Surface receptor Toso controls B cell–mediated

regulation of T cell immunity

Jinbo Yu, … , Niko Föger, Kyeong-Hee Lee
J Clin Invest. 2018;128(5):1820-1836. https://doi.org/10.1172/JCI97280.

Research Article Immunology Inflammation

The immune system is tightly controlled by regulatory processes that allow for the
elimination of invading pathogens, while limiting immunopathological damage to the host. In
the present study, we found that conditional deletion of the cell surface receptor Toso on B
cells unexpectedly resulted in impaired proinflammatory T cell responses, which led to
impaired immune protection in an acute viral infection model and was associated with
reduced immunopathological tissue damage in a chronic inflammatory context. Toso
exhibited its B cell–inherent immunoregulatory function by negatively controlling the pool of
IL-10–competent B1 and B2 B cells, which were characterized by a high degree of self-
reactivity and were shown to mediate immunosuppressive activity on inflammatory T cell
responses in vivo. Our results indicate that Toso is involved in the
differentiation/maintenance of regulatory B cells by fine-tuning B cell receptor activation
thresholds. Furthermore, we showed that during influenza A–induced pulmonary
inflammation, the application of Toso-specific antibodies selectively induced IL-10–
competent B cells at the site of inflammation and resulted in decreased proinflammatory
cytokine production by lung T cells. These findings suggest that Toso may serve as a novel
therapeutic target to dampen pathogenic T cell responses via the modulation of IL-10–
competent regulatory B cells.

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RESEARCH ARTICLE The Journal of Clinical Investigation   

Surface receptor Toso controls B cell–mediated

regulation of T cell immunity
Jinbo Yu,1,2 Vu Huy Hoang Duong,1,2 Katrin Westphal,1,2 Andreas Westphal,1,2 Abdulhadi Suwandi,3 Guntram A. Grassl,3
Korbinian Brand,2 Andrew C. Chan,4 Niko Föger,1,2 and Kyeong-Hee Lee1,2
Inflammation Research Group, 2Institute of Clinical Chemistry, and 3Institute of Medical Microbiology and Hospital Epidemiology and German Center for Infection Research (DZIF), Hannover Medical School,
1

Hannover, Germany. 4Research, Genentech, South San Francisco, California, USA.

The immune system is tightly controlled by regulatory processes that allow for the elimination of invading pathogens,
while limiting immunopathological damage to the host. In the present study, we found that conditional deletion of the cell
surface receptor Toso on B cells unexpectedly resulted in impaired proinflammatory T cell responses, which led to impaired
immune protection in an acute viral infection model and was associated with reduced immunopathological tissue damage
in a chronic inflammatory context. Toso exhibited its B cell–inherent immunoregulatory function by negatively controlling
the pool of IL-10–competent B1 and B2 B cells, which were characterized by a high degree of self-reactivity and were shown
to mediate immunosuppressive activity on inflammatory T cell responses in vivo. Our results indicate that Toso is involved
in the differentiation/maintenance of regulatory B cells by fine-tuning B cell receptor activation thresholds. Furthermore, we
showed that during influenza A–induced pulmonary inflammation, the application of Toso-specific antibodies selectively
induced IL-10–competent B cells at the site of inflammation and resulted in decreased proinflammatory cytokine production
by lung T cells. These findings suggest that Toso may serve as a novel therapeutic target to dampen pathogenic T cell
responses via the modulation of IL-10–competent regulatory B cells.

Introduction of Bregs is complicated by the description of multiple different

A key feature of the immune system is the maintenance of a del- Breg subsets with partially overlapping phenotypes and surface
icate balance between protecting against infectious agents and marker characteristics in different mouse and human model
minimizing immune-mediated tissue damage. To achieve this systems (13). Until now, no Breg-defining transcription factor or
critical equilibrium, tight regulatory mechanisms have evolved unique lineage marker has been identified, and Bregs are mainly
to control protective immune responses and to avoid misguided defined by their ability to secrete IL-10. The nature and origin of
or excessive inflammation. It is now well established that a subset Bregs are still controversial, and it is unclear whether they repre-
of CD4+ T cells, termed regulatory T cells, exhibit immunosup- sent a distinct B cell lineage or a dynamic cellular state.
pressive function and are crucial for the maintenance of normal Toso, also known as Faim3 (Fas apoptosis inhibitory molecule
immune homeostasis by controlling inflammation and prevent- 3) or FcμR (Fc receptor for IgM), is a type I transmembrane protein
ing autoimmunity (1). More recently, it has been recognized belonging to the immunoglobulin gene superfamily. Expression
that B cells can also negatively regulate T cell responses in an of Toso is restricted to lymphoid organs, where it is particularly
antibody-independent manner. A suppressive role of B cells in highly expressed in B cells. In humans, a tight association of
pathogenic T cell responses was already suggested in 1996 by Jan- Toso overexpression with B cell malignancy has been observed
eway and colleagues, who showed that B cell–deficient mice exhib- in patients with chronic lymphocytic leukemia (14–16). Toso was
ited exacerbated disease in experimental autoimmune enceph- originally identified as a surface molecule with negative regu-
alomyelitis (EAE) (2). An increasing number of reports has since latory function on lymphocyte apoptosis (17, 18). Subsequent-
indicated immunoregulatory function of B cells in various disease ly, additional studies have identified Toso as an Fc receptor for
models, including T cell–dependent autoimmune models (3–8), soluble IgM (Fcμ receptor) (19, 20). More recently, it has been
transplantation (9), inflammation (10), and cancer (11), which has demonstrated that Toso physically interacts with membrane IgM-
led to the concept of regulatory B cells (Bregs). Bregs exhibit their containing B cell antigen receptor (BCR) complexes on the surface
regulatory function primarily via the release of IL-10, although of mature B cells and/or within the trans-Golgi network of devel-
other mechanisms may also be involved (12). In vivo identification oping B cells (21, 22). Functionally, increasing evidence suggests
that Toso serves as a physiologically important immunoregulatory
molecule for B and T cells. Toso-deficient mice have been reported
Authorship note: JY and VHHD contributed equally to this work. NF and KHL are co–senior to show enhanced serum levels of IgM and IgG autoantibodies
authors.
(21, 23–25), which, however, are not associated with autoimmune
Conflict of interest: The authors have declared that no conflict of interest exists.
Submitted: September 5, 2017; Accepted: February 13, 2018.
pathology (26). Furthermore, studies on Toso-deficient mice have
Reference information: J Clin Invest. 2018;128(5):1820–1836. revealed strong immunoprotective function of Toso in a model of
https://doi.org/10.1172/JCI97280. Listeria infection (27) and during lymphocytic choriomeningitis

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The Journal of Clinical Investigation     RESEARCH ARTICLE

