T-cell dysfunction in glioblastoma: Applying a new framework

23 A functional, replete T cell repertoire is an integral component to adequate immune surveillance and to the initiation and maintenance of productive anti-tumor immune responses. 25 Glioblastoma (GBM), however, is particularly adept at sabotaging anti-tumor immunity, eliciting 26 severe T cell dysfunction that is both qualitative and quantitative. Understanding and countering 27 such dysfunction are among the keys to harnessing the otherwise stark potential of anti-cancer 28 immune-based therapies. While T cell dysfunction in GBM is long described, newer immunologic 29 frameworks now exist for re-classifying T cell deficits in a manner that better permits their study 30 and reversal. Herein, we divide and discuss the various T cell deficits elicited by GBM within the 31 context of the five relevant categories – Senescence, Tolerance, Anergy, Exhaustion, and 32 Ignorance. Categorization is appropriately made according to the molecular bases of dysfunction. 33 Likewise, we review the mechanisms by which GBM elicits each mode of T cell dysfunction and discuss the emerging immunotherapeutic strategies designed to overcome them.


INTRODUCTION 37
For more than a century, many have advanced an intimate role for the immune system in 38 restricting cancer development. As early as 1909, Paul Erlich stipulated the actuality of "immune 39 surveillance," proposing that aberrant cells continuously arise during growth and development in 40 a manner that would ultimately result in an enormous frequency of cancers if not for the host's 41 immunologic defense mechanisms (1). Conversely, Erlich postulated that cancer instead emerges 42 when these aberrant cells outstrip and escape normal immune-surveillance function, winning the 43 advanced age (15). Telomere shortening is also seen in states of chronic infection, such as HIV, 92 (16) and in chronic inflammatory states (17), as often seen with cancer. Whether T cells in patients 93 with GBM demonstrate decreased telomere lengths and corresponding senescent states remains an 94 active area of investigation. 95 Phenotypic indicators of T cell senescence include CD57, a well-known marker of terminal 96 differentiation in human T cells (18), as well as loss of the co-stimulatory molecules CD27 and 97 CD28 (19). These changes correlate with critical telomere shortening and loss of telomerase 98 activity. CD57 + CD27 + T cells have recently been categorized as incompletely differentiated 99 tumor-infiltrating lymphocytes (TILs), which maintain the ability to proliferate after T cell 100 receptor (TCR) stimulation but become senescent with further antigenic exposure (20). In GBM, 101 immunosenescence of the CD4 + compartment has been correlated with poor prognosis: overall 102 survival is significantly shorter in GBM patients with higher levels of CD4 + CD28 -CD57 + T cells 103 (21). 104 Immunosenescence, albeit not specifically T cell senescence, is perhaps also reflected in 105 thymic senescence, a mode of dysfunction characterized by involution of the thymus. Thymic 106 involution is a natural byproduct of aging but also accompanies states of chronic inflammation 107 such as those seen with obesity, viral infection, and malignancy (22). It inevitably leads to a 108 decrease in the output of immature T cells, also termed "recent thymic emigrants" (RTE) (23). 109 rearrangement has occurred and is, therefore, considered a reliable tool for tracking and 115 quantifying RTE as a surrogate of thymic activity (25). As an extension, the absence of detectable 116 TREC can serve as a marker of thymic senescence. The aforementioned study showed that within 117 GBM patients (comparisons to controls were never made), TREC levels correlated with the clinical 118 outcome of GBM better than did patient age, with lower TREC levels predicting poorer clinical 119 outcomes. Additionally, numbers of RTE maintained a stronger correlation with predicted clinical 120 outcomes in vaccinated GBM patients than did immunological parameters, such as IFN-γ 121 production (24). These findings were corroborated preclinically in studies that demonstrated 122 decreased thymic function and decreased output of RTEs in murine models of intracranial glioma 123 (26). In these murine models of glioma, thymic atrophy appeared to be secondary to increased 124 Notch-1 and Jagged-1 signaling, resulting in the induction of apoptosis of thymocytes (27). Negative selection occurs in the thymus, where developing T cells expressing TCRs with overly 139 high affinity for self-antigen/MHC complexes are necessarily eliminated (29). The process is not 140 exhaustive, however, and self-reactive T cells, particularly those possessing specificity for organ-141 specific antigens not presented in the thymus, have the potential to elude elimination and gain 142 access to the peripheral circulation (30). As a result, numerous mechanisms for the peripheral 143 enforcement of tolerance have evolved to prevent continuous T cell self-reactivity and auto-144 immunity (Fig 2). Modes of peripheral T cell tolerance include peripheral deletion (31), 145 suppression by regulatory T cells (Treg) (32), and the activation of imprinted programs forcing T 146 cells into a hyporesponsive state (33), (34). GBM exhibits the noteworthy capacity to usurp each 147 of these tolerizing mechanisms, preventing an effective anti-tumor response. Understanding the 148 molecular mechanisms underlying such subterfuge will permit future strategies for breaking T cell 149 tolerance to cancer antigens while avoiding concomitant autoimmune damage. 150 151

