IFN-producing killer dendritic cells are antigen-presenting cells endowed with T-cell cross-priming capacity

Abstract

IFN-producing killer dendritic cells (IKDC) represent a recently discovered cell type in the immune system that possesses a number of functions contributing to innate and adaptive immunity, including production of type 1 and 2 IFNs, interleukin (IL)-12, natural killing, and ultimately antigen presentation to naïve T cells. Here, we compared in vitro and in vivo responses of mouse IKDC, conventional dendritic cells (DC), and natural killer (NK) cells to murine cytomegalovirus infection and found distinct functions among these cell subsets. Upon recognition of infected fibroblasts, IKDC, as well as NK, produced high level of IFN-gamma, but unlike NK, IKDC simultaneously produced IL-12p40 and up-regulated MHC class II (MHC-II) and costimulatory molecules. Using MHC-II molecule expression as a phenotypic marker to distinguish activated IKDC from activated NK, we further showed that highly purified MHC-II(+) IKDC but not NK cross-present MHC class I-restricted antigens derived from MCMV-infected targets to CD8(+) T cells in vitro and in vivo. Our findings emphasize the unique nature of IKDC as a killer antigen-presenting cell directly linking innate and adaptive immunity.

Figure 1. Genomic characterization of MHC-II processing machinery

A, Heatmaps showing the expression levels of the probe sets for the mRNA associated with MHC-II processing pathway in IKDC, NK, and CDC from spleen (two independent samples 1 and 2) and LN (two pooled samples) of BALB/c mice. The color scheme is based on the base 2 logarithmic scale. B, Fold changes of the probe set signals for IKDC versus NK and IKDC versus CDC. Up-regulated and down-regulated genes are highlighted in green and red, respectively. The criterion of significance was set as the posterior probability >0.5. Fold changes highlighted in grey are not statistically significant. Absolute value of each probe set is provided in Table S1. C, Protein extracts from cell-sorted IKDC and NK were tested by western blot for Ii and Legumain expression. Tubulin detection is shown as control.

Figure 2. IKDC up-regulated MHC-II upon recognition of MCMV-infected targets

A, confocal microscopy of C57BL/6 IKDC (CD11c^intB220⁺CD49b⁺), NK (CD11c^-B220^-CD49b⁺), and CDC (CD11c^hiB220^-CD49b^-) labeled with anti-IA/IE (red) and anti-CD49b (green) antibodies. Blue, nuclei. B, IKDC, NK, and CDC shown in A were stained by ICS for I-A/E (see legend) or isotype-matched control (shaded histogram) and expression was assessed by FACS. C, BALB/c CD11c⁺ (including CDC and IKDC) and CD11c^- (including NK) splenocytes were incubated 12 h with MCMV-infected or mock-treated fibroblasts. MHC-II expression was assessed by FACS on NK (CD49b⁺ cells from the CD11c^- fraction), IKDC (CD49b⁺ from the CD11c⁺ fraction), and CDC (CD11c^hiCD49b^- from the CD11c⁺ fraction).

Figure 3. Upon recognition of MCMV-infected fibroblasts, IKDC differentiated into mature MHC-II^hi APC endowed with CD4⁺ T-lymphocyte stimulatory properties

A, C57BL/6 CD11c⁺ and CD11c⁻ splenocytes were incubated with MCMV/GFP- or MCMV/OVA-infected fibroblasts. CDC (CD11c^hiNK1.1⁻MHC-II^hi), IKDC (CD11c⁺NK1.1⁺MHC-II⁺) and NK (CD11c⁺NK1.1⁺MHC-II⁻) were FACS-sorted from gates 1, 2 and 3, respectively, and incubated with OVA-specific OT-II CD4⁺ T-cells. IL-2 was measured by ELISA in day 3 culture supernatants. ODN CpG 1668 was added or not to the culture to stimulate the APCs. APC were also incubated in presence of OVA_323–339 to stimulate OT-II. B, Similar presentation assay was performed with HA antigenic model. The purity of sorted populations used in this assay is shown in Figure S3. C, Co-stimulatory molecules expression on CD11c⁺NK1.1⁺MHC-II⁺ IKDC (open) versus CD11c⁺NK1.1⁺MHC-II⁻ NK (light grey) and CD11c^hiNK1.1⁻MHC-II^hi CDC (dark grey) in presence of infected fibroblasts.

