We have shown that i.v. exposure to MHC-mismatched donor PBMC using a novel, mild conditioning protocol results in B-cell tolerance with transient T-cell unresponsiveness. Here we show that B-cell tolerance is stable and allo-Ab cannot be induced with repeated exposure to donor cells.

MGH miniature swine conditioned with CD3-immunotoxin, low-dose (100cGy) total body irradiation (TBI), and a short, 45 day course of cyclosporine A, received cells i.v. from a haplo- or fully MHC-mismatched donor. Weekly immunophenotyping was performed. Hematopoietic cells from donor and third-party MHC-mismatched pigs were injected subcutaneously (s.c.) +/- complete Freund's adjuvant (CFA) monthly, starting 60 days following conditioning. Control animals were challenged in the same manner. The draining lymph nodes were analyzed by Histology and bulk RNAseq. Allo-Ab was measured by flow cytometry and complement dependent Ab mediated cellular cytotoxicity assay.

Phenotypic analysis of recipient cells during conditioning revealed that most cell populations, including CD4+ Foxp3+ Treg cells, were only partially depleted and quickly recovered to baseline. Allo-Ab responses were not detected even after repeated s.c. injections of donor cells. The addition of CFA provoked a transient, weak IgM response suggestive of an extrafollicular B-cell response with no germinal center development. Control animals consistently developed Allo-Ab responses against donor cells. Histologic analysis of draining lymph nodes shows similar morphology and presence of active Germinal centers in both groups. However, preliminary analysis reveals an abundance of FoxP3+ cells in tolerant animals. This suggests that donor cells trigger a germinal center reaction in tolerant animals that may be controlled by memory T-follicular regulatory cells. Further histologic and transcriptome analysis of draining LN cells are currently underway and may yield additional insights into the mechanical underpinnings.

Stable B-cell tolerance to donor antigens across fully MHC-mismatched barriers can be induced by a novel, mild conditioning protocol that does not result in prolonged T-cell depletion. We are currently investigating the mechanism of this phenomenon. Understanding immunomodulatory mechanisms involved in controlling B-cell responses to transplantation antigens could lead to significant advances in improving transplantation outcomes.

CITATION INFORMATION: Schuetz C., Climov M., Paril A., Wang Z., Andrews Roy A., Navarro Alvarez N., Matar A., Sachs D., Duran-Struuck R., Huang C. Immunomodulation of Alloantibody Responses Am J Transplant. 2017;17 (suppl 3).

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