Antigen-affinity controls pre-germinal centser B cell selection by promoting Mcl-1 induction through BAFF receptor signaling

Upon antigen encounter, the responsive B cell pool undergoes stringent selection which eliminates cells with low B cell receptor (BCR) affinity. Already before formation of the germinal center, activated B cells of low-affinity are negatively selected in a process that is molecularly not well understood. In this study, we investigated the mechanism behind pre-GC affinity-mediated B cell selection. We applied affinity mutants of HEL antigen and found that rapidly after activation B cells become highly dependent on the cytokine BAFF. Moreover, expression of BAFF receptor CD268 is regulated in a BCR-affinity dependent fashion. High affinity responses via BAFF correlated with PI3K activation, which controlled expression of the pro-survival protein Mcl-1, and thereby increased survival. In the presence of excess BAFF, or in absence of the Mcl-1 antagonist Noxa, more low-affinity B cells survived the first two days after antigen encounter. This resulted in increased numbers of antigen-specific B cells of low affinity upon immunization and reduced the overall affinity of cells that contributed to the germinal center reaction. Our findings elucidate a crucial molecular pathway of B cell selection in the earliest phases of activation by identifying a novel link between BCR affinity and BAFF-R signaling towards Mcl-1.

The humoral immune response provides lasting protection against (re)-infection. Upon pathogen encounter, antigen-specific B cell clones are selected from a vast pool of cells, each one unique based on its antigen receptor. The minimal ligand-affinity of this B cell receptor (BCR) required for cell activation is relatively low and each pathogen therefore stimulates many cells 1,2 . To prevent sub-optimal B cells from consuming precious nutrients and cytokines, the antigen-responsive cell pool is subject to selection for only those cells with the highest specificity 3 . This process is most rigorous in the germinal center (GC), a structure which arises several days after antigen encounter 4 . Here, the overall antigen affinity of the responsive B cell pool is rapidly increased through active editing of the BCR via somatic hypermutation 3,5 . Cells of reduced affinity are eliminated via apoptosis in a Darwinian selection process that ensures only survival of the fittest clones 3,5 .
To ensure an efficient GC-reaction, the number of clones that is allowed to enter this structure must be restricted 6 . The activated B cell pool is therefore subject to antigen-affinity based selection from the earliest stages of B cell activation 7 . This selection appears to be independent of an intrinsic survival rheostat, but is driven by the competitive pressure of other activated B cell clones 1 . In absence of competing clones with a higher affinity, even cells of very low affinity are able to generate B cell responses of equal magnitude as high-affinity cells. However, when high-and low-affinity B cells are competing, high-affinity cells predominate in the antibody-producing cell pool upon immunization 1 . This system ensures that at all times a B cell response of the highest affinity is generated, independent of the initial affinity of the B cell pool.
CD4 T cell help 7 plays an important role in affinity-based selection in the pre-GC stage. Within 6 hours after antigen recognition, activated B cells move to the border of B and T cell follicles 8 . B cells of reduced affinity take up less antigen than high-affinity cells, resulting in a reduced number of non-self peptides presented in MHC-II molecules to CD4 T cells 7 . Thus, high-and low-affinity B cells actively compete with each other for T cell-derived help. However, the nature of this help and whether T cell help is the only mechanism of pre-GC B cell selection, is currently unknown.

Results
BCR affinity differentially regulates expression of CD25 and CD268. To investigate the role of antigen affinity on the survival of B cells in the first days after activation, we used murine B cells expressing a transgenic antigen receptor (MD4) that recognizes Hen Egg Lysozyme (HEL). B cells were cultured in the presence of purified HEL, or HEL mutants carrying two (HEL2x) or three (HEL3x) substitutions, resulting in a 250 and 13.000 fold lower affinity for MD4 respectively 13 . When cultured in the presence of antigen alone, rapid activation-induced cell death occurred, which emphasized the requirement for a cofactor. Whereas LPS was the most potent stimulus for activation of B cells, from various cytokines implied in B cell survival, BAFF (Also known as BlyS) was shown to be the most efficient cofactor in this culture system (Supplementary Figure 1a). To study the effects of affinity on early B cell behavior we cultured cells therefore in the presence of low amounts of BAFF, and a concentration range of various HEL antigens.
