A detailed description of the roles of Vpu as an antagonist of the restriction factor tetherin, which prevents viral budding and release from infected cells, and in the regulation of host transmembrane proteins, are described elsewhere in this edition (Neil lab review). Its anti-tetherin activity also implicates Vpu in innate intracellular signaling pathways, because tetherin also acts as a PRR that activates signaling cascades upon recruitment of HIV virions (84, 85). Intriguingly, another study examining tetherin signaling demonstrated that tetherin has a long and a short isoform, that are, as for MAVS, derived from alternate start codons (243, 244). Also like MAVS, the long form can activate an innate immune signal whereas the short form cannot. Intriguingly, Vpu preferentially targets the long signaling form of tetherin, despite the fact that the short form is competent for tethering newly formed virions (243). In the light of these data, we consider that the name tetherin is rather misleading, in fact, being tethered may not be disadvantageous to HIV-1, particularly given this feature aids cell-to-cell spread in T cells (245). Indeed, preferential targeting of the long form of tetherin by HIV-1 suggests that it is signaling and its consequences that exert the dominant pressure on the virus. This is consistent with a model in which the most important feature of restriction factors is their PRR function, which can protect many cells through initiating IFN responses, rather than their restriction function, which is focused on individual viral particles. Having said this, tetherin signaling may be a recent adaptation given that simian tetherin variants were found to be unable to activate signaling when expressed in 293T cells (84). Of course, these proteins may signal in their cognate species and the role of signaling in viral restriction by tetherin requires further study. Furthermore, cell-free virus is required for transmission and therefore antagonism of tetherin by Vpu is critical. Tethered viruses may also enhance antibody-dependent cell-mediated cytotoxicity (246).

In contrast to other accessory proteins, Vpu is exclusively encoded by HIV-1 lineage viruses and is absent from HIV-2 clades. It is not packaged inside the virion, and its Rev-dependent expression occurs late in the viral lifecycle (49). Vpu potently inhibits NF-κB activation and ensuing ISG expression; this likely requires viral integration in primary myeloid and CD4+ T cells (62, 247, 248). As mentioned earlier, activation of NF-κB occurs downstream of multiple innate sensing pathways to drive antiviral gene expression. Paradoxically, NF-κB activation is also implicated in driving HIV-1 and HIV-2 proviral transcription (249). Thus, primate lentiviruses may encode factors to closely regulate NF-κB activation at different stages in the lifecycle to strike a balance between shutting down antiviral responses and augmenting viral gene expression. In particular Nef and Tat, expressed at high levels early in the viral lifecycle, have been shown to increase virus replication by promoting NF-κB activation (250, 251). Intriguingly, Vpu's role as an antagonist of innate immune signaling is independent of tetherin antagonism and is apparently conserved between all lineages of SIV and HIV-1 containing Vpu (except HIV-1 group N) (62, 248). Several reports show that Vpu disrupts NF-κB activation downstream of a range of exogenous and overexpression stimuli that are not specifically related to innate signaling (62, 84, 85, 248). Besides NF-κB, there are also conflicting data regarding antagonism of IRF3 by Vpu (248, 252, 253). It seems likely that host cell type and activation status may significantly impact the role of Vpu in the context of intracellular innate immune responses to HIV, which has yet to be fully elucidated.

#### FUTURE PERSPECTIVES

The last decade has seen an extraordinary expansion in our understanding of HIV and its interaction with the cellautonomous innate immune system, especially pertaining to the field of DNA sensing. We are beginning to understand the complexities of the cellular responses to HIV-1 and the subtleties of HIV evasion strategies in different cell types. Viruses are the masters of compromise, able to switch roles between viral accessory proteins or finely tune their behavior with as little as single amino acid changes. In our view, particularly pertinent studies are those that explain the differences in cofactor requirements or innate evasion strategies in cell lines versus primary cells. Of course, cell lines make tractable models for HIV infection, but we must remember that, in many cases, this is because of defects in cell-autonomous innate immunity related to their cancer origins, and so we must be cautious in interpreting experiments studying tropism in cells that cannot mount authentic responses. Also important are studies that take into account the fact that cells communicate through cytokine and cGAMP secretion (63, 72). Humanized mouse models are now allowing sophisticated and relatively cheap *in vivo* investigation of HIV therapeutics and innate responses (254–256). Similar studies considering innate immunity may eventually be more informative for HIV-1 than simian models given that simian lentiviruses, such as the Vpx encoding SIVmac, have different cellular tropisms and likely innate immune relationships with their hosts. Mouse models may be particularly effective for the study of HIV-1 tropism in human T cells *in vivo*, which must be activated for HIV-1 replication *in vitro*, thereby masking innate responses and cytokine secretion profiles in response to the virus itself.

It is clear that HIV adaptation to host defenses influences the HIV-1 lifecycle at every stage. As our understanding of HIV-1 innate immune evasion increases, comparative studies between the different SIV and HIV strains are becoming increasingly informative and may shed light on the determinants of pandemicity. Manipulation of these critical host–virus interactions offers tantalizing opportunities for development of novel therapies. However, excitement at the prospect of translation of our rapidly expanding knowledge base must be tempered by the contradictions and uncertainties in the field; there is still much to be understood. The literature tells us that there is great diversity in the innate immune capacities of each cell type relevant to HIV infection. One major and evolving challenge is understanding the dynamic relationship between intracellular immunity and the specific circumstances of each individual cell or cell population: cell location, cell-cycle status, relative cytokine exposure, for example, will variegate each cell's interaction with HIV. We view HIV as the ultimate tool for molecular cell biology, which, correctly deployed, will teach us a great deal of fundamental human biology and continue to transform our understanding of health and disease leading to innovative new tools and therapeutics.

## AUTHOR CONTRIBUTIONS

All authors contributed to conception, drafting, and final approval and are accountable for the integrity of the final manuscript. RS and LT contributed equally to this article.

### ACKNOWLEDGMENTS

Work in the Towers lab is funded by a Senior Biomedical Research Fellowship from the Wellcome Trust to GT (108183/Z/15/Z),

### REFERENCES

an ERC Advanced grant from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007–2013)/ERC grant agreement number 339223 and the National Institute for Health Research (BRC317). LT is a Henry Wellcome Post Doctoral Fellow funded by the Wellcome Trust and the Royal Society, DF is a Wellcome Trust Clinical PhD Fellow (205236/Z/16/Z), and HK is a Medical Research Council UK funded PhD student. The authors are grateful to all members of the Towers group for their constructive input to this review.

and tissues: implications for HIV-1 restriction. *Nucleic Acids Res* (2010) 38(13):4274–84. doi:10.1093/nar/gkq174

237. Dehe PM, Gaillard PH. Control of structure-specific endonucleases to maintain genome stability. *Nat Rev Mol Cell Biol* (2017) 18(5):315–30. doi:10.1038/ nrm.2016.177

238. Fregoso OI, Emerman M. Activation of the DNA damage response is a conserved function of HIV-1 and HIV-2 Vpr that is independent of SLX4 recruitment. *MBio* (2016) 7(5):e01433-16. doi:10.1128/mBio.01433-16

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Sumner, Thorne, Fink, Khan, Milne and Towers. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

#### *Dimitra Peppa1,2\**

*1Division of Infection and Immunity, University College London, London, United Kingdom, 2Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom*

Human cytomegalovirus (HCMV) has been closely associated with the human race across evolutionary time. HCMV co-infection is nearly universal in human immunodeficiency virus-1 (HIV-1)-infected individuals and remains an important cofactor in HIV-1 disease progression even in the era of effective antiretroviral treatment. HCMV infection has been shown to have a broad and potent influence on the human immune system and has been linked with the discovery and characterization of adaptive natural killer (NK) cells. Distinct NK-cell subsets, predominately expressing the activating receptor NKG2C and the marker of terminal differentiation CD57, expand in response to HCMV. These NK-cell populations engaged in the long-lasting interaction with HCMV, in addition to characteristic but variable expression of surface receptors, exhibit reduced expression of signaling proteins and transcription factors expressed by canonical NK cells. Broad epigenetic modifications drive the emergence and persistence of HCMV-adapted NK cells that have distinct functional characteristics. NKG2C+ NK-cell expansions have been observed in HIV-1 infected patients and other acute and chronic viral infections being systematically associated with HCMV seropositivity. The latter is potentially an important confounding variable in studies focused on the cellular NK-cell receptor repertoire and functional capacity. Here, focusing on HIV-1 infection we review the evidence in favor of "adaptive" changes likely induced by HCMV co-infection in NK-cell subsets. We highlight a number of key questions and how insights into the adaptive behavior of NK cells will inform new strategies exploiting their unique properties in the fight against HIV-1.

Keywords: human immunodeficiency virus, human cytomegalovirus, natural killer cells, NKG2C, CD57, adaptive

### INTRODUCTION

Natural killer (NK) cells are a diverse group of innate lymphocytes residing at the crossroads of innate and adaptive immunity (1). Their remarkable effector agility is achieved via expression of a wide array of receptors and integration of signals that are finely attuned to ensure self-tolerance, while permitting effective responses against viral assaults and tumor transformation. In addition to important immunoregulatory functions (2, 3), a number of murine studies support that NK cells can acquire immunological memory similarly to B and T cells (4–7). While antigen-specific NK responses have been documented in mice and more recently in primates (8), clear evidence for NK-cell memory

#### *Edited by:*

*Nina Bhardwaj, Icahn School of Medicine at Mount Sinai, United States*

#### *Reviewed by:*

*Amir Horowitz, Icahn School of Medicine at Mount Sinai, United States Domenico Mavilio, Università degli Studi di Milano, Italy*

> *\*Correspondence: Dimitra Peppa<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to HIV and AIDS, a section of the journal Frontiers in Immunology*

*Received: 30 June 2017 Accepted: 29 September 2017 Published: 17 October 2017*

#### *Citation:*

*Peppa D (2017) Natural Killer Cells in Human Immunodeficiency Virus-1 Infection: Spotlight on the Impact of Human Cytomegalovirus. Front. Immunol. 8:1322. doi: 10.3389/fimmu.2017.01322*

in humans is lacking. The NK-cell compartment in humans displays phenotypic and functional heterogeneity encompassing populations at various stages of maturation with distinct receptor combinations (9–11). In recent years, it has become apparent that variegated expression of inhibitory and activating receptors at the single cell level leads to a more diverse NK-cell repertoire than previously envisaged. Cytometry by time-of-flight has enabled us to profile the healthy human NK-cell repertoire, uncovering between 6,000 and 30,000 unique NK-cell subsets per individual (12). This observed diversity is generated by a combination of factors including genetic contributions (13, 14), along with differentiation in reprogramming in response to local tissue milieu (15) and infections/environmental factors (12). The substantial influence of environmental factors is supported by twin studies demonstrating that non-heritable factors exert a more profound and cumulative influence compared to heritable traits (16, 17). One such factor is human cytomegalovirus (HCMV), a widespread β-herpesvirus with a prevalence ranging from 40 to 100% depending on age, socioeconomic factors, and geographical region (18). In immunocompetent hosts, HCMV infection is usually subclinical leading to latency, whereas in immunosuppressed patients, including human immunodeficiency virus-1 (HIV-1) infected and transplant patients, it remains a significant cause of morbidity and potentially life threatening complications (18). HCMV has a broad impact on immunity (16) and has recently been associated with the expansion of adaptive or memory-like NK-cell subsets (19, 20).

In the context of HIV infection, HCMV is a highly prevalent (21) and well-recognized opportunistic pathogen responsible for significant morbidity and mortality prior to the introduction of antiretroviral treatment (ART) (22, 23). However, despite the roll-out of effective ART, HCMV remains a significant cofactor in HIV-1 disease progression (24–26), displaying a strong association with systemic inflammation (27, 28), cardiovascular disease (29, 30), reduced immune resilience (31), and immune senescence (27). A recent report has highlighted the role of HCMV replication in intestinal barrier dysfunction in asymptomatic HIV-1 infection and contribution to persistent immune activation (32). It is thus highly relevant to increase our understanding of the complex inter-relationship between HCMV and HIV-1 and of the effects that it bears on the effector immune response. The recent identification of distinct NK-cell subsets with adaptive properties induced by HCMV has raised a number of intriguing questions, including the ability of other viruses to induce them and their physiological relevance in different disease settings. Here, we summarize findings on the molecular signature of HCMV-adapted NK cells and discuss how NK-cell phenotypic and functional features described in HIV-1 infection could partly reflect the immunological fingerprint of HCMV.

#### FEATURES OF CMV-ADAPTED NK CELLS—EMPHASIS ON HCMV

Evidence from both murine and human studies has demonstrated an important role for NK cells in antiviral defense against herpesviruses, in particular HCMV (33), reinforced by elaborate viral evasion strategies (34).

Although NK cells have been originally described to represent short-lived innate lymphocytes, they can exhibit persistent memory in response to infections. This is best exemplified by mouse CMV (MCMV) infection, where naive NK cells that express Ly49H, recognizing the virally encoded glycoprotein m157, were reported to clonally expand and to subsequently contract forming a pool of long-lived memory cells (6). MCMVprimed memory NK cells mount a robust response upon secondary challenge with enhanced interferon-γ (IFN-γ) secretion and cytotoxicity (6), but display reduced "bystander" functionality to heterologous infection suggesting the specialized nature of these cells (35).

