Immunological Memory Sleep After Vaccination Boosts

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Sleep after Vaccination Boosts Immunological Memory Tanja Lange, Stoyan Dimitrov, Thomas Bollinger, Susanne Diekelmann and Jan Born This information is current as of September 28, 2021. J Immunol 2011; 187:283-290; Prepublished online 1 June 2011; doi: 10.4049/jimmunol.1100015 http://www.jimmunol.org/content/187/1/283 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Sleep after Vaccination Boosts Immunological Memory

Tanja Lange,*,† Stoyan Dimitrov,*,1 Thomas Bollinger,‡ Susanne Diekelmann,* and Jan Born*

Sleep regulates immune functions. We asked whether sleep can influence immunological memory formation. Twenty-seven healthy men were vaccinated against hepatitis A three times, at weeks 0, 8, and 16 with conditions of sleep versus wakefulness in the following night. Sleep was recorded polysomnographically, and hormone levels were assessed throughout the night. Vaccination-induced Th cell and Ab responses were repeatedly monitored for 1 y. Compared with the wake condition, sleep after vaccination doubled the frequency of Ag-specific Th cells and increased the fraction of Th1 cytokine-producing cells in this population. Moreover, sleep markedly increased Ag-specific IgG1. The effects were followed up for 1 y and were associated with high sleep slow-wave activity during the postvaccination night as well as with accompanying levels of immunoregulatory hormones (i.e., increased growth hormone and prolactin but decreased cortisol release). Our findings provide novel evidence that sleep promotes human Th1 immune responses, implicating a critical role for slow-wave sleep in this process. The proinflammatory milieu induced during this sleep stage Downloaded from apparently acts as adjuvant that facilitates the transfer of antigenic information from APCs to Ag-specific Th cells. Like the nervous system, the immune system takes advantage of the offline conditions during sleep to foster adaptive immune responses resulting in improved immunological memory. The Journal of Immunology, 2011, 187: 283–290.

he immune system and the CNS share the ability to re- we show for the ﬁrst time that sleep indeed enhances the vaccine-

