182? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 119? ? ? Number 1? ? ? January 2009
Infiltration of CD4+ lymphocytes into the brain
contributes to neurodegeneration in a mouse
model of Parkinson disease
Vanessa Brochard,1,2 Béhazine Combadière,3 Annick Prigent,1,2 Yasmina Laouar,4
Aline Perrin,1,2 Virginie Beray-Berthat,1,2 Olivia Bonduelle,3 Daniel Alvarez-Fischer,1,2
Jacques Callebert,5 Jean-Marie Launay,5 Charles Duyckaerts,1,2 Richard A. Flavell,4,6
Etienne C. Hirsch,1,2 and Stéphane Hunot1,2
1INSERM, UMR S679, Experimental Neurology and Therapeutics, Hôpital de la Salpêtrière, Paris, France. 2Université Pierre et Marie Curie — Paris 06,
UMR S679, Paris, France. 3INSERM U543, Université Pierre et Marie Curie — Paris 06, Paris, France. 4Department of Immunobiology,
Yale University School of Medicine, New Haven, Connecticut, USA. 5CR Claude Bernard, IFR6, Service de Biochimie, Hôpital Lariboisière,
Assistance Publique — Hôpitaux de Paris, Paris, France. 6Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, USA.
Parkinson disease (PD) is a neurodegenerative disorder characterized
by the loss of dopamine-containing neurons (DNs) in the substan-
tia nigra (SN) pars compacta (SNpc). Except for rare genetic forms,
PD is a sporadic condition of unknown cause. Yet, several scenarios
regarding the mechanisms by which DNs degenerate have been sug-
gested, including mitochondrial dysfunction, oxidative stress, and
the impairment of protein degradation machinery (1). In addition to
these well-established pathomechanisms, there is mounting evidence
from epidemiological, postmortem, and animal studies to suggest
that innate neuroinflammatory processes associated with glial cell
activation participate in the progression of DN cell death (2–6).
Far more enigmatic is the putative role of the adaptive immune
system in PD pathogenesis. While several changes in cellular and
humoral immune responses have been described in the peripheral
immune system of PD patients, it remains unclear whether these
alterations are primarily involved in the etiogenesis of PD or sim-
ply reflect secondary consequences of nigrostriatal pathway injury
(5). Up to now, the hypothesis that the cellular arm of the adap-
tive immune system plays a role in neurodegeneration has been
hampered by the fact that no clear demonstration of a prominent
involvement of leukocytes at the site of neuronal injury has been
provided in PD. Yet, the reports many years ago that cytotoxic T cells
(Tc) can be found in massive numbers in the SN of a patient with PD
(7) and that the density of IFN-γ–positive cells is markedly increased
in brains of patients of with PD (8), suggest that T cell brain recruit-
ment may be associated with nigrostriatal pathway injury in PD. In
support of this view is the demonstration that the accumulation of
T cells in the brain can be stimulated in mice by 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine–induced (MPTP-induced) DN injury (9).
Yet, whether this immune response truly contributes to neurodegen-
eration and, if so, by what mechanisms remains to be established.
In this study, we sought to determine whether PD is associated
with leukocyte brain infiltration within the affected SN region
and, if so, whether this process contributes to DN degeneration.
We found that both CD8+ and CD4+ T cells significantly invade
the SN in postmortem specimens from patients with PD and in
MPTP-intoxicated mice. We also show that removal of CD4+ but
not of CD8+ T cells in mice greatly reduced MPTP-induced nigros-
triatal DN cell death. Finally, we further demonstrate that the del-
eterious activity of infiltrated CD4+ T cells involved the Fas/FasL
pathway but not Ifn-γ production.
Lymphocyte brain infiltration in PD patients. To test the possibility
that leukocytes infiltrate the brain parenchyma of PD patients,
we performed immunohistochemical staining for various leuko-
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Nonstandard?abbreviations?used: BBB, blood-brain barrier; DN, dopamine-con-
taining neuron; GFAP, glial fibrillary acidic protein; gld, FasL-mutated generalized
lymphoproliferative; MPP+, 1-methyl-4-phenylpyridinium; MPTP, 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine; PCNA, proliferating cell nuclear antigen; PD, Parkinson
disease; Rag1, recombinase activating gene 1; SN, substantia nigra; SNpc, SN pars
compacta; α-Syn, α-synuclein; Tc, cytotoxic T cell; Tcrb, T cell receptor β chain; TH,
Citation?for?this?article: J. Clin. Invest. 119:182–192 (2009). doi:10.1172/JCI36470.
Related Commentary, page 13
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
cyte-specific markers (10) on postmortem human brain (for anti-
body description, see Supplemental Table 1; supplemental mate-
rial available online with this article; doi:10.1172/JCI36470DS1).
Positive staining was observed for CD8 and CD4 but not for B
cell and NK cell markers. Within the SNpc, CD8+ and CD4+ T
cells were found either in close contact with blood vessels or in
the vicinity of melanized DNs (Figure 1, A and B). These cells
displayed a typical lymphocyte morphology that was further
confirmed by electron microscopy analysis. At the ultrastructural
level, parenchymal CD8+ T cells displayed small cytoplasmic vol-
ume, membrane-type CD8 staining, and, in some cases, uropod-
like cytoplasmic extension, usually involved in T cell motility
(Figure 1C). Importantly, quantitative analysis revealed a sig-
nificant increase (10-fold in average) in the density of CD8+ and
CD4+ T cells in the SNpc but not in the nonlesioned red nucleus
from PD patients compared with age-matched control subjects
(Figure 1D and Supplemental Table 2).
MPTP-induced DN lesion stimulates extravasation of lymphocytes into
the brain. The above data suggest that migration of peripheral lym-
phocytes within the CNS is associated with nigrostriatal pathway
injury in PD. Yet, although postmortem studies in humans are
essential to establish that immune cell infiltration is characteristic
Lymphocyte infiltration in brains of patients of with PD. (A) CD8 and (B) CD4 immunostaining with hematein counterstain on mesencephalic
transverse sections from PD patients. CD8+ or CD4+ T cells (arrows) are found in close contact with blood vessels or have migrated deep into
the brain parenchyma close to neuromelanin-containing DNs (arrowheads). Note that brain staining for CD79α and CD20 (B cells) and CD57
(NK cells) was not detected in either group of patients. All antibodies were previously tested on human tonsil tissue sections taken as a positive
control, and all of them gave the expected cellular staining (Supplemental Figure 1). Scale bars: 250 μm (upper panel in A and upper-left panel
in B); 30 μm (dashed boxes); 15 μm (upper-right panel in B). (C) Ultrastructural view of an infiltrated CD8+ T cell in the SNpc from a parkinsonian
patient. Parenchymal CD8+ T cells display a small cytoplasmic volume, membrane-type CD8 staining (arrowheads), and a uropod-like cytoplas-
mic extension, usually involved in T cell motility (arrow). Per, pericyte; BM, basal membrane; e, endothelial cell; m, mitochondria; n, nucleus.
Scale bar: 4 μm. (D) Density of infiltrated CD8+ (left panel) and CD4+ T cells (right panel) in the SNpc of parkinsonian patients (n = 14 and 9 for
CD8 and CD4 staining, respectively) and age-matched control subjects (n = 4 and 7 for CD8 and CD4 staining, respectively). Bars represent the
mean density. *P < 0.05, **P < 0.001 compared with controls (Student’s t test).
184? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
of the disease, they cannot provide information about the dynam-
ic and functional relevance of such mechanisms. To address this
issue, we conducted in vivo experiments using the well-character-
ized MPTP-intoxicated mouse model of PD (1). Although it is not
a phenocopy of human disease, this noninvasive model provides a
unique means to study non-cell autonomous pathomechanisms,
as it recapitulates many of the hallmarks of PD, including DN loss,
attenuation of striatal dopamine, and glial cell–associated inflam-
matory processes. To easily track the migration of peripheral leu-
kocytes independently of their phenotypic traits, we first used a
passive transfer strategy that consisted of transferring GFP-tagged
T or B cells into recombinase activating gene 1–deficient (Rag1-
deficient) mice, which lack mature lymphocytes. Recipient animals
with reconstituted lymphoid organs were then treated with MPTP
or saline solution (Figure 2A and Supplemental Figure 2). Unlike
GFP+ B cells (data not shown), we found GFP+ T cells in the SNpc
MPTP-induced nigrostriatal path-
way injury stimulates T cell brain
infiltration in mouse. (A) Schematic
representation of the experimental
approach (see Methods). The green
circle within reconstituted mice refers
to the lymphoid compartments replen-
ished with GFP+ cells. (B) GFP+ T
cell infiltration in the SNpc and (C)
striatum following MPTP intoxication.
Numerous GFP+ T cells can be seen
in the SNpc 2 days after MPTP but
not saline exposure. GFP+ T cells are
also found in the striatum from intoxi-
cated mice, though there are far fewer
than in the SNpc. Scale bars: 300 μm
(B); 100 μm (C); 10 μm (insets). (D)
Immunofluorescent staining for TH,
Glut1, CD8, or CD4. Note that infiltrat-
ed GFP+ T cells are clustered within
the SNpc in close proximity to TH+
DNs. Transmigrated GFP+ T cells are
not found in the lumen of Glut1+ blood
vessels (arrows) and consist of both
CD8+ and CD4+ T cell subsets. Scale
bars: 20 μm. (E) T cell brain infiltra-
tion in MPTP-treated C57BL/6 inbred
mice. CD3 immunostaining show-
ing numerous T lymphocytes within
the SNpc (dashed line) from MPTP-
treated mice (left panel). Scale bar:
100 μm. Kinetic of nigral CD4+ and
CD8+ T cell (insets) density after MPTP
exposure (right panel). S, saline. Data
points represent the mean ± SEM.
*P < 0.01 compared with saline- and
MPTP-treated mice at day 4 after
MPTP exposure; †P < 0.01 compared
with saline-treated controls (Tukey
post-hoc analysis). Scale bars: 50 μm.
(F) Double immunofluorescence
staining for PCNA and GFP and
GFAP or Mac1. Note that PCNA+
cells in the SNpc never colocalize
with GFP+ or GFAP+ cells, whereas
they superpose perfectly with Mac-1+
(arrows). Scale bar: 50 μm.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
and striatum after MPTP treatment (Figure 2, B and C). Most, if
not all, other brain structures but DN-containing regions were
devoid of T cell infiltrate, indicating the region-specific extrava-
sation of these immune cells (Supplemental Figure 3). Brain sec-
tions immunostained for tyrosine hydroxylase (TH) show GFP+ T
cells within the SNpc clustered in close proximity to TH-express-
ing DNs (Figure 2D). Furthermore, infiltrated T cells had clearly
transmigrated through the brain parenchyma, since most of them
were found to be excluded from the lumen of blood vessels iden-
tified by Glut1 immunostaining (Figure 2D). Finally, both CD8+
and CD4+ T cell subtypes were found to be recruited (Figure 2D).
To ensure that the infiltration of T cells into the brain observed
in our graft-based experimental model was not merely an arti-
fact related to the methodology, we looked at leukocyte trans-
migration in MPTP-intoxicated C57BL/6J inbred mice, using
immunohistochemistry for the T cell–specific marker CD3. In
these conditions, a similar T cell infiltration process was observed
(Figure 2E). When compared with the time course of glial cell
activation, CD3+ T cell brain infiltration was found to arise after
the increase of CD11b+ microglial cells (i.e., 12 hours post-MPTP
intoxication) but concomitantly with astrogliosis (Supplemental
Figure 4). Consistent with our postmortem findings, nigral CD8+
T cells were more abundant than CD4+ T cells, and transmigra-
tion occurred at day 2 after treatment, increased continuously for
up to 7 days, and had totally ceased at 21 days (Figure 2E). Such
dynamics are therefore compatible with a possible role of activated
microglial cells in the brain region–specific recruitment of T cells.
The continuous nigral accumulation of T cells indicates that
lymphocytes could clonally expand into the brain parenchyma. To
test such a possibility, we surveyed the expression of proliferating
cell nuclear antigen (PCNA), taken as a marker of dividing cells, at
0.5, 1, 2, 4, and 7 days after the last MPTP injection. As shown in
Figure 2F, while we observed numerous PCNA+ cells in the SNpc
after MPTP intoxication, none of them were found to colocalize
with either GFP+ T cells or glial fibrillary acidic protein–positive
(GFAP-positive) astrocytes at all the time points analyzed. Instead,
most of these cells also expressed the myeloid marker CD11b, sug-
gesting that infiltrated macrophages and/or resident microglial
cells multiply at the site of injury. Thus, accumulation of T cells
may indicate a continuous extravasation process, at least up to 7
days after neurotoxin exposure. Yet, the number of CD3+ T cells
within the SNpc was found to dramatically decrease shortly after
this accumulation (i.e., at day 9) (Supplemental Figure 5A), raising
the possibility that they could be rapidly eliminated by apoptotic
cell death due to regulatory mechanisms aimed at terminating the
immune response. To test this, we looked for morphological crite-
ria as well as molecular changes indicative of apoptosis in the entire
SNpc and ventral tegmental area of animals sacrificed 7 and 9 days
after systemic MPTP administration. Immunohistochemistry for
CD3 coupled with thionin counterstaining revealed the presence
of apoptotic cells characterized by shrinkage of cellular body, chro-
matin condensation, and presence of distinct, round, well-defined
chromatin clumps (Supplemental Figure 5B). Whereas apoptotic
cells were found in all MPTP-treated mice analyzed at day 7 and
9 (n = 10 and 5, respectively), they were not observed in saline-
injected controls. In few instances, these cells have retained some
CD3 staining, attesting their T cell origin (Supplemental Figure
5C). Additional evidence for T cell apoptosis was obtained from
double immunofluorescence staining for CD3 and activated cas-
pase-3. Thus, some CD3+ T cells were found to be positive for the
p17 fragment of activated caspase-3 (Supplemental Figure 5D).
Since a previous report indicated that the blood-brain barrier
(BBB) may be dysfunctional in PD patients (11), we examined
whether MPTP-associated BBB disruption could account for a
passive entry of lymphocytes into the brain. A time course of BBB
leakage from 6 hours to 7 days after MPTP administration was
determined by assessing serum albumin and immunoglobulin
extravasation. Our data reveal that MPTP induces a widespread
and transitory serum protein leakage (detectable at 6 hours but
absent from 12 hours after MPTP treatment), contrasting there-
fore with the cell type– and region-specific leukocyte extravasa-
tion process (Figure 3A). To further explore the mechanism of T
cell extravasation, we surveyed the expression of ICAM-1/CD54,
Mechanism of lymphocyte entry into the brain. (A) Immunofluores-
cent staining for serum albumin on brain sections (forebrain and hind-
brain levels are shown) from mice sacrificed 6 hours after the last
MPTP or saline injection. Patches of staining (arrows) are detected in
various brain areas at 6 hours following MPTP exposure but not after
saline treatment. The dashed line indicates the boundary between
the striatal and cortical areas. CTX, cortex; STR, striatum; RMC,
magnocellular part of the red nucleus; VTA, ventral tegmental area;
SNC, substantia nigra compacta; SNR, substantia nigra reticulata;
Aq, aqueduct of Sylvius; cp, cerebral peduncle. Scale bar: 300 μm.
(B) Immunostaining for CD54/ICAM-1 (red) on mesencephalic sec-
tions from MPTP-intoxicated GFP+ T cell–reconstituted Rag–/– mice.
The expression pattern of CD54 overlaps with that of the GFP+ T cell
infiltrate within the SNpc (dashed line) (left panel). At higher mag-
nification (right panel), CD54 expression is visible on GFP+ T cells
(arrows), blood vessels (asterisks), and branched glial cells (arrow-
heads). Scale bars: 300 μm (left panel); 10 μm (right panel).
