Vol. 57, No. 10INFECTION AND IMMUNITY, OCt. 1989, p. 3181-31880019-9567/89/103181-08$02.00/0Copyright © 1989, American Society for Microbiology

Plasmodium falciparum Schizont Sonic Extracts SuppressLymphoproliferative Responses to Mitogens and

Antigens in Malaria-Immune AdultsELEANOR M. RILEY,* OUSMAN JOBE, MICHAEL BLACKMAN, HILTON C. WHITTLE,

AND BRIAN M. GREENWOODMedical Research Council Laboratories, P.O. Box 273, Fajara, Near Banjul, The Gambia

Received 27 October 1988/Accepted 22 June 1989

Celular immune responses to malaria antigens are suppressed during acute Plasmodium falciparuminfection, and evidence from both murine and human studies suggests that parasite-derived factors may bedirectly immunosuppressive. In this study we have shown that P. falciparum schizont sonic extract will suppressin vitro lymphoproliferative responses to purified malaria antigens and other soluble antigens. The degree ofsuppression appears to correlate with the level of the lymphoproliferative response to the schizont preparationand is correspondingly more marked in malaria-immune donors than in nonimmune individuals. The effect canbe transferred with primed mononuclear cells and is partially abrogated by removal of CD8' lymphocytes. Thesuppressive component of the schizont preparation is nondialyzable and partially heat labile and comigrateswith hemoglobin-derived proteins in the molecular mass range 10 to 20 kilodaltons.

Nonspecific immunosuppression is a well-recognized fea-ture of acute falciparum malaria infection in humans (20) andis associated with increased susceptibility to concomitantbacterial infections and reduced humoral immune responsesto vaccination (3, 10). Recent evidence suggests that there isalso specific suppression of T-cell-mediated responses tomalaria antigens during acute infection, but that T-cellresponses to many other antigens are not affected (2, 6, 11,14).Lymphocytotoxic antibody formation (21), increased fre-

quency of CD8+ T (suppressor) cells (19), and defectiveactivation of T helper cells (15, 17) have all been implicatedin specific suppression of T-cell reactivity, and serum frommalaria patients has been shown to suppress cell-mediatedimmune responses in vitro (11, 14). Circulating malariaantigens have been found in acutely ill and convalescentmalaria patients (22), and it has been suggested that thesecontribute to suppression of in vitro lymphoproliferation (1).We have shown previously (12) that lymphocytes of malaria-immune adults respond in an antigen-specific manner topurified soluble malaria antigens but that crude Plasmodiumfalciparum schizont sonic extract is nonspecifically mitoge-nic for immune and nonimmune cells. To determine whetherthis mitogenic preparation is also immunosuppressive, wehave investigated the effects of adding a sonicated schizontpreparation to mononuclear cell cultures in an in vitrolymphoproliferation assay.

MATERIALS AND METHODSSubjects. Venous blood samples were collected from 22

malaria-immune adults living in a rural Gambian villagewhere transmission of falciparum malaria is intense andseasonal (4). Samples were collected at the end of the dryseason, when malaria transmission is low. All subjects wereaparasitemic at the time of sampling. Each individual wasbled on at least two occasions. Control samples were ob-tained from six nonimmune Europeans who had been livingin malaria-endemic countries for less than 3 years, who had

* Corresponding author.

been taking regular proguanil chemoprophylaxis, and whohad not experienced symptomatic malaria infection.Lymphocyte cultures. Blood (20 ml) was diluted in heparin-

ized RPMI 1640 (Flow Laboratories, Irvine, United King-dom), and mononuclear cells were isolated by buoyant-density centrifugation (Lymphoprep, Nygaard, Norway) asdescribed previously (11). Cells (2 x 105), in 150 ,ul ofcomplete tissue culture medium containing 10% nonimmune(European) human serum, were placed in each well of around-bottom microdilution plate (Linbro Chemical Co.,New Haven, Conn.). Then 20 ,ul of schizont sonic extractand 20 pd of mitogen or purified soluble antigen were addedto triplicate wells. Control wells received 20 1±l of culturemedium or 20 ,ul of uninfected erythrocyte antigen. Plateswere incubated at 37°C in 5% CO2 for 5 days, and then thesamples were radiolabeled; harvesting took place 18 h later.Cellular incorporation of [3H]thymidine was measured byliquid scintillation counting.

