CpG-oligodeoxynucleotides co-stimulate primary T cells in the absence of antigen-presenting cells

0014-2980/99/0404-1209$17.50+.50/0© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999

CpG-oligodeoxynucleotides co-stimulate primary Tcells in the absence of antigen-presenting cells

Sylvia Bendigs1, Ulrich Salzer1, Grayson B. Lipford1, Hermann Wagner1 andKlaus Heeg2

1 Institute of Medical Microbiology, Immunology and Hygiene, Technische Universität München,München, Germany

2 Institute of Medical Microbiology and Hygiene, Philipps-Universität Marburg, Marburg,Germany

CpG-containing oligodeoxynucleotides (CpG-ODN) act as powerful adjuvant during in vivoinduction of T cell responses. While CpG-ODN directly activate antigen-presenting cells(APC) and thus exert an extrinsic activity on T cells, it is unclear whether they directly affectT cells (intrinsic activity). Here we analyze the effects of CpG-ODN on T cells in an APC-freecell culture. We report that CpG-ODN co-stimulate T cells provided they were triggered viatheir TCR. CpG-ODN induced IL-2 production, IL-2 receptor expression and thus prolifera-tion. Proliferation was blocked by cyclosporin A or anti-IL-2 monoclonal antibodies (mAb)but not by anti-IL-4 mAb. Moreover, CpG-co-stimulated T cells differentiated into cytolytic Tlymphocytes in vitro. Of note, IL-2-driven growth of primed T cells was not affected by CpG-ODN. Co-stimulation was also operative in T cells from CD28−/− mice and in TCR-transgenicT cells stimulated with peptide. CpG-ODN-mediated co-stimulation of T cells in vitro maythus explain part of the potent adjuvant effects of CpG-ODN in vivo.

Key words: CpG-oligodeoxynucleotide / T cell / Co-stimulation / Immunostimulatory DNA / CD28

Received 24/8/98Revised 28/12/98Accepted 30/12/98

[I 18721]

Abbreviations: TCR-tg: LCMV-TCR-transgenic CpG-ODN: CpG-oligodeoxynucleotide CsA: Cyclosporin A DC:Dendritic cell ODN: Oligodeoxynucleotide

1 Introduction

Bacterial DNA and synthetic CpG-oligodeoxy-nucleotides (CpG-ODN) derived thereof exert powerfuladjuvant activity by promoting adaptive immuneresponses. Adjuvanticity includes augmentation of spe-cific antibodies [1–4], of IFN- + [4, 5], as well as the gener-ation of cytolytic T cell responses to proteinaceous anti-gens [1, 4]. In addition, bacterial DNA and CpG-ODNmay instruct ongoing immune responses to Th1-typereactivity [1, 6–8], as shown in murine models of allergy[9] and lethal leishmaniasis [10]. During lethal leishmanialinfection, CpG-ODN even re-directed established Th2responses to protective Th1 responses [10].

The immunostimulatory property of bacterial DNA isattributed to unmethylated CpG sequences which occurin vertebrate DNA at low frequencies [11]. Synthetic

CpG-ODN share and probably mimic the stimulatoryactivity of bacterial DNA [12]. We have shown that CpG-ODN activate APC, such as immature dendritic cells(DC), in a CD40-CD40 ligand (CD40L)-independent fash-ion to express co-stimulatory molecules (i.e. B7-2,CD40) and to secrete co-stimulatory effector cytokineslike TNF and IL-12 [13, 14]. CpG-ODN are taken up bymacrophages and DC into early endosomes, triggeringwithin minutes the SAPK/JNK stress kinase pathwayand nuclear transcriptional activity of AP-1 and NF- O B[15, 16]. These events are followed by initiation of cyto-kine mRNA transcription and up-regulation of co-stimulatory molecules [14, 17]. The impressive adjuvanteffects of bacterial DNA and CpG-ODN may thus beattributed to their capacity to activate APC (extrinsiceffect) [14, 15, 17]. CpG-ODN, however, could alsodirectly influence T cell responsiveness (intrinsic effect).

