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Kinetics and Organ Distribution of
IL-17-Producing CD4 Cells in Proteolipid
Protein 139−151 Peptide-Induced
Experimental Autoimmune
Encephalomyelitis of SJL Mice
Harald H. Hofstetter, Klaus V. Toyka, Magdalena
Tary-Lehmann and Paul V. Lehmann
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2007 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2007; 178:1372-1378; ;
doi: 10.4049/jimmunol.178.3.1372
http://www.jimmunol.org/content/178/3/1372
The Journal of Immunology
Kinetics and Organ Distribution of IL-17-Producing CD4 Cells
in Proteolipid Protein 139 –151 Peptide-Induced Experimental
Autoimmune Encephalomyelitis of SJL Mice1
Harald H. Hofstetter,*† Klaus V. Toyka,† Magdalena Tary-Lehmann,* and Paul V. Lehmann2*
I
nterleukin-17 is a potent proinflammatory cytokine produced
by activated memory T cells. It was first identified as a rodent
cDNA transcript, termed CTLA8, isolated from an activated
T cell hybridoma (1). Its initial characterization demonstrated
that IL-17 promotes the production of other cytokines and chemokines from a variety of cell types and acts as a chemoattractant for monocytes and neutrophils. Furthermore, IL-17 was
shown to induce IL-6 in an inflammatory environment (2) and
to induce the up-regulation of costimulatory molecules such as
ICAM-1 (3).
Although proinflammatory in nature, IL-17 does not appear to fit
the “Th1/Th2” categorization (4). In patients with autoimmune disease, some T cell clones were found to coexpress IFN-␥ and IL-17,
whereas other clones do not coexpress IL-17 with either IFN-␥ or
IL-4 (5). IL-17-producing T cells have been shown to coexpress
GM-CSF under certain conditions (6). Unlike IFN-␥-producing
“Th1” cells whose differentiation is IL-12 dependent, differentiation into IL-17-expressing memory/effector T cells was shown to
be independent of IL-12 but dependent on IL-23 (7, 8). Therefore,
it appears that IL-17-producing T cells are an independent effector
cell lineage that has been designated “Th17” (4, 9). Th17 cell
differentiation was shown to depend on costimulation by CD28
and ICOS (9) but to be independent of the transcription factors
STAT1, STAT4, STAT6, or T-bet (4). The development of Th-17
*Department of Pathology, Case Western Reserve University, Cleveland, OH 44106;
and †Clinical Research Group for Multiple Sclerosis, Department of Neurology, University of Würzburg, Würzburg, Germany
Received for publication November 23, 2005. Accepted for publication November
15, 2006.
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 by a travel scholarship of the European Neurological
Society (to H.H.H.) and a nonrestricted research grant by Biogen Idec Deutschland (to
H.H.H. and K.V.T.). This work was also supported by grants to P.V.L. from the
National Institutes of Health (NS-39434) and to M.T.L. (AI-47756).
2
Address correspondence and reprint requests to Dr. Paul V. Lehmann, Department
of Pathology, Wolstein Building, Room 5129, Case Western Reserve University,
Cleveland, OH 44106-4943. E-mail address: [email protected]
www.jimmunol.org
cells from naive precursor cells is inhibited by IFN-␥ as is the
function of CD4 memory/effector cell lineages that secrete IL-17
(Th-17)3 (9). These data not only confirm that Th-17 cells are a
unique Th cell lineage that mediates tissue inflammation (4),
but that they also provide a basis for understanding how disrupted IFN-␥ production enhances the development of a pathogenic Th17 effector cell response exacerbating autoimmune
disease.
IL-17’s pleiotropic biologic properties and the associations with
several human autoimmune diseases and their animal models indicate that this cytokine may play a critical role in T cell mediated
inflammation. Overproduction of IL-17 has been associated with
several diseases in which T cells are thought to be significantly
involved such as transplant rejection (10), systemic sclerosis (11),
inflammatory bowel disease (12), rheumatoid arthritis, and its animal model, collagen-induced arthritis (13). IL-17-producing T
cells have been demonstrated in the joints of patients with RA. In
collagen-induced arthritis, the blockage of IL-17 was shown to
ameliorate and even prevent pathology (14). In multiple sclerosis,
elevated levels of IL-17 were detected in patient cerebrospinal
fluid (15), and a gene microarrays study of chronic CNS lesions
showed that among chronic inflammation associated molecules,
IL-17 was most prevalent (16).
