Figure 2S 4 7 A - C 080125 CSCs 080418 CSCs - + IFN-a 48 h + IFN-a 48 h + IFN-a 72 h 6 + IFN-a 72 h 3 5 MRFI 4 2 3 2 1 1 0 0 MHC I MHC II MICA MICB ULBP-1 ULBP-2 ULBP-3 ULBP-4 MHC I MHC II MICA MICB ULBP-1 ULBP-2 ULBP-3 ULBP-4 7 B 13 080125 FBS - D 080418 FBS - + IFN-a 48 h 12 + IFN-a 48 h + IFN-a 72 h + IFN-a 72 h 6 080125 FBS 11 10 5 9 8 4 7 6 3 MRFI 5 4 2 3 2 1 1 0 0 MHC I MHC II MICA MICB ULBP-1 ULBP-2 ULBP-3 ULBP-4 MHC I MHC II MICA MICB ULBP-1 ULBP-2 ULBP-3 ULBP-4 Molecule Molecule FIGURE 4S FIGURE 5S Panel A Panel B FIGURE 6S A B C D Supplemental Results Table 1S. Modulation by IFN-α of APM in GBM CSC and FBS tumor cell lines. Molecule * Cell line IFN-α‡ HLA β2-m# HLA LMP TAP1 TAP2 class II A A HC§ 2 7 10 080125 CSCs - 1∞ (1) 3 (65) 2 (91) 1 (2) 6 (47) 2 (61) 1 (3) 1 (2) 1 (3) + 2 (81) 11 (80) 13 (99) 1 (3) 8 (88) 4 (91) 1 (2) 1 (3) 2 (68) 080125 FBS - 2 (81) 4 (63) 4 (83) 1 (3) 6 (80) 3 (67) 2 (86) 1 (3) 2 (75) + 2 (99) 14 (90) 7 (97) 5 (75) 7 (100) 6 (98) 2 (90) 1 (4) 3 (87) 080418 CSCs - 2 (51) 1 (1) 1 (3) 2 (47) 2 (83) 2 (54) 1 (4) 1 (2) 1 (3) + 2 (81) 3 (76) 5 (75) 2 (50) 2 (83) 3 (71) 1 (3) 2 (87) 1 (2) 080418 FBS - 1 (3) 3 (70) 2 (88) 1 (4) 3 (87) 2 (76) 1 (3) 1 (3) 1 (2) + 2 (78) 7 (98) 5 (99) 2 (94) 5 (100) 3 (100) 1 (4) 2 (100) 1 (2) 070104 CSCs - 1 (2) 1 (3) 1 (3) 2 (78) 1 (3) 1 (2) 1 (3) 1 (3) 1 (2) + 2 (98) 8 (100) 10 (88) 4 (89) 3 (98) 3 (94) 1 (4) 2 (86) 2 (79) * expression of APM molecules was evaluated by intracellular staining and cytofluorimetric analysis; ‡ cells were treatead or not (+/-) for 72 h with 1000 IU/ml of IFN-α; # β-2 microglobulin; § β-2 microglobulin-free HLA-A heavy chain; ∞ values are indicated as ratio between the mean of fluorescence intensity of cells stained with the selected mAb and that of the negative control; bold values indicate significant MRFI (≥ 2). The numbers in parenthesis represent the percentage of positive cells. The experiment has been repeated three times; SD for each value ≥ 0.8 ≤ 4. Table 2S . Expression of proteasome and APM-associated chaperon molecules in GBM CSC and FBS tumor cell lines. Cell line Molecule * Delta MB1 Z Calnexin Calreticulin ERp57 Tapasin 0627 CSCs 7 ‡ (100) 1 (3) 1 (5) 20 (100) 2 (92) 2 (67) 2 (86) 0627 FBS 1 (1) 1 (2) 1 (3) 3 (99) 1 (3) 1 (2) 1 (2) 080125 CSCs 16 (85) 1 (2) 1 (2) 49 (99) 2 (85) 1 (3) 2 (99) 080125 FBS 1 (5) 1 (4) 1 (5) 28 (100) 3 (95) 1 (2) 1 (3) 080418 CSCs 8 (100) 1 (5) 1 (4) 13 (100) 2 (99) 1 (5) 1 (4) 080418 FBS 7 (89) 1 (5) 1 (4) 16 (98) 3 (98) 1 (3) 1 (5) 080201 CSCs 36 (99) 3 (98) 3 (98) 55 (100) 10 (98) 3 (98) 7 (97) 080201 FBS 16 (98) 3 (98) 6 (98) 80 (100) 9 (98) 2 (97) 5 (98) 071011 CSCs 17 (98) 3 (87) 3 (75) 100 (100) 15 (98) 3 (87) 7 (76) 071011 FBS 16 (100) 1 (4) 2 (87) 20 (100) 4 (87) 1 (3) 2 (98) 080325 CSCs 10 (98) 3 (98) 4 (98) 200 (100) 5 (99) 2 (95) 5 (98) 070104 CSCs 1 (3) 2 (93) 1 (4) 52 (100) 2 (88) 1 (4) 1 (3) 1869 EBV-B 2 (99) 3 (89) 2 (82) 100 (91) 2 (94) 2 (81) 2 (70) * expression of APM molecules was evaluated by immunofluorescence on permeabilized cells and cytofluorimetric analysis; ‡ values are indicated as ratio between the mean of fluorescence intensity of cells stained with the selected mAb and that of the negative control; bold values indicate significant MRFI (≥ 2). The numbers in parenthesis represent the percentage of positive cells. The experiment has been repeated three times; SD for each value ≥ 1.2 ≤ 4. Table 3S . Modulation by IFN-α of the expression of proteasome and APM-associated chaperon molecules in GBM CSC and FBS tumor cell lines. Molecule * Cell line IFN-α ‡ Delta MB1 Z Calnexin Calreticulin ERp57 Tapasin 080125 CSCs - 16# (85) 1 (2) 1 (5) 49 (100) 2 (95) 1 (2) 2 (3) + 22 (100) 1 (3) 1 (4) 73 (100) 2 (100) 1 (2) 2 (2) 080125 FBS - 1 (5) 1 (4) 1 (5) 28 (100) 3 (95) 1 (2) 1 (3) + 10 (99) 1 (2) 2 (99) 30 (100) 3 (98) 2 (89) 1 (2) 080418 CSCs - 8 (100) 1 (5) 1 (4) 13 (100) 2 (99) 1 (5) 1 (4) + 8 (100) 1 (4) 1 (3) 13 (100) 3 (100) 8 (99) 1 (3) 080418 FBS - 7 (89) 1 (5) 1 (4) 16 (98) 3 (98) 1 (3) 1 (5) + 9 (100) 1 (3) 1 (3) 26 (100) 3 (99) 1 (2) 1 (3) 070104 CSCs - 1 (3) 2 (93) 1 (4) 52 (100) 2 (88) 1 (4) 1 (3) + 13 (99) 1 (3) 1 (4) 27 (100) 3 (98) 1 (3) 1 (3) * expression of APM molecules was evaluated by immunofluorescence on permeabilized cells and cytofluorimetric analysis; ‡ cells were treatead or not (+/-) for 72 h with 1000 IU/ml of IFN-α; # values are indicated as ratio between the mean of fluorescence intensity of cells stained with the selected mAb and that of the negative control; bold values indicate significant MRFI (≥ 2). The numbers in parenthesis represent the percentage of positive cells. The experiment has been repeated three times; SD for each value ≥ 1 ≤ 4. Table 4S. Modulation of MHC and APM molecules by 5-Aza CdR treatment of GBM and FBS tumor cell lines Cell line 5-Aza-CdR * Molecule ‡ MHC II MICA MB1 LMP10 TAP1 080125 CSCs - 1# (2) 1 (4) 1 (2) 1 (3) 1 (2) + 1 (2) 1 (3) 2 (98) 1 (3) 1 (3) 080125 FBS - 1 (2) 3 (95) 1 (3) 1 (4) 1 (2) + 1 (3) 3 (98) 3 (90) 2 (80) 2 (88) 080418 CSCs - 1 (2) 1 (2) 1 (5) 1 (4) 1 (3) + 1 (3) 1 (3) 1 (3) 1 (3) 1 (2) 080418 FBS - 1 (3) 3 (95) 3 (98) 4 (97) 1 (3) + 1 (3) 4 (99) 6 (100) 6 (100) 6 (99) 070104 - 2 (18) 1 (3) 2 (93) 1 (3) 1 (3) + 5 (87) 1 (4) 2 (97) 1 (2) 1 (3) 1869 EBV-B - 12 (96) 1 (3) 3 (89) 3 (91) 4 (95) + 20 (100) 1 (3) 5 (100) 4 (100) 8 (100) * 5-Aza-2'-deoxycytidine (5μM) was added to culture medium for 4 days; ‡ molecule expression was assessed by surface (MHC II and MICA) or intracellular staining (MIB, LMP10 and TAP1) of cells with specific antibodies (see Material and Methods) and cytofluorimetric analysis. # values are indicated as ratio between the mean of fluorescence intensity of cells stained with the selected mAb and that of the negative control; bold values indicate significant MRFI (≥ 2). The numbers in parenthesis represent the percentage of positive cells. The experiment has been repeated three times; SD for each value ≥ 1 ≤ 4. Table 5S. Expression of immune response inhibitory molecules by GBM CSC or FBS tumor cell lines Cell line Molecule* CTLA-4 PD-1 PD-L1 PD-L2 070104 CSC 2‡ (69) 5 (95) 12 (98) 1 (4) 080125 CSCs 2 (81) 2 (94) 10 (99) 1 (3) 080125 FBS 2 (65) 3 (96) 19 (99) 2 (70) 080418 CSCs 2 (55) 2 (85) 7 (99) 1 (2) 080418 FBS 1 (5) 1 (2) 9 (98) 1 (3) 080201 CSCs 2 (77) 3 (95) 11 (99) 1 (3) 080201 FBS 2 (95) 2 (97) 31 (98) 2 (93) 071011 CSCs 2 (42) 2 (59) 4 (97) 1 (2) 071011 FBS 1 (5) 1 (4) 2 (50) 1 (3) 080325 CSCs 2 (62) 2 (56) 4 (94) 1 (3) SW480 col 2 (59) 2 (80) 6 (98) 1 (1) 1061 mel 2 (54) 1 (1) 6 (99) 1 (2) 1869 EBV-B 1 (1) 2 (83) 5 (97) 1 (2) * expression of the indicated molecules was evaluated by immunofluorescence and cytofluorimetric analysis; ‡ values are indicated as ratio between the mean of fluorescence intensity of cells stained with the selected mAb and that of the negative control (MRFI); bold values indicate significant MRFI (≥ 2). The numbers in parenthesis represent the percentage of positive cells. The experiment has been repeated three times with SD for each value ≥ 0.6 ≤ 4. SW480 col and 1061 mel, that are a colorectal and a melanoma cell lines, respectively, were used in the experiment as not GBM tumor cells. Table 6S. Genes differentially expressed between CSC and FBS, p<0.005. Genes are ranked according to fold change. Fold- Paramet Permutati change Gene symbol Description UG cluster ric p- on p-value (CSC/FB value S) oligodendrocyte transcription OLIG1 factor 1 (OLIG1), mRNA.