mertk-apc (fab8912a)  (R&D Systems)

Journal: bioRxiv

Article Title: Single-cell transcriptomics reveals heterogenous thymic dendritic cell subsets with distinct functions and requirements for thymocyte-regulated crosstalk

doi: 10.1101/2023.12.18.572281

Figure Lengend Snippet: (a) Flow cytometric gating strategy to identify distinct DC subsets shown in . (b) UMAP visualization of scRNA-seq transcriptional data from pooled FACS-sorted cDC subsets, colored by their hashtag labels. (c) Same UMAP plot as (b), colored by transcriptionally distinct cDC clusters defined by the Seurat pipeline. (d) Dot plot showing normalized expression levels (z-score) of select genes associated with the hashtagged FACS-sorted cDC subsets. Expression is normalized per row (representing a gene) across different FACS-sorted cDC subsets. (e) Frequency of each transcriptionally distinct cDC cluster contained within the FACS-sorted hashtagged cDC subsets. (f) Immunofluorescence images of MertK + macrophages and CD63 + SIRPα + MerTK - aDC2s in thymic sections (representative of N=3 independent experiments). Centroids are placed over CD63 + SIRPα + cells that also express MertK: the frequency of MertK-expressing cells among CD63 + SIRPα + myeloid cells was used to correct aDC2 densities from data as in (h). Scale bar, 250µm. Lines demarcate the cortex (C), medulla (M), and CMJ in the thymic sections. Centroids with 40% opacity were overlaid on the quantified cells. (g) Immunofluorescence of DC subsets (representative of N=3 independent experiments), identified by the indicated markers. White boxes indicate the regions of interest displayed in . Scale bars, 1,000µm. (h) Quantification of cell densities of each of the indicated DC subsets in distinct anatomical regions of the thymus, based on histocytometry analyses of data as in (f-g). Data are compiled from N=3 independent experiments. Box plots represent mean ± SD. Statistical analysis was performed using Tukey’s Honest Significant Difference test, where *p<0.05, **p<0.01. (i) Quantification of cortical cell densities of each of the indicated DC subsets, based on histocytometry analyses of data as in (f-g). Data are compiled from N=3 independent experiments. Box plots represent mean ± SD.

Article Snippet: To stain for macrophages, slides were stained with Mertk-APC (D5MMER, Invitrogen), CD63-PE-DazzleRed594 (NVG-2, Biolegend) and SIRPα-Biotin (P84, Biolegend) overnight at 4°C.

Techniques: Expressing, Immunofluorescence

Journal: bioRxiv

Article Title: Single-cell transcriptomics reveals heterogenous thymic dendritic cell subsets with distinct functions and requirements for thymocyte-regulated crosstalk

doi: 10.1101/2023.12.18.572281

Figure Lengend Snippet: (a) Merged UMAP visualization of 34,750 cells depicting major cell continents in WT, MHC-II -/- , B2m -/- and Rag2 -/- HAPC scRNA-seq datasets. Data are compiled from N=2 independent experiments (n=2 mice). (b) Feature plots displaying expression levels of select genes associated with distinct HAPC subtypes – B cells ( Cd19 ), cDC ( Itgax, zbtb46 ), pDC ( Siglech, Itgax ), Macrophages and Monocytes ( MertK, Csf1r ) and granulocytes ( Camp ). Residual thymocytes included in the sorted cells were identified by Lck expression. (c) Heatmap displaying normalized expression of the top 10 enriched genes in each transcriptionally distinct cDC cluster from . (d) Feature plots showing scRNA-seq expression levels of (top) B2m transcripts in WT versus B2m -/- cDCs and (bottom) H2-Aa transcripts in WT versus MHC-II -/- cDCs overlaid on the UMAP visualization from . (e) Feature plots showing expression levels of Cd4 and Cd8a transcripts in thymocytes from WT versus Rag2 -/- mice overlaid on the UMAP visualization in (a). (f) Quantification of the frequencies of cDC1, cDC2, aDC1, aDC2, and Il15ra hi aDC subsets identified in from scRNA-seq transcriptional profiling of thymic DCs in the four indicated genotypes. Data are compiled from 2 independent experiments per genotype (n=2 mice). Bars represent mean ± SEM and symbols represent individual mice.

