Our previous work has revealed the ability of CD11b to regulate BCR signaling and control autoimmune disease in mice. However, how CD11b regulates the immune response under normal conditions remains unknown. Through the use of a CD11b knockout model on a nonautoimmune background, we demonstrated that CD11b-deficient mice have an elevated Ag-specific humoral response on immunization. Deletion of CD11b resulted in elevated low-affinity and high-affinity IgG Ab and increases in Ag-specific germinal center B cells and plasma cells (PCs). Examination of BCR signaling in CD11b-deficient mice revealed defects in association of negative regulators pLyn and CD22 with the BCR, but increases in colocalizations between positive regulator pSyk and BCR after stimulation. Using a CD11b-reporter mouse model, we identified multiple novel CD11b-expressing B cell subsets that are dynamically altered during immunization. Subsequent experiments using a cell-specific CD11b deletion model revealed this effect to be B cell intrinsic and not altered by myeloid cell CD11b expression. Importantly, CD11b expression on PCs also impacts on BCR repertoire selection and diversity in autoimmunity. These studies describe a novel role for CD11b in regulation of the healthy humoral response and autoimmunity, and reveal previously unknown populations of CD11b-expressing B cell subsets, suggesting a complex function for CD11b in B cells during development and activation.

Bcells are mediators of humoral immunity and can secrete Abs that are able to bind their cognate Ags at sites distal from the B cell itself (13). Ab production by B cells is a tightly regulated process. There are several mechanisms that exist to keep B cell activation in check, such as negative selection/deletion, induction of anergy, and re-editing of the BCR (4). The BCR signaling cascade is one of the major mechanisms for controlling B cell activation. The Src-family kinases LYN, BLK, and CSK serve as BCR inhibitory signals. LYN enacts inhibition by recruitment and activation of inhibitory surface receptors FcγR2B and CD22 (5). The cytoplasmic tail domains of molecules then recruit and activate Src homology domain-containing inositol polyphosphate 5-phosphotase 1 (SHIP-1) and SH-2-containing protein tyrosine phosphatase 1 (SHP-1), which inactivate phosphorylation activation signals downstream of the BCR, including PIP3 and Syk, thereby reducing the strength of the B cell activation signal (68). These events also determine the magnitude and longevity of the resulting humoral response (9). Proper control of these signals is crucial to maintain a healthy level of Ab response that otherwise can lead to autoimmune or immunodeficient disease if regulation is overactive or deficient, respectively (10, 11).

CD11b, the 165-kDa integrin alpha M, noncovalently associates with CD18 to form αMβ2, also known as macrophage-1 Ag or CR3. CR3 is a type I membrane glycoprotein and is one of the four members of the leukocyte-restricted β2-integrin family (1215). CD11b is conventionally considered a myeloid cell marker. However, the association of CD11b/integrin alpha M mutation with systemic lupus erythematosus, a disease mainly driven by T and B cell autoimmunity, suggests a potential function in lymphocytes as well. One early study found that CD11b is expressed more on CD27+IgD memory B cells than other peripheral B cell populations. These cells were demonstrated to migrate in vitro more effectively than CD11b subsets, suggesting a role in homing ability for memory B cells (16). A small subset of B1 B cells, roughly 10%, expresses CD11b. These cells were found to specialize in stimulation and expansion of CD4+ T cells and produced far less IgM than the majority CD11b subset (17). A CD11b-expressing population of plasma cells (PCs) has also been described. These intestinally located PCs reside in the Peyer’s patch and produce higher amounts of IgA Ab than CD11b cells (18). Our previous study demonstrated that CD11b acts as a negative regulator of BCR-mediated autoreactive B cell activation/tolerance (19). However, the autoimmune setting is highly irregular. Alterations to homeostasis and bias of an autoimmune genetic background can skew the normal function of this regulatory factor. How CD11b regulates the immune response under normal conditions remains unknown.

In this study, we examined the role of CD11b on B cell activation and Ab production in a normal, nonautoimmune setting. We also explored whether CD11b regulates BCR repertoire and diversity using CD11b reporter mice. Our data suggest that CD11b critically regulates humoral response during immunization in a nonautoimmune background. Through the use of a CD11b reporter, we reveal a dynamic expression of CD11b on various B cell subsets between naive and immune conditions. In addition, CD11b expression on PCs plays a critical role in BCR repertoire selection and diversity during autoimmunity. These data describe a novel role for CD11b in regulating a healthy humoral response and suggest a function in controlling BCR signaling and repertoire selection at multiple stages of B cell development in health and autoimmunity.

Ig transgenic (Tg) mice expressing the Vh of an anti-NP (4-hydroxy-3-nitrophenylacetyl) Ab have been fully backcrossed onto C57BL/6 background as described previously (20). CD11b-deficient (CD11b knockout [KO]) mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and interbred with NP Tg mice to generate NP CD11b KO mice. For the comparison experiments, NP WT and NP CD11b KO mice on a C57BL/6 background were bred in the same animal facility. CD11b-FRT reporter mice and CD11b flox/flox (CD11bfl/fl) mice were generated by Biocytogen (Wakefield, MA), and both strains were on a C57BL/6 background. To generate myeloid cell or B cell–specific CD11b-deleted mice, we bred LysM-Cre mice or CD19-Cre mice (Jackson Laboratory) with CD11bfl/fl mice to generate myeloid cell or B cell–specific conditional KO (cKO) mice (CD11bfl/flLysMCre/+ and CD11bfl/flCD19Cre/+), respectively. Lupus-prone CD11b reporter ABIN1-FRT mice were generated by interbreeding CD11b FRT mice with ABIN1[D485N] inactive knock-in mice. The ABIN1[D485] mouse model was a gift from Dr. Philip Cohen (University of Dundee, Dundee, U.K.). Generation and characterization of lupus phenotypes in ABIN1[D485N] mice were previously described (21). All animals were maintained under specific pathogen-free conditions and handled in accordance with the protocols approved by the Institutional Animal Care and Use Committee of the University of Louisville.

