RESA to human T cells. Variations in responsiveness induced by antigen presenting cells from different but MHC class II identical donors

Immunology Letters, 43 (1994) 59-66 0165-2478/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved IMLET 2245

Presentation of the Plasmodium falciparum antigen Pf155/RESA to human T cells. Variations in responsiveness induced by antigen presenting cells from different but MHC class II identical donors Marita Troye-Blomberg a.., Jean-Paul Lepers b, Katarina Sj/Sberg a, Lucie Rahalimalala b, .~Lke Larsson a, Olle Olerup a and Peter Perlmann a a Department of Immunology, Stockholm University, 106 91 Stockholm, Sweden; b lnstitut Pasteur de Madagascar, B.P. 1274, Antananarivo 101, Madagascar; and c Center for BioTechnology, Karolinska Institute, Huddinge Hospital, Huddinge, Sweden (Received 30 June 1994; accepted 26 August 1994)

Key words: Plasmodium falciparum; Response variation; MHC class II different but identical donors

I. Summary The antibody response in humans naturally primed to a malaria vaccine candidate antigen (Pf155/RESA) is genetically regulated. Here, the impact of antigen presenting cells (APC) on the control of in vitro T-cell responses induced by Pf155/RESA or synthetic peptides corresponding to its major Pf155/RESA epitopes was studied. T cells and APC were from the peripheral blood of monozygotic or dizygotic twins and their age matched siblings, all living in the central highlands of Madagascar. When induced to proliferate (thymidine incorporation) in vitro by antigenic peptides, the T-cell responses varied less within the twin pairs than between them and their siblings or the entire group, implying that they were genetically regulated. Occasional MHC class II associations of some of the responses were weak and did not reflect underlying MHC class II restrictions. When T cells and APC from different but MHC class II identical donors were incubated in various combinations, antigen charged APC from homologous donors induced in vitro T-cell proliferation which differed from that induced by the T-cell donors' own APC. Pretreatment of the APC with either paraformaldehyde or anti-class II antibodies inhibited or abolished this antigen dependent T-cell proliferation. The results

* Corresponding author: Marita Troye-Blomberg, Department of Immunology, Stockholm University, S-106 91 Stockholm, Sweden. SSDI0165-2478(94)00163-4

suggest that the observed differences in T-cell responses induced by APC from different donors reflect differences at the level of these cells. Whether they reflect differences in the proteases involved in antigen processing, in the costimulatory signals provided by the APC to the T cells or in the secretion of other regulatory factors remains to be elucidated.

2. Introduction Efforts to develop malaria vaccines are presently focusing on subunit vaccines, i.e., vaccines in which the immunogens consist of selected parasite antigens or epitopes containing fragments of these. While subunit vaccines have been reported to be efficient [1-3] they are also afflicted with several problems which are not commonly encountered--at least not to the same extent --with vaccines based on attenuated or killed pathogens. Thus, genetic factors restricting host responses to foreign antigens may be of major importance. One potentially limiting factor with such immunogens is that they may only bind efficiently to a restricted number of HLA alleles and, therefore, may not induce proper responses in a substantial proportion of a target population (MHC-linked non-responsiveness) [4-6]. However, as immunity even to a "simple" subunit vaccine is multifactorial, unresponsiveness may reflect control at several different levels, involving genes operating independently or in concert with the MHC system [7]. We have previously investigated the natural antibody 59

response in malaria primed humans to defined epitopes of the Plasmodium falciparum antigen Pf155/RESA [8] considered to be a candidate for a vaccine against the asexual blood stages of this parasite [9]. In vitro studies using congenic mice have demonstrated that both T- and B-cell responses to defined epitopes of the Pf155/RESA antigens are restricted by the MHC class II system [6]. However, in human outbred populations no consistent MHC class 1I restrictions of anti-Pf155/ RESA peptide specific T- and B-cell responses have been seen [10,11]. This is not surprising in view of the extensive polymorphism of the MHC class 11 system which is twice as extensive in West Africans as in North European Caucasians [12]. However, in neither were there any obvious MHC class 11 restrictions seen when antibody responses to Pf155/RESA were measured in naturally primed monozygotic twins from Liberia and Madagascar [13]. Nevertheless, these studies showed that formation of IgG antibodies to Pf155/ RESA was genetically controlled, a control apparently reflecting the impact of factors encoded by genes outside the MHC class I1 region. In this paper we present the results of an investigation in which we have compared proliferative T-cell responses to defined Pf155/RESA epitopes in vitro in monozygotic twins with that of HLA class II identical individuals. Moreover, we have studied how the proliferative T-cell responses are affected by antigen presenting cells obtained from different but MHC class I1 identical donors.

