Published May 8, 2015, doi:10.4049/jimmunol.1500082 The Journal of

Thymic Involution Perturbs Negative Selection Leading to Autoreactive T Cells That Induce Chronic Inflammation

Brandon D. Coder, Hongjun Wang,1 Linhui Ruan,2 and Dong-Ming Su

Thymic involution and the subsequent amplified release of autoreactive T cells increase the susceptibility toward developing , but whether they induce chronic inflammation with advanced age remains unclear. The presence of chronic low- level proinflammatory factors in elderly individuals (termed inflammaging) is a significant risk factor for morbidity and mortality in virtually every chronic age-related disease. To determine how thymic involution leads to the persistent release and activation of autoreactive T cells capable of inducing inflammaging, we used a Foxn1 conditional knockout mouse model that induces accel- erated thymic involution while maintaining a young periphery. We found that thymic involution leads to activation shortly after thymic egress, which is accompanied by a chronic inflammatory phenotype consisting of cellular infiltration into non– lymphoid tissues, increased TNF-a production, and elevated serum IL-6. Autoreactive T cell clones were detected in the periphery of Foxn1 conditional knockout mice. A failure of negative selection, facilitated by decreased expression of Aire rather than impaired generation, led to autoreactive T cell generation. Furthermore, the young environment can reverse age-related regulatory T cell accumulation in naturally aged mice, but not inflammatory infiltration. Taken together, these findings identify thymic involution and the persistent activation of autoreactive T cells as a contributing source of chronic inflammation (inflammaging). The Journal of Immunology, 2015, 194: 000–000.

hronic inflammation is a ubiquitous feature of the aging cancers including colitis-associated colon cancer and hepatocellular process and implicated in virtually every age-related carcinoma (5). IL-6 and TNF-a are the most predictive inflamma- C disease (1, 2). The term “inflammaging” describes the tory biomarkers and are highly correlated with “all-cause” mor- low-level, chronic, and systemic proinflammatory state that bidity and mortality in the elderly (6). Although the etiology accompanies advanced age in the absence of infection (1). Even of inflammaging is not fully understood, the source of proin- though clinical manifestations are not obvious, the presence flammatory factors is primarily attributed to the combination of proinflammatory factors such as IL-6, TNF-a,IL-1,and of cellular senescence–induced senescence-associated secretory C-reactive protein are associated with the severity, incidence, and phenotype (SASP) and the persistent activation of immune cells mortality of cardiovascular diseases such as atherosclerosis (1, 2, 7).

by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. and myocardial infarction (3), neurodegenerative diseases such as The persistent activation of immune cells is hypothesized to Parkinson’s disease (4) and Alzheimer’s disease (1), and late-life result from chronic cell death and self-debris serving as damage- associated molecular patterns, leading to innate immune cell activation (1, 8) or repeated life-long exposure to latent viral Department of Cell Biology and Immunology, University of North Texas Health infections, such as CMV (9, 10), continuously activating both Science Center at Fort Worth, Fort Worth, TX 76107 innate and adaptive immune responses. However, it is not known 1Current address: Department of Histology and Embryology, Jilin Medical College, Jilin, People’s Republic of China. whether thymic involution can lead to the persistent activation of 2Current address: First Affiliated Hospital, Wenzhou Medical University, Wenzhou, T cells that are capable of inducing inflammaging. People’s Republic of China. The is a primary lymphoid organ comprised of cortical

http://classic.jimmunol.org Received for publication January 13, 2015. Accepted for publication April 14, 2015. and medullary thymic epithelial cells (cTECs and mTECs) re- This work was supported by National Institute of and Infectious Diseases/ sponsible for the development of and the generation National Institutes of Health Grants R01AI081995 (to D.-M.S.) and 3R01AI081995- of central toward self-tissues. Thymic-driven 03S1 (to B.D.C.). central immune tolerance is accomplished through two mecha- D.-M.S. and B.D.C. conceived and designed the experiments; B.D.C., L.R., and nisms: the elimination of autoreactive T cell clones via the process H.W. performed the experiments and analyzed the data; and D.-M.S. and B.D.C. wrote the paper. of negative selection (11), and the generation of natural regulatory

Downloaded from Address correspondence and reprint requests to Dr. Dong-Ming Su, Department of T cells (nTregs) via diverted differentiation (12, 13). These two Cell Biology and Immunology, University of North Texas Health Center at Fort processes depend on the TCR–self-peptide–MHC avidity and Worth, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107. E-mail address: signal strength, where a weak signal leads to survival [email protected] (14), a strong signal leads to (15), and a moderate The online version of this article contains supplemental material. signal plus (IL-2) leads to nTreg differentiation (16, 17). Abbreviations used in this article: Aire, gene; cKO, condi- The efficiencies of negative selection and the differentiation of tional knockout; cTEC, cortical ; Ctr, control; DP, double- positive; F-cKO, Foxn1 conditional knockout; fx, loxP-floxed Foxn1 (Foxn1 flox); nTregs are dependent on the production and presentation of tissue- IRBP, interstitial retinol-binding protein; mOVA, membrane-bound OVA; mTEC, specific Ags on MHC, which is, in part, regulated by the auto- medullary thymic epithelial cell; nTreg, natural regulatory T cell; RTE, recent thymic emigrant; SASP, senescence-associated secretory phenotype; SP, single- immune regulator gene (Aire) in mTECs (18–22). positive; TEC, thymic epithelial cell; Teff, T effector cell; Tg, transgenic; TM, The thymus undergoes a progressive and age-related involution T tamoxifen; Treg, regulatory T cell; uCreER , ubiquitous promoter-driven Cre- attributed to the deterioration of the thymic microenvironment (23), recombinase and estrogen-receptor fusion protein; WT, wild-type. which is made of an integrated three-dimensional meshwork of Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 cTECs and mTECs, where TEC differentiation is regulated by the

