Jonathan Charaix

Alexia Borelli

Jérémy C Santamaria

Lionel Chasson

Matthieu Giraud

Arnauld Sergé

Magali Irla
email: magali.irla@inserm.fr

Recirculating Foxp3+ regulatory T cells are restimulated in the thymus under Aire control

Keywords: Autoimmune regulator, Autoimmunity, Medullary thymic epithelial cells, Foxp3 + Regulatory T cells, Thymus

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

CD25 + Foxp3 + regulatory T cells (Treg) constitute a distinct subset of CD4 + T cells endowed with suppressive functions. By maintaining immune tolerance, Treg critically prevent the emergence of autoimmune and inflammatory diseases. The vast majority of Treg is produced in the thymic medulla where they arise from two distinct developmental programs involving CD25 + Foxp3 -(CD25 + TregP) and CD25 -Foxp3 lo (Foxp3 lo TregP) precursors [START_REF] Owen | Thymic regulatory T cells arise via two distinct developmental programs[END_REF][START_REF] Lio | A two-step process for thymic regulatory T cell development[END_REF][START_REF] Santamaria | Regulatory T Cell Heterogeneity in the Thymus: Impact on Their Functional Activities[END_REF][START_REF] Marshall | Differential requirement for IL-2 and IL-15 during bifurcated development of thymic regulatory T cells[END_REF]. For a long-time, thymic CD25 + Foxp3 + Treg were thought to exclusively correspond to developing cells. However, recent studies have shown that circulating CD25 + Foxp3 + Treg have the ability to migrate back into the thymus [START_REF] Thiault | Peripheral regulatory T lymphocytes recirculating to the thymus suppress the development of their precursors[END_REF][START_REF] Cowan | CCR7 Controls Thymus Recirculation, but Not Production and Emigration, of Foxp3(+) T Cells[END_REF][START_REF] Yang | Both retention and recirculation contribute to long-lived regulatory T-cell accumulation in the thymus[END_REF]. Thus, thymic CD25 + Foxp3 + Treg is a heterogeneous population containing both developing and recirculating cells [START_REF] Santamaria | Regulatory T Cell Heterogeneity in the Thymus: Impact on Their Functional Activities[END_REF]. Interestingly, recirculating Treg show suppressive properties similar to their splenic counterparts [START_REF] Yang | Both retention and recirculation contribute to long-lived regulatory T-cell accumulation in the thymus[END_REF]. They exhibit an activated and differentiated phenotype and were found to negatively regulate the de novo generation of CD25 + Foxp3 + Treg through IL-2 consumption [START_REF] Thiault | Peripheral regulatory T lymphocytes recirculating to the thymus suppress the development of their precursors[END_REF].

The recirculation of peripheral Foxp3 + Treg in the thymus is mediated at least by two chemokine receptors, CCR6 and CXCR4 [START_REF] Thiault | Peripheral regulatory T lymphocytes recirculating to the thymus suppress the development of their precursors[END_REF][START_REF] Cowan | Aire controls the recirculation of murine Foxp3(+) regulatory T-cells back to the thymus[END_REF]. Interestingly, the entry of peripheral Ccr6 -/-Treg into the thymus is impaired, suggesting that CCR6, expressed by effector/memory T cells, is involved in the recirculation of Foxp3 + Treg from the periphery to this organ [START_REF] Cowan | Aire controls the recirculation of murine Foxp3(+) regulatory T-cells back to the thymus[END_REF]. Furthermore, the expression of the CCR6 ligand, CCL20, is regulated by the Autoimmune regulator (Aire) [START_REF] Cowan | Aire controls the recirculation of murine Foxp3(+) regulatory T-cells back to the thymus[END_REF]. Aire is mainly expressed by a subset of medullary thymic epithelial cells (mTEC) commonly called mTEC hi , characterized by a CD80 hi MHCII hi phenotype. Furthermore, upon recirculation into the thymus, a subset of B cells has been described to upregulate Aire [START_REF] Yamano | Thymic B Cells Are Licensed to Present Self Antigens for Central T Cell Tolerance Induction[END_REF]. Independent reports have described that CD25 + Foxp3 + Treg are reduced in the thymus of Aire KO mice [START_REF] Aricha | The susceptibility of Aire(-/-) mice to experimental myasthenia gravis involves alterations in regulatory T cells[END_REF][START_REF] Lei | Aire-dependent production of XCL1 mediates medullary accumulation of thymic dendritic cells and contributes to regulatory T cell development[END_REF]. Accordingly, Aire expression was proposed to direct autoreactive CD4 + thymocytes into the Treg cell lineage [START_REF] Malchow | Aire Enforces Immune Tolerance by Directing Autoreactive T Cells into the Regulatory T Cell Lineage[END_REF]. Interestingly, in the perinatal life, Aire promotes the generation of a distinct population of Foxp3 + Treg, which persists in adults and prevents the emergence of autoimmunity [START_REF] Yang | Immune tolerance. Regulatory T cells generated early in life play a distinct role in maintaining self-tolerance[END_REF]. In C57BL/6 mice, Aire-deficiency is associated with mild autoimmunity characterized by lymphocyte infiltrations and autoantibody production targeting several organs such as the pancreas, eyes, salivary glands, liver and lungs [START_REF] Anderson | Projection of an immunological self shadow within the thymus by the aire protein[END_REF][START_REF] Jiang | Modifier loci condition autoimmunity provoked by Aire deficiency[END_REF][START_REF] Kuroda | Development of autoimmunity against transcriptionally unrepressed target antigen in the thymus of Aire-deficient mice[END_REF][START_REF] Ramsey | Aire deficient mice develop multiple features of APECED phenotype and show altered immune response[END_REF]. In human, autosomal recessive mutations in the Aire gene induce a life threatening pathology called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), also known as autoimmune polyendocrine syndrome-type 1 (APS-1) [START_REF] Consortium | An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHD-type zinc-finger domains[END_REF][START_REF] Nagamine | Positional cloning of the APECED gene[END_REF]. Interestingly, numbers, activation and suppressive functions of Foxp3 + Treg are altered in APECED patients [START_REF] Ryan | CD4+CD25+ T-regulatory cells are decreased in patients with autoimmune polyendocrinopathy candidiasis ectodermal dystrophy[END_REF][START_REF] Kekäläinen | A defect of regulatory T cells in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy[END_REF][START_REF] Laakso | Regulatory T cell defect in APECED patients is associated with loss of naive FOXP3(+) precursors and impaired activated population[END_REF].

Although thymic Foxp3 + Treg have been found to be more heterogeneous than previously thought [START_REF] Santamaria | Regulatory T Cell Heterogeneity in the Thymus: Impact on Their Functional Activities[END_REF], this emerging population of recirculating peripheral Treg remains poorly described. In particular, the mechanisms that control their suppressive phenotype in the thymus are unknown. In this study, we show that Aire regulates the suppressive properties of recirculating CCR6 + Treg, independently of the remodeling of the 3D organization of the thymic medulla where they reside [START_REF] Thiault | Peripheral regulatory T lymphocytes recirculating to the thymus suppress the development of their precursors[END_REF]. High-throughput sequencing revealed that recirculating CCR6 + Treg in the thymus of Aire KO mice have impaired expression of several genes associated with Treg suppressive functions and helper T cell polarization. Accordingly, recirculating CCR6 + Treg from Aire KO mice failed to attenuate the severity of multiorgan autoimmunity, demonstrating that their suppressive activity is impaired. Moreover, the adoptive transfer of splenic Foxp3 + Treg in Aire KO recipients resulted in an impaired suppressive signature upon their recirculation in the thymus. By using bone marrow (BM) chimeras, we demonstrate that Aire expressed by mTEC rather than by thymic B cells is responsible for the suppressive properties of recirculating CCR6 + Treg. Finally, recirculating Treg were found in close contact with Aire + mTEC and restimulated in an antigen-specific manner. Altogether, our data reveal that Aire confers to mTEC the capacity to control the restimulation of recirculating CCR6 + Treg in the thymus.

Materials and methods

Mice

CD45.1 WT (B6.SJL-PtprcaPepcb/BoyCrl, Stock n°002014, Charles River), CD45.2 WT (Stock n°000664, Charles River), CD45.1/2 WT, CD45.2 Aire KO [START_REF] Sansom | Population and single-cell genomics reveal the Aire dependency, relief from Polycomb silencing, and distribution of self-antigen expression in thymic epithelia[END_REF], Rag2 KO [START_REF] Shinkai | RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement[END_REF], CD45.1 Foxp3 eGFP mice [START_REF] Wang | Th2 lymphoproliferative disorder of LatY136F mutant mice unfolds independently of TCR-MHC engagement and is insensitive to the action of Foxp3+ regulatory T cells[END_REF], OTII [START_REF] Barnden | Defective TCR expression in transgenic mice constructed using cDNA-based alpha-and beta-chain genes under the control of heterologous regulatory elements[END_REF] and Rip-mOVA [START_REF] Kurts | Constitutive class I-restricted exogenous presentation of self antigens in vivo[END_REF] were on a C57BL/6J background. Rip-mOVA x OTII mice were backcrossed on a Rag2 KO background. All mice were maintained under specific pathogen-free conditions at the Centre d'Immunologie de Marseille-Luminy (CIML, France). Standard food and water were given ad libitum. Males and females were used at the age of 5 days, 9 days, 6 weeks and 1 year. Chimeras were generated between 6 to 10 weeks of age. All experiments were done in accordance with national and European laws for laboratory animal welfare (EEC Council Directive 2010/63/UE) and the Marseille Ethical Committee for Animal experimentation no. 14.

BM chimeras

BM chimeras were generated by injecting intravenously (i.v.) 5.10 6 BM cells from CD45.1 Foxp3 eGFP mice into lethally irradiated CD45.2 Aire WT or Aire KO recipients (two doses of 500 rads, 8h apart, X-ray using a RS-2 000

Irradiator; Rad Source Technologies). Similarly, 5.10 6 BM cells from either CD45.2 Aire WT or Aire KO mice were injected into lethally irradiated CD45.1/2 WT recipients. Mice were analyzed 6 weeks post-reconstitution.

Foxp3 + Treg adoptive transfer 5.10 5 cell-sorted splenic congenic CD45.1 CD4 + CD25 + Foxp3 eGFP Treg were i.v injected into sublethally irradiated CD45.2 Aire WT or Aire KO recipient mice (one dose of 500 rads, X-ray using a RS-2 000 Irradiator; Rad Source Technologies). Mice were analyzed 1-week post-Treg adoptive transfer.

Multi-organ autoimmunity experiments

Rag2 KO recipients were injected i.v. with 3.10 6 CD4 + CD25 + Treg-depleted splenocytes purified from CD45.1 WT mice. Four weeks later, 15.10 4 splenic CD4 + CD25 hi Treg or 10.10 4 recirculating thymic CCR6 + CD4 + CD25 + Treg from Aire WT or Aire KO mice were adoptively transferred. Mice that did not receive any Treg were used as controls.

Peripheral tissues were harvested and analyzed three weeks later.

Cell isolation

Thymic Treg, splenic Treg and Treg-depleted splenocytes were isolated by scratching the thymus or the spleen on a 70-μm mesh. Red blood cells were lysed with RBC lysis buffer (eBioscience). Thymic and splenic Treg cells were pre-enriched by depleting CD8 + and CD19 + cells using biotinylated anti-CD8α (clone 53.6.7; BD Biosciences) and anti-CD19 (clone 1D3; BD Bioscience) antibodies with anti-biotin microbeads by AutoMACS using the Deplete program (Miltenyi Biotech). Recirculating thymic Treg were sorted as CCR6 + CD4 + CD8 -CD25 + cells and splenic Treg as CD4 + CD25 hi cells using a FACSAria III cell sorter (BD Biosciences). Splenocytes were depleted of CD4 + CD25 + Treg using a FACSAria III cell sorter (BD Biosciences). Thymus were digested at 37°C in HBSS medium containing Liberase TM (50 µg/ml; Roche) and DNase I (100 μg/ml; Roche) until complete tissue digestion. Total mTEC (CD45 -EpCAM + UEA-1 + Ly51 -/lo ) or mTEC hi (CD45 -EpCAM + UEA-1 + Ly51 -/lo CD80 hi ) were pre-enriched by depleting CD45 hematopoietic cells using anti-CD45 magnetic beads (Miltenyi Biotec) by AutoMACS with the DepleteS program and sorted using a FACSAria III cell sorter (BD Biosciences).

In vitro co-culture assays 3.10 4 cell-sorted splenic CD4 + CD25 hi Treg from Aire WT or Aire KO mice were co-cultured for 24h with 6.10 3 CD45 - EpCAM + UEA-1 + Ly51 -/lo mTEC from Aire WT or Aire KO mice in RPMI (ThermoFisher) supplemented with 10% FBS (Sigma Aldrich), L-glutamine (2 mM, ThermoFisher), sodium pyruvate (1 mM, ThermoFisher), 2mercaptoethanol (2×10 -5 M, ThermoFisher), penicillin (100 IU per ml, ThermoFisher), streptomycin (100 μg per ml, ThermoFisher) and mouse IL-2 (40 ng/ml, Immunotools). For antigen-specific co-culture assays, 5.10 3 cellsorted splenic CD4 + CD25 hi Treg from Rip-mOVA x OTII x Rag2 KO mice were co-cultured for 24h with 1.10 3 CD45 - EpCAM + Ly51 -/lo Aire eGFP mTEC from Aire het or Aire KO mice previously loaded with OVA323-339 peptide (5µg/ml, Polypeptide group) for 2h.

Flow cytometry

Cells were stained with standard procedures using antibodies listed in Table S2. For intracellular staining with anti-Foxp3, anti-GITRL and anti-OX40L antibodies, cells were fixed, permeabilized and stained with the Foxp3 staining kit according to the manufacturer's instructions (eBioscience). Stained cells were analyzed with FACSCanto II and LSR II (BD Biosciences) and data were analyzed using FlowJo software (Tree Star).

Quantitative RT-PCR

Total RNA was extracted with TRIzol (Invitrogen) and cDNA was synthesized with random oligo dT primers and Superscript II reverse transcriptase (Invitrogen). Quantitative PCR was performed with SYBR Premix Ex Taq Master Mix (Takara) on an ABI 7500 fast real-time PCR system (Applied Biosystem). The results were normalized to actin mRNA expression. A list of primer sequences is provided in Table S3.

RNA-sequencing experiments

5.10 4 CCR6 + Treg were cell-sorted from the thymus of 6 week-old Aire WT and Aire KO mice. Two biological replicates were prepared for each condition. The total RNA was extracted using the RNeasy Micro Kit (Qiagen) and treated with DNase I. RNA-seq libraries were prepared using the TruSeq Stranded mRNA kit (Illumina) and sequenced with the Illumina HiSeq 2000 machine to generate datasets of single-end 50bp reads. The reads were mapped to the mouse reference genome (mm10) using TopHat2 (version 2.0.12) (28), then counted using Cufflinks or Cuffdiff (version 2.2.1) [START_REF] Trapnell | Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation[END_REF][START_REF] Trapnell | Differential analysis of gene regulation at transcript resolution with RNA-seq[END_REF] and the mm10 genome GTF gene annotation file. In addition to read counting, Cuffdiff performs between-sample normalization and was used to calculate the differential gene expression and its statistical significance in Aire WT vs Aire KO Treg by using the default "pooled" dispersion method that applies to experiments having few (≥2) biological replicates per condition. Expression levels generated by Cufflinks, as fragments per kilobase of transcript per million mapped reads (FPKM), were processed by the Matrix2png program [START_REF] Pavlidis | Matrix2png: a utility for visualizing matrix data[END_REF] to generate heatmaps of gene expression levels. The dataset generated in this study are available in the Gene Expression Omnibus (GEO) database under accession number GSE188419. The expression of Tnfsf4 and Tnfsf18 was analyzed in RNA-sequencing dataset from Aire KO mTEC hi (GSE87133).

Immunofluorescence staining

Thymi were collected from 9 day-, 6 week-and 1 year-old Aire WT or Aire KO mice and fat tissues removed to avoid any interference with the 3D reconstitution process. Organs were longitudinally included in O.C.T (Sakura Finetek), frozen at -80 °C and cut in 20-µm-thick slices. Thymic sections were fixed with 2% paraformaldehyde, then saturated with 3% BSA and 0.01% Triton X100 in 0.1 M Tris HCl buffer. Sections were next stained for 45 min with rabbit anti-keratin 14 or with anti-Aire Alexa Fluor 488 (5H12; eBioscience), anti-Foxp3 PE (FJK-16s; eBioscience) and anti-CD73 Alexa Fluor 647 (TY/11.8; Biolegend) in hybridization buffer (1% BSA and 0.02% Triton X100 in 0.1 M Tris HCl, pH 7.4). Keratin 14 staining was revealed with Cy3-conjugated anti-rabbit (Invitrogen). Sections were counterstained with DAPI (1 µL/mL, BioLegend) and mounted with Mowiol (Calbiochem). For 3D reconstruction, images were acquired with a slide scanner (Panoramic SCAN II; 3D Histech). Confocal microscopy was performed with a LSM 780 confocal microscope (Carl Zeiss Microscopy).

Detection of immune infiltrates and autoantibodies

Peripheral tissues were fixed in buffered 10% formalin solution. 4 µm-thick paraffin-embedded sections were counterstained with hematoxylin and eosin. Autoantibody production was assessed by immunostaining organ sections from Rag2 KO mice with the sera (1/80) of analyzed mice. Autoantibodies were revealed with FITCconjugated goat anti-mouse IgG. Sections were counterstained with DAPI and mounted with Mowiol (Calbiochem). All images were acquired with a slide scanner (Panoramic SCAN II; 3D Histech) and analyzed with ImageJ software (National Institutes of Health) to compute the mean fluorescence intensity of each image.

3D reconstitution

For 3D reconstitution, images from the entire thymus of 9 day-, 6 week-and 1-year-old Aire WT and Aire KO mice were processed with For3D software as previously described [START_REF] Irla | Three-dimensional visualization of the mouse thymus organization in health and immunodeficiency[END_REF][START_REF] Serge | For3D: Full Organ Reconstruction in 3D, an Automatized Tool for Deciphering the Complexity of Lymphoid Organs[END_REF]. Briefly, images were smoothed by median and Gaussian filtering and medulla volumes were determined using ImageJ and Matlab (The Mathworks) software.

Medullary islets identified in the 3D structures were measured individually using ImageJ and were color-coded using Imaris (Bitplane).

Statistical analysis

Statistics were performed with GraphPad Prism 9.1 software. Normal distribution of the data was assessed using d'Agostino-Pearson omnibus normality test. Statistical significance was then assessed using unpaired Student's t test for two normal distributions, Mann-Whitney test for two non-normal distributions or Kruskal-Wallis test for more than two distributions. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001. All bar graphs show mean ± SEM, unless mentioned.

Results

Aire regulates the recirculation of peripheral CD25 + Foxp3 + Treg in the thymus, independently of the remodeling of the 3D organization of the medulla throughout life

Considering the importance of Aire in the induction of self-tolerance [START_REF] Perniola | Twenty Years of AIRE[END_REF], we first assessed whether it could be involved in the topology of the thymic medulla. For this, we compared WT (Aire WT mice) with Aire-deficient mice in which the first exon of the Aire locus was replaced by the sequence of enhanced green fluorescent protein (eGFP) (Aire KO mice) [START_REF] Sansom | Population and single-cell genomics reveal the Aire dependency, relief from Polycomb silencing, and distribution of self-antigen expression in thymic epithelia[END_REF]. Thymic sections of 9 day-, 6 week-and 1-year-old, either Aire WT or Aire KO mice were stained with the keratin-14 mTEC-specific marker [START_REF] Klug | Interdependence of cortical thymic epithelial cell differentiation and T-lineage commitment[END_REF] and reconstructed with our in-house dedicated "Full organ reconstruction in 3D" (For3D) software (33) (Fig. 1A-C and Movies S1-6). Interestingly, the number of medullary islets in Aire WT mice diminished between 9 days and 6 weeks of age while it increased between 6 weeks and 1 year of age (Fig. 1D). Furthermore, the total and individual medullary volumes increased between 9 days and 6 weeks while it decreased between 6 weeks and 1 year (Fig. 1E,F). Nevertheless, the central compartment of ~1 mm 3 was observed at all ages analyzed (Fig. 1F) These observations reveal that the medulla organization is dynamic throughout life. In comparison to Aire WT mice, the thymi of Aire KO mice show a similar dynamic medulla organization characterized by normal numbers of medullary islets and total medullary volumes with the presence of a major compartment (Fig. 1A-C,F). These results indicate that the dynamic remodeling of the 3D organization of the thymic medulla throughout life is not regulated by Aire.

Because medulla formation correlates with Foxp3 + Treg emergence during ontogeny [START_REF] Fontenot | Developmental regulation of Foxp3 expression during ontogeny[END_REF], we next analyzed whether Aire deficiency could affect their development in neonates of 5 days of age. In contrast to CD25 + TregP, frequencies and numbers of Foxp3 lo TregP and mature CD25 + Foxp3 + Treg were diminished in the thymus of 5-dayold Aire KO mice compared to their respective counterparts (Fig. 2A). This decrease was also observed in 6-weekand 1-year-old Aire KO mice (Fig. 2B,C). Since mature CD25 + Foxp3 + Treg contain both developing and recirculating cells in the adult thymus, we used the key thymus-homing chemokine receptor CCR6 to distinguish developing (CCR6 -) and recirculating (CCR6 + ) mature Treg [START_REF] Cowan | CCR7 Controls Thymus Recirculation, but Not Production and Emigration, of Foxp3(+) T Cells[END_REF]. Interestingly, both numbers of CCR6 -and CCR6 + mature Treg were reduced in Aire KO mice compared to Aire WT mice at 6 weeks and 1 year of age (Fig. 2D,E). Furthermore, 1year-old Aire WT mice showed a marked reduction in numbers of CCR6 -and CCR6 + mature CD25 + Foxp3 + Treg compared to 6-week-old Aire WT mice, which reflects the effect of thymic involution on Treg cells in normal conditions. To date, two chemokine receptors, CCR6 and CXCR4 have been implicated in the recirculation of peripheral Treg into the thymus [START_REF] Thiault | Peripheral regulatory T lymphocytes recirculating to the thymus suppress the development of their precursors[END_REF][START_REF] Cowan | Aire controls the recirculation of murine Foxp3(+) regulatory T-cells back to the thymus[END_REF]. Strikingly, the expression of their respective ligands, Ccl20 and Cxcl12, was substantially reduced in Aire KO mTEC hi (Fig. 2F), consistently with the altered recirculation of peripheral Treg in the thymus. Altogether, these results show that Aire controls both the development and recirculation of Foxp3 + Treg throughout life. Thymic CCR6 + Treg from Aire KO mice show an impaired effector and suppressive phenotype Because Aire controls the recirculation of CCR6 + Treg, we made the hypothesis that it could also control their suppressive properties. To test this hypothesis, we first measured the expression level of several genes associated with Treg suppressive functions in purified CCR6 + CD4 + CD25 + single-positive (SP) thymocytes that correspond to CD25 + Foxp3 + Treg (Fig. S1). Interestingly, whereas Foxp3 level was normal, the expression of Klrg1, a marker of terminally differentiated Treg [START_REF] Cheng | IL-2 receptor signaling is essential for the development of Klrg1+ terminally differentiated T regulatory cells[END_REF], was reduced in CCR6 + Treg from 6-week-old Aire KO mice (Fig. 3A).

Accordingly, the expression of genes encoding for the inhibitory cytokine Il10, the cytolytic molecules Gzmb and Fasl, Lag3-associated with dendritic cell modulation, as well as the ectoenzymes Entpd1 (CD39) and Nt5e (CD73), implicated in target cell metabolic disruption, was reduced in CCR6 + Treg of Aire KO mice. A similar altered suppressive signature was also observed in CCR6 + Treg purified from the thymus of 1-year-old Aire KO mice (Fig.

S2).

To further determine the impact of Aire on the functional properties of CCR6 + Treg, we analyzed their gene expression profile by high-throughput RNA-sequencing (Fig. 3B,C). Genes showing a significant variation (p ≤ 0.05) in gene expression between Aire WT and Aire KO CCR6 + Treg with a fold change difference > 2 or < 0.5 were considered as up-and down-regulated, respectively. We identified a total of 2 634 upregulated genes reaching significance for 1 060 of them (Cuffdiff p < 0.05) in Aire WT CCR6 + Treg compared to their Aire KO counterparts (Fig. 3B). Moreover, only 1 175 genes were downregulated with 301 of them reaching significance (Cuffdiff p < 0.05). Thus, the expression of Aire in the thymus upregulates three times more genes than it downregulates in recirculating CCR6 + Treg. In accordance with the altered Treg suppressive signature observed by qPCR (Fig. 3A), we found that Il10, Gzmb, Fasl, Lag3, Entpd1 and Nt5e were downregulated in CCR6 + Treg of Aire KO mice (Fig. 3C and Table S1). Furthermore, the expression of Prdm1 (Blimp-1), which characterizes effector Th-like Treg [START_REF] Cretney | Differentiation and function of Foxp3(+) effector regulatory T cells[END_REF] and the terminally differentiated markers Klrg1 and Tigit [START_REF] Cheng | IL-2 receptor signaling is essential for the development of Klrg1+ terminally differentiated T regulatory cells[END_REF][START_REF] Joller | Treg cells expressing the coinhibitory molecule TIGIT selectively inhibit proinflammatory Th1 and Th17 cell responses[END_REF] was reduced in CCR6 + Treg of Aire KO mice compared to their Aire WT counterparts. Strikingly, CCR6 + Treg of Aire KO mice also expressed lower levels of several genes associated with their suppressive signature such as Ctla4 and Lgals1 (galectin-1) implicated respectively in dendritic cell modulation and target cell apoptosis [START_REF] Fallarino | Modulation of tryptophan catabolism by regulatory T cells[END_REF][START_REF] Garín | Galectin-1: a key effector of regulation mediated by CD4+CD25+ T cells[END_REF]. The expression of Tbx21 and Irf4, encoding for transcription factors associated with Th1-and Th2-like Treg, as well as Pparg and Id2 genes associated with fat-resident effector Treg (42) was also diminished. Accordingly, the expression of the chemokine receptors Cxcr3 of Th1-like, Ccr4 and Ccr8 of Th2-like as well as Ccr1 and Ccr2 of fat-resident Treg [START_REF] Sawant | Once a Treg, always a Treg?[END_REF], implicated in effector Treg migration to the inflammatory site, was reduced in CCR6 + Aire KO Treg. Altogether, these results indicate that Aire expression is crucial for the effector and suppressive properties of recirculating CCR6 + Treg in the thymus.

Recirculating CCR6 + Treg from Aire KO mice fail to attenuate the severity of multi-organ autoimmunity Because numbers and suppressive signature of recirculating CCR6 + Treg were reduced in the thymus of Aire KO mice, we analyzed whether these defects would be also observed in the periphery. Blood and splenic CD4 + Foxp3 + Treg from 6-week-and 1-year-old Aire KO mice showed similar frequencies, numbers and expression levels of suppressive genes compared to their Aire WT counterparts (Fig. S3). We next assessed the ability of peripheral and thymic CCR6 + Treg from Aire KO mice to dampen the severity of multiorgan autoimmunity. To this end, CD4 + CD25 + Treg-depleted splenocytes from CD45.1 WT mice were transferred into Rag2 KO lymphopenic recipients. Four weeks later, splenic Treg or thymic CCR6 + Treg purified from either Aire WT or Aire KO mice were adoptively transferred into these recipients (Fig. 4A and Fig. S4A). Signs of autoimmunity in peripheral tissues were visualized by histology and quantified by flow cytometry three weeks later. Rag2 KO mice that did not receive any Treg were used as controls. Accordingly with the normal suppressive signature of splenic Treg from Aire KO mice (Fig. S3), Rag2 KO mice adoptively transferred with these cells show tissue infiltration levels similar to mice transferred with Aire WT splenic Treg (Fig. 4B,C). In marked contrast to mice that received Aire WT thymic CCR6 + Treg, mice adoptively transferred with thymic CCR6 + Treg from Aire KO mice failed to attenuate T-cell infiltration in several peripheral tissues despite similar numbers of CD45.2 donor Foxp3 + Treg in lymph nodes (Fig. 4D,E and Fig. S4B,C). Examination of CD45.1 donor cell infiltration by flow cytometry revealed that 100% of Rag2 KO mice that received Aire KO thymic CCR6 + Treg showed a high infiltration level in the pancreas and salivary glands, 83% in eyes, 33% in the lung and liver as well as 16% in the kidney (Fig. 4E). In contrast, only 16% of Rag2 KO mice transferred with Aire WT thymic CCR6 + Treg showed a high infiltration in the liver. Furthermore, flow cytometry analysis showed that thymic CCR6 + Treg of Aire KO mice were unable to prevent the infiltration of CD45.1 CD4 + and CD8 + T cells in the pancreas and eyes as well as CD45.1 CD8 + T cells in salivary glands (Fig. S4D). Finally, immunostaining of Rag2 KO tissue sections with sera from these mice revealed higher levels of autoantibodies against the pancreas and salivary glands in mice transferred with Aire KO thymic CCR6 + Treg than in mice injected with Aire WT thymic CCR6 + Treg (Fig. 4F,G). Thus, thymic CCR6 + Treg of Aire KO mice failed to attenuate the severity of multiorgan autoimmunity, demonstrating that their suppressive activity was impaired.

Aire expression in the thymic stroma controls the suppressive signature of recirculating CCR6 + Treg

Since Aire expression is not restricted to mTEC but was also found in thymic B cells (9), we then investigated its respective contribution in the stromal and hematopoietic compartments to control the recirculation and suppressive properties of CCR6 + Treg. To determine the role of Aire in hematopoietic cells, we generated BM chimeras by reconstituting lethally irradiated CD45.1/2 WT recipients with either CD45.2 Aire WT or Aire KO BM cells (Fig. S5A). Six weeks later, Aire KO BM chimeras did not show major defects in total CD19 + B220 + B cells, neither in the IgD -and IgD + B cell subsets, both described to express Aire (9) (Fig. S5B,C). Overall, frequencies and numbers of CD25 + TregP, Foxp3 lo TregP, CCR6 -and CCR6 + mature CD25 + Foxp3 + Treg were also normal (Fig. S5D,E). We next cell-sorted CCR6 + Treg from the thymus of these BM chimeras and analyzed their suppressive signature.

Recirculating Treg from both chimeras exhibited a similar expression of suppressive genes that was altered in CCR6 + Treg of Aire KO mice (Fig. S5F). Thus, Aire expression in thymic B cells is unlikely involved in Treg development, recirculation and suppressive signature.

We then analyzed whether Aire expression in stromal cells controls CCR6 + Treg functional suppressive properties.

To this end, we generated BM chimeras in which lethally irradiated CD45.2 Aire WT or Aire KO mice were reconstituted with CD45.1 Foxp3 eGFP BM cells (Fig. 5A). Six weeks later, similar numbers of CD4 + SP thymocytes of CD45.1 donor origin were observed in Aire WT and Aire KO recipients (Fig. 5B). Although numbers of CD25 + TregP and Foxp3 lo TregP were also similar in both groups, frequencies and numbers of mature CD25 + Foxp3 + Treg were specifically reduced in Aire KO chimeras (Fig. 5C). This defect was attributable to diminished frequencies and numbers of CCR6 + Treg (Fig. 5D). Recirculating CCR6 + mature CD25 + Foxp3 + Treg of CD45.1 origin were then cell-sorted from the thymus of both chimeras and analyzed for the expression of several genes associated with Treg effector functions. Whereas Foxp3 level was normal, the expression of Klrg1, Il10, Tgfb1, Gzmb, Fasl, Lag3, Entpd1 and Nt5e was reduced in Aire KO chimeras compared to control chimeras (Fig. 5E). Altogether, these results

show that whereas Aire in hematopoietic cells is dispensable, its specific expression in stromal cells controls both recirculation and suppressive properties of CCR6 + Treg.

Antigen-specific restimulation of recirculating Treg by Aire + mTEC

To further decipher the impact of Aire-expressing mTEC in the suppressive signature of recirculating Treg, we used an in vitro co-culture setup of peripheral Treg and mTEC purified from Aire WT or Aire KO mice. Compared to Aire WT splenic Treg co-cultured with Aire WT mTEC, the expression of Tgfb1, Gzmb, Fasl, Entpd1 and Nt5e was reduced in Aire WT splenic Treg co-cultured with Aire KO mTEC (Fig. 6A). Moreover, Aire KO splenic Treg co-cultured with Aire WT mTEC upregulated the expression of these genes in contrast to Aire KO splenic Treg co-cultured with Aire KO mTEC. Thus, as compared to their Aire WT counterparts, Aire KO mTEC failed to enhance suppressive signatures of both Aire WT and Aire KO splenic Treg.

We then analyzed the in vivo effect of the Aire KO thymic stroma in regulating the suppressive signature of peripheral WT Treg. To this end, splenic Treg purified from CD45.1 Foxp3 eGFP congenic mice were adoptively transferred into either Aire WT or Aire KO recipients (Fig. 6B). One week later, we observed reduced frequencies and numbers of donor CD45.1 Foxp3 eGFP Treg in the thymus of Aire KO mice as compared to Aire WT mice (Fig. 6C).

Moreover, the remaining donor Foxp3 eGFP Treg that recirculated back to the thymus exhibited an impaired suppressive signature (Fig. 6D). These results not only confirm that Aire favors the recirculation of peripheral Treg in the thymus but also highlight its key role in enhancing their suppressive properties. Thus, we made the hypothesis that recirculating Treg could be restimulated by Aire + mTEC. To test this, WT thymic sections were first stained for Aire, Foxp3 and CD73, the latter being a reliable marker of recirculating Treg [START_REF] Owen | Thymic regulatory T cells arise via two distinct developmental programs[END_REF][START_REF] Peligero-Cruz | IL18 signaling promotes homing of mature Tregs into the thymus[END_REF].

Of note, anti-CCR6 antibody was not used in this experiment because it failed to give any signal in our hands. Interestingly, we observed that both developing CD73 -and recirculating CD73 + Treg were found in close proximity to Aire + mTEC (Fig. 6E). These results suggest that recirculating Treg could be restimulated by establishing antigen-specific contacts with Aire + mTEC. To test this hypothesis, splenic OTII Treg from Rip-mOVA x OTII mice were co-cultured with OVA323-339-loaded Aire het (Aire eGFP/WT ) or Aire KO (Aire eGFP/eGFP ) mTEC hi (Figure S6).

Compared to unstimulated OVA-specific Treg, Aire KO mTEC hi were able to activate OTII Treg but to a lesser extent than Aire het mTEC hi (Fig. 6F). These results demonstrate that Aire + mTEC can stimulate peripheral Treg in an antigen-specific manner and this ability is impaired in the absence of Aire. Furthermore, as compared to OTII Treg co-cultured with Aire het mTEC hi , the expression of Il10, Tgfb1, Gzmb, Fasl, Lag3, Entpd1 and Nt5e was reduced in OTII Treg co-cultured with Aire KO mTEC hi (Fig. 6G), indicating that this antigen-specific restimulation is much less efficient. We thus analyzed MHCII expression in mTEC from Aire KO mice. In accordance with a previous study [START_REF] Anderson | The cellular mechanism of Aire control of T cell tolerance[END_REF] and the pro-apoptotic role of Aire [START_REF] Gray | Proliferative arrest and rapid turnover of thymic epithelial cells expressing Aire[END_REF], we found increased frequencies of MHCII + mTEC in these mice (Fig. S7A). We then made the hypothesis that costimulatory signals may be implicated in the phenotype observed.

