SUPPLEMENTARY METHODS
Calculation of delta CT CD1d from qRT-PCR of 20 Primary Medulloblastoma
mRNA expression of housekeeping gene HPRT from qRT-PCR of all 20 samples of primary MB samples was normalized using the median CT of HPRT. Correction factor was calculated for each tumor (Median HPRT – HPRT CT for each sample). Corrected CTCD1d for each tumor was calculated (CT CD1d + correction factor). The highest corrected CT CD1d was used as the reference CT of CD1d. Delta CT of CD1d of each tumor was calculated (Reference CT CD1d – Individual CT CD1d).
SUPPLEMENTARY FIGURES
Supplementary Figure 1: FACs analysis of a fresh primary CD1d-medulloblastoma (A, C) Using CD1d and CD45 antibody. Y-axis representative of CD1d expression, x-axis representative of CD45 expression. (A) Tumor cells are CD45- and some tumor cells are CD1d+. (C) Some lymphocytes (CD45+) are CD1d+. (B) Using CD1d and CD68 antibody. Y-axis representative of CD1d expression, x-axis representative of CD68 expression. Macrophages are CD68+. Majority of cells expressing CD1d in medulloblastoma are CD68+ macrophages. (D) Using NKT 6B11 and CD3 antibody. Y-axis representative of NKT 6B11 (expressed exclusively by NKT cells), x-axis representative of CD3 expression. T cells (CD3+) infiltrate medulloblastoma, very few are NKT cells.
Supplementary Figure 2: Kaplan Meier curves showing association of survival outcome in medulloblastoma with CD1d expression by IHC (A-B) and CD163L1 (membrane form of CD163) expression by microarray (C-D). (A) Overall survival (OS) (p=0.02), (B) Event-free survival (EFS) (p=0.06) with different degree of CD1d staining, (C) OS (p=0.05), (D) EFS (p=0.03) with different degree of CD163 gene expression
Although both the OS and EFS were better for the group of patients with marked CD1d-expressing MBs (5-year OS and EFS, 100% and 100% respectively, p=0.02 and p=0.06 respectively) compared to the groups with moderate or mild CD1d expression, we are not able to explain why the group of patients with moderate CD1d-expressing MBs had a worse outcome (5-year OS and EFS, 43.8% and 45% respectively) compared to the group of patients with mild CD1d expression (5-year OS and EFS, 91.5% and 71.6% respectively). Further molecular subgroup sub-analyses are limited by the small sample size.
Contrary to the prior study [3], we found that marked CD1d protein expression is associatedwith better event-free survival. These differences may be attributable to the use of mRNA versus protein expression for correlation between the two studies, and different patient cohorts that are both relatively small.
Supplementary Figure 3: qRT-PCR of T-cell-receptor- and V24J18 mRNA in 20 primary pediatric medulloblastoma tumors demonstrating that medulloblastomas are barely infiltrated by V24-invariant natural killer T-cells. Y-axis representative of delta-CT value of T-cell-receptor- expression, x-axis representative of delta-CT value of V24J18 expression. Most tumors have low mRNA expression of V24J18 reflective of low NKT cell infiltration as indicated.
Supplementary Figure 4: CD1d is expressed in some medulloblastoma cell lines. (A-B) FACs analysis of medulloblastoma cell lines using CD1d and CD3 antibody. Y-axis representative of CD3 expression, x-axis representative of CD1d expression.(A) D283-MED (no CD1d expression), (B) MED-8A (CD1d expression).
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