Our study compares for the first time the simultaneous quantitative expression of HLA-A and -B alleles on undifferentiated pluripotent embryonic stem cells, more differentiated multipotent stem cells and terminally-differentiated lineage-specific cells with different fates in body compartments.
Allele specific HLA cell-surface expression was markedly different between cells at different stages of differentiation and maturation. Pluripotent hESC are known to express low levels of cell-surface HLA class I based on the staining patterns obtained after incubation with a W6/32 pan HLA class I antibody 
. Basak et al. have described low constitutive levels of HLA-A2 cell-surface expression on the H9 human embryonic stem cell line 
while the surface expression of HLA B or Cw have, to the best of our knowledge, not been investigated. Low but detectable expression of HLA-A, -B, and -C mRNA has been reported as judged by quantitative RT-PCR 
, but this does not imply expression on the protein level because many post transcriptional factors are needed for successful expression and some potentially important ones like TAP1, TAP2, LMP2 and LMP7 have been reported missing at the mRNA level in the HS293 embryonic stem cell line 
. Our results show that classical HLA class I cell surface expression on hESC does not comprise expression of the HLA-B locus, at least not of the alleles studied in the two hESC lines here. However, after stimulation of hESC with IFNγ the expression of HLA-A alleles increased to high levels as we have previously observed it in multipotent hMSC 
, while only a modest induction of HLA-B was seen. This indicates that the antigen-presentation pathway(s) required for generation, transport and expression of HLA molecules on the cell surface are readily inducible in hESC. The mechanism behind the differential constitutive expression of HLA-A, but not -B remains to be elucidated but could relate to different dependencies on the peptide loading complex 
hMSC are multipotent cells that are developmentally more differentiated than hESC. As we have recently reported, hMSC do express high levels of HLA-A, but the expression of HLA-B is substantially down-regulated, though detectable in most cases 
. Both the examined HLA-A and HLA-B alleles retained the ability to become induced upon IFNγ stimulation as reported previously 
. Furthermore, after hMSC cell lines were subjected to in vitro
differentiation according to established validated procedures into some of their downstream lineages (adipocytes and osteoblasts), they showed a marginal reduction in the cell-surface expression levels of HLA-A (though only statistically significant for osteoblast differentiation) when compared to their levels in their parental hMSC lines. This is in accordance with a previous report based on HCA2 and HCA10 antibodies 
that detect multiple loci of HLA class I molecules 
hHSC represent another class of multipotent cells that share the local bone marrow microenvironment with the hMSC. hHSC showed a strong HLA-A cell-surface expression comparable to that found on the surface of mature T and B lymphocytes, and comparatively higher than multipotent hMSC of bone marrow origin. This indicates that cells of hematopoietic lineages are programmed to express high levels of classical HLA class I proteins very early during hematopoiesis. Indeed, a recent study demonstrated that hemangioblasts, a precursor of hHSC and endothelial cells, show an increase in HLA-A2 expression compared with hESC, and this expression increased dramatically as cells differentiated into hHSC 
in accordance with our results. Interestingly, we find that HLA-B alleles are also relatively strongly expressed on hHSC though still lower expressed than HLA-A. Thus, it is evident that hHSC express three times more of the HLA-A alleles compared to HLA-B alleles.
Greene JM et al. 
recently studied the relative expression of HLA transcripts in blood leukocytes by pyrosequencing and found that whereas the class I loci contributed differently, an almost equal contribution was found from alleles on the same locus and this contribution varied very little among lymphocyte subsets. Though not incompatible with this, our protein expression data is somewhat in contrast to this, because we found that mature T lymphocytes (CD4+
) exhibited different patterns of expression depending on the type of individual alleles as well as their body compartment of origin. Again, this suggests that post-transcriptional mechanisms are likely to affect HLA class I expression in allele-specific ways. Moreover, the difference in expression between peripheral blood and BM-derived T lymphocytes suggests that HLA surface expression may even be influenced by signals exerted on them by the compartment where they are located even in the absence of overt inflammation or immunization. However, it is surprising that cell surface expression of the HLA-B8 allele remained low in CD4+
lymphocytes regardless of their origin, while its expression was moderately high in B lymphocytes. This suggests that HLA-B8 allele might be regulated differently from other alleles.
Locus or even allele-specific regulation of classical HLA class I expression may prove important for understanding T cell and NK cell responsiveness in several tissues. The density of expressed peptide-loaded HLA molecules encoded by an individual allele may impact the outcome of an immune response just as their affinity for a given T-cell receptor 
. This opens for an alternative explanation for the phenomenon that certain HLA alleles are associated with clearing of certain viral infections e.g. HCV 
and with slow progression of other infections, e.g. HIV 
The finding that HLA-A is preponderant in most cells studied could be related to the clinical observation in allogeneic bone marrow transplantation that mismatches to HLA-A is tolerated more poorly than mismatches in HLA-B alleles 
. However, our finding that HLA-A was down-regulated during differentiation of hMSC toward osteoblasts may be promising for future stem cell therapies using in vitro
Overall, our findings show that expression of cell-surface HLA-A and -B alleles are regulated individually through different mechanisms in normal human cells, according to the cell type, differentiation state or their location in the body. Future studies should address the specific mechanisms governing allele-specific HLA expression.