Innate lymphoid cells in HIV pathogenesis and in the human female genital tract

HIV enters the female genital tract during sexual contact, via semen, and needs to overcome innate immune barriers to establish infection. These barriers include epithelial cells, mucus and secretions, which have been demonstrated to inactivate the virus and prevent viral access into the mucosa ( 3 ). Epithelial barrier function is considered a major contributing factor to HIV prevention, and pathological conditions that alter epithelial barrier integrity (such as inflammation, dysbiosis, and other sexually transmitted infections) increase the risk for HIV infection ( 5 – 8 ). Mechanisms that reinforce barrier protection should reduce HIV acquisition risk ( 9 , 10 ). There are also several innate immune populations with the ability to interact and inactivate HIV ( 11 ). For example, genital neutrophils rapidly release NETs that inactivate the virus and prevent HIV infection in vitro ( 12 , 13 ). Dendritic cells also capture HIV and produce chemokines and antimicrobials with anti-HIV activity shortly after exposure ( 14 – 17 ). However, little is known about other innate immune cell populations and their antiviral properties in the FGT, including ILCs. Despite evidence for innate immune cell mediated antiviral activity in the FGT, the in vivo consequences of these in vitro findings and their effectiveness in preventing HIV acquisition are difficult to evaluate. Innate cells also are drivers of inflammation, particularly under pathological conditions of dysbiosis or genital infection ( 18 , 19 ), and inflammation in turn is another critical factor that increases HIV susceptibility ( 20 ). Persistent genital inflammation mediates epithelial barrier disruption and attraction of target immune cells that support viral replication. Another critical factor that modifies inflammation, immune composition, and epithelial integrity is the microbiome ( 21 ). Alterations to the vaginal microbiome, even at the subclinical level, increase the risk for HIV acquisition ( 22 ), a topic recently reviewed elsewhere ( 23 ). Investigation into the mechanisms that control inflammation and immune populations in the FGT will inform how to reduce the risk of HIV acquisition. Importantly, immune cell distribution and function in the FGT is regulated by multiple physiological factors, including sex hormones (endogenous and exogenous), menstrual cycle, pregnancy, menopause and aging, placing women at different risk for HIV acquisition throughout their lifespan ( 2 , 3 , 24 – 28 ). Understanding these dynamic changes is important to develop universal prevention strategies.

To date, the role of human ILCs in HIV pathogenesis has only been investigated in the context of chronic HIV infection, but their potential contribution to mucosal HIV acquisition remains unknown. Only a limited number of studies using nonhuman primates have examined the involvement of ILCs in mucosal SIV acquisition and/or protection after vaccination. A recent study in a nonhuman primate model showed an increase in different ILC populations in rectal and vaginal mucosal surfaces after vaccination with ΔV1 SIV, particularly at 12 weeks post-immunization. ILCs exhibited increased reactivity when stimulated with gp120 peptides, showing a higher frequency of IL-13+ ILC2, IL17+ NKp44+ ILCs and IL-17+ ILC3, which were associated with reduced risk of viral acquisition, and of IFN-y+ ILC1s, which correlated with control of viral load ( 88 ). A previous study using replicating adenovirus (Ad)-SIV recombinants for mucosal priming also demonstrated that IL-17+ NKp44+ ILCs were associated with reduced viral acquisition and lower viremia after SIV infection ( 89 ). These findings indicate that the DNA/ALVAC/gp120-based vaccination effectively generates a mucosal ILC response able to reduce SIV acquisition and enhance control of viral load ( 88 ). However, another ILC subset (NKG2A-NKp44- double-negative ILCs) correlated with increased risk for SIV acquisition and higher viral loads, suggesting differential contribution of ILC subsets to mucosal HIV pathogenesis ( 88 , 89 ). Importantly, these studies in NHP models lay the groundwork for investigating how human ILCs could generate protective mucosal responses to HIV. Overall, the role that human mucosal ILCs play during HIV acquisition is a significant gap in our knowledge. Finally, it remains unclear whether ILCs can be infected by HIV. Some ILC1s have been described to express CD4, the HIV entry receptor, in the human gut, spleen, blood, and liver ( 90 ). Further, one study demonstrated that blood ILC1s could be infected with HIV in vitro and in a humanized mice model ( 90 ). Further research needs to determine if human mucosal ILCs can support viral replication in vivo .

