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. 2001 Jun;107(12):1599-606.
doi: 10.1172/JCI12432.

A viral protein that selectively downregulates ICAM-1 and B7-2 and modulates T cell costimulation

L Coscoy  1 , D Ganem
Affiliations

A viral protein that selectively downregulates ICAM-1 and B7-2 and modulates T cell costimulation

L Coscoy et al. J Clin Invest. 2001 Jun.

Abstract

Kaposi's sarcoma-associated (KS-associated) herpesvirus (KSHV) is a B-lymphotropic agent linked to AIDS-related lymphoproliferative disorders and KS. We and others have earlier identified two viral genes, K3 and K5, that encode endoplasmic reticulum proteins that downregulate surface MHC-I chains by enhancing their endocytosis. Here we have examined the ability of these proteins to influence the disposition of other host surface proteins implicated in immune recognition and activation. We report that K5, but not K3, expression in BJAB cells dramatically reduces ICAM-1 and B7-2 surface expression; B7-1 expression is unaffected. This K5-induced reduction can be reversed by coexpression of a dominant negative mutant of dynamin, indicating that the loss of ICAM and B7-2 surface expression is due to their enhanced endocytosis. This downregulation is functionally significant, because K5-transfected B cells show substantial impairment in their ability to induce T cell activation. K5 is thus the first example of a viral modulator of immunological synapse formation and T cell costimulation. We propose that its expression reduces T cell responses to KSHV-infected B cells early in infection, thereby diminishing antiviral cytokine release and the production of stimulatory signals for CTL generation.

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Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
(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.
Figure 3
Figure 3
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.
Figure 4
Figure 4
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.
Figure 5
Figure 5
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.
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