PDGFA-associated protein 1 protects mature B lymphocytes from stress-induced cell death and promotes antibody gene diversification

Delgado-Benito et al. identify the protein Pdap1 as a regulator of mature B cell homeostasis. Pdap1 protects mature B cells against chronic induction of the integrated stress response and promotes both their survival and antibody gene diversification by CSR and SHM.


Introduction
The diversity of our Ig gene repertoire is the result of antibody diversification reactions occurring at different stages of B lymphocyte development (Dudley et al., 2005;Methot and Di Noia, 2017). Developing B cells in the bone marrow randomly assemble different gene segments (known as variable, V; diversity, D; and joining, J, genes) at the Ig heavy (Igh) and light (Igl) chain loci via V(D)J recombination (Roth, 2014;Dudley et al., 2005). This process generates unique antibody gene receptors with the potential to collectively recognize a formidable number of antigens. Mature B cells further diversify their Ig genes in the periphery via somatic hypermutation (SHM) and class switch recombination (CSR; Pavri and Nussenzweig, 2011;Methot and Di Noia, 2017). SHM introduces point mutations into the variable V(D)J region of the Ig genes to generate higher-affinity variants. CSR recombines the Igh constant (C) regions to replace the C portion of the IgM heavy chain with one of the alternative isotypes (IgG, IgA, and IgE), thus diversifying the Ig effector function. SHM and CSR are crucial to mount protective humoral responses, as evidenced by primary human immunodeficiency syndromes that are caused by defects in these reactions (Durandy et al., 2013).
SHM and CSR are both dependent on the B cell-specific enzyme activation-induced cytidine deaminase (AID; Revy et al., 2000;Muramatsu et al., 2000). AID deaminates cytosine residues to uracil in single-stranded DNA stretches at the variable regions of both Igh and Igl loci during SHM, and within special recombining elements (switch [S] regions) of the Igh during CSR Chaudhuri et al., 2003;Petersen-Mahrt et al., 2002;Pham et al., 2003;Sohail et al., 2003;Ramiro et al., 2003;Dickerson et al., 2003;Matthews et al., 2014;Methot and Di Noia, 2017). The resulting U:G mismatches are differentially processed to generate either mutations in the variable regions or DNA breaks in the S regions (Peled et al., 2008;Methot and Di Noia, 2017;Matthews et al., 2014). AID expression is induced when resting mature (naïve) B cells are activated by the antigen and T cell interactions (Zhou et al., 2003;Sayegh et al., 2003;Gonda et al., 2003;Cunningham et al., 2004;Dedeoglu et al., 2004;Muramatsu et al., 1999). Antigen stimulation reprograms naïve B cells to exit the quiescent state, expand their cellular biomass, and undergo a proliferative burst within transient and anatomically distinct structures in secondary lymphoid organs called germinal centers (GCs; Cyster and Allen, 2019;Victora and Nussenzweig, 2012). AID expression peaks in GC B cells (Cattoretti et al., 2006;Crouch et al., 2007;Roco et al., 2019). The GC reaction represents the end stage of B cell development, as GC B cells differentiate into memory B cells or long-lived plasma cells that secrete high-affinity antibodies.
The integrated stress response (ISR) is a homeostatic program activated by a variety of physiological and pathological stresses to promote cellular recovery (Ron and Walter, 2007;Pakos-Zebrucka et al., 2016). These stresses include both cellintrinsic and -extrinsic stimuli, such as ER stress, mitochondrial dysfunction, hypoxia, and amino acid deprivation (Quirós et al., 2017;Harding et al., 1999Harding et al., , 2003Dever et al., 1992;Rzymski et al., 2010;Ye et al., 2010). All forms of stress converge into the phosphorylation of the α subunit of the eukaryotic translation initiation factor 2 (eIF2α) on serine 51 (Donnelly et al., 2013). This event causes a reduction in global protein synthesis while allowing the preferential translation of few selected genes including the ISR effector activating transcription factor 4 (Atf4; Hinnebusch, 2000;Harding et al., 2000;Scheuner et al., 2001;Lu et al., 2004). Atf4 induces the transcriptional up-regulation of stress-responsive genes and rewires cell metabolism toward the recovery of cellular homeostasis . The inhibition of general protein translation in the early stage of the ISR is followed by a later phase of translational recovery, which restores protein synthesis once the stress is resolved to support cell survival (Brostrom and Brostrom, 1998;Novoa et al., 2003;Kojima et al., 2003;Marciniak et al., 2004;Brostrom et al., 1989;Ma and Hendershot, 2003).
Although the ISR is an adaptive program meant to restore cellular homeostasis and promote cell survival, under conditions of severe or prolonged stress, it induces cell death by activating proapoptotic pathways (Zou et al., 2008;Puthalakath et al., 2007;Teske et al., 2013;Wang et al., 2009;Ohoka et al., 2005;Gupta et al., 2012;Hiramatsu et al., 2014;Marciniak et al., 2004). Furthermore, the sustained protein synthesis during chronic stress causes proteotoxicity and leads to cell death Krokowski et al., 2013). Here, we identified Pdap1 as an essential regulator of mature B cell physiology that functions by countering chronic activation of the ISR. Pdap1 ablation in mature B cells causes sustained expression of Atf4, long-term induction of the ISR transcriptional program, and cell death. Furthermore, Pdap1 is essential for efficient induction of AID expression and physiological levels of CSR and SHM.

