Journal: The Journal of Experimental Medicine

Article Title: CX 3 CR1 + mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22

doi: 10.1084/jem.20140678

Figure Lengend Snippet: Increased ILC3 production of IL-22 in mild to moderate IBD correlates with presence of fecal stream. (A) LPMCs isolated from descending colon biopsies from patients with endoscopically mild to moderate Crohn’s’ disease ( n = 8, gray) or ulcerative colitis ( n = 6, black; Table S1 ), as well as age-matched non-IBD control patients undergoing routine screening colonoscopy ( n = 8, white), were stimulated ex vivo with PMA/ionomycin and evaluated by intracellular cytokine staining for expression of IL-17 and IL-22. The percentage of CD3 + or CD3 − fraction expressing IL-17 or IL-22 is indicated. *, P ≤ 0.05, two-tailed Student’s t test. Black bars represent the geometric mean. (B) Expression of c-Kit and CD56 in electronically gated CD3 − IL-22 + (black lines) and CD3 − IL-22 − (gray) LPMCs. (C) Expression of RORγt by c-Kit + CD56 + LPMCs. Lin − cells (CD14/CD19/CD3/CD11b/CD11c/TCRγδ − ; Fig. S3 A ) were stained with antibodies to surface markers c-Kit and CD56 and to intracellular RORγt. Lin − CD56 + c-Kit + ILC3 (black line) were compared with Lin − CD56 + c-Kit − NK cells (gray) for RORγt expression. (D) Surface staining of Lin − c-Kit + ILCs for the indicated markers (black line) compared with isotype control (gray) and all live LPMCs (dotted line) (E) LPMCs stained for intracellular IL-22 after stimulation with IL-23 for 3 h (solid line) or with control media (dotted line). Cells shown were gated on Lin − CD56 + c-Kit + . The isotype control is in gray. (F) CD11c + MHCII + human colonic APCs were electronically gated for expression of CD103 and CD14. One of three donors is shown. (G) Lamina propria cells from biopsy samples of tissue exposed (prediversion) or not exposed to the fecal stream (post-diversion) were cultured for 3 h and ILC3 production of IL-22 was assessed by flow cytometry. (left) Result from one representative donor. (right) Percentage of IL-22 + ILCs in afferent (Pre) and efferent (Post) limbs of three diverted patients. **, P ≤ 0.01, two-tailed Student’s t test. Black bars represent the geometric mean.

Article Snippet: Staining of human cells was performed with c-Kit-e450 (104D2), CD56-PECy5.5 (CMSSB), HLA-DR-PE (LN3), CD11c-FITC (3.9), CD3-e780 (UCHT1), CD19-PerCP5.5 (HIB19), CD103 PECy7 (B-Ly7), CD14 PE (61D3), CD86-PE (IT2.2), CD123 PE (6H6), CD11b PE (CBRM1/5), and RORγt (AFKJS-9) obtained from eBioscience; CD161-FITC, CD83-PE, CD11c PE (555392), CD127 FITC (M21), CCR6-biotin (11A9) were purchased from BD; BDCA-1-APC were obtained from Miltenyi Biotec; CD45-APC (4505) and CD64 (6404) were obtained from Invitrogen; NKp44-APC (p44-8.1) was purchased from R&D Systems; and BDCA-3 APC (M80) was purchased from BioLegend.

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

Journal: The Journal of Experimental Medicine

Article Title: CX 3 CR1 + mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22