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500082 2 INFLAMMAGING RELATED TO THYMIC INVOLUTION

Foxn1 gene (24). It has been reported that defects in mTEC as well as sera) of recipient RAG2/2 mice were collected for analysis of structure and the loss of Aire can affect the maintenance of central inflammatory cell infiltration. immune tolerance (25–27) by leading to the generation of fewer Thymic lobe kidney capsule transplantation (28) or deficient nTregs (29), and thereby increasing the incidence of autoimmune disease. However, the mechanisms through which The surgical operation of the kidney capsule transplantation was performed as previously described (31). Intact newborn mouse thymic lobes of fx/fx- thymic involution impacts the two mechanisms of central toler- uCreERT and fx/fx-only with and without mOVA-Tg were directly trans- ance (negative selection and nTregs) are not fully understood. planted into young host OT-II+ TCR-Tg mice. Three days after the graft, Furthermore, whether thymic atrophy alone leads to the release of the host mice were i.p. injected with TM (1 mg/10 g body weight/d) for 3 autoreactive T cells that become persistently activated immune consecutive days to induce deletion of the Foxn1 gene. Two weeks after the last TM injection, the grafted thymi were isolated for FACS analysis of cells and contribute to inflammaging remains unclear. CD4 and CD8, as well as the TCR-Tg (Va2Vb5) marker. In this study, we focus on the involvement of thymic involution in inflammaging by utilizing a loxp-Foxn1–ubiquitous promoter- Specific autoreactive T cell detection model: interstitial driven Cre-recombinase and estrogen-receptor fusion protein retinol-binding protein (IRBP) P2 immunization and (uCreERT) conditional knockout (F-cKO) mouse model, which P2-tetramer enrichment of IRBP-specific T cells induces accelerated thymic involution of a fully matured thymus The fx/fx-uCreERT (F-cKO) or fx/fx-only (FF-Ctr) mice (6 wk old) were while maintaining a young periphery (30), and a naturally aged given 33 TM i.p. injections to induce deletion of the Foxn1 gene. Four C57BL/6 mouse model. The F-cKO mouse model allows for the weeks after the last TM injection, mice were immunized by s.c. injection m m inducible deletion of Foxn1 after the thymus has fully matured, of 100 g IRBP (aa 294–306) P2 peptide emulsified in 100 lCFA.Ten days following immunization, cells from lymph nodes and spleen of the either by administering tamoxifen (TM) or the slow leakage of mice were harvested for IRBP–P2–I-Ab–tetramer (allophycocyanin la- uCreERT, resulting in accelerated epithelial-driven thymic atrophy beled) enrichment with anti-allophycocyanin microbeads and MACS col- that is comparable with thymic epithelium dysfunction observed umns (Miltenyi Biotec), according to published protocols (32). Positively selected cells were counted and then stained with Abs for flow cytometry. in naturally aged C57BL/6 mice (24, 30). Although the slow b T P2–I-A tetramer was generated by the National Institutes of Health Tet- leakage of uCreER results in weak deletion of genomic Foxn1 at ramer Core Facility and provided by Dr. Mark Anderson (University of ∼1 mo of age (24), observable biological effects including the loss California, San Francisco). of Foxn1 expression, thymic involution, mTEC disruption, and Flow cytometry assay thymic dysfunction do not become apparent until ∼3–9 mo of age (24) or until induced with the administration of TM (30). We Single-cell suspensions were prepared from the thymus and spleen of mice demonstrate that thymic involution disrupts central immune tol- using a 70-mm cell strainer. Spleen cells were erythrocyte depleted with erance and results in the release of autoreactive T cells to the RBC lysing buffer (Sigma-Aldrich, catalog no. R7757) and washed with staining buffer. Samples were then treated with Fc receptor blocking Ab periphery. Furthermore, shortly after thymic egress, these auto- 2.4G2. Samples were then stained with specific Ab of cell surface cluster reactive T cells gain the activated immune cell phenotype and of differentiation markers and or fixed with 2% paraformaldehyde and induce systemic low-grade inflammation that is indicative of permeabilized with Triton X-100, as previously reported (33), followed by inflammaging. Finally, we determined that the mechanism re- intracellular staining for Bim (Cell Signaling Technology, no.2819s), Ki67, Foxp3 (eBioscience, no. 12-5773-82), and Aire (eBioscience, no. 50- sponsible for the thymic involution–driven breakdown of immune 5934-80). TECs were digested following previously published methods

by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. tolerance results from perturbed negative selection and a reduction (33) and then stained with surface and intracellular Abs. Fluorochrome- in the mTEC expression of Aire rather than defects in the gen- conjugated Abs (clone) CD4 (GK1.5), CD8 (53-6.7), CD44 (IM7), Ki67 eration of Tregs. Taken together, these results identify thymic (16A8), TNF-a (MP6-XT22), CD25 (PC61), TCR Va2 (B20.1), TCR Vb5 involution as a contributing source of inflammaging and a poten- (MR9-4), CD45 (30-F11), MHC class II (M5/114.15.2), Ly51 (6C3), EpCAM (G8.8), and GITR (YGITR 765) were purchased from BioLegend. tial therapeutic target for age-related chronic inflammation. Flow cytometry was performed using an LSR II flow cytometer (BD Biosciences) and data were analyzed using FlowJo software. Materials and Methods isolation from non–lymphoid organs and flow Mice, crossbreeding, and animal care cytometry–based Treg analysis