Results
Pdap1 is required for efficient CSR To identify novel modulators of mature B cell physiology, we performed loss-of-CSR screens via somatic gene targeting by CRISPR-Cas9 in the B cell lymphoma line CH12. CH12 cells share several properties of resting B cells and have been used to study key aspects of late B cell differentiation, including B cell activation, antibody diversification by CSR, and immunoglobulin secretion (Arnold et al., 1983;Stockdale et al., 1987;Wiest et al., 1990;Corley et al., 1985;Bishop and Haughton, 1986;Nakamura et al., 1996;Ovnic and Corley, 1987;Fagone et al., 2007;Gass et al., 2002;Dufort et al., 2014;LoCascio et al., 1984aLoCascio et al., , 1984b. Upon cytokine stimulation, CH12 cells express AID and undergo CSR to IgA with high efficiency (Nakamura et al., 1996). CRISPR-Cas9-mediated deletion of factors required for CSR reduces the efficiency of isotype switching in this cell line (Delgado-Benito et al., 2018). Among all tested candidates, targeting of Pdap1 resulted in a considerable reduction of CSR (Fig. 1, A and B). To confirm the results of the loss-of-CSR screen, we generated Pdap1-deficient CH12 clonal derivative cell lines, which included both indel knockout (Pdap1 −/− ) and in-frame deletion mutant (Pdap1 mut ) clones (Fig. 1,C and D). In agreement with the CSR defect observed in bulk CH12 cultures, CSR was impaired in Pdap1-deficient clonal derivatives ( Fig. 1 E). We concluded that Pdap1 supports efficient CSR in CH12 cells.
Pdap1 is a 28-kD phosphoprotein highly conserved in vertebrates. It was originally identified as a casein kinase II substrate and a weak interactor of platelet-derived growth factor A (PDGF-A; Shen et al., 1996;Fischer and Schubert, 1996). More recently, Pdap1 was described as an RNA-binding protein in several RNA-protein interactome studies (Castello et al., 2012(Castello et al., , 2016Trendel et al., 2019;Baltz et al., 2012;Iadevaia et al., 2020). However, the precise cellular functions of Pdap1 and its involvement in adaptive immunity are unknown.
To verify Pdap1 involvement in CSR in vivo and elucidate the underlying mechanism, we generated a mouse model bearing a conditional Pdap1 F allele and bred it to Cd19 Cre/+ mice to specifically ablate Pdap1 expression at the early stages of B cell differentiation (Rickert et al., 1997;Fig. S1, A and B). B cell development was largely unaffected in Pdap1 F/F Cd19 Cre/+ mice (Fig. S1, C and D). Although incomplete Cd19 Cre -mediated recombination of Pdap1 F in B cell progenitors could still mask a potential phenotype, these results suggest that Pdap1 is dispensable for V(D)J recombination and early B cell differentiation. However, we observed a pronounced reduction in the number of splenic resting mature B cells (Fig. S1 E). To confirm the intrinsic CSR defect caused by Pdap1 deficiency, we isolated resting splenocytes from Pdap1 F/F Cd19 Cre/+ mice and monitored their capability to undergo CSR upon in vitro stimulation. We assessed CSR under conditions that induce switching to IgG1, IgG3, IgG2b, and IgA and found that Pdap1-deficient B cells displayed reduced levels of CSR for all tested isotypes (Fig. 2, A-D). Western blot (WB) analysis confirmed the near-complete abrogation of Pdap1 expression in these cells (Fig. 2 E). Altogether, these data indicate that Pdap1 is required for efficient CSR in primary B cells irrespective of the stimulation condition. Top right: Summary graph for three independent experiments using gPdap1-1/3 (individually or in pooled format) or Nickases-a/c (individually). Controls for gRNA-nucleofected CH12 were cells electroporated with either empty vector or gRNAs against random sequences not present in the mouse genome (gCtrl). A previously described gRNA against the CSR factor 53BP1 (Delgado-Benito et al., 2018) was used as a positive control for loss-of-CSR function. Bottom: WB analysis of whole-cell extracts from CH12 cultures nucleofected with Nickase-a to -c. Triangles indicate twofold dilution. Asterisk denotes aspecific bands used for internal normalization of protein levels. N, Nickase. (C) Graph summarizing CSR efficiency of activated CH12 clonal cell lines derived by nucleofection of CH12 cultures with Nickase-a/c and single-cell sorting. Each symbol in the graphs indicates a single-cell clonal derivative. (D) Left: Genomic scars of the targeted alleles in the selected Pdap1-deficient CH12 clonal derivatives. fs, frameshift; PTC, premature termination codon; Δ, bp deletion; het, heterozygous configuration (different indels causing fs and PTC at the two Pdap1 alleles); homo, homozygous configuration. Right: Representative WB analysis of WT and Pdap1-deficient CH12 cell lines. R1 and R2 are WT clonal derivatives generated by targeting CH12 with random sequences not present in the mouse genome. mut, mutated. Asterisk denotes aspecific bands. Numbers underneath the blot indicate relative quantification of Pdap1 signal. (E) Left: Representative flow cytometry plots measuring CSR to IgA in activated CH12 cell lines of the indicated genotypes. WT controls included both the parental CH12 cell line (WT) and the random clonal derivatives R1 and R2. Right: Summary graph for four independent experiments. Significance in B, C, and E was calculated with the Mann-Whitney U test, and error bars represent SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.  BAFF,TGFβ,. Numbers in the plots refer to the percentage of switched cells (= IgG1 + /IgG3 + /IgG2b + /IgGA + cells). Right: Summary graph for at least six mice per time point per genotype. (E) WB analysis of whole-cell extracts from B cells of the indicated genotypes 96 h after stimulation with LPS and IL-4 (left) or LPS, BAFF, and TGFβ (right). 1 and 2 indicate two different Pdap1 F/F Cd19 Cre/+ mice. Triangles indicate threefold dilution. Asterisk denotes aspecific bands. Numbers underneath the blots indicate relative quantification of Pdap1 signal. Significance in A-D was calculated with the Mann-Whitney U test, and error bars represent SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. See also Figs. S1 and S2.
proliferation capabilities of splenocyte cultures by cell tracking dye dilution. The CellTrace Violet dilution profiles of Pdap1 F/F Cd19 Cre/+ B cells were indistinguishable from the control counterparts under all stimulation conditions (Fig. S2, A-D). Furthermore, class switching was reduced in Pdap1-deficient B cells independently of the number of cell divisions (Fig. S2 E). We concluded that Pdap1 is dispensable for B cell proliferation, and that the CSR defect of Pdap1 F/F Cd19 Cre/+ B cells is not due to reduced proliferation capabilities.
The efficiency of CSR is directly linked to the levels of AID expression Takizawa et al., 2008;Teng et al., 2008). Furthermore, AID targeting is dependent on noncoding transcription across the S regions (germline transcription; GLT), which exposes single-stranded DNA stretches that are the substrate of AID-mediated deamination (Chaudhuri et al., 2003;Dickerson et al., 2003;Ramiro et al., 2003). Therefore, we monitored Aicda mRNA and GLT levels in activated B cells by quantitative RT-PCR (qPCR) analysis. We found that Aicda transcript levels were consistently reduced in Pdap1 F/F Cd19 Cre/+ B cells compared with controls across all stimulation conditions (Fig. 3 A). Transcription of donor Sμ region was not affected by Pdap1 deletion (Fig. 3 B), whereas acceptor S region transcription exhibited a varied phenotype, with reduced levels of GLTγ1 and GLTα, and minimally affected or unaltered expression for GLTγ3 and GLTγ2b, respectively (Fig. 3 C). Analogously, analysis of Aicda and germline transcripts in Pdap1-deficient CH12 cell lines showed reduced Aicda mRNA levels but unaffected Sμ and Sα region transcription (Fig. S3, A-C). In agreement with the reduction of Aicda mRNA, Pdap1deficient B cells expressed lower levels of AID protein upon activation compared with control cells (Fig. 3 D).
AID-mediated deamination in the donor Sμ and recombining acceptor S region leads to the formation of DNA double-strand breaks (DSBs), which are ultimately repaired by end joining to ensure productive CSR events (Matthews et al., 2014;Boboila et al., 2012). The reduced AID expression and isotype-specific GLT phenotypes of Pdap1-deficient B cells converge into a pre-DSB defect, since DSB formation at S regions relies on both AID levels and efficient targeting. These results, however, did not exclude the possibility that Pdap1 might play an additional role during CSR downstream break formation, for example in repair of S region DSBs. To test this possibility, we overexpressed AID in Pdap1-deficient B cells and monitored the levels of CSR (Fig. 3  E). To this end, we transduced control and Pdap1 F/F Cd19 Cre/+ splenocytes with a construct expressing AID fused to the hormone-binding domain of the modified estrogen receptor (AID-ER). Provision of 4-hydroxytamoxifen (4-HT) induces the translocation of the cytoplasm-accumulated AID-ER fusion protein into the nucleus and initiates CSR. We found that 4-HT addition rescued the CSR defect of Pdap1 F/F Cd19 Cre/+ cells to levels equivalent to AID-ER-reconstituted Aicda −/− splenocytes in cultures stimulated to undergo class switching to IgG2b (Fig. 3 E), which exhibited unaffected GLTγ2b (Fig. 3 C). In contrast, the rescue of CSR was not significant in cells activated to switch to IgG1, with severely reduced GLTγ1 in the absence of Pdap1 (Fig. 3, C and E), and therefore impaired in AID targeting even under conditions of increased AID levels. Altogether, these data indicate that the CSR defect of Pdap1-deficient B cells is caused by impaired formation of AID-induced DSBs, which is caused primarily by reduced AID expression, with the additional contribution of impaired GLT in an isotype-specific manner.
Pdap1 has been recently identified in RNA-protein capture experiments as an RNA binding protein (Castello et al., 2012(Castello et al., , 2016Trendel et al., 2019;Baltz et al., 2012;Iadevaia et al., 2020). Therefore, we considered the possibility that Pdap1 might contribute to the posttranscriptional regulation of AID expression. Because Pdap1 is localized primarily in the cytoplasm (Fig. S4), we first tested whether Pdap1 controls the degradation of Aicda mRNA. To do so, we treated Cd19 Cre/+ and Pdap1 F/F Cd19 Cre/+ splenocyte cultures with Actinomycin D to inhibit the de novo transcription of AID and measured Aicda mRNA decay by qPCR. We found that depletion of Pdap1 did not affect the kinetics of Aicda mRNA degradation (Fig. 4, A and B). This result also indirectly shows that Pdap1 deficiency does not alter decapping or deadenylation of Aicda transcripts since a defect in any of these processes would result in accelerated decay. We concluded that Pdap1 is dispensable for Aicda transcript stability. Next, we monitored Aicda transcript splicing. Several splice variants for AID have been identified, with the full-length transcript being the only splice isoform encoding a functional AID protein (van Maldegem et al., 2009(van Maldegem et al., , 2010Sala et al., 2015). To assess whether Pdap1 modulates Aicda splicing, we performed RNA next-generation sequencing (RNA-seq) of activated B cells induced to undergo CSR to either IgG1 or IgG2b and analyzed the pattern of Aicda exon usage. We found no difference in the profile of Aicda exon usage between Cd19 Cre/+ and Pdap1 F/F Cd19 Cre/+ B cells ( Fig. 4 C). Furthermore, primary Aicda transcript levels in Pdap1 F/F Cd19 Cre/+ splenocytes were reduced compared with Cd19 Cre/+ cells to an extent similar to that observed for the fulllength spliced product (Figs. 4 D and 3 A). We concluded that ablation of Pdap1 does not affect Aicda mRNA splicing. Altogether, these findings indicate that Pdap1 supports physiological levels of AID expression, and that the CSR defect of Pdap1-deficient B cells is caused, at least in part, by impaired induction of AID expression following activation rather than a defect in Aicda posttranscriptional regulation.

