Generation and characterization of malaria-specific human CD8+ lymphocyte clones: effect of natural polymorphism on T cell recognition and endogenous cognate antigen presentationby liver cells

Malaria-specific human CD8+ T lymphocyte clones

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Generation and characterization of malaria-specific human CD8+ lymphocyte clones: effect of natural polymorphism on T cell recognition and endogenous cognate antigen presentation by liver cells Anilza Bonelo1,2, Danila Valmori3, Frederic ´ Triponez4, Jean-Marie Tiercy5, Gilles 4 4 Mentha , Jose´ Oberholzer , Patrick Champagne6, Jackie F. Romero1, Fulvio Esposito7, Issa Nebie´ 8, Catherine Barbey9, Pedro Romero3, Socrates Herrera2, Giampietro 1 Corradin1 and Jose´ Alejandro Lopez ´ 1

Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland Instituto de Inmunolog´ıa, Universidad del Valle, Cali, Colombia 3 Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne branch, Lausanne, Switzerland 4 Clinic et Polyclinic of Digestive and Transplant Surgery, University Hospital of Geneva, Geneva, Switzerland 5 Transplantation Immunology Unit, Hopital ˆ Cantonal Universitaire, Geneva, Switzerland 6 Laboratory of Immunology, Institut de Recherches Cliniques de Montreal, ´ Montreal, ´ Canada 7 Dipartimento di Biologia Moleculare, Cellulare e Animale, Universit a´ di Camerino, Camerino, Italy 8 Centre National de Recherche et de Formation sur le Paludisme, Ouagadougou, Burkina Faso 9 Immunology and Allergology Division, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland 2

CD8+ cytolytic T lymphocytes (CTL) play a fundamental role in the clearance of malaria parasites from the liver in mouse models. In humans, however, only low levels of parasite-specific CD8+ T lymphocytes have been observed in individuals living in endemic areas. In the present study, we identified high levels of circulating CD8+ T lymphocytes specific for a previously described HLA-A2-restricted CTL epitope of the circumsporozoite (CS) protein of Plasmodium falciparum in an adult living in Burkina Faso, as evidenced by IFN- + ELISPOT assay and MHC-tetramer technology. After cloning by limiting dilution culture, T cell recognition of natural CS variants of P. falciparum was studied. The results demonstrate that naturally occurring variations drastically affect residues critical for T cell recognition as only two out of nine sequences analyzed were efficiently recognized by the CTL clones. These clones were also used to analyze T cell recognition of the endogenously presented cognate antigen. We observed efficient antigen recognition of both HLA-A*0201-transfected murine antigen presenting cells and liver cells from HLA-A*0201/Kb-transgenic mice upon infection with recombinant vaccinia virus encoding the CS protein (WR-CS). More importantly, we demonstrate for the first time efficient recognition of WR-CS-infected human liver cells. Key words: Malaria / Human CTL clone / Polymorphism / Hepatocyte / TCR recognition

1 Introduction The control of malaria infection is a complex task in which various components of the immune system are engaged. Sterile immunity in malaria has been success-

[I 20974] Abbreviations: P-ES: Pre-erythrocytic stage CS: Circumsporozoite WR-CS: Recombinant vaccinia virus encoding the CS protein TAP: Transporter associated with antigen processing I 2 m: g 2-microglobulin © WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000

Received Accepted

9/6/00 24/7/00

fully obtained, both in animal models and in humans by immunization with irradiated sporozoites [1, 2]. In mice, protection has also been achieved after vaccination with synthetic peptides or DNA encoding proteins from the pre-erythrocytic stage (P-ES) of the parasite [3–7]. In these systems, protection appears to be the result of activation of humoral [8] or T cell-mediated immune responses or both [9–12]. Specific CD8+ T lymphocytes play a prominent role in the achievement of sterile immunity, as adoptive transfer of epitope-specific CD8+ T lymphocyte clones can protect mice against challenge with Plasmodium berghei [13] and P. yoelii [14] sporozoites. In 0014-2980/00/1111-3079$17.50 + .50/0

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human volunteers immunized with irradiated P. falciparum sporozoites, CTL responses to several P-ES epitopes have been detected [15], suggesting that P-ESspecific CTL could contribute to protective immunity against this otherwise deadly parasite infection.

