Parasite Immunology, 2010, 32, 494–502
DOI: 10.1111/j.1365-3024.2010.01206.x
Frequency of specific CD8+ T cells for a promiscuous epitope derived from Trypanosoma cruzi KMP-11 protein in chagasic patients P. LASSO,1 D. MESA,1 A. CULLAR,2 F. GUZMN,3 N. BOLAOS,4 F. ROSAS,5 V. VELASCO,5 M. DEL CARMEN THOMAS,6 M. C. LOPEZ,6 J. M. GONZALEZ4 & C. J. PUERTA1 1 Laboratorio de Parasitologa Molecular, Pontificia Universidad Javeriana, Bogot, Colombia,2Grupo de Inmunobiologa y Biologa Celular, Pontificia Universidad Javeriana, Bogot, Colombia, 3Laboratorio de Gentica e Inmunologa Molecular, Pontificia Universidad Catlica de Valparaso, Chile,4Grupo de Ciencias Bsicas Mdicas, Facultad de Medicina, Universidad de los Andes, Bogot, Colombia,5Fundacin Clnica Abood Shaio, Bogot, Colombia, 6Instituto de Parasitologa y Biomedicina Lpez Neyra, CSIC, Granada, EspaÇa
SUMMARY The K1 peptide is a CD8+ T cell HLA-A*0201-restricted epitope derived from the Trypanosoma cruzi KMP-11 protein. We have previously shown that this peptide induces IFN-c secretion by CD8+ T cells. The aim of this study was to characterize the frequency of K1-specific CD8+ T cells in chagasic patients. Nineteen HLA-A2+ individuals were selected from 50 T. cruzi infected patients using flow cytometry and SSP-PCR assays. Twelve HLA-A*0201+ noninfected donors were included as controls. Peripheral blood mononuclear cells were stained with HLA-A2-K1 tetramer, showing that 15 of 19 infected patients have K1-specific CD8+ T cells (0Æ09–0Æ34% frequency) without differences in disease stages or severity. Of note, five of these responders were A*0205, A*0222, A*0226, A*0259 and A*0287 after molecular typing. Thus, a phenotypic and functional comparison of K1-specific CD8+ T cells from non-HLA-A*0201 and HLA-A*0201+ infected patients was performed. The results showed that both non-HLA-A*0201 and HLAA*0201+ individuals have a predominant effector memory CD8+ T cell phenotype (CCR7), CD62L)). Moreover, CD8+ T cells from non-HLA-A*0201 and HLA-A*0201+ individuals expressed IL-2, IFN-c and perforin without any differences. These findings support that K1 peptide is a proCorrespondence: Concepcin Puerta, PhD, Laboratorio de Parasitologa Molecular, Departamento de Microbiologa, Facultad de Ciencias, Pontificia Universidad Javeriana, Cra. 7a No. 43-82, Ed. 52, Of. 608, Bogot, Colombia (e-mail:
[email protected]). Disclosures: None Financial support: Colciencias, Research Project No. 1203-33318692 Received: 17 July 2009 Accepted for publication: 24 January 2010
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miscuous epitope presented by HLA-A2 supertype molecules and is highly recognized by chagasic patients. Keywords CD8+ T cell, Chagas’ disease, HLA-A2 supertype, Trypanosoma cruzi
INTRODUCTION Chagas’ disease is primarily a neglected tropical disease caused by the hemoflagellate Trypanosoma cruzi, affecting 28 million people in Latin-America where insects from the Reduviidae family constitute the parasite’s main vector (1). As a huge proportion of migrating people is infected, this parasitic disease is also a threat for nonendemic countries where blood transfusion, congenital transmission and organ transplants are the main routes of transmission (2). An acute phase is developed after the infection has been acquired in which a nonsterile immune response controls parasite levels (3,4). Individuals thus progress to a life-long chronic phase with a low level of parasitaemia and around 60–70% of them remain asymptomatic (indeterminate chronic phase) and 30–40% of them develop cardiac or digestive manifestations having variable intensity depending on several factors, including the type of infecting T. cruzi strains (3–6). Trypanosoma cruzi adopts an intracellular replication form during its life-cycle, which (as shown later) induces a CD8+ T cell immune response through cytokine production and cytotoxic activity, this being crucial for controlling the infection (7,8). CD8+ T cell-depleted mice develop earlier and more severe T. cruzi infection (9). However, only a few human CD8+ T cell parasite-specific epitopes have been described to date because of the parasite’s genetic complexity, a high degree of polymorphism among 2010 Blackwell Publishing Ltd
