B cells are capable of independently eliciting rapid reactivation of encephalitogenic CD4 T cells in a murine model of multiple sclerosis

Recent success with B cell depletion therapies has revitalized efforts to understand the pathogenic role of B cells in Multiple Sclerosis (MS). Using the adoptive transfer system of experimental autoimmune encephalomyelitis (EAE), a murine model of MS, we have previously shown that mice in which B cells are the only MHCII-expressing antigen presenting cell (APC) are susceptible to EAE. However, a reproducible delay in the day of onset of disease driven by exclusive B cell antigen presentation suggests that B cells require optimal conditions to function as APCs in EAE. In this study, we utilize an in vivo genetic system to conditionally and temporally regulate expression of MHCII to test the hypothesis that B cell APCs mediate attenuated and delayed neuroinflammatory T cell responses during EAE. Remarkably, induction of MHCII on B cells following the transfer of encephalitogenic CD4 T cells induced a rapid and robust form of EAE, while no change in the time to disease onset occurred for recipient mice in which MHCII is induced on a normal complement of APC subsets. Changes in CD4 T cell activation over time did not account for more rapid onset of EAE symptoms in this new B cell-mediated EAE model. Our system represents a novel model to study how the timing of pathogenic cognate interactions between lymphocytes facilitates the development of autoimmune attacks within the CNS.


Introduction
Multiple sclerosis (MS) is a debilitating autoimmune disease of the central nervous system (CNS) with an unknown etiology despite being the subject of intense study for over a century [1]. MS is characterized by the chronologically and spatially distinct formation of lesions ("plaques") comprised of cellular and humoral inflammation, demyelination, and axonal damage. Experimental autoimmune encephalomyelitis (EAE) is the main animal model for MS used to investigate the cellular mechanisms of disease as well as to develop new MS treatments [2,3]. Early experiments with EAE identified the CD4 T cell as both necessary and sufficient for disease and prompted further investigation into the characteristics of MHCII+ antigen presenting cells (APCs) responsible for the regulation of CD4 T cell behavior during neuroinflammation [4].
In MS, B cell depletion therapies (BCDTs) have recently been shown to be highly effective at amelioration of disease [5,6]. BCDT reduces relapses and decreases inflammatory lesions [5,7] but does not affect cerebral spinal fluid (CSF) levels of immunoglobulin nor deplete the long-lived antibody-secreting plasma cells from within the CSF or other tissues [8,9]. Various approaches with BCDT in EAE also demonstrate that B cells can have an enormous influence on cognate encephalitogenic T cell pathogenicity and highlight the importance of antibodyindependent B cell functions for the pathogenesis of CNS autoimmunity [10][11][12]. B cells are not highly phagocytic yet are very efficient at presenting antigens acquired via receptor-mediated endocytosis [13][14][15]. Although the target antigens for MS are unknown, recombinant B cell receptors (BCRs) derived from CSF-localized B cell clones exhibit specificity for myriad CNS components [16][17][18][19]. Through the process of linked recognition, a non-auto-reactive B cell could still present self-peptide antigens associated with internalized immune complexes to activate auto-reactive CD4 T cells [20]. Understanding how B cell-mediated antigen presentation influences neuro-inflammation and tolerance in MS could lead to potent and more specific immunomodulatory therapies.
Our previous work demonstrated that B cells are capable of serving as the only APC during passive EAE [15]. However, transgenic mice with elevated B cell specificity for MOG (IgH MOG mice) crossed to mice expressing MHCII exclusively by CD19 + B cells (CD19-B MHCII mice)referred to as CD19-B MHCII xIgH MOG mice because CD19 Cre drives MHCII expression on B cells-develop passive EAE with a statistically significant and reproducible delay in onset compared to WT mice [15]. Thus, B cells are capable of propagating auto-antigen-specific CNS demyelination on their own but may be limited in their efficiency as APCs. To explore the kinetics of B cell cognate interactions during EAE, B MHCII xIgH MOG mouse were bred to a Tamoxifen (Tam)-inducible CD20 Tam-Cre murine reagent generated by Shlomchik and colleagues [21]. By carefully controlling of the timing of cognate interactions between B cells and CD4 T cells during EAE, we have observed a rapid induction of disease, demonstrating that B cells can efficiently drive auto-reactive CD4 T cell responses targeting the CNS.

