Human B Cells Secrete Granzyme B When Recognizing Viral Antigens in the Context of the Acute Phase Cytokine IL-21¹

Full activation of virus-specific CTL normally requires the uptake of viral Ags by APC, migration of these APC into the draining lymph nodes, and processing and presentation of Ags by the APC in the context of MHC molecules, a complex process that can take up to several days (1, 2). Therefore a high rate of viral replication may pose a considerable challenge for an antiviral T cell response. In contrast to CTL, B cells can recognize Ags in an immediate and MHC-independent manner. Furthermore, BCR can recognize a larger variety of potential Ags than TCR, including peptide, carbohydrate, glycolipid, and nucleic acid Ags (3, 4).

Granzymes such as granzyme B (GrB)⁴ represent a major constituent of the granules of CTL (5). The ligation of the TCR by MHC-presented Ag on the target cell, in combination with secondary signals including IL-2, IL-15, and CD28 activation, results in the degranulation of cytotoxic granules and the release of effector molecules such as GrB into the secretory synapse. CTL can deliver GrB to targets such as virus-infected cells, thereby effectively inducing apoptosis (6). Apart from CTL and NK cells, no other normal human lymphocyte population is currently known to produce and secrete active GrB in physiological settings.

IL-21, a recently discovered member of the IL-2 family of cytokines, is secreted mainly by CD4⁺T, NKT, and Th17 cells (7, 8) and is preferentially produced in the acute phase of viral infections (9, 10, 11). Part of its pleiotropic effects on B, T, and NK cells (12, 13, 14) is the induction of gene transcription for granzymes A and B in CTL (15, 16). We have recently demonstrated that the stimulation of malignant B cells with IL-21 and either immunostimulatory CpG oligodeoxynucleotide or Abs activating the BCR results in GrB secretion by these B cells as well as in GrB-dependent apoptosis of malignant bystander B cells (17).

Motivated by these observations, we now investigated whether nonmalignant B cells would exhibit similar responses and whether natural stimuli such as viral Ags could also induce GrB expression in B cells. We demonstrate in this study, for the first time, that viral Ags can indeed induce the secretion of active GrB by human B cells in the context of the acute phase cytokine IL-21. These findings suggest that GrB secretion by B cells may be part of an early immune response during the acute phase of certain viral infections. Our results also demonstrate that B cells may contribute to the elevated GrB serum levels found in the early phase of various viral diseases (18).

The present study was approved by the Ethics Committee at the University of Ulm (Ulm, Germany). Peripheral blood from healthy individuals, tick-borne encephalitis virus (TBEV)-vaccinated healthy subjects (2 wk after vaccination with FSME Immun; Baxter Vaccines), or unvaccinated control subjects (never TBEV-vaccinated) was collected after obtaining informed consent. Alternatively, tonsillar tissue was obtained from children undergoing tonsillotomy or tonsillectomy. Immediately after removal, the tissues were minced and a cell suspension was prepared using a BD Medimachine (BD Biosciences). PBMC or tonsillar mononuclear cells were immediately isolated by Ficoll density gradient centrifugation according to standard procedures. For most experiments, CD19⁺ B cells were magnetically purified (>99% purity) using the B cell isolation kit II according to the manufacturer’s instructions (Miltenyi Biotec). In some experiments cells were sorted based on the expression of surface markers including CD19, CD27, CD38, and IgD as indicated on a FACSAria flow cytometer (BD Biosciences). The cells were cultured in AIM-V medium (Invitrogen) and incubated in 96-well plates (1 × 10⁶ cells/ml and 200 μl/well, if not stated otherwise) at 37°C and 5% CO₂ atmosphere in the presence of various reagents as indicated.