Two ligands of the Tumor Necrosis Factor Superfamily (TNFSF) have been described to be constitutively expressed by mTEC and participate in thymic Treg development [START_REF] Mahmud | Costimulation via the tumor-necrosis factor receptor superfamily couples TCR signal strength to the thymic differentiation of regulatory T cells[END_REF]. Interestingly, we found that mTEC hi expressed reduced levels of Tnfsf4 (OX40L, Aire WT : 2.32 FPKM vs Aire KO : 0.37) and Tnfsf18 (GITRL, Aire WT : 2.92 vs Aire KO : 0.81) in Aire KO mice (Fig. S7B). This result was also confirmed at the protein level by flow cytometry (Fig. S7C). These observations are consistent with the reduced cellularity of Foxp3 lo TregP and CD25 + Foxp3 + Treg in Aire KO mice (Fig. 2A-C). Further investigations are needed to decipher the role of these two TNFSF ligands in the biology of recirculating Treg in the thymus. Altogether, our data reveal that Aire + mTEC, through antigen-specific restimulation, are responsible for the strong suppressive signature of recirculating Treg.

Discussion

Our study demonstrates that Aire expression by mTEC promotes the suppressive properties of recirculating CCR6 + Treg independently of the dynamic remodeling of the medullary 3D organization, where recirculating Treg reside.

We previously described that the medulla of 6-week-old young adult mice is complex with a large central compartment surrounded by hundreds of individual islets [START_REF] Irla | Three-dimensional visualization of the mouse thymus organization in health and immunodeficiency[END_REF]. Nevertheless, determining whether this topology varies throughout life and whether Aire is implicated remained open issues. Interestingly, we found that 9-dayand 6-week-old WT mice show ~1 000 and ~400 medullary islets, respectively. Given that individual islets arise from a single progenitor [START_REF] Rodewald | Thymus medulla consisting of epithelial islets each derived from a single progenitor[END_REF], these observations suggest that during thymic development, they grow and fuse together, leading to a reduced number of islets in young adult mice compared to neonates. In 1-year-old mice, the number of medullary islets increases to reach ~1 000 islets, as observed in neonates. Considering that the medulla topology is governed by crosstalk with autoreactive CD4 + thymocytes (32, 49-52), the high islet number observed in aged mice could be due to a suboptimal cellular crosstalk due to reduced cellularity of CD4 + thymocytes linked to age-related thymic involution. Although Aire plays multiple roles in T-cell tolerance induction [START_REF] Perniola | Twenty Years of AIRE[END_REF], our results show that it does not shape the 3D organization of the thymic medulla. This is consistent with the fact that the clonal deletion of autoreactive CD4 + thymocytes is impaired in Aire KO mice [START_REF] Anderson | The cellular mechanism of Aire control of T cell tolerance[END_REF][START_REF] Liston | Aire regulates negative selection of organ-specific T cells[END_REF], which consequently leads to an effective medulla organization.

Several studies have shown that mTEC are implicated in Foxp3 + Treg development [START_REF] Cowan | The thymic medulla is required for Foxp3+ regulatory but not conventional CD4+ thymocyte development[END_REF][START_REF] Aschenbrenner | Selection of Foxp3+ regulatory T cells specific for self antigen expressed and presented by Aire+ medullary thymic epithelial cells[END_REF][START_REF] Malchow | Aire-dependent thymic development of tumor-associated regulatory T cells[END_REF]. Nevertheless, the specific role of Aire in thymic Treg heterogeneity throughout life remained to be defined. Interestingly, we found that Aire controls the cellularity of Foxp3 lo TregP and mature CD25 + Foxp3 + Treg in 5-day-, 6-week-and 1-year-old mice. This is illustrated by decreased numbers of Foxp3 lo TregP and CD25 + Foxp3 + Treg in Aire KO mice, which could not be due to impaired medulla organization since no defect was observed at this level. Given that CD25 + TregP and Foxp3 lo TregP show distinct developmental pathways that give rise to CD25 + Foxp3 + Treg with non-overlapping regulatory activities (1), our results provide new insights in the role of Aire in the emergence of Foxp3 lo TregP. Furthermore, Aire regulates the pool of recirculating CCR6 + Treg throughout life. Interestingly, CCR6 + Treg from Aire KO mice expressed reduced levels of several genes associated with their polarization and suppressive functions.

Importantly, they express normal levels of Foxp3, indicating that they remain engaged in the Treg cell lineage. In accordance with the defective suppressive signature of CCR6 + Treg from Aire KO mice, we found that the adoptive transfer of these cells failed to attenuate the severity of multiorgan autoimmunity. In contrast to their thymic counterparts, splenic Treg of Aire KO mice show a protection similar to Aire WT splenic Treg, consistently with their normal suppressive signature. Altogether, these results indicate that Aire is crucial for the suppressive functions of recirculating CCR6 + Treg in the thymus.

Although Aire is expressed by a subset of recirculating thymic B cells (9), we found that its absence in hematopoietic cells had no impact neither in Treg development nor in CCR6 + Treg recirculation and suppressive signature. Considering the weak expression of Aire in thymic B cells compared to mTEC, it is not surprising that Aire deficiency in hematopoietic cells does not control the cellularity and suppressive signature of CCR6 + Treg. In marked contrast, beyond controlling the recirculation of CCR6 + Treg, BM chimeras in Aire KO recipients revealed that Aire expression in stromal cells is responsible for their highly suppressive phenotype.

Interestingly, recirculating Foxp3 + Treg were observed in close proximity to Aire + mTEC, similarly to developing Foxp3 + Treg. The reduced expression of Ccl20 and Cxcl12, likely responsible for the lower amount of recirculating Treg in these mice as previously reported [START_REF] Thiault | Peripheral regulatory T lymphocytes recirculating to the thymus suppress the development of their precursors[END_REF][START_REF] Cowan | Aire controls the recirculation of murine Foxp3(+) regulatory T-cells back to the thymus[END_REF], could contribute to a lesser stimulation of recirculating Treg by mTEC. However, in vitro co-culture experiments suggest that defective chemoattraction is unlikely responsible for the phenotype observed in Aire KO mice. Furthermore, in vitro co-culture assays revealed that Aire + mTEC were capable to activate peripheral Treg in an antigen-specific manner. Our results suggest that peripheral Treg could regulate de novo Treg development not only by competing for IL-2 (5, 57) but also for the cognate self-antigen. In the absence of Aire, in vitro co-culture assays and in vivo adoptive transfer experiments demonstrate that recirculating Treg activation and suppressive signature were altered. Therefore, our results unravel that Aire + mTEC control the activated and differentiated phenotype of recirculating Treg upon their entry into the thymus. A possible explanation could be that Aire controls co-stimulation signals that are responsible for the effector phenotype of recirculating Treg. In addition, Aire could also modulate indirectly CCR6 + Treg suppressive properties through other mechanisms such as the medullary positioning of XCR1 + type 1 conventional dendritic cells by controlling the production of the chemokine XCL1 [START_REF] Lei | Aire-dependent production of XCL1 mediates medullary accumulation of thymic dendritic cells and contributes to regulatory T cell development[END_REF]. Moreover, further investigations are required to determine the fate of CCR6 + Treg in the thymic medulla. Three possibilities can be envisaged: CCR6 + Treg (1) become long-term resident cells, (2) migrate back to the periphery or (3) die by apoptosis.

This study ameliorates our understanding on recirculating Treg in the thymus, which remain poorly described to date. In summary, it identifies that Aire controls the suppressive properties of recirculating CCR6 + Treg in the thymus. It also assigns a new role for Aire in conferring to mTEC the aptitude to restimulate recirculating Treg.

Thus, this study furthers our understanding on the mechanisms allowing recirculating Treg to fine-tune de novo Treg production. Finally, our results are expected to contribute to a better understanding in Treg deficiencies observed in the human pathology APECED. B Scatterplot representations of log10 gene expression levels (FPKM) in recirculating CCR6 + Treg from Aire WT and Aire KO mice. For representation purposes, expression values of genes below 0.01 were assigned to 0.01. Genes with fold difference > 2 and p-adj < 0.05 were considered as up-or down-regulated genes (red and green dots, respectively). RNA-seq was performed on 2 independent biological replicates derived from two mice. Aire + mTEC hi were identified as EpCAM + Ly51 -/lo CD80 + Aire eGFP cells and purified from Aire het (Aire eGFP/WT ) and Aire KO (Aire eGFP/eGFP ) mice. Table S1. FPKM values of RNA-seq data derived from thymic CCR6 + Treg from Aire WT and Aire KO mice.

Legends

C

Fig. 1 F

 1 Fig.1The medullary topology is dynamic throughout life, independently of Aire expression.

Fig. 2 A

 2 Fig. 2 Developing CCR6 -and recirculating CCR6 + Treg are reduced in Aire KO mice throughout life.

Fig. 3

 3 Fig.3The suppressive signature of recirculating CCR6 + Treg is impaired in the thymus of Aire KO mice.

  Heatmap of selected genes down-regulated in recirculating CCR6 + Aire KO Treg (Fold Change > 2) compared to their CCR6 + Aire WT counterparts and involved in Treg suppressive functions, Treg Th-like, helper T cell polarization, fat and muscle Treg. Two biological replicates are shown for each condition.

Fig. 4 A

 4 Fig. 4 The adoptive transfer of recirculating CCR6 + Treg of the Aire KO thymus fails to protect from multiorgan autoimmunity.

Fig. 5 ABE

 5 Fig. 5 Aire deficiency in the thymic stroma impairs the recirculation and the suppressive signature of CCR6 + Treg.

Fig. 6 .ACDFG

 6 Fig. 6. Aire + mTEC control the activation and suppressive signature of recirculating Treg through antigendependent contact.

Fig. S1

 S1 Fig. S1 Gating strategy used to purify recirculating CCR6 + Treg in the thymus.

Fig. S2

 S2 Fig. S2 The suppressive signature of thymic CCR6 + Treg from 1-year-old Aire KO mice is altered.

Fig. S3 SplenicABCD

 S3 Fig. S3 Splenic Aire KO Treg show a normal suppressive signature throughout life.

Fig. S5 AF

 S5 Fig. S5 Aire expression in hematopoietic cells does not control the recirculation and suppressive signature of thymic CCR6 + Treg.

Fig. S6

 S6 Fig. S6 Gating strategy used to purify Aire + mTEC hi .

Fig. S7 A

 S7 Fig. S7 Aire KO mTEC express reduced levels of OX40L and GITRL.

Aire WT lobe 1 Aire WT lobe 2 Aire KO lobe 1 Aire KO lobe 2 *

 1212 Figure 2

  Figure 5

  Fig. S3

Table S1 Gene Aire WT mice Aire KO mice

 S1

	Il10	3.09 ± 0.44	0.31 ± 0.19
	Gzmb	25.57 ± 6.90	4.83 ± 1.84
	Fasl	1.35 ± 0.66	0.92 ± 0.15
	Lag3	4.29 ± 0.53	2.07 ± 0.45
	Entpd1	10.57 ± 0.19	5.43 ± 0.38
	Nt5e	69.74 ± 11.42	59.41 ± 7.12
	Prdm1	18.78 ± 3.56	2.84 ± 0.06
	Klrg1	60.16 ± 9.26	12.96 ± 2.67
	Tigit	77.14 ± 16.33	23.54 ± 1.83
	Ctla4	259.10 ± 19.43	156.50 ± 2.15
	Lgals1	421.5 ± 20.12	244.0 ± 3.59
	Tbx21	5.22 ± 1.64	2.2 ± 0.32
	Irf4	20.71 ± 1.10	13.22 ± 1.40
	Pparg	2.54 ± 0.09	0.87 ± 0.39
	Id2	192.5 ± 25.10	34.59 ± 2.99
	Cxcr3	58.30 ± 14.08	14.94 ± 2.71
	Ccr4	35.47 ± 5.00	11.08 ± 0.12
	Ccr8	256.6 ± 35.04	136.7 ± 3.35
	Ccr1	3.67 ± 1.31	0.34 ± 0.13
	Ccr2	39.94 ± 2.88	7.26 ± 0.64

Acknowledgements

We are grateful to Georg Holländer (University of Basel, Switzerland) and Bernard Malissen (CIML, Marseille, France) for providing us Aire KO and Foxp3 eGFP mice, respectively. We thank the CIML flow cytometry, histology, PICSL imaging facility of the CIML (ImagImm) and animal facility platforms for technical support. We thank Cloé Zamit (CIML, France) for help with mouse genotyping.

