Enhanced T-cell immunity to osteosarcoma through antibody blockade of PD-1/PD-L1 interactions

Abstract

Osteosarcoma is the most common bone cancer in children and adolescents. Although 70% of patients with localized disease are cured with chemotherapy and surgical resection, patients with metastatic osteosarcoma are typically refractory to treatment. Numerous lines of evidence suggest that cytotoxic T lymphocytes (CTLs) limit the development of metastatic osteosarcoma. We have investigated the role of PD-1, an inhibitory TNFR family protein expressed on CTLs, in limiting the efficacy of immune-mediated control of metastatic osteosarcoma. We show that human metastatic, but not primary, osteosarcoma tumors express a ligand for PD-1 (PD-L1) and that tumor-infiltrating CTLs express PD-1, suggesting this pathway may limit CTLs control of metastatic osteosarcoma in patients. PD-L1 is also expressed on the K7M2 osteosarcoma tumor cell line that establishes metastases in mice, and PD-1 is expressed on tumor-infiltrating CTLs during disease progression. Blockade of PD-1/PD-L1 interactions dramatically improves the function of osteosarcoma-reactive CTLs in vitro and in vivo, and results in decreased tumor burden and increased survival in the K7M2 mouse model of metastatic osteosarcoma. Our results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma should be pursued as a therapeutic strategy.

FIGURE 1.

PD-L1 ⁺ and PD-1 ⁺ expression in human metastatic osteosarcoma tissue. A, Paraffin-embedded human primary and metastatic osteosarcoma tissue was stained with 1:200 PD-L1 antibody (Abcam ab174838), detected using a Alexa Fluor 488 tagged secondary, (B) or 1:200 PD:1 antibody (Abcam ab52587), detected using Texas Red tagged secondary, compared with isotype staining. Leica TCS SP5 confocal microscope was used to detect and produce images. Images are maximum intensity projections of several 0.8 mm confocal sections. Counterstained with DAPI 1:2000. Scale bars, 50 µm. Twelve of 16 pediatric patients (approximately 64%) with metastatic disease were PD-L1 ⁺ . Thirteen of 16 pediatric patients (approximately 73%) were PD-1 ⁺ . These sections are 4 rep-resentative patient samples. C, Multiple fields of view from patients stained with anti-CD8 and anti-PDL1 show a strong correlation between PD-L1 ⁺ expression and number of CD8-infiltrating T cells.

FIGURE 2.

CTLs slow progression of metastatic osteosarcoma. Balb/cJ mice were injected with 10⁶ K7M2 osteosarcoma cells post-CD8 depletion. Survival was significantly decreased in mice depleted of CD8 cells (dashed line), compared with wild-type mice (solid line). Log-rank (Mantel-Cox) test showed a significant P-value <0.0001. CD8-depleted group had an n = 10, whereas the wild-type K7M2 injected group had an n = 55.

FIGURE 3.

Expression of PD-L1 and PD-1, on K7M2 cells and tumor-infiltrating lymphocytes, in a mouse model of metastatic osteo-sarcoma. A, PD-L1 is expressed on K7M2 osteosarcoma cell line, and upregulated after implantation. K7M2 cells were stained with anti-PD-L1 antibody (black) or isotype control staining (IgG2a λ, line). MFI for each peak is indicated on graph, showing significant differences between isotype control and PD-L1. In addition, 10⁶ K7M2 cells were injected into a Balb/cJ mouse, and stained for PD-L1 (black). After implantation, a subpopulation of cells (approximately 30%) expressed high levels of PD-L1. B, Immunofluorescent detection of PD-1. Mouse metastatic osteosarcoma tissue was stained with PD-1 antibody, and compared with isotype staining. Leica TCS SP5 confocal microscope was used to detect and produce images. Images are maximum intensity projections of several 1 µm confocal sections. Counterstained with DAPI 1:2000. Scale bars, 50 µm. C, 10⁶ K7M2 cells were injected into Balb/cJ mice. Lymphocytes were isolated and stained with anti-PD-1 and anti-CD8 antibodies and compared with PD-1 expression on lymphocytes isolated from spleen. Flow data were analyzed using FlowJo8.8. D, Multiple mice showed the similar levels of PD-1 ⁺ CD8 ⁺ TILs. Unpaired t test gave a P-value significance of <0.0001 comparing TIL CD8 (black) to CD8 isolated from spleen (open). ****P < 0.0001.

FIGURE 4.

