Conferral of enhanced natural killer cell function by KIR3DS1 in early human immunodeficiency virus type 1 infection

A flurry of recent reports on the role of activating and inhibitory forms of the killer cell immunoglobulin-like receptors (KIR) in natural killer (NK) cell activity against human immunodeficiency virus type 1 (HIV-1) have yielded widely divergent results. The role of the activating NK receptor encoded by the KIR3DS1 allele and its putative ligands, members of the HLA class I Bw4Ile80 cluster, in early HIV-1 disease is controversial. We selected 60 treatment-naïve adults for study from the OPTIONS cohort of individuals with early HIV-1 infection in San Francisco. We performed NK cell functional assays measuring gamma interferon (IFN-γ) and CD107a expression by NK cells in the unstimulated state and after stimulation by the major histocompatibility complex class I-deficient 721.221 B-lymphoblastoid cell line. In addition, we measured CD38 expression (a T-cell activation marker) on T and NK cells. Persons who have at least one copy of the KIR3DS1 gene had higher IFN-γ and CD107a expression in the unstimulated state compared to those who do not possess this gene. After stimulation, both groups experienced a large induction of IFN-γ and CD107a, with KIR3DS1 carriers achieving a greater amount of IFN-γ expression. Differences in effector activity correlating with KIR3DS1 were not attributable to joint carriage of HLA Bw4Ile80 and KIR3DS1. We detected a partial but not complete dependence of KIR3DS1 on the members of B*58 supertype (B*57 and B*58) leading to higher NK cell function. Possessing KIR3DS1 was associated with lower expression of CD38 on both CD8⁺ T and NK cells and with a loss or weakening of the known strong associations between CD8⁺ T-cell expression of CD38 mean fluorescence intensity and the HIV-1 viral load. We observed that possessing KIR3DS1 was associated with higher NK cell effector functions in early HIV-1 disease, despite the absence of HLA Bw4Ile80, a putative ligand of KIR3DS1. Carriage of KIR3DS1 was associated with diminished CD8⁺ T-cell activation, as determined by expression of CD38, and a disruption of the traditional relationship between viral load and activation in HIV-1 disease, which may lead to better clinical outcomes for these individuals.NK cell function is regulated by a family of receptors encoded by the killer cell immunoglobulin-like receptor (KIR) genes (18, 33). Within the KIR family, certain genes encode inhibitory receptors that recognize HLA class I ligands (i.e., HLA-Bw4 or HLA-C), whereas other KIR genes encode activating receptors which are not completely known. Studies on the role of KIRs in human immunodeficiency virus (HIV) disease have focused on the activating receptor encoded by the KIR3DS1 allele. However, recent genetic association and functional studies of KIR and HIV disease have yielded widely disparate results on the role of KIR3DS1 and its putative ligands, a subset of HLA class I-B alleles referred to as Bw4Ile80. The Bw4Ile80 cluster is a subset of HLA-B alleles that bear an isoleucine at position 80 in the α-1 helix, on the rim of the peptide-binding cleft. The inhibitory receptors encoded by KIR3DL1 alleles, which are highly related in the extracellular domains to the activating receptor encoded by KIR3DS1, specifically recognize HLA-Bw4 ligands (5). Because of this similarity, KIR3DS1 has been assumed to also recognize Bw4Ile80 ligands. In 2002, Martin et al. reported that HIV-infected individuals in the Multicenter AIDS Cohort Study possessing the KIR3DS1 allele demonstrated significantly delayed progression to AIDS, provided that the individuals also expressed a Bw4Ile80 allele (20).In 2005, Gaudieri et al. reported on the association of the entire KIR gene cluster and HLA class I in HIV disease progression in an Australian HIV cohort (8, 9). These authors observed a trend toward slowed CD4⁺ T-cell percent loss among those who carried both Bw4Ile80 and KIR3DS1 (8). However, this trend was not statistically significant, and Gaudieri et al. simultaneously observed an acceleration of time to AIDS (1987 definition) among joint KIR3DS1 and Bw4Ile80 carriers. In 2006, Qi et al. published a follow-up report from the Multicenter AIDS Cohort Study cohort documenting an association between the coexpression of KIR3DS1 and Bw4Ile80 and enhanced protection against certain opportunistic infections in HIV-infected individuals (26), an effect partially attributed to very modest differences in viral load. In 2007, our group observed that KIR3DS1 gene carriage was associated with higher CD4⁺ T-cell counts and hence protection against HIV type 1 (HIV-1) progression in early disease (4); however, we observed that this effect was not attributable to differences in the viral load and further was independent of Bw4Ile80. In other words, our analyses suggested that the KIR3DS1 and Bw4Ile80 genes were each associated with protection against HIV disease but via different mechanisms.Until recently, it was not clear if KIR3DS1 was expressed on the surface of NK cells; however, two recent reports have conclusively established that KIR3DS1 is expressed on NK cells (6, 24) and that expression is dose dependent, with higher expression for homozygotes. These studies also demonstrated that KIR3DS1 recognizes neither HLA-Bw4 nor HLA-Bw6 ligands, at least when these major histocompatibility complex (MHC) class I molecules are expressed on Epstein-Barr virus-transformed B-lymphoblastoid cell lines. Similarly, an independent study by another group reported that KIR3DS1 fails to bind to soluble Bw4Ile80 tetrameric complexes (10). In contrast, Alter et al. have presented results from in vitro cytotoxicity assays suggesting that target cells possessing HLA-Bw4Ile80 are better targets for NK cells possessing KIR3DS1 (1); however, no evidence was provided to confirm a physical interaction between the KIR3DS1 and HLA-Bw4 proteins.Here, we present a study of the NK cell phenotype and function in 60 treatment-naïve, recently HIV-1-infected persons with defined HLA-B and KIR3DS1/KIR3DL1 allotypes. We also measured the expression of CD38 on NK cells and CD8⁺ T cells, a widely used marker of disease progression and virulence in HIV research and a marker of immune activation. The expression of CD38, as measured by flow cytometry, is known to be elevated on CD8⁺ T cells in HIV disease, reaching steady-state levels in early HIV-1 infection (7), and predicts disease progression independently of the viral load (19). The individuals studied were selected from our recent genetic association study of KIR and HLA among 255 recently HIV-1-infected persons (4), in which KIR3DS1 carriage alone was associated with higher CD4⁺ T-cell counts, despite the absence of a difference in the viral loads. On the basis of these clinical findings, we performed this study to determine whether persons who carried the KIR3DS1 gene had enhanced NK cell phenotypic and functional profiles and if these profiles were further enhanced by carriage of the putative KIR3DS1 ligands encoded by HLA-Bw4Ile80 alleles. Flow cytometry-based detection of KIR3DS1 has been hampered by the absence of a monoclonal antibody that can bind to KIR3DS1 specifically and not cross-react with the related KIR3DL1 proteins (25). Hence, we used genotypic KIR assignments for our analyses rather than flow cytometry-based methods.