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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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Tim-3 expression is dysregulated in HIV-1 infection. (A) Flow cytometry gating strategy: Gates are set to include CD3- lymphocytes and exclude dead cells (viability marker+). NK cell subsets are defined by the expression of CD56 and CD16, and Tim-3+ NK cells by using appropriate fluorescence-minus-one controls. The four right flow cytometry panels show representative examples of Tim-3+ bulk and CD56bright, CD56dim and CD56neg NK cells in an HIV-1 negative subject (HIV-), an individual with late primary untreated infection, an untreated chronic HIV-1 progressor, and a HAART-treated HIV-1+ patient. Percentages of Tim-3+ bulk NK cells, and MFI of Tim-3 on CD56bright NK cells are indicated on the top left of the main panel and on the top right of the CD56bright NK cell gate, respectively. (B) Dot plots represent the percentage of Tim-3+ NK cells from 13 healthy individuals and 85 HIV-1-infected subjects (C), including 14 with early untreated HIV-1 infection, 54 with chronic untreated HIV-1 infection (blue, 17 viremic controllers; green, 17 elite controllers; red, 20 untreated progressors), and 17 with HAART-treated HIV-1 infection. (D) Percentages of Tim-3 + NK cells in CD56bright (CD3-CD56+CD16-), CD56dim (CD3-CD56+CD16+) and CD56neg (CD3-CD56-CD16+) NK cells in 13 HIV-1 negative and 85 HIV-1-infected subjects. (E) MFI of Tim-3 on CD56bright NK cells in 13 healthy individuals and 14 subjects with early infection, 54 with chronic untreated HIV-1 infection, including 17 viremic controllers (blue, VC), 17 elite controllers (green, EC), 20 untreated progressors (red, CU), and 17 with HAART-treated HIV-1 infection (CT). Horizontal lines indicate the median percentages. Statistical differences with p < 0.05 are indicated and were determined using the Mann–Whitney t test to compare phenotype frequencies between two groups.
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Figure 1: Tim-3 expression is dysregulated in HIV-1 infection. (A) Flow cytometry gating strategy: Gates are set to include CD3- lymphocytes and exclude dead cells (viability marker+). NK cell subsets are defined by the expression of CD56 and CD16, and Tim-3+ NK cells by using appropriate fluorescence-minus-one controls. The four right flow cytometry panels show representative examples of Tim-3+ bulk and CD56bright, CD56dim and CD56neg NK cells in an HIV-1 negative subject (HIV-), an individual with late primary untreated infection, an untreated chronic HIV-1 progressor, and a HAART-treated HIV-1+ patient. Percentages of Tim-3+ bulk NK cells, and MFI of Tim-3 on CD56bright NK cells are indicated on the top left of the main panel and on the top right of the CD56bright NK cell gate, respectively. (B) Dot plots represent the percentage of Tim-3+ NK cells from 13 healthy individuals and 85 HIV-1-infected subjects (C), including 14 with early untreated HIV-1 infection, 54 with chronic untreated HIV-1 infection (blue, 17 viremic controllers; green, 17 elite controllers; red, 20 untreated progressors), and 17 with HAART-treated HIV-1 infection. (D) Percentages of Tim-3 + NK cells in CD56bright (CD3-CD56+CD16-), CD56dim (CD3-CD56+CD16+) and CD56neg (CD3-CD56-CD16+) NK cells in 13 HIV-1 negative and 85 HIV-1-infected subjects. (E) MFI of Tim-3 on CD56bright NK cells in 13 healthy individuals and 14 subjects with early infection, 54 with chronic untreated HIV-1 infection, including 17 viremic controllers (blue, VC), 17 elite controllers (green, EC), 20 untreated progressors (red, CU), and 17 with HAART-treated HIV-1 infection (CT). Horizontal lines indicate the median percentages. Statistical differences with p < 0.05 are indicated and were determined using the Mann–Whitney t test to compare phenotype frequencies between two groups.

