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. 2004 Nov 11;64(5):722–729. doi: 10.1136/ard.2004.024455

Analysis of the CD8+ T cell response to the G1 domain of aggrecan in ankylosing spondylitis

J Zou 1, H Appel 1, M Rudwaleit 1, A Thiel 1, J Sieper 1
PMCID: PMC1755485  PMID: 15539415

Abstract

Background: CD4+ T cell responses to the G1 domain of aggrecan in patients with ankylosing spondylitis (AS) were recently reported. Whether such an immune response can be seen in the CD8+ subpopulation has not yet been determined.

Objective: To determine if HLA-B27 restricted G1-specific CD8+ T cells are present in AS and to analyse immunodominant CD8+ T cell epitopes.

Methods: Peripheral blood mononuclear cells of 45 patients with AS were stimulated with overlapping 18-mer peptides covering the whole G1 protein. Results were compared with those for patients with rheumatoid arthritis (RA) and healthy controls. For epitope analysis, G1-specific interferon gamma positive (IFNγ+) T cells were isolated by magnetic activated cell sorting. After in vitro expansion, CD8+ T cells were restimulated with 14 subpools of G1 peptides. T cells responding to G1 peptide subpools were quantified by flow cytometry according to IFNγ secretion. Predicted peptides were subsequently confirmed by stimulation with single peptides.

Results: G1-specific CD8+ T cell responses were found in 29/45 (64%) patients with AS, 18/35 (51%) patients with RA, but not in healthy controls. Five CD8+ T cell epitopes were identified as immunodominant in five patients. However, the T cell response was not HLA-B27 restricted. Nonamer peptides with an HLA-B27 binding motif did not induce a T cell response.

Conclusion: A G1 peptide-specific CD8+ T cell response is present in AS but also in patients with RA. It does not seem to be HLA-B27 restricted. Whether such a response has a role in the pathogenesis of AS needs clarification.

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Figure 1.

Figure 1

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 Percentage of patients with AS (64%), with RA (51%), or healthy controls (0%) responding to in vitro stimulation with the whole peptide pool of overlapping G1 peptides of the proteoglycan aggrecan. Response was measured by IFNγ production of CD8+ T cells after antigen-specific stimulation in comparison with stimulation without antigen. For more details see "Patients and methods" section. Analysis was done with whole PB.

Figure 2.

Figure 2

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 Example of an antigen-specific response to the whole peptide pool of aggrecan derived G1 peptides compared with stimulation without antigen in a patient with AS. After staining for T cell surface markers and intracellular cytokines a gate for CD8+ T cells was set. The percentage of IFNγ/CD69 double positive cells of the CD8+ T cell subpopulation is indicated. Stimulation with SEB was used as a positive control.

Figure 6.

Figure 6

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 Example of 18-mer peptides derived from the G1 domain of aggrecan inducing an antigen-specific CD8+ T cell response but not CD4+ T cell response compared with stimulation without antigen (Ag) in patient 1. After staining for T cell surface markers and intracellular cytokines a gate for CD8+ or CD4+ T cells was set. The percentage of IFNγ/CD69 double positive cells of the CD8+ or CD4+ T cell subpopulation is indicated.

Figure 7.

Figure 7

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 Antigen-specific CD8+ T cell response to 18-mer single peptides (peptides 16,17) which cannot be blocked by an anti-HLA B27 antibody in patient 1. T cells were taken from PB.

Figure 3.

Figure 3

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 Example of an antigen-specific IFNγ secretion assay after in vitro stimulation with aggrecan G1 peptides compared with stimulation without antigen in a patient with AS. After stimulation, IFNγ+ T cells were isolated twice by MACS. For more details see "Patients and methods" section.

Figure 4.

Figure 4

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 (A) Example of a patient with AS (patient 1) with an antigen-specific CD8+ T cell response to the subpools of peptides (pools 1, 8, 10, 11) derived from the G1 domain of aggrecan compared with stimulation without antigen. After staining for T cell surface markers and intracellular cytokines a gate for CD8+ T cells was set. The percentage of IFNγ/CD69 double positive cells of the CD8+ T cell subpopulation is indicated. Peptide subpools 1, 8, 10, and 11 were positive, indicating that the crossing peptides 9, 16, 17, and 24 might be stimulatory peptides. (B) Example of a patient with AS (patient 1) with an antigen (Ag)-specific CD8+ T cell response to single peptides 16 and 17, but not to peptides 9 and 24 and control peptide, derived from the G1 domain of aggrecan compared with stimulation without Ag. After staining for T cell surface markers and intracellular cytokines a gate for CD8+ T cells was set. The percentage of IFNγ/CD69 double positive cells of the CD8+ T cell subpopulation is indicated.

Figure 5.

Figure 5

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 Example of a patient with AS with an antigen-specific CD8+ T cell response to 18-mer single peptide 17, but not to a nonamer peptide that is part of this 18-mer peptide, derived from the G1 domain of aggrecan, compared with stimulation without antigen (Ag).

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