Congruent with animal models, HCMV infection has been shown to induce an adaptive reconfiguration of the NK-cell compartment. Seminal work by Lopez-Botet's group described a higher proportion of NK cells expressing the DAP-12 coupled NKG2C receptor in healthy individuals seropositive for HCMV (36, 37). These observations have been extended to hematopoietic stem cell transplantation (38, 39) and solid organ transplantation (40). Expansion of these subpopulations of NK cells and their subsequent longevity resembled clonal expansion of adaptive immune cells. Expanded NKG2C+ NK cells display a differentiated phenotype characterized by expression of CD57, increased expression of the inhibitory CD85j (38, 40), and a preferential oligoclonal pattern of inhibitory killer immunoglobulin receptors (KIRs) for self HLA-C1 and/or C2 allotypes (41, 42). In addition, they lack NKG2A, the inhibitory counterpart of NKG2C sharing specificity for HLA-E, and express lower levels of natural cytotoxicity receptors (NCR: NKp30 and NKp46) (36), CD161, CD7, and Siglec-7 (43–45) and have higher expression of CD2 involved in their activation (46, 47). Expression of other receptors such as NKG2D is maintained (36). The phenotypic hallmarks of adaptive NK cells are summarized in **Figure 1**. Of note, the magnitude of the HCMV imprint on NK-cell subsets varies within seropositive individuals (i.e., the NKG2Cbright phenotype is found in 50% of HCMV+ individuals) and the adaptive NKG2C+ compartment can persist in high frequencies for years (41). Subclinical or tissue specific reactivations of HCMV during latency may contribute to the maintenance of NK+NKG2C+ pool in addition to NKG2C copy number and age-related changes in NK-cell differentiation (48, 49). The exact ligand involved in recognition and the cellular mechanisms driving the expansion of NKG2C+ NK cells are yet to be elucidated. It remains unclear whether this is mediated through interaction with its cellular ligand HLA-E alone, HLA viral loaded peptide or an unknown ligand of host or viral origin (41, 50–52).

The large phenotypic heterogeneity of adaptive NK cells extending beyond the NKG2C+ subset, is illustrated by the detection of NK-cell subsets sharing numerous attributes of adaptive NK cells in individuals independent of NKG2C or in the absence of NKG2C (KLRC2-deficient individuals) and in transplant recipients of NKG2C null grafts (41, 47, 53). Strikingly, these HCMV-driven expansions encompass activating KIRs (53), suggesting their potential role in the recognition and response to HCMV.

Further reports described a subset of human NK cells deficient for the adaptor protein FcεRI-γ, which was strongly associated with HCMV seropositivity (54). FcεRI-γ− NK cells share a lot of the characteristics of adaptive NK cells, respond robustly to CD16 stimulation (55) and similar to NKG2C+ cells display more vigorous effector responses to HCMV-infected targets, but only in the presence of HCMV-specific antibodies (54, 56). NK cells lacking FcεRI-γ expand in response to HCMV-infected targets accentuated by the presence of anti-HCMV antibody, highlighting the role of specific humoral immunity in also favoring their preferential expansion (57–59). Interestingly, these cells also responded to herpes-simplex virus-1 (HSV-1)-infected targets in the presence of HSV-1 plasma (54) demonstrating cross-protection to other viruses. The enhanced effector function of this subset was attributed to selective and more potent signaling through the CD3ζ chain, which has three immunoreceptor tyrosine-based activation motifs. Subsequently, CD2 has been identified as a key co-stimulatory receptor synergizing with CD16 to stimulate increased cytokine production in adaptive NK cells (47). Global epigenetic profiling has identified commonalities between adaptive NK cells and memory CD8 T cells (58, 60). These adaptive NK cells are marked by DNA methylation silencing of the transcription factor, promyelocytic leukemia zinc finger (PLZF), as well as stochastic down-regulation of several signaling molecules, such as Syk, EAT-2, and DAB-2 (58, 60). PLZF is known to interact with several target genes, including IL12RB2, IL18RAP, and KLRB1 (61), explaining the lack of responsiveness to IL12/ IL18 stimulation (58). However, in comparison to conventional NK cells, adaptive NK cells display augmented IFN-γ and TNF-α production when triggered via antibody-dependent cellular cytotoxicity (ADCC); the hypomethylated IFN-γ and tumor necrosis factor (TNF) regulatory regions in adaptive NK cells provide a mechanism for increased cytokine production (58, 60). Interestingly, adaptive NK cells display reduced degranulation toward activated autologous T cells (58), which may impact on the regulation of immune responses.

Taken together, these results suggest the heterogeneity and functional specialization of adaptive NK cells in the immunosurveillance of infected cells and functional bias toward ADCC (**Figure 1**). Whereas the expansion of adaptive NK cells may serve as a strategy to control HCMV, during its life long interaction Peppa HIV-1/HCMV Co-Infection and NK Cells

with the host, it remains unclear whether other viral infections can induce adaptive properties in NK cells. Although potential cross-reactivity of adaptive NK cells could confer an advantage in the tumor setting such as reduced relapse risk in leukemia patients (62, 63), their role in the control of heterologous infections or post vaccination is less well defined (64, 65).

### SKEWING AND ADAPTATION OF NK CELLS TO HIV-1 INFECTION: THE CONFOUNDING EFFECT OF HCMV

Accumulating data support an important role for NK cells in the control of HIV-1 infection and protection against disease acquisition (66–68). These stem from elegant genetic studies linking specific KIR/HLA combinations with HIV-1 outcome (66, 67), functional studies where protective KIR alleles are associated with enhanced NK-cell cytolytic function *in vitro* (69) and evidence of KIR-facilitated immune pressure on HIV-1 to escape NK-cell recognition (70). However, chronic HIV-1 infection is known to alter NK-cell composition and effector function. This has been documented by a number of studies with often conflicting results, which can be attributed to a number of factors including the influence of immunogenetics, disease state, and the cross-sectional nature of studies. The latter have not always adequately controlled for a number of confounding factors such as age, gender, ethnicity, and HCMV serostatus among HIV-1-infected and HIV-1-negative controls. Given the high prevalence of HCMV co-infection within HIV cohorts and the profound skewing and adaptation of NK cells to HCMV, this is an important variable to consider when interpreting findings.

HIV-1 viremia is associated with a significant and pathological redistribution of the NK compartment with the emergence of an aberrant CD56−CD16+ NK-cell subset (71, 72). This rare population displays phenotypic perturbations, including downregulation of the activating NCRs, and features in common with mature CD56dim NK cells (72, 73). It has been proposed to represent an activated subset generated from chronic target engagement with impaired function. Recent studies have demonstrated that a decreased expression of the c-lectin-type inhibitory receptor, Siglec-7, on NK cells occurs early during HIV-1 infection and precedes the loss of CD56 (74). Expression of Siglec-7 is not affected in long-term non-progressors (LTNP), and ART leads to a progressive restoration of NK-cell subsets (74). Paralleling the observations in HIV-1 infection, HCMV reactivation in patients undergoing umbilical cord blood transplantation has been shown to induce the expansion of the CD56−/CD16+/Siglec-7− NK-cell subset (38). The expansion of hypofunctional CD56− NK cells following HCMV reactivation likely occurs when T-cell immunity is impaired and may also reflect the modulating effects of HCMV. It remains to be determined whether the CD56−/CD16+ subset represents a subgroup of NK cells with adaptive features that has become anergic following repeated stimulation.

A number of other studies have reported a variable degree of perturbations in the NK-cell repertoire consistent with a dichotomous effect of viremia, including down-regulation of activating NK-cell receptors and up-regulation of expression of inhibitory NK receptors (iNKRs) (75–77). Collectively, these changes have been described to contribute to defective NK-cell function described in HIV-1 infection (76, 77). Although the HCMV serostatus is not always considered in these studies, it is plausible that these changes are biased by HCMV co-infection and possible reactivation with increasing immunosuppression. Along these lines, the observed down-regulation of NCRs, stable expression of NKG2D, and higher levels of CD85j and skewing of inhibitory KIRs (although not consistently reported) bear phenotypic resemblance to NK-cell subsets with adaptive features described in HCMV infection. NK cells in HIV-1 infection exhibit a higher ratio of CD57+ to CD57− due to the loss of CD57− cells in comparison to healthy controls; however, this comparison may be confounded by the HCMV status of these individuals, which was not reported (78). A shift toward a more mature terminally differentiated NK-cell phenotype is nonetheless supported by a study of HIV-1 infected individuals on effective ART, demonstrating that HCMV accelerates age-related increases in CD57 expression (79).

The most convincing evidence of the impact of HCMV coinfection on the NK-cell repertoire in HIV-1 infection comes from reports on NKG2C expression. Guma et al. originally proposed that HCMV co-infection is responsible for the expansions of NKG2C+ NK cells encountered in HIV-1 infected individuals (80). These findings were further supported by additional studies when the HCMV serostatus was taken into consideration (81, 82). The dramatic expansion of NKG2C+ NK cells in HIV-1 infected individuals was accompanied by a decrease in the expression of NKG2A leading to a low NKG2A/C+ NK-cell ratio; these changes were attributed to concomitant infection and/or HCMV reactivation rather than being a consequence of HIV-1 infection alone (82). A number of reports describe NKG2C+ NK-cell expansions in several acute and chronic viral infections, being systematically associated with HCMV co-infection (83–86). Although the relative increase in the proportions of NKG2C+ NK cells between HIV-1-infected and HIV-1-uninfected HCMV seropositive individuals varies between studies and cohorts (80, 81), the data suggest that the impact of HCMV exposure is potentially greater in HIV-1 infection. It has been suggested by animal models that the differentiation of adaptive NK cells is driven by inflammation (87). Thus, it is plausible that adaptive NK-cell expansions may be inflated in HIV-1 infected individuals, as a result of lack of immune control, ongoing immune activation and higher infectious burdens, including HCMV. One could speculate that the size of the HCMV imprint represents a compensatory mechanism in antiviral defense especially when T-cell-mediated control is impaired (88). It remains uncertain whether HCMV reactivation occurs alongside acute infection or alternatively whether pre-existing HCMV primed NK-cell subsets expand in response to secondary viral infection alone. HIV-1 causes down-regulation of HLA-A, B while retaining HLA-E expression (89, 90), similar to HCMV maintaining/ stabilizing HLA-E expression (91, 92). Thus, a direct effect of HIV-1 on NKG2C+ NK-cell expansion is conceivable. The recently reported down-regulation of HLA-C by most primary HIV-1 clones (93) raises questions about the ability of HCMV expanded NKG2C+ NK cells, preferentially expressing self-HLA-C KIRs, to recognize "missing-self " on HIV-infected targets compared to mature educated NK cells.

Open questions remain regarding not only the mechanism but also the clinical implications of such HCMV-NK-cell interaction in terms of protection against acquisition and HIV-1 disease progression. NKG2C deletions have been linked to a higher risk of contracting HIV-1, in addition to accelerated disease progression and elevated pre-treatment viral load (94). Although these findings are interesting, this study did not report and correct for the influence of HCMV co-infection. One could speculate that the expansion of NKG2C+ NK cells in HCMV seropositive individuals may confer protection against primary HIV-1; this notion is however not supported by some older observations that prior infection with HCMV is associated with low CD4 count, progression to AIDS and increased mortality (95). It has been suggested that maturation leads to divergence and increased NK-cell receptor diversity was found to be associated with an increased risk of HIV-1 acquisition in a small cohort of high-risk women (96). Given that viral challenge may increase receptor diversity, further work is required to determine whether this represents reduced plasticity to new challenging pathogens or whether it is linked to other immune characteristics such as exhaustion. Recently, a subpopulation of PD1+ NK cells, mainly composed of fully mature NK cells, has been described in HCMV+ individuals (97). It would be of interest to assess whether NK cells expanded in HCMV/ HIV-1 co-infection succumb to continuous stimulation and examine the factors that may contribute to the induction of PD1 in this setting. PD1 signaling could therefore down-regulate not only T-cell-mediated responses but also innate responses, and this mechanism may be particularly prominent in HIV-1 infection (98).

Conversely, a link between a mature NK-cell compartment (CD57+) and decreased levels of viral load and immune activation at the time of the primary HIV-1 infection has been reported. Those patients with a mature NK profile at inclusion showed a better early response to ART in comparison to patients with an immature NK profile (99). However, the HCMV serostatus of these individuals is not recorded and the status of NK cells at the point of infection is not known. Whether mature CD57+ or NKG2C+CD57+ NK cells represent adaptive NK cells that contribute directly to better virus control during acute HIV-1 infection and how their role evolves during chronic infection remain unclear.

In agreement with the findings in HCMV seropositive individuals, an NK-cell population that lacks FcεRI-γ expression and has superior ADCC activity has been identified in HIV-1 viremic individuals and shown to persist following virological suppression with ART (100, 101). This subset shares some phenotypic characteristics with adaptive NK cells induced by HCMV (100). Although this subset is associated with HCMV antibody levels in the general population, in HIV-1-infected individuals correlates with inflammatory markers (100). The long-term effects of expansion of FcεRI-γ-deficient NK cells in HIV-1 infection needs to be further elucidated given a possible role in tumor surveillance. Nonetheless, the identification of a subset with enhanced ADCC activity in HIV-1 infection has potentially important implications for the design of vaccine strategies aimed at generating ADCCpromoting antibody responses.

These collective data demonstrate that a number of the phenotypic NK-cell features described in HIV-1 bear the trademarks of HCMV infection (**Figure 2**). With increased definition of the assortment of NK-cell subsets with adaptive features driven by HCMV infection and the increased appreciation of HCMV in driving ongoing immune activation even during effective ART, it would be important to reassess the NK-cell repertoire composition, their response potential in different phases of infection and stimulus-dependent functional properties. A comprehensive analysis of the transcriptional signatures and epigenetic modifications of NK cells in HIV-1 infection is lacking and worth exploring.

#### CONCLUDING REMARKS AND FUTURE PERSPECTIVES

The potent effector function of NK cells and the rapidity of NK-cell response have identified them as key areas for research. Recent reports about the diversity of NK-cell repertoire and ability to assume adaptive features in response to HCMV infection and even display memory-like responses to cytokines (102) and antigen-specific responses in primates (8) have opened up prospects for the generation of new therapies. HCMV co-infection is highly prevalent in HIV-1 infected cohorts and remains an important cofactor in disease progression even in the era of ART. Both HIV-1 and HCMV as well as immune activation can further shape NK-cell responsiveness and differentiation. It is therefore important to capture the diversity of the NK-cell repertoire and identify potentially novel adaptive signatures of NK-cell subsets with preserved activation pathways. Whereas a number of questions remain regarding the epigenetic diversification,

development, and persistence of NK cells with adaptive properties, elucidating how clonal NK-cell populations can be directed or reshaped will critically inform our ability to harness NK cells toward a therapeutic goal.

