Most aspects of physiology, including immunity, present 24-h variations called circadian rhythms. In this review, we examine the literature on the circadian regulation of CD8+ T cells, which are important to fight intracellular infections and tumors. CD8+ T cells express circadian clock genes, and ∼6% of their transcriptome presents circadian oscillations. CD8+ T cell counts present 24-h rhythms in the blood and in secondary lymphoid organs, which depend on the clock in these cells as well as on hormonal rhythms. Moreover, the strength of the response of these cells to Ag presentation varies according to time of day, a rhythm dependent on the CD8+ T cell clock. The relevance of CD8+ T cell circadian rhythms is shown by the daily variations in the fight of intracellular infections. Such a circadian regulation also has implications for cancer, as well as the optimization of vaccination and immunotherapy.

CD8+ T cells are potent cells of the adaptive immune system able to eradicate intracellular infections, control chronic infections, and eliminate tumors. Following recognition of a peptide fragment of the Ag presented by MHC class I molecules on APCs in the lymph nodes draining the site of infection, the few naive CD8+ T cells expressing a TCR specific for the Ag will undergo massive proliferation. This is accompanied by their differentiation into effector CD8+ T cells armed to control the infectious agents or the tumors. These effector functions include the production of cytokines (IL-2, IFN-γ, and TNF-α) and cytotoxic molecules (perforin and granzymes). The cytotoxic activity of CD8+ effectors, via granzyme secretion or Fas ligand–Fas interaction, will mediate the killing of infected and cancer cells. Following pathogen clearance, 90–95% of the effector CD8+ T cells will die during the contraction phase while the remaining 5–10% will further differentiate into memory CD8+ T cells. These memory CD8+ T cells will provide long-term protection against reinfection and cancer relapse (1). The understanding of all of the physiological and molecular events controlling CD8+ T cell response is pivotal to develop better vaccination and cancer immunotherapy strategies. Recent years have shown that circadian rhythms are among the physiological processes that influence the CD8+ T cell response.

All aspects of physiology vary according to the time of day and night. Although such rhythms in physiology appeared during evolution as an adaptation to the cycles of the environment, they are not occurring passively, as a direct response to day/night cycles. Instead, these rhythms, called circadian rhythms, are generated by circadian clocks within the organism (2). These circadian clocks can run with a period of ∼24 h, even in the absence of timing cues from the environment (although environmental cues can reset the endogenous clocks, as happens for example when we recover from jetlag) (3). The molecular mechanism of circadian clocks, located in most cell types throughout the body, is based on transcriptional–translational feedback loops involving a number of circadian clock genes, in which clock proteins regulate their own expression in a molecular cycle that lasts ∼24 h (4). In the main feedback loop, transcription factors CLOCK and BMAL1 together activate the expression of Period (Per)1/Per2 and Cryptochrome (Cry)1/Cry2, whose protein products form complexes and repress the activity of CLOCK/BMAL1, and thus their own expression (2, 4). As a result, many clock genes show a circadian rhythm in their mRNA and protein expression. Moreover, because CLOCK/BMAL1 and other clock transcription factors are more or less active across the 24-h cycle, they can bind other loci in the genome and regulate thousands of other genes in a rhythmic fashion: these are called clock-controlled genes, and the fraction of transcripts showing a 24-h rhythmicity ranges from 4 to 20% depending on the organs and cell types (5).

The immune system is not an exception within the circadian system: all immune cell types express clock genes and thus possess their own endogenous clocks (6, 7). As a consequence, many studies have uncovered 24-h rhythms in most immune functions, including leukocyte migration, cytokine and chemokine expression, phagocytosis, or allergic reactions (68). As many other immune processes and contexts would deserve being studied under a circadian angle, the readers can find some tips in designing such experiments under Circadian toolbox for immunologists below. Within the organism, the various tissue clocks are forming a network, with a master clock in the suprachiasmatic nucleus in the brain, and clocks throughout the body sending rhythmic cues, for example in the form of rhythmic humoral molecules (2). With this in mind, a circadian rhythm displayed by a certain tissue or cell type could be due to the clock within these cells, or to factors external to this particular tissue (such as rhythmic hormones, neuronal cues, rhythmic metabolites), or a combination of the two. Also of note, 24-h rhythms in immune processes (and more generally physiological processes) might be driven by rhythms of food availability, sleep/wake cycles, or other rhythmic external cues; oscillations termed circadian rhythms are those that persist in the absence of external timing cues. In this review, we focus on the regulation of CD8+ T cells by circadian clocks. The reader is referred to other recent reviews for a broader and comprehensive overview of the circadian regulation of immune functions (8, 9).

