LAMP-2-deficient human B cells exhibit altered MHC class II presentation of exogenous antigens

Abstract

Major histocompatibility complex (MHC) class II molecules present antigenic peptides derived from engulfed exogenous proteins to CD4(+) T cells. Exogenous antigens are processed in mature endosomes and lysosomes where acidic proteases reside and peptide-binding to class II alleles is favoured. Hence, maintenance of the microenvironment within these organelles is probably central to efficient MHC class II-mediated antigen presentation. Lysosome-associated membrane proteins such as LAMP-2 reside in mature endosomes and lysosomes, yet their role in exogenous antigen presentation pathways remains untested. In this study, human B cells lacking LAMP-2 were examined for changes in MHC class II-restricted antigen presentation. MHC class II presentation of exogenous antigen and peptides to CD4(+) T cells was impaired in the LAMP-2-deficient B cells. Peptide-binding to MHC class II on LAMP-2-deficient B cells was reduced at physiological pH compared with wild-type cells. However, peptide-binding and class II-restricted antigen presentation were restored by incubation of LAMP-2-negative B cells at acidic pH, suggesting that efficient loading of exogenous epitopes by MHC class II molecules is dependent upon LAMP-2 expression in B cells. Interestingly, class II presentation of an epitope derived from an endogenous transmembrane protein was detected using LAMP-2-deficient B cells. Consequently, LAMP-2 may control the repertoire of peptides displayed by MHC class II molecules on B cells and influence the balance between endogenous and exogenous antigen presentation.

Figure 1

Exogenous antigen presentation is defective in LAMP-2-deficient Danon B-LCL. (a) Cell lysates were prepared from Danon or wild-type Frev B-LCL, the proteins were resolved by gel electrophoresis, and immunoblotted with an antibody to LAMP-2 or LAMP-1. The blot was stripped and reprobed with an antibody to GAPDH. Data are representative of at least two independent experiments. (b) LAMP-2-deficient DB.DR4 or wild-type 7C3.DR4 cells were incubated with various concentrations of human serum albumin antigen overnight and then cultured with HSA_64–76-specific T cells to measure MHC class II presentation. (c, d) DB.DR4 or wild-type Frev cells were incubated with various concentrations of human IgG antigen overnight and then cultured with either κI_188–203-specific T cells (c) or κII_145–159-specific T cells (d) to measure MHC class II presentation. Neither DB.DR4 nor Frev expressed endogenous IgG κ light chain. Data are representative of three independent experiments, and the error bars indicate the mean T-cell activation ± the standard deviation. Note that the size of the symbols at some points masks the error bars.

Figure 2

Comparable expression of MHC class II messenger RNA and protein in Danon and wild-type B-LCL. (a) Total RNA was extracted from various wild-type or Danon B-LCL, complementary DNA was synthesized, and quantitative reverse transcription–polymerase chain reaction analysis performed using primers specific for HLA-DRα chain or for GAPDH as a control. Data are representative of the fold difference in messenger RNA expression observed in three independent experiments. (b) Wild-type Frev or LAMP-2-deficient DB.DR4 cells were incubated with L243 antibody to detect total surface HLA-DR and then stained with a fluorescein isothiocyanate-conjugated F(ab′)₂ fragment of goat anti-mouse IgG secondary antibody. The mean fluorescence intensity (MFI) as measured by flow cytometry indicates the level of surface HLA-DR, and data are the average MFI of three independent experiments. (c) Cell lysates were prepared from LAMP-2-deficient DB.DR4 or wild-type Frev B-LCL, the proteins resolved by gel electrophoresis under non-reducing conditions to preserve MHC class II dimers, and immunoblotted with an antibody to HLA-DRα chain. Data are representative of at least five independent experiments. The ratios of HLA-DRαβ dimers to GAPDH as a loading control were 1·5 and 1·3 for DB.DR4 and Frev, respectively. (d) Various wild-type or Danon B-LCL were first permeabilized and then incubated with L243 or W6/32 antibody to detect total intracellular HLA-DR or MHC class I molecules, respectively. Cells were then stained with a phycoerythrin-conjugated F(ab′)₂ fragment of rabbit anti-mouse immunoglobulin secondary antibody. The MFI as measured by flow cytometry indicates the levels of total surface or intracellular MHC class I or class II molecules. Data are the average MFI of three independent experiments. (e) Wild-type Priess and Frev expressing endogenous HLA-DR4 and wild-type 7C3.DR4 or LAMP-2-deficient DB.DR4 transfectants were incubated with 3.5.9-13F10 antibody to detect surface HLA-DR4β chains and then stained with a Cy2-conjugated F(ab′)₂ fragment of donkey anti-rat IgG secondary antibody. The MFI as measured by flow cytometry indicates the level of surface HLA-DR4, and data are representative of more than three independent experiments. (f) Various wild-type or Danon B-LCL were first permeabilized and then incubated with MaP.DM1 (top panel) or a monoclonal antibody to HLA-DO (bottom panel) to detect total intracellular HLA-DM or HLA-DO molecules, respectively. Cells were then stained with a phycoerythrin-conjugated F(ab′)₂ fragment of rabbit anti-mouse immunoglobulin secondary antibody. The MFI as measured by flow cytometry indicates the levels of total intracellular HLA-DM or HLA-DO molecules. Data for HLA-DM staining are the average MFI of three independent experiments while the data for HLA-DO staining are a representative experiment.

