Published November 16, 2018, doi:10.4049/jimmunol.1500497 The Journal of Immunology

Nonredundant Roles of IL-21 and IL-4 in the Phased Initiation of B Cells and Subsequent Self-Renewal Transitions

David G. Gonzalez,*,† Christine M. Cote,* Jaymin R. Patel,‡ Colin B. Smith,x Yuqi Zhang,{ Kevin M. Nickerson,‖ Tingting Zhang,* Steven M. Kerfoot,# and Ann M. Haberman*,**

We examined the unique contributions of the cytokines IL-21 and IL-4 on germinal center (GC) initiation and subsequent maturation in a murine model system. Similar to other reports, we found T follicular helper cell expression of IL-21 begins prior to T follicular helper cell migration into the B cell follicle and precedes that of IL-4. Consistent with this timing, IL-21 signaling has a greater influence on the perifollicular pre-GC B cell transition to the intrafollicular stage. Notably, Bcl6hi B cells can form in the combined absence of IL-21R– and STAT6-derived signals; however, these nascent GC B cells cease to proliferate and are more prone to apoptosis. When B cells lack either IL-21R or STAT6, aberrant GCs form atypical and centrocytes that differ in their phenotypic maturation and costimulatory molecule expression. Thus, IL-4 and IL-21 play nonredundant roles in the phased progression of GC B cell development that can initiate in the combined absence of these cytokine signals. The Journal of Immunology, 2018, 201: 000–000.

ffective immune responses to pathogens and vaccines chemotactic propensities and supply the cytokines IL-4 and critically depend on germinal centers (GCs) to generate IL-21 to GC B cells (3, 4). E long-lived, high-affinity memory B cells and plasma Our prior studies examining these early events leading to GC cells (1, 2). The current model of GC development envisions that initiation indicate that B cells dwell at the follicular boundary and activated B cells, after interacting with cognate T cells, commit between follicles for several days, while in close and recurring to one of several potential fates: They either re-enter the follicle contact with differentiating T cells. Within this milieu, the first and commit to the formation of a new GC, or they migrate away events of GC B cell differentiation and Tfh cell maturation occur, from the follicle and differentiate into short-lived Ab-forming including elevated expression of Bcl6, a transcriptional repressor cells or early memory B cells. Similarly, a proportion of acti- required for their formation (5–10). Bcl6 controls GC B cell dif- by guest on October 1, 2021. Copyright 2018 Pageant Media Ltd. vated CD4+ T cells differentiate into a helper cell subset, known ferentiation by regulating cell cycle genes, repression of terminal as T follicular helper (Tfh) cells, that have rewired follicular differentiation factors, and suppression of some signaling path- ways, including BCR signaling (8, 11). Repressed target genes in mice include CD38, a member of an ectoenzyme family (12), and CD23. However, the extent of repression of potential Bcl6 target *Department of Immunobiology, Yale University, New Haven, CT 06519; genes is influenced by the composition of Bcl6 corepressor †Department of Genetics, Yale University, New Haven, CT 06519; ‡Molecular, Cel- lular and Developmental Biology, Yale University, New Haven, CT 06519; xDepart- complexes that can differ functionally (13). ment of Radiology, University of Pittsburgh Medical Center, Pittsburgh, PA 15213; Following interactions with Ag-specific T cells at the follicle {Department of Surgery, Yale-New Haven Hospital, New Haven, CT 06519; ‖De- # periphery, Bcl6-expressing B cells committed to the GC pathway

http://classic.jimmunol.org partment of Immunology, University of Pittsburgh, Pittsburgh, PA 15261; Depart- ment of Microbiology and Immunology, Schulich School of Medicine and Dentistry, re-enter the follicle, divide at an extremely rapid rate, and form very Western University, London, Ontario N6A 5C1, Canada; and **Department of Lab- large aggregates that organize into mature GCs with micro- oratory Medicine, Yale School of Medicine, New Haven, CT 06519 anatomically distinct compartments. The light zone (LZ) is dis- ORCIDs: 0000-0002-0106-6461 (D.G.G.); 0000-0002-4380-4805 (J.R.P.); 0000- 0002-4523-7752 (C.B.S.); 0000-0002-0114-2671 (K.M.N.); 0000-0002-5401- tinguished by the presence of follicular dendritic cells, a stromal 0373 (S.M.K.). cell subset, the fine processes of which form a reticular network Received for publication March 2, 2015. Accepted for publication October 19, 2018. with high levels of the complement receptor CD35 (14). The LZ Downloaded from This work was supported by National Institutes of Health (NIH)/National Institute of also harbors a high density of Tfh cells. Much of the terminology Arthritis and Musculoskeletal and Skin Diseases, Rheumatic Diseases Core Centers used to describe murine GC B cells is based on the appearance of Grant P30AR053495-07 and NIH/National Institute of Allergy and Infectious Dis- ease Grants R01AI080850 and R21AI101704. S.M.K. and T.Z. were supported by a tonsillar GCs that harbor “centrocyte” LZ B cells that are typically fellowship from the Canadian Institutes of Health Research. smaller and “centroblast” dark zone (DZ) B cells that are larger Address correspondence and reprint requests to Dr. Ann M. Haberman, Department and more often mitotic (15). Whereas centrocytes are predomi- of Immunobiology, Yale University School of Medicine, P.O. Box 208011, New lo hi Haven, CT 06520-8011. E-mail address: [email protected] nantly CXCR4 CD86 and express higher levels of CD40 and CD86, centroblasts are characterized as typically CXCR4hi and The online version of this article contains supplemental material. CD86lo (16). Intravital imaging studies of Tfh contacts with LZ Abbreviations used in this article: DKO, double knockout; DZ, dark zone; GC, germinal center; LZ, light zone; MFI, mean fluorescence intensity; NP, hapten B cells (17, 18) coupled with prior observations of interzonal (4-hydroxy-3-nitrophenyl) acetyl; NP-OVA, NP-haptenated OVA; OT-II, OVA-specific migrations (19–21) support a model of GC B cell dynamics in TCR-transgenic; p.i., postimmunization; Tfh, T follicular helper; WT, wild-type. which Tfh cell engagement of centrocytes within the LZ instructs Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50 a rewired transcriptional program that propels them to migrate to

