16-0452-82

antibody from Invitrogen Antibodies

Targeting: PTPRC CD45, GP180, LCA, T200

Provider product page for 16-0452-82

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Antibody Data
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Product number: 16-0452-82 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD45R (B220) Monoclonal Antibody (RA3-6B2), Functional Grade, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The RA3-6B2 monoclonal antibody reacts with exon A-restricted isoform of mouse CD45, a 220 kDa surface molecule. CD45R/B220 epitope is mainly expressed by the B cell lineage from early Pro-B to mature B cells. However, some activated T cells, lymphokine activated killer cells (LAK), NK cell progenitors in the bone marrow, and T cells of the lpr/lpr mutant mouse also express this antigen. Applications Reported: The RA3-6B2 antibody has been reported for use in flow cytometric analysis. RA3-6B2 has also been reported in functional studies of B cells. Applications Tested: The RA3-6B2 antibody has been tested by flow cytometric analysis of mouse splenocytes and can be used at less than or equal to 0.5 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. Storage and handling: Use in a sterile environment. Filtration: 0.2 µm post-manufacturing filtered. Purity: Greater than 90%, as determined by SDS-PAGE. Endotoxin Level: Less than 0.001 ng/µg antibody, as determined by LAL assay. Aggregation: Less than 10%, as determined by HPLC.
Reactivity: Human, Mouse
Host: Rat
Isotype: IgG
Antibody clone number: RA3-6B2
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: 4° C

PTPRC protein structure - 16-0452-82 shown in red.

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Experimental details: Figure 4 Double immunofluorescence localisation of B220 (Green) and NF-kappaB p65 (Red) in HCV-Tg mice and the fractionation analysis of mouse tissues. A : Co-localisation of NF-kappaB p65 immunoreactivity with B220 is indicated by arrows. (a-b) Cells double-positive for B220 and NF-kappaB in the control mouse (CD19cre). (c-d) Cells double-positive for B220 and NF-kappaB in the asymptomatic HCV-Tg mouse (RzCD19cre). (e-f) Cells double-positive for B220 and NF-kappaB in the lymphomatous HCV-Tg mouse (RzCD19cre). B : Quantitative analysis of the ratio of double-positive cells among B220-positive cells in each HCV-Tg mouse. Bar graph indicates the percentage of cells with NF-kappaB p65 nuclear translocation in B220-positive cells. C : Bar graph shows the ratio of double-positive cells within the B220-positive cells in normal, asymptomatic and lymphomatous HCV-Tg mice. Ho: Hoechst33342 Data are presented as means +- S.E., * P

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Experimental details: Figure 7 Marginal zone B cell regions were narrowed in Ziz2 KO mice. (A) Spleen sections were stained with anti-B220 (Red) and anti-CD169 (Green) antibodies for the MZ B cell region (B220-positive region outside CD169-positive cells). All mice were 10 weeks old. Scale bars: 100 mum. (B) MZ B cell regions were narrower in Ziz2 and Ziz3 KO mice than in wild type mice. (C) The proliferative activity of MZ B cells in response to LPS was not altered in both KO mice. Three mice per group were used. Data from three independent experiments (one mouse per group per experiment was used) were summarized. (D) The migratory activity of MZ B cells was analyzed using a transwell and flow cytometry. Activity against BLC or SDF1 (SDF) was not altered in both KO mice. Four mice per group were used. Each plot indicates data from one mouse. Data from four independent experiments (one mouse per group per experiment was used) were summarized. 2KO: Ziz2 KO. 3KO: Ziz3 KO. ***: P < 0.0001.

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Experimental details: Figure 3 GPR183 and 7alpha,25-OHC Promote ILC3 Localization to CPs and ILFs (A) Distribution of Gpr183 +/+ and Gpr183 -deficient hematopoietic cells in the colon of mixed bone marrow chimeras. Bone marrow cells from Gpr183 +/+ or Gpr183 -/- mice (CD45.2 + ) were mixed 9:1 with B6 cells (CD45.1 + ) and injected into irradiated Rag1 -/- recipients (CD45.1 + ) for the generation of bone marrow chimeras. Sections were stained for detection of Gpr183 +/+ and Gpr183 -/- cells (CD45.2, red) or B6 cells (CD45.1, green). Nuclei were visualized by DAPI staining (blue). Scale bars on the right (white) represent 100 mum. (B) Distribution of Gpr183 +/+ and Gpr183 -deficient ILCs in the colon of mixed bone marrow chimeras. Bone marrow cells from Gpr183 +/+ or Gpr183 -/- mice (CD90.2 + ) were mixed 9:1 with B6 cells (CD90.1 + ) and injected into irradiated B6 recipients (CD90.1 + ) for the generation of bone marrow chimeras. Colon sections were stained for detection of Gpr183 +/+ and Gpr183 -/- (CD90.2, red) or B6 (CD90.1, green) ILCs. Scale bars (red) represent 100 mum. The lower panel shows the number of clusters in Gpr183 +/+ -B6 and Gpr183 -/- -B6 chimeras consisting of CD90.2 + (red) or CD90.1 + (green) ILCs. Data are represented as means +- SEM. p value by two-way ANOVA. (C and D) Distribution of donor-derived ILC3s (RORgammat-GFP + , green) in the colon (C) and small intestine (D) of bone marrow chimeras. Bone marrow cells from Rag1 -deficient Rorc(gamma

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Experimental details: Figure 3. Localization of intestinal IgD + B Cells. (A) Representative photomicrographs of small intestinal sections stained with DAPI- and fluorophore-conjugated Ab for IgD (FITC), IgA (PE), and B220 (APC) as indicated from GF ( n = 4) and conventionalized SW littermates (Col, n = 4). Photomicrographs shown are representative of one of two independent experiments. (B) Dot plot showing the number of LP IgA + cells and IgD + B220 + cells per high power field (200x) per GF ( n = 4; 10 fields/mouse) and conventionalized SW littermates (Col; n = 4; 11 fields/mouse) described as in A. (C) Dot plots showing the perpendicular distance (see Materials and methods) between individual LP IgA + cells ( n = 85 for GF; n = 971 for Col), IgD + B220 + cells ( n = 59 for GF; n = 108 for Col), and the serosal surface from GF ( n = 4) and colonized SW littermates (Col, n = 4) described as in A. (D) Representative photomicrographs of small intestinal sections stained as in A from SPF SW mice at the age of postnatal day 21 ( n = 3), 42 ( n = 3), and 120 ( n = 3). Negative control uMT mice ( n = 2) are also shown. White arrows indicate IgD + B220 + cells. Bars, 60 um. Photomicrographs are representative images from one of three independent experiments for each time point and two independent experiments for uMT mice. (E) Dot plot showing the number of IgA + cells and IgD + B220 + cells per high power field (200x) from SPF SW mice and uMT mice described as

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Experimental details: Fig. 1 Gdpd3 is implicated in CML disease initiation in vivo. a Diagram of pathways of lysophospholipid biosynthesis. G3P is converted into LPAs, and LPAs are then converted into phospholipids by the addition of polar bases via the Kennedy (de novo) pathway. The Lands'' cycle (remodelling pathway) generates lysophospholipids of distinct composition by substituting fatty acid ester and polar base groups of phospholipids. Lysophospholipase D Gdpd3 converts lysophospholipids back into LPAs by catalysing hydrolysis (magenta dotted line). (PC Phosphatidylcholine, PS Phosphatidylserine, PE Phosphatidylethanolamine, PI Phosphatidylinositol, LPC Lysophosphatidylcholine, LPS Lysophosphatidylserine, LPE Lysophosphatidylethanolamine, LPI Lysophosphatidylinositol). b qRT-PCR determination of Gdpd3 mRNA expression in LT-stem (LT), CD48, MPP, and LK cells (see Supplementary Fig.2) isolated from Gdpd3 +/+ tet-CML-affected (SCL-tTA + TRE-BCR-ABL1 + ) mice (one male, six females) or normal littermate (SCL-tTA + ) mice (four males, four females). Data are the mean ratio +- s.d. of transcript levels normalised to Actb ( n = 3 biologically independent samples) ( P -value, unpaired two-sided Student''s t -test). c Quantitation of the colony-forming capacity of Gdpd3 +/+ CML-LSK cells that were transduced with/without Cy3-labelled siRNA targetting mouse Gdpd3 mRNA (mGdpd3 siRNA #1 or #3). Cy3 + and Cy3 - CML-LSK cells were purified at 3 days post-transduction and plated in semi-solid methylcellulo

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Experimental details: Fig. 7 Lgr4/Gpr48 is involved in CML stem cell self-renewal in vivo. a RNA-Seq determinations of mRNA levels of the indicated GPCR family genes in LT-CML stem cells isolated from Gdpd3 +/+ tet-CML-affected mice (22 males, 12 females) and Gdpd3 -/- tet-CML-affected mice (five males, five females). Results are expressed as FPKM (see Methods). Data are the mean FPKM +- s.d. ( n = 3 biologically independent samples) ( P -value, unpaired two-sided Student''s t -test). Results of the MA-plot and GO term enrichment analyses for these RNA-Seq data are shown in Supplementary Fig. 11a, b . b Quantitation of the colony-forming capacity of Gdpd3 +/+ CML-LSK cells that were transduced with/without Cy3-labelled siRNA targetting mouse Lgr4/Gpr48 mRNA (mLgr4 #3 or Lgr4 #4). Cy3 + and Cy3 - CML-LSK cells were purified at 3 days post-transduction and plated in a semi-solid methylcellulose medium. Data are the mean colony number +- s.d. ( n = 3) and are representative of three biologically independent experiments. ( P -value compared with control, unpaired two-sided Student''s t -test). The relevant FACS data are shown in Supplementary Fig. 12 . c Absolute numbers of LT-CML stem cells isolated from BM of the two hind limbs of Lgr4 +/+ tet-CML-affected mice (four males, one female) and Lgr4 Gt/Gt tet-CML-affected mice (five females) ( n = 5 biologically independent samples). Data are the mean absolute numbers +- s.d. of LT-CML stem cells ( P -value, unpaired two-sided Student''s t -test). (See S

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Experimental details: 5 Peripheral blood cytometry. A,B. A significant positive gene dose-dependent effect was observed for (A) white blood cells, and (B) platelet counts at 12 weeks of age (mean +- SEM; Df11(1)/+, N = 21; WT, N = 41; Dp11(1)/+, N = 20 ; *P < 0.05, **P < 0.01, ***P < 0.001). C-F. Flow cytometry revealed a significant positive gene dose-dependent effect for percentage (F) CD8+, but not (C) B220+, (D) Mac1+, (E) CD4+ positive cells in peripheral blood (mean +- SEM; Df11(1)/+, N = 19; WT, N = 58; Dp11(1)/+, N = 25; *P < 0.05, **P < 0.01).

