A three-colour flow cytometry technique for measuring trophoblast intracellular antigens: the relative expression of TAP1 in human cytotrophoblast and decidual cells.

Flow cytometry is conventionally used to measure cell-surface antigen expression. However, many antigens are found within the cytoplasm, and it is necessary to fix and permeabilize cells to enable antibodies to gain access to them. In this study we have established the conditions for studying intracellular antigens in human trophoblast cells by flow cytometry using an antibody to TAP1 (a key molecule in the process of Class I MHC assembly). We have previously shown by immunocytochemistry that TAP1 expression is apparently greater on Class 1 positive extravillous cytotrophoblast than on any other fetal or maternal tissue. However, as immunohistochemistry is not quantitative we have used three-colour flow cytometry to measure the expression of TAP1 in different trophoblast populations. Villous and extravillous cytotrophoblast were identified in first trimester and term placental and decidual digests on the basis of their expression of cytokeratin and Class I MHC antigens. The level of expression of TAP1 for each population was investigated using a commercial kit that determines the number of antibody-binding sites per cell. TAP expression was found to be three- to fivefold higher in extravillous cytotrophoblast, confirming our previous findings. The techniques developed here are directly applicable to the measurement of other intracellular molecules in trophoblast, in particular cytokines.

[1]  J. Bowen,et al.  Recognition of trophoblast HLA class I molecules by decidual NK cell receptors--a review. , 2000, Placenta.

[2]  C. Jones,et al.  Studies of mesenchymal cells from 1st trimester human placenta: expression of cytokeratin outside the trophoblast lineage. , 1999, Placenta.

[3]  T. Elliott.  How does TAP associate with MHC class I molecules? , 1997, Immunology today.

[4]  A. McMichael,et al.  The human major histocompatibility complex class Ib molecule HLA‐E binds signal sequence‐derived peptides with primary anchor residues at positions 2 and 9 , 1997, European journal of immunology.

[5]  P. Le Bouteiller,et al.  Interferon‐γ rescues HLA class Ia cell surface expression in term villous trophoblast cells by inducing synthesis of TAP proteins , 1996, European journal of immunology.

[6]  H. Rammensee,et al.  Nonclassical HLA-G molecules are classical peptide presenters , 1996, Current Biology.

[7]  J. Bajorath,et al.  The membrane-bound and soluble forms of HLA-G bind identical sets of endogenous peptides but differ with respect to TAP association. , 1995, Immunity.

[8]  J. Dausset,et al.  Soluble HLA-G molecule. An alternatively spliced HLA-G mRNA form candidate to encode it in peripheral blood mononuclear cells and human trophoblasts. , 1995, Human immunology.

[9]  S. Ferrone,et al.  Serum HLA class I antigens: markers and modulators of an immune response? , 1995, Immunology today.

[10]  R. Tampé,et al.  Expression of TAP1 by human trophoblast , 1995, European journal of immunology.

[11]  E. Filvaroff,et al.  Soluble form of an HLA-B7 class I antigen specifically suppresses humoral alloimmunization. , 1994, Human immunology.

[12]  Michael G. Ormerod,et al.  Flow Cytometry: A Practical Approach , 1994 .

[13]  I. Sargent,et al.  Antigenic heterogeneity of human cytotrophoblast and evidence for the transient expression of MHC class I antigens distinct from HLA-G. , 1993, Placenta.

[14]  N. Zavazava,et al.  Soluble MHC class I antigens (sHLA) and anti-HLA antibodies in heart and kidney allograft recipients. , 1993, Tissue antigens.

[15]  P. Cresswell,et al.  Cytokines increase transporter in antigen processing-1 expression more rapidly than HLA class I expression in endothelial cells. , 1992, Journal of immunology.

[16]  D. Barlow,et al.  Isolation and purification of human endometrial stromal and glandular cells using immunomagnetic microspheres. , 1992, Human reproduction.

[17]  I. Sargent,et al.  Isolation and characterisation of a subpopulation of human chorionic cytotrophoblast using a monoclonal anti-trophoblast antibody (NDOG2) in flow cytometry. , 1992, Journal of reproductive immunology.

[18]  D. Daya,et al.  The use of cytokeratin as a sensitive and reliable marker for trophoblastic tissue. , 1991, American journal of clinical pathology.

[19]  I. Sargent,et al.  Purification of human cytotrophoblast from term amniochorion by flow cytometry. , 1990, Placenta.

[20]  I. Sargent,et al.  Flow cytometric characterisation of cell populations in human pregnancy decidua and isolation of decidual macrophages. , 1990, Journal of immunological methods.

[21]  S. Fisher,et al.  A class I antigen, HLA-G, expressed in human trophoblasts. , 1990, Science.

[22]  A. Daar,et al.  The human Thy-1 molecule. , 1989, Immunology series.

[23]  A. McMichael,et al.  Evidence for a novel HLA antigen found on human extravillous trophoblast and a choriocarcinoma cell line. , 1986, Immunology.

[24]  J. Bulmer,et al.  Antigen expression by trophoblast populations in the human placenta and their possible immunobiological relevance. , 1985, Placenta.

[25]  L. Bobrow,et al.  Monoclonal antibody to cytokeratin for use in routine histopathology. , 1984, Journal of clinical pathology.

[26]  A. McMichael,et al.  Class 1 major histocompatibility complex antigens on human extra-villous trophoblast. , 1984, Immunology.

[27]  H Stein,et al.  Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). , 1984, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[28]  C. Redman,et al.  Human reproductive immunology--elucidation by monoclonal antibody techniques. , 1983, Obstetrics and gynecology annual.

[29]  M. Wilchek,et al.  The use of the avidin-biotin complex as a tool in molecular biology. , 2006, Methods of biochemical analysis.

[30]  L. Lampson,et al.  Two populations of Ia-like molecules on a human B cell line. , 1980, Journal of immunology.

[31]  C. Barnstable,et al.  Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis , 1978, Cell.