ALBERT

Description: Abstract 3905 Immune regulation is central for the development of an efficient cellular immune response. Both Treg cells and plasmacytoid DC can suppress T cell proliferation in a granzyme B (GzmB)-dependent and perforin-independent manner. In the present study we found that, depending on stimulation with interleukin (IL-) 21, B cells (BC) can also express GzmB and effectively suppress T cell proliferation. GzmB expression in BC is enhanced by BC receptor engagement, and is suppressed by CD40 ligation. Since CD4+ T cells are a main source of IL-21, we tested whether they can induce GzmB in BC. We found that incompletely activated CD4+ T cells, but not fully activated T cells induce GzmB in co-cultured BC. Using confocal microscopy, we showed that BC-derived GzmB is enzymatically active and that GzmB+ BC transfer GzmB to CD4+ T cells. Furthermore, GzmB+ BC decreased CD4+ T cell expression of the TCR-zeta chain, a GzmB target, which is required for T cell proliferation. Our results suggest BC may regulate cellular adaptive immune responses by Treg cell-like mechanisms. Inhibition of BC-derived GzmB may represent a novel strategy to induce more effective and comprehensive cellular immune responses. Disclosures: No relevant conflicts of interest to declare.

Description: 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.

Description: B cells are not currently known to be capable of producing granzyme B or being cytotoxic. We recently found that human B cells activated with Interleukin 21 (IL-21) and antibodies to the B cell receptor (BCR) or immunostimulatory oligonucleotides (CpG ODN), can produce granzyme B. Further studies were done to assess the biological and potential therapeutic significance of this finding. Granzyme B ELISpot, intracellular staining for granzyme-B, quantitative real time RT-PCR for granzyme B messenger RNA and gene expression array confirmed B cells obtained from the peripheral blood of normal individuals (Fig. 1) and many B cell lines including Namalwa, Daudi, Ramos and EBV-transformed lymphoblasts, can be induced to produce granzyme B. This granzyme B is functional as demonstrated by cleavage of a granzyme B-sensitive colorimetric substrate. IL-21 based treatment also increased the transcription of the gene for perforin and the production of Interferon-γ in select B cell populations. We conclude that IL-21 based therapy can induce B cells to produce functional granzyme B and other components known to be present in the cytotoxic granules of CTL and NK cells. These unexpected findings could have significant implications on our understanding of the role of B cells in immune regulation and for a variety of immune phenomena including auto-, cancer and infectious immunity. Figure 1. Interleukin 21 (IL-21) induces de-novo synthesis of Granzyme B by activated B cells. Peripheral blood mononuclear cells (PBMC) were stimulated with the above depicted agents for 18 hours. After further incubtion with Brefeldin A for 4 hours cells were harvested, fixed, permeabilized, stained with PE- and PE-Cy5-labeled antibodies against Granzyme B and CD19, and analyzed flowcytometrically. Figure 1. Interleukin 21 (IL-21) induces de-novo synthesis of Granzyme B by activated B cells. Peripheral blood mononuclear cells (PBMC) were stimulated with the above depicted agents for 18 hours. After further incubtion with Brefeldin A for 4 hours cells were harvested, fixed, permeabilized, stained with PE- and PE-Cy5-labeled antibodies against Granzyme B and CD19, and analyzed flowcytometrically.

Description: Interleukin 21 (IL-21), a recently discovered cytokine with structural homology to IL-2, IL-4 and IL-15, has pleiotropic effects on lymphocyte populations including NK, T and B cells and is currently undergoing early clinical evaluation. We explored the effect of the combination of IL-21 and immunostimulatory CpG ODN on B chronic lymphocytic leukemia (B-CLL), and other CD5-positive B cells. IL-21 plus CpG ODN were synergistic in their ability to induce apoptosis of the B-CLL cells, and also induced production and secretion of granzyme B from the B-CLL cells. B-CLL cells treated with IL-21 and CpG ODN were capable of inducing apoptosis of untreated autologous B-CLL cells. This bystander killing was inhibited by anti-granzyme B antibodies. The effect was observed in all cases of CD5-positive B-CLL, but not in CD5-negative B-CLL samples. IL-21 plus CpG ODN also induced granzyme B production and apoptosis of benign CD5-positive B1 cells obtained from umbilical cord blood. In contrast, the number of CD5-negative B2 cells increased in the same samples during in vitro culture, resulting in a decreased ratio of CD5-positive to CD5-negative cord blood B cells (Fig. 1). Our results indicate the combination of IL-21 and CpG ODN is able to induce apoptosis of both benign and malignant CD5-positive B cells. Given the suspected role of B1 cells in autoimmune diseases, our findings could have important implications for the understanding of their pathogenetic mechanisms. These results might also open new avenues for the development of novel therapies for both autoimmune dieseases and CD5-positive B-CLL. Figure 1. IL- 21 and CpG ODN therapy selectively eliminates CD5 positive B cells in cord blood. Figure 1. IL- 21 and CpG ODN therapy selectively eliminates CD5 positive B cells in cord blood.

