A viral protein that selectively downregulates ICAM-1 and B7-2 and modulates T cell costimulation (original) (raw)
K5 but not K3 downregulates B7-2 and ICAM-1. To address the possibility that K3 or K5 could modulate other APC components implicated in immune activation, we transduced a B cell line (BJAB) with retroviral vectors expressing K3 or K5. Mass cultures of each transduction, representing pools of more than 100,000 colonies of primary transductants, were selected for assay. Each pool displayed strong downregulation of MHC-I and β2-microglobulin (β2m), as judged by flow cytometry. (Figure 1). These cells were then assayed for surface levels of B7-1 (CD80), B7-2 (CD86), and ICAM-1 (CD54). As shown in Figure 1, K3 expression did not influence the surface distribution of any of these molecules. However, K5 expression diminished B7-2 surface expression 60- to 100-fold, while it had no impact on B7-1 surface accumulation. Cells expressing K5 displayed an equally large decrement in ICAM-1 surface staining. These decrements were specific: neither K5 (Figure 1) nor K3 (data not shown) reduced the surface levels of MHC-II, CD43, CD58, CD40, CD48, CD33, CD 22, CD20, CD19, or CD18. Thus, this downregulation is not the result of global redistribution of all polypeptides from the BJAB cell membrane. It is also not the result of K5 overexpression: levels of K5-specific mRNA are, in fact, lower in these lines than in lytically induced BCBL-1 cells (L. Coscoy, unpublished data). While this manuscript was under review, similar results were reported by Ishido et al. (39).
Surface expression of molecules associated with T cell activation. BJAB cells were transduced with PMX-pie (as control), PMX-FlagK5, or PMX-HaK3 retroviral vectors. Cells were stained with mAb’s directed against B cell membrane proteins associated with T cell activation (MHC-II, B7-2, B7-1, ICAM-1, CD58, CD45, and CD43) as well as other B cell membrane proteins as control (CD48, CD40, CD33, CD22, CD20, CD19, CD18, β2m). Bound Ab’s were revealed by an R-phycoerythrin–conjugated goat anti-mouse Ab, and fluorescence intensity was monitored by flow cytometry. WT, wild-type.
To explore the fate of the B7 and ICAM chains, we asked if the disappearance of these chains from the cell surface, like that of MHC-I and β2m chains, was due to enhanced endocytosis. To do this we asked if surface levels of the downregulated proteins could be restored by coexpression of a dynamin dominant negative mutant. Dynamin is an essential participant in endocytosis, and its inhibition selectively blocks the endocytic pathway. The dynamin K44E mutation is negative for dynamin function and strongly inhibits endogenous (wild-type) dynamin function (40), making it a useful reagent for this purpose. Figure 2a shows that, in the presence of K44E, but not wild-type dynamin, surface levels of both B7-2 (CD86) and ICAM-1 (CD54) are restored to nearly normal levels. This effect is specific, since overexpression of K44E does not affect the cell surface expression of other molecules, for example CD58 (Figure 2a).These data indicate that, as for MHC-I, the downregulatory effect of K5 protein is exerted chiefly through enhanced endocytosis of its ICAM and B7 targets.
(a) Endocytosis of B cell membrane proteins in K5 expressing BJAB. BJAB cells expressing K5 were transfected with 20 μg of an expression vector for a Ha-tagged dynamin transdominant mutant (known to specifically block endocytosis events) or for an expression vector for the Ha-tagged wild-type dynamin (as control). Forty-eight hours after transfection, cells were incubated with mAb directed against either MHC-I, B7-2, ICAM-1, or CD58 and bound mAb’s were revealed by incubation with an R-phycoerythrin–conjugated goat anti-mouse Ab. Cells were then fixed, incubated in presence of saponin with a fluorescein-conjugated mAb directed against the Ha tag, and analyzed by flow cytometry. Results represent the cell surface expression of MHC-I, B7-2, ICAM-1, and CD58 proteins in the dynamin-transfected cells. (b) BJAB cells expressing K5 or control cells were incubated for 12 hours in presence of 50 mM ammonium chloride. Cells were then fixed, incubated with mAb’s against B7-2 and ICAM-1 in the presence of saponin, and analyzed by flow cytometry.