2 Results

P-ES-specific CTL-mediated protection would imply that specific CD8+ T cells recognized parasite-derived peptides on infected hepatocytes, presented in the context of HLA class I molecules [16]. Indeed, CD8+ T cell recognition of parasite-derived antigens endogenously presented by liver cells has been demonstrated in mouse models [16, 17] whereas the demonstration of this phenomenon in human liver cells has remained elusive to date. Among the P-ES antigens expressed by P. falciparum, the circumsporozoite (CS) protein is the most widely studied and was the first protein identified as a target of protective immune responses [18]. Sequence analysis of CS proteins obtained from different field isolates and laboratory strains [19, 20] allowed the characterization of two regions of extensive polymorphism located near the C-terminal end, termed Th2R and Th3R. Strikingly, all of the nucleotide substitutions encode amino acid changes. We have previously identified an HLA-A*0201-restricted CD8+ T cell epitope within region Th2R [21]. A current hypothesis suggests that this polymorphism may be a mechanism for immune evasion developed by infecting plasmodia [22].

In a previous study we had detected circulating CD8+ T cells specific for peptide Pf CS 7G8 334–342 (YLKKIKNSL) in individuals living in a malaria-endemic area of Burkina Faso [21]. In a more recent study, we detected CD8+ T lymphocytes specific for a highly homologous sequence found in the NF54 isolate (Pf CS 327–335 YLNKIQNSL) in 23.5% of the 17 HLA-A*0201 donors living in the same area [25]. The frequency of Pf CS 327–335-specific CD8+ T cell precursors detected after one cycle of in vitro peptide stimulation was rather low for most of the donors with the exception of donor BF7. To assess the frequency of Pf CS 327–335-specific CD8+ T cell precursors in the peripheral blood of donor BF7, CD8+ T cells secreting IFN- + specifically in response to peptide Pf CS 327–335 were enumerated by ELISPOT as illustrated in Fig. 1. The calculated frequency of circulating Pf CS 327–335-specific CD8+ T cells for patient BF7 was 1:129 CD8+ T cells. Pf CS 327–335-specific CD8+ T cells were efficiently expanded by in vitro stimulation with synthetic peptide and their frequency increased to

Previous reports on malaria-specific immune responses in humans pointed out the low frequency of Plasmodium-specific CD8+ T lymphocytes in naturally exposed individuals [15, 23, 24]. As a consequence of the difficulty of isolating specific CD8+ T lymphocytes present at low frequencies, no stable malaria-specific CTL lines or clones had been generated before. We obtained similar results in a recent study in Burkina Faso. Low frequencies of CD8+ T cells specific for a panel of P. falciparumderived HLA-A2-binding peptides were observed in the majority of the individuals analyzed [25]. As an exception, a high frequency of CD8+ T lymphocytes specific for some of the analyzed peptides including peptide Pf CS 327–335 were detected in peptide-stimulated PBMC from one of these donors (BF7). In the present study, we have used monoclonal populations of specific CD8+ T lymphocytes isolated from PBMC of donor BF7 to study T cell recognition of natural variants of the antigenic sequence found in different isolates of P. falciparum. The results demonstrate that naturally occurring variations very often affect residues critical for T cell recognition. Specific CD8+ T lymphocyte clones were also used to analyze T cell recognition of the endogenously presented cognate antigen. Importantly, we demonstrated efficient recognition of human liver cells infected with recombinant vaccinia virus encoding the CS protein (WR-CS).

