Binding Assay, Expressing

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: C1q binds to CD33M and CD33m isoforms on the cell surface. ( A ) A representative flow cytometry plot showing binding of biotin C1q (20 ug/ml) to HEK293T cells transfected with plasmids encoding CD33m and CD33M and control mock plasmid. Cells were incubated with C1q as outlined in Materials and Methods. Numbers in graphs = % positive/MFI. Debris/dying cells was excluded on the basis of forward and side scatter. ( B ) Pooled flow cytometry data illustrating binding of C1q (at comparable levels) on the surface of CD33m/M transfected HEK293T cells and a lack of C1q binding on mock transfected cells. For A and B, N = 3. ( C ) Representative slot blot showing that C1q binds to cell-associated CD33m, CD33M or LAIR-1 in a dose dependent manner. Protein prepared from whole cell lysates (HEK293T cells transfected with plasmid encoding CD33m, CD33M or LAIR-1) was immobilized on membranes at increasing doses; biotin-whole C1q was in solution at 10 ug/ml. HSA = human serum albumin control. Protein concentration was calculated by the BCA method. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). ( D ) Pooled data analyses illustrating dose related interactions between biotin-C1q and cell-associated CD33m or CD33M represented in ( C) . ( B , D ), the mean ± SE of pooled data is shown; for all proteins and doses, N = 3–5. *P = < 0.05, **P = <0.01, ***P < 0.001. One-way ANOVA followed by Tukey’s pairwise multiple comparison was used.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Flow Cytometry, Binding Assay, Transfection, Plasmid Preparation, Incubation, Dot Blot, Protein Concentration

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: C1q triggers CD33 ITIM phosphorylation and CD33-LAIR-1 physical associations in human monocytes. Monocytes were either untreated (utx), treated with whole C1q, gC1q, C1q CLR or pervanadate (PV) as detailed in Materials and Methods. ( A , B ) Immunoblot analyses showing increased phosphorylated (p) CD33 after treatment with C1q and gC1q, but not C1q CLR. CD33 was immunoprecipitated with anti-CD33M antibody; tyrosine phosphorylation of CD33 (pCD33M) was detected with anti-phosphotyrosine (4G10) antibody. The membrane was stripped and re-probed with anti-CD33 WM53 antibody (CD33M). Arrows denote molecular weight of 67kD. Bar graphs denote pooled data expressed as fold change relative to total CD33 and corresponding statistical analyses. One-way ANOVA with post-hoc Tukey multiple comparisons was used to determine significance. N = 4 for A; N = 3 for B. ( C ) The addition of whole C1q (20 ug/ml) prompts concurrent increases in pLAIR-1 and pCD33 in a phospho-immunoreceptor array. ( D ) C1q CLR “tails” (20 ug/ml) do not elicit increases in pCD33M in the phospho-immunoreceptor array. Bar graphs represent fold change (plus/minus C1q), calculated from the mean pixel density of duplicates, as determined by densitometry analysis. Corresponding C1q minus/plus array data is located below each bar graph in C and D. Data from a typical array are shown. N = 3 for C and D. ( E ) Proximity ligation assay, performed on freshly isolated human monocytes as detailed in materials and methods, showing that whole C1q is required for CD33-LAIR-1 crosslinking. Red fluorescent dots represent molecular associations between CD33 and LAIR-1; blue represents nuclear staining with DAPI. CLR and gC1q represent the C1q collagen tail and globular heads of C1q, respectively. Original magnification = 60X. One of three representative experiments is shown.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Western Blot, Immunoprecipitation, Molecular Weight, Proximity Ligation Assay, Isolation, Staining

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Masking of CD33C2 domains impedes C1q binding on the cell surface. ( A , B ) Increased detection of the CD33C2, but not the CD33V domain or LAIR-1, occurs after sialidase treatment of THP-1 cells. Cells were treated with sialidase (20 mU/10 6 cells) for 60 min at 37 °C in a humidified incubator, resuspended twice in PBS, then in staining buffer for detection of CD33C2 domains (HIM3-4), CD33V domains (WM53) and LAIR-1. A = Pooled data, N ≥ 4. B = Representative experiment from A; gates were set according to isotype Ab controls. ( C ) Unmasking of CD33C2 domains with sialidase correlates with increased binding of C1q on THP-1 cells; N = 3–4. ( D ) Reduced levels of sialic acid on the THP-1 surface after sialidase treatment as revealed by decreased binding to Sambucus nigra lectin (SNL) (5 ug/ml). N = 5. ( E ) Pooled flow cytometry data showing highly expressed CD33C2 and CD33V domains and LAIR-1 on freshly isolated normal monocytes. Results represent mean +/− SEM. N = 3–4. For ( A , C , D ) solid bars = no sialidase; open bars = sialidase. Two-tailed Student’s t test was used to determine significance.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Binding Assay, Staining, Flow Cytometry, Isolation, Two Tailed Test

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Maturing DCs exhibit decreases in LAIR-1 and CD14 along with a reduced ability to detect CD33C2 epitopes. ( A , B ) Decreased expression of LAIR-1 and CD14 on DCs. ( C , D ) Pooled flow cytometry data showing CD33C2 domains (detected with mAb HIM3-4) and CD33V domains (detected with mAb WM53) on freshly isolated monocytes and DCs obtained after 5–6 days of culture with 50 U/ml rGM-CSF and 50 ng/ml rIL-4. ( E ) Pooled data showing co-detection of CD33C2 and CD33V domains on fresh monocytes and day 6 DCs. ( F ) Representative dot plot comparing co-detection of CD33C2/V epitopes on freshly isolated monocytes and day 6 DCs; gates were set based on isotype controls. (G ) Increased levels of sialic acid on immature DCs detected with Sambucus nigra lectin (SNL) linked to PECy7. For ( A – E ), N = 3–5; for (G), N = 2. Two-tailed Student’s t test was used to determine significance. Solid bars represent day 0; hatched bars represent day 6.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Expressing, Flow Cytometry, Isolation, Two Tailed Test