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γδ T Cells Regulate the Extent and Duration
of Inflammation in the Central Nervous
System by a Fas Ligand-Dependent
Mechanism
Eugene D. Ponomarev and Bonnie N. Dittel
J Immunol 2005; 174:4678-4687; ;
doi: 10.4049/jimmunol.174.8.4678
http://www.jimmunol.org/content/174/8/4678
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References
The Journal of Immunology
␥␦ T Cells Regulate the Extent and Duration of Inflammation
in the Central Nervous System by a Fas Ligand-Dependent
Mechanism1
Eugene D. Ponomarev and Bonnie N. Dittel2
A
rare, but diverse, population of lymphocytes, ␥␦ T cells
have been shown to regulate a variety of immune responses, including those associated with autoimmunity
(1–3). The prevailing view is that ␥␦ T cells bridge the innate and
adaptive immune responses, primarily by exerting specific functions, which are determined by the tissue and local microenvironment in which they reside (3). Thus, they have been shown to
perform multiple functions during the immune response, with the
ability to both reduce and exacerbate inflammation (1–3). ␥␦ T
cells constitute a small proportion of circulating lymphocytes, and
during infectious disease or autoimmunity they have been shown
to migrate into the injured site and are thought to regulate the
nature of the inflammatory response (1–3).
Mechanisms of immunoregulatory functions by ␥␦ T cells include the production of chemokines and cytokines and cytotoxicity
(2, 3). One of the best-studied cytotoxicity mechanisms in ␥␦ T
cells is the induction of the Fas (CD95) apoptotic pathway in target
cells. The engagement of Fas with its counterreceptor, Fas ligand
(FasL),3 leads to cell death via apoptosis of the Fas-expressing
target (4, 5). ␥␦ T cells have been shown to kill a variety of target
cells via Fas/FasL. ␥␦ T cells are known to express FasL at sites
of inflammation (6 – 8), including the CNS (9). Much of what is
known about the functions of Fas/FasL have been learned using lpr
Blood Research Institute, Blood Center of S.E. Wisconsin, Milwaukee, WI 53201
Received for publication July 8, 2004. Accepted for publication February 1, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by a grant from the Wadsworth Foundation and
Grant RG 3299-A-2 from the National Multiple Sclerosis Society.
2
Address correspondence and reprint requests to Dr. Bonnie N. Dittel, Blood Research Institute, Blood Center of S.E. Wisconsin, P.O. Box 2178, 8727 Watertown
Plank Road, Milwaukee, WI 53201-2178. E-mail address: [email protected]
3
Abbreviations used in this paper: FasL, Fas ligand; BM, bone marrow; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; MS, multiple
sclerosis; MBP, myelin basic protein; SA, streptavidin; wt, wild type.
Copyright © 2005 by The American Association of Immunologists, Inc.
and gld mice, which carry spontaneous mutations in the Fas and
FasL genes, respectively (10). Mice carrying either mutation are
susceptible to dysregulation of homeostasis within the immune
system that can result in autoimmunity (10, 11).
In the animal model of the human autoimmune CNS demyelinating disease multiple sclerosis (MS) (12), experimental autoimmune encephalomyelitis (EAE), a role for both Fas and FasL has
been indicated in the pathogenesis of disease (13–19). EAE is an
inflammatory autoimmune disease of the CNS that is associated
with an ascending paralysis, demyelination, and accumulation of
cellular infiltrates containing primarily macrophages and ␣␤ T
cells as well as B cells and ␥␦ T cells. EAE is induced by the
priming of Th1 T cells to myelin self-Ags by immunization or by
the adoptive transfer of myelin-specific encephalitogenic Th1 T
cells (13). In EAE, using the adoptive transfer model, a role for Fas
expression in the CNS has been shown to be important for the
development of EAE (13–15). Several studies, including our own,
also demonstrated a role for FasL expression in mice in the resolution of EAE disease symptoms (13–15). EAE disease symptoms
induced by adoptive transfer in gld mice were more severe and
were associated with the sustained presence of infiltrating cells
during the late/chronic stage of disease, suggesting that a FasLexpressing cell in the host is required for the effective elimination
of the infiltrating encephalitogenic T cells (13–15). The phenotype
of the FasL-expressing cell could not be determined by the above
studies.
A role for ␥␦ T cells in EAE has been suggested, but the mechanism of their regulation has not been elucidated because contradictory results have been reported when EAE was induced in ␥␦ T
cell-deficient rodents. In B10.PL mice deficient in ␥␦ T cells, we
have found that IFN-␥ expression in the CNS is reduced during
early EAE, and this correlated with an inability to recover from
EAE (20). This result is consistent with several studies that also
observed aggravation of the severity of EAE in ␥␦ T cell-deficient
mice (21, 22); however, a reduction in the severity of EAE has also
been observed (23, 24).
0022-1767/05/$02.00
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␥␦ T cells have been shown to regulate immune responses associated with inflammation, but the mechanism of this regulation is
largely unknown. Using the experimental autoimmune encephalomyelitis (EAE) model of the human CNS autoimmune disease
multiple sclerosis, we demonstrate that ␥␦ T cells are important regulators of CNS inflammation. This was shown using ␥␦ T
cell-deficient mice that were unable to recover from EAE. The chronic disease was accompanied by a prolonged presence of both
macrophages and lymphocytes in the CNS. This extended inflammatory response was due to alterations in both cell proliferation
and death. In mice lacking ␥␦ T cells, proliferation of encephalitogenic T cells was 3-fold higher, and caspase activity, indicating
apoptosis, was 2-fold lower compared with those in control mice recovering from EAE. ␥␦ T cell-deficient mice reconstituted with
wild-type ␥␦ T cells recovered from EAE and resolved inflammation in the CNS, whereas mice reconstituted with Fas liganddysfunctional ␥␦ T cells did not. Thus, ␥␦ T cells regulate both inflammation in the CNS and disease recovery via Fas/Fas
ligand-induced apoptosis of encephalitogenic T cells, and a quick resolution of inflammation in the CNS is essential to prevent
permanent damage to the CNS resulting in chronic disease. The Journal of Immunology, 2005, 174: 4678 – 4687.
The Journal of Immunology
Materials and Methods
Mice
B10.PL (H-2u) and B6.129P2-Tcrdtm1Mom mice were purchased from The
Jackson Laboratory. The myelin basic protein (MBP)-TCR transgenic mice
expressing a TCR transgene specific for the acetylated NH2-terminal peptide of MBP (Ac1–11) were generated as previously described (13).
