Figure 1.
Batf3−/− mice are susceptible to EAE.
(A) Clinical course of EAE in C57BL/6 mice, Batf3−/− mice (C57BL/6 background), and Csf2rb−/− mice. Data are combined from two experiments (n = 10 mice per group). Points represent the mean clinical score. For clarity, error bars are not displayed. (B) ELISPOT assays on popliteal lymph node cells at day 7 following footpad immunization (MOG35–55 peptide in CFA) and restimulation with MOG35–55 peptide. Data are from one of two similar experiments (n = 3 per group). Mean ± SEM, p values by unpaired student's t test.
Figure 2.
Batf3-dependent dermal CD11blow/−Langerin+ DCs develop in Csf2rb−/− mice, but lack expression of CD103.
(A) FACS analysis of SDLN (inguinal) DCs from C57BL/6, Batf3−/− (C57BL/6 background), and Csf2rb−/− mice. Left plots are gated on CD11chigh cells. The second column is gated on migratory (DEC205+CD8α−) DCs. The third, fourth, and fifth columns are gated on Langerin+ migratory DCs. Numbers represent the percentage of cells within the indicated gates. MFI are shown for the indicated gates. Data are representative of eight to ten individual inguinal lymph nodes from four to five mice per genotype over two experiments. (B) FACS analysis of SDLN (inguinal) DCs from C57BL/6, Batf3−/−, and Csf2rb−/− mice. Left plots are gated on CD11chigh cells. Middle plots are gated on migratory (DEC205+CD8α−) DCs. Right plots are gated on all Langerin+ migratory DCs, independent of expression of CD11b. Numbers represent the percentage of cells within the indicated gates. Data are representative of inguinal lymph nodes from at least five mice per genotype for all stains except CD24, which has been performed on one mouse per genotype. (C) Absolute cell numbers per individual inguinal lymph nodes calculated from the FACS analysis in (A). Langerhans cells were gated as Langerin+CD11b+EpCAMhigh DCs, and dermal Langerin+ DCs were gated as Langerin+CD11blow/−EpCAMmid DCs. Horizontal bars represent the geometric mean; p values by unpaired student's t test. (D) Clinical course of EAE in 129S6/SvEv mice and Batf3−/− mice (129S6/SvEv background). Data are from one of three similar experiments (n = 5 mice per group). Points represent the mean clinical score. For clarity, error bars are not displayed.
Figure 3.
GM-CSF controls the level of CD103 expression on Batf3-dependent CD11blow/− peripheral DCs.
(A) FACS analysis of lung DCs from C57BL/6, Batf3−/− (C57BL/6 background), and Csf2rb−/− mice. Left plots are gated on live, single cells. The second column of plots is gated on CD11c+CD45.2+ cells, consisting of a mixture of lung macrophages and DCs. Note that in Csf2rb−/− mice, lung macrophages lack CD11c expression [38], and therefore this gate includes only DCs. The third and fourth columns of plots are gated on I-Abhigh autofluorescencelow DCs. Numbers represent the percentage of cells within the indicated gates. Data are representative of six individual lungs from three mice per genotype over two experiments. (B) FACS analysis of kidney DCs from C57BL/6, Batf3−/− (C57BL/6 background), and Csf2rb−/− mice. Left plots are gated on live, single cells. The second column of plots is gated on CD11chighCD45.2+ cells, consisting of DCs. The third column of plots is gated on CD11chighI-Ab+ DCs. Numbers represent the percentage of cells within the indicated gates. Data are representative of six individual kidneys from three mice per genotype over two experiments.
Figure 4.
GM-CSF induces CD103 expression on Flt3L-derived CD8α-equivalent DCs.
FACS analysis of developing DCs in bone marrow cells from C57BL/6, Batf3−/− (C57BL/6 background), and Csf2rb−/− mice cultured with Flt3L for 9 days, with or without the addition of GM-CSF to the culture on day 7. The left column is gated on live single cells, the second on CD11c+CD45RA− cDCs, the third on I-Ab+ cDCs, and the fourth on CD24highSirpαlow cells. Numbers represent the percentage of cells within the indicated gates. Data are representative of bone marrow cells from two mice per genotype over two experiments.