Antibody-Dependent Cellular Cytotoxicity and NK Cell-Driven Immune Escape in HIV Infection: Implications for HIV Vaccine Development

The HIV-1 genome is malleable and a difficult target tot vaccinate against. It has long been recognised that cytotoxic T lymphocytes and neutralising antibodies readily select for immune escape HIV variants. It is now also clear that NK cells can also select for immune escape. NK cells force immune escape through both direct Killer-immunoglobulin-like receptor (KIR)-mediated killing as well as through facilitating antibody-dependent cellular cytotoxicity (ADCC). These newer finding suggest NK cells and ADCC responses apply significant pressure to the virus. There is an opportunity to harness these immune responses in the design of more effective HIV vaccines

Influence of Cytokines on HIV-Specific Antibody-Dependent Cellular Cytotoxicity Activation Profile of Natural Killer Cells

There is growing interest in HIV-specific antibody-dependent cellular cytotoxicity (ADCC) as an effective immune response to prevent or control HIV infection. ADCC relies on innate immune effector cells, particularly NK cells, to mediate control of virus-infected cells. The activation of NK cells (i.e., expression of cytokines and/or degranulation) by ADCC antibodies in serum is likely subject to the influence of other factors that are also present. We observed that the HIV-specific ADCC antibodies, within serum samples from a panel of HIV-infected individuals induced divergent activation profiles of NK cells from the same donor. Some serum samples primarily induced NK cell cytokine expression (i.e., IFNγ), some primarily initiated NK cell expression of a degranulation marker (CD107a) and others initiated a similar magnitude of responses across both effector functions. We therefore evaluated a number of HIV-relevant soluble factors for their influence on the activation of NK cells by HIV-specific ADCC antibodies. Key findings were that the cytokines IL-15 and IL-10 consistently enhanced the ability of NK cells to respond to HIV-specific ADCC antibodies. Furthermore, IL-15 was demonstrated to potently activate “educated” KIR3DL1+ NK cells from individuals carrying its HLA-Bw4 ligand. The cytokine was also demonstrated to activate “uneducated” KIR3DL1+ NK cells from HLA-Bw6 homozygotes, but to a lesser extent. Our results show that cytokines influence the ability of NK cells to respond to ADCC antibodies in vitro. Manipulating the immunological environment to enhance the potency of NK cell-mediated HIV-specific ADCC effector functions could be a promising immunotherapy or vaccine strategy

The antibodies involved in the antibody dependent cellular cytotoxicity immune response in HIV

© 2012 Dr. Gamze IsitmanBackground: Antibodies that mediate killing of Human Immunodeficiency Virus (HIV) infected cells by NK cells could be a very useful component of a successful HIV vaccine strategy. Antibody dependent cellular cytotoxicity (ADCC) targets HIV-infected cells by utilising the Fc region of specific antibodies bound to NK cells. However, the role of ADCC responses in preventing HIV-1 remains controversial. Previous studies of ADCC activity have been hampered by difficulties identifying and mapping these responses. An intracellular cytokine staining (ICS) technique developed in our laboratory allows us to detect and map HIV-specific ADCC via detecting activation of NK-cells by ADCC on small volumes of blood. There is an imperative to study the antibodies involved in inducing a HIV-specific ADCC response and use this knowledge to develop effective HIV vaccines. Methods: ADCC responses to overlapping HIV-1 consensus peptide pools were analysed using an ICS assay measuring NK cell activation in 83 anti-retroviral therapy (ART)-naïve HIV-infected subjects followed prospectively for 3 years. We mapped 32 responses to individual consensus HIV subtype B 15mer peptides within the Pol protein. The ADCC-assay was also used to analyse autologous virus-derived peptide epitope responses and compared to consensus derived peptides sequence, across a titration of peptide concentrations, to study escape from ADCC immune response. Isolation of the antibodies responsible for Env and Vpu-specific ADCC responses were purified using affinity chromatography and assessed for their viral inhibition activity, purity and neutralisation activity. We also evaluated bulk antibody production methods using EBV transformation of B cells, Mass Spectrometry of the purified ADCC antibodies and the use of fluorochrome bound peptides to identify and select the ADCC-specific B cells. Results: From the 83 subjects 32 Pol-specific responses were identified. Of these 12 have been mapped to regions of Pol and 2 subjects were identified with ADCC-specific responses to 2 different highly conserved Pol epitopes. Fifty-four subjects recognised Env peptides. From 11 mapped Env responses studied, 6 showed a loss of recognition of autologous virus-derived peptides. This suggested a potential pressure ADCC responses apply on HIV. Purification of Env and Vpu-specific ADCC antibodies proved that ADCC inducing antibodies inhibited viral replication in the Antibody-Dependent Cellular Viral Inhibition (ADCVI) assay and they did not involve neutralising activity. Experiments to determine the most optimal ADCC-specific B cell isolation technique indicated that the use of dual fluorochrome labelled peptides offered the most promising results. Conclusions: Targeting ADCC to more conserved proteins, such as Pol and Vpu proteins may be a more effective ADCC-based vaccine approach. Escape from Env-specific ADCC may, unless broad or directed to rare conserved regions within Env, limit the utility of Env-specific ADCC in controlling or preventing HIV. Development of methods to isolate and generate large amounts of ADCC-specific antibodies will assist in defining the utility of HIV-specific ADCC responses and their role in limiting S/HIV infection. The most effective ADCC epitopes can then be engineered into novel HIV vaccines

NK Cells Expressing the Inhibitory Killer Immunoglobulin-Like Receptors (iKIR) KIR2DL1, KIR2DL3 and KIR3DL1 Are Less Likely to Be CD16+ than Their iKIR Negative Counterparts.

