Qualitative and Quantitative Effects of CD28/B7-Mediated Costimulation on Naive T Cells In Vitro1

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Cells In VitroCD28/B7-Mediated Costimulation on Naive T Qualitative and Quantitative Effects of

Christoph Burkhart and David C. WraithShivanthi P. Manickasingham, Stephen M. Anderton,

http://www.jimmunol.org/content/161/8/38271998; 161:3827-3835; ;J Immunol 

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, 25 of which you can access for free at: cites 43 articlesThis article

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1998 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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Qualitative and Quantitative Effects of CD28/B7-MediatedCostimulation on Naive T Cells In Vitro1

Shivanthi P. Manickasingham, Stephen M. Anderton, Christoph Burkhart, andDavid C. Wraith 2

The CD28/B7 system provides costimulatory signals necessary for optimal T cell activation. We have examined the effects ofblocking B7.1 and/or B7.2 in an in vitro system using TCR transgenic T cells specific for myelin basic protein. Activation of naiveT cells was found to be B7.2 dependent and not dependent on the presence of B7.1 molecules. However, increasing the strengthof signal through the TCR using peptide analogues with higher affinity for MHC compensated for blockade of B7.2 molecules,suggesting that signal 1 alone can be sufficient for the activation of naive T cells. The role of B7 molecules in the differentiationof T cells was further investigated by restimulating T cells with fresh APC and peptide in B7-sufficient conditions. A down-regulation of IL-2 and IFN- g production by T cells primed in the presence of anti-B7.2 mAb was partially overcome when highaffinity peptide analogues were used to restimulate T cells. In contrast, a significant down-regulation of the differentiation of cellsproducing Th-2 cytokines was observed in the presence of anti-B7 Abs. Differentiation of IL-4-secreting cells was influenced byboth B7.1 and B7.2, while IL-5 secretion was totally dependent on B7.2. These results suggest that B7-mediated costimulation isessential for the development of Th-2-associated cytokines, the absence of which cannot be overcome by increasing the strengthof the signal through the TCR. The Journal of Immunology,1998, 161: 3827–3835.

Full activation of naive CD41 T cells is believed to requirethe engagement of the TCR by specific Ag in the contextof MHC class II molecules in addition to an Ag-nonspe-

cific interaction referred to as costimulation. The best character-ized costimulatory pathway involves the CD28-B7 family of co-stimulatory molecules. CD28 is expressed on the majority of naiveand memory T cells (1), and signaling through this molecule isthought to permit the activation of Ag-specific T cells that wouldotherwise enter a state of anergy or nonresponsiveness (2). The B7family of costimulatory molecules, B7.1 and B7.2, belong to the Igsuperfamily and were initially thought to be only expressed on“professional” APC. Later studies have shown that B7 moleculesare also expressed on murine activated T cells creating a highlycomplex system (3). B7.1 and B7.2 also bind to CTLA-4, a ho-mologue of CD28 (4), although recent studies indicate thatCTLA-4 delivers a negative signal to activated T cells (5).

Blockade of the CD28/B7 pathway using CTLA-4Ig, a solublefusion protein comprising the extracellular domain of CTLA-4 andthe Fc portion of the IgG1 molecule, has been shown to block anumber of in vitro proliferative responses to allogeneic Ags andself-MHC restricted Ags. CTLA-4Ig treatment has been shown toprevent xenograft rejection and prolong allograft survival (6, 7) inaddition to having significant effects on the experimental course of

several autoimmune diseases (8, 9). However, various studies us-ing CD28-deficient mice have shown that although Ab responseswere depressed, T cell proliferation could be induced after in vitropriming (10). In addition, recent studies using the nonobese dia-betic (NOD)3 mouse as a spontaneous model of diabetes haveshown that T cells from CD282/2 mice were capable of prolifer-ating and producing IL-2 in response to the autoantigen GAD65

(9). These results indicate that cellular immunity was, to someextent, functional in CD28-deficient mice.

Recently, there has been considerable interest in whether B7.1and B7.2 play distinct roles in the differentiation of Th subsets(11–13). CD41 Th cells, upon antigenic stimulation, differentiateinto subpopulations producing distinct spectra of cytokines andhaving separate effector functions (14). Th1 cells are characterizedby IL-2, TNF-b, and IFN-g production, thereby inducing a de-layed-type hypersensitivity response in addition to IgG2a produc-tion (15, 16). Th2 responses, on the other hand, are characterizedby IL-4, IL-5, and IL-10 production and provide effective help forhumoral immunity (especially the production of IgE and IgG1 iso-types) and also possibly suppression of Th1-type responses (15,16). Various recent reports suggest that B7.1 and B7.2 play distinctand differential roles in the induction of Th1 and Th2 responses.The differential expression of B7.1 and B7.2 in addition to the factthat these molecules bind to distinct determinants on CD28 suggestthat functional differences may also exist. In fact, murine B7.1 andB7.2 only share 25% amino acid homology and have marked dif-ferences in their cytoplasmic domains (17). In addition, murineB7.2 is expressed constitutively on professional APC and is rap-idly induced in response to various stimuli (18, 19). B7.1, on theother hand, is expressed much later after activation and also atlower levels than B7.2 (19). However, studies into the role(s) ofCD28/B7 molecules in the differentiation of Th cells have yielded

Department of Pathology and Microbiology, University of Bristol School of MedicalSciences, Bristol, United Kingdom

Received for publication December 19, 1997. Accepted for publication June 2, 1998.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the Wellcome Trust and a fellowship fromthe Deutsche Forschungsgemeinschaft (to C.B.).2 Address correspondence and reprint requests to Prof. David C. Wraith, Departmentof Pathology and Microbiology, University of Bristol School of Medical Sciences,University Walk, Bristol, United Kingdom BS8 1TD.

