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Galectin-9/TIM-3 interaction regulates virus-specific primary and memory CD8 T cell response.

Sehrawat S, Reddy PB, Rajasagi N, Suryawanshi A, Hirashima M, Rouse BT - PLoS Pathog. (2010)

Bottom Line: In support of this, we show that animals unable to produce Gal-9, because of gene knockout, develop acute and memory responses to HSV that are of greater magnitude and better quality than those that occur in normal infected animals.The increased effector T cell responses led to significantly more efficient virus control.Our results indicate that manipulating galectin signals, as can be achieved using appropriate sugars, may represent a convenient and inexpensive approach to enhance acute and memory responses to a virus infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee, USA.

ABSTRACT
In this communication, we demonstrate that galectin (Gal)-9 acts to constrain CD8(+) T cell immunity to Herpes Simplex Virus (HSV) infection. In support of this, we show that animals unable to produce Gal-9, because of gene knockout, develop acute and memory responses to HSV that are of greater magnitude and better quality than those that occur in normal infected animals. Interestingly, infusion of normal infected mice with alpha-lactose, the sugar that binds to the carbohydrate-binding domain of Gal-9 limiting its engagement of T cell immunoglobulin and mucin (TIM-3) receptors, also caused a more elevated and higher quality CD8(+) T cell response to HSV particularly in the acute phase. Such sugar treated infected mice also had expanded populations of effector as well as memory CD8(+) T cells. The increased effector T cell responses led to significantly more efficient virus control. The mechanisms responsible for the outcome of the Gal-9/TIM-3 interaction in normal infected mice involved direct inhibitory effects on TIM-3(+) CD8(+) T effector cells as well as the promotion of Foxp3(+) regulatory T cell activity. Our results indicate that manipulating galectin signals, as can be achieved using appropriate sugars, may represent a convenient and inexpensive approach to enhance acute and memory responses to a virus infection.

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Related in: MedlinePlus

Galectin-9 induces apoptosis of TIM-3 expressing CD8+ T cells in vitro and its expression is up regulated in the lymphoid organs after HSV infection.PLNs single cell suspension isolated 6 dpi from HSV infected animals were incubated for 5 hr with varying concentrations of galectin-9 in the absence or the presence of α-lactose. The experiments were repeated multiple times with similar results. A. Representative FACS plots showing the expression of TIM-3 and annexin-V on gated CD8+ T cells under indicated incubation conditions are shown. B. The bar diagram shows the numbers of TIM-3+CD8+ T cells as calculated from A. C. Representative FACS plots showing the expression of Kb-gB-Tet and annexin-V on gated CD8+ T cells under indicated incubation conditions are shown. D. The bar diagram shows the numbers of TKb-gB-Tet+CD8+ T cells as calculated from C. E. TIM-3 and annexin-V expression on gated CD8+ T cells isolated from PLNs of HSV infected mice at 6 dpi and incubated for 5 hr with different concentrations of galectin-3 is shown. F. The bar diagram shows the numbers of TIM-3+CD8+ T cells as calculated from E. G. Immunoblots showing galectin-9 expression in the homogenates of isolated PLNs (upper panel) and spleens (middle panel) obtained from HSV infected animals at different time points are shown. Expression of β-actin as sample loading control is shown in the bottom panel. (lane # 1, day 0; #2, day 2, #3, day 3.5, #4, day 5, #5, day 8 and #6, recombinant galectin-9). H. Galectin-9 concentrations as measured by sandwich ELISA using anti-Gal-9 mAb (1A2) in the PLN homogenates is shown. The experiments were performed three times and three animals were sacrificed at each time point.
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ppat-1000882-g003: Galectin-9 induces apoptosis of TIM-3 expressing CD8+ T cells in vitro and its expression is up regulated in the lymphoid organs after HSV infection.PLNs single cell suspension isolated 6 dpi from HSV infected animals were incubated for 5 hr with varying concentrations of galectin-9 in the absence or the presence of α-lactose. The experiments were repeated multiple times with similar results. A. Representative FACS plots showing the expression of TIM-3 and annexin-V on gated CD8+ T cells under indicated incubation conditions are shown. B. The bar diagram shows the numbers of TIM-3+CD8+ T cells as calculated from A. C. Representative FACS plots showing the expression of Kb-gB-Tet and annexin-V on gated CD8+ T cells under indicated incubation conditions are shown. D. The bar diagram shows the numbers of TKb-gB-Tet+CD8+ T cells as calculated from C. E. TIM-3 and annexin-V expression on gated CD8+ T cells isolated from PLNs of HSV infected mice at 6 dpi and incubated for 5 hr with different concentrations of galectin-3 is shown. F. The bar diagram shows the numbers of TIM-3+CD8+ T cells as calculated from E. G. Immunoblots showing galectin-9 expression in the homogenates of isolated PLNs (upper panel) and spleens (middle panel) obtained from HSV infected animals at different time points are shown. Expression of β-actin as sample loading control is shown in the bottom panel. (lane # 1, day 0; #2, day 2, #3, day 3.5, #4, day 5, #5, day 8 and #6, recombinant galectin-9). H. Galectin-9 concentrations as measured by sandwich ELISA using anti-Gal-9 mAb (1A2) in the PLN homogenates is shown. The experiments were performed three times and three animals were sacrificed at each time point.

