Populations Among Germinal Center B Cells Contribution of DOCK11 To

Contribution of DOCK11 to the Expansion of Antigen-Specific Populations among Germinal Center B Cells

Akihiko Sakamoto and Mitsuo Maruyama Downloaded from ImmunoHorizons 2020, 4 (9) 520-529 doi: https://doi.org/10.4049/immunohorizons.2000048 http://www.immunohorizons.org/content/4/9/520 This information is current as of October 2, 2021. http://www.immunohorizons.org/

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Adaptive Immunity

Contribution of DOCK11 to the Expansion of Antigen-Specific Populations among Germinal Center B Cells

Akihiko Sakamoto* and Mitsuo Maruyama*† *Department of Mechanism of Aging, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511, Japan; and †Department of Aging

Research, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan Downloaded from

ABSTRACT

Germinal centers (GCs) are a structure in which B cell populations are clonally expanded, depending on their affinities to Ag. Although http://www.immunohorizons.org/ we previously isolated a characteristic protein called dedicator of cytokinesis 11 (DOCK11) from GC B cells, limited information is available on the roles of DOCK11 in GC B cells. In this study, we demonstrate that DOCK11 may contribute to the expansion of Ag- specific populations among GC B cells upon immunization of mice. The lack of DOCK11 in B cells resulted in the lower frequency of Ag-specific GC B cells along with enhanced apoptosis upon immunization. Under competitive conditions, DOCK11-deficient B cells were dramatically prevented from participating in GCs, in contrast to DOCK11-sufficient B cells. However, minor impacts of the DOCK11 deficiency were identified on somatic hypermutations. Mechanistically, the DOCK11 deficiency resulted in the suppression of B cell–intrinsic signaling in vitro and in vivo. Although DOCK11 expression by B cells was required for the induction of T follicular helper cells at the early stages of immune responses, minor impacts were identified on the expansion of Ag-specific populations among GC B cells. Thus, DOCK11 appears to contribute to the expansion of Ag-specific populations among GC B cells through the stimulation of B cell–intrinsic signaling. ImmunoHorizons, 2020, 4: 520–529. by guest on October 2, 2021

INTRODUCTION rho family GTPase cell division cycle 42 (CDC42), resulting in cytoskeletal reorganization (13, 14). Because the cytoskeleton plays Germinal centers (GCs) are a structure in which B cell populations an important role in humoral immune responses (15–18), DOCK11, are clonally expanded, depending on their affinities to Ag (1–4). similar to CDC42 (19–22), may play some roles in B cells. Upon Ag binding, B cells are activated and proliferate to form GCs. Among DOCK-D family proteins (23–25), DOCK10 and Ig genes are somatically hypermutated, resulting in the generation DOCK11 are expressed by B cells (12, 26). We and other groups of BCR with high affinities to Ag. High-affinity B cell clones are previously reported that the lack of DOCK10 caused mild to no selectively expanded with help from T follicular helper (Tfh) cells defects in the development of B cells or humoral immune (5–11). Thus, GCs are an important structure for the selection of responses (26–29). Similarly, the lack of DOCK11 caused only high-affinity B cell clones. mild, if any, defects in the development of B cells (28). Limited We previously isolated a characteristic protein called dedicator information is currently available on the roles of DOCK11 in of cytokinesis 11 (DOCK11, also known as Zizimin2) from GC B cells humoral immune responses, including the clonal expansion of GC (12). As a guanine nucleotide exchange factor, DOCK11 activated the B cell populations.

Received for publication June 7, 2020. Accepted for publication August 13, 2020. Address correspondence and reprint requests to: Dr. Mitsuo Maruyama, Department of Mechanism of Aging, National Center for Geriatrics and Gerontology, 7-430 Morioka-cho, Obu, Aichi 474-8511, Japan. E-mail address: [email protected] ORCID: 0000-0003-0788-3646 (A.S.). This work was partially supported by research funding for longevity sciences from the National Center for Geriatrics and Gerontology (30-41 to A.S. and 29-26 and 19-1 to M.M.). Abbreviations used in this article: AF, Alexa Fluor; Cg1, Cg1, IgG1 C region; CDC42, cell division cycle 42; CGG, chicken g globulin; DOCK11, dedicator of cytokinesis 11;

GC, germinal center; Igk, Ig L chain k; KO, knockout; NP, 4-hydroxy-3-nitrophenylacetyl; Tfh, T follicular helper; VH186.2, IgH V region 186.2. The online version of this article contains supplemental material. This article is distributed under the terms of the CC BY-NC 4.0 Unported license. Copyright © 2020 The Authors

520 https://doi.org/10.4049/immunohorizons.2000048

ImmunoHorizons is published by The American Association of Immunologists, Inc. ImmunoHorizons CONTRIBUTION OF DOCK11 TO Ag-SPECIFIC GC B CELLS 521

