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Increased remissions in multiple sclerosis (MS) during pregnancy suggest that elevated levels of sex steroids exert immunoregulatory activity. Estrogen (E2=17β-estradiol) protects against experimental autoimmune encephalomyelitis (EAE), but the cellular basis for E2-induced protection remains unclear. Studies demonstrate that depletion of B cells prior to induction of EAE exacerbates disease severity, implicating regulatory B cells. We thus evaluated pathogenic and E2-induced protective mechanisms in B cell deficient (µMT−/−) mice. EAE-protective effects of E2 were abrogated in µMT−/−) mice, with no reduction in disease severity, cellular infiltration or pro-inflammatory factors in the CNS compared to untreated controls. E2 treatment of WT mice selectively up-regulated expression of PD-L1 on B cells and increased the percentage of IL-10-producing CD1dhighCD5+ regulatory B cells. Upregulation of PD-L1 was critical for E2-mediated protection since E2 did not inhibit EAE in PD-L1−/− mice. Direct treatment of B cells with E2 significantly reduced proliferation of MOG35–55-specific T cells that required ERα. These results demonstrate for the first time a requirement for B cells in E2-mediated protection against EAE involving direct E2 effects on regulatory B cells mediated through ERα and the PD-1/PD-L1 negative co-stimulatory pathway. E2-primed B cells may represent an important regulatory mechanism in MS and have strong implications for women receiving current MS therapies that cause B-cell depletion.
Subjects with MS, a debilitating neurological and inflammatory autoimmune disease, often show clinical improvement during pregnancy, followed by temporary post-partum exacerbations [1,2]. Levels of sex steroids mirror these improvements during pregnancy, suggesting an immunoregulatory role. Treatment with pregnancy level estriol (E3) reduces central nervous system (CNS) lesions . EAE, an animal model of MS, disease is associated with production of pro-inflammatory cytokines, IFN-γ and IL-17 [4,5] and recovery with anti-inflammatory cytokines, IL-4 and IL-10 [6,7]. As in humans with MS, EAE severity in guinea pigs, rats and rabbits improves during pregnancy . We have previously demonstrated that relatively low doses of 17β-estradiol (E2) and E3 confer potent protection against clinical and histological signs of EAE [8,9]. The immunosuppressive effects of E2 are mediated via specific receptors and depend on the regulated expression and cellular distribution of these receptors , particularly estrogen receptor-α (ERα) that mediates the major therapeutic effects of E2 . The expression of ERs on various immune cells, including T and B lymphocytes and myeloid antigen presenting cells (APCs), suggests that therapeutic effects of E2 may involve direct receptor binding. However, EAE-protective effects of E2 were not diminished in ERα-depleted encephalitogenic T cells, obviating direct E2 effects on these cells . E2 treatment increased percentage and activity of CD4+Foxp3+ regulatory T cells , suggesting their possible involvement in E2-mediated protection in EAE.
Recent studies suggest that B cells are important for EAE pathogenesis [14–16]. Myelin oligodendrocyte glycoprotein (MOG)-specific antibodies can enhance demyelination and inflammation in EAE, although they are not essential for EAE induction and progression . While studies in double transgenic mice with MOG-specific T and B cell receptors show that B cells can function as APCs during EAE initiation , studies using B cell-deficient mice demonstrate that mice develop a severe non-remitting form of EAE [15,16,19]. CD20 antibody–mediated B cell depletion before EAE induction results in exacerbation of disease symptoms and increased encephalitogenic T cell influx into the CNS . These studies led to the identification of IL-10 producing CD1dhighCD5+ regulatory B cells [6,7], which when depleted, resulted in increased disease severity. The regulatory B cells were maximally protective during early EAE initiation, with no obvious role during disease progression . The role of regulatory B cells in E2-mediated protection against EAE has not been evaluated, and is the focus of the current study.
Our results demonstrate that in the absence of B cells, protective effects of E2 against EAE are completely lost, resulting in severe cellular infiltration and higher levels of proinflammatory cytokines/chemokines in the CNS. Treatment with E2 prior to EAE induction selectively up-regulated levels of PD-L1+ and CD1dhighCD5+ B cells populations. The up-regulation of PD-L1 was critical for E2-mediated protection since the absence of PD-L1 resulted in loss of protection against EAE. Therefore we conclude that B cells are indispensible mediators of immunomodulatory effects of E2, with the likely involvement of regulatory B cells.. These results have significant implications in the context of MS pathogenesis.
