Abstract
NK cell functions are tightly regulated by the balance between the inhibitory and stimulatory surface receptors. We investigated the surface expression of galectin-9 (Gal-9) and its function in NK cells from HIV-infected individuals on antiretroviral therapy, long-term nonprogressors, and progressors compared with healthy controls. We also measured the expression of TIGIT and TIM-3 on different NK cell subpopulations and compared their functionality to Gal-9+ NK cells. Our data demonstrated significant upregulation of Gal-9 on NK cells in HIV-infected groups versus healthy controls. Gal-9 expression was associated with impaired expression of cytotoxic effector molecules granzyme B, perforin, and granulysin. In contrast, Gal-9 expression significantly enhanced IFN-γ expression in NK cells of HIV-1–infected individuals. We also found an expansion of TIGIT+ NK cells in HIV-infected individuals; however, dichotomous to Gal-9+ NK cells, TIGIT+ NK cells expressed significantly higher amounts of cytotoxic molecules but lower IFN-γ. Moreover, lower expression of cytotoxic effector molecules in Gal-9+ NK cells was associated with higher CD107a expression, which suggests indiscriminate degranulation. Importantly, a positive correlation between the plasma viral load and Gal-9+ NK cells was observed in progressors. Finally, we found that a cytokine mixture (IL-12, IL-15, and IL-18) can improve effector functions of Gal-9+ NK cells in HIV-infected individuals, although, such an effect was observed for Gal-9− NK cells, as well. Overall, our data highlight the important role of Gal-9 in dysfunctional NK cells and, more importantly, a dichotomy for the role of Gal-9 versus TIGIT and suggest a potential new avenue for the development of therapeutic approaches.
Introduction
NK cells play a crucial role as antiviral effectors of the innate immune system, and their deficiency has been associated with an enhanced susceptibility to viral infections (1). Recent studies have shed light on the adaptive features of NK cells with a memory-like phenotype against pathogens (2), enforcing an even greater role for these cells in chronic conditions such as HIV infection.
HIV-1 infection is associated with changes in the relative proportions of subpopulations, phenotypes, and functions of NK cells as the disease advances. Although acute HIV infection has been associated with a marked increase in the absolute number of CD56dimCD16+ NK cells and a decline in CD56bright NK cells, the ratio of these NK cell subpopulations normalizes in patients on antiretroviral therapy (ART) (3). However, in the setting of untreated HIV with ongoing viral replication, abnormal proportions of the NK cell subpopulations persist that significantly impact the anti-HIV activity of NK cells as a whole (4). Expansion of the CD56−CD16+ NK cell subset, which prevents a decline in the number of cytotoxic NK cells (CD56dim), is one such example (5). The downregulation of CD56 is one of many alterations in NK cell surface markers observed in HIV-1–infected individuals (5). Furthermore, HIV infection of monocytes/macrophages in vitro can induce the dysregulated release of several matrix metalloproteinases (MMPs) such as MMP-9 (6), which cleaves CD16 on NK cells, impeding their ability to kill via Ab-dependent cellular cytotoxicity (7).
Additionally, HIV-1 replication alters the expansion of activating and inhibitory killer-cell Ig-like receptors (KIRs); higher expression of inhibitory KIRs and lower expression of activating KIRs lead to NK cell dysfunction and defective antiviral activity (8). For example, upregulation of the inhibitory KIR2DL3 contributes to an increase in mother-to-child HIV transmission (9), whereas higher expression of activating KIRs, such as KIR3DS1 and its interaction with HLA-Bw4, is associated with slower progression to AIDs (10, 11). NK cells also express a broad range of other receptors defined as the natural cytotoxicity receptors (e.g., NKp30, NKp44, and NKp46) and the C-type lectin receptors (e.g., NKG2A, NKG2C, and NKG2D) (12), and HIV-1 infection is known to alter the expression levels of these activating/inhibitory receptors. In addition, HIV-1 infection impairs cytokine (e.g., IFN-γ and TNF-α) production, which limits NK cell priming and impairs their ability to eliminate HIV-infected CD4+ T cells via NKp46- and NKG2D-mediated mechanisms (13).
Apart from inhibitory KIRs, NK cells express several other o-inhibitory receptors that are postulated to impair NK cell function, such as programmed cell death protein 1 (PD-1), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and T cell Ig and mucin domain 3 (TIM-3) (14, 15). The role of these coinhibitory receptors in T cell exhaustion in the context of chronic viral infections and cancer is extensively documented (16–19). Although transient upregulation of these coinhibitory receptors is required for immune homeostasis, their persistent expression is associated with defective T cells and NK cell effector functions (14, 18). For instance, upregulation of PD-1 on NK cells results in decreased degranulation capacity and IFN-γ expression (20), and overexpression of TIGIT on NK cells is associated with impaired cytolytic activity (21, 22) and lower IFN-γ expression (23). In line with these observations, a recent study investigating NK cell TIGIT expression in a cancer model showed that TIGIT blockade restored NK cell degranulation and cytokine production capabilities (24). Unlike TIGIT and PD-1, the role of TIM-3 in NK cell function is not well defined. TIM-3 is reported to be a marker of NK cell maturation because it gets upregulated following NK cell stimulation, and NK cells expressing TIM-3 have higher degranulation and cytokine production (25). However, other studies have reported TIM-3 as an NK cell exhaustion marker and poor prognostic factor in patients with melanoma and lung cancers (26, 27). On the contrary, stimulation of TIM-3 with its ligand galectin-9 (Gal-9) can increase the cytotoxic ability of NK cells as assessed by the levels of IFN-γ expression (28). Given the nonselective binding of TIM-3 and binding to multiple ligands, the different effects of TIM-3 are postulated to be dependent on the density and type of ligand associating with TIM-3 (15). Nonetheless, the role of TIM-3 as an inhibitory receptor on NK cells requires further investigation.
