Aug 27, 1992 - stimulation and rest, two T-cell lines were analyzed. Both lines were ... by infection and drug cure, as no specific antigen or vaccine is known to.
Vol. 61, No. 5
INFECTION AND IMMUNITY, May 1993, p. 1958-1963 0019-9567/93/051958-06$02.00/0 Copyright © 1993, American Society for Microbiology
An Approach to Development of Specific T-Lymphocyte Lines by Use of Preprocessed Antigens in Plasmodium vinckei vinckei Murine Malaria GLENN M. WASSERMAN,"2 SANJAI KUMAR,`* JEFFREY AHLERS,3 FRED RAMSDELL,4 JAY A. BERZOFSKY,3 AND LOUIS H. MILLER' Laboratory ofMalaria Research' and Laboratoty of Cellular and Molecular Immunology,4 National Institute of Allergy and Infectious Diseases, and Section on Immunogenetics and Vaccine Research, National Cancer Institute,3 Bethesda, Maryland 20892, and Medical Research Fellowship, Walter Reed Army Institute of Research, Washington, D. C. 203072 Received 27 August 1992/Accepted 5 February 1993
The development of parasite-specific T-cell lines represents one approach to the potential identification of relevant immunogens in erythrocytic malarial infection. However, the use of parasitized-erythrocyte lysates as antigens inhibits the proliferation of T cells. To circumvent this problem, we preincubated antigen-presenting cells (APCs) from spleens of malaria-naive, BALB/c mice with a Plasmodium vinckei vinckei (hereafter referred to as P. vinckei)-parasitized erythrocyte lysate. APCs were subsequently irradiated and washed prior to being incubated with T lymphocytes from P. vinckei-immune, histocompatible mice. After 8 to 10 cycles of antigenic stimulation and rest, two T-cell lines were analyzed. Both lines were predominantly CD4+. Proliferation assays demonstrated marked lymphocyte blastogenesis to syngeneic but not allogeneic APCs that had preprocessed malarial antigen. Antigen incubated directly with T cells and nonpulsed APCs in vitro did not result in T-cell proliferation. Assays of interleukin-2 (IL-2), IL-4, IL-5, and gamma interferon were compatible with one cell line being predominantly TH1 and the other being TH2. Thus, APCs that have preprocessed malarial antigen and are free of extraneous parasite material induce highly reactive, antigen-specific, major histocompatibility complexrestricted T-cell lines that functionally appear capable of inducing humoral and/or cell-mediated immunity.
Malaria remains a leading cause of infant and child mortality worldwide, with an estimated 100 to 500 million cases and 2.5 million deaths annually. Younger children, pregnant women, and nonimmune migrants represent the vast majority of clinical cases and deaths (26, 38, 41). Methods to produce an effective malaria vaccine have continued to be elusive (4, 14, 27). Plasmodium vinckei vinckei (hereafter referred to as P. vinckei) murine malaria is a useful model for studying immune mechanisms that must act quickly to protect against death (22). BALB/c mice inoculated with parasitized erythrocytes (PRBC) develop greater than 90% parasitemia and die within 7 to 10 days. At present, immunity to erythrocytic P. vinckei infection can only be induced by infection and drug cure, as no specific antigen or vaccine is known to induce immunity. This immunity is mediated in part by CD4+ T cells. Selective depletion of CD4+ T cells in immune mice abrogates immunity that can be reconstituted by adoptive transfer of CD4+ T cells from immune, syngeneic mice. CD4+ T cells from nonimmune, syngeneic mice do not reconstitute immunity (22). One strategy in the search for identifying protective malarial antigens is to develop highly antigen-responsive T-cell lines (2, 36) and, subsequently, clones (3, 35) that could be screened for the ability to confer protective immunity on adoptive transfer into a nonimmune animal model. These
human malarias, as has been demonstrated with the circumsporozoite protein in sporozoite immunity (10, 27). This strategy has been used successfully with influenza virus (42), Rickettsia tsutsugamushi (21), Mycobacterium tuberculosis (23), Mycobacterium leprae (30), and Leishmania major (35, 36), all of which are intracellular organisms that require cellular immunity for protection. How then does one generate antigen-specific T-cell lines to plasmodial antigens? Data obtained with Plasmodium berghei (16, 20) in mice and Plasmodium vivax (15) and Plasmodium falciparum (33) in humans suggest that parasite-derived factors, associated in some species with PRBC membranes (16, 20, 33), inhibit T-cell activation. Attempts to induce T-cell proliferation in vitro by incubating T cells from a P. vinckei-immune BALB/c mouse with syngeneic, antigen-presenting cells (APCs) and a P. vinckei-infected erythrocyte (RBC) lysate resulted in diminished T-cell proliferation (unpublished data). Consequently, allowing APCs to preprocess malarial antigen and washing extraneous material from the medium before incubating these APCs with immune T cells may (i) diminish the T-cell-inhibitory factors present in the lysate and (ii) enable a more rapid presentation of processed malarial antigen to T cells (6, 7, 39). We hypothesized that this method of inducing stimulation of T cells to P. vinckeiPRBC antigens would enhance T-cell reactivity to and specificity for these antigens.