Flow cytometry
Spleens were harvested from Avertin-anesthetized mice and single cell suspensions were treated with ACK erythrocyte lysis buffer. Mice were perfused with 25mL of ice-cold PBS and CNS tissues were isolated. Mononuclear cells were purified from homogenized brains and spinal cords by centrifugation for 30 min in 30% Percoll (GE Healthcare) solution as previously reported [22].

Histology and immunofluorescence
Mice were sacrificed and perfused with 25mL ice cold PBS followed by 20mL 4% paraformaldehyde (Sigma-Aldrich). Spinal cord tissue was removed from the vertebrae and then fixed in 4% paraformaldehyde for more than 12 hours, followed by dehydration in 30% sucrose for 48 hours. The tissue was embedded in optimal cutting temperature media (TissueTek, Torrence, CA) and cut at 8-10μm thick sections using a Leica CM1900 cryostat (Germany). The WUSM Developmental Biology Histology & Microscopy Core stained sections with Luxol Fast Blue to detect myelin. Slides were examined by light microscopy using a Nikon 90i motorized upright digital microscope with camera and Metamorph software (Molecular Devices). Sections were also stained to quantify myelinated white matter using goat anti-MOG (Invitrogen) with isotype control goat IgG2a (abcam) and secondary antibody donkey anti-goat Alexa-555 (Invitrogen). Slides were mounted with Fluoroshield Mounting Medium with DAPI (abcam). Slides were examined by immunofluorescent microscopy using a Nikon 90i motorized upright digital microscope with CoolSNAP ES 2 camera (Photometrics). Inflammation score was recorded by blinded observer using the following scale system: 0 = normal, no inflammation; 1 = mild inflammation with a few small regions of increased cellularity; 2 = moderate inflammation with one large lesion or several smaller inflammatory foci; 3 = severe inflammation with dense parenchymal infiltration and many large lesions; 4 = massive inflammation in which most of the white matter has dense cellularity. Demyelination was quantified by blinded calculation of lesion area in thoracic spinal cord sections using Metamorph software (Molecular Devices, Inc).

Statistical analysis
All data generated are reported as mean ± the standard error of the mean (SEM). Unpaired ttests were used for comparison of cellular infiltrates. Time-to-EAE-onset incidence curves were compared by Log-rank (Mantel-Cox) tests. Group effects were compared via analysis of variance (ANOVA) with Tukey's test or Kruskal-Wallis test with Dunn's test for multiple comparisons when assumptions were not violated. Mann-Whitney tests with two-tailed P values were performed for flow cytometry assays quantifying lymphocytes in the CNS. All statistical analyses were completed using PRISM 7 software (GraphPad).

B cell antigen presentation is insufficient to initiate spontaneous autoimmune CNS demyelination
Increasing the frequency of MOG-specific B cells in 2D2 mice, in which T cell receptors are highly specific for MOG, routinely leads to spontaneous inflammatory demyelination within the spinal cord and optic nerve, indicating that naïve MOG-specific B and T cells collaborate to induce neuro-inflammation [24,25]. Additionally, transgenic SJL/J mice with MOG-specific T cells require an intact B cell compartment for susceptibility to spontaneous EAE [26].
To test the hypothesis that antigen presentation by B cells can independently trigger spontaneous EAE, we crossed CD19-B MHCII xIgH MOG with 2D2 mice and salvaged 2D2 CD4 T cell development in progeny by thymus transplant. While both 2D2 and 2D2xIgH MOG mice with WT MHCII expression spontaneously developed optic neuritis, only 2D2xIgH MOG mice developed spontaneous EAE, confirming previously published data (Table 1). However, 2D2xCD19-B MHCII xIgH MOG mice were completely resistant to both optic nerve inflammation and spontaneous EAE (Table 1) despite a substantial reconstitution of the CD4 T cell compartment with MOG-specific T cells as reported [23]. These results suggested that antigen-specific B cells alone were not sufficient to elicit CD4 T cell-dependent spontaneous inflammatory demyelination within the CNS, even when a high frequency of cognate, auto-reactive T cells were present. When these data are considered in conjunction with our previous observation that EAE is delayed in CD19-B MHCII xIgH MOG mice [15], it suggests that B cells are intrinsically less efficient than other APCs in their ability to initiate and direct CD4 T cell auto-reactivity to MOG.

