A B cell–driven EAE mouse model reveals the impact of B cell–derived cytokines on CNS autoimmunity

Significance

In multiple sclerosis (MS), increasing evidence suggests that B cells may contribute to pathogenesis via antigen presentation and production of proinflammatory cytokines. However, these B cell effector functions are not featured well in classical experimental autoimmune encephalomyelitis (EAE) mouse models. Here, we developed a cotransfer EAE model, where autoreactive B cells activate cognate T cells and thereby provide the critical stimulus for disease development. As our model allows for B cell manipulation prior to transfer, we found that overexpression of the proinflammatory cytokine IL-6 by autoreactive B cells leads to an accelerated EAE but does not affect the T cell response. In summary, we generated a tool to dissect pathogenic B cell effector functions in CNS (central nervous system) autoimmunity.

Abstract

In multiple sclerosis (MS), pathogenic T cell responses are known to be important drivers of autoimmune inflammation. However, increasing evidence suggests an additional role for B cells, which may contribute to pathogenesis via antigen presentation and production of proinflammatory cytokines. However, these B cell effector functions are not featured well in classical experimental autoimmune encephalomyelitis (EAE) mouse models. Here, we compared properties of myelin oligodendrocyte glycoprotein (MOG)-specific and polyclonal B cells and developed an adjuvant-free cotransfer EAE mouse model, where highly activated, MOG-specific induced germinal center B cells provide the critical stimulus for disease development. We could show that high levels of MOG-specific immunoglobulin G (IgGs) are not required for EAE development, suggesting that antigen presentation and activation of cognate T cells by B cells may be important for pathogenesis. As our model allows for B cell manipulation prior to transfer, we found that overexpression of the proinflammatory cytokine interleukin (IL)-6 by MOG-specific B cells leads to an accelerated EAE onset accompanied by activation/expansion of the myeloid compartment rather than a changed T cell response. Accordingly, knocking out IL-6 or tumor necrosis factor α in MOG-specific B cells via CRISPR-Cas9 did not affect activation of pathogenic T cells. In summary, we generated a tool to dissect pathogenic B cell effector function in EAE development, which should improve our understanding of pathogenic processes in MS.

Multiple sclerosis (MS) is an autoimmune disease characterized by chronic inflammation targeting the myelin sheath of the central nervous system (CNS) and resulting in demyelination, axonal damage and loss. Until recently, MS and its animal model experimental autoimmune encephalomyelitis (EAE) were considered to be primarily T cell–driven, with T helper (Th)1 and Th17 cells as the main orchestrators of pathogenic inflammatory processes. However, it has become clear that B cells are involved in the pathogenesis as well. B cells are chiefly recognized for their ability to produce and act via antibodies, and indeed, the presence of antibodies as oligoclonal bands in the cerebrospinal fluid (CSF) of people with MS (pwMS) was one of the first biomarkers used for diagnosis of MS (1). Furthermore, ectopic lymphoid follicle-like structures have been observed in the CNS of pwMS as well as in some EAE mouse models and have been associated with cortical lesions underneath (2–4). These structures contain clusters of B cells and plasma cells, indicating that these cell types are recruited to the site of inflammation during EAE and may contribute to disease pathogenesis. Autoreactive antibodies recognizing aquaporin-4 or MOG have a well described pathognic role in the MS-related diseases neuromyelitis optica spectrum disorder and myelin oligodendrocyte glycoprotein (MOG) associated disease (reviewed in ref. 5). Although no definite autoantigen could be identified in MS so far, antibodies and complement are often deposited in active MS lesions, and some pwMS benefit from plasmapheresis suggesting that production of (auto)reactive antibodies can contribute to pathology in MS (6, 7). However, the efficacy of the anti-CD20 B cell–depleting therapies rituximab and ocrelizumab in relapsing-remitting MS, which spare antibody-secreting cells (ASCs) and leave antibody titers mostly stable (8–10), suggests that other B cell effector functions also play a role in disease development. Thus, it has been hypothesized that presentation of autoantigen to T cells in the context of proinflammatory cytokine production is a pathogenic B cell effector function in MS. In EAE, antigen presentation by B cells was sufficient to induce disease by reactivation of mainly Th1 cells, but also Th17 cells in the CNS (11, 12). Additionally, different cytokines were found to be expressed by B cells in the context of MS and EAE: B cells from peripheral blood of pwMS produce increased amounts of the proinflammatory cytokines interleukin (IL)-6, tumor necrosis factor (TNF)α, and lymphotoxin but lower levels of antiinflammatory IL-10, which may lead to the activation of pathogenic T cells (13–15). Furthermore, IL-6 secretion by B cells was shown to be crucial for differentiation of pathogenic Th17 cells and disease induction in EAE (12, 16). B cells of pwMS were also shown to produce granulocyte-macrophage colony-stimulating factor (GM-CSF) suggesting an additional role in the stimulation of proinflammatory myeloid cell responses (17). However, it remains unclear whether proinflammatory cytokines are preferentially produced by autoreactive B cells or independent of B cell antigen-specificity. Furthermore, there are open questions regarding the mechanism of action of B cell–derived proinflammatory cytokines in vivo including i) the stimulus for their production /release and ii) the main target cells. To address these questions in a systematic approach, we have compared properties of MOG-specific and polyclonal B cells and developed an adjuvant-free cotransfer EAE model, in which MOG-specific B cells provide the critical stimulus for disease development. As our model allows for manipulation of the B cells prior to transfer, it is a suitable tool for mechanistic investigations into pathogenic B cell effector functions. Thus, in proof-of-principle experiments, we investigated the roles of the proinflammatory cytokines IL-6 and TNFα in B cell pathogenicity in CNS autoimmunity.

Results

MOG-Specific IgH^MOG B cells Show an Increased Marginal Zone (MZ) B cell Compartment and A More Proinflammatory Cytokine Profile.

To investigate potential differences between autoreactive and polyclonal B cells, we made use of IgH^MOG mice, which carry the heavy chain from the MOG-specific antibody 8.18c5 as a knock-in (18). Thus, about 90% of their B cells recognize MOG via their BCR, as determined by MOG-protein-tetramer staining (19), and secrete high levels of MOG-specific antibodies. IgH^MOG mice show no signs of spontaneous EAE, however, when crossed with 2D2 mice, which carry MOG-specific TCR transgenic CD4⁺ T cells, about 60% of animals spontaneously develop severe EAE indicating that antigen-specific B and T cells cooperate to induce autoimmunity (20, 21). Since CD80 expression on B cells was enhanced in sick 2D2xIgH^MOG mice, and since IgH^MOG B cells could present MOG and induce proliferation and activation of MOG-specific T cells in vitro, it was suggested that B cells contribute to spontaneous disease development through their antigen-presenting cell (APC) function (20, 21). Whether IgH^MOG B cells promote pathogenesis also by secretion of cytokines has not been investigated.

