Batf3-Dependent CD11blow/− Peripheral Dendritic Cells Are GM-CSF-Independent and Are Not Required for Th Cell Priming after Subcutaneous Immunization

Dendritic cells (DCs) subsets differ in precursor cell of origin, functional properties, requirements for growth factors, and dependence on transcription factors. Lymphoid-tissue resident CD8α+ conventional DCs (cDCs) and CD11blow/−CD103+ non-lymphoid DCs are developmentally related, each being dependent on FMS-like tyrosine kinase 3 ligand (Flt3L), and requiring the transcription factors Batf3, Irf8, and Id2 for development. It was recently suggested that granulocyte/macrophage colony stimulating factor (GM-CSF) was required for the development of dermal CD11blow/−Langerin+CD103+ DCs, and that this dermal DC subset was required for priming autoreactive T cells in experimental autoimmune encephalitis (EAE). Here, we compared development of peripheral tissue DCs and susceptibility to EAE in GM-CSF receptor deficient (Csf2rb−/−) and Batf3−/− mice. We find that Batf3-dependent dermal CD11blow/−Langerin+ DCs do develop in Csf2rb−/− mice, but that they express reduced, but not absent, levels of CD103. Further, Batf3−/− mice lacking all peripheral CD11blow/− DCs show robust Th cell priming after subcutaneous immunization and are susceptible to EAE. Our results suggest that defective T effector priming and resistance to EAE exhibited by Csf2rb−/− mice does not result from the absence of dermal CD11blow/−Langerin+CD103+ DCs

Zbtb46 expression distinguishes classical dendritic cells and their committed progenitors from other immune lineages

Distinguishing dendritic cells (DCs) from other cells of the mononuclear phagocyte system is complicated by the shared expression of cell surface markers such as CD11c. In this study, we identified Zbtb46 (BTBD4) as a transcription factor selectively expressed by classical DCs (cDCs) and their committed progenitors but not by plasmacytoid DCs (pDCs), monocytes, macrophages, or other lymphoid or myeloid lineages. Using homologous recombination, we replaced the first coding exon of Zbtb46 with GFP to inactivate the locus while allowing detection of Zbtb46 expression. GFP expression in Zbtb46(gfp/+) mice recapitulated the cDC-specific expression of the native locus, being restricted to cDC precursors (pre-cDCs) and lymphoid organ- and tissue-resident cDCs. GFP(+) pre-cDCs had restricted developmental potential, generating cDCs but not pDCs, monocytes, or macrophages. Outside the immune system, Zbtb46 was expressed in committed erythroid progenitors and endothelial cell populations. Zbtb46 overexpression in bone marrow progenitor cells inhibited granulocyte potential and promoted cDC development, and although cDCs developed in Zbtb46(gfp/gfp) (Zbtb46 deficient) mice, they maintained expression of granulocyte colony-stimulating factor and leukemia inhibitory factor receptors, which are normally down-regulated in cDCs. Thus, Zbtb46 may help enforce cDC identity by restricting responsiveness to non-DC growth factors and may serve as a useful marker to identify rare cDC progenitors and distinguish between cDCs and other mononuclear phagocyte lineages

Peripheral CD103+ dendritic cells form a unified subset developmentally related to CD8α+ conventional dendritic cells

Although CD103-expressing dendritic cells (DCs) are widely present in nonlymphoid tissues, the transcription factors controlling their development and their relationship to other DC subsets remain unclear. Mice lacking the transcription factor Batf3 have a defect in the development of CD8α(+) conventional DCs (cDCs) within lymphoid tissues. We demonstrate that Batf3(−/−) mice also lack CD103(+)CD11b(−) DCs in the lung, intestine, mesenteric lymph nodes (MLNs), dermis, and skin-draining lymph nodes. Notably, Batf3(−/−) mice displayed reduced priming of CD8 T cells after pulmonary Sendai virus infection, with increased pulmonary inflammation. In the MLNs and intestine, Batf3 deficiency resulted in the specific lack of CD103(+)CD11b(−) DCs, with the population of CD103(+)CD11b(+) DCs remaining intact. Batf3(−/−) mice showed no evidence of spontaneous gastrointestinal inflammation and had a normal contact hypersensitivity (CHS) response, despite previous suggestions that CD103(+) DCs were required for immune homeostasis in the gut and CHS. The relationship between CD8α(+) cDCs and nonlymphoid CD103(+) DCs implied by their shared dependence on Batf3 was further supported by similar patterns of gene expression and their shared developmental dependence on the transcription factor Irf8. These data provide evidence for a developmental relationship between lymphoid organ–resident CD8α(+) cDCs and nonlymphoid CD103(+) DCs

Bcl11a regulates the expression of <i>Flt3</i> and <i>Il7r</i>.

