Eur. J. Immunol. 1990. 20: 307-315

K. Rebecca Jonesoo, Julian K. HicklingA, Geoffrey A. T. Targetto and John H. L. PlayfairO Department of Immunologyo, University College and Middlesex School of Medicine, Molecular Immunology LaboratoryA, Imperial Cancer Research Fund, Lincoln’s Inn Fields and Immunology Group, Department of Medical Parasitologyo, London School of Hygiene and Tropical Medicine, London

Nonimmune T cell response to I! falciparum

Polyclonal in vitro proliferative responses from nonimmune donors to Plasmodium falciparum malaria antigens require UCHLl+ (memory) T cells* The in vitro polyclonal proliferative responses of peripheral blood mononuclear cells to whole blood stage parasites or fractionated antigens from the human malaria parasite Plasmodium falciparum were studied. Cells from healthy laboratory donors who had never been exposed to malaria antigens in vivo consistently proliferated to l? falciparum antigens, as did cord blood mononuclear cells. This response was only observed in sheep rosette-positive cells in the presence of adherent cells and was inhibited by NH&I, indicating a requirement for antigen processing. The proliferative response was strongest at day 6 and was dependent on the presence of cells expressing high levels of CD45 180-kD isomer (UCHL1 monoclonal antibody), a marker for activated or memory cells, but not for CD45R (SN130 monoclonal antibody) a marker for naive or unprimed Tcells. This suggests a similarity to the recall response to tuberculin antigen. These results suggest that the proliferative response to malaria antigens observed previously and described as a nonspecific mitogenic response may be a cross-reactive response to epitopes shared between l? falciparum and other common immunogens. This would explain the establishment of T cell clones to malaria antigens from such donors, but might suggest that the epitopes to which such clones are specific may be of questionable protective or diagnostic use.

1 Introduction Analyzing the specificity of in vivo primed lymphocytes to malaria Ag may yield information useful in identifying protective Ag for vaccine development against this important disease of man. However, this strategy is complicated by the fact that lymphocytes from nonimmune individuals also proliferate in vitro to malaria Ag. Human PBMC and cord blood mononuclear cells (CBMC) have been shown to proliferate in response to infected monkey RBC [l], and monkey lymphocytes respond to Plasmodium falciparum-infected human cells [2]. In addition there are several reports of polyclonal nonimmune human PBMC responding to P falciparum-infected human RBC [3-5].The nature of these short-term polyclonal Tcell responses was not analyzed; the T cells may have been responding in an Ag-specific manner, or in response to T cell-specific mitogens similar to PHA, or the bacterial protein superantigen toxins such as staphylococcal enterotoxins A and B (SEA/SEB) or toxic shock syndrome toxin 1 (TSST-1) [6]. [I 77051

*

307

This work was supported by the Medical Research Council of Great Britain and the Imperial Cancer Research Fund.

Correspondence:K. Rebecca Jones, Immunologic Pharmaceutical Corporation, 855 CaliforniaAvenue, Palo Alto, CA 94304, USA Abbreviations: CBMC: Cord blood (mononuclear cells) DMF: Dimethyl formamide E+: Sheep red blood cell rosettepositive cells HIS: Hyperimmune serum NCP Nitrocellulose paper SEAISEB: Staphylococcal enterotoxins A or B 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

In addition, T cell clones have been generated to purified asexual stage, and sexual stage Ag from l? falciparum [7-91. These clones could be generated at a similar high frequency as those from immune, i.e. in vivo primed individuals.They produced IL 2, IFN y, and required MHC class 11-matched APC [S]. Synthetic peptides containing predicted T cell epitopes from l? falciparum antigens have also been used to generate Tcell clones [lo, 111. Again, clones were generated at a similar frequency from immune and nonimmune donors. The ability to establish long-term clonal responses from nonimmune individuals suggests that either T cells were being primed in vitro or, alternatively, Tcells specific for Ag cross-reactive with those present in l? falciparum were being expanded. Activated or memory humanTcells can be characterized by increased expression of LFA-3, CD2, LFA-1, CDw29 (mAb 4B4) and CD45 180-kD isomer (recognized by mAb UCHLl), expression of which has been shown t o increase 29-fold, making UCHLl suitable for the depletion of Tcells specific for recall Ag [12]. Indeed PBMC depleted of UCHLl+ cells show reduced responses to PPD or tetanus toxoid but maintained responses to PHA and alloreactivity 1131. The aim of this study was to determine whether the phenomenon of in vitro T cell proliferation from nonimmune subjects to €? falciparum Ag is due to (a) the presence of a nonspecific mitogen such as the lectin PHA, (b) a mitogenic response similar to that induced by the bacterial toxins which cross-link T cells and MHC in a nonspecific fashion [6], (c) an Ag-specific reaction induced by in vitro priming or 0014-2980/90/0202-0307$02.50/0

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K. R. Jones, J. K. Hickling, G. A. T. Brgett and J. H. L. Playfair

(d) an Ag-specific proliferation induced in Tcells primed in vivo to cross-reactive Ag between malaria and other unknown immunogens. Proliferation assays used to study the polyclonal response during the first week of activation established that the kinetics of the response resemble a response to specific Ag, rather than a nonspecific mitogenic response, and the dependence of the response on SRBC rosette-positive T cells in the presence of APC was NH4Cl sensitive. Furthermore the response was shown to require nonadherent cells expressing UCHLl [ 131, suggesting that the response seen is memory cell dependent and, therefore, probably due to cross-reacting epitopes on malaria Ag.