spond to external stimuli and to form memory (1). Vac- driven induction of immunological memory in the expansion, http://www.jimmunol.org/ T cination induces a multistep immune response eventually contraction, and maintenance phase as reflected by increased leading to differentiation of Ag-specific memory T and B cells as formation of Ag-specific Th cells and IgG1. effectors of immune protection (2, 3). After vaccination, APCs take up the Ag and migrate to lymphatic tissues to present part of the Ag at the cell surface to Th cells, thereby forming the so-called Materials and Methods Subjects immunological synapse and initiating the transfer of the antigenic information into long-term storage in Ag-specific T and B cells (1, Participants were 27 healthy, nonsmoking young men with a mean (6 SEM) 3). Formation of the immunological synapse represents an early age of 26.1 6 0.7 y (body weight: 81.4 6 1.9 kg; body mass index: 24.5 6 stage in the immune response that determines quantity and quality 0.5). They presented with a normal nocturnal sleep pattern and did not take by guest on September 28, 2021 any medication at the time of the experiments. None had a medical history of immunological memory, and it is at this stage adjuvants act to of any chronic disease or mental disorder. Acute illness was excluded enhance the immunogenicity of the vaccine (4). by physical examination and routine laboratory investigation, including Sleep generally regulates immune functions in a supportive chemistry panel, C-reactive protein concentration ,6 mg/l, and a WBC , manner (5–7). Sleep impacts APC and Th cell traffic and facili- count 9000/ml. Naive immune status against HAV and hepatitis B virus was explored by medical history and vaccination card and confirmed by tates the release of endogenous adjuvants like growth hormone HAV–Ab levels ,5 IU/ml and hepatitis B virus surface Ag (HBs)–Ab (GH), prolactin, and APC-derived IL-12, whereas it lowers the re- levels of 0 IU/ml, respectively. In addition, HBs and hepatitis B core–Ab lease of immunosuppressive hormones like cortisol (7–9). In this were negative in all subjects. study, we used an experimental vaccination approach against The men were synchronized by daily activities and nocturnal rest. They hepatitis A virus (HAV) to examine whether these effects of sleep had a regular sleep–wake rhythm for at least 6 wk before the experiments. All subjects were adjusted to the experimental setting by spending at least can boost immunological memory formation. To our knowledge, one adaptation night in the laboratory that took place before the experiment proper. The presence of signs of sleep disturbances including apnea and nocturnal myoclonus was excluded by interview and by recordings during this adaptation night. For private reasons, three subjects dropped off *Department of Neuroendocrinology, University of Lu¨beck, 23538 Lu¨beck, Ger- at the second vaccination and two further subjects at the third vaccination. many; †Department of Internal Medicine, University of Lu¨beck, 23538 Lu¨beck, Germany; and ‡Department of Microbiology and Hygiene, University of Lu¨beck, Twelve subjects were available at a follow-up examination 1 y later. The 23538 Lu¨beck, Germany study was approved by the Ethics Committee of the University of Lu¨beck. 1 All men gave written informed consent prior to participating in accordance Current address: Department of Psychiatry, University of California, San Diego, La with the Declaration of Helsinki. Jolla, CA. Received for publication January 4, 2011. Accepted for publication May 3, 2011. Experimental design and procedure at days of vaccination This work was supported by a grant from the Deutsche Forschungsgemeinschaft The participants were randomly assigned to a condition of sleep and a (SFB 654: Plasticity and Sleep). condition of continuous wakefulness (wake). Both groups were comparable Address correspondence and reprint requests to Dr. Tanja Lange, Department for age, body weight, and body mass index (p . 0.5). Each participant in of Neuroendocrinology, University of Lu¨beck, Ratzeburger Allee 160, Haus 50, both groups was injected with a vaccine into the deltoid muscle of the 23538 Lubeck, Germany. E-mail address: [email protected] ¨ nondominant arm at 8 AM three times (i.e., at weeks 0, 8, and 16), with 1 The online version of this article contains supplemental material. ml of a hepatitis A vaccine that was combined with a hepatitis B vaccine Abbreviations used in this article: Cy7, cyanin 7; EEG, electroencephalography; GH, (Twinrix, 720 IU HAV, 20 mg HBs; GlaxoSmithKline Biologicals, Rix- growth hormone; HAV, hepatitis A virus; HBs, hepatitis B virus surface Ag; REM, ensart, Belgium) (see Fig. 1B for study protocol). All primary vaccinations rapid eye movement; SWA, slow-wave activity; SWS, slow-wave sleep. were performed in February and March, and the third shot was done in June or July 2007, with no difference in timing between the sleep and wake Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 groups (p . 0.6). After immunizations, subjects were instructed to refrain www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100015 284 SLEEP AND IMMUNOLOGICAL MEMORY from physical or psychological stress and not to nap during the daytime. surface Ag (VP1–VP4, attenuated strain HM175, final concentration for Adherence to these instructions was confirmed by questionnaires. The each peptide 1.8 mg/ml, PEPscreen; ProImmune, Oxford, U.K.). The HBs participants returned to the laboratory at 9 PM for preparing blood sam- peptide pool consisted of 54 peptides of 15 aa overlapping by 11 made pling and, in the regular sleep condition, for standard polysomnographic from HBs (adw serotype, final concentration for each peptide 3 mg/ml, recordings. PEPscreen; ProImmune). Sleep in the sleep group was allowed between 11 PM (lights off) and 6:30 After a stimulation period of 6 h cells were surface-stained for CD3 AM (lights on) in the morning. In the wake condition, subjects stayed awake and CD4 for 15 min at room temperature. After washing, fixation, and in bed during this period in a half-supine position, watching TV, reading, permeabilization according to the manufacturer’s instructions (Cytofix/ listening to music, and talking to the experimenter at normal room light Cytoperm Kit; BD Biosciences), cells were stained intracellularly for (∼300 lx). Subjects of the wake condition were not allowed to sleep until 8 cytokines and CD154/CD40L (which detects both intracellular and surface PM the following day. For hormonal analyses, blood was sampled re- Ag) (12, 13). The following combination of mAb reagents were used in the peatedly during the experimental nights via an i.v. forearm catheter, which main experiment: CD3 PerCP, CD4 PE, CD154/CD40L allophycocyanin, was connected to a long, thin tube and enabled blood collection from an IL-4 PE-cyanin 7 (Cy7), IFN-g allophycocyanin-Cy7, IL-2 FITC, and adjacent room without the subject’s awareness or disturbing sleep. To TNF-a PE-Cy7. The Abs were all bought from or prepared as custom prevent clotting, ∼500 ml saline solution was infused throughout the ex- conjugates (IL-4 PE-Cy7 [clone 8D4-8], IFN-g allophycocyanin-Cy7 perimental period in both conditions. [clone B27]) by BD Biosciences. A minimum of 200,000 CD3+CD4+ All subjects rated their sleepiness (Stanford Sleepiness Scale) and mood events were collected for each sample on a FACSCanto (BD Biosciences). [using the short form of the German version of the Multidimensional Mood Data were analyzed using FACSDiva software. The FACS plots are dis- Questionnaire (10)] in the morning of the first night after the vaccinations. played as 5% probability with outliers. As expected, participants of the wake group scored higher in subjective For each sample, the percentages of total CD40L+ cells or CD40L+ cells ratings of sleepiness and lower in mood than the sleep group (p , 0.05 for coexpressing different cytokines in relation to total CD3+CD4+ cells were the three vaccinations). determined. Data for stimulation with anti-CD28/anti-CD49d alone were

