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Regulatory T Cells in Central Nervous
System Injury: A Double-Edged Sword
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J Immunol 2014; 193:5013-5022; Prepublished online 15
October 2014;
doi: 10.4049/jimmunol.1302401
http://www.jimmunol.org/content/193/10/5013
http://www.jimmunol.org/content/suppl/2014/10/15/jimmunol.130240
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Copyright © 2014 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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Supplementary
Material
James T. Walsh, Jingjing Zheng, Igor Smirnov, Ulrike
Lorenz, Kenneth Tung and Jonathan Kipnis
The Journal of Immunology
Regulatory T Cells in Central Nervous System Injury: A
Double-Edged Sword
James T. Walsh,*,†,‡,x Jingjing Zheng,*,†,{ Igor Smirnov,*,† Ulrike Lorenz,‖,#
Kenneth Tung,#,** and Jonathan Kipnis*,†,‡,x
A
cute injury to the CNS evokes cellular and molecular
responses that lead to secondary neurodegeneration,
a process of sustained neuronal degeneration (1). Accompanying this period of secondary degeneration is a coordinated immune response to the trauma, including chemotaxis of
microglia to ATP released from the damaged cells (2) and directed
migration of both the innate and adaptive immune cells to the
injury site due to chemokine signals (3). The dogma that this infiltration of immune cells into the injury site was a detrimental
response has been challenged by the finding that neuronal survival
could be improved by boosting T cell activity rather than by its
suppression (4–6), although the phenotype of protective T cells
after CNS injury, and particularly the role of regulatory T (Treg)
cells in this process, is still a matter of debate (7–10).
*Center for Brain Immunology and Glia, School of Medicine, University of Virginia,
Charlottesville, VA 22908; †Department of Neuroscience, School of Medicine, University of Virginia, Charlottesville, VA 22908; ‡Graduate Program in Neuroscience,
University of Virginia, Charlottesville, VA 22908, xMedical Scientist Training Program, School of Medicine, University of Virginia, Charlottesville, VA 22908; {Institute
of Neurosciences, Fourth Military Medical University, Xi’an 710038, China; ‖Beirne
Carter Center for Immunology Research, University of Virginia, Charlottesville, VA
22908; #Department of Microbiology, Immunology, and Cancer Biology, School of
Medicine, University of Virginia, Charlottesville, VA 22908; and **Department of
Pathology, School of Medicine, University of Virginia, Charlottesville, VA 22908
Received for publication September 9, 2013. Accepted for publication September 20,
2014.
This work was supported by National Institute of Neurological Disorders and Stroke,
National Institutes of Health Grant NS061973 (to J.K.).
Address correspondence and reprint requests to Dr. Jonathan Kipnis, University of
Virginia, 409 Lane Road, Charlottesville, VA 22908. E-mail address: kipnis@virginia.
edu
The online version of this article contains supplemental material.
Abbreviations used in this article: ATRA, all-trans retinoic acid; dCLN, deep cervical
lymph node; DTx, diphtheria toxin; hCD2, human CD2; iTreg, Treg cells in vitro;
RGC, retinal ganglion cell; SDLN, skin draining lymph node; Teff, T effector; Treg,
T regulatory.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302401
Naturally occurring Treg cells, which express the transcription
factor Foxp3 (11–13), have been intensively studied for their
ability to suppress adaptive immune responses (14–17). This subset of T cells, which develops with high avidity to self-Ags, is
especially important in controlling autoimmunity (18). Therefore,
it has been proposed that Treg cells mediate their actions by attenuating both protective and inflammatory postinjury immune
responses, and thus either exacerbating (19) or ameliorating (20)
neuronal degeneration. Despite these studies, the exact mechanism
of their action in the injured CNS remains unclear.
Recently, the heterogeneity of macrophages has come to light,
with two general classes being described as classically or alternatively activated (21). Although classically activated macrophages express high levels of proinflammatory cytokines such as
TNF and IL-1b, and exhibit a robust respiratory burst (22), alternatively activated (tissue-building) macrophages express high
levels of arginase-1 and several factors that play a role in promoting tissue homeostasis and recovery from insults (23). Several
studies have shown the neuroprotective ability of alternatively
activated macrophages in CNS injury (24–26), but what leads to,
and sustains, this phenotype is unclear in the context of CNS
trauma.
In this study, we show that the regulation of the T cell response
to CNS injury is taking place in the draining deep cervical lymph
nodes (dCLN) rather than at the site of injury. In line with this,
surgical resection of the dCLN results in impaired neuronal survival. We show that removal of Treg cells that leads to exaggerated
response of T effector (Teff) cells is associated with reduction in
alternatively activated macrophages at the site of injury and leads
to impaired neuronal survival. Exogenous supply of activated Treg
cells, however, results in suppression of neuroprotective IL-4–
producing T cells, and consequently also results in suppression of
alternatively activated macrophages at the site of injury. Thus,
both depletion and addition of Treg cells are detrimental for
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Previous research investigating the roles of T effector (Teff) and T regulatory (Treg) cells after injury to the CNS has yielded
contradictory conclusions, with both protective and destructive functions being ascribed to each of these T cell subpopulations. In
this work, we study this dichotomy by examining how regulation of the immune system affects the response to CNS trauma. We
show that, in response to CNS injury, Teff and Treg subsets in the CNS-draining deep cervical lymph nodes are activated, and
surgical resection of these lymph nodes results in impaired neuronal survival. Depletion of Treg, not surprisingly, induces a robust
Teff response in the draining lymph nodes and is associated with impaired neuronal survival. Interestingly, however, injection of
exogenous Treg cells, which limits the spontaneous beneficial immune response after CNS injury, also impairs neuronal survival.
