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Keywords:

  • Autoimmunity;
  • B cells;
  • EAE;
  • MS;
  • Regulatory T cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and Methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

B cells and regulatory T (Treg) cells can both facilitate remission from experimental auto immune encephalomyelitis (EAE), a disease of the central nervous system (CNS) used as a model for multiple sclerosis (MS). Considering that B-cell-depletion therapy (BCDT) is used to treat MS patients, we asked whether Treg-cell activation depended on B cells during EAE. Treg-cell proliferation, accumulation in CNS, and augmentation of suppressive activity in the CNS were normal in B-cell-deficient mice, indicating that B cells are not essential for activation of the protective Treg-cell response and thus provide an independent layer of regulation. This function of B cells involved early suppression of the encephalitogenic CD4+ T-cell response, which was enhanced in B-cell-deficient mice. CD4+ T-cell depletion was sufficient to intercept the transition from acute-to-chronic EAE when applied to B-cell-deficient animals that just reached the peak of disease severity. Intriguingly, this treatment did not improve disease when applied later, implying that chronic disability was ultimately maintained independently of pathogenic CD4+ T cells. Collectively, our data indicate that BCDT is unlikely to impair Treg-cell function, yet it might produce undesirable effects on T-cell-mediated autoimmune pathogenesis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and Methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Experimental autoimmune encephalomyelitis (EAE) is a T-cell-mediated inflammatory disease associated with the development of demyelinating lesions in central nervous system (CNS). It is widely used as an animal model for relapsing-remitting multiple sclerosis (RR-MS) [[1]]. EAE can be induced in genetically susceptible animals by immunization with myelin antigens such as myelin oligodendrocyte glycoprotein (MOG). After onset of paralysis, animals can spontaneously recover from disease, providing a model to investigate how remission from disease flares might be regulated during RR-MS.

Remission from EAE involves IL-10-producing B cells and CD4+Foxp3+ regulatory T (Treg) cells [[2, 3]]. These two protective cell types might be impaired in RR-MS because Treg cells from RR-MS patients were less suppressive, and B cells produced less IL-10, than the corresponding cells from healthy individuals [[4, 5]]. Furthermore, standard treatments for RR-MS (IFN-β and glatiramer acetate) can enhance the suppressive functions of Treg cells or B cells [[5-7]]. It might also be relevant that helminth parasites can increase the regulatory functions of Treg and B cells, and reduce rate of relapses in RR-MS patients [[8, 9]]. The specific functions of Treg cells and B cells in limitation of autoimmune flares are still poorly understood. B cells and Treg cells are required during different phases of EAE for control of pathogenesis, with an early involvement of B cells and a later action of Treg cells [[10]]. It has been proposed that the beneficial functions of B cells involve a positive effect on Treg-cell-mediated protective activities, that is, B cells might facilitate remission from EAE by promoting the suppressive function of Treg cells. Supporting this notion, expression of Foxp3 messenger RNA (mRNA) was transiently reduced in CNS of B-cell-deficient mice, compared with control animals, in a model of EAE induced by adoptive transfer of encephalitogenic T cells [[11]].

The notion that B cells instruct the protective function of Treg cells during autoimmune disease is of considerable importance, given that B cells can also be pathogenic during RR-MS and B-cell depletion therapy (BCDT) is becoming a possible treatment for RR-MS patients [[12]]. It is likely that current BCDT approaches deplete both the pathogenic and protective functions of B cells [[13]]. To characterize the interrelation between B cells and Treg cells in the context of an autoimmune disease of the CNS, we analyzed how B-cell deficiency affected homeostasis and activation of Treg cells during EAE.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and Methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

B-cell deficiency is associated with reduced CD103 Treg-cell frequency in secondary lymphoid organs

Recovery from EAE requires regulatory mechanisms mediated by B cells and Treg cells [[2, 3]]. This is illustrated by the fact that mice deficient in B cells (JHT mice, which have a deletion of JH-Eμ sequences of the IgH locus.) and mice having a defective Treg-cell compartment similarly developed chronic forms of EAE upon immunization with MOG (35–55) peptide, while wild-type (WT) mice eventually entered in remission from disease (Fig. 1A). The similar phenotype of JHT and anti-CD25 (clone PC61) treated mice led us to ask if B-cell deficiency was associated with an abnormal Treg-cell function in JHT mice.