Mentions: The immunoregulatory molecule Tim-3, previously associated with T cell exhaustion and anergy, was recently shown to mark NK cell maturation and to suppress cell-mediated toxicity[21]. As progressive HIV-1 infection leads to the accumulation of dysfunctional NK cells[41,42], we first examined whether surface expression of Tim-3 protein on NK cells was altered in individuals at different stages of HIV-1 infection when compared to healthy subjects (Table 1). As demonstrated in previous studies, our data highlight a high degree of variation between the frequencies of Tim-3-expressing NK cells in HIV-1 negative individuals (median, 73.36; interquartile range [IQR], 57.80-80.14)[21] (Figure 1). Similar heterogeneity of Tim-3 expression on NK cells was observed in HIV-1-infected individuals (median, 59.77; IQR, 47.80-66.04), yet percentages of Tim-3+ NK cells were significantly decreased in HIV-1 infection compared to healthy subjects (Figure 1A and B). Cell surface density of Tim-3 on NK cells, as measured by the mean fluorescence intensity (MFI), trended to be lower in HIV-1 infection compared to HIV-1-negative controls, however the difference did not reach statistical significance (HIV-1-infected subjects: median, 497; IQR, 387–622; healthy controls: median, 600; IQR, 457.3-805.5; p = 0.1). A substantial decrease in the number of NK cells expressing Tim-3 was observed as early as in primary HIV-1 infection, and appeared to be partially restored by highly active antiretroviral therapy (HAART) (Figure 1C). The lowest proportions of Tim-3+ NK cells were found in viremic controllers (p = 0.002 and p = 0.02 compared to healthy controls and untreated progressors, respectively), yet there was an overall high degree of variability in Tim-3 expression among these subjects (median, 48; IQR 38.7-64.3), which could not be attributed to differences in any of the demographic or clinical data analyzed (data not shown). Interestingly, elite controllers had similar percentages of Tim-3+ NK cells (median, 58.2; IQR, 47–62.7) as untreated subjects with chronic viremic HIV-1 infection (median, 62, IQR, 55.1-67).


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)

Tim-3 expression is dysregulated in HIV-1 infection. (A) Flow cytometry gating strategy: Gates are set to include CD3- lymphocytes and exclude dead cells (viability marker+). NK cell subsets are defined by the expression of CD56 and CD16, and Tim-3+ NK cells by using appropriate fluorescence-minus-one controls. The four right flow cytometry panels show representative examples of Tim-3+ bulk and CD56bright, CD56dim and CD56neg NK cells in an HIV-1 negative subject (HIV-), an individual with late primary untreated infection, an untreated chronic HIV-1 progressor, and a HAART-treated HIV-1+ patient. Percentages of Tim-3+ bulk NK cells, and MFI of Tim-3 on CD56bright NK cells are indicated on the top left of the main panel and on the top right of the CD56bright NK cell gate, respectively. (B) Dot plots represent the percentage of Tim-3+ NK cells from 13 healthy individuals and 85 HIV-1-infected subjects (C), including 14 with early untreated HIV-1 infection, 54 with chronic untreated HIV-1 infection (blue, 17 viremic controllers; green, 17 elite controllers; red, 20 untreated progressors), and 17 with HAART-treated HIV-1 infection. (D) Percentages of Tim-3 + NK cells in CD56bright (CD3-CD56+CD16-), CD56dim (CD3-CD56+CD16+) and CD56neg (CD3-CD56-CD16+) NK cells in 13 HIV-1 negative and 85 HIV-1-infected subjects. (E) MFI of Tim-3 on CD56bright NK cells in 13 healthy individuals and 14 subjects with early infection, 54 with chronic untreated HIV-1 infection, including 17 viremic controllers (blue, VC), 17 elite controllers (green, EC), 20 untreated progressors (red, CU), and 17 with HAART-treated HIV-1 infection (CT). Horizontal lines indicate the median percentages. Statistical differences with p < 0.05 are indicated and were determined using the Mann–Whitney t test to compare phenotype frequencies between two groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3750478&req=5

