Programmed cell death 1 suppresses B-1b cell expansion and long-lived IgG production in response to T cell-independent type 2 antigens.
ABSTRACT B-1b cells play a key role in producing Abs against T cell-independent type 2 Ags. However, the factors regulating Ab production by this unique B cell subset are not well understood. In this study, a detailed analysis of the B cell response to 2,4,6-trinitrophenol (TNP)-Ficoll was performed using normal mice. TNP-Ficoll delivered i.p. or i.v. induced rapid Ag-specific B-1b cell activation, expansion, isotype switching, and plasmablast/plasma cell differentiation. Ag-specific B-1b cell numbers peaked at day 5 and then gradually declined in the spleen but remained elevated in the peritoneal cavity beyond 40 d postimmunization. In addition to expressing CD43, CD44, and CD86, Ag-activated B-1b cells transiently expressed programmed cell death 1 (PD-1), which functionally suppressed BCR-induced B-1b cell in vitro proliferation when additional costimulatory signals were lacking. Inhibiting PD-1:PD-1 ligand interactions during TNP-Ficoll immunization significantly enhanced Ag-specific B-1b cell expansion and the frequency of IgG isotype switching and plasmablast/plasma cell differentiation. Remarkably, PD-1 mAb blockade during the first week following immunization resulted in significantly increased numbers of both splenic and bone marrow Ag-specific IgG3-secreting cells, but not IgM-secreting cells, at both early (day 5) and late (week 6) time points. Moreover, Ag-specific serum IgG3 levels, as well as IgG2c, IgG2b, and IgA levels, remained significantly elevated in PD-1 mAb-treated mice relative to control Ab-treated mice for ≥6 wk postimmunization. Thus, PD-1:PD-1 ligand interactions occurring shortly after initial T cell-independent type 2 Ag encounter play a critical role in suppressing Ag-specific B-1b cell expansion and the development of long-term IgG-producing bone marrow and spleen cells.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: There are multiple, distinct B-cell populations in human beings and other animals such as mice. In the latter species, there is a well-characterized subset of B-cells known as B1 cells, which are enriched in peripheral sites such as the peritoneal cavity but are rare in the blood. B1 cells can be further subdivided into B1a and B1b subsets. There may be additional B1 subsets, though it is unclear if these are distinct populations or stages in the developmental process to become mature B1a and B1b cells. A limitation in understanding B1 subsets is the relative paucity of specific surface markers. In contrast to mice, the existence of B1 cells in human beings is controversial and more studies are needed to investigate the nature of these enigmatic cells. Examples of B1b antigens include pneumococcal polysaccharide and the Vi antigen from Salmonella Typhi, both used routinely as vaccines in human beings and experimental antigens such as haptenated-Ficoll. In addition to inducing classical T-dependent responses some proteins are B1b antigens and can induce T-independent (TI) immunity, examples include factor H binding protein from Borrelia hermsii and porins from Salmonella. Therefore, B1b antigens can be proteinaceous or non-proteinaceous, induce TI responses, memory, and immunity, they exist in a diverse range of pathogenic bacteria, and a single species can contain multiple B1b antigens. An unexpected benefit to studying B1b cells is that they appear to have a propensity to recognize protective antigens in bacteria. This suggests that studying B1b cells may be rewarding for vaccine design as immunoprophylactic and immunotherapeutic interventions become more important due to the decreasing efficacy of small molecule antimicrobials.Frontiers in Immunology 01/2014; 5:535.
- [Show abstract] [Hide abstract]
ABSTRACT: Formation of the splenic marginal zone (MZ) depends on the alternative NF-κB signaling pathway. Recently, we reported that unrestricted activation of this pathway in NF-κB2/p100-deficient (p100(-/-) ) knock-in mice alters the phenotype of MZ stroma and B cells. Here, we show that lack of the p100 inhibitor resulted in an expansion of both MZ B and peritoneal B-1 cells. However, these cells failed to generate proliferating blasts in response to T-cell-independent type 2 (TI-2) antigens, correlating with dampened IgM and absent IgG3 responses. This phenotype was in part due to increased activity of the NF-κB subunit RelB. Moreover, p100(-/-) →B6 bone marrow chimeras were more susceptible to infection by encapsulated Streptococcus pneumoniae bacteria, pathogens that induce TI-2 responses. In contrast to the TI-2 defect, p100 deficiency did not impair immune responses to the TI-1 antigen LPS and p100(-/-) MZ B cells showed normal antigen transportation into B-cell follicles. Furthermore, p100(-/-) MZ B and B-1 cells failed to respond to TI-2 antigens in the presence of wild-type accessory cells. Thus, NF-κB2/p100 deficiency caused a predominant B-cell-intrinsic TI-2 defect that could largely be attributed to impaired proliferation of plasmablasts. Importantly, p100 was also necessary for efficient defense against clinically relevant TI-2 pathogens.European Journal of Immunology 11/2013; · 4.52 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Ingestion of B. anthracis spores causes gastrointestinal (GI) anthrax. Humoral immune responses, in particular, IgA secreting B-1 cells, play a critical role in the clearance of GI pathogens. Here, we investigated whether B. anthracis impacts the function of colonic B-1 cells to establish active infection. GI anthrax infection led to significant inhibition of immunoglobulins (e.g., IgA) and increased program death-1 (PD-1) on B-1 cells. Furthermore, infection also diminished type 2 innate lymphoid cells (ILC2) and their ability to enhance differentiation and immunoglobulin production by secreting IL-5. Such B-1 cell and ILC2 dysfunction is potentially due to cleavage of p38 and Erk1/2 MAPK in these cells. Conversely, mice that survived infection generated neutralizing antibodies via the formation of robust germinal center B cells in Peyer's patches and had restored B-1 and ILC2 function. These data may provide additional insight for designing efficacious vaccines and therapeutics against such a deadly pathogen.The Journal of Infectious Diseases 05/2014; · 5.85 Impact Factor
of June 13, 2013.
This information is current as
Cell-Independent Type 2 Antigens
Production in Response to T
Cell Expansion and Long-Lived IgG
Programmed Cell Death 1 Suppresses B-1b
Karen M. Haas
2011; 187:5183-5195; Prepublished online 14
, 36 of which you can access for free at:
cites 69 articles
is online at:
The Journal of Immunology
Information about subscribing to
Submit copyright permission requests at:
Receive free email-alerts when new articles cite this article. Sign up at:
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists, Inc. All rights reserved.
Copyright © 2011 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month by
The Journal of Immunology
by guest on June 13, 2013
The Journal of Immunology
Programmed Cell Death 1 Suppresses B-1b Cell Expansion
and Long-Lived IgG Production in Response to
T Cell-Independent Type 2 Antigens
Karen M. Haas
B-1b cells play a key role in producing Abs against T cell-independent type 2 Ags. However, the factors regulating Ab production
by this unique B cell subset are not well understood. In this study, a detailed analysis of the B cell response to 2,4,6-trinitrophenol
(TNP)-Ficoll was performed using normal mice. TNP-Ficoll delivered i.p. or i.v. induced rapid Ag-specific B-1b cell activation,
expansion, isotype switching, and plasmablast/plasma cell differentiation. Ag-specific B-1b cell numbers peaked at day 5 and then
gradually declined in the spleen but remained elevated in the peritoneal cavity beyond 40 d postimmunization. In addition to
expressing CD43, CD44, and CD86, Ag-activated B-1b cells transiently expressed programmed cell death 1 (PD-1), which
functionally suppressed BCR-induced B-1b cell in vitro proliferation when additional costimulatory signals were lacking. Inhibit-
ing PD-1:PD-1 ligand interactions during TNP-Ficoll immunization significantly enhanced Ag-specific B-1b cell expansion and the
frequency of IgG isotype switching and plasmablast/plasma cell differentiation. Remarkably, PD-1 mAb blockade during the first
week following immunization resulted in significantly increased numbers of both splenic and bone marrow Ag-specific IgG3-
secreting cells, but not IgM-secreting cells, at both early (day 5) and late (week 6) time points. Moreover, Ag-specific serum IgG3
levels, as well as IgG2c, IgG2b, and IgA levels, remained significantly elevated in PD-1 mAb-treated mice relative to control Ab-
treated mice for ‡6 wk postimmunization. Thus, PD-1:PD-1 ligand interactions occurring shortly after initial T cell-independent
type 2 Ag encounter play a critical role in suppressing Ag-specific B-1b cell expansion and the development of long-term IgG-
producing bone marrow and spleen cells.The Journal of Immunology, 2011, 187: 5183–5195.
niae, an important cause of localized and systemic life-threatening
infections (1). TI-2 Ags, such as pneumococcal polysaccharides,
are often carbohydrate structures consisting of repeating epitopes
that extensively cross-link Ag-specific BCRs and induce Ab pro-
duction in the absence of MHC class II-restricted T cell help (2).
Numerous pathogens are known (3–9) or suspected (4) to display
TI-2 Ags. Ab responses to TI-2 Ags are elicited rapidly, yield
persistent titers, and may offer significant protection. For example,
the Pneumovax vaccine, composed of 23 pneumococcal poly-
saccharides, provides significant protection against invasive pneu-
mococcal disease in adults, with titers lasting for ∼10 y (10). None-
theless, TI-2 Ags can often present unique challenges to vaccine
development, including modest Ag-specific IgG production and
impaired Ab responses in neonates (11–13). Thus, a better un-
umoral immune responses to T cell-independent (TI)
type 2 (TI-2) Ags are critical for protective immunity to
encapsulated bacteria, such as Streptococcus pneumo-
derstanding of the mechanisms regulating TI-2 Ab production is
necessary to develop enhanced TI-2 Ag-based vaccines.
Ab responses to TI-2 Ags differ in multiple respects to those
elicited by T cell-dependent (TD) Ags. Importantly, the B cell
subsets producing Ab in response to TI-2 versus TD Ags differ.
B-1b cells produce Ab responses to classical carbohydrate TI-2
Ags, including pneumococcal polysaccharide (14), nitrophenol
(NP)-Ficoll (15), and a-1,3 dextran (16), as well as protein-based
TI Ags present on clinically relevant pathogens (17–20). B-1a and
marginal zone (MZ) B cells also contribute to TI Ab production
(21–23). This is in contrast to TD Ab responses, in which follic-
ular B cells largely contribute to Ab production. The accessory
signals required for optimal TD and TI-2 Ab responses also differ.
TI-2 Ab responses can ensue in the absence of cognate T cell help,
whereas TD Ab responses are dependent on T cell-derived signals.
