Expansion of KSL cells in VV-infected mice is MyD88-dependent. (A): Expansion of KSL cells from WT and MyD88^−/− mice. WT and MyD88^−/− mice were infected for 2 days, and lineage-depleted bone marrow (BM) cells were used to compare the KSL populations. Numbers in dot plots represent the percentages of lineage-depleted cells. The bar graph illustrates the average number of KSL cells in mock- and VV-infected mouse BM from three independent experiments. In each experiment, BM cells were pooled from three mice per group. Asterisks denote a statistically significant decrease in KSL cells of the MyD88^−/− group compared with the WT group (p < .01). (B): The bar graph shows mean ± SD of CD34⁺Flt3⁻ and CD34⁺Flt⁺ KSL cells in BM of VV-infected WT and MyD88^−/− mice 2 days postinfection. Results are from three independent experiments. In each experiment, BM cells were pooled from three mice per group. An asterisk denotes a statistically significant difference in CD34⁺Flt⁻ KSL cells of the MyD88^−/− group compared with the WT group (p < .05). (C): Detection of viral E3L protein expression in KSL cells. BM cells were harvested from VV-infected WT and MyD88^−/− mice 2 days postinfection, and lineage-negative cells were stained with the TW3.2 antibody. (D): Cell cycle analysis. Lineage-depleted BM cells were prepared from WT or MyD88^−/− mice 2 days postinfection and used for cell-cycle analysis as described in Materials and Methods. Data shown are electronically gated Lin⁻Sca-1⁺Kit⁺ subsets. Similar results were obtained from two independent experiments. (E): Differential responses to toll-like receptor ligands. Sorted KSL cells from WT and MyD88^−/− mice were cultured on irradiated feeder cells for 7 days with PG or LPS. KSL expansion was determined by flow cytometry by gating on Lin⁻Sca-1⁺c-Kit⁺ cell population. Abbreviations: FSC, forward scatter; KO, knockout; KSL, c-Kit⁺Sca-1⁺Lin⁻; LPS, lipopolysaccharide; PG, peptidoglycan; PI, propidium iodide; SSC, side scatter; VV, vaccinia virus; WT, wild-type.

VV infection influences DC development. (A): Effect of VV infection on DC differentiation during ex vivo culture. BM cells were prepared from WT and MyD88^−/− mice 2 days postinfection and cultured in the presence of GM-CSF (top) or Flt3-L (bottom) for 5 and 12 days, respectively, as described in the Materials and Methods. Numbers in dot plots represent the percentages of NK1.1⁻CD3⁻CD11c⁺ gated cells. The bar graph illustrates the average number of live cells at the end of the culture determined by trypan blue staining. Data are the average of three independent experiments using four mice per group. Asterisks denote statistically significant differences in the cell number between VV-infected WT and MyD88^−/− mice (p < .01). (B): Effect of in vivo infection of VV on DC subsets. BM cells harvested from WT or MyD88^−/− mice 2 days postinfection were used for DC subset analysis. The cells in the dot plot were gated on NK1.1⁻CD3⁻CD11c⁺ cells and the numbers indicate the percentage of total BM populations. Bar graph shows the average in the percentage of CD11c⁺ cells from three independent experiments by using four mice per group. (C): IFN-β production by BM DC. BM cells were harvested from WT or MyD88^−/− mice 2 days postinfection and cultured for 12 days in the presence of Flt3-L. Supernatants were collected to measure IFN-β by enzyme-linked immunosorbent assay. Data are the average of three independent experiments. ** indicates p < .01. Abbreviations: BM, bone marrow; GM-CSF, granulocyte macrophage-colony-stimulating factor; IFN, interferon; KO, knockout; pDC, plasmacytoid dendritic cells; VV, vaccinia virus; WT, wild-type.