We found that no Treg accumulate at the site of CNS injury, and that changes in Treg numbers do not alter the amount of
infiltration by other immune cells into the site of injury. The phenotype of macrophages at the site, however, is affected: both
addition and removal of Treg negatively impact the numbers of macrophages with alternatively activated (tissue-building) phenotype. Our data demonstrate that neuronal survival after CNS injury is impaired when Treg cells are either removed or added.
With this exacerbation of neurodegeneration seen with both addition and depletion of Treg, we recommend exercising extreme
caution when considering the therapeutic targeting of Treg cells after CNS injury, and possibly in chronic neurodegenerative
conditions. The Journal of Immunology, 2014, 193: 5013–5022.
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neuronal survival after injury through regulation of macrophage
phenotype.
Materials and Methods
Animals
Female C57BL/6 (stock 000664) and UBC-GFP (stock 004353) mice were
purchased from The Jackson Laboratory (Bar Harbor, ME); DEREG mice
were a gift of T. Sparwasser (Institute of Infection Immunology, Twincore,
Germany) (16); KN2 mice were a gift of M. Mohrs (Trudeau Institute,
Sarnac Lake, NY) (27). All animals were housed in temperature- and
humidity-controlled rooms, maintained on a 12-h/12-h light/dark cycle
(lights on 7:00 A.M.), and age matched in each experiment. All strains
were kept in identical housing conditions. All procedures complied with
regulations of the Institutional Animal Care and Use Committee at the
University of Virginia.
Treg CELLS IN CNS TRAUMA
3 3 106 cells/ml was incubated in T cell culture media supplemented with
1 mg/ml anti-CD3 (clone 145-2C11; American Type Culture Collection
stock CRL-1975; Ab grown and isolated by UVA lymphocyte culture
center) and 1 mg/ml anti-CD28 (clone 37.51; Bioxcell, stock BE0015-1).
For Treg cells in vitro (iTreg) cultures, the media was supplemented with
10 nM ATRA (Fisher), 5 ng/ml TGF-b (PeproTech), and 250 U/ml IL-2
(R&D Systems). The cultures were maintained for 5 d before CD4+ T cells
were isolated using magnetic bead separation (Miltenyi Biotec) and
injected i.v. into C57BL/6J mice.
Flow cytometry
Retrograde labeling of retinal ganglion cells
Macrophage-skewing assay
Mice were anesthetized, and the skull was exposed and immobilized in
a stereotactic device. Holes were drilled in the skull above the superior
colliculus (bilaterally 2.9 mm caudal to bregma and 0.5 mm lateral to
midline). A quantity amounting to 1 mL 4% Fluoro-gold was injected 2 mm
below the meningeal surface at a rate of 0.5 ml/min using a Hamilton
syringe and an automatic injector. The dye was allowed to diffuse into the
tissue for 1 min before the syringe was removed. The scalp was then sutured closed, and the mice were allowed to recover on warming pads at
37˚C before returning them to their cage.
Bone marrow was isolated from wild-type mice and cultured on untreated
petri dishes in DMEM/F12 containing 10 ng/ml M-CSF (eBioscience), 10%
FCS, L-glutamine, and pen-strep (Invitrogen). The media was changed
every 3 d, and macrophages were used after 8 d in vitro. The day before the
macrophages were used, they were replated on tissue culture–treated
24-well plates. CD4+ T cells from injured or uninjured deep cervical or
skin draining lymph nodes (SDLN) were isolated using magnetic bead
separation (Miltenyi Biotec) and incubated at 1 3 106 cells/well in complete macrophage media. Twenty-four hours after addition of T cells, the
macrophages were washed five times with PBS to remove the nonadherent
T cells, and RNA was isolated from the macrophages.
In vivo drug treatment
Optic nerve injury
Mice were subjected to an optic nerve injury 3 d after stereotactic surgery.
Briefly, mice were anesthetized with a 1:1:8 mixture of ketamine:xylazine:
saline. An incision was made in the connective tissue above the sclera. The
venous sinus around the optic nerve was retracted to expose the optic nerve,
and the nerve was crushed using N5 self-closing forceps 2 mm behind the
globe for 3 s. The mice were then allowed to recover at 37˚C on a warming
pad before returning to their cages.
Retina excision
Mice were enucleated, and the cornea removed at the corenal limbus. The
lens and the underlying vitreous were removed with forceps. The retina was
separated from the sclera and pigment epithelium. Four cuts were made
toward the optic disc, and the retina was mounted on nitrocellulose paper and
fixed in 4% paraformaldehyde overnight. Pictures of all four quadrants of the
retina were taken at equal distances from the optic disc of the retinas using an
Olympus IX-71 microscope. The pictures were then counted by a blinded
observer to determine the number of retinal ganglion cells (RGCs).
