Immune Netw.  2016 Apr;16(2):134-139. 10.4110/in.2016.16.2.134.

Enhanced Anti-tumor Reactivity of Cytotoxic T Lymphocytes Expressing PD-1 Decoy

• Shin JH
• Park HB
• Choi K

• KMID: 2168054
• DOI: http://doi.org/10.4110/in.2016.16.2.134

Abstract

Programmed death-1 (PD-1) is a strong negative regulator of T lymphocytes in tumor-microenvironment. By engaging PD-1 ligand (PD-L1) on tumor cells, PD-1 on T cell surface inhibits anti-tumor reactivity of tumor-infiltrating T cells. Systemic blockade of PD-1 function using blocking antibodies has shown significant therapeutic efficacy in clinical trials. However, approximately 10 to 15% of treated patients exhibited serious autoimmune responses due to the activation of self-reactive lymphocytes. To achieve selective activation of tumor-specific T cells, we generated T cells expressing a dominant-negative deletion mutant of PD-1 (PD-1 decoy) via retroviral transduction. PD-1 decoy increased IFN-γ secretion of antigen-specific T cells in response to tumor cells expressing the cognate antigen. Adoptive transfer of PD-1 decoy-expressing T cells into tumor-bearing mice potentiated T cell-mediated tumor regression. Thus, T cell-specific blockade of PD-1 could be a useful strategy for enhancing both efficacy and safety of anti-tumor T cell therapy.

Keyword

PD-1; PD-L1; T lymphocytes; PD-1 decoy

MeSH Terms

Adoptive Transfer
Animals
Antibodies, Blocking
Autoimmunity
Cell- and Tissue-Based Therapy
Humans
Lymphocytes
Mice
T-Lymphocytes
T-Lymphocytes, Cytotoxic*
Zidovudine
Antibodies, Blocking
Zidovudine

Figure

• 

  Figure 1 Overexpression of PD-1 decoy increases IFN-γ secretion from T cells. Activated B6 splenic T cells were transduced with retrovirus carrying either a control vector (pMIG-w) or PD-1 decoy and rested for 3 days in the absence of stimulation. (A) Retroviral transduction efficiency was measured by flow cytometry using GFP. The GFP positive populations were gated and the expression levels of PD-1 were analyzed. PD-1 expression in the control represents the levels of endogenous PD-1, while the PD-1 decoy sample shows the levels of both endogenous and the decoy receptor. (Filled gray area: Isotype control, Percentage of GFP positive cells indicated inside histograms) (B) GFP positive T cells were sorted and stimulated with anti-CD3 in the presence of irradiated splenocytes for 48 hours. IFN-γ in the cultured supernatants was measured by ELISA (Student's t-test, *p<0.05, **p<0.01). Results are representative of 3 independent experiments.

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  Figure 2 Enhanced anti-tumor reactivity of OT-I T cells carrying PD-1 decoy. OVA-specific CD8 T cells (OT-I cells) were transduced with retroviruses carrying control vector or PD-1 decoy as described in the methods and materials. (A) Transduction efficiency and PD-1 decoy expression of OT-I cells were measured as described in Fig. 1A. (B) Expression of PD-L1 on OVA-expressing tumor cell lines was analyzed by flow cytometry. (A-B, Gray filled area: Isotype control, Percentage of GFP positive cells indicated inside histograms) (C) The retrovirus-transduced OT-I cells were co-cultured with OVA-expressing tumor cell lines for 24 hours, and IFN-γ in the cultured supernatants was measured by ELISA (Student's t-test, ns; not significant, *p<0.05, **p<0.01, ****p<0.0001). The transduced OT-I cells were rested for 3 days in the absence of stimulation and sorted by GFP expression before co-culture with the tumor cells. Results are representative of 2~3 independent experiments (A-C).

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  Figure 3 The PD-1-CD28 chimera does not improve the anti-tumor reactivity of T lymphocytes compared to PD-1 decoy. B16 melanoma antigen (gp100)-specific CD8 T cells (Pmel-1 cells) were transduced with retroviruses carrying PD-1-CD28 chimera or PD-1 decoy. (A) Expression levels of PD-1-CD28 chimera and PD-1 decoy in GFP-positive Pmel-1 populations were analyzed by flow cytometry. (B) B16 melanoma cells were either left untreated or treated with IFN-γ (20 ng/ml) for 48 hours. Then, PD-L1 expression was determined by flow cytometry. (C) Retrovirus-transduced GFP positive Pmel-1 cells were sorted by flow cytometry. The sorted cells (1×105) were co-cultured with IFN-γ-treated B16 melanoma cells (1×104) for 48 hours. IFN-γ in the cultured supernatants was quantified by ELISA (Student's t-test, *p<0.05, **p<0.01, ***p<0.001).

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  Figure 4 PD-1 decoy potentiates anti-tumor therapeutic efficacy of adoptively transferred T cells. B6 mice were injected with OVA-expressing E.G7 cells (2×106) subcutaneously. After 7 days, the mice were either left untreated or intravenously injected with control vector (pMIG-w)-transduced (2×10 6) or PD-1 decoy-transduced (2×106) OT-I cells. The retrovirus-transduced OT-I cells were rested for 3 days before adoptive transfer. The mean tumor size of 10 mice per group was recorded (*p=0.0039, Wilcoxon matched-pairs test). Results are representative of 2 independent experiments.

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