Pdap1 deficiency in B cells induces the Atf4 stress response transcriptional program
To define the mechanism responsible for the defective induction of AID expression in Pdap1-deficient cells, we compared the transcriptome profiles of Cd19 Cre/+ and Pdap1 F/F Cd19 Cre/+ splenocytes stimulated with either LPS and IL-4 or LPS, B cellactivating factor (BAFF), and TGFβ (Table S1). The use of two stimulation conditions allowed us to zoom in common differentially regulated pathways and added a temporal dimension to the experiment, since LPS-BAFF-TGFβ-stimulated splenocytes proliferate faster than LPS-IL-4-activated ones (Fig. S2 F). To identify differentially expressed genes, we set the significance level of false discovery rate (FDR) to <0.05 (Fig. 5 A). The number of differentially regulated genes was higher in LPS-IL-4-stimulated cultures than in LPS-BAFF-TGFβ-activated cells (1,227 versus 173). Furthermore, the number of genes upregulated in Pdap1 F/F Cd19 Cre/+ was considerably higher than the , and Igγ1/Igγ3/Igγ2b/Igα (C) GLT levels in B cells activated to undergo CSR to the corresponding isotypes. The schematic representations on the left in B and C indicate the location of primers used to analyze the germline transcripts. Graphs summarize four to eight mice per genotype per stimulation condition. One control mouse (Cd19 Cre/+ or Pdap1 F/F ) within each experiment was assigned an arbitrary value of 1. (D) Representative WB analysis of splenocytes of the indicated genotypes 48 h after stimulation with LPS and IL-4. 1 and 2 indicate two different mice per genotype. Triangles indicate threefold dilution. Arrowhead and asterisk denote AID and aspecific band, respectively. Numbers underneath the blot indicate relative quantification of AID signal. (E) Summary graphs showing CSR to IgG2b (top) and IgG1 (bottom) following transduction of splenocytes of the indicated genotypes with either empty vector (EV) or an AID-ER-expressing retroviral construct. Graphs summarize three to eight mice per genotype analyzed in three (IgG2b) or four (IgG1) independent experiments. Different colors are used to group samples based on genotype (red for Aicda −/− , black for controls, and green for Pdap1 F/F Cd19 Cre/+ ). Empty and filled column bars indicate samples infected with EV and AID-ER constructs, respectively. Significance in A, C, and E was calculated with the Mann-Whitney U test. Error bars represent SD. ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. See also Fig. S3.
down-regulated ones in LPS-BAFF-TGFβ-stimulated cultures (124 up-versus 49 down-regulated) but was evenly distributed between the two categories in the LPS-IL-4 stimulation condition (561 up-versus 666 down-regulated).
Pathway enrichment analysis of the down-regulated genes in Pdap1 F/F Cd19 Cre/+ B cells in the LPS-IL-4 dataset identified the protein translation-related categories "ribosome," "ribosome biogenesis in eukaryotes," and "RNA transport" as the most significant categories (Fig. 5 B). Furthermore, analysis of individual genes showed that the most significantly down-regulated ones under both stimulations were factors essential for CSR and/ or genes highly expressed or induced in activated B cells (e.g., Aicda, Batf, Ccl22, Ighg3, and Tmem176b;  Table S1). In contrast, the top up-regulated categories in both datasets were related to metabolic pathways of amino acid and aminoacyl-tRNA biosynthesis (Fig. 5 B). Among the most significantly upregulated genes, we found key enzymes for the biosynthesis of asparagine (Asns), cysteine (Cth), and serine (Psat1, Psph), glutathione metabolism (Chac1), amino acid transporters (Slc1a4, Slc6a9), and several other metabolic and cytoprotective genes (  Table S1).
These data indicate that, in addition to the inhibition of key factors of the CSR program, Pdap1 deficiency in activated B cells is accompanied by transcriptional changes that reflect initial repression of global protein synthesis, up-regulation of cellular stress response genes, and metabolic rewiring to aid translational recovery. These changes represent key aspects of the transcriptional program of the ISR (Ron and Walter, 2007;Pakos-Zebrucka et al., 2016). To confirm the activation of the ISR in Pdap1-deficient B cells, we monitored the kinetics of eIF2α We concluded that Pdap1 deficiency results in up-regulation of the ISR core component Atf4. Altogether, these data indicate that Pdap1 ablation in mature B cells induces Atf4-dependent expression of stress response genes and down-regulation of the CSR program.