The demonstration of a circadian rhythm of Per2 expression in mouse lymph node explants initially raised the possibility that T and B lymphocytes, the main constituents of lymph nodes, possess an endogenous circadian clock (10). The circadian expression of several other clock genes was subsequently confirmed (11). The studies that evaluated whether T cells express circadian clock genes in a rhythmic manner first focused on mouse and human CD4+ T cells (12). The expression of the molecular clockwork in CD8+ T cells was expected given the observations that the magnitude of the CD8+ T cell response to TCR triggering in vitro or Ag presentation in mice varies according to the time of day (11). In 2015, using Per1-Venus reporter mice, Hemmers and Rudensky confirmed Per1 expression in mouse splenic CD8+ T cells, although with only a minor variation during 24 h (13). More recently, using PER2:luciferase reporter mice (knock-in mice that express a fusion of the PER2 and luciferase proteins), we have shown sustained rhythmic expression of PER2 protein in CD8+ T cells cultured in vitro during several days (14). However, the amplitude of the rhythm was lower than what has been reported for other cell types, including immune cells such as macrophages (10) and dendritic cells (14). Nevertheless, this demonstrates that CD8+ T cells possess an endogenous and cell-autonomous circadian clock. The presence of an endogenous clock within human CD8+ T cells still needs to be confirmed.

It has long been recognized that lymphocyte counts in the human peripheral blood vary according to the time of day (1519). Both CD4+ and CD8+ T cell counts are rhythmic, with peak levels during the night that decline in the morning and stay low during the day. This is true for naive, effector, and memory CD4+ and for naive CD8+ T cell subsets (16, 18, 19). Human effector CD8+ T cells show an opposite rhythm, with peak cell counts during the day (18). The variation in naive T cell counts in human blood is controlled by the circadian production of cortisol that regulates CXCR4 expression on T cells, which then influences their migration to the bone marrow or other sites that produce CXCL12, the ligand for CXCR4 (16, 18). In contrast, the rhythm of effector CD8+ T cells is controlled by catecholamines (adrenaline, noradrenaline) (18).

In nocturnal rodents, lymphocyte counts in the blood are high during the day, which corresponds to the resting phase of these animals. More specifically, CD4+ and CD8+ T cell numbers in the mouse blood are highest at zeitgeber time (ZT)4–5 (beginning of the day) and lowest at ZT16 (beginning of the night) (20, 21). In contrast, an opposite rhythm of CD4+ and CD8+ T cells is observed in the lymph nodes, with maximal levels in the night. This suggests that in mice, T cells redistribute from the blood to lymph nodes during the night (22, 23). Interestingly, the rhythm of cell numbers in the lymph nodes is maintained in constant darkness (dark/dark [DD]), suggesting that it is controlled by the circadian clock (22). Accordingly, the T cell–specific genetic ablation of Bmal1 (an essential clock gene) abolished the oscillation of CD4+ and CD8+ T cell distribution in lymph nodes (22). Importantly, note that our group did not observe a rhythm of CD8+ T cell counts in both the lymph nodes and the spleen of mice in constant darkness (11, 14). One possible explanation is that Druzd et al. (22) only reported total cell counts in the lymph nodes in DD. Furthermore, another group did not observe variation in total CD8+ T cell counts in mediastinal lymph nodes following influenza infection of mice under a light/dark (LD) cycle (24). In rats kept in LD, CD8+ T cell counts in lymph nodes were shown to have daily variation (25). Altogether, these studies provide strong evidence for a rhythm of CD8+ T cell counts in blood, although contradictory results have been reported for secondary lymphoid organs. Therefore, further studies are needed to confirm whether CD8+ T cell distribution within lymph nodes and spleen varies during the day and whether this is regulated by the circadian clock.

A few mechanisms have been proposed for the daily variation of lymph node CD8+ T cell counts. First, in mice the migration and egress of T cells to and from the lymph nodes are influenced by the time of day. At the molecular level, the time-of-day difference in migration to lymph nodes is explained by a rhythm of CCR7 expression by CD8+ T cells and the CCR7 ligand CCL21 by lymph nodes whereas rhythmic egress is controlled by variation in the expression of S1PR1 (a receptor for the chemoattractant S1P that promotes egress to efferent lymphatic vessels, where expression of S1P is highest). Furthermore, the rhythm of CCR7 and S1PR1 expression in CD8+ T cells is abolished in T cell–specific Bmal1 knockout (KO) mice (22), suggesting a direct control by the circadian clock of these cells.

