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: The EMBO Journal

Article Title: Transcriptional and chromatin profiling of human blood innate lymphoid cell subsets sheds light on HIV ‐1 pathogenesis

doi: 10.15252/embj.2023114153

Figure Lengend Snippet: A CD127 + and CD127 − cells from Lin − CD56 − population after gating on lymphoid, singlet, live, CD45 + cells of PBMCs. Lineage (Lin) markers include antibodies against: CD3, CD4, TCRαβ, TCRγδ, CD19, CD20, CD22, CD34, FcεRIα, CD11c, CD303, CD123, CD1a, and CD14. B Heatmap of differentially expressed genes by RNA‐Seq, sorted Lin − CD56 − CD16 − CD127 + versus Lin − CD56 − CD16 − CD127 − cells, from PBMCs of five donors (log2 fold change > 1, P adj < 0.01 determined by DESeq2, the postsort profiles are shown in Appendix Fig ). C Normalized counts of Lin − CD56 − CD127 + (red) and Lin − CD56 − CD127 − (blue) cell‐related genes from (B) by DESeq2 ( n = 5). D, E Reactome analysis based on enriched transcripts of Lin − CD56 − CD127 + cells (D) or Lin − CD56 − CD127 − cells (E). F Gating of Lin − CD56 hi NK, Lin − CD56 dim NK, Lin − CD56 − NK cells, and Lin − CD56 − CD16 − CD127 + ILCs. G The indicated populations in (F) were detected with isotype controls or antibodies against TBX21, CRTH2, and RORγT. Data information: All data were generated using blood from healthy HIV‐1‐negative donors.

Article Snippet: UCB was processed as previously described and underwent CD3 T‐cell depletion (Brehm et al , ).

Techniques: RNA Sequencing, Generated

Journal: The EMBO Journal

Article Title: Transcriptional and chromatin profiling of human blood innate lymphoid cell subsets sheds light on HIV ‐1 pathogenesis

doi: 10.15252/embj.2023114153

Figure Lengend Snippet:

Article Snippet: UCB was processed as previously described and underwent CD3 T‐cell depletion (Brehm et al , ).

Techniques: Control, Sequencing, Cell Stimulation, Multiplex Assay, Purification, Cell Isolation, Software

Journal:

Article Title: Inflammatory cytokines in small intestinal mucosa of patients with potential coeliac disease

doi: 10.1046/j.1365-2249.2002.01798.x

Figure Lengend Snippet: Monoclonal mouse anti-human antibodies (MoAbs) used in this study

Article Snippet: CD3 , CD3-T cell receptor , 1:200 , Becton-Dickinson, San Jose, CA, USA.

Techniques: