The PD-1 regulatory axis inhibits T cell independent B cell memory generation and reactivation

Abstract

The inability of T cell-independent type 2 antigens (TI-2 Ags) to induce recall responses is a poorly understood facet of humoral immunity, yet critically important for improving vaccines. Using normal and V_HB1–8 transgenic mice, we demonstrate B cell-intrinsic PD-1 expression negatively regulates TI-2 memory B cell (B_mem) generation and reactivation in part, through interacting with PDL1 and PDL2 on non-Ag-specific cells. We also identified a significant role for PDL2 expression on B_mem in inhibiting reactivation and Ab production, thereby revealing a novel self-regulatory mechanism exists for TI-2 B_mem. This regulation impacts responses to clinically relevant vaccines, as PD-1 deficiency was associated with significantly increased Ab boosting to the pneumococcal vaccine following both vaccination and infection. Notably, we found a B cell-activating adjuvant enabled even greater boosting of protective pneumococcal polysaccharide-specific IgG responses when PD-1 inhibition was relieved. This work highlights unique self-regulation by TI-2 B_mem and reveals new opportunities for significantly improving TI-2 Ag-based vaccine responses.

Introduction

Humoral responses to pathogen-displayed polysaccharides are critical for protection against infection. These polysaccharides often behave as T cell-independent type 2 antigens (TI-2 Ag) which by definition, are able to elicit rapid antibody (Ab) production in the absence of classical cognate T cell help or other strong innate stimuli but fail to do so in infants and in Xid mice lacking functional Btk (1). Pathogenic bacteria, including Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Salmonella typhi, and Bacillus anthracis, bear TI-2 Ags as capsule components (1–5). Fungi, parasites, and viruses also display TI-2 Ags (2, 6–10). Understanding the regulation of natural and vaccine-acquired humoral immunity to TI-2 Ags is critical for developing improved treatments and vaccines for TI-2 Ag-bearing pathogens.

A key aspect of TI-2 responses that is poorly understood relates to the pathways that regulate recall responses. Some TI Ags encountered on gram negative bacteria can induce plasmablast-like memory cells with the capacity for expansion and increased Ab production upon pathogen re-exposure (11, 12); however, this does not readily occur with native polysaccharides. The refractoriness of polysaccharide-specific Ab responses to booster immunization with native polysaccharides has long been recognized (13–15). Although TI-2 Ags (pneumococcal polysaccharide, meningococcal polysaccharide, and haptenated Ficoll, etc.) were originally not thought to generate memory B cells (B_mem), several early studies using normal mice (16, 17) and more recent studies using adoptive transfers of transgenic (Tg) B cells expressing a high affinity NP-specific BCR (V_HB1–8) have shown classical non-Ab-secreting B_mem develop following haptenated-Ficoll immunization (18, 19). Our work shows these Tg B_mem display a phenotype similar to T cell-dependent (TD) memory B cells (20, 21), including CD80 and PDL2 expression (19). The extent to which these markers are also expressed by naturally occurring TI-2 memory cells is not known but is important to consider as each receptor interacts with potent immunoinhibitory molecules. Studies using V_HB1–8 Tg mice indicate Ag-specific IgG-dependent suppression of Ag-specific memory B cells is a major mechanism contributing to failed boosting with TI-2 Ags (18, 19). However, the extent to which other regulatory pathways contribute to this regulation is not clear.

Overcoming impaired boosting to TI-2 Ags is a significant challenge that must be overcome in order to develop efficacious TI-2 Ag-based vaccines. Establishment of high titers as well as functional B_mem which can respond to secondary immunization and/or infection are necessary to achieve the highest level of protection. Protein conjugation to bacterial polysaccharides is one strategy that enables significant boosting in young children (except perhaps with serotype 3 pneumococcal polysaccharide-conjugates (15)). However, the cost of conjugate vaccines continues to increase as new polysaccharides are included, raising concerns about the feasibility of deploying them in developing countries (22). Concerns related to eliciting and maintaining high titers to an increasing number of distinct polysaccharides conjugated to the same protein have also not yet been addressed. Relative to young children, in adults, pneumococcal polysaccharide-conjugate vaccines do not boost as well and overall Ab responses have been reported to be similar to those elicited to native pneumococcal polysaccharides (23). Finally, it is not clear if the B_mem elicited by conjugate vaccines are adequately responsive to native polysaccharides which may be encountered during infections. Hence, alternative approaches to enhancing these vaccine responses may be required under some circumstances. Indeed, the use of B cell-activating adjuvants (24, 25) along with other immunomodulators represent strategies to improve TI-2 Ag-specific B_mem formation and responsiveness to secondary Ag encounter in some individuals.

Our previous work demonstrated PD-1 suppresses primary Ab responses to TI-2 Ags (26, 27). However, the extent to which the PD-1:PD-1 ligand (PDL) pathway regulates the formation of TI-2 B_mem and their reactivation potential has not been investigated. In the current study, we used the V_HB_1–8 Tg system as well as normal B cells to assess the role PD-1 and its ligands, PDL1 and PDL2, play in TI-2 B_mem formation and regulation. Our work demonstrates a key role for the PD-1:PD-L regulatory axis in limiting memory B cell formation largely due to interactions between B cell-expressed PD-1 and non-Ag-specific B cell expressed PDL1. However, PD-1-mediated suppression of B_mem activation, division, and Ab production following boosting may also involve interactions between PD-1 upregulated on activated B_mem and B_mem-expressed PDL2, in addition to interactions with non-B_mem cell-expressed PDL1 and to a lesser extent, non-B_mem cell-expressed PDL2. Thus, TI-2 B_mem may engage in a novel autoregulatory mechanism involving PD-1-PDL2 interactions. Inhibition of this pathway in the context of vaccination with adjuvanted polysaccharide vaccines yields high Ag-specific IgG production with significant boosting and enhanced protective capacity, demonstrating inhibition of TI-2 Ag boosting by Ag-specific IgG can be overcome by disrupting PD-1-mediated inhibition in combination with B cell-activating adjuvants. Thereby, our findings hold significance for developing alternative and improved polysaccharide vaccine formulations, especially for at-risk populations in which T cell help is impaired.

Materials and Methods

Mice

WT (CD45.2 and CD45.1 congenic) and V_HB1–8 Tg (B1–8hi IgH transgenic knock-in; B6.129P2-Ptrpca Igh^tm1Mnz/J) mice on a C57BL/6 background were from Jackson Laboratory. PD-1^−/− (28), PDL1^−/− (29), and PDL2^−/− (30) mice were on a C57BL/6 background and obtained as previously described (27). PD-1^−/− mice were crossed to CD45.1^+/+ V_HB1–8 Tg mice to yield CD45.2^+/+ PD-1^−/−V_HB1–8 Tg mice. Mice were housed under specific pathogen free conditions, except during infection experiments. Mice were used at 2–4 months of age and were age-matched for experiments. All studies and procedures were approved by the Wake Forest Animal Care and Use Committee.