Peripheral T Cell Deletion 152
The most obvious method for evading T cells is perhaps to eliminate them, a capacity 153 recognized in GBM dating to the late 1990s. First described in melanoma, this mechanism for 154 eliciting T cell apoptosis involves a FasL-mediated deletion of invading lymphocytes (35), which 155 has subsequently been described in GBM (36,37). Both CD4 + and CD8 + T cells demonstrate 156 increased susceptibility to apoptosis in GBM patients, with those T cells expressing Fas-L having exhaustion (discussed later) may have unwittingly contributed significantly to the observations of 161 early apoptosis in this study. 162 163

Regulatory T cells 164
Regulatory T cells (Treg) contribute substantially to peripheral tolerance by suppressing T 165 cell antigen-specific responses. Treg are a subset of CD4 + T cells expressing the transcription factor 166 Foxp3 (40). In a tumor setting, Treg potently suppress anti-tumor responses and promote tolerance 167 through secretion of the Th2-polarizing immunoregulatory cytokines, TGF-β and IL-10 (41). 168 These, in turn, limit T cell IL-2 and IFN-γ production (42)  As alluded to earlier, T cell anergy has been a frequently mis-applied term, often serving 272 as a "black box" for T cell dysfunction in the GBM and other cancer literature. Broadly, T cell 273 anergy describes a mechanism by which lymphocytes become perpetually inactive following an 274 Research.
on July 19, 2018. © 2018 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Author Manuscript Published OnlineFirst on March 28, 2018; DOI: 10.1158/1078-0432.CCR-18-0047 antigen encounter (Fig 3). Anergy was initially described in 1908, when Von Pirquet noted the 275 loss of delayed type hypersensitivity (DTH) responses to tuberculin in individuals infected with 276 measles (77). The same observation was made later in 1972 in patients with GBM, when they 277 failed to respond to dinitrochlorobenzene (78). Alternatively, in 1980, anergy was used to describe 278 the functional inactivation of B cells after tolerance induction with repeated antigen administration 279 (79). Here, the term "clonal anergy" emerged due to the nature of the lost antigen-specific 280 response. Presently, the term anergy is used to describe two separate phenomena: clonal or "in 281 vitro" anergy, and adaptive tolerance or "in vivo" anergy (34). Although different modes of 282 dysfunction, both terms encompass impairments to IL-2 production and T cell proliferation. 283 Ultimately, however, clonal anergy and adaptive tolerance are distinct biochemical states: clonal 284 anergy results primarily from defective co-stimulation resulting in RAS/MAPK dysfunction (80) 285 whereas adaptive tolerance results from continuous low levels of antigen exposure and deficient 286 Zap70 kinase activity, promoting impaired mobilization of calcium and NF-kB. It is important to 287 note that anergy has primarily been studied in CD4 + T cells. Therefore, while many of its features 288 may overlap with those of tolerance and exhaustion, these latter programs have been studied in 289 more detail in CD8 + T cells, as will be discussed further. 290 Clonal T cell anergy has been shown to be a long-lived defect in cell-cycle progression and 291 effector function and, while predominately irreversible, some have reported a degree of correction 292 with strong stimuli. More specifically, anergic T cells produce negligible amounts of IL-2, which 293 is crucial for clonal expansion; however, addition of high levels of exogenous IL-2 can sometimes 294 reverse the phenotype (81). The source of decreased IL-2 production is decreased IL-2 295 transcription, secondary to further defects in the upstream mitogen-activated protein kinase 296 (MAPK) family (82). Diminished IL-2 production in GBM patients was first noted when 297 Research.
on July 19, 2018. © 2018 American Association for Cancer clincancerres.aacrjournals.org Downloaded from peripheral blood lymphocytes (PBL) were found to have fewer phytohemagglutinin (PHA)-298 responsive cells, and these PHA-activated cells produced significantly lower levels of IL-2 as 299 compared to healthy controls (83). Subsequently, however, this phenomenon was attributed at least 300 in part to increased Treg activity in patients with GBM (thereby making it more reversible), and is, 301 therefore, perhaps more closely associated with T cell tolerance than with anergy, the latter term 302 likely then having been misapplied in the study (by the same authors as this review) (44). Although The role of adaptive tolerance in GBM and other cancers is less clear at this time, and it 314 may be difficult to truly distinguish from other modes of dysfunction. Defects in Zap70 kinase 315 have been found to be a key instigator of adaptive tolerance in T cells (80); this same mechanism, 316 however, has also been implicated in T cell exhaustion (84), which similarly results from 317 continuous low levels of antigen exposure. Likewise, the transcription factor nuclear factor of 318 activated T cells (NFAT), downstream of Zap70, plays a primary role in both exhaustion and 319 adaptive tolerance (85). In a B16 melanoma model, one study showed that T cells from NFAT-1 320 Research.
on July 19, 2018. © 2018 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. deficient mice were resistant to tumor-induced anergy, resulting in delayed tumor appearance and 321 slowed tumor growth (86). We propose that the terms "in vivo anergy" and "adaptive tolerance" 322 might best be absorbed into the definition of T cell exhaustion, as the mechanisms of these 323 processes appear to be the same in both CD4 + and CD8 + T cells. Using one term to describe the 324 phenomenon of chronic and, perhaps, suboptimal antigen exposure leading to defects in calcium 325 mobilization via NF-kB and NFAT will facilitate discussion and reviews on this topic, as well as 326 effective targeting strategies. 327