To test our hypothesis that presence of B cells is obligatory for E2-mediated protection against EAE, female µMT−/− mice were used. Age-matched WT and µMT−/− mice were sham-treated (Control) or implanted with E2 pellets, 8 days prior to immunization with mMOG35–55 peptide with CFA/Ptx. The EAE disease course was followed for 26 days after immunization. As expected, the control WT and the µMT−/−mice demonstrated an early onset of EAE, at days 12 and 11 respectively, and exhibited a steady increase in the disease scores by day 15 post-immunization (Fig. 1A). Although E2-implanted µMT−/−mice demonstrated a significant delay in the onset of EAE (Day 15 post-immunization), they completely lost E2-mediated protection by day 18, with their disease scores being comparable with their respective sham-treated counterparts by day 19. As indicated in Table 1, WT controls and control and E2-implanted µMT−/− mice demonstrated a high incidence of EAE in each of 3 independent experiments, with WT E2-implanted mice showing complete protection against EAE. Due to late onset of disease in E2-implanted µMT−/− mice, their Cumulative Disease Index (CDI) was lower that of Control WT and µMT−/− mice; however their peak disease scores were comparable with those of the controls (Table 1). Spinal cords sections were assessed for extent of inflammation and demyelination in CNS of control and E2-implanted WT and µMT−/−mice. As established earlier , the spinal cords of WT control mice demonstrated leukocyte infiltration along with demyelination (Fig. 1B). Spinal cord sections from E2-implanted µMT−/− mice also demonstrated massive leukocyte infiltration with several foci of inflammation (indicated by arrows, Fig. 1B) along with severe demyelination similar to control µMT−/− mice. However, spinal cord sections from the WT E2-treated mice showed no obvious signs of inflammation and demyelination and appeared healthy. Hence, the requirement of B cells in E2-mediated protection vs. EAE is indicated. To test whether B cells are sufficient to restore E2-mediated protection, we adoptively transferred B cells from naïve WT mice into µMT−/− mice. The recipients were either sham-treated or E2-implanted a day after the transfers and EAE was induced 7 days later. Preliminary data (Fig. 1C) indicates that µMT−/− that received B cells and E2-implants were significantly protected from EAE as compared to the sham-treated recipients. Taken together, these results clearly demonstrate that B cells are indispensible for E2-mediated protection against EAE.
Since, E2-implanted µMT−/− mice demonstrated a severe clinical disease; we evaluated the immune cell trafficking into the CNS of these mice. Leukocytes isolated from brains of sham-treated (control) and E2-implanted WT and µMT−/− mice were stained for several cell surface markers. Brains of control and E2-implanted µMT−/− mice not only demonstrated higher numbers of total leukocytes as compared to E2-implanted WT mice (Fig. 2A), but also had a significant increase in percentages of CD4+ and CD8+ T cells as compared to brains of WT E2-implanted mice (Fig. 2B). Brains of E2-implanted µMT−/− mice demonstrated significant increase in percentages of infiltrating macrophages (CD11b+CD45high) and dendritic cells (CD11c+CD45+), which participate in disease pathogenesis through effects on blood brain barrier permeability, antigen presentation and immune regulation (Fig. 2B). WT E2-implanted mice demonstrated significantly less infiltration of T cells, macrophages and dendritic cells (Fig. 2B).
Chemokines and their receptors play a critical role in trafficking of leukocytes under inflammatory conditions. Chemokines like CCL2 (MCP-1) and CCL5 (RANTES) influence T cell and macrophage migration . CXCL13 is a chemoattractant for B cells and activated T cells with monocytes/macrophages being its cellular source in CNS during EAE disease . CXCR5 is the receptor for CXCL13, expressed by most B cells, subsets of CD4+ T cells and some DCs . To evaluate the role of chemokines and their receptors and also inflammatory cytokines IL-1β and IL17, responsible for the trafficking of inflammatory immune cells into CNS real time PCR was performed on RNA isolated from spinal cords. As indicated in Fig. 2C, spinal cords of both sham-treated and E2-implanted µMT−/−mice demonstrated a significant increase in mRNA expression of CCL2, CCL5, CXCL13, IL-1 β and IL17. Expression of CXCR5 by µMT−/−mice, despite the absence of B cells is indicative of presence of CD4+ T and dendritic cells in spinal cords. Thus, E2-implanted µMT−/− mice exhibit strong inflammatory immune responses in their CNS as compared to E2-implanted WT mice, which correlate with their high clinical disease scores.
