Dale Kalamasz

Positive selection of viable cell populations using avidin-biotin immunoadsorption.

We have developed a new method for the selective enrichment of lymphoid subpopulations from dog and human bone marrow and peripheral blood. A mononuclear cell preparation was treated successively with monoclonal antibody, biotinylated goat anti-mouse immunoglobulin and passed over a column containing avidin linked to polyacrylamide or Sepharose beads. Adherent cells were recovered by mechanical agitation and analyzed by immunofluorescence staining and fluorescence-activated cell sorter analysis. Dog bone marrow mononuclear cells were treated successively with the antibody 7.2, which recognizes the Ia-antigen, 1:500 dilution of biotinylated goat anti-mouse immunoglobulin and passed over avidin-Biogel (1 mg/ml) at a flow rate of 3.0 ml/min. Enrichment from a starting population that was 24.4 +/- 9.1% 7.2-positive to 78.3 +/- 6.8% 7.2-positive adherent cell population was observed with 47.7 +/- 7.8% recovery of 7.2-positive cells. Human bone marrow mononuclear cells were treated successively with the T cell antibody Leu-4 followed by 1:500 dilution of B-GAMIg and passed over a column of avidin-Biogel (1 mg/ml) at a flow rate of 1.5 ml/min. Enrichment from 7.2 +/- 3.3% Leu-4-positive cells in the starting cell population to 73.1 +/- 6.8% Leu-4-positive cells in the adherent cell population with total recovery of Leu-4-positive cells averaging 64.0 +/- 12.7%. Human bone marrow mononuclear cells positively selected with antibody Leu-4 or another T cell antibody, Leu-5 had a markedly enhanced response to the T cell mitogen, phytohemagglutinin compared to untreated bone marrow. Enrichment of a subpopulation of lymphocytes from dog peripheral blood mononuclear cells has been accomplished using antibody DT2, which reacts with a broad spectrum of dog lymphocytes. Nonspecific cell binding is primarily limited to granulocytes and monocytes. Future work is being directed at improving recovery of positively selected cells, reducing nonspecific cell binding and applying the technique to the selective enrichment of hematopoietic stem cells from bone marrow.

Optimization of human T-cell expansion ex vivo using magnetic beads conjugated with anti-CD3 and anti-CD28 antibodies

T-cell receptor engagement and accompanying costimulatory signals control the level of activation and functional potential of individual T cells. The authors previously developed a novel technology in which human T cells are activated and expanded in culture ex vivo using anti-CD3 and anti-CD28 monoclonal antibodies covalently linked to superparamagnetic beads (Xcyte™ Dynabeads®). In this study the addition of N-acetyl l-cysteine (NAC) to the cultures markedly increased the expansion of T cells from human peripheral blood mononuclear cells without diminishing cell function. NAC increased the rate of T-cell division, reduced apoptosis, and increased the percentage of antigen-specific memory T cells in the cultures. The effect of varying the ratio of beads to T cells (1:10–10:1) at culture initiation was also evaluated. Polyclonal T cells were expanded at all bead-to-T cell ratios tested (range 1:10–10:1). While high bead-to-T cell ratios (5:1 and 10:1) deleted, low ratios (1:10 and 1:5) preserved memory T cells directed against cytomegalovirus, Epstein-Barr virus, and influenza virus antigens. Adding more anti-CD3/anti-CD28 beads during the culture led to further expansion of T cells. Experiments also revealed that reducing the amount of anti-CD3 antibodies relative to the amount of anti-CD28 antibodies on the beads favored the proliferation of antigen-specific T cells. In summary, these data indicate that T cell-stimulating effects of anti-CD3/anti-CD28 beads can be further manipulated to control the expansion of antigen-specific memory T cells and can be used to rapidly expand antigen-specific T cells ex vivo for potential clinical applications.