To investigate the role of FAK in CD4 T cells, we first measured Fak expression in different CD4 T cell subsets. Naïve CD4 T cells were isolated from the spleens of C57BL/6 mice and cultured for 3 days under specific subset-polarizing conditions. FAK expression was highest at both mRNA and protein levels in Th17 cells among the various subsets ( Figure 1A ). Next, we explored the effect of FAK on CD4 T cell differentiation using a gene deletion system. We isolated naïve CD4 T cells from Fak^fl/fl mice and introduced a Cre-expressing retroviral vector (RV-Cre) into the cells to induce gene deletion ( Figure 1B ). We examined the effect of FAK deficiency on CD4 T cell differentiation by stimulating the control or RV-Cre-transduced cells under various subset-polarizing conditions. Since RV-Cre contains a GFP-coding sequence (36), the transduced cells can be identified by fluorescence during FACS analysis. Deletion of Fak reduced Th17 cell differentiation and slightly increased Treg cell differentiation ( Figure 1C ). Differentiation into Th1 and Th2 cells was not affected. These results suggest that FAK specifically affects Th17 cell differentiation.

To further investigate the role of FAK in Th17 and Treg cell differentiation, we measured subset-specific genes in sorted GFP+ cells from control and RV-Cre-transduced cells using flow cytometry and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Fak-deficiency reduced expression of IL-17A, while that of FOXP3 increased markedly in Th17 cells ( Figures 2A, B ). The transcript levels of Rorc and Il23r also decreased in RV-Cre-transduced Th17 cells ( Figure 2B ). Additionally, to examine whether FAK has the same effect on pathogenic Th17 cells, we stimulated RV-Cre-transduced CD4 T cells under pathogenic Th17-polarizing conditions and measured expression of Il17a and Foxp3 ( Supplementary Figure S1 ). Consistent with data from conventional Th17 cells, Fak-deficient pathogenic Th17 cells showed reduced expression of Il17a, but increased expression of Foxp3. To further confirm the effects of FAK in Th17 cells, we used an shRNA-mediated knockdown method. ShRNA-mediated knockdown of FAK ( Figure 2C ) reduced expression of Il17a ( Figure 2D ), but increased that of Foxp3 ( Supplementary Figure S2 ). Taken together, these data indicate that FAK is required for Th17 cell differentiation in vitro.

Next, we conducted RNA-sequencing (RNA-seq) to identify altered genes in RV-Cre-transduced (GFP+) Th17 cells ( Figure 2E and GEO accession # GSE298541). Expression of 1,720 genes increased, while that of 1,489 genes decreased, in RV-Cre-transduced Th17 cells compared with control Th17 cells ( Figure 2E ). Gene ontology analysis revealed significant changes in multiple biological processes in RV-Cre-transduced Th17 cells, including “cell cycle”, “cell development”, “nuclear division”, “locomotion”, and “immune response” ( Figure 2F ). Moreover, differentially expressed gene analysis, focusing on genes related to immune or inflammatory responses ( Figure 2G ), revealed that expression of Th17-related genes such as Il17a, Il17f, Rorc, and Cysltr1 (37) by RV-Cre-transduced Th17 cells decreased, while that of Treg-related genes such as Tnfrsf18 and Tnfrsf4 increased. Taken together, these data strongly suggest that FAK is expressed at high levels by Th17 cells, and essential for expression of signature genes associated with Th17 cells.

To further investigate the role of FAK in vivo, we generated Fak conditional KO mice by crossing Fak^fl/fl mice with transgenic mice expressing Cre recombinase under the control of the Rorc promoter (hereafter referred to as Fak^fl/flRorc^cre mice). To assess the efficiency of Fak deletion in Th17 cells, we measured Fak mRNA expression across each polarized CD4 T cell subset. Fak expression was greatly reduced in Th17 cells, while it remained intact in other subsets ( Supplementary Figure S3 ). Naïve CD4 T cells from WT and Fak^fl/flRorc^cre mice were stimulated under various polarizing conditions and their gene expression was measured by RT-qPCR. Expression of subset-specific markers by Th1 (Ifng), Th2 (Il4), and Treg (Foxp3) cells remained intact, regardless of Fak deletion ( Figure 3A ). Importantly, the level of Il17a mRNA ( Figure 3A ), and the percentage of IL-17A+ cells ( Figure 3B ), fell markedly in the Fak^fl/flRorc^cre Th17 cell population compared with the control (WT) Th17 cell population.

