Human thymus contains IFN-α–producing CD11c–, myeloid CD11c+, and mature interdigitating dendritic cells (original) (raw)
Isolation of three distinct thymic DC populations. A multistep isolation method was developed including (a) depletion of sheep erythrocyte rosette-forming cells on a Ficoll-density gradient; (b) magnetic bead depletion of B cells (CD19+), T-lineage cells (CD3+, CD7+), monocytes (CD14+), and NK cells (CD56+); and (c) fluorescence-activated sorting of cells negative for CD3, CD7, CD14, and CD19 (27 ± 15%; range 13.7–54%; n = 8) on the basis of CD11c expression and either intermediate or high levels of HLA-DR. As illustrated in Figure 1a, three Lin– HLA-DR+ populations were isolated: HLA-DRint CD11c– (R2, 25.5 ± 12% of Lin– cells; range 13.6–50.8%; n = 8), HLA-DRint CD11c+ (R3, 10.2 ± 4%; range 5.4–16.7%; n = 8), and a population expressing high levels of both HLA-DR and CD11c (R4, 4.5 ± 2%; range 0–6.7%; n = 8). Freshly isolated HLA-DRint CD11c– cells displayed a characteristic plasma cell–like morphology (Figure 1, b and e) resembling the plasmacytoid T cells (8), whereas both CD11c+ populations possessed an irregular outline and a polylobulated nucleus (Figure 1, c, d, f, and g). HLA-DRint CD11c+ cells had a heterogeneous morphology, and 50% of them had the features of the one presented in Figure 1f. Numerous fine dendrites characterized the HLA-DRhi subset (Figure 1d).
Purification of thymic DC subsets. (a) DC-enriched Lin– cells (R1) contain HLA-DRint CD11c– pDC (R2), HLA-DRint CD11c+ imDC (R3), and HLA-DRhi CD11c+ mDC (R4) subpopulations. FSC, forward scatter; PE-Cy5, phycoerythrin–cyanin 5.1. (b–d) Giemsa staining. (e–g) Transmission electron microscopy. Bar = 1 μm. (h–j) HLA-DR (red) and DC-LAMP (blue) stainings. Original magnification (b–d, h–j): ×1,000.
We next tested the capacities of the three HLA-DR+ populations to stimulate CD4+ CD45RA+ allogeneic T cells. All three subsets induced naive T-cell proliferation, thereby demonstrating that they all exhibited dendritic cell function (Figure 2). We accordingly refer to immature HLA-DRint CD11c–, HLA-DRint CD11c+, and mature HLA-DRhi CD11c+ subpopulations as plasmacytoid DCs (pDCs), immature DCs (imDCs), and mDCs, respectively.
Thymic DCs induce proliferation of CD4+ CD45RA+ T cells. A total of 5 × 104 PB naive CD4+ T cells were cocultured with graded doses of fresh mDCs, imDCs, pDCs, or IL-3–treated pDCs, and monocyte-derived DCs (Mo-DC) as positive control, for 5 days. Results are expressed as mean ± SD of triplicate cultures and are representative of three experiments.
Phenotypic analysis of thymic DC populations. To characterize the thymic DC subsets with a panel of PE-conjugated mAb’s (Figure 3), we modified the isolation procedure described here by omitting the use of anti-CD11c PE. The modification exploited the similar expression of CD11c and CD13 on thymic DC subsets. Thus, Lin– cells (identified using PC5-conjugated anti-CD3, -CD7, -CD14, and -CD19 mAb’s) were sorted into either HLA-DRint or HLA-DRhi (using a FITC-conjugated mAb). The sorted cells were devoid of CD3-, CD14-, CD19-, and CD56-contaminating cells (data not shown) and also did not express IL-7Rα and CD34, excluding thymocyte or progenitor contamination. All the sorted cells expressed high levels of CD40 and CD4, but not CD8α. HLA-DRint cells contained (a) pDCs, a lymphoid-related population; CD13–, CD33lo, CD11c–, CD45RO–, CD45RA+, IL-3Rα+, and CD86lo, CD83–; and (b) imDCs, a myeloid-related population; CD13+, CD33+, CD11c+, CD45RO+, CD45RA–, IL-3Rα–, and CD86lo, CD83–. The HLA-DRhi mDC/IDCs had a broad range of staining intensity with CD13 and CD11c mAb’s and expressed a phenotype of mDCs indicated by high levels of HLA-DR and expression of CD86 and CD83. Only mDC/IDCs were labeled intracellularly by an mAb specific for DC-LAMP, a newly described mDC-specific lysosomal marker (14) (Figure 1, h–j). Unlike blood DC subsets, thymic DCs were CD62L–, suggesting that, as previously observed in tonsils, emigration of circulating cells from blood into peripheral lymphoid organs results in CD62L cleavage (9). Finally, all three subsets expressed low levels of CD1a, intermediate levels of GMCSF-Rα, and heterogeneous levels of the T cell–related markers CD2, CD5, and CD7.