Pdap1 supports survival of mature B cells
The ISR is an adaptive pathway meant to restore cellular homeostasis following cell intrinsic and extrinsic stresses (Harding et al., 1999(Harding et al., , 2000Brostrom et al., 1996;Dever et al., 1992;Ron and Walter, 2007;Pakos-Zebrucka et al., 2016). However, when the stress is severe in either intensity or duration and overwhelms the response adaptive capacity, the ISR activates the apoptotic cell death program (Pakos-Zebrucka et al., 2016). Furthermore, down-regulation studies have implicated Pdap1 in cell survival and apoptosis resistance of cancer cell lines (Sharma et al., 2016;Weston et al., 2018). Therefore, we tested whether activation of the stress response in the absence of Pdap1 would induce apoptosis in B cells. To do so, we monitored caspase activation in splenocytes by CaspGLOW staining. Caspase activity increased after stimulation and was higher in Pdap1-deficient cells compared with controls at 24 and 48 h after activation (Fig. 6 A). Accordingly, despite the unaffected proliferation capability (Fig. S2), the number of live cells in Pdap1 F/F Cd19 Cre/+ splenocyte cultures was reduced 48 and 72 h after activation (Fig. 6 B). Finally, no differences were detected in mitochondrial mass or respiration capacity compared with controls ( Fig. 6 C and Fig. S5 C), thus indicating that activation of the ISR is not caused by mitochondrial disfunction. However, Pdap1 F/F Cd19 Cre/+ cultures displayed an increased proportion of cells with low mitochondrial membrane potential at early time points after activation (24 and 48 h; Fig. 6 D). Hence, a fraction of Pdap1-deficient B cells may undergo mitochondrial depolarization, an event usually associated with apoptosis induction (Gottlieb et al., 2003). Next, we tested whether Pdap1 deletion would affect the survival of activated B cells in vivo. To this end, we measured the percentage of GC B cells in Peyer's patches of Cd19 Cre/+ and Pdap1 F/F Cd19 Cre/+ mice. Peyer's patches are specialized secondary lymphoid tissues that line the wall of the small intestine. Because of their chronic exposure to an enormous variety of food-and microbiome-derived antigens, Peyer's patches display continual GC activity and are key to the induction of mucosal IgA antibody responses (Reboldi and Cyster, 2016). We found that the percentage of GC B cells was reduced in Peyer's patches of Pdap1 F/F Cd19 Cre/+ mice compared with the control group (Fig. 6 E). In agreement with the CSR defect exhibited by Pdap1 F/F Cd19 Cre/+ splenic B cells following activation in vitro (Fig. 2), the percentage of switched IgA GC B cells was considerably decreased in Pdap1 F/F Cd19 Cre/+ mice ( Fig. 6 E). Altogether, these data suggest that Pdap1 is required to support survival of activated B cells.
Pdap1 is dispensable for plasma cell differentiation Plasma cell differentiation represents the terminal phase of B cell development and is regulated by a transcriptional program that represses B cell identity while promoting the expression of plasma cell signature genes Shi et al., 2015;Minnich et al., 2016). This process is accompanied by massive expansion of the ER and the up-regulation of molecular chaperone and folding enzyme expression. The expansion of the secretory network is essential to accommodate the demands of increased immunoglobulin synthesis and secretion and is dependent on the activation of the unfolded protein response (UPR; Zhang et al., 2005;Gass et al., 2002Gass et al., , 2008Iwakoshi et al., 2003;van Anken et al., 2003;Shaffer et al., 2004;Taubenheim et al., 2012;Todd et al., 2009). The UPR is a ubiquitous signaling network that senses and responds to the accumulation of misfolded proteins in the ER are up-or down-regulated in Pdap1 F/F Cd19 Cre/+ cells are highlighted in red or purple, respectively. Data summarizes results from three mice per genotype per stimulation condition and is presented as log2(RPM) (reads per gene per million mapped sequence reads) values. (B) Pathway enrichment analysis (KEGG pathways) of the differentially regulated genes (FDR < 0.05) from A. The number of differentially regulated genes in each category is indicated. (C) Volcano plots displaying differentially expressed genes between control and Pdap1-deficient splenocytes activated with LPS and IL-4 (right) or LPS, BAFF, and TGFβ (left). The red and purple dots represent transcripts up-and down-regulated in Pdap1 F/F Cd19 Cre/+ cells, respectively, with FDR < 0.05 and expression fold change > 2 (up-regulated) or 1.7 (down-regulated) in both stimulation conditions. The names of the down-regulated and the 30 most significantly up-regulated genes within each stimulation condition are indicated in each graph. The fold change threshold for down-regulated genes was set to 1.7 to include genes yielding a biologically relevant effect even with less pronounced variations in expression levels (e.g., Aicda, haploinsufficient gene). (D) Representative WB analysis of splenocytes of the indicated genotypes before (resting B cells) and after 24-96-h activation with LPS and IL-4. Data are representative of two mice per genotype, and quantification values of WB signals are indicated underneath the corresponding blots. See also Fig. S5 and Table S1.  (Ron and Walter, 2007;Walter and Ron, 2011;Schröder and Kaufman, 2005). This response is mediated by three ERresident proteins which, in addition to Perk, comprise the inositol-requiring protein kinase/endoribonuclease-1 (Ire1) and Atf6 (Tirasophon et al., 1998;Wang et al., 1998;Haze et al., 1999Haze et al., , 2001. The UPR ultimately reestablishes protein homeostasis by integrating Perk-eIF2α-dependent attenuation of global protein synthesis with Ire1-and Atf6-mediated transcription of factors and enzymes that increase the protein folding and degradation capabilities of the ER. The Ire1 and Atf6 branches of the UPR are activated during normal differentiation of naïve B cells into plasma cells, and the Ire1 arm is essential for the expansion of their secretory network (Zhang et al., 2005;Gass et al., 2002Gass et al., , 2008Iwakoshi et al., 2003;van Anken et al., 2003;Aragon et al., 2012). In contrast, the Perk arm of the UPR is dispensable for plasma cell development (Zhang et al., 2005;Gass et al., 2008). Furthermore, this pathway is suppressed during differentiation of naïve B cells into plasma cells (Ma et al., 2010;Gass et al., 2008;Zhang et al., 2005), and unrestricted Perk-eIF2α-Atf4 signaling in activated B cells blocks the formation of plasma cells (Zhu et al., 2019). Given the up-regulated expression of Atf4 in Pdap1-deficient B cells, we considered the possibility that plasma cell development might be impaired in the absence of Pdap1. To this end, we analyzed the plasma cell compartment of Pdap1 F/F Cd19 Cre/+ mice. We found that the percentage and number of plasma cells (CD138 + TACI + ) in bone marrow and spleen of unimmunized mice was similar between control and Pdap1 F/F Cd19 Cre/+ mice (Fig. 7 A).  Activation of naïve B cells with specific combinations of stimuli in vitro induces differentiation into plasmablasts. Plasmablasts are proliferating antibody-secreting cells (ASCs) whose differentiation is driven by the same transcriptional program responsible for plasma cell development . Therefore, we tested the formation of plasmablasts (B220 + CD138 + ) in vitro under the same stimulation conditions used for our previous analyses. We observed no significant difference in the percentage of plasmablasts between controls and Pdap1 F/F Cd19 Cre/+ cultures (Fig. 7 B). Accordingly, we did not detect any apparent change in the activation of the plasmablast and plasma cell gene signature between the two genotypes in the RNA-seq analysis (Table S1). We concluded that Atf4 up-regulation in activated Pdap1 F/F Cd19 Cre/+ B cells does not inhibit the generation of plasmablasts and plasma cells, and that Pdap1 is not required for the B cell developmental program controlling plasmablast and plasma cell formation and identity.
The ER expansion and increase in protein processing and folding capabilities of plasma cells are regulated by the transcription factor Xbp1-s (spliced Xbp1; Shaffer et al., 2004). Xbp1 deficiency in B cells does not interfere with the development of plasma cells but impairs immunoglobulin secretion because of defective expansion of the secretory pathway (Hu et al., 2009;Todd et al., 2009;Taubenheim et al., 2012;Shaffer et al., 2004). Xbp1-s is expressed as a result of an unconventional splicing event mediated by Ire1 that removes a 26-nt segment from Xbp1 mRNA (unspliced Xbp1 transcript, Xbp1-u), thus changing the transcript reading frame in the resulting spliced Xbp1 transcript (Xbp1-s; Fig. 7 C; Yoshida et al., 2001). Xbp1-s translocates into the nucleus and activates the transcription of genes responsible for the expansion of the secretory pathway (Shaffer et al., 2004;Tellier et al., 2016). We found no difference in Xbp1 mRNA splicing and Xbp1-s expression levels between control and Pdap1 F/F Cd19 Cre/+ splenocyte cultures (Fig. 7, C and D). These results suggest that the transcriptional program controlling ER network expansion in plasma cells is not affected by Pdap1 deletion. Finally, ELISpot analysis did not detect any significant difference in the number or spot size of ASCs between the two groups ( Fig. 7 E), thus indicating that antibody production is not affected by Pdap1 deficiency. We concluded that Pdap1 is dispensable for the differentiation programs that establish plasma cell identity and function under steady-state conditions.

