CD95 helps the identification of T_(TN) natural counterpart in vivo, in healthy donors and in recipients of allogeneic HSCT. Circulating CD95⁺CD45RA⁺CD62L⁺ cells express higher levels of CD45R0 compared with their CD95⁻ counterpart: (A) representative FACS plots and (B) its quantification. Functional validation of CD95⁺CD45RA⁺CD62L⁺ cells: purified CFSE-labeled CD95⁺ and CD95⁻ CD45RA⁺CD62L⁺ were challenged with decreasing doses of anti-CD3 antibody with or without costimulation. (C) Representative FACS plots of CFSE dilution and (D) quantification of CFSE dilution. (E) Proposed model for T_SCM in vivo generation after HSCT: the conditioning regimen causes severe host lymphopenia, creating a milieu in which infused allogeneic lymphocytes are exposed to elevated levels of IL-7 and IL-15. Conditioning regimen favors systemic inflammation, leading to the activation of antigen-presenting cells (APCs), which become costimulation competent. (F) Representative plots of circulating CD8⁺ lymphocytes subpopulations (left) and CD95 expression on CD45RA⁺CD62L⁺ putative naive CD8⁺ cells (right) from 2 representative HSCT recipients. Gray histograms represent fluorochrome-matched isotype controls. (G) Proportion of CD45RA⁺CD62L⁺ CD8⁺ T lymphocytes expressing CD95 (T_SCM) in HSCT recipients and healthy controls. (H) Proportion of CD45RA⁺CD62L⁺CD95⁺ lymphocytes gated on CD8⁺ T cells in HSCT recipients and healthy controls. (I) CD95 expression on antigen-specific T cells. Circulating T lymphocytes are gated on the CD45RA⁺CD62L⁺ population (top left plot) and then CMV pp65-specific and polyclonal CD8⁺ T cells are identified (bottom left plot). CD95 expression is enriched on CMV pp65-specific (top right plot) compared with polyclonal (bottom right plot) CD45RA⁺CD62L⁺ lymphocytes. (J) Scatter plot showing CD95 differential expression on CMV pp65-specific and polyclonal CD45RA⁺CD62L⁺ lymphocytes, gated on CD8⁺. (K) Scatter plot showing expression levels of CD45R0 on CMV pp65-specific CD45RA⁺CD62L⁺CD95⁺ lymphocytes and naive CD45RA⁺CD62L⁺CD95⁻ lymphocytes, gated on CD8⁺.