mouse cd3 t cell column enrichment kit  (R&D Systems)

Journal: Gene Therapy

Article Title: Immunosuppression overcomes insulin- and vector-specific immune responses that limit efficacy of AAV2/8-mediated insulin gene therapy in NOD mice

doi: 10.1038/s41434-018-0052-5

Figure Lengend Snippet: AAV2/8-insulin-based therapy elicits a cellular immune response against the vector and the transgene in NOD diabetic mice. Livers of NOD mice treated with AAV2/8-HLP-hINSco (indicated with AAV2/8) show signs of T cell infiltration observed in pre-diabetic NOD mouse pancreata. a Pancreas sections from a 8-week-old control NOD female pancreas ( a , top panel), a 30-day AAV2/8-insulin-treated C57BL/6 ( a , middle panel) and a 30-day AAV2/8-insulin-treated NOD ( a , bottom panel) were stained for insulin (red) and glucagon (green), and CD3 (yellow) to show β-cell destruction in treated mice. b Livers from AAV2/8-HLP-hINSco -injected C57BL/6 ( b , left) and NOD ( b , right) mice were stained for insulin and CD3 to test for T cell infiltration. c Livers from AAV2/8-HLP-hINSco-injected NOD mice were stained for insulin and CD8. Blue shows nuclear DAPI staining. Scale bar 50 μm. d, e Ex vivo stimulated splenocytes from AAV2/8-HLP-hINSco -treated NOD mice and C57BL/6 controls for IFN-γ ELISPOT assay. Cells from NOD and C57BL/6 mice were harvested after 30 days ( d ) or more than 200 days ( e ) from the day of the injection of the AAV2/8-insulin vector. Cells were stimulated in vitro for 40 h with CD8 + immunodominant peptides (either InsB 15–23 peptide for NOD or insulin K b -restricted epitope A 12–21 containing peptide for C57BL/6) or the AAV8 capsid-specific CD8 + T cell peptide NSLANPGIA and the number of resulting IFN-γ spots was counted with an ELISPOT reader. f Levels of anti-inflammatory cytokines in NOD mice compared to C57BL/6. Splenocytes from C57BL/6 and NOD mice were harvested at the end of a 30-day treatment with AAV2/8-HLP-hINSco 5 × 10 9 vg and stimulated with PMA/Iono for 48 h. Supernatants were then collected and tested for IL-10 production. g Mouse anti-AAV8 ELISA performed on blood plasma of diabetic, healthy and 5 × 10 9 vg AAV2/8-HLP-hINSco-treated NOD and C57BL/6 mice. Treated mice were tested for anti-AAV8 antibodies 30 days after the beginning of the therapy. The absorbances at 405 nm correlate with the concentration of the antibody. *( p ≤ 0.05), **( p ≤ 0.01), ***( p ≤ 0.001), unpaired Student’s t -test. Data shown are expressed as mean ± SE and are representative of two independent experiments

Article Snippet: T cells were isolated with the Mouse CD3 + T cell column enrichment kit (R&D) and plated at the concentration of 3 × 10 5 cells/ 50 μl/well, and analysed using the Mitostress kit (Agilent technologies) according to the manufacturers’ instructions.

Techniques: Plasmid Preparation, Staining, Injection, Ex Vivo, Enzyme-linked Immunospot, In Vitro, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: PLoS ONE

Article Title: Interferon Regulator Factor 8 (IRF8) Limits Ocular Pathology during HSV-1 Infection by Restraining the Activation and Expansion of CD8 + T Cells

doi: 10.1371/journal.pone.0155420

Figure Lengend Snippet: (A) IRF8 fl/fl mice were cross-bred with CD4-Cre mice to generate mice with deletion of IRF8 in the CD4 and CD8 compartments (IRF8KO) and the IRF8 fl/fl /Cre alleles were identified by PCR analysis of mouse tail genomic DNA. Presence of the 314bp band indicates Irf8 -floxed DNA, while the 214bp band is consistent with size of the endogenous Irf8 gene sequence in the wild-type mouse genome. (B, C) Naïve CD8 + T cells isolated from the spleen were activated with anti-CD3/CD28 for 3 days and analyzed for IRF8 expression by (B) RT-PCR or (C) western blotting. (D) The TCR-activated WT or IRF8KO CD8 + T cells were also analyzed by the thymidine incorporation assay. (E) Naïve T cells isolated from spleen were analyzed by FACS. Numbers in quadrants indicate percentages of T cells expressing the cell surface markers, CD44 or CD62L. Data represent at least 3 independent experiments.

Article Snippet: The T-lymphocytes were isolated by negative selection by using a mouse CD3 T cell enrichment column kit (R & D Systems).

Techniques: Sequencing, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Thymidine Incorporation Assay

Journal: PLoS ONE

Article Title: Interferon Regulator Factor 8 (IRF8) Limits Ocular Pathology during HSV-1 Infection by Restraining the Activation and Expansion of CD8 + T Cells

doi: 10.1371/journal.pone.0155420

Figure Lengend Snippet: (A) WT mice were challenged with HSV-1 by corneal scarification and we titrated the gB-tetramer to analyze and characterize the virus-specific CD8 + T cell responses in the spleen. (B-D) IRF8KO and WT mice were infected with HSV-1 by corneal scarification and cells isolated from the (B) spleen, or (C) PBMC (C, D) LN on day 8 p.i were analyzed by FACS using the gB-tetramer. The cells were gated on CD3/CD8 and numbers in quadrants indicate percentages of CD8 + T cells expressing CD44 and CD8 + gB-tetramer + T cells expressing IFN-γ. (E) IFN-γ expression by CD8 + T cells was analyzed by real-time qPCR. Data represent at least 3 independent experiments.

Article Snippet: The T-lymphocytes were isolated by negative selection by using a mouse CD3 T cell enrichment column kit (R & D Systems).

Techniques: Virus, Infection, Isolation, Expressing