Tamoxifen-inducible systems for induction of MHCII expression
By breeding the IAß b stop flox/flox xIgH MOG mouse to Tam-inducible CD20 Tam-Cre mice, we aimed to temporally regulate MHCII expression exclusively by B cells. To assess the efficiency of recombination at the IA-ß locus following Tam administration, peripheral blood was collected from CD20-B MHCII xIgH MOG and UBC MHCII mice prior to oral gavage with Tam and at various time points thereafter. MHCII expression was undetectable prior to Tam treatment for both CD20-B MHCII xIgH MOG and UBC MHCII mice similar to the two hour timepoint ( Fig 1A). 52.3 ± 4.7% (Mean ± SEM) of B cells from CD20-B MHCII xIgH MOG mice expressed MHCII 24 hours after Tam administration ( Fig 1A). After 72 hours, the mean frequency of MHCII+ B cells in CD20-B MHCII xIgH MOG spleens (89.3 ± 1.4%) was similar to that of WT (84.5 ± 1.8%) and CD19-B MHCII xIgH MOG mice (94.3 ± 1.5%) ( Fig 1B). Following Tam administration, fewer B cells in UBC MHCII mice expressed MHCII compared to WT B cells, yet MHCII was induced on other APC subsets in UBC MHCII mice ( Fig 1B).

B cell-mediated EAE is delayed in CD20-B MHCII xIgH MOG mice
Based on the high efficiency of MHCII induction by Tam administration, we hypothesized that expression of MHCII by B cells in CD20-B MHCII xIgH MOG mice would manifest EAE similar to CD19-B MHCII xIgH MOG mice. Thus, we treated CD20-B MHCII xIgH MOG mice and UBC MHCII mice with Tam three days prior to the adoptive transfer of encephalitogenic T cells.
In WT mice, adoptive transfer of encephalitogenic CD4 T cells typically resulted in clinical and pathological signs within 10 days (Fig 2A). The time to onset and disease course for WT and UBC MHCII mice treated with Tam prior to the adoptive transfer of CD4 T cells were not significantly different ( Fig 2B and 2C). As previously reported, CD19-B MHCII xIgH MOG mice exhibited a statistically significant delay in the day of onset of EAE compared to WT mice, with EAE signs beginning on average at 15.0 ± 1.0 days following transfer of donor T cells [15]. CD20-B MHCII xIgH MOG mice treated with Tam before T cell transfer exhibited similar disease kinetics, with an average day of EAE onset of 15.8 ± 0.9 days (Fig 2B and 2C). Hence, in our Tam-inducible MHCII system, B cell-restricted MHCII expression requires an extended period of time to facilitate disease mediated by encephalitogenic CD4 T cells compared to a full complement of MHCII+ APC subsets. These results indicate that the conditional and temporal regulation of MHCII in vivo is suitable for investigating the timing of B cell involvement during EAE.

B cells are capable of inducing accelerated onset EAE
Antigen acquisition and presentation by B cells is implicated as a critical precursor to CD4 T cell activation and demyelination in B cell-dependent models of EAE [27][28][29]. It is possible for UBC MHCII mice treated with Tam one week after T cell transfer was 6.0 ± 0.4 days post Tam, whereas the average for mice treated with Tam after two or three weeks post T cell transfer was 5.6 ± 0.3 days and 5.7 ± 0.3 days post Tam, respectively (Fig 3A and 3B). Surprisingly, we observed a rapid onset of EAE signs for CD20-B MHCII xIgH MOG mice that was dependent on the length of time between T cell transfer and MHCII induction (Fig 3A and 3B). The mean day of onset for CD20-B MHCII xIgH MOG mice was significantly delayed compared to UBC MHCII mice when Tam was administered at either week one or week two after T cell transfer ( Fig 3A). However, when mice received Tam three weeks after CD4 T cell transfer, the mean time to EAE onset for CD20-B MHCII xIgH MOG and UBC MHCII mice was not significantly different ( Fig 3B). These results demonstrate that B cell-mediated EAE has the capacity to develop more quickly than even the conventional WT passive EAE model. Thus, our Taminducible MHCII expression system provides a means to assess the kinetic differences in neuro-inflammation coordinated by antigen-specific B cells.