Description: Abstract 2675 Poster Board II-651 Interleukin 21 (IL-21) is a novel and highly promising cytokine for the treatment of neoplastic and infectious diseases. Recently, IL-21 has been identified as inducer of plasma cell differentiation. Here we show that CD40 ligand is critically involved in this process and that, in its absence, human B cells differentiate into granzyme B (GzmB)-secreting cytotoxic cells rather than plasma cells. GzmB expression and secretion by human B cells was demonstrated by FACS analysis, ELISpot, ELISA, Sensizyme, Western immunoblotting, RT-PCR, and spinning disk confocal microscopy. GzmB secretion requires the presence of IL-21 and B cell receptor engagement, and depends on phosphorylation of JAK1/3 and STAT3. CD40 ligation effectively suppresses GzmB secretion by B cells, suggesting GrB-secreting B cells play a role in the early phase of inflammatory processes, before CD40 ligand-expressing T cells are present. Of note, ex-vivo re-stimulation of B cells from recently vaccinated individuals with inactivated viruses also induces GzmB expression. GzmB is enzymatically active and GzmB-secreting B cells induce apoptosis in various tumor cell lines, a process we were able to visualize by using spinning disk confocal microscopy. Our data reveal an as yet unrecognized role of IL-21-activated B cells, which involves GzmB secretion and cellular cytotoxicity. Our findings may have implications for the understanding of tumor immunosurveillance and early anti-viral immune responses, and may open novel approaches for the immunotherapy of neoplastic and viral diseases. Disclosures: No relevant conflicts of interest to declare.

Description: Despite their low frequency of 0,1 - 0,5% in the peripheral blood, human plasmacytoid dendritic cells (pDC) are important modulators of adaptive immune responses and have meanwhile taken the step from the bench to promising initial tumor vaccination studies. PDC express high levels of the IL-3-receptor CD123, MHC class II and blood dendritic cell antigens (BDCA)-2 and -4. Apart from secretion of IFN-alpha and their capacity to rapidly initiate antigen-specific CD8+ T cell responses by cross-presentation, they produce large amounts of the serine protease granzyme B (GrB). GrB exhibits various non-cytotoxic functions including T cell regulation and the support of antigen processing and phagocytosis. We therefore hypothesized that pDC may use their GrB for both T cell regulation and for antigen uptake and processing. PDC were isolated using magnetically labeled antibodies against CD123, and cultured in the presence of various stimuli. Here, we demonstrate that pDC are potent producers of enzymatically active GrB, which reached maximal concentrations up to two logs higher than those produced by cytotoxic cells including CTL and NK cells. The strongest inducers of GrB in pDC were IL-3 and IL-10, whereas toll-like receptor (TLR)-7 or TLR-9 ligands such as CpG ODN or imiquimod as well as CD40 ligand suppressed GrB induction. We demonstrate that pDC-derived GrB can suppress effector T cell proliferation by GrB-mediated degradation of the T cell receptor zeta-chain. Moreover, we tested the uptake of fluorescently labeled antigenic material into pDC after lysis of tumor cells by freeze-thaw cycles and irradiation. These studies revealed a correlation between pDC GrB expression and antigen uptake, with GrBhigh pDC taking up significantly more antigenic material than GrBlow pDC. Our data support a novel concept, in which pDC acquire their maximal immunogenic potential in a two-step activation process: First, high levels of GrB are induced by IL-3 and IL-10, thereby supporting antigen processing and uptake into pDC, while preventing precocious activation of T cells. Subsequently, TLR agonists and CD40 ligand turn off GrB in pDC and initiate the transfer of MHC-antigen complexes and co-stimulatory molecules to the pDC surface, resulting in highly immunogenic pDC. Our data may be of translational relevance for the further development of novel DC vaccination strategies in tumor therapy. Disclosures No relevant conflicts of interest to declare.