To discriminate between both possibilities, we used areductionistic in vitro approach and analyzed CpG-ODNeffects on T cells in an APC-free system. Here we showthat CpG-ODN provide co-stimulation (signal 2) to Tcells subjected to TCR ligation (signal 1). Phenotypically,

Eur. J. Immunol. 1999. 29: 1209–1218 Oligonucleotides co-stimulate T cells 1209

Figure 1. CpG-ODN provide co-stimulation to T cells. Puri-fied T cells were stimulated at 20 000 cells/well on anti-CD3-coated microtiter plates. Cultures were supplemented with10 ? g/ml LPS, 1 ? g/ml ConA, 10 U/ml recombinant humanIL-2, ODN 1668 or ODN 1628 (black bars) as indicated (Ø,cultures stimulated with anti-CD3 only). After 4 days the pro-liferative response was recorded. The values represent thecpm ± SD of four replicate cultures.

CpG-mediated co-stimulation substituted APC-dependent T cell co-stimulation via CD28, in that itinduced IL-2 secretion, IL-2R up-regulation and thus Tcell proliferation. In addition, CPG-ODN-co-stimulated Tcells developed cytolytic effector function. These datashow that CpG-ODN directly co-stimulate T cells (intrin-sic effect) independent of their ability to stimulate APC(extrinsic effect).

2 Results

2.1 Synthetic CpG-ODN co-stimulate T cells

To analyze whether CpG-ODN intrinsically affect T cells,a stimulation protocol was set up devoid of APC. T cellswere highly enriched by magnetic cell sorting and stimu-lated in microtiter plates with immobilized anti-CD3 mAb(signal 1). Without addition of supplements signal 1failed to induce T cell proliferation (Fig. 1). Lack of prolif-eration in the presence of LPS or ConA indicated that theculture system was devoid of APC. The fact that exoge-nously added IL-2 was capable of inducing sustainedproliferation indicated that signal 1 was operative(Fig. 1). Addition of CpG-ODN (ODN 1668 and ODN1628) to the anti-CD3-stimulated T cells induced dose-dependent proliferative responses that were about 50 %of the IL-2-driven maximal responses, confirming ourprevious report [1]. Since the culture system was devoidof APC, we concluded that CpG-ODN directly co-stimulate T cells (intrinsic effect).

Next we tested different CpG-containing ODN for theirability to co-stimulate T cells (Table 1). Besides ODN1628 and ODN 1668, the ODN IL-12p40, ODN IL-13 andODN SP1 were effective. ODN IL-12p40 has been shownto induce preferentially IL-12 while lacking TNF-inducingactivity [13]. ODN YB-1 (DNA consensus binding site forthe cellular YB-1 protein [18]) and ODN 1720 [19] whichcontains an inverted CpG-dinucleotide were negative(Table 1). The latter results imply that CpG-ODN exert Tcell co-stimulation in a sequence-specific fashion andthat phosphothioate modification does not account forthe effects.

The co-stimulatory effect of CpG-ODN was analyzed indetail. Resting peripheral T cells were negativelyenriched for CD4+ and CD8+ T cell subsets and thereafterstimulated with immobilized anti-CD3 mAb (Fig. 2 B, F),anti-CD28 mAb (Fig. 2 C, G), a combination of both mAb(Fig. 2 D, H) or without mAb (Fig. 2 A, E). Part of thesecultures were supplemented with CpG-ODN 1668 andafter 4 days proliferative responses were recorded. Over-all the results allowed several conclusions. First, in theabsence of TCR stimuli, neither CD4+ nor CD8+ T cellswere activated by CpG-ODN (Fig. 2 A, E, C, G). Thus,unlike macrophages and DC which are a priori sensitiveto CpG-ODN, T cells require TCR-mediated stimuli (sig-

Table 1. Sequence dependency of CpG-ODN-mediated Tcell co-stimulationa)

Stimulationanti-CD3 plus

ODN sequence 5'-3' T cellco-stimulationcpm ±SD

None (negative control) 499 356

IL-2 (positive control) 77 802 1 420

ODN 1668 TCC ATG ACGTTC CTG ATG CT

30 583 7 750

ODN 1628 GGG GTC AACGTT GAG GGG GG

70 481 2 164

ODN IL-12p40 AGC TAT GACGTT CCA AGG

45 275 5 384

ODN IL-13 GGA ATG ACGTTC CCT GTG

40 632 7 859

ODN SP1 TCG ATC GGGGCG GGG CGA GC

64 565 4 002

ODN YB-1 AGC CAT CCCTTC CCT TTT

856 497

ODN 1720 TCC ATG AGCTTC CTG ATC CT

257 133

a) Purified T cells (10 000) were stimulated withimmobilized anti-CD3 mAb and ODN at 3 ? M in fourreplicate cultures as indicated. After 4 days the cultureswere pulsed with [3H] thymidine and the proliferativeresponse was recorded.