In the current study, we examined the impact of the DOCK11 CD21/CD35 allophycocyanin (clone no. 7E9, 0.25 mg/ml; Bio- deficiency on the formation of GC B cells upon immunization. Legend), anti-CD23 PE (clone no. B3B4, 0.25 mg/ml; BioLegend), Because DOCK11 was found to contribute to the expansion of anti-CD38 PE-Dazzle594 (clone no. 90, 0.5 mg/ml; BioLegend), Ag-specific populations among GC B cells, the underlying anti-CD44 FITC (clone no. IM7, 0.63 mg/ml; BioLegend), anti- mechanisms were examined in more detail. CD45R (B220) PE-Cy7 (clone no. RA3-6B2, 0.25 mg/ml; Bio- Legend), anti-CD45.1 (Ly5.1) FITC or allophycocyanin-Cy7 (clone no. A20, 1.25 mg/ml; BioLegend), anti-CD45.2 (Ly5.2) MATERIALS AND METHODS Brilliant Violet 605 (clone no. 104, 0.25 mg/ml; BioLegend), anti-CD62L Alexa Fluor (AF) 700 (clone no. MEL-14, 0.63 mg/ml; Mice BioLegend), anti-CD95 allophycocyanin (clone no. REA453, All mice were maintained on a C57BL/6 background under specific 0.15 mg/ml; Miltenyi Biotec), anti-CD185 (CXCR5) biotin pathogen-free conditions. Dock11 knockout (KO) (28), Cd19-Cre (clone no. L138D7, 1.25 mg/ml; BioLegend), anti-CD279 (PD1) (30), IgG1 C region (Cg1)-Cre (31), Dock11 flox (28), Cdc42 flox (32), PE (clone no. 29F.1A12, 0.5 mg/ml; BioLegend), anti-Foxp3 AF 647 and B1-8f mice (33) were described previously. Ly5.1 mice were (clone no. MF23, 0.5 mg/ml; BD Biosciences), anti-IgG1 FITC or BV605 fl obtained from Sankyo Labo Service (Tsukuba, Japan). Dock10 ox (clone no. A85-1, 0.5 mg/ml; BD Biosciences), anti-IgG1 PE- Downloaded from mice were recovered from frozen embryos by the German Dazzle594 (clone no. RMG1-1, 0.5 mg/ml; BioLegend), anti-Igk AF Research Center for Environmental Health (Neuherberg, 700 (clone no. RMK-45, 1.25 mg/ml; BioLegend), antiphosphorylated Germany) and backcrossed to C57BL/6 mice for more than Bruton tyrosine kinase (BTK) (Y223) BV421 (clone no. N35-86, 10 generations. Each genotype was elucidated by PCR. All 1:400; BD Biosciences), antiphosphorylated spleen tyrosine kinase animal experiments were performed using 2- to 4-mo-old male (SYK)(Y352)PE(cloneno.17A/P-ZAP70, 1:400; BD Biosciences), and female mice with the approval of the Institutional Review Ghost Dye Red 780 (1:1000; Tonbo Biosciences), NP23 PE (0.5 mg/ml; http://www.immunohorizons.org/ Board at the National Center for Geriatrics and Gerontology. LGC Biosearch Technologies), propidium iodide (1 mg/ml; Sigma- Aldrich), streptavidin Brilliant Violet 605 (0.25 mg/ml; Bio- Immunization Legend), and Zombie NIR (1:1000; BioLegend). Apoptotic cells Primary immunization was performed by an i.p. injection of 50 mg were identified using a CaspGLOW fluorescein active caspase of 4-hydroxy-3-nitrophenylacetyl (NP) coupled to Ficoll (NP53-Ficoll; staining kit (BioVision, Milpitas, CA). Intracellular staining LGC Biosearch Technologies, Teddington, Middlesex, U.K.), was performed using a Foxp3/transcription factor staining 100 mg of alum-precipitated chicken g globulin (CGG) (Calbiochem, buffer set (Thermo Fisher Scientific, Waltham, MA), according currently Sigma-Aldrich, St. Louis, MO), or 100 mgofalum- to the manufacturer’s instructions. A flow cytometric analysis was precipitated NP33-CGG (LGC Biosearch Technologies). Secondary performed on a Gallios cytometer (Beckman Coulter, Indianapolis, immunization was performed by an i.p. injection of 50 mgof IN) equipped with a bandpass filter (605BP30; Omega Optical, by guest on October 2, 2021

