High surface expression of programmed death 1 (PD-1) is associated with T-cell exhaustion; however, the relationship between PD-1 expression and T-cell dysfunction has not been delineated. We developed a model to study PD-1 signaling in primary human T cells to study how PD-1 expression affected T-cell function. By determining the number of T-cell receptor/peptide-MHC complexes needed to initiate a Ca(2+) flux, we found that PD-1 ligation dramatically shifts the dose-response curve, making T cells much less sensitive to T-cell receptor-generated signals. Importantly, other T-cell functions were differentially sensitive to PD-1 expression. We observed that high levels of PD-1 expression were required to inhibit macrophage inflammatory protein 1 beta production, lower levels were required to block cytotoxicity and IFN-γ production, and very low levels of PD-1 expression could inhibit TNF-α and IL-2 production as well as T-cell expansion. These findings provide insight into the role of PD-1 expression in enforcing T-cell exhaustion and the therapeutic potential of PD-1 blockade.
HIV-1 specific T cell response; TCR signaling; peptide counting.
Conflict of interest statement
The authors declare no conflict of interest.
Development of a model to study PD-1 signaling and function in primary human T cells. (
A) RNA encoding both chains of the A2-SL9–specific TCR was transfected into resting primary human CD8 T cells, and, after overnight culture, the ability to bind SL9 tetramer was measured by flow cytometry. ( B) aAPCs were generated by transducing K562s with HLA.A2, the DsRed -SL9 minigene, or PD-L1 as indicated. Single-cell clones of each aAPC were expanded, and expression of each introduced construct was validated by flow cytometry. K.A2 cells are depicted as a long dashed line, K.A2.SL9 DsRed as a short dashed line, and the K.A2.SL9-DsRed.PD-L1 as a solid line. Isotype staining (gray shading) served as a negative control for A2 staining. ( C) Cells described in A were labeled with CFSE and cocultured with the indicated aAPC at a 2:1 ratio. CFSE dilution was measured by flow cytometry after 5 d of culture. As a positive control, T cells also were stimulated with CD3/CD28 beads at a 3:1 ratio. ( D) T cells described in A were cocultured with K. A2 DsRed. SL9 (solid lines) at a 1:2 ratio or with CD3/CD28 beads (long dashed lines) at a 1:3 ratio for 3 d and were stained with indicated antibodies. Mock TCR-transfected T cells incubated with KT. A2 DsRed. SL9 aAPCS served as control (gray shading).
PD-1 inhibits Ca
2+ flux in a dose-dependent manner. ( A) RNA (10 µg) encoding both chains of the A2-SL9–specific TCR was mixed with 0 (thin solid line), 0.125 (short dashed line), 1 (long dashed line), or 10 (thick solid line) µg RNA encoding PD-1 and was transfected into resting primary human CD8 T cells. After overnight culture, the ability to bind SL9 tetramer ( Upper) and PD-1 expression ( Lower) were measured by flow cytometry. Untransfected cells served as negative control (gray shading). ( B) PD-1 geometric mean fluorescent intensities are plotted from the data gathered in A. ( C) T cells pulsed with the calcium-sensitive dye Fura-2 were injected into the chamber containing poly- l-lysine–anchored K.A2.SL9.PD-L1 aAPCs, and the 510-nm emissions excited by 340-nm and 380-nm light were captured immediately for 45 min at 5-s intervals. Images show the composition of a bright-field image overlaid with a transparent color scale of the ratio of fluorescence emissions at 340 and 380 nm of Fura-2. The ratio correlates with intracellular calcium concentration: Green indicates resting (low) concentration, and red indicates high intracellular calcium concentration. The pictures show 200-s snapshots after mixing of cells transfected with A2-SL9–specific TCRs alone or with a mixture of different amounts of PD-1 with K.A2 DsRed.PD-L1 (see Movies S1– S4). ( D) Plot showing the number of T cells that fluxed Ca 2+ at least once during the 45-min experiment as a function of PD-1 expression. An increase in the ratio of 510-nm emission excited by 340 nm to that excited by 380 nm indicated an increased intracellular calcium level. T cells with a ratio more than two times that before stimulation were considered to have fluxed calcium. Data are representative of three individual experiments.
