Role of autoreactive Tc17 cells in the pathogenesis of experimental autoimmune encephalomyelitis

Abstract

Background

The pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE—an animal model of MS) is primarily mediated by T cells. However, recent studies have only focused on interleukin (IL)‐17‐secreting CD4⁺ T‐helper cells, also known as Th17 cells. This study aimed to compare Th17 cells and IL‐17‐secreting CD8⁺ T‐cytotoxic cells (Tc17) in the context of MS/EAE.

Methods

Female C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein peptides 35–55 (MOG_35–55), pertussis toxin, and complete Freund's adjuvant to establish the EAE animal model. T cells were isolated from the spleen (12–14 days postimmunization). CD4⁺ and CD8⁺ T cells were purified using isolation kit and then differentiated into Th17 and Tc17, respectively, using MOG35–55 and IL‐23. The secretion levels of interferon‐γ (IFN‐γ) and IL‐17 were measured via enzyme‐linked immunosorbent assay using cultured CD4⁺ and CD8⁺ T cell supernatants. The pathogenicity of Tc17 and Th17 cells was assessed through adoptive transfer (tEAE), with the clinical course assessed using an EAE score (0–5). Hematoxylin and eosin as well as Luxol fast blue staining were used to examine the spinal cord. Purified CD8⁺ CD3⁺ and CD4⁺ CD3⁺ cells differentiated into Tc17 and Th17 cells, respectively, were stimulated with MOG_35–55 peptide for proliferation assays.

Results

The results showed that Tc17 cells (15,951 ± 1985 vs. 55,709 ± 4196 cpm; p < 0.050) exhibited a weaker response to highest dose (20 μg/mL) MOG_35–55 than Th17 cells. However, this response was not dependent on Th17 cells. After the 48 h stimulation, at the highest dose (20 μg/mL) of MOG_35–55. Tc17 cells secreted lower levels of IFN‐γ (280.00 ± 15.00 vs. 556.67 ± 15.28 pg/mL, p < 0.050) and IL‐17 (102.67 ± 5.86 pg/mL vs. 288.33 ± 12.58 pg/mL; p < 0.050) than Th17 cells. Similar patterns were observed for IFN‐γ secretion at 96 and 144 h. Furthermore, Tc17 cell‐induced tEAE mice exhibited similar EAE scores to Th17 cell‐induced tEAE mice and also showed similar inflammation and demyelination.

Conclusion

The degree of pathogenicity of Tc17 cells in EAE is lower than that of Th17 cells. Future investigation on different immune cells and EAE models is warranted to determine the mechanisms underlying MS.

INTRODUCTION

Multiple sclerosis (MS) is an inflammatory disease that primarily damages the white matter of the central nervous system (CNS); however, its pathogenesis remains unclear. In the past few decades, MS and experimental autoimmune encephalomyelitis (EAE) have been recognized as typical CD4⁺ T cell-mediated autoimmune diseases. Multiple studies, including our previous research, have demonstrated the involvement of CD8⁺ T cells in different stages of MS/EAE and have shown that the number of CD8⁺ T cells is greater than that of CD4⁺ T cells in the brain lesion area, perivascular cuff, and white matter area. Given their critical role in the pathogenesis of MS, CD8⁺ T cells may be a significant target for MS/EAE treatment.

Interleukin (IL)−17⁺ CD4⁺ T-helper (Th17) cells and their related antibodies have the potential for treating autoimmune diseases, including MS/EAE. IL-17 and Th17-related therapies like secukinumab, ixekizumab, and brodalumab have been approved for the treatment of other diseases; however, clinical trials of these antibodies for MS have failed. Therefore, other T cell subsets, particularly CD8⁺ T cells and their subsets, should be considered.

Recent studies have identified several subsets of CD8⁺ T cells, including T cytotoxic (Tc) cells, such as Tc1 (dominant IFN-γ-secretion), Tc2 (dominant IL-4 and IL-5-secretion), Tc9 (dominant IL-9-secretion), Tc17 (dominant IL-17-secretion), and regulatory T (Treg) cells (dominant IL-10 and/or Foxp3 expression). Among them, Tc17 cells, discovered in 2009 in humans and mice, are now a focal point in global immunology research, with an emphasis on their role in disease pathogenesis and development of potential new treatment methods for tumor immunity, organ rejection following transplantation, acquired immunodeficiency syndrome, and autoimmune diseases.

