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 potential role of antibody dependent cellular cytotoxicity in protection from HIV-1

© 2013 Dr. Leia Helen WrenPublications included in thesis:Wren, L. H., Chung, A. W., Isitman, G., Kelleher, A. D., Parsons, M. S., Amin, J., et al. (2012). Specific antibody-dependent cellular cytotoxicity responses associated with slow progression of HIV infection. Immunology, 138(2), 116-123. DOI: 10.1111/imm.12016Madhavi, V., Navis, M., Chung, A., Isitman, G., Wren, L., De Rose, R., et al. (2013). Activation of NK cells by HIV-specific ADCC antibodies: role for granulocytes in expressing HIV-1 peptide epitopes. Human Vaccines & Immunotherapeutics, 9( 5). DOI: 10.4161/hv.23446Wren, L., Parsons, M. S., Isitman, G., Center, R. J., Kelleher, A. D., Stratov, I., et al. (2012). Influence of cytokines on HIV-specific antibody-dependent cellular cytotoxicity activation profile of natural killer cells. PLoS ONE, 7(6), e38580. DOI: 10.1371/journal.pone.0038580Parsons, M. S., Wren, L., Isitman, G., Navis, M., Stratov, I., Bernard, N. F., et al. (2012). HIV infection abrogates the functional advantage of natural killer cells educated through KIR3DL1/HLA-Bw4 interactions to mediate anti-HIV antibody-dependent cellular cytotoxicity. Journal of Virology, 86(8), 4488-4495. DOI: 10.1128/JVI.06112-11Kramski, M., Lichtfuss, G. F., Navis, M., Isitman, G., Wren, L., Rawlin, G., et al. (2012). Anti-HIV-1 antibody-dependent cellular cytotoxicity mediated by hyperimmune bovine colostrum IgG. European Journal of Immunology, 42.2771-2781. DOI: 10.1002/eji.201242469Chung, A. W., Navis, M,, Isitman, G., Wren, L., Silvers, J., Amin, J., et al. (2011). Activation of KN cells by ADCC antibodies and HIV disease progression. Journal of Acquired Immune Deficiency Syndrome, 58(2),127-131. DOI: 10.1097/QAI.0b013e31822c62b9Wren, L., & Kent, S. J. (2011). HIV vaccine efficacy trials: glimmers of hope and the potential role of antibody-dependent cellular cytotoxicity. Human Vaccines, 7(4), 466-473. DOI: 10.4161/hv.7.4.14123Background: Antibodies (Ab) mediating cellular cytotoxicity (ADCC) could potentially provide protection from HIV-1 acquisition in a vaccine setting. ADCC Abs mediate the cytolysis of HIV-1 infected target cells through the cross-linking of the Fc portion of the Abs by effector cell populations bearing Fc receptors (FcRs) such as NK cells. ADCC activated effector cells also secrete pro-inflammatory cytokines, which helps to recruit other immune cells to aid in the clearance of infected cells. ADCC responses occur naturally in HIV-1 positive people and have often, but not always, been associated with slower progression of HIV-1 to AIDS. In addition, ADCC responses were elicited by the recent RV144 HIV-1 vaccine trial carried out in Thailand. This trial provided a modest 31.2% reduction in HIV-1 acquisition in the vaccinated cohort. By studying ADCC responses elicited naturally and by vaccination, the potential of ADCC responses to protect from HIV-1 acquisition can be explored. Furthermore a better understanding of ADCC targets and methods for manipulating or enhancing this potentially beneficial immune response will enable the development of more effective HIV-1 vaccines. Methods: ADCC responses against whole Env glycoprotein (gp) 140 and multiple linear HIV-1 overlapping peptide pools were analysed in 65 Long term slow progressors (LTSP) and 74 subjects with progressive HIV-1 disease. Studies were carried out using an assay that assessed ADCC activity based on the activation of NK cells. We evaluated the effect of Ab purification from the plasma of HIV+ subjects and the effect of a number of HIV-relevant soluble factors on the activation profile of healthy donor NK cells using the NK cell activation ADCC assay. This assay was used in conjunction with a rapid fluorescent ADCC (RFADCC) assay, which measures ADCC based on target cell cytolysis, to assess ADCC responses elicited by the phase II RV135 and phase III RV144 HIV-1 vaccine trials. Results: We found that the breadth but not the magnitude of ADCC responses was correlated with slow progression of HIV-1. In addition regulatory/accessory HIV-1 proteins were targeted more frequently in LTSPs. ADCC responses targeting HIV-1 Vpu were over represented in LTSPs and Vpu-specific ADCC Abs were mapped to 3 distinct epitopes within the protein. The cytokines interleukin (IL)-15 and IL-10 were found to consistently enhance both the cytokine production and degranulation of ADCC activated NK cells. Envspecific ADCC responses were detected in plasma samples from the RV135 and RV144 trials, consistent with recent reports in the literature, although improved methods will be needed to map epitope-specific responses. Conclusions: Broader ADCC responses may play a role in long-term control of HIV-1 progression in a subset of LTSPs. Conserved ADCC epitopes within HIV-1 Vpu may represent potentially beneficial vaccine targets. The cytokine milieu influences the ADCC effector cell response and those involved with enhancing the magnitude of ADCC responses may be useful vaccine adjuvants. Improvements to the breadth, specificity and durability of the HIV-1-specific ADCC responses elicited by the RV144 trial could improve vaccine efficacy to the point where it would be a viable vaccine to administer to large human populations

