Journal: Cell Reports

Article Title: CD4 + T Cells Recognize Conserved Influenza A Epitopes through Shared Patterns of V-Gene Usage and Complementary Biochemical Features

doi: 10.1016/j.celrep.2020.107885

Figure Lengend Snippet:

Article Snippet: Anti-human CD8 allophycocyanin-vio770 (clone BW135/80) , Miltenyi Biotec , Cat#170-081-073.

Techniques: Staining, Virus, Recombinant, Enzyme-linked Immunospot, Sequencing, Expressing, Plasmid Preparation, Software

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A and B) Flow cytometry analysis of Ahr expression (GFP) in CD8 + T cells of Ahr +/+ and Ahr dCAIR/+ mice was performed. Histogram plot of GFP( Ahr ) in CD8 + T cells isolated from spleen (Sp), peripheral lymph node (pLN), mesenteric lymph node (mLN), small intestine lamina propria (LPL), or intraepithelial lymphocytes (IEL) (A). Quantification of ΔgMFI in Ahr dCAIR/+ compared with GFP-negative Ahr +/+ mice (B). (C and D) Flow cytometry quantification of ΔgMFI of Ahr -GFP in different CD8 + T cell populations isolated from LPL including CD44 − CD62L + (T naive ), CD127 − KLRG1 − (T EE ), CD127 − KLRG1 + (T SLE ), CD127 + KLRG1 − (T MPE ) (C), and CD44 + CD62L + (T CM ), CD44 + CD62L − (T EM ), and CD69 + CD103 + (T RM ) (D). Data are shown as mean ± SEM (n = 3 mice per group). Data are representative of two independent experiments. See also .

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: Flow Cytometry, Expressing, Isolation

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A–E and H) RNA-seq analysis of IEL resident CD8 + T cells isolated from Ahr f/f and Ahr f/f Cd8 cre mice and statistics (q values shown) were calculated via DESeq2 differential expression analysis, and FPKM values were quantified using RSEM. MA plot highlighting genes upregulated (red) and downregulated (blue) in Ahr f/f Cd8 cre compared with Ahr f/f (A). Gene set enrichment analysis of resident and circulating core gene signatures (B). Heatmap depicting (fold change >1.5) genes enriched in respective signatures (C). FPKM expression values for transcriptional regulators (D) and secreted factors associated with cell function (E). (F and G) Flow cytometry quantification of granzyme B protein levels of IEL resident CD8 + T cells isolated from Ahr f/f and Ahr f/f Cd8 cre mice. The percentages (F) and total cell number (n = 5 mice per group) (G) are shown. Data are representative of two independent experiments. (H) FPKM expression values for proliferation, cell cycle, and apoptosis genes. (I–K) Naive CD8 + T cells were isolated from Ahr +/+ and Ahr − / − mice and then subjected to in vitro T RM -like differentiation culture conditions. RNA was isolated for qRT-PCR expression analysis of Ahrr (I). Flow cytometry quantification of CD69 + CD103 + in vitro T RM -like cells (J) and CD44 + CD62L + in vitro T CM -like population frequency (K). Data are shown as mean ± SEM (n = 3 mice per group). Data are representative of three independent experiments. See also , , and .

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: RNA Sequencing, Isolation, Quantitative Proteomics, Expressing, Cell Function Assay, Flow Cytometry, In Vitro, Quantitative RT-PCR

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A and B) ChIP-seq of Ahr was performed using in vitro T RM -like CD8 + T cells. Analysis of Ahr binding was performed, and pie chart of peak annotation (A) as well as top seven enriched transcription factor motifs (B) are shown. (C and D) Binding and Expression Target Analysis (BETA) was performed to integrate RNA-seq and ChIP-seq data. Visualization of transcription factor activating/repressive function prediction (C) and rank product volcano plot depicting top direct target candidates (D). (E and F) Ahr − / − in vitro T RM -like CD8 + T cells transduced with retroviral constructs encoding MIG-EV (empty vector), MIG-Ahr, MIG-Y9A, or MIG-DbHLH. The cells were treated with DMSO or FICZ on day 3. On day 5, CD69 and CD103 expression was analyzed by flow cytometry (E), and RNA was isolated for qRT-PCR expression analysis of Ahr direct target gene Ahrr (F). Data are shown as mean ± SEM (n = 3 technical replicates per group). Data are representative of two independent experiments. (G) Flow cytometry analysis of CD69 and CD103 expression in Ahr − / − in vitro T RM -like CD8 + T cells transduced with retroviral constructs encoding MIG-EV, MIG-Ahr, hCD2-EV, or hCD2-Blimp1. Data are representative of two independent experiments. See also .

