As opposed to the peritumor regions, the amount of CD3+ T cells and the amount of PD-L1+ cells were positively correlated in the intratumor regions (Figure 1E, remaining)

As opposed to the peritumor regions, the amount of CD3+ T cells and the amount of PD-L1+ cells were positively correlated in the intratumor regions (Figure 1E, remaining). Syngeneic mouse model tests proven that TAMs indicated PD-L1 and tumors treated with anti-PD-L1 demonstrated smaller sized diameters than those treated with IgG. In these mice, anti-PD-L1 treatment improved activation markers in intratumoral Compact disc8+ T cells and decreased how big is the TAM inhabitants. Regarding nivolumab-treated individuals, three of eight individuals taken care of immediately the anti-PD-1 treatment. The percentage of Ki-67-positive CD8+ and CD4+ T cells was higher in responders than non-responders after nivolumab. Overall, PD-L1 manifestation on TAMs may be targeted by immune-based HCC treatment, and ICI treatment leads to the reinvigoration of tired Compact disc8+ T cells in HCC. < 0.001) (Shape 1ACE). Specifically, Compact disc3+ T cells and Compact disc68+ macrophages had been confirmed to become distributed in various patterns, and PD-L1 was indicated in an identical pattern to Compact disc68 (Shape 1A). Moreover, the amount of PD-L1-expressing cells favorably correlated with the amount of Compact disc68+ macrophages (Shape 1D middle), however, not with the amount of Compact disc3+ T cells (Shape 1D remaining). The amount of PD-L1+ cells in the intratumoral area demonstrated no significant relationship with the amount of Compact disc68+ macrophages (Shape 1E middle). As opposed to the peritumor areas, the amount of Compact disc3+ T cells and the amount of PD-L1+ cells had been favorably correlated in the intratumor areas (Shape 1E, remaining). Finally, we compared the amount of PD-L1+ cells in the peritumor and intratumor areas with the focus of serum alpha fetoprotein (AFP) and verified that there is no relationship (Shape 1D, figure and right 1E, right). Open up in another home window Shape 1 correlations and Patterns of Compact disc3, Compact disc68, and PD-L1-expressing cells in human being HCC cells: (A) a representative design of Compact disc3, Compact disc68, and PD-L1 manifestation in human cells acquired through liver organ resection; (B,C) the amount of Compact disc3+ T cells, Compact disc68+ macrophages, and PD-L1+ cells situated in peritumoral and intratumoral area. *** < 0.001; (DCE) relationship of Compact disc3+ T cells, Compact disc68+ macrophages, serum AFP, and PD-L1+ cells situated in peritumoral and intratumoral area (= 33). Abbreviations: AFP, alpha fetoprotein; HCC, hepatocellular carcinoma; PD-L1, designed loss of life ligand 1. 2.2. Improvement in Compact disc4+ and Compact disc8+ T Cell Features after PD-L1 Manifestation Blockade on M2 Macrophages Following, we researched whether Compact disc8+ and Compact disc4+ T cell features are induced upon the blockade of PD-L1 manifestation on M2 macrophages. We isolated PBMCs from healthful donor blood and stained them with Compact disc3 and Compact disc14 microbeads for magnetic cell sorting. Compact disc14+ cells were polarized into M2 macrophages through treatment with M-CSF and IL-4 after that. After polarization, Compact disc3+ T cell co-culture tests had been performed. In co-cultures, we noticed functional enhancements from the Compact disc8+ T cells co-cultured with PD-L1-pretreated M2 macrophages. The amounts of Compact disc8+ IFN-+ T and Compact disc8+ TNF-+ T cells considerably elevated by 5% to 10% and 8% to 10%, respectively (Amount 2A,B). Furthermore, PD-1 and Compact disc69 expression considerably elevated on PMA/Ionomycin-activated Compact disc8+ T cells after PD-L1 blockade on M2 macrophages (Supplementary Amount S1A,B). In keeping with the observations reported for Compact disc8+ T cells, PMA/Ionomycin-activated Compact disc4+ INF-+ T cells elevated by around 8% to 14%, as the Compact disc4+ TNF-+ T cell people increased by around 7% to 9% (Amount 2C,D). Further, Compact