3. Materials and Methods

3.1. Study subjects Venous blood samples were obtained from 60 donors (age, 2-35 years; 24 males and 36 females) living in three villages in the central highland of Madagascar. The donors comprised of 20 twin pairs of whom 14 were monozygotes, established by RFLP analysis as described previously [11,13]. Sex and age distribution of the twins were similar, with 11 of the pairs below 14 years of age. For each twin pair, one sibling of similar age (age difference < 4 years) was also included. P. falciparum exposure and housing were similar for the donors. Two groups of HLA class I1 (DR and DQ) identical individuals [13], as analysed by PCR amplification with sequence specific primers [12,13], were bled at a second occasion. All donors had anti-P, falciparum serum antibodies, when tested by conventional immunofluorescence. Approximately 40% had antiPf155/RESA antibodies as determined by erythrocyte 60

membrane immunofluorescence (EMIF) [8], ranging in titres from 1 / 5 0 to > 1/5000.

3.2. Preparation and fractionation of peripheral blood lymphocytes Thirty ml of venous blood were drawn into heparinized tubes. Mononuclear cells were isolated by gelatine sedimentation, carbonyl iron treatment, and FicollIsopaque (Pharmacia, Uppsala, Sweden) centrifugation [14]. The T cells were >_ 98% pure and free of surface immunoglobulin positive B-cells. Sheep erythrocytes attached to T lymphocytes were eliminated by osmotic lysis [15].

3.3. Adherent cells These were obtained from peripheral blood mononuclear cells by incubation for 1-2 h at 37°C in petri dishes in 50% heat-inactivated human AB + serum. Non-adherent cells were washed off and adherent cells were recovered after overnight incubation at 4°C. The adherent cell fraction was depleted of T cells by rosette formation as above.

3.4. Treatment of adherent cells (1) To prevent uptake in some experiments, adherent cells were treated for 5 min at room temperature with 1% paraformaldehyde in Hanks' balanced salt solution [16]. The paraformaldehyde was removed by washing the macrophages three times with Hanks' balanced salt solution. After the third wash, the macrophages were incubated in tissue culture medium at 37°C for 30 min. The cells were then washed three more times and were used without further treatments. (2) To investigate the effect of anti-class II antibodies on antigen presentation to the T cells a monoclonal antibody at a predetermined optimal concentration (5 /zg/ml) was added to the adherent cells for 2 h before addition of antigens and T ceils. The source of monoclonal antibody was ammonium sulphate precipitated and protein A purified culture supernatant from a hybridoma, denoted IVA 12, obtained from the American Type Culture Collection. This antibody recognises monomorphic structures on all MHC class 11 DR, DQ (and DP) /3-chains [17].

3.5. Cell cultures A total number of 5000 adherent cells was seeded into round-bottomed microtiter plates (Linbro Chemical Co., New Haven, CT) and prepulsed for 4 h with 0.2 ml of antigen (1 /xg/ml for r-Pf155/RESA, 1 /xM for the peptides and 10 /zg/ml for leucoagglutinin (La), re-

spectively) in triplicate wells in complete tissue culture medium (HEPES-buffered RPMI 1640, Biocult Laboratories, Paisley, UK), supplemented with 2 mM Lglutamine, 25 /.tg/ml gentamicin and 10% heat-inactivated human AB + serum. Antigen free medium controls were included in all experiments. Supernatants were removed and replaced with fresh tissue culture medium without antigen and 2 × 105 autologous or MHC class II identical T cells were then added to each well. Proliferation was assayed 5 days later by determining incorporation of 3H-thymidine (Radiochemical Centre; Amersham, UK; sp. act., 7.0-7.8 Ci/mmol; 1 /xCi/well for 16 h. Results were expressed as a stimulation index (SI), defined as mean c.p.m, of test cultures/ mean c.p.m, of control cultures. Mean incorporation in the absence of antigen in T cells was 666 cpm ( + 64 SEM; 95% confidence limits 535-796).