3 Abbreviations used in this paper: NOD, nonobese diabetic; EAE, experimental au-toimmune encephalomyelitis; PE, phycoerythrin.

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00

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contradictory results. Studies by Kuchroo et al. have shown thatblockade of B7.1 ameliorated experimental autoimmune enceph-alomyelitis (EAE), a Th1-mediated disease, whereas blockade ofB7.2 exacerbated this disease (20). In contrast, anti-B7.2 treatmentwas seen to depress the development of diabetes, also a Th1-me-diated disease, in the NOD mouse, whereas anti-B7.1 exacerbateddisease (21). These findings suggest that the relationship betweenB7.1/B7.2 and cytokine development is not as straightforward aspreviously thought.

In this study we have examined the effects of blocking B7.1and/or B7.2 on priming and subsequent Th cell differentiation in anin vitro system using TCR transgenic T cells specific for the my-elin basic protein-derived peptide Ac1-9. We have tried to ascer-tain whether costimulation is only required under suboptimal lev-els of T cell activation by using altered peptide ligands of the Agthat bind with increasing affinity to class II MHC. The relativeaffinity of these analogues, which consisted of alanine (4A) ortyrosine (4Y) at position 4 instead of wild-type lysine, was ana-lyzed by Fugger et al. (22). Their results showed that whereasAc1-9 (4Y) had a relative binding affinity 50- to 100-fold greaterthan that of Ac1-9 (4A), the binding affinity of Ac1-9 was so weakthat it was immeasurable. These APL have allowed us to examinewhether the requirement for costimulation could be bypassed byincreasing the strength of signaling to the T cell via the TCR.These studies reveal a differential requirement for costimulationfor Th1 vs Th2 responses. There is a requirement for costimulationvia B7.2 for Th1 responses that can be overcome by increasing thestrength of the signal delivered via the TCR. Th2 responses are, onthe other hand, more strictly dependent on costimulation via bothB7.1 and B7.2.

Materials and MethodsMice

Mice were bred and maintained at the Department of Pathology and Mi-crobiology (Bristol, U.K.). Generation of the Tg4 TCR transgenic mouse,which expresses a TCR specific for the immunodominant Ac1-9 epitope ofMBP, has been described previously (23). Transgenic T cells express theTCR-ab (Va4, Vb8.2) of the Ac1-9-specific T cell hybridoma 1934.4derived from the encephalitogenic T cell clone PJR-25 (24). These micewere used at 8 to 14 wk of age. Expression of the TCR was evaluated bytwo-color flow cytometry of peripheral blood using anti-CD4 (clone H129.19, Sigma) and an anti-Vb8 mAb (F23.1). B10.PL mice were used asa source of I-Au-expressing APC.

Peptide Ags

The acetylated N-terminal peptide of murine MBP (Ac1-9AcASQKRPSQR) and the high affinity analogues with alanine and tyrosinesubstituted for wild-type lysine at position 4 (4A and 4Y, respectively)were synthesized using F-moc chemistry on an AMS 422 multiple peptidesynthesizer (Abimed, Lagenfeld, Germany).

Tissue culture medium and reagents

Cultures were maintained in Iscove’s modified Dulbecco’s medium (LifeTechnologies, Paisley, U.K.) supplemented with 5% FCS (Sigma, Poole,U.K.), 2 mM L-glutamine, 100 U/ml penicillin, 100mg/ml streptomycin,and 53 1025 M 2-ME (all from Life Technologies).

Primary stimulation of TCR transgenic splenocytes

Splenocytes from TCR transgenic mice were cultured in 96-well plates(Falcon, Becton Dickinson, Milton Keynes, U.K.) at 23 105 cells/well inthe presence of various concentration of Ac1-9 or its analogues and thefollowing Abs at 20mg/ml: anti-B7.1 (clone 1G10), anti-B7.2 (clone GL-1;PharMingen, San Diego, CA), or rat IgG2a (clone IR418; Serotec, Oxford,U.K.). Cells were assayed for proliferation and cytokine production asdescribed below.

Secondary stimulation of TCR transgenic T cells

Splenocytes were cultured in duplicate wells at 23 105 cells/well in 24-well plates (Falcon) with 4mg/ml of Ac1-9 and anti-B7 Abs or rat IgG2a(isotype control) at 20mg/ml for 48 h. Cells were purified on a Nycoprep1.077 g/ml animal gradient (Nycomed, Oslo, Norway), washed, and re-stimulated in the absence of B7 Abs with irradiated B10.PL APC andpeptides Ac1-9 or Ac1-9 (4A or 4Y analogues) at a range of concentra-tions. Proliferation and cytokine production were assessed (daily over a4-day period).

