UCL Cancer Institute, University College London, London, United Kingdom.
Address correspondence to: Chris Boshoff, UCL Cancer Institute, University College London, London, United Kingdom, WC1E 6BT. Phone: 44.20.7679.6850; Fax: 44.20.7679.6817; E-mail: firstname.lastname@example.org.
Published February 21, 2011 - More info
Kaposi sarcoma–associated herpesvirus (KSHV) is a B-lymphotropic virus whose primary site of replication is the oropharynx. KSHV can infect both T and B cells from primary tonsillar explant cultures. However, T cells do not support lytic replication, while B cells spontaneously produce substantial amounts of infectious virus. Here, we provide evidence for a mechanism by which activated T cells may promote or stabilize latency of KSHV infection in B cells. When mixed cultures of B cells and activated T cells were exposed to KSHV, little spontaneous virus production was observed. Removing T cells from the mix or treating the mixed culture with immune suppressants enhanced virus production. Adding back activated T cells to purified infected B cells efficiently suppressed KSHV production, primarily due to CD4+ T cells. This suppressive activity required T cell activation and direct cell-cell contact, but not prior exposure to KSHV antigen. Suppression was not MHC restricted and did not result in killing of the target cell. We therefore propose that oropharyngeal T cells activated by a variety of stimuli can recognize ligands on infected target B cells, leading to signaling events that prevent spontaneous lytic activation and promote latent infection in this compartment.
Jinjong Myoung, Don Ganem
Kaposi sarcoma–associated herpesvirus (KSHV; also known as HHV8) is the causative agent of two B cell tumors, multicentric Castleman disease (MCD) and primary effusion lymphoma (PEL). However, little is known about the nature of the specific B cell subtype(s) most susceptible to infection. Identifying these cells would provide direct insight into KSHV transmission and virus-induced transformation. To identify this subset and to determine whether infection alters its cellular phenotype, we exposed human tonsillar cells to KSHV and characterized infected cells using high-throughput multispectral imaging flow cytometry (MIFC). Stable expression of the virally encoded latency-associated nuclear antigen (LANA), a marker of latent KSHV infection, was observed predominantly in cells expressing the l light chain of the B cell receptor. These LANA+ B cells proliferated and exhibited similarities to the cells characteristic of MCD (IgMl-expressing plasmablasts), including blasting morphology with elevated expression of Ki67, variable expression of CD27, and high levels of IgM and IL-6 receptor. Furthermore, the proportion of infected cells showing a blasting phenotype increased upon addition of exogenous IL-6. Our data lead us to propose that oral transmission of KSHV involves the latent infection of a subset of tonsillar IgMl-expressing B cells, which then proliferate as they acquire the plasmablast phenotype characteristic of MCD.
Lynn M. Hassman, Thomas J. Ellison, Dean H. Kedes
Kaposi sarcoma herpesvirus (KSHV) is specifically associated with Kaposi sarcoma (KS) and 2 B cell lymphoproliferative diseases, namely primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). KS, PEL, and MCD are largely incurable and poorly understood diseases most common in HIV-infected individuals. Here, we have revealed the role of viral FLICE-inhibitory protein (vFLIP) in the initiation of PEL and MCD by specifically expressing vFLIP at different stages of B cell differentiation in vivo. Mice showed MCD-like abnormalities and immunological defects including lack of germinal centers (GCs), impaired Ig class switching, and affinity maturation. In addition, they showed increased numbers of cells expressing cytoplasmic IgM-λ, a thus far enigmatic feature of the KSHV-infected cells in MCD. B cell–derived tumors arose at high incidence and displayed Ig gene rearrangement with downregulated expression of B cell–associated antigens, which are features of PEL. Interestingly, these tumors exhibited characteristics of transdifferentiation and acquired expression of histiocytic/dendritic cell markers. These results define immunological functions for vFLIP in vivo and reveal what we believe to be a novel viral-mediated tumorigenic mechanism involving B cell reprogramming. Additionally, the robust recapitulation of KSHV-associated diseases in mice provides a model to test inhibitors of vFLIP as potential anticancer agents.
Gianna Ballon, Kang Chen, Rocio Perez, Wayne Tam, Ethel Cesarman
Kaposi sarcoma herpesvirus (KSHV), a human gammaherpesvirus, is the etiological agent for the endothelial-derived Kaposi sarcoma (KS) and also for certain lymphoproliferative disorders. In these lymphoproliferations, the KSHV-infected cells carry the stigmata of B lymphocytes, with plasmablastic features. The JCI has published three manuscripts addressing key questions related to B cell infection and viral latent expression in B cells. Myoung and Ganem provide evidence that CD4+ lymphocytes suppress KSHV replication, promoting latency in B cells; Hassman and colleagues show that KSHV infection drives plasmablast differentiation in a subset of IgM+ λ light chain–expressing cells; and Ballon and colleagues describe the in vivo transdifferentiation of B lymphocytes by KSHV-encoded viral FLICE-inhibitory protein (vFLIP).
