From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
IMMUNOBIOLOGY
Self-antigen presentation by mouse B cells results in regulatory T-cell induction
rather than anergy or clonal deletion
Sara Morlacchi,1 Cristiana Soldani,1 Antonella Viola,1,2 and Adelaida Sarukhan1
1Istituto
Clinico Humanitas, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Milan, Italy; and 2Department of Translational Medicine,
University of Milan, Milan, Italy
Multiple mechanisms operate to ensure
T-cell tolerance toward self-antigens.
Three main processes have been described: clonal deletion, anergy, and deviation to CD4ⴙ regulatory T cells (Tregs)
that suppress autoreactive T cells that
have escaped the first 2 mechanisms.
Although it is accepted that dendritic
cells (DCs) and B cells contribute in maintaining T-cell tolerance to self-antigens,
their relative contribution and the processes involved under physiologic condi-
tions remain only partially characterized.
In this study, we used different transgenic
mouse models to obtain chimeras where
a neo self-antigen is expressed by thymic
epithelium and/or by DCs or B cells. We
found that expression of cognate ligand
in the thymus enhances antigen-specific
FoxP3ⴙ cells independently of whether
the self-antigen is expressed on thymic
epithelium or only on DCs, but not on
B cells. On the contrary, self-antigen expression by B cells was very efficient in
inducing FoxP3ⴙ cells in the periphery,
whereas self-antigen expression by DC
led mainly to deletion and anergy of
antigen-specific FoxP3ⴚ cells. The results presented in this study underline
the role of B cells in Treg induction and
may have important implications in clinical protocols aimed at the peripheral expansion of Tregs in patients. (Blood. 2011;
118(4):984-991)
Introduction
T-cell tolerance toward self-antigens is achieved through a series of
mechanisms that have evolved to keep autoimmunity in check.
Three main processes, which start in the thymus but continue in the
periphery, have been described: clonal deletion of cells reactive to
proteins present in the thymus, including peripheral tissue antigens
expressed by thymic epithelial cells and blood-borne antigens;
anergy induction as a result of antigen recognition under noncostimulatory or inhibitory conditions that remain only partially
understood; and the generation of regulatory T cells (Tregs) that
further limit peripheral reactivity toward self. The relative contribution of each of these mechanisms to the maintenance of T-cell
tolerance has been subject of some debate.1 However, it is clear that
the breakdown of any of these mechanisms, which start in the
thymus and continue in the periphery, can lead to the development
of autoimmune disease, even though the kinetics, severity, and
target organs may be different. Thus, the generation and maintenance of FoxP3⫹ Tregs have been shown to be critical in avoiding
severe lymphoproliferative disease and extensive multiorgan infiltration throughout the entire life span of the organism.2 Similarly,
mice deficient for Fas or Fas ligand, and thus incapable of
efficiently deleting T cells in the periphery, develop lymphoproliferation and generalized autoimmunity,3,4 although apoptosis has
been reported to be dispensable in T-cell tolerance induction to a
systemic self-antigen.5 Recently, E3 ubiquitin ligases, such as
Cbl-b, Itch, Grail, and TRAF6, have been implicated in the
development and maintenance of T-cell anergy, and mice deficient
for these genes display resistance to anergy induction6,7 and
enhanced susceptibility to spontaneous8,9 and induced10,11 autoimmune diseases. However, some genes typically expressed by
anergic cells are also expressed by Tregs,12 and it has been difficult
to dissociate anergy induction from Treg induction in vivo and
determine their relative contribution in the establishment of
self-tolerance.
Tolerance induction to target antigens may represent a therapeutic strategy in the treatment of autoimmune diseases and allergy.
Thus, it is important to understand the conditions by which these
different processes occur and, in particular, the role of the different
populations of antigen-presenting cells in tolerance induction
(regulatory cell induction vs anergy vs deletion). The critical role of
dendritic cells (DCs) not only in the induction of efficient immune
responses but also in the maintenance of T-cell tolerance, via the
cross-presentation of tissue-restricted self-antigens and their transport to the lymph nodes under steady-state conditions, has been
well documented.13 A large body of recent experimental data
suggest that antigen presentation by DCs under noninflammatory
conditions leads to the generation or expansion of Tregs,14,15
although the exact mechanisms involved remain only partially
understood.
B cells are considered to be poorly immunogenic antigenpresenting cells, and it was reported that animals can be rendered
tolerant to antigens presented by naive B cells.16 More recent
studies show that B cells can positively or negatively regulate
T cell-mediated responses through antibody-independent mechanisms and modulate the development, proliferation, and survival of
Tregs.17 Indeed, B cells can efficiently generate Tregs in vitro18-20
but, on the other hand, they were reported to induce deletion of
self-reactive T cells in vivo.21 Thus, the role of B cells in induction
Submitted February 10, 2011; accepted May 18, 2011. Prepublished online as
Blood First Edition paper, June 7, 2011; DOI 10.1182/blood-2011-02-336115.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2011 by The American Society of Hematology
984
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
of T-cell tolerance to self-antigens under physiologic conditions,
and in particular, in the induction of Tregs, remains poorly defined.
In this study, we sought to determine the role of DCs and B cells
in the induction of T-cell tolerance toward a neo–self-antigen in
vivo. We show that antigen presentation by these 2 different cell
types results in distinct types of tolerance. Presentation by DCs
favors deletion and anergy, whereas presentation by B cells leads to
peripheral Treg induction.
Methods
Mice
Balb/c (H-2d) mice were from Charles River Laboratories. TCR-HA
transgenic mice expressing a T-cell receptor-␣␤ (TCR␣␤) specific for
peptide 111–119 from influenza virus hemagglutinin (HA) presented by
I-Ed have been previously described22 and are on the Balb/c background.
These mice were crossed with mice expressing influenza HA under the
control of the ubiquitous pgk promoter to generate TCR-HAxpgk-HA
double-transgenic mice23 or to mice expressing influenza HA under the
control of the Igk L chain promoter and enhancer to generate TCRHAxIg-HA mice.23 FoxP3-GFP knock-in mice (C.Cg-Foxp3tm2Tch/J)
were purchased from The Jackson Laboratory and backcrossed onto the
TCR-HAxIg-HA and TCR-HAxpgk-HA mice. Ig-HA mice were also
backcrossed onto Rag⫺/⫺ Balb/c mice. All mice were used between
6 and 10 weeks of age. Procedures involving animals and their care
conformed to institutional guidelines (authorization no. 11/2006-A from
the Italian Ministry of Health) in compliance with national (4D.L. N.116,
GU, suppl 40, 18-2-1992) and international law and policies.24 All
efforts were made to minimize the number of animals used and their
suffering.
