Introduction

Antigen-presenting dendritic cells (DC) are bone marrow-derived migratory cells distributed sparsely but widely inlymphoid and non-lymphoid tissues.1 Their function is con-sidered to be the collection of foreign antigens in peripheralsites and then, after migration to lymphoid tissue, the initia-tion of immune responses by presentation of processedpeptide antigens to T cells.1–4 However, there is now evidencefor heterogeneity in the lineage origin and functional proper-ties of DC.5 Our laboratory has proposed that a group ofCD8α-bearing DC found in mouse thymus and spleen are of lymphoid origin,6–8 rather than the myeloid origin wellestablished for conventional DC.9,10 We also have evidencethat these putative lymphoid-derived or lymphoid-related DCin mouse spleen have distinct regulatory effects on the T cellsthey activate. These effects include initiation of Fas-mediateddeath of activated CD4 T cells11 and a restriction of endoge-nous IL-2 production by activated CD8 T cells.12 The presentpaper concerns the regulation of T cell cytokine output.

We initially observed that the proliferative response ofCD8 T cells stimulated by either alloantigens or defined anti-gens was limited if the antigen was presented by CD8α+

splenic DC, compared with the more extended proliferativeresponse to CD8α– splenic DC.12 This was shown to be dueto the low level of IL-2 production induced in the CD8 T cellsby CD8α+ DC, compared with that induced by CD8α– DC.12

The basis of this restricted IL-2 production was not the Fas-mediated T cell death observed with CD4 T cells.11,12 The

difference in IL-2 production was not due to veto signalstransmitted by the CD8α molecule itself,12 nor to differencesin signals from conventional costimulator molecules, such asB7–1 or B7–2,13 nor to soluble factors produced by the DC,13

nor to the differential survival of the CD8α– DC.13 A new, asyet unidentified, signalling system appears to be involved. Inview of the potential importance of DC control of T cellcytokine output, we have extended the study to othercytokines and to CD4 T cells.

We now report that the nature of the stimulating DC deter-mines the level of production of a spectrum of cytokines, notjust of IL-2, and that this regulation of cytokine productionapplies to CD4 as well as CD8 T cells. We also report that,although IL-2 is essential for the initial proliferation of bothCD4 and CD8 T cells in response to DC stimulation, thecontrol over cytokine production is independent of IL-2availability and is separate from the signals that activate T cells into cycle.

Materials and Methods

Animals

Normal mice of the C57BL/6J Wehi (B6) (H-2b), BALB/c (H-2d)and CBA/CaH Wehi (CBA) (H-2k) strains and mutant mice ofC3H/HeJ.lpr (C3H.lpr) (H-2k) strain were bred under specificpathogen-free conditions at the Walter and Eliza Hall Institute animalfacility (Melbourne, Vic., Australia). C57BL/6 IL-2Rα null mice14

were purchased from The Jackson Laboratory (Bar Harbor, ME,USA), then bred and maintained under clean conventional condi-tions. Female CBA mice at 6–12 week of age, female C3H.lpr miceat 4–5 weeks of age or male and female IL-2Rα null mice at5–8 weeks of age were used for purification of responder T cells.Female B6 or BALB/c mice at 5–7 weeks of age were used forpurification of stimulatory DC.

Immunology and Cell Biology (2000) 78, 214–223

Research Article

Regulation of T cell cytokine production by dendritic cells

VADIM KRONIN, 1 HUBERTUS HOCHREIN, 1 KEN SHORTMAN 1 and ANNE KELSO 2

1The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria and 2The Queensland Institute ofMedical Research, Brisbane, Queensland, Australia

Summary Previous work has established that the dendritic cells (DC) of mouse spleen regulate the IL-2 production, and hence the extent of proliferation, of the CD8 T cells they activate. It is now reported here that interaction of primary CD8 T cells with splenic CD8α– DC induced much higher production of IL-3, IFN-γ andgranulocyte-macrophage colony-stimulating factor (GM-CSF), as well as IL-2, than did interaction with CD8α+

splenic DC. Furthermore, the CD8α– DC also induced higher levels of IL-2, IL-3 and IL-10 production in primaryCD4 T cells, compared with that induced by CD8α+ DC. These quantitative differences did not involve qualitativeshifts in the type of cytokine produced. Interleukin-4 production remained low in all the primary T cell culturesand restimulation experiments in secondary cultures did not reveal any bias in the cytokine production profile.When exogenous IL-2 was added to the primary cultures to ensure equal proliferation in response to CD8α– orCD8α+ DC, the higher level of production of IL-3, IFN-γ and GM-CSF induced by CD8α– DC was maintained.Thus, this general control of T cell cytokine production by splenic DC involves factors additional to those thatgovern activation of T cells into cell cycle.

Key words: cell-to-cell interactions, cytokine, dendritic cell, T lymphocyte.

Correspondence: K Shortman, The Walter and Eliza Hall Instituteof Medical Research, Post Office Royal Melbourne Hospital, Vic.3050, Australia. Email: shortman@wehi.edu.au

Received 30 August 1999; accepted 20 December 1999.

