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IMMUNOBIOLOGY The forkhead FOXP1 represses human differentiation

Martine van Keimpema,1 Leonie J. Gr¨uneberg,1 Michal Mokry,2 Ruben van Boxtel,3 Menno C. van Zelm,4 Paul Coffer,5 Steven T. Pals,1,* and Marcel Spaargaren1,*

1Department of Pathology, Lymphoma and Myeloma Center Amsterdam (LYMMCARE), Academic Medical Center, Amsterdam, The Netherlands; 2Department of Pediatric Gastroenterology, University Medical Center Utrecht, Utrecht, The Netherlands; 3Hubrecht Institute for Developmental Biology and Stem Cell Research, and University Medical Center Utrecht, Utrecht, The Netherlands; 4Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands; and 5Department of Cell Biology, University Medical Center Utrecht, Utrecht, The Netherlands

Key Points Expression of the forkhead transcription factor FOXP1 is essential for early B-cell devel- opment, whereas downregulation of FOXP1 at the germinal center (GC) stage is required for • Aberrant expression of GC B-cell function. Aberrantly high FOXP1 expression is frequently observed in diffuse FOXP1 in human MBCs large B-cell lymphoma and mucosa-associated lymphoid tissue lymphoma, being associ- represses their ability to ated with poor prognosis. Here, by expression analysis upon ectopic overexpression differentiate into PCs. of FOXP1 in primary human memory B cells (MBCs) and B-cell lines, combined with chro- • Human IgG1 MBCs combine matin immunoprecipitation and sequencing, we established that FOXP1 directly represses PRDM1 IRF4 XBP1 lower FOXP1 expression expression of , ,and , transcriptional master regulators of plasma cell (PC) differentiation.Inaccordance, FOXP1isprominently expressed inprimary humannaive and with a higher propensity to MBCs, but expression strongly decreases during PC differentiation. Moreover, as compared differentiate as compared with 1 1 1 with immunoglobulin (Ig) M MBCs, IgG MBCs combine lower expression of FOXP1 with an IgM MBCs. enhanced intrinsic PC differentiation propensity, and constitutive (over)expression of FOXP1 in B-cell lines and primary human MBCs represses their ability to differentiate into PCs. Taken together, our data indicate that proper control of FOXP1 expression plays a critical role in PC differentiation, whereas aberrant expression of FOXP1 might contribute to lymphomagenesis by blocking this terminal B-cell differentiation. (Blood. 2015;126(18):2098-2109) Introduction

The hallmark of antibody-mediated adaptive immunity is the gener- program, including BCL6, PAX5, SpiB, and BACH2. These tran- ation of plasma cells (PCs) producing high titers of antigen-specific scription factors inhibit differentiation of activated B cells, allowing antibodies. Upon the first encounter with antigen and T-cell help, naive sufficient time for affinity maturation and CSR to occur. They act pre- B cells give rise to a primary immune response. This response results in dominantly by repressing the factors required for PC differentiation.4 the generation of short-lived antibody-secreting plasmablasts and ger- As such, PC differentiation involves the tight control of expression and minal center (GC) B cells. In the GCs, the B cells acquire somatic coordinated interplay between these transcriptional activators and re- mutations in the variable region of the immunoglobulin and can pressors, including several double-negative feedback mechanisms, for undergo class switch recombination (CSR), whereby the immuno- instancePAX5andBCL6repressingBLIMP1expression,andvice globulin constant region is switched from immunoglobulin (Ig) M to versa.10-13 Aberrations in genes that regulate PC differentiation, IgG, IgA, or IgE.1 Cells that acquire somatic mutations that improve such as translocations of PAX5 and BCL6,amplification of SPIB, antigen-binding affinity gain a survival advantage and emerge from and loss-of-function deletions or mutations in PRDM1, are frequently GCs as long-lived memory B cells (MBCs) or PCs, which maintain present in B-cell non-Hodgkin lymphomas.14-17 These aberrations serum immunoglobulin levels.2 After reexposure to their cognate an- stall further differentiation of the B cells, thereby contributing to tigen, MBCs can rapidly differentiate to PCs, resulting in a fast second- lymphomagenesis.18,19 ary response. TheforkheadtranscriptionfactorFOXP1hasbeenshowntobe Differentiation of B cellsinto PCs is regulated by a complex network essential for early B-cell development.20 Recurrent chromosomal trans- of transcription factors. IRF4, BLIMP1 (encoded by the PRDM1 gene), location involving FOXP1 in diffuse large B-cell lymphoma (DLBCL) and XBP1 are essential drivers of PC differentiation and immunoglob- and mucosa-associated lymphoid tissue lymphoma, and the frequent ulin secretion,3,4 IRF4 being able to drive expression of BLIMP1,5-8 aberrantly high FOXP1 expression in these lymphomas, which is as- which in turn induces expression of XBP1.9 Induction of PC differen- sociated with poor prognosis, suggest that FOXP1 also exerts functional tiation requires an active suppression of the B-cell roles in mature B cells.21-24 In accordance, we recently demonstrated

Submitted February 1, 2015; accepted August 7, 2015. Prepublished online as There is an Inside Blood Commentary on this article in this issue. Blood First Edition paper, August 19, 2015; DOI 10.1182/blood-2015-02- The publication costs of this article were defrayed in part by page charge 626176. payment. Therefore, and solely to indicate this fact, this article is hereby *S.T.P. and M.S. contributed equally to this study. marked “advertisement” in accordance with 18 USC section 1734. The online version of this article contains a data supplement. © 2015 by The American Society of Hematology

2098 BLOOD, 29 OCTOBER 2015 x VOLUME 126, NUMBER 18 From www.bloodjournal.org by guest on July 14, 2019. For personal use only.

BLOOD, 29 OCTOBER 2015 x VOLUME 126, NUMBER 18 FOXP1 REPRESSES PLASMA CELL DIFFERENTIATION 2099 that FOXP1 overexpression in primary human B cells cooperates with ELISPOT nuclear factor kB pathway activity to promote B-cell survival.14,25 26 IgG and IgM enzyme-linked immunospot (ELISPOT) assays were performed Furthermore, a recent study by Sagardoy et al showed that FOXP1 using IgG and IgM ELISpot kits (Mabtech) according to the manufacturer’s expression is temporarily repressed at the GC stage, which is needed for instructions. appropriate GC B-cell function.26 However, potential functions of FOXP1 in differentiation of post-GC B cells have not yet been assessed. ELISA Here, we show that FOXP1 directly represses expression of essential Enzyme-linked immunosorbent assay (ELISA) was performed essentially as drivers of PC differentiation, such as PRDM1, IRF4,andXBP1;that described.34 Details are described in the supplemental Methods. FOXP1 is prominently expressed in human naive, GC, and MBCs but IgG isotype ELISA was performed using the human IgG subclass profile not in PCs; and that reduced expression of FOXP1 is required for ELISA kit (Invitrogen) according to the manufacturer’s instructions. efficient PC differentiation. Flow cytometry