Fluorochrome-labeled mAbs against NK1.1, Gr-1, CD3, CD19, CD11b, CD22, CD23, CD21/35, IgD, CD38, CD80, CD43, IgM, B220, GL7, CD138, anti-rabbit IgG, and viability detection Ab were purchased from BioLegend (San Diego, CA). NP-CGG (chicken γ globulin conjugate), NP-PE, NP23-BSA, and NP2-BSA were purchased from Biosearch Technologies (Petaluma, CA). Abs against Phospho-Zap-70/Syk, phospho-Lyn, and SHP-1 were obtained from Cell Signaling Technology (Beverly, MA). Anti-mouse IgM-HRP and IgG-HRP were purchased from Southern Biotech (Birmingham, AL).

For Th1-type immunization, NP WT and NP CD11b KO mice were immunized by s.c. injection with a solution of 50 μg of NP-CGG in CFA at a 1:1 ratio. After 14 d, mice were boost immunized with 50 μg of NP-CGG in IFA at a 1:1 ratio. Sera were collected before and at day 21 postimmunization. Anti-NP Ab was measured using parallel ELISAs in which wells were coated with 1 μg of NP23-BSA or NP2-BSA to detect low-affinity or high-affinity Abs, respectively. Similarly, CD11b control or cKO mice were immunized by s.c. injection with 100 μg of OVA in CFA at a 1:1 ratio and boosted with 100 μg of OVA in IFA after 14 d. Sera were collected before and at day 21 postimmunization. Anti-OVA Ab titers were measured via ELISA using a 96-well plate coated with 1 μg of OVA. FRT mice were immunized by s.c. injection with 100 μg of OVA or with the same volume of PBS in CFA at a 1:1 ratio and boosted with 100 μg of OVA in IFA after 14 d. Sera were collected before and at day 21 postimmunization. Anti-OVA Ab titers were measured via ELISA using a 96-well plate coated with 1 μg of OVA. The OD value read at 450 nm was used to determine the overall affinity. For Th2-type immunization, NP WT or NP CD11b KO mice were immunized via i.p. injection of 50 μg of NP20–29-CGG in Alum adjuvant at a 1:1 ratio. CD11b FRT Reporter mice were immunized via i.p. injection of 100 μg of OVA peptide in Alum adjuvant at a 1:1 ratio. Sera were collected before and at given time points after immunization. Anti-NP and anti-OVA titers were measured via ELISA using a 96-well plate coated with 1 µg of NP22-BSA or OVA and a standard ELISAMAX kit (BioLegend).

Single-cell suspensions were blocked in the presence of anti-CD16/CD32 at 4 °C for 15 min and stained on ice with the appropriate Abs in PBS buffer. The samples were acquired using BD FACSCanto cytometer (BD Bioscience, San Jose, CA) and analyzed using FlowJo software (Tree Star, Ashland, OR). Sorting was performed using FACSAria III (BD Biosciences) or MoFlo XDP (Beckman Coulter). Cell purity was confirmed at greater than 90% via flow cytometry.

Cells were prewarmed in a 37°C incubator for 2 h and stimulated for 10 or 30 min at 37 °C with FITC-labeled anti-IgM F(ab′)2 (Jackson ImmunoResearch Laboratories). Both stimulated and unstimulated cells were fixed with 1.5% paraformaldehyde. Cells were then permeabilized using permeability buffer and intracellularly stained with Abs against SHP-1, CD22, CD11b, Phospho-Zap-70/Syk, and phospho-Lyn in staining buffer at 4°C overnight. Alexa Fluor 647 donkey anti-rabbit IgG was used as secondary Ab and incubated with cells in the dark at room temperature for 30 min. The samples were acquired using the Amnis ImageStream system (Amnis Corporation) and analyzed using IDEAS analysis software (Amnis Corporation). The degree of colocalization was analyzed using the similarity feature.

Draining lymph nodes (LNs) from OVA/CFA/IFA-immunized control and CD19-cre;CD11bfl/fl cKO mice were embedded in OCT medium (Tissue-Tek OCT compound 4583; SAKURA) by using a dry ice–cooled 2-methylbutane (Sigma-Aldrich) bath. Cryosections (7 μm) were fixed in −20°C acetone for 20 min and air-dried at room temperature. Sections were blocked with 20% FBS in PBS for 1 h at room temperature and then were stained with the following Abs at 4°C overnight: FITC-B220, PE-GL7, and allophycocyanin-CD3 (BioLegend) at 1:100 dilution. After three washes with PBS, slides were mounted with Fluoro-Gel (Electron Microscopy Sciences). Images were acquired at 10× magnification using Nikon Confocal Microscope (Nikon).

Sorted CD11b+ and CD11b PCs from CD11b reporter ABIN1 mice (4 mo old; n = 4) were frozen in TRIzol (Life Technologies) for RNA extraction. IgG 5′ RACE-expressed Ab repertoire libraries were constructed using the SMARTer Mouse BCR Profiling Kit (catalog number [Cat. No.] 634422; Takara Bio, Mountain View, CA, USA) following the manufacturer’s instructions, with 500 ng of RNA input per library. The quality and size of each IgG library was assessed using the Agilent 2100 Bioanalyzer High Sensitivity DNA Assay Kit (Cat. No. 5067-4626; Agilent). Libraries were then pooled to 10 nM and sequenced on the Illumina MiSeq platform using the 600-cycle MiSeq Reagent Kit v3 (2 × 300 bp, paired-end; Cat. No. MS-102-3003; Illumina).