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500497 2 ROLES OF IL-21 AND IL-4 IN GC B CELL DYNAMICS

the DZ as centroblasts. During extensive clonal expansion, GC absence of k expressing B cells. Hapten-specific mice were crossed with strains of mice either harboring a homozygous deletion of the transcription B cells with higher affinity BCR variants are enriched, eventually tm1Gru differentiating further into long-lived plasma cells and memory factor Stat6 (no. 5977; B6.129S2(C)-Stat6 /J; The Jackson Laboratory or a homozygous deletion of the receptor for the cytokine IL-21 (44) to be cells, a process that is only observed when effective cognate Tfh used as sources of hapten-specific B cells lacking the ability to signal cell responses are also evoked (2, 22). through Stat6 or IL-21, respectively. IL-21R2/2 mice were generated at The formation of small GCs that fail to mature in the absence of a Lexicon Pharmaceuticals, provided by ZymoGenetics (a Bristol-Myers –adaptive immune response calls into question the absolute Squibb Company), and backcrossed 12 generations onto the C57BL/6 background prior to crossing with hapten-specific strains. The lack of requirement of Th cell–derived cytokines for the onset of GC IL-21R expression in IL-21R2/2 mice was confirmed by PCR analysis and B cell differentiation (23). T cell support for GCs is known to flow cytometry. depend on the cytokines IL-21 and IL-4. In the absence of sig- OVA-specific TCR-transgenic (OT-II) mice (no. 4194; Tg(TcraTcrb) naling through either IL-4 or IL-21 receptors in B cells, GC for- 425Cbn/J; The Jackson Laboratory were used as a source of carrier- mation occurs in vivo, but the resulting GCs are diminished in size specific T cells. Hapten- or carrier-specific mice were further crossed with strains of mice expressing fluorescent proteins within all nucleated (24–27). In the case of IL-21, smaller GCs are thought to occur cells that produced either DsRed under control of the b-actin promoter from a direct effect on B cells that results in reduced Bcl6 levels [no. 6051; Tg(CAG-DsRed*MST)1Nagy/J; The Jackson Laboratory] or within GC B cells (24, 25). In vitro, both IL-21 and IL-4 can enhanced GFP via the ubiquitin promoter (no. 4353; Tg(UBC-GFP) enhance Bcl6 transcription and/or translation in B cells (28, 29). 30Scha/J; The Jackson Laboratory). Mice carrying a transgene encoding a TCR specific for an LCMV epitope (SMARTA) were used as recipients Both cytokines are also known to influence the formation of Ab- for cell transfers (45). Some experiments used wild-type (WT) C57BL/6 secreting cells (30–35). Interestingly, Tfh cells can differ in their mice as recipients (no. 664; The Jackson Laboratory). All mice had been cytokine expression profiles at different stages in their differenti- fully backcrossed onto the C57BL/6 background and were maintained ation and at distinct locations within lymphoid tissue (35–37). under specific pathogen–free conditions. All experiments were approved by the Yale University Institutional Animal Care and Use Committee. Whereas Tfh cells that emerge early in an immune response VNP Homozygosity of the Igh gene and the Jk knockout allele was de- predominantly express IL-21 but substantially less IL-4, the re- tected by PCR, as described previously (7). Homozygosity of the Stat6 verse is true of distal LZ Tfh cells within fully mature GCs (37). knockout allele was detected by the presence of a ∼380 bp PCR product IL-4R and IL-21R complexes activate distinct signaling pathways; (mutant) and the absence of a ∼275 bp PCR product (WT) with the fol- IL-21 predominantly activates STAT1 and STAT3, whereas IL-4 lowing primer sequences: Stat6 WT, 59-AAGTGGGTCCCCTTCACTCT- 39; Stat6 common, 59-ACTCCGGAAAGCCTCATCTT-39; and Stat6 and IL-13 signal uniquely via STAT6 (38, 39). mutant, 59-AATCCATCTTGTTCAATGGCCGATC-39. Homozygosity of In this study, we examined the earliest stages of GC B cell the IL-21R knockout allele was detected by the presence of a ∼380 bp formation, its reliance on IL-21 and IL-4 signaling, and its tem- PCR product (mutant) and the absence of a ∼280 bp PCR product (WT) poral relationship to the expression of these cytokines by Tfh cells. with the following primer sequences: IL-21R mutant, 59-GCAGCGCA- 9 9 9 Our results support a multiphased model of GC B cell development. TCGCCTTCTATC-3 and 5 -GAAGTTCTGCACAGTGTCTAGC-3 ; hi IL-21R WT locus, 59-CTCCAAAGGGAGGGATCAGAAC-39 and 59- Notably, we found that Bcl6 -nascent GC B cells still develop in GAAGTTCTGCACAGTGTCTAGC-39. OT-II mice were screened for the the combined absence of both IL-21– and IL-4–derived signaling presence of the TCR transgene by detection of a PCR product with the but are unable to progress further to form a fully mature GC. following primer sequences: OT-II, 59-GCTGCTGCACAGACCTACT-39 Similar to prior reports (36, 37, 40), we observed IL-4 and IL-21 and 59-CAGCTCACCTAACACGAGGA-39. SMARTA mice were screened for the presence of the TCR transgene by detection of a ∼170 bp PCR by guest on October 1, 2021. Copyright 2018 Pageant Media Ltd. to be differentially expressed by emerging Tfh cells; IL-21 ex- product with the following primer sequences: Va2, 59-ATAAAAAGGAA- pression is evident as early as 2–3 d post immunization, whereas GATGGACGATT-39 and 59-TGGGGCTGACTGATACCG-39. increased IL-4 expression becomes apparent when Tfh cells reside within follicles. Consistent with this timing, we found that B cell– Adoptive transfers and immunizations intrinsic IL-21 signaling played a stronger role than IL-4 during Hapten-specific B cells were enriched from the spleens of B1-8+ Jk2/2 mice the transition from the perifollicular pre-GC B cell phase to the by immunomagnetic purification with the EasySep Negative Selection intrafollicular phase. Mouse B Cell Enrichment Kit (STEMCELL Technologies). T cells were isolated from the spleens and lymph nodes of OT-II mice with the EasySep Our results further indicate that IL-21 and IL-4 play nonre- Negative Selection Mouse CD4+ T Cell Enrichment Kit (STEMCELL dundant roles in subsequent GC B cell maturation and self-renewal Technologies). For all flow cytometry, sorting, and histology experiments, 6 6 http://classic.jimmunol.org in vivo. In the absence of either of these signaling pathways, GC 3 3 10 B cells and 1 3 10 T cells were transferred prior to immuni- B cells form aberrantly and are compromised in their self-renewal zation. To ensure consistency in the cell transfer of the donor cell pop- ulations, all recipients of a time course experiment received their in that environment. IL-21R– and STAT6-deficient GC B cells are transferred cells on the same day. Cells were injected i.v. into SMARTA atypical, differing in their phenotypic maturation and costimula- recipients that were immunized 1–5 d later such that the tissue harvest, tory molecule expression. Whereas IL-21 signaling is requisite to staining, and flow cytometric analysis for the entire time course study were properly instruct the centrocyte to centroblast transition, IL-4– begun on the same day. In some experiments, WT C57BL/6 mice were used as recipients instead. For immunizations, NP-haptenated OVA Downloaded from dependent signaling has a greater influence on the acquisition of (NP-OVA) was precipitated in alum at 0.25 mg/ml and 50 mg was injected the centrocyte state. Therefore, in the absence of either of these i.p. The succinic anhydride ester of Nitrophenyl (Biosearch Technologies) + signaling pathways, Bcl6 B cells can form; however, their failure was conjugated to OVA (Sigma-Aldrich) in-house. In some transfer stud- to establish and maintain a properly functioning GC has revealed ies, 3 mg BrdU in 200 ml PBS was injected i.v. in the tail vein 6 h before nonredundant roles for IL-21 and IL-4 signaling in the phased sacrifice. progression of GC B cell development. Ab reagents Abs with the following specificities were used for flow cytometry and Materials and Methods histology: GFP-FITC (goat; Rockland Immunochemicals), CD38-PE/Cy7 Mice and genotyping and -Pacific Blue (90; BioLegend), CD45R-PE, -BV421, -PE/Cy7, and -APC/Cy7 (RA2-6B2; BioLegend), Bcl6-AL647 (K112-91; BD Phar- B1-8 mice (41) were crossed to a homozygous deletion of the Jk locus mingen), Bcl6-AL647 (7D1; Santa Cruz Biotechnology), activated cas- (42). The B1-8 K/I gene carries the Vh 186.2 IgH derived from the B1-8 pase-3–PE (C92-605; BD Pharmingen), BrdU-bi and -AL647 (3D4; hybridoma, which generates a BCR with moderate affinity for the hapten Phoenix Pharmaceuticals), CD4-BV421 (GK1.5; BioLegend), ICOS-APC (4-hydroxy-3-nitrophenyl) acetyl (NP) when paired with l1orl3 L chains (C398.4A; BioLegend), CD62L-APC/Cy7 (Mel-14; BioLegend), (43). B1-8 cells obtained from mice with a homozygous deletion of the Jk CCR7-PE (4B12; BioLegend), CD86-BV605 (GL1; BD Biosciences), locus are, thus, highly enriched for the anti-NP specificity because of the CD40-bi (HM40-3; eBioscience), CXCR4-PE (L276F12; BioLegend), The Journal of Immunology 3

