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Dysregulated Tim-3 expression on natural killer cells is associated with increased Galectin-9 levels in HIV-1 infection.

Jost S, Moreno-Nieves UY, Garcia-Beltran WF, Rands K, Reardon J, Toth I, Piechocka-Trocha A, Altfeld M, Addo MM - Retrovirology (2013)

Bottom Line: Natural killer (NK) cells constitutively express high levels of Tim-3, an immunoregulatory molecule recently proposed to be a marker for mature and functional NK cells.Plasma concentrations of Gal-9 were higher in HIV-1-infected individuals than in healthy individuals.In vitro, Gal-9 triggered Tim-3 downregulation on NK cells as well as NK cell activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ragon Institute of MGH, MIT and Harvard, 400 Technology Square, Cambridge, MA 02139, USA.

ABSTRACT

Background: Natural killer (NK) cells constitutively express high levels of Tim-3, an immunoregulatory molecule recently proposed to be a marker for mature and functional NK cells. Whether HIV-1 infection modulates the expression of Tim-3 on NK cells, or the levels of its ligand Galectin-9 (Gal-9), and how signaling through these molecules affects the NK cell response to HIV-1 remains inadequately understood.

Results: We analyzed Tim-3 and Gal-9 expression in a cohort of 85 individuals with early and chronic HIV-1 infection, and in 13 HIV-1 seronegative control subjects. HIV-1 infection was associated with reduced expression of Tim-3 on NK cells, which was normalized by HAART. Plasma concentrations of Gal-9 were higher in HIV-1-infected individuals than in healthy individuals. Interestingly, Gal-9 expression in immune cells was significantly elevated in early infection, with monocytes and dendritic cells displaying the highest expression levels, which correlated with HIV-1 viral loads. In vitro, Gal-9 triggered Tim-3 downregulation on NK cells as well as NK cell activation.

Conclusions: Our data suggest that high expression levels of Gal-9 during early HIV-1 infection can lead to enhanced NK cell activity, possibly allowing for improved early control of HIV-1. In contrast, persistent Gal-9 production might impair Tim-3 activity and contribute to NK cell dysfunction in chronic HIV-1 infection.

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Incubation with soluble Gal-9 increases NK cell function and decreases surface expression of Tim-3. (A) Dot plots represent the percentages of CD107a + NK cells and the MFI of Tim-3 on NK cells from 8 healthy individuals upon pre-activation with 1 ng/mL of IL-15 and IL-18 overnight followed by stimulation with either 0.9 μg/mL of soluble Gal-9 for another 16 h or K562 target cells at an effector:target ratio of 10:1 for 6 h. Representative primary flow cytometry panels show CD107a upregulation (upper panel) and Tim-3 expression (lower panel) on NK cells that were left unstimulated, or were activated as indicated. (B) Unstimulated or Gal-9-activated NK cells were divided into Tim-3bright, Tim-3dim and Tim-3low/neg so that the bright and the low/neg subpopulations each consistently represents about 25% of the bulk NK cells, and subsequently analyzed for CD107a expression. Representative primary flow panel shows an example of subdivision of NK cells according to Tim-3 expression. Percentages of positive NK cells and median fluorescence intensity are indicated. Histograms display CD107a upregulation in each subset following incubation with soluble Gal-9. Horizontal lines indicate the median percentages. Statistically significant difference reached when p < 0.05 is indicated.
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Figure 4: Incubation with soluble Gal-9 increases NK cell function and decreases surface expression of Tim-3. (A) Dot plots represent the percentages of CD107a + NK cells and the MFI of Tim-3 on NK cells from 8 healthy individuals upon pre-activation with 1 ng/mL of IL-15 and IL-18 overnight followed by stimulation with either 0.9 μg/mL of soluble Gal-9 for another 16 h or K562 target cells at an effector:target ratio of 10:1 for 6 h. Representative primary flow cytometry panels show CD107a upregulation (upper panel) and Tim-3 expression (lower panel) on NK cells that were left unstimulated, or were activated as indicated. (B) Unstimulated or Gal-9-activated NK cells were divided into Tim-3bright, Tim-3dim and Tim-3low/neg so that the bright and the low/neg subpopulations each consistently represents about 25% of the bulk NK cells, and subsequently analyzed for CD107a expression. Representative primary flow panel shows an example of subdivision of NK cells according to Tim-3 expression. Percentages of positive NK cells and median fluorescence intensity are indicated. Histograms display CD107a upregulation in each subset following incubation with soluble Gal-9. Horizontal lines indicate the median percentages. Statistically significant difference reached when p < 0.05 is indicated.

