anti mouse cd8 α ab  (Bio X Cell)

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

Article Title: Adeno-Associated Virus Capsid Structure Drives CD4-Dependent CD8 + T Cell Response to Vector Encoded Proteins

doi: 10.4049/jimmunol.0803965

Figure Lengend Snippet: Kinetics of T cell activation following i.m. injection of AAV2/8, and AAV2/rh32.33. C57BL/6 mice were injected with 1011 GC of AAV.nLacZ in the left tibialis anterior. At days 7, 28, and 63 postinjection, mice were sacrificed and lymphocytes harvested from spleen. IFN-γ-producing T cell responses to AAV capsid (A) and nLacZ transgene (B, primary axis) were determined by IFN-γ ELISPOT. At various time points postinjection, lymphocytes were isolated from whole blood and stained with a PE-conjugated H-2Kb-ICPMYARV tetramer together with a FITC-conjugated anti-CD8 Ab to determine the percentage of nLacZ-specific CD8+ T cells in the total CD8+ T cell population (B, secondary axis). Data are mean ± SD for four mice per group. Statistical analysis compared AAV2/8 with AAV2/rh32.33 at day 28. *, p < 0.001, by unpaired Student’s t test.

Article Snippet: Following the stimulation, cells were washed and stained with FITC-conjugated anti-mouse CD8 α (Ly-2) Ab (BD Pharmingen) for 30 min at 4°C.

Techniques: Activation Assay, Injection, Enzyme-linked Immunospot, Isolation, Staining

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

Article Title: Adeno-Associated Virus Capsid Structure Drives CD4-Dependent CD8 + T Cell Response to Vector Encoded Proteins

doi: 10.4049/jimmunol.0803965

Figure Lengend Snippet: Cellular infiltration in skeletal muscle following i.m. injection of AAV2/8 or AAV2/rh32.33 expressing nLacZ. Day 28 postinjection of 1011 GC into the right tibialis anterior of C57BL/6 mice, skeletal muscle was harvested for X-Gal histochemical stain (A) and confocal microscopy (B and C). Sections were stained with DAPI (B), and CD8-TRITC shown in blue, CD4-Cy5 in green, and Foxp3-FITC Abs in red (C). Representative sections are from four mice per group.

Article Snippet: Following the stimulation, cells were washed and stained with FITC-conjugated anti-mouse CD8 α (Ly-2) Ab (BD Pharmingen) for 30 min at 4°C.

Techniques: Injection, Expressing, Staining, Confocal Microscopy

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

Article Title: Adeno-Associated Virus Capsid Structure Drives CD4-Dependent CD8 + T Cell Response to Vector Encoded Proteins

doi: 10.4049/jimmunol.0803965

Figure Lengend Snippet: Impairment of CD4, CD40L, or CD28 ablates the IFN-γ-producing T cell response to AAV2/rh32.33.nLacZ and restores β-gal expression in skeletal muscle. C57BL/6, CD40L−/−, and CD28−/− mice were injected with 1011 GC of vector in combination with either the CD4-depleting Ab GK1.5, CD40L blocking Ab MR-1, or the CD40 agonist FGK45. Mice were sacrificed 28 days postinjection with vector, and lymphocytes were isolated from the spleen. Following stimulation with the AAVrh32.33 capsid library or the nLacZ dominant peptide, T cell responses were determined by IFN-γ ELISPOT (primary axis). Lymphocytes isolated from whole blood were stained using the PE-conjugated H-2Kb-ICPMYARV tetramer together with a FITC-conjugated anti-CD8 Ab to determine the percentage of nLacZ-specific CD8+ T cells in the total CD8+ T cell population (secondary axis). X-Gal histochemical staining was performed on sectioned skeletal muscle. Representative sections from four mice per group are shown. Data are mean ± SD for four mice per group, and represent five independent experiments performed using common controls. Statistical analysis compared AAV2/rh32.33 alone in wild-type C57BL/6 mice with AAV2/rh32.33 given in combination with GK1.5, MR-1, FGK45 in CD40L−/− and CD28−/− mice. *, p < 0.001, by ANOVA Student-Newman-Keuls test.

