Accumulation of B1-like B cells in transgenic mice over-expressing catalytically inactive RAG1 in the periphery

Abstract

Summary During their development, B lymphocytes undergo V(D)J recombination events and selection processes that, if successfully completed, produce mature B cells expressing a non‐self‐reactive B‐cell receptor (BCR). Primary V(D)J rearrangements yield self‐reactive B cells at high frequency, triggering attempts to remove, silence, or reprogramme them through deletion, anergy induction, or secondary V(D)J recombination (receptor editing), respectively. In principle, expressing a catalytically inactive V(D)J recombinase during a developmental stage in which V(D)J rearrangement is initiated may impair this process. To test this idea, we generated transgenic mice expressing a RAG1 active site mutant (dnRAG1 mice); RAG1 transcript was elevated in splenic, but not bone marrow, B cells in dnRAG1 mice relative to wild‐type mice. The dnRAG1 mice accumulate splenic B cells with a B1‐like phenotype that exhibit defects in B‐cell activation, and are clonally diverse, yet repertoire restricted with a bias toward Jκ1 gene segment usage. The dnRAG1 mice show evidence of impaired B‐cell development at the immature‐to‐mature transition, immunoglobulin deficiency, and poorer immune responses to thymus‐independent antigens. Interestingly, dnRAG1 mice expressing the anti‐dsDNA 3H9H56R heavy chain fail to accumulate splenic B1‐like cells, yet retain peritoneal B1 cells. Instead, these mice show an expanded marginal zone compartment, but no difference is detected in the frequency of heavy chain gene replacement. Taken together, these data suggest a model in which dnRAG1 expression impairs secondary V(D)J recombination. As a result, selection and/or differentiation processes are altered in a way that promotes expansion of B1‐like B cells in the spleen.

Accelerated progression of chronic lymphocytic leukemia in Eμ-TCL1 mice expressing catalytically inactive RAG1

“…15 Em-TCL1 and dnRAG1 mice, 12 both on C57Bl/6 backgrounds, were crossed to generate cohorts of wild-type (WT), dnRAG1, Em-TCL1, and DTG mice as described in supplemental Methods (available on the Blood website). SCID mice (B6.CB17-prkdc scid / SzJ) were purchased from The Jackson Laboratory (Bar Harbor, ME).…”

“…We recently generated dnRAG1 mice expressing a catalytically inactive, but DNA-binding competent, form of RAG1 in the periphery which show evidence of impaired secondary V(D)J recombination that occurs in response to self-reactivity. 12 Interestingly, these animals develop a progressive, antigen-dependent, accumulation of CD5…”

“…12 The indolent accumulation of CD5 1 B cells in dnRAG1 mice is The online version of this article contains a data supplement.…”

“…15 The CLL cells emerging in Em-TCL1 mice phenotypically resemble those accumulating in dnRAG1 mice, but arise with delayed kinetics compared with dnRAG1 mice. 12,15 We hypothesized that if molecular defects which promote benign CD5 …”

“…cDNA was prepared from DNAse I-treated RNA using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA) and subjected to PCR using primer sets for Prl2a1, Il10, Sox4, Rgs13, Sell, and b-actin (primer sequences available in supplemental Methods) with SYBR Green PCR Master Mix on an ABI 7500 Fast instrument (Applied Biosystems) as described. 12 Relative gene expression levels were calculated using the DDC T method using b-actin as the calibrator.…”