HIV is a chronic viral infection that, if untreated, is associated with the destruction of CD4+ T cells, rendering the infected individual immune-compromised and susceptible to opportunistic infections and tumors. Chronic immune activation is one of the hallmarks of HIV infection and directly correlates with disease progression.1 More recently, immune exhaustion, the functional impairment of CD4+ and CD8+ T cells, has emerged as a potential contributor to HIV pathogenesis.2 In addition to CD4+ and CD8+ T cells, innate immune cells including monocytes, NK cells, NKT, and γδ T cells have also been reported to alter in number and function in HIV-infected individuals.3–7 Innate immune effector cells are the first line of defense against infection. They play a crucial role in containing invading pathogens and driving adaptive immunity; thus, their impairment would further contribute to the immune-compromised state of HIV infection.7,8
The T-cell immunoglobulin- and mucin-domain–containing molecule-3 (Tim-3) is a transmembrane protein of the immunoglobulin superfamily, preferentially expressed on differentiated TH1 cells.9,10 It is also found on many other cell types including innate immune cells, such as monocytes,11 NK cells,12,13 NKT cells,14 and γδ cells.15 Tim-3 was initially identified on interferon gamma (IFNγ)-producing CD4+ T cells (TH1) and cytotoxic CD8+ T cells (TC1),10 and shown to have a negative regulatory function, abrogating TH1- and TC1-driven immune responses.16,17 Interaction of Tim-3 on T cells with its ligand galectin-9 results in the selective loss of IFNγ-producing cells,17–19 and, a reduction in Tim-3 levels enhances IFNγ secretion.18,20–22 In vivo blockade of Tim-3 exacerbates allergic encephalomyelitis and type 1 diabetes,10,23 and in T cells isolated from multiple sclerosis patients, Tim-3 expression is downregulated, and IFNγ secretion is increased compared with healthy controls,18 further supporting its role as a negative regulator of immune responses.
Although several studies have demonstrated that Tim-3 is an inhibitory molecule on TH1 and TC1 T cells, the role of Tim-3 in innate immunity is not fully understood. Tim-3 is highly expressed on some innate immune cells, including monocytes, dendritic cells, and NK cells. It has been suggested that Tim-3 may help promote the transition from innate to adaptive immunity by enhancing proinflammatory responses, phagocytosis, and cross-presentation.24–27 However, more recent data suggest, that similarly to T cell responses, Tim-3 may serve a regulatory function in innate immune responses.11,13,28
Increased expression of Tim-3 on T cells has been reported in chronic viral infections that are associated with T-cell dysfunction, including HIV.29–32 Tim-3 expressing CD8+ and CD4+ T cells from chronic viremic HIV(+) donors were found to be dysfunctional, and blocking Tim-3 restored proliferative ability and IFNγ secretion to these cells.30 The initiation of combination antiretroviral therapy (cART) in chronic progressive HIV-1 infection has been correlated with a decline in Tim-3 expression on T cells in some, but not, all individuals.30 However, these findings have yet to be extended to innate cell subsets. Characterizing Tim-3 expression on innate cells is of particular importance in HIV(+) individuals because blocking Tim-3 is being suggested as a potential therapeutic avenue.30,33
In this study, we investigate Tim-3 on innate immune cell subsets in chronic HIV(+) individuals pre-cART and post-cART therapy. We report that HIV infection is associated with elevated levels of Tim-3 on innate lymphocytes but not on monocytes. Innate cells from HIV-infected donors were significantly less responsive to an inflammatory stimulus that decreased Tim-3 on monocytes and increased Tim-3 on NK, NKT, and γδ T cells from HIV(−) controls. The responses of all subsets were restored to some extent with cART but only reached control levels in monocytes and NK cells. During HIV infection, Tim-3 expression in the innate immune compartment, and its modulation in response to cART, is therefore not uniform among cell types.