As these signals drive somatic hypermutation, class switching,
and B memory cell formation, TI-2 Ags, as well as TI-1 Ags
(supplying additional activating signals), induce limited-affinity
maturation and isotype switching (IgG3 in mouse and IgG2 in hu-
man) and an unconventional type of memory (15–17, 24). Hence,
the factors modulating TI-2 Ag-dependent B cell activation, pro-
liferation, isotype switching, and differentiation may differ from
those involved in TD Ag-dependent B cell responses.
Humoral responses to TI-2 Ags also rely heavily on distinct
BCR-signaling pathways (25, 26), as well as key regulators of
these pathways. Numerous cell surface receptors that regulate
BCR signaling, including programmed cell death 1 (PD-1), have
been implicated in regulating TI-2 Ab responses. PD-1, a member
of the B7/CD28 family, is expressed by Ag-specific B cells shortly
after TI-2 Ag immunization (27) and is well-documented to neg-
atively regulate AgR signaling on both B and T cells following
engagement of its ligands, PD-L1 and PD-L2 (28, 29). PD-12/2
mice generate enhanced IgG3 production in response to the TI-2
Department of Microbiology and Immunology, Wake Forest University School of
Medicine, Winston-Salem, NC 27157
Received for publication July 8, 2011. Accepted for publication September 8, 2011.
This work was supported in part by National Institutes of Health Grant
R21AI095800-01 and Wake Forest School of Medicine.
Address correspondence to Dr. Karen M. Haas, Department of Microbiology and
Immunology, Wake Forest University School of Medicine, Room 5112, Gray Build-
ing, Winston-Salem, NC 27157. E-mail address: firstname.lastname@example.org
The online version of this article contains supplemental material.
Abbreviations used in this article: 7AAD, 7-aminoactinomycin D; ASC, Ab-secreting
cell; BM, bone marrow; cRPMI, complete RPMI; Ft-LPS, Francisella tularemia
LPS; MZ, marginal zone; NP, nitrophenol; PAMP, pathogen-associated molecular
pattern; PD-1, programmed cell death 1; PDL, programmed cell death 1 ligand;
TD, T cell dependent; TI, T cell independent; TI-2, T cell independent type 2;
by guest on June 13, 2013
Ag, DNP-Ficoll, and exhibit multiple immune abnormalities, in-
cluding moderate myeloid and lymphoid hyperplasia, hyperre-
sponsive B cells, and decreased CD5 expression on peritoneal B-1
cells that may be due to dysregulated CD5 expression and/or in-
creased B-1b cell numbers (29). It is unclear whether increased
TI-2 Ab responses in PD-12/2mice are due to one or more of
these preexisting abnormalities or due to PD-1 regulatory effects
that occur at the time of immunization. Thus, the role of PD-1:
PD-1 ligand (PDL) interactions in regulating TI-2 Ab responses
Studies investigating factors regulating TI-2 Ag responses have
used pathogen-derived Ags, including pneumococcal polysac-
charides, as well as synthetic Ags, such as haptenated Ficoll,
which, in contrast to pathogen-derived Ags, are free of contami-
nating pathogen-associated molecular patterns (PAMPs) that can
supply additional immunomodulatory signals. The synthetic Ag,
2,4,6-trinitrophenol (TNP)-Ficoll, an inert copolymer of sucrose
and epichlorohydrin conjugated to TNP, has been used for decades
as a prototypic TI-2 Ag. Recent studies using knockout mice with
deficiencies in select B cell populations suggested that MZ B cells
play a key role in the humoral immune response to TNP-Ficoll (30,
31). Nonetheless, because mice lacking this subset remain able to
produce anti–TNP-Ficoll Ab responses (27), alternative popula-
tions may participate in humoral responses to this commonly
In this study, Ag-specific B cell activation, expansion, differ-
entiation, and Ab production in response to TNP-Ficoll were ex-
amined using normal mice. In contrast to BCR transgenic mice,
normal mice are advantageous in that they express a broad Ab
repertoire and unaltered B cell subset distribution, both of which
may be important factors in shaping TI-2 Ab responses. Impor-
tantly, in the current study, Ag-specific B-1b cells were found to be
a major B cell population that responded to TNP-Ficoll, regardless
of immunization route. In response to immunization, Ag-specific
B-1b cells selectively increased in number; expressed multiple
markers of activation, including PD-1; and underwent isotype
switching and expressed CD138, a marker of plasmablast/cell
differentiation. Data generated using a mAb to block PD-1 from
interacting with its ligands at the time of immunization provide
evidence that PD-1:PDL interactions suppress Ag-specific B-1b
cell expansion, isotype switching, and overall B cell Ab pro-
duction in response to TI-2 Ags. Collectively, the results of this
study support a key role for PD-1 in regulating B-1b cell responses
and long-lived Ab production against TI-2 Ags.
Materials and Methods
Experiments were performed on 2–3-mo-old wild type C57BL/6 mice (The
Jackson Laboratory, Bar Harbor, ME) or CD192/2mice (14) housed under
specific pathogen-free conditions. All studies and procedures were ap-
proved by the Wake Forest University Animal Care and Use Committee.
Immunizations, ELISAs, and ELISPOTs
TNP-Ficoll–specific B cell expansion and PD-1 upregulation experiments
were performed on mice immunized i.p. or i.v. with 50 mg TNP65-Ficoll
(Biosearch Technologies, Novato, CA). For serum Ab analyses, mice were
immunized i.p. with 25 mg TNP65-Ficoll. Ags were diluted in sterile PBS
and injected in a final volume of 200 ml. In some experiments, mice were
administered PD-1 mAb (RMP1-14; low endotoxin/no azide format;
BioLegend, San Diego, CA) or control rat IgG (Southern Biotechnology
Associates, Birmingham, AL) in 200 ml sterile PBS via i.p. injection with
200 mg mAb on day 0 and 100 mg mAb on days 3 and 5.
ELISAs were as described (27, 32). Serum samples were diluted in TBS
containing 1% BSA (Sigma Chemical, St. Louis, MO). TNP-specific Ab
levels were measured by adding diluted serum samples to plates that had
been coated with 5 mg/ml TNP-BSA (Biosearch Technologies) in 0.1 M
borate buffered saline overnight at 4˚C. Alkaline phosphatase-conjugated
polyclonal goat anti-mouse IgM, IgG1, IgG2c, IgG2b, IgG3, IgG, and IgA
Abs (all from Southern Biotechnology Associates) and p-nitrophenyl
phosphate (Sigma) were used to detect Ag-specific Ab.
ELISPOTs were performed on total splenocytes and bone marrow
(BM) cells. ELISPOT 96-well plates (Immobilon P; Millipore, Billerica,
MA) were precoated with TNP-BSA (5 mg/ml) in PBS overnight at 4˚C,
washed two times with PBS, and blocked for 1 h at 37˚C with complete
RPMI (cRPMI) 1640 containing 10% FCS (Life Technologies BRL). Cells
were plated at a concentration of 106to 107cells/ml in cRPMI 1640 con-
taining 10% FCS and cultured for 18 h. Alkaline phosphatase-conjugated
polyclonal goat anti-mouse IgM and IgG3 Abs (Southern Biotechnology
Associates) were used in conjunction with NBT/5-bromo-4-chloro-3-indolyl
phosphate substrate (Promega, Madison, WI), according to the manufac-
turer’s instructions. Membranes were dried, and spots were enumerated.
Abs and immunofluorescence analysis
lysis buffer (0.15 M NH4Cl/0.01 M KHCO3). Peritoneal cells were isolated
by lavaging the peritoneal cavity with 10 ml PBS. Single-cell blood, spleen,
lymph node, and peritoneal cavity leukocyte suspensions (2 3 107/ml) were
incubated in PBS containing 2% bovine calf serum with 20 mg/ml TNP30–
Ficoll–Fluorescein8(Biosearch Technologies) for 30 min at room temper-
ature, followed by subsequent staining with fluorochrome-labeled mAbs
on ice for 25 min. Biotinylated- or fluorochrome-conjugated Abs and
secondary-detection reagents used included anti-mouse IgM and IgG3
(Southern Biotechnology Associates); Abs reactive with mouse CD1d
(1B1), CD5 (53-7.3), B220 (RA3-6B2), CD11b (M1/70), CD23 (B3B4),
CD19 (6D5), CD44 (IM7), and CD86 (GL1) (all from BioLegend); CD19
(ID3), CD80 (16-10A1), CD21/35 (7G6), and PD-1 (J43) mAb (all from
eBioscience); and CD138 (BD Biosciences). Cells were analyzed using
FACSCalibur and FACSCantoII flow cytometers (Becton Dickinson, San
Jose, CA). Positive and negative cell populations were determined using
unreactive isotype-matched Abs (BioLegend and eBioscience), and data
were analyzed using FlowJo analysis software (Tree Star).
B cell-proliferation assays
CD52B cell subsets were purified from peritoneal cavity lavage by a
negative-depletion procedure. Macrophages were removed by plate ad-
herence in RPMI 1640 containing 5% FCS (1 h at 37˚C, 5% CO2). Non-
adherent cells were depleted of Thy1.2+cells using magnetic bead
depletion (Dynal). Thy1.22cells were further depleted using biotinylated
F4/80, GR1, DX5, and CD5 mAbs (BioLegend) in conjunction with
magnetic depletion using Biotin binder beads (Dynal). In some experi-
ments, CD11b+B cells were further purified using Miltenyi bead purifi-
cation. Purities were typically ∼85–95% B cells. Purified peritoneal B cells
were labeled with CFSE (0.6 mM) using Vybrant’s CFDA SE Cell Tracer
Kit (Invitrogen), according to the manufacturer’s instructions. Cells (2 3
106/ml) were cultured in cRPMI 1640 medium containing 10% FCS (Life
Technologies Certified serum, Invitrogen) for 4 d in medium alone or in
the presence of 1 mg/ml biotinylated F(ab9)2anti-mouse IgM Ab (Jackson
ImmunoResearch, West Grove, PA). In some cultures, 2 mg/ml bio-
tinylated PD-1 mAb (J43; eBioscience) or biotinylated Armenian hamster
IgG (eBioscience) was added, along with 5 mg/ml streptavidin (Sigma).
LPS (Escherichia coli 0111:B4; Sigma) and anti-mouse CD40 (HM40-3;
BD Biosciences) were also used. Cells were harvested on day 4 and stained
with fluorochrome-labeled mAbs against CD11b and B220, as well as 7-
aminoactinomycin D (7AAD) and Annexin V-PE (BD Biosciences). An
equal number of CD11b+B220+events were collected using a FACSCali-
bur instrument, and data were analyzed using FlowJo analysis software.
Data are shown as mean 6 SEM. Differences between sample means were
assessed using the Student t test.