Ab repertoire sequencing data processing and analysis were carried out using R packages within the Immcantation Portal. After sample demuxing, initially processing was conducted using the Presto software package (22). In brief, reads were trimmed to Q = 20, and only reads in which the IgG primer could be identified were retained. Read mate-pairs were assembled using pairSeq and assemblePairs functions, attempting de novo assembly first and then using blastn-guided assembly if there was insufficient base pair overlap between reads. A maximum overlap error rate of 0.1 between paired reads was allowed, and assembled reads <400 bp were discarded. Duplicate sequences within each library were collapsed, maintaining sequences represented by >1 read. IgH variable (IGHV), diversity, and joining gene assignments were determined using IgBlast (23). The germline database used included C57BL/6 germline sequences available in the ImMunoGeneTics Information System database (24); the sequence for the gene IGHV1-2 was added manually. Clones were defined across CD11b and CD11b+ IgG repertoires from each animal using Change-O (25), based on IGHV/J gene assignments and junction sequence similarity (Hamming distance = 0.15). Clonal abundance curves for each IgG repertoire were estimated using the Alakazam package (26), with 200 bootstrap realizations, based on methods described by Chao et al. (27, 28). Repertoire diversity curves were estimated for each repertoire using the alphaDiversity function, with 200 bootstrap realizations. A one-sided paired t test was used to assess differences in the Simpson’s index (p < 0.05). Evidence for selection on IGHV sequences within each repertoire and animal was assessed using baseline and testBaseline functions within the shazam package (29). The RDI (repertoire dissimilarity index) (30) was estimated within each cell subset using the calcRDI function. RDI values were compared between cell types using a one-way ANOVA. The sequence data have been deposited in the Sequence Read Archive BioProject under accession number PRJNA706399.

Data are shown as mean ± SEM unless otherwise indicated. Statistical differences were analyzed by using a two-tailed unpaired Student t test. Differences were considered significant at the level p < 0.05. Statistical analysis was performed with GraphPad Prism software.

To examine the effect of CD11b deficiency on B activation and Ab production in normal background mice, we crossed global CD11b KO mice with NP Ig Tg mice. These mice carry a prerecombined BCR H chain variable locus that has affinity for the hapten NP, allowing for a greater scale response after immunization. In addition, this system allows NP-specific B cells to be identified. CD11b deficiency in KO B cells was confirmed by Western blot analysis (Supplemental Fig. 1A). NP WT and NP CD11b KO mice were immunized with 50 μg of NP-CGG following the CFA-IFA protocol (Th1 type). Sera were collected before and at day 21 postimmunization, and anti-NP Ab response (high-affinity and low-affinity IgG levels) was measured by ELISA. Compared with preimmunization, both WT and KO mice showed the increased anti-NP IgG levels at day 21 postimmunization (Fig. 1A). In addition, the titers of both low-affinity and high-affinity anti-NP IgG in KO mice were higher than those in WT mice, indicating a dysregulation of the B cell response to Ag immunization in the absence of CD11b. We then examined the frequency of total PCs (B220dimCD138+), plasmablasts (B220hiCD138+), and germinal center (GC) B cells (B220+GL7hi), as well as frequency of NP-specific PCs, plasmablasts, and GC B cells in this model. The gating strategy was shown in Supplemental Fig. 1C. In the spleen, the frequency of PCs, plasmablasts, and GC B cells was comparable in both NP CD11b KO and WT mice (Fig. 1B). However, when examining the NP-specific cell population, we found the frequency of NP+ PCs in CD11b KO mice was higher than that in WT mice, although NP+ plasmablasts and NP+ GC B cells were comparable between WT and CD11b KO mice (Fig. 1C). Unlike the spleen, CD11b KO mice had increased total plasmablasts and GC B cells in draining LNs compared with WT mice (Fig. 1D). In addition, the NP-specific subsets had an even stronger phenotype in draining LNs. The frequencies of NP+ PCs, NP+ plasmablasts, and NP+ GC B cells in CD11b KO mice were significantly higher than those in WT mice (Fig. 1E).

FIGURE 1.

CD11b-deficient mice have enhanced Ab production and higher number of Ag-specific PCs and GC B cells after immunization. NP WT and NP CD11b KO mice were immunized s.c. with a solution of 50 μg of NP-CGG emulsified in CFA at a 1:1 ratio. After 14 d, mice were boost immunized with 50 μg of NP-CGG in IFA at a 1:1 ratio. (A) Sera were collected before and at day 21 postimmunization. Anti-NP IgG levels with different affinity were measured by ELISA with NP2-BSA (high-affinity) and NP23-BSA (low-affinity)-coated plates. OD value was measured at 450 nm. Spleen (B and C) and draining LNs (D and E) were harvested on day 21 postimmunization. FACS analysis of total (B and D) and NP-specific (gated on NP+ population, C and E) PCs, plasmablasts, and GC B cells are shown. Representative dot plots and summarized data are shown. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (unpaired two-tailed Student t test). Each point represents an individual mouse.

FIGURE 1.

CD11b-deficient mice have enhanced Ab production and higher number of Ag-specific PCs and GC B cells after immunization. NP WT and NP CD11b KO mice were immunized s.c. with a solution of 50 μg of NP-CGG emulsified in CFA at a 1:1 ratio. After 14 d, mice were boost immunized with 50 μg of NP-CGG in IFA at a 1:1 ratio. (A) Sera were collected before and at day 21 postimmunization. Anti-NP IgG levels with different affinity were measured by ELISA with NP2-BSA (high-affinity) and NP23-BSA (low-affinity)-coated plates. OD value was measured at 450 nm. Spleen (B and C) and draining LNs (D and E) were harvested on day 21 postimmunization. FACS analysis of total (B and D) and NP-specific (gated on NP+ population, C and E) PCs, plasmablasts, and GC B cells are shown. Representative dot plots and summarized data are shown. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (unpaired two-tailed Student t test). Each point represents an individual mouse.

