Circadian lifestyle determinants of immune checkpoint inhibitor efficacy

Immune Checkpoint Inhibitors (ICI) have revolutionised cancer care in recent years. Despite a global improvement in the efficacy and tolerability of systemic anticancer treatments, a sizeable proportion of patients still do not benefit maximally from ICI. Extensive research has been undertaken to reveal the immune- and cancer-related mechanisms underlying resistance and response to ICI, yet more limited investigations have explored potentially modifiable lifestyle host factors and their impact on ICI efficacy and tolerability. Moreover, multiple trials have reported a marked and coherent effect of time-of-day ICI administration and patients’ outcomes. The biological circadian clock indeed temporally controls multiple aspects of the immune system, both directly and through mediation of timing of lifestyle actions, including food intake, physical exercise, exposure to bright light and sleep. These factors potentially modulate the immune response also through the microbiome, emerging as an important mediator of a patient’s immune system. Thus, this review will look at critically amalgamating the existing clinical and experimental evidence to postulate how modifiable lifestyle factors could be used to improve the outcomes of cancer patients on immunotherapy through appropriate and individualised entrainment of the circadian timing system and temporal orchestration of the immune system functions.


Introduction
Discoveries in immunotherapy have revolutionised cancer treatment.In 2018, James Allison and Tasuku Honjo won the Nobel Prize in Medicine for their work investigating the proteins CTLA-4 and PD-1 (1).Found on T cells, these proteins acted as checkpoint molecules, moderating T cell activation and preventing overactivation.As T cells are also involved in immunosurveillance of cancer cells (2), tumours can exploit CTLA-4 and PD-1 expression to evade host immune response.Inhibiting these checkpoint molecules can therefore enhance the antitumour immune response (3).
The antibodies subsequently developed to target checkpoint molecules and block their function are referred to as immune checkpoint inhibitors (ICI) and are the most widely used form of immunotherapy in cancer clinics.Currently, ICI are licensed to treat a wide array of cancers, including melanoma, lung, head and neck, renal, mesothelioma, breast, oesophageal, gastric, colorectal, biliary tract and urothelial carcinomas (4).
However, primary or acquired resistance remains a problem even with ICI (5).In addition, over activation of T cells can endanger self-tolerance, with the unavoidable risk of developing potentially life-threatening autoimmune adverse effects even years following initial treatment (6,7).
In patients unlikely to respond to ICI therapy, accurate prediction of efficacy and tolerability would allow clinicians to minimise adverse effects and delays to these inherently time-pressured treatment plans.For many reasons, including difficulties developing appropriate invitro assays, the determinants of ICI efficacy, tolerability, and deleterious interactions are not fully understood, but are generally appreciated to be multifactorial and likely involve both modifiable and non-modifiable factors.
Many biomarkers both from the original tumour and circulating cells have emerged as areas of research interest into the impact of ICI efficacy and/or tolerability (8,9).Alongside these factors, recent reports have highlighted the relevance of the circadian timing system (10).In turn, the CTS function is influenced by a host of lifestyle factors (11,12).Lifestyle factors are of particular interest to clinicians, as they allow outcomes to not only be predicted, but to be potentially manipulated as well.This aspect of non-pharmacological interventions in oncology is indeed rising growing interest recently (13,14).In this review, we critically summarise existing evidence on key lifestyle factors of interestdiet, physical exercise, and bright light exposurewith regards to ICI efficacy, through CTS manipulation, and impact on the immune system and the microbiome.We then discuss how these factors all interact to form a complex web which, with further understanding, may be manipulated by the empowered patient in conjunction with clinicians and various specialised healthcare professionals to optimise response to cancer immunotherapy (15, 16).