Figure 1: Tim-3 expression is dysregulated in HIV-1 infection. (A) Flow cytometry gating strategy: Gates are set to include CD3- lymphocytes and exclude dead cells (viability marker+). NK cell subsets are defined by the expression of CD56 and CD16, and Tim-3+ NK cells by using appropriate fluorescence-minus-one controls. The four right flow cytometry panels show representative examples of Tim-3+ bulk and CD56bright, CD56dim and CD56neg NK cells in an HIV-1 negative subject (HIV-), an individual with late primary untreated infection, an untreated chronic HIV-1 progressor, and a HAART-treated HIV-1+ patient. Percentages of Tim-3+ bulk NK cells, and MFI of Tim-3 on CD56bright NK cells are indicated on the top left of the main panel and on the top right of the CD56bright NK cell gate, respectively. (B) Dot plots represent the percentage of Tim-3+ NK cells from 13 healthy individuals and 85 HIV-1-infected subjects (C), including 14 with early untreated HIV-1 infection, 54 with chronic untreated HIV-1 infection (blue, 17 viremic controllers; green, 17 elite controllers; red, 20 untreated progressors), and 17 with HAART-treated HIV-1 infection. (D) Percentages of Tim-3 + NK cells in CD56bright (CD3-CD56+CD16-), CD56dim (CD3-CD56+CD16+) and CD56neg (CD3-CD56-CD16+) NK cells in 13 HIV-1 negative and 85 HIV-1-infected subjects. (E) MFI of Tim-3 on CD56bright NK cells in 13 healthy individuals and 14 subjects with early infection, 54 with chronic untreated HIV-1 infection, including 17 viremic controllers (blue, VC), 17 elite controllers (green, EC), 20 untreated progressors (red, CU), and 17 with HAART-treated HIV-1 infection (CT). Horizontal lines indicate the median percentages. Statistical differences with p < 0.05 are indicated and were determined using the Mann–Whitney t test to compare phenotype frequencies between two groups.
Mentions: The immunoregulatory molecule Tim-3, previously associated with T cell exhaustion and anergy, was recently shown to mark NK cell maturation and to suppress cell-mediated toxicity[21]. As progressive HIV-1 infection leads to the accumulation of dysfunctional NK cells[41,42], we first examined whether surface expression of Tim-3 protein on NK cells was altered in individuals at different stages of HIV-1 infection when compared to healthy subjects (Table 1). As demonstrated in previous studies, our data highlight a high degree of variation between the frequencies of Tim-3-expressing NK cells in HIV-1 negative individuals (median, 73.36; interquartile range [IQR], 57.80-80.14)[21] (Figure 1). Similar heterogeneity of Tim-3 expression on NK cells was observed in HIV-1-infected individuals (median, 59.77; IQR, 47.80-66.04), yet percentages of Tim-3+ NK cells were significantly decreased in HIV-1 infection compared to healthy subjects (Figure 1A and B). Cell surface density of Tim-3 on NK cells, as measured by the mean fluorescence intensity (MFI), trended to be lower in HIV-1 infection compared to HIV-1-negative controls, however the difference did not reach statistical significance (HIV-1-infected subjects: median, 497; IQR, 387–622; healthy controls: median, 600; IQR, 457.3-805.5; p = 0.1). A substantial decrease in the number of NK cells expressing Tim-3 was observed as early as in primary HIV-1 infection, and appeared to be partially restored by highly active antiretroviral therapy (HAART) (Figure 1C). The lowest proportions of Tim-3+ NK cells were found in viremic controllers (p = 0.002 and p = 0.02 compared to healthy controls and untreated progressors, respectively), yet there was an overall high degree of variability in Tim-3 expression among these subjects (median, 48; IQR 38.7-64.3), which could not be attributed to differences in any of the demographic or clinical data analyzed (data not shown). Interestingly, elite controllers had similar percentages of Tim-3+ NK cells (median, 58.2; IQR, 47–62.7) as untreated subjects with chronic viremic HIV-1 infection (median, 62, IQR, 55.1-67).

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