Antibodies and reagents
The clonotypic 6.5 monoclonal antibody, which recognizes the transgenic
TCR-HA, was produced in our laboratory and was used coupled to biotin or
phycoerythrin. All other antibodies for flow cytometry were purchased from
BD Biosciences PharMingen. The polyclonal Ki67 monoclonal antibody
was purchased from Abcam. Cells were analyzed on a flow cytometer
(FACSCanto; BD Biosciences). Cell sorting was performed using a
FACSAria (BD Biosciences). FACS data were analyzed using Diva
software and FlowJo software version 7.2.5. The HA peptide (SVSSFERFEIFPK) was purchased from Invitrogen. Vybrant CFDA-SE cell tracer kit
(Invitrogen) and CellVue Maroon (Molecular Targeting Technologies) were
used according to the manufacturer’s instructions.
In vitro proliferation and suppression assays
For in vitro experiments, total lymph node and spleen suspensions from the
different transgenic or chimeric mice were stained with CD4 and
6.5 antibodies and sorted on a FACSAria to obtain HA-specific 6.5⫹
T conventional (Tc; CD4⫹ 6.5⫹ GFP⫺) or 6.5⫹ Tregs (CD4⫹ 6.5⫹ GFP⫹)
cells, respectively. DCs were obtained from the spleen of Balb/c mice by
positive selection with anti-CD11c microbeads (Miltenyi Biotec). All
assays were performed in complete Dulbecco modified Eagle medium
(Lonza), supplemented with 2-mercaptoethanol (Invitrogen) and 10%
FCS. A total of .5 ⫻ 104 Tc cells were incubated with DCs (1.5 ⫻ 104) in
flat-bottom 96-well plates in the absence or presence of Tregs at a
Tc/Treg ratio of 1:1 or 2:1. After 2 days of culture, supernatants were
collected for the quantification of cytokines, and 1 ␮Ci 3H-methylthymidine was added for an additional 16 hours. All conditions were
performed in triplicates. Some experiments were performed staining
T cells with CellVue Maroon, and the proliferation was analyzed by flow
cytometry after 3 days of coculture. For experiments shown in Figure
3A, spleen cell suspensions were obtained by collagenase digestion,
stained with CD11c and CD19 antibodies, and sorted electronically.
B CELLS vs DCs IN SELF-TOLERANCE INDUCTION
985
They were then cocultured in vitro with CD4⫹6.5⫹ T cells from
TCR-HA single transgenic mice.
Bone marrow chimeras
pgk-HA or Balb/c recipient mice were irradiated 700 cGy and reconstituted
with bone marrow obtained from TCR-HA FoxP3-GFP mice (2 ⫻ 106
cells), together with an equal amount of bone marrow from Rag⫺/⫺, Rag⫺/⫺
Ig-HA mice or 0.5 to 0.7 ⫻ 106 CD19⫹ cells sorted from the bone marrow
of an Ig-HA mouse. Mice were kept on antibiotics for 2 weeks after transfer.
Reconstitution was confirmed after 7 weeks, and mice were analyzed
between 8 and 12 weeks after transfer. In all chimeric mice, the percentage
of B cells in the spleen was comparable at the time of analysis
(71.8% ⫾ 1.6%).
Adoptive transfer experiments
CFSE-labeled CD4⫹6.5⫹ T cells from a Thy1.1 TCR-HA mouse (1 ⫻ 106)
were injected into pgk-HA, Ig-HA, or Balb/c recipients (Thy1.2). Some
Balb/c recipients had been reconstituted 7 to 8 weeks earlier with pgk-HA
bone marrow. Recipient mice were killed 3 days after adoptive transfer, and
cell suspensions from lymph node and spleen were analyzed by flow
cytometry.
Immunohistochemistry
Frozen spleen sections (10 ␮m) from the different chimeric mice were fixed
with 4% formalin, rehydrated with PBS, and permeabilized with PBS/
Tween 0.1%. Then the slides were incubated with the anti-FoxP3 Alexa647conjugated (1:50; BD Biosciences) and with the primary antibody antiCD19 (1:50; eBioscience) that was revealed with the Alexa488 anti–rat
antibody (1:500; Invitrogen). After washes, nuclei were counterstained with
Hoechst 33258 (1 ␮g/mL) and mounted with ProLong (Invitrogen). Negative controls included slides incubated with the secondary antibodies alone.
Acquisition of images was made by confocal microscopy Fluoview
FV1000 (Olympus) with an oil immersion objective (60 ⫻ 1.4 NA
Plan-Apochromat; Olympus). Images were processed using Adobe Photoshop 9.0.2. The statistical analysis was performed using Student unpaired
2-tailed t test, ANOVA 1-way test analysis with Tukey Multiple Comparison Test. For each specimen, Foxp3⫹ cells were counted in randomly
selected CD19 positive follicles at 20⫻ magnification. Values are expressed
as mean ⫾ SE.
Statistical analysis
Results are expressed as mean ⫾ SD. Student t test was performed where
indicated.
Results
The pattern of self-antigen expression determines the fate of
antigen-specific T cells
Although it is well established that Treg selection is positively
regulated by expression of the agonist ligand in the thymus,25,26
Tregs can also be generated in periphery,27 through processes that
are not fully understood. Here, we took advantage of 2 mouse
models-pgk-HA and Ig-HA mice-expressing the neo–self-antigen
HA in the thymus. The pgk-HA mouse expresses HA on thymic
epithelium, although expression on other cell types in periphery has
not been explored. The Ig promoter in Ig-HA mice drives expression of HA in B cells,28 but also in DCs29 and thymic epithelium.26
These 2 different transgenic mice were backcrossed onto mice
expressing a transgenic TCR (recognizable by the clonotypic
antibody 6.5) specific for the immunodominant peptide of HA in
the context of IEd molecules. In addition, in this study, all
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
986
MORLACCHI et al
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
Figure 1. HA expression in thymus induces negative selection of HA-specific T cells and an increase in HA-specific FoxP3ⴙ cells, but with a
different age-dependent kinetics depending on
expression of the HA transgene. (A) Primary
and secondary lymphoid organs from 12-week-old
TCR-HA, TCR-HAxpgk-Ha, and TCR-HAxIg-HA mice
were analyzed for FoxP3 and 6.5 expression among
gated CD4⫹ cells. Shown are representative dot plots,
with the percentage of total 6.5⫹ cells among CD4⫹
cells (value outside the parentheses) and the percentage of FoxP3⫹ cells among CD4⫹6.5⫹ cells (value in
parentheses). (B) The same data are presented for a
pool of 12-week-old mice, with an average of 3 mice
per time point. **P ⬍ 0.01. ***P ⬍ .001. (C) Absolute
numbers of 6.5⫹FoxP3⫹ cells and of total 6.5⫹ cells in
periphery (lymph nodes and spleen) of the 3 different
types of mice vary with the age. Each time point
represents 2 to 5 mice. (D) A representative image
of the mesenteric lymph nodes (mLN) and spleen
of a 16-week-old TCR-HA, TCR-HAxpgk-HA, and
TCR-HAxIg-HA mouse.
