Lineage- and sublineage-specific chimerism after nonmyeloablative HSCT in IPEX syndrome. Flow cytometric cell sorting of leukocyte lineages and lineage-specific chimerism analyses were performed regularly after hematopoietic stem cell transplantation as described elsewhere.⁶  Gating strategy: Syto41/CD45RO fluorescein isothiocyanate (FITC), CD3 peridinin chlorophyll protein (PerCP), CD4 allophycocyanin (APC), CD25 phycoerythrin (PE), analyzed and sorted on a FACSAria (Becton Dickinson, Sunnyvale, CA). Acquisition and data analysis was performed with FACSDiva (Becton Dickinson) software. Monoclonal antibodies (mAb) used were CD3 (UCHT1), CD4 (MT310), CD8 (DK25), CD14 (TÜK4), CD15 (C3D1), CD19 (HD37), CD45 (T29/33; all from Dako, Glostrup, Denmark); CD3 (SK7), CD14 (MoP9), CD33 (P67.6), CD38 (HB-7), CD45 (2D1), CD56 (NCAM16.2; all from Becton Dickinson); and CD19 (J4.119) and CD45RA (2H4; both from Coulter, Krefeld, Germany). The purity of the sorted blood cell populations shown in panel A was ≥ 98% as confirmed by rerunning the stained, sorted samples. (A) The proportion of donor cells (shown as percent donor chimerism, y-axis) is shown over time after transplantation (x-axis). ★ indicates 2 of 3 occasions within the 6th year after HSCT that included more in-depth sublineage chimerism analyses as shown in panels B and C. indicates the occasion when the latest bone marrow aspiration was performed with practically identical chimerism results as shown for simultaneously analyzed peripheral blood except that CD34⁺ cells, which were not detectable in the periphery, were of 8% to 14% donor origin (not shown). (B) This diagram shows more detailed sublineage chimerism analyses including the CD4⁺CD25^neg, CD4⁺CD25^med, and CD4⁺CD25^high T cells as indicated, performed at the latest 3 visits at 5, 5.5, and 6 years after transplantation (mean + error bars = range). (C) The plot is representative of 2 specimens from 2 independent outpatient visits at 5.5 and 6 years after HSCT, which were analyzed for lineage-specific and subpopulation-specific chimerism by fluorescence-activated cell sorting and short tandem repeat–polymerase chain reaction (STR-PCR). It shows the CD3/CD4-gated fractions of CD25^neg/CD25^med/CD25^high cells with their STR-PCR peaks (insert; r, recipient; d, donor; “control” showing both individual and the common STR-marker peaks). CD4⁺ T cells comprised 58% of all T cells, CD4⁺CD25^high were approximately 1% of all CD3⁺CD4⁺ cells (shown as “++”), CD4⁺CD25^med were 16% of CD3⁺CD4⁺ (marked “+”). As indicated visually by the donor peak (“d”) in the insert, CD4⁺CD25^high cells were 91% (± 6%) donor-derived, whereas CD25^med were 5% (± 6%), and CD4⁺CD25^neg 3% (± 6%) donor-derived and thus > 89% to 91% recipient (“r”). STR-PCR chimerism results of granulocytes, monocytes, NK and B cells were all < 6% donor-derived (ie, complete recipient chimerism; not shown). Alleles were quantified by capillary electrophoresis and fluorescence-based quantification using the ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).