Human plasmacytoid dendritic cells (pDCs) are crucially involved in the modulation of adaptive T-cell responses in the course of neoplastic, viral, and autoimmune disorders. In several of these diseases elevated extracellular levels of the serine protease granzyme B (GrB) are observed. Here we demonstrate that human pDCs can be an abundant source of GrB and that such GrB ؉ pDCs potently suppress T-cell proliferation in a GrBdependent, perforin-independent manner, a process reminiscent of regulatory T cells. Moreover, we show that GrB expression is strictly regulated on a transcriptional level involving Janus kinase 1 (JAK1), signal transducer and activator of transcription 3 (STAT3), and STAT5 and that interleukin-3 (IL-3), a cytokine secreted by activated T cells, plays a central role for GrB induction. Moreover, we find that the immunosuppressive cytokine IL-10 enhances, while Toll-like receptor agonists and CD40 ligand strongly IntroductionPlasmacytoid dendritic cells (pDCs) represent a central link between innate and adaptive immunity and play a crucial role in viral, autoimmune, and neoplastic diseases. [1][2][3][4] One of their most prominent features is the ability of pDCs to produce and secrete large amounts of type I interferons (IFNs), thereby initiating and orchestrating antiviral immune responses. 1,5 pDCs can also function as antigen-presenting cells and stimulate effector T-cell responses. 6 However, pDC effects on T-cell subsets can include both T-cell activation (immunogenic function) 7 and induction of T-cell anergy (tolerogenic function). 7,8 pDCs therefore play an important role in fine-tuning cellular immune responses depending on the microenvironment. Several studies suggest that both the activated T cell-derived cytokine interleukin-3 (IL-3) 9 and the immunosuppressive cytokine IL-10 induce a rather tolerogenic pDC phenotype associated with suppression of T-cell responses. [10][11][12] In contrast, activation of pDCs in the presence of ligands for the pDC-characteristic Toll-like receptors (TLRs) TLR7 and TLR9 1 and for CD40 10 results in an immunogenic phenotype with such pDC triggering a proinflammatory immune response including T-cell activation and cytotoxicity. 6,13,14 Several inflammatory diseases including viral and autoimmune diseases have been found to be associated with elevated levels of extracellular granzyme B (GrB). Granzymes including GrB have been found to be locally elevated in bronchoalveolar lavage fluid from patients with chronic allergic asthma and in synovial fluid from patients with rheumatoid arthritis. 15 Infections with cytomegalovirus after renal transplantation, with the dengue fever virus or with HIV, have been associated with high serum levels of GrB. 15 Granzymes such as GrB represent a major constituent of the granules of cytotoxic cells, including cytotoxic T lymphocyte (CTL) and natural killer (NK) cells. The classical function of GrB is induction of apoptosis in target cells recognized by CTLs. 16,17 Evidence is growing that apart from its cytotoxic effec...

Plasmacytoid dendritic cell (PDC)-derived IFN-α plays a central role in antiviral defense and in Th1-driven autoimmune diseases, such as systemic lupus erythematosus (SLE). In the current study, we explored how PGE2 effects the phenotype of PDCs from healthy and SLE subjects. Although PGE2 is considered to mediate mainly proinflammatory effects, we show that PGE2 and PG analogs potently inhibit secretion of IFN-α by TLR-activated PDCs. This effect is mainly mediated by PG receptors E-prostanoid 2 and E-prostanoid 4 and involves inhibition of IFN regulatory factor 7 expression. Of note, profound IFN-α inhibition by PGE2 is also seen in PDCs from SLE subjects, independent of age, disease activity, and therapy. We show that TLR9-activated PDCs treated with PGE2 exhibit DC2-like characteristics with enhanced expression of CD86 and CD62L, and decreased expression of CD80 and MHC class I. Consequently, PGE2-treated PDCs suppress secretion of Th1 cytokines by T cells while increasing the secretion of Th2 cytokines. Prevention of CpG-induced CD62L downregulation by PGE2 suggests that it may induce the retreat of PDCs from inflamed tissues. Our data on the effects of PGE2 on PDCs may explain occasional reports about the induction of SLE-like symptoms by cyclooxygenase inhibitors as well as improvement of such symptoms by treatment with PG analogs. In conclusion, our data suggest that PGE2 and certain PG analogs, some of which are already in clinical use, should be evaluated as a novel and inexpensive treatment approach for patients with SLE and other IFN-α–dependent, Th1-driven autoimmune diseases.

