Reappraisal of the relationship between the HIV-1-protective single-nucleotide polymorphism 35 kilobases upstream of the HLA-C gene and surface HLA-C expression

Abstract

Previous studies have found an association between a single-nucleotide polymorphism 35 kb upstream of the HLA-C locus (-35 SNP), HLA-C expression, and HIV-1 set point viral loads. We show that the difference in HLA-C expression across -35 SNP genotypes can be attributed primarily to the very low expression of a single allelic product, HLA-Cw7, which is a common HLA type. We suggest that association of the -35 SNP and HIV-1 load manifests as a result of linkage disequilibrium of this polymorphism with both favorable and unfavorable HLA-C and -B alleles.

FIG. 1.

Staining of 220.221AEH cells and PBMC with DT9 and HLA-E-specific antibodies. (A) Staining of the 220.221AEH cell line, showing unstained cells (filled black area) and cells stained with MEM-E/06 (filled brown area), MEM-E/08 (red line), 3D12 (green line), and DT9 (blue line). (B) Representative staining of CD3⁺ lymphocytes (gating on appropriate forward and side scatter, live cells, and CD3 expression) from a healthy volunteer (HLA-A2, -B35, -B44, -Cw4, and -Cw5), showing unstained cells (filled black area) and cells stained with MEM-E/06 (filled brown area), MEM-E/08 (red line), 3D12 (green line), DT9 (blue line), and an IgG2b isotype control (filled gray area).

FIG. 2.

Characterization of monoclonal antibodies DT9, 3D12, MEM-E/06, and MEM-E/08 against 97 common HLA class I allotypes. Binding was determined for DT9 (A), MEM-E/06 (B), MEM-E/08 (C), and 3D12 (D), using beads coated with recombinant HLA-A (blue), -B (green), and -C (red) proteins, and normalized relative to the binding of W6/32 (the horizontal line corresponds to the level of binding seen with W6/32). Values shown represent the averages for three runs; the maximum standard errors of the means were 0.06 (DT9), 0.05 (MEM-E/06), 0.012 (MEM-E/08), and 0.01 (3D12). The order of the HLA allotypes tested (along the x axis) was as follows: A0101, -0201, -0203, -0206, -0301, -1101, -1102, -2301, -2402, -2403, -2501, -2601, -2901, -2902, -3001, -3002, -3101, -3201, -3301, -3303, -3401, -3402, -3601, -4301, -6601, -6602, -6801, -6802, -6901, and -7401, B8001, -0702, -0801, -1301, -1302, -1401, -1402, -1501, -1502, -1503, -1510, -1511, -1512, -1513, -1516, -1801, -2705, -2708, -3501, -3701, -3801, -3901, -4001, -4002, -4006, -4101, -4201, -4402, -4403, -4501, -4601, -4701, -4801, -4901, -5001, -5101, -5102, -5201, -5301, -5401, -5501, -5601, -5701, -5703, -5801, -5901, -6701, -7301, -7801, -8101, and -8201, and Cw0102, -0202, -0302, -0303, -0304, -0401, -0501, -0602, -0702, -0801, -1203, -1402, -1502, -1601, -1701, and -1802.

FIG. 3.

Direct DT9 staining of live CD3⁺ T lymphocytes from −35 SNP homozygotes. PBMC from HIV-1-uninfected (A and B) and chronically HIV-1-infected (C and D) subjects were stained with DT9. DT9 median fluorescence intensities (MFI; y axis) were corrected for background autofluorescence of unstained cells. −35 SNP genotypes are indicated on the x axis. In panels B and D, “other” refers to −35 TT individuals who were not homozygous for HLA-Cw7. P values were calculated using the Mann-Whitney test.

FIG. 4.

Indirect staining of lymphocytes with 3D12, DT9, and W6/32 antibodies. Representative staining results are shown for CD3⁺ lymphocytes (gated on appropriate forward and side scatter, live cells, and CD3 expression) from two HIV-1-uninfected subjects: a −35 TT subject (homozygous for HLA-Cw*07) (A) and a −35 CC subject (HLA-Cw*05/06) (B). Data are shown for unstained cells (filled black area), 3D12 staining (green), DT9 staining (blue), and W6/32 (pan-class I) staining (gray).

FIG. 5.

Indirect staining of lymphocytes from 30 healthy volunteers, using 3D12 and DT9. (A) Paired comparisons of MFI of unstained cells and cells stained with 3D12. (B and C) Indirect staining (with 3D12 and DT9, respectively) of live CD3⁺ lymphocytes, corrected for background autofluorescence of unstained cells. (D) Correlation of background-corrected DT9 and 3D12 staining. (E) Ratios of HLA-C levels (3D12 staining subtracted from DT9 staining) to total class I protein levels (W6/32 staining). For panels B to E, red and half-red symbols denote individuals homozygous and heterozygous for HLA-Cw*07, respectively. Statistical significance was calculated using the Mann-Whitney test.