T Cell Dysfunction in Epstein-Barr Virus-Associated Lymphoma

The Epstein-Barr virus (EBV) is thought to be one of tumorigenic viruses, which can be detected in many kinds of lymphomas, such as extranodal NK/T-cell lymphoma and Burkitt lymphoma. Those EBV positive lymphomas are also called EBV-associated lymphomas. Some studies showed that patients with EBV-associated lymphoma had a worse prognosis. Dysfunction of T cells may contribute to failure of immune surveillance and tumorigenesis. Therefore, this study was aimed to investigate T cell dysfunction in patients with EBV-associated lymphoma. Peripheral blood was collected from 20 patients with EBV-associated lymphoma and 10 healthy donors (healthy EBV seropositive donors) at the Peking University First Hospital. First, the phenotypes and ratios of PBMCs were analyzed using flow cytometry. Compared with healthy donors, the proportion of CD3⁺ T cells were little lower in the patients, but had no significantly difference (healthy donors vs patients, 47.82±6.85% vs 34.43±3.63%, p=0.09). The ratios of CD4⁺ T cells / CD8⁺ T cells were significantly decreased in the patients, the proportion of CD4⁺ T cells in CD3⁺ T cells were decreased (healthy donors vs patients, 59.40±3.84% vs 29.46±2.84%, p<0.0001), and CD8⁺T cells in CD3⁺T cells were increased (healthy donors vs patients, 33.94±3.60% vs 57.36±2.59%, p=0.0003) in the patients. NK cells had no significantly difference between the two groups (healthy donors vs patients, 9.74±3.68% vs 10.37±2.16%, p=0.88). Further, an enzyme-linked immunospot assay revealed that the production of IFN-γ in T cells was remarkably reduced upon stimulation with EBV mixed peptides, suggesting EBV specific T responses were insufficient in the patients with EBV-associated lymphoma. To study the mechanism of T cells dysfunction in EBV-associated lymphoma, multiple inhibitory receptors were analyzed. Compared with healthy donors, we found that CD4⁺ T cells and CD8⁺ T cells expressed higher levels of PD-1, LAG-3, TIM-3 and CTLA-4 in the patients, suggesting T cells were exhausted in the patients with EBV-associated lymphoma. Furthermore, the T-bet^hi PD1^mid exhausted T cells which can be rescued by blockade of PD1 pathway and the EOMES^hi PD1^hi exhausted T cells which can not be rescued by the blockade of PD1 pathway were both higher in the patients with EBV-associated lymphoma. CD57+KLRG-1+CD160+CD28- were markers of T cell senescence, we found this subset cells were also a little higher in patients, suggesting that T cell senescence might be another contributor of T cell dysfunction in the patients with EBV-associated lymphoma. In addition, some studies have demonstrated that exhausted T cells do not derive from the 'senescent' KLRG-1+ subset of effector T cells but rather from the CD127+ subset of memory precursors that are present in the effector phase. We found the proportion of CD127⁺ T cells reduced significantly in the patients, further suggesting memory T cells decreased and exhausted T cells increased in the patients with EBV-associated lymphoma. All together, we demonstrated here that T cell dysfunction was the predominant feature of the patients with EBV-associated lymphoma, including the T cells ratio imbalance and T cell exhaustion. Our study contributes to understand the pathogenesis of EBV-associated lymphoma and suggests that immune checkpoint blockade may be warranted.