Cells were stained with anti-human IgM or IgG (both from Southern Biotech), CD38 (BD), or CD20 conjugated with PE or APC and analyzed on a Materials and methods FACSCanto. For intracellular staining the Foxp3/transcription factor staining buffer set (ebioscience) and anti FOXP1-APC (R&D), CD19-APC-H7, Constructs CD27-FITC, and IgM-V450 (all from BD), and IgG-PE were employed. Retroviral construct FOXP1-LZRS-IRES-YFP was described previously.25 LZRS-IRES-YFP empty vector and LZRS-BCL6-IRES-GFP were kindly provided by Dr H. Spits.27 Results B-cell isolation FOXP1 represses expression of PC signature genes and is Buffy coats were obtained from Sanquin blood bank (Amsterdam, the Netherlands). prominently expressed in all human mature B-cell subsets MBCs were isolated as previously described.25 IgM1 MBCs were isolated 1 1 2 2 except for PCs by FACSAria sorting of the CD19 CD27 IgG IgA population, using an allophycocyanin (APC)-conjugated antibody against CD19 (BD), an fluores- Gene expression microarray analysis of primary human MBCs, retro- – cein isothiocyanate (FITC) conjugated antibody against CD27 (BD), and virally transduced with LZRS-FOXP1-IRES-YFP to constitutively over- phycoerythrin (PE)–conjugated antibodies against IgG and IgA (Southern 1 1 express FOXP1 or with “empty” expression vector (LZRS-IRES-YFP) Biotech), and IgG MBCs were isolated by FACSAria sorting of the CD19 25 1 2 2 as a negative control, revealed that FOXP1-downregulated genes CD27 IgM IgA population, using antibodies against CD19, CD27, and PE- fi conjugated antibodies against IgM and IgA (Southern Biotech). Tonsils were were enriched for a previously de ned signature of genes highly ex- 35,36 P 5 obtained from children undergoing routine tonsillectomy as previously pressedinPCs(PC-2, .0035; Figure 1A). Among these described.25 Tonsillar B-cell subsets were isolated by FACSAria sorting using genes were PRDM1, IRF4,andXBP1, important transcriptional drivers an PE-conjugated antibody against CD19 (DAKO), an FITC-conjugated anti- of PC differentiation. body against IgD (Southern Biotech), and an APC-conjugated antibody FOXP1hasbeenshowntobeexpressedinhumantonsillarB-cell against CD38 (BD). subsets, but its expression and role in terminally differentiated B cells (ie, PCs) has not been investigated.26 To compare expression levels of Cell culture and retroviral transductions FOXP1 in human tonsillar B-cell subsets and PCs, we separated human 1 1 2 fi CD19 tonsil B cells into naive B cells (IgD CD38 ), transitional For in vitro PC differentiation studies, puri ed human B-cell subsets were 1 1 2 1 cultured28 and transduced,25 as described. Bcells(IgD CD38 ), GC B cells (IgD CD38 ), class-switched MBCs 2 2 2 11 SKW6.4 and OCI-Ly7 cells were induced to differentiate as previously (IgD CD38 ), and PCs (IgD CD38 ) by cell sorting. Expression described.29,30 Details are described in the supplemental Methods (see the Blood levels of FOXP1 were determined by quantitative real-time PCR and Web site). western blotting (Figure 1B). Naive B cells, GC B cells, and MBCs showed prominent expression of FOXP1 messenger RNA (mRNA) and Microarray analysis, ChIP-seq, and qRT-PCR . Notably, FOXP1 protein was also prominently expressed in 1 2 31 human peripheral blood CD19 CD27 (mainly naive B cells) and Microarray analysis, chromatin immunoprecipitation and sequencing 1 1 (ChIP-seq),32 RNA isolation, complementary DNA synthesis, and quantita- CD19 CD27 (MBCs) B-cell subsets. In contrast, levels of FOXP1 tive reverse-transcription polymerase chain reaction (qRT-PCR)33 were transcripts and protein were very low in PCs. The same samples were also performed essentially as described.25 Details are described in the supple- analyzed for mRNA levels of genes that are known to be regulated during mental Methods. PC differentiation. As expected, expression levels of BCL6, PAX5, and SpiB were high in GC B cells but low in PCs, whereas the levels of Luciferase assay BLIMP1, IRF4, and XBP1 were highest in PCs (Figure 1C). The observed low expression of FOXP1 in PCs, combined with the repression The BLIMP1-pGL3 construct (Addgene) was used for the luciferase-reporter of the PC gene signature by FOXP1, suggests that FOXP1 downreg- assay. For details, see supplemental Methods. ulation might be required for PC differentiation, whereas aberrantly high expression of FOXP1 might prevent PC differentiation. Immunoblotting

Samples were applied on a 10% sodium dodecyl sulfate polyacrylamide gel FOXP1 arrests differentiation of B-cell lines into electrophoresis gel and blotted with rabbit anti FOXP1 (Abcam or Cell antibody-secreting cells and directly represses expression of Signaling), mouse-anti-BCL6 (BD), mouse-anti b-actin or mouse-anti- important transcriptional drivers of differentiation b-tubulin antibodies (Sigma), followed by horseradish peroxidase– conjugated goat anti-rabbit or goat anti-mouse and developed by enhanced To investigate the putative repressiveroleofFOXP1inPCdifferen- chemiluminescence (Amersham Pharmacia). tiation, we first assessed the effects of ectopic FOXP1 overexpression From www.bloodjournal.org by guest on July 14, 2019. For personal use only.

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Figure 1. FOXP1 represses the PC gene signature and is expressed in human mature B-cell subsets but not in PCs. (A) Expression of genes that belong to the PC-2 PC gene signature35,36 and were significantly repressed by FOXP1 in microarray analysis of 2 independent experiments of primary human MBCs, transduced with FOXP1 or control vector. Data are represented as z scores. The lower panel shows the mean relative expression values of the gene set. (B-C) Human CD191 tonsil B-cell subsets, that is, naive (NBC) (IgD1CD382), transitional (TBC) (IgD1CD381), GC B (IgD2CD381), class-switched MBCs (IgD2CD382), and PCs (IgD2 CD3811), and peripheral blood B-cell subsets (MBC [CD271] and naive enriched [CD272]) were sorted. (B) Gene and protein expression levels of FOXP1 were analyzed in tonsillar and peripheral blood B-cell subsets. Gene expression levels in tonsillar B-cell subsets were quantified by qRT-PCR and normalized to expression levels in naive B cells. Means 6 standard error of the mean (SEM) of 4 independent experiments are shown. Significant differences compared with naive B cells are indicated (1 sample t test, **P , .01). FOXP1 protein expression levels were analyzed by immunoblotting; b-actin was used as loading control. Representative blots of 2 independent experiments are shown. (C) Gene expression levels of BCL6, SPIB, PAX5, PRDM1, IRF4,andXBP1,wereanalyzedintonsillar samplesbyqRT-PCR.Expressionlevelswerenormalizedto expression levels in naive B cells. Means 6 SEM of 4 independent experiments are shown. Significant differences compared with naive B cells are indicated (1 sample t test, *P , .05; **P , .01; ***P , .001).

