To the Editor: Plasmacytoid dendritic cells (pDCs) produce inflammatory cytokines upon infection with various pathogens, but can also induce production of Foxp3+ regulatory T cells (Treg).1 Recently, Lombardi et al.2 reported that expression of the α- and β-subunits of CD8 could be used to differentiate three stable subsets of pDC. The subsets expressing CD8α alone or in combination with CD8β were tolerogenic, inducing Treg cells in a mouse model of airway hyperreactivity, whereas CD8α−β−pDC were immunogenic, robustly secreting inflammatory cytokines that exacerbated disease.2 In contrast to these results, we found that CD8 expression by mature pDC is not stable but, rather, is inducible after toll-like receptor (TLR) stimulation, thereby indicating that CD8 expression does not delineate discrete and stable pDC subsets. Purified CD8α−β− pDCs were cultured in the presence or absence of TLR agonists CpG (TLR9), R848 (TLR7), or media alone. Approximately 40% of pDCs cultured in media alone expressed CD8β (Figure 1a). In response to either CpG or R848, a significant proportion of CD8α−β− pDCs upregulated both CD8α and CD8β expression (Figure 1a and b). To determine whether the increase in expression of CD8α and CD8β was associated with cellular activation, we assessed the expression of major histocompatibility complex (MHC) class II and mPDCA-1, both of which are upregulated upon activation of pDCs.1, 3, 4 CD8α+β+ pDCs from the cells cultured with CpG (Figure 1c) or R848 (not shown) had greatly elevated levels of both MHC class II and mPDCA-1 compared with CD8α−β− pDCs in the same cultures or cultured in media alone. Furthermore, we found using an IL12p40-yellow fluorescent protein reporter mouse that expression of IL12p40 following CpG stimulation was restricted to CD8α+ pDC (Figure 1d) with little interleukin (IL)12 produced directly by CD8α− pDCs (Figure 1d).

Figure 1
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pDCs upregulate CD8α and CD8β upon activation in vitro. (a) pDCs were identified by flow cytometry as mPDCA-1+, CD11cint cells and assessed for cell surface CD8α and CD8β (unsorted). Sorted CD8− pDCs (sorted) were cultured for 16 h in media only, 0.5 μM CpG 1668, or 25 μg ml−1 R848, as indicated. (b) Proportion of pDCs that were CD8α+β+ after culture of CD8− pDCs as described in panel a. (c) Cell surface MHC-II (left) and mPDCA-1 (right) on CD8α− pDCs after incubation in media alone (broken line), CD8α−β− pDCs (solid line), and CD8α+β+ (gray filled) pDCs after overnight treatment with CpG 1668. (d) YFP expression by semipurified pDCs isolated from IL12p40-YFP reporter mice5 incubated in media alone or CpG 1668 as described in panel a. Data are representative of three independent experiments. ***P<0.001; ****P<0.0001 for proportions of CD8α+β+ pDCs from seeded CD8− pDCs by two-tailed Mann–Whitney U-test. IL, interleukin; MHC, major histocompatibility complex; pDC, plasmacytoid dendritic cell; YFP, yellow fluorescent protein.

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There have been other reports of pDCs modulating expression of CD8α upon stimulation with TLR agonists.3, 4 Therefore, we suggest that expression of CD8 subunits by pDC mirrors cellular activation rather than defining stable mature subsets with different activities. Lombardi et al.2 observed differential cytokine expression by CD8-expressing pDCs. This may be because CD8-expressing pDC had been activated prior to treatment in vitro with agonists and thus were partially ‘‘exhausted’’ and unable to be fully restimulated to produce cytokines. Whether the pDC acquire a tolerogenic phenotype after their initial activation and upregulation of CD8α/β remains to be fully elucidated.