subtracted from the percentages for the Ag-specific stimulation. Downloaded from Sleep, slow-wave activity, and hormones Measurement of the Ag-specific Ab response Sleep was recorded by standard polysomnography including the electroencephalography (EEG) (from C3 and C4, referenced to an electrode HAV- and HBs-specific Ab of the subtypes IgG1, IgG2, IgG3, and IgG4 attached to the nose), electromyography, and electrooculography. EEG were determined by ELISA. Microtiter plates were either precoated with signals were filtered between 0.15 and 35 Hz and sampled at 200 Hz. HAV (anti-HAVenzyme immunoassay; Mediagnost, Reutlingen, Germany) Subsequent 30-s epochs of polysomnographic recordings were scored or coated with HBs (incubation of 96 well microtiter plates (Nunc, offline by two experienced technicians according to standard criteria as Wiesbaden, Germany) with 0.04 mg HBs (Aldevron, Nu¨rnberg, Germany) http://www.jimmunol.org/ wake, sleep stages 1–4, and rapid eye movement (REM) sleep (11). Sleep for 9 h at 4˚C). After washing and blocking, serum was added at four stages 3 and 4 were combined to slow-wave sleep (SWS). dilutions (1:5, 1:25, 1:125, and 1:625) for 2 h and thereafter mouse anti- Determination of slow-wave activity (SWA) was based on power spectral human IgG subtype biotin-conjugated Ab (Sigma-Aldrich, Steinheim, analyses. For these analyses, first, all epochs with artifacts and arousals were Germany) for 1 h. Then, avidin–HRP conjugate (Sigma-Aldrich) was rejected semiautomatically (movement artifacts in the electromyography added for 30 min, followed by incubation with the enzyme substrate tet- exceeding 650 mV followed by visual inspection). Fast Fourier trans- ramethylbenzidine (Invitrogen, Karlsruhe, Germany) for 5 min. The re- formation, applied to successive 10.24-s epochs (2048 data points, Han- action was stopped with 100 ml2MH2SO4 (Merck, Darmstadt, Germany). ning window), was performed on all artifact-free epochs scored as non- OD was measured at a 450-nm wavelength with a microtiter plate reader REM sleep stages 2–4. SWA was defined by the mean power density (MRXII; Dynex, Denkendorf, Germany). For each IgG subtype, OD was within 0.68–1.17 Hz, covering the slow oscillation frequency band. Data then determined as weighted average across all dilutions. In addition to by guest on September 28, 2021 were averaged per subject across the entire night and across both recording IgG subtype analyses, total HBs-Ab were assessed at week 20 by quan- sites (C3, C4). Because of technical problems in two subjects, SWA could titative microparticle enzyme immunoassay (AxSYM AUSAB; Abbott, not be analyzed. Wiesbaden, Germany) to assess the responder status of each subject GH, prolactin, and cortisol were measured in serum using a commer- according to the recommendations of the permanent commission for cial assay (Immulite; DPC-Biermann, Bad Nauheim, Germany). Epine- vaccinations (14). phrine and norepinephrine were measured in plasma by standard HPLC Statistical analyses (ChromSystems, Munich, Germany). Serum/plasma samples were kept frozen at 270˚C until assay. Because hormonal levels were remarkably Data are presented as means 6 SEM. Normal distribution was confirmed stable for each subject across the three nights, for further analyses data by the Kolmogorov–Smirnov test for all variables of interest, except for were collapsed across these nights. the fraction of cytokine positive cells within the population of Ag-specific Th cells. These values were therefore log transformed prior to statistical Follow up procedures analyses. Analyses of differences between the effects of sleep versus wake relied on ANOVAwith a group factor sleep/wake and a repeated measures During the 7 d after each inoculation subjects were monitored with respect factor time covering the different times of measurement after vaccination to activities, stress, core body temperature and potential side effects. Blood (immune response: weeks 0–20; hormones: 11 PM–7 AM; n = 22). Age and samples to measure Ag-specific responses were drawn at 8 AM on 13 body weight were added as covariates in the analyses of the Th cell and occasions: immediately before and 1, 2, and 4 wk after each inoculation Ab response. The ANOVA model was used also to specify subsequently and again 1 y after the first inoculation (i.e., at weeks 0, 1, 2, 4, 8, 9, 10, 12, effects at single time points and for time periods (i.e., for average values 16, 17, 18, 20, and 52). At these time points, subjects were also asked with over respective periods after the first [weeks 0–8], second [weeks 8–16], respect to the occurrence of stressful events, infections, side effects and and third inoculation [weeks 16–20]), for the entire period throughout sleep habits during the prior week(s). weeks 0–20, and for the 1-y period (weeks 0–52), when main effects for 3 Measurement of the Ag-specific Th cell response the sleep/wake factor or sleep/wake time interactions were revealed to be significant. Where appropriate, df were corrected following the PBMCs were immediately isolated from 30 ml heparinized blood by Greenhouse–Geisser procedure. Ficoll-Paque gradient centrifugation in Leucosep tubes (Greiner Bio- Stepwise and backward regression analyses were calculated to assess One, Frickenhausen, Germany) according to the manufacturer’s instruction. relationships between the HAV-specific Th cell response and sleep stages PBMCs were washed twice and then left in tissue culture medium RPMI and Th cell response and hormones, respectively. Pearson’s correlation 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 0.1 coefficient was used for subsequent correlation analyses. A p value #0.05 mg/ml streptomycin (all from Sigma-Aldrich, Seelze, Germany). Five was considered significant. million cells in 0.5 ml medium were placed in 50 ml polypropylene tubes (Sarstedt, Nu¨mbrecht, Germany) and stimulated at 37˚C and 5% CO2 for 6 h with a pool of HAV surface peptides, a pool of HBs peptides, or co- Results stimulation and brefeldin A alone. Costimulation consisted of anti-CD28 Clinical course after vaccinations and anti-CD49d (BD Biosciences, San Jose, CA), both at 1 mg/ml, and was—like brefeldin A (10 mg/ml; Sigma-Aldrich)—included in all stim- All vaccinations were well tolerated. Subjects reported only minor ulations. The HAV peptide pool consisted of 73 (VP1), 53 (VP2), 59 side effects like pain at the injection site and fatigue, and these (VP3), and 3 (VP4) peptides of 15 aa overlapping by 11 made from HAV reports did not differ for the three vaccinations. As expected, The Journal of Immunology 285 reported fatigue was higher in the wake than sleep condition in the Sleep boosts the Th1 immune response developing after morning after the first postvaccination night (p = 0.02). Ques- vaccination tionnaires did not reveal any difference between the sleep and wake In addition to the increase in Ag-specific Th cells after vaccination, groups with regard to self-reported activities or stressful events we monitored the functional aspect of cytokine expression in these during the respective follow-up periods after the vaccination. cells. We found that vaccination induced a general predominance of Sleep proinflammatory and Th1 cytokine activity that primarily supports cellular adaptive immune responses (IL-2 . IFN-g TNF-a .. Sleep variables confirmed that subjects slept normally under lab- IL-4) (Supplemental Fig. 1). Specifically, vaccination induced oratory conditions with SWS predominant during the first night- a marked but short-lived increase in the fraction of IFN-g+ cells half and REM sleep dominating the second night-half. Table I within the CD40L+ Th cell subset, reaching a maximum 2 wk after summarizes sleep variables of all three nights with no significant the first Ag inoculation (collapsed across sleep and wake con- differences from session to session. ditions: 38.0 6 3.5 versus 19.2 6 2.7% at baseline, n = 27; p , 0.001), whereas the fraction of IL-2+ Th cells gradually rose after Sleep enhances the HAV-specific Th cell response to each vaccination reaching an ∼2-fold increased level at weeks 18– vaccination 20 (71.2 6 1.6 versus 43.0 6 5.7% at baseline, n = 22; p , 0.001). + Vaccination elicited a robust increase in CD40L HAV-specific In addition, we observed significant increases in the fraction of Th cells, which was already evident 2 wk after each inoculation TNF-a+ and IL-4+ Th cells, although these were less pronounced and reached a maximum between 2 and 4 wk after completion of (Supplemental Fig. 1). the three-shots schedule (collapsed across sleep and wake con- Interestingly, in subjects who slept the night after the first in- Downloaded from ditions: 0.124 6 0.014 versus 0.012 6 0.001% measured at oculation, the transient vaccination-induced increase in the fraction baseline, n = 22; p , 0.001). Although the Ag-specific Th cell of IFN-g+ cells within the CD40L+ Th cell subset at weeks 0–8 response to HAV vaccination has not been characterized pre- was significantly more pronounced than in those subjects who had viously, the magnitude of the response and its time course were stayed awake on this night (32.2 6 2.6 versus 22.9 6 3.1%, n = comparable in size with those following HBs vaccination (15; see 27; p=0.03) (Supplemental Fig. 1). The boosting effect of sleep also results below), and it clearly shows the phases of expansion on vaccine-induced increases in HAV-specific Th cells was indeed http://www.jimmunol.org/ (weeks 1 and 2, 8–10, and 16–18) and contraction/maintenance most prominent in the subset of IFN-g+CD40L+ Th cells (Fig. 2B, (weeks 2–8, 10–16, and 18–52) (see also Supplemental Fig. 4). 2C). Importantly, when the participants had slept on the night after inoculations, the increase in HAV-specific Th cells was clearly Sleep enhances the HAV-specific IgG1 response enhanced when compared with the condition of continuous Promoting both cellular and humoral defense mechanisms, the Th wakefulness. Overall (i.e., across weeks 0–52), we found a .2- cell response to vaccination represents a most immediate indicator fold higher percentage of Ag-specific Th cells during the sleep of effective antigenic memory formation (2, 3). Nevertheless, in than during the wake condition (0.123 6 0.022 versus 0.060 6 addition to the Th cell response, we determined the Ag-specific 0.009%, n = 12; p = 0.001) (Figs. 1A,2A). This boosting effect of B cell response by measuring Ag-specific IgG titers, which are by guest on September 28, 2021 sleep became significant already 8 wk after the first inoculation commonly used for quantification of the Ag-specific immune re- (0.031 6 0.005 versus 0.019 6 0.001% in the wake group, n = 27; sponse in the clinical setting. As expected (16), we found opso- p=0.04) and was most prominent after the second and third nizing HAV-specific IgG1 and IgG3, which markedly increased at inoculations with a maximum at weeks 18–20 (0.161 6 0.024 weeks 12 and 20 (p , 0.01) (Fig. 3A,3B). In contrast, HAV- versus 0.093 6 0.007%, n = 22; p = 0.001) (Fig. 1A). The en- specific IgG2 and IgG4 remained unchanged. When subjects slept hancement in HAV-specific Th cells was remarkably persistent. during the nights after inoculations, analogous to the Th cell re- We still observed doubling of HAV-specific Th cells in the sleep sponse, IgG1 levels were significantly higher than in those sub- group in the maintenance phase [i.e., 1 y after initial vaccination jects who had stayed awake at weeks 8–16 (p , 0.05). Indeed, (0.099 6 0.022 versus 0.044 6 0.008%, n = 12; p =0.004)] enhancing effects of sleep on Ab titers followed those on Th cells, (Fig. 1A). as indicated by significant correlations between IgG1 levels at

