HIV Protective KIR3DL1/S1-HLA-B Genotypes Influence NK Cell-Mediated Inhibition of HIV Replication in Autologous CD4 Targets

<div><p>Carriage of the genetic combination encoding a high expression inhibitory Killer Immunoglobulin-like Receptor (KIR)3DL1 with its ligand, HLA-B*57 (<i>*h/*y+B*57</i>) is associated with slower time to AIDS and better HIV viral load control than being a <i>Bw6</i> homozygote (<i>Bw6hmz</i>). Natural Killer (NK) cells from <i>*h/*y+B*57</i> carriers receive potent educational signals through HLA-B*57 KIR3DL1 ligation leading to high functional potential. NK cells from <i>Bw6hmz</i> are not educated through KIR3DL1 because Bw6 antigens do not interact with this inhibitory receptor. To better understand the impact of KIR/HLA combinations on NK cell mediated anti-viral activity we measured NK cell mediated inhibition of HIV replication in autologous infected CD4 (iCD4) cells by assessing the frequency of p24 positive CD4 targets and supernatant levels of HIV p24 longitudinally in the presence versus absence of NK cells. Forty-seven HIV uninfected subjects were studied, including carriers of <i>*h/*y+B*57</i>, a low expression <i>KIR3DL1</i> genotype with <i>HLA-B*57</i> termed <i>*l/*x+B*57</i>, a genotype designated <i>3DS1+*80I</i> and <i>Bw6hmz</i>. NK cells from <i>*h/*y+B*57</i> carriers, like those from <i>3DS1+*80I</i> subjects, inhibited HIV replication in autologous iCD4 cells better than those from <i>Bw6hmz</i> and <i>*l/*x+B*57</i> carriers. Cell contact between NK and iCD4 cells activated NK cells to inhibit viral replication in a non-contact dependent fashion through secretion of CC-chemokines. iCD4 stimulated NK cells from <i>*h/*y+B*57</i> and <i>3DS1+*80I</i> carriers produced higher levels of CC-chemokines than those from <i>Bw6hmz</i> or <i>*l/*x+B*57</i> carriers. Higher levels of CC-chemokines were produced by KIR3DL1⁺ than KIR3DL1⁻ NK cells. We conclude that NK-mediated inhibition of viral replication in autologous iCD4 cells is partially due to a block at the level of HIV entry into new targets by secreted CC-chemokines.</p></div

NK cells from subjects positive for <i>*h/*y+B*57</i> secrete more CC-chemokines in response to autologous HIV infected CD4 (iCD4) cells than those from <i>Bw6hmz</i>.

<p>Box and whisker plots show the levels of CCL3 (A), CCL4 (B) and CCL5 (C) secreted over 3 days into the supernatant of cultures of NK cells and autologous iCD4 cells at a 10∶1 ratio from individuals positive for <i>*h/*y+B*57</i> (n = 7) or from <i>Bw6hmz</i> (n = 10). The line in each box represents the median value, the lower and upper limits of the boxes the 25% and 75% quartiles and the whiskers the minimum and maximum values for each group. Lines linking groups indicate comparisons where medians were significantly different.</p

NK cells inhibit HIV replication in autologous HIV infected CD4 T cells in a contact dependent manner.

<p>(A) Flow plots show the frequency of p24 positive CD4 cells from a single individual cultured for 7 days under the following conditions: uninfected CD4 T cells cultured alone, infected CD4 (iCD4) cells cultured alone, iCD4 cells cultured with autologous NK cells in the same well at a 10∶1 NK∶iCD4 ratio (NK+iCD4), iCD4 cells and NK cells cultured in separate transwell chambers at a 10∶1 NK∶iCD4 ratio (NK/iCD4 TW), iCD4 cells cultured alone in the upper chamber of a transwell with NK cells and iCD4 cells cultured together in the lower transwell chamber at a 10∶1 NK∶iCD4 ratio (iCD4 TW), iCD4 cells cultured with NK cells in the same transwell chamber at a 10∶1 NK∶iCD4 cell ratio (NK+iCD4 TW). (B) Bar graphs show the frequency of HIV infected cells on days 3, 7 and 10 under the same culture conditions as described in (A) for up to 12 individuals. One subject was positive for <i>*h/*y+B*57</i>, 7 were <i>3DS1+*80I</i>, 2 were <i>Bw6hmz</i>, 1 was <i>3DS1+Bw4 not *80I</i> and 1 was <i>3DL1hmz+*80I</i> (not B*57). Bar height and error bars represent the mean and the standard error of the mean for each group. Lines linking bars indicate comparisons where means are significantly different. “*” = a p-value<0.05, “**” = a p-value of <0.01.</p

NK cells from subjects carrying <i>*h/*y+B*57</i> and <i>3DS1+*80I</i> suppress viral replication better than those from <i>Bw6hmz</i> and <i>*l/*x+B*57</i> carriers.

