NLGP mediated tumor growth restriction and CD8⁺ T cells.

<p>Mice were inoculated with Sarcoma 180 tumor cells (1×10⁶ cells/mice); after the formation of palpable tumor, mice were treated with NLGP (25 µg) once in a week, and four injections in total. <b>A.1</b>. Different immune organs (Blood, Spleen, TDLN, VDLN) and tumors were harvested on day 21 after initiation of therapy, lymphocytes were isolated, labeled with CD8-PE and percentage of CD8⁺ T cells were analyzed by flow cytometry, along with blood and spleen cells from naive mice. *<i>p</i><0.001; **<i>p</i><0.05. <b>A.2</b>. Representative dot plots of CD8 positivity of TILs harvested from tumor of PBS- and NLGP-treated mice. <b>B</b>. Tumor volume of PBS- and NLGP-treated mice was plotted against percentage of CD8⁺ cells. <b>C</b>. Presence of CD8⁺ T cells was detected on cryosections of tumor tissues from either PBS or day 21 NLGP-treated mice by immunohistochemistry. <b>D.1</b>. MNCs from TDLN and tumors were harvested from mice with day 21 tumors and stained for CD8⁺CD69⁺. *<i>p</i><0.05. <b>D.2</b>. CD8⁺ cells within MNCs of PBS- and NLGP-treated mice were gated and CD69⁺ cells were identified within this gated population as presented by dot plots. <b>D.3</b>. MNCs from TDLN and tumors were harvested from mice with day 21 tumors and stained for CD8⁺CD44⁺. <b>D.4</b>. A representative dot plot for CD8⁺CD44⁺ cells within MNCs of PBS- and NLGP-treated mice. <b>D.5</b>. MNCs from TDLN and tumors were harvested from mice with day 21 tumors and stained for CD8⁺CD62L⁺ cells. <b>E.1</b>. Expression of cytokine receptors, like, IL-7R, IL-4R and IL-2R, on cells from TDLN and TIL of NLGP-treated mice was determined flow cytometrically. <b>E.2</b>. Expression of T cell exhaustion markers, like, lag3, pd1 and tim3, in cells from TDLN and TIL of NLGP-treated mice was determined at transcriptional level by RT-PCR. <b>F</b>. CD8⁺ cells were depleted in NLGP-treated mice, along with PBS (negative) and NLGP (positive) treatment into two other groups as controls. Tumor growth curve is presented. *<i>p</i><0.001, in comparison to NLGP-treated group.