HIV replication and immune activation are critically intertwined. Immune activation has been recognized as a significant factor in the destruction of the immune response and rapid HIV disease progression 1 , but the relationship between immune activation and susceptibility to HIV infection is not well defined. In vitro studies demonstrated that quiescent CD4+ T cells can be infected by HIV, but viral replication is inefficient 2 . As such, HIV preferentially establishes productive infection in activated T cells 3 4 . One reason for this preference is the large number of host factors required for efficient HIV replication 5 6 7 , which are primarily expressed in activated cells. Based on these observations, it is reasonable to hypothesize that individuals with lower levels of immune activation would have lower susceptibility to HIV infection. Indeed, in the Pumwani HESN cohort, reduced susceptibility of unstimulated PBMC to in vitro HIV infection was linked to low levels of activated CD4+ CD69+ T cells and elevated levels of immunosuppressive Tregs 8 , in agreement with earlier data showing elevated HIV susceptibility of lymphocytes from individuals with high immune activation 9 . These data support a previous study, which showed that unstimulated cells from HESN had reduced susceptibility to infection compared to controls, but levels of infection were comparable when cells were pre-activated with a mitogen 10 . In addition to these in vitro data, evidence gathered from various models support this immune quiescence hypothesis, as detailed in the sections below.

To evaluate the role of T cell activation on resistance to infection, T cells were phenotyped in HESN and HIV-negative, yet susceptible, CSW from the Pumwani cohort. Reduced CD69+ CD4+ and CD8+ T cells were observed in HESN when compared with controls, indicating low levels of T cell activation 17 .

Further evidence of low T cell activation in HESN participants of the Pumwani cohort is provided by a study in which microarray technology was implemented to investigate CD4+ T cell function 18 . These analyses revealed that unstimulated CD4+ T cells from HESN had a lower level of gene expression than high risk HIV-negative controls, suggesting a quiescent cellular state. A subsequent study confirmed these observations by examining gene expression in whole blood 19 . Genes involved in T cell receptor signaling and host factors required for HIV replication were among the underexpressed genes in HESN 18 19 . Unstimulated cells from HESN were also found to secrete lower levels of cytokines ex vivo compared to control groups, but this difference in cytokine secretion was not observed following stimulation 18 , indicating that HESN have a low baseline level of cellular activation, but respond normally to stimulation and are not immunosuppressed. Likewise, a recent study demonstrated reduced production of proinflammatory cytokines and chemokines by unstimulated lymphoctyes from HESN relative to low-risk HIV-negative controls 20 . While the majority of cytokines were comparable between groups following stimulation of peripheral blood mononuclear cells (PBMC), IL-17 and IL-22 remained lower in HESN compared to controls, suggesting blunted Th17 responses in HESN 20 .

Evidence for T cell immune quiescence in HESN is not limited to the Pumwani cohort. For instance, low frequencies of CD4+ and CD8+ T cells expressing the activation markers HLA-DR, CD38, CD70 and Ki67 were identified in HESN men who have sex with men (MSM) 21 , and studies of serodiscordant couples found reduced expression of CD38 22 , HLA DR and CCR5 10 on CD4+ T cells from the uninfected partner. Lymphocytes isolated from HESN CSW from a cohort in Côte d’Ivoire expressed lower levels of CD69 and reduced proinflammatory cytokines and chemokines following allostimulation compared to low-risk HIV-negative controls 23 . Other HESN studies of CSW from the Central African Republic and intravenous drug users in Vietnam showed that despite higher CD8+ T cell activation, there were significantly fewer CCR5+ CD4+ T cells in the blood of HESN 10 24 . Also, independent studies of uninfected hemophiliacs who were exposed to HIV-contaminated blood products 25 and of high-risk MSM 21 found reduced lymphoproliferation in HESN compared to healthy controls. Together, these studies from a variety of cohorts showed a common phenotype of reduced systemic immune activation in HESN.

