Article Figures & Data
Figures
- FIG 1
B-1 cells are highly susceptible to FrMLV infection and are required for robust infection in the popliteal lymph nodes (pLNs). (A) Total numbers of GFP+ FrMLV-infected cells (LTR-GFP) in the B-1 (CD19+ IgDlo CD43+), B-2 (CD19+ IgDhi), and CD4+ T (CD3+ CD4+) cell populations within pLNs (n = 4) of wild-type C57BL/6J (B6) mice, 3 dpi after subcutaneous (s.c.) challenge. (B) Analyses of data presented in panel A showing the percentage of FrMLV-infected cells in pLNs (n = 4) at 3 dpi (s.c.) within each indicated cell population. (C) Gating strategy for characterizing the B-2, B-1a, and B-1b cell populations in the pLNs of B6 and bumble (bmb) mice. N.D., not detected in 5 out of 8 lymph nodes analyzed. (D) Number of B-1a, B-1b, B-2, CD4+ T cells, CD11b+ cells, and subcapsular sinus macrophages (SCS Mϕ; CD169+ CD11b+) in pLNs (n = 4 to 8) of uninfected B6 and bmb mice. (E) Mean fluorescence intensity of CD169 staining in SCS Mϕ (CD169+ CD11b+) in pLNs (n = 4 or 6) of uninfected B6 and bmb mice. ns, P > 0.05. (F) Percentages of cells capturing Gag-GFP-labeled FrMLV particles 1 h p.i. (hpi) (s.c.) in pLNs (n = 6) of B6 and bmb mice. ns, P > 0.05. (G) FrMLV-infected cells (glycoGag+) at indicated time points in pLNs (n = 4 to 10) of B6 and bmb mice after s.c. infection. *, P = 0.0159; ****, P < 0.0001. Statistical comparisons were performed using nonparametric Mann-Whitney tests (two-tailed).
- FIG 2
Interleukin-10 (IL-10) does not significantly contribute to the observed reduction of FrMLV infection in bumble (bmb) mice after subcutaneous challenge. (A) Percentage of IL-10+ cells (n = 6) from pLNs of FrMLV-infected B6 and bmb mice (s.c., 5 dpi) after ex vivo stimulation with FrMLV-specific peptide for 44 h and phorbol myristate acetate (PMA)/ionomycin for the last 4 h. (B) IL-10 concentration (pg/ml) was determined by ELISA in the supernatants of ex vivo-cultured cells isolated from pLNs (n = 5 or 6) of FrMLV-infected B6 and bmb mice (s.c., 5 dpi) after stimulation with FrMLV-specific peptide for 24 h. (C) Total numbers of FrMLV-infected (GFP+, LTR-GFP) cells in the pLNs (n = 6) of B6 and IL-10−/− mice (s.c., 5 dpi). ns, P > 0.05. Statistical comparisons were performed using nonparametric Mann-Whitney tests (two-tailed).
- FIG 3
CD8+ T cell activity and neutralizing antibody responses do not contribute to reduced FrMLV infection in bumble mice. (A) FrMLV-infected cells in the pLNs (n = 4) of B6 and bumble (bmb) mice treated with isotype or CD8+ T cell-depleting antibodies (s.c., 5 dpi). (B to D) Percentage of CD8+ T cells that are CD107a+ (surface staining) after stimulation with FrMLV-specific peptide for 17 h (B) or granzyme A+ (C) or granzyme B+ (D) in pLNs (n = 5) of FrMLV-infected B6 and (bmb) mice (s.c., 6 dpi). Cells from pLNs from uninfected (UI) B6 and bmb mice are shown as controls. ns, P > 0.05. (E) FrMLV-neutralizing activity in serially diluted sera from B6 and bmb mice (n = 7 to 9) at indicated time points after FrMLV infection. (F) Neutralizing titers (IC50) in sera from the experiment shown in panel E. ns, P > 0.05. Statistical comparisons were performed using nonparametric Mann-Whitney tests (two-tailed).
- FIG 4
Adoptively transferred wild-type B-1 cells restore FrMLV infection in bumble (bmb) mice. (A) Experimental design for determining the effect of adoptively transferred wild-type B-1 cells on FrMLV infection in bmb mice. Representative flow cytometry plots of cells from a pLN 1 day after adoptive transfer (s.c. hock). The numbers of B-1 cells that homed to the pLN (shown below the DsRed+ gate) when increasing numbers of adoptively transferred B-1 cells were used (top). (B and C) FrMLV-infected cells (GFP+ [B] or glycoGag+ [C]) in the pLNs (n = 4 to 9) of bmb mice adoptively transferred with the indicated numbers of B-1 cells or B-2 cells isolated from B6 or IL-10−/− mice at 3 dpi (B) and 5 dpi (C). Infection levels in the pLNs of B6 and bmb mice determined in parallel are shown as controls. ns, P > 0.05. Statistical comparisons were performed using nonparametric Mann-Whitney tests (two-tailed).