NP33-CGG. Brattleboro, VT). Cell sorting was performed on a FACSAria II cytometer (BD Biosciences). MACS A single-cell suspension was prepared by passing tissues through Quantitative RT-PCR a 100-mm nylon cell strainer. RBC lysis was performed in lysis RNA was extracted with a TRI reagent (Molecular Research buffer (150 mM NH Cl, 14 mM NaHCO ,and2mMEDTA).To 4 3 Center, Cincinnati, OH), followed by reverse transcription to isolate B cells, cells were incubated with anti-CD43 microbeads cDNA with ReverTra Ace transcriptase (Toyobo, Osaka, Japan). (Miltenyi Biotec, Bergisch Gladbach, Germany). More than 90% Quantitative PCR was performed using a THUNDERBIRD SYBR purity was achieved, as measured by flow cytometry. To enrich GC mix (Toyobo). The primers used were as follows: 59-GCTTGACAG B cells, cells were incubated with anti-CD4 biotin (clone no. GK1.5, CATGGCCAAAA-39 and 59-AACGCTGAACACCCACTAGG-39 for 0.63 mg/ml; BioLegend, San Diego, CA), anti-CD8a biotin (clone Dock11 (34); 59-TAGTGCCACCTCCTGCTCACT-39 and 59- no. 53-6.7, 1:800;Tonbo Biosciences, San Diego, CA), anti–TER-119 CAACAATTCCACGTGGCAGCC-39 for Aicda (35); and 59-AGT biotin (clone no. TER-119, 0.63 mg/ml; BioLegend), anti-CD38 biotin CCCTGCCCTTTGTACACA-39 and 59-GATCCGAGGGCCTCACTA (clone no. 90, 1.25 mg/ml; BD Biosciences, San Jose, CA), and, in AAC-39 for 18S ribosomal RNA (34). All samples were run in some cases, anti–Ig L chain k (Igk) biotin (clone no. RMK-12, duplicate on a PikoReal real-time PCR system (Thermo Fisher 1.25 mg/ml; BioLegend) and anti-CD45.1 (Ly5.1) biotin (clone Scientific). The DD cycle threshold method was applied for the no. A20, 1.25 mg/ml; BioLegend), followed by an incubation relative quantification of RNA expression levels. with antibiotin microbeads (Miltenyi Biotec).

Flow cytometry Preparation of NP-BSA The following Abs and reagents were used for ﬂow cytometry: For ELISA of anti-NP Abs, varied concentrations of 4-hydroxy-3- anti-BCL6 BV421 (clone no. K112-91, 1:400; BD Biosciences), anti- nitrophenylacetic acid succinimide ester (LGC Biosearch Tech- CD4 PE-Cy7 (clone no. GK1.5, 0.25 mg/ml; BioLegend), anti-CD19 nologies) were conjugated to BSA, according to the manufacturer’s allophycocyanin-Cy7 (clone no. 6D5, 0.25 mg/ml; BioLegend), anti- instructions. The resultant NP-BSA conjugates were puriﬁed

https://doi.org/10.4049/immunohorizons.2000048 522 CONTRIBUTION OF DOCK11 TO Ag-SPECIFIC GC B CELLS ImmunoHorizons through a PD-10 desalting column (Cytiva, Marlborough, MA). The follicular B cells. Naive follicular B cells were isolated from spleens conjugation ratio was determined by the absorbance value at 430 nm. of naive mice (Supplemental Fig. 1A). B1-8 IgH forms an NP- specific BCR when combined with an Ig L chain l (39). To obtain ELISA Ag-specific GC B cells, B1-8 IgH-carrying mice (33) were i.p. Serum was collected from mice under anesthesia with isoflurane. immunized with alum-precipitated NP-CGG. NP-specificIgG1+ For sandwich ELISA, 96-well plates were coated with 2 mg/ml GC B cells were then isolated from spleens (Supplemental Fig. 1B).

NP20-BSA or NP4-BSA. Serially diluted samples were loaded, The isolation of GC B cells was confirmed by the upregulation followed by incubations with 1 mg/ml anti-IgG1 biotin (clone no. of activation-induced cytidine deaminase (40) (Fig. 1A). In A85-1; BD Biosciences) and streptavidin HRP (1:3000; Cytiva). contrast, the expression levels of DOCK11 in NP-specificIgG1+ GC Color development was performed with tetramethylbenzidine B cells were decreased to 27% of those in naive follicular B cells and stopped by acidification. Absorbance at 450 nm was measured (Fig. 1B). on a 680 microplate reader (Bio-Rad Laboratories, Hercules, CA). Dose-response curves were analyzed, using R software (R Founda- Impact of the DOCK11 deficiency on the frequency of tion for Statistical Computing, Vienna, Austria) (36). Ab concentra- Ag-specific GC B cells

tions were determined, based on the EC50 values of dilution factors. Although DOCK11 was originally isolated from GC B cells (12), the Downloaded from N1G9 anti-NP IgG1 (37) was used as a standard. expression levels of DOCK11 were rather downregulated in GC B cells as compared with those in naive follicular B cells. Thus, Adoptive transfer DOCK11 seemed to be dispensable in GC B cells. To examine B1-8 IgH-bearing B cells were isolated by MACS using spleens whether the remaining expression of DOCK11 is still required in pooled from three or more mice. The numbers of NP-binding cells GC B cells, we generated mice lacking DOCK11 in B cells. Dock11 were enumerated by flow cytometry. Adoptive transfer was flox mice (28) were crossed with Cd19-Cre mice (30). After http://www.immunohorizons.org/ performed by an i.v. injection of B cells, including 1 3 105 of NP- specific cells per recipient.

