Surface phenotype of IKDCs. Splenic DCs were isolated and enriched as described in the Materials and methods. The enriched DC preparation was stained with anti-CD11c-Cy7-PE, anti-CD45R (B220-FITC), and anti-CD49b (DX5-biotin), followed by the secondary reagent SA-Alexa Fluor 594, and then analyzed by flow cytometry. Dead cells were stained with PI. (A) Gating for cDCs (CD11c^hiCD45R⁻) and pDCs (CD11c^intCD45R⁺) without excluding CD49b⁺ cells. (B) Gating for CD49b⁺ within the DC-enriched preparation. (C) Expression of CD11c and CD45R on the CD49b⁺ cells, showing the gates used for IKDC and CD49b⁺DC isolation. (D) The expression of NK1.1, NKG2D, and MHC class II on splenic DC subsets. For NK1.1 expression analysis, splenic DC preparations were stained with anti-NK1.1 (PK136-APC), anti-CD45R (B220-PE), anti-MHC class II (M5/114-FITC), anti-CD11c (N418-Alexa Fluor 594), and anti-CD49b (DX5-biotin; followed by secondary reagent SA-Cy7PE). For NKG2D expression analysis, splenic DC preparations were stained with anti-NKG2D (CX5-biotin), anti-CD49b (DX5-FITC), anti-CD11c (N418-Alexa Fluor 594), and anti-CD45R (B220-PE), followed by SA-Cy7-PE secondary reagent. For MHC class II expression analysis, splenic DC preparations were stained with anti-MHC class II (M5/114-FITC), anti-NK1.1 (P136-APC), anti-CD11c (N418-Alexa-594), and anti-CD45R (B220-PE). Based on the expression of CD11c, CD45R, and CD49b, subsets were defined as IKDC (CD11c^intCD45R⁺CD49b⁺), CD49b⁺DC (CD11c^hiCD49b⁺), pDC (CD11c^intCD45R⁺CD49b⁻), and cDC (CD11c^hiCD45R⁻CD49b⁻). Spleen suspensions, which were depleted of red blood cells using 1.091 g/cm³ Nycodenz density centrifugation, were the source of NK cells. Splenocytes were stained with anti-NK1.1 (PK136-APC), anti-CD49b (DX5-Biotin; followed by secondary reagent SA-Cy7-PE), and anti-CD3 (KT3-Alexa-594), then NK cells sorted as NK1.1⁺CD49b⁺CD3⁻. The continuous line represents staining on gated cells and the dotted line represents background.

IKDCs kill YAC-1 and CHO-K1 target cells. Splenic DCs were enriched and sorted based on their expression of CD11c, CD45R, and CD49b, as described in the Materials and methods. CD11c^hiCD45R⁻ (cDC), CD11c^intCD45R⁺CD49b⁻ (pDC), and CD11^intCD45R⁺CD49b⁺ (IKDC) were purified and incubated at various ratios with ⁵¹Cr-labeled YAC-1 (A) and CHO-OVA (B) target cells in a 4-h ⁵¹Cr release assay, or in a 5-h ⁵¹Cr release assay at an E/T ratio of 2:1 (C). Lysis at each effector to target (E/T) cell ratio was determined as described in the Materials and methods. Each assay point was done in triplicate or duplicate, and the result is representative of a minimum of two experiments. Error bars indicate the SEM.

IKDCs cultured in the presence of OVA protein or peptide do not induce naive OVA-specific transgenic T cells to proliferate. Splenic pDCs (CD11c^intCD45R⁺CD49b⁻), cDCs (CD11c^hiCD45R⁻CD49b⁻), IKDCs (CD11c^intCD45R⁺CD49b⁺), and NK cells (CD49b⁺NK1.1⁺CD3⁻) were isolated and purified by flow cytometry. The purified cells (10⁴) were cocultured for 3 d (A and B) with graded doses of class II–restricted OVA_323-339 peptide and 5 × 10⁴ CFSE-labeled OT-II cells in the absence (A) or presence (B) of CpG and GM-CSF. APCs and CFSE-labeled OT-II cells were also incubated for 3 and 5 d with 0.5 mg/ml whole OVA protein in the absence (C) or presence (D) of CpG and GM-CSF. The proliferative response of OT-II cells was enumerated by flow cytometry, as described in the Materials and methods. Sample points were prepared in duplicates, and results are representative of several experiments.

IKDCs stimulated with CpG do not up-regulate MHC-class II and normally die in culture. IKDC and pDCs were isolated from B6 and Bcl-2 transgenic B6 mice. Cells (1–2 × 10⁴) were cultured in the presence of CpG and GM-CSF (which enhances the survival of DCs). Cells were enumerated and assessed for the level of MHC class II expression on day 0 and 2. (A) Percentage recovery of IKDCs and pDCs from B6 and Bcl-2 transgenic mice after 2 d in culture with CpG and GM-CSF. Data represents two pooled experiments, and the error bars indicate the SEM. (B) The levels of MHC class II expression before and after activation with CpG and GM-CSF using Bcl-2 transgenic cells to extend cell survival. The solid line represents staining on gated cells, and the dashed line represents background. Histograms presented are representative of three independent experiments.

IKDCs fail to present the antigen of target cells that they have lysed. Splenic pDCs (CD11c^intCD45R⁺CD49b⁻), cDCs (CD11c^hiCD45R⁻CD49b⁻), IKDCs (CD11c^intCD45R⁺CD49b⁺), and NK cells (CD49b⁺NK1.1⁺CD3⁻) were isolated and purified by flow cytometry. These cells (10⁴) were cocultured with graded numbers of CHO-OVA cells and either 5 × 10⁴ CFSE-labeled OT-II (A and B) or OT-I (C and D) transgenic T cells in the absence (A and C) or presence (B and D) of CpG and GM-CSF. The proliferative response of the transgenic T cells was enumerated by flow cytometry, as described in the Materials and methods. Sample points were prepared in duplicate, and the presented data is representative of a minimum of two experiments.

