Use of CRISPR/Cas9 for the Modification of the Mouse Genome.

The use of CRISPR/Cas9 to modify the mouse genome has gained immense interest in the past few years since it allows the direct modification of embryos, bypassing the need of labor-intensive procedures for the manipulation of embryonic stem cells. By shortening the overall timelines and reducing the costs for the generation of new genetically modified mouse lines (Li et al., Nat Biotechnol 31: 681-683, 2013), this technology has rapidly become a major tool for in vivo drug discovery applications.

Ontogenic, Phenotypic, and Functional Characterization of XCR1(+) Dendritic Cells Leads to a Consistent Classification of Intestinal Dendritic Cells Based on the Expression of XCR1 and SIRPα.

In the past, lack of lineage markers confounded the classification of dendritic cells (DC) in the intestine and impeded a full understanding of their location and function. We have recently shown that the chemokine receptor XCR1 is a lineage marker for cross-presenting DC in the spleen. Now, we provide evidence that intestinal XCR1(+) DC largely, but not fully, overlap with CD103(+) CD11b(-) DC, the hypothesized correlate of "cross-presenting DC" in the intestine, and are selectively dependent in their development on the transcription factor Batf3. XCR1(+) DC are located in the villi of the lamina propria of the small intestine, the T cell zones of Peyer's patches, and in the T cell zones and sinuses of the draining mesenteric lymph node. Functionally, we could demonstrate for the first time that XCR1(+)/CD103(+) CD11b(-) DC excel in the cross-presentation of orally applied antigen. Together, our data show that XCR1 is a lineage marker for cross-presenting DC also in the intestinal immune system. Further, extensive phenotypic analyses reveal that expression of the integrin SIRPα consistently demarcates the XCR1(-) DC population. We propose a simplified and consistent classification system for intestinal DC based on the expression of XCR1 and SIRPα.

Expression of XCR1 Characterizes the Batf3-Dependent Lineage of Dendritic Cells Capable of Antigen Cross-Presentation.

Cross-presentation of antigen by dendritic cells (DCs) to CD8(+) T cells is a fundamentally important mechanism in the defense against pathogens and tumors. Due to the lack of an appropriate lineage marker, cross-presenting DCs in the mouse are provisionally classified as "Batf3-IRF-8-Id2-dependent DCs" or as "CD8(+) DCs" in the spleen, and as "CD103(+)CD11b(-) DCs" in the periphery. We have now generated a mAb to XCR1, a chemokine receptor which is specifically expressed on CD8(+) DCs and a subpopulation of double negative DCs in the spleen. Using this antibody, we have determined that only XCR1(+)CD8(+) (around 80% of CD8(+) DCs) and their probable precursors, XCR1(+)CD8(-) DCs, efficiently take up cellular material and excel in antigen cross-presentation. In lymph nodes (LNs) and peripheral tissues, XCR1(+) DCs largely, but not fully, correspond to CD103(+)CD11b(-) DCs. Most importantly, we demonstrate that XCR1(+) DCs in the spleen, LNs, and peripheral tissues are dependent on the growth factor Flt3 ligand and are selectively absent in Batf3-deficient animals. These results provide evidence that expression of XCR1 throughout the body defines the Batf3-dependent lineage of DCs with a special capacity to cross-present antigen. XCR1 thus emerges as the first surface marker characterizing a DC lineage in the mouse and potentially also in the human.

Superior antigen cross-presentation and XCR1 expression define human CD11c+CD141+ cells as homologues of mouse CD8+ dendritic cells.

In recent years, human dendritic cells (DCs) could be subdivided into CD304+ plasmacytoid DCs (pDCs) and conventional DCs (cDCs), the latter encompassing the CD1c+, CD16+, and CD141+ DC subsets. To date, the low frequency of these DCs in human blood has essentially prevented functional studies defining their specific contribution to antigen presentation. We have established a protocol for an effective isolation of pDC and cDC subsets to high purity. Using this approach, we show that CD141+ DCs are the only cells in human blood that express the chemokine receptor XCR1 and respond to the specific ligand XCL1 by Ca2+ mobilization and potent chemotaxis. More importantly, we demonstrate that CD141+ DCs excel in cross-presentation of soluble or cell-associated antigen to CD8+ T cells when directly compared with CD1c+ DCs, CD16+ DCs, and pDCs from the same donors. Both in their functional XCR1 expression and their effective processing and presentation of exogenous antigen in the context of major histocompatibility complex class I, human CD141+ DCs correspond to mouse CD8+ DCs, a subset known for superior antigen cross-presentation in vivo. These data define CD141+ DCs as professional antigen cross-presenting DCs in the human.

Selective expression of the chemokine receptor XCR1 on cross-presenting dendritic cells determines cooperation with CD8+ T cells.

The expression of the chemokine receptor XCR1 and the function of its ligand XCL1 (otherwise referred to as ATAC, lymphotactin, or SCM-1) remained elusive to date. In the present report we demonstrated that XCR1 is exclusively expressed on murine CD8(+) dendritic cells (DCs) and showed that XCL1 is a potent and highly specific chemoattractant for this DC subset. CD8(+) T cells abundantly secreted XCL1 8-36 hr after antigen recognition on CD8(+) DCs in vivo, in a period in which stable T cell-DC interactions are known to occur. Functionally, XCL1 increased the pool of antigen-specific CD8(+) T cells and their capacity to secrete IFN-gamma. Absence of XCL1 impaired the development of cytotoxicity to antigens cross-presented by CD8(+) DCs. The XCL1-XCR1 axis thus emerges as an integral component in the development of efficient cytotoxic immunity in vivo.