Figure 3

Endocytosis and MHC class II distribution in Danon and wild-type B-LCL. (a) Wild-type 7C3.DR4 or LAMP-2-deficient DB.DR4 B-LCL were incubated with fluorescein isothiocyanate (FITC) -albumin for 0 or 120 min at 37° and uptake was measured by flow cytometry. Data are representative of two independent experiments. (b) Wild-type Frev or LAMP-2-deficient DB.DR4 B-LCL were incubated with LysoTracker Red at 37° overnight to label acidic organelles, permeabilized, and then incubated with FITC-conjugated L423 antibody to detect HLA-DR. The results are representative of three independent experiments. (c) Wild-type Frev or LAMP-2-deficient DB.DR4 B-LCL were first permeabilized and then incubated with FITC-conjugated L423 antibody to detect HLA-DR and AlexaFluor647-conjugated-LAMP-1 Ab to detect LAMP-1. The results are representative of two independent experiments.

Figure 4

Efficient presentation of an endogenous antigen by Danon B-LCL. Variable numbers of DB.DR4 or 7C3.DR4 cells were cultured with HLA-A_52–70-specific, HLA-DR4-restricted T cells to measure MHC class II presentation of endogenous HLA-A. As a control, the HLA-DR4 transfected murine B cell CH27.DR4 was left untreated or incubated with 10 μm HLA-A_52–70 peptide and then cultured with HLA-A_52–70-specific, HLA-DR4-restricted T cells. Data are representative of three independent experiments, and the error bars indicate the mean T-cell activation ± the standard deviation.

Figure 5

LAMP-2-deficient DB.DR4 cells fail to efficiently present exogenous peptides to CD4⁺ T cells. (a) LAMP-2-deficient DB.DR4 or wild-type Priess cells were left untreated (UT) or incubated with 10 μm exogenous GAD_273–285 peptide for 4 hr and then cultured with GAD_273–285-specific T cells to measure MHC class II presentation. (b) DB.DR4 or wild-type 7C3.DR4 cells were left untreated (UT) or incubated with 10 μm exogenous HSA_64–76 peptide for 4 hr and then cultured with HSA_64–76-specific T cells to measure MHC class II presentation. (c, d) DB.DR4 or wild-type Frev cells were left untreated (UT) or incubated with 10 μm or 100 μm exogenous κI_188–203 peptide (c) or κII_145–159 peptide (d) for 4 hr and then cultured with κI_188–203-specific or κII_145–159-specific T cells, respectively, to measure MHC class II presentation. Data in (a–d) are representative of three independent experiments, and the error bars indicate the mean T-cell activation ± the standard deviation.

Figure 6

Acidic pH restores exogenous peptide presentation by LAMP-2-deficient DB.DR4. (a) LAMP-2-deficient DB.DR4 or wild-type 7C3.DR4 cells were left untreated (UT) or incubated overnight with 100 μm biotinylated κI_188–203 peptide. Lysates were prepared and added to plates coated with an anti-HLA-DR4 antibody to capture HLA-DR4 molecules in the lysates. The binding of biotinylated κI_188–203 peptide to captured HLA-DR4 was measured using europium-strepavidin. The results are the average of three independent experiments, and the error bars indicate the standard deviation ± between the experiments. (b, c) DB.DR4 or wild-type Frev cells were left untreated (UT) or incubated with 10 μm exogenous κI_188–203 peptide (b) or 10 μm exogenous κII_145–159 peptide (c) in either cell culture medium (pH 7) or in sodium phosphate/citric acid buffer (pH 5·5) for 4 hr and then cultured with κI_188–203-specific or κII_145–159-specific T cells, respectively, to measure MHC class II presentation. Data in (b, c) are representative of three independent experiments, and the error bars indicate the mean T-cell activation ± the standard deviation. (d) DB.DR4 or Frev cells were left untreated (UT) or treated with a NaCl/citric acid buffer to strip surface MHC class II before incubation with 10 μm exogenous κI_188–203 peptide for 4 hr. Cells were then cultured with κI_188–203-specific T cells to measure MHC class II presentation. Data are representative of two independent experiments, and the error bars indicate the mean T-cell activation ± the standard deviation.