Activated Mouse B Cells Lack Expression of B Magdalena Hagn, Gabrielle T. Belz, Axel Kallies, Vivien R. Sutton, Kevin Y. Thia, David M. Tarlinton, Edwin D. This information is current as Hawkins and Joseph A. Trapani of October 1, 2021. J Immunol 2012; 188:3886-3892; Prepublished online 16 March 2012; doi: 10.4049/jimmunol.1103285 http://www.jimmunol.org/content/188/8/3886 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Activated Mouse B Cells Lack Expression of

Magdalena Hagn,*,† Gabrielle T. Belz,‡ Axel Kallies,‡ Vivien R. Sutton,*,† Kevin Y. Thia,*,† David M. Tarlinton,‡ Edwin D. Hawkins,*,†,1 and Joseph A. Trapani*,†,x,1

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 phys- iologically relevant stimuli but were unable to detect either GrB expression or its proteolytic activity, even when Ag-specific trans- genic 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 or murine g-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 Downloaded from 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. The Journal of Immunology, 2012, 188: 3886–3892.

ranzyme B (GrB) is a 32-kDa protease that is In human B cells, GrB is induced by IL-21 and is strongly a key constituent in secretory granules of NK cells and enhanced by simultaneous BCR cross-linking. Notably, exposure to G CTLs (1, 2). Five different human , seven in viral Ags can substitute for artificial BCR cross-linking and en- http://www.jimmunol.org/ rats and 10 in mice, have been described to date (3, 4). Classi- hance the IL-21–induced GrB secretion from B cells of vaccinated cally, GrB is released via granule exocytosis toward target cells human donors (16). In a subsequent study, CD40L was found to such as tumor or virus-infected cells where it induces play a determining role in this process: B cells secreted GrB in its by cleaving the BH3-only proapoptotic protein Bid and/or pro- absence but differentiated into plasma cells when it was present cessing of the effector caspase-3 (5, 6). The pore-forming mol- (17). Moreover, malignant human B cells (chronic lymphocytic ecule plays a major role in this process as it facilitates leukemia, B-CLL) secrete low levels of GrB upon triggering by delivery of granzymes into the target cell (7–9). In mice, GrB either BCR cross-linking or CpG oligodeoxynucleotide (CpG

expression has to date been described in T cells, NK cells, and ODN) combined with IL-21. These B cells were found to induce by guest on October 1, 2021 their thymic precursors (10). In contrast, recent evidence has apoptosis in bystander B-CLL cells in a GrB-dependent manner linked human GrB to a broader spectrum of cells including (18). Furthermore, a significant correlation between GrB and IL- CD34+ hematopoietic progenitor cells (11), (12), 21 serum levels has been found in patients with the autoimmune (13), mast cells (14), plasmacytoid dendritic cells (15), disease systemic lupus erythematosus. In these patients, GrB was and B cells (16). constitutively expressed in CD5+ but not CD52 B cells (19). These various studies have revealed a novel function for IL-21 and strongly suggest a role for GrB expression in B cells, which *Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Victo- may contribute to immunosurveillance, early antiviral immune ria 3002, Australia; †Sir Peter MacCallum Department of Oncology, The University response, and autoimmune pathogenesis. Furthermore, we hy- of Melbourne, Melbourne, Victoria 3010, Australia; ‡The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3052, Australia; and xVictorian pothesized that the induced expression of GrB in B cells upon BCR Comprehensive Cancer Centre, Melbourne, Victoria 3052, Australia cross-linking and exposure to IL-21 might play a role in the 1E.D.H. and J.A.T. contributed equally to this work. regulation of contraction of germinal center responses by elimi- Received for publication November 17, 2011. Accepted for publication February 14, nating helper populations such as follicular T helper cells. At this 2012. stage, however, there is little experimental evidence for the This work was supported by Grant HA 6136/1-1 from the Deutsche Forschungsge- physiological importance of GrB expression in B cells