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Rescue of exhausted CD8 T cells by PD-1–targeted therapies is CD28-dependent

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Science  31 Mar 2017:
Vol. 355, Issue 6332, pp. 1423-1427
DOI: 10.1126/science.aaf0683
  • Fig. 1 B7-costimulation is necessary for rescue of virus-specific CD8 T cells after PD-1 blockade during chronic LCMV infection.

    (A) Experimental layout. In (B) and (C), mice received CTLA-4-Ig, and in (D) to (J), mice received anti-B7-1 and anti-B7-2 blocking antibodies during the course of anti-PD-L1 treatment. (B) Frequencies of LCMV-DbGP276–specific CD8 T cells in the spleen. Data are representative of three independent experiments, with at least four mice per group. (C) Numbers of LCMV-DbGP276–specific CD8 T cells in the spleen. Data show one representative experiment of three independent experiments, with at least four mice per group. Error bars indicate SEM. (D) Numbers of LCMV-DbGP33 and LCMV-DbGP276–specific CD8 T cells in different organs. Data show combined data from two of three independent experiments, with three to five mice per group. Error bars indicate SEM. (E) Ki-67 expression on LCMV-DbGP276–specific CD8 T cells in the spleen. Data are representative of three independent experiments, with three to five mice per group. (F) Frequencies of splenic LCMV-DbGP276–specific CD8 T cells expressing granzyme B. Data show one representative experiment of three independent experiments, with three to five mice per group. Error bars indicate SEM. (G) Numbers of IFN-γ–producing CD8 T cells in the spleen after ex vivo restimulation with the indicated peptides. Data show combined data from two of three independent experiments, with three to five mice per group. Comparisons are between treated groups and untreated mice. Error bars indicate SEM. (H) Frequencies of CD8 T cells producing IFN-γ in the spleen after ex vivo restimulation with a pool of LCMV peptides. Data are representative of three independent experiments, with three to five mice per group. (I and J) Viral titer in (I) lung and (J) liver, as quantified by means of plaque assay. PFU, plaque forming units. Data show combined data from two of three independent experiments, with three to five mice per group. Error bars indicate SEM. (K) Experiment layout for (L) and (M). (L) Frequency of P14 cells in spleen. Data show combined data from two of three independent experiments, with three or four mice per group. Error bars indicate SEM. (M) Frequency of P14 cells producing IFN-γ after ex vivo restimulation with LCMV GP33 peptide. Data show combined data from two of three independent experiments, with three or four mice per group. Error bars indicate SEM. Analysis of variance (ANOVA) with Sidak’s correction for multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001. ****P < 0.0001. NS, not significant.

  • Fig. 2 Cell-intrinsic requirement of CD28 expression for exhausted CD8 T cell expansion upon PD-1 blockade.

    (A) Experimental layout for (B) and (C). (B) CD28 and Ki-67 expression on P14 CD28f/f CreERT2neg and P14 CD28f/f CreERT2+, in the spleen of a representative mouse for each group (n = 9 mice). (C) Summary of data as in (B). Graph shows frequencies of cells expressing Ki-67 in the spleen among each indicated population. Data show one representative experiment of three independent experiments, with at least three mice per group. Error bars indicate SEM. (D) Experimental layout for (E) to (H). (E) Frequencies of cells expressing Ki-67 in the spleen among each indicated population, 9 days after anti-PD-L1 treatment. Data show combined data from two independent experiments with at least three mice per group. Error bars indicate SEM. (F) Frequencies of cells expressing Ki-67 in the spleen among each indicated population, 14 days after anti-PD-L1 treatment. Data show combined data from two out of three independent experiments with at least three mice per group. Error bars indicate SEM. (G) Gating strategy and Ki-67 expression on splenocytes from a representative mouse treated with anti-PD-L1 for 14 days, as in (F). (H) Frequency of CD28neg cells among each population of Cre+ cells as indicated, 14 days after anti-PD-L1 treatment. Data show combined data from two experiments out of three independent experiments, with at least three mice per group each. Error bars indicate SEM. (C), (E), (F), (H) unpaired t test; **P < 0.01, ***P < 0.001. NS, not significant.

  • Fig. 3 Effectiveness of PD-1 therapy for control of CT26 tumor relies on the CD28/B7 pathway.

    Mice were depleted of CD4 T cells for the duration of the experiment. CT26 tumor–bearing mice were enrolled into different treatment groups as indicated. (A) Individual tumor growth, represented by tumor volume. (Insets) Ratio of mice that experienced tumor progression in each treatment group. Shaded gray area indicates duration of treatment. Data show one representative experiment out of three independent experiments. (B) Survival curves from data in (A). Data show one representative experiment (9 or 10 mice per group) out of three independent experiments. Comparisons are by log-rank (Mantel-Cox) test *P < 0.05. (C) Percentage of mice unable to control tumor growth. Data show summary of three independent experiments (n = 26 to 28 mice per treatment group). Error bars indicate SEM. ANOVA with Sidak’s correction for multiple comparisons; **P < 0.01, ***P < 0.001. NS, not significant.

  • Fig. 4 PD-1+ CD8 T cells that proliferate in the peripheral blood of lung cancer patients receiving PD-1 therapy express CD28.

    (A) Overview of study design. (B) Ki-67 and PD-1 expression on CD8 T cells from two representative patients (Pt) out of 13 patients with increased CD8 T cell responses after PD-1–targeted therapy. (C) As in (B), but showing HLA-DR and CD38 expression. Dot plots at the far right were gated on posttreatment Ki-67+ PD-1+ CD8 T cells, as indicated in (B). (D) CD28 expression on Ki-67+ PD-1+ CD8 T cells in posttreatment samples, as gated in (B) (n = 13 patients with least a twofold increase from baseline in the frequency of Ki-67+PD-1+ CD8 T cells).

Supplementary Materials

  • Rescue of exhausted CD8 T cells by PD-1–targeted therapies is CD28-dependent

    Alice O. Kamphorst, Andreas Wieland, Tahseen Nasti, Shu Yang, Ruan Zhang, Daniel L. Barber, Bogumila T. Konieczny, Candace Z. Daugherty, Lydia Koenig, Ke Yu, Gabriel L. Sica, Arlene H. Sharpe, Gordon J. Freeman, Bruce R. Blazar, Laurence A. Turka, Taofeek K. Owonikoko, Rathi Pillai, Suresh S. Ramalingam, Koichi Araki, Rafi Ahmed

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Supplementary Text
    • Figs. S1 to S9
    • References (27–30)

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