3.6. Antigens and peptides Recombinant and purified Pf155/RESA lacking approximately 30% of the N terminus of the natural protein was kindly provided by P. Schools and D. Pye, CSL Limited, Melbourne and R. Anders, WEHI, Victoria 3050, Australia. Peptides, 15-22 amino acids in length and representing repeated sequences of Pf155/ RESA [18,19] were used. Briefly, the protected resins were prepared in groups of 100 by the method of simultaneous multiple peptide synthesis [20]. Typical purities of the crude peptides ranged from 65-95%. All peptides were desalted before use and tested for cytotoxicity. None of the peptides used were toxic.

3.7. Serology A small aliquot of plasma was obtained from each sample and used for determination of P. falciparum antibodies by conventional immunofluorescence and for determination of anti-Pf155/RESA antibodies in erythrocyte membrane immunofluorescence [8]. Antipeptide antibodies were determined by ELISA as described [19].

3.8. HLA class H typing Genomic HLA class II (DR and DO) typing was performed on DNA from peripheral blood cells using restriction fragment length polymorphism (RFLP) analysis [12,21]. The restriction enzyme used for digestion of DNA was TaqI and the eDNA probes used for hybridisation were specific for DRB, DQA and DQB, respectively. Allelic TaqI DRB, DQA and DQB RFLP patterns are given in local Roman letter nomenclature.

The associated serologically defined DR and DQ specificities are also given in the text and Tables. In experiments in which MHC class II compatible APC and T cells from different donors were mixed, the identity of selected donors was ascertained by PCR amplification with sequence specific primers as described in [22,23].

3.~ Smtis~cal analys~ Statistical calculations were performed on loge-transformed SI values. The variance in the proliferative T-cell responses was calculated by using a program for one-way analysis of variance (ANOVA; Statgraphics, STCS, Rockville, MD, USA). Similarly, a monozygotic twins vs. siblings variance was calculated by comparing the means of the monozygotic twins with the value of the siblings. The possible impact of the number of HLA class II differences between monozygotic twin pairs and the corresponding sibling on the peptide specific proliferative T-cell responses was investigated by linear regression analysis. The number of haplotype differences (0, 1 or 2) was used as independent variable and the squareroot of the SD, calculated on mean logo(SI) of the twins and the log~(SI) of the sibling, was used as dependent variable for each set of twins/sibling.

4. Results

4.1. Variances of proliferative T-cell responses within and between Malagasy groups We have previously reported that antibody levels to intact Pf155/RESA and to some of its major B-cell epitopes in monozygotic twins are more concordant than those between the pairs or between the pairs and their age matched siblings or unrelated controls [13]. Similar results have now been obtained for proliferative T-cell responses induced by two synthetic peptides representing immunodominant amino acid sequences from either the central or the C-terminal repeat region of the antigen [24,25]. As seen from Table 1, the variation in the responses to either one of the two peptides increased with decreasing consanguinity of the cell donors. However, as the responses to the short peptides in these naturally primed individuals were low, larger donor materials would have been required to evaluate the significance of the differences in variation between the groups. Nevertheless, the differences in variation were statistically significant for peptide 1 when the monozygotic twins were compared with "all donors" (P < 0.01) and for peptide 2 when the twins were compared with their siblings (P < 0.025). Simi61

larly, of the small group of dizygotic twin pairs available (6) only three responded positively to the peptides, thus making statistical analysis impossible. No associations were seen between the proliferative T-cell responses and the donors' age.

