B cell depletion enhances T regulatory cell activity essential in the suppression of arthritis.
ABSTRACT The efficacy of B cell-depletion therapy in rheumatoid arthritis has driven interest in understanding the mechanism. Because the decrease in autoantibodies in rheumatoid arthritis does not necessarily correlate with clinical outcome, other mechanisms may be operative. We previously reported that in proteoglycan-induced arthritis (PGIA), B cell-depletion inhibits autoreactive T cell responses. Recent studies in B cell-depletion therapy also indicate a role for B cells in suppressing regulatory mechanisms. In this study, we demonstrate that B cells inhibited both the expansion and function of T regulatory (Treg) cells in PGIA. Using an anti-CD20 mAb, we depleted B cells from mice with PGIA and assessed the Treg cell population. Compared to control Ab-treated mice, Treg cell percentages were elevated in B cell-depleted mice, with a higher proportion of CD4(+) T cells expressing Foxp3 and CD25. On a per-cell basis, CD4(+)CD25(+) cells from B cell-depleted mice expressed increased amounts of Foxp3 and were significantly more suppressive than those from control Ab-treated mice. The depletion of Treg cells with an anti-CD25 mAb concurrent with B cell-depletion therapy restored the severity of PGIA to levels equal to untreated mice. Although titers of autoantibodies did not recover to untreated levels, CD4(+) T cell recall responses to the immunizing Ag returned as measured by T cell proliferation and cytokine production. Thus, B cells have the capacity to regulate inflammatory responses by enhancing effector T cells along with suppressing Treg cells.
- SourceAvailable from: Damian Maseda[Show abstract] [Hide abstract]
ABSTRACT: B cells mediate multiple functions that influence immune and inflammatory responses in rheumatoid arthritis. Production of a diverse array of autoantibodies can happen at different stages of the disease, and are important markers of disease outcome. In turn, the magnitude and quality of acquired humoral immune responses is strongly dependent on signals delivered by innate immune cells. Additionally, the milieu of cells and chemokines that constitute a niche for plasma cells rely strongly on signals provided by stromal cells at different anatomical locations and times. The chronic inflammatory state therefore importantly impacts the developing humoral immune response and its intensity and specificity. We focus this review on B cell biology and the role of the innate immune system in the development of autoimmunity in patients with rheumatoid arthritis.Expert Review of Clinical Immunology 04/2014; · 2.89 Impact Factor
Thesis: PhD thesis[Show abstract] [Hide abstract]
ABSTRACT: Autoimmune diseases such as rheumatoid arthritis (RA) or multiple sclerosis (MS) are commonly regarded as complex or multifactorial diseases. This complexity regards to effector mechanisms involved in pathologic manifestations, and also to the diversity of genetic and environmental factors that predispose individuals to such diseases. Identification of genetic traits becomes relevant to better understand the progression of these diseases, enabling the development of new therapies. Autoantibody formation against cartilage structures (e.g. collagen type II, CII), anti-citrullinated proteins (ACPA) and anti-Fc domains of other antibodies (rheumatoid factors, RF) are pathogenic and typically observed in RA patients. It is thus important to investigate their role in the disease development. In study I we evaluated the usefulness of a particular outbred stock of mice, the Northport heterogeneous stock (HS), in the study of genetic associations of different animal models. We observed that HS mice were suitable for studying disease models of MS, while being limited to study certain RA models, due to the absence of particular major histocompatibility complex (MHC) alleles. By introducing an arthritis permissive MHC H-2q allele, in study II we made use of the best characterized animal model of RA in mice (collagen-induced arthritis, CIA), and evaluated the genetic associations of autoantibody production during the course of the disease. The genetic associations with RF and ACPA production were evident and clearly distinct from anti-CII antibody responses. Amongst several identified quantitative trait loci (QTLs), we distinguished the Fc gamma receptor (FcγR) and immunoglobulin heavy chain (IgH) loci as the most central QTL regulating autoantibody formation. The Cia9 congenic fragment confirmed our FcγR association, while the involvement of the IgH locus on specific antigen recognition was thoroughly investigated in study III. Here we identified different germ-line polymorphisms controlling the antibody production and recognition of a specific CII epitope, named J1. Finally, in study IV, Cia37 congenic mice were used to investigate the role of vitamin D receptor (VDR) polymorphisms in arthritis susceptibility. The influence of vitamin D on cytokine secretion and the VDR gene expression profile observed, strongly implicate the VDR and vitamin D as regulators of autoimmunity in mice. In summary, several genetic associations as well as mechanistic hypothesis involving autoantibody formation are described in this thesis. We hope these findings can be of use for better understanding the pathology of RA, as well as for the development of new therapeutics to treat RA patients.11/2012, Degree: PhD, Supervisor: Rikard Holmdahl