http://classic.jimmunol.org All animal experiments were in compliance with protocols approved by the from non–lymphoid organs (liver, lung, and salivary gland) Institutional Animal Care and Use Committee of the University of North were isolated by two-layer density gradient centrifuge on Lympholyte-M Texas Health Science Center, in accordance with guidelines of the National (Cedarlane Laboratories, Burlington, ON, Canada, no. CL5031) at 2000 Institutes of Health. Various gene-manipulated mouse colonies (all on rpm for 15 min. The cells between the two layers were collected for Treg a C57BL/6 background) and their crossbreeding schemes are listed in cell staining. Thymocytes were isolated by cell strainer. Single-cell sus- T Supplemental Table I. They are the Foxn1 cKO (fx/fx-uCreER mice with pensions were stained with fluorochrome-conjugated anti-mouse cluster of induced Foxn1 deletion via TM treatment, termed F-cKO) (30), fx/fx-only differentiation Abs (BioLegend). Intracellular detection of Foxp3 with PE- (without uCreERT, same as wild-type [WT] in Foxn1 expression, termed 2 2 conjugated anti-Foxp3 (clone FJK-16s, eBioscience) and detection of Bim Downloaded from / FF-control [Ctr]) (30), Rag2-GFP reporter, RAG knockout, membrane- with anti-Bim followed by secondary allophycocyanin-conjugated anti- bound OVA (mOVA) transgenic (Tg), OT-II TCR Tg, and autoimmunity rabbit were performed on fixed and permeabilized cells via Cytofix/ 2/2 regulator gene (Aire) knockout mice. Approximately one- to 2-mo-old Cytoperm (eBioscience). Data were acquired and analyzed as above. F-cKO mice display very faint deletion of Foxn1 exons 5 and 6 as detected by PCR, but they do not differ from fx/fx-only control mice in Foxn1 Treg suppressive function assay expression, mTEC maturation, thymic size, and other factors (24). Fol- T lowing induced Foxn1 deletion via TM, ∼1- to 2-mo F-cKO mice display fx/fx-uCreER and fx/fx-only mice (3–4 mo old) were treated with TM i.p. very strong deletion of Foxn1 exons 5 and 6 and undergo accelerated (F-cKO and FF-Ctr). Two to 3 wk following the final injection, spleens thymic involution (30). Mouse ages are indicated in each figure legend, were harvested and passed through a cell strainer to obtain a single-cell defined as young (1–2 mo old) and aged (18–22 mo old) groups. Aged WT suspension. The samples were erythrocyte depleted and washed, and then mice were purchased from the National Institute on Aging. stained with anti-CD4 and anti-CD25. Cells were sorted on a BD Influx cell sorter (BD Biosciences) and collected into two groups: Tregs (CD4+ + + 2 4 Adoptive transfer CD25 ) or T effector cells (Teffs; CD4 CD25 ). Teffs (5 3 10 )/Tregs (2.5 3 104) were cocultured in 96-well U-bottom plates with 5 3 104 Erythrocyte-depleted spleen cells from young and aged WT mice or young irradiated APCs (splenocytes from C57BL/6 mice), 1 mg/ml anti-CD3ε, F-cKO mice were i.v. injected through the retro-orbital route into young 2 mg/ml anti-CD28, and RPMI 1640 medium totaling 100 ml per well for 2/2 7 RAG mice (2.5 3 10 cells per recipient mouse). Sixty days after the 72 h. Proliferation of cells was determined using CellTiter 96 AQueous One injection, the representative tissues (such as the lung, liver, salivary gland, solution reagent (Promega, catalog no. TB245) following the Promega The Journal of Immunology 3

protocol: adding 20 ml solution per well for the last 2 h of culture, and following specific tetramer enrichment (41). To expand and detect absorbance was measured at 490 nm using an ELISA 96-well plate reader autoreactive T cells, we first induced thymic involution through (BioTek ELx800). Foxn1-cKO. One month following induction of thymic involution, 2 2 H&E staining for visualizing lymphoinfiltration we immunized F-cKO, FF-Ctr, and Aire / (positive control)

2/2 mice with an IRBP peptide-2 (P2) and 10 d later pooled Following adoptive transfer into young RAG mice, salivary glands were + harvested and fixed, cut into 5-mm-thick sections, and stained with H&E. lymph nodes and spleen to enumerate CD4 T cells reactive to the P2 peptide by P2–I-Ab tetramer enrichment. F-cKO mice showed Statistical analysis a significant expansion of activated P2–I-Ab autoreactive T cells, For evaluation of group differences, the unpaired two-tailed Student t test which were mostly undetectable in FF-Ctr littermate controls was used assuming equal variance. A p value ,0.05 was considered sta- (Fig. 2A, 2B). This likely explains the source of activated RTEs in tistically significant. our Foxn1-cKO thymic involution model as autoreactive T cell clones responding to peripheral self-. Results Next, we wanted to know whether the activated immune cell Newly released T cells from the atrophied thymus acquire an phenotype resulting from thymic involution was sufficient to in- activated immune cell phenotype duce an elevated proinflammatory state. We focused on the cyto- Recent evidence suggests that persistently activated immune cells kines IL-6 and TNF-a, which are the most indicative of poor may be a prime cause of age-related chronic inflammation, but the prognosis in chronic age-related disease (2, 6). One month fol- mechanisms leading to the chronic activation of immune cells are lowing induction of thymic involution, we observed a 2-fold in- not fully understood (6, 9, 10). Utilizing our previously described crease in the percentage of CD4+TNF-a+ T cells in F-cKO mice loxp-Foxn1-uCreERT conditional knockout mouse model (F-cKO) compared with FF-Ctr littermate controls (Fig. 2C). Additionally, for accelerated thymic involution (30), we sought to determine the concentration of IL-6 in the serum was found to be 2-fold whether thymic atrophy could lead to activated T cells without any higher in F-cKO mice (Fig. 2D) compared with FF-Ctr littermate additional stimuli. Administration of TM into ∼1- to 2-mo-old controls, implicating thymic involution in both local and systemic F-cKO mice allows for accelerated thymic involution that would inflammation. This low-grade (just above baseline), but signifi- not emerge from the slow leakage of uCreERT until ∼3–9 mo of cant, inflammatory state found in the periphery of Foxn1-cKO age (24, 30). To differentiate T cells that have egressed from the mice is consistent with the conditions described in inflammaging. atrophied thymus versus those that had egressed prior to thymic We also found pathological changes in multiple organs, such as involution, we crossed our Foxn1-cKO mice with Rag2 promoter– inflammatory cell infiltration in the liver, lung, pancreas, brain, and driven GFP-expressing mice, which identifies newly generated salivary glands of these Foxn1-cKO mice reported in our previ- T cells or recent thymic emigrants (RTEs) (34–36). The GFP ously published study (42), which confirms the presence of an signal is turned on in thymocytes undergoing RAG-dependent autoreactive activated immune cell phenotype resulting from TCR recombination and persists for up to 2 wk following thy- thymic involution, although neither dominant clinical manifes- mic egress (35). tations nor full-blown autoimmune disease can be observed in Generally, RTEs are not in a highly active state because they these mice. Taken together, these results indicate that thymic in-

by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. have yet to encounter their cognate Ags. As expected, using TM to volution alone is sufficient to induce a chronic and low-grade conditionally knockout the Foxn1 gene in young fx/fx-uCreERT inflammatory state. mice (F-cKO) to induce accelerated thymic atrophy (30) resulted Conditional Foxn1 knockout impairs clonal deletion of in fewer CD4+ and CD8+ RTEs owing to decreased thymic output single-positive thymocytes resulting from defects in negative (Fig. 1A, box in top left panel, Supplemental Fig. 1A). However, selection within this reduced population of F-cKO RTEs, there was a higher proportion of CD44hi cells (Fig. 1B, left panels, Supplemental The detection of autoreactive T cells in the periphery of Foxn1-cKO Fig. 1B), which represent Ag-experienced T cells (37), and mice indicates that thymic involution results in the impairment of these F-cKO RTEs were more proliferative (Fig. 1B, Supplemental central immune tolerance, disrupting either negative selection or http://classic.jimmunol.org Fig. 1B, Ki67+ cells shown in red histograph in right panels). the function/generation of nTregs. We first wanted to focus on the We further ascertained that the RTEs undergoing proliferation functional ability of the Foxn1-cKO atrophied thymus to execute (Ki67+) were CD44hi RTEs (Fig. 1A, red boxes, 1C, right panel). negative selection and remove self-reactive TCRs from the de- These mice are housed together in a clean environment and are veloping TCR thymocyte pool. As we previously reported (30), specific pathogen-free. Taken together, these results demonstrate conditional knockout of Foxn1 in TECs of young mice results in that even in the absence of infection, CD4+ and CD8+ RTEs derived a significant decrease of total thymocyte number and causes thy-