1210 S. Bendigs et al. Eur. J. Immunol. 1999. 29: 1209–1218

Figure 2. CpG-ODN co-stimulate anti-CD3-triggered Tcells.CD4+ and CD8+ T cells were negatively selected and platedat 20 000 cells per well in non-coated (A, E), anti-CD3-coated (B, F), anti-CD28-coated (C, G) or anti-CD3-plusanti-CD28-coated (D, H) microtiter plates. As indicated,10 U/ml IL-2 or ODN 1668 (black bars) were added (Ø, cul-tures with no supplementation). After 4 days the proliferativeresponse from four replicate cultures each was recorded.

Figure 3. Kinetics of CpG-ODN-mediated co-stimulation.Purified T cells and T cell subsets were negatively selectedand plated on anti-CD3-coated microtiter plates at 7 500cells/well. IL-2 (10 U/ml; Æ ), LPS (10 ? g/ml; | ) or ODN 1628(1 ? M; , ) were added. One group was stimulated with anti-CD3 and anti-CD28 antibodies ( ? ). After the time pointsindicated the proliferative responses of four replicate cul-tures each were recorded.

nal 1) in order to develop sensitivity to CpG-ODN(Fig. 2 B, F). Second, anti-CD28-mediated stimuli alonefailed to induce sensitivity to CpG-ODN (Fig. 2C, G).Third, when T cells were co-stimulated via cross-linkingof CD28, addition of CpG-ODN did not enhance the pro-liferative responses induced (Fig. 2 D, H). Fourth, CpG-ODN enhanced more effectively proliferative responsesof CD8+ T cells (Fig. 2 B, F). We also determined thekinetics of the proliferative responses co-stimulated byCpG-ODN. The results (Fig. 3) revealed that the kineticsof proliferation of CpG-ODN-co-stimulated T cells weresimilar to those of anti-CD28-co-stimulated T cells, yetthe magnitude of proliferation was lower (Fig. 3). Of note,peak values for CD4+ T cells were reached late (day 5 to6) whereas CD8+ T cells peaked already at day 3.

2.2 CpG-ODN induce IL-2 production, IL-2-drivengrowth and cytolytic activity in anti-CD3-stimulated T cells

Activation of T cells is a tightly regulated process thatconsecutively involves IL-2R expression and IL-2 pro-duction, thus triggering IL-2-driven growth. We therefore

tested whether CpG-ODN influence these restrictionpoints, i.e. whether they confer IL-2-driven growth toanti-CD3-stimulated T cells. We first determined expres-sion of IL-2R § -chains on T cells after anti-CD3 stimula-tion in the presence of CpG-ODN (Fig. 4). The resultsdetailed in Fig. 4 A and B indicate that exogenouslyadded IL-2 but not LPS caused up-regulation of IL-2R § .However, if CpG-ODN were added, a dose-dependentup-regulation of IL-2R § was observed on CD4+ T cellsand to a greater extent on CD8+ T cells (Fig. 4 C, D). Wealso analyzed whether CD3-stimulated T cells produceIL-2 when co-stimulated by CpG-ODN. IL-2 productionwas induced in a dose-dependent fashion by ODN 1628


Figure 4. IL-2R expression after CpG-ODN co-stimulation.Purified T cells were negatively selected and cultured at20 000 cells/well on anti-CD3-coated microtiter plates withthe reagents indicated (10 U/ml IL-2, 10 ? g/ml LPS) (A, B).ODN 1628 was added at 2 ? M and 0.4 ? M (C, D). After 24 h12 individual culutures were pooled and subsequentlystained. Cells were gated on forward and side scatter andthe IL-2R § expression on 10 000 gated CD4+ or CD8+ T cellswas recorded. The shaded areas represent the IL-2Rexpression of CD4+ T cells (A, C) or CD8+ T cells (B, D) stimu-lated with anti-CD3 mAb only.