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Experimental details: Figure 4 Lung leukocyte infiltration during influenza infection is comparable between PAD4 WT and KO mice. Experimental setup as described in Figure 2 . (A-C) Five mice per group were analyzed at d3 or d7 post infection. Leukocytes were isolated from infected lungs. (A) Total cell numbers of lung-infiltrating leukocytes at d3 and d7 p.i. Each symbol represents an individual mouse, filled squares represent PAD4 WT, open squares depict PAD4 KO mice. (B+C) The subsets of infiltrating leukocytes were enumerated by flow cytometry both at d3 p.i. (B) and d7 p.i. (C). Gated populations are indicated at the bottom of the graph. Total numbers of infiltrating cells are shown. WT mice are depicted in white bars, KO mice as grey bars. Bars represent mean + SEM. (D) Lungs for histological examination were harvested at d8 p.i. (5 mice/group). Leukocyte infiltration was assessed on H&E stained paraffin sections. Sections from two representative mice per group are shown. The scale bar indicates 200 um. Data is representative of two independent experiments.

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Experimental details: Figure 2 B cell development is disrupted at different stages in Ikzf1 DeltaF1/DeltaF1 and Ikzf1 DeltaF4/DeltaF4 mice. ( a ). B-cell development was analyzed in whole bone marrow from 6- to 8-week-old mice. One representative experiment out of three or more is shown. Staining is shown for total bone marrow in row 1, pre-pro B to large pre-BII (B220 + CD43 + ) cells in row 2, lineage (Lin) - B220 + CD43 + CD19 - cells (for analysis of pre-pro B) in row 3, and small pre-BII to mature B (B220 + CD43 - ) cells in row 4. The average percentage of the indicated population in total bone marrow is shown. Lineage markers included CD3, TER119, Gr-1, and CD11b. ( b ) Absolute numbers of the indicated populations in total bone marrow of wild-type (n=13), Ikzf1 DeltaF1/DeltaF1 (n=6) and Ikzf1 DeltaF4/DeltaF4 (n=6-8) mice are shown. Each symbol represents an individual mouse and bar shows the mean. * P

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Experimental details: Extended Data Figure 2 Age-related changes in ATMs in response to fasting, related to Figure 1 a , Representative gating strategy for ATM analyses. b , Representative contour plots of CD206 and CD11c expression within F4/80 + CD11b + ATMs from VAT of fed or fasted mice. Values represent mean for each condition from 2 individual experiments. Exact Ns are displayed in table 1. c, Quantification of CD11c + CD206 - (top) and CD11c + CD206 + (bottom) populations from panel b, expressed as a percentage of total F4/80 + CD11b + cells. Data are represented as mean+-SEM and pooled from two independent experiments. Each symbol represents an independent biological sample pooled from 1-4 mice; exact Ns are displayed in figure and on table 1. Tukey''s Test; P value ***

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Experimental details: Figure 2. CHD4 Is Essential for Early B Cell Development (A) Mature B cells are absent in spleens of Mb1 cre/wt Chd4 fl/fl mice. Splenocytes were stained with B220 and CD19 and analyzed using flow cytometry (top panel). Absolute numbers of total splenocytes and CD19 + B220 + B cells from each mouse strain are shown (n = 3; **p < 0.005) (bottom left). The spleen from a representative 10-week-old Mb1 cre/wt Chd4 fl/fl mouse was compared with littermate control ( Chd4 fl/fl ) (bottom right). (B) CHD4 deletion impairs early B cell development in the bone marrow. Flow cytometric analysis shows expression of IgM and B220 in the bone marrow. Cellularity of bone marrow is shown on the right side of representative flow plots. BM cells were stained with c-Kit and CD25 and analyzed for pre-B (IgM - B220 + CD25 + c-Kit - ) and pro-B (IgM - B220 + CD25 - c-Kit + ) cells. The red box represents the parent gate for analysis of pre-B and pro-B cells. n = 5; **p < 0.005. Furthermost right panel shows PCR-based genotyping and loss of the fl cel Chd4 allele in total bone marrow cells and sorted pro-B cells. Genotyping PCR from two independent experiments is shown. (C) Loss of p53 does not restore B cell developmental defects in Mb1 cre/wt Chd4 fl/fl mice. Absolute numbers of bone marrow from each mouse strain are shown (n = 4; **p < 0.005) (left). Flow cytometric analysis shows expression of IgM and B220 expression or CD25 and c-Kit in bone marrow (BM) (right). (D) Transgenic expression of Bcl2

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Experimental details: Figure 6. CHD4 Is Dispensable for Plasma-blast Differentiation (A and B) Naive splenic B cells from control ( CHD4 fl/fl ) or CHD4 KO ( Cd19 cre/wt Chd4 fl/fl ) mice were stimulated in vitro with LPS alone or LPS plus IL-4 for 96 h, and formation of B220 lo CD138 + plasmablasts was examined. (A) Representative flow cytometry data. (B) Frequency of B220 lo CD138 + plasmablast for- mation in vitro (n = 5). (C-L) Mice were intravenously injected with 50 mg LPS or PBS (mock) and analyzed at day 5. (C) Representative flow cytometry plot for B220 lo CD138 + plasmablasts. Mock represents Chd4 fl/fl p53 fl/fl animals injected with PBS. (D) Frequency of plasmablasts (B220 lo CD138 + of live cells). (E) Absolute number of plasmablasts (B220 lo CD138 + live cells). (F) Intracellular staining of CHD4 within plasma- blast population (B220 lo CD138 + of live cells). (G) Representative flow cytometry plot of IgG3 intracellular staining of B220 lo CD138 + live cell population. Mock represents Chd4 fl/fl p53 fl/fl animals injected with PBS. (H) Frequency of IgG3 + plasmablasts (amongst B220 lo CD138 + live cells). (I) MFI of CHD4 expression in IgG3 positive plasmablasts. (J) Representative flow cytometry plot for IgG3 cells within the B220 + live cell population. Mock represents Chd4 fl/fl p53 fl/fl animals injected with PBS. (K) Frequency of IgG3 CSR upon LPS administration among total B220 + cells. (L) Intracellular staining of CHD4 in IgG3-positive B cells. Mice used in (C-L): n = 3 contro

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Experimental details: Fig. 4 Analysis of B cell development in bone marrow and spleen. A. FACS analysis of BM B cells. BM cells from WT and RhoF KO mice (6-7 weeks old, n = 5, male: 40%) were stained with antibodies against CD43, B220, BP1, and CD24 and analyzed by flow cytometry. The number shows the percentage (mean +- standard deviation) of the indicated subpopulation within the parent population; Fraction A (germline pro-B cells), fraction B (DJ-rearranged pro-B cells), fraction C (Early pre-B cells), fraction D (Late pre-B cells), fraction E (newly formed B cells), and fraction F (follicular-type recirculating B cells). The figures are representatives of three independent experiments. B. FACS analysis of B cells from the spleen. Splenocytes from WT and RhoF KO mice (6-7 weeks old, n = 6, male: 33%) were stained with antibodies against CD23, CD21, and IgM and analyzed by flow cytometry. The number shows the percentage (mean +- standard deviation) of the indicated subpopulation within the parent population; T1 (Transitional 1), T2 (Transitional 2), Fo (mature follicular B cells), and MZ (MZ B cells). C. The number of B cell subsets in BM was calculated by multiplying the total number of viable (trypan blue negative) BM cells by the fraction of the target population in viable (7AAD negative) cells. The data are shown as mean +- standard deviation (6-7 weeks old, n = 5, male: 40%). White bars: WT. Black bars: RhoF KO. D. The number of B cell subsets in the spleen was also calculated by multiplyin

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Experimental details: Figure 2 Loss of Gata5 from renal endothelial cells leads to renal alterations. ( a ) GATA5 is expressed in the kidney as assessed by western blot performed on total kidney extracts. ( b ) Gata5 is essentially expressed in the glomeruli as assessed by qPCR on isolated glomeruli (Glo) and microdissected tubules (Tub) from Wt mice kidneys. ( n =3-5 per group). The results are reported as mean+-s.e.m. * P

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Experimental details: Figure 5 NG2-cre, but not NG2-cre ERTM , targeted cells are the source of Scf in the bone marrow (a) Whole-mount sternum from NG2-cre/ iTdTomato/ Scf-GFP mice, anti-VE-cadherin. Representative images from 3 mice. Scale bars, 20 mum. (b) Representative FACS plot showing percentage of NG2-cre/ iTdTomato + cells within CD45 - TER119 - CD31 - Scf-GFP + cells. n=3 mice. (c-e) Analyses of LepR-cre/ Scf fl/- mice. (c) Numbers of HSCs (left) in BM and LSK cells in spleen (right). n=4 mice for cre (-), n=3 mice for cre (+). (d) FACS analyses of HSC (CD150 + CD48 - LSK) cell cycle with Ki-67 and Hoechst 33342 staining. n=5 mice for cre (-), n=6 mice for cre (+). (e) HSC localization relative to arterioles. Error bars: n=3 mice. P value has been calculated using n=272 HSCs for cre (-), 293 HSCs for cre (+) pooled from 3 mice per group. P =0.3402. (f-i) Analyses of NG2-cre/ Scf fl/- mice. (f) Numbers of total BM cells (left) and CD150 + CD48 - LSK HSCs (right) in BM. n=5 mice for cre (-), n=7 mice for cre (+). (g) Percentages of donor-derived cells after competitive reconstitution. n=5 mice for cre (-), n=7 mice for cre (+). (h) FACS analyses of HSC cell cycle with Ki-67 and Hoechst 33342 staining. n=6 mice for cre (-), n=7 mice for cre (+). (i) HSC localization relative to arterioles. Error bars: n=3 mice. P value has been calculated using n=224 HSCs for cre (-), 274 HSCs for cre (+) pooled from 3 mice per group. P =0.2872. (j-l) Analyses of NG2-cre ERTM / Scf fl/- mice. (j) Absolute nu

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Experimental details: Figure 2 Splenic T cell hyperplasia and T cell size in p27 -/- Lck-Bcl-2 mice. A) Total splenic cells from mice of the indicated genotypes were isolated and stained with anti-B220-PE and anti-CD3-FITC antibodies as described in the Materials and Methods . The percentage of CD3-positive cells and the total number of splenic T cells (total # of splenic cells X %CD3) is shown. The data are representative of at least three mice from each genotype. B) The Mean Forward Scatter of CD3 positive splenic T cells was determined by staining with an anti-CD3 antibody as described in the Materials and Methods . The Mean+-SD of at least 5 mice is shown for the genotypes indicated. * P

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Experimental details: Figure 4 Loss of memory B cells in the absence of Atg7 ( a , b ) DUMP - B220 + IgG1 + CD38 + NP + memory B cells in the spleen of B/ Atg7 -/- mice or WT controls eight weeks after immunization. Unimmunized mice were used as negative controls (a). Total NP-specific memory B cells in the spleen are presented in (b). ** P

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Experimental details: Figure 4 Global transcription changes in p21 Cip1 -deficient mice. ( A ) Differentially expressed genes (DEG) in the liver of p21KO compared to WT mice under ad libitum feeding conditions and upon 24 h fasting. Data were obtained from RNA-Seq analysis of 2-3 male mice per group. Red circles correspond to upregulated and green circles to downregulated genes. Values were considered significant when FDR value was q < 0.05. ( B ) Heat map of significantly altered pathways in the liver in the same samples as in panel A. Results were obtained from GSEA and considered significant when FDR < 0.25. ( C ) Representative GSEA plots of immune-related pathways downregulated in fasted p21KO mice compared to fasted controls. FDR < 0.25. ( D ) Relative amount of CD45 + immune cells in the liver of ad libitum fed and 24 h fasted WT and p21KO mice. Values correspond to average +- s.d. (n = 6 males, 12-16 weeks old). ( E ) Representative pictures of F4/80 stained liver sections of WT and p21KO mice under feeding conditions, 24 h and 48 h fasting. Bars correspond to 50 mum. Statistical significance was determined by two-way ANOVA and Bonferroni post-hoc test: **p < 0.01. See also Figure S3 .

