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Vitamin B1 Supports the Differentiation of T Cells through TGF-β Superfamily Production in Thymic Stromal Cells

Summary Homeostatic generation of T cells, which occurs in the thymus, is controlled at least in part by endogenous cytokines and ligands. In addition, nutritional factors are other key regulators for the homeostasis of host immunity, but whether and how nutrition affects the homeostatic generation of thymocytes remains to be established. Here, we showed that vitamin B1 deficiency resulted in a bias toward the maturation of γδ thymocytes accompanied by decreased differentiation into double-positive thymocytes during thymic involution. These events were mediated through the increased production of TGF-β superfamily members due to the accumulation of branched-chain α-keto acids in thymic stromal cells. These findings revealed essential roles of vitamin B1 in the appropriate differentiation of T cells through the metabolism of thymic stromal cells.


INTRODUCTION
The thymus is the primary lymphoid organ for generating T cells, a process that is regulated through the interaction of endogenous molecules with thymocytes (e.g., Notch and T cell receptor [TCR]) and stromal cells (e.g., Delta ligand and self-peptides on major histocompatibility complex [MHC]) (Hogquist and Jameson, 2014;Takahama, 2006). Initially, thymocytes lack cell surface expression of both CD4 and CD8 (i.e., double-negative [DN] cells) and develop from stage DN1 to DN4 as TCR expression increases due to the interaction between Notch on thymocytes and Delta ligand on thymic stromal cells (Takahama, 2006). The strength of the TCR signal from the MHC on thymic stromal cells with Delta-Notch interaction drives the transition to CD4 + CD8 + double-positive (DP) thymocytes (Hogquist and Jameson, 2014;Takahama, 2006). Moreover, transforming growth factor (TGF)-b superfamily members, including TGF-b1, and Activin A from thymic stromal cells arrest the development of thymocytes by preventing their progression from the DN to the DP stage (Licona-Limó n et al., 2009;Takahama, 2006) and induce the apoptosis of DP thymocytes (Szondy et al., 2003). Furthermore, a strong TCR signal and high TGF-b superfamily activity during the DN stage accelerate the development or maturation of gd thymocytes (Hogquist and Jameson, 2014;Woolf et al., 2007).
T cells have metabolic heterogeneity in regard to their various functions and development stages (Bantug et al., 2018;Buck et al., 2015). Indeed, naive T cells are relatively dependent on bioenergetic catabolism, using oxidative phosphorylation and fatty acid oxidation for homeostatic maintenance (Bantug et al., 2018;Buck et al., 2015). In contrast, to meet metabolic demands, activated and proliferating T cells generally depend on anabolism characterized by high mechanistic target of rapamycin (mTOR) activity and fatty acid synthesis and catabolism through glycolysis and amino acid metabolism (Bantug et al., 2018;Buck et al., 2015). Recently, it was reported that the homeostatic production of thymocytes was regulated through the metabolic states of mTOR in both thymocytes (Yang et al., 2018) and thymic stromal cells (Wang et al., 2016).
Accumulating evidence suggests that nutritional factors are other key regulators of T cell development and differentiation. Indeed, malnutrition induces thymic involution (Savino et al., 2007), and several nutrients, including zinc and vitamin A, are necessary for the maintenance of thymus (Cunningham-Rundles et al., 2005). Regarding vitamin A, retinoic acid signaling in thymic epithelial cells (TEC) is required to maintain Vitamin B1 (thiamine) is an essential nutrient for the central metabolism rooted in pyruvate, branched amino acids, and ribose 5-phosphate as well as the citric acid cycle (Dhir et al., 2019). We previously reported that naive B cells in Peyer patches rely metabolically on the citric acid cycle, which has a high requirement for vitamin B1, to produce ATP for energy . In contrast, IgA-producing plasma cells in the intestinal lamina propria utilize the glycolytic pathway for ATP production and thus have a decreased requirement for vitamin B1 . Indeed, mice maintained on a vitamin B1deficient diet showed significant reduction of naive B cells in Peyer patches without remarkable changes in IgA-producing plasma cells in the intestinal lamina propria . Given that Peyer patches are the site in naive B cells for the class switching of IgM to IgA, especially to intestinal antigens, maintaining mice on a vitamin B1-deficient diet resulted in impaired intestinal IgA responses against orally immunized vaccine antigens .
In the current study, we explored the immunologic roles of vitamin B1 in T cell development in thymus and found that vitamin B1 was highly required for appropriate production of TGF-b superfamily members from thymic stromal cells. This process was controlled through the metabolism of branched-chain amino acids.