Antibodies and fluorescent reagents for cell purificationand analysis

The mAb derived from the following hybridoma clones were usedfor magnetic bead depletion: anti-CD3, KT3-1.1;15 anti-CD4,GK1.5;16 anti-CD8 α-chain, 53-6.7;17 anti-Thy 1.2, 30-H12;17 anti-IL-2Rα, PC61;18 anti-Gr-1, RB68C5;19 anti-Mac-1, M1/70.15;20 anti-macrophage Ag, F4/80;21 anti-B220, RA36B2;22 anti-erythrocyte Ag,TER-119 (provided by Dr T Kina, Chest Disease Research Institute,Kyoto University, Kyoto, Japan); anti-FcRII, 2.4G2;23 anti-MHCclass II, M5/114;24 and anti-CD44, 1 M7.81.25 All the listed mAbwere used as either ascites fluid or culture supernatants. The mAbused for immunofluorescent staining were the following: phycoery-thrin-conjugated anti-CD8 α-chain, 53-6.7 (PharMingen, San Diego,CA, USA) and anti-CD11c, N418,26 purified and conjugated withFITC in this laboratory.

Isolation of responder T cells

T cells were isolated from the lymph nodes (LN) using an immuno-magnetic bead depletion procedure. The cell suspensions were firstincubated with a mixture of the following mAbs at saturating levels:anticlass II MHC, M5/114; anti-erythrocyte Ag, TER-119; anti-CD44, 1M7.81 to select for naive T cells; and either anti-CD8 α-chain, 53-6.7; or anti-CD4, GK1.5 for isolation of CD4 T cells andCD8 T cells, respectively. The cells were then washed, after whichthe coated cells and B cells were removed with anti-IgG-coupledmagnetic beads using an 8:1 ratio of beads to cells and 1:1 mixtureof antirat and antimouse IgG-coupled Dynabeads (Dynal Inc., Oslo,Norway). The recovered T cells populations contained > 98% CD4+

cells or > 97% CD8+ cells, respectively.

Isolation of dendritic cells

Dendritic cells were isolated based on a procedure originallydescribed elsewhere,27 with some modifications.12 Briefly, thespleens were pooled from groups of 16 mice, cut into fragments andthen digested with collagenase (1 mg/mL; type II, Worthington Bio-chemicals, Freehold, NJ, USA) and DNase at room temperature for25 min, followed by EDTA treatment for the next 5 min to disruptDC–T cell complexes. All remaining procedures were at 4°C. Thelow density cells were enriched by centrifugation for 10 min at1700 g in Nycodenz (1.077 g/cm3, pH 7.2, 0.308 osmolar, NyegaardDiagnostics, Oslo, Norway). The low-density cells were then incu-bated for 30 min with a mixture of mAbs consisting of anti-CD3;anti-CD4; anti-Thy-1.2; anti-IL-2Rα; anti-Gr-1; anti-Mac-1α; anti-macrophage, F4/80; anti-B220; anti-erythrocyte; and anti-FcR II atpretitrated levels. Anti-Thy-1.2 and anti-Mac-1α were used at a lowconcentration that would only remove cells expressing high levels ofthese antigens. After incubation, the coated non-DC were depletedwith anti-IgG-conjugated magnetic beads using a 1:1 mixture ofantirat IgG and antimouse IgG beads, either at 8:1 (Dynabeads) or at10:1 (Paesel and Lorei, Hanau, Germany) bead-to-cell-ratio. Theremaining cells were stained with fluorochrome-conjugated anti-CD11c and anti-CD8α mAb and propidium iodide (to exclude deadcells). Populations of > 95% pure CD11c+CD8α+ and CD11c+CD8α–

DC were isolated by sorting on modified dual-laser FACS II (BectonDickinson, San Jose, CA, USA) and then immediately suspended inculture medium. Because some DC bear relatively high levels ofMac-1α, FcRII or IL-2Rα, antibodies against these antigens wereomitted from the depletion cocktail in some test experiments. Thisled to a 30% increase in DC yield and a higher proportion of CD8α–

DC in the final preparation. However, the results for differential proliferation and IL-3 cytokine production were unaltered.

Mixed leucocyte culture

Purified T cells (2 × 104) were mixed with 125–2000 DC in 200 µLof medium contained in wells of 96-well V-bottom plates and cul-tured for 1–6 days (37°C, in a humidified 10% CO

2-in-air incubator).

The culture medium was a modified RPMI-1640 medium describedelsewhere.12 In some experiments, murine rIL-2 was added into thecultures, at 100 U/mL.

Restimulation of T cells in culture

The primary mixed leucocyte cultures consisted of C3H lpr CD4 Tcells or CBA CD8 T cells incubated with CD8α+ or CD8α– B6 DC,as described earlier. After 4 (CD8 T) or 5 (CD4 T) days, the cellsfrom 20 cultures were pooled and dead cells were removed by centrifugation in Nycodenz medium (1.091 g/cm3, 4°C, mouseosmolarity). The recovered viable cells were cultured at 2 × 104 cells/well in modified RPMI-1640 medium, in the U-bottomed wells of96-well plates that had been coated previously with both anti-CD3and anti-CD28 mAbs (kindly provided by A Strasser, The Walter andEliza Hall Institute, Melbourne, Vic., Australia). The precoating wasachieved by incubating the wells overnight at 4°C with 50 µL/wellPBS containing mAb KT3 (10 µg/mL) and mAb 37N5 (10 µg/mL).After 48 h of incubation at 37°C to reactivate the T cells, super-natants were harvested and the levels of cytokines were measured.Effective restimulation was monitored in separate cultures by [3H]-thymidine (TdR) uptake.