Mentions: Cross-linking of Tim-3 has been shown to inhibit NK cell cytotoxic functions in vitro[21]. Based on our observation of increased levels of Gal-9 in natural HIV-1 infection, we sought to next investigate whether Gal-9 could modulate NK cell function in vitro. To address this question, we compared NK cell function following treatment with soluble Gal-9 to that upon exposure to major histocompatibility complex (MHC)-deficient target cells (i.e. K562 cells). Incubation with Gal-9 triggered NK cell activation, as measured by CD107a surface expression, which occurred concomitantly with a decreased surface expression of Tim-3 (Figure 4A). Percentages of Tim-3+ NK cells were also diminished upon Gal-9 stimulation, although to a lesser extent (data not shown). Treatment with soluble Gal-9 did not lead to a significant increase in production of IFN-γ (unstimulated: median, 0.7; IQR 0.42-0.94; Gal-9; median, 1.014; IQR, 0.3893- 2.033), as compared to incubation with K562 cells (median, 9.7; IQR, 5.08- 17.13; p = 0.008 vs. unstimulated and p = 0.004 vs. Gal-9). In order to further understand the function executed by Tim-3 in the NK cell response to Gal-9, we analyzed changes in CD107a expression on NK cells bearing high (Tim-3bright), medium (Tim-3dim) or low/no (Tim3low/neg) levels of Tim-3, and observed that following incubation with Gal-9, CD107a upregulation on Tim3low/neg NK cells was enhanced compared to that of Tim-3bright and Tim3dim NK cells (Figure 4B). Tim-3bright NK cells were enriched in CD56bright NK cells which might intrinsically have defective degranulation properties. Therefore, we quantified CD107a upregulation on Tim-3bright, Tim-3dim and Tim-3low/neg CD56dim NK cells, and found that upon Gal-9 stimulation, the activity of CD56dim NK cells expressing high amounts of Tim-3 was significantly reduced compared to those expressing dim (p < 0.0001) and low (p < 0.0001) levels, showing that CD56bright NK cells were not introducing a bias in our findings (data not shown).


Dysregulated Tim-3 expression on natural killer cells is associated with increased Galectin-9 levels in HIV-1 infection.

Jost S, Moreno-Nieves UY, Garcia-Beltran WF, Rands K, Reardon J, Toth I, Piechocka-Trocha A, Altfeld M, Addo MM - Retrovirology (2013)

Incubation with soluble Gal-9 increases NK cell function and decreases surface expression of Tim-3. (A) Dot plots represent the percentages of CD107a + NK cells and the MFI of Tim-3 on NK cells from 8 healthy individuals upon pre-activation with 1 ng/mL of IL-15 and IL-18 overnight followed by stimulation with either 0.9 μg/mL of soluble Gal-9 for another 16 h or K562 target cells at an effector:target ratio of 10:1 for 6 h. Representative primary flow cytometry panels show CD107a upregulation (upper panel) and Tim-3 expression (lower panel) on NK cells that were left unstimulated, or were activated as indicated. (B) Unstimulated or Gal-9-activated NK cells were divided into Tim-3bright, Tim-3dim and Tim-3low/neg so that the bright and the low/neg subpopulations each consistently represents about 25% of the bulk NK cells, and subsequently analyzed for CD107a expression. Representative primary flow panel shows an example of subdivision of NK cells according to Tim-3 expression. Percentages of positive NK cells and median fluorescence intensity are indicated. Histograms display CD107a upregulation in each subset following incubation with soluble Gal-9. Horizontal lines indicate the median percentages. Statistically significant difference reached when p < 0.05 is indicated.
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Related In: Results  -  Collection