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strains and the previously described impairment of CD8+ T cell immune response during chronic Chagas’ disease (10–12). These class I restricted epitopes are encoded in different genes such as: amastigote-stage surface protein-1 (ASP-1), ASP-2, trypomastigote-form surface antigen-1 (11,13–15), kinetoplastid membrane protein-11 (KMP-11) (16), calcium-binding protein (11), ribosomal P2b protein (17), cruzipain and FL-160 flagellar protein (18). One of the antigens, T. cruzi KMP-11, is an abundant protein, expressed during the three stages of the parasite’s life-cycle, especially in insect-developed forms (19,20). This protein is able to elicit both B- and T-cell lymphoproliferation and cytotoxic responses (21–24). Immunization of HLA-A2 ⁄ Kb transgenic mice with the recombinant fusion protein encompassing the 70 kDa T. cruzi heat shock protein (HSP70) and KMP-11 sequences induced a cytotoxic response against human cells expressing KMP-11 protein (21) and protected immunized mice after parasite challenge (22). An HLA-A*0201-restricted cytotoxic epitope (termed K1) has been identified in the N-terminal region of the KMP-11 protein encompassing amino acids 4–12 (21). Our group has recently used ELISPOT assays to demonstrate that CD8+ T cells from chagasic patients recognize the K1 peptide within the context of the HLA-A*0201 molecule and specifically secreted IFN-c (16). This finding has suggested that the K1 peptide is efficiently processed, presented and recognized by CD8+ T lymphocytes during the natural course of Chagas’ disease. Studying specific K1 CD8+ T cells from chagasic HLA-A*0201+ donors has been expanded in the present work using a tetramer staining approach. Of special interest, our results indicate that the K1 peptide is highly recognized by infected patients in the context of other HLA-A2 subtypes besides the HLAA*0201 ones. It is also shown that K1 peptide-specific CD8+ T cells display a phenotype compatible with differentiated effector memory T cells (TEM). Thus, these cells are able to produce IL-2, IFN-c and perforin in response to K1 peptide irrespective of their HLA-A2 allele. These findings suggest that the K1 peptide is a promiscuous epitope presented by HLA-A2 supertype molecules.
MATERIALS AND METHODS Study population Four groups of donors were enrolled in the study; all of them were volunteers who had signed the informed consent form. Fifty chagasic donors who were anti-T. cruzi antibody-positive in both immunofluorescence assays and ELISA tests (1) were clinically evaluated at Fundacin Abood Clnica Shaio and the Instituto Nacional de Salud in Bogot, Colombia and classified according to the 2010 Blackwell Publishing Ltd, Parasite Immunology, 32, 494–502
Frequency of CD8+ T cells for K1 peptide
Kuschnir grading system (25). Twenty-one of those T. cruzi infected individuals were classified as being indeterminate chagasic patients or G0 stage [normal electrocardiography (ECG) and no major findings arising during clinical examination] and 29 as cardiac chronic chagasic patients, having different disease severity levels as follows: 13 G1 (abnormal ECG results), 11 G2 (abnormal ECG results and cardiac enlargement) and 5 G3 (abnormal ECG results, cardiac enlargement and clinical signs of heart failure). The group of healthy donors (HD) consisted of 20 sera-negative individuals who have always resided in nonendemic areas and exhibited normal ECG results and clinical examination, having similar ages to those of the chagasic donors. Approximately 20 mL of blood was obtained from all individuals by venipuncture in heparinized tubes to obtain peripheral blood mononuclear cells (PBMCs), EDTA-tubes for DNA extraction and tubes without anticoagulant for serological tests (Vacutainer; Beckton-Dickinson, San Jos, CA, USA). This study was approved by the Research and Ethics Committees from the Universidad Javeriana’s Science Faculty and Fundacin Abood Clnica Shaio.