transgenic mice were on a FoxP3-EGFP background, to identify
and isolate FoxP3⫹ cells by GFP expression.
Both TCR-HAxpgk-HA and TCR-HAxIg-HA mice, despite
thymic deletion of HA-specific T cells because of negative
selection, are known to generate HA-specific Tregs.30 Indeed,
compared with the TCR-HA single transgenic mouse, mice having
thymic expression of HA showed enhanced selection of HA-specific
FoxP3⫹ thymocytes (Figure 1A top row). This was also true for the
periphery, where, despite the strong deletion of CD4⫹6.5⫹ cells
observed for both types of double-transgenic mice (Figure
1A bottom row; Figure 1B left graph), the percentage of FoxP3⫹
cells among the 6.5⫹ population was significantly higher than in the
single transgenic mice (Figure 1A bottom row; Figure 1B right
graph). It is interesting to note that, in the periphery of single
transgenic TCR-HA mice, there were more CD4⫹6.5⫹FoxP3⫹ cells
in the periphery with respect to the thymus, suggesting either
expansion of thymic-derived Tregs in periphery or their de novo
generation, even in the absence of the cognate antigen. This is true
both for the percentage of FoxP3⫹ cells among CD4⫹6.5⫹ cells, as
observed in Figure 1 (thymus vs lymph nodes) and for the absolute
numbers of CD4⫹6.5⫹FoxP3⫹ cells (8.37 ⫾ 1.3 ⫻ 104 cells in the
thymus vs 85.5 ⫾ 30.7 ⫻ 104 cells in the lymph nodes plus spleen,
reflecting an average 10-fold increase). Interestingly, when comparing absolute numbers of CD4⫹6.5⫹ cells in secondary lymphoid
organs (lymph nodes and spleen) of the 3 types of mice, we
observed an age-dependent accumulation in the number of
FoxP3⫹6.5⫹ cells in TCR-HAxIg-HA mice (Figure 1C left graph).
Indeed, the number of total CD4⫹6.5⫹ cells accumulated with age
(Figure 1C right graph), and this increase was in great part because
of a striking increase in total cellularity of secondary lymphoid
organs of TCR-HAxIg-HA mice, clearly evident from the age of
12 weeks (Figure 1D). In contrast, this increase in total cellularity
was not observed for TCR-HAxpgk-HA mice. Indeed, the absolute
number of FoxP3⫹6.5⫹ and total CD4⫹6.5⫹ cells in TCR-HA
single transgenic mice or TCR-HAxpgk-HA mice tended to
decrease with age, independently of FoxP3 expression (Figure 1C)
because the Tc/Treg ratio remained relatively constant with age
(data not shown).
We then compared the functional properties of HA-specific
FoxP3⫹ and FoxP3⫺ cells obtained from these mice. By staining
cells with a cell dye, we could determine by flow cytometry the
division of CD4⫹6.5⫹FoxP3⫹ and FoxP3⫺ cells in total lymph
node cell preparations, when stimulated with different peptide
doses in vitro. The 6.5⫹FoxP3⫹ cells generated in the 3 different
mice were capable of proliferating at a comparable level because of
exogenous interleukin-2 (IL-2) provided by the 6.5⫹FoxP3⫺ cells
present in the same well. In sharp contrast, 6.5⫹FoxP3⫺ T cells
from TCR-HAxIg-HA mice did not divide at any peptide dose
(Figure 2A). Furthermore, very low interferon-␥ levels were
detected in the supernatant of TCR-HAxIg-HA cultures compared
with those from TCR-HA and TCR-HAxpgk-HA mice (Figure 2B).
These data strongly suggested that HA-specific conventional
T cells were in a state of anergy in TCR-HAxIg-HA mice but they
were functional in TCR-HAxpgk-HA mice. To understand whether
this different proliferative capacity of FoxP3⫺ cells was the result
of differences in the suppressive capacity of their FoxP3⫹ counterparts, we sorted CD4⫹6.5⫹FoxP3⫺ and FoxP3⫹ cells from both
mice and stimulated them in vitro with the HA peptide. As shown in
Figure 2C, FoxP3⫺ cells from TCR-HAxpgk-HA mice proliferated
similarly to control HA-specific T cells and, as expected, the
FoxP3⫹ cells did not proliferate because of the absence of
exogenous IL-2. In contrast, FoxP3⫺ cells from TCR-HAxIg-HA
mice were unable of responding in vitro to peptide stimulation,
similar to their FoxP3⫹ counterparts (Figure 2C).
Overall, these data suggested that self-antigen-specific cells
undergo a different fate depending on the pattern of expression of
the self-antigen.
Under homeostatic conditions, HA is expressed and presented
by splenic B cells and DCs in Ig-HA mice.29 As a result, sorted
CD19⫹ or CD11c⫹ cells from the spleen of Ig-HA mice induced
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
Figure 2. Different behavior of HA-specific FoxP3ⴚ cells in TCR-HAxpgk-Ha and
TCR-HAxIg-HA mice. (A) Total lymph node suspensions from TCR-HA, TCR-HAxpgk-HA,
or TCR-HAxIg-HA mice were stained with cell-vue Maroon and incubated in vitro with
different peptide doses. Proliferation of CD4⫹6.5⫹FoxP3⫺ and FoxP3⫹ cells was
determined by flow cytometry. (B) Interferon-␥ present in the supernatant from the
same cultures was determined by enzyme-linked immunosorbent assay. Results
correspond to the pool of triplicate wells, with cells obtained from one mouse of each
genotype. Two independent experiments were performed with similar results.