To test whether there are already fundamental differences in the B cell phenotype, activation status, or cytokine profile in IgH^MOG mice, we analyzed B cell subsets in the spleen by flow cytometry. We detected a twofold increase of IgM^high CD1d⁺ MZ B cells in the spleens of heterozygous and homozygous IgH^MOG mice, both in frequency and total number (Fig. 1 A–C), whereas numbers of follicular B cells were comparable in WT and IgH^MOG mice (Fig. 1D). Identification of MZ B cells as IgM^highCD21^highCD23^low B cells showed a similar increase in IgH^MOG mice (SI Appendix, Fig. S1 A and B). Immunofluorescent stainings of spleen sections showed that the general position of MZ B cells is similar in WT and IgH^MOG mice and that MZ B cells merely formed a thicker ring around the follicle in IgH^MOG mice compared to WT mice (SI Appendix, Fig. S1C). Since MZ B cells were shown to not only respond to T cell–independent antigens but also to interact with CD4⁺ T cells (22, 23), we tested the ability of IgH^MOG B cell subsets to activate CD4⁺ T cells in an in vitro coculture system. While MOG-specific 2D2 CD4⁺ T cells were not able to respond to MOG protein antigen in the absence of APCs, proliferation was efficiently induced in the presence of both follicular and MZ B cells indicating that both IgH^MOG follicular and MZ B cells can contribute to T cell activation (Fig. 1E). MZ B cells are known to produce more cytokines, especially more IL-6 than follicular B cells (24). We therefore tested whether the observed changes in the B cell subset distribution in IgH^MOG mice affected their cytokine production. Indeed, IgH^MOG B cells produced significantly more IL-6 and TNFα than WT B cells after stimulation with anti-CD40 and lipopolysaccharide (LPS) (Fig. 1 F and G). To test whether the increased cytokine levels could be explained by the increased numbers of MZ B cells, we sorted follicular and MZ B cells based on their expression of IgD, IgM, and CD1d (Fig. 1A) and stimulated them separately. As expected, IL-6 was produced by the majority of MZ B cells (60 to 80% as compared to 10 to 30% in follicular B cells), but the frequency of IL-6⁺ B cells was enhanced in both follicular and MZ B cells from IgH^MOG mice as compared to WT follicular and MZ B cells, respectively (Fig. 1H). TNFα was produced by similar fractions of follicular and MZ B cells and again the frequency of TNFα-producers was increased in IgH^MOG mice in both B cell subsets (Fig. 1I). These data show that compared to polyclonal B cells, MOG-specific IgH^MOG B cells have a more proinflammatory cytokine profile to which both follicular and an expanded population of MZ B cells contribute.

Fig. 1.

IgH^MOG mice show an increased MZ B cell compartment and a more proinflammatory cytokine profile. Splenocytes of WT, heterozygous IgH^MOG(+/–), and homozygous IgH^MOG mice were isolated and analyzed by flow cytometry. (A) Identification of IgD^high follicular (Fol) B cells and IgM^high CD1d⁺ MZ B cells in the spleens of WT, heterozygous IgH^MOG(+/–), and homozygous IgH^MOG mice. (B) Frequency and absolute numbers of (C) MZ B cells or (D) follicular (Fol) B cells in WT, IgH^MOG(+/–), or IgH^MOG mice. Horizontal lines indicate means. Dots represent individual mice. **P < 0.01 and ***P < 0.005, one-way ANOVA with Tukey post-test. (E) Fol B cells and MZ B cells were sorted and cocultured with Cell Trace Violet (CTV)-labeled CD4⁺ T cells isolated from 2D2 mice at a 1:1 ratio in the presence of 50 µg/mL mMOG protein. CD4⁺ T cell proliferation was measured by CTV dilution after 4 d of coculture. Histograms show CTV dilution within the CD4⁺ cell population cultured without B cells (black) or in the presence of Fol B (green) or MZ B cells (orange) and are representative of three independent experiments. Dotted histograms show the respective condition without the addition of MOG antigen. (F and G) Total B cells or (H and I) B cell subsets were isolated or sorted from the spleens of WT or IgH^MOG mice and stimulated with anti-CD40 and LPS for 24 h. Prior to FACS staining, the cells were stimulated with PMA, ionomycin, and brefeldin A for 4 h. Dots represent individual mice. (F–I) *P < 0.05 and **P < 0.01, paired t test.

An Adjuvant-Free B cell–Dependent Transfer EAE Model to Investigate B cell Effector Functions during Disease Initiation.

To gain mechanistic insight and evaluate the impact of B cell–derived cytokine production in the context of autoantigen presentation, an in vivo EAE model is required where B cells act as APCs and can be easily manipulated. However, classical EAE models either operate independently of B cells or — in the case of the double-transgenic 2D2xIgH^MOG model — disease develops spontaneously at a very young age, and specific questions can only be addressed via crossings to knock-out (KO) lines. Thus, we developed an adjuvant-free B:T cell cotransfer EAE model, in which autoreactive B cells provide the critical stimulus for disease development and can be genetically modified to dissect the mechanisms of B cell pathogenicity. We employed the induced germinal center B cell (iGB) culture system (25) to activate, expand, and manipulate B cells. Primary B cells are cultured on fibroblastic feeder cells expressing CD40L and BAFF (40LB cells), which leads to extensive B cell proliferation and expansion (25). After 3 d, B cells acquire a germinal center (GC) phenotype characterized by the upregulation of the GC markers Fas and GL7. Addition of IL-4 further induces isotype switching from IgM to IgG1 (25). Importantly, upon adoptive transfer the iGB cells differentiate into memory-like B cells in vivo and are able to interact with T cells upon a secondary challenge with T cell–dependent antigen (25).

