Figure 2
Figure 2. Impact of LMP-1 aggregation, signaling, and the immunoproteasome on presentation of LMP-1 epitopes through the MHC class I pathway. (A) Schematic representation of LMP-1 constructs used in the study. Expression constructs encoding either the full-length LMP-1 (referred to as wtLMP-1) or a series of deletion/point mutants expressing different forms of LMP-1 protein were used. The deletion mutant was designed to express LMP-1 protein in which the first transmembrane domain was deleted (referred to as Δ1-43LMP-1). The expression vector with point mutations within the CTAR domains of LMP-1 (referred to as PQT > AAA, Y384G, or PQT > AAA/Y384G) blocks TRAFF/TRADD binding and NF-κB activation.8,18 (B) C33A cells were transiently transfected with the expression vectors encoding wtLMP-1, Δ1-43LMP-1, Y384G, PQT > AAA, and PQT/AAA/Y384G. These cells were incubated with YLQQNWWTL-specific T cells at a responder-to-stimulator ratio of 10:1 and then assessed for intracellular IFN-γ production by flow cytometry (the first 3 amino acids of the sequences are underlined to indicate the abbreviation used for each epitope in the figure). Data represent the percentage of IFN-γ-producing YLQQNWWTL-pentamer–positive cells. (C) Intracellular location was assessed by fluorescent microscopy. (D) HEK293 and C666.1 cells were transiently transfected with vectors encoding wtLMP-1 or Δ1-43LMP-1 fused to GFP. Transfected cells were incubated with either YLQQNWWTL-specific T cells (HEK293 cells) or IALYLQQNW-specific T cells (C666.1) at responder-to-stimulator ratio of 10:1 and then assessed for intracellular IFN-γ production by flow cytometry. Data represent the percentage of IFN-γ-producing YLQ-pentamer–positive cells or IAL-specific cells. (E) LMP-1-GFP expression in epithelial cells transfected with expression vectors encoding wtLMP-1 or Δ1-43LMP-1 fused to GFP. (F) YLQQNWWTL-specific T cells were incubated with T1 and T2 cells at responder-to-stimulator ratios of 10:1, 20:1, and 40:1 and then assessed for intracellular IFN-γ production by flow cytometry. Data represent the percentage of YLQQNWWTL-pentamer–positive cells producing IFN-γ. (G) HLA-matched LCL and Δ1-43LMP-1-transfected C33A cells were treated with the proteasomal inhibitors, lactacystin and MG132 (as a control C33A cells were also treated with the lysosomal inhibitor chloroquine), and then incubated with YLQQNWWTL-specific T cells at a responder-to-stimulator ratio of 10:1. Data represent the percentage of YLQQNWWTL-pentamer–positive cells producing IFN-γ.

Impact of LMP-1 aggregation, signaling, and the immunoproteasome on presentation of LMP-1 epitopes through the MHC class I pathway. (A) Schematic representation of LMP-1 constructs used in the study. Expression constructs encoding either the full-length LMP-1 (referred to as wtLMP-1) or a series of deletion/point mutants expressing different forms of LMP-1 protein were used. The deletion mutant was designed to express LMP-1 protein in which the first transmembrane domain was deleted (referred to as Δ1-43LMP-1). The expression vector with point mutations within the CTAR domains of LMP-1 (referred to as PQT > AAA, Y384G, or PQT > AAA/Y384G) blocks TRAFF/TRADD binding and NF-κB activation.8,18  (B) C33A cells were transiently transfected with the expression vectors encoding wtLMP-1, Δ1-43LMP-1, Y384G, PQT > AAA, and PQT/AAA/Y384G. These cells were incubated with YLQQNWWTL-specific T cells at a responder-to-stimulator ratio of 10:1 and then assessed for intracellular IFN-γ production by flow cytometry (the first 3 amino acids of the sequences are underlined to indicate the abbreviation used for each epitope in the figure). Data represent the percentage of IFN-γ-producing YLQQNWWTL-pentamer–positive cells. (C) Intracellular location was assessed by fluorescent microscopy. (D) HEK293 and C666.1 cells were transiently transfected with vectors encoding wtLMP-1 or Δ1-43LMP-1 fused to GFP. Transfected cells were incubated with either YLQQNWWTL-specific T cells (HEK293 cells) or IALYLQQNW-specific T cells (C666.1) at responder-to-stimulator ratio of 10:1 and then assessed for intracellular IFN-γ production by flow cytometry. Data represent the percentage of IFN-γ-producing YLQ-pentamer–positive cells or IAL-specific cells. (E) LMP-1-GFP expression in epithelial cells transfected with expression vectors encoding wtLMP-1 or Δ1-43LMP-1 fused to GFP. (F) YLQQNWWTL-specific T cells were incubated with T1 and T2 cells at responder-to-stimulator ratios of 10:1, 20:1, and 40:1 and then assessed for intracellular IFN-γ production by flow cytometry. Data represent the percentage of YLQQNWWTL-pentamer–positive cells producing IFN-γ. (G) HLA-matched LCL and Δ1-43LMP-1-transfected C33A cells were treated with the proteasomal inhibitors, lactacystin and MG132 (as a control C33A cells were also treated with the lysosomal inhibitor chloroquine), and then incubated with YLQQNWWTL-specific T cells at a responder-to-stimulator ratio of 10:1. Data represent the percentage of YLQQNWWTL-pentamer–positive cells producing IFN-γ.

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