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ß T Cell Response to Toxoplasma gondii in Previously Unexposed Individuals¹

Carlos S. Subauste^1,*,, Franklin Fuh, Rene de Waal Malefyt and Jack S. Remington^,§

^* Division of Infectious Diseases, University of Cincinnati College of Medicine, Cincinnati, OH 45267; Research Institute Palo Alto Medical Foundation, Palo Alto, CA 94301; DNAX Research Institute of Molecular and Cellular Biology, Inc., Palo Alto, CA 94304; and ^§ Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, CA 94305

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mechanisms by which T cells from previously unexposed hosts respond in vitro to certain intracellular pathogens remain to be fully understood. We report and characterize the in vitro reactivity to Toxoplasma gondii of human ß T cells from T. gondii-seronegative individuals. Resting ß T cells from these individuals proliferated in response to PBMC infected with T. gondii or pulsed with T. gondii lysate Ags. This was accompanied by an increase in the percentage of CD4⁺ ß T cells. Purified CD4⁺ ß T cells but not CD8⁺ ß T cells proliferated in response to these T. gondii preparations. Both CD4⁺ ß T cells with naive (CD45RA⁺) and memory (CD45RO⁺) phenotypes from adults as well as ß T cells from T. gondii-seronegative newborns proliferated after incubation with T. gondii. This ß T cell response to the parasite was inhibited by anti-HLA-DR mAb and to a lesser degree by anti-HLA-DQ mAb. Use of paraformaldehyde-fixed PBMC completely abrogated the proliferation of ß T cells, indicating the need for processing of T. gondii Ags. Analysis of the TCR Vß expression did not show evidence for restriction in TCR Vß usage during T. gondii stimulation of ß T cells. ß T cells secreted significant amounts of IFN- after incubation with T. gondii-infected monocytes. This rapid and remarkable ß T cell response may play an important role in the early events of the immune response to T. gondii.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is well recognized that T cell-mediated immunity plays a central role in the host response to intracellular pathogens (1). The generation of a T cell response against conventional microbial Ags requires processing of these Ags and their presentation in association with MHC molecules on the surface of Ag-presenting cells (2). This would be followed by Ag recognition at the level of the highly variable complementarity determining region (CDR)³ 3 of the TCR and clonal expansion of Ag-specific T cells (3). In general, prior in vivo priming to the Ag is required to detect in vitro T cell responses to conventional Ags (4).

Certain microbial Ags can induce a T cell response without following the pattern of events described above. The best characterized example are the so-called superantigens. Superantigens directly bind MHC class II molecules and the TCR in regions outside the peptide-binding groove and the CDR3, respectively (4, 5, 6). Provided that the T cells bear the appropriate ß- or -chain of the TCR, superantigens can stimulate CD4⁺, CD8⁺, and T cells in vitro without prior in vivo priming (6). However, there appear to be other situations in which microbial Ags induce an in vitro response by T cells from previously unexposed hosts following mechanisms that remain to be fully characterized (7, 8, 9).

Toxoplasma gondii is an obligate intracellular protozoan against which T cell-mediated immunity can confer protection (10, 11). Indeed, T. gondii has become an important opportunistic pathogen in patients with deficiencies in cell-mediated immunity (12). We previously demonstrated that human peripheral blood T cells from both T. gondii-seropositive and T. gondii-seronegative individuals proliferate in response to the parasite (13). This response was accompanied by activation and expansion of T cells (13). However, given the fact that T cells represent a small subset of the T cell population present in peripheral blood (14), we considered that the remarkable levels of T. gondii-mediated T cell proliferation observed with T cells from seronegative individuals might be due to a concomitant response of other subsets of T cells. In the present study, we demonstrate in vitro reactivity to T. gondii of presumably unprimed CD4⁺ ß T cells from T. gondii-seronegative adults and newborns. In addition, we demonstrate that ß T cells produce IFN- in response to T. gondii, an effector function that may be critical to the early immune response to the parasite. This rapid and remarkable ß T cell response may play an important role in the early events of the immune response to T. gondii.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Study population

Buffy coats from heparinized blood of healthy volunteer donors were obtained from the Stanford Blood Bank (Stanford, CA). Samples of umbilical cord blood were obtained from placentas of healthy newborns at Stanford Children’s Hospital. Serologic tests for detection of T. gondii IgG and IgM Abs were performed in all samples of blood (15). Except when noted, samples used had no demonstrable T. gondii IgG or IgM Abs. In some experiments, blood from chronically infected but otherwise healthy adults was used (positive anti-T. gondii IgG and negative anti-T. gondii IgM).

