Cross-reactivity with self-antigen tunes the functional potential of naïve B cells specific for foreign antigens

Abstract

Upon antigen exposure, naïve B cells expressing B cell receptors (BCR) able to bind antigen can undergo robust proliferation and differentiation that can result in the production of antibody-secreting and memory B cells. The factors determining whether an individual naïve B cell will proliferate following antigen encounter remains unclear. In this study, we found that polyclonal naïve murine B cell populations specific for a variety of foreign antigens express high levels of the orphan nuclear receptor Nur77, which is known to be up-regulated downstream of BCR signaling as a result of cross-reactivity with self-antigens in vivo. Similarly, a fraction of naïve human B cells specific for clinically-relevant antigens derived from respiratory syncytial virus (RSV) and HIV-1 also exhibited an IgM^LOW IgD⁺ phenotype, which is associated with self-antigen cross-reactivity. Functionally, naïve B cells expressing moderate levels of Nur77 are most likely to proliferate in vivo following antigen injection. Together, our data indicate that BCR cross-reactivity with self-antigen is a common feature of populations of naïve B cells specific for foreign antigens and a moderate level of cross-reactivity primes individual cells for optimal proliferative responses following antigen exposure.

INTRODUCTION

During hematopoietic development, B cell progenitors undergo VDJ recombination in order to generate immunoglobulin (Ig) expressed on the cell surface as a B cell receptor (BCR) (1). The random nature of this process gives way to the broad spectrum of receptor specificities needed to recognize diverse pathogens but can also yield cells expressing receptors that bind self-antigens. In order to prevent antibody-mediated autoimmunity, self-reactive B cells are subject to tolerance mechanisms including receptor editing, clonal deletion, and the restriction of functional responsiveness, termed anergy (2). The clones that survive these checkpoints comprise the repertoire of naïve B cells that may respond to vaccination or infection (3). There is growing evidence suggesting that antibody responses are hindered by peripheral tolerance mechanisms due to cross-reactivity of antigen-specific BCR with host-derived self-antigens. Studies of individual antibody lineages have identified isolated cases where such cross-reactivity may influence activation of particular clones (4–10). Additionally, several studies have found increased protective antibody responses in animals and patients prone to autoimmunity (11–13). However, it is not clear from these examples how universal this feature may be for naïve B cell populations targeting different foreign antigens.

Such studies pose a number of technical difficulties, as they require analysis of rare antigen-specific B cells prior to somatic hypermutation (SHM). Previous work has shown that as many as 20% of the BCRs in the naïve human repertoire exhibit detectable reactivity with self-antigens (14). In this work however, the specificity of these B cells was not fully defined. More recently, Zikherman and colleagues used a reporter mouse in which BCR signaling in response to self-antigen binding was assessed by the expression of eGFP as a surrogate for the expression of the orphan nuclear receptor Nur77 (15–18). These studies revealed that mature follicular B cells expressed a range of Nur77, and high levels were associated with self-antigen reactivity and diminished function (15). Importantly, while other factors such as CD40 and Toll-like receptor signaling could result in modest Nur77^eGFP upregulation in vitro, Nur77^eGFP expression can be detected in naïve B cells at steady state in the absence of these signals (16). These studies also revealed that Nur77^eGFP expression was low in naïve MD4 B cells, which express a transgenic BCR specific for the foreign antigen hen egg lysozyme (15, 16). These data may indicate that naïve B cells specific for foreign antigens exhibit low cross-reactivity with self-antigens. However, this conclusion is drawn upon the examination of a single BCR, while there are potentially hundreds of thousands of unique BCRs able to bind a given antigen. Furthermore, the MD4 BCR was originally isolated from an animal post-immunization following germinal center selection and extensive somatic hypermutation, which may have reduced cross-reactivity from the unmutated BCR from which the MD4 antibody was derived. A more recent study analyzed B cells with a fixed VH186.2 transgenic heavy chain specific for the foreign antigen 4-hydroxy-3-nitrophenyl (NP) paired to different non-transgenic light chains. In this context, positive selection was more evident in cells expressing BCR that exhibited higher self-antigen reactivity(18). These data examining oligoclonal populations of B cells indicates that mild self-reactivity is a key part of B cell development and suggests that non-transgenic polyclonal populations of foreign antigen-specific naïve B cells would have similar features.

A recent study examining the reactivity of foreign and self-reactivity of antibodies produced by cultures of thousands of single the transitional and mature human B cells may have shed light on this question (19). These data revealed that on average, half of the B cells binding both foreign and self-antigens were deleted between the transitional and mature stage (19). Importantly, self-antigen cross-reactivity was never completely removed from the foreign antigen-specific populations, and the extent of deletion appeared to vary from antigen-to-antigen (19).

In this study, we used antigen-specific enrichment to analyze populations of naïve murine B cells specific for both model antigens as well as pathogen-derived antigens and found that Nur77^eGFP was expressed similarly amongst most populations. Unexpectedly, these levels were indistinguishable from the larger population of naïve B cells whose specificities were unknown. Similarly, naïve human B cells specific for Influenza virus, RSV, or HIV-1 proteins contained B cells with the IgD⁺ IgM^LOW phenotype that has been previously reported to contain human self-antigen-reactive B cells (20, 21). Importantly, the level of self-antigen cross-reactivity appears to influence the likelihood that a naïve B cell will respond to antigen immunization. Naïve antigen-specific B cells with high Nur77^eGFP levels were ~2-fold less likely to proliferate in response to antigen injection compared to their counterparts that expressed moderate levels of Nur77^eGFP. However the cells with low Nur77^eGFP levels were also less likely to proliferate compared to the population expressing moderate levels of Nur77^eGFP, indicating that some level of cross-reactivity with self-antigen may support a cell state optimally poised to proliferate during an immune response. Together, our data suggest that all polyclonal populations of B cells specific for foreign antigens contain BCRs with a range of self-antigen reactivities that tunes cells for optimal function.

MATERIALS AND METHODS

Animals

Six to fourteen week-old male and female mice were used for experiments. CD45.2 (C57BL/6), CD45.1 congenic (B6.SJL-Ptprca Pep3b/BoyJ), IgH^a congenic (B6.Cg-Gpi1^a Thy1^a Igh^a/J), Nur77^eGFP (C57BL/6-Tg(Nr4a1-EGFP/cre)820Khog/J) (22), MD4 (C57BL/6-Tg(IghelMD4)4Ccg/J) (4) and Rag1^−/− (B6.129S7-Rag1tm1Mom/J) (23) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). IgH^a and Nur77^eGFP were backcrossed to generate IgH^a/a Nur77^eGFP mice before use. MD4 and Rag1^−/− were backcrossed to generate MD4 Rag1^−/−. animals were maintained in a specific pathogen-free facility in accordance with Fred Hutchinson Cancer Research Center Institutional Animal Care and Use Committee approval and NIH guidelines.

Antigens

Purified R-Phycoerythrin (PE) and allophycocyanin (APC) were purchased from ProZyme and ovalbumin was purchased from Millipore Sigma. Biotinylated peptides (GGGEQKLISEEDLGGG) conjugated to 4-Hydroxy-3-nitrophenyl (NP-biotin) or 2,4 Dinitrophenyl (DNP-biotin) were purchased from GenScript.