Human IL-21 was purchased from BioSource International and used at a final concentration of 50 ng/ml if not stated otherwise. Human IL-1β (100 ng/ml), IL-2 (500 U/ml), IL-3 (100 ng/ml), IL-4 (500 U/ml), IL-6 (200 ng/ml), IL-7 (25 ng/ml), IL-8 (50 ng/ml), IL-9 (25 ng/ml), IL-10 (25 ng/ml), IL-11 (200 ng/ml), IL-12 (50 ng/ml), IL-15 (50 ng/ml), GM-CSF (500 U/ml), G-CSF (1500 U/ml), M-CSF (10 ng/ml) and TNF-α (200 ng/ml) were all purchased from PeproTech. IL-23 (50 ng/ml) was purchased from BD Biosciences. IL-18 (40 ng/ml) was purchased from MBL International. IFN-α (100 U/ml) and IFN-γ (100 U/ml) were purchased from PBL InterferonSource. Human IL-22 (100 ng/ml) was purchased from RDI Research Diagnostics. Leukemia-inhibiting factor (10 ng/ml) was purchased from AbD Serotec. TBEV Ags were applied as inactivated TBEV (strain Neudörfl) adsorbed to 0.35 mg of Al(OH)₃ (FSME Immun; Baxter). Pure Al(OH)₃ was purchased from Sigma-Aldrich. For Ag-independent BCR stimulation (anti-BCR), affinity-purified rabbit F(ab′)₂ against human IgA/IgG/IgM (H and L chains) were used (Jackson ImmunoResearch Laboratories) at 6.5 μg/ml. The JAK inhibitor pyridone 6 (P6) was purchased from Calbiochem. Cycloheximide, an inhibitor of eukaryotic protein translation, was purchased from Sigma-Aldrich. CD40 ligand (CD40L) was purchased from Axxora and used according to the manufacturer’s instructions along with an enhancer.

For FACS analysis, cells were harvested at the indicated time points and stained as described previously (19). FITC-, PE-, PE-Cy5-, PE-Cy7- or allophycocyanin-labeled Abs to CD19, CD20, CD27, CD38, IgD, MHC I, MHC II, CD69, CD54, CD80, IFN-γ, and CD86 were purchased from BD Biosciences. Abs to perforin, granulysin, TRAIL, and Fas ligand (FasL) were purchased from eBioscience. PE-labeled anti-granzyme A, PE- or allophycocyanin-labeled anti-GrB (clone GB12), and the corresponding isotype controls were purchased from Invitrogen. For flow cytometric intracellular GrB detection, cells were incubated at 1 × 10⁶/ml for 16 h, brefeldin A (Epicentre Technologies) added to a final concentration of 1 μg/ml, and cells cultured for four more hours. Intracellular staining was performed using a fixation and permeabilization buffer (An Der Grub Bio Research). Briefly, cells were washed and resuspended in fixation buffer, incubated for 15 min at room temperature, and washed with PBS. Cells were then resuspended in permeabilization buffer and anti-GrB or isotype control mAb were added. After another 15 min of incubation at room temperature, cells were washed with PBS. For flow cytometric detection of apoptosis, cells were stained with annexin V (BD Biosciences) for 15 min at room temperature. Propidium iodide at 1 μg/ml was added just before flow cytometric analysis. Flow cytometric analyses were performed on a FACScan or a FACSCalibur flow cytometer (BD Immunocytometry Systems) and the data were analyzed using FlowJo software version 8.7.1 (Tree Star).

For detection of intracellular GrB activity, an intracellular fluorogenic GrB substrate was used and analyzed by flow cytometry as described earlier (OncoImmunin) (20). For a demonstration of the enzymatic activity of secreted granzyme B in the supernatants of stimulated B cells, we used a highly specific granzyme B activity assay according to the manufacturer’s specifications (SensiZyme; Sigma-Aldrich). Briefly, GrB from supernatants and GrB standards derived from active human recombinant GrB (Alexis) were captured on a 96-well plate by an Ab-based precipitation step, followed by the cleavage of substrate A by captured granzyme B. After the addition of substrate B, the accumulating chromogenic product was detected in a Dynatech MR 7000 ELISA reader at 405 nm.

