A method for detecting intracellular perforin in mouse lymphocytes.

Cytotoxic lymphocytes destroy pathogen-infected and transformed cells through the cytotoxic granule exocytosis death pathway, which is dependent on the delivery of proapoptotic granzymes into the target cell cytosol by the pore-forming protein, perforin. Despite the importance of mouse models in understanding the role of cytotoxic lymphocytes in immune-mediated disease and their role in cancer immune surveillance, no reliable intracellular detection method exists for mouse perforin. Consequently, rapid, flow-based assessment of cytotoxic potential has been problematic, and complex assays of function are generally required. In this study, we have developed a novel method for detecting perforin in primary mouse cytotoxic T lymphocytes by immunofluorescence and flow cytometry. We used this new technique to validate perforin colocalization with granzyme B in cytotoxic granules polarized to the immunological synapse, and to assess the expression of perforin in cytotoxic T lymphocytes at various stages of activation. The sensitivity of this technique also allowed us to distinguish perforin levels in Prf1(+/+) and Prf1(+/-) mice. This new methodology will have broad applications and contribute to advances within the fields of lymphocyte biology, infectious disease, and cancer.

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.

Activated mouse B cells lack expression of granzyme B.

Recently, it has been reported that human B cells express and secrete the cytotoxic protease granzyme B (GrB) after stimulation with IL-21 and BCR cross-linking. To date, there are few clues on the function of GrB in B cell biology. As experimental transgenic murine systems should provide insights into these issues, we assayed for GrB in C57BL/6 B cells using an extensive array of physiologically relevant stimuli but were unable to detect either GrB expression or its proteolytic activity, even when Ag-specific transgenic BCRs were engaged. Similar results were also obtained with B cells from DBA/2, CBA, or BALB/c mice. In vivo, infection with either influenza virus or murine γ-herpesvirus induced the expected expression of GrB in CTLs, but not in B cell populations. We also investigated a possible role of GrB on the humoral immune response to the model Ag 4-hydroxy-3-nitrophenylacetyl-keyhole limpet hemocyanin, but GrB-deficient mice produced normal amounts of Ab with typical affinity maturation and a heightened secondary response, demonstrating conclusively the redundancy of GrB for Ab responses. Our results highlight the complex evolutionary differences that have shaped the immune systems of mice and humans. The physiological consequences of GrB expression in human B cells remain unclear, and the current study suggests that experimental mouse models will not be helpful in addressing this issue.

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.

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.

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.

B cell-derived circulating granzyme B is a feature of acute infectious mononucleosis.

Granzyme B (GzmB) is a serine protease best known for inducing target cell apoptosis when released by cytotoxic T lymphocytes (CTLs) or natural killer cells with pore-forming perforin. As a result, GzmB detected in the serum of virus-infected individuals has typically been attributed to these sources. Here, we show that patients with recently diagnosed infectious mononucleosis caused by Epstein-Barr virus (EBV) have high circulating levels of GzmB that may be derived from infected B cells early in course of disease. We recently reported that human B cells from healthy donors secrete active GzmB when stimulated in vitro through B-cell receptor (BCR) ligation and interleukin (IL)-21. We found that infecting B cells with EBV greatly amplified GzmB secretion in response to the same stimuli, but the expression was terminated once the infection had become latent. Our results represent a rare instance of GzmB expression by non-CTL/natural killer cells in the context of infection with a human pathogen.

A colorimetric assay that specifically measures Granzyme B proteolytic activity: hydrolysis of Boc-Ala-Ala-Asp-S-Bzl.

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB's preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

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.

Why do human B cells secrete granzyme B? Insights into a novel B-cell differentiation pathway.

B cells are generally believed to operate as producers of high affinity antibodies to defend the body against microorganisms, whereas cellular cytotoxicity is considered as an exclusive prerogative of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). In conflict with this dogma, recent studies have demonstrated that the combination of interleukin-21 (IL-21) and B-cell receptor (BCR) stimulation enables B cells to produce and secrete the active form of the cytotoxic serine protease granzyme B (GrB). Although the production of GrB by B cells is not accompanied by that of perforin as in the case of many other GrB-secreting cells, recent findings suggest GrB secretion by B cells may play a significant role in early antiviral immune responses, in the regulation of autoimmune responses, and in cancer immunosurveillance. Here, we discuss in detail how GrB-secreting B cells may influence a variety of immune processes. A better understanding of the role that GrB-secreting B cells are playing in the immune system may allow for the development and improvement of novel immunotherapeutic approaches against infectious, autoimmune and malignant diseases.