Table I. Sleep parameters

First Night (min) Second Night (min) Third Night (min) r Valuea Total sleep time 440.2 6 3.6 447.4 6 5.0 420.2 6 18.0 0.16 Wake 52.7 6 12.1 20.4 6 6.1 20.9 6 6.5 20.28 Stage 1 32.1 6 2.8 31.7 6 3.1 23.8 6 2.8 20.47 Stage 2 221.1 6 14.4 242.2 6 9.1 228.7 6 13.0 0.11 Stage 3 43.6 6 4.5 47.2 6 4.8 48.9 6 4.5 0.05 Stage 4 14.6 6 5.5 16.1 6 5.9 10.7 6 6.2 0.74* SWS 58.2 6 6.8 63.3 6 6.6 59.6 6 8.7 0.60(*) REM 73.1 6 3.7 84.5 6 5.8 83.0 6 9.6 20.25 Sleep onset latency 35.1 6 4.0 25.8 6 2.9 21.7 6 5.11 20.01 SWA (mV2) 101.1 6 13.7 102.5 6 11.4 109.5 6 20.6 0.72* Mean 6 SEM total sleep time and time (in min) spent awake (wake), in non-REM sleep stages S1–S4, SWS (equivalent to S3 plus S4) and in REM sleep during the nights after inoculations in the sleep condition. Sleep onset latency (in min) is indicated with reference to 11 PM (lights off). Bottom line indicates SWA (power density in the 0.68–1.17 Hz frequency band during S2 and SWS, in mV2). Right column indicates correlation coefficients between respective sleep parameter (collapsed across the participant’s three nights) and HAV-specific Th cells at weeks 18–20 (n = 9). Note most consistent relationship with SWS stage 4 and SWA. aSignificant coefficients are bold. *p , 0.05, (*)p = 0.09. 286 SLEEP AND IMMUNOLOGICAL MEMORY

FIGURE 1. Sleep enhances the HAV-specific Th cell response to vaccination. A, Emergence of CD40L+ HAV-specific Th cells (percentage of total Th cells) after HAV vaccination (three shots at weeks 0, 8, and 16 – vertical syringes) in two groups of subjects who either slept (d) or stayed awake (s) in the night following inoculations. y-axis is log transformed. Means 6 SEM are indicated. n = 12–27 for both groups. **p , 0.01, *p , 0.05, (*)p , 0.1 for comparisons between sleep and wake conditions. B, Study design. Two groups of subjects were tested who either slept (sleep group, n = 13) or stayed awake (wake group, n = 14) on the night after inoculations with HAV vaccine, taking place three times (i.e., at weeks 0, 8, and 16). Immediately before vaccinations, 1, 2, and 4 wk after each vaccination, and again 1 y after the Downloaded from first inoculation, blood was sampled to assess the Ag- specific Th cell response. The Ab response was assessed every 4 wk and again 1 y after the first inoculation. The schema below the study design provides more details about the procedure on each of the 3 d of inoculation. Inoculation took place at 8 AM. Subjects of the sleep group slept between 11 PM and 6:30 AM. http://www.jimmunol.org/ Subjects of the wake group stayed awake until 8 PM on the following day. Dots indicate time points of blood sampling. Sleep was monitored by polysomnography. by guest on September 28, 2021 week 12 and CD40L+ Th cells at week 10 (r = 0.69, n = 19; p , 19). We sampled blood during the nights after vaccination (every 0.01), confirming the view that Th1 cells drive the production of 1.5 h) to assess a potential impact of these hormones on the HAV- IgG1 (16, 17). specific Th cell response (Supplemental Fig. 2). As expected, sleep induced a profound increase in concentrations of GH and SWA predicts the HAV-specific Th cell response to vaccination prolactin when compared with wakefulness (p , 0.001). The in- We next investigated which processes during sleep may account for crease in GH occurred during periods of SWS dominating the the enhanced HAV-specific Th cell response after vaccination. For early night. In contrast, sleep was associated with decreased cor- this purpose, we examined by regression analyses whether certain tisol nadir levels during the early SWS-rich night-half (p , 0.02). sleep parameters predicted the later outcome of the immune re- This decrease was compensated by enhanced morning cortisol sponse as reflected by the number of HAV-specific Th cells. (As levels after sleep (p = 0.008). Sleep also diminished catechol- the relevant sleep parameters were stable across the three post- amine levels, which was somewhat more robust for norepineph- inoculation nights, their mean values were used for this analysis.) rine (p , 0.001) than for epinephrine (p = 0.006). To be noted, We found the highest correlations between the time spent in SWS although relatively increased compared with sleep, plasma con- stage 4 and the percentage of HAV-specific Th cells at weeks 18–20 centrations of epinephrine and norepinephrine during nocturnal of the vaccination schedule (r = 0.74; p = 0.02) as well as 1 y later wakefulness (12.8 6 1.1 and 263.7 6 18.7 pg/ml, respectively) (r = 0.90; p = 0.02) (Table I). Likewise, increased EEG SWA were still distinctly below the levels observed during daytime during non-REM sleep was significantly associated with increased waking (measured before vaccinations, 32.6 6 3.3 and 392.9 6 HAV-specific Th cell percentages at weeks 18–20 (r = 0.72; p = 33.2 pg/ml; n = 14; p , 0.001 for both comparisons) and were far 0.03) and 1 y later (r = 0.94; p=0.005) (Fig. 4A). Other measures, from reaching any level commonly associated with acute stress including REM sleep-related parameters, were not predictive (p . (20). This in conjunction with the finding that nocturnal average 0.20) (Table I). These data demonstrate a strikingly strong asso- concentrations of the primary stress hormone cortisol were closely ciation between adaptive immune responses and SWS. comparable between the sleep and wake conditions (p = 0.81) clearly excludes that responses to vaccination in the wake con- The endocrine milieu during SWS predicts the HAV-specific Th dition were substantially affected by acute stress. cell response to vaccination To dissociate the relative contributions of these hormones to the Brain–immune interactions during sleep are conveyed mainly via HAV vaccination response, we performed regression analyses on endocrine pathways, apart from direct neuronal influences on average concentrations of the hormones between 0:30 and 2 AM lymphatic tissue (1, 18). Immunostimulating hormones like GH (i.e., the time interval with predominant SWS and most prominent and prolactin as well as immunosuppressive hormones such as hormonal changes for all five hormones) (Supplemental Fig. 2). cortisol and catecholamines are strongly regulated by sleep (7, 8, The percentage of HAV-specific Th cells was used as dependent The Journal of Immunology 287