Assessment of proliferation by [3H]-thymidine uptake

After the indicated culture time, 37 kBq [3H]-TdR (Amersham,Buckinghamshire, UK) was added into the wells and culture contin-ued for the next 6 h, after which the contents of the wells were trans-ferred to glass fibre filters (Inotech, Dottikon, Switzerland). Thelevel of [3H]-TdR incorporation was measured using a gas phasescintillation beta counter (Berthold, Wildbad, Germany) andexpressed as c.p.m.

Bioassay of cytokines in culture supernatants

Culture supernatants were pooled from 10 to 20 wells and the levelsof cytokines were measured as follows.

Interleukin-2 This procedure was done as described in detail else-where.28 Briefly, 4000 cells of a IL-2-dependent cytotoxic T lym-phocyte line, CTLL, were incubated with serial dilutions of culturesupernatants in 100 µL DMEM-FCS medium in flat-bottom wells for16–24 h, after which 18.5 kBq [3H]-TdR was added into each wellfor 4–6 h. Incorporated radioactivity was measured in a liquid scin-tillation β-counter. Titers were determined from dose–responsecurves, by comparison with calibration curves obtained using puri-fied human rIL-2 (Cetus Corp., Emeryville, CA, USA). Values givenare in World Health Organization (WHO) International Units.

Interleukin-3 and granulocyte–macrophage colony stimulatingfactor This procedure was done as described in detail elsewhere.29

Briefly, 200 FDC-P1 cells were seeded into the wells of Terasakiplates in 5 µL DMEM containing 5% FCS and either: (i) no addi-tives; (ii) the neutralizing anti-GM-CSF mAb 22E9;30 (iii) the neu-tralizing anti-IL-3 mAb 19B3;31 or (iv) both anti-GM-CSF andanti-IL-3 mAb. Serially diluted supernatants from the cultures wereadded to these plates at 5 µL per well. Recombinant murine GM-CSFand IL-3 (Biogen SA, Geneva, Switzerland) were used as standards.

DC regulate T cell cytokine production 215

After 48 h of incubation, the proliferation of FDC-P1 cells wasscored microscopically and cytokine levels were determined by com-parison of the sample and the standard titration curves. The sensitiv-ity of both GM-CSF and IL-3 detection was approximately 1 pg/mL.

γ-Interferon The procedure was done as described by Kelso28 usingB lymphoma cell line WEHI-279, for which proliferation is inhibitedby IFN-γ. WEHI-279 cells, 104 per well, were incubated with culturesupernatants in the presence and absence of neutralizing anti-IFN-γmAb (Ρ4–6 Α2). After 3 days, the number of cells was quantifiedusing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) assay. Titers were determined from the dose–responsecurves, standardized by comparison with recombinant rIFN-γ(Genentech Inc, South San Francisco, CA, USA) and expressed inGenentech units. The activities in supernatants were attributed toIFN-γ if they were inhibited by anti-IFN-γ mAb by at least 90%.

Enzyme-linked immunosorbent assay for cytokines inculture supernatants

Cytokines were assayed by a standard two-site ELISA. Briefly, 96-well flexible polyvinyl chloride Dynatech Microtiter plates(Dynatech Laboratories, Chantilly, VA, USA) were coated with theappropriate purified capture mAb, namely JES6-1A12.9 (antimouseIL-2), BVD4-1D11 (antimouse IL-4), TRFK5 (antimouse IL-5),JES5-2 A5.1 (antimouse IL-10) or R4-6A2 (antimouse IFN-γ).Cytokine binding to these mAbs was then detected with the appro-priate biotinylated detection mAb, namely JES6-5H4.1 (antimouseIL-2), BVD6-24G2.3 (antimouse IL-4), TRFK4 (antimouse IL-5),SXC-1 (antimouse IL-10) or XMG1.2 (antimouse IFN-γ). The abovehybridomas were kindly provided by Dr John Abrams (DNAXResearch Institute, Palo Alto, CA, USA). A readout was thenobtained by using a streptavidin–horseradish peroxidase conjugate(PharMingen) and a substrate solution containing 548 µg/mL 2,2′-Azino-bis(3-ethylbenz-thiazoline-6-sulphonic acid (ABTS; SigmaChemical Co., St Louis, MO, USA) and 0.001% hydrogen peroxide(Ajax Chemicals, Auburn, NSW, Australia) in 0.1 mol/L citric acidpH 4.2, followed by scanning at OD

414/492 nmon a Multiscan

MCC/340 MKII plate reader (Titertek Instruments Inc., Huntsville,AL, USA).

Cytokine levels were quantified by reference to standard curvesproduced with recombinant mouse IL-2, recombinant mouse IL-4and recombinant mouse IL-5 produced by transfectant lines providedby Dr Fritz Melchers (The Basel Institute for Immunology, Basel,Switzerland), recombinant mouse IL-10 (produced by transfectantline 20H11, supplied by DNAX Research Institute) and recombinantmouse IFN-γ (PharMingen).

