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.

Recently, we reported that IL-21 induces granzyme B (GzmB) and GzmB-dependent apoptosis in malignant CD5 1 B cells from patients with chronic lymphocytic leukemia. Several autoimmune diseases (AD) are associated with enhanced frequencies of CD5 1 B cells. Since AD are also associated with elevated IL-21 and GzmB levels, we postulated a link between CD5 1 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 1 SLE B cells constitutively express GzmB. IL-21 directly induced GzmB expression and secretion by CD5 1 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 1 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 1 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. IntroductionHuman B cells expressing the common T-cell antigen CD5 are hypothesized to represent a unique B-cell population and are mainly found in fetal cord blood and in serous cavities of healthy adult individuals [1,2]. Given that CD5 1 B cells are the major source of poly-reactive natural antibodies, these cells have long been suspected of playing a pathogenic role in the development of autoimmune diseases (AD) such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) or psoriasis [3][4][5]. 2060Meanwhile it is known that autoimmune antibodies are also produced by CD5 À B cells, and that CD5 1 B cells are neither necessary nor sufficient for the development of autoimmunity [2,6]. In contrast, recent findings suggest that CD5 1 B cells can even act to prevent inflammatory and AD [3,[6][7][8], although the exact mechanisms by which they may control autoimmunity remain elusive.IL-21, a type I cytokine that shares the common cytokine receptor g-chain with IL-2, IL-4, IL-7, IL-9 and IL-15 and is produced by CD4 1 T cells, NKT cells and Th17 cells [9][10][11][12]. It exhibits pleiotropic effects on a range of lymphoid lineages and is involved in the pathogenesis of autoimmunity [10,11,13,14]. Recently, we demonstrated that in the absence of CD40 ligation, IL-21 can induce large amounts of the serine protease granzyme B (GzmB) in peripheral B cells [15]. GzmB represents a major component of the granules of NK cells and CTL [16]. Classically, GzmB has been linked primarily to the induction of apoptosis in target cells after attack by cytotoxic cells. It is known that GzmB is produced by a variety of other cell types including plasmacytoid dendritic cells [17], B cells [15,18], basophils [19], mast cells [20] and CD34 1 hematopoietic progenitor cells [21]. Consequently, the spectr...

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.

CD5+ B1 cells are thought to play a key role in innate immune responses by secreting natural immunoglobulins. Here we demonstrate that human B1 cells (B1C) are able to express the pro‐apoptotic serine protease granzyme B (GzmB) in response to viruses including tick‐borne encephalitis, rabies and hepatitis B virus. We reveal that this response depends on B1C receptor stimulation, TLR9 engagement and interleukin 21 (IL‐21), a cytokine primarily found in the acute phase of viral infections. Up to 35% of freshly isolated B1C directly respond with GzmB expression to stimulation with inactivated viruses in the presence of IL‐21. Of note, GzmB is secreted by B1C in an enzymatically active state, reaching levels comparable to those secreted by cytotoxic cells and its induction requires activation of similar signaling pathways as in CTL and NK cells. Our findings suggest GzmB secretion by B1C represents a novel innate immune response mechanism. GzmB‐secreting B1C may play a role in the early anti‐viral immune defense, and may contribute to elevated serum GzmB levels found in viral diseases. Further studies will elucidate whether B1C cells exhibit cytotoxicity towards virus‐infected or tumor cells.

3906 Interleukin 21 (IL-21) and CpG oligodeoxynucleotides (CpG ODN) are two novel and highly promising agents for the treatment of hematological diseases. Recently, we reported that IL-21 and CpG ODN induce granzyme B (GrB) and GrB-dependent apoptosis in malignant B cells from patients with B chronic lymphocytic leukemia (B-CLL), but not in healthy peripheral B cells. Here we further characterized the factors accountable for the different apoptotic response of B-CLL cells versus normal B cells. GrB induction in B-CLL cells after stimulation with IL-21 and CpG ODN was associated with upregulation of molecules, which are normally involved in the differentiation of cytotoxic T lymphocytes (CTL) including T-bet, perforin, monokine-induced by interferon-gamma (MIG) and IP-10, a finding not observed in normal healthy B cells. Furthermore, the induction of GrB in B-CLL cells by IL-21 and CpG ODN required signaling via a JAK/STAT-dependent pathway, as suggested by upregulation of phosphorylated STAT1/3. Finally, stimulation of B-CLL cells with IL-21 and CpG ODN upregulated molecules involved in cell adhesion (CD54), antigen presentation (MHC class I), co-stimulation (CD40, CD86) and GrB uptake (CD222, mannose-6-phosphate receptor), suggesting B-CLL cells activated with IL-21 and CpG ODN are able to contact other immune cells and may be able to reabsorb secreted GrB. In summary, B-CLL cells can express factors involved in cytotoxic differentiation of CTL as well as GrB in response to stimulation with IL-21 and CpG ODN. Our data provide novel insights into the aberrant signaling state of B-CLL cells and may pave the way for the development of novel immunotherapeutic treatment approaches for B-CLL. Disclosures: No relevant conflicts of interest to declare.