CD4+CD25+ T regulatory (Treg) cells expressing the Foxp3 transcription factor have been shown to be present in the CNS during the autoimmune disease experimental autoimmune encephalomyelitis (EAE) and can inhibit EAE clinical disease by an IL-10-dependent mechanism. In addition, IL-10 expression in the CNS late in the EAE disease course has been attributed to recovery. However, it is not known how Treg cells and IL-10 expressions are regulated during EAE. We have previously shown a requirement for B cells in recovery from EAE and here investigated whether this was due to a deficiency in Treg cells and IL-10 in the CNS. We found that B cell deficiency resulted in a delay in the emergence of Foxp3-expressing Treg cells and IL-10 in the CNS during EAE, but not in the periphery. Reconstitution with wild-type B cells resulted in disease recovery and normalized IL-10 and Foxp3 expression. However, reconstitution with B7-deficient B cells did not. Furthermore, we show that IL-10 and Foxp3 expression is enhanced in CNS nonencephalitogenic T cells. These data suggest a novel mechanism whereby B cells regulate CD4+CD25+ Treg cells via B7 and subsequently enter the CNS and suppress autoimmune inflammation, mediating recovery.

Multiple sclerosis (MS)3 is an inflammatory disease that affects the CNS and is considered to be a CD4+ Th1-mediated autoimmune disease modeled in the mouse as experimental autoimmune encephalomyelitis (EAE) (1). Myelin basic protein (MBP) is a myelin Ag thought to induce autoimmune responses in both MS disease and EAE (1). In EAE, immunization of B10.PL mice (H-2u) with the NH2-terminal acetylated peptide of MBP (Ac1–11) results in an acute clinical disease course in which the mice spontaneously recover and do not relapse (2). However, if they are deficient in B cells, we have previously shown that B10.PL mice are unable to resolve clinical disease symptoms and exhibit a chronic disease course (2). A similar result was reported using myelin oligodendrocyte glycoprotein-induced EAE in the C57BL/6 (H-2b) mouse (3). In this same study, it was shown that B cell production of IL-10 and expression of CD40 was required for recovery (3). These data suggest that B cells interact with CD40L-expressing T cells to provide the CD40 costimulatory signal. In addition, B cells can provide CD28 costimulation to T cells via the B7 molecules B7.1 (CD80) and/or B7.2 (CD86). This is supported by a study in which CD40L-deficient mice were shown to be resistant to EAE, a mechanism suggested to be due to a lack of up-regulation of the B7 molecules (4). Indeed, mice deficient in both B7 molecules were shown to have attenuated EAE induced by adoptive transfer of encephalitogenic T cells (5). However, these mice also have a B7 deficiency in the CNS, which was shown to lead to decreased survival of T cells in the target organ (6). Little is known about the role of the various B7 expressing cell populations in EAE, which include B cells.

In addition to B cell production of IL-10 (3), a role for IL-10 production in the CNS has been suggested to be important for recovery from EAE. IL-10-deficeint mice exhibit a severe EAE disease course (7) and IL-10 expression in the CNS is highest at the peak of disease and during the recovery phase (8, 9). Additional evidence supporting a regulatory role for IL-10 in EAE comes from studies in which IL-10 was expressed in the CNS, resulting in the inhibition of EAE (10). In the CNS, cellular sources of IL-10 include microglial cells, astrocytes, and T cells (9). Within the T cell population, CD4+CD25+ T regulatory (Treg) cells have been shown to produce IL-10 in the CNS (11) and suppress EAE (11, 12), with one report demonstrating a dependence upon IL-10 (13). The development and function of the CD4+CD25+ Treg population requires the expression of the Foxp3 transcription factor (14, 15), making Foxp3 expression the best marker of this Treg population. However, it is unknown how Treg cells are regulated during EAE.

In this study, we investigated whether the chronic EAE clinical disease course that occurs in B cell deficient-B10.PL (μMT) mice was due to a deficiency in Treg cells in the CNS. We found that, in the absence of B cells, the up-regulation of IL-10 expression and the emergence of Treg cells detected by Foxp3 expression in the CNS during EAE were delayed and did not reach maximal levels until the wild-type (WT) mice exhibited clear signs of clinical recovery. Both IL-10 and Foxp3 expression in WT mice reached maximal levels in the CNS at the peak of disease just before spontaneous recovery. We next investigated the mechanism whereby B cells regulated EAE and found that their expression of B7 was essential for clinical recovery as well as the up-regulation of IL-10 and Foxp3 in the CNS. Furthermore, we found that both IL-10 and Foxp3 expression was enhanced in the nonencephalitogenic CNS T cell population. These data are supportive of a model whereby B cells regulate EAE clinical recovery by interacting with CD4+CD25+ Treg cells through a B7-dependent mechanism that causes the cells to mobilize and migrate into the CNS, resulting in disease resolution via their production of IL-10. Because a short delay in the appearance of Treg cells in the CNS in the B cell-deficient mice was sufficient to prevent recovery, Treg cell therapy in MS is an attractive possibility for the treatment of newly diagnosed patients or shortly after relapse just before any permanent neurological damage.

B10.PL (H-2u) WT mice were purchased from The Jackson Laboratory or generated locally. B cell-deficient B10.PL mice (μMT) and MBP-TCR transgenic mice expressing a TCR transgene specific for the acetylated NH2-terminal peptide of MBP (Ac1–11) bound to I-Au have been described (2, 16), respectively. The μMT mice were backcrossed to B10.PL for more than eight generations (2). B6.129S4-Cd80tm1ShrCD86tm1Shr/J mice deficient in both B7.1 (CD80) and B7.2 (CD86) (B7−/−) (17) were purchased from The Jackson Laboratory on the C57BL/6 background and backcrossed to B10.PL for three generations and then intercrossed to generate H-2u knockout mice. Animals were housed at the Biomedical Research Center of the Medical College of Wisconsin, Milwaukee, WI. All animal protocols were approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee.