on in vitro differentiation of B-cell lines. The Epstein-Barr virus (EBV)- ELISPOT (Figure 2B). Moreover, qRT-PCR analysis showed transformed B-cell line SKW6.4 and the GC-DLBCL cell line OCI- repression of PRDM1, IRF4,andXBP1 induction, as well as the active Ly7 can be induced to differentiate into antibody-secreting cells splice variant of XBP1, in FOXP1-transduced OCI-Ly7 cells, whereas through stimulation with cytokines.29,30 When cultured in normal cul- downregulation of PAX5 and BCL6 was not affected (Figure 2C). ture medium, these cell lines do not secrete immunoglobulins and ex- ChIP-seq analysis in OCI-LY7, as well as other DLBCL cell lines, press low levels of PRDM1, IRF4,andXBP1. Stimulation of these cell revealed that FOXP1 binds in the vicinity of the transcription start sites lines with interleukin (IL) 21 and CD40 ligand (CD40L)–expressing (TSSs)ofthePRDM1, IRF4,andXBP1 genes (Figure 3). De novo motif L cells (OCI-Ly7) or IL-21 alone (SKW6.4) rapidly induced immuno- search verified the presence of forkhead binding motifs in the FOXP1 globulin secretion and the expression of PC-specific genes. Interestingly, binding regions in each of these genes (data not shown).25 Direct binding ectopic overexpression of FOXP1 in these cell lines (Figure 2A), and repression of the BLIMP1 promoter by FOXP1 was confirmed cultured under these differentiation-inducing conditions, strongly by a luciferase-reporter assay (Figure 3C). Together, these data repressed immunoglobulin secretion, as determined by ELISA and indicate that FOXP1 inhibits the capacity of OCI-LY7 and SKW6.4 From www.bloodjournal.org by guest on July 14, 2019. For personal use only.

BLOOD, 29 OCTOBER 2015 x VOLUME 126, NUMBER 18 FOXP1 REPRESSES PLASMA CELL DIFFERENTIATION 2101

Figure 2. FOXP1 represses PC differentiation of SKW6.4 and OCI-LY7 cells. (A-C) SKW6.4 and OCI-Ly7 cells were cultured without stimulation or were stimulated to differentiate with IL-21 and CD40L-L cells (OCI-Ly7) or IL-21 alone (SKW6.4) and were transduced with FOXP1-IRES-YFP or control empty vector. Four (OCI-Ly7) or 6 (SKW6.4) days after transduction, YFP1 cells were sorted. (A) Gene expression levels of FOXP1 as determined by qRT-PCR analysis in sorted cells. Expression levels were normalized to b-actin and then to expression levels in stimulated, control-transduced cells. Means 6 SEM of 3 independent experiments are shown. (B) Equal numbers of sorted cells were cultured and analyzed by ELISA (left) or ELISPOT (right). Equal numbers of sorted cells (50 000) were cultured for an additional 24 hours. Thereafter, the supernatants were collected, and IgM protein levels were analyzed by ELISA. Levels were normalized to levels in stimulated, control-transduced cells. Means 6 standard deviation (SD) of 5 (OCI-Ly7) or 4 independent experiments are shown. (left). Equal numbers of sorted cells were plated onto membranes in serial dilutions and cultured for an additional 18 hours, after which numbers of IgM-secreting cells were determined by ELISPOT. Spot numbers were normalized to numbers in stimulated, control-transduced cells. Means 6 SD of 3 independent experiments are shown (right). (C) Gene expression levels of BCL6, PAX5, PRDM1, IRF4, and XBP1 in OCI-LY7 were analyzed by qRT-PCR. Expression levels were normalized to b-actin and then to expression levels in stimulated, control-transduced cells. Means 6 SEM of 3 independent experiments are shown (1 sample t test, *P , .05; **P , .01; ***P , .001). cells to differentiate into antibody-secreting cells with typical PC 3 to 4 days of culture in the presence of IL-21 and IL-2 only. Indeed, features, by directly repressing the upregulation of PRDM1, the expression of B-cell-specific genes (ie, BCL6, PAX5,andSpiB), IRF4,andXBP1. including FOXP1, gradually declined, whereas the expression of PC-specific genes (ie, PRDM1, IRF4,andXBP1) increased during Constitutive FOXP1 (over)expression represses PC consecutive days of the PC differentiation assay (Figure 4A). differentiation of primary human MBCs Furthermore, at the end of the culturing period, a large proportion 2 1 Next, we wanted to assess whether preventing downregulation of of the cells had acquired a PC phenotype (CD20 CD38 )and FOXP1 might impair differentiation of primary human B cells secreted immunoglobulins. toward PCs. To this end, CD271 MBCs were sorted from human Ectopic overexpression of FOXP1 in these primary human B cells peripheral blood and cultured under conditions previously de- (Figure 4B), cultured under PC differentiation–inducing conditions, scribed to drive differentiation toward immunoglobulin-secreting resulted in strong repression of PRDM1, IRF4,andXBP1 as determined PCs28: MBCs were first cultured for 5 days on irradiated CD40L- by qRT-PCR, whereas downregulation of PAX5 and BCL6 was not expressing L cells in the presence of IL-21 and IL-2, followed by affected (Figure 4B). A similar repression of the PC signature genes was From www.bloodjournal.org by guest on July 14, 2019. For personal use only.

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Figure 3. FOXP1 binds in the proximity of the TSS of PRDM1, IRF4, and XBP1. (A) Tracks showing locations of FOXP1 ChIP-seq peaks in the proximity of the TSS of the PRDM1, IRF4, and XBP1 genes in OCI-Ly7 and 3 other DLBCL cell lines. Called peaks are indicated by black bars above the tracks. (B) ChIP with an antibody against FOXP1 in the DLBCL cell lines OCI-LY7, OCI-LY3, and OCI-LY10, followed by quantitative PCR for regions in the proximity of the PRDM1, XBP1, and IRF4 genes in which FOXP1 ChIP-seq peaks were observed in multiple cell lines (regions are indicated in panel A by red bars above the tracks). Hemoglobin beta (HBB) was taken along as a negative control region. Means 6 SEM of at least 3 independent experiments are shown. For all sites analyzed, significant differences were observed between FOXP1 and IgG immunoprecipitation (IP). Significant differences for FOXP1 binding between a specific region and the control region (HBB) are indicated (t test, *P , .05; **P , .01). Primers are listed in supplemental Table 1. (C) A luciferase-reporter construct driven by the PRDM1 promoter was cotransfected in HEK293T cells with a renilla expression vector and increasing (left) or fixed amounts (right) of a FOXP1 expressing vector (FOXP1), or with the empty control vector (ctrl). Values were corrected for renilla luminescence (transfection efficiency) and normalized to expression in control-transduced cells. Means 6 SD of a representative experiment of 2 independent experiments performed in triplicate (left), or the means 6 SEM of 5 independent experiments (right), are shown. observed in B cells transduced with BCL6 (Figure 4B), an established primary B cells resulted in a strong reduction inthe formation of CD202 inhibitor of in vitro PC differentiation37 (Figure 4C). Moreover, similar CD381 PCs (Figure 4C). Importantly, we have previously shown that to overexpression of BCL6, ectopic overexpression of FOXP1 in these FOXP1 overexpression does not affect expansion, survival, or From www.bloodjournal.org by guest on July 14, 2019. For personal use only.