Next, we induced EAE, a Th17-driven model of MS (38, 39), by immunizing both WT control and Fak^fl/flRorc^cre mice with myelin oligodendrocyte glycoprotein (MOG35-55) peptide plus pertussis toxin. Clinical scores revealed that Fak deficiency attenuated autoimmune-related symptoms ( Figure 3C ). Histological examination of spinal cords using hematoxylin and eosin (H&E) staining and myelin basic protein (MBP) immunohistochemistry revealed significantly less inflammatory cell infiltration and reduced myelin damage in Fak^fl/flRorc^cre mice ( Figures 3D, E ). Flow cytometry analysis of CD4 T cells infiltrating the central nervous system (CNS) (spinal cord) of control and Fak^fl/flRorc^cre mice revealed that the percentage of IL-17A+ cells was lower in the CNS of Fak^fl/flRorc^cre mice ( Figure 3F ), while the percentage of FOXP3+ cells remained unchanged ( Figure 3F ). Furthermore, the percentage and number of CD4 cells and IL-17+ cells were reduced in the CNS-infiltrating mononuclear cells from Fak^fl/flRorc^cre mice compared with the control mice ( Figures 3G, H ). The percentage of IFNγ+ cells slightly increased but their number decreased ( Figure 3I ). The percentage of Foxp3+ cells remained unchanged, but their number decreased ( Figure 3J ). We also measured expression of various signature genes in CNS-infiltrating mononuclear cells by RT-qPCR. The findings revealed that cells from Fak^fl/flRorc^cre mice showed lower expression of Th17 signature genes such as Il17a, Rorc, and Il23r, as well as the Th1-related gene Ifng, with no alteration in expression of Foxp3 ( Figure 3K ). Collectively, these data demonstrate that FAK plays a critical role in an animal model of Th17-mediated autoimmune diseases.

Next, to assess the time-dependent kinetics of Fak expression in Th17 cells, we cultured naïve CD4 T cells for 3 days under Th17-polarizing conditions and harvested them at different time points. We then measured the levels of Fak transcripts, as well as those of Th17 signature genes, using Th0 cells as a control ( Figure 4A ). Fak expression was induced after 18 h of cultivation, and was highest at 24 h. Expression of Rorc, Il17f, and Il17a peaked at 18, 24, and 48 h, respectively. The level of Rorc transcripts peaked at 18 h, followed by Il17f, Il17a, and Fak. RORγT (encoded by Rorc), induced during the early differentiation stage, regulates expression of Th17 signature genes such as Il17a, Il17f, and Il23r (13). To test whether FAK expression is regulated by RORγT, we introduced a Rorc-expressing vector into Th17 cells ( Figure 4B ). Ectopic expression of Rorc increased Il17a expression but had no effect on Fak expression in Th17 cells. We then assessed the effect of RORγT functional inhibition using GSK805, a well-characterized RORγT inhibitor (40). Consistent with a previous report (40), GSK805 markedly suppressed Il17a expression, whereas Fak expression remained unchanged ( Figure 4C ). These results suggest that FAK is not a direct downstream target of RORγT.

STAT3 is a key signaling component for Th17 cell differentiation, and is activated by IL-6 (12). Once activated, STAT3 forms a homodimer with another STAT3 molecule and translocates into the nucleus to induce expression of Th17 signature genes. Typically, activation of STAT3 is triggered by JAK, located downstream of the IL-6 receptor (41); however, recent studies revealed that STAT3 can also be activated by direct interaction with FAK, in lymphatic endothelial cells and cancer cells (42–44). To assess whether FAK affects activation of STAT3 in Th17 cells, we measured pSTAT3 levels in Fak-deficient Th17 cells. Fak-deficient Th17 cells showed decreased levels of pSTAT3, but increased levels of pSTAT5 ( Figure 4D ). As STAT3 and STAT5 act reciprocally, and IL-2 is a key cytokine required for STAT5 activation (45, 46), we next examined expression of Il2 in Fak-deficient Th17 cells to determine whether FAK affects the reciprocal roles of STAT3 and STAT5 ( Figure 4E ). Il2 expression was elevated in Fak-deficient Th17 cells, along with pSTAT5 levels. Taken together, these data indicate that Fak-deficiency reduces activation of STAT3, but induces phosphorylation of STAT5 leading to IL-2 cytokine production by Th17 cells.

