Considerations for successful cancer immunotherapy in aged hosts

Accepted Manuscript Considerations for successful cancer immunotherapy in aged hosts

Vincent Hurez, Álvaro Padrón, Robert S. Svatek, Tyler J. Curiel PII: DOI: Reference:

S0531-5565(17)30415-1 doi:10.1016/j.exger.2017.10.002 EXG 10167

To appear in:

Experimental Gerontology

Received date: Revised date: Accepted date:

30 May 2017 30 September 2017 3 October 2017

Please cite this article as: Vincent Hurez, Álvaro Padrón, Robert S. Svatek, Tyler J. Curiel , Considerations for successful cancer immunotherapy in aged hosts. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Exg(2017), doi:10.1016/j.exger.2017.10.002

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ACCEPTED MANUSCRIPT Considerations for successful cancer immunotherapy in aged hosts Vincent Hurez1, Álvaro Padrón1, Robert S. Svatek2,3, Tyler J. Curiel1,3-5,*

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Department of Medicine, University of Texas Health San Antonio, TX 78229

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Department of Urology, University of Texas Health San Antonio, TX 78229

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The UT Health Cancer Center, University of Texas Health San Antonio, TX 78229

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Department of Microbiology, Immunology & Molecular Genetics, University of Texas Health San Antonio, TX

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78229 5

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Running title: Aging and cancer immunotherapy

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The Barshop Institute for Aging and Longevity Studies, University of Texas Health San Antonio TX 78229

Keywords: Aging, immunity, immunotherapy, cancer

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Abbreviations used: ARID, Age Related Immune Dysfunction; BCG, Bacille Calmette-Guérin; CAR, chimeric

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antigen receptor; CDMRP, Congressionally Directed Medical Research Program; CD40L, CD40 ligand; CpG, poly-(cysteine 5’ to guanine); CRISPR, clustered regularly interspaced short palindromic repeats; CTLA-4,

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cytotoxic T lymphocyte antigen-4; DC, dendritic cell; DNA, deoxyribonucleic acid; ICAM-1, intracellular

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adhesion molecule-1; IL, interleukin; Lag3, lymphocyte activation gene 3; M1, type 1 (macrophage); M2, type 2 (macrophage); MDSC, myeloid derived suppressor cell; MHC, major histocompatibility complex; mTOR,

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mammalian target of rapamycin; PD-1, programmed death-1; PD-L1, programmed death ligand-1; Th17, T helper cell 17; TGF-β, transforming growth factor-β; Tim3, T-cell immunoglobulin and mucin-domain containing-3; TNF-α, tumor necrosis factor-α; Treg, regulatory T cell

Financial support: Tyler Curiel (CA170491, CA54174, CDMRP, The Holly Beach Public Library, The Owens Foundation, The Barker Foundation and the Skinner endowment).

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ACCEPTED MANUSCRIPT *To whom correspondence should be addressed: Tyler Curiel, MD, MPH, Department of Medicine, University of Texas Health San Antonio, STRF MC 8252, 8403 Floyd Curl Drive, San Antonio, TX 78229-3900, USA. Phone: 210-562-4083, E-mail: [email protected]

Abstract

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Improvements in understanding cancer immunopathogenesis has now led to unprecedented successes in

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immunotherapy to treat numerous cancers. Although aging is the most important risk factor for cancer, most

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pre-clinical cancer immunotherapy studies are undertaken in young hosts. This review covers age-related immune changes as they affect cancer immune surveillance, immunopathogenesis and immune therapy

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responses. Declining T cell function with age can impede efficacy of age-related cancer immunotherapies, but examples of successful approaches to breach this barrier have been reported. It is further recognized now that

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immune functions with age do not simply decline, but that they change in potentially detrimental ways. For example, detrimental immune cell populations can become predominant during aging (notably pro-

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inflammatory cells), the prevalence or function of suppressive cells can increase (notably myeloid derived

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suppressor cells), drugs can have age-specific effects on immune cells, and attributes of the aged microenvironment can impede or subvert immunity. Key advances in these and related areas will be reviewed

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as they pertain to cancer immunotherapy in the aged, and areas requiring additional study and some

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speculations on future research directions will be addressed. We prefer the term Age Related Immune

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Dysfunction (ARID) as most encompassing the totality of age-associated immune changes.

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ACCEPTED MANUSCRIPT 1.0 INTRODUCTION The immune system can identify (and eliminate) cells expressing specific antigens with remarkable sensitivity. Tumors are theoretically highly antigenic tissues owing to their various mutations. Antigenicity, the expression of antigens, nonetheless does necessarily equal clinically useful immunogenicity (the capacity of antigens to provoke useful immunity). Thus, spontaneous rejection of clinically apparent tumors is a rare event. Work over

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the past 15 years shows how endogenous immunity fails to eradicate clinically through a multiplicity of factors

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including immunoediting (discussed below), from tumor-driven inflammation and immune dysfunction, and

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because tumor rejection antigens can be self antigens that are protected by autoimmune defense mechanisms [1, 2]. Recent work has advanced thinking towards anti-cancer immunotherapies with high potential for

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efficacy, which has now clearly been demonstrated clinically. As hosts age, these already-formidable barriers

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to treatment success become compounded, and additional barriers can emerge [3-6].