T. gondii and infection

Tachyzoites of the RH strain were obtained from infected monolayers of human foreskin fibroblasts as well as from peritoneal fluid of mice as previously described (13). To obtain toxoplasma lysate Ags (TLA), mammalian cell-free RH tachyzoites released from infected human foreskin fibroblasts were resuspended in sterile distilled water and subjected to three freeze-thaw cycles. After reconstitution with sterile 10x PBS, cellular debris was pelleted by centrifugation at 900 x g for 20 min. Supernatant was collected and stored at -70°C and used as TLA. Antigenic preparations were devoid of detectable levels of endotoxin (<10 pg/ml) using a Limulus amebocyte lysate assay (Sigma). PBMC or monocytes were incubated overnight with UV-attenuated tachyzoites at a ratio of 2 or 4 parasites per cell, respectively. This resulted in a rate of infection of approximately 10% for PBMC and 40% for monocytes (mainly, one organism per cell).

Purification of ß T cells

PBMC and cord blood mononuclear cells (CBMC) were isolated by centrifugation on Ficoll-Hypaque gradients (Pharmacia LKB Biotechnology, Piscataway, NJ). To purify ß T cells, nylon wool-nonadherent PBL were incubated with saturating concentrations of the following mAb: anti-CD16 (Medarex, Annandale, NJ), anti-CD56 (Becton Dickinson, San Jose, CA), anti-CD19 (Coulter Cytometry, Hialeah, FL), and anti- TCR (anti-TCR1, generous gift from Dr. Michael Brenner). Anti-glycophorin A mAb (10F7 MN, American Type Culture Collection (ATCC), Rockville, MD) was added to remove erythroblasts present in CBMC. To obtain purified populations of CD4⁺TCR-ß⁺ and CD8⁺TCR-ß⁺ T cells, anti-CD8 (OKT8, ATCC) and anti-CD4 (OKT4, ATCC) were added, respectively, to the combination of mAb mentioned above. Anti CD45RO (UCHL-1, Immunotech, Westbrook, ME) and anti-CD45RA (ALB11, Immunotech) mAbs were used to obtain purified CD4⁺CD45RA⁺ and CD4⁺CD45RO⁺ T cells, respectively. Magnetic beads coated with anti-mouse IgG (Dynal, Great Neck, NY) were added at a ratio of 10 beads per cell. Rosetting cells were removed with a magnet (Dynal). Addition of magnetic beads was repeated once for purification of CD4⁺CD45RA⁺ and CD4⁺CD45RO⁺ cells. These protocols resulted in populations that were either >99% CD3⁺TCR-ß⁺, >98% CD4⁺TCR-ß⁺, >98% CD8⁺TCR-ß⁺, >98% CD4⁺CD45RA⁺TCR-ß⁺, or >98% CD4⁺CD45RO⁺TCR-ß⁺ cells as determined by flow cytometry.

Purification of monocytes

PBMC were incubated with the following mAb (from Becton Dickinson except when indicated): anti-CD2, anti-CD3, anti-CD8, anti-CD19 (Coulter Cytometry), anti-CD56, anti-CD66b (Immunotech), and anti-glycophorin A. After addition of magnetic beads coated with anti-mouse IgG (Dynal), rossetting cells were removed with a magnet. Populations obtained were >96% pure for monocytes by microscopic examination of Giemsa-stained cytocentrifuge preparations. In addition, cytofluorometric analysis indicated that >92% of the cells were CD14⁺, with <0.5% CD3⁺, <0.5% CD19⁺, <0.5% CD56⁺, and <2% CD66b⁺ cells.

