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Amastigote Surface Proteins of Trypanosoma cruzi Are Targets for CD8⁺ CTL¹

Department of Cellular Biology, University of Georgia, Athens, GA 30602

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Amastigotes of Trypanosoma cruzi express surface proteins that, when released into the host cell cytoplasm, are processed and presented on the surface of infected cells in the context of MHC class I molecules to be recognized by CD8⁺ CTL. To further understand the role of CTL in T. cruzi infection, we used the available MHC class I peptide binding motifs to identify potential CTL target epitopes in two recently described T. cruzi amastigote surface proteins, ASP-1 and ASP-2. The predicted amino acid sequences of ASP-1 and ASP-2 were screened for H-2^b allele-specific class I peptide motifs, and four peptides (PA11, PA12, PA13, and PA14) and six peptides (PA5, PA6, PA7, PA8, PA9, and PA10) were synthesized from ASP-1 and ASP-2, respectively. The majority of the peptides bound to some degree to H-2^b class I MHC molecules, and six of 10 of the peptides stimulated spleen cells from T. cruzi-infected mice to lyse target cells sensitized with the homologous peptides. Short term T cell lines specific for three of these peptides also lysed T. cruzi-infected target cells. These results demonstrate that ASP-1 and ASP-2 are targets of in vivo generated CTLs and that this CTL response induced by T. cruzi infection is parasite and peptide specific, MHC restricted, and CD8 dependent.

	Introduction

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Chagas’ disease, the result of infection with the parasitic protozoan Trypanosoma cruzi, is a major health problem in Latin America. An estimated 16 to 18 million people are infected, and an additional 90 million are at risk of infection with T. cruzi. Current chemotherapies for treating the infection are inadequate, and immunoprophylactic regimens to prevent infection are nonexistent. Immune control of T. cruzi requires the actions of multiple mechanisms, including a strong humoral immune response, a potent type 1 cytokine production and the activation of CD8⁺ T cells for recognition of parasite-infected host cells (reviewed in 1 . To date, few vaccination protocols have been shown to be effective in eliciting the cadre of responses necessary to protect hosts from infection, and fewer still parasite molecules have been identified that might be the target of such protective responses. One potential explanation for this situation is that most studies of potential vaccine candidates have focused on molecules expressed in the insect stage epimastigotes or on blood-form trypomastigote stages of the parasite. In contrast, the intracellular amastigote stage has been relatively poorly studied for the identification of molecules that might be the target of protective immune responses.

The recent demonstration of the critical importance of CD8⁺ T cells in immunity to T. cruzi has focused attention on immune recognition of the parasite-infected host cell, and thus on amastigote-derived peptides that might be involved in this recognition (2, 3, 4, 5, 6, 7). CD8⁺ T cells are major constituents of the inflammatory foci in T. cruzi-infected tissues (8, 9, 10, 11, 12), and an intact CD8⁺ T cell compartment is required for mice to survive the acute stage of T. cruzi infection (2, 4, 6, 7). We also reported that CD8⁺ T cell lines specific for a peptide from the TSA-1³ protein could transfer to naive mice protection from lethal infection with T. cruzi (13), providing the first evidence for the immunoprotective capacity of T. cruzi Ag-specific CD8⁺ T cells. In an attempt to identify additional CD8⁺ T cell target molecules that might also be capable of inducing protection to T. cruzi infection, we began a project to clone genes expressed in the intracellular amastigote stage. This effort focused on the identification of amastigote proteins that were surface anchored by glycosylphosphatidylinositol (GPI) linkages, since previous studies had shown that GPI-anchored proteins were released by amastigotes into the host cell cytoplasm (14) and from this site of release entered the class I MHC processing and presentation pathway (15).