Since IFN-␥ and TNF are the “classic” proinflammatory cytokines, it was widely held that they are the central effector molecules of T cells in experimental autoimmune encephalomyelitis
(EAE). This notion was clearly challenged by the finding that
IFN-␥ knockout (KO) mice, as well as TNF KO mice, are highly
susceptible to EAE (17). It also has been shown that IL-12 KO
mice are able to maintain a vigorous inflammation in the CNS,
characterized by strong production of IL-17 and GM-CSF in the
target organ itself (18). Furthermore, evidence has accumulated
3
Abbreviations used in this paper: Th-17, CD4 memory/effector cell lineages that
secretes IL-17; drLN, draining lymph node; EAE, experimental autoimmune encephalomyelitis; KO, knockout; PLP, proteolipid protein; PLPp, proteolipid protein peptide 139 –151; PTX, pertussis toxin.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
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In experimental autoimmune encephalomyelitis (EAE), the production of proinflammatory cytokines by neuroantigen-specific T
cells is thought to initiate and maintain the inflammatory autoimmune pathology. Because gene knockout strategies have shown
that IFN-␥ and TNF are not essential for EAE development, there is increasing interest in establishing the role of other proinflammatory cytokines, primarily IL-17 in EAE. We used an IL-17 ELISPOT assay to track the neuroantigen-specific IL17-producing T cells at single-cell resolution in various organs of SJL mice undergoing PLP 139 –151-induced EAE. Overall,
the migration patterns and population kinetics of the PLP 139 –151-specific IL-17-producing CD4 cells were reminiscent of
the IFN-␥-producing cells, with the exception of IL-17 producers far outnumbering the IFN-␥ and IL-2 producers in the
inflamed CNS. The selective enrichment of IL-17-producing CD4 cells in the CNS is suggestive of the pathogenic role of an
independent (non-Th1) IL-17-producing proinflammatory effector T cell class in EAE. The Journal of Immunology, 2007,
178: 1372–1378.
The Journal of Immunology
Materials and Methods
Animals, Ags, and treatments
Female SJL/J mice at age 6 – 8 wk were purchased from The Jackson
Laboratory and maintained in the specific pathogen-free animal facility
of Case Western Reserve University. PLP 139 –151 peptide (PLPp)
(HSLGKWLGHPDKF) was synthesized by Princeton Biomolecules.
IFA was purchased from Invitrogen Life Technologies. PLPp/CFA was
prepared by mixing PLPp in CFA yielding a final concentration of 1 mg/
ml. PLPp/CFA was injected subcutaneously at two different sites of the
flank. Pertussis toxin (PTX) (200 ng; List Biological Laboratories) was
injected twice i.p., in 500 ␮l of saline, first at the time of the CFA immunization and the second time 24 h later. Mice were assessed daily for the
development of paralytic symptoms, and the severity of disease was recorded according to a standard scale: grade 1, floppy tail; grade 2: hind leg
weakness; grade 3, full hind leg paralysis; grade 4, quadriplegia; and grade
5, Death. Access to food and drinking water was ensured for all mice,
including those with paralysis.
Cell preparations and purifications from organs
Single-cell suspensions from the various immune organs and the CNS were
prepared as described previously (24). The cells were counted by trypan
blue exclusion and 106 spleen-, 5 ⫻ 105 draining lymph nodes (drLN)-, or
1.5–2.5 ⫻ 105 CNS-derived mononuclear cells were plated. PLPp was
tested at 20 ␮g/ml or in serial dilutions in the concentrations specified.
Cultures containing medium alone functioned as the negative control. Single-cell suspensions were tested either as bulk populations or as purified
cell fractions, as specified. Subpopulations of T cells were isolated using
commercially available murine T cell isolation columns (R&D Systems),
following the instructions of the manufacturer. Eluted cells were washed,
counted by trypan blue exclusion and resuspended at appropriate concentrations for use in the various assays. Cell separations routinely yielded
90 –95% purity as controlled by FACS analysis.
