Thymic Epithelial Cell Support of Thymopoiesis Does Not Require Klotho Yan Xing, Michelle J

Thymic Epithelial Cell Support of Thymopoiesis Does Not Require Klotho Yan Xing, Michelle J. Smith, Christine A. Goetz, Ron T. McElmurry, Sarah L. Parker, Dullei Min, Georg A. This information is current as Hollander, Kenneth I. Weinberg, Jakub Tolar, Heather E. of September 28, 2021. Stefanski and Bruce R. Blazar J Immunol published online 29 October 2018 http://www.jimmunol.org/content/early/2018/10/28/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 29, 2018, doi:10.4049/jimmunol.1800670 The Journal of Immunology

Thymic Epithelial Cell Support of Thymopoiesis Does Not Require Klotho

Yan Xing,*,1 Michelle J. Smith,*,†,1 Christine A. Goetz,*,† Ron T. McElmurry,* Sarah L. Parker,* Dullei Min,‡ Georg A. Hollander,x,{ Kenneth I. Weinberg,‡ Jakub Tolar,* Heather E. Stefanski,*,2 and Bruce R. Blazar*,†,2

Age-related thymic involution is characterized by a decrease in thymic epithelial cell (TEC) number and function parallel to a disruption in their spatial organization, resulting in defective thymocyte development and proliferation as well as peripheral T cell dysfunction. Deficiency of Klotho, an antiaging gene and modifier of fibroblast growth factor signaling, causes premature aging. To investigate the role of Klotho in accelerated age-dependent thymic involution, we conducted a comprehensive analysis of thymopoiesis and peripheral T cell homeostasis using Klotho-deficient (Kl/Kl) mice. At 8 wk of age, Kl/Kl mice displayed a severe reduction in the number of thymocytes (10–100-fold reduction), especially CD4 and CD8 double-positive cells, and a reduction Downloaded from of both cortical and medullary TECs. To address a cell-autonomous role for Klotho in TEC biology, we implanted neonatal thymi from Klotho-deficient and -sufficient mice into athymic hosts. Kl/Kl thymus grafts supported thymopoiesis equivalently to Klotho- sufficient thymus transplants, indicating that Klotho is not intrinsically essential for TEC support of thymopoiesis. Moreover, lethally irradiated hosts given Kl/Kl or wild-type bone marrow had normal thymocyte development and comparably reconstituted T cells, indicating that Klotho is not inherently essential for peripheral T cell reconstitution. Because Kl/Kl mice have higher levels of serum phosphorus, calcium, and vitamin D, we evaluated thymus function in Kl/Kl mice fed with a vitamin D–deprived diet. We http://www.jimmunol.org/ observed that a vitamin D–deprived diet abrogated thymic involution and T cell lymphopenia in 8-wk-old Kl/Kl mice. Taken together, our data suggest that Klotho deficiency causes thymic involution via systemic effects that include high active vitamin D levels. The Journal of Immunology, 2018, 201: 000–000.