2.1 Isolation and characterization of peptide Pf CS 327–335-specific human CD8+ T lymphocyte clones

Fig. 1. IFN- + production measured by the ELISPOT technique. (A) Frequency of IFN- + production: ex vivo ELISPOT with PBMC from donor BF7 in duplicate (left side) or after one round of in vitro stimulation with peptide Pf CS 327–335 (right side). (B) Staining of specific CD8+ T lymphocytes with HLA A*0201/ Pf CS 327–335 tetramers. Lymphocytes derived from (HLA-A*0201) donor BF7 were stained with Pf CS 327–335-specific tetramer (PE labeled) and anti-CD8+ (FITC labeled) specific antibody, control (left); in a polyclonal culture after one round of in vitro stimulation with the nonapeptide Pf CS 327–335 (middle) or clone BF2–65 (right).

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1:3 CD8+ T cells after a single round of peptide stimulation (Fig. 1). These data were confirmed by staining cultures after the first stimulation with specific HLA-A*0201 tetramer as shown in Fig. 1. In this assay, over 90% cultured cells and 98% of clone cells were stained by the specific tetramer. Several specific monoclonal T lymphocyte populations were obtained by limiting dilution culture in the presence of irradiated allogeneic PBMC, PHA and cytokines as previously described [26]. The cell line (BF7) from wich the clones were obtained expressed preferentially V g 22 as well as all of the clones isolated. Fig. 2 illustrates the cytolytic activity of a representative clone (BF2–65) that was able to lyse T2 target cells in the presence but not in the absence of peptide Pf CS 327–335. Typically, 50%

Fig. 2. Recognition of peptides from various P. falciparum natural isolates and laboratory strains. (A) Lysis of T2 target cells in the presence or absence of exogenously added synthetic nonapeptides S1 to S9 was measured by 51Cr-release assay at different lymphocyte (clone BF2–65) to target cell ratios. (B) Relative antigenic activities of synthetic nonapeptides S1, S3 and S6 were assessed in the presence of increasing peptide concentrations with a lymphocyte to target cell ratio of 10:1. The amino acid sequences of nonapeptides S1 to S9 are presented in Table 1.

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maximal specific lysis was obtained at a 1:1 E:T cell ratio (Fig. 2A). In peptide titration experiments, 50% maximal specific lysis was obtained at a peptide concentration of approximately 0.1 nM (Fig. 2B).

2.2 Natural CS polymorphisms in P. falciparum isolates have a profound impact on recognition by Pf CS 327–335-specific CTL Peptide Pf CS 327–335 NF54 strain (YLNKIQNSL) is located within the polymorphic region Th2R of the CS protein. Eight additional variant sequences (listed in Table 1) have been identified from both natural isolates and laboratory strains of P. falciparum [20]. To assess the impact of natural polymorphism on antigen presentation and recognition, we first analyzed the capacity of each single variant peptide to bind to HLA-A*0201. Peptide binding was evaluated using functional assays based on peptide-induced HLA-A*0201 cell surface expression on the transporter associated with antigen processing (TAP)-deficient human cell line T2 as previously described [27]. As illustrated in Table 1, seven of the nine variant sequences tested were able to bind to HLA-A2 with an intermediate affinity (relative binding affinity 0.3-0.1) as compared to a high-affinity binder peptide from influenza matrix protein (Flu MA 58–66) used as an internal reference. The other two variant sequences (S2 and S4) were low-affinity binders (relative binding affinity X 0.03). The recognition of variant sequences by a specific CTL clone (BF2–65) was then evaluated in a chromiumrelease assay (Fig. 2A). The level of lysis obtained with the NF54-derived sequence was detected only with two other peptides (S3 and S6); the remaining sequences induced low levels of specific lysis (Table 1). Similar results were obtained with several other clones. Peptide titration experiments showed that peptides S3 and S6 elicited 50% of specific lysis at 0.5 nM and 5 nM, respectively (Fig. 2B). Peptide recognition was also evaluated by IFN- + ELISPOT that confirmed the results obtained in the chromium-release assay. Peptides S1, S3 and S6 induced high levels of specific IFN- + production, whereas peptides S2, S4, S5, S7, S8 and S9 induced only low levels of IFN- + production though clearly above the background levels observed in this assay (Fig. 3).