virus infection (28). Toso-deficient mice are also largely resis- Reduced production of these important antiviral cytokines (IFN-γ
tant to the development of EAE and exhibit reduced pathogenic and TNF-α) by Toso –/– T cells was also observed in the spleen of
T cell responses (29). The mechanism underlying the phenotypic infected animals, irrespective of whether mice were infected with
defects of Toso-deficient mice remains a controversial issue, and a high dose (1,000 PFU) or a low dose (50 PFU) of influenza virus
models involving different effector mechanisms and different (Supplemental Figure 1, B–G, and data not shown), the latter not
immune cell types have been proposed (21, 22, 27, 29). Particular- inducing any mortality in WT or Toso –/– mice.
ly, it is unclear whether the effects of Toso on tolerance in the B Thus, although T cells in Toso –/– mice were capable of being
cell compartment are interrelated with impaired immune protec- activated and expanding in response to viral infection, these
tion in Toso-deficient mice. T cells were compromised in mounting an efficient antiviral
We demonstrate here that the specific deletion of Toso on B cytokine response.
cells results in impaired antiviral T cell responses. We provide Conditional deletion of Toso in B cells results in impaired protec-
evidence that links this immunoregulatory function of B cells on tive T cell immunity and limits immunopathological tissue damage.
T cell immunity to a specific set of IL-10–competent B cells. Our To assess whether reduced production of proinflammatory cyto-
data show that these Bregs are negatively regulated by Toso and kines by T cells in Toso –/– mice is a T cell–intrinsic defect that
exhibit high prevalence for self-reactivity. Thus, via control of depends on the specific deletion of Toso in T cells, or whether
the pool of Bregs, Toso exhibits a dual role in immune homeo- this is an indirect effect mediated by other cell types, such as
stasis: it maintains normal self-tolerance within the B cell com- antigen-presenting dendritic cells or B cells, we used a conditional
partment and, at the same time, ensures protective T cell immu- gene targeting approach. To this end, we crossed Tosof/f mice onto
nity against infection. different Cre-recombinase–expressing transgenic mouse lines —
CD4-Cre mice, CD11c-Cre mice, and CD19-Cre mice — to specifi-
Results cally delete Toso in T cells, dendritic cells, and B cells, respectively
Toso deficiency results in increased mortality and reduced produc- (Supplemental Figure 2).
tion of proinflammatory cytokines by T cells upon influenza infec- Upon influenza A infection, virus-induced mortality, as well as
tion. To assess the impact of Toso on immune responses during IFN-γ and TNF-α production by T cells, was not affected by abla-
acute viral infection, we intranasally infected WT and Toso –/– tion of Toso in T cells (Tosof/f/CD4-Cre+/– mice) (Figure 2A and
mice with 1,000 PFU of influenza virus strain A/PR8 (H1N1). Supplemental Figure 3, A–D). Overall survival and T cell responses
Whereas 84% of WT animals survived infection, Toso –/– mice were also normal in Tosof/f/CD11c-Cre+/– mice (Figure 2B and
exhibited significantly increased mortality; most died between Supplemental Figure 3, E–H), indicating that conditional deletion
days 10 and 15 postinfection (p.i.), and only 23% survived (Figure of Toso in dendritic cells does not compromise their capacity to
1A). Pulmonary viral titers in the bronchoalveolar lavage fluid efficiently prime T cell activation. To our surprise, increased virus-
were comparable between WT and Toso –/– mice at day 4 p.i., indi- induced lethality and impaired production of proinflammatory
cating normal viral replication and infectivity, but were relatively cytokines by T cells were only observed upon specific deletion of
increased in Toso –/– mice during the clearance phase (day 7 p.i.) Toso in B cells (Tosof/f/CD19-Cre+/– mice) (Figure 2, C–G). Simi-
(Supplemental Figure 1A; supplemental material available online larly to straight Toso –/– mice (Figure 1A), most Tosof/f/CD19-Cre+/–
with this article; https://doi.org/10.1172/JCI97280DS1). Thus, mice died between days 10 and 15 p.i. (only 11% survival), whereas
increased influenza-induced mortality of Toso –/– mice was asso- 88% of CD19-Cre+/– control mice survived infection (Figure 2C).
ciated with delayed viral clearance. Also, the functional ability of CD4+ and CD8+ T cells to produce
Antiviral immunity and recovery from influenza infection IFN-γ and TNF-α was significantly impaired in Tosof/f/CD19-
are largely dependent on effector T cell responses (30, 31), which Cre+/– mice compared with CD19-Cre+/– control mice (Figure 2,
usually peak around days 9–10 p.i., just when Toso –/– mice start D–G). These data strongly indicate that the functional defect of T
to become moribund. We thus next examined virus-specific T cells in Toso-deficient mice is not a T cell–intrinsic phenotype, but
cell responses in Toso –/– mice. Viral antigen-specific CD4+ and rather is indirectly induced by Toso-deficient B cells. Upon influ-
CD8+ T cell populations were enumerated in the lungs of infected enza infection, Tosof/f/CD19-Cre+/– mice also had significantly
animals at day 9 p.i. by tetramer staining for the immunodom- more CD4+ T cells expressing PD-1 (Figure 2H and Supplemental
inant CD4 T cell epitope NP311–325/I-Ab (NP311) or the CD8 T Figure 4A), an inhibitory surface receptor that has been associated
cell epitope NP366–374/Db (NP366). Both frequency and absolute with T cell exhaustion (32). Interestingly, increased PD-1 expres-
numbers of virus-specific NP311-tetramer–positive CD4+ T cells sion on CD4+ T cells upon Toso deletion in B cells correlated with
and NP366-dextramer–positive CD8+ T cells were compara- increased expression of its cognate ligand PD-L2 on CD19+ B cells
ble between WT and Toso –/– mice (Figure 1, B and C), indicating (Figure 2I and Supplemental Figure 4B).
normal antigen-specific priming and clonal expansion of virus- We further extended our studies on the B cell–specific role of
specific T cells in Toso –/– mice. Toso to a model of infection-induced intestinal pathology, using
Effector T cells contribute to viral control and elimination infection with attenuated Salmonella Typhimurium. In this model,
by the production of potent proinflammatory cytokines such as chronic Salmonella infection of the murine gastrointestinal tract
TNF-α and IFN-γ. The percentage as well as absolute numbers is associated with severe immunopathology that manifests in tis-
of IFN-γ– and TNF-α–producing T cells from lungs of influenza sue fibrosis and extensive damage to the gut tissue along with the
A–infected mice was significantly reduced in Toso –/– mice with expression of a characteristic Th1-dominated inflammatory cyto-
both CD4+ and CD8+ T cells being affected (Figure 1, D–G). kine profile (33). Using this model, conditional deletion of Toso on

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Figure 1. Toso deficiency results in increased

mortality and reduced production of inflammato-
ry cytokines by T cells upon influenza infection.
WT and Toso–/– (KO) mice were infected i.n. with
1,000 PFU influenza virus strain A/PR8 (H1N1).
(A) Survival of mice was monitored over time. n
= 13 per genotype; **P < 0.005; log-rank test. (B
and C) Lung cells were isolated at day 9 p.i., and
the frequency and number of virus-specific I-Ab/
NP311–325 (NP311) tetramer–positive CD4+ T cells (B)
and Db/NP366–374 (NP366) dextramer–positive CD8+
T cells (C) were quantified. (D–G) Lung cells isolat-
ed on day 9 p.i. were restimulated ex vivo, and the
number and frequency of IFN-γ–producing (D and
E) and TNF-α–producing (F and G) CD4+ T cells (D
and F) and CD8+ T cells (E and G) were quantified
by intracellular cytokine staining. (B–G) Each sym-
bol represents an individual mouse; horizontal
lines indicate the mean ± SEM. (B and C) n = 6;
(D–G) n = 5. *P < 0.05; **P < 0.01; ***P < 0.001;
Student’s t test. Data are representative of at
least 4 independent experiments.

B cells in Tosof/f/CD19-Cre+/– mice led to impaired production of the antiinflammatory cytokine IL-10. To this end, purified B cells
TNF-α by CD4+ and CD8+ T cells, which was associated with sig- from WT and Toso –/– mice were treated for 24 hours with BAFF,
nificantly attenuated overall cecal pathology (mainly attributable LPS, anti-CD40, or anti-IgM or were control treated. Most inter-
to reduced tissue damage in the epithelium and the mucosa) and estingly, Toso –/– B cells exhibited a strongly increased capacity
relative protection from weight loss compared with CD19-Cre+/– to produce IL-10 when compared with WT B cells (Figure 3A).
control mice (Supplemental Figure 5). Depending on the stimulus, splenic B cells from Toso –/– mice
Together, our data suggest that while expression of Toso induced up to 4-fold higher frequency and numbers of IL-10–
on B cells is associated with enhanced protective T cell immu- producing cells (Figure 3, B and C). Increased production of
nity during acute infection, it may also contribute to T cell– IL-10 by B cells from Toso-deficient mice is a B cell–intrinsic
mediated immunopathological tissue damage under chronic phenotype, as it was specifically observed upon conditional
inflammatory conditions. deletion of Toso in B cells (Tosof/f/CD19-Cre+/– mice), whereas
Toso deficiency results in increased numbers of IL-10–producing B cells from Tosof/f/CD4-Cre+/– mice and Tosof/f/CD11c-Cre+/–
B cells. The unexpected finding that T cell effector function is mice exhibited normal IL-10 production (Figure 3, D and E). B
indirectly regulated by Toso expression on B cells prompted us cell cytokine production was, however, not generally affected by
to analyze functional characteristics of Toso-deficient B cells. Toso deficiency, as activation-induced TNF-α production was
Here, we first assessed the capacity of Toso –/– B cells to produce normal in Toso –/– B cells (Supplemental Figure 6).