B10.PL-TCR␦⫺/⫺ (TCR␦⫺/⫺) mice were produced in our breeding colony
by backcrossing B6.129P2-Tcrdtm1Mom mice onto B10.PL for three generations, then intercrossing to generate homozygous mice carrying the indicated gene disruption. The B10.PL-gld mice were generated as previously described (13).
Peptides and Abs
The MBP Ac1–11 peptide (Ac-ASQKRPSQRSK) was generated as previously described (13). The anti-mouse Abs CD11b-PE, CD4-FITC,
CD45-FITC, TCR␤-PE, CD25-PE, and CD69-PE and streptavidin
(SA)-CyChrome were purchased from eBioscience. The anti-mouse
Abs ␥␦ TCR-FITC, anti-V␤8.2-biotin, anti-FasL (Armenian hamster
IgG1), anti-Fas, and anti-Armenian hamster IgG1-biotin were purchased from Pharmingen. Anti-mouse F4/80-biotin and SA-PE were purchased from Caltag Laboratories. Anti-mouse CD11b-PE-Cy5 and Armenian hamster IgG1 isotype control were purchased from Biolegend. Clone
2.4G2 was purchased from American Tissue Culture Collection.
EAE induction
EAE was induced by the adoptive transfer of MBP-specific encephalitogenic T cells generated as previously described (13). Briefly, 1 ⫻ 106
activated MBP-TCR T cells were i.v. injected into sublethally irradiated
(360 rad) 5- to 8-wk-old B10.PL and TCR␦⫺/⫺ mice. Individual animals
were assessed daily for symptoms of EAE and scored using a scale from 1
to 5 as follows: 0, no disease; 1, limp tail and/or hind limb ataxia; 2, hind
limb paresis; 3, hind limb paralysis; 4, hind and fore limb paralysis; and 5,
death.
Mononuclear cell isolation and flow cytometry
Mononuclear cells were isolated from the CNS of B10.PL or TCR␦⫺/⫺
mice with EAE on days 0, 7, 10, 15, 25, and 40 from mice perfused with
25–30 ml of cold PBS. The brains and spinal cords were homogenized, cell
suspensions were incubated with 0.5 mg/ml collagenase type II (SigmaAldrich) at 37°C for 30 min, and mononuclear cells were isolated using
40/70% discontinuous Percoll gradients. Total cell numbers were determined by counting on a hemocytometer, and viability was assessed by
trypan blue exclusion. For the purification of ␥␦ T cells, total mononuclear
cells were isolated on day 18 after EAE induction and stained with anti-␥␦
TCR-FITC and CD11b-CyChrome, and the CD11b⫺␥␦ TCR⫹ cells were
sorted using a FACSAria (BD Biosciences). Two-color flow cytometry
using anti-CD45-FITC and anti-CD11b-PE or anti-CD4-FITC and antiTCR␤-PE with anti-CD4-FITC was conducted using total mononuclear
cell preparations. Three-color flow cytometry using anti-CD45-FITC, antiCD11b-PE-Cy5, and anti-F4/80-biotin combined with SA-PE, CD11b-PECy5 and ␥␦ TCR-FITC, TCR␤-FITC and anti-CD25-PE, or anti-CD69-PE
were conducted using total mononuclear cell preparations. FasL expression
was assessed using a three-step process as follows: 1) anti-TCR␤-FITC or
anti-␥␦ TCR-FITC and anti-CD11b-PE-Cy5 and anti-FasL, 2) antiArmenian hamster IgG1-biotin, and 3) SA-PE. For all Ab stainings, FcR
were first blocked with anti-mouse FcR (2.4G2). Ab incubations were conducted on ice, and the cells were fixed in 1% paraformaldehyde and analyzed using a FACScan or LSR II (BD Biosciences).
BrdU labeling
For labeling of proliferating cells in vivo, groups of four or five B10.PL or
TCR␦⫺/⫺ mice on days 10, 15, and 21 after EAE induction by adoptive
transfer were injected i.p. with 1 mg of BrdU (Sigma-Aldrich) 14 h before
isolation of brain mononuclear cells. Freshly isolated mononuclear cells
from each experimental group were pooled and stained with anti-CD4-PE
and anti-V␤8.2-biotin combined with SA-CyChrome. Subsequently BrdU
incorporation into the cellular DNA was detected using the BrdU flow kit
(BD Biosciences) according to the manufacturer’s instructions. Briefly,
cells were fixed, permeabilized, treated with DNase, and incubated with
anti-BrdU-FITC. The samples were kept on ice and immediately analyzed
by three-color flow cytometry.
Caspase apoptosis assay
Analysis of apoptosis of V␤8.2⫹ T cells isolated from the CNS was performed using the caspase 3-specific fluorogenic substrate PhiPhilux-G2D2
purchased from OncoImmune. Freshly isolated CNS mononuclear cells
from four or five B10.PL or TCR␦⫺/⫺ mice on days 10, 15, and 21 after
EAE induction by adoptive transfer were pooled and incubated with
PhiPhilux-G2D2 dissolved in RPMI 1640 medium containing 10% FBS at
37°C for 1 h according to the manufacturer’s instructions. The cells were
washed twice with ice-cold buffer containing 2% FCS and 0.05% sodium
azide. FcR were blocked for 15 min on ice before staining with anti-V␤8.2FITC. After washing, the cells were kept on ice and immediately analyzed
by three-color flow cytometry. Necrotic and dead cells were excluded from
the light scatter gate using propidium iodide. Manipulations for mononuclear cell isolation were performed on ice within 3– 4 h using 40/70%
discontinuous Percoll gradients without collagenase treatment. For the apoptosis of encephalitogenic T cells, MBP-TCR T cells used for EAE induction were labeled with 0.3 ␮M SNARF-1 (Molecular Probes) dissolved
in PBS at 37°C for 30 min. After three washes, 20 ⫻ 103 cells were placed
in round-bottom microtiter plates in the presence of medium alone, anti-Fas
mAb (20 ␮g/ml), or 100 ⫻ 103 ␥␦ T cells isolated from the CNS of either
B10.PL or B10.PL-gld mice 18 days after EAE induction. After culture for
4.5 h at 37°C and 5% CO2, the cells were labeled with PhiPhilux-G1D2 as
described above, and SNARF-1⫹ target cells were gated and analyzed for
fluorescence.
BM chimeras
Mixed BM chimeras were generated by transferring 4 ⫻ 106 total BM cells
from B10.PL, TCR␦⫺/⫺, or B10.PL-gld mice into sublethally irradiated
(360 rad) TCR␦⫺/⫺ or B10.PL mice and were allowed to reconstitute for
6 wk.
