NC bp. b 1481 bp
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1 Kcna3 NC p 1481 p 346 p c *** *** d relative expression CD4 T cells Kcna1 Kcna2 Kcna3 Kcna4 Kcna Kcna6 Kcna7 Kcnn4 relative expression CD8 T cells Kcna1 Kcna2 Kcna3 Kcna4 Kcna Kcna6 Kcna7 Kcnn4 e % of max Kv1.3 Supplementary Figure 1. Generation of Kcna3 -/- rats. (a) Zinc finger nuclease (ZFN) strategy for deletion of Kcna3 gene. Kcna3 gene consists of single exon, so two pairs of ZFNs were designed to cleave together in order to generate an approximately 1.2 k deletion etween the cut sites (NCBI genomic coordinates in reference sequence NC_11). Primers flanking each ZFN site were designed to test individual NHEJ activity (green arrows) as well as paired together (red arrows) to screen for deletion mutations etween the two ZFN sites. () Deletion screen to identify Kcna3 -/- founder rats. Expected wild-type and = 1481 p, expected deletion etween ZFNs = 346 p. (c) Sequence confirmation of Kcna3 deletion in rat #17. ZFN targeted deletion of Kcna3 resulted in 2 stop codons, indicated y red asterisks. (d) K + channel expression in and Kcna3 -/- CD4 + (left panel) and CD8 + (right panel) T cells. Expression of Kv1.3 (Kcna3), other Kv1 family memers (Kcnax), and KCa3.1 (Kcnn4) was measured in ex vivo purified T cells. Relative gene expression was determined y normalizing to housekeeping gene Rpl19. Data are shown as mean ± s.d. (e) Kv1.3 surface protein expression is asent on Kcna3 -/- rat CD4 + T cells. Blue histogram represents Kv1.3 expression on CD4 + T cells, red histogram denotes ; gray filled histogram represents staining with isotype control antiody.
2 2.4 (3.6) spleen LN lood thymus 6.1 (2.9) 18. (.7) 7. (1.) 2.4 (.2) 7.3 (.2) 11.2 (.1) 33.4 (1.) 47.9 (.) 2.4 (4.2) 19.2 (.6) 19.4 (2.2) 1.9 (.3) 49.4 (1.) CD8 8.6 (3.) 69.3 (.9) 68.1 (.7) 32. (.) CD4 spleen # CD4 + T cells (x1 6 ) # CD4 T cells # CD8 T cells # CD4+CD8+ # B cells T cells 2 # CD8 + T cells (x1 6 ) n.d. # B cells (x1 6 ) 1 1 LN # CD4 + T cells (x1 6 ) # CD8 + T cells (x1 6 ) n.d. # B cells (x1 6 ) thymus # CD4 + T cells (x1 6 ) # CD8 + T cells (x1 6 ) # CD4 + CD8 + T cells (x1 6 ) # B cells (x1 6 ) c thymus LN spleen % CD4 T cells % CD8 T cells % CD4+CD8+ % B cells 4 T cells % CD4 + CD8 + T cells n.d. n.d. % B cells % B cells % B cells
3 Supplementary Figure 2. Immune cell distriution in Kcna3 -/- rats. (a) T cell frequencies in Kcna3 -/- rats. Flow cytometry staining was used to define CD4 + and CD8 + T cell populations from spleen, pooled LN, lood and thymus. Data are shown for live CD3 + gated cells. Data are representative of 4 individual Kcna3 -/- and littermate rats. Frequencies of specific cell populations are shown for indicated gated population as mean ± s.d. Flow cytometric analysis was used to enumerate T cell and B cell populations () or determine relative cell frequencies (c) in spleen, LN and thymus and Kcna3 -/- rats. Individual animals (n = 4 iological replicates per group) are represented y discrete symols and are shown with mean ± s.d.. No statistically significant differences as determined y Student s t test were oserved etween and for any cell population. n.d. denotes cell suset not determined.