Article Snippet: To stain for macrophages, slides were stained with Mertk-APC (D5MMER, Invitrogen), CD63-PE-DazzleRed594 (NVG-2, Biolegend) and SIRPα-Biotin (P84, Biolegend) overnight at 4°C.

Techniques: Expressing

Journal: Nature Communications

Article Title: Altered function and differentiation of age-associated B cells contribute to the female bias in lupus mice

doi: 10.1038/s41467-021-25102-8

Figure Lengend Snippet: RNA-seq was performed on sorted CD11c + and CD11c − GL7 + CD38 lo B cells (gated on B220 + splenocytes) from aged (24+wk) female DKO (F) mice. a Volcano plot showing genes differentially expressed ( p < 0.01 after Benhamini–Hochberg false discovery rate (FDR) was used to correct for multiple comparisons) between CD11c + B220 + GL7 + CD38 lo (CD11c + ) and CD11c − B220 + GL7 + CD38 lo (CD11c − ) cells. b Plot showing the enrichment of the ABC geneset from DKO mice in CD11c + B220 + GL7 + CD38 lo cells. c Heatmap showing the expression of GC B cell target genes in CD11c + and CD11c − B220 + GL7 + CD38 lo cells. d Representative plots and quantifications of pre-GC B cells (BCL6 mid IRF4 + ) and GC B cells (BCL6 hi IRF4 − ) among CD11c + CD19 + GL7 + Fas + (CD11c + ; red) and CD11c − CD19 + GL7 + Fas + (CD11c − ; black) cells from aged (24+wk) DKO(F) mice. Data show mean ± SEM; n = 6 from 2 independent experiments; p -value by paired two-tailed t -tests. e Plot showing the enrichment of a pre-GC geneset (GSE12845) in CD11c + B220 + GL7 + CD38 lo cells. f Representative histograms and quantifications of the phosphorylation of SYK(Y352) and LYN(Y416) in CD11c + CD19 + GL7 + Fas + cells (CD11c + ; red), CD11c − CD19 + GL7 + Fas + (CD11c − ; black) cells from aged (24+wk) DKO(F) mice. CD19 + CD11c + CD11b + (ABCs; green) CD19 + GL7 − Fas − cells (gray) are shown as control. Data show mean ± SEM; n = 4 from 3 independent experiments; p -value by paired two-tailed t -test. g Plot showing the top pathways upregulated in CD11c − GL7 + CD38 lo cells (black) and CD11c + GL7 + CD38 lo cells (red) by GSEA. Dotted line indicates significance threshold at FDR q < 0.25. h Representative histogram and quantification of Ki67 expression in the indicated populations from DKO(F) mice. Data show mean ± SEM; n = 5 from 3 independent experiments; p -value by paired two-tailed t -tests. i Heatmap showing the expression of genes related to apoptotic cell clearance in CD11c + and CD11c − GL7 + CD38 lo cells from DKO ( F) mice. j Representative histogram and quantification of MerTK expression on the indicated populations from aged (24+wk) DKO(F) mice. Data show mean ± SEM; n = 8 from 4 independent experiments; p -value by paired two-tailed t -tests.

Article Snippet: Antibodies to Foxp3-APC (FJK-16s; 1:100), IgD-FITC (11-26; 1:500), IgM-PE/Cy7 (II/41; 1:1000), IRF4-FITC (3E4; 1:200), IRF8-PE (V3GYWCH; 1:200), Ki67-PE (solA15; 1:800), MerTK-APC (DS5MMER; 1:200), and PD1-PB (J43; 1:200) were obtained from eBioscience.