NP650 was created by first conjugating streptavidin-APC (PJ27S, ProZyme) to DyLight 650 NHS ester following the manufacturer’s instructions (Thermo Fisher Scientific). The molar concentration of streptavidin-APC-DyLight 650 was calculated by measuring the absorbance of streptavidin-APC at 280 nm using the extinction coefficient of 0.268 μM⁻¹cm⁻¹. The resulting streptavidin-APC-DyLight 650 was mixed at a molar ratio of 1 to 6 with NP-biotin and incubated for 30 minutes on ice. The resulting conjugate was purified from excess NP-biotin using a 100 kDa Amicon Ultra size exclusion column (Millipore Sigma) and the concentration determined as described for streptavidin-APC-DyLight 650.

RSV prefusion and postfusion F antigens, HIV-1 Env 426C^TM4ΔV1−3, and influenza HA stem were produced as described previously (24–26).

Antigen Tetramers

Purified antigens were biotinylated using an EZ-link Sulfo-NHS-LC-Biotinylation kit (Thermo Fisher Scientific) using a 1 to 1.3 molar ratio of biotin to antigen. Unconjugated biotin was removed by centrifugation using an Amicon Ultra size exclusion column (Millipore Sigma) with a smaller molecular weight cutoff than the antigen. To determine the average number of biotin molecules bound to each antigen streptavidin-PE (PJRS25, ProZyme) was mixed with a fixed amount of biotinylated antigen in increasing concentrations and incubated at room temperature for 30 minutes. Samples were run on an SDS-Page gel (Bio-Rad Laboratories) and transferred to nitrocellulose prior to incubation with streptavidin-Alexa Fluor 680 (Thermo Fisher Scientific) diluted 1:10,000 in 1x DPBS containing 0.2% Tween-20 (Millipore Sigma) to determine the ratio at which there was excess biotinylated antigen available for streptavidin-Alexa Fluor 680 to bind. To make tetramers, biotinylated antigens were mixed with streptavidin-PE or streptavidin-APC (PJ27S, ProZyme) at the ratio determined above, or at a 5 to 1 ratio using the biotin concentration provided by the manufacturer. Following a 30 minute incubation on ice, unconjugated biotinylated antigen was often removed by several rounds of dilution and concentration using a 100 kDa Amicon Ultra (Millipore Sigma) or 300 kDa Nanosep centrifugal devices (Pall Corporation). Tetramers were stored at 1 μM in 1x DPBS at 4°C or 1x DPBS containing 50% glycerol at −20°C prior to use. Control PE594, PE650 and APC755 tetramers were created by mixing a biotinylated control antigen with streptavidin-PE preconjugated with DyLight 594 (PE594) or DyLight 650 (PE650), and streptavidin-APC preconjugated with DyLight 755 (APC755) following the manufacturer’s instructions for DyLight conjugation (Thermo Fisher Scientific). On average, PE594/PE650 and APC755 contained 4 – 8 DyLight molecules per PE or APC. The concentration of each tetramer was calculated by measuring the absorbance of PE at 565 nm combined with an extinction coefficient of 1.96 μM⁻¹cm⁻¹, or APC at 650 nm using an extinction coefficient of 0.7 μM⁻¹cm⁻¹.

Murine single cell suspensions

The spleen and inguinal, axillary, brachial, cervical, mesenteric, and periaortic lymph nodes from individual mice were pooled and dissociated with forceps prior to incubation with 0.8 mg/mL Dispase (Thermo Fisher Scientific), 0.2 mg/mL Collagenase P (Roche) and 0.1 mg/mL of DNase I (Roche) in RPMI (Thermo Fisher Scientific) for 20 min at 37°C. Tissue fragments were next forced through a 100 micron mesh to create single cell suspensions, which were filtered using another 100 micron mesh to remove tissue debris and centrifuged at 300xg for five minutes at 4°C.

Human single cell suspensions

Venipuncture was used to obtain blood from healthy, HIV-seronegative adult volunteers enrolled in the General Quality Control study in Seattle, WA. This protocol was approved by the Fred Hutch institutional review board and informed consent was obtained from all study participants before enrollment into the parent protocols. PBMCs were isolated from whole blood using Accuspin System Histopaque-1077 (Millipore Sigma) and the resulting cell fraction washed in 1xDPBS and centrifuged at 300xg for five minutes at 4°C. The supernatant was aspirated and the cell pellet resuspended in 10% Dimethylsulfoxide in heat-inactivated Fetal Bovine Serum and cryopreserved in liquid nitrogen before use.

Antigen-specific B cell enrichment

Antigen-specific B cells were enriched using previously described protocols (27–30). For labeling with fluorescent model antigens, 0.2 mL samples containing 1–2 × 10⁸ cells were incubated with 1 pmole of APC, NP650 or PE for 25 minutes on ice in FACS buffer (1x DPBS containing 1% heat-inactivated newborn calf serum) containing 2 μg of anti-Fc receptor antibody 2.4G2 (Bio X Cell) for murine cells, or 2.5% heat-inactivated mouse serum (Thermo Fisher Scientific) and 2.5% heat-inactivated rat serum (Thermo Fisher Scientific) for human cells. For antigen tetramer labeling, cells were first incubated with 1–3 pmole of tetramer containing a control antigen conjugated to PE650, PE594 and/or APC755 for 10 minutes on ice prior to incubation with 1 pmole of APC- or PE-conjugated antigen tetramer for 25 minutes on ice.

After the incubation, 15 mL of FACS buffer was added and the sample centrifuged at 300xg for five minutes at 4°C. The supernatant was discarded and the pellet resuspended prior to the addition of 25 μL of anti-APC and/or 25 μL of anti-PE microbeads (Miltenyi Biotec). Following a 15 – 30 minute incubation on ice, 5 mL of FACS buffer was added and the sample was passed over a magnetized LS column (Miltenyi Biotec). The tube and column were washed once with FACS buffer and then removed from the magnetic field. Five mL of FACS buffer was pushed through the column with a plunger twice to elute column-bound cells.

Flow Cytometry and Cell Counts

Cells from the column-bound fraction and 1/40th of the column flow through fractions were incubated in 50 μL of FACS buffer containing a cocktail of antibodies and a 0.05 μL/mL fixable viability dye (FVD) eFluor 506 (eBioscience), FVD eFluor 780 (eBioscience), or Ghost Dye Violet 510 (Tonbo Biosciences) for 30 minutes on ice. Antibodies for murine experiments included various combinations of 2 μg/mL GL7 Fitc (GL7, BD Biosciences), 2 μg/mL anti-IgD PerCP-Cy5.5 (11–26c.2a, BD Biosciences), 4 μg/mL anti-IgM^b BV650 (AF6–78, BD Biosciences), 4 μg/mL anti-Igλ BV421 or BUV395 (R26–46, BD Biosciences), 2 μg/mL anti-Igκ BV605 (187.1, BD Biosciences), 2 μg/mL anti-CD9 PE (eBioKMC8, eBioscience), 2 μg/mL anti-CD19 BUV395 or BUV737 (1D3, BD Biosciences), 2 μg/mL anti-CD21 PE-Cy7 (eBio8D9, eBioscience), 2 μg/mL anti-CD23 BV786 (B3B4, BD Biosciences), 2 μg/mL anti-CD38 Pacific Blue (90, BioLegend), 2 μg/mL anti-B220 BV711 or BV786 (RA3–6B2, BD Biosciences), 2 μg/mL anti-CD45.1 PE-eFluor 610 (A20, eBioscience), 2 μg/mL anti-CD45.2 PerCP-Cy5.5 (104, eBioscience), 2 μg/mL anti-CD45.2 PE-Cy7 (104, BioLegend), 4 μg/mL anti-CD79b BUV661 (HM79b, BD Biosciences), 2 μg/mL anti-CD93 BV421 or BV650 (AA4.1, BD Biosciences), 2 μg/mL anti-CD3 BV510 (145–2C11, BD Biosciences), 2 μg/mL anti-F4/80 BV510 (BM8, BioLegend), and 2 μg/mL anti-Gr-1 BV510 (RB6–8C5, BD Biosciences). Following surface staining, samples were centrifuged for five minutes at 300xg and supernatant discarded. For intracellular Ig assessment, samples were resuspended and incubated with 250 μL of Cytofix/Cytoperm (BD Biosciences) for 30 minutes on ice. Four mL of Permeabilization buffer (BD Biosciences) was added and samples were centrifuged for five minutes at 400xg and supernatant discarded. Samples were resuspended with 40 μg/mL F(ab’)2 goat anti-Ig H+L Alexa Fluor 350 and incubate for 30 minutes on ice. Four mL of Permeabilization buffer (BD Biosciences) was added and samples were centrifuged for five minutes at 400xg and supernatant discarded.