Second, glucocorticoids, hormones that are secreted by the adrenal glands in a circadian manner, were shown to regulate the expression of the IL-7 receptor α-chain (CD127) on murine CD4+ and CD8+ T cells. The expression of CD127, the receptor for the T cell survival cytokine IL-7, is expressed at its highest level at ZT16 (early night) on CD8+ T cells from the spleen. The daily rhythm of CD127 expression by CD8+ T cells was abolished in mice deficient for glucocorticoid receptor (GR) expression in T cells (Nr3c1fl/fl-Cd4-cre+) and in mice in which the GR-responsive enhancer of Il7ra was inactivated. These genetic manipulations also abolished the rhythm of CD8+ T cell counts in the blood, lymph nodes, spleen, and Peyer’s patches (23). The effect of the rhythmic daily expression of CD127 in CD8+ T cells on their daily oscillation in lymphoid organs was not solely due to an impact on survival but was mediated by the induction of the expression of CXCR4, the receptor for CXCL12 (produced in the lymph nodes and bone marrow), by IL-7 signals (23).

Third, a daily variation in lymphocyte counts (CD4+ and B cells; CD8+ T cells were not evaluated) in mouse lymph nodes was shown to be regulated by the circadian release of noradrenaline from adrenergic nerves, which innervate lymphoid organs, via β-adrenergic receptor signaling in lymphocytes (26). Previous work from the same group had shown that β-adrenergic receptor signaling enhances the responsiveness of the chemokine receptors CCR7 and CXCR4 (27). Whether adrenergic signals also influence the daily rhythm of CD8+ T cells in lymph nodes was not reported by Suzuki and colleagues (27) but is likely, given that β-adrenergic receptor signaling impacts the response of CD8+ T cells to CCL21 and CXCL12, the ligands for CCR7 and CXCR4, respectively.

In summary, several mechanisms converge to promote the retention of T cells in lymphoid organs at the night onset (Fig. 1): 1) glucocorticoids and inputs from adrenergic neurons affect T cell retention via the regulation of CXCR4 expression and signaling, respectively; 2) adrenergic signals and the clock transcription factor BMAL1 regulate CCR7 signaling and expression, respectively; and 3) BMAL1 controls S1PR1 expression.

FIGURE 1.

Circadian rhythm in CD8+ T cells. The presence of the molecular clock within CD8+ T cells combined with the effects of rhythmic circulating molecules on CD8+ T cells induces a rhythmicity of the expression of hundreds of clock-controlled genes (CCGs) and of signaling pathways within CD8+ T cells. This circadian regulation impacts CD8+ T cell abundance in the blood as well as the magnitude of the response of CD8+ T cells following Ag recognition. In the right panel, the size of items denotes relative abundance or activity. SCN, suprachiasmatic nucleus; CORT, corticosterone or cortisol; NA, noradrenaline.

FIGURE 1.

Circadian rhythm in CD8+ T cells. The presence of the molecular clock within CD8+ T cells combined with the effects of rhythmic circulating molecules on CD8+ T cells induces a rhythmicity of the expression of hundreds of clock-controlled genes (CCGs) and of signaling pathways within CD8+ T cells. This circadian regulation impacts CD8+ T cell abundance in the blood as well as the magnitude of the response of CD8+ T cells following Ag recognition. In the right panel, the size of items denotes relative abundance or activity. SCN, suprachiasmatic nucleus; CORT, corticosterone or cortisol; NA, noradrenaline.

