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# A fresh look at a neglected regulatory lineage: CD8+Foxp3+ Regulatory T cells

Adrian Liston, Meryem Aloulou

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## To cite this version:

Adrian Liston, Meryem Aloulou. A fresh look at a neglected regulatory lineage: CD8+Foxp3+ Regulatory T cells. Immunology Letters, 2022, 247, pp.22-26. 10.1016/j.imlet.2022.05.004. hal-03988581

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Submitted on 14 Feb 2023

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# A fresh look at a neglected regulatory lineage: CD8⁺Foxp3⁺ Regulatory T cells

Adrian Liston<sup>a, *,</sup> Meryem Aloulou<sup>a, b, *</sup>

## ARTICLE INFO

**Keywords:**

Foxp3

Treg

CD8 Treg

Immune regulation

## ABSTRACT

CD4⁺Foxp3⁺ Regulatory T cells (Tregs) are essential for maintaining self-tolerance and are increasingly recognised to have important roles in tissue homeostasis and repair. In the CD8 compartment an analogous Foxp3⁺ population is present, which shares phenotypic aspects with the more common CD4⁺Foxp3⁺Treg population. While oft neglected for their low frequency, there is increasing evidence that these CD8⁺Foxp3⁺ cells are bona fide regulatory cells, with both shared and distinct characteristics from their CD4⁺ analogue. Here we focus on the evidence for a regulatory function of CD8⁺Foxp3⁺ cells, and the potential unique role this neglected lineage may play in immune homeostasis and disease prevention.

## 1. Introduction

Of the multiple T cell lineages reported to have suppressive or regulatory properties, the majority of research has concentrated on the CD4⁺Foxp3⁺ regulatory T cell subset. The discovery of mutations in FOXP3 in severe lymphoproliferative autoimmune disease in humans (IPEX syndrome) [1] and mice (scurfy) [2], coupled with the identification of Foxp3 as the master regulator of the CD4⁺CD25⁺ suppressive population [3–6], provided a consistent phenotype and plethora of tools for studying this key subset. Since these discoveries, CD4⁺Foxp3⁺ Tregs have been intensively investigated and are now considered the archetypal regulatory lineage. By contrast, the CD8⁺Foxp3⁺population has remained poorly investigated, including their biology, origin, and, indeed, whether they have analogous suppressive functions. The key reason for this paucity of research is the extreme low frequency of CD8⁺Foxp3⁺ cells, ~0.1% of CD8⁺T cells in mice and ~0.3% in humans [7]. In this review, we will summarize the key findings to date and explore the knowledge gaps on CD8⁺Foxp3⁺ cells, in comparison to the CD4⁺Foxp3⁺ Treg counterpart.

### 1.1. CD8⁺Foxp3⁺ Treg identity

In CD4⁺ T cells, Foxp3 amplifies the molecular characteristics of the proto-Treg and stabilizes their identity [4,8]. Expression of functional Foxp3 protein in CD4⁺ Tregs is required for the sustained function as

regulatory cells, and competitive expansion [4,9]. Foxp3 does not, however, coordinate the entire Treg signature in CD4⁺T cells, with analysis of CD4⁺ Tregs with non-functional Foxp3 protein showing a partial retention of the transcriptional signatures and surface protein identity, features conferred through TCR engagement during Treg development in the thymus before Foxp3 expression [8]. The contribution of Foxp3 to the phenotype of the CD8⁺Foxp3⁺ lineage remains less clear. This question was partially addressed in mice that allow "wanna-be" Tregs to be followed, based on GFP expression in the absence of functional Foxp3 [10]. The CD8⁺Foxp3⁺GFP⁺ population was detected at similar frequencies to that of CD8⁺Foxp3⁺ cells in wildtype mice, suggesting that the expression of functional Foxp3 protein in CD8⁺Foxp3⁺ cells does not confer the competitive advantage observed in CD8⁺Foxp3⁺cells [11].