We cultured WT or IgH^MOG B cells in the iGB culture system for 3 d. iGB cells were loaded with MOG protein for 3 h and then transferred together with unmanipulated MOG-specific 2D2 CD4⁺ T cells into Rag1-KO mice that lack endogenous B and T cells (Fig. 2A). Importantly, only mice that received MOG-specific IgH^MOG iGB cells developed clinical signs of EAE, whereas WT iGB recipients stayed completely healthy throughout the observation period of 30 d indicating that B cell antigen specificity is required to activate 2D2 CD4⁺ T cells and induce disease (Fig. 2B and SI Appendix, Fig. S2A). Transferred iGB cells could be partly recovered from both IgH^MOG and WT iGB recipients and were found in the spleen and several LNs but did not migrate to the CNS in sick IgH^MOG iGB recipients (SI Appendix, Fig. S2B), suggesting that they perform their pathogenic effector function in the periphery. In contrast, MOG-specific CD4⁺ T cells migrated efficiently to the CNS of IgH^MOG iGB recipients where they produced mainly IFNγ indicating that after activation, 2D2 CD4⁺ T cells primarily differentiate into Th1 cells in our experimental system (Fig. 2C). Phenotypic analysis revealed that iGB cells down-regulated Fas and GL7 expression after adoptive transfer (SI Appendix, Fig. S2C), as described previously for iGB cells that displayed a memory B cell phenotype upon transfer in vivo (25). In contrast, the frequency of IgG1⁺ B cells remained stable or even increased after adoptive transfer (Fig. 2 D and E). Interestingly, the frequency of IgG1⁺ B cells in spleen and mesenteric lymph nodes (mLNs) was significantly higher in IgH^MOG iGB recipients than in WT iGB recipients and a similar trend could be observed in cervical (c)LNs (Fig. 2E). In line with this, we detected high titers of MOG-specific IgG1 antibodies in the serum of IgH^MOG iGB recipients but not in the serum of WT iGB recipients (Fig. 2F). Together, these data indicate that IgH^MOG iGB cells can provide the critical stimulus to activate 2D2 CD4⁺ T cells and induce disease in our cotransfer EAE model and that antigen-specific B:T cell interaction in this system takes place primarily in the periphery, with a potential focus on spleen and mesenteric LNs.

Fig. 2.

An adjuvant-free B cell–dependent adoptive cotransfer model to investigate B cell effector functions during EAE. (A) Experimental outline. WT or IgH^MOG B cells were activated and expanded in the iGB culture system for 3 d. Then, the cells were loaded with MOG protein for 3 h, and 1.5 × 10⁷ iGB cells were adoptively transferred together with 5 × 10⁶ unmanipulated 2D2 CD4⁺ T cells into Rag1-KO recipients. (B) EAE incidence in Rag1-KO mice that received either WT or IgH^MOG iGB cells together with 2D2 CD4⁺ T cells. Clinical data are shown for 3 mice (WT) or 7 mice per group (IgH^MOG) pooled from 2 independent experiments. **P < 0.01 Mantel–Cox log-rank test. (C) IFNγ and IL-17A expression by CD4⁺ T cells in the CNS of sick IgH^MOG iGB recipients at the peak of the disease. Flow cytometry plots are representative for n = 7 mice pooled from 2 independent experiments. (D) Representative flow cytometry plots and (E) quantification of IgG1⁺ B cells before and after adoptive transfer in different organs analyzed at the peak of disease (IgH^MOG iGB) or at the end of the observation period (WT iGB). Horizontal lines indicate means. Dots represent individual mice. *P < 0.05, unpaired t test. iLN, inguinal lymph node; cLN, cervical lymph node; mLN, mesenteric lymph node. (F) MOG-specific IgG1 antibodies in serum samples (1:1,000 diluted) from iGB recipients were measured by ELISA. Results are shown as optical density (OD). Bars indicated means. Dots represent individual mice. ****P < 0.0001, unpaired t test.

Disease Development Is Independent of High MOG-Specific IgG1 Antibody Titers.

Since IgH^MOG iGB cells produced very high levels of MOG-specific IgG1 antibodies in vivo after adoptive transfer (Fig. 2F), we asked whether MOG-specific IgG1 antibodies mediated pathogenicity of IgH^MOG iGB cells in our adoptive cotransfer system. Thus, we cultured IgH^MOG iGB cells in the absence of IL-4, to avoid isotype switching to IgG1. Proliferation rates of the B cells in iGB culture were similar in the absence or presence of IL-4 (Fig. 3C), however, iGB cells cultured without IL-4 showed lower levels of Fas and especially GL7, indicative of a less advanced GC phenotype (Fig. 3A). As expected, cells cultured in the absence of IL-4 did not switch their antibody isotype, but remained IgM^high, whereas 10 to 20% of IgG1⁺ iGB cells could be observed in the presence of IL-4 (Fig. 3B). When switched vs. unswitched IgH^MOG iGB cells were cotransferred with 2D2 CD4⁺ T cells into Rag1-KO recipients, both populations could induce EAE with slight, but not significant differences in disease incidence and time point of onset, and with similar severity (Fig. 3 D and E). MOG-specific IgG1 levels were still detectable in the serum of mice receiving unswitched IgH^MOG iGB cells, but were significantly reduced compared to mice receiving switched IgH^MOG iGB cells (Fig. 3F). Interestingly, mice that developed EAE upon transfer of unswitched IgH^MOG iGB cells showed slightly higher MOG-specific IgG1 titers than mice from the same group that remained healthy (Fig. 3F). In contrast, MOG-specific IgM levels in the serum were similar between unswitched and switched IgH^MOG iGB recipients and no disease-status dependent differences were observed for MOG-specific IgM titers in any of the two groups (Fig. 3G). Taken together, these data indicate that disease development in our B cell–dependent EAE model is independent of high MOG-specific IgG1 antibody titers.

Fig. 3.

Disease development is independent of high MOG-specific IgG1 antibody titers. IgH^MOG B cells were cultured in the iGB culture system for 3 d in the absence or presence of IL-4. Then, the cells were loaded with MOG protein and adoptively transferred together with unmanipulated CD4⁺ T cells into Rag1-KO recipients. (A) Fas and GL7 and (B) IgG1 expression in iGB cells before adoptive transfer. (C) Expansion of unswitched (without IL-4, black) or switched (+ IL-4, red) iGB cells after 3 d of culture. Data are shown as mean + SEM pooled from 5 independent experiments. (D) EAE incidence in Rag1-KO mice that received either unswitched (without IL-4, black) or switched (+IL-4, red) IgH^MOG iGB cells. Clinical data are shown for 10 mice (unswitched) or 8 mice per group (switched) pooled from 2 independent experiments. (E) EAE scores in sick mice receiving either unswitched or switched IgH^MOG iGB cells. (F) MOG-specific IgG1 and (G) IgM levels in the serum of mice receiving unswitched (black) or switched (red) IgH^MOG iGB cells. Antibody levels are shown separately for mice that developed EAE (solid lines) and mice that remained healthy (dashed lines). Dots indicate means ± SEM for indicated numbers of mice per group. *P < 0.05, unpaired t test.

IL-6 Production by Antigen-Specific B cells Can Enhance Their Ability to Induce EAE.

