Immunology 1998 93 341–349

The CD4+ T-cell response to protein immunization is independent of accompanyingIFN-c-producing CD8+ T cells

A. G. DOYLE, L. RAMM & A. KELSO Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital,Queensland, Australia

SUMMARY

By virtue of their strong bias towards production of interferon-c (IFN-c), CD8+ T cells have thepotential to promote the development of type 1 immune responses. We have previously shownthat the CD4+ T-cell response to immunization with the protein antigen keyhole limpethaemocyanin ( KLH ) has a mixed interleukin-4 (IL-4)/IFN-c production profile. Here we showthat this immunization regimen also stimulates accumulation in the draining lymph nodes ofCD8+ T cells, which preferentially contain IFN-c mRNA ex vivo and secrete IFN-c protein invitro. This provides a model to test whether CD8+ cell-derived IFN-c participates in the normalcontrol of the immune response to a non-viable exogenous antigen. To investigate regulation ofthe anti-KLH response by the CD8+ population or IFN-c produced by this or other cell types,mice were administered depleting antibodies. Depletion of CD8+ cells had no effect on thefrequency of clonogenic KLH-specific CD4+ T cells, the IL-4/IFN-c profiles of their progeny, orthe isotype profiles of the serum antibody response to KLH. In contrast, IFN-c neutralizationdiminished cell accumulation in the lymph nodes and reduced both the frequency of KLH-specificCD4+ T cells that gave rise to IFN-c-producing clones and serum titres of KLH-specific IgG2aand IgG3. Therefore, despite the potential for cross-regulation, the CD4+ T-cell response to thisimmunogen is independent of the IFN-c-skewed CD8+ response.

INTRODUCTION tion14 and graft-versus-host disease.15 It is likely that theseeffects are not entirely attributable to cytotoxic T-lymphocyteWhen stimulated in vitro, CD8+ T cells possess the capacity(CTL) activity and that cross-regulation of CD4+ T cells byto produce a large array of cytokines, including interferon-cCD8+ T-cell-derived IFN-c is involved.(IFN-c), granulocyte–macrophage colony-stimulating factor

Although more commonly induced by antigens derived(GM-CSF), tumour necrosis factor-a (TNF-a), interleukin-2from intracellular sources, such as foreign major histocompat-(IL-2), IL-3, IL-4, IL-5, IL-6 and IL-10.1,2 However, theiribility complex (MHC) or intracellular pathogens, there haveresponses in vivo are more restricted, with a propensity towardsbeen a number of recent reports of activation of classproduction of IFN-c. Indeed, examples of expression of typeI-restricted CD8+ T cells in response to exogenous anti-2 cytokines by murine CD8+ T cells in vivo or directly ex vivogens.16–19 This raises the possibility that CD8+ cell-mediatedare scarce.3–6 A significant consequence of the proclivity ofenhancement of IFN-c production is a normal control mechan-CD8+ T cells for IFN-c production in vivo could be theism for the immune response against both classes of antigen.potentiation of IFN-c production by CD4+ T cells. The pres-In the present study we show that immunization with theence of IFN-c during CD4+ T-cell priming can bias towardsexogenous protein keyhole limpet haemocyanin (KLH )development of type 1 responses by promoting its owninduces an increase in CD8+ cell numbers concomitant withsecretion by CD4+ effector cells, by suppressing proliferationthe CD4+ T-cell response. The latter response is characterizedof IL-4-secreting cells and via augmentation of IL-12 and itsby a high precursor frequency of antigen-specific IL-4-secretingreceptor.7–9 The presence of CD8+ T cells has been correlatedcells and a lower precursor frequency of IFN-c secretors,with a strong type 1 response or suppressed type 2 responsewhich correlates functionally with the KLH-specific antibodyin a number of in vivo experimental systems, including IgE

production in response to antigen delivered by aerosol or with response dominated by IgG1 with lower levels of IgE andricin,10,11 intracellular parasite infections,12,13 allograft rejec- IgG2a.20 Given the cross-regulatory properties ascribed to

IL-4 and IFN-c, evolution of this mixed response to a moreReceived 29 July 1997; revised and accepted 8 October 1997. skewed cytokine profile might be predicted, as observed for

example during leishmania infection.21 However, the mixedCorrespondence: Prof. A. Kelso, Queensland Institute of MedicalIL-4/IFN-c profile is maintained throughout the response.20Research, Post Office Royal Brisbane Hospital, Queensland 4029,