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Figure 4. FOXP1 inhibits PC differentiation of primary human MBCs. (A) CD191CD271 MBCs were sorted from human peripheral blood and cultured under conditions that promote PC differentiation. Gene expression levels of FOXP1, BCL6, PAX5, SPIB, PRDM1, IRF4,andXBP1 were analyzed by qRT-PCR in samples of cells that were cultured for 2, 5, or 8 days after sorting. Expression levels were normalized to levels in cells that were cultured for 2 days. Means 6 SEM of 3 independent experiments are shown. (B) CD191CD271 MBCs were sorted from human peripheral blood and transduced with FOXP1-IRES-YFP, BCL6-IRES-GFP, or control empty vector and cultured under conditions that promote PC differentiation. Six days after transduction, yellow fluorescence protein (YFP)- or green fluorescence protein (GFP)-positive cells of cultures transduced with FOXP1, BCL6, or control vector were sorted (left). Gene expression levels of PRDM1, IRF4, XBP1, FOXP1, BCL6,andPAX5 were analyzed by qRT-PCR. Expression levels in FOXP1- and BCL6-transduced cells were normalized to b-actin and then to expression levels in control-transduced cells. Means 6 SEM of 5 independent experiments are shown (right). Representative example of FOXP1 and BCL6 overexpression in primary YFP1 or GFP1 B cells as determined by immunoblotting. b-actin was used as loading control. (C) Six days after transduction, YFP- or GFP- positive cells were analyzed for CD20 and CD38 surface expression by flow cytometry. Representative density plots of 1 out of 13 independent experiments are shown (left). Percentages of CD381CD202 cells in FOXP1- and BCL6-transduced cultures were normalized to control cultures. Means 6 SD values of 13 independent experiments are shown (right). proliferation in primary B cells at these culture conditions,25 In conclusion, FOXP1 directly represses the expression of strongly indicating that the observed effects reflect inhibition of PC important transcriptional master regulators of PC differentiation differentiation. and prevents differentiation of primary human MBCs toward PCs, From www.bloodjournal.org by guest on July 14, 2019. For personal use only.

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Figure 5. FOXP1 overexpression specifically inhibits formation of IgG- but not IgM-secreting PCs. (A-B) CD191CD271 MBCs were sorted from human pe- ripheral blood and transduced with FOXP1-IRES-YFP, BCL6-IRES-GFP, or control empty vector and cultured under conditions that promote PC differentiation. Six days after transduction, YFP- or GFP-positive cells of cultures transduced with FOXP1, BCL6, or control vector were sorted. (A) Equal numbers of sorted cells (50 000) were cultured with IL-21 and IL-2 for an additional 24 hours. Thereafter, the supernatants were collected, and IgM and IgG protein levels were ana- lyzed by ELISA. Levels were normalized to levels in control-transduced cells. Means 6 SD of at least 4 independent experiments are shown. (B) Equal num- bers of sorted cells were plated onto membranes in serial dilutions and cultured with IL-21 and IL-2 for an additional 18 hours, after which numbers of IgM- or IgG-secreting cells were determined by ELISPOT. Spot numbers were normalized to numbers in stimu- lated, control-transduced cells. Means 6 SD of 3 in- dependent experiments are shown (1 sample t test, *P , .05; **P , .01; ***P , .001).

indicating that FOXP1 downregulation is essential for efficient PC were IgM1 MBCs,attheendoftheassay3%to9%ofthecellsshowed differentiation. surface IgG or IgA expression (supplemental Figure 1). Thus, although CSR did indeed occur, the percentage of the cells that switched was too FOXP1 overexpression specifically inhibits formation of IgG- small to account for the observed reduced formation of IgG-secreting but not IgM-secreting PCs PCs upon FOXP1 expression. Moreover, the switching efficiency was not affected by ectopic overexpression of FOXP1 (supplemental Figure 1). Because FOXP1 inhibits differentiation of primary human MBCs to- ward PCs, as determined by transcriptional and phenotypic parameters, IgG1 MBCs combine a reduced expression of FOXP1 with an we anticipated that ectopic expression of FOXP1 in MBCs cultured enhanced propensity to differentiate as compared with under PC-promoting conditions would also result in decreased levels of IgM1 MBCs secreted immunoglobulins. Our culture conditions indeed resulted in secretion of IgM and IgG from untransduced cells (Figure 5). Unex- Our results show that ectopic overexpression of FOXP1 specifically pectedly, however, whereas IgG secretion was strongly reduced upon represses formation of IgG-secreting PCs in our in vitro culture system, ectopic overexpression of FOXP1, the levels of secreted IgM were which cannot be explained by impaired class switching. To find a not affected (Figure 5A). In contrast, in the culture supernatant of possible explanation for the differential effect of ectopic FOXP1 BCL6-transduced MBCs significantly lower levels of both IgG and overexpression on the formation of IgM- vs IgG-secreting PCs, we IgM were detected. Notably, FOXP1 overexpression reduced the assessed the expression levels of FOXP1 in IgM1 and IgG1 MBCs. secretion of all IgG isotypes (IgG1-4; data not shown). Additional Interestingly, FOXP1 mRNA and protein expression levels were higher 1 1 1 ELISPOT analysis showed that FOXP1 overexpression also exclu- in IgM MBCs as compared with IgG MBCs,aswellasthe2(IgM ) sively results in a reduction of the number of IgG-secreting cells being cell lines that displayed impaired PC differentiation upon FOXP1 formed, whereas BCL6 overexpression caused a reduction in the overexpression (Figure 6A-B). Intracellular flow cytometry analysis 1 number of IgM-secreting cells as well (Figure 5B). These results imply demonstrated that the IgM MBCs uniformly express higher levels that, in contrast to BCL6, overexpression of FOXP1 in primary B cells ofFOXP1ascomparedwithIgG1 MBCs (Figure 6C and supplemental only impairs the formation of IgG-secreting PCs. Figure 2). Therefore, it is tempting to suggest that the (already) high The observed exclusive inhibition in formation of IgG-secreting endogenous expression of FOXP1 in IgM1 MBCs may preclude further PCs by FOXP1 could very well be because of repression of CSR in our repression of their differentiation upon ectopic expression of FOXP1. culture system, especially because the majority of the isolated MBCs If the level of FOXP1 expression in MBCs would indeed control their (6 65%) are unswitsched and express IgM. To assess if MBCs dis- ability to differentiate into PCs, there should be an intrinsic difference in 1 1 played CSR in our culturing system and whether this is affected by differentiation propensity of IgM (FOXP1high)vsIgG (FOXP1low) ectopic overexpression of FOXP1, we specifically sorted unswitched MBCs. To investigate this, IgG1 (IgM2IgA2) or IgM1 (IgG2IgA2) (IgG2IgA2) MBCs and cultured them under the same PC-inducing MBCs were sorted and cultured under PC differentiation-inducing 2 1 conditions as before. Whereas at the start of the assay .99% of the cells conditions. Strikingly, the percentage of CD20 CD38 PCs formed From www.bloodjournal.org by guest on July 14, 2019. For personal use only.