2 Materials and methods 2.1 l? fakiparum antigen preparation I! fakiparum blood stage parasites were cultured in vitro by a modification of the method of Trager and Jensen [14]. Briefly, NF54 strain parasites [15], an airport infection of probable West African origin, were grown in tissue culture flasks in FWMI 1640 (Flow Labs., Irvine, Scotland), with Hepes (Flow; 25 mM), sodium hydrogencarbonate (Sigma, St. 'Louis, MO; 0.25%), L-glutamine (Flow, 4 mM) and gentamycin (Gibco, Grand Island, NY, 50 pg/ml), supplemented with 10% heat-inactivated human A+ pooled serum (North London Blood Transfusion Service, London, GB), at a 2%-4% hematocrit in O+ human RBC washed free of leukocytes and anticoagulant (North London Blood Transfusion Service). A continuous flow apparatus was used for large-scale production of Ag, with constant replenishment and removal of gassed (British Oxygen Company, Harlow; GB 5% 02,5% C02,90% N2) medium at 37°C. Sterile parasite Ag were enriched from an 8% asynchronous parasitemia (with a < 0.1% gametocytemia). Following washing, the FU3C were lysed in 0.04% (w/v) white saponin (BDH, Poole, Dorset, GB), for 5 min, followed by three washes in serum-free FWMI 1640 (Gibco). Parasite Ag concentration was expressed as the concentration of free parasites as estimated using a hemocytometer. After four cycles of freezing and thawing in liquid nitrogen, parasites were stored at - 20 "C in RPMI 1640 at 1 x 109/mluntil use, and were active at stimulating PBMC for at least 6 months. Uninfected O+ and A+ RBC were treated in the same way to provide control Ag.

2.2 Antigens, mitogens and inhibitors The recall Ag PPD [16] was obtained from Evans Medical, Horsham, GB; 100O00 U/ml, and used at concentrations indicated. The mitogenic lectin PHA (Wellcome Diagnostics, Beckenham, GB) was used at 1 pg/ml. The superantigen SEB (Sigma) was used at 10 pg/ml and 100 ng/ml. To inhibit endosome-dependent antigen processing [171 PBMC were pretreated with 10 mM NH4CI (Sigma) for 2 h at 37 "C, then antigen or mitogen was added and the cells were incubated as for standard proliferation assays. The alternative lysosomotrophic agent chloroquine was unsuitable for such assays since exposure of PBMC to chloroquine (Sigma, at 1 m ~ for ) prolonged periods inhibited their proliferative response even to PHA. Whole asynchronous f? fakiparum parasites at 2%-5'%0 parasitemia were

Eur. J. Immunol. 1990. 20: 307-315

obtained direct from culture in O+ RBC, washed three times, counted and resuspended at the stated parasite density in the proliferation assays. 2.3 Preparation of PBMC

Venous blood (50-100 ml) from healthy laboratory volunteers (adults between the ages of 2540 years), or cord blood (University College Hospital, London), was collected onto preservative-free heparin (10 U/ml; CP Pharmaceuticals,Wrexham, GB) , diluted with an equal volume of RPMI 1640 and layered onto Histopaque (Sigma). Following centrifugation (400 x g, 22 "C for 30 min), the cells were washed, and resuspended at the required concentration in RPMI 1640, supplemented with L-glutamine and gentamycin as above, and with 10% autologous plasma. 2.4 Proliferation assays PBMC, CBMC, or other test cells in the presence of irradiated (2500 rad) adherent cells were incubated at 5 X 104-2 x lo5cells/well in round or flat-bottom 96-well plates (Nunclon), with the appropriate test Ag for 5 days (unless indicated otherwise) at 37 "C, 5% C02 in air. Wells were then pulsed for 18 h with [methyL3H]dThd (1 pCi/well; TRK 120 Amersham International, Amersham, GB). Cells were harvested onto glass fiber filters (Titertek, Flow Labs., Rockville, MD), using a semi-automatic harvester (Skatron Lier, Norway), and dThd incorporation determined using a liquid scintillation counter (Packard, Downers Grove, IL).

2.5 Fractionation of malaria Ag To prepare Ag for Tcell blotting [18, 191, pelleted parasite Ag was solubilized in NP40 (BDH) in the presence of protease inhibitors [20], and run on 5%-15% gradient SDS-PAGE (LKB, Rockville, MO; 2001 Vertical electrophoresis unit) under nonreducing conditions. The Ag were then electroblotted, using a semi-dry blotter (LKB 2117 Novablot), onto nitrocellulose paper (NCP; Schleicher and Schuell, Keene, NH; 0.45 pm). Unstained high molecular mass markers (200,116,97,66,42 kDa; Sigma),were run in parallel and stained with Amido black. Fractions (1 cm long),were cut from a l-cm wide strip of separated Ag, and cut further into l-mm strips. Each 1cm2was solubilized in 5 ml DMSO, dialyzed against PBS for 30 min to precipitate Ag-carrying nitrocellulose particles, washed, and sterilized by irradiation. Ag concentration was expressed using a modification of the method of Abou-Zeid [18] by Dr. D. Ulaeto as nitrocellulose area/well; each fraction was assumed to contain 100 mm2. Control fractions were prepared in the same way from uninfected, RBC.