## AUTHOR CONTRIBUTIONS

The author confirms being the sole contributor of this work and approved it for publication.

### REFERENCES

## ACKNOWLEDGMENTS

My apologies to colleagues whose work could not be cited due to space limitations.

## FUNDING

This work was supported by an MRC grant MR/M008614/1 awarded to DP.

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**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer AH and handling editor declared their shared affiliation.

*Copyright © 2017 Peppa. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# The Dynamic Interplay between HIV-1, SAMHD1, and the Innate Antiviral Response

*Jenna M. Antonucci, Corine St. Gelais and Li Wu\**

*Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, United States*

The innate immune response constitutes the first cellular line of defense against initial HIV-1 infection. Immune cells sense invading virus and trigger signaling cascades that induce antiviral defenses to control or eliminate infection. Professional antigenpresenting cells located in mucosal tissues, including dendritic cells and macrophages, are critical for recognizing HIV-1 at the site of initial exposure. These cells are less permissive to HIV-1 infection compared to activated CD4+ T-cells, which is mainly due to host restriction factors that serve an immediate role in controlling the establishment or spread of viral infection. However, HIV-1 can exploit innate immune cells and their cellular factors to avoid detection and clearance by the host immune system. Sterile alpha motif and HD-domain containing protein 1 (SAMHD1) is the mammalian deoxynucleoside triphosphate triphosphohydrolase responsible for regulating intracellular dNTP pools and restricting the replication of HIV-1 in non-dividing myeloid cells and quiescent CD4+ T-cells. Here, we review and analyze the latest literature on the antiviral function of SAMHD1, including the mechanism of HIV-1 restriction and the ability of SAMHD1 to regulate the innate immune response to viral infection. We also provide an overview of the dynamic interplay between HIV-1, SAMHD1, and the cell-intrinsic antiviral response to elucidate how SAMHD1 modulates HIV-1 infection in non-dividing immune cells. A more complete understanding of SAMHD1's role in the innate immune response to HIV-1 infection may help develop stratagems to enhance its antiviral effects and to more efficiently block HIV-1 replication and avoid the pathogenic result of viral infection.

Keywords: HIV-1, sterile alpha motif and HD-domain containing protein 1, infection, innate immunity, myeloid cells

#### INTRODUCTION

Innate immunity is the cell-intrinsic defense mechanism that senses incoming pathogens and is characterized by type-I interferon (IFN-I) induction and the release of inflammatory cytokines that upregulate antiviral IFN-stimulated genes (ISGs) (1, 2). The activation of the innate response to pathogens is dependent on cellular pattern recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs), including viral structures or nucleic acids. Interferon-inducible protein IFI16 and cyclic GMP-AMP synthase (cGAS) are cytosolic sensors of HIV-1 that detect viral DNA (3, 4). Recognition of PAMPs results in induction of IFN-I and ISGs to control initial infection and spread, while the concomitant induction of the inflammatory response and cytokines can initiate

#### *Edited by:*

*Persephone Borrow, University of Oxford, United Kingdom*

#### *Reviewed by:*

*Jonathan Paul Stoye, Francis Crick Institute, United Kingdom Stephen Kent, University of Melbourne, Australia*

> *\*Correspondence: Li Wu<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to HIV and AIDS, a section of the journal Frontiers in Immunology*

*Received: 30 September 2017 Accepted: 30 October 2017 Published: 10 November 2017*

#### *Citation:*

*Antonucci JM, St. Gelais C and Wu L (2017) The Dynamic Interplay between HIV-1, SAMHD1, and the Innate Antiviral Response. Front. Immunol. 8:1541. doi: 10.3389/fimmu.2017.01541*

**78**

adaptive immune responses (5, 6). Modulation of IFN-I activation is essential for viral clearance. However, overstimulation of IFN pathways can lead to inflammatory autoimmune disease (7).

HIV-1 is sensitive to ISGs and the IFN-induced antiviral response; so it is not surprising that HIV-1 is a poor inducer of IFN (8). HIV-1 benefits from evading innate immune activation and utilizes a variety of tactics to escape detection (9, 10). Professional antigen-presenting cells located in mucosal tissues, including dendritic cells (DCs) and macrophages, are critical for recognizing HIV-1 at the site of initial exposure. However, these cells are less permissive to HIV-1 infection compared to activated CD4+ T-cells, mainly due to host restriction factors that control the establishment or spread of viral infection. Several host proteins can restrict HIV-1 at various points in the viral lifecycle, including APOBEC proteins, TRIM5α, and tetherin (11–13). However, HIV-1 can exploit innate immune cells and their cellular factors to avoid detection and clearance by the host immune system (13).

SAMHD1 is host protein capable of blocking replication of retroviruses and several DNA viruses in cells (14–18). SAMHD1 is constitutively expressed at various levels in all cell types and highly expressed in myeloid lineage and resting CD4+ T-cells (14, 15, 19). IFN-I treatment increases SAMHD1 expression in certain cell types with low endogenous SAMHD1 levels (20, 21). SAMHD1 has been implicated as a negative regulator of the IFN-I inflammatory response (22–24), however, the underlying mechanism is not fully understood. While HIV-2 encodes the SAMHD1 antagonist Vpx, the more pathogenic HIV-1 does not. It was hypothesized that HIV-1 lacks a countermeasure against SAMHD1 because it is beneficial for infection. In this review, we will discuss the contributions of SAMHD1 to both the direct restriction of HIV-1 and to the modulation of the antiviral innate response and to analyze the hypothesis that HIV-1 restriction by SAMHD1 leads to a diminished induction of innate immunity.

#### INNATE IMMUNE SENSING OF HIV-1

Although HIV-1 can be sensed by the innate immune system, the prevailing theory is that HIV-1 avoids immune surveillance through poor replication in immune cells causing ineffective triggering of innate cytosolic sensors (25). Several studies have identified the molecular basis of cytosolic sensors important for targeting viral pathogens. Here, we focus on HIV-1 DNA as a trigger of the innate antiviral response. After sensing viral DNA, cGAS generates the second messenger, cyclic guanosine monophosphate-adenosine monophosphate (26–28), that activates the stimulator of IFN genes (STING) (6). STING activation leads to phosphorylation of TANK-binding kinase 1 (TBK1) and the subsequent phosphorylation and dimerization of IFNregulatory transcription factors IRF3 and IRF7. Nuclear translocation of the IRF3/IRF7 homo-or-hetero dimers will activate IFN-I gene expression (**Figure 1**). This signaling cascade results in an upregulation of IFN-I and ISGs as a defense against viral infection (29, 30). Reverse transcribed HIV-1 DNA was identified as the trigger to the cGAS-STING pathway (3). Although cGAS is the primary sensor of cytosolic viral DNA, IFI16 can also act as a sensor of HIV-1 single-stranded DNA that induces an IFN-β response in macrophages by a cGAS-STING-dependent pathway (4).

### INTRODUCTION TO SAMHD1

Human SAMHD1 is a 626-amino acid protein containing an N-terminal nuclear localization signal followed by a sterile-alpha motif and histidine/aspartic acid (HD) domain. SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) (33, 34) that converts dNTPs into the constituent deoxynucleoside and inorganic triphosphate upon stimulation by dGTP or GTP (33–35). SAMHD1 and ribonuclease reductase, the enzyme responsible for *de novo* dNTP synthesis through the conversion of ribonucleotide diphosphates to deoxyribonucleotides (36), are allosterically regulated to achieve balanced intracellular dNTP levels in a cell-cycle-dependent manner (37). During G1 to S-phase transition in actively proliferating cells, ribonuclease reductase expression increases, leading to expansion of the dNTP pool to facilitate DNA synthesis (38, 39). The activity of SAMHD1 is activated by high dNTP levels, and degradation of nucleic acids in the absence of DNA replication protects the cell from innate immune activation and cancer development (40, 41). Mutations in SAMHD1 that affect its enzyme activity are associated with Aicardi-Goutières syndrome (AGS), an encephalopathic autoimmune disease characterized by symptoms mimicking chronic viral infection (22). The accumulation of intracellular dNTPs caused by mutations in the genes encoding proteins involved in nucleic acid metabolism, including SAMHD1 and TREX1 (42), are sensed by PRRs, resulting in aberrant production of IFN-I (43). AGS patients present with increased production of IFN-α, the chemokine most characteristic of congenital virus infection. AGS patients with SAMHD1 mutations can present with signs of lupus erythematosus, with many symptoms mimicking those of HIV-1 infection (22, 44). Furthermore, cells isolated from AGS patients with homozygous *SAMHD1* mutation revealed that SAMHD1-deficient monocytes supported productive infection by HIV-1 (20), suggesting a link between SAMHD1 function in both autoimmunity and HIV-1 restriction.

Long interspersed element 1 (LINE-1) is the only autonomous and active human retroelement capable of producing new genomic insertions through its endogenous endonuclease and reverse transcriptase activities (45, 46). A study on AGS-related SAMHD1 mutations indicate that all disease-related mutations reduced LINE-1 inhibition in dividing cells (47). Recent work suggests that SAMHD1 potently blocks LINE-1 transposition in cycling cells by triggering the sequestration of LINE-1 ORF1p into stress granules (48). Impaired inhibition of LINE-1 retrotransposition may lead to triggering of the autoimmune response by stimulating toll-like receptors (TLRs) (49), although this has not been confirmed. Impaired dNTPase activity and LINE-1 suppression by mutant SAMHD1 could explain the chronic inflammatory response characteristics of AGS disease. These studies outlining the pathogenic effect of SAMHD1 deficiency on autoimmune disease implicate SAMHD1 as a negative regulator of the innate immune system.

response. Expression of ISGs allows for immune activation and the induction of an antiviral state of the cell. gRNA, HIV-1 genomic RNA; cDNA, complementary

DNA; vRNA, viral RNA; dsDNA, double-stranded DNA; STING, stimulator of IFN genes; the letter P indicates phosphorylation.

### SAMHD1-MEDIATED HIV-1 RESTRICTION

HIV-1 replicates inefficiently in non-diving cells, such as quiescent CD4+ T-cells, DCs, and monocytes. HIV-1 infection can be enhanced in these cells by Vpx, an accessory protein encoded by HIV-2 and certain lineages of simian immunodeficiency viruses (SIVs) (50, 51). This hinted at the existence of a cellular restriction factor counteracted by Vpx (50). SAMHD1 was identified as the mystery HIV-1 restriction factor by a mass spectrometry analysis of cellular proteins immunoprecipitated from cells expressing Vpx (14, 15). Vpx interacts with the C-terminal domain of SAMHD1, thereby initiating proteasomal degradation by an E3 ubiquitin ligase complex, and relieving SAMHD1-mediated lentiviral restriction (14, 15, 52, 53).

The mechanism and modulation of SAMHD1-mediated HIV-1 restriction is an area of intense scrutiny (**Figure 2**). Overexpression of SAMHD1 in PMA-treated monocytic U937 cells results in a depletion of dNTP levels (54). It was later confirmed that SAMHD1 restricts the replication of retroviruses and several DNA viruses by depleting the concentration of intracellular dNTPs to levels insufficient to support viral DNA synthesis (14–18, 54, 55). Structural studies strengthened a model of nucleotide-dependent tetramer assembly of SAMHD1 (56–58), where GTP binds to guanine-specific allosteric sites and dNTP binds to non-specific activator sites, initiating the formation of enzymatically active tetramers with the catalytic core of the HD domain (33, 34, 37, 59). Moreover, binding of single-stranded nucleic acids (ssNAs) to the dimer–dimer interface of SAMHD1 inhibits the formation of the catalytically active tetramer (60).

As SAMHD1 is also highly expressed in activated CD4+ T-cells that support productive infection, several studies demonstrated posttranslational modification as a means of mechanistic regulation of SAMHD1 function in restricting HIV-1. SAMHD1 is phosphorylated at several residues; however, phosphorylation of threonine 592 was identified as essential for the negative

modulation of its HIV-1 restriction activity (61–65) and tetramer formation (66, 67). SAMHD1 is phosphorylated by cyclindependent kinase 1 (CDK1) and CDK2 in complex with cell cycle regulatory protein cyclin A. This regulation of SAMHD1 function is associated with the cell cycle, as CDK1 and cyclin A are highly expressed in dividing cells. Furthermore, S-phase requires elevated dNTP levels, indicating modulation of the dNT-Pase activity of SAMHD1 during the cell cycle (64). SAMHD1 protein levels may be altered during various stages of the cell cycle depending on different cell types (68, 69). Interestingly, proliferation-induced oxidation of SAMHD1 by hydrogen peroxide reversibly inhibits its dNTPase activity through the formation of tetramer-inhibiting disulfide bonds (70), suggesting a dynamic structure-based regulatory mechanism of SAMHD1's dNTPase activity that is influenced by the cell cycle (**Figure 2**).

deoxynucleosides; PPPs, triphosphate; two linked letters S indicate the disulfide bond.

Although the accepted consensus is that SAMHD1 restricts HIV-1 infection through the depletion of intracellular dNTPs, several studies suggested the existence of an additional yet-undiscovered mechanism of SAMHD1-mediated retroviral restriction. This undefined antiviral activity appears to be dependent on phosphorylation (61, 63, 65) and is not fully dependent on low dNTP levels (71). SAMHD1 acts as a ssNA binding protein that degrades single-stranded DNA and RNA *via* a metal-dependent 3′–5′ exonuclease activity *in vitro* (72–74). It has been suggested that SAMHD1 utilizes its nucleic acid binding potential to exert a ribonuclease activity against incoming HIV-1 genomic RNA in a phosphorylation-dependent manner (75). SAMHD1 was shown to restrict retroviruses though degradation of HIV-1 RNA in human monocyte-derived macrophages (MDMs), monocytes, and CD4+ T-cells (75, 76). It was proposed that SAMHD1 degrades incoming HIV-1 gRNA, thereby restricting infection and preventing innate immune sensing of viral nucleic acids. However, recent studies have been unable to confirm the controversial findings (55, 77–79). As a nuclear-localized protein (80), incoming viral genomic RNA would be inaccessible by SAMHD1 for hydrolysis. Additional studies showed that dNTPase inactive SAMHD1 mutant retained exonuclease activities *in vitro*, indicating the exonuclease activity could not be attributed to the known dNTP-binding active site (77). Seamon et al. (77) suggested that the nuclease activity attributed to SAMHD1 was due to contamination during purification. Cell-based assays also failed to recapitulate the findings, thereby confirming the lack of SAMHD1 RNase activity to restrict HIV-1 in infected cells (55, 78). Ryoo et al. suggested that the differences in experimental conditions are responsible for the conflicting results, including a shorter infection time and the use of RNaseH-defective reverse transcriptase (81). They further identified SAMHD1 as a phosphorolytic not hydrolytic ribonuclease (82).