Since high levels of MOG-specific IgG antibodies were not required for iGB cell pathogenicity in our adoptive cotransfer EAE model, we focused on B cell APC function in the context of proinflammatory cytokine production as the pathogenic B cell effector function. Previous reports have described IL-6 expression by B cells in EAE and MS, and IL-6 plays a crucial role in differentiation of pathogenic Th17 cells (12, 14, 16, 26, 27). Moreover, we found that MOG-specific IgH^MOG B cells produce more IL-6 than polyclonal B cells (Fig. 1 F and H), and thus we sought to determine whether and how the more proinflammatory cytokine profile contributes to pathogenicity of IgH^MOG B cells. Phenotypic analysis suggested that CNS-infiltrating CD4⁺ T cells primarily differentiate into Th1 cells in our adoptive transfer model, but we also detected a small population of IFNγ⁺ IL-17A⁺ cells (Fig. 2C). Thus, we speculated that increased levels of iGB cell–derived IL-6 during priming of the T cell response may promote differentiation of Th17 cells and thereby affect the disease course.

To test this hypothesis in vivo, we took advantage of the high B cell proliferation rates in iGB culture and developed a protocol to manipulate iGB cells via retroviral transduction prior to adoptive transfer. B cells were cultured in the iGB culture system for 48 h and then transduced using murine stem cell virus (MSCV) retrovirus to overexpress IL-6 (Fig. 4A). After transduction, iGB cells express IL-6 together with eGFP from an internal ribosome entry sequence (IRES) allowing us to monitor transduction efficacy (SI Appendix, Fig. S3A). Due to high B cell proliferation rates during iGB culture, we could transduce B cells with very high efficiency ranging between 30 to 60 % in both WT and IgH^MOG iGB cells, as determined by GFP expression on day 5 of the culture (Fig. 4B). Following transduction, IL-6 was actively secreted by the transduced iGB cells, as very high IL-6 concentrations could be measured in the culture supernatants (Fig. 4C). GFP levels of IL-6-overexpressing WT or IgH^MOG iGB₆ cells or IgH^MOG iGB cells transduced with an empty vector (iGB_GFP) remained stable in vivo upon adoptive cotransfer with 2D2 CD4⁺ T cells into Rag1-KO recipients (SI Appendix, Fig. S3B). Furthermore, iGB₆ cells continued to produce IL-6 in vivo, as measured in the serum of both IgH^MOG iGB₆ and WT iGB₆ recipients whereas only very low levels of IL-6 were detectable in the serum of IgH^MOG iGB_GFP recipients (Fig. 4D). Importantly, IgH^MOG iGB₆ recipients developed EAE with comparable severity but approximately 1 wk earlier than mice that received IgH^MOG iGB_GFP cells (Fig. 4E and SI Appendix, Fig. S3C). However, WT iGB₆ cells were also able to induce EAE with similar time of onset, severity, and incidence as IgH^MOG iGB_GFP cells (Fig. 4E and SI Appendix, Fig. S3C). Surprisingly, IL-6 overexpression in transferred iGB cells did not lead to an enhanced Th17 response in CNS-infiltrating 2D2 CD4⁺ T cells since these cells still produced mainly IFNγ and the frequencies of IFNγ⁺ IL-17A⁺ and IL-17⁺ CD4⁺ T cells were similar between all experimental groups (Fig. 4F). Thus, the question remained how IL-6 overexpression by iGB cells enhances their disease-inducing capacity. Macroscopic examination revealed that both WT iGB₆ and IgH^MOG iGB₆ recipients had enlarged spleens and significantly increased numbers of splenocytes compared to IgH^MOG iGB_GFP recipients (SI Appendix, Fig. S3D and Fig. 4G). In line with that, spleen cell numbers directly correlated with IL-6 levels in the serum of all mice irrespective of the experimental group (SI Appendix, Fig. S3E). When analyzing the cellular composition of the enlarged spleens of iGB₆ recipients, we detected an expansion of the CD11b⁺ myeloid compartment with about 80% of splenocytes expressing CD11b in both IgH^MOG iGB₆ and WT iGB₆ recipients (Fig. 4H). This expanded CD11b⁺ myeloid population was mainly composed of Ly6G⁺ neutrophils and Ly6C⁺ inflammatory monocytes (Fig. 4I and SI Appendix, Fig. S3F). To investigate which cells are able to sense and respond to IL-6 in our adoptive transfer system, we measured IL-6Rα expression in different populations. At day 8 after transfer, just before onset of EAE symptoms, IL-6Rα was not expressed by CD4⁺ T cells or transferred iGB cells but only by CD11b⁺ cells. Among the different CD11b⁺ myeloid cell populations, IL-6Rα was mainly expressed by Ly6C^high monocytes, followed by regular monocytes and DCs (Fig. 4 J and K). Notably, the levels of IL-6Rα on inflammatory monocytes were significantly down-regulated in WT iGB₆ recipients compared to IgH^MOG iGB_GFP recipients. Furthermore, as they have a comparable time of disease onset we have compared CD11b⁺ cells from WT iGB₆ recipients and IgH^MOG iGB_GFP recipients on day 8 by bulk RNA sequencing to analyze the effects of IL-6 overexpression on the myeloid compartment (28). Thereby, we confirmed that IL-6 strongly induces proliferation/expansion of the myeloid compartment, as we detected upregulation of many cell cycle–associated genes such as Ccne2, Cdc6, and Cdk2 in CD11b⁺ cells from WT iGB₆ recipients (SI Appendix, Fig. S3G). Consistent with the FACS data (Fig. 4K), downregulation of IL-6Rα was also detected on the transcriptomic level and in addition, we found upregulation of genes that have been described to be induced by IL-6 such as Ccr5 (29) (SI Appendix, Fig. S3H). Interestingly, transcripts associated with antigen presentation (MHC-II, CD74) were down-regulated in CD11b⁺ cells from WT iGB₆ recipients compared to IgH^MOG iGB_GFP recipients. Taken together, IL-6 overexpression by iGB cells does not promote Th17 differentiation but instead induces a massive expansion of myeloid cells in the periphery associated with an earlier disease onset. This highly proinflammatory environment may lead to T cell activation even in WT iGB₆ recipients in the absence of MOG-specific iGB cells. Nevertheless, the fact that IgH^MOG iGB₆ cells were able to induce EAE about 1 wk earlier than both WT iGB₆ cells and IgH^MOG iGB_GFP cells indicates that both antigen-specificity and IL-6 expression add up to enhanced B cell pathogenicity in our adoptive cotransfer EAE model.

Fig. 4.