Results
Mice deficient in ␥␦ T cells exhibit a chronic EAE disease
course
To assess the role of ␥␦ T cells in the EAE disease course of
B10.PL mice (H-2u), we generated B10.PL mice deficient in ␥␦ T
cells (TCR␦⫺/⫺). EAE was induced in B10.PL and TCR␦⫺/⫺ mice
by the adoptive transfer of Ac1–11-specific CD4 T cells generated
from MBP-TCR transgenic mice (13). B10.PL and TCR␦⫺/⫺ mice
exhibited an identical early disease course, with 100% of the mice
in each group succumbing to disease with an average day of onset
on day 10 (Fig. 1 and Table I). After a 12-day disease course,
including onset and the effector phase, B10.PL mice spontaneously
recovered, with full recovery observed before day 40 (Fig. 1). In
contrast, TCR␦⫺/⫺ mice did not undergo any signs of recovery
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In the present study we show a careful analysis of the nature of
the CNS inflammation associated with EAE in B10.PL mice deficient in ␥␦ T cells compared with control mice. In our past studies using the EAE model in B10.PL mice, we showed that EAE
disease in B10.PL mice is associated with complete recovery from
disease (13). However, in this study, when B10.PL mice were
rendered deficient in ␥␦ T cells, the mice were unable to recover
from EAE, exhibiting a long term chronic disease course that was
accompanied by a prolonged presence of inflammatory cells in the
CNS. In determining the mechanism of the extended inflammation,
we found that the presence of higher numbers of inflammatory
cells in the CNS of ␥␦ T cell-deficient mice was due to both enhanced cell proliferation and increased survival of encephalitogenic T cells. Reconstitution of ␥␦ T cell-deficient mice with wildtype (wt) ␥␦ T cells by bone marrow (BM) transplantation
reversed both the chronic disease course and the sustained presence of inflammatory infiltrates in the CNS. In contrast, reconstitution with ␥␦ T cells from gld donor mice had no effect on either
EAE disease parameter. Thus, these data suggest that ␥␦ T cells
promote the resolution of inflammation in the CNS by inducing
apoptosis of encephalitogenic T cells through a FasL-dependent
mechanism. Furthermore, these data show the importance of a
quick resolution of inflammation in the CNS, because a prolongation of only 10 days resulted in chronic disease, suggesting that
permanent damage to the nervous system had occurred.
4679
4680
␥␦ T CELLS REGULATE EAE INFLAMMATION VIA Fas/FasL
Table I. Summary of the EAE disease course in B10.PL and TCR␦⫺/⫺
micea
FIGURE 1. Comparison of EAE clinical course in B10.PL and
TCR␦⫺/⫺ mice. EAE was induced by the i.v. adoptive transfer of 1 ⫻ 106
MBP-TCR T cells into sublethally irradiated B10.PL or TCR␦⫺/⫺ recipient
mice. Individual mice were evaluated daily starting on day 5 after transfer,
and the daily scores from 20 B10.PL (E) and 20 TCR␦⫺/⫺ (F) mice in four
groups were averaged.
Cellular infiltrate in the CNS is increased and prolonged in
TCR␦⫺/⫺ mice during EAE
In addition to ascending paralysis, EAE is characterized by accumulation of inflammatory cells in discrete lesions in the CNS. In
B10.PL mice with acute disease, the inflammatory infiltrate increases steadily throughout the effector phase of disease and then
gradually disappears during the recovery phase. To determine
whether chronic disease in TCR␦⫺/⫺ mice was associated with
increased cell numbers in the CNS, we isolated total mononuclear
cells from the CNS of B10.PL and TCR␦⫺/⫺ mice throughout the
EAE disease course and quantitated the absolute number of infiltrating cells, macrophages, lymphocytes, and CD4 T cells. The
cells were analyzed for the expression of CD11b, CD45, CD4, and
TCR␤, and macrophages (CD11b⫹CD45high) were distinguished
from lymphocytes (CD11b⫺CD45high) and resident microglial
cells (CD11b⫹CD45low) by the expression level of CD11b and
CD45. In normal control mice, very few macrophages (Fig. 2B)
and lymphocytes (Fig. 2C) were present in the CNS before the
induction of EAE. To confirm that a CD45high expression pattern
detected macrophages, we directly compared CD11b⫹ cells for the
expression of CD45 (Fig. 2E) or F4/80 (Fig. 2F) and found them
to be identical. Of the few lymphocytes present, about one-half are
CD4 T cells (Fig. 2D). Upon EAE induction in the B10.PL mouse,
the absolute number of infiltrating cells in the CNS paralleled the
EAE disease course (Fig. 1) and increased steadily until the peak
of disease was reached on day 15 (Fig. 2A). As the mice recovered
from disease symptoms, the number of infiltrating cells also declined, as seen on day 25, and cell numbers remained slightly
elevated on day 40, even though disease symptoms had subsided
(Fig. 1). TCR␦⫺/⫺ mice also had a steady increase in the number
of infiltrating cells, which reached maximum at the peak of disease
on day 15 and was similar in number to that in B10.PL mice (Fig.
2A). However, the cellular infiltrate was sustained through day 25,
and a statistically significantly larger number of cells remained in
the CNS ( p ⬍ 0.008) compared with B10.PL mice (Fig. 2A). In
addition, the sustained infiltration was accompanied by sustained
disease symptoms (Fig. 1). The cellular infiltrate in the CNS of
TCR␦⫺/⫺ mice eventually subsided and was not significantly different from that in control mice on day 40 (Fig. 2A). These data
suggest that ␥␦ T cells play an important role in regulating the
TCR␦⫺/⫺
20
100
9.9 ⫾ 0.4
0.4 ⫾ 0.12
35.2 ⫾ .1
20
100
9.9 ⫾ 0.4
1.8 ⫾ 0.12c
57.6 ⫾ 2.1c
a
Graded disease score as described in Materials and Methods.
Represents the percentage of mice that developed a clinical disease score of at
least 1.
c
p ⬍ 0.0001 compared to B10.PL.
d
The cumulative disease score was calculated by adding the disease score from
the day of onset to day 40. The values shown are the mean of all mice with disease
in each group.
b
extent of the inflammatory response in the CNS. In addition to
regulating inflammation during recovery, ␥␦ T cells may play a
role early in disease before disease onset, as indicated by a reduced
number of infiltrating cells in the CNS of TCR␦⫺/⫺ mice on day
7, just before disease onset, compared with that in B10.PL mice
( p ⫽ 0.05).