T cell proliferation

Twenty-four hours after the in vitro splenocyte assay was set up, cells werepulsed with 0.5mCi of [3H]thymidine for 14 to 18 h. Thymidine incorpo-ration was measured on a liquid scintillation beta counter (1450 Microbeta,Wallac, Milton Keynes, U.K.) and expressed as mean counts per minute.

Cytokine assay

Cytokine release was measured using a cell-based ELISA described byBeech et al. (25). The following cytokine-specific capture Abs were usedto coat microtiter plates: JE56-1A12 (anti-IL-2), 11B11 (anti-IL-4),TRFK4 (anti-IL-5), or R4-6A2 (anti-IFN-g). Splenocytes, previously stim-ulated with peptide and anti-B7 mAb, were added at approximately 13105 cells/well and incubated for an additional 18 to 24 h. On the indicateddays after culture, specifically bound cytokines were quantified using thefollowing biotinylated secondary Abs: E56-SH4 (anti-IL-2), BVD6-24G2(anti-IL-4), TRFK5 (anti-IL-5), or XMG1.2 (anti-IFN-g; all from Phar-Mingen) followed by extravidin peroxidase (Sigma). The level of eachcytokine was calculated using standard curves, obtained from knownamounts of recombinant mouse cytokines (IL-4: Genzyme, Boston, MA;IL-2, IL-5, and IFN-g: PharMingen).

Flow cytometric analysis of B7 expression on T cells

Splenocytes were cultured as previously at 23 105 cells/well in 24-wellplates (Falcon) with 4mg/ml of Ac1-9 and biotinylated anti-B7 Abs or ratIgG2a (isotype control) at 20mg/ml for 48 h. Cells were purified on aNycoprep 1.077 g/ml animal gradient (Nycoprep) and washed, and subse-quent binding of any biotinylated Ab was visualized with streptavidin-PE(Sigma). Cells were double stained with anti-CD3 (clone 29B, Sigma) andanti-Ia (clone MRC OX-6, Serotec, Oxford), flow cytometric analysis wasperformed with a FACScan flow cytometer (Becton Dickinson, MountainView, CA), and data analyzed using CellQuest software (BectonDickinson).

ResultsThe series of experiments described below were all performed atthe same time and are therefore directly comparable. However, forthe sake of clarity, results obtained with wild-type Ac1-9 will bediscussed first followed by those generated with the Ac1-9 ana-logues 4A and 4Y.

Influence of costimulation on the primary response of transgenicT cells in vitro

Naive spleen cells from the Tg4 transgenic mouse were stimulatedin vitro with peptide Ac1-9 alone or in the presence of a control ratIgG2a, anti-B7.1, anti-B7.2, or a combination of both anti-B7 Abs.Results obtained with control rat IgG2a were equivalent to thosegenerated without Ab. The concentration of Ab used was titratedand shown to be saturating (data not shown). T cell activation wasmeasured by proliferation and cytokine production. There was noproduction of either IL-4 or IL-5, characteristic of the Th2 subsetof cells (data not shown), following primary stimulation in vitro inthe absence of anti-B7 mAbs. The level of proliferation appearedto correlate with IL-2 production, and both were dependent oncostimulation via the B7.2 molecule (Fig. 1,A andB). Abs directedto B7.1 did not have a significant effect in isolation, but actedsynergistically with anti-B7.2 Abs in reducing both IL-2 produc-tion and proliferation. IFN-g was produced at normal levels in thepresence of anti-B7.1 Abs (Fig. 1C). Production of this cytokine

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was, however, completely inhibited by anti-B7.2 Abs. These re-sults show clearly that the primary response of naive T cells invitro is dependent on costimulation by B7.2 molecules.

Influence of costimulation on the differentiation of Th subsets invitro

Tg4 spleen cells were cultured in the presence of both Ac1-9 andanti-B7 Abs and were subsequently restimulated with Ag and freshAPC after 2 to 3 days. It is clear from Figure 2,b andc, that theB7.1 molecule did not affect the differentiation of IL-2- or IFN-g-secreting cells at higher Ag concentrations. B7.1 was, however,shown to play a role in the differentiation of IFN-g-secreting cellsat the lowest Ag dose tested and had a similar effect in two of threeexperiments. On the other hand, B7.2 contributed to the generationof both IL-2- and IFN-g-secreting cells, and this effect was mostmarked at low doses of Ag. Despite the influence of B7.2 on IL-2production, blockade of this molecule had only a marginal effecton proliferation (Fig. 2a). There was no evidence of synergy be-tween B7.1 and B7.2 molecules, with, if anything, enhancement ofIL-2 production and proliferation at certain Ag concentrations inthe presence of anti-B7.1 Abs.

The differentiation of IL-4-secreting cells was partially reducedin the presence of either B7.1 or B7.2 Abs (Fig. 2d). In addition,there were two notable observations. First, the effect of the B7.2molecule was most marked at a low Ag concentration. Secondly,there was no evidence for a synergistic effect of the two anti-B7Abs on the differentiation of IL-4-secreting cells.