Description: Recently, we and others found that B cells differentiate into regulatory B cells (Breg) in response to interleukin (IL-)21. Of note, the key characteristic of human IL-21-induced Breg is expression of the serine protease granzyme B (GrB), whereas murine Breg, which require both IL-21 and CD40 ligand (CD40L) for their induction, predominantly express the immunosuppressive cytokine IL-10. Using two different disease models and various immunological methods, we further characterized the conditions leading to Breg differentiation in humans. Here, we demonstrate that in humans CD40L determines whether IL-21 induces differentiation of B cells into plasma cells (CD40L presence) or into GrB+ Breg (CD40L absence), which can directly control T cell proliferation by GrB-dependent degradation of the T cell receptor z-chain. Furthermore, we show that GrB+ Breg are circulating at high frequencies in the peripheral blood of untreated, highly viremic HIV patients, but not in healthy subjects. Of note, HIV-infected CD4+ T cells express IL-21, but not CD40L, and induce a GrB+ regulatory phenotype in healthy third party B cells in vitro. Consequently, addition of CD40L multimers can compensate for this insufficient T helper cell function, resulting in increased plasma cell/Breg ratios. Moreover, we investigated a patient with a congenital defect of Nuclear-Factor-kappa-B-Essential-Modulator (NEMO), which is essential for normal CD40 signaling. Even in the presence of viral infections, when CD4+ T helper cells from such patients are highly activated with strong expression of IL-21, they are not able to establish sufficient antibody responses. Instead, we found this patient to almost exclusively harbor B cells with a regulatory phenotype including high basal levels of GrB. When untreated NEMO B cells were co-cultured with allogeneic T cells from a healthy third party donor, these T cells failed to proliferate and to survive in response to a 6-day stimulation with anti-CD3/CD28 antibodies, an effect not observed with B cells from healthy donors. Since NEMO B cells lack normal CD40 signaling, our findings unequivocally demonstrate that in contrast to murine Breg IL-21-dependent induction of human Breg can occur in a CD40-independent fashion. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2674 Poster Board II-650 Human plasmacytoid dendritic cells (pDC) play a central role in regulating 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. We found that human pDC can be an abundant source of GrB based on FACS analysis, ELISpot, ELISA, Sensizyme, Western immunoblotting, RT-PCR, and fluorescence microscopy. GrB is actively secreted by pDCs and reaches maximal levels up to two logs higher than those produced by classical GrB producers such as CTL or NK cells. However, pDC GrB production is not accompanied by perforin secretion. Spinning disk confocal microscopy revealed that GrB+ pDC bind to and transfer active GrB to T cells. Importantly, this GrB transfer induces a suppression of T cell proliferation in a GrB-dependent, perforin-independent manner, a process reminiscent of regulatory T cells. GrB expression in pDC is regulated on a transcriptional level by JAK1, STAT3 and STAT5. IL-3 and IL-10 enhance GrB production by pDCs while GrB production is inhibited by toll-like-receptor agonists and CD40 ligand. These findings suggest that GrB production by pDCs is involved in the complex interactions between pDC and T cells and that GrB-secreting pDC may play a regulatory role related to anti-tumor immunity, anti-viral immune responses, and autoimmune processes. Disclosures: No relevant conflicts of interest to declare.

Description: B cells are not currently known to produce granzyme B (GrB) in (patho-) physiological settings. We recently reported that B-chronic lymphocytic leukemia cells and normal B cells treated with interleukin-21 (IL-21) and anti-B cell receptor antibodies (anti-BCR) produce functional GrB. Here we demonstrate for the first time that viral antigens can also induce specific peripheral B lymphocytes to produce and secrete substantial amounts of active GrB. Using FACS, ELISpot, immunofluoresence and western blot we show that B cells from subjects recently vaccinated against tick-borne encephalitis virus (TBEV) but not unvaccinated subjects respond to viral TBEV antigens with GrB secretion in a dosedependent manner. This response is direct and occurs only in the presence of co-activation with certain IL-2 family cytokines such as IL-21. Similar results were found with other viruses including hepatitis B and rabies. GrB production in B cells required activation of JAK1 and STAT3 and inhibition of JAK1 by pyridone 6 completely abrogated GrB induction by viral antigens or anti-BCR. Our findings suggest GrB secretion by B cells may be part of a novel, anti-viral immune response mechanism. Further studies will elucidate whether or not granzyme B-secreting B cells can act as cytotoxic cells towards virus-infected cells.