4 (RLU) PBS PBS OVA OVA (pg/ml) PBS PBS OVA OVA Supplementary Figure 3. OVA-specific in vitro recall responses from DTH rats. (lue) or Kcna3 -/- (green) OVA-immunized rats were susequently challenged with OVA (filled symols) or PBS (open symols). (a) and () responses of DLN and spleen cells (1:1 ratio) harvested from animals receiving secondary challenge with PBS or OVA were determined after 3-day stimulation with OVA. Individual iological replicates (n = 4 per group) are shown with mean ± s.d.
5 % CD4+CD2+ % CD8 T cells T cells % CD4 T cells % T cells # live cells % CD4+CD2+ % CD8 T cells T cells % CD4 T cells % T cells # live cells # live cells (x1 6 ) % T cells % T cells % CD4 + CD2 + T cells # live cells (x1 6 ) % CD4 + CD2 + T cells spleen spleen OVA 1 spleen OVA 3 # live cells (x1 6 ) % T cells % CD4 + CD2 + T cells # live cells (x1 6 ) % T cells % CD4 + CD2 + T cells DLN DLN OVA 1 DLN OVA 3 # live cells (x1 6 ) % T cells % CD4 + CD2 + T cells # live cells (x1 6 ) % T cells % CD4 + CD2 + T cells Supplementary Figure 4
6 Supplementary Figure 4. Immunophenotyping of rat spleen (a) and DLN () cells following OVA immunization. Flow cytometric analysis was used to enumerate T cell populations from and Kcna3 -/- rats following primary immunization with OVA or after 3 rounds of OVA immunization. Individual animals (n = 4 iological replicates per group) are represented y discrete symols and are shown with mean ± s.d. No statistically significant differences as determined y Student s t test were oserved etween and.
7 Ct value ex vivo 1 TT 4 TT RPL19 KCNA1 KCNA2 KCNA3 KCNA4 KCNA KCNA6 KCNA7 KCNN4 Relative expression (compared to ex vivo) TT 4 TT KCNA1 KCNA2 KCNA3 KCNA4 KCNA KCNA6 KCNA7 KCNN4 Supplementary Figure. K + channel expression in repeatedly stimulated TT-specific human T cells. Gene expression of Kv1 family memers (KCNAx) and KCa3.1 (KCNN4) was measured in primary tetanus toxoid-stimulated T cells (1 TT) or T cells that underwent four rounds of TT stimulation (4 TT). (a) Raw Ct values, with only Kv1.3 and KCa3.1 having Ct values less than 33. () Relative expression of K + channels shown in comparison to ex vivo T cells. Data are shown as mean ± s.d. of triplicate measurements from one representative experiment. denotes elow limit of detection.
8 CD8 gate CD4 gate SSC CD FSC CD4 GAD6 peptide como -34 CD4 gate -1% -11% CD2 CFSE CD8 gate CD2 CFSE % -6% c GAD6 protein CD4 gate CD2 CFSE CD8 gate CD2 CFSE % -12% -79% -13% Supplementary Figure 6. inhiits Type 1 diaetes autoantigen-specific CD4 and CD8 T cell proliferation. a. CD4 + and CD8 + T cell gating strategy. Live lymphocyte gate was set ased on forward scatter (FSC) vs. side scatter (SSC). CD4 + and CD8 + gates were then set ased on CD4 and CD8 expression, respectively. PBMC from HLA-DR4 + T1D donors were stimulated with a comination of HLA-DR4-restricted GAD6 peptides (, n = 1 iological replicates) in the asence or presence of either 1 nm or 1 µm -34, or PBMC from non-hla- or HLA-typed T1D donors were stimulated with GAD6 protein (c, n = 13 iological replicates). responses were determined at day 7 y CFSE dilution of CD4 + - or CD8 + -gated cells. Representative data from one donor are shown. Percentages indicated in red denote % reduction in proliferation relative to -treated cells.