(C) CD4⫹6.5⫹FoxP3⫹ and FoxP3⫺ cells from TCR-HAxpgk-HA (left graph) or
TCR-HAxIg-HA (right graph) mice were electronically sorted and incubated in vitro
with splenic-derived DCs and different peptide doses. FoxP3⫺ cells from a TCR-HA
single transgenic mouse were used as control of proliferation. Thymidine was added
after 48 hours of culture and left for an additional 16 hours. Experiments were
performed in triplicate and 1 representative experiment of 3 is shown.
proliferation of HA-specific T cells in vitro (Figure 3A). In
contrast, neither DCs nor B cells from pgk-HA mice were capable
of inducing HA-specific proliferation in vitro, unless peptide was
added to the culture (Figure 3A). However, CFSE-labeled HAspecific cells transferred into pgk-HA recipients did show some in
vivo proliferation, even at levels much lower than those observed
when the same cells were transferred into Ig-Ha recipients (Figure
3B). This proliferation was in great part because of antigen
presentation by nonhematopoietic cells, such as liver sinusoidal
endothelial cells,31 because HA-specific T cell transfer into chimeric recipients, where only the hematopoietic compartment was of
pgk-HA origin, resulted in very poor T-cell proliferation (Figure
3B, last row). However, we cannot completely discard that, under
certain inflammatory conditions, HA can be presented by DCs in
pgk mice because bone marrow-derived DCs from pgk mice were
able to induce HA-specific T-cell proliferation when stimulated by
lipopolysaccharide, albeit at levels lower than those elicited by
DCs from Ig-HA mice (supplemental Figure 1, available on the
Blood Web site; see the Supplemental Materials link at the top of
the online article).
Self-antigen expression on B cells or DCs induces Tregs
development or T-cell deletion/anergy, respectively
Based on the differences observed between the 2 double-transgenic
mouse models described, we tried to further dissect the role of
B cells versus DCs in tolerance induction toward self-antigens. For
this, we generated bone marrow chimeras (Figure 4A) where
B CELLS vs DCs IN SELF-TOLERANCE INDUCTION
987
HA-specific T cells (bone marrow from TCR-HA, FoxP3-GFP
mice) would develop in a context with no HA expression (with
Rag⫺/⫺ bone marrow, in a Balb/c recipient/ctrl chimera), HA
expression on thymic epithelium (with Rag⫺/⫺ bone marrow in a
pgk-HA recipient/T chimera), on thymic epithelium plus DCs (with
Rag⫺/⫺Ig-HA bone marrow in a pgk-HA recipient/TD chimera), or
on thymic epithelium plus B cells (with CD19⫹ cells from Ig-HA
bone marrow in a pgk-HA recipient/TB chimera). Based on the
results described in Figure 3B, HA expression by nonhematopoietic cells in the periphery of the pgk-HA recipients cannot be
excluded, but one would expect it to remain constant between the
T, TD, and TB chimeras. In all chimeras, the TCR-HA FoxP3-GFP
bone marrow generated a population of TCR transgenic T cells
together with non–HA-expressing B cells and DCs. HA-expressing
DCs or B cells were generated by the coadministration of
RAG⫺/⫺IgHA bone marrow (TD chimeras) or CD19⫹ cells from
Ig-HA bone marrow (TB chimeras), respectively.
The different chimeras were analyzed 8 to 12 weeks after
reconstitution. As shown in Figure 4B and C, the presence of HA in
the thymus (chimeras T, TD, and TB) resulted in a partial deletion
of 6.5⫹ cells compared with the control (ctrl chimera) but enhanced
selection of CD4⫹6.5⫹FoxP3⫹ thymocytes. In the periphery, the
same tendency was seen, both in lymph nodes and in spleen.
However, chimeras expressing HA on both thymic epithelium and
B cells (TB chimeras) displayed the higher percentage and absolute
numbers of FoxP3⫹ HA-specific T cells in secondary lymphoid
organs (Figure 4C).
Figure 3. HA presentation by hematopoietic cells in the periphery of TCRHAxIg-HA but not in TCR-HAxpgk-HA mice. (A) CD11c⫹ and CD19⫹ cells were
electronically sorted from Ig-HA and pgk-HA mice, and different amounts of cells were
tested for their capacity to induce HA-specific proliferation in vitro. Peptide
(p, 0.1 ␮g/mL) was added in 1 condition as positive control. (B) CD4⫹6.5⫹ cells from a
Thy1.1 TCR-HA mouse were CFSE-labeled and transferred (1 ⫻ 106) into Balb/c,
Ig-HA, or pgk-HA recipients. Balb/c mice reconstituted 7 weeks before with pgk-HA
bone marrow (last column) were also used as recipients. Three days after transfer,
the proliferation of CD4⫹Thy1.1⫹ cells in the lymph nodes was determined by flow
cytometry. Representative dot plots are shown, and the percentage of Thy1.1⫹ cells
within the CD4⫹ gate is indicated. Two mice per group and 2 independent
experiments were performed with similar results.
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
988
MORLACCHI et al
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
These results indicate that self-antigen expression by B cells is
very efficient in promoting FoxP3⫹ T cells, whereas self-antigen
expression by DCs results in T cell anergy.
Figure 4. Development and function of HA-specific FoxP3ⴙ and FoxP3ⴚ cells in
the different chimeras expressing HA in the thymus. Thymi, lymph nodes, and
spleen cell suspensions from the different chimeras illustrated in panel A were
analyzed for 6.5 and GFP expression within the CD4⫹ gate. Shown are representative dot plots (B) as well as the percentage of 6.5 cells in the CD4 gate, of FoxP3⫹
cells in the CD4⫹6.5⫹ gate and the absolute numbers of CD4⫹6.5⫹FoxP3⫹ cells in
lymph nodes and spleen (C). Three mice per group were analyzed. (D) The mean
fluorescence intensity of GFP within the CD4⫹6.5⫹GFP⫹ gate is shown.