Natural Killer (NK) cell education, which requires the engagement of inhibitory NK cell receptors (iNKRs) by their ligands, is important for generating self-tolerant functional NK cells. While the potency of NK cell education is directly related to their functional potential upon stimulation with HLA null cells, the influence of NK cell education on the potency of the antibody dependent cellular cytotoxicity (ADCC) function of NK cells is unclear. ADCC occurs when the Fc portion of an immunoglobulin G antibody bridges the CD16 Fc receptor on NK cells and antigen on target cells, resulting in NK cell activation, cytotoxic granule release, and target cell lysis. We previously reported that education via the KIR3DL1/HLA-Bw4 iNKR/HLA ligand combination supported higher KIR3DL1+ than KIR3DL1- NK cell activation levels but had no impact on ADCC potency measured as the frequency of granzyme B positive (%GrB+) targets generated in an ADCC GranToxiLux assay. A lower frequency of KIR3DL1+ compared to KIR3DL1- NK cells were CD16+, which may in part explain the discrepancy between NK cell activation and target cell effects. Here, we investigated the frequency of CD16+ cells among NK cells expressing other iNKRs. We found that CD16+ cells were significantly more frequent among NK cells negative for the inhibitory KIR (iKIR) KIR2DL1, KIR2DL3, and KIR3DL1 than those positive for any one of these iKIR to the exclusion of the others, making iKIR+ NK cells poorer ADCC effectors than iKIR- NK cells. The education status of these iKIR+ populations had no effect on the frequency of CD16+ cells

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

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

CD16 expression on total CD56⁺ NK cells populations expressing all combinations of iKIRs and on CD56^dim single iNKR expressing NK cell populations.

<p><b>(A)</b> The frequency of KIR⁺ (expressing any Boolean combination of KIR2DL1 (2DL1), KIR2DL3 (2DL3), or KIR3DL1 (3DL1]) subsets in CD16^- and CD16⁺ total CD56⁺ NK cell populations. <b>(B)</b> The gating strategy for assessing the frequencies of single NKG2A, 2DL1, 2DL3, and 3DL1 and CD16 positive CD56^dim NK cells. <b>(C)</b> Frequencies of CD16 positive CD56^dim NK cells that are single positive for NKG2A (n = 26), 2DL1 (n = 25),2DL3 (n = 22), and 3DL1 (n = 22) or negative for all iNKR tested (iNKR^-). Wilcoxon tests were used to determine significance of within subject differences for the indicated NK subsets. Each data point represents results a separate individual. Bar height and error bars represent the median and interquartile range for the data set. Significant values are shown; “**” = p< 0.01; “***” = p< 0.001; “****” = p< 0.0001.</p

Differential NK cell activation patterns by HIV-specific ADCC.

<p>The ability of NK cells to respond to anti-HIV ADCC antibodies was assessed using a flow-based assay. (A) Stimulated cells were stained with fluorochrome conjugated antibodies against CD3, CD2, CD56, CD107a and IFNγ. After collection on a FACS II Canto, lymphocytes were gated upon and NK cells were identified as CD3⁻CD2⁺CD56⁺. Cells within the NK cell population were assessed for IFNγ production and CD107a expression prior to and following activation. (B) Zebra plots depict examples of the diverse anti-HIV ADCC responses obtained when NK cells from a common donor are stimulated with sera from different HIV-infected individuals in the presence of Env peptides (top) or gp140 protein (bottom). The numbers in the quadrants represent the percentages of responding NK cells that are mediating IFNγ⁺CD107a⁻, IFNγ⁻CD107a⁺ and IFNγ⁺CD107a⁺ responses. (C) The pie chart on the left illustrates the frequency with which IFNγ dominant, CD107a dominant and even response profiles were observed, when sera samples from 32 HIV-infected individuals were used to stimulate NK cells from a common donor in the presence of gp140 protein. The pie chart on the right depicts the same analysis for stimulation with Env peptides. (D) The box and whiskers plot depicts the assessment of the relationship between the ability of different sera to induce diverse anti-HIV ADCC functional profiles against HIV gp140 and the magnitude of the total ADCC response. The total percent of NK cell activation was compared between sera that induced “even” or “skewed” ADCC responses, using a T-test. (E) The scatter plot illustrates the relationship between the levels of sera anti-HIV gp140 IgG and the percent of total NK cells, from a common donor, activated by the sera in the whole blood ADCC assay in the presence of gp140. This correlation was assessed with the Spearman correlation. (F) The scatter plot depicts the assessment of the impact of the level of sera-associated anti-HIV gp140 IgG on the functional profile induced by different sera, evaluated by a T-test.</p

Activation of NK Cells by ADCC Responses During Early HIV Infection

Partial control of HIV occurs during acute infection, although the mechanisms responsible are poorly understood. We studied the ability of antibody-dependent cellular cytotoxicity (ADCC) antibodies in serum to activate natural killer (NK) cells in longitudinal samples from 8 subjects with well-defined early HIV infection who controlled viremia to low levels. NK cell activation by ADCC antibodies to gp140 Env proteins was detected in half of the subjects at the first time point studied, a mean of 111 d after the estimated time of infection. In contrast, ADCC-mediated NK cell activation in response to linear HIV peptides evolved more slowly, over the first 2 y of infection. Our studies suggest that HIV-specific ADCC responses to conformational epitopes occur early during acute HIV infection, and broaden to include linear epitopes over time. These findings have implications for the immune control of HIV