The MBP Ac1–11 peptide (Ac-ASQKRPSQRSK) was generated by the peptide core of the Blood Research Institute (BloodCenter of Wisconsin, Milwaukee, WI). The 2.4G2 hybridoma was purchased from American Tissue Culture Collection. Abs to the mouse proteins Vβ8.2-biotin, IFN-γ-PE, and BrdU-FITC, as well as rat IgG2b and streptavidin (SA)-PE-Cy5 were purchased from BD Pharmingen. SA-allophycocyanin-Cy7 was purchased from BioLegend. Abs to the mouse proteins CD4-FITC, CD4-PE, CD11b-PE, CD11b-FITC, TCRβ-FITC, CD25-PE-CY5, and Foxp3-PE, as well as rat IgG1-PE and rat IgG2a-PE, were purchased from eBioscience.

EAE was induced in WT and μMT mice by the adoptive transfer of MBP-specific encephalitogenic T cells generated as previously described (16). Briefly, 0.5–1 × 106 activated MBP-TCR T cells were i.v. injected into sublethally irradiated (344–360 rad) mice that were at least 6 wk old. Individual animals were assessed daily for symptoms of EAE and scored using a scale from 1 to 5 as follows: 0, no disease; 1, limp tail and/or hind limb ataxia; 2, hind limb paresis; 3, hind limb paralysis; 4, hind and fore limb paralysis; and 5, death.

Mixed BM chimeras were generated by transplanting various donor BM into sublethally irradiated (360 rad) recipient mice (donor→recipient) such that the level of chimerism was ∼50%. The level of chimerism was determined by transplanting with GFP donor BM and assessing the level of GFP expression in the recipient mice. Chimeras were generated by transplanting WT mice with WT BM (WT→WT) or transplanting μMT mice with BM from μMT (μMT→μMT), WT (WT→μMT), or B7−/− (B7−/−→ μMT) mice. After at least a 6-wk reconstitution, EAE was induced in the chimeras by the adoptive transfer of 0.5 × 106 encephalitogenic MBP-TCR T cells. In some experiments, B cell reconstitution was determined by measuring the percentage of splenocytes expressing IgG/M, which was 40–60% at the end of the experiment. We did not observe a significant difference in B cell reconstitution in the various groups of mice. There were no B cells detectable in the spleen via IgG/M staining in μMT and μMT→μMT chimeric mice.

Mononuclear cells were isolated from the CNS of WT or μMT mice with EAE at peak of disease from three to five mice perfused with 15–25 ml of cold PBS as described (18). The brains and spinal cords were homogenized, and mononuclear cells were isolated using 40/70% discontinuous Percoll gradients (Sigma-Aldrich). Total cell numbers were determined by counting on a hemocytometer, and viability was assessed by trypan blue exclusion.

Mononuclear cells were isolated from the CNS of three or four WT and μMT mice at peak of disease following EAE induction. FcR were blocked with anti-mouse FcR (2.4G2), followed by staining with the anti-mouse mAb CD4-FITC, CD11b-PE, and Vβ8.2-biotin followed by SA-PE-Cy5. CD11b cells were gated for CD4 and sorted into Vβ8.2+ and Vβ8.2 populations using a FACSAria cell-sorting system (BD Biosciences).

For labeling of proliferating cells in vivo, 3–4 WT or μMT mice with EAE were injected i.p. with 1 mg of BrdU (BD Bioscience) 12–18 h before the isolation of brain mononuclear cells at the peak of the disease. BrdU incorporation into the cellular DNA was detected using the BrdU flow kit (BD Biosciences) as described (18, 19). Cells were cell surface stained with CD4-PE, CD25-PE-CY5, and Vβ8.2-biotin followed by SA-PE-Cy7 before permeabilization and staining with anti-BrdU-FITC. Analysis was performed using a LSR II and FACSDiva software (BD Biosciences).

For intracellular staining of cytokines or FoxP3, mononuclear cells were isolated from the CNS of 3–5 WT or μMT mice with EAE at peak of disease. For detection of IFN-γ, FcR were blocked before cell surface staining with CD4, CD25, and Vβ8.2 and then fixed and permeabilized using the Cytofix/Cytoperm kit according to the manufacturer’s instructions (BD Pharmingen) as described (20). Permeabilized cells were then stained with anti-IFN-γ-PE. For detection of Foxp3, FcR were blocked followed by cell surface staining for CD4, CD25, and Vβ8.2. Foxp3 staining was subsequently performed using a kit purchased from eBioscience. Samples were analyzed using a LSR II and FACSDiva software.

For immunofluorescent staining, longitudinal sections of the spinal cord and sagittal sections of the brain and spinal cord were generated as previously described (21). Frozen sections were generated from perfused mice at peak of EAE disease and after recovery from WT, μMT, or chimeric mice. Frozen sections 10-μm thick were blocked with newborn calf serum and serially stained with anti-CD11b-PE and anti-TCRβ-FITC.

Mice were perfused by intracardial injection with PBS. Spinal cords and superficial cervical lymph nodes were isolated and homogenized and total RNA was isolated using TRIzol (Invitrogen Life Technologies). For sorted T cell populations, RNA was isolated using the Dynabeads mRNA DIRECT micro Kit (Dynal Biotech), according to the manufacturer’s instructions. cDNA was generated as described (20). Hypoxanthine phosphoribosyltransferase primers were used to ensure the quality of the sample before real-time reactions (22). IL-10, IFN-γ, and Foxp3 mRNA were quantitated by real-time PCR using SYBR Green as the detection agent as described (20). The PCR was performed with the iCycler iQ system (Bio-Rad). All components of the PCR mix were purchased from Bio-Rad and used according to the manufacturer’s instructions. Cycler conditions were one amplification cycle of denaturation at 95°C for 5 min followed by 40 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min. Specificity of the RT-PCR was controlled by the generation of melting curves. IL-10, IFN-γ, and Foxp3 expression threshold values were normalized to GAPDH expression using standard curves generated for each sample by a series of four consecutive 10-fold dilutions (1–1 × 103) of the cDNA template. For all reactions, each condition was performed in triplicate. Data analysis was performed using iQ Cycler analyzing software. The GAPDH and IFN-γ primers have been described (20). The sense The sense IL-10 primer was 5′-GGTTGCCAAGCCTTATCGGA-3′ and the antisense primer was 5′-ACCTGCTCCACTGCCTTGCT-3′ (23). The sense Foxp3 primer was 5′-GGCCCTTCTCCAGGACAGA-3′ and the antisense primer was 5′-CTGATCATGGCTGGGTTGT-3′ (24).