For human PBMC experiments, cells were labeled with various combinations of 6.24 μg/mL anti-IgM FITC (G20–127, BD Biosciences), 0.3 μg/mL anti-IgD PerCP-Cy5.5 (IA6–2, BD Biosciences), 25 μg/mL anti-Igλ Pacific Blue (MHL-38, BioLegend), 2.5 μg/mL anti-Igκ BV786 (G20–193, BD Biosciences), 50 μL/mL of anti-CD10 BUV395 (HI10a, BD Biosciences), 20 μL/mL of anti-CD19 PE-Cy7 (HIB19, BD Biosciences), 100 μL/mL of anti-CD19 BUV496 (SJ25C1, BD Biosciences), 20 μg/mL anti-CD20 eFluor 450 (2H7, eBioscience), 25 μL/mL anti-CD20 BUV395 (2H7, BD Biosciences), 50 μL/mL of anti-CD27 BV650 or BV480 (L128, BD Biosciences), 20 μg/mL anti-CD79b APC/Fire750 (MHL-38, BioLegend), 60 μL/mL of anti-CD3 BV711 (UCT1, BD Biosciences), 60 μL/mL of anti-CD14 BV711 (MφP9, BD Biosciences), and 2.8 μg/mL of anti-CD16 BV711 (3G8, BioLegend). Following surface staining, samples were centrifuged for five minutes at 300xg and supernatant discarded. Human samples were resuspended with 250 μL of Cytofix/Cytoperm (BD Biosciences) or 2% paraformaldehyde and incubated for 15 minutes on ice prior to the addition of 4 mL of FACS buffer. Human samples were centrifuged for five minutes at 400xg, and supernatant discarded.

Prior to flow cytometry, both human and murine samples were resuspended in FACS buffer containing 20,000 Fluorescent AccuCheck counting beads (Thermo Fisher Scientific) to calculate cell numbers. Flow cytometry was performed on a 5-laser (355nm, 405nm, 488nm, 561nm, 640nm) LSR II, LSRFortessa or FACSymphony (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR).

Sorting of Nur77^eGFP-expressing cells

Naïve follicular B cells were enriched from pooled spleen and lymph nodes from 18–20 mice using a B cell negative selection kit (Miltenyi Biotec, Auburn, CA) supplemented with Biotin-conjugated anti-CD93 (BD Biosciences) to eliminate transitional B cells (31). The B cells were washed in warm RPMI medium, adjusted to a final concentration of 2 × 10⁷ cells/mL in pre-warmed RPMI medium, and incubated with 5 μM CellTrace Violet (CTV) (Thermo Fisher Scientific) for 12 minutes at 37°C prior to the addition of media containing 10% newborn calf serum. Following washing, cell surface marker staining was performed as described above. B cells expressing different levels of Nur77^eGFP were sorted by a FACS Aria II cell sorter (BD Biosciences) using SSC-H/SSC-W-based duplet discrimination, followed by gating on CD9⁻ CD3⁻ F4/80⁻ Gr-1⁻ FVD eFluor 780⁻. CD9 was utilized to exclude marginal zone cells (32).

In vitro stimulation

Purified CTV-labeled B cells were adjusted to a concentration of 2×10⁶ cells/mL in RPMI (ThermoFisher Scientific, Logan, UT) containing 10% fetal bovine serum (ThermoFisher Scientific), 100 units/mL penicillin (Thermo Fisher Scientific), 100 μg/mL streptomycin (Thermo Fisher Scientific), 2 mM L-glutamine (ThermoFisher Scientific), and 27.5 μM 2-mercaptoethanol (Millipore Sigma). Five mL of cells were added per well of a 6-well flat bottomed plate and cultured in the presence or absence of 1–10 μg/mL F(ab’)₂ goat anti-mouse Ig H+L (Jackson Immunoresearch) for 72 hours at 37°C. Following incubation, cell surface marker staining was performed as described above prior to analysis of CTV dilution in gated CD19⁺ CD3⁻ Gr-1⁻ F4/80⁻ FVD eFluor 780⁻ cells using flow cytometry.

Adoptive transfer

One-three x 10⁶ of FACS-purified B cells in 0.1 mL of 1x DPBS were injected retro-orbitally into CD45.1⁺ recipient mice. The following day animals were injected subcutaneous in the base of the tail with 50 μL of CFA (Millipore Sigma) mixed 1 to 1 with 1xDPBS with or without 62.5 pmoles of NP650. Seven days following antigen injection, NP650⁺ cells were enriched and CD45.2⁺ CD45.1⁻ donor B cells were assessed using flow cytometry as described above.

Sorting of single Env-specific naïve B cells

Single human B cells were isolated using a FACSARIA II cell sorter (BD Biosciences) following tetramer enrichment and cell surface marker staining using human PBMCs as described above. Specifically, Env-specific B cells were sorted using SSC-H/SSC-W-based duplet discrimination, followed by gating on CD19⁺ CD20⁺ CD3⁻ CD14⁻ CD16⁻ FVD⁻ B cells that bound HIV-1 Env-PE (or APC) tetramers but not PE650 (or PE594 or APC755) tetramers containing control antigens. Single cells were sorted into individual wells of 96-well PCR plates (Eppendorf) containing 10 μL/well of ice-cold lysis buffer containing 0.25 μL 12.5 U RNase out (Thermo Fisher Scientific), 2.5 μL 5x SuperScript IV First Strand Buffer (Thermo Fisher Scientific), 0.625 μL 0.1M DTT (Thermo Fisher Scientific), 0.3125 μL 10% Igepal detergent (Millipore Sigma), and 6.625 μL DEPC treated water. Plates were sealed with adhesive PCR plate seals (Thermo Fisher Scientific), centrifuged briefly and immediately frozen on dry ice before storage at −80°C.

