Chronic ethanol induces inhibition of antigen-specific CD8�

but not CD4� immunodominant T cell responses followingListeria monocytogenes inoculation

Prajwal Gurung,*,† Betty M. Young,* Ruth A. Coleman,* Susan Wiechert,* Lucas E. Turner,*Nancy B. Ray,*,1 Thomas J. Waldschmidt,*,† Kevin L. Legge,*,† and Robert T. Cook*,2

*Department of Pathology and †Interdisciplinary Program in Immunology, Carver College of Medicine, University ofIowa, Iowa City, Iowa, USA

Abstract: Chronic ethanol consumption resultsin immunodeficiency. Previous work withchronic ethanol-fed mice has shown reducedsplenic weight and cellularity, including reducednumbers of CD8� T cells. However, antigen-specific CD8� and CD4� T cell responses inchronic ethanol-fed mice have been studied rel-atively little. We have used an attenuated Listeriamonocytogenes strain DPL 1942 (LM �actA) toinoculate mice and subsequently used CD4� andCD8� immunodominant peptides of LM to mea-sure the CD4� and CD8� T cell responses afterchronic ethanol exposure. We found no majordifferences between control and ethanol-fedmice in the kinetics and persistence of antigen-specific CD4� T cells in response to an immuno-dominant LM peptide, as measured by intracel-lular IFN-� staining. In contrast to CD4� re-sponses, three methods of in vitro antigenpresentation indicated that the primary responseof CD8� T cells to several different epitopes wasreduced significantly in mice chronically fed eth-anol. Antigen-specific CD8� T cells were alsoreduced in chronic ethanol-fed mice during thecontraction phase of the primary response, andmemory cells evaluated at 29 and 60 days afterinoculation were reduced significantly. BrdUproliferation assays showed that in vivo prolifer-ation of CD8� T cells was reduced in ethanol-fedmice, and IL-2-dependent in vitro proliferationof naive CD8� T cells was also reduced. In con-clusion, these results suggest that antigen-spe-cific CD4� T cell responses to LM are affectedlittle by chronic ethanol consumption; however,antigen-specific CD8� T cell responses are re-duced significantly, as are in vivo and in vitroproliferation. The reduction of antigen-specificCD8� T cells may contribute strongly to theimmunodeficiency caused by ethanol abuse. J.Leukoc. Biol. 85: 34–43; 2009.

Key Words: CD8� T cells � attenuated Listeria � �actA � intra-cellular bacteria

INTRODUCTION

Chronic excessive ethanol consumption in humans has longbeen known to increase the severity of several diseases includ-ing a prominent increase of infectious diseases such as pneu-monia and tuberculosis [1–10]. Modeling of ethanol-inducedimmune dysfunction to explain the increased infectious dis-eases has included work with rodents after ethanol adminis-tration and study of the direct effects of ethanol on immunecells in vitro. Studies with acute and chronic ethanol-fedC57BL/6 and BALB/c mice have shown that there is a signif-icant decrease in splenic cellularity, CD4� T cell numbers,CD8� T cell numbers, B cell numbers, and NK cell numbers[9, 11–15]. Several investigators have reported in work withrodent splenocytes or human peripheral blood mixed T cellsthat short-term or acute ethanol exposures resulted in de-creased T cell proliferation in response to mitogens [16–22].Although a lack of proliferative response to added IL-2 wascommonly reported, the protocols varied widely, renderingcomparisons difficult. In general, there was no consistent re-duction in IL-2 production nor any change in IL-2R expres-sion. Studies with chronic alcoholic patients [23] and mice feda liquid ethanol diet up to 13 weeks [24] were reported to showa decrease in Th1 immunity and increased Th2 responses [23,24]. Other studies with short-term ethanol-fed C57BL/6 micehave suggested that ethanol consumption increases cytokineimbalance to favor Th2 responses by enhancing Th2 and/or bysuppressing Th1 function [14, 23–25].

Although cytokine imbalance studies and evaluation of theresponse to mitogens are of potential value, direct studies ofthe effects of chronic ethanol on the levels of antigen-specificCD4� or CD8� T cell responses have been lacking. Specifi-cally, to model more accurately the changes occurring inchronic human alcoholics, a clear distinction between antigen-

1 Current address: Department of Internal Medicine, University of Iowa,Carver College of Medicine, Iowa City, IA 52242, USA.

2 Correspondence: Department of Pathology, University of Iowa, CarverCollege of Medicine, Iowa City, IA 52242, USA. E-mail: robert-cook@uiowa.edu

Received February 11, 2008; revised August 29, 2008; accepted August 29,2008.