186?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
an immunoglobulin-like cell adhesion molecule, involved in
leukocyte adherence and transendothelial migration at sites of
inflammation (12). Whereas CD54 was barely detectable in con-
trols (data not shown), we observed a marked increase in CD54
immunostaining within the SNpc at the site of T cell infiltration
following MPTP intoxication (Figure 3B). Blood vessels, glial cells,
and T cells were all intensely positive, indicating possible cross-
interactions among these cellular actors.
Mice deficient in cell-mediated immunity are more resistant to MPTP.
Investigations dedicated to unraveling the role of infiltrated T
cells in various models of neuronal injury have led to opposite
conclusions (13, 14). To determine whether T cell accumulation
in the SNpc following MPTP intoxication is beneficial or harm-
ful to DNs, we compared the effects of the toxin in T cell receptor
β chain mutant mice lacking mature T lymphocytes (Tcrb–/–) (15)
and in their WT littermates. We found that whereas only 72% of
the nigral TH+ DNs survived MPTP injection in WT mice, 91% of
these neurons survived in Tcrb–/– mice treated with a similar MPTP
regimen (Figure 4, A and B). Importantly, the decrease in MPTP-
induced DN loss in Tcrb–/– mice correlated with a reduction in the
number of infiltrated T cells to a level similar to that of saline-
injected animals (Figure 4C). Residual T cell infiltration in Tcrb–/–
mice may come from the small number (about 8% of the WT level)
of cells remaining in the thymus of these mutants (15).
Given the apparent harmful behavior of infiltrated T cells, we
next investigated whether CD8+ and/or CD4+ T cells could medi-
ate such a detrimental function. To that end, we intoxicated mice
homozygous for either a targeted mutation of Cd8a or Cd4, char-
acterized by a deficiency in functional Tc and Th, respectively (16,
17). Whereas MPTP induced a comparable level of DN cell death in
Cd8a–/– mice as in WT littermates, we observed a marked reduction
in TH+ cell loss in Cd4–/– mice (Figure 4, A and B). Interestingly, the
rate of DN survival in Cd4–/– mice was comparable to that of Tcrb–/–
animals, suggesting that most of the deleterious outcome associ-
ated with infiltrated T cells is mediated by the CD4+ Th subset. In
support of this idea, susceptible WT and Cd8a–/– mice exhibited a
similar number of infiltrated CD4+ T cells following MPTP expo-
sure (Figure 4C). However, there were no significant differences
in the extent of loss in striatal levels of dopamine, 3-4-dihydroxy-
phenylacetic acid (DOPAC), and homovanillic acid (HVA) between
Mice deficient in CD4+ T cells are more resistant to MPTP. (A) Quantification of TH+ DNs in the SNpc at day 7 after MPTP (4 × 18 mg/kg) or saline
treatment in WT, Tcrb–/–, Cd8a–/–, and Cd4–/– mice. Bars represent the mean number of total nigral TH+ DNs. Open symbols indicate saline-treated
animals and filled symbols indicate MPTP-treated animals. Each symbol represents 1 individual animal. A significant protection against MPTP-
induced DN cell loss is observed in Tcrb–/– and Cd4–/– mice but not in Cd8a–/– animals as compared with their WT littermates. *P < 0.05 compared
with MPTP-treated WT and Cd8a–/– mice; †P = 0.956 compared with MPTP-treated WT littermates (Tukey post-hoc analysis). Nissl+ cell counts
confirmed that TH+ cell loss was not a consequence of downregulated expression of TH (data not shown). (B) Representative photomicrographs
of mesencephalic sections immunostained for TH with Nissl counterstain from saline- or MPTP-treated WT and lymphocytic deficient mice. Insets
show Mac-1+ microglial cells (arrows) in the SNpc from the same animals. Scale bar: 300 μm; 100 μm (insets). (C) Quantification of the total num-
ber of infiltrated CD4+ and CD8+ T cells in the SNpc from MPTP-treated WT and lymphocytic deficient mice. MPTP-treated Cd8a–/– mice display
as many CD4+ T cells as MPTP-treated WT littermates. **P < 0.05 compared with saline-injected WT mice (Mann-Whitney U test). ††P < 0.001
compared with MPTP-treated WT and Cd8a–/– mice; #P < 0.001 compared with MPTP-treated WT mice (Dunn test). ND, not detected.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
mutant mice and their WT littermates after the administration
of MPTP, implying that striatal T cell infiltration is not a major
component of dopaminergic fibber injury (Table 1). Importantly,
reduced MPTP metabolism in mutant mice is not likely to account
for the observed neuroprotection, as striatal 1-methyl-4-phenylpyr-
idinium (MPP+) content after a single systemic MPTP injection did
not differ between genotypes (Table 2). Moreover, consistent with
the partial resistance of Tcrb–/– and Cd4–/– mice to MPTP, midterm
(2–7 days) microglial cell activation was almost completely abol-
ished in mutant mice (Figure 4B).
The Fas/FasL pathway is required for CD4 T cell–dependent DN toxicity.
The finding that CD4-deficient mice are partially protected against
MPTP-induced injury suggests that Th-mediated deleterious mech-
anisms are engaged and instrumental in DN demise. Cytokines,
including IFN-γ, and membrane bound ligands, such as FasL, rep-
resent 2 major classes of molecules mediating effector functions
of CD4 Th (e.g., macrophage activation). Interestingly, it has been
shown that IFN-γ was a contributing factor in the death of DNs
by regulating microglial activity (18). In fact, we found that IFN-γ
expression was upregulated in the ventral midbrain 2 days after
MPTP exposure, which coincided with the first wave of CD4+ T cell
brain infiltration (Supplemental Figure 6A). To analyze the putative
deleterious role of lymphocyte-derived IFN-γ, we passively trans-
ferred total splenocytes isolated from either WT or IFN-γ–deficient
mice into recipient Rag1–/– animals. Consistent with the deleterious
role of T cells as previously evidenced in Tcrb–/– mice, we noticed
that MPTP-induced injury in immunodeficient Rag1–/– mice was
significantly less severe than in WT littermates (Figure 5, A and B).
Importantly, this partial resistance was completely reversed when
spleen cells from either WT or Ifng–/– mice were passively transferred
prior to MPTP intoxication (Figure 5A). Thus, lymphocyte-derived
IFN-γ is not likely to be required for T cell–mediated DN cell death.
In support for this assertion, Ifng–/– mice and their WT littermates
were found to be equally sensitive to MPTP-induced DN cell death
using an identical intoxication paradigm (Supplemental Figure
6B). Therefore, we next considered the involvement of a Fas-based
mechanism. To test this, we took advantage of the FasL-mutated
generalized lymphoproliferative (gld) mice that bear a point muta-
tion in the extracellular domain of FasL (CD95L), resulting in a
dramatic decrease of affinity to its receptor Fas (19). Thus, we com-
pared the MPTP susceptibility of Rag1–/– recipients that received
spleen cells from either gld or C57BL/6J donors (designated gld rec.
Rag1–/– and WT rec. Rag1–/–, respectively). In WT rec. Rag1–/– mice,
MPTP caused as much DN loss as in WT animals (Figure 5, A–C).
In sharp contrast, both nonreconstituted Rag1–/– and gld rec. Rag1–/–
mice exposed to a similar dose of MPTP exhibited a significantly
smaller reduction in the number of nigral DNs (Figure 5, B and C).
The protection gained in gld rec. Rag1–/– mice was consistently with-
in a similar range to that observed in Cd4–/– animals (Figure 4A and
Figure 5B). Furthermore, this protective effect was not likely due to
altered extravasation potency of gld-derived splenocytes, as a simi-
lar number of brain infiltrated CD4+ T cells were observed between
MPTP-treated WT rec. Rag1–/– and gld rec. Rag1–/– animals (mean
number of CD4+ T cells in the SNpc ± SEM, 81.6 ± 22.5 and 95.5 ± 38.2,
respectively; P = 0.55, Mann-Whitney U test). Together, these results
suggest that CD4+ Th-mediated DN cell death requires the expres-
sion of a functional FasL but not IFN-γ.
Mounting evidence supports the notion that innate immunity sig-
nificantly contributes to dopaminergic neurodegeneration in PD
(1). By contrast, although altered cellular and humoral functions
have been reported in the peripheral immune system of PD patients,
the role of adaptive immunity in the pathogenesis of this disorder
has remained much more elusive (5, 20). Among these peripheral
immune changes, the significant increased ratio of CD8+ Tc to CD4+
Th and of IFN-γ–producing to IL-4–producing T cells suggests the
existence of a disease-associated shift to a Tc1/Th1-type immune
response, which may reflect and/or contribute to the harmful brain
inflammatory reaction (21). Nonetheless, a role for the cellular arm
of the adaptive immune system in neurodegeneration is curbed by
the fact that no clear demonstration of a prominent involvement of
leukocytes at the site of neuronal injury has been provided in PD.
In this study, we present evidence that peripheral T cells migrate
to and accumulate in the brain parenchyma during parkinsonism.
Importantly, we have shown that CD4+ but not CD8+ T cells are del-
eterious to DNs, which suggests that the adaptive immune system
may contribute to disease progression in PD.
It is now well established that the CNS is not only continuously
monitored by T cells (22) but also massively invaded by peripheral
leukocytes in neuropathological circumstances (23). In agreement
with the seminal but rather preliminary observation by McGeer
and colleagues, showing a substantial presence of CD8+ T cells
in the brain from one PD case (7), we now provide strong quan-
titative evidence from a large number of individuals that both
CD8+ and CD4+ T lymphocytes markedly accumulate in the SNpc
Striatal monoamine levels (pM/mg tissue)
WT (n = 5)
Tcrb–/– (n = 3)
Cd8a–/– (n = 3)
Cd4–/– (n = 3)
WT (n = 5)
Tcrb–/– (n = 4)
Cd8a–/– (n = 4)
Cd4–/– (n = 4)
82.3 ± 7.5
85.4 ± 12.7
84.6 ± 4.1
65.0 ± 8.9
15.4 ± 1.9
15.8 ± 3.0
16.0 ± 1.0
15.7 ± 2.3
10.1 ± 1.0
9.5 ± 1.6
9.9 ± 0.9
8.7 ± 1.4
1.7 ± 0.3
2.8 ± 0.4
4.9 ± 1.6
3.5 ± 0.5
1.8 ± 0.2
2.5 ± 0.3
2.2 ± 0.3
3.0 ± 0.5
3.2 ± 0.4
3.7 ± 0.5
3.2 ± 0.1
4.5 ± 0.6
Striatal dopamine, DOPAC, and HVA levels in WT, Tcrb–/–, Cd8a–/–, and
Cd4–/– mice at 7 days after the last MPTP injection do not differ (P > 0.05;
Kruskal-Wallis test) between groups. Data represent mean ± SEM for the
indicated number of mice. DOPAC, 3-4-dihydroxyphenylacetic acid; HVA,
Striatal MPP+ content after MPTP injection
7.09 ± 2.95
6.00 ± 2.55
7.89 ± 2.17
5.29 ± 2.16
Striatal MPP+ levels in WT, Tcrb–/–, Cd8a–/–, and Cd4–/– mice at 90 min-
utes after a single MPTP injection do not differ (P = 0.87; Kruskal-Wallis
test) between groups. Data represent mean ± SEM for 5 mice per group.
188?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
of PD patients. Consistent with a previous report (9), we found
that experimental lesion of the nigrostriatal pathway in MPTP-
intoxicated mice results in a similar brain accumulation of T cells.
Yet, brain accumulation of T cells following MPTP intoxication
was not restricted to the SNpc. Indeed, other catecholaminergic
areas known to be moderately injured following MPTP treatment
(24) were also invaded, albeit to a lower extent. This extravasation
of lymphocytes is not likely to be a generalized nonspecific leu-
kocyte response, as (a) other leukocyte subsets, including B cells
and NK cells, were not detected in the lesioned SNpc, and (b) an
increased occurrence of T cells was not observed in nonlesioned
brain areas, suggesting that this process does not simply reflect an
overall enhancement of lymphocyte patrolling. Altogether, these
findings indicate that, as with the glial cell reaction previously
described, T cell brain infiltration in PD most certainly represents
a secondary and highly regulated pathogenic event associated with
neuronal cell death. Such a cellular and site-specific brain immune
response is not likely to be caused
by a massive disruption of the BBB,
for which little evidence exists in PD
(25, 26) and which remains contro-
versial in MPTP-intoxicated mice
(27, 28). We showed a widespread
BBB leakage that occurred shortly
(6 hours) after neurotoxin expo-
sure and was rapidly resolved (by 12
hours after MPTP treatment), well
before lymphocytic invasion. Taken
together, these findings argue that
lymphocyte brain infiltration is not
a passive phenomenon. Instead,
molecular and cellular changes
associated with neuronal injury are
likely to regulate this site-specific
brain recruitment of T cells. Pos-
sible mechanisms may involve early
microglial cell activation and innate
neuroinflammatory processes that
could modify the local microenvi-
ronment. In line with this, we found
an upregulated expression of the
ICAM-1 adhesion molecule on both
capillaries and glial cells, which may
participate in the attachment of leu-
kocytes to the vascular endothelium
and their diapedesis (29). Interest-
ingly, a similar ICAM-1 overex-
pression was recently described in
both the SN of patients with PD
and MPTP-intoxicated monkeys
(30). Although the level of T cell
brain accumulation was found to
be higher in the disease model than
in PD patients, one has to consider
that the human disease progresses
over several decades, whereas it only
lasts 1 to 2 weeks in mouse model.
Moreover, investigations performed
on human postmortem tissue most
often address late pathomecha-
nisms and can not therefore predict the intensity of the lympho-
cyte infiltration process, which might be greater during earlier
stages of the disease. Given that incidental Lewy body disease is
considered by some authors to be a presymptomatic form of PD
(31), it might be valuable to explore such lymphocytic invasion in
those patients. Finally, it is worth noting that the distribution of T
cell accumulation within the SNpc from PD patients is much more
heterogeneous than that in MPTP mice. Indeed, in few instances,
as shown in Figure 1, infiltrating T cells in brains of patients of
with PD were found to be clustered around the few remaining
pigmented DNs, whereas they were virtually absent in advanced
depigmented areas. Such distribution suggests that the number of
T cells reaching the target neurons is conceivably substantial and
strengthens their possible involvement in DN cell demise.
A major contribution of our study is the demonstration that
infiltration of T cells into the brain actively participates in DN
degeneration. A decreased vulnerability of DNs to MPTP toxicity
FasL, but not IFN-γ, is required for T cell–mediated DN toxicity in MPTP mouse. (A) Quantification of TH+
DNs in the SNpc at day 7 after MPTP (4 × 18 mg/kg) or saline treatment in WT and Rag1–/– mice recon-
stituted with spleen cells from either C57BL/6 inbred or Ifng–/– donors. All groups of animals are equally
sensitive to MPTP-induced DN injury. *P < 0.05 compared with their saline counterparts; †P < 0.05
compared with their saline counterparts but not different from MPTP-treated WT mice (Tukey post-
hoc analysis). (B) Quantification of TH+ DNs in the SNpc at day 7 after MPTP (4 × 18 mg/kg) or saline
treatment in nonreconstituted Rag1–/– mice (non rec. Rag1–/–) and in Rag1–/– mice reconstituted with
spleen cells from either C57BL/6 or gld donors (WT rec. Rag1–/– and gld rec. Rag1–/–, respectively).
Nonreconstituted Rag1–/– mice and gld rec. Rag1–/– mice are partially protected against MPTP-induced
DN loss as compared with WT rec. Rag1–/– animals. **P < 0.01 compared with MPTP-intoxicated WT
rec. Rag1–/– mice (Tukey post-hoc analysis). (A and B) Open symbols indicate saline-treated animals
and filled symbols indicate MPTP-treated animals. Each symbol represents 1 individual animal. Bars
represent the mean number of total nigral TH+ DNs. (C) Representative photomicrographs of mesen-
cephalic sections immunostained for TH with Nissl counterstain from saline- or MPTP-treated WT rec.
Rag1–/– and gld rec. Rag1–/– mice. Scale bar: 300 μm.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
associated with the removal of mature T lymphocytes was achieved
by 2 different genetic immune defects (Rag1–/– and Tcrb–/–), thus
supporting a role for the cellular arm of the adaptive immune sys-
tem in experimental parkinsonism. Our results are in agreement
with a recent study, showing a similar neuropathological improve-
ment in MPTP-intoxicated SCID mice (32). Notably, our data fur-
ther reveal that different T cell subsets do not contribute equally to
DN cell death. Indeed, while the predominance of infiltrated CD8+
over CD4+ T cells was consistently observed both in PD patients
and MPTP-intoxicated mice, removal of the CD8+ T cell subset in
Cd8a–/– mice did not mitigate MPTP injury. In sharp contrast, the
Th deficiency achieved in Cd4–/– mice conferred as much neuropro-
tection as in Rag1–/– and Tcrb–/– animals, suggesting that CD4+ T
cells mediate most if not all the deleterious activity associated with
the adaptive immune response.
Since infiltrated T cells highly express ICAM-1 as well as lym-
phocyte function-associated antigen-1 (LFA-1) and CD44 (9, 30),
we believe that most of them could be recruited from an activated/
memory population. A mechanism of peripheral leukocyte activa-
tion secondary to MPTP intoxication has recently been proposed
(32). In this model, nitrotyrosine modification within α-synuclein
(α-Syn) promotes an antigen-specific T cell response in draining
lymph nodes. After migrating into the injured brain areas, these
antigen-specific T cells could undergo further activation and
promote microglial cell–dependent neurodegeneration through
cytokine release. Although the differential contribution of CD8+
versus CD4+ T cells in this deleterious immune response was not
addressed by the authors, their prediction analysis of α-Syn–specif-
ic T cell epitopes suggested a significant potential for CD4+ T cells
of mice expressing IAk or IAb MHC class II molecules to respond to
nitrated epitopes of α-Syn. These data, taken in conjunction with
our present findings, support a model in which MPTP-induced
brain antigen modification most likely generates a secondary and
harmful Th response contributing to DN degeneration.
Looking at the cytotoxic mechanism mediated by the CD4+ T
cell response following nigrostriatal pathway injury, we found
that T cell expression of FasL but not IFN-γ was required. It has
been shown that IFN-γ is critical in microglial-mediated loss of
DNs in MPTP-intoxicated mice (18). Yet, using 2 different experi-
mental approaches, i.e., total and cell-specific IFN-γ deletion,
we were unable to show a major role for this proinflammatory
cytokine in DN cell demise. While the reason for such a discrep-
ancy is still not clear, a lack of IFN-γ expression in our disease
model could not be incriminated. Indeed, in agreement with a
previous report (33), we were able to detect a rise in IFN-γ follow-
ing MPTP-induced nigrostriatal pathway injury. Whether differ-
ent treatment protocols lead to distinct involvement of IFN-γ in
disease models will need further clarification.
The requirement of FasL in CD4+ T cell–mediated cytotoxicity is
consistent with the finding that Fas-deficient mice display attenu-
ated MPTP-induced DN loss (34), although another study has
come to a different conclusion (35). Whereas, in the context of
antigen presentation, the Fas/FasL pathway has been implicated in
the deletion of activated macrophages, thereby contributing to the
resolution of inflammation (36), recent evidence suggests that this
pathway may instead induce proinflammatory cytokine responses
in tissue macrophages (37). CD4+ Th FasL-mediated activation of
microglial cells could therefore participate in the inflammatory
reaction and DN degeneration. T cell–derived FasL may also medi-
ate inflammatory responses in astrocytes, which are known to be
particularly resistant to Fas-mediated cell death and express pro-
inflammatory cytokines and chemokines upon Fas ligation (38).
In line with this, Fas expression has been shown to be upregulated
on these glial cells both in PD patients and in the MPTP model
(34, 39). Alternatively, infiltrated CD4+ T cells may also induce DN
cell demise through cell-cell contact. Such a mechanism has previ-
ously been shown in vitro, in which antigen primed CD4+ or CD8+
T cells induced neuronal death independently of antigen presen-
tation and involved cell surface expression of FasL by activated
T cells (40). Further investigations will be required to determine
whether or not CD4+ T cell–mediated cytotoxicity relies strictly on
MHC-dependent antigen presentation.
In our disease model, the finding that harmful lymphocyte
response is CD4 T cell–dependent but IFN-γ–independent raises
important but still unresolved concerns about the phenotypic
characteristics of these CD4 T cells. Although these observations
would not favor the contribution of a Th1-type response, one
can not exclude the possibility that CD4+ Th1 cells may mediate
cytotoxicity independently of IFN-γ as previously reported (40, 41).
In particular, the role of other proinflammatory Th cytokines, such
as TNF-α in T cell–mediated DN cell death, should also be consid-
ered as a potential deleterious mechanism, even though the role of
this factor is still debated (42–44). Finally, another tantalizing pos-
sibility could be the involvement of an alternative inflammatory
CD4 T cell population, namely, the recently described, Th17 cells.
A growing body of evidence indicates that Th17 cells play a criti-
cal function not only in protection against microbial challenges
but also in many organ-specific autoimmune diseases, including
multiple sclerosis and its disease model experimental autoimmune
encephalomyelitis (45). In line with this, it has recently been shown
that Th17 cells have the potential of killing neurons, probably
through the granzyme B cytolytic enzyme system (46). Whether
such a CD4 T cell subset is implicated in parkinsonism as well
merits further considerations.
It is worth noting that the CD4+ T cell response to neurodegenera-
tive processes can differently affect disease outcome. Indeed, while
in the MPTP mouse model of PD, CD4+ T cells are likely harmful to
DNs (Figure 4A and ref. 32), it has recently been documented that
this T cell population can provide supportive neuroprotection in
animal model of inherited amyotrophic lateral sclerosis (ALS), by
stimulating the trophic properties of glia (47). Interestingly, such
pathological improvement can be recapitulated by delivering activat-
ed regulatory or effector T cells to ALS mice but not by Copolimer-1
immunization, indicating that antigen-driving Th2/Th3 responses
are impaired in this animal model (48). Thus, different neurode-
generative contexts could result in distinct T cell responses, which
in turn may positively or negatively influence disease progression.
Undoubtedly, a better phenotypic characterization of these CD4+
T cell subsets should improve our understanding of the role of the
adaptive immune system in various neurodegenerative conditions.
In summary, we have shown that peripheral T cells infiltrate
the brain parenchyma at the site of neuronal injury both in PD
patients and in experimental parkinsonism. We have demonstrat-
ed that this cell-mediated immune response contributes to DN cell
degeneration through a CD4+ T cell–dependent Fas/FasL cytotoxic
pathway. Our results, together with recent investigations (32), pro-
vide further rationale for targeting the adaptive arm of the immune
system in PD. Therapeutic strategies may involve developing vac-
cines for antigens that promote cell-mediated antiinflammatory
responses (49, 50) as well as blocking the migration of immune
190?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
cells across the BBB (51). Besides providing potential neuropro-
tective benefits for the remaining DNs, these immunomodulatory
strategies may also be of interest for cell transplantation therapies,
as long-term survival of grafted DNs, which eventually undergo
PD-related pathological changes over time, could be jeopardized
by such an immune-related hostile microenvironment (52).