Pdap1 is required for efficient SHM of Ig loci
Aicda is a haploinsufficient gene, and Aicda +/− mice exhibit defects in both antibody diversification reactions initiated by AID, CSR, and SHM Sernández et al., 2008;Takizawa et al., 2008). Given the reduced levels of AID expressed by Pdap1 F/F Cd19 Cre/+ B cells following activation, we considered the possibility that Pdap1 depletion might also affect SHM. To test this hypothesis, we sequenced the intronic regions downstream of the J H 4 and J K 5 elements in GC B cells sorted from Peyer's patches of aged Pdap1 F/F Cd19 Cre/+ mice (Jolly et al., 1997). Because of their chronic exposure to microbial antigens, GC B cells in Peyer's patches accumulate a high number of mutations over time (González-Fernández et al., 1994). Accordingly, we found that GC B cells from control mice displayed a high mutation frequency at both J H 4 and J K 5 intronic regions (Fig. 8, A-D). The vast majority of sequences contained mutations (86%, 79 of 92 for J H 4, and 83%, 104 of 125 for J K 5), and a considerable portion of B cells were heavily mutated (Fig. 8, E and F). In contrast, Pdap1 F/F Cd19 Cre/+ GC B cells exhibited a significantly lower mutation frequency at both J H 4 and J K 5 introns (Fig. 8, A-D). The distribution of mutations per sequence was skewed toward an increase in the proportion of sequences bearing zero or fewer than five mutations (50%, 54 of 107 for J H 4, and 65%, 83 of 127 for J K 5), whereas the number of highly mutated clones was concomitantly reduced (Fig. 8, E and F). In agreement with a role of Pdap1 in promoting AID expression rather than the processing of AID-induced lesions, the profile of mutations was not affected by Pdap1 deletion (Fig. 8, G and H).
Altogether, these findings indicate that ablation of Pdap1 impairs the efficiency of SHM of heavy and light chain Ig loci. Therefore, Pdap1 is required to support both antibody diversification reactions occurring in mature B cells, namely CSR and SHM.