CD83-bi (Michel-19; BioLegend), Mouse IgD-V450 (11-26c.2a; BD generated (Superscript VILO cDNA Synthesis Kit; Invitrogen), the IL-4 Biosciences), and CXCR5-PE (2G8; BD Pharmingen). The following Abs and IL-21 genes relative to the b-actin genes were amplified (M 3000p; were purified and conjugated in-house: CD4-Pacific Blue and -AL680 StrataGene) with the following primers, as described previously: IL-21 (46) (GK1.5), CD45R-AL647 (RA2-6B2), IgD-bi (11-26c), CD35-bi (8C12), and IL-4 (7). Relative gene expression was calculated as the log2 difference and CD23-AL680 (B3B4). Anti–FITC-AL488 (goat polyclonal; Invi- between the cycle threshold value for the gene of interest and b-actin. trogen), streptavidin-BV421 and -BV605 (BioLegend), and streptavidin- AL555, and -PECy7 (Invitrogen) were used as secondary reagents. Statistical analysis Prism software (GraphPad) was used to graph the data and to calculate Immunofluorescent histology statistical significance using an unpaired Student t test with a two-tailed Portions of excised spleens were fixed with 1% paraformaldehyde-lysine- 95% confidence interval unless otherwise indicated. ANOVA followed periodate solution and frozen in O.C.T. (Tissue-Tek) after passage through by Student t test with a Bonferroni correction was applied to multiple sucrose gradient solutions. Cryostat sections (7 mm) were blocked in PBS comparisons. containing 1% BSA, 0.1% Tween 20, and 10% rat serum prior to staining with combinations of the Abs described above. Prior to staining with anti- Results BrdU, some sections were permeabilized with 0.1% Triton X-100 in 0.1% IL-21 gene expression by the Tfh cell population precedes sodium citrate buffer, followed by DNase (Bovine Pancreas D5025; migration into the B cell follicle; elevated IL-4 expression is Sigma-Aldrich ) digestion to reveal BrdU epitopes. Stained sections were most prominent at mature GC time points mounted with Prolong Gold anti-fade mounting medium (Invitrogen). Separate images for each fluorochrome were acquired with an automated To assess the influence of IL-21 and IL-4 on the initial stages of GC wide-field microscope (Nikon Eclipse Ti) and a CCD camera (QImaging B cell differentiation, we examined the kinetics of Tfh cell cytokine Retiga 2000R) with NIS-Elements software. Emitted light was collected through 440/40– or 460/50–, 525/50–, 605/70–, and 700/75–nm bandpass expression during the early stages of an adaptive immune response. filters. Final processing to overlay single-channel images was performed For these studies, we employed a well-characterized adoptive with Adobe Photoshop. transfer system. To track individual Ag-specific T cells in vivo, purified hapten-specific B cells obtained from B1-8+/+ Jk2/2 mice Flow cytometry were adoptively transferred together with OVA-specific (OT-II) Spleen cell suspensions were initially blocked with an anti-FcgR (CD16/32 T cells that expressed GFP under the direction of the ubiquitin 2.4G2) in PBS containing 3% FCS before further incubation with a promoter. SMARTA transgenic mice that harbor an irrelevant combination of the indicated reagents. Dead cells were excluded on the basis of epithelial membrane Ag staining or LIVE/DEAD Fixable Aqua TCR transgene were used as recipients to eliminate the endoge- (Molecular Probes). Intracellular staining of Bcl6 and activated caspase-3 nous T cell response to OVA (45). Recipients received an i.p. was done using the BD Cytofix/Cytoperm Fixation/Permeabilization So- immunization with NP-OVA in alum, and splenocytes were pre- lution Kit (BD Biosciences). Staining with anti-BrdU required that sam- pared for sorting 1 to 11 d later. T cells were sorted based on ples were fixed and permeabilized in a three-step process: an initial fixation expression of the classical Tfh cell markers CXCR5 and ICOS with 1% paraformaldehyde, a second fixation and permeabilization in + + 2 ethanol, and a final fixation and permeabilization in 1% paraformaldehyde such that an Ag-specific Tfh cell population (GFP CD4 CD45R containing 0.05% Tween 20. This was followed by DNase digestion to CXCR5hi ICOShi) and the nonresponding endogenous T cell pop- reveal BrdU epitopes for staining. Flow cytometry was performed on an ulation (GFP2 CD4+ CD45R2 CD62Lhi) were collected separately. LSR II cytometer (Becton Dickinson) and analyzed with FlowJo software IL-21 gene expression (Fig. 1A) was significantly increased as (Treestar). A total of 1 3 106 events was collected per sample. After doublet discrimination and gating on live cells, populations, as indicated in early as 2 d postimmunization (p.i.) in the differentiating Tfh cell by guest on October 1, 2021. Copyright 2018 Pageant Media Ltd. the figure legends, were assessed. Fluorescence Minus One Controls were population when compared with nonresponding endogenous used to guide further analysis of viable cells after doublet discrimination. T cells. This increased further 3 d p.i. and remained elevated Quantitative PCR through day 11 p.i., consistent with previous reports on IL-21 expression kinetics (37, 47). By contrast, IL-4 gene expression RNA was isolated (RNeasy Plus Kit; QIAGEN) from cells sorted based on (Fig. 1B) was delayed relative to IL-21 and could only be detected expression of the classical Tfh cell markers CXCR5 and ICOS such that an Ag-specific Tfh cell population (GFP+ CD4+ CD45R2 CXCR5hi ICOShi) 3 d p.i. and, similar to prior observations (36, 37, 40), becomes and the nonresponding endogenous T cell population (GFP2 CD4+ CD45R2 predominant only at a mature GC time point. The dominance of CD62Lhi) were collected separately for each sample. After cDNA was IL-21 at the early day 2 time point suggests that IL-21 may play a http://classic.jimmunol.org Downloaded from