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In vitro T cell proliferation

The presence of the molecular clockwork within CD8+ T cells raised the possibility that it would influence the response of CD8+ T cells. We have observed a circadian rhythm of CD8+ T cell proliferation when we stimulated CD8+ T cells collected from mouse lymph nodes during a 24-h DD cycle. Following in vitro anti-CD3 stimulation, faster proliferation was observed when CD8+ T cells were sampled at late day or night when compared with cells taken earlier in the daytime (11). This proliferation rhythm was abolished when the T cells were from mice with a dominant negative form of the transcription factor CLOCK (11). Interestingly, no rhythm of CD8+ T cell proliferation was observed when cells were stimulated with PMA and ionomycin, indicating that the rhythm of T cell proliferation relies on proximal TCR signaling (11). The protein level of ZAP70, a kinase controlling proximal TCR signaling, was shown to vary in a circadian manner in resting T cells, further supporting the notion that proximal TCR signaling is regulated by the circadian clock (11). Whether the human CD8+ T cell response to in vitro TCR stimulation is influenced by the circadian clock is unknown but is likely, as this was reported for human CD4+ T cells (12, 19).

In vivo response to vaccination

To determine whether the in vivo CD8+ T cell response varies depending on the time of the day, mice were vaccinated at ZT6 and ZT18 with bone marrow–derived dendritic cells (BMDCs) pulsed with class I–restricted OVA peptide and matured with LPS. This strategy was chosen to avoid any confounding effects of possible daily rhythm of APC functions (Ag processing and presentation, expression of costimulatory ligands, and production of cytokines). Using this vaccination strategy, which primes CD8+ T cell response within the spleen, we showed that the level of CD8+ T cell expansion was influenced by the time of vaccination with a twice higher expansion when mice were vaccinated at ZT6 (midday) compared with ZT18 (midnight) (11). These observations suggested that in vivo CD8+ T cell priming and expansion following antigenic recognition has a daily rhythm. To further show that CD8+ T cell priming is gated by the circadian clock, similar experiments were performed in constant darkness during a 24-h period. CD8+ T cell priming and expansion following BMDC vaccination showed circadian variation with a peak of the T cell response at circadian time (CT)6 (middle of subjective night), similar to what was observed under an LD cycle (14). Furthermore, deletion of the essential clock gene Bmal1 in mature CD8+ T cells abolished the rhythm of T cell expansion, demonstrating that this circadian variation is controlled by the clock intrinsic to CD8+ T cells (14). The production of IFN-γ by CD8+ T cells following BMDC vaccination was also circadian, but this reflected the difference of CD8+ T cell expansion and therefore suggests that the circadian clock mostly influences T cell proliferation and not acquisition of effector function (14). Moreover, the effect of the circadian time on the CD8+ T cell response was not due to difference in CD8+ T cell numbers in the spleen at the time of vaccination, as in our hands we did not observe circadian variation of T cell numbers in the spleen (14). Moreover, we observed the highest response at CT6, which does not correspond to the peak of CD8+ T cell numbers in the spleen and lymph nodes reported by others (22, 23). The influence of the circadian rhythm on the level of CD8+ T cell expansion is biologically relevant, as it impacted the ability to control a challenge with a lethal dose of the intracellular bacteria Listeria monocytogenes encoding OVA, the Ag used for the BMDC vaccination (14).

The fact that the CD8+ T cell response was affected by the time at which vaccination was done suggests that circadian regulation is at the level of T cell priming. To understand how priming was affected by the time of vaccination, the transcriptome of naive CD8+ T cells sampled from mice at different circadian times was analyzed. This revealed that 5.9% of protein-coding transcripts showed a circadian rhythm. Pathway analysis on the rhythmic transcripts revealed a signature of genes involved in TCR signaling, including ZAP70 and the Akt-mTOR pathway, suggesting that naive CD8+ T cells are more ready to respond to Ag presentation at certain times of the day (Fig. 1) (14). The difference in CD8+ T cell response to BMDC vaccination was already apparent at day 3 postvaccination: at that early stage, Ag-specific CD8+ T cell expansion was already seen in mice vaccinated at CT6 but not yet when mice were vaccinated at CT18. Furthermore, 3 d after BMDC vaccination, the TCR-induced activation markers CD69 and CD5 were expressed at higher levels by Ag-specific CD8+ T cells vaccinated at CT6 compared with CT18. These observations are consistent with the notion that CD8+ T cells are more sensitive to TCR signaling at certain times of the day. In support of this, increased phosphorylation of Akt and S6 following a short (30-min) in vitro antigenic stimulation was observed when naive CD8+ T cells were collected at CT6 than CT18. Altogether, these data indicate that the circadian clock influences the transcriptome of CD8+ T cells, allowing for better signaling following TCR triggering during the daytime (14).