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Experimental details: Figure 7. NOX2 and FOXC1 Are Important for the Growth of Primary Human Myeloid Leukemia Cells (A) The Cancer Genome Atlas (TCGA) database for AML was used to analyze the expression levels of several NADPH-dependent oxidases (NOX1-NOX5), accessory subunits (NCF1, NCF2, NCF4, NOXA1, NOXO1, p22Phox, RAC1, and RAC2) as well as related oxido-reductase enzymes (dual-oxidases 1,2, A2). The reads per kilobase of transcript per million mapped reads (RPKM) values for each gene in a total of 188 AMLs are shown. (B) RNA-seq analysis was performed on functionally validated leukemic stem cells isolated from human primary AMLs. The RPKM value for each gene is shown. Unpublished data are used with permission. Additional supporting data are shown in Figure S7A . (C) Equal numbers of control or shNOX2-transduced primary AML cells were cultured in vitro in the presence of 10 ng/mL of IL-3, SCF, and FL3, and the relative percent expansion is reported for each specimen. n = 3, mean +- SD. *p < 0.05; **p < 0.01. (D) Control and shNOX2 primary AML cells were purified and cultured in vitro for 12 days. Annexin V, DAPI staining was performed to evaluate the degree of apoptotic cell death. A representative flow plot (left) and quantitation of 3 technical triplicates (right) are shown. Additional data are shown in Figure S7B . (E) The relative expression level of NOX2 , CEBP epsilon, Elane , and CTSG is shown in AML specimens in which NOX2 was knocked down using shRNAs. Additional supporting data are p

Supportive validation

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Experimental details: Figure 6 The characterization of Il-2 producers regulated by FX and TAC. FX stimulated Il-2 production in CD45R + CD25 + ILCregs. Some Il-2 + CD45R + CD25 + ILCregs were observed in TAC treated skin and others were CD45R - CD25 - cells. The panel is focused on the dermis.

Supportive validation

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Experimental details: Fig. 4. Macrophage-derived MCPIP1 regulates B-lymphocyte expansion in lymphoid organs. (A-D) Spleen sections were stained with anti-mouse IRF4, BLIMP1, CD95 and B220 antibodies and quantified by Adobe Photoshop software as percentage of positively stained high-power field (HPF) from eight mice per group; *** P

Supportive validation

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Experimental details: Fig. 1 Ubiquitous miR-210 over-expression attenuates the inflammatory response after hindlimb ischemia. A Representative immunofluorescence staining for CD45 (green), a common leukocyte marker expressed in all nucleated hematopoietic cells, of gastrocnemius muscle sections of Wt and Tg-210 mice, 7 days after ischemia. Nuclei were stained by Hoechst (blue). Images are presented as merge. Magnification x400. Calibration bar 50 um. B Quantification of CD45-positive cells/mm 2 ( n = 5, test T * P < 0.03). C Representative immunofluorescence staining for F4/80 (green), a pan-macrophage marker, in gastrocnemius muscle sections of Wt, and Tg-210 mice, 7 days after ischemia. Nuclei were stained by Hoechst (Blue). Images are presented as merge. Magnification 63 x 0.5. Calibration bar 50 um. D Quantification of F4/80-positive macrophages/mm 2 ( n = 6-7, ** P < 0.009). In both A , C insets show magnification of the indicated areas.

Supportive validation

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Experimental details: Figure 6 Assessments of organ damage and mouse metabolites during HLSA or recovery from HLSA. A , upper left panel , MMP-1, IL-1beta, and CRP levels in plasma had no significant change between the control group, HLSA group, and R-HLSA group. Upper right panel , CK-MB as a marker of heart injury in the plasma had no significant change between the control group, HLSA group, and R-HLSA group. Lower left panel , creatinine and BUN as markers of kidney injury in the plasma slightly increased in the HLSA group and returned to normal level after recovery from HLSA. Lower right panel , AST and ALT as markers of liver injury in the plasma showed no change between the control group, HLSA group, and R-HLSA group. Data were presented as mean +- SD (N = 6; ANOVA: ** p < 0.01). B , upper panel , food consumption and water consumption 3 days before and 3 days after a single ATP-induced HLSA depicted no change. Data were presented as mean +- SD (N = 6; ANOVA: p > 0.05). Lower panel , metabolomics analysis of scores plot from PCA analysis based on 1 H NMR data from the plasma, brain, liver, and kidney of the control group, HLSA group, and R-HLSA group. The PCA score showcased clusters correspond to metabolic patterns in different groups, with each point representing one sample. Circles represent 95% confidence interval for each score in each group (see also Figs. S5-S8 ). C , draining lymph nodes of mice that underwent HLSA once a day for 10 consecutive days were analyzed with FACS. CD44 and

Supportive validation

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Experimental details: Figure 5 CD4 surface expression of CD45RB and global glycosylation are altered in SLE123 mice. ( A and B ) Cell surface expression of CD45RB and total CD45 were measured by flow cytometry on CD4 + T cells. SLE123 demonstrated a downregulation of CD45RB on the cell surface as compared with B6 CD4 + T cells. There was no difference in pan-CD45 expression. Quantified in B . ( C ) CD45 is composed of an intracellular region that controls cytoskeletal binding and its phosphatase activity. The extracellular domain is composed of a region with fibronectin repeats that is heavily N-glycosylated. An alternatively spliced region is heavily O-glycosylated, and this region imparts unique functions to each CD45 isoform. Additionally, a sialic acid residue on the B portion of this region is essential for the binding of therapeutic aCD45RB. ( D and E ) We utilized lectins to detect the level of alpha-2,3-linked sialylation (MALII), O-linked glycosylation (Jacalin), and N-linked glycosylation (PHA-L) in CD4 + T cells from B6 and SLE123 mice. We determined SLE123 CD4 + T cells had reduced levels of alpha-2,3-linked sialic acids and O-glycosylation and an increase in N-glycosylation. Quantified in E . ( F ) Utilizing an antibody that detects a desialylated form of anti-CD45RB, we determined SLE123 CD4 + T cells possessed increased binding of this antibody compared with B6. ( G and H ) To determine the binding of therapeutic aCD45RB to B6 and SLE123 CD4 + T cells, we incubated splenocytes from

Supportive validation

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Experimental details: Figure 2. pIgR -/- mice spontaneously develop COPD-like lung pathology and adaptive immune activation. ( A ) Representative image of emphysema in an 18-month-old pIgR -/- and age-matched WT mouse (hematoxylin and eosin, scale bar = 50 mum). ( B ) Measurement of mean linear intercept (MLI), a morphometric measurement of emphysema, in WT and pIgR -/- mice at the indicated ages. ( C ) Representative image of small airway wall thickening in an 18-month-old pIgR -/- and age-matched WT mouse (Masson's trichrome, scale bar = 50 mum). ( D ) Measurement of VV airway , a morphometric measurement of small airway wall thickness, in WT and pIgR -/- mice at the indicated ages. ( E ) Example of a tertiary lymphoid structure (TLS) in an 18-month-old pIgR -/- mouse as indicated by immunostaining for B220. Scale bar = 50 mum. ( F ) Morphometric analysis of TLS area in lungs of WT or pIgR -/- mice at the indicated ages. ( G-J ) Quantification of total, CD45 + , CD19 + , CD3 + , CD4 + , and CD8 + cells in the lungs of 18-month-old WT and pIgR -/- mice by flow cytometry. ( B and D ) * = p

Supportive validation

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Experimental details: Fig. 7 Upregulation of PD-L1 by MTHFD2 is required for tumorigenesis. a - b A total of 2 x 10 6 ( a ) or 5 x 10 6 ( b ) Pan02 cells stably infected with lentivirus carrying indicated shRNAs or exogenous expressed PD-L1, were subcutaneously injected into athymic nude mice ( a ) or C57 mice ( b ), tumor volume was calculated every 7 days. Tumor xenografts at the 28th day in nude mice ( a ) or the 21th day in C57 mice ( b ) were shown. Data represent the means +- s.e.m ( n = 8 mice per group); p value (Student's t test, two-sided) with control is presented. c , PD-L1 mRNA levels in tumor tissues in C57 mice were analyzed by real-time PCR. The values are presented as mean +- s.e.m ( n = 3); p values (Student's t test, two-sided) with control or the indicated groups are presented. d The lysates of 8 pooled tumor tissues in C57 mice were subjected to immunoblotting analyses using the indicated antibodies. e Immunohistochemical staining was performed on tumor sections in C57 mice with anti-CD8 antibody. Representative images are shown. Scale bars, 50 mum. Histological semi-quantification was performed. f Cells digested from indicate tumor tissues in C57 mice were stained with anti-CD45 antibody and subjected to flow cytometric analyses (also see Supplementary Fig. 7b ). Representative images (1 out of 3 experiments) are shown. g A schematic model showing the role of MTHFD2 in tumor immune evasion. MTHFD2 promotes PD-L1 mediated tumor immune evasion through the folate-cycle-UTP-UDP-G

Supportive validation

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Experimental details: Figure 6 Colonic innate and adaptive immune cell responses following peroral C. coli infection of human microbiota-associated TLR4-deficient IL10 -/- mice. IL10 -/- mice (open symbols) and TLR4-deficient IL10 -/- mice (TLR4 -/- IL10 -/- ; closed symbols) with a human gut microbiota were either perorally infected with C. coli (circles) or received vehicle (mock; diamonds) on days 0 and 1 by gavage. Upon necropsy on day 21 post-infection, the average counts of ( A ) macrophages and monocytes (F4/80 + ), ( B ) T lymphocytes (CD3 + ), ( C ) regulatory T cells (FOXP3+) and ( D ) B lymphocytes (B220+) were microscopically determined in six high power fields (HPF, 400 times magnification) per mouse in immunohistochemically stained colonic paraffin sections. Medians (black bars) and numbers of examined mice (in parentheses), as well as levels of significance ( p -values) determined by the one-way ANOVA test and Tukey's post-correction or the Kruskal-Wallis test and Dunn's post-correction, are shown. Data were pooled from three independent experiments.