Immunohistochemical staining of optic nerve tissue
For arginase-1 staining, mice were perfused transcardially with ice-cold
PBS containing 4 U/ml heparin and then with 4% paraformaldehyde.
Eyes were enucleated and frozen on dry ice in OCT. The 10-mm sections
were cut on a Lyca cryostat and mounted on gelatin-coated slides. Sections
were then stained for arginase-1 (Santa Cruz Biotechnology; clone V20),
CD68 (BioLegend; clone FA11), Iba1 (Biocare Medical; polyclonal), and
GFP (Abcam; polyclonal). For CD4 and CD11b staining, mice were perfused transcardially with ice-cold PBS containing 4 U/ml heparin. Eyes
were enucleated and frozen on dry ice in OCT. The 10-mm sections were
cut on a Lyca cryostat and mounted on gelatin-coated slides. Slides were
postfixed in 3:1 acetone:ethanol at 4˚ before staining with the flowing Abs:
CD4-FITC (eBioscience; clone GK-1.5), CD11b (BioLegend; clone M1/
70), and Foxp3-biotin (eBioscience; clone FKJ-16s). For Foxp3 and CD4
costaining, CD4 was detected with an anti-fluorescein secondary Ab (Life
Technologies) and FoxP3 was detected with Alexfluor 594–conjugated
streptavidin (Jackson Immunochemical).
dCLN removal
Mice were anesthetized with a 1:1:8 mixture of ketamine:xylazine:saline. A
10-mm incision was made midline above the trachea. Salivary glands and
sternocleidomastoid muscles were retracted bilaterally to expose the dCLN.
dCLN were removed using Dumont forceps, and the skin was sutured
closed. Mice that received sham surgery had their dCLN exposed, and then
the skin was sutured. The mice were allowed to recover on a 37˚C warming
pad before returning to their cages. Mice were allowed to recover from
surgery for at least 2 wk before optic nerve injury.
Production of bone marrow chimeras
C57BL/6 mice underwent split-dose lethal irradiation (350 rad, then 950 rad
48 h later). Bone marrow cells were isolated by flushing out the femur and
tibia of UBC-GFP mice. A total of 1 3 107 bone marrow cells was injected
into the irradiated mice 3 h after the last irradiation. Mice were allowed to
reconstitute for 6 wk before being used in experiments.
T cell cultures
For Teff cultures, total lymph nodes were dissected, and a single-cell
suspension was made by mashing through a 70-mm mesh. A total of
Results
A CD4+ T cell response in the CNS-draining dCLN after CNS
injury
To determine where the immune response to CNS injury was
occurring, we first examined CNS-draining dCLN as compared
with SDLN (axillary and inguinal) for T cell activation and proliferation upon CNS injury. We found an increase in the number and
percentage of CD4+ T cells and a concurrent reduction in the
percentage of CD8+ T cells in CNS-draining dCLN (Fig. 1A–C).
No change in the number or percentage of CD4+ T cells was
observed in the SDLN (Fig. 1D–F). When the induced CD4+
T cells were examined for subpopulation (Treg versus Teff), both
activated Teff (CD4+CD25+Foxp32) and Treg (CD4+Foxp3+) cells
were increased in the dCLN after the injury (Fig. 1G–I), but not in
the SDLN (Fig. 1J–L). To determine whether the immune response in the dCLN was playing an important role in the response
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A total of 200 mg all-trans retinoic acid (ATRA; Fisher) was dissolved in
corn oil and injected i.p. every other day starting 3 d before injury.
Diphtheria toxin (DTx) was dosed at 40 mg/kg in PBS and was injected
into C57BL/6 or DEREG mice 2 d before optic nerve injury and on the day
of optic nerve injury. A total of 250 mg anti-CD25 (clone PC-61) was
injected into mice 8 d before optic nerve injury.
Axillary and inguinal lymph nodes or dCLN were isolated and mashed
through a 70-mm strainer in PBS containing 1% BSA and 2 mM EDTA.
The following Abs were used, and are all from eBioscience, unless otherwise noted: Foxp3-Alexa 488, CD4-PerCP Cy5.5, TCR-b allophycocyanin eFluor780, CD45-allophycocyanin, CD8-eFluor 450, CD25-PE
(BD Bioscience), IL-4 PE, IFN-g allophycocyanin, and CD69-PE Cy7. For
intranuclear staining, the cells were fixed overnight in Foxp3 Fix/Perm
buffer (eBioscience) before incubating with Foxp3 Ab in FACS buffer
containing 0.3% saponin (Fisher). For intracellular staining, mice were
injected with 3 mg brefeldin A 5 h before they were sacrificed. The cells
were stained for extracellular markers before they were fixed in the fixation
buffer (eBioscience) for 30 min, then permeabilized and stained for intracellular Ags in FACS buffer containing 0.3% saponin.
The Journal of Immunology
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to CNS injury, we used an optic nerve injury model, in which
RGCs are prelabeled with the neuronal tracer Fluoro-gold and then
the optic nerve is injured and the number of surviving RGCs in the
retina is quantified (Fig. 1M). This injury leads to a decrease in the
number of RGCs in mice that underwent the dCLN removal from
those that received a sham surgery (Fig. 1N), whereas their contralateral uninjured retinas did not display a loss of RGCs (Fig. 1O).