Nevertheless, few studies have reported the role of Tc17 cells in MS/EAE. These cells have been detected in the peripheral blood (PB), cerebrospinal fluid (CSF), and active lesions in patients with MS. Dimethyl fumarate (DMF) treatment for MS can reduce the proportion of Tc17 cells in PB mononuclear cells, decrease the ratio of the regulatory factor RORC to TBX21. Unlike Tc1 cells, Tc17 cells are not cytotoxic; however, they can reduce the expression of T-bet and Eomes (Tc1 cell-specific transcription factors) and increase the expression of IFN-γ, cytotoxic granzyme B, and perforin.

In our previous study, we demonstrated that CD8⁺ T cells purified from EAE mice exhibited antigenic specificity and induced encephalitis, similar to our findings on CD8⁺ T cells in experimental autoimmune uveitis (EAU). Interestingly, antigen-specific CD8⁺ T cells also express IL-17 in EAU. While IL-23 can induce antigen-specific Th17 cells in EAU, TGF-β, and IL-6 can only induce non-antigen-specific Th17 cells and are not pathogenic. Although our research on EAU provides a good idea and methodological basis for this study, the results for EAE and EAU may differ owing to differences in target organs and antigens. Therefore, further research and validation using the EAE model are necessary.

In this study, we aimed to investigate the role of Tc17 cells in EAE, examine the cytological characteristics and pathogenicity of Tc17 cells, and compare Tc17 and Th17 cells. Our findings provide new research directions for understanding the pathogenesis of MS/EAE and developing targeted cell therapies. Hence, this study has significant theoretical implications and potential applications.

METHODS

Ethics approval

All experiments were conducted at the Science and Technology Innovation Center of the Hunan University of Chinese Medicine in Changsha, China. The experimental procedures were approved by the Ethics Committee of the Hunan University of Chinese Medicine (Approval No. LL2022050501). The animal treatments were conducted in accordance with the guidelines provided by the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all necessary steps were taken to minimize pain and suffering of the animals.

Selection of animals

Specific pathogen-free female C57BL/6 (B6) mice (age: 10–14 weeks; n = 20 or 60 per experiment; three experiments in total) were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd. (Changsha, Hunan, China; License no. SCXK [Hunan] 2019-0004) and housed in the Center of Experimental Animals at the Hunan University of Chinese Medicine (License no. SYXK [Hunan] 2019-0009). The mice were housed individually in cages with independent ventilation systems at a temperature of 20°C–26°C and relative humidity of 40%–60%. They had free access to food and water, and steps were taken to ensure minimal pain and suffering. The sample size was determined based on the experimental design requirements.

Antigen-induced EAE (aEAE) models

The aEAE models were established in our previous study. Mice were anesthetized by intraperitoneal injection of pentobarbital (0.2 mL/kg) per our previous protocol. To actively induce disease, mice were subcutaneously injected with a 200-mL emulsion containing 200 µg of myelin oligodendrocyte glycoprotein peptides 35–55 (MOG_35–55; amino acids 35–55 of bovine MOG; Sigma-Aldrich; purified using high-performance liquid chromatography; purity >95%) and complete Freund's adjuvant Sigma-Aldrich, St. Louis, MO, USA) over six sites at the tail base and on the flank. At 0 and 24 h after immunization, mice were intraperitoneally injected with pertussis toxin (List Biological, Campbell, CA). EAE scores were assigned on a scale of 0–5 as follows: 0, no obvious changes in motor functions of the mouse compared with nonimmunized mice; 1, limp tail; 2, limp tail and weakness of hind limbs; 3, limp tail and complete paralysis of hind limbs (most common) or limp tail with paralysis of one front and one hind limb; 4, complete hind limb and partial front limb paralysis; and 5, death or euthanasia due to severe paralysis.