hymic aging is a major contributing factor to immuno- homeostatic oligoclonal expansion of T cells in the periphery logical senescence as the thymus begins to involute with (4, 5). These dynamic changes result in an increased susceptibil- T the onset of puberty, leading to a progressive defect in the ity to infections (6, 7), suboptimal responses to vaccines (8–12), ability to generate naive T cells (1, 2). The primary elements of and an increased risk to develop cancer and autoimmune diseases by guest on September 28, 2021 the thymic microenvironment affected by age-related involution (13–15). The age-related decline in thymopoietic activity (1) is are structural thymic epithelial cells (TECs). TECs are replaced by especially apparent in patients who have undergone chemotherapy adipocytes and peripheral lymphocytes, resulting in decreased (16) or allogeneic hematopoietic stem cell (HSC) transplantation thymopoietic activity and T cell selection (3). Consequently, both (17). The necessary preparative regimen with cytotoxic chemo- quantity and quality of the T cell repertoire are affected with therapy and/or radiation severely damages the thymus, the recovery decreased de novo T cell generation, which is compensated by a of which is extremely limited in aged individuals (18, 19). To study the process of aging in mice, Klotho-deficient (Kl/Kl) *Division of Blood and Marrow Transplantation, Department of Pediatrics, Masonic animals have been used, as they grow normally up to 3 wk of age Cancer Center, University of Minnesota, Minneapolis, MN 55455; †Center for Im- and then begin to show premature aging phenotypes (20). Klotho munology, University of Minnesota Medical School, Minneapolis, MN 55455; ‡Division of Stem Cell Transplantation and Regenerative Medicine, Department of encodes a b-glucuronidase–related molecule in two separate iso- Pediatrics, Stanford Medicine, Stanford University, Palo Alto, CA 94304; xDepart- forms, transmembrane and secreted; the transmembrane molecule { ment of Biomedicine, University of Basel, 4056 Basel, Switzerland; and Department serves as a coreceptor for fibroblast growth factor 23 (FGF23) by of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Headington, Oxford OX3 9DS, United Kingdom transporting this cytokine to its receptor, FGFR1c, and thereby 1Y.X. and M.J.S. contributed equally to this work. regulating mineral metabolism (21–23). Klotho is expressed in the 2H.E.S. and B.R.B. contributed equally to this work. kidney and parathyroid gland, and the secreted form can also be found in the blood, cerebrospinal fluid, and urine (24). FGF23 ORCIDs: 0000-0003-2150-1448 (Y.X.); 0000-0002-8790-0874 (G.A.H.); 0000-0002- 0957-4380 (J.T.); 0000-0001-7527-9315 (H.E.S.); 0000-0002-9608-9841 (B.R.B.). suppresses phosphate reabsorption and vitamin D synthesis in the Received for publication May 11, 2018. Accepted for publication September 28, kidney, causing negative phosphate balance because of both its 2018. phosphaturic hormone function and its function as a counter- This work was supported by the National Institutes of Health Grants P01 CA065493, regulatory hormone for vitamin D (24). The secreted form of R01 AI081918, and 2P01 CA065493, and the Children’s Cancer Research Fund. Klotho inhibits insulin growth factor 1 signaling and confers in- Address correspondence and reprint requests to Dr. Bruce R. Blazar, University of creased resistance to oxidative stress (25–27). Mice transgenic for Minnesota, 420 Delaware Street, SE, Mayo Mail Code 109, Minneapolis, MN 55455. E-mail address: [email protected] Klotho live 20–30% longer than wild-type (WT) controls (28), Abbreviations used in this article: BM, bone marrow cell; cTEC, cortical TEC; whereas the protein’s absence results in an advanced aging syn- DN, double-negative; DP, double-positive; FGF23, fibroblast growth factor 23; drome resembling progeria. Multiple organs are affected in Kl/Kl HSC, hematopoietic stem cell; Kl/Kl, Klotho-deficient; mTEC, medullary TEC; SP, mice, resulting in growth retardation, pituitary abnormalities, ar- single-positive; TEC, thymic epithelial cell; Treg, regulatory T cell; WT, wild-type. teriosclerosis, ectopic calcification of various organs, osteoporosis, Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50 skin atrophy, emphysema, and atrophy of both the genital organs