Discussion
In this study, we report the identification of a novel factor required for mature B cell homeostasis and function. Pdap1-deficient B cells develop normally but display reduced cell viability at the mature stage both in vitro and in vivo. Furthermore, they fail to efficiently induce AID expression and cannot support physiological levels of CSR and SHM.
Pdap1 deficiency in mature B cells phenocopies key aspects of the cellular response to chronic stress. Stress-induced eIF2α phosphorylation drives the inhibition of global protein synthesis, while allowing the preferential translation of stress-responsive genes like Atf4 (Harding et al., 2000;Hinnebusch, 2000;Scheuner et al., 2001;Lu et al., 2004). The transcriptional program induced by Atf4 comprises cytoprotective genes as well as the up-regulation of biosynthetic pathways to reprogram cellular metabolism and aid homeostasis recovery. This anabolic program encompasses metabolic pathways of amino acid biosynthesis and the expression of amino acid transporter and aminoacyl-tRNA synthetase genes, and supports recovery of translation under conditions of acute stress Rendleman et al., 2018;Quirós et al., 2017;Krokowski et al., 2013;Han et al., 2013). However, during chronic stress, Atf4-dependent increase in protein synthesis leads to proteotoxicity, oxidative stress, and cell death Krokowski et al., 2013). Pdap1-deficient B cells express Atf4 and exhibit the same transcriptional signature of Atf4-mediated translational recovery (Fig. 5, Fig. S5, and Table S1). Furthermore, the expression of this anabolic program correlates with increased apoptosis of Pdap1 F/F Cd19 Cre/+ B cells. Therefore, our data are consistent with a model in which ablation of Pdap1 in activated B cells leads to cell death via induction of an Atf4dependent chronic stress response. In addition, resting B cells from Pdap1-deficient mice displayed phosphorylation of Perk and its downstream target eIF2α as well as Atf4 expression, thus indicating constitutive activation of the Perk-mediated ISR pathway. This result is in agreement with the reduced number of resting B cells isolated from spleens of Pdap1 F/F Cd19 Cre/+ (Fig.  S1 E) and indicates that Pdap1 deficiency is also detrimental in naïve B cells. Therefore, Pdap1 is required to protect B lymphocytes from stress-induced cell death under both resting and activated conditions. Interestingly, while Atf4 expression increased steadily over time after activation in Pdap1-deficient B cells, phosphorylation of Perk and eIF2α was no longer detectable. This observation has two important implications. First, the up-regulation of Atf4 expression in Pdap1-ablated cells is uncoupled from Perk-eIF2α phosphorylation following activation. This result is in agreement with the identification of a phospho-eIF2α-independent regulation of Atf4 expression observed during conditions of prolonged cellular stress (Guan et al., 2014). This regulation is likely under the control of mTORC1, which is activated in B cells after stimulation (Powell et al., 2012;Limon and Fruman, 2012;Iwata et al., 2017), and has been shown to promote Atf4 translation in a phospho-eIF2α-independent manner in other cellular contexts (Park et al., 2017;Ben-Sahra et al., 2016). Second, the dephosphorylation of Perk and eIF2α occurs at early time points after activation. This observation implies the existence of an active mechanism operating immediately upon B cell activation that physiologically inhibits the Perk-eIF2α signaling cascade. In line with this point, although mature B cells retain the potential to activate all three arms of the UPR in response to pharmacological triggers of the pathway, the Perk branch is suppressed during normal differentiation of activated B cells into plasma cells (Ma et al., 2010;Gass et al., 2008;Zhang et al., 2005). Moreover, nonphysiological Perk-eIF2α-Atf4 signaling after B cell activation even blocks the formation of plasma cells (Zhu et al., 2019). In Pdap1 F/F Cd19 Cre/+ splenocytes, the Perk-eIF2α-Atf4 pathway is already active under resting conditions, and the increased loss of mitochondrial membrane potential and apoptosis were observed at early, but not late, stages after activation. The earlier kinetics of Atf4 induction in our experimental conditions would explain why Pdap1 deficiency leads to cell death of naïve and early activated B cells but does not affect the plasma cell compartment. It is important to note that the analysis of plasma cell differentiation and function was performed under steady-state conditions; therefore, we cannot completely exclude the possibility that Pdap1 deficiency might affect plasma cell differentiation following immunization. The activation of the Atf4-dependent stress response in Pdap1 F/F Cd19 Cre/+ B cells was accompanied by down-regulation of key factors driving antibody diversification, including AID (Muramatsu et al., 2000;Revy et al., 2000) and the transcription factor Batf, which has been shown to directly control AID expression and other aspects of the CSR program (Ise et al., 2011). Defective induction of Aicda expression in activated Pdap1-deficient B cells was common to all stimulation conditions (Figs. 3 A and 5 C) and likely represents the dominant cause for the reduced CSR and SHM efficiency. Aicda is an haploinsufficient gene, and the efficiency of CSR is directly proportional to AID levels. Mice heterozygous for a null Aicda allele display defects in both CSR and SHM Sernández et al., 2008;Takizawa et al., 2008). Analogously, Pdap1 deficiency reduced Aicda expression to approximately half of WT levels in activated B cells and is associated with a similar reduction in the efficiency of both antibody diversification reactions. Furthermore, overexpression of AID in LPS-BAFF-TGFβ-stimulated splenocytes, which did not exhibit any additional defect in GLT, rescued the IgG2b CSR defect of Pdap1 F/F Cd19 Cre/+ B cells to a considerable extent.
Pdap1 might control activation of the ISR and the antibody diversification program via independent mechanisms. We found that Pdap1 is localized primarily in the cytoplasm in different cell types. Considering this observation and that Pdap1 was recently described as a RNA-binding protein (Castello et al., 2012(Castello et al., , 2016Iadevaia et al., 2020;Trendel et al., 2019;Baltz et al., 2012), it is tempting to hypothesize that it might play a role in mRNA turnover. In regard to AID, however, we showed that Pdap1 promotes its efficient expression but is dispensable for Aicda transcript stability. Therefore, a potential involvement of Pdap1 in the regulation of AID levels via control of mRNA turnover would occur indirectly, e.g., via modulation of the expression of intermediate factors. Although we cannot exclude the possibility that Pdap1 might regulate the expression of AID independently from its protective role against chronic ISR activation, we favor the hypothesis that the two processes are tightly linked. It is intriguing to speculate that the sustained induction of the ISR in Pdap1 F/F Cd19 Cre/+ B cells might actively interfere with the CSR and SHM programs. In this respect, limiting AID expression under conditions of cellular stress might represent a mechanism to alleviate the additional burden imposed by AID-induced genotoxic stress on activated B cells. Analysis of humoral responses upon genetic deletion of ISR components on a Pdap1deficient background would provide the basis for a new exciting line of investigation into the relationship between response to cellular stresses and antibody gene diversification in mature B cells.

Mouse strains
Cd19 Cre (Cd19 tm1(cre)Cgn ; Rickert et al., 1997) and Aicda −/− (Aicda tm1Hon/tm1Hon ; Muramatsu et al., 2000) mice were previously described and were maintained on a C57BL/6 background. The conditional Pdap1 F allele bears LoxP sites flanking Exon 2 (ENSMUST00000031627.8) and was generated by CRISPR-Cas9-mediated knock-in microinjection of single-cell embryos on a C57BL/6N strain background (Max Delbrück Center [MDC] Transgenics platform). Germline transmission was confirmed, and positive pups were bred with Cd19 Cre mice to generate Pdap1 F/F Cd19 Cre/+ mice. Mice were maintained in a specific pathogen-free barrier facility under standardized conditions (20°C ± 2°C; 55 ± 15% humidity) on a 12-h light/dark cycle. 7-27wk-old mice of both genders were used in age-matched groups for the experiments. Controls included littermates and mice from inbred strains. All experiments were performed in compliance with European Union directive 2010/63/EU and in agreement with Landesamt für Gesundheit und Soziales directives (Berlin, Germany). Primers used for genotyping the Pdap1 F allele are listed in Table S2.