4.2. Associations between Pf155 / RESA induced T-cell proliferation and MHC class II type In our previous studies we attempted to establish the MHC class II associations of P f 1 5 5 / R E S A peptide specific proliferative T-cell responses but no significant associations could be demonstrated [10,11] However, these studies were done with outbred populations, and the great MHC class II polymorphism could be one of the reasons for the lack of demonstrable MHC class II associations. To overcome this problem a similar analysis was performed with a group of genetically more homogenous Malagasy donors. As reported previously [13], among these donors two MHC class II haplotypes were found at elevated frequencies: thus, 7.5% were homozygous for the haplotype X X I V / X X I V - I I / I I - V / V (DRwl2/wl2-DQw7/w7) and 18% carried the haplotype I I / X X I V - I / I I - I / I I (Drwl5 / wl2-DQwl / w6). Linear regression analysis was performed with the square root of the differences in the proliferative responses to short synthetic peptides between monozygotic twin pairs and their siblings as dependent variable and the number of haplotype differences (0, 1 or 2) between them as independent variables. The peptides used correspond to three immunodominant T-cell epitopes of Pf155/RESA. No associations were found with the peptide (EENVEHDA) 3 and the cross-reacting peptide EENVEHDA(EENV) 2 representing immunodominant sequences from the C terminus [19]. However, there was a weak but statistically significant correlation ( P < 0.016) between the proliferative response to the peptide (DDEHVEEPTVA) 2, representing 2 copies of an immunodominant sequence from the central repeat block of P f 1 5 5 / R E S A and the MHC class II haplotypes expressing the allele XXIV (DRwl2).

4.3. Presentation of Pf155 /RESA to autologous or homologous T cells by MHC class II identicalantigen presenting cells (APC) The major regulatory factors of the antigen induced T-cell proliferation seen in these studies did not appear to reflect MHC class II restrictions. It is well established that environmental factors such as exposure could influence T-cell responses. In the present material no associations between the level of proliferative T-cell responses and age were observed (not listed). In other systems it has been shown that genes acting in APC such as macrophages can determine the immunological outcome of host-parasite interactions [26,27]. By taking advantage of the existence of the two groups of apparently identical MHC class II haplotypes present in our study population, a possible regulatory impact of APC on T-cell proliferation could be investigated. Therefore, blood was taken at a second occasion from 5 donors homozygous for the haplotype X X I V / X X I V - I I / I I - V / V (DRw12/w12-DQ7/DQ7) and 5 donors with the haplotype I I / X X I V - I / I I - I / I I ( DRwl 5 / wl2-DQwl / w6). An apparent MHC class II identity of the donors within these two groups was also ascertained by PCR [22,231 PBL were separated into adherent cells, used as APC, and highly purified T cells. These cells were mixed in various criss-cross combinations within the MHC class II identical groups. The APC were prepulsed with P f 1 5 5 / R E S A before addition of the T cells. Fig. 1 shows the typical results of an experiment in which APC from one donor (no. 2) were used to present antigen to his own or 4 other individuals' T cells. In parallel, the T cells of the latter were also stimulated with autologous APC. As can be seen, the APC from donor 2 induced proliferation both in his own T cells and in those from 3 other individuals who were low responders when antigen was presented by their own APC. Fig. 1 also shows that the T cells from the monozygotic twins reacted similarly when stimulated with the homologous APC from donor 2. This

TABLE 1 VARIANCES OF PROLIFERATIVET-CELL RESPONSES WITHINAND BETWEENMALAGASYDONOR GROUPS Peptide

pl: (DDEHVEEPTVA)2 p2: (EENVEHDA)3

(455-476) (893-916)

Within monozygotic twin pairs MS DF 0.149 14 0.170 13

Monozygotic twins vs. siblings MS DF 0.328 14 0.547 13

All donors MS 0.487 0.270

DF 61 57

Variance (MS, mean squares) in proliferativeT-cell responses within the groups. DF, degrees of freedom. Peptides pl and p2 corresponding to sequences in Pf155/RESA are given in one letter code. Nos in parenthesesgive position in Pf155/RESA polypeptide. 62