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Figure 1. Frequencies of Treg cells in secondary lymphoid organs of B-cell-deficient JHT mice. (A) EAE was induced in C57BL/6 mice (black squares), JHT mice (open triangles), and C57BL/6 mice pretreated by i.v. injection of 200 μg anti-CD25 mAb (PC61) 3 days prior to immunization (gray circles). Data show mean EAE scores ± SEM. (B) CD4+ splenocytes of naïve 6–10-week-old adult C57BL/6 and JHT mice were analyzed for surface CD25 and intracellular Foxp3 expression. (C–E) The frequencies of Foxp3+ Treg cells (C) among CD4+CD8 T cells in thymus, and (D, E) among CD4+ T cells in (D) spleen, and (E) LNs of naïve adult C57BL/6 (black squares) and JHT (open triangles) mice are shown. (F) CD4+ splenocytes of naïve C57BL/6 and JHT mice were analyzed for intracellular Foxp3 and surface CD103 expression. (G–I) Frequencies of CD103 and CD103+ Treg cells (G) among CD4+CD8 T cells in thymus, and (H, I) among CD4+ T cells in (H) spleen, and (I) LNs of naïve adult C57BL/6 (black bars) and JHT (white bars) mice. Data are representative of (B, F) or show mean + SEM (C–E, G–I) of data pooled from at least three independent experiments (four mice per group per experiment). ***p < 0.0001, unpaired t-test.

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To assess the role of B cells in Treg-cell homeostasis, we measured frequencies of Foxp3+ cells among CD4+ T cells in naïve adult JHT and WT mice. JHT mice had lower Treg-cell frequencies in spleen and lymph nodes (LNs) but not in thymus compared with WT mice (Fig. 1B–E; Supporting Information Fig. 1) [[14]]. Similar results were obtained using μMT mice as an alternative strain of B-cell-deficient mice, or B-cell-deficient offspring from (JHT × C57BL/6) F1 intercrosses compared with WT littermate controls, indicating that this phenotype was not due to particularities in the genetic background of JHT mice but was directly associated to the lack of B cells (Supporting Information Fig. 2 and 3). Intriguingly, Treg-cell frequency was normal in spleen and LNs of 2-week-old JHT mice, indicating that B cells were selectively required for Treg-cell homeostasis in the adult mouse (Supporting Information Fig. 4). The peripheral Treg-cell compartment can be subdivided into two different subsets according to the expression of the integrin CD103 (αEβ7) [[15]]. Analysis of the frequencies of these Treg-cell subsets in adult JHT and WT mice revealed that B-cell deficiency selectively affected CD103 but not CD103+ Treg cells in secondary lymphoid organs (Fig. 1F–I).

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Figure 2. Phenotype and suppressive capacity of Treg cells from B-cell-deficient JHT mice. Surface expression of (A, B) CD25, (C, D) CD62L,+ (E, F) GITR, and (G, H) intracellular staining of CTLA-4 for CD4+ T-cell subsets in LNs of naïve 6–10-week-old C57BL/6 (black bars) and JHT (white bars) mice. (A, C, E, G) Representative stainings for LN cells from naïve C57BL/6 mice are shown. (B, D, F, H) Histograms show mean ± SEM of data pooled from two independent experiments (three mice per group per experiment). (I) In vitro suppression assay for CD4+CD25+CD103 and CD4+CD25+CD103+ Treg cells isolated by flow cytometry from pooled spleens and LNs of naïve C57BL/6 (black squares and diamonds, respectively) and JHT (open triangles and circles, respectively) mice. The assays included 2 × 104 CD4+CD25 responder T cells from naïve C57BL/6 mice, 1 × 105 irradiated C57BL/6 splenocytes, and 0.1 μg/mL anti-CD3. Data are shown as mean + SEM of n = 3 replicates and are representative of three independent experiments. *p < 0.05, unpaired t-test.

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Figure 3. Activation of Treg cells in vivo upon induction of EAE. C57BL/6 (black bars) and JHT (white bars) mice were treated with 1 mg BrdU via the intraperitoneal route on days 0, 4, or 8 after EAE induction. dLNs were analyzed 5 h after BrdU injection. (A) Representative staining for BrdUincorporation by T cells in dLNs of C57BL/6 mice at day 8. (B, C) BrdUincorporation by (B) CD4+Foxp3+CD103, and (C) CD4+Foxp3+CD103+ cells is shown. Data are shown as mean + SEM of data pooled from two independent experiments with three mice per group per experiment. (D) The suppressive capacity of CD4+CD25+ Treg cells from dLNs of C57BL/6 (black squares) and JHT (open triangles) mice on day 8 after EAEinduction. 2×104 CD4+CD25 T cells from naïve C57BL/6 mice were stimulated with 0.1 μg/mL anti-CD3 in presence of 1 × 105 irradiated splenocytes and increasing numbers of Treg cells. Proliferation was measured by incorporation of 3H-thymidine after 64 h of culture. Data are shown as mean + SEM of n = 3 replicates and are representative of three individual experiments.