HLA-A2 typing by flow cytometry HLA-A2 typing was performed by incubating 50 lL of peripheral blood with BB7Æ2 FITC monoclonal antibody conjugated for 20 min at 4C, followed by additional 15-min incubation after having lytic buffer added, and washing with PBS 1X (26). From the total of 70 individuals studied, 27 (38Æ57%) expressed the HLA-A2 molecule as judged by flow cytometry. Additionally, four more individuals were detected by SSP-PCR as being positive for the HLA-A2 allele, giving a total of 31 HLA-A2+ individuals (44Æ29%). Such discrepancy can be explained by the fact that the BB7Æ2 monoclonal antibody would not be able to recognize all A2 alleles as these molecules’ great polymorphism is concentrated in the a1 and a2 domains of the H chain and BB7Æ2 recognizes overlapping epitopes in the a2 domain (26). HLA-A2+ donors were distributed as follows according to disease stage: 9 indeterminate (G0) and 10 chronic chagasic patients (G1 = 4, G2 = 3, G3 = 3). Twelve of the 20 HD were HLA-A2+ (Table 1). HLA-A2-positive donors were further subtyped by PCR for HLA-A*0201.
HLA-A*0201 typing by PCR A total of 50–150 ng genomic DNA was extracted from peripheral blood using a GFX genomic blood DNA purification kit (Amersham Biosciences, Piscataway, NJ, USA); this was amplified in 25 lL reaction mix using 296 (5¢-GTGGATAGAGCAGGAGGCT-3¢) and 302 (5¢-CCAA
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Table 1 General characteristics of HLA-A2+ studied individuals Clinical HLA-A*0201 HLA-A*02 Code Age Gender statusa (SSP-PCR) (SSP UniTray) 025 055 056 057 060 064 082 089 092 065 026 038 070 052 066 075 005 030 054 001 002 004 009 010 011 017 018 020 031 032 033
61 41 61 34 62 54 62 50 36 56 48 65 64 64 44 51 64 58 46 45 48 25 52 29 67 40 41 38 38 24 27
M M F F F F M F F F F M F F F F M F F M M F F F M F F F F F M
G0 G0 G0 G0 G0 G0 G0 G0 G0 G1 G1 G1 G1 G2 G2 G2 G3 G3 G3 HD HD HD HD HD HD HD HD HD HD HD HD
+ + + + + + ) ) ) ) + + + + + ) + + + + + + + + + + + + + + +
ND ND ND ND ND ND A2 (A*0259)+ A32 A2 (A*0259)+ A24 A2 (A*0222)+ A24 A2 (A*0287)+ A43 ND ND ND ND ND A2 (A*0205*0226) ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
a
Clinical status of infected individuals. Levels of Chagas disease’s severity according to Kuschnir grading system in G0, G1, G2, or G3 (25); HD, healthy donor; ND, not determined.
GAGCGCAGGTCCTCT-3¢) sequence-specific primers for HLA-A*0201 using a Stomacher 3500 Thermal Cycler, PTC 100 (MJ research, Watertown, MA, USA) (27). The expected-489 bp amplified product was separated on ethidium-bromide stained 1Æ5% agarose gels. Further confirmation was made for some discordant flow cytometry and PCR HLA-A2 typing results using HLA-A SSP-PCR (Biotest, Landsteinerstr, Dreieich, Germany) and HLA-A*02 SSP UniTray (Dynal Invitrogen Corporation, Brown Deer, WI, USA) kits, following manufacturer’s protocols.