Immunizations, ELISAs, in vivo mAb blockade, and Streptococcus pneumoniae challenge

Mice were immunized with 1, 10, or 25 μg NP₄₀-AECM-Ficoll or TNP₆₅-AECM-Ficoll (Biosearch Technologies) in 200 μl PBS i.p. as indicated. Mice were immunized with vaccine-grade Pneumovax 23 (Merck, Whitehouse Station, NJ) containing 0.125 μg each of 23 pneumococcal polysaccharide (PPS), or Prevnar-13 (Pfizer, formerly Wyeth Pharmaceuticals, New York, NY) containing ~0.1 μg of each of 13 PPS, as previously described (19, 31). In some experiments, adjuvant containing 20 μg Salmonella minnesota monophosphoryl lipid A (MPL), 20 μg synthetic cord factor (trehalose-6,6’-dicorynomycolate (TDCM)) in 0.5% squalene/0.05% Tween-80 (Sigma) was mixed with Ags prior to injections (24). In select experiments, mice were administered bromodeoxyuridine (BrdU; 0.8 mg/ml) in drinking water for 5 days post NP-Ficoll immunization or from weeks 5 to 6 post immunization. NP-, TNP-, PPS3-, and Pneumovax-specific ELISAs were performed as previously described, with NP-specific IgM^a and IgG2a (selectively produced by Tg B cells) specifically measured in some experiments (19, 24). Standard curves were used to approximate Ag-specific Ab concentrations, as previously described (19, 24). PD-L mAb blockade was performed by administering PDL1 (10F.9G2), PDL2 (TY25), or rat IgG control mAbs (all from BioXcell; InVivo mAb) i.p. on d1 (200 μg), d3 (200 μg), and d5 (100 μg) post immunization as previously described (26, 27). Immune mice were infected intranasally (i.n. 10⁷ CFU) or i.p. with serotype 3 WU2 strain S. pneumoniae (10⁴ CFU) and monitored every 12 hours for signs of distress as previously described (31, 32). Ab levels were measured 14 days later. For passive serum transfer experiments, CD19^−/− mice were administered 0.5 μl pooled sera pre-mixed with 200 CFU WU2 in 100 μl PBS i.p.

B cell phenotyping

Single-cell suspensions (2 × 10⁷/mL) in PBS containing 2% newborn calf serum were incubated with Fc Block (eBioscience) for 15 minutes, followed by staining with fluorochrome-conjugated Abs: CD19, PDL2, CD73, PD-1 (from BD Biosciences), and CD138 and CD86 or CD44 (from eBioscience); B220, CD80, CD11b, CD21/35, CD45.1, CD45.2 (BioLegend), pooled rat anti-mouse-IgG1, −IgG2b, −IgG2a (omitted specifically for Tg cell staining due to CD45 mAb reactivity), and −IgG3 Abs (Southern Biotech), and NP₄₀-APC for 30 min at room temperature, followed by washing and fixing in 1.5% buffered formaldehyde (19). As we previously reported (19), we detect ~90% of IgG-switched Tg B cells using staining for IgG1, 2b, and 3. BrdU-labeled DNA was detected as previously described (27). Fluorochrome-labeled isotype controls were used to determine background staining levels of NP-specific B cells. Adoptively transferred Tg memory B cells were identified in recipient mice as CD45.1⁺ (or CD45.2⁺) NP-APC⁺CD19⁺FSC^low CD138^negCFSE^low cells. Cells were analyzed using a FACSCantoII or FortessaX20 cytometer (BD Biosciences) with FSC-A/FSC-H doublet exclusion. Data were analyzed using FlowJo analysis software (Tree Star).

Adoptive transfer experiments

V_HB1–8 Tg cells from single cell splenocyte and peritoneal lavage preparations were subjected to CD43 negative selection (Dynal), CFSE-labeled (1 μM CFDA-SE; InvivoGen), and transferred i.v. (5 × 10⁵) and i.p., respectively, into sex-matched wild type CD45.2⁺ C57BL/6 recipients. PD-1^−/−V_HB1–8 Tg cells were transferred into CD45.1⁺ C57BL/6 WT recipients, unless otherwise indicated. In transfers of memory cells, CD43⁻ B cells were further depleted of any CD138⁺ cells and in some cases, selected for IgG⁺ cells using biotinylated anti-mouse IgG1, 2a, 2b, 3 mAbs (clones RMG1–1, RMG2a-62, RMG2b-1, and RMG3–1, respectively (BioLegend)) in conjunction with Miltenyi streptavidin bead purification.

In vitro B cell activation assays

Peritoneal cavity cells containing memory B cells derived from V_HB1–8 Tg mice were harvested 2–3 months post immunization (i.p.) with 25 μg NP-Ficoll. In some experiments, B cells were selected using CD19⁺ beads (Miltenyi). Cells were CFSE-labeled and cultured for 4 days in complete RPMI + 10% FCS (1×10⁶/ml) in media alone or with 5 ng/ml NP₄₀-Ficoll, with either 2 μg/ml rat IgG2b (LTF-2), rat anti-mouse PDL1 (10F.9G2), or rat anti-mouse PDL2 (TY25; all InVivo mAbs from BioXcell). On day 4, cells were stained in culture wells with fixable Live/Dead dye, NP-APC, and fluorochrome-labeled mAbs to detect CD138, IgM, IgG, and CD45.1 as well as Countbright beads (ThermoFisher) for enumeration. Cells were harvested from wells, washed, fixed in 1.5% buffered formaldehyde and analyzed by flow cytometry.

Statistical analyses

Data are shown as means ± SEM. Differences between sample means were assessed using Student’s t-test or one-way ANOVA with Tukey’s post-hoc analysis. Survival analysis of Kaplan Meier curves was performed using the Log-rank test.

Results

TI-2 B_mem generated in normal mice express a phenotype similar to V_HB1–8 Tg B_mem cells

WT recipients of naïve CFSE-labeled V_HB1–8 Tg (CD45.1⁺) CD80^negPLD2^neg B cells develop a CD19⁺CD138^negCFSE^loFSC^loB220^hiCD80⁺PDL2^+/− population following NP-Ficoll immunization (Fig. 1A), as we previously published (19). We assessed whether B_mem could be identified in normal non-Tg mice using these markers. Six weeks following immunization with 10 μg NP₄₀-Ficoll, WT mice harbored a population of endogenous NP-specific B cells bearing a CD19⁺CD138^negFSC^loCD80⁺PDL2^+/− phenotype (Fig 1B). CD80⁺PDL2⁺ NP-specific B cells are scarce in naïve WT mice, but can be found for months following immunization (Supplemental Fig. 1A–B), consistent with what we have observed for adoptively transferred V_HB1–8 Tg B cells (19). Moreover, IgG⁺CD138^neg NP-specific B cells present in normal immune WT mice are largely CD80⁺PDL2^+/− (Fig. 1C), consistent with a B_mem phenotype. Non-Ag-specific IgG⁺CD138^neg B cells in these mice also express CD80, with a small fraction expressing PDL2 (Fig. 1D). Relative to non-class switched NP-specific CD138^neg B cells, IgG⁺ NP-specific CD138^neg B cells express higher levels of CD80, PDL2, CD73, and CD21/35, but have comparable cell size (FSC) and B220 expression (Fig. 1E). Collectively, this data supports NP-Ficoll immunization elicits the formation of TI-2 B_mem in WT mice with a CD19⁺CD138^negFSC^loB220^hiCD80⁺PDL2^+/−CD73^+/−CD21/35^lo/hi phenotype, similar to that described for high affinity V_HB1–8 Tg memory B cells (19). Thus, V_HB1–8 Tg NP-specific B_mem adequately model the surface phenotype of normal NP-specific B_mem generated in response to NP-Ficoll and these B cells may therefore be subject to similar modes of regulation by surface regulators (ie., PDL2, CD80, CD73, etc.).

Figure 1. Identification of TI-2 B_mem in V_HB1–8 Tg and normal mice.

A-E) Spleen B cells from WT mice immunized with 10 μg NP₄₀Ficoll were assessed for NP binding and expression of memory markers 6 weeks later. A) WT recipients of CFSE-labeled V_HB1–8 Tg (CD45.1⁺) B cells were immunized with NP₄₀Ficoll. NP-specific CD45.1⁺CD19⁺CD138^negCFSE^loFSC^lo B cells from immune WT recipients and naïve V_HB1–8 Tg mice were analyzed for CD80 and PDL2 expression or isotype control binding. B-C) Endogenous NP-specific CD138^neg B cells from WT mice immunized with NP₄₀Ficoll were analyzed for CD80 and PDL2 expression (B), including expression on gated IgG⁺ B cells (C). D) CD80 and PDL2 expression on non-NP binding IgG⁺CD138^neg B cells in WT mice. Isotype control binding by CD19⁺ NP-specific B cells is indicated in panels A-D. E) Expression of markers on endogenous NP-specific IgG⁺CD138^neg B cells in immunized WT mice. Results are representative of at least 3 independent experiments, each performed with 4 or more mice per analysis.