Gal-9 is a β-galactoside–binding lectin that is ubiquitously expressed intracellularly in many tissues as well as various immune cells (29). There are multiple reported receptors for Gal-9, including protein disulfide isomerase (PDI), CD44, CD137, TIM-3, and IgE, highlighting its potential versatile roles in different physiological and pathological conditions (29–31). Initially discovered as an eosinophil chemoattractant, Gal-9 has a wide range of immunomodulatory roles, including cell adhesion, migration, and apoptosis (29, 32). Moreover, Gal-9 is made on free ribosomes, and it is secreted nonclassically as it does not contain an endoplasmic reticulum signaling sequence (32, 33). We and others have shown increased soluble Gal-9 levels in the plasma of HIV-infected individuals (34, 35). In addition, we have reported that recombinant Gal-9 (rGal-9) prevents HIV-1 replication by interacting with TIM-3 on activated CD4+ T cells (34). In line with our observations, others have reported that rGal-9 by upregulation of APOBEC3G reduces the infectivity of HIV in CD4+ T cells (36). Contrary to these reports, rGal-9 contributes to promote HIV cell entry by modulating the T cell surface reduction-oxidation state through interactions with PDI (30, 34).
We and others have shown that regulatory T cells also express Gal-9 (17, 37), which interacts with TIM-3 to suppresses Ag-specific CD8+ T cell effector functions (16). By binding TIM-3, rGal-9 enhances cytokine production and degranulation of primed NK cells (28). In contrast, another study reported that rGal-9 impairs NK cell functions by downregulating multiple genes associated with cell-mediated cytotoxicity and decreases IFN-γ production (38). These reports indicate that Gal-9 plays an important role in HIV pathogenesis. Therefore, we aimed to study the role of surface Gal-9 expression on NK cells in HIV patients. We hypothesized that HIV-1 infection may enhance Gal-9 expression on NK cells, which impairs their functionality. We determined the expression of surface Gal-9 on NK cells in different HIV-infected patient populations (those on suppressive ART, long-term nonprogressors [LTNPs] not on ART, and HIV progressors [Ps] not on ART) and compared them with HIV-uninfected healthy controls (HCs). In addition, the functionality of Gal-9+ versus Gal-9− NK cells was investigated by examining the expression of perforin, granzyme B (GzmB), granulysin (GNLY), and IFN-γ. Moreover, we evaluated the expression levels of other important coinhibitory receptors (TIGIT and TIM-3) on NK cells and compared their functionality to Gal-9+ NK cells. Finally, the impact of cytokine mixture stimulation on the functionality of Gal-9+ versus Gal-9− NK cells was studied.
Materials and Methods
Study participants
The study cohort was composed of four groups for a total of 141 subjects: 34 HIV seronegative HCs; 72 HIV patients currently undergoing ART; 15 LTNPs, which are individuals who have been infected with HIV for more than 11 y, have a CD4 count of >400 cells/μl blood, and a plasma viral load of either undetectable or <10,000 copies/ml in the absence of ART; and 20 Ps, who have a CD4 count of <400 cells/μl blood and a plasma viral load >10,000 copies/ml in the absence of ART.
Ethics statement
This study was approved by the institutional research review boards at the University of Alberta, and written informed consent was obtained from all the participants in the study (protocol numbers Pro000046064 and Pro000070528). In addition, frozen PBMCs for the Ps group were obtained from the Center for AIDS Research–University of Washington, Seattle.
Human sample collection and processing
PBMCs were isolated from the fresh blood using Ficoll-Paque gradients and cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich). In some studies, total NK cells were negatively isolated from fresh PBMCs according to the manufacturing instructions (STEMCELL Technologies), with a purity exceeding 95% (Supplemental Fig. 1A).
Flow cytometry analysis
Fluorophore-conjugated Abs with specificity against human cell Ags and cytokines were purchased from BD Biosciences, Thermo Fisher Scientific, or BioLegend. The used Abs were as follows: anti–Gal-9 (9M1-3), anti-TIGIT (MBSA43), anti–TIM-3 (7D3), anti-CD3 (HIT3a), anti-CD16 (eBioCB16, 3G8, B73.1), anti-CD56 (CMSSB, B159), anti-CD14 (M5E2), anti-CD19 (HIB19), anti-GzmB (GB11), anti-perforin (dG9), anti-GNLY (RB1), anti–IFN-γ (B27), anti-CD107a (H4A3), anti-NKG2D (1D11), anti-NKp30 (p30-15), anti-NKp44 (P44-8), CD44 (G44-26), CD137 (4B4-1), and anti-NKp46 (9-E2). LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (L34966; Thermo Fisher Scientific) was used to exclude the dead cells. Apoptotic assay was performed using Annexin V (BD Biosciences). Cells after fixation with paraformaldehyde (2%) were acquired using Fortessa-X20 or BD LSR Fortessa-SORP (BD Biosciences). All data were analyzed using the FlowJo Software (version 10).
Image stream cytometry
ImageStream analysis was performed on surface-stained PBMCs that were fixed with 4% paraformaldehyde. Over 15,000 images were collected using Amnis ImageStream Mark II (EMD Millipore Sigma). The analysis was performed by choosing an aspect ratio >0.8, using cells that were in focus, and gating for NK cells (CD3−, CD56+/−, CD16+/−). Afterwards, the Gal-9+ population was gated on total NK cells.
Cell culture studies
In some studies, total PBMCs were stimulated with a cytokine mixture containing IL-12 (10 ng/ml), IL-15 (20 ng/ml), and IL-18 (100 ng/ml). Stimulated and unstimulated PBMCs with cytokines were cultured for 24 h with 5% CO2 in the presence of anti-CD107a (563869; BD Biosciences). At the 20-h mark, GolgiStop (BD Biosciences) was added, and 4 h later, cells were subjected to surface and intracellular staining. In other experiments, PBMCs were cultured for 24 h in the presence or absence of lactose (30 mM), after which, they were subjected to staining for GzmB, perforin, CD107a, and IFN-γ. Other than these situations, we measured the expression of inhibitory receptors, GzmB, perforin, GNLY, and IFN-γ on fresh cells.
Gene expression analysis
RNA was extracted from the enriched NK cells obtained from the HCs and HIV-infected individuals on ART. The resulting cDNA (5 ng/μl) was used as a template for TaqMan quantitative PCR (Applied Biosystems) with the gene expression probe assays Lgals9 (QT00014273; Qiagen) and β2-macroglobulin (QT00088935; Qiagen) as a housekeeping gene. The mRNA from HCs was used as a reference group for the fold change calculations, in which the gene expression of the targeted genes was calculated by the 2−ΔΔcycle threshold method.