FIGURE 1. PLPp-elicited IL-17 is produced by CD4 cells as measured
by ELISPOT. CD4 cells were purified from spleen cells of SJL mice immunized with PLPp/CFA/PTX. The specified numbers of CD4 cells were
plated in serial dilution with a constant number (500,000/well) of irradiated
naive SJL spleen cells. An IL-17 ELISPOT assay was performed in the
presence of PLPp (at 20 ␮g/ml) as specified in Materials and Methods.
Each data point represents the mean spot number obtained in triplicate
wells; SD was ⬍20% of the mean for all data points (data not shown). One
of two experiments with similar results is shown.
bated at 37°C, 5% CO2 for 20 h (IFN-␥, IL-2, and IL-17). After washing
with PBS, detection Abs were added overnight at 4°C. XMG1.2-biotin (BD
Pharmingen) was used for IFN-␥, rat anti-mouse IL-2-biotin (JES6-5H4;
BD Pharmingen) was used for IL-2, and rat anti-mouse IL-17-biotin
(TC11-8H4.1; BD Pharmingen) was used for IL-17. The plate-bound second Ab was then visualized by adding streptavidin-alkaline phosphatase
(DakoCytomation) and NBT/5-bromo-4-chloro-3-indolyl phosphate substrate (Bio-Rad). Plates were dried overnight and images of the ELISPOT
wells were captured with an ImmunoSpot Series 3B Analyzer (Cellular
Technology). Image analysis of the ELISPOT results was performed with
the ImmunoSpot 3.2 Analysis Software (Cellular Technology).
Results
PLPp/CFA/PTX immunization primes CD4 cells that
produce IL-17
CD4-positive cells were purified from spleen cells of mice immunized with PLPp/CFA. The purified CD4 cells were serially diluted
on an APC layer consisting of a constant number of naive irradiated syngeneic spleen cells. As shown in Fig. 1, the numbers of spots
elicited by PLPp was linearly proportional to the number of CD4 cells
plated. The linear function seen between the number of IL-17 spots
and CD4 cells plated on a constant number of splenic APC strongly
argues against bystander reactions by non-CD4 cells. Bystander
reactions that CD4 cells induce, e.g., by cytokine production typically result in parabolic curves—as we have seen for IL-4 produced by mast cells in ELISPOT assays (25). PLPp peptide also
induced IL-17 spots in bulk spleen cells of PLP-immunized mice
(see Figs. 3–5), but IL-17 was not induced by PLPp or control Ags
HEL and OVA in spleen or lymph node cells of nonimmunized
mice that contain eosinophils and other cell types of the innate
immune system that may be able to produce IL-17. Therefore, the
IL-17 detected in bulk cell populations was produced by Ag-specific memory CD4 cells.
Cytokine measurements by ELISPOT and computer-assisted
ELISPOT image analysis
Before the onset of EAE, high frequencies of IL-17-producing
PLPp-specific T cells accumulate in the immune periphery
ImmunoSpot M200 plates from Cellular Technology were coated overnight with the capture Abs in sterile PBS. R46A2, at 4 ␮g/ml (BD Pharmingen), was used for IFN-␥, JES6-1A12 at 4 ␮g/ml (BD Pharmingen) for
IL-2, and TC11-18H10.1 at 2 ␮g/ml (BD Pharmingen) was used for IL-17.
The plates were blocked for 1 h with sterile PBS containing 1% BSA and
washed three times with sterile PBS. Cells were plated in HL-1 medium
(BioWhittaker) with and without PLPp. Subsequently plates were incu-
SJL mice were immunized with PLPp in CFA and with PTX following the protocol that induces EAE in these mice (see Materials
and Methods): in our colony, ⬎90% of the mice developed EAE
with the onset of disease between days 11 and 12, and a mean
score of 2.7 at the peak of the acute disease on day 20 —the typical
disease course in our colony is shown in Fig. 2. On day 8, in the
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that treatment with anti-IFN-␥ Abs can have an EAE-deteriorating
effect (19). In contrast to the classic “Th1” effector arm, there is
increasing evidence that the IL-17-IL-23 pathway plays a critical
role in EAE. IL-23 KO mice are resistant to the induction of EAE
(20)—this finding implicates IL-17-producing T effector cells because IL-23 production by cells of the innate immune system is
required for the differentiation of IL-17-producing T cells. Moreover IL-6 KO mice are resistant to EAE development (21). Also
this finding suggests the involvement of IL-17 producing effector
cells: IL-17 activates cells of the local inflammatory environment
to produce IL-6 (2).