TNP-specific B cell activation and expansion following TNP-
As early as 3 d post–TNP-Ficoll immunization, significant increases
in both the frequency and number of TNP-specific (B220+) B cells
were observed in both the peritoneal cavity and spleen (Fig. 1A),
as previously demonstrated (27). Five days postimmunization, Ag-
specific B cell frequencies and numbers peaked in the spleen (Fig.
5184 PD-1 REGULATION OF B-1b CELL RESPONSES TO TI-2 Ags
by guest on June 13, 2013
cytometric analysis and enumeration of TNP30-Fl-Ficoll–binding (Ag-specific) cells from naive and immune mice (50 mg TNP65-Ficoll administered i.p.).
A, Left panels, Representative flow cytometric analysis of Ag-specific B220+splenic and peritoneal B cells in naive and immune (day-3) mice. Right panels,
Frequencies and numbers of splenic and peritoneal Ag-binding B cells from days 0–35 postimmunization (n $ 5 mice/group). B, Phenotype of splenic and
peritoneal Ag-specific B cells in naive (shaded graphs) and immune (day-5; thick line) mice. Isotype control binding for Ag-specific B cells from immune
mice is indicated by the dashed line. C, B220 and CD11b expression by blood, spleen, peritoneal cavity, and lymph node Ag-specific cells at days 0 and 5
postimmunization. D, CD5 and CD11b expression on spleen Ag-specific B cells at days 0 and 35 postimmunization. E, Frequencies and numbers of Ag-
specific splenic and peritoneal B-2 (B220+CD11b2CD52), B-1b (B220+CD11b+CD5lo-neg), and B-1a (B220+CD11b+CD5+) cells in naive and immune
mice. Activation marker (CD43, CD44, CD80, and CD86; F) and IgG3 (day-5; G) expression by Ag-specific CD11b+and CD11b2B cells in spleen (F, G)
and peritoneal cavity (G) following TNP-Ficoll immunization. Isotype-control binding is shown for Ag-specific cells from immune mice (F). All results are
representative of data obtained with at least three mice/group. Data in A and E represent mean (6 SEM). *p , 0.05, naive versus immune mice.
Ag-specific B cell phenotype, activation, differentiation, and expansion kinetics in response to TNP-Ficoll immunization. A–G, Flow
The Journal of Immunology 5185
by guest on June 13, 2013
1A). However, by 35 d postimmunization, splenic Ag-specific B cell
numbers were only increased ∼20% over numbers in naive animals
(Fig. 1A). In contrast, elevated Ag-specific peritoneal B cell fre-
quencies and numbers did not decrease following the day-5 time
point, but they remained significantly increased over naive levels
beyond 35 d postimmunization. The increases observed in TNP-
Ficoll–binding B cells following immunization were likely due to
Ag-specific binding as opposed to FcR binding of TNP-specific Ab,
because stripping B cells of any FcR-bound Ab by 3 min of in-
cubation with 50 mM glycine buffered saline (pH = 3) (33) did not
significantly alter the frequency of TNP-Ficoll–binding B cells (99 6
4% of no-treatment control, n = 4). Thus, TNP-Ficoll immuni-
zation rapidly increased Ag-specific B220+B cell numbers in the
spleen and peritoneal cavity, with numbers gradually contracting
in spleen but remaining elevated in the peritoneal cavity 5 wk after
The phenotype, activation, and differentiation status of Ag-
specific B cells were assessed following TNP-Ficoll immuniza-
tion. Relative to Ag-binding B cells in naive mice, Ag-specific
splenic and peritoneal B cells in immune mice (day 5) had in-
creased forward scatter and side scatter, as well as increased CD86,
CD44, and CD43 expression, indicative of activation (Fig. 1B,
data not shown). In addition, Ag-specific splenic B cells in im-
mune mice had unchanged CD1dintexpression, reduced levels
of CD21/35, CD23, and B220, and increased levels of CD19. A
fraction of Ag-specific B220+B cells in the spleen had also un-
dergone isotype switching to IgG3 and expressed CD138, in-
dicative of plasmablast differentiation (Fig. 1B, data not shown).
Thus, TNP-Ficoll immunization induces Ag-specific peritoneal
and splenic B cell activation and differentiation, with a substantial
population of splenic Ag-specific B cells expressing a B220lo
Ag-specific B-1b cells are a major B cell population
responding to TNP-Ficoll immunization
The B220loCD19hiCD1dintCD21/35lophenotype is common to
B-1 cells. Thus, the expression pattern of additional B-1 markers,
CD11b and CD5, was examined for Ag-specific cells. In naive
mice, Ag-specific peritoneal B cells were mostly CD11b+and
expressed either intermediate (CD5int) or very low levels of CD5
(CD5neg-lo), characteristic of B-1a and B-1b cells, respectively
(Fig. 1B), whereas naive splenic TNP-specific B cells were CD52
and CD11b2. Following i.p. immunization, CD11b+Ag-specific
B cell frequencies increased in the peritoneal cavity, spleen, blood,
and lymph nodes (Fig. 1B, 1C). These cells had decreased B220
expression levels. In addition, increases in Ag-specific B cells in
the spleen and peritoneal cavity expressing a CD5neg-lophenotype
were observed following immunization (Fig. 1B). Importantly,
the level of CD5 expressed by Ag-specific B cells from immune
mice was lower than that expressed by peritoneal B-1a cells and
was comparable to levels present on peritoneal B-1b cells (Fig.
1B, Supplemental Fig. 1A). Interestingly, Ag-specific B cells
expressing CD5 were not found in naive CD192/2mice, but they
were identified in TNP-Ficoll–immunized CD192/2mice (Sup-
plemental Fig. 1B), which lack B-1a cells (14). Thus, B cells
responding to TNP-Ficoll express a CD11b+CD5neg-lophenotype.
The phenotype of Ag-specific B cells (CD11b+B220loCD19hi
CD1dintCD21/35loCD5neg-lo) responding to TNP-Ficoll was simi-
lar to that of peritoneal B-1b cells. Therefore, changes in Ag-
specific B-1b cell frequencies, activation, isotype switching, and
differentiation were specifically evaluated following TNP-Ficoll
immunization. Relative to naive numbers, Ag-specific B-1b cell
numbers were significantly increased in spleen (16-fold) and
peritoneal cavities (3-fold) 5 d following immunization, in con-
trast to Ag-binding B-1a and B-2 cells, which were not signifi-
cantly altered (Fig. 1E). Ag-specific peritoneal B-1b cell numbers
were increased further (12-fold) at day 35. Ag-specific CD11b-
expressing CD52cells were still detectable in the spleen 35 d
postimmunization (Fig. 1D) and were still significantly greater
in frequency and number (2.5-fold) than in naive mice at this
time point (Fig. 1E). Ag-specific CD11b+(B-1b) B cells were
the predominant subset activated by immunization, as evidenced
by increased levels of CD43, CD44, CD86, and CD80 expression
on this subset relative to CD11b2B cells (Fig. 1F). Moreover, Ag-
specific B-1b cells selectively underwent isotype switching to
IgG3, and these cells were found in both the spleen and peritoneal
cavity (Fig. 1G). Finally, Ag-specific CD138+CD11b+plasma-
blasts were observed in the spleen but not the peritoneal cavity.
Nonetheless, a large fraction of Ag-specific B220loCD138+cells
were CD11b2(data not shown), consistent with the loss of this
marker upon differentiation to Ab-secreting cells (ASCs) (34–36).
Thus, Ag-specific B-1b cells become activated, expand, undergo
class switching, and differentiate into splenic ASCs in response to
Ag-specific B-1b cells are the major B cell population
responding to TNP-Ficoll immunization, regardless of the route
of Ag delivery
To assess whether the Ag-specific B cell responses to TNP-Ficoll
were dependent on the route of Ag delivery, mice were immunized
with TNP-Ficoll by i.v., s.c., or i.p. injection. Five days post-
immunization, Ag-specific peritoneal and spleen B cell frequen-
cies, numbers, and phenotypes were analyzed. Increases in total
Ag-specific splenic B cell frequencies over naive mice (0.47 6
0.02%; p , 0.05) were not significantly different between mice
immunized i.v. (1.15 6 0.13%) or i.p. (1.00 6 0.04%; data not
shown). Similarly, the increases observed in total Ag-specific
peritoneal B cell frequencies over naive mice (0.75 6 0.08%;
p , 0.05) were not significantly different between mice immu-
nized i.v. (1.60 6 0.22%) or i.p. (1.67 6 0.31%; data not shown).
Subcutaneous immunization elicited weaker, but nonetheless signif-
icant, increases in Ag-specific splenic B cell frequencies (0.63 6
0.11% versus 0.47 6 0.02% for naive; p , 0.05) and increases
in Ag-specific peritoneal B cell frequencies (1.05 6 0.12% ver-
sus 0.75 6 0.08% for naive). Thus, i.p. and i.v. immunizations
elicit similar increases in Ag-specific splenic and peritoneal
B cell frequencies 5 d following immunization, whereas s.c. im-
munization elicits a weaker response.
Ag-specific B cell phenotypes were assessed to determine
whether the Ag-specific B cell subset(s) responding to TNP-Ficoll
were altered by the route of Ag delivery. As shown in Fig. 2A and
2B, the frequencies of CD21intCD1dint, CD21loCD1dint, CD21lo-int
CD1dlo, and CD21hiCD1dhiAg-binding splenic B cells were ex-
amined. Significant increases in CD21loCD1dint
B cells (.4-fold) and CD21intCD1dloAg-specific B cells ($2-
fold) were observed relative to naive mice (Fig. 2B). These
increases were comparable between mice immunized i.p. or i.v.
(p . 0.05). Ag-specific CD21intCD1dint(mainly follicular B cells)
and CD21hiCD1dhi(MZ B cells) frequencies were not signifi-
cantly changed 5 d following i.p. or i.v. TNP-Ficoll immunization.
Moreover, CD11b+Ag-specific cells appeared in CD21loCD1dint
and CD21lo-intCD1dlopopulations following immunization, re-
gardless of whether TNP-Ficoll was delivered by i.p. or i.v. route
(Fig. 2C). In contrast, Ag-specific CD21intCD1dintand MZ B cell
populations exhibited minimal CD11b expression following im-
munization. Notably, no significant difference was observed in
the frequencies of Ag-specific CD11b2splenic B cells in mice
immunized i.p. versus i.v. (data not shown). Thus, i.p. and i.v.