Funding

This work was supported by institutional grants from INSERM, CNRS and Aix-Marseille Université. The Immune Tolerance and T-Cell Differentiation laboratory received funding from the ARC Foundation (PJA20171206491 to M.I.), CoPoC-proof of concept (MAT-PI-17326-A-01 to M.I.), a prematuration grant from A*MIDEX, a French "Investissements d'avenir" program (LTalpha-Treg to M.I.) and Agence Nationale de la Recherche (grant ANR-19-CE18-0021-01, RANKLthym to M.I.). We also acknowledge financial support from France Bio Imaging (ANR-10-INBS-04-01) and France Génomique national infrastructure, funded as part of the "Investissements d'Avenir" program managed by the ANR (ANR-10-INBS-0009). J.C. and A.B. were supported by a PhD fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche et de l'Innovation (MESRI).

Data availability

Code availability

Not applicable

Author contributions

JC, AB, JCS, LC, and MI conducted the experiments, analyzed and interpreted the data. MG and AS analyzed the data. JC, AB, JCS and MI wrote the manuscript. MI initiated, supervised and conceived the study.

All data generated or analyzed during this study are included in this published article and its supplementary information files. The dataset generated in this study are available in the Gene Expression Omnibus (GEO) database under accession number GSE188419.

Declarations Conflict of interests

The authors declare that they have no conflict if interest.

Ethical approval

All experiments were done in accordance with national and European laws for laboratory animal welfare (EEC Council Directive 2010/63/UE) and the Marseille Ethical Committee for Animal experimentation.

Consent to participate

Not applicable.

Consent to publication

Not applicable.

Table S2. List of antibodies used for flow cytometry.

Table S3. List of primers used for RT-qPCR. Movie S1. 3D rotation of Aire WT thymic lobe of 9-day-old mouse, with medullary compartment colored according to their volume.

Aire WT thymic lobe (DAPI, blue) of a 9-day-old mouse is rendered in 3D with medullary compartments (Keratin 14, pseudo-colors) encoded according to medullary volumes from cyan (smallest medullae) to magenta (largest medullae). All axes are graduated with 500-µm grid spacing. Movie S2. 3D rotation of Aire KO thymic lobe of 9-day-old mouse, with medullary compartment colored according to their volume.

Aire KO thymic lobe (DAPI, blue) of a 9-day-old mouse is rendered in 3D with medullary compartments (Keratin 14, pseudo-colors) encoded according to medullary volumes from cyan (smallest medullae) to magenta (largest medullae). All axes are graduated with 500-µm grid spacing.

Movie S3. 3D rotation of Aire WT thymic lobe of 6-week-old mouse, with medullary compartment colored according to their volume.

Aire WT thymic lobe (DAPI, blue) of a 6-week-old mouse is rendered in 3D with medullary compartments (Keratin 14, pseudo-colors) encoded according to medullary volumes from cyan (smallest medullae) to magenta (largest medullae). All axes are graduated with 500-µm grid spacing.

Movie S4. 3D rotation of Aire KO thymic lobe of 6-week-old mouse, with medullary compartment colored according to their volume.

Aire KO thymic lobe (DAPI, blue) of a 6-week-old mouse is rendered in 3D with medullary compartments (Keratin 14, pseudo-colors) encoded according to medullary volumes from cyan (smallest medullae) to magenta (largest medullae). All axes are graduated with 500-µm grid spacing.

Movie S5. 3D rotation of Aire WT thymic lobe of 1-year-old mouse, with medullary compartment colored according to their volume.

Aire WT thymic lobe (DAPI, blue) of a 1-year-old mouse is rendered in 3D with medullary compartments (Keratin 14, pseudo-colors) encoded according to medullary volumes from cyan (smallest medullae) to magenta (largest medullae). All axes are graduated with 500-µm grid spacing. Movie S6. 3D rotation of Aire KO thymic lobe of 1-year-old mouse, with medullary compartment colored according to their volume.

Aire KO thymic lobe (DAPI, blue) of a 1-year-old mouse is rendered in 3D with medullary compartments (Keratin 14, pseudo-colors) encoded according to medullary volumes from cyan (smallest medullae) to magenta (largest medullae). All axes are graduated with 500-µm grid spacing.


Corrine K Hanson

Sathish Kumar

Jiujiu Yu

Maëlle Quéré
email: maelle.quere@univ-lorraine.fr

Jean-Ma Alberto
email: jean-marc.alberto@univ-lorraine.fr

Franck Brol
email: franck.broly@chru-lille.fr

Sébastien Hergalant
email: sebastien.hergalant@inserm.fr

Christo Christov
email: christo.christov@univ-lorraine.fr

Guillaume Gauchotte
email: guillaume.gauchotte@univ-lorraine.fr

Jean-Louis Guéant
email: jean-louis.gueant@univ-lorraine.fr

Farès Namour

Shyue-Fang Battaglia-Hsu
email: shyue-fang.battaglia@univ-lorraine.fr

ALDH1L2 Knockout in U251 Glioblastoma Cells Reduces Tumor Sphere Formation by Increasing Oxidative Stress and Suppressing Methionine Dependency

Keywords: tumor sphere, glioblastoma, methionine, ROS, ALDH1L2

Previously, the in vitro growth of cancer stem cells in the form of tumor spheres from five different brain cancer cell lines was found to be methionine-dependent. As this earlier work indicated that ALDH1L2, a folate-dependent mitochondria aldehyde dehydrogenase gene, is upregulated in glioblastoma stem cells, we invalidated this gene using CRISPR-cas 9 technique in this present work. We reported here that this invalidation was effective in U251 glioblastoma cells, and no cas9 off target site could be detected by genome sequencing of the two independent knockout targeting either exon I or exon III. The knockout of ALDH1L2 gene in U251 cells rendered the growth of the cancer stem cells of U251 methionine independent. In addition, a much higher ROS (reactive oxygen radicals) level can be detected in the knockout cells compared to the wild type cells. Our evidence here linked the excessive ROS level of the knockout cells to reduced total cellular NADPH. Our evidence suggested also that the cause of the slower growth of the knockout turmor sphere may be related to its partial differentiation.

Background

Methionine is the precursor of the universal methyl donor, S-adenosyl-methionine, for most methylation reactions taking place inside cells. Methionine cycle and folate cycle together constitute the key components of the one carbon (1C) metabolism used by cells to feed 1C units to various intermediate metabolites for biological processes [START_REF] Sanderson | Nutrient Availability Shapes Methionine Metabolism in P16/MTAP-Deleted Cells[END_REF]. 1C metabolism takes place in both cytosol and mitochondria, and these two compartments are linked through formate produced by the mitochondria folate cycle. Once produced, mitochondrial formate moves freely into cytoplasm for conversion to downstream cytosolic folates for de novo synthesis of either methionine or nucleic acids [START_REF] Yang | Serine and One-Carbon Metabolism in Cancer[END_REF]. One major byproduct of folate cycle is NADPH [START_REF] Fan | Quantitative Flux Analysis Reveals Folate-Dependent NADPH Production[END_REF], a crucial electron donor powering cellular level anabolic and redox reactions.

Metabolic reprogramming in pluripotent stem cells and cancer stem cells (CSC) share many similarities. Among them, the methionine-dependent growth [START_REF] Shiraki | Methionine Metabolism Regulates Maintenance and Differentiation of Human Pluripotent Stem Cells[END_REF][START_REF] Lamb | Targeting Tumor-Initiating Cells: Eliminating Anabolic Cancer Stem Cells with Inhibitors of Protein Synthesis or by Mimicking Caloric Restriction[END_REF] and the hypersensitivity to reactive oxygen species (ROS), the latter due to reprogramming of NADPH metabolic pathway that renders these cells highly sensitive to oxidative stress [START_REF] Ju | NADPH Homeostasis in Cancer: Functions, Mechanisms and Therapeutic Implications[END_REF]. In fact, ROS level plays a key role in the control of stem cell fate as a high ROS level can trigger senescence and death [START_REF] Bigarella | Stem Cells and the Impact of ROS Signaling[END_REF], whereas a lower ROS level can help to preserve stemness [START_REF] Liao | Antioxidants Inhibit Cell Senescence and Preserve Stemness of Adipose Tissue-Derived Stem Cells by Reducing ROS Generation during Long-Term in Vitro Expansion[END_REF] and cause tumor radioresistance [START_REF] Diehn | Association of Reactive Oxygen Species Levels and Radioresistance in Cancer Stem Cells[END_REF]. It is worth noting that the ROS signaling in CSC is especially dynamic, shaped not only by the constantly changing microenvironment but also by the metabolic plasticity of the CSC. Such plasticity is likely a key adaptive mechanism of these cells to ensure their survival. Our previous work suggested that 1C metabolism takes part in this adaptive response to maintain the CSC population in U251 glioblastoma cells.

The ROS scavenging aldehyde dehydrogenases (ALDH) have been used as markers for stem cells [START_REF] Vassalli | Aldehyde Dehydrogenases: Not Just Markers, but Functional Regulators of Stem Cells[END_REF]. These enzymes have multiple functional roles in CSCs, including also aldehyde detoxification and retinoic acid signaling [START_REF] Muralikrishnan | Targeting Aldehyde Dehydrogenases to Eliminate Cancer Stem Cells in Gynecologic Malignancies[END_REF]. They are implicated in therapeutic resistance to chemotherapy [START_REF] Tanei | Association of Breast Cancer Stem Cells Identified by Aldehyde Dehydrogenase 1 Expression with Resistance to Sequential Paclitaxel and Epirubicin-Based Chemotherapy for Breast Cancers[END_REF][START_REF] Duong | Aldehyde Dehydrogenase 1A1 Confers Intrinsic and Acquired Resistance to Gemcitabine in Human Pancreatic Adenocarcinoma MIA PaCa-2 Cells[END_REF], and pharmacological inhibitors of ALDH activity can abolish stem cell characteristics [START_REF] Rausch | Synergistic Activity of Sorafenib and Sulforaphane Abolishes Pancreatic Cancer Stem Cell Characteristics[END_REF]. ALDH1L2, 10-formyltetrahydrofolate dehydrogenase and a mitochondrial 1C cycle folate enzyme, can reduce ROS production and promote metastasis in certain contexts [START_REF] Piskounova | Oxidative Stress Inhibits Distant Metastasis by Human Melanoma Cells[END_REF]. In the neuro-ichthyotic syndromes, the only known human diseases associated with ALDH1L2 mutations, the loss of ALDH1L2 activity causes diminished NADPH production, elevated ROS, increased oxidative stress, and impaired mitochondrial function [START_REF] Sarret | Deleterious Mutations in ALDH1L2 Suggest a Novel Cause for Neuro-Ichthyotic Syndrome[END_REF]. NADPH combats oxidative stress and acts either by generating reduced glutathione (GSH) or as a direct antioxidant [START_REF] Kirsch | a Directly Operating Antioxidant?[END_REF]. Thus, a loss of function of ALDH(1L2) can exacerbate oxidative stress and mitochondrial dysfunction.

1C metabolism plays a pivotal role in stem cell proliferation as methionine cycle is capable of providing both methionine and SAM for the proliferation of these cells, and folate cycle can supply NADPH for antioxidative protection against ROS. In earlier work, the methionine growth dependency of the CSC of U251 glioblastoma was attributed to reduced 5-methyl-tetrahydrofolate [START_REF] Zgheib | Folate Can Promote the Methionine-Dependent Reprogramming of Glioblastoma Cells towards Pluripotency[END_REF]. Comparative transcriptomics between the differentiated and CSC U251 cells revealed potential activation of the mitochondrial folate cycle in CSC as several key mitochondria folate enzymes, including ALDH1L2, are upregulated while several cytosolic folate enzymes are downregulated in these CSC cells.

The objective of this current work was thus to determine the role played by the upregulated ALDH1L2 in CSC obtained in vitro in the form of tumor sphere (TS). To achieve this, we invalidated ALDH1L2 using a CRISPR Cas9 system. As ALDH1L2 converts mitochondrial 1C donor 10-formylTHF to CO 2 and THF, we expected that its knockout will help to preserve the 1C units, and hence render the growth of CSC methionineindependent. In addition, because the cofactor needed for this ALDH1L2-dependent oxidation of mitochondrial 10-formylTHF is NADP + , we expected that this knockout will reduce the cellular content of NADPH. As NADPH is a major reducing power for anti-oxidative defense, we expected to see an increased ROS level in the CSC knockout cells.

