Figure 2
Failure of tolerance induction of anti-Gal–producing B cells inGalT−/−Cr2−/− mice. (A) Anti-αGal IgM titers of GalT−/− or GalT−/−Cr2−/− mice receiving conditioning alone or conditioning with wild-type BMT. Serum levels of anti-αGal IgM were measured in GalT+/+ control mice, conditioned (control; ▴, n=5) or chimeric (BMT; ▵, n=5) GalT−/− mice, and conditioned (control; ▪, n=4) or chimeric (BMT; □, n=6) GalT−/−Cr2−/− mice 6 weeks after BMT and at 10 weeks, 8 days following immunization with RRBCs. P < .05 comparing chimeric and control GalT−/−; P > .05 (NS) comparing chimeric and control GalT−/−Cr2−/− mice, chimeric GalT−/− and GalT−/−Cr2−/− mice, or conditioned GalT−/− and GalT−/−Cr2−/− mice at 6 weeks after BMT. P > .05 comparing chimeric and control GalT−/−Cr2−/− mice after rabbit RRBC immunization. P < .02 comparing chimeric GalT−/−Cr2−/− mice and GalT−/− mice and comparing conditioned GalT−/−Cr2−/− mice and GalT−/− mice after RRBC immunization. Data shown are individual animals in a single experiment. (B) ELISpot assay was used to detect anti-αGal (IgM + IgG)–producing cells. PerC cells prepared from conditioned and chimeric GalT−/− mice (□, n=3; ▪, n=4; respectively) and conditioned and chimeric (BMT) GalT−/−Cr2−/− mice (▵, n=3; ▴, n=4; respectively) were cultured with LPS for 8 days and used in ELISpot assays. P < .001 comparing chimeric and control GalT−/− mice (†) and P < .05 comparing chimeric GalT−/− and GalT−/−Cr2−/− mice (*) at 8 × 105 and 1.6 × 105 cells-per-well dilutions. There was no statistical difference between chimeric and control GalT−/−Cr2−/− mice (‡) at the 8 × 105 cells-per-well dilution, but P < .05 at 1.6 × 105 cells per well. The frequencies obtained from BSA-coated plates were subtracted from those obtained with Gal-BSA–coated plates. The average frequencies of spot-forming cells detected on BSA-coated plates at 8 × 105 cells per well were 5.2 ± 3.0, 7.1 ± 1.5, 12.6 ± 4.2, and 10.0 ± 5.0 in BMT GalT−/−, BMT GalT−/−Cr2−/−, conditioned GalT−/−, and conditioned GalT−/−Cr2−/− groups, respectively. Data shown are mean ± SD in each group.

Failure of tolerance induction of anti-Gal–producing B cells inGalT−/−Cr2−/− mice. (A) Anti-αGal IgM titers of GalT−/− or GalT−/−Cr2−/− mice receiving conditioning alone or conditioning with wild-type BMT. Serum levels of anti-αGal IgM were measured in GalT+/+ control mice, conditioned (control; ▴, n=5) or chimeric (BMT; ▵, n=5) GalT−/− mice, and conditioned (control; ▪, n=4) or chimeric (BMT; □, n=6) GalT−/−Cr2−/− mice 6 weeks after BMT and at 10 weeks, 8 days following immunization with RRBCs. P < .05 comparing chimeric and control GalT−/−; P > .05 (NS) comparing chimeric and control GalT−/−Cr2−/− mice, chimeric GalT−/− and GalT−/−Cr2−/− mice, or conditioned GalT−/− and GalT−/−Cr2−/− mice at 6 weeks after BMT. P > .05 comparing chimeric and control GalT−/−Cr2−/− mice after rabbit RRBC immunization. P < .02 comparing chimeric GalT−/−Cr2−/− mice and GalT−/− mice and comparing conditioned GalT−/−Cr2−/− mice and GalT−/− mice after RRBC immunization. Data shown are individual animals in a single experiment. (B) ELISpot assay was used to detect anti-αGal (IgM + IgG)–producing cells. PerC cells prepared from conditioned and chimeric GalT−/− mice (□, n=3; ▪, n=4; respectively) and conditioned and chimeric (BMT) GalT−/−Cr2−/− mice (▵, n=3; ▴, n=4; respectively) were cultured with LPS for 8 days and used in ELISpot assays. P < .001 comparing chimeric and control GalT−/− mice (†) and P < .05 comparing chimeric GalT−/− and GalT−/−Cr2−/− mice (*) at 8 × 105 and 1.6 × 105 cells-per-well dilutions. There was no statistical difference between chimeric and control GalT−/−Cr2−/− mice (‡) at the 8 × 105 cells-per-well dilution, but P < .05 at 1.6 × 105 cells per well. The frequencies obtained from BSA-coated plates were subtracted from those obtained with Gal-BSA–coated plates. The average frequencies of spot-forming cells detected on BSA-coated plates at 8 × 105 cells per well were 5.2 ± 3.0, 7.1 ± 1.5, 12.6 ± 4.2, and 10.0 ± 5.0 in BMT GalT−/−, BMT GalT−/−Cr2−/−, conditioned GalT−/−, and conditioned GalT−/−Cr2−/− groups, respectively. Data shown are mean ± SD in each group.

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