Vitamin B1 Supports the Homeostatic Development of T Cells in the Thymus
First, we evaluated whether dietary vitamin B1 affects T cell development in the thymus. Macroscopic analysis revealed that mice maintained on vitamin B1-deficient chow demonstrated remarkable decreases in the volume and weight of thymus and the total number of thymus cells, namely, thymic involution, compared with mice maintained on control chow ( Figures 1A and 1B). Accordingly, the amount of vitamin B1 in thymus gradually decreased during the 3 weeks after dietary vitamin B1 was discontinued ( Figure 1C). Flow cytometric analysis indicated that the proportion of DP thymocytes was decreased preferentially (Figure 1D). In contrast, vitamin B1 deficiency did not affect the overall proportion of TCRgd + cells among DN thymocytes ( Figure 1E), but the proportion of cells that transitioned from immature, CD44 -CD24 + cells to the mature, CD44 + CD24subset of TCRgd + DN thymocytes was increased ( Figure 1F).
Given that increased expression of RUNX3 reportedly induces the maturation of DN TCRgd + thymocytes in thymus (Woolf et al., 2007), we assessed Runx3 mRNA expression in DN thymocytes by using qRT-PCR analysis. Runx3 mRNA expression levels in DN thymocytes were upregulated in vitamin B1-deficient mice compared with the control group ( Figure 1G). These results suggest that vitamin B1 is required to control RUNX3 expression and thus maintain appropriate generation of DP and DN TCRgd + thymocytes.

High Requirement for Vitamin B1 in Thymic Stromal Cells
We investigated whether vitamin B1 affects thymocytes directly. When purified DN thymocytes were cocultured with the bone marrow-derived OP9-DL1 cell line as stromal cells, they developed to single-positive and DP thymocytes (Lai et al., 2010). A similar pattern emerged when cultures were treated with oxythiamine, a vitamin B1 inhibitor ( Figure 2A). Therefore, these results suggested that vitamin B1 inhibition did not directly affect thymocyte development in vitro, unlike in vivo vitamin B1 deficiency. Moreover, because we hypothesized that OP9-DL1 cells incubated with oxythiamine might not sufficiently recapitulate the in vivo thymic stromal environment in vitamin B1-deficient mice, we assessed the mRNA expression levels of thiamine transporter 1 (THTR1) (Dutta et al., 1999) in the OP9-DL1 cells and thymic stromal cells. Indeed, the levels of Thtr1 mRNA were significantly higher in thymic stromal cells than in OP9-DL1 cells ( Figure S1). We then used immunohistochemistry to examine thiamine transporter 1 (THTR1) levels and showed that a population of EpCAM + stromal cells expressed higher levels of THTR1 than did CD45 + thymocytes ( Figure 2B). Given these findings, we then adopted a fetal thymic organ culture (FTOC) system to better mimic the thymic environment in vivo. Although treatment with oxythiamine did not significantly alter the proportion of DP thymocytes that developed from DN1-3 thymocytes ( Figure S2), the proportion of CD24 -, mature DN TCRgd + thymocytes increased ( Figure 2C). This result indicates that the thymic environment created by stromal cells requires vitamin B1 for the appropriate control of thymocyte development. Both TGF-bs and Activin A (also known as Inhba), which belongs to the TGF-b superfamily, reportedly block the developmental transition of thymocytes from the DN to DP stage (Licona-Limó n et al., 2009;Takahama et al., 1994) and induce the apoptosis of DP thymocytes (Szondy et al., 2003). Moreover, RUNX3 transcription is upregulated by TGF-b superfamily members through the phosphorylation of smad2 and smad3 (Klunker et al., 2009;Reis et al., 2013). Prompted by these reports and our current findings, we evaluated the effect of vitamin B1 deficiency on the production of TGF-b superfamily members in thymic stromal cells. Compared with the control group, thymic stromal cells from the vitamin B1-deficient condition had increased levels of Tgfb2, Gdf10, and Inhba mRNA ( Figure 3A); mRNA transcript levels did not differ between the control and vitamin B1-deficient groups for any other TGF-b superfamily member ( Figure S3). In addition, flow cytometric analysis consistently demonstrated that vitamin B1 deficiency increased the proportion of TCRgd + thymocytes expressing phosphorylated smad2 and smad3; these signaling (B) total number of thymocytes at days 3, 7, 14, and 21 of feeding mice a vitamin B1deficient diet (VB1-) or control diet (Con); and (C) thymic vitamin B1 concentration at days 3, 7, 14, and 21 of feeding mice a vitamin B1-deficient diet (VB1-) relative to control diet. FACS plots of (D) CD4 and CD8a on live thymocytes gated on 7-AAD -, (E) of TCRgd + cells among DN thymocytes, and (F) of CD44 and CD24 among DN-TCRgd + thymocytes after 3 weeks of VB1-or Con diet. Scale, 1 cm. Horizontal lines indicate median values. p values were obtained by using the Mann-Whitney U-test (*p < 0.05). The data shown are reproducible and are representative of two to five independent experiments. (G) The levels of Runx3 mRNA in DN and DP thymocytes sorted from the VB1-and Con groups are shown. Horizontal lines indicate median values. p values were obtained by using the Mann-Whitney U-test (*p < 0.05).