Proliferation and activation of B cells, as measured by CFSE dilution and GL7 upregulation respectively, correlated strictly with antigen concentrations. When using antigen of reduced affinity, higher concentrations were required to achieve similar effects (Fig. 1a, Supplementary Figure 1b). The EC50 for CFSE dilution (0.58, 24 and 256 ng/ml for HEL, HEL2x and HEL3x respectively) was comparable to that of GL7 induction (0.69, 34 and 213 ng/ml for HEL, HEL2x and HEL3x). In contrast, whereas the EC50 of survival was not significantly different from that of proliferation and activation for high-affinity stimulation (1.60 ng/ml), it was five to ten fold higher for HEL3x and HEL2x respectively (487 and 1003 ng/ml for HEL2x and HEL3x) (Fig. 1a, Supplementary Figure 1b). This indicates that low-affinity antigens are incapable of inducing a requisite factor for activated B cell survival, also under conditions that can fully activate B cells, which is not directly dependent on the level of proliferation.
Previously, we have shown in T cells that antigen-affinity closely correlates with IL-2Rα (CD25) expression, and potentiates sensitivity to the survival cytokine IL-2 14,15 . We hypothesized that B cell receptor affinity might similarly correlate with expression of a receptor for a pro-survival cytokine. Differences in survival following high-and low-affinity stimulation were only observed in the first two days after activation (Fig. 1b), marking the time frame in which this survival factor operates. At this time point, various receptors were induced in response to antigen-stimulation, but only BAFF-R (CD268) and IL-2Rα (CD25) correlated with antigen-affinity (Fig. 1c). Further analysis revealed that expression of the IL-2Rα subunit peaked at 24 hours after stimulation, whereas CD268 expression had not yet reached a maximum after 48 hours (Fig. 1d). BCMA and TACI, the two additional receptors that are able to bind BAFF were not regulated in an antigen-affinity dependent fashion (Fig. 1c). Thus, antigen affinity positively correlates with B cell survival in the first two days after activation and with induction of BAFF-R and IL-2Rα .

BAFF, but not IL-2 mediates activated B cell survival in an antigen-dependent fashion.
To investigate the impact of BAFF and IL-2 on survival, activated B cells were stimulated in the presence of either cytokine and viability was assessed after two days. Even high concentrations of IL-2 failed to improve survival (Fig. 2a). In contrast, low concentrations of BAFF achieved a strong survival increase of activated B cells, which was most prominent after high-affinity stimulation (Fig. 2b). When high concentrations of BAFF were used, low-but not high-affinity B cells showed a further increase of viability, indicating that BAFF becomes limiting for survival of low-affinity cells. As reported previously 16 , BAFF promoted survival of both resting and antigen-stimulated cells. However, the increase in survival mediated by BAFF was significantly higher for activated cells, indicating that antigen-encounter makes B cells more dependent on this cytokine to sustain viability (Fig. 2c).
To investigate the impact of BAFF on activated B cell survival in a model independent of recombinant cytokine, 3T3 cells were generated that express a form of human BAFF on their membrane that cannot be cleaved off ( Supplementary Figure 1c-e). HEL TG B cells were cultured on these cells in the presence of high-or low-affinity antigen. 3T3 cells expressing BAFF (3TBAFF) increased B cell survival significantly over cells cultured on 3T3 cells transduced with an empty vector (3TEV). Notably, high-affinity B cells showed significantly better survival than low-affinity cells when cultured in the presence of 3TBAFF cells (Fig. 2d).
To investigate whether the BAFF receptor is induced through transcriptional upregulation, we performed qPCR analysis of BAFF-R mRNA in HEL TG B cells stimulated in vitro with HEL mutants. This showed that Scientific RepoRts | 6:35673 | DOI: 10.1038/srep35673 BAFF-R transcription was induced in an affinity-dependent fashion (Fig. 2e). In summary, activated B cells induce expression of the BAFF-R in an affinity-dependent fashion, which sensitizes cells to BAFF-mediated survival.