or B cell- meinschaft (to M.H.). E.D.H. and J.A.T. are supported by a Peter Doherty fellowship mediated cytotoxicity. Hence, the aim of the current study was and a program grant, respectively, from the National Health and Medical Research Council of Australia. Further support was received from the National Health and to establish a reliable mouse model further to study this interesting Medical Research Council of Australia (to A.K., G.T.B., and D.M.T.), the Sylvia and set of observations. Charles Viertel Foundation and the Howard Hughes Medical Institute (to G.T.B.), and the Australian Research Council Future Fellowship (to A.K.). Address correspondence and reprint requests to Dr. Edwin D. Hawkins and Prof. Materials and Methods Joseph A. Trapani, Peter MacCallum Cancer Institute, St. Andrew’s Place, East Mice, in vivo immunization, and viral infections Melbourne, VIC 3002, Australia. E-mail addresses: [email protected] (E.D.H.) and [email protected] (J.A.T.) Mice 6 to 10 wk of age were used in all experiments and studies performed The online version of this article contains supplemental material. according to the institutional guidelines for animal experimentation of the Abbreviations used in this article: CpG ODN, CpG oligodeoxynucleotide; GrB, Peter MacCallum Cancer Centre and The Walter and Eliza Hall Institute of granzyme B; HEL, hen egg lysozyme; NP–KLH, 4-hydroxy-3-nitrophenylacetyl Medical Research. C57BL/6, BALB/c, DBA/2, and CBA mice were pur- coupled to keyhole limpet hemocyanin. chased from The Walter and Eliza Hall Institute of Medical Research (Melbourne, VIC, Australia). Granzyme B cluster-deficient mice (B6. 2/2 2/2 Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 GrzmB ) and HEL Rag SHH/+ SHL/- Rag mice (SWHel) were bred www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103285 The Journal of Immunology 3887 and maintained at the Peter MacCallum Cancer Centre. In vivo immuni- 10% FBS. Cells were plated on flat-bottom 96-well plates at 100,000 cells/ zation was administered by i.p. injection, with 50 mg of 4-hydroxy-3- well/200 ml (spleen) or 25,000 cells/well/200 ml (lymph node) in the nitrophenylacetyl coupled to keyhole limpet hemocyanin (NP–KLH; The presence of reagents as follows: recombinant mouse IL-21 (100 ng/ml) and Walter and Eliza Hall Institute of Medical Research) in the molar ratio of IL-4 (500 U/ml) (R&D Systems, Minneapolis, MN); CpG ODN 1668 (59- 13:1 as described previously (20), with alum as an adjuvant. Secondary TCCATGACGTTCCTGATGCT-39; GeneWorks, Thebarton, SA, Aus- responses were generated by i.p. injection 45 d later of 10 mg NP–KLH tralia) was used at a final concentration of 1 mM; LPS derived from Sal- suspended in PBS. For viral infections, mice were infected intranasally monella typhosa (Sigma-Aldrich, St. Louis, MO) and anti-CD40 (FGK4.5; with 104.5 PFU HKx31 (H3N2) influenza virus (21) or with 3 3 104 PFU The Walter and Eliza Hall Institute of Medical Research) were at 20 mg/ murine g-herpesvirus-68–OVA (MHV–OVA) (22). ml. BCR engagement in naive B cells was achieved by using Abs against IgM (clone b.7.6) and IgD (clone 1.19) at 10 mg/ml (kindly provided by Cell culture of mouse B cells Prof. Phil Hodgkin, The Walter and Eliza Hall Institute of Medical Re- On day 10 (NP–KLH) or day 5 and day 10 (viral infections) after im- search). For in vivo immunization experiments with NP–KLH, some cells m munization, spleens and mediastinal lymph nodes were harvested from were restimulated in vitro with plate-bound NP13–BSA at 5 g/ml. In euthanized mice. Mononuclear cells were prepared from lymph nodes after SWHEL-transgenic B cell cultures, hen egg lysozyme (HEL) was used for red cell lysis and B cells purified from the spleen. B cell enrichment (96– direct ligation of the BCR (100 ng/ml; Sigma-Aldrich). 99%) was achieved using a negative selection with Ab-coated beads (no. NP ELISA 130-090-862; Miltenyi Biotec, Bergisch Gladbach, Germany). NK cells were isolated from spleens of either B6.GrzmB2/2 or C57BL/6 mice using Serum of immunized B6.GrzmB2/2, C57BL/6, or naive mice was collected MACS-based negative selection (Miltenyi Biotec) with subsequent sepa- from the retro-orbital sinus on day 10, 14, and 21 after injection or on day ration on an autoMACS sorter. Media for primary cell culture consisted of 8 after boost (day 53). Concentrations on NP-specific total or high-affinity RPMI 1640 medium supplemented with 2 mM glutamine, 50 mM2- Abs were determined by ELISA. Briefly, 96-well flat-bottom plates were mercaptoethanol, 100 mM nonessential amino acids, 100 U/ml penicillin, coated with 10 mg/ml NP13–BSA and NP14–BSA (for total Ab) and NP1–

