Methods and compositions for treating thymic atrophy

Abstract

Methods and compositions are directed to a solving the problem of thymus involution / atrophy by stimulating the maintenance or regeneration of the thymus and / or ameliorating atrophy of the organ. Therapeutic composition(s) containing one or more of an autophagy activating agent, a TORC1 activator or a FGFRlc (Fibroblast Growth Factor Receptor 1c) agonist, and / or an antioxidant can be administered to a subject for thymic regeneration or amelioration of thymic involution.

Description

METHODS AND COMPOSITIONS FOR TREATING THYMIC ATROPHYRELATED APPLICATION

[0001] This Application is an International Application claiming priority to U.S. Provisional Patent Application No. 63 / 691,233 filed September 5, 2024 and 63 / 699,120 filed 9 / 25 / 2024 which are incorporated by reference in their entirety.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under grant Nos. AI121367, AG081709, and AI154109 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] A sequence listing is being submitted electronically with this application. The sequence listing is incorporated herein by reference. The sequence listing that is contained in the file named "UTSKP0575" which is 3 kilobytes (as measured in Microsoft Windows®) and was created on 9 / 4 / 2025.FIELD

[0004] Embodiments are generally related to the field of medicine and immunology, in particular thymus function.BACKGROUND

[0005] T lymphocytes are critical mediators of the adaptive immune response; however, they are continuously lost and therefore must be replaced throughout the lifespan. New T cells are generated in the thymus by periodic recruitment of thymus seeding progenitors (Goldschneider et al., The Journal of experimental medicine 163, 1-17, 1986; Foss et al., The Journal of experimental medicine 193, 365-74, 2001). Mutually inductive signaling, where thymic stromal cells and progenitors interact to drive differentiation, occurs in the unique stromal microenvironment of the thymus directs these multi-potent progenitors along a well-characterized program of differentiation, proliferation, and selection to generate a self-tolerant, self-restricted T cellpopulation (Petrie and Zuniga-Pflucker, Annual review of immunology 25, 649-79, 2007; Klug et al., PNAS USA 95, 11822-27, 1998; Klein et al., Nat Rev Immunol 14, 377-91, 2014). The stromal microenvironment is comprised of various types of thymic epithelial cells (TECs) as well as mesenchymal, neural, vascular, and non-lymphoid hematopoietic cells (e.g., dendritic cells and macrophages). TECs generate most of the known inductive signals required for T cell differentiation in the thymus, as well as a variety of chemokines and other cytokines required to direct the critical spatial migration of progenitors throughout the various anatomical regions of the thymus (Petrie and Zuniga-Pflucker, Annual review of immunology 25, 649-79, 2007).

[0006] Rearrangement of T cell receptor (TCR) gene segments during T cell development allows the generation of a broad spectrum of TCR specificities, estimated to consist of over 108unique TCRs in humans (Qi et al. PNAS USA 111, 13139-44, 2014). This maximizes the diversity of antigens that T cells can recognize, including the potential to respond to emerging pathogens to which our species has never been exposed. Since each T cell generally has one TCR specificity, this diversity depends on the number of T cells generated in the thymus. However, careful analysis of histological data from human thymus (Steinmann et al., Scand J Immunol 22, 563-75, 1985), estimates of thymic output in humans based on T cell receptor excision circle (TREC) analysis (Douek et al.. Nature 396, 690-95, 1998; Jamieson et al., Immunity 10, 569-75, 1999; Thome et al., Sci Immunol 1, 2016), and mathematical modeling (Bains et al., J Immunol 183, 4329-36, 2009), indicate that peak thymus output occurs in the first few years of life, though thymic output continues through the 9th decade of life (Mitchell et al., Clinical and experimental immunology 161, 497-503, 2010). This pronounced age-related atrophy results in a reduction in production of new, naive T cells (Hartwig and Steinmann, Meeh ageing and develop 75, 151-56, 1994; Aspinall et al., Journal of compar pathology 142 Suppl 1, SI 11-15, 2010; Cepeda and Griffith, Exper gerontology 105, 113-17, 2018), which in turn diminishes TCR diversity with age in mice and humans, as decreased thymic generation of new T cells is compensated by homeostatic proliferation of memory T cells (Haynes et al., Immunologic research 22, 253-61, 2000; Hale et al., PNAS USA 103, 8447-52, 2006; Ernst et al. J Immunol 145, 1295-1302, 1990; Utsuyama et al., Meeh of ageing and develop 63, 57-68, 1992). Resulting immunodeficiencies include decreased vaccine responsiveness and tumor surveillance (Palmer et al., PNAS USA 115, 1883-88, 2018), as well as diminished response to infection, especially new viral infections, including SARS-CoV-2 (Nikolich-Zugich et al., Geroscience 42, 505-14, 2020; Xiao et al., Nat Aging 3,418-35, 2023; Nikolich-Zugich and Rudd, Current opinion in immunology 22, 535-40, 2010). The narrowing of the naive T cell repertoire that results from thymic atrophy restricts the magnitude of the T cell response to new infections by reducing the number of naive T cells capable of mounting a robust response against a given antigen (Nikolich-Zugich and Rudd, Current opinion in immunology 22, 535-40, 2010).

[0007] Paradoxically, aging is also associated with increases in circulating autoantibodies, as well as increased susceptibility to some autoimmune disorders (Cooper and Stroehla, Autoimmun Rev 2, 119-25, 2003; Goronzy and Weyand, Cellular and molecular life sciences: CMLS 69, 1615- 23, 2012). However, the underlying mechanisms regulating these increases are not fully elucidated. Age-associated thymic atrophy has been considered a likely contributor to declines in central T cell tolerance induction (Muller and Pawelec, Ageing research reviews 23, 116-23, 2015), and several studies have demonstrated age-associated decreases in thymic function in addition to loss of mass (Ki et al., Cell Rep 9, 402-15, 2014; Baran-Gale et al., Elife 9, 2020). Diminished expression of tissue-restricted self-antigen (TRA) genes in thymic antigen-presenting cells, critical for tolerizing developing T cells, has been reported in mice and humans. This reduction contributes to declines in central T cell tolerance induction. (Klein et al., Nat Rev Immunol 14, 377-91, 2014; Griffith et al., Aging cell 11, 169-77, 2012; Baran-Gale et al., Elife 9, 2020; Bredenkamp et al., Development 141, 1627-37, 2014; Cepeda et al., Cell Reports 22, 1276- 87, 2018; Gies et al., Journal of allergy and clinical immunology 139, 1049-52 el012, 2017; Derbinski et al., Journal of exper medicine 202, 33-45, 2005).

[0008] Due to the broad sequelae of thymic atrophy, many studies have focused on regeneration of the thymus, which can be induced experimentally in a number of ways, most commonly by surgical or chemical androgen ablation (Sutherland et al., J Immunol 175, 2741-53, 2005; Min et al., Clinical immunology 118, 117-23, 2006; Duah et al., Frontiers in immunology 12, 706244, 2021). The thymus retains a remarkable capacity to regenerate after removal of a negative stimulus (Sutherland et al., J Immunol 175, 2741-53, 2005), or provision of a positive stimulus (Dudakov et al., Science 336, 91-95, 2012; Chaudhry et al., Immunological reviews 271, 56-71, 2016; Majumdar and Nandi, Scand J Immunol, 2017; Rode and Boehm, PNAS USA 109, 3463-68, 2012; Chu etal., Blood 112, 2836-46, 2008; Montecino-Rodriguez et al., Endocrinology 139, 4120-26, 1998; Rossi et al., Blood 109, 3803-11, 2007), but regeneration achieved by approaches published to date are transient, and age progressively limits the size reached at returnto steady state (Cepeda and Griffith, Experimental gerontology 105, 113-17, 2018). Nonetheless, even transient regeneration has positive impacts on immunity to microbial infections and tumor surveillance (Heng etal., PLoS One 7, e42677, 2012), and exploring novel approaches to increase thymic activity represents an active area of research (Dudakov et al., Science 336, 91-95, 2012; Hun et al. Biomaterials 118, 1-15, 2017; Parent et al., Cell Stem Cell 13, 219-29, 2013; Shukla et al., Nat Methods 14, 531-38, 2017; Sun et al., Cell Stem Cell 13, 230-36, 2013; Bredenkamp et al., Regen Med 10, 317-29, 2015; Garfin et al., Journal of experimental medicine 210, 1087-97, 2013; Shah et al., Nat Biotechnol 37, 293-302, 2019; Kim et al., J Immunol 194, 4784-95, 2015; Fahy et al., Aging cell 18, el3028, 2019). Recent and on-going clinical trials have demonstrated transient reversal of aging indicators in T cells using growth hormone (GH) in aged men (Fahy et al., Aging cell 18, el3028, 2019). However, as in animal models of regeneration, the impacts on T cell immunity are transient and decline within one year (Fahy et al., Aging cell 18, el3028, 2019). Thus, safe and durable approaches for improving thymus function with age hold strong potential for extending the healthspan (Kinsella and Dudakov, Frontiers in immunology 11, 1745, 2020; Alawam et al., Frontiers in immunology 11, 858, 2020), but have not yet been established. The proposed studies explore novel approaches to address the significant public health concern represented by waning T cell immunity in the elderly.

[0009] There remains a need for additional compositions and methods for ameliorating thymus involution / atrophy .SUMMARY

[0010] Methods and compositions described herein provide a solution to the problem of thymus involution / atrophy by stimulating the maintenance or regeneration of the thymus and / or ameliorating atrophy of the organ. Therapeutic composition(s) containing one or more of an autophagy activating agent, a FGFRlc (Fibroblast Growth Factor Receptor 1c) agonist, and / or an antioxidant can be administered to a subject for thymic regeneration or amelioration of thymic involution.

[0011] Certain embodiments are directed to methods for thymus regeneration or amelioration of thymic atrophy in a subject, comprising administering to the subject a therapeutically effective amount of one or more therapeutic agent selected from (i) an autophagy activating agent, (ii) a TORC1 activator or a mT0RC2 activator or a FGFRlc (Fibroblast Growth Factor Receptor 1c)agonist, and / or (iii) an antioxidant. In certain aspects the autophagy activating agent is rilmenidine or an analog thereof. In other aspects the T0RC1 activator or a mT0RC2 activator or FGFRlc (Fibroblast Growth Factor Receptor 1 c) agonist agent is FGF21 or an analog thereof. In certain aspects the antioxidant agent is Vitamin C (Ascorbic Acid), Vitamin E (Tocopherols and Tocotrienols), Beta-Carotene, Selenium, Glutathione, Coenzyme Q10 (CoQlO), Alpha-Lipoic Acid, Resveratrol, Curcumin, Polyphenols, Flavonoids (e.g., quercetin, catechins), Anthocyanins (found in berries), and / or N-Acetylcysteine (NAC). The agents can be administered in a sequence or simultaneously, independently or in a co-formulation. In certain aspects the composition(s) is administered via intravenous injection or intrathymic administration. In certain aspects one or more composition is a sustained release composition. The dosage regimen for each agent independently can be from 5 to 200 mg daily, weekly or monthly. The subject can be a mammal (e.g., a human, a companion animal, or livestock) having or at risk of having thymic atrophy; or an aging mammal; or a mammal at risk of exposure to or in an environment known to harbor a pathogen, e.g., microbial, fungal, parasite, etc.; or a subject at risk or diagnosed with cancer. Thymus regeneration can be measured by an increase in T-cell receptor excision circles (TRECs) or thymic epithelial space.

[0012] Other embodiments are directed to a pharmaceutical composition(s) for thymus regeneration, comprising independently or co-formulated (i) an autophagy activating agent, (ii) a TORC1 activator or a FGFRlc (Fibroblast Growth Factor Receptor 1c) agonist, and / or (iii) an antioxidant and a pharmaceutically acceptable carrier. The (i) an autophagy activating agent, (ii) a TORC1 activator or a FGFRlc (Fibroblast Growth Factor Receptor 1c) agonist, and / or (iii) an antioxidant are present in a ratio optimized for synergistic thymus regeneration effects, such as a 1 / 1 / 1, 1 / 1 / 2, 1 / 1 / 3, 1 / 1 / 4, 1 / 1 / 5, 1 / 2 / 1 / , 1 / 2 / 2, 1 / 2 / 3, 1 / 2 / 4, 1 / 2 / 5, 1 / 3 / 1, 1 / 3 / 2, 1 / 3 / 3, 1 / 3 / 4, 1 / 3 / 5,1 / 4 / 1, 1 / 4 / 2, 1 / 4 / 3, 1 / 4 / 4, 1 / 4 / 5, 1 / 5 / 1, 1 / 5 / 2, 1 / 5 / 3, 1 / 5 / 4, 1 / 5 / 5, 2 / 1 / 1, 2 / 1 / 2, 2 / 1 / 3, 2 / 1 / 4, 2 / 1 / 5,2 / 2 / 1, 2 / 2 / 3, 2 / 2 / 4, 2 / 2 / 5, 2 / 3 / 1, 2 / 3 / 2, 2 / 3 / 3, 2 / 3 / 4, 2 / 3 / 5, 2 / 4 / 1, 2 / 4 / 2, 2 / 4 / 3, 2 / 4 / 4, 2 / 4 / 5, 2 / 5 / 1,2 / 5 / 2, 2 / 5 / 3, 2 / 5 / 4, 2 / 5 / 5, 3 / 1 / 1, 3 / 1 / 2, 3 / 1 / 3, 3 / 1 / 4, 3 / 1 / 5, 3 / 2 / 1, 3 / 2 / 2, 3 / 2 / 3, 3 / 2 / 4, 3 / 2 / 5, 3 / 3 / 1,3 / 3 / 2, 3 / 3 / 4, 3 / 3 / 5, 3 / 4 / 1, 3 / 4 / 2, 3 / 4 / 3, 3 / 4 / 4, 3 / 4 / 5, 3 / 5 / 1, 3 / 5 / 2, 3 / 5 / 3, 3 / 5 / 4, 3 / 5 / 5, 4 / 1 / 1, 4 / 1 / 2,4 / 1 / 3, 4 / 1 / 4, 4 / 1 / 5, 4 / 2 / 1, 4 / 2 / 2, 4 / 2 / 3, 4 / 2 / 4, 4 / 2 / 5, 4 / 3 / 1, 4 / 3 / 2, 4 / 3 / 3, 4 / 3 / 4, 4 / 3 / 5, 4 / 4 / 1, 4 / 4 / 2,4 / 4 / 3, 4 / 4 / 5, 4 / 5 / 1, 4 / 5 / 2, 4 / 5 / 3, 4 / 5 / 4, 4 / 5 / 5, 5 / 1 / 1, 5 / 1 / 2, 5 / 1 / 3, 5 / 1 / 4, 5 / 1 / 5, 5 / 2 / 1, 5 / 2 / 2, 5 / 2 / 3,5 / 2 / 4, 5 / 2 / 5, 5 / 3 / 1, 5 / 3 / 2, 5 / 3 / 3, 5 / 3 / 4, 5 / 3 / 5, 5 / 4 / 1, 5 / 4 / 2, 5 / 4 / 3, 5 / 4 / 4, 5 / 4 / 5, 5 / 5 / 1, 5 / 5 / 2, 5 / 5 / 3,5 / 5 / 4, or any values or ranges there between.

[0013] Other embodiments are directed to use of a combination of two or more of (i) an autophagy activating agent, (ii) a TORC1 activator or a mT0RC2 activator or a FGFRlc (Fibroblast Growth Factor Receptor 1c) agonist, and / or (iii) an antioxidant in the manufacture of a medicament for thymus regeneration in a subject. In particular for treatment of infections or cancer.

[0014] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

[0015] The term “thymus” as used herein refers to a gland located in the upper chest, just behind the sternum and in front of the heart. The thymus plays a vital role in the development of the immune system. Its primary function is to produce T-lymphocytes or T cells, which are critical components of the adaptive immune system. The thymus is divided into two lobes, each surrounded by a capsule. Inside, it has a cortex (outer layer) and a medulla (inner layer). This structure supports the maturation process of T cells.

[0016] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0017] Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0018] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.”

[0019] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps.

