Differential natural killer cell-mediated inhibition of HIV-1 replication based on distinct KIR/HLA subtypes

Abstract

Decline of peak viremia during acute HIV-1 infection occurs before the development of vigorous adaptive immunity, and the level of decline correlates inversely with the rate of AIDS progression, implicating a potential role for the innate immune response in determining disease outcome. The combined expression of an activating natural killer (NK) cell receptor, the killer immunoglobulin-like receptor (KIR) 3DS1, and its presumed ligand, human leukocyte antigen (HLA)-B Bw4-80I, has been associated in epidemiological studies with a slow progression to AIDS. We examined the functional ability of NK cells to differentially control HIV-1 replication in vitro based on their KIR and HLA types. NK cells expressing KIR3DS1 showed strong, significant dose- and cell contact-dependent inhibition of HIV-1 replication in target cells expressing HLA-B Bw4-80I compared with NK cells that did not express KIR3DS1. Furthermore, KIR3DS1+ NK cells and NKLs were preferentially activated, and lysed HIV-1 infected target cells in an HLA-B Bw4-80I-dependent manner. These data provide the first functional evidence that variation at the KIR locus influences the effectiveness of NK cell activity in the containment of viral replication.

Figure 1.

NK cells are able to mediate inhibition of HIV-1 replication in a contact-dependent manner. (A) The line graph depicts the mean change in the viral load (log10 p24 Gag in supernatant) observed in a single subject from three separate experiments in wells containing CD4⁺ T cells alone (continuous black line), and NK cells and autologous CD4⁺ T cells at a ratio of 10:1 (short dashed black line), 1:1 (long dashed gray line), and 1:10 (long dashed black line). (B) The figure represents a log reduction in p24 Gag production at different NK cell/CD4⁺ T cell ratios (10:1, 1:1, and 1:10) for days 3, 7, 10, and 14 (n = 5). (C) The whisker box plots show the percentage of p24 Gag⁺ CD3⁺ T cells at days 3, 7, 10, and 14 in wells containing NK cells at different NK cell/CD4⁺ T cell ratios (10:1, 1:1, and 1:10; n = 6). (D) The figure represents the log inhibition of HIV-1 replication observed on days 3, 7, 10, and 14 in wells where NK cells and autologous HIV-1–infected CD4⁺ T cells were co-cultured together (gray) or separated in a transwell experiment (black) at an NK cell/CD4⁺ T cell ratio of 10:1 (n = 5). (E) The whisker box plots show the log inhibition of HIV-1 replication observed on days 3, 7, 10, and 14 in wells where NK cells and autologous HIV-1–infected CD4⁺ T cells were co-cultured together (gray) at an NK cell/CD4⁺ T cell ratio of 10:1, or where the supernatant was transferred from co-cultured cells to autologous HIV-1–infected CD4⁺ T cells (black; n = 5). Data represent the mean of at least three experiments and standard deviations.

Figure 2.

NK cells derived from subjects with KIR3DS1 and HLA-B Bw4-80I suppress viral replication more effectively in vitro. (A) The dot plot demonstrates the heterogeneity of viral suppression (given as log inhibition of p24 Gag production) observed in the total cohort of 36 study subjects at an NK cell/CD4⁺ T cell ratio of 10:1 (n = 36). Horizontal bars represent the mean log inhibition. (B) The whisker box plots represent the log inhibition observed in subjects with KIR3DS1 and HLA-B Bw4-80I (black; n = 8), HLA-Bw4 alone (light gray; n = 10), KIR3DS1 alone (white; n = 8), and individuals that express neither of these two alleles (dark gray; n = 10), where NK cells and CD4⁺ T cells were co-cultured at an E/T ratio of 10:1. Data represent the mean and SDs. **, P < 0.001; *, P < 0.05.

Figure 3.

NK cells expressing KIR3DS1 inhibit HIV-1 replication more effectively and respond stronger to Bw4-80I⁺ HIV-1–infected target cells. (A) The flow plots demonstrate the segregation of KIR3DL1⁺ NK cells (1), KIR3DS1⁺/KIR3DL1⁻ NK cells (2), and NK cells expressing neither of these receptors (3), using the combination of the z27 and DX9 antibodies for a subject that does not encode KIR3DS1 (left) and one that does encode KIR3DS1 (right). Numbers represent the proportion of events present within that quadrant. (B). The dot plot represents the log inhibition of HIV-1 replication (given as log inhibition of p24 Gag production) in co-cultures of autologous HIV-1–infected Bw4-80I⁺ CD4⁺ T cells with bulk NK cells, sorted populations of KIR3DS1⁺/3DL1⁻ NK cells, KIR3DL1⁺ NK cells, and NK cells expressing neither of these markers at an NK cell/CD4⁺ T cell ratio of 10:1. All experiments were performed in triplicate in five different KIR3DS1⁺/KIR3DL1⁺ subjects. Horizontal bars represent the mean log inhibition.

Figure 4.

KIR3DS1⁺ NK cells are activated and lyse HIV-1–infected cells in the presence of HLA-B Bw4-80I alleles. (A) Using the combination staining of DX9 and z27, gates were set along the z27 axis to gauge the level of degranulation on z27^hi (DX9⁺z27⁺ or KIR3DL1⁺), z27^dim (DX9⁻z27⁺ or KIR3DS1⁺), or z27^neg (DX9⁻z27⁻ or double negative) NK cells after stimulation with (a) medium alone, (b) K562, (c) 221 cells, (d) uninfected autologous CD4⁺ T cells, (e) autologous HIV⁺ CD4⁺ T cells, and (f) PMA/ionomycin. NK cells and target cells were plated at a ratio of 10:1 for all experiments. (B) The line graphs show the percentage of CD107a⁺ NK cells derived from KIR3DS1⁺/HLA-B Bw4⁺ (black lines) and KIR3DS1⁺/HLA-B Bw6⁺ (gray lines) individuals after the culture of NK cell populations in the presence of autologous HIV-1–uninfected or –infected CD4⁺ T cells (at a ratio of 10:1). Gating strategies were used to calculate the percentage of CD107a⁺ NK cells within the KIR3DS1⁺ NK cell population (z27⁺DX9⁻; top) or within the KIR3DL1⁺ NK cell population (z27⁺DX9⁺; bottom). Only KIR3DS1⁺/3DL1⁻ NK cells co-cultured with Bw4⁺ HIV-1–infected CD4⁺ T cells significantly (P = 0.03) up-regulated CD107a expression. (C) The whisker box plot represents the percentage of untransfected 221 cells that were lysed in a 6-h chromium assay using bulk NK cells derived from KIR3DS1⁺ subjects (light gray; n = 6) and KIR3DS1⁻ individuals (dark gray; n = 6). NK cells were co-cultured with Cr⁵¹-labeled targets at a ratio of 20:1. (D) The bar graph displays the ratio of the percent lysis of HIV-1–infected versus –noninfected, HLA-transfected 221 cells (with 52% of cells staining for p24 by intracellular flow at the maximal time of infection) by a KIR3DS1-transfected NKL (NKL/221 target cell, 20:1; n = 3). Data represent the mean of at least three experiments and standard deviations.