100 mg/ml streptomycin, 1 mM sodium pyruvate, 10 mM HEPES, and BSA and NP2–BSA (for high-affinity Ab) and incubated overnight at 4˚C. Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. Human but not mouse B cells express and secrete active GrB after stimulation of the B cell and the IL-21 receptors. (A) Freshly isolated B cells from peripheral blood of healthy donors (human) or splenic B cells from C57BL/6 mice were incubated for 16 h with anti-BCR, IL-21, or the combination. After culture for 4 h with brefeldin A, cells were harvested, fixed and permeabilized, and stained for intracellular GrB. Cells were analyzed by FACS. Dot plots show GrB+ B cells. (B) Splenic NK cells were isolated from wild-type C57BL/6 or B6.GrzmB2/2 mice and activated for 4 d with IL-2. Shown are intracellular staining for GrB and analysis by FACS. Dot plots indicate percentages of GrB+ B cells. (C) Upper panel, Purified B cells (.99.5%) and non-B cells from healthy human individuals were cultured for 3 d at 1 3 106 per well in the presence of IL-21, anti-BCR, or PHA. After culture, cells were lysed, and ASPase activity of equal amounts of protein was determined at OD405 nm. The error bars represent SEM of data pooled from two in- dependent experiments. Lower panel, Isolated splenic B cells from C57BL/6 mice were incubated for 3 d with anti-CD40, anti-IgM, and anti-IgD in the presence of IL-21. Activated T cells from C57BL/6 mice served as positive control and activated T cells from a B6.GzmA/B2/2 mouse as negative control. After culture, cell lysates were prepared, protein amounts adjusted, and ASPase activity determined. Shown is one representative plot of two independent experiments. The error bars represent SEM of intraexperimental duplicates. (D) Purified human and mouse B cells (2 3 106) were cultured overnight as indicated. Total RNA was isolated and real-time RT-PCR for GrB mRNA performed. Relative quantity compared with non-B cells (upper panel) or wild- type (WT) NK cells (lower panel) was determined by the comparative CT method. Bar graphs represent means of two individual experiments for each mouse (n = 2). Error bars indicate SEM. 3888 MOUSE B CELLS DO NOT EXPRESS GRANZYME B

Table I. Primers used for PCR Intracellular GrB staining After stimulation, brefeldin A (1 mg/ml; A.G. Scientific, San Diego, CA) Gene Sequence (59 to 39) was added for 4 h, and cells were harvested and stained as described Human GrB F: CCATCCAGCCTATAATCCTA previously (16). For human studies, a PE-labeled Ab to GrB (clone GB11; R: CCTGCACTGTCATCTTCACCT Sanquin, Amsterdam, The Netherlands) was used, and for mouse B cells, Human RPL-32 F: GCGTAACTGGCGGAAACCCA an Alexa Fluor 647 mouse anti-human Ab (clone GB11; BD Biosciences, TTGTGAGCGATCTCGGCACA San Jose, CA). To exclude a lack of sensitivity for the Ab to detect GrB in R: 2/2 Mouse GrB F: TGTTTTCTCTGCCATCTGCTCTC mice, in all experiments NK cells from either C57BL/6 or B6.GrzmB R: GCTTTGTAAAAGTCTCCAGCCTGTG were activated over 4 d with IL-2 in 24-well plates at 700,000 cells/ml/well Mouse RPL-32 F: TTCCTGGTCCACAATGTCAAG (1000 U/ml) and used as staining controls (Fig. 1B). Cell surface markers R: TGTGAGCGATCTCAGCAC were analyzed for some mouse experiments using B220–FITC, CD3–Pa- cific blue, or CD138–PE (BD Biosciences). Flow cytometry was per- F, forward primer; R, reverse primer. formed on a FACSCanto or LSR device (BD Biosciences) and data analyzed using FlowJo software (version 8.8.7; Tree Star, Stanford, CA). Standards and serum samples were added serially diluted in blocking GrB secretion and activity assay buffer (PBS, 0.05% Tween-20, 0.6% milk, and 1% FBS) and incubated at room temperature overnight. Plates were then washed and incubated for C57BL/6 B cells were isolated and stimulated