Downloaded from from the atrophied thymus take on an activated immune cell mic atrophy. We observed a dramatic reduction in the percentage phenotype shortly after encountering the peripheral environment. and total cellularity of the CD4+CD8+ double-positive (DP) thy- Therefore, these activated T cells are potentially autoreactive mocyte subpopulation (Fig. 3A, 3B) in the F-cKO thymus com- T cells. pared with the FF-Ctr. Because of the highly stochastic nature of TCR rearrangement (43), one would presume that a diminished Thymic involution results in autoreactive T cells and chronic DP subpopulation would be accompanied by equally reduced CD4 low-grade inflammation and CD8 single-positive (SP) subpopulations. However, we found To verify whether activated RTEs derived from the atrophied that the total numbers of CD4 and CD8 SP thymocytes were not thymus were responding to peripheral self-antigens, we employed reduced in the F-cKO thymus compared with the FF-Ctr, and the an IRBP immunization model to detect and amplify autoreactive frequency of SP thymocytes is increased in the F-cKO atrophied T cell clones within a polyclonal T cell repertoire. IRBP is an eye thymus (Fig. 3A, 3B). The increased proportion of SP thymocytes protein expressed in mTECs as a tissue-specific Ag under the suggests that the F-cKO cannot efficiently perform clonal deletion control of the autoimmune regulator gene Aire (32, 38–40). Au- (negative selection) resulting from the loss of Foxn1 in TECs. toreactive T cells can be induced to incite autoimmune uveitis in We next attempted to investigate the mechanisms leading to the Aire-deficient mice and is detected in the spleen and lymph nodes accumulation of SP thymocytes in the Foxn1-cKO thymus. Aire, 4 INFLAMMAGING RELATED TO THYMIC INVOLUTION

FIGURE 1. Newly released CD4+ T cells from the atrophied thymus acquired an activated immune cell phenotype. F-cKO and FF-Ctr (fx/fx-uCreERT and fx/fx mice, respectively) were crossed with Rag2-GFP mice and then Foxn1fx/fx deletion was induced in 6-wk- old adult mice with i.p. injected TM. Fourteen days later, peripheral splenocytes were freshly isolated and stained with CD4, CD44, and Ki67 Abs, and CD4+ GFP+ cells were defined as CD4+ RTEs. (A) Repre- sentative dot plots show CD44hiKi67+ cell gates (red boxes) in CD4+ RTEs from F-cKO (top panels) and FF-Ctr (bottom panels)mice.(B) Representative histo- grams of CD44hi (left panels)andKi67+ (right panels) in CD4+ RTEs from F-cKO (top panels)andFF-Ctr (bottom panels)mice.(C) Summarized results of per- centage CD44hi, Ki67+,andCD44hiKi67+ DP cells in CD4+ RTEs (from left to right panels). A Student t test was used to determine statistical significance between groups. All data are expressed as means 6 SEM. Data are pooled from at least three independent experiments (n =numberanimals). by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. http://classic.jimmunol.org

which regulates expression of tissue-specific Ags, is one key progenitors seed the kidney-grafted thymic lobes and factor in determining the efficiency of negative selection (21, 44). give rise to thymocytes with TCRs that are specific for OVA, MHC +

Downloaded from We previously published that Aire mTECs are reduced following class II restricted (CD4), and bind MHC/OVA complexes with the loss of Foxn1 (42). In the present study, we further found that a strong affinity. Thymocytes possessing TCRs that bind to MHC/ the reduction of Aire+ mTECs was not simply due to the reduction OVA complexes with too strong of an affinity will be signaled to of total mTECs, but is actually due to a reduction in the frequency undergo clonal deletion. In our system, host mice do not receive of Aire+ mTECs among all mTECs (Fig. 3C, 3D), as well as any irradiation as in the conventional bone marrow transplantation a reduction in the mTEC expression of Aire on a per cell basis, method. Therefore, there is minimal risk for damage to the stromal measured by mean fluorescence intensity (Fig. 3E). This disrup- cell niche that may affect Ag presentation. RIP-mOVA mice have tion in Aire expression following the loss of Foxn1 potentially been crossbred with Foxn1-cKO mice yielding F-cKO-mOVA, FF- perturbs negative selection by impairing the expression of Aire- mOVA, and FF-Ctr offspring. Fetal thymic lobes of equal size and dependent self-antigens (42). cellularity (data not shown) were harvested from F-cKO–mOVA, To find direct evidence that the loss of Foxn1 impairs the clonal FF-mOVA, and FF-Ctr newborns and then transplanted under the deletion of autoreactive thymocytes, we used the OT-II–RIP– kidney capsule of young adult OT-II Tg mice. Three days after the mOVA model of Aire-dependent negative selection. RIP-mOVA+ transplantation surgery, i.p. injections of TM were administered to mTECs express chicken OVA as a mock self- under the the OT-II host mice for 3 successive days to induce the Foxn1 control of the RIP by an Aire-dependent mechanism (19, 45). OT-II conditional knockout in the kidney-grafted thymus. Two weeks after The Journal of Immunology 5