After induction in periphery (spleens and draining lymph nodes), pathogenic T cells migrate to the CNS to initiate inflammatory process responsible for clinical signs of EAE . To evaluate MOG-specific responses generated in spleens, mononuclear cells from sham-treated and E2-implanted WT and µMT−/−mice were cultured and supernatants were collected for cytokine expression. As expected sham-treated WT mice produced higher levels of IL-17, TNF-α and MCP-1 (Fig. 3A). But splenocytes of both the sham-treated and E2-implanted µMT−/− mice produced either equivalent (IL-17) or higher (TNF-α & MCP-1) levels of proinflammatory cytokines as compared to WT sham-treated mice. Levels of IL-17 and MCP-1 produced by splenocytes of WT E2-implanted mice were significantly lower than other 3 groups of mice (Fig. 3A). Although IFN-γ and IL-6 levels in splenocytes were higher in sick sham-treated WT and sham and E2-treated µMT−/− mice, these levels were not significantly different from those in the E2-treated WT mice (data not shown). In addition to higher pro-inflammatory environment, T cells from splenocytes of µMT−/− mice also demonstrated higher proliferative responses (Fig 3B). Hence, absence of B cells abrogates immunosuppressive and protective effects of 17β-estradiol by demonstrating not only higher demyelination and infiltration of immune cells in the CNS but also a high pro-inflammatory environment in the periphery (spleens).
E2, a pleiotropic regulator, may directly or indirectly affect signaling pathways in different cells and tissues . Since, B lymphocytes express intracellular estrogen receptors (ER-alpha and beta) ; it was of relevance to determine whether immunomodulatory effects of E2 are mediated via its direct or indirect action on B cells. E2 effects on the ability of cultured B cells to present antigen to MOG-specific T cells were tested. Purified splenic B cells from naïve WT and ERα−/− mice, pretreated with or without pregnancy levels of E2  (2500 pg/ml), were co-cultured with purified CD4+ T cells from naïve 2D2Tg mice. As shown in Fig. 4, E2-treated B cells only from WT mice suppressed the proliferative responses of T cells while B cells from ERα−/− mice failed to do so, thus, indicating that E2 can act directly on B cells to modulate their function.
Since, E2 can act directly on B cells, as demonstrated above; we evaluated B cell phenotype in the protected WT E2-implanted mice. As demonstrated in our concurrent study (Offner, manuscript submitted), PD-L1 was significantly up-regulated on B cells in E2-implanted Foxp3-DTR mice, in which development of lethal EAE was attenuated. PD-1/PD-L interactions, comprising an important negative co-stimulatory pathway, could thus constitute an alternate pathway through which B cells can suppress T cell responses. We monitored the expression of PD-L1 on B cells of splenocytes from sham-treated and E2-implanted WT mice, over a time course from Day 0 to Day 20 post-immunization. As demonstrated in Fig. 5A. & B percentages of PD-L1+ B cells (Supporting Information Fig. 6A) in spleens of E2-treated WT mice increased significantly post-immunization as compared to their sham-treated counterparts. PD-L1 expression in spleens of µMT−/− mice (either sham or E2-treated) was significantly lower as compared to WT mice (data not shown); indicating that PD-L1 expression in conjunction with B cells could play a critical role in E2-mediated protection. To test this hypothesis, we evaluated EAE severity in sham-treated and E2-implanted PD-L1−/− mice. As shown in Fig. 5C & Table 2, E2-treatment did not protect PD-L1−/− mice that developed EAE disease scores equivalent to those of sham-treated WT and PD-L1−/− mice. Thus, PD-L1 is a critical co-stimulatory molecule in E2-mediated protection against EAE.
In a recent study, CD1dhighCD5+ B cells were characterized as an immunosuppressive high IL-10-producing regulatory subpopulation . To elucidate whether these cells contributed to E2-mediated protection, CD1dhighCD5+ B cells were enumerated in spleens of either sham-treated or E2-implanted WT mice, over a time course from Day 0 to Day 20 post-immunization. Although no differences were observed on Day 0 (7 days after implantation of E2 pellets) spleens of E2-implanted WT mice demonstrated a significant increase in the percentage of this population both on Day 8 and Day 20 post-immunization (Fig. 6A & B). Moreover, day 20 splenocytes from WT E2-implanted mice demonstrated a remarkable increase in production of IL-10, in contrast to background levels of IL-10 produced by splenocytes from control or E2-treated µMT−/− mice (Fig. 6C). IL-10 is proposed to be an immunomodulator of EAE [6,7]. Hence the protective nature of E2-treated B cells, as demonstrated in E2-implanted WT mice, can be further attributed, at least in part, to this IL-10-producing CD1dhighCD5+ B cell sub-population.