Statistical analysis
The p values displayed in cumulative flow cytometry plots were determined by nonparametric Mann–Whitney U test. When more than two groups were compared, one-way ANOVA followed by Tukey test was used to compare the results. Prism Software was used for statistical analysis. Results are presented as mean ± SEM with a p value <0.05 being considered as statistically significant.
Results
Frequency of NK cell subpopulations in different HIV-1–infected groups
To determine possible changes in the NK cell frequency in HIV-infected individuals, we analyzed NK cell subpopulations in different HIV-infected individuals (on ART [72 patients], Ps [20 patients], and LTNPs [15 patients] compared with HCs [34 individuals]). For consistency throughout the study, we decided to divide NK cells into three subpopulations (CD16+CD56−, CD16+CD56+, and CD56+CD16−) as shown in Fig. 1A. Although NK cells can be further subdivided into other subpopulations (e.g., CD56dim, CD56bright), it was difficult to quantify expression of inhibitory receptors on such small subpopulations. Therefore, the gating strategy of NK cells was adopted from other reports (23, 39) as shown in Supplemental Fig. 1B. Based on these criteria, we found that the percentages of CD56+ NK cells were significantly increased in HIV-1–infected individuals on ART, Ps, and LTNPs compared with HCs (Fig. 1B). In contrast, the percentages of CD56+CD16+ NK cells were significantly lowered in Ps and LTNPs compared with HCs (Fig. 1B). In addition, percentages of CD56+CD16+ NK cells were significantly declined in Ps versus LTNPs and HCs. In a way, ART maintained the frequency of CD56+CD16+ NK cells as there was no difference in their frequency compared with HCs (Fig. 1B). Furthermore, we observed a significant reduction in the frequency of CD16+ subpopulation in patients on ART compared with HCs and Ps, whereas the percentages of CD16+ NK cells were similar in HCs and LTNPs (Fig. 1B).
Frequency of NK cell subpopulations and expression levels of Gal-9, TIGIT, and TIM-3 on NK cells in different HIV-1–infected groups.
(A) Representative plots of NK cell subpopulations (CD16+, CD56+CD16+, and CD16+) in patient on ART, a P, an LTNP, and an HC. (B) Cumulative data showing the percentages of NK cell subpopulations in HIV patients on ART, Ps, LTNPs, and HCs. (C) Representative plots showing expression of Gal-9, TIGIT, and TIM-3 on NK cells. (D) Cumulative data showing percentages of CD56+ NK cells expressing either Gal-9, TIGIT, or TIM-3. (E) Cumulative data showing percentages of CD56+CD16+ NK cells expressing either Gal-9, TIGIT, or TIM-3. (F) Cumulative data showing percentages of CD16+ NK cells expressing either Gal-9, TIGIT, or TIM-3. Error bars indicate mean ± SEM, and each point represents an individual.
The expression pattern of Gal-9, TIGIT, and TIM-3 on NK cell subpopulations in HIV-infected individuals on ART versus HCs
Then we measured the surface expression of Gal-9, TIGIT, and TIM-3 on NK cells in different HIV-infected individuals. We observed that Gal-9 was significantly upregulated on the surface of CD56+ and CD56+CD16+ but without any changes in CD16+ NK cells in HIV-infected individuals on ART (Fig. 1C–F). TIGIT was significantly overexpressed on CD56+ NK cells but remained similar to the levels that were seen in HCs for CD56+CD16+ and CD16+ NK cells in this HIV-infected group (Fig. 1C–F). Interestingly, the percentages of TIM-3+CD56+ NK cells were significantly decreased in HIV-infected individuals on ART compared with HCs; however, the frequency of TIM-3–expressing CD56+CD16+ and CD16+ NK cells remained unchanged in HIV-infected individuals on ART (Fig. 1C–F).
The expression pattern of Gal-9, TIGIT, and TIM-3 on NK cell subpopulations in HIV-infected Ps and LTNPs versus HCs
The frequency of NK cell subpopulations in Ps and LTNPs was compared with HCs. As shown in Fig. 2A, 2B, the percentages of CD56+ NK cells were significantly higher in both Ps and LTNPs compared with HCs, but percentages of CD56+CD16+ NK cells were significantly lower in Ps and LTNPs versus HCs. However, no significant difference in the frequency of CD16+ NK cells was noted between these three groups (Fig. 2A, 2B). We further analyzed the expression levels of Gal-9, TIGIT, and TIM-3 on NK cell subpopulations in Ps compared with HCs. Other than a significant upregulation of Gal-9 on CD56+ and CD56+CD16+ NK cells compared with HCs, no substantial changes in TIGIT or TIM-3 expression levels were noted on NK cell subpopulations (CD56+, CD56+CD16+, and CD16+) in Ps versus HCs (Fig. 2C–F). Notably, the pattern of expression for Gal-9, TIGIT, and TIM-3 on NK cells from LTNPs was different than other patient subgroups: 1) CD56+ NK cells had significantly higher levels of Gal-9 and TIGIT compared with HCs (Fig. 2G) and 2) CD56+CD16+ NK cells had elevated levels of Gal-9, TIGIT, and TIM-3 compared with HCs (Fig. 2H). Although there was no difference in the frequency of Gal-9+CD16+ cells, we found significantly higher percentages of TIGIT+CD16+ and TIM-3+CD16+NK cells in LTNPs versus HCs (Fig. 2I). These observations demonstrate a unique phenotype for NK cell subpopulations in Ps versus LTNPs.
The frequency of NK cell subpopulations and expression pattern of Gal-9, TIGIT, and TIM-3 on NK cell subpopulations in HIV-infected Ps and LTNPs versus HCs.
(A) Cumulative data showing percentages of CD56+, CD56+CD16+, and CD16+ NK cells in Ps versus HCs. (B) Cumulative data showing percentages of CD56+, CD56+CD16+, and CD16+ NK cells in LTNPs versus HCs. (C) Representative plots of Gal-9, TIGIT, and TIM-3 expression on NK cells in a P versus an HC. (D) Cumulative data showing percentages of Gal-9+–, TIGIT+-, and TIM-3+–expressing CD56+, (E) CD56+CD16+, and (F) CD16+ NK cells in HCs versus Ps. (G) Cumulative data showing percentages of Gal-9+–, TIGIT+-, and TIM-3+–expressing CD56+, (H) CD56+CD16+, and (I) CD16+ NK cells in HCs versus LTNPs.