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We next examined whether global CD11b deficiency has similar effects on Th2-type immunization. To this end, WT or CD11b KO NP Tg mice were immunized with NP20–29-CGG in Alum adjuvant. At day 14, anti-NP IgG level was elevated in CD11b KO mice compared with WT controls. Baseline IgM Ab was also increased in these mice (Fig. 2A). Although we did not observe any significant change in total GC B cells, NP-specific GC B cells were significantly increased in CD11b KO mice (Fig. 2B). Total PCs were also increased in addition to the NP-specific subset (Fig. 2C). Increases in both of these populations suggest CD11b expression on B cells is able to limit the high-affinity Ab response, although there is a bystander effect at PC stage. Taken together, these data demonstrate an Ag-restricted negative-regulatory role for CD11b on the maintenance of PC and GC B populations, which impacts the magnitude of the Ab response.

FIGURE 2.

Global CD11b deficiency alters the Th2 humoral response. NP WT and NP CD11b KO mice were immunized with 50 μg of NP-CGG suspended in Alum via i.p. injection. (A) Serum was collected on day 14 postimmunization. Anti-NP IgG and IgM Ab was measured via ELISA in plates coated with 1 μg/well of NP-BSA. OD was measured at 450 nm. Data are mean ± SEM. (B and C) FACS analysis of splenic GC B cells (B) and PCs (C) from day 14 postimmunization mice. Representative dot plots and summarized results are shown. *p < 0.05, **p < 0.01, ****p < 0.0001 (unpaired two-tailed Student t test). FSC, forward scatter; SSC, side scatter.

FIGURE 2.

Global CD11b deficiency alters the Th2 humoral response. NP WT and NP CD11b KO mice were immunized with 50 μg of NP-CGG suspended in Alum via i.p. injection. (A) Serum was collected on day 14 postimmunization. Anti-NP IgG and IgM Ab was measured via ELISA in plates coated with 1 μg/well of NP-BSA. OD was measured at 450 nm. Data are mean ± SEM. (B and C) FACS analysis of splenic GC B cells (B) and PCs (C) from day 14 postimmunization mice. Representative dot plots and summarized results are shown. *p < 0.05, **p < 0.01, ****p < 0.0001 (unpaired two-tailed Student t test). FSC, forward scatter; SSC, side scatter.

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Ag-driven BCR signaling plays a critical role in the initiation of B cell activation and differentiation into Ab-secreting PCs (31). Previous studies have shown that the BCR signaling is regulated by the availability of Ags, as well as costimulatory and coinhibitory receptors, such as CD79, CD22, CD45, and Galectin-9 (32), through the activation of the tyrosine kinase Syk and Lyn or SHP-1 (33, 34). Activation of Syk is associated with BCR stimulatory receptor, while Lyn activation and SHP-1 negatively regulate BCR signaling. To determine whether CD11b deficiency directly regulates BCR signaling as we previously demonstrated in autoreactive B cells (19), B cells from draining LNs of immunized WT or CD11b KO NP Tg mice were stimulated with or without FITC-labeled anti-IgM F(ab′)2 fragment to mimic BCR crosslinking. Cells were then analyzed using ImageStream. Percent of colocalization was determined by the similarity score. As shown in (Fig. 3A, unstimulated CD11b KO B cells exhibited more BCR-pSyk colocalizations than WT B cells. On BCR crosslinking, more BCR-pSyk colocalization was noticed at 30 min. In addition, there was more BCR-pSyk colocalization in CD11b KO B cells compared with WT B cells. In contrast, WT B cells exhibited more BCR-pLyn colocalizations compared with B cells without CD11b (Fig. 3B). In addition, with the BCR stimulation, CD11b KO B cells showed a blunted response in BCR-pLyn colocalization. BCR ligation resulted in the association of pSyk, pLyn, and SHP-1 from BCR in both groups. There was no significant difference in the colocalization of BCR-SHP-1 between the two groups (Fig. 3C). The differential colocalizations of pSyk and pLyn with BCR in B cells from immunized NP WT mice versus NP CD11b KO mice suggest that CD11b serves as a negative regulator in BCR signaling, which could inhibit activation signals, such as pSyk, and promote inhibitory signals, such as pLyn.

FIGURE 3.

The loss of CD11b alters BCR signaling. NP WT and NP CD11b KO mice were immunized s.c. with a solution of 50 μg of NP-CGG emulsified in CFA at a 1:1 ratio. After 14 d, mice were boost immunized with 50 μg of NP-CGG in IFA at a 1:1 ratio. Draining LN cells at day 21 postimmunization were stimulated with 20 μg/ml anti-IgM F(ab′)2-FITC for given times and then stained with Abs against B220, phospho-Lyn, phospho-Syk, and SHP-1. BCR colocalization with pSyk (A), pLyn (B), and SHP-1 (C) was measured via ImageStream, gated on B220+ cells, and defined by a similarity score. Representative images and summarized percentages of colocalized cells are shown. Each point represents an individual mouse sample. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01 (unpaired two-tailed Student t test). BF, bright field.

FIGURE 3.

The loss of CD11b alters BCR signaling. NP WT and NP CD11b KO mice were immunized s.c. with a solution of 50 μg of NP-CGG emulsified in CFA at a 1:1 ratio. After 14 d, mice were boost immunized with 50 μg of NP-CGG in IFA at a 1:1 ratio. Draining LN cells at day 21 postimmunization were stimulated with 20 μg/ml anti-IgM F(ab′)2-FITC for given times and then stained with Abs against B220, phospho-Lyn, phospho-Syk, and SHP-1. BCR colocalization with pSyk (A), pLyn (B), and SHP-1 (C) was measured via ImageStream, gated on B220+ cells, and defined by a similarity score. Representative images and summarized percentages of colocalized cells are shown. Each point represents an individual mouse sample. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01 (unpaired two-tailed Student t test). BF, bright field.