Reverse transcriptase polymerase chain reaction assay forIL-2 mRNA

The IL-2 mRNA expression in the CD8 T cell–DC cultures wasdetermined at several time points using reverse transcriptase poly-merase chain reaction (RT-PCR). Messenger RNA from these cultures was isolated using a QIAGEN RNeasy Kit according tomanufacturer’s instructions (QIAGEN Pty Ltd, Clifton Hill, Vic.,Australia). The first-strand cDNA was synthesized using a ReverseTranscription System (Promega, Armidale, NSW, Australia). Theeffective cDNA concentrations were usually equalized for thepaired CD8α+ and CD8α– DC-stimulated CD8 T cells by makingcDNA dilutions to yield the same intensities for a β-actin PCRproduct after 30 cycles. Using these pretitrated cDNA concentra-tions, PCR for IL-2 was performed with 25 or 30 cycles: 30 cycles

was used to visualize IL-2 mRNA bands at the earliest timepoints,whereas 25 cycles was adequate after 1 day of culture. The PCRconditions for β-actin and IL-2 were initially 94°C for 30 s, 60°Cfor 30 s, 72°C for 1 min and a final 5 min step at 72°C. The primersused were for IL-2 5′-CCCACTTCAAGCTCCACTTC-3′ and 5′-TCCACCACAGTTGCTGACTC-3′ (PCR product: 389 bp) and forβ-actin 5′-GTGGGCCGCTCTAGGCACCAA-3′ and 5′-CTCTT-TGATGTCACGCACGATTTC-3′ (PCR product: 540 bp). The PCRproducts were segregated in a 1.5% agarose gel, stained with ethi-dium bromide and photographed under ultraviolet illumination.

Results

Both CD4 and CD8 T cells produce lower levels of IL-2 inresponse to CD8+ DC than in response to CD8– DC

We have shown previously that CD8 T cells produce onlyvery low levels of IL-2 in culture supernatants in response to CD8α+ splenic DC and, as a consequence, proliferation is restricted.12 In contrast, CD8α– DC induce greater IL-2 production and a more extended proliferative response. Pro-liferation of CD4 T cells stimulated by CD8α+ DC was alsoreduced compared with proliferation stimulated by CD8α–

DC, but this was due to the Fas-mediated apoptosis of theCD4 T cells.11 In contrast to the situation with CD8 T cells,IL-2 was usually not limiting proliferation in cultures of CD4T cells.11 However, although IL-2 was not limiting, itremained possible that CD8α+ DC nevertheless induce lowerlevels of IL-2 production by CD4 T cells than do CD8α– DC.To check this possibility, we directly compared the level ofIL-2 in supernatants of cultures of CD4 and CD8 T cellsresponding to allogeneic CD8α– and CD8α+ DC. Because theFas-mediated apoptosis of CD4 T cells when stimulated byCD8α+ DC would confuse the analysis of direct regulation ofIL-2 production, we used Fas-deficient CD4 T cells from lprmice to eliminate these complications. We also ensured thatthe responding T cells were all naïve and not previously acti-vated by depleting the small proportion of T cells expressingthe CD44 activation marker.

As shown in Fig. 1, stimulation of CD8 T cells withCD8α– DC produced much higher levels of IL-2 in theculture supernatants than did stimulation with CD8α+ DC, inconfirmation of our previous findings. In the case of CD4 Tcells, although the overall production of IL-2 was muchhigher, there was also a higher level of IL-2 in the super-natants of cultures stimulated with CD8α– DC than in thosestimulated with CD8α+ DC. These twofold to fourfold dif-ferences in IL-2 production by CD4 T cells were seen in allexperiments and were detected as early as day 2 of the cul-tures. These findings using the IL-2-dependent CTLL line asa readout were confirmed using an ELISA for IL-2 levels(data not shown). The findings with CD4 and CD8 T cellswere also confirmed using the DC isolation procedure, whichomitted the depletion of cells bearing high levels of Mac-1,FcRII and IL-2Rα and which gave higher DC recoveries.This eliminated the possibility that selective loss of certainDC subsets during isolation had distorted the results. Finally,similar differences were obtained using normal as well as lprCD4 T cells. However, as argued earlier, low IL-2 productiondue to a regulatory mechanism could not be distinguishedfrom low IL-2 production caused by cell death when normalCD4 T cells were used.

V Kronin et al.216

DC regulate T cell cytokine production 217

In parallel experiments (data not shown), we confirmedour earlier findings11,12 that proliferation was generallysimilar in cultures of lpr CD4 T cells stimulated with eitherCD8α+ or CD8α– DC, whereas a marked difference wasalways seen in the extent of proliferation of CD8 T cells.Thus, the reduced level of IL-2 produced in response toCD8α+ DC was nevertheless adequate for CD4 T cell prolif-eration in culture, but became limiting later in culture whenCD8 T cells were used.

Interleukin-2 is critical for T-cell proliferation even atearly stages of the culture

Although proliferation is curtailed at later times due to lackof IL-2, CD8+ DC do induce substantial proliferation of allo-geneic CD8 T cells at the early stages of the culture. In fact,up to day 2.5–3 proliferation induced by CD8+ DC is at leastas good as proliferation induced by CD8– DC12 and thenumber of T cells in the cultures was similar.12 However, noIL-2 was detected in the supernatants of cultures of CD8 Tcells stimulated by CD8+ DC,12 suggesting that the early proliferation of T cells may be dependent on other cytokines.To check this hypothesis, we used IL-2Rα null mice14 as thesource of the responding T cells. In the absence of the α-chain of IL-2R, murine IL-2Rβγ complexes fail to bind IL-2with sufficient affinity to allow signalling from IL-2.14,32,33

Naïve, non-activated CD4 T cells and CD8 T cells werepurified from the LN of either normal C57BL/6 mice ormutant IL-2Rα null B6 mice and their proliferative responsesto allogeneic CD8α– and CD8α+ DC were compared (Fig. 2).Responses of both CD4 and CD8 IL-2Rα null T cells werevery low at all culture times and were strongly diminishedcompared with the responses of normal T cells, regardless ofthe type of stimulating DC. These results indicate that IL-2 isthe crucial cytokine for T cell proliferation, even at the earlystages of culture and even with CD8+ DC. Presumably theCD8 T cells stimulated by CD8α+ DC did make a low levelof IL-2, sufficient to produce a short burst of proliferation,but not sufficient to accumulate in the culture supernatantand sustain continued expansion.