[0020] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a chemical composition and / or method that “comprises” a list of elements (e g., components or features or steps) is not necessarily limited to only those elements (or components or features or steps), but may include other elements (or components or features or steps) not expressly listed or inherent to the chemical composition and / or method.

[0021] As used herein, the transitional phrases “consists of’ and “consisting of’ exclude any element, step, or component not specified. For example, “consists of’ or “consisting of’ used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of’ or “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0022] As used herein, the transitional phrases “consists essentially of’ and “consisting essentially of’ are used to define a chemical composition and / or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

[0023] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.DESCRIPTION OF THE DRAWINGS

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be betterunderstood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0025] FIG. 1A-1B. Thymic atrophy is responsive to redox state in a stromal dependent manner. (A) Drinking water containing the antioxidants N-acetylcysteine (NAC) or L-ascorbic acid (ASC) was given from weaning; thymus weight was measured at 10 weeks. Mice supplemented with antioxidant compounds exhibited significantly larger thymuses than WT controls (values indicate mean SD for five animals, except for young control thymus, which represents historical average. (B) Weight of kidney, liver, heart, spleen, body fat, and lean mass.

[0026] FIG. 2A-2B. Dietary and genetic antioxidant supplementation rescues CD8 T cell responsiveness to the influenza virus immunodominant epitope, NP366-374 at 6 months of age. (A) A representative gating strategy used to identify tetramer-positive cells from bronchoalveolar lavage (BAL). BAL cells were stained with APC-conjugated NP366-374 / Db tetramer, PE- conjugated PA224-233 / Db tetramer, anti-CD8 fluorescein isothiocyanate (FITC) and PE-Cy5- conjugated anti -CD 19 and anti-CD4 (“lineage cocktail” channel). (B) BAL was harvested 10 days after influenza virus infection (5 WK WT; n=9) or from naive individual young mice (5 WK NAIVE; n=6) and older mice given either control untreated water (6 MO WT; n=6) or NAC- supplemented water from weaning (6 MO NAC; n=6) and older mCat Tg mice (6 MO mCat Tg; n=5). Symbols represent individual mice. Horizontal bars indicate one-way ANOVA; p< 0.05.

[0027] FIG. 3A-3B. Increased basal autophagy increases thymus size and may rescue clonal deletion of self-specific T cells in 6-month-old mice. (A) total thymus cellularity in 6-month old WT (filled circles, n=5) and 6-month old BecnlKI / KI mice (open hexagons, n=4) and (B) frequencies of cleaved caspase-3+ cells undergoing clonal deletion (gated on lineage(-) signaled singlets, but without CCR7 staining) in young (5-week old) WT (open circles, n=9), young Becnl KI / KI mice (open hexagons, n=5), or 6-month old WT (striped circles, n=9), or 6-month old BECN1KI / KI mice (striped hexagons, n=2).

[0028] FIG. 4. Overexpression of FGF21 in LPOFGF21 KI mTECs results in increased thymus size and diminishes age-associated thymic atrophy. (A-B) Thymus weight / body weight ratios (somatic index) and total thymus cellularity were determined in LPOWT and LPOFGF21 KI female mice at 1 month, 3 months, and 12 months of age (n=5-9 mice per group). (C-D) Thymocyte CD4-CD8- DN, CD4+CD8+ DP, CD4+ SP, and CD8+ SP subset frequency (C) and total cell number (D), as well as the frequency and total number of cTEC (CD45.2-, EpCAM+,Ly51+) and mTEC (CD45.2-, EpCAM+, Ly51 -), were quantified in 1-month-old (n=5-10) (top) and 13-month-old (bottom) (n=5-6) LPOWT and LPOFGF21 KI female mice. For (A-D) data were analyzed via Student’s t-tests. ns= p>0.05, *= p<0.05, **= p<0.005, ***= p<0.001, ****= p<0.0001. Mean and SEM are indicated by horizontal lines. Each symbol represents an individual mouse. Data are representative of three or more experiments.

[0029] FIG. 5A-5J. FGF21 overexpression in mTECs mitigates waning naive T cell frequency and T cell responsiveness to influenza infection in older mice. (A-B) Representative gating strategy showing isolation of CD62L+, CD44hi naive T cells and CD62L+, CD44LO memory T cells from (A) CD3+, CD8+ and (B) CD3+, CD4+ splenocytes in 2-month-old and 12-month-old LPOWT and LPOFGF21 mice. (C-D) (C) CD8+ and (D) CD4+ naive:memory ratios were quantified in splenocytes of 2-month-old LPOWT (n=5 per group) and LPOFGF21 (n=5 per group) and 12-month-old LPOWT (n=6) and LPOFGF21 mice (n=6). Data were analyzed via Student's t-tests. ns= p>0.05, **= p.

[0030] FIG. 6A-6E. Persistent medullary TEC-driven overexpression of FGF21 protects against age-associated impairments in clonal deletion and development of peripheral autoimmunity. (A) Gating strategy used to detect clonal deletion among early (CCR7 ) and late (CCR7+) lineage- (CD19; CD25’, TCRyS', NK1. E) signaled (CD5+, TCRp+) T cells. (B-C) The frequency of cleaved caspase 3+cells was quantified among early and late signaled T cells in 2- month-old LPOWTand LPOFGF21 / FGF21homozygous mice (n=5 per group) and in 12-month-old LPOWTand LPOFGF21 / FGF21homozygous mice (n=5 per group). Data were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test. Horizontal lines indicate significance (p<0.0001). (D) The presence of antinuclear antibodies was assessed in serum from 12-month-old LPOWTand LPOFGF21mice (n=8 per group). Serum was incubated on HEp-2-coated slides, followed by immunostaining with anti-mouse IgG FITC. The number of animals that tested positive for ANAs are indicated in the IgG channel image. Images are at 20x magnification. Scale bar= 50 pm. (E) Liver, lung, and salivary tissue were harvested from 12-month-old LPOWTand LPQFGF2I (n=7 pergroup)anc] evaluated for the presence of lymphocytic infiltrates via H&E staining (indicated via arrows). The number of mice testing positive for infiltrates are indicated in each tissue. Scale bar= 50 pm (20x).

[0031] FIG. 7A-7E. Paracrine thymic FGF21 signaling increases cTEC size during aging. (A- D) Representative maximum intensity Z-Stack 3D projections taken from 60 pm optical stacks(20x magnification) from 1 -month-old and 6-month-old FoxN 1CreR26ContettlLPOWTand FoxN 1CreR26ConfetlLPOFGF21thymic sections (n=3-4 mice per group). Scale bar= 50 pm. (E) cTEC area (pm2) was quantified from Z-Stack projections from at least 3 biological replicates in each group from (A-D) via blinded analysis with Imaged software. Each symbol represents a single cTEC. Red horizontal lines represent mean ± SEM. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons. ****=p<0.0001.

[0032] FIG. 8. Combination treatment with NAC, Rilmenidine, and FGF21 overexpression delays thymus atrophy. Mice from 5 to 10 weeks of age were treated with Rilmenidine (i.p. injections), NAC (in drinking water), and FGF21 (genetic overexpression), showing greater delay in atrophy with all three agents.*

[0033] FIG. 9. Relative thymus size (left) and % Naive (CD44-CD62L+) in splenic CD8 T cells in 6-month-old WT mice after 4 weeks of treatment with Rilmenidine (0.072mg / ml) +NAC (15mg / ml) (n=3) or untreated drinking water (n=4). Symbols represent individual mice. Horizontal bars indicate Student's t-test p<0.05. Mean and SEM are indicated by horizontal bars in each group.

[0034] FIG. 10. NAC +Rilmeni dine does not impair clonal deletion. Clonal deletion was assessed in 6-month-old WT mice treated with NAC+Rilmenidine in drinking water for 4 weeks and in untreated controls, and no significant difference was found using Student's t-test.

[0035] FIG. 11. Improved tumor control and ICB efficacy when thymus atrophy is delayed in aged LPOFGF21mice. Young (1 month) or older (12 month) WT (black / blue) and older LPOFGF21KI mice were challenged with B16 melanoma cells on day 0, followed by anti-PDLl (solid symbols) or isotype control (open symbols) injections as indicated (top.) Tumor volume was measured during the challenge and ICB treatment and tumor weight was measured at the end of the experiment on day 14 (middle.) The frequencies of TCR0+ cells among viable singlets within the tumor (bottom left), PD-1 + CD8+ T cells among all T cells (bottom , middle), and CD101+ cells among all PD-1 + CD8+ T cells (bottom, right) on Day 14 are displayed.DESCRIPTION

[0036] The following discussion is directed to various embodiments of the invention. The term “invention” is not intended to refer to any particular embodiment or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure,including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be an example of that embodiment and not intended to imply that the scope of the disclosure, including the claims, is limited to that embodiment.

[0037] A significant barrier to identifying effective approaches for thymus regeneration is a lack of understanding of the mechanisms that regulate thymus atrophy and growth. Despite the critical roles played by thymic stromal cells (TSCs) in steady-state T cell differentiation, their biology is still relatively under-characterized. This is due, in part, to the fact that stromal cells are very rare, representing less than 1% of total thymic cellularity. Isolation of stromal cells requires extensive enzymatic treatments that have been shown to induce important changes in their biology. Indeed, even simple removal of stromal cells from their native three-dimensional architecture induces profound changes. Although these mechanisms are still unclear, it is known that the primary targets of both age-induced atrophy and experimental thymic regeneration are stromal, rather than lymphoid cells (Hakim et al., J Clin Invest 115, 930-39, 2005), particularly cortical stromal cells (Griffith et al., Aging cell 11, 169-77, 2012). Factors regulating the persistence of TSCs relatively obscure.

[0038] It was found that TSCs, especially those in the cortex, express conspicuously low levels of the hydrogen peroxide (H2C>2)-quenching enzyme catalase, acquire high levels of oxidative damage (including high levels of 8-hydroxy-2-deoxyguanosine), and have greater sensitivity to reactive oxygen species (ROS)-induced mitochondrial damage relative to lymphocytes from the same thymus. TSCs, particularly in the cortex, are continuously exposed to developing T cells that are undergoing high rates of cell division. As a result, stromal cells, unlike lymphoid cells, which quickly exit the cell cycle and emigrate, persist in a state of exposure to the cell-permeable products of metabolism and cell division, including H2O2, and may therefore experience particularly high ROS levels. A similar scenario has been demonstrated in the bone marrow (BM), where Cx43- dependent channels facilitate transfer of ROS from proliferating hematopoietic stem cells to adjacent BM stromal cells, a function critical for hematopoietic regeneration. The unusually highly oxidative environment present in the thymus suggests that there may be a physiological function for low catalase expression in TSCs.

[0039] Among ROS, H2O2 has a relatively long half-life and is cell permeable, making it an important secondary messenger in maintaining cellular homeostasis. Several studies have shownthat moderate levels of ROS play crucial physiological roles in many different biological processes, both at the transcriptional and translational levels, and can promote autophagy specifically in several ways. Upon nutrient starvation, H2O2 not only acts as an early inducer of autophagy but also as the main transducer of intracellular signals promoting autophagy. In the presence of H2O2, inactive AMP-activated protein kinase (AMPK) is converted to active AMPK, leading to the formation of the Unc-51-like autophagy-activating kinase 1 (ULK1) complex, which is required for autophagosome formation. H2O2 also initiates oxidation of autophagy-related (Atg) 4, which leads to the inactivation of its delipidating activity on LC3, further promoting the structural integrity of the mature autophagosome. EhChcan also initiate autophagy by altering the thiol redox state, by inhibiting phosphoinositide 3-kinases / protein kinase B / mechanistic target of rapamycin (PI3K / Akt / mT0R) signaling, and by inducing beclin-1 expression, which leads to the formation of a complex between beclin-1 and a class III PI3K, an important step for initiating autophagy.

[0040] Recent work has established that TSCs exhibit an unusually high level of basal autophagy in the steady state and that constitutive autophagy in TSCs is required for self-antigen presentation and central T cell tolerance induction. However, the mechanisms regulating constitutive autophagy in TSCs are not well understood. Together, these observations led to the hypothesis that the high levels of H2O2 in TSCs established by low catalase expression may represent the mechanism promoting the conspicuously high levels of basal autophagy required for self-antigen presentation and T cell tolerance induction.I. Thymus Stabilization and Regeneration

[0041] In certain aspects methods and / or compositions are described for the treatment of thymus involution / atrophy by stimulating the maintenance or regeneration of the thymus and / or ameliorating atrophy of the organ. Therapeutic compositions containing one or more (independently formulated or co-formulated or combinations thereof) of (i) an autophagy activating agent, a FGFRlc (Fibroblast Growth. Factor Receptor 1 c) / P-Klotho agonist, and / or (ii) an autophagy activating agent, and / or (iii) an antioxidant can be administered or co-administered to a subject for thymic regeneration or amelioration of thymic involution.A. Autophagy Activation

[0042] The unusually highly oxidative environment present in the thymus (Griffith et al., Cell reports 12, 1071-79, 2015; Liu et al., Nature 459, 387-92, 2009), together with apparent impairment of negative selection in even young mCat Tg mice suggested that there may be a physiological function for low catalase expression in thymic stromal cells (TSCs) related to promoting negative selection and tolerance in developing T cells. Among reactive oxygen species (ROS), H2O2has a relatively long half-life and is cell permeable, making it an important secondary messenger in maintaining cellular homeostasiss2. Several studies have shown that moderate levels of ROS play crucial physiological roles in many different biological processes, both at the transcriptional and translational levels (Holmstrom and Finkel, Nature reviews Molecular cell biology 15, 411-21, 2014; Turpaev, Biochemistry (Moscow) 67, 281-92, 2002), and can promote autophagy specifically in several ways. Upon nutrient starvation, H2O2 not only acts as an early inducer of autophagy, but also as the main transducer of intracellular signals promoting autophagy (Filomeni et al., Autophagy 6, 999-1005, 2010; Filomeni et al., Cell Death & Differentiation 22, 377-88, 2015; Scherz-Shouval et al., The EMBO journal 26, 1749-60, 2007; Scherz-Shouval and Elazar, Trends in cell biology 17, 422-27, 2007). TSCs exhibit an unusually high level of basal autophagy in the steady state (Mizushima et al., Mol Biol Cell 15, 1101-11, 2004; Nedjic et al., Nature 455, 396-400, 2008), and that constitutive autophagy in TSCs is required for self-antigen presentation and central T cell tolerance induction (Nedjic et al., Nature 455, 396-400, 2008; Aichinger et al, Journal of experimental medicine 210, 287-300, 2013). However, the mechanisms regulating high constitutive autophagy in TSCs are not understood. It is contemplated that the high levels of H2O2 in TSCs established by low catalase expression may represent the mechanism promoting the conspicuously high levels of basal autophagy required for self-antigen presentation and T cell tolerance induction.

[0043] Autophagy recycles organelles that have been damaged by oxidative damage, this treatment alone may diminish oxidative damage well enough alone to prevent atrophy / allow for regeneration without the negative side effects found with antioxidant treatment alone. In certain aspects an autophagy activating agent can be formulated in a composition and administered to a thymus. In certain aspects the autophagy activating agent is rilmenidine. Activation of autophagy helps to recycle organelles that have been damaged by oxidative damage, this treatment alone may diminish oxidative damage well enough alone to prevent atrophy / allow for regeneration.

