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. 2011 Dec 1;187(11):5745-55.
doi: 10.4049/jimmunol.1102105. Epub 2011 Oct 21.

Influence of galectin-9/Tim-3 interaction on herpes simplex virus-1 latency

Pradeep B J Reddy  1 , Sharvan Sehrawat, Amol Suryawanshi, Naveen K Rajasagi, Sachin Mulik, Mitsuomi Hirashima, Barry T Rouse
Affiliations

Influence of galectin-9/Tim-3 interaction on herpes simplex virus-1 latency

Pradeep B J Reddy et al. J Immunol. .

Abstract

After HSV-1 infection, CD8(+) T cells accumulate in the trigeminal ganglion (TG) and participate in the maintenance of latency. However, the mechanisms underlying intermittent virus reactivation are poorly understood. In this study, we demonstrate the role of an inhibitory interaction between T cell Ig and mucin domain-containing molecule 3 (Tim-3)-expressing CD8(+) T cells and galectin 9 (Gal-9) that could influence HSV-1 latency and reactivation. Accordingly, we show that most K(b)-gB tetramer-specific CD8(+) T cells in the TG of HSV-1-infected mice express Tim-3, a molecule that delivers negative signals to CD8(+) T cells upon engagement of its ligand Gal-9. Gal-9 was also upregulated in the TG when replicating virus was present as well during latency. This could set the stage for Gal-9/Tim-3 interaction, and this inhibitory interaction was responsible for reduced CD8(+) T cell effector function in wild-type mice. Additionally, TG cell cultures exposed to recombinant Gal-9 in the latent phase caused apoptosis of most CD8(+) T cells. Furthermore, Gal-9 knockout TG cultures showed delayed and reduced viral reactivation as compared with wild-type cultures, demonstrating the greater efficiency of CD8(+) T cells to inhibit virus reactivation in the absence of Gal-9. Moreover, the addition of recombinant Gal-9 to ex vivo TG cultures induced enhanced viral reactivation compared with untreated controls. Our results demonstrate that the host homeostatic mechanism mediated by Gal-9/Tim-3 interaction on CD8(+) T cells can influence the outcome of HSV-1 latent infection, and manipulating Gal-9 signals might represent therapeutic means to inhibit HSV-1 reactivation from latency.