With Th17 cells emerging as an independent lineage of effector
cells in EAE, we have set out to characterize them. We used an
IL-17 ELISPOT assay that permits the tracking of these cells in
different tissues at single-cell resolution, establishing their relative
and absolute numbers in the respective tissues at various stages of
the disease. In addition, we have used this approach to establish the
functional avidity of the PLP 139 –151-specific IL-17-producing
CD4 cells in different organs and at various time points of the
disease. Because “Th1” and “Th2” cells express different chemokine and homing receptors and follow distinct differentiation and
migration patterns in the body (22, 23) and because IFN-␥-producing (“Th1” cells) and IL-17-producing (“Th-17”) effector cells
seem to belong to different lineages (4), it was essential to explore
whether the migration rules established previously for the IFN-␥producing cells (24) also apply for the IL-17-producing effector
cells in EAE.
1373
1374
IL-17-PRODUCING T CELLS IN EAE
builds up in the immune periphery before these cells migrate to the
CNS in significant numbers.
During acute EAE, the frequency of PLPp-specific
IL-17-producing cells is highest in the CNS, but the vast
majority of such cells continues to reside in the spleen
FIGURE 2. Clinical course of PLPp-induced EAE in our SJL colony.
Female SJL mice were immunized with PLPp/CFA/PTX, and the disease
severity was scored daily following the standard scale specified in Materials and Methods. In the representative experiment shown here, 12 mice
were immunized. The mean disease score and the SD for these mice is
shown.
FIGURE 3. Organ distribution of PLPp-specific cytokine-producing cells on day 8 after PLPp
immunization. A, Frequencies of the cytokine producing cells per million cells tested. SJL mice
were immunized with PLPp/CFA/PTX, and on day
8, cells were harvested from drLN, spleens, and
the CNS (as described in Materials and Methods).
None of the mice tested at this time displayed
signs of clinical EAE. For the ELISPOT measurements, 106 spleen-, 5 ⫻ 105 drLN-, or 1.5–2.5 ⫻ 105
CNS-derived mononuclear cells were plated. IL17, IFN-␥, and IL-2 assays were done in parallel in
the presence or absence of PLPp (20 ␮g/ml).
Spleen and drLN cells were tested in three replicate wells—for these cells, data points show the
mean spot number for the specified cytokine measured in the triplicate PLPp stimulated wells for an
individual mouse with the number of spots in the
medium control well subtracted. CNS assays were
performed in single wells for PLPp and medium
control due to the low number of cells obtained.
Spot numbers are normalized for 1 million cells of
each organ. B, The total number of cytokine-producing cells is shown for each individual mouse
and the specified organs as obtained by multiplying the frequencies of PLPp-induced cytokine
spots for each mouse with the number of cells obtained from the respective organs.
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“pre-EAE onset stage,” PLPp induced IL-17-producing cells were
detected in the drLN and in the spleen but not in the CNS (Fig.
3A). Within each group of mice, the frequency of IL-17 producing
PLPp-specific cells was comparable in each organ to the numbers
of IL-2- and IFN-␥-producing PLPp-specific cells (Fig. 3A). Multiplying the frequencies of PLPp specific cells with the total number of cells obtained from an organ permitted us to calculate the
absolute number of PLPp-specific CD4 cells in each organ (Fig.
3B); on day 8, the vast majority of PLP-p specific IL-17-, IL-2-,
and IFN-␥-producing cells was recovered from the spleen. The
data show that a considerable PLPp-specific effector cell mass
EAE onset typically occurred on day 11–12. We sacrificed mice on
day 12, and tested their drLN cells, spleen cells and the CNS
isolates as outlined above. During acute EAE, PLPp-specific IL-17
producing cells became detectable in the CNS, occurring there in
higher frequencies than in the drLN and spleen (Fig. 4A). This
organ distribution pattern was also observed for IFN-␥- and IL-2producing PLPp-specific T cells. While IL-17 producing cells only
slightly outnumbered IFN-␥-producing cells in the spleen and
drLN. In the CNS a clear dominance of IL-17 producers was seen
over IFN-␥ and IL-2 producers. Fig. 4B shows the absolute numbers of PLPp-specific cells in each of these organs. Despite their
high frequency in the CNS, the vast majority of the PLPp-specific
cytokine-producing cells continued to be present in the spleen. Therefore, only a minor fraction of the primed neuroantigen-specific T cell
pool migrated to the target organ. In the spleen of individual mice
the frequencies of IL-17- and IL-2-producing T cells were regularly higher than that of the IFN-␥-producing T cells. Because
these frequency differences of cytokine producing cells were seen
in parallel experiments involving the same cell material, this finding suggests that IL-17 expressing cells do not coexpress IFN-␥ or
IL-2—a notion that has been confirmed by others using independent approaches (4).