derived from the influenza virus58–66 matrix protein (GILGFVTTL) (11) as control, were synthesized by the National Institute of Health (NIH) Tetramer Facility (Atlanta, USA). PBMCs were adjusted to 1 · 106 cells per tube and stained with tetramers at 0Æ5 lg ⁄ mL plus antiCD3-PerCP and anti-CD8-FITC (BD Biosciences, San Jos, CA, USA) for 20 min in the dark at room temperature. After washing with staining buffer (1% foetal bovine serum in PBS), cells were fixed with 1% paraformaldehyde in PBS. At least 50 000 events, gated for CD3+ CD8+ T cells, were acquired and analysed using a FACSCalibur II flow cytometer and CellQuest Pro software (BD, Bioscience, San Jose, CA, USA).
T cell phenotyping and function For immunophenotyping, two million PBMCs were stained with anti-CD3 (APC), anti-CD8 (APC-Cy7), antiCCR7 (PE Cy7), anti-CD62L (APC) and the K1 tetramer (PE) for 30 min at room temperature (all antibodies were purchased from BD Biosciences and BD Pharmingen, San Diego, CA, USA). Subsequently, cells were washed twice with 2 mL of PBS 1X (Cambrex Bio Science, Rockland, ME, USA) and fixed with 0Æ5% paraformaldehyde in PBS. To determine intracellular perforin expression, after surface antigens staining, CD8+ T cells were permeabilized with Cytofix ⁄ Cytoperm (BD Pharmingen), washed twice with Perm ⁄ WashTM (BD Pharmingen) and stained with anti-perforin FITC or mouse IgG2b j isotype control for 30 min at 4C. Intracellular cytokines were detected in PBMCs stimulated with K1 peptide (10 lg ⁄ mL) in the presence of CD28 (1 lg ⁄ mL), and CD49d (1 lg ⁄ mL) for 12 h at 37C. The last 9 h of culture were performed in the presence of brefeldin A (10 lg ⁄ mL) (BD Pharmingen). Then, cells were permeabilized and stained with anti-IFNc (PE Cy7) or anti-IL-2 (APC) for 30 min at 4C. To evaluate the cytotoxic activity, anti-CD107a and anti-CD107b FITC were added to the PBMCs prior to stimulation. In each experiment, nonstimulated cells were used as negative control and Staphylococcal enterotoxin B (3Æ7 lg ⁄ mL) as positive control. Data were acquired in a FACSCanto II flow cytometer and analysed using FACS Diva (BD Biosciences) software. Results were expressed as percentages, mean fluorescence intensity (MFI) and integrated mean fluorescence intensity (iMFI); the latter was obtained multiplying the frequency by the MFI of tetramer-positive CD8+ T cells (28).
Tetramer staining HLA-A2 PE-labelled tetramers loaded with K1, a peptide derived from T. cruzi KMP-114-12 protein (TLEEFSAKL) (20) or with a modified Flu-MP peptide (Flu-MP*),
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Statistical methods Differences among groups were assessed by Student’s t-test when n ‡ 10 and by Mann–Whitney test when 2010 Blackwell Publishing Ltd, Parasite Immunology, 32, 494–502
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n < 10. GraphPad InStat 3Æ0 statistics software was used for both analyses. P < 0Æ05 was considered statistically significant.
RESULTS Population HLA-A2 subtyping Since the K1 peptide has been described as being an HLA-A-0201-restricted epitope, different HLA-A2 patients were typed by flow cytometry (data not shown) and subtyped by PCR using specific primers for HLA-A*0201. Analysis of the 31 HLA-A2+ individuals showed that 26 of them (83Æ87%) were subtyped as being HLA-A*0201+, distributed as follows: 6 indeterminate and 8 chronic chagasic, 3 G1, 2 G2 and 3 G3 patients, plus 12 HD (Table 1). Other HLA-A2 alleles were analysed by SSP Uni trayPCR as five T. cruzi infected donors were negative for HLA-A*0201; the following alleles were found in these five donors: A*0205, A*0222, A*0226, A*0259 and A*0287 (Table 1).
