6 research outputs found

    Neem leaf glycoprotein prevents post-surgical sarcoma recurrence in Swiss mice by differentially regulating cytotoxic T and myeloid-derived suppressor cells

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    <div><p>Post-surgical tumor recurrence is a common problem in cancer treatment. In the present study, the role of neem leaf glycoprotein (NLGP), a novel immunomodulator, in prevention of post-surgical recurrence of solid sarcoma was examined. Data suggest that NLGP prevents tumor recurrence after surgical removal of sarcoma in Swiss mice and increases their tumor-free survival time. In NLGP-treated tumor-free mice, increased cytotoxic CD8<sup>+</sup> T cells and a decreased population of suppressor cells, especially myeloid-derived suppressor cells (MDSCs) was observed. NLGP-treated CD8<sup>+</sup> T cells showed greater cytotoxicity towards tumor-derived MDSCs and supernatants from the same CD8<sup>+</sup> T cell culture caused upregulation of FasR and downregulation of cFLIP in MDSCs. To elucidate the role of CD8<sup>+</sup> T cells, specifically in association with the downregulation in MDSCs, CD8<sup>+</sup> T cells were depleted <i>in vivo</i> before NLGP immunization in surgically tumor removed mice and tumor recurrence was noted. These mice also exhibited increased MDSCs along with decreased levels of Caspase 3, Caspase 8 and increased cFLIP expression. In conclusion, it can be stated that NLGP, by activating CD8<sup>+</sup> T cells, down regulates the proportion of MDSCs. Accordingly, suppressive effects of MDSCs on CD8<sup>+</sup> T cells are minimized and optimum immune surveillance in tumor hosts is maintained to eliminate the residual tumor mass appearing during recurrence.</p></div

    CD8<sup>+</sup> T cells downregulate MDSCs in Fas dependent pathway.

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    <p>(A) Percentage of Annexin V-PI<sup>+</sup> MDSCs within the blood of PBS, NLGP, CD8<sup>+</sup> T cell depleted NLGP immunized mice (n = 6). (B) Flow cytometric assessment of Gr1<sup>+</sup>FasR<sup>+</sup> MDSCs in post-surgery PBS-, NLGP-treated mice with or without CD8<sup>+</sup> T cell depletion. (C) Expression of FasL within CD8<sup>+</sup> T cells in mice with tumor surgery in PBS and NLGP immunized mice. (D) Flow cytometric assessment of Caspase 3 within Gr1<sup>+</sup> MDSCs in PBS, NLGP and CD8 depleted NLGP immunized mice. (E) Protein level expression of Caspase 3, Caspase 8 and cFLIP within MDSCs from PBS, NLGP and CD8 depleted NLGP immunized surgically tumor removed mice. (n = 6, in each group). (F) Experimental design with MDSCs and CD8<sup>+</sup> T cells. (G1) Expression of FasL within NLGP-treated CD8<sup>+</sup> T cells. (G2) Expression of cFLIP and FasR within MDSCs in the presence and absence of supernatants from NLGP-treated CD8<sup>+</sup> T cells, with or without IFNγ neutralization. (H) Assessment of the cytotoxic potential of NLGP-treated CD8<sup>+</sup> T cells towards tumor-derived MDSCs, in the presence of Brefeldin A and Concanamycin A. (**<i>p</i><0.001,*<i>p</i><0.01). (n = 3, in each group). Bar diagrams along with representative figures are present in each case (A-C).</p

    NLGP mediated downregulation of regulatory cells is CD8<sup>+</sup> T cell dependent.

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    <p>(A) Flow cytometric assessment of the status of TAMs (CD11b<sup>+</sup>F4/80<sup>+</sup>), DC2s (CD11c<sup>+</sup>IL-10<sup>+</sup>), Tregs (CD4<sup>+</sup>CD25<sup>+</sup>Foxp3<sup>+</sup>) and MDSCs (Gr1<sup>+</sup>CD11b<sup>+</sup>) in pre- and post-surgical S180 tumor bearing mice (n = 6). (B) Status of regulatory cells (TAMs, DC2s, Tregs, MDSCs) in post-surgery PBS, NLGP, CD8<sup>+</sup> T cell depleted NLGP immunized mice (n = 6). (C) RT-PCR analysis to assess the expression of suppressive molecules present in MDSCs in surgically tumor removed PBS, NLGP and CD8<sup>+</sup> T cell depleted NLGP immunized cohorts (n = 6). (D) Gene expression profile of molecules responsible for MDSC’s differentiation in NLGP and CD8<sup>+</sup> T cell depleted NLGP immunized surgically tumor removed mice (n = 6). (E) RT-PCR analysis of S100A8 and S1001A9 molecules responsible for MDSCs trafficking in PBS, NLGP and CD8<sup>+</sup> T cell depleted NLGP immunized surgically tumor removed mice (n = 6). (F) Status of CD8<sup>+</sup> Ki67<sup>+</sup> T cells after co culture with MDSCs isolated from PBS, NLGP, CD8+ T cell depleted NLGP mice. Representative figures along with bar diagram showing mean relative expression of three individual mice in each group are presented. (**<i>p</i><0.001,*<i>p</i><0.01).</p

    Primer list<sup>*</sup>.

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    <p>Primer list<sup><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175540#t001fn001" target="_blank">*</a></sup>.</p

    Recurrent tumor growth and survival of Swiss mice with post-surgery NLGP treatment.

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    <p>(A) Experimental design showing sarcoma inoculation, NLGP treatment and blood collection. (B) Recurrent tumor growth curve in pre- and post-surgery phases of mice with or without NLGP treatment (n = 9). (C) Representative photographs of tumor free and tumor bearing mice in the NLGP and PBS groups, respectively, in the post-surgical period. (D) Survival of mice undergoing surgery followed by NLGP treatment (n = 9) (**<i>p</i><0.001).</p

    CD8<sup>+</sup> T cells play an important role in NLGP mediated prevention of tumor recurrence.

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    <p>(A1) Status of CD8<sup>+</sup> T cells in PBS and NLGP immunized mice after tumor inoculation (n = 9). (B1) Percent positive CD8<sup>+</sup> T cells in PBS and NLGP immunized mice after surgery (n = 9). A representative figure in both cases is shown in right upper corner panel (A2, B2). (C) Expression of CD69 on CD8<sup>+</sup> T cells in post-surgical PBS- and NLGP-treated mice (n = 9). (D) Flow cytometric analysis of Granzyme B on CD8<sup>+</sup> T cells in post-surgical PBS- and NLGP-treated mice (n = 9). Bar diagrams along with representative figures in right panel are shown (C,D). (E) Experimental design showing sarcoma inoculation, CD8<sup>+</sup> T cell depletion, NLGP immunization and blood collection. (F) Circulating CD8<sup>+</sup> T cell status following <i>in vivo</i> depletion of same cells. (G) Table showing number of recurrent tumor bearing and tumor free mice. (H) Tumor growth curve of recurrent tumor bearing mice in CD8<sup>+</sup> T cell depleted NLGP immunized mice (n = 6). (I) Survivability curve in NLGP-treated post-surgery mice with or without CD8<sup>+</sup> T cell depletion (n = 6). (J) RT-PCR analysis of the expression of IFNγ, Perforin and Granzyme B gene expression profile in partial CD8<sup>+</sup> T cell depleted post-surgery NLGP-treated mice. The bar diagram represents the mean ± SD of three individual observations from each group at each time point (**<i>p</i><0.001,*<i>p</i><0.01).</p
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