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800670 2 TEC SUPPORT OF THYMOPOIESIS DOES NOT REQUIRE Klotho and the thymus (20). Interestingly, mice that are FGF23 deficient pressure (30); and demonstrate an increased risk for stroke and or Kl/Kl have phenotypes similar to one another. These deficits coronary artery disease (31). Polymorphisms in KLOTHO (loss of can be alleviated by reversing the effects of hyperphosphatemia function) have been associated with an increased risk for osteo- either genetically or by diet, suggesting a link between aging and porosis and spondylosis (32), and reduced KLOTHO protein ex- phosphate (24). pression has been noted in patients with chronic renal failure (33). The Kl/Kl mouse model has provided insight into the process of Although the effects of Klotho deficiency on kidney develop- aging in humans. Indeed, human KLOTHO shares 86% amino ment and function, mineral metabolism, and bone maintenance are acid identity with its mouse ortholog (29). Individuals homozy- well studied in Kl/Kl mice, the direct effect of Klotho deficiency gous for KLOTHO variants that disrupt the molecule’s trafficking on the TECs is unknown. We therefore sought to determine and catalytic functions experience a decreased life expectancy whether the effects of Klotho on thymic aging are cell intrinsic or (29); have increased cardiovascular risk factors, such as elevated reflect a systemic metabolic consequence of a lack of the Klotho high-density lipoprotein cholesterol levels and high systolic blood protein. Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Profound thymus involution in Klotho-deficient (Kl/Kl) mice at 8 wk of age. (A) A representative image of the thymus from Kl/Kl and littermate control mice at 4 and 8 wk of age. (B) Quantification of total thymocytes in Kl/Kl mice and littermate controls. (C) A summary of percentages of thymocyte subpopulations, including DN (lineage2CD42CD82), DP (CD4+CD8+), CD4SP (TCRb+CD4+CD82), and CD8SP (TCRb+CD42CD8+) cells. (D) A graph shows quantification data of absolute numbers of thymocyte subpopulations. (E) Frequencies of semimature (CD69+MHC class I2) and mature (CD69+/2MHC class I+) subpopulations in CD4SP cells. (F) Quantification of absolute numbers of semimature and mature CD4SP in the thymi. Each symbol in the graphs represents an individual mouse (n $ 5); small horizontal lines indicate the group mean (6SD). *p , 0.05. ns, not significant (p $ 0.05). The Journal of Immunology 3

Materials and Methods T cell precursors from the blood and control their T cell lineage Mice commitment, differentiation, and selection (37). The earliest stages of intrathymic T cell development are marked by the absence of B6.Cg-Foxn1 nu/J mice were purchased from The Jackson Laboratory and were used at 8–12 wk of age. Kl/+ mice (B6-CD45.2+) were generously provided by CD4 and CD8 expression as well as other lineage markers (des- the University of California, Davis, Mutant Mouse Regional Resource Centers ignated double-negative [DN] cells). These immature DN cells are and were intercrossed (Kl/+ by Kl/+) in our animal colony under the guidance located in the cortex, where they significantly expand in number + of in-house veterinary staff. B6-Ly5.2/Cr (B6-CD45.1 ) mice were purchased before they acquire the concomitant expression of both CD4 and from the National Cancer Institute and were used at 7 wk of age. Mice were CD8 (double-positive [DP]) and express an ab T cell Ag receptor housed in a specific pathogen-free facility and used with the approval of the University of Minnesota Institutional Animal Care and Use Committee. For (TCR). DP thymocytes constitute the most abundant subpopula- vitamin D experiments, breeders were fed and pups were maintained on a tion of thymocytes and are subjected first to a process of positive vitamin D–deprived diet (TD 89123) purchased from Harlan Teklad. selection, which assures that thymocytes express a TCR with Flow cytometry and Klotho expression sufficient affinity for a peptide–MHC complex expressed on cortical TECs (cTECs) (38). Positively selected thymocytes differ- Mouse thymus, spleen, and lymph node were processed into single-cell entiate into TCRb+ CD4 and CD8 single-positive (SP) cells, suspensions and analyzed by flow cytometry. TECs were isolated as pre- respectively, and migrate to the medulla, continuing their selection viously described (34). Dead cells were stained by a fixable viability dye conjugated to eFluor 780 (eBioscience). Fixation and intracellular/ and maturation (39, 40). Self-reactive thymocytes are either intranuclear staining were performed using the eBioscience Foxp3 staining kit or BD Fixation/Permeabilization Solution Kit. The following Abs