FIGURE 2. Autoreactive T cells and chronic low- grade inflammation following thymic involution. One month following TM treatment for the conditional knockout of Foxn1,(A) F-cKO, FF-Ctr, and Aire2/2 (positive control) mice were immunized with IRBP P2 peptide and lymphocytes were harvested 10 d later for flow cytometry following P2 tetramer enrichment of specific T cells. Plots are pregated on CD3+CD4+ events, and CD44hiP2-tetramer+ population is dis- played in red boxes. (B) Calculated absolute number of P2-specific CD3+CD4+CD44hi peripheral T cells is shown in a bar format. (C) Freshly isolated splenocytes from FF-Ctr and F-cKO mice without immunization were costimulated with anti-CD28 and anti-CD3ε in culture for 4.5 h, and then TNF-a production was assessed by intracellular staining. (D) Sera from FF-Ctr and F-cKO mice in (C) were collected and IL-6 was measured by ELISA. A Student t test was used to determine statistical significance between groups. All data are expressed as means 6 SEM. Data are pooled from three independent experiments (each symbol represents an animal). by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. http://classic.jimmunol.org the final TM injection, the grafted thymic lobes were isolated for Thymic involution does not impair the generation or function of FACS analysis (Fig. 4C). As expected, OT-II hosts grafted with nTregs newborn thymic lobes from FF-Ctr mice that completely lack the The manifestation of autoreactive inflammation in the Foxn1-cKO mOVA transgene contain a normal proportion of OT-II–specific mouse model of accelerated thymic involution suggests a break- + 2 a b + top panel CD4 SP thymocytes (CD4 CD8 V 2V 5 ) (Fig. 4A, , down in the maintenance of central immune tolerance. We revealed

Downloaded from 4B, filled circles); when the OT-II progenitors were seeded into measureable dysfunction in the process of negative selection in the the grafted FF-mOVA thymus that carries the mOVA transgene Foxn1-cKO thymus. We next addressed whether Foxn1-cKO–driven and WT levels of Foxn1 (not atrophied), then strong negative thymic involution alters the other arm of central immune tolerance, selection was observed in OT-II CD4 SP thymocytes (Fig. 4A, that is, the generation of nTregs. Several studies have shown that the middle panel, 4B, filled squares). However, when the OT-II disruption of thymic medullary microstructure andareductionin progenitors seeded into the grafted F-cKO–mOVA thymus, + which carries the mOVA transgene and the Foxn1-cKO–induced medullary size led to the generation of fewer or deficient CD4 nTreg thymic atrophy, the proportion of CD4+CD82Va2Vb5+ cells cells (29). Despite the severe depletion of overall thymic cellularity, was increased (Fig. 4A, bottom panel, 4B, open squares) com- there was not a significant difference (slightly increased) in the total + 2 + + pared with FF-Ctr thymus, indicating that the OT-II CD4 SP numbers of thymic CD4 CD8 CD25 Foxp3 nTregs (Fig. 5A, 5C) thymocytes in F-cKO–mOVA thymus evade clonal deletion. in the F-cKO thymus compared with FF-Ctr. Instead, the frequency of These results provide direct evidence that the loss of Foxn1 and thymic nTregs was increased in the F-cKO mice (Fig. 5B). These subsequent thymic involution perturbs negative selection, lead- findings suggest thymic involution does not impair the production of ing to the survival of SP thymocytes that potentially recognize nTregs and may actually be a contributing factor toward the age- self-antigen. related accumulation of Tregs (46). 6 INFLAMMAGING RELATED TO THYMIC INVOLUTION

FIGURE 3. Clonal deletion of SP thy- mocytes was impaired in the Foxn1-cKO atrophied thymus. (A and B) Five days after inducing Foxn1fx/fx deletion with TM in 6-wk-old FF-Ctr and F-cKO mice, thymoyctes were freshly isolated for cell surface stain- ing of CD4 versus CD8. (A) Representative flow cytometry plots show CD4+ and CD8+ SP gates of F-cKO (right panel) and FF-Ctr (left panel) mice. (B) Summarized results of absolute cell numbers per thymus (left panel) and percentage of each subpopula- tion in total thymocytes (right panel). Open bars represent FF-Ctr mice; filled bars rep- resent F-cKO mice. Each group contained seven animals. (C and D) Thymic epithelial cells were enzymatically digested from F-cKO and FF-Ctr thymi and intracellularly stained for Aire Ab. (C)Flowcytometrywas used to gate on mTECs (CD452, MHC class II+,EpCAM+, Ly512), (D) Summarized results of the percentage of Aire+ mTECs, by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. and (E) the mean fluorescence intensity (MFI) of Aire+ mTECs. A Student t test was used to determine statistical significance between groups. All data are expressed as means 6 SEM. Data are pooled from at least three independent experiments with a total of n = 7 animals per group. http://classic.jimmunol.org Downloaded from

Irrespective to their numbers, Tregs must possess adequate Foxn1-cKO atrophied thymus to suppress the proliferation of suppressive capabilities to sustain immune tolerance. Whereas Teffs. Tregs (CD4+CD25+) were isolated from the spleens of many laboratories have shown that aged Tregs retain their sup- either F-cKO or FF-Ctr mice, and Teffs (CD4+CD252)were pressive function (46–49), others have reported a functional de- isolated from the spleens FF-Ctr mice (Fig. 5D). They were cline from thymic involution–derived Tregs (29, 50). To deter- cocultured in the presence of irradiated APCs and CD3ε and mine whether Foxn1-cKO–induced thymic atrophy impairs nTreg CD28 Abs. We did not find any differences in the suppressive function, we tested the ability of peripheral Tregs derived from the ability of Tregs coming from either F-cKO or FF-Ctr mice The Journal of Immunology 7

FIGURE 4. mOVA-expressing Foxn1-cKO thymi do not efficiently delete OT-II TCR Tg thymocytes. mOVA-expressing mice were crossed with F-cKO and FF-Ctr to generate F-cKO–mOVA, FF-Ctr-mOVA, or FF-Ctr (without mOVA expression) genotypes. New- born thymi of equal size from each genotype were grafted separately under the kidney capsule of OT-II host mice, which were then injected i.p. with TM to induce loxp-floxed Foxn1 deletion in the grafted thymi. Two weeks following kidney capsule transplantation, grafted thymi were harvested and stained with CD4, CD8, Va2, and Vb5 Abs. (A) Representative dot plots of thymocytes from the grafted thymi gated on CD4 SP thymocytes (left panels) and then the OT-II TCR+ (Va2+Vb5+) population (right panels). (B) Summa- rized results of the percentage of CD4SP-Va2+Vb5+ thymocytes of FF-Ctr (d), FF-Ctr-mOVA (n)or F-cKO-mOVA (N) grafted thymi. (C) Image of grafted thymus under OT-II kidney capsule 2 wk postsurgery. A Student t test was used to determine statistical signifi- cance between groups. All data are expressed as means 6 SEM. Data are pooled from three independent