2.3 Peptide Pf CS 327–335-specific CTL efficiently recognize the cognate antigen endogenously presented by liver cells The ability of peptide Pf CS 327–335-specific CTL to recognize the cognate antigen endogenously presented by either transfected murine or human liver cells was evalu-

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Table 1. Sequences from CS protein of various P. falciparum isolates Sequence

Isolatesa)

No. of substitutions

HLA-A*0201 binding relative activity b)

Recognition % lysisc)

S1

YLNKIQNSL

NF54, 427

0

0.12

100

S2

YLKKIKNSI

7G8

3

X 0.03

9

S3

YLKKIQNSL

WEL, It2G1, T9–101, T4R

1

0.25

83

S4

YLKTIQNSL

LE5

2

0.03

0

S5

YLKTIKNSL

T9–98,366a,399,406,419

3

0.10

10

S6

YLQKIQNSL

366b

1

0.20

91

S7

YLQKIKNSL

406, 419

2

0.30

22

S8

YLNTIQNSL

427

1

0.20

4

S9

YLQKIRNSL

366c

2

0.28

14

1.00

NA

Code

Flu MA

GILGFVFTL

d)

NA

None

0

a) [20]. b) Relative activity referred to the binding of MA influenza virus peptide. c) Numbers represent the percent specific lysis at the highest E:T ratio tested (30:1) minus the percent specific lysis obtained in the absence of peptide. d) NA: Non applicable.

ated by assessing the specific IFN- + secretion induced by liver cells infected with WR-CS. To validate the use of murine cells as APC, we first assessed the ability of human CTL clones to lyse transfectant murine EL-4 HLA-A*0201/Kb cells. These cells were very efficiently lysed in the presence of peptide Pf CS 327–335 but not

in the absence of peptide. More importantly, human CTL clones did not lyse untransfected EL-4 cells even in presence of peptide (Fig. 4A). Peptide antigen presentation by EL-4 HLA-A*0201/Kb cells was also confirmed by IFN- + ELISPOT assay (Fig. 4B). Having verified the ability of human CTL to recognize specifically peptide antigen presented by HLA-A*0201/ Kb-positive murine target cells, we then assessed the ability of human CTL to recognize the naturally processed CS antigen. As illustrated in Fig. 5A, a significant level of IFN- + production was induced by liver cells from HLA-A*0201/Kb-transgenic mice infected with WR-CS as compared to wild-type WR. Similarly, human liver cells from HLA-A*0201 donors efficiently induced IFN- + production by Pf CS 327–335-specific CTL following infection with WR-CS but not with WR (Fig. 5B).

3 Discussion

Fig. 3. IFN- + production measured by the ELISPOT technique. Recognition of nonapeptides S1 to S9 from various P. falciparum natural isolates and laboratory strains presented by T2 cells to CTL clone BF2–65. T2 cells were pulsed with or without synthetic peptides.

In this study we report the generation and functional characterization of monoclonal CD8+ human T lymphocyte clones specific for the 337–335 peptide from the CS protein of P. falciparum derived from a donor presenting an exceptionally high frequency of peptide-specific circulating CD8+ lymphocytes. Indeed, whereas high fre-

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Fig. 4. MHC class I restriction of the clone BF2–65. EL4 HLA-A*0201/Kb and EL4 and human T2 target cells pulsed with or without Pf CS 327–335 peptide were incubated with clone BF2–65. Antigen recognition was evaluated by 51Crrelease assay (A) or ELISPOT (B).