To determine whether T cells from the injured dCLN displayed
a different phenotype after CNS injury than the SDLN, we used
flow cytometry to analyze the intracellular cytokines produced in
the lymph nodes after injury. T cells from the dCLN displayed
higher levels of IL-4 after optic nerve injury than those from the
SDLN (Fig. 2A, 2B). To examine whether this phenotype is induced by the injury, we used KN2 reporter mice that express
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FIGURE 1. The dCLN display an immune response after CNS injury, and their resection exacerbates neuronal survival. (A) Flow cytometry of CD4+ and
CD8+ lymphocytes in the dCLN from uninjured mice or from mice 5 d postinjury. Numbers indicate percentage of CD4+ and CD8+ T cells, as a percentage
of TCR-b+ cells. (B and C) Frequency of CD4+ and CD8+ as a percentage of TCR-b+ lymphocytes (B) and number of CD4+TCR-b+ T cells (C) in the
dCLN, as quantified by flow cytometry 5 d after injury. n = 3 per group. Representative of .3 experiments. *p , 0.05, **p , 0.01 Student t test. (D) Flow
cytometry of CD4+ and CD8+ lymphocytes in the SDLN of uninjured mice or from mice 5 d postinjury. Numbers indicate percentage of CD4+ and CD8+
T cells, as a percentage of TCR-b+ cells. (E and F) Frequency of CD4+ and CD8+ T cells, as a percentage of TCR-b+ cells (E) and number of CD4+ T cells
(F) in the SDLN 5 d postinjury, as quantified by flow cytometry. n = 3 per group. Representative of .3 experiments. Student t test. (G) Flow cytometry of
CD4+ lymphocytes in the dCLN of uninjured mice or from mice 5 d postinjury. Numbers indicate percentage of activated Teff and Treg cells, as a percentage
of CD4+ cells. (H and I) Frequency of Treg (H) and Teff (I) cells in the dCLN as a percentage of the uninjured dCLN. n = 6 per group. Representative of two
experiments. *p , 0.05 Student t test. (J) Flow cytometry of CD4+ lymphocytes in the SDLN of uninjured mice or from mice 5 d postinjury. Numbers
indicate percentage of activated Teff and Treg cells, as a percentage of CD4+ cells. (K and L) Frequency of Treg (K) and Teff (L) cells in the SDLN as
a percentage of the uninjured SDLN. n = 6 per group. Representative of two experiments. Student t test. (M) Representative images of Fluoro-gold–stained
retinas from uninjured or injured eyes. Insets are at original magnification 340, larger are at original magnification 310. Boxes represent fields counted for
RGC quantification. (N) Neuronal survival of mice receiving sham surgery or undergoing dCLN removal 2 wk prior to injury, as assessed by Fluoro-gold
staining. Survival is quantified as a percentage of control survival. n = 11 sham and 12 dCLN removed. Representative of two experiments. **p , 0.01
Student t test. (O) RGC counts of the contralateral uninjured retina of mice receiving sham surgery or undergoing dCLN removal 2 wk prior to injury, as
assessed by Fluoro-gold staining. RGC counts are quantified as a percentage of the control. n = 11 sham and 12 dCLN removed. Representative of two
experiments. Student t test.
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human CD2 (hCD2) when IL-4 is being translated. In these mice,
hCD2 expression is seen in CD44+ memory T cells (Fig. 2C), and
this hCD2 expression is induced after injury in the dCLN after
injury, but not in the SDLN (Fig. 2D). To determine whether
T cells induced after optic nerve injury in the draining lymph
nodes are capable of supporting alternative activation of macrophages, we isolated T cells from injured and uninjured dCLN and
SDLN and cocultured them with a pure population of bone marrow–derived macrophages. T cells from the dCLN of optic nerve–
injured mice were able to support an alternative activation phe-
Treg CELLS IN CNS TRAUMA
notype of bone marrow macrophages in vitro, whereas T cells
obtained from SDLN of injured mice or from dCLN of uninjured
mice were unable to promote this alternatively activated (tissuebuilding) phenotype of macrophages (Fig. 2E). This suggests that
the injury indeed induces Teff cells in the draining dCLN that are
capable of promoting a neuroprotective macrophage phenotype.