Fig. 1

### 1.2. CD8⁺Foxp3⁺ cell differentiation

The majority of CD4⁺Foxp3⁺ Tregs are produced by the thymus, and are selected through high-affinity interactions with self-peptide/MHC-II complex within the thymus [12,13]. This imparts a propensity for recognizing self-antigens in the periphery [14], a critical requirement for implementing immune regulation. Nonetheless, under specific conditions, Treg can also be induced in the periphery from naïve conventional CD4⁺T (CD4⁺Tconv) cells [15], and can even be specific for

<footnote><sup>a</sup> Babraham Research Campus, The Babraham Institute, Cambridge CB22 3AT, United Kingdom</footnote>

<footnote><sup>b</sup> Toulouse Institute for Infectious and Inflammatory Diseases (INFINITY), INSERM UMR1291, CNRS UMR, Toulouse 5051, France</footnote>

<footnote>* Corresponding authors at: Babraham Research Campus, The Babraham Institute, Cambridge CB22 3AT, United Kingdom.
E-mail addresses: adrian.liston@babraham.ac.uk (A. Liston), meryem.aloulou@babraham.ac.uk (M. Aloulou).</footnote>
<figure><img src="image_6.png" /><figcaption>**Fig. 1.** Key phenotypic markers for CD8⁺ and CD4⁺ Foxp3⁺ Tregs. A non-exhaustive schematic overview of molecules expressed by CD8⁺ and CD4⁺ Foxp3⁺ Tregs and variance on their level expression. MHC-I restricted CD8⁺Foxp3⁺ cells express high levels of CD103, CD122, NOX2 and Bim and low levels of IL2R, CTLA-4, GARP and Mcl1 compared to the MHC-II restricted CD4⁺Foxp3⁺ Tregs. Abbreviations–MHC–Major histocompatibility complex; TCR–T cell receptor; IL2R–Interleukin-2 receptor; CTLA-4–Cytotoxic T lymphocyte-associated antigen 4; Foxp3–Forkhead box P3; IL-2R–Interleukin-2 receptor; GITR–Glucocorticoid-induced tumor necrosis factor receptor; ICOS–Inducible T cell co-stimulator, IL7R–Interleukin-7 receptor; LAG-3–Lymphocyte activation gene-3; NOX2–Nicotinamide adenine dinucleotide phosphate oxidase 2; CXCR5–C-X-C chemokine receptor type 5; CCR7–C–C chemokine receptor type 7; GARP–Glycoprotein A repetitions predominant; TGF-β–Transforming growth factor beta; CCL4–Chemokine (C–C motif) ligands 4; IL-10–Interleukin 10; Mcl-1–Myeloid cell leukemia sequence 1; Bim–bcl-2-like protein 11.</figcaption></figure>

foreign epitopes [16–18]. Thus, while CD4⁺Foxp3⁺ Tregs have a distinct TCR repertoire, there is some overlap with conventional CD4⁺ cells [19, 20]. The origin and TCR repertoire on CD8⁺Foxp3⁺ cells, by contrast, have barely been investigated. While Foxp3⁺ cells are observed among single positive CD8 thymocytes [7,11], an exclusive thymic origin is not proposed; indeed, several studies have shown peripheral CD8⁺Foxp3⁺ cell generation in different contexts, such as for maintenance of peripheral tolerance to tissue-specific antigen expressed by either intestinal epithelial cells in the gut [21] or in transplanted tissue during bone marrow [22,23] or skin [24] transplantation.

*In vitro* and *in vivo* induced CD8⁺Foxp3⁺ cells have been generated by conventional CD8 T cell activation in the presence of TGF-β1 [11] or following TCR activation in the absence of optimal co-stimulation, both in human and mouse [21,24–30]. These approaches are similar to those used for generation of induced CD4⁺Foxp3⁺ Tregs, suggesting common, or overlapping, induction pathways. The similarity in induction response is also reflected in the methylation signature of *Foxp3*. The epigenetic regulation of the *Foxp3* locus allow the discrimination between the thymic and peripheral CD4⁺Foxp3⁺ Tregs [31] The stable Foxp3 expression on thymic CD4⁺Foxp3⁺ Tregs is achieved by epigenetic modulation of the Treg-specific demethylated region (TSDR), a non-coding sequence within the *Foxp3* gene locus. While the methylation status of TSDR haven't been investigated in thymic CD8⁺Foxp3⁺ cells, *in vitro*-induced CD8⁺Foxp3⁺ Tregs contain a fully methylated TSDR, comparable to induced CD4⁺Foxp3⁺ Tregs [11]. To these extents, the origin of CD4⁺ and CD8⁺ Tregs appear comparable.