protective T cells could then be used in a proliferation assay with subfractions of malarial antigen to identify relevant T-cell epitopes (11, 13, 14, 19, 23). Protective epitopes identified in this manner may have homologous antigens in *
MATERIALS AND METHODS Parasite. P. vinckei (ATCC 30091; American Type Culture Collection, Rockville, Md.) was maintained as a cryopreserved stabilate in liquid nitrogen and by continuous transfer
Corresponding author. 1958
VOL. 61, 1993
of PRBC intraperitoneally to naive mice at 5- to 7-day intervals. Parasite preparations were free of pathogens, including virus elevating lactate dehydrogenase levels. Mice. Female BALB/c and B10.BR mice obtained from the National Cancer Institute Breeding Facility were maintained under standard conditions at the National Institute of Allergy and Infectious Diseases- and American Association for the Accreditation of Laboratory Animal Care (AALAC)approved animal facility. Mice were free of virus elevating lactate dehydrogenase, Mycoplasma pneumoniae, mouse encephalomyelitis virus, Sendai virus, and other pathogens. Naive BALB/c mice were made immune against challenge with intraperitoneal or intravenous inoculation of P. vinckeiPRBC by repeated infection and drug cure (22). Mice were treated with 0.5 mg of chloroquine intraperitoneally (Winthrop-Breon, New York, N.Y.) for 3 days when their parasitemia reached 10 to 50% (day 4 or 5). Mice were reinfected 2 weeks later and drug cured. A third inoculation of PRBC without drug cure confirmed that all the mice were protected. Antigen lysates. Whole P. vinckei and P. berghei antigen lysates were prepared from parasitized blood with greater than 70% parasitemia. A small amount of heparin was used to prevent clotting. PRBC consisting of schizonts and free merozoites were washed free of plasma and adjusted to 109 schizonts per ml before disruption by seven cycles of freezing and thawing. A control RBC lysate adjusted to 109 cells per ml was prepared in an identical manner from nonparasitized, naive BALB/c mice. Lysates were stored at -70°C until use. APCs. APCs were prepared from single-cell suspensions of whole BALB/c mouse spleens and represented a total population of spleen cells. RBCs were removed by hypotonic shock. The spleen cells were irradiated at 3,300 rads and washed in complete medium (42% RPMI, 42% EagleHanks amino acid medium, 10% fetal calf serum, 2 mM L-glutamine, 100 jig of penicillin per ml, 100 ,ug of streptomycin per ml, 2 x 10' M 2-mercaptoethanol, 10 mM N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES]). APCs that preprocessed malarial antigen were prepared by gently rocking APCs at 107/ml with PRBC or RBC equivalents of antigen lysate per ml in complete medium for 12 to 24 h at 37°C in a humidified, 6% CO2 environment before cells were irradiated and washed free of residual antigen. T-cell lines. Spleen cells from mice immune to P. vinckei were passed over prepared nylon wool columns and plated in 24-well plates (Costar, Cambridge, Mass.). Initial concentrations of cells were 4 x 106 T cells and 3 x 106 APCs in 2 ml of complete medium. After two cycles of stimulation and rest, the concentrations were adjusted to 4 x 105 T cells and 5 x 106 APCs. T-cell lines were maintained in 14-day cycles consisting of 5 days of stimulation with antigen and 9 days of rest without antigen stimulation. T cells were stimulated by the addition of 5 x 106 P. vinckei-pulsed APCs per well. The rest phase was achieved by separating harvested T cells from APCs by differential sedimentation over Lympholyte M (Cedarlane, Hornby, Ontario, Canada) and adding to the T cells appropriate concentrations of fresh APCs, not exposed to antigen, in 24-well plates. T-cell line 2 also received rat T-cell monoclone (Collaborative Research, Bedford, Mass.) as a source of interleukin-2 (IL-2); this monoclone was adjusted to a concentration of 10% at the beginning of the rest phase. Phenotypic analysis. T-cell lines were phenotyped by use of one-color and two-color flow microfluorometry (Epics profile analyzer from Coulter, Hialeah, Fla., and BD 440