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Figure 2. Conditional deletion of Toso in

B cells results in increased mortality and
reduced T cell cytokine responses upon
influenza infection. (A–G) Mice were
infected i.n. with 1,000 PFU influenza virus
strain A/PR8 (H1N1). (A–C) Survival of mice
was monitored over time. (A) CD4-Cre+/–
and Tosof/f/CD4-Cre+/– mice (n = 9). (B)
CD11c-Cre+/– and Tosof/f/CD11c-Cre+/– mice
(n ≥14). (C) CD19-Cre+/– mice and Tosof/f/
CD19-Cre+/– mice (n ≥8). **P < 0.005; log-
rank test. (D–G) Lung cells isolated on day
9 p.i. were restimulated ex vivo, and the
number and frequency of IFN-γ–producing
(D and E) and TNF-α–producing (F and
G) CD4+ T cells (D and F) and CD8+ T cells
(E and G) were quantified by intracellular
cytokine staining. (H and I) CD19-Cre+/–
mice and Tosof/f/CD19-Cre+/– mice were
infected i.n. with 50 PFU influenza virus
strain A/PR8 (H1N1). On day 7 p.i., spleens
were analyzed for frequency and number of
PD-1–positive CD4+ T cells (H) and PD-L2–
positive CD19+ B cells (I). (D–I) Each symbol
represents an individual mouse; horizontal
lines indicate the mean (± SEM). (D–G)
n = 5; (H and I) n = 4. *P < 0.05; **P < 0.01;
***P < 0.001; Student’s t test. Data are
representative of at least 3 independent
experiments.

While only small numbers of IL-10–competent B cells are between WT and Toso –/– mice upon influenza A infection (Sup-
found in naive mice, number and frequency of IL-10–producing plemental Figure 7). Finally, upon acute infection with influenza
cells expand considerably upon influenza A infection (Figure 3, A virus, significantly higher frequency and numbers of IL-10–
F and G). Importantly, also under these conditions of an in vivo competent B cells were detected in the lungs of Tosof/f/CD19-
viral infection, splenic B cells from Toso –/– mice had a significantly Cre+/– mice compared with CD19-Cre+/– control mice (Figure 3H).
higher capacity to produce IL-10, as compared with B cells from Thus, taken together, our data suggest that Toso expression on B
WT mice (Figure 3, F and G). Total B cell counts in the spleen cells exhibits a B cell–intrinsic negative regulatory function on the
and numbers of GL7+CD95+ germinal center B cells were similar capacity of these cells to produce the antiinflammatory cytokine

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Figure 3. Toso deficiency results in increased numbers of IL-10–producing B ally confirming the different nature of these two different B cell
cells. (A–C) Purified B cells from WT and Toso–/– (KO) mice were treated with
subsets. Marginal zone (MZ) precursor B cells, which have been
BAFF, LPS, αCD40, or αIgM for 24 hours. For the last 5 hours, cells were stim-
ulated with PMA/ionomycin in the presence of brefeldin A (BFA)/monensin implicated in IL-10 production in a model of experimental arthri-
and subsequently analyzed for IL-10 production. (A) Representative flow tis (4), were also largely unable to produce IL-10 in our system
cytometric analysis. (B and C) Bar graphs show frequency (B) and absolute (Supplemental Figure 8, B–E).
numbers (C) of IL-10–positive B cells. Data are mean ± SEM from 2 cultures Further phenotypical analysis showed that the B220lo B1 B
derived from different mice. (D and E) B cells from mice with straight and
cell population expresses high levels of CD5 and CD43, typical for
conditional Toso knockout, as well as the indicated control mice, were
stimulated for 5 hours with LPS and PMA/ionomycin in the presence of B1a B cells, while IL-10–competent B220hiCD1d+ B2 cells exhibit
BFA/monensin. Frequency (D) and number (E) of IL-10–positive B cells were a CD5intIgMhiIgDloCD21hiCD23lo phenotype reminiscent of MZ B
determined by intracellular cytokine staining. (F and G) WT and Toso–/– (KO) cells (Figure 4H and Supplemental Figure 8F). IL-10–incompetent
mice were infected i.n. with 50 PFU influenza virus strain A/PR8 (H1N1). B220hiCD1d– cells were mainly IgMloIgDhiCD21intCD23hi follicular
At the indicated days p.i., splenocytes were restimulated ex vivo, and the
B cells (Figure 4H).
frequency (F) and number (G) of IL-10–positive CD19+ B cells were quantified
by intracellular cytokine staining. (H) CD19-Cre+/– mice and Tosof/f/CD19-Cre+/– Detection of IL-10–producing B cells from influenza-infected
mice were infected i.n. with 1,000 PFU influenza virus strain A/PR8 (H1N1). mice required short-term ex vivo restimulation. Here, we observed
Lung cells isolated on day 9 p.i. were restimulated ex vivo, and number and that IL-10–producing B1 and B2 B cell subsets are substantially
frequency of IL-10–positive CD19+ B cells were quantified by intracellular expanded in both lung and spleen from influenza A–infected mice
cytokine staining. Each symbol represents an individual mouse; horizontal
compared with uninfected control mice (Supplemental Figure 9,
lines indicate the mean ± SEM. (D and E) n = 3–7; (F–H) n = 4–5. *P < 0.05;
**P < 0.01; ***P < 0.001; Student’s t test. Data are representative of at least A–C). Moreover, IL-10–producing B2 B cells (B2/IL-10 cells) from
3 independent experiments. infected mice were also characterized by high expression of CD1d,
and, under such an inflammatory context, IL-10–producing B1
and B2 B cells showed strong expression of Tim-1, CD73, and FasL
IL-10 both under steady-state conditions and in an inflammatory (Supplemental Figure 9, D and E).
setting during viral infection. Most importantly, in Toso –/– mice both of the IL-10–
Phenotypic characteristics of IL-10–competent B cell subsets. We competent B cell subsets (B200hiCD1d+ B2 cells and B220lo B1a
next extended our analysis of IL-10–producing B cells onto different cells) were significantly increased in frequency and numbers
B cell subsets. Flow cytometric analysis of B cells from Vert-X IL-10 (Figure 4I). Interestingly, however, on a per-cell basis, the cell-
reporter mice — an IL-10-IRES-GFP knock-in mouse strain — intrinsic capacity to produce IL-10 was comparable between the
showed that, upon LPS induction, essentially all splenic B1 cells respective B cell subsets from WT and Toso –/– mice (Figure 4E).
(CD19+B220lo), as well as a small but significant fraction of B2 Thus, Toso –/– IL-10–competent B cells appeared to be functionally
cells (CD19+B220hi), produced IL-10, as indicated by GFP expres- normal, but were present in significantly higher quantities upon
sion (Supplemental Figure 8A). Thus, IL-10–competent B cell genetic ablation of Toso.
subsets reside within both B1 and B2 B cell compartments. IL-10– Suppressive function of IL-10–producing Bregs. IL-10–producing
producing B1 cells exhibited a CD5hi phenotype and showed low B cells have been described as Bregs that exhibit immune regula-
expression for CD1d, while, in line with previous reports (34), IL-10– tory functions and can downmodulate T cell responses and inflam-
producing B2 B cells were mainly characterized as CD1dhiCD5int cells. matory reactions (35, 36). Thus, to demonstrate immunosup-
Importantly, intracellular IL-10 staining of activated WT and Toso –/– pressive activity of IL-10–producing B1 and B2 B cell subsets, we
B cells revealed that both of these IL-10–producing B cell populations performed in vitro T cell–B cell coculture experiments. To this end,
— IL-10–producing B1a cells (B220lo) and IL-10–producing B2 cells B cells from Vert-X IL-10/GFP reporter mice were purified by FACS
(B220hi) — were markedly increased in B cell cultures from Toso –/– into IL-10–producing B1 B cells (B220loGFP+; “B1/IL-10 cells”) and
mice, irrespective of whether IL-10 production was induced by treat- B2 B cells (B220hiGFP+; “B2/IL-10 cells”), as well as GFP-negative
ment with BAFF/IL-21, LPS, or CpG oligonucleotides (Figure 4, A–C). control B cells (B220hiGFP–; B2/effector cells). Sorted B cell popu-
Based on B220 surface staining and the characteristic expres- lations were added to cultures containing purified naive CD4+ or
sion of CD1d on IL-10–producing B2 B cells, we were able to CD8+ T cells. Cultures were stimulated with anti-CD3 mAbs, and
efficiently identify IL-10–competent B1 and B2 B cell subsets proinflammatory cytokine production by CD4+ and CD8+ T cells
within untreated naive CD19+ B cells. High-purity cell sorting was assessed. The frequency of IFN-γ+ and TNF-α+ CD8+ T cells
experiments showed that more than 90% of naive sorted B220lo was significantly reduced in cocultures with B1/IL-10 cells and
B1 cells could be induced to produce IL-10 (Figure 4, D and E). B2/IL-10 cells, compared with cultures with B2/effector control
IL-10–competent B cells were also highly enriched within naive cells (Figure 5, A and B). A similar suppressive effect of IL-10–
B220hiCD1d+ B2 B cells, where approximately 50% of sorted cells producing B1 and B2 B cell subsets was observed in cocultures with
could be induced to express IL-10 (Figure 4E). In contrast, the naive CD4+ T cells (Figure 5C).
large population of B220hiCD1d– B2 B cells was largely unable Next, we assessed whether freshly isolated naive B220lo B1a
to produce IL-10. We also examined whether IL-10–compe- cells and B220hiCD1d+ B2 cells can also exhibit immunosup-
tent B220hiCD1d+ B2 cells are interrelated with B220hiAA4.1+ pressive function on T cell immunity. For these experiments on
transitional B2 cells; however, these two B2 B cell subsets were nonactivated B cell subsets, we performed adoptive transfer of
clearly distinct by CD1d versus AA4.1 surface expression (Figure purified naive B cell subsets followed by in vivo viral challenge.
4F), and, importantly, IL-10 production could not be induced in We adoptively transferred 1 × 106 FACS-sorted CD19+B220lo B1a
B220hiAA4.1+ transitional B cells (Figure 4G), thus also function- cells, CD19+B220hiCD1d+ B2 B cells, and CD19+B220hiCD1d– B2

