Journal: The Journal of Experimental Medicine

Article Title: Pivotal roles of CD8 + T cells restricted by MHC class I–like molecules in autoimmune diseases

doi: 10.1084/jem.20060936

Figure Lengend Snippet: Expression of distinct MHC molecules on splenocytes. (a) Splenocytes from the indicated mouse strains were stained with 28-8-6 and 25-9-17 antibodies. K b−/− D b−/− CIITA −/− and β 2 m −/− CIITA −/− mice did not express MHC class Ia or MHC class II molecules. (b) Expression of Qa-1 b , Qa-2, and H2-M3 on splenocytes as revealed by staining with 6A8.6F10.1A, 1-1-2, and H2-M3.130 antibodies, respectively. Black lines represent isotype control and red lines show expression of class I–like molecules. (c) MHC class II–restricted CD4 + T cells or MHC class Ia–restricted CD8 + T cells cannot survive in K b−/− D b−/− CIITA −/− (TKO) mice. TCR Tg CD4 + T cells (OT-II) or CD8 + T cells (OT-I) were purified, CFSE stained, and adoptively transferred to RAG-2 −/− or K b−/− D b−/− CIITA −/− mice. 1 wk later, spleens of these mice were evaluated for the presence of CFSE-labeled T cells.

Article Snippet: Fluorochrome-conjugated antibodies against H-2K b /D b (28-8-6), I-A b (25-9-17), CD3 (145.2C11), TCRαβ (H-57), CD8α (53-6.7), CD4 (GK1.5), pan-NK marker CD49b (DX5), CD44 (IM7), β7 integrin (M293), CD122 (TM-β1), and Ly6C (AL-21); anti-IgG, anti-BrdU–FITC, biotin-conjugated anti–Qa-1 b (6A8.6F10.1A), and anti–Qa-2 (1-1-2); and purified anti-H2-M3 (H2-M3.130 ) antibodies were purchased from BD Biosciences.

Techniques: Expressing, Staining, Purification, Labeling

Journal: Nature Biomedical Engineering

Article Title: Immune-privileged tissues formed from immunologically cloaked mouse embryonic stem cells survive long term in allogeneic hosts

doi: 10.1038/s41551-023-01133-y

Figure Lengend Snippet: a . Structure of representative piggyBac (PB) or Sleeping Beauty (SB ) plasmids used to express the CDS of eight immunomodulatory genes ( Ccl21, Pdl1, Fasl, Serpinb9, H2-M3, Cd47, Cd200 , and Mfge8) and luciferase in FailSafe B6 mESCs. Gene expression driven by a CAG promoter and linked by an IRES to an eGFP fluorophore, or Puromycin or Neomycin resistance genes. The entire expression cassette is flanked by inverted terminal repeat of piggyBac or Sleeping Beauty transposons. The NCBI Refseq protein ID encoded by each gene CDS is listed in Supplementary Table . b . Method 1) Failsafe B6 mESCs were transfected with a PB plasmid encoding an eGFP-linked Pdl1 , then a clone with high eGFP high expression was isolated. This was followed by co-transfection of PB transposon plasmids encoding Ccl21, Fasl, Serpinb9, H2-M3, Cd47, Cd200, Mfge8 and luciferase and then drug selection with Puromycin. Method 2) FailSafe B6 mESCs were simultaneously transfected with PB plasmids encoding Pdl1, Fasl, Cd47, and Cd200 , followed by drug selection with Puromycin and then a FACS sort on clones with high expression of each factor based on antibody staining and and eGFP for Pdl1 . Bulk sorted cells then transfected with SB transposons encoding Ccl21, Mfge8, SerpbinB9, H2-M3 and luciferase, followed by drug selection with G418. Both methods resulted in a polyclonal pool that were used to establish clonal lines and analyzed by RT-qPCR for transgene expression levels.

Article Snippet: For both mESCs on coverslips and tissue sections, primary antibodies were used against mouse PD-L1 (Novus, NBP1-43262AF488), CD47 (BD Biosciences, 740055), CD200 (BD Biosciences, 565544), H2-M3 (BD Biosciences, 551769), FASL (Novus, NBP1-97519), SERPINB9 (HycultBiotech, HP8035), CCL21 (R&D, AF457) and MFGE8 (Biolegend, 518603).

Techniques: Luciferase, Expressing, Transfection, Plasmid Preparation, Isolation, Cotransfection, Selection, Clone Assay, Staining, Quantitative RT-PCR

Journal:

Article Title: T lymphocyte mediated lysis of mitomycin C treated Tenon's capsule fibroblasts

doi: 10.1136/bjo.2002.007708

Figure Lengend Snippet: (Top) The effect of Fas blocking (M3) antibody on Fas induced Jurkat T cell apoptosis. Jurkat T cells were incubated in 10 mg/ml anti-Fas M3 for 30 minutes before the addition of 100 ng/ml of anti-Fas IgM. Apoptosis was measured with the lactate dehydrogenase release assay. Anti-Fas M3 significantly inhibits anti-Fas IgM induced apoptosis (p<0.01). An isotype control antibody (M33) did not inhibit Fas induced apoptosis. The data shown are representative data from two separate experiments performed in quadruplicate. (Bottom) The effect of Fas blocking M3 antibody on T cell /fibroblast co-culture. Tenon’s fibroblasts were pre-incubated with no antibody, M3 or M33 (10 mg/ml) for 30 minutes then treated with mitomycin C (0.4 mg/ml for 5 minutes). PHA activated T cells were then added (E:T = 20:1). Viable fibroblast number was counted at 48 hours with the modified LDH release assay. M3 had little effect on T cell mediated killing. Values were shown as a percentage of the control fibroblast number. The data shown are representative of three separate experiments performed in quadruplicate.

Article Snippet: Fas receptor blockade was performed using anti-Fas M3 (Immunotech) which binds to the receptor and prevents receptor activation.

Techniques: Blocking Assay, Incubation, Release Assay, Co-Culture Assay, Modification, Lactate Dehydrogenase Assay