Characteristics of Immune Reconstitution by Mixed Umbilical Cord Blood Transplantation In Mice: Predominant Engraftment of Major Histocompatibility Complex (MHC)-Matched Umbilical Cord Blood Stem Cell In Recipients' Bone Marrow.
Authors: Nakano Akira , Sato Hideaki , Wakayama Ayumi , Shinohara Nobukata , Ito Kyoko , Kashiwakura Ikuo , Ito Koichi 
Published: October 2019

Abstract
Abstract 3721

Background:
Currently, umbilical cord blood cells (UCBCs) are used as the primary source of hematopoietic stem cells (HSCs) for transplantation instead of bone marrow cells (BMCs). UCBC transplantation has several advantages over BMC transplantation, including the much larger size of the available donor pool, the rich proportion of hematopoietic progenitor cells, and the low content of mature T cells that might cause a graft-versus-host reaction. However, the limited quantity of cord blood samples that can be obtained from a pregnant woman is considered to be a disadvantage of UCBC transplantation. To overcome quantity limitations, the use of mixed cord blood might be necessary. Due to the lack of appropriate animal models, the analyses of the differentiation capacity of mixed UCBCs in recipients have been limited to in vivo xenogeneic experiments and clinical observations. In this study, we evaluated the reconstitution and functioning of immune systems induced by mixed UCBC transplantation in mice.

Method:
To deplete NK cells, female C57BL/6 (B6) [H-2b] recipient mice were intraperitoneally administered rabbit anti-asialo GM1 antibody 1 day before transplantation. On the following day, the B6 recipients irradiated with a radiation dose of 9 Gray were transplanted with 3 different combinations of mixed UCBC {[1] GFP-Tg B6 (H-2b) and C3H (H-2k); [2] GFP-Tg B6 (H-2b) and BALB/c (H-2d); and [3] C3H (H-2k) and BALB/c (H-2d)}, with each combination containing equal number of cells. Engraftment of cells in the recipients' peripheral blood was detected by flow cytometric analysis using a specific antibody against lineage markers such as CD3e (T cells), CD45R/B220 (B cells), CD11b (macrophages), or Ly-6G (granulocytes) at intervals of 4 weeks for 16 weeks. In addition, the donor origin of each lineage population was determined by H-2Kk and/or H-2Kd antibody staining. GFP+ lineage cells were found to have B6 donor origin. The dermis samples harvested from the B6, C3H, and BALB/c mice were simultaneously skingrafted on the shaved backs of the recipients at over 16 weeks after transplantation. After the recipients rejected the skin graft, alloreactive T cell and antibody responses were examined to evaluate the functional maturity of the reconstituted immune system in the recipients.

Result:
The survival rates of the recipient mice at 16 weeks after transplantation were as follows: (1), 73%, 8/11; (2), 92%, 12/13; and (3), 100%, 1/1. Flow cytometric analysis showed that cells of all lineages were reconstituted by only GFP+ cells in almost all the B6 recipients transplanted with mixed UCBCs: (1), 50%, 4/8 and (2), 100%, 12/12. This findings indicated predominant bone marrow engraftment of UCBC-HSCs from MHC-matched B6 donors. Furthermore, the allogeneic UCBCs in the recipients transplanted with combination [3] were eliminated from the recipients, and their lineage cells (GFP− H-2Kk− H-2Kd−) in peripheral blood were derived from the B6 recipient's own X-ray-resistant HSC in the bone marrow. The recipient mice in which the immune system was reconstituted by UCBC-HSCs from the B6 donors accepted the skin graft from the B6 donors, but completely rejected the skin grafts from the C3H and BALB/c donors. This finding indicates that both CD8+ killer and CD4+ helper T cells were functionally mature in the recipients. Furthermore, the functional competence of both cellular and humoral immunity in the recipients that rejected the skin grafts from the C3H and BALB/c donors was determined by evaluating alloreactive T cell and antibody responses against H-2k and H-2d in an in vitro experiment.

Conclusion:
This study showed the promising potential of transplantation of mixed UCBCs for achieving high survival rates and functional immune reconstitution. We found that only MHC-matched HSCs were involved in hematopoiesis in the recipients' bone marrow. This finding suggests the presence of a surveillance system in bone marrow with the ability to distinguish self from non-self with different MHC antigens.

Disclosures:
No relevant conflicts of interest to declare.