Proliferation assays

ß T cells were cultured in either 24-well or round-bottom 96-well plates (Limbro; ICN Pharmaceuticals, Costa Mesa, CA) at 5 x 10⁵ cells/ml in complete medium consisting of RPMI 1640 supplemented with 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 10% Sabin-Feldman dye test-negative pooled human AB⁺ serum (Irvine Scientific, Santa Ana, CA). In vitro stimulation of ß T cells was conducted as previously described (13). Except when noted, ß T cells were incubated with -irradiated (3000 rad) autologous PBMC that had been either infected with UV-attenuated tachyzoites of T. gondii (5 T cells:1 infected PBMC) (13) or incubated with previously determined optimal concentrations of TLA (10 µg/ml), tetanus toxoid (10 µg/ml; Massachusetts Public Health Biologic Laboratory, Boston, MA), staphylococcal enterotoxin B (SEB) (0.1 µg/ml; Toxin Technology, Sarasota, FL), toxic shock syndrome toxin-1 (TSST-1; 1 µg/ml; Toxin Technology), or PHA (0.25 µg/ml) (Wellcome Diagnostics, Dartford, U.K.). Unless otherwise stated, when stimulated with T. gondii, tetanus toxoid, SEB, TSST-1, or allogeneic PBMC, ß T cells were cultured for 7 days at 37°C, 5% CO₂; they were cultured for 3 days when stimulated with PHA. In some experiments, PBMC incubated overnight with T. gondii or control PBMC were fixed with 1% paraformaldehyde as described (16). Briefly, after washing to remove serum, cells were resuspended in 1% paraformaldehyde in PBS for 5 min at 37°C. Reaction was stopped by adding cold 0.15 M glycine. After three washes, cells were resuspended in complete medium and incubated for at least 1 h at 37°C. This was followed by a final wash before incubation with ß T cells.

Cells were pulsed with 1 µCi of [³H]thymidine during the last 18 h of in vitro stimulation and harvested as previously described (13). Results are presented as mean cpm ± SD of triplicate wells. Stimulation indices were calculated by dividing the cpm of cultures stimulated with T. gondii by cpm of cultures without T. gondii.

Inhibition experiments using mAb

In some experiments, ß T cells were incubated with saturating concentrations of either anti-CD4 (OKT4; IgG2b, ATCC), anti-CD8 (OKT8; IgG2, ATCC), or isotype control mAbs (PharMingen, San Diego, CA) for 30 min on ice before adding PBMC and antigenic preparations. When anti-MHC class II mAbs were used, PBMC were incubated with saturating concentrations of either anti-HLA-DR (L243; IgG2a, ATCC), anti-HLA-DQ (SPV-L3; IgG2a, generous gift from Dr. Hans Yssel), or isotype control mAbs (PharMingen) for 30 min on ice before adding T cells.

Analysis of Vß expression

PBMC (2.5 x 10⁶/ml) were stimulated with either tachyzoites of T. gondii (1 tachyzoite/PBMC) or TLA (10 µg/ml) for 7 days; or SEB (100 ng/ml) or PHA (0.25 µg/ml) for 4 days. Cells were then washed and cultured for another 48 h in the presence of IL-2 (120 IU/ml; Chiron, Emeryville, CA) as previously described (17). Before culture, and after in vitro stimulation, cells were analyzed by two-color cytofluorometry as described below.

FACS analysis

To determine purity and phenotypic composition of T cell preparations, cells were incubated for 30 min at 4°C with the following mAbs in PBS containing 1% FBS and 0.1% sodium azide: anti-TCR-ß, anti-TCR-, anti-CD3, anti-CD4, anti-CD8, anti-CD16, anti-CD56 (all from Becton Dickinson), anti-CD45RA, and anti-CD45RO (Immunotech).

Flow cytometry was also used to analyze the TCR repertoire. This was done by staining cells with either phycoerythrin (PE)-conjugated anti-CD4 or PE-conjugated anti-CD8 mAb and one of the following FITC-conjugated mAb directed against the following Vß-chains (from Immunotech except when noted): Vß2, Vß3.1 (T Cell Diagnostics, Woburn, MA), Vß5.1, Vß5.2, Vß8, Vß12, Vß13.1, Vß13.6, Vß16, Vß18, Vß21.3, Vß22. Samples were analyzed on a FACScan cytofluorometer (Becton Dickinson). Isotype control mAb were used to assess background fluorescence. Resting T cells and blasts were gated according to forward angle and 90° light scatter patterns.

Cytokine assays

Purified ß T cells (1 x 10⁶/ml) were incubated with either T. gondii-infected or uninfected purified monocytes (5 x 10⁵/ml). Supernatants collected at 24, 48, and 72 h were used to measure concentrations of IL-2, IL-4, and IFN-, respectively, by ELISA (13). Data is presented as mean of triplicate wells ± SEM. None of the cytokines assayed were detected in supernatants obtained from wells that lacked T cells and contained only monocytes with or without T. gondii tachyzoites.