doi: 10.1084/jem.20140678

Figure Lengend Snippet: CX 3 CR1 + MNP-derived TL1A synergizes with IL-23 and IL-1β to induce IL-22 in intestinal ILC3. (A) Gene-set enrichment analysis was used to determine whether the indicated disease-related SNP were differentially expressed between CD14 + MNPs and CD103 + DCs. Significance was estimated using the hypergeometric cumulative distribution, with a raw p-value cutoff of 0.05 for differential expression. Data were averaged from two independent donors. (B) B cells (CD3 − CD19 + ), CX 3 CR1 + MNPs, CD103 + DCs, and Ly6C + monocytes were sorted from the intestinal lamina propria of Cx3cr1 GFP/+ and quantitative PCR for TL1A was performed. Relative quantitation was performed by ΔCt normalized to GAPDH expression. Data are from two biological replicates performed with two technical replicates. *, P ≤ 0.05. Two-tailed Student’s t test. (C–E) Sorted intestinal ILCs from mice (C and E) or cultured human intestinal ILCs (D) were stimulated with media alone, IL-1β, or IL-23 with or without TL1A as indicated for 18 h. Brefeldin was added to the cultures 4 h before intracellular cytokine staining for IL-22 (C and D) or GM-CSF (E). Data are representative of six independent experiments. (F–H) Sorted intestinal ILCs were transfected with siRNA targeting Tnfrsf25 or a scramble control. (F) Knockdown efficiency was assessed after 24 h by flow cytometry comparing scramble control (solid line) with Tnfrsf25 siRNA. One of two representative experiments is shown. ILCs were then cultured with media alone (-) or IL-23 and TL1A or co-cultured with CX 3 CR1 + MNPs with or without LPS as indicated for an additional 18 h. (G) IL-22 production was measured by intracellular flow cytometry. Brefeldin was added to the cultures 4 h before intracellular cytokine staining. Data are representative of two independent experiments. (H) IL-22 secretion by samples from G were assessed by ELISA, performed in duplicate, before addition of Brefeldin. *, P ≤ 0.05; **, P ≤ 0.01. Two-tailed Student’s t test. Error bars represent SEM.

Article Snippet: Staining of human cells was performed with c-Kit-e450 (104D2), CD56-PECy5.5 (CMSSB), HLA-DR-PE (LN3), CD11c-FITC (3.9), CD3-e780 (UCHT1), CD19-PerCP5.5 (HIB19), CD103 PECy7 (B-Ly7), CD14 PE (61D3), CD86-PE (IT2.2), CD123 PE (6H6), CD11b PE (CBRM1/5), and RORγt (AFKJS-9) obtained from eBioscience; CD161-FITC, CD83-PE, CD11c PE (555392), CD127 FITC (M21), CCR6-biotin (11A9) were purchased from BD; BDCA-1-APC were obtained from Miltenyi Biotec; CD45-APC (4505) and CD64 (6404) were obtained from Invitrogen; NKp44-APC (p44-8.1) was purchased from R&D Systems; and BDCA-3 APC (M80) was purchased from BioLegend.

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitation Assay, Two Tailed Test, Cell Culture, Staining, Transfection, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: TRACE-Seq Reveals Clonal Reconstitution Dynamics of Gene Targeted Human Hematopoietic Stem Cells

doi: 10.1101/2020.05.25.115329

Figure Lengend Snippet: CD34 + enriched cord blood-derived HSPCs were cultured in HSPC media containing SCF, FLT3L, TPO, IL-6, and UM-171 for 48h, electroporated with Cas9 RNP (HBB sgRNA), transduced with AAV6 donors (either BC or non-BC), and cultured for an additional 48h prior to intrafemoral transplant into sublethally irradiated NSG mice (total manufacturing time was less than 96 hours). 16-18 weeks post transplantation, total BM was collected and analyzed for engraftment by flow cytometry, sorted on lineage markers, and sequenced for unique barcodes. Two independent experiments were performed to assess reproducibility of identifying clonality of gene-targeted HSPCs. a Total human engraftment in whole bone marrow, (as measured by proportion of human HLA-ABC + cells). b Multilineage engraftment of human CD19 + , CD33 + , and HSPCs (CD19 - CD33 - CD10 − CD34 + ). c Genome editing efficiency in each indicated sorted human lineage subset as determined by NGS (HR reads / [sum of HR reads + unmodified reads]). d Barcodes from each subset were sorted from largest to smallest by percentage of reads. Depicted are the numbers of most abundant, unique barcode alleles comprising the top 50% and top 90% of reads from each lineage of all mice transplanted with BC donor edited HSPCs. Mean ± SEM genomes analyzed from each group: CD19 + : 8500 ± 1000, CD33 + : 8800 ± 800, HSPC: 1500 ± 500 (see ). e Correlation between numbers of high confidence barcodes (> 0.5%) in lymphoid (grey) and myeloid (black) compartments and total human engraftment (as percent of human and mouse BM-MNCs). Lymphoid and myeloid values plotted for n=9 primary engrafted mice and n=1 secondary engrafted mouse. f Correlation between numbers of high confidence barcodes (> 0.5%) in lymphoid (grey) and myeloid (black) compartments and HR adjusted engraftment ([human engraftment] x [lineage specific engraftment] x [HR efficiency]). Lymphoid and myeloid values plotted for n=9 primary engrafted mice and n=1 secondary engrafted mouse. g Numbers of high confidence barcodes from each mouse which contribute to lymphoid only (CD19 + ), myeloid only (CD33 + ), or both lineages. High confidence barcodes: barcodes with at least 0.5% representation (see Extended Data Figure 1 ). All points represent individual mice, with the exception of panels e-g (where barcodes from each mouse are separated based on lineage contribution). Error bars depict mean ± SEM. p values reflect 2-tailed t-test.