FIGURE 2. Sleep boosts the Th1 immune response developing after vaccination. Average frequencies of CD40L+ HAV-specific Th cells (percentage of total Th cells) (A) and average frequencies of these cells (CD40L+ HAV-specific Th cells) (B) that were specifically positive for the cytokines of interest (i.e., from left to right, IFN-g, IL-2, TNF-a, and IL-4). Means 6 SEM values are shown for the sleep (n) and wake conditions (N) during respective time intervals after inoculations: that is, weeks 0–8, weeks 8–16, weeks 16–20, and for the whole 1-y observation period Downloaded from (weeks 0–52); n = 12–27 for both groups. ***p , 0.001, **p , 0.01, *p , 0.05 for comparisons between sleep and wake condition. C, Representative dot plots of CD40L+ Th cells after HAV-specific in vitro stimulation at week 18 in one subject from the sleep and wake group, respectively. FSC, forward scatter; SSC, side scatter. http://www.jimmunol.org/ by guest on September 28, 2021

variable. We found that GH, prolactin, and cortisol significantly schedule (collapsed across sleep and wake conditions: 0.100 6 contributed to the fully developed HAV-specific Th cell response 0.017 versus 0.004 6 0.001% measured at baseline, n = 22; p , as measured at weeks 18–20, as well as to the response 1 y later, 0.001), with this pattern well agreeing with a previous report (15). explaining 48% (p = 0.002) and 56% (p = 0.02) of the variance, Compared with wakefulness, sleep on the nights after HBs vac- respectively. Adding epinephrine or norepinephrine concentration cination significantly enhanced both the CD40L+ HBs-specific Th to the analyses did not increase explained variance, which agrees cell response and the Ab response, although overall, these effects with previous findings showing that the effects of catecholamines appeared to be less consistent than for the HAV vaccination. The on the vaccination response are inconsistent (18). The combined enhancing effect of sleep on the frequency of HBs-specific Th action of GH, prolactin, and cortisol on the formation of the HAV- cells became significant after the second inoculation (p = 0.05, for specific immune response is best described by an “adjuvant fac- weeks 8–16) and continued during weeks 16–20 after the third tor” summarizing the average hormone concentrations (0:30–2 inoculation (p = 0.05) (Supplemental Fig. 3A). At the follow-up AM) during postinoculation nights by the formula GH 3 prolactin/ 1 y later, HBs-specific Th cell percentages in the sleep group were cortisol. This factor showed correlations as high as r = 0.71 (p , still on average distinctly higher than in the wake group, but 0.001) and r = 0.80 (p = 0.002) with the percentage of HAV- this difference failed to reach significance (0.057 6 0.018 versus specific Th cells at weeks 18–20 and week 52, respectively (Fig. 0.037 6 0.010%, n = 12; p = 0.18). 4B). These analyses suggest that GH, prolactin, and cortisol syn- In line with previous reports (21), the Ab response to HBs ergistically contribute to the sleep-dependent enhancement of the vaccination was restricted to the IgG1 subtype reaching peak vaccination response. concentrations toward the end of the 20-wk vaccination period. When the participants slept on the nights following vaccination, HBs-specific Th cell and Ab response this peak was distinctly enhanced (14.25 6 2.34 versus 7.21 6 The use of a combined hepatitis A/B vaccine allowed us to directly 1.72, n = 22; p = 0.04) (Supplemental Fig. 3B). This sleep-induced compare the response to HAV with that to HBs. Data regarding the increase in HBs-specific IgG1 was remarkably persistent and was response to HBs essentially confirmed findings after HAV vacci- also revealed at the follow-up examination 1 y later (p = 0.02) nation, and therefore, the figures are presented as supplemental (Supplemental Fig. 3B). Assessment of HBs-specific total IgG at material (Supplemental Fig. 3). Vaccination induced an increase in the end of the vaccination period (week 20) revealed that three CD40L+ HBs-specific Th cells, which started 2 wk after the first, subjects failed to reach the clinical criterion Ab level of 100 mIU/ and already 1 wk after second and third inoculation, respectively, ml IgG (indicating sufficient immunity) and had to undergo ad- and reached a maximum 2 wk after completion of the three-shot ditional inoculation. All three subjects were in the wake group. 288 SLEEP AND IMMUNOLOGICAL MEMORY Downloaded from

FIGURE 3. Sleep enhances the HAV-specific IgG1 response. A,HAV- specific IgG1 between weeks 8 and 16 were significantly higher in subjects who had slept the nights after inoculation (d) than in subjects who had stayed awake these nights (s). Mean 6 SEM extinction values (OD) are indicated; n = 10–23 for both groups. *p , 0.05, (*)p , 0.1 for com- http://www.jimmunol.org/ parisons between sleep and wake condition. B, IgG2, IgG3, and IgG4, which were not affected by sleep, are shown collapsed across sleep and wake conditions (gray circles). ##p , 0.01, #p , 0.05 for comparison with baseline; note, a significant response to HAV vaccination is revealed only for IgG1 and IgG3 with a first maximum at week 12 and a second at week 20.