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ABSTRACT: For the sake of therapy of diabetes, it is critical to understand human beta cell function in detail in health and disease. Current studies of human beta cell physiology in vivo are mostly limited to immunodeficient mouse models, which possess significant technical limitations. This study aimed to create a new model for the study of human islets through induction of transplant tolerance in immunosufficient mice. B6 diabetic mice were transplanted with human islets and treated with anti-CD45RB. To assess whether anti-CD45RB-induced transplant tolerance requires B cells, B6 recipients received additional anti-CD20 or B6μMT−/− mice were used. For some anti-CD45RB-treated B6μMT−/− mice, additional anti-CD25 mAb was applied at the early or late stage post-transplant. Immunohistology was performed to show the Foxp3 cells in grafted anti-CD45RB/anti-CD20-treated Foxp3-GFP B6 mice. The results showed that anti-CD45RB alone allowed indefinite graft survival in 26.6% of B6 mice, however 100% of xenografts were accepted in mice treated simultaneously with anti-CD20, and 88.9% of xenografts accepted in anti-CD45RB-treated μMT−/− mice. These μMT−/− mice accepted the islets from another human donor but rejected the islets from baboon. Additional administration of anti-CD25 mAb at the time of transplantation resulted in 100% rejection, whereas 40% of grafts were rejected while the antibody was administrated at days 60 post-transplant. Immunohistologic examination showed Foxp3+ cells accumulated around grafts. We conclude that induction of tolerance to human islets in an immunosufficient mouse model could be generated by targeting murine CD45RB and CD20. This new system will facilitate study of human islets and accelerate the dissection of the critical mechanisms underlying islet health in human disease.Xenotransplantation 07/2014; · 2.57 Impact Factor
The Journal of Immunology
B Cell Depletion Enhances T Regulatory Cell Activity
Essential in the Suppression of Arthritis
Keith M. Hamel,* Yanxia Cao,†Susan Ashaye,* Yumei Wang,†Robert Dunn,‡
Marilyn R. Kehry,‡Tibor T. Glant,xand Alison Finnegan*,†
The efficacy of B cell-depletion therapy in rheumatoid arthritis has driven interest in understanding the mechanism. Because the
decrease in autoantibodies in rheumatoid arthritis does not necessarily correlate with clinical outcome, other mechanisms may be
operative. We previously reported that in proteoglycan-induced arthritis (PGIA), B cell-depletion inhibits autoreactive T cell
responses. Recent studies in B cell-depletion therapy also indicate a role for B cells in suppressing regulatory mechanisms. In this
study, we demonstrate that B cells inhibited both the expansion and function of T regulatory (Treg) cells in PGIA. Using an anti-
CD20 mAb, we depleted B cells from mice with PGIA and assessed the Treg cell population. Compared to control Ab-treated mice,
Treg cell percentages were elevated in B cell-depleted mice, with a higher proportion of CD4+T cells expressing Foxp3 and CD25.
On a per-cell basis, CD4+CD25+cells from B cell-depleted mice expressed increased amounts of Foxp3 and were significantly more
suppressive than those from control Ab-treated mice. The depletion of Treg cells with an anti-CD25 mAb concurrent with B cell-
depletion therapy restored the severity of PGIA to levels equal to untreated mice. Although titers of autoantibodies did not recover
to untreated levels, CD4+T cell recall responses to the immunizing Ag returned as measured by T cell proliferation and cytokine
production. Thus, B cells have the capacity to regulate inflammatory responses by enhancing effector T cells along with suppress-
ing Treg cells.The Journal of Immunology, 2011, 187: 4900–4906.
T cells, B cells, neutrophils, and macrophages, although the precise
contribution of each of these cell populations is unclear (1). There
is a renewed interest in the involvement of B cells in RA based on
the clinical efficacy of B cell-depletion therapy with anti-CD20
Ab (rituximab) (2, 3). B cell depletion reduces rheumatoid factor
and anti-CCP Abs; however, variable decreases in these Abs
irrespective of improvement in clinical disease activity suggests
additional mechanisms for efficacy (4, 5). Removal of autoreactive
B cells participating in Ag presentation, costimulation, and cyto-
kine production likely play a role, but these have not been com-
pletely elucidated in RA (6). In other autoimmune diseases and in
an animal model of autoimmune disease, the T cell compartment
is altered after B cell depletion, resulting in reduced T cell acti-
vation and cytokine production (7–12).
Our murine model of RA, proteoglycan-induced arthritis
(PGIA), is similar to human disease by several criterion including
clinical assessment, radiographic analysis, scintigraphic bone
heumatoid arthritis (RA) is a debilitating inflammatory
disease of the synovial joints mediated by chronic acti-
vation of several different cell populations including
scans, laboratory tests, and histological assessment of diarthrodial
joints (13–18). In this model, proteoglycan (PG)-specific B cells
are required as Ab-secreting cells as well as Ag-specific APCs
(19). Secondary signals delivered by B cells through CD80/CD86
are essential for autoreactive T cell activation and the develop-
ment of arthritis (20). B cells are required to maintain chronic
inflammation as anti-CD20 mAb treatment inhibits arthritis,
T cell proliferation, and cytokine production (12).