Blockade of B7.1 molecules resulted in a modest reduction inthe differentiation of IL-5-secreting cells. In marked contrast, the dif-ferentiation of these cells was completely blocked by anti-B7.2 Abs.

The combined results shown in Figure 2 clearly distinguish theinfluence of B7.1 and B7.2 on the differentiation of Th1 and Th2cells. B7.1 blockade did not affect Th1 cell differentiation and yetconsistently reduced the levels of Th2-associated cytokines on re-stimulation in the absence of anti-B7 Abs. B7.2 blockade reducedthe levels of both Th1- and Th2-associated cytokines. Most nota-bly, however, the differentiation of IL-5-producing cells as op-posed to IL-4-producing cells was completely dependent on co-stimulation by B7.2.

Antigenic peptides with increasing affinity for MHC influencethe primary response of T cells in vitro

The primary response of naive Tg4 T cells to peptide Ac1-9 wasinhibited by coculture with an anti-B7.2 mAb (Fig. 1). The depen-dence of the primary response on costimulation was further testedthrough the use of peptides with higher affinity for MHC. Theresults shown in Figure 3 emphasize that the primary response ofTg4 T cells does not depend on B7.1. It is clear, however, that theinhibition of proliferation and both IL-2 and IFN-g production byanti-B7 Abs in the primary response (Fig. 1,A–C) could be par-tially overcome by increasing the affinity of Ac1-9 analogues fortheir MHC restriction element (Fig. 3). Neither the intermediate(4A) nor the high affinity (4Y) analogue could, however, com-pletely restore the ability of cells to fully respond to Ag in vitro.Interestingly, stimulation of cells with the higher affinity peptidesfailed to elicit secretion of IL-5, even in the absence of anti-B7Abs. The highest concentration of the 4Y analogue stimulated se-cretion of IL-4, the levels of which were on the threshold of de-tection (data not shown). This low level of IL-4, however, wasinhibited by the anti-B7.2 mAb (data not shown).

These results show that the primary response of naive Tg4 cellsin vitro is predominantly of the Th1 type. Weak responses to an-tigenic stimuli were dependent on costimulation by B7.2, but wereindependent of B7.1. The dependence on costimulation via B7.2was partially overcome through the use of more highly antigenicpeptide Ags.

T cells primed in the absence of B7 molecules reveal adifferential pattern of dependence on costimulation for thesecretion of Th1- and Th2-associated cytokines whenrestimulated using peptides with increasing affinity for MHC

Both the primary response and differentiation of IL-2-secretingcells in the presence of Ac1-9 were B7.2 dependent. This depen-dence on B7.2 for differentiation of IL-2-secreting cells was, how-ever, totally overcome when peptides with higher affinity for MHC(peptides 4A and 4Y) were used in the restimulation studies (Fig.4, c andd). This is in contrast to what was seen in the presence ofwild-type Ac1-9, where differentiation of IL-2-secreting cells wasdependent on B7.2 (Fig. 2b). The addition of Abs to B7.1 and B7.2led paradoxically to an increase in the level of IL-2 detected. Thismight be explained by less vigorous growth of these cultures. Thisis not supported, however, by the data shown in Figure 4,a andb,for B7.1. The differentiation of T cells capable of proliferating on

FIGURE 1. Primary proliferative T cell responses and cytokine produc-tion of Tg4 transgenic T cells. Splenocytes were cultured at 23 105 cells/well in the presence of 20mg/ml of the indicated anti-B7 mAb(s) or theisotype control and varying concentrations of Ac1-9. T cell proliferationwas measured by pulsing the plates with 0.5mCi of [3H]thymidine duringthe final 12 h of a 24-h culture (A). Cytokine levels were quantified by cellcapture ELISA harvested at 24 h (B) or 48 h (C). Results are the means ofduplicate wells and are representative of two experiments.

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secondary stimulation with Ag and fresh APC was partially inhib-ited by primary culture only in the presence of anti-B7.2 and notthe anti-B7.1 mAb.

The secretion of IFN-g remained B7.2 dependent despite re-stimulating these cells using peptides with high affinity for MHC.Whereas the use of the higher affinity peptides caused only a slightincrease in the level of IFN-g produced by control cultures, a sig-nificant increase in cytokine levels was observed in the presence ofanti-B7.2 mAb compared with the same concentration of wild-type

peptide (Fig. 2c). There was no evidence for a role of B7.1 in thisprocess, nor was there significant enhancement of the response inthe presence of the anti-B7.1 mAb.

The secretion of IL-4 in control cultures was increased by theaddition of a peptide, Ac1-9(4Y), with high affinity for MHC (Fig.5b) compared with the wild-type peptide (Fig. 2d). The depen-dence on both B7.1 and B7.2 was not overcome by addition ofhigh affinity peptides (Fig. 5,a andb); if anything, it was furtherenhanced.

