Children with endemic Burkitt lymphoma are deficient in EBNA1-specific IFN-gamma T cell responses

Abstract

Endemic Burkitt lymphoma (eBL) is the most common childhood cancer in equatorial Africa and is linked to Epstein-Barr virus (EBV) and Plasmodium falciparum coinfections early in life. Epstein-Barr nuclear antigen 1 (EBNA1) is the sole viral latent antigen expressed in BL tumors. Loss of EBNA1-specific immune surveillance could allow eBL emergence. Therefore, EBNA1-specific T cell responses were analyzed by IFN-gamma ELISPOT in Kenyan children with eBL and compared to healthy children with divergent malaria exposure. Significantly fewer children with eBL, 16% (7/44) had EBNA1-specific IFN-gamma responses in contrast to healthy children living in a malaria holoendemic area or in an area with sporadic malaria transmission, 67% (40/60) and 72% (43/60) responders, respectively (p < 0.003). Children with eBL maintained IgG(1) dominated antibody responses to EBNA1 similar to healthy children suggesting a selective loss of IFN-gamma secreting EBNA1-specific T cells in the presence of intact humoral immunity. CD8(+) T cell responses to EBV lytic and latent antigens not expressed in the tumors were similarly robust in eBL patients compared to healthy children. In addition, CD4(+) T cell responses to a malaria protein, merozoite surface protein 1, were present in lymphoma patients. This study demonstrates a selective loss of EBNA1-specific T cell responses in children with eBL and suggests a potential immunotherapeutic target for this EBV-associated lymphoma.

Figure 1. Comparison of the magnitude of IFN-γ ELISPOT responses in children with eBL and healthy children

The magnitude of IFN-γ ELISPOT responses was compared between children with eBL (n=44) and healthy children from Kisumu (n=60) and Nandi (n=60) for EBNA1 total peptide pool (EATP) and EBNA1 sub-pools, (i.e., EAI, EAII, EAIII, EAIV, EAV) and for malaria antigen MSP1 (3D7). IFN-γ spot forming units (SFU) minus PBS control SFU per 1x10⁶ PBMCs are displayed (range of SFU in PBS wells was 0-12 SFU/10⁶ PBMCs). Only responses above background are shown. Each dot represents an individual sample. Lines indicate the mean and SEM for each pool tested. There were significantly lower EATP levels in eBL children compared to Nandi (p<0.001) and Kisumu (p<0.001) children, and Kisumu and Nandi children did not differ from each other (p=0.562). Significantly lower responses for children with eBL were also observed for EBNA1 sub-pools EAI, EAIII and EAV whereas there were no differences in responses for EAII and EAIV. For MSP1, children with eBL had significantly higher IFN- γ responses compared to Kisumu and Nandi children (p<0.05)

Figure 2. Mean EBNA1 IgG1 subclass levels in different populations

(A) Mean EBNA1 IgG1 levels in plasma were determined by ELISA for healthy Kisumu and Nandi children, children with eBL and healthy adults. Scatter plot show individual points for IgG1 titer and SEM is indicated for each group. (B) Frequency of EBNA1 IgG subclass positive samples. The frequency of response is shown for each EBNA1 IgG isotype tested.

Figure 3. Mean EBV viral load stratified by EBNA1-specific IFN-γ ELISPOT response among eBL patients and healthy children with divergent malaria exposure histories

EBV viral load was natural log transformed and then the mean EBV viral load was determined. The mean EBV viral load was compared within each group of children (eBL, Kisumu and Nandi) stratified by EBNA1-specific total pool (EATP) IFN-γ responders and non-responders (indicated as EBNA1 IFN-γ response). A near-significant trend emerged for eBL and Kisumu (but not the Nandi children). EBV viral load tended to be higher in children without IFN-γ responses to EBNA1 in these two groups.