5186PD-1 REGULATION OF B-1b CELL RESPONSES TO TI-2 Ags
by guest on June 13, 2013
TNP-Ficoll immunization elicits similar increases in CD11b+
Ag-specific splenic B cells that coexpress a CD21loCD1dintor
Consistent with the results above, i.v. and i.p. immunization
resulted in similar increases in Ag-specific splenic B-1b (CD19+
CD11b+CD5neg-lo) cell frequencies and numbers (Fig. 2D; .10-
fold) over naive mice. Subcutaneous immunization also increased
Ag-specific splenic B-1b cell frequencies and numbers over naive
mice, albeit to a lesser extent (data not shown). Remarkably, i.v. and
i.p. immunization induced similar increases in Ag-specific perito-
neal B-1b cell frequencies, although the numbers were slightly
higher in i.p. immunized mice (Fig. 2D). As shown in Fig. 2E,
similar frequencies of the Ag-specific splenic B cell pool had un-
dergone isotype switching to IgG3 in i.p. and i.v. immunized mice.
Moreover, regardless of the route of immunization, the majority of
Ag-specific IgG3+B cells coexpressed CD11b+(Fig. 2D). In ad-
dition, similar frequencies of the Ag-specific splenic B cell pool
expressed CD138 in i.p. and i.v. immunized mice (Fig. 2F), and no
differences were observed in the frequencies of Ag-specific CD138+
cells coexpressing CD11b. Thus, i.p. and i.v. TNP-Ficoll immuni-
zations stimulated similar increases in Ag-specific B-1b cell fre-
quencies and numbers in multiple tissues and induced similar
degrees of IgG3 isotype switching and plasmablast/cell differenti-
ation, with comparable participation by Ag-specific B-1b cells.
PD-1 expression is induced on Ag-specific B-1b cells in vivo
Ag-specific B cells express PD-1 3 d following TNP-Ficoll im-
munization (27). As shown in Fig. 3, PD-1 upregulation is largely
analysis and enumeration of splenic and peritoneal Ag-specific B cells from naive and immune mice (50 mg TNP65-Ficoll administered i.p. or i.v.; day 5).
Representative flow cytometric analysis (A) and frequencies (B) of Ag-specific splenic B cells in naive and immune mice expressing a CD21intCD1dint,
CD21loCD1dint, CD21lo-intCD1dlo, and CD21hiCD1dhi(MZ B) phenotype. C, CD11b expression by CD21intCD1dint, CD21loCD1dint, CD21lo-intCD1dlo, and
CD21hiCD1dhiAg-specific cells. D, Frequencies and numbers of splenic and peritoneal Ag-specific B-1b (CD19+CD11b+CD5lo-neg) cells in naive and
immune mice. E, IgG3 expression by Ag-specific splenic B (CD19+) cells (upper panels) and CD11b expression by IgG3+Ag-specific cells (middle
panels). Frequencies of CD19+Ag-specific cells (“Total”) and CD19+CD11b+Ag-specific cells (“CD11b+”) expressing IgG3 are shown for immune mice.
F, CD138 expression by Ag-specific splenic B cells (upper panels) and CD11b and B220 expression by CD138+Ag-specific cells (middle panels).
Frequencies of B220+Ag-specific cells (“Total”) and B220+CD11b+Ag-specific cells (“CD11b+”) expressing CD138 are shown for immune mice. Results
are representative of data obtained with at least three mice/group. Data in B and D–F represent mean (6 SEM). *p , 0.05, naive versus immune mice.
Ag-specific B-1b cells participate in the response to TNP-Ficoll, regardless of whether Ag is delivered i.p. or i.v. A–F, Flow cytometric
The Journal of Immunology 5187
by guest on June 13, 2013
confined to CD11b-expressing (B-1) cells. PD-1 upregulation on
Ag-specific peritoneal B cells was observed as early as 1 d post-
immunization and appeared on blood and spleen B-1b cells by
2 d postimmunization. PD-1 expression levels were highest be-
tween days 2 and 3, decreased by day 5, and were undetectable
by day 9 postimmunization. A similar trend was observed for
CD44 expression (data not shown). Thus, PD-1 is selectively and
transiently induced on Ag-specific B-1b cells following TNP-
PD-1–BCR coengagement suppresses BCR-induced B-1b cell
Similar to in vivo-expression kinetics, PD-1 is induced on cultured
purified peritoneal B-1b cells and spleen B cells between days 1
and 2 post-BCR activation, with peak expression observed on day
3 (Fig. 4A). CD5 expression was similarly induced on BCR-
activated spleen B cells and B-1b cells purified by negative bead
selection (Fig. 4B, Supplemental Fig. 1C, data not shown) or
FACS sorting (Supplemental Fig. 1D, 1E). CD5 expression was
also induced on FACS-purified CD192/2peritoneal B-1b cells
(Supplemental Fig. 1E). Culturing cells in the presence of 5 mg/ml
LPS had little effect on BCR-induced PD-1 expression levels in
B-1b cells and only slightly reduced expression on splenic B cells
(Fig. 4C). Similar to these results with LPS, TNFR superfamily
members (i.e., BlyS receptors and CD40) do not modulate BCR-
induced PD-1 upregulation on B-1b cells (27). In contrast, LPS
suppressed CD5 upregulation on BCR-activated B-1b cells (Fig.
4B), consistent with that previously reported for spleen B cells
(37). Thus, BCR signaling induces PD-1 expression on B-1b cells,
with secondary signals supplied by LPS and TNFR family mem-
bers having little effect on PD-1 upregulation.
To determine the functional consequences of PD-1 engagement
on B-1b cell proliferation induced by BCR signaling, a biotinylated
PD-1 mAb was used in combination with streptavidin to crosslink
PD-1 independently or with the BCR using biotinylated F(ab9)2
goat anti-mouse IgM. Independent PD-1 cross-linking during
BCR activation had little effect on B-1b cell or spleen B cell
proliferation elicited either by anti-IgM or LPS (data not shown).
However, cocross-linking PD-1 with IgM during B cell activation
significantly reduced B-1b cell and spleen B cell proliferation, as
measured by reduced division indices (average number of cell
divisions of entire population) relative to cultures in which a bio-
tinylated isotype control mAb was used in place of PD-1 mAb
(Fig. 4D, 4E). The frequencies of B-1b cells characterized as vi-
able (Annexin V2/7AAD2), early (Annexin V+/7AAD2), or late
in vivo. B220+Ag-specific peritoneal, spleen, and blood CD11b2and
CD11b+cells were evaluated for PD-1 expression (thick line) by flow
cytometry on days 0, 1, 2, 3, 5, 9, and 14 postimmunization with 50 mg
TNP65-Ficoll. Isotype control binding by Ag-binding cells is indicated by
the shaded area.
PD-1 expression is induced on Ag-specific B-1b cells
liferation but not survival. A, BCR-induced PD-1 surface expression on
purified peritoneal B-1b cells and spleen B cells. B cells were cultured with
5 mg/ml goat anti-mouse IgM F(ab9)2for the indicated number of days,
harvested, stained with PD-1 mAb, and analyzed by flow cytometry. The
shaded graphs indicate isotype control binding by activated B cells (d2).
PD-1 expression by unstimulated (d3) medium (Med.)-cultured B cells (far
left panels). BCR-induced CD5 (B) and PD-1 (C) upregulation on B-1b
cells (B, C) and splenic B cells (C) activated with 5 mg/ml goat anti-mouse
IgM F(ab9)2in the presence or absence of 5 mg/ml LPS. The shaded graphs
indicate isotype control staining in C. Proliferation and survival in BCR-
activated B-1b cells (D, F) and spleen B cells (E) in the presence and
absence of BCR-PD-1 cocross-linking. Purified CD52peritoneal B or
spleen B cells were labeled with 0.5 mM CFSE and cultured in the pres-
ence of 1 mg/ml biotinylated goat anti-mouse IgM F(ab9)2along with 2 mg/
ml biotinylated Armenian hamster anti-mouse PD-1 (J43) or biotinylated
Armenian hamster IgG control. Streptavidin (5 mg/ml) was added to cul-
tures as a cross-linking agent. At day 4, cells were harvested and stained
with fluorochrome-labeled mAbs against CD11b and B220, with CFSE
loss assessed in the B220+CD11bintperitoneal population (D, F) or splenic
B220+population (E). Apoptosis was assessed using 7AAD and Annexin
V-PE staining. Mean (6 SEM) division indices were calculated from at
least three experiments using FlowJo analysis software (D, E). *p , 0.05,
differences between means. Proliferation for CFSE-labeled peritoneal B-
1b (G) and spleen B cells (H), cultured as in D and E, along with anti-
CD40 (0.5 mg/ml; HM40-3) or LPS (1 mg/ml). Symbols in H represent
division indices for individual mice.
PD-1–BCR coengagement suppresses B-1b cell pro-
5188 PD-1 REGULATION OF B-1b CELL RESPONSES TO TI-2 Ags
by guest on June 13, 2013
(Annexin V+/7AAD+) apoptotic cells were similar between cul-
tures in which PD-1 was cocross-linked with the BCR compared
with cultures in which biotinylated isotype control mAb was used
in place of PD-1 mAb (Fig. 4F). Thus, reduced proliferation was
not due to decreased survival, because the viabilities of B-1b cell
cultures (as well as spleen B cell cultures) subjected to anti–IgM-
PD-1 mAb cross-linking versus anti–IgM-control mAb cross-
linking were not significantly different within experiments (B-1b
cell cultures: 45.6 6 16% versus 41.9 6 13.8%, n = 3 experi-
ments, p . 0.05, paired t test; spleen B cell cultures: 22.4 6 4.7%
versus 23.6 6 6%, n = 5; p . 0.05, paired t test). Finally, co-
stimulation supplied by either CD40 (Fig. 4G, 4H) or LPS (Fig.
4H) prevented PD-1 inhibitory effects on BCR-induced pro-
liferation in both B-1b (Fig. 4G, data not shown) and spleen cells
(Fig. 4H). Thus, PD-1 coengagement with the BCR exerts an in-
hibitory effect on BCR-induced B cell proliferation, but not sur-
vival, which can be overcome by costimulation.
PD-1 mAb blockade significantly increases Ag-specific B-1b
cell numbers and differentiation following TI-2 Ag
Given the expression pattern of PD-1 by Ag-specific B-1b cells
following TNP-Ficoll immunization (Fig. 3) and its inhibitory
effects on primary B cell proliferation (Fig. 4) (38), the effect of
blocking PD-1 from interacting with its ligands during TI-2 Ag
immunization was assessed. This was accomplished using the PD-
1–blocking mAb, RMP1-14. Following TNP-Ficoll immunization,
mice receiving rat IgG control Ab had significantly increased Ag-
specific peritoneal B cell frequencies (1.7-fold) and numbers (2.6-
fold) relative to naive mice (Fig. 5A). As expected, this increase
was largely attributed to significantly increased Ag-specific B-1b
cells, because significant increases were not observed in B-1a
or B-2 subsets (Fig. 5B). However, mice receiving the PD-1–
blocking mAb following immunization had significantly greater
increases in Ag-specific peritoneal B cell frequencies (2.4-fold)
and numbers (4.4-fold) relative to control mice (Fig. 5A). PD-1
mAb treatment significantly increased Ag-specific peritoneal B-1b
cells over mice treated with control Ab 5 d postimmunization, but
it had no effect on other Ag-binding B cell subsets (Fig. 5B).
