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 z-chain. Mechanistic investigations into how IL-21 induced GrB expression in B cells to confer Breg function revealed a CD19

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...

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, 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.

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...

The role of B cells in tumor infiltrations is controversially discussed. 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. Since GrB-mediated effector T cell modulation is known from regulatory T cells (Treg) and plasmacytoid dendritic cells, we hypothesized the existence of similar mechanisms in B cells. Here we show that IL-21 induces B cells expressing high levels of GrB and controlling T cell proliferation by GrB-dependent degradation of the T cell receptor ζ-chain. Detailed characterization of IL-21-induced GrB+ B cells reveals a CD19+CD38+CD1d+IgM+CD147+ phenotype and expression of additional regulatory molecules including IL-10, CD25 and IDO. Of note, IL-21-mediated GrB induction integrates both BCR- and TLR-mediated signals and is enhanced in the presence of B cell CD5 expression. This is the first report demonstrating that IL-21 induces GrB+ human regulatory B cells, which can be 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.

<p>MOV file - 3136K, Supplemental Video 1. B cells transfer active GrB to T cells.</p>

<p>PDF file - 182K, Supplemental Figure 1. B cells activated with IL-21 and anti-BCR secrete enzymatically active granzyme B. Supplemental Figure 2. Interleukin 21 in the absence of B cells does not suppress T cell proliferation. Supplemental Figure 3. GrB-secreting B cells do not induce apoptosis in T cells. Supplemental Figure 4. IL-10, CD25 and IDO are expressed on IL-21-induced GrB+ Breg, but not on GrB- B cells. Supplemental Figure 5. IL-2 retitration has no significant effect on the suppression of CD4+ T cell proliferation by GrB-secreting B cells. Supplemental Figure 6. IL-21-induced GrB secretion by B cells depends on TLR7 and TLR9 signaling. Supplemental Figure 7. B cells in the microenvironment of solid tumors sporadically express IL-10.</p>