Figure 5. CpG-ODN induce IL-2 production and IL-2-drivengrowth. (A) Purified T cells were stimulated at 20 000 cells/well with ODN 1668 and ODN 1628 as indicated (Ø, anti-CD3 stimulation only). After 4 days the supernatant of fourreplicate cultures was collected and the IL-2 content wasdetermined by ELISA. (B) Purified T cells (15 000/well) wereincubated in four replicate cultures on anti-CD3-coatedmicrotiter plates with 10 U/ml IL-2 or ODN 1628 at 3 ? M and1 ? M. Then anti-IL-2 mAb (S4B6) at 5 ? g/ml or anti-IL-4mAb (11B11) at 3 ? g/ml were added as indicated. After 3days the proliferative response was recorded.

and ODN 1668 (Fig. 5 A). As expected, the growth of theCpG-co-stimulated T cell cultures was inhibited in thepresence of anti-IL-2 mAb but not by anti-IL-4 mAb(Fig. 5 B). To test whether CpG-co-stimulated T cellswould gain cytolytic effector function, we harvested cul-tures after 4 days and tested for lytic activity againstanti-CD3-expressing hybridoma and NK-sensitive YAC-1 target cells (Fig. 6). Cultures that were supplementedwith IL-2 (Fig. 6 C) or ODN 1628 (Fig. 6 A) developedstrong cytolytic activity; the cytotoxicity induced by ODN1668 was lower (Fig. 6 B). Of note, the cytotoxicity wasTCR dependent since YAC-1 cells were not lysed, impli-cating that no NK-like activity was induced by CpG-ODN.

Cyclosporin A (CsA) blocks the Ca2+-dependent phos-phatase calcineurin, a critical step in TCR-initiated sig-naling [20, 21]. To analyze whether perturbation of TCR-mediated signaling (signal 1) would affect developmentof sensitivity to CpG-ODN, we analyzed the influence ofCsA on the growth of CpG-ODN-co-stimulated T cells.

The results revealed that CsA blocked proliferation of Tcells at very low concentrations (25 ng/ml) (Table 2). Inaddition, CpG-ODN did not affect IL-2-driven T cellgrowth of secondary T cells (data not shown).

Collectively the results suggest that CpG-ODN co-stimulate TCR-cross-linked T cells to express IL-2R andto produce IL-2 causing IL-2-driven growth and todevelop cytolytic effector funtion. In these respects theresponse of T cells to CpG-ODN appears phenotypicallysimilar to CD28-mediated co-stimulation. If so, CpG-ODN ought to substitute for CD28-mediated signaling inCD28-deficient mice. To address this issue, T cells fromCD28+/+ or CD28−/− mice were stimulated with anti-CD3plus CpG-ODN and thereafter the proliferative responseswere recorded (Table 3). While T cells from CD28−/− micewere unresponsive to co-stimulation with anti-CD28, thesame cells responded to co-stimulation with CpG-ODN1628 but not to ODN ATT-Rep (Table 3). The magnitudeof the proliferative response induced with ODN 1628,however, was lower compared to IL-2-driven responses


Figure 6. CpG-ODN induce cytolytic activity. Purified T cellswere stimulated at 20 000 cells/well on anti-CD3-coatedmicrotiter plates with ODN 1628 (A), ODN 1668 (B), 10 U/mlIL-2 (C) or without supplementation (D). After 4 days cellsfrom replicate cultures were harvested and serially diluted(three replicas) in microtiter plates. To each well 1 000 51-Cr-labeled target cells ( Æ , anti-CD3 hybridoma 3C11; | , YAC-1) were added. After 4 h the supernatant was removed and+ -irradiation was measured.

or to that of T cells derived from wild-type CD28+/+ mice.ODN ATT-Rep (included as negative control) failed to co-stimulate CD28+/+ and CD28−/− T cells (Table 3).