Frequency of K1 peptide-specific CD8+ T cells A tetramer consisting of Flu-MP* peptide ⁄ HLA-A*0201 was used as positive control. Flu-MP*-specific percentages in CD8+ T cells ranged from 0% to 0Æ36% in all the HLA-A2+ population. There was no statistically significant difference when healthy and infected groups were compared (P > 0Æ05), indicating that chronic parasite
Frequency of CD8+ T cells for K1 peptide
infection did not modify this antigen-specific cell population (Figure 1a and b). A cut-off point for HLA-A2 ⁄ K1 tetramer CD8+ T cells was fixed at 0Æ076%, after determining the average background value (0Æ046%) plus three standard deviations (0Æ010%) in 12 HLA-A2+ HD who had no anti-T. cruzi antibodies and had never lived in Chagas’ disease endemic areas. K1-specific cells were found in 77Æ78% (7 ⁄ 9) of indeterminate and 80% (8 ⁄ 10) of chronic chagasic patients, 0Æ16% (€SD = 0Æ1) and 0Æ16% average (€SD = 0Æ07) respectively (Figure 1c and d). According to the disease’s stage and severity, K1-specific CD8+ T cells were found in 2 ⁄ 4 of G1, 3 ⁄ 3 of G2 and 3 ⁄ 3 of G3 donors, having averages of 0Æ15% (€SD = 0Æ04), 0Æ22% (€SD = 0Æ06) and 0Æ15% (€SD = 0Æ02) respectively. Notably, the five infected HLA-A2+ patients having a different haplotype to HLA-A*0201 were shown to have CD8+ T cells also specifically recognizing the K1 peptide, having values (0Æ20 € SD = 0Æ11) similar to those observed for HLA-A*0201+ infected patients (0Æ14 € SD = 0Æ08). These patients’ clinical status was G0 for three patients and G1 and G2 for one patient.
Phenotypic and functional characterization To address whether K1-specific CD8+ T cells from HLAA*0201+ and HLA-A2+ non-HLA-A*0201 infected patients exhibit the same phenotypic and functional characteristics, the expression of CCR7 and CD62L, and the pro-
Figure 1 Frequency of CD8+ T cells specific for Flu-MP* (a) and K1 (c) peptides determined by soluble tetramer assay. Noninfected HLA-A*0201+ donor cut-off point = 0Æ076%. A representative dispersogram from one patient for Flu-MP* (b) and K1 (d) peptides is shown. 2010 Blackwell Publishing Ltd, Parasite Immunology, 32, 494–502
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Figure 2 Phenotypic characterization of CD8+ T cells specific for K1 peptide determinated by flow cytometry. Expression of CCR7 and CD62L, double positive cells: central memory T cells (TCM) and double negative cells: effector memory T cells (TEM). No statistically significant differences between A*0201+ and non-A*0201 infected patients, P > 0Æ05. Statistically significant differences are observed between the phenotype of central memory and effector memory T cells (P < 0Æ05), showing the predominant effector memory T cell phenotype in both, non-HLA-A*0201 and HLA-A*0201+ infected donors.
duction of IL-2, IFN-c and perforin, a pore-forming protein closely linked to cytolysis (29), were analysed. It was found that the population of K1 peptide-specific CD8+ T cells was predominately TEM (CCR7), CD62L)) cells irrespective of the HLA-A*0201 molecule presence (Figure 2, Table 2). Secretion of IL-2 was detected in K1 peptide-specific CD8+ T cells from both HLA-A*0201+
Table 2 Phenotypical characterization of K1 peptide-specific CD8+ T cells % of K1 Tet+ CD8+ T cells Code 025 056 057 055 064 038 052 066 054 030 082 092 065 075
Clinical statusa
CD62L+, CCR7+
CD62L), CCR7)
G0 G0 G0 G0 G0 G1 G2 G2 G3 G3 G0 G0 G1 G2
2Æ95 5Æ88 17Æ02 14Æ63 4Æ30 4Æ54 0Æ40 16Æ16 43Æ87 17Æ30 0Æ88 1Æ30 7Æ08 39Æ17
74Æ55 58Æ82 42Æ55 43Æ90 53Æ76 67Æ04 90Æ12 38Æ38 9Æ18 40Æ38 76Æ00 80Æ22 48Æ67 13Æ40
a Clinical status of infected individuals. HLA-A2+ ⁄ non-HLAA*0201 patients are in bold.