For preincubation of cells with schizont antigen, 1.5 x 106cells were suspended in 1 ml of culture medium and 100 ,ul ofschizont sonic extract or uninfected erythrocyte sonic ex-tract was added. After 2 days, cells were washed three timesin medium and 103 preincubated cells were added to eachwell of an autologous cell suspension, together with 20 ,ul ofantigen or mitogen. The plates were then incubated foranother 6 days before harvesting was carried out. Cell- andantigen-free supernatants were prepared by incubatingwashed, preincubated cells in fresh culture medium for 24 h.

Depletion of CD8 cells. CD8-positive cells were removedfrom the mixed mononuclear cell preparation by rosettingwith antibody-coated bovine erythrocytes (9). Briefly,mononuclear cells were incubated for 45 m with mousemonoclonal anti-CD8 antibody (OKT8; Ortho Diagnostics,Inc., Raritan, N.J.), washed thoroughly, and combined withgoat anti-mouse immunoglobulin-coated bovine erythro-cytes. After incubation for 45 min on ice, the cell pellet wasresuspended in fresh culture medium, and CD8+ rosettedcells were separated from CD8- nonrosetted cells by Lym-phoprep centrifugation. The CD8-depleted cell fraction washarvested from the interface. The efficiency of CD8+ deple-

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FIG. 1. Lymphoproliferative response to schizont sonic extract and control uninfected erythrocyte sonic extract. Each point representsthe mean counts in triplicate wells for each individual. Horizontal bars represent mean counts for each group. Symbols: 0, immune donors;0, nonimmune donors. Numbers along the bottom represent the amount of schizont sonic extract added: 0, none (i.e., background countsof unstimulated wells); 10, 10 ±g/ml added; 50, 50 ,ug/ml added; U, uninfected erythrocyte antigen (50 ,ug/ml) added. *, Insufficient cells wereavailable from three individuals for testing with uninfected sonic extract.

tion was checked by immunofluorescent staining of the cellfractions, and the percentage of CD8+ cells was typicallyreduced from 25 to 30% in the undepleted preparation toapproximately 3 to 4% in the depleted preparation. Theproportion ofCD8+ cells in the rosetted fraction ranged from81 to 93%.

Antigens and mitogens. Phytohemagglutinin (PHA) (DifcoLaboratories, Detroit, Mich.) was added to cells at a finalconcentration of 12 ,ug/ml. Candida albicans extract (10%,wt/vol; Hollister Stier, Elkhart, Ind.) was used at a finaldilution of 1:80. Purified soluble malaria antigen, affinitypurified against polyclonal immune serum from a continuousculture of a Tanzanian isolate of P. falciparum (7), was a giftfrom P. H. Jakobsen, Statens Seruminstitut, Copenhagen,Denmark, and was used at a final concentration of 37.5,ug/ml. This antigen preparation consists of at least sevenseparate soluble exoantigens (molecular masses rangingfrom 30 to 300 kilodaltons). The preparation has previouslybeen shown to induce proliferation of cells from immuneindividuals but not of cells from nonimmune individuals (12).Crude schizont antigen was obtained from a continuousculture of a Gambian isolate (G372) of P. falciparum. Sch-izont enrichment was performed by Plasmagel (LaboratoireRoger Bellon, Nevilly Sur Seine, France) sedimentation, andthe enriched fraction was washed three times in serum-freeparasite culture medium, sonicated for 30 s in a PG100Ultrasonic disintegrator at 150 W (MSE, Crawley, UnitedKingdom), and stored at -20°C. The sonic extract was usedat a final protein concentration of either 50 or 10 ,ug/ml. A

similar preparation of uninfected erythrocytes was used as acontrol.Column fractionation of schizont preparation. Schizont