Tumor immune surveillance is part of larger scheme of immunoediting [7], easily remember as involving three

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E’s [8]. The first “E” is elimination of cancer cells as they initially emerge. The second “E” is equilibrium, in

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which malignant cells mutate to evolve immune elimination as the immune system evolves to attack new mutations. Selective pressure during equilibrium provoke tumor antigenic evolution that finally allows immune

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escape, the third “E”. In escape, the tumor can evade immune defenses and become clinically manifest, a

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process seen in mouse models [9-11] and in humans[12-14].

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The original six fundamental cancer hallmarks [15] did not include lack of immune rejection, which was corrected with the revision to eight fundamental hallmarks [16]. Generalized, chronic inflammation [17, 18] is also considered a cancer hallmark [19], as well as stem cell features [20], genomic instability [21], and abnormal vasculature [22].

These cancer hallmarks (particularly immune rejection and chronic inflammation) affect age-specific immunotherapy development. Age effects on immunity extend far beyond simple declines in functions or reductions in cell numbers. We propose the term “Age Related Immune Dysfunction” (ARID) to encompass the 3

ACCEPTED MANUSCRIPT full range of age-related alterations in immunity with advancing age. The following sections address major topics relating to age effects on cancer immunotherapy.

1.1 TUMOR-SPECIFIC T CELLS T cell immunity wans with age owing to various processes affecting T cell numbers, diversity, phenotype and

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function [23]. As an example, CD45RA+CD62L+CD27+CD28+ naive T cells are generated in thymus throughout

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life, but production decreases with age [24], and thus T cell repertoire diversity declines with age as overall

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peripheral T cell numbers stay essentially the same [25]. Hematopoietic stem cell generation of T cell precursors [26] also declines. While naïve T cells decrease, there is a concomitant increase in memory T cells

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with age. Apparently terminally differentiated effector T cells (based on flow cytometry phenotype), including virus-reactive cells, with greatly reduced T cell receptor repertoire diversity and compromised potential to

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proliferate specifically increase [27]. T cell signaling, including through the T cell receptor also declines with

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age [28].

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Strategies to reduce age-related defects and improve T cell immunity have been tested. CD4+ T cell functions decline with age, but such loss can be partially overcome using cytokines including tumor necrosis factor

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(TNF)-α or interleukin (IL)-6 [29]. T cell priming (the activation of naïve, antigen-specific T cells) is defective in

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age, but can be improved with agonist αCD137 antibodies [30]. Tumor-specific immunity can also be improved. For example, tumor control and anti-tumor T cell differentiation through OX40 signals decreases with age [31,

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32], but aged mice can be induced to mount protective antitumor immunity in a lymphoma model using an agonist αCD40 antibody [33]. We found that aged mice develop anti-tumor immunity comparable to that in young mice against B16 melanoma, a poorly immunogenic and highly aggressive tumor, by simultaneous reduction of age-increased suppressive myeloid and dysfunctional regulatory T cells [5], which is discussed in detail below.

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ACCEPTED MANUSCRIPT T cells (not necessarily cancer-specific) can improve host response to anti-cancer immunotherapy [34]. It has been proposed that improving the function of aging T cells could improve cancer chemotherapy responses in the aged [35], although this concept has not been tested to our knowledge.

1.2 REGULATORY T CELLS

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Regulatory T cells (Tregs) mediate significant tumor immune dysfunction. Thus, inhibiting Treg function or

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reducing their numbers is under study as cancer immunotherapy [36-39]. Reports of Treg effects in age-related

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immune dysfunction are contradictory. Some studies showing show no changes or lessened Treg contributions [40, 41], whereas other studies find increased age-related Treg function and/or prevalence in humans and

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mice [42-45]. Treg prevalence in lymphoid organs but not thymus or blood have been reported in aged mice

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[5, 46, 47].

Age effects on Treg functions depends on context and the specific function investigated. Some functions could

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be reduced [48], such as suppressing delayed type hypersensitivity [44], or inhibiting Th17 immunity [46]. Aged

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Tregs mediate equal or greater suppression versus young mice in some reports [5, 49]. Treg data in elderly humans is limited, but they could increase in with age in blood [50, 51]. Tregs from young and elderly persons

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inhibited T cell proliferation similarly, but IL- 10 (an anti-inflammatory cytokine) was lower in aged Tregs [52].

