Fig 2: The Cdk4/6 inhibitor PD enlarges HSCs, causing their decline in reconstitution potential.(A) Mean volume (fl) of HSCs isolated from mice treated with vehicle (n = 6), Cdk4/6 inhibitor (PD, n = 10), RAP (n = 4), or Cdk4/6 inhibitor + RAP (n = 5) for 85 days (? = difference). Same control as in Fig. 1A. (B) DNA damage in CometChip: percentage tail DNA of HSCs (%) isolated from mice treated with vehicle, Cdk4/6 inhibitor (PD), RAP, or Cdk4/6 inhibitor + RAP (n = 552). (C) Reconstitution assay: CD45.2 mice were treated with vehicle (n; donors = 5, recipients = 8), Cdk4/6 inhibitor (PD, n; donors = 5, recipients = 7), RAP (n; donors = 5, recipients = 8), or Cdk4/6 inhibitor + RAP (n; donors = 5, recipients = 5) for 85 days before their HSCs were isolated for transplantation into lethally irradiated CD45.1 recipient mice. No drug treatment was performed after the reconstitution. Percentage (%) of donor-derived white blood cells in recipient and slope of reconstitution kinetics were determined over time. (D) Experimental strategy to determine the role of cell size for HSC fitness: If size determinates fitness, then similarly sized HSCs are expected to exhibit a similar reconstitution potential, irrespective of whether HSCs were treated with vehicle or Cdk4/6 inhibitor (PD). (E) Reconstitution assay: 600 M- or XL-sized HSCs of CD45.2 donor mice treated with vehicle (n; donors = 6, recipients =7) or Cdk4/6 inhibitor (PD, n; donors = 9, recipients M = 12, XL = 9) were transplanted into lethally irradiated recipient mice (CD45.1), which were not treated with drugs after the reconstitution. Percentage (%) of donor-derived white blood cells in recipients and slope of reconstitution kinetics were determined over time.

Fig 3: CD169 Limits Dissemination of FVC into Erythroblast Niche of the Splenic Red Pulp(A and B) Merged immunostaining images of splenic tissue sections from B6 and CD169-/- mice 5 dpi after s.c. administration (2 × 106 IU) of Ypet expressing FVC (green). B cells, erythroblasts, and metallophilic macrophages were identified using antibodies to surface markers B220 (blue), CD71 (red), and CD169 (pink), respectively. The B cell follicular area (white pulp) and extrafollicular erythroblast rich areas (red pulp) are demarcated by dashed white lines. Magnified images of merged and individual channels of insets are shown on the right.(C) Merged immunostaining images of splenic tissue sections from B6 and CD169-/- mice 5 dpi after r.o. administration of FVC (2,500 SFFU). Metallophilic macrophages lining the white pulp and FVC-infected cells were identified using antibodies to surface marker CD169 (red) and viral protein Glycogag (green).(D) Electron tomography of a spleen section from CD169-/- mice for an experiment as in (A). The image shows a cluster of clonally expanded FVC-infected erythroblasts (labeled E). Insets show details from serial tomographic reconstructions, demonstrating nascent viruses (red arrowheads) budding from the surfaces or invaginations of infected erythroblasts.See also Figures S2 and S3 and Video S1. Scale bars as indicated.

Fig 4: B cell production of IL-27, not cross-presentation, contributes to CD8 T cell vaccine responses(A) B cells or splenocytes enriched for DCs were pre-incubated with protein or peptide for 1 h and then washed thoroughly before combining with proliferation dye-labeled OT1s. OT1 proliferation was determined by proliferation dye dilution 72 h after incubation with the indicated cell type.(B–D) Bone marrow chimeras were generated by reconstituting H-2Kb/Kbm8CD45.1/2 F1 mice with a mixture of µMT-/-CD45.1/2 + WTCD45.2/2, or H-2Kbm8CD45.2/2 bone marrow. Chimeric mice were immunized 10 weeks after reconstitution and analyzed for Ag-specific CD8 T cell responses 7 days later. (B) Total splenic tetramer+ CD8 T cells. (C) Tetramer+ cells as a percentage of CD8 T cells. (D) CD127+ cells as a percentage of tetramer+ cells.(E) The percentage of cells producing IL-27 was tracked over time in B cells (CD19+B220+), CD19+B220- cells, and XCR1+ DCs (B220-CD11chighXCR1+) using IL-27p28-GFP reporter mice after combined-adjuvant immunization.(F–J) Bone marrow chimeras were generated by reconstituting WTCD45.1/2 mice with a mixture of µMT-/-CD45.1/2 + WTCD45.2/2, Ebi3-/-CD45.2/2, or p28-/-CD45.2/2 bone marrow. Chimeric mice were immunized 10 weeks after reconstitution. (F) Tetramer+ cells as a percentage of grafted CD8 T cells in the blood 7 days after vaccination. (G) CD127+ cells as a percentage of tetramer+ cells on day 7. (H) Tetramer+ cells as a percentage of grafted CD8 T cells 3 months after vaccination. (I) Secondary expansion of Ag-specific CD8 T cells in the blood defined as tetramer+ percentage of CD8 (pre-challenge) divided by tetramer+ percentage of CD8 (post-challenge). (J) LM CFUs in the liver of mice 4 days post-challenge.Data in (A) are representative of 3 experiments. Data shown in (B)–(D) and (F)–(J) are means ± SEM from 2 combined experiments. Data shown in (E) are means ± SEM; n = 3 mice per group, from 3 experiments combined. Significance was defined by two-tailed, unpaired Student’s t tests, where *p < 0.05, **p < 0.01, and ***p < 0.001.See also Figure S6.

Fig 5: Fc?RIIB deficiency in MDSCs inhibits tumorigenesis. (A) C57BL/6 (WT) or Fc?RIIB-/- (KO) mice were injected subcutaneously with 106 MC38 cells. Tumor growth was monitored at indicated time points, n = 5. (B) WT or KO mice were sacrificed at day 14 post-xenograft of MC38 cells and tumors were analyzed by FCM for the frequency of CD4+ and CD8+ T cells. Shown data are representative from two independent experiments, n = 5 per group. (C, D) The frequency of IFN-?-producing (C) and GzmB-producing (D) CD8+ T cells in the MC38 tumor from WT and Fc?RIIB-KO mice were determined by FCM. (E) The frequency of tumor-infiltrating MDSCs in WT or KO tumor was assessed 14 days after MC38 tumor inoculation. (F) The expression of Gr-1 (green) in MC38 tumor tissue sections from WT or KO mice was detected by immunofluorescence. Scale bars: 50 µm. (G) gMDSCs and mMDSCs frequency in CD11b+ cells from WT or KO tumor tissues were analyzed by FCM. (H, I) PD-L1 (H) and Arg1 (I) expression in gMDSCs and mMDSCs from WT or KO tumor tissues were analyzed by FCM; n = 4. (J-L), Irradiated WT mice (CD45.1) were i.v. injected with WT (CD45.2) or Fc?RIIb-/- (CD45.2) BM Cells for BM reconstitution assay. Six weeks after BM chimaera reconstitution, mice were injected subcutaneously with 1 × 106 MC38 cells. Tumor size was monitored over time (J), n = 5; The frequency of tumor-infiltrating MDSCs, n = 5 (K), CD8+ T cells and IFN-?-producing CD8+ T cells, n = 5 (L) in tumor from BM chimeric mice was assessed. Data are expressed as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, by Mann-Whitney test.