Two lymphotropic human herpesviruses are linked to lymphoma development: EBV and Kaposi sarcoma herpesvirus (KSHV). The mechanisms by which EBV infects B lymphocytes and induces their differentiation and proliferation are reasonably well understood (1). In vitro, EBV infection of human primary B cells causes the establishment of latent infection in a fraction of cells exposed to virus, cellular transformation, and the outgrowth of indefinitely proliferating B lymphoblastoid cell lines. In contrast, the lack of B cell systems available for the study of KSHV in vitro and in vivo has hampered our understanding of the natural life cycle of KSHV in B cells and of KSHV-induced B cell lymphoproliferations. The JCI has now published three papers (2–4) that reveal provocative findings regarding KSHV and B cell infection and function.
The main route for infection by EBV and KSHV is via saliva. EBV enters tonsillar B cells via the CD21 receptor and steers the differentiation of pregerminal naive B lymphocytes toward memory cells by way of viral latent transcripts. The presence of KSHV in saliva (5) and in tonsillar and peripheral CD19+ B cells (6) and the inefficient in vitro infection of primary nonstimulated B lymphocytes from PBMCs prompted the groups of Don Ganem (2) and Dean Kedes (3) to utilize primary tonsillar explants to study KSHV infection ex vivo. Previously, efficient productive or lytic infection of IL-4 and CD40 ligand–activated PBMC-derived B lymphocytes and infection of B lymphocytes from tonsils were demonstrated (7). It is unclear whether activation of B lymphocytes results in the upregulation of surface molecules required for KSHV infection, for example, heparin sulfate (8) and DC-SIGN (CD209) (7), and/or whether such activation triggers signaling pathways that encourage viral entry and intracellular transport (9).
Myoung and Ganem showed that exposure of primary human tonsillar explants to KSHV virions results in infection of B and T lymphocytes, with B lymphocytes producing substantial amounts of infectious virions (2). Strikingly, and in contrast to exposure of B lymphocytes to EBV, KSHV displays predominantly lytic infection in tonsillar-derived B lymphocytes. This spontaneous lytic viral reactivation of infected B lymphocytes was suppressed when the investigators added activated T lymphocytes from tonsillar explants. However, these activated CD4+ T lymphocytes did not induce B lymphocyte cytolysis and were not dependent on autologous T lymphocytes being used. Thus, the suppression of spontaneous viral lytic cycle entry in B lymphocytes was MHC unrestricted and not dependent on killing of target cells. Treatment of mixed cultures with the T cell inhibitor, cyclosporine, abrogated the inhibition of lytic replication. Myoung and Ganem found that activated viable T lymphocytes require physical contact with the infected B lymphocytes to inhibit lytic virus replication. They therefore proposed that unidentified effector T cell surface ligands are responsible for T cell–target cell recognition and might trigger an exocytosis event in the effector T cells, releasing factors to the KSHV-infected B lymphocytes.
These in vitro findings contrast with what we have learned about primary EBV infection (10): the current paradigm is that lack of functional T lymphocytes, for example, induced by iatrogenic or acquired immunosuppression, leads to the in vivo outgrowth of latent infected B lymphocytes and subsequent EBV-driven lymphoproliferations such as posttransplant lymphoproliferative disease. Myoung and Ganem propose that T lymphocyte activation is necessary to block KSHV lytic reactivation in B lymphocytes, promoting latent infection (Figure 1).