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Figure 4. Phenotypic characteristics of IL-10–competent B cell subsets. (A–C) Purified B cells from WT and Toso–/– (KO) mice were cultured for 16 hours with
BAFF plus IL-21 (A), LPS (B), or CpG oligonucleotides (C). For the last 5 hours, cells were treated with PMA/ionomycin in the presence of BFA/monensin and
CD19+ B cells analyzed for IL-10 production. (D) B220 versus CD1d staining on naive CD19+ B cells from WT and Toso–/– (KO) mice. (E) B220hi B2-CD1d– B cells
(black), B220hi B2-CD1d+ B cells (blue), and B220lo B1 B cells (red) from WT and Toso–/– (KO) mice were purified by FACS. Cells were stimulated for 16 hours
with LPS plus PMA/ionomycin/BFA/monensin during the last 5 hours and subsequently analyzed for IL-10 production. (F) CD19+B220hi B cells were analyzed
for CD1d versus AA4.1 (CD93) staining to identify AA4.1–CD1d– B2-effector cells, AA4.1+ transitional B2 cells (B2-trans), and CD1d+ B2-Bregs. (G) AA4.1–CD1d–
B2-effector cells, AA4+ B2-transitional cells, and CD1d+ B2-Bregs were purified from IL-10/GFP reporter (Vert-X) mice by flow cytometric cell sorting. Cells
were treated for 16 hours with LPS plus PMA/ionomycin during the last 5 hours and analyzed for GFP (IL-10) expression. (H) Flow cytometric analysis of naive
B cells from C57BL/6J mice. Left panel is gated on CD19+ B cells and shows gating for total B2 cells (B220hi), B220hiCD1d– B2 cells, B220hiCD1d+ B2 cells, and
B220lo B1 cells. FACS profiles on the right show expression of IgM versus IgD (top panel) and CD23 versus CD21 (bottom panel) on the indicated B cell subsets.
(I) Number and frequency of splenic B220lo B1 B cells and B220hiCD1d+ B2 B cells in WT and Toso–/– (KO) mice. Each symbol represents an individual mouse;
horizontal lines indicate the mean ± SEM. n = 5; **P < 0.01; Student’s t test. Data are representative of at least 3 independent experiments.

B cells from untreated Toso –/– mice into C57BL/6 recipient mice. nificantly increased (Supplemental Figure 12, A and B). This was
One day after adoptive transfer, mice were intranasally infected further accompanied by an increase in CD21hiCD23lo MZ B cells
with influenza A, and cytokine responses in lung T cells were (Supplemental Figure 12, C and D), suggesting enhanced differen-
analyzed on day 9 p.i. (Figure 5D). In animals that had received tiation toward this particular B cell subset, which is also consistent
CD19+B220hiCD1d– cells, virus-induced TNF-α or IFN-γ produc- with increased numbers of MZ-like B2-Bregs in Toso –/– mice.
tion by CD4+ and CD8+ T cells was comparable to that in normal In addition to slightly increased surface expression of IgM, we
control animals that had not received any transferred cells (“no also noted that overall CD21 and CD23 expression levels, as well
transfer”; Figure 5, E–H). Adoptive transfer of naive CD19+B220lo as CD62L surface levels, were slightly downregulated in Toso –/– B
B1a cells, however, had clear suppressive effects on virus-induced cells, further indicating alterations in peripheral B cell maturation/
cytokine production by CD4+ and CD8+ T cells (Figure 5, E–H). differentiation (Supplemental Figure 12, E–H). Expression of CD19,
Reduced TNF-α and IFN-γ production by T cells was also observed B220, IgD, MHC-II, and CD44 was not affected by Toso deficiency
upon transfer of naive CD19+B220hiCD1d+ B2 B cells, although its (Supplemental Figure 12H).
effects were less pronounced than those of CD19+B220lo B1a cells, Furthermore, higher numbers of B220lo B1a cells were found
consistent with the greater enrichment of IL-10 competency of not only in the spleen of Toso –/– mice (Figure 4I and Supplemen-
CD19+B220lo B1a cells (Figure 4E). Based on their immunoregula- tal Figure 13, A and B), but also in the peritoneal cavity, where an
tory function, CD19+B220lo B1a cells are here termed as “B1-Bregs” increase in B1a cells was accompanied by a corresponding decrease
and CD19+B220hiCD1d+ B2 cells as “B2-Bregs.” Adoptively trans- in B1b cells (Supplemental Figure 13, C and D). Analysis of perito-
ferred naive B1- and B2-Bregs isolated from IL-10–/– mice did neal B cells revealed that B1a cells have an extremely high capacity
not exhibit measurable suppressive activity on proinflammatory to produce IL-10, whereas B1b cells are substantially less potent in
cytokine production by CD4+ and CD8+ T cells during influenza IL-10 production (Supplemental Figure 13, E and F). Altogether, our
A infection (Supplemental Figure 10), suggesting that IL-10 is a analysis of B cell development in Toso –/– mice suggests that Toso
critical effector molecule of Bregs in vivo, although the involve- is dispensable for B cell development in the BM, but fine-tunes the
ment of alternative effector mechanisms, such as CD73-mediated maturation/differentiation of specific B cell subsets in the periphery.
adenosine generation, which has been reported to be affected in Self-reactivity of regulatory B cell subsets. Development of
IL-10–deficient B cells (37), cannot be fully ruled out. peripheral B cell compartments is tightly regulated, and even
Our findings on adoptive transfer of as little as 1 × 106 cells small changes in B cell maturation/differentiation may alter the
demonstrate that naive B1-Bregs and B2-Bregs can both act as balance of peripheral B cell tolerance. We thus next examined
physiological regulators of T cell function by suppressing virus- serum antibody levels and autoantibody production in Toso –/–
induced T cell cytokine production during acute influenza A infec- mice. Basal levels of serum IgM and IgG were comparable between
tion. Moreover, these data also provide a mechanistic explanation 8- to 10-month-old WT and Toso –/– mice (Figure 6A). However,
for the impaired T cell responses in Toso –/– mice during influenza A consistent with published reports (21, 23–25), we detected
infection, which are likely caused by the higher numbers of immu- elevated serum titers of autoreactive antibodies in Toso –/– mice.
nosuppressive B1 and B2 Breg subsets in these mice. IgM and IgG antibodies directed against dsDNA or ssDNA were
B cell development in Toso-deficient mice. Toso surface expres- significantly increased in sera from aged Toso –/– mice compared
sion is not restricted to B1 and B2 immunoregulatory B cells, but with WT controls (Figure 6, B and C), supporting a role of Toso in
Toso is rather expressed on all peripheral B cells, with relatively the maintenance of self-tolerance.
highest expression on B220hiCD1d– effector (follicular) B cells To elucidate the major source of self-reactive antibodies in
(Supplemental Figure 11A). Thus, how Toso specifically affects Toso –/– mice, we analyzed the capacity of different B cell subsets
the generation of regulatory B cell subsets is still puzzling. Con- to produce autoantibodies. High-purity sorted B1- and B2-Bregs
sistent with previous reports (24, 25) and the absence of Toso sur- from Toso –/– mice exhibited strong production of anti-dsDNA and
face expression in early developmental B cell stages, we observed anti-ssDNA antibodies upon in vitro cultivation with LPS, while,
normal development of B cells in the bone marrow of Toso –/– mice in marked contrast, B2-effector cells and AA4.1+ transitional B2 B
(Supplemental Figure 11, B–G). Analysis of splenic B cells from cells were largely unable to produce self-reactive antibodies (Fig-
Toso –/– mice revealed reduced frequency of mature IgMloIgDhi B ure 6D). Efficient production of autoantibodies was not a specific
cells, while the population of IgMhiIgDhi transitional B cells was sig- feature of Toso –/– Bregs, but was similarly observed in WT B1- and