Australia. Due to their preferential production of IFN-c it is possible

© 1998 Blackwell Science Ltd 341

A. G. Doyle et al.342

that CD8+ cells might contribute to maintenance of IFN-c and 5% heat-inactivated fetal calf serum (FCS; CSL Ltd,production by CD4+ cells in this response and in other non- Melbourne, Australia). For bulk culture, draining lymph nodepolarized immune responses. We therefore investigated cells were incubated at 106/ml in 200-ml volumes in microtitrewhether the IFN-c-biased CD8+ cell response associated with wells. Stimulation with anti-CD3 antibody was achieved byKLH immunization is necessary for the establishment of the precoating flat-bottomed wells with the anti-CD3 mAbcharacteristic mixed IL-4/IFN-c CD4+ cell response. 145-2C11 at 10 mg/ml.22 Antigen-stimulated cultures were per-

formed in U-bottomed wells with 5×105 irradiated syngeneicspleen cells (3000 rads) and KLH hydrolysate (100 mg/ml ),MATERIALS AND METHODSprepared by boiling a 20 mg/ml solution of KLH/1 NaOH

Immunization for 2 min and neutralizing with HCl. Culture supernatantsFemale 6–8-week-old C57BL/6 mice purchased from the were harvested after 24 hr for cytokine analysis.Animal Resources Centre (Perth, Western Australia) were For limiting dilution analyses (LDA), titrated doses ofimmunized subcutaneously at the base of the tail with 100 mg CD4+ or CD8+ cells were cultured in U-bottomed wells inalum-precipitated KLH (Calbiochem, La Jolla, CA) in 100 ml microtitre plates (30–44 wells/dose) in the presence or absencebalanced salt solution. of KLH (40 mg/ml ), with 5×105 irradiated syngeneic spleen

cells (3000 rads) and 120 IU/ml human recombinant IL-2T-cell preparation

(Cetus Corp., Emeryville, CA). Control wells lackingCell suspensions were prepared from inguinal and para-aortic

responder cells were included in each plate. After 10–14 dayslymph nodes by forcing through a stainless steel mesh. Afterincubation, cultures were scored microscopically for lympho-washing and centrifugation over Ficoll–Paque (Pharmacia,cyte growth. By this time, proliferating lymphocytes wereUppsala, Sweden), cells were stained with phycoerythrin(PE)-clearly distinguishable as a halo of viable cells around theconjugated anti-CD4 (GK1.5) and fluorescein isothiocyanatepellet of dead irradiated filler cells. Wells with greater than 50(FITC)-conjugated anti-CD8 (53.6) monoclonal antibodyviable lymphocytes were scored as positive for growth. Cultures(mAb) (both from Becton Dickinson, San Jose, CA) thenwere then washed and restimulated with concanavalin Aresuspended in balanced salt solution with 1 mg/ml propidium(10 mg/ml ) (Pharmacia) to induce cytokine synthesis. Cultureiodide. Single-positive CD4+ and CD8+ cells were isolatedsupernatants were collected for cytokine assays 24 hr later.20using a FACS Vantage (Becton Dickinson, Sunnyvale, CA).

A large forward scatter/side scatter gate was employed toCytokine assaysinclude small lymphocytes and larger activated cells and blastIL-4 was measured by enzyme-linked immunosorbent assaycells. Dead and damaged cells were excluded on the basis of(ELISA) based on the protocol of Sander et al.,25 using thepropidium iodide uptake and low forward scatter. BymAb BVD4-1D11 (5 mg/ml ) in carbonate buffer (pH 9·6) forre-analysis, purity of sorted cells was at least 96% and generally

>98%. CD44 staining was performed in conjunction with coating, and biotinylated BVD6-24G2 (0·3 mg/ml ) mAb ineither CD4 or CD8 staining, using biotinylated IM7.8 block solution [mouse tonicity phosphate-buffered saline(Pharmingen, San Diego, CA) followed by FITC-conjugated (PBS) with 0·1% skim milk powder, 0·05% Tween-20 and 1%streptavidin (Caltag, San Francisco, CA) and anti-CD4 or FCS] followed by streptavidin–horseradish peroxidase con-anti-CD8 antibody conjugated to phycoerythrin. jugate (Vector, Burlingame, CA) for detection. Absorbance