Treg depletion can improve anti-tumor immunity and immunotherapy outcomes [37, 39], but reported Treg

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depletion effects in cancer immunotherapy in aged hosts are conflicting [53, 54]. One mouse study linked impaired tumor rejection to increased Tregs. In this study, αCD25 depleted Tregs and improved anti-cancer immunity [54]. We depleted Tregs in mice bearing B16 melanoma using denileukin diftitox [5]. Denileukin diftitox similarly depleted Tregs in young and aged mice, but it delayed tumor growth and augmented tumorspecific immunity only in young mice. Denileukin diftitox altered interferon-γ and IL-17 producing T cells distinctly in young versus aged mice. CD11b+Gr-1hi myeloid derived suppressor cells (MDSC) were more numerous and suppressive in aged tumor-bearing aged mice, and depleting Tregs increased MDSC numbers even further. 5

ACCEPTED MANUSCRIPT We added anti-Gr-1 antibody to deplete MDSC along with Treg depletion, and restored anti-tumor immunity in the aged mice that slowed tumor growth comparable to Treg depletion in young hosts. Anti-Gr-1 antibody did not further augment anti-tumor immunity or tumor control in young mice as denileukin diftitox did not increase their MDSC. Dual Treg plus MDSC depletion increased interferon-γ-producing CD4+ and CD8+ T cells, and

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CD8+ tumor-specific T cells in aged mice, mirroring the effects on these T cell subsets seen in young mice

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treated with Treg depletion alone. To our knowledge this is the first cancer immunotherapy that treats aged, but

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not young hosts [5]. Figure 1 outlines the differences in young versus aged mice accounting for differential treatment outcomes. Detailed data on age effects on human T cell subsets in responses to cancer

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immunotherapy are not yet reported to our knowledge.

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Age effects of Tregs in human cancer immunotherapy are not yet specifically reported to our knowledge. In

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209 melanoma patients receiving αCTLA-4 immunotherapy, high Tregs (and also eosinophils and

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lymphocytes) correlated with better clinical outcomes [55], but age was not specifically assessed as a variable.

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1.3 OTHER CELLS

Lower T cell function and chronic, low-level age-related inflammation can increase suppressive myeloid cells.

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Thus, targeting aged innate cells could improve anti-tumor T cell functions in elderly cancer patients [56].

1.3.1 Dendritic cells

Dendritic cells (DCs) are antigen-presenting cells important in promoting T cell immunity [57]. The local tumor environment favors increasing numbers of dysfunctional DC that hinder anti-tumor immunity and immunotherapy outcomes [58, 59].

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ACCEPTED MANUSCRIPT DC precursors in blood and Langerhans dendritic cells in skin, conventional DC in other organs and DC generation from bone marrow can all decrease with advancing age, which could contribute to reduced protective immunity with age. By contrast, other DC subsets, notably those with potential autoimmune reactivity can increase in certain animal models of autoimmunity, which could help explain the age-related propensity for

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autoimmunity, described in detail in [60, 61].

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Distinct DC functions with age can be decreased or unchanged depending on the function, such as reduced

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alloreactivity in a mixed lymphocyte reaction, reduced T cell activation molecules (e.g., MHC class II, ICAM-1), reduced migration and antigen capture capacity, and reduced cytokine or chemokine production, reviewed

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extensively in [62]. Some of these effects are DC-intrinsic whereas others (such as reduced migration) could be attributable to defective host factors. If declining or altered DC functions reduce T cell function or promote

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inflammation, that could increase age-related cancer risk. Augmenting the capacity of DC to present antigens, a function that can decline with age, induces potent tumor-specific cytotoxic T cell activities. CD40L or agonist

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anti-CD40 antibodies augment DC activation in human and animal studies [63]. A vaccine consisting of CD40L

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hooked to specific cancer antigens showed promise in older cancer patients [64].

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In aged humans, reduced Langerhans DC have also been reported [65]. The two principal circulating human

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DC subsets (myeloid and plasmacytoid) also appear to decline in numbers and function with age as does DC generation from bone marrow, all effects similar to those seen in aged mice, reviewed in [62]. Interestingly,

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although in situ DC from the aged humans have apparent functional defects, when DC are generated from aged precursors (such as monocyte-derived DC), their numbers and functions appear similar to those from young precursors. Thus, DC-based cancer immunotherapies based on generation from precursor cells could be a viable strategy in aged cancer patients. The effects of age on tumor microenvironment-driven DC dysfunction in humans are not reported to our knowledge, but would be useful to know.

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ACCEPTED MANUSCRIPT 1.3.2 Macrophages Myelopoiesis increases in age [66], leading to increased myeloid cell numbers. Macrophages are important elements of tumor stroma and contribute to tumor-associated immune dysfunction [67]. Tumor macrophages can be pro-inflammatory M1 or anti-inflammatory M2 macrophages, and these phenotypes can interconvert. M1 macrophages produce pro-inflammatory cytokines including TNF-α and IL-12 that augment anti-tumor

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immunity. M2 macrophages are generally anti-inflammatory and produce cytokines such as IL-10, TGF-β that

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promote tumorigenesis or angiogenesis [67].

Macrophages increase in lymphoid organs of aged mice [68]. M1 macrophage functions could increase

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because of increased age-related reactive oxygen species, but others report reduced M1 macrophage function with age [69], perhaps from IL-10 producing M2 macrophages that increase with age [67]. Tumor-associated

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macrophages from aged mice produce high levels of transforming growth factor (TGF)-β that can be immunosuppressive, consistent with an M2-type phenotype, whereas young macrophages do not [68].