Methods

Generation of ALDH1L2 CRSPR Knockouts (KO)

To create ALDH1L2 KO cells, U251 were transfected with the lentivirus particles. This was followed by cloning. For each isolated clone, ALDH1L2 gene deletion was evaluated by targeted Sanger sequencing as well as genome sequencing (Supplemental Methods).

Whole Genome Sequencing (WGS)

Whole genome sequencing (WGS) and primary analysis of the results were performed by BGI (Beijing Genomics Institute, Shen-Zhen, China). The secondary and tertiary analyses (alignment of reads on the reference human genomic sequence, detec-tion_annotation_visualization of variants) were carried out (Supplemental Methods).

Metabolites Analysis

Metabolites analysis including Methionine, Homocysteine, SAM, SAH, total Cysteine, Cystathionine, total Glutathione, Choline, Betaine, Glycine, Serine, methyl-THF and formyl-THF were realized by LCMS (LCMS 8045, Shimadzu, Kyoto, Japan) on a Kinetex column (Kinetex 00D-4462-EO). These measurements were performed as detailed in the Supplemental Methods section.

Statistical Analysis

Data generated by cell experiments were analyzed with ANOVA and Student's t-test and were presented as mean ± standard error of the mean (s.e.m). All the reported p-values are two-sided, and p-values < 0.05 are considered statistically significant.

Results

CRISPR-Cas9 Targeting Either Exon 1 or Exon 3 of ALDH1L2 Abolishes ALDH1L2 Expression

In a previous study comparing stem-like verse differentiated glioblastoma cells established under in vitro culture conditions as tumor sphere (TS), we observed differential folate enzyme expressions, with tumor spheres showing higher mitochondria folate activity [START_REF] Zgheib | Folate Can Promote the Methionine-Dependent Reprogramming of Glioblastoma Cells towards Pluripotency[END_REF]. The higher expression of the mitochondrial ALDH1L2 in TS was particularly noted. In the present study, we hypothesized that ALDH1L2 is crucial for the formation of tumor spheres in glioblastoma.

To test, we produced two lines of ALDH1L2 KO U251 glioblastoma cells using the CRISPR-Cas 9 targeting exon 1 and exon 3 of the human ALDH1L2, respectively. Sanger sequencing of the ALDH1L2 KO clones showed bi-allelic disruption of the targeted exon 1 and 3. For exon 1, we identified two INDELs. In Figure 1II, their Cas 9 break points are indicated by " " on the wild type sequence. The mutant sequences diverge from that of the wild type by a deletion of 42 bp in one and 62 bp in the other clone. The 42 bp deletion includes the 24 bp at the end of exon 1 and the 18 bp at the beginning of the intron 1 (IIb). The 62 bp deletion includes the 33 bp at the end exon 1 and 29 bp at the beginning of intron 1 (IIc). The consequences of these deletions on the protein synthesis are unpredictable since they include the 5 end of intron 1. For exon 3, two clones were also identified, carrying a 14 pb and a 23 bp deletion, respectively. In Figure 1IId, the positions of the two deletions are marked by " " and " " on the wild type sequence. The final sequences of the exon 3 deletion mutants are shown in e and f, with " " and " " indicating the Cas 9 breaking points. Both exon 3 deletions were mapped and expected to yield the same truncated protein consequent to a stop codon generated at position 113. We further confirmed the relative ALDH1L2 expressions in the wild type and mutant clones under differentiated monolayer and non-adherent TS culture conditions using the Simple Western WES TM protein analysis platform as well as immunohistological staining (Figure 1III).

To determine the off-target sites of the selected CRISPR-Cas9 ALDH1L2 knockout cells, we performed whole genome sequencing. After mapping sequence reads to the reference genome, we used several software programs to examine the patterns of sequence alignment both near the on-target site and other sites in the genome to determine the possible presence of cas9 off-targets. Analyses of the reads spanning the cleavage site bound by CRIPSR-Cas9 in ALDH1L2 exons 1 and 3 revealed the same indels identified by the Sanger sequencing. Nucleotide variations were also observed in several other regions. Closer inspection of the reads alignment there revealed that they are all located in regions of poor sequence quality, as observed as well in the wild type cells. Thus, the staggered alignment (see Supplement Figure S1) at these other sites is not due to the "off-target" effect of the CRIPSR-cas9 system, but rather to incorrect base callings.

Tumor Sphere (TS) Growth of the ALDH1L2 KO Cells are Slower and Less Methionine-Dependent

Armed with the ALDH1L2 knockout clones, we examined the growth of TS derived from these cells either with or without exogenous methionine. We confirmed here that wild type U251 TS growth indeed requires methionine; however, this dependency is abolished in KO cells for both exon 1 (AL1) and exon 3 (AL3) KO cells (Figure 2III). As 0.01 mM appeared to be the optimal methionine concentration for wild type U251 TS growth, we compared the third passage of TS growth during an 8-day period under the condition of either methionine-free (MET-) or with 0.01 mM methionine (MET+) (Figure 2Ia). Specifically, on the 8th day, the impact of methionine on TS growth was compared. With MET-(Figure 2Ic), the total TS numbers obtained were 3.09 ± 0.89 (WT as 100%) vs. 8.76 ±

SAM 4 M R L R R G S Q A L R R F S T G M L R R G M L R R G S Q A intron 1 shortened intron 1 shortened A E A Y R S V G A E L N V A E A Y R A E A

Tumor Sphere (TS) Growth of the ALDH1L2 KO Cells Are Slower and Less Methionine-Dependent

Armed with the ALDH1L2 knockout clones, we examined the growth of TS derived from these cells either with or without exogenous methionine. We confirmed here that wild type U251 TS growth indeed requires methionine; however, this dependency is abolished in KO cells for both exon 1 (AL1) and exon 3 (AL3) KO cells (Figure 2III). As 0.01 mM appeared to be the optimal methionine concentration for wild type U251 TS growth, we compared the third passage of TS growth during an 8-day period under the condition of either methionine-free (MET-) or with 0.01 mM methionine (MET+) (Figure 2Ia). Specifically, on the 8th day, the impact of methionine on TS growth was compared. With MET-(Figure 2Ic), the total TS numbers obtained were 3.09 ± 0.89 (WT as 100%) vs. 8.76 ± 1.03 (AL1, 284%) and 9.79 ± 1.37 (AL3, 317%) (p < 0.05 for AL1 and p < 0.05 for AL3, respectively). With MET+, the total TS numbers obtained were 18.55 ± 2.68 (WT as 100%) vs. 15.63 ± 2.41 (AL1, 84%) and 11.85 ± 0.52 (AL3, 64%) (p = 0.46 and 0.07, respectively) (Figure 2Id MET+). We also compared the ratio of TS number (MET+)/TS number (MET-) for each cell line (Figure 2Ib). For wild type, this MET+/MET-ratios were 7.83 ± 3.59, while, for the two knockout cells, they were 1.89 ± 0.45, and 1.27 ± 0.21, respectively (n = 3, p < 0.05). These analyses all confirmed that wild type TS cells are much more methionine-dependent than either of the knockout cells. In addition to the TS number, we noted that the TS formed from the ALDH1L2 KO cells were generally smaller than those observed from the wild type cells (Figure 2II; 194,830 µm 3 and 229,430 µm 3 vs. 610,190 µm 3 , n = 27 and n = 28 vs. n = 32 for AL1, AL3 and U251wt, respectively; p < 0.03).

1.03 (AL1, 284%) and 9.79 ± 1.37 (AL3, 317%) (p < 0.05 for AL1 and p < 0.05 for AL3, respectively). With MET+, the total TS numbers obtained were 18.55 ± 2.68 (WT as 100%) vs. 15.63 ± 2.41 (AL1, 84%) and 11.85 ± 0.52 (AL3, 64%) (p = 0.46 and 0.07, respectively) (Figure 2Id MET+). We also compared the ratio of TS number (MET+)/TS number (MET-) for each cell line (Figure 2Ib). For wild type, this MET+/MET-ratios were 7.83 ± 3.59, while, for the two knockout cells, they were 1.89 ± 0.45, and 1.27 ± 0.21, respectively (n = 3, p < 0.05). These analyses all confirmed that wild type TS cells are much more methionine-dependent than either of the knockout cells. In addition to the TS number, we noted that the TS formed from the ALDH1L2 KO cells were generally smaller than those observed from the wild type cells (Figure 2II; 194,830 µm 3 and 229,430 µm 3 vs. 610,190 µm 3 , n = 27 and n = 28 vs. n = 32 for AL1, AL3 and U251wt, respectively; p < 0.03).

Figure 2. Tumor spheres (TS) derived from U251 ALDH1L2 KO cells display not only decreased growth but also methionine-independent growth. TS growth in methionine free (MET-) vs. methionine (0.01 mM)-containing (MET+) media were compared between KO vs. wt cells. In (Ia), in MET-, TS of the KO cells grew better than that of the wt. In (Ib), the TS growth ratio was calculated as TS growth in MET+) divided by that in MET-. The higher the ratio, the more the methionine growth dependency. In (Ic), TS growth in MET-was normalized to that of the wt TS in MET-. In (Id), TS growth in MET+ was normalized to that of the wt TS MET+. In (II), analyses of the computer-assisted reconstruction of sphere volumes revealed that ALDH1L2 KO has smaller "tumor sphere volume". A, A1, A2 = wt cells. B, B1, B2 = AL1 cells. C, C1, C2 = AL3 cells. A1, B1, C1 are results of segmentation. A2, B2, C2 are results of 3D reconstructions: axes values are in pixels, and images are not to scale, so size differences can be appreciated visually in A1, B1, C1 but are best demonstrated by measured volumes: A = 1,857,000 µm 3 , B = 258,000 µm 3 , C = 206,000 µm 3 . D: Spheroid volumes versus wt (µm 3 ), mean ± SD, * p < 0.003, Student t-test. In (III), ALDH1L2 KO suppressed U251 TS growth methionine dependency. For each cell type, mean ± SE of three independent experiments (each done in quadruplicate) is shown (three biological replicates each include four technical replicates). wt: wild type, AL1: U251 cells ALDH1L2 exon 1 KO, AL3: U251 cells ALDH1L2 exon 3 KO.

ALDHL2 Knockout U251 Cells Have Reduced NADPH but Higher Cytosolic Methyl Donors in Tumor Spheres

In mitochondria, a number of substrates such as serine provide 1C units to produce mitochondrial 10-formyl-THF for downstream processes to either be oxidized to CO2 with simultaneous reduction of NADP+ to NADPH, or be transformed into formate, which, in Tumor spheres (TS) derived from U251 ALDH1L2 KO cells display not only decreased growth but also methionine-independent growth. TS growth in methionine free (MET-) vs. methionine (0.01 mM)-containing (MET+) media were compared KO vs. wt cells. In (Ia), in MET-, TS of the KO cells grew better than that of the wt. In (Ib), the TS growth ratio was calculated as TS growth in MET+) divided by that in MET-. The higher the ratio, the more the methionine growth dependency. In (Ic), TS growth in MET-was normalized to that of the wt TS in MET-. In (Id), TS growth in MET+ was normalized to that of the wt TS MET+. In (II), analyses of the computer-assisted reconstruction of sphere volumes revealed that ALDH1L2 KO has smaller "tumor sphere volume". A, A1, A2 = wt cells. B, B1, B2 = AL1 cells. C, C1, C2 = AL3 cells. A1, B1, C1 are results of segmentation. A2, B2, C2 are results of 3D reconstructions: axes values are in pixels, and images are not to scale, so size differences can be appreciated visually in A1, B1, C1 but are best demonstrated by measured volumes: A = 1,857,000 µm 3 , B = 258,000 µm 3 , C = 206,000 µm 3 . D: Spheroid volumes versus wt (µm 3 ), mean ± SD, * p < 0.003, *** p < 0.001, Student t-test. In (III), ALDH1L2 KO suppressed U251 TS growth methionine dependency. For each cell type, mean ± SE of three independent experiments (each done in quadruplicate) is shown (three biological replicates each include four technical replicates). wt: wild type, AL1: U251 cells ALDH1L2 exon 1 KO, AL3: U251 cells ALDH1L2 exon 3 KO.