BCR sequencing and analysis

Reverse transcription was performed using SuperScript IV (Thermo Fisher Scientific) as previously described (33, 34). Briefly, 3 μL of reverse transcription reaction mix consisting of 1.5 μL of 50 μM random hexamers (Thermo Fisher Scientific), 0.4 μL 25 mM dNTPs (Thermo Fisher Scientific), 0.5 μL 10 U SuperScript IV RT, and 0.6 μL water was added to each well containing a single sorted B cell in 10 μL lysis buffer and incubated at 50°C for 1 hr. Following reverse transcription, 2 μL of cDNA was added to 19 μL of PCR reaction mix containing 0.2 μL 0.5 U HotStarTaq Polymerase (Qiagen), 0.075 μL 50 μM 3’ reverse primers, 0.115 μL 50 μM 5’ forward primers, 0.24 μL 25 mM dNTPs, 1.9 μL 10x Buffer (Qiagen), and 16.5 μL water. The PCR program for IgM/IgG and Igκ was 50 cycles of 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 55 seconds, followed by 72°C for 10 minutes. The PCR program for Igλ was 50 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 55 seconds, followed by 72°C for 10 minutes. After the first round of PCR, 2 μL of the PCR product was added to 19 μL of the second round PCR reaction so that the final reaction contained: 0.2 μL 0.5 U HotStarTaq Polymerase, 0.075 μL 50 μM 3’ reverse primers, 0.075 μL 50 μM 5’ forward primers, 0.24 μL 25 mM dNTPs, 1.9 μL 10x Buffer, and 16.5 μL water. PCR programs were the same as the first round of PCR. Five μL of the PCR product was run on an agarose gel to confirm the presence of a ~500 base pair heavy chain band or 450 base pair light chain band. Five μL from PCR reactions showing the presence of heavy or light chain amplicons was mixed with 2 μL of ExoSAP-IT (Thermo Fisher Scientific) and incubated at 37°C 15 min, followed by 80°C for 15 min to hydrolyze excess primers and nucleotides. Hydrolyzed second round PCR products were sequenced by Genewiz with the respective reverse primer and sequences were analyzed using IMGT/V-Quest to identify V, D and J gene segments. Sequences with more than five mutations or less than 85% V gene similarity were excluded from Supplemental Table 1.

Expression of human BCRs

Paired heavy chain VDJ and light chain VJ sequences that lacked signs of somatic hypermutation from Env-specific or control B cells of unknown specificity were cloned into pTT3-derived expression vectors containing the human IgG1, Igκ or Igλ constant regions (25) using In-Fusion cloning (Clontech) and sequences confirmed using Sanger sequencing (Genewiz). Expression vectors were transfected into 293F cells and antibodies purified from culture supernatant using protein A columns (Thermo Fisher Scientific) followed by media exchange into 1x DPBS in house or by the Fred Hutch Antibody Development Shared Resource.

Bio-Layer Interferometry (BLI) Analysis

BLI using the Octet.Red instrument (Forte Bio) were performed at room temperature with shaking at 1,000 RPM following the manufacturer’s instructions. Briefly, 100 μg/mL of cloned antibodies were loaded on anti-human IgG Fc capture biosensors (Forte Bio) for 240 seconds followed by a wash step in kinetics buffer (1X DPBS, 0.01% BSA, 0.02% Tween 20, and 0.005% NaN_3, pH 7.4) for 60 seconds. After washing, 1 μM HIV-1 Env was associated for 300 seconds followed by dissociation into kinetics buffer for an additional 300 seconds. The HIV-1 envelope-specific antibody VRC01 was used as a positive control in all experiments, and the RSV-specific antibody palivizumab was used as a negative control.

HEp-2 binding assay

The ZEUS IFA ANA HEp-2 Test System was used following the protocol recommended by the manufacturer (Zeus Scientific) with minor changes. Briefly, HEp-2-coated slides from the kit were incubated with 25 μL of antibody at 0.1 mg/mL for 30 minutes at room temperature. Slides were then washed three times in 1x DPBS followed by incubation of 25 μL of 0.01 mg/mL goat anti-human IgG Alexa Fluor 594 (Thermo Fisher Scientific) in 1x DPBS for 30 minutes at room temperature in the dark. After washing three times with 1x DPBS, slides were coated in 50% glycerol and coverslips were applied. The EVOS Cell Imaging System (Thermo Fisher Scientific) was used to acquire images, which were analyzed using ImageJ to determine the average Alexa Fluor 594 fluorescence per HEp-2 cell.

Statistical Analysis

Prism software (GraphPad) was used to calculate p values using an unpaired two-tailed t test, a ratio paired two-tailed t test, or one-way ANOVA followed by Dunnett’s multiple comparison test.

RESULTS

Nur77^eGFP expression by naïve murine B cells specific for model antigens

To assess the degree to which cross-reactivity with self-antigen could exist within polyclonal naïve B cells specific for foreign antigens, we measured Nur77^eGFP expression in rare naïve follicular antigen-specific B cells using previously described antigen-specific enrichment approaches (27–30). In our initial experiments, we analyzed B cells specific for a complex model antigen we called NP650, which contains four 4-hydroxy-3-nitrophenyl (NP) molecules bound to streptavidin-allophycocyanin (APC) and ~6 molecules of DyLight 650. In Nur77^eGFP transgenic mice, a relatively large population, ~0.4%, of naïve follicular B cells bound NP650, which was increased to ~18% following enrichment using anti-APC microbeads (Fig. 1A, B). The binding of NP650 was confirmed to be BCR-mediated, since MD4 Rag1^−/− B cells expressing a transgenic BCR specific for hen egg lysozyme did not bind this antigen above the limit of detection even with enrichment (Fig. 1B). Additionally, we confirmed that immunization with NP650 in CFA resulted in activation and differentiation of B cells specific for NP, APC and DL650 when these populations were assessed individually (Fig. S1).

Figure 1. Expression of Nur77^eGFP in naïve follicular B cells specific for a model antigen.

(A) Representative flow cytometric analysis of CD19⁺ B220⁺ CD23⁺ CD21/35^MID CD3⁻ Gr-1⁻ F4/80⁻ FVD⁻ follicular B cells. (B) Representative flow cytometric analysis of NP650 binding to follicular B cells from pooled spleen and lymph node samples from MD4 Rag1^−/− and Nur77^eGFP transgenic animals with or without enrichment of NP650-binding cells with APC-specific microbeads prior to analysis. The fraction “depleted” of NP650-binding cells with anti-APC microbeads is also shown. (C) Nur77^eGFP expression by NP650⁺ and NP650^NEGATIVE follicular B cells from Nur77^eGFP and GFP^NEGATIVE control. (D) Combined data from seven experiments displaying the gMFI of Nur77^eGFP within NP650⁺ follicular B cells normalized to the NP650^NEGATIVE follicular B cells from individual mice using enrichment (n=18) or following incubation with 5 nM of NP650 at 37°C (n=6) for 3 hours. (E) Nur77^eGFP gMFI of NP650⁺ follicular B cells from individual samples (n=5) where one fraction was enriched for NP650 as described in panel B, and a second fraction was labeled with NP650 immediately before analysis. The line connects matched samples from two combined experiments. (F) Representative gating of B cells binding low, mid and high levels of NP650. (G) Combined data from four experiments displaying the Nur77^eGFP gMFI of follicular B cells binding low, mid and high levels of NP650 normalized to the NP650^NEGATIVE B cells from individual mice using the enrichment protocol on ice or following incubation at 37°C (n=6) for three hours. The bars in D and G represent the means and p values (*, p<0.002; ***, p<0.0001) were determined using an unpaired two-tailed t test.

