Dorit Fabricius

Granzyme B produced by human plasmacytoid dendritic cells suppresses T cell expansion

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 GrB-dependent, 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 inhibit, GrB secretion by pDCs. GrB-secreting pDCs may play a regulatory role for immune evasion of tumors, antiviral immune responses, and autoimmune processes. Our results provide novel information about the complex network of pDC-T-cell interactions and may contribute to an improvement of prophylactic and therapeutic vaccinations.

Interleukin 21-induced granzyme B-expressing B cells infiltrate tumors and regulate T cells

The pathogenic impact of tumor-infiltrating B cells is unresolved at present, however, some studies suggest that they may have immune regulatory potential. Here, we report that the microenvironment of various solid tumors includes B cells that express granzyme B (GrB, GZMB), where these B cells can be found adjacent to interleukin (IL)-21-secreting regulatory T cells (Treg) that contribute to immune tolerance of tumor antigens. Because Tregs and plasmacytoid dendritic cells are known to modulate T-effector cells by a GrB-dependent mechanism, we hypothesized that a similar process may operate to modulate regulatory B cells (Breg). IL-21 induced outgrowth of B cells expressing high levels of GrB, which thereby limited T-cell proliferation by a GrB-dependent degradation of the T-cell receptor ζ-chain. Mechanistic investigations into how IL-21 induced GrB expression in B cells to confer Breg function revealed a CD19(+)CD38(+)CD1d(+)IgM(+)CD147(+) expression signature, along with expression of additional key regulatory molecules including IL-10, CD25, and indoleamine-2,3-dioxygenase. Notably, induction of GrB by IL-21 integrated signals mediated by surface immunoglobulin M (B-cell receptor) and Toll-like receptors, each of which were enhanced with expression of the B-cell marker CD5. Our findings show for the first time that IL-21 induces GrB(+) human Bregs. They also establish the existence of human B cells with a regulatory phenotype in solid tumor infiltrates, where they may contribute to the suppression of antitumor immune responses. Together, these findings may stimulate novel diagnostic and cell therapeutic approaches to better manage human cancer as well as autoimmune and graft-versus-host pathologies.

Human B Cells Secrete Granzyme B When Recognizing Viral Antigens in the Context of the Acute Phase Cytokine IL-21

Human B cells are currently not known to produce the proapoptotic protease granzyme B (GrB) in physiological settings. We have discovered that BCR stimulation with either viral Ags or activating Abs in the context of the acute phase cytokine IL-21 can induce the secretion of substantial amounts of GrB by human B cells. Importantly, GrB response to viral Ags was significantly stronger in B cells from subjects recently vaccinated against the corresponding viruses as compared with unvaccinated subjects. GrB-secreting B cells featured a homogeneous CD19+CD20+CD27−CD38−IgD− phenotype, improved survival, and enhanced expression of costimulatory, Ag-presenting and cell-adhesion molecules. B cell-derived GrB was enzymatically active and its induction required the activation of similar signaling pathways as those in CTLs. Our findings suggest that GrB-secreting B cells support the early antiviral immune response against viruses with endosomal entry pathways, thereby counteracting overwhelming viral replication at the beginning of an infection until virus-specific T cells from draining lymph nodes arrive at the site of infection. Our data may also explain the elevated serum GrB levels found in the early phase of various viral diseases.

Prostaglandin E2 Inhibits IFN-α Secretion and Th1 Costimulation by Human Plasmacytoid Dendritic Cells via E-Prostanoid 2 and E-Prostanoid 4 Receptor Engagement

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 B cells differentiate into granzyme B-secreting cytotoxic B lymphocytes upon incomplete T-cell help

Recently, CD4+ T helper cells were shown to induce differentiation of human B cells into plasma cells by expressing interleukin (IL‐)21 and CD40 ligand (CD40L). In the present study we show, that in the absence of CD40L, CD4+ T cell‐derived IL‐21 induces differentiation of B cells into granzyme B (GzmB)‐secreting cytotoxic cells. Using fluorescence‐activated cell sorting (FACS) analysis, ELISpot and confocal microscopy, we demonstrate that CD4+ T cells, activated via their T‐cell receptor without co‐stimulation, can produce IL‐21, but do not express CD40L and rapidly induce GzmB in co‐cultured B cells in an IL‐21 receptor‐dependent manner. Of note, we confirmed these results with recombinant reagents, highlighting that CD40L suppresses IL‐21‐induced GzmB induction in B cells in a dose‐dependent manner. Surprisingly, although GzmB‐secreting B cells did not express perforin, they were able to transfer active GzmB to tumor cell lines, thereby effectively inducing apoptosis. In contrast, no cytotoxic effects were found when effector B cells were activated with IL‐2 instead of IL‐21 or when target cells were cultured with IL‐21 alone. Our findings suggest GzmB+ cytotoxic B cells may have a role in early cellular immune responses including tumor immunosurveillance, before fully activated, antigen‐specific cytotoxic T cells are on the spot. CD40 ligand determines whether IL‐21 induces differentiation of B cells into plasma cells or into granzyme B‐secreting cytotoxic cells.

