Abstract
CMV reactivation after stem cell transplantation can be treated with CMV-specific T cells but current in vitro techniques using dendritic cells as antigen-presenting cells are time-consuming and expensive. To simplify the production of clinical grade CMV specific T cells, we evaluated gene-modified activated T cells (T-APC) as a reliable and easily produced source of APC to boost CD4 and CD8 T cell responses against the immunodominant CMV antigen pp65. Peripheral blood mononuclear cells from CMV seropositive donors were activated for 2–3 days in complete medium (IMDM, AB serum, glutamine, and antibiotics) supplemented with 0.8 mg/ml phytohaemagglutinin and 100 IU IL-2/ml. The cells were transduced with Phoenix-A derived recombinant virus encoding pp65 in retronectin-coated 6-well plates, and further expanded in anti-CD3 plus CD28-coated flasks for 3–7 more days. Cultured cells expressed high levels of HLA-DR, and the costimulatory molecules CD80 and CD86. Autologous PBMC (0.5 – 1.0 x 106 cells) were stimulated with 106 irradiated (25 Gy) transduced T-APC in a 24-well plate. After 1–3 days IL-2 and IL-7 were added to a final concentration of 20 IU/ml and 10 ng/ml, respectively. Two weeks later the T cell lines were tested for antigen specificity using the flow cytometric intracellular detection of interferon-gamma following stimulation for 6 hours with a pp65 peptide library of 15-mers, overlapping by 11 amino acids. This technique induced a 135-fold (median; range, 20–120,000) and a 255-fold (median; range, 17–20,000) expansion of pp65-specific CD4 and CD8 responder cells, respectively, in 10/10 seropositive donors (figure). To further improve proliferation, CD25-expressing T regulatory cells were removed from the PBMC at the start of the culture by immunomagnetic depletion (Miltenyi). In 7/10 donors, CD25 depletion resulted in increased CD4 and/or CD8 responder numbers (p>0.05; Mann Whitney paired t-test). Median increase in responder cell numbers was 4.25-fold (range, 1.4–6) for CD4+ T cells, and 4.2-fold (range, 3–7.5) for CD8+ T cells. These data indicate that T-APC efficiently boost pp65-specific CD4 and CD8 T cell numbers to clinically useful levels and that removal of CD25-expressing cells can further augment the total yield of antigen-specific T cells in most donors. The approach has the advantage of using a single leukocyte collection from the donor to generate large numbers of CMV-specific T cells within a total 3 week culture period using only one stimulation of antigen.
Fold-change in the total number of pp-65 specific CD4 and CD8 T cells from PBMC
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