Comparative analyses of B cell populations in trout kidney and mouse bone marrow: establishing "B cell signatures"

Abstract

This study aimed to identify the frequency and distribution of developing B cell populations in the kidney of the rainbow trout, using four molecular B cell markers that are highly conserved between species, including two transcription factors, Pax5 and EBF1, recombination-activating gene RAG1, and the immunoglobulin heavy chain mu. Three distinct B cell stages were defined: early developing B cells (CLP, pro-B, and early pre-B cells), late developing B cell (late pre-B, immature B, and mature B cells), and IgM-secreting cells. Developmental stage-specific, combinatorial expression of Pax5, EBF1, RAG1 and immunoglobulin mu was determined in trout anterior kidney cells by flow cytometry. Trout staining patterns were compared to a well-defined primary immune tissue, mouse bone marrow, and using mouse surface markers B220 and CD43. A remarkable level of similarity was uncovered between the primary immune tissues of both species. Subsequent analysis of the entire trout kidney, divided into five contiguous segments K1-K5, revealed a complex pattern of early developing, late developing, and IgM-secreting B cells. Patterns in anterior kidney segment K1 were most similar to those of mouse bone marrow, while the most posterior part of the kidney, K5, had many IgM-secreting cells, but lacked early developing B cells. A potential second B lymphopoiesis site was uncovered in segment K4 of the kidney. The B cell patterns, or "B cell signatures" described here provide information on the relative abundance of distinct developing B cell populations in the trout kidney, and can be used in future studies on B cell development in other vertebrate species.

Figure 1. Expression levels of trout RAG-1 and EBF proteins using western blot analysis

For RAG-1 analysis,1 × 10⁶ cells per sample were loaded. For EBF analysis, 100μg per sample of protein lysate was loaded. Immune cells were freshly isolated from trout blood (PBL), spleen (SPL), and anterior kidney (K1). Molecular weight markers are shown on the right.

Figure 2. One-color flow cytometric analysis of trout anterior kidney and mouse bone marrow

A. Dot plots indicating the major cell populations in each tissue, and the gated lymphoid population based on size (FSC) and complexity (SSC). B and C. Contour graphs showing relationship between cell size (FSC) and expression of molecular B cell markers immunoglobulin mu (HC mu), Pax5, EBF, or RAG-1. A control antibody, an Alexa555-conjugated goat anti-rabbit IgG (C555), is shown in the right panel for each species. Percentages are mean values (see Table II). Arrows indicate two populations based on cell size: low FSC and high FSC. B. Trout K1 contour graphs (N=5). C. Mouse BM contour graphs (N=5).

Figure 3. Two-color flow cytometric analyses of mouse bone marrow cells using known mouse B cell markers in combination with molecular B cell markers

A. Contour graphs, using mouse cell surface marker B220 in combination with EBF, RAG, and Pax5. B. Contour graphs, using mouse cell surface marker CD43 in combination with HCmu, CD11b, and Pax5. Numbers are mean percent gated cells with SE in parentheses (N=3).

Figure 4. Qualitative comparison of mouse and trout expression patterns by two-color flow cytometry

Shown are contour graphs using combinations of two molecular B cell markers: EBF1 and RAG, HCmu and Pax5, and EBF1 and Pax5. The relevant populations are boxed and labeled. Arrow indicates a Pax5+/mu– population of cells (see text). Control antibodies C555 and C647 are shown on the right. A. Contours using mouse BM cells, B. Contours using trout K1 cells.

Figure 5. Frequency and distribution of late developing and IgM-secreting plasma cells in the trout kidney

Pax5 and HCmu antibodies were used in two-color flow cytometry. A. Contour graph of K1 to illustrate the relevant cell populations. Right graphs (B and C): Frequency of cells (as percent of total lymphoid cells) on the Y-axis; distribution in kidney segments K1–K5. Means and SE are shown. B. IgM+ plasma cells (N=5). C. Late developing B cells (N=5).

Figure 6. Frequency and distribution of early developing B cells in the trout kidney

Expression patterns of EBF1 or RAG1 expressing cells, using flow cytometry. A. Using EBF and RAG1 as markers. Percent EBF+/RAG1+ cells in each segment is shown on the Y-axis; means and SE are indicated (N=3). B. Using RAG1 and HCmu as markers. Distribution of RAG1+/HCmu– cells. Means and SE as shown (N=4).

Figure 7

Model illustrating the predicted distribution of B cell populations in the trout kidney.