Mobilized Multipotent Hematopoietic Progenitors Stabilize and Expand Regulatory T Cells to Protect Against Autoimmune Encephalomyelitis

Achieving immunoregulation via in vivo expansion of Foxp3+ regulatory CD4+ T cells (Treg) remains challenging. We have shown that mobilization confers to multipotent hematopoietic progenitors (MPPs) the capacity to enhance Treg proliferation. Transcriptomic analysis of Tregs co-cultured with MPPs revealed enhanced expression of genes stabilizing the suppressive function of Tregs as well as the activation of IL-1β–driven pathways. Adoptive transfer of only 25,000 MPPs effectively reduced the development of experimental autoimmune encephalomyelitis (EAE), a pre-clinical model for multiple sclerosis (MS). Production of the pathogenic cytokines IL-17 and GM-CSF by spinal cord-derived CD4+ T-cells in MPP-protected recipients was reduced while Treg expansion was enhanced. Treg depletion once protection by MPPs was established, triggered disease relapse to the same level as in EAE mice without MPP injection. The key role of IL-1β was further confirmed in vivo by the lack of protection against EAE in recipients of IL-1β–deficient MPPs. Mobilized MPPs may thus be worth considering for cell therapy of MS either per se or for enrichment of HSC grafts in autologous bone marrow transplantation already implemented in patients with severe refractory multiple sclerosis.


INTRODUCTION
Autologous transplantation of hematopoietic progenitors (HSCs) is being performed in patients with severe, refractory, autoimmune diseases and singularly, multiple sclerosis (1)(2)(3)(4). Although beneficial effects of the procedure have been ascribed to the resetting of a naïve, non-activated immune system, as well as a rebound of diverse regulatory cell subsets (5), the hypothesis that selective HSC subsets might exert an active immunoregulatory role should not be neglected.
Hematopoietic stem cells and progenitors are present in the bone marrow (BM) at various stages of differentiation. Longterm HSC (LT-HSC) and short-term HSC (ST-HSC) are e n d o w e d w i t h s e l f -r e n e w i n g p o t e n t i a l a n d u p o n transplantation replenish on the long-term or on the shortterm, respectively, the entire blood system. They give rise to a series of multipotent hematopoietic progenitors (MPP) with decreasing self-renewal capacity, that differentiate toward committed progenitors and more mature cells of the various hematopoietic lineages. Four MPP subsets have been identified (MPP1-4) that, in addition to their c-kit and Sca-1 expression shared with LT-and ST-HSCs, can be distinguished immunophenotypically by the gain of expression of CD34, CD48, and CD135 (Flt3), the Flt3-L receptor and loss of CD150 expression (6).
Taking into consideration that HSCs are increasingly isolated from the peripheral blood after mobilization, the possibility that mobilized HSCs exhibit immunoregulatory properties was explored. Using a cocktail of hematopoietic growth factors composed of G-CSF and Flt3-ligand (Flt3-L) for a synergistic HSC mobilization from the BM to the periphery, we could show previously that MPP were enriched by mobilization and acquired the capacity to enhance the proliferation of TCR-activated Foxp3 + Tregs (7,8). The promotion of Treg expansion also took place in vivo, and accounted for the protection against spontaneous type 1 diabetes (TID) in Non Obese Diabetic (NOD) mice induced by adoptive transfer of as few as 10,000 mobilized MPP per mouse. Both contact-and soluble factordependent mechanisms (7,8) were involved in this acquired property of mobilized MPP.
Tregs play an important role in the control of tolerance in multiple sclerosis as well as in its animal model of experimental autoimmune encephalomyelitis (EAE). Considerable efforts are being produced aiming at promoting expansion of Tregs in vitro with IL-2, rapamycin, activation with anti-CD3/CD28 mAbcoated beads (9,10) or with minute foreign antigen doses (11), for subsequent administration to patients with autoimmune diseases. Notably, Treg cell therapy may require billions of cells (12). Promoting the expansion of Tregs directly in vivo thus represents a therapeutic strategy worth of interest. Interleukin-2 (IL-2) at low dose has been demonstrated to expand preferentially Treg and numerous trials in a host of clinical settings are underway (13). However, it remains interesting to develop alternative strategies susceptible to confer highly selective expansion of Treg with no expansion of effector T-cells. We therefore investigated whether adoptive transfer of mobilized MPP could be used to protect against EAE by selectively promoting in vivo Treg expansion.
We herein report that MPP promote Treg proliferation and survival both in vitro and in vivo. Transcriptomic analysis demonstrated that Treg co-cultured with MPP display enhanced expression of several genes contributing to the stabilization of their regulatory function, reduced apoptosis and enhanced plasticity enabling them to effectively control neuro-inflammation. Adoptively transferred mobilized MPP effectively reduce the EAE score by an IL-1b-driven mechanism. Furthermore, the key role of Treg in the protection against EAE conferred by MPPs was demonstrated by disease relapse occurring upon depletion of Treg undertaken once protection was established. Therefore, in vivo expansion of functional Treg can be efficiently induced by MPP, and MPPbased cell therapy could represent a therapeutic strategy against autoimmune diseases either per se or as a an enrichment of autologous HSCT, already implemented in patients with severe multiple sclerosis (1)(2)(3)(4)(5).