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To further confirm these findings, we purified B cells from draining LNs of immunized NP Tg mice and then stimulated with FITC-labeled anti-IgM F(ab′)2 fragment. These B cells were also stained with CD11b Ab to differentiate CD11b+ from CD11b B cells. As shown in Supplemental Fig. 2A, unstimulated CD11b B cells exhibited more BCR-pSyk colocalizations than CD11b+ B cells. On BCR crosslinking, there was more BCR-pSyk colocalization in CD11b B cells compared with CD11b+ B cells. In contrast, CD11b+ B cells exhibited more BCR-pLyn colocalizations compared with CD11b B cells with the BCR stimulation (Supplemental Fig. 2B). These data are largely consistent with CD11b WT and KO B cells shown in (Fig. 3. We also examined colocalization between BCR and negative regulator CD22. We found that CD11b B cells showed decreased BCR-CD22 colocalization on BCR crosslinking (Supplemental Fig. 2C). Collectively, these data suggest that CD11b serves as a checkpoint molecule in regulating BCR signaling.

Our previous study has shown CD11b expression on B cells (19). However, expression of CD11b at each stage of B cell development is not well characterized. To examine this, we generated a CD11b-reporter mouse model that adds a GFP tag to the CD11b promoter site (Supplemental Fig. 3A). We first sought to investigate any changes in CD11b expression under immune conditions compared with the naive state, and a Th2-type immunization was used. Beginning at the early stages of development, we examined the prepro-B (fraction A), pro-B (fraction B), pre-B (fraction C), and immature B cell (fraction D) populations in the bone marrow (BM). Expression was greatest at the earliest prepro-B cell stage and increased even further after immunization (Supplemental Fig. 3B, 3C). In the intermediate pro-B and pre-B stages, the CD11b expression was minimal, averaging less than even 1% of the total population. By the immature B cell phase, expression elevated slightly and was once again increased in the immune setting (Supplemental Fig. 3C). As a whole, the developmental stages of B cells have minimal levels of CD11b expression compared with other subsets.

In the peripheral lymphoid organs, we revealed CD11b expression in the follicular (FO) and marginal zone (MZ) B cells of the spleen. The FO B compartment revealed a low but very well-defined population of CD11b-expressing cells (Fig. 4A). Interestingly, expression decreased by nearly half after immunization. The MZ CD11b+ subset was larger and also showed a similar decrease in immunized mice. Also within the spleen, we examined GC B cells to reveal an average of 2% of naive GC B cells express CD11b, which decreased by nearly half postimmunization (Supplemental Fig. 3D). Post-GC long-lived memory B cells reside in the spleen as well. CD11b expression in these cells increased dramatically after Th2 immunization (Fig. 4B). Among the B cell subsets examined, the PCs carry the most consistently high amounts of CD11b expression. An average of 12% of splenic-resident short-lived PCs express CD11b in the naive state, which decreased after immunization (Fig. 4C). Surprisingly, the opposite effect was observed in BM resident long-lived PCs. Here, CD11b expression averaged at roughly 8% increased after immunization (Fig. 4D). The opposing shift in expression under immunization conditions indicates a differential role for CD11b in these two closely related but distinct populations. We also used Th1-type immunization and observed similar decreased CD11b expression in PC and GC B cells on immunization (Fig. 4E). The varying changes in expression across all of these B cell subsets outline a complex and dynamic role for CD11b regulation.

FIGURE 4.

CD11b is dynamically expressed on different B cell subsets. CD11b FRT reporter mice were immunized with 50 μg of NP-CGG suspended in Alum via i.p. injection. Mice were sacrificed at day 14. Unimmunized mice (naive) were used as controls. (A) CD11b expression on splenic FO and MZ B cells. Gating strategy, representative dot plots, and summarized data are shown. (B) CD11b expression on splenic memory B cells. Gating strategy, representative dot plots, and summarized data are shown. (C) CD11b expression on PCs. Gating strategy, representative dot plots, and summarized data are shown. (D) CD11b expression on PCs from BM. Gating strategy, representative dot plots, and summarized data are shown. (E) CD11b FRT reporter mice were immunized by s.c. injection with 100 μg of OVA or with the same volume of PBS in CFA at a 1:1 ratio and boosted with 100 μg of OVA in IFA after 14 d. Draining LNs were harvested on day 21 postimmunization. CD11b expression on PC or GC B cells is shown. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (unpaired two-tailed Student t test).

FIGURE 4.

CD11b is dynamically expressed on different B cell subsets. CD11b FRT reporter mice were immunized with 50 μg of NP-CGG suspended in Alum via i.p. injection. Mice were sacrificed at day 14. Unimmunized mice (naive) were used as controls. (A) CD11b expression on splenic FO and MZ B cells. Gating strategy, representative dot plots, and summarized data are shown. (B) CD11b expression on splenic memory B cells. Gating strategy, representative dot plots, and summarized data are shown. (C) CD11b expression on PCs. Gating strategy, representative dot plots, and summarized data are shown. (D) CD11b expression on PCs from BM. Gating strategy, representative dot plots, and summarized data are shown. (E) CD11b FRT reporter mice were immunized by s.c. injection with 100 μg of OVA or with the same volume of PBS in CFA at a 1:1 ratio and boosted with 100 μg of OVA in IFA after 14 d. Draining LNs were harvested on day 21 postimmunization. CD11b expression on PC or GC B cells is shown. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (unpaired two-tailed Student t test).

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CD11b is conventionally considered as a marker for myeloid cells. Although in our studies we used dump gating strategy to gate out myeloid cells, T cells, and NK cells, it is possible that some myeloid cells remain in the gated cells. To further investigate the role of CD11b on different subsets of cells, we generated CD11bfl/fl mice using CRISPR-Cas9 technology and then bred these mice with LysM Cre mice to establish a CD11b cKO model that specifically depletes CD11b expression in myeloid cells. We found a roughly 75% reduction of CD11b expression on myeloid cells (Fig. 5A). These mice were immunized with OVA using the CFA/IFA protocol (Th1 type). Unlike our previous results, no changes were observed in the IgG Ab response between the control and the CD11b cKO mice (Fig. 5B). The frequencies of PCs and GC B cells in the spleen (data not shown) or draining LNs were unaffected (Fig. 5C). We further immunized these mice using Th2-type immunization. Both mouse strains produced larger amounts of anti-OVA IgG; however, no difference was observed between the two groups (Fig. 5D). In addition, the frequency of PCs and GC B cells was comparable in the spleen of both mouse strains (Fig. 5E). These results suggest that CD11b deficiency on myeloid cells does not alter the humoral immune response.