Interleukin-2 mRNA transcripts are found in CD8 T cellsstimulated by both CD8+ and CD8– DC

As a further test of whether IL-2 production is initiated inCD8 T cells early in culture by both CD8+ and CD8– DC, theinduction of IL-2 mRNA was checked. RNA was extractedfrom CD8 T cells harvested after culture with CD8+ or CD8–

DC, and RT-PCR was performed using primers specific forIL-2 or for β-actin as a control for the efficiency of RNAextraction and amplification (Fig. 3). With a relatively highsensitivity PCR assay, RNA transcripts for IL-2 were alreadydetected at day 1 of culture, with both CD8+ and CD8– DC asstimulators. The level of transcripts increased by day 2 andpersisted at this level on day 3, but IL-2 mRNA was notdetected in the CD8 T cells from day 4 cultures. The appar-ent level of IL-2 mRNA was initially highest with CD8α+ DCstimulators, but then, as mRNA levels increased, was severalfold higher at days 2 and 3 in cultures stimulated with CD8–

DC than in those stimulated with CD8+ DC. The presence ofexogenous IL-2 in the culture supernatants, which gaveenhanced proliferation and ensured equivalent proliferationin both cultures, did not alter this pattern of IL-2 mRNAexpression (Fig. 3).

Thus, these results support the conclusions from Fig. 2 thatIL-2 is produced in both cultures and accounts for the initiallyequivalent proliferative response shown in Figs 2,3. As theresponse proceeds, a modest enhancement in IL-2 mRNAtranscription in cultures stimulated by CD8α– DC (Fig. 3)becomes a very large difference in supernatant IL-2 levels(Fig. 1). This is presumably because the total level of IL-2produced by CD8 T cells is relatively low compared with thatproduced by CD4 T cells (Fig. 1), and the reduced amount isall consumed by day 3 in cultures stimulated by CD8+ DC.There could also be some regulation of IL-2 secretion, as wellas the control of transcription. The final result is at day 4, thatwhen there is no further production of mRNA for IL-2(Fig. 3), the supply of IL-2 is insufficient to sustain prolifera-tion in cultures initially stimulated by CD8+ DC. In contrast,the initial excess production of IL-2 when CD8– DC are stim-ulators permits a sustained proliferation at day 4 of culture.

Figure 1 The production of IL-2 in cultures of CD8 (a) or CD4 (b) T cells stimulated by allogeneic CD8α+ (s) and CD8α– (d) den-dritic cells (DC). The DC were isolated from spleens of B6 mice; 1000 DC were used per culture. CD8 and CD4 T cells were isolatedfrom lymph nodes of C3H.lpr or CBA mice, respectively; 2 × 104 T cells were used per culture. Culture supernatants were pooled from 10to 20 wells and used for assays of the levels of IL-2, using an IL-2-dependent cell line bioassay. No IL-2 was detected in cultures of DCor T cells alone. The results in each panel represent the data pooled from two experiments. The levels of IL-2 in the cultures of CD8 Tcells stimulated by CD8α+ DC were below the sensitivity limit of the assay.

Dendritic cells regulate the production by CD8 T cells ofa wide range of cytokines

As we have shown before12 and confirmed in this paper(Fig. 1), the nature of the stimulating DC determines the levelof IL-2 production by CD8 T cells. The question was, there-fore, whether DC can also regulate the production ofcytokines other than IL-2. To address this question, naïveCD8 T cells purified from the LN of CBA mice were stimu-lated with either CD8α+ or CD8α– DC from spleens of B6mice and the levels of cytokines in the supernatants weremeasured. In parallel experiments, T-cell proliferation inthese cultures was also measured. As shown in Fig. 4 (leftcolumn), the production of IL-3, IFN-γ, and GM-CSF wasmuch higher in cultures of CD8 T cells stimulated by CD8α–

DC than in those stimulated by CD8α+ DC. In no case couldthese cytokines be detected in cultures of the DC alone. Theproduction by CD8 T cells of a wide range of cytokines there-fore appeared to be determined by the stimulating DC. The

levels of IL-4 and IL-5 in these primary cultures were alsoassessed, but none were detected within the sensitivity of theassays.

