However, ~70% of B6 AM14 V8/V8 mice had substantial numbers of MZ B cells as shown by the canonical CD23int CD21+ phenotype (Fig. defense. Introduction B cells expressing the transgene-encoded AM14 BCR, specific for IgG2a, have provided a critical tool for defining the role of nucleic acid sensing TLRs in the activation IDH-305 of autoreactive B cells (1C5). The AM14-producing hybridoma was originally isolated from a diseased MRL/lpr mouse and this antibody is considered a rheumatoid factor since it binds to autologous MRL IgG2a, as well as BALB/c IgG2a (6C8). AM14 binds monomeric IgG2a with relatively low affinity (Kd=~2.2 10?6 M, (7), and as a result, AM14 B cells are essentially non-responsive (ignorant) to normal circulating levels of IgG2a (6) and do not proliferate or exhibit any evidence of anergy in non-autoimmune mice. Immune complexes (ICs) consisting of IgG2a-bound proteins also fail to induce AM14 B cell proliferation. By contrast, IgG2a autoantibody/autoantigen (autoAbs/autoAgs) ICs that incorporate DNA or RNA and therefore carry their own autoadjuvant are potent inducers of AM14 B cell proliferation (1, 2, 5). We have extensively characterized B cells derived from BALB/c AM14 IDH-305 V8 site-directed transgenic mice and shown that these highly allelically restricted B cells are limited to the follicular (FO) B cell compartment; hence these mice lack marginal zone (MZ) B cells (9). The IgG2a monoclonal autoAb PL2C3 binds to both DNA and RNA associated proteins and induces proliferation of Tlr9?/? and Tlr7?/?, but not double deficient, AM14 V8 B cells. Intriguingly, BCR/TLR7 and BCR/TLR9 activation of AM14 V8 B cells leads to distinct functional outcomes (3). To better define the receptor specific signaling cascades, we backcrossed the AM14 and V8 transgenes to C57BL/6J (B6) mice so that we could take advantage of tissue specific deleter strains only available on a B6 background. Unexpectedly, we found that B6 AM14 V8 B cells are hyporesponsive to anti-IgM stimulation, readily differentiate to a MZ B cell phenotype, and undergo extensive receptor editing. These outcomes reflect the capacity of the AM14 V8 BCR to respond to a cross-reactive antigen expressed by B6 but not BALB/c mice. Materials and Methods Animals BALB/c AM14 V8 site-directed transgenic (sdTg) mice have been described previously (9, 10). BALB/c and C57BL/6J (B6) wild type mice, BALB/c Rag2?/? and B6 Rag1?/?, BALB/c and B6 CD45.1 mice were obtained from Jackson Lab. B6 mice homozygous for the AM14 heavy and heterozygous for the V8 light chain (V8/+) were provided by Dr. M. Shlomchik (University of Pittsburgh). These mice were intercrossed to generate AM14 homozygous and V8 homozygous (V8/ V8) mice. AM14 V8/ V8 mice were crossed to B6 Rag1?/? (Jackson Lab), B6 Btk?/? (11) or B6 Unc93B13d/3d, kindly provided by Dr. IDH-305 B. Beutler (12). F1 offspring were intercrossed to obtain B6 AM14 V8/+ Rag1?/?, AM14 V8/+ Btk?/? and AM14 V8/+ Unc93B13d/3d. All mice were bred and maintained at the Department of Animal Medicine of the University of Massachusetts Medical School in accordance with the regulations of the American Association for the Accreditation of Laboratory Animal Care. Cell culture Splenic B cells were positively selected with B220 magnetic beads (BD Biosciences) and then cultured in RPMI/5% heat-inactivated FCS as described previously (9) with the following ligands: 1 g/ml CpG 1826 (s-oligodeoxynucleotide, Idera Pharmaceuticals), 15 g/ml goat anti-mouse IgM F(ab)2 (Jackson ImmunoResearch), or 1 g/ml mAb PL2C3 (13). B cell ABL1 proliferation was assessed by 3H-thymidine incorporation at 30 hr or fluorescent dye dilution IDH-305 at 72 hr. BLyS was provided by Human Genome Sciences and added to selected experimental groups as mentioned at a final concentration of 50 ng/ml. Inhibitor (Inh) 18 (3 CCT GGA TGG GAA CTT ACC GCT GCA 5), an inhibitory ODN, was used at 1 g/ml to block TLR9.