Autoreactive T cells that escape central tolerance are regulated
in the periphery by T regulatory (Treg) cells (21). Treg cells are
divided into natural Treg cells that are induced in the thymus and
inducible Treg cells that are activated in the periphery. Treg cells
are characterized by high expression of CD25 and the transcrip-
tion factor Foxp3, which is essential for Treg cell activity (22–24).
Treg cells quell inflammation using several suppressive mecha-
nisms that are mediated through both soluble and membrane-
bound factors (25). In a number of autoimmune diseases, there
are documented defects in Treg cell numbers and function that
could potentially allow effector T (Teff) cell escape from sup-
pression by the production of proinflammatory cytokines by either
Teff cells or APCs (26–30). IFN-g produced by Th1 cells and IL-
6, IL-21, TGF-b produced by APCs that promote Th17 cells in-
hibit Treg cell differentiation (31–35). Thus, an inappropriate
balance between Teff and Treg cells permits autoreactive re-
A direct impact of B cells on Treg cell activity is not clearly
defined, although some recent work suggests that there may be
a link. In a mouse model of Crohn’s disease, B cells exacerbate
ileitis through suppression of Treg cell function (36). Suppression
of disease in B cell-depleted nonobese diabetes and thyroiditis is
accompanied by an increase in CD25+Foxp3+Treg cells (37–39).
In clinical studies, Foxp3 mRNA transcripts and Treg cell num-
bers are elevated in lupus patients following B cell-depletion
therapy, whereas in another study, Treg cells of patients with id-
iopathic thrombocytopenic purpura are defective prior to treat-
ment, but had restored capacity to suppress after B cell-depletion
*Department of Immunology/Microbiology, Rush University Medical Center, Chi-
cago, IL 60612;†Section of Rheumatology, Department of Internal Medicine, Rush
University Medical Center, Chicago, IL 60612;‡Biogen Idec, San Diego, CA 92122;
andxDepartment of Orthopedic Surgery, Rush University Medical Center, Chicago,
Received for publication June 22, 2011. Accepted for publication August 20, 2011.
This work was supported by Grant AR47657 from the National Institutes of Health
Address correspondence and reprint requests to Dr. Alison Finnegan, Section of
Rheumatology, Department of Medicine, Rush University Medical Center, 1735 West
Harrison Street, MC 109, Chicago, IL 60612. E-mail address: alison_finnegan@rush.
Abbreviations used in this article: DDA, dimethyldioctadecyl; hCD20, human CD20;
mCD20, mouse CD20; PG, proteoglycan; PGIA, proteoglycan-induced arthritis; RA,
rheumatoid arthritis; Teff, effector T; Treg, T regulatory; WT, wild-type.
therapy (40, 41). Treg cells were also increased in rituximab-
responsive cryoglobulinemia vasculitis patients (11). Based on
these data, we were interested in determining if the Treg cells are
involved in the suppression of arthritis after B cell depletion.
In this study, we report that the inflammatory environment in
autoimmune arthritis determines Treg cell activity. In B cell-
depleted mice, there was a significant decrease in Teff cell ac-
tivity that corresponded to an increase in the percentage of CD4+
CD25+Foxp3+T cells and in the level of Foxp3 protein expres-
sion. CD4+CD25+Treg cells from B cell-depleted mice were also
significantly more effective at suppressing PG-specific CD4+
T cell proliferation than Treg cells from control mice. Treg cells
contributed to the inhibition of PGIA as Treg cell suppression in
B cell-depleted mice was completely abolished by the simulta-
neous depletion of Treg cells with anti-CD25 mAb. Recovery of
arthritis severity in mice depleted of both B cells and Treg cells
was accompanied by the restoration of PG-specific CD4+T cell
responses. These data demonstrate that the activity of B cells in
autoimmune arthritis tips the balance toward inflammatory Teff
cells and away from Treg cell control of inflammation.
Materials and Methods
Mice, Ag, and assessment of arthritis
BALB/c wild-type (WT) mice were obtained from the National Cancer
Institute (Bethesda, MD). BALB/c IFN-g–deficient mice were obtained
from The Jackson Laboratory (Bar Harbor, ME). BALB/c B cell-deficient
mice (JHD) were provided by Dr. Mark Shlomchik (Yale University, New
Haven, CT). Female mice (.3 mo) were immunized i.p. with 150 mg
human PG in 2 mg dimethyldioctadecyl (DDA)-ammonium bromide ad-
juvant (Sigma-Aldrich, St. Louis, MO) in 200 ml PBS (pH 7.2) and
boosted on day 21 with 100 mg PG in DDA as previously described (42).