Accelerated EAE is not due to immune cell trafficking to the CNS prior to Tam administration
A low frequency of CNS MHCII+ B cells can support neuro-inflammation in CD19-B MHCII x-IgH MOG mice with EAE [15]. Localization to the CNS compartment prior to Tam administration would give a small number of B cells ready access to both MOG antigens and cognate T cells primed to respond rapidly upon MHCII expression by the B cells. Hence, antigen-specific lymphocytes entering the CNS over time prior to the induction of MHCII expression could explain the increasingly rapid onset of EAE signs observed in CD20-B MHCII xIgH MOG mice. To assess this possibility, the spinal cords of CD20-B MHCII xIgH MOG mice and UBC MHCII mice were examined for inflammation and demyelination before and after treatment with Tam three weeks post-T cell transfer. Histological examination of these tissues did not reveal any signs of inflammation or demyelination prior to Tam treatment (Fig 4A). Both CD20-B MHCII xIgH MOG mice and UBC MHCII mice that developed EAE after receiving Tam on week three post-T cell transfer had similar inflammatory foci (Fig 4A and 4B). Semi-quantitative assessment of inflammation of the spinal cord at the level of the brainstem and cervical, thoracic, and lumbar regions of the spinal cord revealed similar degrees of inflammation ( Fig  4C). However, we observed a significant difference in the extent of inflammation between the genotypes at the thoracic level ( Fig 4B). The difference in area of demyelinated white matter within the thoracic spinal cords of CD20-B MHCII xIgH MOG and UBC MHCII mice with EAE was significantly different, though this could be due to a higher mean EAE score for the UBC MHCII mice (3.33 ± 0.4 vs. 2.4 ± 0.3) (Fig 4D). These observations were verified by flow cytometric analyses of the composition of infiltrating mononuclear cells in brain and spinal cord tissues examined prior to Tam treatment at various time points post T cell transfer (Fig 5 and S1 Fig). UBC MHCII , CD20-B MHCII xIgH MOG , and MHCII deficient Cre -IAß b stop flox/flox xIgH MOG littermate control mice were harvested one, two, or three weeks post T cell transfer but prior to Tam treatment and brains and spinal cords were analyzed by flow cytometry to detect infiltrating B cells and donor CD4 T cells. Prior to Tam treatment, these recipient mice are all MHCIIdeficient and analysis of donor T cell cytokine production after incubation in different genotypes does not indicate that the genotype of the recipient influences encephalitogenicity over time (Fig 6 and S2 Fig). While the mean frequency of B cells in the brain at week two post cell transfer was significantly different compared to week one post cell transfer (p < 0.01), there was no difference at week three and no overall increase in the frequency of lymphocytes in the CNS over time prior to Tam treatment ( Fig 5A). Altogether, very few infiltrating lymphocytes were detected in the brain and spinal cord before MHCII expression was induced, and the cellular composition of CNS tissues from experimental mice was comparable to the number of B cells and endogenous CD4 T cells detected in the CNS of naïve WT mice (S1 Fig). Taken together this suggests that the rapid onset of disease is not a result of anticipatory migration of CD4 T or B cells into the CNS. Flow cytometry of CNS tissues harvested from CD20-B MHCII x-IgH MOG mice three days post-EAE onset showed that the frequency of B cells in the brains and spinal cords tended to be more variable when Tam is administered soon after T cell transfer (Fig 5B-5D). Additionally, although there is a higher frequency of B cells in the CNS of CD20-B MHCII xIgH MOG mice with EAE when treated with Tam two weeks after T cell transfer (Fig 5C), the difference is not apparent when treated with Tam three weeks after T cell transfer (Fig 5D). These results suggest that the rapid onset of EAE signs observed in CD20-B MHCII x-IgH MOG mice treated with Tam weeks after T cell transfer is not the result of enhanced localization of B cells or T cells in the CNS compartment.