Because we observed that T cells in the draining lymph node
were able to promote an alternative activation of macrophages, we
addressed a possibility that T cells are controlling the phenotype
of the infiltrating monocytes/macrophages. Arginase-1–expressing
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FIGURE 2. CNS injury promotes a milieu conducive to alternative activation of macrophages in the dCLN. (A) Gating strategy and representative
staining of IL-4 production by CD4+ T cells in the draining dCLN and SDLN after CNS injury. (B) Quantification of the mean fluorescence intensity of
IL-4 staining of CD4+ T cells in the dCLN or SDLN. n = 4 per group. Representative of two experiments. ***p , 0.001 Student t test. (C) Representative
staining of hCD2 (IL-4) production by CD4+ T cells in the draining dCLN in injured and uninjured mice. (D) Quantification of the number of hCD2+
staining of CD4+ T cells in the dCLN or SDLN in uninjured and injured mice. n = 5 per group. Representative of two experiments. *p , 0.05 Student
t test. (E) arg1 mRNA expression of bone marrow–derived macrophages that had been cocultured with CD4+ T cells from the indicated lymph nodes of
mice with or without optic nerve injury for 24 h. n = 3 per group. Representative of .3 experiments. *p , 0.05 one-way ANOVA with Bonferroni’s
posttest. (F) Representative images of injured optic nerves of GFP⇒C57BL/6 bone marrow chimeras stained for arginase-1 and Iba1. Arrowheads point to
GFP2 radio-resistant microglia, whereas arrows point to infiltrating macrophages (scale bars, 100 mm). (G) Quantification of percentage of Iba1+ cells in
the injured optic nerve that are GFP+arginase-1+, GFP+arginase-12, GFP2arginase-1+, and GFP2arginase-12. n = 3 per group. *p , 0.05 one-way
ANOVA with Bonferroni’s posttest. (H) Quantification of the percentage of GFP+ and GFP2 cells that are arginase-1+ in the injured optic nerve. n = 3 per
group. ***p , 0.001 Student t test.
The Journal of Immunology
that their phenotype switch took place in the periphery prior to
infiltration rather than in the CNS parenchyma. These results are
in line with previous findings, suggesting that monocytes with an
alternatively activated phenotype are arriving from a periphery
through a unique path into the injured CNS (25).
Depletion of Treg exacerbates neurodegeneration after CNS
injury
The contribution of different subsets of T cells to neuronal survival
after CNS injury has been intensively studied (4, 5, 19, 31, 32), yet
their role in this postinjury neuronal survival remains controversial
(8, 10, 33). Because Treg cells are known to exert asymmetric
control of T cell responses in nonpathological situations (34), we
tested the hypothesis that Treg cells were responding to injury in
the draining lymph nodes, where they controlled the phenotype
of Teff cells. We used DEREG mice (16), which express the
DTx receptor under the Foxp3 promoter to assess the effect of
Treg depletion on neuronal survival. Treatment of these mice with
40 mg/kg DTx 2 d before injury completely eliminates Treg cells in
the bloodstream (Supplemental Fig. 2A). Seven days after injury,
the DEREG mice treated with DTx still displayed decreased
numbers of Treg cells in their CNS-draining dCLN (Fig. 3A) and
an increase in the number of activated Teff cells in the dCLN and
SDLN (Fig. 3B, Supplemental Fig. 2B).
FIGURE 3. Alleviation of Treg suppression after CNS injury leads to a reduced neuronal survival after optic nerve injury. (A and B) Bar graphs represent
quantification of flow cytometry analysis of the dCLN of DEREG or wild-type littermates treated with DTx 2 d before injury and on the day of injury,
showing percentage of CD25+Foxp3+ Treg cells (A) and of CD25+Foxp32 Teff cells (B), graphed as a percentage of TCR-b+CD4+ cells (n = 12 wild-type and
9 DEREG treated mice. Representative of three experiments. ***p , 0.001, *p , 0.05 Student t test (C) Neuronal survival after optic nerve injury in
DEREG and wild-type mice injected with 40 mg/kg DTx 2 d before injury and on the day of injury. Survival is quantified as a percentage of control survival.
n = 19 wild type and 25 DEREG. Representative of three experiments. *p , 0.05 Student t test. (D) Quantification of the number of CD4+ T cells found in
the injury site of DEREG mice treated with DTx normalized to the number of CD4+ T cells found in the injury site of C57BL/6 mice treated with DTx.
n = 3 per group. Representative of two experiments. Student t test. (E) Quantification of the number of CD11b+ cells found in the injury site, normalized to
the number of CD11b+ T cells found in the injury site of C57BL/6 mice treated with DTx. n = 3 C57BL/6 treated with DTx and 9 DEREG treated with
DTx. Representative of two experiments. Student t test. (F) Representative images of CD68 and arginase-1 in injured optic nerve of DEREG and wild-type
mice treated with two doses of 40 mg/kg DTx. Scale bars, 100 mm. (G) Arginase-1+ area graphed as a percentage of CD68+ area in C57BL/6 or DEREG
mice treated with DTx. n = 3 C57BL/6 treated with DTx and 8 DEREG treated with DTx. *p , 0.05 Student t test. (H) Quantitative PCR for arg1 of optic
nerves of C57BL/6 or DEREG mice treated with 40 mg/kg DTx 2 d before injury and on the day of injury normalized to arg1 expression in the contralateral
uninjured nerve. n = 7 C57BL/6 treated with DTx and 4 DEREG treated with DTx. Representative of two experiments. *p , 0.05 Student t test.
