Improving umbilical cord blood processing to increase total nucleated cell count yield and reduce cord input wastage by managing the consequences of input variation.

BACKGROUND AIMS: With the rising use of umbilical cord blood (UCB) as an alternative source of hematopoietic stem cells, storage inventories of UCB have grown, giving rise to genetically diverse inventories globally. In the absence of reliable markers such as CD34 or counts of colony-forming units, total nucleated cell (TNC) counts are often used as an indicator of potency, and transplant centers worldwide often select units with the largest counts of TNC. As a result, cord blood banks are driven to increase the quality of stored inventories by increasing the TNC count of products stored. However, these banks face challenges in recovering consistent levels of TNC with the use of the standard protocols of automated umbilical cord processing systems, particularly in the presence of input variation both of cord blood volume and TNC count, in which it is currently not possible to process larger but useable UCB units with consequent losses in TNC. METHODS: This report addresses the challenge of recovering consistently high TNC yields in volume reduction by proposing and validating an alternative protocol capable of processing a larger range of units more reliably. RESULTS: This work demonstrates improvements in plastic ware and tubing sets and in the recovery process protocol with consequent productivity gains in TNC yield and a reduction in standard deviation. CONCLUSIONS: This work could pave the way for cord blood banks to improve UCB processing and increase efficiency through higher yields and lower costs.

Flow cytometry assessment of apoptotic CD34+ cells by annexin V labeling may improve prediction of cord blood potency for engraftment.

BACKGROUND: Nonviable CD34+ cells are commonly assessed by standard flow cytometry using the nuclear stain 7-aminoactinomycin D (7AAD). 7AAD, however, only detects necrotic and late apoptotic cells, not earlier apoptosis, which engraft poorly in animal models of cord blood (cord) transplantation. The standard method, therefore, may overestimate engraftment potency of cord units under certain conditions. STUDY DESIGN AND METHODS: To detect apoptotic events, costaining with 7AAD and annexin V (AnnV), in parallel with the quantitative, standard enumeration, was used. Cord units were assessed before and after cryopreservation using both staining methods and colony-forming units (CFU) to determine if graft potency can be predicted using a "functional flow cytometry" approach. RESULTS: Significant numbers of CD34+ AnnV+ events were found within the 7AAD-gated population. Nonapoptotic cell dose (CD34+ AnnV-) correlated well with CFUs in both a small-scale (n = 10) and a large-scale banking study (n = 107). Finally, following samples postthaw with time showed increasing numbers of apoptotic CD34+ cells and consequently the AnnV assessed dose was better at predicting the CFU compared with just the standard enumeration. CONCLUSION: Defining the apoptotic population of CD34+ cells improved the prediction of CFU, making this method a rapid test of potency for assessment of cord units for clinical use.

The role of CD4+ T cell help in cancer immunity and the formulation of novel cancer vaccines.

Recent years have seen the unprecedented surge of interest in the role of CD4+ T cells and the role they play in the development of the immune response. In this symposium review, we examine the evidence for this and discuss their functions, particularly in respect to the cancer immunology, including CD4+CD25+ cells (Treg).

Peptide immunisation of HLA-DR-transgenic mice permits the identification of a novel HLA-DRbeta1*0101- and HLA-DRbeta1*0401-restricted epitope from p53.

Because of the central role of CD4(+) T cells in antitumour immunity, the identification of the MHC class II-restricted peptides to which CD4(+) T cells respond has become a priority of tumour immunologists. Here, we describe a strategy permitting us to rapidly determine the immunogenicity of candidate HLA-DR-restricted peptides using peptide immunisation of HLA-DR-transgenic mice, followed by assessment of the response in vitro. This strategy was successfully applied to the reported haemaglutinin influenza peptide HA(307-319), and then extended to three candidate HLA-DR-restricted p53 peptides predicted by the evidence-based algorithm SYFPEITHI to bind to HLA-DRbeta1*0101 (HLA-DR1) and HLA-DRbeta1*0401 (HLA-DR4) molecules. One of these peptides, p53(108-122), consistently induced responses in HLA-DR1- and in HLA-DR4-transgenic mice. Moreover, this peptide was naturally processed by dendritic cells (DCs), and induced specific proliferation in the splenocytes of mice immunised with p53 cDNA, demonstrating that immune responses could be naturally mounted to the peptide. Furthermore, p53(108-122) peptide was also immunogenic in HLA-DR1 and HLA-DR4 healthy donors. Thus, the use of this transgenic model permitted the identification of a novel HLA-DR-restricted epitope from p53 and constitutes an attractive approach for the rapid identification of novel immunogenic MHC class II-restricted peptides from tumour antigens, which can ultimately be incorporated in immunotherapeutic protocols.