OPEN ACCESS
iScience 23, 101426, September 25, 2020 3 iScience Article molecules are induced by the TGF-b superfamily ( Figure 3B). These results suggest that vitamin B1 modulates the expression of Tgfb2, Gdf10, and Inhba in thymic stromal cells, thus maintaining the homeostatic generation of DP and TCRgd + thymocytes through the phosphorylation of smad2 and smad3.

Vitamin B1 Promotes the Metabolism of Branched-Chain a-Keto Acids in Thymic Stromal Cells
Vitamin B1 is well known as a necessary coenzyme for catalyzing the conversion of pyruvate to acetyl-CoA, of a-ketoglutarate to succinyl-CoA, of branched-chain a-keto acids (BCKAs) to branched-chain acyl-CoA, and of ribose 5-phosphate to glyceraldehyde 3-phosphate (Manoli and Venditti, 2016;Whitfield et al., 2018). Therefore, we wondered what metabolic changes might occur in thymic stromal cells. Metabolomic analysis using ion chromatography with Fourier transform mass spectrometry (IC-MS) showed that iScience Article ketoleucine and ketoisoleucine, which are BCKAs that are generated as intermediate metabolites from branched-chain amino acids (BCAAs), were increased in the supernatant of thymic stromal cells that had been treated with oxythiamine; however, metabolites associated with the citric acid cycle and glycolysis remained unchanged (Figures 4 and S4).

BCKAs Induced Excessive Production of TGF-b Superfamily Members
We investigated whether the BCKAs that accumulated in thymic stromal cells due to vitamin B1 insufficiency could increase the quantities of mRNAs encoding TGF-b superfamily members. Culturing thymic stromal cells in media containing ketoleucine and ketoisoleucine increased the mRNA expression of Inhba, Tgfb2, and Gdf10 ( Figures 5 and S5). These results suggest that vitamin B1-mediated metabolism of BCAAs, especially BCKAs, controls the production of TGF-b superfamily members in thymic stromal cells.
Activin A, TGF-b, and GDF10 act as ligands for the TGF-b superfamily receptors ALK4 and ActRIIA (to Activin A and GDF10), ALK5 (to TGF-b), and others (Heldin and Moustakas, 2016), whereas K02288 inhibits various TGF-b superfamily receptors (e.g., ALK2, ALK1, ALK6, ALK3, ActRIIA, ALK4, and ALK5) (Sanvitale et al., 2013). These activities allowed us to use FTOC to evaluate the effect of K02288 on the oxythiamine-induced maturation of DN gd thymocytes. Treatment with K02288 canceled the oxythiamineinduced excessive maturation of DN gd thymocytes in FTOCs ( Figure 6). These results suggest that impaired thymocyte differentiation in the vitamin B1-deficient condition was mediated by TGF-b superfamily members.

Changes in Peripheral T Lymphocytes
We next investigated the effect of vitamin B1 deficiency on the number of peripheral T lymphocytes in the gut, the site of numerous TCRgd + T lymphocytes, under immunologically naive conditions. Flow cytometric analysis revealed that vitamin B1 deficiency increased the proportion of CD8aa TCRgd + T lymphocytes in the small intestine ( Figure 7A). A similar increase occurred in the CD8aa subpopulation of TCRb + T  Figure 7B), whereas vitamin B1 deficiency decreased the number of CD4 TCRb + T lymphocytes in the small intestine ( Figure 7C). In addition, we found that the numbers of CD4 and CD8a IFN-g + TCRb + T lymphocytes, which are the main functional subsets in spleen (Saxena et al., 2012), were reduced in the spleens of vitamin B1-deficient mice ( Figure S7). These results suggest that vitamin B1 is required for the appropriate differentiation and function of T lymphocytes in peripheral tissues.

