1d). Some of the lineage markers used for rhesus macaque cells differed from those used for human cells. CD20 replaced CD19 for staining of rhesus B cells as mentioned above. CD56 was excluded from the rhesus staining

panel because it is expressed both on rhesus natural killer cells and on subpopulations of monocytes and mDCs.14,15,38,39 The total DC population was further subdivided into mDCs and pDCs based on their expression of CD11c and CD123, respectively (Fig. 1d). For these stainings, the same clones of antibodies worked well for both human and rhesus DCs. We found no significant difference in the percentage of rhesus pDCs (0·07 ± 0·06%) and human pDCs (0·16 ± 0·28%) see more of total PBMCs (P = 0·145) (Fig. 1e). In contrast, the percentage of rhesus mDCs (0·31 ± 0·19%) was lower than of human mDCs (0·83 ± 0·63%) (P = 0·0003). These levels are comparable to values reported in previous studies.14,15,26,40,41

We next compared the proliferative response of rhesus and human B cells to selected TLR Birinapant datasheet ligands (TLR3, 7/8, 9 ligands) in vitro. We first analysed the proliferation of B cells in total PBMC cultures induced by the three distinct classes of CpG ODN (A, B and C), the imidazoquinoline compound 3M-0012 referred to as TLR7/8-L binding both TLR7 and TLR8, and polyI:C binding TLR3. Proliferation was measured using thymidine incorporation at day 5 of culture. Both human and rhesus B cells express TLR8 and TLR9 but not TLR3 and TLR7.26,42 According to this expression pattern, we observed that all the CpG classes and TLR7/8-L induced significant proliferation compared with unstimulated cultures in both the rhesus and human culture systems (Fig. 2a,b). In contrast, poly I:C did not induce proliferation. CpG class B and C as well as TLR7/8-L induced the strongest proliferation both in rhesus and human cultures. However, while CpG C was significantly more potent in its ability to induce

proliferation in rhesus cultures than the other ligands, CpG B was superior in the human cultures, consistent with previous reports.2,43 The proliferative response was also examined using CFSE dilution allowing us to determine the identity of the proliferating cells (Fig. 2a,b, histograms). The vast majority of cells that divided within the nearly PBMCs were found to be CD20+ and CD19+ B cells in the rhesus and human cultures, respectively (data not shown), indicating that mainly B cells proliferated in response to these TLR ligands. In general, rhesus B cells showed lower proliferative capacity compared with human B cells, as found by both detection methods. Human B cells also exhibited somewhat higher spontaneous proliferation in the unstimulated cultures. Taken together, we concluded that rhesus macaque and human B cells proliferated in response to the same TLR ligands, with only CpG B and CpG C displaying a difference in rank order.