The circadian timing system
The human body has an inherent timekeeping ability.Its internal 'clock' is thought to have evolved thanks to the survival advantage conferred by the ability to predict bodily requirements and adapt accordingly (17,18).Circadian (i.e., with a period of about 24 hours) rhythms reflect the nature of the world humans have evolved inbeing that environmental properties change with time in a predictable pattern based on the Earth's rotation.In humans, the CTS hierarchically involves a central pacemaker, the suprachiasmatic nucleus in the ventral hypothalamus, and peripheral oscillators, temporally coordinated by hormonal, neural and physiological cues (19).Timing within cells themselves involve transcription-translation feedback loops and posttranslation modifications involving a set of core clock genes, which encode proteins with limited half-lives (17).The rhythmic oscillations in core clock genes coordinate, in a tissue specific function and directly and indirectly, circadian transcription of selected genes, which ultimately engender variation in cellular functions over the 24-hour period, including cancer-and immunity-related hallmarks (20)(21)(22).
Although the CTS does not require external input, it can be entrained using external stimuli, such as light exposure.Other stimuli which can entrain the CTS include feeding times, exercise, and social schedules (11,12,(23)(24)(25)(26). Consequentially, manipulation of exposure to rhythmic entraining cues can be used to enhance or shift the CTS function (27), with potential benefit for patients' wellbeing (28).
Timekeeping behaviour is also important to the immune system.Intrinsic clocks have been demonstrated to be present in a number of innate immune cells, causing rhythmic gating of function as well as regulating temporal spatial abundance (29).Natural killer cell cytolytic activity was found to be suppressed in correlation with altered clock gene expression in rats experiencing a simulation of chronic shift-lag, which was also associated with increased lung tumour growth (30).Additionally, experimental disruption of host circadian rhythms has shown to create an immunosuppressive remodelling in the tumour microenvironment, promoting cancercell proliferation and metastatic spread (31, 32).Evidence of circadian rhythmicity has also been found in the adaptive immune system in regulating CD8 + T-cell and dendritic cell differentiation and trafficking, with implications in cancer immunotherapy (29,33).Studies in night shift work in humans corroborate experimental evidence on a negative effect of circadian disruption on immune system physiology (34).
The circadian rhythmicity of the innate and adaptive immune system ensures proportionate responses to infections, whereas dysregulation presents acutely in an inflammatory cytokine syndrome or manifests long-term as chronic inflammatory conditions, with relevant therapeutic implications in oncology as well as in many other medical conditions and procedures (35,36).
The taxonomic composition of the microbial ecosystem, principally but not solely in the gut, has been associated with the incidence and clinical course of many different diseases, as well as with response to specific treatments (37).The mechanisms involved, which can display circadian oscillations (38, 39), include direct vagal stimulation, inflammation processes, and production of cytokines and metabolites (38,40).Of paramount interest here is the growing evidence of the impact of the gut microbiota on ICI efficacy (41).Alongside this, the CTS and microbiome have been demonstrated to have intertwined relationships illustrated best by research showing how in combination they can synchronize bidirectionally the body's metabolic response to diet (42) as well as light (43), exercise (44) and socialisation (45).Thus, the gut microbiome, itself potentially modifiable through iatrogenic interventions (46) takes a pivotal role in the rhythmic interplay among malignant processes, metabolism and immunity (13,38,(47)(48)(49).Indeed, as a developing theme, the gut microbiome has been demonstrated as having effects on innate immunity, adoptive immunity and intriguingly direct within the tumour microenvironment (50).
The link between the immune system and the CTS has been used to investigate potential ways of optimising response to various cancer treatments, including ICI.In particular, the time of day of ICI administration has been shown to be an independent prognostic factor for overall survival in several cancer types, with consistent findings disfavouring late afternoon administration (51).As the CTS can be entrained through modifications to lifestyle determinants, it therefore stands to reason that via the CTS certain lifestyle modifications could ultimately positively impact overall survival in cancer patients receiving ICI (Figure 1).