#### THE INTERSECTION OF HIV-1, SAMHD1, AND THE INNATE ANTIVIRAL RESPONSE

SAMHD1 cDNA was originally identified as a ortholog of the mouse IFN-γ-induced gene *Mg11* in human DCs (83). A link to the innate immune response was strengthened by the discovery that cytokines, including toll-like agonists and IFNs, can induce SAMHD1 expression (84, 85). Cell lines treated with IFN-I (21, 86) and human primary monocytes treated with IFN-α and IFN-γ (20, 84, 87) show enhanced expression of SAMHD1. While SAMHD1 is highly expressed in MDMs, monocytederived dendritic cells (MDDCs), and primary CD4+ T-cells, IFN treatment does not increase SAMHD1 protein levels further (21, 88–90). However, treatment of MDMs and MDDCs with IFN-I results in reduced phosphorylation of SAMHD1 at residue T592 (61), indicating a shift from catalytically inactive to active SAMHD1. Interestingly, the *SAMHD1* promoter is a direct target of IRF3. The overexpression and activation of IRF3 enhances SAMHD1 promoter activity in HeLa cells (86).

HIV-1 does not trigger a sterilizing immune response (91) and is a poor activator of inflammatory pathways (8), resulting in an impaired response to HIV-1 and the development of persistent infection. The DC response to HIV-1 infection contributes to this dysfunctional immune response (92). Myeloid cells constantly sample the cellular environment to identify pathogens and send out danger signals in the form of IFN-I. DCs are essential for activating the adaptive immune response to infection, as maturation leads to T-cell responses through antigen priming (91, 93). Interestingly, HIV-1 infects DCs without activating an effective antiviral response. As SAMHD1 limits HIV-1 cDNA synthesis in myeloid cells (14, 54), it was hypothesized that degradation of SAMHD1 by Vpx in DCs would result in productive HIV-1 infection and the synthesis of viral proteins that would directly enter antigen presentation, thereby strengthening the T-cell response to infection (94). This could be why the *vpx* gene was lost from the ancestor of HIV-1 during the coevolution of primate SAMHD1 and lentiviruses (95).

Vpx-mediated degradation of SAMHD1 in DCs leads to enhanced HIV-1 infection, and studies in primary MDMs and MDDCs indicate that Vpx-mediated SAMHD1 degradation results in cGAS stimulation and IRF3 activation (3). Early work suggested that enhanced infection by SAMHD1 depletion leads to DC maturation (94). A study utilizing coculture of autologous activated CD4+ T lymphocytes with SAMHD1-deficient MDDCs infected with primary clinical HIV-1 isolates indicated enhancement of both infection and IFN response (96). Interestingly, cocultured primary T-lymphocytes, but not HIV-1, trigger a decrease in SAMHD1 expression in MDDCs independent of dNTP levels (96). This study suggests that crosstalk between lymphocytes and DCs induces downregulation of SAMHD1 expression, a requirement for stimulation of HIV-1 production in DCs, thereby inducing the innate sensing of HIV-1 and DC maturation (96).

Conversely, recent work indicates that DC maturation, measured by CD83 and CD86 expression, does not occur in SAMHD1-deficient cells due to additional manipulation of the innate immune system by HIV-1 (97). HIV-1 suppresses TLRinduced maturation of DCs independent of SAMHD1 expression, although Vpx-mediated depletion of SAMHD1 enhanced the effect of HIV-1 infection on lipopolysaccharide-induced DC maturation (97). Vesicular stomatitis virus G-proteinpseudotyped HIV-1 suppressed maturation similar to strains containing HIV-1 envelope protein, suggesting that viral replication, not envelope-receptor interactions, is required for suppression of maturation (97). Removing the SAMHD1-mediated block of reverse transcription resulted in a stronger suppression of maturation. Although infection and subsequent innate immune sensing in DCs is blocked by SAMHD1, HIV-1 maintains an additional SAMHD1-independent mechanism of suppressing DC maturation through downregulation of TLRs (97).

Two additional models suggest that, in MDDCs, HIV-1 attempts to hide its genomic RNA and newly synthesized cDNA from cytosolic sensors by obstructing the nucleic acids using viral capsid. The models differ with respect to the effect of recruitment of cellular cyclophillins and cleavage and polyadenylationspecific factor 6 (CPSF6) by capsid. One model suggests increased cyclophillin A (CypA) binding to the capsid increases sensitivity to innate sensing (94), while another proposes CypA binding coordinates uncoating, reverse transcription, and nuclear import of the preintegration complex (98), all to minimize the exposure of viral nucleic acids to cytosolic sensors. Future work is needed to clarify the contribution of CypA and SAMHD1 to the negative regulation of the innate immune response in myeloid cells to provide insight into HIV-1 mechanisms of evasion.

Non-cycling CD4+ T-cells and macrophages are less permissive to HIV-1 because of SAMHD1. However, during HIV-1 infection *in vivo,* activated CD4+ T-cells and macrophages are infected due to phosphorylation of SAMHD1. Although cytosolic HIV-1 DNA is abundant in these permissive cells, a cell-autonomous IFN response is not triggered (99). This is due at least in part to host protein TREX1. As a single-stranded DNA exonuclease, TREX-1 digests cytoplasmic DNA from retroviral DNA intermediates, thereby preventing the activation of mislocalized DNA by an innate immune sensor (99). Cytosolic HIV-1 DNA is accumulated in HIV-1 infected TREX1-deficient CD4+ T-cells and macrophages, which leads to inhibition of TBK1-dependent IFN-I response (99). This suggests a competition between two DNA sensors: cGAS leading to antiviral effects, and TREX1 leading to enhanced viral replication (100).

#### REMAINING QUESTIONS

Although it is clear that HIV-1 utilizes a variety of mechanisms to evade myeloid cell activation, controversial questions still exist. Conflicting reports could be due to the use of different cell types, and the differential use of clinical HIV-1 isolates, replicationcompetent lab strains, or pseudotyped virus. It is essential to confirm experimental findings with primary cells that accurately recapitulate *in vivo* mucosal infection sites. Further understanding of the strategies HIV-1 utilizes to evade the innate response will allow for better ideas on how to increase the innate immune response to HIV-1.

The existence of a yet-undiscovered mechanism of HIV-1 restriction that is dependent on phosphorylation cannot be overlooked (63). Pretreatment MDMs with Vpx enhances the rate of HIV-1 cDNA synthesis (101), suggesting that the decrease in reverse transcription kinetics conferred by SAMHD1-mediated modulation of dNTP levels negatively regulates the rate of proviral DNA synthesis in non-dividing cells. When transcription is silenced, integrated proviral DNA can lead to latency (102). Although SAMHD1 is highly expressed in cells purported to harbor latent provirus (19, 103) and the HIV-1 proviral promoter is activated by transcription factors (104), the effect of SAMHD1 expression on latency development or reversal has not been explored. It is possible that SAMHD1 utilizes its nucleic acid binding ability to restrict HIV-1 infection postintegration, although a recent study confirmed SAMHD1 exerts no effect on HIV-1 Gag synthesis, viral particle release, and virus infectivity in 293T cells transfected with a proviral DNA construct (55). SAMHD1 may exert a direct effect on proviral DNA through binding, as purified recombinant SAMHD1 was shown to bind *in vitro* transcribed fragments of *gag* and *tat* cDNA (72), or indirect effects may occur due to SAMHD1 modulation of inflammatory pathways. It is plausible that suppression of latency reactivation by SAMHD1 would further prevent activation of the innate antiviral response. Although viral nucleic acids can be sensed by IFI16 or cGAS in the absence of SAMHD1 (24, 105), whether other pro-inflammatory pathways are affected by SAMHD1 expression remains unknown.

Discovering the mechanisms used by HIV-1 to avoid innate immune sensors is critical for the design of new therapies to eradicate HIV-1 infection. Therapeutic strategies aiming to inhibit host factors that promote HIV-1 replication and to stimulate the immune response could diminish viral infection and transmission. Current work aims to determine whether a role exists for drugs targeting SAMHD1. Expression of SAMHD1 can increase the susceptibility of HIV-1 to nucleoside reverse transcriptase inhibitors by reducing the levels of competitive dNTPs (106–109), suggesting modulation of SAMHD1 function may be a means to enhance drug effectiveness. Conversely, as SAMHD1 expression enables immune evasion by HIV-1 (13), it is tempting to hypothesize that SAMHD1 could be used as a drug target to

#### REFERENCES

enhance the innate immune response to viral infection. However, research is just beginning to uncover mechanisms to modify the dNTPase activity of SAMHD1 (110, 111). Importantly, as an ISG and a negative regulator of the innate immune system, SAMHD1 may be involved in an unknown negative feedback loop aimed at modulating the complex and delicate system of inflammatory pathways.

The effect of SAMHD1 on IFN-I induction during viral infection should be further studied *in vivo*. Although initial robust IFN-I responses can lead to an upregulation of antiviral genes and a block in infection, chronic immune hyperactivity could lead to desensitization of IFN-I and an eventual suppression of antiviral gene expression. This phenotype was observed when Sandler et al. manipulated the IFN-α2a response to SIV infection in rhesus macaques (112). The dismantling of the antiviral state after long-term IFN-α2a treatment led to an increase in SIV reservoir size and an accelerated CD4+ T-cell loss (112). Studies are necessary to determine whether stimulation of the IFN-I response through inhibition of SAMHD1 function leads to chronic inflammation and progression to AIDS *in vivo*.

### CONCLUSION

The identification of SAMHD1 as a regulator of the innate immune response to viral infection has led to the development of an exciting field of research. The structural and functional studies of SAMHD1 connect the physiology of HIV-1 infection to the innate antiviral response and the dynamic regulatory mechanisms in cells. Further work will aid in the development of stratagems to enhance the antiviral effects of the intrinsic immune system.

### AUTHOR CONTRIBUTIONS

JA wrote the manuscript with input and edits from CG and LW.

#### ACKNOWLEDGMENTS

We thank the Wu lab members for valuable discussions.

### FUNDING

This work was supported by a grant (AI104483) from the National Institutes of Health (NIH) to LW. LW is also supported in part by NIH grants (AI120209 and GM128212). JA was supported by C. Glenn Barber funds from the College of Veterinary Medicine at The Ohio State University.

catalysis and restriction. *PLoS Pathog* (2015) 11:e1005194. doi:10.1371/ journal.ppat.1005194

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Antonucci, St. Gelais and Wu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Natural Killer Cell Interactions with Classical and Non-Classical Human Leukocyte Antigen Class I in HIV-1 Infection

*Angelique Hölzemer1,2\*, Wilfredo F. Garcia-Beltran3 and Marcus Altfeld2,4*

*1 First Department of Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 2German Center for Infection Research (DZIF), Partner site Hamburg-Lübeck-Borstel-Riems, Hamburg, Germany, 3Harvard Medical School, Boston, MA, United States, 4 Institute for Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany*

#### *Edited by:*

*Persephone Borrow, University of Oxford, United Kingdom*

#### *Reviewed by:*

*Amir Horowitz, Icahn School of Medicine at Mount Sinai, United States Simon Kollnberger, Cardiff University, United Kingdom*

*\*Correspondence:*

*Angelique Hölzemer<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to HIV and AIDS, a section of the journal Frontiers in Immunology*

*Received: 05 July 2017 Accepted: 24 October 2017 Published: 14 November 2017*

#### *Citation:*

*Hölzemer A, Garcia-Beltran WF and Altfeld M (2017) Natural Killer Cell Interactions with Classical and Non-Classical Human Leukocyte Antigen Class I in HIV-1 Infection. Front. Immunol. 8:1496. doi: 10.3389/fimmu.2017.01496*

Natural killer (NK) cells are effector lymphocytes of the innate immune system that are able to mount a multifaceted antiviral response within hours following infection. This is achieved through an array of cell surface receptors surveilling host cells for alterations in human leukocyte antigen class I (HLA-I) expression and other ligands as signs of viral infection, malignant transformation, and cellular stress. This interaction between HLA-I ligands and NK-cell receptor is not only important for recognition of diseased cells but also mediates tuning of NK-cell-effector functions. HIV-1 alters the expression of HLA-I ligands on infected cells, rendering them susceptible to NK cell-mediated killing. However, over the past years, various HIV-1 evasion strategies have been discovered to target NK-cell-receptor ligands and allow the virus to escape from NK cell-mediated immunity. While studies have been mainly focusing on the role of polymorphic HLA-A, -B, and -C molecules, less is known about how HIV-1 affects the more conserved, non-classical HLA-I molecules HLA-E, -G, and -F. In this review, we will focus on the recent progress in understanding the role of non-classical HLA-I ligands in NK cell-mediated recognition of HIV-1-infected cells.

