In this study, we investigated the T cell dysfunction in EBV associated lymphoma by comparing to healthy donors. We observed that EBV-specific cytotoxic T cells reduced significantly in patients than in healthy donors and displayed features of exhaustion and senescence.
Exhausted T cells usually express higher level of inhibitory receptors such as PD1, CTLA4, LAG3 and Tim3[22–24]. PD1 is a member of the B7 receptor family which plays an important role in the immune response, and transmits regulatory signals to inhibit T cell activation and proliferation, thereby mediating immune escape of tumor cells. EBV-infected lymphoma cells have been reported to express PD-L1, a ligand for PD1[31, 32]. High expression of PD1 on the surfaces of CD4+T and CD8+T were found in the humanized mouse models of EBV-positive DLBCL (diffuse large B-cell lymphoma) and the peripheral blood of EBV-positive DLBCL patients[33, 34]. In a humanized mouse model, PD-1 blockade greatly enhanced the ability of T cells to produce the anti-tumor cytokine IFN-γ in response to EBV peptides and decreased the growth of EBV-induced lymphomas. Our results also showed higher expression of the inhibitory receptors, especially PD1, CTLA4, Tim3 and LAG3 on peripheral CD4+ and CD8+ T cells in the patients with EBV-associated lymphoma than healthy donors, suggesting T cells exhaustion. Furthermore, our study suggested that T cell exhaustion existed widely across different types of lymphoma, including not only EBV-positive diffuse large B-cell lymphoma, but also EBV-associated Extranodal NK/T-cell lymphoma, Angioimmunoblast T-cell lymphoma and Hodgkin's lymphoma.
Several studies found a variety of transcription factors including Eomes, T-bet, Blimp-1, BATF, FoxO1andVHL were expressed in exhausted T cells.T-bet could decrease the expression of inhibitory receptors during chronic viral infection, resulting in more slightly exhausted CD8+ T cells. The transcription factors T-bet and EOMES together define and maintain exhausted T cell subsets both in mice and humans. Two distinct types of exhausted T cells exist, including the progenitor and terminal subsets, which is confirmed in both mouse and human. The progenitor subset featuring T-bethiPD1mid. has strong proliferative capacity and can be transformed into a terminal subset with the chronic stimulation of antigen[35, 36]. The terminal subset with the phenotype of EOMEShiPD1hi express higher levels of inhibitory receptors with reducing co-production of antiviral cytokines. The T-bethiPD1mid exhausted T cells can be rescued by blockade of PD1 pathway while the EOMEShiPD1hi exhausted T cells cannot. In this study, we found higher proportion of exhausted CD4+ and CD8+ T cells of both phenotypes in patients with EBV-associated lymphoma than in healthy donors.
Checkpoint inhibitors can partially reverse the T cell exhaustion of malignant tumors and have been applied in these malignant tumor diseases treatment. PD1 inhibitors have good therapeutic effect in a variety of lymphomas, especially in Hodgkin's lymphoma with a slightly higher EBV-positive rate.Dramatic responses to pembrolizumab (PD1 blockade) were observed with an overall response rate of 100% in EBV-positive metastatic gastric cancer.These phenomena illustrate that EBV may also have an aggravating effect on T cell exhaustion. Therefore, immunological checkpoint inhibitor may achieve certain efficacy in patients with EBV-associated lymphoma. Our study found that peripheral blood T cells from patients with EBV-associated lymphoma expressed multiple inhibitory receptors simultaneously, including PD1, CTLA4, LAG3, TIM3, and had both types of exhausted T cells (T-bethiPD1mid and EOMEShiPD1hi exhausted T cells). Previous studies have found that the ability to inhibit tumor growth using PD1 blockade in combination with CTLA4 blockade is stronger than one of the checkpoint inhibitors alone in NSG mice with EBV-positive DLBCL. CTLA4 blockade could increase the frequency of IFN-γ and granzyme-B expressing CD8+ T cells independently of T-bet by selectively inhibiting the accumulation of Eomesodermin mRNA and protein. The EOMEShiPD1hi exhausted T cells may be rescued by CTLA4 blockade which compensate for the killing effect of PD1 blockade on the above two subsets of exhausted T cells. Using multiple immune checkpoint inhibitors to treat EBV-associated lymphoma may more completely reverse T cell exhaustion and achieve better efficacy.
A study defined that a subset of exhausted CXCR5+CD8+ T cells plays an important role in the control of viral replication during chronic viral infection. In HIV patients, they found the number of CXCR5+CD8+ T cells was inversely correlated with viral load. The CXCR5+ population displayed better treatment than the CXCR5− population when adoptively transferred to chronically infected mice and showed the synergistic result on decreasing viral load when combined with anti-PD-L1 treatment. In this study, we found the CXCR5+CD8+ T cells reduced in EBV-associated lymphoma patients, suggesting the anti-viral immune response was weakened in EBV-associated lymphoma.
Senescence and exhausted T cells do have some similarity in certain aspects of functionality but they are not entirely the same[14, 39]. Low expression of CD28 and high expression of CD57 and KLRG-1 are considered to be the key markers that distinguish T cell senescence from T cell exhaustion[40, 41]. Some studies have demonstrated that exhausted T cells do not derive from the “senescent” KLRG-1+ effector T cells but from the memory precursors in the effector phase[40, 41]. In our study, we found that CD28 expression was decreased, KLRG-1and CD57 expression were significantly increased in peripheral blood CD4+ T cells and CD8+ T cells in patients with EBV-associated lymphoma. Therefore, T cell senescence might be another contributor of T cell dysfunction in the patients with EBV-associated lymphoma.
More EBV antigen-specific cytotoxic T cells in healthy donors were found in healthy donors than in patients with EBV-associated lymphoma, suggesting insufficient EBV specific T responses in patients. Donor lymphocyte infusion can not only increase the recipient's EBV antigen-specific T lymphocytes directly, but also reverse the T cell exhaustion to promote the tumor-killing ability of the immune system[8, 42]. Donor lymphocyte infusion has been used in the treatment of EBV-associated lymphoma and has achieved good efficacy[43, 44]. Our study found less EBV antigen-specific T lymphocytes and T cell exhaustion in patients with EBV-associated lymphoma, suggesting donor lymphocyte infusion could be a good treatment strategy of EBV-associated lymphoma.