Combined positive and negative cell selection from allogeneic peripheral blood progenitor cells (PBPC) by use of immunomagnetic methods

Abstract

Summary:Twenty-four mobilized peripheral blood products from healthy donors for allogeneic transplantation were positively selected for CD34 + cells and depleted of CD4 + and CD8 + cells (+/− selection) by combining clinical grade immunomagnetic methods. A sequential, 'two-step' strategy combining positive selection of CD34 + cells by use of the Isolex 300i (versions 1 and 2) device and T cell depletion (TCD) using the MaxSep device and a simultaneous, 'one-step' method of CD34 + cell selection and TCD using the Isolex 300i (software versions 1 and 2) have been investigated. Using these magnetic bead separation systems, two groups of sequential +/− selection (Isolex 300i version 1/MaxSep and Isolex 300i version 2/MaxSep) and two groups of simultaneous +/− selection (Isolex 300i versions 1 and 2) were analysed. In the sequential +/− selection, logarithms of TCD (CD3 + cell depletion) obtained by the positive selection step had median values of 3.7 with the version 1 (n = 5) and 4.5 with version 2 software of the Isolex 300i (n = 5) (P = 0.07). Version 2 also gave a higher CD34 + cell purity and yield than did version 1 (92% vs 77%, P Ͻ 0.05 and 55% vs 34%, P = 0.3, respectively). Additional TCD obtained in the second step with the MaxSep device for the two groups had a median value of 0.9 log and 7% CD34 + cell losses. In the simultaneous +/− selection, the Isolex 300i version 2 (n = 10) gave a median TCD of 5.1 log and version 1 (n = 4) of 4 log (P Ͻ 0.005). Higher CD34 + cell purity and yield were also obtained with version 2 than with version 1 (97% and 76%, P Ͻ 0.005 and 57% and 39%, P = 0.07, respectively). These data indicate that simultaneous, 'one-step' +/− selection in the Isolex 300i version 2 achieves a high TCD with a high CD34 + cell purity and an acceptable CD34 + cell yield. Bone Marrow Transplantation ( The development of HLA-mismatched or haploidentical donor stem cell transplantation requires a high degree of T cell depletion (TCD) while preserving an adequate number of CD34 + cells in the graft in order effectively to prevent acute graft-versus-host disease (aGVHD) and ensure engraftment. [1][2][3] Several reasons make CD34 positive cell selection methods (positive selection, + selection) particularly attractive to deplete T cells especially in peripheral blood progenitor cell (PBPC) grafts: they can be applied to the large numbers of cells present in PB, they are able to preserve adequate numbers of progenitors after selection, thus maintaining the potential benefits of a PBPC transplant in this setting and they passively deplete the elevated numbers of T cells present in the PB grafts.Several methods are available for positive selection. 4-7 The immunomagnetic technique, which is now exploited by the Isolex 300i cell selection technology (Nexell Therapeutics, Irvine, CA, USA), involves specific binding of target cells by a mouse anti-CD34 MoAb, 9C5. The target cells-9C5 complexes are then captured by sheep antimouse IgG-coated paramagnetic beads (IB) (SAM-M450; Dynabeads, Dynal, Oslo, Norway)...

The beneficial role of inhibitory KIR genes of HLA class I NK epitopes in haploidentically mismatched stem cell allografts may be masked by residual donor‐alloreactive T cells causing GVHD

“…All transplants were performed using granulocyte colony‐stimulating factor‐mobilized peripheral blood stem cells (23). T‐cell depletion for the prevention of GVHD was achieved by positive selection of CD34 + cells using Dynal immunomagnetic beads (24) (Isolex, Baxter healthcare, Deerfield, IL) or Milenyi's CliniMACS (Miltenyi Biotec GmbH, Bergisch Gladback, Germany) (25). Median mononuclear cell count, CD34 + , and CD3 + cell doses were 11.5 (range 1.5–60.2) × 10 8 /kg, 8.6 (range 1.9–31.5) × 10 6 /kg, and 10.8 (range 0.9–50) × 10 4 /kg, respectively.…”

A novel high throughput immunomagnetic cell sorting system for potential clinical scale depletion of T cells for allogeneic stem cell transplantation

“…Honrnick et al [28] suggested that the depletion of HLA class II+ cells from the irradiated stimulator cells could increase the sensitivity and specificity of the LDA. Second, it has been suggested that a correction factor could be used to relate T-cell content calculated by flow cytometry as compared to LDA, thereby eliminating the variability between flow cytometry and LDA [22,23]. Such an example of a correction factor is the cloning efficiencies, which for our T cell depletion studies is given by (summarized in Table 3):…”

“…In addition to flow-cytometric assessment of the extent of T cell depletion, we also performed mixed lymphocyte cultures and limiting dilution assays to assess residual T cell alloreactivity after T cell depletion. We used the methodology of Kawanishi et al [22] and Kernan et al [23]. Briefly, the cell suspension in which the presence of T-cells was to be tested was referred to as the responders, or "A" cells, and the stimulator cells as "B".…”

Combined isolation of CD34+ progenitor cells and reduction of B cells from peripheral blood by use of immunomagnetic methods

“…Our laboratory has also tested this strategy to purge T cells from allogeneic grafts, with excellent results too. 41 Nevertheless, the efficiency of the simultaneous positive and negative selection approach with the Isolex 300i to purge B cells should be evaluated in a larger number of procedures.…”