EBNA1-specific T cells from patients with multiple sclerosis cross react with myelin antigens and co-produce IFN-gamma and IL-2

Abstract

Symptomatic primary Epstein-Barr virus (EBV) infection and elevated humoral immune responses to EBV are associated with an increased risk of developing multiple sclerosis (MS). We explored mechanisms leading to this change in EBV-specific immunity in untreated patients with MS and healthy virus carriers matched for MS-associated HLA alleles. MS patients showed selective increase of T cell responses to the EBV nuclear antigen 1 (EBNA1), the most consistently recognized EBV-derived CD4(+) T cell antigen in healthy virus carriers, but not to other EBV-encoded proteins. In contrast, influenza and human cytomegalovirus-specific immune control was unchanged in MS. The enhanced response to EBNA1 was mediated by an expanded reservoir of EBNA1-specific central memory CD4(+) T helper 1 (Th1) precursors and Th1 (but not Th17) polarized effector memory cells. In addition, EBNA1-specific T cells recognized myelin antigens more frequently than other autoantigens that are not associated with MS. Myelin cross-reactive T cells produced IFN-gamma, but differed from EBNA1-monospecific cells in their capability to produce interleukin-2, indicative of a polyfunctional phenotype as found in controlled chronic viral infections. Our data support the concept that clonally expanded EBNA1-specific CD4(+) T cells potentially contribute to the development of MS by cross-recognition of myelin antigens.

Figure 1.

Selective increase in rVV-EBNA1 recognition in MS. PBMCs from 24 MS patients and 24 healthy virus carriers were separately infected with rVV constructs expressing the EBV latent antigens EBNA1, 2, 3A, 3B, 3C, LMP1, and LMP2A, as well as the lytic EBV-encoded protein BZLF1. (A) Approximately 10–15% of PMBCs infected with the LMP1-encoding rVV consistently showed intracellular expression of LMP1 by flow cytometry, indicating that rVVs achieve EBV antigen expression in PBMCs. (B) EBNA1 protein expression in rVV-EBNA1ΔGA infected PBMCs compared with a fusion protein consisting of the C terminus of EBNA1 (aa 400–641) coupled to the heavy chains of an antibody (IgG-EBNA1). (C) IFN-γ specific ELISPOT responses in PBMCs to the indicated EBV antigens, to the virus vector backbone (rVV-TK⁻), and to influenza A virus (A/Aichi/68; H3N2) infection. Bars represent means. *, P = 0.005. (D) IFN-γ –specific ELISPOT responses to the indicated antigens, but in CD8-depleted PBMCs. *, P = 0.003.

Figure 2.

Increased CD4⁺ T cell proliferation to EBNA1, but not HCMV-pp65 or influenza, in MS. PBMCs from 24 MS patients and 24 healthy virus carriers were stimulated with overlapping peptide libraries spanning the C-terminal domain of EBNA1 (aa 400–641), with the entire sequence of the HCMV-encoded pp65 protein (aa 1–561), and by influenza A virus infection. Antigen-specific proliferative responses were determined in a flow cytometry–based CFSE-dilution assay. (A) An analysis of a representative blood donor. (B) Summary of all positive responses. *, P = 0.004. Bars represent means.

Figure 3.

Phenotype of EBNA1-specific T cell subsets is indicative of frequent antigen recognition in MS. The phenotype of EBNA1-specific T cells was assessed on CFSE^low cells proliferating in response to stimulation with the EBNA1 peptide library by CD27, CD28, CD62L, and CXCR3 flow cytometric staining in 24 MS patients and 24 healthy virus carriers. (A) Frequencies of the indicated subsets in MS patients (MS) and healthy controls (HD). (B) Analysis of a representative patient with MS.

Figure 4.

Elevated titers of EBNA1-specific IgG1 antibodies in MS. EBNA1-specific IgG isotype-specific titers were determined by ELISA and compared with HCMV-EA IgG responses. The presented data summarize 20 MS patients and 16 healthy virus carriers. *, P = 0.02. Error bars represent the SD.

Figure 5.

Autoantigen-recognition of EBNA1-specific T cells. Cross-reactivity of EBNA1-specific T cells to myelin antigens or proinsulin was analyzed. (A) Frequencies of EBNA1-specific TCLs generated from 6 patients with MS and 6 healthy EBV carriers recognizing peptides of myelin antigens or proinsulin. *, P = 0.01. (B) Frequencies of autoantigen-recognizing EBNA1-specific T cells in MS patients compared with healthy virus carriers. (C) Stimulation indices (SI) after EBNA1 stimulation of EBNA1-monospecific, EBNA1-specific myelin-reactive, and EBNA1-specific insulin-reactive T cells in patients and controls. (D) Serological clonality analysis by flow cytometric staining for TCR-Vβ3, 1, 5S1, 2, 5S2, 5S3, 6S7, 7, 8, 9, 11, 13S1, 12, 13S6, 16, 14, 17, 18, 1S3, 20, 22, and 23. Depicted are the results for one representative TCR Vβ3⁺ CD4⁺ T cell clone. (E) Cytokine profile of cross-reactive versus monospecific EBNA1-specific T cells. IL-2: *, P < 0.0001. MIP-1β: *, P = 0.02. Error bars represent the SD.