Antibody data
- Antibody Data
- Antigen structure
- References [30]
- Comments [0]
- Validations
- Flow cytometry [1]
- Other assay [31]
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- Product number
- 64-0452-82 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- CD45R (B220) Monoclonal Antibody (RA3-6B2), Super Bright™ 645, 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: This RA3-6B2 antibody has been reported for use in flow cytometric analysis. Applications Tested: This RA3-6B2 antibody has been tested by flow cytometric analysis of mouse splenocytes. This 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. Super Bright 645 is a tandem dye that can be excited with the violet laser line (405 nm) and emits at 645 nm. We recommend using a 660/20 bandpass filter. Please make sure that your instrument is capable of detecting this fluorochrome. When using two or more Super Bright dye-conjugated antibodies in a staining panel, it is recommended to use Super Bright Complete Staining Buffer (Product # SB-4401) to minimize any non-specific polymer interactions. Please refer to the datasheet for Super Bright Staining Buffer for more information. Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Protect this vial and stained samples from light. Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically. Excitation: 405 nm; Emission: 645 nm; Laser: Violet Laser Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company.
- Reactivity
- Human, Mouse
- Host
- Rat
- Isotype
- IgG
- Antibody clone number
- RA3-6B2
- Vial size
- 100 µg
- Concentration
- 0.2 mg/mL
- Storage
- 4° C, store in dark, DO NOT FREEZE!
Submitted references Lenvatinib for effectively treating antiangiogenic drug-resistant nasopharyngeal carcinoma.
Transient expansion and myofibroblast conversion of adipogenic lineage precursors mediate bone marrow repair after radiation.
A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system.
High-throughput analysis of lung immune cells in a combined murine model of agriculture dust-triggered airway inflammation with rheumatoid arthritis.
Role of CXCL5 in Regulating Chemotaxis of Innate and Adaptive Leukocytes in Infected Lungs Upon Pulmonary Influenza Infection.
Polyvinyl alcohol hydrolysis rate and molecular weight influence human and murine HSC activity ex vivo.
Type 2 diabetic mice enter a state of spontaneous hibernation-like suspended animation following accumulation of uric acid.
Metabolic preconditioning in CD4+ T cells restores inducible immune tolerance in lupus-prone mice.
Targeting Ovarian Carcinoma with TSP-1:CD47 Antagonist TAX2 Activates Anti-Tumor Immunity.
Impaired HA-specific T follicular helper cell and antibody responses to influenza vaccination are linked to inflammation in humans.
Hypoxia-induced miR-210 modulates the inflammatory response and fibrosis upon acute ischemia.
Monocyte-derived dendritic cells link localized secretory IgA deficiency to adaptive immune activation in COPD.
The folate cycle enzyme MTHFD2 induces cancer immune evasion through PD-L1 up-regulation.
Murine myeloid cell MCPIP1 suppresses autoimmunity by regulating B-cell expansion and differentiation.
Ibrutinib does not prevent kidney fibrosis following acute and chronic injury.
Proteostasis in dendritic cells is controlled by the PERK signaling axis independently of ATF4.
Histone deacetylase 3 represses cholesterol efflux during CD4(+) T-cell activation.
Myeloid Cell CK2 Regulates Inflammation and Resistance to Bacterial Infection.
Shp1 Loss Enhances Macrophage Effector Function and Promotes Anti-Tumor Immunity.
Ischemia Reperfusion Injury Triggers CXCL13 Release and B-Cell Recruitment After Allogenic Kidney Transplantation.
Pituitary Adenylate Cyclase-Activating Polypeptide Alleviates Intestinal, Extra-Intestinal and Systemic Inflammatory Responses during Acute Campylobacter jejuni-induced Enterocolitis in Mice.
The Host-Specific Intestinal Microbiota Composition Impacts Campylobacter coli Infection in a Clinical Mouse Model of Campylobacteriosis.
The lysophospholipase D enzyme Gdpd3 is required to maintain chronic myelogenous leukaemia stem cells.
Toll-Like Receptor-4 Dependent Intestinal and Systemic Sequelae Following Peroral Campylobacter coli Infection of IL10 Deficient Mice Harboring a Human Gut Microbiota.
