Mariel Jais

DC-SIGN Is a Receptor for Human Herpesvirus 8 on Dendritic Cells and Macrophages

Human herpesvirus 8 (HHV-8) causes Kaposi’s sarcoma and pleural effusion lymphoma. In this study, we show that dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN; CD209) is a receptor for HHV-8 infection of myeloid DCs and macrophages. DC-SIGN was required for virus attachment to these cells and DC-SIGN-expressing cell lines. HHV-8 binding and infection were blocked by anti-DC-SIGN mAb and soluble DC-SIGN, and mannan, a natural ligand for DC-SIGN. Infection of DCs and macrophages with HHV-8 led to production of viral proteins, with little production of viral DNA, similar to HHV-8 infection of vascular endothelial cells. Infection of DCs resulted in down-regulation of DC-SIGN, a decrease in endocytic activity, and an inhibition of Ag stimulation of CD8+ T cells. We propose that DC-SIGN serves as a portal for immune dysfunction and oncogenesis caused by HHV-8 infection.

Human Herpesvirus 8 Infects and Replicates in Primary Cultures of Activated B Lymphocytes through DC-SIGN

ABSTRACT Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposis sarcoma, primary effusion lymphoma, and some forms of multicentric Castlemans disease. Although latent HHV-8 DNA can be detected in B cells from persons with these cancers, there is little information on the replication of HHV-8 in B cells. Indeed, B cells are relatively resistant to HHV-8 infection in vitro. We have recently shown that DC-SIGN, a C-type lectin first identified on dendritic cells (DC), is an entry receptor for HHV-8 on DC and macrophages. We have also demonstrated previously that B lymphocytes from peripheral blood and tonsils express DC-SIGN and that this expression increases after B-cell activation. Here we show that activated blood and tonsillar B cells can be productively infected with HHV-8, as measured by an increase in viral DNA, the expression of viral lytic and latency proteins, and the production of infectious virus. The infection of B cells with HHV-8 was blocked by the pretreatment of the cells with antibody specific for DC-SIGN or with mannan but not antibody specific for xCT, a cystine/glutamate exchange transporter that has been implicated in HHV-8 fusion to cells. The infection of B cells with HHV-8 resulted in increased expression of DC-SIGN and a decrease in the expression of CD20 and major histocompatibility complex class I. HHV-8 could also infect and replicate in B-cell lines transduced to express full-length DC-SIGN but not in B-cell lines transduced to express DC-SIGN lacking the transmembrane domain, demonstrating that the entry of HHV-8 into B cells is related to DC-SIGN-mediated endocytosis. The role of endocytosis in viral entry into activated B cells was confirmed by blocking HHV-8 infection with endocytic pathway inhibitors. Thus, the expression of DC-SIGN is essential for productive HHV-8 infection of and replication in B cells.

Monofunctional and polyfunctional CD8+ T cell responses to human herpesvirus 8 lytic and latency proteins.

ABSTRACT Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposis sarcoma, primary effusion lymphoma, and multicentric Castlemans disease. It is postulated that CD8+ T cell responses play an important role in controlling HHV-8 infection and preventing development of disease. In this study, we investigated monofunctional and polyfunctional CD8+ T cell responses to HHV-8 lytic proteins gB (glycoprotein B) and K8.1 and latency proteins LANA-1 (latency-associated nuclear antigen-1) and K12. On the basis of our previous findings that dendritic cells (DC) reveal major histocompatibility complex (MHC) class I epitopes in gB, we used a DC-based system to identify 2 novel epitopes in gB, 2 in K8.1, 5 in LANA-1, and 1 in K12. These new HHV-8 epitopes activated monofunctional and polyfunctional CD8+ T cells that produced various combinations of gamma interferon, interleukin 2, tumor necrosis factor alpha, macrophage inhibitory protein 1β, and cytotoxic degranulation marker CD107a in healthy HHV-8-seropositive individuals. We were also able to detect HHV-8-specific CD8+ T cells in peripheral blood samples using HLA A*0201 pentamer complexes for one gB epitope, one K8.1 epitope, two LANA-1 epitopes, and one K12 epitope. These immunogenic regions of viral lytic and latency proteins could be important in T cell control of HHV-8 infection.