Supportive validation

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Experimental details: Figure 6 Serum CXCL13 levels in mouse ktx. Post ktx levels of serum CXCL13 at day 1 were significantly increased compared to baseline. A higher increase was observed in longer cold ischemia time (30 vs. 60 min cold ischemia time). Isogenic ktx with prolonged cold ischemia time of 60 min had significantly lower CXCL13 levels compared to allogenic ktx (A) . PAS stain at day 7 revealed enhanced cell infiltration in allogenic compared to isogenic ktx (B) . Double staining for CD3+ T-lymphocytes (green) and CD45R+ B-cells (red) was performed at day 7. More interstitial CD3+ T-lymphocytes were observed in allografts compared to isografts. Allografts exhibited scattered B-cells in interstitial tissue, but also clusters of CD45R+ cells. Isografts showed only few B cells in the interstitium at day 7 (B) (bar: 100 mum, n = 6 per group, one-way ANOVA * p < 0.05; ** p < 0.01; *** p < 0.001). BL, baseline.

Supportive validation

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Experimental details: Figure 7 Colonic immune cell responses upon oral C. coli infection of microbiota-depleted IL-10 -/- mice that had been challenged with human or murine fecal microbiota transplantations. On days -7, -6, and -5, mice were subjected to murine (open symbols) or human (closed symbols) fecal microbiota transplantations and challenged with a C. coli patient isolate (circles) or vehicle as mock controls (diamonds) on days 0 and 1 by gavage. On day 21 post-infection, the average numbers of ( A ) macrophages and monocytes (F4/80+), ( B ) T lymphocytes (CD3+), ( C ) regulatory T cells (FOXP3+), and ( D ) B lymphocytes (B220+) were determined in the large intestinal mucosa and lamina propria out of 6 high-power fields (HPF, 400x magnification) per mouse. Medians (black bars), significance levels ( p -values calculated by the Kruskal-Wallis test and Dunn's post-correction), and total numbers of mice under investigation (in parentheses) are shown. Results from four experiments were pooled.

Supportive validation

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Experimental details: Figure 5 Innate and adaptive immune cell responses in the colon following PACAP treatment of C. jejuni infected secondary abiotic IL-10 -/- mice. Mice were perorally infected with C. jejuni strain 81-176 by gavage on day (d) 0 and d1, and subjected to intraperitoneal treatment with either synthetic PACAP or vehicle (mock) from d2 until d5 post-infection. On d6 the average numbers of ( A ) macrophages and monocytes (F4/80 + ), ( B ) T lymphocytes (CD3 + ), ( C ) regulatory T cells (FOXP3 + ) and ( D ) B lymphocytes (B220 + ) were quantitated in six high power fields (HPF) of colonic paraffin sections applying immunohistochemistry. Naive mice were used as negative controls. Box plots indicate the 75th and 25th percentiles of the median (black bar within boxes). The total range, the significance levels ( p values calculated by the one-way ANOVA test followed by the Tukey post-correction test for multiple comparisons) and the total numbers of mice under investigation (in parentheses) are indicated. Results pooled from four independent experiments are shown.

Supportive validation

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Experimental details: Figure 1 Ibrutinib promotes renal fibrosis after unilateral ureteric obstruction (UUO). ( A ) Design of the study. ( B - E ) Representatives images and quantifications of Fibronectin ( B ), Sirius Red ( C ), B220 ( D ) and F4/80 ( E ) staining in sham and UUO mice receiving ibrutinib (Ibru) or vehicle (Veh). Data are represented as means +- SEM. *p < 0.05 (Mann-Whitney test; n = 6 mice per group); ns, not significant.

Supportive validation

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Experimental details: Figure 3 Ibrutinib promotes rhabdomyolysis-induced renal fibrosis. ( A ) Design of the study. ( B ) Kaplan-Meier survival curves after glycerol injection in mice receiving ibrutinib (Ibru) or vehicle (Veh) from day 3. ( C ) Blood urea nitrogen (BUN) 20 days after glycerol injection. ( D - G ) Representatives images and quantification of Sirius Red ( D ), Fibronectin ( E ), F4/80 ( F ) and B220 ( G ) staining 20 days after glycerol injection in mice receiving ibrutinib (Ibru) or vehicle (Veh) from day 3. Values are expressed as mean +- SEM. * p < 0.05, compared with the vehicle group (Mann-Whitney test, n = 5-10 mice); ns, not significant.

Supportive validation

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Experimental details: Fig. 2 Generating immuno-CRISPR (iCR) mice and evaluating the CRISPR-mediated modifications by sequencing. ( A ) Model describing the experimental setup where CD45.1 + Lin- BM cells were modified by CRISPR targeting Zap70 and grafted into irradiated CD45.2 + recipients. ( B ) Flow cytometry analysis of cells in the blood of Zap70 iCR mice and WT control mice eight weeks post transplantation. Cells gated on viable, CD45.1+, single lymphocytes. ( C ) Quantification of B and T cells in the blood of WT and Zap70 iCR mice in (B). ( D ) Analysis of the level of mutations in the sgRNA targeted Zap70 region in the BM cells used for transplantation, total cells from the blood, as well as in B and T cells sorted from the spleen of Zap70 iCR mice and WT control mice 8 weeks after engraftment. ( E ) Model describing the experimental setup where a secondary transplantation was used to amplify the population of successfully modified mice. ( F ) Analysis of the level of mutations in the sgRNA targeted GFP region in blood cells of the GFP iCR mice four weeks after transplantation, in an experiment with low efficiency. One mouse showed good knockout efficiency (labeled in orange) and was used as BM donor for secondary transplantation. ( G ) Kinetics of the level of mutations of GFP in the secondary iCR mice. ( H ) As examples, representative flow cytometry plots and ( I ) GFP + cell population percentage of alveolar macrophages, B1 cells, neutrophils and germinal center B cells from secondary

Supportive validation

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Experimental details: Figure 4 Proof-of-mechanism study for TAX2 use as an anti-tumor immunomodulatory drug. The effects of TAX2 peptide treatment on the activation of an anti-tumor immune response were investigated in two syngeneic models of epithelial ovarian carcinoma. We inoculated 5 x 10 6 ID8 cells either subcutaneously or intraperitoneally to C57BL/6JRj mice in order to mimic primary tumor growth ( a - h ) and metastatic dissemination (peritoneal carcinosis, i - l ), respectively. TAX2 treatments (30 mg * kg -1 BW) vs. vehicle injections (0.9% NaCl) were performed thrice weekly for 4 and 8 weeks for the s.c. and i.p. ID8 models, respectively ( n = 11 per group for both models). ( a - h ) IHC staining of s.c. ovarian carcinoma allografts for the analysis of the vascular/lymphovascular features, as well as immune-cell-infiltration profile. ( a ) Representative microphotograph for CD31 immunostaining of intra-tumor vascular structures. Histogram displays results of automated quantification for the percent of CD31-positive pixels across the whole tumor section (mean +- SEM, Mann-Whitney U test; n.s., not significant). ( b ) Lymphatic vessel density analysis. Microscopic views are shown, while histogram displays the number of Lyve-1-positive functional lymphatic vessels in 5 high power fields (HPF), as determined by a pathologist who was blinded to the treatment (mean +- SEM, Mann-Whitney U test, * p < 0.05). ( c - f ) Macroscopic views (x20 magnification) of s.c. tumor allograft sections after

Supportive validation

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Experimental details: Figure 8. Increased IL-2 production impairs T follicular helper (Tfh) cell formation and the germinal centre response. Assessment of the Tfh cell and germinal centre response in Il2 cre/+ ; Rosa26 stop-flox-Il2/+ transgenic mice that do not switch off IL-2 production, and Il2 cre/+ ; Rosa26 +/+ control mice 12 days after influenza A infection. Flow cytometric contour plots ( A ) and quantification of the percentage of CXCR5 high PD-1 high Foxp3 - CD4 + Tfh cells in the mediastinal lymph node ( B ) and spleen ( C ). Flow cytometric contour plots ( D ) and quantification of the percentage of Bcl6 + Ki67 + B220 + germinal centre B cells in the mediastinal lymph node ( E ) and spleen ( F ). The height of the bars indicates the median, each symbol represents one mouse, data are pooled from two independent experiments. p-Values calculated between genotype groups by Mann-Whitney U test.

Supportive validation

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Experimental details: Fig. 2. Effect of different PVA hydrolysis rates and molecular weights on ex vivo cultured human cord blood CD34 + hematopoietic stem and progenitor cells. a , Human cord blood CD34 + cells were cultured in PVA media and cell numbers were counted at day 14. Three independent experiments were performed with cord blood CD34 + cells derived from different donors. Cell cultures were started from 7 x 10 3 (Exp.1) or 5 x 10 3 (Exp. 2 and 3) CD34 + cells. Mean +- S.D is shown. Statistical significance was calculated using ANOVA followed by Tukey-Kramer; *p < 0.05, **p < 0.01. b , Proliferation of the two different CD34 + cell fractions after 7-day culture in PVA-containing media. Fifty cells were sorted into four wells in a 96-well plate and cell numbers in each well were counted at 7-day. Statistical significance was calculated using ANOVA and Tukey-Kramer. **p < 0.01.

Supportive validation

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Experimental details: Figure 8 Establishment of adaptive immunity in the infected lungs of WT and CXCL5 -/- mice. (A) Lung lobe sections from WT and CXCL5 -/- mice were stained at 10 d.p.i. with an anti-B220 antibody and hematoxylin (x30 magnification). (B) The positively stained area (iBALT structure) in each lung lobe section was calculated using CaseViewer software (3DHISTECH). The total area of iBALT per lung section was calculated from 4 mice per group (3 lung lobe sections per mouse). (C) Lung sections were stained with anti-B220 (red) and anti-CD4 (yellow) antibodies for immunofluorescence detection. The immunofluorescence sections were counterstained with DAPI (blue). (D) GO functional analysis of upregulated genes in the infected lungs of CXCL5 -/- mice compared to WT mice at 7 and 10 d.p.i. The methods and criteria for filtration of differentially expressed genes are described in the MATERIALS AND METHODS section. (E) Mice were infected with H1N1 or H3N2 (3000 CCID 50 ), and the body weight loss (n=12) of the mice was monitored until 2 weeks post infection. WT-1 H1N1: WT mice infected with H1N1; WT-1 H3N2: WT mice infected with H3N2; CXCL5 -/- 1 H1N1: CXCL5 -/- mice infected with H1N1; CXCL5 -/- 1 H3N2: CXCL5 -/- mice infected with H3N2; WT-2 H1N1: WT mice recovered from H1N1 infection and infected with H1N1; WT-2 H3N2: WT mice recovered from H1N1 infection and infected with H3N2; CXCL5 -/- 2 H1N1: CXCL5 -/- mice recovered from H1N1 infection and infected with H1N1; CXCL5 -/- 2 H3N2: CXC