sonic extract was centrifuged at high speed to remove anyprecipitated components, and the supernatant (with 10%sucrose added) was run over a Sephacryl S-300 SF column(Pharmacia, Uppsala, Sweden) in phosphate-buffered sa-line-0.5% fetal bovine serum at a speed of 5 ml/h. Thirty10-min fractions were collected, and every four consecutivefractions were pooled and sterilized by filtration through afilter (pore size, 0.22 ,um). The pooled fractions were com-pared with crude sonic extract, sonic extract supernatant,and sonic extract precipitate in standard proliferation as-says. The molecular weights of the pooled fractions wereestimated by protein electrophoresis.

Analysis of data. Responses are expressed either as thou-sands of counts per minute (kcpm) or as Akcpm, whichrepresents the counts in the wells with antigen minus thecounts in the control wells without antigen. The effect ofadding schizont sonic extract to the culture is expressed asthe counts in the wells with antigen and sonic extract minusthe counts in the wells with antigen only. Differences be-tween results for wells with and without schizont sonicextract are analyzed by a paired t test.

RESULTS

Cells from a group of 14 immune donors were used inexperiments 1 to Sa, inclusive. Cells from two of these

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FIG. 2. Effect of schizont sonic extract on the lymphoproliferative response to PHA. Akcpm represents [(kcpm PHA + schizont) minus(kcpm schizont)]. Each point represents the mean counts in triplicate wells for each individual. Horizontal bars represent the mean countsfor each group. Symbols: 0, immune donors; 0, nonimmune donors. Numbers along the bottom represent the amount of schizont sonicextract added: 0, none (PHA alone); background values are 1.6 + 0.9 kcpm in immune donor cells and 2.2 ± 0.6 kcpm in nonimmune donorcells); 10, 10 ,ug/ml added to PHA; 50, 50 ,ug/ml added to PHA; U, PHA plus uninfected erythrocyte antigen (50 ,ug/ml). *, Insufficient cellswere available from two donors for testing with uninfected sonic extract.

subjects and from eight new subjects were used in experi-ment Sb.

(i) Lymphoproliferative response to schizont sonic extract(experiment 1). At a concentration of 50 ,ug/ml, the schizontsonic extract causes significant proliferation of cells fromboth immune and nonimmune donors (P <0.005) (Fig. 1). Ata concentration of 10 ,ug/ml, the sonic extract inducesproliferation of cells from some immune donors but not ofcells from nonimmune controls. The control sonic extract ofuninfected erythrocytes did not cause proliferation of cellsfrom either group of donors, regardless of whether the highor the low concentration was used.

(ii) Effects of adding schizont sonic extract to antigen- ormitogen-stimulated lymphocyte cultures (experiment 2). Ex-periment 2a measured the response to PHA. The effect ofadding schizont sonic extract to PHA-stimulated cells wasstudied by comparing the counts in the wells containing PHAand sonic extract with those in the wells containing PHAalone (Fig. 2). For cells from nonimmune donors, addition of50 ,ug of schizont sonic extract per ml (but not 10 ,ug/ml)significantly depressed the response to PHA (P < 0.01). Forcells from immune donors, addition of either 10 or 50 ,ug ofschizont sonic extract per ml significantly depressed theresponse to PHA (P < 0.025 and P < 0.005 respectively).The control sonic extract had no suppressive effect. Re-sponses to PHA alone were somewhat higher in malaria-immune African donors than in the nonimmune European

donors, but values for both groups were within the normalrange for 7-day PHA stimulations in our laboratory (E. M.Riley et al., unpublished data).Experiment 2b measured the response to C. albicans

antigen. For cells from the nonimmune donors, responses toC. albicans were significantly decreased by the addition of50 ,ug of schizont sonic extract per ml (P < 0.05); addition of10 ,ug of schizont sonic extract per ml also decreased theresponse to C. albicans antigen, but the effect was notstatistically significant (P > 0.05) (Fig. 3). The controluninfected-erythrocyte sonic extract had no effect on theresponse to C. albicans. For cells from the immune group,both concentrations of schizont antigen, but not the controlpreparation, markedly depressed the responses to C. albi-cans antigen (P < 0.005). This is not simply an effect ofadding excess antigen to the culture, since higher doses of C.albicans antigen or mixtures of C. albicans antigen and PHAdid not cause suppression of the response (data not shown).Experiment 2c measured the response to purified soluble

