Defective Th1 and Th2 cytokine synthesis in the T-T cell presentation model for lack of CD40/CD40...

0014-2980/98/1111-3552$17.50+.50/0 © WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998

Defective Th1 and Th2 cytokine synthesis in the T-Tcell presentation model for lack of CD40/CD40ligand interaction

Lucrezia De Vita1, Daniele Accapezzato1, Giorgio Mangino1, Stefania Morrone2,Isabella Santilio1, Marco Antonio Casciaro1, Danila Fava1, Guglielmo Bruno1,Gianfranco Del Prete3, Angela Santoni2 and Vincenzo Barnaba1, 4

1 Fondazione Andrea Cesalpino, Istituto di Clinica Medica, Universita di Roma “La Sapienza”,Rome, Italy

2 Dipartimento di Medicina Sperimentale, Universita di Roma “La Sapienza”, Rome, Italy3 Istituto di Medicina Interna e Immunoallergologia, Universita di Firenze, Florence, Italy4 Istituto Pasteur-Cenci Bolognetti, Rome, Italy

In this study, T or NK cell clones used as antigen-presenting cells (T- or NK-APC) wereshown to be significantly less efficient than professional APC in inducing Th1 and Th2 cyto-kines by antigen-specific T cell clones. This phenomenon was not related to a limitedengagement of TCR by T-APC, since comparable thresholds of TCR down-regulation wereshown when antigen was presented by either T-APC or professional APC. Rather, the stimu-latory T-APC weakness was due to their inability, because they are CD40–, to provide theappropriate co-stimuli to responder T cells both indirectly via IL-12, and partially via directCD40L triggering on T cells. Indeed, the simultaneous addition of IL-12 and reagents directlyengaging CD40L on responder T cells restored T cell cytokine synthesis when antigen waspresented by T-APC. In addition, either IL-12 production or blocking of T cell cytokine syn-thesis by anti-IL-12 p75 antibodies was evident only when professional APC were used inour antigen-specific system. The down-regulation of cytokine synthesis in the system of T-Tcell presentation could represent a novel mechanism of immune regulation, which may inter-vene to switch off detrimental Th1- or Th2-mediated responses induced by antigen presen-tation among activated T cells infiltrating inflamed tissues.

Key words: Antigen presentation / Th1/Th2 / Co-stimulatory molecules / Cytokine

Received 26/6/98Revised 4/8/98Accepted 10/8/98

[I 18551]

Abbreviations: DC: Dendritic cells CD40L: CD40 ligandHBenvAg: Hepatitis B envelope antigen MFI: Mean fluores-cence intensity T-APC: T cell clones as APC NK-APC: NKcell clones as APC BCECF-AM: 2’,7-bis-(carboxyethyl)-5(6’)-carboxyfluorescein

1 Introduction

Optimal antigen-elicited T cell activation resulting in pro-liferation and cytokine production is absolutely depen-dent on professional APC such as dendritic cells (DC),through the productive engagement of MHC-peptide/TCR and of co-stimulatory molecule receptor/ligandpairs [1, 2]. Upon activation, T cells express CD40 ligand(CD40L), a member of the TNF cytokine superfamily,

whose receptor, CD40, is a 50-kDa transmembrane pro-tein expressed on a wide variety of cells [3–9]. CD40ligation has been demonstrated to be essential for bothCD40+ cell activation/differentiation and IL-12 produc-tion [3, 4, 10–12]. In this connection, recent data suggestthat activated T cells stimulate IL-12 production by DCvia CD40/CD40L interaction, thus favoring the develop-ment of Th1 responses [10–14]. On the other hand, onlya limited number of reports suggest the possibility thatCD40/CD40L interaction can also function in the oppo-site direction, promoting T cell activation through CD40Lsignaling [15, 16]. Recently, it has been reported thatresting T cells, activated by anti-CD3 mAb, could pro-duce IL-4 or IFN- + when co-triggered via CD40L eitherby specific antibodies or by the synergistic effect ofCD40-transfected P815 cells and IL-12, respectively [17,18]. Although it has been clearly established that IL-12[19] is the most powerful inducer of Th1 cell develop-