Light exposure
Photic signals from the retina to the suprachiastmatic nucleus (SCN) encodes time of day information regarding the environmental surroundings (52).Photic signalling integrated in the SCN modify cellular and molecular activity of astrocytes, neurones and synchronises peripheral clock activity of organs (52, 53).The SCN interacts with peripheral clocks via inputs from the endocrine and adrenergic nervous system (54) resulting in activation of immune cells (55).
However, photic signalling information can be altered in cancer patients because of insufficient physiological bright light exposure during the day and experience of artificial light at night (ALAN).For example, ALAN affects both innate and adaptive immune function in invertebrates, birds, and rodents with robust pineal melatonin rhythm (43).Abnormal photic exposure not consistently encoding time-of-day information can affect innate and adaptive immune activity (44,45,56,57), potentially impacting the efficacy of ICI.Although the impact of circadian photic schedule on ICI remains putative to date, in other conditions such as in psychiatry, light-based chronotherapy has shown positive therapeutic interactions with pharmacological and other behavioural interventions (58-60).Although prone to intrinsic constraints in the anatomical region of measured light exposure, contemporary digital tools allow for circadian evaluation of light exposure schedule and intensity, with potential cancer chronoimmunotherapeutic overtones (61,62).
It is shown trafficking of immune cells are affected in a time of day dependent fashion (63, 64) Photic exposure entrains immune cell trafficking via the adrenergic nervous system (63,64).No direct evidence exists to our knowledge on the impact of photic scheduling and entraining effects on outcomes of ICI, chemotherapy or other targeted therapies used in cancer.There are experimental and clinical data to support the impact of daytime bright light exposure and avoidance of artificial light at night, on both the immune and microbiome activity (43, 65-68).For instance, in the absence of light, the sympathetic nervous system triggers the pineal gland to produce melatonin, which synchronises SCN activity to peripheral clocks of immune cells (57, 69).Melatonin regulates innate, cellular and humoral responses of the immune system through modulating production of cytokines and oxidative stress (57).Additionally, melatonin acts as an immunostimulant under basal or immunosuppressed conditions, providing more effective early immune response against external stressors, such as viruses or parasites (70).However, in transient or chronically exacerbated immune response states, melatonin exerts negative regulation and can be regarded as an anti-inflammatory molecule (71).

Physical exercise
Physical exercise has been shown to strongly entrain the human circadian timing system, particularly through its effects on skeletal muscle and the cardiovascular system (82,83).Furthermore, peripheral clocks within cardiovascular cells are key for modulating endothelial function, vasodilation, resistance, blood pressure, heart rate and several other key functions (84).Aerobic exercise induces neuroendocrine changes including increased production and release of melatonin and lower cortisol levels at night.This allows resynchronisation of the circadian clock, resulting in better sleep quality, lower blood pressure and heart rate (82).
Entrainment of the circadian rhythm via exercise occurs even through low-intensity exercise and may be partly driven by changes in body temperature during physical activity (85).The degree to which physical exercise increases the body temperature is also dependent on the circadian phase, being larger in the rest phase than in the active phase (84).Moreover, combining photic cues, and non-photic exercise cues, has been shown to result in entrainment of the human clock at a faster rate than those with limited exercise (82).
Altogether, appropriate physical exercise in terms of timing, intensity, duration and type, adapted to the individual constraints of cancer patients, can be exploited to entrain the CTS and increase the robustness of circadian rhythms.Mobile health devices can lend useful tools to implement tailored circadian-based exercise schedules, even in cancer patients on ICI (16,86,87).
Although no direct evidence on the impact of physical exercise on outcomes on ICI is available to date, its impact on both the immune system and the microbiome supports its individualised manipulation to try to increase circadian-based ICI efficacy.Indeed, physical exercise has several immunomodulatory effects, including immune cell mobilisation in the blood, particularly PD-1+ CD8+ T cells redirected to peripheral tissues, which are crucial for host defence against tumours (88,89).
An additional study analysing mice with pancreatic cancer demonstrated an improved responsiveness to immunotherapy in mice that exercised regularly, compared to those that did not.Mice who had regular exercise also had a greater antitumor response and an increased volume and influx to tumours of NK and CD8+ T cells (90).
Moreover, regular exercise influences the gut/brain axis, leading to an anti-inflammatory, immunoregulatory state and enriched gut microflora diversity (91).Indeed, multiple factors have been associated with intestinal dysbiosis in cancer patients (92), and physical exercise, alongside diet, could be a potentially modifiable element to ameliorate the gut microbiome in order to maximise benefit from ICI.