Although Ag-specific B-1b cell frequencies increased 2.6-fold in
control immune mice, they increased 4-fold in mice receiving PD-
1 mAb. As observed in earlier experiments (Fig. 1E), increases
in Ag-specific peritoneal B-1b cells were still observed out to
40 d following immunization (Fig. 5C). However, mice receiving
PD-1–blocking mAb (at days 1, 3, and 6) exhibited significantly
greater increases in Ag-specific peritoneal B-1b cell frequencies
and numbers relative to mice receiving control Ab (Fig. 5C).
Notably, PD-1 mAb treatment did not influence overall total peri-
toneal B-1b cell frequencies or numbers on days 5 and 40 (Fig.
5D, data not shown). Finally, PD-1 mAb treatment resulted in
significantly increased Ag-specific IgG3+peritoneal B cell fre-
quencies and numbers 5 d postimmunization (Fig. 5F), nearly all
of which expressed CD11b (Fig. 5E). Thus, blocking PD-1 in-
teractions with its ligands during TNP-Ficoll immunization signif-
icantly and selectively increased Ag-specific peritoneal B-1b cell
numbers and IgG3+B cells.
Immunization-induced increases in Ag-specific total B and
B-1b cell frequencies and numbers were not significantly altered
in blood or spleen by PD-1 mAb treatment relative to control Ab-
treated mice 5 or 40 d postimmunization (Fig. 5G, 5H, data
not shown). However, 5 d postimmunization, CD138 (marking
plasmablast/plasma cell differentiation) was expressed by a sig-
nificantly greater frequency of Ag-specific B cells in mice that
received PD-1 mAb compared with mice that received control Ab
(Fig. 5I). Approximately one third of these Ag-specific CD138+
plasmablasts expressed CD11b in both treatment groups at day 5
(data not shown). Moreover, although overall Ag-specific splenic
B cell frequencies were not altered by treatment, the frequency
of remaining Ag-specific splenic B cells that were IgG3+at
40 d postimmunization was significantly increased (2-fold) in
mice that had received PD-1 mAb blockade (0.41 6 0.06%, n = 4)
versus control Ab (0.2 6 0.03%, n = 4; p , 0.05, data not shown).
Thus, blocking PD-1:PDL interactions significantly increased
isotype switching in Ag-specific B cells and the frequency of Ag-
specific B cells committed to producing Ab.
PD-1 mAb blockade significantly increases IgG production
against TI-2 Ags
Because blocking PD-1 interactions with its ligands significantly
promoted increases in Ag-specific B-1b cell numbers, IgG
switching, and differentiation following immunization, the effect
of PD-1 mAb blockade on TI-2 Ab production was assessed. As
shown in Fig. 6, PD-1 blockade had no effect on TNP-specific IgM
levels following TNP-Ficoll immunization. However, PD-1 mAb
blockade significantly enhanced Ag-specific IgG levels. IgG3
production, the dominant isotype produced in response to TNP-
Ficoll, was significantly increased by PD-1 mAb blockade, as
were Ag-specific IgG1, IgG2b, IgG2c, and IgA levels. Notably,
Ag-specific IgG3 and IgG2b levels remained significantly aug-
mented at 6 wk postimmunization in mice that had received
transient early PD-1 mAb blockade. Thus, blocking PD-1:PDL
interactions during the first week following immunization signif-
icantly enhanced the production of isotype-switched Ag-specific
Abs, with IgG3 and IgG2b levels remaining increased up to 6 wk
PD-1 mAb blockade significantly increases both early and
long-term splenic and BM IgG3-producing cells in response to
Evidence suggests that TI-2 Ag-activated, as well as TI-pathogen
activated, B-1b cells may predominantly secrete long-term Ab as
plasmablasts within the spleen (15, 16, 20, 39), although B-1b
cells may also differentiate into long-lived BM plasma cells (40).
ELISPOTs were performed to assess Ab production by splenic
and BM plasma(blast) cells at both early and late time points fol-
lowing TNP-Ficoll immunization. At 5 d postimmunization, TNP-
secreting IgM and IgG3 ASC spleen and BM numbers were sig-
nificantly increased over naive mice (Fig. 7A). Relative to day-5
ASC numbers, day-40 IgM and IgG3 splenic ASC numbers were
diminished, whereas BM ASC numbers were increased (Fig. 7B).
Therefore, given the previous estimation of total BM cellularity in
mice (1.5 3 108) (41) relative to spleen (1 3 108), BM ASCs
likely make a substantial contribution to TNP-specific Ab levels.
Notably, at both time points, IgG3 ASCs accounted for $75% of
splenic IgG ASC numbers and ∼50% of BM IgG ASCs, indicating
this was the major IgG isotype produced in response to TNP-
Ficoll (data not shown). Thus, TNP-Ficoll immunization induces
rapid induction of splenic ASCs and long-term production of IgM
and IgG3 Ab by both splenic and BM ASCs.
The effect of blocking PD-1:PDL interactions, during the first
week following immunization, on ASC numbers at both early (day
5) and late (day 40) time points was assessed. PD-1 mAb treatment
during the first week following TNP-Ficoll immunization signif-
icantly increased ($ 2-fold) IgG3-secreting cells in spleen and
BM at both time points relative to control Ab-treated mice (Fig.
7A, 7B). In contrast, PD-1 mAb blockade did not significantly
alter Ag-specific splenic IgM-producing cell numbers relative
to control Ab-treated mice at either time point (Fig. 7A, 7B),
The Journal of Immunology5189
by guest on June 13, 2013
cells. A–H, Ag-specific B220+frequencies and numbers at days 5 and 40 in mice immunized with 50 mg TNP65-Ficoll i.p. and administered PD-1 (RMP1-
14; black bars) or rat IgG control (gray bars) Abs (200 mg on day 1 and 100 mg on day 3; 100 mg was also given on day 5 for the 40-d experiment). Ag-
specific cells and CD11b, CD5, IgG3, and CD138 expression were assessed by flow cytometry. Peritoneal Ag-specific B220+B cell frequencies and
numbers (A) and Ag-specific peritoneal B-1a, B-1b, and B-2 subset frequencies (B) in naive mice and 5 d postimmunization in mice treated with PD-1 mAb
or rat IgG control. C, Ag-specific peritoneal B-1b cell frequencies and numbers in naive and immune mice 40 d postimmunization. D, Total peritoneal B-1b
cell frequencies at days 0, 5, and 40 postimmunization. Ag-specific IgG3+CD11b+peritoneal B cell frequency plots (E) and Ag-specific B220+IgG3+cell
frequencies and numbers 5 d postimmunization (F). Blood (G) and spleen (H) Ag-specific B-1b cell frequencies and numbers at days 0 and 5 post-
immunization. I, CD138+B220+cell frequencies within the Ag-specific B cell population. The first plot demonstrates the gating strategy for Ag-binding
cells. Data represent mean (6 SEM) (n $ 3–4 mice/group). *p , 0.05.
Blocking PD-1:PDL interactions during TI-2 Ag immunization increases the frequency of Ag-specific B-1b cells, IgG3+cells, and CD138+B
5190PD-1 REGULATION OF B-1b CELL RESPONSES TO TI-2 Ags
by guest on June 13, 2013
although there was a trend toward increased IgM-secreting BM
cells at both time points. Moreover, anti-TNP–specific IgG and
IgG3 levels, but not IgM levels, were significantly increased in 7-d
cultures of spleen cells from PD-1 mAb-treated mice relative to
control Ab-treated mice (Fig. 7C), consistent with increased IgG-
secreting cell frequencies found in these mice. Thus, blocking PD-
1:PDL interactions during the early stages of TI-2 Ag encounter
significantly increased the number of IgG3-secreting splenic and
BM ASCs found at both early (day 5) and late (day 40) time points
In this study, nontransgenic normal mice were used to analyze
the activation, phenotypic alterations, and expansion kinetics of
Ag-specific B cells in response to a defined prototypic TI-2 Ag.
B cells responding to TNP-Ficoll expressed a phenotype consistent
with activated B-1b cells, as was true for isotype-switched Ag-
specific B cells (Fig. 1). Importantly, immunization induced Ag-
specific B-1b cells to selectively upregulate PD-1 (Fig. 3). PD-1
was shown in multiple studies to suppress Ag-specific T cell
zation significantly increases the production of isotype-switched Ab. TNP-
specific serum IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, and IgA levels in
mice immunized with 25 mg TNP-Ficoll and administered PD-1 or rat IgG
control Abs, as in Fig. 5. Data represent mean (6 SEM) (n $ 3–4 mice/
group). Similar results were obtained in an independent immunization
experiment. *p , 0.05, control Ab versus PD-1 mAb.
Blocking PD-1:PDL interactions during TI-2 Ag immuni-
early and long-term Ag-specific IgG3-secreting cell numbers in spleen and
BM. TNP-specific IgM and IgG3-secreting cells (ASCs) in spleen and BM
5 days (A) and 40 days (B) postimmunization, as determined by ELISPOT.
C, TNP-specific IgM, IgG, and IgG3 levels secreted by total splenocytes
(harvested 5 d postimmunization) cultured in cRPMI 1640 + 10% FCS for
7 d. Culture supernatants were diluted 1:3 in TBS containing 1% BSA and
assessed for TNP-specific Abs by ELISA. Mice were immunized and
administered PD-1 or rat IgG control Abs, as in Fig. 5. Data represent
mean (6 SEM) (n $ 3–4 mice/group). *p , 0.05, control Ab versus PD-1
mAb. Although not indicated, in all cases Ag-specific ASC numbers and
anti-TNP Ig levels were significantly higher in immune mice compared
with naive mice. N.D., not detected.
Transient blockade of PD-1:PDL interactions increases both
The Journal of Immunology5191
by guest on June 13, 2013
expansion and function (42) and, in the current study, was shown
to similarly inhibit AgR-induced proliferation of B-1b cells
in vitro (Fig. 4). PD-1 expression by Ag-specific B-1b cells may
similarly contribute to suppression of Ag-specific B-1b cell ex-
pansion and/or Ab production in vivo, because PD-1 mAb block-
ade significantly enhanced Ag-specific B-1b cell numbers, IgG3
switching, and Ab production (Figs. 5–7). Collectively, the results
of this study demonstrated a key role for the PD-1:PDL regula-
tory axis in controlling B-1b cell responses and IgG production
to TI-2 Ags.