ILCs are a heterogenous population of tissue-resident lymphocytes that contribute to tissue homeostasis and immune defense through cytokine production ( 29 ). ILCs are highly diverse and plastic, adjusting to their tissue environment ( 30 – 32 ). ILCs have been primarily studied in mouse models, but human ILCs are distinct from their murine counterparts through surface marker expression, cytokine responsiveness, and distribution, making the study of human ILCs critical to understanding mucosal immunity ( 33 , 34 ). ILCs can be broadly divided into cytotoxic NK cells and helper ILCs ( 35 ), the latter is the focus of this review. Helper ILCs are generally identified as lineage negative cells that express the IL7 receptor (CD127). Based on additional surface markers ILCs are traditionally classified into three major subsets: ILC1, ILC2 and ILC3 that respectively mirror Th1, Th2, and Th17 CD4+ T helper cell cytokine profile and function ( 34 ) ( Table 1 ). However, ILCs are known to transition between subsets and modify their functional phenotype, allowing adjustments to their changing tissue environment. For example, RNA velocity analysis revealed an intermediate ILC3-ILC1 cluster that indicated conversion to ILC1s in human tonsils (Cella et al 2019). Tissue-specific adaptations and rare presence make ILC characterization difficult and highlights the need to define human ILC subsets in their tissue of residence through combined protein and RNA analyses. ILC1s produce IFN-y and TNF and rely on T-box transcription factor 21 (TBX21 encoding T-BET) in humans. ILC1s can be distinguished from NK cells through expression of the Homolog of Blimp1 (HOBIT), and lack of expression of Eomesodermin (EOMES), which is present in NK cells ( 36 ). However, recent advances demonstrate that ILC1s cannot be defined by a single marker or transcription factor, and that HOBIT is not expressed in all ILC1s, specifically the PRDM1+ CD103+ intraepithelial ILC1 subpopulation (ieILC1) ( 36 ). ILC1s have been identified in most human tissues, including the liver, spleen, lung, tonsil, adipose tissue, gastrointestinal tract, and uterus, but adaptations are reported depending on the tissue of residence ( 37 – 39 ). For example, an intraepithelial subset of ILC1s in tonsils and intestinal tract can express NKp44, a natural cytotoxicity receptor (NCR) normally expressed by ILC3s ( 32 , 36 , 40 ). ILC1s have been involved in antiviral mucosal protection in mice. For example, murine ILC1s in the oral mucosa produce IFN-y in the absence of infection, creating an antiviral tissue environment ( 41 ). In another study, lung ILC1s produced IFNG and mediated antiviral responses influenza infection ( 42 ). The role of ILCs in tissue-specific antiviral protection has been excellently reviewed recently ( 43 ). ILC2s secrete IL-4, IL-5, IL-13, IL-9, and amphiregulin and require the transcription factor GATA-3. Overall, ILC2s are less abundant in human tissues than their murine counterparts and are mostly described in the lung and skin under pathological inflammatory conditions and allergies ( 33 , 44 , 45 ). In the homeostatic gut, ILC2s are hardly present and only detectable during severe inflammation ( 46 ). A subset of ILC2s have been described to have regulatory effects similar to regulatory T cells. IL-10 producing ILC2s that suppressed IL-5 producing ILC2s were found in the nasal polyps of patients with chronic rhinosinusitis ( 47 ). Whether these regulatory ILC2s arise in other chronic inflammatory conditions is unknown. Recently, a study illustrated that peripheral ILC2s can secrete granzyme B (GZMB) and directly lyse cells by inducing pyroptosis and apoptosis, illustrating their possible therapeutic role in tumor treatment ( 48 ). Group 3 ILCs produce IL-22 and IL-17, express RORyt, and have been distinctly separated into ILC3s (NCR+ILC3s and NCR-ILC3s) and lymphoid tissue inducer (LTI)-like cells. Murine NCR+ILC3s express NKp46, while human NCR+ILC3s express NKp44 instead. Unlike NKp44+ ILC1s, NKp44+ ILC3s are not always characterized as intraepithelial ( 49 ). Like other ILC subsets, ILC3s have tissue specific phenotypic profiles with ILC3s from mucosal tissues differing greatly in their cell surface markers from ILC3s in the lymph nodes, tonsils, and spleen ( 33 , 50 ). In humans, ILC3s are most frequent in the intestinal mucosa where they mediate mucosal epithelial protection. NKp44+ ILC3s contribute to intestinal barrier integrity through IL-22 secretion, which induces antimicrobial peptide production and tight junction formation by epithelial cells ( 49 , 51 ). NKp44- ILC3s in the intestinal mucosa can also produce IL-17 in response to IL-23, and production of these cytokines is reliant on mTOR complex 1 (mTORC1) metabolic reprogramming signaling ( 52 – 54 ). Current knowledge on ILC3-mediated pathogen responses comes primarily from mouse models, but little is known about human ILC3s. In mice, intestinal ILC3s produce IL-22 mediated by IL-18 in response to C. rodentium infection, which contributes to decreased vulnerability and inflammation ( 55 , 56 ). Additionally, IFN-y+ ILC3s clear Chlamydia muridarum from the murine large intestine, playing an important role in pathogen clearance ( 57 ). In a study of tuberculosis, ILC3s accumulated rapidly in Mycobacterium tuberculosis -infected lungs and coincided with the accumulation of alveolar macrophages. The authors determined that ILC3s were needed for macrophage recruitment, inducible bronchus-associated lymphoid tissues formation, and M. tuberculosis control during early infection ( 58 ). One challenge to identifying and studying ILC3s, specifically in humans, is their plasticity to convert to ILC1s. This is often driven through environmental cues that can become more abundant during infections and tissue damage ( 59 ). However, the implication of mucosal ILC plasticity in response to pathogens remains poorly understood. LTIs are grouped into the ILC3 category as they also express RORyt and similar surface markers such as CCR6 ( 60 ). LTIs or LTI-like cells are involved in formation of secondary lymphoid organs during fetal development in mice, and they produce IL-22 and IL-17 ( 61 ). Unlike other ILC3s, LTIs do not express NKp44 or NKp46 ( 62 ). Currently, it is not known about human LTIs and whether LTIs might be involved in pathogen response at mucosal surfaces.