The Journal of Immunology
1375
After recovery from EAE, considerable numbers of
IL-17-producing PLPp-specific cells persist in the
immune periphery, but not in the CNS
After the acute onset of PLPp-induced EAE, the SJL mice went
into remission around day 38, after which they developed a relapsing-remitting disease course— by day 56 their clinical disease
stabilized around an average score of 1 (Fig. 2). On day 56, drLN,
spleens, and cells isolated from the CNS were tested as above. No
PLPp-specific IL-17 (or IFN-␥ or IL-2)-producing cells were detected in the CNS of such mice (Fig. 5). IL-17 (and IFN-␥ and
IL-2)-producing cells continued to be detectable in the spleens, but
their frequencies were reduced compared with those seen on days
8 and 12 (Fig. 5A vs Figs. 3A and 4A). It is likely that by this time
the first wave of effector T cells (that had been engaged by peripheral immunization) has exhausted and that the second wave T
cells (that have been engaged by determinant spreading) drive the
autoimmune process in the CNS. Relative to the earlier time
points, the reactivity in drLN has also declined by day 56. The
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FIGURE 4. Organ distribution of PLPpspecific cytokine producing cells on day 12
after PLPp immunization. A, Frequencies of
cytokine-producing cells. B, Total number of
cytokine-producing cells is shown. Legend
to Fig. 2 applies, with the exception that the
cells were tested on day 12 after immunization, and that all of the mice displayed clinical EAE with a grade ⬎2. Note that in A,
the scale on the y-axis is lined up to 800/
million, above which a logarithmic scale was
used to accommodate the high frequencies
of IL-17-producing cells in the CNS.
absolute numbers of cytokine producing cells on day 56 showed
that the majority of the PLPp-specific T cells producing either
cytokine continued to be present in the spleen (Fig. 5B).
The disappearance of cytokine-producing cells from drLN and the
CNS, along with the decreased numbers in the spleen, suggest that the
PLPp-specific T cell pool has undergone contraction over the 56-day
observation period. To assess the magnitude of this contraction, we
calculated the total clonal mass for each mouse and each time
point, consisting of the sum of PLPp-specific cytokine producing
cells recovered from spleen, drLN and the CNS. The results are
shown in Fig. 6A. The absolute numbers of the PLPp-specific IL17-producing cells averaged 37,327 ⫾ 8,913 on day 8, 43,686 ⫾
10,687 on day 12, and 23,200 ⫾ 9,346 on day 56. The decline in
numbers from day 12 to day 56 reached statistical significance
( p ⬍ 0.01). Also, the number of IFN-␥- and IL-2 producing cells
declined by day 56 to ⬃50% of the day 12 value. Therefore, an
exhaustion of the PLPp-specific T cell population occurred, but it
affected only approximately half of the T cell pool.
1376
IL-17-PRODUCING T CELLS IN EAE
FIGURE 5. Organ distribution of PLPp-specific cytokine producing cells on day 56 after PLPp immunization. Legend to Fig. 2 applies, with the exception that
the cells were tested on day 56 after immunization, and
that all of the mice displayed stable EAE scores for ⬎10
days before testing.
