Marcie R. Finney

Influence of human leucocyte antigen disparity and graft lymphocytes on allogeneic engraftment and survival after umbilical cord blood transplant in adults

The dose of graft‐nucleated cells and CD34+ haematopoietic progenitor cells are predictors of allogeneic engraftment and survival in umbilical cord blood (UCB) recipients. In this single institution prospective phase II trial, flow cytometric analyses of CD34+ progenitor and lymphocyte populations in unmodified single unit human leucocyte antigen (HLA)‐disparate UCB grafts infused into 31 consecutive adults (median age 41 years, range 20–64) receiving myeloablative conditioning were compared with clinical outcomes. Median infused UCB graft‐nucleated cells and CD34+ dose was 2·2 × 107/kg and 1·2 × 105/kg respectively. Day to absolute neutrophil count ≥0·5 × 109/l with full donor chimerism averaged 27 d (range 12–41). Univariate analyses demonstrated that UCB graft‐infused cell doses of CD34+ (P = 0·015), CD3+ (P = 0·024) and CD34+HLADR+CD38+ progenitors (P = 0·043) correlated with neutrophil engraftment. This same analysis did not demonstrate a correlation between CD34+ (P = 0·11), CD3+ (P = 0·28) or CD34+HLADR+CD38+ (P = 0·108) cell dose and event‐free survival (EFS). High‐resolution matching for HLA‐class II (DRB1) resulted in improved EFS (P = 0·02) and decreased risk for acute graft‐versus‐host disease (GVHD) (P = 0·004). Early mortality (prior to post‐transplant day +28) occurred in three patients, while 26 patients achieved myeloid engraftment. These results suggest that UCB graft matching at DRB1 is an important risk factor for acute GVHD and survival, while higher UCB graft cell doses of CD34+, committed CD34+ progenitors and CD3+ T cells favourably influence UCB allogeneic engraftment.

Umbilical cord blood-selected CD133+ cells exhibit vasculogenic functionality in vitro and in vivo

BACKGROUND AIMS Current clinical trials utilize non-selected bone marrow (BM) mononuclear cells (MNC) to augment vasculo genesis within ischemic vascular beds. Recent reports have identified a diminished number and function of hemat-opoietic stem cells (HSC) from aged and diseased patients. Umbilical cord blood (UCB) provides a potential robust allo-geneic source of HSC for therapeutic vasculogenesis. METHODS MNC and magnetically isolated CD133(+) cells were assessed for viability (trypan blue) and surface phenotype (flow cytometry). To test in vivo functionality of the cells, NOD/SCID mice underwent ligation of the right femoral artery followed immediately by cell injection. Blood flow recovery, necrosis, BM engraftment of human cells and histologic capillary density were determined. Cells were tested for potential mechanisms mediating the in vivo effects, including migration, cytokine secretion and angiogenic augmentation (Matrigel assays). RESULTS Surface expression analysis showed CD31 (PECAM) expression was greatly increased in UCB CD133(+) cells compared with BM MNC. At 28 days, perfusion ratios were highest in animals receiving UCB CD133(+) cells, while animals receiving BM CD133(+) cells and BM MNC demonstrated perfusion ratios statistically higher than in animals treated with cytokine media alone. Animals receiving CD133(+) cells showed a statistically higher capillary density, reduced severe digit necrosis and increased engraftment in the BM than animals treated with unselected BM MNC. In vitro studies showed equivalent migration to stromal-derived factor-1 (SDF-1), increased production of tumor necrosis factor alpha (TNF-alpha) and increased branch points with the co-incubation of CD133(+) cells with human umbilical vein endothelial cells (HUVEC) in the Matrigel angiogenesis assay. CONCLUSIONS Taken together, UCB CD133(+) cells exhibit robust vasculogenic functionality compared with BM MNC in response to ischemia.

Allogeneic transplantation of multiple umbilical cord blood units in adults: role of pretransplant-mixed lymphocyte reaction to predict host-vs-graft rejection.

Allogeneic transplantation of multiple umbilical cord blood units in adults: role of pretransplant-mixed lymphocyte reaction to predict host-vs-graft rejection

Regulation of IL-2 expression by transcription factor BACH2 in umbilical cord blood CD4+ T cells

On activation, umbilical cord blood (UCB) CD4+ T cells demonstrate reduced expression of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), whereas maintaining equivalent interleukin-2 (IL-2) levels, as compared with adult peripheral blood (PB) CD4+ T cells. Nuclear factor of activated T cells (NFAT1) protein, a transcription factor known to regulate the expression of IL-2, TNF-α and IFN-γ, is reduced in resting and activated UCB CD4+ T cells. In contrast, expression of Broad-complex-Tramtrack-Bric-a-Brac and Cap‘n’collar homology 1 bZip transcription factor 2 (BACH2) was shown by gene array analyses to be increased in UCB CD4+ T cells and was validated by qRT-PCR. Using chromatin immunoprecipitation, BACH2 was shown binding to the human IL-2 proximal promoter. Knockdown experiments of BACH2 by transient transfection of UCB CD4+ T cells with BACH2 siRNA resulted in significant reductions in stimulated IL-2 production. Decreased IL-2 gene transcription in UCB CD4+ T cells transfected with BACH2 siRNA was confirmed by a human IL-2 luciferase assay. In summary, BACH2 maintains IL-2 expression in UCB CD4+ T cells at levels equivalent to adult PB CD4+ T cells despite reduced NFAT1 protein expression. Thus, BACH2 expression is necessary to maintain IL-2 production when NFAT1 protein is reduced, potentially impacting UCB graft CD4+ T-cell allogeneic responses.