VV infection alters the CMP and CLP compartments. (A): Lineage-depleted bone marrow (BM) cells from mock- or VV-infected mice at the indicated time points after infection were used to assess CMP and CLP populations. The numbers in dot plots are the percentages of lineage-depleted cells. The bar graph shows the cell numbers of CMP and CLP in BM of mock- and VV-infected mice from three independent experiments. In each experiment, BM cells (two femurs and two tibias) were pooled from four mice per group. Asterisks denote a statistically significant difference in CMP and CLP populations of the VV-infected group compared with the mock-infected group (* and ** indicate p < .05 and p < .01, respectively). (B): More differentiated myeloid cells in VV-infected BM. BM cells were isolated from mock- and VV-infected mice at the indicated times followed by staining. Fluorescence-activated cell sorting profiles shown are representative examples from a single experiment, and the number indicates the percentage of CD11^intGr-1^int. Similar results were obtained from a total of three experiments. (C): Peripheral blood cells were prepared from mock- and VV-infected mice at the indicated time points and stained with antibodies to identify myeloid (CD11b⁺, F4/80⁺, or Gr-1⁺) and lymphoid (B220⁺, CD19⁺, CD3⁺, or NK1.1⁺) cells. Numbers shown in the bottom graph are the number of myeloid cells (neutrophils and monocytes) (mean ± SD; n = 4 mice per group) and lymphoid cells (B and T cells) (mean ± SD; n = 4 mice per group). Asterisks denote statistically significant differences in myeloid and lymphoid populations of the VV-infected group compared with the mock-infected group (* and ** indicate p < .05 and p < .01, respectively). (D): Lineage-depleted BM cells were prepared from WT or MyD88^−/− mice after 2 days of infection and analyzed for CMP and CLP compartments. The numbers shown in dot plots are the percentages of CMP and CLP of the lineage-depleted cell population. Dot plots are representative of three independent experiments, and the average cell numbers shown in bar graphs are from three independent experiments. In each experiment, BM cells (two femurs and two tibias) were pooled from three mice per group. ** indicates p < .01. Abbreviations: CLP, common lymphoid progenitor; CMP, common myeloid progenitor; FSC, forward scatter; IL, interleukin; KO, knockout; VV, vaccinia virus; WT, wild-type.

VV infection results in increased KSL cells in bone marrow (BM). (A): Differential expansion of BM cells upon VV infection. BM cells were isolated from M- and VV-infected mice at 1, 2, and 7 D postinfection, and lineage⁺ cells were depleted followed by staining with antibodies recognizing Sca-1 and c-Kit. The numbers in dot plots are the percentages of lin⁻ cells. The bar graph shows the mean ± SD of KSL cells from a total of three independent experiments. In each experiment, BM cells (two femurs and two tibias) were pooled from four mice per group. Asterisks denote a statistically significant increase in KSL cells of the VV-infected group compared with the M-infected group (p < .01). (B): Comparison of different subpopulations of BM cells. Sca-1⁺c-Kit⁺, Sca-1⁺c-Kit⁻, and Sca-¹⁻c-Kit⁺ cells in (A) were used to determine CD34 and Flt3 expression. The frequencies of CD34⁺Flt3⁻ and CD34⁺Flt⁺ cells in these subpopulations in M- and VV-infected mice at indicated time point are shown. (C): Increase in the cell number of CD34⁺Flt3⁻ cells. The bar graph shows the average cell number of CD34⁺Flt3⁻ and CD34⁺Flt⁺ populations shown in (B). Asterisks denote a statistically significant increase in CD34⁺Flt3⁻ KSL cells of VV-infected mice compared with M-infected mice (* and ** indicate p < .05 and p < .01, respectively). (D): Detection of the VV gene product. Polymerase chain reaction was performed with RNA from Lin⁻ BM cells that were prepared from VV-infected mice 24 hours postinfection. Houskeeping gene, HPRT, was used as control for RT-PCR reaction. (E): Determination of cell proliferation. Lin⁻ cells were isolated from BM of M- and VV-infected mice for 1 D. Cells were then cultured in vitro for 1 and 2 D and analyzed as described in Materials and Methods. Abbreviations: D, days; KSL, c-Kit^hiSca-1⁺Lin⁻; M, mock; HPRT, hypoxanthine-guanine phosphoribosyl transferase; VV, vaccinia virus.