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. Author manuscript; available in PMC: 2024 Jan 15.
Published in final edited form as: J Immunol. 2023 Jan 15;210(2):148–157. doi: 10.4049/jimmunol.2200538

Type I IFN receptor signaling on B cells promotes antibody responses to polysaccharide antigens

M Ariel Spurrier 1,*, Jamie E Jennings-Gee 1,*, Karen M Haas 1
PMCID: PMC9812919  NIHMSID: NIHMS1849873  PMID: 36458995

Abstract

We previously reported monophosphoryl lipid A (MPL) and synthetic cord factor, trehalose-6,6’-dicorynomycolate (TDCM) significantly increases antibody (Ab) responses to T cell independent type 2 antigens (TI-2 Ags) in a manner dependent on B cell-intrinsic TLR4 expression, as well as MyD88 and TRIF proteins. Given the capacity of MPL to drive type I IFN production, we aimed to investigate the extent to which type I IFN receptor (IFNAR) signaling was required for TI-2 responses and adjuvant effects. Using Ifnar1−/− mice and IFNAR1 Ab blockade, we found IFNAR signaling is required for optimal early B cell activation, expansion, and Ab responses to non-adjuvanted TI-2 Ags, including the pneumococcal vaccine. Further study demonstrated B cell-intrinsic type I IFN signaling on B cells was essential for normal TI-2 Ab responses. In particular, TI-2 Ag-specific B-1b cell activation and expansion were significantly impaired in Ifnar1−/− mice; moreover, IFNAR1 Ab blockade similarly reduced activation, expansion, and differentiation of IFNAR1-sufficient B-1b cells in Ifnar1−/− recipient mice, indicating B-1b cell-expressed IFNAR supports TI-2 Ab responses. Consistent with these findings, type I IFN significantly increased survival of TI-2 Ag-activated B-1b cells ex vivo as well as promoted plasmablast differentiation. Nonetheless, MPL/TDCM adjuvant effects, which were largely carried out through innate B cells (B-1b and splenic CD23− B cells), were independent of type I IFN signaling. In summary, our study highlights an important role for B-1b cell-expressed IFNAR in promoting responses to non-adjuvanted TI-2 Ags, but nonetheless demonstrates adjuvants which support innate B cell responses may bypass this requirement.

Keywords: adjuvant, pneumococcal vaccine, innate B cell, antibody, T cell-independent antigen, type I IFN

Background

Pneumococcal vaccines consist of S. pneumoniae-derived capsular polysaccharides (PPS) which behave as T cell-independent type 2 antigens (TI-2 Ags). Pneumovax®23, composed of 23 types of PPS, provides protection against invasive pneumococcal disease in adults for up to 10 years, with an estimated efficacy of 60-70% (1). Adjuvants for native polysaccharide vaccines have not been used in the clinic. Alum does not boost primary Ab responses to TI-2 Ags (2) and poorly boosts responses to PPS-conjugates (3). Earlier work with Ribi adjuvant, consisting of Salmonella typhimurium monophosphoryl lipid A (MPL) and Mycobacterium cord factor in squalene elicited increased primary PPS-specific Ab responses in mice (4, 5), but is not suitable for use in humans due to its toxicity. We recently demonstrated low-toxicity Salmonella minnesota MPL and synthetic cord factor analog trehalose dicorynomycolate (TDCM) emulsified in squalene significantly increased Ag-specific IgM and IgG levels in response to PPS and haptenated-Ficoll (6). The mechanisms by which this adjuvant carries out its effects have not been fully elucidated but is important for informed design of adjuvants for use with polysaccharide Ags in humans.

We previously reported MPL/TDCM induced type I IFN production in vivo (7), likely through the effects of MPL on TLR4-TRIF activation of type I IFN production (8). Importantly, a previous study demonstrated the TLR3 agonist, poly(I:C), increased Ab responses to NP-Ficoll via a mechanism dependent upon IFNAR signaling and follicular B cells (9). To further understand the mechanisms by which MPL/TDCM increases Ab responses to TI-2 Ags, we investigated the importance of type I IFN and B cell subsets in adjuvant effects. We unexpectedly found a role for B cell-intrinsic IFNAR expression in supporting B cell responses to non-adjuvanted TI-2 Ags. B-1b cells, which express significantly higher levels of IFNAR than B-2 cells, were particularly dependent on type I IFN signaling for optimal activation, survival, expansion, and differentiation in response to TI-2 Ags. However, B-1b cell development did not appear to be impacted by IFNAR deficiency. In contrast to reported adjuvant effects of poly(I:C) on follicular (CD23+) B cell responses to TI-2 Ags, we found MPL/TDCM adjuvant effects were largely carried out through innate B cells (B-1b and CD23− splenic B cells). However, type I IFNAR signaling was not required for MPL/TDCM adjuvant effects. These findings highlight the importance of type I IFN in supporting B-1b cell Ab responses to non-adjuvanted TI-2 Ags, but reveal B cell-activating adjuvants may activate alternative pathways supportive of activation, survival, and differentiation that ultimately overcome the requirement for IFNAR-derived signals.

Methods

Mice

Wild-type (WT), mumt (Ighmtm1Cgn), CD19Cre(B6.129P2(C)-Cd19tm1(cre)Cgn/J) and B1-8hi IgH knock-in (VHB1-8 Tg) mice were on a C57BL/6 background (Jackson Laboratories). Ifnar1−/− and floxed ifnar1fl/fl (Ifnar1tm1Uka) mice were on a C57BL/6 background and kindly provided by Dr. Erik Barton (originally from Drs. Herbert Virgin and Ulrich Kalinke, respectively) and used as previously described (7), with Cd19tm1(cre)Cgn/J mice crossed with Ifnar1fl/fl mice to generate heterogeneous CD19-Cre+/−/Ifnar1fl/+ breeders. These breeders were used to generate male mice deficient for IFNAR on B cells (CD19-Cre+/−/Ifnar1fl/fl), male mice expressing heterozygous IFNAR levels on B cells (CD19-Cre+/−Ifnar1fl/+), and male littermate controls (CD19-Cre+/−) for experiments. Homozygous WT and Ifnar1−/− breeders were used to generate mice for experiments, the majority of which used male mice. Studies including female mice used sex-matching among groups. Studies used age-matched mice housed in an SPF facility and were approved by Wake Forest School of Medicine’s Animal Use Committee.