The division of labor among B cell subsets in Ab production
against TI-2 Ags is not completely clear, with B-1b cells, B-1a
cells, MZ B cells, and even follicular B cells (43), under certain
circumstances, implicated in producing TI-2 Ab responses. Work
by multiple laboratories demonstrated a key role for B-1b cells in
producing Abs in response to defined TI-2 Ags, including type 3
pneumococcal polysaccharide (14, 40), NP-Ficoll (15), and a-1,3
dextran (16); additional TI Ags (17–19); and the Gal a1-3Galb1–
4GlcNAc carbohydrate epitope involved in transplant rejection
(34). Nonetheless, MZ B cells and B-1a cells may also contribute
to Ab production against these and other TI-2 Ags (16, 30, 31),
including phosphorylcholine when either displayed on bacteria
(21) or on Ficoll as a TI Ag (14). These subsets may respond
differentially to Ag and/or additional cues to produce Abs (23,
44). The present study demonstrates that the Ag-specific B cells
that rapidly become activated, expand, and/or mobilize in re-
sponse to TNP-Ficoll are CD11b+CD19hiCD21loCD44+CD86+
CD80+CD1dintCD232B220loCD43+PD-1+CD52/lo(Fig. 1), con-
sistent with an activated B-1b cell phenotype (Fig. 4) (27). Im-
portantly, CD5 was expressed by Ag-specific B-1b cells at a lower
level than that found for B-1a cells (Fig. 1B, Supplemental Fig.
1A) and was present on Ag-specific B cells from immunized (but
not naive) CD192/2mice (Supplemental Fig. 1B), which lack
B-1a cells but produce near-normal Ab responses to TNP-Ficoll
(14, 27). Increased CD5 expression is observed on BCR-activated
spleen B cells (Fig. 4B) (37) and bead-purified B-1b cells (Fig. 4B,
Supplemental Fig. 1C). Because CD5loB cells could have
remained following bead selection, wild-type and CD192/2CD52
B-1b cells were also FACS sorted to high purity and were simi-
larly found to express increased CD5 levels with BCR activation
(Supplemental Fig. 1D, 1E). Thus, low CD5 expression on Ag-
specific B-1b cells following immunization may be indicative
of AgR-mediated B-1b cell activation. Alternatively, it remains
possible that TNP-Ficoll induces expansion of Ag-specific CD5lo
B-1 cells as opposed to increasing expression of CD5 on Ag-
activated B-1b cells. Interestingly, Francisella tularemia LPS
(Ft-LPS), an extremely weak TLR4 agonist, elicits a similar Ag-
specific TI response to that observed with TNP-Ficoll, with i.p.
immunization leading to the appearance of a population of Ag-
specific IgDloCD21lo-intCD232CD138+/2CD5+cells in the spleen
(22). Whether the B cell populations responding to these two
different Ags are related or distinct is unclear. CD5 expression by
Ag-activated B-1b cells may be transient, because CD5 expression
was not found on the Ag-specific B-1b cells remaining in the
spleen 5 wk postimmunization (Fig. 1D), and previous work by
Hayakawa et al. (45) demonstrated that anti-TNP ASCs in the
spleen are CD52following TNP-Ficoll immunization. Similarly,
Cole et al. (22) reported diminished CD5 expression as Ft-LPS–
specific B cells differentiated to plasma cells. Notably, CD5 ex-
pression on Ag-specific B-1b cells may be suppressed in the
context of infection, because PAMPs, such as LPS, may inhibit
CD5 upregulation on Ag-activated B-1b cells (Fig. 4B) as ob-
served for spleen B cells (Fig. 4B) (37). Finally, Ag-specific
CD11b+B cells expressed IgG3 and CD138 following immuni-
zation, demonstrating the potential for B-1b cells to undergo
isotype switching and produce Ab in response to signals from a
synthetic TI-2 Ag alone, consistent with previous findings using
adoptive-transfer experiments (14). Thus, B-1b cells become ac-
tivated, expand, undergo isotype switching, and differentiate into
Ab-producing cells in response to TNP-Ficoll.
In addition to examining the phenotype of responding B cells,
the dynamics of expansion, differentiation, and contraction of Ag-
specific B cells in response to TNP-Ficoll was investigated. Sig-
nificant increases in CD11b+B cell frequencies and numbers
responding to TNP-Ficoll were observed in the peritoneal cavity,
spleen, blood, and lymph nodes, with peak increases typically
observed at day 5. This is likely explained by mobilization of
responding peritoneal B-1b cells, as well as expansion of these
and other nonperitoneal B-1b cells, found in tissues, such as
lymph nodes (14, 15). Indeed, the fact that i.p. and i.v. immuni-
zation elicited similar increases in Ag-specific B-1b cells (Fig. 2)
highlights a potential role for nonperitoneal B-1b cells in TI-2
Ab responses, as supported by other findings (19). Following peak
expansion, Ag-specific B cell numbers and frequencies gradually
declined in the spleen, although frequencies remained elevated
over naive mice, and Ag-specific B-1b cells expressing low levels
of CD11b were still present 5 wk postimmunization (Fig. 1A, 1D,
1E). This is not unexpected because CD11b expression diminishes
on B-1 cells outside of the peritoneum (35, 36). It is possible that
these remaining cells are long-lived plasmablasts, memory cells,
or cells continuing to participate in the primary response to TNP-
Ficoll, which may resist degradation in vivo. In a previous study,
Maclennan and colleagues (15) used immunohistochemistry to
demonstrate that NP-reactive B cells can persist in splenic tissue
of Rag-12/2mice as either plasma cells or plasmablasts for $2
mo in response to NP-Ficoll, which is known to involve B-1b cells
(46). Infection models using Enterobacter cloacae (16) and Bor-
relia hermsii (17) similarly demonstrated that B-1b cells may
yield a form of unconventional memory that may be attributed to
the presence of long-lived splenic B-1b plasmablasts. Nonetheless,
it is evident from the present study that, although splenic ASCs
produce the majority of TI-2 Ab in the early stages of the immune
response, BM ASCs make a substantial contribution to persistent
TI-2 Ab levels. Importantly, the degree to which B-1b cells con-
tribute to BM ASCs in response to TNP-Ficoll was not resolved
by the current study. Notably, B-1b cells were shown to give
rise to BM ASCs in response to PPS-3 (40), and BLIMP-1+B-1
B cells can seed the long-lived ASC compartment (47). However,
whether B-1b cell-derived BM ASCs represent plasmablasts or
fully differentiated BM plasma cells is not clear, given a recent
report demonstrating that plasmablasts also reside in the BM (48).
In contrast to results with spleen B-1b cells, Ag-specific peritoneal
B-1b cell numbers remained elevated at peak levels up to ∼6 wk
following immunization (Figs. 1E, 5C). This long-term mainte-
nance of peritoneal Ag-specific B-1b cells following immuniza-
tion has not been observed (49) or examined (50, 51) in previous
studies using BCR transgenic mouse strains to examine B cell
expansion in response to purified TI-2 Ags, although it was
reported for a-1,3 dextran-specific B-1b cells in VHJ558 Tg mice
following E. cloacae challenge (16). Moreover, accumulation and
maintenance of Ft-LPS–specific peritoneal B-1a cells 2 mo fol-
lowing Ft-LPS immunization were reported by Cole et al. (22).
The explanation and significance of this finding are unclear, but
studies examining the functionality and role of these peritoneal
cells are underway. In summary, Ag-specific B-1b cell numbers
significantly increased in multiple tissues during the first week
following TI-2 Ag immunization and, with the exception of the
peritoneal B cells, gradually declined thereafter. Further studies
5192PD-1 REGULATION OF B-1b CELL RESPONSES TO TI-2 Ags
by guest on June 13, 2013
aimed at examining the signals controlling Ag-specific B-1b cell
expansion, contraction, and differentiation, as well as long-term
maintenance, are warranted.
The results of this study demonstrated a significant role for
PD-1:PDL interactions in regulating B-1b cell function. First, BCR
cross-linking induced PD-1 expression on B-1b cells, and PD-1
significantly inhibited BCR-induced proliferation when cocross-
linked with the BCR, without enhancing apoptosis (Fig. 4), in
a manner similar to that observed for splenic B cells (Fig. 4)
(38). Second, Ag-specific B-1b cells in spleen, peritoneal cavity,
and blood were specifically induced to express PD-1 following
immunization, with the highest levels observed between days
2 and 5. Finally, interfering with PD-1:PDL interactions by ad-
ministering a PD-1–blocking mAb between days 1 and 6 post-
immunization significantly increased Ag-specific B-1b cell num-
bers, the frequency of Ag-specific B-1b cells switching to IgG3,
the frequency of Ag-specific splenic CD138+cells, and Ag-
specific IgG production by both splenic and BM ASCs (Figs. 5,
7). These increases were accompanied by significant increases in
Ag-specific serum IgG, as well as IgA, levels (Fig. 6). Although it
is possible that anti-TNP BM and spleen ASCs are largely derived
from Ag-specific B-1b cells, it remains unresolved whether this is
the case, as well as whether increased BM or spleen ASCs ob-
served with PD-1 mAb blockade are due to expanded peritoneal
B-1b cells. The fact that transient PD-1 mAb blockade applied
during the first week of immunization resulted in significantly
increased Ag-specific B-1b cell numbers and IgG-producing ASCs
at both early and late time points suggested that PD-1:PDL
interactions play a critical role in inhibiting the early immune
response to TI-2 Ags and that the splenic and BM IgG ASCs
generated during the early response to TI-2 Ags are long-lived.
