Epstein-Barr virus (EBV) can be found latently infecting Reed-Sternberg (RS) malignant cells in approximately 50% of classical Hodgkin lymphoma (cHL) patients in Brazil. EBV signaling leads to a disbalance between effector and regulatory CD4 T lymphocytes in the tumor microenvironment, promoting the immune evasion of RS malignant cells. However, little is known about these lymphocytes subpopulations in the peripheral blood of patients with cHL and how treatment can modify this regulatory/effector ratio. In this study, we analyzed the regulatory and effector CD4+ subpopulations in peripheral blood in patients with EBV related and non-related cHL and the impact of treatment on these cells.
This is an open multicentric study and, so far, we included 35 patients from December 2009 to December 2011. Blood was drawn at diagnosis and after completion of treatment (1 to 4 months). Eighteen healthy blood donors volunteers were recruited as controls. Quantification of regulatory and effector T lymphocytes was done by flow cytometry using CD3, CD8, CD4, CD25, Foxp3, GITR, CD127 and interleukin-17 (IL17) antibodies and correlated to phenotypic and clinical parameters in uni- and multivariate models pre and post-treatment. In this study, only cHL patients whose histology could be confirmed and EBV association established were studied. All patients were HIV negative and received ABVD chemotherapy protocol and radiotherapy if necessary.
From the 35 cHL patients, 17 were EBV related and 18 EBV non-related. The percentage of CD4+CD25highFoxP3+ and CD4+GITR+ at diagnosis was significantly different from healthy controls (median 1.04 vs 0.26, p=0.02; 4.2 vs 2.2, p=0.003; respectively). CD4+CD127+ T lymphocytes were not different from controls (p=0.3). Additionally, CD4+ T lymphocytes with effector phenotype (CD4+IL17+) were significantly increased in cHL patients compared with controls (0.42 vs 0.13, p<0.001). When we compared pre-treatment values of regulatory and effector CD4+ T lymphocytes with post-treatment values, we did not find any statistical difference. Interestingly, post-treatment values were not statistically different from healthy controls. There was no difference on regulatory and effector CD4+ T lymphocytes in the EBV related and non-related cHL patients. Regarding patient's baseline characteristics, patients with advanced disease and B symptoms presented with increased CD4+CD25highFoxP3+ and CD4+GITR+ T lymphocytes (p=0.03 and p=0.01, respectively).
Our results demonstrate that patients with cHL presented with increased CD4+CD25highFoxP3+, CD4+GITR+ and CD4+IL17+ at diagnosis compared with healthy controls. Also, treatment had no impact on these CD4+ T lymphocytes populations. Probably, the moment blood was drawn after completion of therapy could have influenced our results as we know that immunological reconstitution in patients with cHL may take several months. Further studies investigating these CD4+ T lymphocytes subpopulations together with functional assays will contribute not only to our understanding on the pathogenesis of cHL but also to the development of therapeutic strategies designed to manipulate regulatory activity. Given that the incidence of EBV-related cHL, disease presentation and severity are different in developing countries than in developed ones, we emphasize the importance of this ongoing Brazilian multicentric project.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.