was read at 415 nm with a reference wavelength of 490 nmReverse-transcription polymerase chain reaction (RT-PCR) after 1 hr incubation with the enzyme substrate 2,2-az-Analysis of cytokine mRNA by RT-PCR was performed inobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) atessentially as described previously.22 Total RNA prepared 0·55 mg/ml in 0·1 citrate (pH 4·4) with 0·1% H2O2. Titres infrom purified CD4+ and CD8+ cells by guanidine bulk culture supernatants were determined from the linearisothiocyanate lysis, and phenol–chloroform extraction was portion of parallel dose–response curves by comparison withreverse-transcribed, cDNA were amplified by two-round nested titrations of baculovirus-expressed recombinant murine IL-4,PCR, and specific products were detected by Southern blot and expressed in arbitrary units (where 1 U/ml is defined ashybridization. PCR samples were amplified for 40 cycles using

the concentration stimulating half-maximal proliferation ofexternal primer pairs, the products were diluted 1533 in H2O,

the IL-4 responsive cell line CT.4S).26 Supernatants fromand 2 ml was used in the second round of 30 cycles with

limiting dilution cultures were assayed at a single concentrationinternal intron-spanning primer pairs. External primers for(50%). Positive supernatants were defined as those whichIL-4, IFN-c and b-actin were included in each first-roundexceeded by at least 3 SD the value for control wells withoutPCR reaction, and each of the respective internal primers inresponders.all second-round reactions. PCR oligonucleotide primers have

Bulk culture supernatants were titrated for IFN-c bybeen described elsewhere.23 All PCR runs included a titrationmeasuring proliferation of the IFN-c-sensitive cell lineof cloned b-actin and cytokine cDNA to monitor cDNAWEHI-279, as described previously.20 IFN-c activity wassensitivity (at least 10−16 g in these experiments) and 10standardized using purified recombinant IFN-c (Genentech,negative control samples. No PCR products were detected inSouth San Francisco, CA) and expressed in antiviral U/ml asthe negative control samples.defined by the supplier. Supernatants from limiting dilutioncultures were assayed at a single concentration (50%). PositiveT-cell culturesupernatants were defined as those which inhibited WEHI-279All cultures were performed in supplemented Dulbecco’s

modified Eagle’s medium24 with 5×10−5 2-mercaptoethanol proliferation by >50%.

© 1998 Blackwell Science Ltd, Immunology, 93, 341–349

Th cytokine response is independent of CD8+ cells 343

CTL assayEffector cells were prepared from draining lymph nodes 5 daysafter immunization. Lymph node cells were either used immedi-ately (ex vivo) or cultured for 5 days at 2×106/ml with 2·5×106irradiated syngeneic spleen cells/ml and KLH hydrolysate(200 mg/ml ) before use. Target cells were prepared by labellingthe thymoma cell line EL-4 (H-2b) by incubation with 100 mCiof Na51CrO4 (Amersham Int., Amersham, UK) for 75 min,washing, and incubating with KLH hydrolysate (200 mg/ml )for 1 hr. Graded numbers of effector cells were incubated with104 labelled target cells, and released 51Cr was measured inthe supernatants after 4 hr.17

In vivo depletion of CD8+ T cells and IFN-c Days after immunization

0 1 2

Cel

l rec

over

y fr

om d

rain

ing

lym

ph n

odes

/mou

se (

×10–6

)

100

10

13 4 5 6 7 8

TotalCD4CD8

Mice were injected intraperitoneally with 700 mg of control orFigure 1. Immunization with KLH increased numbers of both CD4+depleting antibody on every third day commencing 4 daysand CD8+ cells in the draining lymph nodes. At the indicated time

prior to KLH immunization. Anti-CD8 (2.43)27 and anti- points after immunization, single-cell suspensions were prepared fromIFN-c ( XMG 1.2)28 mAb were purified using protein inguinal and para-aortic lymph nodes, and total viable cells wereG–Sepharose (Pharmacia). Purified rat IgG (Sigma, St Louis, enumerated. Proportions of CD4+ and CD8+ cells were determinedMO) was used as the control treatment. by flow cytometry. Values are arithmetic means±SD determined in

five experiments, each with pooled draining lymph nodes from twoSerum immunoglobulin ELISA mice.Titres of KLH-specific immunoglobulins were determined byELISA.20 Briefly, serially diluted serum samples were incu- maximum sensitivity, IL-4 mRNA was restricted in four outbated in KLH-coated wells, and bound immunoglobulin was of five experiments to the CD4+ population, with expressiondetected with biotinylated isotype-specific antibodies first detected 3 days after immunization. In one experiment(Southern Biotechnology Associates, Birmingham, AL) fol- IL-4 mRNA was detected in CD8+ cells at days 3, 5 and 8.lowed by horseradish peroxidase-coupled streptavidin, then In contrast, IFN-c mRNA was detected in both CD4+ andABTS substrate (as per IL-4 ELISA). To avoid competition CD8+ populations throughout the time–course (Fig. 2a).for antigen by more abundant IgG antibodies, KLH-specific IFN-c mRNA was similarly detectable after one round ofIgE was measured using biotinylated antigen.29 PCR (data not shown). IFN-c production by CD8+ cells from

unimmunized mice was detectable only in the subpopulationStatistical analysis bearing high levels of CD44 (Fig. 2b), which is a stable markerStatistical significance was assessed by Mann–Whitney test.

