Supplementary Data

Figure 2S

43210

76543210

080125 CSCs
080418 CSCs

+ IFN-a 48 h + IFN-a 72 h

13 12 11 10
9

-+ IFN-a 48 h + IFN-a 72 h

080125 FBS
080125 FBS
080418 FBS

+ IFN-a 48 h + IFN-a 72 h
6

543210

876543210

Molecule
Molecule

Supplemental Results

Table 1S. Modulation by IFN-α of APM in GBM CSC and FBS tumor cell lines.

Molecule ^*

class II

080125 CSCs

080125 FBS
-+-

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) 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
080418 FBS
-+-

2 (81) 3 (76) 5 (75) 2 (50) 2 (83) 3 (71) 1 (3) 2 (87) 1 (2)
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)

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.

0627 CSCs 7 ^‡(100) 1 (3) 1 (5) 20 (100)

0627 FBS 1 (1) 1 (2) 1 (3) 3 (99)

080125 CSCs 16 (85) 1 (2) 1 (2) 49 (99)

080125 FBS 1 (5) 1 (4) 1 (5) 28 (100)

2 (85)

1 (3) 1 (2)

2 (99)
3 (95)

1 (5) 1 (3)
1 (4) 1 (5)

071011 FBS 16 (100) 1 (4) 2 (87) 20 (100)

080325 CSCs 10 (98) 3 (98) 4 (98) 200 (100) 070104 CSCs 1 (3) 2 (93) 1 (4) 52 (100)
1869 EBV-B 2 (99) 3 (89) 2 (82) 100 (91)
4 (87) 5 (99) 2 (88) 2 (94)

2 (95) 5 (98)

1 (4) 1 (3)

2 (81) 2 (70)
2 (98)

* 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 ^*

16^#(85) 1 (2) 1 (5) 49 (100) 22 (100) 1 (3) 1 (4) 73 (100)
1 (5) 1 (4) 1 (5) 28 (100)
10 (99) 1 (2) 2 (99) 30 (100)
2 (95)
2 (100)
3 (95) 3 (98)

1 (2) 2 (3) 1 (2) 2 (2) 1 (2) 1 (3)

2 (89) 1 (2)

080125 FBS
+

080418 CSCs
080418 FBS
-+-

8 (100) 1 (5) 1 (4) 13 (100) 8 (100) 1 (4) 1 (3) 13 (100)
7 (89) 1 (5) 1 (4) 16 (98)
9 (100) 1 (3) 1 (3) 26 (100)
2 (99)

1 (5) 1 (4)

3 (100) 8 (99) 1 (3)
3 (98)

1 (3) 1 (5) 1 (2) 1 (3)

3 (99)

1 (4) 1 (3) 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 ≤

Table 4S. Modulation of MHC and APM molecules by 5-Aza CdR treatment of GBM and FBS tumor cell lines

080125 CSCs
080125 FBS
-+-

1^#(2)

1 (2) 1 (3)
1 (4) 1 (3)
1 (2)

2 (98)

1 (3)
1 (3) 1 (3) 1 (4)

2 (80)

1 (2) 1 (3) 1 (2)

2 (88)
3 (95) 3 (98)

3 (90)

080418 CSCs
080418 FBS
-+-

1 (2) 1 (3) 1 (3) 1 (3)
1 (2) 1 (3)
1 (5) 1 (3)
1 (4) 1 (3)
1 (3) 1 (2) 1 (3)

6 (99)
3 (95) 4 (99)
3 (98)
6 (100)
4 (97)
6 (100)

2 (18)

1 (3) 1 (4)

2 (93)

1 (3) 1 (2)
1 (3) 1 (3)

12 (96)

1 (3) 1 (3)

* 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

070104 CSC

2^‡(69)

080125 CSCs
080125 FBS

2 (81)

2 (94)

10 (99)

080418 CSCs
080418 FBS

9 (98)

080201 CSCs
080201 FBS

071011 CSCs
071011 FBS

2 (50)

080325 CSCs SW480 col
1061 mel

2 (62) 2 (59) 2 (54)
2 (56) 2 (80)

4 (94) 6 (98) 6 (99)

1 (3) 1 (1) 1 (2)

1869 EBV-B

* 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

UG cluster ric p- value
Permutati change on p-value (CSC/FB
S)

oligodendrocyte transcription factor 1 (OLIG1), mRNA. ubiquitin specific peptidase 30 (USP30), mRNA. calsequestrin 2 (cardiac muscle) (CASQ2), mRNA.

OLIG1 USP30 CASQ2

2077 1593

Hs.486434 45281 Hs.57975 4.00E-07

906 134
SPARC-like 1 (hevin) (SPARCL1), transcript variant 2, mRNA.

SPARCL1
CA10
Hs.62886

84
996 828 carbonic anhydrase X (CA10), transcript variant 3, mRNA. ADAM metallopeptidase domain 19 (meltrin beta) (ADAM19), transcript variant 2, mRNA. 3-hydroxy-3-methylglutarylCoenzyme A synthase 1 (soluble) (HMGCS1), transcript variant 2, mRNA. SRY (sex determining region Y)- box 8 (SOX8), mRNA. ribosomal protein S6 kinase, 90kDa, polypeptide 2

Hs.463466 18432

HMGCS1 SOX8
Hs.397729

53
691 684

Hs.243678 16431

(RPS6KA2), transcript variant 2, mRNA. dipeptidyl-peptidase 4 (DPP4), mRNA. periostin, osteoblast specific factor (POSTN), transcript variant 2, mRNA.

RPS6KA2
DPP4
Hs.719131

8
655 583

Hs.368912 25174

779
57
569

CDNA FLJ32758 fis, clone
1857 hCG_1999814 TESTI2001792 collagen, type VIII, alpha 2 (COL8A2), mRNA.

Hs.666511 22226

538 506

COL8A2 EMP2
Hs.353001 43215

2117 epithelial membrane protein 2 (EMP2), mRNA.

Hs.531561

guanine nucleotide binding protein (G protein), gamma 7 (GNG7), mRNA.

PREDICTED: misc_RNA
LOC346329 (LOC346329), miscRNA. arylsulfatase D (ARSD), transcript

variant 1, mRNA.

Hs.528631 29984 Hs.567236 507

823 shroom family member 2
SHROOM2 (SHROOM2), mRNA. olfactory receptor, family 51, subfamily B, member 2

410 410 381

OR51B2 ST3GAL6 SIPA1L2

(OR51B2), mRNA.

Hs.680163 44691

1307
9.00E-04
38
ST3 beta-galactoside alpha-2,3- sialyltransferase 6 (ST3GAL6), mRNA. signal-induced proliferationassociated 1 like 2 (SIPA1L2), mRNA.

Hs.148716 Hs.715656

7475 1074
CD164 molecule, sialomucin (CD164), transcript variant 5, mRNA. calumenin (CALU), transcript variant 1, mRNA.

CD164 CALU

Hs.520313 1.10E-06 1.00E-04

Hs.718425 7871 799

381 377 paroxysmal nonkinesigenic dyskinesia (PNKD), transcript variant 2, mRNA.

369 kelch domain containing 8A
KLHDC8A (KLHDC8A), mRNA.

Hs.10414

169
25
RAB11 family interacting protein
RAB11FIP4 4 (class II) (RAB11FIP4), mRNA. Hs.406788 43079 chromosome 11 open reading
352 348

C11orf66

frame 66 (C11orf66), mRNA. trinucleotide repeat containing 6B (TNRC6B), transcript variant 2, mRNA.

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