FIGURE 1. Kinetics of Tfh cell IL-21 and IL-4 gene expression. (A and B) GFP+ OVA-specific T cells and nonfluorescent NP-specific B cells were transferred into nonresponding SMARTA recipients, which were subsequently immunized i.p. with NP-OVA in alum. Spleens were harvested at indicated times p.i., and two populations of T cells, Tfh (GFP+ CD4+ CXCR5hi ICOShi) and nonresponding host naive T cells (GFP2 CD4+ CD62Lhi), were sorted. cDNA was generated from mRNA, and the expression of the IL-21 (A) and IL-4 (B) genes relative to b-actin and normalized to naive T cell levels was determined by quantitative PCR. Each data point represents an individual mouse, except for the day 1 and day 2 Tfh cell populations. Because of the recovery of very small numbers at these early time points, two mice were pooled for each data point. Shown is a representative experiment of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. 4 ROLES OF IL-21 AND IL-4 IN GC B CELL DYNAMICS

larger role in influencing early B cell differentiation, whereas IL-4 Tfh cells becomes more prominent after T cell migration to the may be more important within the mature intrafollicular GC. follicle interior. Our previous report on the microanatomic location of Tfh cells using this same adoptive transfer system found that the initial Nascent GC B cell development occurs in the combined development of Tfh cells began at the periphery of B cell follicles absence of IL-21R and STAT6 signaling 2–3 d p.i., prior to their migration into the follicle interior shortly To evaluate potential differences in the B cell–intrinsic roles of thereafter (7). Based on this timing with our transfer system, IL-21 IL-21 and IL-4 signaling during initial GC development, B1-8+/+ expression initiates when Tfh cells are completing their residency Jk2/2 mice were crossed to IL-21R2/2 mice or STAT62/2 mice, at the follicle periphery, whereas an increase in IL-4 expression by the latter of which are unable to signal through IL-4 or IL-13 by guest on October 1, 2021. Copyright 2018 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from