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Figure 6. IgG1 MBCs display reduced expression of FOXP1 and an enhanced propensity to differentiate as compared with IgM1 MBCs. (A-B) IgM1 (IgG2IgA2) and IgG1 (IgM2IgA2) MBCs were sorted from human peripheral blood and cultured for 2 days after which FOXP1 gene expression levels in these cells, and in OCI-Ly7 and SKW6.4 cells, were compared by qRT-PCR (A) or by immunoblotting (B). (A) Expression levels are normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT) and then to levels in IgM1 MBCs. Means 6 SEM of 3 independent experiments are shown. (B) FOXP1 protein expression levels were determined by immunoblotting. b-tubulin levels were determined as loading control. FOXP1 and b-tubulin levels were quantified using Image Studio Lite software, and the ratio of FOXP1 over b-tubulin expression was determined for each lane. A representative blot of 2 independent experiments is shown. (C) PBMCs were isolated from human peripheral blood and stained for surface expression of CD19, CD27, IgM, and IgG, as well as intracellular expression of FOXP1. Intracellular FOXP1 expression in CD191CD271IgM1 MBCs and CD191CD271IgG1 MBCs was analyzed by flow cytometry. A histogram representative of 4 independent experiments (left), and the mean relative mean fluorescence intensity (MFI) (geometric mean) values 6 SD of the IgM1 and IgG1 MBCs (n 5 4; right) are shown. MFI values were normalized to values in IgM1 MBCs (1 sample t test, ***P , .001). A specificity control for the intracellular FOXP1 staining is shown in supplemental Figure 2B. (D-E) The sorted cells were cultured under conditions that promote PC differentiation. (D) Eight days after sorting, cells were analyzed for CD20 and CD38 surface expression by flow cytometry. Representative density plots of 1 out of 3 independent experiments are shown (left). Percentages of CD381 cells were normalized to IgM1 MBCs. Means 6 SD values of 3 independent experiments are shown (right). Levels were normalized to levels in IgM1 MBCs (1 sample t test, ***P , .001). (E) Gene expression levels of FOXP1, BCL6, PAX5 SPIB, BACH2, PRDM1, IRF4, and XBP1 were analyzed by qRT-PCR at the start (2 days after sorting) and end (8 days after sorting) of the PC differentiation assay. Expression levels were normalized to expression levels in IgM1 MBCs that had been cultured for 2 days. Means 6 SEM of 2 independent experiments performed in triplicate are shown. From www.bloodjournal.org by guest on July 14, 2019. For personal use only.

2106 VAN KEIMPEMA et al BLOOD, 29 OCTOBER 2015 x VOLUME 126, NUMBER 18

Figure 7. FOXP1 overexpression inhibits differen- tiation of IgG1 MBCs but cannot further repress differentiation of IgM1 MBCs. IgM1 (IgG2IgA2) and IgG1 (IgM2IgA2) MBCs were sorted from human peripheral blood and transduced with FOXP1-IRES- YFP, BCL6-IRES-GFP (except for IgG1 MBCs), or control empty vector and cultured under conditions that promote PC differentiation. (A) Equal numbers of sorted cells (50 000) were cultured with IL-21 and IL-2 for an additional 24 hours. Thereafter, the supernatants were collected, and IgM and IgG protein levels were ana- lyzed by ELISA. Levels were normalized to levels in control-transduced cells. Means 6 SD of 3 indepen- dent experiments are shown. (B) Equal numbers of sorted cells were plated onto membranes in serial dilutions and cultured with IL-21 and IL-2 for an additional 18 hours, after which numbers of IgM- or IgG-secreting cells were determined by ELISPOT. Spot numbers were normalized to numbers in stimu- lated, control-transduced cells. Means 6 SD of 3 indepen- dent experiments are shown (1 sample t test, *P , .05; **P , .01; ***P , .001).

was indeed much higher in cultures started from IgG1 MBCs than from of IgG1 vs IgM1 MBCs, which is due to the reduced FOXP1 expres- 1 IgM1 MBCs (Figure 6C). Furthermore, also the decrease in MBC- sion in IgG MBCs. specific genes and the concomitant increase in PC-specific genes during culturing was more pronounced in cultures started from IgG1 MBCs (Figure 6D). Of interest, thus far, dissimilar differentiation propensities between IgM1 and IgG1 MBCs have only been reported upon antigen Discussion stimulation38,39; however, in our culture system the B-cell antigen FOXP1isprominentlyexpressedinmatureBcellsandisapotential (BCR) is not activated by antigen. Thus, our results indicate oncogene in B-cell non-Hodgkin lymphomas; however, the functions the existence of an antigen/BCR-independent, intrinsic difference in in of FOXP1 in mature B cells and B-cell lymphomagenesis have not yet vitro differentiation propensity of IgM1 vs IgG1 MBCs. 1 been fully explored. Here, by gene expression microarray and ChIP-seq Importantly, whereas FOXP1 expression levels were lower in IgG analysis, we demonstrated that FOXP1 directly represses expression of MBCs compared with IgM1 MBCs, no significant differences in multiple master regulators of PC differentiation. Furthermore, we es- BCL6 expression were observed for other established repressors ( , tablished that FOXP1 expression strongly declines upon PC differen- PAX5 SpiB Bach2 PRDM1 IRF4 XBP1 , , )ordrivers( , , ) of PC differen- tiation, that constitutive FOXP1 overexpression represses the ability of BACH2 tiation (Figure 6D-E). In our analysis, we also included ,which SKW6.4 and OCI-LY7 cells, as well as primary human MBCs, to dif- has recently been implicated in dissimilar differentiation potentials of ferentiate into antibody-secreting PCs; and that IgG1 MBCs combine 1 “ ” 40 murine naive B cells vs IgG MBCs (see Discussion ) ; however, reduced expression of FOXP1 with an enhanced intrinsic PC differen- whereas we did observe higher BACH2 expression levels in human tiation propensity as compared with IgM1 MBCs. naive B cells as compared with human MBCs (supplemental Figure 3), 1 FOXP1 overexpression in primary human MBCs and in the BACH2 expression levels did not differ between human IgM and DLBCL cell line OCI-Ly7 resulted in reduced expression of PRDM1, 1 fi 41 IgG MBCs, neither in directly ex vivo puri ed cells (supplemental IRF4,andXBP1, important drivers of PC differentiation and immuno- Figure 3) nor after 2 days of in vitro culture (Figure 6D). To assess if the globulin secretion (Figures 2C and 4B). Notably, FOXP1 overex- reduced levels of FOXP1 may be critically required for the enhanced 1 pression did not affect the downregulation of PAX5 and BCL6 during differentiation propensity of IgG MBCs, we ectopically overex- PC differentiation of OCI-LY7 (Figure 2C) and primary human MBCs 1 2 2 pressed FOXP1 in sorted IgG (IgM IgA ) MBCs. Indeed, this (Figure 4B). This indicates that the observed FOXP1-mediated re- resulted in reduced (IgG) immunoglobulin secretion and reduced pression of IRF4, PRDM1, and XBP1 is the cause rather than a formation of IgG-secreting PCs (Figure 7A-B). In contrast, PC forma- reflection of the impaired PC differentiation, and that FOXP1 can 1 tion in sorted IgM MBCs, which already express relatively high levels repress PC differentiation despite downregulation of PAX5 and BCL6 of FOXP1 and display a reduced PC differentiation propensity, (ie, the effect of PAX5 or BCL6 downregulation is overruled by could not be further repressed by ectopic FOXP1 overexpression, FOXP1 expression). Furthermore, ChIP-seq and ChIP-PCR analysis whereas BCL6 overexpression repressed differentiation of both showed direct binding of FOXP1 in the vicinity of the TSSs of IRF4, MBC subsets (Figure 7A-B). Taken together, our data reveal an PRDM1,andXBP1 in OCI-Ly7 and other DLBCL cell lines enhanced BCR-independent intrinsic PC differentiation propensity (Figure 3A). Moreover, BLIMP1-promoter luciferase-reporter assays From www.bloodjournal.org by guest on July 14, 2019. For personal use only.