Early events after EBV and KSHV infection of tonsillar cells. (A) EBV is amplified by permissive epithelial cells (lytic infection) and infects mucosal naive B cells. The viral default pathway in B cells is latent infection, where EBV persists as an episome (red circle). A minority of infected B cells are transformed (TrB). In infectious mononucleosis, a significant expansion of transformed lymphoblastoid cells occurs. Anti-EBV antigen CD4+ and CD8+ T cells control the proliferation of transformed cells. EBV persists in B lymphocytes, as part of the long-lived memory B cell pool (MeB). (B) Early events during KSHV infection are less established. It is uncertain whether B cells become infected after amplification of KSHV in epithelium. Data presented in this issue suggest that KSHV induces significant spontaneous lytic replication ex vivo in tonsillar-derived B cells (2). This lytic infection is suppressed when infected B cells come in contact with activated CD4+ T cells. The suppression of spontaneous viral lytic cycle entry in B cells is MHC unrestricted and not dependent on killing of target cells. New data also suggest that the primary target for KSHV in tonsillar explants could be IgM+ memory B cells (3). The majority of KSHV latent infected B cells after exposure of tonsillar explants to virus express IgMλ. KSHV induces plasmablastic differentiation of these IgMλ+ cells. An enrichment of IgMλ-expressing plasmablasts also occurs in inducible vFLIP knockin mice, targeting vFLIP to different stages of B cell proliferation (4). Such IgMλ+ infected plasmablasts are thought to cause KSHV-related MCD. Lytic infected cells are indicated by disrupted nuclear membranes.
It is unclear whether B lymphocytes display de novo entry into the lytic cycle (i.e., the lytic cycle being the default pathway) or whether high level spontaneous reactivation occurs from latency. It will be of significant interest to know whether these intriguing findings occur in vivo and whether this suppression of lytic reactivation is restricted to primary infection within the tonsillar microenvironment.
Prior to the introduction of effective anti-HIV treatment, it was noted that nearly 50% of those who acquired KSHV after HIV infection went on to develop Kaposi sarcoma (KS). Thus, being exposed to KSHV in the setting of a damaged T lymphocyte immune response would result in a higher KSHV viral load set point and an increased risk of developing KS. The findings by Myoung and Ganem could help explain this observation: a diminished T lymphocyte response upon primary KSHV infection would result in enhanced spontaneous B lymphocyte reactivation and a higher viral load.
B cell neoplasia associated with KSHV infection includes the monoclonal primary effusion lymphoma (PEL) (11) and a plasmablastic variant of multicentric Castleman disease (MCD) (12), a polyclonal neoplasm. In both these tumors, KSHV infection is associated with preterminally differentiated plasma cells with either cytoplasmic or surface immunoglobulins. Mature B cells exhibit allelic exclusion in which only a single class of Ig heavy chain and a single class of light chain, κ or λ, is expressed. Light chain restriction in a B cell population is usually considered proof of monoclonality and therefore cancer. However, KSHV-infected plasmablasts in MCD are an exception, in which polyclonal expansion of IgMλ light chain–expressing KSHV-infected plasmablasts is the hallmark (refs. 13–15 and Figure 2). This is a curiosity, as almost all other lymphoproliferative disorders demonstrate both κ and λ light chain restriction, and no functional difference between these light chains is identified. This observation suggests that KSHV either preferentially infects λ light chain–expressing lymphocytes, that λ light chain cells provide a survival and/or proliferation advantage to KSHV-infected B cells, or that KSHV preferentially steers expansion of λ-expressing cells.
KSHV-related plasmablastic MCD. (A) KSHV-LANA–positive cells are scattered throughout the mantle zone of a lymph node affected by MCD. (B) Higher magnification of MCD mantle zone. The KSHV-positive cells (black arrowheads) are large, have prominent nuclei with 1 or 2 nucleoli, and resemble plasmablastic cells. Lymphocytes surrounding the plasmablasts stain negative for KSHV. Reprinted with permission from ref. 13 (copyright 1999, National Academy of Sciences, USA). Original magnification, ×140.
The Kedes group investigated whether KSHV preferentially infects plasmablasts or infects a less differentiated cell and actively drives them toward a plasmablast phenotype (3). In a series of elegant experiments, Hassman et al. employed multispectral imaging flow cytometry, which combines the high-throughput power of flow cytometry with the morphological and subcellular spatial detail of multicolor fluorescent imaging to identify and characterize KSHV-infected cells. First they demonstrated that within ex vivo suspensions of human tonsillar cells, KSHV infection (as measured by cells expressing the latency-associated nuclear antigen [LANA]) preferentially occurred in B cells. Even though both light chain subsets were present in the tonsillar cultures in comparable proportions, LANA-positive cells were almost exclusively observed in the λ subset. They show further that KSHV infection drives the proliferation of IgMλ tonsillar B cells. In addition, infected cells acquired phenotypic changes mimicking MCD plasmablasts, including the blasting morphology, IgM expression, and high levels of IL-6 receptor expression.
Hassman et al. raise an intriguing, but plausible possibility: rather than targeting naive B cells, KSHV preferentially infects and drives the proliferation of IgM+ memory B cells. Such cells are present in tonsils and spleen and become IgMhi, Ki67+, and CD27+ during plasmablast differentiation (16). Such differentiation might be triggered by KSHV-induced NF-κB activation (see below), and such a phenotype is compatible with the plasmablasts present in their infected tonsillar cultures and in MCD.