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macrophages (M2 type) have been previously described to support neuronal survival after CNS injury (24–26, 28). Indeed,
using immunohistochemistry of injured optic nerves, we demonstrate that arginase-1 expression is induced in the injury site
(Fig. 2F), whereas there is no detectable arginase-1 staining in
the uninjured optic nerves (Supplemental Fig. 1A). We established GFP⇒C57BL/6 bone marrow chimeric mice (29), whose
peripheral immune system is replaced by the GFP+ bone marrow,
but that have a significant number of GFP2 microglia in the optic
nerve (Supplemental Fig. 1B). Therefore, despite the issues inherent with bone marrow transplantation following irradiation
(30), the large majority of the engrafted Iba1-positive cells in the
uninjured CNS of chimeric mice are of non-GFP origin. It is,
therefore, conceivable to assume that an increase of GFP+ cells
after injury results primarily from their peripheral recruitment of
monocyte-derived macrophages, although, as stated above, the
procedure has its limitation. In the chimeric mice, most of the
GFP+Iba1+ cells in the site of the injury were arginase-1 positive,
suggesting that at least the majority of the infiltrating cells were
highly skewed after injury. However, significantly fewer of the
radio-resistant GFP2Iba1+ microglia were arginase-1 positive,
suggesting that macrophages infiltrating from the periphery are the
primary source of alternatively activated myeloid cells (Fig. 2G,
2H). This preferential skew of infiltrating myeloid cells suggests
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The mRNA expression of arg1, the gene for arginase-1, was reduced in DEREG mice, confirming the decrease in alternatively
activated macrophages after Treg depletion (Fig. 3H).
Exogenous Treg cells inhibit a beneficial response to CNS injury
A complete depletion of Treg cells using DEREG mice resulted in
impaired outcome of CNS injury in our optic nerve crush injury
model (Fig. 3C). However, the question still remains whether increased activity of Treg cells would conversely offer a benefit after
CNS injury. First, we tested the physiological outcome of Treg
manipulation via potentiation of Treg-suppressive function by
treating mice with ATRA, which induces differentiation of Treg
cells (37), stabilizes the Treg phenotype (38), and makes Treg cells
more suppressive (38). As expected, treatment of mice with ATRA
increased the Treg population in the dCLN after injury (Fig. 4A),
but surprisingly not in the SDLN (Supplemental Fig. 4A), and
resulted in a decrease of activated Teff cells in the dCLN and
SDLN (Fig. 4B, Supplemental Fig. 4B). Interestingly, and in line
with some reports (19) but contrary to other previous findings (7),
mice treated with ATRA exhibited decreased neuronal survival
compared with vehicle-treated mice (Fig. 4C), suggesting that
induction of highly suppressive Treg cells limits the protective Teff
responses. To rule out a possible in vivo effect of ATRA on cells
other than T cells, we differentiated iTreg using ATRA and TGFb
(Supplemental Fig. 4C). Injection of iTreg cells into CNS-injured
mice also resulted in an increase in Treg cells (Fig. 4D) and attenuation of their activated Teff response to injury in the dCLN
(Fig. 4E), but no change in the number of Treg cells and Teff cells
in the SDLN (data not shown) and a reduction in neuronal survival
(Fig. 4F). Mice treated with Teff cells (that were activated without
TGF-b and ATRA, and that contained only ∼3% Foxp3+ Treg
FIGURE 4. Potentiation of Treg function impairs neuronal survival after optic nerve injury. (A and B) Bar graphs represent quantification of flow
cytometry analysis of the dCLN of wild-type mice treated with vehicle or ATRA showing percentage of CD25+Foxp3+ Treg cells (A) and of CD25+Foxp32
Teff cells (B), graphed as a percentage of TCR-b+CD4+ cells. n = 7 vehicle treated and n = 9 ATRA treated. Representative of two experiments. *p , 0.05
Student t test. (C) RGC survival in wild-type mice treated with vehicle or ATRA. Survival is quantified as a percentage of control survival. n = 7 vehicle and
n = 9 ATRA. Representative of two experiments. *p , 0.05 Student t test. (D and E) Bar graphs represent quantification of flow cytometry analysis of dCLN
of wild-type mice treated with vehicle or 1 3 106 exogenous Treg cells 1 d before injury and 1 d after injury, showing percentage of CD25+Foxp3+ Treg cells
(D) and of CD25+Foxp32 Teff cells (E), graphed as a percentage of TCR-b+CD4+ cells. n = 12 Treg cells injected and n = 13 vehicle injected. *p , 0.05
Student t test. (F) Neuronal survival in wild-type mice injected with vehicle, 1 3 106 Treg or 1 3 106 Teff cells 2 d before injury and on the day of injury.
Survival is quantified as a percentage of control survival. n = 13 vehicle injected, 12 Treg cell injected, and 7 Teff cell injected. Representative of two
experiments. *p , 0.05 one-way ANOVA with Bonferroni’s posttest.
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To test the effect of Treg cell depletion on CNS injury, we again
used the optic nerve crush injury model. As expected from previous studies (35), DEREG mice treated with DTx, and thus depleted of Treg cells, showed a decrease in the number of surviving
RGCs 7 d after injury, as compared with wild-type mice treated
with DTx (Fig. 3C). We examined the contralateral retina of injured mice (Supplemental Fig. 2C) and histological sections of
uninjured mice treated with DTx (Supplemental Fig. 2D), which
did not display any loss of RGCs or immune cell infiltrate,
suggesting DTx by itself did not have destructive effects on
uninjured CNS tissue. Furthermore, there was no difference in
neuronal survival in C57BL/6 mice treated with saline or DTx
(Supplemental Fig. 2E), confirming that DTx treatment was not
causing nonspecific effects at the dose that we are using. To further establish the role of depletion of Treg cells, we used an antiCD25 Ab, which depletes Treg cells (that express high levels of
CD25) (36). In mice that were Treg depleted, there was a decrease
in the number of CD25+Foxp3+ Treg cells in the dCLN even 7 d
after optic nerve injury and a corresponding decrease in neuronal
survival (Supplemental Fig. 2F, 2G), consistent with the results
seen in DEREG mice.