3552 L. De Vita et al. Eur. J. Immunol. 1998. 28: 3552–3563

Figure 1. T-APC or NK-APC are less efficient than professional APC in inducing cytokine synthesis by antigen-specific T cells.HLA-DRB1*1501-restricted cloned T cells (VB3G4 clone), specific for HBenvAg(193–207) peptide, were cultured (3 × 104/well) withirradiated autologous DC (1 × 104/well) or T-APC or NK-APC (1 × 105/well), which were previously pulsed with increasing concen-trations of peptide. Supernatants were collected after 48 h of culture and the concentrations of IFN- + or IL-4 were determined byELISA. Cytokine concentrations are expressed as mean of triplicate determinations. Data are from one representative experimentout of four using clone VB3G4. Comparable results were obtained with four different T cell clones.

ment [13, 20–24], recent findings have demonstratedthat it can also prime in vitro a T cell population charac-terized by secretion of both IL-10 and IFN- + [25, 26], withpotential down-regulatory effect on Th1 responses [27].Furthermore, it has been reported that IL-12, althoughable to induce either enhanced or transient IFN- + pro-duction in antigen-stimulated Th1 or Th2 cell clones,respectively [19, 28, 29], fails to inhibit Th2-type cyto-kines once these responses are established [30]. More-over, IL-12 seems to potentiate IL-4 production byestablished T cell clones, further suggesting a dichotomyof IL-12 function on resting or activated T cells [31].

Herein, we compared the different capabilities of CD40+

or CD40– APC secreting or not IL-12 in inducing Th1 orTh2 cytokines by antigen-specific T cell clones, and veri-fied whether either IL-12 or CD40L or other molecules,such as CD28, are involved in this function. Moreover,we wondered which implications activated T cells, whichare CD40– and unable to produce IL-12, may have whenthey function as APC [32] in the context of inflamed tis-sues, where antigen presentation may occur among infil-trating T cells.

2 Results

2.1 Activated T or NK cells, used as APC, areless efficient than professional APC ininducing cytokine production by antigen-specific T cell clones

DC used in both this and the following experiments werein an immature phase when they could up-regulateantigen-presenting functions following cognate interac-tions with activated T cells [11]. Fig. 1 shows that a repre-sentative hapatitis B envelope antigen (HBenvAg)(193–207)-specific CD4+ T cell clone produced IFN- + or IL-4 in adose-dependent fashion in response to peptide pre-sented by the different types of APC. As expected [33],DC as APC were more efficient than T- or NK-APC, whichdrastically down-modulated both IFN- + and IL-4 synthe-sis. This effect was observed particularly at peptide con-centrations lower than 1 ? g/ml and was more stringentfor IL-4 than for IFN- + synthesis. Comparable resultswere obtained by using four different responder T cellclones. As evident in Table 1, when APC were pulsedwith 0.01 ? g/ml peptide, only DC or PBMC, but not T- orNK-APC provided the appropriate stimuli required for anefficient production of the cytokines tested, and in par-ticular of IL-4 and IL-5.

Eur. J. Immunol. 1998. 28: 3552–3563 Defective cytokine synthesis in the T-T cell presentation model 3553

Table 1. Effect of different APC types on cytokine production by antigen-specific T cell clones

Responder Tcell clone

MHC restrictionelement

HBenvAg peptide APC IL-4 pg/ml IL-5 pg/ml IFN- + pg/ml

VB3G4 DRB1*1501 193–207 DC 3 648a) 2266a 11 100a)