IL-6 production by antigen-specific B cells can enhance their ability to induce EAE. (A) Experimental outline. WT or IgH^MOG B cells were cultured in the iGB culture system. After 48 h, the cells were retrovirally transduced overnight and then cultured on fresh feeder cells. Two days after transduction, the cells were loaded with MOG protein and adoptively transferred together with unmanipulated 2D2 CD4⁺ T cells into Rag1-KO mice. (B) Frequency of successfully transduced GFP⁺ cells on day 5 of iGB culture in WT (filled bars) or IgH^MOG iGB cells (open bars). (C) IL-6 concentration in the culture supernatants measured by ELISA. Striped bars represent IL-6 levels in cultures transduced with an empty eGFP vector. Data are shown as mean + SEM from 2 to 5 experiments. (D) IL-6 levels measured in the serum of mice receiving IL-6 overexpressing (iGB₆) IgH^MOG or WT iGB cells or IgH^MOG cells transduced with an empty vector (iGB_GFP). (E) EAE incidence is shown for 7 mice per group pooled from 2 independent experiments. *P < 0.05_, Mantel–Cox log-rank test with adjusted P values for multiple comparisons using the Holm–Sidak method. (F) Frequency of IFNγ⁺, IFNγ⁺ IL-17A⁺, and IL-17A⁺ CD4⁺ T cells in the CNS of iGB recipients analyzed at the peak of the disease. (G) Quantification of the total number of spleen cells and (H) the frequency of CD11b⁺ myeloid cells in the spleen of iGB recipients. Horizontal lines indicate means. Dots represent individual mice. *P < 0.05 and ****P < 0.0001, one-way ANOVA with Tukey post-test. (I) Distribution of different cell types in the CD11b⁺ population shown as mean from 4 mice per group. (J) IL-6Rα expression in different cell populations in the spleen of IgH^MOG iGB_GFP mice 8 d post-transfer. Histograms are representative for n = 3 mice. (K) Quantification of the frequency of IL-6Rα⁺ cells within the different CD11b⁺ myeloid cell populations. Dots represent individual mice. *P < 0.05, unpaired t test.

IL-6 and TNFα Production by iGB Cells Is Not Crucial For EAE Development.

IgH^MOG B cells express more proinflammatory cytokines such as IL-6 and TNFα than WT B cells (Fig. 1 F–I) and IL-6 overexpression in IgH^MOG iGB cells leads to earlier disease onset in our adoptive cotransfer EAE model (Fig. 4E). However, it is not clear whether the production of IL-6 and TNFα is required for the pathogenicity of IgH^MOG iGB cells. To answer this question, we established a protocol for the CRISPR-Cas9-mediated KO of these cytokines in IgH^MOG iGB cells. IgH^MOG mice were crossed to R26-Cas9 mice, which express the Cas9 protein constitutively under control of the ROSA26 (R26) promoter. Three single guide (sg)RNAs were designed to target the Il6 and Tnf genes, respectively, and cloned into a MSCV vector, which allowed for GFP reporter expression and puromycin selection (SI Appendix, Fig. S4A). First, R26-Cas9 × IgH^MOG B cells were transduced in the iGB system, cultured with puromycin and analyzed at different time points after transduction. Puromycin selection led to the outgrowth of a very pure culture of transduced iGB cells, as more than 80 % of cells were GFP⁺ on day 3 and more than 90 % on day 4 after transduction (SI Appendix, Fig. S4B). KO efficiency was determined by flow cytometry or ELISA and revealed successful KO with all three designed sgRNAs but was most efficient with sg1 for both TNFα and IL-6 (Fig. 5 A and B). For adoptive transfer, cells were selected with puromycin for 2 d (Fig. 5C). On day 3 after transduction puromycin-enriched cells (SI Appendix, Fig. S4C) were cotransferred with 2D2 CD4⁺ T cells into Rag1-KO recipients. Mice receiving IgH^MOG iGB cells expressing TNFα.sg1 or IL-6.sg1 developed disease with similar incidence and severity as mice receiving IgH^MOG iGB cells expressing a nontargeting (NT) sgRNA (Fig. 5D and SI Appendix, Fig. S4D) indicating that neither IL-6 nor TNFα expression by MOG-specific iGB cells was required for EAE development. In line with this, no differences in the cytokine profile of the CNS-infiltrating 2D2 CD4⁺ T cells could be observed (Fig. 5E). Nevertheless, we could demonstrate a robust and stable KO of the targeted cytokines even after recovery of the transferred iGB cells ex vivo (Fig. 5 F–I). Thus, IL-6 and TNFα production by IgH^MOGiGB cells is not necessary for disease induction in our adoptive cotransfer EAE model. In summary, we have successfully generated an important tool to manipulate the disease-inducing B cells prior to adoptive transfer. This can be employed to further investigate molecular players in pathogenic B cell effector function in EAE development.

Fig. 5.

IL-6 and TNFα production by iGB cells is not required for EAE induction. (A and B) R26-Cas9 × IgH^MOG B cells were cultured in the iGB culture system and transduced with MSCV retrovirus. After transduction, the cells were cultured on fresh feeder cells in the presence of IL-21 and puromycin. (A) Representative flow cytometry plots showing the frequency TNFα production by transduced iGB cells after 3 d of puromycin selection. (B) IL-6 KO efficiency was tested by measuring IL-6 levels in the culture supernatants on day 3 or 4 after transduction. Data are shown from one representative experiment out of three independent experiments. (C) Experimental outline for adoptive transfer. R26-Cas9 × IgH^MOG B cells iGB cells were transduced of day 2 of culture, and the cells were then enriched by puromycin selection in the presence of IL-4 for 3 d. Then, the cells were loaded with MOG protein and injected together with unmanipulated 2D2 CD4⁺ T cells into Rag1-KO mice. (D) EAE incidence is shown for 6 to 8 mice per group pooled from 2 independent experiments. (E) Cytokine production by CD4⁺ T cells in the CNS of iGB recipients analyzed at the peak of the disease. Horizontal lines indicate means. Dots represent individual mice. (F and G) Representative flow cytometry plots and (H and I) quantification of TNFα and IL-6 production by iGB cells recovered from the spleens of sick recipients at the peak of the disease. Horizontal lines indicate means. Dots represent individual mice. **P < 0.01 and ****P < 0.0001, one-way ANOVA with Tukey post-test. NT CTRL, nontargeting control.