RESULTS

Time–course of cell accumulation in draining lymph nodes

Immunization with KLH was shown previously to lead to anincrease in total cell recoveries from the draining lymphnodes.20 Figure 1 shows the rapid increase observed in CD4+and CD8+ cells, with numbers of both populations maximalby about day 3. Significance values for the increase in CD8+cell numbers relative to unimmunized controls were: day 3,P<0·005; day 5, P<0·05; day 8, P<0·005. The proportion ofCD4+ cells bearing high levels of the activation marker CD44increased from 16% at day 0 to 31% at day 5, while for CD8+cells the proportions remained essentially unchanged at 24%CD44hi on day 0 and 22% on day 5 (mean of two experiments,data not shown). Cell accumulation in the lymph nodes wasdependent on the presence of KLH in the inoculum, as lymphnodes from mice injected with alum alone were indistinguish-

Tota

lC

D44

lo

CD

44hi

CD4+ CD8+Day 0

IFN-c

IL-4

b-actin

(b)

Tota

lC

D44

lo

CD

44hi

CD4+ cells CD8+ cellsDay

IFN-c

IL-4

b-actin

(a)0 1 3 5 8 0 1 3 5 8

able from unimmunized controls in terms of cell numbers andFigure 2. Time–course of expression of IL-4 and IFN-c mRNA bycomposition (data not shown).CD4+ and CD8+ cells from draining lymph nodes following immuniz-ation with KLH. (a) mRNA was extracted from 5×105 freshly purifiedcells, subjected to cDNA synthesis, and 10% of the cDNA amplifiedCytokine production by draining lymph node cellsby PCR. Specific products were detected by Southern hybridization.

The cytokine profiles of CD4+ and CD8+ cells from the RT-PCR sensitivity for cloned cDNAs was 10−18 g for b-actin anddraining lymph nodes of KLH-immunized mice were compared 10−16 g for IL-4 and IFN-c. (b) mRNA extracted from 5×104 purifiedby mRNA analysis directly ex vivo and by measurement of CD44lo or CD44hi ( lowest and highest 30% of expression distribution)

day 0 cells was subjected to RT-PCR as above.protein secretion in vitro. Using two-round PCR to achieve

© 1998 Blackwell Science Ltd, Immunology, 93, 341–349

A. G. Doyle et al.344

Days after KLH immunization

0 1 2U

nits

of c

ytok

ine/

106 c

ells

1

0·1

0·013 4 5 6 7 8

CD4CD8

10

0·1

1

10

100

1000

0 1 2 3 4 5 6 7 8

(a) (b)

Figure 3. Time–course of expression of IL-4 and IFN-c protein by CD4+ and CD8+ cells from draining lymph nodes followingimmunization with KLH. Secreted IL-4 (a) and IFN-c (b) were measured in the supernatants of anti-CD3 mAb-stimulated bulkcultures of CD4+ and CD8+ cells isolated at the indicated times after priming with KLH (dashed line=detection threshold).Geometric mean values±SD were calculated from five experiments, each with pooled draining lymph nodes from two mice.

of previous antigenic stimulation,30 presumably in this case enhanced clonogenicity of naive CD8+ cells by 2·2-fold (meanof three experiments, data not shown). However, thewith antigens other than KLH. Corresponding with the mRNA

data, the CD44hi fraction produced 73 times more bioactive IL-4/IFN-c profile was opposite to that seen in the CD4+population following immunization: the CD8+ cells that wereIFN-c/cell than the CD44lo population when stimulated by

solid-phase anti-CD3 antibody (data not shown). clonogenic in the presence of KLH gave rise predominantlyto IFN-c-producing clones (54%), with IL-4 detected onlyThe mRNA expression data correlated with production of

IL-4 and IFN-c protein in bulk cultures with solid-phase anti- very rarely (ratio of IL-4:IFN-c producers <0·007). LDAdisplayed single-hit kinetics as shown previously.20CD3 antibody (Fig. 3). This stimulus was used because

antigen-induced secretion of IL-4 or IFN-c was at or belowthe limit of detection, as reported previously.20,26 With anti-

