Immunological behavior of enhanced green fluorescent protein (EGFP) as a minor histocomaptibility 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 F1 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.