disc4+ T cells demonstrated a rise in the appearance of PD-1 and Compact disc69 after PD-L1 appearance blockade on M2 macrophages (Supplementary Amount S1C,D). Open up in another window Amount 2 Functional improvement of Compact disc8+ and Compact disc4+ T cells after co-culture with anti-PD-L1-treated macrophages: (A,B) appearance and MFI of (A) IFN-, and (B) TNF-, in Compact disc8+ T cells when Compact disc3+ T cells had been co-cultured with PD-L1-obstructed macrophages (= 3) * < 0.05, ** < 0.01; (C,D) appearance and MFI of (C) IFN-, and (D) TNF- in Compact disc4+ T cells when Compact disc3+ T cells had been co-cultured with PD-L1-obstructed macrophages (= 3) * < 0.05, ** < 0.01; (E) test schedule for parting of T cells and macrophages in the tissues obtained by hepatic resection; (F,G) differential appearance of IFN- and TNF- in Compact disc8+ and Compact disc4+ T cells when Compact disc3+ T cells had been co-cultured with PD-L1-obstructed Compact disc206+ macrophages and control cells from individual tissues obtained through liver organ resection. Abbreviations: IFN-, interferon-; MFI, mean fluorescence strength; PD-L1, programmed loss of life ligand 1; TNF-, tumor necrosis aspect-. Next, we performed very similar tests using Compact disc206+ macrophages and Compact disc3+ T cells newly isolated in the HCC tissues of an individual. PD-L1 appearance was obstructed on Compact disc206+ macrophages using anti-PD-L1 antibody, and these cells had been co-cultured with PMA/Ionomycin-activated Compact disc3+ T cells (Amount 2E). We discovered that the populace of Compact disc8+ INF-+ T cells elevated by around 8% to 13%, while that of Compact disc4+ INF-+ T cells elevated by.We demonstrated which the function of Compact disc8+ and Compact disc4+ T cells was restored by inhibiting the appearance of PD-L1 in macrophages. significant upsurge in T cell efficiency following the pretreatment of M2 macrophages with anti-PD-L1. Syngeneic mouse model tests showed that TAMs portrayed PD-L1 and tumors treated with anti-PD-L1 demonstrated smaller sized diameters than those treated with IgG. In these mice, anti-PD-L1 treatment elevated activation markers in intratumoral Compact disc8+ T cells and decreased how big is the TAM people. Regarding nivolumab-treated sufferers, three of eight sufferers taken care of immediately the anti-PD-1 treatment. The percentage of Ki-67-positive Compact disc4+ and Compact MX1013 disc8+ T cells was higher in responders than nonresponders after nivolumab. General, PD-L1 appearance on TAMs could be targeted by immune-based HCC treatment, and ICI treatment leads to the reinvigoration of fatigued Compact disc8+ T cells in HCC. < 0.001) (Amount 1ACE). Specifically, Compact disc3+ T cells and Compact disc68+ macrophages had been confirmed to end up being distributed in various patterns, and PD-L1 was portrayed in an identical pattern to Compact disc68 (Amount 1A). Moreover, the amount of PD-L1-expressing cells favorably correlated with the amount of Compact disc68+ macrophages (Amount 1D middle), however, not with the amount of Compact disc3+ T cells (Amount 1D still left). The amount of PD-L1+ cells in the intratumoral area demonstrated no significant relationship with the amount of Compact disc68+ macrophages (Amount 1E middle). As opposed to the peritumor locations, the amount of Compact disc3+ T cells and the amount of PD-L1+ cells had been favorably correlated in the intratumor locations (Amount 1E, still left). Finally, we compared the amount of PD-L1+ cells in the peritumor and intratumor locations with the focus of serum alpha fetoprotein (AFP) and verified that there is no relationship (Body 1D, correct and MX1013 Body 1E, correct). Open up in another window Body 1 Patterns and correlations of Compact disc3, Compact disc68, and PD-L1-expressing cells in individual HCC tissue: (A) a representative design of Compact disc3, Compact disc68, and PD-L1 appearance in human tissue acquired through liver organ resection; (B,C) the amount of Compact disc3+ T cells, Compact disc68+ macrophages, and PD-L1+ cells situated in intratumoral and peritumoral area. *** < 0.001; (DCE) relationship of Compact disc3+ T cells, Compact disc68+ macrophages, serum AFP, and PD-L1+ cells situated in peritumoral and intratumoral area (= 33). Abbreviations: AFP, alpha fetoprotein; HCC, hepatocellular carcinoma; PD-L1, designed loss of life ligand 1. 