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B-cell deficiency does not affect Treg-cell function in naïve adult mice

We then examined whether lack of B cells influenced the functional properties of Treg cells in naïve adult mice. CD25 (Fig. 2A and B), CD62L (Fig. 2C and D), GITR (Fig. 2E and F), and+ CTLA-4 (Fig. 2G and H) were expressed similarly by CD103 Treg cells from JHT and WT mice. CD103+ Treg cells from JHT mice expressed modestly higher amounts of CD25 than Treg cells from WT mice (Fig. 2B), but otherwise displayed a normal phenotype (Fig. 2D, F, and H). In addition, we tested the inhibitory activity of CD103 and CD103+ Treg cells from C57BL/6 and JHT mice in a classical in vitro suppression assay, using CD4+CD25 T cells as responder T cells. We could confirm that CD103+ Treg cells have a higher suppressive effect than CD103 Treg cells in vitro [[15]], however without any difference between cells from WT or JHT mice (Fig. 2I; Supporting Information Fig. 5). CD4+CD25 T cells from JHT mice had a similar propensity to be regulated by Treg cells compared to CD4+CD25 T cells from C57BL/6 mice (Supporting Information Fig. 6). Taken together, our results show that Treg cells maintain normal suppressive functions in the absence of B cells.

Normal Treg-cell activation in draining LNs of JHT mice after EAE induction

To measure Treg-cell activation in draining lymph nodes (dLNs) upon EAE induction, we quantified proliferation of CD103 and CD103+ Treg cells on days 0, 4, and 8 after EAE induction using short in vivo pulses with bromodeoxyuridin (BrdU) (Fig. 3A). In naïve mice, CD103+ Treg cells displayed a stronger proliferation than CD103 Treg cells, indicating a higher level of basal activation (Fig. 3B and C) [[16]]. Induction of EAE markedly increased CD103 Treg-cell proliferation, while the division rate of CD103+ Treg cells remained unchanged (Fig. 3B and C). Notably, B-cell deficiency did not alter CD103 or CD103+ Treg-cell proliferation at any time point tested. In addition, Treg cells taken from dLNs of WT or JHT mice on day 8 after immunization were similarly suppressive in vitro (Fig. 3D). Thus, Treg-cell activation proceeds normally in B-cell-deficient mice after EAE induction.

Normal Treg-cell accumulation in CNSofJHT mice during EAE

During EAE, Treg cells enter the CNS, where they may locally regulate pathogenic inflammation [[3]]. Accumulation of Treg cells in CNS was detectable in both WT and JHT mice by day 8 after immunization, before mice showed sign of disability, and increased until day 21, when mice reached peak of disease severity (Fig. 4A and B). A detailed kinetic analysis revealed comparable absolute numbers of CD103 and CD103+ Treg cells in CNS of JHT and C57BL/6 mice throughout the disease course (Fig. 4A and B). The CNS Treg-cell compartment progressively became enriched in CD103+ cells in the two strains of mice, and JHT mice transiently displayed a slightly higher frequency of CD103+ Treg cells among CD4+Foxp3+ T cells than C57BL/6 mice on day 21 (Fig. 4C). Considering frequencies of Treg cells (defined as Foxp3+, or CD103Foxp3+, or CD103+Foxp3+ cells) among CD45+CD4+ T cells, we found similar values in CNS of WT and JHT mice, except on days 21 and 35 when JHT mice had modestly higher percentages of CD103+ Treg cells than control mice (Fig. 4D–F). From these data, we conclude that Treg cells normally accumulate in CNS of JHT mice during EAE.