DISCUSSION
Here we showed that vitamin B1 modulates the metabolism of BCKAs and consequent production of TGF-b superfamily members in thymic stromal cells; these activities are required to maintain the appropriate differentiation of DP and gd thymocytes ( Figure 8). These findings extend current knowledge by revealing the novel role of vitamin B1 as an essential micronutrient for homeostatic development of immune cells.
Our current study showed that thymic stromal cells-but not CD45 + thymocytes-express THTR1 ( Figure 2). Notably, THTR1 was not expressed in all thymic stromal cells ( Figure 2B). In addition to cTEC and mTEC, recent studies using single-cell RNA sequencing have indicated that EpCAM + thymic stromal cells . The expression level of p63 in TECs is negatively correlated with that of FoxN1, one of the main differential transcription factors in TECs (Burnley et al., 2013). Therefore, the THTR1 content of TECs might be orchestrated through FoxN1induced downregulation of p63 and thus upregulation of Sp1. Furthermore, compared with other cTECs and mTECs, the MHC class II high cTECs among EpCAM + thymic stromal cells show greater expression of FoxN1 mRNA (Nowell et al., 2011;O'Neill et al., 2016); this result implies that FoxN1-initiated control of THTR1 expression might be upregulated in MHC class II high cTECs.
We found that vitamin B1 insufficiency increased BCKA levels and the production of some TGF-b superfamily members, including Inhba, Gdf10, and Tgfb2, in thymic stromal cells (Figures 3 and 5). Ketoleucine increased intracellular Ca 2+ levels in rat cerebral cortex in vitro (Funchal et al., 2005). Moreover, increased levels of intracellular Ca 2+ , which induces TGF-b in the 3T3TbRII cell line (Xiao et al., 2008), activated p38 MAPK and ERK1/2 in bone marrow macrophages in vitro (Zhou et al., 2010). Why Inhba, Gdf10, and Tgfb2 mRNAs specifically are increased in thymic stromal cells remains unknown at this point. However, our current findings suggest that BCKAs, including ketoleucine and ketoisoleucine, may increase intracellular Ca 2+ in thymic stromal cells, thus inducing MAPK-and ERK-mediated mRNA expression of TGF-b superfamily members, including Inhba, Gdf10, and Tgfb2.
We showed that dietary deficiency of vitamin B1 decreased the number of DP thymocytes and increased the numbers of mature gd thymocytes in mice; these changes were associated with the increased expression of Runx3 mRNA and the phosphorylation of smad2 and smad3 in thymocytes (Figures 1 and 3). In support of our current findings, previous studies showed that various TGF-b superfamily members, including Activin A, GDF10, and TGF-b2, induce the phosphorylation of Smad2 and Smad3 (Heldin and Moustakas, 2016) and upregulate the expression of RUNX3 (Jin et al., 2004;Reis et al., 2013) and that TGF-b2 induces the RUNX3-mediated maturation of gd thymocytes (Woolf et al., 2007). In addition, TGF-b and Activin A Consistent with the in vivo results, treatment with oxythiamine increased the proportion of CD24mature DN gd thymocytes ( Figure 2C) but did not significantly alter the proportion of DP thymocytes that developed from DN1-3 thymocytes ( Figure S2). As an explanation of this apparent discrepancy, previous studies showed that fetal TECs had a greater rate of cell growth than those from adult thymus (Cowan et al., 2019) and that increased cell growth activity was associated with low dependency on BCAA catabolism in cardiomyocytes (Shao et al., 2018). Thus, under vitamin B1-deficient conditions, BCKA accumulation likely was lower in FTOCs than in adult thymus. In support of this notion, exogenous addition of BCKAs reduced the proportion of DP thymocytes in FTOCs ( Figure S6). Furthermore, given that TGF-b receptors (i.e., Tgfbr1, Tgfbr2) were more highly expressed in TCRgd + thymocytes than in DP thymocytes (Do et al., 2010), DN gd thymocytes might have greater sensitivity to the oxythiamine-induced production of BCKAs and TGF-b superfamily ligands.
In line with the immunologic phenotypes in the thymus, vitamin B1 deficiency decreased the population of DP thymocyte-derived CD4 TCRb + T lymphocytes in the gut ( Figure 6). In contrast, we noted increases in the numbers of TCRgd + T lymphocytes and CD8aa TCRb + T lymphocytes ( Figure 6), which developed from DN thymocytes through TGF-b-mediated RUNX3 expression (Konkel et al., 2011;Pobezinsky et al., 2012). Moreover, we showed that vitamin B1 deficiency led to a decrease in IFN-g + TCRb + T lymphocytes (Figure S7). Given that pyruvate dehydrogenase (PDH) induces the production of acetyl-CoA to induce the transcription of IFN-g transcription (Peng et al., 2016) and that vitamin B1 is an essential co-enzyme for PDH , vitamin B1 deficiency plausibly impaired PDH activity and consequently inhibited IFN-g production.
Together, our current findings indicate that vitamin B1 is required for the appropriate differentiation of thymocytes, especially the development of DN cells into DP or gd thymocytes. This regulation is mediated through control of the production of TGF-b superfamily members including Activin A, GDF10, and TGF-b2; this regulation is achieved by promoting the metabolism of BCKAs in thymic stromal cells. These findings provide new evidence of vitamin B1-mediated interaction between stromal and immune cells for the appropriate development of thymocytes.