malaria antigen. Responses to the purified P. falciparumantigen were low in the nonimmune control cells, and thedifference between antigen-stimulated and unstimulatedcells was not significant (P > 0.1). However, cells from threeindividuals in this group did give minimal responses to theantigen, and these responses were completely abrogated byaddition of schizont antigen (Fig. 4). For the cells from theimmune group, responses to the purified P. falciparum

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FIG. 3. Effect of schizont sonic extract on the lymphoproliferative response to C. albicans antigen. Akcpm represents [(kcpm C. albicans+ schizont) minus (kcpm schizont)]. Each point represents the mean counts in triplicate wells for each individual. Horizontal bars representthe mean counts for each group. Background values are as in Fig. 2. Symbols: 0, immune donors; 0, nonimmune donors. Numbers alongthe bottom represent the amount of schizont sonic extract added: 0, none (C. albicans antigen alone); 10, 10 ,ug/ml added to C. albicansantigen; 50, 50 ,ug/ml added to C. albicans antigen; U, C. albicans antigen plus uninfected erythrocyte antigen (50 ,ug/ml). *, Insufficient cellswere available from one individual for testing with uninfected sonic extract.

antigen varied. However, addition of either concentration ofsonic extract strongly depressed these responses (P <0.005). Again, addition of the control sonic extract had nosuppressive effect.

(iii) Preincubation of cells with schizont sonic extract andtheir effects on antigen-stimulated lymphocytes (experiment3). To investigate the possibility that suppression was cellmediated, we investigated the effects of preincubating cellswith schizont sonic extract (or a control antigen from unin-fected erythrocytes) and adding these cells to autologouscultures. We added 103 preincubated cells to 2 x 105autologous cells and assessed the responses to C. albicans ormalaria antigens after 6 days. Cells from eight immune

donors were used, and responses were analyzed by a pairedt test (Fig. 5). The proliferative responses to both C. albicansand purified malaria antigens were significantly decreased bythe addition of cells preincubated with schizont sonic extract(P < 0.025). Cells preincubated with control erythrocytesonic extract had no effect; antigen responses in thesecultures were not significantly different from those in cul-tures in which cells were preincubated in medium alone or inwhich no preincubated cells were added (data not shown).Supernatants from the same preincubated cells were alsotested for suppressive activity as follows: 100 p1l of cell- andantigen-free supernatant from washed, preincubated cellswas added to 2 x 105 autologous cells, and the proliferative

TABLE 1. Effect of removing CD8+ cells from the cell population prior to culture with schizont antigen

CD8+ cells present CD8+ cells depleted

Antigen Mean Akcpm + SEM' Mean Akcpm + SEM'Control +Schizont Suppression Control +Schizont Suppression

(erythrocytes) antigen (erythrocytes) antigen

PHA 33.3 + 3.0 15.9 + 4.7 47.7 46.8 ± 7.3 23.9 ± 2.7 48.9C. albicans 27.3 ± 9.3 17.8 ± 7.6 34.8 22.6 ± 7.8 21.5 ± 6.7 4.8Malaria (purified) 26.3 ± 5.9 3.9 ± 2.9 85.1 18.4 ± 3.9 10.3 ± 5.1 44.0

a n = 6. Mean ± SEM background values in unstimulated wells are 3.9 ± 0.4 with CD8+ cells present and 3.5 ± 1.1 with CD8+ cells depleted.