Article Snippet: MNCs were harvested by ficoll gradient centrifugation and human hematopoietic cells were identified by flow cytometry using the following antibody cocktail: HLA-A/B/C FITC (clone W6/32, Biolegend), mouse CD45.1 PE-CY7 (clone A20, Thermo Scientific), CD34 APC (clone 581, Biolegend), CD33 V450 (clone WM53, BD Biosciences), CD19 Percp5.5 (clone HIB19, BD Biosciences), CD10 APC-Cy7 (HI10a, Biolegend), mTer119 PeCy5 (clone Ter-119, Thermo Scientific), and CD235a PE (HIR2, Thermo Scientific).

Techniques: Derivative Assay, Cell Culture, Transduction, Irradiation, Transplantation Assay, Flow Cytometry

Journal: bioRxiv

Article Title: TRACE-Seq Reveals Clonal Reconstitution Dynamics of Gene Targeted Human Hematopoietic Stem Cells

doi: 10.1101/2020.05.25.115329

Figure Lengend Snippet: Representative HBB gating strategy (upper) and sort purity (lower) for CD19 + , CD33 + , and HSPCs (CD19 - CD33 - CD10 − CD34 + ) populations.

Article Snippet: MNCs were harvested by ficoll gradient centrifugation and human hematopoietic cells were identified by flow cytometry using the following antibody cocktail: HLA-A/B/C FITC (clone W6/32, Biolegend), mouse CD45.1 PE-CY7 (clone A20, Thermo Scientific), CD34 APC (clone 581, Biolegend), CD33 V450 (clone WM53, BD Biosciences), CD19 Percp5.5 (clone HIB19, BD Biosciences), CD10 APC-Cy7 (HI10a, Biolegend), mTer119 PeCy5 (clone Ter-119, Thermo Scientific), and CD235a PE (HIR2, Thermo Scientific).

Techniques:

Journal: bioRxiv

Article Title: TRACE-Seq Reveals Clonal Reconstitution Dynamics of Gene Targeted Human Hematopoietic Stem Cells

doi: 10.1101/2020.05.25.115329

Figure Lengend Snippet: a Top: Schema of AAVS1 locus and AAV6 donor containing SFFV-BFP-[barcode]-pA expression cassette. Bottom: Barcode region depicted in red, with a maximum theoretical diversity of 3 = 531,441. b Recovery of barcodes from untreated genomic DNA containing 1, 3, 10, and 30 individual plasmids containing BFP barcodes. c Total human engraftment in whole bone marrow collected 16-18 weeks post transplantation (expressed as proportion of human HLA-ABC + cells), left, and genome editing efficiency as measured by percentage of BFP+ cells by flow cytometry, right. d Multi-lineage engraftment of human CD19 + and CD33 + lineages, left, with respective genome editing efficiencies, right. Error bars depict mean ± SEM. e Example of gating on highly engrafted mouse on %BFP + within each lineage (left) and representative gating strategy and sort purity (right). f Barcodes from each subset were sorted from largest to smallest by percentage of reads. Depicted are the numbers of most abundant, unique barcode alleles comprising the top 50% and top 90% of reads from each lineage of all mice transplanted with BC donor edited HSPCs. Mean ± SEM genomes analyzed from each group— CD19 + : 7900 ± 1500, CD33 + : 10000 ± 3000 (see ).