Using another vaccination model, Silver et al. (28) have reported a stronger T cell response and IFN-γ production when mice were vaccinated with OVA in the presence of the adjuvant CpG oligodeoxynucleotides at ZT19 (night) when compared with ZT7 (day). This contrasts with the observation that the CD8+ T cell response to BMDC vaccination is highest at ZT6/CT6 (midday) (11, 14). However, a direct comparison of these two studies is difficult given that only the total T cell response, and not the CD8+ T cell response, was reported by Silver et al. (28). Furthermore, their mode of immunization does not allow isolation of the effect of the circadian clock on the CD8+ T cell response, as the expression of the ligand for CpG oligodeoxynucleotide, TLR9, is rhythmic, peaking at ZT19 (28). Therefore, the enhanced T cell response following vaccination at ZT19 might result from a better response of innate cells and APCs at ZT19, which would then impact the T cell response.

The daily and/or circadian variation of CD8+ T cell response will likely influence the response of humans to vaccination. Until now, no studies have directly evaluated whether the CD8+ T cell response to vaccines is influenced by the time of vaccination. However, several studies have revealed that the time at which vaccination is done has an impact on the protective response developed in humans (reviewed in Ref. 7). Following influenza vaccination, some but not all studies reported that the time of vaccination influences the level of Ab produced in response to the vaccines (2931). The Ab response to hepatitis A vaccine was also influenced by the time of day but only in male subjects (31), whereas no effect was observed following hepatitis B vaccination and hexavalent vaccination (against diphtheria, tetanus, poliomyelitis, Haemophilus influenzae type B, hepatitis B, and Pneumococcus) (32, 33). More recently, studies have also uncovered a time-of-day dependence of the Ab response to COVID-19 vaccines, although with different peak times, whereas others have not observed a time-of-day effect (3437). As a CD8+ T cell response is induced by some of these vaccines and contributes to the protection against infection, it will be important to evaluate whether the time of vaccination influences the human CD8+ T cell response.

Response to Listeria monocytogenes

L. monocytogenes infection is a classical model to study the CD8+ T cell response. This bacteria replicates into the cytoplasm, allowing for Ag presentation by MHC class I molecules and induction of a CD8+ T cell response. The circadian rhythm of the CD8+ T cell response to infection with L. monocytogenes was studied by several groups. Following infection of mice with L. monocytogenes, it was first reported that the production of IFN-γ by CD8+ T cells at day 6 postinfection was higher when infection was done at ZT8 than ZT0 (late and early in daytime, respectively), suggesting a rhythm of the CD8+ T cell response (38). Unfortunately, no data on the expansion of Ag-specific CD8+ T cells, using tetramer staining, were reported, preventing the determination of whether the daily variation was due to differences in CD8+ T cell expansion or effector differentiation (38). These results agree with the rhythm of CD8+ T cell response to BMDC vaccination (14), further supporting the influences of the circadian clock on the CD8+ T cell response.

In contrast, no daily rhythm of the CD8+ T cell response to Listeria infection was observed in another study (13). In this report, the response to L. monocytogenes expressing OVA was evaluated using the adoptive transfer of wild-type and Bmal1-deficient OT-I TCR transgenic T cells, expressing a TCR specific for the OVA257–264 peptide presented by the MHC class I molecule Kb, into congenic recipients, followed by infection at ZT2 (early day) or ZT14 (early night). At day 7 postinfection, the authors did not observe any impact of the time of infection on the IFN-γ and TNF-α production by the adoptively transferred OT-I CD8+ T cells, with no influence of Bmal1 (13). However, they observed a decreased IL-2 production by OT-I T cells when mice were infected at ZT14 compared with ZT2, a difference present in both wild-type and Bmal1−/− OT-I T cells (13). Unfortunately, only the percentage of cytokine producing OT-I CD8+ T cells was reported. Considering our results using BMDC vaccination where we did not observe a variation in the proportion of cells producing cytokines within the Ag-specific CD8+ T cells, but a difference in expansion (14), it is still possible that the expansion of OT-I CD8+ T cells would be affected by the time of Listeria infection. Furthermore, the time points used for the Listeria infection (13) might have prevented detection of the rhythm of CD8+ T cell response, as these are time points where Nobis et al. (14) did not observe a difference in the CD8+ T cell response to vaccination.