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FIG. 4. Effect of schizont antigen on the lymphoproliferative response to purified P. falciparum soluble antigen. Akcpm represents [(kcpmpurified P. falciparum + schizont) minus (kcpm schizont alone)]. Each point represents the mean counts in triplicate wells for each individual.Horizontal bars represent the mean counts for each group. Background values are as in Fig. 2. Symbols: 0, immune donors; 0, nonimmunedonors. Numbers along the bottom represent the amount of schizont sonic extract added: 0, none (P. falciparum antigen alone); 10, 10 ,ug/mladded to P. falciparum antigen; 50, 50 ,ug/ml added to P. falciparum antigen; U, P. falciparum antigen plus uninfected erythrocyte antigen(50 ,ug/ml). *, Insufficient cells were available from two donors for testing with uninfected sonic extract.

response to purified antigen was assessed after 6 days. Therewas no evidence that such supematants were able to sup-press antigen-induced proliferation (data not shown).

(iv) Effects of removing CD8+ cells from the mononuclearcell population prior to antigen stimulation (experiment 4). Todetermine whether the cells involved in mediating suppres-sion were typical CD8+ (T suppressor) cells, we removedCD8+ cells from the cell preparations from malaria-immunedonors before setting up the lymphoproliferative assays.Removal of CD8+ cells made no overall difference to thedegree of suppression of responses to PHA, but suppressionof responses to purified soluble malaria antigen and, to alesser extent, to C. albicans antigen, was markedly reducedin the absence of CD8+ cells (Table 1).

(v) Preliminary characterization of the immunosuppressivecomponent of P. falciparum (experiment 5). In experiment 5a,

lymphoproliferative assays were repeated with cells from sixindividuals by using schizont sonic extract which had beendialyzed (dialysis membrane molecular mass cutoff, 12 to 14kDa) or heat treated (100°C for 5 min) instead of using theuntreated schizont sonic extract. The results are shown inTable 2. Dialysis of schizont antigen made no difference toits suppressive potential, but heat treatment of the antigendid partially abrogate both its mitogenic and suppressiveeffects.

In experiment 5b, Sephacryl S-300 column fractions weretested for their ability to induce lymphoproliferation and tosuppress PHA responses in cells from 10 individuals. Ultra-centrifugation of the sonic extract produced a highly sup-pressive supernatant and a somewhat less suppressive pre-cipitate (Table 3). Proliferative responses to most of thecolumn fractions were minimal, and when these fractions

TABLE 2. Effect of dialyzed and heat-treated schizont antigen on the lymphoproliferative responseto mitogens and other purified antigens

Mean Akcpm + SEM (% suppression)aAntigen

PHA C. albicans antigen Purified malaria antigen

None 14.4 ± 2.5 14.2 + 3.0 7.5 + 3.250 ,ug of schizont antigen/ml 5.8 ± 2.9 (59.9%) 7.5 ± 3.1 (47.1%) 0.1 ± 0.1 (98.7%)Heat-treated schizont antigen 9.2 ± 5.6 (35.9%) 10.0 ± 2.4 (29.8%) 2.9 ± 0.9 (61.3%)Dialyzed schizont antigen 6.6 ± 2.4 (53.9%) 6.6 t 2.2 (53.9%) 0.2 ± 0.2 (97.5%)

a Akcpm, Counts in stimulated wells minus background counts in unstimulated wells for cells from six immune donors. Background value (with no antigen)is 2.8 + 0.6.

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FIG. 5. Effect of adding cells preincubated with either sonicated, schizont-infected erythrocytes or with uninfected erythrocyte sonicextract to lymphoproliferation assays. Each point represents the mean counts in triplicate wells for each individual. (a) C. albicansantigen-stimulated cultures; (b) purified P. falciparum antigen-stimulated cultures. U, Cells preincubated with 10 ,ug of uninfected erythrocytesonic extract per ml; 10, cells preincubated with 10 ,ug of schizont sonic extract per ml. Akcpm is value for antigen-stimulated wells minusvalue for unstimulated control wells.

were added to PHA-stimulated cells, no suppression of thePHA response was observed. However, fractions 15 to 18(which contained most of the hemoglobin and other pig-ments) induced significant proliferative responses in cellsfrom some donors and markedly suppressed the response to