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infected patients (3 of 10 with a range of frequency of 0Æ11–4Æ50%) and HLA-A2+ non-HLA-A*0201 infected patients (4 of 4 with a range of frequency of 1Æ27– 1Æ76%). There were no differences in the percentage, MFI or iMFI when both groups were compared (Figure 3a, Table 3). IFN-c production was detected with a range of frequency of 0Æ42–5Æ49% in 6 of 10 HLA-A*0201+ infected patients as well as in 3 of 4 HLA-A2+ nonHLA-A*0201 infected patients with a range of frequency of 0Æ13–1Æ64%. There were no differences in all analysed parameters (Figure 3b, Table 3). Finally, when perforin expression was compared between HLA-A*0201+ and non-HLA-A*0201 infected patients, it was observed that K1 peptide-specific CD8+ T cells from 9 of 9 HLA-A*0201+ infected patients and 4 of 4 HLA-A2+ non-HLA-A*0201 infected patients secreted this cytokine with a range of frequency of 9Æ61–83Æ41% and 10Æ91–61Æ29% respectively. No differences between both groups in their percentage, MFI or iMFI (Figure 3c, Table 3), were observed. A representative cytokine and perforin staining is shown in Figure 3d.
DISCUSSION The chronic parasite infections mainly found in tropical countries represent a global health threat because of their burden and socioeconomic impact, besides the lack of effective treatment for some of them and the unavailability of vaccines (30). A better comprehension of the defence mechanism could favour the development of therapies based on immune approaches. Both host innate and specific immune responses are crucial for controlling parasitic infections such as Chagas’ disease (31). T. cruzi infection triggers several effector mechanisms like lytic antibodies for controlling parasite blood stages or CD8+ and CD4+ T cells for controlling intracellular stages. The lack of one of these cellular mechanisms in animal models can result in uncontrolled or lethal acute infection (7,32,33). CD8+ T cells can act by secreting cytokines or displaying cytotoxic activity via perforin ⁄ granzyme (8). However, experimental data suggest that chronic infections with long and continuous antigenemia can induce a certain degree of dysfunction in the lymphoid cell compartments (34) associated with the deletion or attrition of different T cell subpopulations (35). Because of the parasite intracellular stage and the CD8+ T cell immune role in animal models, several studies have described HLA class I-restricted CD8+ T cell epitopes in T. cruzi proteins, but still in low numbers (13,36). Generation of CD8+ T cell epitopes is influenced by several factors including abundance of the targeted protein, 2010 Blackwell Publishing Ltd, Parasite Immunology, 32, 494–502
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Frequency of CD8+ T cells for K1 peptide
Figure 3 Functional characterization of CD8+ T cells specific for K1 peptide determinated by flow cytometry. (a) Frequency, mean fluorescence intensity (MFI) and integrated mean fluorescence intensity (iMFI) of the IL-2 (a), IFN-c (b) and perforin (c) production in: HLA-A*0201(-) patients (white bars) and HLA-A*0201(+) patients (grey bars). (d) Representative flow cytometry profiles of IL-2, IFN-c and perforin expression in K1 peptide-specific CD8+ T cells. No statistically significant differences between HLA-A*0201+ and non-HLAA*0201 infected patients, P > 0Æ05.