Statistical analysis

Statistical significance was assessed by unpaired Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
ß T cells from T. gondii-seronegative individuals proliferate in response to the parasite

The proliferative response of resting ß T cells to T. gondii was studied. Figure 1, A through D, shows that not only ß T cells from healthy individuals chronically infected with T. gondii but also ß T cells from T. gondii-seronegative donors proliferated when incubated with autologous PBMC infected with T. gondii or pulsed with TLA. However, whereas ß T cells from both groups of donors exhibited a remarkable proliferative response at the higher doses of parasite Ags, only ß T cells from seropositive individuals had significant proliferation at the lowest doses of parasite Ags. Proliferative response to T. gondii occurred regardless of whether tachyzoites were obtained from human foreskin fibroblasts or peritoneal cavities of infected mice (data not shown). Furthermore, no T cell proliferation was detected when either tachyzoite-free peritoneal lavage fluid or lysate from uninfected foreskin fibroblasts were used instead of T. gondii preparations (data not shown). Although ß T cells from every seronegative donor tested (n = 10) proliferated in response to T. gondii, this response varied among individuals (T. gondii-infected cells: mean SI = 68.0, range 17.8–199.1; TLA-pulsed cells: mean SI = 51.1, range 11.2–137.6).

View larger version (12K): [in this window] [in a new window]   FIGURE 1. ß T cells from both T. gondii-seronegative (A and C) and T. gondii-seropositive (B and D) individuals proliferate in response to T. gondii. ß T cells were incubated with either decreasing concentrations of T. gondii-infected PBMC (decreasing infected PBMC:T cell ratios) (A and B) or uninfected PBMC plus decreasing concentrations of TLA (C and D). Results are representative of three independent experiments.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Time course of proliferation of ß T cell from T. gondii-seronegative individuals in response to T. gondii, exogenous superantigen (TSST-1), recall Ag (tetanus toxoid), mitogen (PHA), or allogeneic PBMC (Allo-PBMC). A, ß T cells were stimulated with autologous PBMC that were either infected with T. gondii (PBMC + Tg), incubated with TLA (PBMC + TLA), or used as controls (PBMC). B, ß T cells were also incubated with autologous PBMC plus either TSST-1 (PBMC + TSST-1), tetanus toxoid (PBMC + Tet Tox), or PHA (PBMC + PHA), or with allogeneic PBMC (Allo-PBMC). Results are representative of three independent experiments.

CD4⁺ ß T cells preferentially respond to T. gondii

To determine whether a particular subpopulation of ß T cells preferentially responds to T. gondii, cytofluorometric analyses of purified resting ß T cells from seronegative individuals were performed before and after in vitro stimulation with T. gondii. Compared with unstimulated ß T cells, stimulation with either PBMC infected with T. gondii or pulsed with TLA resulted in an increase in the percentage of CD4⁺ T cells (Table I). Furthermore, more than 94% of the ß T cell blasts obtained after in vitro stimulation with parasite Ags were CD4⁺. These results were not caused by a nonspecific response of CD4⁺ T cells due to in vitro culture conditions, since stimulation of ß T cells with PHA did not result in an increase in the percentage of CD4⁺ T cells. The lack of a significant CD8⁺ T cell response to T. gondii was not due to failure of the experimental conditions to provide stimulus to CD8⁺ T cells, since incubation of ß T cells from chronically infected individuals with cells that contained intracellular tachyzoites induced a significant CD8⁺ ß T cell blast population (Table I).

View this table: [in this window] [in a new window]   Table I. Phenotypic composition of ß T cells stimulated with either T. gondii or PHA1

View larger version (15K): [in this window] [in a new window]   FIGURE 3. CD4+ T cells are the subset of ß T cells from T. gondii-seronegative individuals that preferentially responds to the parasite. A, ß T cells were incubated with T. gondii-infected PBMC (PBMC + Tg), PBMC plus TLA (PBMC + TLA), or control PBMC in the presence of either isotype control mAb (IgG2b), anti-CD4 mAb, or anti-CD8 mAb. B, ß CD4+, ß CD8+, or unseparated ß T cells were incubated with T. gondii-infected PBMC (PBMC + Tg), PBMC plus TLA (PBMC + TLA), or control PBMC. Results are representative of three independent experiments.