Limitations of the Study
This study demonstrated that vitamin B1 was necessary for the appropriate metabolic functions in thymic stromal cells for the homeostatic differentiation of T cells. Although we proposed that a specific subset of thymic stromal cells expressing high levels of THTR1 were responsible for it, we could not specify them due to the experimental restriction of anti-THTR1 antibody.

Materials Availability
New unique reagents were not generated in this study.

Data and Code Availability
The data in this study are available from the corresponding author on request.

ACKNOWLEDGMENTS
We thank RIKEN BRC CELL BANK for providing the OP9-DL1 cell line. We also thank our laboratory members for their helpful support. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (

DECLARATION OF INTERESTS
The authors declare that they have no conflict of interest. The proportion of IFN-g + gated on CD4 or CD8ab TCRb + T lymphocytes in spleen at 3 weeks after mice began to receive vitamin B1-deficient (VB1-) or control (Con) chow. Horizontal lines indicate median values. P values were obtained by using the Mann-Whitney U-test (*, P < 0.05). Data shown are reproducible and representative two independent experiments. * *
Vitamin B1-deficient and control diets with chemically defined components were purchased from Oriental Yeast, as previously described . Assay for vitamin B1 concentration Vitamin B1 concentration was measured by using VitaFast Vitamin B1 (r-Biopharm) according to a previous study . Briefly, the vitamin B1 concentration in a water extract of thymus was assessed by vitamin B1-dependent growth of Lactobacillus fermentum.

Isolation of single cells
Lymphocytes were separated from small intestinal lamina propria and intestinal epithelium, as previously described . Briefly, small intestinal tissue from which Peyer's patches had been removed was rinsed in RPMI 1640 medium and cut into small pieces; cells were dislodged by incubating tissue pieces for 15 min in RPMI 1640 medium containing 0.5 mM EDTA and 2% neonatal calf serum. The remaining tissue pieces then were digested for 15 min in 1.0 mg/mL collagenase; this step was repeated. Solutions containing the cells obtained after EDTA exposure (containing intestinal epithelial lymphocytes) and those released after collagenase treatment (containing lymphocytes from the lamina propria) were passed through a 100-µm cell strainer and left them as separate cell populations, and then underwent centrifugation through a discontinuous Percoll (GE Healthcare) gradient; lymphocytes were isolated at the interface between the 40% and 75% Percoll layers.
For analysis of thymocytes only, thymic cells were dissociated by passage through a 100-µm cell strainer before centrifugation. For analysis of both thymocytes and stromal cells, these cells were separated from thymus as previously described with modifications (Miller et al., 2018;Seach et al., 2012).
Briefly, thymus was cut into small pieces and stirred for 30 min at 37 ˚C in 0.3 mg/mL collagenase in RPMI 1640 medium containing 2% neonatal calf serum.
The cells were dissociated further by passage through a 100-µm cell strainer and then subjected to centrifugation through a discontinuous Percoll gradient.
Fetal thymic organ culture (FTOC) The protocol was modified from that previously described (Hirano et al., 2015).
Stained cells underwent flow cytometry, and data were analyzed by using FlowJo software.