High antigen levels do not preclude B-cell tolerance induction to alpha1,3-Gal via mixed chimerism

Abstract

Background: Studies of bone marrow transplantation (BMT) from wild-type mice or rats to alpha1,3-galactosyltransferase (GalT) knockout mice have demonstrated that induction of mixed chimerism tolerizes not only T cells, but also natural antibody-producing B cells, even across xenogeneic barriers. Given that rodent cells express lower levels of the alphaGal epitope than the more clinically relevant porcine species, the consequences of exposure to cells expressing high levels of alphaGal on the ability to induce B-cell tolerance are unknown.

Methods: The effects on chimerism and anti-alphaGal B-cell tolerance of an i.p. injection of 10(9) porcine RBC were evaluated in GalT knockout mice receiving wild-type allogeneic BMT after non-myeloablative conditioning with T-cell depleting monoclonal antibodies, thymic irradiation, and low-dose total body irradiation.

Results: Achievement of mixed chimerism and tolerance of anti-alphaGal-producing B cells was not affected by exposure to high-density alphaGal at the time of BMT. The absence of induced anti-alphaGal or anti-pig antibody responses in conditioned control mice suggested that the B-cell xeno-response to pig is T-cell-dependent.

Conclusion: High alphaGal density on pig cells might not preclude the ability to achieve tolerance of pre-existing alphaGal-reactive human B cells via induction of mixed chimerism. This strategy has the potential to induce B-cell tolerance to non-alphaGal epitopes, against which natural antibodies have been found in the sera of healthy humans.

Figure 1. Immunization of naïe GalT KO mice by i.p. injection of GalT KO or WT pig RBC

(A) FCM analysis of αGal expression on different cell types. Open histogram represents the isotype control, grey histogram represents IB4 stain. (B) Titer of anti-αGal IgM in the serum was calculated as described in Methods section (ELISA) and is shown before and after the immunization of GalT KO mice with either WT (●) or GalT KO (○) pig RBC. (C–D) Analysis of anti-xeno and anti-αGal IgM in the sera is shown over time in regard to the i.p. immunization. Anti-pig IgM and anti-αGal IgM binding on pig RBC were analyzed by FCM. Each symbol represents an individual animal. (C) WT (■) or GalT KO (□) pig RBC were incubated with sera collected from GalT KO mice immunized with WT pig RBC and counterstained with anti-IgM. Dotted line represents the MCF obtained when WT pig RBC were incubated with serum from a naive WT mouse. (D) WT (■) or GalT KO (□) pig RBC were incubated with sera from GalT KO mice immunized with GalT KO pig RBC and counterstained with anti-IgM. Dotted line represents the MCF obtained when WT pig RBC were incubated with serum from a naive WT mouse. One representative experiment of 2 is shown (n=23 animals/group/experiment).

Figure 2. Non-myeloablative conditioning regimen impairs the immunization of naïe GalT KO mice by WT or GalT KO pig RBC

GalT KO mice underwent conditioning as in the Methods section followed by i.p. injection with either WT (A) or GalT KO (B) pig RBC on Day 0. (A) WT (■) or GalT KO (□) pig RBC were incubated with sera collected from GalT KO mice immunized with WT pig RBC, counterstained with anti-IgM and analyzed by FCM. (B) WT (■) or GalT KO (□) pig RBC were incubated with sera collected from GalT KO mice immunized with GALT KO pig RBC, counterstained with anti-IgM and analyzed by FCM. Dotted line represents the MCF obtained when WT pig RBC were incubated with serum from a naive WT mouse.

Figure 3. Exposure to high levels of αGal does not impair establishment of mixed chimerism in GalT KO recipient mice

GalT KO mice underwent conditioning as described in Methods section, followed by i.v. injection of allogeneic GalT⁺ B10.A BMT (□) and i.p. injection of either WT (Δ) or GalT KO (▼) pig RBC on Day 0. Percentages of donor WBC are shown for the Mac-1⁺ (A), B220⁺ (B), CD4⁺ (C), and CD8⁺ (D) lineages at the indicated time points Mean (±SEM) of two experiments are shown with 8 to 13 animals per group

Figure 4. Exposure to high levels of αGal does not impair αGal-bearing-B cell tolerance after establishment of mixed chimerism in GalT KO recipient mice

(A) Chimeric mice (empty symbols) and conditioned control mice (filled symbols) were analyzed 17 to 19 weeks post-BMT, 8 days after i.p. injection with rabbit RBC. B cell tolerance was assessed in the spleen by Elispot assay revealing specific αGal IgM production. (B) Serum was collected from chimeric mice (white bars) or conditioned control mice (black bars) before (i.e. 16 or 18 weeks after BMT) (plain bars) and 8 days after i.p. immunization with rabbit RBC (i.e. 17 or 19 weeks post BMT) (hatched bars) and specific anti-αGal IgM titer was analyzed by Elisa. Sera from mixed chimeras mice receiving an i.p. injection of either WT (C) or GalT KO (D) pig RBC and BMT on D0 were analyzed by FCM at the indicated time points. WT (■) or GalT KO (□) pig RBC were incubated with sera and counterstained with anti-IgM-biotin and a streptavidin-conjugated fluorochrome.