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: ChIP-sequencing, In Vitro, Binding Assay, Expressing, RNA Sequencing, Transduction, Retroviral, Construct, Plasmid Preparation, Flow Cytometry, Isolation, Quantitative RT-PCR

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A–F) Analysis of antigen-specific (OTI) CD8 + T cell response in the IEL during oral L . m .-OVA infection was performed as depicted in the schematic (A). Flow cytometry quantification of the percentage of Ahr +/+ vs. Ahr − / − OTI cells on day 9, 20, and 34 post infection (B). Flow cytometry quantification of memory precursor populations based on expression of CD127 and KLRG1 (C) as well as CD69 and CD103 (D) in Ahr +/+ vs. Ahr − / − OTI cells present in the IEL on day 9 post infection. Flow cytometry analysis of CD45.1 and GzmB gated on OTI cells depicting CD45 . 1 + Ahr +/+ (CD45.1/.2) and CD45 . 1 − Ahr − / − (CD45.2/.2) OTI cells production of granzyme B on day 34 post infection (E). Quantification of granzyme B + OTI T cells in the mice of indicated genotypes (F). (G–J) L . m .-OVA re-infection was performed and Ahr +/+ vs. Ahr − / − OTI IEL resident CD8 + T cells analyzed on day 3 post re-infection. Flow cytometry analysis of CD45.1 and CD45.2 depicting percentage of Ahr +/+ (CD45.1/.2) and Ahr − / − (CD45.2/.2) OTI IEL resident CD8 + T cells as well as (G) quantification of percentage are shown (H). Flow cytometry analysis (I) and quantification (J) of granzyme B production in analyzed cells. Data are compiled from two independent experiments and shown as mean ± SEM (n = 3–6 replicates per group). See also .

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: Infection, Flow Cytometry, Expressing

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A–D) scRNA-seq analysis of Ahr +/+ (n = 2) vs. Ahr − / − (n = 2) TIL CD8 + T cells was performed. UMAP dimensionality reduction and cluster visualization (left) as well as pie chart frequency depiction (right) color-coded to represent cluster ID (A). Pseudotime visualization (B) and quantification (C). Differential gene expression depicted via color intensity as average expression and circle size as percent expressed in CD8 + T cells (D). (E–H) Ahr f/f and Ahr f/f Cd8 cre mice were inoculated subcutaneously with B16F10 mouse melanoma and tumor size monitored (E). At endpoint, tumor weight was quantified (F), and TILs were isolated for flow cytometry analysis. Pie chart visualization depicting polyfunctionality of TIL CD8 + T cells in mice with indicated genotypes (G). Polyfunctionality score quantification (triple-positive plus double-positive minus triple-negative divided by total cells) of TIL CD8 + T cells isolated from Ahr f/f and Ahr f/f Cd8 cre tumor-bearing mice (H). Data are compiled from two independent experiments and are shown as mean ± SEM (n = 6 to 7 replicates per group). See also .