Discussion The immunoregulatory functions of sleep are not well understood. by guest on September 28, 2021 By comparing the immune response after HAV vaccinations in healthy men who either slept or stayed awake in the night following FIGURE 4. SWA and adjuvant factor are strong predictors of the HAV- inoculations, we provide, to our knowledge, first-time evidence specific Th cell response to vaccination. A, Correlation coefficients be- that sleep acts like an adjuvant to enhance the Ag-specific Th cell tween sleep SWA (averaged across the three postinoculation nights) and + and Ab response. Sleep SWA and the associated hormonal milieu frequency of CD40L HAV-specific Th cells at (from left to right) weeks 8, 12, 16, 18, and 20 and 1 y after HAV vaccination. Below, scatter plots of in the postvaccination night were strong predictors of the sub- + sequent Th cell response. the correlations for the frequency of CD40L HAV-specific Th cells (percentage of total Th cells) for subjects of the sleep (d) and wake (s) Mounting an adaptive immune response that eventually results in condition at weeks 18–20 (left panel) and week 52 (right panel). B, Cor- Ag-specific memory is a slow multistep process taking a minimum relation coefficients between average GH, prolactin, and cortisol concen- of several days. Our findings of an enhanced Ag-specific Th cell trations during the early, SWS-rich part (0:30–2 AM) of postinoculation response after sleep occurring within 24 h postvaccination indicates nights and frequency of HAV-specific Th cells at weeks 8, 12, 16, 18, and an effect during the early stages of the immune response, most 20 and 1 y after HAV vaccination. (Analyses performed across sleep and likely on APC–Th cell interactions taking place during this time wake groups.) Note most robust correlations for the adjuvant factor de- window in lymphatic tissues (3, 4). Our analyses of sleep and scribing the synergistic action of the three hormones of interest by the endocrine activity suggest a scenario in which SWS plays a key formula GH 3 prolactin/cortisol. Underneath, scatter plots of the corre- role (Supplemental Fig. 4). SWS, a brain state hallmarked by EEG lations between the adjuvant factor and the frequency of HAV-specific Th SWA, stimulates release of immunostimulating hormones like GH cells (percentage of total Th cells) at weeks 18–20 (left) and week 52 (right). ***p , 0.001, **p , 0.01, *p , 0.05. and prolactin and inhibits immunosuppressive cortisol (7, 8, 19). These hormones are not only uniquely regulated by sleep but also affect the interaction between APC and Th cell and the response to tin strongly enhance APC-derived IL-12 that, along with Ag sti- vaccination, which is increased by GH and prolactin but reduced mulation, is an important signal switching Th cell differentia- by cortisol (22–24). Interestingly, such hormonal effects appear to tion toward increased Th1 cytokine production (9, 17, 25, 26). In be most pronounced within the first 24 h after inoculation (i.e., the line, sleep during the nights following the three inoculations im- time window of interest in the current study) (23, 24). proved the development of adaptive Th1 immune responses at The sleep-associated hormonal changes prompt a shift of the two functional levels. First, once early differentiation of IFN-g– type 1/type 2 cytokine balance toward enhanced activity of producing HAV-specific Th cells (i.e., within 2 wk) is increased, proinflammatory and Th1 cytokines (TNF-a, IL-12, IL-2, and IFN- sleep promotes IFN-g–dependent effector functions. Second, g) favoring cellular aspects of adaptive immune responses over through increased formation of IL-2 expressing HAV-specific Th activity of anti-inflammatory or Th2 cytokines (like IL-10, not cells, sleep enhances T cell survival, which enforces the induction measured in this study, and IL-4) (7, 8). Sleep as well as prolac- of Ag-specific memory (2, 15). The Journal of Immunology 289

Our findings support a concept in which SWS establishes neurobehavioral domain where SWS and underlying SWA have a unique endocrine milieu that exerts adjuvant activity by stimu- been identified as a brain state critically supporting the transfer of lating the production of proinflammatory cytokines, eventually information about episodes experienced during prior wakefulness enhancing the development of stable adaptive immunity both at the from temporary to long-term stores (31, 41). Beyond adding to an T and B cell sites. Interestingly, proinflammatory cytokines and integrative concept of the memory function of sleep, our obser- muramyl dipeptide (like many other vaccine adjuvants) have SWS- vation has a number of immunological and clinical implications. promoting properties speaking for the presence of a feed-forward Indicating a supporting effect of sleep on adaptive immunity, our loop where proinflammatory cytokines consolidate central nervous findings can explain immunodeficiency and poor responsiveness SWS during an ongoing immune response (27, 28). Animal data to vaccination in the course of aging known to be accompanied confirm such an assumption, because the amount of SWS predicts by a particular decline in SWS, GH, prolactin, and APC-derived the survival rate after pathogen challenge in rabbits (29). Although IL-12 (7, 42–45). Considering the stability of sleep characteristics, REM sleep deprivation is known to impact immune parameters, as observed in this study across the three postvaccination nights, some of which are also involved in regulating adaptive immune they highlight the contribution of habitually good sleep to im- responses (30), correlation analyses of the current study argue munocompetence and longevity in successful aging (44, 46). In against REM sleep playing a major role for immunological the past decades, modern lifestyle led to a decrease in average memory formation. Further studies are needed to determine the sleep duration also in young and middle-aged subjects. Cross- specific functions of REM sleep in this process, which, given that sectional and longitudinal epidemiological studies indicate that REM sleep follows SWS, could complement effects initiated this sleep curtailment relates to negative health outcomes that may during the prior periods of SWS (as it was hypothesized for involve immune pathways (46). Interestingly, habitual short sleep Downloaded from memory formation in the neurobehavioral domain) (31). duration is associated with an increased susceptibility to experi- The observed sleep-induced increases in HAV- and HBs-specific mental rhinovirus infection and predicts poor Ab titers and poor IgG1 explain observations from two previous studies in humans in clinical protection after three doses of hepatitis B vaccination in which under conditions of sleep restriction or deprivation total IgG 125 middle-aged men and women (47, 48). Hence, the suppress- was slightly reduced shortly after vaccination (influenza and HAV) ing effects of acute experimental sleep loss on immunological

