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

Abstract

Programmed cell death-1 (PD-1)-targeted therapies enhance T cell responses and show efficacy in multiple cancers, but the role of costimulatory molecules in this T cell rescue remains elusive. Here, we demonstrate that the CD28/B7 costimulatory pathway is essential for effective PD-1 therapy during chronic viral infection. Conditional gene deletion showed a cell-intrinsic requirement of CD28 for CD8 T cell proliferation after PD-1 blockade. B7-costimulation was also necessary for effective PD-1 therapy in tumor-bearing mice. In addition, we found that CD8 T cells proliferating in blood after PD-1 therapy of lung cancer patients were predominantly CD28-positive. Taken together, these data demonstrate CD28-costimulation requirement for CD8 T cell rescue and suggest an important role for the CD28/B7 pathway in PD-1 therapy of cancer patients.

Fig. 1

B7-costimulation is necessary for rescue of virus-specific CD8 T cells following PD-1 blockade during chronic LCMV infection. (A) Experimental layout. In B and C mice received CTLA-4-Ig, and in D-J mice received anti-B7-1 and anti-B7-2 blocking antibodies during the course of anti-PD-L1 treatment. (B) Frequencies and (C) numbers of LCMV-D^bGP276-specific CD8 T cells in the spleen. (D) Numbers of LCMV-D^bGP33 and LCMV-D^bGP276-specific CD8 T cells in different organs. (E) Ki-67 expression on LCMV-D^bGP276-specific CD8 T cells in the spleen. (F) Frequencies of splenic LCMV-D^bGP276-specific CD8 T cells expressing granzyme B. (G) Numbers of IFN-γ–producing CD8 T cells in the spleen after ex vivo restimulation with the indicated peptides. Comparisons are between treated groups and untreated mice. (H) Frequencies of CD8 T cells producing IFN-γ in the spleen after ex vivo restimulation with a pool of LCMV peptides. (I) Viral titer in lung and (J) liver, as quantified by plaque assay. PFU = plaque forming units. (K) Experiment layout for L and M. (L) Frequency of P14 cells in spleen. (M) Frequency of P14 cells producing IFN-γ after ex vivo restimulation with LCMV GP33 peptide. C and F show data from one representative experiment, and D, G, I, J, L and M show combined data from two experiments. Data are representative of at least 3 independent experiments, with 3-5 mice per group. ANOVA with Sidak’s correction for multiple comparisons. *P<0.05, **P<0.01, ***P<0.001. ****P<0.0001. Error bars indicate standard error of the mean (SEM). 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 CD28^f/f CreERT2^neg and P14 CD28^f/f CreERT2+, in the spleen of representative mice. (C) Frequencies of cells expressing Ki-67 in the spleen among each indicated population. Summary of data as in B. Data from B and C are from one representative experiment of 3 independent experiments, with at least 3 mice per group. (D) Experimental layout for E–H. (E) Frequencies of cells expressing Ki-67 in the spleen among each indicated population, 9 days after anti-PD-L1 treatment. (F) as in E, but 14 days after anti-PD-L1 treatment (G) Gating strategy and Ki-67 expression on splenocytes from a representative mouse treated with anti-PD-L1 for 14 days. (H) Frequency of CD28^neg cells among each population of Cre+ cells as indicated, 14 days after anti-PD-L1 treatment. Data from E-H are from two or three independent experiments, with at least three mice per group. C, E, F, H unpaired t-test, **P<0.01, ***P<0.001. Error bars indicate SEM. NS = not significant.

Fig. 3

Effectiveness of PD-1 therapy for control of CT-26 tumor relies on the CD28/B7 pathway. Mice were depleted of CD4 T cells for the duration of the experiment. CT-26 tumor bearing mice (mean volume of about 150 mm³) were enrolled into different treatment groups as indicated. (A) Individual tumor growth, represented by tumor volume. Insets on each panel show ratio of mice that experienced tumor progression. Shaded grey area indicates duration of treatment. (B) Survival curves from data in A. Comparisons are by log-rank (Mantel-Cox) test *P<0.05. A and B show one representative experiment out of three. (C) Percentage of mice unable to control tumor growth. Summary of three independent experiments with 7–12 mice per group in each experiment. ANOVA with Sidak’s correction for multiple comparisons. *P<0.05, **P<0.01, ***P<0.001. Error bars indicate SEM. 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) with significant CD8 T cell responses after PD-1 targeted therapy. (C) as in B, but showing HLA-DR and CD38 expression. Dot plots in the right were gated on post-treatment Ki-67+ PD-1+ CD8 T cells as indicated in B. (D) CD28 expression on Ki-67+ PD-1+ CD8 T cells in post-treatment samples as gated in B. N=13, patients with least a two-fold increase from baseline in the frequency of Ki-67+PD-1+ CD8 T cells.