Study Population and Ethics Statement
The University Health Network and University of Toronto Ethics Review Boards approved this study. The study participants gave written informed consent. HIV-1 infected [HIV(+)] treatment-naive participants were recruited at the Maple Leaf Medical Clinic, Toronto, Canada. HIV(+) participants were chronic progressors (HIV-infected for >1 year, with CD4+ T-cell count decline of >50 cells per cubic millimeter per year). Venous blood samples were collected just before the initiation of cART therapy (M0) and at 6 months (M6) post-cART. Clinical data for the cohort are in Table 1 (CD4+ counts, viral loads).
Controls were HIV-negative [HIV(−)], recruited in the same demographic area with a similar age and sex profile. Each pair of HIV(−) and HIV(+) samples was collected at the same time and processed identically. When possible, each HIV(+) donor was matched to the same HIV(−) individual for the 2 time points.
A full assessment of the innate cell subsets for our participants has been reported elsewhere.34
Multicolor Flow Cytometry
All the experiments were performed with freshly isolated peripheral blood mononuclear cells (PBMCs), purified from venous blood samples using Ficoll gradients. To determine cellular phenotypes, freshly isolated PBMCs were immediately stained with conjugated monoclonal antibodies to CD14, CD3, CD56, γδ (BioLegend), and Tim-3 (R&D). BD FACS lysing solution (BD Biosciences) was used to fix cells and lyse contaminating red blood cells (RBCs). Cells were analyzed using an FACSCanto or LSRII (BD Biosciences) within 24 hours after fixing. A minimum of 100,000 CD3+ events were collected for each sample. Data were analyzed using FlowJo (Tree Star).
Plasmodium falciparum (ITG strain) was cultured as previously described.35 Cultures were treated with Mycoplasma-Removal Agent (MP Biochemicals) and routinely tested negative for Mycoplasma by polymerase chain reaction. Cultures were synchronized using alanine lysis and trophozoite-stage cultures were used in all assays.
For stimulation assays, freshly isolated PBMCs were plated at 1 × 106/mL of media (RPMI-1640, 10% fetal calf serum, minimal nonessential amino acids, sodium pyruvate, 2β-ME, gentamycin) in 24-well plates, and cultured with 3 × 106 malaria parasites (∼10% parasitemia) as described in Ref. 36 or equivalent hematocrit levels of uninfected RBCs for 48 hours. A ratio of 3 parasites to 1 PBMC represents a parasitemia level of 15,000 parasites per microliter36 and was chosen because it recapitulates a number of the clinical signs associated with malaria disease.37–39 The cells were collected at 48 hours for analysis of cell surface markers. Malaria parasites can stimulate monocytes, NK, NKT, and γδ T cells in vitro in a physiological manner.34,36 We have previously reported that coculture of HIV(−) PBMCs with malaria parasites leads to tumor necrosis factor (TNF) and IFNγ production by monocytes, NK, NKT, and γδ T cells, as assessed by intracellular cytokine staining.34
Wilcoxon match pairs tests were used to compare baseline differences in cell subset Tim-3 expression on matched HIV(+) and HIV(−) samples. For the stimulation assays, the percentage change over the RBC control was calculated for each malaria parasite stimulated sample. Comparisons were made between the HIV(−) group (pooled between the 2 time points) and the HIV(+) treatment-naive and post-cART groups. Kruskal–Wallis test with the Dunn multiple comparisons posttest was used to assess differences between the 3 groups.
Tim-3+ NK Cells, NKT, and γδ T Cells, but Not Monocytes, Are Increased in HIV(+) Individuals
Tim-3 has been shown to be overexpressed on T cells in chronic HIV infection where it has been associated with a dysfunctional phenotype,30 but its expression on most innate immune cells has not previously been reported. To determine Tim-3 expression on innate cells in HIV infection, we measured levels of Tim-3+ monocytes (CD3−CD14+), NK cells (CD3−CD56hi and CD3−CD56lo), NKT cells (CD3+CD56+), and γδ T cells (CD56−CD3+γδ+) by flow cytometry, using freshly isolated PBMCs from HIV(+) participants pre-cART and post-cART and compared them with HIV(−) controls (Fig. 1). Of the cell types studied, monocytes had the greatest expression of Tim-3, with >80% of monocytes being Tim-3+ (Fig. 1A, left panel). Both the percentage of Tim-3+ monocytes and the levels of Tim-3 on monocytes [Tim-3 mean fluorescence intensity (MFI)] were similar between HIV(−) controls and HIV(+) donors pre-cART and post-cART (Fig. 1A, P > 0.05).