[0044] Rilmenidine, chemically known as N-(dicyclopropylmethyl)-4,5-dihydro-l,3-oxazol- 2-amine, is marketed under various brand names including Albarel, Hyperium, Iterium, and Tenaxum. Rilmenidine is an antihypertensive drug that acts primarily as an II -imidazoline receptor agonist, which helps in reducing blood pressure. Substitutes or analogs for rilmenidine, can include moxonidine, an II -imidazoline receptor agonist used for hypertension; clonidine, an alpha-2 adrenergic receptors with some activity on imidazoline receptors as well.Rilmenidine

[0045] Substitute compounds for rilmenidine include, but are not limited to Rapamycin, PP242, Torin 1 , Metformin, BH3 mimetics (ABT-737), Xestospongin B, L-NAME, Clonidine, PI-103 hydrochloride, Lithium (L-690330), Carbamazepine, Resveratrol, Verapamil, EGFR antagonists (erlotinib hydrochloride). Sodium valproate, Spermidine, Vinblastine, Nocodazole, Bafilomycin .Al, Chloroquine, Hydroxychloroquine, and Spautin-1.B. FGFRlc (Fibroblast Growth Factor Receptor 1c) Agonist (TORC1 activator)

[0046] Many growth factors promote thymus growth (IGF1, GH, KGF, many of these in clinical trials), but what is special about FGF21 is that it is not a mitogen, but does apparently increase the size of individual thymic epithelial cells to protect thymus size. FGF21, or Fibroblast Growth Factor 21, is a protein that in humans is encoded by the FGF21 gene. FGF21 is involved in glucose and lipid metabolism, promoting insulin sensitivity, and affecting energy homeostasis. It acts as a stress hormone, particularly in response to fasting, starvation, or ketogenic diets, helping the body to adapt to these conditions by promoting the use of fat for energy. FGF21 acts through the FGFRlc (Fibroblast Growth Factor Receptor 1c) and the co-receptor -klotho, which is necessary for its metabolic activities. FGF21 activates TORC1, thus other TORO activators can be used as a substitute or analog of FGF21.

[0047] mTOR is a serine / threonine protein kinase belonging to the PI3K-related protein kinase (PIKK) family68, 69 and is the main catalytic subunit in two distinct complexes designated mTOR complex 1 (mTORCl) and mTOR complex 2 (mTORC2). These complexes integrateenvironmental cues and result in both distinct and common cellular outcomes, with significant crosstalk between mTORCl and mT0RC2 signaling pathways. mTORCl responds to inputs such as energy status, nutrients, growth factors, oxygen, and stress, and promotes biosynthetic pathways and cell growth. It also inhibits autophagy and other catabolic processes. mT0RC2 is thought to be activated primarily by growth factor signaling, and promotes anabolic metabolism, proliferation, and survival. Notably, mT0RC2 signaling also regulates cytoskeletal organization. mTOR signaling is a high priority area of interest in longevity research, and long-term systemic inhibition of mTOR activity by high dose rapamycin administration, which inhibits both mTORCl / 2 activity, has been shown to decrease thymus size.

[0048] In contrast, short-term treatment with selective mTORCl inhibitors has been shown to improve influenza vaccination responses and reduce infection after a rest period in clinical trials. The expression patterns of soluble ligands capable of activating the mTOR pathway were either absent or unchanged during aging and regeneration in cortical stromal cells, making autocrine signaling unlikely. However, expression of known TEC regulators IGF1 and FGF21 were both diminished with age, and dynamically regulated in medullary stromal cells during regeneration. Fgf21 expression has previously been reported in thymic stromal cells and mTECs in particular, and expression of its obligate coreceptor bKlotho (Klb) has been reported in cTEC, but not in other thymic subsets. Expression o Fgf21, but no KI h, declines with age, and transgenic overexpression of Fgf21 in the liver delays thymic atrophy in mice. FGF21 signaling promotes mTOR signaling, and has also been shown to regulate cell morphology by promoting the extension of cellular processes critical to the functions of dendrites in the brain, in immature neurons during traumatic brain injury, and in glial cells of the retina.

[0049] Analogs or related compounds that have been studied or developed for similar functions (TORC1 activation) include (i) FGF21 Variants: LY2405319 (PF-05231023) is an FGF21 analog developed by Eli Lilly, which has been investigated for its potential in treating type 2 diabetes and obesity; AKR-001 is an analog of FGF21, which has been explored for its metabolic benefits; (ii) FGF19 and FGF23 are members of the FGF family that also play roles in metabolic processes; (iii) Pegylated FGF21, e.g., BMS-986036 (Pegbelfermin), is a pegylated form of FGF21, which increases its half-life in the body, making it a candidate for treating non-alcoholic steatohepatitis (NASH); (iv) small molecule activators, while not analogs in the strict sense, are small molecules that aim to activate pathways similar to those influenced by FGF21; and / or (v)antibody-based therapies can target FGF21 receptors or related pathways to mimic or enhance FGF21 activity. Each of these analogs or related compounds aims to leverage the metabolic benefits of FGF21.

[0050] mT0RC2 plays a role in cell growth, metabolism, and survival. While mTORCl has been extensively studied and has well-known inhibitors like rapamycin, mT0RC2 is less understood but equally important. Analogs or related compounds that have been studied or developed for similar functions (TORC2 activation) include (i) Growth Factors such as TGF0, Insulin and IGF-1 (Insulin-like Growth Factor 1); (ii) Phospholipids such as Phosphatidic Acid (PA); (iii) Sphingolipids such as sphingosine 1 -phosphate (SIP), (iv) AMPK activating agents such as metformin, troglitazone, pioglitazone, rosiglitazone, resveratrol, quercetin, genistein, epigallocatechin gallate, berberine, curcumin, ginsenoside Rbl, a-lipoic acid, and ciyptotanshinone; and other small molecules such as SC-79 (CAS 305834-79-1).

[0051] One example of FGF21 is human FGF21 (GenBank accession AAQ89444.1) which has the amino acid sequence MDSDETGFEH SGLWVSVLAG LLGACQAHPI PDSSPLLQFG GQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSR FLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPR GPARFLPLPGLP PALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (SEQ ID NO: 1).C. Antioxidant

[0052] Thymus atrophy can be caused at least in part by oxidative damage to stromal cells, and that genetic (mCat Tg mice) or dietary (NAC or Ascorbic Acid (vitamin C)) approaches for increasing antioxidant activity can delay thymus atrophy. Delay in thymic atrophy can be achieved by dietary or genetic approaches for increased antioxidant activity and can improve the T cell response to flu infection in older mice. However, these approaches exacerbate, rather than rescue, age-associated defects in thymus selection against self-reactive T cells. This means this approach (antioxidant treatment alone) may cause unwanted autoimmunity. Data from other groups support the notion that mitigating oxidative damage would improve thymus function, including recent studies on calorie restriction.

[0053] In certain aspects an antioxidant is administered in combination with other agents described herein. Some common antioxidants that can be used in aspects of the current invention include Vitamin C (Ascorbic Acid), Vitamin E (Tocopherols and Tocotrienols), Beta-Carotene,Selenium, Glutathione, Coenzyme Q10 (CoQlO), Alpha-Lipoic Acid, Resveratrol, Curcumin, Polyphenols, Flavonoids (e g., quercetin, catechins), Anthocyanins (found in berries), and / or N- Acetylcysteine (NAC). These antioxidants are often recommended or used in various forms such as dietary supplements, foods, or even in medical treatments for conditions where oxidative stress plays a role, like cardiovascular diseases, cancer, neurodegenerative diseases, and aging-related conditions.D. Combination therapy

[0054] The overall idea is that both oxidative damage and loss of growth factor signaling promote thymus atrophy. Boosting antioxidant activity alone mitigates oxidative damage, but decreases basal autophagy, and therefore stromal cell antigen presentation, and the T cell tolerance that that antigen presentation usually induces. By adding autophagy activators to antioxidants, protection from oxidative damage can be maintained in conjunction with prevention of autophagy impairment. That reduction of FGF21 signaling within the thymus contributes to atrophy, so boosting two or three of the pathways together provides a combination that results in thymic regeneration or amelioration of thymic involution / atrophy.

[0055] As used herein, “administering in combination”, “co-administration” or “combination therapy” is understood as administration of two or more active agents using separate formulations or a single pharmaceutical formulation, or consecutive administration in any order such that, there is a time period while both (or all) active agents overlap in exerting their biological activities. It is contemplated herein that one active agent (e.g., autophagy activating agent) can improve the activity of a second therapeutic agent to the activities of the second therapeutic agent or can have a synergistic effect with the second therapeutic agent. “Administering in combination” does not require that the agents are administered at the same time, at the same frequency, or by the same route of administration. As used herein, “administering in combination”, “co-administration” or “combination therapy” includes administration of a composition comprising one or more agents with one or more additional agents.E. Combination with Immune Checkpoint Inhibitor Therapy

[0056] Immune checkpoint inhibitor (ICI) therapy is a form of cancer immunotherapy. The therapy targets immune checkpoints, key regulators of the immune system that when stimulatedcan dampen the immune response to an immunologic stimulus. Some cancers can protect themselves from attack by stimulating immune checkpoint targets. Checkpoint therapy can block inhibitory checkpoints, restoring immune system function, and permitting the immune system to respond to the cancer. The use of thymus regeneration before; during; after; before and during; before and after; during and after; or before, during and after immune check point inhibition for cancer therapy is contemplated.

[0057] Key immune checkpoint inhibitors target the molecules CTLA4, PD-1, and PD-L1. PD-1 is the transmembrane programmed cell death 1 protein (also called PDCD1 and CD279), which interacts with PD-L1 (PD-1 ligand 1, or CD274). PD-L1 on the cell surface binds to PD1 on an immune cell surface, which inhibits immune cell activity. Among PD-L1 functions is a key regulatory role on T cell activities. It appears that (cancer-mediated) upregulation of PD-L1 on the cell surface may inhibit T cells that might otherwise attack. Antibodies that bind to either PD-1 or PD-L1 and therefore block the interaction may allow the T-cells to attack the tumor. A number of ICI therapies targeting these molecules have been approved for a wide range of uses, and more therapies and cancer targets are under investigation. Approved ICIs include ipilimumab (targeting CTLA-4); nivolumab, pembrolizumab, and cemiplimab (targeting PD-1); and atezolizumab, avelumab, and durvalumab (targeting PD-L1).

[0058] In certain aspects the thymic treatments described herein can be used in combination with ICI, for example the thymic treatment(s) described herein can be used in combination with ipilimumab (targeting CTLA-4); nivolumab, pembrolizumab, and cemiplimab (targeting PD-1); and atezolizumab, avelumab, durvalumab (targeting PD-L1) and / or other immune checkpoint inhibitors. Accordingly, the present invention provides a method of treatment of a cancer, the method comprising administering a composition(s) containing one or more of an autophagy activating agent, a TORC1 activator or FGFRlc (Fibroblast Growth Factor Receptor 1c) agonist, and / or an antioxidant can be administered to a subject for thymic regeneration or amelioration of thymic involution and an immune checkpoint inhibitor to a patient, preferably a human patient, in need thereof, thereby treating cancer. The immune checkpoint inhibitor may comprise an immunoglobulin molecule, preferably an antibody, targeting an immune checkpoint molecule. By “targeting” is meant that the immunoglobulin molecule is an agonist of the immune checkpoint molecule, and / or that it specifically binds to the immune checkpoint molecule to block activation of the immune checkpoint, thereby enhancing immune function or response. The immunecheckpoint molecule may be selected from CTLA-4, PD-1, and PD-L1 . In preferred embodiments the immune checkpoint molecule is PD-1. In some embodiments, a plurality of immune checkpoint molecules may be targeted; for example, CTLA-4 and PD-1, or CTLA-4 and PD-L1, or CTLA-4 and PD-1 and PD-L1; preferably CTLA-4 and PD-1. In some embodiments, the immune checkpoint inhibitor comprises a monoclonal antibody which specifically binds CTLA-4, or which specifically binds PD-1, or which specifically binds PD-L1. Examples of such monoclonal antibodies include pembrolizumab, nivolumab, ipilimumab, avelumab, atezolizumab, durvalumab, cemiplimab (REGN2810), camrelizumab (SHR1210), envafolimab (KN035), sintilimab (1131308), spartalizumab (PDR001), tislelizumab (BGB-A317), prolgolimab (BCD- 100), toripalimab (JS001), dostarlimab (TSR-042, WBP-285), tremelimumab (ticilimumab, CP- 675,206). Particularly preferred combinations include composition(s) containing one or more of an autophagy activating agent, a TORC1 activator or a FGFRl c (Fibroblast Growth Factor Receptor lc) / p-Klotho agonist, and / or an antioxidant can be administered to a subject for thymic regeneration or amelioration of thymic involution and atezolizumab; composition(s) containing one or more of an autophagy activating agent, a TORC 1 activator or a FGFR lc (Fibroblast Growth Factor Receptor 1 c) / p-Klotho agonist, and / or an antioxidant can be administered to a subject for thymic regeneration or amelioration of thymic involution and pembrolizumab; composition(s) containing one or more of an autophagy activating agent, a TORC1 activator or a FGFRlc (Fibroblast Growth Factor Receptor 1c) / p-Klotho agonist, and / or an antioxidant can be administered to a subject for thymic regeneration or amelioration of thymic involution and nivolumab and ipilimumab; composition(s) containing one or more of an autophagy activating agent, a TORC1 activator or a FGFRlc (Fibroblast Growth Factor Receptor 1 c) / p-Klotho agonist, and / or an antioxidant can be administered to a subject for thymic regeneration or amelioration of thymic involution and durvalumab; and composition(s) containing one or more of an autophagy activating agent, a TORC1 activator or a FGFRl c (Fibroblast Growth Factor Receptor 1c) / p- Klotho agonist, and / or an antioxidant can be administered to a subject for thymic regeneration or amelioration of thymic involution and dostarlimab. In some embodiments, the immune checkpoint inhibitor comprises a peptide inhibitor of PD-1 / PD-L1 interaction, or a small molecule inhibitor. Examples of such include AUNP12, CA-170, and BMS-986189. The composition(s) containing one or more of an autophagy activating agent, a TORC1 activator or a FGFRlc (Fibroblast Growth Factor Receptor 1 c) / 'P-Klotho agonist, and / or an antioxidant can be administered to a subject forthymic regeneration or amelioration of thymic involution and the immune checkpoint inhibitor may be administered concurrently, separately or sequentially. Multiple administrations of either the composition(s) containing one or more of an autophagy activating agent, a TORC1 activator or a FGFRlc (Fibroblast Growth Factor Receptor 1c) / p-Klotho agonist, and / or an antioxidant can be administered to a subject for thymic regeneration or amelioration of thymic involution, or the immune checkpoint inhibitor, or both, may be given. Other administration schedules may be used.

[0059] The present invention provides a novel combination therapy for the treatment of thymic involution / atrophy, comprising administration of a combination of these agents to synergistically enhance efficacy by targeting multiple pathways involved in thymic involution / atrophy. These agents can be formulated independently or co-formulated into various pharmaceutical forms such as tablets, capsules, injectables, or transdermal patches, ensuring bioavailability and patient compliance.II. Pharmaceutical Compositions and Modes of Administration

[0060] The pharmaceutical compositions described herein may be administered to a subject in any suitable formulation. These include, for example, liquid, semi-solid, and solid dosage forms, The preferred form depends on the intended mode of administration and therapeutic application.

[0061] In certain embodiments the composition is suitable for oral administration. In certain embodiments, the formulation is suitable for parenteral administration, including topical administration and intravenous, intraperitoneal, intramuscular, intrathymic, and subcutaneous, injections. In a particular embodiment, the composition is suitable for intravenous administration.

[0062] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. For intravenous administration, the formulation may be an aqueous solution. The aqueous solution may include Hank's solution, Ringer's solution, phosphate buffered saline (PBS), physiological saline buffer or other suitable salts or combinations to achieve the appropriate pH and osmolarity for parenterally delivered formulations. Aqueous solutions can be used to dilute the formulations for administration to the desired concentration. The aqueous solution may contain substances which increase the viscosity of the solution, such as sodium carboxymethyl cellulose, sorbitol, or dextran. In some embodiments, the formulation includes a phosphate buffer saline solution which contains sodium phosphate dibasic, potassium phosphate monobasic, potassium chloride, sodium chloride and water for injection.

[0063] Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear, or nose. Formulations suitable for oral administration include preparations containing an inert diluent or an assimilable edible carrier. The formulation for oral administration may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.

[0064] As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, body weight, the severity of the affliction, and mammalian species treated, the particular compounds employed, and the specific use for which these compounds are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine methods, for example, human clinical trials, animal models, and in vitro studies.

[0065] Local Delivery of Compositions. In particular embodiments of the present invention, one or more compositions is administered locally to treat thymic atrophy. Particular embodiments are directed to the delivery of one or more compositions described herein in a sustained release composition and / or configuration, particularly for intrathymic administration. Nevertheless, more than one administration of the therapeutic agents described herein may be required. In specific embodiments, there is an intrathymic drug delivery, such as using a device to elute the drug in a periodic manner; in a sustained low dose manner; by intrathymic injection or perfusion. The agent may be delivered in a sustained release formulation, again by an intrathymic route.