were purchased from BD Biosciences: TCRb (H57-597), TCRgd (GL-3), Downloaded from and EpCAM (G8.8). The following Abs were purchased from eBioscience: CD3 (145-2C11), CD4 (GK1.5), CD11c (N418), CD25 (PC61.5), CD45R (B220, RA3-6B2), MHC class II (M5/114), and Ki67 (SolA15). The following Abs were purchased from BioLegend: CD8 (53-6.7), CD11b (M1/70), CD44 (IM7), CD45 (30-F11), CD62L (MEL-14), CD122 (TM-b1), Gr-1 (RB6-8C5), Ly-51 (6C3), NK-1.1 (PK136), and TER-119 (TER-119). UEA-1 was purchased from Vector Laboratories. Data were collected using a BD LSR II flow cytometer and were analyzed using http://www.jimmunol.org/ FlowJo version 10 (Tree Star). The TaqMan Gene Expression Assay (assay identifiction number: Mm00502000_m1; Thermo Fisher Scientific) was used for quantification of Klotho gene expression. Neonatal thymus transplantation Mice heterozygous for Klotho were mated overnight and then separated. At the time of harvest, neonate pups were screened for Klotho via PCR. WT or Kl/Kl thymi were placed under the kidney capsule of B6.Cg-Foxn1nu/J mice in the previously described manner (35, 36). by guest on September 28, 2021 Bone marrow transplantation B6-CD45.1+ recipients were lethally irradiated using 1100 cGy total body irradiation by x-ray 1 d before infusion. On the second day, bone marrow cells (BM) were harvested from Kl/Kl mice and littermates. Mature T cells were removed from donor BM using anti-CD4 and anti-CD8 Abs, and low- toxicity rabbit complement was given i.v. at a cell dose of 1 3 107. Immunofluorescence staining Thymi were harvested and snap frozen in O.C.T. compound. Frozen sections (8 mm) were cut using a CM1900 cryostat (Leica Biosystems). Slides were dried for 30 min and then were immerged in acetone for 5 min at room temperature. The sections were blocked in PBS with 3% BSA (PBSB) for 1 h at room temperature and stained with the rabbit anti-mouse K5 polyclonal Ab (MBL International) and rat anti-mouse K8 mAb (TROMA-I; Developmental Studies Hybridoma Bank, University of Iowa), followed by DyLight 550 donkey anti-rabbit IgG Ab and DyLight 650 donkey anti-rat IgG (Invitrogen). ProLong Gold antifade reagent (Invi- trogen) was used to prevent photobleaching. Images were obtained using a microscope (DM5500B; Leica Biosystems) with a camera (DFC340FX; Leica Microsystems) operating with the Leica Application Suite Advanced Fluorescence (Leica Microsystems) software and analyzed using ImageJ (National Institutes of Health) software. Statistical analyses Prism software (GraphPad) was used for statistical analysis. Data sets were compared using an unpaired Mann–Whitney U test. Data are shown as 6 , mean values SD. Significance was defined as p 0.05. FIGURE 2. Reduction of TECs in 8-wk-old Kl/Kl mice. (A) A graph shows the absolute number of TECs in the thymus of Kl/Kl and control Results mice at 4 and 8 wk of age. (B) Frequencies of TEC subpopulations cTEC 2 + + 2 Kl/Kl mice showed profound involution of thymus at young (UEA-I Ly51 ) and mTEC (UEA-I Ly51 ). (C) Quantification of cTECs adult age and mTECs in Kl/Kl mice and littermate control at 8 wk of age. Each symbol in the graphs represents an individual mouse (n $ 5); small hor- The thymus is the primary lymphoid organ for the development of izontal lines indicate the group mean (6SD). *p , 0.05. ns, not significant T cells by providing a unique microenvironment able to attract (p $ 0.05). 4 TEC SUPPORT OF THYMOPOIESIS DOES NOT REQUIRE Klotho eliminated by negative selection (clonal deletion) or differentiate mice (Fig. 1D). Semimature SP thymocytes were barely detected into regulatory T cells (Tregs) (41). Subsequently, thymocytes are in 8-wk-old Kl/Kl mice (Fig. 1E, 1F, Supplemental Fig. 1E), selected for tolerance to tissue-restricted Ags but are responsive to suggesting that de novo generation of SP thymocytes was severely foreign Ags presented by the individual’s MHC haplotype (39). blocked (42). Thus, the thymus of 8-wk-old Kl/Kl mice was pre- We investigated the thymic microenvironment of Kl/Kl mice maturely involuted to an extent only observed in older, physio- from 4 wk of age, shortly after which these mice begin to display logically aged mice. signs of advanced aging (20). At 4 wk of age, the thymi of Kl/Kl mice were comparable to those of their WT littermates in size, Thymus involution in 8-wk-old Kl/Kl mice is associated with a cellularity, and subpopulation distribution (Fig. 1A–C). Kl/Kl severe reduction in TECs mice at 8 wk of age (young adult) displayed a significantly re- To assess the effect of Klotho on TECs, both cTECs and medullary duced thymic size when compared with that of WT littermate TECs (mTECs) were evaluated. They are defined by Ly51 ex- controls (Fig. 1A) and a profound decrease of thymocytes in total pression and binding capacity to the lectin UEA-1. cTECs are cellularity (Fig. 1B). Flow cytometry revealed that the frequency critical for T cell progenitor expansion and positive selection of of DP thymocytes was significantly decreased but that of DN, thymocytes, whereas both cTECs and mTECs affect the negative CD4SP, and CD8SP thymocytes remained unchanged in 8-wk-old selection of self-reactive thymocytes (43). To address whether Kl/Kl mice when compared with age-matched controls (Fig. 1C, TEC development and maintenance are impaired in the absence Supplemental Fig. 1A–D). All thymocyte subpopulations had, of Klotho, we assessed TEC cellularity and frequencies in 4- and however, a significantly reduced cellularity in 8-wk-old Kl/Kl 8-wk-old Kl/Kl mice. Total TEC cellularity and the frequencies of Downloaded from http://www.jimmunol.org/