For flow cytometric detection of secreted GrB, we established a biotin-NeutrAvidin-based GrB secretion assay. Briefly, 3 μl of 1 M DTT (Pierce) was added to 1 mg of anti-human GrB Ab (clone GB10; Mabtech) in 0.5 ml of PBS with 10 mM EDTA. After incubation for 1 h at 4°C, excess DTT was removed by gel chromatography (Sephadex G25; Pharmacia). Subsequently, 0.5 mg of NeutrAvidin (Pierce) in 200 μl of PBS was added to 50 μg of succinimidyl-4-(N-maleimidomethyl)-1-carboxylate (Thermo Scientific) in 20 μl of DMSO, and the mixture was incubated for 30 min at room temperature. The activated NeutrAvidin was purified on Sephadex G25 and eluted in PBS/EDTA. Reduced Ab and activated NeutrAvidin were mixed in a molar ratio of 2:1. After 1 h at 20°C, the reaction was stopped by adding N-ethylmaleimide at 10 μg/ml (Pierce). Then B cells from healthy subjects were highly purified (>99.5%) and biotinylated using a solution of 200 μl of sulfosuccinimidyl-6-(biotinamido)-hexanoate (pH 8.4) at 1 mg/ml (EZ-link; Pierce) and incubated for 10 min at room temperature. Then cells were washed twice with 50 ml of PBS containing 0.5% BSA (PBS/BSA), counted, and resuspended in 4 ml of PBS/BSA. After another washing step with PBS/BSA, cells were incubated for 20 min at room temperature with the anti-GrB Ab-NeutrAvidin conjugate, washed again, and resuspended in AIM-V medium (Invitrogen). Subsequently, cells were allowed to capture secreted GrB on their surface by incubating them for 48 h in 96-well plates (1 × 10⁶ cells/ml and 200 μl/well) at 37°C and 5% CO₂ atmosphere in the presence of IL-21 (50 ng/ml) and anti-BCR (6.5 μg/ml). Then cells were harvested and stained with a PE-labeled detection Ab to human GrB (clone GB12; Invitrogen) and analyzed by FACS.

A human GrB ELISPOT kit was purchased from Cell Sciences and PVDF-bottom, 96-well plates were from Millipore and used following the manufacturer’s instruction. Briefly, plates were prepared by adding the capture Ab and blocking with 2% skim milk in PBS; the cells were then plated in AIM-V medium at 1 × 10⁵ per 100 μl per well in the presence of various agents as indicated and cultured for 16 h. After culture, the detection Ab was added and the plates were incubated for 1.5 h. Streptavidin-alkaline phosphatase was distributed, and the plates were incubated for 1 h. Finally, BCIP/NBT buffer (5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium substrate) was added and color was allowed to develop for 10 min at room temperature, followed by rinsing with distilled water. The plates were dried completely and the spots were read on an ImmunoSpot series 1 analyzer using ImmunoSpot 3 software, both from CTL Cellular Technology.

B cells from healthy individuals were magnetically isolated to a purity of >99.6% and incubated for 15 h with either no treatment or in the presence of IL-21 (50 ng/ml), anti-BCR (6.5 μg/ml), or both. After incubation, the cells were counted and mRNA from 5 × 10⁵ cells per sample was prepared using Dynabeads mRNA DIRECT kit (Invitrogen). For the PCR, 5 μl of the generated cDNA was used per 25-μl reaction with recombinant Taq DNA polymerase (0.02 U/μl; Invitrogen). Primers used for GrB were as follows: forward primer, 5′-CCATCCAGCCTATAATCCTA-3′, reverse primer 5′-CCTGCACTGTCATCTTCACCT-3′ (Biomers.net). The housekeeping gene RPL32 was run simultaneously as a standard. Five microliters of the reaction products were run on a 1.5% agarose gel along with a 100-bp ladder.

B cells from healthy subjects were isolated and incubated for 5, 10, and 20 min (for JAK and STAT detection) or overnight (for GrB detection) at 1 × 10⁶ cells and 500 μl/well with IL-21 and anti-BCR in the presence or absence of P6 (1 μM). Cell lysates were prepared and Western immunoblotting was performed as described previously (21). After washing and blocking, primary Abs were added followed by a washing step and incubation with the secondary Ab conjugated to HRP for 1 h. Primary Abs against GrB, pJAK1, pTYK2, and pSTAT3 (where “p” indicates phosphorylated), and actin were from Cell Signaling Technology, the Ab against pJAK3 was from Santa Cruz Biotechnology, and the Ab against pSTAT1 was from BioSource International. Secondary Abs were anti-goat IgG from Sigma-Aldrich and anti-rabbit IgG and anti-mouse IgG from GE Healthcare.

