Pathogenesis of Endemic Burkitt's Lymphoma: The Role of T-Cell Responses

Abstract

The mechanisms underlying the deficiency in cellular immunity to eBL and its development have not been conclusively investigated. This study sought to examine T cell phenotypes and responses in eBL. Participants recruited for this study included; 19 healthy controls, 21 eBL patients and 26 malaria patients, PBMCs were isolated from peripheral blood samples collected from participants and combinations of T-cell subset, activation marker (CD25, CD69, CD95, HLA-DR), IFN-γ-, IL-4- or FoxP3-specific monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE) or PE-Cy5 were used to in surface or intracellular staining of the PBMCs. Intracellular staining was done using FoxP3 staining buffer set. For intracellular staining for cytokines, PBMCs were stimulated with EBNA1 peptide pool and PHA according to the protocol for intracellular cytokine analysis. Cytokine levels in plasma were measured by ELISA. Both absolute numbers and frequency of lymphocytes were significantly lower in malaria patients compared to eBL patients (p<0.0001 and p<0.0001, respectively) as well as healthy controls (p<0.0001 and p<0.0001, respectively). The percentage T cells in population of lymphocytes were lower in both BL and malaria patients compared to healthy controls (BL vs Controls, p=0.003; Malaria patients vs Control, p=0.002). It was also found that the CD4/CD8 ratio was significantly higher in BL patients compared to controls and malaria patients (BL vs Controls, p=0.007; BL vs Malaria patients, p=0.033). Whereas the median frequency of CD4CD95+ cells did not differ between malaria patients and controls (p= 0.462) nor between eBL and malaria patients (p=0.054), it was higher in eBL patients compared to controls (p=0.014). Generally, CD4+ and CD4FoxP3+ cells expressed more IFN-γ in controls than in eBL patients. CD4+ and CD4+FoxP3+ T cells from healthy controls had higher IFN-γ REI to EBNA1 compared to those from eBL patients. Malaria patients were found to have higher frequency of γδ+ cells compared to eBL patients (p=0.001) and healthy controls (p=0.014). The frequency of the CD3+ γδ+ cells was lower in eBL patients compared to healthy controls (p=0.004), making it the lowest among the categories. Similar to CD4+ T cells, Vδ1+ T cells in healthy controls expressed more IFN-γ to all stimulants than those from eBL patients. But unlike CD4+ cells, IFN-γ REI to EBNA1 by Vδ1+ T cells was similar between patients and controls. Additionally, higher frequency of Vδ1+ T cells expressed IFN-γ compared to CD4+ cells. Examining the frequency of T reg cells, it was observed that frequency of CD4+CD25hi+FoxP3 was higher in both malaria (p=0.000) and eBL (p=0.022) patients compared to healthy controls. The frequencies of CD4+CD25hi+Foxp3- cells were also higher in both malaria (p=0.012) and eBL (p=0.035) patients compared to controls. Th1 and Th2 profile of CD4+ and Vδ1+ cells were also examined. Whereas REI for IFN-γ and IL-4 in CD4+ cells were similar in health controls, the frequency of IFN-γ expression was higher than IL-4 expression to all stimulants. Likewise REI for IFN-γ and IL-4 in CD4+ cells were similar in eBL patients, just as in health controls but the expression of IL-4 by CD4+ cells to EBNA1 was higher than IFN-γ. The frequency of Vδ1+ cells expressing IFN-γ to EBNA1 and REI for IFN-γ, were higher than those for IL-4 in controls. Similarly, REI for IFN-γ to EBNA1 was higher than REI for IL-4 in eBL patients. However, the frequency of cells expressing IL-4 to both EBNA1 was higher than those expressing IFN-γ in the patients. Levels of peripheral blood TNF-α was significantly lower in eBL patients compared to healthy controls (P=0.002). Conversely, plasma level of IL-10 was significantly higher in eBL patients than in healthy controls (p=0.036). Put together, the data suggest that reduced Th1 response in eBL might be generally due to the prevailing Th2 micro-environment, elevated levels of T reg cells and specifically upregulation of CD95 expression by CD4+ cells. It also indicates that malaria may contribute to the development of eBL by skewing of immune responses in favour of Th2, generation of T reg cells and T cell exhaustion.