Human cartilage PG was purified from joint replacement surgeries by
procedures approved by the Institutional Review Board of Rush University
Medical Center (Chicago, IL) as previously described (43). All animal
experiments were approved by the Animal Care and Use Committee at
Rush University Medical Center. Arthritic scores were assessed by blinded
observers three times per week and evaluated by the extent of erythema
and swelling of each paw on a scale of 0–4 providing a maximum score for
each mouse of 16. Scoring of each paw was as follows: 0, normal; 1, mild
erythema and swelling of several digits; 2, moderate erythema and
swelling; 3, more diffuse erythema and swelling; and 4, severe erythema
and swelling of complete paw with ankylosis. Arthritis score represents the
mean 6 SEM of the data.
Abs, treatments, and B and Treg cell depletion
B cell depletion was performed by a single i.v. injection of 250 mg anti-
mouse CD20 (mCD20) mAb (18B12, IgG2a; Biogen Idec, San Diego, CA)
or the control anti-human CD20 (hCD20) mAb (2B8; Biogen Idec), which
has no cross-reactivity to the mouse CD20 molecule as described pre-
viously (12). Depletion of Treg cells was achieved by weekly i.p. injec-
tions of 500 mg anti-mouse CD25 mAb (PC 61.5.3, rat IgG1) (BioXCell,
West Lebanon, NH) or control anti-HRPN mAb (rat IgG1) (BioXCell). All
Ab treatments of PG-immunized mice began 4 d after the second PG-DDA
Treg flow cytometry
Spleen and lymph nodes of anti-mCD20 mAb and anti-hCD20 mAb-treated
mice were harvested after PG-DDA immunization. Single-cell suspen-
sions of each tissue were immunostained using anti-CD3 FITC, anti-CD4
PerCP-Cy5.5, and anti-CD25 allophycocyanin (BD Biosciences, San Jose,
CA) with anti-Foxp3 PE (eBioscience, San Diego, CA). Using an FACS-
Canto II (BD Biosciences), stained cells were acquired and analyzed with
FACSDiva software (BD Biosciences). Data represent the mean 6 SEM.
In vitro Treg cell suppression assays
Spleens were harvested from untreated WTor WT treated with anti-mCD20
mAb or anti-hCD20 mAb mice after PG-DDA immunization. Splenic ef-
fector CD4+T cells (Teff) from untreated arthritic WT mice were isolated
using AutoMACS separation with negative selection microbeads (Miltenyi
Biotec, Bergisch Gladbach, Germany). Splenic Treg cells from arthritic
mice treated with anti-mCD20 mAb or anti-hCD20 mAb were CD25
positively selected from negatively isolated CD4+T cells (Miltenyi Bio-
tec). Varying concentrations of Treg cells (0–1.25 3 105) were cocultured
with Teff cells (1.25 3 105) and mitomycin C-treated WT naive splenic
APCs (2.5 3 105) with or without PG (10 mg/ml) in RPMI 1640 media
containing 5% FCS, 100 mg/ml penicillin, 100 mg/ml streptomycin, and
2 mM L-glutamine (complete media) in quadruplicate in 96-well Falcon
plates (Fisher Scientific, Fair Lawn, NJ). WT naive spleen cells were in-
cubated at 1 3 107cells/ml with 25 mg/ml mitomycin C (Sigma-Aldrich,
St. Louis, MO) for 30 min to inactivate proliferation. Proliferation was
measured by [3H]thymidine incorporation overnight of a 5-d culture. Data
represent the mean 6 SEM.
Detection of anti-PG Abs by ELISA
Mice were bled from the orbital plexus for serum at 4, 8, and 11 wk after
the first PG-DDA immunization. Anti-mouse PG and anti-human PG Abs
in serum samples were determined by ELISA. Individual mouse serum
samples and internal standard samples (pooled sera from arthritic mice)
culture 96-half-area-well plates (Costar, Corning, NY) that were coated
overnight with 0.5 mg human PG or 0.75 mg native mouse PG in carbonate
buffer. Known concentrations of plate-bound unlabeled murine IgG1 and
IgG2a Ab (Southern Biotechnology Associates, Birmingham, AL) without
plate-bound PGs were used as standard curves. Unlabeled plate-bound Abs
in standard curve wells or serum Abs bound to PG-coated wells were
detected using HRP-labeled anti-mouse IgG1 or IgG2a (Zymed, San
Francisco, CA) secondary Abs with o-phenylenediamine. Spectropho-
tometer readings at 490 nm determined colorimetric changes and con-
centrations of anti-PG serum Abs. Data represent the mean 6 SEM.
