Figure 1. Titration of HFE plasmid in T-cell antigen recognition mediated by HLA-A2. 293-A2 cells were transfected with decreasing doses of HLA-A1, or HFE ...
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European Journal of Immunology Supporting Information for DOI 10.1002/eji.201343955 Alexandre Reuben, Mikael Manuela M. Santos and Rejean Lapointe ¨ Phenix, ´ ´ The WT hemochromatosis protein HFE inhibits CD8+ T-lymphocyte activation
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eji-journal.eu
Reuben et al. Supporting Information
gp100209-217
ITDQVPFSV
0.5 µg
10000
HFEC282Y
HFEH63D
0
HFEWT
5000 HLA-A1
MIP-1β (pg/ml)
0.25 µg
HFEC282Y
0
HFEH63D
5000 HFEWT
HFEC282Y
HFEH63D
HFEWT
5000
**
15000
10000
HLA-A1
MIP-1β (pg/ml)
**
HLA-A1
MIP-1β (pg/ml)
**
15000
10000
0
n.s.
n.s.
*
15000
n.s.
n.s.
n.s.
0.125 µg
Figure 1. Titration of HFE plasmid in T-cell antigen recognition mediated by HLA-A2. 293-A2 cells were transfected with decreasing doses of HLA-A1, or HFE variant plasmids and constant dose of gp100 plasmid. MIP-1β secretion by gp100209-217-specific CD8+ T lymphocytes following co-culture with transfected 293-A2 cells. MIP-1β statistics were calculated after combining values from three independent experiments. Amino acid sequence of the tested epitope is highlighted below the name of the epitope. Data are shown as mean ± SEM of two replicates from a single experiment representative of three experiments performed. Multiple comparisons were evaluated by 1-way ANOVA, followed by the Bonferroni multiple comparison test. *P< 0.05, and **P< 0.01 when compared to HLA-A1; n.s. - not significant.
gp100209-217
ITDQVPFSV n.s.
IFN-γ (pg/ml)
2000
*
1500 1000 500
HFEC282Y
HFEWT
GFP
0
Figure 2. HFE inhibits T-cell antigen recognition mediated by HLA-A2 in MelS-FB melanoma cells. IFN-γ secretion by gp100209-217-specific CD8+ T lymphocytes following co-culture with MelS-FB cells co-transfected with GFP or HFE variants and gp100 plasmids. Amino acid sequence of the tested epitope is highlighted at the top of the figure. Data are shown as mean ± SEM of two replicates from a single experiment representative of two experiments performed. Multiple comparisons were evaluated by 1way ANOVA, followed by the Bonferroni multiple comparison test. *P< 0.05 when compared to GFP; n.s. - not significant.
gp100209-217 ITDQVPFSV n.s.
IFN-γ
(% of HLA-A1)
125
****
100 75 50 25 0 cis
trans
HLA-A1
cis
trans
HFEWT
gp100209-217 Figure 3. HFE must be expressed in cis of antigen to affect CD8+ T-lymphocyte activation. 293-A2 cells were transfected with HLA-A1 or HFEWT plasmids. In parallel, 293-A2 cells were transfected with exclusively HLA-A1 or HFEWT plasmids, or exclusively gp100 antigen plasmid. Both HLA-A1 or HFEWT-transfected and gp100transfected 293-A2 cell populations were mixed at a 1:1 ratio and IFN-γ secretion by gp100209-217-specific CD8+ T lymphocytes following co-culture with transfected 293-A2 cells was quantified. Amino acid sequence of the tested epitope is highlighted at the top of the figure. Data are shown as mean ± SEM of two replicates from a single experiment representative of two experiments performed. Multiple comparisons were evaluated by 1way ANOVA, followed by the Bonferroni multiple comparison test. ****P< 0.0001 when compared to HLA-A1; n.s. - not significant.
Pan-MHC I
-β2-m
B
+β2-m
100
100
80
80
Events
Events
A
60 40
60 40 20
20
0
0 0
102
103
104
HLA-ABC
105
0 102
103
104
HLA-ABC
105
Figure 4. MHC I surface expression is restored by β2-m co-transfection. MHC I expression in 293-A2 cells co-transfected with HLA-A1, or HFE variants A) without or B) with β2-m plasmids. Results are representative of three similar experiments performed. Grey-filled histograms represent unstained controls. HLA-A1 = black, HFEWT = red, HFEH63D = blue, HFEC282Y = green.
Figure 5. HFEWT does not alter the expression of MHC I chaperones involved in the peptide loading complex. 293-A2 cells were transfected with HLA-A1, or HFE variant plasmids in duplicates. Protein levels of MHC I chaperones were quantified by western blotting. β-actin was used as a loading control. Results are representative of at least two similar experiments performed.
HLA-A1 Endo H PNGase F
HFEWT HFEC282Y
-
+
-
-
+
-
-
+
-
-
-
+
-
-
+
-
-
+
HLA-A1 1 2 3 4 5 6 7 8 9 Figure 6. MHC I glycosylation is unaffected by HFEWT. We evaluated the sensitivity of MHC I to Endo H (lanes 2, 5, 8) and PNGase F (lanes 3, 6, 9) glycosidases in 293-A2 cells transfected with untagged HLA-A1, HFEWT, or HFEC282Y plasmids and HLA-A1 FLAG. Protein extracts left undigested are shown in lanes 1, 4, and 7. Extracts were evaluated by western blot and probed with anti-FLAG antibody. Results are representative of three similar experiments performed.
Untransfected
Transfected
100
Events
80 60 40 20 0 0 101
102 GFP
103
104
Figure 7. Transfection efficiency in 293-A2 cells. 293-A2 cells were transfected with the GFP plasmid. Results are representative of three similar experiments performed. Grey histogram represents untransfected control.
