Although it is recognized that PDC play a critical role in the link between innate and adaptive immunity and that their numerical and functional dysfunction contributes to HIV pathogenesis (12
), study of the fate of PDC in HIV infection has been hampered by the difficulty of monitoring the PDC throughout the body. Likewise, it is difficult to study PDC in human hosts at the earliest periods after infection with HIV. Thus, an animal that allows study of PDC function in the context of immunodeficiency virus infection is very much needed. The present study was undertaken to determine the extent to which macaque PDC are similar to their human counterparts.
We used reagents that we routinely use in the study of human PDC to further describe the rhesus macaque PDC. Others have reported that macaque PDC, like their human counterparts, can be identified by using four markers: lineage, HLA-DR, CD123, and CD11c (8
). We demonstrate here that the macaque PDC, like their human counterparts, can be identified by using our two-color scheme (9
), which utilizes CD123 and HLA-DR only. Using a four-color flow cytometer, defining the PDC by two colors, opens up two additional channels for additional studies, such as intracellular analysis of IFN-α, chemokines, or IRF-7. Two existing antibodies frequently used to identify and/or isolate human PDC, namely, BDCA-2 and BDCA-4, however, failed to react with the macaque PDC PDC phenotype, and frequencies were found to be similar within macaque PBMC obtained from freshly isolated blood, PBMC separated from heparanized blood that had been shipped overnight, and in cryopreserved, thawed PBMC. The macaque, however, had a significantly lower percentage of PDC in the peripheral blood than human donors. In addition, we observed more variability in the expression of HLA-DR by the macaque than the human PDC, but this did not interfere with our ability to identify the PDC. By using Giemsa stain, isolated PDC were indistinguishable from human PDC.
In addition to their phenotypic similarity to human PDC, the macaque PDC within the PBMC vigorously produced IFN-α in response to stimulation with HSV, as measured both by total IFN-α activity in an IFN bioassay and by ELISPOT analysis with human IFN-α specific reagents. Although the levels of IFN in supernatants of HSV and SV-stimulated samples were statistically indistinguishable, the ELISPOT frequencies of the IPC were lower in macaques than in humans. The lower frequency of HSV-responsive IPC, as measured by ELISPOT, is consistent with the observation that the monkeys had a lower percentage of PDC among PBMC than humans. The ability of the gated PDC to produce IFN-α, as measured as the percent PDC positive for intracellular IFN-α, was statistically equivalent between monkeys and humans, indicating that, as we previously demonstrated in humans (12
), not all PDC respond to HSV with IFN production, a finding that has also been seen with human PDC stimulated with the TLR7 agonist, imiquimod (18
). The markedly lower frequency of SV-responsive IPC in monkeys compared to humans may reflect limitations to the ELISPOT assay. SV is known to induce both PDC and monocytes to produce IFN-α, with the monocytes expressing 5- to 10-fold lower expression of IFN-α on a per-cell basis than the PDC (13
). In the ELISPOT, this is seen by a mixture of small (monocyte-derived) and large (PDC-derived) spots. The number of smaller, monocyte-derived IFN-α spots was noticeably lower in the macaque than in the human, perhaps reflecting spots that were too dim to detect, thus limiting the usefulness of the ELISPOT assay for detecting SV-induced IPC.
Also similar to the human PDC, macaque PDC produced both CXCL10/IP-10 and CCL4/MIP-1β, as well as TNF-α, in response to HSV. Thus, as in humans, the macaque PDC are uniquely poised to interact with other cell types such as NK cells and T cells (28
) and to link innate and adaptive immune responses (24
). Overall, the similarity of macaque PDC to human PDC in response to HSV demonstrates the usefulness of the macaque model for the study of PDC.
Coates et al. studied PDC in Flt3L-treated macaques (8
). Although growth factors such as Flt3L may be useful in therapeutics, we have demonstrated that fresh, untreated PDC can be functionally studied in the macaque model. In addition, we were able, by using the two-color scheme to identify PDC, to demonstrate that macaque PDC, like their human counterparts, produce IFN-α, IP-10, MIP-1β, and TNF-α in response to viral stimulation. The similarity in cytokine production of macaque to human PDC further establishes the macaque model as a good system for studying PDC.
In addition to the phenotypic and functional similarities between the macaque and human PDC, the macaque PDC, again similar to human PDC (9
), were found to express high levels of the transcription factor IRF-7 compared to other peripheral blood cell types. In humans, we have demonstrated that this IRF-7 can be rapidly translocated to the nucleus of PDC after stimulation with HSV. We postulate that this high constitutive IRF-7 is what makes PDC such exquisite “professional IFN-producing cells” (40
In conclusion, the macaque PDC model provides a valuable system to study these important cells in a nonhuman primate setting. Furthermore, the similarity between SIV and HIV pathogenesis in rhesus macaques and humans, respectively, provides a useful model in the macaque for studying HIV pathogenesis. Studies are currently under way to evaluate the PDC system in the context of acute and chronic immunodeficiency virus infection. The demonstration of the macaque as a good model for PDC study will hopefully permit the elucidation of the role of PDC in viral pathogenesis, as well as in other human diseases.