In contrast, the populations of the hepatic NKT cells stained with CD69 or CD25 in the septic mice were higher than that in the sham (Fig
In contrast, the populations of the hepatic NKT cells stained with CD69 or CD25 in the septic mice were higher than that in the sham (Fig. CD8+ T cell numbers were significantly increased, whereas NKT cell numbers were reduced, but more activated with increased CD69 and CD25 expression. In the septic lungs, the CD4+ T and CD8+ T cell numbers showed no significant change, whereas they were severely reduced in the septic blood. Overall, this study provides important information on the alterations of different T-cell subsets in various tissues after sepsis. test. Differences in values were considered significant if < 0.05. 3. Results 3.1. CD4+CD8+ DP cells decrease in the thymus after sepsis To determine the impact of sepsis on T-lymphocytes, we compared the frequency and total Evacetrapib (LY2484595) numbers of T cell subsets in various organs between sham and septic mice at 20 h after CLP. We first prepared single-cell suspensions from the thymus and analyzed thymic subsets by flow cytometry. Total lymphocyte count showed a significant reduction by 73% in the septic thymi compared to sham (Fig. 1A). Among the thymic T cell subpopulations DP (CD4+CD8+) subset was significantly decreased in frequency by 30% (Fig. 1B and ?and1C)1C) and severely depleted in absolute cell numbers by 81%, compared to sham (Fig. 1D). However, the frequency of DN (CD4?CD8?), and CD4+SP and CD8+ SP thymic subsets was significantly increased by 3.5-, 3.7-and 3.0-fold, respectively, because of the decreased percentage of DP subset (Fig. 1B and ?and1C).1C). The absolute cell numbers of these subsets were not affected and were comparable to those in the sham (Fig. 1D). Thus, CLP-induced sepsis results in thymic hypocellularity due to a major loss of DP thymocyte subset without affecting the other thymic subsets. Open in a separate window Figure 1 Hypocellularity and CD4+ CD8+ double positive (DP) lymphocyte subset depletion in the thymi of septic mice(A) Total numbers of thymocytes isolated from the thymi harvested after 20 h from sham- HDAC6 or CLP-operated mice are shown. (B) Flow cytometric analysis of surface CD4 and CD8 expression on the gated live lymphocytes isolated from the sham and CLP thymi. Numbers adjacent to the outlined areas in the representative dot plots show the percentage of various thymic lymphocyte subsets as indicated. DP indicates CD4+CD8+ double positive thymocytes, DN indicates CD4?CD8? double negative thymocytes, CD4SP indicates CD4+ single positive thymocytes, and CD8SP indicates CD8+ single positive thymocytes. Data are representative of three independent analyses with a total of four to five Evacetrapib (LY2484595) mice in each group. (CCD) The graphs show the percentage (C) and absolute cell numbers (D) of the indicated thymic lymphocyte subsets. Data expressed as mean SEM (n = 5 per group). < 0.05 versus sham. 3.2. CD4+ and CD8+ T cell numbers decrease, Tregs frequency increases and na?ve and Tcm CD4+ T cell numbers decrease in the spleen after sepsis Lymphocytes isolated from the spleen of sham and septic mice were stained with anti-CD4 and anti-CD8 Abs. Total splenic lymphocyte count showed a significant reduction by 41% in the septic mice compared to sham (Fig. 2A). No significant differences in CD4+ and CD8+ T-cell frequency were noticed at 20 h after CLP as compared to the shams (Fig. 2B and ?and2C),2C), although we noticed a decreasing trend in CD4 T cell frequency (Fig. 2C). However, CD4+ and CD8+ T cell numbers in the septic spleens were significantly decreased by 45% and 36%, respectively (Fig. 2D). Open in a separate Evacetrapib (LY2484595) window Figure 2 T cell depletion and increased frequency of CD4+ CD25+ regulatory T cells (Tregs) in the spleens of septic mice(A) Total numbers of splenocytes isolated from the spleens harvested after 20 h from sham- or CLP-operated mice are shown. (B) Flow cytometric analysis of surface CD4 and CD8 expression on the gated live lymphocytes isolated from the Evacetrapib (LY2484595) sham and CLP spleens. Numbers adjacent to the outlined areas in the representative dot plots show the percentage of CD4+ T and CD8+ T cell subsets as indicated. Data are representative of three independent analyses with a total of four to five mice in each group. (CCD) The graphs show the.