ALDHL2 Knockout U251 Cells Have Reduced NADPH but Higher Cytosolic Methyl Donors in Tumor Spheres

In mitochondria, a number of substrates such as serine provide 1C units to produce mitochondrial 10-formyl-THF for downstream processes to either be oxidized to CO 2 with simultaneous reduction of NADP+ to NADPH, or be transformed into formate, which, in turn, is integrated into a cytosolic folate cycle (see Figure 1I). These competing pathways are carried out by two distinct mitochondrial enzymes ALDH1L2 and MTHFD1L, respectively. This bifurcation is a key mechanism determining the fate of 1C units. In tumor spheres of the ALDH1L2 knockout cells, this bifurcation is expected to tilt towards the production of mitochondria formate, leading to reduced mitochondria NADPH production while simultaneously fueling the generation of cytosolic 10-formyl THF to supply the cytosolic methionine cycle. These predictions were confirmed for TS cells (Figure 3). Relative to wild type TS, ALDH1L2 knockout TS have significantly less total cellular NADPH (347.7 vs. 84.5 and 74.1 umol/g protein, respectively, for wt and AL1 and AL3 KO; n = 3, p < 0.01). As a result, the relative differences in the normalized TS to adherent NADPH ratio are greater than those not normalized (3.10 vs. 0.53 and 0.42, respectively for wt and AL1 and AL3 KO; n = 3, p < 0.0005). Similar changes in NADP + were not observed; furthermore, we observed, in general, more 100-fold lower NADP + level in these three types of cells, and no significant difference between TS vs. ML or WT vs. KO cells (n = 3, p = 0.7). We expected also increased levels of the cytosolic folates. This was indeed the case. More whole cell folate isoforms such as formyl THF (0.001 vs. 0.010 and 0.009 µmol/g protein for wt vs. AL1 and AL3 KO, respectively n = 3, p < 0.007) and methyl THF (0.004 vs. 0.05 and 0.01 µmol/g protein for wt vs. AL1 and AL3 KO, n = 3, p < 0.0007) were detected along with increased levels of methionine, serine and glycine. The latter increases in 1C-bearing amino acids suggest that ALDH1L2 in cancer stem cells consumes an important amount of 1C units for the production of NADPH.

Nutrients 2022, 14, x FOR PEER REVIEW 6 of 11 turn, is integrated into a cytosolic folate cycle (see Figure 1I). These competing pathways are carried out by two distinct mitochondrial enzymes ALDH1L2 and MTHFD1L, respectively. This bifurcation is a key mechanism determining the fate of 1C units. In tumor spheres of the ALDH1L2 knockout cells, this bifurcation is expected to tilt towards the production of mitochondria formate, leading to reduced mitochondria NADPH production while simultaneously fueling the generation of cytosolic 10-formyl THF to supply the cytosolic methionine cycle. These predictions were confirmed for TS cells (Figure 3). Relative to wild type TS, ALDH1L2 knockout TS have significantly less total cellular NADPH (347.7 vs. 84.5 and 74.1 umol/g protein, respectively, for wt and AL1 and AL3 KO; n = 3, p < 0.01). As a result, the relative differences in the normalized TS to adherent NADPH ratio are greater than those not normalized (3.10 vs. 0.53 and 0.42, respectively for wt and AL1 and AL3 KO; n = 3, p < 0.0005). Similar changes in NADP + were not observed; furthermore, we observed, in general, more 100-fold lower NADP + level in these three types of cells, and no significant difference between TS vs. ML or WT vs. KO cells (n = 3, p = 0.7). We expected also increased levels of the cytosolic folates. This was indeed the case. More whole cell folate isoforms such as formyl THF (0.001 vs. 0.010 and 0.009 µmol/g protein for wt vs. AL1 and AL3 KO, respectively n = 3, p < 0.007) and methyl THF (0.004 vs. 0.05 and 0.01 µmol/g protein for wt vs. AL1 and AL3 KO, n = 3, p < 0.0007) were detected along with increased levels of methionine, serine and glycine. The latter increases in 1C-bearing amino acids suggest that ALDH1L2 in cancer stem cells consumes an important amount of 1C units for the production of NADPH.

ALDH1L2 KO Cells Bear More ROS Despite No Change in Glutathione Content

We subsequently determined the level of GSH and total glutathione as their changes can signal their involvement in NADPH-linked anti-oxidative response of the mitochondria. As shown in Figure 3II, no significant differences were detected between the three

ALDH1L2 KO Cells Bear More ROS despite No Change in Glutathione Content

We subsequently determined the level of GSH and total glutathione as their changes can signal their involvement in NADPH-linked anti-oxidative response of the mitochondria. As shown in Figure 3II, no significant differences were detected between the three cell types in either TS or differentiated ML cells. As in normal cells, the redox homeostasis of the mitochondria is maintained mainly by glutathione, glutaredoxin, and thioredoxin systems, and we examined these systems in the U251 cell transcriptomes obtained from our previous study comparing the TS cells and the differentiated ML cells. No difference in the transcription of TXN, GSR and GPX family genes was found. No change was also found for another known redox buffer system peroxiredoxin III and its regulator sulfiredoxin [START_REF] Toledano | Keeping Oxidative Metabolism on Time: Mitochondria as an Autonomous Redox Pacemaker Animated by H2O2 and Peroxiredoxin[END_REF]. The mitochondrial SOD2, however, was notably increased in the TS cells of U251, suggesting its involvement as the main antioxidative mechanism in TS cells of U251. In addition, we also examined the expression for the NOX genes since they may cause increased H 2 O 2 and a free oxygen radical level. Their transcription levels were nondetectable.

The impact of reduced NADPH levels on overall ROS levels of the ALDH1L2 KO TS cells were examined as ROS has been established as a negative regulator for stemness genes in both normal as well as cancer stem cells including glioblastoma stem-like cells [START_REF] Pourbagher | Downregulation of Stemness Genes and Induction of Necrosis in Rat LA7 Cancer Stem Cells Induced Tumors Treated with Starved Fibroblasts Culture Supernatant[END_REF][START_REF] Buccarelli | Elesclomol-Induced Increase of Mitochondrial Reactive Oxygen Species Impairs Glioblastoma Stem-like Cell Survival and Tumor Growth[END_REF]. We adapted a commercially developed non-fluorescence cell permeable ROS detection dye broadly used to detect the ROS in live cells [START_REF] Mertens | Age-Dependent Instability of Mature Neuronal Fate in Induced Neurons from Alzheimer's Patients[END_REF]. It reacts directly with wide varieties of reactive species like hydrogen peroxide, peroxynitrite and hydroxyl radicals, and resulting in products emitting green fluorescence capable of detection by microscopy to indicate the cellular contents of ROS/RNS (see Supplemental Methods). As shown in Figure 3II, the ROS level in wild type is significantly less than that observed in both AL1 and AL3 knockout cells in both ML and TS states (9.15 vs. 26.97 and 14.91 for ML cells (n = 5 vs. 6 and 5 in wt vs. AL1 and AL3 KO, respectively; p < 0.004) and 8.61 vs. 22.82 and 21.50 for TS cells (n = 8 vs. 13 and 6 in wt vs. AL1 and AL3, respectively; p < 0.007).

Mitochondria Morphological Studies

Previous evidence has suggested that, for non-neoplastic stem cells, there is a close connection between stemness and mitochondrial morphology and function, with fewer mitochondria and less developed cristae (hence, less OXPHOS metabolism) associated with less differentiated cells [START_REF] Loureiro | Mitochondrial Biology in Cancer Stem Cells[END_REF]. In cancer stem cells, although mitochondria dysfunction is linked to the so-called hallmarks of cancer [START_REF] Galluzzi | Mitochondrial Gateways to Cancer[END_REF][START_REF] Kroemer | Tumor Cell Metabolism: Cancer's Achilles' Heel[END_REF], no fixed metabolic phenotype is associated with cancer stems cells. For example, cancer stem cells of the glioblastoma [START_REF] Janiszewska | Imp2 Controls Oxidative Phosphorylation and Is Crucial for Preserving Glioblastoma Cancer Stem Cells[END_REF] along with lung cancer [START_REF] Ye | Mitochondrial and Energy Metabolism-Related Properties as Novel Indicators of Lung Cancer Stem Cells[END_REF] and pancreatic ductal adenocarcinoma [START_REF] Sancho | MYC/PGC-1α Balance Determines the Metabolic Phenotype and Plasticity of Pancreatic Cancer Stem Cells[END_REF] have been found to exhibit OXPHOS metabolic phenotype while breast cancer [START_REF] Ciavardelli | Breast Cancer Stem Cells Rely on Fermentative Glycolysis and Are Sensitive to 2-Deoxyglucose Treatment[END_REF], colon cancer [START_REF] Emmink | The Secretome of Colon Cancer Stem Cells Contains Drug-Metabolizing Enzymes[END_REF], and glioblastoma U87 cells [START_REF] Zhou | Metabolic Alterations in Highly Tumorigenic Glioblastoma Cells: Preference for Hypoxia and High Dependency on Glycolysis[END_REF] exhibit glycolytic phenotype. Tumor tissue types and tumor microenvironment may thus all contribute to the metabolic plasticity of the cancer stem cells.

Here, to know the metabolic phenotype of the U251 cells and the effect of knocking out ALDH1L2, we compared the morphology of the mitochondria of the wild type vs. the knockout cells. As shown in Figure 4, in wild type U251 cells, the mitochondria of the TS cells is much more fragmented than those of the ML cells. This difference between mitochondria network of the TS vs. ML cells appears to diminish with the knockout of the ALDH1L2. In knockout TS cells, the organization of the mitochondria network looks more like that of the ML cells. Besides this difference, we noted that the TS of the knockout cells exhibits a much greater tendency to differentiate into ML cells when the TS was placed on poly-lysine coated cover glass (data not shown). Within less than 30 min, the TS of the knockout flattened out onto the cover slide, with a fully developed mitochondria network identical to that of the ML cells.

Conclusions

One key feature of cancer cells is metabolic reprogrammation. The molecular details of the reprogramming of 1C metabolism in cancer are not well known. Even fewer studies have been published on the mechanisms by which altered 1C metabolism may influence the fate of cancer stem cell (CSC). Using a 3D in vitro culture technique [START_REF] Weiswald | Spherical Cancer Models in Tumor Biology[END_REF], we studied the CSC of the glioblastoma in the form of tumorospheres (TS), and found a profound alteration in 1C metabolism at the level of a mitochondria folate cycle [START_REF] Zgheib | Folate Can Promote the Methionine-Dependent Reprogramming of Glioblastoma Cells towards Pluripotency[END_REF]. This earlier work revealed that mitochondria folate enzyme ALDH1L2 is much more expressed in TS than in differentiated glioblastoma cells; in addition, the growth of the TS cells is much more methionine dependent than that of the differentiated cells. To understand these differences, we created the knockout of ALDH1L2. Here, we report the creation and the consequences of the ALDH1L2 knockout in U251 cells.

ALDH1L2 protein is a 1C unit spender. This mitochondria folate-dependent aldehyde dehydrogenase takes the 1C from the formyl group of its immediate substrate 10-formylTHF and converts it into CO2. As this conversion requires NADP as an electron acceptor, it produces NADPH as a consequence. A knockout of this enzyme may thus abolish the methionine-dependence of the TS growth. Our data here showed that this is indeed the case. Such reprogramming of 1C metabolism in wild type TS cells is expected to improve their reducing power for anabolic reactions and redox balance, albeit it can end up with a reduced cellular proliferation if exogenous methionine is not provided. The carbon wasting of ALDH1L2 thus explains the dependence of TS cells on methionine.

