HLA class I deficiencies due to mutations in subunit 1 of the peptide transporter TAP1

Abstract

The transporter associated with antigen processing (TAP), which is composed of two subunits (TAP1 and TAP2) that have different biochemical and functional properties, plays a key role in peptide loading and the cell surface expression of HLA class I molecules. Three cases of HLA class I deficiency have previously been shown to result from the absence of a functional TAP2 subunit. In the present study, we analyzed two cases displaying not only the typical lung syndrome of HLA class I deficiency but also skin lesions, and found these patients to be TAP1-deficient. This defect leads to unstable HLA class I molecules and their retention in the endoplasmic reticulum. However, the absence of TAP1 is compatible with life and does not seem to result in higher susceptibility to viral infections than TAP2 deficiency. This work also reveals that vasculitis is often observed in HLA class I-deficient patients.

Figure 1

Absence of maturation and instability of HLA class I molecules in HLA class I–deficient patients. (a) BRE and TND-3 cells from HLA class I–deficient patients and MRC-5 HLA class I⁺ control cells were metabolically labeled, lysed, and HLA class I molecules immunoprecipitated with W6/32 (pan anti–HLA class I heavy chains). Immunoprecipitates were treated (+N) or not (–N) with neuraminidase and analyzed by IEF. (b) BRE and TND-3 (HLA class I^–) and TND-4 (HLA class I⁺) cells were metabolically labeled for 20 min and chased for 0, 1, 2, or 4 hours. HLA class I molecules were immunoprecipitated with W6/32, treated (+) or not (–) with Endo H, and separated by SDS PAGE. (c) Cells were metabolically labeled for 30 min and chased for 30 min, and HLA class I molecules were immunoprecipitated with B1G6 (anti-β2m) and adsorbed on protein–agarose beads. The adsorbed proteins were further incubated for 0, 30, or 60 min at 37°C in lysis buffer, and the beads were washed. After treatment (+N) or not (–N) with neuraminidase, the immunoprecipitated proteins were eluted and analyzed by IEF. IEF, isoelectric focusing gel electrophoresis; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; β2m, β2-microglobulin.

Figure 2

BRE and TND-3 cell lines are TAP1 deficient. (a) Cells were labeled with the MAB W6/32 and analyzed by flow cytometry. Closed histograms, isotype control. Thick lines: left, BRE fibroblast cell line from patient 1; right, TND-3 EBV-B cell line from patient 2. Dotted lines: left, STF-1 TAP2-deficient fibroblast cell line; right, ST-EMO TAP2-deficient EBV-B cell line. Thin lines: left, STF-6 normal fibroblast cell line; right, ST-EAH normal EBV-B cell line. (b) Cells were infected overnight with TAP1 or TAP2 recombinant vaccinia viruses, labeled with W6/32, and analyzed by flow cytometry. Left, BRE cells from patient 1; right, TND-3 cells from patient 2. (c) BRE and TND-3 cells were incubated overnight at 26°C in culture medium (RPMI 1640 supplemented with 10% FCS; Life Technologies) in the presence of 100 μg/ml synthetic peptides, then labeled with MAB W6/32 and analyzed by flow cytometry. Closed histograms, isotype control. Thick lines, incubation without peptide. Thin lines: P1, HLA-A2402-specific peptide; P2, HLA-A2601-specific peptide. (d) Molecular genetic characterization of the mutation. Upper sequence, normal TAP1 sequence; lower sequences, corresponding TAP1 sequences in patients 1 and 2. G indicates the exon 2 nucleotide involved in the splicing reaction between exon 1 and exon 2, and arrows point to the positions of the two mutations.