Galectin-9/TIM-3 Interaction Regulates Virus-Specific Primary and Memory CD8+ T Cell Response

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 α-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

An approach to control relapse of inflammatory lesions after discontinuation of primary therapy.

Long-term treatment with the fungal metabolite drug FTY720 (Fingolimod) was shown to be highly effective in controlling viral immunopathological lesions. However, in this report we show that the anti-inflammatory effect of FTY720 in herpes simplex virus-1 (HSV-1) induced ocular inflammation is lost upon the discontinuation of treatment and lesions rapidly recurred. The lesions that developed after FTY720 treatment withdrawal involved mainly Th17 cells rather than Th1 cells explained in part by differential expression of surface CD103, an integrin that permits migration of effector cells to inflammatory sites. The expression of IL-6, a proinflammatory cytokine involved in the generation of Th17 cells, was found to be increased in FTY treated mice as compared to controls and this effect could be abrogated upon administration of neutralizing antibody to IL-6. Furthermore, IL-17RKO mice failed to show the recurrence of stromal keratitis (SK) lesions upon FTY720 withdrawal. These results indicate that approaches such as neutralization of proinflammatory cytokines might be considered along with FTY720 treatment if interruption of drug therapy becomes necessary

CD4+ T Cells Are Required for the Priming of CD8+ T Cells following Infection with Herpes Simplex Virus Type 1▿ †

The role of CD4+ helper T cells in modulating the acquired immune response to herpes simplex virus type 1 (HSV-1) remains ill defined; in particular, it is unclear whether CD4+ T cells are needed for the generation of the protective HSV-1-specific CD8+-T-cell response. This study examined the contribution of CD4+ T cells in the generation of the primary CD8+-T-cell responses following acute infection with HSV-1. The results demonstrate that the CD8+-T-cell response generated in the draining lymph nodes of CD4+-T-cell-depleted C57BL/6 mice and B6-MHC-II−/− mice is quantitatively and qualitatively distinct from the CD8+ T cells generated in normal C57BL/6 mice. Phenotypic analyses show that virus-specific CD8+ T cells express comparable levels of the activation marker CD44 in mice lacking CD4+ T cells and normal mice. In contrast, CD8+ T cells generated in the absence of CD4+ T cells express the interleukin 2 receptor α-chain (CD25) at lower levels. Importantly, the CD8+ T cells in the CD4+-T-cell-deficient environment are functionally active with respect to the expression of cytolytic activity in vivo but exhibit a diminished capacity to produce gamma interferon and tumor necrosis factor alpha. Furthermore, the primary expansion of HSV-1-specific CD8+ T cells is diminished in the absence of CD4+-T-cell help. These results suggest that CD4+-T-cell help is essential for the generation of fully functional CD8+ T cells during the primary response to HSV-1 infection

Dendritic Cells Are Required for Optimal Activation of Natural Killer Functions following Primary Infection with Herpes Simplex Virus Type 1▿

Natural killer (NK) cells play an important role in the optimal clearance of herpes simplex virus type 1 (HSV-1) infection in mice. Activated NK cells function via cytokine secretion or direct cytolysis of target cells; dendritic cells (DCs) are thought to make critical contributions in the activation of both of these functions. Yet, the magnitude and physiological relevance of DC-mediated NK cell activation in vivo is not completely understood. To examine the contribution of DC help in regulating NK cell functions after infection with HSV-1, we utilized a transgenic mouse model that allows the transient ablation of DCs. Using this approach, it was found that the gamma interferon (IFN-γ) expression potential of NK cells is quantitatively and qualitatively impaired in the absence of DCs. With regard to priming of NK cytolytic functions, the ablation of DCs did not significantly affect cytotoxic protein expression by NK cells. An in vivo cytolytic assay did, however, reveal impairments in the magnitude of NK cell cytotoxicity. Overall, this study provides direct evidence that functional DCs are required for optimal IFN-γ expression and cytolytic function by NK cells following infection with HSV-1

Continued therapy with FTY720 reduces the severity of SK lesions while discontinued treatment leads to relapse.

<p>C57BL/6 mice infected with 1×10⁴ PFU of HSV-1 were treated with FTY720 with various concentrations (0.03, 0.3 and 3.0 mg/Kg body weight) starting from 24 h pi. (A–B) FTY720 treatment depletes T cells from the periphery. (A) FACS plots showing the frequencies of CD4⁺ and CD8⁺ T cells from peripheral blood of FTY720 treated and control mice gated on total live cell population. (B) Bar graphs represents the frequencies of CD4⁺ and CD8⁺ T cells in blood isolated from FTY720 treated and control mice. (C) SK disease progression was measured in FTY720 and control groups at different time points till day 15 pi. (n = 5 to 6 mice per group). D–E. SK lesion (D) and angiogenesis (E) scores in different groups of mice on 15 dpi is shown. One-way ANOVA with Bonferroni’s multiple comparison test was used to calculate significance between control and FTY720 treated groups. (F) Bar graph depicts disease incidence (SK score >2.0) in various groups of mice measured on day 15 pi. Data represents mean ± SEM of at least two independent experiments. (G) FACS plots represent the frequencies of CD4⁺ T cell infiltration in various groups of mice. Numbers in the plots represent the cumulative data with SEM in indicated groups of mice. (H) Bar graphs showing the number of CD45⁺ CD11b⁺ double positive cells (macrophages/monocytes/dendritic cells) and CD45⁺CD11b⁺Gr1⁺ neutrophils in the corneas of different groups. Data represent means ± SEMs of at least two independent experiments. Statistical significance was calculated by one-way ANOVA (*p≤0.05, **p≤0.01, and ***p≤0.001).</p

FTY720 treatment delays viral clearance from infected corneas and enhance IL-6 expression.

