#OA2-AM25-ST-28 - Low Affinity RBC Autoreactive B Cells Escape Deletion in the Bone Marrow of Mice

Loss of tolerance to self-antigens on RBCs can result in autoimmune hemolytic anemia (AIHA).  B cells express their particular antibody as a surface receptor (B cell receptor (BCR)) and are deleted during development in the bone marrow if their BCR reacts strongly with self-antigen. Murine models have shown robust deletion of RBC autoreactive B cells during development in the bone marrow, but used methods that only detect high affinity autoreactive B cells.  In contrast, studies in mice with normal immune systems show that RBC autoreactive B cells are present and can be simulated into IgG secreting plasma cells.  As a resolution to this apparent contradiction, we hypothesized low affinity RBC autoreactive B cells escape deletion in the bone marrow.

Study

Design/Methods: IgHEL mice are BCR transgenic such that every B cell has a fixed specificity for hen egg lysozyme (HEL).  The widely used HOD transgenic mouse expresses an RBC specific model antigen (HOD) that contains HEL and is reactive with the IgHEL BCR with a high affinity.  Three new transgenic mice were generated with specific mutations in the HEL portion of the HOD transgene that lowered the affinity for IgHEL 80, 13000, or 620000 fold, respectively.  Each mouse (named for their affinity: HODHi, HODMed, HODLo) were crossed with IgHEL mice to generate an “RBC autoreactive” animal.  B cell development was measured in each crossed strain by flow cytometry in the bone marrow and spleen and compared to IgHEL mice (no HOD) as a baseline for normal B cell development. 

Results/Findings: Compared to IgHEL mice with no autoantigen, the total number of B cells in the bone marrow decreased (2.4 fold, < 0.0001) in HODHi and (2.2 fold, < 0.0001) in HODMed mice, respectively.  In both strains a major decrease was observed in the transition from Pro B cells into Mature B cells (a known point for deletion of autoreactive B cells). In contrast, there was no significant difference in the number of B cells in HODLo mice and no decrease in the Pro B cell transition to Mature B cells.  However, compared to IgHEL mice, HODLo autoreactive mice showed a 4.9 fold increase in the splenic T3 B cell population (p value 0.0002); no significant difference were seen in T3 B cells in the other strains. A total of 4-8 mice were tested for each strain in several batches and at different times.  Age and sex of mice was equivalent in all groups. 

Conclusions: The current findings demonstrate that low affinity RBC autoreactive B cells escape deletion.  Such cells could differentiate into high affinity autoantibody secreting cells through normal B cell affinity maturation.  The finding of increased T3 B cells only in HODLo autoreactive mice is of potential importance since T3 B cells can represent a negative regulatory population that actively suppresses autoimmunity and future studies should test this hypothesis.