Diet
Our eating habits generally follow a broad pattern that repeats every 24 hours.This pattern will vary from person to person and culture to culture, however commonly it may include three meals of various composition at a similar time each day.Interactions between the CTS and diet can therefore be divided into those relating to meal timing and those related to meal composition.Evidence suggests both these factors interact with the CTS (25, 93).
Food is one of the main synchronisers of the peripheral clocks (94).Both meal timing and meal composition can disrupt and reprogramme the CTS by altering clock gene expression, causing reorganisation of liver metabolic pathways and altered pancreatic insulin secretion (95, 96).Thus, with regards to circadian entrainment, both timing, including fasting duration, and composition of the meal are relevant and could be potentially manipulated for therapeutic purposes.Modern digital tools can provide monitoring capability of feeding and fasting habits over the 24-h period and a way to behavioural dietary interventions (16,86,(97)(98)(99).
Furthermore, diet can influence markers of immune function, with an association between diet and incidence of several immunemediated diseases including allergy, diabetes, and cancer reported (100).Moreover, fasting can influence immune responses in tumour-laden mice, with twice-monthly fasting resulting in higher white blood cell count and reduction in neoplasms despite no change in calorie intake (101).The influence of circadian dietary pattern on the immune system has also been explored, with studies showing associations between circadian feeding cycle, fasting period and alterations in both adaptive and innate immune response, with potential therapeutic implications (56,102,103).
Modifying diet also affects the gut microbiome, with different diets associated with noticeably different abundances and diversity of gut microbiota (104)(105)(106).
Interactions between outcomes on ICI and diet are thought to often occur via the microbiome, with studies reporting correlations between diversity and relative abundance of specific species, such as Akkermansia and Ruminococcaceae (107-109).
Contrarily to light exposure and physical exercise, there is clinical evidence on the impact of diet type on ICI efficacy.Although there is some discordance between studies, and heterogeneity with regards to cut-offs, adherence and duration of particular diets, disease types and clinical outcomes, there is an overall trend towards better outcomes associated with what is regarded as an healthy diet in humans in general by the WHO (110).For instance, high amount of fruit and vegetable, and low amount of dairy portions, were significantly associated with clinical benefit from ICI therapy (111).Specifically, increased fibre intake (threshold of 20g per day or more), higher adherence to a Mediterranean diet (rich in whole grains, fish, nuts, fruit, and vegetables, and low in red and processed meat), and a periodic fasting-mimicking diet (consisting of a nutritional composition that mimics fasting) displayed beneficial impact in patients receiving ICI in various studies (112)(113)(114).
Moreover, normal (> 30 ng/dL) vitamin D3 levels, whether naturally-occurring or through oral supplementation, were associated with significantly better outcomes (115).Furthermore, experimental evidence suggests an impact of ketogenic (low carbohydrate, low protein, and high fat) diet, of dietary aminoacid restriction and of polyphenols administration on ICI efficacy (116-118).
Interestingly, defecation frequency was also relevant, with emptying bowels less than daily associated with poor response to ICI (111).