Purification of MOG_35–55-specific CD4⁺ and CD8⁺ T cells

This procedure was performed according to our previously published protocol. The EAE mice were killed 14 days after immunization using sodium pentobarbital (50 mg/kg; Sinopharm Chemical Reagent Co., Beijing, China, 50 mg/kg), and T cells were isolated from the spleen using a nylon wool column (Kisker, Steinfurt, Germany). CD4⁺ and CD8⁺ T cells were purified from the spleen using CD4 and CD8 isolation kits (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), respectively. The spleen cells were incubated in 10 mL of CD8 MicroBeads and 90 mL of buffer per 1 × 10⁷ cells for 15 min in a refrigerator (2°C–8°C). The buffer was added to obtain a final volume of 500 mL, and 5 × 10⁷ cells were obtained. Thereafter, the cells were separated into bound and unbound cells on a magnetic separator column (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and washed with 15 mL of medium according to the manufacturer's protocol. The flow-through fraction containing CD4- or CD8-enriched cells was collected, and the purity of the isolated cell fraction was determined using flow cytometry.

In accordance with our previously published protocol, we double-stained aliquots of 2 × 10⁵ cells with combinations of allophycocyanin–fluorescein isothiocyanate- or phycoerythrin-conjugated monoclonal antibodies against CD3, CD4, or CD8 following the manufacturer's instructions (Biolegend, San Diego, CA, USA). Data collection and analysis were performed using a BD LSRFortessa 4 flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (FlowJo Co., San Diego, CA, USA).

Preparation of MOG_35–55-specific Th1, Tc1, Th17, and Tc17 cells

For the preparation of Th1 and Tc1 cells, 1 × 10⁷ purified CD4⁺ and CD8⁺ T cells were mixed with 2 mL of Roswell Park Memorial Institute (RPMI) 1640 medium per well in a six-well plate (Costar; Corning, NY, USA) and then stimulated with 20 mg/mL MOG_35–55 in the presence of 1 × 10⁷ mitomycin C (MMC; Med Chem Express NJ, USA)-treated syngeneic spleen cells as antigen-presenting cells (APCs). Activated lymphoblasts were isolated after 2 days using density-gradient centrifugation (Lymphocyte Separation) and cultured in RPMI 1640 medium containing 10 ng/mL IL-2 (USCN Co., Wuhan, Hubei, China, 10 ng/mL).

Similarly, for the preparation of Th17 and Tc17 cells, 1 × 10⁷ purified CD4⁺ and CD8⁺ T cells in 2 mL of RPMI 1640 medium per well in a six-well plate were stimulated with 20 mg/mL MOG_35–55 in the presence of 1 × 10⁷ MMC-treated syngeneic APCs plus 10 ng/mL IL-23 (R&D Systems, Inc., Minneapolis, MN). Activated lymphoblasts were isolated after 2 days using density-gradient centrifugation and cultured in RPMI 1640 medium containing 10 ng/mL IL-23.

Proliferation assay

Per our previously published protocol, MOG_35–55-specific Th1, Tc1, Th17, and Tc17 cells were prepared and seeded in 96-well plates at 4 × 10⁵ cells/well. The cells were cultured in 200-mL medium with or without MOG_35–55 in the presence of MMC-treated syngeneic APCs (1 × 10⁵) at 37°C for 48 h. [³H]-thymidine incorporation during the last 8 h was assessed using a microplate scintillation counter (Packard; PerkinElmer, Meriden, CT, USA). The proliferative response was expressed as the mean count per minute ± standard deviation of triplicate determinations.

Enzyme-linked immunosorbent assay (ELISA)

As previously described, 1 × 10⁶ MOG_35–55-specific Th1, Tc1, Th17, and Tc17 cells mixed with 1 mL of RPMI 1640 medium per well in a 24-well plate (Costar; Corning, Corning, NY, USA) were stimulated with 20 mg/mL MOG_35–55 in the presence of MMC-treated syngeneic spleen cells as APCs. After 48, 96, and 144 h, IFN-γ and IL-4 secretion in the supernatants of the four aforementioned MOG_35–55-specific T cell cultures was measured using commercially available ELISA kits (USCN Co., Wuhan, Hubei, China).

