HMGB1 and C1q inhibit plasticity of macrophages. DC differentiation induced by GM-CSF and IL-4 was assessed by flow cytometry. Monocytes were treated with C1q and/or HMGB1 for 24 hours (day 0) and then further cultured with GM-CSF and IL-4 for 2 days (day 2). (A) High levels of CD14 and LAIR-1 denote suppression of DC differentiation. Data represent the mean ± SEM of 3 independent experiments. Statistical analysis was performed by one-way ANOVA. ns, not significant. *P < .05; **P < .01; ***P < .001. (B) Transcription of Mer and CD163 was measured by qPCR. Data represent the mean ± SEM of 3 independent experiments in which each condition was tested in triplicate. Statistical analysis was performed by one-way ANOVA. ns, not significant. *P < .05; **P < .01; ***P < .001. (C) Mixed lymphocyte reaction. Monocytes were exposed to HMGB1 (3 μg/mL), C1q (25 μg/mL), or both for 24 hours in X-Vivo 15 serum-free medium, washed, and further incubated for 2 days in X-Vivo 15 medium. Cell Trace Violet–stained allogeneic primary human CD4 T cells and anti-CD3 (1 μg/mL) were added (2:1). T cells alone in the presence of anti-CD3 antibody or T cells with antibodies to CD3/CD28 were negative and positive controls, respectively. After 4 days, the nonadherent cells, which stained with anti-CD4 antibodies, were assessed by flow cytometry. Live CD4⁺ T cells were analyzed. Bar graphs represent the mean ± SEM of percentage having undergone division (left) and division index (right). N = 3. *P < .05; **P < .01; ***P < .001 (one-way ANOVA).