Immunophenotype of isolated thymic DC subsets analyzed by flow cytometry. Thymic DCs were sorted into Lin– (PE-Cy5) HLA-DRint (FITC) and Lin– HLA-DRhi subsets. Anti–CD13-PE-Cy5 labeling of HLA-DRint cells clearly resolved two distinct populations. CD13+ HLA-DRint, CD13– HLA-DRint, and CD13+ HLA-DRhi DCs were analyzed using PE-conjugated mAb’s for the expression of a number of lymphoid, myeloid, costimulatory, and adhesion markers. Data shown are representative of three experiments. Ag, antigen.
Thymic mDCs have the features of IDCs. Purification of the mature thymic DC subset enabled their extensive characterization. In addition to CD83, CD86, and DC-LAMP proteins, mDCs were shown to express mRNA characteristic of mature IDCs. Depending on their maturation or activation stage, DCs produce a series of soluble molecules implicated in the initiation of the immune response. IL-12p40 mRNA was strongly detected in mDC, which also expressed DC-LAMP mRNA, and a faint band of IL-12p40 mRNA was also observed in imDCs but not in pDCs (Figure 4). Two chemokines, TECK and TARC, have been shown to be expressed by murine thymic DCs (33, 34). Both TECK and TARC mRNA were restricted to the mDC subset, although they were barely detectable in the total thymocyte population. The expression of the chemokine receptor is also strictly regulated during the maturation process of DCs in vivo. For example, CCR7 has been described only in mDCs (35). In line with the mature phenotype of the HLA-DRhi CD11c+ DC, CCR7 mRNA was only found in mDCs. A disintegrin metalloproteinase designated decysin (36) was demonstrated to be highly induced upon spontaneous or CD40-dependent maturation of CD11c+ imDC subset. The mDC subset displayed decysin mRNA expression, in contrast to the imDCs and pDCs. Taken together, these observations showed that HLA-DRhi population expressed all the properties of mDCs and corresponded to the thymic IDCs.
Thymic mDCs express genes specific to IDCs. Fresh thymic DC subsets, in vitro–matured pDCs, and total thymocytes were analyzed for expression of mRNA for IL-12p40, DC-LAMP, TECK, TARC, CCR7, decysin, and β-actin.
Thymic DCs are localized in the medulla. The anatomic localization of the three DC subsets was analyzed by in situ stainings using either immunohistochemistry or immunofluorescence. Figure 5, a–f, shows that DC-LAMP+ cells and IL-3Rα+ CD45RA+ cells are both located in the medulla. As with the phenotype observed for freshly isolated mDCs, all DC-LAMP+ cells coexpressed the mDC markers CD40 and CD86 (Figure 5, e and f). Two-color immunofluorescence analyses of thymus sections further clearly demonstrated that DC-LAMP+ cells were CD45RA– and IL-3Rα– (Figure 5, i and l). Different levels of CD11c intensity on DC-LAMP+ cells could suggest heterogeneity among this population (Figure 5o). The CD11chi DC-LAMP– cells could be either imDCs, macrophages, or granulocytes, cell types that in humans also express CD11c. Using anti–DC-LAMP and anti-CD68 mAb’s, we showed that CD68+ macrophages did not express DC-LAMP (Figure 5b), confirming that DC-LAMP was uniquely expressed in mature thymic IDCs. It appears that unlike T cells, both thymic IL-3Rα+ CD45RA+ pDCs and DC-LAMP+ mDC/IDCs were located in the medulla, regardless of their stage of differentiation.