overnight with anti-IgM plus 4 h at room temperature with a goat anti-mouse IgG1 HRP secondary Ab anti-IgD (= anti-BCR), anti-CD40 plus IL-4 with or without IL-21. The next (Southern Biotech, Birmingham, AL). After washing, tetramethylbenzi- day, cells were cultured for 4 h in the presence of PMA (1 mg/ml) and dine substrate (Sigma-Aldrich) containing 0.01% H2O2 (Merck, Darm- ionomycin (10 mg/ml), and culture supernatants were collected. Addi- stadt, Germany) was added, the reaction stopped with 1 M H2SO4, and tionally, whole-cell lysates were prepared and normalized for protein plates were analyzed in an ELISA plate reader (VersaMax PLUS ROM content. GrB activity was determined by the hydrolysis of synthetic pep- v.1.21; Molecular Devices, Sunnyvale, CA) at 450 nm. tide thiobenzylester substrate (Asp-ase activity, Boc-Ala-Ala-Asp-S-Bzl; Downloaded from SM Biochemicals), as described previously (25, 26). Supernatants of IL-2– Human B cell culture stimulated NK cells or lysates of activated T cells from either C57BL/6 or B6.GrzmAB2/2 were used as controls. T cells (2.5 3 107 cells/ml in 6- Human B cells were isolated using a MACS-based kit with negative se- well plates) were stimulated with IL-2 (100 U/ml), IL-7 (2 ng/ml), and lection (no. 130-091-151; Miltenyi Biotec). B cells (purity .99%) were purified anti-CD3 and anti-CD28 (100 ng/ml; BD Biosciences) for 3 d. resuspended in AIM-V medium (Invitrogen, Carlsbad, CA) and cultured Cells were split on day 2 when fresh IL-2 was added. on 96-well round-bottom plates at 200,000 cells/well/200 ml, if not stated http://www.jimmunol.org/ otherwise. The following reagents were used as in vitro B cell stimuli: Real-time RT-PCR human recombinant IL-21 (50 ng/ml) was purchased from Invitrogen and 6 affinity purified rabbit F(ab9)2 against human IgA plus IgG plus IgM (H Murine or human B cells (2 3 10 ) were isolated and activated as described plus L) was used for polyclonal BCR stimulation at 6.5 mg/ml (anti-BCR; earlier. After culture, total RNA was obtained using the RNeasy mini kit Jackson ImmunoResearch Laboratories). Non-B cells (= B cell-depleted (Qiagen, Doncaster, Australia) according to the manufacturer’s methods. fraction) were activated with a combination of 100 U/ml human RNA was reverse-transcribed using M-MLV Reverse Transcriptase (RNase recombinant IL-2 (Proleukin; National Cancer Institute, Bethesda, MD) H Minus, Point Mutant) and oligonucleotide primer (both from Promega, and PHA [1 mg/ml (23, 24)]. Madison, WI). Real-time quantitative PCR was performed using an by guest on October 1, 2021

FIGURE 2. Mouse B cells do not express GrB after in vivo infection with influenza virus. Mice (n = 4) were infected intranasally with 104.5 PFU HKx31 influenza virus. Mediastinal lymph nodes and spleens were harvested on day 5 (n = 2) and day 10 (n = 2) postinfection. Organs of two naive mice served as a baseline control. Mononuclear cells (lymph nodes) and B cells (spleen, purity .98%) were isolated and cultured. Brefeldin A was added for 4 h im- mediately after isolation [ex vivo, (A, B)] or after stimulation for 16 h with LPS, anti-CD40 plus IL-4, and CpG ODN in the presence or absence of IL-21. (C) After culture, cells were harvested and stained for CD3, CD19, and B220 (lymph nodes) and intracellular GrB (lymph nodes and spleen). (A) Dot plots indicate baseline GrB expression of one representative naive mouse or mice after 5 d and 10 d postinfection, respectively. The upper panels show gating strategy for CD3+ T cells and B220+ B cells. The middle and lower panels illustrate percentages of GrB+ cells as analyzed by FACS. (B) Line graph depicts average ex vivo GrB expression of B and T cells from lymph node and splenic B cells of two mice 6SEM. (C) Bar graphs show percentage cells with GrB expression after 16 h of culture with reagents as indicated. Error bars indicate SEM of data from two mice. The Journal of Immunology 3889