FIGURE 2. The generation of Bcl6hi Ag-specific B cells is reduced in the absence of either IL-21R– or STAT6-dependent signals because of reduced proliferation. (A–D) Flow cytometry analysis of splenic Ag-specific B cells. GFP+ hapten-specific B cells (WT, IL-21R2/2,STAT62/2,orIL-21R2/2STAT62/2) and nonfluorescent OVA-specific T cells were transferred into nonresponding SMARTA or C57BL/6 recipients prior to i.p. immunization with NP-OVA in alum. Transfers and immunizations were timed so that analysis was performed on the same day for all time points. Recipients received an i.v. injection of 3 mg BrdU 6 h before harvest to label cells actively proliferating. The legend shown in (A) applies to data plots in (C)–(D). (A) Shown is the percentage of Bcl6hi Ag-specific B cells (CD45R+ CD42 GFP+). Each data point represents an individual mouse. Shown is a representative experiment of three inde- pendent experiments. (B) Ag-specific B cell expansion is reduced in the absence of either IL-21R or STAT6 signaling at the onset of the follicular GC response 4 d p.i. Shown is the average number of GFP+ Ag-specific B cells per 1 3 106 total splenocytes as determined by flow cytometry. Data shown are pooled from three separate experiments. (C) Ag-specific B cell proliferation is reduced in the absence of IL-21R and STAT6 signaling. Shown is the percentage of BrdU+ CD38lo Ag-specific B cells. Shown is a representative experiment of three independent experiments. (D) The absence of either IL-21R– or STAT6-dependent signaling does not affect the amount of apoptotic cell death present during the early GC response as determined by activated caspase-3 staining. When both IL-21R– and STAT6-dependent signaling are knocked out, however, cell death is found to increase in the CD38lo sub- population. *p , 0.05, **p , 0.01, ***p , 0.001. n.s., not significant. The Journal of Immunology 5

receptors that uniquely require STAT6 for downstream signaling IL-21R2/2 B cells (day 4) than the STAT6-deficient population (48). WT, IL-21R2/2,STAT62/2, or IL-21R2/2STAT62/2 (day 6) (Fig. 2C). (double knockout [DKO]) NP-specific B cells were adoptively We also evaluated the amount of apoptotic cell death based on transferred with OVA-specific T cells into either SMARTA or activated caspase-3 staining by flow cytometry. The extent of WT C57BL/6 recipients. Similar results were observed with ei- apoptotic cell death within the CD38lo population of IL21R- or ther type of recipient (data not shown). Recipients were immu- STAT6-deficient B cells was not significantly different (Fig. 2D), nized with NP-OVA in alum i.p. 2–6 d prior to analysis. suggesting that the decrease in Ag-specific B cell numbers more The kinetics of Bcl6 expression by transferred Ag-specific likely results from a reduced proliferative rate rather than an in- B cells was determined by flow cytometry of splenocytes creased rate of cell death. However, CD38lo DKO B cells showed (Fig. 2A, Supplemental Fig. 1). Two days p.i., Bcl6 expression a significant increase in the amount of cell death, indicating that levels within the NP-specific B cell population was slightly but either IL-21R– or STAT6-dependent signaling is sufficient to at universally shifted (∼23 -fold increase, Supplemental Fig. 1), least transiently promote survival independently. That the rapid unrelated to an increase in cell size (data not shown). It was not proliferative capacity typical of day 6 WT GC B cells is not until 3 d p.i. that a distinct Bcl6hi population became apparent, achieved unless B cells are sufficient in both the IL-21 and IL-4 presumably representing GC-committed B cells. Indeed, this co- signaling pathways indicates that these cytokines synergize in a incided with the onset of CD38 downregulation on Bcl6hi but not B cell–intrinsic fashion to evoke a response that is distinct from Bcl6lo WT NP-specific B cells (Supplemental Fig. 2). Importantly, that observed with either independently. Bcl6hi cells were evident within IL-21R2/2 STAT62/2 (DKO) To further assess the physical location of proliferating B cells, B cell populations at this early stage of the response (Fig. 2A). immunofluorescence histology of splenic tissue sections was Therefore, IL-21 and IL-4 signaling is not required for early performed (Fig. 3). Based on BrdU incorporation, proliferative formation of pre-GC B cells prior to follicular localization. By day Ag-specific B cells appropriately homed to the periphery of fol- 4 p.i., significantly reduced numbers of Bcl6hi cells were observed licles regardless of genotype 2 d p.i. (Fig. 3A, 3C). By 3 d p.i., in the IL-21R2/2 and DKO responses, whereas the defect in the Ag-specific B cells, regardless of genotype, could be found with STAT62/2 B cell response was less severe (Fig. 2A and Table I). upregulatedBcl6atthefollicleperiphery(Fig.3A,3D).By6dp.i., By day 6 p.i, a time point at which WT GCs are established, the GFP+ Bcl6hi B cells deficient in either IL-21R or STAT6 were STAT62/2 response was also significantly defective compared observed within follicles harboring newly formed GCs. Whereas with WT response although not to the level of the IL-21R2/2 or the GCs observed in the recipients of WT B cells were domi- DKO response (Fig. 2A). Therefore, expansion of the Ag-specific nated by the transferred GFP+ Ag-specific B cells, the small GCs GC population is compromised among STAT62/2 B cells and that did form with IL-21R– or STAT6-deficient B cells had a more severely compromised in the IL-21R2/2 and DKO pop- mixed composition that included some recipient-derived B cells ulations. Together, these results suggest that B cell differentiation that may have gained a competitive advantage under these to the Bcl6hi state can initially occur in the combined absence of conditions (Fig. 3B). Thus, although an initial increase in Bcl6 IL-4 and IL-21, but that further expansion is limited beyond the levels can occur independently of IL-21R and STAT6 signaling, nascent state. propagation within GCs requires both.