[0066] One or more compositions can be provided by sustained release systems, by encapsulation or by implantation devices. The compositions may be administered by bolus injection or continuously by infusion / perfusion, or by implantation device. Where an implantation device is used, the device may be implanted into or near the thymus. The injections may be given as a one-time treatment, repeated (daily, weekly, monthly, annually etc.) to achieve the desired therapeutic effect.

[0067] Preparation of composition(s) can involve the formulation with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that may provide controlled or sustained release of one or more agent, which may then be delivered via a depot injection. Implantable drug delivery devices may be used to introduce the desired composition. For example, a method of local administration of one or more compositions of the invention may be delivered by an osmotic pump. In additional embodiments, drug encapsulation systems are utilized that provided sustained release, including the following: liposomes, biodegradable microspheres, cylinders, and capsules, such as those made of lactic and glycolic acid and hydrogels. In further embodiments, membrane-enclosed reservoir devices, monolithic systems, and nano-particles may be employed.

[0068] In certain embodiments, one or more compositions are delivered orally. In certain embodiments, one or more compositions are administered parenterally. In certain embodiments, one or more compositions are delivered by injection or infusion. In certain embodiments, one or more compositions are delivered topically including transmucosally. In certain embodiments, one or more compositions are delivered by inhalation. In one embodiment, the compositions provided herein may be administered by injecting directly to a thymus. In some embodiments, one or more compositions may be administered by intravenous injection or intravenous infusion. In certain embodiments administration is systemic. In certain embodiments administration is local.

[0069] A “subject” to be treated by the methods of the invention can mean either a human or non-human animal, preferably a mammal, more preferably a human. In certain embodiments, a subject has a detectable or diagnosed disorder (e.g., cancer, immunodeficiency or the like) or thymic condition (e.g., thymic involution / atrophy) prior to initiation of treatments using the methods of the invention. In embodiments, the subject is a non-human mammal. In embodiments, the subject is a non-human mammal such as a non-human primate (e.g., monkeys, apes), ungulate(e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e g., rabbit).

[0070] “Therapeutically effective amount” means the amount of a compound(s) that, when administered to a patient for treating a disorder or condition, is sufficient to effect such treatment for the disorder or condition. When administered for preventing a disorder or condition, the amount is sufficient to avoid or delay onset of the disorder or condition. The “therapeutically effective amount” will vary depending on the compound, the disorder or condition, and its severity and the age, weight, etc., of the pati ent / subject to be treated. A therapeutically effective amount need not be curative. A therapeutically effective amount need not prevent a disorder or condition from ever occurring. A therapeutically effective amount is an amount that will at least delay or reduce the onset, severity, or progression of a disease or condition, or in some aspects regenerate, at least partially, thymic mass.

[0071] As used herein, “treatment”, “treating” and cognates thereof refer to the medical management of a subject with the intent to improve, ameliorate, stabilize, prevent or cure a disease, pathological condition, or disorder. This term includes active treatment (treatment directed to improve the disease, pathological condition, or disorder), causal treatment (treatment directed to the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed for the relief of symptoms), preventative treatment (treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition (e g., infection), or disorder); and supportive treatment (treatment employed to supplement another therapy).III. Examples

[0072] The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.EXAMPLE 1AUTOPHAGY ACTIVATION

[0073] The inventors have evidence, based on Beclin 1 knock in mice, that increased autophagy alone delays thymus atrophy. Recent work demonstrates that age-associated atrophy does indeed diminish central T cell tolerance induction. A study revealed that despite the significantly diminished lymphopoietic capacity in the aged thymus, it continues to serve a protective role against infection, cancer, and autoimmunity in adults. Given that retaining even a thymic “remnant” in adulthood had significant protective capacity (Kooshesh et al. N Engl J Med, 389:406-17, 2023), supports the notion that increasing thymus function in older adults holds strong potential for significantly increasing the human healthspan.

[0074] The concept that autophagy activation may increase thymus size is based on the recent finding that autophagy in thymic stromal cells is redox regulated. This observation revealed the reason that increased antioxidant activity both delays thymus atrophy and improves anti-viral T cell responses, while also impairing central T cell tolerance induction. Together, these studies revealed a combination of increased antioxidant and autophagy activity as a novel potential therapeutic approach for improving T cell function and tolerance in older animals.

[0075] Young stromal gene expression signature is not restored by androgen ablation-induced regeneration of organ size, suggesting an androgen-independent cause for age-associated atrophy. When computational deconvolution was used to investigate changes in stromal gene expression during aging and thymus regeneration, we found that a number of critical stromal functions are impaired during aging, including loss of tissue-restricted antigen (TRA) gene expression by medullary TECs (mTECs), which is critical for deleting potentially autoreactive cells and inducing central self-tolerance in the thymus, and this functional decline was not reversed by castration. We found that regeneration is very short-lived (lasting only a few weeks), and generally does not reverse the stromal dysfunctions induced by aging. Thus, the transient nature of thymus regeneration appears to result from the fact that the stromal cells in the regenerated thymus are fundamentally indistinct from those in the atrophic thymus. These observations suggest that increasing the size of the aged thymus could be detrimental if it allows increased production of autoreactive T cells, and therefore illustrate the significance of evaluating this possibility for new approaches to thymus regeneration. They also highlight the need to understand the mechanisms regulating atrophy in order to restore not only thymus size, but also its function.

[0076] Low antioxidant expression and high oxidative damage in cortical thymic stromal cells promote age-associated thymic atrophy. Our studies show that cortical thymic stromal cells are deficient in the peroxide quenching enzyme catalase, acquire high levels of damage consistent with ROS exposure, and have greater sensitivity to ROS relative to lymphocytes from the same thymus. Supplementing drinking water with the antioxidant N-acetyl cysteine (NAC) or ascorbic acid significantly preserved the size of the thymus with age, while no effects were found on any other organ tested, or on body composition (Griffith et al., Cell reports 12, 1071-79, 2015). A genetic complementation approach was pursued using transgenic mice expressing a mitochondria-targeted human catalase transgene under the control of a ubiquitous (chicken P -actin) promoter (mCat Tg)(Schriner et al., Science 308, 1909-11, 2005). Genetic complementation of catalase activity significantly delays thymic atrophy, and that stromal expression of the transgene was sufficient for this effect (Griffith et al., Cell reports 12, 1071-79, 2015). We next tested whether the delay in thymic atrophy imparted by increasing antioxidant activity results in increased thymus function in aging by testing its effects on T cell responsiveness and tolerance. Supplementing antioxidant activity delays waning anti-viral T cells responses. Although the numbers of PA-specific T cells were maintained at 6 months of age, as reported for 18-month-old mice (Yager et al., Journal of experimental medicine 205, 711-23, 2008), NP-specific T cells were significantly decreased by approximately 3 -fold in the 6 month-old mice. The decline in NP-specific T cell frequency was rescued by the expression of the catalase transgene. Similar rescue of NP-specific T cell frequency was achieved by dietary antioxidant supplementation when we treated WT mice with NAC beginning at weaning. We found that both NP and PA-specific T cells were significantly diminished in the thymus 14 week old mice relative to those at 5 weeks of age. NP-specific T cell numbers were significantly higher in thymi from 14 week old mCat Tg mice relative to age- matched controls, while PA-specific T cell numbers did not significantly increase in mCat Tg mice (Hester et al. Cell Rep 38, 110363, 2022), suggesting that delaying thymus atrophy by increasing antioxidant activity can increase the output, and therefore the diversity, of naive T cells capable of responding to infection.

[0077] Supplementing antioxidant activity exacerbates age associated declines in central tolerance induction. To assess the impact of aging on thymic selection, we employed a recently reported flow cytometric approach to measure clonal deletion in the polyclonal T cell population based on identification of cleaved caspase 3 among signaled (TCRhiCD5hi) thymic T cells (afterexclusion of cells expressing CD25, NK1.1 , and TCRg / d)(Breed et al., J Jmmunol 202, 3226-33, 2019). Although the frequencies of CD4+CD8+ double positive (DP) lymphocytes that had been signaled and were available for positive selection were similar in 6-month old WT mice relative to 5-week old WT animals, we found that the expression of CD5 within that signaled population was diminished in the older mice. Within the DP “signaled” population, the frequency of cells undergoing clonal deletion was decreased. We likewise found decreased clonal deletion among SP4 and SP8 T cells. Among non-signaled cells, we found a decrease in the frequency of cells undergoing death by neglect in the older mice. Together, these studies suggest that the efficiency of clonal deletion of self-specific T cells declines with age, concomitant with diminished TCR signaling as indicated by CD5 expression. In contrast to the effect on thymus size, none of the age- associated perturbations in selection were rescued in age-matched mCat Tg mice (Hester et al. Cell Rep 38, 110363 (2022).

[0078] To further test the impact of age-associated declines in thymic TRA gene expression on negative selection, we focused on T cells recognizing a model TRA encoded by the Apolipoprotein B (ApoB) gene. By 6 months of age, the thymus has already deteriorated significantly, having lost approximately 75% of its cellularity relative to its peak size in 5-week old mice (Griffith et al., Cell reports 12, 1071-79, 2015; Dominguez-Gerpe and Rey-Mendez, Microscopy research and technique 62, 464-76, 2003). Thus, barring changes in selection efficiency, the total number of T cells recognizing any given antigen would likewise be expected to decline by about 75%. When we measured the total number of T cells recognizing a representative foreign antigen, a variant of peptide 52-68 of the lEa chain (Dongre etal., European journal of immunology 31, 1485-94, 2001) using an MHCII tetramer, designated 2W:I-Ab, we found that the total number of T cells recognizing 2W was indeed decreased significantly (>2- fold) in 6 month old mice relative to mice at 5 weeks of age (Hester et al. Cell Rep 38, 110363, 2022). In contrast, the number of T cells recognizing the ApoBpe self-antigen did not change at 6 months of age. These results are consistent with the notion that the levels of ApoBpe-specific T cells generated in the aged thymus are maintained in older animals due to a loss of negative selection and a consequent failure of clonal deletion with aging. The fact that the increase in selfspecific T cells in mCat Tg thymi was even greater in magnitude than the increase in T cells recognizing foreign antigens suggested that negative selection was impaired in mCat Tg mice relative to WT.

[0079] To investigate the impact of increased antioxidant activity on autophagy, we used an autophagy reporter mouse model (GFP-LC3 Tg mice) in which transgenic expression of a GFP- LC3 fusion protein allows fluorescent labeling of autophagosomes (Mizushima et al., Mol Biol Cell 15, 1101-11, 2004). The GFP-LC392 reporter mice were used to show that high levels of basal autophagy in cTECs and DCs are regulated in part by high levels of H2O2 present in these populations at the steady state (Semwal et al., PNAS USA 119, e2204296119, 2022). Overexpression of the antioxidant catalase in transgenic mice (mCat Tg) caused decreases in H2O2 levels in cTECs and DCs (Semwal el al., PNAS USA 119, e2204296119, 2022), which in turn diminished autophagy, measured by GFP MFI. Testing whether potential downstream effects of catalase expression on T cell development were dependent on changes in autophagy required a means of independently manipulating autophagy levels in catalase transgenic mice. To accomplish this, we employed a recently developed transgenic mouse model in which a Phel21 Ala mutation was introduced to the Beclin 1 gene (BecnlF121A / F121A)(Fernandez et al., Nature 558, 136-40, 2018). This mutation inhibits the interaction of Becnl with its negative regulator BCL2, resulting in increased basal autophagy. BecnlF121A / F121 A KI (herein referred to as BecnlKVKI) mice were crossed with GFP-LC3 reporter mice to allow autophagy measurement. To test whether the reduced autophagy in mCat Tg mice could be rescued by introducing the BecnlKI allele, we crossed mCat Tg mice with BecnlKEKI GFP-LC3 transgenic mice to generate mCat Tg mice expressing both BecnlKI and GFPLC3 alleles (mCat Tg:BecnlKI / KI:GFP-LC3). We found that the decreased autophagy we observed in mCat Tg stromal cells was significantly rescued by expression of two BecnlKI alleles in DCs. The decreased autophagy we observed in mCat Tg cTECs was partially rescued by expression of two BecnlKI alleles in cTECs, and there were no significant differences between BecnlKI / KI and either mCat Tg or mCat non-Tg groups. To compare our flow cytometric approach to a microscopy-based approach for evaluating autophagy, we compared autophagic punctae in GFP-LC3 transgenic mice on the mCat Tg and mCat non-Tg background. We found that mCat Tg thymi had a lower frequency of GFP+ punctae per cortical area relative to mCat non-Tg mice, consistent with decreased autophagy in mCat Tg animals. To distinguish between diminished autophagosome formation and increased autophagosome degradation, we crossed mCat Tg mice to a second autophagy reporter strain bearing the RFP- GFP-LC3 fusion gene (Li et al., J Am Soc Nephrol 25, 305-15, 2014) to evaluate both autophagosomes and autolysosomes. Since GFP fluorescence is quenched in the autolysosomewhile RFP fluorescence persists, autophagosomes (GFP+RFP+) and autolysosomes (GFP-RFP+) may be distinguished in this model. When we compared GFP and RFP expression in cTECs from mCat Tg and non-Tg mice, we found that the decrease in GFP fluorescence in mCatTg mice reflected a marked increase in GFP- RFP- double negative cells not undergoing autophagy, rather than an accumulation of GFP-RFP+ cells with high frequencies of autolysosome activity.

[0080] To address whether the diminished autophagy we found in mCat Tg thymic stromal cells was physiologically relevant for negative selection of potentially autoreactive T cells, we again used a flow cytometric approach to measure thymocyte clonal deletion, but in these studies we further divided signaled cells into early (CCR7-) or late (CCR7+) stages (Hu et al., Frontiers in immunology 6, 398, 2015; Kurobe et al., Immunity 24, 165-77, 2006; Nitta et al., PNAS USA 106, 17129-33, 2009; Ueno et al., Journal of experimental medicine 200, 493-505, 2004). We found that the frequency of both early and late clonally deleted cells declined in mCat Tg mice relative to non-Tg mice, and was rescued in mCat Tg:BecnlKiKimice, although the magnitude of the decrease and rescue among early (CCR7-) signaled thymocytes was greater than that in late (CCR7+) signaled thymocytes. This suggests a catalase-mediated decline in clonal deletion that has a greater impact during the early stages of negative selection than during later stages. These results support the notion that the redox status of thymic stromal cells promotes high basal autophagy within these populations that in turn promotes clonal deletion in developing T lymphocytes. Since autophagy is critical for antigen presentation, especially during MHC class II presentation to CD4+ T cells (Aichinger et al, Journal of experimental medicine 210, 287-300, 2013; Paludan et al., Science 307, 593-96, 2005), diminished autophagy could be predicted to cause a particular decrease in the efficiency of negative selection of CD4 T cells. We measured the frequency of single positive (SP) CD4 and CD8 T cells in mCat Tg mice, and found an increase in the frequency of SP CD4+ T cells in mCat Tg thymi relative to those from non-Tg mice. This increase was rescued in mice bearing the Been IKI allele, indicating a potential autophagy-mediated increase in deletion of potentially autoreactive CD4+ T cells in mCat Tg mice bearing the BecnlKi allele.

[0081] Our hypothesis predicts that over time, mCat Tg mice would show signs of increased autoimmunity due to escape of self-reactive T cells into the periphery. To test this, we performed blinded evaluation of serum from mCat Tg mice at 6-8 months age to detect the presence of antinuclear antibodies (ANA). We found an increase in the frequency of ANA+ serum in mCat Tgmice (9 / 14) relative to age-matched WT mice (3 / 13), which was rescued in mCat Tg:BecnlKi / Ki mice (1 / 7). We also performed blinded evaluation of lymphocytic infdtrates in the lungs and livers of WT, mCat Tg, and mCat Tg:Becnl Ki / Kimice using hematoxylin and eosin (H&E) staining. We found an increase in the presence of lymphocytic infdtrates in lungs and livers of 6-8 month old mCat Tg mice relative to WT (5 / 7 and 0 / 5 each, respectively). This increase was also partially rescued in mCat Tg : Been 1KI / KI mice (1 / 7 and 2 / 7 in lung and liver, respectively). Together these data suggest that immune tolerance is impaired in mCat Tg mice and that it is rescued by increasing basal autophagy in mCat Tg mice bearing the Becnl KI allele (mCat Tg:BecnlKi / Ki).