VB3G4 193–207 PBMC 1 250 4 414 7 120

VB3G4 193–207 T-APC 380 320 3 322

VB3G4 193–207 NK-APC 320 472 3 110

VB3D11 DPXXXX 193–207 DC 5 622 7 430 8 227

VB3D11 193–207 PBMC 5 305 6 878 6 560

VB3D11 193–207 T-APC 523 363 2 899

VB3D11 193–207 NK-APC 584 618 3 200

VB3B8 DRB1*1501 344–360 DC 1 229 850 12 020

VB3B8 344–360 PBMC 1 106 900 12 862

VB3B8 344–360 T-APC 535 X 20 1 600

VB3B8 344–360 NK-APC 342 X 20 1 048

VB1E9 DQXXXX 344–360 DC 1 320 2 153 3 320

VB1E9 344–360 PBMC 1 115 1 812 2 612

VB1E9 344–360 T-APC 111 110 631

VB1E9 344–360 NK-APC 117 172 801

a) T cells were stimulated with 0.01 ? g/ml peptide and autologous APC. After 48 h SN were collected and tested by ELISA.Background response in the absence of antigen was for all clones X 20 pg/ml.

2.2 The lower T-APC efficiency in inducingcytokine synthesis is not caused by a lowerTCR occupancy

We next investigated whether the lower ability of T-APCin inducing cytokine production in comparison with pro-fessional APC reflected a lower TCR occupancy. Wetook advantage of recent findings demonstrating thatTCR down-regulation is a consequence of its specificligation and can be used to measure the number of TCRtriggered by antigen-pulsed APC [34, 35]. Thus, we com-pared the TCR down-regulation and the levels of cyto-kine production in a T cell clone (VB3G4) specific for theHBenvAg(193–207) peptide and the HLA-DRB1*1501restriction element, stimulated by increasing peptideconcentrations presented by either DC (HLA-DR MFI:1,231) or T-APC (HLA-DR MFI: 477). In these experi-ments, to minimize the differences in APC functionbetween DC and T-APC, recently activated cloned Tcells (i.e. a few days after restimulation) were used asAPC, when they are able to synthesize a high rate ofMHC class II molecules [32]. T cells were then separatedfrom contaminating feeder cells on multistep Percoll gra-

dients, recovering G 99 % T cells as shown by FCM withanti-CD3 mAb. Fig. 2 shows that these T-APC, however,provided a lower cytokine induction than professionalAPC, although they induced comparable levels of TCRdown-regulation, ruling out the possibility that this defectwas related to a lower TCR occupancy by T-APC.

2.3 Professional APC provide in trans the co-stimulatory signals restoring the ability of T-or NK-APC to induce optimal cytokineproduction

We tested the capacity of allogeneic (HLA-DRB1*1501-negative) professional APC (either PBMC or DC) to pro-vide as bystander the appropriate co-stimuli required forefficient cytokine production, when added in cultures inwhich an HLA-DRB1*1501-restricted T cell clone wastriggered by autologous T-APC pulsed with 0.1 ? g/mlpeptide, a concentration previously demonstrated toprovide a decreased cytokine synthesis when presentedby T- or NK-APC (Fig. 1). Under these conditions, theinability of responder T cell clones to efficiently synthe-


Figure 2. TCR down-regulation and cytokine production inT cells antigen-stimulated by DC or T-APC. Cloned VB3G4cells (1.5 × 105/well) were cultured for 5 h with autologousirradiated DC or T-APC (3 × 105/well), previously pulsed withincreasing concentrations of peptide and treated withBCECF-AM during the last 10 min. Then SN were collectedfor assaying cytokine production, while cells were tested forTCR down-regulation as described in Sect. 4.4. Data arefrom one representative experiment out of seven using cloneVB3G4.

size both IFN- + and IL-4 was reverted by co-stimulationby each of professional APC tested (Fig. 3). A hierarchy inthis function was definitely evident, since DC were themost efficient co-stimulators. On the contrary, noimprovement in cytokine synthesis was observed whenallogeneic T-APC were added to the system as co-stimulators. The same resuls were obtained whenantigen-specific T cell clones were triggered by autolo-gous peptide-pulsed NK-APC (data not shown). Alto-gether these results suggested that T- or NK-APC areunable to provide the appropriate co-stimulus for an effi-cient induction of cytokine production and that this is aprivileged function of professional APC.

