Ability of human T lymphocytes to adhere to vascular endothelial cells and to augment endothelial permeability to macromolecules is linked to their state of post-thymic maturation

The accumulation of mononuclear cells at sites of chronic inflammation is dependent on a number of factors including localized adherence of lymphocytes to vascular endothelial cells (EC), cytokine-mediated increased adhesiveness of endothelium, chemotactic factors and endothelial permeability. The present study investigates two of the above attributes of lymphocyte-EC interaction: namely, the ability of maturationally distinct subpopulations of human T lymphocytes to adhere to vascular EC and to increase vascular endothelial permeability to macromolecules in an in vitro model. Thus, human T lymphocytes were separated into CD4+ CD8-helper/inducer, CD4- CD8+ cytotoxic/suppressor, CD29+ CD45RA- CD45RO+ memory, and CD29- CD45RA+ CD45RO- naive/virgin T subpopulations, were activated with PHA and PMA, and then examined for their adherence to EC and also for their effect on endothelial permeability. Upon activation, cells within each of the above four subpopulations exhibited increased adherence to EC. In contrast, resting CD29+ CD45RA- CD45RO+ memory T lymphocytes exhibited two to three times greater ability to adhere to EC than their CD29- CD45RA+ CD45RO- naive/virgin counterparts. Consistent with their increased adherence to EC, CD29+ CD45RO+ memory T lymphocytes, when activated, significantly increased endothelial permeability to albumin. Although activated CD45RA+ naive T lymphocytes exhibited increased adherence to EC, these cells failed to increase significantly endothelial permeability. Similar to their polyclonal counterparts, Ag-specific CD4+ CD29+ CD45RO+ T cell clones, but not their actively released mediators, also increased endothelial permeability via a noncytolytic mechanism(s). This ability of CD29+ CD45RO+ memory T lymphocytes to augment endothelial permeability may facilitate their transendothelial migration into extravascular space. These observations may provide additional insights into molecular mechanism(s) underlying pathophysiology of localized chronic inflammatory responses in general and more specifically selective accumulation of CD29+/CD45RO+ memory T lymphocytes at sites of chronic inflammation such as rheumatoid synovium.     

Generation of CD4(+)CD45RA(+) effector T cells by stimulation in the presence of cyclic adenosine 5'-monophosphate-elevating agents

After TCR cross-linking, naive CD4(+)CD45RA(+) T cells switch to the expression of the CD45RO isoform and acquire effector functions. In this study we have shown that cAMP-elevating agents added to anti-CD3- and anti-CD28-stimulated cultures of T lymphocytes prevent acquisition of the CD45RO(+) phenotype and lead to the generation of a new subpopulation of primed CD4(+)CD45RA(+) effector cells (cAMP-primed CD45RA). These cells displayed a low apoptotic index, as the presence of dibutyryl cAMP (dbcAMP)-rescued cells from CD3/CD28 induced apoptosis. Inhibition of CD45 splicing by dbcAMP was not reverted by addition of exogenous IL-2. cAMP-primed CD45RA cells had a phenotype characteristic of memory/effector T lymphocytes, as they showed an up-regulated expression of CD2, CD44, and CD11a molecules, while the levels of CD62L Ag were down-regulated. These cells also expressed the activation markers CD30, CD71, and HLA class II Ags at an even higher level than CD3/CD28-stimulated cells in the absence of dbcAMP. In agreement with this finding, cAMP-primed CD45RA cells were very efficient in triggering allogenic responses in a MLR. In addition, cAMP-primed CD45RA cells produce considerable amounts of the Th2 cytokines, IL-4, IL-10, and IL-13, whereas the production of IFN-gamma and TNF-alpha was nearly undetectable. The elevated production of IL-13 by neonatal and adult cAMP-primed CD45RA cells was specially noticeable. The cAMP-dependent inhibition of CD45 splicing was not caused by the production of immunosuppressor cytokines. These results suggest that within the pool of CD4(+)CD45RA(+) cells there is a subpopulation of effector lymphocytes generated by activation in the presence of cAMP-elevating agents.     

CD4+ T cells from CD4C/HIVNef transgenic mice show enhanced activation in vivo with impaired proliferation in vitro but are dispensable for the development of a severe AIDS-like organ disease

The cellular and molecular mechanisms of dysfunction and depletion of CD4+ T lymphocytes over the course of human immunodeficiency virus type 1 (HIV-1) infection are still incompletely understood, but chronic immune activation is thought to play an important role in disease progression. We studied CD4+ T-cell biology in CD4C/HIV transgenic (Tg) mice, in which Nef expression is sufficient to induce a severe AIDS-like disease including a preferential decrease of CD4+ T cells. We show here that Nef-expressing Tg CD4+ T cells exhibit an activated/memory-like phenotype which appears to be independent of antigenic stimulation, as documented in experiments involving breeding with AD10 TcR Tg mice. In addition, in vivo bromodeoxyuridine incorporation showed that a larger proportion of Tg than non-Tg CD4+ T cells entered the S phase. However, in vitro, Tg CD4+ T cells were found to have a very limited capacity to divide in response to stimulation with anti-CD3 and anti-CD28 or in allogeneic mixed leukocyte reactions. Interestingly, despite these observations, the deletion of Tg CD4+ T cells had little impact on the development of other AIDS-like organ phenotypes. Thus, the Nef-induced chronic activation of CD4+ T cells may exhaust the T-cell pool and may contribute to the thymic atrophy and the low number of CD4+ T cells observed in these Tg mice.     