Alterations in Cholesterol Metabolism Restrict HIV-1 Trans Infection in Nonprogressors

ABSTRACT HIV-1-infected nonprogressors (NP) inhibit disease progression for years without antiretroviral therapy. Defining the mechanisms for this resistance to disease progression could be important in determining strategies for controlling HIV-1 infection. Here we show that two types of professional antigen-presenting cells (APC), i.e., dendritic cells (DC) and B lymphocytes, from NP lacked the ability to mediate HIV-1 trans infection of CD4+ T cells. In contrast, APC from HIV-1-infected progressors (PR) and HIV-1-seronegative donors (SN) were highly effective in mediating HIV-1 trans infection. Direct cis infection of T cells with HIV-1 was comparably efficient among NP, PR, and SN. Lack of HIV-1 trans infection in NP was linked to lower cholesterol levels and an increase in the levels of the reverse cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) in APC but not in T cells. Moreover, trans infection mediated by APC from NP could be restored by reconstitution of cholesterol and by inhibiting ABCA1 by mRNA interference. Importantly, this appears to be an inherited trait, as it was evident in APC obtained from NP prior to their primary HIV-1 infection. The present study demonstrates a new mechanism wherein enhanced lipid metabolism in APC results in remarkable control of HIV-1 trans infection that directly relates to lack of HIV-1 disease progression. IMPORTANCE HIV-1 can be captured by antigen-presenting cells (APC) such as dendritic cells and transferred to CD4 helper T cells, which results in greatly enhanced viral replication by a mechanism termed trans infection. A small percentage of HIV-1-infected persons are able to control disease progression for many years without antiretroviral therapy. In our study, we linked this lack of disease progression to a profound inability of APC from these individuals to trans infect T cells. This effect was due to altered lipid metabolism in their APC, which appears to be an inherited trait. These results provide a basis for therapeutic interventions to control of HIV-1 infection through modulation of cholesterol metabolism. HIV-1 can be captured by antigen-presenting cells (APC) such as dendritic cells and transferred to CD4 helper T cells, which results in greatly enhanced viral replication by a mechanism termed trans infection. A small percentage of HIV-1-infected persons are able to control disease progression for many years without antiretroviral therapy. In our study, we linked this lack of disease progression to a profound inability of APC from these individuals to trans infect T cells. This effect was due to altered lipid metabolism in their APC, which appears to be an inherited trait. These results provide a basis for therapeutic interventions to control of HIV-1 infection through modulation of cholesterol metabolism.

Human Herpesvirus 8 Infects and Replicates in Langerhans Cells and Interstitial Dermal Dendritic Cells and Impairs Their Function.

ABSTRACT The predominant types of dendritic cells (DC) in the skin and mucosa are Langerhans cells (LC) and interstitial dermal DC (iDDC). LC and iDDC process cutaneous antigens and migrate out of the skin and mucosa to the draining lymph nodes to present antigens to T and B cells. Because of the strategic location of LC and iDDC and the ability of these cells to capture and process pathogens, we hypothesized that they could be infected with human herpesvirus 8 (HHV-8) (Kaposis sarcoma [KS]-associated herpesvirus) and have an important role in the development of KS. We have previously shown that HHV-8 enters monocyte-derived dendritic cells (MDDC) through DC-SIGN, resulting in nonproductive infection. Here we show that LC and iDDC generated from pluripotent cord blood CD34+ cell precursors support productive infection with HHV-8. Anti-DC-SIGN monoclonal antibody (MAb) inhibited HHV-8 infection of iDDC, as shown by low expression levels of viral proteins and DNA. In contrast, blocking of both langerin and the receptor protein tyrosine kinase ephrin A2 was required to inhibit HHV-8 infection of LC. Infection with HHV-8 did not alter the cell surface expression of langerin on LC but downregulated the expression of DC-SIGN on iDDC, as we previously reported for MDDC. HHV-8-infected LC and iDDC had a reduced ability to stimulate allogeneic CD4+ T cells in the mixed-lymphocyte reaction. These results indicate that HHV-8 can target both LC and iDDC for productive infection via different receptors and alter their function, supporting their potential role in HHV-8 pathogenesis and KS. IMPORTANCE Here we show that HHV-8, a DNA tumor virus that causes Kaposis sarcoma, infects three types of dendritic cells: monocyte-derived dendritic cells, Langerhans cells, and interstitial dermal dendritic cells. We show that different receptors are used by this virus to infect these cells. DC-SIGN is a major receptor for infection of both monocyte-derived dendritic cells and interstitial dermal dendritic cells, yet the virus fully replicates only in the latter. HHV-8 uses langerin and the ephrin A2 receptor to infect Langerhans cells, which support full HHV-8 lytic replication. This infection of Langerhans cells and interstitial dermal dendritic cells results in an impaired ability to stimulate CD4+ helper T cell responses. Taken together, our data show that HHV-8 utilizes alternate receptors to differentially infect and replicate in these tissue-resident DC and support the hypothesis that these cells play an important role in HHV-8 infection and pathogenesis.