To determine whether ␥␦ T cells differentially regulated the extent of lymphocyte and macrophage populations in the CNS during
EAE, we quantitated the number of each cell population and found
that both macrophage (Fig. 2B) and lymphocyte (Fig. 2C) populations remained elevated on day 25 in TCR␦⫺/⫺ mice compared
with control mice, with p values of 0.05 and 0.02, respectively. To
further differentiate the lymphocyte population, we quantitated the
number of CD4 T cells in the CNS that were also retained in the
CNS on day 25 at a statistically significant higher level compared
with that in control mice ( p ⫽ 0.004; Fig. 2D). We also confirmed
that ␥␦ T cells were present in the CNS during EAE and exhibited
a similar kinetic pattern of emergence and decline, with 18 ⫾ 6,
40 ⫾ 7, 20 ⫾ 6, and 4 ⫾ 2 ⫻ 103 ␥␦ T cells present in the CNS
on days 7, 15, 25, and 40, respectively. ␥␦ T cells were not detectable in the CNS before EAE induction by flow cytometry;
however, they were detectable by PCR (20).
Proliferation of encephalitogenic T cells in TCR␦⫺/⫺ mice is
sustained during the late stages of EAE
The sustained presence of T cells in the CNS of TCR␦⫺/⫺ mice
suggests that ␥␦ T cells function to regulate the extent and/or duration of the inflammatory response. We reasoned that the prolonged presence of T cells in the CNS could occur by two mechanisms, either enhanced or sustained cell proliferation and/or a
delay or inhibition of cell death. To determine whether cell proliferation was altered, we compared T cell proliferation in the CNS
in B10.PL control mice and TCR␦⫺/⫺ mice by measuring BrdU
incorporation. Mice were i.p. injected with BrdU 14 h before the
isolation of total mononuclear cells from the CNS, and BrdU incorporation was measured by flow cytometry. We examined BrdU
incorporation in the two major populations of T cells in the CNS:
CD4⫹V␤8.2⫹ encephalitogenic T cells (Fig. 3, A–F) and
CD4⫹V␤8.2⫺ nonencephalitogenic T cells (Fig. 3, G–L). V␤8.2 is
the TCR ␤-chain expressed by the MBP-TCR transgenic T cells
used to induce EAE. For V␤8.2⫹ T cells, the number of dividing
cells was equal 10 days after EAE induction in B10.PL and
TCR␦⫺/⫺ mice (Fig. 3, A and D, respectively). In wt V␤8.2⫹ cells,
the number of dividing cells was decreased by ⬎50% on days 15
and 21 (Fig. 3, B and C, respectively), whereas the number did not
substantially decrease in the TCR␦⫺/⫺ mice (Fig. 3, E and F, respectively). This analysis was performed three times, and the average cumulative data are presented in Fig. 3M, showing that the
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(Fig. 1). This lack of recovery is reflected in the average day 40
disease score of 1.8 for the TCR␦⫺/⫺ mice, whereas the B10.PL
mice had a score of 0.4 ( p ⬍ 0.0001; Table I). Likewise, the
cumulative disease score for TCR␦⫺/⫺ mice was significantly
higher ( p ⬍ 0.0001) than that for B10.PL mice (58 vs 35, respectively; Table I). These data clearly demonstrate a potential regulatory role for ␥␦ T cells in the recovery from EAE.
No. of mice
Incidence (%)b
Average day onset ⫾ SE
Average day 40 disease score ⫾ SE
Average cumulative disease score
⫾ SEd
B10.PL
The Journal of Immunology
4681
decreased number of proliferating B10.PL V␤8.2⫹ T cells vs the
same cell population in TCR␦⫺/⫺ mice during the day 15 and 21
time points is a consistent observation. The reduction in T cell
proliferation in B10.PL mice during recovery from EAE (Fig. 3M)
is consistent with the loss of total CD4 T cells from the CNS
during this time period (Fig. 2D). Likewise, the prolonged presence of CD4 T cells in the CNS on day 25 (Fig. 2D) is consistent
with the sustained level of proliferation of encephalitogenic T cells
in TCR␦⫺/⫺ mice (Fig. 3M).
To determine whether ␥␦ T cells also regulated the proliferation
of nonencephalitogenic T cells in the CNS, we analyzed BrdU
incorporation in CD4⫹V␤8.2⫺ T cells in the same mononuclear
cell preparations. In both B10.PL and TCR␦⫺/⫺ mice on day 10,
nonencephalitogenic T cells underwent proliferation, but at a rate
⬃50% lower than encephalitogenic T cells (Fig. 3G). As with
encephalitogenic T cells, the percentage of BrdU⫹ cells decreased
during the EAE time course in both types of mice (Fig. 3, G–I and
J–L). However, there was no difference in the percentage of pro-
liferating cells in B10.PL compared with TCR␦⫺/⫺ mice at any
time point examined (Fig. 3, G–L). For the V␤8.2⫺ T cells, the
average of three experiments is shown in Fig. 3N and is consistent
with the data shown in the contour plots. BrdU incorporation of
CD4⫹ cells was not different in the spleens of the same B10.PL
and TCR␦⫺/⫺ mice (data not shown), indicating that ␥␦ T cells do
not regulate bystander proliferation of nonencephalitogenic T
cells, but, rather, specifically regulate the proliferation of the Agspecific encephalitogenic T cells in the CNS.
Survival of encephalitogenic T cells in TCR␦⫺/⫺ mice is
enhanced during EAE
In addition to a higher level of encephalitogenic T cell proliferation, the sustained levels of CD4 T cells in the CNS of TCR␦⫺/⫺
mice could also be due to increased cell survival. This possibility
was examined by measuring the level of caspase activity in encephalitogenic T cells during the EAE disease course. The presence of caspase activity indicates that an apoptotic pathway has
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FIGURE 2. Quantitation of the absolute number of macrophages and lymphocytes in the CNS of B10.PL and TCR␦⫺/⫺ mice with EAE. EAE was
induced in B10.PL (f) and TCR␦⫺/⫺ (o) mice as described in Fig. 1, and total mononuclear cells were isolated from the CNS on days 0, 7, 10, 15, 25,
and 40 after EAE induction. The isolated cells were analyzed for the expression of CD45 and CD11b or TCR␤ and CD4 by two-color flow cytometry. A,
The absolute number of infiltrating mononuclear cells was determined by multiplying the total cell count obtained by counting on a hemocytometer by the
percentage of CD45high cells determined by flow cytometry (excluding CD11b⫹CD45low resident microglial cells) and then dividing by the number of mice
in each group. B, The percentage of macrophages was determined by flow cytometry by gating on CD11b⫹CD45high cells, and the absolute number of
macrophages was determined by multiplying the percentage of macrophages by the absolute number of mononuclear cells obtained in A. C, The total
number of lymphocytes was determined as described for B gating on CD45highCD11b⫺ cells. D, The total number of CD4⫹ lymphocytes was determined
as described for B gating on TCR␤⫹CD4⫹ cells. Each bar represents the average of three separate experiments, with each individual observation containing
pooled cells from four or five mice, with the SE given. The asterisk above the bar indicates a statistically significant increase (p ⬍ 0.05) from the control
B10.PL group. The plus sign above the bar indicates a statistically significant decrease (p ⬍ 0.05) from the control B10.PL group. E and F, Total
mononuclear cells isolated from the CNS on day 15 after EAE induction were analyzed by two-color flow cytometry for the expression of CD11b and either
anti-CD45 (E) or F4/80 (F), and data are shown as two-color contour plots with the percentage of positive cells indicated in the corner of each quadrant.
