HIV protective KIR3DL1/S1-HLA-B genotypes influence NK cell-mediated inhibition of HIV replication in autologous CD4 targets

Abstract

Carriage of the genetic combination encoding a high expression inhibitory Killer Immunoglobulin-like Receptor (KIR)3DL1 with its ligand, HLA-B*57 (*h/*y+B*57) is associated with slower time to AIDS and better HIV viral load control than being a Bw6 homozygote (Bw6hmz). Natural Killer (NK) cells from *h/*y+B*57 carriers receive potent educational signals through HLA-B*57 KIR3DL1 ligation leading to high functional potential. NK cells from Bw6hmz 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 *h/*y+B*57, a low expression KIR3DL1 genotype with HLA-B*57 termed *l/*x+B*57, a genotype designated 3DS1+*80I and Bw6hmz. NK cells from *h/*y+B*57 carriers, like those from 3DS1+*80I subjects, inhibited HIV replication in autologous iCD4 cells better than those from Bw6hmz and *l/*x+B*57 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 *h/*y+B*57 and 3DS1+*80I carriers produced higher levels of CC-chemokines than those from Bw6hmz or *l/*x+B*57 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.

Figure 1. NK cells inhibit HIV replication in autologous HIV infected CD4 T cells in a contact dependent manner.

(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 *h/*y+B*57, 7 were 3DS1+*80I, 2 were Bw6hmz, 1 was 3DS1+Bw4 not *80I and 1 was 3DL1hmz+*80I (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.

Figure 2. Infected CD4 T cells stimulate autologous NK cell to produce CC-chemokines.

Levels of CCL3 (A), CCL4 (B) and CCL5 (C) secreted into supernatants after days 1, 2 and 3 under various culture conditions were assessed by ELISA. Shown are results for NK cells cultured with infected CD4 cells (iCD4) at a 10∶1 ratio with 100 international units (IU)/ml of human recombinant IL-2 (IL-2) (NK+iCD4+IL-2, n = 33 observations), NK cells cultured with 100 IU/ml IL-2 (NK+IL-2, n = 24 observations), NK cells cultured with uninfected CD4 and 100 IU/ml of IL-2 (NK+CD4+IL-2, n =  9 observations) and iCD4 with 100 IU/ml IL-2 (iCD4+IL-2, n = 11 observations). Data points and error bars represent the mean and the standard error of the mean for the groups. Results were generated using subjects with the following KIR/HLA genotypes; *h/*y+B*57 (n = 4), 3DS1+*80I (n = 5), Bw6hmz (n = 4), *l/*x+B*57 (n = 4) and other KIR/HLA (n = 5). Samples were tested on up to 4 occasions in separate experiments. (D) Percent inhibition of viral replication on days 3, 7 and 10 of an NK cell autologous iCD4 cell co-culture (10∶1 ratio) in the absence of anti-CC-chemokine neutralizing antibodies (nAbs), in the presence of anti-CCL3, anti-CCL4 or anti-CCL5 nAbs, individually, or together. Results were generated using subjects with the following KIR/HLA gentotypes; *h/*y+B*57 (n = 2) and 3DS1+*80I (n = 5). Data points and error bars represent the mean and standard error of the mean of values for the groups.

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

The box and whisker plots show the percent viral inhibition observed when NK cells from subjects positive for *h/*y+B*57 (n = 7) 3DS1+*80I (n = 9), Bw6hmz (n = 10) and *l/*x+B*57 (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.

Figure 4. NK cells from subjects positive for *h/*y+B*57 secrete more CC-chemokines in response to autologous HIV infected CD4 (iCD4) cells than those from Bw6hmz.

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 *h/*y+B*57 (n = 7) or from Bw6hmz (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.

Figure 5. Percent of CCL3+ and CCL4+ NK cells and NK cell subsets following stimulation with autologous HIV infected CD4 (iCD4) cells.

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 *h/*y+B*57 (n = 7) *l/*x+B*57 (n = 4) and Bw6hmz (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.