We next compared the level of Nur77^eGFP in naïve follicular B cells that bound NP650 compared to cells that were NP650^NEGATIVE (Fig. 1C). This analysis revealed a roughly 1:1 ratio of Nur77^eGFP geometric mean fluorescence intensities (gMFI) between the two populations (Fig. 1D). These results raised the concern that the enrichment methodology resulted in an upregulation of Nur77^eGFP expression. For the enrichment protocol, fluorescent antigens are bound to BCRs expressed by antigen-specific B cells, followed by magnetic enrichment using microbeads specific for the fluorochrome. While the labeling and enrichment procedure is carried out largely on ice, it is possible that BCR signaling and subsequent Nur77^eGFP upregulation could occur during short portions of this 2–3 hour procedure when the cells are not on ice. Against this notion, Nur77^eGFP was not upregulated in enriched NP650-specific B cells compared to a portion of the sample that was analyzed immediately following NP650 labeling without enrichment (Fig. 1E). In contrast, Nur77^eGFP upregulation was present in NP650-specific B cells if the samples were incubated with NP650 at 37°C for three hours (Fig. 1D). Nur77^eGFP expression was correlated with the level of NP650 bound by the cells when the samples were incubated at 37°C for three hours, but not when samples were kept on ice (Fig. 1F, G). Together, these data indicate that Nur77^eGFP expression is not altered by antigen-specific enrichment, and instead reflective of in vivo BCR signaling.

We next assessed whether B cells specific for individual components of NP650 expressed either high or low levels of Nur77^eGFP. This did not appear to be the case since the NP-specific and APC-specific naïve follicular B cells expressed indistinguishable Nur77^eGFP gMFI and robust coefficient of variation (rCV) compared to their counterparts that did not bind these antigens (Fig. 2A–D). These results appear to conflict with the results of a recent study that found low Nur77^eGFP levels in NP-specific B cells utilizing a transgenic heavy chain paired to endogenously produced lambda (Igλ) light chains (18). In agreement with this report, we found that Nur77^eGFP levels were slightly lower in NP-specific naïve follicular B cells utilizing Igλ light chains (Fig. 2E, F). This did not appear to be unique to NP-specific cells since Nur77^eGFP expression was slightly lower in all naïve follicular B cells utilizing Igλ light chains, including those specific for the model antigen 2,4-Dinitrophenol (DNP) (Fig. 2E, F). Lower Nur77^eGFP expression in B cells utilizing Igλ light chains likely resulted from slightly lower surface BCR expression compared to their counterparts utilizing Igκ light chains, which was detected when surface levels of CD79b (Igβ) were assessed (Fig. 2G, H). Similarly, human Igλ⁺ naïve follicular B cells expressed slightly lower CD79b levels compared to their Igκ⁺ counterparts (Fig. S2). Differences in Nur77^eGFP expression between B cells expressing Igλ and Igκ light chains were eliminated when normalized to the level of surface BCR (Fig. 2I, J).

Figure 2. Expression of Nur77^eGFP in naïve follicular B cells specific for model antigens.

Representative flow cytometric analysis of (A) APC-specific and PE-specific B cells, and (B) DNP and NP-specific B cells within CD19⁺ B220⁺ CD23⁺ CD21/35^MID CD3⁻ Gr-1⁻ F4/80⁻ FVD⁻ follicular B cells from pooled spleen and lymph node samples from Nur77^eGFP transgenic animals enriched with APC-specific and PE-specific microbeads prior to analysis. The control PE594 and APC755 tetramers are included in B in order to gate out B cells specific for streptavidin, APC and PE (28). The percentage of B cells in the enriched fraction that are within each gate are shown on each plot and the bold percentages represent the frequency of B cells binding the tetramer within the entire unfractionated sample. (C & D) Combined data from 14 experiments displaying the (C) gMFI and (D) rCV of Nur77^eGFP within the listed antigen-specific follicular B cells normalized to antigen^NEGATIVE follicular B cells from individual IgH^b or congenic IgH^a mice (n=8–18) within the enriched fraction. (E) Representative flow cytometric analysis of Igκ and Igλ expression by NP-specific, DNP-specific and antigen^NEGATIVE follicular B cells. (F) Combined data from two experiments displaying the gMFI of Nur77^eGFP within the listed population of Igκ⁺ or Igλ⁺ follicular B cell population from individual mice (n=8) normalized to antigen^NEGATIVE follicular B cells. (G) Representative flow cytometric analysis and (H) gMFI of surface CD79b expression on Igκ⁺ or Igλ⁺ B cells. Lines in H connect data points from individual mice (n=10) from two experiments. (I) Representative flow cytometric analysis and (J) gMFI of Nur77eGFP by Igκ⁺ or Igλ⁺ B cells when normalized to the level of CD79b expressed by each cell. Lines in J connect data points from individual mice (n=8) from two experiments. The p values (*, p<0.05; **, p<0.01; ***, p<0.001) in C and F were determined using an unpaired two-tailed t test and H using a ratio paired two-tailed t test.

Overall, the expression of Nur77^eGFP in DNP-specific naïve follicular B cells was also similar to the DNP^NEGATIVE population (Fig. 2B–D). Unexpectedly, increased expression of Nur77^eGFP was detected in naïve follicular B cells specific for the model antigen R-phycoerythrin (PE) (Fig. 2A, C). Previous work has demonstrated that the PE-specific population is large and dominated by a large fraction of cells utilizing V_H1–81 in IgH^b mouse strains such as C57BL/6 (35). The increased expression of Nur77^eGFP appeared linked to the usage of V_H1–81 since the PE-specific population from IgH^a mice expressed similar levels of Nur77^eGFP compared to the APC-specific and antigen^NEGATIVE populations (Fig. 2C). Together, these data suggest that a range of cross-reactivity with self-antigens is common within populations of B cells specific for foreign antigens.

Nur77^eGFP expression by naïve murine B cells specific for antigens derived from human pathogens

We next considered whether these results were applicable to antigens derived from human pathogens. For this, we examined the populations of B cells specific for an influenza hemagglutinin (HA) stem antigen (26), an HIV-1 envelope (Env) antigen called 426C^TM4ΔV1-V3 (25), and antigens representing the prefusion and postfusion forms of the RSV F protein (24, 36) using tetramer enrichment. We found that the follicular B cells specific for influenza HA stem, HIV-1 Env, RSV prefusion F, and postfusion F expressed a similar level of Nur77^eGFP (Fig. 3). These data suggested that a range of self-antigen cross-reactivity is a common feature amongst populations of naïve B cells specific for pathogen-derived antigens.

Figure 3. Expression of Nur77^eGFP in naïve follicular B cells specific for pathogen-derived antigens.

Representative flow cytometric analysis of (A) Influenza HA stem-specific and HIV-1 Env-specific B cells, and (B) RSV prefusion F-specific and postfusion F-specific B cells within CD19⁺ B220⁺ CD23⁺ CD21/35^MID CD3⁻ Gr-1⁻ F4/80⁻ FVD⁻ follicular B cells from pooled spleen and lymph node samples from Nur77^eGFP transgenic animals enriched with APC-specific and PE-specific microbeads prior to analysis. The control PE594 and APC755 tetramers are included in order to gate out B cells specific for purification tags, trimerization domains, streptavidin, APC and PE (28). The percentage of B cells in the enriched fraction that are within each gate are shown on each plot and the bold percentages represent the frequency of B cells binding the tetramer within the entire unfractionated sample. (C & D) Combined data from four experiments displaying the (C) gMFI and (D) rCV of Nur77^eGFP within the listed antigen-specific follicular B cells normalized to the antigen^NEGATIVE follicular B cells from individual mice (n=8) within the enriched fraction.