Keywords: HIV-1, innate immunity, natural killer cells, killer cell immunoglobulin-like receptor, human leukocyte antigen class I, human leukocyte antigen-F, human leukocyte antigen-E

## INTRODUCTION

Untreated HIV-1 infection will lead to progressive, severe, and mostly fatal immune deficiency in the vast majority of individuals. Protective HIV-1 immunity is observed in a small subset of subjects whose immune system can naturally control HIV-1 infection and who are termed "elite controllers." Despite intense research in this area over the past decades, the correlates leading to protective immunity are still insufficiently understood. Host genetics alone can only explain approximately 20% of the variable outcomes between individuals observed in the natural course of infection (1). Nonetheless, a consistently documented key genetic determinant of HIV-1 control is the presence of particular human leukocyte antigen (HLA) class I alleles. This strong association between classical HLA-I alleles and HIV-1 disease outcome has been identified in genome-wide association studies (1, 2) as well as in large cohorts studying the immunogenetics of HIV-1 disease (3, 4). The protective effects of certain HLA-I alleles have mostly been attributed to enhanced CD8+ T lymphocyte-mediated immunity (5–7). HLA-I presentation of HIV-1 epitopes derived from conserved sequences of HIV-1 to CD8+ T cells can pressure the virus to select for mutations in these epitopes, but viral escape can be associated with costs in viral fitness (8). Indeed, early CD8+ T-lymphocyte responses contribute to the initial drop in HIV-1 peak viremia and with this, first HIV-1 escape mutations arise (9). Other protective factors in HIV-1 infection include enhanced proliferation potential of T lymphocytes (10, 11), polyfunctional immune responses (12, 13), variations in host restriction factors (14), and variants in HIV-1 coreceptors, in particular, of CCR5 (15, 16).

Over the past years, the role of antiviral innate immune responses mediated by natural killer (NK) cells in HIV-1 infection has been increasingly appreciated (17, 18). *In vitro,* NK cells can inhibit HIV-1 replication in autologous CD4+ T cells as effectively as CD8+ T cells (19). Additionally, the strong protective effect of host HLA-I alleles on disease progression has been linked to receptor families recognizing HLA-I. These include killer-cell immunoglobulin-like receptors (KIRs), predominantly expressed on NK cells (20), and leukocyte immunoglobulin-like receptors (LILRs), expressed on professional antigen presenting cells such as dendritic cells (DCs), monocytes, macrophages, and B cells, but also on T cells and NK cells (21). Indeed, accumulating data from population studies have identified certain *KIR*, *LILR*, and *HLA-I* allele combinations associated with slower HIV-1 disease progression (22–24), which has helped decipher a further piece of complex host genetics in HIV-1 disease variability.

Natural killer cells comprise 5–15% of the circulating lymphocytes (25) and their role in controlling viral infections has been long established (26). Two major subsets exist: CD56bright CD16dim/neg and CD56dimCD16pos NK cells (25). These differ in their expression of key NK-cell receptors, response to soluble factors and cellular targets, capacity for cytotoxicity, and production of immunomodulatory cytokines (27). NK cells are a crucial first line of defense that detect infected cells before antigen sensitization has occurred (28, 29), and therefore, they precede adaptive immunity in the early phases of HIV-1 infection. Indeed, there is evidence that the early events following infection prior to the development of a specific immune response can determine the viral set point and influence the clinical course of infection (30). In acute HIV-1 infection, a rapid expansion occurs in predominantly cytotoxic CD56dim NK cells, prior to CD8+ T cell expansion (31). On the other hand, in chronic HIV-1 infection, a redistribution of NK cells toward less functional subsets can be observed (32–35) and the presence of persistent viremia appears to deteriorate NK-cell function (19, 34, 36). Overall, the full extent of receptor-ligand interactions between NK cells and HIV-1–infected target cells in HIV-1 infection leading to either NK-cell expansion/killing or exhaustion is highly complex and not yet fully understood.

Natural killer cells, as members of the innate immune system, express a plethora of germline-encoded receptors, and their effector function is determined by integration of inhibitory and activating NK-cell receptor signaling, whereby inhibitory signals tend to be dominant (27). Major NK-cell receptor families are (i) natural cytotoxicity receptors (i.e., NKp46, NKp44, and NKp30), which deliver mainly activating signals, (ii) the KIR family, encompassing inhibitory and activating members and monitoring HLA-I, (iii) the C-type lectins with activating natural killer group 2D (NKG2D) and the heterodimers NKG2A-CD94 and NKG2C-CD94, and (iv) the FcγRIIIa receptor (CD16), which can bind to the Fc-region of IgG antibodies. Critical activating signals can also be delivered by other coreceptors including 2B4, DNAM-1, or CD2 (37, 38). Differential expression of activating and inhibitory receptors allows for a certain degree of specificity and shaping of NK-cell function in response to different stimuli. Ultimately, the stochastic expression of receptors on each NK cell leads to substantial NK-cell diversity and determines the differential response to target cells (39, 40).

HIV-1–infected cells can become vulnerable to NK cellmediated killing by upregulation of stress signals recognized by activating NK-cell receptors and/or by downregulation of inhibitory NK-cell-receptor ligands. Of note, signaling *via* the FcγRIIIa receptor (CD16), which mediates antibody-dependent cellular cytotoxicity (ADCC), is sufficient to induce NK-cell activation on its own (37). However, the strength of CD16-mediated activation is dependent on tuning of NK-cell responsiveness through inhibitory interactions of KIR or NKG2A with HLA class I (41, 42). Stress ligands upregulated on HIV-1–infected cells are the major histocompatibility complex (MHC) class-I-chain-related proteins (MIC-) A and -B, the UL16-binding proteins (ULBPs) 1–3, which are the ligands for the activating NKG2D receptors (43, 44), and a yet unknown ligand for NKp44 (45, 46). In turn, HIV-1 encodes for multiple accessory proteins with pleiotropic functions to overcome host restriction factors and host immune responses (47–49). The upregulation of stress ligands such as ULBPs and MIC-A/B is counteracted *via* HIV-1 Nef (50) and the ligands for coactivating receptors such as NTB-A and DNAM-1 are downregulated *via* HIV-1 Vpu and partially Nef (51–53). The impact of HIV-1 Nef and Vpu on HLA class I expression will be discussed later. In this review, we will focus on the recent progress in understanding the interplay of HLA-I with HLA-I binding NK-cell receptors, and how this interaction either limits HIV-1 replication or is exploited by the virus to enhance pathogenesis.

### KIR–HLA Interactions in HIV-1 Disease Progression and Acquisition

Classical and non-classical HLA-I genes (also known as HLA-Ia and HLA-Ib, respectively) are located within the MHC region p21.3 on chromosome 6, the most polymorphic region of the human genome. An extensive amount of allelic variation occurs within the region encoding for classical HLA-I genes (54). In contrast, non-classical HLA-I alleles display varying degrees of oligomorphism. To date, the classical *HLA-A*, *HLA-B*, and *HLA-C* loci comprise >10,000 alleles encoding for 8,662 distinct proteins, whereas the non-classical *HLA-E*, *HLA-F*, and *HLA-G* loci combined encode for 101 alleles and only 30 proteins (The Immuno Polymorphism Database, as of July 2017) (55, 56).

Classical HLA-I is ubiquitously expressed on nucleated cells. Given that the primary function of HLA-I is to present peptides derived from degradation of intracellular proteins (57), it is not surprising that variations mainly occur in regions surrounding the peptide-binding groove (58) so as to maximize diversity of peptides presented across different gene and allele products. Under pathologic conditions such as malignant transformation or infection with intracellular pathogens, HLA-I presents antigenic peptides and thereby can elicit an immune response *via* HLA-I restricted cytotoxic CD8+-T cells. Historically, it was thought that NK cells only respond to changes in surface levels of classical HLA class I [to missing-self (59)], but there is increasing evidence that KIR can bind differentially depending on the HLAclass I presented peptide (60–65).

In 2007, the first genome-wide association studies reported three protective single-nucleotide polymorphisms (SNPs) in HIV-1 disease (2). The presence of these SNPs was associated with lower viral set point in chronically HIV-1–infected subjects and together explained almost 15% of interindividual disease variability. Strikingly, all three SNPs were located in the MHC region of chromosome 6, emphasizing the crucial role of HLA class I in HIV-1 infection. The first SNP is in high linkage disequilibrium with HLA-B\*57, a second SNP was located 35bp upstream of the HLA-C locus, and results in higher HLA-C expression levels. The last SNP was linked to an RNA polymerase subunit, ZNRD1 and affected the time to AIDS progression. Subsequent genome-wide association studies confirmed the first two SNPs and identified six additional SNPs associated with HIV-1 disease control in two different ethnic cohorts. Again, all SNPs were concentrated around the HLA-I region (1). Accordingly, the strongest HLA class I protective effects so far are reported for HLA-B\*57 (66, 67) and HLA-B\*27 (4, 68); two HLA class I alleles carrying the serologically defined Bw4 motif (determined by the amino acids 77–83). There is a strong association of HLA-Bw4 homozygosity with the ability to suppress viral replication of HIV-1 and with delayed time to AIDS progression (69).

The genes encoding for KIRs are located within the leukocyte receptor cluster on Chromosome 19q13.4, which additionally encodes Ig-like transcripts (ILTs) [also termed leukocyte Ig-like receptors (LIRs)], and leukocyte-associated inhibitory receptors (70). The KIR locus exhibits substantial polymorphism, in its degree only second to the MHC region in the human genome (71). KIRs can be subdivided into two different classes: KIRs with two extracellular Ig-like domains (KIR2Ds) and those with three domains (KIR3Ds). These Ig-like domains are classified as D0, D1, or D2. Type 1 KIR2Ds contain a D1 domain distal to a D2 domain, type 2 KIR2Ds (KIR2DL4 and KIR2DL5) have a D0–D2 domain organization, and KIR3Ds have all three domains as D0–D1–D2. In general, KIR2Ds bind to HLA-C and KIR3D bind to HLA-A and B-ligands (72, 73). Regarding signaling capacity, a long cytoplasmic tail (KIR-L) renders the KIR inhibitory as it contains immune tyrosine inhibitory motifs (ITIMs), whereas a short cytoplasmic tail (KIR-S) associates to adaptor molecules such as DAP12 and delivers activating signals (74). An exception to this is KIR2DL4, which holds an ITIM in its long cytoplasmic tail, but also associates with activating adaptor elements (73). KIRs are a major receptor family on NK cells, but are also expressed on CD4+ and CD8+ T cells (both αβ and γδ T cells) (75–80). Of note, expression of inhibitory KIR on T cells is increased following chronic immune activation, as was observed in the case of CMV reactivation in a posttransplantation setting (81, 82). Increased KIR expression on bulk CD8+ T cells in HIV-1 infection has been reported, but barely detectable KIR expression was described, when investigating HIV-specific CD8+ T cells (83, 84). Overall, little is known about a modulation of KIR-expression on T cells with or without CMV reactivation in HIV-1–infected subjects.

#### KIR3DS1/KIR3DL1 and HLA-Bw4I80

The first study associating KIRs to HIV-1 control came from the laboratory of Mary Carrington in 2002. This study showed that possessing *KIR3DS1* and an *HLA-B* allele with a Bw4 motif and an isoleucine at position 80 (HLA-Bw4I80) was associated with slower progression to AIDS, when compared to patients having only one or none of these alleles (22). A follow-up analysis by the same group reported a protective effect of combined KIR3DS1 and HLA-Bw4I80 against development of certain opportunistic infections in HIV-1–infected patients, also after controlling for presence of protective (e.g., *HLA-B\*57* and *HLA-B\*27*) and deleterious (*HLA-B\*35*) alleles (85). The *KIR3DS1/KIR3DL1* locus is unique in that it encodes functionally divergent alleles (86). The inhibitory KIR3DL1 binds to HLA-I allotypes that possess a Bw4 motif (HLA-Bw4, which can derive from *HLA-A* or *HLA-B* alleles). Polymorphisms in position 80 of these HLA-Bw4 molecules have been shown to modulate the strength of binding to KIR3DL1 (87, 88). In addition, the interaction of KIR3DL1 with HLA-Bw4 is sensitive to the sequence of the HLA-Bw4–presented peptide (61). Contrary to KIR3DL1, a ligand for its activating counterpart, KIR3DS1, remained initially unknown.

In a cohort of recently infected individuals, Barbour et al. did not detect a synergistic protective effect of *KIR3DS1* and HLA-Bw4I80 assessing viral load and CD4+ T cell loss. Nonetheless, encoding for at least one *KIR3DS1* allele was associated with higher CD4+ T cell counts and encoding for HLA-Bw4I80 alleles correlated with lower viral load, suggesting a protective, but independent effect of *KIR3DS1* and HLA-Bw4I80 (89). A further epidemiologic study reported that HIV-1 viral load at set point correlated positively with the number of *KIR3DS1* gene copies in the presence of HLA-B Bw4I80 ligands. Higher copy numbers of the *KIR3DL1* gene also correlated with lower viral set point in the presence of HLA-Bw4I80 and at least one copy of *KIR3DS1* (90). In addition, a study by Jiang et al. (91) in a Chinese cohort showed that *KIR3DS1/KIR3DL1* heterozygotes were enriched in HLA-Bw4I80–bearing long-term non-progressors with higher CD4+ T cell counts and decreased viral loads as compared to KIR3DL1 homozygotes or individuals without HLA-Bw4I80 (91).

As KIRs are predominantly expressed on NK cells, Martin et al.'s first report associating a KIR to an outcome in HIV-1 infection (22) triggered multiple studies on NK-cell functionality attempting to elucidate the underlying protective mechanism of *KIR3DS1* in combination with HLA-Bw4I80 in HIV-1 disease. In line with the epidemiological data, functional studies reported that NK cells derived from donors possessing *KIR3DS1* combined with HLA-Bw4I80 inhibited viral replication in infected autologous CD4+ T cells more potently than NK cells from donors having either or neither allele. Sorted KIR3DS1+ NK cells degranulated significantly more in response to HIV-1–infected HLA-Bw4–expressing CD4+ T cells compared to infected HLA-Bw6+ CD4+ T cells (92). A second study showed that NK cells from individuals encoding for *KIR3DS1* displayed enhanced cytotoxic function compared to NK cells from individuals without *KIR3DS1*, but this was independent of the presence of HLA-Bw4I80 (93). Also, in acutely HIV-1–infected subjects a preferential expansion of KIR3DS1+ NK cells—and to a lesser extent KIR3DL1+ NK cells—was observed, which persisted only in subjects bearing HLA-Bw4I80 (94). Morvan et al. reported an expansion of KIR3DS1+ NK cells in response to various non-specific stimuli, but KIR3DS1+ NK-cell function was not influenced by the presence of HLA-Bw4 in this setting. Nonetheless, the frequency of KIR3DS1+ NK cells and KIR3DS1 expression levels on NK cells were higher in healthy subjects with HLA-Bw4I80 than in those without HLA-Bw4I80 (95). Furthermore, HIV-1 viral inhibition assays demonstrated that in individuals encoding HLA-Bw4, having one copy of *KIR3DS1* and one or more copies of *KIR3DL1* resulted in increased antiviral capacity of bulk NK cells compared to individuals containing either *KIR3DS1* or *KIR3DL1* alone, which displayed the lowest amounts of viral inhibition (90). No differences were seen in HLA-Bw6 homozygous donors, whose NK cells had poor antiviral capacity. Having increasing copy numbers of *KIR3DL1* was correlated with elevated *KIR3DS1* transcript and frequency of KIR3DS1 expression on NK cells. Interestingly, this hinted at a *KIR3DL1*-related mechanism regulating the peripheral expansion and functionality of KIR3DS1+ NK cells (90). A more recent study reported that NK cells from *KIR3DS1* and HLA-Bw4I80 cocarriers produced higher levels of chemokines after cell contact with infected CD4+ T cells than NK cells derived from HLA-Bw6 homozygous donors, leading to superior inhibition of viral replication (96).