- FIG 5
Cell-intrinsic TLR7-mediated sensing of FrMLV and autocrine type I interferon signaling contributes to susceptibility of B-1 cells. (A and B) Total numbers of FrMLV-infected cells (glycoGag+) (A) or FrMLV-infected B-1 cells (glycoGag+ CD19+ IgDlo CD43+) (B) at 3 and 5 dpi (s.c.) in pLNs (n = 4 to 9) of B6 and Tlr7−/− mice. (C) FrMLV-infected cells in the pLNs (n = 2 to 6) of bumble (bmb) mice adoptively transferred with the indicated numbers of B-1 cells from B6 or Tlr7−/− mice at 5 dpi (s.c.). Infection levels in the pLNs (n = 10) of B6 and bmb mice determined in parallel are shown for statistical comparison. ns, P > 0.05. (D and E) Total numbers of FrMLV-infected cells (glycoGag+) (D) or FrMLV-infected B-1 cells (glycoGag+ CD19+ IgDlo CD43+) (E) at 3 and 5 dpi (s.c.) in pLNs (n = 4 or 10) of B6 and Ifnar1−/− mice. (F) FrMLV-infected cells in the pLNs (n = 7 to 8) of specified mouse strains after reconstitution with (4 × 105) B-1 cells from B6 or Ifnar1−/− mice at 5 dpi (s.c.). Infection levels in the pLNs (n = 10) of B6, bmb, and Ifnar1−/− mice determined in parallel are shown as controls. ns, P > 0.05. Statistical comparisons were performed using nonparametric Mann-Whitney tests (two-tailed).
- FIG 6
TLR7–IFN-I axis promotes B-1 cell activation and FrMLV infection. (A and B) Gating strategy (A) and numbers (B) of CD69+ cells in infected (glycoGag+) and uninfected (glycoGag–) B-1 cell populations (CD19+ IgDlo CD43+) in the pLNs (n = 6) of FrMLV-infected B6 mice (s.c., 5 dpi). (C) Numbers of CD69+ B-1 cells in pLNs (n = 6) of FrMLV-infected B6, Tlr7−/−, and Ifnar1−/− mice (s.c., 5 dpi). Cells from pLNs (n = 2) of uninfected (UI) B6, Tlr7−/−, and Ifnar1−/− mice are shown as controls. Statistical comparisons were performed using nonparametric Mann-Whitney tests (two-tailed).
- FIG 7
B-1 cells promote FrMLV infection of B-2 cells. (A) Temporal analyses of the indicated FrMLV-infected cell types in pLNs (n = 3 or 4) of B6 and bumble (bmb) mice (s.c., 3 and 5 dpi). (B and C) Analyses of indicated FrMLV-infected (GFP+, LTR-GFP for 3 dpi, glycoGag+ for 5 dpi) cell types in the pLNs (n = 3 to 6) of bmb mice with the indicated numbers of adoptively transferred B-1 cells (s.c., 3 dpi). Infection levels in the pLNs of B6 and bmb mice determined in parallel are shown for statistical comparison. ns, P > 0.05. Statistical comparisons were performed using nonparametric Mann-Whitney tests (two-tailed).
- FIG 8
FrMLV-infected cells localize to the medullary region of popliteal lymph nodes. (A) Merged images of immunostained pLN tissue cryosections from a B6 mouse adoptively transferred with GFP+ B-1 cells (green) and infected with FrMLV (5 dpi, s.c.) (glycoGag+, white). B cells, CD4+ T cells, and CD169+ macrophages were identified using antibodies for surface markers B220 (blue), CD4 (blue), and CD169 (red), respectively. Scale bars, 100.00 μm. (B) Merged images of medullary regions containing infected cells (glycoGag+, red, 5 dpi, s.c.) in immunostained pLN tissue cryosections from B6 mice adoptively transferred with GFP+ B-1 cells (green). B cells and CD4+ T cells were identified using antibodies for surface markers B220 (blue) and CD4 (blue), respectively. Scale bars, 50.00 μm. (C) Histograms compiled from 36 or 15 FrMLV-infected (GFP+ glycoGag+) B-1 cells in medullary regions of two pLNs (as shown in panel B) (5 dpi, s.c.), indicating a distribution of the number of infected B or CD4+ T cells located proximally to infected B-1 cells. The proximity was defined as located within a 15-μm distance.
- FIG 9
Scheme showing a hypothetical model of FrMLV infection in B-1 cells and spread to B-2 cells. Lymph-borne FrMLV is captured by CD169+ subcapsular sinus macrophages (SCS Mϕ) in the pLN. Panel 1, B-1 cells acquire viral particles when interacting with FrMLV-laden SCS macrophages (6). Panel 2, TLR7 expressed by B-1 cells senses viral RNA upon viral entry and induces downstream signaling through adaptor protein MyD88, which culminates in the synthesis of IFN-I. IFN-I signaling activates B-1 cells and enhances their susceptibility to FrMLV. Panel 3, Productively infected B-1 cells function as cellular superspreaders for the subsequent expansion of FrMLV in B-2 cell populations.