In another study, it was reported that CD8+ T cell expansion in the spleen was higher when mice were infected with Listeria at ZT16 (compared with ZT4), which correlated with the time of the day where more CD8+ T cells were present in the spleen (23). Moreover, the authors showed that ablation of the rhythm of CD8+ T cell numbers present in the spleen, using mice deficient for GR expression in T cells or mice in which the GR-responsive enhancer of Il7ra has been inactivated, also abrogated the rhythm of the CD8+ T cell response to Listeria infection (23).

Although the results presented by the different groups seem contradictory, the use of different time points may explain the differences. Alternatively, these differences might also result from the infectious dose of bacteria, the route of administration, and the use or not of adoptively transferred CD8+ T cells. Therefore, further studies using several infection time points during 24 h will be needed to clarify the circadian regulation of the CD8+ T cell response to Listeria infection.

Response to viral infection

As described above, CD8+ T cells are important to target virus-infected cells. The CD8+ T cell response to influenza virus was shown to be influenced by the time at which the infection was done in mice (22). A 3-fold increase in IFN-γ–producing CD8+ T cells within the lungs was observed when infection was done at ZT8 (daytime) versus ZT20 (nighttime). However, whether this was a consequence of differences in CD8+ T cell expansion or their migration to the lungs was not addressed (22). In another study, no variation in the number of activated CD8+ T cells in the mediastinal lymph nodes and the lungs following influenza infection was observed at ZT11 and ZT23 (24). Unfortunately, the authors did not evaluate Ag-specific CD8+ T cells and did not report the production of IFN-γ by Ag-specific CD8+ T cells and therefore might have missed the daily rhythm of CD8+ T cell response to influenza infection.

The response of CD8+ T cells to lymphocytic choriomeningitis virus was similar when infection was done in mice at ZT1 or ZT13. This is true for both the expansion of CD8+ T cells specific for the gp33 and np396 epitopes and for their ability to produce IFN-γ and TNF-α (13). Moreover, the T cell–specific deletion of Bmal1 did not impact the CD8+ T cell response to lymphocytic choriomeningitis virus (13). However, the use of only two time points might have led the authors to miss the peak of the CD8+ T cell response.

Future studies are needed to define the extent of regulation of the CD8+ T cell response to infection by the circadian clock. This will require the use of more than two time points during 24 h and a comprehensive analysis of the Ag-specific CD8+ T cell response (identification of Ag-specific CD8+ T cells with tetramer staining and analysis of effector functions). Experiments performed in constant darkness will be necessary to determine the circadian nature of the rhythms. However, one should keep in mind that other immune cell types might respond in a circadian manner and that this may influence the CD8+ T cell response. There might be, for example, a circadian regulation of CD4+ T cells, which might lead to a rhythm of the help to CD8+ T cells. Therefore, the intrinsic role of the circadian clock in CD8+ T cells can be revealed using mouse models in which key components of the molecular clockwork are inactivated solely within CD8+ T cells such as when Bmal1 floxed mice are cross to E8I-cre mice.

It will also be important to identify the molecular bases of the control of the CD8+ T cell response by the circadian clock. In particular, experiments should define the genes directly regulated by clock transcription factors and how the identified genes control the CD8+ T cell response.

Another key possible impact of circadian rhythms relates to the response of CD8+ T cells following vaccination of human subjects. Current evidence points to an impact of the time of day on the Ab response, but knowledge on the effect on CD8+ T cells is lacking. This is particularly important for vaccines aimed at protecting against viral infections.

CD8+ T cells also play crucial roles during the antitumor immune responses and contribute to certain autoimmune diseases. However, the impact of circadian regulation of these responses have yet to be studied. This is highly relevant as tumor incidence is higher in shift workers (39, 40). Also, no studies yet have addressed a possible circadian regulation of CD8+ T cell exhaustion, which would be relevant for cancer immunotherapy. Indeed, circadian rhythm affects tumor development, an effect that may impact the antitumor immune response and response to immunotherapy (4147).

This section explains key nomenclature and concepts in chronobiology. It also presents considerations to be taken when performing circadian experiments.

  • ZT0 is the time of lights on and ZT12 is the time of lights off for animals on a 12-h light/12-h dark cycle. This is also referred as the LD cycle (Fig. 2A).

  • Under constant darkness conditions (DD), circadian time CT0–12 is the subjective day (rest period in nocturnal rodents, equivalent to the night in the previous LD cycle), and CT12–24 is the subjective night (active period, equivalent to the day in the previous LD cycle) (Fig. 2A).