Immunizations and ELISAs

Mice were immunized with 10 μg TNP65-Ficoll, 1 to 5 μg NP40-Ficoll, or the equivalent of 0.125 to 1 μg each PPS within Pneumovax23 (Merck). In one case, a mixture of PPS (type 3, 4, 6B, 8, 9N, 12F, 14, 19F, 23F; ATCC) was used to generate Pneumovax9 due to a Pneumovax23 vaccine shortage. MAR1-5A3 monoclonal Ab mouse IFNAR-1 (BioXcell) or control mouse IgG (Jackson Immunoresearch) was administered i.p. (200 μg on d0 and 100 μg on d2 and 4 of immunization) to block IFNAR1 signaling where indicated. Adjuvant containing 20 μg Salmonella minnesota MPL, 20 μg trehalose-6,6‘-dicorynomycolate (TDCM) in 0.5% squalene/0.05% Tween-80 or 2% squalene/0.2% Tween-80 (Sigma) for intraperitoneal (i.p.) and intramuscular (i.m.) injections, respectively, was mixed with Ag prior to injection. ELISAs were as previously described (6, 10, 11). TNP- and PPS-specific Ig levels were estimated using a standard curve generated using anti-mouse Ig (H+L) coated wells in conjunction with mouse IgM and IgG isotype standards (Southern Biotechnology Associates). NP-specific Ig concentrations were estimated using NP-specific IgM and IgG standard curves as previously described (11).

Flow cytometry

Peritoneal cells were harvested using 10 ml of DPBS to lavage the peritoneal cavity. Blood was collected in heparin, with PBMC purified using a 1083 Ficoll density gradient (Sigma). Spleen and bone marrow suspensions were lysed in ammonium chloride lysis buffer, followed by washing in staining buffer (PBS containing 2% newborn calf serum). Cells were resuspended in staining buffer and pre-incubated with 0.5 μg/ml FcBlock (eBioscience) and stained with fluorochrome-conjugated mAbs (Biolegend, eBioscience, and BD Biosciences): CD19 (1D3), CD11b (M1/70), CD5 (53-7.3), CD23 (B3B4), CD21/35 (7E9), CD138 (281-2), CD86 (GL1), CD19 (1D3), CD11b (M1/70), CD45R/B220 (RA3-6B2), rat anti-mouse IgG (pooled rat anti-mouse IgG1, IgG2b, IgG2a, IgG3; Southern Biotechnology Associates) and Live/Dead fixable aqua stain. Countbright beads (Thermofisher) were included for enumeration. After staining, samples were washed and fixed in 1.5% buffered formaldehyde. For TNP- and NP-specific B cell staining, cells were incubated with TNP(65)-FL-AECM-Ficoll (20μg/ml; Biosearch Technologies Inc.) or NP40-allophycocyanin and anti-mouse CD45.1 (12). Intracellular Ki-67 (solA15) and rabbit anti-active Caspase-3 (BD Biosciences) staining was performed according to manufacturer’s instructions (eBioscience™ Fix/Permeabilization kit). Cells were analyzed using a BD LSR Fortessa X20 (Becton Dickinson) and data was analyzed using FlowJo analysis software (Tree Star).

Bone marrow chimeras

WT recipient mice were lethally irradiated (950 rad; single dose Cesium137 gamma irradiator) and reconstituted i.v. with 107 total bone marrow cells consisting of muMT bone marrow mixed with either WT or Ifnar1−/− bone marrow in a 90:10 ratio five to six hours later, as previously described (13). Recipient mice were maintained on Septra 1 week prior to irradiation and 2 weeks afterwards. Mice were rested for four weeks prior to immunization.

Adoptive transfer experiments

Naïve splenic and peritoneal B cells were purified from VHB1-8 Tg mice using CD43 bead depletion (Dynal) since CD43 is largely lacking on naïve NP-specific B1b, marginal zone and follicular B cells from these mice (11). CD23+ and CD23− B cells were further purified using Miltenyi beads. B-1b cells were further enriched using CD11b Miltenyi beads. VHB1-8 Tg B cells were transferred i.v. into CD45.2+ mice unless otherwise indicated. Naïve spleen B cells were purified from WT mice in a similar manner. WT peritoneal B cells were obtained using EasySep pan B cell isolation (StemCell) combined with biotinylated anti-F4/80 and CD23 mAbs. A second purification step was used for B-1b cell enrichment using biotinylated Abs against CD5, F4/80, and GR1 in conjunction with streptavidin Dynabeads (>90% CD5−CD19+ B cell purity; 70-85% CD11b+). Recipient muMT mice were reconstituted with cells i.v.

Ex vivo cultures of VHB1-8 Tg B cells

Naïve CD43− VHB1-8 Tg spleen (1 x 107) and peritoneal (5 x 105) B cells were purified as described above and adoptively transferred into Ifnar1−/− recipient mice. Mice were immunized with 25 μg NP40-Ficoll the next day. Splenocytes were harvested and placed in culture with media alone or with type I IFNα/β (600 U/ml; Murine Interferon Alpha/Beta, NR-3082 obtained through BEI Resources, NIAID, NIH). Twenty-four hours later, cells were harvested and stained.

Statistical analyses

Data are shown as means ± SEM. Unless indicated otherwise, differences between sample means were assessed using Student’s t test. Where indicated, comparisons among multiple groups were made using one-way ANOVA with Tukey’s post hoc test and comparisons over time used repeated measures two-way ANOVA (Prism7 software).

