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1 8F4-Specific Lysis, % 1 UPN1 UPN3 8 UPN7 6 Pearson r =.69 UPN2 UPN5 P (one-tailed) = UPN8 n = 9 2 UPN9 UPN4 UPN F4, % Max MFI Supplementary Figure S1. AML samples UPN1-UPN9 show variable level of susceptibility to 8F4-mediated lysis in vitro. Cells from HLA-A2+ (filled circles) and HLA- A2- (open circles) patients were stained with Alexa 647-conjugated 8F4 as described in Methods. To test susceptibility to 8F4-mediated complement-dependent cytotoxicity (CDC, 8F4-specific Lysis), same samples were incubated with 8F4 or control antibodies in presence of rabbit complement. After one hour cells were stained with aqua viability marker. Viability was analyzed by flow cytometry for in presence of BD counting beads. 8F4 MFI (% of Max) and 8F4-Specific Lysis (%) were calculated as indicated in Methods. Filled circles show HLA-A2 + AML samples, open circles show show HLA-A2 - AML samples. One representative experiment out of three is shown.!
2 UPN1 AML cells! UPN1+IgG! UPN1+8F4! Control mouse! 4 weeks! UPN5 AML cells! UPN5+IgG! UPN5+8F4! Control mouse! UPN6 AML cells! UPN6+IgG! UPN6+8F4! Control mouse! <FL 9 Log>: CD45 M APC A <FL 8 Log>: CD45 APC e Bone marrow! Spleen! Liver! 3 weeks! Bone marrow! Spleen! 3 weeks! Supplementary Figure S2. 8F4 Ex vivo treatment prevents engraftment of primary HLA-A2+ AML in NOD scid/hla-a2 mice. HLA-A2 + AML (UPN1, UPN5) and HLA-A2 negative AML control (UPN6) were co-incubated with (2 µg/ml) 8F4 or isotype control mouse IgG2a anti-klh monoclonal antibody (IgG) and intravenously injected into sublethally-irradiated recipient mice. Three to four weeks later, mouse tissues were analyzed for the presence of leukemia cells. Flow cytometry plots depict leukemia cells in tissues of mice that received AML, treated ex vivo with 8F4 or control antibody. AML cells ( + /mocd45 - ) are shown within the gate in each plot. Freshly thawed AML is shown in left panels, and cells from control mice that did not receive AML are shown in right panels Bone marrow!
3 A NOD scid/hla-a2 FMO! NOD scid/hla-a2! NOD/scid! F4 (PE)! B B6/HLA-A2 FMO! B6/HLA-A2! B6! F4 (Alexa 647)! Supplementary Figure S3. PR1/HLA-A2 is expressed in bone marrow of HLA-A2 transgenic mouse models. Bone marrow cells of NOD scid/hla-a2 (A) and B6/HLA-A2 (B) were stained with fluorophore-conjugated 8F4, and analyzed by flow cytometry. Bone marrow cells of non-hla-a2- transgenic NOD scid and B6 were used for control, respectively. Histograms show 8F4 staining of live singlet mononuclear cells, identified based on scatter characteristics. FMO (Fluorescence minus one) - bone marrow cells cells were stained with the same antibody panel except for 8F4.!
4 Serum 8F4, µg/ml! 2-phase exponential elimination model (R 2 =.95): t 1/2 (fast) = 2.9 hours t 1/2 (slow) = 93.4 hours Supplementary Figure S4.! Serum 8F4 binding concentration after single iv injection (1 mg/kg) in NSG mice. Serum 8F4 concentration was determined by PR1/HLA-A2 ligand ELISA by measuring OD from duplicate wells at the indicated time points. Concentration was determined from a standard curve. Each data point represents mean concentration ± SEM (n=3 mice per time point). The data was analyzed according to a two-phase exponential decay model with Prism 6 (Macintosh) software. Half-life was determined according to the model of double exponential elimination model.! Hours Time, hours!
