Innovative Two-Step Negative Selection of Granulocyte Colony-Stimulating Factor–Mobilized Circulating Progenitor Cells: Adequacy for Autologous and Allogeneic Transplantation
Authors: Rambaldi Alessandro , Borleri Gianmaria , Dotti Gianpietro , Bellavita Piermario , Amaru Ricardo , Biondi Andrea , Barbui Tiziano 
Published: October 2019

Abstract
A major obstacle in purifying either autologous or allogeneic hematopoietic stem cells from granulocyte colony-stimulating factor (G-CSF) mobilized circulating progenitor cells (CPC) is represented by the huge cellularity present in each apheretic product. To obtain a significant debulking of unwanted cells from the leukapheresis, we developed a modified protocol of immune rosetting whereby human ABO-Rh– compatible red blood cells (RBCs) are treated with chromium chloride and then coated with murine monoclonal antibodies (MoAbs) against leukocyte antigens. When experiments were performed with leukaphereses obtained from normal donors or from T-cell acute lymphoblastic leukemia (T-ALL) patients, RBCs were coated with murine MoAbs against human mature myeloid cells (CD11b) and T cells (CD6); whereas, in the case of patients with B-precursor ALL, B-cell non-Hodgkin's lymphoma (B-NHL), or multiple myeloma (MM), RBCs were coated with anti-CD11b only. After incubation with CPC, rosetting cells (myeloid precursor cells, granulocytes, monocytes, and T cells) were removed by Ficoll-Hypaque density gradient centrifugation with a blood cell processor apparatus, COBE (Lakewood, CO) 2991. After this step, a significant reduction of the initial cellularity was consistently obtained (range, 72% to 97%), whereas the median absolute recovery of the CD34+ cells was above 85% (range, 64 to 100), with a 10-fold relative enrichment ranging from 3% to 41%. In a second step, CPC can be further purged of contaminating T or B cells by incubation with lymphoid-specific magnetic microbeads (anti-CD2 and -CD7 to remove T cells; anti-CD19 to remove B cells) and elution through a type-D depletion column (composed of ferromagnetic fiber) inserted within a SuperMACS separator device (Miltenyi Biotech, Bergisch-Gladbach, Germany). By this approach, a highly effective (three to four logs) T-cell depletion was achieved in all experiments performed with normal donors or T-ALL patients (median loss of CD3+cells: 99.8% [range 99.2 to 100]) and an equally efficient B-cell depletion was obtained from B-precursor ALL, B-NHL, or MM patients. At the end of the procedure the T- or B-cell depleted fraction retained a high proportion of the initial hematopoietic CD34+ stem cells, with a median recovery above 70% (range 48% to 100%) and an unmodified clonogenic potential. In five patients (two follicular NHL and three ALL) the purified fraction of stem cells was found disease free at the molecular level as assessed by polymerase chain reaction (PCR) analysis of the t(14;18) chromosome translocation or clono-specific DNA sequences of IgH or T-cell receptor γ and δ chain genes. Purified autologous and allogeneic CPCs were transplanted in three and six patients, respectively, who showed a prompt and sustained hematologic engraftment. In conclusion, this method represents a simple and reproducible two-step procedure to obtain a highly efficient purging of T or B cells from G-CSF expanded and mobilized CPCs. This approach might lead to the eradication of the neoplastic clone in the autologous stem cell inoculum as well as for T-cell depletion during allogeneic transplantation.