BCR affinity primes cells for BAFF-induced Mcl-1 protein upregulation. Survival of activated B
cells depends on the differential regulation of several members of the Bcl-2 family of pro-and anti-apoptotic proteins. Antigen recognition is associated with upregulation of the pro-survival proteins Bcl-XL and Mcl-1, whereas Bcl-2 is down-modulated 10 . Especially loss of Mcl-1 is associated with rapid cell death of activated B cells 10 . Therefore, we analyzed protein expression of these three molecules in the context of antigen-affinity dependent activation. Stimulation with HEL proteins resulted in a rapid downregulation of Bcl-2. In contrast, we observed a strong induction of Mcl-1 which correlated positively with BCR affinity, especially after 48 hours (Fig. 3a, Supplementary Figure 2a). Bcl-XL levels were also induced, but at a later time point after antigen stimulation. These findings prompted us to further investigate antigen affinity-mediated Mcl-1 induction.
BAFF has been shown to induce Mcl-1 in resting B cells and promote their survival 17 . Indeed, we observed that BAFF or antigen alone resulted in a modest increase of Mcl-1 levels. Reminiscent of the induction of BAFF-R in activated B cells, we observed a much stronger induction of Mcl-1 in the presence of both antigen and BAFF (Fig. 3b). Importantly, Mcl-1 levels correlated strongly with antigen-affinity in the presence of BAFF and were much lower when BAFF was omitted (Fig. 3c, Supplementary Figure 2b). Protein levels of pro-apoptotic Bcl-2  were stimulated for 24 h with 100 ng/ml HEL, 100 ng/ml BAFF or both (n = 3) (c) HEL TG B cells were stimulated with 100 ng/ml HEL, HEL2x or HEL3x in the presence or absence of 100 ng/ml BAFF (n = 3). Protein levels were quantified using densitometry and mean levels normalized for β -actin are shown in comparison to T = 0. (d) HEL TG B cells were stimulated with 100 ng/ml HEL, HEL2x or HEL3x in the presence 100 ng/ml BAFF or with 100 ng/ml HEL3x in combination with 500 ng/ml BAFF (HiBaff). Total cell lysates were then probed by western blot for the indicated proteins. β -Actin was used as a loading control. Affinity 'scale bars' correspond with high (HEL), middle (HEL2x) or low (HEL3x) affinity ligands used for stimulation (n = 3). Values show means ± sem. *P < 0.05 (Student's t-test and ANOVA with Bonferroni's post-testing). family member Bim, which have previously been shown to increase in response to cytokine deprivation 18 , were not reduced in the presence of BAFF (Fig. 3c). Moreover, Mcl-1 levels could be increased in cells stimulated with low-affinity antigen through addition of extra BAFF (Fig. 3d, Supplementary Figure 2c). In summary, the strength of BCR triggering is transduced via BAFF signaling to adjust Mcl-1 protein levels.
BCR affinity promotes B cell survival by PI3K-Akt-GSK3β signaling in response to BAFF. In activated B cells BAFF mediated survival was shown to depend on PI3K signaling 19,20 . In addition, it is known that PI3K signaling can control Mcl-1 protein levels. Mcl-1 is targeted for proteasomal degradation following phosphorylation by GSK3β . PI3K phosphorylates and activates Akt, which in turn phosphorylates and inhibits GSK3β , thus promoting in Mcl-1 stabilization 21 . We tested on several levels of this signaling cascade whether it is responsible for the affinity-controlled survival B cells following BAFF stimulation. First, inhibition of PI3K signaling using a Pan-PI3K inhibitor or CAL-101, a specific PI3Kδ inhibitor, resulted in a concentration-dependent reduction in survival, both for high-and low-affinity B cells. Viability correlated closely with Mcl-1 protein levels (Fig. 4a,b). Second, in presence of equal amounts of BAFF, low affinity B cells demonstrated reduced capacity to phosphorylate Akt, even though it did not reach statistical significance (Fig. 4c). However, considering the major impact of Akt-inhibition on B cell survival (Fig. 4a,b), even small changes in Akt phosphorylation appear to have a large biological impact. This is possibly due to amplification of the signal downstream in the signaling cascade.
Third, B cells stimulated in vitro with low-affinity antigen demonstrated moderately reduced levels of pGSK3β . Low-affinity B cells stimulated with high amounts of BAFF, in contrast, showed increased levels of phosphorylated GSK3β , which closely correlated with Mcl-1 protein (Fig. 4d). Fourth, blocking GSK3 activity in antigen-stimulated B cells, increased Mcl-1 levels in a concentration-dependent fashion (Fig. 4e). In summary, Mcl-1 protein levels are regulated through the PI3K signaling cascade in activated B cells in response to BAFF. Low-affinity B cells are less responsive to BAFF and therefore have reduced capacity to induce the PI3K signaling cascade.