Analysis of *MHCI*, *MHCII*, *β2m*- and *CD1d*-deficient mice demonstrates that the cells are bona fide MHCI-restricted CD8⁺ T cells [32].

However, the extremely low numbers of CD8⁺Foxp3⁺ cells have precluded deep investigation of the TCR repertoire in freshly *in vivo* isolated cells. To overcome their low number, studies have been performed in the context of experimental graft-versus-host disease (GVHD), known to promote conversion of conventional CD8 T cells into CD8⁺Foxp3⁺ cells, even greater than observed for induced CD4⁺Foxp3⁺ Tregs [22,23,33]. In this system the mesenteric lymph nodes develop as the anatomic site where CD8⁺Foxp3⁺ cells emerge under the influence of recipient dendritic cells and TGF-β [23]. The TCR repertoire of *in vivo*-generated induced CD8⁺ or CD4⁺ Foxp3⁺ Tregs sorted from this context show that CD8⁺Foxp3⁺ cells displayed greater TCR repertoire sharing with conventional CD8⁺ T cells than is observed between regulatory and conventional CD4⁺ T cells [26]. This suggests limited TCR distinctiveness in the CD8⁺Foxp3⁺ cell compartment, however, a key caveat is that this result may represent a feature of the particular inductive environment of GVHD, rather than a generalizable trait of CD8⁺Foxp3⁺ cells. The TCR repertoire of endogenous CD8⁺Foxp3⁺ cells, and the implications thereof for differentiation and function, remain one of the key unanswered questions (Box 1).

### 1.3. Evidence for suppressive function of CD8⁺Foxp3⁺ cells

Suppressive capacity is the most important functional trait of Tregs, making them a unique subtype distinct from other T cells. Based on several studies, Foxp3⁺CD8⁺ cells have validated suppressive functions. This feature has been reported for CD8⁺Foxp3⁺ cells from distinct origins, either naturally-derived or *in vivo* and *in vitro* induced cells. The comparison of *in vitro* suppressive capacity of murine CD8⁺ and CD4⁺Foxp3⁺ Tregs show either equivalent [7,22,32], or greater [23,26] efficacy compared to CD4⁺Foxp3⁺ Tregs, most likely dependent on the origin of CD8⁺Foxp3⁺ cells investigated.

CD8⁺Foxp3⁺ cells appear highly potent in exerting class I-restricted suppression. This suppression is likely mediated in an antigen-specific manner, as shown by superior *in vitro* suppressive capacity of induced CD8⁺Foxp3⁺ cells, *in vivo*-generated from bone-marrow transplanted mice, in the presence of stimulating dendritic cells bearing host MHCI. By contrast, CD4⁺Foxp3⁺ Tregs suppress proliferation to a similar degree, regardless of the stimulating dendritic cells [23]. Notably, the suppressive capacity of CD8⁺Foxp3⁺ cells appears to be dependent upon Foxp3, as silencing Foxp3 with small interfering RNA abrogated the *in vitro* ability to suppress anti-DNA antibody production and CD4⁺T cells proliferation in a lupus mouse model [30].

Similar to the murine results, human CD8⁺FOXP3⁺ cells exhibit suppressive function, whether tested as *in vivo* freshly isolated cells or *in vitro* induced cells. The first evidence of their suppressive function *in vitro* was described on a subset of single positive CD8⁺ thymocytes [34]. Peripheral CD8⁺FOXP3⁺ cells expanded from type 1 diabetics under low dose IL-2 [35] or Teplizumab, a humanized anti-CD3 antibody, treatments [25,27] display a suppressive activity to T cell responses against beta-cell antigen *in vitro*. Similarly, in multiple sclerosis patients, CD8⁺FOXP3⁺ cells from peripheral blood and cerebrospinal fluid recognize and *in vitro* suppress the autologous autoreactive CD4 T cells by activating Indoleamine 2,3-dioxygenase (IDO) activity and reducing co-stimulatory molecules on dendritic cells [36]. As in the murine context, this suppression is abrogated by FOXP3 silencing using siRNA [36]. In cancer, the human CD8⁺FOXP3⁺ cells isolated from prostate, ovarian or colorectal cancers display a potent *in vitro* suppressive function [37–39]. While the CD8 T cell compartment is largely known for its cytotoxic effector function, this mechanism has not been reported on CD8⁺FOXP3⁺ cells in most studies, except in *Mycobacterium bovis* bacillus Calmette–Guérin (BCG)-infected patients. In this context, CD8⁺FOXP3⁺ cells display cytotoxic function in addition to their suppressive function, killing BCG-infected autologous macrophages but not uninfected target cells, while also inhibiting T cell proliferation in an antigen-nonspecific manner [40]. CD8⁺FOXP3⁺ cells dominate the suppressive landscape, compared to CD4⁺FOXP3⁺ Tregs, after *in vitro*
live *Mycobacterium* bacillus Calmette–Guérin activation of human cells [41]. Together, these results demonstrate a suppressive function for CD8⁺FOXP3⁺ cells, in both mouse and human, consistent with a regulatory lineage, albeit one with distinct features from the archetypal CD4 Treg population.