2.6 T cell blotting analysis Ag-carrying nitrocellulose particles were incubated with PBMC or CBMC, in round-bottom 96-well plates (Nunclon), at various nitrocellulose dilutions (optimum 0.2-1 mm*/well), under the same conditions as in prolifer-

Eur. J. Immunol. 1990. 20: 307-315

Nonimmune T cell response to l? falciparum

ation assays with soluble Ag. Uninfected RBC Ag blotted onto NCP and NCP with no extraneous Ag were used as negative controls; unfractionated PPD blotted directly onto nitrocellulose was used as a positive control.

309

and the SRBClysed withTris (0.017 M),NH&I (0.144 M) in distilled water. 2.9 Depletion of T cell subsets

2.7 Western blotting Narrow strips were cut from the blot adjacent to the strip used for Tcell assays. These were blocked with 5% BSA (Sigma) in PBS with 0.05% Tween 20 (BDH) for 4 h, and incubated overnight with hyperimmune serum (HIS; M O O of a pool of seven sera from immune adults in Papua New Guinea, a gift from Dr. P. Graves), or mAb (1/500 of mAb 89.1, or 111.2 [21] a gift from Dr. M. Lockyer) overnight. Blots were washed, incubated with 1/2000 goat anti-mouse Ig (Southern Biotechnology, Birmingham, AL) or antihuman IgG alkaline phosphate-conjugated second antibody (ICN Immunobiologicals, Costa Mesa, CA), and developed with nitroblue tetrazolium, 90 mg/ml in 70% DMF, Sigma) and BCIP (5-bromo-4-chloro-3-indoyl phosphatetoluidine salt, 15 mg in 1 ml DMF, Bio-Rad, Richmond, CA) in 100 ml of sodium carbonate buffer, pH 9.8. 2.8 SRBC rosetting

Nonadherent PBMC were incubated with mAb UCHLl or mAb SN130, (1 ml culture SN/107cells) on ice. Cells (1 x 108/ml)were washed, and incubated on ice for 20 min with magnetic beads coated with sheep anti-mouse IgG mAb at a ratio of 4 beaddcell (Dynal, Oslo, Norway; M-450). Free cells were separated from rosettes using a magnet (Dynal), transferred to a fresh tube and the depletion procedure was repeated. The purity of depleted populations was analyzed by FCM. Cells were incubated with mAb culture SN followed by 1/20 goat anti-mouse-FITC mAb (Dako, Golstrup, Denmark), and fixed with 100 ~ 1 4 % formaldehyde (BDH) in RPMI 1640 with 10% FCS. Analysis was performed on a Becton Dickinson (Mountain View, CA) FACScan. Antibodies were UCHLl [22], anti-CD3; UCHL1, anti-CD45 180 isomer [23] (both gifts from Professor I? Beverley); SN130 [24]. anti-CD45R was a gift from Professor G. Janossy, Royal Free Hospital, London; and Tac, anti-IL 2R (Becton Dickinson).

Adherent cells were removed from PBMC, by incubation for 90 min in RPMI 1640 with 10% autologous plasma in petri dishes (Sterilin, Richmond, GB), at 37 "C. Washed nonadherent cells were resuspended at 1 x lo7 cells/ml RPMI 1640, and 4.5 ml of 2% untreated SRBC, and 3.5 ml of 4.3% autologous plasma in RPMI 1640 were added to each 1ml of cell suspension. Rosetting cells were incubated for 30 min at 37 "C, centrifuged for 5 min at 400 x g and 4"C, and incubated at 4°C for 30 min. Rosettes were separated from E- cells by centrifugation over Histopaque,

3 Results 3.1 Proliferation of PBMC and CBMC to P.fakiparum PBMC from healthy laboratory volunteers never exposed to malaria parasites were stimulated with asexual stage malaria Ag to determine whether proliferation of PBMC from nonimmune individuals [3-51 could be reproduced (Fig. 1). PBMC proliferated in vitro to whole blood stage

Table 1. Time course of proliferative response to mitogen (PHA), superantigen (SEB) and recall antigen (PPD) compared with l? falciparum by PBMC from donors not exposed to malaria in vivo Proliferation (cpm x Day 4

Day 6

Exp.

Day 2

Medium only

1 2

0.65 (0.12) 0.49 (0.07)

0.37 (0.04) 0.41 (0.10)

0.52 (0.04) 0.67 (0.05)

PHA 1 yg/ml

1 2

66.11 (20.12) 54.91 (1.26)

87.42 (2.16) 68.67 (1.26)

53.50 (1.78) 54.78 (3.42)

SEB 10 yglml

1 2

55.18 (2.24) 47.28 (2.73)

87.92 (7.06) 77.41 (0.69)

124.87 (1.54) 126.21 (4.32)

SEB 100 pglml

1 2

26.21 (0.83) 26.23 (2.08)

89.88 (4.63) 74.15 (1.48)

177.19 (3.31) 114.33 (7.53)

PPD 250 Ulml

1 2

0.68 (0.08) 1.33 (0.37)

3.89 (0.49) 9.06 (1.82)

