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Messi M, Giacchetto I, Nagata K, Lanzavecchia A, Natoli G, Sallusto F. is usually absent in all resting T cells, was up-regulated in both CD45RA+ and CD45RO+ T cells as a result of stimulation with anti-CD3 and anti-CD28 and measured the relative expression of these two cytokine receptors on the total CD4+ T cell population and on the fraction destined to become the Th1 subpopulation. In summary, we have established that the expression of the IL-12 and IL-18 receptors are expressed preferentially on CD45RA+ reverted and CD45RO+ effector memory CD4+ cells, and that upon T cell receptor (TCR) stimulation a significant proportion of both CD45RA+ and CD45RO+ cells, not destined to become Th1 cells, are refractory to stimulation via the TCR and do not up-regulate the IL-12 and IL-18 receptors. MATERIALS AND METHODS Samples Heparinized blood was obtained from 22 Gusb healthy volunteers who gave informed consent. Tonsils (= 15) were obtained at routine tonsillectomy (median age 10, range 4C34). The study was approved by the ethics committee of the Royal Free Hospital and Hospital Germans Trias i Pujol. Isolation and stimulation of lymphocyte subpopulations Mononuclear cells were separated by Ficoll Hypaque density gradient. Two aliquots of the cell suspension were incubated with either monoclonal anti-CD45RA or anti-CD45RO-coated MACS beads (Dako, High Wycombe, UK) according to the manufacturer’s instructions (Milteny Biotec, Bisley, Surrey, UK). Total mononuclear cells and the depleted populations were suspended at 2 106 cells/ml in supplemented medium and were stimulated with anti-CD28 (1 and CD45ROmemory populations As shown in Table 1 and Fig. 1, the majority of CD4+ T cells expressing the IL-12R1 and IL-18 chains were of the CD45RO phenotype. Nevertheless, 65% and 10% of CD4+ and CD45RA+ T cells in adult peripheral blood also expressed the IL-12R1 and IL-18R subunits. These lymphocytes were distributed equally between the naive CD45RA+ (CD62L+, CD27+ and CCR7+) and the memory CD45RA+ (CD62LC, CD27C and CCR7C) populations (Fig. 2). The observed CD45RA+ populations defined by CD62L+, CD27+ and CCR7+ overlapped significantly, covering 87 22%, 92 23% and 87 25% of the CD45RA+. As some CD45RA+ cells can also co-express CD45RO, we JNJ 26854165 also analysed the expression of CD62L+, CD27+ and CCR7+ around the CD45ROC populations with very similar results (Fig. 2). The IL-12 and IL-18 receptors were absent from the recent thymic emigrants, which were defined by their expression of CD45RA and CD31. Open in a separate window Fig. 1 Up-regulation of regulatory cytokine receptors IL-12R1, IL12R2 and IL-18R on CD3 gated T cell and isolated CD45RA+ and CD45RO+ subsets. (a) Comparative dot plots representing the expression of IL-12R1 (CD212), IL-12R2 and IL-18R on CD3+ peripheral blood T cells from adults. Cells were stimulated with anti-CD3 and anti-CD28 and subsequently harvested and analysed at 0 h (left panel), 24 h (centre panel) and 48 h (right panel), respectively. Cells were stained for CD3, CD4, IL-12R1 JNJ 26854165 or IL-18R or IL-12R2 and analysed by flow cytometry as described in Material and methods. (b) Comparative graphs representing the expression of IL-12R1, IL-12R2 and IL-18R on CD45RA+ (dotted JNJ 26854165 bars) and CD45RO+ (black bars) CD4+ T cells from adults and from cord blood. Cells were stimulated with anti-CD3 and anti-CD28 and harvested subsequently and analysed at 0, 24, 48 and 72 h (days 1, 2, 3 and 4, respectively). Cells were stained as above. The physique shows that there is an up-regulation of all three regulatory cytokine receptors in a proportion of the CD3+CD4+ CD45RA+ and CD45RO+ populations. Open in a separate window Fig. 2 Distribution of IL-12R1, IL-18R, CCR7 on CD45RA+/CD45RO+ CD4+ lymphocytes. Top panels: comparative dot plots representing the expression of CCR7 on CD45RA+ (a) and CD45RO+ (b) CD4 lymphocytes. Bottom panels: comparative dot blots representing the distribution of IL-12R1 (c) and IL-18R (d) on CCR7+ and CCR7C populations on CD4+ CD45RA+ peripheral blood gated T cells from adults. Cells were stained for CD4, CD45RA or CD45RO and CCR7 (a,b) or IL-12R1 JNJ 26854165 or IL-18R, CCR7, CD4 and CD45RA (c,d) and analysed by flow cytometry as described in Material and methods. Note that a small proportion of CD45RA negative CD4+ cells express CCR7 (a,b) JNJ 26854165 and that approximately 50% of these cells are also positive for the IL-12 and IL-18 receptors (c and d). Table 1 Relative expression of IL-12R1 and IL-18R on CD4+ T cells expressing different CD45 isoforms from adult blood, cord blood and tonsil CD45RAcells expressed reduced levels of the IL-12 receptor compared to that observed in the adult population while the levels of IL-18R remained similar As expected, due.