5 Blood chimerism, % A. Peripheral Blood Chimerism! UPN9! 1mg/kg, 3x/ week, 2 weeks Weeks Tissue engraftment, % B. Tissue Engraftment! BM SP LV Supplementary Figure S5. 8F4 treatment does not affect the growth of established HLA-A2 negative leukemia (UPN9) in NSG. 8F4 treatment (1mg/kg 3 times weekly) was started at week 19 when AML engraftment was confirmed.(a) AML engraftment was monitored by flow cytometry analysis of human cells ( + /HLA-ABC + / - ) in the peripheral blood. (B)Three days after the sixth dose of 8F4, AML persistence was confirmed by FACS in bone marrow (BM), spleen (SP) and liver (LV).
6 Blood chimerism, % UPN1!.5 mg/kg and 2. mg/kg; 1x/week *** *** Time post transplant, weeks pre- Rx 2. mg/kg 2. mg/kg.5 mg/kg IgG 8F4! Supplementary Figure S6. Low dose 8F4 prevents growth of established leukemia. AML (UPN1, 7 x 1 6 cells) were injected iv (tail vein) into NSG mice and engraftment was confirmed by ~1-fold increase of AML in peripheral blood (.17% ±.8, n=4 on day 7; 1.7% ±.67, n=4 on day 14). Starting on day 15, mice were treated once weekly with low-dose 8F4 (.5 mg/kg or 2 mg/kg) or IgG isotype control (2 mg/kg). The percentages of gated human cells (mo CD45 - /hu CD45 + /HLA- A2 + ) are shown. Data shown is mean percent ± SEM of AML chimerism for each treatment group (n=2-5);! *** p<.5.!!
7 IgG-treated! 8F4-treated! Spinal cord, lumbar region, x4! 2µm l------l 2µm l------l Brain, x1! 1 mm l l 1 mm l l Supplementary Figure S7. Persistence of AML (UPN8) in central neural system (CNS) of 8F4treated mouse. Representative tissue histology (H&E staining) shows extensive AML infiltration in parenchyma and meninges (black arrow) of the brain and spinal cord, and into the other tissues surrounding the CNS, regardless of treatment.!
8 Live Dead Aqua- - + Lin F4 - treated <APC-Cy7-A>: MCD # Cells <PE-Cy7-A>: CD <Pacific Blue-A>: HCD <PE-Cy5-A>: LIN ( ) <PE-A>: CD38 IgG - treated <APC-Cy7-A>: MCD # Cells <PE-Cy7-A>: CD <Pacific Blue-A>: HCD <PE-Cy5-A>: LIN ( ) <PE-A>: CD38 Supplementary Figure S8. Phenotype of residual human cells in bone marrow of mice, transplanted with AML UPN2, and treated with 8F4 or IgG, as shown on Figure 2. Cells were transplanted into secondary NSG, as shown on scheme Figure 4B. 16 weeks after secondary transplant tissues of recipients were analyzed (See Figure 4C)
9 A! Cells, x1 3 /mm WBC 1 mg/kg, 3x /week, total 1 Cells, x1 3 /mm GRA 1 mg/kg, 3x /week, total 1 g/dl HGB 1 mg/kg, 3x /week, total 1 Cells, x1 3 /mm PLT 1 mg/kg, 3x /week, total Time post first treatment, weeks Time post first treatment, weeks Time post first treatment, weeks Time post first treatment, weeks B! Mouse:! Treatment:! B6! B6/HLA-A2! B6/HLA-A2! NT! NT! 8F4! 15 1 Granulocytes, % SSC-A FSC-A SSC-A FSC-A SSC-A FSC-A Mouse Treatment B6 B6/HLA-A2 B6/HLA-A2 NT NT 8F GR1 high, % % of Max % of Max % of Max GR GR GR-1 Mouse Treatment B6 B6/HLA-A2 B6/HLA-A2 NT NT 8F4 Supplementary Figure S9. Multiple doses of 8F4 transiently reduce normal hematopoiesis in HLA-A2 transgenic mice. C57BL/6-Tg(HLA-A2.1)1Enge/J (B6/HLA-A2) mice received 8F4 (1 mg/kg, 3 times per week for total 1 intravenous injections)! starting day. (A) Complete blood count (CBC) was assessed before 8F4 and then weekly beginning 1 week after the1 th dose. Data are expressed as mean ± SEM of n=3 mice. (B) At week 16 8F4-treated mice (n=3) were sacrificed and bone marrow cells were analyzed for presence of granulocytes by flow cytometry. For control, 5 untreated B6/HLA-A2 and 4 B6-WT mice were analyzed. Top panels show representative scatter plots for each group, second row shows overlays of histograms for each group. Right panels show mean±sem for each group.!