MHC class II molecules are required for T. gondii-mediated ß T cell proliferation

In view of the preferential response of CD4⁺ T cells, experiments were conducted to determine whether T. gondii-mediated T cell proliferation in seronegative individuals was dependent on MHC class II molecules. mAb to MHC class II molecules inhibited the proliferative response of ß T cells to T. gondii (Fig. 4). The degree of inhibition varied depending on the mAb tested. Anti-HLA-DR mAb (L243) induced 89.9% inhibition (range, 81.9–100%; p 0.0003) of the proliferation in response to infected cells and 83.8% inhibition (range, 71.8–100%; p 0.01) of the proliferation in response to TLA. In contrast, anti-HLA-DQ (SPV-L3) induced 15% inhibition (range, 3.8–27.5%) (p range, 0.6–0.02) of the proliferation in response to infected cells and no inhibition (0%) of that in response to TLA (n = 3). These results were not due to a nonspecific inhibitory effect of the anti-MHC class II mAb used, since none of these mAb induced significant inhibition of the proliferation of ß T cells to PHA (data not shown). Thus, these results demonstrate that the proliferative response of ß T cells from seronegative donors to T. gondii requires MHC class II molecules, in particular HLA-DR molecules.

View larger version (17K): [in this window] [in a new window]   FIGURE 4. Anti-MHC class II mAb inhibit proliferation of ß T cells in response to T. gondii. ß T cells were incubated with either T. gondii-infected PBMC (PBMC + Tg), PBMC plus with TLA (PBMC + TLA), or control PBMC in the presence of either anti-HLA-DR, anti-HLA-DQ, or isotype control mAb (IgG2a). Results are representative of three independent experiments.

CD4⁺CD45RA⁺ and CD4⁺CD45RO⁺ T cells from seronegative donors were purified to determine their role in the ß T cell proliferation to T. gondii. Whereas CD4⁺CD45RO⁺ T cells proliferated in response to a recall Ag (tetanus toxoid), CD4⁺CD45RA⁺ failed to proliferate, confirming their naive status (Fig. 5). Incubation with either T. gondii-infected PBMC or allogeneic PBMC induced proliferation of both CD4⁺CD45RA⁺ and CD4⁺CD45RO⁺ T cells.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Both CD45RA+CD4+ and CD45RO+CD4+ ß T cells from T. gondii-seronegative adults proliferate in response to T. gondii. T cells were incubated with autologous PBMC that had been either infected with T. gondii (PBMC + Tg), incubated with tetanus toxoid (PBMC + Tet Tox), or used as controls (PBMC) or with allogeneic PBMC (Allo-PBMC). Results are representative of three independent experiments.

View larger version (12K): [in this window] [in a new window]   FIGURE 6. ß T cells from T. gondii-seronegative healthy newborns proliferate in response to T. gondii. T cells were incubated with either T. gondii-infected autologous CBMC (CBMC + Tg), CBMC plus tetanus toxoid (CBMC + Tet Tox), control CBMC, or allogeneic PBMC (Allo-PBMC). Results are representative of three independent experiments.

Processing of T. gondii Ag(s) is required for ß T cell proliferation

To determine whether processing of T. gondii Ag(s) is necessary to induce an ß T cell response, proliferation assays were conducted using either paraformaldehyde-fixed or untreated PBMC (Fig. 7). The complete inhibition of ß T cell proliferation in response to tetanus toxoid induced by fixation of PBMC demonstrated that, as previously reported (20), the protocol of fixation resulted in inhibition of Ag processing (Fig. 7A). However, despite the use of fixed PBMC, ß T cells still exhibited a remarkable proliferative response to a superantigen (TSST-1) that does not require Ag processing. Use of fixed PBMC completely abrogated the proliferative response of ß T cells to T. gondii. The lack of T cell proliferation in response to the parasite did not appear to be caused by impaired recognition of T. gondii Ags-MHC molecules due to the fixation protocol. T cell proliferation was observed after stimulation with either T. gondii-infected PBMC or PBMC preincubated with T. gondii and then fixed with paraformaldehyde (Fig. 7B). In parallel experiments, ß T cells failed to proliferate in response to paraformaldehyde-fixed PBMC plus TLA but proliferated when incubated with fixed PBMC plus a superantigen (SEB) (Fig. 7B). In addition, stimulation with paraformaldehyde-fixed T. gondii-infected PBMC has allowed us to generate T. gondii-reactive ß T cell lines (21). Taken together, our results indicate the need for processing of T. gondii Ags for induction of an ß T cell response.