4682
␥␦ T CELLS REGULATE EAE INFLAMMATION VIA Fas/FasL
been activated, and the cell is in the process of undergoing cell
death. Caspase activity was examined by flow cytometry using the
caspase 3-specific substrate PhiPhilulx-G2D2, which becomes fluorescent upon cleavage by activated caspase 3 (25). The negative
control consisted of unstained cells without the addition of PhiPhilulx-G2D2 as shown for the day 10 point in B10.PL and TCR␦⫺/⫺
mice (Fig. 5, B and F, respectively). When caspase activity was
examined on day 10 after EAE induction, ⬎50% more encephalitogenic T cells in B10.PL wt mice compared with TCR␦⫺/⫺ mice
were positive for caspase activity (Fig. 4A), a difference that is
statistically significant ( p ⫽ 0.03). A less pronounced difference
was observed on day 15 (Fig. 4A). However, again on day 21, there
was a statistically significance difference in caspase activity ( p ⫽
0.04), with 25% of the encephalitogenic T cells in B10.PL mice
undergoing apoptosis and 14% in TCR␦⫺/⫺ mice (Fig. 4A). A
representative experiment of three is shown in Fig. 4, B–I, demonstrating the fluorescence intensity of the cleaved caspase substrate in V␤8.2⫹ encephalitogenic T cells on day 10 (C and G), day
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FIGURE 3. Comparison of BrdU incorporation in
CD4 T cells in the CNS of B10.PL and TCR␦⫺/⫺ mice
during EAE. EAE was induced in B10.PL (A–C, G–I)
and TCR␦⫺/⫺ (D–F, J–L) mice as described for Fig. 1,
and total mononuclear cells were isolated from the CNS
of mice 10 (A, D, G, and J), 15 (B, E, H, and K), and 21
(C, F, I, and L) days later. Mice were i.p. injected with
1 mg of BrdU 14 h before isolation of mononuclear
cells, which were analyzed for incorporation of BrdU
and expression of CD4 and V␤8.2 by three-color flow
cytometry. A–L, Two-color contour plots are shown
with BrdU incorporation on the x-axis and CD4 expression on y-axis. The percentages of CD4⫹V␤8.2⫹ gated
(A–F) and CD4⫹V␤8.2⫺ gated (G–L) T cells that incorporated BrdU are indicated on the contour plots. The
data shown are from one representative experiment of
three, with each individual observation containing
pooled cells from four or five mice. M and N, The percentages of V␤8.2⫹ (M) and V␤8.2⫺ (N) T cells from
B10.PL (f) and TCR␦⫺/⫺ (o) mice showing BrdU incorporation are shown, with each bar representing the
average of three separate experiments and with each individual observation containing pooled cells from four
or five mice, with the SE given.
15 (D and H), and day 21 (E and I) in B10.PL and TCR␦⫺/⫺ mice
after EAE induction, respectively.
We found that ⬎90% of V␤8.2⫹ cells in the CNS are also
CD4⫹, making any contribution of CD8 T cells to the caspase
analysis minimal. In addition, no difference was observed in
caspase activity in CD4 T cells in the spleens of B10.PL and
TCR␦⫺/⫺ mice with EAE (data not shown). Thus, these data suggest that the sustained presence of T cells in the CNS of TCR␦⫺/⫺
mice during the timeframe of recovery for B10.PL mice is a combination of higher cell proliferation (Fig. 3M) and lower rate of cell
death (Fig. 4A) compared with the same cells in B10.PL mice.
Recovery from EAE and resolution of cellular infiltrates in CNS
requires expression of FasL by ␥␦ T cells
The reduction in caspase activity in TCR␦⫺/⫺ mice on day 21 after
the induction of EAE indicates that the elimination of encephalitogenic T cells in the CNS occurs by apoptosis. However, the
The Journal of Immunology
4683
mechanism of apoptotic cell death could not be determined because caspase is activated by a variety of signals leading to apoptosis, including Fas/FasL. To determine whether ␥␦ T cells could
be mediating the apoptosis of encephalitogenic T cells via a Fas/
FasL mechanism, we reconstituted TCR␦⫺/⫺ mice with ␥␦ T cells
that were able to express either functional or dysfunctional FasL
(gld). This was accomplished by generating mixed BM chimera
mice by transplanting sublethally irradiated TCR␦⫺/⫺ mice with
BM from either wt B10.PL (wt3 TCR␦⫺/⫺) or B10.PL-gld
(gld3 TCR␦⫺/⫺) mice. In these chimera mice, the emerging ␥␦ T
cell populations will express either wt FasL (B10.PL donor BM) or
a nonfunctional FasL (gld donor BM), whereas the ␣␤ T cells will
be mixed, with ⬃50% of the cells being recipient in origin (data
not shown). ␥␦ T cell reconstitution was evident 4 wk post-transplant, as indicated by the presence of ␥␦ T cells in the intestinal
intraepithelial lymphocytes cell population (data not shown).
First, we determined whether the reconstitution of ␥␦ T cells in
TCR␦⫺/⫺ mice would revert their chronic EAE disease course
(Fig. 1). As shown in Fig. 5A, TCR␦⫺/⫺ mice reconstituted with
BM from B10.PL wt mice (wt3 TCR␦⫺/⫺) were able to recover
from disease symptoms and exhibited a similar disease course
compared with the control chimeras, where B10.PL mice were
transplanted with B10.PL BM (wt3wt). In contrast, TCR␦⫺/⫺
mice reconstituted with BM from gld mice were unable to resolve
EAE disease symptoms and exhibited a chronic disease course
similar to that observed in TCR␦⫺/⫺ mice reconstituted with BM
from TCR␦⫺/⫺ (TCR␦⫺/⫺3 TCR␦⫺/⫺; Fig. 5A). These data show
that recovery from EAE requires the expression of FasL by ␥␦ T
cells. We next examined whether ␥␦ T cells in the CNS of mice
with EAE expressed FasL, and found that ␥␦ T cells isolated from
the CNS of mice with EAE during the recovery phase of disease
expressed detectable FasL (Fig. 5E) at a level slightly higher than
that expressed by ␣␤ T cells in the CNS of the same mice (Fig.
