CD4+ T Cell Effects on CD8+ T Cell Location Defined Using Bioluminescence

T lymphocytes of the CD8+ class are critical in delivering cytotoxic function and in controlling viral and intracellular infections. These cells are “helped” by T lymphocytes of the CD4+ class, which facilitate their activation, clonal expansion, full differentiation and the persistence of memory. In this study we investigated the impact of CD4+ T cells on the location of CD8+ T cells, using antibody-mediated CD4+ T cell depletion and imaging the antigen-driven redistribution of bioluminescent CD8+ T cells in living mice. We documented that CD4+ T cells influence the biodistribution of CD8+ T cells, favoring their localization to abdominal lymph nodes. Flow cytometric analysis revealed that this was associated with an increase in the expression of specific integrins. The presence of CD4+ T cells at the time of initial CD8+ T cell activation also influences their biodistribution in the memory phase. Based on these results, we propose the model that one of the functions of CD4+ T cell “help” is to program the homing potential of CD8+ T cells

Correlation Of Ultrasound-Induced Hemolysis With Cavitation Detector Output In Vitro

A 20-MHz passive acoustic detector was used to quantify the amount of transient acoustic cavitation occurring in a sample exposed to intense pulsed ultrasound, A dilute suspension of human erythrocytes with and without a microbubble echo-contrast agent was exposed in vitro to 500 W/cm(2) (SPPA) ultrasound of center frequency 1 MHz and tone burst duration 20, 100, 200, 500 and 1000 mu s at a pulse repetition frequency of 20 Hz. Inertial cavitation occurring within the sample, as measured by the temporal average of the detector output, correlated well with hemolysis, suggesting that violent bubble collapse is responsible for cell damage, The result also raises the prospect of cavitation monitoring as a possible predictor of adverse bioeffects when echo-contrast agents are used clinically. (C) 1997 World Federation for Ultrasound in Medicine & Biology

Effect of a Stabilized Microbubble Echo Contrast Agent on Hemolysis of Human Erythrocytes Exposed to High Intensity Pulsed Ultrasound

Microbubble contrast agents have been shown to enhance ultrasonic cell lysis in vitro when exposed to continuous-wave ultrasound having spatial peak temporal average (SPTA) intensities of a few W/cm2. The response is strongly dependent upon the hematocrit (HCT) of the cell sample; detectable cell lysis essentially disappears as the HCT approaches 5%-10%. This study was conducted to determine whether high intensity pulsedsound is an effective lytic agent in the presence of preexisting potential cavitation nuclei (Albunex® contrast agent). Human erythrocytes weresuspended in autologous plasma to HCTs ranging from 1%–40%. Suspensions were exposed or sham exposed for 60 seconds to focused, pulsed ultrasound. The pulse duration was 1 msec, and the pulse repetition frequency was 20 Hz. The pressure amplitudes, spatial peak pulse average (SPPA) intensity, and SPTA intensity were 4.7 MPa peak positive pressure, -2.7 MPa peak negative pressure, 420 W/cm2, and 8.5 /cm2, respectively. Samples were exposed to ultrasound in a dialysis membrane exposure vessel rotating at 200 rpm. When included in the erythrocyte samples, the Albunex concentration was 35 μL/mL suspension. Significant ultrasound-induced hemolysis in the absence of Albunex was observed only at the lowest HCT value tested (1%). In the presence of Albunex significant cell lysis was observed at all tested HCT values. The relative fraction of cells lysed by the combination of ultrasound exposure and Albunex diminished with increasing HCT, but the number of cells lysed per sample was nearly constant over the range of 5%–40% HCT. The ultrasound exposure parameters used in this study differ substantially from those associated with diagnostic imaging equipment; it is not valid to infer from the present results that the use of Albunex in diagnostic applications will induce or enhance hemolysis in vivo

Follicular Lymphoma Tregs Have a Distinct Transcription Profile Impacting Their Migration and Retention in the Malignant Lymph Node

<div><p>We have previously shown that regulatory T cells (Tregs) infiltrating follicular lymphoma lymph nodes are quantitatively and qualitatively different than those infiltrating normal and reactive nodes. To gain insight into how such Treg populations differ, we performed RNA sequence (RNAseq) analyses on flow sorted Tregs from all three sources. We identify several molecules that could contribute to the observed increased suppressive capacity of follicular lymphoma nodal tregs, including upregulation of CTLA-4, IL-10, and GITR, all confirmed by protein expression. In addition, we identify, and confirm functionally, a novel mechanism by which Tregs target to and accumulate within a human tumor microenvironment, through the down regulation of S1PR1, SELL (L-selectin) and CCR7, potentially resulting in greater lymph node retention. In addition we identify and confirm functionally the upregulation of the chemokine receptor CXCR5 as well as the secretion of the chemokines CXCL13 and IL-16 demonstrating the unique ability of the follicular derived Tregs to localize and accumulate within not only the malignant lymph node, but also localize and accumulate within the malignant B cell follicle itself. Such findings offer significant new insights into how follicular lymphoma nodal Tregs may contribute to the biology of follicular lymphoma and identify several novel therapeutic targets.</p></div

Gene list categories.

<p>Gene list categories.</p

Characteristics of significant gene lists and functionally relevant genes.

<p>A. IPA enriched functions for significant genes for the three pairwise comparisons. Functions that had a B-H corrected enrichment p-value less than 0.00001 for at least one comparison are shown. Functions were clustered to aid in visualization based on their patterns of category membership (a function can be associated with or more broader category) using one minus the kappa statistic for distance (dendrogram not shown). In the heat map, functions within each cluster (delineated by white lines) are sorted by size, their significance in the FL vs. NLN gene list, and then the significance in the RLN vs. FL gene list. No enriched functions were found at this significance level for the RLN vs. NLN list at this level of significance (middle column). B. Heat map of genes in categories from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155347#pone.0155347.t001" target="_blank">Table 1</a> that had a pairwise difference with FDR < 0.1 for at least one comparison. Color maps to a gene’s z-score based on log-transformed data (computed independently for each gene). Genes and samples (within groups) are clustered using Euclidean distance and average linkage. Circles next to the heat map indicate whether the gene had a FDR < 0.1 (unfilled) or < 0.05 (filled) for the specified comparison.</p

FL Tregs secrete significantly higher levels of selected cytokines and chemokines than NLN Tregs.

<p>Tregs were sorted from FL (n = 10) and NLN (n = 5) samples and were cultured for six hours in serum free media with added antiCD3/CD28. Culture supernatants were collected and analysed for IL-10, IL-16, CCL3, CCL4, and CXCL13 by luminex. Assays were performed in duplicates. Line segments represent mean values for each group. Values below the limit of detection are set to “0”. Significance indicated by asterisks (p < 0.05) determined by Wilcoxon rank sum tests with permutations to compute p-values.</p