Adoptive-transferred EAE models (tEAE)

For adoptive transfer, Th1, Tc1, Th17, and Tc17 cells from MOG_35–55-immunized B6 mice prepared p.i. at day 12 were exposed to immunizing peptides for 3 days in the presence of a medium containing IL-2 or IL-23 (10 ng/mL). Activated T-cell blasts were separated by Ficoll gradient centrifugation and 5 × 10⁶ activated T cells were adoptively transferred to each naive B6 mouse. The cell suspensions were prepared in 0.2 mL of phosphate-buffered saline, as described previously.

Histological study

Spinal cord sections were prepared from tissues collected from tEAE animals. Spinal cord tissues were fixed in 4% ice-cold paraformaldehyde/methanol solution and embedded in paraffin before being sectioned (5-mm-thick) using a microtome for staining with hematoxylin and eosin (H&E) and Luxol fast blue (LFB).

Statistical analysis

The data are represented as mean ± standard deviation and were analyzed using SPSS software (SPSS Statistics for Windows, version 18.0, SPSS Inc., IBM, Almonk, NY, USA) and GraphPad Prism 8.0 plotting. The data were first tested for normality and homogeneity of variance. If both conditions were met, one-way analysis of variance (ANOVA) and two-way ANOVA were used for multiple group comparisons, and the least significant difference t test was used for pairwise comparison. Non-normally distributed data are represented as (median [Q1, Q3]) and compared between multiple groups using Kruskal–Wallis H test or compared in pairs using Mann–Whitney U test. Statistical significance was defined as p < 0.050.

RESULTS

Tc17 cells are MOG_35–55 specific

Four types of T cells (Th1, Tc1, Th17, and Tc17) were assessed for their antigen-specific functions using a proliferation assay. The results presented in Figure  and Table  demonstrated that the MOG_35–55 peptides exhibited a potent stimulatory effect on all four types of T cells at the highest dose (20 μg/mL, p < 0.050) but not at lower doses (0.2 or 2 μg/mL, p = 0.741 or p = 0.502) or in the absence of the peptides (0 μg/mL p = 0.262). This suggests that all four types of T cells were specific to MOG_35–55, in line with our earlier reports.

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Table 1 Proliferation assay of all four kinds of T cells.

Items	Th17	Tc17	Th1	Tc1	F	p
MOG35-55 0 mg/mL	2780.00 ± 868.85	2176.67 ± 55.08	2200.00 ± 167.03	2290.00 ± 200.75	1.164	0.382
MOG35-55 0.2 mg/mL	3080.00 ± 997.45	2680.00 ± 396.61	2973.33 ± 362.95	3580.00 ± 552.45	1.061	0.418
MOG35-55 2.0 mg/mL	5146.7 ± 701.17	5440.00 ± 995.19	3956.67 ± 640.81	5516.67 ± 338.43	3.128	0.088
MOG35-55 20.0 mg/mL	55709 ± 4196.09	15951.00 ± 1985.30	55630.33 ± 4077.21	15368 ± 3532.62	126.430	<0.050

At the highest dose (20 μg/mL) of MOG_35–55 peptide, all four types of T cells displayed distinct responses, as depicted in Table . The counts per minute of Th1 (55,630 ± 4077) and Th17 cells (55,709 ± 4196)—derived from CD4⁺ T cells—were considerably higher than those of Tc1 (15,368 ± 3533; Th1 vs. Tc1, p < 0.050) and Tc17 (15,951 ± 1985; Th17 vs. Tc17, p < 0.050)—derived from CD8⁺ T cells (Figure ). These findings are consistent with those of our previous studies. There were no significant differences between Th1 and Th17 cells (Th1 vs. Th17: p = 0.979) or between Tc1 and Tc17 cells (Tc1 vs. Tc17: p = 0.846) (Figure ). This led us to investigate the differences between Th1 and Th17, as well as Tc1 and Tc17. To this end, we examined the cytokine profiles of all four types of T cells.

Cytokine profiles of MOG_35–55-specific Tc17 and Th17 cells are similar, but Tc17 profiles are weaker than those of Th17 cells

To determine the cytokine profiles of activated Th1, Tc1, Th17, and Tc17 cells, we used ELISA to measure the cytokine levels in the culture supernatants of these cells at three time points (48, 96, and 144 h) poststimulation.

