Analysis of Splenic Hematopoietic Cells in MCMV Infected Mice

For all analyses, mice were infected with 3.2 × 105 pfu MCMV-Smith or MCMV-GFP.
(A) Spleens from mock and MCMV-infected mice were analyzed by flow cytometry 48 h and 72 h post-infection. The changes in the granulocyte (gran,Gr-1hi F4/80−), macrophage (macro, Gr-1lo F4/80hi CD11clo) and DC (DC, CD11chi F4/80lo) populations were analyzed. Infection with either MCMV-Smith or MCMV-GFP induced the recruitment of granulocytes, the loss and/or alteration in the cell surface phenotype of macrophages (F4/80 downregulation and CD11b upregulation, see also (B)), and loss/alteration of DC. Also note the increase of autofluorescent cells (moving along the diagonal of the dot plot) in MCMV-infected mice.
(B) The expression of CD11b on macrophage (Gr-1lo F4/80hi CD11clo) and “MCMV-macrophage” (Gr-1lo F4/80int CD11clo) populations was analyzed in mock and MCMV-infected mice at 48 h post-infection (similar results were also seen at 72 h, unpublished data). The dot plots show that the expression of F4/80 decreased on the cell population presumed to be splenic macrophages in MCMV-infected mice (i.e., “MCMV-macrophages”) as reported previously [29], while the expression of GR-1 in these cells remained the same. However, the altered expression of CD11b in macrophages from these three experimental groups also raised the possibility that this is a distinct cell population only seen in the spleen of MCMV-infected mice, potentially recruited from the blood or bone marrow. Mock (red), MCMV-Smith (blue), and MCMV-GFP (green). (C) The percentage of GFP-expressing cells was determined in individual cell populations from spleens of MCMV-GFP–expressing mice (a summary of data is presented in Table 1). For this analysis, GFP-expressing cells were defined as cells that displayed higher levels of fluorescence in the FL-1 channel when compared with mice infected with MCMV-Smith virus (not mock-infected mice). The increase in the autofluoresence occurred in DC, granulocyte, and NK cell populations following infection with MCMV-Smith. DC and granulocytes were phenotypically defined as in (A). For analysis of macrophages, FL-1 fluorescence displayed in histograms was analyzed by gating on the macrophages (Gr-1lo F4/80hi CD11clo) in mock-infected mice and compared with the “MCMV-macrophages” (Gr-1lo F4/80int CD11clo). GFP expression in NK cells (DX5+CD3−), and lymphocytes (72 h only) was also analyzed. The higher percentage of cells expressing GFP as fraction of the total splenocytes observed at 72 h after infection (9.9%) compared with 48h (1.6%) is considered in the context of the increased lymphocyte death at this time point (shown in Figure 4B). The high level of FL-1 autofluorescence in the DC from mice infected with MCMV-Smith at both 48 h and 72 h post-infection may be due to activation and/or apoptosis of these cells, and a change in the “tightness” of this gated population is evident in MCMV-infected mice. Virtually all splenic DC matured (defined by upregulation of CD80, CD86, CD40, and MHC II) upon infection with MCMV at this dose by 16 h post-infection (unpublished data). Granulocytes expressed higher levels of GFP at 72 h compared with 48 h after infection, suggesting that MCMV-GFP replicates in these cells. However, increased phagocytosis of GFP protein or GFP-expressing cells by granulocytes cannot be excluded. Data shown is from splenocytes pooled from two individually infected mice, and is representative of the results seen in three independent experiments.
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