Understanding the mechanistic basis of the protective effect of *KIR3DS1* has proven difficult, as multiple attempts had failed to demonstrate a functional interaction of KIR3DS1 with its putative HLA-Bw4 ligand (74, 97) or for that matter, an interaction with any ligand. To add an additional layer of complexity, the combined genotype of high expressing *KIR3DL1\*h* alleles and HLA-Bw4I80 (in particular *HLA-B\*57*) conferred strong protection toward HIV-1 disease progression (23, 98). Indeed, increased target cell cytotoxicity was observed in NK cells derived from elite controllers with protective *KIR3DL1\*h/\*y* receptor genotypes along with its HLA-Bw4I80 ligand (99). As *KIR3DS1* homozygosity is rare, in the majority of studies investigating *KIR3DS1* and HLA-Bw4 epistasis, *KIR3DS1*-bearing subjects possessed also *KIR3DL1* as a potential confounding variable.

Protection by an inhibitory KIR in HIV-1 disease seems counterintuitive, but might be mediated through a process called NK-cell licensing or education. Expression of an inhibitory KIR during NK-cell development provides strong inhibitory signals in response to its specific HLA-I ligand, ensuring self-tolerance. This allows NK cells to acquire enhanced cytotoxic function, which becomes apparent once exposed to missing or altered self (100). KIR3DL1 allotypes indeed differ in their inhibition of NK-cell function, with an overall trend toward increasing inhibitory capacity in high-expressing KIR3DL1 allotypes (101). Thus, a potential explanation is that presence of high-expression *KIR3DL1\*h* alleles together with HLA-Bw4I80 determines the increased cytotoxicity of KIR3DL1+ NK cells toward HIV-1–infected targets (taking into account that HLA-B is downregulated *via* actions of the HIV-1 accessory protein Nef) (23). Indeed, a study in slow progressors to AIDS reported increased polyfunctionality of NK cells from donors carrying the *KIR3DL1\*h/\*y* allele together with its *HLA-B\*57* ligand compared to HLA-Bw6 homozygous donors (102). Boudreau et al. recently demonstrated functionally that killing of HIV-1–infected targets *via* KIR3DL1+ NK-cells was dependent on the strength of NK-cell education *via* distinct combinations of KIR3DL1 and HLA-Bw4, with highest cytotoxicity mediated by high-expressing KIR3DL1 and HLA-Bw4I80 interactions (103). Moreover, NK cell education not only leads to enhanced functionality (104), but signaling through inhibitory KIRs on NK cells can additionally promote NK-cell survival (105), potentially leading to accumulation of educated NK cells expressing inhibitory receptors in chronic viral infection.

Supplementary evidence comes from studies in highly exposed HIV-1 seronegative individuals. One study reported a significant overrepresentation of *KIR3DS1* homozygosity in high-risk uninfected individuals compared to seroconverted individuals, independent of HLA-Bw4I80 (106). This group also reported an association of the KIR3DL1\*h/\*y-HLA-B\*57 combined genotype with protection from HIV-1 acquisition (107). Another study showed enrichment of the HLA-Bw4 carrier–*KIR3DS1* homozygous genotype in HIV-1-exposed seronegative subjects (108). In summary, whereas the results from epidemiological studies are not clear-cut, these studies point toward a potential dual effect of *KIR3DS1* (with or without HLA-Bw4I80) on both the course of HIV-1 infection and HIV-1 acquisition.

#### HLA-C and KIR2Ds

Genome-wide association studies have clearly implicated the *HLA-C* locus in HIV-1 control, identifying a protective SNP associated with higher HLA-C expression levels (1, 2). Interestingly, HLA-C surface expression levels are only 10% of surface levels of HLA-A and -B (109), and *HLA-C* alleles demonstrate less polymorphism compared to *HLA-B* (56). Nonetheless, individuals with high HLA-C expression levels have been shown to have a higher likelihood of mounting an HLA-C-restricted CD8+ T-cell response (110) and exhibit higher mutation rates in HLA-C–presented HIV-1 epitopes, indicating CD8+ T-cell pressure *via* HLA-C (111). However, given that virtually all individuals encode for KIRs (i.e., KIR2Ds) able to recognize cognate HLA-C molecules, it was proposed that NK cells might play an additional role in mediating the protective effect of higher HLA-C expression. Inhibitory KIR2DL1 binds to HLA-C group 2 allotypes (HLA-C2, which contain Asn77 and Lys80), whereas inhibitory KIR2DL2 and KIR2DL3, which are allelic products of the same *KIR2DL2/3* locus, bind to HLA-C group 1 allotypes (HLA-C1, which contain Ser77 and Asn80). Notably, KIR2DL3 also recognizes HLA-B\*46:01 due to an intergenic miniconversion between HLA-B\*15:01 and HLA-C\*01:02 (65). It was long believed that while HIV-1 Nef downregulated HLA-A and HLA-B surface expression to avoid recognition by cytotoxic CD8+ T cells (112), it spared HLA-C surface expression to ensure inhibition of NK cells *via* engagement of KIR2DL. This paradigm—which initially arose from studies performed with lab-adapted HIV-1 strains—was recently revised, when Apps et al. demonstrated that HLA-C is downregulated by HIV-1 Vpu variants derived from most primary HIV-1 isolates. HIV-1 Vpu-mediated downregulation of HLA-C was shown to subsequently impair the ability of HLA-C–restricted CD8+ T cells to inhibit viral replication (113). Regarding NK-cell function, it was reported earlier that expression of HLA-C (and HLA-E) on activated, HIV-1–infected CD4+ T cells impaired NK-cell killing, whereas blocking the HLA-C interaction with KIR2D enhanced NK-cell cytotoxicity toward HIV-1–infected CD4+ T-cell blasts (114, 115). During primary HIV-1 infection, KIR2DL+ NK-cell frequencies increased with the presence of their cognate HLA-C ligand (e.g., KIR2DL1+ NK cells expanded in HLA-C2 homozygous individuals) and exhibited more polyfunctional responses, presumably due to a licensing effect (116). Downmodulation of HLA-C by various HIV-1 strains resulted in reduced binding of KIR2Ds to HIV-1– infected cells. Moreover, NK cells were able to sense alterations in HLA-C expression as measured by differing degrees of HIV-1-replication inhibition. Yet, remaining HLA-C surface levels were sufficient to inhibit antiviral function of licensed KIR2DL+ NK cells (encountering their cognate HLA-C ligand) compared to unlicensed NK cells (117). Thus, although NK cells licensed through inhibitory KIR2D exhibit increased functionality against HLA-I–deficient target cells, first reports indicate that this subset does not have superior antiviral function against HIV-1–infected targets expressing self-HLA-C.

#### The Role of HIV-1 Peptides in KIR:HLA-I Interactions

HIV-1 exhibits an extraordinary ability to adapt to and evade host immune responses. The constant battle of the immune system attacking the virus and the virus evading leads to an extremely rapid accumulation of HIV-1 variants and quasispecies that, at least partially, escape from immune pressure (118, 119). Analyzing the major mechanisms of HIV-1 evasion and sites of sequence mutations provides direct insights into where the human immune system is able to apply critical pressure on the virus. A particular example is the rapid increase in HIV-1 mutations in HLA-I presented epitopes recognized by cytotoxic CD8+ T cells (CTL), which allows the virus to overcome adaptive immune pressure. These mutations can abrogate CTL recognition, but sometimes also impair viral replication (120). By now, a substantial body of evidence from structural (62, 63) and functional studies (60, 61, 65, 121–123) shows that KIR binding is modulated by the sequence of HLA-I–presented peptides, and in particular, C-terminal residues of these peptides. Unlike T cells, NK cells have germ-line encoded receptors that do not undergo recombination nor are they "specific" at discriminating self from non-self peptides (27). Instead, they have a moderate degree of peptide "sensitivity," mediated in large part by KIR:HLA-I interactions, which allows NK cells to monitor for changes in the peptide repertoire expressed by target cells. In fact, common HIV-1 sequence variants can modulate binding of inhibitory KIR to HLA-I, and by this means modulate NK-cell function (61, 124, 125), which has also been demonstrated in the case of SIV (121). Alternatively, NK cells may respond to altered MHC-I peptide processing following induction of the immunoproteasome in response to viral infection. IFN-γ stimulation results in increased cleavage of peptides after hydrophobic and basic residues. Thereby, it alters the C-terminus of available peptides for HLA class I presentation [reviewed in Ref. (126)], which may ultimately affect KIR binding to HLA-I:peptide complexes presented on the cell surface of stressed cells.

Viral variants arising due to CTL-mediated pressure can in turn impact KIR recognition by (i) impairing binding to inhibitory KIRs (61, 127), (ii) reducing HLA-C surface levels (128), or (iii) enhancing binding to inhibitory KIRs directly, a mechanism termed as "double-escape" (129). Furthermore, several amino acid polymorphisms in the viral genome, which showed a significant enrichment in subjects having a specific *KIR* gene, have been identified (130). As one example, a polymorphism in the overlapping reading frame of *vpu* and *env* was associated with the presence of *KIR2DL2* in HIV-1–infected subjects. Antiviral activity of KIR2DL2+ NK cells against this viral variant was reduced *in vitro* (130). However, a role for HLA-I in this process could not be determined due to small sample size. A subsequent study in a larger cohort of HIV-1 clade C–infected individuals identified two viral sequence variants, that were significantly enriched in individuals in the presence of the combined *KIR2DL3–HLA-C\*03:04* genotype. One of the variants (Tgag303V) was contained within a CTL epitope and located at the C-terminal end of the nonamer (YVDRFFKVL), but did not mediate escape from recognition by HLA-C\*03:04-restricted CTLs compared to the wild-type sequence (131). This viral variant, however, enhanced binding to KIR2DL3 and inhibited KIR2DL3+ NK cells *in vitro* (132). Overall, these studies support the concept of KIR-mediated selection pressure on HIV-1 as an additional source driving viral evolution. Furthermore, a recent report showed that binding of KIR2DL2/3 to HLA-C1 allotypes is more selective to presented peptides than KIR2DL1 binding to HLA-C2 (60), further enhancing our mechanistic understanding of KIR:peptide:HLA-I interactions. Moreover, this study showed that certain peptides (including an HIV-1 Gag peptide) allow for binding of KIR2DLs to non-canonical HLA-C molecules (60). Taken together, whereas NK cells are not able to distinguish between self- and non-self peptides, KIR binding to HLA-I is certainly sensitive to changes in the peptide sequence presented on HLA-I molecules. This may in turn facilitate recognition of HIV-1–infected cells, potentially not only *via* presentation of viral peptides but also due to stressed-induced changes in the HLA-I-presented peptide repertoire.

Of note, the majority of studies evaluating the peptide sensitivity of KIR:HLA-I interactions to date have relied on external labeling with peptides, but overall, the abundance of viral peptides eluted from HLA-I compared to self-peptides is low (133, 134). Yet, antagonist peptides (i.e., peptides that are presented by HLA-I but abrogate KIR binding) can significantly interfere with KIR clustering at immune synapses and override NK-cell inhibition (135, 136). Therefore, HIV-1 infection may lead to NK-cell activation by causing a shift between antagonist and agonist peptides. Consequently, further investigations on how HIV-1 infection changes the HLA-I–presented peptide repertoire and how this impacts NK-cell function are needed. Nonetheless, CTL pressure on viral sequence appears to be dominant, as the first escape mutations arise after peak viremia and following expansion of HIV-1–specific CTLs (9). KIR are also expressed on T cells and can modulate CTL activity (75, 83); therefore, a potential role of KIR+ T cells in explaining *KIR/HLA* disease associations has to be considered. Overall, studies suggest a complex interplay between innate and adaptive immune pressures in driving HIV-1 sequence evolution, with HLA-I being central to the interaction with KIRs and TCRs.

