PZTD mice have a PD-L2-ZsGreen-TdTomato-diphtheria toxin receptor cassette inserted after the stop codon in exon 5 of the programmed cell death 1 ligand 2 (Pdcd1lg2) gene. Specifically, the cassette includes (from 5' to 3') a floxed cassette containing a IRES sequence fused to a green fluorescent protein (ZsGreen) after the stop codon in exon 5 of the Pdcd1lg2 gene, and an FRT-flanked neomycin-resistance gene (Neo) and a BGH polyadenylation sequence inserted after exon 6. A duplication of exon 5 was inserted after the 3' loxP site, and an IRES and diphtheria toxin receptor (DTR) sequence were inserted after the stop codon in the duplicate exon 5, followed by a self-cleaving peptide sequence and a red fluorescent protein sequence (TdTomato). Pdcd1lg2 encodes PD-L2, one of two ligands for PD-1 (programmed cell death protein 1) that is only expressed on macrophages and dendritic cells upon activation, and the PD-L2+ subpopulation of peritoneal B-1a cells termed L2pB1 cells. L2pB1 cells differ from L2nB1 cells in their immunoglobulin repertoire, expression of interleukin 10, and their capacity to phagocytose phosphatidylcholine. PD-1 is an inhibitory receptor expressed on activated lymphocytes where it regulates tolerance and autoimmunity by negative regulation of T cell responses. Homozygous and heterozygous PZTD knock-in mice are viable and fertile. In the targeted allele, the IRES sequence links Pdcd1lg2 and the ZsGreen gene so that ZsGreen is expressed whenever Pdcd1lg2 is expressed. FACS analysis of peritoneal lymphocytes in naϊve wildtype, heterozygous, and homozygous littermates reveals specific ZsGreen fluorescence only in PD-L2-expressing B-1a cells, while expression is not seen in T cells, B-2 B cells, or other cells. In naϊve mice, no PD-L2-expressing macrophages are observed. A small number of L2pB1 cells are detectable outside the peritoneal cavity, including in the spleen and thymus, as expected. When bred to CD19-Cre mice (see Stock No. <a href="https://www.jax.org/strain/006785">006785</a>), with B cell-specific cre expression, the floxed neo and ZsGreen genes are permanently deleted only in CD19+ B cells, while PD-L2-expressing macrophages and dendritic cells still express ZsGreen. Removal of the floxed region allows the expression of the IRES-DTR-2A-TdTomato cassette. Upon deletion of the floxed region by Cre recombinase, the PD-L2 gene now ends at the stop codon of the duplicated exon 5, and PD-L2+ cells are highly susceptible to diptheria toxin (DT), allowing the depletion of L2pB1 cells. In order to continue labeling L2pB1 cells with a fluorescent protein after the depletion of the ZsGreen gene, a TdTomato was inserted downstream of the DTR gene, linked in frame by a self-cleaving 2A peptide. As a result, after Cre excision of the floxed region, PD-L2, DTR, and TdTomato are expressed simultaneously but independently.

PZTD mice have a PD-L2-ZsGreen-TdTomato-diphtheria toxin receptor cassette inserted after the stop codon in exon 5 of the Pdcd1lg2 gene. These mice may be useful for visualizing and specific depletion of L2pB1 cells, and they provide a tool for studying the immune regulatory functions of L2pB1 cells in health and disease.

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