day 8 EB cells
4.4. Expression of MHC class I molecules by ES and EB cells
CD8+ CTL recognize with their T cell receptor (TCR) short peptides derived from intracellular antigens presented by MHC I molecules on the surface of nucleated cells. Therefore, the expression of MHC I molecules on the cell surface is an absolute prerequisite for the recognition and elimination of cells infected with intracellular pathogens by antigen specific CTL. In previous studies, murine ES cells were found to be devoid of MHC I molecules while human ES cells expressed low levels (26, 49, 50).
The expression level of MHC class I molecules on the ES cell lines and on EB cells used in this study was measured both at the mRNA and the protein level. PCR analysis revealed that undifferentiated ES cells express readily detectable levels of mRNA coding for the heavy chain of H2-Kb MHC class I molecules. In EB cells, expression of MHC I mRNA was enhanced between days 2 to 12 of differentiation (Fig. 7A). Expression of mRNA coding for β2-microglobulin (β2M) which is required for the stabilisation of the complex of MHC heavy chain and antigenic peptide was detected in both ES and EB cells (Fig. 7A).
Western blot analysis revealed that in lysates of undifferentiated ES cells, only a faint signal for H2-Kb molecules was detectable. However, H2-Kb was detectable already on day 2 of differentiation in EB cells and the signal strength increased further over the next days of differentiation (Fig. 7B).
IFN-γ enhances the expression of MHC class I molecules in somatic (51, 52) as well as in embryonic cells that express IFN-γ receptors on their surface (27, 50). To assess whether IFN-γ enhances the level of MHC I expression in the ES and EB cells from various differentiation stages used in this study, these cells were incubated with 20 ng/ml IFN-γ for 48 h. As shown in figure 7A and 7B, treatment with IFN-γ did neither enhanced the expression of MHC class I mRNA nor MHC I protein in undifferentiated ES cells as assessed by RT-PCR and Western blot analysis, respectively. However, from day 2 of differentiation, EB derived cells treated with IFN-γ exhibited strongly enhanced expression of MHC I heavy chain at the protein level (Fig. 7B).
Western blot analysis detects the total contents of cellular MHC class I protein without allowing differentiation between the fractions of MHC class I
molecules present on the cell surface, i.e. the fraction relevant for recognition by T cells, and within intracellular compartments. Therefore, surface expression of MHC class I molecules on ES and EB cells was determined by flow cytometry.
As expected from the Western blot data, no significant expression of MHC class I molecules on undifferentiated ES cells was detected by flow cytometry (Fig. 7C). However, surface expression of MHC class I molecules was detectable in EB cells from day 2 of differentiation onwards. Treatment with IFN-γ strongly enhanced the expression of MHC class I molecules in EB derived cells from day 4 of differentiation onwards to a level almost comparable to that of the B6SV fibroblasts serving as positive controls (Fig. 7C).
Finally, MHC I expression on the surface of ES and EB cells infected with LCM virus was assessed to exclude that infection with LCM virus might interfere with the expression of MHC I molecules. As shown in figure 7D, infection with LCM virus did neither in undifferentiated ES cells nor in EB cells influence the expression of MHC class I molecules as detected by flow cytometry (Fig. 7D).
Spontaneous expression of low level of MHC I molecules and strong induction of MHC I expression on EB cells by IFN-γ are, in addition to the productive infection of these cells with the LCM virus, essential prerequisites for recognition of target cells by antigen specific CTL. Nevertheless, the poor lysis of ES and EB cells infected with the LCM virus by highly active LCM virus specific CD8+ CTL might be due to low expression of MHC class I molecules or an immature antigen processing machinery in these cells.
Figure 7: MHC class I expression in ES and EB cells
(A) Expression of mRNA coding for the MHC class I heavy chain, β2-microglobulin, and Oct4 in IFN-γ-treated and control CGR8 ES and EB cells as detected by RT-PCR. IFN-γ was added to a final concentration of 20 ng/ml for 48 h prior to mRNA isolation. β-actin was amplified as a loading control.
(B) Western Blot analysis of MHC I heavy chain, β2-microglobulin and Oct4 proteins in CGR8 ES and EB cells from various differentiation stages. ES and EB cells were left untreated or incubated with 20 ng/ml IFN-γ for 48 h prior to preparation of cell lysates. H-2Kb heavy chain was detected by using anti-H-2Kb heavy chain-specific serum. β2-microglobulin, Oct4 and β-actin were detected using specific monoclonal antibodies.
(to be continued) A
B
MHC class I HC Oct4
ß-Actin ß-2m Interferon γ: - + - + - + - + - + - +
ES cells
Embryoid bodies
day 2 day 4 day 6 day 8 day 12
MHC class I HC Oct4
Oct4
ß-Actin ß-2m Interferon γ: - + - + - + - + - + - +
ES cells
Embryoid bodies
day 2 day 4 day 6 day 8 day 12
Interferon γ
H2Kb
ß-Actin ß2m Oct4
ES cells day 2 day 4 day 6 day 8 day 10 day 12 day 16 - + - + - + - + - + - + - + - +
Embryoid bodies
Interferon γ
H2Kb
ß-Actin ß2m Oct4
ES cells day 2 day 4 day 6 day 8 day 10 day 12 day 16 - + - + - + - + - + - + - + - +
Embryoid bodies
H2Kb
ß-Actin ß2m Oct4
ES cells day 2 day 4 day 6 day 8 day 10 day 12 day 16 - + - + - + - + - + - + - + - +
Embryoid bodies
(C) Flow cytometry analysis of MHC class I expression on the surface of undifferentiated αPIG ES and EB cells at different stages of differentiation. B6SV fibroblasts (from H-2b C57BL/6 mice) were used as a positive control. IFN-γ was added to a final concentration of 20 ng/ml for 48 h. Single cell suspensions of ES or EB cells were stained with monoclonal antibody specific for H-2Kb (clone AF6-88.5, BD Pharmingen) or isotype control antibodies and were analyzed by FACScan and CellQuest software.
