Female C57BL/6 mice (6–8 weeks) were purchased from the Vital River Laboratory Animal Technology Corporation (Beijing, China). The OT-II TCR transgenic mice were a gift from Guixiu Shi (University of Xiamen, China). All mice were bred in the specific pathogen-free facility of Chengdu Medical College, and the experimental protocols were approved by the Animal Care and Use Committee of Chengdu Medical College. All experimental animal protocols were followed regarding the national requirements for animal ethics.

Female C57BL/6 mice (6–8 weeks) were anesthetized and euthanized by cervical disconnection. The femur and tibia bones were isolated aseptically, and then washed once with 75% alcohol, and three times with cold phosphate-buffered saline (PBS). After the ends of the bones were cut, a 1 ml sterile syringe was used for flushing out the bone marrow cells with 5 ml of cold PBS. Then, the cell suspensions were passed through a nylon mesh to remove small pieces of bone and debris. Subsequently, the single bone marrow cells were washed with cold PBS, and 1 × 10⁷ cells were plated in 10 ml RPMI 1640 medium containing 10% FBS, penicillin, and streptomycin supplemented with 20 ng/ml recombinant murine GM-CSF and 10 ng/ml recombinant murine IL-4 (PeproTech). Then, half of the medium was displaced every 2 days. On day 5, the cells were collected, and CD11c+MHCII+ DCs were sorted using a BD FACSJazz cell sorter (BD Biosciences). The sorted cells were plated in a 24-well plate and treated with 1, 4, and 16 μM K313 (# 5939009, ChemBridge Corp, San Diego, CA, USA) ( Figure 1 ), and DMSO-treated cells were used as vehicle control. After 6 h, 100 ng/ml LPS was added to stimulate the maturation of BMDCs.

Ethical approval was obtained through the Ethical Review Committee of Chengdu Medical College, and informed consent of all participating subjects was obtained. The protocol of generating human DCs from human blood mononuclear cells [peripheral blood mononuclear cells (PBMCs)] has been described (Nair et al., 2012). In short, peripheral blood was directly drawn into vacuum blood collection tubes containing sodium heparin, and PBMCs were isolated using a density gradient centrifugation on Ficoll-Paque Plus solution (Dakewei, Beijing, China). CD14+ monocytes were sorted using a BD FACSJazz cell sorter and cultured in RPMI 1640 medium containing 10% FBS, penicillin, and streptomycin, supplemented with 40 ng/ml recombinant human GM-CSF and 20 ng/ml recombinant human IL-4 (PeproTech) for 5 days. The culture medium, including supplements, was refreshed on day 3. On day 5, the cells were plated in a 24-well plate and treated with 1, 4, and 16 μM K313. DMSO-treated cells were used as vehicle control. After 6 h, 200 ng/ml LPS was added to stimulate the maturation of DCs.

On day 6, the cultured cells were collected and washed once with cold PBS. After blocking with anti-mouse CD16/CD32 (Mouse BD Fc Block, BD Pharmingen) or human BD Fc Block (BD Pharmingen), the cells were labeled with the following antibodies: PE-Cy7-anti-mouse CD11c (clone: HL3), FITC-anti-mouse CD40 (clone: 3/23), FITC-anti-mouse CD80 (clone: 16-10A1), PE-anti-mouse CD86 (clone: GL-1), Alexa Fluor647-anti-mouse CCR7 (clone: 4B12) (BioLegend) or APC-anti-human CD80 (clone: 2D10), PE-anti-human CD86 (clone: BU63), FITC-anti-human CD40 (clone: 5C3), BV421-anti-human CD14 (clone: MΦP9). After staining on ice for 30 min and washing once with cold PBS, the cells were detected by a flow cytometer (ACEA Biosciences Quanteon NovoCyte) and the data were analyzed by ACEA Novo Express software.

To detect the survival of K313-treated BMDCs, the apoptosis of cells from the culture on day 6 was analyzed by flow cytometry using a PE-Annexin V Apoptosis Detection Kit I (BD Pharmingen) according to the corresponding protocol; cells were cultured and treated with K313 as described above (Method 2.2). To test whether the phagocytosis of DCs was influenced by K313, the CD11c+MHC II+ DCs pretreated with K313 were prepared as per the previous method. FITC-Dextran (40,000 Da, Sigma) was incubated with the cells at 37°C for 30 min and stopped by cold PBS. Cells incubated in FITC-Dextran solution at 4°C were used as a negative control. Then, the cells were washed once and analyzed using a flow cytometer.

The sorted BMDCs were cultured and treated as per the above method. Finally, the prepared cells were pulsed with OVA_323-339 for 1 h. Meanwhile, splenic cells were isolated from female OT-II mice (6–8 weeks). After a grinding step, red blood cell lysis buffer was added to remove the red blood cells; then, the cells were washed twice with cold PBS to prepare the single-cell suspension. The naïve CD4+ T cells were purified using a Naive CD4+ T Cell Isolation Kit (Miltenyi), according to the manufacturer's instructions. Subsequently, the purified cells were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen) following the standard protocols, and then co-cultured with OVA_323-339 pulsed BMDCs in a 24-well plate for stimulating antigen-specific T cell proliferation. The ratios of DCs: T was 1:10, 1:30, and 1:100, the number of T cells was the same in each groups, with a proportional change in the number of DCs. After 5 days, the cells were collected, stained with CD4-PE, and detected using a flow cytometer.