The NF-κB pathway plays a crucial role in various biological processes in CD4 T cells, including survival, maintenance, and differentiation (47). In a resting state, NF-κB is located in the cytosol, where it is bound and masked by the inhibitor of NF-κB (IκB). Upon stimulation, IκB kinase (IKK) becomes activated and phosphorylates IκB, triggering its degradation. The released NF-κB then translocates to the nucleus and activates the expression of its target genes. Notably, NF-κB serves as a direct substrate for FAK (48), and FAK regulates activation of the NF-κB pathway in non-immune cells (49, 50). Based on this knowledge, we formulated a hypothesis that FAK might affect Th17 cell differentiation through the NF-κB pathway. To test the hypothesis, we first examined the effect of Fak deficiency on the NF-κB pathway in Th17 cells. Upon stimulation with anti-CD3/CD28, the NF-κB pathway was activated in WT Th17 cells, but not in Fak-deficient Th17 cells ( Figure 5A ). Notably, cytosolic IκB levels did not change, and translocation of RelA into the nucleus decreased, in Fak-deficient Th17 cells compared with WT Th17 cells. Additionally, phosphorylation and degradation of IκB were downregulated ( Figure 5A ). It is noteworthy that the protein SOCS3 inhibits both the JAK/STAT (51, 52) and NF-κB signaling pathways (53) by promoting protein degradation. Expression of SOCS3 at both the protein ( Figure 5A ) and RNA ( Figure 5B ) levels increased in Fak-deficient Th17 cells, suggesting disruption of both the STAT3 and NF-κB signaling pathways.

Several STAT3 and NF-κB binding sites have been identified in the Il17a promoter and the intergenic regions between the Il17a and Il17f genes (54, 55). These binding sites play a crucial role in transcriptional regulation of Il17a expression in response to various stimuli. Therefore, we conducted chromatin immunoprecipitation (ChIP) assays to investigate whether FAK affects binding of RelA to the regulatory regions of the Il17a and Il17f genes. We observed RelA binding to the promoter and distal regulatory regions of the Il17a gene including -5kb, +10kb, and +28kb regions (56, 57), and the promoter and +5kb enhancer region of the Il17f gene in Th17 cells ( Figure 5C ); however, deletion of Fak led to a significant reduction in RelA binding to the Il17a promoter, whereas binding to the Il17f promoter remained unaltered ( Figure 5C ). Additionally, binding of RelA to the promoter of Socs3 was also inhibited in Fak-deficient Th17 cells ( Figure 5C ). These results suggest that FAK regulates RelA binding to the Il17a and Socs3 genes.

Next, to investigate the effect of FAK on activity of the Il17a promoter, we conducted a transient reporter assay. EL4 mouse thymoma cells were transfected with reporter constructs containing the Il17a promoter, with or without Fak siRNA, and then treated with phorbol 12-myristate 13-acetate (PMA) and ionomycin ( Figure 5D ). The results showed that the promoter activity of Il17a was significantly reduced upon siRNA-mediated knockdown of FAK expression ( Figure 5D ). A similar effect was observed after treatment with an NF-κB inhibitor ( Figure 5E ). To further explore the relationship between FAK and NF-κB during activation of the Il17a promoter, FAK-knockdown Th17 cells were exposed to an NF-κB inhibitor. Interestingly, the NF-κB inhibitor did not have any additional effects on reducing the activity of the Il17a promoter, suggesting that FAK and NF-κB are part of the same signaling pathway that regulates Il17a promoter activation ( Figure 5F ). To validate these findings, we used Rela-deficient cells. Naïve CD4 T cells were isolated from Rela^fl/fl mice and transduced with RV-Cre under Th17-polarizing conditions. The resulting Rela-deficient Th17 cells exhibited a significant reduction in IL-17A expression at both the protein ( Figure 5G ) and mRNA levels ( Figure 5H ). Expression of Th17-related genes Rorc and Il23r also decreased ( Figure 5H ). However, treatment with a specific FAK inhibitor PND1186, a pyridine reversible inhibitor of FAK (58), did not alter expression of IL-17A in Rela-deficient Th17 cells ( Figure 5I ). In summary, these results suggest that FAK plays a critical role in Th17 cell differentiation by facilitating recruitment of RelA, an NF-κB subunit, to the Il17a promoter and distal regulatory regions and stimulating the promoter activity.