by guest on October 1, 2021. Copyright 2018 Pageant Media Ltd. Proliferation rate and extent of apoptosis of Ag-specific B cells CD38 and CD23 expression differences of emergent GC B cells in the absence of IL-21R– and STAT6-dependent signaling To assess the extent of maturity of emerging GC B cells, the extent The total numbers of Ag-specific B cells (Fig. 2B) were compa- of suppression of CD38 and CD23 surface membrane levels was rable regardless of genotype from the naive time point through 3 d assessed by flow cytometry. CD38 and CD23 are expressed at p.i. This indicates that survival of naive B cells in the recipient high levels by naive follicular B cells, and these expression levels host was not affected in the absence of IL-21R– or STAT6- are maintained upon initial Bcl6 elevation during pre-GC B cell dependent signals, consistent with previous reports (25, 26), and formation. At mature GC time points within WT mice, CD38 that initial expansion of responding B cells occurs independently levels are reduced on both centroblasts and centrocytes, whereas

http://classic.jimmunol.org of these signals at the follicle periphery. To determine whether the CD23 levels are typically higher in centrocytes (49, 50). An reduction in Ag-specific B cell numbers observed 4–6 d p.i. was increase in Bcl6 levels coincides with reduced CD38 levels in due to a change in proliferation rate within the GC compartment, WT GC B cells regardless of CD23 levels (Fig. 4A, 4B). Con- the extent of incorporation of the nucleotide analogue BrdU dur- sistent with previous reports on the effect of IL-21 on GC B cells ing DNA replication was assessed by flow cytometry. Consistent (24, 25), the Bcl6hi IL-21R–deficient and DKO populations, al- with the timing of the diminution of responding B cell numbers, though expressing levels higher than naive B cells, nevertheless + lo

Downloaded from a decrease in BrdU CD38 B cells was evident earlier among displayed lower Bcl6 mean fluorescence intensity (MFI) levels

Table I. Bcl6hi Ag-specific B cell numbers

No. of Bcl6hi Ag-Specific B Cells per 103 Total Ag-Specific B Cellsa

Genotype Naive Day 2b Day 3b Day 4b Day 6b WT 9.9 6 6.3 6.6 6 2.2 37.8 6 9.6 230.1 6 37.5 363.7 6 26.8 IL-21R2/2 6.1 6 3.4 3.9 6 1.2 15.2 6 3.2 66.6 6 13.9** 46.7 6 10.1*** STAT62/2 3.4 6 1.2 2.4 6 0.9 23.1 6 4.3 224.8 6 47.5 106.3 6 9.0*** IL-21R2/2 STAT62/2 3.5 6 1.8 1.7 6 2.2 14.5 6 5.3 41.9 6 3.9** 30.0 6 4.4*** Indicated significance based on comparison with WT population at each given time point. Shown is a representative experiment of three independent experiments. Data shown as mean 6 SEM. (n = 4 or 5). aAg-specific B cells were defined as GFP+ B220+ CD42 splenic mononuclear cells. bDays p.i. with NP-OVA in alum i.p. **p , 0.01, ***p , 0.001. 6 ROLES OF IL-21 AND IL-4 IN GC B CELL DYNAMICS by guest on October 1, 2021. Copyright 2018 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from

FIGURE 3. Appropriate Ag-specific B cell homing and increased Bcl6 expression can occur in the absence of IL-21R– and STAT6-dependent signals. (A–D) Immunofluorescence staining of spleen sections from C57BL/6 mice that received an adoptive transfer of GFP+ NP-specific B cells (WT, IL-21R2/2, STAT62/2, or IL-21R2/2STAT62/2) and nonfluorescent OVA-specific T cells. Recipients were immunized 1–5 d after transfer, and tissue was harvested for analysis 2–6 d p.i. Mice received an i.v. injection of 3 mg BrdU 6 h before harvest. Sections were stained for B220 to highlight B cell follicles, GFP and BrdU to identify actively proliferating cells, and Bcl6. Scale bar, 100 mm. Representative images from one of three independent experiments for each time point are shown. (A) Histology time course to evaluate arrival of Ag-specific B cells to the B cell follicle after adoptive transfer (naive), homing to bridging channels (day 2), increased levels of Bcl6 (day 3), and GC formation (day 6). (B) Higher magnification images (Figure legend continues) The Journal of Immunology 7

FIGURE 4. CD23 and CD38 expression levels of Bcl6hi Ag-specific B cells. (A–E) Flow cytometry analysis of splenic Ag-specific B cells. Cell transfers

by guest on October 1, 2021. Copyright 2018 Pageant Media Ltd. and immunizations were performed as described in Fig. 2. Plots shown are representative of three independent experiments with each data point repre- senting an individual mouse. (A and B) CD38 downregulation directly correlates with an increase of Bcl6 in responding Ag-specific B cells. Transferred GFP+ B cells were analyzed based on subset gating with CD23 and CD38 (A). One representative plot from a naive and day 6 time point is shown. A histogram of Bcl6 expression (B) color coordinated with the subset-gating scheme from (A) shows that increased Bcl6 protein levels are directly co- ordinated with a reduction in CD38 levels at this time point. (C) In the absence of IL-21R–dependent signaling, expression of Bcl6 in Ag-specific B cells is reduced. Shown is the MFI of Bcl6 staining for CD38lo GFP+ Ag-specific B cells. Day 9 and 11 time points are from an independent experiment. (D) In the absence of IL-21R–dependent signaling, expression of CD23 is elevated in Bcl6hi Ag-specific B cells. Shown is the MFI of CD23 staining for Bcl6hi Ag- specific B cells. The day 6 and 11 time points are from an independent experiment. (E) In the absence of either IL-21R– or STAT6-dependent signaling, CD38 downregulation in Bcl6hi Ag-specific B cells is reduced. Shown is the MFI of CD38 staining for Bcl6hi Ag-specific B cells. The day 6 and 11 time points are from independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. http://classic.jimmunol.org compared with WT GC B cells at 6–11 d p.i. However, Bcl6 MFIs B cells, indicating that IL-4 signaling in the absence of IL-21 results are not significantly different in STAT6-deficient B cells (Fig. 4C). in CD23 overexpression, but the absence of both does not during The phenotype of the signaling-deficient Bcl6hi B cells indicates GC development. Notably, the opposite relationship was observed that the GC B cells that form do so aberrantly. We found that in with CD38 expression levels. CD38 levels among STAT62/2 Bcl6hi the absence of IL-21R signaling the distribution of CD23hi versus B cells was significantly higher than that of WT Bcl6hi Bcells lo lo hi