We previously reported that actively induced EAE in B cell-deficient B10.PL mice (μMT) by immunization with the Ac1–11 MBP peptide resulted in a chronic clinical disease course (2). In the present study, examining the mechanism of how B cells functionin disease recovery, we chose to induce EAE by passive transfer of in vitro activated encephalitogenic T cells because of the predictable and consistent disease course that we obtain by this method (16). In addition, in our adoptive transfer model the encephalitogenic T cells generated from MBP-TCR transgenic mice are primed in vitro, avoiding any effects that in vivo priming may have in the chimeric mice used in throughout this study. Thus, we first determined whether passive EAE in μMT mice also resulted in chronic disease. As shown in Fig. 1 A, the WT B10.PL mice completely recovered from EAE while the μMT mice exhibited a chronic disease course, with both groups having similar days of onset and a parallel disease course until the peak of the disease in WT mice. These results are consistent with our previous study using active induction showing a regulatory role for B cells in recovery from EAE (2).

FIGURE 1.

Passive EAE induction in μMT mice results in chronic disease but not increased encephalitogenic T cell proliferation or IFN-γ production in the CNS. A, EAE was induced in WT (▪) and μMT (▿) mice, the disease course was scored starting on day 6 after transfer, and the average daily score of 10 mice per group from three separate experiments is shown. B, At the peak of EAE disease, WT and μMT mice were i.p. injected with 1 mg of BrdU 12–18 h before isolation of CNS mononuclear cells and analyzed for incorporation of BrdU and expression of CD4 and Vβ8.2 by flow cytometry. The absolute number ± S.E. of CD4+Vβ8.2+ cells that incorporated BrdU incorporation is shown, with each bar representing the average of three separate experiments with each individual observation containing pooled cells from three mice. C, At peak of EAE disease, sagittal brain stem and longitudinal spinal cord frozen sections were generated and stained with anti-CD11b-FITC (green) and anti-TCRβ-PE (red) and are shown at ×100 original magnification, representing one representative mouse of two in each group. D and E, EAE was induced in WT (▪) and μMT (□) mice and total RNA was isolated from the spinal cord (D) and cervical lymph node (E) on days 10, 14, 21, and 28 or 32 following EAE induction. cDNA was generated and IFN-γ expression was analyzed by real-time PCR. Quantitative PCR results are presented as a ratio of the number of specific copies to the number of GAPDH copies. Each bar represents pooled RNA from two or three mice in experiment 1 (left panels) and three mice in experiment 2 (right panels). The day 0 time point is from mice in which EAE was not induced and ND indicates nondetectable levels of cytokine expression. F, Mononuclear cells were isolated from the CNS of WT (▪) and μMT (□) mice at peak of disease and analyzed for cell surface expression of CD4 and Vβ8.2 and for IFN-γ production by intracellular cytokine staining. The absolute number ± S.E. of CD4+Vβ8.2+ cells expressing IFN-γ is shown, with each bar representing the average of three separate experiments and each individual observation containing pooled cells from three mice.

FIGURE 1.

Passive EAE induction in μMT mice results in chronic disease but not increased encephalitogenic T cell proliferation or IFN-γ production in the CNS. A, EAE was induced in WT (▪) and μMT (▿) mice, the disease course was scored starting on day 6 after transfer, and the average daily score of 10 mice per group from three separate experiments is shown. B, At the peak of EAE disease, WT and μMT mice were i.p. injected with 1 mg of BrdU 12–18 h before isolation of CNS mononuclear cells and analyzed for incorporation of BrdU and expression of CD4 and Vβ8.2 by flow cytometry. The absolute number ± S.E. of CD4+Vβ8.2+ cells that incorporated BrdU incorporation is shown, with each bar representing the average of three separate experiments with each individual observation containing pooled cells from three mice. C, At peak of EAE disease, sagittal brain stem and longitudinal spinal cord frozen sections were generated and stained with anti-CD11b-FITC (green) and anti-TCRβ-PE (red) and are shown at ×100 original magnification, representing one representative mouse of two in each group. D and E, EAE was induced in WT (▪) and μMT (□) mice and total RNA was isolated from the spinal cord (D) and cervical lymph node (E) on days 10, 14, 21, and 28 or 32 following EAE induction. cDNA was generated and IFN-γ expression was analyzed by real-time PCR. Quantitative PCR results are presented as a ratio of the number of specific copies to the number of GAPDH copies. Each bar represents pooled RNA from two or three mice in experiment 1 (left panels) and three mice in experiment 2 (right panels). The day 0 time point is from mice in which EAE was not induced and ND indicates nondetectable levels of cytokine expression. F, Mononuclear cells were isolated from the CNS of WT (▪) and μMT (□) mice at peak of disease and analyzed for cell surface expression of CD4 and Vβ8.2 and for IFN-γ production by intracellular cytokine staining. The absolute number ± S.E. of CD4+Vβ8.2+ cells expressing IFN-γ is shown, with each bar representing the average of three separate experiments and each individual observation containing pooled cells from three mice.