FIGURE 5.

CD11b deficiency in myeloid cell does not impact the Ab response on immunization. (A) Efficiency of LyzMcre-driven deletion of CD11b. Spleen (left) and peripheral blood (PB) (right) from CD11bfl/fl and CD11fl/flLyzMCre/+ mice were stained with anti-CD11b Abs. Representative dot plots are shown. (B) CD11bfl/fl and CD11bfl/flLyzMCre/+ mice were immunized by s.c. injection with 100 μg of OVA in CFA and boosted with 100 μg of OVA in IFA after 14 d. Sera were collected before and at day 21 postimmunization. Anti-OVA IgG was measured via ELISA at given dilutions in plates coated with 1 μg/well of OVA. (C) LNs were harvested on day 21 postimmunization. Representative FACS analysis of PCs and GC B cells and summarized data are shown. (D) CD11bfl/fl and CD11bfl/flLyzMCre/+ mice were immunized with 50 μg of OVA suspended in Alum via i.p. injection. Sera were collected at day 14. Anti-OVA IgG was measured by ELISA. (E) Spleens from Alum-immunized mice at day 14 were stained for PC and GC B cells. Representative dot plots and summarized data are shown. Each dot represents one individual mouse sample.

FIGURE 5.

CD11b deficiency in myeloid cell does not impact the Ab response on immunization. (A) Efficiency of LyzMcre-driven deletion of CD11b. Spleen (left) and peripheral blood (PB) (right) from CD11bfl/fl and CD11fl/flLyzMCre/+ mice were stained with anti-CD11b Abs. Representative dot plots are shown. (B) CD11bfl/fl and CD11bfl/flLyzMCre/+ mice were immunized by s.c. injection with 100 μg of OVA in CFA and boosted with 100 μg of OVA in IFA after 14 d. Sera were collected before and at day 21 postimmunization. Anti-OVA IgG was measured via ELISA at given dilutions in plates coated with 1 μg/well of OVA. (C) LNs were harvested on day 21 postimmunization. Representative FACS analysis of PCs and GC B cells and summarized data are shown. (D) CD11bfl/fl and CD11bfl/flLyzMCre/+ mice were immunized with 50 μg of OVA suspended in Alum via i.p. injection. Sera were collected at day 14. Anti-OVA IgG was measured by ELISA. (E) Spleens from Alum-immunized mice at day 14 were stained for PC and GC B cells. Representative dot plots and summarized data are shown. Each dot represents one individual mouse sample.

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To better elucidate the cell-specific importance of CD11b in this phenotype, we generated a CD19-cre;CD11bfl/fl cKO model. CD11b depletion efficiency in B cells was evaluated with Western blot analysis. B cells from CD11b cKO mice expressed substantially lower CD11b compared with control B cells (Supplemental Fig. 1B). These mice were then immunized with 100 μg of OVA in the CFA/IFA protocol. Twenty-one days after immunization, anti-OVA IgG Abs increased significantly in CD19-cre;CD11bfl/fl cKO mice compared with those from control mice (Fig. 6A), mimicking the phenotype of global CD11b KOs. We also performed IgG subtype analysis. IgG2b levels were significantly increased in the cKO mice compared with control mice; IgG1 levels were comparable between the two groups. The frequency of GC B cells and PCs was also examined in the spleen and LN. Neither GC B cells nor PCs were altered in the spleen (data not shown). However, compared with CD11bfl/fl control mice, CD19-Cre;CD11bfl/fl cKO mice had a significant increase of PCs and GC B cells in the draining LNs (Fig. 6B). The increased GC formation in the draining LNs was also revealed in CD19-Cre;CD11bfl/fl cKO mice assessed by confocal microscopy (Fig. 6C). Similarly, elevated Ab production was also seen in Th2-type immunization in cKO mice compared with control mice (Fig. 6D). These results further demonstrate the importance of CD11b expression on B cells in regulating the humoral immune response in a nonautoimmune setting.

FIGURE 6.

Loss of CD11b in B cells leads to increased serum Ab, as well as PCs and GC formation. CD11bfl/fl and CD11bfl/flCD19Cre/+ mice were immunized by s.c. injection with 100 μg of OVA in CFA and boosted with 100 μg of OVA in IFA after 14 d. (A) Sera were collected before and at day 21 postimmunization. Anti-OVA IgG, IgG1, and IgG2b were measured by ELISA. (B) Draining LNs were harvested on day 21 postimmunization. FACS analysis of PCs and GC B cells was performed. Representative dot plots and summarized data are shown. (C) Draining LNs from control and cKO mice were snap frozen in the OCT medium and cryosectioned. Slides were then stained with Abs against B220, GL7, and CD3. Scale bars: 100 μm. (D) CD11bfl/fl and CD11bfl/flCD19Cre/+ mice were immunized with 50 μg of OVA suspended in Alum via i.p. injection. Sera were collected at day 14. Anti-OVA IgG and IgM were measured by ELISA. *p < 0.05, **p < 0.01 (unpaired two-tailed Student t test).

FIGURE 6.