Supportive validation

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Experimental details: Figure 3. Histone deacetylase 3 (HDAC3)-deficient CD4 + T cells are capable of differentiation, but produce fewer cells than wild-type (WT). ( a ) In vitro differentiation assays were performed to examine differentiation into the T h 1, T h 2, T h 17, and T reg lineages characterized by transcription factor expression. Splenocytes were harvested and magnetically enriched for naive (CD44 - ) CD4 + T cells by negative selection. Cells in all assays were stimulated with 2 ug/ml plate-bound alphaCD3 and 0.5 ug/ml alphaCD28 for 4 days. For T h 1 differentiation, 1 ug/ml alphaIL-4 antibody and 10 ng/ml of IL-12 were added to the media. For T h 2 differentiation, 1 ug/ml of each alphaIFNgamma and alphaIL-12 antibody, as well as 10 ng/ml of IL-4 was added to the media. For T h 17 differentiation, media was supplemented with 10 ug/ml of alphaIFNgamma and alphaIL-4 antibody as well as 10 ng/ml of rIL-23, 5 ng/ml TGF-beta1, and 20 ng/ml IL-6. For T reg differentiation, media was supplemented with 10 ug/ml alphaIFNgamma and alphaIL-4 antibody as well as 2 ng/ml TGF-beta1, and 2 ng/ml interleukin-2 (IL-2). Unstimulated control samples did not receive alphaCD3/alphaCD28 stimulation, but did receive 10 ng/ml IL-7 to maintain cell survival during culture. Bar plots on the right show % of cells from the total culture that are positive for the transcription factor +- standard deviation (SD), total cell number +- SD, and geometric mean of expression +- SD ( n = 2-4 mice/group from two to three

Supportive validation

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Experimental details: Figure 6 MALP ablation blocks bone marrow recovery after radiation. ( A ) Representative fluorescence images of Td + cells, Perilipin + LiLAs, and CD45 + hematopoietic cells in femoral bone marrow of Adipoq/Td/DTR mice after receiving 2 weeks of vehicle (Veh) or DT injections with or without prior radiation. Scale bar: 20 mum (top), 100 mum (middle), and 20 mum (bottom). ( B ) Quantification of CD45 + cells per bone marrow area. n = 3-6 mice/group. ( C ) Bone marrow cells were flushed from femurs and counted. n = 3-8 mice/group. ( D ) Cell counts of hematopoietic lineage cells in the bone marrow. n = 3-11 mice/group. B cells = B220 + , T cells = CD3 + , myeloid cells = Gr1 + and/or Mac1 + . ( E ) Cell counts of HSPCs. n = 3-11 mice/group. LK, Lineage - cKit + , LSK, Lineage - Sca1 + cKit + , SLAM LSK, Lineage - Sca1 + cKit + CD48 - CD150 + , MPP, Lineage - Sca1 + cKit + CD48 + CD150 - . ( F ) Representative fluorescence images of Adipoq/Td/DTR femoral bone marrow with Emcn staining (vessels). Arrows point to Td + pericytes. Scale bar: 20 mum. ( G ) Quantification of bone marrow vessel diameter, density, and area. ( H ) The number of pericytes per vessel length (VL) was measured. n = 3-4 mice/group. ( I ) The percentage of Emcn + endothelial cells in bone marrow was measured by flow cytometry. n = 3-4 mice/group. ( J ) qRT-PCR analysis of hematopoietic and angiogenic factors in sorted Td - and Td + cells from bone marrow before and after radiation. n = 4 mice/group. Statistica

Supportive validation

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Experimental details: Figure 2. The miR-92 deficient mir-17-92 cooperates with c-Myc to promote highly aggressive B-lymphomas. ( A ) The percentage of IgM positive and IgM negative B-lymphomas was calculated for each genotype ( Emu-myc/MSCV, n = 10 ; Emu-myc/17-92, n = 9; Emu-myc/17-92Delta92, n = 10; Emu-myc/17-92Mut92, n = 10). ( B ) The Emu-myc/17-92Mut92 and Emu-myc/17-92Delta92 mice developed high grade B-lymphomas that were frequently disseminated into the liver. When compared to Emu-myc/MSCV and Emu-myc/17-92 mice, Emu-myc/17-92Mut92 and Emu-myc/17-92Delta92 lymphomas gave rise to more liver dissemination, as indicated by H&E and B220 staining. ( C ) Emu-myc/17-92Mut92 and Emu-myc/17-92Delta92 lymphomas exhibited a decreased apoptosis compared to Emu-myc/MSCV or Emu-myc/17-92 lymphomas. Representative lymphomas were stained for H&E, cleaved caspase-3 and PCNA. Arrow, 'starry sky' feature of apoptotic lymphoma cells; arrowhead, apoptotic cells with positive staining for cleaved caspase-3; scale bar, 50 mum. ( D and E ) Apoptosis was quantitatively measured in representative lymphomas of each genotype using the 'starry sky' features ( D ) and cleaved caspase-3 staining ( E ). *p < 0.05, **p < 0.01, ***p < 0.001. DOI: http://dx.doi.org/

Supportive validation

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Experimental details: Lenvatinib insignificantly affects the immune microenvironment in NPC in humanized NSG mice. A Schematic diagram of the establishment of humanized NSG mice. B Representative FACS analysis of human CD45 + cells in mouse peripheral blood. Human CD45 + cell percentage greater than 25% was considered successful in modeling. C - E Tumor growth ( C ), tumor weights ( D ) were measured in vehicle-, anti-VEGF-, and lenvatinib-treated NPC tumors. The tumor inhibition ratio were calculated ( E ) ( n = 3 samples per group). F Representative micrographs of Ki67 + proliferative cells and cleaved caspase-3 + apoptotic cells in vehicle-, anti-VEGF-, and lenvatinib-treated NPC tumors. Scale bar = 50 mum. Quantification of Ki67 + , cleaved caspase-3 + signals, and PA index in vehicle-, anti-VEGF-, and lenvatinib-treated NPC tumors. ( n = 8 random fields per group) G Representative micrographs of CD31 + microvessels and CA9 + hypoxic areas in vehicle-, anti-VEGF-, and lenvatinib-treated NPC tumors. Scale bar in upper panel = 100 mum, scale bar in lower panel = 50 mum. Quantification of CD31 + tumor vessel parameters and CA9 + signals in vehicle-, anti-VEGF-, and lenvatinib-treated NPC tumors ( n = 8 random fields per group). H Quantification of hCD45 + hCD14 + population, hCD45 + hCD19 + population, hCD45 + hCD3 + population, and hCD45 + hCD56 + population in the NPC TME ( n = 3 samples per group). I Quantification of mCD45 + mCD11b + mF4/80 + population, mCD45 + mB220 + population, mCD45 + mC

Supportive validation

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Experimental details: Figure 2 Differentiation of iPSCs into Mphi (A) Schematic representation of the three step EB-based protocol employed for hematopoietic differentiation. (1) embryoid bodies (EBs) are formed for 8 days in the presence of 10 ng/ml IL-3 with 30 ng/ml SCF added from day 5 onward; (2) EBs are dissociated to single cells and CD41 + cells are isolated by fluorescence-activated cell sorting; and (3) terminal differentiation is achieved by 10-15 days of adherent culture in the presence of 30% M-CSF containing supernatant (L929 cells). (B) Representative picture of EBs at day 8. Scale bar, 500 mum. (C) Flow cytometric analysis of CD41 expression on day 8 and representative picture of May-Grunwald/Giemsa-stained cytospins of CD41 + cells. Scale bar, 50 mum. (D) Approximately 10-fold increase in cell number during differentiation (independent experiments, n = 3, mean +- SD). (E and F) Representative pictures of (E) BMlin - -Mphi and (F) CD45.1 iPSC-Mphi in bright-field and in May-Grunwald/Giemsa-stained cytospins. Scale bars, 100 mum and 20 mum. (G and H) Exemplary flow cytometry plots for expression of surface markers F4/80, CD11b, CD14, CD11c, Gr-1, CD45.1, CD45.2, CD41, CD3, and B220 on BMlin - -Mphi (G) and CD45.1iPSC-Mphi (H).

Supportive validation

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Experimental details: Figure 5 Differentiation of miPAP1 into Mphi (A and B) Representative picture of (A) bright-field EBs at day 8 of differentiation (scale bar, 500 mum) and (B) flow cytometric analysis of CD41 expression. (C) Representative pictures of (upper picture; scale bar, 100 mum) bright-field microscopy and (lower picture; scale bar, 20 mum) May-Grunwald/Giemsa-stained cytospins of miPAP1-Mphi. (D) Flow cytometric analysis of surface marker expression (data for F4/80, CD11b, CD14, CD11c, Gr-1, CD45.1, CD45.2, CD41, CD3, and B220).

Supportive validation

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Experimental details: Figure 6 Disease Modeling of herPAP Using miPAP iPSCs (A) Number of CD41 + progenitor-derived colonies in methylcellulose-based clonogenic assays employing complete (IL-6, erythropoietin, SCF, IL-3, and supplemented with 20 ng/ml human G-CSF + 20 ng/ml murine GM-CSF) or basic medium (50 ng/ml murine GM-CSF only; independent experiments, n = 3 miPAP1 and 2, n = 2 CD45.1 iPSC, mean +- SD) and representative pictures of colonies. Scale bars, 500 mum. (B) GM-CSF clearance assay comparing miPAP-Mphi with CD45.1(10.3) iPSC-Mphi, BMlin - -Mphi, and no cells incubated with 2 ng/ml GM-CSF: at indicated time points (0, 4, 10, 24, and 30 hr) GM-CSF concentrations in supernatants were analyzed by ELISA, normalized to 0 hr (independent experiments, n = 3 BMlin - and CD45.1 iPSC, n = 2 miPAP1 and 2, mean +- SD). (C and D) Flow cytometry plots of STAT5 phosphorylation levels upon stimulation with mGM-CSF (C) and (D) summary of mean fluorescence intensity (MFI) data (independent experiments, n = 3 BMlin - and CD45.1 iPSC, n = 2 miPAP1 and 2, mean +- SD). ns, not significant; ** p

Supportive validation

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Experimental details: Fig 6 Tn P acts systemically during the induction phase modulating DCs and induces regulatory cells. Splenocytes from EAE mice treated with vehicle or Tn P (n = 5/group) were isolated at day 7, and analyzed. A ) The MFI of MHC class II, CD40, CD80, CD86 in cDC (CD11c+CD11b+) and B ) MFI of PDL-1 and PDL-2 in pDC (CD11c+B220 low ) were analyzed by flow cytometry (50,000 events). C ) The percentage of CD4+ cells, D ) and the MFI of CD18, CD40L, and CD69 in the CD4+ gate were analyzed by flow cytometry. E , F , G ) The percentages of FOXP3-positive CD4+CD25+ Treg, IL-4-positive CD4 Th2 cells and CD5-positive CD19+CD1d+ Breg cells were analyzed by flow cytometry. Values in the bar graphs are the mean +- SEM. * p < 0.05 and ** p < 0.01 compared with vehicle-treated EAE-mice.

Supportive validation

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Experimental details: mTORC1 inactivation in osterix-expressing cells results in decreased B-cell populations in the BM. ( A ) General images for H&E-stained femur BM sections of control and Osx-Raptor mice. Microphotographs were taken at x10 magnification. ( B ) BM cellularity in control and Osx-Raptor mice ( n = 19 control, n = 17 Osx-Raptor). ( C ) FACS plots of total B lymphocytes (B220 + CD19 + ) in the BM of control and Osx-Raptor mice. ( D ) FACS plots of T lymphocytes (CD3 + ) in the BM of control and Osx-Raptor mice. ( E ) FACS plots of granulocytes (CD11b + Gr-1 + ) in the BM of control and Osx-Raptor mice. ( F ) Total percentages of B lymphocytes, T lymphocytes, and granulocytes. Numbers in each region represent the percentage of cells in the total population ( n = 4 control, n = 5 Osx-Raptor). Both male and female mice at 3 weeks of age were used for experiments. Error bars in bar graphs are SE; *** p < 0.001.