Besides the normalized methionine dependence, the TS of the ALDH1L2 knockouts exhibit two other features suggestive of the possibility that these KO TS cells are less stemlike than the wild type TS cells. First, the morphological difference between mitochondria

Conclusions

One key feature of cancer cells is metabolic reprogrammation. The molecular details of the reprogramming of 1C metabolism in cancer are not well known. Even fewer studies have been published on the mechanisms by which altered 1C metabolism may influence the fate of cancer stem cell (CSC). Using a 3D in vitro culture technique [START_REF] Weiswald | Spherical Cancer Models in Tumor Biology[END_REF], we studied the CSC of the glioblastoma in the form of tumorospheres (TS), and found a profound alteration in 1C metabolism at the level of a mitochondria folate cycle [START_REF] Zgheib | Folate Can Promote the Methionine-Dependent Reprogramming of Glioblastoma Cells towards Pluripotency[END_REF]. This earlier work revealed that mitochondria folate enzyme ALDH1L2 is much more expressed in TS than in differentiated glioblastoma cells; in addition, the growth of the TS cells is much more methionine dependent than that of the differentiated cells. To understand these differences, we created the knockout of ALDH1L2. Here, we report the creation and the consequences of the ALDH1L2 knockout in U251 cells.

ALDH1L2 protein is a 1C unit spender. This mitochondria folate-dependent aldehyde dehydrogenase takes the 1C from the formyl group of its immediate substrate 10-formylTHF and converts it into CO 2 . As this conversion requires NADP as an electron acceptor, it produces NADPH as a consequence. A knockout of this enzyme may thus abolish the methionine-dependence of the TS growth. Our data here showed that this is indeed the case. Such reprogramming of 1C metabolism in wild type TS cells is expected to improve their reducing power for anabolic reactions and redox balance, albeit it can end up with a reduced cellular proliferation if exogenous methionine is not provided. The carbon wasting of ALDH1L2 thus explains the dependence of TS cells on methionine.

Besides the normalized methionine dependence, the TS of the ALDH1L2 knockouts exhibit two other features suggestive of the possibility that these KO TS cells are less stemlike than the wild type TS cells. First, the morphological difference between mitochondria of the wt vs. that of the KO TS is indicative of the fact that the metabolic phenotype of the former may be less OXPHOS (oxidative phosphorylation) than that of the latter, as the former shows a far-more extensive mitochondrial network. In fact, when placing the tumor spheres onto cover glasses coated with polylysine for observation under a microscope, the KO TS quickly attached onto the cover glass, and flattened into adherent monolayer-like cells with an extensive mitochondria network; no similar transformation was observed for wt TS. Second, consistent with the morphological finding, using Oxygraphy, we found the metabolic phenotype of the knockout tumorospheres tend to be more OXPHOS as the Bioenergetic Health Index [START_REF] Chacko | The Bioenergetic Health Index: A New Concept in Mitochondrial Translational Research[END_REF] is significantly higher in KO vs. wild type cells (data not-shown).

In conclusion, our evidence reported here supports the notion that the upregulation of ALDH1L2 is key to the formation of cancer stem cells of the U251 glioblastoma cells, and its function appears to be related to the production of NADPH via the utilization of 1C-units delivered by mitochondria 10-formyl THF. As this conversion consumes 1C-units, it reduces the level of mitochondria formate, and hence the level of cytosolic formate, the 1C-unit donor needed for cytosolic methionine synthesis. This leads to the methionine dependence of the CSC formation reported in our earlier work [START_REF] Zgheib | Folate Can Promote the Methionine-Dependent Reprogramming of Glioblastoma Cells towards Pluripotency[END_REF]. We also suggest that the increase in NADPH production generated by the upregulation of ALDH1L2 is used to reduce the ROS level, a condition needed to maintain the stemness characteristic of the cancer stem cells.

Figure 1 .

 1 Figure 1. ALDH1L2 KO U251 glioblastoma cells were generated using the CRISPER-Cas9 system (I) One carbon metabolism features linked mitochondrial and cytosolic pathways; folate and méthionine cycles are intertwinned in the cytosol. THF: TetraHydroFolate, 5,10-meTHF: 5,10 methylene THF, 10-fTHF: 10-formylTHF, 5-mTHF: 5 methyl THF, SAM: S-adenosyl methionine, SAH: S-adenosyl homocysteine. (II) Genomic Sanger sequencing of individual deletion clones shows bi-allelic disruption of targeted exons 1 and 3. For exon 1, the new sequences diverge from the wild type sequence by deletions of 42 and 62 bp. Wild type sequence is shown in (a) with arrows indicating the breaking points. Sequencing of the 42 deletion including 24 bp of exon 1 and 18 bp of intron 1 is shown in (b) and sequencing of the 62 bp deletion including 33 bp of exon 1 and 29 bp of intron 1 in (c). The consequences of these deletions on protein synthesis are unpredictable since they carry away the 5′ end of intron 1. For exon 3, the divergence is due to deletions of 14 and 23 bp. Wild type sequence is shown in (d) with top and bottom arrows indicating the 14 and 23 bp deletion boundaries inside exon 3. Sequencing of the 14 and 23 deleted exon 3 is shown in (e) and (f) with arrows indicating the breaking points. Both deletions were mapped and confirmed to yield the same truncated protein with a stop codon generated at codon 113 of the newly synthesized proteins. Sequences targeted by the sgRNA are underlined in solid lines and PAM sequences in dashed line. Amino acid sequences are depicted in single letter code above nucleotide sequences. (III) WES TM analysis of ALDH1L2 protein in U251cells in the upper panel shows that ALDH1L2 is detected in wt TS but not in the deletion clones. It is of note that ALDH1L2 is weaky expressed in the wt ML. Immunohistolological staining of the ALDH1L2 in wt and knockout cells in the lower panel. Abbreviations, wt: Wild Type; ML: MonoLayer Cells; TS: Tumor Spheres; KO: knockout.

Figure 1 .

 1 Figure 1. ALDH1L2 KO U251 glioblastoma cells were generated using the CRISPER-Cas9 system (I) One carbon metabolism features linked mitochondrial and cytosolic pathways; folate and méthionine cycles are intertwinned in the cytosol. THF: TetraHydroFolate, 5,10-meTHF: 5,10 methylene THF, 10-fTHF: 10-formylTHF, 5-mTHF: 5 methyl THF, SAM: S-adenosyl methionine, SAH: S-adenosyl homocysteine. (II) Genomic Sanger sequencing of individual deletion clones shows bi-allelic disruption of targeted exons 1 and 3. For exon 1, the new sequences diverge from the wild type sequence by deletions of 42 and 62 bp. Wild type sequence is shown in (a) with arrows indicating the breaking points. Sequencing of the 42 deletion including 24 bp of exon 1 and 18 bp of intron 1 is shown in (b) and sequencing of the 62 bp deletion including 33 bp of exon 1 and 29 bp of intron 1 in (c). The consequences of these deletions on protein synthesis are unpredictable since they carry away the 5 end of intron 1. For exon 3, the divergence is due to deletions of 14 and 23 bp. Wild type sequence is shown in (d) with top and bottom arrows indicating the 14 and 23 bp deletion boundaries inside exon 3. Sequencing of the 14 and 23 deleted exon 3 is shown in (e,f) with arrows indicating the breaking points. Both deletions were mapped and confirmed to yield the same truncated protein with a stop codon generated at codon 113 of the newly synthesized proteins. Sequences targeted by the sgRNA are underlined in solid lines and PAM sequences in dashed line. Amino acid sequences are depicted in single letter code above nucleotide sequences. (III) WES TM analysis of ALDH1L2 protein in U251cells in the upper panel shows that ALDH1L2 is detected in wt TS but not in the deletion clones. It is of note that ALDH1L2 is weaky expressed in the wt ML. Immunohistolological staining of the ALDH1L2 in wt and knockout cells in the lower panel. Abbreviations, wt: Wild Type; ML: MonoLayer Cells; TS: Tumor Spheres; KO: knockout.

Figure 2 .

 2 Figure 2.Tumor spheres (TS) derived from U251 ALDH1L2 KO cells display not only decreased growth but also methionine-independent growth. TS growth in methionine free (MET-) vs. methionine (0.01 mM)-containing (MET+) media were compared KO vs. wt cells. In (Ia), in MET-, TS of the KO cells grew better than that of the wt. In (Ib), the TS growth ratio was calculated as TS growth in MET+) divided by that in MET-. The higher the ratio, the more the methionine growth dependency. In (Ic), TS growth in MET-was normalized to that of the wt TS in MET-. In (Id), TS growth in MET+ was normalized to that of the wt TS MET+. In (II), analyses of the computer-assisted reconstruction of sphere volumes revealed that ALDH1L2 KO has smaller "tumor sphere volume". A, A1, A2 = wt cells. B, B1, B2 = AL1 cells. C, C1, C2 = AL3 cells. A1, B1, C1 are results of segmentation. A2, B2, C2 are results of 3D reconstructions: axes values are in pixels, and images are not to scale, so size differences can be appreciated visually in A1, B1, C1 but are best demonstrated by measured volumes: A = 1,857,000 µm 3 , B = 258,000 µm 3 , C = 206,000 µm 3 . D: Spheroid volumes versus wt (µm 3 ), mean ± SD, * p < 0.003, *** p < 0.001, Student t-test. In (III), ALDH1L2 KO suppressed U251 TS growth methionine dependency. For each cell type, mean ± SE of three independent experiments (each done in quadruplicate) is shown (three biological replicates each include four technical replicates). wt: wild type, AL1: U251 cells ALDH1L2 exon 1 KO, AL3: U251 cells ALDH1L2 exon 3 KO.

Figure 3 .

 3 Figure 3. ALDH1L2 knockout U251 cells have lower NADPH level (I), no difference in GSH ratio (reduced GSH/total GSH) (II), and higher one carbon metabolites, including methionine, 5, methyltetrahydrofolate and formyl-tetrahydrofolate (III), as well as larger ROS production (IV). Tumor spheres were collected at passage 3 and adherent cells at 80% confluency. AL1 and AL3 were compared to U251 wild type, n = 6, ** p < 0.01, *** p < 0.001, AL1 = ALDH1L2 exon1 KO, AL3 = ALDH1L2 exon 3 KO.

IVFigure 3 .

 3 Figure 3. ALDH1L2 knockout U251 cells have lower NADPH level (I), no difference in GSH ratio (reduced GSH/total GSH) (II), and higher one carbon metabolites, including methionine, 5, methyltetrahydrofolate and formyl-tetrahydrofolate (III), as well as larger ROS production (IV). Tumor spheres were collected at passage 3 and adherent cells at 80% confluency. AL1 and AL3 were compared to U251 wild type, n = 6, ** p < 0.01, *** p < 0.001, AL1 = ALDH1L2 exon1 KO, AL3 = ALDH1L2 exon 3 KO.

Figure 4 .

 4 Figure 4. ALDH1L2 knockout alters mitochondrial morphology. ALDH1L2 knockout led to a visible change to the morphology of the mitochoindria of the KO TS cells. Relative to control wt TS cells, the mitochondria of the KO TS cells have a larger network-like structure, where, in wide type TS cells, the mitochochondria are much more fragmented with an oval-like shape. Mitochondria in U251 tumor spheres and in adherent U251 cells were labeled with green fluroescent protein (GFP) fused to the leader sequence of E1 alpha pyruvate dehydrogenase and observed under a confocal microscope (see supplemental Methods).

Figure 4 .

 4 Figure 4. ALDH1L2 knockout alters mitochondrial morphology. ALDH1L2 knockout led to a visible change to the morphology of the mitochoindria of the KO TS cells. Relative to control wt TS cells, the mitochondria of the KO TS cells have a larger network-like structure, where, in wide type TS cells, the mitochochondria are much more fragmented with an oval-like shape. Mitochondria in U251 tumor spheres and in adherent U251 cells were labeled with green fluroescent protein (GFP) fused to the leader sequence of E1 alpha pyruvate dehydrogenase and observed under a confocal microscope (see Supplemental Methods).

Supplementary Materials:The Supplementary Materials information can be downloaded at: https: //www.mdpi.com/article/10.3390/nu14091887/s1.

Acknowledgments:

We thank Dejian Ren (Department of Biology, University of Pennsylvania) for his help in the construction of the CRISPR knockouts. We also thank La Ligue Contre Le Cancer, Université de Lorraine (BMS, projet incitatif 2019), as well as INSERM for the financial supports. The PhD fellowship granted to Maelle Quere was given by the Université de Lorraine (2018-2021).

Data Availability Statement: All data are available upon request.

Conflicts of Interest:

The authors declare no conflict of interest.