Results

Type I IFN signaling is required for optimal Ab responses to TI-2 Ags

Before investigating the role of type I IFN signaling in MPL/TDCM adjuvant effects, we first investigated whether type I IFN played a role in regulating Ab responses to TI-2 Ags. As shown in Fig. 1A, mice lacking IFNAR1 and thus functional IFNAR (Ifnar1−/−) exhibited a significant reduction (~30%) in TNP-specific IgM and IgG levels 7 days post TNP-Ficoll immunization, although responses approximated WT levels at later time points. However, in response to Pneumovax23 immunization, Ifnar1−/− mice produced significantly less (50%) IgM and IgG to serotype 3 polysaccharide (PPS3) found within the vaccine as well as significantly less IgM and IgG reactive with total Pneumovax23, and these differences were sustained (Fig. 1B–C). Administration of an IFNAR1 blocking mAb to WT mice at the time of immunization was also found to significantly reduce PPS3 and Pneumovax23-specific IgM responses 5 days post immunization, and PPS-specific IgG levels, although low, were also reduced (Fig. 1D). Thus, IFNAR supports optimal TI-2 Ab responses, and is particularly important for optimal IgM and IgG responses to pneumococcal polysaccharides.

Figure 1. Type I IFN receptor signaling is required for optimal Ab responses to TI-2 Ags.

Figure 1.

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A-C) WT and Ifnar1−/− mice were immunized with 10 μg TNP65-Ficoll or Pneumovax23 containing 1 μg each PPS. Serum IgM and IgG levels reactive with TNP-BSA (A), PPS3 (B), and whole Pneumovax23 (C) were determined by ELISA (n≥5 mice/group). Results are representative of those obtained in 4 independent experiments. D) WT mice (n=4-5 mice/group) were immunized with a mixture of 0.125 μg each PPS (3, 4, 6B, 8, 9N, 12F, 14, 19F, 23F; “Pneumovax9”) on day 0. Mice received an IFNAR1 blocking mAb or control mouse IgG i.p. (200 μg on day 0 and 100 μg on d2 and 4) and PPS3- and whole Pneumovax9-specific IgM and IgG levels were measured on day 5. Asterisks (*) indicate significant differences between Ab levels between groups.

B cell subset development is normal in Ifnar1−/− mice

As alterations in innate B cell subsets can lead to defective TI-2 Ab responses, we assessed B cell subsets in Ifnar1−/− mice. As shown in Fig. 2, we found total B cell frequencies and numbers in spleen and peritoneal cavity were similar between WT and Ifnar1−/− mice. Furthermore, we did not find differences in B cell subsets. Peritoneal B-2 cell and spleen follicular B cell frequencies and numbers were similar in WT and Ifnar1−/− mice as were innate peritoneal B-1a and B-1b and splenic marginal zone B cell frequencies and numbers (Fig. 2). Splenic B-1a cell frequencies and numbers were also similar between WT and Ifnar1−/− mice (Supplemental Fig. 1A). Thus, B-1 and B-2 B cell subset development and maintenance appears normal in Ifnar1−/− mice.

Figure 2. Normal B cell subset distribution in IFNAR−/− mice.

Figure 2.

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Spleen and peritoneal B cell subsets were analyzed in naïve WT and Ifnar1−/− mice. The mean frequency of total spleen B (CD19+), follicular B (FOB:CD21intCD23+), marginal zone B (MZB: CD21hiCD23−) and CD21loCD23lo cells among total leukocytes and numbers are indicated in the top panels. The frequency of total peritoneal B (CD19+), B-2 (CD11b−), B1 (CD11b+), B1a (CD11b+CD5+), and B1b (CD11b+CD5−) cells among total leukocytes and numbers are indicated in the bottom panels (n=5 mice/group).

Type I IFN signaling on B cells is required for optimal Ab responses to TI-2 Ags

IFNAR expression is ubiquitous and hence, altered TI-2 Ab responses in Ifnar1−/− mice could be due to lack of IFNAR on one or more cell types. We investigated the importance of B cell-expressed IFNAR in TI-2 Ab responses by crossing Ifnar1fl/fl mice with CD19-Cre transgenic mice to generate mice lacking IFNAR only on CD19-expressing B cells (CD19-Cre+/−Ifnar1fl/fl) or expressing heterozygous IFNAR expression on B cells (CD19-Cre+/−Ifnar1fl/+). As shown in Figure 3, CD19-Cre+/− mice produced lower Ab responses to TNP-Ficoll and PPS relative to WT mice and were therefore used to draw comparisons with CD19-Cre+/−Ifnar1fl/fl mice. In response to TNP-Ficoll, CD19-Cre+/−Ifnar1fl/fl mice produced significantly less TNP-specific IgG relative to CD19-Cre+/− and CD19-Cre+/−Ifnar1fl/+ mice (Fig. 3A). In response to Pneumovax23, CD19-Cre+/−Ifnar1fl/fl mice produced significantly less PPS3-specific IgM, and PPS3- and Pneumovax23-specific IgG levels were 50% decreased relative to CD19-Cre+/− mice (Fig. 3B–C). Interestingly, CD19-Cre+/−Ifnar1fl/+ mice expressing heterozygous IFNAR levels on B cells produced TI-2 Ab levels similar to CD19-Cre+/− mice, suggesting heterozygous IFNAR expression on B cells is sufficient to support B cell responses to TI-2 Ags, whereas selective deficiency of IFNAR on B cells results in impaired Ab responses to TI- 2 Ags.

Figure 3. Type I IFN receptor signaling on B cells is required for optimal Ab responses to TI-2 Ags.

Figure 3.