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Because EAE disease induction was not compromised in μMT mice induced with neither immunization nor passive transfer, these data suggest that a B cell deficiency does not affect T cell responses at the level of initiation of disease. However, we and others have shown that T cell responses in μMT mice are affected compared with their WT counterparts (2). Thus, we examined whether the passively transferred encephalitogenic (CD4+Vβ8.2+) T cells had altered effector function in the CNS of μMT mice. Using BrdU to measure proliferation in vivo, we found that at the peak of EAE disease there was no difference in the absolute number of proliferating encephalitogenic T cells in WT vs μMT mice (Fig. 1,B). In addition, there was no significant difference in the absolute number of CNS mononuclear cells or CD4+ T cells in the two groups of mice (data not shown). When we examined the intensity and location of inflammatory lesions in the two groups of mice, we detected similar lesion patterns containing macrophage/microglia and T cells in the lumbar spinal cord and brain stem (Fig. 1 C). We also detected infiltrates in the meningeal regions of the brain in both groups of mice, but not in the brain parenchyma (data not shown).

We next examined the expression of the encephalitogenic T cell effector cytokine IFN-γ during the EAE disease course in the spinal cord and cervical lymph node. In two independent experiments, the increase in expression of IFN-γ in the spinal cord at the onset of the disease and the subsequent decrease during recovery was similar in WT and μMT mice (Fig. 1,D). This change in IFN-γ production during the EAE disease course is consistent with our previous published findings (20). We did not detect a differential change in IFN-γ expression in the cervical lymph nodes during EAE, and there was no consistent difference in expression between the WT and μMT mice (Fig. 1,E). We used intracellular cytokine staining to confirm that similar numbers of CD4+Vβ8.2+ encephalitogenic T cells in the CNS of WT and μMT mice produced IFN-γ at peak of disease (Fig. 1 F). Overall, these data suggest that the chronic EAE disease phenotype observed in μMT mice is not due to a dysregulation in encephalitogenic T cell effector function or their migration into the CNS.

Because the production of IL-10 by B cells has been shown to be required for recovery from EAE (3) (M. K. Mann and B. N. Dittel, unpublished observations), we examined whether a dysregulation in IL-10 production could be detected in the CNS (Fig. 2,A) and the cervical lymph nodes (Fig. 2,B) of μMT mice as compared with WT mice. We performed two separate experiments with similar results using the same cDNA samples as for Fig. 1, D and E. Because it is not known when the B cell-derived IL-10 is required during EAE and because it is known that IL-10 mRNA expression peaks in the CNS at the time of disease recovery (8, 9), we examined IL-10 expression throughout the EAE disease course using real-time PCR. On day 0 before EAE induction, IL-10 mRNA levels in the spinal cord were near the level of detection in both WT and μMT mice (Fig. 2,A). Likewise, in the cervical lymph nodes, the levels were expressed at similar levels (Fig. 2,B). In WT mice, IL-10 levels in the spinal cord were only slightly increased on the day of onset (day 10) but reached maximal levels at the peak of disease on day 14, which represented a ∼100-fold increase (Fig. 2,A). The levels then declined during the recovery phase (day 21) and showed a further reduction once the mice showed signs of total recovery (day 28) (Fig. 2,A). IL-10 mRNA levels were also detected in the spinal cords of μMT mice with EAE, but the expression was delayed and did not reach maximum until day 21, a level that was increased by ∼13-fold (Fig. 2,A). In μMT mice, the maximal level of IL-10 detected was 10-fold lower than in the WT mice (Fig. 2 A), suggesting that B cells are either producing IL-10 in the CNS or are regulating its expression.

FIGURE 2.

Delayed expression of IL-10 in spinal cord of μMT mice during EAE. IL-10 expression in the spinal cord (A) and cervical lymph node (B) of WT (▪) and μMT (□) mice with EAE was examined using the same cDNA preparations as for Fig. 1, D and E. Quantitative PCR results are presented as a ratio of the number of specific copies to the number of GAPDH copies.

FIGURE 2.

Delayed expression of IL-10 in spinal cord of μMT mice during EAE. IL-10 expression in the spinal cord (A) and cervical lymph node (B) of WT (▪) and μMT (□) mice with EAE was examined using the same cDNA preparations as for Fig. 1, D and E. Quantitative PCR results are presented as a ratio of the number of specific copies to the number of GAPDH copies.

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In the cervical lymph nodes of both WT and μMT mice, IL-10 levels were fairly consistent between time points in both sets of mice and any variability between the mice was not consistent in the two experiments (Fig. 2 B), suggesting that IL-10 immune regulation is specific to the CNS microenvironment during EAE.

The reduced and delayed IL-10 expression in the spinal cords of μMT mice suggest that B cells may regulate an IL-10-producing Treg cell population. Because Foxp3+CD4+CD25+ Treg cells from the CNS have been shown to produce IL-10 and suppress EAE (11), we examined whether this population of cells was altered in the CNS during EAE. We first examined whether μMT mice have normal numbers of Treg cells by measuring Foxp3 mRNA. To perform this analysis we used the same cDNA samples as for Fig. 1, D and E. In unmanipulated animals the levels of Foxp3 message were similar in the WT and μMT mice in both the spinal cord (Fig. 3,A) and the lymph node (Fig. 3 B). As with IL-10, the expression of Foxp3 was 100-fold higher in the lymph node as compared with the spinal cord. Thus, a B cell deficiency does not alter the development or maintenance of this population of Treg cells.

FIGURE 3.

FoxP3 expression is delayed in μMT spinal cord during EAE. Foxp3 expression in the spinal cord (A) and cervical lymph node (B) of WT (▪) and μMT (□) mice with EAE was examined using the same cDNA preparations as for Fig. 1. Quantitative PCR results are presented as a ratio of the number of specific copies to the number of GAPDH copies.

FIGURE 3.

