Constitutive HERV-K18 SAg expression restricted to thymocyte lineage. Thymic HERV-K18 expression was addressed by RPA and FACS analysis on total thymocytes and thymocyte subsets. (A) Shown is an RPA of whole thymus tissue from 5 donors using HERV-K18 SAg and human TBP control probes. The HERV-K18 SAg probe generates a full-length protected fragment from HERV-K18 transcripts and smaller internally digested fragments from other HERV-Ks. (B) Thymocyte suspensions from 4 donors (donors 6-9) were incubated with an isotype control (gray shaded curve) and with monoclonal antibodies against HERV-K18 (open curve), which were subsequently revealed by FITC-labeled secondary antibodies. (C) RPA was performed on whole thymus tissue, on single-cell suspensions, and on FACS-sorted samples. The cell fraction obtained after the elution of thymocytes is labeled stroma. Thymocyte suspensions were FACS sorted into the immediate precursors of DP cells (ISP = CD4⁺CD8^-CD3^-), into immature DP thymocytes (CD4⁺CD8⁺), and into mature SP thymocytes (CD4^-CD8⁺, CD4⁺CD8^-). (D) Freshly isolated thymocytes were labeled with isotype control antibodies (gray shaded curve) and monoclonal anti–HERV-18 antibodies (open curve). Gating on thymocyte subsets was based on CD4/CD8 expression, and exclusion of dead cells, which allowed to us address HERV-K18 expression of thymocyte subsets.