Diminished proliferation of naïve B cells expressing high levels of Nur77^eGFP in vitro

B cells expressing BCRs able to bind self-antigens often exhibit reduced responsiveness to BCR stimulation (4–10). Previous work demonstrated that calcium signaling in response to BCR stimulation was reduced in B cells expressing high levels of Nur77^eGFP compared to cells expressing low levels (15). Here, we assessed whether this reduced calcium flux was mirrored by reduced proliferation of FACS-purified mature follicular B cells expressing high levels of Nur77^eGFP following BCR stimulation with anti-Ig in vitro. For this, we incubated FACS-purified naïve B cells expressing high, mid and low levels of Nur77^eGFP with anti-Ig for three days in vitro and measured CellTrace Violet (CTV) dilution. In these experiments, we found that 39% of cells expressing high levels of Nur77^eGFP had diluted CTV following stimulation with 2 μg/mL anti-Ig for three days in vitro (Fig. 4A–C). In contrast, 61% of B cells expressing low levels of Nur77^eGFP had low CTV levels in response to the same amount of anti-Ig (Fig. 4B–C). In contrast, 44% of B cells expressing moderate levels of Nur77^eGFP had low CTV levels in response to the same amount of anti-Ig (Fig. 4B–C). These differences in CTV dilution were diminished as the concentration of anti-Ig was increased (Fig. 4C), indicating that strong BCR stimulation could overcome proliferative defects. Similar differences in proliferation were detected when B cells binding NP650 were examined within populations expressing high or low levels of Nur77^eGFP (Fig. 4D). These data suggest that Nur77^eGFP expression is a surrogate for reduced function in B cells specific for foreign antigens.

Figure 4. In vitro proliferation of naïve follicular B cells expressing different levels of Nur77^eGFP.

(A) Representative flow cytometric analysis of FACS-purified Nur77^eGFP-Low, FACS-purified Nur77^eGFP-Mid and Nur77^eGFP-High naïve follicular B cells and the unsorted fraction of B cells. (B) B cells expressing different levels of Nur77^eGFP were FACS-purified and labeled with CTV and cultured in vitro for 72 hours in the presence or absence of 2 μg/mL of anti-Ig prior to flow cytometric analysis of CTV dilution within gated CD19⁺ CD3⁻ Gr-1⁻ F4/80⁻ FVD⁻ B cells. (C) Combined data from three experiments displaying the average frequency ± SD of CTV^LOW cells following in vitro culture with 2, 5, 10, or 25 μg/mL of anti-Ig for 72 hours. (D) Displays the average frequency ± SD of CTV^LOW cells within NP650⁺ B cells from the experiments described in C. The p values (*, p<0.05; **, p<0.01; ***, p<0.001) were determined using an unpaired two-tailed t test.

Diminished response of naïve B cells expressing high and low levels of Nur77^eGFP in vivo

The modest in vitro response of B cells expressing high levels of Nur77^eGFP suggested that these cells may respond poorly to antigen immunization in vivo. To test this, 1–3 × 10⁶ FACS-purified mature CD45.2⁺ follicular B cells that expressed high, moderate, or low levels of Nur77^eGFP were adoptively transferred into CD45.1⁺ recipients one day prior to injection of NP650 in complete Freud’s adjuvant (CFA), or CFA alone. We utilized NP650 for these experiments because the frequency of B cells able to bind this combination antigen is large enough to reliably detect hundreds of these cells within the CD45.2⁺ donor population after transfer (Fig. 5A). Seven days after the injection of CFA alone, the differences in Nur77^eGFP expression were maintained within the three groups of NP650-specific cells (Fig. 5B). Following the injection of NP650 in CFA, the majority of donor NP650-specific B cells in all three groups had fully diluted levels of CTV compared to their counterparts in control mice injected with CFA alone (Fig. 5C, D), indicating proliferation. However, significantly fewer, 66%, of NP650-specific cells that expressed high levels of Nur77^eGFP had low CTV compared to the 79% of donor cells that expressed low levels of Nur77^eGFP (Fig. 5C, D). Unexpectedly, NP650-specific B cells that expressed moderate levels of Nur77^eGFP contained the highest frequency of CTV^LOW cells, 88% (Fig. 5D). These data suggest that the expression of moderate levels of Nur77^eGFP corresponds with optimal function.

Figure 5. In vivo proliferation of naïve B cells expressing different levels of Nur77^eGFP.

Naïve follicular B cells expressing low, mid or high levels of Nur77^eGFP were FACS-purified, CTV labeled, and adoptively transferred into CD45.1⁺ recipient mice one day prior to subcutaneous injection of 62.5 pmoles NP650 in CFA or CFA alone. Seven days later, pooled spleen and lymph nodes from individual mice were analyzed following simultaneous NP650 and CD45.2-based enrichment. (A) Representative flow cytometric analysis of donor CD45.2⁺ CD45.1⁺ NP650⁺ B cells from enriched fractions. (B) Nur77^eGFP gMFI in donor NP650⁺ B cells in mice seven days after the injection of CFA. Representative of two similar experiments. (C) Representative CTV dilution seven days after the injection of NP650 in CFA or CFA alone within donor NP650⁺ B cells expressing different levels of Nur77^eGFP at the time of transfer (D) Combined data from four experiments showing the percentages of CTV^LOW donor NP650⁺ B cells in each group. (E) Combined data from four experiments showing the percentages of donor B cells that were CTV^HIGH NP650⁺ following injection of NP650 in CFA or CFA alone. (F) Same data as E expressed as a percentage of cells that remained CTV^HIGH NP650⁺ in animals injected with NP650 in CFA compared CFA alone. (G) The percentage of cells that remained CTV^HIGH NP650⁺ is plotted vs the percentage of CTV^LOW cells. The bar graphs in D, E, and F represent the mean ± SD (n=9–12) and p values (*, p<0.05; **, p<0.01; ***, p<0.001) in B, D, E, and F were determined using an unpaired two-tailed t test.

An increase in the percentage of CTV^LOW cells could be a reflection of a higher number of divisions made by the naïve cells that were activated in response to antigen. In these experiments, the responding cells in each group were essentially fully CTV diluted (Fig. 5C), indicating seven or more cell divisions. These data could reflect differences in migration of activated cells out of the spleen and lymph nodes, but the differences in calcium signaling reported previously (15) led us to hypothesize that differences in the frequency of CTV^LOW cells resulted from differences in the number of naïve B cells that participated in the response and divided at least once. The frequency of naïve B cells that failed to divide can be determined by comparing the frequency of antigen-specific donor B cells that are CTV^HIGH in mice injected with and without antigen (30). Notably, naïve CTV^HIGH B cells binding low levels of NP650 were excluded from these analyses because previous experiments revealed that the frequency of these cells was unaltered by NP650 injection (Fig. S3). Within recipient animals receiving Nur77^eGFP-High cells, ~0.19% of donor B cells remained undivided and CTV^HIGH in animals injected with NP650 in CFA, compared to ~0.26% in animals injected with CFA alone (Fig. 5E). While this decrease was not significant, it suggested that ~3/4 (19% / 26%) of Nur77^eGFP-High NP650-specific naïve B cells fail to proliferate in response to immunization (Fig. 5F). In contrast, only 45% of NP650-specific naïve B cells that expressed moderate levels of Nur77^eGFP failed to proliferate (Fig. 5E, F). An intermediate frequency, 61%, of NP650-specific naïve B cells that expressed low levels of Nur77^eGFP failed to proliferate (Fig. 5E, F). Importantly, the average frequencies of naïve B cells that failed to proliferate inversely correlated with the average frequencies of CTV diluted cells (Fig. 5G). Together, these data indicate that naïve foreign antigen-specific cells expressing moderate levels of Nur77^eGFP are more than twice as likely to respond to antigen immunization compared to their counterparts expressing high levels of Nur77^eGFP.

