Background Donor-specific antibodies (DSA) generated in recall responses are produced by long-lived plasma cells (L-PC) residing in the bone marrows (BM). Mechanisms of L-PC activation, colony expansion and niche of antibody secretion, however, are poorly understood. Here we report on a work to define recall DSA secreting cells in the spleens.

Methods A mouse model of allosensitization employing a C57BL/j6 mouse as recipient of skin graft from C57BL/j6-tg-HLA.A2 mouse is used. To induce recall antibody response pre-sensitized recipient is re-immunized with a 2^nd skin allograft at day 90 Ptx. Serum DSA IgG levels are measured in a flow-cytometric antibody binding assay. DSA secretion by PC is studied in a mixed cell culture antibody binding assay, which measures DSA secreted by the spleen/BM cells binding to HLA.A2 expressed on the target cells (CCL120.1). B cell and PC in the spleens and the BM are examined in multiparameter flow cytometry. CD138⁺ cells in the spleens are studied in an immunofluorescent microscopy with fluorescent imaging analysis.

Results Serum DSA IgG levels significantly increased (p<0.01 vs. control) at day 7 following re-sensitization, indicating a rapid development of recall IgG response. High levels of DSA IgG binding to the HLA.A2+ target cells (49.4+-24 MFI at day 14) were present in splenocyte cultures as compared to that from the BM cell cultures (23.4+-7.4 MFI, p=0.02). Increases in CD38^dimCD138⁺B220^– plasma cell population (p<0.05) in the spleens, but not in the BM were detected in FACS analysis. Immunofluorescent microscopy found a significant increase in CD38^dimCD138⁺ plasma cells in the peri arteriolar sheet (PALS) of the spleens, indicating migration of PCs from the blood. In addition, treatment with the Bruton tyrosine kinase inhibitor (ibrutinib) significantly reduced the CD138⁺ population (p<0.01 vs. control) in the spleens and suppressed DSA IgG in the blood (p=0.05).

Conclusion Allosensitization led to expansion of CD38^dimCD138⁺ cells in the PALS, which correlated with increased DSA IgG secretion by splenic PC during recall responses. The results suggest that the spleen is an important niche for colony expansion of long-lived plasma cells and recall antibody production. Thus, peripheral lymphoid organs such as the spleen may well serve as a pharmaceutical target for recall alloantibody suppression.

CITATION INFORMATION: Kim I, Chai N.-N, Klein A, Jordan S, Wu G. Recall Alloantibody Production Is Driven by CD138⁺ Cells Proliferating in the Spleens: An Indication for Long-Lived Plasma Cell Activities and Potential Therapeutic Target. Am J Transplant. 2016;16 (suppl 3).

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