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. 2013 Jan;65(1):186-96.
doi: 10.1002/art.37732.

A novel human autoantigen, endothelial cell growth factor, is a target of T and B cell responses in patients with Lyme disease

Elise E Drouin  1 , Robert J Seward, Klemen Strle, Gail McHugh, Kianoosh Katchar, Diana Londoño, Chunxiang Yao, Catherine E Costello, Allen C Steere
Affiliations

A novel human autoantigen, endothelial cell growth factor, is a target of T and B cell responses in patients with Lyme disease

Elise E Drouin et al. Arthritis Rheum. 2013 Jan.

Abstract

Objective: Autoantigen presentation by HLA-DR molecules is thought to be a central component of many autoimmune diseases, but identifying disease-relevant autoantigens has been a difficult challenge. In this study we aimed to identify autoantigens in patients with antibiotic-refractory Lyme arthritis, in which infection-induced autoimmunity is thought to play an important role.

Methods: Using tandem mass spectrometry, naturally presented HLA-DR self peptides from a patient's synovium were identified, synthesized, and reacted with his peripheral blood mononuclear cells (PBMCs). Immunoreactive peptides and their source proteins were then tested for T and B cell responses using large numbers of patient cells or sera.

Results: Of 120 HLA-DR-presented self peptides identified from one patient, one peptide derived from endothelial cell growth factor (ECGF) caused his PBMCs to proliferate. T and B cell responses to ECGF occurred systemically in ∼10-30% of patients with early or late manifestations of Lyme disease, primarily in those with refractory arthritis-associated HLA-DR alleles, such as DRB1*0101 and 0401. Compared with patients with antibiotic-responsive arthritis, those with antibiotic-refractory arthritis had significantly higher concentrations of ECGF in synovial fluid (P<0.0001) and more often had ECGF antibody reactivity. Among non-antibiotic-treated historical patients who developed arthritis, 26% had ECGF reactivity, which often developed before the onset of arthritis and was associated with significantly longer courses of arthritis.

Conclusion: T and B cell responses to ECGF occur in a subset of patients with Lyme disease, particularly in those with antibiotic-refractory arthritis, providing the first direct evidence of autoimmune T and B cell responses in this illness.

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Figures

Figure 1
Figure 1
An overview of the isolation and identification of in vivo HLA-DR presented peptides from patients’ synovial tissue. Antibiotic-refractory Lyme arthritis usually manifests as one swollen knee (A). In those cases in which therapeutic arthroscopic synovectomies are performed, 20 to 60 g of inflamed synovial tissue and subcutaneous fat are removed (B). Immunohistologic staining of the synovial tissue shows marked exogenous expression of HLA-DR molecules (C). HLA-DR complexes are immunoprecipitated (IP) from synovial cell lysates (D). HLA-DR presented peptides are eluted and identified by tandem mass spectrometry (E). In this figure, the LC/MS/MS spectrum of the ECGF340-355 peptide is shown.
Figure 2
Figure 2
Screening of in vivo presented HLA-DR-peptides identified from the synovial tissue of one patient for T cell autoreactivity using the patient’s own PBMC. All 120 non-redundant HLA-DR-presented peptides identified were synthesized and tested in sets of three (2 μM of each peptide). T cell proliferation was measured using a standard 3H-thymidine incorporation assay. A positive result was defined as a proliferative response > 2 times background (no antigen control).
Figure 3
Figure 3
Testing of PBMC from healthy control subjects, or patients with RA, EM, or antibiotic-responsive or antibiotic-refractory Lyme arthritis for T cell recognition of ECGF peptides. PBMC were incubated with individual ECGF peptides (1 μM), PHA (positive control) or no peptide (negative control), and assayed by IFN-γ ELISpot assay. The results were quantified using a stimulation index (SI) defined in the Methods section along with the sequences of the peptides. A positive response was defined as a SI 3 standard deviations above the mean of healthy control subjects (the area above the shaded region). The 5 peptides predicted to be promiscuous binders were tested in all patients or control subjects. Due to limited availability of cells, the 2 peptides predicted to be non-promiscuous binders (ECGF220-234 and ECGF302-316) were tested in only a subset of patients or control subjects (15 of 18 healthy control subjects, none of the 12 RA patients, 18 of 19 patients with EM, 7 of 27 antibiotic-responsive arthritis patients, and 11 of 37 antibiotic-refractory arthritis patients).
Figure 4
Figure 4
IgG anti-ECGF autoantibody responses in the sera of healthy control subjects, or patients with EM, antibiotic-responsive or antibiotic-refractory Lyme arthritis, or RA as determined by ELISA and immunoblotting. For the ELISA, plates were coated with recombinant human ECGF then incubated with serum samples (diluted 1:100) (A). For immunoblotting, recombinant human ECGF was electrophoresed through acrylamide gels and transferred to nitrocellulose membranes. Membrane strips were then incubated with serum samples (diluted 1:100). Band intensity was determined by densitometry (B).
Figure 5
Figure 5
Correlation of clinical course with IgG anti-ECGF antibody responses as determined by ELISA in a non-antibiotic-treated historic patient followed for 6 years throughout his entire disease course. He initially had EM followed several months later by a brief episode of arthritis and then prolonged arthritis in a hip for 4 years. The antibody response to ECGF correlated with the 2 episodes of arthritis, and was highest during the period of prolonged arthritis. The shaded region represents the range of values found in healthy control subjects as shown in Figure 4.
Figure 6
Figure 6
Detection of ECGF protein in joint fluid and synovial tissue. ECGF concentrations in joint fluid of patients with antibiotic-responsive or antibiotic-refractory Lyme arthritis, or RA were measured by sandwich ELISA (A). Representative serial synovial tissue sections from a patient with antibiotic-refractory Lyme arthritis or RA stained with anti-ECGF or isotype control antibodies (B).
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