Decreased NK cell frequency and function is associated with increased risk of KIR3DL allele polymorphism in simian immunodeficiency virus-infected rhesus macaques with high viral loads

Abstract

NK cells have been established as an important effector of innate immunity in a variety of viral infections. In HIV-1 infection in humans, alterations of NK cell function, frequency, and expression of various NK receptors have been reported to be associated with differential dynamics of disease progression. Expression of certain alleles of KIR3DL and KIR3DS receptors on NK cells was shown to correlate with levels of virus replication. In the SIV-infected rhesus macaque (RM) model of AIDS, several families of killer inhibitory Ig-related receptors (KIR receptors) corresponding to their human counterparts have been characterized, but only at the level of individual sequence variants. Here we define 14 different alleles of KIR3DL expressed among 38 SIV-infected RM, characterized by either high or low levels of SIV replication, by analyzing multiple sequences from individual animals and show an unequal distribution of certain alleles in these cohorts. High levels of SIV replication were associated with significant increases in KIR3DL mRNA levels in addition to decreases in both the frequency and function of NK cells in these animals. The higher frequency of inheritance of two KIR3DL alleles characterized by a single nucleotide polymorphism 159 H/Q was associated with RM that exhibited high plasma viral load. This data for the first time defines multiple alleles of KIR3DL in RM and shows an association between virus control, NK cell function and genetic polymorphisms of KIR receptors.

FIGURE 1

NK cell surface receptors. NK cells express a variety of cell surface markers which can elicit negative or positive signals. The negative signal generating/inhibitory molecules include 1) the Ig superfamily molecules such as the killer cell Ig-like receptors with long cytoplasmic tails (KIRxDL), lymphocyte inhibitory receptors (LIRs) and sialic acid binding Ig-like lectins (SIGLECs), and 2) the C-type lectin receptors, which include NKG2a/CD94 heterodimer. The positive signal generating/activating receptors include 1) the ITAM-bearing molecules CD16, p30, and p46 (ITAM associated with FcRγ and the CD3ε); 2) ITAM bearing molecules NKG2c/CD94 heterodimer, KIRDS, and p44 (ITAM-bearing DAP12); and 3) the non-ITAM bearing receptors CD2, NKG2D (associated with DAP10), and 2B4 (associated with SAP polypeptide). NCR, natural cytotoxicity receptor.

FIGURE 2

KIR3DL expression in RMs pre- and post-SIV infection. Real-time PCR analysis for the levels of expression of KIR3DL cDNA in purified NK cells from 6 RM each from LVL and HVL cohorts. In each, animal quantitation was performed in cells pre- and post-SIV infection and results are expressed as fold change post-SIV vs pre-SIV.

FIGURE 3

Phylogenetic identification of KIR3DL alleles in RMs. Full-length sequences of KIR3DL from each animal were assigned into groups, based on the sequence similarity (>98%) and consensus sequences were generated representing “primary” individual alleles. These primary alleles were then compared between the animals based on the phylogenetic relationship (>98% similarity) and 14 alleles were defined within the population. The tree shown reflects phylogenetic relationship between the alleles formed by inidividual primary alleles (e.g., mm1–2al) and several individual sequences (xxxx.seq).

FIGURE 4

Sequences of KIR3DL alleles in RMs. Alignment of protein sequences of 14 KIR3DL alleles (allele 1–14; GenBank accession numbers FJ562108 through FJ562121) and newly identified variant A. The three extracellular domains (D0, D1, and D2) and stem sequences are shown.

FIGURE 5

Sequences of new KIR3DL variants. Nucleotide sequences of newly identified variants B–N are aligned with the sequence of allele 8: A, variants B and C; B, variants D and M; C, variants F and H; D, variants E, K, and I; and E, variants J, G, L, and N. Partial alignments of each set of variants are shown, depicting the part of the sequence with major variation against the allele 8. Sequence numbering reflects AF334616.

FIGURE 5

Sequences of new KIR3DL variants. Nucleotide sequences of newly identified variants B–N are aligned with the sequence of allele 8: A, variants B and C; B, variants D and M; C, variants F and H; D, variants E, K, and I; and E, variants J, G, L, and N. Partial alignments of each set of variants are shown, depicting the part of the sequence with major variation against the allele 8. Sequence numbering reflects AF334616.

FIGURE 6

Sequences of the most prevalent KIR3DL alleles in RMs. Alignment of protein sequences of RM KIR3DL alleles 13, 14, and 8, together with comparison to the sequence of human KIR3DL (Hu KIR3DL; NM_013289) and RM consensus sequence (RM consensus). The three extracellular domains, stem, and cytoplasmic tail sequences are shown.

FIGURE 7

NK cell function in RMs pre- and post-SIV infection. A, Purified NK cells from SIV naive (SIV uninfected) and majority of SIV-infected animals from both HVL and LVL cohorts were analyzed for their lytic activity. Means of each group are displayed. B, PBMC from RM pre-and post-SIV infection were analyzed in two cohorts—high viral load (HVL) and low viral load (LVL)—for frequency of NK cells (CD3⁻CD8⁺). Relative change in NK cell frequency for each animal is first calculated as frequency post-SIV/frequency pre-SIV and means of these relative individual changes are displayed for each cohort.

FIGURE 8

Phylogenetic relationship between previously defined KIR3DL variants and newly defined alleles. The phylogenetic distance tree depicts the relationship between the 11 previously described full-length KIR3DL variants (mm KIR3DL 1–11) (31) and 14 newly defined alleles (3DLDNAAllele 1–14).