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Table 3 Production of intracellular cytokines and perforin by K1 peptide-specific CD8+ cells IL-2 Code 025 056 057 055 064 038 052 066 054 030 082 092 065 089 075
Clinical statusa G0 G0 G0 G0 G0 G1 G2 G2 G3 G3 G0 G0 G1 G1 G2
IFN-c
%
MFI
0Æ00 0Æ00 0Æ00 0Æ00 0Æ11 1Æ72 0Æ00 4Æ50 0Æ00 0Æ00 – 1Æ67 1Æ76 1Æ27 1Æ54
0Æ00 0Æ00 0Æ00 0Æ00 12436Æ00 4514Æ00 0Æ00 1936Æ00 0Æ00 0Æ00 – 5434Æ00 9050Æ00 11119Æ0 7126Æ00
iMFI 0Æ00 0Æ00 0Æ00 0Æ00 1367Æ96 7764Æ08 0Æ00 8712Æ00 0Æ00 0Æ00 – 9058Æ48 15946Æ10 14132Æ25 10952Æ66
Perforin
%
MFI
0Æ00 0Æ00 0Æ53 0Æ00 0Æ42 2Æ15 0Æ89 5Æ49 2Æ17 0Æ00 – 0Æ15 0Æ13 0Æ00 1Æ64
0Æ00 0Æ00 1018Æ00 0Æ00 1104Æ00 1296Æ00 1241Æ00 968Æ00 982Æ00 0Æ00 – 2341Æ00 1206Æ00 0Æ00 1023Æ00
MFI 0Æ00 0Æ00 539Æ54 0Æ00 463Æ68 2786Æ40 1104Æ49 5314Æ32 2130Æ94 0Æ00 – 341Æ79 155Æ57 0Æ00 1675Æ67
%
MFI
45Æ60 35Æ49 9Æ61 10Æ18 – 44Æ63 22Æ75 83Æ41 24Æ87 48Æ81 27Æ65 15Æ90 10Æ91 – 61Æ29
1556Æ34 2279Æ80 1357Æ88 535Æ83 – 2177Æ52 1144Æ39 2134Æ89 1583Æ96 1360Æ52 1244Æ20 641Æ60 1852Æ11 – 3003Æ45
iMFI 70969Æ10 80910Æ10 13049Æ23 5454Æ75 – 97182Æ72 26034Æ87 178071Æ17 39393Æ09 66406Æ98 34402Æ13 10201Æ44 20206Æ52 – 184081Æ45
a Clinical status of infected individuals. MFI, mean fluorescence intensity; iMFI, integrated mean fluorescence intensity. HLA-A2+ ⁄ nonHLA-A*0201 patients are in bold.
proteosome enzymatic cutting and peptide affinity for its respective MHC molecules (37). Even more, in the case of T. cruzi, the identification of specific CD8+ T cell epitopes could be hampered by the parasite genetic polymorphism (38) and proteomic complexity that includes at least 12 000 proteins gathered into super-families (39,40). Our group has previously shown that the nonapeptide K1, located at the N-terminal T. cruzi KMP-11 protein is an HLA-A*0201 restricted epitope recognized by natural parasite exposed CD8+ T cells (16,21). Herein, a K1 ⁄ HLA-A*0201 tetramer was used to determine the percentages of circulating CTL precursor cells in T. cruzi infected patients. With this more sensitive approach, it was identified that 79% of infected donors had K1 peptide positive cells, a higher percentage than the 16Æ6% detected by ELISPOT in HLA-A*0201 chagasic patients (16). This marked difference is also a result of the strict statistical analysis used for selecting positive donors in the IFN-c ELISPOT study (16) and the fact that not all the K1peptide-specific CD8+ T cells are able to produce IFN-c (see below, Table 3). Martin et al. (13) have shown that trans-sialidase, a protein family encoded by more than 1400 polymorphic genes, is a frequent CD8+ T cell epitope found in Chagas’ patients. Indeed, there is a frequency of 0Æ15–0Æ4% specific CD8+ T cells in 5 of 8 chronic chagasic patients for the HLA-A2 restricted peptides, Ts38 (FANHKFTLV) and Ts44 (FANYKFTLV) when tetramer approach was used (13). A similar tetramer positive cells percentage (0Æ22%) has also been reported in one chronic chagasic donor with