BCR sequencing BCR sequencing was performed, as previously described (38), with some modifications. Briefly, NP-specificIgG1+ GC B cells were single-cell sorted into 10 ml of RNase-free water containing 50 ng of carrier RNA (Thermo Fisher Scientific). Gene segments of IgH

Vregion186.2(VH186.2) linked to Cg1wereampliﬁed, using a SuperScript IV one-step RT-PCR system (Thermo Fisher Scientiﬁc). by guest on October 2, 2021 Primers for nested PCR were as follows: 59-TTCTTGGCAGCAA

CAGCTACA-39 (VH186.2 sense), 59-GGATCCAGAGTTCCAGGT CACT-39 (Cg1 external antisense), and 59-GGAGTTAGTTTGGG CAGCAG-39 (Cg1 internal antisense).

Cell stimulation To stimulate B cells, DMEM was supplemented with 10% heat- inactivated FBS, 1 mM sodium pyruvate, 10 mM HEPES, 1% nonessential amino acids, 100 U/ml penicillin, 100 mg/ml streptomycin, and 52 mM 2-ME. B1-8 IgH-bearing B cells were incubated at 37°C with or without 0.2 mg/ml NP53-Ficoll. The stimulation was stopped by directly adding twice the volume of fi ice-cold xation/permeabilization working solution in a Foxp3/ FIGURE 1. Downregulation of DOCK11 in GC B cells. ff transcription factor staining bu er set. The expression levels of activation-induced cytidine deaminase (A) and Statistical analysis DOCK11 (B) by the indicated splenic B cells were measured by quan- Statistical analyses were performed using R software. A p value titative RT-PCR and normalized to those of 18S ribosomal RNA. GC B ,0.05 was considered to be significant. cells were isolated from the spleens of B1-8 IgH-carrying mice 14 d postimmunization with alum-precipitated NP-CGG. Gating strategies for naive follicular B cells (B220+CD19+CD23+CD21–) and NP-specific RESULTS IgG1+ GC B cells (B220+CD19+CD95+CD38–NP+IgG1+Igk–) are shown in Supplemental Fig. 1A, 1B, respectively. Each symbol represents an Downregulation of DOCK11 in GC B cells individual mouse. Horizontal lines represent geometric means. Data are To identify the roles of DOCK11 in GC B cells, we compared the pooled from two independent experiments, using six or more mice per expression levels of DOCK11 in GC B cells with those in naive experimental group. ***p , 0.001, **p , 0.01 (Welch t test).

https://doi.org/10.4049/immunohorizons.2000048 ImmunoHorizons CONTRIBUTION OF DOCK11 TO Ag-SPECIFIC GC B CELLS 523 immunization with NP-CGG, the numbers of NP-specific IgG1+ GC B cells were enumerated by flow cytometry (Fig. 2A). NP-specificIgG1+ GC B cells were similarly formed to Downloaded from http://www.immunohorizons.org/

FIGURE 3. Impact of the DOCK11 deficiency after the B cell activation stage. (A) Numbers of NP-specific and whole IgG1+ GC B cells (B220+CD19+CD95+CD38–IgG1+) in the spleens of the indicated strains 14 d postimmunization with NP-CGG. Each symbol represents an individual mouse. Horizontal lines represent geometric means. Data are by guest on October 2, 2021 pooled from four independent experiments, using eight or more mice per experimental group. (B) Frequencies of NP-specific cells among IgG1+ GC B cells in (A). (C) Levels of NP-specific IgG1 in serum of the indicated strains 14 d postimmunization with NP-CGG, as measured by ELISA. Each symbol represents an individual mouse. Horizontal lines represent means. Data are pooled from two independent experiments, using nine or more mice per experimental group. **p , 0.01, *p , 0.05 (Welch t test). cKO, conditional KO. FIGURE 2. Impact of the DOCK11 deficiency on the specificity of GC B cells. (A) Gating strategies for NP-specific IgG1+ GC B cells. The left contour plotsaregatedonB220+ cells. Numbers show the percentages of cells the levels of those in Cd19-Cremice(Fig.2B).Incontrast,the in each gate. (B) Numbers of NP-specific and whole IgG1+ GC B cells frequencies of NP-specific cells among IgG1+ GC B cells were (B220+CD19+CD95+CD38–IgG1+) in the spleens of the indicated strains decreased in DOCK11-deficient mice (Fig. 2C). Thus, the 14 d postimmunization with NP-CGG. Each symbol represents an individual DOCK11 deficiency appeared to affect the frequency of Ag- mouse. Horizontal lines represent geometric means. Data are pooled from specific populations among GC B cells. three independent experiments, using eight or more mice per experimental The frequencies of Ag-specific GC B cells were further group. (C) Frequencies of NP-specific cells among IgG1+ GC B cells in (B). examined using mice lacking other DOCK11-related proteins, (D) Gating strategies for apoptotic cells among NP-specific IgG1+ GC B including another DOCK-D family protein DOCK10 and the cells. The contour plots are gated on B220+CD19+CD95+CD38–NP+IgG1+ DOCK11 substrate CDC42 (12, 14). Dock10 and Cdc42 flox mice cells. Numbers show the percentages of cells in each gate. (E) Frequencies (32) were crossed with Cd19-Cre mice. After immunization with of apoptotic cells among NP-specific IgG1+ GC B cells in (D). Each symbol NP-CGG, NP-specificIgG1+ GC B cells were similarly formed in represents an individual mouse. Horizontal lines represent means. Data are these mice (Supplemental Fig. 2A). The frequencies of NP-specific pooled from two independent experiments, using four or more mice per cells among IgG1+ GC B cells were decreased in CDC42-deficient experimental group. *p , 0.05 (Welch t test). cKO, conditional KO. mice but not in DOCK10-deficient mice (Supplemental Fig. 2B).