Figure 4. IKDC activated by MCMV-infected fibroblasts simultaneously produced IFN-γ and IL-12p40

A, C57BL/6 CD11c⁺ and CD11c⁻ fractions were incubated 12h with MCMV-infected fibroblasts. IFN-γ and IL-12p40 were detected by ICS. In the CD11c⁺ fraction, CD11c⁺NK1.1⁻ included CDC and PDC whereas CD11c⁺NK1.1⁺ included IKDC (MHC-II⁺). In the CD11c⁻ fraction, the majority of the CD11c⁻NK1.1⁺ NK upregulated CD11c after co-culture with infected fibroblasts and were CD11c⁺NK1.1⁺MHC-II⁻. The dot plots are representative of at least 3 experiments. B, CD11c⁺NK1.1⁺ splenocytes from Trif^Lps2/Lps2MyD88^−/− and IL-12p40^−/− mice were assessed for IFN-γ by ICS. Where indicated, rmIL-12 (100 pg/ml) was added.

Figure 5. IKDC cross-presented viral antigens derived from MCMV-infected fibroblasts

A, IKDC (CD11c^intNK1.1⁺CD49b⁺I-Ab^hi, red), CDC (CD11c^hiNK1.1⁻CD49b⁻I-Ab^hi, black) and NK (CD11c^intNK1.1⁺CD49b⁺I-Ab⁻, green) were cell sorted from 12h co-culture of CD11c⁺ (IKDC and CDC) or CD11c⁻ (NK) fractions with MCMV-infected primary fibroblasts. Left dot plots represent an overlay of the CD49b/NK1.1 staining for different populations. The percentages of each population are indicated above each of the two gates CD49b⁺NK1.1⁺ (IKDC and NK) and CD49b⁻NK1.1⁻ (CDC). The differential expression of CD11c and I-Ab used to sort IKDC, CDC, and NK is shown in right dot plots. B, Expression of MHC-I (H-2k^b), B220, and CD40 by purified IKDC, CDC and NK. Percentages of cells expressing CD40 are indicated. C, IKDC and CDC, but not NK, cross-present OVA derived from MCMV/OVA-infected fibroblasts. Each cell population was mixed with CFSE-labeled CD8⁺ OT-I at a ratio of 1APC:3T-cells. CFSE dilution was assessed by FACS after 4 day-incubation. Percentages of proliferating T-cells within gate are indicated for cultures containing stimulators previously incubated with MCMV/OVA (black)- or MCMV/GFP (grey)-infected fibroblasts.

Figure 6. IKDC derived from MCMV-infected mice cross-presented viral antigens to CD8⁺ T-cells

C57BL/6 mice were infected i.p. with 4×10⁵pfu MCMV/OVA or MCMV/GFP. Splenic IKDC (CD11c^intCD49b⁺I-Ab^hi), CDC (CD11c^hiCD49b⁻I-Ab^hi) and NK (CD11c^intCD49b⁺I-Ab⁻) were sorted at day 2 p.i. A, Purity of FACS-sorted IKDC (red), CDC (black), and NK (green). Left dot plots represent an overlay of the CD49b/CD11c staining for these populations. The percentages of each population are indicated above each of the two gates CD49b⁺CD11c^int (IKDC and NK) and CD49⁻CD11c^hi (CDC). The differential expression of MHC-II (I-Ab) used to sort IKDC, CDC and NK is shown in overlaid histograms. B, Each population was stained for MHC-I (H2-Kb), CD40, NKG2D, and KLRG1. C, DEAD assay. Ex vivo sorted cell populations were mixed with CFSE-labeled CD8⁺ OT-I at a ratio of 1APC:3T-cells. CFSE dilution was assessed by FACS after 4d-incubation. Percentages of proliferating T-cells within gate are indicated for cultures containing stimulators derived from mice infected with MCMV/OVA (black) or MCMV/GFP (grey).