Block Of PD-1/PD-L1 Axis Enhanced Homeostatic Proliferation-driven Antitumor Immunity During Recovery From Lymphopenia

BackgroundIt is well known that lymphopenia is followed by spontaneous expansion of the remaining T cells in the periphery to restore the original T-cell pool size and maintain homeostasis.Lymphopenia-induced homeostatic proliferation of T cells is driven by the recognition of self-MHC/peptide ligands in the absence of foreign antigens or inflammatory signals.However,several studies have also shown that the integration of other immunotherapeutic strategies might be necessary to successfully eradicate preexisting malignant tumors,because homeostatic proliferation-driven antitumor responses seem to rapidly decline in association with tumor growth.In the present study,we analyzed the dynamic change and connection of functional antitumor immunity and PD-1 expression on T cells when the number of T cells recovered from lymphopenic conditions.Next,we explore whether and how PD-1 induced T-cell hyporesponsiveness and promoted tumor-induced immune tolerance in the later stage of homeostatic proliferation-driven antitumor responses.In addition,anti-PD-1/PD-L1 therapy,such as anti-PD-1 antibodies and Crispr/Cas9 knockout system,were used in our study and evaluate the effects of block of PD-1/PD-L1 axis in homeostatic proliferation-driven antitumor immunity in lymphopenic mice.This study is not only important for elucidating the mechanism of the wane of homeostatic proliferation-driven antitumor responses,but also provides a scientific basis for developing novel combined treatment of anti-PD-1/PD-L1 therapy and non-myeloablative dose of radiotherapy.Materials and Methods1.We first examined whether homeostatic proliferation of T cells could induce antitumor immunity in lymphopenic mice.9Gy or 6.5Gy irradiation or CTX chemotherpy are common means of inducing lymphodepletion or lymphopenia in mice.On the second day of irradiation or chemotherpy,C57BL/6 mice were injected s.c.with B16 melanoma cells shortly after the irradiation,and marrow cells or T cells were infused,then tumor growth was monitored and recorded.2.We analyzed the dynamic change and connection of functional antitumor immunity and PD-1 expression on T cells when the number of T cells recovered from lymphopenic conditions.We analyzed the effector function of tumor-specific T cells on days 7,14,and 21 using flow cytometry and IFN-y ELISpot.We conducted time course experiments to determine the kinetics of PD-1 expression on CD8+T cells during reconstitution of the lymphopenic environment.To address whether cell death was involved in the progression of homeostatic proliferation,CD8+donor cells stained with Annexin-V and BCL-2 were analyzed by flow cytometry on day 21 after transfer.3.We next evaluated the therapeutic efficacy of anti-PD-1 antibody during reconstitution of the lymphopenic environment.IL-7 or IL-15 plays an important role in the homeostatic expansion and survival of T cells and indicates the trend of tenting into memory T cells,so we analyzed whether the anti-PD-1 antibody treatment can increase the expression levels of IL-7R(CD127)and IL-15R(CD122)in CD8+ T cells.In order to explore whether anti-PD-1 antibody treatment can promote homeostatic proliferation-driven T cells to turn into memory T cells,the ratio of memory T cells were analyzed on day 21 after transfer.To evaluate whether block of PD-1/PD-L1 axis can enhance the antigen recognition ability of HP cells,functional status of DC cells were assessed using flow cytometry on day 14 after transfer.4.To further assess the role of PD-L1 on homeostatic proliferation-driven antitumor immunity,we next used Crispr/Cas9 system targeting PD-L1 gene for the generation of B16 cell lines with constitutive knockout of PD-L1 expression.Then PD-L1 knocking out B16 cells(B16-PD-L1)and B16 cells transfected with a control vector(B16-NC)were inoculated into immunocompetent C57BL/6 mice.Crispr/Cas9 RNPs targeted PD-1 gene were electroporated into nucleus to specifically knockout the PD-1 gene of T cells.The knockout efficiency was detected by T7E1 assays.Then the PD-1 knockout T cells were transferred into lymphopenic mice after 6.5Gy total body irradiation.Results.1.The lymphocyte infusion into nonirradiated naive mice showed little antitumor effect at day 14 after the tumor injection.The infusion of T cells suppressed B16 s.c.tumor growth in sublethally irradiated mice.The antitumor effect of sublethal irradiation combined with infusion of CD8+T-cell-depleted splenocyte suspension was significantly reduced with respect to standard sublethal irradiation and immune splenocyte treatment.Transferred CD4+and CD8+T cells underwent several rounds of proliferation in secondary lymphoid organs 10 days after their transfer into sublethally irradiated mice.2.Barely detectable before transfer,PD-1 expression slightly increased at day 7,peaked at day 21,and returned to low levels by day 35.The expression of BCL-2 significantly decreased while the percentage of apoptosis T cells increased on day 21 after transfer.These results suggested that T cells transferred to lymphopenic host highly expressed PD-1 and tend to apoptosis on day 21 of homeostatic proliferation.3.Anti-PD-1 antibody treatment significantly inhibited tumor growth and extended mouse survival time in immune reconstitution mice.The percentage of IFN-?-releasing CD8+ T cells on Day21 was significantly increased in lymphopenic mice treated with anti-PD-1 antibody after T cell infusion.The expression levels of IL-7R and IL-15R were significantly increased after anti-PD-1 antibody administration.It indicated the survival of T cell in the later stage of homeostatic proliferation.CD3+CD8+CD44[high]CD62L[high],as T central memory(TCM)cells,were significantly increased in lymphopenic mice combined treated with anti-PD-1 antibody and T cell infusion.The expression of CD80,CD86 and I-A/I-E in DC cells were significantly increased lymphopenic mice combined treated with anti-PD-1 antibody and T cell infusion when compared with anti-PD-1 antibody or T cell infusion alone.4.When PD-L1 knocking out B16 cells(B16-PD-L1)and B16 cells transfected with a control vector(B16-NC)were inoculated into immunocompetent C57BL/6 mice,knockout of PD-L1 significantly decreased tumor burden in irradiated mice after T cells infusion.Tumor growth in lymphopenic mice intravenously infused PD-1 knockout T cells were significantly inhibited compared with transferring naive T cells into lymphopenic mice or transferring PD-1 knockout T cells into immunocompetent mice.ConclusionsIn this study,we found that PD-1/PD-L1 axis played an important role in T-cell hyporesponsiveness in later stage of T-cells homeostatic proliferation.Block of PD-1/PD-L1 axis remarkably augmented homeostatic proliferation-driven antitumor immunity in three ways.That is promoting the recognition of tumor associated antigen(TAAs),enhancing cell killing of T cells and inducing tumor-specific CTL convert to memory T cells during the three stage of homeostatic proliferation of T cells.Finally,we found that Crispr/Cas9-mediated knockout of PD-1 in T cells significantly enhanced homeostatic proliferation-driven antitumor responses and inhibited tumor growth.These findings provided a scientific basis for developing novel combined treatment of anti-PD-1/PD-L1 therapy and non-myeloablative dose of radiotherapy.