IFN-γ secretion

At the three time points, IFN-γ secretion from all four types of MOG_35–55-peptide-activated T cells was increased only at the highest dose (20.0 μg/mL) of MOG_35–55 peptide (p < 0.050) but not at the lower doses (0.2 or 2.0 μg/mL, p = 0.144 or p = 0.170) or in the absence of peptide (0 μg/mL, p = 0.135) (p > 0.050, e.g., at 48 h) (Figure ). These results are similar to those of our previous studies.

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Interestingly, after 48 h poststimulation, IFN-γ secretion (from Th1 (1113.33 ± 64.30 pg/mL) and Th17 (556.67 ± 15.28 pg/mL) cells was substantially higher than that from Tc1 (563.35 ± 15.28; Th1 vs. Tc1: p < 0.050) and Tc17 (280.00 ± 15.00; Th17 vs. Tc17: p < 0.050) cells, respectively (Table  and Figure ). Similar patterns were observed for IFN-γ secretion at 96 and 144 h (Table  and Figure , respectively). These results are similar to those of our previous studies.

Table 2 IFN-γ secretions of all four kinds of T cell.

Items	Th17	Tc17	Th1	Tc1	F	p
48 h
MOG35–55 0 mg/mL,	40.67 ± 16.77	27.33 ± 6.43	48.33 ± 12.58	27.33 ± 6.43	2.482	0.135
MOG35–55 0.2 mg/mL	52.33 ± 22.90	31.00 ± 6.08	52.33 ± 15.04	29.00 ± 7.00	2.394	0.144
MOG35–55 2.0 mg/mL	55.33 ± 21.57	35.67 ± 1.53	53.67 ± 15.18	32.67 ± 8.74	2.166	0.170
MOG35–55 20.0 mg/mL	556.67 ± 15.28	280.00 ± 15.00	1113.33 ± 64.30	563.35 ± 15.28	303.360	<0.050
96 h
MOG35–55 0 mg/mL	40.67 ± 16.77	27.33 ± 6.43	59.00 ± 8.54	27.33 ± 6.43	6.172	0.018
MOG35–55 0.2 mg/mL	52.33 ± 22.90	31.00 ± 6.08	68.67 ± 9.02	29.00 ± 7.00	6.163	0.018
MOG35–55 2.0 mg/mL	65.67 ± 19.14	35.33 ± 6.43	70.00 ± 10.00	32.67 ± 8.74	7.929	0.009
MOG35–55 20.0 mg/mL	826.67 ± 30.55	234.33 ± 22.28	1470.00 ± 81.85	866.67 ± 15.28	365.546	<0.050
144 h
MOG35–55 0 mg/mL	40.67 ± 16.77	27.33 ± 6.43	59.00 ± 8.54	27.33 ± 6.43	6.172	0.018
MOG35–55 0.2 mg/mL	52.33 ± 22.90	33.00 ± 8.19	68.67 ± 9.02	33.33 ± 1.16	5.226	0.027
MOG35–55 2.0 mg/mL	65.67 ± 19.14	58.00 ± 7.21	90.00 ± 10.00	52.00 ± 10.58	5.301	0.026
MOG35–55 20.0 mg/mL	1136.67 ± 126.62	633.33 ± 30.55	2586.67 ± 80.83	1300.00 ± 87.18	53.242	<0.050

Moreover, at the highest dose (20 μg/mL) of MOG_35–55 peptide, IFN-γ secretion by Th1 cells was considerably higher than that by Th17 cells (Th1 vs. Th17: p < 0.050, at 48, 96, and 144 h), as well as by Tc1 and Tc17 cells (Tc1 vs. Tc17: p < 0.050 at 48, 96, and 144 h) (Figure ).

IL-17 secretion

At the three time points, IL-17 secretion from all four types of MOG_35–55-peptide-activated T cells was increased only at the highest dose (20.0 μg/mL) of MOG_35–55 peptide (p < 0.050) but not at lower doses (0.2 or 2.0 μg/mL) or in the absence of peptide (0 μg/mL, p = 0.382) (p > 0.050, e.g., at 48 h). These results are similar to those of our previous study.