SYTO9 green fluorescent nucleic acid stain (Invitrogen) in a Corbett Rotor we analyzed the cells by flow cytometry, we found that unlike Gene 6000 cycler (Corbett Life Sciences, Concorde, Australia). Primers human B cells, murine B cells did not express GrB (Fig. 1A), used for PCR (all from Sigma) are presented in Table I. although they were activated and underwent multiple rounds of Stimulated non-B cells served as a positive control for human experi- ments and activated C57BL/6 NK cells for mouse B cells. Samples were run division after 48 h as demonstrated by dilution of CFSE (Sup- in duplicate and mRNA expression of GrB normalized to RPL-32 using plemental Fig. 1). Furthermore, extending cultures for up to Rotor Gene 6000 Software (version 1.7) and Microsoft Excel 2004. The 3 d was not sufficient to induce GrB expression (data not shown). (2ΔΔCt) relative expression levels to positive controls were calculated as 2 . Similar results were found in B cells derived from BALB/c, CBA, Statistics or DBA/2 mice (Supplemental Fig. 2). In all experiments, IL-2– activated NK cells from either B6.GrzmB2/2 or C57BL/6 mice 6 Data are expressed as mean SEM. Statistical differences between the were used as negative or positive staining controls, respectively means of two data groups was determined by using the paired or unpaired Student t test, and p values ,0.05 were considered significant. (Fig. 1B). Human GrB was enzymatically active when secreted into B cell culture supernatants (17) and thus was confirmed once again by testing whole-cell lysates of activated human B cells for Results cleavage of the highly specific Ala-Ala-Asp substrate (Fig. 1C, Stimulation with IL-21 and BCR cross-linking induces GrB in upper panel); however, neither proteolytic activity of cell lysates human but not in murine B cells (Fig. 1C, lower panel) nor secretion of active GrB in cell culture In previous reports, we demonstrated that the IL-2 family supernatants (Supplemental Fig. 3) was detected when murine IL-21 in combination with BCR ligation induces expression and B cells were stimulated in a similar fashion. To determine whether secretion of the GrB in human B cells (16, 17, 19). the GrB gene is transcribed in mouse B cells, we measured GrB These studies suggested that GrB might play a significant role in mRNA in stimulated B cells. In humans, GrB mRNA is not Downloaded from the early anti-viral immune response and the control of autoim- constitutively expressed but requires IL-21 and engagement of the munity. To enable more detailed studies of the underlying mech- BCR (Ref. 16 and Fig. 1D, upper panel). In mice, however, GrB anisms, we investigated the induction of GrB in CFSE-labeled mRNA levels were not significantly different from those of GrB- B cells from C57BL/6 mice in response to anti-CD40 plus IL-4 deficient mice, in which the gene has been largely deleted (Fig. 1D, and BCR stimulation in the presence or absence of IL-21. When lower panel, Table I). Similar results were obtained for GrA (whose http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. In vivo immunization with NP–KLH does not induce GrB in mouse B cells, and GrB deficiency does not alter B cell responses. C57BL/6 (n = 9) and B6.GrzmB2/2 (n = 9) mice were injected i.p. with the T cell-dependent Ag NP–KLH, and B cell responses assessed on day 10. B cells from immunized mice (wild-type and knockout) and one naive control were isolated from spleen. Brefeldin A was added immediately after isolation (A) or after 16 h of culture with LPS, anti-CD40 plus IL-4, CpG ODN, and plate-bound NP–KLH, with or without IL-21 (B). After culture, cells were harvested, fixed and permeabilized, and stained for intracellular GrB. Data shown are one representative set from two independent experiments. (A) B cells and non-B cells of immunized and naive mice were stained ex vivo for baseline GrB expression and analyzed by flow cytometry. Dot plots show percentage of GrB in splenic B cells from one representative mouse (n = 4). (B) B cells were cultured for 16 h with several B cell stimuli as indicated above. Dot plots show percentage of GrB+ B cells in primary response to Ag of one representative wild-type and one knockout mouse (n = 4). (C and D) Sera of immunized C57BL/6 and B6.GrzmB2/2 mice were collected on day 10 (n = 5), day 14 (n = 7), and day 21 (n = 4) after injection with 50 mg NP–KLH (C) or on day 8 after boost (day 53 after immunization, n =4)(D). Bar graphs show total Ab (left panels) and high-affinity Ab (right panels) to NP as detected by ELISA. The error bars represent SEM of data pooled from two independent experiments. One naive C57BL/6 mouse served as negative control (data not shown). 