## 1.4. Molecular mechanisms of suppressive function

CD4⁺FOXP3⁺ Tregs suppress using a variety of molecular mechanisms [42]. Analysis of the CD8⁺FOXP3⁺ cell transcriptome, using the *in vivo* induced GVHD model, demonstrated a more similar transcriptome profile to CD4⁺FOXP3⁺ Tregs than to conventional CD8⁺T cells [26], suggesting the potential for shared molecular mechanisms. *In vitro* assays suggest that CD8⁺FOXP3⁺ cells function mainly via contact-dependent processes [25,27,29]. Compared to CD4⁺FOXP3⁺ Tregs, CD8⁺FOXP3⁺ cells express equivalent GITR and CD127 lower CD25, CTLA-4 and GARP, and elevated CD103, CD122 and ICOS [7,22, 23]. Several of these molecules are biologically active, similar to CD4⁺FOXP3⁺ Tregs, with CD8⁺FOXP3⁺ cells recognized to confer immunosuppressive effects through both contact-dependent and independent mechanisms, including as CTLA4 [34], cytokines IL-10 and TGF-β [25] and cAMP [43]. However, the variance on the expression of these suppressive molecules between CD4⁺ and CD8⁺FOXP3⁺ cells suggest a differing pathway of function. NADPH Oxidase 2 (NOX2), essential for host defense and immunoregulation, has been reported in CCR7⁺CD8⁺FOXP3⁺ cells in human [43]. Interestingly, only a small proportion of CD4⁺FOXP3⁺ Tregs have a signal for NOX2 expression, making NOX2 a distinct CD8⁺FOXP3⁺ cell feature. NOX2 is particularly critical for the immunosuppressive function of human CCR7⁺CD8⁺FOXP3⁺ cells, where the release of exosomes containing NOX2 inhibit the expansion of the CD4 T cell by inhibiting TCR activation [43]. NOX2 expression was reported inversely correlating with age, underlying an age-related failure of CD8⁺FOXP3⁺ cell function [43]. Another potential mediator of suppression by CD8⁺FOXP3⁺ cells is CC chemokine ligand-4 (CCL4), identified in human granulomatous lesions of BCG-infected lymph nodes [40]. In this system, CCL4 was an effector molecule secreted by *ex vivo* LAG3⁺CD8⁺FOXP3⁺ cells following *in vitro* stimulation, with the addition of anti-CCL4 blocking antibodies inhibiting the observed suppression of T cell proliferation [40]. The relative importance of these different molecular mediators of suppression remains, however, undefined, and likely varies based on the biological context.

## 1.5. A putative role for CD8⁺FOXP3⁺ cells in disease

Key evidence for a role of CD8⁺FOXP3⁺ cells in disease is based on correlative data linking a change in frequency to disease state. CD8⁺FOXP3⁺ cell numbers are reported as reduced in immunological diseases such as multiple sclerosis [36,44], allergic asthma [45] and Giant Cell Arteritis [43,46], suggesting a role in tolerance induction. In Giant Cell Arteritis, a functional defect in the suppressive function of CD8⁺FOXP3⁺ cells is observed, and is related to excessive Notch4 signaling restricting NOX2-exosome release [46]. On the other side, elevated CD8⁺FOXP3⁺ frequencies have been reported in chronic infectious diseases, such as HIV [47], and various cancers [37–39,48–51], potentially aiding persistence of infection or limiting anti-tumor immunity. While a number of these associations may remain correlative rather than function in nature, the potential role for CD8⁺FOXP3⁺ cells in the pathophysiology of human disease has only just started to be explored.