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: Gene Expression, Expressing, Isolation, Flow Cytometry

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A–C) Human peripheral blood (PBMC) vs. IEL CD8 + T cells were analyzed via flow cytometry. Staining of CD45RA and CD45RO (top), CD103 and CD69 (bottom) (A), as well as T-BET and AHR (B) in tissue fractions as indicated in the figure. Quantification of AHR protein levels (gMFI) in human PBMC, LPL, and IEL CD8 + T cells (C). Data are compiled from three independent experiments and shown as mean ± SEM (n = 4 replicates per group). (D–G) Human naive CD8 + T cells were isolated from PBMCs and then subjected to in vitro T RM -like differentiation culture conditions. The cells were given differentiation cytokines and treated with DMSO (control), FICZ, or CH223191 on day 2. The assay was collected on day 4, and flow cytometry quantification of CD103 expression was performed (D). Data are compiled from three independent experiments and are shown as mean ± SEM (n = 4 replicates per group). RNA was isolated for qRT-PCR expression analysis of AHR (E), AHR direct target gene AHRR (F), and GZMB (G). Data are shown as mean ± SEM (n = 3 technical replicates per group). Data are representative of three independent experiments.

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: Flow Cytometry, Staining, Isolation, In Vitro, Control, Expressing, Quantitative RT-PCR

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet:

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: Virus, Isolation, Recombinant, SYBR Green Assay, DNA Library Preparation, Reverse Transcription, Staining, Transgenic Assay, Plasmid Preparation, Software

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: A IOD of ACAT2 expression in CC tissues and adjacent tissues was examined using immunohistochemical staining ( n = 47 biologically independent samples). IOD of DHCR7 B and MSMO1 C expression in CC patients with high ( n = 27 biologically independent samples) or low ( n = 20 biologically independent samples) expression of ACAT2 was examined using immunohistochemical staining. The number of activated CD8 T cells (CD8A + GZMB + ) D or activated NK cells (CD56 + GZMB + ) E infiltrated in the tumor tissues of patients with high ( n = 27 biologically independent samples) and low ACAT2 ( n = 20 biologically independent samples) expression was detected. Data represent mean ± SEM. Statistical analysis was performed using the paired A or unpaired ( B – E ) t-test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: ACAT2 expression in HCeEpiC and CC cell lines was examined using RT-qPCR A and Western blot analysis B ( n = 5 independent experiments). C ACAT2, DHCR7, and MSMO1 expression in CC cells after infection with Scramble-sh, ACAT2-sh #1, and ACAT2-sh #2 was examined using Western blot analysis ( n = 5 independent experiments). D Detection of total cholesterol, free cholesterol, and cholesteryl ester levels in CC cells ( n = 5 independent experiments). The proliferation of CC cells was examined using CCK8 ( E ) and colony formation assays F (n = 5 independent experiments). G CC cells were co-cultured with (E: T = 3:1) with NK cells or CD8 T cells for 6 h, respectively, and the death of CC cells was detected ( n = 5 independent experiments). H IFN-γ and GZMB released from immune cells in a co-culture system with CC cells were examined using ELISA ( n = 5 independent experiments). Data represent mean ± SEM. Statistical analysis was performed using the one-way ( A , B ) or two-way ( C - H ) ANOVA, followed by Tukey’s multiple comparisons test ( A – H ).

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Infection, Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: A ACAT2 knockdown efficiency in U14 cells was examined using western blot analysis ( n = 10 independent experiments). B Volume changes of transplanted tumors in mice subcutaneously inoculated with U14 cells (n = 10 animals). C The images and weight of the tumors harvested on day 21 ( n = 10 animals). D Protein expression of ACAT2, MSMO1, DHCR7, and PCNA in transplanted tumors was examined using western blot analysis ( n = 10 animals). E Detection of total cholesterol, free cholesterol, and cholesteryl ester levels in transplanted tumors ( n = 10 animals). The gating strategy for GZMB + NK cells and CD8 + T cells F and quantification G were analyzed using flow cytometry ( n = 10 animals). H Survival of mice over 60 days after subcutaneous inoculation of U14 cells was analyzed using the log-rank test ( n = 20 animals). Data represent mean ± SEM. Statistical analysis was performed using the one-way ( A , C , E , G ) or two-way ( B , D ) ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Knockdown, Western Blot, Expressing, Flow Cytometry