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Figure 5. Suppressive function of IL-10–producing Bregs. (A–C) Bregs sup- survival upon treatment with other stimuli, such as LPS or BAFF
press inflammatory cytokine production in T cells in vitro. B cells from IL-10/
plus IL-21 (Supplemental Figure 15, F and G).
GFP reporter (Vert-X) mice were treated for 16 hours with LPS, and PMA/
ionomycin was added during the last 5 hours. B2/effector cells (B220hiG- As Toso interacts with the membrane BCR complex (22) and
FP–), B2/IL-10 cells (B220hiGFP+), and B1/IL-10 cells (B220loGFP+) were then given the effects of Toso deficiency on B2-effector cell survival/
purified by FACS and were subsequently cocultured with naive CD8+ T cells proliferation, we examined the role of Toso in proximal BCR-
(A and B) or naive CD4+ T cells (C). Cultures were stimulated with anti-CD3 mediated signaling events and early markers of B cell activation.
for 48 hours and restimulated with PMA/ionomycin in the presence of BFA/
Upon stimulation with anti-IgM, Toso-deficient B2-effector cells
monensin for 5 hours. Percentage of IFN-γ–producing (A) and TNF-α–pro-
ducing (B and C) T cells was determined by intracellular cytokine staining. exhibited slightly impaired and less sustained phosphorylation of
(D–H) Bregs suppress inflammatory cytokine production in T cells during the tyrosine kinase Btk, indicating attenuated BCR signaling (Fig-
antiviral immune response in vivo. (D) Experimental model. Briefly, naive ure 6J). Lower Btk activation correlated with reduced induction
CD19+B220hiCD1d– B2 B cells (gray), CD19+B220hiCD1d+ B2 B cells (blue), and of activation markers, such as CD25, CD69, and CD86, in Toso-
CD19+B220lo B1a cells (red) were purified from Toso–/– (KO) mice by FACS and
deficient B cells (Figure 6, K and L, and Supplemental Figure
were adoptively transferred into C57BL/6J mice. Mice were infected i.n. with
1,000 PFU influenza virus strain A/PR8 (H1N1). On day 9 p.i., lung cells were 15H). It is noteworthy that IgM-mediated induction of activation
isolated and analyzed for cytokine staining. (E–H) Number and frequency markers was not fully impaired upon Toso deletion, but Toso –/–
of TNF-α–producing (E and F) and IFN-γ–producing (G and H) CD4+ T cells cells rather exhibited a relative shift in IgM receptor responsive-
(E and G) and CD8+ T cells (F and H). Mice that had not received adop- ness (Figure 6L and Supplemental Figure 15H), indicating altered
tively transferred cells (no transfer; open squares) but were also infected
signaling/activation thresholds. Lower BCR-mediated induction
with influenza were used as positive control; uninfected mice served as a
negative control (open circles). Data are expressed as mean ± SEM; symbols of activation markers was also observed in Toso –/– B2-Bregs and
represent individual mice. (A–C) n = 3; (E–H) n = 4–9. *P < 0.05; **P < 0.01; B2-transitional cells, while, owing to their cell type–intrinsic low
***P < 0.001; 1-way ANOVA and Dunnett’s post hoc test. Data are represen- BCR responsiveness, B1-Bregs largely failed to respond to anti-
tative of at least 3 independent experiments. IgM stimulation (Figure 6K). Together, these data indicate that
Toso acts as a signal amplifier to fine-tune BCR responsiveness.
Anti-Toso treatment modulates IL-10–competent B cell numbers at
B2-Bregs (Supplemental Figure 14A). B2-effector cells and AA4.1+ sites of inflammation and results in impaired T cell responses upon influ-
transitional B2 B cells exhibited only minimal autoantibody pro- enza infection. Given the negative regulatory effect of Toso on IL-10–
duction under these conditions, even though they showed effi- competent B cells and their immunosuppressive function on T cell
cient blast formation and expression of the plasma cell marker immunity, we sought to investigate the effects of anti-Toso antibody
CD138 (Supplemental Figure 14B). Together, these data indicate treatment on B and T cell responses in the influenza-induced lung
that self-reactive B cells are highly prevalent among B1-Bregs and inflammation model and to evaluate its potential for immunomod-
B2-Bregs. Interestingly, IL-10–competent immunoregulatory B ulatory therapeutic applications. To induce virus-mediated lung
cell numbers rise as mice age, and increased numbers of Bregs in inflammation, mice were infected intranasally with influenza A.
Toso –/– versus WT mice become even more pronounced in older One day before infection and on days 2 and 5 p.i., mice were treat-
mice (Figure 6, E and F). It is thus likely that the higher numbers ed with either anti-Toso mAb or control IgG, and B cells in inflamed
of Bregs in Toso –/– mice are responsible for increased autoantibody lungs were analyzed on day 9 p.i. Notably, Toso is highly expressed
levels in Toso –/– mice. on essentially all peripheral B cells; however, in vivo anti-Toso mAb
Toso fine-tunes B cell antigen receptor responsiveness. Regulation application did not result in B cell depletion, as comparable numbers
of B cell antigen receptor (BCR) signaling is a critical determinant of B cells were detected in lungs of anti-Toso and control IgG–treated
of peripheral B cell differentiation/maintenance and the estab- mice (Figure 7A). Treatment with anti-Toso mAb did, however,
lishment of tolerance (38–41). We thus next evaluated the effects induce a striking increase in both frequency and absolute num-
of Toso deficiency on BCR responsiveness and B cell activation/ bers of IL-10–competent B cells in lungs from influenza-infected
survival. Reflecting their different maturation/differentiation state, animals (Figure 7B). Importantly, consistent with our findings on
overall responsiveness to IgM receptor triggering varied consider- Toso-deficient mice, increased numbers of IL-10–competent B cells
ably among different CD19+ B cell subtypes. Upon anti-IgM stimula- correlated with impaired T cell responses at sites of inflammation,
tion, B2-effector cells (B220hiAA4.1–CD1d–) proliferated vigorously, as virus-induced TNF-α and IFN-γ production by CD4+ and CD8+
while B2-Bregs (B220hiCD1d+) and the IL-10–incompetent popu- T cells was significantly reduced in lungs of mice that had received
lation of transitional B2 cells (B220hiAA4.1+), which both express Toso mAb compared with control IgG–treated mice (Figure 7, C–F).
high levels of IgM, rather showed induction of cell death. B1a B cells Together, these data re-emphasize the immunoregulatory role of
(B220loAA4.1–CD1d–; B1-Bregs) were largely unresponsive to anti- Toso during inflammatory disease. Furthermore, as Toso is con-
IgM triggering (Figure 6G and Supplemental Figure 15, A–E). served between mice and humans and is also expressed on human
Toso –/– B2-effector cells showed a dose-dependent reduction B cells (ref. 19 and Supplemental Figure 16), the data also suggest
in anti-IgM–induced cell proliferation, which was primarily asso- that Toso may provide a promising therapeutic target to modulate
ciated with reduced cellular survival, while, similar to previous IL-10–competent B cell compartments and to dampen excessive T
reports (25), the actual rate of cell divisions of live cells was com- cell responses at local sites of inflammation.
parable between WT and Toso –/– cells (Figure 6, H and I). Toso
deficiency had, however, no effects on anti-IgM–induced survival/ Discussion
apoptosis of B1-Bregs, B2-Bregs, or B2-transitional cells (Supple- Using conditional gene deletion, we here demonstrate that
mental Figure 15, A and B), and Toso –/– B cells also showed normal impaired antiviral T cell responses upon influenza A infection in