Mice
Wild type C57BL/6J, C57BL/6 Foxp3-GFP-KI, and C57BL/6 IL-1b −/− (obtained from CDTA, Orleáns, France) mice were bred in our animal facility under specific pathogen-free conditions. Live animal experiments were conducted according to the EU Directive 2010/63/EU for animal experiments under an animal study proposal approved by the Paris Descartes University Ethical Committee for Animal Experimentation and the French Ministry of Research and Higher Education, number 3846-2015070622031545v4.

EAE Induction
Active EAE was induced in 10-to 12-week-old female mice by s.c. immunization at two sites, upper back and lower back, with 200 mg myelin oligodendrocyte glycoprotein (MOG)  peptide (MEVGWYRSPFSRVVHLYRNGK) emulsified in CFA containing 400 mg heat-killed Mycobacterium tuberculosis H37Ra (Hooke Laboratories, Lawrence, MA, USA), on day 0. Additionally, mice received 300 ng pertussis toxin (Hooke Laboratories, Lawrence, MA, USA) i.p. in 0.1 ml/mouse on days 0 and 1. Clinical signs of EAE were assessed daily with a 0-to 5-point scoring system, as follows: 0, no obvious changes in motor function compared to non-immunized mice; 0.5, tip of tail is limp; 1, limp tail; 1.5, limp tail and hind leg inhibition; 2, limp tail and weakness of hind legs; 2.5, limp tail and dragging of hind legs; 3, limp tail and complete paralysis of hind legs or paralysis of one front and one hind leg; 3.5, limp tail and complete paralysis of hind legs, in addition to: mouse is moving around the cage, but when placed to its side, is unable to right itself; 4, limp tail complete hind leg and partial front leg paralysis; 4.5, complete hind leg and partial front leg paralysis, no movement around the cage, mouse is not alert; 5, mouse does not move any more in the cage. Mice with score ≥4 for two consecutive days and mice with score 5 were euthanized. Disease scores over the course of the 35-d experiments were totalized for each animal, and the mean for the experimental group was expressed as a cumulative EAE score. injected s.c. for four consecutive days with human recombinant G-CSF (200 mg/kg/day) (Zarzio 48 MU/0.5 ml, Sandoz) and recombinant murine Flt3L (20 mg/kg/day) (Immunotools, Friesoythe, Germany). Total splenocytes were magnetically sorted for c-kit + cells with an automated magnetic cell sorter (Robosep, StemCell Technologies, Vancouver, BC, Canada), further stained with the mAbs directed against CD34 (BD Biosciences, Le Pont de Claix, France), Sca-1 (anti-Ly6A/E) and CD11b (eBioscience, ThermoFisher Scientific, Illkirch, France), and electronically sorted into c-kit h i g h Sca-1 high CD34 + CD11b -/low cells with the FACS Aria II cell sorter (BD Biosciences). Each mouse received intravenously 25,000 cells of the above subset at the same day of immunization with MOG 35-55 /CFA. Intravenous treatment with anti-CD25 mAb (PC61) (200 mg/ml) or the control isotype antibody was performed at day 17 of the disease, once protection by MPP was established.

