Figure 4
Figure 4. Requirement for nonhematopoietic CR1/CR2 for maximal anti-Gal responses and for suppression of anti-αGal Ab production via mixed chimerism induction. (A,D) ELISA assays. Serum after secondary nonmyeloablative conditioning of control and mixed chimeric (BALB/c secondary BMT donors) GalT−/−Cr2+/+ → GalT−/−Cr2+/+ (S+H+, groups 1 and 2), GalT−/−Cr2−/− → GalT−/−Cr2−/− (S−/H−, groups 3 and 4), GalT−/−Cr2−/− → GalT−/−Cr2+/+ (S+H−, groups 5 and 6), and GalT−/−Cr2+/+ → GalT−/−Cr2−/− (S−/H+, groups 7 and 8) radiation chimeras (where S denotes the Cr2 gene status of the nonhematopoietic stromal compartment and H denotes the Cr2 gene status of the hematopoietic compartment) was tested 8 days after sensitization with RRBCs for anti-αGal IgM (A) and IgG (D). Increased titers of anti-αGal IgM and IgG were detected following immunization in conditioned S+/H+ and S+/H− mice as well as conditioned S−/H− and S−/H+ mice. Levels of anti-αGal IgM Ab were significantly reduced in S+/H+ and S+/H− allogeneic BMT recipients compared with conditioned controls but not in S−/H− and S−/H+ recipients. *Statistically significant differences. There were 4 mice in group 1, 6 in group 2, 4 in group 3, 3 in group 4, 3 in group 5, 3 in group 6, 2 in group 7, and 3 in group 8. Data shown are mean ± SD. (B-C) ELISpot assays. Spleen cells (B) and PerC cells (C) from secondary conditioned and αGal+ BALB/c BMT GalT−/−Cr2−/−/GalT−/− radiation chimeras were tested by ELISpot assay 8 days after RRBC sensitization. PerC cells were cultured with LPS for 8 days before assessment in ELISpot assay. Anti-Gal Ab–producing cells were detected among splenocytes from conditioned control but not αGal+ BMT S+/H+ and S+/H− recipient mice. Anti-Gal Ab–producing B cells were also detected in conditioned S−/H− and S−/H+ recipient mice, but their numbers were not reduced by the presence of αGal+ mixed chimerism in S−/H− and S−/H+ recipient mice. *Statistically significant differences. The spot numbers obtained from BSA-coated plates were subtracted from those obtained from Gal-BSA–coated plates. The average frequencies against BSA were 10.1 ± 6.2, 0.1 ± 0.3, 2.3 ± 1.7, 1.8 ± 1.1, 6.2 ± 6.1, 0.3 ± 0.5, 6.2 ± 2.4, 4.2 ± 0.3, and (C) 16.7 ± 6.4, 1.0 ± 1.3, 12.7 ± 5.1, 9.0 ± 4.3, 17.5 ± 6.5, 0.3 ± 0.5, 10.1 ± 2.0, 9.8 ± 2.0 spots per million in groups 1 to 8, respectively. There were 5 mice in group 1, 8 in group 2, 4 in group 3, 5 in group 4, 3 in group 5, 3 in group 6, 2 in group 7, and 2 in group 8. Data shown are mean ± SD.

Requirement for nonhematopoietic CR1/CR2 for maximal anti-Gal responses and for suppression of anti-αGal Ab production via mixed chimerism induction. (A,D) ELISA assays. Serum after secondary nonmyeloablative conditioning of control and mixed chimeric (BALB/c secondary BMT donors) GalT−/−Cr2+/+ → GalT−/−Cr2+/+ (S+H+, groups 1 and 2), GalT−/−Cr2−/− → GalT−/−Cr2−/− (S−/H−, groups 3 and 4), GalT−/−Cr2−/− → GalT−/−Cr2+/+ (S+H−, groups 5 and 6), and GalT−/−Cr2+/+ → GalT−/−Cr2−/− (S−/H+, groups 7 and 8) radiation chimeras (where S denotes the Cr2 gene status of the nonhematopoietic stromal compartment and H denotes the Cr2 gene status of the hematopoietic compartment) was tested 8 days after sensitization with RRBCs for anti-αGal IgM (A) and IgG (D). Increased titers of anti-αGal IgM and IgG were detected following immunization in conditioned S+/H+ and S+/H− mice as well as conditioned S−/H− and S−/H+ mice. Levels of anti-αGal IgM Ab were significantly reduced in S+/H+ and S+/H− allogeneic BMT recipients compared with conditioned controls but not in S−/H− and S−/H+ recipients. *Statistically significant differences. There were 4 mice in group 1, 6 in group 2, 4 in group 3, 3 in group 4, 3 in group 5, 3 in group 6, 2 in group 7, and 3 in group 8. Data shown are mean ± SD. (B-C) ELISpot assays. Spleen cells (B) and PerC cells (C) from secondary conditioned and αGal+ BALB/c BMT GalT−/−Cr2−/−/GalT−/− radiation chimeras were tested by ELISpot assay 8 days after RRBC sensitization. PerC cells were cultured with LPS for 8 days before assessment in ELISpot assay. Anti-Gal Ab–producing cells were detected among splenocytes from conditioned control but not αGal+ BMT S+/H+ and S+/H− recipient mice. Anti-Gal Ab–producing B cells were also detected in conditioned S−/H− and S−/H+ recipient mice, but their numbers were not reduced by the presence of αGal+ mixed chimerism in S−/H− and S−/H+ recipient mice. *Statistically significant differences. The spot numbers obtained from BSA-coated plates were subtracted from those obtained from Gal-BSA–coated plates. The average frequencies against BSA were 10.1 ± 6.2, 0.1 ± 0.3, 2.3 ± 1.7, 1.8 ± 1.1, 6.2 ± 6.1, 0.3 ± 0.5, 6.2 ± 2.4, 4.2 ± 0.3, and (C) 16.7 ± 6.4, 1.0 ± 1.3, 12.7 ± 5.1, 9.0 ± 4.3, 17.5 ± 6.5, 0.3 ± 0.5, 10.1 ± 2.0, 9.8 ± 2.0 spots per million in groups 1 to 8, respectively. There were 5 mice in group 1, 8 in group 2, 4 in group 3, 5 in group 4, 3 in group 5, 3 in group 6, 2 in group 7, and 2 in group 8. Data shown are mean ± SD.

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