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. 2018 Apr 10;9(27):19177-19191.
doi: 10.18632/oncotarget.24951.

PD-L1 Expression in Medulloblastoma: An Evaluation by Subgroup

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Free PMC article

PD-L1 Expression in Medulloblastoma: An Evaluation by Subgroup

Allison M Martin et al. Oncotarget. .
Free PMC article

Abstract

Background: This study evaluated the expression of PD-L1 and markers of immune mediated resistance in human medulloblastoma (MB), the most common malignant pediatric brain tumor.

Results: Overall levels of PD-L1 in human MB were low; however, some cases demonstrated robust focal expression associated with increased immune infiltrates. The case with highest PD-L1 expression was a sonic hedgehog (SHH) MB. In cell lines, SHH MB, which are low-MYC expressing, demonstrated both constitutive and inducible expression of PD-L1 while those in Group 3/4 that expressed high levels of MYC had only inducible expression. In vitro, IFN-γ robustly stimulated the expression of PD-L1 in all cell lines while radiation induced variable expression. Forced high MYC expression did not significantly alter PD-L1.

Methods: Human MB tumor samples were evaluated for expression of PD-L1 and immune cell markers in relation to molecular subgroup assignment. PD-L1 expression was functionally analyzed under conditions of interferon gamma (IFN-γ), radiation, and MYC overexpression.

Conclusions: MB expresses low levels of PD-L1 facilitating immune escape. Importantly, TH1 cytokine stimulation appears to be the most potent inducer of PD-L1 expression in vitro suggesting that an inflamed tumor microenvironment is necessary for PD-1 pathway activation in this tumor.

Keywords: B7-H1; PD-1; PD-L1; brain tumor; medulloblastoma.

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare competing financial interests. Charles G. Drake has consulted; for Amplimmune, Bristol Myers Squibb (BMS), Merck, Novartis, Regeneron and Roche-Genentech and Michael Lim has consulted for Aegenus, BMS, and Regeneron all of whom have either anti-PD-1 or anti- PD-L1 reagents in various stages of clinical development. In addition, Dr. Lim has consulted for Oncorous and Boston Biomedical, companies that are developing other immune based anti-cancer treatments. Dr. Drake has received sponsored research funding from BMS. Dr. Lim has received sponsored research. funding from Arbor, Aegenus, Altor, BMS, Immunocellular, Celldex, and Accuray. The corresponding author, Dr. Martin, has no conflicts to declare.

Figures

Figure 1
Figure 1. PD-L1 expression and immune infiltrates by subgroup
Medulloblastoma samples were scored for tumor cell PD-L1 expression, the degree of CD3+ and CD68+ tumor infiltrating immune cells (TIL/TIM), and IBA-1+ cells and compared between SHH and Group 3/4 MB Subgroups. (IBA-1 available for JHH cohort only).
Figure 2
Figure 2. Differential expression pattern of PD-L1 and immune infiltrates in SHH vs group 3/4 MB
Immunohistochemistry of PD-L1, CD3, and CD68 in two representative cases from SHH and Group 3/4 with the highest degree of PD-L1 expression in their respective subgroups. SHH case 36 (top row in A, B, C) had >2% PD-L1 positive staining overall and images represent an area of intense focal staining. Group 3/4 case 25 (bottom row in D, E, F) had 1–2% PD-L1 positive staining overall but did not demonstrate any single area of intense focal staining as seen in Case 36. Isotype antibody staining for these cases is available in Supplementary Figure 1. All images 400× original magnification.
Figure 3
Figure 3. IBA-1 staining reveals heavy microglial infiltration in SHH MB with many microglia co-expressing PD-L1
Immunohistochemistry of one representative SHH MB, 18872, stained for IBA-1 (A) and PD-L1 (B). Many of the IBA-1 expressing microglial cells are also PD-L1+. All images 400× original magnification.
Figure 4
Figure 4. Medulloblastoma cell lines express PD-L1 in a subgroup dependent manner
PD-L1 expression in low and high MYC MB cell lines. Paraffin embedded cell line pellets stained for PD-L1 in DAOY (A) and D425-MED (D). Histograms of PD-L1 expression by flow cytometry using phycoerythrin (PE) conjugated MIH1 clone of PD-L1 (EBioscience) with and without IFN-γ stimulation in DAOY (B), UW228 (C), D425-MED (E), and D283-MED (F).
Figure 5
Figure 5. MYC overexpression in DAOY (YM21) does not alter PD-L1 expression
Dot Plots of PD-L1 expression by flow cytometry using PE-conjugated MIH1 clone of PD-L1 (EBioscience) in YM21 construct made by overexpressing MYC in DAOY via stable lentiviral transfection.
Figure 6
Figure 6. Radiation induces PD-L1 in most medulloblastoma cell lines
Bar graph depicts percent PD-L1 positive cells above baseline as determined by flow cytometry using PE-conjugated MIH1 clone of PD-L1 (EBioscience) in DAOY (A), UW228 (B), D283-MED (C), and D425-MED (D) after irradiation with 2, 5, or 10 Gy at 2, 4, 8, 24, and 48 hours. IFN-γ values were obtained without radiation. Each bar graph depicts findings from 2 different experiments where all data points were repeated. Error bars represent standard deviation of the mean. Levels of PD-L1 that were comparable to those induced by IFN-γ included UW228 hours 2–24 at 2, 5 and 10 Gy and D283-MED at hour 8, 10 Gy. All other values were significantly less than those induced by IFN-γ as determined by 2-way ANOVA corrected for multiple comparisons. Full statistical analysis available in Supplementary Table 1-1–1-4.

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