https://doi.org/10.4049/immunohorizons.2000048 524 CONTRIBUTION OF DOCK11 TO Ag-SPECIFIC GC B CELLS ImmunoHorizons

A Collectively, the DOCK11-CDC42 axis may contribute to the frequency of Ag-specificGCBcells. We then examined the impact of the DOCK11 deficiency on the apoptosis of Ag-specific GC B cells. Apoptotic cells were identified by binding of a fluorophore-conjugated VAD-FMK inhibitor to activated caspases (Fig. 2D). After immunization of DOCK11-deficient mice with NP-CGG, NP-specificIgG1+ GC B cells contained larger numbers of apoptotic cells than those BCfrom Cd19-Cre mice (Fig. 2E). Thus, DOCK11 may be required for the survival of Ag-specificGCBcells.

Impact of the DOCK11 deficiency after the B cell activation stage We then investigated the stage at which the DOCK11 deficiency ff fi may a ect the frequency of Ag-speci cGCBcells.Cg1-Cre mice Downloaded from are a strain in which the expression of Cre recombinase is induced by transcription of the Cg1(31).Reflecting the finding that Ig class- DEswitching starts as early as the B cell activation stage (41), Cre-mediated recombination occurs within a few days after immunization (31). Therefore, by crossing Dock11 flox mice with Cg1-Cre mice

instead of Cd19-Cre mice, one would be able to examine the http://www.immunohorizons.org/ impact of the DOCK11 deficiency in GC B cells while DOCK11 expression is maintained in naive B cells. After immunization of the resultant mice with NP-CGG, NP-specificIgG1+ GC B cells were similarly formed to the levels of those in Cg1-Cre mice (Fig. 3A). In contrast, the frequencies of NP-specific cells among F G IgG1+ GC B cells were decreased in mice lacking DOCK11 by Cg1- Cre recombinase (Fig. 3B). These results indicate that the lack of DOCK11 after the B cell activation stage still affects the frequency of Ag-specific populations among GC B cells.

We next examined whether the serum Abs were reflected by by guest on October 2, 2021 the frequency of Ag-specific GC B cells. After immunization with ffi FIGURE 4. Impact of the DOCK11 deficiency on the expansion of NP-CGG, serum levels of both high- and low-a nity Abs were Ag-specific populations among GC B cells. decreased in mice lacking DOCK11 by Cg1-Cre recombinase (Fig. (A) Experimental outline to examine the impact of the DOCK11 de- 3C). Additionally, the ratios between the levels of high- and low- ffi ficiency on the selection of GC B cells. (B) Contour plots showing a nity Abs were also decreased, suggesting that DOCK11 donor-derived NP-specific cells (B220+CD38+CD95–NP+Ly5.1–)among expression after the B cell activation stage may contribute to ffi naive B cells from mice given transfers with B1-8 IgH-bearing B cells the a nity maturation of Abs. of the indicated Dock11 genotypes. Contour plots are gated on – fi B220+CD38+CD95 cells. Detailed gating strategies are shown in Impact of the DOCK11 de ciency on the expansion of fi Supplemental Fig. 3B. Numbers show the percentages of cells in each Ag-speci c populations among GC B cells fi gate. (C) Frequencies of donor-derived NP-specific cells among The speci cities of GC B cells are assessed through competition naive B cells in (B). Each symbol represents an individual mouse. among B cell clones (3). Because DOCK11 appearedto contribute to Horizontal lines represent means. Data are from four independent experiments, using five or more mice per experimental group. (D) independent experiments, using five mice per experimental group. (F) Contour plots showing donor-derived NP-specific IgG1+ GC B cells Frequencies of B cell clones carrying the indicated numbers of nucleotide + – + + + + – + (B220 CD38 CD95 NP IgG1 Ly5.2 Ly5.1 ) from mice given the mutations on the VH186.2 region among donor-derived NP-specific IgG1 transfer with the B1-8 IgH-bearing B cells of the indicated Dock11 GC B cell clones in (E). Numbers in the centers represent the numbers of genotypes,followedbyanimmunizationwithNP-CGG.Contour clones sequenced. Means and SEM of the nucleotide mutations are shown – plots are gated on B220+CD38 CD95+NP+IgG1+ cells. Detailed gating below the pie chart. p = 0.070 (Welch t test). (G) Frequencies of high- strategies are shown in Supplemental Fig. 3C. Numbers show the per- affinity clones carrying a W33L mutation among donor-derived NP- centages of cells in each gate. (E) Frequencies of donor-derived cells specific IgG1+ GC B cell clones in (E). Numbers in the centers represent among NP-specific IgG1+ GC B cells in (D). Each symbol represents an the numbers of clones sequenced. p = 0.38 (Fisher exact test). ***p , 0.001 individual mouse. Horizontal lines represent means. Data are from two (Welch t test). WT, wild-type.