Isolation of Immune Cells from the Spinal Cord
Spinal cord isolated from control and MPP-recipient mice were incubated for 30 min in digestion buffer of DNAse and Liberase (27 WU/ml) in PBS 1× at 37°C, mixing every 5 min. EDTA (500 ml, 100 mM) was added for 1 min to end the digestion. Cells were passed through a 100 mm cell strainer, using a syringe plunger (back side) to smash the tissue. A Percoll separation was performed resuspending the cells first in 3 to 5 ml of 40% Percoll and underlayed with the same volume of 70% Percoll (in PBS) followed by centrifugation for 35 min at 1300g (2800 rpm) without brake. Cells at the interface were collected with a Pasteur pipette and diluted 10 times with complete medium RPMI 10% fetal cell serum (FCS), centrifuged and resuspended in 2 to 3 ml of complete medium.

Microarray Experiment
To analyze the possible mechanisms involved in mouse CD4 + CD25 high regulatory T-cells (Treg cells) expansion promoted by mobilized MPP (MPP), we evaluated the transcriptomes of activated Treg extracted from simple culture (Treg control group) or from co-culture with MPPs (Treg + MPP). Treg and MPP were sorted as previously described. Treg were cultivated for 3 days with anti-CD3 and anti-CD28 alone or together with freshly purified MPP. After 3 days, CD4 + cells (Treg) were sorted for extraction of RNA using RNeasy Micro kit (QIAGEN). Its quality was verified in an Agilent Bioanalyzer. Total RNA was amplified and converted to biotinylated cRNA according to the manufacturer's protocol (Illumina TotalPrep RNA Amplification Kit; Ambion). Paired biological replicates (3 for each group) were hybridized to the Sentrix BeadChips Array mouse WG-6 v2 (Illumina) and gene expression analysis was performed using GeneChip Mouse Genome 430 2.0 arrays (Affymetrix), as recommended by the manufacturer.

Transcriptomics
Fluorescence values corresponding to raw expression data for each 6 samples were extracted from CEL files using the R oligo package (https://bioconductor.org/) with the corresponding platform definitions (pd.mouse430.2). Probe annotations were added using affycoretools (Bioconductor) with the mouse4302.db database. Internal positive and negative controls, and ambiguous or unknown probes were removed, which left 39.444 probes, corresponding to 21,108 unique and well-annotated genes. Briefly, quality control steps, data normalization, unsupervised explorations and functional annotations were conducted as described previously [https:// doi.org/10.1007/s12035-018-1128-3]. Statistical analyses were achieved using linear modeling with empirical Bayes, p values were computed by applying a moderated two-way t-test and adjusted for false discovery rate (FDR) following the Benjamini-Hochberg procedure. Hierarchical clustering heat maps were obtained on gene-median-centered data with uncentered correlation as similarity metric. Volcano plot were rendered using EnhancedVolcano (Bioconductor). Additional pathway analyses were performed with the Reactome platform (https:// reactome.org) and with ReactomePA (Bioconductor) for overrepresentation tests in mouse. Target enrichment were obtained with EnrichR [https://doi.org/10.1093/nar/gkw377]. For all experiments, an FDR or q-value < 0.05 indicated statistical significance.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism (GraphPad Software, La Jolla, CA). Disease curves were analyzed using two-way ANOVA test, with Bonferroni posttest. Cell proportions were analyzed using one way ANOVA with Bonferroni or Tukey's post-test. Data are shown as mean ± s.e.m. P < 0.05 was considered statistically significant.

Mobilized MPP Characterization
C-kit + spleen cells derived from mobilized C57Bl/6 mice were sorted for c-kit hi Sca-1 hi CD34 + CD11b − cells ( Figure 1A). Using SLAM markers (14), FACS analysis revealed that 80% of the mobilized sorted cells displayed a CD48 + CD150 − CD135 − (Flt3 − ) phenotype ( Figure 1B), and thereby corresponded to the described MPP3 subset, reported to be prone to myeloid differentiation (15). Reflecting the continuum of the MPP differentiation process, approximately 20% CD150 + cells were detectable among the mobilized c-kit + Sca-1 + CD34 + CD11b − cells, most probably corresponding to progenitors at the MPP2 (CD48 + CD150 + CD135 − ) stage. To assess the capacity of the sorted mobilized progenitors to differentiate into multiple hematopoietic lineages, in keeping with their MPP phenotype, we cultured them on OP-9 and OP-9D4 stromal cells, the latter permitting differentiation into the T-cell lineage that requires Notch pathway stimulation conferred by the expression of the Notch ligand Delta-4. A myeloid differentiation bias was also  noted upon in vitro differentiation of the mobilized MPP. Yet, their differentiation potential into both myeloid and lymphoid hematopoietic lineages remained detectable ( Figure 1C).