Antigen specificity of CD8+ cellsCD3 mAb stimulation both the CD4+ and CD8+ populationsproduced detectable IFN-c, whereas IL-4 production was The issue of the antigen specificity of CD8+ cells from lymphrestricted mainly to CD4+ cells (significant IL-4 production nodes of immunized mice was explored further by assayingwas seen in CD8+ cells from day 3 onwards in one experiment for cytokine production by freshly purified cells in antigen-out of five). Immunization with KLH stimulated increased stimulated bulk cultures. A hydrolysate of KLH was used inIFN-c production by the CD4+ population, peaking at around order to generate peptides that could be presented on MHCday 5. Secretion of IFN-c by the CD8+ population was class I molecules. Figure 5a shows that CD8+ cells from bothconsistently greater than the CD4+ population, and on a per naive and day 5-immunized mice produced small quantites ofcell basis was only marginally enhanced by immunization. IFN-c but that this was not significantly enhanced in theHowever, the total IFN-c-producing potential in draining presence of antigen. Similarly, effector CD8+ cells used directlylymph nodes was elevated because CD8+ cell numbers ex vivo, or cultured for 5 days to enrich antigen-reactive cells,increased over the time–course. IL-4 production by the CD4+ did not lyse KLH hydrolysate-loaded target cells morepopulation became detectable 1–3 days after immunization, efficiently than unloaded target cells (Fig. 5b). In addition topeaking at day 5. the KLH hydrolysate, other antigen preparations such as

intact KLH and an endopeptidase digest of KLH, and othermethods to introduce KLH into the class I presentation

Precursor frequency analysis of cytokine-secreting CD4+ and pathway including electroporation and liposome- and osmoticCD8+ cells shock-mediated methods31 to load antigen into spleen or EL-4

stimulator cells, did not demonstrate antigen-specific stimula-Limiting dilution analysis (LDA) provided additionaltion of IFN-c production or CTL activity (data not shown).information on antigen specificity and the frequency of KLH-Likewise, titres of other cytokines, IL-3 and IL-4, were noresponsive cells. The trends observed by LDA (Fig. 4) werehigher in the presence of antigen than in its absence.consistent with the population data in Figs 2 and 3. Cloning

of most CD4+ cells was antigen specific, as indicated by theapproximately 10-fold enhancement of cloning efficiencies in

Effects of in vivo CD8+ T-cell depletion and anti-IFN-cthe presence of KLH. The majority of CD4+ clones produced

antibody treatmentIL-4, with a smaller proportion producing IFN-c (ratio 4·351).For CD8+ cells, antigen-specificity was less clear: in four To explore whether CD8+ cells influence the response of

CD4+ T cells to KLH, mice were depleted of CD8+ cells andexperiments clonogenicity of day 7-immunized CD8+ cells inthe presence of KLH was only enhanced 2·1-fold relative to their responses to KLH were analysed. An advantage of this

approach is the potential to identify a role for CD8+ cells,control cultures with no antigen, and KLH addition similarly

© 1998 Blackwell Science Ltd, Immunology, 93, 341–349

Th cytokine response is independent of CD8+ cells 345

Growth

prec

urso

r fr

eque

ncy

%

1

0·1

0·01Growth IL-4 IFN-c

10

0·1

1

10

0·01

CD4+ LN cells CD8+ LN cells

Growth Growth IL-4 IFN-c

–KLH +KLH –KLH +KLH

Figure 4. Converse IL-4/IFN-c profiles of day 7 KLH-primed CD4+ and CD8+ lymph node cells revealed by limiting dilutionanalysis. Cells purified from draining lymph nodes 7 days after immunization were cultured at limiting dilution with accessorycells and IL-2 in the presence or absence of antigen. After 10–14 days, cultures were scored for proliferative expansion, thenrestimulated with concanavalin A for 24 hr to determine the frequency of precursors that gave rise to clones producing IL-4 andIFN-c. Each point represents a separate precursor frequency estimate (~, geometric mean; M, detection threshold).

whether or not they are KLH-specific. IFN-c, a candidateeffector in any regulatory activity of the CD8+ cells, wasdepleted in a separate group of mice to investigate whetherthe parameters examined were dependent on IFN-c.