The majority of HIV transmissions occur at the genital mucosa. As such, mucosal immunology is a central issue in studies of HIV susceptibility. Evaluations of inflammation in the female genital tract have demonstrated immune quiescence at the mucosal level. For instance, cervical mononuclear cells (CMC) from HESN from the Pumwani cohort were shown to have reduced expression of genes encoding proinflammatory cytokines compared to high-risk HIV-negative CSW from the same cohort 20 . Of note, reduced cervical expression of IL-17 and IL-22 were observed in that study, consistent with observations from the peripheral blood. Th17 cells expressing these cytokines may be an important target for HIV infection, as these cells are enriched in the CMC population and are preferentially depleted in HIV-infected individuals 26 .

CMCs from HESN from the Pumwani cohort were also observed to have reduced expression of genes encoding various pattern recognition receptors (PRRs), including toll-like receptor (TLR)-2, TLR-4, TLR-7, TLR-8, RIG-I and MDA5. HESN also had reduced expression of UNC93B, which is involved in trafficking of TLRs within the cell. CMCs also produced low basal levels of cytokines in culture, but were highly responsive to stimulation with the TLR7/8 ligand ssRNA40, suggesting that despite low levels of PRRs at baseline, HESN are capable of mounting an appropriate anti-viral response 27 .

Consistent with reduced proinflammatory cytokine expression by CMCs, reduced expression of IL-1α, IL-8, MIG and IP-10 were found in HESN cervicovaginal lavage (CVL) samples compared to high-risk HIV-negative controls 28 . MIG and IP-10 are both IFNγ-inducible chemokines that bind the receptor CXCR3, inducing recruitment of activated T cells. Lower levels of these chemokines would result in a decreased recruitment of activated T cells to the FGT. Consistent with this, a trend toward reduced proportions of cervical HLA DR + CD8+ T was observed in HESN 28 .

Overall, these studies described HESN individuals with lower levels of inflammation at the FGT that may result in fewer HIV target cells at the point of first encounter with HIV.

As described in the sections above, elevated levels of immune activation are associated with increased risk of HIV infection, and protection from infection correlates with immune quiescence. This is marked by reduced T cell activation, reduced gene transcription, reduced lymphoproliferation, reduced peripheral and mucosal cytokine and chemokine production, elevated Tregs, elevated mucosal anti-proteases and reduced IRF-1 expression and IFNγ-responsiveness. Previous HESN studies have demonstrated HIV-specific T cell responses, both systemically and mucosally 48 . Taken together, these observations can be used to frame a model of reduced susceptibility to infection. In HIV-susceptible individuals, HIV exposure leads to penetration of the mucosal barrier and establishment of infection in a small focus of CD4+ target cells. Inflammation resulting from infection drives infiltration of activated target cells, which fuel propagation of infection and dissemination to lymphoid tissues. Once dissemination occurs, the infection cannot be cleared (Figure 1A). In contrast, in HESN, innate molecules in the FGT mucosa may limit establishment of the initial focus of infection through direct anti-viral activity and maintenance of mucosal integrity. While it is likely that some viral particles penetrate the epithelial barriers and gain access to target cells in the submucosa, Tregs and antiproteases maintain low levels of T cell activation, leading to reduced availability of activated CD4+ target cells. Consequently, the target cell population is limited to resting T cells, which do not effectively support viral replication. Reduced expression of proinflammatory cytokines and chemokines prevents infiltration of activated target cells. As such, the focus of viral replication remains small and unproductive in HESN, allowing innate or adaptive cells, such as NK or HIV-specific CTL, opportunity to clear the low-level infection and prevent dissemination (Figure 1B).

The immune quiescence model is supported by studies of mucosal simian immunodeficiency virus (SIV) transmission in non-human primates (NHP). In acute infection of rhesus macaques, a small founder population of infected cells is established in the submucosa within the first few days following viral challenge. This founder population is comprised of mainly resting CD4+ T cells, as these cells outnumber activated cells. Local expansion of the founder population occurs through low-level viral replication in resting cells. However, the infection is fuelled by an influx of activated target cells in the inflammatory infiltrate resulting from the innate response to SIV 49 . In this way, SIV exploits the inflammatory immune response to gain access to target cells for viral replication and dissemination of infection. Interestingly, topical administration of the anti-inflammatory compound glycerol monolaurate blunts the inflammatory response, preventing infiltration of activated target cells to the FGT, resulting in protection from SIV infection 50 . Furthermore, vaccination of macaques with a tolerogenic vaccine prior to SIV exposure resulted in development of CD8+ Tregs that suppressed CD4+ T cell activation and prevented the establishment of SIV infection 51 .