FIGURE 2. Restimulation of Tg4 T cells withwild-type Ac1-9. Splenocytes were primed in thepresence or the absence of anti-B7 mAb and Ac1-9(0.4 mg/ml). Viable cells, recovered 3 days afterpriming, were restimulated with a range of Ac1-9concentrations and B10.PL APC in the absence ofanti-B7 mAbs. Proliferation was measured (a) as de-scribed in Figure 1. Peak cytokine production, quan-tified by cell capture ELISA, was measured at 24 h(b andd), 48 h (c), and 72 h (e). The results are themeans of duplicate wells and are representative ofthree experiments.

FIGURE 3. Primary proliferative T cell re-sponses and cytokine production of Tg4 transgenicT cells following stimulation with Ac1-9 analogues.Whole splenic cells were stimulated as described inFigure 1. Cells were activated with a range of con-centrations of Ac1-9 analogues, 4A (a, c, ande) and4Y (b, d, and f). Proliferation and peak cytokineproduction were measured as described in Figure 1.The results are the means of duplicate wells and arerepresentative of three experiments.

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The partial dependence on B7.1 molecules, shown for peptidesAc1-9 (Fig. 2e) and Ac1-9(4A) (Fig. 5c), of IL-5 producing cellswas totally overcome by using the highest affinity peptide Ac1-9(4Y) (Fig. 5d). In marked contrast to Th1 cells, the results shownin Figure 5,c andd, emphasize the B7.2 dependence of Th2 celldifferentiation. Even with saturating doses of the highest affinitypeptide (Ac1-9 4Y), the differentiation of IL-5-secreting cells re-mained totally B7.2 dependent.

These results reveal distinct patterns of dependence on costimu-lation for the differentiation of the Th1 and Th2 subsets of Tg4 Tcells. Differentiation of cells secreting Th1 cytokines was B7.2dependent and B7.1 independent. The B7.2 dependence for differ-entiation of IL-2-secreting cells could be overridden by peptides ofhigher affinity for MHC. In addition, B7.2 dependence for differ-entiation of IFN-g-secreting cells could be partially overcome byhigher affinity peptides. The results of these studies distinguish thecostimulation dependence for differentiation of cells producing ei-

ther IL-4 or IL-5. IL-4-producing cells were only partially depen-dent on either B7.1 or B7.2, and this could not be overcome withhigh affinity peptides. The differentiation of IL-5-producing cellswas, by contrast, totally dependent on B7.2. Compared with wild-type Ac1-9, the high affinity peptides overcame the dependence onB7.1 for IL-5 secretion. However, there remains an absolute re-quirement for costimulation through B7.2 for IL-5 secretion, asevidenced by the fact that addition of the higher affinity peptide didnot induce an overall increase in levels of IL-5 by T cells previ-ously primed in the absence of B7.2 molecules.

Expression of B7 molecules on untreated and treated T cells

The expression of B7 molecules on activated murine T cells hasbeen reported recently (3). It is possible that the effects we haveobserved on proliferation and cytokine induction in anti-B7-treatedcultures may be due to direct signaling of T cells by these intactAbs rather than simply blockade of costimulatory molecules on

FIGURE 4. Proliferation and Th-1-associated cy-tokine production following restimulation of Tg4 Tcells with Ac1-9 analogues (4A and 4Y). Spleno-cytes were primed as described in Figure 2. Viablecells, recovered 2 or 3 days after priming, were re-stimulated with a range of concentrations of Ac1-9analogues 4A (a,c, ande) and 4Y (b, d, andf) withB10.PL APC in the absence of anti-B7 mAbs. Pro-liferation was measured (a and b) as described inFigure 1. Peak cytokine production, quantified bycell capture ELISA, was measured at 24 h (c andd)and 48 h (e and f). The results are the means of du-plicate wells and are representative of threeexperiments.

FIGURE 5. Th-2-associated cytokine productionfollowing restimulation of Tg4 T cells with Ac1-9analogues (4A and 4Y). Splenocytes were primedand restimulated as described in Figure 4 with theAc1-9 analogues 4A (a andc) and 4Y (b andd) withB10.PL APC in the absence of anti-B7 mAbs. Peakcytokine production, quantified by cell captureELISA, was measured at 24 h (a andb) and 72 h (candd). The results are the means of duplicate wellsand are representative of three experiments.

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APC. However, for this to occur, the T cell would have to exhibitan activated phenotype after incubation with peptide and anti-B7Abs despite not having received costimulatory signals from splenicAPC. In fact, incubation of splenic cultures with anti-B7.2 or anti-B7.1 plus anti-B7.2 Abs resulted in a significant down-regulationof T cell proliferation and IL-2 production (Fig. 1). These T cellsprobably exhibit a null phenotype rather than an activated pheno-type, thereby precluding T cell expression of B7 molecules. How-ever, to clarify this, we assessed the expression of B7 molecules onT cells after treating whole splenic cells with biotinylated anti-B7Abs and peptide. Expression of B7 molecules on T cells and APCswas visualized 48 h after culture by double staining with strepta-vidin-PE- and FITC-conjugated anti-CD3 followed by flow cyto-metric analysis. Splenic cells activated in the absence of anti-B7Abs displayed a mean channel number of 22.5 and 28.4 for ex-pression of B7.1 and B7.2 molecules, respectively, on CD31 cells(Table I). These T cells had clearly up-regulated expression of B7molecules compared with control, naive T cells, which displayedmean channel numbers of 9.6 (B7.1) and 11.2 (B7.2). T cells thathad been stimulated with Ag in the presence of anti-B7.1, anti-B7.2, or both Abs displayed mean channel numbers between 7.7and 12.6. This shows that in our culture system, T cells requiredcostimulation via B7.1 or B7.2 to up-regulate expression of B7molecules on their own surfaces.

