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Clinical and Vaccine Immunology, April 2006, p. 530-533, Vol. 13, No. 4
1071-412X/06/$08.00+0     doi:10.1128/CVI.13.4.530-533.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

ESAT-6 Peptide Recognition by Bovine CD8⁺ Lymphocytes of Naturally Infected Cows in Herds from Southern Italy

Istituto Zooprofilattico Sperimentale della Sicilia, Via G. Marinuzzi 3, 90100 Palermo, Italia,¹ Dipartimento di Biopatologia e Metodologie Biomediche, Università degli Studi di Palermo, Corso Tukory 211, 90100 Palermo, Italia,² TB Research Group, Veterinary Laboratories Agency, Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom³

Received 9 December 2005/ Returned for modification 14 January 2006/ Accepted 16 February 2006

	ABSTRACT

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The aim of this study was to define epitopes of Mycobacterium bovis from ESAT-6 (early secretory antigen of 6 kDa) recognized by CD8⁺ T lymphocytes from cows naturally infected with Mycobacterium bovis. We found that bovine CD8⁺ T cells recognized 10 out of 11 ESAT-6 peptides tested.

	TEXT

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Bovine tuberculosis (bTB) remains a major disease of domestic cattle and also constitutes a significant human health risk, particularly in developing countries. Eradication programs for this disease are based on the skin test with bovine purified protein derivative and the slaughter of test-positive animals (field reactors). To improve the specificity of bovine purified protein derivative and to further vaccine development, the definition of more-specific antigens and a better understanding of antituberculous immune responses in cattle are research priorities. Therefore, the aim of this study was to define epitopes from one such antigen, ESAT-6 (early secretory antigen of 6 kDa), recognized by bovine CD8⁺ T lymphocytes, using samples obtained from animals naturally infected with Mycobacterium bovis from Sicily, Italy. Confirming earlier studies from other geographical locations (1, 7, 8, 9, 10), our results obtained after testing Sicilian skin test reactor cattle with confirmed bTB with the BOVIGAM gamma interferon (IFN-) assay demonstrated that a pool of 11 ESAT-6-derived synthetic peptides (see sequences below) was recognized with high responder frequency by 73% of the 152 animals with confirmed bTB tested (data not shown). In contrast, none of the noninfected cattle responded to stimulation with the peptide pool compared to 5% of these animals responding to tuberculin, thereby confirming the improved test specificity associated with ESAT-6 (data not shown).

(This research was conducted by P. Macaluso and Marco Pio La Manna in partial fulfillment of the requirements for a Ph.D. degree from Università degli Studi di Palermo, Palermo, Italy.)

It has been shown that CD4⁺ T cells predominate early after infection, while CD8⁺ T cells become apparent at later infection stages (4, 6). With a view to study the epitopes recognized by bovine CD8⁺ T cells after M. bovis infection, we analyzed CD8⁺ T-cell responses after stimulation with ESAT-6-derived synthetic peptides. Responses after peptide stimulation were assessed by fluorescence-activated cell sorting (FACS) analysis of intracytoplasmic IFN- production. For that purpose, heparinized blood samples were drawn from 30 cattle of different breeds and cross-breeds, from different parts of Sicily, Italy, that had been designated tuberculin test reactors following skin testing with the single intradermal comparative cervical tuberculin test. The skin tests were performed as specified in reference 2, and M. bovis infection was confirmed by bacterial culture from pooled head lymph nodes and/or by the presence of gross pathological lesions found at necropsy. Blood samples were collected 10 to 20 days after the skin tests, and all samples were processed within 24 h of sampling. Eleven ESAT-6-derived peptides (Louisiana State University Health Care Center, New Orleans; 16-mers overlapping by 8 amino acids) were used in this study. Peptide purity (minimum, >90%) and sequence fidelity were confirmed by analytical reverse-phase high-performance liquid chromatography and mass spectrometry, respectively. The following peptides were used in this study: 6- 45, MTEQQWNFAGIEAAAS; 6-46, AGIEAAASAIQGNVTS; 6-47, AIQGNVTSIHSLLDEG; 6-48, IHSLLDEGKQSLTKLA; 6-49, KQSLTKLAAAWGGSGS; 6-50, AAWGGSGSEAYQGVQQ; 6-51, EAYQGVQQKWDATATE; 6-52, KWDATATELNNALQNL; 6-53, LNNALQNLARTISEAG; 6-54, ARTISEAGQAMASTEG; and 6-55, QAMASTEGNVTGMFA.

Two peptides representing a murine H-Y-D^b CD8 epitope (WMHHNMDLI) and a murine immunodominant CD8⁺ T-cell epitope from the 38-kDa protein of Mycobacterium tuberculosis (DQVHFQPLPPAVVSKDSALI) were used as control peptides (Chem Progress, Milan, Italy). They were used at the same concentration of ESAT-6 peptides. Peripheral blood mononuclear cells (PBMC) (10⁶/ml) were incubated with ESAT-6-derived peptides (5 µg/ml), and 5 µM monensin (Sigma, St. Louis, Missouri) was added after 6 h of culture. After an additional culture period of 18 h, cells were harvested and labeled with phycoerythrin (PE)-conjugated anti-bovine CD8 monoclonal antibody (MAb) (clone CC63; mouse immunoglobulin G2a [IgG2a]) (Serotec, Oxford, United Kingdom). Cells were fixed in phosphate-buffered saline containing 4% paraformaldehyde, permeabilized using the Leucoperm kit (Serotec, United Kingdom), and then stained for intracytoplasmic bovine IFN- using a fluorescein isothiocyanate (FITC)-labeled MAb against bovine IFN- (clone CC302; mouse IgG1). FACS analysis was carried out on a FACScalibur using CELL-Quest software (Becton Dickinson). FITC-labeled mouse IgG1 (Serotec, United Kingdom) was used as the isotype control for the anti-bovine IFN- MAb, and PE-labeled mouse IgG2a (Serotec, United Kingdom) was used as the isotype control for the anti-CD8 MAb. Data are presented as the means of three replicate determinations per animal tested. This analysis was performed in 30 cows with confirmed bovine tuberculosis.