X P. Supplementary Figure 1. Nature Medicine: doi: /nm Nilotinib LSK LT-HSC. Cytoplasm. Cytoplasm. Nucleus. Nucleus
a b c Supplementary Figure 1 c-kit-apc-eflu780 Lin-FITC Flt3-Linc-Kit-APC-eflu780 LSK Sca-1-PE-Cy7 d e f CD48-APC LT-HSC CD150-PerCP-cy5.5 g h i j Cytoplasm RCC1 X Exp 5 mir 126 SPRED1 SPRED1 RAN P SPRED1
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Supplementary Fig. S1. Evaluation of the purity and maturation of macrophage cultures tested by flow cytometry. The lymphocytic/monocytic cellular fraction was isolated from buffy coats of healthy donors
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well for 2 h at rt. Each dot represents an individual mouse and bar is the mean ±
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Supplemental Figure 1 1a 1c PD-1 MFI fold change 6 5 4 3 2 1 IL-1α IL-2 IL-4 IL-6 IL-1 IL-12 IL-13 IL-15 IL-17 IL-18 IL-21 IL-23 IFN-α Mut Human PD-1 promoter SBE-D 5 -GTCTG- -1.2kb SBE-P -CAGAC- -1.kb
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SUPPLEMENTARY FIGURE 1
SUPPLEMENTARY FIGURE 1 A LN Cell count (1 ) 1 3 1 CD+ 1 1 CDL lo CD hi 1 CD+FoxP3+ 1 1 1 7 3 3 3 % of cells 9 7 7 % of cells CD+ 3 1 % of cells CDL lo CD hi 1 1 % of CD+ cells CD+FoxP3+ 3 1 % of CD+ T
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Supplemental Figures Supplemental Figure 1: Representative FACS data showing Concurrent Brain cell type Acquisition using either Percoll PLUS (top row) or myelin removal beads (bottom two rows). Debris
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SUPPLEMENTARY METHODS
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VUmc Basispresentatie
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Supplementary material page 1/10
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a 10 4 Link et al. Supplementary Figure 1 Nature Immunology: doi: /ni.1842 Cells per mouse ( 10 5 ) TRPV2KO anti-gr1 anti-gr anti-f4/80
a 10 4 WT 10 4 TRPV2KO 10 3 10 3 anti-gr1 10 2 10 1 anti-gr1 10 2 10 1 10 0 10 0 10 1 10 2 10 3 10 4 anti-f4/80 42.3 45.2 10 0 10 0 10 1 10 2 10 3 10 4 anti-f4/80 10 4 10 4 40 42.5 anti-cd11b 10 3 10 2
Figure S1. Western blot analysis of clathrin RNA interference in human DCs Human immature DCs were transfected with 100 nm Clathrin SMARTpool or
Figure S1. Western blot analysis of clathrin RNA interference in human DCs Human immature DCs were transfected with 100 nm Clathrin SMARTpool or control nontargeting sirnas. At 90 hr after transfection,
Phenotype Determines Nanoparticle Uptake by Human
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Nature Immunology: doi: /ni.3412
Supplementary Figure 1 Gata1 expression in heamatopoietic stem and progenitor populations. (a) Unsupervised clustering according to 100 top variable genes across single pre-gm cells. The two main cell
Fisher et al. Supplemental Figure 1
Supplemental Figure 1 A TNF IL-1 IL-6 CCL2 CCL5 CXCL10 pg/mg total protein 50 30 10 4,000 3,000 2,000 1,000 n.d. 1 1 14,000 12,000 10,000 8,000 6,000 4,000 2,000 6,000,000 CT26 5,000 16,000 B16 4,000 12,000
Figure S1. Gating strategy used in NK cells and γδ T lymphocytes coculture An example of flow cytometry analysis shows the gating of NK cells and γδ
Figure S1. Gating strategy used in NK cells and γδ T lymphocytes coculture An example of flow cytometry analysis shows the gating of NK cells and γδ T lymphocytes used in all NK activation and cytotoxicity
Supplemental Information. CD4 + CD25 + Foxp3 + Regulatory T Cells Promote. Th17 Cells In Vitro and Enhance Host Resistance