Next, we reconfirmed Gal-9 expression on different NK cell subpopulations by using ImageStream (Supplemental Fig. 1C). In addition, we measured Gal-9 mRNA in total NK cells from HIV-infected individuals on ART versus HCs and noted no significant difference in Gal-9 mRNA levels in NK cells from HIV patients versus HCs (Supplemental Fig. 1D).
Gal-9+ compared with Gal-9− NK cells express significantly lower levels of GzmB and perforin, which is in contrast to TIGIT+ NK cells
Next, we investigated the effector functions of NK cells in regard to the surface expression of Gal-9, TIGIT, and TIM-3 in HIV-infected individuals on ART. We found that overexpression of Gal-9 was associated with impaired GzmB expression in different NK cell subpopulations (CD56+, CD56+CD16+, and CD16+) (Fig. 3A–D). In contrast, overexpression of TIGIT on NK cells was associated with significantly higher GzmB expression capacity (Fig. 3A–D). The expression of TIM-3 was not associated with any significant impact on GzmB expression ability in CD56+ and CD16+ NK cells but was associated with a decline in GzmB expression in only CD56+CD16+ NK cells (Fig. 3B–D).
Expression of GzmB in Gal-9+, TIGIT+, and TIM-3+ NK cell subpopulations compared with their negative counterparts in different HIV-infected groups.
(A) Representative plots of GzmB expression in Gal-9+/Gal-9− versus TIGIT+/TIGIT− NK cells. (B) Cumulative data comparing GzmB expression in Gal-9+/Gal-9−, TIGIT+/TIGIT−, and TIM-3+/TIM-3− among CD56+, (C) CD56+CD16+, and (D) CD16+ NK cells from HIV-infected individuals on ART. (E) Cumulative data showing GzmB expression among CD56+ NK cells in regard to the expression of Gal-9, TIGIT, and TIM-3 in HIV-infected individuals of ART, Ps, and LTNPs.
We then compared the effects of Gal-9, TIGIT, and TIM-3 expression on NK cell function in all HIV-infected groups (ART treated, Ps, LTNPs). In all patients, we observed that overexpression of Gal-9 was associated with a significant decline in GzmB expression in CD56+, CD56+CD16+, and CD16+ NK cells (Fig. 3E, Supplemental Fig. 1E, 1F). In contrast, overexpression of TIGIT was associated with enhanced GzmB production ability (Fig. 3E, Supplemental Fig. 1E, 1F). We did not find any significant difference in GzmB expression in TIM-3+ and TIM-3− NK cells (CD56+, CD16+CD56+, and CD16+) in HIV-infected individuals on ART (Fig. 3E, Supplemental Fig. 1E, 1F), but in CD16+CD56+ NK cells from Ps and LTNPs, the expression of TIM-3 was associated with significantly higher levels of GzmB expression (Fig. 3E). However, TIM-3 levels were not associated with GzmB expression in CD56+ and CD16+ NK cells from Ps and LTNPs (Supplemental Fig. 1E, 1F).
Similar to GzmB, we found that overexpression of Gal-9 on NK cells (CD56+, CD56+CD16+, and CD16+), from HIV-infected individuals on ART, was associated with impaired perforin expression (Fig. 4A–D), whereas the opposite was observed for TIGIT expression (Fig. 4A–D). Although TIM-3 was not associated with perforin expression in CD56+ and CD16+ NK cells, TIM-3 expression levels did correlate with increased perforin expression in CD56+CD16+ NK cells in HIV-infected individuals on ART (Fig. 4A–D).
Expression of perforin in Gal-9+, TIGIT+, and TIM-3+ NK cell subpopulations compared with their negative counterparts in different HIV-infected groups.
(A) Representative plots of perforin expression in Gal-9+/Gal-9− versus TIGIT+/TIGIT− NK cells. (B) Cumulative data comparing perforin expression in Gal-9+/Gal-9−, TIGIT+/TIGIT−, and TIM-3+/TIM-3− among CD56+, (C) CD56+CD16+, and (D) CD16+ NK cells from HIV-infected individuals on ART. (E) Cumulative data showing perforin expression among CD56+CD16+ NK cells in regard to the expression of Gal-9, TIGIT, and TIM-3 in HIV-infected individuals of ART, Ps, and LTNPs.
Similar to GzmB, we found that in all HIV-infected groups (ART treated, Ps, or LTNPs), overexpression of Gal-9 was associated with a significant decline in perforin expression in all NK cell subpopulations (Fig. 4E, Supplemental Figs. 1G, 2A), but overexpression of TIGIT was associated with significantly greater perforin expression (Fig. 4E, Supplemental Figs. 1G, 2A). The effects of TIM-3 expression on different NK cell subpopulations was not consistent; we found low TIM-3 expression was associated with reduced perforin expression in NK cells from only LTNPs but not Ps or HIV patients on ART (Fig. 4E, Supplemental Figs. 1G, 2A). Therefore, our data indicate an interesting dichotomy for Gal-9+– versus TIGIT-expressing NK cells in HIV-infected individuals.
Impaired cytolytic activity of Gal-9+ NK cells in HIV-infected individuals
Coordinated action of perforin and GzmB is a requirement for optimal NK cell–mediated cytotoxicity (40). Therefore, we decided to determine how the expression of Gal-9 and TIGIT correlated with the coexpression of GzmB and perforin in different NK cell subpopulations in HIV-infected individuals. We observed that expression of Gal-9 was associated with a significant decline in coexpression of GzmB and perforin in all NK cell subpopulations (Fig. 5A–D). In sharp contrast, TIGIT expression on NK cells was associated with a significant increase in coexpression of GzmB and perforin in NK cells (Fig. 5A–D). The effects of TIM-3 expression on coexpression of these cytolytic molecules was negligible (data not shown). We further analyzed for the coexpression of TIGIT, Gal-9, and TIM-3 on NK cells but, surprisingly, we did not observe any substantial coexpression for these inhibitory molecules on NK cells (Fig. 5E, 5F). These observations indicated heterogeneity of NK cells in HIV-infected individuals.
Impaired cytolytic activity of Gal-9+ NK cells.