To further analyze whether ODN-mediated co-stimulation also holds true for T cells stimulated via MHCand peptide, we used T cells from LCMV-TCR-transgenic (TCR-tg) mice [22]. To exclude indirect effectsof ODN on T cells via stimulation of APC, T cell-depeleted splenocytes were fixed with 0.01 % glutaral-dehyde. Fixation completely abolished the ability of APCto produce IL-12 upon stimulation with either ODN 1668

Table 2. CsA inhibits CpG-ODN-induced T cell co-stimulationa)

Responder Stimulation No CsA cpm (± SD) 25 ng/ml CsA cpm (± SD) 100 ng/ml CsA cpm (± SD)

T cells anti-CD3anti-CD3 + IL-2

anti-CD3 + ODN 1628

600 ± 8479 018 ± 7 75659 202 ± 1 240

ND11 072 ± 797

47 ± 17

ND1 170 ± 505

16 ± 8

CD4+ anti-CD3anti-CD3 + IL-2

anti-CD3 + ODN 1628

433 ± 8611 101 ± 2 053

5 140 ± 780

ND371 ± 140

29 ± 11

ND68 ± 1421 ± 16

CD8+ anti-CD3anti-CD3 + IL-2

anti-CD3 + ODN 1628

474 ± 30383 691 ± 2 86650 293 ± 1 382

ND24 892 ± 2 929

558 ± 902

ND4 058 ± 1 031

49 ± 37

a) T cell subsets were negatively selected and stimulated as indicated at 6 500/well. ODN 1628 was added at a concentrationof 1 ? M. The proliferative response of four replicate cultures was recorded at day 4 (T cells, CD8+ T cells) or day 5 (CD4+ Tcells).

or ODN 1628 (Fig. 7 A). When fixed APC were loadedwith peptide and used as stimulator cells for TCR-tg Tcells only a marginal stimulation was recorded (Fig. 7 B).Addition of IL-2 caused a proliferative response in a pep-tide dose-dependent manner, indicating that antigenpresentation was operative on fixed APC. ODN 1628 andto a lesser extent ODN 1668 induced proliferation(Fig. 7 B). Thus ODN also co-stimulate TCR-tg T cellswhen activated via MHC plus peptide.

3 Discussion

The adjuvant activity of bacterial DNA and CpG-ODNincludes proliferative T cell responses and CTLresponses to proteinaceous antigens [1, 4], IFN- + syn-thesis [4, 5] and production of specific antibodies with abias for Th1-oriented Ig isotypes [1, 4, 6–8, 10]. Howbacterial DNA and CpG-ODN support clonal expansionof antigen-specific T cells is unclear, but either potentia-tion of APC function and/or direct action on T cells rep-resent two mutually non-exclusive possibilities. Supportfor potentiation of APC function (termed extrinsic effect)is provided by our recent demonstration that bacterialDNA and CpG-ODN cause in vitro maturation of imma-ture DC both in terms of up-regulation of MHC class II,B7.2, CD40 molecules as well as by induction of cyto-kine synthesis such as IL-12, IL-6 and TNF [13, 14].These CpG-ODN-driven events induce acquisition ofprofessional APC functions [14].

Evidence for a direct effect of CpG-ODN on T cells sub-jected to TCR ligation is described here. While immuno-stimulatory CpG-ODN fail to activate resting T cellsdirectly, they effectively co-stimulate T cells subjected toanti-CD3-mediated TCR occupancy (signal 1). T cell co-stimulation by ODN was also operative in T cells from


Figure 7. CpG-ODN co-stimulate TCR-tg T cells triggeredvia MHC and peptide. (A) Splenocytes were either treatedwith 0.01 % glutaraldehyde (black bars) or medium (graybars), irradiated and then seeded at 105/well. Cells werethen stimulated with ODN 1668 or ODN 1628 as indicated.Supernatant was removed 22 h later and tested for IL-12p40content by ELISA. (B) Purified T cells (15 × 103) from TCR-tgmice were cultured together with 105 fixed stimulator cellsand different amounts of the LCMV peptide. IL-2 was addedat 10 U/ml, ODN were added at 1 ? M. After 3 days the prolif-erative response was recorded.

Table 3. CpG-ODN co-stimulate T cells from CD28−/− micea)

Responder cells

Stimulation CD28+/+

T cellsCD28+/+

CD4+CD28+/+

CD8+CD28−/−

T cellsCD28−/−

CD4+CD28−/−

CD8+

anti-CD3 70 59 33 340 408 248

anti-CD3 + LPS 47 49 26 802 312 307

anti-CD3 + IL-2 26 259 24 893 42 747 37 871 61 380 53 087

anti-CD3 + 1628(2 ? M)

24 237 5 365 26 069 7 575 4 212 10 677

anti-CD3 + ATTRep(2 ? M)

60 60 39 863 590 452

anti-CD3 + anti-CD28 62 478 38 687 53 127 316 199 145

a) T cells subsets from CD28−/− or wild-type mice were negatively selected and stimulated with the reagents as indicated at6 000/well. After 4 days (T cells, CD8+ T cells) or 5 days (CD4+ T cells) the proliferative response (cpm) of four replicatecultures was recorded.