What is the fate of the chains that have undergone endocytosis? Figure 2b shows that the reduced levels of surface staining of both molecules is mirrored by a reduced level of intracellular accumulation of the chains, as revealed by flow-cytometric analysis in the presence of saponin. This suggests that the chains underwent degradation after internalization. This degradation can be partially relieved by exposure of the cells to ammonium chloride, which raises endolysosomal pH and impairs protease activity in this compartment (Figure 2b). This suggests that most newly internalized chains are degraded by endolysosomal proteases.
Functional significance of K5-mediated B7-2 and ICAM downregulation. We next tested the functional significance of ICAM-1 and B7-2 downregulation in T cell stimulation accompanying antigen presentation. Since K5 does not function in murine cells (ref. 35; L. Coscoy, unpublished data), we were unable to use standard in vitro assays involving antigen-specific stimulation of primary T cells from TCR-transgenic mice. Therefore, we used instead a model system that employs cultured human T and B cell lines and replaces conventional antigen with staphylococcal superantigen (41). Superantigens mimic conventional antigens in that they lead to functional interactions of TCR with MHC molecules; however, they have the advantage of allowing recognition of multiple TCR v-regions, producing strong T cell responses. Our assay measures the ability of BJAB cells (bearing or lacking K5 genes) to stimulate Jurkat T cells in the presence of SEE. Activation is assayed by the upregulation of several transcription factors known to be stimulated by T cell-APC engagement, including NFAT and AP-1. Since their upregulation is resistant to CTLA4-Ig, NFAT and AP-1 activation reflect stimulatory pathways other than those mediated by B7-CD28 (42), most likely those mediated by TCR-based ITAM signaling. (These pathways, in turn, are known to be augmented by ICAM/LFA-1 interactions.) To assay for B7-CD28–dependent pathways, we took advantage of the fact that such pathways activate a composite transcription factor–binding site known as RE/AP (42). Luciferase genes under the control of the RE/AP element faithfully recapitulate CD28-dependent costimulation, and their expression is blocked by CTLA4-Ig (42). In fact, upregulation of this reporter correlates closely with enhanced IL-2 release during T cell activation (42).
Accordingly, Jurkat cells were transiently transfected with luciferase reporters under the control of either the RE/AP, NFAT, or AP-1 regulatory elements from the IL-2 promoter. Sixteen hours after transfection, these Jurkat cells were stimulated by incubation with SEE plus control BJAB cells or K5-expressing BJAB, and luciferase activity was measured 12 hours later. As shown in Figure 3a, incubation of transfected Jurkat cells with BJAB or BJAB-K5 alone without SEE was insufficient for activation of the RE/AP-dependent promoter. As expected, activation of this reporter was strongly induced by the combination of BJAB and SEE. However, this activation was greatly reduced (60–80%) by the expression of K5 in the BJAB line, indicating that reduction in B7-2 strongly attenuated T cell activation. There was, however, a small amount of residual RE/AP activation observed in the presence of K5 (Figure 3a). Unlike primary B cells, which do not express B7-1 in the resting state, BJAB cells constitutively express both B7-1 and B7-2; we speculated that this residual activation was due to the presence of B7-1 on the BJAB cell surface. Consistent with this, this residual RE/AP activity could be ablated by incubating the BJAB-K5/Jurkat cocultures with anti–B7-1 Ab (Figure 3a). Thus, K5 expression effectively removes virtually all functional B7-2 from the cell surface. The significance of the remaining B7-1 will be considered in the Discussion section.
T cell activation by K5 expressing BJAB cells. (a) Twenty-four hours after transfection with a RE/AP reporter plasmid, Jurkat cells were stimulated by incubation with BJAB cells and SEE. Jurkat activation was quantified by luciferase assays 16 hours after stimulation. Each luciferase assay was done in triplicate on at least three different independent transfection experiments. For blocking conditions, BJAB cells were preincubated with Ab’s 30 minutes before addition of Jurkat cells and SEE. (b) Jurkat cells were transfected with NFAT, AP-1, or NF-κB reporter plasmid and stimulated as in a. Stimulation with TPA/ionomycin was used as positive control. (c) Jurkat cells were left unstimulated, stimulated with PMA/iono, or stimulated with BJAB cells and SEE. After 16 hours, cells were stained with an R-phycoerythrin–conjugated anti-CD19 Ab and a fluorescein-conjugated anti-CD69 Ab. Jurkat cells were identified as being negative for CD19 (a B cell marker, present at the cell surface of BJAB cells). Histograms represent CD69 expression on Jurkat cells.