BLOOD, 29 OCTOBER 2015 x VOLUME 126, NUMBER 18 FOXP1 REPRESSES PLASMA CELL DIFFERENTIATION 2107 confirmed direct repression of the PRDM1gene by FOXP1 by binding higher antigen affinity of IgG vs IgM BCRs39,43,47-50; however, our to its promoter region. Together, our results indicate that FOXP1 can results, obtained in a culture system in which the BCR is not activated directly repress transcription of IRF4, PRDM1,andXBP1, irrespective by antigen, indicate that antigen-/BCR-independent factors (ie, differ- of PAX5 and BCL6, thereby repressing PC differentiation. Because ential expression of FOXP1) also contribute to these differences. Future FOXP1 protein expression is temporarily lowered at the GC stage,26 it studies may reveal the molecular mechanisms underlying this differ- is tempting to speculate that the extent of FOXP1 upregulation at the ential expression of FOXP1 among human B-cell and MBC subsets. end of the GC stage might contribute to the fate of a GC , that is, In accordance with our findings, in a recent study Zuccarino-Catania whether it differentiates into either a PC (no or weak FOXP1 upreg- et al demonstrated that functionally distinct murine MBC subsets can be ulation) or an MBC (strong FOXP1 upregulation). defined by expression of surface independent of immuno- Strikingly, FOXP1 overexpression inhibited terminal differentia- globulin isotype, which indicates that the immunoglobulin isotype is tion of primary human IgG1, but, in contrast to BCL6, not of IgM1 just a surrogate marker for MBC subsets and that their terminal dif- MBCs (Figures 5 and 7). This difference does not appear to rely on ferentiation potential might, for example, rather be determined by intrinsic functional differences between the IgM vs the IgG BCR dissimilar transcription factor expression.51 Likewise, Kometani et al because FOXP1 could also inhibit PC differentiation of IgM- recently reported that although the IgG cytoplasmic tail enhances expressing B-cell lines (Figure 2). Rather, the divergent effects on IgG1 signaling upon BCR cross-linking,47-50 this alone is insufficient to 1 and IgM MBCs might be the consequence of the dissimilar levels confer the heightened differentiation activity of murine IgG1 MBCs, of endogenous FOXP1 protein expression, which is higher in IgM1 compared with naive B cells.40 Rather, repression of the transcription MBCs than in IgG1 MBCs and the IgM1 cell lines studied factor Bach2 in antigen-experienced B cells predisposes IgG1 MBCs (Figure 6A-B). The high(er) endogenous expression of FOXP1 in to differentiate into PCs.40 Our data clearly establish that reduced 1 1 IgM MBCs appears to be sufficient to fully exert the differentiation expression of FOXP1 in human IgG MBCs is required for their inhibiting potential of FOXP1, because, in contrast to ectopic over- differentiation into PCs, at least in an in vitro culture system. However, expression of BCL6, ectopic overexpression of FOXP1 cannot further whether the higher FOXP1 expression in IgM1 B cells is also sufficient 1 reduce the PC differentiation of IgM MBCs. Notably, an isotype- to repress their differentiation propensity, by directly preventing or specific effect on PC differentiation has previously also been described delaying upregulation of differentiation master regulators (Figure 6), for the transcription factor STAT3: mice in which this gene is knocked hastobedeterminedinfurtherstudies. out in B cells showed a specific defect in T-cell–dependent IgG PC In conclusion, we showed that proper control of FOXP1 expression formation.42 However, FOXP1 and STAT3 do not seem to act via is critical for MBC fate and PC differentiation. From an oncological sequential pathways as STAT3 knockdown in DLBCL cell lines did not perspective, failure to downregulate FOXP1 expression, because of affect FOXP1 expression and p-STAT3 expression levels were not aberrant, constitutively high, expression of FOXP1 by chromosomal affected by FOXP1 overexpression in primary B cells or DLBCL cell translocations or trisomy 3,14,21,22,52,53 will block B-cell differentiation, lines (data not shown). similar to other established oncogenes and tumor suppressor genes, In line with our finding that FOXP1 tempers differentiation of such as BCL6, SpiB, SOX11, and PRDM1.14,16,54-56 Combined with 1 1 MBCs, we found that IgG MBCs, which have a twofold lower its recently reported antiapoptotic actions25 (observedinbothIgM and expression of FOXP1 as compared with IgM1 MBCs,haveaclearly IgG1 MBCs; data not shown), this would render FOXP1 a highly increased propensity to differentiate in our in vitro culture system. efficacious oncogene for B-cell lymphomagenesis. From an immuno- Previously, differences in the differentiation potential of MBC subsets logic perspective, expression of FOXP1 in MBCs might prevent pre- have been described, but, to the best of our knowledge, exclusively in mature differentiation of these cells; specifically, the high expression systemsinwhich,incontrasttoourdifferentiation system, the BCR was levels of FOXP1 in IgM1 MBCs could be involved in the reduced triggered by antigenic stimulation.38,39 In these mouse studies, it has propensity of this MBC subset to undergo terminal differentiation, which been shown that IgG1 MBCs rapidly differentiate into antibody- is suggested to be important for long-lasting immunity,43 whereas secreting PCs upon antigenic challenge and do not reenter GCs. In reduced expression of FOXP1 in IgG1 MBCs allows their efficient contrast, IgM1 MBCs can return to the GC, whereas only a small pro- terminal differentiation into antibody-producing PCs. portion of these cells differentiate into PCs.38,39 These unique properties of IgM1 MBCs may be important during reinfection with a mutated version of the original pathogen; by reentering the GC these MBCs could acquire additional mutations in their BCR that would result in Acknowledgments a new high-affinity response to the mutated antigen.43 Moreover, because of their long life span, IgM1 MBCs may provide long-lasting The authors thank Berend Hooibrink and Toni van Capel for fl immunity, serving as a reservoir of humoral immune memory against uorescence-activated cell sorter sorting; Jan Koster and Richard aspecific antigen, responding when switched immunoglobulin-positive Volckman for providing help with microarray analysis; Edwin B cells and serum immunoglobulin levels drop below useful levels.39,43 Cuppen, Ewart de Bruijn, Nico Lansu, and Wensi Hao for deep se- 1 1 Between human IgM and IgG MBCs, a similar difference has quencing; and Sander Boymans for mapping of the sequencing data. been proposed to exist: based on replication history and immunoglob- This work was supported by a grant from the Dutch Cancer Society. ulin somatic hypermutation profiles, it has been deduced that CD271 IgM1 MBCs are derived from primary GC responses, whereas at least part of the switched MBCs seem to have undergone multiple rounds of Authorship GC passage.44 Furthermore, clonally related IgM1 and IgG1 Bcells have been found in human GC and peripheral blood, suggesting that Contribution: M.v.K. designed the research, performed experiments, 1 human IgM MBCs can indeed reenter GCs, undergo CSR, and exit as analyzed data, designed figures, and wrote the manuscript; L.J.G., 1 switched MBCs.45,46 Traditionally, these differences between IgM M.M., and R.v.B. performed experiments and analyzed data; M.C.v.Z. 1 and IgG MBCs have been ascribed to differences in signaling and P.C. interpreted data and cosupervised part of the study; S.T.P. through the unique cytoplasmic tails of IgM and IgG BCR and the cosupervised the study and reviewed the manuscript; and M.S. designed From www.bloodjournal.org by guest on July 14, 2019. For personal use only.