Discussion

In MS and EAE, pathogenic T cell responses are known to be key drivers of autoimmune inflammation, but increasing evidence suggests an additional role for B cells. However, mechanisms, effector functions, and specificities conferring B cell pathogenicity are not clear. Importantly, especially the efficacy of B cell depletion therapy via anti-CD20 antibodies in pwMS, which spares ASCs (8–10), strongly suggests that cellular effector functions of B cells are crucial for disease. To investigate how B cells contribute to pathogenesis, we first compared properties of polyclonal WT B cells and MOG-specific B cells from IgH^MOG mice. Interestingly, analysis of B cell subsets revealed increased numbers of MZ B cells in the spleen of IgH^MOG mice (Fig. 1 A–C and SI Appendix, Fig. S1 A and B). An expanded MZ B cell compartment has been described in several B cell transgenic or knock-in lines including for example mice with B cells specific for hen-egg-lysozyme (MD2, MD4 C57BL/6 and SW_HEL C57BL/6 (30, 31). Thus, the increased number of MZ B cells in IgH^MOG mice may be a result of the reduced repertoire diversity in transgenic and heavy chain knock-in mice. However, an expanded/activated MZ B cell compartment was also described in several autoimmune conditions in mice and humans. For instance, MZ B cells were shown to be reactive against type II collagen and are involved in the initiation of autoimmune processes in collagen-induced arthritis (CIA) (32, 33). The autoimmune potential of MZ B cells may be explained by their functional properties: MZ B cells express high levels of MHC-II, CD80, and CD86 making them highly efficient APCs (23, 34). In fact, presentation of self-antigen to T cells by MZ B cells was demonstrated in animal models of CIA, type 1 diabetes and systemic lupus erythematosus (32, 35, 36). In line with this, MZ B cells isolated from IgH^MOG mice were able to present MOG to 2D2 CD4⁺ T cells and stimulate T cell proliferation in our in vitro coculture experiments (Fig. 1E). In addition, MOG-specific MZ B cells showed a more proinflammatory cytokine profile with elevated production of IL-6 and TNFα (Fig. 1 H and I). Similar to our findings, enhanced IL-6 secretion by MZ B cells was also found in CIA and mouse models of Sjögren's syndrome (37, 38). Furthermore, IL-6-expressing MZ B cells were increased in actively induced EAE (16). Importantly, in IgH^MOG mice, IL-6 and TNFα were not only increased in MZ B cells but also in follicular B cells (Fig. 1 F and G), indicating that both the expansion of the MZ B cell compartment, as well as the more proinflammatory cytokine profile of both follicular and MZ B cells may contribute to their autoimmune potential.

To gain mechanistic insight and evaluate the impact of B cell–derived cytokine production in the context of autoantigen presentation in vivo, we developed an adoptive cotransfer EAE model, in which B cells provide the critical stimulus for disease development and can be genetically modified to dissect the mechanisms of B cell pathogenicity. By cotransferring highly activated MOG-specific IgH^MOG B cells and unmanipulated MOG-specific 2D2 CD4⁺ T cells, we were able to induce EAE in a B cell adoptive transfer model that does not require additional adjuvant and thus avoids a broad activation of innate immune cells. To obtain highly activated B cells, we employed the iGB culture system generating activated GC B cells, which differentiate into memory B cells upon in vivo transfer and express higher levels of MHC-II and CD80 than naïve B cells (25). To make use of this potential APC function, the expanded iGB cells were loaded with MOG protein prior to adoptive transfer. Only MOG-specific IgH^MOG iGB cells could stimulate 2D2 CD4⁺ T cells to induce EAE, whereas WT iGB cells were not able to do so (Fig. 2B). Although these experiments do not ultimately prove that the transferred IgH^MOG iGB cells indeed act as APCs in this setting, we hypothesize that only MOG-specific iGB cells are able to bind and process their cognate antigen MOG and present it to 2D2 T cells. In line with this, others have shown that MOG-specific B cells can serve as APCs in EAE: Molnarfi et al. demonstrated that B cell APC function is required for EAE induced by immunization with human MOG in CFA and is independent of MOG-specific antibodies (12). Furthermore, B cell antigen presentation is sufficient for EAE induction in mice, in which MHC-II is expressed exclusively by B cells (11).

On the other hand, MOG-specific antibodies, which are secreted in high levels by the iGB cells in our system (Fig. 2F), could also contribute to their pathogenicity. To determine the contribution of MOG-specific IgG1 antibodies to disease development, we compared the pathogenic potential of unswitched IgM⁺ IgH^MOG iGB cells and partly switched IgH^MOG iGB cells (10 to 20 % IgG1⁺): both were able to induce EAE with similar onset, incidence and severity (Fig. 3 D and E) indicating that high levels of MOG-specific IgG1 antibodies are not critical for disease development in our cotransfer model, although this does not rule out a contribution of low levels of MOG-specific IgGs to pathogenesis. Notably, others have reported disease-enhancing effects of MOG-specific IgG antibodies. For instance, Flach et al. used MOG-specific B and T cell cotransfer into C57BL/6 mice followed by active immunization and showed that MOG-specific antibodies enhance antigen presentation by CNS-resident APCs and facilitate T cell reactivation directly in the CNS (39). However, in this setting, the use of adjuvant leads to a broad activation of the myeloid compartment, which in turn primes the MOG-specific T cells in the periphery and also readily reactivates T cells in the CNS — a process that can be amplified by antibody-opsonized antigen. In contrast, in our model autoreactive B cells are specifically activated while the innate immune cells are less involved, which may explain why the presence of high levels of MOG-specific IgG1 did not further enhance disease development in our model. Interestingly, we found slightly increased IgG1 titers in sick vs. healthy recipients of unswitched iGB cells (Fig. 3F), as well as in spleens and mLNs of IgH^MOG iGB recipients compared to WT iGB recipients (Fig. 2E). This also supports the hypothesis that direct B:T cell interactions occur in our model leading to T cell activation and subsequently to continued isotype switching of the B cells in response to T cell–derived stimuli.