View larger version (17K): [in this window] [in a new window]   FIGURE 7. T. gondii Ags require processing by APCS to induce proliferation of ß T cells from T. gondii seronegative individuals. A, T cells were incubated with either unfixed PBMC or paraformaldehyde-fixed PBMC (fPBMC) plus either TLA, tetanus toxoid (Tet Tox), or TSST-1. B, T cells were incubated with either unfixed PBMC, T. gondii-infected PBMC (PBMC + Tg), or PBMC plus TLA, or with paraformaldehyde-fixed PBMC (fPBMC), fixed T. gondii-infected PBMC (f[PBMC + Tg]), or fixed PBMC plus either TLA or SEB. Results are representative of three independent experiments.

The effects of incubation of PBMC with either T. gondii tachyzoites, TLA, SEB, or PHA on TCR Vß expression was analyzed by flow cytometry. Stimulation with PHA did not selectively expand any of the Vß studied, since comparison of PHA-induced T cell blasts with freshly isolated T cells showed similar patterns of Vß expression (Table II). As previously described (22), SEB induced expansion of Vß3.1- and Vß12-bearing T cells. Both PHA and SEB had similar effects on CD4⁺ and CD8⁺ ß T cells. Thus, for each of the Vß studied, the percentage of CD4⁺ and CD8⁺ ß T cells remained unchanged (data not shown). In contrast, only 1 to 2% of the blast populations induced after incubation with either tachyzoites or TLA were CD8⁺TCR-ß⁺. Therefore, we concentrated our analysis on CD4⁺ ß T cells.

T. gondii stimulates production of IFN- by resting ß T cells from seronegative individuals

To study the production of cytokines by ß T cells from T. gondii-seronegative donors, either uninfected or T. gondii-infected purified autologous monocytes were incubated with resting ß T cells. Whereas incubation with uninfected monocytes did not result in production of IFN-, significant amounts of this cytokine were produced after stimulation with infected monocytes (Fig. 8). Incubation of ß T cells with uninfected monocytes plus PHA resulted in production of significant amounts of IFN- and IL-2, while the production of IL-4 was either low or below detectable levels. Neither IL-2 nor IL-4 were detected after stimulation with infected monocytes (data not shown).

View larger version (21K): [in this window] [in a new window]   FIGURE 8. ß T cells from T. gondii-seronegative donors produce IFN- in response to T. gondii. ß T cells were incubated with either T. gondii-infected autologous monocytes (Mono + Tg) or uninfected monocytes (Mono). After 72 h, supernatants were collected and IFN- was measured by ELISA. Results are representative of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our initial demonstration that ß T cells from seronegative individuals react to T. gondii in vitro suggested the possibility that the parasite might contain T cell mitogens. However, this hypothesis is not supported by our data. The inhibitory effect of anti-MHC class II mAb on the T. gondii-mediated ß T cell proliferation, the requirement for Ag processing in order for these cells to respond to the parasite and the changes in the TCR Vß repertoire induced by stimulation with the parasite argue against the presence of a T. gondii T cell mitogen in the experimental system we studied.

Despite the absence of serologic evidence of infection with T. gondii, we considered that prior in vivo exposure to cross-reactive T cells epitopes from other immunogens ubiquitously present in the environment could have resulted in clonal expansion of ß T cells that could recognize T. gondii. This possibility was addressed by studying the in vitro reactivity of CD45RA⁺ and CD45RO⁺CD4⁺ T cells. The expression of these isoforms of the CD45 molecule has been associated with naive (CD45RA⁺) and memory (CD45RO⁺) CD4⁺ T cells (23). Our demonstration that both CD45RA⁺ and CD45RO⁺ cells proliferated in response to the parasite, whereas only CD45RO⁺ T cells responded to a recall Ag, does not support this hypothesis. However, it is important to keep in mind that the induction of expression of memory surface phenotype after T cell activation may not be permanent since reversion to a naive surface phenotype appears to occur (24). Nevertheless, the response of neonatal ß T cells to T. gondii strongly argues against prior exposure to epitopes that cross-react with T. gondii as the mechanism responsible for ß T cell reactivity to the parasite.