by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. experiments (each symbol represents one animal). http://classic.jimmunol.org Downloaded from (Fig. 5E). Additionally, there were no measureable differences unclear. As shown in Fig. 5A–C, thymic involution increased the in the functional marker GITR (glucocorticoid-induced TNF frequency of thymic nTregs, but this result did not appear to carry receptor family-related gene) (12, 51) between F-cKO and FF- over into the periphery. We did not observe any peripheral accu- Ctr Tregs (Supplemental Fig. 2). These data suggest that the mulation, including splenic Tregs (Figs. 6A, 6B) and nonlym- progressive loss of Foxn1 and subsequent thymic involution phoid (lung, liver, salivary gland) Tregs, in the F-cKO mice does not impair the suppressive capacity of Tregs. (Supplemental Fig. 3). To better answer whether thymic involution imparts any intrinsic The peripheral environment rather than the involuted thymus changes onto nTregs that could lead to their peripheral accumu- determines the age-related accumulation of peripheral Tregs lation, we performed an adoptive transplantation by infusing a pool Natural aging induces the accumulation of Tregs (the frequency is of WT naturally aged (.18 mo) T cells into young RAG2/2 mice, significantly increased in aged mice and humans) (46), which has which lack T and B cells. Two months after transfer, we found that been attributed to decreased levels of the proapoptotic gene Bcl2 the age-associated accumulation (frequency and relative ratio) of family member Bim (52–54); however, whether thymic involution aged donor Tregs was decreased, matching the levels of the young influences age-related accumulation of peripheral Tregs remains donor (6 wk) WT control (Fig. 6C, 6D). Furthermore, the di- 8 INFLAMMAGING RELATED TO THYMIC INVOLUTION

FIGURE 5. Thymic involution does not im- pair the production or function of nTregs. Two to 3 wk after inducing Foxn1fx/fx deletion with TM in F-cKO or FF-Ctr mice (3–4 mo old) thymoyctes were freshly isolated for cell sur- face staining of CD4, CD8, CD25, and intra- cellular staining of Foxp3 (A–C), or freshly isolated erythrocyte-depleted spleen cells were sorted for Tregs (CD4+CD25+) and Teffs (CD4+ CD252) and then cocultured to determine the suppressive function of Tregs on Teffs (D and E). (A) Representative dot plots show nTregs (CD4+CD25+Foxp3+) in the thymi of F-cKO and FF-Ctr mice. Summarized results show the percentage (B) and absolute number (C)of thymic nTregs. (D) Representative dot plots before and after sorting of Tregs and Teffs from the spleen of F-cKO and FF-Ctr mice. (E) Summarized results show suppressive function of Tregs from F-cKO (dark bar) and FF-Ctr mice (light bar), and proliferation of Teffs from F-cKO (dark striped bar) and FF-Ctr mice (light striped bar). A Student t test was used to de- termine statistical significance between groups. All data are expressed as means 6 SEM. Data are pooled from three independent experiments with six animals/group (A–C) and three ani- mals/group (D and E). by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. http://classic.jimmunol.org

minished expression of Bim observed in the aged Treg population pendent on age-related changes to the peripheral environment is was restored and equivalent to young mice (Fig. 6E). These results unclear. To test whether the young microenvironment can protect suggest that the peripheral microenvironment, rather than the against age-induced autoreactive T cells, we adoptively trans- 2/2

Downloaded from thymus (RAG mice only have a rudimentary thymus), is re- ferred either WT aged (.18 mo) or WT young (,6 wk) spleno- sponsible for the accumulation of Tregs with age. Moreover, the cytes into young RAG2/2 hosts. Two months after the infusion, young peripheral microenvironment is capable of reducing a pre- we observed that young hosts infused with young splenocytes viously accumulated Treg pool. generally appeared healthy, but young hosts infused with aged splenocytes showed inflammatory infiltration around salivary Young microenvironment cannot reverse age-induced vessels with severity that progressively worsened as the age inflammatory infiltration of the donor increased. The 18 mo donor was more likely to Our previous publications have shown that thymic involution leads present with a single inflammatory foci, and hosts given .22 mo to inflammatory infiltration in the lung, pancreas, liver, and most donor cells were likely to display multifoci infiltration (Fig. 7A). frequently in the salivary gland (42). However, we determined that To eliminate the effect of peripheral age-related factors (thymus the increased frequency of thymic nTregs does not persist into the independent) on inflammatory infiltration, we infused a pool of periphery of Foxn1-cKO mice, and the age-related accumulation T cells from young F-cKO or FF-Ctr splenocytes into young of peripheral Tregs can be reversed by the young microenviron- RAG2/2 mice. Although Foxn1-cKO mice lack any peripheral ment (Fig. 6). Whether inflammatory infiltration is an intrinsic age-related modifications not directly resulting from thymic defect among T cells derived from an involuted thymus or de- involution, we found inflammatory cell infiltration in the salivary The Journal of Immunology 9

FIGURE 6. The peripheral environ- ment rather than the atrophied thymus determines the age-related accumulation of peripheral Tregs. (A) Representative dot plots show the percentage of periph- eral Tregs (CD4+CD25+Foxp3+) from the spleens of FF-Ctr and F-cKO mice. (B) Summarized results of percentage peripheral Tregs in FF-Ctr and F-cKO mice. (B–F) Young or aged erythrocyte- depleted spleen cells from WT mice were adoptively transferred into young RAG2/2 mice. The peripheral Tregs were analyzed before and 8 wk after the trans- plantation via flow cytometry. (C) Representative dot plots show Treg gates before (top panels)andafter(bottom panels) the transfer. (D) Top panel shows percentage Foxp3+ cells in CD4+ spleno- cytes; bottom panel shows relative ratio of Foxp3+ cells in CD4+ splenocytes. (E) Representative histogram of Bim+ Tregs in young and aged mice before and after the transfer. (F) Relative mean fluo- rescence intensity (MFI) of Bim within Tregs before and after the transfer. A Student t test was used to determine statis- tical significance between groups. All data by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. are expressed as means 6 SEM (n = 5–11/ group). Data are pooled from at least three independent experiments. http://classic.jimmunol.org

glands of the young RAG2/2 host mice infused with F-cKO (atrophied mation in the absence of overt/acute infection with advanced age,