In SIV-infected animals, SIV-specific CD8+ T cells are detectable by the second week of infection. However, infection is well established by this time, such that the ratio of HIV-specific CD8+ T cells to infected target cells is too low to prevent further viral replication and dissemination 52 . Nonetheless, mucosal HIV-specific T cells present at the time of inoculation have been shown to be protective against simian-human immunodeficiency virus (SHIV) challenge 53 . We propose that in HESN, mucosal HIV-specific T cells, which have been previously identified in HESN 54 , are present early enough to clear the initial focus of infection prior to expansion and dissemination.

Data that conflict with the immune quiescence hypothesis appear in reports of elevated immune activation as a correlate of protection in HESN. Elevated levels of T cell activation and cytokine production have been found in systemic or mucosal samples from cohorts of HESN injection drug users (IDU) 24 55 56 and uninfected partners of HIV + patients 24 55 56 57 . However, in the Vietnamese HESN IDU cohort 24 , activation of the CD8+ and NK T cell compartments occurred at the same time as reduced levels of CD4+ CCR5+ T cell targets. This suggests the possibility that in certain cohorts, CD4+ T cell quiescence can occur concomitantly with activation of other immune cell subsets. It is possible that other disparate findings may be reconciled by differences in the site or intensity of HIV exposure, presence of other immunogenic factors or differences in assays used to characterize immune activation.

An important consideration is the translation of the immune quiescence hypothesis into interventions that prevent HIV infection. Our model suggests that inducing immune quiescence at the site of HIV exposure will reduce the number of activated target cells, thereby preventing infection or limiting it to small foci of infected resting target cells, which can be cleared by mucosal HIV-specific T cells or innate mechanisms. A practical method of inducing immune quiescence in the mucosa may be a microbicide that incorporates anti-inflammatory mediators into the formulation, perhaps in combination with antiretroviral compounds that have shown moderate efficacy in recent trials 14 . This approach has been suggested based on results of functional studies performed in the wake of the CAPRISA 004 trial 15 . Suitable compounds would be drugs that are already approved by the FDA, have outstanding safety records, have a demonstrated ability to reduce excessive immune activation, and are affordable for those in developing countries. There are a number of potential candidate anti-inflammatory compounds, such as type I interferon blockers 58 , cyclooxygenase type 2 inhibitors 59 , statin inhibitors 60 , chloroquine 61 62 and others, that address some or all of these criteria. An advantage of the approach of inducing the IQ phenotype is that it is targeting the host immune system and not the virus, thereby limiting any pressure on HIV to mutate to evade the mechanism.

In implementing induction of immune quiescence as a strategy to reduce HIV transmission, caution needs to be taken to ensure that the limitation of excessive mucosal T cell activation does not result in the induction of an inability to mount a mucosal immune response. Indeed, it may seem counter-intuitive to try to limit excessive immune activation on the one hand, while at the same time inducing anti-viral immune responses with an HIV vaccine. However, these goals are not incongruent. Limiting HIV target cells by inducing IQ in the FGT can be accomplished on its own, independent of an HIV vaccine. Should this strategy be used in conjunction with an HIV vaccine, it would be important to activate and educate the immune system with the vaccine first, prior to inducing IQ in the FGT. It is important to recall that the induction of IQ is not equivalent to the induction of anergy. As shown, individuals with high levels of quiescent T cells are capable of responding to recall antigen or mitogens 63 . Indeed, in the Pumwani HESN cohort we observe a quiescent phenotype 63 and HIV-specific immunity 64 65 . Therefore, HIV-specific immunity in the context of decreased generalized T cell immune quiescence is not only highly plausible, but it has been observed. As such, candidate formulations designed to induce IQ will need to be tested for the ability to reduce immune activation and inflammation without interfering with HIV-specific CD8+ T cell responses or innate antiviral mechanisms such as NK cells.