NK cells also expressed high levels of Tim-3. NK cells can be separated into 2 groups based on CD56 expression levels. CD56hi NK cells are thought to be mainly a cytokine producing subset, whereas the CD56lo NK cells are thought to be mainly cytotoxic. CD56lo NK cells expressed high levels of Tim-3, and both the percentage of Tim-3+ cells and the levels of Tim-3 were similar between HIV(−) controls and HIV(+) donors pre and post-cART (Fig. 1B, P > 0.05). However, both the percentage of Tim-3+ and the levels of Tim-3 were significantly higher on CD56hi NK cells from HIV(+) treatment-naive donors compared with HIV(−) controls (Fig. 1C). Eighty-eight percent of HIV(+) individuals (14/16) had greater percentages of Tim-3+ CD56hi NK cells (Fig. 1C, left panel, P = 0.01) and 88% had increased MFI (Fig. 1C, right panel, P = 0.001). However, after 6 months of cART, this difference was diminished. Only 56% (9/16) of HIV(+) participants had greater percentages of Tim-3 expressing cells compared with HIV(−) controls (Fig. 1C, left panel, P = 0.18), and Tim-3 MFI levels were similar between the 2 groups (Fig. 1C, right panel, P = 0.31).
Tim-3 expression was the lowest on NKT and γδ T cells, although HIV(+) donors had significantly higher levels of Tim-3+ NKT (Fig. 1D, left panel, P = 0.001) and γδ T cells (Fig. 1E, left panel, P = 0.002) compared with HIV(−) controls. The levels remained higher after 6 months of cART (Fig. 1E, left panel, P = 0.01 and P = 0.003, respectively). Similar trends were observed for Tim-3 MFI on these cell types (Figs. 1D, E, right panels).
In summary, untreated HIV infection was associated with an increase in Tim-3-expressing CD56hi NK cells, NKT, and γδ T cells, as well as an over-expression of Tim-3 in these subsets. Neither of these observations were true for monocytes or CD56lo NK cells.
The Impact of an Inflammatory Stimulus on Tim-3 Expression on Innate Immune Cells
We next investigated if an inflammatory stimulus would differentially affect Tim-3 expression on innate cells from HIV(+) vs uninfected donors. PBMCs were stimulated for 48 hours with live human malaria parasites [P. falciparum (ITG strain) infected RBC]. Human malaria parasites were chosen as an inflammatory stimulus because they are known to stimulate monocytes, NK cells, NKT cells, and γδ T cells in vitro in a physiologically relevant manner.34,36,40–42 Additionally, because both HIV and malaria are global health priorities and coinfection with HIV and malaria is becoming a growing concern, understanding how innate cells respond to malaria in the presence of HIV is of clinical relevance.
Although the baseline percentage of Tim-3+ monocytes did not differ between HIV(+) donors and HIV(−) controls (Fig. 1A), their response to stimulation differed significantly. As previously reported with Toll-like receptor (TLR) ligand stimulation,43 stimulation with malaria parasites led to a decrease in Tim-3+ monocytes from HIV(−) controls (Fig. 2A). However, this was not observed with monocytes from treatment-naive HIV(+) donors (Fig. 2A). After stimulation, there was an overall 66% decrease in the median number of Tim-3+ monocytes from HIV(−) controls, compared with only a 13% decrease in monocytes from treatment-naive HIV(+) donors (P < 0.01). After 6 months of cART, monocytes from HIV(+) donors responded similarly to HIV(−) controls (Fig. 2A), with a median decrease in Tim-3+ monocytes of 46%.
