Patients with Epstein Barr virus-positive lymphomas have decreased CD4(+) T-cell responses to the viral nuclear antigen 1

Abstract

Epstein Barr virus (EBV) causes lymphomas in immune competent and, at increased frequencies, in immune compromised patients. In the presence of an intact immune system, EBV-associated lymphomas express in most cases only 3 or fewer EBV antigens at the protein level, always including the nuclear antigen 1 of EBV (EBNA1). EBNA1 is a prominent target for EBV-specific CD4(+) T cell and humoral immune responses in healthy EBV carriers. Here we demonstrate that patients with EBV-associated lymphomas, primarily Hodgkin's lymphoma, lack detectable EBNA1-specific CD4(+) T-cell responses and have slightly altered EBNA1-specific antibody titers at diagnosis. In contrast, the majority of EBV-negative lymphoma patients had detectable IFN-gamma expression and proliferation by CD4(+) T cells in response to EBNA1, and carry EBNA1-specific immunoglobulins at levels similar to healthy virus carriers. Other EBV antigens, which were not present in the tumors, were recognized in less EBV positive, than negative lymphoma patients, but detectable responses reached similar CD8(+) T cell frequencies in both cohorts. Patients with EBV-positive and -negative lymphomas did not differ in T-cell responses in influenza-specific CD4(+) T cell proliferation and in antibody titers against tetanus toxoid. These data suggest a selective loss of EBNA1-specific immune control in EBV-associated lymphoma patients, which should be targeted for immunotherapy of these malignancies.

Figure 1. EBNA1 specific CD4⁺ T cell production of IFNγ among patients and healthy volunteers

EBNA1 specific CD4⁺ T cells were analyzed by intracellular IFNγ staining. CD4⁺ T cell responses in response to Medium (no stimulus), Staphylococcal enterotoxin B (SEB), influenza A viral particle infection (FLU), and EBNA1 peptides (EBNA1) are shown. A. Whole blood assay after gating on lymphocytes, based on size, and on CD4⁺ T cells. The frequency of IFNγ⁺CD4⁺ T cells is indicated. CD4⁺ cells (x-axis) and intracellular cytokine staining for INFγ (y-axis) are depicted. The top row represents detected IFNγ responses from one of twenty EBV-positive carriers. The second row represents an EBV-seronegative individual. The third row represents one of 17 EBV-seropositive patients with EBER-negative lymphomas and the bottom row is representative of one of 9 patients with an EBER positive lymphoma. Gates for IFNγ positive cells are based on isotype controls. B. Graph depicting the average frequency of CD4⁺ T cell production of IFNγ in response to the indicated antigens HCMV derived CD8⁺ T cell epitopes (CMV) was used as an additional negative control.

Figure 2. EBNA1 specific CD4⁺ T cell proliferation among patients and healthy volunteers

EBNA1 specific CD4⁺ T cells were analyzed by CFSE proliferation assays. CD4⁺ T cell responses in response to Medium (no stimulus), Staphylococcal enterotoxin B (SEB), influenza A viral particle infection (FLU), and EBNA1 peptides (EBNA1) are shown. CFSE dilution, or proliferation, was determined after gating on lymphocytes, based on size. The frequency of CFSE dilute (proliferating) CD4⁺ T cells is indicated. Intensity of CFSE (x-axis) and CD4⁺ cells (y-axis) are depicted. The top row represents proliferation responses from one out of twenty EBV-positive carriers, samples from an EBV-seronegative individual are shown in the second row. The third row represents one of 16 EBV-seropositive patients with EBER-negative lymphomas and the bottom row is representative of one of 8 patients with an EBER positive lymphoma. Gates for CFSE dilute or proliferating cells are shown.

Figure 3. Immunoglobulin titers to EBNA1 and tetanus toxoid among patients and healthy carriers

Average immunoglobulin G isotype titers from the healthy and patient cohorts are shown in this figure. A. Average immunoglobulin titers to EBNA1 are compared from 20 healthy EBV carriers, 25 EBV-seropositive patients with EBER-negative lymphomas and 19 patients with EBER positive lymphomas. Ratios of patients or volunteers with positive or detectable titers are outlined in table below graph. Although all three cohorts had similar IgG1, IgG2 and IgG3 titers, only patients from the EBV positive lymphoma cohort had a detectable EBNA1 specific IgG4 titer. This was statistically significant compared to both healthy and EBV-negative lympohoma cohorts (p < 0.005). B. Average immunoglobulin titers to tetanus toxoid are compared from 12 healthy EBV-seropositive volunteers, 25 EBV-seropositive patients with EBER-negative lymphomas and 19 patients with EBER positive lymphomas. Ratios of patients or volunteers with positive or detectable titers are outlined in table below graph. All three cohorts had similar IgG1, IgG2, IgG3 and IgG4 titers,