  • (Fig. 2B presents some key characteristics of biological rhythms: period, amplitude, phase, mesor.

  • The presence of a rhythmic process in experiments done under an LD cycle is not necessarily indicative of a circadian rhythm. Indeed, the rhythm could be driven by the external LD changes (3).

  • To be considered as circadian, a rhythm should be endogenous, i.e., it can occur in the absence of external timing cues such as light (48). As such, experiments aiming at demonstrating that a rhythm is controlled by the circadian clock have to be done in DD over several (e.g., four or six) CTs during a 24-h cycle. Experiments done with only two time points (e.g., early day and early night) present limitations: even when there is a difference between time points, one cannot talk of a “rhythm,” only a time-dependent difference; and when there is no difference, it cannot be concluded that there is no rhythm because the selected time points might be on the shoulders of the rhythm (see blue dots in (Fig. 2B).

  • When performing a circadian analysis using mouse models, it is important to put the mice in LD for at least 2 wk (or even longer when the LD cycle has been shifted by 12 h), ideally in a room with no light infiltration from outside or in lightproof ventilated cabinets, to synchronize the rhythm of the animals. Then the mice are released in constant darkness (DD) and the circadian experiments are done 2–3 d later. This is important to prevent the possible influence of the previous LD cycle that could still be present at day 1. Note that over days the internal time (phase) of animals will deviate from the clock hour, as the endogenous period of mice is not exactly 24 h. Furthermore, the procedures should be done in the dark using vision googles or in dim red light to prevent any acute effect of light exposure.

  • Bmal1 is the only clock gene whose deletion on its own leads to complete loss of circadian rhythms (49). Because of this, Bmal1 deletion is often used to ablate clock function. Full body Bmal1 KO mice suffer from many health problems (e.g., behavioral, metabolic, early aging) (4952). Therefore, the use of tissue-specific Bmal1 KO is often used instead, using floxed mice (53) crossed to relevant cre-deleter strains. This also has the advantage of allowing definition of the role of the cell-intrinsic clock in the circadian rhythm that is studied in a particular cell type. Residual circadian rhythms following tissue-specific Bmal1 KO would indicate that other rhythmic cues, external to the tissue, are involved.

  • Circadian studies in human are usually done using a constant routine protocol, which consists in keeping the subjects awake under dim light, limiting their activity including a semirecumbent posture, and providing isocaloric hourly snacks (48). This minimizes environmental factors that could mask the measured circadian parameters. As in animal studies, rhythms observed in human subjects under conditions where there are external time cues (e.g., LD cycle, meals) cannot be termed circadian.

FIGURE 2.

Basic aspects and nomenclature of biological rhythms. (A) Schematic of light/dark (LD) cycle and constant darkness (dark/dark [DD]) conditions. Under LD conditions, ZT0 is the time of lights on, and ZT12 is the time of lights off for animals on a 12-h light/12-h dark cycle. In DD, CT0–12 is the subjective day and CT12–24 is the subjective night. (B) Key characteristics of biological rhythms.

FIGURE 2.

Basic aspects and nomenclature of biological rhythms. (A) Schematic of light/dark (LD) cycle and constant darkness (dark/dark [DD]) conditions. Under LD conditions, ZT0 is the time of lights on, and ZT12 is the time of lights off for animals on a 12-h light/12-h dark cycle. In DD, CT0–12 is the subjective day and CT12–24 is the subjective night. (B) Key characteristics of biological rhythms.

Close modal

CD8+ T cells possess an endogenous circadian clock that affects several aspects of T cell physiology. The abundance of CD8+ T cells in blood and secondary lymphoid organs varies according to the time of day. This circadian rhythm is controlled both by the endogenous clock within CD8+ T cells and by circulating factors expressed in a circadian manner. Furthermore, the endogenous CD8+ T cell clock impacts the response of CD8+ T cells to vaccination and infection. Further studies are needed to fully appreciate the impact of circadian rhythms on CD8+ T cell responses in diseases and to uncover how the clock acts at the molecular levels with CD8+ T cells.

We thank laboratory members for helpful discussions.

This work was supported by grants from the Canadian Institutes of Health Research.

Abbreviations used in this article:

BMDC

bone marrow–derived dendritic cell

CT

circadian time

DD

dark/dark

GR

glucocorticoid receptor

KO

knockout

LD

light/dark

Per

Period

ZT

zeitgeber time

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The authors have no financial conflicts of interest.