(E) CD4⫹6.5⫹GFP⫹ and GFP⫺ cells from lymph node suspensions of each type of
chimera were electronically sorted and tested in vitro for their suppressive and
proliferative capacity, respectively. For the suppression assay, naive 6.5⫹ cells were
incubated with Balb/c splenic DCs in the absence or presence of peptide (0.1 ␮g/mL)
and in the absence or presence of GFP⫹ cells from the different chimeras. For the
proliferation assay, GFP⫺ cells from the different chimeras were coincubated with
DCs and peptide. The experiment was performed in triplicate, and 2 independent
experiments gave similar results.
We next tried to determine whether Tregs generated in the
different types of chimeras were functional. The intensity of
expression of FoxP3 is considered to be a marker for suppressive
capacity, and decreased Foxp3 expression results in the development of an aggressive autoimmune syndrome, although anergic
properties of the cells are maintained.32 Interestingly, Tregs from
the TB chimeras were those with the highest FoxP3 expression
(Figure 4D). CD4⫹6.5⫹FoxP3⫹ cells obtained from the different
chimeras were all capable of suppressing HA-specific T-cell
proliferation in vitro with comparable efficiency (Figure 4E).
Remarkably, FoxP3⫺ T cells sorted from the different chimeras
behaved very differently; whereas HA-specific T cells from T and
TB chimeras were capable of proliferating in vitro on antigenic
stimulation, those from TD chimeras were not (Figure 4E).
Figure 5. Development and function of HA-specific FoxP3ⴙ and FoxP3ⴚ cells in
the absence of HA expression in the thymus. Thymi, lymph nodes, and spleen
cells suspensions from the different chimeras illustrated in panel A were analyzed for
6.5 and GFP expression within the CD4⫹ gate. Shown are representative dot plots
(B) as well as the percentage of 6.5 cells in the CD4 gate, of FoxP3⫹ cells in the
CD4⫹6.5⫹ gate, and the absolute numbers of CD4⫹6.5⫹FoxP3⫹ cells (C). A total of
3 mice per group were analyzed. (D) Immunofluorescence staining for CD19 (green),
Foxp3 (purple), and nuclei (blue) in spleen sections of the different chimeras. The
graphs represent the quantification of Foxp3⫹ cells in CD19⫹ follicules. Bar
represents 50 ␮m. (E) The percentage of intracellular Ki67⫹ cells within the FoxP3⫹
and FoxP3⫺ CD4⫹6.5⫹ cells was determined by flow cytometry. (F) CD4⫹6.5⫹GFP⫹
and GFP⫺ cells from lymph node and spleen suspensions of each type of chimera
were electronically sorted and tested in vitro for their suppressive and proliferative
capacity, respectively, as described in Figure 4.
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
Self-antigen expression on thymic epithelium is not necessary
for the enhancement of Treg generation by B cells
To determine whether antigen expression by B cells alone (ie, no
HA expression on thymic epithelium) would also be efficient in
promoting FoxP3⫹ T cells, we performed more experiments with
bone marrow chimeras (Figure 5A). This time, all recipients were
Balb/c-and thus did not express HA on thymic epithelium, but we
reconstituted them with TCR-HA Foxp3EGFP bone marrow
together with either DCs (bone marrow from Rag⫺/⫺IgHA mice,
D chimera) or B cells (CD19⫹ cells from IgHA bone marrow,
B chimera) expressing HA. Figure 5B-C shows that HA expression
by DCs induced very efficient thymic deletion of CD4⫹6.5⫹ cells,
as previously reported,29 whereas HA expression by B cells did not
induce significant thymic deletion. These data are in agreement
with the fact that B cells represent a very small population in the
thymus and do not delete CD4⫹ T cells efficiently.33 In accordance,
HA mRNA expression was only detected in the D chimera
(supplemental Figure 2). Furthermore, these results indicate that
very little B cell–derived HA antigen, if at all, is being crosspresented by DCs, at least in the thymus. As a result, the percentage
of FoxP3⫹ cells among CD4⫹6.5⫹ thymocytes was higher in the
D chimera than in the other mice (Figure 5C). However, results
were different in periphery, where the percentage of FoxP3⫹ cells
among CD4⫹6.5⫹ cells increased significantly in the B chimera
(Figure 5B-C) and their absolute numbers were significantly higher
than those in the D chimera (Figure 5C). Indeed, the absolute
numbers of CD4⫹6.5⫹FoxP3⫹ cells in the D chimera were very
low because of a significant deletion of HA-specific cells, both in
thymus and the periphery. In vitro proliferation experiments
performed with DCs sorted from the spleens of B and D chimeras
confirmed that DCs from D mice expressed and presented HA
antigen, as expected, whereas DCs from B mice were not capable
of inducing T-cell proliferation, at least at detectable levels, thus
excluding cross-presentation of B cell–derived HA antigen also in
the periphery (supplemental Figure 3).
Immunohistochemical analysis revealed different distribution
of Tregs in spleens (Figure 5). In control mice, the great majority of
CD3 (not shown) and FoxP3 cells (Figure 5D) were outside the
B-cell zones. In D chimeras, some FoxP3⫹ cells were inside the
B-cell zones but basically remained at the margins, which are rich
in follicular DCs.34 In contrast, B chimeras showed accumulation
of FoxP3⫹ cells within the B-cell zones. These data were confirmed
by a quantitative analysis of FoxP3⫹ cell distribution within splenic
B-cell zones.
To understand whether in the B chimera B cells induced
expansion of thymic-derived FoxP3⫹ cells or their de novo
generation, we analyzed T-cell proliferation by staining for Ki67, a
cell proliferation marker.35 Independently of FoxP3 expression,
CD4⫹6.5⫹ cells from the B chimeras were similar to those obtained
from control mice in terms of in vivo proliferation, suggesting that
the increase in the CD4⫹6.5⫹FoxP3⫹ cell compartment observed in
B chimeras is the result of de novo generation of Tregs in periphery
(Figure 5E) rather than their expansion. We also observed that in
D chimeras HA specific T cells were proliferating more in vivo than
their “naive” counterparts, independently of their FoxP3 expression, suggesting that antigen presentation by DCs induces constitutive proliferation even under homeostatic conditions.
Finally, the suppressive capacity of FoxP3⫹ T cells generated in
B and D chimeras was confirmed in in vitro experiments performed
after sorting of FoxP3⫹ and FoxP3⫺ cells (Figure 5F). This
experiment also confirmed that HA-specific T cells obtained from
B CELLS vs DCs IN SELF-TOLERANCE INDUCTION
989
mice that express the antigen on DCs (D chimera) are unable to
proliferate (Figure 5F).
