Copy number variation of KIR genes influences HIV-1 control

Abstract

A genome-wide screen for large structural variants showed that a copy number variant (CNV) in the region encoding killer cell immunoglobulin-like receptors (KIR) associates with HIV-1 control as measured by plasma viral load at set point in individuals of European ancestry. This CNV encompasses the KIR3DL1-KIR3DS1 locus, encoding receptors that interact with specific HLA-Bw4 molecules to regulate the activation of lymphocyte subsets including natural killer (NK) cells. We quantified the number of copies of KIR3DS1 and KIR3DL1 in a large HIV-1 positive cohort, and showed that an increase in KIR3DS1 count associates with a lower viral set point if its putative ligand is present (p = 0.00028), as does an increase in KIR3DL1 count in the presence of KIR3DS1 and appropriate ligands for both receptors (p = 0.0015). We further provide functional data that demonstrate that NK cells from individuals with multiple copies of KIR3DL1, in the presence of KIR3DS1 and the appropriate ligands, inhibit HIV-1 replication more robustly, and associated with a significant expansion in the frequency of KIR3DS1+, but not KIR3DL1+, NK cells in their peripheral blood. Our results suggest that the relative amounts of these activating and inhibitory KIR play a role in regulating the peripheral expansion of highly antiviral KIR3DS1+ NK cells, which may determine differences in HIV-1 control following infection.

Figure 1. KIR and HLA-B interactions.

An NK cell can express KIR3DL1 alone, KIR3DS1 alone, both receptors, or neither. KIR3DL1 is an inhibitory receptor and has been shown to interact with HLA-Bw4-80I and with HLA-Bw4-80T. KIR3DS1 is an activating receptor and may interact with HLA-Bw4-80I, possibly through an indirect mechanism. An NK cell can be activated to lyse an infected cell via either the activating of KIR receptor signaling or the dampening of inhibitory KIR activity.

Figure 2. Association between KIR3DS1 and KIR3DL1 effective counts and viral load at set point.

(A–B) The effective counts of KIR3DS1 and KIR3DL1-surface associate with viral load (VL) at set point. (C–D) The insets show the association between the raw KIR3DS1 or KIR3DL1-surface count, for the subset of patients from graphs A and B where the effective counts equal zero. An effective count of zero can be due to the absence of the KIR receptor or due to the absence of the HLA-Bw4 ligand. The raw count does not associate with viral load at set point when the effective count for KIR3DS1 or KIR3DL1-surface equals zero. Error bars show one standard deviation.

Figure 3. Interaction between KIR3DL1 and KIR3DS1.

(A) Test of whether KIR3DL1 status influences the effect of KIR3DS1 on viral load. (B) Test of whether KIR3DS1 status influences the effect of KIR3DL1 on viral load.

Figure 4. NK cell inhibition of HIV-1 replication in vitro.

The inhibitory capacity of NK cells from HIV-negative donors with different combinations of KIR3DL1 and/or KIR3DS1 was tested in an NK cell inhibition assay. NK cells derived from individuals who express a KIR3DS1 and two KIR3DL1 exhibit a remarkably robust capacity to inhibit HIV-1 replication in vitro if HLA-Bw4-80I is present (mean inhibition = 88%, n = 5), but not if HLA-Bw4-80I is absent (mean inhibition = 13%, n = 3) (p<0.05). Similarly, NK cells derived from individuals who express one KIR3DL1, one KIR3DS1, and HLA-Bw4-80I also inhibit HIV-1 replication (mean inhibition = 42%, n = 19) much better than individuals with HLA-Bw4-80I and two KIR3DL1 (mean inhibition = 6%, n = 10), HLA-Bw4-80I and two KIR3DS1 (mean inhibition = 6%, n = 4), or individuals who do not have HLA-Bw4-80I (mean inhibition = 8%, n = 35) (p<0.005) (* p<0.05, ** p<0.005, *** p<0.0005).

Figure 5. Expression patterns of effective KIR3DS1 and effective KIR3DL1.

Increasing the copies of effective KIR3DL1 in the presence of effective KIR3DS1 results in elevated KIR3DS1 transcript levels, and an increased frequency of NK cells expressing KIR3DS1 in the peripheral blood. (A) The levels of KIR3DS1 and KIR3DL1 transcripts were assessed by quantitative PCR analysis in purified populations of NK cells, demonstrating that individuals who express increasing copies of effective KIR3DL1 in the presence of an effective KIR3DS1 possess increasing amounts of KIR3DS1 transcripts, but not KIR3DL1 transcripts (n = 5 in each group). The level of KIR3DS1 transcripts (B) but not KIR3DL1 (C) correlates with the level of in vitro NK-cell-mediated inhibition. (D) Furthermore, raw flow cytometric data from two representative individuals show that the protective genotype of one effective KIR3DS1 and two effective KIR3DL1 is associated with an elevated frequency of KIR3DS1+ NK cells (58%) in the peripheral blood. Z27, on the y-axis, stains for both KIR3DS1 and KIR3DL1, and DX9 on the x-axis stains for just KIR3DL1. (E) These same individuals show a trend towards an accumulation of KIR3DS1+ NK cells, but not KIR3DL1+ NK cells, in the peripheral circulation compared to individuals who have fewer copies of KIR3DL1 in the presence of an effective KIR3DS1 gene count (n = 5 in each group) (* p<0.05).