The thymic medulla contains both pDCs and mDCs. All DC-LAMP+ cells were located in the medulla (M), identified by the presence of Hassall’s corpuscles (HC), either using anti–DC-LAMP antibody revealed in blue (a, d, and f) or in red (b, c, and e). Double stainings identified DC-LAMP+ cells coexpressing CD40 (blue, e) and CD86 (red, f), characteristic of mDCs that are clearly distinct from CD68+ macrophages (blue, b). CD45RA+ (blue, c) IL-3Rα+ (red, d) pDCs are also located in the medulla and are distinct from mature CD45RA– IL-3Rα– CD11c+ DC-LAMP+ DCs (i, l, and o). Patterns of the individual colors are depicted separately for anti–DC-LAMP (red, g and m; or green, j), -CD45RA (h), -IL3Rα (k), and -CD11c (n). M, medulla; C, cortex; HC, Hassall’s corpuscles. Original magnification: (a, f) ×200; (b–e, g–o) ×400.
Thymic pDCs express lymphoid-associated transcripts. IL-3Rα mRNA was selectively present in pDC (Figure 6a), whereas the abundance of GM-CSFR transcripts was comparable in the three DCs subsets (data not shown). Because the thymus contains precursor cells capable of giving rise to DCs and to T, B, and NK cells (16, 18, 19), we investigated in purified thymic DCs the expression of lymphoid-related transcripts. Figure 6a shows that although the three DC subsets lacked CD3γ, CD19, RAG-1, and RAG-2 mRNA, pDCs expressed pre-Tα and λ-like 14.1 mRNA, that are T and B progenitor genes (37, 38). Moreover, while searching for pDC specific markers (M.-C. Rissoan, unpublished data), we found a tag sequence corresponding to the Ets family transcription factor Spi-B (39) that has been described as exclusively expressed in lymphoid cells (40). As shown in Figure 6a, thymic pDCs expressed high levels of Spi-B transcripts that are, unlike pre-Tα and λ-like transcripts, also found in mDCs. The levels of Spi-B mRNA expression in the different thymic DC subsets, was analyzed by quantitative PCR. Approximately tenfold higher levels of Spi-B mRNA were observed in mDCs than in imDCs, which increased a further tenfold in pDCs (Figure 6b). It has been hypothesized that murine CD8α+ FasL-expressing DCs negatively regulate T-cell activation through Fas-FasL–induced apoptosis (41). None of the human thymic DC subsets expressed FasL mRNA (Figure 6a). Overall, expression of lymphoid specific genes in thymic pDCs further argues in favor of their lymphoid origin. In addition, the presence of both myeloid and lymphoid markers within the thymic IDC population suggest that IDCs are heterogeneous.
Thymic pDCs express lymphoid-associated transcripts. (a) Fresh thymic DC subsets, IL-3/CD40L–treated pDCs, and total thymocytes were analyzed for expression of mRNA for IL-3Rα, CD3γ, CD19, RAG-1, RAG-2, pre-Tα, λ-like 14.1, Spi-B, FasL, and β-actin. (b) Expression levels of Spi-B mRNA in subsets of thymic DCs. Spi-B/β-actin ratios were determined by quantitative RT-PCR. Standard curves were calculated in which the first point of the curve containing the highest amount of cDNA was set arbitrarily to 100 U for β-actin and to 560,000 U for Spi-B. Each symbol represents an independent experiment.