doi: 10.1189/jlb.0208101

34 Journal of Leukocyte Biology Volume 85, January 2009 0741-5400/09/0085-0034 © Society for Leukocyte Biology

specific CD4� and CD8� T cell responses after chronic etha-nol exposure is needed, including measurement of T cellexpansion and contraction phases and the development ofmemory levels. As a clinically relevant model of cell-mediatedimmunity, we have chosen the T cell response to Listeriamonocytogenes (LM), which has been characterized extensivelyin mice [26–29]. The early CD8� T cell response to LM hasbeen shown to be critical for development of sterilizing immu-nity [30]. Previous studies with virulent LM in ethanol-admin-istered mice and rats consistently have shown increased num-bers of bacteria in different organs and the inability to clear theinfection compared with controls [7, 10, 31–33]. In the currentwork, for evaluation of CD8� T cell responses, we have used anattenuated strain of LM, DPL 1942, which is unable to nucleatecellular actin (�actA) and is cleared within 2 days by water-and ethanol-administered mice. Attenuated LM �actA inocu-lation allows consistent evaluation of antigen-specific T cellresponses in water- and chronic ethanol-fed mice, without theexcess mortality of ethanol-fed mice demonstrated with thevirulent LM [7]. A recombinant attenuated LM strain [LM�actA Leishmania homologue of the receptor for activated Ckinase (LACK)], which expresses LACK antigen, was used todetect antigen-specific CD4� T cell responses in BALB/cfemale mice. Two immunodominant epitopes, Listeriolysin OAA #91–99 (LLO91–99; GYKDGNEYI) of LM for specificCD8� T cells [29] and LACK AA #158–173 (LACK158–173;FSPSLEHPIVVSG) of Leishmania major for specific CD4� Tcells, were used to assay the T cell responses [34]. Previousstudies about the time course of antigen-specific CD8� T cellresponses in normal mice using LM �actA have shown that theantigen-specific response peaks at 7 days after inoculation,contracts by Day 12, and reaches early memory phase by Day29 [26–28]. Here, we asked if the time course of the antigen-specific T cell response is similar in control and ethanol-consuming mice; whether the resulting number of antigen-specific T cells is reduced; whether cytokine production by Tcells is reduced or altered, favoring a Th2/T cytotoxic 2 (Tc2)response in chronic ethanol-consuming mice following LM�actA inoculation; and whether T cells from chronic ethanolmice proliferate normally.

MATERIALS AND METHODS

Mice and ethanol administration

Female BALB/c mice were obtained from the National Cancer Institute (Fred-erick, MD, USA) at 5–7 weeks of age. Mice were housed in barrier facilities inthe Animal Care Unit at the University of Iowa (Iowa City, IA, USA). Uponreceipt, mice were acclimatized for at least 1–2 weeks, and each lot wasdivided randomly into two groups to maintain lot and age-matched water(control) and ethanol groups. Starting at ages 8–10 weeks and using a well-described protocol, pharmaceutical-grade ethanol was administered in double-distilled water at 10% (w/v) for 2 days, 15% for 5 days, and finally, 18% toBALB/c mice as the only source of drinking water [9, 11, 12, 35]. Ethanol-fedmice were kept at their final concentration throughout the remainder of theexperiment, and the durations of ethanol exposures indicated were the numberof weeks mice were exposed to the final ethanol concentration. The mice werefed laboratory chow (Teklad NIH-31-irradiated, Harlan, Indianapolis, IN,USA) ad libitum, and control mice were given the same double-distilled waterused for mixing ethanol solutions. With this ethanol administration protocol,

mice were visibly inebriated in the early morning following nocturnal drinking,and blood alcohol levels were as high as 400 mg/dL, with much lower levelslater in the day [9]. All procedures were carried out under a protocol approvedby the Animal Care Committee of the University of Iowa.

Bacterial preparation and infectionLM �actA and LM �actA LACK were the kind gifts of John T. Harty(University of Iowa). They were grown to log phase (OD 600: 0.06–––0.09) intryptic soy broth (TSB) media. Bacteria were diluted to 5 � 105 cfu in 0.3 mL(LM �actA) or 1 � 107 cfu in 0.3 mL (LM �actA LACK) sterile saline (HospiraInc., Lake Forest, IL, USA) and injected i.p. into control and ethanol-consum-ing mice. Serial dilutions of the same LM �actA cultures used for injectionswere plated in TSB agar culture plates and grown overnight at 37°C, andcolonies were counted to determine independently the number of injectedbacteria. LM �actA were not detectable in spleens or livers of control orethanol-fed mice 2–3 days after inoculation, as determined by standard organculture and cfu assays.

Cell harvest and cultureLM �actA-inoculated water control or ethanol-consuming mice were killed atdifferent time-points, and the spleens were harvested. Spleens were homoge-nized between glass slides and suspended in enriched RPMI media, whichcontained sterile-filtered RPMI 1640 (Invitrogen, Carlsbad, CA, USA), 10%FCS (Invitrogen), 1% 200 mM L-glutamine (Invitrogen), 0.1% 50 mM 2-ME,and 0.1% 50 mg/mL gentamycin (Sigma-Aldrich, St. Louis, MO, USA). Thetotal number of splenocytes/spleen was determined by counting cells in ahemocytometer.

In vitro antigen presentationThe APC present in whole splenocytes, purified and activated normal B cells,or P815 cells (a mastocytoma cell line, ATCC #TIB-64TM) were used as APC.B cells were purified from whole splenocytes using a B cell isolation kit(Miltenyi Biotec, Auburn, CA, USA). B cells were cultured with LPS at 1�g/mL at 37°C overnight for activation. After overnight culture, B cells werewashed 2� with PBS and then reconstituted at 2 � 106/mL. LLO peptide (10�M; LLO91–99, was added to 2 � 106 B cells/mL and incubated at 37°C for 1 h.Peptide-loaded B cells were washed 2� with PBS. For P815 assay, 10 �MLLO91–99 peptide was added to 5 � 106 P815 cells/mL and incubated at 37°Cfor 1 h followed by two washes to load P815 cells. CD8� T cells were enrichedusing the CD8�� cell isolation kit (Miltenyi Biotec). Activated B cells or P815cells loaded with the LLO91–99 peptide were used as APC at the specifiedratios. Purified CD8� T cells and peptide-loaded APC were cultured for 6 h in thepresence of Brefeldin-A, and the responding CD8� T cells were determinedby intracellular IFN-� staining. In some assays, peptide at a concentrationof 10

�7M (LLO91–99, p60217–225, p60449–457) or 25 �g/mL (LACK158–173)

was added directly to whole splenocytes in culture to assay antigen-specificCD8� or CD4� T cells, respectively, after presentation by homologous,splenic APC.