9 GAD6 como HA peptide GAD6 como HA peptide c 1 d GAD6 como CMV PepMix GAD6 como CMV PepMix Supplementary Figure 7. inhiits GAD6-specific T1D T cells ut not autologous HA- or CMV-specific T cells. PBMC from T1D donors (n = 9) were stimulated with a comination of four HLA-DR4-restricted GAD6 peptides. and responses were compared to HA (a, ) or CMV (c, d) stimulation in the asence or presence of either 1 nm (a, c) or 1 µm -34 (, d). Data shown are % inhiition of proliferation response as determined y 3 H-thymidine incorporation after 4 days primary in vitro stimulation and % inhiition of production after 3 days stimulation. Colored symols and corresponding lines represent each individual donor.
10 HA CMV Tetanus toxoid p<.1 p< HA CMV p<.1 Tetanus 8 p<.1 p<.1 p<.1 toxoid p=.4 p< Supplementary Figure 8. T1D donor pathogen-specific T cell responses are not inhiited y. PBMC from T1D donors were stimulated with HA peptide (n = 1 iological replicates), CMV PepMix (n = 9) or tetanus toxoid (n = 9) in the asence or presence of either 1 nm or 1 µm -34. (a) Inhiition of proliferation response as determined y 3 H-thymidine incorporation after 4 days primary in vitro stimulation. () Inhiition of production after 3 day stimulation. Data are shown as individual data points together with mean ± s.d. Statistically significant differences are denoted with p-values as determined y Student s t test.
11 (cpm) 2 1 stim: 2 stim: (pg/ml) stim: 2 stim: (cpm) 2 2 stim: 3 stim: (pg/ml) stim: 3 stim: c (cpm) 2 3 stim: 4 stim: (pg/ml) stim: 4 stim: Supplementary Figure 9.
12 Supplementary Figure 9. Sensitivity to channel lockers is dependent on repeated stimulation and is not affected y prior exposure to inhiitor. (a) Primary (1 ) T cell stimulation with TT was performed in the asence or presence of either 1 nm or 1 µm -34. After 4 days in culture, inhiitor was washed out, cells were rested for 3 days, then restimulated a second time (2 stim) with TT and irradiated autologous PBMC in the presence of the indicated inhiitor. responses (upper panels) were determined y addition of 3 H-thymidine one day prior to liquid scintillation counting which was performed on day 4. concentrations (lower panels) in supernatants harvested 3 days after stimulation were determined y ELISA. Data are shown as mean ± s.d. of duplicate wells. () 2 TT-specific T cells sujected to inhiitor washout and 3 TT stimulation. (c) 3 TT-specific T cells sujected to inhiitor washout and 4 TT stimulation.
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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: Cryopreservation alters CD62L expression by CD4 T cells. Freshly isolated (left) or cryopreserved PBMCs (right) were stained with the mix of antibodies described
Supporting Information Table of Contents
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Supplementary Information:
Supplementary Information: Follicular regulatory T cells with Bcl6 expression suppress germinal center reactions by Yeonseok Chung, Shinya Tanaka, Fuliang Chu, Roza Nurieva, Gustavo J. Martinez, Seema
Nature Biotechnology: doi: /nbt Supplementary Figure 1. Binding capacity of DNA-barcoded MHC multimers and recovery of antigen specificity
Supplementary Figure 1 Binding capacity of DNA-barcoded MHC multimers and recovery of antigen specificity (a, b) Fluorescent-based determination of the binding capacity of DNA-barcoded MHC multimers (+barcode)
% of Cells A B C. Proliferation Index. T cell count (10 6 ) Division Index. % of Max CFSE. %Ki67+ cells. Supplementary Figure 1.