FIGURE 3. Stromal cells from Klotho-deﬁcient and -sufﬁcient thy-

mus showed comparable ability to by guest on September 28, 2021 support T cell development after thymus transplantation. (A) Repre- sentative images of thymus grafts under kidney capsule in athymic host mice 8 wk after transplantation. (B) Quantification of total thymocytes of Klotho-deficient and -sufficient thymus grafts in the recipients 8 wk after transplantation. (C) Frequencies and cellularity (D) of thymocyte subpopulations in Klotho-deficient and -sufficient thymus grafts 8 wk after transplantation. (E) Frequencies and cellularity (F) of T cell subsets in the spleen from athymic recipients with Klotho-deficient and -sufficient thymus grafts 8 wk after transplantation. Each symbol in the graphs represents an individual mouse (n $ 4); small horizontal lines indicate the group mean (6SD). ns, not significant (p $ 0.05). The Journal of Immunology 5 their individual subsets were comparable at 4 wk of age to those of peripheral T cell reconstitution in both groups of transplanted WT or heterozygous littermate controls (Fig. 2A, 2B). In contrast, mice (Fig. 3E, 3F). In aggregate, these results demonstrated that 8-wk-old Kl/Kl mice showed a significant reduction in the number non-hematopoietic thymic stromal cells, including TECs, do not of total TECs (Fig. 2A), affecting especially mTECs (Fig. 2B, rely on Klotho expression for the organs’ thymopoietic activity. 2C). These data suggest that a Klotho deficiency intrinsically or extrinsically reduced TEC cellularity in 8-wk-old animals. Selective Klotho deficiency in BM does not impair thymocyte differentiation TECs do not require Klotho expression to support thymopoiesis Although mature blood cells, including T cells, do not express To determine the expression of Klotho in TECs, we performed Klotho, findings from the transplant experiments did not formally lo hi RT-PCR on sorted cTEC, mTEC , and mTEC cells in both WT exclude the requirement of Klotho for progenitor cells to commit to and Kl/Kl animals. Klotho expression was found in both mTEC a T cell fate and differentiate into mature thymocytes able to compartments but was minimal in the cTEC compartment in WT promote TEC maturation via thymus cross-talk. To examine this animals. As expected, there was no expression in the TECs of possibility, lethally irradiated H-2–matched WT mice were grafted Kl/Kl animals. Thymic involution is the result of cell-autonomous with either Klotho-deficient or -proficient HSCs and analyzed changes in cell maintenance as a function of age but can also 8 wk later (Fig. 4A). Recipients rescued with Kl/Kl HSCs dis- occur as a result of systemic abnormalities via various paracrine played thymocyte cellularity and differentiation comparable to effector mechanisms. To distinguish between these two explana- mice grafted with WT HSCs (Fig. 4B–D). These results demon- tions for the observed presenescent changes in thymic cellularity, strate that thymic Klotho expression in thymocytes is dispensable we grafted thymic lobes from either neonatal Kl/Kl or WT mice for repopulation of the T cell lineage in irradiated hosts. Downloaded from under the kidney capsule of haploidentical athymic [i.e., nude, Foxn1nu/nu (44)] recipients and compared their growth and dif- Thymus involution and peripheral T cell lymphopenia in 8-wk-old ferentiation. Foxn1nu/nu mice are homozygously deficient for Kl/Kl mice are averted by vitamin D deprivation the expression of functional Foxn1, a master regulator of TEC Previous studies have shown that elimination of vitamin D in growth, differentiation, and function of TEC, and therefore athy- Kl/Kl mice diet corrected the hypervitaminosis D typically ob-