Detection of antigen-specific cellsFresh splenocytes or purified CD8� T cells were stained with the LLO91–99-loaded class I MHC pentamer (Proimmune, UK), according to the manufac-turer’s instructions, to detect antigen-specific CD8� T cells. As a secondmethod for determining antigen-specific T cells, splenocytes or T cells werepresented peptide at 37°C in the presence of Brefeldin A (BD Biosciences/PharMingen, San Diego, CA, USA) for 6 h, permeabilized, and stained intra-cellularly for IFN-� to detect antigen-specific CD8� or CD4� T cells asdescribed below. Class I-specific immunodominant LLO91–99 peptide andsubdominant p60219–225 and p60449–457 peptides (Biosynthesis, Inc., Lewis-ville, TX, USA) were used for antigen-specific CD8� T cells, and classII-specific LACK158–173 peptide (Biosynthesis, Inc.) was used for measuringantigen-specific CD4� T cells by intracellular cytoplasmic staining.

Surface marker staining, intracellular staining,antibodies, and analytic flow cytometryFor staining of surface markers, cells were incubated with fluorochrome-conjugated mAb at room temperature. After 30 min, the cells were washed with

Gurung et al. Chronic ethanol and CD8� T cell responses 35

PBS � 0.1% sodium azide and then fixed with 1% formaldehyde in PBS �0.1% sodium azide.

For intracellular staining, first, surface markers were stained as just de-scribed. The cells were then washed 2� with staining buffer [0.1% heat-inactivated FCS (Invitrogen) in PBS�0.1% sodium azide] and incubated withcytofix/cytoperm (BD Biosciences/PharMingen) for 20 min at 4°C. Cells werewashed 2� with perm/wash buffer (BD PharMingen) and then stained withfluorochrome-conjugated mAb for intracellular staining by incubating for 30min at 4°C. Cells were washed 2� with perm/wash buffer and then fixed instaining buffer. Antigen-specific CD8� and CD4� T cells were analyzed usinga BD FACSCalibur flow cytometer, as described previously in detail [9, 36].For detection of low-frequency events, up to 106 cells were analyzed for eachcondition. All antibodies used in the current work were anti-mouse mAb.FITC-conjugated anti-CD4, -CD8, and -CD44 mAb; PE-conjugated anti-CD8,IFN-�, and CD122/IL-2R� mAb; PerCP-conjugated anti-CD4 and -CD8 mAb;and allophycocyanin-conjugated anti-CD4 and -CD8 and BrdU mAb were allpurchased from BD Biosciences.

Secreted cytokines

Splenocytes (2�106) were cultured with CD8-specific LLO91–99 peptide for 8and 22 h, and supernatants were collected and frozen at –73°C. Supernatantswere analyzed using a mouse Th1/Th2 cytokine cytometry bead array kitexactly as described by the manufacturer (BD Biosciences/PharMingen). Cy-tokines were analyzed using a BD FACSCalibur flow cytometer.

BrdU proliferation assays

Control and ethanol-fed mice were inoculated with LM �actA (5�105cfu/0.3mL); BrdU (Sigma-Aldrich, Steinheim, Germany; 5 mg in 0.5 mL PBS) wasadministered at the same time i.p., and in some experiments, BrdU wasadministered 5 days after LM �actA inoculation. After BrdU administration,BrdU was also added in some experiments, as indicated, to drinking water ofcontrols and to ethanol in water, both at 1.5 mg/mL. Mice were killed 7 dayspost-LM �actA inoculation, and spleens were harvested. Whole splenocyteswere cultured for 6 h in the presence of Brefeldin A, permeabilized, andstained for intracellular BrdU exactly as instructed by the manufacturer’smanual (BD Biosciences/PharMingen). Stained splenocytes were analyzedusing a BD FACSCalibur flow cytometer.

In vitro proliferation

T cells from naive control or chronic ethanol-fed BALB/c female mice wereused for in vitro proliferation assays. Single-cell suspensions of splenocyteswere depleted of B220� cells by positive selection using magnetic separationbeads and columns (Miltenyi Biotec) to enrich for T cells, which were thenstained with FITC-CD44 and PE-CD8 antibody. CD8�CD44low (naıve pheno-type) T cells were sort-purified by standard methods using a BD FACSDiva cellsorter. Sort-purified CD8� T cells were cultured with soluble anti-CD3 mAb at1 �g/mL, IL-12p70 (eBioscience, San Diego, CA, USA) at 6 U/mL, IFN-�(Accurate Chemical and Scientific Corp., Westbury, NY, USA) at 100 U/mL,and various concentrations of IL-2 (PharMingen) as indicated for 3 days.IL-12p70 and IFN-� were used in these experiments to achieve optimumproliferation, as determined in control experiments not shown. After 3 days,cells were pulsed with tritiated 3H-thymidine and harvested after 6 h. Prolif-eration of cells, as determined by the uptake of 3H-thymidine, was analyzed byliquid scintillation counting by standard methods (PerkinElmer Life andAnalytical Sciences, Shelton, CT, USA).

Statistics

All results are expressed as mean SE. The Mann-Whitney nonparametric testwas performed for comparison of water and ethanol groups. A two-tailedunpaired t-test (Welch corrected) was also performed if sufficient numberswere present to evaluate normality to evaluate the significance of the meandifference between water and ethanol groups. One-way ANOVA was performedto determine statistical significance when more than two groups were present.All P values shown are two-tailed, and P values less than 0.05 (P0.05) wereconsidered significant. All statistical analyses were performed using the InStatprogram, Version 3.0a (GraphPad Software, Inc., San Diego, CA, USA).