## 1.6. Potential for therapeutic utility

The association of CD8⁺FOXP3⁺ cell-deficiency with multiple immunological disorders has spurred an interest in induced CD8⁺FOXP3⁺ cells as a potential cell therapy. Human CD8⁺FOXP3⁺

cells are induced *in vitro* in the presence of TGF-β and rapamycin along with anti-CD3/28 and IL-2. These cells have *in vitro* suppressive activity, with stable inhibitory ability in presence of pro-inflammatory cytokines [52]. Functionally-active CD8⁺FOXP3⁺ cells can also be induced through coculture with allogeneic CD40-activated B cells [53]. A likely candidate for adoptive cell transfer of induced CD8⁺FOXP3⁺ cells is GVHD, based on the extensive *in vivo* functionality observed in this model. Bone-marrow transplantation induces *in vivo* CD8⁺FOXP3⁺ cell induction, which are able to prevent GVHD mortality at least as efficiently as induced CD4⁺FOXP3⁺ Tregs [22,26], with synergistic effects observed between the two [33]. Similar effects are observed through the adoptive cell transfer of in vitro-induced CD8⁺FOXP3⁺ cells [53]. A key advantage of CD8⁺FOXP3⁺ cells over CD4⁺FOXP3⁺ Tregs in this context may be the superior preservation of the graft-versus-leukaemia effect [53–55]. Efficacy of adoptive cell transfer of CD8⁺FOXP3⁺ cells may be limited by instability under inflammatory contexts [55], however, this can be countered to an extent through the addition of vitamin C to *in vitro* CD8⁺FOXP3⁺ polarizing conditions, which increase DNA methylation of the *Foxp3* locus [55]. These results suggest that CD8⁺FOXP3⁺ cell therapy may be a useful therapeutic approach to counter GVHD while preserving graft-versus-leukaemia.

## 1.7. Homeostasis of CD8⁺FOXP3⁺ cells

A key feature of the CD8⁺FOXP3⁺ population is the relative scarcity compared to the more plentiful CD4⁺ counterpart. In part, this may be due to the increased expression of Bim, an apoptotic molecule, compared to CD4⁺FOXP3⁺ Tregs [26]. The relative expression of the pro-survival and apoptotic proteins, Mcl1 and Bim, respectively, is a key contributor to the homeostatic set-point of CD4⁺FOXP3⁺Treg frequency [56]. This ratio is tipped towards a pro-proliferative and anti-apoptotic state by IL-2 receptor signaling in CD4⁺FOXP3⁺ Tregs [56]. CD8⁺FOXP3⁺ cells, by contrast, have increased expression of Bim and reduced expression of Mcl1, with no difference observed in the expression of other regulator of cell death such as Bcl2, Puma, Bad, Bak, or Caspase 3 [7,26]. The apparent conversation of the IL2-Mcl1-Bim axis across CD4⁺ and CD8⁺FOXP3⁺ cells raise the question of why CD8⁺FOXP3⁺ cells remain so rare. One explanation would be relative insensitivity of CD8⁺FOXP3⁺ cells to IL2. However, CD8⁺FOXP3⁺ cells express the high affinity trimeric IL2 receptor, and *in vitro* have only slightly lower sensitivity to IL2 than CD4⁺FOXP3⁺ Tregs, being 20- to 40-fold more sensitive than conventional CD8⁺T cells [7]. The 2-4-fold reduced sensitivity of CD8⁺FOXP3⁺ T cells to IL2 compared to their CD4⁺FOXP3⁺ Treg counterpart correlates with two-fold lower levels of basal CD25 expression [7]. However, clinical trials using low dose IL2 therapy demonstrate successful expansion of both CD4⁺ and CD8⁺FOXP3⁺ Tregs in humans and mice, demonstrating the capacity of exogenous IL2 to drive homeostatic expansion despite the slightly lower CD25 expression [7,35,57]. Recent experiments may shed light on this apparent discordance. A transgenic system for inducing cell lineage-specific IL-2 expression has allowed rewiring of IL2 production to diverse leukocyte sources [32]. Using this system, we found that IL2 sourced from B cells, using *Cd19CreRosall2* mice, induced dramatic expansion of CD8⁺FOXP3⁺ Tregs but not CD4⁺FOXP3⁺ Tregs, while IL2 sourced from T cells or dendritic cells drove the reverse expansion. Further investigation of CD8⁺FOXP3⁺ cells showed enrichment for CXCR5 and PD1 expression, a phenotype shared with follicular helper CD4 T (Tfh) cells [58], and anatomical enrichment in the B cell zone at steady state [32]. This suggests that CD8⁺FOXP3⁺ cells, being excluded from the IL2 produced in T cell zones, are kept in IL2 starvation-mediated scarcity in the homeostatic state. Notably, both clinical IL2 injection and *Cd19CreRosall2* mice result in parallel expansion of both CD8⁺FOXP3⁺ cells and the ILC2-eosinophil axis, suggesting that exogenous IL2 voids the anatomical segregation of endogenous T cell-derived IL2, allowing both populations to expand. While T cells are major producer of IL2 during homeostasis, IL2-producing B cells arise in
both humans [59] and mice [60,61] in certain contexts. This anatomical colocalisation of CD8⁺Foxp3⁺ cells, and a paucity derived from B cell-dependency, may provide a tantalizing clue towards a potential divergence in suppressor function between the CD4⁺ and CD8⁺ Foxp3⁺ lineages.