T cell proliferation and assessment of Th1 and Th17 cytokines
Spleens were isolated from individual untreated mice or mice treated with:
anti-mCD20 mAb; anti-hCD20 mAb; anti-mCD20 mAb with anti-CD25
mAb; anti-mCD20 mAb with rat IgG1 anti-HRPN mAb; anti-CD25
mAb; or rat IgG1 anti-HRPN mAb after PG-DDA immunizations. Iso-
lated CD4+T cells (2.5 3 105) as described above were cultured with
mitomycin C-treated naive total spleen APCs (2.5 3 105) with or without
PG (10 mg/ml) for 5 d. Proliferation was measured by [3H]thymidine in-
corporation as described above. Data represent the mean 6 SEM. Super-
natants were removed from similarly established cultures at day 4 and
cytokines analyzed for IL-17 or IFN-g concentrations, respectively, with
ELISA kits (IFN-g [BD Biosciences] or IL-17 [R&D Systems, Minneap-
olis, MN]) according to the manufacturer’s instructions. Data represent the
mean 6 SEM.
The Mann–Whitney U test was used to compare nonparametric data for
statistical significance. Significance is based on a p value , 0.05.
Depletion of Treg cells prior to immunization but not after
Previous studies have demonstrated that inflammatory conditions
can limit Treg cell activity. To determine if the inflammatory
environment in arthritis affects the function of Treg cells, we
assessed Treg cell activity in PGIA. Treg cells were depleted prior
to PG-DDA immunization or 4 d after the second PG-DDA im-
munization when anti-PG–specific B and T cell responses have
developed (12). BALB/c mice were depleted of Treg cells by
treatment with anti-CD25 mAb i.p. every 7 d. CD25+Foxp3+Treg
cells were depleted in the blood, spleen, and lymph nodes as
measured by Foxp3+and CD25+staining of CD4+cells (data not
shown). Depletion of Treg cells 3 d prior to PG-DDA immuni-
zation resulted in the early onset of PGIA with enhanced arthritis
severity (Fig. 1A, 1B). However, depletion of Treg cells 4 d after
the second PG/DDA immunization at a time point when arthritis
was initiated had no affect on PGIA (Fig. 1C, 1D). Although
disease severity was not exacerbated, splenic CD4+T cells from
Treg cell-depleted mice displayed a significant increase in PG-
specific proliferation and IFN-g production than CD4+T cells
from control Ab-treated mice (Fig. 1E, 1F). However, the IL-17
The Journal of Immunology4901
secretion from CD4+T cells was suppressed (Fig. 1G) likely due
to the ability of IFN-g to suppress IL-17 production. These data
indicate that Treg cells are able to inhibit already primed Teff
cells. However, the inability of Treg cell depletion to exacerbate
arthritis after immunization suggests that the number or potency
of Treg cells may be reduced in the inflammatory environment.
Alternatively arthritis is at maximum severity and cannot be fur-
ther increased in absence of Treg cells.
CD4+CD25+Treg cell number and function increase in
B cell-depleted mice
B cell depletion is well documented to suppress autoimmune dis-
ease. Reduction in autoantibodies does not always correlate with
inhibition of disease, suggesting other mechanisms may be in-
volved. As described previously, depletion of B cells with anti-
CD20 mAb treatment in early PGIA suppressed arthritis severity
and inhibited PG-specific CD4+T cell proliferation and IFN-g and
IL-17 cytokine production (12). Thus, the reduction in Teff cell
responses may shift the balance away from Teff cells and toward
Treg cells. To determine if the reduction in Teff cell activity in
B cell-depleted mice was in part due to an increase in Treg cells,
we analyzed spleens and lymph nodes of mice after treatment with
either anti-CD20 mAb or control mAb at a time point when B cell
numbers had recovered. The percentages of CD4+T cells were
similar in anti-CD20 mAb and control mAb-treated mice. How-
ever, there was a significant increase in the percentage of CD4+
Foxp3+CD25+cells (Fig. 2A–C) in the spleen and lymph node and
a significant increase in the expression of Foxp3 (reflected in the
mean fluorescent intensity) in Treg cells of the lymph nodes of
B cell-depleted mice (Fig. 2D, 2E). This increase in the percent-
age of Treg cells was due to a reduction in CD4+T cell numbers,
not an increase in Treg cell numbers (Fig. 2F), resulting in a re-
duction in the ratio of CD4+T cells to CD4+CD25+Foxp3+Treg
cells in B cell-depleted mice (Fig. 2G).