Immunity, Volume 34 Supplemental Information D4 + D25 + + Regulatory T ells Promote Th17 ells In Vitro and Enhance Host Resistance in Mouse andida albicans Th17 ell Infection Model Pushpa Pandiyan, Heather
Islet viability assay and Glucose Stimulated Insulin Secretion assay RT-PCR and Western Blot
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Supporting Information
Supporting Information Chan et al. 1.173/pnas.9654916 A Patient B Xenograft C * remaining feature of normal lymph node * * * D lymphocytes Infiltrating transitional carcinoma cells E Enlarged axillary
Supplementary Figure 1: Expression of NFAT proteins in Nfat2-deleted B cells (a+b) Protein expression of NFAT2 (a) and NFAT1 (b) in isolated splenic
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Evaluation of directed and random motility in microslides Assessment of leukocyte adhesion in flow chambers
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Supplementary information
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Nature Immunology: doi: /ni Supplementary Figure 1
Supplementary Figure 1 A β-strand positions consistently places the residues at CDR3β P6 and P7 within human and mouse TCR-peptide-MHC interfaces. (a) E8 TCR containing V β 13*06 carrying with an 11mer
activation with anti-cd3/cd28 beads and 3d following transduction. Supplemental Figure 2 shows
Supplemental Data Supplemental Figure 1 compares CXCR4 expression in untreated CD8 + T cells, following activation with anti-cd3/cd28 beads and 3d following transduction. Supplemental Figure 2 shows the
MATERIALS AND METHODS. Neutralizing antibodies specific to mouse Dll1, Dll4, J1 and J2 were prepared as described. 1,2 All
MATERIALS AND METHODS Antibodies (Abs), flow cytometry analysis and cell lines Neutralizing antibodies specific to mouse Dll1, Dll4, J1 and J2 were prepared as described. 1,2 All other antibodies used
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Supplementary Information Memory-type ST2 + CD + T cells participate in the steroid-resistant pathology of eosinophilic pneumonia Naoko Mato 1, 2, Kiyoshi Hirahara 2, Tomomi Ichikawa 2, Jin Kumagai 2,
Online Appendix Material and Methods: Pancreatic RNA isolation and quantitative real-time (q)rt-pcr. Mice were fasted overnight and killed 1 hour (h)
Online Appendix Material and Methods: Pancreatic RNA isolation and quantitative real-time (q)rt-pcr. Mice were fasted overnight and killed 1 hour (h) after feeding. A small slice (~5-1 mm 3 ) was taken
Supplementary Figure 1. Successful excision of genes from WBM lysates and
Supplementary Information: Supplementary Figure 1. Successful excision of genes from WBM lysates and survival of mice with different genotypes. (a) The proper excision of Pten, p110α, p110α and p110δ was
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Supplementary Table 1
Supplementary Table 1 Flow Cytometry Antibodies Antibody Fluorochrome Clone Vendor CD45 PE-cyanine 7 30-F11 D ioscience CD3 Pacific lue 17A2 iolegend (San Diego, CA) CD11b APC M1/70 iolegend (San Diego,
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Blocking antibodies and peptides. Rat anti-mouse PD-1 (29F.1A12, rat IgG2a, k), PD-
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Nature Medicine: doi: /nm.2109
HIV 1 Infects Multipotent Progenitor Cells Causing Cell Death and Establishing Latent Cellular Reservoirs Christoph C. Carter, Adewunmi Onafuwa Nuga, Lucy A. M c Namara, James Riddell IV, Dale Bixby, Michael