As our analysis did not provide any evidence for reduced Treg-cell accumulation or adverse alteration in the composition of the Treg-cell infiltrate in the CNS of JHT mice, we next asked if lack of B cells influenced Treg-cell activity in CNS during EAE. First, we analyzed the proliferation of Treg cells in CNS on day 21 after EAE induction using BrdU. CD103 Treg cells had a slightly higher cycling rate in JHT than in C57BL/6 mice, while CD103+ Treg cells behaved similarly in the two types of mice (Fig. 4G). To investigate the suppressive capacity of CNS Treg cells, JHT mice were backcrossed with a reporter Foxp3-eGFP gene-targeted mouse, which allowed isolation of CNS Treg cells in an unambiguous manner [[17]]. Foxp3-eGFP+CD4+ Treg cells isolated from CNS on day 21 after immunization were markedly more suppressive than Treg cells from secondary lymphoid organs (spleen and LNs) of naïve mice (Fig. 4H). This enhanced regulatory activity was, however, similar for Treg cells from Foxp3-eGFP and JHT × Foxp3-eGFP mice, showing that CNS Treg cells gained higher suppressive capacity independently of B cells (Fig. 4H). Taken together, our results show that Treg cells accumulated in CNS and acquired enhanced suppressive functions normally in the absence of B cells.

WT mice with reduced frequency of CD103 Treg cells enter remission from EAE

Naïve adult JHT mice showed reduced frequency of CD103 Treg cells in secondary lymphoid organs (Fig. 1). Lack of remission from EAE in JHT mice could result from this reduction or from a lack of B cells per se. To distinguish between these two possibilities, we prepared mice having a reduced CD103 Treg-cell frequency but a normal B-cell compartment. These mice were obtained by treating C57BL/6 mice with 200-μg anti-CD25 (clone PC61) antibody. On day 30 after anti-CD25 administration, C57BL/6 mice showed reduced frequency of CD103 Treg cells while CD103+ Treg cells had returned to control levels (Fig. 5A–C). At that time, CD103 Treg cells were present at lower frequency in PC61-treated C57BL/6 mice than in JHT mice (Fig. 5B). Upon EAE induction, the long-term depleted C57BL/6 mice displayed a higher maximum disease score than control mice, but nonetheless underwent remission from disease (Fig. 5D). To further evaluate the role of CD103 Treg cells during EAE in a complementary approach, we adoptively transferred CD103 Treg cells isolated from spleen and LNs of naïve C57BL/6 mice into JHT mice. Such reconstitution did not restore remission from disease in recipient mice, but lowered their maximal disease score (Fig. 5E and F). Administration of control CD4+CD25 T cells had no effect (Fig. 5E and F). We conclude from these experiments that reduction in CD103 Treg-cell frequency contributes to exacerbated disease severity, but is not alone responsible for the lack of remission from disease in JHT mice. These data suggest that B cells control recovery from EAE via a different mechanism than by promoting Treg-cell function.

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Figure 4. Infiltration of Treg cells into the CNS during EAE. Lymphocytes from pooled brains and spinal cords of C57BL/6 (black bars) and JHT(white bars) were isolated at the indicated time points after EAE induction and analyzed by flow cytometry. (A–B) Absolute numbers of (A) CD45highCD4+Foxp3+CD103 and (B) CD45highCD4+Foxp3+CD103+ Treg cells in the CNSof C57BL/6 (black bars) and JHT(white bars) mice. (C) The frequencies of CD103+ Treg cells among CD45highCD4+Foxp3+ Treg cells in the CNS are shown. (D–F) Frequencies of (D) Foxp3+, (E) Foxp3+CD103, and (F) Foxp3+CD103+ Treg cells among CD45highCD4+ cells are shown. Data are shown as mean ± SEM of data pooled from three independent experiments with three mice per experiment for each time point. (G) Proliferation of Foxp3+CD103 and Foxp3+CD103+ Treg cells in the CNS was analyzed on day 21 after EAE induction 5 h after BrdU injection. (H) The suppressive capacity of CD45highCD4+GFP+ Treg cells from CNS of Foxp3-eGFP (black squares) and B-cell-deficient JHT ×Foxp3-eGFP (open triangles) mice on day 21 after EAE induction was assessed using CD4+CD25+ Treg cells from spleen and LNs of naïve C57BL/6 mice as controls (black circles). 2 × 104 CD4+CD25 T cells from naïve C57BL/6 mice were stimulated with 0.1 μg/mL anti-CD3 in the presence of 1 × 105 irradiated splenocytes and the indicated numbers of Treg cells. Proliferation was measured by incorporation of 3H-thymidine after 64 h. Data are shown as mean ± SEM of n = 3 replicates and are representative of three individual experiments. *p < 0.05, unpaired t-test.