quencies of antigen-specific cells have been reported in response to viruses (influenza, EBV, HIV) [28–30] and, in certain cases, tumors [31], low frequencies of Plasmodium-specific CD8+ T lymphocytes have been thus far detected in naturally exposed individuals [23, 24]. These low responses could be related to the limited persistence of liver-stage antigen expression in vivo (5-9 days for P. falciparum). This hypothesis is supported by the observation that donors immunized with irradiated sporozoites (which may remain in the liver for up to 6 months) develop higher levels of parasite-specific CD8+ T lymphocyte responses. In addition, sterile immunity (which is achieved by this form of immunization but in general not in natural infections) is rapidly and completely abolished after parasite clearance by primaquine treatment in the mouse model [32]. The results reported here show that high levels of circulating CD8+ T lymphocytes directed against a single antigenic determinant from the P-ES can occasionally be found in a natural setting, possibly as a consequence of a massive antigen load and/or a vigorous expansion of a limited number of precursor T cells. Although it is difficult

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Fig. 5. Recognition of cognate antigen endogenously processed and presented by liver cells. Liver cells were infected with vaccinia virus containing P. falciparum CS protein (WRCS) or wild type (WR). IFN- + produced by clone BFA-13 was evaluated by ELISPOT upon incubation with HLA-A*0201/Kb murine liver cells and EL4 A*0201/Kb cells (A) or human HLA A*0201 liver cells (B).

to ascertain if the presence of high levels of circulating parasite-specific CD8+ T cells result in sterile immunity in this particular donor, the relevance of Pf CS 327–335specific CD8+ T lymphocytes in protection is suggested by two findings. First, Pf CS 327–335-specific CD8+ T cells recognized the antigen with high avidity, as demonstrated by the recognition of peptide at 10–11M and by the recognition of recombinant virus-infected target cells. Second, ex vivo ELISPOT analysis showed that Pf CS 327–335-specific CD8+ T cells were able to efficiently produce IFN- + , a cytokine which, together with TNF- § , has been shown to be crucial in CD8+ T lymphocyte immunity to malaria, mediating the elimination of intracellular parasites through the stimulation of NO production [16, 33–35]. The high “in vitro” growth potential of Pf CS 327–335specific CD8+ T lymphocytes suggests that they could be memory cells. Indeed, whereas pathogen-specific naive precursors are, in general, undetectable ex vivo, terminally differentiated effector cells exhibit very limited growth potential. While this point could not be established in the present study the recent development of

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HLA-A*0201 tetrameric complexes containing antigenic peptides could allow, in future studies, the accurate phenotyping of Plasmodium-specific lymphocytes present in the blood of exposed individuals. Pathogens can attempt to evade immune recognition by expressing mutated antigens. This phenomenon is particularly pronounced in the case of immune responses to immunodominant epitopes. Epitope variation can result in the lack of antigen recognition (escape) or in the induction of altered responses that do not eliminate the pathogen. In previous studies, polymorphism in an HLAB35 CTL epitope located in the C-terminal region of the CS protein has been shown to negatively influence T cell recognition [22]. Some of the amino acid variations affected HLA binding, whereas others more directly affected TCR contact residues by generating antagonistic peptides [36, 37]. Similarly, in the present study, we could show that Pf CS 327–335-specific CD8+ T lymphocytes fully crossrecognized only two out of eight different variants from both natural isolates and laboratory strains of P. falciparum. Although two of the poorly cross-reactive variants exhibited decreased binding affinity for HLA-A*0201, the lack of recognition could not be simply explained by decreased binding since CD8+ T cell response to one of the variants (7G8, peptide S2) was clearly detected in previous studies in human donors [21, 25]. CD8+ T cell-mediated protection against malaria implies that Plasmodium-specific T lymphocytes recognize the cognate antigen presented on the surface of infected hepatocytes in the context of the appropriate HLA class I molecule. Thus, it was important to assess whether peptide Pf CS 327–335-specific CTL could recognize the cognate antigen endogenously processed and presented by liver cells. Previous reports in the literature have shown that HLA-A*0201-restricted human CTL can recognize antigens presented by APC bearing HLAA*0201/Kb chimeric molecules [38]. We confirmed these results and showed that malaria-specific CD8+ T lymphocyte clones recognize endogenous antigens expressed in liver cells. This finding is in line with previous observations demonstrating antigen presentation of malaria antigens by liver cells to CD8+ T lymphocytes in a murine model [16]. More importantly, in this report, we demonstrate for the first time that human liver cells present endogenously processed antigens to malariaspecific human CD8+ T lymphocytes.