Representative FACS panels are shown in Fig. 1, generated with PBMC obtained from animal 4857. In this animal, CD8-positive IFN--positive cells were found when PBMC were stimulated with peptides 6-45 (2.2%) and 6-51 (1.8%), while no such cells were induced after exposure to peptide 6-53. As shown in Fig. 1, the number of IFN--positive CD8-negative cells (10.3% in response to peptide 6-45 and 8.6% in response to peptide 6-51) was larger than that of IFN-⁺ CD8⁺ T cells, a result consistent with the notion that CD8-positive IFN--positive cells are not the dominant T-cell subset responding to stimulation with ESAT-6-derived peptides. Indeed, it is likely that CD4⁺ and/or T cells represent the main source of IFN- in these experiments. Nevertheless, our experiments demonstrated that CD8⁺ T cells make a significant contribution to the ESAT-6-induced immune response in tuberculous cattle. Identical experiments were performed in 29 additional cattle with confirmed bTB, with the results summarized in Table 1. Interestingly, all ESAT-6-derived peptides, with the exception of peptide 6-53, were able to induce IFN- responses of CD8⁺ T cells, with individual peptides recognized with responder frequencies ranging from 10 to 36% (Table 1).

View larger version (13K): [in this window] [in a new window]   FIG. 1. Intracytoplasmic IFN- in CD8-positive cells of PBMC from cow 4857 stimulated in vitro with different ESAT-6 peptides. The skin of the cow was analyzed by the single intradermal comparative cervical tuberculin test. PBMC from 30 naturally infected cows in vitro reacting to mycobacterial antigens and recognizing ESAT-6 sequences with different patterns were exposed in vitro to ESAT-6 peptides for 6 hours. After the cells were washed, they were stained with PE-labeled anti-bovine CD8 (CD8 PE) and with FITC-labeled anti-bovine IFN-. The isotype control shown was stained with FITC- and PE-labeled isotype antibodies. Data shown were reproduced in 30 animals tested, with differences in the percentages of CD8+ IFN-+ cells of less than 10%. The proportion of CD8-positive cells for each staining varied from 18 to 38%. For negative controls and cutoff values to determine positive samples, CD8+ IFN-+ cells were tested by incubating PBMC from each cow with peptide 6-53 (mean of 0.21% of CD8+ IFN-+ cells), with the H-Y-Db nonamer (mean of 0.28% of CD8+ IFN-+ cells), and with the 38-kDa protein of Mycobacterium tuberculosis (mean of 0.31% of CD8+ IFN-+ cells). Samples were considered positive when the proportion of double-fluorescent cells was more than 1% after subtraction of means for negative controls. A total of 100,000 events were analyzed gating live cells.

View this table: [in this window] [in a new window]   TABLE 1. Percentages of cows with CD8-positive IFN--positive cells in response to ESAT-6 epitopes

Although CD8-positive cells are not the dominant T-cell subset, our data nevertheless suggest that the peptide set recognized by CD8-positive cells from infected cows was almost identical to that recognized by CD4⁺ T cells (6, 7, 11). The actual epitopes displayed by bovine leukocyte antigen class I molecules and subsequently recognized by CD8⁺ T cells await definition and will almost certainly be distinct from the epitopes recognized by CD4⁺ T cells present on the same peptides. One can envisage that CD8⁺ T cells recognizing mycobacterial epitopes in the context of major histocompatibility complex class I molecules will contribute to the overall IFN- response postinfection by, for example, compensating for the inhibition of major histocompatibility complex class II expression caused by mycobacterial compounds associated with decreased processing and presentation of mycobacterial antigens to human HLA-DR-restricted CD4⁺ T cells (3, 5). We did not determine in this study whether the CD8⁺ T cells activated by ESAT-6-derived epitopes consist solely of /ß or whether CD8⁺ / T-cell receptor-positive T cells are also contributing to the overall response. Although bovine CD8⁺ / T cells constitute only a minor subpopulation of the / cells in peripheral blood compared to the dominant CD8^– peripheral / T-cell population, the possibility that they play a role in the described CD8⁺ T-cell response cannot be formally ruled out.

In conclusion, our results have demonstrated a role of ESAT-6-specific CD8⁺ T cells in the response to M. bovis infection of cattle, which could have a substantial contribution to protective immunity.

	ACKNOWLEDGMENTS

 
This work was supported by grants from the Ministero della Salute (grant R6D060P1DNTB01 to S.R., F.V., G.S., and F.D.) and the Ministero dell'Università e della Ricerca Scientifica (grant ex-60% 2002 to G.S. and A.B.). P.M. and M.P.L.M. were supported by grants from the Ministero dell'Università e della Ricerca Scientifica as Ph.D. students in Immunopharmacology.

	FOOTNOTES

 
* Corresponding author. Mailing address: Dipartimento di Biopatologia e Metodologie Biomediche, Università degli Studi di Palermo, Corso Tukory 211, 90100 Palermo, Italia. Phone: 39 091 6555939. Fax: 39 091 6555924. E-mail: sireci{at}unipa.it.

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