Downloaded from thymus) splenocytes, but infiltration was not observed in hosts infused have been defined (7, 9, 55–57), the precise etiology and mecha- with FF-Ctr (normal thymus) splenocytes (Fig. 7B). nisms responsible for inflammaging are largely unknown (1). Cur- Taken together, these results indicate that unlike the age-related rent knowledge of the etiology of inflammaging pinpoints latent accumulation of Tregs, inflammatory infiltration from the naturally viral infections giving rise to the persistent activation of immune aged or Foxn1-cKO atrophied thymus cannot be reversed by the cells (1), and SASP expressing senescent tissue cells (58), as young peripheral microenvironment. This indicates that inflam- sources of a chronic inflammation. However, the predisposition matory infiltration is an intrinsic defect in T cells imprinted during toward autoimmunity in the elderly and the contribution of auto- development in the involuted thymus. reactive T cells in generating a systemic inflammatory environ- ment are widely overlooked in the prevention and treatment of Discussion inflammaging. In this study, we identified thymic involution re- Virtually every chronic age-related disease emerges under con- sulting from the loss of Foxn1 as an additional source of activated ditions of chronic inflammation, that is, inflammaging, which immune cells (autoreactive T cells) that lead to a state of per- provides a highly significant risk factor for both morbidity and sistent and low-grade immunopathology with elevated proin- mortality in the elderly. Although the characteristics of inflam- flammatory cytokines. Newly generated T cells that have just maging, which describes a chronicsystemiclow-gradeinflam- exited the atrophied thymus of Foxn1-cKO mice adopt an acti- 10 INFLAMMAGING RELATED TO THYMIC INVOLUTION

FIGURE 7. Young microenviron- ment does not reverse age-induced inflammatory infiltration. (A) Repre- sentative images of H&E-stained salivary tissue slides from young RAG2/2 mice, which were trans- planted with splenocytes from donor young (6-wk-old) and aged (18- or 20-mo-old) C57BL/6 WT mice for 8 wk. Healthy refers to the absence of inflammatory infiltration; single fo- cus refers to one inflammatory-infil- trating cluster being found in one tissue slide; multi foci indicates that there were more than one inflam- matory-infiltrating cluster found in one tissue slide. Yellow arrows point out infiltrating clusters; n = animal numbers at the indicated age. (B) Representative images of H&E- stained salivary tissue slides from young RAG2/2 mice, which were transplanted with splenocytes from F-cKO (middle and right panels) and FF-Ctr (left panel) mice for 8 wk. Yellow arrows point out inflamma- tory infiltrating clusters. There were at least five animals in each group producing consistent results, and data are representative of at least three independent experiments.

vated and proliferative phenotype shortly after contacting the impairment associated with thymic atrophy may result from the periphery, despite never encountering foreign Ag. Additionally, diminished expression of Aire in mTECs, which supports our

by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. we were able to detect autoreactive T cells that specifically re- previous finding of decreased tissue-specific Ag expression in the spond to the peripheral self-antigen IRBP in the periphery of Foxn1-cKO thymus (42). Although there is evidence to support Foxn1-cKO mice, which suggests that T cell activation and elevated both Aire-dependent (21, 44) and Aire-independent (60) nTreg proinflammatory cytokines are the result of self-antigen stimulation. development, we did not observe any defects in the ability of the Interestingly, Foxn1-cKO mice do not develop overt autoimmune atrophied thymus to generate nTregs, despite the diminished ex- disease that would account for the elevated inflammatory milieu, pression of Aire in Foxn1-cKO thymi. It is possible that the low but rather present with general and relatively mild infiltration in level of Aire present in the atrophied thymus is sufficient to multiple non–lymphoid organs (42). Furthermore, it is unlikely that support nTreg development but is not sufficient to support the extrinsic aging factors cause low-level T cell activation, as these strong avidity necessary for the clonal deletion of autoreactive http://classic.jimmunol.org inflammatory T cells cannot be rejuvenated by the young micro- thymocytes. Additionally, it is possible that nTreg generation is environment. Because these inflammatory T cells do not encounter maintained in the Foxn1-cKO thymus by Aire-independent thymic foreign Ag, they likely result from defects in the thymic-driven dendritic cells (22). We previously published that thymic dendritic generation and maintenance of immune tolerance. cell accumulation shifts from mTEC to cTEC regions in the Identification of activated autoreactive T cells in the periphery atrophied thymus (42). However, further work needs to be done to of Foxn1-cKO mice is evidence of defects in the maintenance of determine whether the loss of Foxn1 and thymic atrophy influence

Downloaded from central immune tolerance. In the present study, we further iden- the functional ability of thymic dendritic cells to facilitate nega- tified impairment in the ability of the Foxn1-cKO involuted thy- tive selection. mus to clonally delete OVATCR Tg thymocytes that recognize the It is unclear whether the loss of Foxn1 affects TCR avidity for endogenous RIP-mOVA Ag. mOVA expression in mTECs is under MHC–self-peptide. The decreased negative selection with en- the control of the RIP and dependent on Aire (19, 45). Complete hanced (at least unimpaired) generation of Tregs in the accelerated knockout of the Aire gene (Aire2/2) disrupts negative selection thymic involution model is probably better explained by an and causes systemic autoimmune disease. Notably, even the par- “avidity window” between negative selection (strong TCR signal tial loss of the Aire gene (heterozygous Aire+/2) leads to a drastic strength) and deviation into the Treg lineage (moderate TCR increase in islet-reactive T cells and progression toward diabetes signal strength) (61); that is, diminishing the avidity of TCR for in an insulin TCR Tg mouse model (59). We previously showed MHC–self-peptide should reduce the clonal deletion of negative mTEC structure disruption and an overall reduction in thymic Aire selection and favor Treg induction in a specific cohort of MHC class expression in the Foxn1-cKO mice (42). We report in the present II–restricted thymocytes. study that the percentage of Aire+ mTECs is diminished in Foxn1- In addition to negative selection, Tregs play a critical role in cKO mice, and the expression of Aire in mTECs is decreased on maintaining immune tolerance by inhibiting the activation of au- a per cell basis. This implies that our observed negative selection toreactive T cells in the periphery. It has been reported that pre- The Journal of Immunology 11