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Detrimental M2 macrophages can be converted to beneficial M1 using IL-12 or CpG plus an αIL-10 receptor

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[70]. Thus targeting tumor-associated age-related M2 macrophages could be useful to treat cancers in aged

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hosts.

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The distribution and some functions of macrophages in aged hosts differs from the young. For example, M1 macrophages with a pro-inflammatory phenotype are increased in liver and fat tissues of normal aged hosts

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versus young hosts whereas anti-inflammatory/immune suppressive M2 macrophages are more prevalent in normal aged versus young hosts in peripheral lymphoid tissues, reviewed in [71]. However, effects in cancer, and whether macrophages from aging hosts are especially prone to promote tumor growth in not fully settled and likely will depend on the specific tumor and/or anatomic site.

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ACCEPTED MANUSCRIPT 1.3.3 Myeloid derived suppressor cells Myeloid derived suppressor cells (MDSC) are immunosuppressive, immature myeloid cells that are elevated in inflammatory diseases including cancers [72-75] and they suppress anti-tumor immunity [76]. MDSCs produce inhibitory molecules (e.g., arginase, IL-10) that inhibit T cell functions, and can promote generation of detrimental M2 macrophages or Tregs [77]. MDSCs increase in blood during human aging [78] and increase

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in lymphoid organs in mice [5, 68]. MDSC are immunopathologic in age including in aged hosts with cancer

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[79, 80]. Treg depletion using denileukin diftitox increased MDSC numbers in aged mice bearing B16

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melanomas, suggesting MDSC control by Tregs [5]. In CT26 colon cancer, an extract of Lentinula edodes mycelia reduced MDSC numbers when combined with a cancer vaccine in aged mice. The proposed

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mechanism was suppression of IL-6 and TNF-α known to promote MDSC [81]. This mycelium extract also improved priming of tumor-specific cytotoxic T cells by the vaccine. Thus, MDSC are an attractive target to

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develop potentially effective approaches to mitigate cancer-associated immune dysfunction in the aged host.

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Age effects of MDSC in human cancer immunotherapy are not yet specifically reported to our knowledge. In

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209 patients receiving αCTLA-4 as melanoma immunotherapy, low baseline blood MDSC (and also lactic acid dehydrogenase and monocytes) correlated with better clinical outcomes[55], but age was not independently

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1.3.4 B cells

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assessed.

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Aging B cells can be dysfunctional to normal immune homeostasis by producing tumor necrosis factor-α that is detrimental [82]. We showed that in aged mice aging-associated PD-L2-expressing B cells enhance anti-tumor immunity to ovarian cancer via Th1 and Th17 induction [83]. Little else regarding B cells in anti-tumor immunity or response to immunotherapy has been reported to our knowledge.

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ACCEPTED MANUSCRIPT 1.3.5 Hematopoietic stem cells As many immune cells come from hematopoietic stem cells, which themselves are subject to aging effects, understanding aging of hematopoietic stem cells could provide insights into improving an aging immune system. Progress on this front has been made [84].

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1.3.6 Novel age-related immune cells

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With age, novel immune cell subsets appear, including those with potential to deviate anti-tumor immunity or

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immunotherapy responses. We described PD-L2-expressing B cells that increase with age and enhance antitumor immunity to ovarian cancer via Th1 and Th17 induction. These cells were not seen in young mice [83].

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CD25lo Tregs appear with age in normal mice [85], in contrast to conventional CD25hi Tregs in young hosts. These are slightly hypofunctional versus CD25hi Tregs but we did not define clear immunopathologic

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significance in cancer immunotherapy of aged mice with B16 melanoma although denileukin diftitox reduced

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their numbers [5].

2.1 Immune checkpoint inhibitors

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2.0 SPECIFIC APPROACHES

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Immune checkpoint receptor blockade is a highly successful immunotherapy strategy for various cancers [86,

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87]. Six distinct checkpoint inhibitor antibody treatments were FDA approved as cancer immunotherapy for a wide variety of cancers at the time of this writing, with more approvals expected soon. Nonetheless, effects in

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elderly patients are little reported.

Immune checkpoint molecules control the magnitude of immune responses either positively (activating) or negatively (inhibiting). Immune checkpoint inhibitor antibodies block negative signals from the immune checkpoints elevated in many cancers, and thus can improve anti-cancer immunity [86, 87]. Inhibitory immune checkpoint molecules increase on aged T cells in humans and mice [5, 88, 89], suggesting increased memorylike T cells that also express these molecules and which are hyporesponsive. Immune checkpoint molecules associated with exhausted (poorly functional) T cells, including PD-1, Lag-3 and Tim-3 increase with age as 10

ACCEPTED MANUSCRIPT well. These and other check point molecules are targets for anti-cancer immunotherapies. Other immune checkpoint receptors such as PD-L1 are highly expressed on young myeloid or B cells, but can be at high levels on tumor cells and aged CD8+ T cells [90]. Age-specific strategies to mitigate inhibitory immune checkpoint signals and reduce hyporesponsiveness or exhaustion of aged T cells without compromising anti-

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tumor effects is an important goal in the development of these modalities.

