anti il 12p70  (R&D Systems)

Journal: bioRxiv

Article Title: NKG2A-mediated immune modulation of natural killer cells by Staphylococcus aureus

doi: 10.1101/2024.11.29.625999

Figure Lengend Snippet: (A) Representative flow cytometry plots depicting expression levels of NKG2A vs CD57 on CD56 dim NK cells from human PBMCs collected from healthy controls that were cultured in medium alone (Unstim) or pretreated with anti-human IL-12p70 or an isotype control antibody (IgG 1 ) before being stimulated with TSST-1 for 48h. (B) Scatter dot plot showing the frequency of NKG2C − NKG2A + (red filled circles), NKG2C + NKG2A - (yellow filled circles), CD57 − NKG2A + (green filled circles), or CD57 + NKG2A + (blue filled circles) subsets among CD56 dim NK cells from human PBMCs collected from healthy controls that were cultured in medium alone (Unstim) or pretreated with anti-human IL-12p70 or an isotype control antibody (IgG 1 ) before being stimulated with TSST-1 for 48h. Each dot represents one donor. Line indicates mean value. (C) Scatter dot plots depicting geometric mean fluorescence intensity (gMFI) of NKG2A (green filled circles), CD16 (yellow filled circles) or CD56 (blue circles filled circles) expression on CD56 dim NK cells from human PBMCs collected from healthy controls that were cultured in medium alone (Unstim) or pretreated with anti-human IL-12p70 or an isotype control antibody (IgG 1 ) before being stimulated with TSST-1 for 48h. Each dot represents one donor. Line indicates mean value. *p < 0.05, **p < 0.01; One-way ANOVA with Tukey’s post-hoc test.

Article Snippet: After further washing, plates were incubated for 1h at RT with a biotin conjugated anti-IL-12p70 detection antibody (1 μg/ml; Mabtech clone MT704) diluted to 1 μg/ml before the process was repeated using streptavidin conjugated to alkaline phosphatase (Mabtech; 1:1000 dilution of stock protein).

Techniques: Flow Cytometry, Expressing, Cell Culture, Control, Fluorescence

Journal: Cell reports

Article Title: Flt3L enhances clonal diversification and selective expansion of intratumoral CD8 + T cells while differentiating into effector-like cells.

doi: 10.1016/j.celrep.2024.115023

Figure Lengend Snippet: Figure 6. Anti-PD-1 treatment expands cytotoxic asGM1posCD8+ T cells (A) Experimental schematic for anti-PD-1 antibody treatment. Panc02-bearing mice were injected intravenously with an isotype Ctrl or anti-PD-1 antibody on days 6, 9, and 12 before being analyzed on day 15. (B) Flow cytometry plots (left) and percentages (right) of intratumoral CD8+ T cells showing asGM1 expression after anti-PD-1 treatment (n = 4). (C) Numbers of asGM1neg (left) and asGM1pos (right) CD8+ T cells normalized to tumor weight in grams after PD-1 blockade (n = 3). (D) Intratumoral CD8+ T cells (2 3 105) were isolated from Panc02-bearing mice treated intravenously with an isotype Ctrl or anti-PD-1 antibody and re-stimulated with anti-CD3/CD28 antibodies plus IL-2. Shown are flow cytometry plots (left), percentages (center, per each population), and numbers (right) of CD8+ T cell populations co-producing TNF-a and IFN-g (n = 5). (E) Flow cytometry plots (left) and percentages (right) of asGM1neg and asGM1posCD8+ T cells co-expressing GzmB and Perf in response to PD-1 blockade (n = 4). (F) Flow cytometry plots (left) and percentages (right) of asGM1posCD8+ T cells from Panc02-bearing WT or Batf3/ mice after anti-PD-1 treatment (n = 3). (G) Flow cytometry histogram (left) and a quantification (right) of CCR7 expression on cDC1s (n = 4). (H and I) Panc02-bearing mice were co-treated intravenously with an isotype Ctrl or anti-PD-1 antibody and intratumorally with an anti-IL-12p70 antibody on days 6, 9, and 12 before being analyzed on day 15 (n = 4). (H) Experimental schematic (left) and tumor weights (right). (I) Percentages of asGM1posCD8+ T cells. Data are representative of at least two independent experiments and presented as mean ± SEM.

Article Snippet: For tumor volume measurement after blocking IL-12, Flt3L Panc02-bearing mice were treated with an isotype control (LTF-2, BioXCell, 30 mg/mouse) or anti-IL-12p70 (R2-9A5, BioXCell, 30 mg/mouse) antibody intratumorally on days 7, 10, 13, 16 and for flow cytom- etry analysis, anti-IL-12p70 or isotype control antibodies were administrated intratumorally in 20 mL of DPBS on days 5, 7, and 9 after tumor inoculation.

Techniques: Injection, Flow Cytometry, Expressing, Isolation, Cytometry

Journal: Nature Communications

Article Title: IL-12 drives the expression of the inhibitory receptor NKG2A on human tumor-reactive CD8 T cells

doi: 10.1038/s41467-024-54420-w

Figure Lengend Snippet: Sorted naïve CD8 T cells were activated with CD3/CD28 beads for 24 hours, rested for 2 days and restimulated with CD3/CD28 beads for 8 days in the presence of the indicated cytokines with or without TGF-β. a Data from a representative donor and ( b ) summary of NKG2A expression after 8 days of culture are shown ( n = 5). c Expression of NKG2A by CD8 T cells after stimulation of PBMC with SEB in the presence of isotype control, α-IL-12p70 Ab, α-CD40 Ab or both. Data from one representative donor are shown. d Summary of ( c ) (left) and percentage inhibition of NKG2A expression by CD8 T cells in the different culture conditions (right) ( n = 4). e Detection of IL-12p70 in the culture supernatants of PBMCs stimulated for 3 days with SEB in presence or not of α-CD40 Ab (left) and percentage inhibition of IL-12p70 secretion (right) ( n = 3). b , d , e Data are from distinct healthy donors. Horizontal lines indicate the mean ± SEM. NS= non-significant; p -values were determined by one-way ANOVA with Tukey’s post hoc test ( b , d , g ).

Article Snippet: For IL-12p70 and CD40L blocking experiments, anti-Human Il-12p70 (R&D Systems, cat #MAB219-100, 10 μg/ml final), anti-CD40 (BioLegend, cat #313019, clone HB14, 10 μg/ml final) or isotype control mouse IgG1 (BioLegend, cat #400166, clone MOPC21, 10 μg/ml final) were added in the corresponding wells.

Techniques: Expressing, Control, Inhibition