Overcoming Immunological Challenges to Helper-Dependent Adenoviral Vector-Mediated Long-Term CFTR Expression in Mouse Airways.
Fucoxanthin Ameliorates Atopic Dermatitis Symptoms by Regulating Keratinocytes and Regulatory Innate Lymphoid Cells.
Requirements for the differentiation of innate T-bet(high) memory-phenotype CD4(+) T lymphocytes under steady state.
Bacteroides fragilis polysaccharide A induces IL-10 secreting B and T cells that prevent viral encephalitis.
The Hematopoietic Oxidase NOX2 Regulates Self-Renewal of Leukemic Stem Cells.
Mitophagy in Intestinal Epithelial Cells Triggers Adaptive Immunity during Tumorigenesis.
Sun Q, Wang Y, Ji H, Sun X, Xie S, Chen L, Li S, Zeng W, Chen R, Tang Q, Zuo J, Hou L, Hosaka K, Lu Y, Liu Y, Ye Y, Yang Y
Cell death & disease 2022 Aug 19;13(8):724
Cell death & disease 2022 Aug 19;13(8):724
Transient expansion and myofibroblast conversion of adipogenic lineage precursors mediate bone marrow repair after radiation.
Zhong L, Yao L, Holdreith N, Yu W, Gui T, Miao Z, Elkaim Y, Li M, Gong Y, Pacifici M, Maity A, Busch TM, Joeng KS, Cengel K, Seale P, Tong W, Qin L
JCI insight 2022 Apr 8;7(7)
JCI insight 2022 Apr 8;7(7)
A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system.
Shen Y, Jiang L, Iyer VS, Raposo B, Dubnovitsky A, Boddul SV, Kasza Z, Wermeling F
Computational and structural biotechnology journal 2021;19:5360-5370
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High-throughput analysis of lung immune cells in a combined murine model of agriculture dust-triggered airway inflammation with rheumatoid arthritis.
Gaurav R, Mikuls TR, Thiele GM, Nelson AJ, Niu M, Guda C, Eudy JD, Barry AE, Wyatt TA, Romberger DJ, Duryee MJ, England BR, Poole JA
PloS one 2021;16(2):e0240707
PloS one 2021;16(2):e0240707
Role of CXCL5 in Regulating Chemotaxis of Innate and Adaptive Leukocytes in Infected Lungs Upon Pulmonary Influenza Infection.
Guo L, Li N, Yang Z, Li H, Zheng H, Yang J, Chen Y, Zhao X, Mei J, Shi H, Worthen GS, Liu L
Frontiers in immunology 2021;12:785457
Frontiers in immunology 2021;12:785457
Polyvinyl alcohol hydrolysis rate and molecular weight influence human and murine HSC activity ex vivo.
Sudo K, Yamazaki S, Wilkinson AC, Nakauchi H, Nakamura Y
Stem cell research 2021 Oct;56:102531
Stem cell research 2021 Oct;56:102531
Type 2 diabetic mice enter a state of spontaneous hibernation-like suspended animation following accumulation of uric acid.
Zhao Y, Cheng R, Zhao Y, Ge W, Yang Y, Ding Z, Xu X, Wang Z, Wu Z, Zhang J
The Journal of biological chemistry 2021 Oct;297(4):101166
The Journal of biological chemistry 2021 Oct;297(4):101166
Metabolic preconditioning in CD4+ T cells restores inducible immune tolerance in lupus-prone mice.
Wilson CS, Stocks BT, Hoopes EM, Rhoads JP, McNew KL, Major AS, Moore DJ
JCI insight 2021 Oct 8;6(19)
JCI insight 2021 Oct 8;6(19)
Targeting Ovarian Carcinoma with TSP-1:CD47 Antagonist TAX2 Activates Anti-Tumor Immunity.
Jeanne A, Sarazin T, Charlé M, Moali C, Fichel C, Boulagnon-Rombi C, Callewaert M, Andry MC, Diesis E, Delolme F, Rioult D, Dedieu S
Cancers 2021 Oct 7;13(19)
Cancers 2021 Oct 7;13(19)
Impaired HA-specific T follicular helper cell and antibody responses to influenza vaccination are linked to inflammation in humans.