RESULTS

Reduced splenic weight, splenic cellularity, and Tcell numbers

Consistent with previously published data for noninoculatedC57BL/6 mice [9, 15], the splenic weight, splenic cellularity,and total CD8� and CD4� T cell numbers were reducedsignificantly in 6-week ethanol-fed female BALB/c mice thatwere inoculated with LM �actA 7 days prior (Fig. 1). Spleenweights, cellularity, and CD8� T cell numbers were reducedsignificantly in mice consuming ethanol for as little as 4 weeksand as long as 44 weeks prior to inoculation. An increasedCD4/CD8 ratio was demonstrated previously for C57BL/6chronic ethanol-fed mice [9]; however, the CD4/CD8 ratios inthe current BALB/c ethanol-administered mice were not dif-ferent from controls at a statistically significant level.

Antigen-specific CD8� T cell responses

Figure 2A shows the flow cytometry pattern of respondingCD8� T cells to P815 cells loaded with or without theLLO91–99 peptide. Figure 2B demonstrates the titration ofvarious CD8:P815 ratios on total CD8� antigen-specificcells. Responding CD8� T cells were significantly lower inethanol-fed mice (Fig. 2C), as total responding cells perspleen and as the percentage responding in vitro. Inocula-tion of bacterial doses as high as 2 � 107 (40-fold) did notresult in any major difference in the response ratio of controland ethanol-fed mice as compared with Figure 2 and Figure3 (dose comparison not shown), although there was somevariation in absolute response levels.

Figure 3A shows the flow cytometry pattern of the CD8� Tcell response with or without the LLO91–99 peptide in nonin-

Fig. 1. Splenic weight, total cellularity, and T cell numbers in chronicethanol-consuming mice. Six-week ethanol-administered and matched watercontrol BALB/c female mice were inoculated with LM �actA. Mice were killed7 days post-LM �actA inoculation, and spleens were harvested. Spleenweights, total splenocytes/spleen, total number of CD8� and CD4� T cells/spleen were measured. N(H2O) � 12; N(EtOH) � 12; **, P 0.01; ***, P 0.001.

36 Journal of Leukocyte Biology Volume 85, January 2009 http://www.jleukbio.org

oculated (control) and 7-day LM �actA LACK-inoculated con-trol and ethanol-fed mice. The response of LLO91–99-specificCD8� T cells for control BALB/c female mice was maximum atDay 7, significant contraction occurred by Day 12, and resid-ual, antigen-specific cells were present at Day 29 (Fig. 3B),similar to the time course for normal mice described by others

[26, 27, 37]. For mice consuming ethanol for 4–6 weeks orlonger, the contraction phase was different, such that the slopeof contraction was flatter as compared with water controls. Thefrequency and total numbers of LLO91–99-specific CD8� Tcells from ethanol-fed mice were reduced at Days 5, 7, 12, and29 after LM �actA inoculation (Fig. 3B). Similar results were

Fig. 2. Antigen presentation assay. (A) MACS-purified CD8� Tcells from 7-day-inoculated, 32-week ethanol-consuming or con-trol BALB/c female mice were cultured with the LLO91–99 peptide-loaded P815 cell at a 5:1 ratio in the presence of Brefeldin A for6 h. As a control, CD8� T cells were cultured with P815 cells notloaded with peptide. Flow cytometry data of one water- andethanol-administered group in the absence or presence of theLLO91–99 peptide. (B) CD8� T cells from 7-day-inoculated, 28-week ethanol-consuming and matched control BALB/c femalemice were cultured with LLO91–99 peptide-loaded P815 cells atdifferent ratios in the presence of Brefeldin A for 6 h at 37°C.

Responding CD8� T cells were analyzed by intracellular IFN-� staining. Control wells contained no P815 cells. (C) Statistical analysis of the datashown in A from all mice in the groups; N(H20) � 9; N(EtOH) � 6; ***, P 0.001.

Fig. 3. Antigen-specific CD8� T cell responses. (A) Representative flow cytometry plots of noninoculated and water- and ethanol-administered CD8� T cells withor without the LLO91–99 peptide at 7 days post-LM �actA LACK inoculation. Flow cytometry plots are gated on CD8� T cells. (B–D) BALB/c female miceconsuming ethanol and water for 4–44 weeks were inoculated with LM �actA or LM �actA LACK; spleens were harvested at different days postinoculation andsplenocytes cultured 6 h. Time course of LLO91–99-specific CD8� T cells as percent responding (B, upper) and total number per spleen (B, lower) in water- andchronic ethanol-consuming mice as determined by intracellular IFN-� staining. Four days, N(H2O) � 4; N(EtOH) � 6. Five days, N(H2O) � 3; N(EtOH) � 4.Seven days, N(H20) � 7; N(EtOH) � 6. Twelve days, N(H2O) � 8; N(EtOH) � 8. Twenty-nine days, N(H2O) � 8; N(EtOH) � 7. (C) p60217–225-specific and(D) p60449–457-specific CD8� T cell responses at 7 days post-LM �actA LACK inoculation; N(H2O) � 7; N(EtOH) � 6; *, P 0.05.

