Cross-reactivity of anti-programmed death ligand 2 polyclonal antibody in mouse tissues

The inhibitory co-receptor programmed death 1 (PD-1, encoded by pdcd1) and its two ligands PD-L1 and PD-L2 comprise an important immune inhibitory signaling pathway for defense against microbes and for self-tolerance. Unlike other members of the B7-CD28 family, expression of PD-L1 and PD-L2 is not limited to the immune system. In this study, we determined that a polyclonal antibody (pAb) (R&D Systems) against extracellular domains of mouse PD-L2 (mPD-L2) could recognize antigen(s) in diverse mouse tissues, including the anterior and intermediate pituitary gland, olfactory bulbs and olfactory epithelium, tongue epithelium, keratinized epithelial cells and skin and whisker hair follicles. These findings differed from previous reports of mPD-L2 localization. Reverse transcription PCR and Western blot analyses, however, were unable to detect any mPD-L2 transcripts or proteins of the 25-kD predicted molecular weight in RNA and protein extracts, respectively, from the above tissues, suggesting that the anti-mPD-L2 pAb cross-reacts with certain novel antigen(s). Developmental studies revealed that the earliest expression of mPD-L2-like antigen was in the olfactory epithelium at embryonic day 12.5 (E12.5). At E14.5, mPD-L2-like antigen was present in the skin, tongue and follicles of the skin and whiskers. The distribution patterns of mPD-L2-like antigen remained similar from E18.5 to adulthood. The results of bioinformatic analysis and other experiments suggested neural cell adhesion molecule and hemicentin-1 as candidate proteins with cross-reactivity to the anti-mPD-L2 pAb. These results demonstrate that care is required in interpreting staining patterns generated when anti-PD-L2 pAb is used to locate PD-L2-expressing cells in the central nervous system and epithelial tissues, such as the olfactory epithelium. In addition, this anti-PD-L2 pAb may be used as an alternative antibody for labeling the olfactory epithelium during embryonic development in mice.