After 48 h, IL-17 secretion from Th1 (238.67 ± 25.72 pg/mL) and Th17 (288.33 ± 12.58 pg/mL) cells was considerably higher than that from Tc1 (96.00 ± 5.29 pg/mL; Th1 vs. Tc1: p < 0.050) and Tc17 (102.67 ± 5.86 pg/mL; Th17 vs. Tc17: p < 0.050) cells, respectively (Table  and Figure ). Similar patterns were observed for IFN-γ secretion at 96 and 144 h, as shown in Table  and Figure .

Table 3 IL-17 secretions of all four kinds of T cells.

Items	Th17	Tc17	Th1	Tc1	F	p
48 h
MOG35–55, 0 mg/mL	21.33 ± 8.08	21.67 ± 9.07	27.33 ± 6.43	24.00 ± 5.29	0.423	0.741
MOG35–55, 0.2 mg/mL	35.00 ± 9.54	25.67 ± 6.03	31.00 ± 6.08	29.00 ± 7.00	0.856	0.502
MOG35–55, 2.0 mg/mL	38.67 ± 6.35	27.00 ± 6.00	33.00 ± 4.36	32.33 ± 7.03	1.610	0.262
MOG35–55, 20.0 mg/mL	288.33 ± 12.58	102.67 ± 5.86	238.67 ± 25.72	96.00 ± 5.29	127.920	<0.050
96 h
MOG35–55, 0 mg/mL	23.33 ± 8.51	18.33 ± 1.53	27.33 ± 6.43	27.00 ± 10.44	0.936	0.467
MOG35–55, 0.2 mg/mL	41.67 ± 7.37	24.33 ± 0.58	32.00 ± 6.08	36.67 ± 2.89	6.516	0.015
MOG35–55, 2.0 mg/mL	54.00 ± 7.21	32.67 ± 3.06	34.33 ± 8.15	39.00 ± 2.65	8.403	0.007
MOG35–55, 20.0 mg/mL	348.33 ± 17.10	128.00 ± 31.05	197.67 ± 7.77	126.33 ± 19.50	76.886	<0.050
144 h
MOG35–55, 0 mg/mL	40.67 ± 16.77	55.00 ± 5.00	27.33 ± 6.43	27.33 ± 6.43	5.378	0.025
MOG35–55, 0.2 mg/mL	54.33 ± 20.65	67.00 ± 11.53	34.67 ± 5.03	29.00 ± 7.00	5.856	0.020
MOG35–55, 2.0 mg/mL	65.67 ± 25.58	67.33 ± 11.02	40.00 ± 4.36	32.00 ± 6.08	4.635	0.037
MOG35–55, 20.0 mg/mL	636.67 ± 55.08	203.33 ± 72.68	496.00 ± 15.10	213.33 ± 47.26	51.137	<0.050

In addition, at the highest dose (20 μg/mL) of the MOG_35–55 peptide, IL-17 secretion from Th1 cells was considerably lower than that from Th17 cells (Th1 vs. Th17: p < 0.050 at 48, 96, and 144 h) and from Tc1 and Tc17 cells (Tc1 vs. Tc17: p < 0.050 at 48, 96, and 144 h) (Figure ).

Adoptive transfer of MOG_35–55-specific Tc17 cells to naïve mice induces tEAE

To determine the role of MOG_35–55-specific Tc17 cells in the pathogenesis of EAE, we induced the disease in wild-type B6 naïve mice by adoptive transfer of MOG_35–55-specific Th1, Tc1, Th17, and Tc17 cells from B6 aEAE mice and then measured the clinical signs using the EAE score assessed by daily checks and histopathology. As shown in Figure , the EAE score after transferring Tc17 cells from immunized mice to naïve mice (Tc17-tEAE) was similar to that after transferring Tc1 cells (Tc1-tEAE). However, these scores were slightly lower than the EAE scores of Th17-tEAE and Th1-tEAE mice.