DiscussionWe have examined the ability of B7.1 and B7.2 costimulatorymolecules to affect the differentiation of Th1 and Th2 subsets invitro. Our results reveal that the use of these costimulatory mole-cules was highly differential and depended on the following fac-tors: 1) the composition of the starting T cell population (i.e., pri-mary response of naive cells or their differentiation), 2) theparticular cytokine assessed, and 3) the affinity of stimulatingpeptide.

First, activation of naive T cells was not observed to be depen-dent on B7.1 molecules. Blocking these molecules had little effecton proliferation or IL-2 or IFN-g production. It is not surprisingthat B7.1 blockade had a minimal effect on the primary T cellresponse to Ac1-9, since B7.1 expression is only found on acti-vated, not on resting, APC (18). Activation of these cells, however,was highly B7.2 dependent, in agreement with previously pub-lished findings that B7.2 is the predominant CD28 ligand early inthe immune response (18, 26). The greater dependence on B7.2compared with B7.1 probably reflects the constitutive, albeit low,level of expression of the former on APC (26). The level of B7.2expression has also been reported to be markedly higher than thatof B7.1 during the course of a primary immune response in vitro(12). Interestingly, activation of naive T cells using peptide ana-logues exhibiting increasing affinity for MHC revealed that thedependence on B7.2 molecules observed with wild-type Ac1-9

was partially overcome using the Ac1-9 analogues 4A and 4Y.Therefore, a lack of costimulation during a primary immune re-sponse in vitro can be counteracted by increasing the strength ofthe activation stimulus through the TCR.

The activation of naive T cells using supraoptimal levels of TCRsignaling in the absence of costimulation has important implica-tions for T cell activation. Our findings are not consistent with theclassical two-signal model for T cell activation, where signals 1and 2 are thought to be essential for full T cell activation, whereasprovision of signal 1 alone results in T cell anergy (27, 28). Thelatter model of T cell activation suggests that signals 1 and 2 arequalitatively different and act in synergy to activate T cells. Wehave found, however, that whereas low levels of TCR stimulationin the absence of CD28 costimulation led to a drastic down-reg-ulation of IL-2 and IFN-g production, an increase in the strengthof signal through the TCR prevented the induction of T cell un-responsiveness. This suggests that the net result of CD28 costimu-lation is to lower the threshold required to trigger naive CD41 Tcells, such that these cells can be efficiently activated by low con-centrations of Ag in the presence of CD28 costimulation. This issimilar to the findings of Viola and Lanzavecchia (29) and Teh andTeh (30). In fact, in the former study it was shown that whereasapproximately 8000 TCRs need to be engaged for T cell activationin the absence of CD28 costimulation, only approximately 1500TCRs are required in the presence of CD28 costimulation (29).This is consistent with our results, where high levels of TCR en-gagement can compensate, to a certain degree, for a lack of co-stimulation for the production of IFN-g and IL-2.

The ability of naive T cells, stimulated in the absence of co-stimulatory molecules to secrete both Th1- or Th2-associated cy-tokines upon subsequent restimulation was assessed using freshAPC (i.e., B7-sufficient conditions). Our results reveal an intrigu-ing pattern of dependence on costimulation by both Th1- and Th2-associated cytokines. We consistently observed that a blockade ofB7.1 molecules did not affect the ability of T cells to secrete IL-2or IFN-g. B7.2, however, had a major role in priming T cells forboth cytokines, in particular IFN-g. This is in agreement with arecent report that B7.2 molecules significantly contribute to theproduction of IFN-g production (12). Even so, the dependence onB7.2 was not obligatory, since restimulating cells (previously ex-posed to wild-type Ac1-9 and anti-B7.2) using high affinity pep-tides could partially overcome the requirement for B7.2. There-fore, increasing the strength of the signal through the TCR duringa secondary encounter with Ag appears to by-pass the initial re-quirement for costimulation to a certain degree. This enabledhigher levels of cytokines to be produced in the absence of B7.2when Ac1-9 analogues (4A and 4Y) were used instead of wild-type peptide.

The dependence on CD28/B7-mediated costimulation for dif-ferentiation of Th2 cytokine-secreting cells was very different fromthat observed for Th1 cells. Whereas B7.1 blockade had little or noeffect on IFN-g or IL-2 production in response to Ac1-9, both IL-4

Table I. Median channel fluorescence intensity of staining showing expression of B7.1 and B7.2 molecules on CD31 spleen cellsa

Ac1-9 Only Untreated Ac1-91 Anti-B7.1 Ac1-91 Anti-B7.2Ac1-9 1 Anti-B7.1 1

Anti-B7.2

Expression of B7.1 on CD31 cells 22.5 9.6 8.6 9.7 7.7Expression of B7.2 on CD31 cells 28.4 11.2 11.7 12.6 8.9

a Splenic T cells were incubated with Ac1-9, biotinylated anti-B7.1 (20mg ml), or anti-B7.2 (20mg ml) or both for 48 h. Dead cells were removed by density gradientcentrifugation, and expression of B7.1 and B7.2 molecules on T cells were visualized by flow cytometry after staining with streptavidin-PE and anti-CD3-FITC. Results arerepresentative of two experiments.

