CCR2 deficiency disrupts the peripheral differentiation of B cells
To investigate whether the lack of CCR2 influences B cell development and differentiation, we first examined B cell subsets using FCM. The CCR2 KO B cells extracted from BM were incubated with anti-BP-1 and -CD24 Abs to distinguish B cell precursors (Fig. 1A-B ). Our results suggest that CCR2 deficiency caused little to no alterations to the development of B cell in BM since the proportion and absolute cell number of precursor B cell subsets in both WT and CCR2 KO mice were similar (Fig. 1C ). However, this was not the case for the peripheral B cell differentiation process. By testing peripheral B cell subsets (Fig. 1D-F ), we discovered that the proportion and cell number of follicular (FO) B cells (B220+IgMlowIgDhigh) as well as the cell number of transitional type-2 (T2) (B220+IgMhighIgDhigh) B cells from the CCR2 KO mice were significantly diminished compared to those observed in WT mice (Fig. 1G-H ). Nevertheless, both the ratio and cell number of transitional type-1 (T1) (B220+IgMlowIgDlow), marginal zone (MZ) (B220+CD23lowCD21high), and germinal center (GC) (B220+GL-7highCD95high) B cells in WT and CCR2 KO mice were comparable (Fig. 1I-K ). Furthermore, no differences were observed for B1a (CD19+IgD-IgM+CD5-CD11b+) and B1b (CD19+IgD-IgM+CD5+CD11b+) B cells (Fig. 1L ) when comparing WT and KO mice (Fig. 1M-N ). To rule out the potential effect of other immune cells on the abnormally differentiated B cells observed in CCR2 KO mice, BM chimeras were generated using irradiated C57BL/6J (CD45.1+) recipients through tail vein injection of BM cells extracted from WT and CCR2 KO (CD45.2+) mice. Thus, we examine the inherent reduction of FO B cells in CCR2 deficient mice (Fig. S1A-H ). Since the kidneys are most susceptible to develop systemic autoimmune disease, we analyzed the glomerulus of CCR2 KO mice and observed a significant increase in IgG immune complex deposition. Additionally, CCR2 KO mice showed increased anti-dsDNA Ab titers (Fig. 1O-P ). While examining other organs, we observed that CCR2 KO mice exhibited more severe lung lymphocyte infiltrations compared to that of WT mice (Fig. 1Q, Fig. S2A ). The transcription factor T-bet expressed by B cell has been previously associated with autoimmunity,25 and as expected, CCR2 KO B cells exhibited higher expression levels of T-bet than those of the WT B cells (Fig. 1R ). Collectively, these findings suggest that CCR2 plays an important role in the integrity maintenance of peripheral B cell differentiation and the regulation the peripheral autoimmunity.