mic and T cell deﬁcient, but they have otherwise intact HSCs. To served in these animals and consequently improved some of their http://www.jimmunol.org/ have a chronotypic comparison with 8-wk-old Kl/Kl mice, thymus disease-related phenotype (25, 45, 46). As our transplantation data grafts and peripheral lymphoid tissues of grafted recipients were indicated that premature thymic involution in Kl/Kl mice was not analyzed 8 wk after transplantation. The size and cellularity of caused by Klotho deﬁciency in either hematopoietic cells or thy- Kl/Kl and WT thymus grafts were comparable (Fig. 3A, 3B) and mic stromal cells, we next investigated whether a reduction in displayed identical proportions and cellularity of the distinct vitamin D levels improved the thymic changes and, as a result, the thymocyte subpopulations (Fig. 3C). The ostensibly normal thy- peripheral T cell compartment of Kl/Kl mice. In this experiment, mopoietic activity of Kl/Kl grafts resulted in a comparable both breeder mice and offspring were maintained on a vitamin by guest on September 28, 2021

FIGURE 4. Klotho-deficient and -sufficient BM showed equivalent capability to generate T cells in the bone marrow chimeras. (A) An experimental schema of making bone marrow chimeras. (B) Quantification of absolute cell numbers of thymocytes in recipients 8 wk after bone marrow transplantation. (C) A summary of percentages of thymocyte subpopulations in the thymi from the hosts. (D) Quantification of absolute numbers of thymocyte subpopulations in the hosts. Each symbol in the graphs represents an individual mouse (n = 3); small horizontal lines indicate the group mean (6SD). *p , 0.05. ns, not significant (p $ 0.05). 6 TEC SUPPORT OF THYMOPOIESIS DOES NOT REQUIRE Klotho

FIGURE 5. Thymus involution in 8-wk-old Kl/Kl mice was abrogated through feeding vitamin D–deprived diet. (A) Cellularity of total thymocytes in the thymi from 8-wk-old

Kl/Kl mice and littermate controls fed Downloaded from with vitamin D–deprived diet and Kl/Kl mice fed with vitamin D–replete diet. (B) Quantiﬁcation of frequencies of thymocyte subpopulations in the indicated mice. Each symbol in the graphs represents an individual mouse

