Chronic antibody-mediated rejection due to anti-human leukocyte antigen (HLA) antibodies (Abs) is an important problem in organ transplantation. The spleen has been reported to be the source of donor-specific antibody (DSA)-producing cells in the anti-donor Ab-producing mouse model. However, the source of anti-HLA Ab-producing cells has not been identified in most humanized mouse models. Therefore, to investigate such anti-HLA Ab-producing cells, we developed an anti-HLA Ab-producing humanized mouse model.

In the first mouse model, severely immunodeficient (NSG) mice were humanized by injection of human cord blood- or bone marrow-derived hematopoietic stem cells (HSCs). After the appearance of T, B, and myeloid cells in the peripheral blood of the humanized mice, peripheral blood mononuclear cells (PBMCs) derived from healthy volunteers were injected in the mice as allo-antigens. Allo-PBMCs were examined by using flow cytometry to distinguish HLA-Ab types between the recipient and donor cells. Before injection of allo-PBMCs, human total IgG and anti-HLA Abs were not detected in the sera of all the mice (n = 32). At 2–4 weeks after injection, we measured human total IgG levels in sera by using the cytometric bead array and anti-HLA class I antibody levels in sera by using a multiple antigen beads-based assay (WAKFlow^R). Non-DSA anti-HLA-Abs were detected in 9 of the 32 mice; however, no DSA were detected. Although the detected anti-HLA Abs were not a donor-specific or cross-reactive group (CREG), we succeeded in developing a humanized mouse model that produced anti-HLA Abs.

In the second mouse model, the same approach was used in naïve NSG mice (n = 34) humanized by injection of PBMCs and then injected with allo-PBMCs. DSA anti-HLA-Abs were detected in 4 mice, and non-DSA HLA-Abs were detected in 16 mice. Among 3 of 4 mice positive for DSA and 14 of 16 mice positive for non-DSA, recipient and donor PBMCs were mix-cultured with human-CD40 ligand expressing mouse feeder cells for 3 days before injection to NSG mice. Thus, the interaction of CD40-CD40L for activation of B cells may be the key mechanism of producing anti-HLA-Abs in a mouse model.

We are now developing a humanized mice model that can produce anti-HLA-Abs and believe that the successful development of such anti-HLA Ab-producing humanized mouse models will enable further development of in vivo patient-specific therapies, including DSA desensitization, for use during organ transplantation.

CITATION INFORMATION: Yanagawa S, Tahara H, Ohdan H. Development of an Anti-HLA Antibody-Producing Humanized Mouse Model. Am J Transplant. 2017;17 (suppl 3).

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