[0082] Redox regulation of autophagy in TSCs is an important regulator of immune tolerance. The studies support the notion that even subtle changes in autophagy impact thymus function, and also demonstrate our ability to measure changes in autophagy in our cells of interest. In addition to its role in antigen presentation, autophagy also protects cells from oxidative damage by promoting turnover of damage organelles (Filomeni et al., Cell Death Differ 22, 377-88, 2015)and may therefore promote the persistence of cTECs by mitigating oxidative damage caused by high H2O2 levels present at the steady state (Griffith et al., Cell reports 12, 1071-79, 2015; Semwal et al., Frontiers in immunology 12, 636072, 2021).

[0083] Together, these results support the hypothesis that a combination therapy incorporating increased antioxidant and autophagy activity could mitigate oxidative damage, while maintaining high levels of autophagy and self-antigen presentation for T cell tolerance. To test whether increased autophagic activity can mitigate thymus atrophy, we have measured thymus size in 6-8- month old BecniFi2iA / iFi2iA mice, and find a significant increase in cellularity in BecniFi2iA / iFi2iA thymi relative to controls. The frequency of cells undergoing clonal deletion likewise appears to be increased in BecniFi2iA / iFi2iA thymi relative to controls, suggesting that age-associated declines in central tolerance induction may be mitigated by increasing autophagy activity. The systemic administration of exogenous pharmaceuticals such Rilmenidine to increase autophagy has not been evaluated for its impacts on thymus function as far as we know.A. Methods

[0084] Rigor and reproducibility: Since thymus atrophy is sexually dimorphic, all aging studies are performed in both male and female cohorts of mice. Regeneration by surgical castration will only be performed in males. Unless otherwise described below (i.e., for pilot studies), 10 miceper group are used. Statistical significance is tested using one-way ANOVA with Tukey’s multiple comparison correction.

[0085] Combining increased antioxidant and autophagy activity will optimally preserve thymus function and prolong thymus regeneration. The rationale being that mitigating oxidative damage in thymic stromal cells by increasing antioxidant activity delays atrophy, increases T cell generation, and increases the frequency of T cells recognizing flu epitopes, but exacerbates age- associated declines in T cell tolerance induction because it diminishes autophagy activity, which is critical for antigen presentation (Hester et al. Cell Rep 38, 110363, 2022; Semwal el al, PNAS USA 119, e2204296119, 2022). This suggests that optimal prevention or reversal of age-associated thymic dysfunction may require prevention of oxidative damage together with complementation of ROS-mediated autophagy. Rilmenidine is an antihypertensive drug with an excellent safety profile that functions as an imidazoline receptor agonist. It has also been used in human clinical trials for the treatment of Huntington’s disease based on its mTOR-independent positive regulation of autophagy activity (Rubinsztein et al., Nat Rev Drug Discov 11, 709-30, 2012). In addition to its efficacy in delaying age-associated atrophy, the capacity of combination NAC / Rilmenidine therapy to delay re-atrophy after regeneration, since the mechanisms regulating that process may be similar to those involved in age-associated atrophy.

[0086] Flow cytometry, immunoassays, microscopy, and in vivo vaccination and infection models in mice are treated with NAC, an autophagy activator (Rilmenidine), both, or vehicle, to evaluate:

[0087] Thymus size, number, frequency, proliferation, survival and development of T cell progenitors (thymocytes). NAC will be administered in drinking water and the Rilmenidine dose, we will use cohorts of 5 mice to compare three doses (administered i.p. 4 times / week at 5, 10 and 20 mg / kg), from weaning to 10 weeks of age, at which time the thymus has lost -50% of its peak cellularity (Griffith etal., Cell reports 12, 1071-79, 2015). Thymus size is evaluated to choose the optimal dose. After treatment (or vehicle) from 2 to 12 months of age, the number and frequency of T cells at various stages in development is evaluated using standard flow cytometry approaches based on surface expression of CD4 and CD8, and DN1-4 CD4-CD8- lineage negative cells will be evaluated based on surface expression of ckit, CD25 and CD44. Proliferation and apoptosis will evaluated by co-staining with anti-Ki67 antibody and Annexin V, respectively.

[0088] Positive and negative selection of thymocytes; In the same groups, we will evaluate positive selection in the polyclonal population as described previously (Semwal etal., PNAS USA 119, e2204296119, 2022). ApoBP:MHCII tetramers are used to measure the number and frequency of APOB-specific conventional T cells and regulatory T cells in the thymus and secondary lymphoid organs (SLO, spleen and lymph nodes) using APOBp: I-Ab tetramers and FoxP3 staining. Previous work also revealed significantly increased functional T cell responsiveness to this model self-antigen when we immunized mice with the APOB peptide in CFA, and then measured the splenic CD4+ T cell responses to APOB by ZFNy ELISpot, even in cells that expressed FoxP3, a mixed phenotype that was rescued in mCat Tg mice (Hester et al. Cell Rep 38, 110363, 2022). We will therefore also use this approach to evaluate self-reactivity to APOB. We will also evaluate the overall frequency of naive and memory T cells in the spleen and lymph nodes (LN) as previously described (Hester et al. Cell Rep 38, 110363, 2022). Since maintenance of naive T cells in LNs varies across the body (Sonar etal., PNAS USA 119, e2121028119, 2022), we will evaluate at least cervical, inguinal, mediastinal, popliteal, mesenteric, brachial, and cutaneous LNs. Serum ANA reactivity, lymphocytic infdtration of lung and liver, and reactivity to self-antigens will be evaluated using self-peptide tetramers and ELISPOT in the same groups of mice (different cohorts). ANA reactivity will be evaluated by blind review in serum (1 : 100 dilution) using FITC conjugated anti-IgG. Samples will be coded and evaluated blindly. To assess immunopathology in mice, lungs and livers will be formalin-fixed and stained with H&E. Sections from at least three different anatomic areas will be examined for lymphocytic infiltration in each tissue. Samples will be coded and evaluated for the presence of lymphocytic infiltrates blinded.

[0089] T cell responsiveness to vaccination and viral challenge. We predict that treatment with NAC and Rilmenidine will increase thymus size, and therefore the diversity of T cells able to respond to vaccination or infection in the periphery. To test this, we will use a heterologous virus for immunization to avoid the complication of cross-neutralizing antibodies to allow direct measurement of the T cell immune responses. At 12 months of age, each group of mice will receive 15 pl of virus at the desired concentration delivered to one of the nostrils using a p20 pipette. Mice will be inoculated with 800 pfu / mouse A / X31 in FBS-free DMEM (or treated with DMEM alone for unvaccinated groups). 4 weeks later mice will be challenged with IxlO4pfu / mouse A / PR8 / 34. Nine days after viral challenge, mice will be weighed and total lymphocytes isolated from the lung (BAL) and single cell suspensions of digested parenchymal lung tissue, mediastinal lymph node,and spleen will be incubated with peptide:MHCI tetramers NPs66-374:Db and PA224-:Db and stained for CD44, CD103, CD69 and CD8. Cells will then be fixed and permeabilized, stained with anti- fFN-y, anti-TNF, and anti-IL-2, and examined by flow cytometry.

[0090] Since lung pathology after influenza infection is exacerbated in older mice (Goplen et al., Sci Immunol 5, 2020), we will evaluate lung pathology at 60 d p i. in separate cohorts of mice. Lungs will be fixed with ImL 10% formalin before H&E stains. Micrographs will be analyzed using ImageJ software to measure total parenchymal area and inflamed areas (% infiltrated with lymphocytes) of the tissues. Monocytes in lungs will be evaluated by flow cytometry using anti- CD64, Siglec-F,CD1 lc,MHCII,CDl lb, and Ly6G as described (Goplen et al., Sci Immunol 5, 2020). Lung fibrosis will be evaluated by measuring collagen content using Mason’s trichrome staining and the hydroxyproline assay as described (Goplen et al., Sci Immunol 5, 2020). b) We will treat 12-month-old mice with NAC and / or Rilmenidine or vehicle as in a) beginning one week prior to thymus regeneration by surgical castration (as previously described (Griffith et al., Aging cell 11, 169-77, 2012) and continue for 3 weeks or 3 months after regeneration, followed by evaluation.

[0091] Reversal of Established Thymic Atrophy. Though we have previously demonstrated that boosting antioxidants and autophagy alone can delay atrophy and dysfunction to varying degrees, until recently we did not have evidence that they could reverse atrophy after it occurred. We now show the first evidence that thymus atrophy can be reversed by exogenous administration of therapeutics that boost antioxidant (NAC) and autophagy activity (Rilmenidine). We treated 6- month-old wild type (WT) mice with NAC+Rilmenidine in drinking water for 4 weeks and found that this increased the relative size of the thymus (FIG. 6, left), and also increased the frequency of naive CD8 T cells in the periphery (FIG. 6, right), while maintaining clonal deletion in thymocytes (FIG. 9), strongly supporting the feasibility that such approaches will improve T cell effector function and tolerance in older animals.EXAMPLE 2FGF21

[0092] Additional studies show that overexpressing FGF21 in the thymus prevents atrophy and has positive effects on T cell immunity to flu in older mice, but does not impair tolerance induction like antioxidant treatment does.

[0093] Consequences of age-associated thymic atrophy include declining T cell responsiveness to pathogens and vaccines, and diminished T cell self-tolerance. Cortical thymic epithelial cells (cTECs) are primary targets of thymic aging, and recent studies suggested that maintenance of their unique labyrinth-like morphology requires mTOR signaling downstream of medullary TEC (mTEC)-derived growth factors. To test this hypothesis, we generated a bicistronic knock-in (KI) mouse model in which the mTOR-activating ligand FGF21 and the fluorescent reporter mCherry are expressed under the control of the Lactoperoxidase gene (LPOFGF21). In addition to salivary gland epithelium, this model directs expression of KI alleles broadly but specifically in mTEC populations within the thymus. We find that mTEC-derived FGF21 promotes temporally distinct patterns of mTORCl and mT0RC2 signaling in cTECs, promotes thymus and individual cTEC growth and maintenance during aging, increases T cell responsiveness to viral infection, and diminishes indicators of peripheral autoimmunity in older mice. These results reveal a molecular mechanism by which paracrine FGF21 signaling regulates thymus size and function throughout the lifespan, as well as potential therapeutic targets for improving T cell function and tolerance in the aging population.

[0094] These findings led us to hypothesize that FGF21 expression by mTECs promotes paracrine activation of mT0RCl / mT0RC2 in cTECs to regulate cell proliferation, individual cell size, and morphology, thereby controlling overall thymus size. To begin to test this hypothesis, we designed a knock-in mouse model in which overexpression of FGF21 is driven broadly in most mTECs. Here we report that paracrine FGF21 signaling activates temporally distinct patterns of mTORCl and mT0RC2 signaling in cTECs, which is sufficient to increase thymus size and function in aging mice, resulting in increased T cell responsiveness to influenza infection as well as decreases in indicators of autoreactivity, relative to age-matched controls.B. Results

[0095] Expression of Fgf21 by mTECs declines with age. Because our previous studies indicated a role iorFgf21 expressed by medullary stromal cells in the regulation of thymus growth, we sought to characterize the stromal cell subsets expressing Fgf21 across the lifespan. Analysis of a previously published single-cell RNA-seq dataset revealed that among the nine subsets of TECs identified, Fgf21 expression was found at the highest levels in mature mTECs, as well as proliferating TECs, which is a rarer population seen predominantly in the early postnatal period.Fgf21 expression in mature mTECs was highest during the early postnatal period and declined after 4 weeks of age, persisting at lower levels through 52 weeks. In contrast, Fgf21 expression in proliferating TECs remained high through middle age. However, since the overall abundance of this population declined significantly following the postnatal period, the total amount of FGF21 produced by this subset of cells would be expected to decline during aging. Fgf21 expression was detected at minimal levels in mature cTECs throughout the first year, and in a small population of structural TEC (sTEC) likely to be found in the medulla based on expression of Enpp282. Thus, single-cell transcriptomic evidence confirmed mature mTECs as a primary source of Fgf21 within the thymus, and also confirmed age-associated declines in Fgf21 expression, consistent with previous reports.

[0096] Identification ofLpo as a candidate mTEC gene. To begin to identify the role of mTEC- derived FGF21 in the regulation of thymus size, we designed a mouse model to drive gene expression broadly, but specifically, in mTECs in the thymus. Using the previously described transcriptional datasets, we applied a series of selection criteria to prioritize broadly expressed mTEC genes. The top candidate genes were ranked based on confidence of medullary stromal expression and relative tissue specificity in a published database of gene expression across a broad range of tissue types (BioGPS GeneAtlas MOE430, germa). To avoid selecting Aire-dependent genes, which are expressed by only a subset of mTECs, typically at low levels, we excluded Aireregulated genes using two previously published gene lists. Among the top candidate genes was Lpo (Lactoperoxidase), which was predicted to have highly specific expression in the thymic medullary stroma, lacrimal, and salivary glands. Immunofluorescence microscopy confirmed that within the thymus, LPO expression was preferentially expressed in the medulla, with a high degree of co-localization with TECs expressing the highest levels of EpCAM, which is characteristic of mTECs, and, importantly, remained highly expressed in the thymus at 12 months of age. Upon validation of Lpo as a candidate for driving gene expression broadly in most mTEC, we designed a bicistronic vector allowing for the insertion of Fgf21 and mCherry cDNA at the end of Exon 13 of the Lpo gene by incorporating P2A and T2A sequences. We then utilized CRISPR-Cas9 to generate founder mice expressing the Lpo-Fgf21 -mCherry knock-in (KI) allele. Sanger sequencing and PCR verification confirmed expression of the knock-in mutation in founders.

[0097] Upon the generation of LPOFGF21knock-in founders, we backcrossed with C57BL / 6 mice to create heterozygous LPOFGF21 / WTand homozygous LPOFGF21 / FGF21experimental mice.Unless otherwise indicated, heterozygous mice (designated LPOFGF21) were used for experiments. We first performed flow cytometric evaluation of mCherry reporter expression among thymic stromal subsets to confirm expression of the KI allele in mTEC subsets. We found mCherry to be expressed specifically in mTEC, and broadly among various mTEC subsets, including UEA1+ mTEC (CD45.2-, EpCAM+, Ly51-, UEA1+), mTEClo (CD45.2-, EpCAM+, Ly51-, MHCIIlo, CD8OI0) and mTEChi (CD45.2-, EpCAMT, Ly51-, MHCIIhi, CD80hi). mCherry was not detected in cTEC (CD45.2-, EpCAM+, Ly51+, UEA1-), eDC (CD45.2+, CD1 lc+, B220-), pDC (CD45.2+, CDl lc+, B220+), endothelial cell (EC) (CD45.2-, EpCAM-, CD31+), pericyte (PC) (CD45.2-, EpCAM-, CD31-, PDGFRab+, CD146+, gp38-), or fibroblast (Fb) (CD45.2-, EpCAM-, CD31-, PDGFRab+, CD146-, gp38+) subsets, consistent with publicly available datasets. Expression of mCherry ranged from 50-75% of all mTEC, and was verified in a proportion of all mTEC subsets evaluated.

[0098] To further evaluate mCherry expression, we performed immunofluorescence microscopy. Thymic sections from 1 -month-old LPOFGF21and LPOWTlittermate controls were stained with fluorescent antibodies targeting K5, K8, DEC 205, Ly51, UEA1, and Aire. Colocalization with medullary markers K5, UEA1, and Aire, but not with cortical markers K8, DEC 205, and Ly51 indicated broad and specific medullary expression of the KI allele in the thymus.