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HLA-A2 restricted peptides for cruzipain [CZ 16–24 (VMACLVPAA), CZ 60–68 (SVFRENLFL)] and FL-160 [FL 457–465 (WLSDCGEAL)] (18). The values found are close in average to the K1 peptide-specific CD8+ T cell frequency described here (0Æ09–0Æ34%) in 15 chronic chagasic donors. Consequently, the dominance of trans-sialidase epitopes in T. cruzi infection is not absolute as other parasite targets, like the K1 peptide, are directly recognized by CD8+ T cells from chagasic patients. High HLA molecule polymorphism can restrict peptide presentation to those having higher binding affinity (41). However, an allele-specific HLA binding peptide can also be presented by other HLA molecules having similar binding specificities grouped into HLA supertypes (42). There are currently nine supertype groups (43) and a T. cruzi immunodominant epitope concentration has been demonstrated in the so-called A2 supertype (14). K1 is a theoretically deduced (data nor shown) and experimentally proved HLA-A*0201 restricted epitope; however, in this study, T cells from 5 HLA-A2+ non-HLA-A*0201 infected patients also recognized this peptide. It should be noted that the cells derived from donors with A*0205 and A*0222 alleles presented the highest K1 tetramer positive cell percentages amongst non-HLA-A*0201 individuals and the later one, the highest percentage among all the 44 T. cruzi infected patients studied here. Interestingly, it is known that nearly 70% of peptides with high affinity for HLA-A*0201 molecules bind at least two other HLA molecules from the A2 supertype (44). 2010 Blackwell Publishing Ltd, Parasite Immunology, 32, 494–502
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Memory T lymphocytes contain distinct populations of central memory (TCM) and effector memory (TEM) cells characterized by distinct homing capacity and effector function (45). Human TCM are memory cells that constitutively express CCR7 and CD62L, two receptors which are required for cell extravasation through high endothelial venules and migration to T cell areas of secondary lymphoid organs. Human TEM are memory cells that have lost the constitutive expression of CCR7 and CD62L and display characteristic sets of chemokine receptors and adhesion molecules that are required for homing to inflamed tissues. Similarly reported by other authors (13,46–48), in this study, it was found that parasite-specific CD8+ T cells had the TEM phenotype. According to the fact that CD8+ TEM carry large amounts of perforin and produce cytokines within hours following antigenic stimulation, K1 peptide-specific CD8+ T cells were able, at a low frequency, to secrete IL-2 or IFN-c, or perforin, at a higher frequency. Of special interest, in both HLA-A*0201+ and non-HLAA*0201 infected patients, the expression of IL-2, IFN-c and perforin by K1 peptide-specific CD8+ T was detected without statistically significant differences in the magnitude (percentage of expressing cells), quality (MFI) or both (iMFI), indicating that these populations are equally functional upon antigen k1 peptide specific stimulation. In conclusion, these results suggest that the K1 peptide is a promiscuous epitope which, having an invariant sequence in all the currently reported strains, may be a good target for developing immune-based prophylactic or therapeutic strategies against Chagas’ disease.
ACKNOWLEDGEMENTS The HLA-A2 ⁄ K1 and HLA-A2 ⁄ Flu-MP* tetramers were generated and kindly provided by the NIH Tetramer Facility. This work was supported by Colciencias Research project No. 1203-333-18692. We thank Zulma Cucunuba from Instituto Nacional de Salud, and Hugo Dez from Laboratorio de Parasitologa Molecular, Pontificia Universidad Javeriana for their collaboration with contacting patients. MCT and MCL were supported by P06-CTS02242 Grant from PAI (Junta de Andaluca) and RICETRD06 ⁄ 0021-0014, Spain.
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