Discussion
Immune checkpoint inhibitors have provided enormous benefit in the management of an ever-expanding array of cancer types, with dramatic increases in overall survival in those who respond.Their current main weaknesses lie in a variable response rate risk of toxicity.Recent studies have consistently reported increased efficacy of ICI therapy when infusions were administered in the morning, and that timing of immunotherapy is an independent prognostic factor for overall survival (10, 51, 119-126).This suggests a link between ICI efficacy and the CTS, which is responsible for circadian variations in many physiological features.In reporting a correlation between time of administration and efficacy, the findings suggest the CTS could be harnessed by clinicians to improve ICI efficacy.In order to do this, it is important a patient's CTS is entrained, as any benefit could be impaired by CTS disruption.Indeed, circadian disruption (evaluated with continuous wrist-actigraphy or with diurnal salivary cortisol slope) has been associated with shorter overall survival in various cancers, but not yet in those treated with ICI (127,128).
Conveniently, the CTS can be entrained by numerous lifestyle factors which have also been shown in studies to have independent effects on the immune system, the microbiome and sometimes on ICI efficacy, as shown above and as brilliantly discussed by others very recently (13).Both ICI therapy and circadian systems are complex, and further research will be needed to better understand the science of their interactions in order to harness this insight for therapeutic purposes.
Although extensive research is aiming at identifying tumourassociated or host-related factors predicting for ICI efficacy or tolerability, most of them are immutable and intrinsic to the patient and the disease, thus potentially impossible to be manipulated (e.g., PD-L1 expression levels) or very hard to be meaningfully modified in a relatively short timeframe (e.g., body mass index) (129).Similarly, the use of some drugs (e.g., antibiotics, proton-pump inhibitors and obviously steroids) has been shown to impair ICI efficacy in retrospective studies (130).Yet, most likely these drugs have been prescribed for a therapeutic reason and arguably it would not be easy to avoid them altogether in clinical practice.
Conversely, lifestyle interventions, including light exposure, physical exercise and diet, could be allegedly manipulated more easily to obtain the maximal therapeutic benefit from ICI (Table 1).Thus, a circadian-based optimisation of entraining cues and timing of administration could safely improve the outcomes of cancer patients treated with ICI.
However, this would require dedicated observational and interventional studies, with a robust translational component, in order to precisely and dynamically personalise lifestyle modifications.Indeed, the intertwining between these factors are multiple and complex, and involve hormonal messaging (e.g., melatonin, Vitamin D), unavoidable interactions (e.g., between outdoors physical activity and exposure to bright sunlight), and indirect microbiome-mediated mechanisms.
Further, they are all intrinsically bound to occur at a certain time of the day, thus impacting on the CTS and its temporal control of the immune system and of pharmacological determinants (136, 137).
Thus, although with this brief overview we have critically discussed photic stimuli, physical exercise and dietary factors, encompassing clinical and experimental findings, we believe that additional research will be of great interest and should be warranted in furthering our understanding of the effects of lifestyle factors on ICI efficacy as a whole, through modulation of the CTS, and the temporal organisation of the immune system and the microbiota.
However, difficulties with this approach should be acknowledged, including the intrinsic heterogeneity in populations, studies and

Intervention Effects
Aspects to be considered/ optimised outcomes, and, for instance, microbiome composition across cohorts (138), as well as the to interventions to factor into a patient's cancer treatment plan.This tolerance is indeed equally relevant when using lifestyle modifications as treatments.Consideration should be given as to the likeliness of patients with cancer and undergoing cancer treatment being able to enact and maintain lifestyle changes without unduly impacting their quality of life.For the patient, the impact of certain lifestyle modifications may not outweigh the possible benefit of increased ICI efficacy in a trade-off which will be personal to the patient.
It is difficult to discuss the potential for lifestyle modifications to optimise cancer treatment further without taking the time to emphasise the importance of the individual patient.Not only will optimisation of cancer treatment have to consider cancer subtype and patient chronotype, but also the patient's symptoms, comorbidities, habits, health beliefs, socio-economic status, social support, self-determination, and values in helping them make informed decisions on how best to utilise lifestyle modifications to optimise their cancer management.Practical implementation of such approaches could also be challenging, without appropriate support.Tellingly, surveys carried out exploring how often patients implement lifestyle changes after cancer diagnosis found that 41 to 65% of patients made dietary changes post-cancer diagnosis and 14 to 27% increased their level of exercise (139,140).Future research could also make use of digital technologies to monitor circadian biorhythm to further refine our understanding of the correlation between the CTS and outcomes on ICI (127).
In summary, building on evidence showing the CTS plays a role in increasing ICI efficacy and circadian disruption have deleterious effect on cancer patients survival, we argue CTS precise and personalised entrainment by lifestyle factors such as photic stimuli, diet composition and timing, and physical exercise could be harnessed to potentially increase ICI efficacy.Conveniently, existing evidence suggests these behavioural interventions shown to improve outcomes on ICIeither directly or via the gut microbiotaregularly are associated with healthier lifestyle habits, with intrinsic health benefits.Combining these findings, the CTS could feasibly be entrained by a patient-tailored combination of lifestyle determinants of ICI efficacy to maximise response, with future research offering patients and clinicians an expanding evidence base on which to draw from.

FIGURE 1 Figure
FIGURE 1Figure showing some of the reported relations between lifestyle factors, the CTS, the microbiome, and ICI efficacy.

TABLE 1 Table
of potential lifestyle interventions, their effects, and clinical considerations for studies/deployment.