Mobilized MPP Enhance Treg Proliferation: Transcriptomic Analysis
Treg sorted from Foxp3-GFP-KI mice and co-cultured in vitro at 1:1 ratio with MPP showed enhanced proliferation, assessed by CFSE dilution over 4 days in response to stimulation by anti-CD3/anti-CD28, compared to Treg cultured alone ( Figure 2A). Furthermore, MPP did not increase the proliferation of activated CD4 + CD25 − cells ( Figure 2B). Thus, MPP specifically increased the in vitro expansion of TCR-activated Treg cells. Neutralization assays demonstrated that the molecular pathways (Jagged-Notch3 and GM-CSF-CD116) previously shown to be implicated in the NOD mouse (7,8) were not implicated in the C57BL/6 strain, nor were TGF-b, IL-10, CD80, CD86, CD40, OX40, ICOS, or GITR on co-cultures, as neutralizing antibodies to these molecules had no significant effect on Treg survival and proliferation. These data raised the question about the mechanism used by MPPs to promote Treg expansion. We therefore performed microarray experiments on sorted Treg after incubation with anti-CD3/anti-CD28 either alone (Treg; N=3) or in presence of MPP during 4 days (Treg+MPP; N=3).
Resulting transcriptomes finally constituted a curated dataset including 39,444 probes, corresponding to 21,108 unique genes. Unsupervised K-means clustering clearly delineated two groups of samples, with Treg on one hand and Treg+MPP on the other ( Figure 3A). These presented with a strongly correlated gene expression profile, divided in two clusters of 4,035 and 4,247 unique genes, down-(cluster c8) and upregulated (cluster c9) in Treg+MPP, respectively. The two dysregulated clusters represented a wide proportion (39.2%) of the whole transcriptome, with a significant differential expression between the two groups ( Figure 3B), thus driving the sample classification. A compilation of the most relevant functional annotations, pathways and transcription factor targets (with GO, Reactome and EnrichR, respectively) were dissected for these two clusters ( Figure 3C). Under-expressed genes in Treg+MPP (cluster c8) were mainly involved in the positive regulation of apoptosis, the Wnt calcium signaling pathway (non-canonical), or in TCR binding and signaling. The cell cycle, especially the replication during the S phase, was also found decreased, as well as the establishment of the immunological synapse through focal adhesion and adherens junction (all FDR < 0.05; Supplementary Table S1). This cluster presented with a large number of genes that are transcription targets of FOXO1 (n=530; q-value = 6.1e-22), an important regulator of cell death acting downstream of CDK1, PKB/AKT1, and STK4/MST1, and was also enriched in targets of FOXP3 (n = 55; q-value = 2.1e-4). Conversely, main over-expressed functions and pathways in Treg+MPP (from cluster c9) included the MHC complex, acute inflammatory response and cytokine-mediated signaling, TNFa on cytokine activity and cytokine-cytokine receptor interaction. Positive T cell differentiation, T cell activation and T cell mediated cytotoxicity were also increased  Figure 1A and Tregs were isolated from Foxp3 eGFP mice as CD4 + CD25 + Foxp3 (GFP) + cells and loaded with Cell-Trace. Tregs were stimulated for 4 days with anti-CD3/anti-CD28 in the absence or presence of MPP at a 1:1 ratio. Cell Trace incorporation was measured in the CD4 + CD25 + gate. (B) Lack of effect of MPP co-cultured with CFSE-incorporated CD4 + CD25conventional T cells (ratio 1:1). ns: Not statistically significant by Student's t-test.
in Tregs cultured with MPPs, as well as TCR negative regulation of apoptosis via IL-2 signaling. Glycolysis was also upregulated (Supplementary Table S2). Transcription targets of MYB, which prevents the differentiation of Tregs into effector T cells (16), and whose deletion leads to fatal immune pathology, were highly enriched in this cluster (n = 284; q-value = 8.7e-10).
In brief, Treg exposed to MPP harbored a "specialized" molecular signature, with a stabilized phenotype Cd4 high , Il2ra high , Foxp3 high , Il7ra−, Ctla4+, Tnfrsf18+, Ikzf2+, Tnfrsf4+, Itgae+, under the transcriptional regulation of increased Satb1, Runx2, Runx3, and Gata3 gene products, switching from FOXO control, with decreased gene expression in Treg+MPP. Moreover, they exhibit enhanced expression of Prdm1 (+1.3-fold; p-value=6e-3), recently reported to prevent methylation of Foxp3 within Treg in central nervous system inflammation (19), and Nr4a (+1.45-fold; p-value = 9e-5), that stabilizes Treg against their differentiation into effector T-cells (20), which is further confirmed by the unchanged Foxp3 expression observed here after contact with MPP. Treg survival is also improved with a finely controlled and reduced apoptosis by means of TCR signaling in response to increased IL-2, the inhibition of NF-kB pathway via the increase of TNFa activity, the FOXO-mediated transcription, and through p21 (Cdkn1a) cell replication inhibiting activity, which has been shown to ultimately promote Treg proliferation through enhanced anti-apoptotic control (21).
MPP exposed Treg also displayed molecular pathways activated by several cytokines (IL1, IL4, IL13, IL10, IL12) and several chemokines acting via STAT proteins (Figure 3; Supplementary Tables S1-S3; Supplementary Figures S1,  S2). For a deeper understanding on IL-1b/TNFa−mediated inflammatory responses, we thus compiled a list of 51 genes involved and interacting with these pathways (from SigDB curated gene sets C2; https://www.gsea-msigdb.org/gsea/ msigdb) along with top correlated genes extracted from gene dendrograms obtained from hierarchical clustering of each down-regulated and upregulated K-means clusters c8 and c9, respectively. Further hierarchical clustering of these 51 genes unravels two clusters of strongly correlated expression patterns, one down-regulated, and one strongly upregulated (up to 20fold), representing an inflammatory-specific signature in Treg +MPP, completely in line with Il1b expression, but not Tnfrsf1a ( Figure 4A). Among the genes dependent of the IL-1b pathways with enhanced expression in Treg co-cultured with MPP were Ifng (IFN-g), Tbx21 and Ccl5 genes. In addition, proinflammatory Myd88 was upregulated and highly correlated with the inflammation signature.
Inflammation also affects hematopoietic progenitors. Stress hematopoiesis, triggered upon mobilization with G-CSF, prompts copious inflammatory cytokine production by MPP, including IL-1b (22), IL-1b expression is enhanced approximately 20 fold in peripheral blood CD34 + cells compared to BM CD34 + cells (23). The role of MPP-derived IL-1b in the promotion of Treg proliferation was confirmed by the reduction of the expansion effect provided by MPP onto Treg in the presence of a neutralizing antibody to IL-1b ( Figure 4B). Moreover, efficiency of MPP isolated from IL-1b KO mice (prepared as in Supplementary Figure S3) in enhancing Treg proliferation in vitro was reduced relative to WT MPP ( Figure  4C). Nevertheless, additional factors besides IL-1b are presumably implicated as the enhanced proliferation effect of MPP was not totally abolished in the absence or the neutralization of this cytokine.