The result of this effort to date has been the cloning of two amastigote surface proteins termed ASP-1 and ASP-2. ASP-1 was identified by screening an amastigote expression library using an amastigote-specific mAb (16). ASP-2 was originally identified as the 83-kDa amastigote protein by Pan and McMahon-Pratt (17) and was cloned based upon published N-terminal amino acid sequence data (18). In this report, we document that, like TSA-1, ASP-1 and ASP-2 are targets of T. cruzi-specific CD8⁺ T cell responses. Peptides from ASP-1 and ASP-2 bind to mouse class I MHC molecules and sensitize cells for lysis by CD8⁺ T cells. ASP-1- and ASP-2-specific CTL also lyse cells infected with T. cruzi, conclusively demonstrating that peptides from these two amastigote stage proteins enter the class I MHC presentation pathway.

	Materials and Methods

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Parasites and mice

The Brazil strain of T. cruzi was used in this study. Blood-form trypomastigotes were maintained in mice, and tissue culture-derived trypomastigotes (TCT) were propagated in Vero cells (CCL 81, American Type Culture Collection, Rockville, MD) as previously described (19). Female C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were infected i.p. with 10³ blood-form trypomastigotes and challenged with 1 x 10⁵ TCT 2 to 5 mo following the primary infection. Spleens were harvested from mice 3 wk to 4 mo postchallenge. Naive mice were not infected or challenged.

Cell culture

Parasites and cell lines were maintained in RPMI 1640 medium (Mediatech, Washington, DC) supplemented with 10% FBS (HyClone Laboratories, Logan, UT), 2 mM L-glutamine (Life Technologies, Gaithersburg, MD), 1 mM sodium pyruvate (Sigma Chemical Co., St. Louis, MO), 50 µM 2-ME (Life Technologies), and 50 µg/ml gentamicin (Life Technologies). The T cell medium (TCM) used for culture of CTL effectors was additionally supplemented with 0.1 mM nonessential amino acids (Life Technologies).

Peptide synthesis

Peptides (Table I) were synthesized by the Molecular and Genetic Instrumentation Facility (University of Georgia, Athens, GA) using a model 350 MPS peptide synthesizer (Advanced ChemTech, Louisville, KY) by standard F-moc-based solid phase chemistry. The control peptides used include peptide 40, a D^b motif-bearing, nine-amino acid peptide from the T. cruzi SA85-1.1 gene (20) that was previously shown not to bind to K^b or D^b molecules (13); AdE1A_234–243, a D^b-binding CTL target epitope from the adenovirus type 5 E1A molecule, residues 234 to 243 (21); OVA_257–264, a K^b-binding CTL target epitope from the OVA molecule, residues 257 to 264 (22); and peptide 77.2, a K^b-binding CTL target epitope from the T. cruzi TSA-1 protein, residues 515 to 522 (13). Lyophilized peptides were dissolved in sterile PBS to 5 mg/ml and were stored at -20°C. Before use, peptides were further diluted in RPMI 1640. Peptides were not toxic to target cell or effector cell cultures.

RMA-S cells (H-2^b; a gift from Dr. Michael Oldstone, Scripps Research Institute, La Jolla, CA) cultured at 37°C in complete RPMI 1640 were adjusted to 1 x 10⁶ cells/ml and transferred to 26°C for at least 16 h. The cells were then harvested, adjusted to 5 x 10⁵/ml, incubated in the presence of peptides at a concentration of 0.005 to 50 µM for 30 min at 26°C, and then shifted to 37°C for 1 to 2 h. The cells were then harvested and washed with cold FACS buffer (1% BSA and 0.05% sodium azide in 1x PBS, pH 7.2–7.4). The ability of peptides to stabilize MHC expression on the RMA-S cells was determined by incubation with a 1/1000 dilution of ascites fluid containing the anti-K^b Y3 Ab (HB 176, American Type Culture Collection, Rockville, MD) or an anti-D^b mAb at 10 µg/ml (clone 28-8.6, PharMingen, San Diego, CA) for 30 min to 1 h on ice followed by an FITC-labeled anti-mouse Ig Ab (Southern Biotechnology Associates, Inc., Birmingham, AL) as the second Ab. The cells were then subjected to flow cytometric analysis on an EPICS Elite (Coulter Electronics, Hialeah, FL).