PD-1 negatively regulates the generation of TI-2 Ab and B_mem through its expression on B cells

Given the capacity for PD-1 to regulate primary Ab responses to TI-2 Ags (26, 27), we assessed whether it also influenced the generation of TI-2 B_mem in non-transgenic mice, defined as the expanded pool of Ag-specific B cells lacking CD138 and expressing IgG. As shown in Fig. 2A, WT mice immunized with 10 μg NP₄₀Ficoll generated a measurable pool of splenic CD138^negIgG⁺ NP-specific B cells that was significantly increased compared to that found in naïve mice, as expected. However, frequencies and numbers of CD138^negIgG⁺ NP-specific splenic B cells were 2-fold higher in immunized PD-1^−/− mice. These cells had a CD80⁺PDL2^+/−FSC^lo phenotype, similar to WT mice. CD138^negIgG^neg NP-specific splenic B cell numbers in immune PD-1^−/− mice were not increased over WT numbers (Fig. 2B). Similar to findings obtained for spleen B cells, NP-specific CD138^negIgG⁺ peritoneal B cell numbers were also significantly increased in immunized PD-1^−/− mice relative to WT mice (3-fold), and CD138^negIgG^neg B cell numbers were also moderately (1.4-fold) increased over WT (Fig. 2C).

Figure 2. PD-1 negatively regulates the generation of TI-2 B_mem through its expression on Ag-specific B cells.

A-C) WT and PD-1^−/− mice immunized with 10 μg NP₄₀Ficoll were assessed for endogenous NP-specific FSC^loCD138^negIgG⁺ and CD138^negIgG^neg B cell frequencies and numbers in spleen (A-B) and peritoneal cavity (C) 3 weeks later (n=4/group). Similar results were obtained in an independent experiment using 4 mice/group. D-E) WT and PD-1^−/− mice immunized with 10 μg NP₄₀Ficoll were fed BrdU in drinking water for 5 days post immunization (D), or between week 5 and 6 post immunization (E), with frequencies of BrdU⁺ cells among splenic NP-specific FSC^loCD138^neg IgM⁺ and IgG⁺ memory cells and among IgM⁺ and IgG⁺CD138⁺ B cells assessed by flow cytometry (n=5–6 mice/group). Frequencies of BrdU⁺ cells among non-Ag- specific IgM⁺ cells are also shown for comparison. F) NP-specific B cell frequencies among splenic and peritoneal B cell populations in naïve V_HB1–8 Tg and PD-1^−/− V_HB1–8 Tg mice. G-I) 4 × 10⁶ CD43⁻CFSE-labeled spleen B cells (i.v.) and 8 × 10⁵ CD43⁻CFSE-labeled peritoneal cavity B cells (i.p.) from V_HB1–8 Tg (CD45.1⁺) or PD-1^−/− V_HB1–8-Tg (CD45.2⁺) mice were transferred into naïve recipient mice (CD45.2⁺ and CD45.1⁺ WT mice, respectively), which were immunized with 10 μg NP₄₀Ficoll one day later. G) NP-specific serum IgM^a and IgG in WT recipients of V_HB1–8 Tg and PD-1^−/− V_HB1–8 Tg B cells. NP-specific IgG in WT mice that had not received cells is indicated by the dotted gray circle. H-I) Total (H) and IgG⁺ (I) V_HB1–8 Tg and PD-1^−/− V_HB1–8 Tg NP-specific CD19⁺CD138^negCFSE^lo FSC^lo memory and CD19⁺CD138⁺ Ab-secreting B cell frequencies in spleen, peritoneal cavity (PerC), bone marrow (BM), and lymph node (LN) of WT recipient mice 3 weeks post immunization (n=4–5/group). Results in G-I were derived from 4–5 mice/group. Similar results were obtained in an independent experiment. Asterisks (*) indicate significant differences between groups (p<0.05). In A, B, H, and I, frequencies are represented as a percentage of total splenic leukocytes.

There were no differences in the frequencies or numbers of CD138^negIgG^neg or CD138^negIgG⁺ NP-specific B cells in naïve PD-1^−/− versus WT mice (Fig. 2A–C), indicating the increased IgG⁺ B_mem in PD-1^−/− mice was not due to an increase in naïve precursors. Significantly increased BrdU labeling of NP-specific B cells in PD-1^−/− mice in the first 5 days post immunization suggested increased IgG⁺ B_mem generation in PD-1^−/− mice was likely due to increased expansion early in the response (Fig. 2D). Consistent with this, BrdU labeling of B_mem between 5 and 6 weeks post immunization revealed little division (4–7% BrdU⁺) in IgG⁺ B_mem with no difference between WT and PD-1^−/− B_mem (Fig. 2E). However, there was a high level of BrdU labeling in the splenic CD138⁺ plasmablast/plasma cell pool at 6 weeks, and a higher frequency of CD138⁺IgG⁺ cells were BrdU⁺ in PD-1^−/− mice. IgG⁺ B_mem remained significantly (3-fold) increased in PD-1^−/− versus WT mice at 6 weeks (p=0.002); however, we did not detect differences in the distribution of CD80⁺, PDL2⁺ or CD80⁺PDL2⁺ IgG⁺ B_mem relative to WT mice. Thus, PD-1 deficiency results in increased expansion of NP-specific IgG⁺ B cells in the early phase of the response to NP-Ficoll and this, as opposed to increased division in the established memory pool, explains increased NP-specific IgG⁺ B_mem cells in PD-1^−/− mice.

In order to determine whether B cell-intrinsic PD-1 expression regulates the generation of TI-2 B_mem, we generated V_HB1–8 Tg mice lacking PD-1 so that adoptive transfers could be used to perform rigorous B_mem analysis. We did not detect differences in the frequencies of NP-specific B cells among splenic and peritoneal B cell populations in naïve V_HB1–8 Tg and PD-1^−/− V_HB1–8 Tg mice (Fig. 2F). We adoptively transferred CD43-depleted CFSE-labeled spleen B cells i.v. and CD43-depleted CFSE-labeled peritoneal cavity B cells i.p. from CD45.1⁺V_HB1–8 Tg or CD45.2⁺PD-1^−/− V_HB1–8 Tg mice into recipient mice (CD45.2⁺ and CD45.1⁺ C57BL/6 WT mice, respectively). Recipient mice were immunized with 10 μg NP₄₀Ficoll on d1. Recipients of PD1^−/− Tg B cells generated significantly increased levels of NP-specific IgG (3-fold) relative to recipients of PD-1^+/+ Tg B cells (Fig. 2G) as well as increased IgM^a (p=0.05) on d10 (the peak of the response). Differences in IgG production were not observed when PD-1^−/− V_HB1–8 Tg B cells were transferred into muMT versus PD-1^−/− muMT mice (Supplemental Fig. 1C). Moreover, differences in Ab production were not observed when PD-1^−/− V_HB1–8 Tg B cells were transferred into WT versus PD-1^−/− mice (Supplemental Fig. 1D), supporting that PD-1 expression on Ag-specific B cells plays a major role in the inhibitory effect PD-1 has in regulating IgG responses to TI-2 Ags.