8

TABLE 2 INHIBITION OF ANTIGEN PRESENTATION BY PARAFORMALDEHYDE FIXED OR ANTI-CLASS II ANTIBODY PRETREATED ANTIGEN PRESENTING CELLS

SI 6

Donor

Antigen

Untreated

31

None r-Pf155 La

685

5.0 17.0

32

None r-Pf155 La

1257

4

None r-Pf155 La

311

2

None r-Pf155 La

170

cpm

4

2

Mz3

Mz4

Dz7

Dz8

Fig. 1. APC from one donor (no. 2) presenting antigen to MHC class II identical T cells. APC were prepulsed with rPf155/RESA (0.2 p,g in 200/zl) for 4 h before addition of purified T cells from MHC class II identical donors (Mz 3,4 = pair of monozygotic twins; Dz 7,8 = dizygotic). T-cell proliferation (thymidine incorporation) was measured after 5 days of incubation. Autologous A P C / T cell combinations were set up in the same way for all donors and run in parallel for comparison. SI = stimulation index. [] = homologous incubations; • = autologous incubations. Mean cpm and range were 493 (231-740) in homologous and 368 (270-489) in autologous incubation. The DRB-DQA-DQB haplotype of the donors was X X I V / X X I V - I I / I I - V / V (ORw12/ w12, BOw7~w7).

appeared not to be the case for the dizygotic twins but more data are needed to establish the significance of this. Similar results were obtained in the reverse experiments as exemplified in Fig. 2 where APC from 5 different donors were prepulsed with antigen and used

PFA-fixed

Anti-class II

cpm

SI

cpm

618

1.6 15.1

715

3.3 22.4

3.3 22.0

769

1.8 22.3

Nd

-

2.5 16.7

608

1.0 18.2

328

1.1 20.4

157

0.8 294.4

SI

Nd 5.8 233.2

-

SI

Adherent cells (APC) were either untreated, pretreated with 1% paraformaldehyde for 15 rain at room temperature or pretreated with a monomorphic anti-class II antibody for 2 h at 37°C before pulsing with antigen or with the mitogen leukoagglutinin (La) for 4 h at 37°C. Then the APC were incubated with 2 × 105 autologous T cells. SI = stimulation index. Nd = not done. (For further explanations see Materials and Methods).

to stimulate T cells from one of them (donor 31). There were no significant differences in the medium controls of either the aut01ogous or the homologous cell mixtures. As can be seen the APC from both donor 30 and 23 which only induced weak proliferation in their own T cells were more successful when mixed with the APC from the unrelated but MHC class II identical donor 31.

SI SI

6

8

6 4 . 31

30

Mz23

Mz24

32

Fig. 2. Antigen presentation to T cells from one donor (no. 31) by MHC class II identical APC. APC from 5 donors were prepulsed with rPf155/RESA before addition of T cells from one MHC class II identical donor (no. 31). Donors 30, 31, 32 were unrelated. Mz 23, 24 = pair of monozygotic twins. For other details see legend to Fig. 1. [] = homologous incubations; • = autologous incubations. Mean cpm and range were 800 (542-980) in homologous and 1047 (6611460) in autologous incubations. The DRB-DQA-DQB haplotype of the donors was I I / X X I V - I / I I - I / I I (DRwl5/w12, DQwl/w6).

pl

p2

p3

Fig. 3. Adherent cells were untreated = • or pretreated [] with 1% paraformaldehyde before pulsing with antigen. Then the APC were incubated with 2 × 10 5 autologous T cells; SI =stimulation index; pl = TVAEEHVEEPTVAEE; p2 = (EENVEHDA) 3 and p3 = EENVEHDA(EENV)2.