Functional avidity can be defined as the peptide dose at which 50%
of the peptide-specific T cells become activated (Keff value). We
tested whether the chronic autoimmune process would lead to a T
cell avidity-based repertoire selection in the IL-17-producing CD4
cell population and/or whether the generation of regulatory T cells
during EAE would result in changes in functional affinity/avidity
of IL-17-producing cells. To address these possibilities, dose-response curves were established for PLPp induced IL-17 ELISPOT
formation, and experiments were performed with pooled spleen
cells of mice that had been immunized 8, 12, and 56 days earlier
(Fig. 6B). The frequencies of maximally inducible IL-17 spots reproduced the data obtained previously: the frequencies of IL-17producing cells was higher on day 12 in the spleen (311/1,000,000)
than it was on day 8 (164/1,000,000), and it was lowest for the day
56 spleens (92/1,000,000). Relative to these numbers that constitute the respective 100% activation value for the dose-response
curve, 50% activation was reached at 3 ␮g/ml for day 8, 3 ␮g/ml
for day12, and 12 ␮g/ml for day 56, respectively. Therefore, there
was a slight shift toward a lower PLPp-specific T cell avidity in the
56-day spleen relative to the earlier time points—a shift that was
reproduced in two independent experiments. However, this shift
is minor in comparison to the ⬃10,000-fold change that we saw
in repertoire selection caused by development of self tolerance
(25). Still, this avidity shift in EAE might accentuate in disease
processes of longer duration than our observation period of
56 days.
Discussion
Previous work from our laboratory and of the community in general has focused on neuroantigen-specific “Th1” cells as the key
effector cells in EAE. In one of these studies (24), we followed the
organ distribution of IFN-␥-, IL-2-, IL-4-, and IL-5-producing T
cells in the course of EAE. The results showed that IFN-␥-producing “Th1 effector cells” readily migrated to the CNS while
IL-2-producing (but IFN-␥-negative) “Thpp cells” migrated to a
lesser extent to the target organ. Different T cell lineages express
unique homing and chemokine receptors, resulting in these cells’
unique migration patterns. In this respect, little is known to date
about the Th-17 effector cell lineage in EAE. Our studies were
designed to fill this gap.
We elected to monitor the Th-17 lineage because there is increasing evidence that these cells play an important role in T cellmediated immune pathology, while evidence is accumulating that
the IFN-␥-producing memory cells might not play the central role
that was originally ascribed to them. IL-17- and IFN-␥-producing
CD4 memory cells appear to belong to different effector cell lineages, the former requiring IL-23, the latter IL-12 for instructed
cytokine differentiation (4, 8). Genetic disruption of IL-23 renders
mice profoundly resistant to EAE (20) while the disruption of elements of the “Th1” pathway: IL-12, TNF, and IFN-␥ itself leads
to increased severity of clinical EAE symptoms (17, 27). Because
different CD4 effector/memory cell lineages, such as “Th1” and
“Th2,” are known to arise under different conditions of instructed
differentiation, as well as to express different chemokine and homing receptors resulting in different migration patterns (23), we were
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The functional avidity of IL-17-producing PLPp-specific cells
does not change significantly during the course of EAE
The Journal of Immunology
interested in establishing whether the IL-17-producing “Th17”
cells would behave differently from the IFN-␥-producing “Th-1”
and the IL-2-producing T cells. (There is evidence that the IL-2producing CD4 memory cells are distinct from the IFN-␥-producing memory cells (25).)
We detected a very strong IL-17 signal when testing freshly
isolated cells without the requirement for additional treatment of
the cells such as IFN-␥ or IL-4 neutralization: the IL-17 spots seen
were comparable in size and density to IFN-␥ spots suggesting
similar per cell productivity rates for both cytokines. We believe
that the reason for this is as follows: when T cells are isolated from
lymphoid tissues they appear to be in a quiescent state, because in
the absence of added Ag (in the medium control wells) no IL-4-,
IFN-␥-, or IL-17-secreting cells are detectable. Even when infiltrating lymphoid cells were isolated from the CNS very few cells
were seen to “spontaneously” produce IFN-␥ (having been recently stimulated by the endogenous autoantigen), and hardly any
cells produced IL-4 (summarized in our Ref. 24). Therefore, the
ELISPOT assays seem to start with resting cells and in a neutral
cytokine environment. Once Ag is provided, the Ag-specific T
cells become activated, and each T cell starts to produce the cytokine it is programmed to produce: Th1 cells producing IFN-␥,
Th17 cells IL-17, and Th2 cells IL-4. In the ELISPOT assay, the
respective cytokine is captured around the secreting cell as it is
being released, before it is diluted in the supernatant, and before
cytokine cross-talk could establish itself.
ELISPOT assays have the unique advantage over intracytoplasmic cytokine staining that they are orders of magnitudes more
sensitive for detecting rare cytokine-expressing/producing cells.