PHA (Table 3). The magnitude of the suppressive effect wassimilar to that seen with crude sonic extract or with sonicextract supernatant. Suppression of PHA responses byfractions 15 to 18 (or the crude schizont sonic extract)correlated with the ability of the cells from each individual to

TABLE 3. Effect of ultracentrifuged and column-fractionated schizont sonic extract on PHA-inducedlymphoproliferation for 10 individual donorsa

Mean kcpm after addition of ultracentrifuged sonic extract Mean kcpm after addition of fractionated' sonic extract

designation PHHAextract +Ae sonic extract sonic extract F15/18 alone PHA + F15/18 % Suppressionetactn supernatant precipitatealone alone sonic extractsueaat peiiaeby F15/18

AG 19.2 8.6 11.3 9.0 10.7 1.0 12.5 35.0SS 25.5 9.3 15.4 7.4 16.3 6.8 12.0 52.9DB 23.8 4.6 13.8 6.8 14.0 18.7 8.4 64.8SC 28.3 10.8 14.5 7.8 11.3 16.5 10.7 61.9YS 15.6 5.2 17.1 9.0 16.6 14.4 10.8 30.6BB 9.7 0.5 11.0 13.6 11.6 0.1 9.9 +2.6JJ 11.5 0.2 11.7 14.7 11.6 0.3 9.8 15.0JS 38.2 0.9 40.9 33.1 38.0 0.2 34.8 8.9MS 12.6 1.0 13.6 9.9 13.3 6.3 10.5 17.0FB 37.3 0.2 32.5 36.3 30.6 0.1 31.9 14.3

a Values are means of triplicate wells (SEM was less than 10%o). Mean + SEM background counts for unstimulated cultures, 2.02 + 0.69.bF15/18, Pool of fractions 15 through 18.

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recognize the fraction (or the sonic extract) in a directproliferation assay (Table 3).Sodium dodecyl sulfate-polyacrylamide gel electrophore-

sis analysis of fractions 15 to 18 (data not shown) revealedseveral distinct bands in the molecular mass range 10 to 20kDa, with a major band at 14 kDa, which probably repre-sents reduced hemoglobin-derived proteins.

DISCUSSION

Antigen-specific suppression of lymphoproliferative re-sponses to purified malaria antigens has been found in bothadults and children who are acutely infected with P. falci-parum malaria (6, 11, 14). In addition, serum or plasma frominfected or recently recovered individuals has been shown tosuppress responses to malaria antigens in both autologous(11) and homologous (16) systems. Since soluble malariaantigens are known to be present in the serum of malariapatients during infection and for some time after treatment(22), it is possible that a soluble parasite-derived substance isdirectly responsible for both these effects. Indeed, Ballet etal. were able to correlate the suppression of in vitro re-sponses to PHA with the presence of circulating malariaantigens (1).

In this study we have examined the immunosuppressiveactivity of a crude preparation of erythrocytes infected withmature P. falciparum schizonts. During a 7-day cultureperiod, the schizont sonic extract severely depressed lym-phoproliferative responses to malaria antigens, as well as(but less severely) responses to unrelated antigens andmitogens. Not only were these effects dependent upon thedose of schizont sonic extract used, but also they were moremarked in cells from malaria-immune donors than in cellsfrom nonimmune donors. The suppressive effect cannot beattributed to a direct toxic effect of the schizont preparation,since cells from many immune donors proliferated well whencultured with the schizont preparation alone. The suppres-sive effect was not seen with a control sonic extract ofuninfected erythrocytes.Minimal responses to purified malaria antigens were seen

in the cells from three of six nonimmune but malaria-exposed control donors, suggesting that these subjects mayhave experienced subclinical malaria infection while takingschizonticidal antimalarial drugs. Also, responses to purifiedmalaria antigens have been found previously in nonimmunedonors and may indicate recognition of cross-reacting anti-gens (5).