Dendritic cell-induced differences in cytokine productionare maintained even when the rate of CD8 T-cell proliferation is equivalent

In confirmation of our previous findings,12 CD8 T-cell pro-liferation was more prolonged in cultures stimulated byCD8α– DC than by CD8α+ DC. It was therefore important toestablish whether the reduced production of cytokines inCD8α+ DC-stimulated cultures simply reflected the reducedproliferation and therefore the reduced numbers of cytokine-producing cells in these cultures, or whether there was somemolecular mechanism determining the differential productionof cytokines by each T cell. To address this question, theexperiments were repeated, but with 100 U/mL exogenous

V Kronin et al.218

Figure 2 Comparison of the proliferation kinetics of normal and IL-2Rα null CD4 (a,c) and CD8 (b,d) T cells stimulated with allogeneicCD8α+ (c,d) and CD8α– (a,b) dendritic cells (DC). (d), IL-2R+ T cells plus DC; (m), IL-2R– T cells plus DC; (h), IL-2R– T cells only.The DC were isolated from spleens of BALB/c mice and 1000 DC were used per culture. CD4 and CD8 T cells were isolated from lymphnodes of either normal B6 or B6 IL-2Rα null mice and used at 20 000 cells per culture. No exogenous cytokines were added. Proliferation of T cells was assessed by [3H]-thymidine (TdR) incorporation after a 6 h pulse. Incorporation of [3H]-TdR was measuredusing gas-phase scintillation counting, which is 30- to 50-fold less sensitive than conventional liquid scintillation counting. The back-ground incorporation of T cells or DC cultured alone was < 50 c.p.m. and at the peaks of proliferation the stimulation indexes for normalCD4 and CD8 T cells were always > 300. The results for each panel are the mean ± SD of data pooled from two experiments with eachindividual experiment involving three cultures per point.

IL-2 added to the cultures. We have shown before12 and con-firmed here (Figs 3,4) that, in the presence of high levels ofexogenous IL-2, CD8α+ DC induce as extensive proliferationof CD8 T cells as do CD8α– DC and the number of CD8 Tcells in the cultures is then similar. The effect of such IL-2addition on cytokine production is shown in Fig. 4 (rightcolumn). Exogenous IL-2 did not increase IL-3 or GM-CSFproduction, but did substantially enhance IFN-γ production.However, even in the presence of exogenous IL-2, when T cellnumbers were if anything marginally higher in cultures stim-ulated by CD8α+ DC (data not shown), the production of IL-3, IFN-γ and GM-CSF was still much higher in cultures ofCD8 T cells stimulated by CD8α– DC than in those stimulatedby CD8α+ DC. Again, no IL-4 or IL-5 could be detected in thesupernatants of these cultures. These results demonstrate thatDC regulate the production of cytokines by CD8 T cells via aprocess that is separate from the signals activating T cells intothe cell cycle or inducing their ongoing expansion.

Dendritic cells regulate the production by CD4 T cells ofa range of cytokines

Because the level of production of IL-2 by CD4 T cells wasshown to be regulated by the stimulating DC, we askedwhether the production of other cytokines by CD4 T cells wassimilarly regulated. Splenic CD8α+ or CD8α– DC were usedto stimulate pure naïve CD4 T cells from the LN of C3H.lprmice, with the culture supernatants collected at various timesand the levels of cytokines determined (Fig. 5). Generally, theproduction of cytokines by CD4 T cells was much higher thanby CD8 T cells, the exception being IFN-γ where, in contrast

to CD8 T cells, production was very low regardless ofwhether CD8α– or CD8α+ DC were used for activation (datanot shown). As with CD8 T cells, no IL-4 or IL-5 wasdetectable by bioassay in any of the cultures of CD4 T cells.In line with the results with IL-2, the production of IL-3 andthe later production of IL-10 was higher in cultures of CD4T cells stimulated by CD8α– DC than in those stimulated byCD8α+ DC (Fig. 5). However, the difference in production ofIL-3 between CD8α– and CD8α+ DC-stimulated cultures wasonly evident when the number of DC used was limiting (500or 1000 DC); in cultures of 2000 DC per well, CD8α+ DCinduced levels of IL-3 almost as high as did CD8α– DC (datanot shown). The one cytokine for which relative levels of production in response to the different types of stimulatingDC was out of line with the results with CD8 T cells, and outof line with other cytokines produced by CD4 T cells, wasGM-CSF (Fig. 5). Production of GM-CSF was usually higherin CD4 T cells when stimulated with CD8α+ DC, comparedwith CD8α– DC, although in occasional experiments the levelwas similar in the two cultures. This contrast between theproduction of GM-CSF and that of IL-2, IL-3 and IL-10 wasobserved even when the same supernatants from the samecultures were assayed. No GM-CSF could be detected in cultures of CD8α– or CD8α+ DC alone, even at a 100-foldhigher DC concentration. It should be noted that the rate ofCD4 T cell proliferation was roughly the same in culturesstimulated by CD8α– or CD8α+ DC, because IL-2 productionwas not as limiting as for CD8 T cell cultures. Accordingly,the control of cytokine production by CD4 T cells alsoappeared to be independent of factors governing T-cell acti-vation or proliferation.

DC regulate T cell cytokine production 219

Figure 3 Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of IL-2 mRNA transcripts in CD8 T cells stimulated byCD8+ (h) or CD8– (j) dendritic cells (DC). (a) No IL-2 added; (b) IL-2 added. The RT-PCR using IL-2 specific primers was performedon CD8 T cells harvested from cultures at the times indicated; the expression of β-actin mRNA was also determined as a control for anyvariation in RNA extraction or PCR efficiency. In this experiment, less cDNA was used for the CD8α+ DC-stimulated sample at day 1,in order to demonstrate the relatively higher IL-2 mRNA in this sample at the earliest timepoint. Thirty PCR cycles were used through-out, to ensure visualization of IL-2 mRNA at day 1. The proliferative response of the cultures at these timepoints is also given to enablea direct comparison to be made; note the change of scale for the cultures with exogenous IL-2. The figure represents one of two RT-PCRassays giving similar results.

