editing fails to remove self-specificity, as may be caused by dnRAG1 expression, the cell may acquire a B1-like phenotype in the spleen and persist there. B cells with less innocuous anti-self specificities, such as Akt inhibitor anti-dsDNA, may not be tolerated in the splenic B1 compartment if editing fails to rescue autoreactivity, but may instead undergo deletion or be sequestered in the MZ compartment.55 If this model is correct, then distinguishing and enumerating putative splenic B1 subsets that arise through lineage-specified or selection-induced mechanisms would be an important focus of future efforts. Although this model provides a reasonable explanation for why splenic B1-like B cells accumulate in dnRAG1 mice, we cannot fully exclude the possibility that alternative, more complicated, scenarios might cause the learn more same outcome. For example, because full-length RAG1 has been shown to interact with other cellular factors and may function as an E3 ubiquitin ligase,60
dnRAG1 expression may cause sequestration or mis-regulated ubiquitylation of cellular factors involved in the V(D)J recombination process, potentially altering the physiology of the cell in a way that promotes differentiation toward a B1-like phenotype. Alternatively, a recurrent illegitimate V(D)J recombination event may be generated during the coincident expression of endogenous RAG1 and transgene-encoded dnRAG1 that promotes splenic B1 B-cell differentiation. However, the failure of splenic B1-like
B cells to accumulate in DTG mice PAK5 expressing both the dnRAG1 and 56Rki transgene is not easily explained in either of these scenarios. Moreover, the latter possibility seems unlikely because we do not detect a recurrent DNA rearrangement involving the heavy or light chain loci by Southern blotting of splenic DNA prepared from dnRAG1 mice (data not shown), but these results cannot fully rule out the possibility that non-immunoglobulin loci are targets of inappropriate V(D)J rearrangement in dnRAG1 mice. For this reason, we currently favour the simpler explanation that dnRAG1 expression interferes with secondary rearrangements associated with receptor editing. This work was supported by grants from the Health Future Foundation, the Nebraska LB506 Cancer and Smoking Disease Research Program, and the Nebraska LB692 Biomedical Research Program. This investigation was conducted in a facility constructed with support from the Research Facilities Improvement Program of the NIH National Center for Research Resources (C06 RR17417-01). The authors declare no conflict of interest. FIGURE S1. dnRAG1 mice bred onto a RAG1-deficient background fail to develop mature lymphocytes.