Isolated B cells from healthy individuals were cultured overnight on 96-well plates at 2 × 10⁵ cells and 200 μl per well in the presence of IL-21 (50 ng/ml) and anti-BCR (6.5 μg/ml). Brefeldin A was added to a final concentration of 1 μg/ml and cells were additionally cultured for 4 h. Subsequently, 1.2 × 10⁶ cells were pooled and transferred into a FACS tube (BD Biosciences). The cells were then surface stained with FITC-labeled Abs to CD19 (BD Biosciences) and intracellularly stained with PE-labeled Abs to GrB or isotype control (both from Invitrogen) as described above for flow cytometry. For signal enhancement, 200 μl of Image-iT FX signal enhancer (Invitrogen, Carlsbad, CA) was used according to the manufacturer’s instructions. After the staining procedure, the cells were transferred to an 8-well Lab-Tek II chamber slide and mounted in the ProLong Gold antifade reagent (Invitrogen). Following the curing time, the cells were examined by fluorescence microscopy.

Data are expressed as means ± SEM. To determine statistical differences between the means of two data columns, the paired Student’s t test was used as appropriate. Pearson’s correlation was used to examine the relationship between two groups of variables. p values were corrected using the Bonferroni method where applicable.

In a previous study we demonstrated that malignant B cells from patients with chronic lymphocytic leukemia produce GrB in response to in vitro stimulation with IL-21 and BCR engagement (17). In the present study we therefore tested whether various B cell subpopulations from healthy subjects are capable of a similar response. Both unfractionated peripheral B cells within PBMC as well as highly purified B cells from healthy individuals were able to produce GrB when stimulated with IL-21 and Abs to the BCR (anti-BCR) (Fig. 1^,A). Similar results were obtained using tonsillar B cells (data not shown). These data suggested that GrB was directly induced in B cells and that no other cells were involved in this process as long as the stimuli of IL-21 and BCR engagement were present. In purified B cells, IL-21 by itself had some GrB-inducing effect that was strongly enhanced in the presence of anti-BCR, suggesting that BCR engagement plays a key role in GrB production by B cells (Fig. 1, A and B). Differentiation of B cells into GrB-producing cells appeared to involve the majority of B cells, because kinetic experiments revealed up to 65% of peripheral B cells staining positive for GrB after 48 h of stimulation (supplemental Fig. 1).⁵ GrB in stimulated B cells could also be visualized using fluorescence microscopy, demonstrating a heterogeneous distribution pattern of GrB within the cytoplasm of B cells (Fig. 1 C).

To test the hypothesis that natural ligands for the BCR are able to substitute for the rather artificial stimulus of anti-BCR, we decided to use an in vivo model that included vaccination against TBEV. Because it is endemic in various parts of Europe, a vaccination against TBEV is recommended and often performed in Southern Germany. For our study we isolated peripheral B cells from healthy donors who had either never been or recently been vaccinated against TBEV. Subsequently, these cells were restimulated in vitro in the presence of IL-21 and TBEV Ags at increasing concentrations. B cells from vaccinated individuals showed a significantly higher spontaneous GrB response in the presence of IL-21 than B cells from unvaccinated individuals. Importantly, GrB response in vaccinated but not in unvaccinated individuals was strongly enhanced by TBEV Ags in a concentration-dependent manner (Fig. 2, A–C). Incubation of B cells with the carrier Al(OH)₃ alone did not enhance GrB response (data not shown). Similar but weaker responses were found in B cells from subjects vaccinated against rabies and hepatitis B upon respective Ag exposures (data not shown). Finally, we tested serum of vaccinated and unvaccinated individuals for in vivo GrB and IL-21 levels. GrB serum levels were significantly higher in vaccinated as compared with unvaccinated individuals and showed a strong correlation with IL-21 serum levels (Fig. 2 D).