TCR-transgenic mice when signal 1 was provided bypeptide and MHC. Inhibition of signal 1 (TCR ligation) byCsA abolished this co-stimulatory activity. Phenotypi-cally similar to CD28-mediated co-stimulation, CpG-ODN triggered up-regulation of IL-2R, secretion of IL-2and thus IL-2-driven growth [23, 24]. Moreover, CpG-ODN-mediated co-stimulation compensates, at least inpart, for the signaling defect characterizing CD28−/−

mouse-derived T cells. Many studies have shown thatCD28 is the principle co-stimulatory receptor for T cellsinteracting with B7 molecules on APC [25, 26]. However,other molecules like CD2, CD5, CD9, CD11a and CD44also have co-stimulatory activity [26]. We show here thatCpG-ODN directly co-stimulate purified CD4+ and CD8+

T lymphocytes in the absence of APC. In contrast toLPS-dependent co-stimulation of T cells [27] APC-derived cytokines are not required. Remarkably, ODN-co-stimulated T cells developed cytolytic effector func-tion in vitro. Cytotoxicity was TCR dependent since anti-CD3-expressing hybridoma cells but not NK-sensitiveYAC-1 target cells were lysed. Hence CpG-ODN-mediated co-stimulation of T cells not only induces acti-vation and clonal expansion of T cells but also differenti-ation into cytolytic effector cells.

The co-stimulatory effects of CpG-ODN on T cells areDNA sequence dependent and independent on phos-phothioate modification of the ODN since several ODN(like ODN ATT-Rep, ODN 1720 or ODN YB-1 as shown inTables 1 and 3) failed to co-stimulate. An importantaspect of our experiments refers to the observation thatCpG-ODN which lack TNF-inducing activity on macro-phages, like ODN IL-12p40 [13], effectively co-stimulateT cells (Table 1). This raises the possibility of devisingadjuvant-ODN sequences for T cell vaccination lacking


potentially harmful side effects such as TNF-mediatedsystemic toxicity [13].

The “two signal” model reflects a simplistic view of pri-mary T cell activation. Lack of co-stimulation is thoughtnot only to prevent productive T cell activation but toinduce T cell anergy or even T cell deletion [28]. The abil-ity of CpG-ODN to directly co-stimulate T cells may be ofrelevance when professional APC are not available. Forexample in the case of tumor cells expressing tumorantigens but lacking co-stimulatory molecules, an APC-independent co-stimulation via CpG-ODN may activateT cell responses (useful effects). On the other hand, theuse of ODN as T cell adjuvant might facilitate an APC-independent activation of autoreactive T cells (harmfuleffects). Clearly these issues need now to be addressedexperimentally.

The mode of action of CpG-ODN on T cells may be dis-tinct from that on other cell types. In contrast to macro-phages or DC, known to be responsive to CpG-ODN [14,15], T cells become sensitive to CpG-ODN only after TCRligation (signal 1). This implies that CpG-ODN sensitivityof T cells for co-stimulation (signal 2) is inducible. Interest-ingly, ongoing experiments reveal that uptake of ODN by Tcells is enhanced after stimulation with anti-CD3 mAb(data not shown). Thus signal 1 may restrict the activity ofODN on T cells. The co-stimulatory activity of CpG-ODNas defined here for T cells may be operative also in othercells of the lymphocytic lineage. Although it was initiallyclaimed that NK cells can be directly stimulated by bacte-rial DNA and CpG-ODN to produce IFN- + [29], recentreports indicate that IFN- + release of NK cells was depen-dent on macrophage-derived IL-12 [30]. These data implythat NK cells respond to bacterial DNA or CpG-ODN onlyafter supplying receptor-mediated signals (IL-12). Thesame might be true for B cells. Although B cells respondto CpG-ODN in whole spleen cell cultures, the prolifera-tive response and IgM secretion is markedly enhancedafter B cell receptor cross-linking [31] and is potentiatedby IFN- + [5]. These results indicate that full sensitivity of Bcells to CpG-ODN-mediated activation might first requireBCR ligation (signal 1). This information led us to a con-ceptional framework that APC such as macrophages andDC are a priori sensitive to CpG-ODN. On the other hand,cells of the lymphocytic lineage such as T cells, B cellsand NK cells respond to CpG-ODN in a co-stimulatoryfashion, i.e. sensitivity to CpG-ODN is inducible uponreceptor occupancy (signal 1).