Role of IL-1b
To investigate whether MPP can expand Treg in vivo and thereby confer protection against immune inflammatory diseases, we chose a Th1/Th17 cell-driven animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). We performed the adoptive transfer of as few as 25,000 mobilized MPP per mouse, cell-sorted as shown in Figure 1A, at day 0 of immunization with the myelin oligodendrocyte glycoprotein peptide (MOG  ) according to the administration protocol outlined in Figure 5A. MPP isolated from WT donors, but not from IL-1b-deficient donors (prepared as in Supplementary  Figure S3), significantly reduced the clinical scores of EAE, relative to mice injected only with PBS ( Figure 5B). Accordingly, both in the periphery (lymph nodes) and in the CNS (spinal cord) of MPP recipients, the CD4 + T-cell production of the major pathogenic cytokines, IL-17, IFN-g and GM-CSF measured at the peak of the disease (day 21) was reduced ( Figures 5C, D), again only in recipients of WT but not of IL-1 b-deficient MPP ( Figure 5B). These data confirm the key role of this cytokine as well in the in vivo protection by MPP against the disease.

Role of IL-1b
Percentages of Foxp3 + Treg were enhanced in gated CD4 + cells in lymph nodes and spinal cord of MPP recipient mice compared to control mice ( Figures 5E, F). Treg proliferation, measured by nuclear staining of Ki67 within gated Foxp3 + CD4 + cells at the peak of the disease tended to be enhanced in the periphery as well as in the spinal cord of MPP recipients relative to control mice with EAE, however without reaching statistical significance ( Figures 5G, H). This in vivo effect on Treg was also IL-1b dependent as, contrary to recipients of WT MPP, recipients of IL-1b KO MPP showed no increase in the percentages nor in the Ki67 expression of Treg in their spinal cord, compared to noninjected EAE controls (( Figures 5E-H).

The Protection Against EAE Conferred by Adoptively Transferred MPP, Once Established, Can Be Abrogated by Treg Depletion
To assess whether Treg that accumulated in MPP recipients played a non-redundant role in the observed protection against disease, MPP recipient mice with established protection were divided into two separate groups (n = 5 mice per group) that received one single injection at day 17 either of the anti-CD25 PC61 mAb (250 mg/mouse, i.p.), which depletes CD25 hi Treg cells or of a control isotype mAb ( Figure 6A). While the same treatments applied to control EAE undergoing mice did not change significantly the disease scores, three days after the mAb injection, disease relapse was observed in the PC61-injected group of MPP recipients which by d25 reached the clinical score of the control EAE group ( Figure 6B). Conversely, the MPP-recipient group injected with the same amount of an isotype mAb did not significantly change its mean clinical score, and remained protected against EAE till the end of the experiment (d30). Therefore, Treg play a key role in the protection conferred by adoptively transferred MPP since their removal, after protection is established, triggers disease relapse.