V Kronin et al.220

Neither CD8α+ nor CD8α– splenic dendritic cells drive T cells towards a Th2 cytokine profile

It was notable that, despite the quantitative differences incytokine production that the CD8α+ versus CD8α– splenicDC induced in both CD4 and CD8 T cells, there was no evi-dence that the DC induced a switch in cytokine productionprofile in these primary cultures. The cytokine profileremained close to a ‘Th1’ type,34 with both IL-2 and IFN-γbeing produced and IL-4 remaining below the detection limit,even in cultures where production was low. A more critical

test of whether differentiation to a different cytokine produc-tion profile had been induced was to isolate the viable cellsat the end of the culture period, to restimulate them non-specifically in a secondary culture and then to sample thesupernatants and determine the resultant cytokine productionpattern. Such results are shown in Table 1.

The intense restimulation with anti-CD3 and anti-CD28induced in the secondary cultures a vigorous proliferativeresponse (data not shown) and an early burst of cytokine production, to levels substantially higher than were found inthe primary cultures with allogeneic DC as stimuli. With CD8

Figure 4 Proliferation and cyto-kine production in cultures of CD8T cells stimulated by CD8α+ andCD8α– dendritic cells (DC), eitherin the absence or in the presence ofexogenous IL-2. The conditionswere similar to those in Figs 1,2for CD8 T cells. The left columnpresents results for the cultureswith no exogenous IL-2 added.The results shown in the rightcolumn correspond to the culturesto which 100 U/mL IL-2 wasadded. No IL-4 could be detectedin either set of cultures at anytimepoint. (a,b), [3H]-thymidineuptake; (c,d), IFN-γ; (e,f), IL-3;(g,h), granulocyte–macrophagecolony stimulating factor (GM-CSF). The level of B6 DC was1000 per culture, except for themeasurements of GM-CSF inwhich the level of DC was 500 perculture. Results are the mean ± SDof data pooled from two separateexperiments, each with three ormore cultures per point. (d,j),Results for CD8α– DC-stimulatedcultures; (s,h), results forCD8α+ DC-stimulated cultures.BMCSU, bone marrow colonystimulating units.

T cells, IL-2 and especially IFN-γ remained the predominantcytokines, with no production of IL-4 or IL-5 being detected.Although CD8 T cells that were initially stimulated withCD8α– DC also produced higher levels of cytokines on non-specific restimulation in the absence of DC, much of the differential between the two lines of cells was lost.

In the case of CD4 T cells, it was possible to detect IL-4and IL-5 production in the secondary cultures. However, IL-2 production was also much higher and IFN-γ was nowdetected at high levels in the secondary cultures. The patternof cytokines was approximately the same whether the initialstimulus in the primary cultures was CD8α+ or CD8α– DC,

DC regulate T cell cytokine production 221

Table 1 Production of cytokines by T cells isolated from primary cultures with DC and then restimulated by anti-CD3 and anti-CD28

CD4 T cells CD8 T cellsCytokine Primary culture with DC Restimulation with mAb Primary culture with DC Restimulation with mAb(pg/mL) CD8α+DC CD8α–DC CD8α+DC CD8α–DC CD8α+DC CD8α–DC CD8α+DC CD8a–DC

IL-2 25 121 4470 ± 1600 5680 ± 2880 < 5 11 320 ± 90 420 ± 40IFN-γ < 150 < 150 68 450 ± 220 38 660 ± 2960 800 1130 42 450 ± 10 320 169 830 ± 13 900IL-4 < 15 < 15 2530 ± 910 2800 ± 240 < 15 < 15 < 15 < 15IL-5 < 15 < 15 43 790 ± 11 930 68 660 ± 24 670 < 15 < 15 < 15 < 15

The dendritic cell (DC) type listed represents those present as allogeneic stimulators in the primary cultures; the restimulation of harvestedviable T cells was with anti-CD3 and anti-CD28 mAbs in the absence of DC addition. The levels of cytokines in the culture superantants weremeasured after 3 days of primary culture or after 2 days of restimulation, using the ELISA assay.

Figure 5 Production of cytokines in cultures of lpr CD4 T cells stimulated by allogeneic CD8α+ (s) and CD8α– (d) dendritic cells(DC). (a), [3H]-thymidine uptake; (b) IL-10; (c), IL-3; (d) granulocyte–macrophage colony stimulating factor (GM-CSF). The conditionswere similar to those in Fig. 1 for CD8 T cells. No exogenous cytokines were added into the cultures. No IL-4 could be detected in thesupernatants of these cultures at any timepoint. The level of B6 DC was 500 DC/well (proliferation and production of IL-10) or 1000DC/well (IL-3 and GM-CSF). The results are the mean ± SD of pooled data from two to four experiments, each with three cultures perpoint. BMCSU, bone marrow colony stimulating units.

and the differences in levels seen in the primary cultureslargely disappeared in the secondary cultures. Overall, therewas no evidence that the differences in IL-2 production notedin the primary cultures were related to an ongoing Th1 to Th2cytokine profile switch.