Ongoing work addressing the signal pathways activatedin APC by CpG-ODN indicates that biologically inactiveODN compete binding of active CpG-ODN on yet ill-defined cell surface structures [16]. Upon cellular uptakethe biology of CpG-ODN appears dependent on acidifi-

cation of endosomes because lysosomotropic drugs(e.g. chloroquine) which prevent endosomal maturationblock CpG-ODN-triggered signals [16, 32]. These eventsinduce within minutes SAPK/JNK activation [16] associ-ated with transcriptional activation of AP-1 and NF O B[15, 16]. Preliminary data have shown that the co-stimulatory activity of ODN is not subject to blockade bychloroquine (data not shown). Together with the depen-dency of co-stimulation on signal 1 these data suggestthat signal pathways of ODN differ between T cells andmacrophages. It will therefore be rewarding to define indetail the rules of signaling of ODN on T cells.

Overall our results suggest that CpG-ODN cause aCD28-like co-stimulation of T cells independent of APC.This intrinsic effect of ODN on T cells may explain, atleast in part, the powerful adjuvanticity of bacterial DNAand CpG-ODN on antigen-specific T cell responses invivo [1, 4]. Furthermore, the efficacy of DNA vaccinespossessing immunostimulatory sequences could be duein part to direct co-stimulation of T cells [33–35].

4 Materials and methods

4.1 Animals

Female 8- to 10-week-old C57BL/6 (H2b) mice were pur-chased from Harlan-Winkelmann (Borchen, Germany).CD28−/− mice [36], a kind gift from Dr. K. Pfeffer, were bred inour animal facility. TCR-tg mice (strain 318) [22] were a gen-erous gift from Dr. H. Pircher, Freiburg.

4.2 Media, mAb and reagents

Cells were cultured in Click’s/RPMI medium supplementedwith 10 % FCS, 1 ? g/ml indomethacine and antibiotics(Biochrom, Berlin, Germany). Phosphothioate-modifiedCpG-ODN were custom synthesized by MWG, Munich, Ger-many. The sequences used were: 1668, 5'-TCC ATG ACGTTC CTG ATG CT-3' [19]; 1628, 5'-GGG GTC AAC GTTGAG GGG GG-3' [29]; IL-12p40, 5'-AGC TAT GAC GTT CCAAGG-3' [13]; IL-13, 5'-GGA ATG ACG TTC CCT GTG-3' [13];SP1, 5'-TCG ATC GGG GCG GGG CGA GC-3' (definedhere); 1720, 5'-TCC ATG AGC TTC CTG ATC CT-3' [19]; YB-1, 5'-AGC CAT CCC TTC CCT TTT-3' [18]; ATT-Rep, 5'-ATTATT ATT ATT ATT ATT AT-3' (control). The bold letters indi-cate the proposed active CpG motifs [19]. The sequence ofODN SP1 is derived from the human SP1 consensus bindingsite. ODN IL-12p40 and ODN IL-13 represent CpG motifsderived from non-coding regions of the human IL-12p40 orIL-13 gene, respectively [13]. mAb (ELISA, anti-CD8, anti-CD4, anti-CD28) and cytokine standards for ELISA werepurchased from Pharmingen, Hamburg, Germany. TheELISA was performed as recommended by the manufac-turer. Anti-CD3 mAb (hybridoma 3C11) and anti-IL-2 mAb


(hybridoma S4B6) were produced using a miniperm in vitrosystem (Heraeus, Hanau, Germany). The concentration ofmAb in supernatants were determined by ELISA. Purifiedanti-IL-4 mAb (11B11) were a generous gift from Dr.M. Röcken, München. Polyclonal anti-hamster Ig antibodieswere purchased from Dianova, Hamburg, Germany. LPSwas bought from Sigma, Deisenhofen, Germany. Recombi-nant human IL-2 was a kind gift from Eurocetus, Amster-dam, The Netherlands. ConA came from Pharmacia,Uppsala, Sweden. CsA was a generous gift from Dr. Borel,Basel, Switzerland. Ficoll (1,077 kg/l) was purchased fromBiochrom, Berlin, Germany. The LCMV glycoprotein 33–41-derived peptide (KAVYNFATM) was custom synthesized byNeosystem Laboratoire (Strasbourg, France).