Although we cannot formally exclude that the levels of
HA expression by DCs and B cells in this transgenic system may be
different and may also play a role, HA mRNA expression by these
cells, determined by real-time PCR, was similar (supplemental
Figure 4).
Altogether, data obtained in B chimeras indicate that, even in
the absence of antigen expression in the thymus, Tregs can be
efficiently generated in periphery by B cells expressing a selfantigen, and this seems to be the result of a de novo generation. In
contrast, self-antigen expression by DCs is very efficient in
deleting antigen-specific T cells and in inducing anergy among the
remaining FoxP3⫺ cells.
Discussion
Treg induction in the thymus can be achieved by epithelial thymic
cells but also by DCs and thus seems to be more dependent on the
maturation state of the thymocyte than on the cell presenting the
antigen.36 We confirm here that expression of agonist ligand in the
thymus, either on thymic epithelial cells or on thymic DCs, leads to
both negative selection of antigen-specific T cells and induction of
antigen-specific Tregs. The stronger thymic deletion observed in
the TCR-HAxIg-HA mice compared with the TCR-HAxpgk-HA
mice (Figure 1A) is because, in the former, HA is expressed by
thymic epithelium and DCs and, as expected, total mRNA HA
expression levels are much higher in Ig-HA mice than in pgk-HA
mice (supplemental Figure 2). It has been recently reported that
thymic expression of cognate antigen can induce IL-17–producing,
antigen-specific T cells in addition to FoxP3⫹ cells.37 Interestingly,
we did indeed find more IL-17–producing cells among CD4 cells in
the thymus of both strains of mice expressing HA antigen in the
thymus (supplemental Figure 5); however, they were not positive
for the HA-specific TCR, indicating that Tregs may favor IL-17–
producing T-cell generation, but independently of their antigen
specificity.
We also show here that, in single transgenic TCR-HA mice, the
percentage and absolute number of HA-specific Tregs increase in
the periphery compared with the thymus as has already been
reported,38 strongly suggesting conversion and/or expansion of
Tregs even in the absence of cognate ligand. When comparing
both types of HA-expressing mice (TCR-HAxpgk-HA and
TCR-HAxIg-HA), we found that the percentage and absolute
numbers of HA-specific cells increased with age in the
TCR-HAxIg-HA mice, whereas it decreased in the TCR and in the
TCR-HAxpgk-HA mice. This increase in absolute numbers with
age was in great part the result of a striking increase in the size of
secondary lymphoid organs in the TCR-HAxIg-HA mice. The
reasons for this are not clear, and no signs of autoimmunity could
be observed in these mice as has been described for TCR-HA
transgenic mice expressing HA antigen under the major histocompatibility complex class II promoter,39 although we do have some
evidence for systemic B-cell activation (A.S., unpublished data, 2003).
Another striking difference between both types of mice was the
fate of the HA-specific FoxP3⫺ cells: GFP⫺ cells from the
TCR-HAxpgk-HA mice were not anergic on further antigenic
stimulation in vitro, in contrast to the GFP⫺ cells from TCRHAxIg-HA mice. The fact that GFP⫺ cells from TCR-HAxpgk-HA
mice express slightly lower levels of the transgenic TCR compared
with their counterparts from TCR-HAxIg-HA mice (Figure 1A)
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
990
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
MORLACCHI et al
may contribute to explain this phenomenon because it has been
reported that down-regulation of TCR levels may contribute to
escape anergy induction.40
The bone marrow chimera experiments performed in this study
demonstrate that different types of antigen-presenting cell can
induce tolerance to self-antigens via different mechanisms. HA
expression on B cells resulted in expansion of HA-specific Tregs,
without the considerable deletion and anergy induction of antigenspecific FoxP3⫺ cells observed on expression of HA by DCs. The
experiments shown in Figure 5 permit us to conclude that the
anergy of 6.5⫹FoxP3⫺ cells from TCR-HAxIg-HA mice and from
the TD chimeras is the result of antigen encounter on DC in the
periphery and not of antigen encounter in the thymus or on
nonhematopoietic cells. Furthermore, they confirm that HA expression by B cells alone was sufficient to induce HA-specific Tregs,
and suggest that this was the result of B-cell/T-cell interactions in
secondary lymphoid structures. The reason we did not observe
higher numbers of 6.5⫹FoxP3⫹ cells in the periphery of TCRHAxIg-HA mice despite expression of HA by B cells is that HA is
also expressed by thymic epithelium and DCs, inducing a strong
deletion of these cells.
The role of B cells in inducing Tregs has been strongly
suggested by other reports. Thus, ␮MT KO B cell-deficient mice
express lower levels of Tregs,38 are less efficient in Treg induction
via oral feeding of antigen,41 and do not recover normally after
acute experimental autoimmune encephalomyelitis induction.42
Furthermore, mouse43 and human19 B cells promote expansion of
allogeneic Tregs ex vivo. The mechanisms involved in such
induction as well as the activation state of the B cells required for
such phenomena remain poorly defined. Although rituximabmediated depletion of B cells in pathogenic conditions has been
reported to ameliorate autoimmune diseases,17 repopulation with
normal B cells led to amelioration of autoimmune diabetes44 and
adoptive transfer of B cells suppressed inflammatory responses in a
mouse model of primary biliary cirrhosis.45 Naive B cells have
been shown to generate Tregs in the presence of a mature
immunologic synapse.18 However, in our TCR-HAxIg-HA mice,
there is evidence of B-cell activation (A.S., unpublished results,
2003), and it has been shown that CD40L-activated human B cells
can efficiently induce expansion of Tregs,46 so it is not clear
whether the activation state of the B cell plays a role. Cytokines,
such as TGF-␤3,47 TGF-␤1,48 and IL-10, produced by follicular
B cells49 have also been reported to be involved in Treg induction
by B cells. The precise mechanisms and molecules involved in our
system are currently under investigation.
In conclusion, although we do not put in doubt that DCs can
generate Tregs, we show that they actually may be more efficient in
inducing tolerance via deletion and anergy of antigen-specific
FoxP3⫺ cells. Contrary to DCs, B cells do not traffic from tissues to
lymph nodes; thus, their capacity to induce Tregs specific for the
nonhematopoietic self may be restricted under physiologic conditions. However, B cells can be made to present antigens via the
administration of chimeric antibodies that target Fc receptors, an
approach that was described to efficiently induce antigen-specific
T-cell tolerance in mice.50 Furthermore, gene therapy approaches in
mice with retroviral vectors that introduce peptide-IgG constructs
in B cells have been shown to successfully reduce the incidence or
onset of different autoimmune diseases.51 Thus, the data presented
in this study may have important implications in clinical protocols
aimed at the peripheral expansion of Tregs in patients.
