Early T cell signalling is reversibly altered in PD-1+ T lymphocytes infiltrating human tumors

Abstract

To improve cancer immunotherapy, a better understanding of the weak efficiency of tumor-infiltrating T lymphocytes (TIL) is necessary. We have analyzed the functional state of human TIL immediately after resection of three types of tumors (NSCLC, melanoma and RCC). Several signalling pathways (calcium, phosphorylation of ERK and Akt) and cytokine secretion are affected to different extents in TIL, and show a partial spontaneous recovery within a few hours in culture. The global result is an anergy that is quite distinct from clonal anergy induced in vitro, and closer to adaptive tolerance in mice. PD-1 (programmed death -1) is systematically expressed by TIL and may contribute to their anergy by its mere expression, and not only when it interacts with its ligands PD-L1 or PD-L2, which are not expressed by every tumor. Indeed, the TCR-induced calcium and ERK responses were reduced in peripheral blood T cells transfected with PD-1. Inhibition by sodium stibogluconate of the SHP-1 and SHP-2 phosphatases that associate with several inhibitory receptors including PD-1, relieves part of the anergy apparent in TIL or in PD-1-transfected T cells. This work highlights some of the molecular modifications contributing to functional defects of human TIL.

Conflict of interest statement

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

Figures

Figure 1. Early T cell signalling is altered in human TIL.

(A) Left, typical example of Ca mobilisation after anti-CD3 stimulation (arrow) in TIL from a NSCLC biopsy, compared to PBT (In this and following figures, each Ca recording corresponds to the average of 10–30 cells). Right, mean Ca measured in TIL (NSCLC n = 23; melanoma n = 4; RCC n = 8) and PBT (n = 5 healthy donors). (B) Examples (left) and mean percentage (right) of Akt phosphorylation elicited by anti-CD3/anti-CD28 stimulation in TIL (n = 7 tumors) and PBT (n = 3 healthy donors). (C) Examples (left) and mean percentage (right) of anti-CD3-induced ERK phosphorylation in TIL (n = 12) and PBT (n = 3). Data represent mean +/− SEM; * p<0.05, ** p<0.01, *** p<0.001 (Student t test).

Figure 2. Interactions between TIL and large cells present in the tumor.

(A) Average Ca responses triggered in TIL by interaction (arrow) with large cells either unloaded (thin line, average from 7 NSCLC tumors) or loaded with SAg (thick line, n = 6 NSCLC tumors) and in PBT (open symbols, n = 15 cells). (B) A typical triple labelling in a NSCLC slice, of TIL (CD3+ in red), monocytic (CD14+ cells in green) and tumor cells (EpCAM+ in blue). Zoom: arrows point to three CD3+-CD14+ conjugates.

Figure 3. TIL show a spontaneous functional recovery in culture.

(A) Typical average Ca responses in ex vivo NSCLC TIL and after 12 h in culture (n = 20–24 cells). (B) Average kinetics of Ca recovery in TIL from melanoma (n = 7), NSCLC (n = 23) and RCC (n = 7). (C) Percentage of phosphorylated ERK (left, n = 13), phosphorylated Akt (middle, n = 7) and IFN-γ producing TIL (right, n = 3), at day 0 and after 24–48 h in culture. Data represent mean +/− SEM ; * p<0.05, ** p<0.01, *** p<0.001 (Student t test).

Figure 4. PD-1, but not its ligands, is frequently expressed in tumor biopsies.

(A) PD-1 expression on PBT, melanoma, Lung specimens (control or tumoral) and RCC (benign and tumoral). PD-1 level is expressed as a ratio relative to the MFI of the control isotype. Bars correspond to the Median; * p<0.05, ** p<0.01, *** p<0.001 (Mann Whitney). (B) Percentage of tumor biopsies showing positivity for PD-L1 or PD-L2 staining (Melanoma n = 15; NSCLC n = 15; RCC n = 12).

Figure 5. PD-1 contributes to the anergic state of TIL.

(A) Ca responses and PD-1 levels measured on ex vivo TIL from 16 NSCLC. Open circles correspond to necrotic tumors. Linear regression curve is shown (r2 = 0.034). (B) Production of IFN-γ by TIL stimulated or not (NS) with anti-CD3/anti-CD28 coated beads. Left, typical dot plot from a NSCLC biopsy. Right, average (n = 3 tumors). Data represent mean +/− SEM; * p<0.05, ** p<0.01, *** p<0.001 (Student t test).

Figure 6. PD-1 expression delivers an inhibitory signal attenuated by sodium stibogluconate.

(A) Typical average Ca responses elicited by anti-CD3 (arrow) in melanoma TIL, treated or not for 20 min with SSG. (B) Average Ca responses in TIL treated or not with SSG (n = 12 tumors). (C) Top: fluorescence images of PD-1 and YFP-transfected PBT. Bottom: typical average Ca responses triggered by anti-CD3 (arrows) in YFP+ and PD-1+ transfectants (left) and in PD1+ transfectants treated or not with SSG (right). (D) Average Ca response (in % of the response in YFP+ cells) in YFP+ and PD-1+ transfectants treated or not with SSG (n = 7 independent experiments). Data represent mean +/− SEM * p<0.05, ** p<0.01, *** p<0.001 (Student t test).