mature thymic aging, induced by a hypomorphic mutation in the impossible, in an 18 mo WT mouse. The strength of our Foxn1- Foxn1 gene (Foxn1D/D mice with a germline mutation), weakens cKO model lies in the ability to induce the involution of a mature the suppressive function of peripheral Tregs (50). However, this is thymus while avoiding the effects of systemic aging. In fact, our contrary to our observation, which shows that Tregs derived model (both TM-induced and slow leakage of uCreERT) under- from the Foxn1-cKO thymus can suppress the proliferation of goes thymic involution, including a loss of Foxn1+ TECs (24, FF-Ctr Teffs just as efficiently as do Tregs derived from the 30), a decline in mature mTEC (24, 30, 42), thymic normal FF-Ctr thymus. Although CD4+CD25+ may not define distribution (42), increased senescent clusters, and increased all Tregs, our results are in agreement with others that show TAp63+ TECs (33) similar to 18 mo C57BL/6 mice. Further- aged Foxp3-GFP Tregs maintain suppressive capabilities (47). more, naturally aged C57BL/6 mice and F-cKO splenocytes that Additionally, recent evidence suggests that there is an age- were transferred into a young environment induced similar in- related accumulation of peripheral Tregs with increased sup- flammatory infiltrates in the salivary gland (Fig. 7). Additionally, pressive function (46). Age-related Treg peripheral accumulation is unlike the prenatal Foxn1 D/D mutation that blocks TEC matura- suggested to result from decreased expression of the proapoptotic tion and reduces Treg function (50), our inducible Foxn1-cKO gene Bcl2 family member Bim (52–54). In the present study, we model of mature thymic atrophy maintains Treg suppressive have shown peripheral Treg accumulation and diminished Bim ex- capacity consistent with naturally aged Tregs (46). However, our pression in naturally aged WT mice can be normalized by the young Foxn1-cKO model undergoes an extremely accelerated thymic peripheral microenvironment and are therefore not due to an intrinsic involution that is dissimilar to the 1–3% shrinkage per year thymic atrophy-related defect, but potentially due to an extrinsic observed in the naturally involuting thymus (68). Furthermore, abnormality in the aged peripheral microenvironment. Although similar to natural age-related thymic involution, the Foxn1-cKO there are many age-related alterations to the peripheral environment, model does not exhibit thymic regrowth that is seen in acute including changes to circulating soluble factors, the stromal com- thymic involution following infection and pregnancy (67). partment, the hematopoietic compartment, and microbiota, the exact However, the Foxn1-cKO model still does not fully recapitulate mechanisms signaling an age-related decrease in Bim and subse- the physiology of the chronic and extremely gradual nature of quent accumulation of Tregs remain unclear. Based on these findings, natural age-related thymic involution. Therefore, the effects of Treg accumulation and function do not appear to significantly con- rapid, as opposed to gradual, involution on defective negative tribute to thymic atrophy-driven inflammaging. selection and the emergence of chronic inflammation cannot be Although direct anti-inflammatory interventions, such as the use eliminated. Although the induced conditional knockout of Foxn1 of low-dose aspirin or statins, to suppress, prevent, and alter the and subsequent accelerated thymic involution in the fully mature state of chronic inflammation hold great promise for treating thymus may not exactly mimic the naturally aged thymus, there are multiple age-related diseases (1), targeting the sources of chronic many molecular, morphological, and functional characteristics inflammation may be the key to enhancing the prognosis of shared between the two. Therefore, the Foxn1-cKO model is useful chronic age-related disease. In this study, we have shown that in to study thymic involution and is applicable for age-related thymic addition to developing a strategy for the elimination of senescent involution with careful consideration for the limitations of the cells to suppress SASP (62), reduction or elimination of autore- animal model.

by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. active T cells that emigrated from the atrophied thymus is par- In summary, we have demonstrated that the steady release of ticularly promising to dampen age-related inflammation. We think autoreactive T cells resulting in tissue-specific inflammation fol- there are two strategies to do so. One is to rejuvenate the atrophied lowing thymic involution contributes to low-grade chronic in- thymus to restore its function, including functional negative se- flammation, known as inflammaging. The observed inflammaging lection and functional generation of naive T cells for TCR di- is the consequence of a T cell predisposition toward autoimmunity versity. This rejuvenation of thymic involution will also improve that arises from a decline in Foxn1 expression leading to the , which is a major source of inflammaging (1). disruption of the steady-state thymic medullary compartment (24, Removal of the atrophied thymus is another strategy that can be 42, 69) and subsequent thymic involution. Thymic involution used to eliminate emigration of the harmful autoreactive T cells. results in the release of autoreactive T cells that become activated http://classic.jimmunol.org This would likely require particular clinical criteria for the surgery shortly after reaching the periphery and produce low levels of in the elderly, such as ease of thymectomy (i.e., the patient is inflammatory cytokines without causing overt autoimmune dis- already having open chest surgery) or if the persistent chronic ease. The autoreactive T cells result from intrinsic defects in inflammation would significantly increase the morbidity of other negative selection, rather than changes to the extrinsically main- diseases. Although vaccination against persistent infections is tained Treg cell pool. Our studies shed new light on the importance another possible strategy to manage the persistent activation of of targeting thymic involution in addition to improving the

Downloaded from immune cells, it does not address autoreactive immune cell peripheral immune microenvironment as a potential treatment for activation. eliminating inflammaging and ultimately reducing morbidity and A caveat to this study is the frequent use of our Foxn1-cKO mortality in chronic age-related disease. mouse model of accelerated thymic involution in place of a true natural age-related atrophied thymus from .18 mo WT mice. Progressive thymic involution is an age-related alteration and its Acknowledgments causes are almost certainly not due to only the loss of one gene. We thank Dr. Mark Anderson (University of California, San Francisco) for This complex and multifaceted nature of thymic involution is providing the IRBP peptide and tetramer, Dr. Rance Berg (University of North Texas Health Science Center) for critical reading of the manuscript, evidenced by the various processes implicated in age-related Dr. Xiangle Sun (University of North Texas Health Science Center) for flow thymic atrophy, including the involvement of other genes such cytometry technical support, and the National Institutes of Health Tetramer as ink4a (63), hormones (64), and adipocyte expansion (65–67). Core Facility for providing the tetramer reagent. However, because thymic involution occurs alongside total body aging, separating the effects of thymic age from systemic aging (which includes the circulation of soluble factors, T cells, and Disclosures dendritic cells back into the thymus) is extremely difficult, if not The authors have no financial conflicts of interest. 12 INFLAMMAGING RELATED TO THYMIC INVOLUTION

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