The present study demonstrates that human pDCs are composed of two subsets, which can be distinguished by the expression of CD2. These two subsets are found not only in blood but also in tonsils. In addition to their circulation in the blood and secondary lymphoid organs such as tonsils, CD2high pDCs can be detected in some tumor biopsies suggesting their potential involvement in tumor immunosurveillance. Both subsets secrete IFN-α and express the cytotoxic molecules Granzyme B and TRAIL. The CD2high pDCs are potent in initiating T cell immune responses. In contrast, the CD2low pDCs appear to display a limited capacity to induce allogeneic T cell proliferation. These different functional properties of CD2high pDCs and CD2low pDCs are associated to distinct transcription profiles, differential secretion of IL12 p40 and with differential expression of co-stimulatory molecule CD80 on activation.
In concordance with other studies, Granzyme B inhibitor does not seem to affect cytotoxic function of pDC. Granzyme B-mediated killing of target cells seems to be completely dependent on the presence of perforin (60
), though alternative mechanism has been suggested (63
). As pDCs do not express perforin, they must either use another protein that can substitute or they use Granzyme for biological function other than killing. Certain acute viral (HIV, EBV) infections seem associated with elevated plasma levels of Granzyme A and B (64
). While it was considered to be associated with an ongoing CTL activation (64
), the source of extracellular Granzyme B in these patients may need to be revisited in the view of our results. Furthermore, elevated Granzyme B plasma levels were also observed in patients with parasite infection with Plasmodium falciparum (65
) and in severe bacterial infection (66
). These in vivo observations are consistent with high secretion triggered in vitro by pDC activation via CD40 and TLR ligands, respectively. Recent studies demonstrate the involvement of Granzyme B in extracellular matrix remodeling via cleavage of vitronectin, laminin and fibronectin (67
). That may lead in vitro to anoikis, cell death due to lack of extracellular contact (67
). This, together with their capacity to secrete Granzyme B, could point to a novel role of pDC in inflammation as well as tumor development and progression.
Thus, the killing mechanism remains to be established. Three recent studies in the human and in the mouse suggested a role of TRAIL-mediated cytotoxicity exerted by cells with pDC properties (68
). Our preliminary results corroborate the potential role of TRAIL (not shown) however the inhibition is not complete suggesting contribution of other mechanisms. The killing is unlikely to be due to contaminating NK cells as it requires high E:T target ratio, is slow (NK cell mediated-lysis usually occurs at 4hrs), we do not detect IFN-γ transcription or secretion as well as we do not do detect perforin (data not shown).
pDCs highly transcribe genes involved in the immune functions such as lysozyme, AXL or fractalkine receptor. Another feature is the expression of two genes linked to Th2 polarization (ALOX5) and Th2 signature such as IgE (RGC32). Thus, CD2high
pDCs might be responsible for the capacity of pDCs to induce type 2 immunity (10
). Conversely, CD2low
pDCs highly transcribe genes involved in cell cycle regulation and in secretory functions. Non-activated CD2high
pDC also show 2-fold higher transcription of CD2 as compared to non-activated CD2low
pDC (Supplementary Fig. 3
). While upon activation both subsets express MHC class molecules and CD83, CD2high
pDCs up-regulate CD80. While both subsets secrete similar levels of cytokines and chemokines such as IL-6, IL-8, TNF alpha, IFN alpha, IP-10, MIP1-α, and MIP1-β on activation, CD2high
pDCs secrete IL-12 p40. Preliminary results show 2-fold enhanced transcription of p19 in microarrays of activated CD2high
pDCs (not shown). Thus, the identification of common and unique transcripts further supports the notion of two pDC subsets in the humans.
The differential expression of lymphoid-related genes (RAG1 and Ig rearrangement products)(71
) or proteins (CD4)(72
) and Ly49Q(73
) has been used to demonstrate the existence of murine pDC subsets. Similar to our results in the human, murine pDC subsets differ in their capacity to trigger allogeneic T cell proliferation and to secrete IL12p70 and IFN-γ (71
pDCs have been shown to arise from both myeloid and lymphoid committed hematopoietic progenitor cells (CMP and CLP, respectively) (74
). The CD2 subset clearly demonstrates the prevalent expression of myeloid related genes thus suggesting that pDCs originating from CMP might have distinct functions than those originating from CLP. Thus, both in human and mouse pDCs constitute a heterogeneous population of cells which display a remarkable array of immune functions. The existence of two pDC subsets might provide a framework for explanation of a diverse array of immune responses elicited by pDCs ranging from CD8+
T cell tolerance/anergy, generation of regulatory T cells, to Th2 polarization.