Although no change in overall numbers of CD4+ T cells
(Fig. 3D) or CD11b+ myeloid cells (Fig. 3E) at the site of injury was found, the phenotype of accumulated macrophages was
altered in DEREG mice treated with DTx. A significant decrease
in arginase-1–expressing CD68 (a marker of activated macrophages) cells was evident (Fig. 3F, 3G), suggesting a decrease in
alternatively activated macrophages (M2 type) after injury in
Treg-depleted mice. No difference in the total amount of CD68+
area was detected (Supplemental Fig. 3A). To confirm the histological observations, we also examined the injured tissue by PCR.
Treg CELLS IN CNS TRAUMA
The Journal of Immunology
compared with ∼85% in Treg-designated culture conditions) did
not show any change in neuronal survival (Fig. 4F), possibly due
to the large number of Teff cells already present in wild-type mice.
Interestingly, 7 d after Treg injection, only very few of the
injected cells were found in the dCLN, despite a substantial increase in overall Treg numbers (Supplemental Fig. 4D). These
results suggest that the injected Treg cells induce endogenous
T cell differentiation, possibly through their high levels of secreted
and membrane-bound TGF-b (39, 40).
Treg cells do not infiltrate the injured CNS after injury
cells accumulating at the site of injury. As with CD4+ T cells,
there was no change in the number of CD11b+ cells that migrated
to the site of injury in Treg-treated mice (Fig. 5F, 5G), further
suggesting that Treg cells are not controlling immune cell migration in this injury model.
To determine whether T cell–derived cytokines are affected by
Treg manipulation after CNS injury, we examined the mRNA expression of genes from the optic nerve of injured mice treated with
either Teff or Treg cells. Mice treated with Treg cells display a
dramatic decrease in the amount of IL-4 mRNA compared with
mice treated with Teff cells (Fig. 6A), suggesting a change in the
Th2 response to damage at the injury site with Treg treatment. To
determine whether these changes are having effects downstream
on myeloid cells after CNS injury, we examined markers of myeloid skewing in the injured optic nerves of mice treated with Treg
cells. Indeed, Treg-treated mice displayed a decrease in the mRNA
expression of alternatively activated macrophage markers arg1
and il10, whereas there was no change in the classical activation
markers nos2 and tnf (Fig. 6B–E). To further demonstrate that
there was a loss of alternative activation of macrophages in Treginjected mice, we examined colocalization between CD68, a
marker of activated myeloid cells, and arginase-1 by immunofluorescence. Although Teff -injected mice displayed marked expression of arginase-1 in the CD68+ fraction 7 d after injury,
injection of Treg cells led to a decrease in arginase-1 expression
by myeloid cells (Fig. 6F, 6G), further demonstrating that both
addition and deletion of Treg cells are detrimental in CNS
trauma through their effects on the innate immune response at
the site of injury.
FIGURE 5. Boost with exogenous Treg cells does not alter immune cell infiltration into the injury site. (A) Representative gates of flow cytometry of
CD4+ and CD8+ lymphocytes in the injured optic nerve 7 d postinjury. Optic nerves were pooled from eight mice, and cells were stained for analysis by
flow cytometry. (B) Representative image from splenic tissue stained for CD4 (green) and Foxp3 (red) (scale bar, 100 mm). (C) Representative image from
spinal cord tissue directly injected (ex vivo) with in vitro induced Treg cells stained for CD4 (green) and Foxp3 (red) (scale bar, 50 mm). (D) Representative
images of CD4+Foxp32 and CD4+Foxp3+ T cells in the optic nerve parenchyma of Teff- and Treg-treated mice (scale bars, 100 mm). (E) Quantification of the
number of CD4+Foxp32 and CD4+Foxp3+ T cells in the optic nerve parenchyma of Teff- and Treg-treated mice (n = 4 mice per group; one-way ANOVA
with Bonferroni’s posttest). (F) Representative images of CD11b+ cells in injury site of the optic nerve of Teff- and Treg-treated mice (scale bars, 100 mm).
(G) Quantification of the number of CD11b+ cells in injury site of the optic nerve of Teff- and Treg-treated mice (n = 9 mice per group; Student t test;
representative of two experiments).
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Because Treg cells are exerting a negative effect on the outcome
to CNS injury, we sought to determine whether Treg cells were
also gaining access to the site of injury. Upon injury, there is the
influx of Teff cells to the CNS parenchyma (Fig. 5A). Despite
being able to visualize Treg cells using Foxp3 immunolabeling in
the spleen (Fig. 5B) and in spinal cords that had been injected
directly with Treg cells (Fig. 5C), we did not see Treg cells in the
parenchyma of the injured optic nerve in animals after exogenous
i.v. injection of Treg cells (Fig. 5D) or in injured wild-type mice,
wild-type mice injected with Teff cells, DEREG mice treated with
DTx, and wild-type mice treated with DTx (data not shown).
