PD-1, PD-L1, PD-L2 expression in the chordoma microenvironment

Abstract

Chordomas are rare malignant tumors that are postulated to arise from remnants of the notochord. Currently, the interaction between chordomas and the host immune system is poorly understood. The checkpoint protein, PD-1 is expressed by circulating lymphocytes and is a marker of activation and exhaustion. Its ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), are expressed on a variety of human cancers; however this pathway has not been previously reported in chordomas. We used flow cytometric and RT-PCR analysis in three established primary and recurrent chordoma cell lines (U-CH1, U-CH2, and JHC7) as well as immunohistochemical analysis of chordoma tissues from 10 patients to identify and localize expression of PD-1 pathway proteins. PD-1 ligands are not constitutively expressed by chordoma cells, but their expression is induced in the setting of pro-inflammatory cytokines in all cell lines examined. In paraffin embedded tissues, we found that tumor infiltrating lymphocytes expressed PD-1 in 3/6 cases. We also found that, although chordoma cells did not express significant levels of PD-L1, PD-L1 expression was observed on tumor-infiltrating macrophages and tumor infiltrating lymphocytes. Our study suggests that PD-1, PD-L1, and PD-L2 are present in the microenvironment of a subset of chordomas analyzed. Future studies are needed to evaluate the contribution of the PD-1 pathway to the immunosuppressive microenvironment of chordomas.

Fig. 1

a Schematic representation of the PD-1–PD-L1 pathway and its modulation by cytokines expressed in the chordoma microenvironment and analysis of the surface expression of PD-L1 by the U-CH1, U-CH2, and JHC7 cell lines with flow cytometry. The blue curve denotes the isotype control and the red line the PD-L1 staining. Expression of PD-L1 was analyzed under basal conditions and following stimulation with IFN-γ and IL-4. The U-CH1, U-CH2, and JHC-7 cell lines all showed basal expression of PD-L1 (column 1) and inducible levels of PD-L1 by IFN-γ (column 2). b Analysis of the surface expression of PD-L2 by the U-CH1, U-CH2, and JHC7 cell lines by flow cytometry. Expression of PD-L2 was analyzed under basal conditions (column 1) and following stimulation with IFN-γ (column 2) and IL-4 (column 3). c RT-PCR demonstrating the relative expression of PD-L1 and PD-L2 in the U-CH1 and U-CH2 cell lines. c The fold expression of mRNA transcripts of PD-L1 and PD-L2 were analyzed in the basal state and following stimulation with IFN-γ and IL-4. Both PD-L1 and PD-L2 were expressed following exposure to IFN-γ in the U-CH1 and U-CH2 cell lines. Exposure to IL-4 increased expression of PD-L2 in the U-CH2 cell line

Fig. 1

a Schematic representation of the PD-1–PD-L1 pathway and its modulation by cytokines expressed in the chordoma microenvironment and analysis of the surface expression of PD-L1 by the U-CH1, U-CH2, and JHC7 cell lines with flow cytometry. The blue curve denotes the isotype control and the red line the PD-L1 staining. Expression of PD-L1 was analyzed under basal conditions and following stimulation with IFN-γ and IL-4. The U-CH1, U-CH2, and JHC-7 cell lines all showed basal expression of PD-L1 (column 1) and inducible levels of PD-L1 by IFN-γ (column 2). b Analysis of the surface expression of PD-L2 by the U-CH1, U-CH2, and JHC7 cell lines by flow cytometry. Expression of PD-L2 was analyzed under basal conditions (column 1) and following stimulation with IFN-γ (column 2) and IL-4 (column 3). c RT-PCR demonstrating the relative expression of PD-L1 and PD-L2 in the U-CH1 and U-CH2 cell lines. c The fold expression of mRNA transcripts of PD-L1 and PD-L2 were analyzed in the basal state and following stimulation with IFN-γ and IL-4. Both PD-L1 and PD-L2 were expressed following exposure to IFN-γ in the U-CH1 and U-CH2 cell lines. Exposure to IL-4 increased expression of PD-L2 in the U-CH2 cell line

Fig. 2

Comparison of PD-L1 and PD-L2 MFI of the U-CH1, U-CH2, and JHC7 cell lines by flow cytometry. MFI values of PD-L1 and PD-L2 were calculated under basal conditions (column 1) and following stimulation with IFN-γ (column 2) and IL-4 (column 3)

Fig. 3

Representative photomicrograph demonstrating the location of PD-1 and PD-L1 expression in the chordoma microenvironment. a PD-L1 expression co-localizing with TILs and TAMs in the same geographic distribution as PD-1 expression. b PD-1 staining on peritumoral and tumor infiltrating lymphocytes c IgG isotype control for PD-L1 staining. ×68 original magnification, all panels