63

4.4. Inhibition of antigen processing and presentation To ascertain that antigen presentation in the present system was dependent on antigen uptake and presentation in association with MHC class II binding, the APC were either treated with paraformaldehyde or with rabbit anti-MHC class II antibodies before pulsing with antigen and incubation with autologous T cells. Paraformaldehyde prevents protein internalization by crosslinking surface molecules [16] while anti-class II antibodies inhibit the interaction of MHC class II molecules with the T-cell receptor [11]. As seen from Table 2, pretreatment of the APC in either way reduced or abolished antigen induced T-cell proliferation. The same results were obtained when pretreated APC were added to homologous but MHC class II identical T cells (not shown). In contrast, proliferation induced by the mitogen leucoagglutinin (La) was not reduced. Thus, T-cell proliferation induced by rPf155/RESA required antigen uptake, probably resulting in antigen processing

8 SI 6

and presentation to the T cells by MHC class II molecules on the APC.

4.5. Presentation of synthetic peptides While antigenic peptides usually need to be internalized and processed by APC in order to activate T cells, shorter peptides may be presented without uptake and intraceUular processing [28]. Peptide responses were usually seen in individuals who responded to the intact P f 1 5 5 / R E S A while the opposite was not always the case. In contrast to what was seen with the intact Pf155/RESA antigen (Table 2) treatment of peptide charged APC with paraformaldehyde gave no consistent inhibition of T-cell activation as shown for donor 32 in Fig. 3. When APC from this donors were charged with either one of two synthetic Pf155/RESA peptides and then incubated with their own T cells or with those from 3 unrelated but MHC class II identical donors, the APC induced significant T-cell responses in some of the homologous combinations even when the T-cells donors' own APC did not (Fig. 4). The same results were obtained with an additional peptide. No significant T-cell stimulation was obtained when peptide charged APC were incubated with anti-MHC class II antibodies (not shown).

5. Discussion

32

24

30

31

8-

SI

32

24

30

31

Fig. 4. APC from one donor (no. 32) presenting peptides to MHC class II identical T cells. APC were prepulsed for 4 h with either peptide TVAEEHVEEPTVAEE (Fig. 3a) or EENVEHDA(EENV) 2 (Fig. 3b) before addition of T cells from unrelated but MHC class II identical donors (nos. 24, 30, 31). For other details see Materials and Methods and legend to Fig. 1. [] = homologous incubations; • = autologous incubations. Mean c.p.m, and range were 1066 (842-1363) in homologous and 945 (666-1257) in autologous incubations. The DRB-DQA-DQB haplotype of the donors was I I / X X I V - I / I I - I / I I

(DQw15/w12, DQwl/ w6).

64

Previous studies of a large number of West Africans for their T-cell responses in vitro (proliferation and/or IFN-T release) to synthetic peptides representing T-cell epitopes of the malaria antigen Pf155/RESA gave no indication of any association between T-cell responses and the donors' HLA DR or DB alleles or DRB-DQADQB haplotypes [10,11]. Preliminary studies of a few twin pairs from Madagascar pointed in the same direction. To more precisely define the basis of the naturally acquired immune responses to malaria we have extended these investigations to include 14 monozygotic twin pairs and age matched siblings living under similar conditions of malaria transmission in Madagascar. Epidemiological details on these donors have been reported [29]. Induction of T-cell proliferation in vitro with antigenic Pf155/RESA peptides resulted in responses which varied less within the twin pairs than between them and their siblings or the entire group, implying that the intensity of the T-cell responses of these donors was genetically regulated as previously also found for their corresponding antibody responses [13]. Further analysis for possible MHC class II associations suggested the existence of a weak but significant correlation between proliferation and the number of shared