The sorted BMDCs were treated and cultured as per the above method. On day 6, the culture medium was collected and centrifuged at 300 g at 4°C for 5 min. The supernatants were gathered for the ELISA test. IL-12, IL-6, IL-10, and TNF-α were measured according to the protocols of the ELISA Kits (BD Pharmingen).

Female C57BL/6 mice (6–8 weeks) were immunized subcutaneously with 200 μg of MOG_35–55 peptide, as previously described (Zhou et al., 2017). Each mouse was injected with 500 ng pertussis toxin (PTx) i.p. on days 0 and 2. The culturing method of BMDCs was conducted as per the above method. On day 5, the sorted DCs were pretreated with K313 (16 μM) for 6 h, then MOG_35-55 (20 μg/ml, Bankpeptide, Hefei, China) and LPS (100 ng/ml) were added to the treated cells for 24 h. The cells were washed and transferred into the EAE mice via tail vein injection (1 × 10⁶ cells/mouse) on days 10, 13, and 16. DCregs induced by 1, 25-Dihydroxyvitamin D₃ (1,25-D3) (10^–8 M; Sigma-Aldrich) were used as a positive control. The development of EAE was observed daily, and clinical scores were made according to the Benson score: 0, no clinical signs; 1, paralyzed tail; 2, loss of coordinated movement, hind limb paresis; 3, both hind limbs paralyzed; 4, forelimbs paralyzed; and 5, moribund (Zhou et al., 2017).

On day 20, the three groups of mice were anesthetized with sodium pentobarbital and perfused with PBS (pH 7.4) and 4% (w/v) paraformaldehyde. Subsequently, 4% (w/v) paraformaldehyde was used to fix spinal cord samples overnight. Through paraffin embedding, slicing, and dewaxing hydration, the coronal sections (5 μm) were stained with hematoxylin and eosin (HE) to examine the extent of cellular infiltration. Meanwhile, the luxol fast blue staining (LFB) method was used to determine demyelination.

The functional phenotypes of splenetic CD4+ T cells from EAE mice were analyzed by flow cytometry. Briefly, splenocytes were treated with anti-mouse CD16/CD32 (Mouse BD Fc Block, BD Pharmingen) and stained with FITC-anti-CD4 (clone:RM4-5), fixed, permeabilized (BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit, BD Pharmingen), and intracellularly stained with PE-anti-IFN-γ (clone: XMG1.2) and Alexa Fluor647-anti-IL-17 (clone: TC11-18H10), followed by flow cytometer (ACEA Biosciences Quanteon NovoCyte). Some splenocytes were stained with FITC-anti-CD4 and APC-anti-CD25 (clone: PC61) (BD Pharmingen), fixed, permeabilized (Transcription Factor Buffer Set, BD Pharmingen), and stained with PE-anti-Foxp3 (clone: R16-715) (BD Pharmingen), followed by flow cytometer analysis of the frequency of Tregs.

The data are expressed as the mean ± SD. Differences between groups were analyzed by repeated analysis of variance (ANOVA) and post hoc Bonferroni correction. All statistical analyses were performed using PRISM 6.0 software (GraphPad Software). A P value of < 0.05 was considered statistically significant.

Phagocytosis is an important characteristic of imDCs. In the pathological process of autoimmunity, DCs recognize and uptake the self-antigens to initiate autoimmune inflammation (Wang et al., 2018). Therefore, whether K313 influenced the phagocytosis of imDCs was tested. The cells were exposed to 1, 4, and 16 μM of K313, while the vehicle group was blank. Results showed that there is no difference in the proportion of FITC-dextran-positive cells between the K313 and vehicle groups. K313 does not affect the phagocytosis of imDCs, even at the highest concentrations of 16 μM ( Figures 2A, C ). Meanwhile, the Annexin V-PE/7-AAD double dyeing was performed to evaluate the apoptosis of DCs stimulated with LPS for 24 h. As shown in Figures 2B, D , K313 did not affect the survival rate of DCs at any concentration, compared to the vehicle group. However, the GM-CSF/IL-4 starved group showed a marked increase in the proportions of early apoptotic cells (Annexin V+ 7-AAD-), and K313 treated DCs showed obvious apoptosis following LPS stimulation for 48 h ( Supplementary Figure 1 ). In the following in vitro and in vivo experiments, the period of LPS stimulation for DC maturation was 24 h. The low cytotoxicity of K313 is evidenced by the above data which is consistent with our previous study (Zhao et al., 2018).