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Histopathological examination revealed slight inflammation (Figure ) and slight demyelination (Figure ) in Th1, Tc1, Th17, and Tc17-tEAE compared to naïve mice (Figure ), consistent with our previous observations. No significant differences were found among the four tEAE groups. This might be because of the following reasons: (1) our T cells were enriched Tc17, not highly purified Tc17, or (2) cytokine secretion might not be consistent with pathology, or it is much more difficult to profile cytokines in induced EAE.

DISCUSSION

Our previous reports have shown that in the EAE model, MOG_35–55 peptide-specific CD8⁺ T cells are antigen-specific and encephalitogenic with IFN-γ production, which are weaker than their CD4⁺ counterparts. However, the CD8⁺ T cell subtype that plays a critical role in the pathogenesis of EAE remains unclear. Currently, the major subsets of CD8⁺ T cells are Tc1, Tc2, Tc9, Tc17, and Treg cells. Among these subsets, Tc17 cells are strongly associated with autoimmune diseases in both human and animal models.

In the present study, we used our already-established strategy for preparing the EAE model to study the role of MOG_35–55-specific Tc17 cells in the pathogenesis of EAE and MS. Here, we focused on Tc17 cells in EAE and used Th17 cells (CD4⁺ counterparts of Tc17) and Tc1 cells (different subsets of CD8⁺) as controls. We found that Tc17 cells responded to MOG_35–55 peptides and secreted IFN-γ and IL-17 and could also induce tEAE in naïve mouse models. However, we observed that the proliferation rate, cytokine secretion, and encephalitogenic activity of Tc17 cells were weaker than those of their Th17 counterparts. Although MOG_35–55-specific Tc17 cells showed similar proliferation rates to our previously explored MOG_35–55-specific Tc1 cells, they exhibited lower IFN-γ secretion, higher IL-17 secretion, and weaker encephalitogenic activity. These results are consistent with our previous findings on MOG_35–55-specific Th1 and Tc1 cells in the EAE model and on interphotoreceptor retinoid-binding protein_1–20-specific Th1 and Th17 cells in the EAU model, both of which were studied in female C57BL/6 mice.

As mentioned previously, similar functional profiles of CD4⁺ and CD8⁺ autoreactive T cells and their subsets were observed in female animal models of closely related autoimmune diseases, such as MS, uveitis, and type I diabetes. Hence, future research into the common mechanisms of these autoimmune diseases, including thymus development, local microenvironments, and estrogen relationships, will be beneficial.

Wagner et al. suggested that classic EAE was dominated by CD4⁺ T cells, whereas atypical EAE was dominated by CD8⁺ T cells, explaining why the classic EAE model did not mimic all features of MS very well. Unfortunately, only a few studies using myelin basic protein in TCR-transgenic 8.8 mice have reported on a CD8⁺ T cell-dominant atypical EAE model. This is a major drawback for research on CD8⁺ T cells, particularly Tc17 cells, in EAE. In addition, studies on different types of EAE models—such as myelin antigen-specific TCR-transgenic mouse EAE models, spontaneous EAE models, humanized mouse EAE models, and nonhuman primate EAE models—might provide new insights into the pathogenesis of the disease.

Although Tc17 cells have similar cytokine profiles to Th17 cells, the two cell types exhibit some differences. In the skin, Tc17 and Th17 cells coexpress retinoic acid receptor-related orphan nuclear receptor gamma (RORγt) and GATA-3 only under antigen stimulation. RORγt regulates IL-6 and IL-23 production via signal transducer and activator of transcription 3 (STAT3); it also regulates IL-17A, IL-17F, IL-23R, and IL-21 production via epigenetic mechanisms. In the thymus, T cell factor-1 suppresses Tc17 cells only at the double-positive stage and suppresses Th17 cells before the CD4 coreceptor expression stage. Interferon regulator factor 3 (IRF3) greatly inhibits Tc17 cell development via RORγt compared to Th17 development. Moreover, DMF affects IL-17 production in murine and human Tc17 cells and to a much lesser extent in Th17 cells. Tc17 and Th17 cells may have different protein kinase B/mammalian target of rapamycin signaling requirements, as well as divergent metabolic requirements. Finally, the plasticity and related functions of Tc17 and Th17 cells are different. Th17 cells can switch to a Th1-like phenotype, resulting in a more pathogenic profile, which has been confirmed by studies on pathological inflammation in the CNS and intestine during bacterial infection or colitis. In contrast, Tc17 cells shift to a Tc1-like phenotype only during CNS autoimmunity. This suggests that the functional specificity of Tc17 and Th 17 cells differs in terms of plasticity toward the type 1 phenotype.