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Figures

Figure 1
Figure 1. Tim-3 expression is up regulated on Kb-gB tetramer specific CD8+ T cells after HSV-1 infection
C57BL/6 WT and Gal-9 KO mice were ocularly infected with 1 ×105 HSV-1 and TGs isolated from four mice at each time point were processed and analyzed by flow cytometry (A) Representative FACS plots depicting the infiltration of macrophages on day 4 pi stained for CD45, CD11b and F4/80 markers in collagenase-digested TG from WT and Gal-9 KO mice. (B) Representative FACS plots depicting the absence of T cells on day 4 pi stained for CD45 and CD3 markers in WT mice. (C) Representative FACS plots showing the infiltration of CD8+ and CD4+ T cells in the TG of WT mice at day 8 pi gated on total CD45 and CD3 population. (D) Representative bar graphs showing the percentage of CD8+ and CD4+ T cells in TG of WT mice on day 8 pi (E) The bar diagrams represents the absolute numbers of CD8+ T cells in WT vs Gal-9 KO mice at different times pi Data represents the mean ± SEM of 3 independent experiments. (F) Frequencies of total CD8+ T cells and Kb-gB-tetramer+ CD8+ T cells expressing Tim-3 in TG of WT mice at different time points pi. Representative FACS plots showing the expression of (G) TIM-3+ on CD45+ CD3+ CD8+ T cells and (H) the frequencies of Tim-3 expressing Kb-gB-tetramer+ specific cells is represented as a bar graph (The cells present in the upper left (Tet+ Tim-3−) and upper right (Tet+ Tim-3+) quadrants are considered as total Tet+ cells and the percentages represented in the bar graphs were calculated from these two quadrants). Representative FACS plots depicting the expression of activation markers. (I) CD69 expression on CD8+ Tim-3+ cells. (J) Bar graph representing the frequencies of CD69+ and Tim-3+ cells (The cells present in the upper left (CD69+ Tim-3−) and upper right (CD69+ Tim-3+) quadrants are total CD69+ cells and the frequencies represented in the bar graphs were calculated from these two quadrants). (K) FACS plot representing CD44 expression on CD8+ Tim-3+ cells. (L) Bar graph represents the frequencies of CD44+ and Tim-3+ cells in the TG resident CD8+ T cells (The cells present in the upper left (CD44+ Tim-3−) and upper right (CD44+ Tim-3+) quadrants are total CD44+ cells and the frequencies represented in the bar graphs were calculated from these two quadrants). (M) Single cell suspensions of cervical lymph node (CLN) obtained from HSV-1 infected mice at different time points pi were stained for CD45, CD3, CD8, Kb-gB tetramer+ and Tim-3 markers. Representative FACS plots showing the expression of Tim-3 marker on CD8+ Kb-gB tetramer+ cells. (N) Representative FACS plots showing the expression of Tim-3 gated on CD45+ CD4+ T cells of WT TG on day 14 pi. Data represents the mean ± SEM of 3 independent experiments. All the data were analyzed with student’s t test.
Figure 2
Figure 2. Gal-9 KO mice show diminished numbers of regulatory T cells in TG
C57BL/6 WT and Gal-9KO mice were infected ocularly with 1 ×105 HSV-1 (RE) and TGs isolated from infected WT and Gal-9 KO mice were analyzed for presence of Foxp3+ CD4+ regulatory T cells. (A) & (B) Representative FACS plots depicting the frequencies of total CD4+ T cells and Foxp3+ T cells in the TG of HSV-1 infected mice on 14 and 32 days pi respectively. (C) and (D) Bar diagrams representing the frequencies and absolute numbers of regulatory T cells in the WT and Gal-9 KO mice on 14 and 32 days pi respectively. Statistical levels of significance were analyzed by student’s t test. Error bars are mean ± SEM.
Figure 3
Figure 3. Gal-9 expression is up regulated in TG after HSV-1 infection
TGs from mice ocularly infected with 1× 105 pfu of HSV-1 were isolated on different times pi and Gal-9 detection and quantification was performed by different methods. (A) TGs isolated from 3 mice at each time point were pooled and Gal-9 expression was detected by western blotting. Immunoblots showing Gal-9 expression at different times pi (lane #1-naive; #2-day 4; #3-day 6; #4-day 14; #5-day 32; and lane 6 recombinant Gal-9. Expression of β-actin as sample loading control is shown in the bottom panel. (B) Gal-9 concentration as measured by sandwich ELISA in the TG homogenates is shown. The experiments were performed three times and four mice were sacrificed at each time point and the pooled TGs were used for the analysis. Statistical levels of signicance were analyzed by student’s t test. Data are represented as mean ± SEM. (C) Gal-9 expression was measured by qPCR in the homogenates of TGs obtained from HSV-1 infected mice at different time points pi. The experiments were repeated three times and three mice were sacrificed at each time point and the individual TGs were used for the RNA extraction. Data are represented as mean ± SEM (D) Representative FACS plots showing the expression of Gal-9 on macrophages stained for CD45, CD11b, F4/80 and Galectin-9 on day 10 pi (E) TGs isolated from HSV-1 infected WT mice at different time points pi were analyzed for macrophage infiltration. Representative FACS plots showing the macrophages stained for CD45, CD11b, F4/80 markers. (F) Neuronal cells in TG express Gal-9 after HSV-1 infection. Representative photomicrographs of double fluorescent immunohistochemistry staining for NeuN+ and Gal-9+ cells in the WT TG isolated from day 14 pi mice.
Figure 4
Figure 4. Gal-9 KO mice mount stronger CD8+ T cell responses in TG compared to WT mice