Mentions: The results of previous in vitro studies have revealed that Gal-9 binding to TIM-3 receptors on some, although not all, T cell subsets causes them to undergo apoptosis [8], [16]. To test the fate of the CD8+TIM3+ population expanded by HSV infection to Gal-9 exposure, PLN cells were collected 6 days pi and exposed in vitro for 5 hrs to a range of concentrations of Gal-9. Subsequently, the cells were analyzed by FACS for changes in the expression levels of TIM-3 and annexin V, the latter indicative of apoptosis [17]. As shown in Fig 3A, approximately 15% of CD8+ T cells were TIM-3+ at the onset of culture and this percentage did not change significantly in the absence of Gal-9. However, Gal-9 addition (at1.0 µM) caused a loss of almost all cells that were TIM-3+ (Fig 3A upper panel). Baseline levels of annexin V+ cells also did not change significantly in the absence of Gal-9 (or in the presence of Gal-3, as shown in Fig 3E-F). However, when optimal amounts of Gal-9 were present, annexin V+ cells increased 15–20% beyond baseline numbers (Fig 3A upper panel and 3B). This number roughly correlated with the fraction of TIM-3+CD8+ T cells that disappeared upon Gal-9 exposure. Under the conditions used, we failed to detect significant numbers of TIM-3+ annexin V+ cells, although at earlier time points some such cells can be demonstrated (data not shown). In additional experiments, we measured the effects of Gal-9 addition on the fate of Tet+ TIM-3+ CD8+ T cells, almost all of which as described previously were TIM-3+. As shown in Fig 3C (upper panel) and 3D, the great majority of Tet+ TIM-3+ T cells were lost after Gal-9 exposure and there was a large increase in annexin V+ T cells.


Galectin-9/TIM-3 interaction regulates virus-specific primary and memory CD8 T cell response.

Sehrawat S, Reddy PB, Rajasagi N, Suryawanshi A, Hirashima M, Rouse BT - PLoS Pathog. (2010)

Galectin-9 induces apoptosis of TIM-3 expressing CD8+ T cells in vitro and its expression is up regulated in the lymphoid organs after HSV infection.PLNs single cell suspension isolated 6 dpi from HSV infected animals were incubated for 5 hr with varying concentrations of galectin-9 in the absence or the presence of α-lactose. The experiments were repeated multiple times with similar results. A. Representative FACS plots showing the expression of TIM-3 and annexin-V on gated CD8+ T cells under indicated incubation conditions are shown. B. The bar diagram shows the numbers of TIM-3+CD8+ T cells as calculated from A. C. Representative FACS plots showing the expression of Kb-gB-Tet and annexin-V on gated CD8+ T cells under indicated incubation conditions are shown. D. The bar diagram shows the numbers of TKb-gB-Tet+CD8+ T cells as calculated from C. E. TIM-3 and annexin-V expression on gated CD8+ T cells isolated from PLNs of HSV infected mice at 6 dpi and incubated for 5 hr with different concentrations of galectin-3 is shown. F. The bar diagram shows the numbers of TIM-3+CD8+ T cells as calculated from E. G. Immunoblots showing galectin-9 expression in the homogenates of isolated PLNs (upper panel) and spleens (middle panel) obtained from HSV infected animals at different time points are shown. Expression of β-actin as sample loading control is shown in the bottom panel. (lane # 1, day 0; #2, day 2, #3, day 3.5, #4, day 5, #5, day 8 and #6, recombinant galectin-9). H. Galectin-9 concentrations as measured by sandwich ELISA using anti-Gal-9 mAb (1A2) in the PLN homogenates is shown. The experiments were performed three times and three animals were sacrificed at each time point.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2865527&req=5

