[Background] Donor specific antibody (DSA) has been causally linked to chronic rejection that accounts for the failure of 20-40% of organ transplants. However, the role of T reg cells on de novo allo-B cell activation and DSA formation has not been fully elucidated.

[Methods] To investigate the role of CD4 T cell subsets on de novo B cell activation after T cell depletion, we further depleted CD4⁺ or CD25⁺ T cells a week after alemtuzumab treatment using anti-CD4 (GK1.5, 100Μg) or CD25 (PC61, 500Μg) mAbs, respectively. In order to test the effect of Treg, either 5×10⁵ CD4⁺CD25⁺ T cells were adoptively transferred or treated with IL-2/IL-2 mAb (JES 6.1) complex for Treg in vivo induction.

[Results] CD52Tg cardiac allograft recipients treated with alemtuzumab alone (n=11) or in conjunction with anti-CD4 (n=6) or CD25 mAbs (n=15) did not show any acute rejection. Alemtuzumab successfully suppressed DSA production in the early phase (2-6 wks) while half of recipients showed elevated DSA production in later phase (7–14 wks). Counterintuitively, recipients treated with additional CD4 mAb showed elevated DSA level at day 50 (MFI fold increase; 3.74±0.88 vs. 2.28±0.88; p=0.03) compared to alemtuzumab control. Additional anti-CD25mAb promoted increased DSA production at 7 weeks (8.36±8.01 vs. 2.29±2.42; p<0.001) and showed significantly increased allo-B cell (H-2K^b/D^b tetramer⁺IgD^–) at day 50 (0.18±0.20 vs. 0.013±0.002%; p<0.05) compared to alemtuzumab control suggesting an early regulatory role of Tregs in this model. However, adoptive transferred T reg cells does not significantly reduce splenic allo-B cell at day 100 (0.025±0.03 vs. 0.075±0.06) and DSA production (2.33±1.46 vs.2.36±1.56). On the other hand, IL-2/IL-2 mAb complex treated recipients showed reduced allo-B cell (0.018± 0.023 vs. 0.075±0.06; p<0.05) as well as DSA production (1.25±0.84 vs. 2.36±1.56; p<0.05) compared to alemtuzumab alone treatment.

[Conclusions] This study showed a regulatory role of CD4 cells on humoral response in alemtuzumab induced AMR model. Depleting CD4 cells or CD25 cell during T cell repopulation interferes with T reg cells contributing to the suppression of early DSA production. In vivo Treg induction showed the possible capacity of Treg on humoral response. However, it may also represent the importance of the Treg prone environment (High IL-2) on suppressing the humoral response since exogenous Treg does not suppress later allo-B cell and DSA production.

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