2108 VAN KEIMPEMA et al BLOOD, 29 OCTOBER 2015 x VOLUME 126, NUMBER 18 the research, supervised the study, analyzed the data, and wrote the Correspondence: Marcel Spaargaren, Department of Pathology, manuscript. Academic Medical Center, University of Amsterdam, Meibergdreef Conflict-of-interest disclosure: The authors declare no competing 9, 1105 AZ Amsterdam, The Netherlands; e-mail: marcel.spaargaren@ financial interests. amc.uva.nl. References

1. McHeyzer-Williams LJ, McHeyzer-Williams MG. 18. Rui L, Schmitz R, Ceribelli M, Staudt LM. endotoxic shock-induced renal inflammation and Antigen-specific memory B cell development. Malignant pirates of the immune system. dysfunction. PLoS One. 2013;8(12):e84479. Annu Rev Immunol. 2005;23(1):487-513. Nat Immunol. 2011;12(10):933-940. 34. Bende RJ, Jochems GJ, Frame TH, et al. 2. Yoshida T, Mei H, D¨ornerT, et al. Memory B and 19. Lenz G, Staudt LM. Aggressive lymphomas. Effects of IL-4, IL-5, and IL-6 on growth and memory plasma cells. Immunol Rev. 2010;237(1): N Engl J Med. 2010;362(15):1417-1429. immunoglobulin production of Epstein-Barr virus- 117-139. infected human B cells. Cell Immunol. 1992; 20. Hu H, Wang B, Borde M, et al. Foxp1 is an 143(2):310-323. 3. Shapiro-Shelef M, Calame K. Regulation of essential transcriptional regulator of B cell plasma-cell development. Nat Rev Immunol. development. Nat Immunol. 2006;7(8):819-826. 35. Guti´errezNC, Ocio EM, de Las Rivas J, et al. 2005;5(3):230-242. Gene expression profiling of B lymphocytes 21. Streubel B, Vinatzer U, Lamprecht A, Raderer M, and plasma cells from Waldenstr¨om’s 4. Nutt SL, Taubenheim N, Hasbold J, Corcoran LM, Chott A. T(3;14)(p14.1;q32) involving IGH and Hodgkin PD. The genetic network controlling macroglobulinemia: comparison with expression FOXP1 is a novel recurrent chromosomal patterns of the same cell counterparts from plasma cell differentiation. Semin Immunol. 2011; aberration in MALT lymphoma. Leukemia. 2005; 23(5):341-349. chronic lymphocytic leukemia, multiple myeloma 19(4):652-658. and normal individuals. Leukemia. 2007;21(3): 5. Klein U, Casola S, Cattoretti G, et al. Transcription 22. Fenton JAL, Schuuring E, Barrans SL, et al. 541-549. factor IRF4 controls plasma cell differentiation and t(3;14)(p14;q32) results in aberrant expression 36. Shaffer AL, Wright G, Yang L, et al. A library of class-switch recombination. Nat Immunol. 2006; of FOXP1 in a case of diffuse large B-cell 7(7):773-782. gene expression signatures to illuminate normal lymphoma. Genes Cancer. 2006; and pathological lymphoid biology. Immunol Rev. 6. Sciammas R, Shaffer AL, Schatz JH, Zhao H, 45(2):164-168. 2006;210(1):67-85. Staudt LM, Singh H. Graded expression of 23. Banham AH, Connors JM, Brown PJ, et al. interferon regulatory factor-4 coordinates isotype 37. Kuo TC, Shaffer AL, Haddad J Jr, Choi YS, Staudt Expression of the FOXP1 transcription factor switching with plasma cell differentiation. LM, Calame K. Repression of BCL-6 is required is strongly associated with inferior survival in Immunity. 2006;25(2):225-236. for the formation of human memory B cells in vitro. patients with diffuse large B-cell lymphoma. J Exp Med. 2007;204(4):819-830. 7. Kwon H, Thierry-Mieg D, Thierry-Mieg J, et al. Clin Cancer Res. 2005;11(3):1065-1072. Analysis of interleukin-21-induced Prdm1 gene 38. Dogan I, Bertocci B, Vilmont V, et al. Multiple 24. Sagaert X, de Paepe P, Libbrecht L, et al. regulation reveals functional cooperation of layers of B cell memory with different effector Forkhead box protein P1 expression in mucosa- STAT3 and IRF4 transcription factors. Immunity. functions. Nat Immunol. 2009;10(12):1292-1299. associated lymphoid tissue lymphomas predicts 2009;31(6):941-952. poor prognosis and transformation to diffuse large 39. Pape KA, Taylor JJ, Maul RW, Gearhart PJ, 8. Ochiai K, Maienschein-Cline M, Simonetti G, et al. B-cell lymphoma. J Clin Oncol. 2006;24(16): Jenkins MK. Different B cell populations mediate Transcriptional regulation of germinal center B 2490-2497. early and late memory during an endogenous and plasma cell fates by dynamical control of immune response. Science. 2011;331(6021): IRF4. Immunity. 2013;38(5):918-929. 25. van Keimpema M, Gr¨unebergLJ, Mokry M, et al. 1203-1207. FOXP1 directly represses transcription of 40. Kometani K, Nakagawa R, Shinnakasu R, et al. 9. Shaffer AL, Shapiro-Shelef M, Iwakoshi NN, et al. proapoptotic genes and cooperates with NF-kBto Repression of the transcription factor Bach2 XBP1, downstream of Blimp-1, expands the promote survival of human B cells. Blood. 2014; contributes to predisposition of IgG1 memory secretory apparatus and other organelles, and 124(23):3431-3440. increases protein synthesis in plasma cell B cells toward plasma cell differentiation. differentiation. Immunity. 2004;21(1):81-93. 26. Sagardoy A, Martinez-Ferrandis JI, Roa S, et al. Immunity. 2013;39(1):136-147. Downregulation of FOXP1 is required during 41. Berkowska MA, Schickel JN, Grosserichter- 10. Shaffer AL, Yu X, He Y, Boldrick J, Chan EP, germinal center B-cell function. Blood. 2013; Wagener C, et al. Circulating human CD27-IgA1 Staudt LM. BCL-6 represses genes that function 121(21):4311-4320. in lymphocyte differentiation, inflammation, and memory B cells recognize bacteria with cell cycle control. Immunity. 2000;13(2):199-212. 27. Scheeren FA, Naspetti M, Diehl S, et al. polyreactive Igs. J Immunol. 2015;195(4): STAT5 regulates the self-renewal capacity and 1417-1426. 11. Lin KI, Angelin-Duclos C, Kuo TC, Calame K. differentiation of human memory B cells and Blimp-1-dependent repression of Pax-5 is 42. Fornek JL, Tygrett LT, Waldschmidt TJ, Poli V, controls Bcl-6 expression. Nat Immunol. 2005; required for differentiation of B cells to Rickert RC, Kansas GS. Critical role for Stat3 in 6(3):303-313. immunoglobulin M-secreting plasma cells. T-dependent terminal differentiation of IgG Mol Cell Biol. 2002;22(13):4771-4780. 28. Schmidlin H, Diehl SA, Nagasawa M, et al. Spi-B B cells. Blood. 2006;107(3):1085-1091. inhibits human plasma cell differentiation by 12. Nera KP, Kohonen P, Narvi E, et al. Loss of Pax5 43. Taylor JJ, Jenkins MK, Pape KA. Heterogeneity in repressing BLIMP1 and XBP-1 expression. Blood. promotes plasma cell differentiation. Immunity. the differentiation and function of memory B cells. 2008;112(5):1804-1812. 2006;24(3):283-293. Trends Immunol. 2012;33(12):590-597. 13. Tunyaplin C, Shaffer AL, Angelin-Duclos CD, 29. Ding BB, Bi E, Chen H, Yu JJ, Ye BH. IL-21 44. Berkowska MA, Driessen GJ, Bikos V, et al. Yu X, Staudt LM, Calame KL. Direct repression of and CD40L synergistically promote plasma cell Human memory B cells originate from three by Bcl-6 inhibits plasmacytic differentiation. differentiation through upregulation of Blimp-1 distinct germinal center-dependent and J Immunol. 2004;173(2):1158-1165. in human B cells. J Immunol. 2013;190(4): -independent maturation pathways. Blood. 2011; 1827-1836. 118(8):2150-2158. 14. Lenz G, Wright GW, Emre NC, et al. Molecular subtypes of diffuse large B-cell lymphoma arise 30. Goldstein H, Kim A. Immunoglobulin secretion 45. Bende RJ, van Maldegem F, Triesscheijn M, by distinct genetic pathways. Proc Natl Acad Sci and phosphorylation of common proteins are Wormhoudt TA, Guijt R, van Noesel CJ. Germinal USA. 2008;105(36):13520-13525. induced by IL-2, IL-4, and IL-6 in the factor centers in human lymph nodes contain responsive human B cell line, SKW6.4. reactivated memory B cells. J Exp Med. 2007; 15. Iqbal J, Greiner TC, Patel K, et al; Leukemia/ J Immunol. 1993;151(12):6701-6711. 204(11):2655-2665. Lymphoma Molecular Profiling Project. Distinctive patterns of BCL6 molecular alterations and their 31. Kocemba KA, van Andel H, de Haan-Kramer A, 46. Seifert M, K¨uppersR. Molecular footprints of functional consequences in different subgroups of et al. The hypoxia target adrenomedullin is a germinal center derivation of human IgM1 diffuse large B-cell lymphoma. Leukemia. 2007; aberrantly expressed in multiple myeloma and (IgD1)CD271 B cells and the dynamics of 21(11):2332-2343. promotes angiogenesis. Leukemia. 2013;27(8): memory B cell generation. J Exp Med. 2009; 1729-1737. 206(12):2659-2669. 16. Mandelbaum J, Bhagat G, Tang H, et al. BLIMP1 is a tumor suppressor gene frequently disrupted in 32. van Boxtel R, Gomez-Puerto C, Mokry M, et al. 47. Martin SW, Goodnow CC. Burst-enhancing role of activated B cell-like diffuse large B cell lymphoma. FOXP1 acts through a negative feedback loop to the IgG membrane tail as a molecular determinant Cancer Cell. 2010;18(6):568-579. suppress FOXO-induced apoptosis. Cell Death of memory. Nat Immunol. 2002;3(2):182-188. Differ. 2013;20(9):1219-1229. 17. Iida S, Rao PH, Nallasivam P, et al. The t(9;14) 48. Horikawa K, Martin SW, Pogue SL, et al. (p13;q32) chromosomal translocation associated 33. Rampanelli E, Dessing MC, Claessen N, et al. Enhancement and suppression of signaling by the with lymphoplasmacytoid lymphoma involves the CD44-deficiency attenuates the immunologic conserved tail of IgG memory-type B cell antigen PAX-5 gene. Blood. 1996;88(11):4110-4117. responses to LPS and delays the onset of receptors. J Exp Med. 2007;204(4):759-769. From www.bloodjournal.org by guest on July 14, 2019. For personal use only.