The above results suggest that FAK regulates differentiation of Th17 cells, as well as Th17-mediated disorders. Therefore, we investigated whether FAK inhibitors prevent or treat diseases associated with Th17 cells. To assess the effect of FAK inhibitors on Th17 cell differentiation in vitro, we cultured naïve CD4 T cells under Th17-polarizing conditions in the presence of varying concentrations of PND1186. Flow cytometry analysis revealed a dose-dependent reduction in expression of IL-17A, accompanied by an increase in FOXP3 expression ( Figure 6A ). Additionally, Treg cells generated by culturing naïve CD4 T cells with PND1186 exhibited a slight increase in the FOXP3+ cell population ( Figure 6B ). The dose-dependent changes were confirmed by RT-qPCR to detect Il17a and Foxp3 transcripts ( Figure 6C ). The increase of Foxp3 mRNA levels was more pronounced than that in the percentage of FOXP3+ cell population ( Figure 6D ). To evaluate whether PND1186 affects Th17 cell death or proliferation, we performed Annexin V and 7-AAD staining to assess apoptosis and measured the Ki-67 expression level as an indicator of proliferative activity. Although a slight increase in apoptosis was observed at the highest concentration, overall cell death was comparable to that of the control ( Supplementary Figure S4A ). Therefore, the reduction in IL-17A+ cell frequency is unlikely to be due to apoptosis. Analysis of Ki-67 mean fluorescence intensity revealed only slightly reduced proliferation of Th17 cells across all inhibitor-treated conditions, suggesting that the effect of PND1186 on Th17 differentiation is not due to its influence on cell proliferation ( Supplementary Figure S4B ). These findings suggest that PND1186 modulates the balance between Th17 and Treg cells. To validate these results, we utilized an independent FAK inhibitor, GSK2256098 (59). Treatment with GSK2256098 also led to a dose-dependent reduction in IL-17A expression at both the protein and transcript levels under Th17-polarizing conditions, and an increase in FOXP3 expression under Treg-polarizing conditions ( Supplementary Figure S5 ). Consistent with the effects observed in Fak-deficient cells ( Figure 4D ), treatment with PND1186 significantly reduced the amount of pSTAT3, but increased that of pSTAT5, in Th17 cells ( Figure 6E ). Moreover, PND1186 reduced IκB phosphorylation and degradation, thereby inhibiting nuclear translocation of RelA ( Figure 6F ), as well as binding of RelA to the Il17a and Il17f promoters and distal regulatory regions ( Figure 6G ). Collectively, these data indicate that specific FAK inhibitors downregulate the STAT3 and NF-κB signaling pathways, thereby repressing differentiation of Th17 cells in vitro.

To investigate the ability of a selective FAK inhibitor to inhibit the development of autoimmune diseases in vivo, we conducted experiments using an EAE model. EAE was induced in C57BL/6 mice, which were then treated daily with PND1186 (intraperitoneal injection; 50 mg/kg) or vehicle control. Remarkably, PND1186 protected mice from EAE, leading to a dramatic delay in disease onset and a notable reduction in disease severity ( Figure 7A ). Histological analysis with H&E and Luxol fast blue staining revealed a marked reduction in inflammatory infiltration and demyelination in the spinal cord of PND1186-treated mice ( Figures 7B, C ). Flow cytometry analysis of the CNS-infiltrating (spinal cord) CD4 T cells revealed a remarkable decrease in the percentage and number of IL-17A+ cells and GM-CSF+ cells, but a slight increase in those of Foxp3+ cells, in PND1186-treated mice compared with those in control mice ( Figure 7D ). Furthermore, administration of PND1186 reduced the percentage and number of CD4 cells, IL-17A+ cells, IFNγ+ cells, and GM-CSF+ cells among CNS-infiltrating mononuclear cells ( Figures 7E–H ). The percentage, but not the number, of Treg cells in the CNS of PND1186-treated mice was higher than that in vehicle controls ( Figure 7I ). Additionally, PND1186 significantly reduced the ratio and number of CNS-infiltrating granulocytes, while the number of CD8 cells and macrophages fell only slightly ( Supplementary Figure S6 ). Consistent with the flow cytometry data, CNS-infiltrating mononuclear cells in PND1186-treated mice exhibited markedly lower levels of Il17a, Rorc, Il23r, and Ifng than those in control mice ( Figure 7J ). Conversely, these cells expressed higher level of Foxp3 mRNA than control cells ( Figure 7J ). Taken together, these results indicate that the selective FAK inhibitor limits progression of EAE by suppressing Th17 differentiation.