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A-C) WT, CD19-Cre+/−, CD19-Cre+/−Ifnar1fl/fl, and CD19-Cre+/−Ifnar1fl/fl mice were immunized as in Figure 1, with serum IgM and IgG levels against TNP (A), PPS3 (B), and total Pneumovax (C) examined on day 21. Asterisks (*, p<0.05) indicate significant differences in values as compared to CD19-Cre+/− mice as assessed by one-way ANOVA with Tukey’s post-hoc analysis. D-F) Irradiated WT mice reconstituted with mixed bone marrow from WT and mumt mice (10:90) or Ifnar1−/− and mumt mice (10:90) were rested for four weeks and then immunized as in A-C, with TNP (D), PPS3 (E), and Pneumovax (F)-specific IgM and IgG assessed on day 27 post immunization (n=7/group). G) WT and Ifnar1−/− mice received 5 x 106 VHB1-8 Tg spleen B cells i.v. and 5 x105 peritoneal cavity B cells i.p. Recipients were immunized with 25 μg NP-Ficoll i.p. one day later and NP-specific IgG levels were assessed (n=4-5 mice/group). Results representative of 2 experiments.

We complemented the above studies by generating radiation bone marrow chimeras. Although bone marrow chimeras may not support complete B cell reconstitution (e.g., fetal-derived B-1 cells), bone marrow progenitors support the eventual development of splenic MZ B cell and peritoneal B-1b cell populations, and to a much lesser extent, B-1a cells ((14, 15) and Supplemental Fig. 1B). PPS-specific Ab responses are therefore reconstituted in bone marrow chimeras, albeit to a lower extent than elicited in non-irradiated animals (ref.(6) and Fig. 3). Given the reconstitution of TI-2 specific Ab responses in bone marrow chimeras, we assessed responses in chimeras constructed by reconstituting irradiated WT mice with bone marrow from B cell-deficient mumt mice mixed with either bone marrow from WT mice or Ifnar1−/− mice (90:10 ratio). As shown in Figure 3D, chimeras with B cells lacking IFNAR produced significantly less IgG (30% reduced) in response to TNP-Ficoll than chimeras reconstituted with WT B cells. IgM and IgG responses to PPS3 were also decreased 3 to 4-fold in chimeras with IFNAR-deficient B cells and in Pneumovax-specific ELISAs, IgG was barely detectable in these chimeras (Fig. 3E–F). Thus, mice with selective IFNAR deficiency on B cells exhibit impaired IgM and IgG responses to TI-2 Ags to a degree similar to mice lacking IFNAR on all cells. Finally, reconstitution of Ifnar1−/− mice with B cells from IFNAR+/+ VHB1-8 Tg mice in which a fraction of B cells (5-10%) co-express the λ1 chain with the VHB1-8 transgene to yield a high affinity receptor specific for NP (11) yielded NP-specific IgG responses that were similar to WT recipients (Fig. 3G), indicating loss of non-B cell IFNAR expression did not noticeably impact this TI-2 Ab response. Thus, B cell-expressed IFNAR is required for optimal responses to TI-2 Ags.

Type I IFN signaling supports early activation and expansion of Ag-specific B-1b cells in response to TI-2 Ags

We assessed the early B cell response to TNP-Ficoll in WT and Ifnar1−/− mice to determine if defects in activation, expansion, and/or differentiation could be identified. As shown in Figure 4A, naïve WT and Ifnar1−/− mice had similar frequencies of TNP-specific CD19+ B cells in the spleen (representative gating strategy shown in Supplemental Fig. 1C). However, 5 days following immunization, TNP-specific CD19+ B cell frequencies expanded 3-fold in WT mice, but only 2-fold in Ifnar1−/− mice (Fig. 4A). We noted similar decreases (~30%) in the frequencies of TNP-specific CD138+ and IgG+CD138+ plasmablasts as well as the frequencies of dividing (Ki67+) TNP-specific B cells, IgG+ B cells, and CD138+ B cells (Fig. 4A).

Figure 4. Impaired activation and expansion of TNP-specific B cells in IFNAR1−/− mice following TNP-Ficoll immunization.

Figure 4.

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(A-B) TNP-specific total (A) and CD11b+ and CD11bneg (B) CD19+ spleen B cells were examined in naïve (n=4-5/group) and TNP-Ficoll-immunized (d5) WT and Ifnar1−/− mice (n=5/group). The mean frequencies of TNP-specific CD19+, CD19+CD138+, CD19+IgG+CD138+, CD19+Ki67+, CD19+ CD138+Ki67+, and CD19+IgG+Ki67+ cells among total splenocytes are indicated. Asterisks (*) indicate significant differences between values for WT and Ifnar1−/− mice (p<0.05).

Alterations in early responses to TI-2 Ags may be attributed to changes in innate B cell responses. CD11b expression is present on B1 cells several days following entry into the spleen (16, 17). Consistent with this, VHB1-8 Tg B-1b cells adoptively transferred into WT recipients express CD11b in the spleen up to 7 days post transfer (6 days post NP-Ficoll immunization), in contrast to adoptively transferred VHB1-8 Tg marginal zone and follicular B cells (Supplemental Fig. 1D). Thus, CD11b expression may be used to distinguish hapten-specific B-1 cells from marginal zone and follicular B cells during the early splenic response to TI-2 Ag, although it may not capture all B-1 cells. As shown in Figure 4B, the frequency of TNP-specific CD19+CD11b+ B cells in the spleen was significantly reduced (two-fold) in Ifnar1−/− relative to WT mice 5 days post immunization. In contrast, no significant difference was observed for frequencies of TNP-specific CD19+CD11b-negative B cells following immunization (Fig. 4B). Cell numbers of splenic TNP-specific CD19+CD11b+ B cells were similarly reduced (>2-fold) in Ifnar1−/− mice (Supplemental Fig. 1E). The frequencies of Ki67+, CD138+, and IgG+ TNP-specific CD19+CD11b+ cells among splenocytes was significantly decreased in Ifnar1−/− mice due to decreased overall TNP-specific CD19+CD11b+ frequencies (Fig. 4B). Overall splenic frequencies of Ki67+, CD138+, and IgG+ TNP-specific CD19+CD11b-negative B cells were not different between Ifnar1−/− and WT mice. Thus, IFNAR deficiency results in reduced participation of CD11b+ B cells in the response to haptenated Ficoll.