T-CELL LINES IN P. VINCKEI MURINE MALARIA
1959
apparatus from Becton Dickinson, Mountain View, Calif., respectively). The following primary monoclonal antibodies (MAbs) were used: (i) CD3+, 500-A2 (17); (ii) CD4+, GK-1.5 (9) and H-129.19 (32); (iii) CD8+, 53-6.7; (iv) -yb, GL-3; and (v) B cells, MRX-OX-20 (Serotec, Oxford, United Kingdom). MAb 500-A2 was detected with fluorescein isothiocyanate-conjugated goat anti-hamster antiserum (Caltag, San Francisco, Calif.). GK-1.5 was detected with fluorescein isothiocyanate-conjugated goat anti-rat immunoglobulin G (Kierkegaard & Perry, Gaithersburg, Md.). MRX-OX-20 was directly labelled with fluorescein isothiocyanate (Serotek). All other MAbs were biotin labelled and detected with allophycocyanin-labelled streptavidin (Caltag). T-lymphocyte proliferation assay. Cultured T cells (104) were plated with 4 x 105 APCs in 200 ,ul of complete medium in 96-well microtiter plates (Costar). A minimum of three wells were used for all test samples and controls. Unless otherwise indicated, cultures were pulsed with 1 ,uCi of [3H] thymidine (Amersham, Arlington Heights, Ill.) on day 4 of culturing and harvested 24 h later by use of an automated cell harvester (Skatron, Sterling, Va.). Radioactivity was measured in a 1205 Beta-plate counter (LKB Wallac, Turku, Finland). Cellular proliferation was expressed as a stimulation index (counts per minute for stimulated sample/counts per minute for controls). Lymphokine assays. Assays were performed on supernatants from 96-well plates that contained 104 cultured T cells and 4 x 105 APCs in 200 Ru of complete medium per well. Wells were used only once. Supernatants were stored at -20°C until use. Gamma interferon (8) and IL-5 (34) were assayed by an enzyme-linked immunosorbent assay (ELISA). IL-2 concentrations were measured by a proliferation assay of IL-2-dependent HT-2A cells (29). IL-4 concentrations were determined by a proliferation assay of an IL-4-dependent T-cell clone, CT.4S (18). Analysis of CT.4S cells by use of recombinant IL-2 and blocking antibodies to murine IL-2 (MAb S4B6 [28]) and murine IL-4 (MAb llB1l [31]) revealed these cells to be minimally responsive to IL-2 at the concentrations observed in this study. Lymphokines were measured against a standard curve generated with recombinant gamma interferon (Genzyme, Boston, Mass.), recombinant IL-2 (Genzyme), recombinant IL-4 (1), and recombinant IL-5 (DNAX, Palo Alto, Calif.). RESULTS Generation of P. vinckei antigen-specific T-cell lines. Splenic T lymphocytes from BALB/c mice immune to P. vinckei were restimulated in vitro with syngeneic, P. vinckei antigen-pulsed APCs. The latter were nonimmune mouse spleen cells that had been incubated with P. vinckei antigen lysate 12 to 24 h prior to irradiation, washed free of remaining antigen lysate, and added to T cells. Cultures were then maintained on 14-day cycles consisting of 9 days of nonexposure to antigen followed by 5 days of stimulation with antigen-pulsed APCs. Two T-cell lines were produced by this technique. As shown in Fig. 1 for T-cell line 2, the numbers of T cells measured at the end of the stimulation phase decreased during the first several cycles of stimulation and rest but then increased greatly. Surface phenotype. After 8 to 10 stimulation-rest cycles, line 1 contained 96% CD4+ and 4% CD8+ T cells, while line 2 consisted of 84.3% CD4+ T cells, 0.3% CD8+ T cells, and 15.4% non-B, non-T cells. Non-B, non-T cells were negative for CD3+, CD8+, yb, and B-cell markers.