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: The proliferation of CC cells was examined using CCK8 A and colony formation assays B ( n = 5 independent experiments). C CC cells were co-cultured with (E: T = 3:1) with NK cells or CD8 + T cells, and the death of CC cells was detected ( n = 5 independent experiments). D IFN-γ and GZMB released from immune cells in a co-culture system with CC cells were examined using ELISA ( n = 5 independent experiments). E TGF-β1 released by CC cells was examined using ELISA ( n = 5 independent experiments). F PD-L1 expression levels in CC cells were observed using immunofluorescence staining ( n = 5 independent experiments). Data represent mean ± SEM. Statistical analysis was performed using the two-way ( A – F ) ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Staining

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: A Volume changes of transplanted tumors in mice subcutaneously inoculated with U14 cells ( n = 5 animals). B The images and weight of the tumors harvested on day 21 ( n = 5 animals). The gating strategy for GZMB + NK cells and CD8 + T cells C and quantification D were analyzed using flow cytometry ( n = 5 animals). Data represent mean ± SEM. Statistical analysis was performed using the one-way ( B , D ) or two-way A ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Human Amniotic MSC Response in LPS-Stimulated Ascites from Patients with Cirrhosis: FOXO1 Gene and Th17 Activation in Enhanced Antibacterial Activation

doi: 10.3390/ijms25052801

Figure Lengend Snippet: Monoclonal antibodies used in flow cytometry.

Article Snippet: CD8 , APC-Cy7 , OKT8 , 1:20 , Caprico Biotechnologies (Norcross, GA, USA).

Techniques: Bioprocessing, Cytometry, Conjugation Assay

Journal: Communications Biology

Article Title: Affinity enrichment of extracellular vesicles from plasma reveals mRNA changes associated with acute ischemic stroke

doi: 10.1038/s42003-020-01336-y

Figure Lengend Snippet: a Schematic diagram representing the workflow for sample processing and release of enriched EVs from the EV-MAP device’s surface. b Fluorescence images after staining the EVs captured on the EV-MAP device’s surface with an APC-labeled secondary antibody, Left—negative control without anti CD8α mAb; c isotype (IgG2B) control; d florescence images of CD8+ EVs captured from cell media. TEM images of: e USER® enzyme and buffer used for EV release from the EV-MAP device’s surface with no EV infusion; f and g EVs captured and released from the MOLT-3 cell culture media. h NTA results ( n = 3) and TEM images showing the number of EVs released during first ( i ) and second ( j ) USER® enzyme release. k Percentage of EVs released during first and second release with USER® enzyme.

Article Snippet: Following EV isolation, devices were incubated with APC conjugated mouse anti-human CD8α mAb (R&D systems/BioTechne) for 40 min. As controls, the same procedure was carried out for an UV/O 3 -modified device without anti-CD8α mAb, but following EV enrichment.

Techniques: Fluorescence, Staining, Labeling, Negative Control, Cell Culture

Journal: Communications Biology

Article Title: Affinity enrichment of extracellular vesicles from plasma reveals mRNA changes associated with acute ischemic stroke

doi: 10.1038/s42003-020-01336-y

Figure Lengend Snippet: a NTA and b , c TEM images of EVs isolated from clinical sample #4 by PEG precipitation and affinity selected with anti-CD8α mAb uisng the 7-bed EV-MAP. d Gel electrophoresis of TRNA size distributions from EVs isolated via EV-MAP and PEG precipitaion for sample #4. e Heat maps presenting the EV mRNA expression profiles for samlpe #4. f NTA results for selected samples 1, 4, 6, and 8. g mRNA expression profiling for selected genes in clinical samples. h Heat map analysis of clinical samples (marked with numbers) and healthy donors (identified with letters). i Principal component analysis for clinical samples (identified with numbers) and healthy donors (identified with letters). j Process flow chart showing the steps and time required for our EV mRNA expression profiling assay that uses the EV-MAP for EV enrichment and subsequent ddPCR quantification of five genes used for AIS diagnostics. The EV-MAP microfluidic for EV enrichment used the 7-bed device and accepted plasma samples with no pre-processing required (see Fig. ). Following enrichment, the EVs were released from the capture surface enzymatically (see Fig. ), or directly lysed on the enrichment bed followed by solid-phase extraction (SPE) of the resulting total RNA (TRNA). mRNA was reverse transcribed and then subjected to ddPCR. Amounts of TRNA used in RT(+/–) are shown in Supplementary Table . The total processing time of this assay is 220 min (3.7 h), including the time for sampling and pipetting.