(A) Representative plots of GzmB/perforin expression in Gal-9+/Gal-9− versus TIGIT+/TIGIT− NK cells. (B) Cumulative data showing coexpression of GzmB and perforin in Gal-9+/Gal-9− versus TIGIT+/TIGIT− among CD56+, (C) CD56+CD16+, and (D) CD16+ NK cells from HIV patients on ART. (E) Representative plots of Gal-9/TIGIT and (F) Gal-9/TIM-3 coexpression in NK cells of an HIV-infected individual. (G) Representative plots of GNLY expression in Gal-9+ versus Gal-9− NK cells and (H) TIGIT+ versus TIGIT− NK cells of an HIV-infected individual. (I) Representative plots of CD107a expression in Gal-9+ versus TIGIT+ NK cells of an HIV-infected individual. (J) Cumulative data showing CD107a expression in Gal-9+/Gal-9− versus TIGIT+/TIGIT− NK cells of HIV patients on ART. (K) Showing the correlation of plasma viral load with the percentages of Gal-9+CD56+ NK cells from Ps. Each point represents data from an individual patient and multiple experiments.
We next investigated the expression of GNLY, which exhibits lytic and cytolytic activities against a wide range of microorganisms and tumor cells (41), and observed a similar pattern to that demonstrated for GzmB and perforin. As shown in Fig. 5G, Supplemental Fig. 2B–D, Gal-9 expression on NK cells was associated with a significant impairment in GNLY expression, whereas the opposite was the case for TIGIT-expressing NK cells (Fig. 5H, Supplemental Fig. 2B–D).
Finally, we sought to determine whether Gal-9+ NK cells produce fewer cytotoxic molecules compared with Gal-9− NK cells or if this reduction is because of constant degranulation. To do this, we investigated the degranulation ability of Gal-9+ versus Gal-9− TIGIT+ versus TIGIT− NK cells by evaluating the expression of lysosomal-associated membrane protein 1 (LAMP1 or CD107a). We found that Gal-9+ NK cells at the baseline had significantly higher levels of CD107a expression compared with Gal-9− and even TIGIT+ NK cells (Fig. 5I, 5J). These observations demonstrate a dichotomy for Gal-9+– versus TIGIT-expressing NK cells and suggest a role for Gal-9 in the impaired lytic and cytotoxic functions of NK cells in HIV-infected individuals. Interestingly, we observed a positive correlation between the plasma viral load and the percentages of CD56+Gal-9+ NK cells in Ps (Fig. 5K); however, this was not the case for LTNPs and ART-treated individuals (data not shown) because of undetectable or very low viral loads. On that note, we did not find such correlation with the percentages of TIGIT+ NK cells in different groups (data not shown).
Gal-9+ compared with Gal-9− NK cells express significantly higher levels of IFN-γ, which is in contrast to TIGIT+ NK cells
NK cells are capable of producing IFN-γ, and the regulation of NK cell IFN-γ production can be modified in the course of chronic conditions (42). We observed that expression of Gal-9 upregulated IFN-γ production in NK cells such that Gal-9+ NK cells (CD56+, CD56+CD16+, and CD16+ subpopulations) produced significantly higher levels of this cytokine compared with their Gal-9− counterparts in HIV-infected individuals on ART (Fig. 6A–C, Supplemental Fig. 2E). Although Gal-9+ CD56+ NK cells from LTNPs and Ps also exhibited significantly higher levels of IFN-γ, this was not the case for CD56+CD16+ NK cells (Fig. 6B, 6C). Additionally, Gal-9+CD16+ NK cells from LTNPs, but not Ps, expressed significantly higher levels of IFN-γ production (Supplemental Fig. 2E). This suggests differential effects of Gal-9–induced IFN-γ expression on NK subpopulations depending on the status of HIV-infected individuals.
Gal-9+ compared with Gal-9− NK cells express significantly higher levels of IFN-γ, which is in contrast to TIGIT+ NK cells.
(A) Representative plots of IFN-γ expression in Gal-9+/Gal-9− versus TIGIT+/TIGIT− NK cells of an HIV-infected individual on ART. (B) Cumulative data showing IFN-γ expression among CD56+ NK cells in regard to the expression of Gal-9, TIGIT, and TIM-3 in HIV-infected individuals on ART, Ps, and LTNPs. (C) Cumulative data showing IFN-γ expression among CD56+CD16+ NK cells in regard to the expression of Gal-9, TIGIT, and TIM-3 in HIV-infected individuals on ART, Ps, and LTNPs.
In contrast, expression of TIGIT was associated with a decline in IFN-γ production ability of different NK cell subpopulations obtained from HIV-infected individuals on ART (Fig. 6A–C). TIGIT did not impact IFN-γ expression in all three NK cell subpopulations from LTNPs and Ps except for CD56+ NK cells in Ps, which was associated with a reduction in IFN-γ production in this group of patients (Fig. 6B, 6C, Supplemental Fig. 2E). In ART-treated patients, TIM-3+ NK cells (both CD56+ and CD16+CD56+) expressed significantly higher IFN-γ levels, whereas this was not the case for CD16+ NK cells (Fig. 6B, 6C, Supplemental Fig. 2E). TIM-3 expression did not affect the IFN-γ production in most NK cell subpopulations in LTNPs and Ps, except for CD16+CD56+ NK cells in Ps, where it was associated with a significantly higher IFN-γ expression (Fig. 6B, 6C, Supplemental Fig. 2E). Our data once again highlight a dichotomy for Gal-9+ and TIGIT+ NK cells in terms of IFN-γ production in the context of HIV infection, whereas the effects of TIM-3 varies depending on the NK cell subpopulations and HIV-infected groups.