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and IL-5 production were partially dependent on B7.1, the absenceof which inhibited cytokine levels to approximately 40 to 60% ofthose observed in control untreated cells. Furthermore, a lack ofB7.2 abrogated IL-5 secretion while significantly reducing levelsof IL-4, suggesting a greater dependence on B7 molecules by Th2subsets compared with Th1 subsets. Interestingly, whereas increas-ing stimulation through the TCR (using high affinity peptides) dur-ing restimulation overcame the requirement for costimulation byTh1 subsets, levels of IL-4 remained diminished even in the pres-ence of high affinity peptides. In addition, IL-4 levels were signif-icantly depressed in the presence of either B7.1 or B7.2 mAbs.However, the presence of both mAbs did not abrogate IL-4 pro-duction completely, leaving residual levels of cytokine secretionthat were unaffected by a lack of CD28/B7-mediated costimula-tion. The question arises as to which cells were capable ofproducing low levels of IL-4 in a CD28/B7-independent fashion.Several possibilities exist. First, it is conceivable that some IL-4-secreting cells, possibly non-T cells, could be costimulation inde-pendent. For example, mast cells have been shown to be an im-portant source of IL-4 and may be indirectly stimulated in thesecultures. Alternatively, certain T cells may have a small but sig-nificant capacity to secrete IL-4 in a costimulation-independentmanner. CD41 NK1.1 cells orgd T cells, both capable of produc-ing IL-4 (31, 32), may have produced small amounts in these cul-tures. Alternatively, all naive T cells may possess a limited capac-ity to secrete modest amounts of IL-4. In support of this, Flavell etal. generated a transgenic mouse expressing the thymidine kinasegene driven by the IL-4 promoter (33). When these transgenicmice were treated with ganciclovir, T cell function was profoundlyaffected, suggesting that the majority of naive T cells expressed thethymidine kinase protein and therefore IL-4 (33).

Whereas IL-4 secretion was shown to be partially dependent onboth B7.1 and B7.2 molecules, analysis of IL-5 production, also aTh2-associated cytokine, revealed very different results. The de-pendence on B7.1, observed during wild-type Ag stimulation andto a lesser extent with Ac1-9(4A), was completely overcome usingpeptide Ac1-9(4Y), which displayed the highest affinity for MHC.The marked dependence on B7.2, however, was emphasized by thefact that blockade of B7.2 during priming abolished IL-5 produc-tion, even when cells were restimulated using supraoptimal levelsof TCR stimulation.

These results indicate that the dependence and utilization ofB7.1 and B7.2 molecules in the differentiation of naive T cells intoTh1 and Th2 subsets differ considerably. In addition, analysis ofcytokine production within these subsets suggests that additionalheterogeneity is present with respect to B7 usage, particularly forthe Th2 subset. These effects were mediated by a lack of CD28-mediated signaling during priming of naive T cells, although it ispossible that the anti-B7 Abs may have been signaling directly byligation of B7 molecules on T cells. However, this is highly un-likely, as T cells that had been primed in the absence of costimu-lation do not produce significant IL-2 or IFN-g and therefore pre-sumably exhibit a nonactivated/null phenotype with little or no B7expression. This point was clarified by assessing the expression ofB7 molecules on T cells after incubation with Ac1-9 and anti-B7Abs. Biotinylated B7 Abs were used for these experiments, suchthat up-regulation of B7 expression on T cells during the cultureperiod could be later visualized by flow cytometry using strepta-vidin-PE-linked Ab. T cells primed in the absence of anti-B7 Absup-regulated the expression of both B7.1 and B7.2 molecules com-pared with that in control T cells incubated in the absence of bothpeptide and anti-B7 Abs. This supports earlier reports that T cellsdo express B7 molecules after activation (3), although more recent

data indicate that T cell expression of B7.2 is found in an alteredhypoglycosylated form that displays reduced binding to bothCD28 and CTLA-4 (34). T cells that had been primed in the pres-ence of anti-B7.1, anti-B7.2, or both Abs were, however, found toexhibit a median channel fluorescence intensity similar to that ofnonactivated T cells. In fact, where both anti-B7 Abs were present,the T cells displayed B7 at lower levels than nonactivated T cells(Table I). It is surprising that splenic cultures incubated with anti-B7.1 Ab, while having little effect on proliferation and cytokineproduction during priming of naive T cells, did not up-regulate theexpression of either B7.1 or B7.2 molecules on CD31 cells. Thereresults imply that interaction of T cells with both B7.1 and B7.2 isrequired for up-regulation of B7 molecules on T cells.

