Selection of CD34+ HSC from Pooled Cord Blood Samples Does Not Result in Co-Purification of Potential Alloreactive CD3+ T Cells.
Abstract Most systems for studying the ex vivo expansion of human UCB-HSC require enrichment of the HSC population, typically by CD34 selection. Because the number of HSC present in a single UCB collection is limited, one approach to increase the total HSC is to pool multiple UCB samples. A potential complicating factor with this method is that alloreactive T lymphocytes present in whole UCB samples would be expected to bind to cells displaying allogeneic HLA, including HSC, and would therefore co-purify with CD34+ UCB-HSC during isolation. Such complexes would be excluded from ISHAGE analysis by the side scatter gate, which is set to exclude aggregates. Alloreactive T cells would be expected to contain cytotoxic T lymphocytes [CTL], which could potentially inhibit or completely abrogate HSC expansion. In order to determine whether CD3+ T lymphocytes co-purify with CD34+ UCB-HSC in pooled samples, UCB pools were prepared containing 2 to 4 UCB samples. CD34+ HSC were isolated by MACS and analyzed by flow cytometry and antibodies to human CD45, CD34 and CD3, with and without ISHAGE gates. Samples of CD34+ HSC purified from a single UCB collection were analyzed concurrently to give the background value for co-purification of syngeneic T cells and formation of T:HSC aggregates in the CD34-selected products. We found a limited number of CD3+ T cells present in CD34+ HSC isolated from single UCB collections [mean = 0.25%, range = 0 – 1.1%]. That value was slightly elevated when pools of UCB were used [mean = 0.44%; range = 0 – 1.9%]. There are few, if any, CD34+/CD3+ cells that can be detected by either the standard ISHAGE gating or by gating merely on CD45+ cells with no side scatter gating [0.02 – 0.04%]. This was true of CD34+ HSC isolated from either pooled UCB or single UCB collections. Based on these results, we conclude that there is not significant co-purification of CD3+ T cells with CD34+ UCB-HSC, and that any such complexes that form are not found at any greater frequency in UCB pools than in single UCB collections.
Selection of Tumor-Specific Cytotoxic T Lymphocytes in Acute Myeloid Leukemia Patients Through the Identification of T-Cells Capable to Establish Stable Interactions With the Leukemic Cells: “Doublet Technology”