Gal-9–mediated impaired NK cell effector functions do not require cell–cell interactions
To determine whether impaired Gal-9+ NK cell effector functions occurred via interactions with TIM-3 or other potential Gal-9 receptors, we blocked Gal-9 using lactose, as we have reported elsewhere (16, 34, 43). Gal-9 blockade did not have any significant impact on NK GzmB, perforin, and CD107a expression (Fig. 7A–C). Similar observations were obtained for IFN-γ production in Gal-9+ versus Gal-9− NK cells (data not shown). These data suggest that Gal-9 expression might be associated with an intrinsic signal rather than cell–cell interactions (e.g., Gal-9:TIM-3). To investigate whether the impaired functionality of NK cells can be improved in vitro, we stimulated NK cells with a cytokine mixture (IL-12, IL-15, and IL-18). We observed that this cytokine mixture significantly enhanced effector functions of Gal-9+ NK cells in terms of GzmB, perforin, IFN-γ production, and degranulation (CD107a) but also significantly increased the activity of Gal-9− NK cells (Fig. 7D–G). These observations demonstrate that the dysfunctional NK cell phenotype mediated by Gal-9 expression is partially reversible. In addition, we investigated whether expression of Gal-9 is associated with apoptosis of NK cells. Although Gal-9 expression has been associated with apoptosis in different cell types (44), we did not find any significant difference between Gal-9+ versus Gal-9− NK cells as measured by Annexin V (Supplemental Fig. 2F, 2G).
Gal-9–mediated impaired NK cell effector functions do not require cell–cell interactions, and Gal-9 associated impairment is reversible by cytokine mixture.
(A) Cumulative data showing expression of GzmB among total Gal-9+/Gal-9− NK cells in the presence or absence of lactose (30 nM). (B) Cumulative data showing expression of perforin among total Gal-9+/Gal-9− NK cells in the presence or absence of lactose (30 nM). (C) Cumulative data showing expression of CD107a among total Gal-9+/Gal-9− NK cells in the presence or absence of lactose (30 nM). (D) Cumulative data showing GzmB expression in total Gal-9+ versus Gal-9− NK cells in the presence or absence of the cytokine mixture (IL-12 [10 ng/ml], IL-15 [20 ng/ml], and IL-18 [100 ng/ml]). (E) Cumulative data showing perforin expression in total Gal-9+ versus Gal-9− NK cells in the presence or absence of the cytokine mixture. (F) Cumulative data showing IFN-γ expression in total Gal-9+ versus Gal-9− NK cells in the presence or absence of the cytokine mixture. (G) Cumulative data showing CD107a expression in total Gal-9+ versus Gal-9− NK cells in the presence or absence of the cytokine mixture. Each point represents data from an individual patient and multiple experiments. (H) Representative plots showing coexpression of Gal-9+ NK cells with CD44 or CD137.
Gal-9 interacts with CD44 on NK cells
Considering the wide range of immunomodulatory functions by Gal-9 in various immune cells, we decided to determine its potential target molecule on NK cells. There is strong evidence that Gal-9 interacts with TIM-3 (16, 34, 45), but it is obvious that Gal-9 has other membrane receptors, such as PDI, CD137, and CD44 (30, 46, 47). We found that Gal-9 mainly coexpressed with CD44 but not with CD137 on NK cells (Fig. 7H). This suggests that the phenotypic characterization of Gal-9+ NK cells could be the result of Gal-9 binding to CD44.
Expression levels of activating receptors on Gal-9+ versus Gal-9− NK cells
To investigate differential activation status of Gal-9+ versus Gal-9− NK cells, we measured the surface expression of NKG2D, NKp30, NKp40, and NKp46. We found no significant difference in the expression of activating receptors on Gal-9+ versus Gal-9− NK cells except for lower expression of NKp46 on Gal-9+ NK cells in HIV-infected individuals on ART (Fig. 8A–C). In addition, the surface expression of activation marker CD38 on Gal-9+ versus Gal-9− NK cells was measured on NK cells. CD38 is involved in the lytic machinery of NK cells and functions as cytotoxic triggers on NK cells (48). Although no significant difference was noted in the expression levels of CD38 on Gal-9+ versus Gal-9− NK cells in patients on ART (Fig. 8D–F) and LTNPs (Fig. 8G), we found a significant reduction in CD38 expression on CD56+CD16+ and CD16+NK cells in Ps (Fig. 8H). We also analyzed the expression levels of CD69 on different NK cell subpopulations, but no significant difference was observed (data not shown). Moreover, we did not observe any significant difference in the expression levels of NKG2D, NKp30, NKp40, NKp44, CD38, and CD69 on TIGIT+ versus TIGIT− NK cells in HIV-infected individuals (data not shown).
Expression pattern of activating receptors on Gal-9+ versus Gal-9− NK cells.
(A) Cumulative data showing expression of NKG2D, NKp30, NKp44, and NK046 in Gal-9+ versus Gal-9− among CD56+ NK cells. (B) Cumulative data showing expression of NKG2D, NKp30, NKp44, and NK046 in Gal-9+ versus Gal-9− among CD56+CD16+ NK cells. (C) Cumulative data showing expression of NKG2D, NKp30, NKp44, and NK046 in Gal-9+ versus Gal-9− among CD16+ NK cells of HIV-infected individuals on ART. (D) Cumulative data showing expression of CD38 in Gal-9+/Gal-9− versus TIGIT+/TIGIT− among CD56+, (E) CD56+CD16+, and (F) CD16+ NK cells from HIV-infected individuals on ART. Each point represents data from an individual patient and multiple experiments. (G) Cumulative data showing expression of CD38 (in percentages) on Gal-9+ versus Gal-9− NK cells subpopulations in LTNPs and (H) Ps.
Discussion
NK cells play a pivotal role in eradication of HIV-infected cells because their activity corresponds both to resistance to HIV infection and negatively to the progression to AIDS (49). One cohort of HIV-exposed but uninfected i.v. drug users were found to have enhanced NK cell cytolytic and cytokine production capabilities in comparison with those who underwent seroconversion (50). Therefore, understanding how HIV-1 infection impacts the reconstitution and function of the heterogenous NK cell pool is important. In this study, we observed that HIV-infected individuals on ART differ in their repertoire of peripheral blood NK cells, with an increase in their CD56+ subset and a decrease in the CD16+ subset. Previous studies indicated that CD56+ NK cells are more-immature precursors compared with the CD16+ subset based on the length of their telomeres and their ability to differentiate into CD56dim/CD16+ NK cells upon activation (51). Importantly, these two subsets have distinct functional properties; the CD56+ subpopulation is more involved in cytokine secretion, whereas the CD16+ subpopulation is more cytolytic because of its ability to function in Ab-dependent cellular cytotoxicity killing and higher perforin and granzymes content (51). In this study, we observed a shift in the NK cell population from a mature and cytotoxic subset (CD16+) to a more-immature (CD56+) population in HIV patients on ART. It is important to note that in HCs, >90% of peripheral blood NK cells consist of CD56dim/CD16+ and <10% comprise the CD56bright/CD56+ subpopulation (52). This ratio is reversed in HIV patients on ART, with the resulting loss of CD56dim/CD16+ cells leading to a decreased ability to kill target cells and failure to upregulate CD107a and IFN-γ upon stimulation with MHC-devoid K562 target cells (3).
