Anti-Human Integrin alpha 4 beta 7 Recombinant Antibody (Etrolizumab)

(A) Upper panels: Flow cytometry of peripheral blood mononuclear cells (PBMCs) from patients with Crohn's disease (CD), ulcerative colitis (UC), and control donors (CON) treated with etrolizumab-s for 24 h at 4 and 37°C. Left: Representative flow cytometry. The frequency of 9D8+ staining on CD4+ T cells is indicated. Right: Quantitative flow cytometry showing surface expression of 9D8 at 37°C relative to expression at 4°C (n = 5 per group). Lower panels: Representative (left) and quantitative flow cytometry (right) of cells cultured at 4 and 37°C without etrolizumab-s treatment. (B) Flow cytometry of PBMCs from CD, UC, and CON donors treated with AF-647-labeled etrolizumab-s. Left: Representative flow cytometry. The frequency of etrolizumab-s+9D8- cells among CD4+ T cells is indicated. Right: Quantitative flow cytometry showing the proportion of these cells at 4°C in relation to 37°C (n = 5 per group). (C) Quantitative flow cytometry of PBMCs treated with AF647-labeled etrolizumab-s at 4 or 37°C and subsequently subjected to acid wash (left) or not (right). n = 5 per group; data are normalized to expression at 37°C. ∗∗∗p < 0.001; ns – not significant.

Lichnog, C., Klabunde, S., Becker, E., Fuh, F., Tripal, P., Atreya, R.,... & Chung, S. (2019). Cellular mechanisms of etrolizumab treatment in inflammatory bowel disease. Frontiers in pharmacology, 10, 39.

(A) Left: Representative images showing localization of the fluorescence signal of AF647-labeled etrolizumab-s after treatment of cells from CD, UC, and CON donors at 4 or 37°C for 24 h. Scale bar: 3 μm. Right: Quantification of mean fluorescence intensity (MFI) of AF647 signal in the cytosol relative to the membrane at 4 and 37°C; n = 5 per group. ∗∗∗p < 0.001. (B) Assessment of etrolizumab internalization with ImageStream®. Representative flow cytometry and microscopy results after cell incubation with AF488-labeled etrolizumab at 4°C (upper row) or 37°C (lower row) and without (left panels) or with (right panels) quench procedure. BF, bright field. Data are representative for two independent experiments with a total of eight samples. (C) Representative images showing localization of AF647 fluorescence signal of etrolizumab-s and AF488 fluorescence signal of EEA (left) and LAMP-1 (right) after cell treatment with etrolizumab-s at 4 or 37°C for 24 h. Scale bar = 3 μm. Images are representative for three independent experiments.

Lichnog, C., Klabunde, S., Becker, E., Fuh, F., Tripal, P., Atreya, R.,... & Chung, S. (2019). Cellular mechanisms of etrolizumab treatment in inflammatory bowel disease. Frontiers in pharmacology, 10, 39.

(A) Representative STED microscopy images showing localization and distribution of β7 integrin on the surface of cells from CD, UC, and CON donors. Additionally, a negative control (NEG) without primary antibody staining is shown. Images are representative for seven independent experiments. (B) Representative images showing etrolizumab-s localization on/in cells incubated with etrolizumab-s at 4°C or cells incubated with etrolizumab-s at 37°C and additionally treated with or without Triton-X. Additionally, a negative control without primary antibody staining is shown. Images are representative for five independent experiments.

Lichnog, C., Klabunde, S., Becker, E., Fuh, F., Tripal, P., Atreya, R.,... & Chung, S. (2019). Cellular mechanisms of etrolizumab treatment in inflammatory bowel disease. Frontiers in pharmacology, 10, 39.

(A) Representative flow cytometry plot showing expression of α4β7 integrin after treatment with etrolizumab at 4 and 37°C. Percentages indicate α4+β7+ and total α4+ cells. (B) Upper panels: Representative flow cytometry plots showing expression of αE integrin after treatment with etrolizumab at 4 and 37°C. Lower panel: Quantification of β7 mean fluorescence intensity (MFI) of β7+ cells (n = 5). ∗∗p < 0.01.

Lichnog, C., Klabunde, S., Becker, E., Fuh, F., Tripal, P., Atreya, R.,... & Chung, S. (2019). Cellular mechanisms of etrolizumab treatment in inflammatory bowel disease. Frontiers in pharmacology, 10, 39.