https://doi.org/10.4049/immunohorizons.2000048 ImmunoHorizons CONTRIBUTION OF DOCK11 TO Ag-SPECIFIC GC B CELLS 525 the frequency of Ag-specific GC B cells, we examined the impact of derived NP-specific IgG1+ GC B cells described above were single- the DOCK11 deficiency on the competition among B cell clones. To cell sorted, followed by sequencing of the VH186.2 region, a region generate DOCK11-deficient B cells with specificity to NP, Dock11 corresponding to specificity to NP (42, 43). Although statistical KO mice (28) were crossed with B1-8 IgH-carrying mice. As a significance was not identified (p = 0.070 in Welch t test), smaller source of a DOCK11-sufficient control, B1-8 IgH-carrying mice numbers of mutations were accumulated among DOCK11- were used. The numbers of NP-specificBcellswereenumerated deficient GC B cells (Fig. 4F). Similarly, smaller numbers of by flow cytometry (Supplemental Fig. 3A). B cells from these mice, high-affinity clones with a W33L mutation (42) were identified including 1 3 105 of NP-specific cells, were transferred into among DOCK11-deficient GC B cells, as compared with those congenic wild-type mice (Fig. 4A). The resultant chimeras would among DOCK11-sufficient counterparts (Fig. 4G). However, no contain donor-derived NP-specific B cells with or without DOCK11 statistical significance was identified (p = 0.38 in Fisher exact test). expression and endogenous DOCK11-sufficient B cells. NP-specific Thus, the impact of the DOCK11 deficiency on somatic hyper- B cells accounted for 0.06% of splenocytes, irrespective of DOCK11 mutations, if any, may be minor. expression on the transferred cells (Fig. 4B, 4C, Supplemental Fig. 3B). After immunization with NP-CGG, DOCK11-sufficient Impact of the DOCK11 deficiency on fi + – donor cells accounted for 89% of NP-speci c IgG1 GC B cells B cell intrinsic signaling Downloaded from (Fig. 4D, Supplemental Fig. 3C). In contrast, DOCK11-deficient DOCK11 was found to contribute to the expansion of Ag-specific counterparts accounted for only 15% of NP-specific IgG1+ GC populations among GC B cells in a cell-intrinsic manner. To B cells (Fig. 4E). Thus, DOCK11-deficient clones were outcompeted examine the impact of the DOCK11 deficiency on B cell–intrinsic by endogenous DOCK11-sufficient clones in a B cell–intrinsic manner. signaling (44), splenic B cells were isolated from B1-8 IgH-carrying Ig genes are somatically hypermutated in GCs, contributing to naive mice or DOCK11-deficient counterparts, followed by stimula- the generation of high-affinity B cell clones. To examine the impact tion with T cell–independent type II Ag NP-Ficoll. In DOCK11- http://www.immunohorizons.org/ of the DOCK11 deficiency on somatic hypermutations, the donor- sufficient B cells, the stimulation induced the phosphorylation of by guest on October 2, 2021

FIGURE 5. Impact of the DOCK11 deficiency on B cell–intrinsic signaling. (A) Histograms of B1-8 IgH-bearing B cells (wild type [WT]) or DOCK11-deficient counterparts (KO) expressing the indicated phosphorylated (p) kinases after the stimulation with or without NP-Ficoll for 5 min. Histograms are gated on B220+Igk– cells. (B) Mean fluorescence intensity (MFI) for the indicated phosphorylated kinases after the stimulation with NP-Ficoll in (A). Each symbol represents an individual mouse from which B cells were isolated. Horizontal lines represent means. Data are pooled from two independent experiments, using six or more mice per experimental group. (C) Gating strategies for NP-specific B cells with GC phenotypes. The left contour plot is gated on CD19+ cells. Numbers show the percentages of cells in each gate. (D and E) Numbers of NP-specific GC-like B cells (D) and frequencies of GC-like cells among NP-specific B cells (E) in spleens from B1-8 IgH-carrying mice (WT) or DOCK11-deficient counterparts (KO) 7 d postimmunization with NP-Ficoll. Each symbol represents an individual mouse. Horizontal lines represent geometric means (D) and means (E). Data are pooled from two independent experiments, using six to seven mice per experimental group. **p , 0.01, *p , 0.05 (Welch t test).