ppat-1000882-g003: Galectin-9 induces apoptosis of TIM-3 expressing CD8+ T cells in vitro and its expression is up regulated in the lymphoid organs after HSV infection.PLNs single cell suspension isolated 6 dpi from HSV infected animals were incubated for 5 hr with varying concentrations of galectin-9 in the absence or the presence of α-lactose. The experiments were repeated multiple times with similar results. A. Representative FACS plots showing the expression of TIM-3 and annexin-V on gated CD8+ T cells under indicated incubation conditions are shown. B. The bar diagram shows the numbers of TIM-3+CD8+ T cells as calculated from A. C. Representative FACS plots showing the expression of Kb-gB-Tet and annexin-V on gated CD8+ T cells under indicated incubation conditions are shown. D. The bar diagram shows the numbers of TKb-gB-Tet+CD8+ T cells as calculated from C. E. TIM-3 and annexin-V expression on gated CD8+ T cells isolated from PLNs of HSV infected mice at 6 dpi and incubated for 5 hr with different concentrations of galectin-3 is shown. F. The bar diagram shows the numbers of TIM-3+CD8+ T cells as calculated from E. G. Immunoblots showing galectin-9 expression in the homogenates of isolated PLNs (upper panel) and spleens (middle panel) obtained from HSV infected animals at different time points are shown. Expression of β-actin as sample loading control is shown in the bottom panel. (lane # 1, day 0; #2, day 2, #3, day 3.5, #4, day 5, #5, day 8 and #6, recombinant galectin-9). H. Galectin-9 concentrations as measured by sandwich ELISA using anti-Gal-9 mAb (1A2) in the PLN homogenates is shown. The experiments were performed three times and three animals were sacrificed at each time point.
Mentions: The results of previous in vitro studies have revealed that Gal-9 binding to TIM-3 receptors on some, although not all, T cell subsets causes them to undergo apoptosis [8], [16]. To test the fate of the CD8+TIM3+ population expanded by HSV infection to Gal-9 exposure, PLN cells were collected 6 days pi and exposed in vitro for 5 hrs to a range of concentrations of Gal-9. Subsequently, the cells were analyzed by FACS for changes in the expression levels of TIM-3 and annexin V, the latter indicative of apoptosis [17]. As shown in Fig 3A, approximately 15% of CD8+ T cells were TIM-3+ at the onset of culture and this percentage did not change significantly in the absence of Gal-9. However, Gal-9 addition (at1.0 µM) caused a loss of almost all cells that were TIM-3+ (Fig 3A upper panel). Baseline levels of annexin V+ cells also did not change significantly in the absence of Gal-9 (or in the presence of Gal-3, as shown in Fig 3E-F). However, when optimal amounts of Gal-9 were present, annexin V+ cells increased 15–20% beyond baseline numbers (Fig 3A upper panel and 3B). This number roughly correlated with the fraction of TIM-3+CD8+ T cells that disappeared upon Gal-9 exposure. Under the conditions used, we failed to detect significant numbers of TIM-3+ annexin V+ cells, although at earlier time points some such cells can be demonstrated (data not shown). In additional experiments, we measured the effects of Gal-9 addition on the fate of Tet+ TIM-3+ CD8+ T cells, almost all of which as described previously were TIM-3+. As shown in Fig 3C (upper panel) and 3D, the great majority of Tet+ TIM-3+ T cells were lost after Gal-9 exposure and there was a large increase in annexin V+ T cells.

Bottom Line: In support of this, we show that animals unable to produce Gal-9, because of gene knockout, develop acute and memory responses to HSV that are of greater magnitude and better quality than those that occur in normal infected animals.The increased effector T cell responses led to significantly more efficient virus control.Our results indicate that manipulating galectin signals, as can be achieved using appropriate sugars, may represent a convenient and inexpensive approach to enhance acute and memory responses to a virus infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee, USA.

ABSTRACT
In this communication, we demonstrate that galectin (Gal)-9 acts to constrain CD8(+) T cell immunity to Herpes Simplex Virus (HSV) infection. In support of this, we show that animals unable to produce Gal-9, because of gene knockout, develop acute and memory responses to HSV that are of greater magnitude and better quality than those that occur in normal infected animals. Interestingly, infusion of normal infected mice with alpha-lactose, the sugar that binds to the carbohydrate-binding domain of Gal-9 limiting its engagement of T cell immunoglobulin and mucin (TIM-3) receptors, also caused a more elevated and higher quality CD8(+) T cell response to HSV particularly in the acute phase. Such sugar treated infected mice also had expanded populations of effector as well as memory CD8(+) T cells. The increased effector T cell responses led to significantly more efficient virus control. The mechanisms responsible for the outcome of the Gal-9/TIM-3 interaction in normal infected mice involved direct inhibitory effects on TIM-3(+) CD8(+) T effector cells as well as the promotion of Foxp3(+) regulatory T cell activity. Our results indicate that manipulating galectin signals, as can be achieved using appropriate sugars, may represent a convenient and inexpensive approach to enhance acute and memory responses to a virus infection.

Show MeSH
Related in: MedlinePlus