HIV-1 adaptation to NK-cell-mediated immune pressure

Abstract

Natural killer (NK) cells have an important role in the control of viral infections, recognizing virally infected cells through a variety of activating and inhibitory receptors. Epidemiological and functional studies have recently suggested that NK cells can also contribute to the control of HIV-1 infection through recognition of virally infected cells by both activating and inhibitory killer immunoglobulin-like receptors (KIRs). However, it remains unknown whether NK cells can directly mediate antiviral immune pressure in vivo in humans. Here we describe KIR-associated amino-acid polymorphisms in the HIV-1 sequence of chronically infected individuals, on a population level. We show that these KIR-associated HIV-1 sequence polymorphisms can enhance the binding of inhibitory KIRs to HIV-1-infected CD4(+) T cells, and reduce the antiviral activity of KIR-positive NK cells. These data demonstrate that KIR-positive NK cells can place immunological pressure on HIV-1, and that the virus can evade such NK-cell-mediated immune pressure by selecting for sequence polymorphisms, as was previously described for virus-specific T cells and neutralizing antibodies. NK cells might therefore have a previously underappreciated role in contributing to viral evolution.

Figure 1. KIR2DL2 associated sequence polymorphisms result in a loss of NK cell inhibition of HIV replication in vitro

(A) The Vpu-Env^WT/WT virus was inhibited more robustly by NK cells derived from a KIR2DL2+ individual (gray lines) compared to the Vpu-Env^V/V virus. NK cells derived from a KIR2DL2^neg (black lines) individual did not inhibit either virus. (B) Similarly, the Vpu-Env^WT/WT virus was inhibited significantly more strongly by NK cells derived from individuals that expressed KIR2DL2 (dark grey bars, n=6) than the Vpu-Env^V/V virus (black bars). NK cells derived from individuals that did not express KIR2DL2 (white bars, n=6) did not significantly inhibit either virus. * p < 0.05; ** p < 0.005; + KIR2DL2^pos; - KIR2DL2^neg.

Figure 2. Amino acid polymorphisms at positions 71/74 in VPU inhibit KIR2DL2, but not KIR2DL3, recognition and binding

The flow cytometric plots depict the frequency of total CD158b (KIR2DL2/2DL3/2DS2)+ NK cells that degranulated following coculture with autologous CD4+ T cells infected with the Vpu-Env^WT/WT virus or the Vpu-Env^V/V virus for 2 representative subjects (left 2DL2+ subjects; right 2DL2- subject; dot plots (A)). Furthermore, the frequency of degranulating CD158+ NK cells of the total CD158+ NK cell population is also represented for both the KIR2DL2+ (top) and KIR2DL2- (bottom) donor in histograms for both the WT/WT (black line) and the V/V virus (grey line) (B). Figure 2 C shows the combined data for NK cell degranulation in 2DL2+ (n=5) and 2DL2- (n=5) individuals. *p < 0.05. The dot plots in figure 2D depict the staining pattern of a KIR2DL2-fusion construct on HIV-infected CD4+ T cells from an HLA-C1/C2 heterozygous donor, and the histogram in figure 2E demonstrates the staining of KIR2DL2- and KIR2DL3-IgG fusion constructs on the same donors for the 2 viral variants. In figure 2F KIR2DL2-IgG- (black) and KIR2DL3-IgG-fusion construct (grey) binding data are summarized for a total of 5 different HLA-C1 heterozygous CD4+ T cell donors for the 2 viral variants in the bar graph. ** p < 0.005.

Figure 3. Two additional KIR2DL2-associated amino acid polymorphisms reduce KIR2DL2-mediated NK cell recognition of virally infected cells

NK cells from KIR2DL2+ individuals (gray bars, n=5) inhibited the replication of the Gag-WT virus and the Nef-WT virus significantly better than the replication of the Gag-V and Nef-V virus (A and C). Similarly, NK cells derived from KIR2DL2+ individuals (gray bars, n=5) were activated significantly more by cells infected with the Gag-WT However, NK cells derived from KIR2DL2- individuals did not inhibit viral replication or degranulate in the response to cells infected with either of the WT or V viruses. * p < 0.05.

Figure 4. KIR2DL2-associated amino acid polymorphisms affect KIR2DL2-, but not KIR2DL3-binding to infected CD4+ T cells

CD4+ T cells infected with the respective variant viruses were stained with KIR2DL2-IgG and KIR2DL3-IgG fusion constructs. The dot plots (A) and histograms (B) depict the staining pattern of KIR2DL2-IgG and KIR2DL3-IgG fusion constructs of HIV-1-infected CD4+ T cells from a HLA-C1/C2 heterozygous donor. The bar graph summarizes KIR2DL2- (black) or KIR2DL3- (grey) IgG fusion construct binding data for 5 different HLA-C1/C2 heterozygous CD4+ T cell donors for the viral variants, as indicated (C). ** p < 0.005