2.2. Improvement in Compact disc8+ and Compact disc4+ T Cell Features after PD-L1 Appearance Blockade on M2 Macrophages Following, we examined whether Compact disc8+ and Compact disc4+ T cell features are induced upon the blockade of PD-L1 appearance on M2 macrophages. We isolated PBMCs from healthful donor bloodstream and stained them with Compact disc14 and Compact disc3 microbeads for magnetic cell sorting. Compact disc14+ cells had been after that polarized into M2 macrophages through treatment with M-CSF and IL-4. After polarization, Compact disc3+ T cell co-culture tests had been performed. In co-cultures, we noticed functional enhancements from the Compact disc8+ T cells co-cultured with PD-L1-pretreated M2 macrophages. The amounts of Compact disc8+ IFN-+ T and Compact disc8+ TNF-+ T cells considerably elevated by 5% to 10% and 8% to 10%, respectively (Body 2A,B). Furthermore, PD-1 and Compact disc69 expression considerably elevated on PMA/Ionomycin-activated Compact disc8+ T cells after PD-L1 blockade on M2 macrophages (Supplementary Body S1A,B). In keeping with the observations reported for Compact disc8+ T cells, PMA/Ionomycin-activated Compact disc4+ INF-+ T cells elevated by around 8% to 14%, as the Compact disc4+ TNF-+ T cell people increased by around 7% to 9% (Body 2C,D). Further, Compact disc4+ T cells demonstrated a rise in the appearance of PD-1 and Compact disc69 after PD-L1 appearance blockade on M2 macrophages (Supplementary Body S1C,D). Open up in another window Body 2 Functional improvement of Compact disc8+ and Compact disc4+ T cells after co-culture with anti-PD-L1-treated macrophages: (A,B) appearance and MFI of (A) IFN-, and (B) TNF-, in Compact disc8+ T cells when Compact disc3+ T cells had been co-cultured with PD-L1-obstructed macrophages (= 3) * < 0.05, ** < 0.01; (C,D) appearance and MFI of (C) IFN-, and (D) TNF- in Compact disc4+ T cells when Compact disc3+ T cells had been co-cultured with PD-L1-obstructed macrophages (= 3) * < 0.05, ** < 0.01; (E) test schedule for parting of T cells and macrophages in the tissues obtained by hepatic resection; (F,G) differential appearance of IFN- and TNF- in Compact disc8+ and Compact disc4+ T cells when Compact disc3+ T cells had been co-cultured with PD-L1-obstructed Compact disc206+ macrophages and control cells from individual tissues obtained through liver organ resection. Abbreviations: IFN-, interferon-; MFI, mean fluorescence strength; PD-L1, programmed loss of life ligand 1; TNF-, tumor necrosis aspect-. Next, we performed equivalent tests using Compact disc206+ macrophages and Compact disc3+ T cells newly isolated.In Vivo Mouse Model An in vivo mouse model was established seeing that described [2] previously. Compact disc8+ T cells was higher in responders than nonresponders after nivolumab. General, PD-L1 appearance on TAMs could be targeted by immune-based HCC treatment, and ICI treatment leads to the reinvigoration of fatigued Compact disc8+ T cells in HCC. < 0.001) (Body 1ACE). Specifically, Compact disc3+ T cells and Compact disc68+ macrophages had been confirmed to end up being distributed in different patterns, and PD-L1 was expressed in a similar pattern to CD68 (Physique 1A). Moreover, the number of PD-L1-expressing cells positively correlated with the number of CD68+ macrophages (Physique 1D middle), but not with the number of CD3+ T cells (Physique 1D left). The number of PD-L1+ cells in the intratumoral region showed no significant correlation with the number of CD68+ macrophages (Physique 1E middle). In contrast to the peritumor regions, the number of CD3+ T cells and the number of PD-L1+ cells were positively correlated in the intratumor regions (Physique 1E, left). Lastly, we