4.3 In vitro cell culture

T lymphocyte subpopulations were prepared by negativeselection. Single-cell suspensions from mesenteric lymphnode cells were first incubated with DYNABEADS coatedwith anti-mouse IgG for 30 min (Dynal, Hamburg, Germany).The rosetted cell population was removed, the cells remain-ing in solution were collected and used as purified T cell pop-ulation. To negatively select CD4+ or CD8+ T cell populations,107 T cells were incubated with 15 ? g anti-CD8 (53672) orwith anti-CD4 antibodies (GK1.5) for 30 min and thenwashed twice. Thereafter cells were rosetted with anti-rat Ig-coupled DYNABEADS. The cell populations were checkedfor purity by cytometry; purity always exceeded 95 %.Round-bottom microtiter plates were incubated with 10 ? g/ml rabbit anti-hamster IgG antibodies overnight. Thereafterthe plates were washed and 1 ? g/ml anti-CD3 mAb and/or1 ? g/ml anti-CD28 mAb (Pharmingen) were incubated over-night. The plates were washed twice and blocked withmedium containing 10 % FCS and used for the T cell stimula-tion assays. To this, four to six replicate cultures of 5 000 to10 000 separated T cells were incubated in 200 ? l mediumcontaining 10 % FCS. ODN were added at initiation of thecell cultures. For stimulation of TCR-tg T cells, splenocytesfrom B6 mice were depleted of T cells by DYNABEADS andthen fixed for 15 min at 37 °C with 0.01 % glutaraldehyde.Stimulator cells were irradiated (12Gy) and then pulsed withLCMV peptide (30 min at 4 °C). In the cultures 105 stimulatorcells and 15 × 103 responder cells were used.

4.4 Proliferation assays

Microcultures were pulsed with [3H] thymidine (14.8 kBq/well) for 6 h. The samples were then collected onto glass fil-ters with a 96-well cell harvester and counted with aMATRIX-96 counter (Packard, Meriden, CT).

4.5 51Cr-release assay

Target cells (anti-CD3-producing hybridoma 3C11 or YAC-1;American Type Culture Collection, Rockville, MD) were

labeled with Na2CrO4 for 90 min at 37 °C and then washedtwice. To replicate serial dilutions of CTL in 100 ? l, the samevolume of target cells (104/ml) was added. CTL and targetcells were incubated for 4 h at 37 °C. Supernatants wereremoved and + -irradiation was measured. Results wereexpressed as percent specific lysis.

4.6 Cytometric analyses

Single-cell suspensions were incubated on ice with the mAbindicated for 30 min (PE-coupled anti-CD4 mAb, biotin-conjugated anti-CD8 mAb and FITC-labeled anti-IL-2R §mAb). Biotin-labeled mAb were visualized with PerCP-coated streptavidin (Becton Dickinson, Heidelberg, Ger-many). Thereafter the cells were washed and fixed with 1 %paraformaldehyde in PBS. Analyses were performed on anEPICS XL cytometer (Coulter, Hialeah, FL). Cells were gatedon forward and side scatter.

Acknowledgments: We thank Mrs. M. Mayer for her excel-lent technical assistance.This work was supported by grants from the BMBF, the Son-derforschungsbereich 391, the Graduiertenkolleg 333 andthe Wilhelm-Sander Stiftung.

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Correspondence: Klaus Heeg, Institute of Medical Microbi-ology and Hygiene, Philipps-Universität Marburg, Pilgrim-stein 2, D-35037 Marburg, GermanyFax: +49-6421-28-6420e-mail: heeg — mailer.uni-marburg.de


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CpG-oligodeoxynucleotides co-stimulate primary T cells in the absence of antigen-presenting cells