2.4 Co-stimulatory signals by B7.1/B7.2molecules are not required for the inductionof cytokine synthesis by antigen-specificCD28– T cell clones

In our system, the incapability of T- or NK-APC to inducean efficient cytokine production by antigen-specific T cellclones was not due to a lack of signals provided by B7.1or B7.2 molecules, as suggested in previous studies [36],for the following reasons. First, both T- (Fig. 4A) and NK-APC (not shown) expressed both CD80 and CD86 mole-cules. Second, T cell clones used in this study, differentlyfrom previous studies [36], were completely CD28 nega-tive (Fig. 4A), ruling out that the B7.1/B7.2 co-stimulationcould be involved in cytokine production, even wheninduced by professional APC. Third, B7.1 and B7.2expression on T cell clones was functional, since theywere powerful stimulators in primary MLR, a phenome-non which was significantly inhibited by the addition of asoluble form of cytotoxic T lymphocyte antigen (CTLA)-4, such as the chimeric CTLA4-IgG1 fusion protein, atthe initiation of the proliferative response (data notshown) [32]. Fourth, the inability to trigger efficient cyto-kine production was not reverted by direct co-stimulation of responding T cells by addition of murine Lcells transfected with human B7.1 in cultures, in whichcloned T cells were used as APC (Fig. 5). That these Lcells are bona fide stimulators is shown by our previousevidence demonstrating that, when co-expressing HLA-A2.1 plus a given peptide, they efficiently primed humanCTL responses (not shown).

2.5 IL-12 restores the inefficient T cell cytokineinduction by antigen-pulsed T- or NK-APCand the simultaneous CD40L signaling helpsthis effect

We reasoned that the inefficient induction of cytokinesynthesis by T- or NK-APC might be due to their incapa-bility, because they are CD40–, either of up-regulating IL-


Figure 3. Bystander allogeneic APC restore cytokine synthesis by T cell clones stimulated by antigen-pulsed T-APC. The HLA-DRB1*1501-restricted VB3G4 clone was cultured with irradiated autologous peptide-pulsed (0.1 ? g/ml) cloned T-APC in thepresence or absence of increasing concentrations of different types of irradiated allogeneic (HLA-DRB1*1501 negative) APC. SNwere collected after 48 h of culture and the concentrations of IFN- + or IL-4 were determined by ELISA. Cytokine concentrationsare expressed as mean of triplicate determinations.

12 syntesis via CD40/CD40L interaction or of directlytriggering responder T cells through CD40L [16–18].Consistent with this possibility were the findings that: (1)IL-12 production was obtained by peptide-pulsed pro-fessional APC, but not by peptide-sensitized T-APC,upon conjugation with an autologous HBenvAg-specificT cell clone (Fig. 6); (2) upon stimulation with differenttypes of APC, responder T cells underwent an antigen-dependent up-regulation of CD40L (Fig. 4B), while eitherthemselves or T-APC remained strictly CD40– (notshown); (3) a significant increase in the production ofboth IFN- + and IL-4 was evident when the heterodimericform of IL-12 (p75) was added to cocultures whereresponding T cells were incubated with peptide-pulsedT-APC (Fig. 7A). Moreover, both cytokines also slightlyincreased when irradiated P815 cells, transfected withhuman CD40 cDNA, were added to the system. Moreimportantly, about 100-fold lower antigen doses wererequired when both CD40+ P815 cells and IL-12 p75were simultaneously added in culture, clearly indicatingthat the former cooperated with the latter in increasingboth IFN- + and IL-4 production by antigen-stimulated Tcell clones. On the contrary, no effect was observed withIL-12 p40, either when it was added alone or togetherwith P815 expressing CD40 (Fig. 7B), supporting theidea that it does not have biological activity in the humansystems [37]. These data rule out the possibility that therescued cytokine induction in this system is caused bythe up-regulation of co-stimulatory molecule expressionand/or of IL-12 synthesis by APC via CD40/CD40L inter-action, since in our system we used CD40– T-APC, whichwere indifferent to stimuli provided by CD40L+ T cellsand unable to produce IL-12 (Fig. 6).