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Figure 6. Self-reactivity of regulatory B cell subsets and altered activation CD5int B2 cells (B2-Bregs) and B220loCD5hi B1a cells (B1-Bregs)
thresholds of Toso-deficient B cells. (A–C) Serum from 8- to 10-month-old
— exhibit immunosuppressive activity on influenza-induced T
female WT and Toso–/– (KO) mice was analyzed for total IgM and IgG levels
(A) and titers of IgM and IgG antibodies against dsDNA (B) and ssDNA (C). cell responses. Adoptively transferred Bregs from IL-10–deficient
n = 6. (D) Sorted B220hiAA4.1–CD1d– B2-effector cells, B220hiCD1d+ B2-Bregs, mice did not exhibit such potent suppressive activity on T cell
B220hiAA4.1+ B2-transitional cells (B2-trans), and B220lo B1-Bregs from cytokine production, suggesting that the immunoregulatory func-
Toso–/– mice were treated for 3 days with LPS. Culture supernatants were tion of Bregs is largely mediated via IL-10. It is, however, conceiv-
analyzed for anti-dsDNA and anti-ssDNA IgM levels (n = 2). (E and F)
able that, depending on the specific inflammatory context, addi-
Frequency (E) and numbers (F) of IL-10–positive splenic B cells from WT and
Toso–/– mice of the indicated ages. n = 3–5. (G and H) Sorted B2-effector tional mechanisms, such as PD-L2/PD-1, CD80/CTLA4, GITR/
cells from WT and Toso–/– (KO) mice were stimulated for the indicated dura- GITRL, or FasL/Fas interactions, secretion of TGF-β or IL-35,
tions with titrated amounts of anti-IgM and analyzed for cell counts or CD73-mediated adenosine generation, also contribute to the
(n = 2) (G) and cell survival (n = 4) (H). (I) Sorted B2-effector cells were inhibitory function of B1- and B2-Bregs (37, 47–51).
labeled with eFluor670, stimulated with anti-IgM, and analyzed for cell
An important but largely unexplored aspect of Bregs is their
division. (J) Sorted B2-effector cells from WT (gray filled) and Toso–/– (KO)
(red line) mice were stimulated for the indicated durations with anti-IgM antigen specificity. We here show that IL-10–competent B1- and
and analyzed for phospho-Btk (p-Btk) staining. (K and L) Sorted B cell B2-Bregs exhibit a high prevalence of autoreactivity and readily
subsets were stimulated for 16 hours with anti-IgM and analyzed for B secrete self-reactive antibodies upon TLR stimulation. Self-reactivity
cell activation markers. (K) Representative flow cytometric histograms for is a typical feature of B1a and MZ B cells, which express polyreactive
CD25 expression. WT, black; Toso–/– (KO), red line; unstimulated control,
BCRs that can bind both to pathogens and to self-antigens (52). Con-
gray filled. (L) Sorted B2-effector cells were analyzed for anti-IgM–induced
upregulation of CD25, CD69, and CD86. Data show the geometric mean sidering this and the striking similarities in surface marker charac-
fluorescence intensity (GeoMFI) (n = 2). Data are expressed as the mean teristics between regulatory B cell subsets and B1a/MZ B cells, it is
± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test. All data are thus an intriguing idea that the majority of Bregs originate from B1a
representative of 2 to 4 independent experiments. and MZ B cells and/or their precursors — the reported phenotypic
diversity of Bregs may be more related to effects of the specif-
ic inflammatory milieu and particular tissue environment and/or
Toso –/– mice were not due to T cell–inherent defects, but rather reflect differences in the cellular activation status. Supporting evi-
were induced by a previously unrecognized role of Toso in B cells. dence for this idea comes from earlier studies that have character-
Our findings thus reveal an unexpected regulatory activity of B ized B1 and MZ B cells as major IL-10–producing B cells and have
cells on T cell function during viral infection. Deletion of Toso on demonstrated regulatory function of these specific B cell subsets
B cells results in a strong increase of IL-10–competent B cells, and, in different models of inflammatory disorders and autoimmunity
as we further demonstrate, this specific subtype of B cells mediates (45, 53–59). Consistent with Bregs being closely related to B1a and
immunosuppressive activity on T cell responses during viral infec- MZ B cells, the observed increase in IL-10–competent B cells in
tion, most likely via an IL-10–dependent mechanism. Immuno- Toso –/– mice correlated with a corresponding increase in B1a and MZ
suppressive function of IL-10 during influenza infection has been B cells. While an increase in B1a cells has also been observed in oth-
demonstrated in studies on IL-10–/– mice (42, 43). Moreover, B cells er Toso-deficient mouse strains, MZ B cell numbers were reported
have been shown to be a relevant source of IL-10 during viral infec- to be either decreased or unchanged (21, 23, 24). Discrepancies in
tion (44). B cells upregulate IL-10 expression during infection with MZ B cell numbers may be due to different targeting strategies and
murine cytomegalovirus, and this B cell–derived IL-10 decreases use of different embryonic stem cell lines (129/Sv vs. B6) and also
virus-specific IFN-γ responses in CD8+ T cells (44). Considering be related to slightly altered expression levels of CD21 and CD23
our data, we thus propose that Toso promotes efficient antivi- in Toso –/– B cells, which complicate MZ B cell identification. Given
ral T cell cytokine responses by restricting the size of the IL-10– their potential to produce self-reactive antibodies, increased num-
competent B cell pool. Specifically, B cell–intrinsic expression of bers of B1a and MZ B cells likely also account for the elevated,
Toso restricted differentiation/maintenance of IL-10–competent albeit nonpathogenic, levels of IgM and IgG autoantibodies that we
B cells in vivo. At the cellular level, Toso –/– Bregs exhibited normal and others have detected in Toso –/– mice (21, 23–25). In the NZB/W
functions. Hence, the impaired T cell responses observed in Toso –/– mouse model of systemic lupus erythematosus, B1 and MZ B cells
mice following influenza infection likely reflect the increased num- are also expanded and spontaneously secrete IgM autoantibodies
bers of Bregs in Toso-deficient mice. and can produce self-reactive, isotype-switched IgG antibodies (60,
The present study demonstrates that IL-10–competent B cells 61). Consistent with a regulatory capacity of B1a and MZ B cells, the
are highly enriched within 2 distinct subpopulations of B cells, number of IL-10–producing B cells was also found to be increased in
the small fraction of splenic B220hiCD1d+CD5int B2 cells and the young NZB/W mice prior to disease onset (62).
fraction of B220loCD5hi B1a cells. These findings are consistent Altogether, these findings suggest that, in analogy to natural
with earlier descriptions of B1a B cells as highly potent producers regulatory T cells (1), Bregs are characterized by self-reactive anti-
of IL-10 (45) and recent reports of an IL-10–competent popula- gen receptors and, upon appropriate activation, utilize the secre-
tion of CD19hiCD1d+CD5+ B cells, termed B10 cells, that exhibits tion of immunomodulatory cytokines, such as IL-10, as a major
immunoregulatory function in models of autoimmunity (6, 8, 34, effector mechanism to prevent excessive inflammatory reactions.
46). Interestingly, IL-10–competent B220hiCD1d+CD5int B cells Our observation that IL-10–producing B cells expand in numbers
share many surface marker characteristics with MZ B cells. and are recruited to the site of inflammation during influenza-
Adoptive transfer of naive B cell subsets showed that both induced pulmonary disease thus suggests a scenario in which rec-
major IL-10–competent B cell compartments — B220hiCD1d+ ognition of self-antigens (which are exposed as a result of tissue

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Figure 7. Immunomodulatory effect of Toso-blocking antibody treatment on influenza-induced lung inflammation. Mice were infected i.n. with influenza
virus strain A/PR8 (H1N1) to induce pulmonary inflammation. On day –1, day 2, and day 5 p.i., mice were treated with anti-Toso mAb or control IgG (200 μg/
mouse; i.v.). Lung cells isolated on day 9 p.i. were restimulated ex vivo and analyzed for cytokine production in T and B cells by intracellular cytokine staining.
(A–C) Quantification of total CD19+ B cells (A) and frequency (B) and number (C) of IL-10–positive B cells in lungs of infected animals. (C–F) Quantification
of frequency and numbers of TNF-α–producing (C and D) and IFN-γ–producing (E and F) CD4+ T cells (C and E) and CD8+ T cells (D and F). (A–F) Each symbol
represents an individual mouse; horizontal lines indicate the mean ± SEM. PBS control, n = 2; influenza A–infected mice, n = 5. *P < 0.05;
**P < 0.01; ***P < 0.001; Student’s t test. Data are representative of 2 independent experiments.