<p>(A) Microarray analysis of sorted GMPs (left) and MPPs (right) from WT and <i>Bcl11a</i>^−/− fetal liver chimeras. (B) Shown is a Venn diagram of probe sets (excluding normalization controls) with a greater than twofold change in expression between WT and <i>Bcl11a</i>^−/− MPPs. (C) Shown are log₂-transformed ratios of gene expression in <i>Bcl11a</i>^−/− MPPs relative to WT MPPs (<i>x</i>-axis) plotted against log₂-transformed ratios of gene expression in WT CDPs relative to WT monocytes (ImmGen; <i>y</i>-axis). For clarity, probe sets with less than twofold changes in expression (log₂-transformed ratios between −1 and 1) along either dimension are omitted (gray). (D) Shown is a heat map of log₂-transformed gene expression in WT and <i>Bcl11a</i>^−/− GMPs and MPPs for probe sets that constitute an ImmGen core cDC signature. Highlighted are genes that show a greater than twofold change in expression between WT and <i>Bcl11a</i>^−/− GMPs (red) or between WT and <i>Bcl11a</i>^−/− MPPs (green).</p

Bcl11a is required for development of lymphoid and DC progenitors in the adult.

<p>(A) Flow cytometry analysis of progenitor populations in lethally irradiated congenic mice reconstituted with WT or <i>Bcl11a</i>^−/− fetal liver cells, analyzed four weeks after transplant. Data are representative of three mice per group. (B) Progenitor populations in WT and <i>Bcl11a</i>^−/− fetal liver chimeras at four weeks after transplant, analyzed by flow cytometry as in (A) and presented as a percentage of total BM cells. Bars represent the mean (± SEM) of three mice per group. (C) CD150 (Slamf1) expression within the donor-derived LSK fraction in WT and <i>Bcl11a</i>^−/− fetal liver chimeras at four weeks after transplant. (D) SL-GMPs in WT and <i>Bcl11a</i>^−/− fetal liver chimeras at four weeks after transplant.</p

Bcl11a is required <i>in vitro</i> for development of Flt3L-derived pDCs and cDCs but not GM-CSF–derived cDCs.

<p>(A) Flow cytometry analysis of pDCs in Flt3L cultures of fetal liver cells. Data are representative of three to four replicates over two experiments. (B) Flow cytometry analysis of pDCs in Flt3L cultures of BM cells derived from fetal liver chimeras. Data are representative of three replicates. (C, D) Flow cytometry analysis of Flt3L-derived cDCs (C) or GM-CSF-derived DCs (D) in cultures of fetal liver cells. Data are representative of three to four replicates over two experiments. (E, F) Counts of total cells and indicated subsets in Flt3L cultures (E) or GM-CSF cultures (F) of fetal liver cells, analyzed by flow cytometry as in (C) or (D), respectively. Bars represent the mean (± SEM) of three to four replicates per group pooled from two experiments.</p

Cytokine signaling in DC development and regulation by Bcl11a.

<p>(A) Flow cytometry analysis of pDCs in WT and <i>Il7r</i>^−/− spleens. Data are representative of four mice per group over two experiments. (B) Flow cytometry analysis of pDCs in WT and <i>Flt3l</i>^−/− spleens. Data are representative of three mice per group over two experiments. (C) Flow cytometry analysis of donor-derived cDCs in the spleen of WT and <i>Bcl11a</i>^−/− fetal liver chimeras, analyzed by flow cytometry as in Fig. 4. Data are representative of three mice per group. (D) Bcl11a binding in the <i>Flt3</i> genomic locus assayed by ChIP-qPCR. Data are represented as fold enrichment as compared to isotype control.</p

Bcl11a deficiency <i>in vivo</i> impairs development of lymphoid and myeloid populations.

<p>(A) Donor-derived lymphoid populations in the spleen of WT and <i>Bcl11a</i>^−/− fetal liver chimeras, analyzed by flow cytometry. Bars represent the mean (± SEM) of three mice per group. (B) Donor-derived myeloid populations in the spleen of WT and <i>Bcl11a</i>^−/− fetal liver chimeras, analyzed by flow cytometry as in Fig. 4. Bars represent the mean (± SEM) of three mice per group.</p

Bcl11a is required for development of lymphoid and DC progenitors in the fetus.

<p>(A) Flow cytometry analysis of progenitor populations in WT and <i>Bcl11a</i>^−/− fetal livers dissected at embryonic day 14.5 (E14.5). Populations are gated as indicated; numbers represent the percentage of cells within the histogram that lie in the indicated gate. Data are representative of two mice per group. (B) Progenitor populations in WT and <i>Bcl11a</i>^−/− fetal livers at E14.5, analyzed by flow cytometry as in (A) and presented as a percentage of total fetal liver cells. Bars represent the mean (± SEM) of two mice per group. (C) CD150 (Slamf1) expression within the LSK fraction in WT and <i>Bcl11a</i>^−/− fetal livers at E14.5. (D) SL-GMPs in WT and <i>Bcl11a</i>^−/− fetal livers at E14.5.</p