In the murine P. berghei system, a factor has been isolatedfrom infected erythrocytes which suppresses primary hu-moral immune responses to T-cell-dependent (but not T-cell-independent) antigens in vivo (8). This factor appears tobe a protein or glycoprotein of relatively low molecular mass(27 kDa). Theander et al. (16) describe a suppressive factorin human malaria serum with a molecular mass 30 to 100 kDawhich is heat labile at 56°C. Our data indicate that thesuppressive factor is soluble, nondialyzable, and partiallyheat sensitive. The suppressive factor comigrates with he-moglobin and hemoglobin-derived pigments and other para-site components in the molecular mass range of 10 to 20 kDa.Further work is now required to define the suppressivemolecule and determine whether it is entirely parasite de-rived or whether altered host components are also involved.

In murine studies, in vivo immunosuppression has beenattributed to T-suppressor-cell activity in some systems andto suppressor macrophages in others (reviewed in reference20). The mechanism of immunosuppression in human P.

falciparum malaria has not been elucidated, but the datapresented here suggest that activation of primed CD8+suppressor cells may be one mechanism. Suppression wasmore complete (with lower doses of schizont antigen) incultures from immune donors than in those from nonimmunedonors. Also, even in the cells from the malaria-immunedonors, the degree of suppression seemed to correlate withthe level of cellular proliferation induced by the schizontpreparation (Table 3). The suppressive effect could be par-tially transferred with prestimulated cells and was abrogatedby CD8+ cell depletion. It seems paradoxical that thefraction of the schizont preparation which is most active insuppressing proliferative responses to mitogens such asPHA is also the fraction which is most potent in activatingcells when presented alone. This raises the possibility thatfractions 15 to 18 may be acting on more than one subset ofcells, some of which proliferate while others (after a lagphase during which induction may occur) inhibit prolifera-tion, via some kind of negative feedback mechanism. If thisis so, the active component of fractions 15 to 18 may in factrepresent a parasite-derived immunoregulatory factor with arole in limiting the T-cell response to malaria antigens.

In the absence of definitive in vitro assays for estimationof clinical immunity to malaria, the extent to which lym-phoproliferation is representative of in vivo cell-mediatedimmunity is uncertain. However, suppression of in vitroresponses does appear to correlate with in vivo immunosup-pression in acute malaria infection (reviewed in reference18), and activation of T suppressor cells has been implicatedin the hyporesponsiveness to malaria antigens seen in para-sitemic individuals (14, 18, 19) and in the regulation ofcellular immune responses in malaria-immune adults (13;H. C. Whittle, K. Marsh, and J. Brown, Clin. Exp. Immu-nol. in press). Attempts to transfer suppression with cell-freesupematants of prestimulated cells were not successful,suggesting that if soluble, cell-derived suppressor factors areinvolved, either they are very labile or their concentration iscritical. Mediators such as prostaglandin, released fromactivated macrophages or other antigen-presenting cells,may also be involved, since we have demonstrated thataddition of indomethacin to cultures from acutely malaria-infected donors will enhance proliferative responses to pu-rified malaria antigens (E. M. Riley, C. MacLennan, D.Kwiatkowski, and B. M. Greenwood, Parasite Immunol., inpress).The ability of P. falciparum-derived antigens to depress

cellular immune responses in humans, both in vitro and invivo, has serious implications for the development of amalaria vaccine. Potential vaccine antigens should bescreened not only for their ability to induce specific antibodyformation but also for their effects on the cellular immunesystem. In particular, the effects of specific purified malariaantigens on the immune response to other, unrelated anti-gens should be investigated, both in vitro and in vivo.

ACKNOWLEDGMENTS

We thank the people of Brefet for their participation and cooper-ation in this study; Idrissa Sambou, Muhammed Singateh, LangBayo, and Lamin Kuyateh for their technical assistance; and KevinMarsh and Dominic Kwiatkowski for useful discussions and criti-cisms.

LITERATURE CITED1. Ballet, J. J., P. Druilhe, P. Brasseur, S. Looareesuwan, P.

Chantavanch, and S. Tharavanl. 1986. Influence of circulatingmalaria antigens on cell mediated immunity in acute Plasmo-

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