Changes in donor T cells over time do not account for rapid onset of B cellmediated EAE
It is possible that donor encephalitogenic CD4 T cells become more pathogenic over time prior to MHCII induction in CD20-B MHCII xIgH MOG mice. To rule out the possible effects of increasing donor CD4 T cell activation, peripheral CD4 T cell markers were assayed by flow cytometry at various time points post-transfer and prior to Tam administration. The mean fluorescence intensity (MFI) of CD44, a marker of effector-memory CD4 T cells, increased over time while CD69, a marker of T cell activation, decreased over time (Fig 6A and 6D). The MFI of two other markers, CD62L, which is expressed by naïve T cells homing to secondary lymphoid tissues, and CD25, expressed by activated T cells and regulatory T cells, also decreased over time in MHCII deficient recipients prior to Tam administration (Fig 6B and  6C). Hence, deviations in activation status were observed in donor CD4 T cells after incubation in MHCII-deficient hosts. However, a mixture of changes, inconsistent with a selection of a singular population of activated encephalitogenic CD4 T cells, was observed after prolonged incubation in the absence of cognate interactions. Subsequently, we reasoned that if the pre-Tam time period induces changes in donor T cells to enhance their encephalitogenic potential, we would then be able to induce rapid-onset EAE in WT or CD19-B MHCII xIgH MOG mice by re-transferring the T cells into new naïve hosts. If the observed changes in donor T cells are responsible for the increasingly rapid EAE onset observed in CD20-B MHCII xIgH MOG mice, donor T cells collected after an initial incubation in an MHCII-deficient host would induce rapid-onset EAE after a subsequent passive transfer into naïve recipients. Therefore, MHCII deficient Cre -IAß b stop flox/flox xIgH MOG mice received encephalitogenic CD4 T cells, which were harvested from spleens and lymph nodes after three weeks of in vivo incubation. These freshly isolated T cells were MACS-purified by CD4 positive selection and immediately transferred into naïve WT or CD19-B MHCII xIgH MOG recipients. The average day of onset was 18.2 for CD19-B MHCII xIgH MOG receiving encephalitogenic CD4 T cells rested for three weeks in a MHCII-deficient environment, similar to the day of onset described previously for CD19-B MHCII xIgH MOG mice receiving freshly in vitro generated donor CD4 T cell lines (Table 2). Likewise, the day of onset for WT and UBC MHCII mice was similar to the day of onset for WT mice whether donor CD4 T cells were rested in vivo for three weeks or transferred immediately. Flow cytometry to detect intracellular cytokines revealed the frequency of IFNγ-producing donor T cells harvested three weeks after transfer into an MHCII-deficient recipient is drastically reduced compared to the frequency of cytokine-producing donor CD4 T cells prior to transfer. This same reduction in IFNγ production was observed in CD4 T cells harvested from CD20-B MHCII xIgH MOG and UBC MHCII mice prior to Tam administration (Fig 6E and S2 Fig). Taken together, the data indicate that prolonged

. Inflammation and demyelination is not evident in spinal cords of encephalitic CD4 T cell recipients prior to Tam administration. (A)
Representative spinal cord sections from recipients of encephalitogenic CD4 T cells (n = 5, CD20-B MHCII xIgH MOG mice; n = 6, UBC MHCII mice) were stained with Luxol Fast Blue, scale bar = 100um; all images generated from 10x magnification. (A) Spinal cords from CD20-B MHCII xIgH MOG mice harvested three weeks after T cell transfer and before Tam administration (left). CD20-B MHCII xIgH MOG mice treated with Tam three weeks after T cell transfer (middle) and harvested three days post EAE onset. UBC MHCII mice treated with Tam three weeks after T cell transfer (right) and harvested three days post EAE onset. (B) Representative spinal cord sections from recipients of encephalitogenic CD4 T cells (at least mice 5 per genotype) were stained with antibodies to detect MOG, scale bar = 100um. Spinal cords from CD20-B MHCII xIgH MOG mice (top left) and UBC MHCII mice (bottom left) harvested three weeks after T cell transfer and before Tam administration. CD20-B MHCII xIgH MOG (top right) and UBC MHCII (bottom right) mice treated with Tam three weeks after T cell transfer and harvested three days post EAE onset. (C) Regions from rostral to caudal sections of spinal cords from CD20-B MHCII xIgH MOG (blue) and UBC MHCII mice (red) were harvested and scored for inflammation. Graph shows mean +/-SEM inflammation scores and Kruskal-Wallis test with Dunn's correction for multiple comparisons was applied. B.S. = brainstem; C = cervical; T = thoracic; L = lumbar. (D) Mean (SD) percent area of demyelinated white matter was quantified for thoracic spinal cord sections from CD20-B MHCII xIgH MOG (blue) and UBC MHCII (red) mice. Significance determined by Mann-Whitney test with two-tailed p value. incubation in a MHCII-deficient environment prior to Tam treatment does not lead to enhanced encephalitogenicity of donor CD4 T cells. Indeed, it is apparent that B cells are sufficient to provide the necessary APC functions to rapidly re-stimulate rested effector memory T cells to initiate EAE symptoms.