5B). Furthermore, we found that the number of ␣␤ T cells expressing CD25 (Fig. 5C) was 2-fold greater than that of ␥␦ T cells
(Fig. 5F), whereas the level of CD69 was identical in the two cell
populations (Fig. 5, D and G, respectively). These results are consistent with those of the Brosan laboratory (9).
To determine whether FasL expression by ␥␦ T cells is also
required for the resolution of inflammatory infiltrates in the CNS
during EAE, we generated identical BM chimeras as those shown
in Fig. 5, isolated mononuclear cells from the CNS of chimera
mice 25 days after EAE induction, and quantitated and phenotypically characterized the cellular infiltrate. We choose the day 25
point, because it is at this point in the EAE disease course that a
statistically significantly greater number of mononuclear cells remain in the CNS of TCR␦⫺/⫺ mice compared with control mice
(Fig. 2A). As shown in Table II, only chimera mice, which either
lacked ␥␦ T cells (TCR␦⫺/⫺3 TCR␦⫺/⫺) or were reconstituted
with FasL dysfunctional ␥␦ T cells (gld3 TCR␦⫺/⫺) did not show
signs of recovery, as indicated by the identical disease score of 2 ⫾
0.1. In contrast, the control chimeras, in which B10.PL mice were
transplanted with either B10.PL BM (wt3wt) or gld BM
(gld3wt), showed signs of recovery, with day 25 disease scores of
1 ⫾ 0.2 and 1.25 ⫾ 0.25, respectively (Table II). The gld3wt
chimeras are a control for the mixed populations of ␣␤ T cells in
the chimeras generated by transplantation with gld BM, resulting
in ⬃50% of the cells being from the gld donor BM. Because these
chimeras are able to recover, the lack of recovery in the
gld3 TCR␦⫺/⫺ chimeras is not due to a loss of FasL function in
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FIGURE 4. Comparison of caspase activity in CD4 T cells isolated from the CNS of B10.PL and TCR␦⫺/⫺ mice with EAE. EAE was induced in B10.PL
(f, B–E) and TCR␦⫺/⫺ (o, F–I) mice as described in Fig. 1, and total mononuclear cells were isolated from the CNS of mice (as described in Fig. 3) 10
(B, C, F, and G), 15 (D and H), and 21 (E and I) days later. Isolated mononuclear cells were evaluated for the expression of V␤8.2 and caspase activity
by three-color flow cytometry gating on V␤8.2⫹propidium iodide⫺ cells. A, The percentage of V␤8.2⫹ T cells exhibiting caspase activity is shown. Each
bar represents the average of three separate experiments, with each individual observation containing pooled cells from four or five mice with the SE given.
The asterisk above the bar indicates a statistically significant decrease (p ⬍ 0.05) from the control B10.PL group. B–I, Two-color contour plots are shown
with PhiPhiloux-G2D2 fluorescence on the x-axis and V␤8.2 on the y-axis (C–E and G–I) or the autofluorescence of unstained cells without the addition
of PhiPhiloux-G2D2 (B and F) as a negative control. The percentage of V␤8.2⫹ T cells exhibiting caspase activity is indicated on the contour plots. The
data shown are from one representative experiment of three performed, with each individual observation containing pooled cells from four or five mice.
4684
␥␦ T CELLS REGULATE EAE INFLAMMATION VIA Fas/FasL
the endogenous ␣␤ T cells or to technical aspects of chimera generation. In addition, TCR␦⫺/⫺ mice transplanted with wt BM
(wt3 TCR␦⫺/⫺) were recovering and had a disease score of 1.1 ⫾
0.3 (Table II).
In addition to disease score, we quantitated the absolute number
of mononuclear cells, macrophages, lymphocytes, CD4 T cells,
and encephalitogenic T cells in the CNS of chimera mice. We
found that chimera mice lacking ␥␦ T cells or reconstituted with
gld ␥␦ T cells had similar numbers of all the characterized cell
types in the CNS (Table II). Similarly, all control chimeras that
exhibited recovery also had similar numbers of the characterized
cell populations in the CNS (Table II). In a direct comparison, the
recovering chimeras had 2.4- to 2.9-fold fewer total mononuclear
cells, 2.9- to 4.2-fold fewer macrophages, 2- to 4-fold fewer lymphocytes, and 2.2- to 4.2-fold fewer CD4 T cells in the CNS on day
25 after EAE induction compared with TCR␦⫺/⫺ mice reconstituted with gld BM (Table II). When we examined encephalitogenic
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FIGURE 5. Comparison of the EAE clinical course
in TCR␦⫺/⫺ mice reconstituted with either wt B10.PL
or B10.PL-gld ␥␦ T cells and phenotype and function of
CNS ␥␦ T cells. A, BM chimeras were generated by
transferring 4 ⫻ 106 total BM cells from B10.PL,
B10.PL-gld, or TCR␦⫺/⫺ mice into sublethally irradiated B10.PL or TCR␦⫺/⫺ recipient mice generating
wt3wt (f), wt3 TCR␦⫺/⫺ (Œ), gld3 TCR␦⫺/⫺ (F),
and TCR␦⫺/⫺3 TCR␦⫺/⫺ (E) chimera mice. After a
6-wk reconstitution, EAE was induced by the i.v. transfer of 1 ⫻ 106 CD4 MBP-TCR T cells, as described in
Fig. 1. The EAE disease course was observed and
scored starting on day 5 after transfer, and the data
shown are the daily score of five mice in each group.
The data are from one representative experiment of
three performed. B–G, Total mononuclear cells from
B10.PL mice isolated 21 days after EAE induction were
analyzed by three-color flow cytometry for the coexpression of ␣␤ TCR (B–D) or ␥␦ (E–G) TCR and FasL
(B and E), CD25 (C and F), or CD69 (D and G). Data
are shown as a histogram after gating of either TCR␤⫹
or TCR␥␦⫹CD11b⫺ cells, with the mean fluorescence
channel intensity (MFI) of the FasL⫹ cells or the percentage of positive CD25 or CD69 cells indicated. The
dotted line represents background staining using an isotype-matched control, and the solid line represents specific staining. The data shown contain pooled cells from
five mice. H–K, SNARF-1-labeled encephalitogenic T
cells were cocultured with medium alone (H), anti-Fas
(I), or ␥␦ T cells isolated from the CNS of either 12 wt
(J) or 12 B10.PL-gld (K) mice 18 d after EAE induction.