2.1.1 αPD-1 antibody

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PD-1 impedes anti-tumor PD-1+ T cells. Monoclonal αPD-1 antibodies (αPD-1) are remarkably effective against many cancers and two distinct αPD-1 antibodies (pembrolizumab and nivolumab) are FDA-approved to

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treat melanoma, renal cell carcinoma, non-small cell lung cancer, Hodgkin’s disease, bladder cancer and head

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and neck cancer.

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PD-1 is largely expressed by effector-memory (CD44hiCD62Llo) T cells that are elevated with age. CD4+PD-1+ T cells in old mice are hypoproliferative, suggesting that PD-1 could contribute to the functional decline in

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effector-memory T cells with age [91, 92]. Although αPD-1 improves the functioning of T cells in aged mice

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[93], this strategy has not been reported as cancer therapy in aged mice to our knowledge. We showed that the mTOR inhibitor rapamycin lowers T cell PD-1 expression with age, and the PD-1+ T cells in rapamycinmice functioned significantly better than PD-1+ T cells in aged mice on control treatment [92],

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suggesting that mTOR inhibition could improve anti-tumor immunity in aged hosts. In this regard, everolimus, an mTOR inhibitor similar to rapamycin, improved B cell immune responses to influenza vaccine in aged humans [94].

2.1.2 αPD-L1 antibody PD-L1 is an immune checkpoint molecule that is a major ligand for PD-1. Like αPD-1, αPD-L1 is thought to work as cancer immunotherapy by shielding PD-1+ anti-tumor T cells from tumor PD-L1 inhibition [87, 95-98]. 11

ACCEPTED MANUSCRIPT Three different αPD-L1 antibodies (atezolizumab, avelumab and durvalumab) are now FDA-approved for cancer treatment.

Most old, naive CD8+ T cells in mice are PD-L1+ versus just 25% in young mice. Aged CD8+ T cells exhibited relatively low proliferation, but αPD-L1 increased proliferation in vitro to a level comparable to young CD8+ T

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cells. αPD-L1 augmented anti-tumor immunity in aged hosts to the level of young hosts in a lymphoma model

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in mice [90].

We showed that αPD-L1 treats B16 melanoma in young mice but fails in aged mice. αPD-L1 treatment efficacy

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was partially restored in combination with αCTLA-4 [99]. The above study [90] tested αPD-L1 in a lymphoma model in Balb/c mice whereas we studied a melanoma in BL6 mice. Thus, either lymphoma versus melanoma,

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or host genetic background among other considerations could help explain different results with αPD-L1 in

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aged hosts.

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2.1.3 αCTLA-4 antibody

Ipilimumab, an anti-CTLA-4 antibody, was the first immune checkpoint inhibitor agent approved by the United

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States Food and Drug Administration, and the first agent in this class that clearly benefits human cancer by

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improving disease-free and overall survival in metastatic melanoma [100]. We showed that αCTLA-4 was the only immunotherapy effective in aged mice as a single agent in B16 melanoma. However, we could not identify

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a clear mechanism based on assessments of T cell activation and cytokine production. Further, αCTLA-4 was less able to reduce Tregs specifically in the tumor compared to young tumor-bearing hosts [99]. This potentially important set of data on this agent merits additional investigations.

In 82 melanoma patients receiving αCTLA-4 as immunotherapy, increases in peripheral blood T cells on treatment predicted good outcomes, but the effects did not reach statistical significance[101]. In 209 melanoma patients receiving αCTLA-4 immunotherapy, low baseline blood MDSC, lactic acid dehydrogenase and monocytes, and high Tregs, eosinophils and lymphocytes correlated with better clinical outcomes[55]. Age was 12

ACCEPTED MANUSCRIPT not independently assessed in either of these two studies. It was just reported [102] that in a cohort of 254 melanoma patients receiving αPD-1 or αPD-L1 antibodies as immunotherapy, there was no treatment or survival response difference by age in cohorts of patients aged <50, 50-64, 65-74 or >75 years. However, individual cohorts were small and likely underpowered, thus without clear ability to exclude age as a factor affecting αCTLA-4 efficacy in human melanoma, a finding that we predict will eventually be made.

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Nonetheless, these are important initial steps in understanding if and how age will alter immune checkpoint

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therapy responses as predicted from mouse studies.

2.2 IMMUNE CHECKPOINT AGONISTS

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Agonists for CD137 and OX40 were described above under T CELLS [30, 33]. These are now in human

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clinical trials for cancer, but not with an age focus to our knowledge.

2.3 ADOPTIVE CELL TRANSFERS

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Adoptive cell transfers of DC or tumor infiltrating lymphocytes were generally poorly effective against cancers.