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Figure 5. Remission from EAE in WT mice with reduced frequency of Foxp3+CD103 Treg cells. (A) C57BL/6 mice were treated with 200 μg anti-CD25 mAb (PC61), and LNs were analyzed at indicated time points to calculate changes in frequencies of CD103 (black circles) and CD103+ (black squares) Treg cells among CD4+ T cells relative to their values in untreated mice. Data are shown as mean ± SEM of data pooled from two independent experiments with three individual mice per group per experiment. (B–C) Frequencies of (B) CD103 and (C) CD103+ Treg cells among CD4+ T cells in LNs of naïve C57BL/6 mice, JHT mice, and C57BL/6 mice that received 200 μg anti-CD25 mAb (PC61) 30 days earlier. (D) EAE was induced in C57BL/6 mice (black squares), JHT mice (white triangles), and C57BL/6 mice treated with PC61 30 days earlier (gray circles). Data are shown as mean + SEM of data pooled from three independent experiments with six mice per group per experiment. (E) Fluorescence-activated cell sorter (FACS) strategy to isolate CD4+CD25+CD103 Treg cells and CD4+CD25 T cells from naïve C57BL/6 mice (spleen and LNs). Numbers indicate frequencies for each population in the FACS plots. (F) CD4+CD25 Tcells (gray triangles) or CD4+CD25+CD103 Treg cells (gray diamonds) were adoptively transferred (1.5 × 106 cells) into naïve JHT mice 4 h prior to EAE induction. Untreated C57BL/6 (black squares) and JHT (white triangles) were included as controls. Data are shown as mean ± SEM of data pooled from three independent experiments with three mice per group per experiment. ***p < 0.0001, unpaired t-test.

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Figure 6. Role of the effector CD4+ T-cell response on EAEdisease course in WT and B-cell-deficient mice. (A, B) Lymphocytes from pooled brains and spinal cords of C57BL/6 (black bars) and JHT (white bars) were isolated at the indicated time points after EAE induction and analyzed by flow cytometry. (A) Absolute numbers of CD4+Foxp3 Teff cells in the CNS of C57BL/6 (black bars) and JHT(white bars) mice at indicated time points are shown. Data are shown as mean ± SEM of data pooled from three independent experiments with three mice per group per time point for each experiment. (B) C57BL/6 (black bars) and JHT(white bars) mice were treated with 1 mg BrdU via the intraperitoneal route on day 21 after EAE induction, and frequencies of proliferating CD45+CD4+Foxp3 Teff cells in the CNS were analyzed 5 h later by flow cytometry. Data are shown as mean + SEM of n = 3 replicates and are representative of three individual experiments with six mice per group. (C) CD4+ cells were isolated from the CNSof C57BL/6 (black squares), and JHT(open triangles) mice on day 21 after EAEinduction. 2 × 104 CD4+ T cells were re-stimulated in vitro with MOG(35–55) in the presence of 1 × 105 irradiated splenocytes. CD4+ T cells from naïve C57BL/6 mice (black circles) were used as a negative control. Proliferation was measured after 64 h by 3H-thymidine incorporation. Data are shown as mean ± SEM of n = 3 replicates and are representative of three individual experiments. (D) EAE was induced in C57BL/6 and JHT mice. A group of C57BL/6 and JHT mice was treated intravenously (i.v.) with 200 μg anti-CD4 mAb (GK1.5) on days 19, 23, 27, and 31 (gray diamonds, and gray triangles, respectively). Another group of C57BL/6 and JHT mice was left untreated (black squares, and open triangles, respectively). (E) EAE was induced in JHT mice. A group of JHT mice was treated i.v. with 200 μg anti-CD4 mAb (GK1.5) on days 37, 41, 45, 49, and 53 (inverted gray triangles), and a control JHT group was untreated (white triangles). (D, E) Data are shown as mean ± SEM of data pooled from three independent experiments with six mice per group per experiment. *p < 0.05, unpaired t-test.

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Effector CD4+ T cells control the transition from acute-to-chronic EAEinB-cell-deficient mice

To precise further the mechanisms driving establishment and maintenance of chronic disease in B-cell-deficient mice, we examined the role of effector CD4+ T (Teff) cells in the two types of mice. First evidence for a stronger autoreactive CD4+ T-cell response in JHT mice was the higher numbers of Teff cells present in the CNS of these mice on day 15 after EAE induction compared with WT animals (Fig. 6A). Furthermore, Teff cells from CNS of JHT mice showed an enhanced proliferation in vivo on day 21 compared to cells from control mice (Fig. 6B). In addition, CNS CD4+ T cells from JHT mice made a stronger proliferative response to MOG (35–55) than cells from WT mice in vitro (Fig. 6C).