(32, 33). Likewise, 7–8 h of sleep deprivation in rodents imme- memory formation observed in the current study seem to apply http://www.jimmunol.org/ diately following antigenic challenge suppressed the secondary also to natural variations in sleep, underscoring the clinical rele- Ab response, an effect that was completely prevented by admin- vance of our findings. Prolonged experimental sleep restriction istration of proinflammatory adjuvants like IL-1 and muramyl likewise consistently revealed a negative impact on immune func- dipeptide (34). However, these human and animal studies focused tions (7, 49). However, as habitual short sleep and experimental on the clonal expansion phase early after vaccination and not on sleep restriction may not always lead to reduced SWS or SWA the contraction/maintenance phase reflecting memory formation (50, 51), such immunosuppressive effects may stem rather from (3). In addition, other animal studies could not confirm these more pronounced endocrine changes like overall increases in results and if antigenic challenge took place after short-term sleep cortisol levels (8). loss even an opposing (i.e., immunoenhancing) effect on early Most important, our findings prompt to consider sleep as well as by guest on September 28, 2021 defense mechanisms became evident (34–36). Overall, the avail- the sleep-induced temporary increase in proinflammatory cytokine able data point out that experimental timing of Ag challenge, sleep and hormone activity as possible tools for optimizing vaccination deprivation, and respective immunological outcome measures is strategies. Especially the synergistic action of multiple proin- essential to uncover the adjuvant like actions of sleep on immu- flammatory factors like GH and prolactin hopefully stimulates nological memory formation. new designs of vaccine adjuvants (52). In this context, additional We were able to show an enhancing effect of sleep also for the hormones that are known to support the immune response after adaptive immune response to HBs. It is difficult to explain that this vaccination and were not investigated in this study, like melatonin enhancement was overall less robust than after HAV vaccination [which presumably was slightly lower in our waking subjects but could be related to differences in Ag uptake, processing, and (26)], could have contributed to the immunosupportive effect of presentation by APCs (37). Interestingly, in comparisons of SWS (53). Whatever the hormonal mediators, our data indicate morning and evening inoculations, Ab titers measured 4 wk later that improving SWS may provide a therapeutic tool for enhancing were revealed to be higher after HAV vaccination in the morning, efficacy of vaccinations. whereas for HBs vaccination, this effect was reversed, with higher Ab titers after vaccination in the evening (38–40). It is likely that Acknowledgments sleep is most effective in supporting antigenic memory formation We thank A. Tschulakow, Z. Dimitrova, D. Efe, U. Ru¨hr, and J. Festring for during a critical time window when the immunological synapse technical assistance and J. Ko¨hl, J. Westermann, J. Textor, W. Solbach, between APC and Th cell is forming. If as a result of the specific L. Marshall, and M. Hallschmid for stimulating discussion. protein structure APC need a longer time for taking up and processing HAV than HBs, then sleep occurring ∼15 h after a morn- Disclosures ing vaccination might optimally cover this critical window for The authors have no financial conflicts of interest. HAV but to a lesser extent for HBs. Our data corroborate the basic concept that sleep serves the References formation of long-term memory, which so far has been elaborated 1. Steinman, L. 2004. Elaborate interactions between the immune and nervous primarily for neurobehavioral memory. In providing evidence that systems. Nat. Immunol. 5: 575–581. the offline period of sleep with distinctly reduced environmental Ag 2. Seder, R. A., P. A. Darrah, and M. Roederer. 2008. T-cell quality in memory and challenge is used to promote the formation of Ag-specific mem- protection: implications for vaccine design. Nat. Rev. Immunol. 8: 247–258. 3. Sallusto, F., A. Lanzavecchia, K. Araki, and R. Ahmed. 2010. From vaccines to ories, our study extends this concept to the immune system. Our memory and back. Immunity 33: 451–463. analyses suggest a key role for SWS in the transfer of antigenic 4. Coffman, R. L., A. Sher, and R. A. Seder. 2010. Vaccine adjuvants: putting innate immunity to work. Immunity 33: 492–503. information from APC into Ag-specific Th cell responses reflecting 5. Benca, R. M., and J. Quintas. 1997. Sleep and host defenses: a review. Sleep 20: memory. Curiously, this observation finds likewise a parallel in the 1027–1037. 290 SLEEP AND IMMUNOLOGICAL MEMORY

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