Double-Unit Umbilical Cord Blood Transplantation Induces MHC-Matched Single-Unit Dominance with Development of Immunocompetent Lymphocytes in Recipient Mice

Double-unit umbilical cord blood cell (dUCBC) transplantation has emerged as an effective strategy for improving the engraftment of umbilical cord blood stem cells in the bone marrow of recipients. Due to a lack convenient animal models, analyses of the differentiation capacity of dUCBC in recipients have been limited to in vivo xenogeneic experiments and clinical observations. In the present study, we evaluated the characteristics of immune reconstitution induced by dUCBC transplantation in mice.

Natural killer cells were depleted from female C57BL/6 (B6) [H-2^b] recipient mice by intraperitoneal administration of rabbit anti-asialo GM1 polyclonal antibody 1 day before transplantation. On the following day, the lethal X-ray-irradiated B6 recipients were given transplants of three different combinations of dUCBC {group (1) GFP-Tg B6 [H-2^b] and BALB/c [H-2^d]; group (2) GFP-Tg B6 [H-2^b] and C3H [H-2^k]; group (3) BALB/c [H-2^d] and C3H [H-2^k]}, each combination containing an equal number of cells. At 16 weeks after transplantation, reconstitution of immune cells was evaluated by flow cytometric analysis utilizing specific antibodies against lineage markers such as CD3 (T cells), CD45R/B220 (B cells), CD11b (macrophages), or Ly-6G (granulocytes). The donor origin of each lineage population was determined by anti-H-2K^k (for C3H) and/or H-2K^d (for BALB/c) antibody staining. GFP⁺ lineage cells were identified as being of B6 donor origin. Skin grafting was then performed in all recipients to assess the functional maturity of the newly developed T and B cells induced by dUCBC transplantation.

The survival rate at 16 weeks after transplantation was 73% (8/11) for case (1), 92% (12/13) for case (2), and 50% (3/6) for case (3). In the great majority of cases (1) and (2), in which dUCBC were administered as a stem cell source, the MHC-matched single unit from GFP-Tg B6 acts as the sole source of long-term hematopoiesis (75% (6/8) for case (1); 100% (12/12) for case (2)). CD3⁺ T cell peripheral blood chimerism from BALB/c was observed in two of the eight B6 survival recipient mice in case (1) at an early stage of hematopoiesis, predicting the long-term engrafting unit. On the other hand, hematopoiesis in case (3) with fully allogenenic dUCBC transplantation was reconstituted by the B6 recipients' own X-ray-resistant hematopoietic stem cells (HSC). Our results indicate that MHC-matched UCBC-HSC predominantly engraft in the recipient's bone marrow after dUCBC transplantation. However, the nature of this selective mechanism remains largely unknown. In all cases, alloreactive cytotoxic cells in recipient may participate in such selection. In dUCBC transplantation, the included allogeneic cells probably act as stimulators for promoting the differentiation and maturation of MHC-matched HSC through activation of certain types of signal transduction (for example, through cytokine secretion). Currently, we are investigating the possible presence of alloreactive cytotoxic cells in bone marrow. Functionally, these recipients were tolerant of skin grafted from B6, whereas they rejected skin from BALB/c and C3H within 20 days, indicating that both CD4⁺ helper and CD8⁺ killer T cells were functionally mature in the recipient mice. Correspondingly, only the alloantibody to BALB/c and C3H was produced in the recipients. One of two chimeric recipient mice in case (1) reacted to only C3H skin with T and B cells.

dUCBC transplantation clearly rescued mice that had been subjected to lethal X-ray irradiation. Furthermore, our observations indicate that T and B cells derived from dUCBCs transplants are immunologically fully competent with the ability to distinguish self from non-self MHC antigens. However, a clear understanding of the mechanisms underlying the predominant engraftment of MHC-matched HSCs in the recipient's bone marrow will be necessary.

No relevant conflicts of interest to declare.