Supportive validation

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Experimental details: B lymphopoiesis in the BM is blocked at the transition from the pro-B to pre-B cell stage in Osx-Raptor mice. ( A , C ) B-cell subpopulations in the BM of control and Osx-Raptor mice were analyzed by FACS. Populations were defined as follows: pre-pro-B cells = B220 + CD19 - CD43 + ; pro-B cells = B220 + CD43 + ; pre-B plus immature B cells = B220 + CD43 - ( n = 6). ( B , D ) The absolute numbers of B-cell subpopulations in the BM of control and Osx-Raptor mice was calculated from FACS. ( E ) Total B-cell proliferation in the BM of control and Osx-Raptor mice was indicated by Frequency of CD19 + EDU + B-cells ( n = 4). ( F ) Apoptosis of total B-cells, pro-B cells, pre-B plus immature B cells in the BM of control and Osx-Raptor mice was assessed by FACS after Annexin-V staining ( n = 4 control, n = 5 Osx-Raptor). Both male and female mice at 3 weeks of age were used for experiments. Error bars in bar graphs are SE; * p < 0.05, *** p < 0.001.

Supportive validation

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Experimental details: IL-7/IL-7R/Stat5 and Erk signaling are dysregulated in different B-cell stages in Osx-Raptor mice. ( A ) IL-7Ralpha expression in B-cell subsets was analyzed by FACS. Subsets were defined as follows: pro-B cells = B220 + CD43 + ; pre-B plus immature B cells = B220 + CD43 - ( n = 4). ( B ) The levels of p-Stat5 in B-cell subsets were analyzed by FACS. ( n = 4) ( C ) The levels of p-Erk in total B-cells were analyzed by Western blot assay. Both male and female mice at 3 weeks of age were used for experiments. Error bars in bar graphs are SE; * p < 0.05; *** p < 0.001. p-Stat5 = phosphorylated Stat5; p-Erk = phosphorylated Erk; MFI = mean fluorescence intensity.

Supportive validation

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Experimental details: B-cell defects in Osx-Raptor mice are non-cell-autonomous. ( A ) Relative Raptor mRNA levels of isolated B220 + B cells from 3-week-old male or female control and Osx-Raptor mice by qPCR ( n = 3). ( B ) FACS plots of B220 + versus GFP + in the BM of 3-week-old control and Osx-Raptor mice ( n = 3). ( C ) Sketch map: Total BM cells from C57BL/6 mice (male mice at 2 months of age) were cultured with CM generated by calvarial osteoblast from 3-day-old control and Osx-Raptor mice with or without IL-7, BM cells co-cultured with control CM supplemented with anti-IL-7 neutralizing antibody. ( D ) FACS analysis of percentages of B cell subsets cultured in CM from calvarial osteoblasts. Subsets were defined as follows: pro-B cells = B220 + CD43 + ; pre-B plus immature B cells = B220 + CD43 - ( n = 4). Error bars in bar graphs are SE; * p < 0.05; ** p < 0.01.

Supportive validation

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Experimental details: Fig. 1 In vivo depletion of Cdkn1a rescues GC formation in Ezh2 -/- mice. Ezh2 fl/fl , Cdkn1a -/- , Ezh2 fl/fl ;Cgamma1-cre and Ezh2 fl/fl ;Cgamma1-cre; Cdkn1a -/- littermate mice were immunized with SRBC to induce germinal center (GC) formation and were killed 10 days later. a Flow cytometry plot of one representative mouse spleen per group. The gated area shows the percentage of GC B cells (GL7 + FAS + ) within live B cells (B220 + DAPI-, see Supplementary Fig. 2A for gating strategy). b Average of GC B populations of each group of mice quantified by flow cytometry as in a ( n = 7 mice per group). c Formalin fixed paraffin embedded splenic tissue was stained for PNA, Ki67, EZH2, and B220. One representative picture of three spleens analyzed per group is shown. d - f Quantification of PNA staining from c ( n = 3 spleens per group). d ""#GC/spleen section"" is the count of all GC per spleen section. e ""GC area/total spleen area"" is the quantified area of each individual GC divided by the total area of the spleen section. f ""Total GC area/total spleen area"" is the sum of all GC quantified areas in a certain section divided by the total area of that spleen section. g Splenocytes were permeabilized and stained for GC B and EZH2 using a fluorochrome-conjugated anti EZH2 antibody. The gated area shows the percentage of GC B cells (GL7 + FAS + ) within live B cells (B220 + DAPI-, see Supplementary Fig. 2A for gating strategy) that are EZH2 positive. The flow plot shown is repre

Supportive validation

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Experimental details: Fig. 3 Characterization of a 3D B cell follicular organoid to model the GC reaction. a Scheme of 3D B cell follicular organoid fabrication. Splenic B cells are co-encapsulated with 40LB stromal cells and IL4 into an RGD-presenting nanocomposite hydrogel that contain gelatin ionically cross-linked with synthetic silicate nanoparticles. b Representative fluorescence pictures of splenic GFP B cells in 3D organoid culture. c Flow cytometry plot of a 3D B cell follicular organoid. The gated area on the top shows the live B cells (B220 + DAPI-) and on the bottom plot, the organoid GC B cells (GL7 + FAS + ) within live B cells. d Average of GC B populations of organoids quantified as in c . e Average of percentage of proliferating organoid GC B populations, quantified as indicated in Supplementary Fig. 4B . f MFI of proliferation dye from e . g Flow cytometry plot of GC B cells stained with annexinV. h Average of percentage of apoptotic GC B cells quantified as in g . i RNA-seq profiles of organoid GC B cells after 4 and 6 days in culture (organoid GCB d4, n = 4 spleens, and d6, n = 3 spleens) were projected into the principal component space defined by GC B cells sorted from immunized mice (in vivo GCB, n = 6 mice), CD138 + plasma cells (in vivo PC, n = 6 mice) and FAS-GL7-IgD + B220 + naive B cells (NB, n = 3 mice). j , k Heat maps of gene expression level of GC B cells showed in i , represented as log2 ratio relative to mean naive B cells j and to mean plasma cells k . PC1/2 = pr

Supportive validation

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Experimental details: Fig. 4 CDKN1A repression by EZH2 is required for GC B cell cycle progression. a - f Organoids were generated using B cells isolated from Ezh2 fl/fl ;Cgamma1-cre, Cdkn1a -/- , Ezh2 fl/fl ;Cgamma1-cre; Cdkn1a -/- and Ezh2 fl/fl control mice ( n = 3 mice per group) and were harvested for flow cytometry analysis after 4 days in culture. a Flow cytometry plots of representative organoids from each genotype. The gated area shows the percentage of organoid GC B cells (GL7 + FAS + ) within live B cells (B220 + DAPI-). b Average of percentage of organoid GC B populations quantified by flow cytometry as in a ( n = 3 biological replicates per group). c Organoids received a BrdU pulse of 2 h before harvest. Cell cycle was analyzed by BrdU staining and 7AAD to measure DNA content. The representative gated area shows the percentage of organoid GC B cells (GL7 + FAS + B220 + DAPI-) that are in S phase (BrdU + ). d Average of percentage of organoid GC B populations in S phase of each group of genotype quantified by flow cytometry as in c ( n = 3 biological replicates per group). e Organoid cells were permeabilized and stained for EZH2 and GC markers GL7, FAS, and B220 to identify organoid GC B cells. The flow cytometry plot shows one representative sample per genotype group. f MFI of EZH2 in organoid GC B cells ( n = 3) quantified by flow cytometry as in e . g - m Organoids were generated using B cells isolated from 3 Cdkn1a +/+ and 3 Cdkn1a -/- mice. g Scheme of EZH2 inhibitor GSK343 and Br

Supportive validation

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Experimental details: Fig. 7 E2F1 is required for GC formation in an EZH2 dependent manner. a - f E2f1 -/- and control WT mice were immunized with SRBC to induce GC formation and were killed 10 days later. a Average of percentage of B cells (B220 + ) within live splenocytes (DAPI-) of each group of mice ( n = 9 per group). b Flow cytometry plot of one representative mouse spleen per group. The gated area shows the percentage of GC B cells (GL7 + FAS + ) within live B cells (B220 + DAPI-, see Supplementary Fig. 2A for gating strategy). c Average of percentage of GC B populations of each group quantified by flow cytometry as in b ( n = 9 per group). d Formalin fixed paraffin embedded splenic tissue was stained for PNA, Ki67, EZH2, phospho Rb Ser780 and B220. One representative picture of 5 spleens analyzed per group is shown. e , f Quantification of PNA staining from d ( n = 5 spleens per group). e ""#GC/spleen section"" is the count of all GC per spleen section. f ""GC area/total spleen area"" is the quantified area of each individual GC divided by the total area of the spleen section. Results shown in a - f are representative of a total of three independent experiments performed with different cohorts of mice. g Bone marrow transplantation was performed using E2f1 -/- and WT donor mice. Each recipient group consisted of 8 mice. BM, bone marrow. h Gating strategy used to analyze the GFP positive GC B cells from transplanted mice. Representative flow cytometry plots showing the gating on GC B cells

Supportive validation

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Experimental details: Figure 1. VEGF and bFGF promote the differentiation of MSCs into EC-like cells. (A) Expression of MSC surface markers was confirmed using flow cytometry. (B) Morphological features of isolated MSCs and MSC-derived EC-like cells following induction as observed under an inverted microscope. The red and white arrows indicate spindle-shaped and polygon-shaped cells, respectively. Magnification, x20. (C) Factor VIII expression was detected in MSC-derived EC-like cells via immunohistochemistry and light microscopy with brown staining indicating the positive signal. VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; MSCs, mesenchymal stem cells; EC, endothelial cell; Ctrl, control; CD, cluster of differentiation.

Supportive validation

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Experimental details: Figure 4 Colonic immune cell responses in multidrug-resistant P. aeruginosa infected mice suffering from chronic colitis. IL-10 -/- mice with chronic colitis were perorally infected with a multidrug-resistant P. aeruginosa (Psae) strain on day (d) 0. Six weeks thereafter (on d42 postinfection; black circles) the average numbers of colonic ( a ) macrophages and monocytes (positive for F4/80), ( b ) T lymphocytes (positive for CD3), ( c ) regulatory T cells (positive for FOXP3), and ( d ) B lymphocytes (positive for B220) were determined from six high power fields (HPF, 400x magnification) per animal in immunohistochemically stained large intestinal paraffin sections. Naive (N) IL-10 -/- mice served as negative controls (open circles). Numbers of mice (in parentheses), medians (black bars) and significance levels (p-values) determined by the Mann Whitney U test are indicated. Data shown were pooled from three independent experiments.