2.6 Blocking effect of antibodies to IL-12 p75 onthe capability of APC to induce cytokineproduction by antigen-stimulated T cellclones

We evaluated the capacity of either anti-IL-12 p75 mAb(20C2) or anti-IL12 p40 mAb (4D6), neutralizing or notIL-12 bioactivity, respectively, in inhibiting the co-stimulation effect of APC on the production of both IFN- +and IL-4. Fig. 8 clearly shows that 20C2, but not 4D6,significantly blocked both IFN- + and IL-4 production by Tcell clones when stimulated by autologous antigen-pulsed DC, but not by autologous antigen-pulsed T-APC,suggesting that only the former are able to produce IL-12. This indirect evidence was confirmed by the findingthat IL-12 p75 was detected only in SN of cultures inwhich T cell clones were stimulated by antigen-pulsedprofessional APC (Fig. 6).

3 Discussion

In this study, we have demonastrated that activated T orNK cells as APC, particularly when pulsed with low anti-gen dose, were significantly less efficient than profes-sional APC in inducing cytokine synthesis by antigen-specific T cell clones. This phenomenon was not due tothe fact that T- or NK-APC, which showed a significantlylower HLA-DR expression than professional APC, couldtrigger fewer TCR than professional APC at low antigenconcentrations [34, 35]. These results suggest that thelower level of specific peptide/class II complexes pro-vided by T-APC is, however, enough for reaching the sat-


Figure 4. Surface phenotype of cloned T cells at day 20 ofculture and antigen-dependent CD40L up-regulation onactivated T cells. (A) The clones used either as APC orresponders, were stained with the mAb described in the fig-ure, and analyzed on a FACScan, using propidium iodide toexclude dead cells. (B) Cloned VB3G4 cells were culturedwith autologous irradiated T-APC, previously pulsed withpeptide and treated with BCECF-AM during the last 10 min.Cells were tested for CD40L expression after 6 h (a), 12 h (b),and 24 h (c) post stimulation. Data are from one representa-tive experiment out of three using clone VB3G4.

uration point of TCR occupancy, and that the latter aloneis not sufficient for a complete T cell activation. In thiscontext, the evidence that the T-APC inefficiency wasrestored by co-stimulation provided by allogeneic pro-fessional APC, clearly indicates that, under suboptimalconditions of T cell activation, T-APC are unable to pro-vide accessory signals required for cytokine productionby T cell clones. Thus, we reasoned that this modelcould be exploited for investigating which co-stimulatorysignals are needed for inducing efficient cytokine pro-duction by T cell clones.

The failing in co-stimultion by T- or NK-APC for an effi-cient cytokine production is not to be attributed to lack ofco-stimulatory B7.1/B7.2 signals, but to their inability,because they are CD40–, to provide the appropriate stim-uli via the CD40/CD40L interaction [10–12]. Consistentwith these evidences were the findings that: (1) IL-12partially restored the inefficient IFN- + and IL-4 produc-tion of responding T cells when low antigen doses werepresented by T-APC; (2) this defect was completely

restored when IL-12 was simultaneously added withCD40+ P815 cells selectively triggering CD40L onresponder T cells; (3) up-regulation of IL-12 synthesisand blocking of T cell cytokine synthesis by anti-IL-12p75 antibodies were only evident when professional APCwere used in our antigen-specific system. Thus, our datasuggest that the interaction between CD40 on profes-sional APC and CD40L on activated T cells delivers sig-nals in the two opposite directions, which result inenhanced production of both Th1- and Th2 cytokines.However, this mechanism should be more stringent forthe cytokine up-regulation of T cell clones with Th1 orTh0 or Th2-line phenotype than for that of well-polarizedTh2 clones, insensitive to IL-12 function for lack of high-affinity IL-12R [23, 24].

