Differential representation of B cell subsets in mixed bone marrow chimera mice due to expression of allelic variants of CD45 (CD45.1/CD45.2)

Abstract

The CD45 congenic marker system is a highly utilized technique to track hematopoietic cells following bone marrow transplantation (BMT), with CD45.1 and CD45.2 being efficiently distinguished by flow cytometry. During the analysis of control mixed BM chimera mice in which lethally irradiated recipients were transplanted with an equal number of BM cells from WT CD45.1 and WT CD45.2 mice, we observed an unequal reconstitution of specific B cell subsets in the bone marrow (BM), lymph node (LN) and spleen. Specifically, in the BM and LN, there was an increase in the percentage of CD45.2 mature B cells. In the spleen, an increase in the percentage of CD45.2 transitional (T) 1 and T2 cells was observed. In contrast, the percentage of splenic CD45.1 marginal zone (MZ) B cells was significantly increased. When we compared the percentage of B cell subsets in unmanipulated WT CD45.1 and WT CD45.2 mice, we found that WT CD45.2 mice had significantly more LN B cells while WT CD45.1 mice exhibited an increase in MZ B cells. These data indicate that the alteration in the ratio of CD45.1 and CD45.2 B cell subsets in mixed chimera mice is a cell-intrinsic effect. Thus whenever the CD45 congenic system is used to track two genetically distinct populations of immune cells WT chimeras must be generated to allow normalization of the experimental data to avoid the reporting of unintentionally skewed data.

Keywords: B cell; Bone marrow transplantation; CD45; Congenic.

Figure 1. In mixed BM chimera mice, CD45.1 and CD45.2 cells contribute differentially in various B cell subsets in the LN and the spleen

Mixed BM chimera mice were constructed by transplanting equally mixed CD45.1 and CD45.2 BM cells (5 × 10⁶ total) into lethally irradiated μMT, WT CD45.1, or WT CD45.2 recipient mice. After a 12–14 week reconstitution, BM (A), LN (B) and splenic (C) B cell subsets were analyzed by flow cytometry. (A) The percentage of CD45.1 and CD45.2 cells in pro-pre, immature, and mature B cells from μMT (left panel), WT CD45.1 (middle panel) and WT CD45.2 (right panel) recipients are shown. (B) Bar graphs represent the percentage of CD45.1 and CD45.2 cells in total LN B cells (B220⁺) from WT CD45.1 (left panel), and WT CD45.2 (right panel) recipients. (C) The percentage of CD45.1 and CD45.2 cells in splenic T1, T2, Fo and MZ B cells from μMT (left panel), WT CD45.1 (middle panel), and WT CD45.2 (right panel) recipient mice are shown. Open and closed bars represent CD45.1 and CD45.2 cells, respectively. Data shown are the mean ± SEM of one representative experiment of two each with five mice per group. *p<0.05, **p<0.01.

Figure 2. CD45.1 mice have a significant increase in the percentage of total LN B cells and a decrease in MZ B cells compared to CD45.2 mice

BM (A), LN (B) and splenic (C) B cell subsets were analyzed from WT CD45.1 (open bar) and WT CD45.2 (closed bar) mice by flow cytometry. The percentage of CD45.1 and CD45.2 BM pro-pre, immature and mature B cells (A; n = 4–5), total LN B cells (B; n = 4–5) and splenic T1, T2, Fo and MZ B cells (C; n = 8–10) of B220-gated cells are shown. Data shown are the mean ± SEM. *p<0.05, ***p<0.001.