Furthermore, there was no difference in the number of CD4+ Teff
cells at the injury site of Treg-treated mice (Fig. 5E). Therefore, it
seems unlikely that acute manipulation of Treg cells in our experimental paradigms is affecting neuronal survival through the
migration of Teff cells into the site of the injury. Next, to determine whether addition of Treg cells could affect monocyte migration to the injured CNS, we quantified the number of CD11b+
5019
5020
Treg CELLS IN CNS TRAUMA
Discussion
There has been an ongoing debate about the role of Treg cells in
CNS injury and in neurodegenerative conditions. We show in this
work that complete depletion of Treg cells using DEREG mice,
a manipulation that has been shown to lead to development of
numerous organ-specific autoimmune diseases (16), leads to increased neurodegeneration after CNS trauma. However, the same
deleterious effect on neuronal survival can be seen when Treg
numbers are increased either by pharmacological compounds or
by exogenous supply of Treg cells. Changes in Treg numbers correlate with the phenotype of macrophages populating the injury
site, with alternatively activated macrophages being spontaneously
induced by the injury, yet inhibited by Treg cell addition or overruled by an exaggerated immune response as a result of Treg cell
depletion.
Early work in models of stroke and Parkinson’s disease showed
that depletion of Treg cells led to increased neurodegeneration,
whereas increases in Treg cell numbers and function improved
disease outcome (10, 41). More recent work, using the same
manipulations, has shown that Treg cells play a detrimental role
after CNS injuries (7), supporting the hypothesis that they are
suppressing a beneficial autoimmune response (19). There are
several factors that may have contributed to these disparate findings. There are technical challenges with the current Treg depletion
strategies that have hindered interpretation of depletion studies,
such as targeting of activated effector cells with anti-CD25
treatment (42, 43). Furthermore, several studies have shown that
Treg cells have the potential to downregulate Foxp3 and become
effector cells, especially when placed in lymphopenic or inflammatory conditions (38, 44, 45), probably due to heterogeneity in
the fate commitment of the Treg cell population (46), complicating
transfer experiments into mice with abnormal adaptive immune
systems. However, the conditions that drive this switch from Treg
to Teff, and relevance of these models in vivo, is still a matter of
debate (47). Although DTx treatment may have indirect effects,
overall the DEREG mouse offers a unique model for Treg deletion
in vivo by both avoiding targeting CD25, indirectly affecting activated Teff, and avoiding cell transfer, which inevitably changes
the cell phenotype. Therefore, although imperfect, DEREG model
presents the best currently available model for Treg depletion.
Our work further addresses the question of how Treg cells are
affecting the outcome from CNS injury. Although we show that
Treg cells have profound effects on neuronal survival from injury,
they are not found at the site of the injury, but are rather enriched
in the draining lymph node. Treg cells are known to exert asymmetric control of T cell responses in nonpathological situations
(34), raising the possibility that these Treg cells are exerting their
action on the phenotype of Teff cells in the draining lymph node.
The Teff cells, in turn, direct the phenotype of the infiltrating innate immune cells. Previous works demonstrated that precursors
for alternatively activated macrophages are recruited to the injured
CNS through a unique path of the choroid plexus (25) and are
coming predetermined to differentiate into alternatively activated
macrophages.
Several studies have shown that alternative activation of macrophages is a beneficial response to CNS injury (25, 28). Tissuebuilding macrophages produce growth factors, such as insulin-like
growth factor 1, vascular endothelial growth factor, TGF-b, and
factors that remodel the extracellular matrix, such as matrix metalloproteinases and resistin-like molecule alpha and promote a
tissue-building phenotype in injured tissue (21). T cells, and
specifically Th2 effector cells, produce several cytokines, such as
IL-4 for example, that can induce alternatively activated macrophages (48). Our results suggest that T cells induced in the CNSdraining dCLN control the phenotype of the infiltrating monocytes,
and future studies need to concentrate on better understanding the
molecular interaction between T cells and myeloid cells that results
in myeloid cells of a particular phenotype to migrate to the site of
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FIGURE 6. Treg cell injection leads to a loss of an alternative activation phenotype of myeloid cells at the site of injury. Optic nerves of Teff and Treg cell–
injected mice were collected 7 d postinjury and examined for expression of the following genes relative to expression of gapdh: (A) il4 (n = 11 per group,
representative of two experiments, *p , 0.05 Student t test), (B) arg1, (C) il10, (D) nos2, and (E) tnf. (B–E) n = 6 per group, *p , 0.05 Student t test. (F)
representative images of Teff and Treg cell–injected mice 7 d after injury stained for arginase-1 (green) and CD68 (red) (scale bars, 100 mm). (G) Arginase-1+
area graphed as a percentage of CD68+ area in Teff and Treg cell–injected mice 7 d postinjury. n = 9 Teff cell injected and 6 Treg cell injected. Representative
of two experiments. **p , 0.01 Student t test.
The Journal of Immunology
Acknowledgments
We thank Shirley Smith for editing the manuscript. We thank the members
of the Kipnis laboratory for valuable comments during multiple discussions
of this work.
Disclosures
The authors have no financial conflicts of interest.
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