What could be the effect of this phenomenon in vivo? Wemay depict the following picture. T cells, once activated(class II+, CD80+, CD86+, CD40L+, CD40–) and differenti-ated [38–40], acquire the capacity to massively invadeinflamed tissues [41, 42]. In these enviroments, theycould present antigen to each other [32], and ultimatelydown-regulate cytokine production, once the concentra-tion of a given dangerous antigen decreases, as it occursin the course of the resolution of an infection. This mayrepresent a novel regulatory mechanism, which may inparticular turn off the possible exorbitant dilatation ofdetrimental Th1- or Th2-mediated effects into inflamedtissues. Moreover, it could play a general role in immuneregulation, since these T cells, not being protected bythe “vital” signals provided by CD40/CD40L interaction,would become vulnerable to both Fas-mediated apopto-sis [43] and the negative CTLA-4 co-stimulation [44].Conversely, this down-regulatory mechanism might beovercome during a chronic inflammatory disease, wherethe persistence of high levels of both a given dangerousantigen and consequently of IL-12 production by directlystimulated tissue DC [45] could sustain the T cell cyto-kine synthesis via the T-T cell presentation mechanism.

4 Material and methods

4.1 Media and reagents

The medium used in this study was RPMI 1640 (Gibco Labo-ratories, Grand Island, NY) supplemented with 2 ? M L-glutamine, 1 % nonessential amino acids, 1 ? M sodiumpyruvate, 100 U/ml penicillin, 100 ? g/ml streptomycin (FlowLab., Irvine, Ayrshire), and 10 % FCS (Hyclone Laboratories,Logan, UT) (RPMI-10 % FCS). The HBenvAg peptides weresynthesized by the solid phase method on an automatedmultiple peptide synthesizer (Abimed, AMS 422, Langenfeld,Germany), using Fmoc chemistry. The purity of peptideswas determined by reverse-phase HPLC. Murine L cells sta-


Figure 5. Co-stimulatory signals by B7.1 molecules do not enhance cytokine synthesis induced by antigen-pulsed T-APC. TheVB3G4 clone was cultured with irradiated autologous T-APC, previously pulsed with increasing concentrations of peptide, in thepresence of 3 × 105/well L cells expressing or not human B7.1. SN were collected after 48 h of culture and the concentrations of IFN-+ or IL-4 were determined by ELISA. Cytokine concentrations are expressed as mean of triplicate determinations. Data are from onerepresentative experiment out of three using clone VB3G4. Comparable results were obtained with two different T cell clones.

Figure 6. IL-12 is proeduced only by professional APC uponantigen-specific interaction with T cells. The VB3G4 clonewas cultured with autlogous DC or T-APC, which were previ-ously pulsed with 1 ? g/ml HBenvAG(193–207) peptide. SN werecollected after 24 h of culture and the concentrations of bothIL-12 p75 and IL-12 p40 were determined by ELISA. Cyto-kine concentrations are expressed as mean of triplicatedeterminations. Data are from one representative experi-ment out of four using clone VB3G4.

bly transfected with a plasmid containing the human B7.1gene were kindly donated by Dr. S. Alberti (Istituto MarioNegri Sud, Santa Maria Imbaro, Chieti, Italy) and obtained asdescribed [46]; FCM analysis showed that transfected Lcells expressed a high mean fluorescence intensity (MFI) forhuman B7.1 (MFI: 543). P815 stably transfected with humancDNA-CD40 was kindly donated by Dr. L. Lanier (DNAX,Research Institute of Molecular and Cellular Biology, PaloAlto, CA) [15] and expressed CD40 with a MFI of 70. Recom-binant IL-4 was a gift from Dr. A. Lanzavecchia (Basel Insti-tute for Immunology, Basel, Switzerland). Human rTNF- §was provided by Hoffmann-La Roche AG (Basel, Switzer-land). Human rIL-12 p40 and IL-12 p75 (Hoffmann-La RocheInc., Nutley, NJ), generated in bacolovirus, were kindly pro-vided by Dr. P. Panina Bordignon (Roche Milano Ricerche,Milan, Italy). The following purified specific mAb were used:anti-CD3 (IgG1, TR66), anti-CD4 (IgG1, 6D10), anti-HLA-DR(IgG2a, L243), anti-HLA-DQ (IgG2a, SPVL3), anti-HlA-DP(IgG1, B7.21) and anti-B7.1 (anti-CD80, IgG2a, B7.24); anti-B70/B7.2 (anti-CD86, IgG2b, IT2.2) was purchased fromPharmingen (San Diego, CA); anti-CD40 (IgG1, mAb89),anti-CD13 (IgG1, SJ.1D1), anti-CD14 (IgG2a, RMO52), anti-CD1a (IgG1, BL6), anti-CD1b (IgG2a, 4.A7.6) and anti-CD1c(IgG1, L161) were purchased from Immunotech S.A., (Mar-seille, France); PE-conjugated anti-CD40L (IgG1, TRAP1)was purchased from Pharmingen (San Diego, CA); anti-CD11a (LFA-1, IgG, TEC-NK2) was purchased from Techno-Genetics, (Milan,Italy); anti-CD54 (ICAM-1, IgG1, 15.2) waspurchased from Sera-Lab, (Crawley Down, GB); anti-CD28(IgM, CK248) was kindly donated by Sandro Poggi (I.S.T.National Institute for Cancer Research, Genova, Italy) [47];anti-IL-12 p40 mAb (IgG1, 4D6) and anti-IL-12 p75 mAb(IgG1, 20C2) non-neutralizing and neutralizing human IL-12bioactivity, respectively, were provided by Dr. P. Panina Bor-dignon (Roche Milano Ricerche) [48]; FITC- and PE-labeled