To address the question whether or not B cell-derived GrB is enzymatically active, we incubated B cells in the presence or absence of IL-21 and anti-BCR. We then added a cell-permeable fluorogenic substrate to the cells that is specifically cleaved by GrB and incubated them for >1 h. Flow cytometric analysis revealed significantly stronger substrate fluorescence in the treated as compared with the untreated control B cells (Fig. 3,A). To determine whether GrB produced by B cells remains inside the cells or is secreted into the environment, we established a FACS-based GrB secretion assay using a surface capture technique as described in Materials and Methods. According to this assay, up to 50% of highly purified B cells stained GrB positive after 48 h of incubation, suggesting that a large portion of the GrB produced by B cells was indeed secreted (Fig. 3,B). Using GrB-specific ELISPOT analyses we were able to confirm these results (Fig. 3,C). Importantly, GrB secretion was completely abrogated by brefeldin A, suggesting that an active transport from the endoplasmic reticulum to the Golgi apparatus was involved in the GrB secretion process (Fig. 3 C). Finally, using a highly specific GrB activity assay that combines an Ab-based precipitation step with substrate cleavage by captured GrB, we confirmed that the secreted GrB in the supernatants was indeed enzymatically active (data not shown).

IL-21 is a recently discovered member of the IL-2 family of cytokines (12). We hypothesized that further cytokines from this and other cytokine families might also be able to induce GrB in B cells and therefore tested a large variety of cytokines alone or in combination with each other. Importantly, no substantial GrB secretion was detected when B cells were stimulated with anti-BCR and members of the IL-2 family other than IL-21, including IL-2, IL-4, IL-7, IL-9, and IL-15 (Fig. 4,A). In contrast, combining IL-2 family cytokines with further cytokines revealed IL-4 and IL-10 as the only combination that induced consistent albeit lower amounts of GrB in BCR-engaged B cells as compared to stimulation with IL-21 (Fig. 4 B and data not shown). IL-21 therefore appears to be the key cytokine for the induction of GrB in BCR-engaged B cells.

Classical cytotoxic cells such as CTL and NK cells possess cytotoxic granules in which they store preformed GrB. B cells, in contrast, do not express GrB without stimulation (Fig. 1,A). Activation of B cells with IL-21 and BCR engagement, however, resulted in strong up-regulation not only of the GrB protein but also of GrB mRNA (Fig. 5,A). This finding suggested that GrB was freshly produced after stimulation of B cells, which was further supported by the fact that the translation inhibitor cycloheximide was a potent suppressor of GrB protein production in B cells (Fig. 5,B). In CTL and NK cells the induction of a cytotoxic response depends on the activation of a series of JAK/STAT family members. Because IL-21 signaling has also been associated with activation of the JAK/STAT pathway (13), we performed Western immunoblotting using purified B cells stimulated with IL-21 and anti-BCR and stained them for pJAK1, pJAK3, pTYK2, pSTAT1, and pSTAT3. As controls we included GrB and actin. We found strong and consistent up-regulation of GrB, pJAK1, and pSTAT3, weak up-regulation of pJAK3, and no up-regulation of pTYK2 or pSTAT1 (Fig. 5,C). We also could demonstrate that the JAK inhibitor P6 was an effective suppressor of GrB secretion by B cells with its IC₅₀ for GrB suppression (17.3 ± 4.6 nM; n = 3) ranging close to the expected IC₅₀ for JAK1 (15 nM) (22) (Fig. 5 D). Consistently, treatment of B cells from vaccinated individuals with P6 completely abrogated the GrB production after restimulation with viral Ags in the presence of IL-21 (supplemental Fig. 2).