FoxP3 expression is delayed in μMT spinal cord during EAE. Foxp3 expression in the spinal cord (A) and cervical lymph node (B) of WT (▪) and μMT (□) mice with EAE was examined using the same cDNA preparations as for Fig. 1. Quantitative PCR results are presented as a ratio of the number of specific copies to the number of GAPDH copies.

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We next examined Foxp3 message at the two sites during the EAE disease course. As with IL-10, Foxp3 message reached maximal levels in the spinal cord at the peak of disease, with a 20-fold increase in message detected (Fig. 3,A). During the recovery process Foxp3 message was reduced but, as with IL-10, did not return to basal levels. Again paralleling IL-10 expression, an increase in Foxp3 expression in the spinal cord of μMT mice was delayed by 7 days, with the levels equal to those of WT mice at the peak of the disease (Fig. 3,A). No consistent change in Foxp3 message was detected in the cervical lymph nodes of either WT or μMT mice (Fig. 3 B).

We next determined which T cell population in the CNS is the major source of IL-10 and Foxp3. We sorted CD4+ encephalitogenic (Vβ8.2+) and nonencephalitogenic T cells (Vβ8.2) from the CNS of WT mice at the peak of disease and measured IL-10 and Foxp3 message level by real-time PCR. The nonencephalitogenic T cells expressed ∼10-fold more IL-10 message and ∼100-fold more Foxp3 message as compared with the encephalitogenic T cells (Fig. 4 A). These data demonstrate that the Treg population is highly enriched in the nonencephalitogenic population of T cells found in the CNS.

FIGURE 4.

IL-10 and Foxp3 expression is highly expressed in CD4+Vβ8.2 T cells in the CNS. A, EAE was induced in WT mice and 14 days later total mononuclear cells were isolated and stained with mAb specific for CD11b, CD4, and Vβ8.2. CD11b cells were gated and CD4+Vβ8.2+ (□) and CD4+Vβ8.2 (▪) T cell populations were obtained by FACS. IFN-γ, IL-10, and Foxp3 message was quantitated as for Fig. 1. One representative experiment of two is shown. B and C, Mononuclear cells were isolated from the CNS of WT and μMT mice at the peak of the disease and analyzed for the expression of CD4, Vβ8.2, CD25, and Foxp3 by flow cytometry. CD4+ cells were gated and analyzed for expression of Foxp3 (y-axis) in combination with CD25 (B) (x-axis) or Vβ8.2 (C) (x-axis). Two-color contour plots are shown as one of two representative experiments with the percentage of CD4-gated cells in each quadrant shown. D, Total splenocytes were isolated from WT and μMT mice and analyzed for the expression of CD4 and Foxp3 as for B and C. CD4+ cells were gated and two-color contour plots are shown with CD4 (x-axis) and Foxp3 (y-axis) from one representative mouse of three in each group. The percentage of CD4-gated cells in each quadrant is shown.

FIGURE 4.

IL-10 and Foxp3 expression is highly expressed in CD4+Vβ8.2 T cells in the CNS. A, EAE was induced in WT mice and 14 days later total mononuclear cells were isolated and stained with mAb specific for CD11b, CD4, and Vβ8.2. CD11b cells were gated and CD4+Vβ8.2+ (□) and CD4+Vβ8.2 (▪) T cell populations were obtained by FACS. IFN-γ, IL-10, and Foxp3 message was quantitated as for Fig. 1. One representative experiment of two is shown. B and C, Mononuclear cells were isolated from the CNS of WT and μMT mice at the peak of the disease and analyzed for the expression of CD4, Vβ8.2, CD25, and Foxp3 by flow cytometry. CD4+ cells were gated and analyzed for expression of Foxp3 (y-axis) in combination with CD25 (B) (x-axis) or Vβ8.2 (C) (x-axis). Two-color contour plots are shown as one of two representative experiments with the percentage of CD4-gated cells in each quadrant shown. D, Total splenocytes were isolated from WT and μMT mice and analyzed for the expression of CD4 and Foxp3 as for B and C. CD4+ cells were gated and two-color contour plots are shown with CD4 (x-axis) and Foxp3 (y-axis) from one representative mouse of three in each group. The percentage of CD4-gated cells in each quadrant is shown.

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We then used intracellular Foxp3 staining to further evaluate the number and phenotype of Foxp3+ cells in the CNS during EAE. We found that at the peak of the disease, the majority of the Foxp3-expressing cells of the CD4 T cell population also expressed CD25 in both WT and μMT mice (Fig. 4B, upper left vs upper right quadrants). Furthermore, a greater percentage of CD4+CD25+ cells in the CNS of WT mice expressed Foxp3 (∼34%), as compared with μMT mice (∼17%) (Fig. 4,B, upper right quadrant). When we examined the CD4+Vβ8.2+ encephalitogenic T cells, we found that only 2–4% of these cells expressed Foxp3 in both the WT and μMT mice (Fig. 4,C, right quadrants). This is substantially lower as compared with 36% of the CD4+Vβ8.2 nonencephalitogenic T cells that express Foxp3 in the WT mice (Fig. 4,C, lower left panel, left quadrants). In μMT mice, only 19.4% of the nonencephalitogenic T cells expressed Foxp3 (Fig. 4,C, lower right panel, left quadrants). This reduction is consistent with the reduced level of Foxp3 message at the same time point (Fig. 3 A). Thus, the primary Foxp3-expressing cells are the nonencephalitogenic T cells, which suggests that B cells regulate the activation and/or mobilization of endogenous Treg cells into the CNS.

We were unable to detect IL-10 production by T cells isolated from the CNS by intracellular cytokine staining as previous reported (11). We contribute this inability to different methodology. In the study by McGeachy et al., T cells isolated from the CNS with PMA and ionomycin before staining for IL-10 were activated in vitro (11). This technique determined the number of T cells in the CNS capable of producing IL-10. However, in our study we were interested determining the number of T cells producing IL-10 in the CNS during EAE; therefore, we examined IL-10 production by T cells directly after isolation from the CNS. Our lack of IL-10 detection was likely due to the fact that IL-10 was being produced at a level below the sensitivity level of the intracellular staining technique. We do not feel that the lack of detection was due to a technical problem, because we have been able to detect IFN-γ and GM-CSF production by T cells directly isolated from the CNS (20).