Loss of CD11b in B cells leads to increased serum Ab, as well as PCs and GC formation. CD11bfl/fl and CD11bfl/flCD19Cre/+ mice were immunized by s.c. injection with 100 μg of OVA in CFA and boosted with 100 μg of OVA in IFA after 14 d. (A) Sera were collected before and at day 21 postimmunization. Anti-OVA IgG, IgG1, and IgG2b were measured by ELISA. (B) Draining LNs were harvested on day 21 postimmunization. FACS analysis of PCs and GC B cells was performed. Representative dot plots and summarized data are shown. (C) Draining LNs from control and cKO mice were snap frozen in the OCT medium and cryosectioned. Slides were then stained with Abs against B220, GL7, and CD3. Scale bars: 100 μm. (D) CD11bfl/fl and CD11bfl/flCD19Cre/+ mice were immunized with 50 μg of OVA suspended in Alum via i.p. injection. Sera were collected at day 14. Anti-OVA IgG and IgM were measured by ELISA. *p < 0.05, **p < 0.01 (unpaired two-tailed Student t test).

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Because we discovered high expression levels of CD11b on PCs, we next examined whether CD11b expression impacted BCR repertoire selection in addition to regulating the overall Ab response. Because autoreactive B cells can spontaneously secrete autoantibodies, we then backcrossed CD11b reporter mice onto ABIN1[D485N] lupus-prone mice. A20-binding inhibitor of NF-κB (ABIN1) is a ubiquitin-binding protein that has been identified as a systemic lupus erythematosus susceptibility locus in genome-wide studies (35, 36). These mice develop autoantibodies, including anti-dsDNA, starting at 3 mo of age and are not subject to the bias of the Faslpr model whose lack of Fas–FasL interaction heavily skews selection of the B cell population (21). We used 4-mo-old ABIN1[D485N] mice and gated on the PCs and found that CD11b was expressed on 20–30% of PCs (Fig. 7A). To examine whether CD11b influenced the B cell repertoire, we sorted CD11b+ and CD11b PCs and performed expressed Ab repertoire sequencing. As shown in (Fig. 7B and (7C, within mice the IgG repertoires of CD11b+ PCs were less diverse than CD11b PCs and tended to be dominated by more highly abundant clones (Supplemental Fig. 4A). This suggests that loss of CD11b on autoreactive B cells may limit the specific selection of particular Ab clones, leading to a more unadulterated and diverse repertoire. This was also suggested via the analysis of somatic hypermutation patterns, as we observed evidence of positive selection in IgG IGHV complementary determining regions among sequences of clonal lineages in CD11b+ cell repertoires (Fig. 7D). Density plots from each mouse were shown in Supplemental Fig. 4B. Finally, examination of global patterns in IGHV gene usage between samples of each group using the RDI also revealed that the repertoires of CD11b PCs were more similar to each other than were repertoires among CD11b+ PCs (p = 0.00003, one-way ANOVA; (Fig. 7E). Taken together, these data strongly suggest that CD11b critically regulates Ab repertoire diversity, abundance, and potentially autoreactivity.

FIGURE 7.

CD11b expression regulates Ab repertoire diversity, abundance, and selection. (A) Gating strategy to sort CD11b+ versus CD11b PCs from ABIN1[D485N] CD11b reporter mice (n = 6). Dump gating (CD3/Gr-1/NK1.1) was used. CD11b+ versus CD11b PCs were subjected to Repseq. (B) Ab repertoire diversity curves for CD11b (red) and CD11b+ (blue) PCs analyzed from four mice. The diversity metric (y-axis) is plotted against diversity orders (q = 0–4, x-axis) for each sample. Shading around individual lines represents the 95% confidence intervals based on 200 bootstrap resamplings of reads from each repertoire. (C) Boxplot showing paired comparison of repertoire diversity estimated at q = 2 (from B; representing Simpson’s index) between CD11b (red) and CD11b+ (blue) PCs within the four mice analyzed (p value provided from one-tailed t test). (D) Density plots representing evidence for selection strength (x-axis) among clones present in CD11b (red) and CD11b+ (blue) PCs grouped across all four mice analyzed, split by complementary determining region and FWR regions within the IGHV segment (p values provided represent a two-sided test of differences between the posterior probability density function of each cell group). (E) Boxplot displaying RDI values for within-group (CD11b; CD11b+) comparisons based on IGHV repertoire-wide gene usage profiles (p value provided from one-way ANOVA).

FIGURE 7.

CD11b expression regulates Ab repertoire diversity, abundance, and selection. (A) Gating strategy to sort CD11b+ versus CD11b PCs from ABIN1[D485N] CD11b reporter mice (n = 6). Dump gating (CD3/Gr-1/NK1.1) was used. CD11b+ versus CD11b PCs were subjected to Repseq. (B) Ab repertoire diversity curves for CD11b (red) and CD11b+ (blue) PCs analyzed from four mice. The diversity metric (y-axis) is plotted against diversity orders (q = 0–4, x-axis) for each sample. Shading around individual lines represents the 95% confidence intervals based on 200 bootstrap resamplings of reads from each repertoire. (C) Boxplot showing paired comparison of repertoire diversity estimated at q = 2 (from B; representing Simpson’s index) between CD11b (red) and CD11b+ (blue) PCs within the four mice analyzed (p value provided from one-tailed t test). (D) Density plots representing evidence for selection strength (x-axis) among clones present in CD11b (red) and CD11b+ (blue) PCs grouped across all four mice analyzed, split by complementary determining region and FWR regions within the IGHV segment (p values provided represent a two-sided test of differences between the posterior probability density function of each cell group). (E) Boxplot displaying RDI values for within-group (CD11b; CD11b+) comparisons based on IGHV repertoire-wide gene usage profiles (p value provided from one-way ANOVA).

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In this work, we used a nonautoimmune setting to observe the effect of CD11b on the healthy humoral response and to identify novel and dynamic CD11b expression on B cell subsets. We observed an increased Ab response in both CD11b global KO and B cell–specific depleted mice, suggesting that this effect is not dependent on the autoimmune nature as we showed previously (19). This phenotype is also seen in both Th1- and Th2-type immunizations. The increase in both switched IgG and nonswitched IgM Ab shows this regulation impacts Ab resulting from GC B cells and extrafollicular B cells. The “choice” between these two maturation pathways is governed by Ag recognition strength, thus implying that B cell CD11b regulation is not dependent on the intensity of BCR–Ag binding (37). However, the NP-specific subpopulation of PCs and GCs was elevated in CD11b KO mice, suggesting that CD11b may have more effect on Ag-specific B cell activation and differentiation.