We next assessed whether B cell-intrinsic PD-1 expression regulated the formation of B_mem. As shown in Fig. 2H, recipients of PD-1^−/− Tg B cells had significantly more NP-specific B cells in spleen, inguinal lymph node (LN), peritoneal cavity (PerC) and bone marrow (BM), as well as significantly increased CD138⁺ Ab-secreting cells (ASC) in spleen, LN, and BM 21 days post immunization. In addition, recipients of PD-1^−/− Tg B cells had significantly more CD138^negCSFE^lo NP-specific memory B cells. The frequencies of spleen and lymph node IgM⁺ B_mem were 2-fold higher and BM frequencies were 25-fold higher, indicating PD-1 inhibits IgM⁺ B_mem generation (Supplemental Figure 1E). The frequency of IgG⁺CD138^neg memory cells was also significantly increased in recipients of PD-1^−/− Tg B cells, with 3-fold higher frequencies in the spleen and 8- and 20-fold higher frequencies in the PerC and BM, respectively (Fig. 2I). At 5 weeks post immunization, PD-1^−/− Tg IgG⁺CD138^neg B cell frequencies remained significantly increased over PD-1-sufficient Tg B cells (Supplemental Fig. 1F); however, the frequencies of CD80⁺PDL2⁺ cells among and PD-1-deficient Tg IgM⁺ and IgG⁺ memory B cells were similar (Supplemental Fig. 1G). Consistent with ELISA results, PD-1^−/− Tg B cells also gave rise to significantly increased CD138⁺IgG⁺ NP-specific ASC frequencies (Fig. 2I), although these frequencies were at least 10-fold lower than CD138^negIgG⁺ NP-specific B cell frequencies. Thus, B cell-intrinsic PD-1 expression inhibits primary NP-specific IgM and IgG production and the generation of both non-switched and IgG⁺ B_mem in response to NP-Ficoll.

PD-1 negatively regulates boosting of TI-2-specific Ab responses

The failure to boost is a key characteristic of TI-2 Ab responses. Ag-specific IgG plays a role in suppressed boosting (18, 19), but it is probable that additional mechanisms are involved. PD-1 is upregulated on naïve and memory V_HB1–8 Tg B cells following NP-Ficoll encounter in vivo (19) and NP-specific B cells in normal mice also upregulate PD-1 in response to both primary and secondary immunization (Supplemental Fig. 1H). Nonetheless, NP-specific B cells in immune mice fail to become properly activated, as evidenced by limited increases in CD86, CD44, size (FSC), and expansion relative to the primary responses made by naïve B cells (Supplemental Fig. 1H–I). Given the upregulation of PD-1 on reactivated B_mem, we next investigated the extent to which PD-1 might suppress recall responses.

To examine whether B cell-intrinsic PD-1 expression suppresses secondary responses in a manner independent of its regulation of B_mem formation, we generated V_HB1–8 Tg and PD-1^−/− V_HB1–8 Tg B_mem in primary WT recipients (19), and then harvested, purified and transferred CD43^negCD138^neg NP-specific memory spleen B cells into naïve mice or mice previously immunized with 1 μg NP-Ficoll (d-35) (Fig. 3A). We compared responses of NP-specific B cells in naïve and immune recipients 4 days post immunization. V_HB1–8 Tg B_mem upregulated CD86 in recipient mice that were immunized for the first time, as did PD-1^−/− Tg B_mem, although PD-1^−/− B cells upregulated CD86 to significantly higher levels (Fig. 3B; gray bars). In contrast, V_HB1–8 Tg B_mem poorly upregulated CD86 in recipient mice that received secondary immunization after B_mem transfer (Fig. 3B, black bars). In fact, levels were not significantly increased over that for immune control recipient mice which had only been immunized 35 days prior and were not boosted (Fig. 3B, white bars). However, PD-1^−/− Tg B_mem in immune recipient mice receiving secondary immunization upregulated CD86 to only a slightly lower degree than PD-1^−/− Tg B_mem in recipient mice that had been immunized for the first time (Fig. 3B, black bars versus gray bars). Four days post immunization, a 4-fold increase in V_HB1–8 Tg and PD-1^−/− V_HB1–8 Tg NP-specific B cell frequencies was observed in naïve recipients that were immunized at the time of transfer (Fig. 3C, gray bars). However, significant increases in NP-specific Tg B cells were not observed in recipients that were previously immunized and boosted, whereas significant increases were observed in boosted memory recipients of PD-1^−/− V_HB1–8 Tg NP-specific B_mem cells, albeit to a lesser extent than that found for naïve recipients receiving Ag for the first time. Significant increases in PD-1^−/− Tg IgG⁺ NP-specific B cell frequencies relative to control mice that were not boosted at the time of transfer were also observed in recipients that were immunized for the first time or boosted, whereas these increases were diminished in recipients of PD-1-sufficient Tg B_mem (Fig. 3D). IgG Ab production from Tg B_mem was too low to distinguish from endogenous responses, but IgM^a responses were evident. IgM^a production was diminished in recipients that had been previously immunized and boosted relative to naïve recipients, consistent with the effect of Ag-specific Ab in suppressing secondary responses (Fig. 3E). Nonetheless, PD-1^−/− Tg B cells generated significantly greater increases in NP-specific IgM^a over non-boosted recipients, indicating B cell-intrinsic PD-1 expression plays a major role in inhibiting secondary memory B cell activation, expansion, and Ab production, even in the presence of Ag-specific Ab.

Figure 3. PD-1 negatively regulates IgG⁺ B_mem expansion, ASC differentiation, and Ab production during boosting.

A-E) CFSE-labeled V_HB1–8 Tg and PD-1^−/− V_HB1–8 Tg B cells (10⁷ spleen and 10⁵ peritoneal B) were transferred into primary WT recipients followed by immunization with 25 μg NP₄₀Ficoll to generate B_mem. On day 35, V_HB1–8 Tg and PD-1^−/− V_HB1–8 Tg CD43^negCD138^negB_mem were CFSE-labeled and transferred into naïve WT recipients or two groups of WT recipients that had been immunized 35 days prior with 1 μg NP₄₀Ficoll (depicted in A). The naïve recipients (gray bars) and one group of previously immunized recipients (black bars) were immunized the next day (day 1) with 1 μg NP₄₀Ficoll. One group of recipient mice that had been immunized 35 days prior to transfer were not boosted (white bars). Five days post transfer, Tg B_mem responses in all three groups were analyzed. B-D) NP-specific B cells in WT recipients of V_HB1–8 Tg or PD-1^−/− V_HB1–8 Tg B_mem were assessed for B) CD86 expression, and increases in total (C) and IgG⁺ (D) B cell frequencies over values in non-boosted recipients of B_mem. E) Increases in NP-specific serum IgM^a in WT recipient mice 5 days post transfer relative to recipient mice that were not immunized on d1. Asterisks indicate significant differences from recipient mice that were not boosted on d1 (n=3–4 mice/group). Hashtags (#) represent differences between comparisons for WT versus PD-1^−/− Tg B cell recipients, as indicated by bars. F-K) V_HB1–8 Tg and PD-1^−/− V_HB1–8 Tg B_mem were generated using intact NP₄₀-Ficoll immunized V_HB1–8 Tg and PD-1^−/− V_HB1–8 Tg mice. On d30, CFSE-labeled IgG⁺CD138^neg B cells from spleen (10⁵) and peritoneal cavities (5 × 10³) were transferred into naïve WT recipients. One recipient group of each was immunized with 1 μg NP₄₀-Ficoll one day later, whereas the other group was not immunized (F). Six days post transfer, NP-specific IgG⁺ B cells (G), IgG⁺ CD138⁺ Ab-secreting cells (H) and NP-specific serum IgG (I), IgM^a (J), and total IgM (K) concentrations were assessed for immune (black bars) and naïve (white bars) recipient mice (n=4/group). In I-K, NP-specific Ig levels are also shown for control naïve and immune mice that did not receive Tg B_mem (“no cells”, n=3 mice/group). Results represent 2 independent experiments. Asterisks indicate significant differences between the indicated groups (p<0.05).