In the CNS, the CD4+ population of cells at the peak of disease represents 15–20% of the total mononuclear cells in the CNS and of these cells 33% are nonencephalitogenic (M. K. Mann and B. N. Dittel, unpublished observations). In our model, we have shown that ∼1 × 106 mononuclear cells are present in the CNS during the peak of disease (19), thus ∼5–7% of the total mononuclear cell population are nonencephalitogenic CD4+ T cells and are potentially Treg cells. Our estimated number of Treg cells in the CNS is similar to that reported in peripheral T cell populations (25).

Because the reduced numbers of Foxp3+ cells in the CNS of μMT mice with EAE could reflect differences in this cell population in the periphery as compared with WT mice, we determined the percentage of splenic CD4+ cells that also express Foxp3 in the two mice. We found that ∼10% of CD4 T cells also expressed Foxp3 in both mice (Fig. 4 D, upper right quadrant). These data confirm the real-time RT-PCR data demonstrating that a B cell-deficiency does not alter the development of Foxp3+ Treg cells in B10.PL mice. However, the μMT background did reduce the absolute number of CD4+ T cells in the spleen by ∼40%. This reduced cellularity was also observed in the CD4+Foxp3+ population where the absolute number was reduced 30% from 2.53 ± 0.3 × 106 in the WT mice to 0.76 ± 0.09 × 106 cells. Because the absolute number of CD4 T cells in the CNS of the two mice did not differ at the peak of EAE disease (data not shown), the reduction in CD4+Foxp3+ cells in the CNS in the μMT mice is not likely due to differences in the population levels in the periphery.

Our data suggest that B cells are active participants in regulating EAE. Because recent work has implicated the involvement of CD28:B7 signaling in the development and maintenance of Treg cells (26, 27), we examined whether B7 on B cells was required for EAE recovery and the mobilization of Treg cells into the CNS. This was accomplished by reconstituting μMT mice with either WT or B7-deficient B cells by transplanting sublethally irradiated μMT mice with BM from either WT (WT→μMT) or B7−/− mice (B7−/−→μMT), generating mixed chimeric mice in which only the B cells are totally of donor origin. When EAE was induced in the chimeric mice, only the mice reconstituted with WT BM were able to recover from EAE (Fig. 5,A). As a control, we also generated chimeric mice by transplanting B7−/− BM into WT mice (B7−/−→WT) to determine whether the reduced numbers of APC, such as dendritic cells, expressing the B7 costimulatory molecules in the (B7−/−→μMT) mixed chimeras were contributing to the inability to recover from EAE. Chimerism in the dendritic cell population was assessed in the (B7−/−→μMT) chimeras by isolating splenic dendritic cells and examining B7.2 expression following overnight activation with LPS. The level of chimerism was ∼50% (data not shown). When EAE was induced in the B7−/−→WT chimeric mice they recovered similarly as the WT→WT control chimeras (Fig. 5 B). These data indicate that reducing the number of dendritic cells that express B7 has no effect on the EAE disease course in our B10.PL model. Thus, these cumulative data demonstrate that B7 expression by B cells is required for recovery from EAE. Furthermore, these data indicate that the lymphoid anatomy in μMT mice is sufficient to allow for the reconstitution of WT B cells.

FIGURE 5.

B7 expression by B cells is required to mediate recovery from EAE. BM chimeric mice were generated as described in Materials and Methods and allowed to reconstitute for at least 6 wk before EAE induction. A and B, Six days after EAE induction the mice were scored for clinical signs of EAE. A, The data shown are the average daily scores of 10 chimeric mice per group from two separate experiments generated by transplanting μMT mice with BM from either WT (♦) or B7−/− (▿) mice. The data shown were generated simultaneously with those of Fig. 1,A. B, The data shown are the average daily scores of 11 WT mice transplanted with WT BM (•) or 13 WT mice transplanted with B7−/− BM (□) in two separate experiments each containing 4–7 mice. C, At the peak of EAE disease, spinal cords were isolated from WT→μMT (upper panels) and B7−/−→μMT (lower panels) chimeric mice. Longitudinal spinal cord sections were stained as for Fig. 2 C. Images are shown at ×100 original magnification and are representative of two mice per group.

FIGURE 5.

B7 expression by B cells is required to mediate recovery from EAE. BM chimeric mice were generated as described in Materials and Methods and allowed to reconstitute for at least 6 wk before EAE induction. A and B, Six days after EAE induction the mice were scored for clinical signs of EAE. A, The data shown are the average daily scores of 10 chimeric mice per group from two separate experiments generated by transplanting μMT mice with BM from either WT (♦) or B7−/− (▿) mice. The data shown were generated simultaneously with those of Fig. 1,A. B, The data shown are the average daily scores of 11 WT mice transplanted with WT BM (•) or 13 WT mice transplanted with B7−/− BM (□) in two separate experiments each containing 4–7 mice. C, At the peak of EAE disease, spinal cords were isolated from WT→μMT (upper panels) and B7−/−→μMT (lower panels) chimeric mice. Longitudinal spinal cord sections were stained as for Fig. 2 C. Images are shown at ×100 original magnification and are representative of two mice per group.

Close modal

Inflammatory lesions in spinal cords of μMT chimeric mice in which B cells were either WT or B7−/− were similar (Fig. 5,C) and resembled those from WT and μMT mice (Fig. 2 C).