59.25 (13.59) 126.75 (25.25)

PPD 125 U/ml

1 2

0.55 (0.06) 1.29 (0.21)

2.85 (0.26) 5.03 (0.82)

35.32 (2.16) 85.85 (4.94)

P falciparum (2 x 106)

1 2

0.73 (0.23) 0.53 (0.05)

6.37 (1.16) 9.45 (0.67)

40.01 (2.55) 54.92 (30.89)

I! falciparum (1 x 106) R falciparum ( 5 x 18)

1 2 1 2

0.68 0.72 0.70 0.57

8.27 4.59 2.91 9.29

44.05 (7.97) 65S6 (8*75) 36.10 (16.40) 43.93 (7.20)

Stimulus

,

(0.16)

(0.08) (0.03) (0.07)

(0.29) (0.27) (1.55) (1.43)

a) Results expressed as cprn ( X 1@), with SD in parentheses from 2 X 18 PBMC from two healthy donors.

K. R. Jones, J. K. Hickling, G. A. T. Targett and J. H. L. Playfair

310 40030

-

-

1

T

r

30000 T

U

u

/A\

day5 day7

Y

2

20000

o.

Eur. J. Immunol. 1990. 20: 307-315

as those stimulated with PPD in contrast to PBMC stimulated with SEB or PHA (results not shown). This suggests that in induction of changes in cell phenotype the polyclonal response of nonexposed PBMC to l? fafciparum Ag resembles a memory T cell response rather than a nonspecific mitogenic response.

U

g

10000

'0

0: 10

I

100

1000

10000 100000 1000000

Parasiteslwell

Figure 1. Proliferation of PBMC at days 5, 6 and 7 from a donor not exposed to malaria in vivo. 5 x 105 cells plated out per flat-bottom well with whole live I? fakiparum (NF54) parasitized O+RBC at4.9%0parasitaemia, (3.7%ring trophozoites, 0.4%late trophozoites and 0.8% schizont stages). An 18-h pulse with labeled dThd was used in each case.

parasites diluted in washed RBC (direct from culture), but not to uninfected RBC, saponin, or saponin-lysed uninfected RBC (results not shown). Peak proliferation was observed on day 6, and was dependent on antigen concentration, although the highest concentrations of whole or saponin-lysed parasites, (results not shown) gave suboptimal proliferation. This was also the case for the proliferative response to PPD (data not shown). Membrane fractions of blood stage Ag prepared by saponin lysis of infected RBC induced a similar response in nonexposed PBMC to those induced by whole parasites (Table 1). Once again peak uptake of [3H]dThd was at day 6, similar to the response to PPD (a recall Ag), but unlike the response to PHA, which peaked at day 2-3 or the superantigen SEB. The responses to l? fafciparum were lower than those to PPD, both being much lower than responses to either PHA or SEB. Similar kinetics were seen with PBMC from a donor who had previously been exposed to I! fafciparum, and with cryopreserved PBMC from a further eight laboratory workers, not exposed to P fafciparum in vivo (results not shown). PBMC were incubated in parallel with proliferation assays with PHA, SEB, PPD and l? fafciparum and stained at 48 and 96 h with mAb to UCHL1, SN130 and IL 2R. Cells stimulated with l? fafciparum Ag showed similar total expression of IL 2R, CD3, CD4, CD8, UCHLl and SN 130

CBMC were also tested for their ability to respond to saponin-lysed parasites. Despite high background counts with CBMC in medium plus autologous plasma, all four samples gave significant proliferation to l? fakiparum (Table 2). Three of the four samples proliferated to PPD, and all four responded to PHA. Cord blood PBMC A and B were analyzed using FACS and were found to have approx. 46% and 30% CD3+ cells, a CD4 :CD8 ratio of approx. 1: 1 and a SN 130: UCHLl ratio of 6 : 1. 11% of the PBMC stained positive with UCHL1, this may have included some non-T cells however, since monocytes stain positive with this marker.

3.2 Proliferation to antigens on nitrocellulose To further characterize the antigens responsible for proliferation of PBMC, cells were stimulated with saponin-lysed parasite Ag separated by SDS-PAGE and blotted onto nitrocellulose. Nitrocellulose alone in the absence of Ag gave a weak stimulation of PBMC proliferation. A strong response was seen with PPD blotted onto NCl? Both the donors tested responded to PHA, soluble PPD and saponin-lysed I! fafciparum parasites, and to PPD blotted onto NCl? In addition, when both donors were tested for proliferation to separated l? falciparum Ag on nitrocellulose, they gave a similar pattern of response. Although some fractions gave proliferation considerably above background for NCP (e.g. A6-All), the high standard deviations reduced the number of significant responses (Table 3). The majority of responses were from fractions A6-All (mol. mass approx. 90 kDa to 346 kDa). As expected the responses were not as high as the response to whole Ag, presumably since the antigen on the nitrocellulose was at a low concentration (as judged by intensity of staining of Amido black, mAb and HIS on Western blotting; results not shown). No significant response was seen to uninfected RBC Ag at similar concentrations of nitrocellulose and similar loading of RBC onto the original

Table 2. Proliferation of CBMC to PHA and R fakiparum antigen

Antigen

Medium only PHA 1 PPD 667 U/ml PPD 333 U/ml PPD 167 U/ml PPD 83 U/ml P f f ) I? f 2 0 PflO Rf5