CD11b+ B cells in peritoneal cavity and spleen express significantly higher (2-fold) levels of IFNAR1 relative to CD11b negative (largely B-2) B cells (Fig. 5A). Given our data supporting normal numbers of peritoneal B-1b cells in Ifnar1−/− mice (Fig. 2) and a role for B cell-intrinsic IFNAR expression in promoting TI-2 Ab responses (Fig. 3), we next sought to determine the extent to which IFNAR expression on B-1b cells regulates early TI-2 Ab responses. To do this, we isolated and adoptively transferred CFSE-labeled peritoneal B-1b cells (CD19+CD11b+CD23neg) from IFNAR-sufficient VHB1-8 Tg mice into Ifnar1−/− mice and treated recipient mice with a control or IFNAR1 blocking or Ab at the time of NP-Ficoll immunization. As shown in Fig. 5B, IFNAR blockade significantly decreased frequencies of CD45.1+ NP-specific B-1b cells in the peritoneal cavity, spleen, blood, inguinal lymph node, and bone marrow on d5 relative to control Ab-treated mice (representative gating strategy shown in Supplemental Fig. 1F). These differences were reflected in decreased CD45.1+ NP-specific cell yields in mice that had received IFNAR1 mAb blockade. However, we did not detect differences in division as measured by CFSE loss (data not shown). IFNAR blockade also significantly decreased CD86 expression levels on CD45.1+ NP-specific peritoneal (4-fold), blood (2-fold), and spleen (1.5-fold) B-1b cells (Fig. 5C). In addition, IFNAR blockade significantly decreased frequencies of IgG+ and CD138+ CD45.1+ NP-specific B-1b cells in peritoneal cavity, spleen, and blood relative to control IgG-treated mice (Fig. 5C). Thus, IFNAR expression by B-1b cells supports activation, expansion, and differentiation in response to TI-2 Ag.

Figure 5. B-1b cell intrinsic IFNAR expression supports activation, expansion, survival, and differentiation to ASC.

Figure 5.

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(A) IFNAR1 expression by peritoneal and splenic CD11b+ and CD11b− CD19+ B cells. Representative staining (left panel) and average mean fluorescence intensities (MFI) are shown for WT and Ifnar1−/− B cells. B-C) Effects of IFNAR1 mAb blockade on VHB1-8 Tg B-1b cells adoptively transferred into Ifnar1−/− mice. Peritoneal B-1b cells (2 x 105, >90% CD19+, >80% CD11b+CD23neg) from VHB1-8 Tg mice were adoptively transferred into Ifnar1−/− mice i.p. Recipient mice were treated with control mouse IgG or an IFNAR1 blocking mAb (200 μg) at the time of NP-Ficoll immunization (5 μg i.p.), and on d2 and 4 (100 μg). Peritoneal cavity (perC), spleen, blood, bone marrow (BM), inguinal (iLN) and mesenteric (MLN) lymph nodes were harvested on d5, with CD45.1+CD19+ NP-specific B cell populations (B), CD86 expression, and CD138+ and IgG+ frequencies and numbers (C) assessed by flow cytometry. Mean cell numbers and frequencies among total leukocytes are shown (n=3-4 mice/group). D) Effects of type I IFN on VHB1-8 CD45.1+CD19+ NP-specific B cell populations in ex vivo cultures. Splenic and peritoneal B cells were adoptively transferred into Ifnar1−/− recipient mice i.v. and i.p., respectively. Five days post immunization with NP-Ficoll, splenocytes were harvested and cultured in the presence or absence of IFN α/β (600 U/ml). CD45.1+CD19+ NP-specific B cells were analyzed 24 hours later for CD11b, IgG, CD138, and intracellular active caspase 3 expression. Results are representative of three similar experiments. Asterisks (*) indicate significant differences between values for WT and Ifnar1−/− mice (A-C) or between medium and IFNα/β cultures(D-F; p<0.05).

To investigate direct effects of type I IFN on B cells responding to TI-2 Ags, we adoptively transferred VHB1-8 Tg B-1b and splenic B cells into Ifnar1−/− mice, and 5 days post immunization, assessed the effects of type I IFN on these cells in vitro. After 24 hours of culture the overall frequencies of total, IgG+, and CD11b+ CD45.1+ NP-specific B cells was not changed by addition of type I IFN (Fig. 5D). However, the frequency of CD138+ cells was significantly increased (3-fold) whereas the frequency of active caspase-3+ staining was significantly decreased among CD45.1+ NP-specific B cells in type I IFN-treated cultures (Fig. 5D). We assessed active caspase-3 staining for CD11b+ (B-1b) and CD11bneg cells and found type I IFN significantly decreased the frequency of staining among CD11b+, but not CD11bneg CD45.1+ NP-specific B cells (Fig. 5E).

Type I IFN significantly increased the frequency of CD138+ cells among CD11b+CD45.1+ NP-specific B cells but had no effect on the frequency of IgG+ cells among CD11b+CD45.1+ NP-specific B cells (Fig. 5F). However, type I IFN significantly decreased caspase 3 staining in both CD138+ and IgG+CD11b+CD45.1+ NP-specific B cell populations. Significantly increased viable CD138+ and IgG+ CD11b+CD45.1+ NP-specific cell counts in these cultures corroborated these findings (Fig. 5F). Thus, type I IFN increases differentiation of TI-2 Ag-activated B cells to CD138+ Ab secreting cells (ASC) and promotes survival of B-1b cells responding to TI-2 Ags.