In addition to quantitative changes in NK cell populations mentioned above, we observed an overexpression of surface Gal-9 on NK cells. Specifically, Gal-9 expression was significantly elevated on NK cells in all HIV-infected individuals (on ART, Ps, and LTNPs) on both the CD56+ and CD56+CD16+ subpopulations. Whether the enhanced Gal-9 expression seen predominantly on more-immature NK cells is playing a role in the inhibition of NK cell maturation merits further investigations. Within the HIV cohort, the only difference in Gal-9 expression was in the CD56+CD16+ population, in which the Ps expressed significantly higher Gal-9 compared with the LTNPs. Given the fact that a positive correlation between the viral load and plasma Gal-9 has been reported (34), higher viremia in Ps may be contributing to the higher surface expression of Gal-9 on NK cells. In agreement, we observed a correlation between the plasma viral load and Gal-9+CD56+ NK cells in Ps. However, the majority of HIV-infected individuals on ART, despite having undetectable viral load, also had high surface Gal-9 expression on their NK cells. Therefore, there might be other mechanisms associated with the elevated Gal-9 expression on NK cells in this population, such as immune activation, which is subsequent to the viral persistence and disease chronicity (53). Upregulation of surface Gal-9 expression on CD56+ and CD56+CD16+ NK cells in different HIV-infected subpopulations excludes the effects of ART on Gal-9 surface expression.
In agreement with a recent report (23), we observed upregulation of TIGIT on NK cells in HIV-infected individuals. In particular we found TIGIT overexpression on CD56+ subpopulation in all HIV-infected groups (e.g., on ART, LTNPs, and Ps) compared with HCs. TIGIT has been reported as a key exhaustion marker on NK cells in cancer (24); therefore, it is not surprising to find it overexpressed on NK cells in chronic conditions, such as HIV infection. Furthermore, upregulation of its ligand, CD155, has been reported to be increased on CD4 T cells in HIV-infected patients (23), enforcing its inhibitory role on NK cell functions. Surprisingly, we observed significantly higher expression of TIGIT on CD16+CD56+ and CD16+ NK cells in LTNPs compared with those on ART, Ps, and HCs. The fact that LTNPs had higher levels of TIGIT than Ps is counterinitiative because LTNPs are spared from disease progression.
Moreover, consistent with a previous study, we found significantly lower TIM-3 expression on CD56+ subpopulation NK cells in HIV patients on ART than HCs (54). Similarly, diminished TIM-3 expression on CD56+ NK cells following ART has been reported (55). Although the role of TIM-3 on NK cells is controversial, several reports indicated that TIM-3 contributes to NK cell activity (25, 28). For instance, high plasma viral load with concurrent chronic inflammation has been associated with NK cell activation and higher TIM-3 expression (56). Nevertheless, this does not explain our observations that NK cells (CD56+CD16+ and CD16+) from LTNPs, despite their low to undetectable viral load, exhibited higher TIM-3 expression compared with those on ART, Ps, and HCs. This discrepancy might be due to the impact of ART on the downregulation of TIM-3 expression or the chronicity of the infection.
It was striking that Gal-9– and TIGIT-expressing NK cells displayed distinct and opposing effector functions. Gal-9+ NK cells, regardless of the HIV-infected subpopulations (e.g., ART treated, Ps, and LTNPs), exhibited impaired cytolytic activity compared with Gal-9− NK cells. In contrast, these Gal-9+ NK cells exhibited greater IFN-γ production compared with their negative counterparts (Fig. 9). Our observations suggest that lower cytolytic granules in Gal-9+ NK cells may impair their ability to kill HIV-1–infected target cells because high granule content and coexpression of GzmB and perforin is essential for NK cell–mediated cytotoxicity (57). In contrast, TIGIT+ NK cells had significantly higher levels of granule contents compared with their TIGIT− siblings and lower IFN-γ than TIGIT− NK cells. Although low IFN-γ expression by TIGIT+ NK cells from HIV-infected patients has been reported (15), it is surprising that NK cells that express TIGIT, the exhaustion marker, exhibit higher expression of cytotoxic mediators than their negative counterparts. However, regulation of NK cells is dependent on an array of activating and inhibitory receptors, and there might be other unexplored activating receptors on the TIGIT+ NK cells that contribute to the increased observed functional phenotype. Our overall observations indicate the presence of two distinct NK cells populations, Gal-9+ and TIGIT+ cells, with dichotomous functionality; Gal-9 expression is associated with impaired cytolytic function but enhanced IFN-γ production, whereas effector functions are reversed for TIGIT-expressing NK cells.
Differential effector functions of Gal-9+ versus Gal-9− NK cells.
HIV infection results in the upregulation of Gal-9 on NK cells. Upregulation of Gal-9 on NK cells is associated with impaired GzmB, perforin, and GNLY expression but enhanced IFN-γ production.
It is worth noting that Gal-9 expression may result in indiscriminate degranulation, as was observed by higher CD107a in Gal-9+ versus Gal-9− NK cells. This raises the possibility that lower cytotoxic contents in Gal-9+ NK cells might be due to their constant degranulation rather than having lower granules content. Why these same cells do not express less IFN-γ is puzzling but may be because they constantly degranulate cytotoxic molecules but not cytokines (58).
Our results do not support a definite role for TIM-3 on NK cell functions, considering the variable effects we observed in different NK cell populations and different HIV groups. For instance, higher expression of TIM-3 on NK cells marks a functionally mature population with high cytotoxicity and cytokine production capability (25). However, such NK cells exhibit impaired function when encountering cells expressing their ligand. Thus, in our study, TIM-3+ NK cells have higher expression levels of GzmB and perforin in the CD56+CD16+ subpopulation, whereas other studies have reported the association of TIM-3 with impaired cytotoxicity and NK cell exhaustion (59).