Retroviral infection
The pMX-AID-ER-IRES-GFP retroviral vector was a kind gift from Qiao Wang (The Rockefeller University, New York, NY). Splenocyte infections were performed as follows. The HEK293T derivative cell line BOSC23 was transfected with pCL-Eco and pMX-IRES-GFP or pMX-AID-ER-IRES-GFP retroviral vectors using FuGENE HD Transfection Reagent (Promega) to generate viral particle-containing supernatants. B cells were activated with 5 µg/ml LPS and 5 ng/ml IL-4 (IgG1) or 5 µg/ml LPS, 10 ng/ ml BAFF, and 2 ng/ml TGFβ (IgG2b) and transduced twice with the viral supernatant, 1 and 2 d after isolation. 4-HT was added 72 h after activation at a final concentration of 0.05 µM, and CSR was assessed 24 h later on the GFP + -gated populations. The pMX-Pdap1-3xFlag construct was generated by cloning the cDNA for human Pdap1 into pMX retroviral vector with a C-terminal 3xFlag tag. Primers used to clone the pMX-Pdap1-3xFlag construct are listed in Table S2. iMEFs infections were performed as indicated above for splenocytes, except that cells were transduced with pCL-Eco and pMX-EV/pMX-Pdap1-3xFlag-viral supernatants four times over 2 d. 48 h after the first transduction, iMEFs were selected using 1.5 µg/ml puromycin (Sigma-Aldrich).

B cell development and differentiation analyses
For analysis of B cell development and differentiation, spleen and bone marrow cell suspensions were incubated with ACK lysis buffer (Thermo Fisher Scientific) for erythrocyte depletion. Subsequently, cells were blocked with TruStain fcX (BioLegend) for 10 min at 4°C and labeled with fluorochrome-conjugated antibodies to determine the surface expression of CD43, IgM, CD21/CD35, and IgD (BD Biosciences), and CD23, CD3, and CD19 (BioLegend) in PBS supplemented with 3% FBS (PBS/FBS) for 20 min at 4°C. Cells were then washed, resuspended in PBS/FBS, and analyzed.
For analysis of plasma cell differentiation in vitro, splenic B cells were isolated by immunomagnetic depletion of CD43 + cells and cultured at a density of 10 6 cells/ml in the presence of either 20 µg/ml LPS (Sigma-Aldrich; L2880-10MG) and 25 ng/ml IL-4, or 20 µg/ml LPS, 10 ng/ml BAFF, and 2 ng/ml TGFβ. To determine the percentage of plasmablasts, cells were harvested after 96 h, blocked with TruStain fcX for 10 min at 4°C, and stained for surface expression of B220 (BioLegend; 103245) and CD138 (BioLegend; 142506) in MACS buffer (PBS supplemented with 0.5% BSA and 2 mM EDTA) for 20 min at 4°C. Cells were then resuspended in FACS buffer (PBS supplemented with 3% FCS and 1 mM EDTA) containing 1 µg/ml propidium iodide and analyzed.
For analysis of the plasma cell compartment in vivo, 9-, 14-, and 25-wk-old mice were sacrificed to isolate the spleen and hind legs. Total splenocytes and bone marrow cells were isolated by smashing the respective organs, and erythrocytes were depleted by incubation with Gey's ABC solution for 3 min on ice. For surface staining, 5 × 10 6 cells were first blocked with TruStain fcX for 10 min at 4°C and then stained for the following markers for 20 min at 4°C in MACS buffer: CD138, TACI (BD PharMingen; 558410), CD93 (BioLegend; 136506), CD19 (Bio-Legend), and B220. Cells were resuspended in FACS buffer containing 1 µg/ml propidium iodide and analyzed.
For ELISpot analysis, MultiScreen HTS -IP plates (Merck) were activated with 35% ethanol in PBS for 1 min, washed three times with PBS, and finally coated with 1 µg/ml anti-Igκ (Southern Biotech) and anti-Igλ (Southern Biotech) in PBS at 4°C overnight. The following day, 2 × 10 5 total splenocytes or BM cells (as well as serial fivefold dilutions) were cultured overnight in RPMI 1640 supplemented with 10% FCS (Sigma-Aldrich) and penicillin-streptomycin. The next day, plates were washed thoroughly six times with PBS containing 0.1% Tween20 and incubated with 1 µg/ml biotinylated antibodies against IgM (Southern Biotech) for 2 h at 37°C. Plates were washed three times under running distilled water and incubated with 0.3 U/ml streptavidin-AP (Roche) for 30 min at room temperature. For detection of spots, plates were washed three times under running distilled water, equilibrated in AP buffer (100 mM Tris-HCl, pH 9.0, 150 mM NaCl, and 1 mM MgCl 2 ), and developed using NBT/BCIP substrate mix (Promega) diluted in AP buffer. Spots were scanned and counted using the ImmunoSpot Series 6 Alfa Analyzer and ImmunoCapture Image Acquisition as well as ImmunoSpot Analysis software (C.T.L.) with the following parameters: sensitivity 176, spot separation 1.00, diffuseness largest, background balance 40, minimum spot size 0.0015 mm 2 , and maximum spot size 0.6976 mm 2 .
For assessment of GC B cells and CSR in vivo, cell suspensions derived from Peyer's patches were blocked with TruStain fcX for 10 min at 4°C. Subsequently, cells were stained with fluorochromeconjugated anti-CD19, anti-B220/CD45R, anti-CD38 (BioLegend), anti-Fas/CD95, and anti-IgA (BD Biosciences) for 20 min at 4°C in PBS/FBS. Cells were washed, resuspended in PBS/FBS, and analyzed. All samples were acquired on a LSRFortessa cell analyzer (BD Biosciences).

Cell proliferation and apoptosis analysis
For cell proliferation analysis by cell tracking dye dilution, primary B cells were pulsed with 5 µM CellTrace Violet (Thermo Fisher Scientific) for 10 min at 37°C. Apoptosis analysis was performed by using CaspGLOW Fluorescein Active Caspase Staining Kit (BioVision) according to the manufacturer's instructions. Samples were acquired on a LSRFortessa cell analyzer (BD Biosciences). Mitochondrial mass and membrane potential were measured via staining with MitoTracker Green and DeepRed (Thermo Fisher Scientific), respectively, according to the manufacturer's instructions. Samples were acquired on a LSRFortessa cell analyzer (BD Biosciences).

