[Background] Lymphocyte depletion prior to transplantation is beneficial in reducing initial maintenance immunosuppression in transplantation. However, despite its excellent efficacy in controlling T cell mediated acute rejection, lymphocyte depletion may promote a humoral response. While T cell repopulation after depletion has been evaluated in many aspects, the B cell response has not been fully elucidated.

[Method] To address this issue, we treated humanized CD52 transgenic mice with alemtuzumab with or without anti-LFA-1 mAb and monitored DSA, allospecific B cells, and cardiac allograft vasculopathy.

[Results] Alemtuzumab induction promoted serum DSA, allo-B cells, and CAV in humanized CD52 transgenic mice after heterotopic heart transplantation. Additional anti-LFA-1 mAb treatment resulted in no changes in graft survival or beating quality compared to alemtuzumab treatment alone. However, DSA production was greatly reduced (Fold increase 4.75±6.9 vs. 0.7±0.5; p<0.01) at POD100 with LFA-1 blockade. In addition, LFA-1 blockade significantly reduced allo-B cells in the spleen at POD 100 (0.07±0.06 vs.0.006±0.002 %; p<0.01), neo-intimal hyperplasia (56±14% vs. 23±13%; p<0.05), arterial disease (77.8±14.2 vs. 25.8±20.1%; p<0.05), and fibrosis (15±23.3 vs. 4.3±1.65%; p<0.05) in this alemtuzumab-induced chronic antibody-mediated rejection (CAMR) model. Surprisingly, serum IL-21 levels were significantly increased in alemtuzumab-treated mice compared to syngeneic controls (8005.3±9766.3 vs. 2.0±6.4 pg/ml; p<0.001), and LFA- blockade reduced the levels of IL-21(8005.3±9766.3 vs. 317.838 pg/ml; p<0.001). In accordance with the increased serum IL-21 level, alemtuzumab treated mice showed hyperplastic germinal center (GC) development, while the supplemental anti-LFA-1 mAb significantly reduced the GC frequency (55±26 vs. 6.2±12% as of PNA⁺ B cell follicle; p<0.01) and size (28.7±18.8 vs. 0.46±1.0% PNA⁺ area; p<0.01).

[Conclusion] We report that the incomplete T cell depletion inside of the GC leads to a systemic IL-21 dominant milieu with hyperplastic GC formation and CAMR. Conventional immunosuppression, such as tacrolimus and rapamycin, failed to reverse AMR, while co-stimulation blockade with LFA-1 corrected the GC hyperplastic response. The identification of IL-21 driven chronic AMR elucidates a novel mechanism that suggests a therapeutic approach with cytolytic induction.

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