(n $ 3); small horizontal lines indi- http://www.jimmunol.org/ cate the group mean (6SD). (C) Im- munofluorescence staining of thymus sections using anti-K5 and anti-K8 Abs and DAPI. Original magnification 310. *p , 0.05. ns, not significant (p $ 0.05). by guest on September 28, 2021

D–deprived diet throughout the experiment. Eight-week-old Kl/Kl survival, thereby contributing to T cell lymphopenia. We there- mice exposed since conception to low vitamin D levels displayed fore probed in Kl/Kl and control mice the cellularity and fre- a total thymus cellularity and thymocyte differentiation compa- quency of CD4 and CD8 SP T (CD4 T and CD8 T) cells in spleen rable to those of age-matched WT animals, whereas Kl/Kl mice andlymphnodes.At8wkofage,Kl/Kl mice fed a vitamin fed with a vitamin D–replete diet displayed, as expected, the D–depleted diet had comparable percentages (Fig. 6A) but de- typical hallmarks of Klotho deﬁciency (Fig. 5A, 5B). The thymus creased numbers (Fig. 6B) of both CD4 T and CD8 T cells architecture of 8-wk-old, conventionally fed Kl/Kl mice showed a compared with controls. structural disorganization, especially of the medulla (Fig. 5C), An increased frequency of CD44hi memory-like cells is a consistent with our previous observations (47). In contrast, the phenotypic hallmark of T cell lymphopenia. Interestingly, Kl/Kl histological structure of the thymus in both Kl/Kl and WT mice mice also may have impaired IL-7–dependent lymphopenia-driven fed with a vitamin D–deprived diet displayed separate and well- differentiation, expansion, or survival of memory-like T cells demarcated cortical and medullary compartments (Fig. 5C). These because of decreased IL-7 production in stromal cells (e.g., BM) data specify that low vitamin D levels correct the thymus phe- in Kl/Kl mice (48). The frequency of CD4 T CD44hi but not notype of Kl/Kl mice and suggest that the premature thymus in- CD8 T CD44hi memory-like T cells was unaffected (Fig. 6C, volution observed in Kl/Kl mice is the result of high systemic Supplemental Fig. 2), although the cellularity of both was reduced levels of vitamin D. in Kl/Kl mice on a vitamin D–depleted diet (Fig. 6D), suggesting a We considered the possibility that hypervitaminosis D in Kl/Kl defect in memory-like T cell support. Moreover, the absolute mice may inhibit the emigration of mature thymocytes to the cellularity of splenic CD4 T and CD8 T cell lymphopenia was periphery, possibly contributing to T cell proliferation and/or comparable between Kl/Kl mice fed a vitamin D–deprived diet The Journal of Immunology 7

FIGURE 6. Reduction of T cells that was found in the periphery in 8-wk-old Kl/Kl mice can be prevented by deprivation of vitamin D. (A) Fre- quency and (B) cellularity of CD4 T and CD8 T cells in the spleen from Kl/Kl mice and littermate controls at 8 wk of age. (C) Frequency and (D) cellularity of CD44hi memory-like T cell subpopulations in the spleen from Kl/Kl mice and littermate controls at 8 wk of age. (E) Frequency and (F) cellularity of CD4 T and CD8 T cells in the spleen from 8-wk-old Kl/Kl mice and littermate controls fed with vitamin D–deprived diet. Each Downloaded from symbol in the graphs represents an individual mouse (n $ 4); small horizontal lines indicate the group mean (6SD). *p , 0.05. ns, not signiﬁcant (p $ 0.05). http://www.jimmunol.org/

