To distinguish these possibilities, we generated chimeras in which CD45

To distinguish these possibilities, we generated chimeras in which CD45.2+Batf3+/+or CD45.2+Batf3-/-bone marrow (BM) was transplanted into lethally irradiated CD45.1+CD45.2+recipients (Fig. in immune responses. Evidence has suggested that CD8+cDCs are important for cross-presentation during infections, but is based on ex vivo analysis (4-6) or in vitro antigen loading (7). Evidence both for and against a role for cross-presentation in responses against tumors has been reported (8-10). Attempts have been made to study the in vivo role of dendritic cells by selective depletion. Diphtheria toxin treatment can deplete all CD11chicells in one transgenic mouse model (11), but affects splenic macrophages and activated CD8+T cells (12). Gene targeting of transcription factors (e.g.,Irf2,Irf4,Irf8,Stat3andId2) has caused broad defects in several DC subsets, T cells and macrophages (13). To identify genes regulating DC development, we performed global gene expression analysis across many tissues and immune cells (fig S1A).Batf3(p21SNFT) (14) was highly expressed in cDCs, with low to absent expression in other immune cells and non-immune tissues. Thus, we generatedBatf3-/-mice lacking expression of the Batf3 protein (fig. S1B-D). In spleens Cintirorgon (LYC-55716) ofBatf3-/-mice we found a selective loss of CD8+cDCs, without abnormalities in other hematopoietic Cintirorgon (LYC-55716) cell types or architecture (Fig. 1,fig. S2-S11). CD8+cDC coexpress DEC205, CD24, and low levels of CD11b (3,15).Batf3-/-mice lacked splenic CD11chiCD8+DEC205+cells (Fig. 1A), showed a loss of CD11chiCD11bdullcells and CD11chiCD8+CD24+cells (Fig. 1B), but had normal populations of CD4+and CD8-CD4-cDC subsets (Fig. 1B). Lymph nodes and thymi ofBatf3-/-mice lacked CD8+DCs but had normal distributions of CD8-CD11c+cells (Fig. 1C). DEC205intand DEC205hiDCs were present in lymph nodes draining the skin ofBatf3-/-mice (Fig. 1C), and showed normal migration from skin to lymph node after topical application of fluorescein-5-isothiocyanate (fig. S3A).Batf3-/-mice had normal development of pDCs (CD11cintCD11b-B220+) (fig. S3B), interstitial DCs of pancreatic islets (CD11c+CD8-) (fig. S3C, D), monocytes, neutrophils (fig. S3E) and SIGN-R1+and MOMA-1+marginal zone macrophages (Fig. Cintirorgon (LYC-55716) 2A). CD8+cDCs developed normally in heterozygousBatf3+/-mice (fig. S4A), and were absent inRag2-/-Batf3-/-mice (fig. S4B). == Fig. 1.Batf3-/-mice selectively lack the CD8+DC subset. == (A) Splenocytes fromBatf3+/+(+/+) orBatf3-/-(-/-) mice were stained for CD11c, CD8 and DEC205. Left panels are gated on live cells. Numbers indicate the percentage of splenocytes within the CD11chiCD8+gate. Right panels are gated on CD11chicells. (B) Splenocytes were depleted of B220+B cells and Thy1.2+T cells and positively selected for CD11c expression by antibody coated magnetic beads (MACS). Cells were then stained for CD11c, CD11b, and either CD8 and CD4 or CD8 and CD24, and analyzed by FACS. Numbers represent the percentage of cells within the indicated gates. (C) Lymph node cells pooled from cervical, axillary and inguinal lymph nodes and depleted of Thy1.2+T cells, or light density cells of the thymus were stained for CD11c, CD45RA CD8, DEC205 or Sirp-. Plots are gated on the indicated populations. == Fig. 2. Functional loss of CD8+cDCs in Batf3-/-mice is cell-intrinsic to the hematopoietic system. == (A) Frozen sections fromBatf3+/+(+/+) orBatf3-/-(-/-) mice were stained for B220 (green) and SIGN-R1 (red) expression (left panels) or for B220 (green) and MOMA-1 (red) (right panels). (B) Irradiated F1(B6.SJL/129SvEv) mice (CD45.1+CD45.2+) were reconstituted with 2 107bone marrow cells fromBatf3+/+(+/+) orBatf3-/-(-/-) CD45.1-CD45.2+mice. After 10 weeks, donor cells (CD45.1-CD45.2+) were analyzed for CD11c, CD8, CD4 and CD24 expression. Shown are plots for CD8 and CD4 (left panels) or CD8 and CD24 (right panels) gated on CD11chidonor-derived cells. Numbers represent the percentage of cells within the indicated gates. (C)Batf3+/+(+/+) orBatf3-/-(-/-) mice were treated i.p. with 10g FL-Fc. After 10 days, splenocytes were enriched for CD11c+by MACS and stained for CD11c, CD8 and B220. Plots are gated on live cells (left) or CD11cintCD8+cells (right). Numbers represent the percentage of cells within the indicated gates. (D)Batf3+/+(+/+) orBatf3-/-(-/-) BM cells were cultured in FL (20 ng/ml) for 9 days, and non-adherent cells analyzed for CD11c, CD45RA, CD24 and Sirp- expression. Plots are gated on live cells (left) or CD11c+CD45RA-cells (right). This loss of CD8+cDCs could result from a cell-autonomous hematopoietic defect or a cell-extrinsic requirement Rabbit Polyclonal to NPY2R forBatf3. To distinguish these possibilities, we generated chimeras in which CD45.2+Batf3+/+or CD45.2+Batf3-/-bone marrow (BM) was transplanted into lethally irradiated CD45.1+CD45.2+recipients (Fig. 2B). Upon reconstitution (fig. S5A), we found CD8+cDCs developed only fromBatf3+/+donor BM cell (Fig. 2B), indicating a cell-intrinsic hematopoietic defect inBatf3-/-mice. Treatment of mice with fms-like tyrosine kinase 3 (flt3) ligand-Fc (FL-Fc) expanded CD8+cDCs, CD8-cDCs and pDCs inBatf3+/+mice, but failed to expand CD8+cDC inBatf3-/-mice (Fig. 2C). In vitro culture.