Multiple sclerosis displays a distinct gender bias towards increased susceptibility and disease severity in females with relapsing-remitting disease. However, women with MS experience remarkable recoveries during pregnancy, indicating a crucial protective role of sex hormones with both estrogen and estriol treatments suggested to have therapeutic potential in treatment of MS. Since MS is often considered to be a T cell-dependent autoimmune disease, the initial focus of immunomodulatory role of estrogen was on T cells. However, our prior studies demonstrated that the protective effects of E2-treatment are not mediated through direct action on the encephalitogenic T cells, and that both immune and CNS cells are required in propagating immunomodulatory effects of E2 . Exact molecular mechanisms in mediating the regulatory effects by E2 in EAE have not yet been identified, thus necessitating an ongoing search to determine the crucial immunomodulatory players. Tregs are prime immunoregulatory candidates since their numbers increased by E2 in EAE-protected mice [13,25,26]. Surprisingly, our contemporary study demonstrated that E2 can fully protect mice against clinical and histological signs of EAE in the absence of Treg cells (Offner, manuscript submitted), suggesting an alternative pathway for E2-mediated protection against EAE. Since the E2-implanted Foxp3-depleted mice demonstrated an increase in PD-L1, CD80 and CD86 expression by B cells, we further investigated the role of B cells in E2-mediated protection against EAE.
Our studies demonstrate for the first time that protective effects of E2 vs. EAE require B cells, and further implicate immunosuppressive IL-10-producing CD1dhighCD5+ B cell subpopulation as a likely contributor. The role of B cells in MS and EAE include both pathogenic and regulatory mechanisms. Multiple studies have demonstrated that B cells are crucial for EAE pathogenesis [14,15]. However, studies have also consistently demonstrated that depletion of CD20+ B cells before EAE induction leads to increased levels of CNS-infiltrating encephalitogenic T cells and enhanced disease severity .
In a similar vein, E2 treatment of the µMT−/− mice delayed but could not inhibit the trafficking of immune cells into the CNS, thus permitting severe inflammatory reactions, demyelination and severe EAE. Besides an increase of the pro-inflammatory chemokines like MCP-1 and RANTES in the CNS, µMT−/− mice demonstrated a significant increase specifically in CXCL13. CXCL13 gains importance in MS since its expression in the intrameningeal follicles rich in B cells  and also within inflammatory cells bearing a macrophage-like morphology from autopsied MS lesions is detected . However, since our study demonstrates higher levels of CXCL13 in absence of B cells, it could be due to subsets of CD4+ T cells which are able to infiltrate the CNS.
B cells contribute to immune responses by functioning as antigen-presenting cells and by secreting cytokines . Thus, in addition to their possible pathogenic role in producing demyelinating antibodies, presenting myelin autoantigens to T cells  or secreting effector cytokines, B cells from MS patients may also secrete anti-inflammatory cytokines ,implicating a potential for modulating disease progression. Particular stages of B cell differentiation are sensitive to effects of E2 [31,32], with ERα being implicated in the development of mature B-lymphocytes . In the present study E2 could modulate T cell proliferative responses by acting on B cells in an ERα-specific manner, consistent with our previous studies that failed to show a direct effect of E2 on T cells.
Our recent studies have demonstrated a critical role for PD-1 in E2-mediated protection of EAE [24–26, 34]. Also, the crucial role of PD-L1 rather than PD-L2 in inhibition of EAE has been demonstrated . Here, we demonstrate a significant increase in the expression of PD-L1 on B cells in E2-treated WT mice after induction of EAE, although no differences were observed after treatment with E2 prior to immunization. Moreover, our study for the first time demonstrates a critical role of PD-L1 in E2-mediated protection, with E2-implanted PD-L1−/− mice showing no protection from clinical and histological EAE. Thus, B cells mediate immunomodulatory effects of E2 on encephalitogenic T cells through ERα and the negative co-stimulatory PD1/PD-L1 pathway.
E2 can best mediate its protective effects when administered prior to EAE initiation rather than during the induction phase post-immunization . Anti-CD20-depletion of B cells prior to EAE induction leads to exacerbation of the disease with increased encephalitogenic T cell infiltration into the CNS while B cell depletion during disease progression suppressed EAE symptoms , thus demonstrating reciprocal roles of B cells in different phases of EAE. Increase in EAE severity upon B cell depletion before disease initiation resulted from depletion of a rare IL-10-producing CD1dhighCD5+ subpopulation of B cells characterized as B regulatory (Breg) cells .