[0099] To quantify gene expression in the LPOFGF21KI thymus, we measured Lpo, Fgf21, and mCherry mRNA in FACS-sorted mTECs as well as whole salivary gland, liver, and lung tissue from 1-month-old LPOFGF21and LPOWTlittermate controls. We detected robust expression of Lpo in the salivary glands (maximally expressing tissue predicted by Biogps.org database) and mTEC of both LPOWTand LPOFGF21mice, suggesting KI alleles do not disrupt Lpo expression. Fgf21 was significantly increased and mCherry was robustly expressed in the salivary glands of LPOFGF21mice. These results were confirmed with immunofluorescence microscopy showing robust expression of LPO and mCherry in salivary gland epithelium of LPOFGF21mice. Fgf21 expression was significantly increased, by about 4-fold, in LPOFGF21mTECs compared to LPOWTlittermate controls. In the liver, Fgf21 was expressed at similar levels in LPOWTand LPOFGF21mice, while Lpo and mCherry mRNA were undetectable, as expected. Since we planned to evaluate T cell responses to influenza, we also measured gene expression in lung. Expression of Lpo, Fgf21, and mCherry were not detectable in the lungs of either LPOWTorLPOFGF21mice. mTEC-driven FGF21 overexpression did not increase circulating FGF21 levels, as serum levels of FGF21 did notsignificantly differ between LPOWTand LPOFGF21mice when measured in 1 -month-old and 13- month-old mice. Taken together, these results indicate faithful expression of the Lpo-Fgf21- mCherry knock-in allele in mTECs and in salivary gland.

[0100] FGF21 overexpression delays thymus atrophy. To determine the effects of constitutive mTEC-mediated FGF21 overexpression on thymus size during aging, we compared thymus somatic index (thymus weight:body weight ratio) in LPOWTand LPOFGF21littermates from 1-14 months of age. While thymus size declined in both LPOWTand LPOFGF21mice with age, the thymus somatic index remained significantly increased in LPOFGF21mice relative to age-matched controls. Body weights did not significantly differ across the lifespan between LPOWTand LPOFGF21mice. Thymus cellularity was increased in both male and female LPOFGF21mice at 1, 3, and 12-14 months of age relative to control littermates, and the average thymus cellularity of 12-14-month old LPOFGF21KI mice was similar to that of 3 -month-old LPOWTlittermates in males and females. We next analyzed the thymic CD4 / CD8 T cell subset frequencies and total numbers in 1 -month- and 12-14-month-oldLPOFGF21mice. The relative frequency of each subset (DN, DP, CD4+ SP, CD8+ SP) did not significantly differ between LPOWTand LPOFGF21mice at any age tested. The frequency of cTECs was increased in 1-month-old LPOFGF21mice relative to controls, but was not significantly different in 12-14-month-old mice. 12-14-month-old LPOFGF21mice had increased total numbers of DN, DP and CD4+ SP lymphocytes (CD8+ SP numbers trended up but increase was not statistically significant), as well as increased cTECs and mTECs, highlighting the effect of FGF21 overexpression on preserving the total number of TEC as well as supporting lymphocyte development.

[0101] Kinetics of mTORCl and mT0RC2 signaling in cTECs. Our prior work and that of others suggested mTOR complexes as potential downstream mediators of FGF21 signaling in cTECs, and transcriptional mTOR targets were dynamically regulated in cortical stroma upon castration-induced regeneration of the thymus. To characterize changes in mTOR pathway activation at the protein level, we first investigated changes in mT0RCl / mT0RC2 signaling in cTECs during aging using a flow cytometry approach. We used antibodies targeting p4E-BPl and pAktS473 to measure activation of mTORCl and mT0RC2, respectively, in cTECs, mTECs, and CD45+ lymphocytes from C57BL / 6 mice. We first compared activation of p4E-BPl and pAktS473 in 3 -week-old mice, representing a period of active thymus growth, and 6-month-old mice, representing a period of age-associated atrophy. We observed a significant age- associateddecline in mTORCl as well as mTORC2 activation in cTECs. We also observed a small but significant decline in p4E-BPl and pAktS473 activity in mTECs, but not in CD45+ lymphocytes. The high mTOR activity in young TEC followed by declines in aging are consistent with previous work indicating roles for mTOR signaling in TEC growth. We next evaluated the impact of mTEC- driven FGF21 overexpression on mT0RCl / mT0RC2 activity during aging. At 2.5 weeks of age, when the thymus is actively growing, LPOFGF21 KI cTECs in males and females showed evidence of significantly increased mTORCl, but not mT0RC2 activity, compared to LPOWTcontrols. At 3 months of age, representing a shift from peak growth to a period of atrophy, LPOFGF21mice had significantly increased pAktS473, but not p4E-BPl, activity. This trend continued at 12 months of age, with mT0RC2, but not mTORCl activity significantly increased in cTECs of LPOFGF21. However, in mTECs there appeared to be only dynamic regulation of mTORCl, with increased p4E-BPl activation in mTECs of 2.5-week-old LPOFGF21mice, but not mT0RC2, activity. In CD45+ lymphocytes we observed no effect of LPOFGF21KI allele expression on mTORC l / mT0RC2 activity, suggesting that FGF21 overexpression driven by mTECs signaled primarily in a paracrine manner to cTECs to affect mTORCl and mT0RC2 activation at distinct times during tissue growth and maintenance. The shift from increased mTORCl to increased mT0RC2 signaling during the transition from early thymus growth to later phases is consistent with our previous observation that expression of the mTORCl regulator Tscl is upregulated after the first few days of thymus growth during regeneration. To test whether Tscl expression was similarly upregulated during later phases of postnatal thymic growth, we compared TSC1 expression in cTECs of 3-week-old, 6-week-old, and 4-month-old LPO'Imice by flow cytometry. Consistent with the kinetics of thymus growth during regeneration, we found that TSC1 expression was low at early growth stages, and steadily increased during aging. To further understand the kinetics of mTOR / TSCl signaling in the cortical stroma in the actively growing / regenerated thymus, we utilized a previously published stromal transcriptome database collected during thymic regeneration to evaluate expression of a manually curated set of genes that are targets of transcription factors (TFs) known to be regulated by mTORCl. These included targets of TFs positively regulated by mTORCl, sterol -regulatory element-binding proteins (SREBPs), T'asn and Acaca (AKA AccT) in the same published dataset described above described above. The expression of these genes increases during early stages of thymic regrowth (peaking around day 10 of regeneration), consistent with positive regulation by mTORCl. Also consistent withincreased mTORCl activity during early phases of thymic regrowth, expression of target genes regulated by a TF inhibited by mTORCl, transcription factor EB (TFEB), Sqstml and Lampl, declines during early phases of thymic regrowth. Repression of genes important for lipid oxidation (Sqstml, Lampl), in favor of those involved in lipid biogenesis (Fasn, Acaca), is consistent with the notion that thymic regrowth requires re-extension of cTEC cellular projections, which would require anabolic metabolism increasing lipid incorporation into extending cellular membranes. Fewer transcriptional targets of mT0RC2 signaling have been identified, however, recent work identified several members of the Hedgehog pathway as targets positively regulated at the transcriptional level by mT0RC2 activity. Of those identified in this study, Gli3 was included in our high-confidence cortical stromal gene list. Consistent with the notion that mTORCl activity is increased early during cTEC growth phases, followed by Tscl -mediated down-regulation that would favor mT0RC2 activity promoting cytoskeletal rearrangements and maintenance of cTEC labyrinth morphology, we find that mTORCl -dependent changes in gene expression begin to revert after about day 10 during regeneration. Conversely, we find that expression of the mT0RC2 target Gli3 begins to increase around day 10. Together, these results are consistent with the hypothesis that FGF21 signaling predominantly mediates mTORCl signaling in cTECs during early phases of thymus growth, while mTORC2-mediated signaling predominates at later stages.

[0102] We next examined whether pharmacological mTOR inhibition mitigates the impact of FGF21 overexpression on thymus size, CD4+CD8+ DP thymocyte number, and mTOR activity in cTECs. 3-week-old LPOWTand LPOFGF21littermates were injected with 4 mg / kg rapamycin or vehicle via intraperitoneal injection every other day for 2 weeks, as previously described. After two weeks of treatment, rapamycin induced a significant decline in the thymus somatic index in both LPOW 1and LPOFGF21mice. Total thymus cellularity was also significantly reduced in rapamycin-treated LPOWTand LPOFGF21mice, abrogating the effects of FGF21 -mediated increases in thymus cellularity. We observed a significant decrease in the frequency of CD4+CD8+ DP lymphocytes and a significant increase in the frequency of CD4-CD8- DN lymphocytes of rapamycin treated LPOWTand LPOFGF21mice, consistent with previous studies. In LPOFGF21knock-in, but not LPOWT mice, we also observed a significant increase in the frequency of CD8+ SP lymphocytes. However, when we compared the total cell number of each of these thymocyte subsets, rapamycin only induced a significant decrease in the total number of CD4+CD8+ DP lymphocytes, and did so in both LPOWTand LPOFGF21mice.

[0103] The total number of DP lymphocytes were reduced to similar levels in LPOWTand LPOFGF21rapamycin treated mice, despite LPOFGF21vehicle-treated mice having a significantly higher number of DP lymphocytes compared to LPOWTvehicle-treated mice. We also observed a decreasing trend in the total number of CD4+ SP lymphocytes in rapamycin-treated LPOFGF21mice, which may reflect increased autophagy following relief of mTORCl inhibition, leading to more MHCII-mediated negative selection of CD4+ SP lymphocytes relative to CD8+ SP lymphocytes.

[0104] When we compared mT0RCl / mT0RC2 signaling in cTECs, mTECs, and lymphocytes in vehicle-treated and rapamycin-treated LPOWTand LPOFGF21mice, we found that rapamycin treatment induced a significant decrease in pS6 (indicator of mTORCl activity) signaling in cTECs, mTECs, and lymphocytes of LPOFGF21mice. Rapamycin treatment promoted a trend toward decreased pS6 activity in cTECs of LPOWTmice but was not found to be statistically significant, while pS6 activity significantly declined in mTECs and CD45+ lymphocytes of LPOWTmice. pAktS473 only decreased in rapamycin-treated cTECs of LPOFGF21mice. Rapamycin treatment has direct negative effects on DP thymocyte survival, but our data are nonetheless consistent with the hypothesis that the effects of FGF21 overexpression on thymus size are mediated through mTOR pathway activation in TECs.

[0105] FGF21 overexpression in mTECs improves T cell responses to influenza. Based upon the observation that FGF21 overexpression boosted thymus size during aging, we next evaluated peripheral changes induced by mTEC-driven FGF21 overexpression. Thymus decline is associated with a decrease in peripheral naive T cells and a homeostatic expansion of memory T cells, predisposing older individuals to new viral infections including influenza and others.

[0106] We first compared the ratio of naive (CD62L+, CD44-): memory (CD62L+, CD44+) CD8+ T cells in the spleens of 2-month-old and 12-month-old LPOWTand LPOFGF21KI mice. While we found no significant difference in the CD8+ naive:memory ratio of 2-month-old mice, we found a significant increase in 12-month-old LPOFGF21mice compared to LPOWTcontrols, suggesting improved export of naive T cells into the periphery of aged LPOFGF21mice. We also found similar increases in the naive:memory ratio of CD4+ splenocytes in aged LPOFGF21mice, consistent with increased export of thymic CD4+ SP T cells seen during aging.

[0107] One well-characterized example of declining T cell immunity in aging is the development of “holes” in the TCR repertoire of T cells specific for flu epitopes. In aged mice, Tcells recognizing the immunodominant flu epitope designated NP366-374 / Db, but not the immunodominant epitope PA224- 233 / Db, significantly decrease in the bronchial alveolar lavage (BAL) 10 days after infection with influenza virus. Generation of such “holes” in the T cell response is thought to be a function of declining thymic output, and such declines in T cell responsiveness have been shown to be improved upon thymus regeneration. To test the functional capacity of peripheral T cells in LPOFGF21mice, we challenged 12-month-old LPOWTand LPOFGF21mice with 103PFU of influenza A (IAV) X31 via intranasal infection, and monitored changes in body weight over the course of ten days. X31 -infected LPOFGF21mice lost significantly less weight relative to control littermates. Similarly, when older 18-month-old LPOFGF21mice were challenged with IAV PR8 (103PFU), we observed a significant reduction in the amount of body weight lost compared to PR8-infected age-matched LPOWTcontrols at 7 dpi, suggesting improved control of infection. Because short-term stressful stimuli such as infection induce rapid thymic atrophy, we compared total thymus cellularity at 10 dpi. We found that infected aged LPOFGF21mice had significantly greater thymus cellularity than infected LPOWTcontrols. Infected LPOFGF21mice had significantly decreased influenza mRNA in the lung compared to infected LPOWTmice at 10 dpi, suggesting improved viral clearance. At 10 dpi bronchi olar lavage (BAL), spleen, and mediastinal lymph nodes (MLN) were also harvested to measure the frequency and total number of influenzaspecific CD8+ T cells via NP366-374 tetramer staining, as previously described.

[0108] Both the frequency and total number of NP-specific CD8+ T cells were significantly increased in the BAL of infected LPOFGF21mice relative to controls, while no difference was observed in the spleen or MLNs of infected mice. These results suggest that FGF21 overexpression increases the number of influenza-specific T cells available to respond to infection in the BAL. Taken together, these observations suggest that FGF21 overexpression in mTECs mitigates waning naive T cell frequency and T cell responsiveness to influenza infection in older mice.

[0109] FGF21 overexpression in mTECs mitigates age-associated impairment of T cell tolerance With the observation that FGF21 overexpression improved the age-associated decline in naive T cells and improved outcomes to influenza infection, we next evaluated age-associated declines in clonal deletion among signaled (CD5+ TCR0+) thymocytes using flow cytometry. We compared both early clonal deletion, occurring among CCR7- signaled thymocytes, and later clonal deletion occurring among CCR7+ thymocytes, in 2-month-old and 12-month-old LPOWT andLPOFGF21 / FGF21homozygous KI mice. As we and others have previously reported we observedan age-associated decline in the frequency of both early and late clonally deleted (cleaved caspase 3+) thymocytes in LPOWTmice. Clonal deletion of early and late signaled thymocytes were both significantly higher in the LPOFGF21mice at both timepoints compared to LPOWTcontrols. Surprisingly, while we observed an age-associated decline in the clonal deletion of late (CCR7+) thymocytes in LPOFGF21mice (though this was less pronounced than the decline in LPOWTmice), early (CCR7 ) clonal deletion was preserved at the level of young mice in 12-month-old LPOFGF21mice, suggesting potential indirect effects of FGF21-mediated paracrine signaling to preserve earlier stages of clonal deletion.

[0110] We next evaluated 12-month-old LPOFGF21mice for the presence of indicators of peripheral autoimmunity. LPOFGF21mice had a lower frequency of antinuclear antibodies (ANAs) in the serum, with only 25% of KI animals testing positive for the presence of antinuclear IgG (compared to 75% of LPOWTanimals). We further investigated the impact of FGF21 overexpression on development of peripheral autoimmunity by evaluating lung, liver, and salivary gland tissue from 12-month-old LPOFGF21mice for the presence of inflammatory lymphocytic infiltrates (defined as >20 lymphocytes per foci). LPOFGF21mice had decreased incidence of lymphocytic infiltrates in the liver and salivary gland, while incidence of infiltrates in the lung generally did not differ from LPOWTmice. These results, together with our peripheral naive T cell and infection data, support the conclusion that mTEC-derived FGF21 overexpression improves thymus function in older mice to preserve T cell output while maintaining negative selection of autoreactive T cells during aging.