At the time of immunization, efficacy of cell depletion wasat least 99% as determined by flow cytometric analysis oflymph node T cells stained with 53.6 anti-CD8 mAb. This levelof depletion was also observed at the day of killing. To checkfor the presence of CD8+ cells binding the depleting antibody,which might mask detection by the 53.6 reagent, staining wasalso performed with goat anti-rat immunoglobulin–FITC, butno positive cells were detected. Anti-IFN-c treatment wasmonitored by measuring IFN-c-inhibitory activity of serum.When sera were titrated against a constant concentration ofIFN-c in the WEHI-279 assay, inhibition of IFN-c activitywas 100-fold to 1000-fold higher in serum of XMG 1.2-treatedmice than in sera of rat IgG-treated control animals.

To investigate the consequences of CD8+ cell depletionand anti-IFN-c mAb treatment, mice were bled and draininglymph node cells were prepared 7 days post-immunization, atime at which a stable IL-4/IFN-c profile has been estab-lished.20 Data on recovery and composition of draining lymphnode cells are shown in Table 1. Total cell recoveries werediminished more than twofold by anti-IFN-c treatment, andthe CD4+:CD8+ cell ratio was slightly elevated comparedwith control mice. In response to CD8+ cell depletion, asmaller diminution of total cells was observed and theproportion of CD4+ cells was elevated, such that theabsolute number of CD4+ cells resembled that in control

E:T ratio

1 10 100

(b)50

40

30

20

10

0

–10

% s

peci

fic ly

sis

+ KLH– KLH+ KLH ex vivo– KLH ex vivo

KLH

–

(a)

10

1

0·1

IFN

-c (

U/1

06 cel

ls)

+– + – +

100No CD8

cellsNaive

CD8 cellsDay 5

CD8 cells

mice (8·9×106±1·6×106 versus control values of 10·3×Figure 5. Analysis of CD8+ cell antigen-specificity. (a) IFN-c secretionby CD8+ cells in bulk culture. CD8+ cells purified from lymph nodes 106±2·2×106; arithmetic mean±SD).of naive or day 5-immunized mice were cultured for 24 hr with As shown in Fig. 6a, the capacity of total lymph node cellsirradiated spleen cells in the presence or absence of KLH hydrolysate. to produce IFN-c in anti-CD3 antibody-stimulated bulk cul-IFN-c in culture supernatants was measured by bioassay. Data tures was markedly diminished (10-fold to 28-fold) by CD8+summarize several experiments, each point representing a separate

cell depletion. This is probably due to removal of the majordetermination (~, geometric mean; M, detection threshold). (b)cellular source of IFN-c rather than an indirect effect onCTL activity of day 5-primed CD8+ cells ex vivo (circles) or expandedCD4+ cells mediated by CD8+ cells or IFN-c, because IFN-cin vitro with irradiated spleen cells and KLH (squares). EL-4 cellsproduction by CD4+ cells was essentially unaltered by CD8+incubated with (filled symbols) or without (empty symbols) KLH

hydrolysate as a source of KLH peptide were used as target cells. cell depletion or anti-IFN-c treatment. Supporting this

© 1998 Blackwell Science Ltd, Immunology, 93, 341–349

A. G. Doyle et al.346

Table 1. Effects of depletion of CD8+ cells or IFN-c on cell yield and CD4/CD8 ratios indraining lymph nodes 7 days after KLH immunization

Draining lymphTreatment* Experiment node cells/mouse % CD4+† %CD8+† CD4+5CD8+Control 1 46×106 20·2 14·3 1·41

2 55×106 21·5 13·2 1·633 56×106 22·2 14·3 1·554 36×106 21·8 15·3 1·42

Anti-CD8 1 36×106 25·0 0·1 2502 28×106 25·3 0·1 2533 39×106 28·0 0·1 2804 27×106 31·0 0·05 560

Anti-IFN-c 1 20×106 23·2 15·6 1·482 21×106 19·8 5·7 3·473 32×106 22·2 9·8 2·274 17×106 21·5 6·7 3·21

*Treatment of mice with anti-CD8 or anti-IFN-c mAb or rat IgG (control ) wascommenced 4 days prior to immunization, and repeated every third day until animals werekilled 7 days after immunization.

†Determined by flow cytometry of viable cells gated on forward scatter, side scatter andpropidium iodide.

and 0·51 for IFN-c depletion. In one experiment, IL-4 wasdetected in supernatants from antigen-stimulated bulk culturesof unfractionated lymph node cells, but no differences wereapparent in the different treatment groups. Expression of IL-4and IFN-c mRNA was also assessed in freshly purified CD4+,CD8+ and unfractionated lymph node cells, but no effects ofdepletion were evident (data not shown).