Downregulation of natural cytotoxicity receptors (NKp30, NKp44, and NKp46) in HIV-infected individuals has already been reported (60), and lower NKp46 has been associated with disease progression (61). Therefore, downregulation of NKp46 in Gal-9+ NK cells implicates that these cells might be less functional in clearing virally infected cells. Although HIV infection in general upregulates surface Gal-9 expression on NK cells in different HIV-infected groups, the consequence of Gal-9 overexpression might not be the same on all NK cells. For instance, expression of Gal-9 was associated with significantly lower CD38 in NK cells from Ps but not in other groups. This suggests that Gal-9+ NK cells from Ps might have more impaired lytic activity than other Gal-9+ NK cells because CD38 triggers cytotoxic functions of NK cells (48).
Because our observations indicated that Gal-9 expression was associated with an intrinsic NK cell dysfunction, we aimed to determine whether the commonly used cytokine mixture (IL-12, IL-15, and IL-18) for NK cell activation (62) can modulate their functions. We observed that this cytokine mixture enhanced NK cell function indiscriminately, suggesting that the impaired effector functions in Gal-9+ NK cells are partially reversible. Furthermore, the same cytokine mixture has been used to enhance NK cell functions in HIV-infected individuals and cancer patients (23, 63).
Although we have defined Gal-9+ as dysfunctional NK cells, one might argue that these cells are functionally different rather than dysfunctional and skewed toward cytokine production rather than directly mediating cell killing. Because IFN-γ can upregulate MHC class I, increase the potential for cytotoxic T cell recognition of infected cells (64–66), activate phagocytic cells, as well as induce an oxidative burst and the recruitment of immune cells to the site of infection, these cells may be viewed as adaptive for controlling HIV replication (67). Although a proinflammatory environment associated with high IFN-γ production is critical for orchestrating a robust immune response to mediate viral control in the early stage of disease, hyperimmune activation and excessive/prolonged production of IFN-γ can lead to systemic inflammation and enhanced HIV progression (68, 69). We propose that overexpression of Gal-9+ on NK cells impairs their cytotoxicity but, at the same time, enables them to secrete more IFN-γ, which may contribute to the increased immune activation seen in chronic stage of HIV infection (70, 71). This is different from the effects of rGal-9, which impairs the function of NK cells (cytotoxicity and IFN-γ production) (38). Considering the wide range of immunomodulatory roles of Gal-9 in various immune and nonimmune cells, our knowledge of underlying signaling events remains limited. We observed that Gal-9 interacts with CD44 on NK cells, which may signal to the epigenetic imprinting of the IFN-γ gene, resulting in enhanced IFN-γ production as reported for T cells (72, 73). However, the impact of such interactions on the expression of cytotoxic molecules merits further investigations.
A limitation of our study is that we extrapolated that Gal-9+ NK cells are less cytotoxic because of the fact that they contained lower GzmB, perforin, and GNLY versus Gal-9− NK cells. However, we were unable to perform a cytotoxicity assay comparing Gal-9+ versus Gal-9− NK cells because of technical issues: NK cells are rare in frequency, and we have been able to obtain a maximum of 400,000 NK cells from 100 million PBMCs. Reisolating Gal-9+ from Gal-9− has been impossible using magnet isolation because of a lack of BSL-2–sorting facility for HIV-infected specimens. We were also using frozen rather than fresh PBMCs for Ps and some LTNPs, of which we had limited cells, and thus we were unable to perform additional studies on these groups. Of note in comparison studies, we did not observe any difference in the expression levels of Gal-9/TIGIT in fresh versus frozen cells. Finally, we were unable to measure the plasma levels of IL-12, IL-15, and IL-18 in the HIV cohort, so we were unable to compare the cytokine mixture concentrations used in vitro with the in vivo levels.
Taken together, our findings revealed that HIV-1 infection differentially impacts NK cell subpopulations in different HIV-infected groups. In addition, we found upregulation of Gal-9+ on NK cells of HIV-infected individuals compared with HCs. More importantly, we observed a dichotomous role for Gal-9+– versus TIGIT+-expressing NK cells in terms of cytotoxic mediator (GzmB, perforin, and GNLY) expression and IFN-γ production (Fig. 9). Therefore, our data provide a novel, to our knowledge, role for Gal-9 in HIV pathogenesis and may help elucidate how the functionality of NK cell subtypes can be enhanced in HIV-infected individuals and other chronic conditions.
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
We thank the University of Alberta Faculty of Medicine and Dentistry’s Applied Genomics Core for assistance. We also thank the University of Alberta Faculty of Medicine and Dentistry’s Flow Cytometry Facility, which has received financial support from the Faculty of Medicine and Dentistry and the Canadian Foundation for Innovation awards to contributing investigators. We also thank HIV-infected individuals from the Northern Alberta HIV Program and the Center for AIDS Research–University of Washington.
Footnotes
This work was supported by an operating grant (Innovative Biomedical and Clinical HIV/AIDS Research) (370867) from the Canadian Institutes of Health Research (CIHR), a CIHR New Investigator Salary Award (360929), and a CIHR Foundation Scheme Grant (353953) (all to S.E.). This work was also supported by a Graduate Studies Entrance Award from the Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry/Alberta Health Services Graduate Student Recruitment Scholarships, and the Faculty of Medicine and Dentistry 75th Anniversary Award (all to M.M.). The Faculty of Medicine and Dentistry 75th Anniversary Award and The Queen Elizabeth II Graduate Scholarship were awarded to S.S.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- ART
- antiretroviral therapy
- Gal-9
- galectin-9
- GNLY
- granulysin
- GzmB
- granzyme B
- HC
- healthy control
- KIR
- killer-cell Ig-like receptor
- LTNP
- long-term nonprogressor
- P
- progressor
- PD-1
- programmed cell death protein 1
- PDI
- protein disulfide isomerase
- rGal-9
- recombinant Gal-9
- TIGIT
- T cell immunoreceptor with Ig and ITIM domain
- TIM-3
- T cell Ig and mucin domain 3.
- Received October 14, 2019.
- Accepted October 31, 2019.
- Copyright © 2019 The Authors
This article is distributed under the terms of the CC BY-NC-ND 4.0 Unported license.