Given the finding that JHT mice have a stronger autoreactive CD4+ T-cell response in CNS than WT mice, we sought to address directly whether CD4+ T cells controlled the initiation of chronic EAE in JHT mice using a depleting anti-CD4 antibody. Treatment with this antibody on days 19, 23, 27, and 31 after immunization restored remission from disease in JHT mice, indicating that CD4+ T cells are necessary to initiate the transition from acute flare to chronic disability (Fig. 6D). In this respect, it was surprising that the number of CNS Teff cells similarly decreased in JHT and C57BL/6 mice between days 21 and 35 after EAE induction (Fig. 6A), suggesting that established EAE was then maintained independently of CD4+ T cells. Indeed, CD4+ T-cell depletion no longer ameliorated disease in JHT mice when started on day 35 postimmunization (Fig. 6E). Taken together, these data suggest that, in addition to Treg cells, B cells intercept the transition from acute-to-chronic pathology by limiting the encephalitogenic CD4+ T-cell response early on, which would otherwise ignite a chronic pathogenic process in the target organ.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and Methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Understanding how Treg cells are activated is of considerable interest given the role of these cells in maintenance of homeostasis and regulation of (auto) immune responses [[18]]. It is also important to investigate the possible functions of B cells in Treg-cell biology, and in autoimmunity, because BCDT is emerging as a treatment for autoimmune diseases. Here, we show that+ B-cell deficiency neither impaired Treg-cell activation in secondary lymphoid organs, nor their accumulation in CNS during EAE. A deficiency in B cells eventually resulted in a lower frequency of CD103 Treg cells in secondary lymphoid organs of adult mice, but this defect was insufficient per se to abrogate the process of remission from disease. Collectively, our findings indicate that Treg cells can be activated normally in absence of B cells, yet this might be insufficient to control disease, stressing the importance of complementary mechanisms of immune regulation provided by B cells.

B-cell-deficient mice had reduced frequencies of CD103 Treg cells in secondary lymphoid organs [[14, 19, 20]]. This defect was detectable in adult but not young animals, implying that B-cell deficiency had to persist for a sufficiently long time for the appearance of this phenotype. B cells could contribute to CD103 Treg-cell homeostasis by secreting IL-2 [[21]], or inducing IL-2 secretion from conventional T cells [[22]], because CD103 Treg cells are particularly dependent on IL-2 (they are more severely reduced than CD103+ Treg cells in IL-2-deficient mice) [[23]]. It is unlikely that B cells maintained CD103 Treg cells by production of TGF-β [[14]] because TGF-β is a major positive regulator of CD103 expression, so we would rather expect a defect in CD103+ Treg cells if this was the case [[24]]. B-cell deficiency did not affect the regulatory function of CD103 and CD103+ Treg cells from lymphoid organs in a steady state, as determined by their suppressive activity in vitro, and their expression of relevant T-cell surface receptors. We also found that B cells were not necessary for Treg-cell activation in secondary lymphoid organs after EAE induction in vivo. These findings indirectly support an important role for dendritic cells (DCs) in driving the Treg-cell response in JHT mice during EAE, which is consistent with the fact that DCs are more potent activators of Treg cells than B cells and macrophages in vitro [[25]], and DCs from immunized mice are stronger Treg-cell stimulators than DCs from naïve mice [[25]].