## 2. Conclusion

Intensive research over the last decades has focused on the CD4⁺Foxp3⁺ Tregs, the archetypic of Regulatory T cells. CD8⁺Foxp3⁺ cells, the analogous Treg subset in the CD8 compartment, has, by contrast, remained neglected, with many fundamental questions remaining unanswered (Box 1). Even the treatment as a single lineage may be in doubt, as CD8⁺Foxp3⁺ cells display different phenotypes when profiled in different contexts. Just in the cancer field alone, CD8⁺FOXP3⁺ cells are identified as CD25⁺CTLA-4⁺CD28⁻ in ovarian cancer [38], PD-1⁻CD127⁻KLRG1⁻ in lung cancer [51], HLA-DR⁺CD38⁺T-bet⁺perforin⁺ in melanoma [48], PD-1⁺CTLA-4⁺TIM-3⁺LAG-3⁺ in cervical cancer [49], CD45RA⁻CD27⁻ gastric cancer [62] and CLTA-4⁺GITR⁺TGF-β⁺ in colorectal cancer [39]. Despite this, there is an emerging consensus of data that these cells form a bona fide regulatory lineage, with suppressive capacity akin in magnitude to the CD4⁺Foxp3⁺ Tregs. While much lower in frequency than the CD4⁺Foxp3⁺ Treg population, dominant suppressive roles for Foxp3⁺CD8⁺ cells can emerge, either through a need for specialised suppressive mechanisms or through the selective expansion under particular biological contexts. This regulatory niche allows the potential utility of CD8⁺FOXP3⁺ cells in a therapeutic context. Indeed, several efficacious treatments, including low-dose IL2 [7,35,57] and Teplizumab [25,27] expand CD8⁺FOXP3⁺ cell numbers, and may work, in part, through this effect. The future clinical testing of *in vitro*-expanded CD8⁺FOXP3⁺ cells may unlock new and innovative uses of this enigmatic population.

Box 1: Key unanswered questions for CD8⁺Foxp3⁺cells

* Are CD8⁺Foxp3⁺cells a distinct and stable lineage of CD8⁺T cells?

* What defines CD8⁺Foxp3⁺cell stability?

* Do CD8⁺Foxp3⁺cells and CD8 Tconv cells share the same TCR repertoire?

* Do CD8⁺Foxp3⁺cells recognize self, non-self or both?

* What is the role of TCR specificity in CD8⁺Foxp3⁺cell development and function?

* Which suppressive mechanisms are used by CD8⁺Foxp3⁺cells?

* Do CD8⁺Foxp3⁺cells control immune response through MHC-I-restricted mechanism?

* What is the relationship between CD8⁺Foxp3⁺cells and B cells?

* Where do the key suppressive functions of CD8⁺Foxp3⁺cells occur?

* What physiological reactions did CD8⁺Foxp3⁺cells evolve to control?

## Conflicts of interest

The authors declare no conflict of interests.

## Acknowledgments

This project was supported by the Biotechnology and Biological Sciences Research Council through Institute Strategic Program Grant funding BBS/E/B/000C0427 and BBS/E/B/000C0428. M.A. was supported by the French MS society (ARSEP).

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