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Transfers of chimeric antigen receptor (CAR) cells show greater promise in hematologic malignancies. To make cell expressing a CAR, the CAR is genetically engineered into lymphocytes that are then transfused into

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the patient [103]. We are unaware of published reports of age effects on immune cells in generating CAR cells,

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aside from understanding that older T cells generally yield fewer CAR T cells versus younger patients following in vitro culture. If aged T cells have reduced cytolytic capacity or homing, they could be less effective as CAR T

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cell cancer immunotherapy. Means to boost aged T cell functions discussed above could potentially boost of CAR T cell efficacy from aged T cells. Natural killer cells and γδ T cells do not require a major histocompatibility match to kill targets cells. Thus, CAR engineered into these cells might be useful to treat aged cancer patients, as CAR NK and γδ T cells are under investigation. Similarly, aged DC with reduced T cell activating or antigen presenting capacities, could limit efficacy when made form aged hosts, although strategies to boost aged DC functions as discussed above could be evaluated.

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ACCEPTED MANUSCRIPT 2.4 mTOR INHIBITORS Mammalian target of rapamycin (mTOR) inhibitors at doses lower than those to induce immunosuppression improve antigen-specific immunity, including tumor-specific immunity [104]. We reported that in aged mice, rapamycin reduces their T cell PD-1 expression and improves their PD-1+ T cell functions [47]. Chronic administration of rapamycin in the eRapa microencapsulated formulation had major effects on many T cell

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functions including increasing IL-17 and reducing interferon-γ production, altering Th (CD4+ T helper)

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polarization in many Th pathways but with little effect on Treg numbers, altering T cell trafficking molecules,

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altering many canonical regulatory pathways, reducing total and memory T cells and had many other significant T cell effects in diverse organs [47]. Surprisingly, effects in all these diverse outcomes were

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generally similar in young and aged hosts and in males and females.

T cell differentiation depends on mTOR, and mTOR suppression can promote Treg function or differentiation

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[92, 105]. Thus, small molecule mTOR inhibitors could blunt anti-tumor immunity by generating detrimental

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Tregs, as Tregs can inhibit anti-tumor immunity [37]. However, in the right settings and at the appropriately low dose, small molecule mTOR inhibitors do yield net anti-tumor immunity benefits [47, 82, 92, 93] and do not

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appear to promote increased Treg numbers or function [92]. We reported that low dose rapamycin is beneficial

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to anti-cancer immunotherapy whereas typical therapeutic rapamycin doses impair it [106].

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Chronic eRapa administration also increased B cell prevalence in spleen and bone marrow of young and aged mice but without significant change in absolute numbers. Follicular and marginal zone B cells were increased and transitional 1 (T1) B cells were decreased. Phenotyping and genomic data suggested that eRapa preserved naıve B cells. Strikingly, eRapa was not consistently anti-inflammatory as expected, but induced or inhibited distinct inflammatory pathways. eRapa appeared to increase numbers and functions on innate lymphoid cells as evidenced by their IL-17 and especially IL-22 production, either of which could be pro-tumor or tumor-protective in distinct settings. Diverse effects on myeloid cells and natural killer cells were also

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ACCEPTED MANUSCRIPT observed. As noted in T cell studies, eRapa effects in all of the distinct cell types, sexes and anatomic compartments were generally similar in young and aged mice [47],

We further found that rapamycin improves γδ T cell-mediated anti-cancer immunity [107]. Thus, mTOR inhibitors might be useful as adjuncts to cancer immunotherapy agents in aged patients, as we showed is

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possible [92] (and see related article in this issue). Rapamycin prolongs life of mice even when given late in

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life [108] which could be due to its cancer prevention effects. However, we unexpectedly found that eRapa

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significantly prolonged life in highly immune deficient mice lacking critical anti-cancer immune defenses [47].

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More more work on mTOR effects is needed.

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2.5 CALORIC RESTRICTION

Similar to rapamycin, caloric restriction suppresses mTOR. Caloric restriction boosted αCD40 treatment of

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breast cancer and sarcoma when used over 6-8 months in mice that were 12 months old. Priming of tumor

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antigen-specific CD4+ but not CD8+ T cells was similar to that seen in young controls [109]. Certain approaches to caloric restriction are tolerated by aged patients, and can improve anti-tumor immunity in young

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subjects [110]. However, mTOR suppression can also reduce immunity in aged hosts [111] and at too late an

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age or at too great a caloric reduction, caloric restriction harms aged hosts. Caloric restriction merits further

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studies to mitigate cancer treatment symptoms, or prevent or treat cancer in aged hosts.

2.6 TOLL-LIKE RECEPTOR AGONISTS Bacille Calmette-Guérin (BCG), an attenuated Mycobacterium bovis is approved by the United States Food and Drug Administration for non-muscle invasive bladder cancer. It potently agonizes Toll like receptors. Although its mechanism of anti-cancer action is not fully understood, it is thought to improve anti-cancer immunity. Observational studies suggest that age influences BCG therapy responses in bladder cancer. Aged bladder cancer patients had increased relapse rates following BCG compared to younger patients in a trial of BCG plus interferon-α immunotherapy [112]. In patients aged 61-70 years old versus those older than 80, 15

ACCEPTED MANUSCRIPT cancer-free survival was 39% versus 61%, respectively, at median follow-up of 24 months. Age was an independent risk for progression in aged bladder cancer patients treated with BCG, and the 2-year progression-free survival was 87% for patients under 75 years versus 65% in patients older than 75 years [113]. In a large cohort of 805 patients with bladder cancer, age had a small (but measurable) effect on reducing BCG therapy response [114]. However, selective surgical management of elderly patients versus

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immune effects and competing morbidities effects are not clear from these studies. Thus, BCG is the only

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agent of which we are aware for which age-specific human immunotherapy effects are extensively reported.

