Search

Prolactin (PRL) has been reported to inhibit apoptosis in various cell types and to serve as a cofactor in the upregulation of CD25 on T cells during activation. We investigated a possible relation between prolactin receptor (PRL-R) or IL-2 receptor alpha (IL-2Ralpha, CD25) expression on circulating T lymphocytes and their apoptosis in patients with breast cancer. Peripheral blood mononuclear cells obtained from 25 patients, 25 normal controls (NC) and three cord blood samples were evaluated for Annexin V binding and expression of CD95, CD25, and PRL-R on CD3(+) T cells by multicolour flow cytometry. Plasma levels of PRL, sCD95L, and sIL-2R were determined in patients and controls and related to T-cell apoptosis. The ability of PRL to protect T cells from apoptosis induced by various agents was also studied. Expression of PRL-R on the surface of T cells was comparable in patients with breast cancer and NC, but PRL plasma levels in patients were significantly lower (P<0.05). In patients, 18+/-11% (mean+/-s.d.) of CD3(+) cells bound Annexin V, compared to 9+/-6% in NC (P<0.0004). Percentages of CD3(+)Fas(+) and CD3(+)CD25(+) cells were higher in the peripheral circulation of patients than NC (P<0.0001 and <0.04, respectively). Levels of sFasL were lowest in plasma of the patients with the highest proportions of CD3(+)Fas(+) T cells. Most T cells undergoing apoptosis were CD3(+)CD25(-) in patients, and the proportion of CD3(+)CD25(-) Annexin V(+) cells was significantly increased in patients compared to NC (P<0.006). Ex vivo PRL protected T cells from starvation-induced or anti-CD3Ab-induced but not from Fas/FasL-dependent apoptosis. These results indicate that expression of CD25 but not of PRL-R on the surface of activated T lymphocytes appears to be involved in modulating Fas/Fas - ligand interactions, which are, in part, responsible for apoptosis of T lymphocytes and excessive turnover of immune cells in the circulation of patients with breast cancer. [ABSTRACT FROM AUTHOR]

CD39 and CD73 are key enzymes in the adenosine (ADO) pathway. ADO modulates pathophysiological responses of immune cells, including B cells. It has recently emerged that a subpopulation of ADO-producing CD39+CD73+B cells has regulatory properties. Here, we define the CD39highsubset of these cells as the major contributor to the regulatory network operated by human B lymphocytes. Peripheral blood B cells were sorted into CD39neg, CD39interand CD39highsubsets. The phenotype, proliferation and IL-10 secretion by these B cells were studied by flow cytometry. 5′-AMP and ADO levels were measured by mass spectrometry. Agonists or antagonists of A1R, A2AR and A3R were used to study ADO-receptor signaling in B cells. Inhibition of effector T-cell (Teff) activation/proliferation by B cells was assessed in co-cultures. Cytokine production was measured by Luminex. Uponin vitroactivation and culture of B cells, the subset of CD39highB cells increased in frequency (p< 0.001). CD39highB cells upregulated CD73 expression, proliferated (approximately 40% of CD39highB cells were Ki-67+and secreted fold-2 higher IL-10 and ADO levels than CD39negor CD39interB cells. CD39highB cells co-cultured with autologous Teff suppressed T-cell activation/proliferation and secreted elevated levels of IL-6 and IL-10. The A1R and A2AR agonists promoted expansion and functions of CD39highB cells. CD39 ectonucleotidase is upregulated in a subset ofin vitro-activated B cells which utilize ADO and IL-10 to suppress Teff functions. Proliferation and functions of these CD39highB cells are regulated by A1R- and A2AR-mediated autocrine signaling. [ABSTRACT FROM AUTHOR]

Abstract: Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4–16 h 51Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3-CD16+CD56+). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies. [Copyright &y& Elsevier]