CB A 5.40 (0.32) 11.04 (8.83) 22.05 (3.97) 11.64 (5.94) 6.31 (1.50) 7.42 (2.22) 25.56 (4.35) 13.81 (1.66) 5.03 (1.16) 4.46 (1.74)

Proliferation (cpm x W)a) CB B CB C

2.81 (0.47) 15.61 (2.96) 14.59 (1.6) 10.72 (3.75) 5.73 (2.06) 5.51 (1.59) 12.85 (3.08) 12.18 (0.24) 8.75 (2.10) 4.78 (1.05)

4.22 (1.40) 33.09 (5.96) 16.47 (3.13) 15.75 (8.66) 15.38 (2.92) 15.36 (6.76) 20.67 (7.43) 32.69 (4.58) 19.09 (9.93) 9.48 (2.08)

CB D

12.08 (3.38) 31.68 (3.48) 13.58 (3.26) 14.78 (3.10) 14.33 (1.58) 11.85 (1.19) 20.22 (1.01) 32.08 (2.25) 20.37 (2.44) 12.88 (3.09)

a) Proliferation expressed as cpm x 10-3 from 6 X 105 CBMC (cord A 2 x 1 8 and cord B 4 X lW), harvested at day 6; figures in brackets represent SD of triplicate wells. b) P f 40,20,10,5,denote 40,20,10 and 5 x 104 saponin-lysed f? fakiparum parasitedwell used as a stimulus.

Eur. J. Immunol. 1990. 20: 307-315

Nonimmune Tcell response to R falciparum

311

Table 3. Tcell blotting of R fakiparum antigens, proliferative responses in PBMCa) Antigen Soluble or on NCP Medium only PPD sol. Agd) P falciparume) NCP no Ag PPD on NCPO P f Alg) PfA2 PfA3 PfA4

I? f A 5 PfA6 PfA7 PfA8 PfA9 P f A10 l? f A l l

Proliferationb) to 1 m m 2 Donor 1 Donor 2

0.14 (0.03)e) 52.49 (4.20) 28.08 (1.69) 1.10 (0.41) 18.57 (7.06) 1.78 (0.97) 0.69 (0.17) 1.75 (0.35) 2.63 (2.15) 1.33 (0.76) 5.28 (3.49) 4.55 (1.45)

*j5.19 (3.06)

Proliferationb)to 0.2 mm2 Donor 1 Donor 2

2.18 (0.39) 12.16 (1.58) 70.87 (2.13) 1.40 (0.14) 48.60(0.49) 3.84 (0.65) 0.72 (0.13) 3.29 (1.09) 3.46 (0.59) NTh) 5.91 (2.42) 4.72 (2.31) 11.48 (5.97) 12.69 (5.71)

0.14 (0.03) 52.49 (4.20)

2.18 (0.39) 12.16 (1.58)

wj w w

32.16 (6.11) 1.03 (0.36) 0.46 (0.13) 1.13 (0.51) 2.46 (1.92) 0.35 (0.31) 4.06 (1.54)

%&3 5.57 (2.06) 7.07 (2.55)

6.26 (2.50)

6

$

1.41 (0.01) 1.42 (0.79) 6.07 (3.94)

NT 3.02 (1.48) 12.53 (5.64) 11.48 (2.07) 16.56 (9.10) 15.72 (4.87) 19.39 (10.67)

Proliferative responses from PBMC from donors not exposed to l? falciparum in vivo after an 18 h pulse with [3H]dThd at day 5-6. Proliferation in cpm x from 2 x 105 cells. SD of triplicates. PPD at 667 Ulml. R falciparum parasites at 2 x l@/well (donor 1) and 4 X 105 (donor 2). 5000 U of PPD blotted on to same area of NCP as l? falciparurn. I! falciparum fractions. NT: not tested. Nitrocelluloseblotted antigens tested at 1mm2and 0.2 mm2/well(antigen load unknown). Al: 25-32 kDa, A2: 32-43 kDa, A 3 43-55 kDa, A4: 55-70 kDa, A5: 70-91 kDa, A 6 91-155 kDa, A T 120-155 kDa, A& 155-199 kDa, A 9 199-266 kDa, A10 266346 kDa, A l k 346 kDa. Figures underlined denote stimulation 2 SD above NCP with no Ag or medium only (which ever is higher).

SDS-PAGE (results not shown). The membrane fractions of E! falciparurn were shown by Western blotting to contain many Ag when stained with HIS from Papua New Guinea, or with mAb to 195-kD merozoite/schizont Ag (89.1 and 111.2), a major immunogen in blood stage malaria parasites (results not shown).

cells gave higher background counts perhaps because the interaction of SRBC with CD2 may stimulate Tcells [25]. Proliferation to both PPD and I? fakiparum required E+ cells, although in some instances this occurred without the addition of irradiated adherent cells, presumably due to the presence of sufficient numbers of contaminating APC in the E+ population (Table 4).

3.3 Characterization of the responding cell population

To contrast the proliferative response of nonimmune PBMC to E! falciparurn with that to mitogens or superantigens (Fig. 2), the lysosomotropic agent NH4Cl was used to inhibit antigen processing and presentation [17].This agent was present throughout the assay and resulted in a reduction of proliferation to both PPD and P fakiparum, but increased responses to PHA and SEB,which have previously been shown not to require processing [ 6 ] .