Type I IFN signaling is not required for MPL/TDCM adjuvant effects on Ab responses to TI-2 Ags

MPL/TDCM induces type I IFN production in vivo and does not optimally enhance TI-2 Ab responses in mice lacking the TIR-domain-containing adapter-inducing interferon-β (TRIF) adapter, which is an important inducer of IFN-β (8, 18). This, along with results from a previous study demonstrating type I IFN-mediated signaling on B cells was critical for poly(I:C)-mediated adjuvant effects in increasing IgG responses to NP-Ficoll (9) raised the possibility that type I IFN signaling was also critical for MPL/TDCM adjuvanticity. Given that IFNAR deficiency resulted in a more significant impairment in Ab responses to PPS relative to haptenated Ficoll and previous work showing that MPL/TDCM has more significant effects on increasing Ab responses to PPS relative to haptenated Ficoll (6), we assessed MPL/TDCM effects on responses of Ifnar1−/− mice to Pneumovax23 delivered i.m. As shown in Fig. 6, Ifnar1−/− mice exhibited significantly impaired PPS3- and Pneumovax23-specific IgM and IgG responses to Pneumovax23 delivered i.m. (Fig. 6), similar to results obtained for i.p. immunization (Fig. 1). However, inclusion of MPL/TDCM yielded significantly increased primary and secondary IgM and IgG responses to PPS3 and Pneumovax23, although IgG responses were variable. Two-way repeated measures ANOVA analyses with vaccine response (Pneumovax23 or Pneumovax23 + adjuvant) and time point as factors indicated both factors and their interaction were significant for WT and Ifnar1−/− groups (p<0.05), and specifically support the conclusion that Ifnar1−/− mice receiving adjuvant had significantly higher overall levels of Pneumovax23- and PPS3-specific IgG and IgM relative to their counterparts that had received Pneumovax23 without adjuvant. Thus, IFNAR is not required for MPL/TDCM adjuvant effects in the context of PPS-specific Ab responses.

Figure 6. MPL/TDCM adjuvant effects on TI-2 Ab responses do not require type I IFN receptor signaling.

Figure 6.

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A-B) WT and Ifnar1−/− mice were immunized i.m. with Pneumovax23 containing 1 μg each PPS either alone or mixed with MPL/TDCM. Serum IgM and IgG levels reactive against PPS3 (A) and whole Pneumovax23 (B) were determined by ELISA. Asterisks (*) indicate significant differences between Ab levels in mice of the same genotype immunized with or without adjuvant (n=5 mice/group) as assessed by Student’s t-test for individual time points. Repeated measures two-way ANOVA using timepoints and individual Ab responses as factors indicated 1) overall levels of Pneumovax23-specific IgG and PPS3-specific IgG and IgM were significantly higher in WT mice that received adjuvanted vaccine versus those that had not (p<0.05) and 2) overall levels of Pneumovax23- and PPS3-specific IgG and IgM were significantly higher in Ifnar1−/− mice that received adjuvanted vaccine versus those that had not (p<0.05).

B-1b and CD23− spleen B cells produce increased Ab responses to NP-Ficoll coadministered with adjuvant, regardless of immunization route

Given the above findings and previous work demonstrating that a type I IFN-activating adjuvant elicited its IFNAR-dependent effects on Ab responses to NP-Ficoll through activating follicular B cells (9), we questioned whether MPL/TDCM enhanced TI-2 Ab responses by supporting Ab production by distinct B cell subsets. We found splenic and peritoneal NP-specific VHB1-8 Tg B cells, particularly CD11b+ peritoneal B-1b cells, were responsive to MPL/TDCM effects on ASC differentiation in the context of TI-2 Ag activation in vitro as evidenced by increased BLIMP1 expression by these cells in the presence of adjuvant (Supplemental Fig. 1G–H).

To determine whether MPL/TDCM elicited its effects through select subsets in vivo, we performed adoptive transfer experiments using B cells from VHB1-8 Tg mice (11). Naïve CD43− peritoneal B-1b cells and CD23+ (enriched for follicular B cells) and CD23− spleen B cells (enriched for marginal zone B cells) from VHB1-8 Tg mice were adoptively transferred i.v. into WT recipients which were then immunized with NP-Ficoll (i.p.). Importantly, λ1-expressing (NP-specific) B cells are similarly represented among these subsets (11). B-1b cells produced the highest level of IgM and IgG in response to NP-Ficoll (Fig. 7A). Inclusion of MPL/TDCM also significantly increased NP-specific IgMa (produced by Tg cells) and IgG (5–fold) by B-1b and CD23− spleen B cells. However, MPL/TDCM had no effect on Ab production by CD23+ spleen cells.

Figure 7. MPL/TDCM promotes increased B-1b and splenic CD23− Ab responses to NP-Ficoll and PPS, regardless of immunization route.

Figure 7.

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CD43− VHB1-8 Tg CD11b+ peritoneal (B-1b) B cells (1.25 x 105 in A, and 5 x 105 in B), splenic CD23+ (5 x 105) and splenic CD23− (5 x 105) B cells were transferred i.v. into WT recipients (n=4-5/group). One day later, recipients were immunized with 1 μg NP-Ficoll i.p. (A) or i.m. (B). NP-specific serum IgMa and IgG levels are shown along with NP-specific IgG levels for immunized mice that did not receive VHB1-8 Tg cells (“no cells”). Results are representative of those obtained in two independent transfer experiments. C-D) WT splenocytes (3 x107) or enriched peritoneal B-1b B cells, splenic CD43−CD23+ and CD43−CD23− B cells (3 x 106) were transferred into muMT recipients (n=4-7/group). One day later, recipients were immunized with Pneumovax23 either alone or with MPL/TDCM i.p. (C) or i.m. (D). An additional group of splenocyte recipients received no immunization (horizontal gray stripe) or MPL/TDCM only (gray fill) in (A). IgM and IgG recognizing PPS3 and total Pneumovax23 were assessed on d10 post immunization. Asterisks (*) indicate significant differences between Ab levels in recipients with and without adjuvant.

We next assessed whether i.m. immunization would impact Ab responses by distinct subsets. In response to NP-Ficoll delivered i.m., B-1b cells produced the highest amount of NP-specific IgMa, whereas CD23− B cells produced the most IgG (Fig. 7B). MPL/TDCM significantly increased NP-specific IgMa production from B-1b cells (1.5-fold) over recipient mice given adjuvant alone. Increases in IgMa in recipients of CD23− and CD23+ cells were moderate. MPL/TDCM increased NP-specific IgG production in recipients of each cell type, with d7 levels increased over 6-fold in B-1b and 9-fold in CD23+ recipients, but less than 2-fold in CD23− B cell recipients. Thus, in the NP-Ficoll immunization system using high affinity VHB1-8 Tg B cells, B-1b and CD23− spleen cells preferentially differentiate into ASC regardless of immunization route and MPL/TDCM further potentiates their level of Ab production, although there is nonetheless some responsiveness to MPL/TDCM in the VHB1-8 Tg CD23+ B cell population following i.m. immunization.