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Nonetheless, firm age-related immune effect conclusions cannot yet be made. As BCG is standard of care immunotherapy and as bladder cancer patients are typically older than for many other cancer types, additional

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studies are warranted.

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3.0 TUMOR MICROENVIRONMENT

The tumor microenvironment includes the tumor itself, infiltrating immune cells, stromal cells and matrix The

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microenvironment

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components.

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aspects

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suggest

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could

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more

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immunosuppressive versus in young hosts [115]. Such aspects include increased M2 macrophages and MDSC that could be attributable to chronic, low grade age-related inflammation. Other factors include tumor

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cells that can attract detrimental MDSC, neutrophils or Tregs that blunt anti-tumor immunity and can generate

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pro-tumorigenic molecules [35, 68]. For example, stromal fibroblasts from older versus younger prostate

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cancer patients generate more pro-inflammatory factors [116].

In a transgenic prostate cancer model and model of benign prostate hyperplasia in mice, there was an agerelated increase in TGF-β1 that further altered local tissue structure suggesting local inflammation [117]. Data aged prostates in normal mice show similar effects [118]. Aged fibroblasts attract CD4+ T cells that support proliferation of prostate epithelial cells. Other cells, including macrophages, CD8+ T cells and neutrophils promote prostate cancer cell proliferation [90].

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ACCEPTED MANUSCRIPT In the microenvironment of aged melanoma, fibroblasts promote melanoma growth and progression. Senescence in aged fibroblasts could drive tumor metastases and angiogenesis by producing frizzled-related protein 2. Older fibroblasts also generate fewer scavengers of reactive oxygen species that can in turn promote more DNA damage [6]. Aged mice receiving agonist αCD40 did not to respond to treatment because of defective CD40 tumor microenvironmental signals, not due to T cell-intrinsic effects [32]. Thus, treatments that

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modify the stromal, could be relevant for elderly cancer patients, an area of investigation that has not seen

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much reported research activity to date.

4.0 IMMUNOTHERAPY TOXICITY MITIGATION

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In aged mice, cancer immunotherapy can be lethal or produce local toxicities or toxicities in distinct organs, in part due to pro-inflammatory cytokines that are generated locally in affected tissues and systemically.

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Macrophages appear to be prime drivers of pro-inflammatory effects over T cells or natural killer cells. Depletion of myeloid cells concomitantly with therapy can mitigate toxicities and yet not compromise treatment

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efficacy significantly. Aged macrophages from mice and from normal humans generally produce higher levels

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of pro-inflammatory cytokines, including TNF-α and IL-6 versus younger macrophages. TNF-α blockade improves survival and reduces toxicities without loss of anti-tumor effects in aged mice [3, 4]. αTNF-α, αIL-6

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and αIL-6R antibodies are all FDA-approved. Thus, this concept merits further investigation for potential rapid

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translation as justified by human data. However, administration of IL-6 and TNF-α administration can improve aged T cell functions [29]. Thus, much additional work is needed to use such approaches safely and effectively

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in cancer immunotherapy strategies.

5.0 MICROBIOME EFFECTS Gut microbes affects systemic immunity and the gut microbial composition is altered by age [119-121]. In two separate papers gut microbes were shown to affect anti-cancer immunotherapy in human subjects being treated with αPD-L1 [122] or αCTLA-4 [123]. At present, there is much justified interest in defining microbial effects on anti-cancer immunity. Age effects in this regard are not yet reported on cancer immunotherapy to our knowledge. As gut microbes can affect anti-cancer treatment outcomes by modulating immunity [124], 17

ACCEPTED MANUSCRIPT including modulating tumor microenvironmental myeloid cells [125], themselves affected with age [92], it is predictable that we will identify age-associated microbial effects on anti-cancer immunotherapy, an area meriting further work.

6.0 CONCLUSIONS

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Cancer immunotherapy has a strong scientific rationale. Recent advances in understanding details of cancer

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driven immune dysfunction have helped develop highly successful, although still imperfect, anti-cancer

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immunotherapies. Much of our understanding of anti-tumor immunity and responses to cancer immunotherapy derives from studies of relatively young hosts. A growing, but still relatively small, body of data is beginning to

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define age effects on cancer immunity and responses to immunotherapies. There are some examples of novel approaches to improving anti-cancer immunotherapy in aged hosts most at risk for cancer and in means to

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mitigate toxicities of immunotherapy with emphasis on age effects.