Because an increase in Foxp3 protein expression in Treg cells
correlates with their suppressor capacity (44), we examined the
suppression potency of Treg cells from B cell-depleted mice in
a suppressor assays. CD4+CD25+Treg cells were isolated from
spleens of PG-immunized mice treated with either anti-CD20
mAb or control Ab. Titrated numbers of CD4+CD25+Treg cells
were cocultured with CD4+CD252Teff cells from arthritic mice
in the presence of PG and mitomycin C-treated naive splenocytes
as APCs. Parallel to the increased levels of Foxp3 expression,
CD4+CD25+cells from B cell-depleted arthritic mice suppressed
PG-specific CD4+T cell proliferation more efficiently at lower
concentrations of Treg cells than those from control Ab-treated
mice (Fig. 2H). At higher concentrations of Treg cells, a saturation
effect on Teff cells by CD4+CD25+cells was observed, as pro-
liferation of Teff cells was similar for cultures containing CD4+
CD25+cells from B cell-depleted or control Ab-treated mice.
These data demonstrate that B cell depletion results in an increase
in the percentage and potency of Treg cells.
We next asked whether the change in Treg cells in B cell-
depleted mice was a consequence of immune activation. In na-
ive mice genetically deficient in B cells, there was the expected
increase in the percentage of CD4+with a concomitant increase
in percentage of CD4+CD25+Foxp3+cells; however, the total cell
number in B cell-deficient mice was reduced, and the number of
CD4+and CD4+CD25+Foxp3+was similar to WT (Fig. 3A, 3B).
Thus, these data suggest that a change in Treg cells in the B cell-
3-wk interval. Mice depleted of Treg cells with anti-CD25 mAb (n = 7) or treated with rat IgG1 (n = 7) were injected i.p. every 7 d beginning either 3 d prior
to the initial PG-DDA immunization (A, B) or 4 d after the second PG-DDA immunization (C, D). Arthritis score (A, C) is the sum of paw inflammation
scores divided by the number of arthritis mice. Incidence (B, D) is the percentage of mice with PGIA. At week 13 after the initial PG-DDA immunization,
spleens were harvested. CD4+T cells were purified from mouse spleens and cocultured with mitomycin C-treated naive total spleen cells with or without
PG (10 mg/ml) for 4 d. Proliferation (E) of CD4+T cells was measured by [3H]thymidine incorporation. Supernatants were harvested and assayed by
ELISA for IFN-g (F) and IL-17 (G). Values are mean 6 SEM and representative of two independent experiments. *p # 0.05.
Depletion of Treg cells prior to immunization but not after accelerates PGIA. BALB/c mice were immunized with PG in DDA twice with a
4902B CELL DEPLETION ENHANCES T REGULATORY CELL ACTIVITY
depleted mice is a consequence of inflammation. To further con-
firm in another system that reduction in inflammation results in an
increase in Treg cells, we assessed Treg cells in PG-immunized
IFN-g2/2mice. We have previously reported that PGIA is sup-
pressed in IFN-g2/2mice (18, 43). In comparing Treg cells in WT
and IFN-g2/2mice, we found the number and percentage of CD4+
CD25+Foxp3+was increased in IFN-g2/2mice (Fig. 3C, 3D).
These data further support that inflammation suppresses Treg cells.
Simultaneous Treg cell and B cell depletion restores PGIA and
the autoreactive CD4+T cell reactivity
The increased suppressor phenotype of Treg cells suggests that the
effectiveness of B cell depletion in PGIA may be in part due to
enhanced Treg cell activity. To test this in vivo, we depleted Treg
cells using anti-CD25 mAb treatment simultaneous with B cell
depletion. The efficacy achieved with B cell depletion was com-
pletely reversed with the concomitant depletion of Tregs as mice
treated with both anti-CD20 and anti-CD25 mAbs experienced
a similar disease course as untreated WT mice (Fig. 4A, 4B). The
absence of a resurgent autoantibody response in B cell-depleted
mice with Treg cell depletion implicated the T cell compartment
in the recovery of disease (data not shown). CD4+T cells isolated
from mice treated with anti-CD20 mAb and anti-CD25 mAb re-
covered their proliferative response to PG (Fig. 4C) as well as
expression of IFN-g and IL-17 (Fig. 4D, 4F) compared with mice
treated with anti-CD20 mAb and rat IgG1 control Ab. These data
demonstrate that Treg cells are a major contributor to the sup-
pression of arthritis in B cell-depleted mice by reducing Teff cell
We and others have suggested that B cell-depletion therapy in
autoimmune diseases is related to the suppression of autoreactive
lymph nodes (B, D) and spleens (C, E) harvested after anti-CD20 mAb (n = 5 to 6) or control Ab (n = 5 to 6) treatment and analyzed by flow cytometry
using anti-CD4, anti-Foxp3, and anti-CD25 fluorochrome-labeled Abs. Treg cells were measured as percentage of CD25+Foxp3+of CD4+cells (B, C).