PD-1:PDL interactions may limit Ag-specific B-1b cell ex-
pansion and IgG production by several mechanisms. Because
in vitro-proliferation assays support an inhibitory role for PD-1 in
regulating BCR-driven proliferation in B-1b cells, simultaneous
interaction between a TI-2 Ag-activated (PD-1–expressing) B-1b
cell and a PDL-expressing cell provides a likely mechanism by
which PD-1:PDL interactions may limit AgR signals that drive
B-1b cell proliferation in vivo. Importantly, it is not clear whether
TNP-specific IgG production by additional B cell subsets is
modulated by PD-1. Because B cell division is required for iso-
type switching (52, 53), PD-1 inhibitory signaling in TI-2 Ag-
activated B cells would, thereby, limit both clonal expansion
and isotype switching. Indeed this is what is observed. Notably,
if TI-2 Ags are associated with PAMPs or elicit T cell help
via protein association, costimulatory signals (e.g., LPS, CD40L)
may help to overcome PD-1 inhibitory signals (Fig. 4G, 4H) or
upregulation in certain cases (38). Although PD-1’s effects have
most often been studied in the context of AgR signaling, PD-1 has
effects on other non-AgR–bearing cell types and, therefore, must
regulate additional signaling pathways. Thus, PD-1 may modulate
B cell survival, proliferation, and/or differentiation independently
of its effects on AgR signaling. Interestingly, PD-1 blockade in
macaques during chronic SIV infection was proposed to increase
SIV Env-binding Ab titers (54), as well as other humoral memory
responses, by preventing deletion of activated PD-1+memory
B cells (55). Although the in vitro results in this study (Fig. 4F)
demonstrated that PD-1–BCR coligation suppressed proliferation
as opposed to survival, it remains possible that PD-1 influences
Ag-specific B-1b cell survival in vivo following activation. Fi-
nally, it remains possible that PD-1, expressed by some other cell
type, regulates B-1b cell responses to TI-2 Ags. For example, PD-
1 expression by follicular Th cells, as opposed to B cells, plays
a significant role in promoting (as opposed to suppressing) ger-
minal center B cell responses and plasma cell formation and,
hence, Ab production, in response to T cell-dependent Ags (56).
Future experiments using PD-1 conditional knockouts or mixed
BM chimeras will be required to test whether PD-1 expression by
B-1b cells contributes to suppression of TI-2 Ab responses.
PD-1 shares functional similarity to other B cell-inhibitory
receptors expressed on B-1b cells, including CD5 and CD22,
which recruit and activate SHP-1 to dampen BCR signaling. Al-
though their expression patterns differ, these receptors are likely
involved in maintaining B cell tolerance, because mice deficient
in any of these receptors have hyperresponsive B cells that pro-
duce autoantibodies (57–66). Interactions between these B cell-
expressed inhibitory receptors and host cell surface ligands likely
function in suppressing Ab responses against self-Ags. Nonethe-
less, PD-1, CD5, and CD22 may have distinct roles in regulating
Ab responses to immunogenic TI-2 Ags versus self-Ags, because
TI-2 Ab responses are augmented in PD-12/2mice (29), are
normal in CD52/2mice (67), and are decreased in CD222/2mice
(63–65, 68). However, recent work by Nemazee and colleagues
(68) indicated that CD22 promotes tolerance to TI-2 Ags that
are decorated with sialic acid (self) ligands. Thus, PD-1 and other
B cell immunoinhibitory receptors may play complex roles in
regulating B cell responses against self-Ags versus foreign TI-2
In summary, this study revealed an immunoinhibitory role for
PD-1:PDL interactions in regulating B-1b cell responses to TI-2
Ags, with particular significance in suppression of long-lasting
Ag-specific IgG production. These findings may have important
implications for current strategies targeting PD-1:PDL interactions
as treatment modalities for multiple diseases and conditions. More
importantly, these findings may have significance for TI-2 Ag-
based vaccine development. TI-2 Ab responses in mice parallel
those in humans (11), and a human B-1 cell counterpart was re-
cently identified (69). Thus, it is possible that PD-1:PDL inter-
actions similarly suppress TI-2 IgG responses in humans. In
contrast to IgM, IgG is produced in limiting quantities against
most TI-2 Ags. Nonetheless, because IgG may elicit enhanced
protection against carbohydrate-bearing pathogens relative to IgM,
future strategies transiently targeting the PD-1:PDL pathway may
provide an opportunity to elicit enhanced IgG-mediated protection
against TI-2 Ag-bearing pathogens.
The author has no financial conflicts of interest.
1. Wuorimaa, T., and H. Ka ¨yhty. 2002. Current state of pneumococcal vaccines.
Scand. J. Immunol. 56: 111–129.
2. Vos, Q., A. Lees, Z. Q. Wu, C. M. Snapper, and J. J. Mond. 2000. B-cell acti-
vation by T-cell-independent type 2 antigens as an integral part of the humoral
immune response to pathogenic microorganisms. Immunol. Rev. 176: 154–170.
3. Wang, T. T., and A. H. Lucas. 2004. The capsule of Bacillus anthracis behaves
as a thymus-independent type 2 antigen. Infect. Immun. 72: 5460–5463.
4. Weintraub, A. 2003. Immunology of bacterial polysaccharide antigens. Carbo-
hydr. Res. 338: 2539–2547.
5. Lee, K. C., C. Shiozawa, A. Shaw, and E. Diener. 1976. Requirement for ac-
cessory cells in the antibody response to T cell-independent antigens in vitro.
Eur. J. Immunol. 6: 63–68.
6. Pinschewer, D. D., A. F. Ochsenbein, A. B. Satterthwaite, O. N. Witte,
H. Hengartner, and R. M. Zinkernagel. 1999. A Btk transgene restores the an-
tiviral TI-2 antibody responses of xid mice in a dose-dependent fashion. Eur. J.
Immunol. 29: 2981–2987.
7. Freer, G., C. Burkhart, I. Ciernik, M. F. Bachmann, H. Hengartner, and
R. M. Zinkernagel. 1994. Vesicular stomatitis virus Indiana glycoprotein as a
T-cell-dependent and -independent antigen. J. Virol. 68: 3650–3655.
8. Mazza, G., D. W. Dunne, and A. E. Butterworth. 1990. Antibody isotype
responses to the Schistosoma mansoni schistosomulum in the CBA/N mouse
induced by different stages of the parasite life cycle. Parasite Immunol. 12: 529–
The Journal of Immunology 5193
by guest on June 13, 2013
9. Parra, C., J. M. Gonza ´lez, E. Castan ˜eda, and S. Fiorentino. 2005. Anti-
glucuronoxylomannan IgG1 specific antibodies production in Cryptococcus
neoformans resistant mice. Biomedica 25: 110–119.
10. Ortqvist, A. 2001. Pneumococcal vaccination: current and future issues. Eur.
Respir. J. 18: 184–195.
11. Gonza ´lez-Ferna ´ndez, A., J. Faro, and C. Ferna ´ndez. 2008. Immune responses to
polysaccharides: lessons from humans and mice. Vaccine 26: 292–300.
12. Weller, S., C. A. Reynaud, and J. C. Weill. 2005. Vaccination against encapsu-
lated bacteria in humans: paradoxes. Trends Immunol. 26: 85–89.
13. Klein Klouwenberg, P., and L. Bont. 2008. Neonatal and infantile immune
responses to encapsulated bacteria and conjugate vaccines. Clin. Dev. Immunol.
14. Haas, K. M., J. C. Poe, D. A. Steeber, and T. F. Tedder. 2005. B-1a and B-1b
cells exhibit distinct developmental requirements and have unique functional
roles in innate and adaptive immunity to S. pneumoniae. Immunity 23: 7–18.
15. Hsu, M. C., K. M. Toellner, C. G. Vinuesa, and I. C. Maclennan. 2006. B cell
clones that sustain long-term plasmablast growth in T-independent extra-
follicular antibody responses. Proc. Natl. Acad. Sci. USA 103: 5905–5910.
16. Foote, J. B., and J. F. Kearney. 2009. Generation of B cell memory to the bac-
terial polysaccharide alpha-1,3 dextran. J. Immunol. 183: 6359–6368.
17. Alugupalli, K. R., J. M. Leong, R. T. Woodland, M. Muramatsu, T. Honjo, and
R. M. Gerstein. 2004. B1b lymphocytes confer T cell-independent long-lasting
immunity. Immunity 21: 379–390.
18. Gil-Cruz, C., S. Bobat, J. L. Marshall, R. A. Kingsley, E. A. Ross,
I. R. Henderson, D. L. Leyton, R. E. Coughlan, M. Khan, K. T. Jensen, et al.
2009. The porin OmpD from nontyphoidal Salmonella is a key target for
a protective B1b cell antibody response. Proc. Natl. Acad. Sci. USA 106: 9803–
19. Colombo, M. J., and K. R. Alugupalli. 2008. Complement factor H-binding
protein, a putative virulence determinant of Borrelia hermsii, is an antigenic
target for protective B1b lymphocytes. J. Immunol. 180: 4858–4864.
20. Alugupalli, K. R. 2008. A distinct role for B1b lymphocytes in T cell-
independent immunity. Curr. Top. Microbiol. Immunol. 319: 105–130.
21. Martin, F., A. M. Oliver, and J. F. Kearney. 2001. Marginal zone and B1 B cells
unite in the early response against T-independent blood-borne particulate anti-
gens. Immunity 14: 617–629.
22. Cole, L. E., Y. Yang, K. L. Elkins, E. T. Fernandez, N. Qureshi, M. J. Shlomchik,
L. A. Herzenberg, L. A. Herzenberg, and S. N. Vogel. 2009. Antigen-specific B-
1a antibodies induced by Francisella tularensis LPS provide long-term pro-
tection against F. tularensis LVS challenge. Proc. Natl. Acad. Sci. USA 106:
23. Baumgarth, N. 2011. The double life of a B-1 cell: self-reactivity selects for
protective effector functions. Nat. Rev. Immunol. 11: 34–46.
24. Obukhanych, T. V., and M. C. Nussenzweig. 2006. T-independent type II im-
mune responses generate memory B cells. J. Exp. Med. 203: 305–310.
25. Patterson, H. C., M. Kraus, Y. M. Kim, H. Ploegh, and K. Rajewsky. 2006. The
B cell receptor promotes B cell activation and proliferation through a non-ITAM
tyrosine in the Igalpha cytoplasmic domain. Immunity 25: 55–65.
26. Fruman, D. A., A. B. Satterthwaite, and O. N. Witte. 2000. Xid-like phenotypes:
a B cell signalosome takes shape. Immunity 13: 1–3.
27. Haas, K. M., J. C. Poe, and T. F. Tedder. 2009. CD21/35 promotes protective
immunity to Streptococcus pneumoniae through a complement-independent but
CD19-dependent pathway that regulates PD-1 expression. J. Immunol. 183:
28. Okazaki, T., and T. Honjo. 2007. PD-1 and PD-1 ligands: from discovery to
clinical application. Int. Immunol. 19: 813–824.
29. Nishimura, H., N. Minato, T. Nakano, and T. Honjo. 1998. Immunological
studies on PD-1 deficient mice: implication of PD-1 as a negative regulator for
B cell responses. Int. Immunol. 10: 1563–1572.
30. Samardzic, T., D. Marinkovic, C.-P. Danzer, J. Gerlach, L. Nitschke, and
T. Wirth. 2002. Reduction of marginal zone B cells in CD22-deficient mice. Eur.
J. Immunol. 32: 561–567.
31. Guinamard, R., M. Okigaki, J. Schlessinger, and J. V. Ravetch. 2000. Absence of
marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral
response. Nat. Immunol. 1: 31–36.