Techniques: RNA Sequencing, Expressing, Two Tailed Test, Phospho-proteomics, Control

Journal: PLoS Pathogens

Article Title: GAS6 signaling tempers Th17 development in patients with multiple sclerosis and helminth infection

doi: 10.1371/journal.ppat.1009176

Figure Lengend Snippet: A ) Representative dot plots showing the three main sub-populations of mononuclear cells based on the expression of CD11b, CD4, and CD1c. TYRO3, AXL, and MERTK expression were evaluated in circulating monocytes (CD11b high CD4 mid ) and DCs (CD1c high CD11b low ) of patients with MS, patients with HIMS, and healthy controls by flow cytometry. B-G ) The percentage of TYRO3-positive as well as the mean fluorescent intensity (MFI) of the receptor on monocytes ( B, C, and D ) and DCs ( E, F, and G ) are graphed. H, I, and J ) The percentage of AXL high as well as the MFI of the receptor on DCs are shown. K, L, and M ) The expression of MERTK on DCs is shown as both percentage and MFI. N, O, and P ) The percentage of circulating CD4 + T cell expressing GAS6 as well as its MFI are shown. Q, R, and S ) The percentage of circulating CD4 + T cell expressing PROS1 and its MFI are shown. Data is presented as a pool of independent samples included in the specific staining (Control N = 21–31; MS N = 10–27; and HIMS N = 11–16). One-way ANOVA with a Fisher post hoc test was performed to determine statistical significances, *p<0.05 **p≤0.01 ****p≤0.001. MS = multiple sclerosis, HIMS = helminth-infected multiple sclerosis.

Article Snippet: Rabbit anti-human TYRO3 (Novus Biological), biotin-conjugated goat anti-human AXL, and APC anti-human MERTK mAb IgG1 (R&D Systems) were used to evaluate TAM receptor expression after fixation and permeabilization (Cytofix/Cytoperm Kit, BD Bioscience, San Diego, CA, USA).

Techniques: Expressing, Flow Cytometry, Staining, Control, Infection

Journal: PLoS Pathogens

Article Title: GAS6 signaling tempers Th17 development in patients with multiple sclerosis and helminth infection

doi: 10.1371/journal.ppat.1009176

Figure Lengend Snippet: The host type 2 immune response is elicited to clear parasitic helminth infections; however, the co-evolution of these parasites has promoted a variety of mechanisms to oppose and redirect immune responses by manipulating immune cell programming and function. Integrating our results with the current knowledge, we propose that gastrointestinal ( 1 ) helminths infections can enhance the regulatory axis of TAM receptors (TYRO3, AXL, and MERTK) in peripheral blood ( 2 ) CD11b high and CD1c high cells, and their ligand GAS6 in CD4 + T cells of patients with MS. This negative regulatory pathway could be essential for dampening co-stimulatory signals (CD40, CD80, and CD86) of antigen-presenting cells and controlling the pathological Th17 (pTh17) response at secondary lymphoid organs ( 3 ). The active chronic infection resets T helper response toward Th2/regulatory signals, limiting pathological Th17 (pTh17) effector response. This reprogramming of CD4 + T cells and GAS6 signaling under a helminth-induced type 2 environment could be essential for maintaining the balance between CD4 + IL-10 + and Th17 cells and reducing inflammatory IL-17 and IFNγ signals in the central nervous system ( 4 ). In summary, GAS6 tempers not only the innate immune response but also regulates Th17 development in an autocrine/paracrine manner.

Article Snippet: Rabbit anti-human TYRO3 (Novus Biological), biotin-conjugated goat anti-human AXL, and APC anti-human MERTK mAb IgG1 (R&D Systems) were used to evaluate TAM receptor expression after fixation and permeabilization (Cytofix/Cytoperm Kit, BD Bioscience, San Diego, CA, USA).

Techniques: Infection