Self-antigen cross-reactivity by human naïve B cells specific for pathogen-derived antigens

Having observed functional impairment in vaccine-relevant populations in mice, we sought to gauge the level of self-antigen cross-reactivity within analogous populations of naïve human B cells specific for pathogen-derived antigens. Unlike Nur77^eGFP expression in mice, high levels of intracellular Nur77 in human B cells does not appear to correlate with self-antigen reactivity (37). Instead, human naïve B cells expressing low levels of IgM have been shown to be enriched for self-antigen-reactive B cells (20, 21), a phenotype commonly exhibited by transgenic B cells specific for self-antigens (38–42). Additionally, both human and murine B cells expressing low levels of IgM have reduced calcium signaling in response to BCR stimulation similar to cells expressing high levels of Nur77^eGFP (15, 20, 21, 43). Overall, naïve follicular murine B cells specific for NP650 or HIV-1 Env expressed indistinguishable levels of IgM compared to cells that did not bind these antigens (Fig. 6A, B). In agreement with published reports (15), naïve B cells of unknown specificity that expressed higher levels of Nur77^eGFP expressed reduced levels of surface IgM (Fig. 6C–E). Similar results were found within murine B cells specific for NP650 or HIV-1 Env (Fig. 6E). Downregulated IgM levels in cells expressing high levels of Nur77^eGFP was mirrored by an overall decrease in surface BCR levels when assessed based upon CD79b staining (Fig. 6F, G). Together, these data suggest that self-antigen binding results in Nur77^eGFP upregulation and a corresponding downregulation of IgM BCRs.

Figure 6. Expression of IgM on naïve follicular B cells specific for model antigens.

Representative flow cytometry analysis and quantitation of (A, B, D, E) IgM and (F, G) CD79b expression by naïve follicular murine B cells specific for NP650 or HIV-1 Env compared to cells that did not bind these antigens. Fluorescence minus one (FMO) controls are displayed for comparison. (C) Representative flow cytometric gating of Nur77^eGFP-Low (red), Nur77^eGFP-Mid (grey) and Nur77^eGFP-High (blue) naïve follicular murine B cells for the analyses conducted in D-G. Data points are from individual mice (B, n=10; E, G, n=8) combined from 2 experiments and p values (*, p<0.05; **, p<0.01; ***, p<0.001) were determined using an unpaired two-tailed t test.

Using antigen tetramer-based enrichment we examined IgM expression in IgD⁺ naïve human B cells specific for HIV-1 Env and RSV F prefusion or postfusion F proteins (Fig. 7A–C). Since most people are naturally infected with RSV early in their lives, we excluded memory B cells and focused upon naïve B cells by gating upon cells that expressed IgD but not CD27 (Fig. 7A). Cells expressing CD10 were also excluded since transitional B cells circulating in the blood express this protein (44). Using this gating strategy, we found that each antigen-specific population expressed a similar level of IgM compared to their counterparts that did not bind antigen (Fig. 7D, E). Similar to murine experiments, low IgM expression was mirrored by low CD79b expression (Fig. 7F, G). These features suggest that populations of B cells specific for foreign antigens exhibit cross-reactivity with self-antigens.

Figure 7. Expression of IgM on naïve human B cells specific for pathogen-derived antigens.

(A) Representative flow cytometric analysis of CD19⁺ CD20⁺ IgD⁺ CD10⁻ CD27⁻ CD3⁻ CD14⁻ CD16⁻ FVD⁻ naïve B cells specific for (B) HIV-1 Env, (C) RSV prefusion F or postfusion F from the fractions enriched using anti-PE and/or anti-APC microbeads from 100 million PBMC. The percentage of B cells in the enriched fraction that are within each gate are shown on each plot and the bold percentages represent the frequency of B cells binding the tetramer within the entire unfractionated sample. (D) Representative IgM expression by HIV-1 Env tetramer-binding naïve B cells compared to Tetramer^NEGATIVE naïve B cells. FMO controls for IgM are also displayed for both populations. (E) Combined data from six experiments displaying the average IgM gMFI within the listed antigen-specific B cell population normalized to the Tetramer^NEGATIVE B cells. Each data point is the average of 2–4 independent assessments of samples from individual donors (n=3–7).

To directly analyze BCR cross-reactivity with self-antigens, we next cloned 27 BCRs from HIV-1 Env-specific B cells and assessed binding to the HEp-2 cell line, a common test of self-antigen reactivity (45). The cloned BCRs were selected from a data set of 103 HIV-1 tetramer-binding B cells from human PBMCs in which paired heavy and light chain sequences were recovered using single cell RT-PCR (Supplemental Table 1). The 27 cloned antibodies exhibited no signs of somatic hypermutation and 24 bound HIV Env above the limit of detection when assessed by Bio-Layer Interferometry (BLI) (Fig. 8A, B). We next assessed self-antigen reactivity of the 24 Env-specific cloned antibodies compared to a set of 18 antibodies cloned from naïve B cells of unknown specificities (Supplemental Table 1) along with an Env-specific positive control, 4E10 (46), and the RSV-specific negative control, palivizumab (47). Using this approach, 8.3% of the Env-specific antibodies bound to HEp-2 cells above background, which was similar to the 11% of antibodies derived from B cells of unknown specificity that also bound (Fig. 8D, E). Together, these data indicate that self-antigen cross-reactivity is a common feature amongst B cells and is composed of a range of affinity within a given population.

Figure 8. Self-antigen cross-reactivity of HIV-1 Env-specific naïve human B cells.

(A) Bio-Layer Interferometry (BLI) measurements of the binding of six antibodies cloned from HIV-1 Env-Tetramer⁺ naïve B cells to purified Env. (B) Frequency of the twenty-seven cloned antibodies that bound to Env above the BLI limit of detection. (C) Representative immunofluorescence images (scale bar, 100 μm) displaying the binding of two Env-specific antibodies derived from naïve B cells, E81G01 and E82A03, to human HEp-2 cells compared to a positive control antibody, 4E10, and a negative control antibody, palivizumab. (D) Mean (± SD) Alexa Fluor 594 fluorescence per HEp-2 cell for 24 Env-specific antibodies and 18 antibodies cloned from naïve B cells of unknown specificity compared to 4E10 and palivizumab. Data was obtained from five independent experiments for each antibody and * denotes antibodies with significantly (*, p<0.01; **, p<0.0001) higher fluorescence compared to palivizumab using one-way ANOVA followed by Dunnett’s multiple comparison test. (E) Percentage of Env-specific and control antibodies from F with significant (p<0.05) binding to HEp-2 cells compared to palivizumab.