Human samples. The study was performed on autopsy brainstem tissue (Hôpital
de la Salpêtrière) from 8 control subjects and 14 parkinsonian patients, which
were well-characterized clinically and neuropathologically. PD patients and
control subjects did not differ significantly in terms of their mean age at death
(PD patients, 77.85 ± 2.11 years of age; controls, 76.62 ± 3.72 years of age;
P = 0.76, Student’s t test; mean ± SEM) or the mean interval from death to the
freezing of tissue (PD patients, 40.55 ± 3.80 hours; controls, 28.10 ± 7.08 hours;
P = 0.11, Student’s t test; mean ± SEM). Experiments using human postmor-
tem material were approved by the Comité de Protection des Personnes review
board (Ile de France 1, Paris, France). Due to limited accessibility to tissue
sections, different numbers of patients were analyzed with respect to each
leukocyte marker. Tissue was fixed in formaldehyde and then embedded in
paraffin. Paraffin-embedded tissues were cut in serial 8-μm thick slices on a
microtome, and sections were recovered on SuperFrost Plus slides (Kindler
O GmbH), before incubation at 56°C for 3 days. Human tonsil slices (Dako)
were used as tissue control for lymphoid marker immunohistochemistry.
Animals. Ten- to twelve-week-old male C57BL/6J mice, weighing 25–30 g
(CERJ) were used. The following strains were obtained from The Jackson Lab-
oratory: B6.129S6-Cd4tm1Knw/J, B6.129S2-Cd8atm1Mak/J, B6.129P2-Tcrbtm1Mom/J,
B6.129S7-Rag1tm1Mom/J, B6.129S7-Ifngtm1Ts/J, B6Smn.C3-Faslgld/J, and
corresponding parental WT inbred C57BL/6J. Mice were kept in a tem-
perature-controlled room (23°C ± 1°C) under a 12-hour light/dark cycle
and had ad libitum access to food and water. All animals were further
genotyped after their sacrifice according to The Jackson Laboratory pro-
tocols. GFP+ cells were isolated from double Tie2-Cre+/ZEG+ transgenic
mice (designated GFP-Tg), in which the GFP reporter gene is expressed in
early hematopoietic cell precursors (53). Animal handling was carried out
according to ethical regulations and guidelines (Guide for the care and use of
laboratory animals. NIH publication no. 85-23. Revised 1985) and the Euro-
pean Communities Council Directive 86/609/EEC. Experiments using ver-
tebrates were approved by the Services Vétérinaires de Paris.
Treatment and tissue preparation. Groups of mice received 4 i.p. injections of
MPTP-HCl (20 mg/kg, except where otherwise stated) at 2-hour intervals
and were sacrificed from 6 hours to 21 days after the last injection. Con-
trol mice received saline solution only. For immunohistochemistry, mice
were euthanized with 100 mg/kg pentobarbital and transcardially perfused
first with 50 ml of heparin solution (5 U/ml) and then with 100 ml of 4%
paraformaldehyde. Brain and spleen free-floating sections were prepared
as described elsewhere (54). For HPLC analysis of MPP+ levels, brains were
rapidly removed from the skull and striata were dissected on humidified
filters at 4°C. Tissues were then immersed in appropriate buffer.
Passive transfers. Single-cell suspensions were prepared from spleen and/or
lymph nodes isolated from Tie2-Cre+/ZEG+, gld, Ifng–/–, or C57BL/6J mice.
GFP+ T and B cells were purified with a cell magnetic separator (MACS,
Miltenyi Biotech) according to the manufacturer’s procedure, using anti-
TCRβ (H57-597), anti-CD3ε (145-2C11), anti-CD4 (H-129.19), anti-CD8α
(53-6.7), and anti-B220 PE-conjugated antibodies (all from BD Biosciences
— Pharmingen) and anti–PE-conjugated MicroBeads (Miltenyi Biotech).
Cells labeled with MicroBeads were retained on the MACS Column while
unlabeled cells passed through. The column was removed from the separa-
tor, and the retained cells were eluted as the enriched, positively selected
cell fraction. The enrichment of cells was confirmed by flow cytometry
analysis using a FACSCalibur (Becton Dickinson) (e.g., more than 95% for
T cells; Supplemental Figure 2A). Recipient Rag1–/– mice received a single
i.v. injection of either 107 GFP+ T cells or 2.5 107 GFP+ B cells or 2 injections,
4 hours apart, of 107 total splenocytes (either gld, Ifng–/–, or WT) in 0.2 ml
phosphate buffered saline solution. Reconstitutions of GFP-expressing T
and B lymphocytes in Rag1–/– recipient mice were checked by FACS analysis
of blood samples collected from reconstituted Rag1–/– recipient mice prior
to MPTP treatment or from lymph nodes after the mice were sacrificed
(Supplemental Figure 2C). Three to four weeks after transfer, mice were
intoxicated with MPTP or received an equal volume of saline solution.
Immunohistochemistry. Human sections were first deparaffinized in 2
changes of xylene for 5 minutes each. They were hydrated in 2 changes
of 100%, 95%, then 75% ethanol and rinsed in distilled water. Antigen
demasking was performed at 90°C–95°C for 30 minutes in citrate buffer,
pH 6, (for CD8, CD20, CD79a, and CD57 staining) or EDTA buffer, pH 8,
for CD4 staining. Sections were then allowed to cool down for 20 minutes
before being rinsed in 0.25 M Tris buffer.
Human sections were pretreated with 0.01% H2O2/20% methanol, fol-
lowed by 0.5% Triton and by normal goat serum (1:30; 30 minutes). They
were then incubated at 4°C for 48 hours with anti-CD8 (1:50; Dako),
anti-CD4 (1:20; Novocastra), anti-CD79α (1:100; Novocastra), anti-CD20
(1:100; L26, Novocastra), or anti-CD57 (1:50; Zymed).
Immunohistochemical staining on mouse brain sections was per-
formed as previously described (51). The following primary antibodies
were used: anti-TH (1:1,000; Peel Freez Biochemicals), anti–Mac-1/CD11b
(1:250; Serotec), anti-GFAP (astrocytes, 1:5,000; Dako), anti-GFP (1:1,000;
Invitrogen), anti-CD3 (1:1,000; Serotec), anti-CD8 (1:100; Serotec), or anti-
CD4 (1:100; Serotec). Staining was revealed by the ABC method (Vector
Laboratories) with 3,3ʹ-diaminobenzidine (DAB) as the peroxidase sub-
strate. Mouse sections were counterstained with thionin solution (Nissl
stain), whereas human sections were counterstained with H&E solution.
For double-staining experiments, brain sections were simultaneously
incubated with 2 primary antibodies developed in different species: anti-
TH (1:1,000; Peel Freez Biochemicals), anti-GFP (1:1,000; Invitrogen),
anti–Glut-1 (1:300; Santa Cruz Biotechnology Inc.), anti-PCNA (1:500;
Dako), anti–Mac-1 (1:250; Serotec), anti-GFAP (1:5000; Dako), anti-CD3
(1:1,000; Serotec), anti-CD8 (1:100; Serotec), anti-CD4 (1:100; Serotec),
anti–ICAM-1 (1:50; Serotec), anti-albumin (1:1,000; Cappel, MP Biomedi-
cals), and anti-p17 caspase-3 (R&D Systems). Sections were then incubated
in specific CY3- or FITC-conjugated secondary antibodies (Jackson Immu-
noResearch Laboratories Inc.) at 1:250 dilution for 90 minutes at room
temperature. For PCNA staining, sections were pretreated in 30% ethanol
for 2 minutes and 70% ethanol at –20°C for 20 minutes. Hoechst nuclear
staining was performed as previously described (53).