Female C57BL/6 mice (6–8 weeks old) were purchased from Daehan Bio Link. Fak ^fl/fl (77), and Rela ^fl/fl (78) mice were purchased from the Jackson Laboratory. All animal experiments were approved by the Sogang University Institutional Animal Care and Use Committee (approval no. IACUCSGU2019_09).

Naïve CD4 T cells were isolated from 6–8-week-old female mice using a Mojosort™ mouse naïve CD4 T cell isolation kit (BioLegend). Purified naïve CD4 T cells were then activated by plate-bound anti-CD3ϵ (10 μg/ml) and soluble anti-CD28 (5 μg/ml) antibodies. The following antibodies and recombinant cytokines were used to differentiate each cell subset. Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml). For pathogenic Th17 cell differentiation, naïve CD4 T cells were cultured with mouse recombinant IL-6 (50 ng/ml), mouse recombinant IL-1β (2 ng/ml), mouse recombinant IL-23 (5 ng/ml, Biolegend), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml). PND1186, GSK2256098, and GSK805 were purchased from Selleck Chemicals and administered at the indicated dosages. All inhibitors were dissolved in DMSO, which served as the vehicle control in all experiments.

Cells were harvested and restimulated for 4 h with the eBioscience™ cell stimulation cocktail (eBioscience). The stimulated cells were then fixed and permeabilized (eBioscience) prior to staining in accordance with the manufacturer’s instructions. Briefly, cells were stained with PerCP/Cy5.5-conjugated anti-IL-17A (BioLegend) and FITC-conjugated anti-FOXP3 (eBioscience) antibodies. For phospho flow cytometry, cells were stimulated for 3 days with Dynabeads™ mouse T-activator CD3/CD28 (Thermo Fisher Scientific) and fixed and permeabilized in fixation buffer (BioLegend), followed by staining with FITC-conjugated anti-pSTAT3 (BD Biosciences) and PerCP/eFluor 710-conjugated anti-pSTAT5 (eBioscience) antibodies. Stained cells were analyzed using Accuri C6 Plus flow cytometer (BD Biosciences). Data were analyzed with Flowjo software.

A total of 1.8 X 10⁶ Phoenix Eco cells were transfected with control MIEG3 (WT) or the RV-Cre retroviral plasmid (36) (a gift from Chen Dong) (2 μg) and the pCL-Eco helper plasmid (1 μg) as previously described (79). Naïve CD4 T cells were activated with plate-bound anti-CD3 and soluble anti-CD28 for 20–24 h. The T cell culture medium was replaced with virus-containing medium (i.e. supernatants from Phoenix Eco cells) supplemented with polybrene (4 μg/ml, Sigma Aldrich) and the culture plates were spun down (1600 x g, 90 min, RT) to facilitate infection. The cells were cultured under appropriate polarizing conditions for 3 days. GFP+ cells were sorted using FACSAria III (BD Bioscience) or FACSAria Fusion (BD Bioscience) and further analyzed.

Fak-shRNA plasmids were cloned into an MSCV-LMP empty vector (GE Healthcare) in accordance with the manufacturer’s instructions. The following 97-mer nucleotides were used as templates: shRNA #1: 5’-TGCTGTTGACAGTGAGCGATGTCTTCAAATGATTGTGTAATAGTGAAGCCACAGATGTATTACACAATCATTTGAAGACACTGCCTACTGCCTCGGA-3’; shRNA #2: 5’-TGCTGTTGACAGTGAGCGACTACTTGATGTTATTGATCAATAGTGAAGCCACAGATGTATTGATCAATAACATCAAGTAGGTGCCTACTGCCTCGGA-3’; and shRNA #3: 5’-TGCTGTTGACAGTGAGCGCACTCAAACAGTGAAGACAAAGTAGTGAAGCCACAGATGTACTTTGTCTTCACTGTTTGAGTATGCCTACTGCCTCGGA-3’. Each shRNA was transduced into CD4 T cells using the retroviral transduction method as described above.