Correction: DC-SIGN on B Lymphocytes Is Required for Transmission of HIV-1 to T Lymphocytes.

In PLoS Pathogens, volume 2, issue 7: DOI: 10.1371/journal.ppat.0020070 Page 3, column 1, line 3 should have included this reference for the Raji-DC-SIGN cells: Wu L, Martin TD, Carrington M, KewalRamani VN (2004) Raji B cells, misidentified as THP-1 cells, stimulate DC-SIGN-mediated HIV transmission. Virology 318: 17-23. The Acknowledgments should have included a thank you to V. N. KewalRamani (National Cancer Institute) for the Raji-DC-SIGN cells.

CD4 regulatory T cells control CD8 T cell responses to human herpesvirus 8 lytic and latency proteins

Methods Longitudinal PBMC samples were obtained from subjects with various outcomes of HHV-8 infection over many years in the Multicenter AIDS Cohort Study. The PBMC were tested by HLA A*0201 multimer staining specific for memory CD8 T cell epitopes of viral lytic and latency proteins, and polyfunctional flow cytometry to detect HHV8-specific, polyfunctional CD8 T cell populations. The effect of Treg was examined by depleting CD4CD25 cells.

Human herpesvirus 8 replicates in primary B lymphocytes and induces polyfunctional cytokine and chemokine responses

Methods B cells were loaded with purified “live” HHV-8, purified UV-light inactivated HHV-8 (UV-HHV-8) or soluble HHV-8 glycoprotein B (gB). HHV-8 replication was measured by real time PCR for viral DNA and intracellular staining (ICS) and flow cytometry for the viral lytic proteins ORF59 and K8.1. ICS was used to assess cellassociated, polyfunctional cytokine-chemokine production, and B cell supernatants were tested for cytokine/ chemokine secretion by Cytometric Bead Array (BD Biosciences).

KSHV interaction with Langerhans and dermal dendritic cells through C-type lectins

The skin contains two types of dendritic cells (DC), Langerhans cells (LC), which reside in the epidermis in close contact with keratinocytes, and dermal dendritic cells (DDC), resident in the dermis. LC and DDC process cutaneous antigens and migrate out of the skin into the draining lymph nodes to present antigens to T and B cells. Recent reports showed that LC and DDC play an important role in certain virus infections, such as HIV-1 and HSV. Because of the strategic position of LC and DDC at mucosal sites of infection and the ability of these cells to capture pathogens, we hypothesized that these cells could be infected with KSHV and have an important role in the development of Kaposi’s sarcoma. We have previously shown that KSHV enters monocytederived dendritic cells (MoDC) through DC-SIGN, resulting in a nonproductive infection. We have now generated LC and DDC from pluripotent cord blood CD34+ precursors by culture with GM-CSF, TNF, and TGF-B to obtain LC, and GM-CSF, TNF, and IL4 to generate DDC. These expressed the typical phenotype of LC, i.e., CD207 pos , CD14 pos , CD11b neg ,C D1a pos , HLADR pos ,D C-SIGN neg , and dermal DC, i.e., DC-SIGN pos , CD14 neg , CD11b pos ,C D1a pos ,H LA-DR pos ,l angerin neg . We found that both LC and DDC supported productive infection with KSHV. Strikingly, while the level of viral DNA replication increased only 4-fold in infected DDC by 24h, we observed a >1 log10 increase in levels of viral DNA in LC. Anti-DC-SIGN mAB inhibited viral infection of DDC as detected by expression of viral proteins and viral DNA, while blocking of langerin on LC did not interfere with viral entry and replication. Infection with KSHV did not alter cell surface expression of langerin on LC, but downregulated expression of DC-SIGN on DDC, as we previously reported for MoDC. Cytokine production in infected LC and DDC was also altered compared to uninfected cells, with an increase in the levels of IL-8, IL-6, and IL-10 in the infected cells. These results indicate that KSHV can target both LC and DDC for productive infection and alter their function, supporting a role for these dermal DC in KSHV infection and pathogen.