Gurung et al. Chronic ethanol and CD8� T cell responses 37

obtained for the kinetics of antigen-specific CD8� T cells inmice consuming ethanol for 19 weeks and 44 weeks beforeinoculation (data not shown). Antigen-specific CD8� T cellswere present even at 60 days (late memory phase) postinocu-lation in control and ethanol-fed mice; similar to the earliertimes, the antigen-specific CD8� T cells were comparativelyreduced in ethanol-fed mice (60 days not shown). Similar to theimmunodominant epitope (LLO91–99), CD8� T cell responsesto subdominant epitopes (p60217–225 and p60449–457) were alsoreduced significantly at Day 7 post-LM �actA LACK inocula-tion in the ethanol-fed mice (Fig. 3, C and D).

Antigen-specific CD4� T cell responses

In contrast to the result with CD8� T cells, the antigen-specificCD4� T cell response at 7 days after LM �actA LACKinoculation was similar between control and ethanol-fed mice(Fig. 4A). In some experiments with long ethanol exposures(�40 weeks ethanol), there was a trend toward reduced totalantigen-specific CD4� T cells per spleen in ethanol-fed mice;

however, no statistical significance was obtained. SolubleIFN-� in the supernatant, produced by antigen-specific CD4�

T cells in response to two subdominant peptides added together(LLO189–200�LLO216–227), was also similar between controland ethanol-fed mice (Fig. 4B). The IFN-� detected in thesupernatants was clearly produced by antigen-specific CD4� Tcells, as no IFN-� was detected in CD4�-depleted cultures inparallel experiments (data not shown). Comparable results withno reduction of antigen-specific CD4� T cells in mice chron-ically fed ethanol were obtained in numerous trials of C57BL/6mice using the immunodominant CD4 epitope (LLO190–201;data not shown).

Cytokine production by CD8� T cells afterLLO91–99 peptide stimulation

Supernatants from whole splenocyte cultures of inoculatedmice that were stimulated with or without the LLO91–99 peptidefor 8 and 22 h were analyzed for Tc1 cytokines (TNF-�, IFN-�,and IL-2) and Tc2 cytokines (IL-4 and IL-5) to determine anyTc1 to Tc2 skewing. Tc1 and Tc2 cytokines were at back-ground levels in supernatants from cultures without theLLO91–99 peptide (Fig. 5A). In the presence of the LLO91–99

peptide, Tc2 cytokines were not detectable in peptide-stimu-lated cells from control or ethanol-fed mice in any experiments.Tc1 cytokines were produced by cells from control and etha-nol-fed groups, albeit less by cells from most ethanol-admin-istered groups (Fig. 5A), during the primary response (7 days).As antigen-specific CD8� T cells were reduced already, re-duced production of these cytokines was expected. IFN-�production was reduced even when normalized to cytokineproduction on an antigen-specific, per-cell basis (Fig. 5B),suggesting a functional defect in antigen-specific CD8� T cellsin addition to their decreased numbers. TNF-�, IFN-�, andIL-2 levels were lower for all 4-, 19-, or 44-week ethanol-fedmice compared with their age-matched controls.

In vivo proliferation of CD8� T cells

BrdU was administered to inoculated mice for 7 days, 2 days,or 1 day (Fig. 6), and the relative proliferation of CD8� T cellswas determined. The percentage of and the total BrdU�CD8�

T cell numbers were significantly lower in ethanol-fed micecompared with their controls (Fig. 6A). Proliferation of antigen-specific CD8� T cells, as determined by double staining forLLO91–99-specific MHC I pentamer or intracellular IFN-� andBrdU, was also reduced in ethanol-fed mice compared withtheir respective controls (Fig. 6B).

CD122 expression and in vitro proliferation ofT cells

CD122 (IL-2R�) is present on CD8� T cells and is importantfor their proliferation and survival. The frequency ofCD8�CD122hi T cells in noninoculated or LM �actA-inocu-lated mice was reduced significantly in ethanol-fed mice (Fig.7, A and B). IL-2-dependent, in vitro proliferation ofCD8�CD44low T cells from naıve mice was also reduced sig-nificantly in the cells from chronic ethanol mice (Fig. 7C). Incontrast and consistent with the normal antigen-specific CD4�

Fig. 4. Antigen-specific CD4� T cells. Chronic ethanol- or water-adminis-tered BALB/c female mice were inoculated with 1 � 107 cfu LM �actA LACK.Mice were killed at 7 days after inoculation, and antigen-specific CD4� T cellresponses were measured in splenocyte cultures. (A) Frequency and totalnumber of LACK158–173-specific CD4� T cells were measured by intracellularcytoplasmic staining for IFN-�. (B) Splenocytes were incubated at the sametime with two CD4-specific subdominant peptides (LLO189–200�LLO216–227)for 96 h, and supernatants were collected and analyzed for IFN-�; N(H2O) �4; N(EtOH) � 4.

38 Journal of Leukocyte Biology Volume 85, January 2009 http://www.jleukbio.org

T cell response in ethanol-fed mice (Fig. 4), CD122 expressionon CD4� T cells from chronic ethanol-fed mice was normal(Fig. 7, A and B). Additionally, multiple proliferation assayshave been carried out for CD4� T cells, and no differencebetween control and ethanol-fed mice has been seen to date(data not shown).

