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. 2017 Jan 15;23(2):454-465.
doi: 10.1158/1078-0432.CCR-16-1163. Epub 2016 Jul 28.

PD-1/PD-L1 Blockade Enhances T-cell Activity and Antitumor Efficacy of Imatinib in Gastrointestinal Stromal Tumors

Adrian M Seifert  1 , Shan Zeng  1 , Jennifer Q Zhang  1 , Teresa S Kim  1 , Noah A Cohen  1 , Michael J Beckman  1 , Benjamin D Medina  1 , Joanna H Maltbaek  1 , Jennifer K Loo  1 , Megan H Crawley  1 , Ferdinand Rossi  1   2 , Peter Besmer  2 , Cristina R Antonescu  3 , Ronald P DeMatteo  4
Affiliations

PD-1/PD-L1 Blockade Enhances T-cell Activity and Antitumor Efficacy of Imatinib in Gastrointestinal Stromal Tumors

Adrian M Seifert et al. Clin Cancer Res. .

Abstract (VSports最新版本)

Purpose: Tyrosine kinase inhibitors are effective in gastrointestinal stromal tumors (GISTs) but often are of transient benefit as resistance commonly develops. Immunotherapy, particularly blockade of the inhibitory receptor programmed death 1 (PD-1) or the ligand programmed death ligand 1 (PD-L1), has shown effectiveness in a variety of cancers. The functional effects of PD-1/PD-L1 blockade are unknown in GISTs VSports手机版. .

Experimental design: We analyzed tumor and matched blood samples from 85 patients with GISTs and determined the expression of immune checkpoint molecules using flow cytometry. We investigated the combination of imatinib with PD-1/PD-L1 blockade in KitV558Δ/+ mice that develop GISTs V体育安卓版. .

Results: The inhibitory receptors PD-1, lymphocyte activation gene 3, and T-cell immunoglobulin mucin-3 were upregulated on tumor-infiltrating T cells compared with T cells from matched blood V体育ios版. PD-1 expression on T cells was highest in imatinib-treated human GISTs. Meanwhile, intratumoral PD-L1 expression was variable. In human GIST cell lines, treatment with imatinib abrogated the IFNγ-induced upregulation of PD-L1 via STAT1 inhibition. In KitV558Δ/+ mice, imatinib downregulated IFNγ-related genes and reduced PD-L1 expression on tumor cells. PD-1 and PD-L1 blockade in vivo each had no efficacy alone but enhanced the antitumor effects of imatinib by increasing T-cell effector function in the presence of KIT and IDO inhibition. .

Conclusions: PD-1/PD-L1 blockade is a promising strategy to improve the effects of targeted therapy in GISTs. Collectively, our results provide the rationale to combine these agents in human GISTs VSports最新版本. Clin Cancer Res; 23(2); 454-65. ©2016 AACR. .

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Conflict of interest statement

The authors have no financial conflict of interest.