Article Snippet: Following the stimulation, cells were washed and stained with FITC-conjugated anti-mouse CD8 α (Ly-2) Ab (BD Pharmingen) for 30 min at 4°C.

Techniques: Expressing, Injection, Plasmid Preparation, Blocking Assay, Isolation, Enzyme-linked Immunospot, Staining

Journal: Nature Communications

Article Title: Rational combination of oncolytic vaccinia virus and PD-L1 blockade works synergistically to enhance therapeutic efficacy

doi: 10.1038/ncomms14754

Figure Lengend Snippet: B6 mice were intraperitoneally inoculated with 5 × 10 5 MC38-luc and treated with VV and/or α-PD-L1 as described. Single cells were made from primary tumours collected from tumour-bearing mice at day 5 post first treatment, blocked with α-CD16/32 Ab and then stained with antibodies against CD45, CD8, CD4, PD-1, ICOS, PD-1, CTLA-4, TIM-3, LAG-3, TIGIT and Foxp3 to determine the quantities of CD8 + T cells ( a ), CD8 + T-cell activation ( b – d ), CD8 + T-cell exhaustion ( e – h ), Treg cells ( i ), CD8 + /CD4 + Foxp3 + T cells ( j ) and CD4 + Foxp3 − T cells ( k , l ) in the TME. Of note, the anti-PD-L1 antibody clone 10F.9G2 was used for therapy while clone MHI5 was used for subsequent phenotypic analysis. Data were analysed using Student's t- test (* P <0.05; ** P <0.01; *** P <0.001).

Article Snippet: Anti-mouse PD-L1 Ab (clone 10F.9G2), α-mouse CD8 Ab (clone 53-6.7), α-mouse CD4 Ab (clone GK1.5) and α-mouse IFN-γ Ab (clone XMG1.2) were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Staining, Activation Assay

Journal: Nature Communications

Article Title: Rational combination of oncolytic vaccinia virus and PD-L1 blockade works synergistically to enhance therapeutic efficacy

doi: 10.1038/ncomms14754

Figure Lengend Snippet: ( a ) B6 mice were intraperitoneally inoculated with 5 × 10 5 MC38-luc cancer cells and treated with VV and/or α-PD-L1 as described. Splenic CD8 + T cells (4 × 10 5 ) were isolated from naive and MC38-luc-bearing mice that received different treatments 18 days post tumour cell injection and restimulated with mitomycin C-treated MC38-luc or B16 cancer cells (4 × 10 4 cells each) in the presence of 4000-rad-irradiated CD8-depleted naive B6 splenocytes (2 × 10 6 ) in 200 μl RPMI-1640 medium supplemented with 10% FBS at 37 °C, 5% CO 2 for 2 days. The concentration of IFN-γ in the culture supernatants was tested by ELISA. The statistical analyses were performed with t -test. ( b ) Naive or MC38-luc-bearing B6 mice with dual treatments, which survived for more than 60 days, were s.c. rechallenged with 1 × 10 6 MC38-luc cancer cells. The primary tumour size was measured and presented here. ( c ) In a separate experiment, B6 mice were inoculated with 5 × 10 5 MC38-luc cells i.p. and treated with VV plus α-PD-L1 or PBS at day 5 post tumour inoculation, α-PD-L1 Ab was injected every 2 days for a total of four times. α-CD8 Ab (250 μg per injection), α-CD4 Ab (150 μg per injection) or α-IFN-γ Ab (200 μg per injection) were intraperitoneally injected into mice to deplete CD8+ T cells, CD4+ T cells or neutralize circulating IFN-γ as scheduled in c , and the overall survival was monitored by Kaplan–Meier analysis and analysed using log rank test ( d ).

Article Snippet: Anti-mouse PD-L1 Ab (clone 10F.9G2), α-mouse CD8 Ab (clone 53-6.7), α-mouse CD4 Ab (clone GK1.5) and α-mouse IFN-γ Ab (clone XMG1.2) were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Isolation, Injection, Irradiation, Concentration Assay, Enzyme-linked Immunosorbent Assay