Generation of effector cells

Peptide-stimulated effector cells were generated by culturing 5 x 10⁶ spleen cells (SC) from T. cruzi-infected mice in 2-ml volumes containing 5 to 10 µM peptide. Following 2 days of culture at 37°C in 6% CO₂, the culture medium was supplemented with 5% rat Con A supernatant (Rat T-STIM culture supplement without Con-A, Collaborative Biomedical Products, Bedford, MA) and incubated for an additional 4 days. In some cases, effector cell populations were further treated to deplete CD8⁺ or CD4⁺ cells by incubating effector cells on ice with 10 µg/ml of anti-CD8 Ab (mAb H35.17.2) (23) or anti-CD4 Ab (mAb GK1.5, American Type Culture Collection TIB 207) followed by a 30-min incubation at 37°C with a 1/6 dilution of rabbit complement (Pel-Freez, Brown Deer, WI). Flow cytometric analysis was performed to ensure effective depletion of the indicated T cell subsets. In experiments using T. cruzi-infected 5A.Kb.3 cells (L cells transfected with the K^b gene; provided by Dr. S. Jameson, University of Minnesota, Minneapolis, MN) as targets, peptide-specific effectors were short term T cell lines generated following 2-wk cycles of stimulation of SC with peptide. For the primary stimulation, 1 x 10⁸ SC from infected mice were cultured in upright 75-cm² flasks containing 25 ml of TCM with 5 µM peptide. Rat Con A supernatant (to a final concentration of 5%) was added after 48-h incubation, and the cells were cultured for an additional 4 days. For the second round of stimulation, the cells were harvested, washed, and cocultured with 1 x 10⁸ irradiated (3000 rad, Gammacell 200 ⁶⁰Co source; Atomic Energy of Canada, Ltd., Ottawa, Canada), naive syngeneic SC in 25 ml of TCM with 5 µM peptide and 5% rat Con A supernatant. After 6 days of culture, viable effector cells were obtained by density gradient centrifugation on Lymphocyte Separation Medium (Organon Teknika, Durham, NC), and their CTL activity was assessed in ⁵¹Cr release assays.

Preparation of target cells

RMA-S cells or P815 cells (H-2^d; American Type Culture Collection TIB 64) were seeded into 24-well plates (10⁶ cells/2 ml/well) and incubated overnight with 1 to 10 µM peptide and 100 µCi of Na₂⁵¹CrO₄ (Amersham Life Science, Inc., Arlington Heights, IL) at 37°C in 6% CO₂. T. cruzi-infected 5A.Kb.3 cells were prepared by incubating cell monolayers (70% confluent) for 18 h with TCT at a 50:1 parasite:host cell ratio. After extensive washing with serum-free RPMI to remove noninvading parasites, cells in the monolayer were harvested by treatment with PBS containing 1 mM EDTA (Life Technologies). The resulting single cell suspensions were washed and labeled with 100 µCi of ⁵¹Cr for 1 h at 37°C. Diff-Quik (Fisher Scientific, Pittsburgh, PA)-stained cytospin preparations of cells indicated that approximately 75% of the cells were infected.