Hill DL, Whyte CE, Innocentin S, Lee JL, Dooley J, Wang J, James EA, Lee JC, Kwok WW, Zand MS, Liston A, Carr EJ, Linterman MA
eLife 2021 Nov 2;10
eLife 2021 Nov 2;10
Hypoxia-induced miR-210 modulates the inflammatory response and fibrosis upon acute ischemia.
Zaccagnini G, Greco S, Longo M, Maimone B, Voellenkle C, Fuschi P, Carrara M, Creo P, Maselli D, Tirone M, Mazzone M, Gaetano C, Spinetti G, Martelli F
Cell death & disease 2021 May 1;12(5):435
Cell death & disease 2021 May 1;12(5):435
Monocyte-derived dendritic cells link localized secretory IgA deficiency to adaptive immune activation in COPD.
Richmond BW, Mansouri S, Serezani A, Novitskiy S, Blackburn JB, Du RH, Fuseini H, Gutor S, Han W, Schaff J, Vasiukov G, Xin MK, Newcomb DC, Jin L, Blackwell TS, Polosukhin VV
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The folate cycle enzyme MTHFD2 induces cancer immune evasion through PD-L1 up-regulation.
Shang M, Yang H, Yang R, Chen T, Fu Y, Li Y, Fang X, Zhang K, Zhang J, Li H, Cao X, Gu J, Xiao J, Zhang Q, Liu X, Yu Q, Wang T
Nature communications 2021 Mar 29;12(1):1940
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Murine myeloid cell MCPIP1 suppresses autoimmunity by regulating B-cell expansion and differentiation.
Dobosz E, Lorenz G, Ribeiro A, Würf V, Wadowska M, Kotlinowski J, Schmaderer C, Potempa J, Fu M, Koziel J, Lech M
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Ibrutinib does not prevent kidney fibrosis following acute and chronic injury.
Belliere J, Casemayou A, Colliou E, El Hachem H, Kounde C, Piedrafita A, Feuillet G, Schanstra JP, Faguer S
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Scientific reports 2021 Jun 7;11(1):11985
Proteostasis in dendritic cells is controlled by the PERK signaling axis independently of ATF4.
Mendes A, Gigan JP, Rodriguez Rodrigues C, Choteau SA, Sanseau D, Barros D, Almeida C, Camosseto V, Chasson L, Paton AW, Paton JC, Argüello RJ, Lennon-Duménil AM, Gatti E, Pierre P
Life science alliance 2021 Feb;4(2)
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Histone deacetylase 3 represses cholesterol efflux during CD4(+) T-cell activation.
Wilfahrt D, Philips RL, Lama J, Kizerwetter M, Shapiro MJ, McCue SA, Kennedy MM, Rajcula MJ, Zeng H, Shapiro VS
eLife 2021 Dec 2;10
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Myeloid Cell CK2 Regulates Inflammation and Resistance to Bacterial Infection.
Larson SR, Bortell N, Illies A, Crisler WJ, Matsuda JL, Lenz LL
Frontiers in immunology 2020;11:590266
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Shp1 Loss Enhances Macrophage Effector Function and Promotes Anti-Tumor Immunity.
Myers DR, Abram CL, Wildes D, Belwafa A, Welsh AMN, Schulze CJ, Choy TJ, Nguyen T, Omaque N, Hu Y, Singh M, Hansen R, Goldsmith MA, Quintana E, Smith JAM, Lowell CA
Frontiers in immunology 2020;11:576310
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Ischemia Reperfusion Injury Triggers CXCL13 Release and B-Cell Recruitment After Allogenic Kidney Transplantation.
Kreimann K, Jang MS, Rong S, Greite R, von Vietinghoff S, Schmitt R, Bräsen JH, Schiffer L, Gerstenberg J, Vijayan V, Dittrich-Breiholz O, Wang L, Karsten CM, Gwinner W, Haller H, Immenschuh S, Gueler F
Frontiers in immunology 2020;11:1204
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Pituitary Adenylate Cyclase-Activating Polypeptide Alleviates Intestinal, Extra-Intestinal and Systemic Inflammatory Responses during Acute Campylobacter jejuni-induced Enterocolitis in Mice.