Discussion
The contributions of antigen specificity, antigen presentation, and cytokine production by individual APC populations on secondary T cell activation are complex and poorly understood. In MS and EAE, variation in T cell activation and effector lineage may be due to subtle differences in the abilities of APCs to acquire and present certain antigens at different times. For example, B6 mice are susceptible to MOG 35-55 peptide-induced active EAE even if MHCII expression is restricted to DCs or if B cells are genetically ablated [23,28,30,31]. However, resistance to MOG protein-induced active EAE can be seen in B cell deficient mice, mice with DC-specific MHCII expression, or mice in which B cells are MHCII deficient [28,32,33]. Taken together, it is apparent that B cell antigen presentation is critical for supporting CNS auto-reactivity to whole protein antigens.
Whether B cells facilitate initial CD4 T cell activation toward protein antigens in EAE has not been directly examined to date. In restricting MHCII expression to B cells, we found that the transgenic combination of B and T cell receptor specificities for MOG no longer evoked spontaneous inflammatory demyelination within the spinal cord (Table 1). While it is possible that 2D2xCD19-B MHCII xIgH MOG mice do not develop spontaneous EAE due to the incomplete reconstitution of CD4 T cells by thymic grafting, our method has been utilized previously with success [23] and wild-type levels of CD4 T cells can be observed in secondary lymphoid tissue 9 weeks post-graft [23]. Other constraints such as a limited humoral MOG response in 2D2xCD19-B MHCII xIgH MOG mice are unlikely to limit inflammatory demyelinating disease, as we believe that soluble MOG-specific antibody is not essential for EAE development. This is based on our observation that passive transfer of MOG-specific antibodies is not sufficient for disease development in CD19-B MHCII mice [15] and the observation by the Zamvil group that mice in which MOG-specific antibody is tethered to B cells (and therefore incapable of being secreted) are susceptible to EAE [33]. While we did not collect serum from our cohort of 2D2xIgH MOG mice, either with or without MHCII expression restricted to B cells, we have detected MOG-specific IgG in CD19-B MHCII xIgH MOG mice, suggesting that antibody production in 2D2xCD19-B MHCII xIgH MOG mice would not be absent or deficient. The lack of spontaneous EAE or optic neuritis in 2D2xCD19-B MHCII xIgH MOG mice is in agreement with the previously reported limitations for B cells to prime CD4 T cells [22,34]. On the other hand, B cells can indeed prime naïve T cells as long as valid B cell antigens are delivered to them [35]. Hence, in the EAE model, limitations in B cell APC function may be rooted in restricted access to relevant auto-antigens sequestered in the CNS. Hence, the responsibility for CD4 T cell activation and initiation of demyelination in spontaneous EAE most likely falls on DCs, which have been found to be capable of independently initiating spontaneous optic neuritis [23]. Whether the physical location of DCs in and around the CNS [36,37] or the intrinsic ability Rapid B cell-mediated EAE for DCs to prime CD4 T cells [38] bestows this capacity for disease initiation is not apparent. Clearly, these features and others are not mutually exclusive.
Given the limitations for B cell antigen presentation in initiating disease, along with the restrictions in eliciting full recall production of cytokines by MOG-specific CD4 T cells in secondary responses [22], we sought to assess the limitations of B cells in supporting cognate CD4 T cell-mediated CNS autoimmunity after priming during EAE. Using a Tam-inducible MHCII expression system, we now show that antigen presentation by B cells is sufficient to induce EAE signs just as rapidly as EAE induced by a full complement of MHCII+ APCs. This result is contrary to our expectations given the reproducible delay in disease onset when B cells function as the sole APC during EAE [15]. Our findings raise questions regarding what drives accelerated disease onset in CD20-B MHCII xIgH MOG mice. One critical component of neuroinflammation worthy of consideration is lymphocyte trafficking. For example, the reduced variability in B cell frequency detected in the CNS of CD20-B MHCII xIgH MOG mice with EAE induced by Tam administration after T cell transfer (Fig 5B-5D) could be explained by chronological changes in the concentration of chemotactic factors driving lymphocytes to the CNS. This theory could also explain the increasingly rapid onset of EAE symptoms observed for CD20-B MHCII xIgH MOG mice treated with Tam after CD4 T cell transfer, as immune cells may traffic to, or organize within, the CNS more quickly if chemokines promoting B cell accumulation were up-regulated.