Downloaded from CD23 CD38 B cells was skewed in favor of a CD23 (Fig. 4E). Thus, an absence of either IL-21 or IL-4 signaling in- phenotype (Fig. 4D), consistent with a previous report of CD23 hibits normal GC B cell differentiation, indicating that these cyto- downregulation after exposure to IL-21 in vitro (31). By contrast, kines play nonredundant roles that are B cell intrinsic. the distribution of CD23 subsets among STAT62/2 GC B cells remained comparable to WT GC B cells. Analysis of the CD23 Aberrant intrafollicular GC formation and skewing of MFI of CD38lo B cells revealed that IL-21R2/2 GC B cells have centroblast and centrocyte phenotypes supra-elevated levels of CD23 that are significantly higher than The elevated CD23 levels in the IL-21R2/2 B cells are reminiscent that of naive B cell populations (Fig. 4D). Interestingly, in the DKO of a phenotypic feature of centrocytes, causing us to question the B cells CD23 levels remain comparable to that of nonresponding relative proportion of centrocytes and centroblasts in the absence

of GCs, indicated in day 6 column by white boxes in (A). (C) Higher magnification images of bridging channels indicated in day 2 column by white boxes. Circles highlight examples to demonstrate proper homing of Ag-specific B cells to the area and active proliferation regardless of genotype. (D) Higher magnification images from B cell follicles indicated in day 3 column by white boxes. Circles highlight examples of high levels of nuclear Bcl6 in Ag-specific B cells regardless of genotype and their proliferation status. 8 ROLES OF IL-21 AND IL-4 IN GC B CELL DYNAMICS by guest on October 1, 2021. Copyright 2018 Pageant Media Ltd.

FIGURE 5. Skewing of centroblast and centrocyte populations in the absence of IL-21R– and STAT6-dependent signaling. (A, B, and E–G) Flow cytometry analysis of splenic Ag-specific (GFP+) B cells. Transfers and immunizations were performed as described in Fig. 2. The legend shown in (B) applies to data plots (E)–(G). Shown is a representative experiment of three independent experiments. (A) Gating strategies for identifying the Bcl6hi GFP+ B cell subset, CXCR4hi and CXCR4lo populations, and centroblasts and centrocytes populations based on CXCR4 and CD86 expression. (B) Percentage of centroblast and centrocyte populations based on the gating of flow cytometry data shown in (A). Each data point represents an individual mouse. (C) Immunofluorescence staining of spleen sections. Sections of tissue obtained 9 d p.i. were stained for IgD+ follicular mantle (blue), GFP and CD35 to http://classic.jimmunol.org identify follicular dendritic cells in the GC LZ (red), and Bcl6 (greyscale). Scale bar, 100 mm. Shown are representative images from one of three in- dependent experiments. (D) Quantification of GC B cell zonal residence and numbers. Shown are the percentages of Bcl6hi GFP+ GC B cells that reside within the LZ 9 d p.i. counted within spleen sections stained as in (C). The number of Bcl6hi Ag-specific B cells per square millimeter of GC were quantified from the same sections with each data point representing an individual GC. Shown is a representative experiment of three independent experiments. (E) Shown is the percentage of Bcl6hi Ag-specific B cells (CD45R+ CD42 GFP+) 9 d p.i. as determined by flow cytometry. Each data point represents an individual mouse. (F) CD40 MFI levels of centrocyte and centroblast populations as gated in (A). Each data point represents an individual mouse. (G) CD86 hi lo A , , , Downloaded from MFI levels of CXCR4 and CXCR4 populations as gated in ( ). Each data point represents an individual mouse. *p 0.05, **p 0.01, ***p 0.001.

of normal cytokine signals. Centroblasts within the DZ are pre- physical distribution comparable to WT B cells (Fig. 5B–D). At dominantly CXCR4hi and CD86lo, whereas centrocytes in the LZ these later time points, the majority of Bcl6hi B cells derived from are typically CXCR4lo and CD86hi. Although the phenotype can the adoptive transfer are found within intrafollicular GCs (Fig. 5D, vary within a zone, these characteristics can be used as a crude 5E, 96.4% 6 1.7% of WT, 93.4% 6 1.9% of IL-21R2/2, and measurement of their relative proportions by flow cytometry 93.0% 6 0.5% of Stat62/2 Bcl6hi B cells). (Fig. 5A). Based on these criteria and using the gating shown in However, with either signaling deficiency, the GC B cells that Fig. 5A, we found there were small but statistically significant form fail to properly adopt other classical characteristics. Those differences between STAT62/2 and IL-21R2/2 Bcl6hi B cells. CXCR4hi IL-21R2/2 centroblasts that do form overexpress CD40 IL-21R2/2 GC B cells have a small shift toward a centrocyte-like and CD86, typically centrocyte features (Fig. 5F, 5G). Conversely, phenotype and are somewhat more likely to be physically located CXCR4lo STAT62/2 centrocytes underexpress CD86, typically a within the LZ (Fig. 5B–D). By contrast, STAT62/2 B cells are centroblast features (Fig. 5F, 5G). Thus, the proper generation of slightly more centroblast-like in their phenotype yet maintain a these GC subsets cannot be achieved without B cell–intrinsic The Journal of Immunology 9