Another potential explanation for our results was that ß T cells underwent in vitro priming during incubation with T. gondii. It has been reported that under certain conditions a primary T cell response can be generated in vitro (25, 26). However, there are significant differences between the T. gondii reactivity of ß T cells that we demonstrated here and the T cell responses in the models in which in vitro priming occurred. Purified dendritic cells instead of PBMC were required as APCs to induce primary T cell sensitization, which translated into Ag-specific proliferation detected 7 days after in vitro stimulation (26). In other models in which PBMC could successfully induce in vitro priming, T cell proliferation was detected only after antigenic restimulation (25). In contrast, our studies using PBMC as APC demonstrate that remarkable ß T cell proliferation was already detectable 3 days after in vitro stimulation with T. gondii-infected cells. Thus, it appears that the mechanism(s) responsible for the in vitro response to T. gondii of ß T cells from unexposed individuals are likely to differ from those that lead to primary in vitro T cell sensitization.

Our initial results revealing the requirement of MHC class II molecules for ß T cell proliferation suggested that these cells might be responding to a T. gondii superantigen. In fact, it has been reported that stimulation of nonimmune murine splenocytes with T. gondii results in an expansion of CD8⁺ T cells that bear Vß5 TCR (27). These results led the investigators to propose that the parasite contains a superantigen for murine T cells (27). Several of our observations indicate the stimulus for the in vitro response of human ß T cells from seronegative humans does not behave like a typical exogenous superantigen. Although there is some controversy regarding the requirement for the processing of certain exogenous superantigens by APCs (28), the large body of evidence indicates that exogenous superantigens do not require processing by APCs to stimulate T cells (20, 29). This conclusion is in sharp distinction to our data, which demonstrates the need for processing of T. gondii Ags to trigger ß T cell proliferation. Whereas exogenous superantigens generally stimulate both CD4⁺ and CD8⁺ T cells (20, 30), here we demonstrate that T. gondii stimulates only human CD4⁺ T cells. The recognition of a superantigen by T cells depends on the expression of the appropriate TCR Vß sequence, with little contribution by other variable components of the TCR (22). The central role that Vß regions have on the T cell stimulation mediated by superantigens is reflected in our demonstration that the T cell blast populations generated by an exogenous superantigen (SEB) had a restricted expression of Vß regions, which was consistent in every donor tested. In contrast, the T cell blast populations induced by stimulation with T. gondii were quite heterogeneous in regard to Vß expression, and there was no consistent pattern of Vß expansion among the different donors tested. The fact that incubation with T. gondii increased the percentages of CD4⁺ T cells that expressed certain Vß regions does not necessarily indicate the presence of a superantigen in the parasite preparations, since imbalances in the Vß repertoire can occur during the immune response to conventional Ags (31). Our results indicate that there is a mechanism(s) involved in the in vitro response of ß T cells from T. gondii seronegative humans that differs from that induced by typical exogenous superantigens. The discrepancies between the results obtained after T. gondii stimulation of T cells from unexposed humans and those obtained using T cells from naive mice may suggest that some of the early immunologic events that occur in mice after exposure to T. gondii do not reflect those that occur in humans.

The explanation for the remarkable in vitro reactivity to T. gondii exhibited by ß T cells from previously unexposed individuals remains to be identified. It is interesting to note that many features of this response resemble that to alloantigens. Mixed lymphocyte reaction is characterized by proliferation of CD4⁺ T cells that is driven by MHC-class II molecules. Using methodology similar to that employed in our studies, the changes in the TCR Vß repertoire during mixed lymphocyte reaction were reported to differ from individual to individual (32). Moreover, studies of TCR repertoire during graft-vs-host disease, an illness triggered by allorecognition, have demonstrated a T cell response that is oligoclonal and not TCR Vß restricted (33, 34). Molecular mimicry has been proposed as a mechanism that may explain allorecognition (35). According to this view, a resemblance between allogeneic MHC molecules and nominal Ag-self MHC complexes would result in self-MHC-restricted T cells recognizing alloantigens. Arguments used to support the molecular mimicry theory (36) include the demonstration of T cell clones with this type of dual specificity (37) and the observation that T cells previously primed in vivo as defined by expression of CD45RO molecules (presumably self-MHC-restricted T cells) recognize alloantigens (38). Of interest in this regard is our demonstration that both CD45RA⁺ and CD45RO⁺ CD4⁺ ß T cells from unexposed individuals responded in vitro not only to alloantigens but also to T. gondii. It remains to be determined whether molecular mimicry is responsible for the in vitro reactivity to T. gondii by ß T cells from unexposed individuals. Of potential relevance to this hypothesis is the demonstration that Plasmodium falciparum-reactive T cell clones recognize bacterial, viral, fungal, and protozoan Ags (39).