EXHAUSTION 329
T cell exhaustion is a hyporesponsive (not unresponsive) state resulting from repeated 330 antigenic exposure under suboptimal conditions (Fig 4)  T cell ignorance results from competent T lymphocytes failing to mount a productive 415 immune response, despite the presence of antigen, due to either anatomical barriers sequestering 416 the antigen from immune surveillance (i.e. immune privileged location) or to antigen expression 417 levels being at insufficient concentrations (Fig 5) (30). In contrast to tolerant T-cells, ignorant T 418 cells are fully functional, though antigen-inexperienced and naïve. If ignorant T cells become 419 exposed to antigen or activated by external stimuli, ignorance can in theory be easily overcome. 420 T cell ignorance in GBM would at first glance appear to be quite relevant, given historic 421 notions of immune privilege within the CNS. The concept of the "immunologic privilege" 422 bestowed upon the brain had its first origins in 1923, when Medawar's experiments showed that 423 foreign homologous tissues grafted to the brain do not provoke an immune response (110). The 424 lack of immunogenicity was believed due to the absence of a brain lymphatic drainage system, the 425 presence of the blood-brain barrier (BBB), and the lack of resident specialized APC within the 426 CNS. With a variety of newer studies highlighting that CNS immune access is not quite so 427 precluded (111) (112), it is now more widely accepted that the brain is more immunologically 428 "distinct" than "privileged," (113), and the contribution of an immunologically distinct CNS to 429 The immune system is increasingly recognized for its role in preventing the development and 453 restraining the progress of cancer. Failure of the immune system to function adequately, whether 454 by immunodeficiency or autoimmunity, is associated with increased prevalence of cancer. 455 Furthermore, many tumors themselves can leave patients in an immune-deficient state, more likely 456 to succumb to infections or other illnesses. Among these is GBM, the most common and the most 457 lethal primary brain tumor. GBM is capable of expertly inhibiting the immune system, eliciting

A B
Research.