Mechanisms of B Cell Tolerance after in Utero Hematopoietic Cell Transplantation

Background: In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative, nonimmunosuppressive transplant approach that results in donor cell engraftment across immune barriers. Although a significant amount of work has investigated the fate of T cells following IUHCT, little attention has been paid to B cell tolerance and the fate of donor derived host reactive or host derived donor reactive B cells following IUHCT. B cell tolerance is broadly believed to occur by a combination of 3 mechanisms: deletion, receptor editing, and functional inactivation (anergy). In the current study we attempt to elucidate the mechanism(s) by which B cell tolerance occurs following IUHCT.

Methods: 10x10⁶ donor bone marrow (BM) cells were injected intravenously via the vitelline vein into gestational day 14 murine fetuses. IUHCT was performed in the congenic (C57Bl/6-GFP [H2K^b] into C57Bl/6 [H2K^b]) and allogeneic (C57Bl/6-GFP into Balb/c [H2K^d]) strain combinations. Naive Balb/c and C57Bl/6 mice served as controls. Mice were sacrificed at day of life 3 (P3), 1 month and 4 months of age at which time their BM, spleen, and serum were harvested. To assess B cell deletion, flow cytometry was used to determine the absolute # and % of host and donor immature and pre B cells in the BM. Additionally, apoptosis of host and donor BM derived B cells was determined by annexin staining. Central receptor editing was evaluated using RT qPCR to measure the amount of Vκ-RS rearrangments in BM pre-B cells. Peripheral receptor editing was studied by calculating the % of λ light chains in mature splenocytes identified by flow cytometry.  Finally, functional inactivation of donor reactive host B cells was assessed by measuring anti-H2K^b serum antibodies (ab) of allogeneic chimeras, naive, and immunized mice at 1 month of age.

Results: The absolute number of BM immature B cells was decreased in allogeneic recipients of IUHCT compared to noninjected Balb/c controls at 1 month of age (fig 1). This effect was lost by 4 months of age. The decrease in B cells resulted primarily from a decrease in immature donor as opposed to host B cells compared to controls (% immature donor B cells in allogeneic recipients vs. controls: 16.2% vs. 39.9%; p<0.0005). Donor B cells in allogeneic chimeras also demonstrated a trend toward increased apoptosis compared to controls (24.8 vs. 18.7%; p=0.2) which was not seen in immature host B cells (18.3 vs. 18.6%; p=0.9). There was no significant decrease in the absolute number of immature B cells or increased apoptosis in congenic recipients compared to uninjected controls. These findings suggest deletion of autoreactivedonor B cells.

Light chain receptor editing involves rearrangements within the κ and λ gene loci and may occur in BM pre-B cells or mature B cells in the spleen. We found no difference in the Vκ-RS rearrangements of pre B cells in allogeneic chimeras and controls at 1 month. In contrast, the quantity of total λ⁺ mature splenic B cells was increased in allogeneic chimeras at P3 (10.8 vs. 8.4%; p=0.02) and resulted from an increased host λ⁺ % compared to controls (10.8 vs. 8.4% p=0.03) suggesting peripheral receptor editing of host cells (fig.2). The λ⁺% increase in allogeneic chimeras was lost by 1 month.

Autoreactive B cells that escape deletion and receptor editing can be functionally inactivated. Neither allogeneic nor naive mice developed ab to H2K^b splenocytes, however, Balb/c mice immunized to H2k^b antigen showed high ablevels (MFI fold change: allo-0.89 naive-1.37 imm-2.77; p<0.05).

Conclusion:

B cell tolerance after IUHCT is achieved by distinct mechanisms for host and donor cell populations. Donor derived host reactive B cells undergo deletion and apoptosis while receptor editing and functional inactivation are the primary mechanisms of B cell tolerance of host derived donor reactive B cells. We hope use this and future studies of antigen specific B cell tolerance to harness the immunologic potential of IUHCT for many hematopoietic and immunologic congenital diseases.