CRISPR-Cas9 gene targeting
For the loss-of-CSR assay in bulk CH12 cultures, three gRNAs against Pdap1 gene were cloned into the U6 cassette of a variant of the original pX330 plasmid (pX330-U6-Chimeric_BB-CBh-hSpCas9, Addgene; 42230) modified to express Cas9 WT -T2A-GFP (kind gift from Van Trung Chu, MDC; gPdap1-1/3), and three gRNA pairs were cloned into tandem U6 cassettes in a mutated version of pX330 expressing Cas9 D10A (Nickase-a/c). CH12 cells were transfected with the Cas9-gRNAs expressing constructs (either individually or in a pooled format) via electroporation with Neon Transfection System (Thermo Fisher Scientific), sorted for GFP-positive cells after 40 h, and left to recover for 72 h before activation for CSR analysis. For the generation of Pdap1-deficient CH12 clonal derivatives, the Nickases-a/c constructs were individually electroporated into CH12. Single GFPpositive cells were sorted in 96-well plates after 40 h, and clones were allowed to grow for 12 d before CSR analysis in 96-well format and expansion of selected clones. Controls for gRNAnucleofected CH12 were cells nucleofected with either empty vector or gRNAs against random sequences not present in the mouse genome (single random gRNA-Cas9 WT as controls for gPdap1-1/3, and random gRNAs pairs-Cas9 D10A as controls for Nickase-a/c). Selected clones R1/R2 and P1/3 were validated at the level of genomic scar and protein expression. The sequences of the gRNAs used in these studies (this paper; Delgado-Benito et al., 2018) are listed in Table S2.
WB analysis WB analysis of protein levels was performed on whole-cell lysates prepared by lysis in radioimmunoprecipitation assay buffer (Sigma-Aldrich) supplemented with Complete EDTA free proteinase inhibitors (Roche). Pierce Phosphatase Inhibitor Mini Tablets (Thermo Fisher Scientific) were added to the lysis buffer for the analysis of phosphorylated Perk and eIF2α. The antibodies used for WB analysis are anti-Pdap1 (Sigma-Aldrich), anti-tubulin (Abcam), anti-β-actin (Sigma), anti-AID (mAID-2; Thermo Fisher Scientific), anti-phospho-Perk (pThr980; Thermo Fisher Scientific), anti-Perk (Cell Signaling Technology), antiphospho-eIF2α (pSer51; Cell Signaling Technology), anti-eIF2α (Santa Cruz), anti-Atf4 (Cell Signaling Technology), and anti-Xbp1-s (Cell Signaling Technology). WB signals were quantified using ImageJ (National Institutes of Health), and normalization was performed against loading controls. qPCR mRNA levels for AID and postspliced germline transcripts were measured as follows. Total RNA was extracted from splenocyte cultures 48 h after activation using TRIzol (Invitrogen) according to manufacturer's instructions and retro-transcribed with SuperScript VILO cDNA Synthesis Kit (Invitrogen). Genomic DNA was removed by RapidOut DNA Removal Kit (Thermo Fisher Scientific). Transcripts were amplified using StepOne-Plus Real-Time PCR System (Applied Biosystems) with Luna Universal qPCR Mastermix (NEB). For analysis of Aicda mRNA decay, Actinomycin D was added to the culture medium 48 h after activation at a final concentration of 10 µg/ml for 0.5, 1, 1.5, 2, 3, and 4 h. qPCR analyses were normalized to Ubc or Gapdh (Aicda mRNA and GLTs) or Gapdh (Aicda mRNA decay). Primers used for qPCR (this paper; Xu et al., 2015;Muramatsu et al., 2000;Albershardt et al., 2012) are listed in Table S2.
For electrophoretic analysis of Xbp1 splicing, Xbp1 transcripts were amplified using the primers listed in Table S2 (Madaro et al., 2013) and either mock-digested or digested with PstI enzyme (NEB) for 30 min at 37°C. The intact spliced (Xbp1-s) transcript RT-PCR product and the unspliced (Xbp1-u) transcript digestion products were visualized on 2% agarose gel.
RNA-seq RNA-seq was performed on three mice per genotype. Splenocytes were cultured in LPS and IL-4 (IgG1) or LPS, BAFF, and TGFβ (IgG2b) for 48 h. Cells were collected by centrifugation, and RNA was extracted with TRIzol (Invitrogen) according to manufacturer's instructions. Ribosomal RNA was depleted using Ribo-Zero Gold rRNA Removal Kit (Illumina). Libraries were prepared with TruSeq Stranded Total RNA Library Prep Kit Gold (Illumina) and run in one lane of a flow cell of HiSeq 4000 (Illumina).
The RNA-seq data were analyzed using the pigx-rnaseq pipeline (Wurmus et al., 2018). STAR (Dobin et al., 2013) mapped the reads to the GRCm38 assembly for mouse by Ensembl, and HTSeq (Anders et al., 2015) was used to count the transcript abundance. Differential expression analysis was done using the DESeq2 (Love et al., 2014) package for R, which uses the Wald test for significance.
For splicing analysis of Aicda and Pdap1 genes, we used the edgeR (Robinson et al., 2010) package for R to determine differentially expressed exons. The annotation was provided by a filtered version of the GRCm38 gene annotation, which contained unique, Havana-annotated exons.

Seahorse assay
Oxygen consumption rate and extracellular acidification rate were measured with the Seahorse XF96 metabolite analyzer using the Seahorse XF Cell Mito Stress Test kit (Agilent). Primary B cells were isolated, counted, and plated at the indicated cell densities directly on Seahorse cell culture plates coated with poly-L-lysine (Sigma-Aldrich). Before measurement, cells were incubated for a maximum of 1 h in Seahorse XF DMEM medium pH 7.4 (Agilent) supplemented with 10 mM glucose (Agilent), 1 mM sodium pyruvate (Agilent), and 2 mM L-glutamine (Agilent).

Statistical analysis
The statistical significance of differences between groups/datasets was determined by the Mann-Whitney U test for all data presented in this study, with the following exceptions. For the RNA-seq analysis, the Wald test of the DESeq2 package for R was used, and we considered genes with an FDR < 0.05 to be significantly differentially expressed. Statistical details of experiments can be found in the figure legends.

Data availability
The deep-sequencing data reported in this paper (RNA-seq) has been deposited in the GEO repository under accession no. GSE141876.
Online supplemental material Fig. S1 shows that Pdap1 is dispensable for B cell development. Fig. S2 shows that Pdap1 is dispensable for B cell proliferation. Fig. S3 shows that Aicda mRNA levels are reduced in Pdap1deficient CH12 cell lines. Fig. S4 shows that Pdap1 is localized primarily in the cytoplasm in B cells and MEFs. Fig. S5 shows the stress response signature of Pdap1-deficient B cells. Table S1 contains the pairwise comparison of differentially regulated genes in control versus Pdap1 F/F Cd19 Cre/+ splenocytes. Table S2 lists the oligonucleotides used in this study.  Table S1 shows a pairwise comparison of differentially regulated genes in control versus Pdap1 F/F Cd19 CRE/+ splenocytes. Table S2 lists the oligonucleotides used in this study.