PD-1 signaling increases the number of engaged TCRs required to initiate a Ca
2+ flux. ( A) T2 cells were transduced with a lentiviral vector encoding PD-L1, and a single clone was expanded and evaluated for PD-L1 expression by flow cytometry. ( B) T2 cells were loaded with the indicated concentrations of biotinylated labeled SL9 peptide and were mixed with CD8 T cells coexpressing intermediate levels of PD-1 and SL9-specific TCR. IFN-γ secretion was measured by ELISA from supernatant collected after 24 h of culture. ( C) Dose response of calcium signals to the number of pMHC complexes at the T-cell/APC interface in the presence and absence of PD-L1. Fura-2 ratios were integrated every 20 s for 10 min after initial calcium release. The integrated calcium signals were normalized to the maximal signal. The number of pMHC complexes was quantified as described in Methods. Each point represents data from a single cell. Data were pooled from more than seven experiments carried out on different days using T cells independently prepared from different donors. The two sets of data (T cells stimulated by PD-L1 − and PD-L1 + aAPCs) are statistically different ( P = 0.008, Mann–Whitney test).
T-cell proliferation is highly sensitive to PD-1–mediated inhibition. (
A) RNA (10 µg) encoding of the A2-SL9–specific TCR was mixed with 0, 0.125, 1, or 10 µg RNA encoding PD-1 and was transfected into equally mixed resting primary human CD8 and CD4 T cells. After overnight culture, PD-1 expression was measured by flow cytometry. Data are representative of three individual experiments. ( B) Cells described in A were stained with CFSE and cocultured with the indicated aAPCs at a 2:1 ratio for 5 d, and CFSE dilution was measured by flow cytometry. As a positive control, T cells also were stimulated with CD3/CD28 beads at a 3:1 ratio. ( C) Percent suppression of T-cell proliferation was calculated as described in Methods and averaged from three independent experiments. Error bars indicate SD ( n = 3). White bars indicate T cells stimulated by K.A2.SL9-dsRED, gray bars indicate T cells stimulated by K.A2.SL9-dsRED PD-L1, and black bars indicate T cells stimulated with K.A2.SL9-dsRED PD-L2. ( D and E) PD-1 ( D) and CTLA-4 ( E) expression was measured after 3-d stimulation with K.A2.SL9 (solid lines) or K.A2.SL9.PD-L1 (long dashed lines).
IL-2 and TNF-α expression are more sensitive to PD-1–mediated inhibition than IFN-γ or MIP-1β production. (
A) Previously stimulated and expanded primary human T cells were transfected with A2-SL9–specific TCR and 0, 0.125, 1, or 10 µg RNA encoding PD-1. After overnight culture, cells were stimulated with the indicated aAPCs for 5 h. The ability of these stimulated CD8 cells to produce MIP1β, IFN-γ, TNF-α, and IL-2 was measured by intracellular cytokine staining. Representative data from donor 1 showing IFN-γ and IL-2 production is plotted in A, and complete data from four donors are plotted in B. Red triangles indicate the samples with K.A2.SL9.PD-L1. Black triangles indicate samples with K. A2. SL9.
High levels of PD-1 are required to interfere with cytotoxicity. (
A and B) An equal mixture of ( A) K.A2.DsRed SL9 and K.A2.GFP or ( B) K.A2.DsRed SL9.PD-L1 and K.A2.GFP was cocultured with primary CD8 T cells transfected with A2-SL9–specific TCRs and 0, 0.125, 1, or 10 µg RNA encoding PD-1 at a 1:5 target–to–T-cell ratio in duplicate. After 24 h of culture, cells were stained by eFluor 780 viability dye to exclude dead cells, and GFP and DsRed expression were measured by flow cytometry. ( C) The percent suppression of T-cell cytotoxicity was calculated as described in Methods and averaged from three independent experiments. Error bars indicate SD ( n = 3).
Strength of PD-1 signaling differentially affects T-cell effector functions. (
A) Development of a model for PD-1 signaling. Primary human T cells were transfected with RNA encoding HIV-1–specific TCRs and a variable amount of PD-1 so that T-cell responses could be studied in a setting where the only variable was PD-1 expression. K562-based artificial antigen-presenting cells (aAPCs) expressing HLA-A2 and the HIV-1–specific epitope in the presence or absence of PD-L1 were constructed to interrogate PD-1 function. ( B) PD-1 signaling makes T cells less sensitive to TCR-generated signals. The peptide-counting assay was performed to determine the number of clustered TCRs required to initiate Ca 2+ flux in the presence or absence of PD-1 engagement. ( C) T-cell functions that are most or least sensitive to PD-1 expression during encounters with aAPCs expressing PD-L1. T cells expressing high levels of PD-1 were largely nonfunctional when stimulated by aAPCs expressing PD-L1, suggesting that PD-1 expression sufficiently explains T-cell dysfunction in response to chronic antigen exposure.
All figures (7)
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Research Support, N.I.H., Extramural
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