B-1b and CD23− spleen B cells produce increased Ab responses to Pneumovax23 coadministered with adjuvant

We next examined effects of MPL/TDCM on responses of B cell subpopulations to the pneumococcal vaccine, Pneumovax23. In muMT mice reconstituted with high numbers of spleen cells (3×107), IgM and IgG levels against Pneumovax23 as well as PPS3 were not significantly different among recipients that received no immunization, Pneumovax only, or MPL/TDCM only (i.p.), whereas mice receiving Pneumovax plus MPL/TDCM i.p. produced significantly increased Pneumovax-specific IgM and IgG (Fig. 7C). PPS3-specific IgM and IgG was also increased, although not significantly. Thus, there is a PPS-specific spleen B cell population that responds to MPL/TDCM adjuvant effects.

When examining sorted B cell populations, we found recipients of B-1b cells produced the highest level of Pneumovax23- and PPS3-specific IgM and PPS3-specific IgG in response to Pneumovax23 i.p. (Fig. 7C). MPL/TDCM significantly increased total Pneumovax23-specific IgM in B-1b, but not in CD23− and CD23+ B cell recipients. Similarly, MPL/TDCM significantly increased PPS3-specific IgM production in B-1b cell recipients, and to a lesser extent in CD23−, but not CD23+ B cell recipients. PPS3- and Pneumovax-specific IgG was low but MPL/TDCM produced moderate increases in some recipients.

In response to Pneumovax23 plus adjuvant delivered i.m., recipients of high dose splenocytes produced moderately increased Pneumovax23- and PPS3-specific IgM, but not IgG (Fig. 7D). Recipients of B-1b cells and splenic CD23− B cells produced significantly increased PPS3- and Pneumovax23-specific IgM in response to adjuvant (Fig. 7D). MPL/TDCM significantly increased PPS3-specific IgG as well as increased Pneumovax23-specific IgG, selectively in B-1b cell recipients. CD23+ B cell recipients were not very responsive. Thus, MPL/TDCM adjuvant effects observed in the context of Pneumovax23 immunization are largely carried out through innate B cell populations.

Discussion

Antibody responses to polysaccharide Ags are critical for protection against encapsulated pathogens. However, our understanding of the factors regulating these responses remains incomplete. Our currrent study reveals several novel findings regarding regulation of TI-2 Ab responses. First, we demonstrate type I IFN signaling on B cells, and in particular, on B-1b cells, promotes optimal Ab responses to TI-2 Ags, including pneumococcal polysaccharides, and does so via support of early B cell activation, survival, and ASC differentiation. Second, we demonstrate an MPL-based adjuvant that significantly augments primary and secondary responses to TI-2 Ags (6), induces type I IFN production in vivo (7), and requires TRIF signaling for optimal adjuvant effects (6), does not require type I IFN for its effects. Finally, we demonstrate MPL/TDCM elicits its adjuvant effects largely by promoting Ab production by innate-like B cells (ie., B-1b and CD23− B cells). Collectively, our results provide valuable information regarding the role of type I IFN in supporting polysaccharide-specific Ab responses as well as critical information that may be leveraged for designing strategies to improve vaccines targeting polysaccharide Ags in the future.

The role of IFNAR in regulating B cell responses is complex based on findings reported for Ifnar1−/− mice and in vitro studies of B cells. We show IFNAR deficiency on a C57BL/6 background does not impact peritoneal or splenic B cell subset frequencies or numbers. An earlier study reported Ifnar1−/− mice on a 129sv background had normal absolute B cell numbers in bone marrow and spleen; however, it indicated the repertoire of immature bone marrow B cells was altered, and further, showed IFN I could modulate the sensitivity of activated B cells to BCR-dependent inhibition of terminal differentiation resulting from LPS or CD40 + IL 4 stimulation (19). In contrast, another study reported that IFN I in conjunction with IL-6 promotes plasma cell differentiation (20). Nonetheless, IFN I protects B cells against apoptosis and has been reported to enhance BCR-mediated activation and proliferation in some studies, but have the opposite effect in others (21–24). Thus, both stimulatory and inhibitory effects of IFN-α/β on in vitro B cell proliferation and Ig production have been reported. In some cases this may be explained by the fact that high dose type I IFN is inhibitory whereas low dose IFN is stimulatory for Ig production by activated B cells (25). Its positive role in supporting TNP- and NP-Ficoll-specific B cell activation and expansion in vivo in the absence of strong type I IFN production is consistent with this notion.

Based on our current findings using mAb blockade in both WT mice and Ifnar1−/− VHB1-8 Tg B cell recipient mice, as well as those obtained using mice selectively lacking type I IFN receptor on B cells (bone marrow chimeras and cre-flox mice), type I IFN regulates B cell TI-2 IgM and IgG responses directly, as opposed to impacting the development of the B cell populations which give rise to these responses. Work on the effects of type I IFN on TI Ab responses has been limited and largely restricted to anti-viral responses. The effect of type I IFN on B cell responses to vesicular stomatitis virus (VSV), which displays highly ordered VSV-G and induces TI IgM responses has been examined in two studies. One study demonstrated IFN-α enhanced VSV-specific B cell IgM production in response to UV-inactivated VSV in vitro and showed that B cell-expressed IFNAR was required for optimal IgM, but not IgG responses, to live VSV in vivo (26). In contrast, in response to noninfective VSV-viral like particles (VLP), IFNAR deficiency significantly reduced TD IgG responses but only slightly reduced IgM responses. This regulation was through B cell-extrinsic IFNAR expression, and the study reported little effect of B cell-expressed IFNAR on the TI Ab response to VSV-VLP (27).