[0111] Paracrine FGF21 signaling increases cTEC size during aging. cTECs form networks of finely branched processes which create the structural niches required for T lymphocyte development and limit the overall size of the thymus. We have previously demonstrated that aging is associated with a marked contraction of the cortical compartment, predominantly resulting from contraction of cTEC size and cortical projections. To address whether paracrine FGF21 signaling to cTECs would protect cell size during aging, we compared cTEC size and morphology between 1 -month-old and 6-month-old LPOWTand LPOFGF21KI mice crossed with FoxNICre R26Confetti reporter mice, which allows for labeling of individual TEC. Qualitative analysis of cTEC morphology in 3D projections from 60 pm optical stacks revealed a significant contracture of cTEC cell size in 6-month-old LPOWT mice, with the most striking changes occurring in the subcapsular zone as we have previously reported. In contrast, cTECs in 6-month-old LPOFGF21mice retained more of their labyrinth morphology, more closely resembling that seen in 1 -month- old mice. Quantification of cTEC cell area revealed that while aging induced a significant reduction in average cTEC size in 6-month-old FoxNICre R26Confetti LPOWTthymi, age- matched FoxNICre R26Confetti LPOFG1,21thymi were protected from decreases in cTEC size. These results, in agreement with our observed increased activation of pAktS473 in cTECs of aged LP0FOF21KI mice, indicate a potential role for FGF21-mediated activation of mT0RC2 in maintenance of cTEC cell morphology and overall size.A. Materials and Methods

[0112] Mice. C57BL / 6 (JAX: 000664) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) at 4-6 weeks of age and subsequently used as breeders. Offspring were subsequently used for experiments between 3 weeks-12 months of age. Sanger sequence-verified LPOFGF21knock-in founders were paired with C57BL / 6 mice as breeders to generate LPOFGF21 / WTheterozygous or LPOFGF21 / FGF21homozygous knock-in (KI) mice for experiments. Non-KI littermate controls were used as WT controls. Mouse genotypes were determined using real time PCR with specific probes designed for the LPOFUF21knock-in mutation (Transnetyx, Cordova, TN). Male and female LPOWTand LPOFGF21KI mice between 2.5 weeks-14 months of age were used for experiments. All mice used in experiments were bred and maintained at The University of Texas Health Science Center at San Antonio animal facility under specific pathogen free conditions. Animal studies and all procedures were approved by the Institutional Animal Care and Use Committee.

[0113] knock-in mice. Guide RNAs targeting Lpo gene stop codon atExon 13 was identified utilizing CRISPOR.Tefor.net guide RNA search tool. Double Strand Break (DSB) of the crRNA, ACCATGGACATGTTCGATGT (SEQ ID NO:2), was located 29bp from the foreign DNA Knock-In locus before the stop codon. The plasmid donor DNA was synthesized from GenScript (Piscataway, NJ); 1,443 base pair foreign DNA composed of T2A-mCherry-P2A- Fgf21 cDNA, flanked with 700bp 5’ Homology Arm and 702bp 3’ Homology Arm was cloned into pUC-57 backbone plasmid. PAM sequence was modified from AGG to AGA in donor DNA to silence Cas9 activity in it. sgRNAs (32ng / pl), donor DNA (15ng / pl), and eSpCas9 Nuclease (50ng / pl) were mixed in injection buffer (lOmM Tris-NaCl, 0.25mM EDTA), and incubated at room temperature for 20 minutes before zygote microinjection session. sgRNAs were synthesizedfrom Synthego (sgRNA with chemical modification), and eSpCas9 was purchased from Millipore- Sigma. PCR verification was performed to confirm successful knock-in mutation insertion in Sanger sequence-verified founders.

[0114] Quantification ofFgf21 expression in TEC subsets. Fgf21 expression was quantified in 9 TEC populations described in a previously published single-cell transcriptome profiling dataset (ArrayExpress, E-MTAB-8560)25. scRNA-seq data obtained from the dataset (relative Fgf21 expression, TEC population frequencies from 1-52 weeks) were analyzed using the Seurat package in R.

[0115] Stromal cell isolation. For thymic stromal cell isolation, thymi were harvested and minced into small pieces with scissors. Fragments were passed several times through a Pl 000 pipette. Thymus pieces were allowed to settle, after which the supernatant containing lymphoid cells was removed. The remaining fragments were digested with 0.125% collagenase D (Roche) for 10 minutes at 37°C. Three additional rounds of mechanical mixing, sedimentation, and removing lymphoid cells were performed, followed by a final digestion in 0.05% trypsin (Sigma). After the final digestion, cells were washed and pelleted by centrifugation, then resuspended in FACS buffer (HBSS, 5% FBS, 0.05% DNAse) containing a cocktail of fluorescent antibodies recognizing Alexa 700-conjugated anti-CD45.2 (Ly-5.2) (Clone 104; Biolegend), PECy-7- conjugated anti-EpCAM (CD326) (Clone G8.8; Biolegend), and biotinylated anti23 Ly-51 (Clone 6C3; Biolegend) / PerCPCy5.5-conjugated streptavidin (Biolegend). For dendritic cell isolation, after the final digestion, all the collected lymphoid cells and stromal cells were combined in one tube, washed, pelleted by centrifugation, and then stained with PerCPCy5.5-conjugated anti- CD45.2 (Clone 30-F-l l; eBioscience), APC-conjugated anti-CDl lc (Clone HL3; BD Pharmingen), FITC-conjugated anti-B220 (Clone RA3-6B2; Biolegend), and PECy7-conjugated anti-EpCAM. For endothelial and mesenchymal (pericyte, fibroblast) cell isolation, stromal cells were stained with biotin labeled anti-PDGFRa and anti-PDGFRb (Clones APA5, APB5; Biolegend), followed by incubation with a cocktail containing BV650-conjugated Streptavidin (Biolegend), FITC-conjugated anti-CD31 (Clone W18222B; Biolegend), APC-conjugated CD 146 (Clone ME-9F1; Biolegend), PE-conjugated gp38 (Clone 8.1.1; Biolegend), Alexa 700-conjugated CD45.2, and PE-Cy7-conjugated anti-CD326. Dead and / or dying cells were excluded by DAPI staining. For intracellular flow cytometry experiments, cells were stained with Zombie Violet viability dye (Biolegend #423113), then subsequently fixed and permeabilized using BDCytofix / Cytoperm kit. Fixed and permeabilized cells were then stained with a cocktail of fluorescent antibodies recognizing APC-conjugated anti-Phospho-AktS473 (Cell Signaling Technology #11962), PE-conjugated anti-Phospho-4EBPlT36 / 45 (Clone V3NTY24; eBioscience), and PE-conjugated anti-Phospho-S6S235 / 236 (Clone cupk43k; eBioscience). To measure TSC1 expression, fixed and permeabilized cells were incubated with pure anti-TSCl (Clone A6F1; Invitrogen), followed by secondary staining with FITC-conjugated Goat AntiMouse IgG (Jackson #115-095-146). Flow cytometry analysis was performed on the Cytek Aurora spectral flow cytometer. Experiments were analyzed using FlowJo software (vl0.9).

[0116] Thymocyte subset isolation. Thymocyte CD4 / CD8 populations were stained by preparing single-cell suspensions from whole thymi crushed against a mesh filter and subsequently filtered. 5 million cells per sample were stained with a cocktail of fluorescent antibodies containing APC-Cy7-conjugated anti-CD4 (Clone 6K1.5; 588 Biolegend) and BUV 395-conjugated anti-CD8 (Clone 53-6.7; BD Biosciences). DAPI staining was used to exclude dead / dying cells. Flow cytometry analysis was performed on the Cytek Aurora spectral flow cytometer. Experiments were analyzed using FlowJo software (vl0.9).

[0117] Gene expression qRT-PCR. Following enrichment of mTECs with collagenase as described above, single-cell suspensions were stained with FITC-conjugated anti-CD45.2 (Clone 104; Biolegend), PE-Cy7-conjugated anti-EpCAM, A647-conjugated anti-Ly51 (Clone 6C3; Biolegend). CD45-, EpCAM+, Ly51- mTECs were subsequently sorted using a BD FACS Aria Fusion cell sorter (BD Biosciences) using the single-cell sorting mode. qRT-PCR was performed as previously described. Briefly, RNA was isolated using the RNAqueousa Micro Kit for sorted mTEC samples (Invitrogen, #AM1931) or the E.Z.N.A.R HP Total RNA Kit (Omega, #R6812) for homogenized whole salivary, liver, or lung tissues per manufacturer’s instructions. cDNA synthesis was performed using a SuperScript VILO cDNA synthesis kit (Invitrogen) per manufacturer’s instructions. cDNA preamplification was performed using the TaqMan PreAmp Master Mix Kit (catalog no. 4384267, ThermoFisher Scientific) per manufacturer’s instructions. Quantitative PCR (cycle 1 : 95°C for 10 min; cycle 2, x40: 95°C for 15 s and 60°C for 1 min) was performed using a BioRad CFX96 Real-Time System / ClOOO Touch Thermal Cycler using presynthesized FAM-MGB TaqMan Gene Expression Assay Probes (ThermoFisher Scientific) to amplify the following genes: Lpo (Assay identification [ID]: Mm00475466_ml), Fgf21 (Assay ID: Mm07297622_gl), mCherry (Assay ID: Mr07319438_mr), and pan_Influenza A (AssayID:Vi99990011 _po). qPCR results were analyzed using Bio-Rad CFX Manager software. Cq values were normalized to Hprt (2-ACq).

[0118] Serum analysis. Analysis of serum FGF21 was performed using the Mouse / Rat FGF- 21 Quantikine ELISA Kit (catalog no. MF2100, R&D Systems) according to the manufacturer’s recommendations. An Agilent Bio-Tek Synergy LX plate reader was used to obtain the optical density (O.D.) set to 450 nm. Wavelength correction was applied by subtracting readings at 540 nm. A standard curve was created using provided standards and subsequently used to determine the relative 614 concentration of FGF21 (pg / mL) in each sample.

[0119] Confocal Immunofluorescence Imaging. To obtain LPO costaining images in C57BL / 6 mice, fresh frozen thymic sections from 3-12-month-old mice were fixed in acetone followed by co-staining with anti-LPO (Proteintech #10376-l-AP) / Goat anti-Rabbit Cy5 (Thermo #A10523) and anti-EpCAM A488 (Clone G8.8; Biolegend). To detect mCherry colocalization in LPOFGF21 mice, thymi from 1 -month-old mice were fixed overnight in 2% paraformaldehyde while rocking at 4°C, followed by a 10-20% sucrose gradient overnight. 10 pm sections were cut on a cryostat and blocked with 5% FBS in PBS prior to costaining with anti-Ly51 A647 (Clone 6C3; Biolegend), anti-DEC 205 A647 (Clone NLDC-45; Biolegend), anti-cytokeratin 8 A488 (Clone EPl 628 Y; Abeam); anti-cytokeratin 5 A647 (Clone EP1601Y; Abeam), anti- Aire A647 (Clone MM-525; BD Biosciences), and UEA1 FITC (Thermo L32476). Sections were stained for 1 hr at room temperature, then washed and coverslipped with Prolong Diamond Antifade Mountant (Thermo #P36961). All images were captured using a Zeiss LSM710 confocal microscope. Endogenous mCherry signal was captured using a 561 nm HeNel laser. DAPI was excited using the 405 nm diode laser, A488 signal was detected using a 488 nm argon laser, and A647 signal was captured using a 633 nm HeNe2 laser. Images were captured at 10X and 20X. Identical laser settings were used to capture images from LPOWTandLPOFGF21tissues. Images were subsequently processed using ImageJ software.

[0120] Confetti cTEC image analysis. Thymic sections from LPOWTand LPOFGF21mice crossed with FoxNICre R26Confetti reporter mice were imaged using a three-stage sequential scan, as previously described. This method allows for maximum detection of three Confetti fluorescent proteins (cytoplasmic YEP and RFP, and membrane CFP) and simultaneous capture of age-associated autofluorescent pigments that can be removed from reporter images. Briefly, the first scan used 405 nm and 458 nm lasers to excite both CFP and autofluorescent pigments, withcollection windows of 463-639 509 nm (for CFP) and 566-628 nm (for autofluorescent pigments). The second scan used a 514 nm laser to excite YFP with a collection window of 519-556 nm, and the third scan used a 561 nm laser to excite RFP with a collection window of 566-628 nm. 20x Z- Stacks were acquired on a Zeiss LSM710 confocal microscope. For full color displays, CFP is presented as blue, YFP as yellow, and RFP as red. For qualitative analysis of cTEC morphology, 3D maximum projections were created in ImageJ with a 0.2 pixel minimum filter using the “3D Project” tool. Following acquisition, cTEC area quantification in this study was performed blinded using ImageJ software. Z-Stack images were processed prior to cTEC area quantification using the Stacks -> Z Project -> Average Intensity function. Z-Stack images were then separated into the 3 acquired reporter channels (RFP, YFP, CFP) and the channel capturing autofluorescent pigments, which was removed from the composite image. Channel signal brightness / contrast were optimized for each image using the Image -> Adjust -> Color balance tool. After signals among the 3 channels were equalized within the image, individual cells (defined by any single color surrounded by cells of other colors) contained wholly within the image volume were identified for quantification. cTEC cell area from freehand selections was measured via the Analyze -> Measure tool. The criteria for selection excluded cell areas <400 pm2or >3000 pm2. At least 10-20 cTECs were measured per Z655 Stack for a total of 20-40 cTECs quantified per individual mouse. Final quantitation represents 1-2 distinct Z-Stacks from at least 3 independent sex-matched thymuses per group at each age.

[0121] Rapamycin-mediated mTOR inhibition. At the time of weaning, 3-week-old LPOWTand LPOFGF21KI mice were injected with 4 mg / kg rapamycin (Thermo Scientific # J62473.EX3) or vehicle via intraperitoneal injection every other day for 2 weeks as previously described. At the time of euthanasia, the thymus was harvested, and mT0RCl / mT0RC2 activation was measured in cTECs, mTECs, and lymphocytes via intracellular flow cytometry as described above. Mouse thymus / body weights and total thymus cellularity via hemocytometer were also determined. Thymocyte CD4 / CD8 subsets were also separately stained as described above.

[0122] Influenza infection. 12-month-old LPOWTand LPOFGF21KI mice were transferred to a BSL-2 facility and allowed to habituate overnight prior to infection. The following day mice were anesthetized with light isoflurane anesthesia and 1000 PFU of mouse-adapted H3N2 influenza (strain A / X-31 H3N2) or H1N1 (strain A / PR8) suspended in 50 pL PBS was intranasally administered to the left nare. Mouse body weights were recorded daily until 10 days post-infection.Bronchoalveolar lavage (BAL) and analysis of influenza-specific T cells. Upon euthanasia of influenza-infected and / or naive control mice, the trachea was exposed and a 2-inch-long (75% polyester, 25% cotton) thread was passed underneath the trachea using curved tweezers. An 18- gauge Insyte™ Autoguard™ shielded I.V. catheter (BD) was inserted into the upper trachea and secured using the thread. Brochoalveolar lavage (BAL) was removed by injecting and aspirating 1.0 mL of PBS using a 1 mL PP / PE syringe (Sigma-Aldrich). BAL was placed in a separate tube on ice and the injection and aspiration procedure was repeated another time. Cells were centrifuged (440 RCF, 5 mins, 4°C), and cells were resuspended in ice-cold FACS buffer (HBSS, 5% FBS, 0.5% DNAse (1 mg / mL), pH 7.2). Dasatinib (Tocris Bioscience) was added to the cells at a concentration of 50 nM and cells were incubated 30 minutes at 37°C. After incubation, cells were cooled on ice for 5 minutes. Fc receptors were blocked with CD16 / CD32 (eBioscience; # 14-0161- 81) for 10 minutes on ice. Cells were then stained with 0.5 pg of NP366-374 / Db (NP) tetramers and incubated protected from light for 1 hour at 25°C. Cells were then washed with sorting buffer and then stained with a cocktail containing fluorescent antibodies conjugated against CD8-V500 (Clone 53-6.7; BD Biosciences), CD4-FITC (Clone RM4-4; Invitrogen), CD90.2-PerCPeFluor710 (Clone 30-H12; Invitrogen), and CD44-BV510 (Clone IM7; Biolegend) on ice for 30 minutes. Cells were washed and suspended in sorting buffer for FACS analysis. NP-tetramer+ cells were analyzed on CD90.2+CD8+ singlet lymphocytes using a Cytek Aurora flow cytometer (Cytek) and FlowJo (BD Biosciences) flow cytometry analysis software.