damage) by autoreactive Bregs induces their immunosuppressive deficiency on B cells have increased numbers of Bregs and, thus, a
activity to dampen the inflammatory response and limit immuno- “dysregulated” system, which results in impaired (“suppressed”)
pathology. We thus propose that the population of Bregs is normally proinflammatory T cell responses (see model in Supplemental Fig-
tightly controlled, to ensure a well-balanced immune response that ure 17). Our data indicate that during acute influenza infection, in
allows for efficient immune protection against pathogens while which antiviral immune protection is largely dependent on effector
minimizing immunopathological tissue damage. Mice with Toso T cells, such suppressed T cell responses are detrimental and are

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The Journal of Clinical Investigation     RESEARCH ARTICLE

associated with impaired immune protection and an increased risk informatics.jax.org/reference/J:123556), or the cd19 promoter [CD19-
of mortality. In contrast, under chronic inflammatory conditions, Cre mice: Cd19tm1(cre)Cgn] (http://www.informatics.jax.org/reference
such as the chronic bacterial-induced model of colitis, in which /J:67676). Vert-X IL-10 reporter mice [B6(Cg)-Il10tm1.1Karp] (http://www.
effector T cells are more associated with immunopathological informatics.jax.org/reference/J:151551) that carry an internal ribosome
tissue damage, higher numbers of Bregs and, thus, impaired T cell entry site (IRES)–enhanced green fluorescent protein (eGFP) fusion pro-
effector function are beneficial, as this limits T cell–mediated tissue tein downstream of exon 5 of the interleukin-10 (Il10) gene were used
destruction (Supplemental Figure 17). for some experiments. Cre-expressing transgenic mouse lines, IL-10
Differentiation and homeostasis of peripheral B cell subsets reporter mice, and IL-10–deficient mice (B6.129P2-Il10tm1Cgn; http://
is critically influenced by signaling through the BCR (38–41). www.informatics.jax.org/allele/MGI:1857199; provided by A. Bleich and
In line with altered peripheral B cell compartments in Toso- I. Brüsch, Hannover Medical School) were all on C57BL/6J background
deficient mice, we here demonstrate that Toso shifts the threshold and were originally obtained from The Jackson Laboratory. Mice were
for BCR-mediated cellular activation/survival pathways. The exact housed in individually ventilated cages under specific pathogen–free
molecular mechanism of how Toso affects BCR responsiveness is conditions in the barrier animal facility at Hannover Medical School.
currently unknown. As an IgM-binding molecule, Toso may interact Mice were infected at 10–13 weeks of age. Controls were sex- and age-
directly with membrane-bound IgM-containing BCR complexes matched. For in vivo influenza infection, mice were anesthetized with
(22) or, alternatively, may indirectly affect B cell signaling via rec- ketamine-xylazine. Virus was diluted in sterile PBS, and mice were
ognition of soluble IgM immune complexes. Increased tonic sig- infected by intranasal (i.n.) administration of a total volume of 40 μl of
naling in Toso-deficient B cells (21) could not be confirmed in our PR8 influenza virus. Mice were monitored daily for weight loss, signs
study and may be related to the unusual occurrence of a lympho­ of illness, and survival. Anti-Toso mAb or control rat IgG (catalog 012-
proliferative disorder in this particular strain of mice, which has not 000-003, Jackson ImmunoResearch) was administered i.v. on days –1, 2,
been observed in any other strain of Toso-deficient mice. and 5 p.i. (200 μg/mouse/time point). To examine the primary immune
Infection with influenza virus is frequently associated with response, cells were harvested from lungs and spleen on the indicated
severe pulmonary immune pathology in human patients. The days postinfection. Bronchoalveolar lavage fluid was collected for viral
anatomical structures in the lung are highly sensitive to tissue titer measurements.
destruction, necessitating a fine balance between pro- and anti- Salmonella infection studies. Streptomycin (20 mg/mouse) was
inflammatory responses during pulmonary infection. In partic- given by oral gavage to mice aged 16 weeks. Twenty-four hours after
ular, after viral clearance, excessive release of proinflammatory antibiotic administration, mice were infected with Salmonella Typh-
cytokines by continually recruited CD8+ T cells can cause severe imurium ΔaroA at a dose of 3 × 106 bacteria in 100 μl HEPES buffer
lung tissue injury. In the present study, we show that during influ- (100 mM, pH 8.0). Control mice (mock infection) were given 100 μl
enza A–induced pulmonary inflammation the application of Toso HEPES buffer. For histopathological analysis, tissues were fixed in
blocking antibody selectively induces IL-10–competent B cells 10% neutral buffered formalin overnight and embedded in paraffin.
at the site of inflammation, an effect that was associated with Cecum sections (5 μm) were deparaffinized and stained with H&E.
reduced production of proinflammatory cytokines by lung T cells. Histological scores in the ceca of infected mice were determined as
These data suggest that clinical targeting of Toso may provide a previously described (63). Briefly, pathological changes were assessed
novel therapeutic approach to control pathogenic T cell responses by evaluation of various parameters such as presence of luminal cells,
via the modulation of IL-10–competent B cell compartments at infiltrating immune cells, crypt abscesses, and the formation of edema
local sites of inflammation. in the respective layer of the intestinal bowel wall including the sur-
face epithelium, mucosa, and submucosa.
Methods Influenza virus. Influenza virus strain A/PR8 (A/Puerto Rico/8/34
Mice and viral infection. Mice with constitutive Toso knockout (Toso –/–) [H1N1]) was obtained from ATCC. Virus was grown in Madin-Darby
and the generation of mice with a conditional floxed Toso allele (Tosof/f canine kidney (MDCK) cells (obtained from ATCC). Viral titers were
mice) have been described before (18). In brief, the targeting vector determined by standard MDCK plaque titration assay. Briefly, serial
was designed to have exons 4–7 flanked by loxP sites. After transfec- 10-fold dilutions of virus stock or bronchoalveolar lavage fluid from
tion into C57BL/6 embryonic stem cells, targeted embryonic stem infected mice were allowed to adsorb onto 90% confluent MDCK cells
cell clones were identified by Southern blotting and were injected into on a 24-well plate. After 2.5 hours of incubation, cells were overlaid
blastocysts. Upon germline transmission the frt-flanked neomycin- with 1.2% Avicel RC-581 (IMDC Deutschland GmbH) in DMEM (Gib-
selection cassette was removed by breeding with C57BL/6 flp deleter co) supplemented with 0.1% BSA, 1% l-glutamine, penicillin, strep-
mice. The resulting floxed-targeted mouse lines were crossed with tomycin, and 1 μg/ml TPCK Trypsin (Thermo Fisher Scientific) and
the EIIa-Cre transgenic mouse line (http://www.informatics.jax.org/ cultured for 24 hours at 37°C in 5% CO2. Cells were washed, fixed, and
reference/J:70555) to obtain Cre-mediated total germline excision of permeabilized, and virus plaques were visualized and enumerated by
exons 4–7 (constitutive Toso knockout). Toso-deficient and Toso-floxed staining with an mAb against influenza A nucleoprotein (AA5H, AbD
mice were backcrossed for more than 8 generations with C57BL/6J. To Serotec). Viral titers were calculated as PFU per milliliter.
obtain cell type–specific conditional deletion, Toso-floxed (Tosof/f) were Flow cytometry (FACS). Single-cell suspensions of spleen, bone
crossed with transgenic mouse lines expressing Cre-recombinase under marrow, and peritoneum were prepared from fresh tissue using stan-
control of the cd4 promoter [CD4-Cre mice: Tg(Cd4-cre)1Cwi] (http:// dard procedures. To isolate pulmonary lymphocytes, lung lobes were
www.informatics.jax.org/reference/J:73127), the cd11c (Itgax) pro- minced and strained through nylon mesh. Following red blood cell
moter [CD11c-Cre mice: Tg(Itgax-cre,-EGFP)4097Ach] (http://www. lysis, cells were blocked with anti-CD16/32 (clone 93, Biolegend) and