Rosetting with SRBC was used to demonstrate the requirement for T cells in the proliferative response of PBMC (Table 4). E- cells did not respond during the 6 days to PPD o r R falciparum, even when adherent cells were present (data not shown).The cells did, however, respond to PHA, possibly suggesting a low level of Tcell contamination. E+

Table 4. Proliferation in SRBC-rosetted cells to l? falciparum Proliferationa) Antigen Medium alone PHA 1 pg/ml PPD 376 U l? f 38d) l? f 19 Pf9

PBMC

E-

0.30 (0.09)o 0.26 (0.07) 58.85 (9.42) 48.70 (10.23) 32.01 (6.08) 0.81 (0.32) 42.47(13.59) 0.28 (0.04) 29.38 (4.99) 0.22 (0.06) 0.27 (0.16) 24.10 (2.65)

E+

E+ and adhb)

0.35 (0.22) 42.73 (9.83) 15% (7.M) 0.22(0.02) 0.37 (0.17) 0.25(0.07)

9.75 (2.54) 72.70 (9.45) 83.33 (21.67) 47.27 (8.04) 49.57 (8.43) 32.42 (3.89)

a) Proliferation expressed as cpm x from 2 X 105 cells (or 1 X 105 E--cells) at day 6. b) Adh: irradiated adherent cells as APC (1 x W/well). c) Figures in brackets represent SD of triplicate wells. d) l? f 38, 19, 9 denote 38, 19 and 9 X 104 saponin-lysedl? falciparurn parasitedwell as stimulating antigen.

312

2501

CI

n 3

Eur. J. Immunol. 1990. 20: 307-315

K. R. Jones, J. K. Hickling, G. A . T. Targett and J. H. L. Playfair

0

250

PHA

2

4

6

8

10

1

0

The purity of the separated cell populations was determined by FCM. UCHLl- depleted populations contained < 5% UCHLl+ cells, expressing low levels of the marker (compared with 37% UCHLl+ cells in the nonadherent population), and enrichment of high expressing SN130+ cells to 95% (from 82% in the nonadherent population). SN130- depleted populations were enriched for high expressing UCHLl+ cells (from 37% to 98%), and had a low level of contaminating low-expressing SN130+ cells (5% above background).

SEB

2

1

4

6

8

10

P. falciparum

4 Discussion

0

2

4

6

8

10

0

2

4

6

8

10

Time (days)

Figure 2. Effect of NH..,CI

on the proliferative response (cpm X lo3) of PBMC harvested at different time points to (a) mitogen PHA (1 pglml), (b) superantigenSEB (10 p,g and 100 ng) , (c) recall antigen PPD (250 Ulml and 63 U/ml) and (d) R falciparum (2 X 106 and 1 X 106 parasiteslml). Solid squares: higher stimulus concentration, no NH4CI. Solid circle: lower stimulus concentration, no NH&I. Open symbols denote proliferation to stimulus in presence of N&Cl (10 mM).

To determine whether the responding T cells from these nonimmune individuals resided in the memory or naive subsets, PBMC were depleted of these populations using the mAb UCHLl and SN130, respectively, prior to stimulation with saponin-lysed F! fulcipurum (Table 5). In two experiments using PBMC from two donors, the response to both PPD and I! fakiparum was reduced when PBMC were depleted of UCHLl+ cells to a mean of 7.9% (for PPD) and 9.4% (for Pfalcipurum) of the response for equivalent numbers of nonadherent cells. In contrast, depletion of SN130+ cells did not cause reduced responses to these antigens. Both depleted subsets proliferated strongly to PHA. Positively enriched populations were not tested, as magnetic particles bound to cells inhibited proliferation.

In this study we have reproduced the previously reported in vitro proliferative response of non-exposed PBMC and CBMC to both whole I! fulcipurum parasites and crude extracts of parasite membranes. The proliferative response to malaria parasites was shown to resemble that to PPD in that it peaked at day 6 after stimulation, and required SRBC rosette-positive cells. Furthermore, the response was shown to be dependent on adherent cells and was NH&l sensitive suggesting a requirement for endosomedependent antigen processing. Depletion of memory (UCHLl+) T cells but not the reciprocal population of naive (SN130+) T cells abrogated the proliferative response. In terms of induction of activation markers, the response also resembled that to PPD rather than to the mitogens tested. The significance and nature of the proliferation of nonimmune PBMC to I! fulcipurum Ag in vitro needs to be understood if Tcell response are to be successfully used to identify parasite antigens which confer true protective immunity to the individual. It was not previously known whether the proliferation to membrane fractions of I! fulciparum was due to the presence of a mitogen similar to PHA, a lectin which binds galactose on the CD3 complex [26],which potentially may have been useful to the parasite for adherence to RBC or endothelium, via protein to carbohydrate binding, and may have had relevance to polyclonal activation of T cells in vivo.

Table 5. Proliferation of UCHL1- and SN130-depleted PBMC to R fulcipurum

Antigen

Exp.

