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: 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 1042 Certain regulatory lymphocyte subpopulations including plasmacytoid dendritic cells and regulatory T cells secrete granzyme B (GrB), thereby suppressing T cell expansion. Recently, we found that B cells can also produce GrB and acquire regulatory potential in response to interleukin (IL-)21. Since HIV has been shown to be associated with elevated serum IL-21 levels, we hypothesized that GrB-expressing B cells may be induced during HIV infection. Here, we show that infection of CD4+ T cells with HIV 1 (NL4-3), but not mock infection, induces strong expression of IL-21 without upregulation of CD40 ligand. Importantly, we further demonstrate that such IL-21+CD40Llow T cells induce GrB in cocultured B cells in an IL-21-dependent fashion, rather than supporting their differentiation into plasma cells. In support of these findings, serum levels of both IL-21 and GrB are significantly higher in HIV-infected patients before HAART as compared to healthy controls. Up to 60% of freshly isolated B cells (36.2% ± 12.9%) from patients infected with HIV, but not B cells isolated from healthy individuals, express GrB. Of note, coculture of HIV-infected CD4+ T cells with GrB+ B cells result in GrB transfer, and strongly suppresses both, proliferation of T cells and viral replication as indicated by significantly reduced p24 levels in coculture supernatants. The observed effects are enhanced by IL-21, and reduced by GrB inhibition. In summary, we demonstrate that HIV infection induces IL-21 in CD4+ T cells, thereby indirectly triggering the development of GrB-secreting B cells with antiretroviral properties. GrB-secreting B cells may play a so far unappreciated role in decelerating HIV expansion, particularly in the early phase of infection. On the other hand, induction of GrB in HIV-specific B cells may interfere with their terminal differentiation into plasma cells, which may explain the lack of an efficient anti-HIV humoral immune response in HIV-infected patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3278 The role of B cells in tumor infiltrations is controversial. Different studies suggest that certain tumor-infiltrating B cell populations exhibit regulatory potential. Here, we demonstrate that the microenvironment of various solid tumors contains granzyme B (GrB)-expressing B cells adjacent to IL-21-providing T cells. GrB-mediated effector T cell modulation is already known from regulatory T cells (Treg) and plasmacytoid dendritic cells. We now show that IL-21 induces B cells to express high levels of GrB and to modulate T cell proliferation by GrB-dependent degradation of the T cell receptor z-chain. Detailed characterization of IL-21-induced GrB+ B cells reveals a CD19+CD38+CD1d+CD147+ phenotype and expression of additional regulatory molecules including IL-10, IDO and CD25. Of note, GrB induction is accompanied by both BCR- and TLR-mediated signals and GrB expression levels are influenced by B cell expression of CD5. In summary, we demonstrate that IL-21 induces GrB-expressing regulatory B cells, which are detected in tumor infiltrations, and which may contribute to the modulation of cellular adaptive immune responses by Treg-like mechanisms. Our findings may stimulate the development of novel diagnostic and cell therapeutic approaches to the management of malignant, autoimmune and graft-versus-host pathologies. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 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.

Description: Abstract 3222 We and others have recently provided evidence that a series of lymphocyte subsets including, plasmacytoid dendritic cells, B cells and regulatory T cells are able to secrete the cytotoxic serine protease granzyme B (GrB) into the extracellular compartment, where it contributes to the suppression of T cell proliferation by a so far undefined GrB-dependent mechanism. For B cells, we found that viral antigens in the context of the acute phase cytokine interleukin (IL-) 21 can potently induce GrB. Here, we demonstrate that infection of CD4+ T cells with HIV-1 (NL4-3), but not mock infection, induces strong expression of IL-21 on both mRNA and protein levels in CD4+ T cells. Moreover, we found that HIV-infected CD4+ T cells are able to induce high levels of GrB in co-cultured B cells and that inhibition of IL-21 with specific antibodies abrogates T cell-mediated GrB induction in B cells. In support of these data, serum levels of both IL-21 and GrB are significantly higher in patients acutely infected with HIV as compared to healthy control subjects. Surprisingly, co-culture of CD4+ T cells with B cells during HIV-1 infection strongly suppressed both, proliferation of T cells as well as virus replication as indicated by significantly reduced p24 levels in culture supernatants. Notably, this effect was enhanced by external stimulation of B cells with IL-21, and was reduced by inhibition of GrB using specific GrB inhibitors. To further explore the underlying mechanisms of our findings, we performed confocal microscopy of T cell-B cell co-cultures and demonstrated that GrB-secreting B cells directly interact with CD4+ T cells, thereby transferring active GrB to them. Moreover, we found that GrB+ B cells decreased CD4+ T cell expression of the T cell receptor-zeta chain, a known GrB target, which is required for T cell proliferation, and known to be suppressed in HIV patients. In summary, we provide evidence that HIV induces the acute phase cytokine IL-21 in infected CD4+ T cells, thereby indirectly triggering the expression of GrB by B cells. GrB-secreting B cells may play a so far unappreciated role in decelerating the expansion of HIV, particularly in the early phase of acutely infected patients. Our study reveals a novel pathogenetic mechanism in HIV infection with potential relevance for the development of novel immunotherapeutic approaches. Disclosures: No relevant conflicts of interest to declare.