monoclonal antibody 20 70  (Hycult Biotech)

Journal: Cells

Article Title: Allergen-Induced C5a/C5aR1 Axis Activation in Pulmonary CD11b + cDCs Promotes Pulmonary Tolerance through Downregulation of CD40

doi: 10.3390/cells9020300

Figure Lengend Snippet: In vitro targeting of C5aR1 strongly enhances the potency of C5aR1 + cDCs to promote CD4 + T cell proliferation through upregulation of CD40. WT mice were treated once with a combination of HDM/OVA (100 μg/40 μg) i.t. C5aR1 + and C5aR1 − CD11b + cDCs from WT mice were FACS-purified from lung tissue 24 h after the treatment. C5aR1 + cDCs were either treated with 5 μg/mL of the C5aR1-specific neutralizing mAb 20/70 or an appropriate isotype control. ( A ) cDCs were co-cultured with CFSE-labeled OVA-specific TCR tg CD4 + T cells in the presence of OVA (10 μΜ) and GM-CSF (20 ng/mL) for four days. Left panel; histogram detailing the T proliferation in the different treatment groups; the grey histogram shows the signal obtained from T cell directly after CFSE labeling; right panel: quantification of T cell proliferation in the different treatment groups. Values shown are the mean ± SEM; n = 12. Data were analyzed by ANOVA, followed by Tukey’s posthoc test; * indicates significant differences between the different treatment groups, **** p < 0.0001. ( B , C ) Evaluation of CD40 ( B ) and MHC-II ( C ) expression 18 h after in vitro targeting C5aR1. Left panels: histograms showing the expression of CD40 ( B ) or MHC-II ( C ) in the presence or absence of C5aR1 targeting, grey histogram = FMO control; right panels: quantitative assessment of CD40 or MHC-II expression in the presence or absence of C5aR1 targeting. Shown is the expression of CD40 or MHC-II (as ΔMFI) in the two treatments groups as mean ± SEM; the ΔMFI is defined as the mean fluorescence intensity of the signal normalized to the FMO control, n = 6–9 per group. Data were analyzed with unpaired t-test; * indicates significance between the two treatment groups, ** p < 0.001. ( D ) Assessment of CD40L targeting on T cell proliferation in C5aR1 + cDCs, in which C5aR1 signaling was blocked. Left panel: histogram showing the impact of C5aR1 blockade vs. C5aR1 and CD40/CD40L blockade on T cell proliferation; the grey histogram shows the signal obtained from T cell directly after CFSE labeling. The graph on the right side shows the quantitative evaluation of the different treatment groups. Values shown are the mean ± SEM; n = 15 per group. Data were compared by ANOVA followed by Tukey posthoc test. * indicates significant differences between the treatment groups; **** p < 0.0001. ( E ) Impact of C5aR1 targeting on T cell proliferation when the antigen concentration is limited. WT mice were treated once with HDM (100 μg) i.t. C5aR1 + cDCs from WT mice were FACS-purified from lung tissue 24 h after the treatment. C5aR1 + cDCs were co-cultured with CFSE-labeled OVA-specific TCR tg CD4 + T cells in the presence of different concentrations of OVA 323–339 peptide (5 μg/mL, 500 ng/mL, 50 ng/mL, 20 ng/mL and 5 ng/mL) and GM-CSF (20 ng/mL). Left panel: frequency of proliferated T cells in response to different concentrations of OVA 323−339 peptide as mean ± SEM, n = 3–9. Data were analyzed by ANOVA, followed by Tukey post-hoc test; * indicates significant differences between the different treatment groups, **** p < 0.0001; right panel: impact of C5aR1 targeting on C5aR1 + cDC-driven T cell proliferation in the presence of low OVA 323–339 peptide concentration, n = 6–10. Data were analyzed by unpaired t test; * indicates significant differences between the two treatment groups; *** p < 0.001.

Article Snippet: The anti-mouse C5aR1 (CD88)-blocking mAb 20/70 was purchased from Hycult Biotech (Uden, The Netherlands).

Techniques: In Vitro, Purification, Cell Culture, Labeling, Expressing, Fluorescence, Concentration Assay