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Comparative Study
. 2019 Jan;99(1):93-106.
doi: 10.1038/s41374-018-0137-1. Epub 2018 Oct 23.

Characterization of immune cell subtypes in three commonly used mouse strains reveals gender and strain-specific variations

Jonathan A Hensel  1 , Vinayak Khattar  1 , Reading Ashton  1 , Selvarangan Ponnazhagan  2
Affiliations
Comparative Study

Characterization of immune cell subtypes in three commonly used mouse strains reveals gender and strain-specific variations

Jonathan A Hensel et al. Lab Invest. 2019 Jan.

Abstract

The lack of consensus on bone marrow (BM) and splenic immune cell profiles in preclinical mouse strains complicates comparative analysis across different studies. Although studies have documented relative distribution of immune cells from peripheral blood in mice, similar studies for BM and spleen from naïve mice are lacking. In an effort to establish strain- and gender-specific benchmarks for distribution of various immune cell subtypes in these organs, we performed immunophenotypic analysis of BM cells and splenocytes from both genders of three commonly used murine strains (C57BL/6NCr, 129/SvHsd, and BALB/cAnNCr). Total neutrophils and splenic macrophages were significantly higher in C57BL/6NCr, whereas total B cells were lower. Within C57BL/6NCr female mice, BM B cells were elevated with respect to the males whereas splenic mDCs and splenic neutrophils were reduced. Within BALB/cAnNCr male mice, BM CD4+ Tregs were elevated with respect to the other strains. Furthermore, in male BALB/cAnNCr mice, NK cells were elevated with respect to the other strains in both BM and spleen. Splenic CD4+ Tregs and splenic CD8+ T cells were reduced in male BALB/c mice in comparison to female mice. Bone marrow CD4+ T cells and mDCs were significantly increased in 129/SvHsd whereas splenic CD8+ T cells were reduced. In general, males exhibited higher immature myeloid cells, macrophages, and NK cells. To our knowledge, this study provides a first attempt to systematically establish organ-specific benchmarks on immune cells in studies involving these mouse strains.

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Figures

Figure 1:
Figure 1:. Immune cell distribution of T cells in C57BL/6NCr, BALB/cAnNCr and 129/SvHsd male and female mice.
Cell suspensions from BM and spleen were isolated from 8-week old mice. The cells were stained with CD3 and CD4 antibodies for CD4+ T cells, CD3 and CD8 antibodies for CD8+ T cells, and CD3, CD4, CD25 and FoxP3 antibodies for T-regs. The cells were then subjected to flow cytometry to determine cell-type percentages. (A) CD4+ T cells in BM, (n≥8). (B) CD4+ T cells in the spleen, (n≥8). (C) CD8+ T cells in BM, (n≥5). (D) CD8+ T in spleen, (n≥6). (E) CD4+ T-regs in BM, (n≥8). (F) CD4+ T-regs in spleen, (n≥7). Results, shown as scatter plots, depict average cell percentages (percent of live cells). Error bars denote SEM. Each dot represents the value from a single mouse. (♂) and (♀) represent male and female mice, respectively.*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p≤0.0001.
Figure 2:
Figure 2:. Immune cell distribution of B cells and NK cells in C57BL/6NCr, BALB/cAnNCr and 129/SvHsd for male and female mice.
Cell suspensions from BM and spleen were isolated from 8-week old mice. The cells were stained with B220 and CD19 antibodies for B cells, and CD3 and NKp46 antibodies for NK cells. The cells were then subjected to flow cytometry to determine cell-type percentages. (A) B cells in BM, (n≥10). (B) B cells in the spleen, (n ≥11). (C) NK cells in BM, (n≥9). (D) NK cells in the spleen, (n≥7). Results are shown as scatter plots depicting average cell percentages (percent of live cells). Error bars denote SEM. Each dot represents the value from a single mouse. (♂) and (♀) represent male and female mice respectively. **p ≤ 0.01, ***p ≤ 0.001, ****p≤0.0001.
Figure 3:
Figure 3:. Immune cell distribution of immature myeloid cells (iMCs), macrophages and myeloid dendritic cells (mDCs) in C57BL/6NCr, BALB/cAnNCr and 129/SvHsd for male and female mice.
Cell suspensions from BM and spleen were isolated from 8-week old mice. The cells were stained with CD11b, F4/80 and CD68 antibodies for macrophages, CD11b and Gr-1 antibodies for iMCs, and CD11b and CD11c antibodies for mDCs. Ly6B was used as negative marker for iMCs. The cells were then subjected to flow cytometry to determine cell-type percentages. (A) iMCs in BM, (n≥7). (B) iMCs in the spleen, (n≥7). (C) Macrophages in BM, (n≥ 6). (D) Macrophages in spleen, (n≥9) (E) mDCs in BM, (n ≥ 10). (F) mDCs in the spleen, (n ≥ 13). Results are shown as scatter plots depicting average cell percentages (percent of live cells). Error bars denote SEM. Each dot represents the value from a single mouse. (♂) and (♀) represent male and female mice, respectively. **p ≤ 0.01, ***p ≤ 0.001, ****p≤0.0001.
Figure 4:
Figure 4:. Immune cell distribution of plasmacytoid dendritic cells in C57BL/6NCr, BALB/cAnNCr and 129/SvHsd male and female mice.
Cell suspensions from BM and spleen were isolated from 8-week old mice. The cells were stained with CD11c, B220 and Siglec H antibodies. CD11b antibody was included as a negative marker for pDCs. The cells were then subjected to flow cytometry to determine cell-type percentages. (A) pDCs in BM, (n≥11). (B) pDCs in the spleen, (n ≥9). Results are shown as scatter plots depicting average cell percentages (percent of live cells). Error bars denote SEM. Each dot represents the value from a single mouse. (♂) and (♀) represent male and female mice respectively.*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p≤0.0001.
Figure 5:
Figure 5:. Immune cell distribution of neutrophils in C57BL/6NCr, BALB/cAnNCr and 129/SvHsd for male and female mice.
Cell suspensions from BM and spleen were isolated from 8-week old mice. The cells were stained with CD11b, Gr-1 and Ly6b antibody. F4/80 antibody was included as a negative marker for neutrophils. The cells were then subjected to flow cytometry to determine cell-type percentages. (A) Neutrophils in BM, (n≥7). (B) Neutrophils in spleen, (n ≥7). Results are shown as scatter plots depicting average cell percentages (percent of live cells). Error bars denote SEM. Each dot represents the value from a single mouse. (♂) and (♀) represent male and female mice respectively.*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p≤0.0001.
Figure 6:
Figure 6:. Strain and gender-specific immune cell distribution of critical cell types in three mouse strains commonly used in pre-clinical research.
(A) Pie charts representing the frequency of CD4+ and CD8+ T cells, Tregs, B cells, NK cells, iMCs, pDCs, mDCs, macrophages, and neutrophils in BM cells for C57BL/6NCr, BALB/cAnNCr, and 129/SvHsd mice. The upper three panels represent distribution in female mice and lower panels depict similar distribution in male mice. (B) Strain and gender-specific immune cell distribution for CD4+ and CD8+ T cells, Tregs, B cells, NK cells, iMCs, pDCs, mDCs, macrophages, and neutrophils in spleens of C57BL/6NCr, BALB/cAnNCr, and 129/SvHsd mice. The upper and lower three panels represent distribution in female and male mice, respectively.
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