BAFF signaling is limiting for survival after low-affinity triggering in vivo.
To confirm whether low-affinity B cells have reduced levels of the BAFF-R and Mcl-1 in vivo, HEL TG B cells were transferred to congenic wild type (WT) recipients. Subsequently, mice were immunized with HEL-Ova or HEL3x-Ova and after two days, levels of Mcl-1 and BAFF-R in B cells were quantified by flow cytometry. Both HEL antigens increased Mcl-1 and BAFF-R expression compared to the bulk pool of recipient B cells, indicating activation of donor cells (Fig. 5a). In accordance with our in vitro observations, high-affinity B cells had increased levels of both Mcl-1 and BAFF-R compared to low-affinity cells (Fig. 5a). To demonstrate a similar regulation of BAFF-R and Mcl-1 in non-transgenic B cells, WT mice were immunized with R-Phycoerythrin (PE). The number of antigen-specific B cells is of very low frequency on day two after antigen injection. We therefore analyzed animals on day twelve after immunization. Since animals only received PE as an antigen, the majority of germinal center cells in spleen will be directed against this protein, which can be visualized by ex vivo staining with PE 6 . Ex vivo stained PE Bright GC cells were considered to be high-affinity cells, whereas PE Dim cells were of low-affinity. We found that both PE Bright and PE Dim germinal center (GL7 + CD38 Dim ) B cells had higher levels of Mcl-1 and the BAFF-R compared to non-GC GL7 Dim cells. PE Bright B cells had significantly higher levels of both molecules then PE Dim germinal center B cells (Fig. 5b).
Our findings suggest that BAFF selectively promotes survival of high-affinity B cells. To test this directly, we made use of BAFF TG mice which secrete high amounts of BAFF into the blood 22 . CFSE-labeled HEL TG B cells were transferred in WT or BAFF TG recipients which were subsequently immunized with HEL-Ova or HEL3x-Ova. Two days later donor cells were quantified in the spleen. WT and BAFF TG mice contained equal numbers of high-affinity cells, but the number of low-affinity B cells was significantly increased in the presence of excess BAFF (Fig. 5c). Both immunogens had activated cells, as observed by induction of Mcl-1. Survival, rather than proliferation was responsible for differences in cell numbers, since cells did not yet show secondary peaks resulting from CFSE dilution (Fig. 5d). Of note, proliferation was readily induced on day 2 after stimulation in vitro (Fig. 1a). This difference is most likely the result from a delay in antigen encounter by B cells in vivo.
To assess the physiological impact of excess BAFF on B cell responses, WT and BAFF TG mice were immunized with TNP-KLH and germinal center B cell numbers were quantified after two weeks. A significant increase of relative and absolute numbers of GC B cell numbers in BAFF TG mice was observed in comparison to WT animals (Fig. 6a). BAFF TG mice have two to three fold more follicular B cells in the spleen (data not shown), whereas the number of GC B cells was increased by 8-9 fold (8.4 fold ± 2.7). This indicates that the increased number of GC cells is not only the result of larger numbers of follicular B cells, but that BAFF availability is indeed limiting for activated B cell expansion in vivo. To investigate whether excess BAFF also impacts the affinity of the antigen-specific B cell pool, WT and BAFF TG mice were immunized with PE. Ex vivo staining revealed that both in spleen and lymph nodes a smaller fraction of GC B cells was able to bind PE with high affinity (Fig. 6b). Antigen titration showed that total B cells from BAFF TG mice more rapidly lost their ability to bind PE (Fig. 6c,d). This indicated that the antigen-specific B cell pool is of reduced overall affinity. In accordance with this observation, the PE-specific antibody response was also of reduced affinity (Fig. 6e), despite the fact that the total IgG pool is enlarged in these animals 22 . To investigate which molecule is responsible for this effect, PE + B cells of WT and BAFF mice were sorted and expression of 40 pro-and anti-apoptotic moles was analyzed by MLPA. However, no significant differences were observed (Supplementary Figure 3). In contrast, Mcl-1 protein levels were significantly higher in PE + cells of BAFF TG mice (Fig. 6f).