DISCUSSION
Mobilized hematopoietic progenitor subsets at the developmental stage of MPP3 as defined by SLAM markers, that remain multipotent although with a clear myeloid developmental bias, displayed the remarkable property to promote the expansion of TCR-activated Treg, without affecting the proliferation of CD4 + CD25cells. The functional outcomes of this property were highly significant, since as few as 25,000 mobilized MPP cells adoptively transferred per recipient conferred protection against EAE, and this protection was highly dependent on Treg as demonstrated by disease relapse upon Treg depletion by the PC61 mAb treatment in MPP-recipient mice.
The molecular mechanism by which C57Bl/6 mice derived MPPs afforded protection against autoimmune disease differs from what we had previously described in spontaneous diabetes in NOD mice in which mobilized MPP triggered both Notchand GM-CSF-dependent signaling (7,8). Genetic variations between strains may account for this inconsistency as it has been shown that GM-CSF effectively enhanced Treg proliferation in both the NOD and BALB/c strains but neither in the CBA nor the C57Bl/6 strains (24). Notably, both GM-CSF overproduction and persistent STAT5 phosphorylation have been reported to be amplified in autoimmune NOD bone marrow cells (25). The latter phenotype can be reproduced in non-autoimmune C57BL/ 6 bone marrow cultures by blocking M-CSF while stimulating with NOD level GM-CSF. Thus, the observed discrepancies between the NOD and the C57BL/6 strains in the molecular mechanisms of MPP on Treg expansion may at least in part originate in this dysregulation of GM-CSF affecting NOD myeloid cell differentiation. We herein show that in C57Bl/6 mice immunized with MOG 35-55 , IL-1b, one of the cytokines reportedly released by stress-activated MPP (22), is a key molecular mediator promoting the expansion of TCR-activated Treg both in vitro and in vivo. IL-1b likewise appears involved in the Treg expansion properties of G-CSF mobilized human MPPs isolated from healthy donors characterized in D'Aveni et al. (submitted). Yet, whether autoimmune patients will exhibit a distinct molecular mechanism for MPP expansion of Treg will have to be explored.
This may appear paradoxical since IL-1b is mostly associated with inflammation in MS and EAE. An absolute requirement of IL-1R was demonstrated for the development of EAE (26)(27)(28)(29). IL-1b is present in MS lesions and IL-1RA moderates the induction of EAE (30,31). Both IL-1b and IL- 1RA appear in white and grey matter lesions in a model of relapsing EAE at sites of active demyelination (30). IL-1b contributes to the generation of GM-CSF + Th17 cells in EAE (28,32). An elevated IL-1b over IL-1RA ratio corresponds to higher risks of developing MS (33). In addition, IL-1b plays a role in glutamate-mediated synaptic excitability and neurotoxicity taking place in EAE (34). Conversely, IL-1b priming reduced EAE severity in rats (28,29,35). Additionally, the capacity of IL-1b to enhance proliferation of TCR-activated Treg, but not of resting Treg, was recently reported in several in vitro studies. IL-1b has been shown to promote TGF-b and IL-2-dependent Foxp3 expression in Treg and to display co-stimulatory effects on their expansion and differentiation (36). It has been proposed that Foxp3 -T cells may be the primary target of IL-1b and actually support Foxp3 + Treg expansion (37). Here, IL-1b produced by mobilized MPP was able to enhance the proliferation and survival of Foxp3 + Tregs isolated from Foxp3-GFP-KI mice, thus devoid of non Treg.
Foxp3 + Treg are endowed with plasticity that enables them to specialize in selectively regulating effector T cell responses (38). Several of the genes dependent of the IL-1b pathways that showed enhanced expression in Treg after interaction with MPP, including Ifng (IFN-g), Tbx21 (39), and Ccl5 (20), have been reported to confer to Treg the capacity to suppress Th1 and Th17 inflammatory cells, most appropriate to control EAE disease. Moreover, they exhibit enhanced expression of Prdm1, recently reported to prevent methylation of Foxp3 within Treg in central nervous system inflammation (19). Indeed, CD4 + T cells of MPP recipients displayed reduced IFN-g, IL-17 as well as GM-CSF cytokine production. This suggests that MPP may confer to Treg the capacity to better suppress effector Tcells with the same gene set expression, i.e. Th1 and Th17 cells (38). Therefore, Treg expanded in contact with MPP have acquired the capacity to provide robust cell therapy against immune inflammatory diseases.