Because IL-10 levels and Foxp3 expression were delayed in μMT mice, we next examined whether B cell expression of B7 was required for the regulation of Treg cells in the CNS during EAE. This was examined using real-time RT-PCR measuring IL-10 and Foxp3 messages in the spinal cord of the chimeric mice at the peak of the disease. This time point was chosen because this is when the greatest difference in message levels was observed between WT and μMT mice (Figs. 2,A and 3,A). The experiment was performed twice with similar results. When μMT mice were reconstituted with WT B cells (WT→μMT) the level of IL-10 message in the spinal cord was normalized and was identical with the control chimeras in which WT mice were transplanted with WT BM (WT→WT) (Fig. 6,A). However, when μMT mice were reconstituted with BM from either μMT (μMT→μMT) or B7−/− (B7−/−→ μMT) mice, IL-10 message levels in the spinal cord were similar in the two chimeras but reduced by ∼10-fold as compared with those in the mice reconstituted with WT B cells (Fig. 6,A). No consistent alteration in the level of IL-10 was observed in the cervical lymph node in the four groups of chimeric mice (Fig. 6 B).

FIGURE 6.

B7 expression by B cells is required to mediate peak expression of IL-10 and Foxp3 in the spinal cord during EAE. A–D, Fourteen days after EAE induction, total RNA was isolated from the spinal cord (A and C) and cervical lymph node (LN) (B and D) of chimeric mice generated by transplanting μMT mice with BM from either WT (WT→μMT) (open bar), μMT (μMT→μMT) (diagonal striped bar), or B7−/− (B7−/−→μMT) (vertical striped bar) mice, or, as a control, WT mice with WT BM (WT→WT) (closed bar). IL-10 (A and B) and Foxp3 (C and D) messages were quantitated as described in Fig. 1. Each bar represents pooled RNA from three mice.

FIGURE 6.

B7 expression by B cells is required to mediate peak expression of IL-10 and Foxp3 in the spinal cord during EAE. A–D, Fourteen days after EAE induction, total RNA was isolated from the spinal cord (A and C) and cervical lymph node (LN) (B and D) of chimeric mice generated by transplanting μMT mice with BM from either WT (WT→μMT) (open bar), μMT (μMT→μMT) (diagonal striped bar), or B7−/− (B7−/−→μMT) (vertical striped bar) mice, or, as a control, WT mice with WT BM (WT→WT) (closed bar). IL-10 (A and B) and Foxp3 (C and D) messages were quantitated as described in Fig. 1. Each bar represents pooled RNA from three mice.

Close modal

In the same chimeric mice we also examined the expression of Foxp3 in the CNS and cervical lymph node. Although no substantial difference was observed in the lymph node (Fig. 6,D), an ∼5-fold reduction in Foxp3 message was observed in the spinal cord of chimeras lacking B cells (μMT→μMT) or those deficient in B7 (B7−/−→μMT), as compared with chimeras with WT B cells (Fig. 6 C). These data suggest that B7 expression by B cells is required to establish a Treg cell IL-10 regulatory network in the CNS that facilitates disease recovery.

In this study, we investigated the mechanism of how B cells regulate the recovery from clinical disease in the B10.PL acute EAE model. We found that mice with an absence of B cells or those with B7-deficient B cells where not only unable to recover from EAE but also had a delay in the expression of IL-10 and the emergence of Foxp3+ Treg cells in the CNS during EAE. The requirement for B7 expression by B cells show that they are active participants in the immune regulatory mechanisms required for the resolution of inflammation in the CNS.

In our original study examining the role of B cells in EAE, we induced EAE by active immunization with the Ac1–11 MBP peptide in the B cell-deficient μMT mice (2). We found that the early phases of the EAE disease course, which are dependent upon the priming of naive T cells, were unaltered in the μMT mice. These data confirmed previous findings that T cell priming did not require B cells (28). Potential caveats to using μMT mice are several reports that have detected altered T cell responses in μMT mice following viral infection. The T cell defects included inability to transfer virus-specific memory, reduced numbers of virus-specific T cells in the CNS following CNS infection, and depletion of virus-specific T cells (29, 30, 31). In these studies, the infections were not lethal and in two of the studies the mice generated Ag-specific T cells capable of clearing the virus (29, 30). Thus even in the absence of B cells, a functional T cell response can be generated. In our current study, we used encephalitogenic T cells from WT mice that were activated in vitro in the presence of B cells. Based on previous observations and data presented in Fig. 1, we do not think that μMT mice have chronic disease due to dysregulation of the encephalitogenic T cell population. In addition, we show that B cells are not required for the development and maintenance of Foxp3+ cells. Rather, we show that B cells regulate the emergence of Treg cells in the CNS in a B7-dependent manner. It is becoming increasingly clear that B cells can regulate immune responses in a variety of ways; thus in their absence T cell defects may vary depending on the experimental condition (32, 33).

Because we used mice deficient in both B7 molecules, we were not able to determine whether B cell expression of B7.1 or B7.2 is critical for their regulatory function. The B7 molecules have two known ligands, CD28 and CTLA-4, both expressed by a variety of T cell populations including Treg cells (34). Although the precise role of B7.1 and B7.2 in immune regulation is not known, evidence has emerged to support a role for B7.1 in Treg cell function (35, 36), as well as CTLA-4 (37, 38). In EAE, the absence of both B7.1 and B7.2 reduced the severity of disease (5). However, when either B7.1 or B7.2 were blocked separately, differential roles were evident with the trend being that the blocking of B7.1 reduced disease severity while the blocking of B7.2 exacerbated disease (39, 40, 41). Because B7 is expressed by all professional APC and the above studies used global blocking strategies, the role of the individual B7 molecules on particular cell populations in EAE is still not clear.