In our study, percentages of this CD1dhighCD5+ Breg cell subpopulation increased significantly in WT E2-implanted mice. Studies in a contact hypersensitivity model showed that transfer of IL-10-producing B cells into B-deficient or B-depleted mice inhibited CD4+ T cell-dependent inflammatory responses . Moreover, suppressive function of CD1dhighCD5+ B cells increased post-immunization  and was dependent on their ability to produce IL-10 , thus indicating that this antigen-induced IL-10-producing Breg cell subset can downregulate T cell activation. Recently, it has been proposed that Breg cells (i.e. IL-10-producing B cells) have a protective role during EAE initiation (congruent with the data in this manuscript), whereas Treg cells have a therapeutic role in the later stages after onset of clinical disease . Further studies would be needed to characterize IL-10 production by the CD1dhighCD5+ B cells. However, an abrogation of IL-10 production after E2 treatment in µMT−/− mice strongly implicates B cells as the source of IL-10 in the WT E2-treatedmice.
In summary, B cells play an indispensible role in E2-mediated protection against EAE. Absence of B cells abrogates the protective effects of E2 and leads to a significantly increased infiltration of inflammatory cells into the CNS of mice with EAE. E2 mediates its immunomodulatory effects on B cells via ERα and PD-L1 and B cells primed with E2 are able to significantly suppress T cell proliferative responses. Finally, this is the first study to implicate CD1dhighCD5+ B cells (IL-10-producing Breg-cells) as possible key contributors induced by E2 treatment. These results suggest an important protective role for E2-primed B-regulatory cells in MS and have strong implications for patients receiving current MS therapies such as Rituximab™ that cause B cell depletion [37,38]. Our study implicates that caution be used during such B cell-depleting therapies since the non-selective elimination of B cells may interrupt the beneficial E2 interactions that could promote anti-inflammatory influence on other immune cells leading to remissions and would preclude potential therapeutic application of E2 given alone or in combination with other regulatory agents .
Wild-type (WT) female C57BL/6 mice were purchased from Harlan Laboratories (Livermore, CA). µMT−/− mice containing no B cells because of targeted disruption of the membrane exon of the immunoglobulin mu chain gene and 2D2 transgenic mice originally obtained from Jackson Laboratories (Bar Harbor, ME), were bred at the Animal Resource Facility at the Portland Veteran Affairs Medical Center. ERα−/− mice also bred in-house, were originally obtained from the Korach laboratory. All mice (on a C57BL/6 background) were used at 7–8 weeks of age and were housed in the Animal Resource Facility at the Portland Veterans Affairs Medical Center in accordance with institutional guidelines. The study was conducted in accordance with National Institutes of Health guidelines for the use of experimental animals, and the protocols were approved by the Institutional Animal Care and Use Committee.
Female WT and µMT−/− mice were implanted with 2.5mg/60-day release 17β-estradiol pellets (Innovative Research of America, Sarasota, FL) or sham-treated (control) one week prior to immunization with 200µg MOG35–55 peptide (PolyPeptide Laboratories, San Diego, CA) in 400µg Complete Freund’s adjuvant (CFA, H37Ra, Difco). Mice received pertussis toxin (Ptx, List Biologicals, Campbell, CA) on the day of immunization (75ng) and 2 days later (200ng). All mice were monitored daily for clinical signs of disease and scored using the following scale: 0 = normal; 1 = limp tail or mild hind limb weakness; 2 = moderate hind limb weakness or mild ataxia; 3 = moderately severe hind limb weakness; 4 = severe hind limb weakness or mild forelimb weakness or moderate ataxia; 5 = paraplegia with no more than moderate forelimb weakness; and 6 = paraplegia with severe forelimb weakness or severe ataxia or moribund condition.
Intact spinal columns removed from mice at the end of the study (i.e. Day 20 post-immunization) were fixed in 10% formalin. Dissectedspinal cords were embedded in paraffin before sectioning. Sections were stained with Hematoxylin and Eosin to assess inflammatory lesions. Transverse sections were stained with a modified eriochrome cyanine (EC) protocol to assess the sparing of the white and gray matter (demyelination), . Slides were analyzed by light microscopy.
10×106 purified splenic B cells from naïve WT mice were transferred into µMT−/− mice, a day before half of the recipients were implanted with E2 pellets. EAE was induced 7 days after the E2 implants and recipient mice were monitored over the next 20 days for clinical disease.