A B C T cell count (1 6 ) 3. 2. 1.. * % of Max CFSE ormoxia ypoxia o Stim. Proliferation Index 2.5 2. 1.5 1. * Division Index 2. 1.5 1..5. D E %Ki67+ cells 1 8 6 4 2 % of Cells 8 ormoxia 6 ypoxia 4 2 *
Supplementary Figure 1. Using DNA barcode-labeled MHC multimers to generate TCR fingerprints
Supplementary Figure 1 Using DNA barcode-labeled MHC multimers to generate TCR fingerprints (a) Schematic overview of the workflow behind a TCR fingerprint. Each peptide position of the original peptide
Sirt1 Hmg20b Gm (0.17) 24 (17.3) 877 (857)
3 (0.17) 24 (17.3) Sirt1 Hmg20 Gm4763 877 (857) c d Suppl. Figure 1. Screen validation for top candidate antagonists of Dot1L (a) Numer of genes with one (gray), two (cyan) or three (red) shrna scored
Supplementary Figure 1. Supernatants electrophoresis from CD14+ and dendritic cells. Supernatants were resolved by SDS-PAGE and stained with
Supplementary Figure 1. Supernatants electrophoresis from CD14+ and dendritic cells. Supernatants were resolved by SDS-PAGE and stained with Coomassie brilliant blue. One µg/ml recombinant human (rh) apo-e
Supplementary Figure 1
Supplementary Figure 1 Identification of IFN-γ-producing CD8 + and CD4 + T cells with naive phenotype by alternative gating and sample-processing strategies. a. Contour 5% probability plots show definition
Type of file: PDF Title of file for HTML: Supplementary Information Description: Supplementary Figures and Supplementary Table.
Type of file: PDF Title of file for HTML: Supplementary Information Description: Supplementary Figures and Supplementary Tale. Type of file: VI Title of file for HTML: Supplementary Movie 1 Description:
SUPPLEMENTARY FIGURES
SUPPLEMENTARY FIGURES Supplementary Figure 1: Chemokine receptor expression profiles of CCR6 + and CCR6 - CD4 + IL-17A +/ex and Treg cells. Quantitative PCR analysis of chemokine receptor transcript abundance
Supplementary Figures
Supplementary Figures Supplementary Fig. 1. Galectin-3 is present within tumors. (A) mrna expression levels of Lgals3 (galectin-3) and Lgals8 (galectin-8) in the four classes of cell lines as determined
Other Clinical Diagnoses
Donor Case ID 14 year old fe npod6342 24 year old npod6367 12 year old fe npod6268 6 year old fe npod69 22 year old fe npod6323 20 year old T1D.6 27 year old T1D.7 30 year old T1D.8 22 year old T1D.9 Duration
Supplementary Information. A vital role for IL-2 trans-presentation in DC-mediated T cell activation in humans as revealed by daclizumab therapy
Supplementary Information A vital role for IL-2 trans-presentation in DC-mediated T cell activation in humans as revealed by daclizumab therapy Simone C. Wuest 1, Jehad Edwan 1, Jayne F. Martin 1, Sungpil
SUPPLEMENTARY MATERIAL
SUPPLEMENTARY MATERIAL Divergent effects of intrinsically active MEK variants on developmental Ras signaling Yogesh Goyal,2,3,4, Granton A. Jindal,2,3,4, José L. Pelliccia 3, Kei Yamaya 2,3, Eyan Yeung
Aquaporin-9-expressing neutrophils are required for the establishment of contact hypersensitivity
Supplemental Material quaporin-9-expressing neutrophils are required for the establishment of contact hypersensitivity atharina Sagita Moniaga 1, Sachiko Watanabe 1, Tetsuya Honda 1, 2, Søren Nielsen 3,
glpx Rv1100 Probe 4.4 kb 75 kda 50 kda 37 kda 25 kda 20 kda
PvuI 2.0 k PvuI PvuI Rv1101c Rv1100 glpx fum 4.4 k PvuI PvuI ΔglpX Rv1101c Rv1100 HygR fum ΔglpX 5 k 4 k 3 k c 75 kda 50 kda 37 kda GLPX Δ Δ/C GLPX 2 k 1.5 k 25 kda 20 kda PRCB 1 k 0.5 k Supplementary
Supplementary Figure 1
d f a IL7 b IL GATA RORγt h HDM IL IL7 PBS Ilra R7 PBS HDM Ilra R7 HDM Foxp Foxp Ilra R7 HDM HDM Ilra R7 HDM. 9..79. CD + FOXP + T reg cell CD + FOXP T conv cell PBS Ilra R7 PBS HDM Ilra R7 HDM CD + FOXP
Supplementary Material