Another mechanism by which IgH^MOG B cells could promote disease development is the production of proinflammatory cytokines in context with APC function. To investigate the role of B cell–derived cytokines in our experimental model, we developed a protocol for the overexpression as well as the CRISPR-Cas9-mediated KO of cytokines in iGB cells prior to adoptive transfer. Since we saw increased IL-6 expression in MOG-specific IgH^MOG B cells (Fig. 1 F and H) and since B cell–derived IL-6 was shown to be important for EAE development in other studies (12, 16, 26, 27), we first determined whether overexpression of IL-6 would further enhance B cell pathogenicity. Furthermore, as IL-6 is one of the key cytokines for differentiation into Th17 cells, we hypothesized that overexpression of IL-6 in the context of antigen presentation by IgH^MOG iGB cells may skew T cell differentiation toward the Th17 lineage. Indeed, IL-6 overexpression by iGB cells led to an earlier disease onset in our experimental system, however, without inducing a demonstrable Th17 response (Fig. 4 E and F). This may be partly due to the use of Rag1-KO mice as recipients, where T cells undergo spontaneous and homeostatic proliferation upon transfer, which goes along with production of IFNγ (40) and thus, a Th1 phenotype may be strongly favored over differentiation and stabilization of Th17 cells. Rather than promoting Th17 cell differentiation, IL-6 overexpression was associated with splenomegaly and expansion/proliferation of myeloid cells in the periphery in our model (Fig. 4 G–I). Analysis of IL-6Rα expression confirmed that CD11b⁺ cells and especially inflammatory monocytes sense and respond to IL-6, whereas T cells and iGB cells do not express IL-6Rα preonset (Fig. 4 J and K). Interestingly, IL-6Rα levels were down-regulated in inflammatory monocytes upon IL-6 overexpression, which may indicate a negative feedback loop in response to IL-6 signaling. Similar to our observations, elevated systemic IL-6 levels in IL-6 transgenic mice led to splenomegaly, but also to expansion of plasma cells (41, 42). Furthermore, transgenic overexpression of IL-6 by CD11c⁺ dendritic cells (DCs) also resulted in a massive expansion of Ly-6G⁺ neutrophils and Ly-6C^high monocytes (43). Most likely, the systemically high IL-6 levels in our cotransfer experiments created a proinflammatory environment, which expanded CD11b⁺ cells and accelerated T cell activation and onset of disease. In this proinflammatory environment even recipients of polyclonal WT iGB₆ cells developed EAE. In that regard, systemically elevated levels of IL-6 may have a similar effect as adjuvant in actively induced EAE models resulting in a broad activation of the myeloid compartment, although the exact mechanism of how the activated and expanded myeloid populations promote pathogenesis remains to be investigated. However, the fact that IgH^MOG iGB₆ cells were superior to both WT iGB₆ cells and IgH^MOG iGB_GFP cells to induce EAE indicates that both the unspecific proinflammatory effect of IL-6 overexpression and the antigen-specificity of the B cells synergize resulting in an earlier disease onset in our system.

We found that the pathogenic effects of IL-6 are mainly associated with expansion of the myeloid compartment rather than shaping the T cell response. Consistent with this, CRISPR-Cas9-mediated KO of IL-6 and also of TNFα in IgH^MOG iGB cells did not affect the pathogenic T cell response nor development of EAE (Fig. 5D), indicating that B cell–derived IL-6 and TNFα are not required for disease induction in our system. In contrast, global IL-6-deficient mice are completely resistant to EAE induced by immunization (44–47). Of note, IL-6 but also TNFα can be secreted by a variety of cells including DCs and macrophages and for TNFα also by T cells, and thus, the loss of B cell–derived IL-6 and TNFα may be compensated by other cells.

In summary, we developed an adoptive cotransfer system, in which only MOG-specific IgH^MOG iGB cells but not polyclonal WT iGB cells are able to interact with MOG-specific 2D2 CD4⁺ T cells to induce EAE. This is a B cell adoptive transfer EAE model that does not require immunization and where B cells provide the critical stimulus for disease development. We are aware that our model also has disadvantages, mainly due to the use of Rag1-KO mice as recipients. The lack of adaptive immune cells in these mice constitutes an artificial environment, which promotes homeostatic proliferation of the transferred T cells, and may facilitate EAE development in general. Furthermore, since we cannot completely exclude a contribution of antibodies and definitely prove B cell APC function, the exact mechanism by which MOG-specific iGB cells induce disease in this model requires further investigation. Nonetheless, we believe that our system offers specific advantages to study the role of B cells in disease pathogenesis: First, our system avoids a broad activation of the myeloid compartment as induced by immunization, but focuses on the cellular effector functions of B cells, which are also implicated in human MS. Second, iGB cells can be manipulated in this system prior to adoptive transfer to investigate B cell effector functions such as antigen presentation and cytokine production. Specifically, genes of interest can be overexpressed or knocked out via CRISPR-Cas9 in iGB cells, and the direct comparison of overexpression and CRISPR-Cas9-mediated KO in the same system may provide important insights to understand molecular players in pathogenic B cell effector function in CNS autoimmunity.

Materials and Methods

Parts of this study are also included in the dissertation of A. S. Thomann (48). Additional methods are described in SI Appendix.

Animals.

C57BL/6 wild-type mice, Rag1-KO mice (B6.129S7-Rag1tm1Mom/J) and 2D2 mice [C57BL/6-Tg(Tcra2D2,Tcrb2D2)1Kuch/J] were purchased from The Jackson Laboratory. CD45.1 mice (B6.SJL-PtprcaPepcb/BoyCrl) were purchased from Charles River. IgH^MOG mice on a C57BL/6 background carrying a rearranged V_HD_HJ_H sequence of the MOG-specific monoclonal antibody 8.18C5 replacing the endogenous IgH-D and IgH-J elements and therefore harboring MOG-specific B cells were described previously (18). IgH^MOG mice were crossed to CD45.1 mice to create IgH^MOG.CD45.1 mice. R26-Cas9 mice [B6.Cg-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J] were obtained from The Jackson Laboratory on a mixed background, backcrossed onto the C57BL/6 background for at least five generations and crossed to IgH^MOG.CD45.1 mice to generate R26-Cas9 x IgH^MOG.CD45.1 mice.

All mice were housed and bred under specific pathogen-free conditions in the Core Facility Animal Models at the Biomedical Center of the Ludwig-Maximilians-Universität München. Animal experiments were designed and performed according to regulations of the animal welfare acts and approved by the animal ethics committee of the state of Bavaria (Regierung von Oberbayern) in accordance with European guidelines.

sgRNA Design.

sgRNAs were designed using the Broad Institute GPP sgRNA Designer (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design). Three sgRNAs per target gene were designed and validated in parallel. sgRNA forward sequences were IL-6.sg1 5′-GTATACCACTTCACAAGTCGG-3′, IL-6.sg2 5′- GCCTACTTCACAAGTCCGGAG-3′, IL-6.sg3 5′-GATGGTACTCCAGAAGACCAG-3′, TNFα.sg1 5′-GTAGACAAGGTACAACCCAT-3′, TNFα.sg2 5′-GAAGAAATCTTACCTACGACG-3′, TNFα.sg3 5′-GCTACTGAACTTCGGGGTGAT-3′, NT CTRL 5′- GCTGCATGGGGCGCGAATCA-3′. For cloning into the BbsI sites of the pMSCV backbone, a CACC overhang was added at the 5′ end of the forward sequence and an AAAC overhang was added at the 5′ end of the reverse sequence.