Unlike monocytes and similar to what has been observed with T cells,10,44 Tim-3 levels on innate lymphocytes increased with stimulation (Figs. 2B–D). However, HIV(+) donors failed to upregulate Tim-3 on CD56hi NK, NKT, and γδ T cells to the same levels as HIV(−) controls after stimulation [Figs. 2B–D, HIV(−) vs HIV(+) M0, P < 0.05]. As seen with baseline Tim-3 levels, the only subset to recover after cART was the CD56hi NK cell subset (Fig. 2B).
Stimulation with malaria parasites led to a 1.6-fold median increase in Tim-3+ CD56hi NK cells in the uninfected group, but only a 0.7-fold increase in the treatment-naive HIV(+) group (Fig. 2C). After 6 months of cART, stimulation with malaria parasites led to a 1.5-fold median increase in Tim-3+ CD56hi NK cells from HIV(+) donors; this was similar to that in the HIV(−) control group and significantly higher than the increase seen before cART (Fig. 2B).
Stimulation with malaria parasites did not lead to any significant changes in Tim-3+ CD56lo NK cells in any of the groups (data not shown).
In the NKT subset (Fig. 2C), stimulation with malaria parasites led to an 8-fold median increase in Tim-3+ cells in the HIV(−) controls. This was significantly higher than in the HIV(+) group, both pre-cART and post-cART (0.36-fold and 1.5-fold median increase respectively).
The γδ T cells responded similarly to the NKT subset (Fig. 2D). Stimulation led to an almost 4-fold median increase in Tim-3+ γδ T cells in the HIV(−) controls, whereas stimulation only led to a 0.8-fold median increase in the treatment-naive HIV(+) donors and a 1.6-fold median increase in the cART-treated group, both significantly lower than the control group.
In summary, in all innate cell subsets studied, Tim-3 responses to an inflammatory stimulus differed significantly between HIV(+) treatment-naive and uninfected donors. Only monocyte and CD56hi NK cell responses recovered fully after 6 months of cART.
Our study demonstrates that chronic untreated HIV infection was associated with higher proportions of Tim-3+ innate lymphocytes, specifically CD56hi NK cells, NKT, and γδ T cells, but normal levels of Tim-3+ monocytes. Furthermore, HIV infection was associated with a failure to upregulate Tim-3 on CD56hi NK cells, NKT, and γδ T cells, and a failure to downregulate Tim-3 on monocytes after an inflammatory stimulus. Initiation of cART resulted in a correction in basal Tim-3 levels only on CD56hi NK cells, and a correction of Tim-3 responses to stimulation in monocytes and CD56hi NK cells. The defects observed in the NKT and γδ T-cell subsets persisted after 6 months of cART.
It has been proposed that Tim-3 serves opposing roles during innate and adaptive immune responses, promoting inflammation in the innate system while limiting inflammation in the adaptive.25 We observed that in the absence of HIV infection, Tim-3 was upregulated on CD56hi NK cells after stimulation. This is in agreement with the findings of a previous study showing that stimulation with IL-12 and IL-18 increases Tim-3 expression on CD56hi NK cells.13 Ndhlovu et al13 further showed that IFNγ production and cytotoxic ability were preferentially associated with the NK cells with the highest Tim-3 levels. It has been suggested that (unlike in T cells where Tim-3 is a marker for dysfunctional or anergic cells), Tim-3 may mark fully mature and functional NK cells. However, Tim-3 can restrain NK cell function when it encounters target cells that express its ligand galectin-9. We observed that HIV(+) treatment-naive donors had a higher basal proportion of Tim-3+ NK cells. This may be the result of chronic stimulation, causing greater maturation/activation of these cells. In fact the activation of NK cells, as assessed by CD38 and HLA-DR expression has been reported in HIV infection.45 However, NK cells from HIV(+) treatment-naive donors failed to upregulate Tim-3 after stimulation to the same levels as controls. This may be due to the greater basal activation state, or may be related to a dysfunctional phenotype. Of note, 6 months of cART was sufficient to correct both the basal Tim-3 level and the activation differences.