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several distinct antigenic determinants present in a polymorphic region. While the latter point is likely to represent a considerable hurdle to overcome, it might be achieved by a careful assessment of the binding to the HLA class I molecule of the natural and experimental variants.

4 Materials and methods 4.1 Peptides Peptides were synthesized by standard solid-phase F-moc chemistry. Purity, as assessed by analytical HPLC and mass spectrometry, was over 90%. Peptide stock solutions (10 mg/ml) were prepared in DMSO and stored at –20°C.

4.2 HLA-A*0201-peptide tetramers HLA-A*0201-CS tetramers were produced as previously described [39]. Briefly, recombinant HLA-A*0201 fused to a C-terminal BirA biotinylation sequence (plasmid provided by Dr. M. M. Davis) and g 2-microglobulin ( g 2 m) chain (plasmid provided by Dr. T. Jardetzky) were expressed in Escherichia coli and isolated from inclusion bodies. HLA-A*0201 chain, g 2 m and peptide were refolded by dilution [40]. The HLAA*0201/Pf CS 327–335/ g 2 m complexes were then enzymatically biotinylated with BirA (Avidity, Denver, CO) at room temperature in the presence of biotin (Sigma, St Louis, MO), ATP (Sigma, St. Louis, MO), Mg2+ (Sigma, St. Louis, MO), and protease inhibitors. Biotinylated monomeric complexes were purified by fast protein liquid chromatography on an ion exchange column and tetramerized by stepwise addition of streptavidin-PE conjugate (Sigma, St. Louis, MO) to a 1:4 molar ratio.

4.3 Flow cytometry analysis Polyclonal cultures of PBMC from donor BF7 were stained after one round of in vitro stimulation with peptide Pf CS 327–335. In addition, established clones were evaluated. Cells were incubated with 0.3 ? g of tetramers in 20 ? l PBS, 5% FCS and 0.2% azide for 1 h at room temperature. Human CD8+-specific FITC-labeled antibody (Becton Dickinson, San Jose, CA) was added and cells were further incubated for 20 min at 4°C. Immediately after washing, cells were analyzed by flow cytometry (FACSCalibur®, Becton Dickinson, San Jose, CA). Data acquisition and analysis were performed using CellQuest software.

4.4 HLA-A*0201/peptide binding assay

Overall, these results underscore the importance of conserved, and possibly subdominant, T cell epitopes as essential components of malaria vaccines or design vaccines aimed to elicit CTL responses directed against

Peptide binding was assessed by measuring the induction of HLA-A*0201 antigen cell surface expression on the TAPdeficient T2 human cell line after overnight incubation with

Eur. J. Immunol. 2000. 30: 3079–3088 exogenous peptides. T2 cells were incubated overnight at room temperature in serum-free medium (X-VIVO 10, Biowhittaker, Walkersville, MD) containing 2 ? g/ml g 2 m (Sigma, St. Louis, MO) in the presence of serial peptide dilutions. After washing, the cells were stained for HLA-A*0201 expression by using FITC-labeled mAb BB7.2 (10 ? g/ml) for 30 min at 4°C and analyzed by flow cytometry (FACScan II, Becton Dickinson, San Jose, CA). The concentration of peptide required to obtain 50% of maximal increase in HLAA*0201 antigen expression was determined from titration curves and normalized to that of the reference Flu MA 58–66 peptide as described before [41].

