MTOR inhibitors [mTORi; Rapamycin, (Rapa)] and calcineurin inhibitors [CNi; Tacrolimus (Tac)] are immunosuppressive drugs commonly used to suppress the rejection of transplanted organs and tissues. Despite their widespread use following transplantation (Tx), the individual effects of these immunosuppressive therapies on alloantibody (alloAb) production postTx is unknown. Our data shows mTORi inhibits postTx alloAb significantly more than CNi. However, the specific cellular targets through which these drugs regulate alloAb production are unclear. Our lab has published a novel mechanism in which CD8⁺ T cells downregulate postTx alloAb production by eliminating IgG1⁺ B cells. We hypothesize that one reason why CNi may be less efficient than mTORi for suppression of alloAb production, is that CNi may suppress CD8-mediated clearance of antibody-producing B cells (thereby resulting in increased alloAb levels). To investigate this hypothesis, we analyzed the effects of Rapa and Tac on alloprimed CD8⁺ T cell killing of alloAb-producing IgG1⁺ B cells. Using in vitro and in vivo cytotoxicity assays, we quantitated IgG1⁺ B cell clearance and corresponding alloAb production. In vitro, alloprimed CD8⁺ T cells were co-incubated with alloprimed IgG1⁺ B cells in the presence of Rapa, Tac, or vehicle control. Tac (10 mM; 9±0.3 lysis) significantly inhibited cytotoxicity as compared to both Rapa (10 mM; 12±1%) and DMSO control (12±0.3% lysis; p<0.02 for both). In the absence of CD8s, DMSO treated B cells exhibited baseline lysis (6±0.3%) and no additional lysis was observed with drugs alone. To test in vivo cytotoxicity, CD8 KO recipients were Tx and cohorts were adoptively transferred (AT) with CD8⁺ T cells (day 0; 10 million cells) and either untreated or treated with Tac (1mg/kg/day; day +6, +7). In vivo cytotoxicity was determined by relative B cell clearance using CFSE^hi stained syngeneic target alloprimed IgG1⁺ B cells and CFSE^lo syngeneic control naïve B220⁺ B cells as previously reported. CD8 KO recipients AT with CD8⁺ T cells induce cytotoxicity towards IgG1⁺ B cells (23.2±0.3%). However, treatment with Tac inhibited CD8-mediated in vivo cytotoxicity (6.6±0.2%, p<0.05). This difference correlated with alloAb levels in Rapa versus Tac treated recipients. These data suggest that Tac suppression of CD8-mediated regulatory mechanisms (CD8 killing of alloprimed B cells) results in higher postTx alloAb levels.

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