Rheumatology Advance Access originally published online on February 28, 2007
Rheumatology 2007 46(5):892-893; doi:10.1093/rheumatology/kel440
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Monoclonal antibody HC10 does not bind HLA-G
Institute of Cell Biology, Histology and Embryology, Center of Molecular Medicine, Medical University of Graz, Harrachgasse 21/VII, 8010 Graz, Austria
Correspondence to: Martin Gauster. E-mail: martin.gauster{at}meduni-graz.at
SIR, Recently, Raine et al. [1] published a study about measuring the ratio of ß2-m associated MHC class I and their free heavy chains (FHCs) on human peripheral blood cells and human placental trophoblast cells. The authors used the monoclonal antibodies (mAbs) W6/32, specific for fully assembled MHC class I molecules and the mAb HC10, specific for FHCs. Flow cytometry analysis revealed higher levels of FHCs on leucocytes of a reactive arthritis (ReA) population than in healthy controls. In addition the authors described a very high relative level of FHCs on placental extravillous trophoblasts (EVTs), suggesting HLA-G to be the main source of FHCs on this cell type. However, the assumption drawn by the authors is misleading, since the employed mAb HC10 is not the appropriate tool to come to such conclusion. It is of particular scientific relevance to comment on this work, since the mAb HC10 binds HLA-B and -C but not to HLA-G.
The used antibody was an integral tool of their study and has an impact on the obtained results. Hence, it is indispensable to clarify the reaction patterns of this mAb in order to avoid misinterpretation of published results and already drawn conclusions. In this context, we demonstrate that HC10 definitely does not react with human HLA-G. We want to refer to some facts about the relevant antibodies (Table 1) and show immunohistochemical data, which might contribute to an adjustment of the results obtained with isolated trophoblasts in their study.
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The well-known and extensively used antibody W6/32 binds a conformational dependent epitope on all HLA class I products [2]. In contrast, mAb HC10 was originally raised against free class I, HLA-B locus heavy chains [3], but was subsequently shown to bind also HLA-C [4, 5]. Another FHC specific mAb, HCA2, raised against HLA-A locus products [6], was not employed by Raine et al. HCA2 recognizes also HLA-G [7], due to an 80% homology between both products and could have served as a good marker in the respective study to measure HLA-G FHCs levels on trophoblasts. In Fig. 1 we show and confirm the abilities of the mAbs W6/32, HCA2, and HC10 to bind HLA-G or not. For that purpose we used the MHC class I negative choriocarcinoma cell line JAR as negative control and JAR cells stable transfected with human genomic HLA-G [8] as a positive control. Staining of these cells revealed that HC10 does not bind to HLA-G overexpressing cells (Fig. 1). In contrast, the mAb 4H84 [9, 10], which is specific for HLA-G as well as the mAbs W6/32 and HCA2, showed intensive staining of JAR-HLA-G cells.
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Since Raine et al. measured the ratio of FHCs and complexed MHC class I on isolated human EVTs, we used human first trimester placenta tissue including cell islands to show HC10 reactivity on EVTs by immunohistochemistry. As indeed shown by several previous studies, EVTs lack classical MHC class I molecules, but express high levels of HLA-G and moderate levels of HLA-C [4, 5, 11]. Though former studies have already shown immunohistochemical data obtained with the mAbs W6/32, HCA2 and HC10, we have recapitulated the immunohistochemistry in combination with the highly HLA-G-specific mAb 4H84 and a sensitive detection system. Thus, we were able to show that EVTs are the only cells in human placenta that express HLA-G (Fig. 1; mAb 4H84). None of the MHC class I products is expressed in villous trophoblasts, as shown by staining with mAb W6/32. The strong HLA-G expression in EVTs can also be demonstrated with the mAb HCA2. The mAb HC10 gives only a moderate staining of EVTs, indicating the expression of HLA-C, because the expression of HLA-B on these cells was excluded previously [12]. The presence of the ß2-m complexed classical HLA-A and -B molecules in the mesenchymal core of the villous chorion is best demonstrated with mAb W6/32 (Fig. 1). However, HLA-B and HLA-A can also be detected with the FHC specific mAbs HC10 and HCA2, respectively.
Taken together, our data strongly substantiate that Raine et al. in their study measured not HLA-G but HLA-C FHCs on isolated trophoblasts. Considering this fact, the results and conclusions obtained in that study have to be reconceived.
The authors have declared no conflicts of interest.
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