Acknowledgments
The authors thank Harald von Boehmer and Marinos Kallikourdis
for critical reading of the manuscript and valuable suggestions,
Ludger Klein for the pgk-HA mice, Chiara Buracchi and Achille
Anselmo for cell sorting, and Marta Lezama for excellent animal
care.
S.M. was supported by Fondazione Cassa di Risparmio delle
Provincie Lombarde (grant 5808/2007). A.V. was supported by
EU-FP7 SYBILLA (grant 201106) and Associazione Italiana
Ricerca sul Cancro. A.S. was supported by Inserm and Fondazione
Fondazione Cassa di Risparmio delle Provincie Lombarde (grant
5808/2007).
Authorship
Contribution: S.M., C.S., and A.S. performed experiments and
analyzed data; S.M. and A.S. designed experiments; and A.V. and
A.S. wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Adelaida Sarukhan, Istituto Clinico Humanitas,
IRCCS, Milan, Italy; e-mail: adelaida.sarukhan@humanitasresearch.it.
References
1. Mathis D, Benoist C. Levees of immunological
tolerance. Nat Immunol. 2010;11(1):3-6.
2. Kim JM, Rasmussen JP, Rudensky AY. Regulatory T cells prevent catastrophic autoimmunity
throughout the lifespan of mice. Nat Immunol.
2007;8(2):191-197.
3. Watanabe-Fukunaga R, Brannan CI, Copeland NG,
Jenkins NA, Nagata S. Lymphoproliferation disorder in mice explained by defects in Fas antigen
that mediates apoptosis. Nature. 1992;356
(6367):314-317.
4. Ramsdell F, Seaman MS, Miller RE, Tough TW,
Alderson MR, Lynch DH. gld/gld mice are unable
to express a functional ligand for Fas. Eur J Immunol. 1994;24(4):928-933.
5. Barron L, Knoechel B, Lohr J, Abbas AK. Cutting
edge: contributions of apoptosis and anergy to
systemic T-cell tolerance. J Immunol. 2008;
180(5):2762-2766.
6. King CG, Buckler JL, Kobayashi T, et al. Cutting
edge: requirement for TRAF6 in the induction of
T cell anergy. J Immunol. 2008;180(1):34-38.
7. Seroogy CM, Soares L, Ranheim EA, et al. The
gene related to anergy in lymphocytes, an E3
ubiquitin ligase, is necessary for anergy induction
in CD4 T cells. J Immunol. 2004;173(1):79-85.
8. Bachmaier K, Krawczyk C, Kozieradzki I, et al.
Negative regulation of lymphocyte activation and
autoimmunity by the molecular adaptor Cbl-b.
Nature. 2000;403(6766):211-216.
9. Fang D, Elly C, Gao B, et al. Dysregulation of
T lymphocyte function in itchy mice: a role for Itch
in TH2 differentiation. Nat Immunol. 2002;3(3):
281-287.
10. Chiang YJ, Kole HK, Brown K, et al. Cbl-b regulates the CD28 dependence of T-cell activation.
Nature. 2000;403(6766):216-220.
11. Nurieva RI, Zheng S, Jin W, et al. The E3 ubiquitin ligase GRAIL regulates T-cell tolerance and
regulatory T cell function by mediating T cell receptor-CD3 degradation. Immunity. 2010;32(5):
670-680.
12. Schwartz RH. T cell anergy. Annu Rev Immunol.
2003;21:305-334.
13. Lutz MB, Kurts C. Induction of peripheral CD4⫹
T-cell tolerance and CD8⫹ T-cell cross-tolerance
by dendritic cells. Eur J Immunol. 2009;39(9):
2325-2330.
14. Kretschmer K, Apostolou I, Hawiger D, Khazaie K,
Nussenzweig MC, von Boehmer H. Inducing and
expanding regulatory T cell populations by foreign
antigen. Nat Immunol. 2005;6(12):1219-1227.
15. Maldonado RA, von Andrian UH. How tolerogenic
dendritic cells induce regulatory T cells. Adv Immunol. 2010;108:111-165.
16. Gilbert KM, Weigle WO. Tolerogenicity of resting
and activated B cells. J Exp Med. 1994;179(1):
249-258.
17. Lund FE, Randall TD. Effector and regulatory
B cells: modulators of CD4(⫹) T cell immunity.
Nat Rev Immunol. 2010;10(4):236-247.
18. Reichardt P, Dornbach B, Rong S, et al. Naive
B cells generate regulatory T cells in the presence of a mature immunologic synapse. Blood.
2007;110(5):1519-1529.
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
19. Chen LC, Delgado JC, Jensen PE, Chen X.
Direct expansion of human allospecific
FoxP3⫹CD4⫹ regulatory T cells with allogeneic
B cells for therapeutic application. J Immunol.
2009;183(6):4094-4102.
20. Zheng J, Liu Y, Lau YL, Tu W. CD40-activated
B cells are more potent than immature dendritic
cells to induce and expand CD4(⫹) regulatory
T cells. Cell Mol Immunol. 2010;7(1):44-50.
21. Frommer F, Heinen TJ, Wunderlich FT, et al.
Tolerance without clonal expansion: selfantigen-expressing B cells program self-reactive
T cells for future deletion. J Immunol. 2008;
181(8):5748-5759.
22. Kirberg J, Baron A, Jakob S, Rolink A, Karjalainen K,
von Boehmer H. Thymic selection of CD8⫹ single
positive cells with a class II major histocompatibility complex-restricted receptor. J Exp Med. 1994;
180(1):25-34.
23. Buer J, Lanoue A, Franzke A, Garcia C,
von Boehmer H, Sarukhan A. Interleukin 10 secretion and impaired effector function of major
histocompatibility complex class II-restricted
T cells anergized in vivo. J Exp Med. 1998;
187(2):177-183.
B CELLS vs DCs IN SELF-TOLERANCE INDUCTION
30.
31.
32.
33.
34.
35.
36.