Supplementary Material HLA-DM Captures Partially Empty HLA-DR Molecules for Catalyzed Peptide Removal Anne-Kathrin Anders, Melissa J. Call, Monika-Sarah E. D. Schulze, Kevin D. Fowler, David A. Schuert,
Supplemental Figure 1. Cell-bound Cetuximab reduces EGFR staining intensity. Blood
Antibody-mediated depletion of CD19-CAR T cells Supplemental 1 Supplemental Materials Supplemental Figure 1. Supplemental Figure 1. Cell-bound Cetuximab reduces EGFR staining intensity. Blood cells were
Cytotoxicity assays. Rory D. de Vries, PhD 1. Viroscience lab, Erasmus MC, Rotterdam, the Netherlands
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SUPPLEMENTARY INFORMATION
Complete but curtailed T-cell response to very-low-affinity antigen Dietmar Zehn, Sarah Y. Lee & Michael J. Bevan Supp. Fig. 1: TCR chain usage among endogenous K b /Ova reactive T cells. C57BL/6 mice
Krishnamoorthy et al.,
Krishnamoorthy et al., c d e ND ND Supplementary Figure 1 RSV-induces inflammation even in the asence of allergen. Tolerized pups were either infected with RSV or not. The mice were sacrificed a week following
tom tom 24hpf tom tom 48hpf tom 60hpf tom tom 72hpf tom
a 24hpf c 48hpf d e 60hpf f g 72hpf h i j k ISV ISV Figure 1. Vascular integrity defects and endothelial regression in mutant emryos. (a,c,e,g,i) Bright-field and (,d,f,h,j) corresponding fluorescent micrographs
Nature Medicine doi: /nm.3957
Supplementary Fig. 1. p38 alternative activation, IL-21 expression, and T helper cell transcription factors in PDAC tissue. (a) Tissue microarrays of pancreatic tissue from 192 patients with pancreatic
sequences of a styx mutant reveals a T to A transversion in the donor splice site of intron 5
sfigure 1 Styx mutant mice recapitulate the phenotype of SHIP -/- mice. (A) Analysis of the genomic sequences of a styx mutant reveals a T to A transversion in the donor splice site of intron 5 (GTAAC
Impaired Response of Lymphocytes from Non-Insulin- Dependent Diabetics to Staphage Lysate and Tetanus Antigen
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1982, p. 109-114 0095-1137/82/010109-06$02.00/0 Vol. 15, No.1 Impaired Response of Lymphocytes from Non-Insulin- Dependent Diaetics to Staphage Lysate and Tetanus
SUPPLEMENT Supplementary Figure 1: (A) (B)
SUPPLEMENT Supplementary Figure 1: CD4 + naïve effector T cells (CD4 effector) were labeled with CFSE, stimulated with α-cd2/cd3/cd28 coated beads (at 2 beads/cell) and cultured alone or cocultured with
Supplementary appendix
Supplementary appendix This appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Supplement to: Chandran SS, Somerville RPT, Yang JC, et al.
Hidenobu Kanda, Rebecca Newton, Russell Klein, Yuka Morita, Michael D. Gunn & Steven D. Rosen
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Application Information Bulletin: Human NK Cells Phenotypic characterizing of human Natural Killer (NK) cell populations in peripheral blood
Application Information Bulletin: Human NK Cells Phenotypic characterizing of human Natural Killer (NK) cell populations in peripheral blood Christopher A Fraker, Ph.D., University of Miami - Miami, Florida
Low Avidity CMV + T Cells accumulate in Old Humans
Supplementary Figure Legends Supplementary Figure 1. CD45RA expressing CMVpp65-specific T cell populations accumulate within HLA-A*0201 and HLA-B*0701 individuals Pooled data showing the size of the NLV/HLA-A*0201-specific
Extracellular vesicles are transferred from melanocytes to keratinocytes after UVA irradiation
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Nature Immunology: doi: /ni Supplementary Figure 1. DNA-methylation machinery is essential for silencing of Cd4 in cytotoxic T cells.
Supplementary Figure 1 DNA-methylation machinery is essential for silencing of Cd4 in cytotoxic T cells. (a) Scheme for the retroviral shrna screen. (b) Histogram showing CD4 expression (MFI) in WT cytotoxic