Spleens and brains from control and E2-treated WT and µMT−/− mice were processed for lymphocyte isolation. Cells were stained with a combination of the following antibodies obtained from BD Bioscience: CD4 (L3T4), CD19 (1D3), CD1d (1B1), CD5 (53-7.3), PD-L1 (MIH5), CD11b (M1/70), CD11c (HL-3), CD45 (30-F11).. The intracellular staining of Foxp3 (MF23) and PD-1 (J43) was completed following overnight incubation in fixation/permeabilization buffer (eBiosciences). Dead cells were gated out using propidium iodide discrimination. Cells were gated on CD19 to determine expression of the CD1dhighCD5+ and PD-L1 populations. Data were collected with CellQuest (BD Biosciences) and FCS Express (De Novo) software on a FACSCalibur (BD Biosciences). Absolute numbers were calculated from live-gated cells.
Single-cell suspensions of spleen cells from WT and µMT−/− mice were cultured in the presence of 25µg/ml MOG35–55 peptide for 48h. Culture supernatants were assessed for cytokine levels using a Luminex Bio-Plex cytokine assay kit (Bio-Rad, Richmond, CA).
Total RNA was isolated from spinal cords of control and E2-treated WT and µMT−/− mice using the RNeasy mini kit protocol (Qiagen, Valencia, CA, USA) and converted into cDNA using oligo-dT, random hexamers, and Superscript RT II (Invitrogen, Grand Island, NY, USA). Reverse transcription-PCR was performed using TaqMan PCR master mix and pre-designed Taqman primers for CCL2, CCL5, CXCL13, CXCR5, IL-1β and IL-17 primers (Applied Biosystems, Foster City, CA, USA). mRNA was quantified in reactions conducted on the ABI Prism 7000 Sequence Detection System (Applied Biosystems) and data represented as relative units compared with the GAPDH reference gene.
Splenocytes, collected from control and E2-treated WT and µMT−/− mice on Day 20 post-immunization, were plated onto 96-well flat bottom plates (400,000 cells per well). Cells were stimulated with 10 or 50µg/ml of MOG35–55 peptide for 72h, and the last 18h in the presence of 3H-Thymidine. Cells harvested onto filter mats were read on Perkin Elmer micro-beta scintillation counter. Stimulation indices were determined by calculating the ratio of antigen specific cpm to medium alone cpm.
Splenic CD19+ B cells and CD4+ T cells were purified using paramagnetic bead-conjugated Abs and AutoMACS (Miltenyi Biotec). CD19+ B cells, isolated from naïve WT and ERα−/− mice (positive fraction) by a CD19 cell isolation kit (Miltenyi Biotec), were pretreated with 2500 pg/ml or no (control) E2 (water soluble 17β-estradiol [Sigma]) for 48h. CD4+ T cells from naïve 2D2Tg mMOG35–55-specific TCR Tg mice, were isolated by a CD4 T cell isolation kit (Miltenyi Biotec) (negative fraction) by depleting non-T cells (positive fraction). Cell purity (>95%) was confirmed using flow cytometry. MOG-specific CD4+ T cells (5 × 104/well) were co-cultured with control or E2-preconditioned B cells, in presence or absence of 25µg/ml mMOG35–55, at 1:2.5 ratio (T:B cells), in triplicate in 96-well culture plates. After 48h, the plates were pulsed for 18h with 3H-thymidine and cells were assessed for uptake of labeled thymidine by liquid scintillation. The data represent the mean ± S.D. of triplicate wells.
Data are reported using GraphPad Prism (v 4.0, San Diego, CA) and expressed as the mean ± SEM. Statistical significance between WT and µMT−/− control and E2-implanted mice was calculated using One way ANOVA with multiple comparison post test (Bonferroni). Statistical differences between disease scores of µMT−/− control and E2-implanted mice in the adoptive transfer experiment were determined by the Mann-Whitney U test. Student t test was used to compare WT control and WT E2-implanted groups (* p ≤ 0.05; ** p≤ 0.01).
The authors wish to thank Dr. Sushmita Sinha and Ms. Sandhya Subramanian for helpful discussions and Ms. Eva Niehaus for assistance with manuscript preparation.
This work was supported by NIH grants NS45445 and NS49210; National Multiple Sclerosis Society grant RG3405-C-6; and the Biomedical Laboratory R&D Service, Department of Veterans’ Affairs.
Conflict of interest: The authors declare no financial or commercial conflict of interest.