#### KIR3DS1 and the Non-Classical HLA-F—A Non-Classical Stress Ligand?

Genetic evidence and functional data not only implicate KIR3DS1 in HIV-1 disease but indicate a widespread effect of KIR3DS1 in autoimmunity, transplantation, cancer, and other viral infections (137). Yet, for years, a definite ligand for this receptor that could account for these effects remained elusive. Only recently, we and others discovered that KIR3DS1 can bind open conformers (OCs) of HLA-F, a non-classical HLA-I molecule (138, 139). This was confirmed *via* surface plasmon resonance (SPR), pull-down experiments, HLA-F tetramer binding studies, as well as KIR3DS1+ reporter cell assays (138, 139). Functionally, HLA-F OCs led to degranulation of KIR3DS1+ NK cells, as well as cytokine production in response to HLA-F (138). HLA-F is a non-classical HLA-I molecule with a unique combination of features. It is (i) highly conserved with one dominant allele (140) [similar to *KIR3DS1* (71)], (ii) displays tight tissue specific regulation, with a mostly intracellular localization (141, 142), and (iii) is expressed on the cell surface of activated lymphocytes (143). HLA-F is known to bind to inhibitory KIR3DL2 (138, 144), as well as inhibitory LILRBs (145, 146), whereas the results on binding of HLA-F to KIRDS4 are conflicting (138, 139, 144, 146). In HLA-F, 5 of 10 residues, which are highly conserved in other HLA class I molecules, are substituted, resulting in an altered peptide groove (146). To date, there has been no structural data published describing the OC of HLA-F. Given that KIR3DS1, KIR3DL2 and KIR3DL1 (albeit weaker), bind to HLA-F, one could imagine a role of the D0 domain in contacting HLA-F OC as the D0 domain enhances KIR3DL binding to HLA class I and mediates binding to a non-HLA class I ligand (72, 147). Nevertheless, the contact residues of KIR3DS1 to OCs of HLA-F conferring specificity and high-affinity of the interaction are entirely unknown to date. SPR data suggest that KIR3DS1 additionally binds to OCs of classical HLA class I, but so far, the functionality of this binding remains to be demonstrated (139).

We previously demonstrated that HIV-1 infection causes upregulation of HLA-F at a transcriptional level in stimulated CD4+ T cells. Therefore, KIR3DS1 binding to HLA-F expressed as a "stressed self " signal on HIV-1–infected cells might explain the superior ability of KIR3DS1+ NK cells to inhibit viral replication in autologous CD4+ T cells (92, 138). Thus, the interaction between KIR3DS1 and HLA-F upregulated on HIV-1–infected cells may have similarities to the well-reported upregulation of stress ligands such as ULPBs and MIC-A/MIC-B in HIV-1 infection, which are in turn recognized by the activating NK-cell receptor NKG2D (43).

Given the identification of HLA-F as a KIR3DS1 ligand, the following question remains unsolved: why is the strong genetic protective effect of *KIR3DS1* observed preferentially in combination with HLA-Bw4I80 in HIV-I infection? We can conceive four potential models that are not mutually exclusive and may explain this phenomenon (**Figure 1**):

from a synergistic but independent effect of KIR3DS1– HLA-F and KIR3DL1–HLA-Bw4I80 interactions*.* Long et al. showed that possessing *KIR3DS1* confers greater NK-cell functionality, also in absence of HLA-Bw4I80 (93). Under this

Figure 1 | Four models with potential mechanisms to explain the underlying protective effect of the combined *KIR3DS1*–HLA-Bw4 genotype in HIV-1 infection. (A) Viral peptides allow for KIR3DS1 binding to HLA-B\*57:01 and trigger natural killer (NK)-cell activation. Presentation of viral peptides (in red) on HLA-B\*57:01 upon HIV-1 infection of target cells (blue) enables binding of KIR3DS1 on NK cells (yellow). The short cytoplasmic tail of KIR3DS1 associates to the adaptor molecule DAP12, which bears two ITAMs. (B) HLA-Bw4 enhances human leukocyte antigen F (HLA-F) expression at the cell surface of HIV-1–infected cells. Open conformers of HLA-F exist as heterodimers with open conformers of HLA-Bw4 on the cell surface of HIV-1–infected cells. This enhances binding and triggering *via* KIR3DS1 on NK cells. (C) KIR3DS1:HLA-F and KIR3DL1:HLA-Bw4 interactions have independent but synergistic protective effects in HIV-1 infection. HIV-1 infection of target cells leads to downregulation of HLA-Bw4 from the cell surface *via* action of the accessory protein Nef. Loss of HLA-Bw4 on the infected cells leads to loss of inhibition *via* KIR3DL1. Simultaneously, cell stress induced by HIV-1 infection leads to upregulation of open conformers of HLA-F, which bind to KIR3DS1 and trigger NK-cell activation. A potential epistatic regulation of *KIR3DS1* gene expression *via* the *KIR3DL1* gene is depicted. (D) KIR3DL1:HLA-Bw4I80 interactions limit KIR3DS1:HLA-Fmediated immune activation. HIV-1 infection directly (and indirectly) causes cellular stress, which in turn upregulates surface expression of HLA-F open conformers on CD4+ T cells and other cell types. OCs of HLA-F bind to KIR3DS1 and trigger NK-cell activation. On the other hand, KIR3DL1 binds to HLA-Bw4 molecules, which are present on HIV-1–infected cells, although at low levels due to HIV-1 Nef-mediated downregulation. Inhibitory signaling *via* KIR3DL1 limits NK-cell activation and inflammatory cytokine production, thus limiting activation *via* KIR3DS1.

model, the most effective NK cells against HIV-1–infected target cells would express both KIR3DS1 and KIR3DL1 and undergo activation *via* KIR3DS1-mediated engagement of HLA-F and KIR3DL1-dependent loss of inhibition due to HLA-B downregulation.

Yet, there is evidence that KIR3DS1 expression and function is not completely independent from KIR3DL1, as KIR3DS1 mRNA, and KIR3DS1+ NK-cell frequency increases with more gene copies of *KIR3DL1* (90). Additionally, *KIR3DS1/KIR3DL1* individuals display superior viral inhibition activity than individuals with either KIR alone in the presence of HLA-Bw4 (90). Thus, there is a possibility of KIR3DL1-mediated epistatic regulation of KIR3DS1 expression and function. However, the existence of a KIR3DS1+KIR3DL1+ coexpressing NK-cell subset has not yet been definitively proven due to the limitations of current anti-KIR antibody cross-reactivity.

(D) *KIR3DL1:HLA-Bw4I80 interactions are necessary to limit KIR3DS1-HLA-F-mediated immune activation*: As chronic viral infections can drive inflammatory processes resulting from persistent immune activation (156), downmodulation of the immune response is important for host homeostasis and preventing immunopathology; especially in HIV-1 infection where immune activation can accelerate disease progression (157). Thus, it is conceivable that inhibition of NK cells *via* KIR3DL1:HLA-Bw4 interactions may be important to counteract an exuberant immune response mediated by KIR3DS1+ NK cells recognizing HLA-F on "stressed"/ infected cells. Moreover, education through inhibitory KIRs has been shown to promote increased survival of iKIR+ NK cells (105). Increased survival of educated KIR3DL1+ NK cells might counteract chronic immune activation that can result in disease progression. In line with this, the study of Martin et al. showed that *KIR3DS1* homozygosity without HLA-Bw4I80 was modestly associated with rapid progression to AIDS (22). Therefore, as supported by mouse models that implicate NK cells as "rheostats" in chronic viral infections (158), combined stimulatory and inhibitory signaling may result in a tunable antiviral response that confers optimal HIV-1 disease control without causing immunopathology.

In summary, our mechanistic understanding of how protection in HIV-1 disease is mediated in the context of combined KIR3DS1 and HLA-Bw4 is still limited and requires further study. Although we focus on NK cells, a potential role for HLA-Bw4I80-restricted CD8+ T cells expressing KIR3DS1 has also to be considered (84). So far, genetic studies of disease susceptibility have been extremely resourceful in guiding our understanding of the mechanisms involved in HIV-1 control. Therefore, HIV-1 disease association studies that are able to tease out the effect of *KIR3DS1* homozygosity in the context of HLA-Bw4I80 would be of great utility, but will require large sample sizes.

#### The Role of Peptide:HLA-F Complex

Major histocompatibility complex class I exists in two biologically relevant conformations on the cell surface: (i) as a membrane-bound heavy chain lacking peptide and β2-microglobulin (β2m) termed open conformer (OC) or (ii) as a trimeric heavy chain:β2m:peptide complex (159). Recently, thermal denaturation assays demonstrated that OCs of HLA-F are more stable (146) than OCs of other HLA-I gene products (160). Earlier findings assessing stability after cold treatment suggested an increased stability of HLA-F OCs compared to open conformers of classical HLA-I (141, 142). This—and the fact that no canonical peptides could be eluted from HLA-F—supported the notion that HLA-F is mainly expressed as an OC devoid of peptide (142, 154).

Recently, the crystal structure of HLA-F (in complex with β2m and peptide) was solved, shedding first light onto the molecular structure of HLA-F (146). Surprisingly, this work showed that HLA-F has a unique peptide-binding grove that resembles the groove of classical HLA-I but does not anchor peptides at their N-terminus, allowing for binding of longer peptides. Indeed, peptides eluted from HLA-F and characterized by mass spectrometry had an extended length distribution compared to classical HLA-I molecules, peaking at 12 amino acids and with peptides up to 30 amino acids observed. This unconventional length rather resembles the length of HLA class II-presented peptides.

Moreover, new insights into the structure and docking mode of LILRB1 interacting with the HLA-F:β2m:peptide complex were gained. The LILR family (also termed LIR, ILT, or CD85) are encoded on chromosome 19 within the leukocyte receptor complex along with the KIR locus. In total, 13 different LILRs have been identified. Similar to KIRs, LILRs can provide an either inhibitory (LILRB) or activating (LILRA) signal, depending on the presence of an ITIM or the association to ITAM-containing adaptor molecules, but also depending on the cellular context (161). LILRB2 is not expressed on NK cells and its implications in HIV-1 disease are reviewed elsewhere (24). LILRB1 recognizes most classical and non-classical HLA-I molecules, except for HLA-E (162–164), given that it binds to the conserved α3 domain of the HLA-I heavy chain as well as β2m (165, 166). Intriguingly, the affinity of LILRB1 to peptide-bound HLA-F:β2m is the highest observed so far compared to other HLA-I ligands (146, 167). LILRB1 is expressed on NK cells in varying percentages (0–50% with high interindividual variability), as well as on T cells and professional antigen presenting cells such as DCs, monocytes/macrophages, and B cells (146, 168). Engagement of LILRB1 *in vitro* leads to inhibition of cytotoxicity and cytokine production in a subset of NK cells (151, 169, 170), but interestingly, LILRB1+ (but not LILRB1– ) NK cells are able to markedly suppress HIV-1 replication in infected monocyte-derived DCs in a manner independent of classical HLA-I (171), hinting at a possible role of HLA-F.

Looking at the binding footprint of LILRB1 on HLA-F, it is improbable that the interaction is sensitive to the nature of the presented peptide—in contrast to certain KIRs. In the case of KIR3DS1, it was shown that KIR3DS1+ reporter cells responded to HLA-F OCs, but were not triggered by peptide-bound HLA-F complexes (146). This could be due to peptide-induced conformational changes in HLA-F structure or direct steric inhibition by the bound peptides. Furthermore, inhibitory KIR3DL2 recognizes OCs of HLA-F or HLA-I and posssibly heterodimers of HLA-F with HLA-I heavy chains, with the latter also being increasingly expressed on activated lymphocytes (144). This raises interesting possibilities for a cell-stress induced conformational change in HLA-F allowing binding to activating receptors, such as KIR3DS1, while abrogating binding to inhibitory receptors, such as LILRB1. Thus, although HLA-F is not expressed on the surface of lymphocytes in a resting state (143), it potentially can exist in various conformations on stressed cells (154) with differential impact on NK-cell function (**Figure 2**).

Figure 2 | The potential impact of HIV-1 infection on expression of non-classical human leukocyte antigen class I (HLA-I) molecules on a target cell and effect on natural killer (NK)-cell receptor binding. (A) *HLA-E.* HIV-1 infection of target cells leaves HLA-E surface levels either unchanged or slightly upregulated. HLA-E can present self-peptides (in black) that bind to the NKG2A:CD94 receptor complex, and inhibit NK-cell function. In the context of HIV-1 infection, HLA-E can present viral or "stress"-self-peptides (in red) that abrogate (or reinforce) binding to NKG2A:CD94 and modulate NK-cell activity. The potential role of viral or "stress" peptides presented on HLA-E that could trigger the activating NKG2C:CD94 receptor complex on NK cells is currently unknown. NKG2C associates to DAP12, an adaptor molecule containing two ITAMs. (B) *HLA-F.* The exact impact of HIV-1 infection on HLA-F surface expression in different cell types needs yet to be established. In general, HLA-F is expressed on activated or stressed cells, potentially in distinct functionally relevant conformations: (i) an open conformer that binds to the activating NK-cell receptor KIR3DS1 or (ii) a β2m-bound complex presenting peptides of unusually long length for HLA-I, which allows binding of the inhibitory receptor LILRB1. (C) *HLA-G:* one study showed downregulation of HLA-G in monocyte-derived macrophages, potentially *via* HIV-1 Vpu, although this has not yet been confirmed in primary cells. Moreover, the functional relevance of HLA-G downregulation in antiviral immune responses has not been established to date, although HLA-G is thought to play a predominantly immunoregulatory role given its interaction with inhibitory receptors. HLA-G can form dimers on the cell surface *via* an interchain α1 disulfide bond, which enhances recognition by inhibitory LILRB1 on NK cells. KIR2DL4 binding to HLA-G remains controversial. KIR2DL4 has a dual activating and inhibitory effect on NK cells, given that its cytoplasmic tail contains an ITIM and it associates to ITAM-bearing FcRγ.

### The Non-Classical HLA-G—An Immune Modulator?

Human leukocyte antigen-G is a non-classical HLA-I that displays a high degree of tissue restriction. It was first discovered in extravillous trophoblast cells in the fetal placenta (172), where HLA-G protein is abundant (173) and since then has been extensively studied in the context of reproduction. Further studies showed that under healthy conditions, HLA-G is expressed in other immune-privileged sites including the cornea (174), thymus (175), nail matrix, and on mesenchymal stem cells (176, 177). Under inflammatory conditions such as CMV infection or within a tumor microenvironment, HLA-G can be expressed on DCs and monocytes/macrophages (178, 179), and is reported to be upregulated in monocytes treated with IFN-γ (a potent inducer of HLA-I and -II expression) as well as IL-10 (180–182). Also, an increasing number of studies shows aberrant HLA-G mRNA expression by tumors (183, 184) [reviewed in Ref. (185)]*,* but some of these findings remain controversial as in other studies no HLA-G protein was identified (179, 186) [reviewed in Ref. (187)]. Overall, there is evidence that HLA-G expression is induced on various immune cells under inflammatory conditions resulting from infections, allergies, or allogeneic stimulation following transplantation (188–191).