Although not much is known about the homeostatic or protective roles of ILCs in the female genital tract, ILCs have been shown to play a central role in maintaining epithelial integrity, immune regulation and display direct or indirect antiviral functions in other mucosal tissues such as the gut, oral mucosa, and the lungs. Considering that HIV is primarily transmitted through mucosal exposure during sexual contact, a better understanding of ILC functions and their antiviral potential in the FGT may open new avenues for prevention. However, considering the additional evidence that ILCs may be involved in inflammatory conditions, careful evaluation is needed to unravel the potential beneficial or detrimental contribution of ILCs to HIV pathogenesis. The characterization, description, and functions of FGT ILCs in homeostasis and HIV infection is an open area for further research.

Women represent half of people living with HIV around the world, but in endemic areas women are disproportionately affected ( 1 ). The main mechanism for HIV transmission is sexual contact, which makes the female genital tract (FGT) a major portal of entry for the virus. The immune system in the female genital mucosa is adapted to balance reproduction and defense, resulting in unique functional specialization that differs from other mucosal surfaces and between species ( 2 ). However, the immune system in the human female genital mucosa remains understudied and critical gaps exist in our understanding of HIV pathogenesis ( 3 ). To prevent HIV infection in women, a deeper understanding of this mucosal system is needed to identify protective innate immune mechanisms. One such critical gap is innate lymphoid cells (ILCs). ILCs are innate lymphocytes that lack antigen-specific receptors or lineage specific markers and specialize in cytokine secretion ( 4 ). ILCs are mainly tissue-resident cells where they contribute to tissue homeostasis, barrier protection and defense from pathogens ( 4 ). While ILCs have been studied in mouse models and some human mucosal surfaces, our understanding throughout the female genital tract is very limited. Further, ILCs role(s) in chronic HIV infection has been briefly investigated, but potential contribution of ILCs to HIV acquisition and HIV pathogenesis in the FGT is completely unknown. The purpose of this review is to report recent advances in our understanding of human mucosal ILCs and HIV infection with a focus on the female genital tract and evidence for a role of ILCs in HIV pathogenesis.