The dominance of peripherally expanded phenotypically memory T cells during immune reconstitution after PTCy primarily derives from the differentiation of transplanted naïve donor T cells. (A) T cells from the spleens and cutaneous (cervical, brachial, axillary, and inguinal) lymph nodes (CLNs) from Thy1.1⁺ or Thy1.2⁺ 8- to 12-week-old female C57BL/6 mice (CD45.1⁻CD45.2⁺) were flow cytometrically sorted to isolate naïve (CD62L⁺CD44⁻) or central/effector memory (CD62L⁺CD44⁺ and CD62L⁻CD44⁺) T cells. (B) Either Thy1.1⁺ naïve and Thy1.2⁺ memory T cells or Thy1.1⁺ memory and Thy1.2⁺ naïve T cells were mixed together at the ratio at which they were sorted (approximately 5:1 naïve to memory). After 7.75 Gy irradiation in 2 divided fractions 8 hours apart, each recipient mouse received 10⁶ total T cells and 10⁷ CD45.1⁺ T-cell–depleted BM cells. Mice were treated with PBS or PTCy 33 mg/kg per day on days +3 and +4, and then (C-F) underwent serial tail bleeds or (G) were euthanized at day +28 for assessment of various organs. Nearly all T cells were donor-derived at all assessments (C), but substantial thymic-dependent T-cell recovery occurred within the first month after transplant (D), leading to a dominance of BM-derived T cells by day +28 to +42. (E) Nearly all of the recovered T cells that were originally transplanted and thus peripherally expanded (not BM-derived) displayed an effector memory (EM) phenotype. (F) Despite this memory phenotype, the vast majority of these cells were derived from naïve T cells that had now assumed an effector memory phenotype. This was true in CD4⁺ and CD8⁺ T cells and in those mice treated with PBS or PTCy. (G) This dominance of naïve-derived T cells was also true in various tissue compartments. MLN, mesenteric lymph nodes.