Electron microscopy. Ultrastructural analysis of T cells in the SNpc was
performed as previously described with minor modifications (55). In brief,
small blocks of mesencephalon containing the SNpc were fixed in a mixture
of 4% paraformaldehyde and 2.5% glutaraldehyde. Sections 50-μm thick
were cut and labeled for CD8 as described above. Following identification
of CD8+ cells, small areas of the sections were then excised and post-fixed in
1% osmium tetroxide for 30 minutes, rinsed in distilled water, dehydrated
in a graded series of ethanol solutions, and embedded in Epon. Ultrathin
(70 nm) sections were cut, counterstained with conventional techniques,
and analyzed with a JEOL 1200EX II electron microscope at 80 kV.
Measurement of striatal dopamine. Striatal levels of dopamine and its metabo-
lites (DOPAC, HVA) were determined by HPLC as previously reported (42).
Measurement of striatal MPP+. Mice were killed 90 minutes after 1 i.p.
injection of 30 mg/kg MPTP-HCl, and their striata were recovered and
processed for HPLC, using UV detection (295-nm wavelength) to measure
MPP+ as described elsewhere (56).
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
Antibody array. C57BL/6J mice (n = 5 per group) were intoxicated with
MPTP and sacrificed from 12 hours to 7 days after the last injection. Con-
trols received an equal volume of saline solution. The expression level of
various cytokines in protein homogenates prepared from the ventral mes-
encephalon was analyzed using a mouse antibody array glass chip (RayBio
Mouse Cytokine Antibody Array G series 1000, RayBiotech Inc.). Incuba-
tion and washes were done according to the manufacturer’s instructions.
Briefly, chip arrays were blocked at room temperature for 30 minutes
before being incubated with 50–100 μl of each sample at room tempera-
ture for 2 hours. Glass chips were then washed and incubated with biotin-
conjugated primary antibody and Alexa Fluor 555–conjugated streptavidin
according to the manufacturer’s instructions. Fluorescence detection and
analysis were performed using an Axon GenePix laser scanner.
Image and data analysis. DAB-immunostained sections were analyzed by
bright-field microscopy, using a Leitz microscope equipped with image
analysis software (Mercator, ExploraNova). TH+ and Nissl+ cell bodies
were quantified stereologically on regularly spaced sections covering the
whole SNpc using the VisioScan stereology tool. The investigator per-
forming the quantification was blinded to the treatment and genotype
groups during the analysis. Fluorescent sections were analyzed on a Zeiss
Axioplan 2 using ExploraNova FluoUp 1.0 software.
Statistics. All values are expressed as the mean ± SEM. Differences in means
between 2 groups were analyzed using 2-tailed Student’s t test or, when
data were not normally distributed, with a nonparametric Mann-Whitney
U test. Differences in means among multiple data sets were analyzed using
1- or 2-way ANOVA with time, treatment, or genotype as the independent
factors. When ANOVA showed significant differences, pair-wise compari-
sons between means were tested by Tukey post-hoc analysis. When data
were not normally distributed, ANOVA on ranks was used (Kruskal-Wal-
lis test followed by pairwise comparison using Dunn test). In all analyses,
P values of less than 0.05 were considered significant (SigmaStat Statistical
Software, Jandel Scientific).
We thank C. Combadière and C. Lobsiger for helpful discussions,
V. Sazdovitch for assistance in collecting postmortem material,
and T. Welte for providing Tie2-Cre+/ZEG+ double transgenic
mice. This work was supported by grants from The Michael J.
Fox Foundation (S. Hunot and E.C. Hirsch), Fondation pour la
Recherche sur le Cerveau (S. Hunot), Fondation France Parkin-
son (V. Brochard), and the German Academic Exchange Service
(D. Alvarez-Fischer). R.A. Flavell is an investigator at the Howard
Hughes Medical Institute. E.C. Hirsch and S. Hunot are investiga-
tors at the Centre National de la Recherche Scientifique (CNRS).
Received for publication June 11, 2008, and accepted in revised
form November 12, 2008.
Address correspondence to: Stéphane Hunot or Etienne C.
Hirsch, INSERM UMR 679, Hôpital de la Salpêtrière, 47 Bd de
l’Hôpital, 75013 Paris, France. Phone: 33-14-21-62-172; Fax:
33-14-42-43-658; E-mail: firstname.lastname@example.org (S. Hunot).
Phone: 33-14-21-62-202; Fax: 33-14-42-43-658; E-mail: etienne.
email@example.com (E.C. Hirsch).
1. Dauer, W., and Przedborski, S. 2003. Parkinson’s dis-
ease: mechanisms and models. Neuron. 39:889–909.
2. Chen, H., et al. 2005. Nonsteroidal antiinflamma-
tory drug use and the risk for Parkinson’s disease.
Ann. Neurol. 58:963–967.
3. Wahner, A.D., Bronstein, J.M., Bordelon, Y.M.,
and Ritz, B. 2007. Nonsteroidal anti-inflamma-
tory drugs may protect against Parkinson disease.
4. Nagatsu, T., Mogi, M., Ichinose, H., and Togari,
A. 2000. Changes in cytokines and neurotroph-
ins in Parkinson’s disease. J. Neural Transm. Suppl.
5. Czlonkowska, A., Kurkowska-Jastrzebska, I.,
Czlonkowski, A., Peter, D., and Stefano, G.B. 2002.
Immune processes in the pathogenesis of Parkin-
son’s disease — a potential role for microglia and
nitric oxide. Med. Sci. Monit. 8:RA165–RA177.
6. Whitton, P.S. 2007. Inflammation as a causative
factor in the aetiology of Parkinson’s disease. Br. J.
7. McGeer, P.L., Itagaki, S., Akiyama, H., and McGeer,
E.G. 1988. Rate of cell death in parkinsonism indi-
cates active neuropathological process. Ann. Neurol.
8. Hunot, S., et al. 1999. FcεRII/CD23 is expressed in
Parkinson's disease and induces, in vitro, produc-
tion of nitric oxide and tumor necrosis factor-α in
glial cells. J. Neurosci. 19:3440–3447.
9. Kurkowska-Jastrzebska, I., et al. 1999. The inflam-
matory reaction following 1-methyl-4-phenyl-1,2,3,
6-tetrahydropyridine intoxication in mouse. Exp.
10. Janeway, C.A., Jr., and Travers, P. 1997. Immunobiol-
ogy: the immune system in health and disease. 3rd edition.
Garland Publishing Inc. New York, New York, USA.
11. Kortekaas, R., et al. 2005. Blood-brain barrier dys-
function in parkinsonian midbrain in vivo. Ann.
12. Engelhardt, B. 2006. Molecular mechanisms
involved in T cell migration across the blood-brain
barrier. J. Neural Transm. 113:477–485.
13. Serpe, C.J., Kohm, A.P., Huppenbauer, C.B., Sand-
ers, V.M., and Jones, K.J. 1999. Exacerbation of
facial motoneuron loss after facial nerve transec-
tion in severe combined immunodeficient (scid)
mice. J. Neurosci. 19:RC7.
14. Chen, Z., Ljunggren, H.G., Zhu, S.W., Winblad, B.,
and Zhu, J. 2004. Reduced susceptibility to kainic
acid-induced excitotoxicity in T-cell deficient CD4/
CD8 (–/–) and middle-aged C57BL/6 mice. J. Neu-
15. Mombaerts, P., et al. 1992. Mutations in T-cell
antigen receptor genes alpha and beta block thy-
mocyte development at different stages. Nature.
16. Fung-Leung, W.P., et al. 1991. CD8 is needed for devel-
opment of cytotoxic T-cells but not helper T-cells.
17. Rahemtulla, A., et al. 1991. Normal development
and function of CD8+ cells but markedly decreased
helper cell activity in mice lacking CD4. Nature.
18. Mount, M.P., et al. 2007. Involvement of interferon-γ
in microglial-mediated loss of dopaminergic neurons.
J. Neurosci. 27:3328–3337.
19. Takahashi, T., et al. 1994. Generalized lymphopro-
liferative disease in mice, caused by a point muta-
tion in the Fas ligand. Cell. 76:969–976.
