Here we show that DMHT requires cell-cell contact and is supported by restricted cell types. These findings reinforce the idea that the cellular environment is an important factor when examining transmission of HIV-1 captured by DC-SIGN [21
]. In addition, we observe that MHC class II molecules are not required for efficient DMHT, suggesting that virus transmission can occur in the absence of the classically defined immune synapse. Despite similar levels of DC-SIGN expression, K562 transfectants were markedly less efficient in the transmission of HIV-1 when compared with Raji cell transfectants. A previous study has implicated cell type differences in the protection and transmission of HIV-1 by THP-1, 293T, and HOS cells transfectants expressing DC-SIGN [11
]. It is likely that the cells identified as "THP-1" in this prior study were actually Raji-derived cells, as THP-1 cells generally do not supportive DMHT [21
]. In contrast to our study, these authors did not observe cell type differences in DMHT of newly captured HIV-1. Instead, cell type differences in HIV-1 transmission were only manifested when target cells were added 2 days after virus inoculation of the DC-SIGN-expressing cells. Although it is possible that K562 cells are especially restrictive in DMHT, it is more likely that differences in assay systems precluded a quantitative comparison of DMHT of newly captured HIV-1 by the 293T and HOS cells in the prior study. We have observed that 293T cells expressing DC-SIGN are significantly less efficient than Raji/DC-SIGN cells in the transmission of newly acquired HIV-1 (data not shown). Notably, K562 cell lines have been used to examine DC-SIGN function in a number of studies [22
]. However, our study represents the first quantitative comparison of DMHT in a single-replication viral cycle between K562 transfectants and other cells. We have observed that K562 cells are not only impaired in DMHT but also that K562 cells can inhibit virus transmission by other cells in a contact-dependent manner. These data provide the first evidence that DC-SIGN transmission of HIV-1 can be regulated in trans
One model to reconcile K562 cell restriction of cell-cell HIV-1 transmission when DC-SIGN is expressed either in cis
or in trans
is that K562 cells express a cell surface molecule that hinders DMHT. It is unlikely that such a molecule competes for occupancy in the DC-SIGN carbohydrate recognition domain, as HIV-1 particles and ICAM-3 bound K562/DC-SIGN and Raji/DC-SIGN cells at comparable efficiencies. In addition, K562 cells did not have a direct detrimental effect on the infectivity of cell-free HIV-1 or on the susceptibility of Hut/CCR5 target cells. Thus, it is more likely that a K562 cell-expressed surface factor influences the fate of DC-SIGN-bound HIV-1 particles or interferes with the formation of an infectious synapse with virus target cells. K562 cells might compete with Hut/CCR5 cells for interaction with the Raji/DC-SIGN donor lines, preventing synaptic transmission of HIV-1 to the Hut/CCR5 cells. Alternatively, the K562 cells might induce the Raji/DC-SIGN cells to traffic HIV-1 to a degradative compartment within the cells, preventing virus transmission. It is unclear whether negative regulation of DMHT by cells in trans
is unique to K562 cells or extends to other cell types, including primary cells. Given the presumed erythrocytic origin of K562 cells, it will be interesting to examine the effect of blood erythrocytes on MDDC-mediated HIV-1 transmission. Notably, we observed that K562 cells express a low level of Duffy antigen/receptor for chemokines (DARC), a promiscuous chemokine receptor that may interact with HIV-1 Env [36
The requirements for DMHT subsequent to HIV-1 binding have not been fully delineated. Others have reported that differentiation of human DCs toward different effector subsets creates cells with different abilities to stimulate HIV-1 replication despite equal levels of DC-SIGN expression [18
]. This study had indicated that ICAM-1 expression on DCs might predict the efficiency of HIV-1 transmission. Prior studies have also indicated that the combined interactions of LFA-1/ICAM-1 and LFA-3/CD2 aid in the efficient HIV-1 replication in cocultured DCs and CD4+ T cells [7
]. Consistent with these studies performed with DCs [8
], we found that HIV-1 transmission mediated by Raji/DC-SIGN cells was impaired when the direct contacts between donor cells and target cells were obstructed by a permeable membrane. Collectively, these data suggest that cell surface ligands could act as cofactors in DMHT and the formation of an infectious synapse.
A preliminary survey of molecules that are expressed on DCs and Raji/DC-SIGN cells and that are important in establishing contact and communication between APCs and CD4+ T cells revealed two potentially significant candidates that are not expressed in K562/DC-SIGN cells, LFA-1 and MHC class II antigens. To investigate the role of MHC class II antigens in the DC-SIGN transmission of infectious HIV-1, we genetically manipulated donor cell lines to alter their MHC class II expression using WT- and DN-CIITA and functionally tested their efficiency of HIV-1 transmission. We observed that HIV-1 transmission mediated by these cell lines was not significantly affected, and coexpression of LFA-1 and MHC class II molecules in K562/DC-SIGN cells was not sufficient to enable efficient HIV-1 transmission by the modified cells. Because of the dominant-negative effect that K562 cells appear to exert on DMHT, it is difficult to conclude the roles of possible positive factors by using these cells. Reduced MHC class II expression in Raji/DC-SIGN/DN-CIITA cells did not have a negative effect on DMHT. However, because MHC class II expression was not fully silenced in Raji/DC-SIGN/DN-CIITA cells, these data do not a priori exclude a contribution of the MHC class II pathway on DMHT.
Despite the presence of a negative factor on K562 cells, K562 cells that express DC-SIGN did modestly stimulate HIV-1 transmission irrespective of MHC class II or LFA-1 expression levels. This stimulation was significantly less than that observed with Raji/DC-SIGN cells, which transmit HIV-1 or other primate lentiviruses at efficiencies comparable to DCs [1
]. Similar transmission results comparing K562/DC-SIGN and Raji/DC-SIGN cells were obtained with virus particles bearing different HIV-1 or SIV Env proteins (data not shown). Because K562/DC-SIGN adsorption of HIV-1 particles was comparable to that of Raji/DC-SIGN cells, this increased virus binding was not predictive of HIV-1 transmission efficiency. These results suggest that the DC-SIGN effect on HIV-1 transmission is more complex than simple virus binding and provide support for a model put forward by Pöhlmann and colleagues that DC-SIGN binding and transmission functions are dissociable [37
The cell type specificity of DC-SIGN function in HIV-1 transmission provides a means to explore this mechanism. There are likely positive as well as negative cellular factors involved in DMHT. The examination of additional cell types, particularly those derived from APCs, and their mechanisms of cell-cell communication and of HIV-1 intracellular trafficking may be useful in identifying features that are required for efficient DC-SIGN-mediated transmission. In addition, understanding the negative regulatory mechanism that cells can exert on DC-SIGN transmission of HIV-1 may facilitate the development of immune-modulating therapies to help prevent the dissemination of HIV-1 by DCs in vivo.