Figure 7. IL-12 p75, but not L-12 p40, enhances induction of both IFN- + and IL-4 synthesis by antigen-pulsed T-APC. TheVB3G4 clone was cultured with irradiated autologous T-APC, previously pulsed with increasing concentrations of peptide, in thepresence of different combinations of irradiated P815 cells expressing or not human CD40 (3 × 105/well) and 20 ng/ml IL-12 p75(A) or IL-12 p40 (B). SN were collected after 48 h of culture and the concentrations of IFN- + or IL-4 were determined by ELISA.Cytokine concentrations are expressed as mean of triplicate determinations. Data are from one representative experiment out offour using clone VB3G4. Comparable results were obtained with four different T cell clones.

goat anti-mouse Ig was purchased from Southern Biotech-nology Associated, Inc. (Birmingham, AL).

4.2 Cultured DC

DC were generated from peripheral blood monocytes usingthe method described by F. Sallusto and A. Lanzavecchia[33]. Briefly, PBMC were isolated on Lymphoprep cushions(LSM, Organon Teknika Corp., Durham, NC) and separatedon multistep Percoll gradients (Pharmacia Fine Chemicals,Uppsala, Sweden). The recovered monocytes (purity G 90 %as shown by FCM with anti-CD14 mAb), were depleted ofcontaminating T cells by immunomagnetic separation withanti-CD4 attached to Dynabeads (Dynal, Oslo, Norway), andcultured at 3 × 105/ml supplemented with 50 ng/ml GM-CSFand 1 000 U/ml IL-4 for 6 days. Phenotypic analysis of these

cells, anaylzed on a FACScan flow cytometer (Becton Dick-inson, Mountain view, CA) as described [49], showed thatthey expressed the different surface molecules tested withthe following MFI: MHC class I molecules (MFI: 756), HLA-DR (MFI: 1 231), HLA-DQ (MFI: 450), HLA-DP (MFI: 156),B7.1 (MFI: 45), ICAM-1 (MFI: 236), LFA-1 (MFI: 344), CD1a(MFI: 240), CD1b (MFI: 120), CD1c (MFI: 141), CD44 (MFI:488), CD44-v9 (MFI: 35), CD14 (MFI: 26).

4.3 Cultured T and NK cells

Well-characterized CD4+ T cell clones specific for HBenvAgpeptides (193–207 and 344–360), selected for their Th0 pro-file of cytokine production, were used as responder cells [49](Table 1). Randomly selected autologous T or NK cell cloneswere used as T-APC or NK-APC. NK cells were obtained by