Caspase activity was analyzed in the SNARF-1⫹ cells,
and the data are shown as a histogram, with PhiPhilouxG1D2 fluorescence on the x-axis and cell counts on the
y-axis. The percentage of cells exhibiting caspase activity is indicated on the histograms.
T cells (CD4⫹V␤8.2⫹) in the chimeras, we found a 2.7- to 5.4-fold
reduction in cell numbers in the control chimeras compared with
the gld3 TCR␦⫺/⫺ chimera mice (Table II). In addition, there was
a statistically significant difference in all parameters examined between the gld3 TCR␦⫺/⫺ and wt3 TCR␦⫺/⫺ chimeras (Table II).
The increased number of encephalitogenic T cells in the CNS of
gld3 TCR␦⫺/⫺ chimera mice on day 25 is consistent with the
reduced apoptosis of these cells observed on day 21 (Fig. 4A).
We next confirmed that ␥␦ T cells isolated from the CNS of
mice with EAE have the capacity to induce the apoptosis of encephalitogenic T cells. By measuring caspase activity, we determined that in vitro activated encephalitogenic T cells alone had a
3% background level of apoptosis (Fig. 5H) and that incubation
with anti-Fas increased apoptosis to 24% (Fig. 5I). The addition of
wt CNS ␥␦ T cells resulted in 14% of the encephalitogenic T cells
undergoing apoptosis (Fig. 5J). This was reduced to 7% when ␥␦
T cells from gld mice were added (Fig. 5K). The 4% apoptosis
The Journal of Immunology
4685
Table II. Summary of disease and cell parameters in BM chimera mice reconstituted with gld BMa
Parameter
wt3wt
wt3 TCR␦⫺/⫺
TCR␦⫺/⫺3 TCR␦⫺/⫺
gld3wt
gld3 TCR␦⫺/⫺
Day 25 average EAE scoreb
Mononuclear cellsd
Macrophagesd
Lymphocytesd
CD4 T cellsd
CD4⫹V␤8.2⫹T cellsd,f
1 ⫾ 0.2
74 ⫾ 12
22 ⫾ 5
39 ⫾ 10
14 ⫾ 4
6⫾1
1.1 ⫾ 0.3
74 ⫾ 3
27 ⫾ 8
25 ⫾ 10
11 ⫾ 2
5 ⫾ 0.5
2 ⫾ 0.1
227 ⫾ 76
95 ⫾ 38
110 ⫾ 38
67 ⫾ 32
52 ⫾ 32
1.25 ⫾ 0.25
92 ⫾ 25
32 ⫾ 5
50 ⫾ 18
21 ⫾ 7
6⫾1
2 ⫾ 0.1c
218 ⫾ 26e
93 ⫾ 19c
99 ⫾ 16c
46 ⫾ 8c
27 ⫾ 5c
a
BM chimeras were generated in as described in Fig. 5 by transferring donor BM into (3) recipient mice. EAE was induced as described in Fig. 1. The data shown are the
average ⫾ SE of three experiments with four to five mice per group observed 25 days after EAE induction.
b
Graded disease score as described in Materials and Methods.
c
p ⬍ 0.04 compared to wt3 TCR␦⫺/⫺.
d
Absolute numbers of mononuclear cell populations isolated from the CNS of the indicated BM chimera mice were determined as described in Fig. 2.
e
p ⫽ 0.005 compared to wt3 TCR␦⫺/⫺.
f
Absolute numbers of CD4⫹V␤8.2⫹ T cells were determined as described in Fig. 2, using two-color flow cytometry gating on CD4 and determining the percentage of V␤8.2⫹
cells.
Discussion
In this study we addressed whether ␥␦ T cells could regulate inflammation associated with an immune response in the absence of
an infectious pathogen. We observed that ␥␦ T cells regulated the
number of inflammatory cells in the CNS both early and late in the
EAE disease course. Early in disease, mice lacking ␥␦ T cells had
both reduced numbers of mononuclear cell infiltrates and apoptotic
cells. Our data also suggest that ␥␦ T cells regulate the resolution
of inflammation late in disease, because mice deficient in ␥␦ T
cells had a prolonged presence of mononuclear cell infiltrates in
the CNS and were unable to recover from EAE. The inability to
down-regulate the inflammatory infiltrate was found to be due to
decreased apoptotic cell death of encephalitogenic T cells. The
higher survival rate of the encephalitogenic T cells was accompanied by an increased percentage of proliferating cells. Finally, we
show that the timely resolution of inflammation in the CNS during
EAE is dependent upon expression of a functional FasL by ␥␦ T
cells. Thus, ␥␦ T cells regulate inflammation in the CNS during
EAE by promoting the death of encephalitogenic T cells via the
Fas/FasL apoptotic pathway.
For our study examining the role of ␥␦ T cells in regulation of
inflammation, we choose EAE in B10.PL mice as our model system because of the predictable and well-characterized inflammatory response that occurs in the CNS. In addition, by inducing EAE
by the adoptive transfer of encephalitogenic T cells primed in
vitro, we were able to eliminate any response or effect of the Mycobacterium tuberculosis in CFA on either ␣␤ or ␥␦ T cells. We
believed that this was important because ␥␦ T cells have been
shown to recognize and respond to mycobacterial heat shock proteins (1, 3, 26). In addition, TCR␦⫺/⫺ mice infected with a low
dose of M. tuberculosis exhibited an altered disease, manifested as
a pyogenic form of the granulomatous response instead of the lymphocytic response seen in controls (27). Also, cross-talk between
␣␤ and ␥␦ T cells is indicated in vivo (28), with each having the
capacity to regulate the responses of the other. Thus, the presence
of CFA could nonspecifically activate and mobilize a population of
regulatory ␥␦ T cells that could influence both ␣␤ T cell priming
and inflammation.
