PD-1 Blockade Can Restore Functions of T-Cells in Epstein-Barr Virus-Positive Diffuse Large B-Cell Lymphoma In Vitro

Lina Quan, Xue Chen, Aichun Liu, Yan Zhang, Xiuchen Guo, Shujie Yan, Yue Liu, Lina Quan, Xue Chen, Aichun Liu, Yan Zhang, Xiuchen Guo, Shujie Yan, Yue Liu

Abstract

Epstein-Barr virus-positive diffuse large B-cell lymphoma (EBV+DLBCL) is an aggressive malignancy that is largely resistant to current therapeutic regimens, and is an attractive target for immune-based therapies. Anti-programmed death-1 (PD-1) antibodies showed encouraging anti-tumor effects in both preclinical models and advanced solid and hematological malignancies, but its efficacy against EBV+DLBCL is unknown. Herein, we performed experiments using co-culture system with T cells and lymphoma cell lines including EBV+DLBCL and EBV-DLBCL [including germinal center B-cell like (GCB)-DLBCL and non-GCB-DLBCL] in vitro. We show that lymphoma cells augmented the expression of PD-1 on T cells, decreased the proliferation of T cells, and altered the secretion of multiple cytokines. However, through PD-1 blockade, these functions could be largely restored. Notbaly, the effect of PD-1 blockade on antitumor immunity was more effective in EBV+DLBCL than that in EBV-DLBCL in vitro. These results suggest that T-cell exhaustion and immune escape in microenvironment is one of the mechanisms underlying DLBCL; and PD-1 blockade could present as a efficacious immunotherapeutic treatment for EBV+DLBCL.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. The comparison of effector memory…
Fig 1. The comparison of effector memory T cells (CD62L-CD45RA-) in lymph node (LN) and peripheral blood (PB) of DLBCL and peripheral blood (PB) of healthy controls (HC).
Dot plot shows CD45RA-CD62L- effector memory CD3+CD4+ and CD3+CD8+ T cells in LN and PB of a DLBCL patient and PB of a healthy person.
Fig 2. The comparison of PD-1 expression…
Fig 2. The comparison of PD-1 expression in lymph node (LN) and peripheral blood(PB) of patients of DLBCL and peripheral blood(PB) of healthy controls (HC).
Shown are the expression of PD-1 on CD4+ and CD8+ T cells in LN and PB of a DLBCL patient and a healthy person.
Fig 3. PD-1 positive T cells are…
Fig 3. PD-1 positive T cells are exhausted T cells.
PD-1 positive T cells express memory marker CD45RO and exhaustion markers CTLA-4. Histograms showing CD45RO and CTLA-4 on intratumoral preexisting CD4+ PD-1+ or CD8+ PD-1+ T cells from DLBCL.
Fig 4. Tumor cells augment the PD-1…
Fig 4. Tumor cells augment the PD-1 expression in activated T cells.
Shown are CD3-positive-gated and the histograms of PD-1 expression on resting T cells, T cells activated by PHA after 24h, T cells co-cultured with OCI-L-y-10, LCL, and SU-DHL-4 overnight from a representative experiment (n = 4).
Fig 5. PD-1 blockade restores the proliferation…
Fig 5. PD-1 blockade restores the proliferation of exhausted T cell resulted by lymphoma cells.
T cells were labeled with CSFE and stimulated with OCI-Ly-10 cells, LCL cells and SU-DHL-4 cells, respectively. Percentages indicate the fraction of cells in the non-dividing peak. Shown are CD3-positive-gated histograms from a representative experiment (n = 4). Tumor cells inhibite T cell proliferation as observed by CSFE staining, and through PD-1 blockade T cell proliferation restored.
Fig 6. PD-1 blockade enhances the activation…
Fig 6. PD-1 blockade enhances the activation of T cells cocultured with allogeneic lymphoma cells (OCI-Ly-10, LCL and SU-DHL-4).
PD-1 blockade affects T cells incubated with PD-L1 positive lymphoma cells (OCI-Ly-10 and LCL). However, incubation of healthy donor T cells with a B-cell lymphoma line that does not express PD-L1 (SU-DHL-4) does not significantly alter cytokine secretion with the blockade of PD-1. Data are represented as mean±SD of quadruplicate experiments. P values shown are for anti-PD-1 antibody versus isotype control antibody. (A) IL-2, (B) IFN-g, (C) TNF-a, (D) IL-10. ***P<0.001.

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