Relative level of Foxp3 protein expression was determined by mean fluorescent intensities (MFI) of CD4+CD25+Foxp3+cells (D, E). Cell number in spleen
(F) of CD4+and CD4+CD25+Foxp3+from control and anti-CD20–treated mice. G, Ratio of the cell number of CD4+to CD4+CD25+Foxp3+. H, CD4+
CD25+cells were isolated and pooled from spleens of two arthritic B cell-depleted (n = 3 groups) or control Ab-treated (n = 3 groups) mice at 21 d or at
peak of inflammation after Ab treatment. Purified CD4+T cells (Teff) from untreated arthritic mice were cocultured for 5 d with mitomycin C-treated naive
splenocytes, PG (10 mg/ml), and titrated number of Treg cells. Proliferation of Teff cells was measured by [3H]thymidine incorporation. Values are mean 6
SEM and representative of two to three independent experiments. *p # 0.05.
CD4+CD25+Treg cell number and function increase in B cell-depleted mice. Representative flow cytometry of Treg cells (A) isolated from
The Journal of Immunology 4903
Teff cells due to the loss of sufficient Ag presentation by B cells
(12, 19, 45). The continual activation of Teff cells in autoimmune
disease indicates a failure to properly control the immune re-
sponse to self-Ags. Treg cells are the main mechanism by which
Teff cells are controlled (46). Accordingly, reduced total numbers
or decreased suppressor capacity of Tregs has been reported in
several autoimmune diseases (46, 47). We found that in PGIA,
depletion of Treg cells led to exacerbated arthritis when depleted
before PG immunization but not after immunization. There are
several possible explanations for these results: Treg cell potency
or number may be reduced despite a clear increase in the Teff
responses (Fig. 1). It is also possible that any exacerbation in
arthritis due to Treg depletion could not be detected as arthritis
was at maximum severity. This study was designed to determine if
the mechanism for suppression of arthritis in B cell-depleted mice
involves Treg cells.
The ability of Th subsets with established effector phenotypes
to convert to anti-inflammatory subsets or Treg cells to convert to
proinflammatory subsets is an area of intense research and debate.
A recent article by Rubtsov et al. (48) demonstrates convincingly
that expression of Foxp3 in committed Treg cells is stable under
physiological and many inflammatory conditions. However, the
authors do mention several situations in which Treg cells may lose
Foxp3 expression, such as cells under certain stress conditions or
those that newly or transiently expressed Foxp3. In fact, epigentic
studies demonstrated that many Th subsets differentiated in vitro
maintain activating histone modifications of genes for master
regulatory transcription factors responsible for other Th subset
phenotypes (49). Adoptive transfer of Foxp3 expressing CD4+
T cells into T cell-deficient hosts demonstrated that Tregs are ca-
pable of losing Foxp3 expression and repopulating the T follicular
helper cell compartment of Peyer’s patches in vivo (50). The
conversion of Treg cells to this proinflammatory phenotype in this
model of homeostatic repopulation required the presence of B cells
and their expression of CD40 (50). We reasoned that the depletion
of B cells and the reduction in Teff cell responses might shift the
balance toward the differentiation of Treg cells. Examination of the
Treg cells in B cell-depleted mice showed that the percentage and
function of Treg cells increased (Fig. 2). Similar increases in Treg
restores PGIA and autoreactive CD4+T cell reactivity.
B cell-depleted mice were treated weekly with anti-
CD25 mAb (n = 7) or rat IgG1 (n = 7) beginning on the
same day as B cell depletion. Arthritis score (A) and
incidence (B) of arthritis. Spleens were harvested 13
wk after the initial PG-DDA immunization. CD4+
T cells were purified from indicated mouse spleens and
cocultured with mitomycin C-treated naive total spleen
cells with or without PG (10 mg/ml). Proliferation (C)
of CD4+T cells was measured by [3H]thymidine in-
corporation during the final 24 h of 5 d cultures. IFN-g
(D) and IL-17 (E) was measured by ELISA of super-
natants of 4 d cultures. Values are mean 6 SEM and
representative of two independent experiments. *p #
Simultaneous Treg and B cell depletion
cells. A and B, Spleen cells from naive WTor B cell-deficient mice (n = 4)
were assessed for number and percentage of CD4+T cells and CD4+
CD25+Foxp3+by flow cytometry. C and D, Spleen cells from PG-immu-
nized WT and IFN-g2/2mice (n = 4) were analyzed for number and
percentage of CD4+T cells and CD4+CD25+Foxp3+by flow cytometry.
Values are mean 6 SD and representative of two independent experiments.
*p # 0.05.
Inflammation suppresses the number and percentage Treg
4904 B CELL DEPLETION ENHANCES T REGULATORY CELL ACTIVITY
cells were reported in B cell-depleted mouse models of NOD and
thyroiditis (39). Therefore, it appears that B cells have a profound
impact on Treg cell presence and effectiveness during inflam-
matory conditions. However, this observation is not universal. In
experimental autoimmune encephalomyelitis, despite the reduction
in Teff responses in B cell-depleted mice after MOG immuniza-
tion, Tregs are not increased, and, in some cases, Treg cells are
reduced (51, 52). There are numerous factors that are involved in
the interplay among B cells, Teff cells, and Treg cells in different
models (i.e., induced versus spontaneous disease models, strain of
mouse, route of Ag exposure, timing of the B depletion, and timing
of Treg analysis).