Osteosarcoma-specific CTLs are deficient in cytokine production. 10⁶ cells were stained for extracellular markers, and then fixed and permeabilized, and stained for IFN-γ, TNF, and IL-2 production. TIL CD8 were incubated with K7M2, 4T1 alone, 4T1 cells pulsed with K7M2 tumor lysate, or K7M2 transduced to be PD-L1 for 4 hours. Flow data were analyzed using FlowJo8.8, gating on the PD-1⁺ CD8 ⁺ population. White bars indicate cytokine production to no additional antigen (TILs incubated alone or with 4T1 not pulsed), whereas black bars indicate cytokine production to osteosarcoma cells (with or without PD-L1 expression) or osteosarcoma antigens. In each case, we are measuring the percentage of PD1 ⁺ CD8 ⁺ T cells that are expressing each of the 3 cytokines. A, Multiple t test comparing IFN-γ production by TIL CD8 alone versus cocultured with K7M2 cells gave a P-value = 0.0046. The t test comparing IFN-γ production by TIL CD8 cocultured with nonpulsed 4T1 versus pulsed 4T1 alone gave a P-value = 0.021. IFN-γ production by TIL CD8 cultured with K7M2 cells compared with 4T1 pulsed with K7M2 lysate gave a P-value = 0.0437. Finally, IFN-γ production by TIL CD8 cultured with K7M2 cells compared with K7M2 that are PD-L1 gave a P-value = 0.001. B, Multiple t test comparing TNF production by TIL CD8 alone versus cocultured with K7M2 cells gave a P-value = 0.009. The t test comparing TNF production by TIL CD8 cocultured with nonpulsed 4T1 versus pulsed 4T1 gave a P-value = 0.05. TNF production by TIL CD8 cultured with K7M2 cells compared with 4T1 pulsed with K7M2 lysate gave a P-value = 0.02. Finally, TNF production by TIL CD8 cultured with K7M2 cells compared with K7M2 with negative PD-L1 expression gave a P-value = 0.002. C, Multiple t test comparing IL-2 production by TIL CD8 alone versus cocultured with K7M2 cells gave a P-value = 0.009. IL-2 production by TIL CD8 cultured with K7M2 cells compared with 4T1 pulsed with K7M2 lysate gave a P-value = 0.025. Finally, IL-2 production by TIL CD8 cultured with K7M2 cells compared with K7M2 with negative PD-L1 gave a P-value = 0.0107. Open bars signify TILs cultured alone, black bars signify TILs + antigen. D, LDH ELISA confirming lack of cytotoxicity through tumor-reactive T cells when cocultured with K7M2 tumor cells. This is compared with a control of 4T1 antigen-presenting cells (APCs) that are PD-L1 , either nonpulsed or pulsed with K7M2 tumor lysate, or K7M2 cells transduced to be PD-L1 . The t test comparing effector responses to K7M2 versus APCs pulsed with K7M2 tumor lysate was significantly different (P < 0.0001). The t test comparing effector responses to K7M2 versus K7M2 for PD-L1 was significantly different (P < 0.0001). *P < 0.05, **P < 0.01, ***P < 0.001 ****P < 0.0001.

FIGURE 5.

Blockade of PD-L1 enhances survival and improves disease state. A, Balb/cJ mice were injected with K7M2. Mice were then given therapeutic anti-PD-L1 antibody injections (10F.9G2) IP over several days, and survival was compared with no treatment group. Survival was significantly increased in mice treated with anti-PD-L1 antibody injections (dashed line), compared with wild-type mice (solid line). Log-rank (Mantel-Cox) test showed a significant P-value = 0.0005. PD-L1 mAb treatment group had an n = 10, and the wild-type K7M2 injected group had an n = 10.B, Implantable K7M2 mice treated with PD-L1 monoclonal antibody had a reduction in number of pulmonary metastatic lesions. Mice treated with PD-L1 antibody had a significant decrease in number of lesions (P < 0.0001). When mice finally succumbed to tumor, metastatic lesions were able to get much larger in size compared with control group (P < 0.0001). Area of lesion, width, and length were measured in millimeters. Volume was calculated using equation (width) × (length/2). ****P < 0.0001.

FIGURE 6.

PD-L1 mAb blockade improves the function of metastatic osteosarcoma–infiltrating CTLs in vivo. K7M2 cells were injected into Balb/cJ mice. Treatment group were started on 200 mg PD-L1 monoclonal antibody (clone 10F.92G) repeated every 3 days for 1000 µg total. TILs were collected at time of death, and incubated with (black) or without (white) K7M2 cell stimulation for 5 hours, with GolgiStop. After incubation, cells were stained for extracellular proteins, then fixed and permeabilized, and stained for TNF, IL-2, and IFN-γ production, and compared with TNF, IL-2, and IFN-γ production from nontreated K7M2 injected mice. Flow data were analyzed using FlowJo8.8, gating on the PD-1 ⁺ CD8 ⁺ population. A, Multiple t test comparing TNF production of TIL CD8 cultured alone or cocultured with K7M2, without PD-L1 treatment, gave a P-value = 0.01. Multiple t test comparing TNF production of TIL CD8 cultured alone or cocultured with K7M2, with PD-L1 treatment, gave a P-value = 0.032. Finally, multiple t test comparing TNF production of TIL CD8 cultured with K7M2 in control versus treated mice gave a P-value = 0.0346. B, Multiple t test comparing IL-2 production of TIL CD8 cultured alone or cocultured with K7M2, without PD-L1 treatment, gave a P-value = 0.009. Multiple t test comparing IL-2 production of TIL CD8 cultured alone or cocultured with K7M2, with PD-L1 treatment, gave a P-value = 0.031. Finally, multiple t test comparing IL-2 production of TIL CD8 cultured with K7M2 in control versus treated mice gave a P-value = 0.0059. Number of mice in osteosarcoma group nontreated, n = 10. Number of mice in osteosarcoma group treated, n = 10. Open bars indicate TIL cultured alone. Black bars indicate TILs cultured with antigen. C, No significant differences were seen when multiple t test compared IFN-γ production of TIL CD8 cultured with K7M2 in control versus treated mice (P = 0.1204). *P < 0.05, **P < 0.01.