Results of limiting dilution analyses of CD4+ cells (Fig. 7a)showed that CD8+ cells have no demonstrable role in theestablishment of the IL-4/IFN-c profile of KLH-specific CD4+precursors. The frequencies of KLH-responsive precursors,IL-4-producing clones and IFN-c-producing clones were simi-lar in control and CD8+ cell-depleted treatment groups. Inagreement with the lack of effect on CD4+ cell function, theisotype profile of KLH-specific antibodies was unchanged byCD8+ cell depletion. In contrast, depletion of IFN-c resultedin a threefold reduction in precursor frequency of IFN-c-producing clones (P<0·026) and this correlated with the

0·1 1 10

(b)

(a)

IFN-c (U/106 cells)

TotalLN cells

TotalLN cells

CD8+

CD4+

ControlAnti-CD8Anti-IFN

1 10 100 1000

decreased production of antigen-specific IgG2a and IgG3Figure 6. Effects of CD8+ cell and IFN-c depletion on IFN-c(Fig. 7b), both of which are IFN-c promoted isotypes.33 Levelssecretion. IFN-c was measured in the supernatants of bulk cultures

of lymph node cells 7 days after KLH immunization. (a) Cells (106/ml ) of KLH-specific IgG2a and IgG3 were both reduced 16-foldwere stimulated for 24 hr with anti-CD3 antibody (*, too few cells to with P<0·0003 and P<0·0005, respectively.assay). (b) Cells (5×106/ml ) were stimulated for 48 hr with unhydro- Our results demonstrate that both CD4+ and CD8+lysed KLH (40 mg/ml ) and irradiated spleen cells. No IFN-c was populations are expanded within the draining lymph nodes indetected in parallel cultures without antigen, or in cultures of APC

response to KLH immunization and that these populationswithout added effector cells (M, detection threshold; SD bars areexhibit distinct IL-4/IFN-c expression patterns, the CD4+shown).cells displaying an IL-4hi/IFN-clo pattern and the CD8+ cellsexpressing IFN-c with essentially no IL-4. The CD8+ responsewas characterized by an absence of demonstrable antigeninterpretation is the observation that IFN-c production wasspecificity. In vivo depletion studies to probe the role of theunaltered in antigen-stimulated bulk cultures of unfractionatedCD8+ population demonstrated that although IFN-clymph node cells (Fig. 6b), in which CD4+ cells were likelyneutralization reduced the precursor frequency of IFN-c-to be the major source of IFN-c.32 Following stimulation withproducing CD4+ clones, depletion of CD8+ cells, a potentiallyanti-CD3 antibody, IL-4 was detectable only in supernatantssignificant source of IFN-c, resulted in no obvious perturbationfrom cultures of purified CD4+ cells. No changes in IL-4of the CD4+ anti-KLH response. Hence, determination of theproduction were induced by depletion, the geometric meanIL-4/IFN-c profile of the CD4+ cell response is dependent onvalues (U/106 CD4+ cells) determined from three experiments

being 0·43 for control treatment, 0·38 for CD8+ cell depletion factors other than the co-elicited CD8+ population.

© 1998 Blackwell Science Ltd, Immunology, 93, 341–349

Th cytokine response is independent of CD8+ cells 347

cytotoxic T cells have been generated from BALB/c mice byimmunization with antigen in complete Freund’s adjuvant.18While we can conclude that the CD8+ population withindraining lymph nodes is expanded as a consequence of immun-ization (Fig. 1) and that this population expresses IFN-c,there was no demonstrable antigen-specific clonal expansionin limiting dilution cultures (Fig. 4), or induction of antigen-specific CTL activity or cytokine production in assays utilizingantigen-presenting cells treated to introduce KLH into theMHC class I presentation pathway. Regardless of the lack ofdetectable KLH-specificity, and whether this merely reflectsinadequate in vitro antigen processing or low assay sensitivity,the capacity to produce IFN-c confers intrinsic immunoregula-tory potential upon the CD8+ population.

