Relative expression levels of the HLA class-I proteins in normal and HIV-infected cells

Abstract

The expression level of HLA class-I proteins is known to influence pathological outcomes: pathogens downregulate HLA to evade host immune responses, host inflammatory reactions upregulate HLA, and differences among people with regard to the steady-state expression levels of HLA associate with disease susceptibility. Yet precise quantification of relative expression levels of the various HLA loci is difficult because of the tremendous polymorphism of HLA. We report relative expression levels of HLA-A, HLA-B, HLA-C, and HLA-E proteins for the specific haplotype A*02:01, B*44:02, C*05:01, which were characterized using two independent methods based on flow cytometry and mass spectrometry. PBLs from normal donors showed that HLA-A and HLA-B proteins are expressed at similar levels, which are 13-18 times higher than HLA-C by flow cytometry and 4-5 times higher than HLA-C by mass spectrometry; these differences may reflect variation in the conformation or location of proteins detected. HLA-E was detected at a level 25 times lower than that of HLA-C by mass spectrometry. Primary CD4(+) T cells infected with HIV in vitro were also studied because HIV downregulates selective HLA types. HLA-A and HLA-B were reduced on HIV-infected cells by a magnitude that varied between cells in an infected culture. Averaging all infected cells from an individual showed HLA-A to be 1-3 times higher and HLA-B to be 2-5 times higher than HLA-C by flow cytometry. These results quantify substantial differences in expression levels of the proteins from different HLA loci, which are very likely physiologically significant on both uninfected and HIV-infected cells.

Fig. 1. Representative flow cytometry staining of each HLA locus on CD4 cells purified from normal donor 2 and infected in vitro with HIV

(A) After gating the CD3+ CD8- population, CD4 downregulation was used to discriminate infected (CD4-) from uninfected (CD4+) cells in a culture (x-axis). Staining for each classical HLA class-I locus was then measured (y-axis). (B) Relative staining of HLA-A (green), HLA-B (red), HLA-C (blue) and isotype control (black) is shown in separate histograms for uninfected (left) and infected (right) cells from the culture.

Fig. 2. Peptides unique to each HLA used for quantitation in a mass spectrometry-based assay

Mature protein sequence of each HLA class-I protein present in the individuals studied is shown. Peptides unique to an HLA locus within this individual that were used for quantitation are highlighted: 3 peptides for HLA-A (yellow), 4 peptides for each of HLA-B (green) and HLA-C (blue) and 2 peptides for HLA-E (red). In the HLA-E sequence X represents either R or G.

Fig. 3. Representative mass spectrometry fragmentation spectrum

The doubly charged HLA-A specific peptide WEAAHVAEQLR, detected for normal PBL donor 1, is shown visualized using Scaffold software. N-terminal b fragment ions are labeled in red, C-terminal y fragment ions are labeled in blue and other fragment ions are labeled in green.