Regulatory DCs exhibited immature phenotypes of lower expression of specific surface markers compared with mature DCs (Hawiger et al., 2001). Of the tested surface markers, CD40, CD80, and CD86, which are co-stimulatory molecules, are indispensable for initiating antigen-specific T cell activation (Rescigno et al., 1998). Therefore, we investigated CD40, CD80, CD86, and CCR7 expression through flow cytometry to evaluate whether K313 affects the maturation of murine and human DCs stimulated with LPS. The results show that the CD40, CD80, and CD86 of murine DCs treated with K313 (4 and 16 μM) decreased, compared with the vehicle group ( Figures 3A–D ). Furthermore, the amount of above-mentioned surface markers gradually decreased, with an increase in K313 concentration ( Figures 3A–C ). Moreover, the expression of CD80, CD86, and CD40 in the 16 μM K313 group was drastically reduced by 66, 41, and 53%, respectively, compared with the vehicle group ( Figures 3A–C ). Similarly, maturation markers (CD40, CD86, and CD80) of human DCs treated with k313 at concentrations of 4 and 16 μM also displayed significantly reduced levels relative to the vehicle control ( Figures 3I–K ). Collectively, the DCs treated with K313 displayed obvious down-regulation of co-stimulatory molecules.

To investigate whether K313 affects the cytokine secretions of BMDCs, the amounts of pro-inflammatory cytokines, such as IL-12, IL-6, TNF-α, and anti-inflammatory cytokine IL-10, were measured using ELISA Kits. The results indicate that the K313-treated DCs secreted increased IL-10 compared with the vehicle group, and the increased expression of IL-10 was positively correlated to the K313 concentration ( Figure 3H ). In contrast, the amount of secreted IL-12 dramatically decreased, and the secretion levels of the cytokine dropped by about seven-fold when DCs were treated with 16 μM K313 ( Figure 3F ). An increased ratio of secreted IL-10: IL-12 was a typical tolerance characteristic of DCregs (Kouwenberg et al., 2016). In keeping with previous findings, DCs treated with 16 μM K313 exhibited a significantly higher ratio of IL-10: IL-12 compared to the vehicle group. In addition, this study showed that the secretion of TNF-α was down-regulated by 16 μM K313 ( Figure 3E ), and treatment with k313 at a higher dose significantly reduced the levels of LPS-stimulated IL-6 ( Figure 3G ). These results suggest that 16 μM K313 can effectively elevate the levels of IL-10, in contrast, to the suppression of the production of IL-12, IL-6, and TNF-α in culture supernatant of BMDCs.

The ability to inhibit autoreactive T cells is an important characteristic of DCregs (Nishimura et al., 2015). In order to test the influence of K313 on the proliferation of antigen-specific CD4+ T cells primed by DCs, K313-modified DCregs were pulsed with OVA_323-339 for 1 h and then co-cultured with purified splenic naïve CD4+ T cells from OT II mice at various DCs: T ratios for 5 days. Finally, the proliferation rate of CFSE-labeled CD4+ T cells was tested by flow cytometry. As expected, the proliferation rate of antigen-specific T cells was positively correlated with the DCs: T ratio and the T cells in the vehicle group showed the most expanded ability in each ratio ( Figure 4A ). Meanwhile, with the increase of K313 treatment concentration in DCs, the proliferation rate of antigen-specific CD4+T cells exhibited a gradual downward trend. ( Figure 4B ). The results suggest that BMDCs treated with K313 can suppress the proliferation of OVA_323-339 peptide-specific CD4+ T cells.

The results of our experiments in vitro show that K313 can induce DCregs, and K313-modified DCregs can suppress the proliferation of antigen-specific CD4+ T cells. To investigate whether DCregs modified by the agent have a therapeutic effect on animal models of autoimmune diseases, K313-modified DCregs loaded or unloaded with MOG_35-55 were transferred into EAE mice through tail vein injection. The results indicate that the group of antigen-loaded DCregs had a milder clinical score than the antigen-unloaded group, and the therapeutic effects of K313-modified DCregs were similar to that of 1, 25-Dihydroxyvitamin D₃-induced DCregs ( Figure 5A ). In addition, HE staining showed that the degree of leukocyte infiltration was dramatically reduced in the antigen-loaded DCregs group ( Figure 5C ), and LFB staining also indicated that there was extensively decreased demyelination in the MOG_35-55-loaded DCregs group ( Figures 5B, D ). Overall, by the adoptive transfer of K313-modified DCregs loaded with MOG_35-55, the development of disease in EAE mice was ameliorated to some extent. To understand the underlying mechanisms by which the direct cell-cell interaction between adoptive transferred DCs and T cells exacerbated the development of EAE, the percentages of splenetic Th1 and Th17 cells and Tregs in the different groups of mice were characterized by flow cytometry. The results show that adoptive transfer with K313-modified DCregs loaded with antigens significantly reduces the percentages of Th1 and Th17 cells and increases the percentage of Tregs compared with the untreated group ( Figure 6 ).