Ex Vivo PD-L1/PD-1 Pathway Blockade Reverses Dysfunction of Circulating CEA-Specific T Cells in Pancreatic Cancer Patients

Abstract

Carcinoembryonic antigen (CEA) is a candidate target for cellular immunotherapy of pancreatic cancer (PC). In this study, we have characterised the antigen-specific function of autologous cytotoxic T lymphocytes (CTL) specific for the HLA-A2 restricted peptide, pCEA691–699, isolated from the peripheral T cell repertoire of PC patients and sought to determine if ex vivo PD-L1 & TIM3 blockade could enhance CTL function. CD8+ T cell lines were generated from peripheral blood mononuclear cells (PBMCs) of 18 HLA-A2+ patients with PC and from 15 healthy controls. In vitro peptide specific responses were evaluated by flow cytometry after staining for intracellular cytokine production and CSFE cytotoxicity assays using pancreatic cancer cell lines as targets. Cytokine secreting functional CEA691-specific CTL lines were successfully generated from 10 of 18PC patients, with two CTL lines able to recognise and kill both CEA691 peptide-loaded T2 cells and CEA+ HLA-A2+ pancreatic cancer cell lines. In the presence of ex vivo PD-L1 blockade, functional CEA691-specific CD8+ T cell responses, including IFN-γ secretion and proliferation, were enhanced and this effect was more pronounced on Ag-specific T cells isolated from tumor draining lymph nodes. These data demonstrate that CEA691-specific CTL can be readily expanded from the self-restricted T cell repertoire of PC patients and that their function can be enhanced by PD-L1 blockade.

Identification and characterization of M6903, an antagonistic anti–TIM-3 monoclonal antibody

“…23 Certain assays have shown that targeting PD-(L)-1 can enhance T cell activation induced by TIM-3 blockade. 9,16,[29][30][31] However, since we did not observe enhanced IFN-γ production with M6903 and anti-PD-L1 combination therapy, the CEF assay may not be the best indicator of PD-(L)-1 blockade-induced IFN-γ production, and TGF-β may contribute significantly in such an assay. In order to confirm the contribution of TGF-β, recombinant human TGF-β1 (rhTGF-β1) was added in the CEF assay and its effect on IFN-γ production and M6903 activity was assessed (Figure 3c).…”

Identification of novel human leukocyte antigen‐A*11:01‐restricted cytotoxic T‐lymphocyte epitopes derived from osteosarcoma antigen papillomavirus binding factor

“…Because the generation of specific CTL was done with peptides but without additional costimulators and antigen‐presenting cells in our experiments, it is possible that CTL induced by these specific peptides are “anergic CTL” or “non‐functional CTL” as has been shown for MART‐1/HLA A2‐specific CTL in both healthy donors and patients . A number of studies have shown that the function of in vitro‐induced CTL may be influenced by a variety of parameters such as the concentration of peptides, costimulation of antigen‐presenting cells, and overexpression of immunosuppressive molecules during in vitro stimulation …”

“…Only QVT-tet-positive CTL were detected (C). Frequency of tetramer-positive cells is shown as the percentage of total CD8 + T cells in vitro-induced CTL may be influenced by a variety of parameters such as the concentration of peptides, costimulation of antigenpresenting cells, and overexpression of immunosuppressive molecules during in vitro stimulation [22][23][24][25][26][27]. Furthermore, we identified a homologous peptide, ASV (R6Q), derived from a microbial ROS family protein.…”

Cytokine-induced killer cells as a feasible adoptive immunotherapy for the treatment of lung cancer

“…Recently clinical trials focused on the tumor site ongoing immune modulation and anti-PD therapy for cancer 26 , 31 . Some research groups attempted combination of anti-PD therapy, which should facilitate adoptive cell immunotherapy reaching its maximal effect to control tumor growth 32 , 33 . During activation and amplification of cytotoxic T lymphocytes in vitro, we monitored the expression level of PD-1 on T cells by flow cytometry using CD279 antibody.…”