Murine Tc17 cells exhibit phenotypic similarities to Th17 cells; however, the cytokine profile of human Tc17 cells remains unclear. A report has shown that some cytokines—such as IFN-γ, tumor necrosis factor-alpha, IL-21, IL-22, GM-CSF, and RORγt and its subfamily homolog RORα—are co-expressed with IL-17 in cultured Tc17 cells. RORγt is a key factor in the development, maintenance, and function of IL-17-producing cells and plays a role in regulating thymopoiesis. Clinical trials of several RORγ inhibitors in patients are ongoing. Other TFs, including STAT3, IRF3, and IRF4, also promote Tc17 cell differentiation.

Different subsets of CD8⁺ T cells are sometimes not stable and differ between Tc1 and Tc17 phenotypes—a phenomenon called “plasticity of Tc17 cells.” For example, the lack of CTLA-4 drives Tc17 cells to downregulate RORγt and IL-17 expression and induces a shift toward Tc1. In contrast, in a mouse model, Tc17 cells exhibited a stable phenotype and mediated protection against fungi and bacteria. Tc17 cells can also switch to the Tc2 phenotype by increasing IL-5 and IL-13 production. Thus, the plasticity of Tc17 cells can be an intriguing and promising target for future studies. TGF-β inhibits various functions of Tc1 cells; however, in the presence of IL-6, TGF-β induces Tc17 cells. In HKx31 (H3N2) influenza A virus-infected Il2^−/−, Il2ra^−/−, Il12^−/−, Blimp1^GFP, and Blimp1^fl/fl mice, Tc17 cells were noncytotoxic and downregulated the expression of T-bet and Eomes compared with Tc1 cells.

CONCLUSIONS

Our results suggest that autoreactive Tc17 cells have unique and independent cytokine profiles, compared to their Th17 counterparts and the Tc1 subset. However, cytokine secretion might not be consistent with pathology, or it is much more difficult to profile cytokines in induced EAE. The pathogenesis of MS is still unclear, and many factors, such as different cells and their subsets, and different kinds of EAE models need to be investigated to determine the mechanisms underlying MS.

AUTHOR CONTRIBUTIONS

Yong Peng received funding support and developed the research hypothesis. Yong Peng, Xiuli Zhang, Yandan Tang, Shunqing He, Guilan Rao, Quan Chen, Yahui Xue, Hong Jin, Shu Liu, Ziyang Zhou, and Yun Xiang finished the experiments and analyzed the data. Yong Peng and Xiuli Zhang wrote the main manuscript. The final manuscript is the product of the joint writing efforts of all authors.

ACKNOWLEDGMENTS

This work was supported by the Scientific Research Project of Hunan Provincial Health Commission, China (No. C2023030765 to Y. P.), Key Plans of Hunan Administration Traditional Chinese Medicine, China (No. A2023039 to Y. P.), University-Hospital Joint-Fund of Hunan University of Chinese Medicine, China (No. 2022XYLH198 to Y. P.), Fund for Creative Research Group of Affiliated First Hospital of Hunan Traditional Chinese Medical College, China (No. 2021B-003 to Y. P.), and Technology Plan Project of Zhuzhou City, Hunan Province, China (No. 2021-009 to Y. P.).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

All data are submitted to Journal.

ETHICS STATEMENT

All experiments were conducted at the Science and Technology Innovation Center of the Hunan University of Chinese Medicine in Changsha, China. The experimental procedures were approved by the Ethics Committee of the Hunan University of Chinese Medicine (Approval No. LL2022050501). The animal treatments were conducted in accordance with the guidelines provided by the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all necessary steps were taken to minimize the pain and suffering of the animals.

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