To specifically determine the extent to which PD-1 regulates the ability of IgG⁺ memory B cells to respond to secondary Ag encounter in the absence of Ag-specific Ab, we again generated V_HB1–8 Tg and PD-1^−/− V_HB1–8 Tg B_mem, this time using intact V_HB1–8 Tg and PD-1^−/− V_HB1–8 Tg mice. On day 30, we positively selected IgG⁺ B cells from spleen and PerC of Tg mice and transferred equal numbers of NP-specific IgG⁺ B_mem into naïve WT recipients (Fig. 3F). We immunized half of the recipients with NP-Ficoll (1 μg) and assessed cell expansion, differentiation, and Ab production 5 days later. As shown in Figure 3G, 5 days following immunization, NP-specific IgG⁺ V_HB1–8 Tg B_mem frequencies increased 2-fold over frequencies in naïve recipients, whereas PD-1^−/− V_HB1–8 Tg memory B cells increased 20-fold. PD-1^−/− Tg B_mem also generated 6-fold greater IgG⁺CD138⁺ ASC (Fig. 3H) and 3-fold greater NP-specific serum IgG (Fig. 3I) over V_HB1–8 Tg B_mem. As expected, NP-specific IgM^a was not detected above background (Fig. 3J) and recipients in all three groups produced comparable levels of NP-specific (endogenous) IgM (Fig. 3K). Collectively, these data demonstrate B cell-intrinsic PD-1 deficiency results in increased TI-2 IgG⁺ B_mem expansion, ASC differentiation, and IgG production.

Finally, we examined the extent to which PD-1 would regulate B_mem reactivation in immune recipients previously immunized with a higher (10 μg) dose of NP-Ficoll. V_HB1–8 Tg B_mem and PD-1^−/− V_HB1–8 Tg B_mem were generated in WT mice and then co-transferred into naïve or previously immunized recipients which were then immunized 1 day after transfer. IgG⁺ PD-1^−/− V_HB1–8 Tg B_mem frequencies were increased 55-fold 4 days following immunization of naïve mice versus 25-fold in previously immunized recipients, relative to B_mem frequencies in recipient mice that were not immunized after transfer (Supplemental Figure 1J). By comparison, WT IgG⁺V_HB1–8 Tg B_mem frequencies increased 30-fold in naïve versus 10-fold in previously immunized recipients. In recipients immunized for the first time, PD-1^−/− V_HB1–8 Tg IgG⁺CD138⁺ ASC cells were increased 60-fold over WT Tg B_mem (Supplemental Fig. 1J). However, PD-1^−/− V_HB1–8 Tg IgG⁺CD138⁺ cells were decreased 30% in recipients that were boosted at the time of transfer. Thus, PD-1^−/− V_HB1–8 Tg B_mem exhibit significantly increased Ag-induced reactivation (ie., expansion and ASC generation in IgG⁺ cells) relative to PD-1-sufficient V_HB1–8 Tg B_mem, but are partially constrained in the memory environment.

PDL1 and PDL2 negatively regulate TI-2 B_mem generation

To determine the extent to which PD-1 ligands, PDL1 and PDL2, regulate B_mem formation to TI-2 Ags, we assessed endogenous B_mem formation in PDL1^−/− and PDL2^−/− mice. Twenty-one days post NP₄₀-Ficoll immunization, the frequency and number of IgG⁺ NP-specific CD138^neg B cells in spleens were significantly increased in PDL1^−/− (2-fold) relative to WT mice (Fig. 4A). The frequencies and numbers of unswitched CD138^neg cells were only ~30% higher in immunized PDL1^−/− mice relative to WT mice (Fig. 4B). PDL2^−/− mice also had significantly increased frequencies (~65% higher) of IgG⁺CD138^neg B cells relative to WT mice (Fig. 4A). Differences were not apparent in the peritoneal cavity among groups of immune mice (Fig. 4A–B). Importantly, differences in NP-specific unswitched CD138^neg or IgG⁺CD138^neg B cells were not observed among WT, PDL1^−/−, and PDL2^−/− naive mice (Fig. 4A–B). Notably, CD80 expression was significantly lower on IgG⁺ B_mem from PDL1^−/− mice relative to WT and PDL2^−/− mice (Fig. 4C)—a finding possibly related to the physical association between PDL1 and CD80 (33). Thus, PDL1 and to a lesser extent, PDL2, significantly limit the generation of IgG⁺ TI-2 B_mem, and PDL1 significantly impacts the phenotype of TI-2 B_mem through promoting CD80 expression.

Figure 4. PDL1 and PDL2 negatively regulate TI-2 B_mem generation.

A-C) Endogenous B_mem responses were assessed in WT, PDL1^−/−, and PDL2^−/− mice 21 days post immunization with 10 μg NP₄₀-Ficoll. Frequencies and numbers of NP-specific spleen and peritoneal cavity IgG⁺CD138^neg B cells (A) and IgG^negCD138^neg B cells (B) and CD80 expression by IgG⁺CD138^neg NP-specific B cells (C) from WT, PDL1^−/− and PDL2^−/− mice (n=4–5 mice/group). Similar results were obtained in an independent experiment using 5 mice/group. D-E) WT, PDL1^−/−, and PDL2^−/− recipients of CFSE-labeled V_HB1–8 Tg naive CD43⁻ spleen (10⁷ i.v.) and peritoneal cavity (5 × 10⁵ i.p.) B cells were immunized with 10 μg NP₄₀-Ficoll. D) Serum NP-specific IgM^a and IgG was quantified in recipient (black icons) and non-recipient control mice (matching gray icons for IgG, d21). E) IgG⁺ CD138^negCFSE^lo V_HB1–8 Tg B_mem numbers in recipient mice 21 days post immunization. F) CD80 expression on IgG⁺ CD138^negCFSE^lo V_HB1–8 Tg B_mem. Results representative of 2 independent experiments (n=8 mice/group). Significant differences between PDL1^−/− or PDL2^−/− and WT mice are indicated by asterisks (*, p<0.05).

PDL1 is constitutively expressed by B cells and PDL2 is upregulated on activated B cells and both are significantly increased on B_mem in TD responses (36). This upregulation occurs on TI-2 Ag-specific B_mem along with other markers, including CD80 and CD73 (19) as well as IL5Rα (CD125) (Supplemental Fig. 1K). To determine whether expression of PDL1 and/or PDL2 on cells other than Ag-specific B cells regulate TI-2 B_mem formation, we transferred naïve CFSE-labeled V_HB1–8 Tg cells into WT, PDL1^−/−, and PDL2^−/− mice and assessed B_mem formation in response to NP-Ficoll immunization. PDL1^−/− recipients produced significantly more NP-specific IgG than WT mice, whereas PDL2^−/− recipients gave intermediate IgG responses (Fig. 4D). IgG⁺ B_mem generation was significantly increased (2-fold) in both PDL1^−/− and PDL2^−/− recipients relative to WT recipients (Fig. 4E). Of note, CD80 expression on V_HB1–8 Tg NP-specific B_mem was not different among WT, PDL1^−/−, and PDL2^−/− recipients (Fig. 4F), indicating B cell-expressed PDL1 supports CD80 expression on TI-2 B_mem. No difference in memory generation was observed when PD-1^−/− mice served as recipients (Supplemental Fig. 1L). Transfer of PD-1^−/− V_HB1–8 Tg B cells into WT, PDL1^−/−, PDL2^−/− recipients also did not produce significant differences in B_mem generation, although B_mem frequencies and numbers were somewhat increased in PDL2^−/− recipients (Supplemental Fig. 1M). These data support that PDL1 and PDL2 limit generation of V_HB1–8 Tg TI-2 IgG⁺ memory B cells due to their expression on a cell type other than V_HB1–8 Tg B cells (ie., endogenous recipient cells) and interactions with PD-1 expressed on B cells. This result does not exclude the possibility that B cell-expressed PDL1 and PDL2 may also regulate these responses, either positively or negatively.