Cloning Procedures.

IL-6 overexpression.

The IL-6 sequence was obtained via PCR-amplification from total complementary DNA isolated from primary mouse cells stimulated with LPS and anti-CD40 using primers, which were designed based on the mRNA reference sequence NM_031168.2 of the NCBI database and contained restriction sites for EcoRI (FWD) and BamHI (REV) (highlighted in bold): IL6 FWD 5′-CGGTAGAATTCAAACCGCTATGAAGTTC-3′, IL-6 REV 5′- ACTAGGATCCTAGGCATAACGCACTAGG-3′. The digested PCR product was ligated into the pMSCV-IRES2-eGFP vector (kindly provided by Gurumoorthy Krishnamoorthy) with T4 ligase and the construct was transformed into competent DH5α bacteria. Correct insertion was confirmed with Sanger sequencing.

CRISPR KO plasmids.

Each pair of sgRNA oligonucleotides was phosphorylated with T4 PNK (New England Biolabs) in T4 ligation buffer (New England Biolabs) for 30 min at 37 °C and then annealed by heating to 95 °C for 5 min and cooling to 25 °C at 5 °C/min. Annealed oligos were diluted 1:200 and ligated into the BbsI-HF-digested MSCV-pklv2-gRNA-puroGFP plasmid (kindly provided by Martin Kerschensteiner) at RT for 10 min. Cloned plasmids were transformed into Stellar™ Competent Cells (Takara) or JM109 Competent Cells (Promega) following the manufacturer’s transformation protocols.

Stimulation of Primary B cells for Intracellular Cytokine Staining.

Mouse B cells were isolated from total splenocytes by positive selection using a biotinylated anti-B220 antibody (clone RA3-6B2, BD Pharmingen) and MojoSort™ Streptavidin Nanobeads (BioLegend) according to the manufacturer’s instructions. If applicable, different B cell subsets were sorted by FACS. Purified B cells or sorted B cell subsets were cultured at 1 to 2 × 10⁶ cells/mL in iGB medium containing 10 µg/mL LPS (Sigma) and 2.5 µg/mL anti-CD40 (1C10, BioLegend) for 24 h. For the last 4 h of stimulation, 50 ng/mL PMA, 1 µg/mL ionomycin, and 5 µg/mL brefeldin A (all Sigma) were added to the cultures. Then, the cells were collected, washed, and stained for flow cytometry analysis.

iGB Cell Culture.

40LB cells (BALB/c3T3 fibroblasts expressing CD40L and BAFF) were described previously (25). The cells were routinely cultured in 40LB medium (Dulbecco’s Modified Eagle’s Medium containing 10% heat-inactivated fetal bovine serum, 1% penicillin–streptomycin, 2 mM L-glutamine, 100 µM nonessential amino acids, 1 mM sodium pyruvate, and 5.72 µM β-mercaptoethanol) and kept subconfluent. One day prior to iGB culture, 40LB cells were seeded at 3.1 × 10³ cells/cm² and allowed to attach overnight. Then, 2.6 × 10⁴/cm² purified B cells (EasySep™ Mouse B Cell Isolation Kit, Stem Cell Technologies) were cultured on 40LB cells with or without 1 ng/mL recombinant mouse (rm)IL-4 (BioLegend) for 3 d (d4 iGB cells). Then, iGB cells were collected by incubation with prewarmed PBS (Sigma/Gibco) containing 0.5% bovine serum albumin (Roth) and 2 mM ethylendiamintetraacetate. For overexpression or CRISPR-Cas9-mediated KO, iGB cells were retrovirally transduced 24 h or 48 h after start of the culture by addition of 2 ng/mL rmIL-4 or 10 ng/mL IL-21, 4 µg/mL polybrene (Sigma), and 1:50-diluted retroviral supernatant to the culture. The next morning, iGB cells were collected, washed, and seeded on fresh feeder cells in the presence of 1 ng/mL rmIL-4 or 10 ng/mL rmIL-21. For CRISPR-Cas-mediated KO, transduced cells were selected by addition of 1.25 to 4 µg/mL puromycin (Invivogen) during the second phase of the culture.

EAE Induction by Adoptive Cotransfer of iGB Cells and MOG-Specific 2D2 CD4⁺ T cells.

iGB cells were collected at indicated time points and washed by centrifugation at 300 g for 10 min at RT. Then, iGB cells were loaded with MOG protein (codon-optimized rodent MOG_1-125, produced in house in HEK-EBNA cells according to the protocol described in Perera et al. JI 2013 (49)) by incubating them at a concentration of 1 × 10⁷ cells/mL in the presence of MOG protein (10 µg/mL) for 3 h at 37 °C. Then, the cells were collected, washed, and filtered through a 70-µm filter, washed two more times, and resuspended in prewarmed PBS. Then, 1.5 to 2.5 × 10⁷ iGB cells/mouse were injected i.p. into Rag1-KO recipients. MOG-specific CD4⁺ T cells were isolated from LNs and spleens of transgenic 2D2 mice either by negative selection using the EasySep™ Mouse CD4+ T Cell Isolation Kit (StemCell Technologies) or the MojoSort™ Mouse CD4 T Cell Isolation Kit (BioLegend) or by positive selection using CD4 (L3T4) MicroBeads (Miltenyi) according to the manufacturer’s instructions. After isolation, CD4⁺ 2D2 T cells were resuspended in PBS, and 4 to 5 × 10⁶ cells/mouse were injected i.p. or i.v. into Rag1-KO recipients. Animals were monitored daily for clinical signs of EAE according to the following scoring criteria: 0, no disease; 0.5, decreased tail tonus; 1, limp tail; 1.5 limp tail and ataxia; 2, hind limb weakness; 2.5, partial hind limb paralysis; 3, complete hind limb paralysis; 3.5, complete hind limb and partial front limb paralysis; 4, complete front and hind limb paralysis; and 5, moribund.

Statistical Analysis.

Statistical analyses were performed using GraphPad Prism version 7. Appropriate statistical tests were selected as indicated in the figure legends. Differences between two groups were determined using two-tailed Student’s t test The statistical differences between more than two groups were calculated using one-way ANOVA with Tukey’s post-test for multiple comparisons. Differences between EAE incidence curves were evaluated using the Mantel–Cox log-rank test with adjusted P values for multiple comparisons using the Holm–Sidak method, when necessary.

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

Bulk RNA sequencing data have been deposited in GEO (GSE245571) (28). All other data are included in the article and/or SI Appendix.