4.5 TCR typing TCR typing was performed as previously described [42]. Briefly, total RNA was purified from 1×106-2×106 T cells using the RNeasy kits from Quiagen (Qiagen, Hilden, Germany). E. coli rRNA (2 ? g) was added to each sample as carrier. RNA was eluted with 45 ? l water and lyophilized. Firststrand cDNA synthesis was performed with 0.5 mM dNTP, 250 ng oligo-dT and 100 U reverse transcriptase (Life Technologies, Basel, Switzerland) in a final volume of 15 ? l. The samples were incubated for 30 min at 37°C, 30 min at 42°C and denatured for 5 min at 95°C. PCR amplification of the cDNA was performed using a radiolabeled constant primer and one primer corresponding to the variable region of the TCR g -chain. Eight microliters of the PCR samples were mixed with an equal volume of formamide/dye loading buffer, heated at 90°C for 2 min and separated on 6% polyacrylamide/urea gels for 4 h. The gels were dried and exposed with one intensifying screen for 3-12 h.

4.6 Cells and tissues The T2 human cell line and mouse EL-4 (H2b thymoma) cells were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS. EL-4 cells transfected with the HLA-A*0201/Kb gene (kindly provided by Dr. Linda Sherman, La Jolla, CA) were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS and G148. Liver cells from HLA-A*0201/Kb-transgenic mice [43] were isolated by perfusion of liver fragments with collagenase and purified on a 60% Percoll gradient following a described protocol [44]. Sixty thousand cells were plated in Lab-Tek chamber slides (Nalgene Nunc International, Naperville, IL) and cultured in hepatocyte attachment medium (Life Technologies, Gaithersburg, MD) containing 10% FCS, 100 ? g/ml penicillin/streptomycin and 2 mM L-glutamine for 48 h at 37°C and 5% CO2. Human liver cells were obtained from informed consenting HLA-A*0201 patients undergoing segmental hepatectomies for liver tumors at the Hopital ˆ Cantonal de Geneve, ´ Clinique et Policlinique de Chirugie Digestive, Geneva, Switzerland. The protocol for the isolation of liver cells has been described elsewhere [45]. Briefly, surgical specimens were perfused under controlled pressure

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with calcium-free medium followed by collagenase infusion. The material recovered from enzymatic disaggregation was washed 3 times. Liver cells (2×104/well) were diluted in RPMI containing 5% FCS and 100 ? g/ml penicillin/streptomycin and distributed in 96-well flat-bottom plates. For liver cell recognition experiments, HLA-A*0201/Kb-transgenic murine or human liver cells were either pulsed with the indicated peptide, or infected with wild-type vaccinia virus (WR) or with a recombinant vaccinia virus encoding the P. falciparum CS protein, gene NF54/3D7 clone (WR-CS) at 5 PFU/cell for 90 min at 37°C. WR and WR-CS were kindly provided by Dr. J. A. Tine, Virogenetics, Troy, New York. After viral infection, liver cells were washed thoroughly (5 times) with DMEM (Life Technologies, Gaithersburg, MD) before addition of CD8+ lymphocytes.

4.7 Generation of Pf CS 327–335-specific CD8+ T lymphocyte clones CD8+ T lymphocyte clones specific for peptide Pf CS 327–335 were obtained from PBMC of the HLA-A*0201 donor BF7, a 32-year-old woman living in Burkina Faso. Infection with P. falciparum is endemic but seasonal in Burkina Faso with a peak of transmission occurring between August and September; samples for the experiments presented here were collected in March 1998 [25]. The donor was free of parasites at the time of the blood collection, as assessed by analysis of Giemsa-stained thick blood smears. CD8+ T lymphocytes were positively selected from total PBMC by magnetic cell sorting (MiniMACS System, Miltenyi Biothech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. CD8+ cells were distributed in 48-well plates together with 106 irradiated autologous PBMC pulsed with Pf CS 327–335 peptide. Cultures were incubated for 10 days at 37°C and 5% CO2 in DMEM (Life Technologies, Gaithersburg, MD) containing 10% human AB serum, 10 mM Hepes, 10 mM pyruvate and 0.1 mM non-essential amino acids (complete medium, CM), supplemented with 10 ng/ml human recombinant IL-7 (kindly provided by Dr. N. Vita, Sanofi Research, France). Human recombinant IL-2 (Glaxo-Welcome, Geneva, Switzerland) was added at 10 U/ml on day 3. IFN- + ELISPOT assay was used to assess peptide-specific activity, as previously described [25]. Monoclonal specific populations were generated from peptide-stimulated cultures by limiting dilution culture in the presence of irradiated allogeneic PBMC, PHA and recombinant human IL-2 as described elsewhere [33]. Isolated clones were further expanded by periodical restimulation (every 3–4 weeks) with irradiated allogeneic PBMC, PHA and recombinant human IL-2.