DISCUSSION

Overall, our results demonstrate that B cells specific for foreign antigen display a breadth of self-reactivity and diminished function that may limit vaccine responses. We initially hypothesized that different populations of B cells would have different levels of cross-reactivity with self-antigens based upon the unique properties of the epitopes displayed by each antigen. While this could still be true, our data suggest that cross-reactivity with self-antigen is generally present amongst polyclonal BCRs specific for foreign antigens including small haptens, large complex antigens, and even rationally designed immunogens. This suggests that cross-reactivity with self-antigens may have more to do with intrinsic biochemical features of the BCRs themselves rather than similarity between the self- and foreign antigens. There are a number of ways this could occur, for example antibodies using V_H4-34 are known to be more self-reactive compared to other alleles (48–50), whereas the unique CDRH3 regions expressed by BCRs using these alleles can mediate binding to diverse sets of foreign antigens. Antibody cross-reactivity to structurally distinct self- and foreign antigens could also occur if different amino acids within the heavy and light chain CDR3s mediated binding to the two antigens.

Uniquely high cross-reactivity with self-antigen within the PE-specific B cell population was surprising. Cross-reactivity with self-antigens was previously reported within the PE-specific memory B cell population, but this was attributed to SHM in the germinal center (51). Our data suggest that this self-reactivity was present in the naïve population, and either not selected against, or increased in the germinal center. Interestingly, increased levels of Nur77^eGFP were not detected in PE-specific B cells in IgH^a mice, which lack the V_H1–81 allele used by the majority of the large PE-specific B cell population in IgH^b C57/BL6 mice (35). This could suggest that V_H1–81 exhibits a mild level of unrecognized self-reactivity with self-antigens that maintains a higher frequency of PE-specific B cells through increased positive selection similar to results obtained from studies of cells expressing a transgenic heavy chain specific for NP (18).

In our analyses we found slightly lower expression of Nur77^eGFP on B cells utilizing Igλ light chains compared to their Igκ counterparts. While we initially hypothesized that this was the result of Igλ light chains having inherently lower levels of self-antigen reactivity since overall, B cells that express low levels of Nur77^eGFP expressed higher levels of CD79b and IgM. This is likely because binding to self-antigens results in Nur77^eGFP upregulation and subsequent down-regulation of surface BCR. However, our data instead suggests that the slightly lower Nur77^eGFP levels in B cells utilizing Igλ, are instead a result of slightly lower BCR expression rather than slightly lower levels of self-antigen reactivity.

Our results contrast with studies of CD4⁺ and CD8⁺ T cells showing that increased cross-reactivity with self-peptide bound to MHC class I was associated with higher CD5 expression and enhanced responsiveness (52, 53). Instead, we find that there is a “sweet spot” in that B cells with too little or too much cross-reactivity with self-antigen function poorly. Moderate signaling in response to self-antigen may provide the level of tonic BCR signaling known to be essential for B cell survival (54). We hypothesize that the differences between B and T cells are the result of differences in positive and negative selection. If T cells with higher affinity for self-antigens are more effectively deleted from the repertoire, the remaining CD5^HIGH T cells may represent the “sweet spot” of reactivity necessary for optimal function. In contrast, mild reactivity with self-antigens appears to increase positive selection of developing B cells (18). The ability of B cells to undergo somatic hypermutation may reduce the need for stringent deletion of cells expressing BCRs with higher affinity for self-antigen. Indeed, naïve B cells with high affinity for both foreign and self-antigens are likely the source of “redeemed” antibodies that appear to lose self-reactivity as a consequence of somatic hypermutation and affinity in the germinal center (55, 56). Together these data suggest that the benefit of redeeming potentially pathogenic antibodies to become protective may outweigh the risks of maintaining B cells cross-reactive with self-antigens. Since TCRs cannot be somatically mutated and redeemed, the benefits of keeping T cells with cross-reactivity to self-peptides/MHC is lower.

During the preparation of this manuscript it was reported that a population of Nur77^eGFP-high CD4⁺ exhibits reduced function (57) similar to what we and others have observed for Nur77^eGFP-high B cells. These Nur77^eGFP-high T cells appear to comprise only a portion of the CD5^HIGH CD4⁺ T cell populations analyzed previously (52), which may have concealed their reduced function. In light of these new data, B and T cells expressing moderate levels of Nur77^eGFP may represent the “sweet spot” for responsiveness to foreign antigens.

One limitation to our analyses of in vivo responsiveness is that we only measured this for B cells specific for our combination antigen NP650. The reason we did not assess multiple antigen-specific populations is because frequency of individual populations is typically below 0.01% of the total B cell population. Below this level we could not obtain enough donor cells sorted upon Nur77^eGFP expression level to reliably detect naïve antigen-specific B cells that failed to respond to immunization. For this reason, we chose to conjugate NP, streptavidin, APC and Dylight 650 to form NP650. Using this combination antigen, we essentially simultaneously probed the response of multiple naïve populations together. Therefore, our data showing that superior response of the naïve B cells expressing moderate levels of Nur77^eGFP isn’t confided to a single antigen-specific population, but reflective of the larger response targeting all of these antigens. While it is possible that one antigen-specific population is dominating this response and skewing our results, we think it is more likely that an unusual response from any one population of antigen-specific B cells in this combination would be masked by the response of the other populations.

In our experiments we also found that naïve B cells binding lower levels of foreign antigen were less likely to proliferate in response to immunization. In fact, we could find no evidence that the third of naïve B cells binding lowest levels of antigen proliferated using these immunization conditions. These results were not surprising and do not mean that these B cells bind antigen with an affinity below the threshold for activation and are therefore irrelevant. However, it is possible that with increased T cell help, increased antigen availability, antigen multimerization, or different adjuvants could induce these lower binding B cells to participate in a relevant immune response. It will also be interesting to determine whether more low affinity B cells participate in the response to antigens provided by infection rather than antigen immunization. In fact, some infections are able to stimulate the activation of B cells expressing BCRs with affinities below the limit of detection for most assays (58).

Our results indicate that two key affinities help control which cells respond to antigen exposure. First, the BCR affinity for the injected antigen must be high enough to allow activation. However, overlaid on top of this is the BCR affinity for self-antigens, where a moderate amount primes cells for optimal responsiveness. Future work is focused upon the elucidation of other factors that tune the functional potential of naïve B cells.

KEY POINTS.

All populations of foreign antigen-specific B cells cross-react with self-antigens

Moderate cross-reactivity to self-antigen promotes optimal naïve B cell response

Non-standard abbreviations

APC

allophycocyanin

APC755

allophycocyanin-DyLight 755

BLI

Bio-Layer Interferometry

CTV

CellTrace Violet

DNP

2,4-Dinitrophenol

Env

HIV-1 Envelope

FVD

fixable viability dye

gMFI

geometric mean fluorescence intensity

HA

hemagglutinin

NP

4-hydroxy-3-nitrophenyl

NP650

4-hydroxy-3-nitrophenyl-streptavidin-allophycocyanin-DyLight 650

PE

R-phycoerythrin

PE594

R-phycoerythrin-DyLight 594

PE650

R-phycoerythrin-DyLight 650

rCV

robust coefficient of variation

RSV

respiratory syncytial virus

SHM

somatic hypermutation