3890 MOUSE B CELLS DO NOT EXPRESS GRANZYME B gene is located on a separate chromosome) and GrC, the gene that 3A). Next, we stimulated B cells from immunized mice for 1 to is tightly linked to the GrB gene (data not shown). 3 d with or without IL-21 and other stimuli such as LPS, CD40 plus IL-4, and CpG ODN 1668 to cover a wide spectrum of B cell Infection with influenza virus does not induce GrB in B cells activators. Direct antigenic restimulation as a highly specific We recently demonstrated that restimulation of Ag-specific B cells stimulus was performed with plate-bound NP–KLH (Fig. 3B, with from previously vaccinated human subjects with viral Ags trig- day 1 shown as a representative time point). Although all stimuli gered GrB expression (16). We therefore tested GrB expression in induced typical morphological changes indicating activation of the the B cells of mice infected with influenza virus. Referring to B cells (Supplemental Fig. 1), we could not detect intracellular previous studies, we hypothesized that GrB expression might GrB at any time point. bridge innate and adaptive immune responses (17). Therefore, we tested an early time point postinfection (day 5) and at a time when GrB is redundant for Ab responses to NP a T cell response would be generated (day 10). As expected, in- The role of GrB in infection, cancer, and has been fection with influenza induced GrB in cytotoxic T cells ex vivo, studied extensively (28–31); however, it is still unclear whether most notably by day 10, but we failed to detect the presence of GrB contributes to humoral immune responses, potentially by GrB in B cells (Fig. 2A, 2B). The effect in T cells was enhanced B cell-induced death of helper cells leading to the contraction of by adding IL-21 and various other stimuli to cell cultures (Fig. 2C the germinal center. Thus, we collected serum from NP–KLH– and data not shown). However, no GrB was detected in B cells at immunized mice on day 10, 14, and 21 after primary immuniza- any time point, irrespective of the stimuli applied. To examine if tion or on day 8 after boost (day 53 after first injection) and tested exposure to persistent viral pathogens was required for GrB ex- for total Ab and high-affinity Abs to NP. We found no significant pression, we tested a second model of infection, murine g-her- difference between wild-type and B6.GrzmB2/2 mice in either Downloaded from pesvirus. Similar to the results obtained with the influenza model, response (Fig. 3C, 3D). T cells expressed GrB after exposure to virus, whereas B cell Direct ligation of SW -transgenic BCRs fails to induce GrB populations did not (Supplemental Fig. 4). HEL in B cells Ag-specific mouse B cells lack expression of GrB As BCR cross-linking appears to play a fundamental role in GrB

We next used the model B cell Ag NP–KLH to address the role of induction in human B cells (16, 17), we applied a transgenic mouse http://www.jimmunol.org/ GrB in the humoral immune response. This model enabled us to model that enabled us to test Ag-specific BCR stimulation. B cells + engage both the B cell and IL-21 receptors and CD4 follicular T from SWHEL-transgenic mice produce Abs of high affinity to HEL helper cells that are known to be crucial for humoral immune and can undergo Ig class switch recombination and somatic responses during the germinal center reaction via the production hypermutation (32). We bred this model onto a RAG2/2 back- of IL-21 (27). Wild-type C57BL/6 and B6.GrzmB2/2 mice were ground so that all B cells expressed a specific BCR against HEL. immunized i.p. with 50 mg NP–KLH Ag and spleens harvested IL-21 and simultaneous expression of CD40L has previously been 10 d after immunization. We first tested baseline GrB expression shown to induce differentiation of B cells to plasma cells in by incubating freshly purified B cells for 4 h with brefeldin A and a CD4+ T cell-dependent manner (33). We recently showed that subsequent testing for intracellular GrB by flow cytometry (Fig. B cells secreted GrB when stimulated by IL-21, but only if CD40 by guest on October 1, 2021