1960
WASSERMAN ET AL.
INFECT. IMMUN.
TABLE 1. Evidence for specificity and major histocompatibility complex restriction in antigen-induced T-cell proliferation
1o1 11
n1.10 o
1
APCs
0 9/
H-2d (no antigen) RBC-pulsed 11-2d P. berghei-pulsed H-2d P. vinckei-pulsed H-2d
E
2
10 8
P. vinckei-pulsed H-2k
Mean + SEM cpma (stimulation index") for line:
1
2
155 + 31 256 ± 85 (1.7) 483 ± 72 (3.1) 1,149 + 160 (7.4) 248 ± 79 (1.8)
484 + 92 439 ± 101 (0.91) 527 + 95 (1.1) 24,508 + 4,166 (50.6) 557 + 84 (1.2)
a [3Hlthymidine was added to cell cultures 96 h after APCs or antigen lysate-pulsed APCs were added to T cells. Cells were harvested 24 h later, and [3H]thymidine incorporation was measured by 1B-irradiation emission. b Stimulation index = cpm of [3H]thymidine in antigen-stimulated wells/ cpm of [3H]thymidine in wells without antigen.
.0
E 106 z
Z 05 0
1
2
3
4
5
6
7
8
9
10
CYCLE
FIG. 1. Numbers of T cells of T-cell line 2 present at the end of the stimulation phase during stimulation-rest cycles. Numbers were adjusted for cells removed for use in experiments.
Proliferative responses of T-cell lines to antigen. Proliferative responses of T-cell lines 1 and 2 were measured after 8 to 10 cycles of stimulation and rest. T-cell proliferation was compared in culture wells that contained P. vinckei antigen preprocessed for presentation to T cells and wells that contained P. vinckei antigen lysate added directly to T cells and APCs (Fig. 2). Both line 1 and line 2 showed a more
marked proliferative response to antigen that had been preprocessed by APCs, compared with the response to P. vinckei antigen lysate left in culture wells. The proliferative responses of both cell lines to P. vinckeipulsed APCs, P. berghei-pulsed APCs, and nonparasitized, RBC-pulsed APCs are shown in Table 1. Neither T-cell line responded to nonparasitized, RBC-pulsed APCs. T-cell line 2 also did not respond cross-reactively to P. berghei-pulsed APCs; however, line 1 cross-reacted weakly. In all experiments, T-cell proliferation in response to P. vinckei-pulsed APCs was more marked for line 2 than for line 1. Major histocompatibility complex restriction. The proliferative responses of both T-cell lines to B1O.BR mouse (H-2k) spleen cells pulsed with P. vinckei antigen lysate in a manner LINE 2
LINE 1 40r
PV-Pulsed APC's _0 PV 107*tni PV IC I
f-PViCn K
x
w
aC z
Q
z
0
4c
-I
4
2
to
(A
DAYS DAYS FIG. 2. Comparison of the T-cell proliferation response induced by P. vinckei (PV) antigen-pulsed APCs with the response induced by P. vinckei antigen lysate (concentrations were 105 to 107 PRBC equivalents per ml) added directly to wells with APCs. The stimulation index (plotted as a function of days in culture) was calculated as indicated in Table 1, footnote b.
T-CELL LINES IN P. VINCKEI MURINE MALARIA
VOL. 61, 1993
1961
TABLE 2. Lymphokines assayed from supernatants following exposure of T cells to APCs or antigen-pulsed APCs Level of the following lymphokine after the indicated treatment': IL-5 (U/ml)r IL_4 (U/ml)d IL-2 (U/ml)c Gamma interferon (ng/ml)A Line and h B
A
C
A
B
C
C
A
B
3 1 1 0.2