As a result of a premature stop codon in exon 6 (192), the cytoplasmic tail of HLA-G is truncated and the heavy chain has a molecular weight of only 39 kDa, compared to the 45 kDa weight of classical HLA class I heavy chain. In total, seven splicing variants of HLA-G have been described (193, 194). The predominant splice variant *in vivo* is HLA-G1, which encodes for the full-length, membrane-bound HLA-G protein (195, 196). Alternatively, soluble HLA-G (sHLA-G) can be generated from three splice variants or *via* proteolysis of the HLA-G1 isoform (197). Interestingly, sHLA-G can confer a protective effect to cells normally permissive to NK-cell killing (197). Apart from HLA-G1, three other alternatively spliced transcripts encode membrane-bound HLA-G, albeit in a truncated form: HLA-G2 lacks the α2 domain, HLA-G4 lacks the α3 domain, and HLA-G3 lacks both the α2 and α3 domains (198). These transcripts were reported to inhibit NK-cell function, although it remains unclear through which NK-cell receptors this occurs (198–200). Moreover, it was suggested that isoforms HLA-G2 and -G3 are expressed in individuals homozygous for the *HLA-G\*0105N* null allele (201), possibly explaining the existence of healthy adults lacking full length HLA-G1 (201). Of note, all splicing variants encode the leader sequence enabling HLA-E expression (202) and thereby their expression in target cells can indirectly inhibit NK cells *via* NKG2A:CD94 (163).

To date, 18 distinct functional proteins of HLA-G have been identified, with the *HLA-G* gene encoding a total of 54 *HLA-G* alleles (including two *HLA-G* null alleles) (56). While most of the polymorphism of classical HLA-I genes lies in the α1 and α2 domains that bear the peptide-binding groove (203), HLA-G has a relatively conserved peptide-binding groove and has allelic variability occurring within the 3′UTR, which is important for posttranscriptional regulation of HLA-G (203, 204). Peptides eluted from HLA-G thus far appear to be derived from a restricted number of proteins (205) and a crystal structure demonstrates that these presented peptides are buried deep within the peptide-binding groove (206). The induction of an HLA-G-restricted CD8+ T cell response against a human cytomegalovirus peptide in mice was described, but the cytolytic capacity of these T cells was limited (207). Overall, it seems that the immune modulatory functions of HLA-G mediated through binding of inhibitory receptors expressed on a variety of immune cells dominates over a potential role in presenting peptides.

#### NK-Cell Receptors Recognizing HLA-G

Human leukocyte antigen-G is recognized by LILRs with greater affinity than HLA-A, -B, or -C molecules (208). In addition, HLA-G is unique in possessing a cysteine at position 42 of its α1 domain, which allows for an unusual conformation of HLA-G as a homodimer of two β2m-associated HLA-G complexes (152, 209, 210) (**Figure 2C**). This conformation dramatically enhances recognition and signaling of LILRB1 (151) and has been demonstrated to occur naturally on trophoblasts (173). Indeed, inhibition of LILRB1+ NK-cell function is sensitive to the conformation of HLA-G, as the heavy chain of HLA-G alone does not inhibit LILRB1+ NK cells (211). Studies measuring inhibition of LILRB1+ NK-cell cytolytic function *via* HLA-G have to account for HLA-E expression as it is upregulated through the HLA-G leader peptide—an exception being the K562 cell line, which does not express HLA-E (212). Independent of HLA-E, HLA-G interferes with immunological synapse formation and inhibits NK-cell cytotoxicity (212, 213). Additionally, Riteau et al. demonstrated that HLA-G expression has a major inhibitory effect on NK cell lysis through LILRB1, also when coexpressed with other HLA-I ligands (214). Besides the inhibitory effect of HLA-G expression on NK-celleffector function itself, HLA-G can impair NK-DC crosstalk. Pretreatment of DCs with sHLA-G leads to reduced activation and IFN-γ production by NK cells (215), while IFN-γ in turn triggers HLA-G surface expression (180, 181). This again supports the notion that HLA-G has tolerogenic properties.

In addition to LILRB1, HLA-G has been proposed to modulate NK-cell function *via* binding KIR2DL4. *KIR2DL4* is a framework gene within the KIR locus and thus is present in virtually all haplotypes, but there is a high frequency of alleles lacking the transmembrane domain or having truncated cytoplasmic tails (216). In peripheral blood, expression of KIR2DL4 is weak and restricted to the CD56bright subset, but can be induced on NK cells *in vitro* with stimulation (217, 218). KIR2DL4 has unique functional properties compared to other receptors of this family. A positively basic arginine residue in the transmembrane domain allows for association with the activating Fc receptor gamma protein (219), while the long cytoplasmic tail contains one immunoreceptor tyrosine-based inhibitory motif (ITIM). This results in mixed activating and inhibitory signaling, which has been shown to occur *in vitro* (219–222). In line with this, crosslinking of KIR2DL4 on peripheral blood NK cells induces IFN-γ production, and (albeit weaker) NK-cell cytotoxicity (217, 218, 223).

Newer reports provide conflicting evidence regarding the interaction of HLA-G with KIR2DL4 (224, 225). Although several groups reported binding using various techniques including cellular transfectants, SPR, and functional assays (169, 226–230), others have failed to reproduce KIR2DL4 binding *via* SPR, tetramers, or functional IFN-γ responses to sHLA-G (210, 231, 232). The crystal structure of the extracellular domains of KIR2DL4 solved by Moradi et al. (233) demonstrated oligomerization of KIR2DL4, uncharacteristic of other KIRs. In this study, no binding of KIR2DL4 to HLA-G was detected *via* SPR (233). An explanation might be that signaling *via* KIR2DL4 only occurs upon concentration of the ligand in endosomes [as discussed in Ref. (187)], since sHLA-G endocytosed into KIR2DL4-containing compartments was shown to induce cytokine secretion of NK cells (229, 234). Regardless of its binding to HLA-G, higher copy numbers of KIR2DL4 have been linked to better survival of CD4+ T cells and increased IFN-γ responses from NK cells during acute SIV infection in rhesus macaques (235).

#### HLA-G Expression in HIV-1

Only a low percentage of immune cells in healthy subjects expresses HLA-G, whereas in HIV-1 infection a substantial upregulation of HLA-G has been observed in both peripheral blood monocytes and T-cell subsets (236). This was later partly attributed to antiretroviral treatment, as frequencies of HLA-G+ monocytes decreased after treatment interruption (237). In fact, nucleoside reverse transcriptase inhibitors were found to increase HLA-G expression, whereas protease inhibitors did not (238). A role for HLA-G+ HIV-1–restricted CD8+ T cells has furthermore been described in HIV-1–infected subjects (239). Contrary to *in vivo* studies of high HLA-G expression levels on monocytes of patients undergoing HAART (236–238), one study showed downregulation of HLA-G1 surface expression in HIV-1–infected monocyte-derived macrophages *in vitro* (240). This downregulation was suggested to be mediated *via* HIV-1 Vpu (240), given that the truncated tail of HLA-G renders it resistant to HIV-1 Nefmediated downregulation (241). Yet, this needs to be confirmed in primary cells. Overall, how HIV-1 directly impacts HLA-G expression in different cell types remains unclear.

In addition to inhibiting DC function *via* LILRB2 (242) and regulating CD4+ T-cell proliferation (243), sHLA-G can inhibit NK-cell killing *in vitro* and may therefore suppress NK-cell function *in vivo* (197). sHLA-G plasma levels change during the course of HIV-1 infection and treatment, as two groups reported high sHLA-G levels in early infection (244) with a significant decrease after treatment initiation (245). In rapid progressors, however, levels of sHLA-G were persistently elevated even despite treatment initiation, while this was not the case for untreated normal progressors and long-term non-progressors (244). Furthermore, sHLA-G levels were higher in patients with opportunistic infections, indicating a potential role of sHLA-G as a surrogate marker of disease progression (246). In a cohort of female commercial sex workers from Benin, HIV-1–infected subjects were reported to have lower levels of sHLA-G in plasma (247) but higher levels of sHLA-G in the genital mucosa (248). Of note, levels of sHLA-G are also in part determined genetically by distinct *HLA-G* alleles (249). Thus, data on sHLA-G levels in HIV-1 infection need to be carefully controlled for confounding factors such as HAART (237, 238), *HLA-G* genetic background (249), sampling site (247, 248), or coinfections (246, 248). In summary, it is not known whether higher sHLA-G levels have direct functional consequences on HIV-1 disease progression *via* modulation of NK and other immune cells, or whether sHLA-G levels are rather a reflection of viremia and the antiviral immune response.

#### Genetic Evidence for a Role of HLA-G in HIV-1 Infection

Although HLA-G polymorphisms are limited, certain HLA-G alleles have been suggested to be involved in susceptibility to HIV-1 infection. In 2004, Matte et al. reported that the *HLA-G\*0105N* allele, a null variant which does not encode functional HLA-G1, was protective in HIV-1 acquisition, whereas the *HLA-G\*01:01:08* allele encoding for full-length HLA-G increased the risk of HIV-1 infection. They formulated the hypothesis that non-functional HLA-G proteins may allow for better NK-cell killing of HIV-1–infected cells (250). This observation was not consistent with findings of subsequent studies, which reported either enrichment of *HLA-G\*0105N* in HIV-1–positive women (251) or did not identify *HLA-G\*0105N* allele as a disease modifying factor (252). Other *HLA-G* alleles identified were *HLA-G\*01:04:04*, which associated with susceptibility to HIV-1 infection, and *HLA-G\*01:01:01*, which was enriched in HIV-1– resistant women (252). One study states that these conflicting findings may be explained by variation of *HLA-G* polymorphisms among different ethnic populations and reports no association of *HLA-G* polymorphisms to HIV-1 susceptibility except in African-American cohorts (253).

As HLA-G is an important player involved in maternal-fetal tolerance, *HLA-G* polymorphisms have been studied in the context of vertical HIV-1 transmission from mother-to-child. Mothers bearing the *HLA-G\*01:03* allele were less likely to perinatally transmit HIV-1 (254). Upregulation of the functional isoform HLA-G1 mRNA in the placenta has been associated with increased risk of HIV-1 mother-to-child transmission (255). Further studies have assessed the risk of variants in the 5′ and 3′UTR of HLA-G, and in particular, the impact of the 14-bp insertion/deletion in the 3′UTR of HLA-G on mother-to-child transmission. In healthy subjects, the 14-bp insertion genotype (ins/ins*)* correlates with lower plasma levels of sHLA-G (256). *In vitro,* transfection of the 14-bp ins/ins HLA-G into K562 cells resulted in increased levels of membrane-bound HLA-G1 expression with higher mRNA stability and lower sHLA-G1 ratio (257). However, studies on the impact of the 14-bp insertion on HIV-1 vertical transmission risk report conflicting results (258–260). In horizontal transmission, the frequency of the 14-bp ins/ins genotype was enriched in HIV-1–infected patients in African (but not European) subjects (261). Overall, population studies attempting to shed light on the question whether functional versus non-functional *HLA-G* alleles are associated with HIV-1 susceptibility have painted an inconsistent picture. Moreover, posttranscriptional regulation of the *HLA-G* gene through variations in the 3′ and 5′ LTR and alternative splicing has to be considered as an important genetic factor modulating HLA-G expression levels in these studies.

### The Oligomorphic Interaction between HLA-E and NKG2:CD94—A Contrast to the Diversified HLA-KIR System

Inhibition of NK cells can be achieved either through highly diversified KIR:HLA-I interactions or through a second inhibitory system indirectly monitoring the level of overall HLA-I expression. This latter inhibitory mechanism is achieved *via* the well-conserved NK-cell receptor–ligand interaction of NKG2A/ CD94 with HLA-E (262). Contrary to other non-classical HLA-I gene products, HLA-E is ubiquitously expressed (263), but at substantially lower levels as compared to classical HLA-A, -B, and -C (264). Its expression is dependent on the expression of other HLA-I, as it presents a nonamer peptide derived from the signal sequence of several HLA-A, -B, and -C gene products as well as HLA-G. HLA-F and HLA-E itself lack an HLA-E–presented leader peptide (265).

Human leukocyte antigen-E has restricted polymorphism with to date only 25 known alleles (56), of which two—HLA-E\*01:01 and \*01:03—are the most frequent in the human population and are believed to be in balancing selection (266, 267). HLA-E\*01:01 encodes for an arginine at position 107 (HLA-ER), whereas HLA-E\*01:03 encodes for a glycine at this position (HLA-EG). This substitution leads to higher surface expression levels of the latter, despite similar intracellular protein levels (160). HLA-E is highly relevant to innate immune responses due to its interaction with heterodimeric NKG2/CD94 type II transmembrane-anchored receptors, which are expressed on a large proportion of NK cells as well as on a subset of CD4+ and CD8+ T cells (268–270).

Natural killer group 2 receptors are a family of C-type lectin receptors encoded within the NK gene complex on chromosome 12p12-13 (271). Almost all NKG2 gene products heterodimerize with CD94, a non-signaling invariant glycoprotein also encoded within the NK gene complex. These include *NKG2A* [which produces NKG2A and NKG2B gene products *via* alternative splicing (272)], *NKG2C*, *NKG2E*, *NKG2F*, and *NKG2H.* The *NKG2D* gene is also located within the NK gene complex, but its gene product has low sequence homology to other NKG2 receptors and forms an NKG2D:NKG2D homodimer (without CD94) that binds to the stress ligands MIC-A, MIC-B, and ULBPs, but not to HLA-E (273). Unlike *KIR* genes*, NKG2* genes exhibit limited polymorphism (262, 274). Aside from being expressed widely on NK cells, they can also be expressed on subsets of T cells (275). Here, we focus on NKG2A:CD94 and NKG2C:CD94 receptor complexes, both of which bind HLA-E but have opposite effects on NK-cell function. While NKG2A signaling inhibits NK-cell cytotoxicity *via* two ITIMs in its cytoplasmic tail (276), NKG2C delivers activating signals through its associated adaptor molecule DAP12 (277).