PDL1 and PDL2 negatively regulate B_mem reactivation and B_mem-expressed PDL2 contributes to inhibition

We next examined the extent to which PDL1 and PDL2 regulate B_mem reactivation. PDL1 and PDL2 are both expressed by TI-2 B_mem, with B-1b cells harboring the greatest frequency of PDL2-expressing B_mem (19). Using an in vitro reactivation assay of V_HB1–8 Tg memory B-1b cells, we assessed the effects of PDL1 and PLD2 mAb blockade on memory B-1b cells. As shown in Fig. 5A, the NP-specific B_mem population was significantly increased in bulk cell cultures in the presence of NP₄₀-Ficoll and control IgG mAb relative to non-stimulated cultures. PD-1 and PDL1+PDL2 mAb blockade significantly increased the number of divided NP-specific B_mem cells (Fig. 5A). The number of NP-specific IgG⁺ and CD138⁺ cells also significantly increased (at least 3-fold) in response to PD-1, PDL1, PDL2 and PDL1+PDL2 mAb blockade relative to control mAb cultures (Fig. 5A). In cultures of purified CD19⁺ B cells containing V_HB1–8 Tg NP-specific B_mem, PDL1 and PDL2 blockade similarly increased the number of NP-specific IgG⁺ cells, CD138⁺ cells, and IgG⁺ CD138⁺ cells relative to control mAb (Fig. 5B). No effect was found for Abs alone in the absence of NP-Ficoll (Supplemental Fig. 1N), and we did not observe effects on PD1^−/− V_HB1–8 Tg B cells (Supplemental Fig. 1O). Thus, this in vitro data suggests PD-1, PDL1, and PDL2 negatively regulate the generation of IgG⁺ and CD138⁺ B cells from TI-2 B_mem following Ag reactivation, and that, along with B cell-expressed PD-1, B cell expression of PDL1 and PDL2 may participate in this inhibition.

Figure 5. PDL1 and PDL2 negatively regulate TI-2 B_mem reactivation, with B_mem-expressed PDL2 playing an important role in inhibition.

A-B) CFSE-labeled total cells (A) or CD19⁺ selected B cells (B) from V_HB1–8 Tg mice immunized (i.p.) 2–3 months prior with 25 μg NP-Ficoll were cultured in media alone or with NP₄₀-Ficoll (1 ng/ml) in combination with control IgG (LTF-2), PD-1 (RMP1–14), PDL1 (10F.9G2), or PDL2 (Ty25) blocking mAbs (2 μg/ml). Numbers of total, divided, IgG class switched and CD138⁺ NP-specific B cells were determined by flow cytometry after 4 days of culture. Significant differences from IgG control cultures are indicated by asterisks (*, p<0.05). Results from one experiment are shown for A and B, with mean numbers (±SEM) for triplicate cultures indicated. Similar results were obtained in 3 independent experiments for both A and B. C-E) As depicted in the schematic shown in C, V_HB1–8 Tg B_mem were generated in WT recipients immunized with 25 μg NP-Ficoll. On day 21, V_HB1–8 Tg B_mem (4 to 10 × 10⁶ total donor PerC cells) were transferred into naïve WT recipients (D, n=5–8 mice/group) as well as PDL1^−/− and PDL2^−/− recipients (E, n=8–10 mice/group). Recipients received 200 μg of PDL1 and/or PDL2 blocking or control IgG mAbs on days 1 and 3 post 1 μg NP₄₀-Ficoll immunization. Serum NP-specific IgG2a was assessed on days 0, 7, 14, and 21. Results are pooled from 2 independent experiments. Ab levels that are significantly different from WT control mAb-treated mice are indicated by asterisks (*, p<0.05) and significant differences between control and PDL mAb-treated mice of the same genotype are indicated by hashtags (#).

Given our in vitro results, we next examined the extent to which PDL1 and PDL2 regulated B_mem reactivation and Ab production in vivo. We transferred naïve V_HB1–8 Tg cells into naïve WT recipients, immunized with NP-Ficoll, and 21 days later transferred V_HB1–8 Tg B_mem (enriched for IgM⁺PDL2⁺ memory B1b cells (19)) into new naïve WT recipients. We then immunized recipient mice, which were given control, PDL1, PDL2 or PDL1+PDL2 blocking mAbs (depicted in Fig. 5C). Given that the total IgG responses fell in the normal range of responses made by non-reconstituted mice, we measured NP-specific IgM^a and IgG2a levels, as these allotypes are only produced by Tg B cells. As shown in Fig. 5D, PDL1 and PLD2 mAb blockade had variable effects on IgM^a secretion by B_mem, with NP-specific IgM^a levels for co-blockade recipients declining more sharply than control mAb-treated mice. Recipients given PDL1 and PDL2 single blockade had lower IgM^a but significantly increased NP-specific IgG2a levels on day 7 relative to control mAb-treated mice (~30-fold). However, PDL1 and PDL2 co-blockade had the most notable effect on IgG2a and increased day 7 levels by 100-fold (Fig. 5D), with levels remaining significantly elevated over that of control mAb recipients out to day 21. We did not detect NP-specific IgG2a in WT recipients of Tg cells that had not been immunized. Thus, PDL1 and PDL2 both inhibit B_mem reactivation and Ab production in vivo, with the strongest increases in IgG production observed when both ligands are blocked.

To determine the extent to which PDL1 and PDL2 expression by Ag-specific B_mem versus other cell types controls B_mem reactivation and Ab production in vivo, we adoptively transferred V_HB1–8 Tg B_mem into PDL1^−/− and PDL2^−/− recipient mice using the same strategy employing blocking mAbs as described above. B_mem produced significantly more IgG2a in PDL1^−/− recipient mice given control mAb relative to WT control recipients (Fig. 5E). However, this was not further increased when PDL1^−/− recipient mice were given PDL1 blocking mAb; in fact, IgG2a levels declined. This result supports Ag-specific B cell-extrinsic PDL1 expression plays a key role in negatively regulating TI-2 B_mem recall responses, whereas Ag-specific B cell PDL1 expression may have a more complex role in regulating responses, one of which may include regulating CD80 expression, which inhibits TI-2 B_mem recall (19). NP-specific IgG2a levels were also increased in PDL2^−/− mice given control mAb relative to WT control mAb-treated recipients (Fig. 5E), indicating non-Ag-specific B cell PDL2 expression also limits B_mem recall responses, either directly or through alterations in the PDL2^−/− environment (34). However, PDL2 blockade significantly increased NP-specific IgG2a responses in PDL2^−/− mice, indicating Ag-specific B_mem-intrinsic PDL2 expression plays an important role in suppressing B_mem IgG production. Collectively, these results support PDL1-mediated inhibition of TI-2 B_mem reactivation primarily occurs through interactions between B_mem and other cell types expressing PDL1, whereas PDL2 expression on B_mem plays a key role in limiting B_mem reactivation.

PD-1 deficiency results in increased boosting to pneumococcal polysaccharides encountered with vaccination and infection

By mechanisms that have not been elucidated, Ag-specific IgG suppresses B_mem from producing Ab in response to secondary Ag encounter (18, 19). Our results presented thus far indicate PD-1 negatively regulates the ability of B_mem to produce IgG in response to secondary Ag encounter when Ag-specific Ig is lacking (Fig. 3I), as well as IgM in weakly immune WT recipients of V_HB1–8 Tg IgM⁺ B_mem (Fig. 3E). We further examined the ability of PD-1^−/− mice to produce increased Ab following boosting using different TI-2 Ags. In response to low dose TNP₆₅-Ficoll, some boosting was observed in WT mice (d40 vs. d30), but PD-1^−/− mice generally produced greater increases in IgG after boosting (Fig. 6A). TNP₆₅-Ficoll generally produced modest boosting in PD1^−/− mice, albeit to a greater extent than in WT mice. Similarly, immunization of V_HB1–8 Tg mice with 25 μg NP-Ficoll followed by boosting with 100 μg NP-Ficoll failed to increase NP-specific IgM and IgG levels, whereas significant increases in both IgM and IgG were observed in PD-1^−/− V_HB1–8 Tg mice (Fig. 6B). In response to PPS3, PD-1^−/− mice generated more IgM following boosting relative to WT mice regardless of route, and in some cases, more IgG based on pairwise analysis, although increases were modest (Fig. 6C).