and control animals (Fig. 6E, 6F). Overall, the data show that skin atrophy, and increased life span (45). Feeding kl/kl mice by guest on September 28, 2021 Kl/Kl animals at 8 wk of age have significant losses of both thy- a vitamin D–deficient diet indeed improved thymic architec- mocytes and TECs and that these deficits can be overcome by ture, TEC differentiation, thymocyte development and matura- decreasing vitamin D levels in vivo. tion, and peripheral T cell reconstitution. Therefore, it may be tempting to speculate that the vitamin D–deficient diet had direct Discussion effects on TECs, despite the fact that the genetic deficiency of Our results demonstrate that thymus function in young Kl/Kl mice Klotho in TECs would be unaffected. Similarly, the data from is unaffected, but severe atrophy with reduced thymopoiesis is kidney capsule implants indicated that the TECs from kl/kl mice observed in these mice by 8 wk of age. This change in thymus are able to develop and support thymopoiesis despite the Klotho function was neither the consequence of a cell-intrinsic loss of deficiency. Taken together, we favor the hypothesis that correc- Klotho expression in hematopoietic cells nor the result of a cell- tion of calcium and phosphorus imbalance and high vitamin D autonomous deficiency in expression of this protein by thymic levels are responsible for improvement in thymopoiesis and TEC stromal cells. Rather, thymic hypocellularity and the loss of a development. regular thymic stromal architecture in Kl/Kl mice are the conse- Both indirect and direct TEC effects of Klotho deficiency can be quence of high vitamin D levels, suggesting that Klotho deficiency envisioned. Metabolic imbalance of calcium, phosphorus, and disrupts thymus function in an indirect, systemic fashion. vitamin D3 may indirectly impair TEC function and development The molecular interactions of FGF23, Klotho, and vitamin D because of apoptosis and stress in the animals. coordinate to regulate phosphate metabolism (24, 46, 49–51). High vitamin D3 levels may indirectly impair TEC development FGF23 decreases renal tubular and phosphate reabsorption and and maturation by interfering with the essential cross-talk sig- stimulates vitamin D3, which results in increased renal Klotho naling mechanisms between TECs and thymocytes needed for synthesis (46). Klotho binds to FGFR1(IIIc), which reduces their mutual development (43, 53). In support of this hypothesis, vitamin D3 synthesis. In Kl/Kl mice, FGF23 is unable to bind thymocytes express the vitamin D receptor according to gene the Klotho transmembrane molecule, precluding its transport expression data (accession no. GSE81163, http://www.ncbi.nlm. to FGFR1c, resulting in a failure to negatively regulate vitamin D3 nih.gov/geo/query/acc.cgi?acc=GSE81163). High vitamin D lev- synthesis via FGF23 receptor-mediated inhibition of 1a-hydrox- els directly impair TEC development because cells within the ylase (52).Thus, high levels of active vitamin D accumulate in thymus that express high levels of vitamin D receptor are sensitive Kl/Kl mice, causing a state of calcium and phosphate imbalance. to vitamin D–induced signaling. In the absence of Klotho, levels To counteract the abnormal calcium and phosphorus state in kl/kl of FGF23 are elevated (54), which may be toxic to developing mice, vitamin D–deficient diets have been tested and have been thymocytes, as it is to the kidney (55), or exert yet unknown ef- shown to ameliorate other consequences related to accelerated fects independent of vitamin D levels. Klotho suppresses NF-kB aging, including decreased ectopic calcification of tissues, lack of translocation and therefore suppresses inflammatory cytokine 8 TEC SUPPORT OF THYMOPOIESIS DOES NOT REQUIRE Klotho production (56), and conversely, Klotho deficiency increases T cells and repertoire constriction predict poor response to vaccination in old primates. [Published erratum appears in 2010 J. Immunol. 185: 4509.] J. proinflammatory cytokines such as IL-6 or TNF-a in monocytes Immunol. 184: 6739–6745. (57), which could be driving thymocyte death, leading to insuffi- 12. Davitt, C. J., and E. C. Lavelle. 2015. Delivery strategies to enhance oral vac- cient support of TECs. cination against enteric infections. Adv. Drug Deliv. Rev. 91: 52–69. 13. Prelog, M. 2006. Aging of the immune system: a risk factor for autoimmunity? Medullary thymocytes also express the vitamin D receptor, and Autoimmun. Rev. 5: 136–139. vitamin D signaling inhibits mitogenic stimulation of these cells (58, 14. 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