<p>The box and whisker plots show the percent viral inhibition observed when NK cells from subjects positive for <i>*h/*y+B*57</i> (n = 7) <i>3DS1+*80I</i> (n = 9), <i>Bw6hmz</i> (n = 10) and <i>*l/*x+B*57</i> (n = 4) are cultured with autologous HIV infected CD4 (iCD4) cells at a ratio of 10∶1 for up to 10 days. The line in each box represents the median value, the lower and upper limits of the boxes the 25% and 75% quartiles and the whiskers the minimum and maximum values for each group; each point is the percent viral inhibition value for a single individual. Lines linking groups indicate comparisons where medians were significantly different. “*” = p<0.05, “**” = p<0.01.</p

Percent of CCL3+ and CCL4+ NK cells and NK cell subsets following stimulation with autologous HIV infected CD4 (iCD4) cells.

<p>CD4 cells infected with HIV and cultured for 7 days were used to stimulate autologous NK cells for 24+ (upper panels) and CCL4+ (lower panels) total NK cells (left), KIR3DL1⁺ (middle) and KIR3DL1⁻ (right) NK cell subsets in subjects positive for <i>*h/*y+B*57</i> (n = 7) <i>*l/*x+B*57</i> (n = 4) and <i>Bw6hmz</i> (n = 9). Each point represents the value for a single individual, the line and error bars through each group show the mean and the standard error of the mean for each data set. Lines linking groups indicate between-group comparisons. “*” = a p-value<0.05, “**” = a p-value of <0.01.</p

Characteristics of study subjects.

<p>Characteristics of study subjects.</p

Frequency of CD16⁺, NKG2A⁺ and CD57⁺ NK cells and their subsets expressing or not CD16.

<p><b>(A)</b> Representative gating strategy for determining the frequency of CD16^+/- cells on CD56^total, CD56^dim, and CD56^bright NK cell populations and on the NKG2A^-/+ and CD57^-/+ subsets of these populations. <b>(B)</b> Frequencies of CD16^+/- cells in the CD56^total, CD56^dim, and CD56^bright NK cell populations. Frequencies of expression of NKG2A <b>(C)</b> and CD57 <b>(E)</b> on CD56^total, CD56^dim, and CD56^bright NK cells. A Friedman test was used to assess the significance of matched between group differences. Frequencies of CD16⁺ and CD16^- cells within the NKG2A⁺ <b>(D)</b> and CD57⁺ <b>(F)</b> CD56^total, CD56^dim, and CD56^bright NK cell populations. Each data point represents results for 1 of 26 separate individuals. Bar height and error bars represent the median and interquartile range for the data set. Wilcoxon tests were used to determine significance of within subject differences for the indicated NK subsets linked by a line connecting the data sets. Significant values are shown; “*” = p< 0.05; “**” = p< 0.01; “***” = p< 0.001; “****” = p< 0.0001.</p

IL-18 and LPS activate caspase-1 and caspase-3 in HT-29 cells.

<p>The cell monolayers were treated with IL-18 (10 ng/ml) or with LPS (10 ng/ml) for 12 hours. Thereafter, the monolayers were washed with PBS, lysed and the activation of the caspases was determined on Western blots by using antibodies specific to the activated forms of the caspases. The Figure shows results from a representative of three independent experiments.</p

The frequency of educated and uneducated CD56^dim NK cell populations expressing CD16 and one of the inhibitory KIR KIR2DL1, KIR2DL3, or KIR3DL1.

<p>The frequency of CD16⁺ cells among educated KIR2DL1⁺ (2DL1) NKG2A^-CD56^dim NK cells from <i>HLA-C2</i> homozygotes (n = 8) versus uneducated <i>HLA-C1</i> homozygotes (n = 11), educated KIR2DL3⁺ (2DL3) NKG2A^-CD56^dim NK cells from <i>HLA-C1</i> homozygotes (n = 10) versus uneducated <i>HLA-C2</i> homozygotes (n = 7), and educated KIR3DL1⁺ (3DL1) NKG2A^-CD56^dim NK cells from <i>Bw4</i> carriers (n = 8) versus uneducated <i>Bw6</i> homozygotes (n = 8). The lines and error bars through the datasets represent medians and interquartile ranges. Mann-Whitney tests assessing the significance of differences in the frequency of CD16⁺, single KIR positive cells in educated versus uneducated NK cell subsets found no significant differences.</p

The effects of HIV and Tat treatments on the secretion of IL-18 and IL-18BP from IEC.

<p>The panels A and B show mean concentrations of IL-18 and IL-18BP, respectively, in the culture supernatants. The vertical line on each bar denotes standard deviation. The Tat used in these experiments was pre-treated with Tat-neutralizing antibodies (Neut Tat) or with control antibodies (Tat).</p