BLOOD, 29 OCTOBER 2015 x VOLUME 126, NUMBER 18 FOXP1 REPRESSES PLASMA CELL DIFFERENTIATION 2109

49. Engels N, K¨onigLM, Heemann C, et al. memory B cell subsets that are independent of 54. Shaffer AL III, Young RM, Staudt LM. Recruitment of the cytoplasmic adaptor Grb2 antibody isotype. Nat Immunol. 2014;15(7): Pathogenesis of human B cell lymphomas. to surface IgG and IgE provides antigen receptor- 631-637. Annu Rev Immunol. 2012;30(1):565-610. intrinsic costimulation to class-switched B cells. 52. Hans CP, Weisenburger DD, Greiner TC, et al. 55. Basso K, Dalla-Favera R. BCL6: master regulator Nat Immunol. 2009;10(9):1018-1025. Confirmation of the molecular classification of the germinal center reaction and key oncogene 50. Liu W, Chen E, Zhao XW, et al. The scaffolding of diffuse large B-cell lymphoma by in B cell lymphomagenesis. Adv Immunol. 2010; protein synapse-associated protein 97 is required immunohistochemistry using a tissue microarray. 105:193-210. for enhanced signaling through isotype-switched Blood. 2004;103(1):275-282. 56. Vegliante MC, Palomero J, P´erez-Gal´anP, et al. IgG memory B cell receptors. Sci Signal. 2012; 53. Staudt LM, Dave S. The biology of human SOX11 regulates PAX5 expression and blocks 5(235):ra54. lymphoid malignancies revealed by gene terminal B-cell differentiation in aggressive 51. Zuccarino-Catania GV, Sadanand S, Weisel FJ, expression profiling. Adv Immunol. 2005;87: mantle cell lymphoma. Blood. 2013;121(12): et al. CD80 and PD-L2 define functionally distinct 163-208. 2175-2185. From www.bloodjournal.org by guest on July 14, 2019. For personal use only.

2015 126: 2098-2109 doi:10.1182/blood-2015-02-626176 originally published online August 19, 2015

The forkhead transcription factor FOXP1 represses human plasma cell differentiation

Martine van Keimpema, Leonie J. Grüneberg, Michal Mokry, Ruben van Boxtel, Menno C. van Zelm, Paul Coffer, Steven T. Pals and Marcel Spaargaren

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