Secondary antigen-specific CD8� Tcell responses

The primary responses of CD8� T cells are inhibited in etha-nol-consuming mice (Fig. 1), and the total number of resultingmemory cells appears to remain low (Fig. 3). As a further testof memory cell numbers, we performed an adoptive transferstudy with cells from 8-week, ethanol-fed and control miceinoculated with LM 44 days before transfer and maintained on

Fig. 5. Cytokine production after CD8-specific LLO91–99 peptide restimula-tion. (A) BALB/c female mice consuming ethanol in water for 4 or 6 weekswere inoculated with LM �actA, and spleens were harvested 7 days postin-oculation. Whole splenocytes (2�106) were cultured with 10�7 M LLO91–99

peptide for 8- and 22-h durations. Supernatants were analyzed using a Th1/Th2 cytokine bead assay. (B) Values from the above experiments were nor-malized to evaluate cytokines produced per antigen-specific CD8� T cells. Fornormalization, ethanol cytokine values were multiplied by an antigen-specificCD8� T cell ratio of water and ethanol; i.e., ratio � percent antigen-specificCD8� T cells (H2O/ethanol). IFN-� values were divided by 10, and IL-2values were multiplied by 10 to fit in the same graph; N(H2O) � 7; N(EtOH) �8; *, P 0.05.

Fig. 6. In vivo CD8� T cell proliferation assays. (A) In vivo total CD8� T cellproliferation assays. (Upper panel) Percent BrdU�CD8� T cells and (lowerpanel) numbers of BrdU�CD8� T cells per spleen. Ethanol- and water-administered BALB/c female mice (9–32 weeks) were inoculated with LM�actA i.p. One hundred sixty-eight hours: BrdU was injected with LM �actAand added to the drinking water or ethanol of water and ethanol mice, whichwere killed 7 days after LM �actA inoculation and BrdU injection; N(H2O) �8; N(EtOH) � 8. Forty-eight hours: BrdU was injected 5 days after LM �actAinoculation and then added to drinking water or ethanol of water and ethanolmice, which were killed 7 days after LM �actA inoculation and 2 days afterBrdU injection; N(H2O) � 4; N(EtOH) � 5. Twenty-four hours: BrdU wasinjected 5 days after LM �actA inoculation, and mice were killed the next day,i.e., 6 days after LM �actA inoculation. BrdU was not added in drinking wateror ethanol; N(H2O) � 4; N(EtOH) � 6. Intracellular cytoplasmic staining wasperformed for BrdU. (B) In vivo antigen-specific CD8� T cell proliferation.Ethanol (14 weeks)-administered BALB/c female mice and water mice wereinoculated with LM �actA. Five days after inoculation, BrdU was administeredas described for the 48-h exposure (A), and splenocytes were harvested at thepeak of the response (Day 7). Whole splenocytes were surface-stained withPerCP-conjugated CD8 mAb and APC-conjugated LLO91–99-loaded class IMHC pentamer and intracellularly stained for incorporated BrdU; N(H20) � 4;N(EtOH) � 4; *, P 0.05; **, P 0.01; ***, P 0.001.

Gurung et al. Chronic ethanol and CD8� T cell responses 39

their respective diets until transfer. Cells were transferred onlyto normal control mice, after which, the recipient mice wereinoculated with LM. The secondary response to the dominantLLO91–99 peptide of donor Thy1.2�CD8� T cells from ethanol-fed mice was reduced significantly in comparison withThy1.2�CD8� T cells from control mice (Fig. 8A), consistentwith the reduction observed previously in 29-day memory cellsof chronic ethanol mice (Fig. 3).

Chronic ethanol-fed mice have reduced LLO91–99- andp60217–225-specific, secondary CD8� T cell responses to LM�actA LACK when compared with normal controls; 3 weeksethanol withdrawal did not reverse the ethanol effects, al-though there was some variability (Fig. 8B and not shown).LACK158 –173-specific CD4� T cell responses were similaramong all three groups (data not shown).

All of the above findings resulted from examination of miceinoculated during chronic ethanol exposure. Using an alterna-tive protocol, control mice were inoculated with LM, followedby extended ethanol drinking and a secondary challenge. LM�actA LACK-immune control mice that were subsequentlyadministered ethanol for 5 weeks and rechallenged with LM�actA LACK trended to a lower CD8� T cell response in theethanol-administered group but did not show statistically sig-nificant reductions of LLO91–99-, p60217–225-, and p60449–457-specific CD8� T cells nor of LACK158–173-specific CD4� Tcells (Fig. 8C, and data not shown).

DISCUSSION

The adverse effect of ethanol consumption on immune functionhas been studied in various models to understand the mecha-nisms involved. We have used a chronic ethanol-in-waterprotocol [35] to understand the effects of chronic ethanolfeeding on adaptive immunity and especially T cells. Consis-tent with previous reports [9, 15, 36], splenic weights, totalcellularity, and CD8� T cell numbers were reduced signifi-cantly in chronic ethanol-fed mice. A major finding was thatantigen-specific CD8� T cell responses to three-peptideepitopes of Listeria were all reduced significantly in micechronically consuming ethanol. These reductions in the etha-nol source CD8� T cell responses are in the same ratio tocontrol responses using three different APC systems for the invitro assay, and it is clear that APC impairment in the in vitroassay does not explain the results. Instead, the sharp reductionin the antigen-specific CD8� T cells reflects compromisedCD8� T cell responses after in vivo inoculation of the ethanol-consuming mice.

