Efficient Human Cytomegalovirus Reactivation Is Maturation Dependent in the Langerhans Dendritic Cell Lineage and Can Be Studied using a CD14⁺Experimental Latency Model

Abstract

Studies from a number of laboratories have shown that the myeloid lineage is prominent in human cytomegalovirus (HCMV) latency, reactivation, dissemination, and pathogenesis. Existing as a latent infection in CD34؉ progenitors and circulating CD14 ؉ monocytes, reactivation is observed upon differentiation to mature macrophage or dendritic cell (DC) phenotypes. Langerhans' cells (LCs) are a subset of periphery resident DCs that represent a DC population likely to encounter HCMV early during primary infection. Furthermore, we have previously shown that CD34؉ derived LCs are a site of HCMV reactivation ex vivo. Accordingly, we have utilized healthy-donor CD34 ؉ cells to study latency and reactivation of HCMV in LCs. However, the increasing difficulty acquiring healthy-donor CD34؉ cells-particularly from seropositive donors due to the screening regimens used-led us to investigate the use of CD14 ؉ monocytes to generate LCs. We show here that CD14 ؉ monocytes cultured with transforming growth factor ␤ generate Langerin-positive DCs (MoLCs). Consistent with observations using CD34 ؉ derived LCs, only mature MoLCs were permissive for HCMV infection. The lytic infection of mature MoLCs is productive and results in a marked inhibition in the capacity of these cells to promote T cell proliferation. Pertinently, differentiation of experimentally latent monocytes to the MoLC phenotype promotes reactivation in a maturation and interleukin-6 (IL-6)-dependent manner. Intriguingly, however, IL-6-mediated effects were restricted to mature LCs, in contrast to observations with classical CD14؉ derived DCs. Consequently, elucidation of the molecular basis behind the differential response of the two DC subsets should further our understanding of the fundamental mechanisms important for reactivation. Human cytomegalovirus (HCMV) represents an opportunistic pathogen that is a major cause of disease in a number of immunocompromised patient populations (30,60). A significant contribution to morbidity in the clinical setting results from the reactivation of latent HCMV, particularly in seropositive bone marrow transplant recipients (40). Although the mechanisms that govern HCMV reactivation have not been fully elucidated, work from a number of laboratories has shown that CD34 ϩ hematopoietic cells, and the early granulocyte-macrophage progenitors derived from them, which are normally resident in the bone marrow are an important site for the carriage of HCMV latency in vivo (27,34,53) and that reactivation is intrinsically linked with the differentiation to a more mature myeloid cell phenotype (19,37,45,56,59,64).Langerhans' cells (LCs) are a unique population of dendritic cells (DCs) resident in the epidermis and a number of mucosal tissues (e.g., nasal, oral, vaginal, and corneal). They are derived from bone marrow progenitors (26) and exhibit a capacity for self-renewal (11, 36), as well as exhibiting prodigious longevity for a DC, with a half-life of up to 78 days documented (62) and, in one case, a donor's LCs were observed to persis...

Circulating Dendritic Cells Isolated from Healthy Seropositive Donors Are Sites of Human Cytomegalovirus Reactivation In Vivo

“…1B). This phenotype was consistent with a more immature dermal, and not Langerhans, DC phenotype (25), similar to that observed for classical MoDCs (20,25), which have been used routinely to study HCMV latency and reactivation. Taken together, these data suggested that the circulating blood DCs isolated were predominantly of a dermal/interstitial-like phenotype, consistent with previous reports for the isolation of myeloid blood DCs from healthy donors (40,41).…”

“…However, crucially, while a wealth of in vitro data supports this prevailing hypothesis, it has never been definitively shown that naturally occurring DCs derived from healthy seropositive individuals are sites of genome carriage and, importantly, sites of HCMV reactivation. Indeed, a previous study of CD11c ϩ dendritic cells isolated from buffy coats of healthy volunteers has suggested that the welldocumented immunoparalysis observed following infection of in vitro-differentiated DCs (20,(30)(31)(32)(33)(34)(35)(36)(37) is not observed when circulating DCs are similarly infected in vitro with HCMV (38). Although these data concern lytic infection, they exemplify the need for a direct analysis of HCMV latency and reactivation in DCs.…”

“…These models have relied on in vitro differentiation to cell types that are defined as dermal (interstitial) or epidermal (Langerhans)-like cell types based on the expression of a panel of cell surface markers identified on corresponding cells directly isolated ex vivo (20,(24)(25)(26)(27)(28)(29). Therefore, these data would predict that circulating DCs were sites of HCMV carriage in vivo and, furthermore, that these cells might be sites of reactivation in vivo.…”

“…Pertinent to this report, a number of studies of both experimental and natural latency have illustrated that the in vitro differentiation of myeloid progenitor cells to terminally differentiated myeloid dendritic cell (DC) phenotypes results in the induction of HCMV reactivation from these latently infected myeloid cell types (5,12,(19)(20)(21)(22)(23). These models have relied on in vitro differentiation to cell types that are defined as dermal (interstitial) or epidermal (Langerhans)-like cell types based on the expression of a panel of cell surface markers identified on corresponding cells directly isolated ex vivo (20,(24)(25)(26)(27)(28)(29).…”

Role of cytomegalovirus on the maturation and function of monocyte derived dendritic cells of liver transplant patients

“…CMV-infected DCs displayed abnormal phenotypic characteristic and stable expression of CD83 as a maturation marker [24,25] . Infectivity of CMV in MoDCs resulted in maturation of the surviving cells, upregulation of the CD86, and down-regulation of MHC-Ⅰ and Ⅱ [21,26] . In this study, results also demonstrated the down-regulation of CD83, CD1a and HLA-DR molecules on MoDCs in CMV reactivated compared to non-reactivated liver transplanted patients.…”