The restimulation studies shown in Figures 2, 4, and 5 suggestthat whereas an increase in the strength of the signal through theTCR can partially compensate for a lack of costimulation duringpriming where Th1-associated cytokines are concerned, Th2-asso-ciated cytokines are far more sensitive to an absence of CD28/B7-mediated costimulation during priming. This may be due to theautocrine nature of IL-4 (35), which would cause the differentia-tion of IL-4-secreting cells to be more sensitive to the initial levelsof T cell priming, including the presence or absence of B7 mole-cules. Although IL-4 was not detected during primary stimulation,it is possible that low levels may have been produced, essential forthe differentiation of naive T cells into Th2 subsets. The presenceof anti-B7 Abs in these primary cultures may inhibit this low levelof IL-4 secretion, thereby drastically down-regulating the produc-tion of IL-4 and IL-5 by T cells upon subsequent encounter withAg. In fact, low levels of IL-4 were detected in primary culturesstimulated with Ac1-9(4Y) and were inhibited in the presence ofanti-B7.2 mAbs. In contrast, Th1-associated cytokines can be in-fluenced a great deal by APC-derived stimuli, i.e., IL-12 produc-tion (36), such that the secretion of IL-2 and IFN-g may be lessdependent on the magnitude of the primary T cell immune re-sponse and the presence of costimulatory molecules.

Our data support a number of recent observations made with invivo disease models where protective Th2-type immune responseswere down-regulated in the absence of B7 molecules. Blockade ofB7.1 and B7.2 down-regulated the mucosal immune response(characterized by IL-4 production and B cell switching to IgE pro-duction) following oral infection of mice with the nematodeHe-ligmosomoides polygyrus(37). Likewise, blockade of B7.2 inhib-ited recruitment of eosinophils (dependent on IL-5 secretion) intothe airway mucosa following allergen exposure (38), further sup-porting our findings that B7.2 blockade abrogated IL-5 production.In addition, T cells from CD282/2 mice, while capable of secret-ing levels of IFN-g comparable to those in wild-type mice, wereunable to produce IL-4 and IL-5 (39). Therefore, the differentiationof naive T cells toward a Th2 phenotype appears to be very muchdependent on and regulated by CD28/B7 engagement.

Several groups have studied the consequences of selectively in-hibiting B7.1 and B7.2 in the in vivo development of autoimmunediseases. Studies made in autoimmune diabetes and EAE demon-strate new levels of complexity regarding the roles of CD28 andB7 interaction in the development of autoimmune diseases.Kuchroo et al. showed that anti-B7.1 inhibited the development ofEAE and blocked the pathogenic Th1 response, whereas anti-B7.2exacerbated EAE and blocked the Th2 response (20). These ob-servations raise the possibility that Th1-driven autoimmune dis-eases such as EAE require CD28-B7.1 interaction. Similar to EAE,murine diabetes, a spontaneous autoimmune disease, is believed tobe promoted by a Th1 response. However, treatment of NOD mice

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with anti-B7.2 mAb blocked the development of diabetes, whereasanti-B7.1 accelerated disease (21). Because the pathology of boththese diseases is mediated by autoimmune Th1 cells, these resultsare difficult to reconcile. There are, however, various possibilitiesthat may account for these observations. First, differences in tissuedistribution may reflect the opposing effects of anti-B7.1 and anti-B7.2 in these autoimmune disease models. While B7.2 is selec-tively up-regulated on islet cells in diabetic mice, B7.1 is prefer-entially expressed in SJL mice during clinical relapses of EAE(40). Secondly, differences in temporal expression of B7.1 andB7.2 may modulate the ongoing immune response, contributing tothe paradoxical effects seen in the above disease models. In addi-tion, the contribution of other cell surface molecules that can in-crease TCR signal strength (e.g., CD2) may influence the patternof dependence on B7.1 and B7.2 (41, 42). Lastly, our results haveclearly demonstrated that the affinity of agonist peptide for MHCcan also drastically influence B7 dependence and cytokine secre-tion. Studies using various Ag concentrations have shown thatwhereas high Ag doses favored the development of Th1 cells only,priming with low doses led to Th2-like responses (43, 44). In thisstudy, increasing the dose and affinity of Ag led to an overallup-regulation of both Th1- and Th2-associated cytokines. How-ever, while the lack of B7 molecules during priming had littleeffect on IL-2 and IFN-g secretion by T cells stimulated with highaffinity peptides, Th2-associated cytokines were drastically down-regulated in the absence of B7 molecules. Therefore, the secretionof Th2-associated cytokines requires signal 2 during priming, theabsence of which cannot be compensated for by increasing thestrength of signal 1 during restimulation. This suggests that signaltransduction products generated by both TCR and CD28 ligationare both distinct and necessary for optimal Th2-associated cyto-kine production. Additionally, these experiments demonstratedthat the state of differentiation at which T cells received CD28-mediated costimulation was crucial for the development of a Th2response. This was evidenced by the fact that Tg4 T cells that hadnot received costimulation during priming, despite being restimu-lated under B7-sufficient conditions and in the presence of highaffinity peptides, down-regulated their production of Th2-associ-ated cytokines. This suggests that signaling via CD28/B7 ligationduring priming of naive T cells may be essential for subsequentTh2 cytokine production.

AcknowledgmentsWe thank Beata Burkhart and Pauline Lowrey for excellent technicalassistance.

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