Supportive validation

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Experimental details: Fig. 1 Loss of miR-143/145 results in reduced LT-HSC frequency. a Frequency of long-term-HSC (LT-HSC, CD45 + EPCR + CD48 - CD150 + ), LSK/short-term HSC (Lin - Sca1 + c-Kit + ), common myeloid progenitors (CMP, Lin - Sca1 - c-Kit + CD34 + CD16/32 lo ), granulocyte-macrophage progenitors (GMP, Lin - Sca1 - c-Kit + CD34 + CD16/32 hi ), and megakaryocyte-erythrocyte progenitors (MEPs, Lin - Sca1 - c-Kit + CD34 - CD16/32 lo ) in the marrow of 8-12-week- old wild-type (WT), miR-143/145 +/- , and miR-143/145 -/- mice, as analyzed by flow cytometry (median +- 1.5 IQR, WT n = 11, 143/145 +/- n = 9, 143/145 -/- n = 8). b Marrow cellularity (2 femurs and 2 tibias) (WT n = 3, 143/145 +/- n = 4, 143/145 -/- n = 8). c Colony-forming unit (CFU) assay of marrow cells from WT and miR-143/145 -/- mice (mean +- SEM, WT n = 11, 143/145 -/- n = 10). d Primary CFU cells were replated in equal proportions per condition, normalized to the number of input cells, to generate secondary CFUs (mean +- SEM, WT n = 9, 143/145 -/- n = 7). e Estimate of HSC frequency in WT and miR-143/145 -/- mice by limiting dilution assay. Shown is a log-fraction plot of the limiting dilution model. The slope of the line is the log-active cell fraction. The dotted lines give the 95% CI

Supportive validation

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Experimental details: Figure 1. Intestinal immune cell responses upon NAP treatment of human microbiota-associated mice suffering from subacute ileitis. Subacute ileitis was induced in human microbiota-associated (hma) mice on day 0 by peroral low-dose T. gondii infection as described in Materials and Methods section. Hma mice were then treated with synthetic NAP (crossed circles) or placebo (PLC; black circles) from day 1 until day 8 postinfection (p.i.). At day 9 p.i., the average numbers of (A) ileal and (B) colonic T lymphocytes (positive for CD3) and of (C) colonic B lymphocytes (positive for B220) were determined microscopically in immunohistochemically stained intestinal paraffin sections from six high power fields (HPF, 400x magnification) per animal. Naive mice (i.e., without ileitis and without treatment; white circles) served as negative controls. Numbers of animals (in parentheses), means, and significance levels ( p values) determined by Mann-Whitney U test are indicated. Data shown were pooled from four independent experiments

Supportive validation

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Experimental details: Figure 4 Surgical denervation of young bone marrow induces premature HSC and niche aging (a) Schematic illustration of surgical denervation experiment. The sciatic and femoral nerves were transected in young (2 months old) C57BL/6 mice and the mice were analyzed 4 months post surgery, at 6 months of age. (b) Absolute numbers of HSCs (lineage - Sca-1 + c-Kit + CD48 - CD150 + ) isolated from Sham and Denervated (Den) femurs (n=7 mice). (c, d) Total peripheral blood (CD45.2 + ), blood myeloid (Mac-1 + CD45.2 + ) and blood B cell (B220 + CD45.2 + ) donor chimerism at the indicated time points post transplantation (c) and bone marrow donor chimerism (Total CD45.2 + (BM); Myeloid (My), Mac-1 + CD45.2 + ; B cell (B), B220 + CD45.2 +; and T cell (T), CD4 + /CD8 + CD45.2 + ) 5 months after primary transplantation of 200 HSCs derived from either sham or denervated (Den) femurs and transplanted in competition with young BM competitor cells (n=4 sham, 6 denervated mice) (left) and 5 months after secondary transplantation of 3 x 10 6 bone marrow from primary recipients (right) (d). (e) Left, representative confocal z -stack projections of HSCs sorted from sham or denervated femurs and stained with Cdc42, Tubulin and DAPI. Scale bar, 10 mum. Right, quantification of the percentage of Cdc42 and Tubulin polarized HSCs out of total HSCs scored (total of 356 sham and 353 denervated HSCs isolated from 4 mice per group). (f) Left, representative confocal z -stack pro

Supportive validation

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Experimental details: Figure 1 ATF3 maintains intestinal homeostasis. (A) Comparison of colon length between naive mice as indicated. (B) Colon crypts from mice were isolated by shaking colon fragments in EDTA and counted under light microscopy. (C) Flow cytometry analysis of Ki67 and CD24 expression in ileum crypts, gated on the CD45 - EpCAM + populations, from the indicated naive mice. (D) Representative micrographs showing intestinal organoids derived from naive mice. (E) Quantitative real-time PCR analysis of cell cycle genes in naive ileum organoids at day 6 of culture (""n"" indicates organoids derived from 4 mice each group). (F) Representative confocal images of whole mount tissues with co-immunofluorescence staining of UEA-1 and WGA in naive ileum villi. Results were from at least two independent experiments and ""n"" refers to the number of mice unless indicated otherwise. All mice were at the age of 2~3 months old when analyzed. Statistical analysis was done using Multiple T -test on Prism software. * P < 0.05, ** P < 0.005, *** P < 0.0005.

Supportive validation

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Experimental details: Figure 7 ATF3 promotes IL-22-induced STAT3 phosphorylation by suppressing phosphatases. (A) Freshly isolated ileum crypts, or (B) ileum organoids at day 6 of culture, were stimulated with IL-22, followed by fixation and intracellular staining of phospho-STAT3, and analyzed by flow cytometry. Western blot analysis of (C) IL-22-stimulated CMT93 cells, or (D) IL-22-stimuated colon fragments isolated from the indicated mice, for the expression of the indicated proteins. (E) Quantitative real-time PCR analysis of IL-22R1 and IL-10R2 mRNA levels in freshly isolated ileum crypts from mice. (F) Flow cytometry analysis of IL-22R1 in freshly isolated ileum crypt cells gated on the CD45 - EpCAM + population. (G,H) Western blot analysis of unstimulated or IL-22-stimulated CMT93 cells for the indicated proteins. ATF3 -/- CMT93 cells with SHP2 knockdown (ATF3 -/- SHP2 KD ) were indicated. Images were representative of four independent experiments (G-H) . Results were from two independent experiments (A-F) . ""n"" refers to the number of mice analyzed (A,B,E,F) . Statistical analysis was done by multiple comparison in Two-way ANOVA test using Prism software. * P < 0.05, ** P < 0.005, *** P < 0.0005.

Supportive validation

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Experimental details: Characterization of the hematopoietic stem cell enriched LES population. (A): Purification of LES cells: Endo + Sca.1 + cells sorted from lineage (B220, CD11b, GR1, Ter119, CD8a, CD4, and CD5) negative population. (B): Percentage of eGFP + LES cells 7 days after transduction at increasing MOI. (C): Percentage of CD45.1 donor-derived cells, in whole blood of primary recipient mice 16 weeks after transplantation and quantified by flow cytometry versus the number of injected cells. (D): Limiting dilution analysis showing the percentage of mice containing fewer than 1% of donor CD45.1 in WBC versus the number of injected cells. Data were obtained by flow cytometry 16 weeks after transplantation. Abbreviations: FSC, Forward Scatter; GFP, green fluorescent protein; LES, Lin - Endoglin + Sca.1 + ; MOI, multiplicity of infection; SSC, Side Scatter.

Supportive validation

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Experimental details: Figure 2 Tpl2 didn't affect peripheral immune activation during FH pathogenesis. The splenic cells or peripheral blood immune cells were isolated from P. acnes -primed WT and Tpl2 -KO mice at day 3, 5, and 7 as described in Materials and methods ( n = 4 mice/group), and subjected for flow cytometry analysis. (A-F) Flow cytometry analysis of the frequencies and absolute numbers of CD4 + T cells, CD8 + T cells, B220 + B cells, CD11c + dendritic cells (A,B) , CD4 + Foxp3 + Treg cells (C,D) , and IFN-gamma- and TNF-alpha-producing pathogenic Th1 cells (E,F) in the spleens of WT and Tpl2 -KO mice at day 7 after P. acnes priming. Data are presented as representative plots of the frequencies of immune cell subpopulations (A,C,E) and a summary graph of the cell frequencies or absolute cell numbers (B,D,F) . (G-J) Flow cytometry analysis of the frequencies and absolute numbers of CD4 + T cells in the spleens (G,H) or peripheral blood (I,J) of WT and Tpl2 -KO mice at day 3, 5, and 7 after P. acnes priming. Data are presented as representative plots of the frequencies of immune cell subpopulations (G,I) and a summary graph of the cell frequencies or absolute cell numbers (H,J) . Results are mean +- SD from three independent experiments.

Supportive validation

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Experimental details: Figure 3 Tpl2 reduced liver infiltration of pathogenic CD4 + T cells. Liver-infiltrating immune cells were isolated from P. acnes -primed WT and Tpl2 -KO mice at day 3, 5, and 7 as described in Materials and methods ( n = 4 mice/group). (A-G) Flow cytometry analysis of the frequencies and absolute numbers of CD4 + and CD8 + T cells, B220 + B cell, CD11c + dendritic cells (A-C) , CD4 + Foxp3 + Treg cells (D,E) , and IFN-gamma- and TNF-alpha-producing pathogenic Th1 cells (F,G) in the livers of WT and Tpl2 -KO mice at day 7 after P. acnes priming. Data are presented as representative plots of the frequencies of immune cell subpopulations (A,D,F) and a summary graph of the frequencies and absolute cell numbers (B,C,E,G) . (H,I) Flow cytometry analysis of the frequencies and absolute numbers of CD4 + T cells in the livers of WT and Tpl2 -KO mice at day 3, 5, and 7 after P. acnes priming. Data are presented as representative plots of the frequencies of immune cell subpopulations (H) and a summary graph of the absolute cell numbers (I) . Results are mean +- SD from three independent experiments. Two-tailed Student's t -tests were performed. * P < 0.05; ** P < 0.01.