Several studies have suggested that NK cell responses are compromised during HIV infection; however, the exact mechanisms underlying the NK cell dysfunction are unknown. Tim-3 expression has been correlated to altered NK cell function in other chronic conditions, including hepatitis B and atherosclerosis.12,46 We have previously observed a defect in malaria stimulation–induced IFNγ and TNF responses from NK cells in our HIV(+) donors that was partially corrected after cART.34 Whether this relates to Tim-3 levels on NK cells requires further investigation.
There are few published reports on the role of Tim-3 in γδ T cells or NKT cells. In pregnant women with preeclampsia, Tim-3 levels were decreased on γδ T cells15 and were associated with increased IFNγ production by these cells. A negative correlation has also been observed between Tim-3 expression and TNF production in NKT cells, suggesting a negative role for Tim-3 on NKT functions.14 In our study, malaria parasite stimulation resulted in increased Tim-3 levels on γδ T cells and NKT cells from uninfected individuals. These cell types were both strong IFNγ and TNF producers.34 However, in treatment-naive HIV(+) individuals, stimulation did not induce an upregulation in Tim-3 in either γδ T cells or NKT cells and cART did not restore this response. Additionally we have recently reported that neither γδ T cells nor NKT cells from HIV(+) individuals produced IFNγ or TNF in response to malaria parasite stimulation pre-cART or post-cART.34 Thus, the high basal levels of Tim-3 on NKT and γδ T cells in our HIV donors may mark dysfunctional cells.
In contrast to the lymphocyte subsets, the proportion of Tim-3+ monocytes was unaffected by HIV infection. However, differences between HIV(+) and HIV(−) controls were observed in stimulation experiments. Stimulation with malaria parasites led to a significant decrease in Tim-3+ monocytes in HIV(−) controls, but not in treatment-naive HIV(+) cases. These differences disappeared after 6 months of cART, at which point monocytes from both HIV(+) and HIV(−) donors downregulated Tim-3 after stimulation. Downregulation of Tim-3 on monocytes has been observed after TLR stimulation (malaria parasites are known TLR stimulators47), and this decrease was associated with an increase in IL-12 production.11 Attenuation of Tim-3 signaling, via antibody blockade or small interfering RNA, increased the production of IL-12 and IL-10 and decreased the expression of PD-1, a negative regulator of monocyte activation.48 In addition, compared with healthy controls, monocytes from hepatitis C patients (a chronic viral infection) failed to downregulate Tim-3 after TLR stimulation and produced less IL-12.43 Thus, Tim-3 on monocytes may act to keep monocytes in check, whereas a reduction in Tim-3 is associated with monocyte activation.28 If this model is correct, then a failure to downregulate Tim-3 on HIV(+) monocytes may be a marker of dysfunctional monocytes. Monocyte and macrophage dysfunction has been described in HIV infection and corroborated by transcriptome analysis. Reduced phagocytosis and antigen presentation, as well as dysregulated inflammatory responses have all been observed.6,49–53
Our study is the first to characterize Tim-3 expression in 4 different innate cell subsets in HIV(+) individuals, both in resting cells and also in response to malaria-infected erythrocytes as an inflammatory stimulus. Our data show that Tim-3 levels and Tim-3 responses to stimuli are altered in innate immune cells in the presence of untreated HIV infection, and are only partially corrected by cART. If Tim-3 is a marker of dysfunction on innate cells, as it has been demonstrated to be on CD4+ and CD8+ T cells, then our data may suggest that untreated HIV infection is associated with a more profound immune deficit that encompasses both adaptive and innate immune cells. Our data clearly indicate that further studies on Tim-3 expression and function in HIV(+) individuals will need to be carried out before stimulating/blocking Tim-3 can be considered a viable therapeutic strategy. An integrated approach that examines the impact of blocking Tim-3 on all major cellular subtypes may be informative in mitigating potential harmful effects that might result from unanticipated manipulation of protective host responses to infection.
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