Article Snippet: Following EV isolation, devices were incubated with APC conjugated mouse anti-human CD8α mAb (R&D systems/BioTechne) for 40 min. As controls, the same procedure was carried out for an UV/O 3 -modified device without anti-CD8α mAb, but following EV enrichment.

Techniques: Isolation, Nucleic Acid Electrophoresis, Expressing, Sampling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-4 diminishes perforin-mediated and increases Fas ligand-mediated cytotoxicity In vivo.

doi: 10.4049/jimmunol.164.7.3487

Figure Lengend Snippet: FIGURE 1. Kinetics of T cell activation markers on T cells. Mice were sacrificed on days 2, 4, 6, 8, and 10 after infection. A total of 2 3106 spleen cells was stained for CD8a and CD44, LFA-1, and L-selectin (A), or CD4, CD8a, and FasL (B). Data represent percent positive T cells; Uninfected mice (M), vvM2 (f), and vvM2/IL-4 (p). Data are representative of five independent experiments with four mice per group.

Article Snippet: Apoptosis assays and enumeration of M2-specific CD81 T cells For enumeration of RSV M2-specific CD81 T cells using H-2Kd tetramers, 1 3 106 cells were stained with PE-labeled influenza NP or RSV M2 tetramers, a gift from Dr. John Altman (Emory University, Atlanta, GA), and allophycocyanin (APC)-conjugated rat anti-mouse CD8a (clone 53- 6.7; PharMingen), FITC-annexin V (R&D Systems, Minneapolis, MN), and vital dye 7-amino-actinomycin D (7-AAD; PharMingen).

Techniques: Activation Assay, Infection, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-4 diminishes perforin-mediated and increases Fas ligand-mediated cytotoxicity In vivo.

doi: 10.4049/jimmunol.164.7.3487

Figure Lengend Snippet: FIGURE 6. Enumeration of RSV M2-specific CD81 T cells using MHC class I tetramers. A total of 1 3 106 cells was stained with PE- labeled influenza NP or RSV M2 tetramers and APC-labeled-CD8, and 100,000 events were collected for flow cytometric analysis. A, Uninfected 1 RSV M2 tetramer. B, vvM2-infected 1 influenza NP tetramer. C, vvM2- infected 1 RSV M2 tetramer. D, vvM2/IL-4-infected 1 RSV M2 tetramer. Separate experiments were performed using FITC-CD4, Cy-Chrome-CD8, and PE-tetramer and showed that only CD81 cells were tetramer double positive (data not shown). Data are representative of two independent ex- periments (n 5 4 and 5). FIGURE 7. Characterization of CD81 T cell death after infection with rVV constructs. Four-color staining of 106 cells with PE-labeled RSV M2 tetramers, CD8-APC, annexin V-FITC, and vital dye 7-AAD was per- formed. Analysis was performed on CD8-positive cells and gated on M2 tetramer-negative (A) or M2-positive populations (B). Cells stained double positive for annexin V and 7-AAD are dead, and annexin V single-positive cells are apoptotic. Percentage of apoptotic population was calculated excluding dead cells. Data are representative of averages taken from five mice.

Article Snippet: Apoptosis assays and enumeration of M2-specific CD81 T cells For enumeration of RSV M2-specific CD81 T cells using H-2Kd tetramers, 1 3 106 cells were stained with PE-labeled influenza NP or RSV M2 tetramers, a gift from Dr. John Altman (Emory University, Atlanta, GA), and allophycocyanin (APC)-conjugated rat anti-mouse CD8a (clone 53- 6.7; PharMingen), FITC-annexin V (R&D Systems, Minneapolis, MN), and vital dye 7-amino-actinomycin D (7-AAD; PharMingen).

Techniques: Staining, Labeling, Infection, Construct