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.

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.

Current subunit vaccines are incapable of inducing Ag-specific CD8⁺ T cell cytotoxicity needed for the defense of certain infections and for therapy of neoplastic diseases. In experimental vaccines, cytotoxic responses can be elicited by targeting of Ag into cross-presenting dendritic cells (DC), but almost all available systems use target molecules also expressed on other cells and thus lack the desired specificity. In the present work, we induced CD8⁺ T cell cytotoxicity by targeting of Ag to XCR1, a chemokine receptor exclusively expressed on murine and human cross-presenting DC. Targeting of Ag with a mAb or the chemokine ligand XCL1 was highly specific, as determined with XCR1-deficient mice. When applied together with an adjuvant, both vector systems induced a potent cytotoxic response preventing the outgrowth of an inoculated aggressive tumor. By generating a transgenic mouse only expressing the human XCR1 on its cross-presenting DC, we could demonstrate that targeting of Ag using human XCL1 as vector is fully effective in vivo. The specificity and efficiency of XCR1-mediated Ag targeting to cross-presenting DC, combined with its lack of adverse effects, make this system a prime candidate for the development of therapeutic cytotoxic vaccines in humans.

Whereas CD4 ⁺ T cells conventionally mediate antitumor immunity by providing help to CD8 ⁺ T cells, recent clinical studies have implied an important role for cytotoxic CD4 ⁺ T cells in cancer immunity. Using an orthotopic melanoma model, we provide a detailed account of antitumoral CD4 ⁺ T cell responses and their regulation by major histocompatibility complex class II (MHC II) in the skin. Intravital imaging revealed prominent interactions of CD4 ⁺ T cells with tumor debris-laden MHC II ⁺ host antigen-presenting cells that accumulated around tumor cell nests, although direct recognition of MHC II ⁺ melanoma cells alone could also promote CD4 ⁺ T cell control. CD4 ⁺ T cells stably suppressed or eradicated tumors even in the absence of other lymphocytes by using tumor necrosis factor–α and Fas ligand (FasL) but not perforin-mediated cytotoxicity. Interferon-γ was critical for protection, acting both directly on melanoma cells and via induction of nitric oxide synthase in myeloid cells. Our results illustrate multifaceted and context-specific aspects of MHC II–dependent CD4 ⁺ T cell immunity against cutaneous melanoma, emphasizing modulation of this axis as a potential avenue for immunotherapies.

DCs often require stimulation from CD4(+) T cells to propagate CD8(+) T cell responses, but precisely how T cell help optimizes the priming capacity of DCs and why this appears to differ between varying types of CD8(+) T cell immunity remains unclear. We show that CD8(+) T cell priming upon HSV-1 skin infection depended on DCs receiving stimulation from both IFN-α/β and CD4(+) T cells to provide IL-15. This was not an additive effect but resulted from CD4(+) T cells amplifying DC production of IL-15 in response to IFN-α/β. We also observed that increased innate stimulation reversed the helper dependence of CD8(+) T cell priming and that the innate stimulus, rather than the CD4(+) T cells themselves, determined how "help'" was integrated into the priming response by DCs. These findings identify T cell help as a flexible means to amplify varying suboptimal innate signals in DCs.