24. EEC Council Directive 86/609, OJ L 358,1,12-121287; NIH Guide for the Care and Use of Laboratory Animals, Unites States National Research
Council 1996.
25. Jordan MS, Boesteanu A, Reed AJ, et al. Thymic
selection of CD4⫹CD25⫹ regulatory T cells induced by an agonist self-peptide. Nat Immunol.
2001;2(4):301-306.
26. Apostolou I, Sarukhan A, Klein L, von Boehmer H.
Origin of regulatory T cells with known specificity
for antigen. Nat Immunol. 2002;3(8):756-763.
27. Apostolou I, Verginis P, Kretschmer K, Polansky J,
Huhn J, von Boehmer H. Peripherally induced
Treg: mode, stability, and role in specific tolerance. J Clin Immunol. 2008;28(6):619-624.
28. Kalberer CP, Reininger L, Melchers F, Rolink AG.
Priming of helper T cell-dependent antibody responses by hemagglutinin-transgenic B cells. Eur
J Immunol. 1997;27(9):2400-2407.
29. Lambolez F, Jooss K, Vasseur F, Sarukhan A.
37.
38.
39.
40.
Tolerance induction to self antigens by peripheral
dendritic cells. Eur J Immunol. 2002;32(9):25882597.
Jooss K, Gjata B, Danos O, von Boehmer H,
Sarukhan A. Regulatory function of in vivo anergized CD4(⫹) T cells. Proc Natl Acad Sci U S A.
2001;98(15):8738-8743.
Thomson AW, Knolle PA. Antigen-presenting cell
function in the tolerogenic liver environment. Nat
Rev Immunol. 2010;10(11):753-766.
Wan YY, Flavell RA. Regulatory T-cell functions
are subverted and converted owing to attenuated
Foxp3 expression. Nature. 2007;445(7129):766770.
Kleindienst P, Chretien I, Winkler T, Brocker T.
Functional comparison of thymic B cells and dendritic cells in vivo. Blood. 2000;95(8):2610-2616.
Crivellato E, Vacca A, Ribatti D. Setting the stage:
an anatomist’s view of the immune system.
Trends Immunol. 2004;25(4):210-217.
Soares A, Govender L, Hughes J, et al. Novel
application of Ki67 to quantify antigen-specific in
vitro lymphoproliferation. J Immunol Methods.
2010;362(1):43-50.
Wirnsberger G, Mair F, Klein L. Regulatory T cell
differentiation of thymocytes does not require a
dedicated antigen-presenting cell but is under
T cell-intrinsic developmental control. Proc Natl
Acad Sci U S A. 2009;106(25):10278-10283.
Marks BR, Nowyhed HN, Choi JY, et al. Thymic selfreactivity selects natural interleukin 17-producing
T cells that can regulate peripheral inflammation.
Nat Immunol. 2009;10(10):1125-1132.
Suto A, Nakajima H, Ikeda K, et al.
CD4(⫹)CD25(⫹) T-cell development is regulated
by at least 2 distinct mechanisms. Blood. 2002;
99(2):555-560.
Rankin AL, Reed AJ, Oh S, et al. CD4⫹ T cells
recognizing a single self-peptide expressed by
APCs induce spontaneous autoimmune arthritis.
J Immunol. 2008;180(2):833-841.
Tanchot C, Barber DL, Chiodetti L, Schwartz RH.
Adaptive tolerance of CD4⫹ T cells in vivo: multiple thresholds in response to a constant level of
antigen presentation. J Immunol. 2001;167(4):
2030-2039.
991
41. Sun JB, Flach CF, Czerkinsky C, Holmgren J.
B lymphocytes promote expansion of regulatory
T cells in oral tolerance: powerful induction by
antigen coupled to cholera toxin B subunit. J Immunol. 2008;181(12):8278-8287.
42. Wolf SD, Dittel BN, Hardardottir F, Janeway CA
Jr. Experimental autoimmune encephalomyelitis
induction in genetically B cell-deficient mice.
J Exp Med. 1996;184(6):2271-2278.
43. Chen X, Jensen PE. Cutting edge: primary B lymphocytes preferentially expand allogeneic
FoxP3⫹ CD4 T cells. J Immunol. 2007;179(4):
2046-2050.
44. Hu CY, Rodriguez-Pinto D, Du W, et al. Treatment
with CD20-specific antibody prevents and reverses autoimmune diabetes in mice. J Clin Invest. 2007;117(12):3857-3867.
45. Moritoki Y, Zhang W, Tsuneyama K, et al. B cells
suppress the inflammatory response in a mouse
model of primary biliary cirrhosis. Gastroenterology. 2009;136(3):1037-1047.
46. Tu W, Lau YL, Zheng J, et al. Efficient generation
of human alloantigen-specific CD4⫹ regulatory
T cells from naive precursors by CD40-activated
B cells. Blood. 2008;112(6):2554-2562.
47. Shah S, Qiao L. Resting B cells expand a
CD4⫹CD25⫹Foxp3⫹ Treg population via
TGF-beta3. Eur J Immunol. 2008;38(9):24882498.
48. Gros MJ, Naquet P, Guinamard RR. Cell intrinsic
TGF-beta 1 regulation of B cells. J Immunol.
2008;180(12):8153-8158.
49. Wei B, Velazquez P, Turovskaya O, et al. Mesenteric B cells centrally inhibit CD4⫹ T cell colitis
through interaction with regulatory T cell subsets.
Proc Natl Acad Sci U S A. 2005;102(6):20102015.
50. Phillips WJ, Smith DJ, Bona CA, Bot A,
Zaghouani H. Recombinant immunoglobulinbased epitope delivery: a novel class of autoimmune regulators. Int Rev Immunol. 2005;24(5):
501-517.
51. Scott DW. Gene therapy for immunological tolerance: using ‘transgenic’ B cells to treat inhibitor
formation. Haemophilia. 2010;16(102):89-94.
From www.bloodjournal.org by guest on March 6, 2016. For personal use only.
2011 118: 984-991
doi:10.1182/blood-2011-02-336115 originally published
online June 7, 2011
Self-antigen presentation by mouse B cells results in regulatory T-cell
induction rather than anergy or clonal deletion
Sara Morlacchi, Cristiana Soldani, Antonella Viola and Adelaida Sarukhan
Updated information and services can be found at:
http://www.bloodjournal.org/content/118/4/984.full.html
Articles on similar topics can be found in the following Blood collections
Immunobiology (5369 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.