Similar to other herpesviruses, only a fraction of KSHV open reading frames are expressed during latency, with the majority being expressed when the virus is triggered to enter the lytic program, resulting in cells exuding infectious virions. Among these latent viral proteins is the viral FADD-like IL-1β–converting enzyme (FLICE/caspase 8) inhibitory protein (vFLIP). Whereas cellular FLIP proteins associate with FADD/DISC, preventing caspase-8–induced apoptosis, vFLIP in KSHV-infected cells, including PEL cells, is mainly associated with the IκB kinase (IKK) complex, leading to IκBα degradation, followed by release of NF-κB (17, 18). Activation of NF-κB is a mechanism exploited by lymphomagenic viruses to prolong cellular survival and to induce proliferation. EBV and human T cell leukemia virus 1 (HTLV-1) encode viral oncoproteins, LMP1 and Tax, constitutively activating NF-κB (19, 20).
The Cesarman group generated inducible vFLIP knockin mice, targeting vFLIP to different stages of B cell proliferation (4). The activation of transgene expression was achieved by crossing the vFLIP knockin mice with mice expressing cre recombinase under the control of either the CD19 or Cγ1 promoter, resulting in vFLIP expression in all CD19+ B cells or more restrictedly, in IgG1+ germinal center (GC) B cells. Within three months, TG mice developed splenomegaly with erased GCs and defective Ig class switch recombination (CSR) and affinity maturation. Within 20 months, most TG animals developed histiocytic/DC sarcoma of B cell origin.
Although these vFLIP TG mice did not develop tumors faithfully mimicking either human MCD or PEL, a number of striking findings were made. First, an enrichment of IgMλ-expressing plasmablasts occurred in all mice. The findings in these transgenic mice support the conclusion that KSHV latent transcripts, including vFLIP, are responsible for the preferential expansion of IgMλ+ cells. This is supported by the findings of Hassman et al., who showed that KSHV selectively established latent (LANA+) infection almost exclusively in Igλ B cells, despite the fact that both Igκ and Igλ B lymphocytes expressed the lytic viral protein PAN after exposure of CD19+ lymphocytes to KSHV (3), and by the discovery that λ-chain–positive B cell development is dependent on NF-κB (21). Second, the Ballon et al. study reveals the in vivo immunological consequences of vFLIP when expressed in B cells. The abrogation of GC formation and inhibition of CSR and affinity maturation by vFLIP could directly contribute to KSHV pathogenesis by curtailing host immunity. Third, this work indicates the in vivo capacity of vFLIP to reprogram/transdifferentiate B cells into histiocytes. This finding supports the clonal B cell origin of histiocytic sarcoma, which can develop in individuals with B cell lymphoproliferations (22), including KSHV-related MCD, and argues that NF-κB signaling contributes directly to the in vivo plasticity of B cells.
These three studies are sound foundations for further investigations of KSHV infection in vivo. Humanized mouse models could be used to test the in vivo relevance of the studies by Myoung et al. and Hassman et al. and to decipher the molecular mechanisms favoring IgMλ B lymphocyte expansion and B cell transdifferentiation (2, 3, 23). Furthermore, the vFLIP transgenic mice described by Ballon et al. could be crossed with mice expressing other KSHV latency–associated transcripts and complement the development of inducible TG mice expressing more than one latent transcript (3). We could also speculate that the activation of vFLIP (with or without other latent viral transcripts) at a more mature stage of B cell differentiation, such as in post-GC B cells, will permit completion of the GC reaction and better recapitulate KSHV-lymphoproliferation development. These three studies are opening up new avenues to explore the immunobiology of KSHV as it relates to its principal reservoir, B lymphocytes.
Chris Boshoff is supported by Cancer Research UK, the Medical Research Council, and the UCL/University College London Hospital Comprehensive Biomedical Research Centre.
Conflict of interest: The author has declared that no conflict of interest exists.
Reference information: J Clin Invest. 2011;121(3):838–841. doi:10.1172/JCI46499.
See the related articles at KSHV infects a subset of human tonsillar B cells, driving proliferation and plasmablast differentiation, Kaposi sarcoma herpesvirus (KSHV) vFLIP oncoprotein induces B cell transdifferentiation and tumorigenesis in mice, and Active lytic infection of human primary tonsillar B cells by KSHV and its noncytolytic control by activated CD4+ T cells.