Human plasmacytoid dendritic cells (pDC) are important modulators of adaptive T cell responses during viral infections. Recently, we found that human pDC produce the serine protease granzyme B (GrB), thereby regulating T cell proliferation in a GrB-dependent manner. In this study, we demonstrate that intrinsic GrB production by pDC is significantly inhibited in vitro and in vivo by clinically used vaccines against viral infections such as tick-borne encephalitis. We show that pDC GrB levels inversely correlate with the proliferative response of coincubated T cells and that GrB suppression by a specific Ab or a GrB substrate inhibitor results in enhanced T cell proliferation, suggesting a predominant role of GrB in pDC-dependent T cell licensing. Functionally, we demonstrate that GrBhigh but not GrBlow pDC transfer GrB to T cells and may degrade the ζ-chain of the TCR in a GrB-dependent fashion, thereby providing a possible explanation for the observed T cell suppression by GrB-expressing pDC. Modulation of pDC-derived GrB activity represents a previously unknown mechanism by which both antiviral and vaccine-induced T cell responses may be regulated in vivo. Our results provide novel insights into pDC biology during vaccinations and may contribute to an improvement of prophylactic and therapeutic vaccines.

Human plasmacytoid dendritic cells (pDC) are important modulators of adaptive T cell responses during viral infections. Recently, we found that human pDC produce the serine protease granzyme B (GrB), thereby regulating T cell proliferation in a GrB-dependent manner. Here, we demonstrate that intrinsic GrB production by pDC is significantly inhibited in vitro and in vivo by clinically used vaccines against viral infections such as tick-borne encephalitis (TBEV). We show that pDC GrB levels inversely correlate with the proliferative response of co-incubated T cells and that GrB suppression by a specific antibody or a GrB substrate inhibitor results in enhanced T cell proliferation, suggesting a predominant role of GrB in pDC-dependent T cell licensing. Functionally, we demonstrate that GrBhigh, but not GrBlow pDC transfer GrB to T cells, and may degrade the zeta-chain of the T cell receptor in a GrB-dependent fashion, thereby providing a possible explanation for the observed T cell suppression by GrB-expressing pDC. Modulation of pDC-derived GrB activity represents a previously unknown mechanism by which both anti-viral and vaccine-induced T cell responses may be regulated in vivo. Our results provide novel insights into pDC biology during vaccinations and may contribute to an improvement of prophylactic and therapeutic vaccines.

3221 Human plasmacytoid dendritic cells (pDC) play a central role in regulating adaptive T cell responses in the course of neoplastic, viral and autoimmune disorders. Recently, we demonstrated that apart from their pro-inflammatory effects, which are mainly mediated by secretion of interferon-alpha (IFN-a), pDC can also exhibit potent anti-inflammatory functions borne by active secretion of granzyme B (GrB), a serine protease classically known from CTL, NK cells and regulatory T cells. Here, we hypothesized, that commonly used anti-viral vaccines may affect pDC on several levels including their immunophenotype as well as their capacity to secrete either IFN-a or GrB. Using various methods including FACS, ELISpot, ELISA, spinning-disk confocal microscopy and RT-PCR, we could demonstrate that various anti-viral vaccines including vaccines against TBEV, yellow fever, polio, measles, rotavirus, varicella and hepatitis B were able to affect pDC by modulating expression of a series of surface molecules involved in cell adhesion, antigen-presentation and co-stimulation. In addition, major differences between the vaccines were found in terms of their effects on secretion of IFN-a and GrB. Interestingly, while only one vaccine, FSME Immun (TBEV) induced substantial IFN-a responses in pDC, all others tested did not. In contrast, virtually all vaccines tested induced more or less strong suppression of GrB secretion by maturing pDC. Of note, pDC that secreted high amounts of GrB induced by far lower allogeneic T cell proliferation as compared to pDC that secreted little or no GrB. Moreover, suppression of pDC-derived GrB by a substrate-specific GrB inhibitor resulted in significant enhancement of T cell proliferation in co-cultures of GrB-secreting pDC with allogeneic T cells. Our data demonstrate 1) that anti-viral vaccines may have distinct effects on both the immunophenotype and the secretory potential of pDC, and 2) that GrB is an important novel variable affecting the capacity of pDC to either trigger or dampen adaptive T cell responses. Our results may have implications for further study of the role pDC play in the regulation of adaptive immune responses, and for the potential application of this knowledge in the development of novel adjuvants admixed to anti-viral vaccines. Disclosures: No relevant conflicts of interest to declare.