Detection of weak receptor–ligand interactions using IgM and J-chain-based fusion proteins

Materials and methods IgM-based fusion proteins were generated by molecular cloning of the IgM backbone DNA sequence into the BTN2A2-IgG or PD-L1-IgG-encoding SigpIg+ vectors upstream of the IgG1 Fc part of the fusion protein. The secretory tailpiece of the human IgM heavy chain was also encoded in the human IgM heavy chain DNA sequence inserted (Mitoma J. et al. Nat Immunol .2007.8: 409-418). Expression of the IgG1 Fc tail was suppressed by including a stop codon at the end of the DNA sequence encoding the IgM domains. Primer sequences used for cloning the IgM heavy-chain were: forward-GTGTGGCGGCCGCTCCTGTGATTGCTGAGCTGC; backward-ATGGATCCTCAGTAGCAGGTGCCAGCTGT. The SigpIg+ vector containing the human IgM heavy chain domains 3-5 is illustrated in Supporting Information Figure 1. The J-chain DNA sequence was obtained from cDNA from the human B cell line Raji and cloned into the pCIPac DNA plasmid (Tregaskes C.A. et al. Dev Comp Immunol .2005.29: 361-374). Primer sequences used for cloning the J-chain were: forward-ATGAAGAACCATTTGCTTTTCTG; backward-TTAGTCAGGATAGCAGGCATCT. IgM or IgG fusion proteins and J-chain were stably or transiently expressed in HEK-293 or HEK-293T cells as indicated. Fusion protein concentrations were measured using a standard ELISA protocol. Flow cytometry analysis of binding of IgG fusion proteins or IgM fusion proteins containing the extracellular domains of PD-L1 or BTN2A2 to enriched, CD3 + primary mouse T cells was performed as follows: Primary mouse CD3 + T cells were enriched from spleen and lymph nodes of C57BL/6 mice by negative selection using a kit from Stem Cell Technologies (London, UK). Enriched cells were more than 95% positive for CD3. Where indicated, T cells were activated for 3 days in T75 flasks coated overnight with 1 μg/ml anti-CD3 clone 2C11 (eBioscience, Hatfield,