[0123] Antinuclear antibody evaluation. The presence of ANAs was evaluated in serum of 10- 12-month-old LPOWT and LPOFGF21 mice. 25 pL serum (1: 100 dilution) was added to HEp-2 slides (MBL International, AN-1012) and incubated at room temperature for 30 minutes. Slides were washed twice and then sample wells were incubated with FITC-conjugated anti-IgG (poly4060, Biolegend) applied at 1 :100 dilution for 30 minutes. Slides were washed and coverslipped with Vectashield Vibrance with DAPI (Vector Laboratories). Images were captured at 10X and 20X magnification using a Zeiss LSM710 microscope. Samples were coded and evaluation was performed blinded. Laser settings for the FITC channel were kept identical between each sample.

[0124] Tissue lymphocytic infiltration studies. Lung, liver, and salivary gland tissue harvested from 10-12-month-old LPOWT and LPOFGF21 mice were fixed for 24 hours in 10% NBF and blocked in paraffin. Slides were cut and stained with H&E. Sections from at least three differentanatomic areas were examined for lymphocytic infiltration in each tissue. Samples were coded and evaluated for the presence of lymphocytic infiltrates blinded.

[0125] Statistics. P-values were calculated in GraphPad Prism 10 software (La Jolla, California) using two-tailed unpaired Student’s t-test, ordinary one way-ANOVA (analysis of variance), or two way-ANOVA. Tukey’s multiple comparisons test was performed following significant one way- and two way-ANOVA tests. P values < 0.05 determined data to be statistically significant, and significance is indicated in figure legends.EXAMPLE 3IMMUNE CHECKPOINT INHIBITION (ICI) THERAPY

[0126] Data shows that the mCat Tg mice may respond better to immune checkpoint inhibition. Although it holds great potential, immune checkpoint inhibition (ICI) therapy is currently effective in a minority of cancer patients, including melanoma patients. Most ICI approaches target activation or reactivation of anti-tumor T cell responses. However, by middle age and beyond, the ages at which most patients receive ICI, the diversity of antigens that the T cell population can recognize is already significantly diminished relative to that of young adults. Although published studies show mixed results regarding the effect of age on ICI, it is important to note that in general these studies compare adult to elderly samples, and since the thymus is already significantly atrophied by adult stages, the impact of thymus size on ICI could not accurately be inferred from these studies. Here, we propose comparing responses in mice at the peak of thymus activity (just after puberty, around 4 weeks of age) to adults at 12 months of age, which is comparable to middle age in humans. The overarching hypothesis of this application is that low T cell receptor diversity in adults is the cause of low ICI efficacy, and that responsiveness can be improved by increasing T cell diversity via thymus regeneration. Notably, relatively low diversity of T cells in adults is further exacerbated in adults who have undergone cytoablative therapy, which damages thymic stromal cells (TSCs) and impairs T cell development.

[0127] Previous studies focused on thymus atrophy revealed that one factor contributing to early atrophy of the thymus was that thymic stromal cells express conspicuously low levels of the hydrogen peroxide quenching enzyme catalase, and this causes TSCs to rapidly accumulate oxidative damage relatively early in life. We found that genetic complementation of catalase activity (in transgenic mice overexpressing a mitochondrially-targeted human catalase under thecontrol of a ubiquitous promoter, mCat Tg delays thymic atrophy, and mitigates a well- characterized age-associate “hole” in the TCR repertoire that develops with age, which is loss of T cells recognizing particular immunodominant influenza epitopes. Because mice with regenerated thymi were unavailable, as a surrogate, we compared a-PD-Ll+ T cell responses to B16 tumors in mice just past puberty (1 month) to adult mice approaching middle age (8 months) on either a WT C57BL6 background (B6, WT) or in congenic B6 mice bearing the mCat transgene (mCat Tg). We predicted that the larger thymus in 8-month-old mCat Tg mice would expand the breadth of the TCR repertoire and increase the number of tumor-specific T cells, similar to the impact of mCat Tg expression on flu-specific T cells. Although statistically significant differences were generally not found in our small pilot study, the data suggest that 8-month-old adult WT mice show significantly diminished responsiveness to oc-PD-Ll treatment relative to their 1 -month-old (peak thymus size) counterparts. Adult mCat Tg mice trended toward improved responses to oc- PD-L1 treatment relative to WT controls. These results are consistent with trends toward age- associated decreases in T cell frequency in tumors in adult WT mice relative to 1-month-olds, and with trends toward increases in age-matched adult mCat Tg tumors relative to WT controls. We used PD1 expression as a proxy for tumor-specific T cells, and likewise saw a trend toward decreases in PD1+ T cell frequency in tumors in adult WT mice relative to 1-month-olds, with trends toward increases in age-matched adult mCat Tg tumors relative to WT controls. These results are consistent with the hypothesis that increasing thymus size and output in adult mice could increase ICI efficacy. Although we did not see statistically significant differences in most cases, it is important to note that our ability to detect differences was hindered by the low number of mice available for these studies.

[0128] Impact of Increased Thymus Size on ICB Efficacy in LPOFGF21 Model. We reasoned that the narrowed TCR repertoire in older animals may limit Immune Checkpoint Blockade (ICB) efficacy. Although the clinical literature is mixed, mouse studies have demonstrated decreased ICB efficacy in aged animals. Notably, most clinical literature compares outcomes in adults over 50, when thymus atrophy is already quite marked, with subjects over 75 years old, possibly masking the impact of thymus function on outcomes. We challenged young WT (1 -month-old, black) and older (12-month-old) WT (red) and LPOFGF21 (blue) mice with B16 melanoma cells, began treatment with anti-PD-Ll (filled symbols) or isotype control (open symbols) when tumors reached ~300mm3. Tumor-infiltrating T cells were evaluated on Day 14 (FIG. 7), and we foundthat while aged WT mice showed significantly faster tumor growth relative to young WT mice prior to ICB treatment (to day 5), and that tumor control prior to treatment was significantly improved in aged LPOFGF21 mice relative to age-matched controls. In addition, ICB was less effective in aged WT mice (where tumors continued to grow, red) relative to young (where tumors shrank, black), and aged LPOFGF21 mice showed improved responses (where tumors stopped growing, blue) relative to age-matched controls. When we examined tumor-infiltrating T cells, we found that aged WT mice had decreased frequencies of CD8+ T cells in the tumor relative to young mice (FIG. 7, bottom left), and this decrease was rescued in aged LPOFGF21 mice, but not WT mice after ICB. Similarly, using PD-1 expression as a proxy for tumor-specific T cells, we found that aged WT mice had decreased frequencies of PD-1+ cells among CD8+ T cells relative to young mice (FIG. 7, bottom middle), and this decrease was rescued in aged LPOFGF21 mice, but not WT mice after ICB. Moreover, using CD101 expression as a marker of exhausted T cells (Tex), we found that aged WT mice had increased frequencies of CDIOI+Tex cells among PD-1+CD8+ T cells relative to young mice (FIG. 7, bottom right), and this increase was rescued in aged LPOFGF21 mice, but not WT mice after ICB, all consistent with improved anti -tumor T cell responsiveness. Though these data are from the LPOFGF21 model, they support the feasibility that increased thymus size could impact anti-tumor responses.

[0129] To test the hypothesis that thymus regeneration will improve a-PDLl efficacy in B16 melanoma tumor-bearing adult mice, (a) At 10 months of age, B6 mice will begin treatment with exogenous FGF21 as described in methods below. At 12 months of age, FGF21 treatment will end, and mice will be challenged with 2.5-3 X IO5B16F10 melanoma cells combined with Matrigel (Corning, 5 mg / ml) injected subcutaneously (s.c.). Tumor volumes will be estimated by measuring length, width, and height using a caliper. When tumors reach ~150 mm3we will begin treatment with a-PD-Ll antibody (or isotype control, 200 pg / mouse every 3 days). We will measure tumor volume and weight, and survival. Mice will be euthanized when the estimated tumor volume reaches 1000 mm3, (b) When mice are euthanized, we will collect the tumor-draining lymph node, spleen, and blood and generate single cell suspensions from each. We will also measure the size of the thymus at euthanasia. Since T helper 2 (Th2), and regulatory CD4 T (Treg), but not Thl responses have been shown to be important for a-PD-Ll and oc-TGFP ICI, we will evaluate the number, frequency, and function of effector and regulatory CD4 T cells using a-CD3 and: Thl : CD4, Tbet, IFNy; Th2: CD4, GATA3, IL-4; Treg: CD4, CD25, FoxP3 with viability dye. Sincestem-like CD8 T cells are primary targets of a-PD-Ll ICI, a-TGFP ICI and combination ICI, we will evaluate their number, frequency and function, along with that of effector and exhausted T cells using a-CD8 and: stem-like: TCF-1, PD1, CD44, Slamf6; Tefr: IFNy, TNF, Granzymes A and B; Tex: CD101 antibodies with viability dye. For cytokine stains cells will then be fixed and permeabilized with Perm Wash Buffer (Biolegend), stained with a-IFN-y, a-TNF, a-Granzyme A and B and be examined by flow cytometry.

[0130] To test the hypothesis that thymus regeneration will improve combination a-PDLl and oc-TGFP efficacy in B16 melanoma tumor-bearing mice, (a) We will compare tumor growth and regression in three additional groups of adult B6 mice that receive a-TGFP treatment (10 mg / kg every 3 days) alone, in combination with thymus regeneration, or in combination with both thymus regeneration and a-PD-Ll treatment, (b) We will phenotype T cells in tissues and blood.B. Methods

[0131] FGF21 (0.25 mg / kg) or vehicle control will first be administered to 12-month-old wild type C57B6 / J mice i.p. At least 20 C57BL / 6J mice (both sexes) will be used per group. Using both sexes is particularly important because most regeneration experiments, and clinical trials, have only included males. Therefore, this study will provide important pre-clinical data regarding efficacy in females. Flow cytometry will be performed. 4-week-old and 10-month old mice are available for purchase from Jackson Laboratories (#000664), so experiments can begin immediately, allowing completion within the grant timeframe. The estimated number of replicates per group was calculated by power analyses based on coefficients of variation in published results and our preliminary data, powered to detect at least 30% difference between groups with confidence levels alpha=0.05 and beta=0.02. Statistical significance will be tested using one-way ANOVA with Tukey’s multiple comparison correction. Experiments will be repeated at least 2-3 times.EXAMPLE 4C OMBIN ALIGN T RE ATMENT

[0132] Mice from 5 to 10 weeks of age have been treated with Rilmenidine (i.p. injections), NAC (in drinking water), and FGF21 (genetic overexpression in the thymus in knock-in mice) andfound that all three agents together delay thymus atrophy more effectively that NAC and Rilmenidine together (FIG. 8).

[0133] Mice in each of the three groups (n=3) were treated with either 10 mg / kg body weight Rilmenidine or vehicle alone by i.p. injection four times per week (Rilm. / Untreated), and received NAC treated (5mg / mL) or untreated water, as indicated. Treatment began at 5 weeks of age and continued to 10 weeks of age, by which time the thymus has lost approximately 50% of its peak cellularity. Treatment with NAC + Rilmenidine resulted in a small increase in thymus cellularity in WT mice, but the increase was not statistically significant. However, the combination of NAC + Rilm. and overexpression of FGF21 in FGF21KI mice resulted in a statistically significant (-50%) increase in thymus cellularity relative to untreated controls. These results suggest that the combination of FGF21, NAC, and Rilmenidine could significantly delay thymus atrophy and age- associated dysfunction.

[0134] In addition to delay of atrophy, the combination of NAC + Rilmenidine has been shown to reverse established atrophy, as detailed in Example 1, with increases in thymus size, naive CD8 T cell frequency, and maintenance of clonal deletion (FIGs. 6 and 9).

Claims

CLAIMS1. A method for thymus regeneration or amelioration of thymic involution in a subject, comprising administering to the subject a therapeutically effective amount of one or more therapeutic agent selected from (i) an autophagy activating agent, (ii) a TORC1 activator or a mT0RC2 activator or a FGFRlc (Fibroblast Growth Factor Receptor 1c) agonist, and / or (iii) an antioxidant.

2. The method of claim 1, wherein the autophagy activating agent is rilmenidine or an analog thereof.

3. The method of claim 2, wherein the analog of rilmenidine is selected from moxonidine, clonidine, rapamycin, PP242, Torin 1, metformin, ABT-737, Xestospongin B, L-NAME, PI-103 hydrochloride, lithium (L-690330), carbamazepine, resveratrol, verapamil, erlotinib hydrochloride, sodium valproate, spermidine, vinblastine, nocodazole, bafilomycin Al, chloroquine, hydroxychloroquine, and spautin-1.

4. The method of any one of claims 1-3, wherein the TORC1 activator or a mT0RC2 activator or FGFRlc (Fibroblast Growth Factor Receptor 1c) agonist agent is FGF21 or an analog thereof.

5. The method of claim 4, wherein the analog of FGF21 is selected from LY2405319 (PF- 05231023), AKR-001, pegbelfermin (BMS-986036), FGF19, FGF23, TGFp, insulin, IGF-1, phosphatidic acid, sphingosine 1 -phosphate, metformin, troglitazone, pioglitazone, rosiglitazone, resveratrol, quercetin, genistein, epigallocatechin gallate, berberine, curcumin, ginsenoside Rbl, a-lipoic acid, cryptotanshinone, and SC-79.

6. The method of any one of claims 1-5, wherein the antioxidant agent is selected from Vitamin C (Ascorbic Acid), Vitamin E (Tocopherols and Tocotrienols), Beta-Carotene, Selenium, Glutathione, Coenzyme Q10 (CoQlO), Alpha-Lipoic Acid, Resveratrol, Curcumin, Polyphenols, Flavonoids, quercetin, catechins, Anthocyanins, and N-Acetylcysteine (NAC).

7. The method of any one of claims 1-6, further comprising administering the agents in a sequence or simultaneously, independently or in a co-formulation.

8. The method of any one of claims 1-7, wherein the administration is via intravenous injection or intrathymic administration.

9. The method of any one of claims 1-8, wherein one or more therapeutic agent is in a sustained release composition.

10. The method of any one of claims 1-9, wherein the dosage regimen for each agent independently is from 5 to 200 mg daily, weekly or monthly.

11. The method of any one of claims 1-10, wherein the subject is a mammal having or at risk of having thymic atrophy, an aging mammal, a mammal at risk of exposure to or in an environment known to harbor a pathogen, or a subject at risk or diagnosed with cancer.

12. The method of any one of claims 1-11, wherein the thymus regeneration is measured by an increase in T-cell receptor excision circles (TRECs) or thymic epithelial space.

13. A pharmaceutical composition for thymus regeneration or amelioration of thymic involution, comprising (i) an autophagy activating agent, (ii) a TORC1 activator or a mT0RC2 activator or a FGFRlc (Fibroblast Growth Factor Receptor 1c) agonist, and / or (iii) an antioxidant, and a pharmaceutically acceptable carrier.

14. The composition of claim 13, wherein the (i) an autophagy activating agent, (ii) a TORC1 activator or a mT0RC2 activator or a FGFRlc (Fibroblast Growth Factor Receptor 1c) agonist, and / or (iii) an antioxidant are present in a ratio of 1 : 1 : 1 to 5 : 5 : 5 or any values or ranges therebetween.

15. The composition of claim 13 or 14, wherein the autophagy activating agent is rilmenidine, the TORC1 activator or mTORC2 activator or FGFRlc agonist is FGF21, and the antioxidant is N-Acetylcysteine (NAC).

16. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 13-15 in combination with an immune checkpoint inhibitor.

17. The method of claim 16, wherein the immune checkpoint inhibitor targets CTLA-4, PD- 1, or PD-L1, and is selected from ipilimumab, nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab, tremelimumab, and dostarlimab.

18. The method of claim 16 or 17, wherein the composition and the immune checkpoint inhibitor are administered concurrently, separately, or sequentially.

19. Use of a combination of (i) an autophagy activating agent, (ii) a TORC1 activator or a mTORC2 activator or a FGFRlc (Fibroblast Growth Factor Receptor 1c) agonist, and / or (iii) an antioxidant in the manufacture of a medicament for thymus regeneration or amelioration of thymic involution in a subject.

20. The use of claim 19, wherein the medicament is for treatment of infections or cancer in the subject.

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