The BCR signaling cascade acts through and is regulated by phosphorylation-controlled proteins such as Syk, Lyn, and SHP-1. In the absence of stimulation, BCRs cluster into autoinhibitory confirmations. Our previous work demonstrated CD11b has a stabilizing effect on CD22, which maintains the inhibitory cluster state to control tonic BCR signaling (38). Binding of Ag causes dispersal of these clusters, opening up ITAMs on the cytoplasmic portions of associated CD79, which in turn recruit downstream signals such as Lyn and Syk (39, 40). Indeed, we observed a significant increased BCR-pSyk colocalization in CD11b-deficient B cells even at the basal level. In contrast, BCR-pLyn colocalization is decreased in CD11b-deficient B cells on BCR activation, demonstrating its involvement in maintaining the pLyn negative regulatory circuit on B cells. Similar to autoreactive B cells (19), we also observed less colocalization between BCR and CD22 in CD11b B cells on BCR crosslink. These observations further confirm the function of CD11b in the regulatory circuit of BCR activation. Whether it is serving a specific regulatory purpose at this stage or simply retaining its function as a permanent feature of the BCR circuit has yet to be determined.

Use of a cell-specific KO model to eliminate B cell CD11b expression was able to replicate the increase in serum IgG and IgM seen in the global KO mice. In addition, the frequency of PCs and GC B cells was also elevated in B cell–specific CD11b-deficient mice. In contrast, usage of myeloid cell–expressed lysosome-cre–driven cKO was unable to replicate any of the enhanced B cell response phenotypes seen in CD11b global KO animals, although previous studies showed that CD11b-expressing myeloid cells are important in the priming and expansion of B cells during an alum immunization reaction (41, 42). Even CD11b-expressing myeloid-derived suppressor cells have been shown to enhance B cell Ab production in certain settings (43, 44). Interestingly, CD11b-expressing myeloid cells were found to suppress lupus-like disease in male, but not female, NZB mice (45). Depletion of CD11b-expressing neutrophils can even cause accelerated disease in both sexes of these mice (46).

CD11b-expressing B cell subsets have been previously identified, namely, in the memory B cell, B1 B cell, and Peyer’s patch resident PC subsets (1618). Using a CD11b reporter, we were able to identify small subsets (2% or less) of CD11b-expressing FO, MZ, and GC B cells, which decreased even further after immunization. The relatively low frequency of these fractions suggests a tightly controlled but necessary role for CD11b in regulating a specific subpopulation of cells. We also confirmed previous reports of a substantial CD11b-expressing fraction of memory B cells, which increased after immunization. Here, CD11b is likely to assist in migration and tissue homing after generation as previously shown, but may also have functions in controlling activation as shown in other subsets (16). Expanding on the CD11b-expressing PCs previously identified in the Peyer’s patch, we observed CD11b+ populations of both splenic and BM resident PCs. In the splenic population, which are typically short-lived PCs, CD11b expression decreased after immunization. The aforementioned CD11b-expressing Peyer’s patch PCs were found to proliferate and produce Ab at a higher rate than CD11b cells (18). Increased CD11b expression may serve as a negative regulator to better control these hyperresponsive cells. Splenic PC CD11b could function in a similar role by tempering the response of newly formed PCs, which carry reduced expression as they complete differentiation and exit the GC. Conversely, long-lived PCs of the BM increased CD11b expression after immunization. Long-lived PCs do not activate and respond to BCR binding Ag or immune complex, but rather exist to provide persistent Ab after initial encounter with Ag (47, 48). Here, high CD11b expression may serve as an additional layer of regulation to prevent BCR signal activation of these cells and maintain a steady state of Ab production. However, the underlying mechanisms that regulate CD11b dynamic expression at different states need to be determined in the future work.

We further demonstrate that CD11b expression is also involved in BCR repertoire selection and diversity. The IgG repertoires of CD11b+ PCs were less diverse than CD11b PCs and were dominated by more highly abundant clones. This suggests that loss of CD11b on autoreactive B cells may lead to a lack of regulatory constraint, limiting the specific selection of particular Ab clones, leading to more diverse repertoire, consistent with our overall conclusion that CD11b is a negative regulator of BCR signaling. This also suggests that CD11b not only regulates the magnitude of Ab production but also impacts on the quality of Ab production via influence on the selection of particular Ab clones. This is important given the fact that protective Abs are critical in eliminating pathogens, while pathogenic autoantibodies promote disease pathogenesis. Understanding how CD11b regulates B cell activation and differentiation in health and disease may provide a novel target for the treatment of infectious diseases and autoimmune disorders.

This work was supported by Department of Health and Human Services (HHS), National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID) grants R21AI124235 and AI124081. C.D., D.T., C.T.W., and J.Y. were supported in part by HHS, NIH, National Institute of General Medical Sciences (NIGMS) grant P20 GM135004.

The sequences presented in this article have been submitted to the Sequence Read Archive BioProject under accession number PRJNA706399.

The online version of this article contains supplemental material.

Abbreviations used in this article

BM

bone marrow

Cat. No.

catalog number

CGG

chicken γ globulin conjugate

cKO

conditional knockout

FO

follicular

GC

germinal center

IGHV

IgH variable

KO

knockout

LN

lymph node

MZ

marginal zone

NP

4-hydroxy-3-nitrophenylacetyl

PC

plasma cell

RDI

repertoire dissimilarity index

SHIP-1

Src homology domain-containing inositol polyphosphate 5-phosphotase 1

SHP-1

SH-2-containing protein tyrosine phosphatase 1

Tg

transgenic

WT

wild-type

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The authors have no financial conflicts of interest.

Supplementary data