Functional inactivation of EBV-specific T-lymphocytes in nasopharyngeal carcinoma: implications for tumor immunotherapy

Abstract

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV) associated malignancy with high prevalence in Southern Chinese. In order to assess whether defects of EBV-specific immunity may contribute to the tumor, the phenotype and function of circulating T-cells and tumor infiltrating lymphocytes (TILs) were investigated in untreated NPC patients. Circulating naïve CD3+CD45RA+ and CD4+CD25- cells were decreased, while activated CD4+CD25+ T-cells and CD3-CD16+ NK-cells were increased in patients compared to healthy donors. The frequency of T-cells recognizing seven HLA-A2 restricted epitopes in LMP1 and LMP2 was lower in the patients and remained low after stimulation with autologous EBV-carrying cells. TILs expanded in low doses of IL-2 exhibited an increase of CD3+CD4+, CD3+CD45RO+ and CD4+CD25+ cells and 2 to 5 fold higher frequency of LMP1 and LMP2 tetramer positive cells compared to peripheral blood. EBV-specific cytotoxicity could be reactivated from the blood of most patients, whereas the TILs lacked cytotoxic activity and failed to produce IFNgamma upon specific stimulation. Thus, EBV-specific rejection responses appear to be functionally inactivated at the tumor site in NPC.

Figure 1. EBV VCA and EA antibody titers and EBV DNA in the serum of NPC patients and healthy EBV carriers.

Antibody titers to EBV VCA IgG and IgA (A) and EA IgG and IgA (B) in the serum of healthy donors (n = 20) and NPC patients (n = 40); EBV DNA load (C) in healthy donors (n = 20) and NPC patients (n = 24); ns =  non significant.

Figure 2. Immunophenotype of PBMCs from healthy donors and NPC patients.

Surface markers analysis was performed by directly immunofluorescence labeling and FACS analysis in PBMCs from healthy donors (n = 20) and NPC patients (n = 40). * P<0.01

Figure 3. Frequency of LMP1 and LMP2 epitope- specific T cells in PBMCs from healthy donors and NPC patients.

The frequency of T cells specific for HLA A2 restricted on LMP1 and LMP2 was detected by EBV tetramer staining and FACS analysis in freshly isolated PBMCs from healthy donors (n = 7) and NPC patients (n = 9). The number of tetramer positive cells in 100 lymphocytes are shown in the figure.

Figure 4. Cytotoxic activity of auto-LCL activated cultures from PBMCs of NPC patients.

Polyclonal CTL cultures were test for cytotoxic activity against a panel of targets including the autologous EBV transformed LCL, PHA blasts, allogenic HLA class I matched or mismatched LCL. A. Representative experiments illustrating three pattern of cytotoxic activity. Pattern I: Lysis of the auto-LCL ≥25% and HLA class I mismatched LCL ≤10%; Pattern II: lysis to both HLA class I matched and mismatched LCL; Pattern III: less than 10% lysis against autologous or allogenic EBV positive or negative targets B. EBV-specific CTL cultures from NPC patients lysed freshly isolated autologous NPC tumor cells. Representative ⁵¹Cr release assays performed with CTL cultures from two NPC patients are shown in the figure.

Figure 5. Frequency of LMP1 and LMP2 epitope- specific T cells in auto-LCL stimulated cultures from healthy donors and NPC patients.

The frequency of T cells specific for HLA A2 restricted epitopes in LMP1 and LMP2 was detected by EBV tetramer staining and FACS analysis in auto-LCL stimulated cultures from healthy donors (n = 4) and NPC patients (n = 9).

Figure 6. Immunophenotype of LCL stimulated cultures and TILs from NPC patients (A).

Surface markers were detected by directly immunofluorescence and FACS analysis in LCL-stimulated cultures (n = 13) and tumor infiltrating lymphocyte (n = 25) expanded for 1 to 4 weeks in IL-2 medium without antigen stimulation. A relative decrease of CD8+ cells and increase of CD4+ cells was observed in TILs compared to LCL-stimulated cultures; both cultures showed high percentage of CD3+CD45RO+ and CD4+CD25+ cells. Frequency of LMP1 and LMP2 epitope-specific T cells in auto-LCL stimulated cultures and TILs from NPC patients (B). LMP1 and LMP2 epitopes specific T cells were detected by EBV tetramer staining and FACS analysis in autologous LCL-stimulated PBMCs (n = 9) and TILs (n = 6) from NPC patients.

Figure 7. Cytotoxic activity of LCL-stimulated cultures and TILs from NPC patients.

The mean ±SD percentage lysis at 10∶1 E∶T ratio of LCL-stimulated cultures (n = 15) and TILs (n = 9) from NPC patients is shown in the figure.