Heimesaat MM, Mousavi S, Kløve S, Genger C, Weschka D, Tamas A, Reglodi D, Bereswill S
Pathogens (Basel, Switzerland) 2020 Sep 30;9(10)
Pathogens (Basel, Switzerland) 2020 Sep 30;9(10)
The Host-Specific Intestinal Microbiota Composition Impacts Campylobacter coli Infection in a Clinical Mouse Model of Campylobacteriosis.
Heimesaat MM, Genger C, Klove S, Weschka D, Mousavi S, Bereswill S
Pathogens (Basel, Switzerland) 2020 Sep 29;9(10)
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The lysophospholipase D enzyme Gdpd3 is required to maintain chronic myelogenous leukaemia stem cells.
Naka K, Ochiai R, Matsubara E, Kondo C, Yang KM, Hoshii T, Araki M, Araki K, Sotomaru Y, Sasaki K, Mitani K, Kim DW, Ooshima A, Kim SJ
Nature communications 2020 Sep 17;11(1):4681
Nature communications 2020 Sep 17;11(1):4681
Toll-Like Receptor-4 Dependent Intestinal and Systemic Sequelae Following Peroral Campylobacter coli Infection of IL10 Deficient Mice Harboring a Human Gut Microbiota.
Kløve S, Genger C, Mousavi S, Weschka D, Bereswill S, Heimesaat MM
Pathogens (Basel, Switzerland) 2020 May 18;9(5)
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Overcoming Immunological Challenges to Helper-Dependent Adenoviral Vector-Mediated Long-Term CFTR Expression in Mouse Airways.
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Fucoxanthin Ameliorates Atopic Dermatitis Symptoms by Regulating Keratinocytes and Regulatory Innate Lymphoid Cells.
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Requirements for the differentiation of innate T-bet(high) memory-phenotype CD4(+) T lymphocytes under steady state.
Kawabe T, Yi J, Kawajiri A, Hilligan K, Fang D, Ishii N, Yamane H, Zhu J, Jankovic D, Kim KS, Trinchieri G, Sher A
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Bacteroides fragilis polysaccharide A induces IL-10 secreting B and T cells that prevent viral encephalitis.
Ramakrishna C, Kujawski M, Chu H, Li L, Mazmanian SK, Cantin EM
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The Hematopoietic Oxidase NOX2 Regulates Self-Renewal of Leukemic Stem Cells.
Adane B, Ye H, Khan N, Pei S, Minhajuddin M, Stevens BM, Jones CL, D'Alessandro A, Reisz JA, Zaberezhnyy V, Gasparetto M, Ho TC, Kelly KK, Myers JR, Ashton JM, Siegenthaler J, Kume T, Campbell EL, Pollyea DA, Becker MW, Jordan CT
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Mitophagy in Intestinal Epithelial Cells Triggers Adaptive Immunity during Tumorigenesis.
Ziegler PK, Bollrath J, Pallangyo CK, Matsutani T, Canli Ö, De Oliveira T, Diamanti MA, Müller N, Gamrekelashvili J, Putoczki T, Horst D, Mankan AK, Öner MG, Müller S, Müller-Höcker J, Kirchner T, Slotta-Huspenina J, Taketo MM, Reinheckel T, Dröse S, Larner AC, Wels WS, Ernst M, Greten TF, Arkan MC, Korn T, Wirth D, Greten FR
Cell 2018 Jun 28;174(1):88-101.e16
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Supportive validation
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- Staining of C57Bl/6 splenocytes with Anti-Mouse CD3 FITC (Product # 11-0032-82) and 0.25 µg of Rat IgG2a K Isotype Control Super Bright 645 (Product # 64-4321-82) (left) or 0.25 µg of Anti-Human/Mouse CD45R (B220) Super Bright 645 (right). Total viable cells were used for analysis.
Supportive validation
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- 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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- 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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- 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
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- 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.
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- 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
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- 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.
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- 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
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- 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
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- 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
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- 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
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- 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.
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- 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.
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- 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.
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- 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.
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- 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.
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- 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.
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- 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
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- 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
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- 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.
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- 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.
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- 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
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- 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
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- 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
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- 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
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- 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
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- 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
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- 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.
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- 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.
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- 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
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- 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