(D) Flow cytometry analysis of MHC I expression on the surface of LCM virus-infected undifferentiated αPIG ES and EB cells at different stages of differentiation. B6SV fibroblasts were used as a positive control. Cells were infected with LCM virus at a MOI of 0.01. After 48 h, single cell suspensions of ES or EB cells were stained with monoclonal antibody specific for H-2Kb (clone AF6-88.5, BD Pharmingen) or isotype control antibodies and analyzed by FACScan and CellQuest software.
All data are representative of at least three independent experiments and were also reproduced with CGR8 ES cells.
C
D
isotype w/o IFNγ
+IFNγ day 2 EBs isotype
w/o IFNγ +IFNγ B6SV fibroblasts
w/o IFNγ +IFNγ isotype
day 6 EBs
ES cells
+IFNγ isotype
w/o IFNγ
w/o IFNγ +IFNγ isotype
day 8 EBs
isotype w/o IFNγ
+IFNγ day 4 EBs
w/o IFNγ +IFNγ isotype
day 14 EBs
w/o IFNγ +IFNγ isotype day 20 EBs
H-2 Kb
counts
isotype w/o IFNγ
+IFNγ day 2 EBs isotype
w/o IFNγ +IFNγ B6SV fibroblasts
w/o IFNγ +IFNγ isotype
day 6 EBs
ES cells
+IFNγ isotype
w/o IFNγ
w/o IFNγ +IFNγ isotype
day 8 EBs
isotype w/o IFNγ
+IFNγ day 4 EBs
w/o IFNγ +IFNγ isotype
day 14 EBs
w/o IFNγ +IFNγ isotype day 20 EBs isotype
w/o IFNγ
+IFNγ day 2 EBs
isotype w/o IFNγ
+IFNγ day 2 EBs isotype
w/o IFNγ +IFNγ B6SV fibroblasts
isotype
w/o IFNγ +IFNγ B6SV fibroblasts
w/o IFNγ +IFNγ isotype
day 6 EBs
w/o IFNγ +IFNγ isotype
day 6 EBs
ES cells
+IFNγ isotype
w/o IFNγ ES cells
+IFNγ isotype
w/o IFNγ
w/o IFNγ +IFNγ isotype
day 8 EBs
w/o IFNγ +IFNγ isotype
day 8 EBs
isotype w/o IFNγ
+IFNγ day 4 EBs isotype w/o IFNγ
+IFNγ day 4 EBs
w/o IFNγ +IFNγ isotype
day 14 EBs
w/o IFNγ +IFNγ isotype
day 14 EBs
w/o IFNγ +IFNγ isotype day 20 EBs
w/o IFNγ +IFNγ isotype day 20 EBs
H-2 Kb
counts
cells infected with LCM virus
100 101 102 103 104
0100
isotype w/o LCMV
+ LCMV B6SV fibroblasts
isotype w/o LCMV
+ LCMV ES cells
isotype w/o LCMV
+ LCMV day 5 EBs
100 101 102 103 104
080
isotype w/o LCMV
+ LCMV day 9 EBs
counts
100 101 102 103 104
064
isotype
w/o LCMV + LCMV ES cells
100 101 102 103 104
064 day 5 EBs isotype
w/o LCMV + LCMV
100 101 102 103 104
080
day 9 EBs isotype
w/o LCMV + LCMV
cells treated with IFN-γγγγand infected with LCM virus
H-2 Kb
cells infected with LCM virus
100 101 102 103 104
0100
isotype w/o LCMV
+ LCMV B6SV fibroblasts
isotype w/o LCMV
+ LCMV ES cells
isotype w/o LCMV
+ LCMV day 5 EBs
100 101 102 103 104
080
isotype w/o LCMV
+ LCMV day 9 EBs
counts
100 101 102 103 104
064
isotype
w/o LCMV + LCMV ES cells
100 101 102 103 104
064 day 5 EBs isotype
w/o LCMV + LCMV
100 101 102 103 104
080
day 9 EBs isotype
w/o LCMV + LCMV
cells treated with IFN-γγγγand infected with LCM virus
H-2 Kb
100 101 102 103 104
0100
isotype w/o LCMV
+ LCMV B6SV fibroblasts
isotype w/o LCMV
+ LCMV ES cells
isotype w/o LCMV
+ LCMV day 5 EBs
100 101 102 103 104
080
isotype w/o LCMV
+ LCMV day 9 EBs
100 101 102 103 104
0100
isotype w/o LCMV
+ LCMV B6SV fibroblasts
isotype w/o LCMV
+ LCMV ES cells
isotype w/o LCMV
+ LCMV day 5 EBs
100 101 102 103 104
080
isotype w/o LCMV
+ LCMV day 9 EBs
counts
100 101 102 103 104
064
isotype
w/o LCMV + LCMV ES cells
100 101 102 103 104
064 day 5 EBs isotype
w/o LCMV + LCMV
100 101 102 103 104
080
day 9 EBs isotype
w/o LCMV + LCMV
100 101 102 103 104
064
isotype
w/o LCMV + LCMV ES cells
100 101 102 103 104
064 day 5 EBs isotype
w/o LCMV + LCMV
100 101 102 103 104
080
day 9 EBs isotype
w/o LCMV + LCMV
cells treated with IFN-γγγγand infected with LCM virus
H-2 Kb
4.5. ES and EB cells loaded with a peptide resembling LCM