PBMC

Medium

1 2 1 2 1 2 1 2 1

0.06 (0.01) 0.06 (0.03) 47.32 (5.21) 20.13 (2.82) 34.15 (1.37) 41.45 (2.07) 53.03 (4.77) 43.56 (4.36) 33.01 (1.98) 6.97 (3.77) 4.28 (0.59) 3.99 (0.28) 2.74 (1.29)

PHA 1 p g / d PPD 2000 U PPD 200 U PPD 20 U P f l x lob=) P f l x 105

1 2 1 2

Proliferationa) UCHLl depl. Non-adhb) 0.09 (0.03) 0.07 (0.02) 40.70 (12.2) 17.69 (0.53) 23.33 (3.27) 42.08 (13.05) 29.33 (4.99) 37.79 (6.80) 14.46 (1.74) 3.48 (1.53) 7.42 (1.63) 3.14 (1.16) 4.69 (0.01)

0.05 (0.02) 0.07 (0.02) 42.92 (1.72) 31.19 (0.69) 2.41 (1.35) 4.32 (1.68) 3.46 (1.11) 2.57 (1.10) 0.16 (0.15) 0.41 (0.27) 1.42 (0.46) 0.18 (0.18) 0.06 (0.03)

SN130 depl.

0.04 (0.02) 0.18 (0.16) 30.79 (1.85) 13.43 (1.88) 25.78 (1.29) 36.06 (5.41) 35.06 (3.51) 37.08 (2.22) 14.46 (1.74) 4.88 (0.59) 5.41 (0.01) 5.60 (0.45) 5.26 (0.21)

a) Proliferation as cpm x 10-3 at day 6 from 1 x lo5 cells/well, figures in brackets are SD of triplicate wells. b) Non-adh: nonadherent cells, UCHLl depl. or SN130 depl.: the remaining cells after mAb UCHLl and SN130 depletion, each subset (including nonadherent cells) reconstituted with 1 x 104 irradiated adherent cells. c) I? f: concentrations of saponinlysed R falciparum parasitedwell.

Eur. J. Immunol. 1990. 20: 307-315 Alternatively the activity might have been similar to the response of non-exposed human and murine T cells to bacterial protein toxins [6]. These response are dependent on MHC molecules but are not MHC restricted, and do not require antigen processing, the superantigen binding both to theTcR and the MHC of an APC [6]. Such a moiety might be present in the parasite as the toxin proposed in the anti-toxic immunity associated with resistance to symptoms seen in highly parasitized children, before specific antiparasitic immunity has developed [27]. Mitogens such as Staphylococcus aureusTSST-1 have been shown to mediate shock in vivo. They can also stimulate human T cells to produce IFN-y, IL 2 and IL 2R, and human monocytes to produce cytokines such as IL 1 and TNF [28]. The kinetics of proliferative response and effect on cell phenotype induced by I? falciparum Ag in PBMC did not resemble those induced by a superantigen. Superantigens have been demonstrated to stimulate greater proliferation in CD45R+ cells than CD45R- cells [29]. This is clearly different from the results presented here, in which the proliferating population to l? falciparum appeared to reside in the CD45R- population. Furthermore, proliferation to l? falciparum in contrast to that to superantigens required antigen processing [6].

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313

fetal circulation to malaria Ag is feasible.The CBMC in this study also responded to PPD (3 out of 4 samples), which according to other reports is dependent on memory cells [16l.These PPD-reactiveTcells could have been induced by cross-reactive common mycobacterial Ag crossing the placenta. It is unlikely that all of the CB samples tested in this study (collected anonymously from healthy infants in a London Hospital), originated from mothers previously exposed to malaria. Thus the response of CBMC to I? falciparum Ag, if also dependent on memory T cells, could have also been due to cross-reactive memory cells to other Ag. We found 11% UCHLl+ cells in the CBMC tested tested here; previously, cord blood has been described as resembling adult blood in its relative proportions of T cell subsets [33]. Placental blood or blood from the first trimester contains no UCHLl+ cells [12,34], whereas a low percentage of CB lymphocytes express UCHLl ligand [34]. It has been suggested that the UCHLl+ cells in CB (which are all CD4+) are the result of priming in utero although the absence of co-expression of IL 2R suggests that the cells are not activated at the time of collection [34].

Depletion of UCHLl+ cells from PBMC prior to stimulation effectively removes memory cells. Preliminary experiments to follow expression of phenotypic markers suggested that the response to I? falciparum resembles that to PPD rather than PHA or SEB. Other early or late acitvation markers could be investigated and may reveal more subtle differences between PPD and l? falciparum stimulation. However, these may be the result of quantitative rather than qualitative differences in stimulation.