B cells can be divided into naive B cells, without any prior Ag contact, or into memory B cells, which have already encountered Ag. Various surface markers can be used to discriminate between these two B cell populations, including surface IgD and CD27 for peripheral B cells, as well as surface IgD and CD38 for tonsillar B cells. Staining of stimulated peripheral B cells for GrB and CD27 suggested that it was mainly B cells with a naive phenotype that expressed GrB (Fig. 6,A). To confirm this, we sorted peripheral and tonsillar B cells according to expressions of IgD, CD27, and CD38, thereby separating naive and memory B cells before stimulating them with IL-21 and anti-BCR (Fig. 6,B and supplemental Fig. 3A). These experiments revealed a higher potential for naive B cells than for memory B cells to differentiate into GrB-producing cells (Fig. 6,C and supplemental Fig. 3B). Further characterization of the phenotype demonstrated that GrB-expressing B cells strongly down-regulated the naive B cell marker IgD as well as the memory B cell marker CD27, whereas CD20 was up-regulated, CD19 remained positive, and CD38 remained negative (Fig. 7,A). GrB-expressing B cells did not express perforin, granzyme A, granulysin, Fas ligand, or TRAIL (data not shown). Instead, they up-regulated several molecules involved in interactions with other immune cells, including costimulatory molecules, the Ag-presenting molecule MHC class II, and the cell adhesion molecule CD54. Overall, GrB-expressing B cells appeared to be in a highly activated state that included an enhanced expression of the early activation marker CD69 (Fig. 7 B) and increased survival rates as compared with untreated B cells (supplemental Fig. 4).

The present study demonstrates for the first time that viral Ags can induce GrB expression in B cells from subjects recently vaccinated against the corresponding viral diseases, an effect not observed in unvaccinated individuals. This response depends on the activation of the BCR and the presence of IL-21. Induction of GrB in B cells involves both transcriptional and translational events and depends on the phosphorylation of JAK1, JAK3, and STAT3. B cell-derived GrB is secreted into the environment and is enzymatically active. Both naive and memory B cells are capable of differentiating into GrB-secreting B cells. Naive B cells from unvaccinated donors seem to exhibit a higher potential in this context, which may be due to a higher constitutive expression of IL-21R by naive B cells as compared with memory B cells (23). However, the involvement of naive vs memory B cell populations may also depend on the presence or absence of a previous antigenic stimulation of B cells. It is thus possible that a higher percentage of memory B cells may be differentiating into GrB-secreting B cells in vaccinated donors. The differentiation process includes the development of a homogeneous CD19⁺CD20⁺CD27⁻CD38⁻IgD⁻ phenotype, increased survival, and enhanced expression of molecules involved in cellular interactions. Our present data therefore suggest that B cell-derived GrB may play a physiological role in cellular antiviral immunity rather than for self-regulatory purposes as shown for activation-induced NK cell death (24).

B cells and T cells exhibit two striking differences in terms of their receptors. TCR recognize their Ags only when presented in the context of MHC molecules and provided by either professional APC or target cells. In contrast, Ag recognition by the BCR is independent of MHC presentation and may theoretically occur in the absence of APCs during the early phase of a viral infection. Furthermore, the BCR can recognize epitopes within larger structures such as virus particles, and the spectrum of Ags recognized is not limited to peptide Ags but also includes carbohydrate, phospholipid, and nucleic acid Ags (3, 4). Not surprisingly in this context, the presence of MHC-independent epitopes in addition to MHC-restricted epitopes could significantly enhance cellular immune responses in a recently described mouse model of human papillomavirus-induced tumors (25). With the intent to combine the advantages of both TCR and BCR, another group designed genetically engineered T cells with chimeric receptors. These T cells, which carry BCRs on the cell surface, connected to the signaling portion of TCRs and showed strongly increased antitumor activity (26, 27). Our present work suggests that the immune system may have evolved a similar mechanism by providing B cells with T cell-like effector molecules.

One possible hypothesis arising from our findings is that GrB-secreting B cells may support a cytotoxic immune response against viral infections. In contrast to CTL and NK cells, B cells do not produce significant amounts of perforin along with GrB. However, a growing body of evidence suggests that GrB-induced apoptosis does not necessarily depend on the presence of perforin (28, 29, 30). GrB uptake into target cells is perforin independent, occurring via mannose-6-phosphate or fluid phase endocytosis (28, 29, 30, 31). It is only its endosomal release into the cytoplasm that requires an endosomolytic agent such as perforin (29, 32). Various viruses are known to carry molecules with such properties to enable their endocytic entry into the cytoplasm after cellular uptake (29, 33, 34, 35). Consequently, a series of studies have shown that virus particles can enable a cytotoxic effect of GrB in the absence of perforin (29, 36).