One common theme among many studies examining the roles of
␥␦ T cells in a variety of infectious disease models is the ability of
␥␦ T cells to regulate the nature of cellular infiltration into the site
of infection. This includes infection with Mycobacterium (27) or
Listeria (29, 30), where lesions associated with disease were altered in the absence of ␥␦ T cells. Also, in a study examining
neurocysticercosis, the number of infiltrating mononuclear cells
was reduced in the brains of TCR␦⫺/⫺ mice (31). These data are
consistent with our observation that the absolute numbers of infiltrating lymphocytes and macrophages were reduced early in the
EAE disease course in TCR␦⫺/⫺ mice (Fig. 2). In addition, in a
study by Rajan et al. (32), lower levels of leukocytes in the CNS
were also observed early in the EAE disease course, although the
phenotype of the cells was not examined. Collectively, these observations suggest that ␥␦ T cells are functionally able to specifically regulate the migration of inflammatory cells into the site of
tissue injury. Presumably, if fewer cells were able to infiltrate the
CNS, then a less severe EAE disease course could be the outcome,
as was observed in several studies (24, 32). However, in our study
using adoptive transfer in the B10.PL TCR␦⫺/⫺ mouse, we saw no
alteration in the early EAE disease course parameters of day of
onset and peak disease score (Table I), even though reduced numbers of mononuclear cells were observed in the CNS (Fig. 2). The
difference between our study and others using adoptive transfer is
the source and potency of the encephalitogenic T cells. Our encephalitogenic T cells are sufficiently potent to induce disease with
5–15 and 50 times fewer cells than the studies by Spahn et al. (24)
and Rajan et al. (32), respectively. Thus, it is likely that we observed a normal early EAE disease course in TCR␦⫺/⫺ mice, because our model requires fewer encephalitogenic T cells to induce
disease.
During EAE onset, the absolute number (Fig. 2) and percentage
of proliferating T cells (Fig. 3) in the CNS are similar in TCR␦⫺/⫺
and wt mice. In contrast, the percentage of apoptosing T cells was
50% less in TCR␦⫺/⫺ mice (Fig. 4). One possible mechanism for
how similar numbers of T cells are present in the CNS when fewer
cells are undergoing cell death in the TCR␦⫺/⫺ mouse is a decrease in the level of infiltration into the CNS. It has been shown
in a model of neurocysticercosis that ␥␦ T cells may promote the
infiltration of lymphocytes and macrophages into the CNS by secretion of cytokines and chemokines (31), which enhance the migration of immune cells from the periphery into the CNS. One
important candidate cytokine that may regulate immune cell migration into the CNS and apoptosis of T cells is IFN-␥. We have
found that the level of IFN-␥ mRNA was significantly reduced in
the CNS of TCR␦⫺/⫺ mice during EAE onset (20). IFN-␥ induces
the expression of the chemokine IFN-inducible protein-10, a
cytokine known to be up-regulated in the CNS in both EAE and
MS (33, 34). A reduced migration of encephalitogenic T cells
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above background with the addition of gld ␥␦ T cells (Fig. 5K) is
probably due to other known ␥␦ T cell killing mechanisms, such as
lymphotoxin or perforin, which are intact in gld ␥␦ T cells (2).
These cumulative data demonstrate that ␥␦ T cells express a functional FasL that is required for recovery from EAE and for timely
resolution of the cellular infiltrate in the CNS of mice with EAE.
4686
Acknowledgments
This work would not have been possible without the wisdom, guidance,
and support of the late Dr. Charles A. Janeway, Jr., to whom we extend our
gratitude. We thank Shelley Morris and Vicki Boelter for assistance with
the animal colony.
Disclosures
The authors have no financial conflict of interest.
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into the CNS of ␥␦ T cell-deficient mice could explain why in
some models of EAE induction, disease was either reduced or
suppressed (24).
A reduction in the percentage of apoptosing encephalitogenic T
cells in TCR␦⫺/⫺ mice compared with control mice was consistent
throughout the EAE disease course, even during recovery on day
21 (Fig. 4). In addition, the level of proliferation of the encephalitogenic T cells on day 21 remained at a higher level than that
observed in control mice (Fig. 3). The higher level of proliferation
is probably due to the greater number of macrophages found in the
CNS serving as APC. The combined effect of decreased cell death
and a higher level of proliferation is consistent with the 7-fold
greater number of CD4 T cells present in the CNS on day 25.
These data suggest that ␥␦ T cells promote the apoptosis of encephalitogenic T cells in the CNS during the recovery phase of
EAE. One possible mechanism that we explored by which ␥␦ T
cells could induce the apoptosis of encephalitogenic T cells is
through Fas/FasL. This possibility was consistent with our previous finding that EAE was more severe in mice expressing a dysfunctional FasL (gld) than in wt mice (13). In our previous study
we were not able to identify the FasL-expressing cell required for
a normal EAE disease course. However, by reconstituting
TCR␦⫺/⫺ mice with ␥␦ T cells from either wt or gld mice, we were
able to demonstrate that both the recovery from EAE (Fig. 5) and
the timely resolution of cellular lesions (Table II) were dependent
upon the expression of a functional FasL by ␥␦ T cells. Our data
demonstrating a role for the Fas/FasL pathway in recovery from
autoimmunity are consistent with the role of ␥␦ T cell FasL-induced apoptosis of target cells expressing Fas in Borrelia-induced
Lyme arthritis in humans (35) and in a mouse model of myocarditis caused by viral infection (6).
In our model, the persistence of neurological clinical symptoms
in TCR␦⫺/⫺ mice was correlated with a sustained presence of both
macrophages and lymphocytes in the CNS on day 25. However,
even though ␥␦ T cell-deficient mice were able to eventually resolve the inflammation (Fig. 2), they remained clinically sick (Fig.
1). The lack of clinical recovery may be explained by two mechanisms: 1) neuronal tissues suffer nonrepairable damage during the
sustained inflammation; or 2) ␥␦ T cells promote the survival and
repair of neuronal tissues during EAE. In addition, ␥␦ T cells seem
to exert regulation of inflammation specifically in the CNS, because we did not find a difference in the rate of apoptosis and
proliferation of CD4 T cells in spleen of TCR␦⫺/⫺ mice with EAE
(data not shown). Thus, the regulation of inflammation in the CNS
is complex and may involve the interaction of ␥␦ T cells with a
variety of cells from both the immune and nervous systems.
Our data suggest that ␥␦ T cells have the capacity to regulate
inflammation at multiple levels during the EAE disease course.
Early in disease, ␥␦ T cells may regulate the infiltration of cells
into the CNS. Late in the disease course, they regulate the duration
of the inflammatory response, which when dysregulated in the
TCR␦⫺/⫺ mice results in chronic disease. This regulation seems to
be an innate function of ␥␦ T cells, because similar functions have
now been observed under a variety of inflammatory conditions,
including as detailed in this study autoimmunity. Because the control of inflammation in the CNS of MS patients is often a therapeutic strategy, and many ␥␦ T cells do not recognize ligands in a
similar manner as the pathogenic CD4 T cells, treatment modalities directly targeting ␥␦ T cells may not only be feasible but also
effective therapies for MS.
␥␦ T CELLS REGULATE EAE INFLAMMATION VIA Fas/FasL
The Journal of Immunology
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