In patient 4, we observed a surprising conversion of the dominant proportion of monocytes from WASP bright to WASP dim 30 to 190 days after HST. It was noteworthy that most of his CD20 + cells were WASP dim. The precise MC status in patient 3, who consistently had WASP dim lymphocytes, is shown in Figure 4. In patients 3 and 4, the monocytes were mostly WASP dim, whereas most of their lymphocytes were WASP bright, except for the CD20 + cells in patient 3. The same phenomenon was observed on a different day after HST in patient 1. Patient 2 had a transient increase in CD4 + WASP dim recipient cells at day 360 after HST, which was correlated with a WASP bright donor CD4 + increment (data not shown). In contrast, most of the lymphocytes were WASP bright donor cells. In patients 2 and 5, we observed stable MC status with regard to monocytes, of which approximately half were found to be recipient cells. The number of WASP dimrecipient lymphocytes and monocytes in patient 1 appeared to diminish gradually, indicating that all hematopoietic cells would eventually be replaced by donor cells. The MC patterns observed varied among the patients, although WASP dim recipient cells were found most frequently in the monocyte population (Table 2). We used 2-color FCM-WASP to follow the 6 patients with MC status for 0.5 to 2.5 years after transplantation (Figure 3). Because anti-WASP mAb belongs to the mouse IgG1 subclass, all antibodies for staining were of either the mouse IgG2 or IgG3 subclass to avoid a cross-reaction.
WASP expression in lymphocytes and monocytes was determined after gating the respective distribution patterns by forward and side scatter (Figure 1A).
#Chimera syndrome software#
Stained cells were analyzed with a FACScan flow cytometer using CellQuest software (Becton Dickinson). The cells were then washed twice and allowed to react with 1:100 diluted fluorescein isothiocyanate–labeled goat anti–mouse IgG1 antibody (Southern Biotechnology Associates) at 4☌ for 30 minutes.
Cells were then washed twice and incubated with 1:100 diluted anti-WASP (3F3-A5) mAb 17 or purified mouse IgG1 (Becton Dickinson, Mountain View, CA) at 4☌ for 30 minutes. After being washed twice, cells were treated with Cytofix/Cytoperm solution from a CytoStain kit (PharMingen, San Diego, CA) at 4☌ for 60 minutes. Briefly, PBMCs were stained for cell-surface antigens by using the following monoclonal antibodies (mAbs): phycoerythrin (PE)–conjugated anti-CD4, anti-CD8, and anti-CD56 (Southern Biotechnology Associates, Birmingham, AL) PE-conjugated anti-CD20 (Beckman-Coulter, Fullerton, CA) and PE-Cy5–conjugated anti-CD45RA and anti-CD45RO (eBioscience, San Diego, CA).
15 We developed 3-color FCM-WASP for further analysis. The methods used for FCM-WASP and 2-color FCM-WASP were reported previously. Our findings may also have important implications for the role of WASP during hematopoietic development. We conclude that FCM-WASP is a potentially useful method for clinical follow-up of WAS patients who have undergone HST. No imbalance in the ratio of naive to memory T cells was observed in WAS patients before HST. We found that, in contrast to WASP bright T cells, most WASP dim T cells remained naive (CD45RA +/RO −) more than a year after HST. Finally, to investigate the naive/memory status of donor and recipient T cells in these patients, 3-color FCM-WASP using anti-CD45RA or CD45RO was performed. However, among PBMCs, recipient cells were most commonly observed in the monocyte population. MC status was observed in every cell lineage examined. Six of the 12 patients with WAS were found to have MC status after HST, whereas others had complete chimera status. Furthermore, with use of 2-color FCM-WASP, the MC status could be characterized by cell lineage. After HST, donor- and recipient-derived peripheral blood mononuclear cells (PBMCs) could be distinguished easily with this method, since the donor cells were WASP bright, whereas the defective recipient cells were WASP dim. In this study, we applied FCM-WASP to evaluate the mixed chimera (MC) status of 12 WAS patients who underwent hematopoietic stem cell transplantation (HST). We previously reported that flow cytometric analysis of intracellular WASP expression (FCM-WASP) was useful in the diagnosis of WAS in patients and carriers. Wiskott-Aldrich syndrome (WAS) is caused by defects in the WAS protein (WASP) gene on the X chromosome.
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