Supportive validation

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Experimental details: Figure 5 MNP FcgammaR A:I Ratio Modulates Intestinal Inflammation (A) Weight loss in co-housed sex-matched WT (black circles, n = 8) and Fcgr2b -/- (red circles, n = 9) mice after a single 6-day course of 2% DSS. Means +- SEM are indicated. Data are representative of 3 independent experiments. (B) Confocal microscopy of colonic CD45 + leukocytes in mice treated as in (A). Data are representative of 2 independent experiments. (C) Flow-cytometric quantification of colonic neutrophil frequency (top) and absolute neutrophil counts (bottom) in WT and Fcgr2b -/- colons at day 25 after aDSS (n = 5-7 per group) or in healthy controls (day 0) (n = 3-5 per group). Medians are indicated. Data are representative of 3 independent experiments. (D) Pro-IL-1beta expression by colonic CD11b + CX3CR1 + MNPs at day 25 after aDSS (n = 5-7 per group). (E) Frequency of pro-IL-1beta expression within the Ly6C hi MHC-II - monocyte and Ly6C + MHC-II int macrophage populations as shown in aDSS (n = 5-7 per group) versus controls (day 0) (n = 3-5 per group). Medians are indicated. Data are representative of 3 independent experiments. (F) Chemokine mRNA levels in flow-sorted Ly6C lo MHC-II hi colonic macrophages at day 21 after aDSS (n = 4-5 per group). Data normalized to WT mRNA levels. Medians are indicated. (G and H) Weight loss (G) and clinical colonic and lymphoid organ features (H) of co-housed sex-matched M-TG (green circles, n = 5 or 6) and N-TG littermate controls (black circles, n = 6) after a

Supportive validation

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Experimental details: Fig. 6 TLR2 + macrophages, pDCs, and PB in the small intestine bind PSA and induce IL-10 secretion. Gating strategy for mononuclear cells isolated from a duodenum (Duod) and b ileum (Ile) of WT and Rag mice analyzed for binding of fluorescent A488-conjugated PSA (left two plots). CD45 - intra-epithelial cells (CD45 - IEC: middle histogram) and CD45 + gated intra-epithelial leukocytes (CD45 + IEL: second right histogram) isolated from the a Duod and b Ile of WT (red) and Rag (blue) mice were analyzed for reactivity to PSA-A488. CD45 + CD11c - B220 + B cells, PDCA1 + B220 + CD11c + pDCs and CD138 + B220 + PB and B220 low PC isolated from a Duod and b Ile of WT mice were analyzed for PSA reactivity (right histogram). Flow cytometry plots show CD11c + PDCA1 - cDC, PDCA1 + B220 + pDC, and B220 + CD19 + B cells isolated from spleen, MLN, PP, IEL, or LP of WT mice were stimulated with c PSA or d LTA-SA (TLR2 agonist) and analyzed for IL-10 expression. e Plots summarize data from c and d and show % IL-10 + B cells (left), cDCs (middle), and pDCs (right) from spleen, MLN, PP, IEL, and LP stimulated with PSA or TLR2 (LTA-SA) ( n = 3 mice). All data show mean +- SEM

Supportive validation

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Experimental details: Figure 2 Analysis of the B lymphocyte populations expanded in TRAF3/BCL2 double-tg mice with lymphoid dyscrasias. Three-color flow-cytometry analysis was performed to determine the phenotype of expanded B lymphocyte populations. Gating of the expanded population was based on the CD45R/B220 and FSC plot of each sample analyzed and is indicated in the figure. The surface molecules analyzed are indicated in the plots, as well as the percentage of cells found in each quadrant. The quadrants settings were selected based on the staining of isotype-controls (not shown). The tissue source where the analyzed lymphocytes were extracted from and the type of B cell malignancy developed by the TRAF3/BCL2 double-tg mice, according to the flow-cytometry and immunohistochemical analysis, is indicated in the figure.

Supportive validation

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Experimental details: Figure 4 Ptpn6 deletion drives robust anti-tumor immunity in two immune-rich syngeneic tumor lines. (A) Flow cytometric analysis of live CD45 + cells from B16F10 melanoma tumors isolated from tamoxifen-treated Ptpn6 fl/fl and Ptpn6 fl/fl ERT2-cre mice at day 14 post tumor implantation. (B) B16F10 tumor volume measurements in tamoxifen-treated Ptpn6 fl/fl ERT2-cre and Ptpn6 fl/fl mice. Each data point represents the average tumor volume of all mice in a given group. Data is representative of three independent experiments with 5-7 mice/group. (C) Shp1 protein relative to total Erk2 protein in peripheral blood cells from indicated mice 14 days after initial tamoxifen dose (200 mg/kg bid for 4 days) and 7 days after B16F10 tumor cells were implanted. Data is representative of at least three independent experiments with 5-7 mice per group. (D) Flow cytometric analysis of live CD45 + cells from E0771 tumors isolated from tamoxifen-treated Ptpn6 fl/fl and Ptpn6 fl/fl ERT2-cre mice at day 19 post tumor implantation. (E) E0771 tumor volume measurements in tamoxifen-treated Ptpn6 fl/fl ERT2-cre and Ptpn6 fl/fl mice. Each data point represents the average tumor volume of all mice in a given group. Data is representative of three independent experiments with 4-5 mice per group. Statistical significance was calculated at each time point using an unpaired t -test. (F) Shp1 protein relative to total Erk2 protein in peripheral blood cells from indicated mice 14 days after initial tamoxifen d

Supportive validation

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Experimental details: Figure 4. K14Cre;fl/fl mice develop esophageal fibrosis and ILD. (A) Masson-Trichrome staining of the lower esophagus of 3-mo-old fl/fl and K14Cre; fl/fl mice. (Bottom) Higher magnification images of the regions indicated by squares in the top panels. Bars: (top) 500 um; (bottom) 200 um. (B) The lower esophagi were cut along the longitudinal direction, spread on sheets, fixed in formalin, and embedded in the paraffin. Masson-Trichrome staining was performed, and the thickness of the lamina propria was evaluated at the ages of 1, 2, and 3 mo. Vertical bars represent lamina propria thickness. Bars, 50 um. (C) The thickness of lamina propria at upper (thoracic) esophagus was evaluated at the age of 3 mo. (D) The thickness of the circular muscle layer was evaluated in the sections of 3-mo-old mice. (E) Hydroxyproline contents of the lower esophagus were compared between these two strains of mice at the age of 3 mo. (F) mRNA levels of type I collagen, cytokines, chemokines, and growth factors in the lower esophagi of 3-mo-old mice were assessed. (G) The lower esophagus sections from 3-mo-old mice were immunohistochemically stained for IL-1beta. Bars, 50 um. (Right) The graph shows the result of semiquantitative scoring of the staining intensity. (H) H&E staining of the lungs from 1-, 2-, and 3-mo-old fl/fl and K14Cre;fl/fl mice. Arrows indicate the peribronchial lymphocyte aggregates. Bars, 200 um. (I) Infiltrating inflammatory cells were immunohistochemically stained for CD3 and

Supportive validation

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Experimental details: Figure 5. K14Cre;fl/fl mice exhibit prominent autoimmunity with Th2/Th17 cell polarization and abnormal B cell activation leading to autoantibody production. (A) A representative image of the gross appearance of spleens and inguinal lymph nodes from 3-mo-old fl/fl and K14Cre;fl/fl mice. (B) The number of splenocytes in 3-mo-old fl/fl and K14Cre;fl/fl mice was counted. n = 4 per each group. (C) Lymphocytes isolated from inguinal lymph nodes were stained for CD3 and CD4 and intracellularly stained for IL-4, IL-17A, and IFN-gamma. (Top) Representative two-dimensional plots for IL-4 and IL-17A in CD3 + CD4 + cells. (Bottom) The percentages of cells positive for IL-4, IL-17A, and IFN-gamma are summarized. n = 7 mice per each group. (D) Splenocytes from 3-mo-old fl/fl and K14Cre;fl/fl mice were stained for B220 and CD19. B220 + CD19 + cells were analyzed for CD19 expression. A representative histogram of mean fluorescence intensity (MFI; top) and the comparison of mean fluorescence intensities (bottom) are shown. n = 4 mice per genotype. (E) Sera from 3- and 8-mo-old mice were analyzed for the concentrations of total IgG, IgM, and IgA. n = 5-7 mice per genotype. Note that the results are shown with log scale. (F) Sera from 1-, 2-, 3-, and 8-mo-old mice were analyzed for the concentration of IL-6. n = 6-7 mice per genotype. (G) B cells were isolated from splenocytes of 3-mo-old mice and cultured for 48 h, either unstimulated or stimulated with anti-CD40 antibody (alphaCD40Ab). The c

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Host humoral immune responses to vector delivery. ( a ) The titer of anti-adenoviral antibodies in mouse bronchoalveolar lavage fluid (BALF). The total anti-Ad antibodies (IgA, IgE, IgGs, IgM) were detected with ELISA in all groups. Data were presented as mean +- SD (standard deviation). ( b ) Neutralizing antibody in mouse BALF. n = 5, #: p < 0.05; **: p < 0.01. ( c ) B-cell presence in mouse lungs. B-cells were detected in mouse lungs with antibodies against B220 by immunohistochemistry staining. The positive cells are shown in brown color. ns, no significant difference.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Development of myeloid cell populations is not detectably altered in MphCK2alpha -/- mice. (A) Schematic of Csnk2a targeting vector. (B) Immunoblots of lysates from BMDMs, splenocytes, or peritoneal cavity cells of LysM cre or LysM cre CK2alpha fl/fl (MphCK2alpha - / - ) mice were probed for CK2alpha or beta-actin as a loading control. n = 2. (C) Splenic myeloid cell populations were stained and analyzed by flow cytometry. Plots depict gating for representative F4/80+ macrophages, Ly6C+ monocytes, and neutrophils (also Ly6G+). (D) Cells from spleen were stained for flow cytometry to enumerate immune cell populations. Data are from pooled experiments, n = 4-12 mice/group. (E) Cells from bone marrow were stained for flow cytometry to evaluate bone marrow precursor populations including common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs), and megakaryocyte-erythrocyte progenitor (MEP). Cells were pre-gated on Lin-(B220, CD3, CD11b, GR-1 and TER-119), Sca-1-, cKit-. Density plots are representative from two experiments.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1. Steady-state Flt3-L BMDCs and splenic DCs display remarkably high levels of eIF2alpha without inhibition of translation. (A) Immunohistochemistry of mouse spleen with staining for CD11c (red) and p-eIF2alpha (green). Scale bar: 200 mum, magnification: 10x. Single color images are shown and merged picture (right row), high level of p-eIF2alpha staining is mostly found co-localizing in cells positive for CD11c+ (DCs, white arrowheads). (B) Immunohistochemistry of mouse spleen in the white pulp for CD11c (red), p-eIF2alpha (green), CD11b (blue), and B220 (turquoise). Scale bars: 50 mum, magnification: 40x. In the upper panel, magnified areas show p-eIF2 detection in cDC2 (CD11c+/CD11b+) and cDC1 (CD11c+/CD11b-). In the lower panel, magnified areas show p-eIF2 detection in pDCs (B220+/CD11c+) and in B cells (B220+ and CD11c-). (C) Relative p-eIF2alpha levels measured by flow in different mouse spleen populations. Statistical analysis was performed by Mann-Whitney test. ** P < 0.01. (D) Levels of p-eIF2alpha and total eIF2alpha were measured in DC populations by immunoblot. Sorted steady-state Flt3-L BMDCs were compared with MEFs and freshly isolated CD8alpha+ T cells stimulated or not with thapsigargin (Tg) for 2 h (200 nM). Ratio of p-eIF2alpha/eIF2alpha is quantified in the graph of the lower panel. (E) Levels of p-eIF2alpha and total eIF2alpha were measured in bulk Flt3-L BMDCs during different days of BM differentiation in vitro. (F) Levels of protein synthesis were

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 1 Agriculture (swine) exposure-related Organic Dust Extract (ODE) induced airway inflammation coupled with Collagen Induced Arthritis (CIA) model. ( A ) Line graph depicts mean with SE bars of arthritis inflammatory score at respective time points from treatment groups. Statistical difference versus sham denoted as ""a"" (p