Human leukocyte antigen (HLA)-G is a non-classical HLA class I molecule thought to play a key role in maternal-fetal tolerance. Although initial studies suggested that HLA-G expression is restricted to extravillous cytotrophoblasts, expression was subsequently reported in a wide variety of other human tissues and tumor cells. However, consensus as to the validity of these collective findings has proven difficult because the antibodies used to define the temporal and spatial expression patterns of HLA-G remain incompletely characterized. The aim of our study was to reassess two of the most widely used HLA-G antibodies (MEM-G/9 and 4H84) in HLA-G-positive (JEG-3 and HLA-G transduced) and -negative (dermal fibroblast, mesenchymal stem cell, K562, and Jurkat) lines using flow cytometry, immunofluorescence, and western blotting. We found that MEM-G/9 recognized HLA-G3 by flow cytometry, indicating that its epitope is present on the α1 domain of HLA-G. Although 4H84 preferably recognized unfolded HLA-G-free chains, it showed strong non-specificity under certain methodological conditions.
"Cell immunostaining was performed as previously described (Zhao et al., 2012b). Briefly, hESC clones grown on matrigelcoated chamber slides (Nalge Nunc International corp, Naperville, IL) were washed with cold PBS once. "
[Show abstract][Hide abstract] ABSTRACT: Human embryonic stem cells (hESC) are capable of extensive self-renewal and expansion and can differentiate into any somatic tissue, making them useful for regenerative medicine applications. Allogeneic transplantation of hESC-derived tissues from results in immunological rejection absent adjunctive immunosuppression. The goal of our study was to generate a universal pluripotent stem cell source by nucleofecting a mutated human leukocyte antigen G (mHLA-G) gene into hESC using the PiggyBac transposon. We successfully generated stable mHLA-G(EF1α)-hESC lines using chEF1α promoter system that stably expressed mHLA-G protein during prolonged undifferentiated proliferation andin differentiated embryoid bodies as well as teratomas. Morphology, karyotype, and telomerase activity of mHLA-G expressing hESC were normal. Immunofluorescence staining and flow cytometry analysis revealed persistent expression of pluripotent markers, OCT-3/4 and SSEA-4, in undifferentiated mHLA-G(EF1α)-hESC. Nucleofected hESC formed teratomas and when directed to differentiate into epidermal precursors, expressed high levels of mHLA-G and keratinocyte markers K14 and CD29. Natural killer cell cytotoxicity assays demonstrated a significant decrease in lysis of mHLA-G(EF1a)-hESC targets relative to control cells. Similar results were obtained with mHLA-G(EF1α)-hESC-derived epidermal progenitors (hEEP). One way mixed T lymphocyte reactions unveiled that mHLA-G(EF1a)-hESC and -hEEP restrained the proliferative activity of mixed T lymphocytes. We conclude that heterologous expression of mHLA-G decreases immunogenicity of hESC and their epidermal differentiated derivatives.
Stem Cell Research 09/2014; 13(2). DOI:10.1016/j.scr.2014.08.004 · 3.69 Impact Factor
"For HLA-E and HLA-G identification mouse monoclonal antibodies against HLA-E (ab2216 clone MEM-E/02: AbCam) and HLA-G (4H84: Exbio, Czech Republic) were used
. MEM-E/02 recognizes denatured HLA-E
[37,38], while 4H84 recognizes denatured HLA-G molecules and also binds to free heavy chains of classical HLA class I molecules
[Show abstract][Hide abstract] ABSTRACT: Background
Evasion of immune surveillance and suppression of the immune system are important hallmarks of tumorigenesis. The goal of this study was to establish distinct patterns that reflect a rectal tumors’ immune-phenotype and to determine their relation to patient outcome.
The study population consisted of 495 Stage I-IV non-preoperatively treated rectal cancer patients of which a tissue micro array (TMA) was available. Sections of this TMA were immunohistochemically stained and quantified for presence of Foxp3+ cells (Tregs) and tumor expression of HLA Class I and non-classical HLA-E and HLA-G. All markers were, separate and combined, analyzed for clinical prognostic value.
Expression of HLA class I (DFS HR 0.637 (0.458-0.886), p = 0.013), Foxp3+ infiltration above median (OS HR 0.637 (0.500-0.813), p < 0.001 and DFS HR 0.624 (0.491-0.793), p < 0.001) and expression of HLA-G (DFS HR 0.753 (0.574-0.989), p = 0.042) were related to a better clinical prognosis. When these markers were combined, patients with 2 or 3 markers associated with poor prognosis (loss of HLA Class I, Foxp3+ below median, and weak HLA-G expression), showed a significantly worse survival (OS and DFS p < 0.001). This immune-phenotype was an independent predictor for DFS (HR 1.56 (1.14-2.14), p = 0.019).
In conclusion, rectal tumors showing loss of HLA class I expression, Foxp3+ infiltration below median and weak HLA-G expression were related to a worse OS and DFS. Combining these immune markers lead to the creation of tumor immune-phenotypes , which related to patient outcome and were significant independent clinical prognostic markers in rectal cancer.
BMC Cancer 07/2014; 14(1):486. DOI:10.1186/1471-2407-14-486 · 3.36 Impact Factor
"We do not know yet if the reaction is equimolar with all HLA-G molecules, and probably some proteins could be underrecognized. For example, it was recently shown by flow cytometry that MEM-G/9 can also react with HLA-G3, but the intensity of the signal is weaker than with HLA-G1 . Some HLA-G complexes are underrecognized by MEM-G/9 and react better with the anti-HLA-G antibody G-233 . "
[Show abstract][Hide abstract] ABSTRACT: Human leukocyte antigen-G (HLA-G) is a low polymorphic nonclassical HLA-I molecule restrictively expressed and with suppressive functions. HLA-G gene products are quite complex, with seven HLA-G isoforms, four membrane bound, and other three soluble isoforms that can suffer different posttranslational modifications or even complex formations. In addition, HLA-G has been described included in exosomes. In this review we will focus on HLA-G biochemistry with special emphasis to the mechanisms that regulate its expression and how the protein modifications affect the quantification in biological fluids.
Journal of Immunology Research 04/2014; 2014:657625. DOI:10.1155/2014/657625 · 2.93 Impact Factor
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