4.8 Chromium-release assay Antigen recognition was assessed using a chromiumrelease assay. Target cells were labeled with 51Cr (Nef Life

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Science Products, Belgium) in Tris-Dulbecco buffer supplemented with 0.2% BSA for 1 h at 37oC. Labeled target cells (1000 cells/well) were then added to varying numbers of effector CD8+ T lymphocytes in V-bottom 96-well microplates in DMEM/5% FCS either in presence or absence of 1 ? M of the peptide. In peptide titration experiments target cells (1000 cells/well) were incubated in the presence of different concentrations of peptide for 1 h before the addition of effector cells. 51Cr release was measured after 4 h culture at 37oC. The percentage lysis was calculated as 100 × (experimental release – spontaneous release)/ (total release – spontaneous release).

4.9 IFN- q ELISPOT assay The IFN- + ELISPOT assay [46] was performed in nitrocellulose-lined 96-well microplates (Millipore MAHA S42, Bedford, MA) using an IFN- + ELISPOT kit (Mabtech Stockholm, Sweden) according to the manufacturer’s instructions. Plates were coated overnight with antibody to human IFN- + and washed 6 times prior to the addition of APC and responder cells. For ex vivo analysis of peptide Pf CS 327–335-specific T cells, highly enriched CD8+ T cells were added to the plates together with the autologous CD8– cell fraction prepulsed with the indicated peptide (1 ? M, in serum-free medium containing 2.5 ? g/ml g 2 m) for 1 h at 37°C. Peptide-stimulated CD8+ T cells or CTL clones were similarly tested using T2 cells (105/well) as APC. In liver cell recognition experiments, T cells were incubated with APC (liver cells) for 4 h before being added to ELISPOT plates. After incubation for 20 h at 37°C, cells were removed and plates were developed with a second (biotinylated) antibody to human IFN- + and streptavidin-alkaline phosphatase conjugate. Spots were counted using a stereomicroscope with a magnification of 15x.

Acknowledgments: We acknowledge the participation of the community of Goundry, Burkina Faso. We gratefully acknowledge N. Montandon for excellent technical assistance. Special thanks to Dr. N. Vita from Sanoffi Research, France, for providing recombinant IL-7, to Dr. J.A. Tine, Virogenetics, Troy, NY, for providing the wild-type and recombinant vaccinia virus, to K. Ellefsen, Drs. R. Sekaly and G. Pantaleo for providing the facilities for tetramer assembling and Dr. L. Renia for help in the hepathocyte cultures. We also thank F. Estevez for critically reading the manuscript. This work was, in part, supported by Swiss National Science Foundation. A. Bonelo was supported by the Instituto Colombiano Francisco Jose´ de Caldas para la Ciencia y la Tecnolog´ıa (COLCIENCIAS) and World Health Organization, Special program for Research and Training in Tropical Diseases (TDR). P. Champagne is supported by a Medical Research Council of Canada doctoral award. J.F. Romero is a recipient of a fellowship from the Swiss National Foundation (MHV No. 32-48977.96).

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Correspondence: Giampietro Corradin, Institute of Biochemistry, University of Lausanne, Ch. de Boveresses 155, CH-1066 Epalinges, Switzerland Fax: +41–21–692 57 05 e-mail: Giampietro.Corradin — ib.unil.ch J. A. Lopez’s ´ present address: Mater Medical Research Institute, Brisbane, Australia