Figure 6. PD-1 negatively regulates boosting to distinct TI-2 Ags.

A) WT and PD-1^−/− mice were immunized with TNP₆₅-Ficoll (1 μg i.p.; 5–6 mice/group), and boosted with an equivalent dose 4 weeks later, with TNP-specific serum IgM and IgG levels immediately prior to (d30) and 10 days after boosting (d40) measured by ELISA. B) V_HB1–8 Tg and PD-1^−/− V_HB1–8 Tg mice were immunized with 25 μg NP-Ficoll on d0 and boosted with 100 μg on d30. Results are pooled from 2 independent experiments. C) WT and PD-1^−/− mice were immunized with 1 μg PPS3 i.p. (4–5 mice/group) or i.m. (5–6 mice/group) and boosted with an equivalent dose 4 weeks later, with PPS3-specific IgM and IgG levels immediately prior to (d30) and 10 days after boosting (d40) measured by ELISA. D) WT and PD-1^−/− mice were immunized once with a 0.5 μg PPS3 dose-equivalent using Pneumovax23 or Prevnar13 (>5 mice/group) and four weeks later infected with serotype 3 Streptococcus pneumoniae strain WU2 via i.p. (10⁴ CFU) or intranasal (10⁷ CFU) route, respectively. Fold-changes in serum PPS3-specific IgM and IgG levels were assessed two weeks following infection relative to levels present immediately prior to infection. In A-D, significant differences from WT mice are indicated by asterisks (*, p<0.05) and significant differences between d30 and d40 values for mice of the same genotype are indicated by hashtags (#).

A failure of B_mem to differentiate into ASC following infection is a problem encountered in infections with encapsulated bacteria, such as Streptococcus pneumoniae. Consistent with this, Pneumovax23- and Prevnar13-immunized WT mice do not exhibit significant increases in PPS3-specific IgM and IgG following serotype 3 infection (Fig. 6D). However, we found PPS3-specific IgG levels significantly increase following S. pneumoniae infections in PD-1^−/− mice (1.5 to 3-fold). These results indicate PD-1 plays a role in inhibiting secondary responses to TI-2 Ags, including pneumococcal polysaccharides, even when encountered in the context of infection.

An adjuvant significantly augments boosting of protective PPS-specific Ab in the context of PD-1 deficiency

Based on the results showing PD-1^−/− mice exhibit PPS3-specific Ab boosting following pneumococcal infections, we hypothesized PD-1 deficiency and pathogen molecular pattern (PAMP) stimulation of TI-2-reactive B cells might enable greater enhances in IgG production relative to either pathway alone. Our previous study demonstrated TLR4-based adjuvants enhance B cell responses to TI-2 Ags, including boosting, through TLR4 expressed on B cells (24). We therefore assessed responses to TNP-Ficoll in WT and PD-1^−/− mice when an MPL/TDCM-containing adjuvant (MT) was included. As shown in Figure 7A, both WT and PD-1^−/− mice generated significantly increased (3 to 4-fold) TNP-specific IgM responses when adjuvant was included. Adjuvant-supported IgM boosting was similar between WT and PD-1^−/− mice. Primary TNP-specific IgG responses were increased with adjuvant in WT and PD-1^−/− mice; however, responses were significantly higher (2-fold) in PD-1^−/− mice relative to WT mice (Fig. 7A). PD-1^−/− V_HB1–8 Tg B cells also produced significantly more (4-fold) NP-specific IgG in WT recipients that were immunized with adjuvant plus NP-Ficoll relative to those immunized with NP-Ficoll alone (Supplemental Fig. 1P), indicating the adjuvant effects on increasing IgG production were also observed when PD-1-deficiency was limited to Ag-specific B cells. Secondary TNP-specific IgG responses were moderately increased with adjuvant inclusion in both WT and PD-1^−/− mice, although responses did not differ, likely due to the high levels of primary Ag-specific IgG that suppress responses. Thus, this data demonstrates PD-1 deficiency results in enhanced adjuvant effects on primary IgG responses to haptenated Ficoll.

Figure 7. An adjuvant significantly augments protective pneumococcal polysaccharide-specific IgG boosting in the context of PD-1 deficiency.

A-C) WT and PD-1^−/− mice were immunized i.p. with 1 μg TNP₆₅-Ficoll, Pneumovax23 containing 0.125 μg each PPS, or these Ags mixed with an adjuvant consisting of 20 μg MPL and TDCM in 0.4% squalene. Mice were boosted on day 29 (5–7 mice/group). TNP (A), Pneumovax (B), and PPS3 (C)-specific IgM and IgG levels were assessed by ELISA. Significant differences between groups are indicated by asterisks (*p<0.05). D) Pooled d56 sera from immunized WT and PD-1^−/− mice was given i.p. (0.5 μl/mouse) along with 200 CFU S. pneumoniae, strain WU-2, i.p. in 100 μl PBS to mice lacking CD19 (n=9 mice/group). Mice were monitored for morbidity and mortality, with survival results analyzed using Kaplan-Meier curves and Log-rank analysis (p value indicates significant difference from non-treated mice). Results are pooled from two independent experiments (n=9 mice/group).

The MT adjuvant increased IgM responses to PPSs within Pneumovax23 to a similar extent in both WT and PD-1^−/− mice (2–5-fold; Fig. 7B). However, the adjuvant significantly augmented Pneumovax-specific IgG responses in PD-1^−/− mice over that found for WT mice. Similar results were obtained when PPS3-specific IgM and IgG responses were analyzed, although differences between adjuvant groups were more striking in the boost response, with PD-1^−/− mice producing as much as 8-fold higher PPS3-specific IgG relative to PD-1^−/− mice that did not receive adjuvant and ~3-fold higher IgG levels relative to WT mice that had received adjuvant (Fig. 7C). We assessed the protective capacity of sera from these mice in a model of lethal serotype 3 S. pneumoniae challenge in CD19^−/− mice, which lack protective natural anti-pneumococcal Ab (35). Significantly increased survival was achieved using optimal (2 μl) quantities of pooled sera from Pneumovax + adjuvant-immunized PD-1^−/− (100%, n=6) and WT (66%, n=6) mice relative to untreated mice (0% survival, data not shown). However, at lower serum transfers (0.5 μl), only sera from Pneumovax + adjuvant-immunized PD-1^−/− mice was protective (Fig. 7D), demonstrating the significantly enhanced protective capacity that is achieved when PD-1-deficiency and adjuvant are combined in the context of Pneumovax immunization. Thus, a B cell-activating adjuvant enhances IgG responses to TI-2 Ags, including protective anti-pneumococcal polysaccharide Ab levels, and does so in a more potent manner when PD-1 inhibition is relieved.

Discussion

The lack of boosting to native polysaccharide-based vaccines following revaccination or infection is a barrier to establishing protection against infectious diseases. Our comprehensive study highlights the critical role the PD1-PDL regulatory pathway plays in negatively regulating recall responses to TI-2 Ags. First, we demonstrate PD-1 and its ligands, PDL1 and PDL2, inhibit the generation of TI-2 B_mem, with the most potent effects observed on IgG⁺ B_mem. This regulation is carried out through interactions between PD-1 upregulated on Ag-activated B-cells and PDL1, and to a lesser extent, PDL2, expressed on other cells. Further, we demonstrate TI-2 IgM⁺ and IgG⁺ B_mem upregulate PD-1 upon secondary Ag encounter and its interactions with either PDL2 expressed on B_mem or PDL1 and/or PDL2 on other cell types, significantly decreases B_mem reactivation, expansion, class-switching, and Ab production. The potential for TI-2 B_mem to suppress their own reactivation through PD-1-PDL2 interactions reveals a novel form of auto-regulation not previously described. Finally, we demonstrate a B-cell activating adjuvant enables significant boosting of protective IgG responses to polysaccharide Ags when PD-1 inhibition is relieved. Collectively, our study 1) demonstrates a central role for B cell-intrinsic PD-1 in negative regulation of B_mem formation and reactivation in response to polysaccharide Ags and 2) reveals combinatorial strategies aimed at relieving PD-1-mediated inhibition and further potentiating polysaccharide-specific B cell activation may improve protective responses to polysaccharide-based vaccines and the infections they are designed to prevent.