IKDCs exposed to two different pathogens do not acquire the ability to stimulate naive T cells into proliferation. Splenic pDCs (CD11c^intCD45R⁺CD49b⁻), cDCs (CD11c^hiCD45R⁻CD49b⁻), IKDCs (CD11c^intCD45R⁺CD49b⁺), and NK cells (CD49b⁺NK1.1⁺CD3⁻) were isolated and purified by flow cytometry. (A and B) These cells were infected in vitro with influenza (PR8 influenza virus), washed, and used at the indicated numbers to stimulate flu-specific transgenic CD4 (HNT; A) and CD8 (CL4) T cells (B). Proliferating T cells were identified and enumerated by flow cytometry. (C and D) Mice were infected with malaria (P. berghei) and killed 3 d later. Spleens from healthy or malaria-infected mice were used to isolate and purify pDCs (CD11c^intCD45R⁺CD49b⁻), cDCs (CD11c^hiCD45R⁻CD49b⁻), IKDCs (CD11c^intCD45R⁺CD49b⁺), and NK cells (CD49b⁺NK1.1⁺CD3⁻), and 10⁴ APCs were cocultured for 3 (C) and 5 d (D) with graded doses of class II-restricted OVA_323-339 peptide and 5 × 10⁴ CFSE-labeled OT-II. Proliferating OT-II were enumerated as indicated in the Materials and methods. The presented data is representative of three independent experiments.

IKDCs are severely reduced in Rag2^−/−Il2rg^−/− mice. Spleens of age- and gender-matched B6 and Rag2^−/−Il2rg^−/− mice were chopped and digested, and lighter density cells were separated using a 1.082-g/cm³ Nycodenz density cut. Irrelevant cell types were removed, as indicated in the Materials and methods. Remaining cells were stained with the following cocktail of fluorescently labeled mAbs: anti-CD11c (N418-Cy7-PE), anti-CD45R-(B220-FITC), anti-NK1.1 (PK136-APC), and anti-CD49b (DX5-biotin, followed by SA-PE secondary; A). Excluded from the analysis were cells that incorporated PI (indicative of dead cells), had large forward scatter width (indicative of cell doublets), and autofluorescent cells (cells that fluoresce in the FL4 channel in the absence of fluorochromes that are detected in FL4). (B) DCs in Rag2^−/−Il2rg^−/− and B6 mice. CD49b⁺ cells (C) or CD49b⁺NK1.1⁺ cells (D) were gated on and analyzed for CD11c and CD45R expression. (E) The total number of DCs, IKDCs, and NK cells were determined and expressed as the ratio of Rag2^−/−Il2rg^−/− to B6. Three independent experiments were conducted using 3–4 Rag2^−/−Il2rg^−/− and B6 mice. Representative data is presented. The ratios of recovered of Rag2^−/−Il2rg^−/− to B6 cells (E) is the mean of the three experiments, and error bars represent the SEM.

IKDCs fail to express the transcription factor PU.1. DCs were isolated from PU.1^GFP and B6 mice, as described in the Materials and methods. Enriched DC preparations or a splenic lymphocyte cell suspension containing NK cells were stained with mAb against CD11c (N418-Alexa-594), CD45R (B220-Cy7-PE), CD49b (DX5-Biotin, followed by SA-PE), and NK1.1 (PK136-APC). The levels of PU.1^GFP expression on gated pDCs (CD11c^intCD45R⁺CD49b⁻), cDCs (CD11c^hiCD45R⁻CD49b⁻), IKDCs (CD11c^intCD45R⁺CD49b⁺), and NK cells (CD49b⁺NK1.1⁺) are represented by the solid lines, and B6 backgrounds are represented by the dashed lines. Data presented is representative of two experiments.

IKDCs and NK cells proliferate, and a proportion express high levels of class II MHC in response to IL-15 and -18, but do not become APCs. Splenic IKDCs (CD11c^intCD45R⁺CD49b⁺), NK cells (NK1.1⁺CD49b⁺CD3⁻), and DCs (CD11c^hiCD49b⁻; 5 × 10³ cells) were cultured in 100 μl of media containing 50 ng/ml IL-18 and 50 ng/ml IL-15. At the indicated time points, cells were counted (A), and level of MHC class II was determined by staining with M5/114-FITC and analysis by flow cytometry (B). (C) Bulk-cultured IKDCs do not activate OT-II cells. IKDCs were cultured as above, harvested on day 4, and determined by flow cytometry to contain 10% MHC class II⁺ cells. 10⁴-cultured IKDCs (containing the equivalent of 10³ MHC class II⁺ cells) or 10³ cells from an enriched DC preparation (DC), were incubated with CFSE-labeled OT-II cells (5 × 10⁴) and 1,000 ng/ml of class II–restricted OVA_323-339 peptide. (D) The levels of the costimulatory markers expressed on cultured MHC class II⁺ and class II⁻ IKDC and NK cells compared with fresh cDCs. Expression profiles were confirmed in two independent experiments. (E and F) Purified MHC class II⁺ IKDCs and NK cells were ineffective at inducing OT-II cell proliferation. MHC class II⁺ and class II⁻ cultured IKDCs and NK cells, and cDCs (10⁴ cells) were incubated with CFSE-labeled OT-II cells (5 × 10⁴) and graded doses of class II–restricted OVA_323-339 peptide (E) or 0.5 mg/ml of whole OVA protein (F) for 4 d. Proliferating OT-II cells were enumerated as indicated in the Materials and methods. The presented data is representative of three independent experiments. Error bars indicate the SEM.