During organ-specific autoimmune diseases, Treg cells infiltrate the inflamed peripheral tissues to locally inhibit pathogenesis [[26]]. Our experiments did not provide evidence that B cells are required for Treg-cell accumulation in CNS during EAE. These results contrast with the findings from Mann and colleagues, who observed a transient reduction in Foxp3 mRNA expression in CNS of B-cell-deficient mice during EAE [[11]]. This might reflect differences in the protocols used to induce disease and to analyze CNS Treg-cell infiltration, or in the genetic background of the mice employed in the two studies. The proportion of CD103+ cells amongst Treg cells progressively increased in CNS during EAE. This enrichment could result from the fact that CD103+ Treg cells express higher amount of CCR6, which facilitates Treg-cell entry in CNS than CD103 cells [[27, 28]]. Alternatively, CD103 could facilitate retention of CD103+ Treg cells in CNS, as shown in models of skin inflammation [[29-31]]. Finally, some CD103 Treg cells may locally convert into CD103+ cells since TGF-β is expressed at areas of inflammation in CNS [[32, 33]]. In WT mice, the accumulation of CD103+ Treg cells in CNS was ultimately associated with recovery from disease, suggesting that CD103+ Treg cells might play an important role in resolution of local pathogenic inflammation in CNS. A similar accumulation of CD103+ Treg cells in CNS of JHT mice was insufficient to drive EAE resolution, implying that B cells provided a nonredundant mechanism of immune regulation.

Our data point to the notion that B cells facilitate recovery from EAE by regulating the encephalitogenic CD4+ T-cell response. CD4+ T cells display a stronger activation status in CNS of JHT than WT mice, and CD4+ T-cell depletion from day 19 on restored recovery from disease in JHT mice. Our results also indicate that at later stages (after day 35 here), disease persistence is not strictly dependent on CD4+ T cells, possibly due to irreversible CNS tissue damage or to a self-perpetuating pathogenic innate immune response in JHT mice. These findings might be relevant to RR-MS because treatment with Campath-1, which depletes T cells and other subsets of immunocytes, is beneficial when applied early but not late during RR-MS, suggesting that CD4+ T cells might contribute to early rather than late phase of RR-MS [[34]]. JHT mice might be useful to compare T-cell responses leading to reversible versus irreversible disability, and to investigate how disease can perpetuate independently of CD4+ T cells. JHT mice could also be useful to test novel therapeutic strategies for treating late stages of RR-MS, when Campath-1 is no longer effective. This model could for instance facilitate the development of novel approaches to repair immune-driven CNS tissue damage.

In conclusion, our data show that Treg-cell activation can proceed normally in the absence of B cells. This conclusion is supported by clinical studies, which showed that patients treated with BCDT did not experience reduction of Treg cells during the period of B-cell depletion, and some patients even showed increased Treg-cell numbers and frequencies in peripheral blood [[35, 36]]. BCDT is often beneficial in autoimmune diseases, yet in few cases it resulted in acute disease exacerbation [[37]]. An important goal of future research will be to identify how B cells can regulate autoimmune T-cell responses independently of Treg cells. B cells could directly affect pathogenic T cells via antigen presentation, or indirectly by promoting the development of other subsets of suppressor T cells such as mucosal-associated-invariant T cells, which are found in elevated amounts in CNS lesions of MS patients, or IL-10-producing Tr1 cells, which have protective functions during EAE [[38-42]]. Alternatively, B cells might inhibit T-cell responses indirectly by suppressing the innate immune system [[43-46]].

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and Methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Mice

Mice were bred under specific pathogen-free conditions at University of Edinburgh (Edinburgh, UK), Bundesinstitut für Risikobewertung (Berlin, Germany), and Centre d'Immunologie de Marseille-Luminy (Marseille, France). EAE was induced by subcutaneous immunization with 50 μg MOG (35–55) peptide in complete Freund's adjuvant (Sigma), with two intravenous (i.v.) injections of 240 ng Pertussis toxin (Sigma) (day 0 and 2), and assessed daily as previously described [[2]]. Experiments were performed according to UK, French, and German legislation.

Lymphocyte isolation from CNS

Mice were perfused with phosphate-buffered saline (PBS). Brain and spinal cord were then removed and digested with 1 mg/mL Collagenase (Worthington Biochemical) and 0.5 mg/mL Dnase (SIGMA-Aldrich) for 30 min at 37°C. Lymphocytes were obtained using a 30% Percoll gradient (GE Healthcare).

Flow cytometry

Crystallizable fragment (Fc) receptors were blocked using anti-FcγR monoclonal antibody (mAb) (clone: 2.4G2). Cells were then stained using mAb for CD45 (30-F11), CD4 (GK1.5), CD8 (53–6.7), CD25 (7D4), CD103 (M290), GITR (DTA-1), CD62L (MEL-14), and CTLA-4 (UC10–4F10–11) from BD Pharmingen (San Diego, CA), or produced in house. Foxp3 staining was done using mouse regulatory T-cell kit (FJK-16s, eBioscience, San Diego, CA, USA). Data were acquired on FACSCalibur (BD Biosciences) and analyzed using FlowJo software (Tree Star Inc.).