In our study, we chose to examine the role of B7 on only B cells in EAE; thus our study is the first to show the role of B7 on a specific cell population in EAE pathogenesis. In addition to B7 expression by B cells, their production of IL-10 has also been shown to be required for EAE recovery (3), the mechanism of which has not been shown. In our model, we have confirmed that B cell production of IL-10 is important for recovery from EAE (M. K. Mann and B. N. Dittel, data not shown). By examining IL-10 levels in both the lymph node and spinal cords of WT and μMT mice, we were able to show that a B cell deficiency does not alter steady-state levels of IL-10 (Fig. 2), suggesting that B cells are not a major source of IL-10 under homeostatic conditions. However, B cells have been shown to produce substantial levels of IL-10 when stimulated through CD40 in the absence of a BCR signal (42). Thus, during EAE B cell-derived IL-10 is likely induced following CD40 engagement by CD40L+ T cells, the most prevalent of which are the transferred encephalitogenic T cells.

In both WT and μMT mice the level of IL-10 is at the level of detection in the spinal cord (Fig. 2,A). Upon EAE induction in WT mice IL-10 levels increased by ∼100-fold, reaching maximum at the peak of disease on day 14 and then decreasing during clinical disease recovery (Fig. 2,A). This observation is consistent with two separate reports on the Lewis rat and SJL mouse, which also showed an increase and then decrease of IL-10 during acute EAE (8, 9). In contrast, maximal expression of IL-10 was delayed in μMT mice by two weeks (Fig. 2 A). This discrepancy in maximal IL-10 levels is not likely due to B cell-derived IL-10 in the CNS, because very few B cells are detectable in the CNS during EAE (11) (M. K. Mann, L. P. Shriver, and B. N. Dittel, unpublished observations). Because we did not observe consistent differences in IL-10 in the cervical lymph nodes between WT and μMT mice, this suggests that IL-10 immune regulation is specific to the CNS microenvironment.

Because IL-10 levels in the spinal cord of B cell-deficient mice were both delayed and reduced, we propose that B cells induce an IL-10-mediated regulatory pathway that is required during disease and, without it, spontaneous recovery is unable to occur. Cell populations in the CNS previously shown to produce IL-10 are astrocytes, microglial cells/macrophages, and T cells (9). Thus, B cells may be interacting with and promoting IL-10 production by one or more of these cell types. Because CD40 expression by B cells is required for disease recovery (3), a finding that we confirmed in our model (M. K. Mann and B. N. Dittel, unpublished observations), we propose that a CD40L+ T cell is the regulatory target. Although Th2 cells have been shown to produce IL-10, they are not prominent in the CNS as indicated by low levels of IL-4 in the CNS during EAE, which is the quintessential Th2 cytokine (M. K. Mann, K. Mares, and B. M. Dittel, unpublished observations). A second T cell population shown to produce high levels of IL-10 in an immune regulatory manner is that of CD4+CD25+ Treg cells (25). These Treg cells have been shown to suppress EAE upon adoptive transfer (12), in an IL-10-dependent manner (13).

When we examined the expression of Foxp3 in the spinal cords of WT vs μMT mice with EAE we found that its increase in expression paralleled that of IL-10 (Fig. 3 A), suggesting that an IL-10-secreting CD25+ Treg population migrates into the CNS during EAE. It is not likely that the Treg cells are expanding in the CNS, because the proliferative potential of these cells is limited. The delayed appearance of the Treg cells in the CNS of μMT mice suggests that B cells are important in their mobilization and perhaps activation. The finding that μMT mice cannot recover from EAE, even though Treg cells are eventually found at normal levels in the CNS, suggests that there is a critical window in which inflammation must be controlled in order for the CNS to recover full function. Th1-mediated autoimmune disorders, like MS and rheumatoid arthritis, are inflammatory in nature and if not controlled properly ultimately lead to irreversible tissue damage. Thus a regulatory role for B cells may be to interact with and mobilize Treg cells early in an immune response to control the level of inflammation.

When we examined T cell populations in the CNS, we found that the nonencephalitogenic cells expressed very high levels of both IL-10 and Foxp3 (Fig. 4). Although it is well known that nonencephalitogenic T cells are very prevalent in the CNS of mice with EAE, their function has remained unknown. Our data would suggest that they contain a population of Foxp3+ IL-10 producing Treg cells, which are important for recovery from EAE. Very few of the encephalitogenic T cells expressed Foxp3, indicating that conversion of these cells into Treg cells is not occurring in our EAE model as has been shown under normal conditions by a B7-dependent mechanism (43, 44).

Our data, combined with previous findings, support a model for B cells in the mobilization of Foxp3-dependent CD4+CD25+ Treg cells into the CNS that suppress EAE via an IL-10-dependent mechanism. We propose that activated CD40L+ encephalitogenic T cells (45) interact with B cells in an Ag-nonspecific manner, providing a CD40 costimulatory signal that induces their expression of both B7 and IL-10. CD40 expression during EAE has been shown to be necessary for B7 induction (4), and B cells receiving CD40 costimulation in the absence of BCR signaling have been shown to produce IL-10 (42). We propose that this B cell then interacts with and activates a CD4+CD25+ Treg cells via B7/CTLA-4 (37, 38). The Treg cells then migrate into the CNS and suppress inflammation via an IL-10-dependent mechanism (13, 46). Our model also supports a role for the required B cell-derived IL-10 in the induction of the Treg cells, because IL-10 has been shown to induce regulatory T cells able to suppress colitis (47). Thus, we demonstrate an essential role for B cells in the rapid induction of a Treg cell network that must be mobilized within a critical window in the EAE inflammatory process to facilitate disease resolution before irreversible tissue damage occurs. Treg cellular therapy in MS patients may be feasible in newly diagnosed disease or early during a relapse.

We thank Shelley Morris for assistance with the animal colony.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported in part by National Multiple Sclerosis Society Grants RG 3299-A-2 and PP0778 and the BloodCenter Research Foundation.

3

Abbreviations used in this paper: MS, multiple sclerosis; BM, bone marrow; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; SA, streptavidin; Treg, T regulatory; WT, wild type.

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