https://doi.org/10.4049/immunohorizons.2000048 526 CONTRIBUTION OF DOCK11 TO Ag-SPECIFIC GC B CELLS ImmunoHorizons kinases, including SYK and BTK (Fig. 5A, 5B). In contrast, the in DOCK11-deficient mice. Collectively, DOCK11 may contribute to phosphorylation of these kinases was suppressed in DOCK11- B cell–intrinsic signaling in vitro and in vivo. deficient B cells. Thus, the DOCK11 deficiency appeared to suppress B cell–intrinsic signaling in vitro. Contribution of DOCK11 expression by B cells to the The impact of the DOCK11 deficiency was then examined in induction of Tfh cells vivo. B1-8 IgH-carrying mice or DOCK11-deficient counterparts Bcell–intrinsic signaling appeared to be responsible for the were i.p. immunized with NP-Ficoll. As previously reported (45), impaired expansion of Ag-specific populations among GC B cells in NP-specific B cells with GC phenotypes were induced in the DOCK11-deficient mice. However, the selection of GC B cells is spleens of DOCK11-sufficient mice (Fig. 5C–E). However, the generallydependent onhelp from Tfh cells (46–48). Therefore, we numbers and frequencies of these GC-like B cells were decreased examined the impact of the DOCK11 deficiency on the induction of Tfh cells. B cell–specificDOCK11-deficient mice and Cd19-Cre mice as a control were immunized with NP-CGG. The numbers of Tfh cells were enumerated in the spleens of these mice (Fig. 6A). The numbers (Fig. 6B) and frequencies of Tfh cells (Fig. 6C) were fi both lower in DOCK11-de cient mice than in Cd19-Cre mice. In Downloaded from contrast, similar numbers and frequencies of CD4+ effector T cells were identified in these mice (Supplemental Fig. 4). These results suggest that DOCK11 expression by B cells is required for the differentiation of Tfh cells among Th cells. To further examine at which stage the induction of Tfh cells

was inﬂuenced, mice lacking DOCK11 expression by Cg1-Cre http://www.immunohorizons.org/ recombinase were used. As mentioned above, DOCK11 expression would start to be deleted at the B cell activation stage (31). Cg1-Cre mice were used as a control. After immunization with NP-CGG, Tfh cells were similarly formed in these mice (Fig. 6D, 6E). Given the timing of the induction of Cg1-Cre recombinase (31), these results indicate that DOCK11 expression by B cells is required for the induction of Tfh cells at the early stages of immune responses.

Impact of decreased Tfh cells on the expansion of

Ag-specific GC B cells by guest on October 2, 2021 Because the induction of Tfh cells was suppressed in DOCK11- deficient mice, we examined the competition of GC B cell clones in these mice. B cell–specific DOCK11-deficient mice and Cd19-Cre mice as a control were immunized with alum-precipitated CGG, followed by the transfer of naive B cells from B1-8 IgH-carrying congenic mice (Fig. 7A). Before secondary immunization, 3–4% of NP-specific cells were identified among the transferred cells (Fig. 7B, 7C). Because GCs are an open structure (49, 50), Ag-specific naive B cells potently participate in ongoing GC reactions (51). After the secondary immunization of Cd19-Cre recipients with NP-CGG, NP-specific cells accounted for 87% of donor-derived IgG1+ GC B cells (Fig. 7D, 7E). Although the frequencies of FIGURE 6. Contribution of DOCK11 expression by B cells on the NP-specific cells were significantly decreased to 79% in DOCK11- induction of Tfh cells. deficient recipients, the difference may be minor. Thus, although (A) Gating strategies for Tfh cells (CD4+CD44+CD62L–CXCR5+FoxP3–) the induction of Tfh cells was suppressed in DOCK11-deficient or PD1+BCL6high (hi) Tfh cells. The leftmost contour plots are gated on mice (Fig. 6B, 6C), the impact on the competition of GC B cell CD4+ cells. Numbers show the percentages of cells in each gate. (B and clones, if any, appeared to be minor. D) Numbers of Tfh and PD1+BCL6hi Tfh cells in the spleens of the High-affinity B cell clones are selectively expanded with help indicated strains 14 d postimmunization with NP-CGG. Each symbol from limited numbers of Tfh cells (46–48). Because the formation represents an individual mouse. Horizontal lines represent geometric of Tfh cells was suppressed in DOCK11-deficient mice, the expansion means. Data are pooled from three independent experiments, using of B cell clones was examined in these mice. The donor-derived NP- three or more mice per experimental group. (C and E) Frequencies specificIgG1+ GC B cells described above were single-cell sorted, + hi + of Tfh and PD1 BCL6 Tfh cells among CD4 T cells in (B) and (D). followed by sequencing of the VH186.2 region. Although slightly **p , 0.01, *p , 0.05 (Welch t test). cKO, conditional KO; i.c., intracellular. smaller numbers of mutations accumulated in donor-derived GC

https://doi.org/10.4049/immunohorizons.2000048 ImmunoHorizons CONTRIBUTION OF DOCK11 TO Ag-SPECIFIC GC B CELLS 527