Hel FIGURE 4. GrB in B cells is not detected by direct ligation of Ig -transgenic BCRs. B cells from SWHEL mice were isolated from spleen, resuspended in B cell media, and cultured for 4 d with LPS, anti-CD40 plus IL-4, and CpG ODN in the presence or absence of IL-21 and HEL, or a combination of both. (A) Intracellular GrB expression was determined on day 1 to day 4 by FACS. Dot plots show one representative experiment on day 1 of four independent experiments. (B) CD138 staining was performed on day 3 and 4 as a positive control to assess engagement of the BCR. Histogram shows expression of CD138 in B cells stimulated with LPS and soluble HEL on day 3. The Journal of Immunology 3891

ligation is absent (17). Similarly, B cells from SWHEL mice dif- and may play a role in reproduction (49), whereas in mice GrN ferentiate into plasma cells when stimulated with LPS, but not (and not GrB) is found in this organ (50). GrB also shows ex- when soluble HEL is added to the culture (34, 35). We hypothe- tensive polymorphism in wild mice (51), whereas only three sized that ligation of the BCR in these cultures might contribute to common human GrB alleles have been identified (52). To exclude the loss of plasma cells by inducing GrB-secreting B cells. B cells strain differences, we also tested CBA, DBA/2, and BALB/c mice, were purified from SWHEL mice, cultured for 4 d with LPS, anti- but we found no expression of GrB. CD40 plus IL-4 and CpG ODN 1668, with or without IL-21 and In conclusion, the current study indicates that findings on GrB soluble HEL. B cells were then stained for intracellular GrB and cannot be generalized from one species to another. The physio- for expression of the plasma cell marker CD138. We observed no logical and pathological relevance of GrB expression in human induction of GrB expression in the presence of HEL (Fig. 4A), B cells still remains unclear, but mouse models are unlikely to although engaging the BCR with soluble HEL induced the ex- provide insight into these processes. Our study demonstrates the pected loss of plasma cells (Fig. 4B). need to develop novel in vivo systems to study human immune responses. Discussion To date, GrB expression in human B cells has been described in Acknowledgments the context of viral infections (16), autoimmunity (19), and cancer We thank Viki Milovac, Sophie Kotsakidis, and Ralph Rossi for skilled as- (18), suggesting a regulatory or modifying role in these diseases. sistance with cytometry experiments and the animal facility staff for main- Yet, exact mechanisms involved in GrB expression of B cells and tenance and housing of mice (all from Peter MacCallum Cancer Centre). the physiological relevance of these findings require further in- Furthermore, we thank Alexander Dobrovic (Department of Pathology, Pe- vestigation. In the absence of a recognized syndrome of human ter MacCallum Cancer Centre) and Dion Kaiserman (Monash University, Downloaded from granzyme deficiency, mouse models have been invaluable for our Melbourne, VIC, Australia) for technical assistance. We also thank Robert understanding of granzyme biology. For example, GrzmB2/2 Brink for supplying HEL-transgenic mice (Garvan Institute, Sydney, NSW, Australia). mice survive most experimental virus infections (36, 37), but the combined loss of GrA and GrB results in a severe immunological defect revealed by infection with poxviruses such as ectromelia Disclosures (38, 39). The authors have no financial conflicts of interest. http://www.jimmunol.org/ We reasoned that if human B cells express GrB, it is likely that certain stimuli will bring about a similar effect in murine B cells. References However, we failed to detect GrB or its proteolytic activity using 1. Hoves, S., J. A. Trapani, and I. Voskoboinik. 2010. The battlefield of perforin/ a variety of assays in mouse B cells stimulated with B cell activators granzyme cell death pathways. J. Leukoc. Biol. 87: 237–243. in combination with IL-21, the most potent inducer of GrB in 2. Afonina, I. S., S. P. Cullen, and S. J. Martin. 2010. 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