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RESEARCH ARTICLE The Journal of Clinical Investigation   

subsequently stained with fluorescent-labeled mAbs summarized in CD40 (10 μg/ml; clone 1C10, Biolegend), anti-IgM [10 μg/ml; goat
Supplemental Table 1. Anti-Toso mAb (rat IgG2a) directed against the anti–mouse F(ab′)2 fragment, Jackson ImmunoResearch], or a combi-
extracellular domain of murine Toso was generated by DNA vaccina- nation of BAFF (200 ng/ml) plus IL-21 (50 ng/ml; Biolegend). PMA
tion (18) and was directly conjugated with DyLight 649 (Thermo Fisher (50 ng/ml; Sigma-Aldrich) and ionomycin (500 ng/ml; Sigma-Aldrich)
Scientific). APC-labeled MHC-II I-Ab/NP311–325 (NP311) tetramer plus brefeldin A and monensin (both eBioscience) were added during
(containing QVYSLIRPNENPAHK of influenza virus nucleoprotein) the last 5 hours of culture. Cells were subsequently analyzed by intra-
was obtained from the NIH Tetramer Core Facility (Emory University cellular flow cytometry.
Vaccine Center, Atlanta, Georgia, USA), and APC-labeled Db/NP366–374 For some experiments, FACS-sorted B cell populations were stim-
(NP366) dextramer (containing ASNENMETM of influenza virus nuc- ulated for the indicated times with titrated amounts of anti-IgM [goat
leoprotein) was purchased from Immudex. For discrimination of live anti–mouse F(ab′)2 fragment, Jackson ImmunoResearch], and cultures
versus dead cells, we used the fixable viability dye eFluor450 (eBiosci- were subsequently analyzed by flow cytometry for cell survival and
ence). For the detection of intracellular cytokines, cells were restim- upregulation of activation markers. To analyze in vitro autoantibody
ulated ex vivo in the presence of brefeldin A and monensin (both production, FACS-sorted B cell populations were stimulated for 3 days
eBioscience). After cell surface staining, cells were fixed with parafor- as indicated. Culture supernatants were then collected for the detec-
maldehyde and permeabilized using Perm/Wash buffer (eBioscience) tion of autoantibodies, and cells were analyzed by flow cytometry.
and stained with mAbs against TNF-α (MP6-XT22), IFN-γ (XMG1.2), For the analysis of B cell signaling, FACS-sorted B effector cells
or IL-10 (JES5-16E3; all Biolegend). Flow cytometric measurements were stimulated with anti-IgM [10 μg/ml; goat anti–mouse F(ab′)2
were performed on a FACSCanto II cell analyzer (BD Biosciences). fragment, Jackson ImmunoResearch]. Stimulation was stopped by
Data were analyzed with FlowJo software (version 9.9.3, Mac; version fixation in paraformaldehyde. Cells were then permeabilized using
10.0.7, PC; Tree Star). Perm/Wash buffer (eBioscience), stained with anti–phospho-Btk/Itk
Cell sorting and adoptive transfer experiments. For high-purity cell (Tyr551, Tyr511; eBioscience), and analyzed by flow cytometry.
sorting experiments, splenic B cells were first enriched by magnetic ELISA and detection of autoantibodies. Serum titers of IgM and
isolation using negative selection (pan–B cell isolation kit; Miltenyi IgG were determined by specific ELISA kits (eBioscience) according
Biotec and Stemcell Technologies). B cell subpopulations were then to the manufacturer’s protocol. To detect autoantibodies in serum
further purified following immunofluorescence surface staining using and cell culture supernatants, high-binding ELISA plates (Greiner)
high-speed flow cytometry cell sorters (FACSAria Fusion and FACSAria were coated overnight with 2 μg/ml ssDNA or dsDNA from calf thy-
IIu, both BD Biosciences). To obtain highly pure B cell subsets with mus (Sigma-Aldrich). ssDNA was obtained by heat denaturation of
minimal contamination by other cell types, we pre-gated on viable dsDNA (95°C, 10 minutes) followed by rapid cooling on ice. Coated
cells that were CD19+, but negative for CD4, CD8, F4/80, and NK1.1. plates were blocked with 1% BSA, 0.5% gelatin in TBS for 2 hours at
Further gating of B cell subsets was primarily based on B220, CD1d, room temperature, and diluted samples were incubated overnight
and AA4.1 surface expression or B220 versus GFP (IL-10) expression. at 4°C in TBS 1% BSA. Bound anti-ssDNA or anti-dsDNA antibod-
Sorted B cell subsets had greater than 98% purity. ies were detected with HRP-conjugated anti-mouse IgG (eBiosci-
For adoptive transfer experiments, FACS-sorted B cell subsets ence) or with anti-mouse IgM-biotin (Jackson ImmunoResearch) and
were immediately transferred i.v. into syngeneic C57BL/6J recipient streptavidin-HRP (R&D Systems) followed by TMB substrate solution
mice (1 × 106 cells per recipient mouse). One day after adoptive trans- (eBioscience). Absorbance was measured at 450 nm.
fer, mice were infected i.n. with influenza virus strain A/PR8. On day In vitro B cell suppression assay. Splenic B cells from Vert-X
9 p.i., lung cells were isolated, restimulated ex vivo, and subjected to IL-10 reporter mice were isolated by positive selection using CD19-
flow cytometric analysis. coupled MicroBeads (Miltenyi Biotec) and were cultured for 16 hours
Ex vivo cell restimulation, cell activation, and B cell cultures. For the with ultrapure LPS (500 ng/ml) plus addition of PMA (50 ng/ml) and
detection of T cell cytokine production, cells were restimulated ex vivo ionomycin (500 ng/ml) during the last 5 hours. B cell cultures were
for 5 hours in the presence of brefeldin A and monensin (both eBiosci- surface-stained, and indicated CD19+ B subpopulations were purified
ence) on tissue culture plates that had been precoated with anti-CD3 with greater than 98% purities by FACS based on B220 versus GFP
(10 μg/ml; clone 145-2C11, eBioscience) and anti-CD28 (2 μg/ml; (IL-10) expression. Naive CD4+ and naive CD8+ T cells were isolated
clone 37.51, Biolegend). Where indicated, splenocytes were restimu- from C57BL/6J WT mice using respective naive T cell isolation kits
lated for 6 hours with influenza A virus peptide of amino acids 366– (Miltenyi Biotec). For suppression assays, FACS-sorted B cell subpop-
374 of the viral nucleoprotein (NP366 peptide), an H-2Kb–restricted ulations were cocultured with purified naive T cells (3 × 105 B cells to 6
epitope that is specific for CD8+ T cells, in the presence of brefeldin × 105 T cells) in 48-well plates that had been precoated with anti-CD3
A and monensin. To assess IL-10 production by B cells during influ- (10 μg/ml; clone 145-2C11, eBioscience) for 48 hours. During the last
enza A virus infection, cells were restimulated ex vivo for 5 hours with 5 hours, PMA/ionomycin plus brefeldin A and monensin was added
PMA (50 ng/ml; Sigma-Aldrich) and ionomycin (500 ng/ml; Sigma- to the cultures. Cells were subjected to surface and intracellular FACS
Aldrich) in the presence of brefeldin A and monensin. Following ex staining and analyzed by flow cytometry.
vivo restimulation, cells were subjected to cell surface and intracellu- Statistics. Data are presented as mean values ± SEM. Statistical
lar FACS staining. analysis was performed using GraphPad Prism (version 6.0h, Mac OS
For the in vitro analysis of IL-10 production by B cells, isolated leu- X). Unless stated otherwise, differences between means were assessed
kocytes or purified B cell populations were cultured for the indicated using 2-tailed Student’s t test. Statistical analysis of Kaplan-Meier sur-
times with ultrapure LPS (500 ng/ml; InvivoGen), CpG oligonucle- vival curves was performed by the log-rank test. A P value of less than
otides (10 μg/ml; InvivoGen), BAFF (200 ng/ml; Biolegend), anti- 0.05 was considered statistically significant.

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The Journal of Clinical Investigation     RESEARCH ARTICLE

Study approval. Animal experiments were performed in accor- Acknowledgments

dance with institutional guidelines and were approved by the local We greatly appreciate the excellent technical support of
authorities (Lower Saxony State Office for Consumer Protection and Martina Krautkrämer and Alibek Galeev. We thank André
Food Safety, Germany). Bleich and Inga Brüsch for providing IL-10–deficient mice. This
work was supported by grants from the DFG (LE1254/2-1 and
Author contributions LE1254/2-2) and the Foundation for Pathobiochemistry and
KHL designed the study. JY and VHHD performed the major- Molecular Diagnostics (SPMD).
ity of the experiments, with specific contributions by AW, AS,
and GAG. Technical assistance was provided by KW. KB pro- Address correspondence to: Kyeong-Hee Lee, Institute of
vided valuable resources. ACC provided key reagents and feed- Clinical Chemistry and Inflammation Research, Hannover
back on the manuscript. NF and KHL supervised the study, ana- Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover,
lyzed data, and wrote the manuscript. Funding acquisition was Germany. Phone: 49.511.532.5285; Email: Lee.Kyeong-Hee@
handled by KHL. mh-hannover.de.

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