CD169+ macrophages are sufficient for priming of CTLs with specificities left out by cross-priming dendritic cells

Abstract

Dendritic cells (DCs) are considered the most potent antigen-presenting cells (APCs), which directly prime or cross-prime MHC I-restricted cytotoxic T cells (CTLs). However, recent evidence suggests the existence of other, as-yet unidentified APCs also able to prime T cells. To identify those APCs, we used adenoviral (rAd) vectors, which do not infect DCs but selectively accumulate in CD169(+) macrophages (MPs). In mice that lack DCs, infection of CD169(+) MPs was sufficient to prime CTLs specific for all epitopes tested. In contrast, CTL responses relying exclusively on cross-presenting DCs were biased to selected strong MHC I-binding peptides only. When both DCs and MPs were absent, no CTL responses could be elicited. Therefore, CD169(+) MPs can be considered APCs that significantly contribute to CTL responses.

Keywords: CD169+ macrophage; CD8 T cell; adenovirus; cross-presentation; dendritic cell.

Fig. 1.

rAd vectors infect MPs in the marginal zone, but not DCs or other cell types. (A) Representative serial micrographs of spleens from C57BL/6 control, Δ-DC, or DTA–treated CD11c-DTR mice analyzed for CD11c, B220 and SIGN-R1, or CD169 or F4/80, respectively. (Scale bar: 200 µm.) (B) Immunofluorescence analysis of spleens from C57BL/6 mice at 48 h after i.v. injection with rAd-GFP. After signal amplification for GFP, spleen sections were analyzed for CD11c and CD169 (Upper) or for CD169 and SIGN-R1 (Lower). (Scale bar: 200 µm.) The white frame (Left) was enlarged for detailed analysis of infected macrophages (Right; arrow: GFP⁺CD169⁺, marginal metallophilic macrophage; arrowhead: GFP⁺SIGN-R1⁺CD169^low, marginal zone macrophage). (C) Similar analysis as shown for B with spleens from Δ-DC mice. (Scale bar: 200 µm.) (D) Surface marker analysis of DCs and CD169⁺ macrophages from spleens of mice injected with LPS. Numbers in histograms indicate mean fluorescent intensities of the respective stainings (mean, n = 3).

Fig. 2.

Δ-DC mice prime normal CTL responses despite the absence of DCs. (A and B) Ag-specific CTLs were detected from spleen cell suspensions at 8 d after i.v. injection of rAd-GP (A) or rAd-OVA (B) using the indicated MHC multimers. Data are gated on CD19⁻CD8⁺ cells from two independent experiments (n = 5 mice) and were analyzed using the unpaired two-tailed Student t test. Frequencies of the respective CD8⁺ T cells are shown in the bar graphs. For functional analyses, spleen cells were cultured in vitro with the indicated peptides in the presence of CD107a-specific mAb and subsequently stained for CD8 and IFN-γ. (C) P14 or OTI T cells (2 × 10⁶) were adoptively transferred into the indicated mouse strains, which were subsequently immunized with rAd-GP or rAd-OVA. The expansion of CD8⁺CD90.1⁺ P14 and OTI T cells was analyzed by flow cytometry 3 d later. Total cell numbers are shown in the bar graph. Data are representative of at least two independent experiments with three mice per group and were analyzed using the unpaired two-tailed Student t test.

Fig. 3.

DCs cross-prime CTLs against high-affinity MHC I/peptide complexes only. (A) Spleens of rAd-GP–infected mice were analyzed as described in Fig. 2 with respective MHC multimers. Pie charts show the frequency of CTLs with the respective specificities in the different mice. Data are representative of more than three independent experiments with three mice per group. (B) Analysis of D^b/GP33- and K^b/GP33-specific T cells in WT and DC-MHCI mice at 8 d after i.v. injection of rAd-GP33. Bar graphs indicate frequencies of specific CTLs as well as total numbers. (C) Adoptive transfer of 2 × 10⁶ CFSE-labeled P14 T cells into mice and immunization with rAd-GP33. Three days later, the CFSE profiles of CD8⁺CD90.1⁺ P14 T cells were monitored; numbers represent SE. Data show percentages from three mice per group and are representative of two independent experiments. Data were analyzed using the unpaired two-tailed Student t test. (D) Pie charts displaying the distribution of CTLs with the indicated specificities at 8 d after immunization with rAd-OVA. Data are representative of more than three independent experiments with three mice per group. (E) Analysis of MHC I expression on DCs and CD169⁺ MPs from spleens of untreated B6 mice, DC-MHCI mice, and MHCI KO mice. Data are from one of three independent experiments with similar results.

Fig. 4.

Only directly infected DCs can prime D^b/GP33-specific T cells. (A) DCs isolated from spleens of B6 or DC-MHCI mice that had been immunized 24 h earlier with i.v. injection of 1 × 10¹⁰ particles of rAd-GP33 or rAd-OVA were cultured with naïve P14 or OT-I T cells. As a positive control, DCs isolated from naïve mice were loaded with pGP33 or pOVA257. Peptide-free DCs from naïve mice served as negative controls. Priming of P14 and OT-I T cells was analyzed on day 4 of culture. Data are representative of two independent experiments with similar results. (B) (Upper) rAd-GFP–infected CD11c⁺ bone marrow DCs were analyzed for GFP expression by flow cytometry. (Lower) Proliferation of CFSE-labeled CD8⁺CD90.1⁺ P14 T cells cocultured with rAd-GP33-transduced DCs was analyzed by flow cytometry. (C) Mice of the indicated strains were infected with 100 pfu of LCMV at 1 d after adoptive transfer of 5 × 10⁵ P14 T cells. CD8⁺CD90.1⁺ P14 T cells were identified by flow cytometry, Bar graphs show frequencies of P14 T cells as well as total cell numbers (n = 3). (D) CFSE dilution of P14 T cells adoptively transferred into WT or DC-MHCI mice at 4 d after immunization with GP33-41 peptide plus LPS (thin line) or LPS alone (thick line). Data are representative of two independent experiments with similar results (n = 3 mice per group) and were analyzed using the unpaired two-tailed Student t test.