Cdk5 disruption attenuates tumor PD-L1 expression and promotes antitumor immunity

Abstract

Cancers often evade immune surveillance by adopting peripheral tissue- tolerance mechanisms, such as the expression of programmed cell death ligand 1 (PD-L1), the inhibition of which results in potent antitumor immunity. Here, we show that cyclin-dependent kinase 5 (Cdk5), a serine-threonine kinase that is highly active in postmitotic neurons and in many cancers, allows medulloblastoma (MB) to evade immune elimination. Interferon-γ (IFN-γ)-induced PD-L1 up-regulation on MB requires Cdk5, and disruption of Cdk5 expression in a mouse model of MB results in potent CD4(+) T cell-mediated tumor rejection. Loss of Cdk5 results in persistent expression of the PD-L1 transcriptional repressors, the interferon regulatory factors IRF2 and IRF2BP2, which likely leads to reduced PD-L1 expression on tumors. Our finding highlights a central role for Cdk5 in immune checkpoint regulation by tumor cells.

Copyright © 2016, American Association for the Advancement of Science.

Figures

Fig. 1. Targeted deletion of Cdk5 in MB results in rejection by CD4+ Tcells

(A) Cdk5 and p35 proteins are expressed in murine and human MB cell lines in vitro. (B) TFS in C57BL/6J mice injected with MM1 crNeg or crCdk5 cells with various depleting antibodies (n = 10 per group). (C) Immunohistochemistry of six clinical MB samples reveals an inverse correlation between tumor Cdk5 expression (top) and CD3+ T cell infiltration (bottom). Pearson correlation = −0.91 (fig. S2D). Scale bars, 100 μm. (D) Tumor-growth kinetics for individual animals in each group from (B). The top right-hand graph shows the center one with an expanded scale. X indicates that an animal was killed because of tumor size or ulceration.

Fig. 2. Disruption of either Cdk5 gene expression or Cdk5 activity suppresses PD-L1 expression that cannot be overcome with IFN-γ stimulation in both human and murine MBs

(A) In vitro mRNA expression in arbitrary units (AU) of PD-L1 by MM1 WT, crCdk5, and crNeg cells with or without 24 hours of IFN-γ stimulation. Values represent the average of three biological replicates ± SD. (B) In vitro PD-L1 surface staining of MM1 WT, crCdk5, crNeg, and crPDL1 cells with or without 24 hours of IFN-γ stimulation. Values represent the average mean fluorescence intensity (MFI) ± SD compared with unstimulated MM1 WT cells over seven or eight replicates. (C) Fold change of surface PD-L1 expression in MM1 WT, shCdk5, and shNS cells over the course of 48 hours of IFN-γ stimulation. (D) MFI of PD-L1 and PD-L2 expressed in MM1 WT, shCdk5, and shNS cells. (E) PD-L1 mRNA expression in MM1 WT cells when treated with roscovitine and stimulated with IFN-γ for 24 hours. AU relative to untreated. DMSO, dimethyl sulfoxide. (F) DAOY and UW228 human MB lines treated with roscovitine and stimulated with IFN-γ for 24 hours. MFI relative to untreated samples. (G) TFS of MM1 crNeg and crPDL1 injected mice over 36 days (n = 10 mice per group). *P < 0.05; **P < 0.01; ***P < 0.001. Significance was determined by two-way ANOVA with Bonferoni posttest (A) and (B), Student's t test (C), or log-rank test (G). Representative experiments are shown in (D) and (E).

Fig. 3. Cdk5 gene silencing alters the IFN-γ signaling pathway and is associated with hyperphosphorylation of IRF2BP2

(A) IFN-γ stimulation of MM1 WT, crCdk5, and crNeg cells for 24 hours. IFNGR downstream mediators STAT1, phosphorylated (p)STAT1, IRF1, IRF2, and IRF2BP2 were assayed. (B) Global quantitative phosphoproteomic analysis (left) of MM1 WT, crCdk5, and crNeg cells shows a change in phosphorylation status of 35 different phosphopeptides found in 18 of the 22 identified proteins (right). Twelve proteins exhibit only increased phosphorylation, three exhibit only decreased phosphorylation, and three have both increased and decreased phosphorylation sites. Phosphoproteomic analysis of three biologic repeats of each cell line. Boxes on the left indicate peptides identified from the highest and lowest phosphorylated species that are magnified on the right.

Fig. 4. Orthotopic Cdk5-deficient tumors exhibit increased PD-L1 staining, CD4+ tumor–infiltrating lymphocytes (TILs), and accumulating infiltrates of CD11b+ populations

(A) Tumors extracted 14 days postinoculation from MM1 WT, shCdk5, or shNS mice stained for PD-L1 expression. Dashed line represents margin between tumor (T) and stroma. Black arrows point to increased PD-L1+ and IBA-1+ cells in the tumor stroma. Scale bars, 400 μm. (B and C) Fluorescence-activated cell sorting (FACS) analysis of MM1 WT (black circle) and MM1 crCdk5 (orange square) tumor infiltrate by percentage of cell type. (D) Ratio of total CD8+:CD4+ cell infiltrate. (E) FACS analysis of the percentage of PD-1+ or PD-L1+ cells in the CD4+ or CD8+ populations. (F) FACS analysis of the percentage of myeloid cells in tumor infiltrate based on differential CD45 staining (left) or Ly6C staining among CD11b+CD45+ cells (right). (G) Percent of total CD11b+ population (left) and subpopulations (right) present in tumor infiltrate that express PD-L1. (H) MFI of PD-L1 expression among CD11b+ total population (left) and subpopulations (right). (B), (C), (D), (E), (F), and (G) were graphed as means ± SD. (H) was graphed as individual MFI with mean indicated. n = 9 per group. Each data point represents pooled samples from three mice. *P < 0.05; **P < 0.01. Significance was determined using the Student's t test.