CD4+ and CD8+ populations from draining lymph nodesof KLH-primed mice expressed distinct profiles of the arche-typal T-helper type-1 (Th1) and Th2 cytokines IFN-c andIL-4. At the mRNA level ex vivo and the level of cytokineprotein secretion in bulk and clonal cultures, the CD8+ cellsdisplayed an IFN-c+/IL-4− profile that contrasted with themixed IL-4+/IFN-c+ response of the CD4+ cells. Given thatthe cytokine milieu during T-cell priming by antigen is con-sidered to be a critical element in determining T-cell cytokineexpression profile, regulatory interactions between the CD4+and CD8+ populations within KLH-primed lymph nodesmight be expected. This is not necessarily dependent upon thepopulations sharing antigen specificity. Precedents for alter-ation of cytokine responses by bystander responses includethe modulation of anti-tetanus toxoid IgG isotypes by viral

(b)

(a)

Control Anti-CD8 Anti-IFN

IgG1 IgG2a IgG3 IgE

1000

100

10

1

0·1

0·01

Ser

um a

nti-K

LH Ig

(arb

itrar

y U

/ml)

Pre

curs

or fr

eque

ncy

(% C

D4+

cel

ls)

1

0·1

0·01

–KLH +KLH

Growth Growth IL-4 IFN-c

infection,38 and induction of IL-5 expression by virus-specificFigure 7. Effects of depletion of CD8+ cells or IFN-c on CD4+ CD8+ cells in the presence of a type-2 CD4+ cell response toprecursor frequencies and KLH-specific immunoglobulin isotypes. ovalbumin.5 Hence, the cytokine expression profile of KLH-Mice were treated with antibody, rat IgG (control ), anti-CD8 mAb

specific CD4+ cells might be influenced by the IFN-c-or anti-IFN-c mAb on every third day commencing 4 days prior toproducing CD8+ population, irrespective of their antigenimmunization with KLH, and serum and lymph nodes were collectedspecificity, and vice versa.7 days after immunization. (a) Precursor frequencies of proliferating

Although the generation of IL-4-producing CD8+ cells isand cytokine-producing CD4+ cells were determined by LDA, as infostered by IL-4 in vitro,39,40 and IL-4-producing CD4+ cellsFig. 4. (b) Serum anti-KLH immunoglobulin titres were measured by

isotype-specific ELISA and expressed in arbitrary units relative to a were present within the draining lymph nodes, we did notstandard serum. Bars represent geometric means. detect IL-4-producing cells consistently in the KLH-primed

CD8+ lymph node population. The CD8+ T-cell responsetherefore did not appear to be regulated by the responding

DISCUSSIONCD4+ T cells. Likewise, there was no evidence of induction ofIL-4 synthesis by CD8+ cells in the presence of IL-4-producingImmunization with alum-precipitated KLH induces a large

accumulation of both CD4+ and CD8+ cells within the CD4+ cells in a contact hypersensitivity model with oppositepatterns of polarized cytokine production41 or in IL-4draining lymph nodes. In this, as in many other responses to

protein antigens, the antigen-reactive CD4+ population has transgenic mice.6 Since most naive CD8+ T cells are able togive rise to type-2 cytokine-producing clones in vitro,23 thisbeen investigated extensively,20,26,34,35 while the elicited CD8+

population and its immunoregulatory potential have been suggests that there are constraints on the generation of IL-4-producing CD8+ cells in vivo, such as anatomical separationlargely overlooked. Our demonstration of the expression of

distinct and opposing cytokine profiles by the CD4+ and of CD4+ and CD8+ cells, or a higher threshold for responsive-ness to IL-4, consistent with the scarcity of reportedCD8+ populations raises the issues of whether the CD8+

T-cell response is antigen-specific, and whether the CD8+ cells demonstrations of such cells in vivo in the mouse.The ratio of CD4+ precursors for IL-4-producing clonescan interact with other cells in the draining lymph nodes,

particularly CD4+ cells, to alter the character of the immune to IFN-c-producing clones (c. 551) is established early in theanti-KLH response (day 3), and contrary to prediction doesresponse.

Exogenous protein antigens can be taken up, processed not become more polarized with time.20 We considered thatCD8+ cell-derived IFN-c might be involved in the maintenanceand presented on MHC class I molecules via described path-

ways,36,37 allowing induction of a specific CD8+ cell of this stable mixed IL-4/IFN-c profile by promoting IFN-cproduction by CD4+ cells, since IFN-c has been shown toresponse.16,18 Hence, it would not be unprecedented to find

antigen-specific CD8+ cells amid the large CD8+ population enhance its own production by CD4+ effectors.7–9 This hypoth-esis, testable by in vivo CD8+ T-cell depletion, is concordantinduced by KLH immunization. Indeed, CD8+ KLH-specific

© 1998 Blackwell Science Ltd, Immunology, 93, 341–349

A. G. Doyle et al.348

Research Trust. We are grateful to Grace Chojnowski for assistancewith the observed expression of IFN-c at early time pointswith flow cytometry and to Dr Frank Carbone for advice on MHC(Figs 2 and 3) before the CD4+ IL-4/IFN-c profile becomesclass I presentation.stable at about day 4.20

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