32. Haas, K. M., M. Hasegawa, D. A. Steeber, J. C. Poe, M. D. Zabel, C. B. Bock,
D. R. Karp, D. E. Briles, J. H. Weis, and T. F. Tedder. 2002. Complement
receptors CD21/35 link innate and protective immunity during Streptococcus
pneumoniae infection by regulating IgG3 antibody responses. Immunity 17: 713–
33. Cobern, L., and P. Selvaraj. 1995. An enzymatic method to determine receptor-
mediated endocytosis. J. Biochem. Biophys. Methods 30: 249–255.
34. Ohdan, H., K. G. Swenson, H. S. Kruger Gray, Y. G. Yang, Y. Xu, A. D. Thall,
and M. Sykes. 2000. Mac-1-negative B-1b phenotype of natural antibody-
producing cells, including those responding to Gal alpha 1,3Gal epitopes in
alpha 1,3-galactosyltransferase-deficient mice. J. Immunol. 165: 5518–5529.
35. Yang, Y., J. W. Tung, E. E. Ghosn, L. A. Herzenberg, and L. A. Herzenberg.
2007. Division and differentiation of natural antibody-producing cells in mouse
spleen. Proc. Natl. Acad. Sci. USA 104: 4542–4546.
36. Kawahara, T., H. Ohdan, G. Zhao, Y. G. Yang, and M. Sykes. 2003. Peritoneal
cavity B cells are precursors of splenic IgM natural antibody-producing cells. J.
Immunol. 171: 5406–5414.
37. Cong, Y. Z., E. Rabin, and H. H. Wortis. 1991. Treatment of murine CD52
B cells with anti-Ig, but not LPS, induces surface CD5: two B-cell activation
pathways. Int. Immunol. 3: 467–476.
38. Zhong, X., C. Bai, W. Gao, T. B. Strom, and T. L. Rothstein. 2004. Suppression
of expression and function of negative immune regulator PD-1 by certain pattern
recognition and cytokine receptor signals associated with immune system dan-
ger. Int. Immunol. 16: 1181–1188.
39. Racine, R., M. Chatterjee, and G. M. Winslow. 2008. CD11c expression iden-
tifies a population of extrafollicular antigen-specific splenic plasmablasts re-
sponsible for CD4 T-independent antibody responses during intracellular
bacterial infection. J. Immunol. 181: 1375–1385.
40. Taillardet, M., G. Haffar, P. Mondie `re, M. J. Asensio, H. Gheit, N. Burdin,
T. Defrance, and L. Genestier. 2009. The thymus-independent immunity con-
ferred by a pneumococcal polysaccharide is mediated by long-lived plasma cells.
Blood 114: 4432–4440.
41. Slifka, M. K., M. Matloubian, and R. Ahmed. 1995. Bone marrow is a major site
of long-term antibody production after acute viral infection. J. Virol. 69: 1895–
42. Keir, M. E., M. J. Butte, G. J. Freeman, and A. H. Sharpe. 2008. PD-1 and its
ligands in tolerance and immunity. Annu. Rev. Immunol. 26: 677–704.
43. Swanson, C. L., T. J. Wilson, P. Strauch, M. Colonna, R. Pelanda, and
R. M. Torres. 2010. Type I IFN enhances follicular B cell contribution to the
T cell-independent antibody response. J. Exp. Med. 207: 1485–1500.
44. Martin, F., and J. F. Kearney. 2002. Marginal-zone B cells. Nat. Rev. Immunol. 2:
45. Hayakawa, K., R. R. Hardy, M. Honda, L. A. Herzenberg, A. D. Steinberg, and
L. A. Herzenberg. 1984. Ly-1 B cells: functionally distinct lymphocytes that
secrete IgM autoantibodies. Proc. Natl. Acad. Sci. USA 81: 2494–2498.
46. Shriner, A. K., H. Liu, G. Sun, M. Guimond, and K. R. Alugupalli. 2010. IL-7-
dependent B lymphocytes are essential for the anti-polysaccharide response and
protective immunity to Streptococcus pneumoniae. J. Immunol. 185: 525–531.
47. Fairfax, K. A., L. M. Corcoran, C. Pridans, N. D. Huntington, A. Kallies,
S. L. Nutt, and D. M. Tarlinton. 2007. Different kinetics of blimp-1 induction in
B cell subsets revealed by reporter gene. J. Immunol. 178: 4104–4111.
48. Racine, R., M. McLaughlin, D. D. Jones, S. T. Wittmer, K. C. MacNamara,
D. L. Woodland, and G. M. Winslow. 2011. IgM production by bone marrow
plasmablasts contributes to long-term protection against intracellular bacterial
infection. J. Immunol. 186: 1011–1021.
49. Whitmore, A. C., H. R. Neely, R. Diz, and P. M. Flood. 2004. Rapid induction of
splenic and peritoneal B-1a cells in adult mice by thymus-independent type-2
antigen. J. Immunol. 173: 5406–5414.
50. Shih, T. A., M. Roederer, and M. C. Nussenzweig. 2002. Role of antigen re-
ceptor affinity in T cell-independent antibody responses in vivo. Nat. Immunol.
51. de Vinuesa, C. G., M. C. Cook, J. Ball, M. Drew, Y. Sunners, M. Cascalho,
M. Wabl, G. G. Klaus, and I. C. MacLennan. 2000. Germinal centers without
T cells. J. Exp. Med. 191: 485–494.
52. Deenick, E. K., J. Hasbold, and P. D. Hodgkin. 1999. Switching to IgG3, IgG2b,
and IgA is division linked and independent, revealing a stochastic framework for
describing differentiation. J. Immunol. 163: 4707–4714.
53. Rush, J. S., M. Liu, V. H. Odegard, S. Unniraman, and D. G. Schatz. 2005.
Expression of activation-induced cytidine deaminase is regulated by cell di-
vision, providing a mechanistic basis for division-linked class switch re-
combination. Proc. Natl. Acad. Sci. USA 102: 13242–13247.
54. Velu, V., K. Titanji, B. Zhu, S. Husain, A. Pladevega, L. Lai, T. H. Vanderford,
L. Chennareddi, G. Silvestri, G. J. Freeman, et al. 2009. Enhancing SIV-specific
immunity in vivo by PD-1 blockade. Nature 458: 206–210.
55. Titanji, K., V. Velu, L. Chennareddi, M. Vijay-Kumar, A. T. Gewirtz,
G. J. Freeman, and R. R. Amara. 2010. Acute depletion of activated memory
B cells involves the PD-1 pathway in rapidly progressing SIV-infected mac-
aques. J. Clin. Invest. 120: 3878–3890.
56. Good-Jacobson, K. L., C. G. Szumilas, L. Chen, A. H. Sharpe, M. M. Tomayko,
and M. J. Shlomchik. 2010. PD-1 regulates germinal center B cell survival and
the formation and affinity of long-lived plasma cells. Nat. Immunol. 11: 535–
57. Hippen, K. L., L. E. Tze, and T. W. Behrens. 2000. CD5 maintains tolerance in
anergic B cells. J. Exp. Med. 191: 883–890.
58. Bikah, G., J. Carey, J. R. Ciallella, A. Tarakhovsky, and S. Bondada. 1996. CD5-
mediated negative regulation of antigen receptor-induced growth signals in B-1
B cells. Science 274: 1906–1909.
59. O’Keefe, T. L., G. T. Williams, F. D. Batista, and M. S. Neuberger. 1999. De-
ficiency in CD22, a B cell-specific inhibitory receptor, is sufficient to predispose
to development of high affinity autoantibodies. J. Exp. Med. 189: 1307–1313.
60. Poe, J. C., S. H. Smith, K. M. Haas, K. Yanaba, T. Tsubata, T. Matsushita, and
T. F. Tedder. 2011. Amplified B Lymphocyte CD40 Signaling Drives Regulatory
B10 Cell Expansion in Mice. PLoS ONE 6: e22464.
61. Nishimura, H., M. Nose, H. Hiai, N. Minato, and T. Honjo. 1999. Development
of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an
ITIM motif-carrying immunoreceptor. Immunity 11: 141–151.
62. Nishimura, H., T. Okazaki, Y. Tanaka, K. Nakatani, M. Hara, A. Matsumori,
S. Sasayama, A. Mizoguchi, H. Hiai, N. Minato, and T. Honjo. 2001. Autoim-
mune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science 291:
63. Otipoby, K. L., K. B. Andersson, K. E. Draves, S. J. Klaus, A. G. Farr,
J. D. Kerner, R. M. Perlmutter, C.-L. Law, and E. A. Clark. 1996. CD22 regulates
thymus-independent responses and the lifespan of B cells. Nature 384: 634–
64. Nitschke, L., R. Carsetti, B. Ocker, G. Ko ¨hler, and M. C. Lamers. 1997. CD22 is
a negative regulator of B-cell receptor signalling. Curr. Biol. 7: 133–143.
65. Sato, S., A. S. Miller, M. Inaoki, C. B. Bock, P. J. Jansen, M. L. K. Tang, and
T. F. Tedder. 1996. CD22 is both a positive and negative regulator of B
5194PD-1 REGULATION OF B-1b CELL RESPONSES TO TI-2 Ags
by guest on June 13, 2013
lymphocyte antigen receptor signal transduction: altered signaling in CD22-
deficient mice. Immunity 5: 551–562.
66. Haas, K. M., S. Sen, I. G. Sanford, A. S. Miller, J. C. Poe, and T. F. Tedder. 2006.
CD22 ligand binding regulates normal and malignant B lymphocyte survival
in vivo. J. Immunol. 177: 3063–3073.
67. Tarakhovsky, A., W. Mu ¨ller, and K. Rajewsky. 1994. Lymphocyte populations
and immune responses in CD5-deficient mice. Eur. J. Immunol. 24: 1678–
68. Duong, B. H., H. Tian, T. Ota, G. Completo, S. Han, J. L. Vela, M. Ota,
M. Kubitz, N. Bovin, J. C. Paulson, and D. Nemazee. 2010. Decoration of T-
independent antigen with ligands for CD22 and Siglec-G can suppress immunity
and induce B cell tolerance in vivo. [Published erratum appears in 2010 J. Exp.
Med. 207: 445.] J. Exp. Med. 207: 173–187.
69. Griffin, D. O., N. E. Holodick, and T. L. Rothstein. 2011. Human B1 cells in
umbilical cord and adult peripheral blood express the novel phenotype CD20+
CD27+ CD43+ CD702. J. Exp. Med. 208: 67–80.
The Journal of Immunology5195
by guest on June 13, 2013