Although naive B cell-deficient mice do not have increased
numbers of Treg cells (Fig. 3) (29), the loss of B cells at the time of
inflammation may result in reduced polarization of naive CD4+
T cells toward effector phenotypes and more toward regulatory
populations. B cell depletion during PGIA results in significantly
reduced PG-specific Th1 and Th17 activation (12). This reduction
in the inflammatory cytokines IFN-g and IL-17 during B cell
depletion likely contributes to the increase in percentage of Treg
cells in the spleens and lymph nodes of arthritic mice depleted of
B cells (Fig. 2B, 2C). Studies show that Th1 IL-12, IFN-g, and
Th2 IL-4 can inhibit TGF-b–induced Treg cell differentiation
of naive Th cells (53). Similarly, a deficiency in IFN-g in PGIA
results in reduction in arthritis and an increase in Treg cells.
Another example in vivo is the inflammation elicited by Toxo-
plasma gondii infection, which induces a substantial Th1 response
that limits Treg cell conversion from naive Th cells and the
maintenance of their regulatory phenotype in vivo (54). The re-
duction in Th17 cells in B-depleted mice with PGIA may also
affect Treg cells because in experimental autoimmune encepha-
lomyelitis, the absence of IL-6 resulted in an overwhelming
adaptive Treg cell response that suppressed the Ag-specific Th17
response and disease manifestation (55).
Along with the increase in the percentage of Treg cells, Foxp3
levels were elevated in CD4+CD25+T cells from mice depleted of
B cells (Fig. 2). The suppressive capabilities of Treg cells have
been directly related to the level of Foxp3 transcripts expressed by
these cells (44). Thus, the increase in Foxp3 expression in B cell-
depleted mice indicated a concurrent amplification of suppressor
function. In vitro proliferation assays confirmed this supposition,
as CD4+CD25+T cells from B cell-depleted mice were signifi-
cantly more effective at suppressing proliferation of CD4+T cells
from arthritic mice in response to restimulation with PG (Fig. 2H).
In the clinic, similar findings have been observed with B cell-
depletion therapy. Elevated levels of Foxp3 mRNA are detected
in PBMCs from lupus patients treated with anti-CD20 mAb (14).
Also, defective Treg cell suppression is restored in idiopathic
thrombocytopenic purpura patients after B cell-depletion therapy
(15). The findings that B cell depletion augments Treg cell Foxp3
expression and suppressor function support a Treg cell-dependent
mechanism of B cell depletion efficacy.
In vivo studies using dual depletion of B cells and Treg cells
resulted in a significant elevation in CD4+T cell responsiveness
to PG as measured by proliferation along with IFN-g and IL-17
production (Fig. 4C–E). The return of PGIA in mice depleted of
both B cells and Treg cells cannot be attributed to be a general
response to Treg cell depletion, as mice treated with anti-CD25
alone did not experience exacerbated arthritis (Fig. 1C, 1D). Treg
cell influence on PGIA has previously been described to be in-
sufficient under normal circumstances and is believed to be the
result of an uncontrolled PG-specific CD4+T cell response (56).
In support of this, Treg cell depletion prior to PG-DDA immuni-
zation and the activation of PG-specific CD4+T cells in PGIAwas
exacerbated with an earlier onset (Fig. 1A, 1B). The reduced
autoreactivity of the CD4+T cells along with the strengthening of
Treg cell populations in B cell-depleted mice allow Treg cells to
control pathogenic inflammation of PGIA. Our demonstration that
Treg cells function to support the efficacy of B cell depletion in
autoimmune arthritis suggests that RA patients receiving B cell-
depletion therapies may benefit from a concomitant therapy that
promotes further Treg expansion such as IL-2/anti–IL-2 com-
plexes (57, 58).
In this study, we demonstrate a role for B cells in limiting Treg
cell suppressor activity in the promotion of inflammation in
a mouse model of autoimmune arthritis, PGIA. In this model,
B cells contribute to an inflammatory environment that inhibits
Treg cell expansion and function and/or promotes the differenti-
ation of naive CD4+T cells preferentially to proinflammatory
effector phenotypes rather than Treg cells. Alternatively, the ef-
ficiency of B cells to act as APCs may expand the pool of auto-
reactive Teff cells beyond the control of Treg cells. Further
elucidation of the mechanisms used by B cells to suppress Treg
cells will have a major impact on the development of new ther-
apies for the treatment of chronic inflammatory diseases.
We thank Dr. Jeffrey Oswald and all of the staff of the Comparative Re-
search Center for expert technical assistance.
The authors have no financial conflicts of interest.
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