reception of both cytokines. A schematic summarizing these re- The results of this study indicate that B cell signaling by these sults is shown in Fig. 6. cytokines may influence GC B cells at different stages. IL-4 and IL-21 are produced with distinct kinetics by T cells with a Tfh cell Discussion phenotype. As shown in this study and similar to previous ob- After engagement with cognate Ag, activated B cells clonally servations, IL-21 expression levels increase 2–3 days after im- expand through multiple rounds of cell division, undergoing a munization, a time period when Tfh cells are predominantly process of differentiation that ultimately results in generation of located at the periphery of follicles, whereas an increase in IL-4 one of several B cell lineages: early memory B cells, short-lived transcription levels is less apparent in our experimental system Ab-secreting cells, or GC B cells. This lineage fate choice is a until a point in time when Tfh cells are primarily intrafollicular complex process, occurring over the course of days during a time (36, 37, 40, 47). Consistent with this, the transition from the period when B cells are in close contact with activated T cells that perifollicular pre-GC B cell state to the intrafollicular stage is are themselves concurrently undergoing differentiation to Th cell unaffected in STAT62/2 B cells. IL-21 signaling, therefore, has a subsets (5–7). The formation of both the GC B cell and Tfh cell greater influence on differentiation at the time of pre-GC B cell re- subsets requires expression of the transcriptional repressor Bcl6 entry to the follicle interior. However, proper GC formation during (8, 9); however, the process leading to B cell commitment to the a subsequent phase within follicles is highly reliant on both cyto- GC lineage is not fully understood. According to contemporary kines. The behavior of IL-21R2/2 B cells during the delayed paradigms, B cell lineage fate is the direct result of instructive intrafollicular expansion phase further stresses the importance of CD40L and cytokine exposure via a sustained immunological IL-4 at a later point. synapse with fully differentiated Th cells, specifically IL-4 and As the GC matures, B cells undergo many rounds of cell division IL-21 derived from Tfh cells in the case of GC formation (35–37, that are interspersed and propelled by contacts with Tfh cells and 51–53). Other innate cell types are also known to produce IL-4, follicular dendritic cells within the LZ (22). A report by Bannard including eosinophils and basophils, and can function as APC to et al. (58) suggests that the transition of LZ resident centrocyte differentiating Th2 cells within mucosal tissues and draining differentiation to that of DZ centroblast results from a timed lymph nodes (54–57). Although they have not been observed program that is instructed through Tfh cell cytokine delivery and within intrafollicular GCs (55), it is unclear whether perifollicular may be resolved through cell division. The results presented in innate cell types have the ability to provide IL-4 directly and this study suggest than an efficient transition between the cen- specifically to activated B cells during early differentiation after troblast and centrocyte states is compromised when the GC ma- immunization. tures under the influence of one cytokine without the other. Our The evidence presented in this study suggests that GC B cell results suggest that IL-21 and IL-4 signaling potentiates different formation is a staged process that can be developmentally arres- aspects of this transition. In the absence of either, intrafollicular ted. In the absence of both IL-21– and IL-4–derived signals, GC B cells, instead, aberrantly form centroblast and centrocyte Ag-specific B cells retain a comparable proliferation rate and form subsets with uncharacteristic phenotypic features. IL-21R– similar numbers of B cells expressing higher levels of Bcl6 during deficient GC B cells have supra-elevated CD23 levels and a the first few days of an adaptive immune response. However, their slightly higher percentage of CXCR4lo centrocyte-like cells within 2/2 hi by guest on October 1, 2021. Copyright 2018 Pageant Media Ltd. further expansion and survival is greatly stunted, indicating that maturing GCs. However, the IL-21R CXCR4 centroblasts this first phase of initial divergence to the GC lineage can occur that do form overexpress CD40 and CD86, typically centrocyte without an obligate subsequent proliferative expansion. Regard- features, suggesting that IL-21 signaling plays a large role in less of their capacity to respond to these cytokines in our exper- instructing the centrocyte to centroblast transition. imental system, Bcl6hi Ag-specific B cells are enabled to re-enter By contrast, IL-4–dependent signaling has a greater influence the follicle interior after their differentiation to this nascent GC on formation of the centrocyte stage. Although STAT62/2 GC state at the follicle periphery. B cells have a normal distribution across the zones of their smaller http://classic.jimmunol.org

FIGURE 6. Schematic of skewed GC B cell transi- tions during self-renewal in the absence of IL-4– or Downloaded from IL-21–dependent signaling. An initial increase in Bcl6 expression by B cells can occur independently of these cytokines; however, proper subsequent GC formation requires responsiveness to both. In the absence of ei- ther, intrafollicular GC B cells, instead, aberrantly form centroblast and centrocyte subsets with unchar- acteristic phenotypic features. Whereas IL-21 signaling plays a larger role in instructing the centrocyte to centroblast transition, STAT6-dependent signaling has a greater influence on centrocyte formation. 10 ROLES OF IL-21 AND IL-4 IN GC B CELL DYNAMICS

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by guest on October 1, 2021. Copyright 2018 Pageant Media Ltd. 44. Bucher, C., L. Koch, C. Vogtenhuber, E. Goren, M. Munger, A. Panoskaltsis-Mortari, tion. [Published erratum appears in 2013 Immunity 39: 1182.] Immunity 39: P. Sivakumar, and B. R. Blazar. 2009. IL-21 blockade reduces graft-versus-host 912–924. http://classic.jimmunol.org Downloaded from