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“Immune decline” is an inaccurate term in reference to the aged immune system, and “immunosenescence”

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and “inflammaging” describe specific attributes of an aged immune system. We propose the term “Age Related Immune Dysfunction” (ARID) to describe the totality of age-related immune changes to include increased or

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decreased numbers of certain immune cells, appearance of novel immune cell populations, increased pro-

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inflammatory or immune suppressive functions, restricted TCR repertoire and the myriad other changes that age brings to immunity. To the extent that underlying ARID can be reversed in aged cancer patients, clinically

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relevant anti-tumor immunity could potentially be achieved as has now clearly been demonstrated.

7.0 CHALLENGES IN DEVELOPING EFFECTIVE AGE-APPROPRIATE CANCER IMMUNOTHERAPY Understanding age effects on tumor-specific immunopathology and related age-specific effects better will help in development of more effective treatments. Individual agents are unlikely to be effective cancer treatments, and thus means to combine agents and approaches optimally needs further definition, including age-specific agents, doses and schedules. Biomarkers that differentiate aged patients with potential to generate tumor18

ACCEPTED MANUSCRIPT specific immunity to selected agents from those patients requiring alternative or adjunctive approaches are also needed. Adjuncts could include adoptive transfer of young or rejuvenated CAR-expressing immune cells, improved antigen presenting cells, thymic transplants, gene therapy to improve specific age-associated defects, microbial enhancements or other approaches.

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Toxicity mitigation strategies that do not negate clinical efficacy are needed, as is means to identify rational

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therapy combinations that minimize use of potentially harmful individual agents while preserving treatment

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efficacy. Studies of age effects of the human gut microbiome are early, but data from them could help understand heterogeneous immunotherapy responses in humans and in pre-clinical models. Aside from the

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gut microbiome, age effects on other microbial populations (e.g., lung, skin) require additional attention. The mutational burden of individual cancers affects efficacy of cancer immunotherapies. This mutational burden is

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dictated by immune editing, among other factors. Thus, we must understand age effects on immune editing and tumor mutations, which information could improve cancer immunotherapy efficacy. Finally, we must

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deepen our knowledge of age effects on tumor stroma, including vasculature and matrix effects that could

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affect immune cell or drug infiltration. As ever more powerful but costly and potentially harmful approaches are brought to bear on ever older cancer patients, economic, feasibility and ethical issues must be addressed in

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greater depth.

Notable similarities in mouse models of the aging immune system with the human immune system include a

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general reduction in important immune cells including T cells and DC, increase in memory phenotype immune cells, reduction in naïve phenotype immune cells, increased MDSC and general increase in inflammatory markers. The ability to respond to vaccines is generally impaired in aged mice and humans. There is little data on effects of age on human responses to cancer immunotherapy including T cell subset-specific data, effects on induction of tumor-specific immune memory, effects of T cell exhaustion and effects of Tregs, MDSC and other immunosuppressive cells and factors on immune and clinical outcomes. The single report thus far on age effects in immune checkpoint blockade in melanoma immunotherapy did not demonstrate an age effect on clinical outcomes, but this study could have been underpowered. Mouse models will be useful to test certain 19

ACCEPTED MANUSCRIPT hypotheses and strategies in approaches to cancer immunotherapy in aged hosts, but it is clear tat much additional human data is also needed to address this problem that we consider to be important, but about which many important top-pevel questions remain unaddressed in detail.

Acknowledgements: VH, RSS and TJC wrote the manuscript. VH and AP performed some experiments

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described.

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Conflict of Interest: The authors declare no financial conflicts.

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Fig. 1. Example of an age-specific immune effect that can be ameliorated to allow effective anti-meanoma immunity. (a) Young mice challenged with B16 melanoma experience increased regulatory T cells (T regs), but not myeloid derived suppressor cells (MDSC), whereas aged mice experience increased Tregs and MDSC. In the aged, the increased MDSC could be due to poor Treg control of them directly, or through indirect mechanisms (denoted by question marks). (b) In young mice, denileukin diftitox (DD) reduces T regs with little MDSC effect, improving antitumour immunity [increased +

interferon (IFN)-γ T cells]. By contrast, in aged mice (c), DD-mediated Treg reduction reduces Tregs but without significant

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increase in IFN-γ T cells. Interleukin (IL)–17 , potentially detrimental T cells, increase, as do deleterious MDSC, thereby inhibiting beneficial anti-tumour immunity. Green anti-tumour immunity means good immunity, whereas red means less effective immunity. (d) By adding anti-granulocyte-differentiation antigen-1 (αGr-1) antibody to DD in aged mice, the MDSC increase is blunted and aged mice now mount anti-tumour immunity comparable to young hosts receiving DD alone, with comparable clinical efficacy. Adding αGr-1 to DD in young hosts does not improve immune or clinical effects

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further, as MDSC were not increased further by DD. The red ‘X’ denotes DD effects to reduce Treg inhibition.

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ACCEPTED MANUSCRIPT Highlights

This article reviews major findings in age effects on the immune system and how they affect the success of cancer immunotherapy. Differences and similarities between mouse models and human data are discussed. This background is

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used to summarize key findings in efforts to use cancer immunotherapy successfully and optimally in aged patients.

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Major reviews are addressed and original source data are discussed. Areas for further investigations and testing are

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discussed.

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