Moreover, they exhibit enhanced expression of Nr4a, that stabilizes Treg against their differentiation into effector Tcells (20). This stabilization is further confirmed by the high and unaffected expression level of Foxp3. Their survival is improved with a reduced apoptosis by means of TCR signaling in response to increased IL-2, the inhibition of NF-kB pathway via the increase of TNFa activity, the FOXO-mediated transcription, and through p21 (Cdkn1a) inhibiting activity. Their cell cycle is also slowed at the end of G1 stage through DNA synthesis inhibition in the S phase, stemming from increased p21 expression (Cdkn1a), which is in turn regulated by RUNX3 and p53. Coupled with a stringent control of apoptotic decrease, a reduced cell cycle by overexpression of Cdkn1a, and independent from Foxp3, ultimately promotes Treg generation, as was reported after ionizing radiation of Langerhans cell (21). Altogether, MPP confer to Treg stability, enhanced survival and a highly functional state along with the phenotype Cd4 high , Il2ra high , Foxp3 high , Il7ra−, Ctla4+, Tnfrsf18+, Ikzf2+, Tnfrsf4+, Itgae+.
Interaction with innate signals was reported to confer to HSC and particularly MPP the capacity to release numerous and copious amounts of cytokines and chemokines (22). Both murine and human HSC stimulated by innate signals contribute to long-term increased anti-infectious response termed trained immunity (40,41). In systemic lupus erythematosus, an autoimmune disease with a humoral component, TLR-stimulated HSC produce IL-17 and IL-21 and expand Th17 and Tc17 cells (42), contributing to disease severity. On the other hand, hematopoietic progenitors further engaged into differentiation, in the myeloid (43), B (44-46) and pDC (Letscher et al., submitted) lineage pathways have instead been conferred immunoregulatory properties upon innate activation. Such innate activated progenitors provide protection against inflammation, autoimmune diseases, but may as well limit anti-cancer defense. They target and suppress effector T cells or favor the accumulation of diverse regulatory cell subsets. Our present data demonstrate that in organ-specific autoimmune diseases such as Type 1 diabetes and EAE, G-CSF mobilized MPP exert unique regulatory properties indirectly via a major mature regulatory cell type, Foxp3 + Treg, that they not only expand in vivo but also confer stabilization and fitness for controlling inflammatory settings.
The data herein presented advocate for the use of mobilized MPP as a promising tool for cell therapy of autoimmune diseases, either per se or as a complement of autologous HSC transplantation already performed in MS patients with severe, non-responsive disease. Although G-CSF was reported to provide protection in two different models of EAE in mice (47,48), in MS patients, mobilization with G-CSF administrated alone caused disease flare (49). However, this deleterious effect could be controlled if G-CSF was associated with steroids (50). The CXCR4 antagonist plerixafor that induces rapid mobilization (51) might be worth evaluating for its potential morbidity in the MS setting and for the capacity of MPP mobilized with this alternative agent to expand Treg to the same extent as those obtained after G-CSF mobilization. In addition, the effectiveness of MPP at different disease stages remains to be evaluated. Indeed, the data presented herein demonstrate the capacity of mobilized MPP to prevent EAE induction. For a putative application to MS, their ability to reduce EAE remains to be evaluated with an adoptive transfer once clinical signs are detectable.