compared the number of PD-L1+ cells in the peritumor and intratumor regions with the concentration of serum alpha fetoprotein (AFP) and confirmed that there was no correlation (Physique 1D, right and Physique 1E, right). Open in a separate window Physique 1 Patterns and correlations of CD3, CD68, and PD-L1-expressing cells in human HCC tissues: (A) a representative pattern of CD3, CD68, and PD-L1 expression in human tissues acquired through liver resection; (B,C) the number of CD3+ T cells, CD68+ macrophages, and PD-L1+ cells located in intratumoral and peritumoral region. *** < 0.001; (DCE) correlation of CD3+ T cells, CD68+ macrophages, serum AFP, and PD-L1+ cells located in peritumoral and intratumoral region (= 33). Abbreviations: AFP, alpha fetoprotein; HCC, hepatocellular carcinoma; PD-L1, programmed death ligand MX1013 1. 2.2. Improvement in CD8+ and CD4+ T Cell Functions after PD-L1 Expression Blockade on M2 Macrophages Next, we studied whether CD8+ and CD4+ T cell functions are induced upon the blockade of PD-L1 expression on M2 macrophages. We isolated PBMCs from healthy donor blood and stained them with CD14 and CD3 microbeads for magnetic cell sorting. CD14+ cells were then polarized into M2 macrophages through treatment with M-CSF and IL-4. After polarization, CD3+ T cell co-culture experiments were performed. In co-cultures, we observed functional enhancements of the CD8+ T cells co-cultured with PD-L1-pretreated M2 macrophages. The numbers of CD8+ IFN-+ T and CD8+ TNF-+ T cells significantly increased by 5% to 10% and 8% to 10%, respectively (Physique 2A,B). Moreover, PD-1 and CD69 expression significantly increased on PMA/Ionomycin-activated CD8+ T cells after PD-L1 blockade on M2 macrophages (Supplementary Physique S1A,B). Consistent with the observations reported for CD8+ T cells, PMA/Ionomycin-activated CD4+ INF-+ T cells increased by approximately 8% to 14%, while the CD4+ TNF-+ T cell population increased by approximately 7% to 9% (Physique 2C,D). Further, CD4+ T cells showed an increase in the expression of PD-1 and CD69 after PD-L1 expression blockade on M2 macrophages (Supplementary Physique S1C,D). Open in a separate window Physique 2 Functional enhancement of CD8+ and CD4+ T cells after co-culture with anti-PD-L1-treated macrophages: (A,B) expression and MFI of (A) IFN-, and (B) TNF-, in CD8+ T cells when CD3+ T cells were co-cultured with PD-L1-blocked macrophages (= 3) * < 0.05, ** < 0.01; (C,D) expression and MFI of (C) IFN-, and (D) TNF- in CD4+ T cells when CD3+ T cells were co-cultured with PD-L1-blocked macrophages (=.CD3+ T cells (anti-CD3 microbeads, 130-050-101, MACS) and CD14+ monocytes (anti-CD14 microbeads, 130-050-201, MACS) were separated from PBMCs using the OctoMACS separator and starting kits (Miltenyi Biotec, Auburn, CA, USA). 4.5. a significant increase in T cell functionality after the pretreatment of M2 macrophages with anti-PD-L1. Syngeneic mouse model experiments demonstrated that TAMs expressed PD-L1 and tumors treated with anti-PD-L1 showed smaller diameters than those treated with IgG. In these mice, anti-PD-L1 treatment increased activation markers in intratumoral CD8+ T cells and reduced the size of the TAM population. Regarding nivolumab-treated patients, three of eight patients responded to the anti-PD-1 treatment. The percentage of Ki-67-positive CD4+ and CD8+ T cells was higher in responders than non-responders after nivolumab. Overall, PD-L1 expression on TAMs may be targeted by immune-based HCC treatment, and ICI treatment results in the reinvigoration of exhausted CD8+ T cells in HCC. < 0.001) (Figure 1ACE). In particular, CD3+ T cells and CD68+ macrophages were confirmed to be distributed in different patterns, and PD-L1 was expressed in a similar pattern to CD68 (Figure 1A). Moreover, the number of PD-L1-expressing cells positively correlated with the number of CD68+ macrophages (Figure 1D middle), but not with the number of CD3+ T cells (Figure 1D left). The number of PD-L1+ cells in the intratumoral region showed no significant correlation with the