Thus, BAFF availability was limiting for the survival of low-affinity B cells early after antigen stimulation. Immunization of mice with excess BAFF therefore reduced the average affinity of the antigen-specific B cell pool, most likely through specific induction of Mcl-1 protein.   low-affinity stimulation, Noxa-deficient B cells contained higher levels of Mcl-1 compared to WT cells (Fig. 7a,b, Supplementary Figure S2d).
To demonstrate that Noxa-deficiency provides a specific survival advantage for low-affinity cells, HEL TG B cells sufficient (Ly5.1/2) and deficient (Ly5.2) for Noxa were mixed in a 1:1 ratio and transferred to WT (Ly5.1) recipients. Subsequently, mice were immunized with HEL-Ova or HEL3x-Ova and the ratio between donor populations was determined two days later. In line with previous findings 6 , Noxa-deficient B cells had a slight survival advantage over WT cells in mice immunized with high-affinity antigens, as the ratio between cells increased by ~1.7 fold. Strikingly, upon low-affinity stimulation, the ratio between WT and Noxa −/− donor B cells on average increased by almost three fold (Fig. 7c).
Thus, the Noxa/Mcl-1 axis controls survival of antigen-specific B cells and drives apoptosis of low-affinity B cells as a result of reduced access to BAFF compared to high-affinity B cells.

Discussion
It has been shown previously that affinity-based B cell selection already takes place in the first days after antigen encounter, on the border between B and T cell regions 4,7 . However, how this earliest phase of selection was mediated mechanistically was unknown. Here we demonstrate that the affinity of BCR triggering controls the ability of B cells to respond to BAFF and that this is an important selection criterion for B cell clones in the first days after antigen encounter. BAFF promotes survival through stimulation of the PI3K signaling pathway and stabilization of the pro-survival protein Mcl-1. Low antigen-affinity results in insufficient access to BAFF and increases cell death upon immunization. Deficiency of Noxa, or excess availability of BAFF allows germinal center entry of more low-affinity clones, thus resulting in an overall reduction of the specificity of the antibody response 6 .
The role of BAFF in mature B cell survival has been studied extensively. Deficiency of BAFF or BAFF-R results in a more than tenfold reduction in peripheral B cells 23,24 as T2 B cells become highly dependent on this cytokine for their survival. This effect is predominantly mediated through the NF-κ B signaling cascade and T2 B cells deficient for IKK1 therefore demonstrate a phenotype that highly resembles that of BAFF −/− mice 19 . Activated B cells further induce expression of the BAFF-R, which mediates survival through the PI3K signaling cascade 20,25 . In previous models, anti-IgM antibodies were used as an activating stimulus, which does not take the varying effect of antigen-affinity on BAFF-R expression levels into account. Rather, the ability of B cells to present antigen to follicular helper T cells in the germinal center and thus gain access to BAFF was therefore postulated as the way in which antigen-affinity is translated into a survival advantage 7,26 . However, BAFF expression is in fact relatively low in the germinal center 26 and somatic hypermutation was only mildly affected in immunization models in which B cells do not have access to BAFF 26 . We show that the capacity of activated B cells to acquire sufficient amounts of BAFF through affinity-dependent BAFF-R induction is of immediate importance for their competitive fitness and controls the subsequent ability to seed germinal centers. In agreement with our findings, BAFF-induced PI3K signaling was shown to be crucial for IgM production upon immunization, whereas IgG1 levels were less dependent on this signaling cascade 19 . In absence of BAFF, deficiency of the PI3K phosphatase PTEN results in an increase of germinal center size, which is predominantly the result of increased numbers of IgM-producing cells and reminiscent of an increase in the number of GC-seeding cells 19 .
Studies using mice deficient for various pro-and anti-apoptotic molecules also indicate a differential control of activated B cell viability before and after germinal center formation. Noxa deficiency does not affect somatic hypermutation upon immunization, but is associated with an increase of germinal center size and reduced antibody affinity as a result of increased numbers of GC-seeding clones 6 . Mice lacking one allele of Mcl-1 have a strong reduction in the number of germinal center B cells, but affinity-based selection in the remaining cells is not affected 10 . In contrast, deficiency of Bim or Puma, two pro-apoptotic proteins which are induced in germinal center B cells, results in a strong reduction of clones carrying somatic hypermutations and an increase in antigen forming cells, whereas relative germinal center size is not affected 12,27 . Bcl-XL appears to play its role much later in the B cell response, when antigen-experienced plasmablasts enter circulation 10 .