⁵¹Cr release assay

⁵¹Cr release assays to assess CTL activity were performed as previously described (24). Briefly, effector cells were washed, diluted with TCM, and plated into triplicate wells of 96-well round-bottom plates (Corning, Corning, NY). Labeled target cells were washed three times with RPMI 1640 and resuspended in TCM, and 5 x 10³ cells were added to the effector cells in a final volume of 200 µl/well at different E:T cell ratios. After a 5-h incubation at 37°C in 6% CO₂, supernatants were harvested using a Skatron MultiHarvesting System (Skatron Instruments, Inc., Sterling, VA) for gamma counting on a Cobra II Autogamma counter (Packard Instrument Co., Downers Grove, IL). The percent specific ⁵¹Cr release was calculated from the mean of triplicate wells as follows: [(experimental counts per minute - spontaneous counts per minute)/(maximum counts per minute - spontaneous counts per minute)] x 100. Maximum and spontaneous ⁵¹Cr release were determined from triplicate wells containing labeled target cells only with no effectors in the presence or the absence of 5% SDS, respectively. SEs for means were generally <6% and were always <10%.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Some of the structural features important for binding of peptides to class I molecules have been reported previously (25, 26). To identify potential class I MHC binding peptides that might be derived from the processing of the T. cruzi ASP-1 and ASP-2 proteins, the predicted amino acid sequences of these proteins were screened for peptide segments that conform to the murine H2-K^b and H2-D^b allele-specific class I peptide binding motifs (Table I). Peptides matching the H2-K^b binding motif contain the primary anchor residues Phe (F) or Tyr (Y) at position 5; Leu (L), Ile (I), Val (V), or Met (M) at position 8 (C terminus); and an auxiliary or secondary anchor residue Y at position 3. Peptides conforming to the H-2D^b binding motif contain Asn (N) at position 5, and M, I, or L at the C terminus (25, 26). Using these predictive models, eight potential K^b-binding peptides and three potential D^b binding peptides were identified in ASP-1, and eight potential K^b-binding peptides and four potential D^b binding peptides were identified in ASP-2. Of these, a total of 10 synthetic peptides were produced, some containing overlapping K^b and D^b motifs (Table I).

RMA-S cells, low H-2^b expressor mutants of the Rauscher virus-induced T lymphoma line RBL-5 (27), express "empty" MHC class I molecules that can be stabilized by exogenous peptides, resulting in surface expression of class I MHC (28, 29). Nonbinding negative control peptides (e.g., Pep40) fail to stabilize cold-induced class I MHC molecules, resulting in low surface staining of MHC, while the positive control peptides OVA_257–264 and AdEIA_234–243 markedly up-regulate surface MHC expression (Fig. 1). Of the three ASP-1 peptides tested, only PA14 bound efficiently to H2-K^b MHC molecules of RMA-S cells compared with the positive control OVA peptide (Fig. 1A). For the six ASP-2-derived peptides, PA6 and PA8 stabilized H2-K^b at a level comparable to that of OVA. In addition, PA5, PA7, PA9, and PA10 demonstrated more modest binding to H2-K^b (Fig. 1A). Peptides PA9, PA10, and PA11, each of which contained overlapping K^b and D^b motif-bearing sequences, also showed significant binding to H2-D^b (Fig. 1B).

View larger version (33K): [in this window] [in a new window]   FIGURE 1. MHC binding activity of ASP-1 and ASP-2 peptides to H2-Kb (A) and H2-Db (B) in a RMA-S stabilization assay. OVA257–264 was used as a positive control Kb stabilizer, and AdE1A234–243 was used as a positive control Db stabilizer. The line drawn in all panels represents the background level of MHC expression in the presence of the negative control peptide, Pep40.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Synthetic peptides from ASP-1 (A) and ASP-2 (B) sensitize target cells for lysis by CTL from T. cruzi-infected mice. SC from C57BL/6J mice infected and rechallenged with T. cruzi and stimulated for 6 days with individual peptides (5 µM) were used as effectors. CTL activity was measured in a 51Cr release assay using RMA-S cells pulsed with homologous peptides or Pep40 (1 µM) at the indicated E:T cell ratios.

View larger version (20K): [in this window] [in a new window]   FIGURE 3. CTL effectors specific for ASP-1 peptide PA14 (A) or ASP-2 peptides PA5, PA6, PA7, and PA8 (B) are present in T. cruzi-infected mice but not in naive mice. SC from immune (infected and rechallenged with T. cruzi) or naive C57BL/6J mice were stimulated for 6 days with individual peptides (10 µM) and then used as effectors. CTL activity was measured in a 51Cr release assay using RMA-S cells pulsed with homologous peptides or Pep40 (10 µM) at the indicated E:T ratios.

