Purpose: Occurring of donor specific antibodies (DSA) is injurious to organ transplant. Development of immunotherapies to suppress DSA, therefore, has been one of the strategic immunosuppression objectives. We recently demonstrated in a proof of concept study that a strong suppression of recall-type DSA is achievable by using a multi-targeting desensitization therapy. Here, we extend our observation to study the dynamic changes of peripheral B lymphocyte subsets induced by the therapy.

Methods: In a mouse model of allo-sensitization C57BL/j6 mice were initially sensitized with a HLA.A2⁺ skin graft. The recipient mice were re-sensitized 90 days later and received a multi-targeting therapy comprised of CD28 costimulatory pathway blockage with CTLA4Ig (abatacept), B cell depletion with anti-CD20 antibody and interleukin 6 signal interruption with anti-IL6 receptor (mMR16-1). Titers of DSA IgG antibodies were measured weekly in a flow cytometric antibody binding assay. Bone marrow cells and splenic mononuclear cells were stained with a panel of cell surface marker antibodies and analyzed in multi-perimeter FACS.

Results: The therapy resulted in DSA IgG suppression at day 7 (80+29 MFI), at day 14 (76+33MFI, p=0.012 vs. control, 332+106MFI) and day 21(73+31MFI, p=0.01 vs. control, 433+70MFI) post re-sensitization. Flow cytometric analysis of splenic lymphocytic cells harvested at Day 14 showed a dynamic change in B-cell subsets in the therapy group when compared to the untreated controls. B-cells (B220⁺) were reduced by 2.3-fold (p<0.01) after treatment with the 3 agents. While early transitional T1 B-cells (sIgM^strong CD93⁺/CD23^– ) did not change, the mature B-cells (CD45R⁺sIgD⁺) in the peripheral pool were reduced by 51-fold (p=0.001) and CD45R⁺/CD23⁺ B-cell subset reduced by 17.8-fold (p=0.001), suggesting a failure of peripheral B-cell maturation. Splenic CD38⁺/CD138⁺ plasma cells were significantly reduced (p=0.002) as well.

Conclusion: Strong suppression of recall DSA responses can be achieved by targeting peripheral B-cell development related pathways. The combined use of CD28 pathway blockade, B-cell deletion and IL6R signal disruption resulted in altered B-cell subset and plasma cell activity that also was manifest by significant reductions in the alloantibody (DSA) responses. This approach may have clinical significance in management of highly-HLA sensitized patients.

CITATION INFORMATION: Kim I., Wu G., Chai N-.N., Klein A., Jordan S. Multi-Targeting Desensitization Therapy Alters Peripheral B-Cell Homeostasis and Suppresses Recall Alloantibody Responses Am J Transplant. 2017;17 (suppl 3).

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