The innate immune effector cell response against HIV-1

Since being identified as the cause of AIDS in 1983, HIV-1 infection has reached pandemic proportions. Despite public awareness about prevention, the growing incidence of HIV-1 infection and the limitations of current antiretroviral therapy underscore the imperative need for a vaccine. Understanding the basis of an immune response that controls infection or provides sterilizing immunity remains a major goal in the search for effective vaccines or immunotherapies. Research into correlates of immunity to HIV-1 have largely focused on CD8+ T cells or neutralising antibodies (NAbs) but to date these responses have not proved effective in containing viral replication in vaccinees who become infected. Natural killer cells (NKs), monocytes (MCs), and neutrophils (PMNs) are cells of the innate immune system with intrinsic cytotoxic function that can be enhanced by antibodies (Abs) in what is termed antibody-dependent cellular cytotoxicity (ADCC). In my studies I investigated the production of PMNs from human stem cells, the elimination of HIV-1 infected cells by these effector cells, the modulation of cellular cytotoxicity by Ab, and characterized how Abs facilitate a potent ADCC response. I developed a novel flow cytometry assay to measure cytotoxic activity against HIV-1 infected CD4+ T cells. Using this, effector cells were shown to have different cytotoxic capacities which were enhanced by Ab. Comparing ADCC mediated by patient serum revealed that higher levels correlated with IgG binding to infected cells. I observed no correlation between serum-mediated ADCC and markers of disease progression including patient status, viral RNA load, CD4+ T cell count, or NAb titers. The data presented here have implications for acquisition and control of early HIV-1 infection by NKs, MCs, and PMNs prior to activation of an adaptive immune response, at later stages in the presence of HIV-1-specific Abs, and are relevant to vaccine-induced anti- HIV-1 Ab-based effector mechanisms.This thesis is not currently available in ORA

NKs, MCs, and PMNs differ in killing activity.

<p>Total area under the curve (AUC) of % ICE curves presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074858#pone-0074858-g003" target="_blank">Figure 3</a> for log₁₀-transformed serum dilutions from 1:20 to 1:62,500 was calculated for all 7 subjects and means calculated. Comparisons of independent groups were made by two-way or one-way ANOVA (A and B, respectively) with Bonferroni post-test. (A) Comparison of AUC for infected cell elimination in the absence of serum or effectors (AUC set at 1), effector cells with control serum, and effector cells with Bpool serum. HIV-1-specific Abs enhance killing by NKs (<i>P</i> < 0.001), MCs (<i>P</i> < 0.01), PMNs (<i>P</i> < 0.05). (B) Comparison of AUC for effector cells with Bpool serum. NKs and MCs mediate similar levels of ADCC; PMNs mediate significantly less ADCC than NKs (<i>P</i> < 0.05).</p

Purity of isolated target and effector cells.

<p>Isolated cells were stained with an appropriate single cell marker for identification. The percent positive populations (B) were assessed on live-gated cells (A): CD4⁺ T cell = CD3^hi, NK = CD56^dim, MC = CD14^hi, PMN = CD66b^hi.</p

NKs, MCs, and PMNs mediate ICE.

<p>Percentage ICE mediated by NKs, MCs, and PMNs in seven subjects, at an effector:target ratio of 10:1 without or with Bpool (HIV⁺) or control (HIV^-) serum at serial 5-fold dilutions starting at 1:20. Each datum point represents the mean of duplicate samples.</p

Lentiviral gene therapy vector with UCOE stably restores function in iPSC-derived neutrophils of a CDG patient

A recent gamma-retroviral clinical Chronic Granulomatous Disease (CGD) gene therapy (GT) trial achieved proof-of-concept but was accompanied by activation of oncogenes and transgene silencing. The ubiquitous chromatin opening element (UCOE) comprises the sequences of two divergently oriented house-keeping gene promoters and is known to have anti-silencing properties. In a screen we identified two novel UCOE constructs that prevent adjacent promoter methylation in P19 cells. Experiments were continued with the shorter UCOE constructs in induced pluripotent stem cells (iPSC) derived from a p47phox-deficient CGD patient. The iPSC line was transduced with the lentiviral GT vectors expressing p47 under the constitutively active SFFV promoter with UCOE element (UCOE_SF) and without UCOE element (SF) adjacent to the SFFV promoter. The iPSC were expanded before propagation towards neutrophils. 20 days after transduction the UCOE_SF vector was protected from methylation in iPSC as previously shown in P19 cells, whereas the SF vector was heavily methylated in iPSC. The UCOE_SF vector maintained stable transgene expression in iPSC, macrophages and neutrophils, whereas the SF vector was strongly silenced. The UCOE_SF vector stably restored ROS production in neutrophils, whereas for the SF vector the count of ROS producing cells was marginal. To conclude, we have shown that the prevention of transgene silencing by UCOE is functionally and mechanistically preserved upon terminal neutrophil differentiation