Kb-gB tetramer specific CD8+ T cell responses in TG of HSV-1 infected mice were compared among age and gender matched HSV infected wild type (WT) and Gal-9 KO animals at 14 and 32 days pi TGs were excised and pooled (n=4) from WT and Gal-9 KO mice, dispersed into single-cell suspensions and stimulated for 6 h with or without HSV-1 gB peptide at 1 µg/ml concentrations, in the presence of brefeldin A. For optimal surface staining of CD107 marker, the FITC conjugated CD107 antibody was added to the culture during stimulation. After incubation, the cells were stained for surface marker CD8 and cytokines IFN-γ, TNF-α and GrB by intracellular staining and analyzed by flow cytometry. Representative FACS plots depicting IFN-γ and TNF-α production in WT vs Gal-9 KO mice on (A) day 14 and (B) day 32. (C–D) Bar diagrams represent mean ± SEM frequencies of TG CD8+ cells producing IFN-γ and TNF-α and the cells producing both IFN-γ and TNF- α. (E) Mean Fluorescence intensity (MFI) of the cytokines produced are shown. Experiments were repeated 3 times and the values are means ± SEM. (F) Gal-9 KO CD8+ T cells proliferate better than WT mice. TG cells were stained with CFSE and cultured for 72 h with or without added CD3/CD28. After incubation, the cells were stained with anti-CD8 α mAb and CFSE staining intensity of CD8+ T cells was analyzed by flow cytometry. A representative histogram shows the extent of CFSE dilution in TG resident CD8+ T cells from WT and Gal-9KO mice gated on CD45 and CD8+ T cells. (G) FACS plots representing the expression of Granzyme B and CD107 by specific TG resident CD8+ T cells in WT and Gal-9 KO mice on day 14 and 32 pi. All of the above experiments were repeated three times with similar results and data represent the mean ± SEM. *P<0.05; **p<0.01; ***p<0.001.
Figure 5
Figure 5. Gal-9 induces apoptosis of Tim-3 expressing TG resident CD8+ T cells
TG single cell suspensions isolated from HSV-1 infected C57BL/6 WT animals on 32 days pi were incubated for 5 h with varying concentrations of rGal-9 in the absence or the presence of α-lactose in a 96 well flat bottomed plate. After incubation the cells were stained for CD8, Tim-3, Kb-gB-tetramer and annexin-V markers. The experiments were repeated multiple times with similar results. FACS plots showing the CD8+ T cells under indicated incubation conditions. (A) Representative FACS plots showing the expression of Tim-3 and annexin-V on gated CD8+ T cells. (B) Representative FACS plots showing the expression of Kb-gB tetramer and annexin-V on gated CD8+ T cells. (C) Representative FACS plots showing the apoptosis gated on total CD8+ T cells in the presence of rGal-9. Lower panels of 5A, 5B and 5C represent the apoptosis of CD8+ T cells in the presence of α-lactose.
Figure 6
Figure 6. Influence of Gal-9/Tim-3 interaction on HSV-1 reactivation
(A) CD8+ T cells in Gal-9 KO TG cultures delays virus reactivation and also reduces the total viral titers. CD8+ T cells present in the TG 32 days after HSV-1 corneal infection can delay HSV-1 reactivation but cannot completely inhibit reactivation. TGs from WT and Gal-9 KO mice were excised 32 day after HSV-1 corneal infection, and suspensions were prepared and pooled. Cultures (1.5 TG equivalent) were incubated in 10% DMEM. Cultures were incubated in medium alone. Samples of culture supernatant fluids were assayed for infectious HSV-1 titers by standard plaque titrations. Pooled data from 3 assays (n = 9) are presented as the Log10 pfu/culture. (B) Addition of rGal-9 to WT TG cultures induced virus reactivation. TG resident CD8+ T cells in day 14 pi inhibits HSV-1 reactivation in ex vivo TG cultures and depletion of these CD8+ T cells triggers virus reactivation. In this experiment, we demonstrate that addition of rGal-9 to day 14 TG cultures induced virus reactivation similar to CD8+ T cell depletion. TGs isolated from WT mice on day 14 pi were used in these experiments. TGs excised on 14 days pi from WT mice were pooled and dispersed into single cell-suspensions. Each experiment was performed in a different plate to avoid cross contamination. Cultures were incubated in culture medium with or without rGal-9 (1 µM) for 8 days. CD8+ T cells were depleted in few wells by using dynabeads. Samples of culture supernatant were collected at 24 h intervals and assayed for infectious virus by plaque titrations. Pooled data from three assays (n=9) are presented as the Log10 pfu/culture. (C) Addition of rGal-9 to the WT 32 day pi ex vivo TG cultures induced enhanced HSV-1 reactivation. TGs isolated from WT mice are used in these experiments. TGs excised from WT mice on 32 days pi were pooled and dispersed into single cell-suspensions. Cultures were incubated in 10% DMEM with or without rGal-9 (1 µM). Samples of culture supernatant were collected at 24 h intervals and assayed for infectious HSV-1 titers by standard plaque titrations. Pooled data from 3 assays (n = 9) are presented as the Log10 pfu/culture. Data is represented as mean ± SEM. *P<0.05; **p<0.01; ***p<0.001.
Figure 7
Figure 7. Viral replication titers in cornea and TG of WT and Gal-9 KO mice
WT and Gal-9KO mice were infected with HSV-1 (RE) at 1 ×105 PFU/eye and mice were sacrificed on days 2, 4, 6 and 8 pi. (A) Eyeballs were homogenized in tissue grinders and subjected to two freeze thaw cycles and viral titers were determined on Vero cells. The viral titers were shown as means ± SEM. (B) TGs were excised, homogenized and subjected to two freeze thaw cycles prior to determination of viral titers by plaque assay. Titers of HSV-1 in cornea or TG of WT and Gal-9 KO mice were not significantly different (p>0.05) at any time point as assessed by Student’s t test.
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