Cells were harvested, washed with ice-cold PBS, and lysed at 4°C for 15 min with RIPA buffer (Sigma Aldrich) containing protease inhibitor (GenDEPOT). To prepare cytosolic and nucleic extracts, cells were harvested, washed, and lysed at 4°C for 10 min with hypotonic buffer containing protease inhibitor. After spinning down the cell suspension, the supernatants were collected for use as cytosolic extracts. The remaining pellets were suspended with RIPA buffer and incubated at 4°C for 15 min. These were used as nucleic extracts. Each extract was mixed with a 5X loading buffer (Thermo Fisher Scientific) and boiled for 5 min. Western blot analysis was performed as described (80). The following antibodies were used: anti-FAK, anti-p65, anti-IκB, anti-pIκB, anti-STAT3, anti-pSTAT3, anti-STAT5, anti-pSTAT5 (all from Cell Signaling Technology), anti-β-Actin (Santa Cruz Biotechnology), anti-SOCS3 (Santa Cruz Biotechnology), and anti-LaminB (Abcam).

Cells were harvested, homogenized in TRI reagent (Molecular Research Center), and total RNA was isolated according to the manufacturer’s instructions. Reverse transcription was performed using TOPscript RT (Enzynomics), and RT-qPCR was performed using TOPreal™ qPCR 2X PreMIX (Enzynomics) for SYBR Green, a TaqMan probe, and a LightCycler 96 (Roche). The primer sequences are listed in Supplementary Table S1 .

Cells (3.0 × 10⁶) were harvested, fixed with 1% formaldehyde solution for 10 min at room temperature, and then subjected to a ChIP assay using the Magna ChIP™ A/G chromatin immunoprecipitation kit (Merck Millipore) according to the manufacturer’s instructions. Cell extracts were immunoprecipitated with either an anti-RelA or normal rabbit IgG (Cell Signaling Technology) antibodies. RT-qPCR was performed using eluted DNA. The primer sequences are listed in Supplementary Table S2 .

The EL4 cells were transfected by electroporation with a pGL3-Il17a promoter vector, a pRL Renilla luciferase reporter vector, and either control or Fak siRNA (Santa Cruz Biotechnology). After transfection, the cells were incubated for 20 h in complete medium. On the following day, cells were stimulated for 4 h with PMA (50 ng/ml) and ionomycin (1 μM). Luciferase activity was measured using a dual-luciferase reporter assay system (Promega). Relative luciferase activity was calculated by dividing the activity of Firefly luciferase by that of Renilla luciferase.

On Day 0, EAE was induced in female C57BL/6 mice (8–10 weeks old) using an EAE induction kit (Hooke Laboratories). For treatment with PND1186, mice were injected intraperitoneally with inhibitor or vehicle solution (50 mg/kg/day) starting from Day 3. Mice were monitored daily to measure the clinical score, which was assessed as follows: 0: no symptoms; 1: limp tail; 2: weakness of hind legs; 3: complete paralysis of hind legs; 4: complete hind leg and partial front leg paralysis; 5: moribund state. CNS-infiltrating cells were isolated as described previously (81). Isolated mononuclear cells were stimulated for 4 h with eBioscience™ cell stimulation cocktail, and then stained with the following antibodies: FITC-conjugated anti-FOXP3 (eBioscience), FITC-conjugated anti-GM-CSF, PE-conjugated anti-IFNγ, PerCP/Cy5.5-conjugated anti-IL-17A, APC-conjugated anti-CD4, FITC-conjugated anti-CD8α, PE-conjugated anti-CD11b, and PerCP/Cy5.5-conjugated anti-GR-1 (all from BioLegend), and APC-conjugated anti-CD45 antibody. Additionally, some of the isolated spinal cords were subjected to H&E and luxol fast blue staining.

Data are presented as the mean ± standard deviation. Statistical differences between the groups were analyzed using the Student t test. P values < 0.05 were considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001).