View larger version (28K): [in this window] [in a new window]   FIGURE 4. CTL specific for ASP-1 and ASP-2 peptides are MHC restricted and CD8+. SC from C57BL/6J mice previously infected and rechallenged at 4.5 mo and 7 wk, respectively, were stimulated in vitro with PA7, PA8, or PA14 (5 µM). After 6 days of culture, the CTL activity of the effector cells was measured against RMA-S (H-2b) or P815 (H-2d) cells pulsed with homologous peptides or Pep40 (5 µM) at the indicated E:T cells ratios (A). Effector cells were treated with mAb H35-17.2 (anti-CD8) plus complement, mAb GK1.5 (anti-CD4) plus complement, or rabbit complement alone and tested for CTL activity against the homologous peptide-pulsed RMA-S cells at a 50:1 E:T cell ratio (B).

View larger version (21K): [in this window] [in a new window]   FIGURE 5. CTL induced by PA8, PA14, and Pep77.2 do not recognize target cells sensitized with homologous peptides from related TS family members. SC from C57BL/6J mice infected and challenged with T. cruzi as described in Figure 4 were restimulated with PA8, PA14, or Pep77.2 (10 µM). The CTL activities of the resulting effectors were determined 6 days later using RMA-S cells pulsed with PA8, PA14, Pep77.2, or Pep40 (10 µM) as targets.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. CTL lines specific for PA7, PA8, and PA14 lyse T. cruzi-infected target cells but not noninfected cells. SC from mice infected and rechallenged with T. cruzi were cultured with the indicated peptides (5 µM) through two rounds of stimulation and tested for the ability to kill 5a.Kbalpha3 cells that were either uninfected or infected with T. cruzi.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We recently identified and cloned the genes encoding two surface proteins expressed by amastigotes of T. cruzi: ASP-1 was obtained by screening an amastigote stage cDNA library with an mAb raised against amastigote extracts (16), and ASP-2 was cloned by PCR using oligonucleotide primers based on the N-terminal amino acid sequence of an 83-kDa amastigote protein (18). Interestingly, despite the use of completely different techniques to identify these two amastigote proteins, both, like TSA-1, are members of subfamily II of the T. cruzi sialidase/TS family (38). In the present study, we report that, also like TSA-1, these two amastigote-stage proteins contain peptide sequences that induce and are the targets of CD8⁺ cytolytic T cells generated during the course of T. cruzi infection.

The screening of the amino acid sequences of ASP-1 and ASP-2 for H-2^b MHC class I binding motifs resulted in the identification of 16 peptides that conform to the H2-K^b binding motifs and seven peptides that conformed to the H2-D^b binding motifs. Multiple peptides from ASP-1 and ASP-2 were found to bind efficiently to H-2^b MHC molecules, confirming the predictive power of a motif-scanning approach to identify such epitopes. However, not all peptides matching the H-2^b binding motifs were able to bind to MHC, and the relative capacity of an individual peptide to stabilize MHC expression in RMA-S cells was not always a good predictor of whether that peptide was processed and presented in the infected cell. This result is in good agreement with those of other studies that have used similar approaches to predict CTL epitopes in various proteins (39).

This study brings to three the number of CTL target molecules identified in T. cruzi. In all three cases the CTL directed against epitopes of TSA-1, ASP-1, and ASP-2 are CD8⁺ and class I MHC restricted. Most importantly, CTL against all three molecules are induced in mice infected with T. cruzi and are able to lyse parasite-infected target cells, verifying that these proteins are released from intracellular parasites and enter the class I MHC processing and presentation pathway in infected host cells. The intracellular release of ASP-1 and ASP-2 had been previously predicted based upon the fact that both molecules are likely to be surface anchored via GPI structures (16, 18), an anchoring mechanism that facilitates protein release by multiple pathways (40). Thus, there appear to be at least two classes of CTL target molecules in T. cruzi, trypomastigote-encoded proteins such as TSA-1 that are released early in the host cell invasion process, perhaps during conversion of trypomastigotes to amastigotes, and amastigote-encoded proteins such as ASP-1 and ASP-2, which may be released throughout the cycle of intracellular development.