Several key time-points in the antigen-specific CD8� T cellresponse after LM �actA inoculation were evaluated byLLO91–99-specific MHC class I pentamer staining (data notshown) and IFN-� intracellular staining after restimulationwith the LLO91–99 peptide. Both readouts demonstrated a sim-ilar reduction in the CD8� T cell response in ethanol-fed mice

Fig. 7. IL-2R expression and in vitro proliferation of CD8� T cells. (A) Histogram of CD122 (IL-2R� chain) expression on CD8� and CD4� T cells from controland 9- to 32-week ethanol-administered BALB/c female mice, as analyzed by flow cytometry. Shaded gray is the isotype control; thick black line is water; and thinline is ethanol CD8� or CD4� T cells. A line across the histogram denotes CD122hi populations. (B) Total percentages of CD8�CD122hi (left) and CD4�CD122hi

(right) T cells are shown as bar graphs. Some mice were inoculated with 5 � 105 cfu LM �actA to generate immune CD8� T cells. No inoculation, N(H2O) �8; N(EtOH) � 6. LM �actA inoculation, N(H2O) � 12; N(EtOH) � 16. (C) In vitro proliferation of splenic CD8�CD44low T cells. Sort-purified CD8�CD44low

T cells from 19-week, ethanol-consuming and control mice were cultured with soluble anti-CD3 mAb, IL-12p70, IFN-�, and increasing concentrations of IL-2 for3 days. After 3 days, cells were pulsed with 3H-thymidine for 6 h, and the uptake of 3H-thymidine was measured using standard liquid scintillation analysis. Oneof two similar experiments. No cyto, No added cytokines; No IL-2, no IL-2 added; and IL-2 (2u), units IL-2 added. *, P 0.05; **, P 0.01; ***, P 0.001.

40 Journal of Leukocyte Biology Volume 85, January 2009 http://www.jleukbio.org

at all time-points when inoculation occurred after 4 weeks ofethanol exposure or greater. Similar to data for normal micepublished by others [26, 27], the maximum response of LM�actA LLO91–99-specific CD8� T cells was seen at Day 7,contraction by Day 12, and measurable early memory phasewas present at Day 29 for control and chronic ethanol-fed mice;the antigen-specific response was still detectable after 60 days.The contraction of antigen-specific CD8� T cell responses inethanol-fed mice was flattened in slope, suggesting a slowerrelative decay rate when compared with control mice, and thetotal number of antigen-specific CD8� T cells was reducedsignificantly in ethanol-fed mice at all time-points (Fig. 3).

We also evaluated the antigen-specific CD4� T cell re-sponse to the LACK158–173 peptide in chronic ethanol-fedBALB/c mice and in contrast to CD8� T cell responses, wereunable to find any significant differences compared with con-trols. However, a trend toward reduced total numbers of anti-gen-specific CD4� T cells in BALB/c mice administered eth-anol for more than 40 weeks was observed (data not shown),and it is possible that some deterioration of CD4� T cellfunction will be found to occur in long-term ethanol exposureprotocols.

Several mechanistic possibilities exist for the reduction inantigen-specific CD8� T cell responses in the chronic ethanol-fed mice. Altered APC functions, including migration, up-regulation of costimulatory molecules, and production of effec-tor cytokines, have been described in the context of various

ethanol-exposure models [19, 25, 36, 38–40]. It is possible,therefore, that there is some contribution of altered APC func-tion in vivo to the reduction of antigen-specific CD8� T cellsin the current work. However, it is clear that the CD8� T cellsof the chronic ethanol-fed mice have intrinsic defects. Thesecells do not proliferate normally in vitro, even when purified asnaıve (from noninoculated mice) CD8� T cells (Fig. 7C), rulingout altered antigen processing/presentation for the effect. An-tigen-specific memory cells raised in ethanol-fed mice did notexpand at a control level, even when transferred to a controlhost (Fig. 8A), and as discussed below, the antigen-specificcells from ethanol-fed mice do not produce normal levels ofIFN-� (Fig. 5) in response to antigenic challenge in vitro.

BrdU incorporation studies were performed to examine theproliferative response of CD8� T cells in vivo. BrdU wasadministered over the entire 7 days of the primary response orin other protocols as only a short pulse of BrdU before eutha-nasia. Similar results were obtained with all different BrdUtime pulses, consistent with reduced proliferation in ethanol-fed mice, in contrast to preferential apoptosis. Direct ex vivoevaluation of apoptosis by Annexin V staining of freshly iso-lated splenocytes also did not reveal any differences betweenCD8� and CD4� T cells from control and ethanol-fed mice(data not shown). These results strongly point to reducedproliferation as the basis for lower CD8� T cell responses,although a role for apoptosis in vivo cannot be excluded

Fig. 8. Secondary CD8� T cell responses to LM �actA LACK reinocula-tion. (A) Thy 1.2 BALB/c control and 8-week ethanol-administered micewere inoculated with LM and continued on their respective diets. Forty-fourdays later, 5 � 106 MACS-purified, splenic CD8� T cells were adoptivelytransferred to Thy1.1 water mice i.v. Recipient Thy1.1 water mice wereinoculated with LM �actA LACK 1 day later, and the secondary response of donor-immune Thy1.2�CD8� T cells was analyzed by intracellular cytoplasmicstaining for IFN-� on Day 5 after inoculation. Thy 1.1 recipients received water source cells N(H2O) � 4 or ethanol source cells N(EtOH) � 6. (B) Water- and46-week chronic ethanol-administered mice were inoculated with LM �actA LACK. Twenty days following inoculation, some ethanol-administered mice werewithdrawn from ethanol for 15 days. All three groups—H2O, EtOH, and EtOH-withdrawn mice—were reinoculated with LM �actA LACK, and the secondaryCD8� T cell responses were measured on Day 5. N(H2O) � 4; N(EtOH) � 5; N(EtOH withdrawn) � 5. (C) Normal water mice (no ethanol history) were inoculatedwith LM �actA LACK. Sixteen days postinoculation, some water mice were started on EtOH. After 5 weeks of EtOH administration, mice were reinoculated withLM �actA LACK, and the secondary CD8� T cell responses were measured on Day 5 by intracellular cytoplasmic staining for IFN-�. N(H2O) � 5; N(EtOH) �3; *, P 0.05. NS, Not significant.