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Figure 4: Incubation with soluble Gal-9 increases NK cell function and decreases surface expression of Tim-3. (A) Dot plots represent the percentages of CD107a + NK cells and the MFI of Tim-3 on NK cells from 8 healthy individuals upon pre-activation with 1 ng/mL of IL-15 and IL-18 overnight followed by stimulation with either 0.9 μg/mL of soluble Gal-9 for another 16 h or K562 target cells at an effector:target ratio of 10:1 for 6 h. Representative primary flow cytometry panels show CD107a upregulation (upper panel) and Tim-3 expression (lower panel) on NK cells that were left unstimulated, or were activated as indicated. (B) Unstimulated or Gal-9-activated NK cells were divided into Tim-3bright, Tim-3dim and Tim-3low/neg so that the bright and the low/neg subpopulations each consistently represents about 25% of the bulk NK cells, and subsequently analyzed for CD107a expression. Representative primary flow panel shows an example of subdivision of NK cells according to Tim-3 expression. Percentages of positive NK cells and median fluorescence intensity are indicated. Histograms display CD107a upregulation in each subset following incubation with soluble Gal-9. Horizontal lines indicate the median percentages. Statistically significant difference reached when p < 0.05 is indicated.
Mentions: Cross-linking of Tim-3 has been shown to inhibit NK cell cytotoxic functions in vitro[21]. Based on our observation of increased levels of Gal-9 in natural HIV-1 infection, we sought to next investigate whether Gal-9 could modulate NK cell function in vitro. To address this question, we compared NK cell function following treatment with soluble Gal-9 to that upon exposure to major histocompatibility complex (MHC)-deficient target cells (i.e. K562 cells). Incubation with Gal-9 triggered NK cell activation, as measured by CD107a surface expression, which occurred concomitantly with a decreased surface expression of Tim-3 (Figure 4A). Percentages of Tim-3+ NK cells were also diminished upon Gal-9 stimulation, although to a lesser extent (data not shown). Treatment with soluble Gal-9 did not lead to a significant increase in production of IFN-γ (unstimulated: median, 0.7; IQR 0.42-0.94; Gal-9; median, 1.014; IQR, 0.3893- 2.033), as compared to incubation with K562 cells (median, 9.7; IQR, 5.08- 17.13; p = 0.008 vs. unstimulated and p = 0.004 vs. Gal-9). In order to further understand the function executed by Tim-3 in the NK cell response to Gal-9, we analyzed changes in CD107a expression on NK cells bearing high (Tim-3bright), medium (Tim-3dim) or low/no (Tim3low/neg) levels of Tim-3, and observed that following incubation with Gal-9, CD107a upregulation on Tim3low/neg NK cells was enhanced compared to that of Tim-3bright and Tim3dim NK cells (Figure 4B). Tim-3bright NK cells were enriched in CD56bright NK cells which might intrinsically have defective degranulation properties. Therefore, we quantified CD107a upregulation on Tim-3bright, Tim-3dim and Tim-3low/neg CD56dim NK cells, and found that upon Gal-9 stimulation, the activity of CD56dim NK cells expressing high amounts of Tim-3 was significantly reduced compared to those expressing dim (p < 0.0001) and low (p < 0.0001) levels, showing that CD56bright NK cells were not introducing a bias in our findings (data not shown).

Bottom Line: Natural killer (NK) cells constitutively express high levels of Tim-3, an immunoregulatory molecule recently proposed to be a marker for mature and functional NK cells.Plasma concentrations of Gal-9 were higher in HIV-1-infected individuals than in healthy individuals.In vitro, Gal-9 triggered Tim-3 downregulation on NK cells as well as NK cell activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ragon Institute of MGH, MIT and Harvard, 400 Technology Square, Cambridge, MA 02139, USA.

ABSTRACT

Background: Natural killer (NK) cells constitutively express high levels of Tim-3, an immunoregulatory molecule recently proposed to be a marker for mature and functional NK cells. Whether HIV-1 infection modulates the expression of Tim-3 on NK cells, or the levels of its ligand Galectin-9 (Gal-9), and how signaling through these molecules affects the NK cell response to HIV-1 remains inadequately understood.

Results: We analyzed Tim-3 and Gal-9 expression in a cohort of 85 individuals with early and chronic HIV-1 infection, and in 13 HIV-1 seronegative control subjects. HIV-1 infection was associated with reduced expression of Tim-3 on NK cells, which was normalized by HAART. Plasma concentrations of Gal-9 were higher in HIV-1-infected individuals than in healthy individuals. Interestingly, Gal-9 expression in immune cells was significantly elevated in early infection, with monocytes and dendritic cells displaying the highest expression levels, which correlated with HIV-1 viral loads. In vitro, Gal-9 triggered Tim-3 downregulation on NK cells as well as NK cell activation.

Conclusions: Our data suggest that high expression levels of Gal-9 during early HIV-1 infection can lead to enhanced NK cell activity, possibly allowing for improved early control of HIV-1. In contrast, persistent Gal-9 production might impair Tim-3 activity and contribute to NK cell dysfunction in chronic HIV-1 infection.

Show MeSH
Related in: MedlinePlus