Since the donors of the PBMC in this study have neither experienced malaria infection nor travelled in malarious areas, the memory Tcells responding to P falciparum must presumably have been primed in vivo to cross-reactiveTcel1 epitopes present on other immunogens. The fact that CBMC are able to respond suggests that individuals may have high frequency circulating memory cells to such Ag present at an early age. Cross-reactivity exists between plasmodia species at both the antibody and the Tcell level [35]. In addition, malaria parasite proteins have been shown to have sequence homology with a number of well-characterized microbial and host cell proteins [36,37]. Further evidence for cross-reactivity could be obtained if T cell clones reactive with malaria Ag were tested with other common microbial Ag. Conserved epitopes may be important for functions shared by malaria parasites and other species, similar to the conservation of heat shock proteins seen in eukaryotes and prokaryotes [38].This conservation of epitopes might conceivably benefit the parasite, by utilising the tolerance of the host to host epitopes, therefore preventing T and B cell reactivity to these epitopes. Shared epitopes between malaria and other pathogens, but not the host, may on the other hand induce an accelerated T cell response on first infection. This may benefit the host if these epitopes are protective, or any subsequent cytokine release is protective. Conversely if the epitopes are nonprotective they may benefit the parasite by producing a T cell response analogous to the polyclonal B cell response seen in l? falciparum malaria [3], which perhaps via the production of cytokines may induce immunopathology or immunosuppression, both of which are features of malaria. In mice the better protection against a non-lethal strain of I? yoelii has been linked to a strong earlyTcel1 response [3]. In contrast, from other studies using a panel of mouse strains, an early strong T cell response correlated with susceptibility to non-lethal I? yoelii malaria [39].

The surprising response of cord blood cells to I? fakiparum Ag is in accordance with previous reports of responses from cells from healthy infants [ l , 3, 321. In endemic areas parasitized RBC have been found in CB, so exposure of the

A study using exo-antigens from I? falciparum separated by gel filtration, has also described a proliferative response in nonimmuneTcells (to Ag of 20 kDa, 70 kDa and 250 kDa) [40]; this contrasts with earlier fractionation data which

A third possibility is that the response might have been due to a specific reaction of Tcells to protein Ag, as a result of in vitro priming of naive memory cells. This has been demonstrated in other Ag systems [30, 311, but in these cases no proliferation was seen in the first 10 days of exposure to Ag (the concentration of which was critical), and priming for subsequent proliferation required representation of Ag with fresh APC. Thus the kinetics of the response were considerably different to those reported here. In addition to the similarity of kinetics of the proliferative response and NH4C1inhibition with the recall antigen PPD, the evidence presented here of dependence on UCHLl+ cells for the response argues for the role of Tcell memory in the proliferation seen [ 131. Human memory T cells can be identified by increased expression of CD45 180-kDa isomer, (the ligand for mAb UCHLl), or CDw29 (the ligand for mAb 4B4), also Pgp-1, LFA-1, LFA-3, Tal and CD2 [121. Naive cells show enhanced CD45R (SN 130,2H4, HB-11 or Leu-18). Once activated in vitro for example by PHA, cells down-regulate CD45R (200 kDa and 220 kDa isomers) and up-regulate the modified 180 kDa variant of this molecule. No conversion of UCHL1+ t o CD45R expressing cells has been reported. This is proposed to represent a maturation of post-thymic T cells primed in vivo by Ag.

314

K. R. Jones, J. K. Hickling, G. A. T. Drgett and J. H. L. Playfair

found stimulatory activity at a wide range of molecular mass peaking at 150 kDa and 200 kDa (28). Interestingly, nonimmune PBMC produced IFN-y in response to these malaria Ag [40], suggestive of a memory response from UCHLl+ cells [29], although other reports have failed to detect I F N y in schizont or malaria SN-stimulated nonimmune PBMC cultures [41]. This latter report failed to correlate the production of this cytokine by immune PBMC with proliferation indices. Malaria exo-antigens have been reported to stimulate nonimmune PBMC to cause CD8+-mediated suppression of PWM-induced B cell proliferation [40], a phenomenon attributed by other investigators to CD4+ CD45R+ cells [42] suggesting that in these exo-antigen-stimulated cultures not only UCHL1+ cells are stimulated. However, earlier studies did show stimulation of production of IgM from nonimmune B cells by malaria SN Ag [3, 431. The discrepancies between the results of Jaureguiberry et al. [40] and ours probably reflect differences in stimulating Ag or culture conditions. The results presented here are in accordance with the work of Sinigaglia et al. [7, 111 and Good et al. [9] who showed that Tcell clones specific for malaria Ag could be raised from nonimmune donor PBMC. In the light of the data presented here and the conclusions drawn from these earlier reports, perhaps at least some of the clones were to epitopes cross-reactive with unknown microbial Ag. It is possible, however, that specific responses in T cells clones may be induced by in vitro priming, since they are generated over a period of several weeks. Our results may also reinforce the view that using the proliferative response of Tcells to P falciparum Ag as an immuno-epidemiological parameter may be fraught with complications depending on the prior exposure of subjects to cross-reactive epitopes. These might be circumvented by reducing the concentration of Ag to detect only highfrequencyTcel1 responses, such as might be present in those individuals recently primed in v i v o with I? falciparum. Alternatively, purified or recombinant Ag could be used as in vifrostimuli if pretested at a range of concentrations with PBMC from non-exposed individuals [1,411.Without these precautions it may not be possible to identify which parasite antigens are important in stimulating a protective immune response. We would like to thank University College Hospital Labour Ward for collecting cord blood, Prof. Peter Beverley (ICRK London), Dr. G. Janossy (Royal Free Hospital, London) and Dr. M. Lockyer (Wellcome Biotechnology) for the gift of monoclonal antibodies, Dr. P Graves for the gift of hyperimmune sera. We are indebted to Steve Eida, Neil Rogers and John Williams for excellent technical assistance, and to Drs. J. R. Lamb, Matthias Merkenschlager, Corinne Ong and David Ulaeto for their advice.

Received June 6, 1989; in revised form September 21, 1989.

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