Age-dependent, polyclonal hyperactivation of T cells is reduced in TNF-negative gld/gld mice

The generalized lymphoproliferative disorder (gld) mouse strain is characterized by severe splenomegaly/lymphadenopathy, the production of autoimmune antibodies, and the appearance of CD4/CD8-negative T cells. An additional TNF deficiency of gld/gld mice attenuates the course of the disorder through a yet-unknown mechanism. In this study, we could demonstrate that the reduced splenomegaly and lymphadenopathy in B6.gld/gld.TNF–/– mice were correlated with a decreased peripheral T cell proliferation rate and a delayed polyclonal activation. A comparative analysis of naïve T cells and memory/effector T cells showed an age-dependent difference in the T cell activation pattern in the spleen of B6.gld/gld and B6.gld/gld.TNF–/– mice. T cells from B6.gld/gld.TNF–/– spleens and lymph nodes showed significantly higher levels of CCR7 and CD62 ligand on their surface compared with B6.gld/gld mice when mice of the same age were compared. Additionally, we found an increased titer of the Th1 cytokine IFN-{gamma} in the serum of B6.gld/gld mice, whereas the concentration of IFN-{gamma} was markedly reduced in the serum of B6.gld/gld.TNF–/– mice. These findings support the hypothesis that increased T cell activation and proliferation in the presence of TNF contribute to the exacerbation of the gld syndrome

Clinical characteristics of HIV-infected donors.

<p>Clinical characteristics of HIV-infected donors.</p

Role of NK cell education in exogenous cytokine stimulation.

<p>Whole blood from nine HLA-Bw4 carriers and five HLA-Bw6 homozygous healthy controls was incubated with ADCC-competent plasma in the presence of HIV-1 Env antigen, with or without exogenous IL-15. (A) CD3⁻ CD56⁺ NK cells were gated upon and assessed for KIR3DL1 expression. (B) KIR3DL1⁺ NK cells from HLA-Bw4 carriers and HLA-Bw6 homozygotes were assessed for their ability to mediate bifunctional (i.e., CD107a⁺IFNγ⁺) anti-HIV ADCC responses in the absence and presence of IL-15. The graph represents the influence of IL-15 on the bifunctionality of NK cells from the KIR3DL1⁺ subset in both groups. The impact of IL-15 on the bifunctionality of these NK cells was assessed using paired T-tests.</p

Consistent effects of IL-10 and IL-15 across numerous donors.

<p>(A) Titration curves depict the effects of adding IL-10 or IL-15 to donor NK cells from three donors, which were all stimulated with the same HIV⁺ plasma in the presence of HIV-1 Env antigen. (B) NK cells from a common donor were stimulated with plasma from 9 HIV-infected individuals in the presence of HIV-1 Env antigen, with or without exogenous cytokines. Graphs on the top demonstrate the consistency of the effect of exogenous IL-10 and IL-15 on bifunctional (IFNγ⁺CD107a⁺) response profiles, regardless of the source of the plasma. Graphs on the bottom depict the consistency of the effect of exogenous IL-10 (50 ng/ml) and IL-15 (5 ng/ml) on the percentage contribution bifunctional responses make to the total NK cell response.</p

Effect of exogenous cytokines on ADCC-induced NK cell activation.

<p>A suite of 7 cytokines and LPS was added separately to a mix of HIV⁺ plasma co-incubated with healthy donor blood and HIV-1 Env antigens. Gated CD3⁻CD2⁺CD56⁺ NK cells were studied for CD107a and IFNγ expression. These experiments were repeated using whole blood from four separate donors (A) Zebra plots depict the anti-HIV ADCC mediated by NK cells when incubated with cytokines in the absence of anti-HIV antibodies and Env antigens, incubated with anti-HIV antibodies and Env antigens with no cytokines added, or with the addition of cytokines. The values shown represent the percentages of total NK cells expressing both IFNγ and CD107a. (B) The effect of all cytokines and LPS on ADCC-driven NK cell activation is shown, expressed as a ratio of the response with cytokine added to the response without cytokine. The effect of cytokine addition on both total CD107a⁺ and total IFNγ⁺ responses is shown. Error bars represent variation between different whole blood donors.</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