Using an identical assay format, but replacing the RE/AP reporter in Jurkat cells with a luciferase reporter dependent upon NFAT activation, we found that substantially less NFAT activity was induced in T cells stimulated by BJAB-K5 cells than by control BJAB cells (Figure 3b). A smaller but still significant inhibition of AP-1 activation was likewise observed when K5 was expressed in the SEE-presenting BJAB cells (Figure 3b). SEE-dependent NF-κB activation was also strongly diminished by K5 expression (Figure 3b). Thus, pathways that are not dependent upon B7-CD28 interactions are also downregulated by K5 expression. ICAM-LFA interactions are known to contribute to some of these activation events (43, 44).
The ability of K5 to impair T cell activation was also assayed by examining surface expression of CD69 in the responding T cell. CD69 is a marker of cell activation that is prominently induced via TCR-mediated signaling. BJAB cells expressing K5 or a control vector were added to Jurkat cells in the presence of SEE, and the levels of CD69 expressed in Jurkat cells were examined by flow cytometry. As shown in Figure 3c, K5 expression in the APCs substantially impaired surface upregulation of CD69 in the responding T cells.
K5 expression does not lead to resistance of cells to NK-mediated cytolysis. In their earlier work, Ishido et al. (39) reported that K5 expression reduced the susceptibility of target cells to killing mediated by the NK cell line YTS. This cell line, derived from a leukemic patient, is very unusual in that it displays expression of CD28 and can recognize B7 molecules on the target cell surface (45). Ishido et al. showed clearly that the escape from YTS killing was due to K5-mediated downregulation of B7-2 and suggested that the function of K5 was to promote escape from NK cell killing (to which KSHV-infected cells would otherwise be susceptible based on the reduction of surface MHC-I molecules mediated by the joint action of K3 and K5) (33–35).
While the escape from YTS killing is clearly documented, it remains unclear whether K5’s prime function in vivo is to mediate escape from NK cell killing more generally. Figure 4 shows that when the NK cell population in the circulation of four independent normal human subjects is examined by flow cytometry, cells bearing NK markers (CD56) lack evidence of CD28 expression. Indeed, similar findings have been observed previously in cultured primary human NK cell lines expanded from the PBMC compartment (46). To determine the functional consequences of K5 expression for NK cell–mediated cytotoxicity, we employed a more representative NK cell line, NKL, in a killing assay. Like primary NK cells, NKL cells do not express surface CD28 (47). As expected, NKL cells efficiently killed control BJAB cells expressing the empty pcDNA3 vector (Figure 5). More importantly, however, BJAB cells engineered to express K5 not only displayed susceptibility to NKL killing but were, in fact, more sensitive to this killing than the controls (Figure 5). This result is in accordance with the diminished levels of MHC-I on such cells that result from the K5-stimulated endocytosis of class I chains (33, 34). While we do not observe strong effects of K5 on protection from NK-mediated cytolysis, we acknowledge that the paucity of cultured NK lines and their uncertain relationship to in vivo NK cells makes it difficult to entirely exclude such effects. Whatever their role in NK biology may be, the best-established functions of B7-2 and ICAM-1 are in the pathway of T cell costimulation, and our data strongly support a major role for K5 in the modulation of this pathway (see below).
Human peripheral blood–derived NK cell lines do not stain for CD28. Human PBMCs were isolated from heparinized venous blood from four healthy adult volunteer donors by Ficoll-density centrifugation. Cells were stained with saturating amount of fluorescein-conjugated anti-CD28, TriColor anti-CD3, and phycoerythrin-conjugated anti-CD56 Ab’s and analyzed by flow cytometry. Dot plots represent the CD28 and CD56 expression status of the CD3-negative population.
NKL mediate MHC-restricted cytotoxicity. Each target cell line was assessed for killing by the NKL cell line at various effector-to-target ratios (721.221 is a human B cell line lacking MHC-I molecules). Cytotoxicity was measured by 51Cr release after 4 hours.