Figures

Figure 1
Figure 1. T cells infiltrating human GIST express high levels of inhibitory receptors compared with blood
(A) CD4+ and CD8+ T cells as a percentage of leukocytes (CD45+ cells) in matched blood and tumor specimens (n = 106) from 85 human GIST patients. (B) Histograms of PD-1, LAG-3, and TIM-3 expression on CD4+ and CD8+ T cells in blood (gray), and tumor (black) from a representative sample. PD-1+, LAG-3+, and TIM-3+ cells as percentage of CD4+ (C) and CD8+ T cells (D) from matched peripheral blood and tumor samples. (E) Linear regression analysis between PD-1 expression on CD4+ and CD8+ T cells in 106 human GIST specimens. Data represent mean ± SEM *P < 0.05.
Figure 2
Figure 2. Patterns of PD-1 expression in T cells in human GIST
(A) Expression of individual or the combination of inhibitory receptors (PD-1, LAG-3, TIM-3) on CD4+ and CD8+ T cells in 63 human GIST specimens by Boolean gate analysis. Data represent mean ± SEM *P < 0.05. (B) Total number of inhibitory receptors expressed by CD4+ (top) and CD8+ T cells (bottom) from the blood (left) and tumor (right). (C) Percentage of PD-1+ cells among CD4+ (top) and CD8+ T cells (bottom) in blood and tumor from untreated (n = 36), imatinib-sensitive (n = 38), and imatinib-resistant (n = 32) GIST specimens. Each dot represents a separate specimen. Median values are marked by a horizontal red line. *P < 0.05. (D) Expression of inhibitory receptors on tumor-infiltrating T cells from GIST patients with multiple metastases (n = 10). Each dot represents a separate tumor specimen.
Figure 3
Figure 3. PD-L1 is expressed in a subset of human GISTs
(A) Representative immunohistochemistry with anti-PD-L1 (clone 5H1) shows membranous and cytoplasmic expression in human GIST (indicated by black arrow; scale bar 50 μm). (B) Freshly isolated KIT- (stroma) and KIT+ (tumor) cells from 2 human GISTs were analyzed for PD-L1 mRNA by real-time PCR. Bars represent mean ± SEM. (C) RNA was isolated from fresh frozen untreated (n = 14), imatinib-sensitive (n = 9), and imatinib-resistant (n = 18) human GISTs and analyzed for PD-L1 mRNA using real-time PCR. Bars represent means. (D) PD-L1 mRNA relative expression in 25 GIST samples with confirmed mutation. Median values are marked by a horizontal red line. (E) Scatter plots to analyze the correlation of PD-L1 mRNA with tumor size and (F) mitotic count. Values are log-transformed.
Figure 4
Figure 4. Imatinib abrogates IFN-γ–induced upregulation of PD-L1 on human GIST cell lines
(A) PD-L1 expression in human GIST cell lines as determined by flow cytometry. Gray histograms represent isotype controls. (B) GIST-T1 cells were treated with either PBS, IFN-γ (100 ng/ml), IFN-γ plus the JAK inhibitor tetracyclic pyridine 6 (P6; 1 μM), or IFN-γ plus imatinib (Im; 100 nM). PD-L1 mRNA relative expression was determined at the indicated time points by real-time PCR. (C) Western blot of GIST-T1 treated as described above for 20 h. (D) GIST-T1 cells were transfected with non-target control siRNA or ON-TARGET plus SMARTpool siRNA (STAT1 siRNA) and then treated with PBS or IFN-γ (100 ng/ml) for 6 h. Western blot analysis demonstrating efficiency of STAT1 knockdown and PD-L1 expression. (E) PD-L1 expression in GIST-T1 cells was measured by PCR. (F) GIST-T1 cells were analyzed for IFNGR1 and IRF1 by real-time PCR after 6 h of the indicated treatment. Data are normalized to control. Data represent mean ± SEM *P < 0.05.
Figure 5
Figure 5. Imatinib modulates IFN-γ–related genes and PD-L1 expression in GIST
(A) Bar graph showing mRNA levels of IFN-γ–related genes determined by microarray analysis of tumors from KitV558Δ /+ mice treated with imatinib for 1 week. Data are shown as log2 fold change compared to vehicle-treated KitV558Δ /+ mice (3 per group). Bars represent mean. (B) Representative immunohistochemistry with anti-PD-L1 of tumors from KitV558Δ /+ mice treated with vehicle or imatinib for 4 weeks (scale bar, 100 μm). (C) Histograms of PD-1 and PD-L1 expression on tumor cells (CD45-Kit+), tumor-associated macrophages (TAMs, CD45+F4/80+CD11b+) and T cell subsets (CD4+ Tconv cells: CD45+CD3+CD4+Foxp3-, Tregs: CD45+CD3+CD4+CD25+Foxp3+, and CD8+ T cells: CD45+CD3+CD8+) in GIST-bearing KitV558Δ /+ mice treated with vehicle or imatinib for 1 week (3–5 per group). Representative plots are depicted with isotype control (gray), vehicle (blue) and imatinib (red). (D) Expression of IFN-γ in stimulated CD8+ T cells from spleens, tumor-draining lymph nodes (TdLNs), and tumors from untreated KitV558Δ /+ mice. CD8+ T cells from the indicated tissues were isolated, stimulated in vitro with PMA and ionomycin, then analyzed by flow cytometry. (E) Effect of PD-1 blockade on cytokine production in T cells. T cells were harvested from the mesenteric lymph node (mLN) from B6 mice (WT) or the tumor from untreated KitV558Δ /+ mice and cultured in vitro in the presence of anti-PD-1 (10 μg/ml) or isotype control. After 48 h, culture supernatant was collected and cytokines were measured by cytometric bead array. Data represent mean ± SEM *P < 0.05.
Figure 6
Figure 6. PD-1/PD-L1 blockade enhances the antitumor effects of imatinib in murine GIST
(A) Tumor weights from KitV558Δ /+ mice (3–5 per group) treated with imatinib (Im) or vehicle (Veh) for 4 weeks. Anti-PD-1 or anti-PD-L1 antibodies or their isotypes (Iso) were given on days 0, 4, 8, and 12. Mice were sacrificed at day 28. (B) Western blot of tumor samples from KitV558Δ /+ mice treated as in A. (C) Bulk tumors were analyzed for expression of Pdl1 and Ido1 by real-time PCR. Data are normalized to control. Data represent mean of at least 3 samples ± SEM *P < 0.05. (D) Tumor weights after 3 months of treatment (3–5 mice per group). Antibodies or isotypes were given as in A. Mice were sacrificed at day 90. (E) Tumor weights from KitV558Δ /+ mice (3–5 per group) treated with imatinib for 1 week. Anti-PD-1 or anti-PD-L1 antibodies were given on days 0, 2, 4, and 6. Mice were sacrificed at day 7. (F) Tumor KIT and TUNEL staining after 1 week of treatment. Scale bars, 50μm. (G) Percentages of intratumoral CD8+ T cells that were Ki67+ or IFN-γ/TNF–producing intratumoral CD8+ T cells after in vitro stimulation with PMA and ionomycin. Data represent means of 3–4 samples ± SEM *P < 0.05. (H) Cytokine array densitometry of tumors from KitV558Δ /+ mice after 1 week of treatment. (I) Tumor weights from KitV558Δ /+ mice treated with 1-MT for 1 week (3–5 per group). Anti-PD-1 antibody was given as in E. Mice were sacrificed at day 7. All tumor weights are shown normalized to vehicle control.
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