number of CD68+ macrophages (Figure 1E middle). In contrast to the peritumor regions, the number of CD3+ T cells and the number of PD-L1+ cells were positively correlated in the intratumor regions (Figure 1E, left). Lastly, we compared the number of PD-L1+ cells in the peritumor and intratumor regions with the concentration of serum alpha fetoprotein (AFP) and confirmed that there was no correlation (Figure 1D, right and Figure 1E, right). Open in a separate window Figure 1 Patterns and correlations of CD3, CD68, and PD-L1-expressing cells in human HCC tissues: (A) a representative pattern of CD3, CD68, and PD-L1 expression in human tissues acquired through liver resection; (B,C) the number of CD3+ T cells, CD68+ macrophages, and PD-L1+ cells located in intratumoral and peritumoral region. *** < 0.001; (DCE) correlation of CD3+ T cells, CD68+ macrophages, serum AFP, and PD-L1+ cells located in peritumoral and intratumoral region (= 33). Abbreviations: AFP, alpha fetoprotein; HCC, hepatocellular carcinoma; PD-L1, programmed death ligand 1. 2.2. Improvement in CD8+ and CD4+ T Cell Functions after PD-L1 Expression Blockade on M2 Macrophages Next, we studied whether CD8+ and CD4+ T cell functions are induced upon the blockade of PD-L1 expression on M2 macrophages. We isolated PBMCs MX1013 from healthy donor blood and stained them with CD14 and CD3 microbeads for magnetic cell sorting. CD14+ cells were then polarized into M2 macrophages through treatment with M-CSF and Rabbit Polyclonal to Glucokinase Regulator IL-4. After polarization, CD3+ T cell co-culture experiments were performed. In co-cultures, we observed functional enhancements of the CD8+ T cells co-cultured with PD-L1-pretreated M2 macrophages. The numbers of CD8+ IFN-+ T and CD8+ TNF-+ T cells significantly improved by 5% to 10% and 8% to 10%, respectively (Number 2A,B). Moreover, PD-1 and CD69 expression significantly improved on PMA/Ionomycin-activated CD8+ T cells after PD-L1 blockade on M2 macrophages (Supplementary Number S1A,B). Consistent with the observations reported for CD8+ T cells, PMA/Ionomycin-activated CD4+ INF-+ T cells improved by approximately 8% to 14%, while the CD4+ TNF-+ T cell populace increased by approximately 7% to 9% (Number 2C,D). Further, CD4+ T cells showed an increase in the manifestation of PD-1 and CD69 after PD-L1 manifestation blockade on M2 macrophages (Supplementary Number S1C,D). Open in a separate window Number 2 Functional enhancement of CD8+ and CD4+ T cells after co-culture with anti-PD-L1-treated macrophages: (A,B) manifestation and MFI of (A) IFN-, and (B) TNF-, in CD8+ T cells when CD3+ T cells were co-cultured with PD-L1-clogged macrophages (= 3) * < 0.05, ** < 0.01; (C,D) manifestation and MFI of (C) IFN-, and (D) TNF- in CD4+ T cells when CD3+ T cells were co-cultured with PD-L1-clogged macrophages (= 3) * < 0.05, ** < 0.01; (E) experiment schedule for separation of T cells and macrophages from your tissues acquired by hepatic resection; (F,G) differential manifestation of IFN- and TNF- in CD8+ and CD4+ T cells when CD3+ T cells were co-cultured with PD-L1-clogged CD206+.Materials and Methods 4.1. these mice, anti-PD-L1 treatment improved activation markers in intratumoral CD8+ T cells and reduced the size of the TAM populace. Regarding nivolumab-treated individuals, three of eight individuals responded to the anti-PD-1 treatment. The percentage of Ki-67-positive CD4+ and CD8+ T cells was higher in responders than non-responders after nivolumab. Overall, PD-L1 manifestation on TAMs may be targeted by immune-based HCC treatment, and ICI treatment results in the reinvigoration of worn out CD8+ T cells in HCC. < 0.001) (Number 1ACE). In particular, CD3+ T cells and CD68+ macrophages were confirmed to become distributed in different patterns, and PD-L1 was indicated in a similar pattern to CD68 (Number 1A). Moreover, the number of PD-L1-expressing cells positively correlated with the number of CD68+ macrophages (Number 1D middle), but not with the number of CD3+ T cells (Number 1D remaining). The number of