Immunological behavior of enhanced green fluorescent protein (EGFP) as a minor histocompatibility antigen with a special reference to skin isograft and specific regulation of local graft-versus-host reaction (GvHR).

Although enhanced green fluorescent protein (EGFP) is widely used as a molecular tag in cell biology, it has become evident that immunogenicity of transgenic or transduced EGFP is important when it applies to transplantation model. Indeed, it appears that applications of EGFP-expressing cells, tissues and organ transplantation are limited in vivo due to the ultimate rejection of the graft. Nevertheless, the immunological behavior of transduced EGFP, in particular, as a minor histocompatibility antigen is not fully understood. Thus employing two strains of EGFP transgenic (Tg) rats generated by the same vector construct, e.g., EGFP-F344 Tg (RT11) and EGFP-DA Tg (RT1a), and its F(1) hybrid with a non-transgenic rat, behavior of EGFP-transgenic antigen(s) was examined by in vivo assays, such as EGFP-transgenic test skin grafts or regulation of EGFP-transgenic lymphocytes. In the latter system, EGFP-specific, T-cell-mediated immune regulation of local graft-versus-host reaction (GvHR) was further investigated with a special reference of in vivo cytotoxic assay, i.e., elimination of colored lymphocytes with either EGFP-incompatible or CFSE-labeled sex-mismatched lymphocytes. We provide evidence that differential immunological behavior of EGFP-transgenic minor histocompatibility antigen was observed in vivo. Thus, immune responses to EGFP-minor histocompatibility antigen(s) were not always accompanied with the rejection of test skin isograft. It only becomes apparent for EGFP-specific elimination and suppression of both systemic and local GvHR induced by EGFP-transgenic T lymphocytes after EGFP-specific sensitization. However, this was not the case where test skin isografting was applied even under extensive sensitization protocols. These findings demonstrate that minor histocompatibility antigen specific immune elimination of EGFP-transgenic T lymphocytes or regulation of local GvHR provides more sensitive and better immune assay systems in vivo than classical test skin isograft systems.

Sensitization to enhanced green fluorescence protein minor histocompatibility antigen by gene transduction into dendritic cells and peritoneal exudate macrophages.

Enhanced green fluorescence protein (EGFP) has been widely applied to gene transduction in cellular and molecular biology as a reporter element. When applied to cell transplantation, it raises fundamental issues concerning cell-associated antigens, in particular, a model of minor histocompatibility antigen(s). Although it is well known that immunological behavior of minor histocompatibility antigens mimic tumor associated antigens (TAA), identified genes coding minor histocompatibility antigens are few and far between. Inasmuch as immunity and tolerance to TAA are provided by immunological behavior of minor histocompatibility antigen such as histocompatibility antigen of the Y chromosome, H-Y, it occurs to us that transgenic as well as transduced EGFP provides a useful model system to be applied to tumor immunology. In this respect, genetic modification of specialized antigen-presenting cells (APC), i.e., dendritic cells (DC), such as gene transduction of EGFP into DC, would provide one of the most important strategies in transplantation as well as tumor immunology inasmuch as DC play a key role in initiating primary immune responses, As far as gene transduction into DC is concerned, others have reported that successful gene transduction occurs in DC by adenoviral vector systems. However, our previous studies concerning EGFP transduction into DC suggested that this view should be carefully examined and interpreted. Employing adenoviral and lentiviral vector systems as well as specialized APC of rat DC and peritoneal exudate macrophages (PEM), EGFP-transduced APC were examined to determine whether and to what extent the EGFP-transduced APC were able to sensitize non-transgenic littermates against transgenic EGFP as antigen(s). Thus EGFP-transgenic cardiac isografts were transplanted to non-transgenic littermates and examined to determine if sensitization of non-transgenic littermate recipients with the EGFP-transduced APC was able to reject the test grafts in an accelerated manner. In this study, we examined this and provide further evidence that widely used viral vector systems are unable to transfer the reporter gene EGFP into mature rat DC generated from bone marrow cells (BMC), driven by Flt3/Flk2 ligand and IL-6. Nevertheless, successful gene transduction was obtained by either applying a lentiviral vector system to the developing DC progenitor cells during a long-term culture of rat BMC or by applying an adenoviral vector system to PEM. Thus, successful gene transduction into specialized APC was verified by in vivo priming of non-transgenic littermates with the EGFP-transduced APC, followed by accelerated rejection of EGFP-transgenic cardiac isografts.