Outcomes of hematopoietic stem cell gene therapy for Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, autoimmunity, and lymphoid malignancies. Gene therapy (GT) to modify autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplantation for patients who lack well-matched donors, avoiding graft-versus-host-disease. We report the outcomes of a phase 1/2 clinical trial in which 5 patients with severe WAS underwent GT using a self-inactivating lentiviral vector expressing the human WAS complementary DNA under the control of a 1.6-kB fragment of the autologous promoter after busulfan and fludarabine conditioning. All patients were alive and well with sustained multilineage vector gene marking (median follow-up: 7.6 years). Clinical improvement of eczema, infections, and bleeding diathesis was universal. Immune function was consistently improved despite subphysiologic levels of transgenic WAS protein expression. Improvements in platelet count and cytoskeletal function in myeloid cells were most prominent in patients with high vector copy number in the transduced product. Two patients with a history of autoimmunity had flares of autoimmunity after GT, despite similar percentages of WAS protein-expressing cells and gene marking to those without autoimmunity. Patients with flares of autoimmunity demonstrated poor numerical recovery of T cells and regulatory T cells (Tregs), interleukin-10-producing regulatory B cells (Bregs), and transitional B cells. Thus, recovery of the Breg compartment, along with Tregs appears to be protective against development of autoimmunity after GT. These results indicate that clinical and laboratory manifestations of WAS are improved with GT with an acceptable safety profile. This trial is registered at clinicaltrials.gov as #NCT01410825.

The clinical significance of PNH-phenotype cells accounting for < 0.01% of total granulocytes detected by the Clinical and Laboratory Standards Institute methods in patients with bone marrow failure.

Small populations of glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells accounting for up to 0.01% of total granulocytes can be accurately detected by a high-sensitivity flow cytometry (FCM) assay established by the Clinical and Laboratory Standards Institute (CLSI method) and have a prognostic value in bone marrow failure (BMF); however, the significance of GPI(-) granulocytes accounting for 0.001-0.009% of granulocytes remains unclear. To clarify this issue, we examined the peripheral blood of 21 BMF patients in whom minor (around 0.01%) populations of GPI(-) granulocytes had been previously detected by a different high-resolution FCM method (OPTIMA method, which defines ≥ 0.003% GPI(-) granulocytes as an abnormal increase) using both the CLSI and OPTIMA methods simultaneously. These two methods detected an "abnormal increase" in GPI(-) granulocytes in 10 patients (48%) and 17 patients (81%), respectively. CLSI detected 0.002-0.005% (median, 0.004%) GPI(-) granulocytes in 7 patients who were deemed positive for PNH-type cells according to the OPTIMA method, which detected 0.003-0.012% (median 0.006%) GPI(-) granulocytes. The clone sizes of GPI(-) cells detected by each assay were positively correlated (r = 0.994, p < 0.001). Of the seven patients who were judged positive for PNH-type cells by OPTIMA alone, five received immunosuppressive therapy, and all of them achieved a partial or complete response. GPI(-) granulocytes detected in BMF patients by the CLSI method should thus be considered significant, even at percentages of < 0.01%.

The GPI-anchored protein CD109 protects hematopoietic progenitor cells from undergoing erythroid differentiation induced by TGF-ß.

Although a glycosylphosphatidylinositol-anchored protein (GPI-AP) CD109 serves as a TGF-ß co-receptor and inhibits TGF-ß signaling in keratinocytes, the role of CD109 on hematopoietic stem progenitor cells (HSPCs) remains unknown. We studied the effect of CD109 knockout (KO) or knockdown (KD) on TF-1, a myeloid leukemia cell line that expresses CD109, and primary human HSPCs. CD109-KO or KD TF-1 cells underwent erythroid differentiation in the presence of TGF-ß. CD109 was more abundantly expressed in hematopoietic stem cells (HSCs) than in multipotent progenitors and HSPCs of human bone marrow (BM) and cord blood but was not detected in mouse HSCs. Erythroid differentiation was induced by TGF-ß to a greater extent in CD109-KD cord blood or iPS cell-derived megakaryocyte-erythrocyte progenitor cells (MEPs) than in wild-type MEPs. When we analyzed the phenotype of peripheral blood MEPs of patients with paroxysmal nocturnal hemoglobinuria who had both GPI(+) and GPI(-) CD34+ cells, the CD36 expression was more evident in CD109- MEPs than CD109+ MEPs. In summary, CD109 suppresses TGF-ß signaling in HSPCs, and the lack of CD109 may increase the sensitivity of PIGA-mutated HSPCs to TGF-ß, thus leading to the preferential commitment of erythroid progenitor cells to mature red blood cells in immune-mediated BM failure.

Resistance of KIR Ligand-Missing Leukocytes to NK Cells In Vivo in Patients with Acquired Aplastic Anemia.

The loss of killer cell Ig-like receptor ligands (KIR-Ls) due to the copy number-neutral loss of heterozygosity of chromosome 6p (6pLOH) in leukocytes of patients with acquired aplastic anemia (AA) may alter the susceptibility of the affected leukocytes to NK cell killing in vivo. We studied 408 AA patients, including 261 who were heterozygous for KIR-Ls, namely C1/C2 or Bw6/Bw4, for the presence of KIR-L-missing [KIR-L(-)] leukocytes. KIR-L(-) leukocytes were found in 14 (5.4%, C1 [n = 4], C2 [n = 3], and Bw4 [n = 7]) of the 261 patients, in whom corresponding KIR(+) licensed NK cells were detected. The incidence of 6pLOH in the 261 patients (18.0%) was comparable to that in 147 patients (13.6%) who were homozygous for KIR-L genes. The percentages of HLA-lacking granulocytes (0.8-50.3%, median 15.2%) in the total granulocytes of the patients with KIR-L(-) cells were significantly lower than those (1.2-99.4%, median 55.4%) in patients without KIR-L(-) cells. KIR2DS1 and KIR3DS1 were only possessed by three of the 14 patients, two of whom had C2/C2 leukocytes after losing C1 alleles. The expression of the KIR3DS1 ligand HLA-F was selectively lost on KIR-L(-) primitive hematopoietic stem cells derived from 6pLOH(+) induced pluripotent stem cells in one of the KIR3DS1(+) patients. These findings suggest that human NK cells are able to suppress the expansion of KIR-L(-) leukocytes but are unable to eliminate them partly due to the lack of activating KIRs on NK cells and the low HLA-F expression level on hematopoietic stem cells in AA patients.