This added sensitivity is critical for the type of studies performed
here because the frequencies of the PLPp-specific cells found in
most organs and at most time points were 1/1,000. We have shown
that in PLPp-immunized mice the IL-17-producing cells are CD4
cells (Fig. 1). This result is consistent with PLPp being prone to
prime CD4 cells (it is an I-As restricted determinant (26)) and with
IL-17 expression being primarily confined to memory T cells,
mostly CD4 cells (4).
Using a newly developed IL-17 ELISPOT assay (27), we provide here a systematic characterization of the population dynamics
of the IL-17 expressing neuroantigen-specific memory/effector
cells in PLP-induced EAE in SJL mice and compare it to that of
the IFN-␥ producing memory/effector cell lineages. Overall, the
IL-17-producing memory cells behaved rather similarly to the
IFN-␥-producing memory cells. Even before the onset of EAE,
high frequencies of PLPp-specific T cells secreting all three cytokines were present in the immune periphery, that is, in the spleen
and the drLN, but none of these cell types had yet infiltrated the
CNS in high numbers. During acute EAE, PLPp-specific cells with
all three cytokine signatures appeared in the CNS in high frequencies, but IL-17-producing cells outnumbered IFN-␥ producers.
This finding might either signify that IL-17-producers (Th-17) are
preferentially recruited to the inflamed CNS or that they preferentially expand in the CNS (24, 28). Alternatively, Th17 cells might
be less susceptible to undergo apoptosis in the CNS than are the
IFN-␥-producing T cells. Whichever of these mechanisms explain
the selective accumulation of the Th17 lineage in the CNS, it is
tempting to postulate that the enrichment of these cells per se enhances their effector potential and hence pathogenicity relative to
the Th-IFN-␥ (Th1) cells.
Despite the high frequencies reached by the PLPp-specific IL-17
cells in the CNS during acute EAE, the vast majority of these cells
continued to reside in the spleen and drLN. The cells present in the
immune periphery seem to form a considerable reservoir from
which the effector cells are recruited to the target organ to participate in the actual autoaggressive inflammatory process. After the
mice recovered from active EAE, considerable numbers of IL-17and IFN-␥-producing PLPp-specific cells persisted in the immune
periphery, but they became undetectable in the CNS. These residual cells did not seem to represent remnants of a clonal selection
process because the functional affinity of the IL-17 producing
PLPp-specific cells did not show major changes in the course of
EAE—although a minor shift toward low avidity repertoires was
seen in the advanced stage of the disease (Fig. 6B) Therefore,
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FIGURE 6. Population dynamics of the PLPp-specific IL-17-producing
cells in the course of EAE. A, The “total clonal mass” of IL-17-producing
cells was calculated for each mouse and each time point by adding the total
number of these cells for spleen, drLN, and the CNS. The mean and SD of
the numbers for each individual mouse is shown for the three time points.
The difference between days 12 and 56 is statistically significant at p ⬍
0.01 using the t test. B, Functional avidity of the PLPp-specific IL-17producing cells over the course of EAE. Spleen cells were obtained on days
8, 12, and 56 after immunization with PLPp (specified by the symbols);
spleens of six mice in each group were pooled. A total of 1 ⫻ 106 spleen
cells/well was plated with the specified concentration of PLPp, and an
IL-17 ELISPOT assay was performed. The maximal number of IL-17 spots
induced was established from the dose-response curve as “100%” along
with the peptide concentration that induced 50% of the maximal IL-17
spots. The results of one of two experiments performed with similar results
are shown.
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1378
major avidity maturation of the autoimmune response did not occur, although it could be expected, if the actually autoaggressive
“high avidity” fraction of the PLPp-specific repertoire was depleted by apoptosis induction in the CNS, and only the low avidity,
and therefore nonautoaggressive clones, were spared.
In summary, although IL-17-producing CD4⫹ cells represent a
different effector cell lineage than the IFN-␥-producing “Th1”
cells, the population dynamics of both cell types appeared to be
rather similar in vivo during the course of EAE. The only major
difference was the preferential enrichment of the IL-17 producers
in the CNS, possibly enhancing their pathogenicity. Therefore, further studies are warranted to elucidate the pluripotent role of Th17
cells in organ-specific autoimmune diseases in general and EAE
and multiple sclerosis in particular.
Acknowledgment
We thank Saada Eid for excellent technical assistance.
Disclosures
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The authors have no financial conflict of interest.
IL-17-PRODUCING T CELLS IN EAE