Our data support a model in which non-B cell APCs prime CD4 T cells toward myelin antigens, enabling subsequent cognate interactions with B cell APCs to support auto-reactive B cell proliferation and differentiation. Clinical studies have demonstrated that memory B cells and plasmablasts are the most common B cell subtype in the CSF of patients with MS [39]. This expansion of antigen-specific B cells during CNS autoimmunity could amplify cognate interactions between dysregulated B and CD4 T cells, which in turn could independently drive neuro-inflammation and relapses at later stages of MS. The high frequency of antigen-specific B cells in our EAE system may imitate the prevalence or expansion of B cells in MS patients [40][41][42]. During an MS remission, previously primed encephalitogenic T cells rest in the periphery (reflected in our EAE model as the period of time prior to Tam treatment). Of note, peripheral B cells from relapsing-remitting MS patients exhibit exaggerated pro-inflammatory cytokine responses that can directly promote T cell activation [43][44][45]. Increased MHCII expression promotes lymphocyte trafficking to the myelin-containing CNS compartment to initiate plaque formation (mimicked by what we observed soon after Tam treatment). It is possible that BCDTs eliminate pathogenic B cell antigen presentation, reducing germinal center  reactions that may be occurring in the periphery and CNS. Studying the interdependency and amplification of pro-inflammatory interactions between dysregulated lymphocytes could lead to more targeted therapeutic interventions that specifically ablate the pathogenic effects of B cells in situ.
The reproducibly rapid onset of EAE that we have demonstrated shows that the time required for B cell trafficking from the periphery to the CNS is not a major contribution to the observed delay in disease onset for CD19-B MHCII xIgr mice. However, our findings raise questions about where antigen presentation needs to occur in the context of B cell-driven CNS autoimmunity. While germinal center reactions mainly occur in the spleen and lymph nodes, various reports describe formation of functional ectopic lymphoid follicles in the CNS of MS patients and in mice with EAE [46][47][48]. Exclusion of B cells from the CNS results in reduced severity of EAE induced by rhMOG yet exacerbates EAE induced by MOG  peptide, indicating that access to the CNS is required for EAE mediated by B cell antigen presentation yet can also be important for regulatory B cells to ameliorate disease [49][50][51]. In contrast, Tesfagiorgis et al. found that antigen-nonspecific B cells are recruited to the CNS compartment while myelin-specific B cells remain in the peripheral draining lymph nodes in a model of active EAE that requires B cells [52]. In our model, the relative infrequency of B cells observed in the CNS of mice that develop EAE may reflect the ease with which MOG-specific B cells capture their soluble protein antigens to present to T cells within the CNS compartment. The formation of ectopic lymphoid follicles in the CNS could create an environment in which very few B cells have ready access to antigen and ample interactions with many cognate encephalitogenic CD4 T cells.
Overall, the results described herein reveal that myelin-specific B cells are not able to support spontaneous EAE, although they have the potential to rapidly induce EAE with similar kinetics as WT APCs. Our new inducible MHCII expression EAE system enables precise control of the timing of cognate interactions between B cells and CD4 T cells, revealing that B cells do not harbor an intrinsic deficit in the promotion of auto-reactive CD4 T cell responses targeting the CNS.