<p>C57BL/6 mice were infected with 1×10⁴ PFU of HSV-1. One group served as infected control and the other was treated with FTY720 (0.3 mg/kg body weight) starting from 24 h pi. (A) Corneal swabs were collected from treated and control groups at various days pi. and virus titers were determined using standard plaque assays using Vero cells. 4–5 mice per group for each time point was used to quantify the viral titers. (B) IL-6 concentration measured by sandwich ELISA in the corneal homogenate samples of FTY720 treated and control groups at different time points are shown. Pooled corneas from each group were used for the ELISA quantification. Bar graphs represent the levels of IL-6 in corneal samples of different groups of mice. Statistical levels of significance were analyzed by a Student t test. Data represent means ± SEM (C). Bar graphs represent the levels of IL-6 in DLNs of different groups of mice. Statistical levels of significance were analyzed by a Student t test. Data represent means ± SEM.</p

Inhibition of Th17 generation prevents recurrence of lesions in HSV-1 infected mice.

<p>C57BL/6 and IL-17 RKO mice were infected with 1×10⁴ PFU of HSV-1. First group of mice were left untreated which served as controls. Second group of mice received FTY720 starting from 24 hrs pi. until 14 days pi. Third group of mice received FTY720 until day 10 pi. and later FTY720 treatment was discontinued. Fourth group of mice received FTY720 until day 10 pi and this group was also treated with IL-6 neutralizing antibody every alternate day until the day 14 pi. Fifth group of mice received only IL-6 neutralizing antibody treatment every alternate day until the day 14 pi. and the disease progression was observed. (A) SK progression was measured in treated and control groups at different time points till day 15 pi. (B) SK lesion scores on day 15 pi. Statistical significance was calculated by one-way ANOVA (*p≤0.05, ** p≤0.01, and ***p≤0.001). (C) Angiogenesis scores were measured in anti-IL-6 Ab treated and control groups on day 15 pi. (n = 5 to 6 mice per group). (D) The comparative analysis of disease progression in WT and IL-17 RKO mice that were given FTY720 treatment or where treatment was withdrawn. SK lesion scores were measured on day 11 and 14 pi. in different groups of mice i.e., infected controls, infected control mice treated with FTY720 (0.3 mg/kg body wt) from day 1–10, IL-17RKO mice and IL-17 RKO mice treated from day 1–10 with FTY720 (0.3 mg/kg body wt) from day 1–10 pi. Per group five mice were used and the experiments were repeated twice. Statistical significance was calculated by one-way ANOVA (*p≤0.05, **p≤0.01, and ***p≤0.001). (E) FACS plots representing the frequencies of IL-17 producing cells isolated from draining lymph nodes of various groups and stimulated in the presence of PMA/Ionomycin. (F) Cumulative data for the production of Th17 in different groups of mice is represented by bar graphs.</p

FTY720 ligation to S1P receptors promotes Tregs and Th17 cell differentiation.

<p>DO11.10 Rag2−/− mice were treated with 0.3 mg/kg body wt of FTY720 for 7 days. Splenocytes were isolated either from FTY720 treated or control animals and their TCR was stimulated under Th1, Th17 or regulatory T cell polarizing conditions in vitro for 5 days. After 5 days of incubation, cells were stained for measuring the production of cytokines and transcription factor Foxp3 by intra cellular/nuclear staining. (A) FACS plots showing the frequencies of IL-17 producing Th17 cells in FTY720 pre-treated and control groups. (B) Bar graphs for the cumulative data on the frequencies of Th17 cells in two groups. (C) Representative FACS plots showing the frequencies of IFN-γ producing Th1 cells in FTY720 pre -treated and control groups. (D) Bar graphs depict the cumulative data of IFN-γ producing cells from different replicates. (E) FACS plots depicting the frequencies of CD25 and transcription factor, Foxp3 staining in differentiated regulatory T cells in FTY720 pre-treated and control groups. (F) Bar graphs represent the cumulative frequencies of Foxp3⁺ cells in different groups. (G) Histograms show the MFI of CD25 and Foxp3 expression in polarized Tregs in control (faint lines) and FTY720 pre treated (thick lines) groups (H) Bar graphs show the cumulative data of the MFI for CD25 and Foxp3 in control and FTY720 pre-treated group. Data represent means ± SEMs of at least two independent experiments. Statistical significance was calculated by one-way ANOVA with *p≤0.05, **p≤0.01, and ***p≤0.001.</p