The fact that all three T. cruzi CTL target molecules identified to date are members of subfamily II of the TS superfamily is notable. While it is possible that this coincidence reflects an immunodominance of TS proteins in the induction of CTL, it seems unlikely that TS molecules are the only or even the best CTL targets on cells infected with T. cruzi. All three of these molecules were originally identified based on their ability to induce Abs (16, 17, 41), not CTL. A strategy to identify trypomastigote or amastigote proteins based on the primary criterion of induction of CTL responses may reveal other, non-TS CTL target molecules in T. cruzi. The fact that the use of three very different methods to identify CTL target molecules resulted in the cloning or characterization of subfamily II TS molecules may also reflect the size of this gene family, which has been estimated to number in the hundreds if not the thousands (20, 42). One interesting explanation for the expression of multiple, apparently enzymatically inactive, TS-like molecules by amastigotes of T. cruzi is that rather than serving as targets for the efficient destruction of infected cells by anti-TS CTLs, they, in fact, are molecules that facilitate evasion of the CTL response. We envision that there are at least two ways in which this evasion could be accomplished: 1) the production of large numbers of distinct peptide epitopes, essentially flooding the system such that the presentation of any one peptide is below the threshold level necessary for the efficient induction of CTL; or 2) the ability of peptides derived from different TS proteins to act as antagonists for T cells specific for other, homologous peptides (43). The existence of such altered peptide ligand effects (44) is made more likely by the finding that the H-2K^b binding and CTL target peptides 77.2 from TSA-1, PA14 from ASP-1, and PA8 from ASP-2 all map to identical regions of their respective molecules. In fact, analysis of the homologous regions of 30 other TS family members for which sequence information is available reveals that the anchor residues required for binding to H-2K^b are conserved in all but two cases, while residues at most other positions in these peptides are highly variable. The possibility that peptides from one TS family member can antagonize responses to peptides from other TS proteins is currently under investigation.

Despite the possibility that the expression of multiple TS family members may decrease the efficiency with which anti-TS molecule CTL responses are generated, TS-specific CTL responses are elicited in vivo and can destroy infected cells in vitro. In addition, preliminary experiments show that PBMC from humans with long term infections with T. cruzi contain readily measurable levels of CTLs to TSA-1-, ASP-1-, and ASP-2-derived peptides (B. Wizel and R. L. Tarleton, unpublished observation), demonstrating that the phenomenon of CTL production in response to TS proteins of T. cruzi is not restricted to murine systems. Finally, adoptive transfer of T cells specific for the TSA-1 peptide Pep77.2 protects naive mice from otherwise lethal infection with T. cruzi (13). The possibility that the induction of strong class I MHC-dependent responses to TS and/or other target molecules presented on infected host cells could provide partial protection from infection and/or disease offers a new avenue for vaccine development in this infection.

	Acknowledgments

 
The authors thank Steve Hilliard for assistance with flow cytometric analysis, Dr. Nisha Garg for advice and help with the figures, and Mark Heiges for excellent technical support.

	Footnotes

 
¹ This work was supported by a grant from the National Institute of Health (AI33060) and a Burroughs-Wellcome Fund Scholar in Molecular Parasitology (to R.L.T.).

² Address correspondence and reprint requests to Dr. Rick L. Tarleton, Department of Cellular Biology, University of Georgia, Athens, GA 30602.

³ Abbreviations used in this paper: TSA-1, trypomastigote surface Ag 1; GPI, glycosylphosphatidylinositol; ASP, amastigote surface protein; SC, spleen cells; TCT, tissue-cultured derived trypomastigotes; TCM, T cell medium; TS, trans-sialidase.

Received for publication July 17, 1997. Accepted for publication October 28, 1997.

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