IFN- plays a critical role in the immune response against T. gondii. This cytokine has been shown to confer protection during both the acute and chronic phases of infection (11, 40, 41). Our results indicate that during the early stages of the immune response, not only NK cells but also ß T cells, the predominant subset of T cells, are an important source of IFN-. It has been proposed that protective immunity to T. gondii is associated with induction of a Th1-type T cell response (42). Thus, the early production of IFN- may confer protection to the host not only because of the direct effects of this cytokine on the growth of intracellular tachyzoites (40) but also because IFN- appears to play a role in promoting the generation of a Th1 cytokine pattern (43). Therefore, the innate capacity of humans to control T gondii infection may be due, at least in part, to the remarkable early ß T cell response that we have demonstrated.

There is evidence that microbial Ags can elicit an immune response without conferring protection against the offending pathogen (44, 45). Although acute infection with T. gondii is usually uneventful in humans, T. gondii successfully avoids elimination from the host, leading to a chronic (quiescent) infection, despite the strong early T cell response elicited by the parasite. Thus, it is conceivable that the early ß T cell response triggered by T. gondii may not be directed against Ags that lead to the elimination of the parasite. Moreover, given that potent polyclonal T cell proliferation can be associated with reduced response to neoantigens (46), the induction of such a massive ß T cell response may interfere, at least temporarily, with the development of protective cell-mediated immunity, allowing the micro-organism to "escape" by forming tissue cysts. In addition, such a response may be involved in the induction of immunosuppression observed during recently acquired T. gondii infection (47, 48). It is also possible that under certain circumstances this ß T cell response may be implicated in some of the manifestations of the disease caused by T. gondii (toxoplasmosis). Approximately 10% of humans acutely infected with the parasite develop a self-limiting illness, usually manifested by lymphadenopathy (49). This form of toxoplasmosis presents with pathologic changes in lymph nodes that may be difficult to distinguish from lymphoproliferative disorders such as lymphoma (50). It remains to be determined whether an exaggerated T cell response or the failure to control this response is involved in the pathogenesis of toxoplasmic lymphadenopathy.

Previous reports on the in vitro response of human T cells to T. gondii did not demonstrate proliferation of T cells from seronegative individuals (51, 52). This apparent discrepancy with our results can be explained by our demonstration of remarkable differences between the proliferative response of T cells from seropositive individuals and that of seronegative individuals to varying concentrations of T. gondii Ag preparations. Studies of T cell-mediated immunity in T. gondii-infected humans and, in particular, in vitro studies aimed at the identification of parasite Ags recognized specifically by T cells from these individuals should be performed using carefully chosen doses of T. gondii Ag preparations. A detailed understanding of the early response of ß T cells to T. gondii, including the identification of the Ag(s) responsible for triggering this phenomenon, is important to the effort to identify protective T. gondii Ags using in vitro assays of T cell function.

	Acknowledgments

 
We thank H. Yssel and M. Brenner for providing reagents.

	Footnotes

 
¹ C.S.S. was supported by National Institutes of Health Grant AI37936-01A1, an AmFAR Grant made in memory of Walter J. Smith, and a grant from the University of California Universitywide AIDS Research Program. This work was supported, in part, by National Institutes of Health Grants AI04717 and AI30230.

² Address correspondence and reprint requests to Dr. Carlos S. Subauste, Division of Infectious Diseases, Department of Medicine, University of Cincinnati College of Medicine, P.O. Box 670560, Cincinnati, OH 45267-0560. E-mail address:

³ Abbreviations used in this paper: CDR3, complementarity determining region 3; CBMC, cord blood mononuclear cells; SEB, staphylococcal enterotoxin B; TLA, toxoplasma lysate Ag; TSST-1, toxic shock syndrome toxin-1; SI, stimulation index.

Received for publication July 15, 1997. Accepted for publication November 26, 1997.

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