The affinity-dependent capacity of B cells to access BAFF in order to stabilize Mcl-1, guarantees that in each B cell response only the cells of highest affinity survive, independent of the absolute level of antigen-affinity. Interestingly, CD8 T cells use a similar mechanism to select for high affinity clones. CD8 T cell receptor affinity correlates with expression of the IL-2 receptor through affinity-dependent induction of the transcription factor T-bet 28 . IL-2, in turn, promotes proliferation and stabilization of Mcl-1 15 . High-affinity T cells therefore have a selective advantage over low-affinity cells in their competition for limiting amounts of IL-2. Mice deficient for Noxa have a reduction in the overall affinity of the effector CD8 T cell pool, though the magnitude of both responses is unaffected 6,15 . We observed that BCR-affinity also positively correlated with expression of CD25 in B cells. In our system IL-2 did not play a role in B cell survival, possibly as a result of differences in the use of PI3K and Stat5 isoforms between B and T cells 29,30 . Nevertheless, for both B and T cells, affinity-sensing of the antigen-responsive pool is mediated by regulation of the Noxa/Mcl-1 axis through differential cytokine receptor expression, thus establishing a common theme.
Surprisingly, mice deficient for BAFF have been reported to form equal numbers of germinal centers as wild type mice in the first week after immunization 23,24 . This apparently contradicts our model of BAFF as an essential molecule for survival of pre-GC B cells. However, since BAFF −/− mice have a strong reduction in mature B cell numbers, the cells that do survive in these animals therefore have abundant access to all other pro-survival factors but BAFF. Moreover, it seems likely that these cells have altered expression levels of pro-survival Bcl-2 family members in order to sustain their vitality in absence of BAFF. Of course, similar limitations apply to our in vivo models that make use of BAFF TG animals. Mice with excess BAFF have reduced negative B cell selection and therefore show B cell hyperplasia from the T2 stage onwards 22 . Increased numbers of antigen-responsive B cells upon immunization may therefore be partially the result of an increase in the broadness of the B cell repertoire. However, HEL TG B cells also showed increased survival after immunization with low-, but not high-affinity antigens when transferred to BAFF TG animals. Thus, BAFF appears to be important for shaping both the naïve and early antigen-responsive B cell repertoire.
In summary, we here uncover a previously unknown link between BCR affinity and BAFF receptor signaling in early B cell survival and selection. Our findings may have important implications for immunization strategies that aim to restrict or broaden the scope of the protective antibody response.

Methods
Mice. Animals were used at 6-12 weeks of age, age-and sex-matched within experiments and were handled in accordance with institutional and international guidelines. Wild type C57BL/6 (B6, strain 00664), HEL TG (strain 002595) and B6 Ly5.1 (strain 002014) mice were purchased from the Jackson Laboratories and kept as breeding colonies in our local animal facility. Noxa −/− mice were a gift from Dr. A. Strasser (WEHI, Melbourne). BAFF TG mice were described previously 22 , and were a gift of Biogen Idec. All mice were either generated in B6 mice or backcrossed at least ten times on this background. All animal experiments were performed after approval of our institute's animal ethics committee (Dierexperimentencommissie AMC). All methods were performed in accordance with national and international guidelines and regulations.
Cell culture. Splenic HEL TG B cells were purified to > 95% purity using the MACS cell separation system (Myltenyi) and anti-CD19 microbeads (Myltenyi). Cells were cultured in RPMI with 10% fetal calf serum (FCS) with HEL proteins that were purified as described 13 . Cells were cultured in combination with indicated cytokines (R&D Systems), LPS (Sigma-Aldrich) or anti-CD40 (Bioceros). For pAkt stainings, cells were deprived from stimuli for 3 h in medium with 1% FCS, followed by stimulation with 500 ng/ml BAFF. Ly294002 (Invitrogen) and CAL-101 (apexbio) were used to inhibit PI3K signaling. Cell division was analyzed by CFSE (Molecular Probes) dilution. To generate 3TBAFF cells, the intracellular and transmembrane domains of CD40L (amino acids 0-112 of NP_000065.1) were fused by overlap extension PCR including a spacer (N-PAAAAAASAAAAAAWVPVAT-C)