Evidence supporting the generation of B_mem in response to TI-2 Ags has been formally shown using adoptive transfers of NP-specific B cells from V_HB1–8 Tg mice (18, 19). Although the factors controlling TI-2 B_mem generation are poorly understood, our work demonstrates B cell intrinsic PD-1, which is transiently upregulated on TI-2 Ag-activated B cells, limits establishment of B_mem along with class switching and IgG production in response to TI-2 Ags (26, 27). Consistent with this, the generation of switched B_mem is most affected by PD-1 regulation. In contrast to findings for TI-2 responses, PD-1 expressed by T cells (T follicular helper and regulatory cells) actually promotes B_mem generation in response to TD Ags whereas B cell-expressed PD-1 appears to play a minor role (36–38). This can be explained by the critical role B cell Ag-receptor signaling has in driving TI-2 Ag-dependent responses where cognate T cell help is lacking. PD-1 co-engagement with the BCR suppresses major signaling processes required for division through recruitment of SHP-2 and/or SHP-1 phosphatases ((26, 39) and unpublished observations). PD-1 may also impact bystander T cell help by suppressing expression of costimulatory molecules such as CD86 (26, 27, 40), as well as the expression of additional receptors required for survival, switching, and differentiation. Our work supports that interactions between Ag-specific B cell-intrinsic PD-1 and B cell-extrinsic PDL1 suppress B_mem generation, although this does not exclude a role for B cell-expressed PDL1. PDL2 expression is much more restricted; however, based on our results, PDL2 also limits B_mem generation. Of note, PDL2^−/− mice have significantly increased levels of IL-5 (34) which supports TI-2 Ag-induced IgG switching and Ab production (1). This could impact B_mem (which express higher levels of IL5Rα) independently of PD-1, as could PDL2 interactions with its alternative ligand, repulsive guidance molecule b (RGMb) (41). Indeed, the trend for increased B_mem generation by PD-1^−/− V_HB1–8 Tg B cells in PDL2^−/− recipients relative to WT recipients raises this as a possibility that warrants further investigation.

PD-1 is also upregulated upon B_mem encounter with TI-2 Ag and thus has the potential to downregulate Ag receptor-induced signals in B_mem upon engagement with PDL1 and/or PDL2, both of which contribute to negative regulation of TI-2 B_mem reactivation. The expression of both PDL1 and PDL2 by TI-2 B_mem renders these cells capable of self-regulation due to homotypic interactions likely involving both Ag recognition and PD-1-PDL engagement. In vitro experiments with purified B cells support this possibility. Evidence for B_mem-intrinsic PDL2 expression in inhibiting B_mem activation was provided by both in vitro experiments and in vivo by using PDL2 ligand blocking mAb in PDL2^−/− mice reconstituted with PDL2⁺ B_mem. While B cell-expressed PDL1 was found to reduce B_mem reactivation in vitro, PDL1 expression on non-Ag-specific B cells appeared to play a more dominant role in vivo, as V_HB1–8 Tg B_mem produced significantly higher levels of Ab in PDL1^−/− relative to WT recipient mice. In fact, PDL1 mAb blockade in PDL1^−/− recipient mice reduced Ab production by Ag-activated PDL1⁺ B_mem. It is possible this could have been due to the depleting effects of 10F.9G2 (a rat IgG2b mAb previously shown to have this effect (42)) on PDL1⁺ B_mem in the PDL1-deficient environment. PDL1 also has the potential to regulate reactivation via its association with CD80 expressed by B_mem. In the absence of PDL1, B_mem express low levels of CD80. The results of our previous study indicated an inhibitory role for CD80 in B_mem reactivation (19). Work on other cell types support a role for PDL1-CD80 in cis interactions in diminishing CD80-CTLA4 and PDL1-PD-1 interactions in trans, but enabling CD80 interactions with CD28 (33, 43). In the case of CD80⁺PDL2⁺ B_mem, PDL1 associated with CD80 on B_mem may shift PD-1-PDL1 interactions to PD-1-PDL2 interactions. PDL2 has a 3-fold higher affinity for PD-1 than PDL1 (44), and this may contribute to greater inhibition than that afforded by PDL1 interactions in the context of homotypic interactions. The extent to which interactions between B_mem-expressed PDL2 and B_mem-expressed PD-1 regulate B_mem reactivation remains to be fully established. Interactions between PD-1 on B_mem and ubiquitously expressed PDL1 provides an additional mechanism by which B_mem interactions with other cell types regulates reactivation. Our work thus far points to a strong inhibitory influence of TNF receptor ligand family members (CD80, PDL1, and PDL2) in TI-2 B_mem reactivation—regulation that is distinct from the roles these ligands have in regulating TD B_mem (21, 36, 45). However, our understanding of the complex interactions among these receptors and their ligands both in cis and in trans, and their impact on the heterogenous TI-2 B_mem population is far from complete. Although the role(s) PD-1 and its ligands play in regulating B cell activation and function have not been fully elucidated, evidence thus far points to a role for PD-1 as a true checkpoint regulator which tempers T cell-independent B cell activation, and in particular IgG production and IgG⁺ B_mem generation, when cognate T cell help or danger signals are lacking.

Ag-specific IgG has been shown to also potently suppress TI-2 B_mem reactivation (18, 19). While the mechanisms responsible have not been fully elucidated, epitope suppression and/or Ag clearance may be involved. In either case, TI-2 Ag crosslinking strength would be diminished and result in ineffective activation, as is supported by our finding of decreased blasting, CD86 upregulation, and expansion by B_mem in the memory environment. Nonetheless, when Ag-specific Ig levels fall, some persisting B_mem become responsive to Ag and produce Ab (19). Increasing Ag dose and/or providing additional B cell activation signals may help achieve the activation required to fully promote Ag-specific B_mem cell division, switching, and differentiation when Ag-specific Ig levels remain high. Consistent with this, priming with low dose TI-2 Ag (haptenated Ficoll) in the presence of the MPL/TDCM-containing adjuvant enables greater boosting when a higher Ag dose is used in conjunction with adjuvant during secondary immunization (24). As evidenced by our study, overriding PD-1 inhibition may also lower the threshold for Ag receptor engagement in both primary and secondary responses. Thus, when combined with a B cell-activating adjuvant or infection bearing B cell-activating PAMPs, the effects of ablating PD-1 inhibition may synergize to significantly increase TI-2 Ag-specific Ab. The increases in pneumococcal polysaccharide-specific IgG achieved using this combinatorial approach were physiologically meaningful in a lethal type 3 S. pneumoniae challenge model, whereby sera from adjuvant-treated PD-1^−/− mice provided complete protection at limiting doses but sera from WT mice was ineffective. In summary, our findings demonstrate a key role for PD-1 and its ligands in regulating the formation and functional reactivation of TI-2 Ag-specific B_mem and highlight new avenues to modulate protective Ab responses against TI-2 Ags relevant for human health.

Key points:

• B cell-intrinsic PD-1 expression inhibits TI-2 B_mem formation and reactivation.

• PDL2 expressed by TI-2 B_mem contributes to self-suppression of reactivation.

• Adjuvant enables robust IgG boosting to PPS in the context of PD-1 deficiency.