Gurung et al. Chronic ethanol and CD8� T cell responses 41

entirely, given the rapid removal of apoptotic cells by macro-phages.

Previous studies of chronic alcoholics have shown that theability of peripheral blood T cells from chronic alcoholics withliver disease to bind IL-2 was reduced when compared withhealthy individuals [41], which is in contrast to studies in ratsthat showed no effect on the ability of IL-2R to bind IL-2 [21].Proliferation and IL-2 production by T cells from acute etha-nol-administered mice with added burn injury were reducedsignificantly when compared with controls [42], as was prolif-eration of T cells from fetal alcohol exposure rats [43]. In thecurrent work, we have examined in vitro proliferation of sort-purified CD8�CD44low T cells from noninoculated mice in anassay designed to test the proliferative response of CD8� Tcells to IL-2, and IL-2-dependent proliferation was reduced innaıve CD8� T cells from chronic ethanol-fed mice at allconcentrations (Fig. 7). Previous studies evaluated low-affinityIL-2R (IL-2R� chain/CD25) on T cells and found no differencebetween the ethanol and control groups [18, 22, 44]. Wemeasured high-affinity IL-2R� (CD122) to evaluate the expres-sion of the IL-2R; interestingly, ex vivo staining demonstratedreduced expression of the IL-2R� chain on CD8� T cells fromchronic ethanol-fed mice, a change not present on CD4� cellsfrom the same mice. It thus appears that one likely mechanismof the reduction of CD8� T cell responses by chronic ethanolconsumption is reduced induction/expression of the high-affin-ity IL-2R on naıve CD8� T cells after antigen exposure andsubsequently, reduced proliferative capacity of those cells. Anadditional feature of CD8� T cell dysfunction in ethanol-fedmice is suggested by the reduction of IFN-� secretion evenafter normalization to the number of antigen-specific CD8� Tcells (Fig. 5). Some degree of dysregulation thus may exist inthe antigen-specific effector cells; we have recently demon-strated a similar result in pulmonary CD8� T cells fromchronic ethanol-fed mice responding to influenza A virus [45].

The secondary responses of LM �actA-immune CD8� Tcells from ethanol-consuming mice after transfer to normalcontrol mice were reduced significantly when compared withLM �actA-immune CD8� T cells transferred from controlmice. This result is consistent with the transfer of a smallerproportion of memory cells from ethanol-fed mice, as impliedby the reduced total number of memory cells assayed previ-ously in such mice 29 days after inoculation (Fig. 3). However,the immune CD8� T cells from donor control mice expandedtwo- to threefold more after inoculation of the recipients whencompared with immune CD8� T cells from ethanol-fed donormice. Thus, an intrinsic defect of memory CD8� T cellsgenerated in mice during chronic ethanol consumption is notexcluded and will be the subject of future studies in thislaboratory. In a different protocol, the secondary response ofCD8� T cells in LM-immune ethanol-fed mice was measuredafter withdrawal from ethanol; the secondary CD8� T cellresponse was not rescued by such withdrawal (Fig. 8B). Theabove results indicate that the decreased primary CD8� T cellresponse resulting from chronic ethanol ingestion at the time ofantigenic exposure results in a reduced memory pool that doesnot recover numerically, even after ethanol is stopped. In aseparate memory protocol, subsequent chronic ethanol feeding

up to 6 weeks had little effect on the functional ability ofpreviously generated LM �actA LACK memory CD8� T cellsto mount a secondary response upon reinoculation with the LM�actA LACK; however, there was a trend toward a total re-duced number of specific CD8� T cells (Fig. 8C). Futurelong-term studies will be required to determine whether longerperiods of ethanol exposures will affect the functional ability ornumbers of memory CD8� T cells originally generated in theabsence of ethanol.

In conclusion, our results show that the antigen-specificCD8� T cell primary response to LM peaks at Day 7, contractsby Day 12, and reaches early memory phase by Day 29 forcontrol and ethanol-fed mice; however, the response curve isflattened, and total numbers are reduced in the ethanol mice.In contrast, antigen-specific CD4� T cell responses are notreduced in the chronic ethanol-fed mice. The total Tc1 cyto-kine production in response to in vitro antigen was reduced inchronic ethanol-fed mice, but Tc1 to Tc2 skewing was not seenin our experimental model. Memory CD8� T cell numbers werealso reduced by chronic ethanol feeding; reduction of second-ary CD8� antigen-specific cell numbers appeared to be de-pendent on the initial reduction occurring during the primaryresponse of CD8� T cells in the ethanol-fed mice. The reduc-tion in the number of antigen-specific CD8� T cells seen inchronic ethanol-fed mice was accompanied by reduced expres-sion of IL-2R� and reduced proliferation in vivo and in vitro,but the in vivo decrease of antigen-specific cells may not belimited to this mechanism.

ACKNOWLEDGMENTS

This work was supported by the Department of Pathology at theUniversity of Iowa and an Interactive Research Program grantincluding National Institutes of Health Grants AA 011405(R. T. C.), AA 014400 (T. J. W.), AA 014406 (Annette J.Schlueter), and AA 014418 (Zuhair K. Ballas) at the Universityof Iowa and AA012450 (Thomas R. Jerrells) at the Universityof Nebraska (Lincoln, NE, USA). We thank Drs. John T. Hartyand Vladimir P. Badovinac at the University of Iowa for kindgifts of LM �actA strains and consultations.

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