Human Plasmacytoid Dendritic Cell Function: Inhibition of IFN-α Secretion and Modulation of Immune Phenotype by Vasoactive Intestinal Peptide

Plasmacytoid dendritic cells (PDC) are considered the main sentinels against viral infections and play a major role in immune tolerance. Vasoactive intestinal peptide (VIP) is a potent immunomodulator, whose role in PDC function is unknown. The present study was designed to investigate whether human PDC express VIP receptors and whether VIP has immunological effects on PDC. Using real-time RT-PCR and immunofluorescence, we demonstrated that VIP receptors VPAC1 and VPAC2 are expressed on PDC. After culturing PDC with VIP and CpG oligodeoxynucleotides for 48 h, expression of surface molecules with significance for PDC-T cell interactions as well as IFN-α secretion were quantified using FACS analysis and ELISA, respectively. For functional assays, CFSE-stained CD4+ T cells were coincubated with differentially treated PDC. T cell proliferation and production of various cytokines were determined by FACS analysis and ELISA. VIP enhanced PDC expression of CD86, MHC II, and CCR7. In contrast, VIP inhibited PDC secretion of IFN-α and expression of Neuropilin-1 and MHC I. The potential of CpG oligodeoxynucleotide-activated PDC to induce proliferation of allogeneic CD4+ T cells was impaired when VIP was present during activation. Furthermore, pretreatment of PDC with VIP resulted in a decrease of the IFN-γ:IL-4 ratio in cocultured T cells, suggesting a modulation of the immune response toward Th2. Taken together, these results strongly suggest that VIP regulates the immunological function of human PDC. VIP may thus be involved in the modulation of immune responses to viral infections as well as in the maintenance of immune tolerance.

CD5+ B cells from individuals with systemic lupus erythematosus express granzyme B

Recently, we reported that IL‐21 induces granzyme B (GzmB) and GzmB‐dependent apoptosis in malignant CD5+ B cells from patients with chronic lymphocytic leukemia. Several autoimmune diseases (AD) are associated with enhanced frequencies of CD5+ B cells. Since AD are also associated with elevated IL‐21 and GzmB levels, we postulated a link between CD5+ B cells, IL‐21 and GzmB. Here, we demonstrate that IL‐21 and GzmB serum levels are highly correlated in subjects with systemic lupus erythematosus (SLE) and that freshly isolated CD5+ SLE B cells constitutively express GzmB. IL‐21 directly induced GzmB expression and secretion by CD5+ B cells from several AD and from cord blood in vitro, and the simultaneous presence of BCR stimulation strongly enhanced this process. Furthermore, IL‐21 suppressed both viability and expansion of CD5+ B cells from SLE individuals. In summary, our study may explain the elevated levels of IL‐21 and GzmB in SLE and other AD. Moreover, our data suggest that IL‐21 may have disease‐modifying characteristics by inducing GzmB in CD5+ B cells and by suppressing their expansion. Our results provide the rationale for further evaluation of the therapeutic potential of IL‐21 in certain AD such as SLE.

CD4+ T Cell–Derived IL-21 and Deprivation of CD40 Signaling Favor the In Vivo Development of Granzyme B–Expressing Regulatory B Cells in HIV Patients

IL-21 can induce both plasma cells and regulatory B cells. In this article, we demonstrate that untreated HIV patients display CD4+ T cells with enhanced IL-21 expression and high in vivo frequencies of regulatory B cells overexpressing the serine protease granzyme B. Granzyme B–expressing regulatory B cells (GraB cells) cells from HIV patients exhibit increased expression of CD5, CD43, CD86, and CD147 but do not produce IL-10. The main functional characteristic of their regulatory activity is direct granzyme B–dependent degradation of the TCR-ζ–chain, resulting in significantly decreased proliferative T cell responses. Although Th cells from HIV patients secrete IL-21 in a Nef-dependent manner, they barely express CD40L. When culturing such IL-21+CD40L− Th cells with B cells, the former directly induce B cell differentiation into GraB cells. In contrast, the addition of soluble CD40L multimers to T cell/B cell cultures redirects B cell differentiation toward plasma cells, indicating that CD40L determines the direction of IL-21–dependent B cell differentiation. As proof of principle, we confirmed this mechanism in a patient lacking intact CD40 signaling due to a NEMO mutation. The majority of peripheral B cells from this patient were GraB cells and strongly suppressed T cell proliferation. In conclusion, GraB cells represent potent regulatory B cells in humans that are phenotypically and functionally distinct from B10 cells and occur in early HIV infection. GraB cells may contribute significantly to immune dysfunction in HIV patients, and may also explain ineffective Ab responses after vaccination. The use of soluble CD40L multimers may help to improve vaccination responses in HIV patients.

B-CLL cells acquire APC- and CTL-like phenotypic characteristics after stimulation with CpG ODN and IL-21

CpG oligodeoxynucleotides (CpG) and IL-21 are two promising agents for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Recently, we reported that the combination of CpG and IL-21 (CpG/IL-21) can induce granzyme B (GrB)-dependent apoptosis in B-CLL cells. Here, we demonstrate that treatment of B-CLL cells with CpG and IL-21 results in the development of antigen-presenting cell (APC)-like cells with cytotoxic features. These properties eventually give rise to B-CLL cell apoptosis, independently of their cytogenetic phenotype, whereas normal B-cell survival is not negatively affected by CpG/IL-21. APC- and CTL-typical molecules found to be up-regulated in CpG/IL-21-stimulated B-CLL cells include GrB, perforin, T-bet, monokine-induced by IFN-γ and IFN-γ-inducible protein 10 (IP-10), as well as molecules important for cell adhesion, antigen cross-presentation and costimulation. Also induced are molecules involved in GrB induction, trafficking and processing, whereas the GrB inhibitor Serpin B9 [formerly proteinase inhibitor-9 (PI-9)] is down-modulated by CpG/IL-21. In conclusion, CpG/IL-21-stimulated B-CLL cells acquire features that are reminiscent of killer dendritic cells, and which result in enhanced immunogenicity, cytotoxicity and apoptosis. Our results provide novel insights into the aberrant immune state of B-CLL cells and may establish a basis for the development of an innovative cellular vaccination approach in B-CLL.

Antiviral Vaccines License T Cell Responses by Suppressing Granzyme B Levels in Human Plasmacytoid Dendritic Cells

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.