Analysis of in vivo Treg-cell proliferation

Mice received an intraperitoneal injection of 1 mg 5-Bromo-2′-deoxyuridine (BrdU), and were sacrificed 5 h later. Cells were stained with the BrdU Flow kit (BD Pharmingen), using the fix/perm buffer from the eBioscience Foxp3 staining kit. Briefly, after staining for surface antigens cells were fixed for 30 min at 4°C, permeabilized, subjected to an additional fixation step, and treated with deoxyribonuclease (Dnase) for 1 h at 37°C. Cells were then stained for BrdU and Foxp3 (FJK-16s, eBioscience) for 30 min at room temperature.

Purification of T-cell subsets by flow cytometry

CD4+ cells were pre-enriched using Dynal CD4+ cell negative isolation kit (Invitrogen). T-cell subsets were then sorted according to expression of CD45, CD4, CD25, and CD103 using FACSAria (BD Biosciences). Dead cells were excluded by propidium iodide (PI). CD45+CD4+GFP+ Treg cells were similarly isolated from CNS of Foxp3-eGFP reporter mice or B-cell-deficient JHT × Foxp3-eGFP mice. Purities were routinely above 95% for Treg-cell subsets and above 98% for CD4+CD25 cells.

In vitro suppression assay

2 × 104 CD4+CD25 T cells isolated from secondary lymphoid organs (spleen and LNs) from naïve C57BL/6 or JHT mice were stimulated in 96-well U-bottom plates with 0.1 μg/mL anti-CD3 (clone 145–2C11) together with 1 × 105 30 Gy-irradiated splenocytes, and increasing numbers of Treg cells as indicated. After 48 h, 1 μCi 3H-thymidine was added, and 3H-thymidine incorporation was measured 16 h later with a Top-count NXT liquid scintillation counter (Perkin Elmer). Data were calculated as percent suppression relative to proliferation of CD4+CD25 T cells in absence of Treg cells.

Antigen-specific CNS CD4+ T-cell re-stimulation

CD45+CD4+ T cells were isolated from CNS by flow cytometry. 2 × 104 CD4+ T cells were stimulated in 96-well U-bottom plates with MOG (35–55) and 1 × 105 irradiated splenocytes. After 48 h, 1 μCi 3H-thymidine was added to the cultures, and 3H-thymidine incorporation was measured 16 h later.

In vivo depletion of Treg-cell and CD4+ T cells

For Treg-cell depletion, C57BL/6 mice were treated by a single i.v. injection of 200 μg anti-CD25 mAb (PC61) in 200 μL PBS either 3 days or 30 days prior to EAE induction. To deplete CD4+ T cells from C57BL/6 and JHT mice during the course of EAE, mice were treated by repetitive i.v. injections of 200 μg anti-CD4 mAb (GK1.5) in 200 μL PBS as indicated.

Statistical analyses

Statistical analyses were performed with GraphPad Prism (GraphPad Software Inc.). *p < 0.05; **p < 0.01; ***p < 0.0001.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and Methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Financial support was from the Hertie-Stiftung, and the Deutsche Forschungsgemeinschaft (SFB-650, TRR-36).

Conflict of interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and Methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

The authors declare no financial interest or commercial conflict of interest.

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  6. Materials and Methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information
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Abbreviations
BCDT

B-cell depletion therapy

dLN

draining lymph node

MOG

myelin oligodendrocyte glycoprotein

RR-MS

relapsing-remitting multiple sclerosis

Teff

effector CD4+ T cell

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and Methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.

FilenameFormatSizeDescription
eji2248-sup-0001-figures1.pdf337K

Supporting Information Fig. 1. Gating strategy for Treg-cell frequency determination.

Supporting Information Fig. 2. Frequencies of Treg cells in secondary lymphoid organs of B-cell-deficient μMT mice.

Supporting Information Fig. 3. Frequencies of Treg cells in secondary lymphoid organs of B-cell-deficient and B-cell-sufficient littermate mice.

Supporting Information Fig. 4. Frequencies of Treg cells in secondary lymphoid organs of 2-week-old C57BL/6 and B-cell-deficient JHT mice.

Supporting Information Fig. 5. Proliferation data of in vitro suppression assay.

Supporting Information Fig. 6. In vitro suppression assay with responder T cells and Treg cells from naive C57BL/6 and JHT mice.

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