DRB-DQA-DQB haplotypes for only one of the three peptides investigated. The results are similar to earlier reports of occasional but weak associations between certain MHC class haplotypes and binding of antibodies to some malaria peptides [10]. The fact that the number of significant associations between immune responses and specific HLA class II haplotypes are few and weak suggests that other factors superimposed on HLA class II restriction are involved in the regulation of these peptide specific T- and B-cell responses. Genetic factors influence disease susceptibility to a wide range of infections [26] including malaria [30,31]. In the latter case, the importance of certain MHC genes for providing resistance to cerebral malaria has recently been demonstrated [31]. However, other genes residing both within- and without the MHC-region have also been shown to determine the immunological outcome of host-parasite interactions. Several of these genes act at the level of the antigen presenting cells [26,27] and may control antigen processing [32-34] or at the level of regulatory factors such as tumor necrosis factor (TNF). The levels of TNF-secretion have been shown to differ in MHC class II identical individuals with and without diabetes associated HLA haplotypes [35]. To investigate if APC could affect our malaria specific proliferative responses we used either autologous or homologous APC, taking advantage of the existence of two groups of apparently HLA class II identical individuals in the study population [13]. The importance of antigen uptake and presentation by APC for the outcome of the T-cell responses was proven by pretreating the former with paraformaldehyde [16] or anti-class II antibodies [11] which abolished antigen dependent but not mitogen dependent T-cell proliferation. Mixing of MHC compatible APC and T cells from different but malaria primed donors showed that the origin of the APC was an important factor in controlling the level of antigen induced T-cell proliferation in vitro. Thus, antigen charged APC from homologous donors sometimes also induced in vitro proliferation of T cells which responded weakly or not at all to their own antigen charged APC. Taken together, these results suggest that some of the differences in T-cell responses seen here reflect differences at the APC level. Similar findings have been reported by others and suggested to be due to differences in proteases involved in the processing of the antigens [36-38]. In the murine system, the importance of processing for determining the repertoire of antigenic peptides available for presentation by MHC class II molecules is well documented [39-43]. In humans, T-cell clones specific for distinct epitopes of tetanus toxoid have been shown to respond differently in vitro when activated by antigen pulsed APC from different

donors. The data were interpreted as reflecting differences in proteases required for processing and presentation of different epitopes [44]. The regulatory impact of APC on T-cell proliferation was seen both with the intact Pf155/RESA polypeptide and with short synthetic peptides used as stimulating antigens. In the latter case only treatment with anti-MHC class II antibodies but not with paraformaldehyde inhibited T-cell activation, in line with the fact that short peptides can be presented to the T-cells without intracellular processing by the APC [28]. However, the finding that the T cell responses to the synthetic peptides also varied greatly when the T cells were confronted with APC from different donors may imply that the antigen presenting activity of the latter cells varied even when intracellular processing did not seem to be involved. Thus, the synthetic peptides could yet have been modified by cell surface bound enzymes which affect antigen presentation in other systems [45,46]. However, since the APC from the different donors may differ in MHC class II synthesis [47], expression of molecules such as B7 delivering co-stimulatory signals required for T-cell activation [48] the interpretation of the results is presently difficult. Moreover, as the responding T cells in these donors are different mixtures of many clones varying in specificities the selection of which is influenced by MHC class II gene products [49], the composition of the T-cell pool will obviously have an essential impact on these in vitro responses. Proliferation of T cells following antigen activation in vitro is commonly used to evaluate cellular immunity associated with infection or vaccination. We have previously shown that proliferation as such is an incomplete measure of cellular sensitization to a given antigen as significant fractions of the T-cell population in any given donor may well respond to antigens by releasing lymphokines such as IFN-y or IL-4 but without proliferating, indicative of a TH1 and TH2 type of response in human malaria [50]. At present, it is not known whether or not lymphokine release from these T-cell subsets is also differentially affected by APC from different donors. In any event, the results presented here indicate that the activity of the APC constitutes an additional factor which has to be considered when assessing the extent of antigen responsiveness by measuring induction of T-cell activation in vitro.

Acknowledgements We thank Prof. Jean Roux (Directeur de l'Institut Pasteur de Madagascar) for providing excellent facilities and Marie Ange and the staff of the malaria unit for technical support. The generous participation of the 65

Malagasy blood donors, which has made this study possible, is gratefully acknowledged. This w o r k was supported b y grants from the U N D P / W H O Special P r o g r a m m e for Research and T r a i n i n g in Tropical Diseases, the Swedish A g e n c y for Research Cooperation with D e v e l o p i n g Countries (SAREC), the French Ministry of Cooperation and Development, the Rockefeller Foundation, the Swedish Medical Research C o u n c i l and the Swedish National Board for Laboratory A n i m a l s .

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