CE Downloaded from DF http://www.immunohorizons.org/ G

FIGURE 7. Impact of decreased Tfh cells on the expansion of Ag-specific GC B cells. (A) Experimental outline to examine the impact of decreased Tfh cells in DOCK11-deficient mice on the selection of Ag-specific cells among transferred congenic B cells. (B) Gating strategies for NP-specific cells (B220+CD95–Ly5.1+NP+IgG1–) among transferred B cells. The left contour by guest on October 2, 2021 plot is gated on B220+ cells. Numbers show the percentages of cells in each gate. (C) Frequencies of NP-specific cells among transferred B cells 1 d posttransfer into the indicated recipients that were immunized with alum-precipitated CGG 14 d before. Each symbol represents an individual mouse. Horizontal lines represent means. Data are pooled from three independent experiments, using five mice per experimental group. (D) Gating strategies for NP-specific IgG1+ GC B cells derived from transferred B cells (B220+CD95+Ly5.1+NP+IgG1+). The leftmost contour plot is gated on B220+ cells. Numbers show the percentages of cells in each gate. (E) Frequencies of NP-specific cells among donor-derived IgG1+ GC B cells 7 d postsecondary immunization of the indicated recipients with NP-CGG. Each symbol represents an individual mouse. Horizontal lines represent means. Data are pooled from two independent experiments, using six mice per experimental group. (F) Frequencies of B cell clones carrying the + indicated numbers of nucleotide mutations on the VH186.2 region among donor-derived NP-specific IgG1 GC B cell clones in (E). Numbers in the centers represent the numbers of clones sequenced. Means and SEM of the nucleotide mutations are shown below the pie chart. p = 0.42 (Welch t test). (G) Frequencies of high-affinity clones carrying a W33L mutation among donor-derived NP-specific IgG1+ GC B cell clones in (E). Numbers in the centers represent the numbers of clones sequenced. p = 0.90 (Fisher exact test). **p , 0.01 (Welch t test).

B cells from DOCK11-deficient recipients (Fig. 7F), similar changes appeared to be required for the survival of Ag-specific GC B cells. did not always occur in the frequency of high-affinity clones with the Through adoptive transfer experiments, DOCK11 was found to W33L mutation (42) (Fig. 7G). Thus, although the induction of Tfh contribute to the expansion of Ag-specific populations among cells was suppressed in DOCK11-deficient mice, the maturation of GC B cells in a cell-intrinsic manner. The DOCK11 deficiency ff GC B cells was not a ected as much as originally expected. resulted in the suppression of B cell–intrinsic signaling in vitro and in vivo. Although DOCK11 expression by B cells was required DISCUSSION for the induction of Tfh cells at the early stages of immune responses, minor impacts were identified on the expansion of In this study, we examined the contribution of DOCK11 to Ag-specific populations among GC B cells. Thus, DOCK11 may the expansion of Ag-specific populations among GC B cells. contribute to the expansion of Ag-specific populations among Immunization of conditional KO strains revealed that DOCK11 GC B cells through the stimulation of B cell–intrinsic signaling.

https://doi.org/10.4049/immunohorizons.2000048 528 CONTRIBUTION OF DOCK11 TO Ag-SPECIFIC GC B CELLS ImmunoHorizons

Although DOCK11 was originally isolated from GC B cells (12), (Wakayama Medical University), A. Takaoka (Hokkaido University), T. Yasuda DOCK11 expression was rather downregulated in GC B cells as (Hiroshima University), and A. Nishikimi (National Center for Geriatrics and compared with that in naive follicular B cells. In our previous Gerontology) for helpful comments and discussion; and N. Ogiso, A. Furio, fi H. Kawasaki, S. Matsubara (National Center for Geriatrics and Gerontology), nding, the expression levels of DOCK11 in GC B cells were and our colleagues for technical assistance. higher than those in non-GC B cells from immunized mice (12). Under this condition, non-GC B cells are comprised of varied types of cells, including activated B cells and Ag-experienced REFERENCES memory B cells. Collectively, immunization appeared to induce the downregulation of DOCK11 in B cells, but the relative 1. De Silva, N. S., and U. Klein. 2015. Dynamics of B cells in germinal centres. Nat. 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