Discussion

These results broaden substantially our original findings thatthe initial contact with either CD8α– or CD8α+ splenic DCdetermines the subsequent IL-2 production of the activatedCD8 T cells and that this in turn determines the extent of theirproliferation later in culture. It is now clear that both DCtypes induce IL-2 production, but only the CD8– DC inducesufficient IL-2 production to sustain proliferation of the CD8T cells beyond day 3 of culture. The regulatory influence ofthe initial DC contact is now seen to extend to the productionof other cytokines by CD8 T cells, with the production of IL-3 being tightly controlled and the production of IFN-γ andGM-CSF also being regulated, but less tightly.

Interestingly, we have now shown that this regulation ofcytokine production extends to the much higher level of IL-2 production by CD4 T cells, although the differencesseen in the culture supernatants are much less dramatic. Thisindicates that CD8α+ splenic DC may have two mechanismsof reducing CD4 T cell IL-2 output: (i) direct regulation ofIL-2 secretion; and (ii) Fas-mediated killing of the T cells.11

In contrast with the effects on CD8 T cells, the reduction ofCD4 T cell cytokine output when CD8+ DC are used as stim-ulators does not usually reduce IL-2 output to the level whereproliferation ceases in culture, although this might occur withlimiting DC numbers. The effect could well be more pro-nounced in vivo. It also extends to other cytokines producedby CD4 T cells, although the effect is not seen, or is evenreversed, with GM-CSF. Independent regulatory control ofparticular cytokines has been noted in other situations.35

It has been argued that DC could direct the cytokine pro-duction profile of T cells and be one of the determinants ofthe Th1 versus Th2 developmental options.34 There is evi-dence that the CD8+ and CD8– DC can, after transfer to mice,bias responding CD4 T cells to a Th1 or a Th2 cytokineprofile, respectively.36,37 The relative level of production ofIL-12 by DC is one mechanism by which this could beachieved. However, it is clear that the striking differences inIL-2 production that we have noted are not accompanied bya switch to IL-4 production and there is little evidence thatthese two populations of splenic DC under these conditionsbias the subsequent cytokine profile on restimulation of thecultured cells. This is in accordance with our previous evidence that the ‘regulatory’ effects could not be obtained bysoluble factors released by the DC and could not be dupli-cated or eliminated by the addition of IL-12 to the cultures.13

It should be emphasized that we have only studied here thetwo main DC populations found in spleen. It is possible thatDC from other sources, such as the additional populations wehave found in lymph nodes,38 would have stronger effects onthe cytokine profile of T cells. The two classes of DC we findin spleen regulate the level, but under these culture conditionsdo not determine the type of cytokine produced.

We do not know at present whether the regulatory effecton cytokine production represents an additional positive

influence from CD8α– splenic DC or a negative, inhibitoryinfluence from CD8α+ splenic DC. The most obvious exper-iments to test this have not given a clear answer, because theeffect is not transmitted by a soluble factor and cannot betransmitted by a third-party cell independent of antigen pre-sentation.13 This suggests that the regulatory influence isimprinted not only on the initial responding T cells, but alsoon their progeny in the primary cultures. However, this isclearly not a permanent state, because we show here that theinfluence of the initial DC contact does not persist once theT cells are recovered from the cultures and restimulated non-specifically via the CD3–TCR complex and via CD28.

The most important aspect of this control over cytokineproduction by DC is that, although activation of the T cell isa prerequisite, the regulation is largely dissociated from thefactors activating T cells into cell cycle or maintaining theirproliferation. The most striking example of this is the regula-tion of CD8 T cell IL-3 production in the presence of exo-genous IL-2; although cell proliferation is equivalent in thetwo cultures, those stimulated by CD8α– DC make highlevels of IL-3 while those stimulated by CD8α+ DC makevery little. This fits with all of our previous evidence that theregulation of IL-2 production by CD8 T cells is not the resultof costimulation via the B7/CD28 system, nor due to negativesignals via CTLA-4, nor is it the result of differences in MHClevels;13 these are all factors that should influence cell acti-vation and proliferation as well as cytokine output. By thesame argument, the effect is unlikely to be a result of differ-ent levels of alloantigen presentation or processing by the twoDC types. The fact that the same effects on CD8 T cell pro-liferation have been seen using more defined antigens andsyngeneic TCR-transgenic T cells also makes this unlikely.12

A study of new molecules expressed by the different DCtypes and further insights into the signalling pathways con-trolling cytokine product should clarify this new mechanismof DC control over cytokine output.

A key issue is the biological importance of this controlmechanism in vivo. At present this is not clear. However,because the cytokine output of T cells has a marked amplify-ing effect on the nature and intensity of an immune response,this is potentially a significant phenomenon. Our culturesystem is very much a reductionist one, involving only theinteraction of two pure cell types in culture. In vivo, we canexpect many other cells in the local environment to influenceT cell cytokine production. However, it is clear that a tightrosette-like interaction between DC and primary T cells isrequired to initiate an immune response, so our culture systemmay be a reasonable model of at least the initial events andpoints to an early step in immune regulation in vivo.

Acknowledgements

This work was supported by the Cooperative Research Centrefor Vaccine Technology, Queensland Institute of MedicalResearch, Brisbane, Queensland, Australia, and by theNational Health and Medical Research Council, Australia.Hubertus Hochrein was supported by a Deutsche KrebshilfeFellowship.

We thank David Vremec and Matthew Krummel forsetting up the ELISA assays for cytokine production andLoretta Coverdale for screening the mutant mice.

V Kronin et al.222

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