Downregulation of the T-cell receptor complex and impairment of T-cell activation by human herpesvirus 6 u24 protein

Abstract

We have performed a screen aimed at identifying human herpesvirus 6 (HHV-6)-encoded proteins that modulate immune recognition. Here we show that the U24 protein encoded by HHV-6 variant A downregulates cell surface expression of the T-cell receptor (TCR)/CD3 complex, a complex essential to T-cell activation and the generation of an immune adaptive response. In the presence of U24, the TCR/CD3 complex is endocytosed but is not recycled back to the plasma membrane. Instead, it accumulates in early and late endosomes. Interestingly, whereas CD3 downregulation from the cell surface is normally associated with T-cell activation, U24 downregulates CD3 independently of T-cell activation. Moreover, we found that U24-expressing T cells are resistant to activation by antigen-presenting cells. HHV-6 has evolved a unique mechanism of inhibition of T-cell activation that may impair the establishment of an adaptive immune response. Furthermore, lymphocyte activation creates an environment favorable to the reactivation and replication of lymphotropic herpesviruses. Thus, by inhibiting T-cell activation, HHV-6 might limit its reactivation and thus minimize immune recognition.

FIG. 1.

Downregulation of TCR/CD3 from the surfaces of U24-expressing Jurkat cells. (A) Jurkat cells were transiently transfected with a bicistronic transcript encoding several HHV-6A gene products (U4, U21, U24, U61, U78) and EGFP. Twenty-four hours posttransfection, surface CD3ɛ in cells displaying GFP expression was analyzed by flow cytometry. (B) Jurkat cells were transiently transfected either with a bicistronic transcript of U24 and EGFP (U24/EGFP) or with EGFP alone. Cells were collected after 24 h, stained for surface CD3ɛ or αβ TCR, and analyzed by flow cytometry. (C) Jurkat cells were transiently transfected with U24/EGFP, collected after 24 h, and stained with antibodies for the indicated cell surface receptors. (D) Jurkat cells were transiently transfected with U24/EGFP or EGFP alone and collected after 24 h. (Left) Cells were fixed and permeabilized to stain for total CD3ɛ and were then analyzed by flow cytometry. (Right) Percentage of CD3 remaining in U24-expressing cells compared to that in EGFP controls. Data used in this analysis were from the mean fluorescence of cells stained with PE-conjugated anti-CD3ɛ antibodies gated on EGFP-expressing cells. Data compiled from at least three consecutive independent experiments were analyzed.

FIG. 2.

U24 does not activate Jurkat T cells. (A) Jurkat cells deficient in either lck or zap70 were transiently transfected with U24/EGFP or EGFP alone. Twenty-four hours posttransfection, levels of surface CD3ɛ in cells displaying GFP expression were analyzed by flow cytometry. (B) Jurkat cells were cotransfected with firefly luciferase driven by promoters essential to T-cell activation and either U24 or vector alone. Experiments were performed in triplicate, and data were normalized with Renilla luciferase. (C) Jurkat cells were transfected with U24/EGFP or EGFP alone and were stained for CD69. Activated EGFP-transfected Jurkat cells were stimulated with PMA and ionomycin for 12 h before staining for CD69.

FIG. 3.

U24 impairs antibody-mediated internalization of CD3ɛ and inhibits recycling. (A) Jurkat cells were transfected with U24/EGFP or EGFP alone. After 24 h, cells were stained at 4°C with PE-conjugated anti-CD3ɛ antibodies, and unbound antibodies were washed away. Cells were then placed at 37°C and allowed to internalize for various times. Surface antibodies were removed with an acid wash, and mean fluorescence was determined by flow cytometry. (B) Jurkat cells were transfected with U24/EGFP or EGFP alone. After 24 h, cells were incubated with PE-conjugated anti-CD3ɛ antibodies at 37°C to allow for internalization. Surface-bound antibodies were stripped with an acid solution, and the cells were again incubated at 37°C. Samples were taken at various time points over a period of 1 h. Cells were again stripped, and the mean fluorescence was determined by flow cytometry.

FIG. 4.

U24 blocks CD3ɛ access to Rab11 recycling endosomes. Cells expressing GFP-Rab proteins and either U24/EGFP (right panels) or EGFP alone (left panels) were sorted for high GFP expression that corresponded to low surface CD3 expression. These cells were then fixed, permeabilized, and stained with an anti-CD3ɛ antibody (OKT3) and a rhodamine-conjugated secondary antibody. Cells were visualized using a Zeiss 510 META/NLO confocal microscope with a 63× objective, and images were magnified six times. Rab5 and Rab4 are markers of early and sorting endosomes, respectively. Rab9 is a marker of late endosomes, and Rab11 is a marker of recycling endosomes. Yellow arrows indicate areas of colocalization, while white arrows indicate areas of noncolocalization.

FIG. 5.

Upregulation of an early T-cell activation marker, CD69, is blocked in U24-expressing cells. Jurkat T cells expressing U24/EGFP or EGFP alone were incubated with SEE-pulsed or nonpulsed BJAB B cells for 6 h. EGFP-positive populations were analyzed for CD69 upregulation by flow cytometry. Max, maximum.