PD-L1+ cells in the intratumoral region showed no significant correlation with the number of CD68+ macrophages (Number 1E middle). In contrast to the peritumor areas, the number of CD3+ T cells and the number of PD-L1+ cells were positively correlated in the intratumor areas (Number 1E, remaining). Lastly, we compared the number of PD-L1+ cells in the peritumor and intratumor areas with the concentration of serum alpha fetoprotein (AFP) and confirmed that there was no correlation (Number 1D, right and Number 1E, right). Open in a separate window Number 1 Patterns and correlations of CD3, CD68, and PD-L1-expressing cells in human being HCC cells: (A) a representative pattern of CD3, CD68, and PD-L1 manifestation in human cells acquired through liver resection; (B,C) the number of CD3+ T cells, CD68+ macrophages, and PD-L1+ cells located in intratumoral and peritumoral region. *** < 0.001; (DCE) correlation of CD3+ T cells, CD68+ macrophages, serum AFP, and PD-L1+ cells located in peritumoral and intratumoral region (= 33). Abbreviations: AFP, alpha fetoprotein; HCC, hepatocellular carcinoma; PD-L1, programmed death ligand 1. 2.2. Improvement in CD8+ and CD4+ T Cell Functions after PD-L1 Manifestation Blockade on M2 Macrophages Next, we analyzed whether CD8+ and CD4+ T cell functions are induced upon the blockade of PD-L1 manifestation on M2 macrophages. We isolated PBMCs from healthy donor blood and stained them with CD14 and CD3 microbeads for magnetic cell sorting. CD14+ cells were then polarized into M2 macrophages through treatment with M-CSF and IL-4. After polarization, CD3+ T cell co-culture experiments were performed. In co-cultures, we noticed functional enhancements from the Compact disc8+ T cells co-cultured with PD-L1-pretreated M2 macrophages. The amounts of Compact disc8+ IFN-+ T and Compact disc8+ TNF-+ T cells considerably elevated by 5% to 10% and 8% to 10%, respectively (Body 2A,B). Furthermore, PD-1 and Compact disc69 expression considerably elevated on PMA/Ionomycin-activated Compact disc8+ T cells after PD-L1 blockade on M2 macrophages (Supplementary Body S1A,B). In keeping with the observations reported for Compact MX1013 disc8+ T cells, PMA/Ionomycin-activated Compact disc4+ INF-+ T cells elevated by around 8% to 14%, as the Compact disc4+ TNF-+ T cell inhabitants increased by around 7% to 9% (Body 2C,D). Further, Compact disc4+ T cells demonstrated a rise in the appearance of PD-1 and Compact disc69 after PD-L1 appearance blockade on M2 macrophages (Supplementary Body S1C,D). Open up in another window Body 2 Functional improvement of Compact disc8+ and Compact disc4+ T cells after co-culture with anti-PD-L1-treated macrophages: (A,B) appearance and MFI of (A) IFN-, and (B) TNF-, in Compact disc8+ T cells when Compact disc3+ T cells had been co-cultured with PD-L1-obstructed macrophages (= 3) * < 0.05, ** < 0.01; (C,D) appearance and MFI of (C) IFN-, and (D) TNF- in Compact disc4+ T cells when Compact disc3+ T cells had been co-cultured with PD-L1-obstructed macrophages (= 3) * < 0.05, ** < 0.01; (E) test schedule for parting of T cells and macrophages through the tissues obtained by hepatic resection; (F,G) differential appearance of IFN- and TNF- in Compact disc8+ and Compact disc4+ T cells when Compact disc3+ T cells had been co-cultured with PD-L1-obstructed Compact disc206+ macrophages and control cells from individual tissues obtained through liver organ resection. Abbreviations: IFN-, interferon-; MFI, mean fluorescence strength; PD-L1, programmed loss of life ligand 1; TNF-, tumor necrosis aspect-. Next, we performed equivalent experiments using Compact disc206+ macrophages and Compact disc3+ T cells newly isolated through the HCC tissues of an individual. PD-L1 appearance was obstructed on Compact disc206+ macrophages using anti-PD-L1 antibody, and these cells had been co-cultured with PMA/Ionomycin-activated Compact disc3+ T cells (Body 2E). We discovered that the populace of Compact disc8+ INF-+ T cells elevated by around 8% to.

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