20. Hunot, S., and Hirsch, E.C. 2003. Neuroinflamma-
tory processes in Parkinson’s disease. Ann. Neurol.
21. Baba, Y., Kuroiwa, A., Uitti, R.G., Wszolek, Z.K.,
and Yamada, T. 2005. Alterations of T-lymphocyte
populations in Parkinson disease. Parkinsonism
Relat. Disord. 11:493–498.
22. Cose, S., Brammer, C., Khanna, K.M., Masopust,
D., and Lefrançois, L. 2006. Evidence that a signifi-
cant number of naive T cells enter non-lymphoid
organs as part of a normal migratory pathway. Eur.
J. Immunol. 36:1423–1433.
23. Togo, T., et al. 2002. Occurrence of T cells in the
brain of Alzheimer’s disease and other neurological
diseases. J. Neuroimmunol. 124:83–92.
24. German, D.C., et al. 1996. The neurotoxin MPTP
causes degeneration of specific nucleus A8, A9 and
A10 dopaminergic neurons in the mouse. Neurode-
25. Farkas, E., De Jong, G.I., de Vos, R.A., Jansen Steur,
E.N., and Luiten, P.G. 2000. Pathological fea-
tures of cerebral cortical capillaries are double in
Alzheimer’s disease and Parkinson’s disease. Acta
26. Haussermann, P., Kuhn, W., Przuntek, H., and Mull-
er, T. 2001. Integrity of the blood-cerebrospinal
fluid barrier in early Parkinson’s disease. Neurosci.
27. Adams, J.D., Jr., Klaidman, L.K., Odunze, I.N., and
Johannenssen, J.N. 1991. Effects of MPTP on the
cerebrovasculature. Int. J. Dev. Neurosci. 9:155–159.
28. Zhao, C., Ling, Z., Newman, M.B., Bhatia, A., and
Carvey, P.M. 2007. TNF-α knockout and minocy-
cline treatment attenuates blood-brain barrier leak-
age in MPTP-treated mice. Neurobiol. Dis. 26:36–46.
29. Springer, T.A. 1994. Traffic signals for lymphocyte
recirculation and leukocyte emigration: the multi-
step paradigm. Cell. 76:301–314.
30. Miklossy, J., et al. 2005. Role of ICAM-1 in persist-
ing inflammation in Parkinson disease and MPTP
monkeys. Exp. Neurol. 197:275–283.
31. Jenner, P., and Olanow, C.W. 1998. Understand-
ing cell death in Parkinson’s disease. Ann. Neurol.
32. Benner, E.J., et al. 2008. Nitrated α-synuclein
immunity accelerates degeneration of nigral dopa-
minergic neurons. PloS ONE. 3:e1376.
33. Ciesielska, A., et al. 2003. Dynamics of expression of
the mRNA for cytokines and inducible nitric syn-
thase in a murine model of the Parkinson’s disease.
Acta Neurobiol. Exp. (Wars.) 63:117–126.
34. Hayley, S., et al. 2004. Regulation of dopaminergic
loss by Fas in a 1-methyl-4-phenyl-1,2,3,6-tetrahy-
dropyridine model of Parkinson’s disease. J. Neurosci.
35. Landau, A.M., et al. 2005. Defective Fas expression
192?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
exacerbates neurotoxicity in a model of Parkinson’s
disease. J. Exp. Med. 202:575–581.
36. Ashany, D., et al. 1995. Th1 CD4+ lymphocytes
delete activated macrophages through the Fas/
APO-1 antigen pathway. Proc. Natl. Acad. Sci. U. S. A.
37. Park, D.R., et al. 2003. Fas (CD95) induces pro-
inflammatory cytokine responses by human
monocytes and monocyte-derived macrophages.
J. Immunol. 170:6209–6216.
38. Choi, C., and Benveniste, E.N. 2004. Fas ligand/
fas system in the brain: regulator of immune and
apoptosis responses. Brain Res. Rev. 44:65–81.
39. Ferrer, I., Blanco, R., Cutillas, B., and Ambrosio, S.
2000. Fas and Fas-L expression in Huntington’s
disease and Parkinson’s disease. Neuropathol. Appl.
40. Giuliani, F., Goodyer, C.G., Antel, J.P., and Yong,
V.W. 2003. Vulnerability of human neurons to T
cell-mediated cytotoxicity. J. Immunol. 171:368–379.
41. Ju, S.T., Cui, H., Panka, D.J., Ettinger, R., and Mar-
shak-Rothstein, A. 1994. Participation of target Fas
protein in apoptosis pathway induced by CD4+
Th1 and CD8+ cytotoxic T cells. Proc. Natl. Acad.
Sci. U. S. A. 91:4185–4189.
42. Rousselet, E., et al. 2002. Role of TNF-alpha recep-
tors in mice intoxicated with the parkinsonian
toxin MPTP. Exp. Neurol. 177:183–192.
43. Ferger, B., Leng, A., Mura, A., Hengerer, B., and
Feldon, J. 2004. Genetic ablation of tumor necro-
sis factor–alpha (TNF-alpha) and pharmacological
inhibition of TNF-synthesis attenuates MPTP tox-
icity in mouse striatum. J. Neurochem. 89:822–833.
44. Sriram, K., Miller, D.B., and O’Callaghan, J.P. 2006.
Minocycline attenuates microglial activation but
fails to mitigate striatal dopaminergic neurotoxici-
ty: role of tumor necrosis factor-alpha. J. Neurochem.
45. Zhu, J., and Paul, W.E. 2008. CD4 T cells: fates,
functions, and faults. Blood. 112:1557–1568.
46. Kebir, H., et al. 2007. Human Th17 lymphocytes
promote blood-brain barrier disruption and
central nervous system inflammation. Nat. Med.
47. Beers, D.R., Henkel, J.S., Zhao, W., Wang, J., and
Appel, S.H. 2008. CD4+ T cells support glial neuro-
protection, slow disease progression, and modify glial
morphology in an animal model of inherited ALS.
Proc. Natl. Acad. Sci. U. S. A. 105:15558–15563.
48. Banerjee, R., et al. 2008. Adaptive immune neu-
roprotection in G93A-SOD1 amyotrophic lateral
sclerosis mice. PLoS ONE. 3:e2740.
49. Benner, E.J., et al. 2004. Therapeutic immunization
protects dopaminergic neurons in a mouse model
of Parkinson’s disease. Proc. Natl. Acad. Sci. U. S. A.
50. Reynolds, A.D., et al. 2007. Neuroprotective activi-
ties of CD4+CD25+ regulatory T cells in an ani-
mal model of Parkinson’s disease. J. Leukoc. Biol.
51. Luster, A.D., Alon, R., and von Andrian, U.H. 2005.
Immune cell migration in inflammation: pres-
ent and future therapeutic targets. Nat. Immunol.
52. Braak, H., and Del Tredici, K. 2008. Assessing fetal
nerve cell grafts in Parkinson’s disease. Nat. Med.
53. Welte, T., et al. 2003. STAT3 deletion during
hematopoiesis causes Crohn’s disease-like patho-
genesis and lethality: A critical role of STAT3
in innate immunity. Proc. Natl. Acad. Sci. U. S. A.
54. Hunot, S., et al. 2004. JNK-mediated induction of
cyclooxygenase 2 is required for neurodegeneration
in a mouse model of Parkinson’s disease. Proc. Natl.
Acad. Sci. U. S. A. 101:665–670.
55. Hunot, S., et al. 1997. Nuclear translocation of NF-
kappaB is increased in dopaminergic neurons of
patients with Parkinson disease. Proc. Natl. Acad.
Sci. U. S. A. 94:7531–7536.
56. Liberatore, G.T., et al. 1999. Inducible nitric oxide
synthase stimulates dopaminergic neurodegenera-
tion in the MPTP model of Parkinson disease. Nat.