DATA AVAILABILITY STATEMENTS
The datasets generated and analyzed for this study can be found in the GEO repository https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE155148. All other data are available upon reasonable request.

ETHICS STATEMENT
The animal study was reviewed and approved by Paris Descartes University Ethical Committee for Animal Experimentation and the French Ministry of Research and Higher Education, number 3846-2015070622031545v4.

ACKNOWLEDGMENTS
Authors are grateful to Jeŕome Meǵret for managing the flow cytometry cell-sorting and to Emilie Panafieu for taking care of the animal housing. Mice in the figures were created with BioRender.com. The content of this manuscript has been published in part as part of the thesis of Sarantis Korniotis (44).

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2020. 607175/full#supplementary-material SUPPLEMENTARY FIGURE 1 | Expression levels of plasticity-and stabilityeffector genes and their relative fold-change in Tregs cultured with MPPs when compared with Tregs alone. Mean expression levels are indicated with the standard deviation for each group (top-right corner). All FDR < 0.05; FC: Fold change; Blue: Treg alone; Orange: Treg+MPP.
SUPPLEMENTARY FIGURE 2 | Expression levels of additional genes of interest in Tregs cultured with MPPs when compared with Tregs alone. Mean expression levels are indicated with the standard deviation for each group. Down-regulated and upregulated genes are all members of K-means clusters c8 and c9, respectively. All p-values < 0.05; Blue: Treg alone; Orange: Treg+MPP. SUPPLEMENTARY FIGURE 3 | Isolation of MPPs derived from IL-1b deficient C57BL/6 donor mice. Splenocytes recovered from G-CSF + Flt3L mobilized mice were magnetically sorted as c-kit+ cells, stained with CD34, Sca-1 and CD11b and sorted as c-kit+ Sca-1+ CD34+ CD11b-/low cells by flow cytometry.
SUPPLEMENTARY TABLE 1 | Functional annotations of the genes in K-means cluster c8, under-expressed in Treg cultured 4 days in presence of MPP versus Treg alone. FDR (false discovery rate obtained by bootstrapping 50 times) cutoff is at 0.05. Enrichments in GO (Gene Ontology) terms as well as gene symbols are provided for each significant GO category. Table is sorted by decreasing enrichment.
SUPPLEMENTARY TABLE 2 | Functional annotations of the genes in K-means cluster c8, over-expressed in Treg cultured 4 days in presence of MPP versus Treg alone. FDR (false discovery rate obtained by bootstrapping 50 times) cutoff is at 0.05. Enrichments in GO (Gene Ontology) terms as well as gene symbols are provided for each significant GO category. Table is sorted by decreasing enrichment.   SUPPLEMENTARY TABLE 3 | Statistics obtained after supervised differential gene expression analyses. Moderated t-test on linear modeling with empirical Bayes was applied on Treg alone vs. Treg+MPP. FDR: False discovery rate by Benjamini-Hochberg. Fold change is indicated in plain and centered on zero.