We therefore believe that in an early phase of certain viral infections, when only a limited number of virus particles and virally infected cells are present at the infection site, specific B cells may detect them via their BCRs. This in turn may result in the up-regulation of adhesion molecules that further support the adherence of such B cells to the infected tissue. In the presence of IL-21-producing cells such as NKT cells (7) or Th17 cells (8), these B cells may then start releasing GrB into this environment. GrB may coenter virus-infected cells and may be coreleased into their cytoplasm along with virus components. Once there, GrB may induce apoptosis before the virus can replicate. As soon as virus-specific T cells arrive on-site and become activated, they could provide signals to the B cells to switch to Ab rather than GrB production. In this context, it is noteworthy that ligation of CD40 inhibits the differentiation of B cells into GrB-secreting B cells (data not shown). Compatible with this observation, another group recently demonstrated that stimulation with IL-21, BCR engagement, and CD40 ligation, but not IL-21 and BCR engagement alone, induced differentiation of B cells into plasma cells (37).

Three additional lines of indirect evidence support our hypothesis for the function of B cell-derived GrB. First, infections with a number of viruses, including CMV, HIV, and flaviviruses such as the Dengue fever virus, were reported to be associated with elevated serum GrB levels, particularly during the acute phases of infection (9, 10, 11) (reviewed in Ref. 18). At the same time, it has been shown that IL-21 is one of the first cytokines to appear in the serum in the course of a viral infection (38). Finally, we demonstrated that in vivo GrB levels were significantly higher in the serum of recently vaccinated as compared with unvaccinated donors and that they highly correlated with serum IL-21 levels in the same donors. All of these observations point toward an interrelationship between viral infections and serum levels of IL-21 and GrB.

Of note, an increasing body of data suggests that apart from their classical proapoptotic effects, granzymes may possess alternative functions as well, particularly when emerging extracellularly. These potential functions may include cytokine-like functions (36, 39), cleavage of surface molecules such as receptors (40, 41), matrix degradation or remodeling (42), immunosuppressive functions (43, 44), or direct destruction of viral proteins important for replication or assembly (reviewed in Refs. 18 and 45). Clearly, future studies of B cell-derived GrB also need to address these potential functions in addition to a prospective cytotoxic effect.

Although it may appear obvious to initiate such studies in a mouse model, we failed to date to detect murine granzyme B in adequately activated murine B cells (B. Jahrsdörfer and D. Fabricius, unpublished observations). Therefore, GrB-secreting B cells may not occur in mice. This does not, however, preclude a physiological role for GrB in human B cells, because it has been shown that murine and human GrB exhibit major structural and functional differences, with murine granzyme B being 30 times less cytotoxic than human GrB (46). Therefore, GrB in general may play different roles in the murine and the human immune systems, a phenomenon that has already been recognized for other cytotoxic molecules such as granulysin (5). This view is further supported by the fact that GrB^−/− mice seem to deal with the many viruses with which they are infected experimentally (1, 47).

In conclusion, we provide evidence that recent stimulation with viral Ags in the form of a vaccination enhances the frequency of human B cells responding with GrB secretion to viral restimulation in the context of the acute phase cytokine IL-21. This process involves engagement of the BCR and depends on the activation of the JAK/STAT pathway. GrB secretion by B cells may represent an early cellular immune response to counteract overwhelming viral replication at the beginning of an infection. The present work may stimulate further studies focusing on the primary function of B cell-derived GrB during the acute phase of viral infections.

We thank M. Rojewski from the Department of Transfusion Medicine for excellent help with cell sorting, A. Westhoff from the Department of Pediatrics for excellent assistance with fluorescence microscopy, and B. Büchele from the Institute of Pharmacology of Natural Products and Clinical Pharmacology for excellent assistance with the Ab-streptavidin conjugation (all from Ulm University, Ulm, Germany). Furthermore, we thank H.-R. Rodewald from the Department of Immunology for permission to use his four-color flow cytometer and B. Böhm from the Department of Medicine II for permission to use his ELISPOT reader (both from Ulm University). Finally we thank S. Blackwell (Holden Comprehensive Cancer Center, University of Iowa) for fruitful discussions and critical review of the manuscript.

The authors have no financial conflict of interest.