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Fig. 1 | Journal of Neuroinflammation

Fig. 1

From: High levels of endothelial ICAM-1 prohibit natalizumab mediated abrogation of CD4+ T cell arrest on the inflamed BBB under flow in vitro

Fig. 1

Inhibition of α4- and β2-integrins is required to inhibit T cell arrest on cytokine stimulated brain-like endothelial cells under flow. A Schematic representation of the Fab arm exchange between natalizumab (NTZ) and another human IgG4 antibody, resulting in a bi-specific IgG4 antibody with only one Fab arm specific for α4-integrins. B Schematic representation of the different NTZ constructs used in the present study. Natalizumab (NTZ) is the humanized anti-α4-integrin IgG4 antibody used in the clinic. The bivalent and monovalent NTZ constructs (bNTZ, 150 kDa; mNTZ, 100 kDa) contain the variable domains of NTZ but have instead a fixed hinge region and a hybrid Fc region formed by an aglycosyl IgG4P CH2 domain and an IgG1 CH3 domain. Point mutations (different from NTZ) within the hinge and CH1 domain are indicated by a yellow and blue star, respectively. Number of arrested human CD4+ Th1 (C) and Th1* (E) cells per field of view (FOV) on 16–24 h pro-inflammatory cytokine (76 IU/mL TNFα + 20 IU/mL IFNγ) stimulated brain-like endothelial cells (BLEC) under flow conditions. Th1 and Th1* cells were pre-incubated with equimolar concentrations of the different NTZ constructs (30 μg/mL bNTZ or 20 μg/mL mNTZ,) and/or with 1 μg/mL of a function-blocking anti-β2-integrin antibody. An isotype control antibody was used as internal control (30 μg/mL Ctrl). Quantification of the post-arrest dynamic behavior of human Th1 (D) and Th1* cells (F) on 16–24 h pro-inflammatory cytokine (76 IU/mL TNFα + 20 IU/mL IFNγ) stimulated BLEC under physiological flow conditions during 30 min of recording. Behavioral categories were subdivided in probing, crawling, stationary (not polarized), and detached and are shown as fraction of all shear-resistant arrested T cells (100%). Within the categories probing and crawling we additionally distinguished the categories with and without subsequent diapedesis. Post-arrest behavior of Th1 and Th1* cells treated with combined bNTZ and anti-β2-integrin antibody could not be analyzed due to the low numbers of arrested T cells. C–F Each figure shows the mean ± SEM of 3 independent experiments. Statistical analysis: one-way ANOVA followed by Tukey’s multiple-comparison test (p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = ****)

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