Localization of recombination activating gene 1/green fluorescent protein (RAG1/GFP) expression in secondary lymphoid organs after immunization with T-dependent antigens in rag1/gfpknockin mice

Abstract

IntroductionB cells are generated in the fetal liver or the adult bone marrow (BM) after immunoglobulin gene rearrangements that create a primary repertoire of random antibody (Ab) specificity. 1-3 B cells that react to various self-antigens (Ags) at immature stages are thought to experience anergy after encountering Ags or to be eliminated by apoptotic mechanisms in the BM microenvironment. [4][5][6] It has been suggested that a substantial proportion of B cells reactive to self-Ags undergoes a process known as receptor editing in which secondary immunoglobulin gene rearrangements replace variable (V) gene segments, generating a new Ab specificity as a part of the primary Ab repertoire. 7-11 B cells recruited to the circulation are stimulated by Ags and helper-T (Th) cells in the T cell area of secondary lymphoid organs. 12-14 Such Ag-driven B cells subsequently undergo rapid proliferation to become centroblasts located in the dark zone of germinal centers (GCs) and then arrest cell cycling and become small centrocytes in the light zone. [15][16][17][18][19][20] Recent studies proposed an interesting model in which mature B cells also undergo a similar process, termed receptor editing, in GCs of secondary lymphoid follicles after stimulation by Tdependent (TD) Ags. [21][22][23][24][25] This notion has been supported by the detection of rag1/rag2 transcripts and by the evidence of immunoglobulin gene rearrangements in situ, suggesting that receptor editing occurs in the GC light zone. 21 In addition, stimulation with anti-CD40 monoclonal antibody (mAb) plus interleukin-4 (IL-4), or lipopolysaccharide (LPS) plus IL-4, induced the up-regulation of rag1/rag2 transcripts and immunoglobulin gene rearrangements in mature surface immunoglobulin M (sIgM) ϩ B cells in vitro. 22,[24][25][26] These results suggested that mature B cells frequently undergo receptor editing in GCs after stimulation with Ags. The receptor editing mechanism might provide a more flexible and diverse Ab specificity response than the one produced solely by the somatic mutation mechanism. The receptor editing in GCs could be a requisite molecular event for an immediate and effective response producing high-affinity Abs against various antigenic stimuli. Alternatively, receptor editing could salvage B cells carrying anti-self specificities.The reactivation of rag gene expression has been suggested as one mechanism for the immunoglobulin gene rearrangements that occur in receptor revision. 27,28 To demonstrate direct in vivo evidence of the induction of RAG molecules in GCs and to measure the frequency of cells undergoing receptor editing (or receptor revision), we examined lymphoid organs for RAG1/green fluorescent protein (GFP) signal using immunized or unimmunized rag1/gfp knockin mice. Unimmunized heterozygous rag1/gfp knockin mice express GFP that can be detected at high sensitivity by microscopic observation of splenic sections. Surprisingly, when we examined GCs of immunized mice, we observed that the RAG1/GFP signal was not significantly indu...

Hematopoietic developmental pathways: on cellular basis

“…In mice Hematopoietic developmental pathways H Iwasaki and K Akashi carrying a green fluorescence protein (GFP) gene knocked into the Rag1 gene locus (Kuwata et al, 1999;Igarashi et al, 2001), a fraction (B5%) of LSK cells express GFP (Igarashi et al, 2002). This population, called as the earliest lymphocyte progenitor (ELP), possesses potent T-, B-and NK-differentiation potential with a weak myeloid colony-forming activity (Igarashi et al, 2002).…”

Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions

“…19), cells were harvested and cytosmears were prepared on silanized slides. For the GCs, C57BL͞6 mice were immunized with trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH; Biosearch), and the spleen sections were prepared as described previously (18). For immunohistological staining, slides were incubated with the mAbs prepared in our laboratory as a control rat mAb of the same isotype recognizing glutathione S-transferase, anti-GANP mAb (29-15; ref.…”

“…Spleen B cells from 7-wk-old C57BL͞6 mice were prepared as described (18). After stimulation of purified B cells in vitro for 48 h with either one of reagents of lipopolysaccharide (LPS; 10 g͞ml; Sigma), anti-IgM Ab (10 g͞ml; ICN), and anti-CD40 mAb (10 g͞ml; LB429; ref.…”

PU.1 Is Involved in the Regulation of B Lineage-associated and Developmental Stage-dependent Expression of the Germinal Center-associated DNA Primase GANP

“…The initiator element, which is present in promoters with or without the TATAA box, is believed to be necessary for positioning the transcription initiation complex in promoters lacking the TATAA box (33). Interestingly, the sequence element 5Ј-CACA(N) 5 GAGNC-3Ј encompassing nucleotides Ϫ174 to Ϫ161 of the ganp promoter shows a 7 of 8-bp identity among 13 nucleotides at its 5Ј and 3Ј ends with the TdT initiator sequence 5Ј-CTCA(N) 5 GAGNC-3Ј (34).…”