Thymic pDCs develop into mature DCs upon stimulation with IL-3 and CD40L. As described in tonsils (8), IL-3 rescued thymic pDCs from apoptosis and allowed their maturation with a rapid upregulation of CD86 (Figure 7a) correlating with a higher T-cell stimulatory activity (Figure 2). However, expression of certain surface molecules varied upon culture conditions. Thymic pDCs cultured in IL-3 or, most strikingly, in IL-3/GM-CSF/TNF-α expressed heterogeneous levels of IL-3Rα, CD11c, CD13, and CD33 (Figure 7a). Stimulation by CD40L together with IL-3 resulted in the differentiation of pDCs into mDCs principally characterized by the induction of DC-LAMP, both at the protein and mRNA levels (Figure 4; and Figure 7, a–c), and maintained expression of IL-3Rα and CD45RA (Figure 7a). The in vitro–matured DCs also accumulated mRNA for IL-12p40, TARC, CCR7, and decysin (Figure 4), markers that are specific for fully mDCs, whereas TECK was not induced. Regarding the lymphoid transcripts, in vitro–matured DCs no longer expressed pre-Tα mRNA, suggesting that pre-Tα is downregulated concomitantly with the acquisition of a mature phenotype. Unlike pre-Tα mRNA, λ-like and Spi-B mRNA were still observed in thymic pDCs after 4 days of culture with IL-3 and CD40L. Taken together, these results show that pDCs mature in vitro into cells exhibiting all the characteristics of mDCs and sharing most markers of thymic IDC. Moreover, the maintained expression of Spi-B in in vitro–matured DCs combined with the presence of Spi-B transcripts in pDCs and mDCs suggests that thymic IDCs may represent a mature stage arising from both the lymphoid-related pDC subset and the myeloid imDC subset.
Phenotype and morphology of thymic pDCs after in vitro–induced maturation. (a) In vitro–cultured pDCs display a phenotype specific for immature (IL-3 or GM-CSF/TNF-α/IL-3) or mature DCs (IL-3/CD40L). Filled histograms represent isotype-matched controls; open histograms represent staining with specific antibodies. (b) IL-3–treated pDCs acquire a dendritic morphology showing strong staining for HLA-DR, and (c) develop into mature DC-LAMP+ DCs upon further CD40 activation. Original magnification (b and c): ×200.
Thymic CD11c– pDCs produce IFN-α in response to influenza virus. It was recently reported that PB pDCs are the natural IFN-α–producing cells (9, 10). To investigate the capacity of thymic pDCs to produce IFN-α, we exposed them to formaldehyde-inactivated influenza virus for 24 hours after cell sorting, and then IFN-α was measured by ELISA. As shown in Figure 8a, production of IFN-α correlated with the enrichment of the pDC fraction. Indeed, total thymic suspensions (thymocytes) did not produce IFN-α, although some IFN-α became detectable after elimination by rosetting of CD2-positive cells, and even more after CD3, CD7, CD14, CD19, and CD56 depletion (DC enriched). Highly purified sorted pDCs produced high levels of IFN-α (2,721 IU/ml) in response to influenza virus, unlike CD11c+ DCs (including imDCs and mDCs). In a separate experiment, we also confirmed that only pDCs and neither imDCs nor mDCs were capable of producing IFN-α (data not shown). The IFN-α–producing capacities of pDCs isolated from thymus, blood, or tonsils were compared. Thymic pDCs produced IFN-α at levels comparable to those obtained with tonsillar pDCs (respectively, 1,371 ± 500 IU/ml [n = 4] and 2,158 ± 590 IU/ml [n = 2]), but lower than those produced by circulating blood pDCs (15,063 ± 257 U/ml, n = 2) (Figure 8b). Monocyte-derived DCs were unable to produce IFN-α under such stimulation (Figure 8b).
Thymic pDCs produce large amounts of IFN-α after being stimulated with influenza virus. Cells (2 × 105) were cultured for 24 hours with inactivated influenza virus. Without influenza virus, IFN activity from all cell types used was less than 5 IU/ml. (a) Mo-DC: monocyte-derived DCs after 6 days of culture with GM-CSF and IL-4. Thymocytes: total thymic suspension. CD2-depleted: thymic mononuclear cells devoid of CD2+ cells by Ficoll Rosetting. DC-enriched: thymic CD2– mononuclear cells that were depleted of Lin+ cells. CD11c– pDC: FACS-sorted Lin– HLA-DRint CD11c– cells. CD11c+ DC: FACS-sorted Lin– HLA-DRint and HLA-DRhi CD11c+ cells. (b) IFN-α production by thymic HLA-DRint CD11c– pDCs and CD4+ CD11c– pDCs purified from blood and tonsils. Each symbol represents an independent experiment.