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The Journal of Immunology, 2007, 178: 1372-1378.
Copyright © 2007 by The American Association of Immunologists, Inc.

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*,{dagger}, Klaus V. Toyka{dagger}, Magdalena Tary-Lehmann* and Paul V. Lehmann2,*

* Department of Pathology, Case Western Reserve University, Cleveland, OH 44106; and {dagger} Clinical Research Group for Multiple Sclerosis, Department of Neurology, University of Würzburg, Würzburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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-{gamma} 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 IL-17-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-{gamma}-producing cells, with the exception of IL-17 producers far outnumbering the IFN-{gamma} 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interleukin-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-{gamma} and IL-17, whereas other clones do not coexpress IL-17 with either IFN-{gamma} or IL-4 (5). IL-17-producing T cells have been shown to coexpress GM-CSF under certain conditions (6). Unlike IFN-{gamma}-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 cells from naive precursor cells is inhibited by IFN-{gamma} 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-{gamma} 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-{gamma} 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-{gamma} 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 that treatment with anti-IFN-{gamma} 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-{gamma}-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-{gamma}-producing cells (24) also apply for the IL-17-producing effector cells in EAE.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
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 µl 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 x 105 draining lymph nodes (drLN)-, or 1.5–2.5 x 105 CNS-derived mononuclear cells were plated. PLPp was tested at 20 µg/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.

Cytokine measurements by ELISPOT and computer-assisted ELISPOT image analysis

ImmunoSpot M200 plates from Cellular Technology were coated overnight with the capture Abs in sterile PBS. R46A2, at 4 µg/ml (BD Pharmingen), was used for IFN-{gamma}, JES6-1A12 at 4 µg/ml (BD Pharmingen) for IL-2, and TC11-18H10.1 at 2 µg/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 incubated at 37°C, 5% CO2 for 20 h (IFN-{gamma}, 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-{gamma}, 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
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.


Figure 1
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  		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 µg/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.

 

Figure 3
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  		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 x 105 drLN-, or 1.5–2.5 x 105 CNS-derived mononuclear cells were plated. IL-17, IFN-{gamma}, and IL-2 assays were done in parallel in the presence or absence of PLPp (20 µg/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.

 

Figure 4
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  		FIGURE 4. Organ distribution of PLPp-specific 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.

 

Figure 5
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  		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.

 
Before the onset of EAE, high frequencies of IL-17-producing PLPp-specific T cells accumulate in the immune periphery

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 "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-{gamma}-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-{gamma}-producing cells was recovered from the spleen. The data show that a considerable PLPp-specific effector cell mass builds up in the immune periphery before these cells migrate to the CNS in significant numbers.


Figure 2
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  		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.

 
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

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-{gamma}- and IL-2-producing PLPp-specific T cells. While IL-17 producing cells only slightly outnumbered IFN-{gamma}-producing cells in the spleen and drLN. In the CNS a clear dominance of IL-17 producers was seen over IFN-{gamma} 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-{gamma}-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-{gamma} or IL-2—a notion that has been confirmed by others using independent approaches (4).

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-{gamma} or IL-2)-producing cells were detected in the CNS of such mice (Fig. 5). IL-17 (and IFN-{gamma} 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 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 IL-17-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-{gamma}- 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.


Figure 6
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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-17-producing 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 x 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.

 
The functional avidity of IL-17-producing PLPp-specific cells does not change significantly during the course of EAE

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-17-producing 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 µg/ml for day 8, 3 µg/ml for day12, and 12 µg/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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
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-{gamma}-, IL-2-, IL-4-, and IL-5-producing T cells in the course of EAE. The results showed that IFN-{gamma}-producing "Th1 effector cells" readily migrated to the CNS while IL-2-producing (but IFN-{gamma}-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 cell-mediated immune pathology, while evidence is accumulating that the IFN-{gamma}-producing memory cells might not play the central role that was originally ascribed to them. IL-17- and IFN-{gamma}-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-{gamma} 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 interested in establishing whether the IL-17-producing "Th17" cells would behave differently from the IFN-{gamma}-producing "Th-1" and the IL-2-producing T cells. (There is evidence that the IL-2-producing CD4 memory cells are distinct from the IFN-{gamma}-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-{gamma} or IL-4 neutralization: the IL-17 spots seen were comparable in size and density to IFN-{gamma} 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-{gamma}-, 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-{gamma} (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-{gamma}, 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-{gamma} producing memory/effector cell lineages. Overall, the IL-17-producing memory cells behaved rather similarly to the IFN-{gamma}-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-{gamma} 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-{gamma}-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-{gamma} (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-17- and IFN-{gamma}-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, 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-{gamma}-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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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). Back

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: pvl2{at}po.cwru.edu Back

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. Back

Received for publication November 23, 2005. Accepted for publication November 15, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Yao, Z., S. L. Painter, W. C. Fanslow, D. Ulrich, B. M. Macduff, M. K. Spriggs, R. J. Armitage. 1995. Human IL-17: a novel cytokine derived from T cells. J. Immunol. 155: 5483-5486. [Abstract]
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