B-1 cells express significantly higher levels of type I IFN receptor than B-2 cells. Thus, it is perhaps not surprising that hapten-specific CD11b+ B-1 cells lacking IFNAR exhibited greater defects in activation, expansion, and survival than CD11bneg B cells. Indeed, IFNAR blockade on NP-specific B-1b cells in vivo significantly decreased their activation and expansion in all tissues examined except MLN. Interestingly, with regard to innate B1 cell production of Abs, IFNAR is required for B1 cell lymph node trafficking and IgM production in response to influenza virus infection due to its role in regulating CD11b conformational changes (28). However, IFNAR is not required for monophosphoryl lipid A-induced tumor-reactive IgM production by B1a cells (7), despite its dependence on the TLR4-TRIF activation pathway (29). We did not find differential representation of NP-specific B-1b cells among tissues (peritoneal cavity, spleen, blood, inguinal lymph node, mesenteric lymph node, or bone marrow) following IFNAR mAb blockade and thus have no evidence to support that trafficking is impaired in the absence of IFNAR signaling in haptenated-Ficoll model system. When taken together, our in vivo and ex vivo experimental results suggest IFNAR supports increased activation of B-1b cells in the context of TI-2 Ag activation, as well as promotes their expansion, likely through activating survival pathways (21). Moreover, our results indicate type I IFN supports differentiation of TI-2 Ag-activated B-1b cells into ASC. Our work does not rule out a role for type I IFN in supporting TI-2 Ab responses by other B cell subsets. Further, although our results suggest IFNAR regulates B cell responses to TI-2 Ag primarily through its expression on B cells, we did find IgG responses were more consistently decreased relative to IgM responses when IFNAR was solely lacking on B cells, suggesting a potential role for IFNAR-derived signals on other cell types in supporting IgM production and/or a possible role for IFNAR in supporting class switching through its survival-promoting effects. Although type I IFN induction of class switching is possible, Stat1 signaling specifically facilitates IgG2a/c switching (30) which generally comprises a minor subclass produced in response to non-adjuvanted TI-2 Ags (6, 11). Interestingly, augmentation of IgG responses to TD Ag adjuvanted by IFNα also requires IFNAR expression on B cells (31), whereas VSV-displaying VLP induce IgG production via a mechanism dependent on IFNAR expression on non-B cells (27). At present, it is not clear whether basal levels of type I IFN contribute to regulation of Ab responses to unadjuvanted TI Ags or if TI-2 Ags induce low levels of type I IFN that may further drive this regulation. It is certainly possible that the cell wall components which contaminate the pneumococcal polysaccharide vaccine induce type I IFN through TLR2 signaling (32). Further work is required to determine whether this is the case.

Although IFNAR1 deficiency yields a distinct phenotype in humans relative to mice at least with regard to mucosal viral infections (33), whether human B cells share similar dependency on type I IFN for optimal TI-2 Ab responses remains unknown. Interestingly, a small sample study reported slightly higher geometric mean titer ratios for Pneumovax23 serotype-specific responses in multiple sclerosis patients treated with type I IFN compared to patients treated with dimethyl fumarate (34). Future work with analysis of larger cohorts of patients either treated with IFN therapeutics or who have developed autoAbs against IFNs, along with studies defining the effects of mutations involved in altered type I IFN pathways (33, 35–37), will likely shed light on this.

We investigated the role type I IFN plays in the adjuvant effects of MPL/TDCM on TI-2 Ab responses in the current study due to: 1) its dependency on TRIF signaling (6), 2) results from a previous study demonstrating the requirement for B cell-expressed IFNAR in the adjuvant effects of poly(I:C) on TI-2 Ab responses (9), and 3) the dependency of non-adjuvanted TI-2 Ab responses on B cell-expressed IFNAR identified herein. Based on the adjuvant effects observed in Ifnar1−/− mice, MPL/TDCM works independently of type I IFN signaling. The TLR4-dependent effects of MPL on activating both MyD88 and TRIF pathways in B cells (6), the latter of which activates NF-kB in addition to type I IFN production, may explain these findings. Our work demonstrates MPL/TDCM significantly enhances Ab responses to polysaccharide Ag largely through its effects on innate-like B cells (ie., B-1b and CD23− spleen B cells). MPL/TDCM adjuvant effects on significantly increased PPS-specific IgG production by B-1b cells to Pneumovax23 delivered i.m. was particularly notable. B-1 and MZ B cells are most often found producing Abs against TI-2 Ags in the absence of adjuvant, so it is perhaps not surprising that this adjuvant further promotes the ability of these B cells to produce more IgM and in some cases IgG, against these polysaccharide Ags (11, 38). In addition to being less responsive to TI-2 Ags relative to marginal zone and B-1 cells, follicular B cells are also less responsive to TLR agonists (39). In contrast to the mechanism defined for poly(I:C) involving B cell-expressed IFNAR (9), the MPL-based adjuvant functions through direct activation of TLR4 on B cells (6). This supports the possibility that combinations of distinct pattern recognition receptor agonists that work through different pathways may be optimized to significantly improve B cell responses to polysaccharide Ags in humans in the future.

Supplementary Material

Supplemental Figure 1

Key points:

  • B-1b cell-intrinsic IFNAR expression promotes TI-2 Ab responses.

  • MPL/TDCM adjuvant effects are independent of type 1 IFN.

  • MPL/TDCM promotes TI-2 Ab responses by innate B cells.

Acknowledgments

This work was supported by NIAID/NIH R01AI18876, R01AI164489, and R21AI144758. MAS was supported by NIH T32AI007401. Research reported in this publication was also supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR001420. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors have no conflicts of interest to disclose.

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Associated Data

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Supplementary Materials

Supplemental Figure 1
NIHMS1849873-supplement-Supplemental_Figure_1.pdf (1.3MB, pdf)

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