Figure 1
Persistent donor NK-cell alloreactivity toward host B cells after haploidentical BMT. (A,B) Haploidentical BMT results in a significant decrease in OVA-specific B cells and anti-OVA antibodies. DBA/1 mice were immunized with OVA, and were either left untreated or were subjected to nonmyeloablative conditioning consisting of low-dose total body irradiation (6 Gy) and a single injection of 0.5 mg of anti-CD40L antibodies (MR1), followed by transplantation with 107 syngeneic, haploidentical (F1→P) or fully MHC-mismatched BM cells from DBA/1, (DBA/1 x C57BL/6) F1 or C57BL/6 donor mice, respectively. The percentage of OVA-specific B cells in spleen was analyzed 2 weeks after BMT. Compared to syngeneic controls, both haploidentical (F1→P) and fully MHC-mismatched BMT recipients showed a significant decrease in the percentage of OVA-specific B cells (P = .008 and P = .0002, respectively). Sera were taken before and after BMT, and tested by ELISA for the presence of anti-OVA IgG2a antibodies. At the start of treatment, no differences in anti-OVA antibody levels were observed between the groups of mice. After treatment, all groups displayed a decrease in anti-OVA antibodies; syngeneic BMT-treated mice showed a decrease in OVA-specific antibody levels of 35%, similar to that of controls (32%), while both haploidentical (F1→P) and fully MHC-mismatched BMT resulted in a strong decrease in antibodies against OVA of 78% (P = .01) and 89% (P = .002), respectively. As negative control, naive mice were analyzed, while immunized mice served as positive control. (N.S. = statistically not significant.) (C) Donor NK cells persistently eradicate host immune cells after haploidentical BMT. At 3 months after BMT, chimeric C57BL/6 mice treated with haploidentical (F1→P) BMT from (BALB/c x C57BL/6) F1 were injected with 107 CFSE-labeled splenocytes (containing 50% to 60% B cells) from syngeneic (CFSEdim) and haploidentical (CFSEhigh) mice mixed at a 1:1 ratio. Before adoptive transfer, NK cells in recipient mice were depleted with 0.25 mg anti-NK1.1 antibodies (PK136), while PBS was used as control. At day +7 after adoptive transfer, the presence of CFSE+ cells in peripheral blood was analyzed by FACS. Error bars indicate mean ± SEM.

Persistent donor NK-cell alloreactivity toward host B cells after haploidentical BMT. (A,B) Haploidentical BMT results in a significant decrease in OVA-specific B cells and anti-OVA antibodies. DBA/1 mice were immunized with OVA, and were either left untreated or were subjected to nonmyeloablative conditioning consisting of low-dose total body irradiation (6 Gy) and a single injection of 0.5 mg of anti-CD40L antibodies (MR1), followed by transplantation with 107 syngeneic, haploidentical (F1→P) or fully MHC-mismatched BM cells from DBA/1, (DBA/1 x C57BL/6) F1 or C57BL/6 donor mice, respectively. The percentage of OVA-specific B cells in spleen was analyzed 2 weeks after BMT. Compared to syngeneic controls, both haploidentical (F1→P) and fully MHC-mismatched BMT recipients showed a significant decrease in the percentage of OVA-specific B cells (P = .008 and P = .0002, respectively). Sera were taken before and after BMT, and tested by ELISA for the presence of anti-OVA IgG2a antibodies. At the start of treatment, no differences in anti-OVA antibody levels were observed between the groups of mice. After treatment, all groups displayed a decrease in anti-OVA antibodies; syngeneic BMT-treated mice showed a decrease in OVA-specific antibody levels of 35%, similar to that of controls (32%), while both haploidentical (F1→P) and fully MHC-mismatched BMT resulted in a strong decrease in antibodies against OVA of 78% (P = .01) and 89% (P = .002), respectively. As negative control, naive mice were analyzed, while immunized mice served as positive control. (N.S. = statistically not significant.) (C) Donor NK cells persistently eradicate host immune cells after haploidentical BMT. At 3 months after BMT, chimeric C57BL/6 mice treated with haploidentical (F1→P) BMT from (BALB/c x C57BL/6) F1 were injected with 107 CFSE-labeled splenocytes (containing 50% to 60% B cells) from syngeneic (CFSEdim) and haploidentical (CFSEhigh) mice mixed at a 1:1 ratio. Before adoptive transfer, NK cells in recipient mice were depleted with 0.25 mg anti-NK1.1 antibodies (PK136), while PBS was used as control. At day +7 after adoptive transfer, the presence of CFSE+ cells in peripheral blood was analyzed by FACS. Error bars indicate mean ± SEM.

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