Article Snippet: MNCs were harvested by ficoll gradient centrifugation and human hematopoietic cells were identified by flow cytometry using the following antibody cocktail: HLA-A/B/C FITC (clone W6/32, Biolegend), mouse CD45.1 PE-CY7 (clone A20, Thermo Scientific), CD34 APC (clone 581, Biolegend), CD33 V450 (clone WM53, BD Biosciences), CD19 Percp5.5 (clone HIB19, BD Biosciences), CD10 APC-Cy7 (HI10a, Biolegend), mTer119 PeCy5 (clone Ter-119, Thermo Scientific), and CD235a PE (HIR2, Thermo Scientific).

Techniques: Expressing, Transplantation Assay, Flow Cytometry

Journal: Nature Communications

Article Title: The TRACE-Seq method tracks recombination alleles and identifies clonal reconstitution dynamics of gene targeted human hematopoietic stem cells

doi: 10.1038/s41467-020-20792-y

Figure Lengend Snippet: CD34 + enriched cord blood-derived HSPCs were cultured in HSPC media containing SCF, FLT3L, TPO, IL-6, and UM-171 for 48 h, electroporated with Cas9 RNP (HBB sgRNA), transduced with AAV6 donors (either BC or non-BC), and cultured for an additional 48 h prior to intrafemoral transplant into sublethally irradiated NSG mice (total manufacturing time was less than 96 h). A total of 16–18 weeks post-transplantation, total BM was collected and analyzed for engraftment by flow cytometry, sorted on lineage markers, and sequenced for unique barcodes. Two independent experiments were performed to assess reproducibility of identifying clonality of gene-targeted HSPCs. a Total human engraftment in whole bone marrow, (as measured by the proportion of human HLA-ABC + cells). b Multilineage engraftment of human CD19 + , CD33 + , and HSPCs (CD19 − CD33 − CD10 − CD34 + ). c Genome editing efficiency in each indicated sorted human lineage subset as determined by NGS (HR reads/[all reads]). d Barcodes from each subset were sorted from largest to smallest by percentage of reads. Depicted are the numbers of most abundant, unique barcode alleles comprising the top 50% and top 90% of reads from each lineage of all mice transplanted with BC donor edited HSPCs. Mean ± SEM genomes analyzed from each group: CD19 + : 8500 ± 1000, CD33 + : 8800 ± 800, HSPC: 1500 ± 500 (see Supplementary Table ). e Correlation between numbers of high confidence barcodes (>0.5%) in lymphoid (gray) and myeloid (black) compartments and total human engraftment (as percent of human and mouse BM-MNCs). Lymphoid and myeloid values plotted for n = 9 primary engrafted mice and n = 1 secondary engrafted mouse. f Correlation between numbers of high confidence barcodes (>0.5%) in lymphoid (gray) and myeloid (black) compartments and HR adjusted engraftment ([human engraftment] x [lineage specific engraftment] x [HR efficiency]). Lymphoid and myeloid values plotted for n = 9 primary engrafted mice and n = 1 secondary engrafted mouse. g Numbers of high confidence barcodes from each mouse which contribute to lymphoid only (CD19 + ), myeloid only (CD33 + ), or both lineages. High confidence barcodes: barcodes with at least 0.5% representation (see Supplementary Fig. ). All points represent individual mice ( n = 9 non-BC treated and n = 10 BC treated biologically independent mice in one independent experiment), with the exception of panels e – g (where barcodes from each mouse are separated based on lineage contribution). Error bars depict mean ± SEM. p values reflect 2-tailed t-test.

Article Snippet: MNCs were harvested by ficoll gradient centrifugation and human hematopoietic cells were identified by flow cytometry using the following antibody cocktail: HLA-A/B/C FITC (1:100, clone W6/32, Biolegend), mouse CD45.1 PE-CY7 (1:200, clone A20, Thermo Scientific), CD34 APC (1:100, clone 581, Biolegend), CD33 V450 (1:100, clone WM53, BD Biosciences), CD19 Percp5.5 (1:50, clone HIB19, BD Biosciences), CD10 APC-Cy7 (1:20, HI10a, Biolegend), mTer119 PeCy5 (1:200, clone Ter-119, Thermo Scientific), and CD235a PE (1:50, HIR2, Thermo Scientific).

Techniques: Derivative Assay, Cell Culture, Transduction, Irradiation, Transplantation Assay, Flow Cytometry