Figure 8. Blocking effect of anti-L-12 p75 antibodies on thecapability of professional APC to induce cytokine productionby antigen-stimulated T cell clones. The VB3G4 clone wascultured with irradiated autologous T-APC or DC [previoslypulsed with 1 ? g/ml HBenvAg(193–207)], in the presence orabsence of 5 ? g/ml anti-IL-12 p75 mAb (20C2, IgG1) or anti-IL12 p40 mAb (4D6, IgG1), neutralizing or not IL-12 bioactiv-ity, respectively. SN were collected after 48 h of culture andthe concentrations of IFN- + or IL-4 were determined byELISA. Cytokine concentrations are expressed as mean oftriplicate determinations. Comparable results were obtainedwith three different T cell clones.

coculturing 4 × 105/ml nylon-nonadherent PBMC with1 × 105/ml irradiated (3 000 rad) RPMI 8866 cells for 10 days,as previously described [50]. On day 10, the cell populationwas 90 % CD56+, CD16+, and CD3– as shown by FCM. Cul-tures were then cloned as previously described [49] toobtain long-term CD3– CD16+ NK clones. In the T-T cell pre-sentation experiments, a non-antigen-specific T cell clonewas used as T-APC to rule out that accessory signals mightbe up-regulated on T-APC via TCR triggering. Both cloned T-APC and NK-APC were taken up 20–25 days after the laststimulation, when contaminating CD40+ APC werecompletely absent (Fig. 1A).

4.4 Antigen-dependent TCR down-regulation andmodification of CD40/CD40L expression

APC (autologous DC or T cell clones) were pulsed for 2 h at37 °C with peptide in complete medium. During the last10 min they were treated with 1 ? M 2’,7-bis-(carboxyethyl)-5(6’)-carbosyfluorescein (BCECF-AM; Calbiochem, SanDiego, CA), and then washed four times [34]. Responder Tcells were mixed with APC at a 1:2 ratio in 200 ? l completemedium in round-bottom microtiter plates, centrifuged toallow conjugate formation and incubated at 37 °C for 5 h. SNwere then collected for assaying cytokine production, whilecells were resuspended, washed in PBS containing 0.5 mMEDTA to dissolve the conjugates, and stained with anti-CD3mAb, followed by a PE-labeled goat anti-mouse Ig. CD3

fluorescence was analyzed on a FACScan, gating out APCtreated with BCECF-AM using both foward scatter/sidescatter (FCS/SSC) parameters and green BCECF fluores-cence. The antigen-dependent up-regulation of CD40 andCD40L on both responder T cells and T-APC were evaluatedfollowing the method previously described for detectingCD3 down-regulation; responder T cells were treated withBCECF-AM, when CD40 expression was detected on T-APC.

4.5 Cytokine production

Cloned T cells (1 x 105) were cultured in 200 ? l RPMI-10 %FCS in 96-well flat-bottom microtiter plates in the presenceor absence of HBenvAg peptide-pulsed APC at 37 °C. After48 h of incubation, SN were collected and tested by sand-wich ELISA for the concentration of IL-4, IL-5, IFN- + (Phar-mingen, San Diego, CA), and IL-12 [51]. In addition, IL-12production by APC was evaluated upon antigen-specificinteraction with specific T cells. Live APC were cultured witha HBenvAg-specific T cell clone in the presence or absenceof the relevant peptide and both the IL-12 p75 and IL-12 p40forms were measured in the culture SN after 12 h. ELISA forIL-12 p75 synthesis by APC were performed as described[52].

Acknowledgments: We thank Lewis L. Lanier and SaverioAlberti for the kind gift of CD40- and B7-transfectants, andPaola Panina Bordignon for the IL-12 assays. This work wassupported by Ministero della Sanita-Instituto Superiore diSanita (I Progetto Epatite Virale and X Progetto AIDS), Minis-tero dell’Universita e della Ricerca Scientifica e Tecnologica40 %, I Progetto Associazione Italiana Sclerosi Multipla(AISM), and Progetto Finalizzato CNR “Biotecnologie”.

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Correspondence: Vincenzo Barnaba, Istituto I ClinicaMedica, Universita di Roma “La Sapienza”, PoliclinicoUmberto I, viale del Policlinico, 155, I-00161 Roma, ItalyFax: +39-6-4940594e-mail: barnaba — uniroma1.it


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