Effector function of resting T cells: activation of synovial fibroblasts

Synovial tissue in rheumatoid arthritis is characterized by infiltration with large numbers of T lymphocytes and APCs as well as hyperplasia of synovial fibroblasts. Current understanding of the pathogenesis of RA includes the concept that synovial fibroblasts, which are essential to cartilage and bone destruction, are regulated by cytokines derived primarily from monocyte-macrophage cells. Recently it has been found that synovial fibroblasts can also function as accessory cells for T cell activation by superantigens and other stimuli. We have now found that highly purified resting T cells, even in the absence of T cell mitogens, induce activation of synovial fibroblasts when cocultured for 6-24 h. Such activation was evident by induction or augmentation of mRNA for stromelysin, IL-6, and IL-8, gene products important in joint inflammation and joint destruction. Furthermore, increased production of IL-6 and IL-8 was quantitated by intracellular cytokine staining and flow cytometry. This technique, previously used for analysis of T cell function, was readily adaptable for assays of synovial fibroblasts. Resting T cells also induced synovial fibroblasts to produce PGE(2), indicating activation of expression of the cyclooxygenase 2 gene. Synergy was observed between the effects of IL-17, a cytokine derived from stimulated T cells that activates fibroblasts, and resting T lymphocytes. Various subsets of T cells, CD4(+), CD8(+), CD45RO(+), and CD45RA(+) all had comparable ability to induce synovial fibroblast activation. These results establish an Ag-independent effector function for resting T cells that is likely to be important in inflammatory compartments in which large numbers of T lymphocytes and fibroblasts can come into direct contact with each other.     

Function of CD8+ T lymphocytes in a self-curing mouse model of visceral leishmaniasis

CD8+ T lymphocytes play an important role in the control of visceral leishmaniasis in non self-cure mice (e.g. BALB/c). In the present study, the mode of action of CD8+ T cells and their in vivo contribution to immunity was addressed in self-curing C57BL/6 mice. During the course of the experimental infection, CD8+ T cells specific for Leishmania infantum (L. infantum) developed and apoptotic cell death subsequently followed. They exhibited perforin-dependent cytotoxicity and a T(C)1 profile characterized by secretion of IFN-gamma and CC chemokines. Despite evidence for activation of CD8+ T lymphocytes, both intravenous and intradermal infection of beta2-microglobulin deficient C57BL/6 mice with L. infantum showed that these knockout animals had similar parasite loads to their wild-type counterpart. Lymphocytes from the beta2-microglobulin deficient mice produced high levels of IFN-gamma, reflecting a T(H)1 response to the parasite, which was apparently sufficient for the immunologic control of the pathogen. Thus, despite their functional activation, CD8+ T lymphocytes do not appear to play a primary role in parasite restraint in the self-curing mouse model of visceral leishmaniasis, as shown using beta2-microglobulin deficient mice which do not produce functional CD8+ T lymphocytes.     

Differences in activation of normal and glycosylphosphatidylinositol-negative lymphocytes derived from patients with paroxysmal nocturnal hemoglobinuria.

In these studies, the role of glycosylphosphatidylinositol (GPI)-anchored surface molecules during T cell activation was investigated in fresh T cells and T cell lines obtained from patients with paroxysmal nocturnal hemoglobinuria. For control, GPI-expressing T cells of the same patients were used. Unstimulated GPI- T cells exhibited significantly reduced surface expression of the activation Ag CD45R0, compared with GPI+ T cells. In addition, in measurements of proliferation, IFN-gamma production, and induction of second messengers such as cytoplasmic Ca2+, CD48- lymphocytes showed a similar response to TCR-specific stimulation, compared with CD48+ lymphocytes. In contrast, stimulation with the lectin PHA produced a decreased response of CD48- lymphocytes in these functions. In addition, stimulation with cross-linked CD59 mAb increased the proliferation of GPI-molecule expressing CD48+ T cell lines only. From these data, it can be concluded that GPI-anchored surface molecules play an important role in T lymphocyte activation.     

The HML-1 antigen of intestinal lymphocytes is an activation antigen.

The Ag recognized by the mAb HML-1 is expressed on more than 90% of human intestinal intraepithelial lymphocytes, whereas in other lymphoid tissues it is rarely or not expressed. In the present study, we have investigated the percentage of HML-1-positive cells in the human intestinal lamina propria and the coexpression of HML-1 with different T cell subset markers. In addition, we studied the inducibility of HML-1 on PBL which normally are HML-1-negative. Flow cytometric analysis of isolated intestinal lamina propria lymphocytes (LPL) showed that about 40% of the cells expressed HML-1, the majority belonging to the CD8-positive subpopulation. Virtually all LPL expressed CD45RO, whereas the percentage of CD29-positive cells was only about 50%, similar to PBL. There were only few cells expressing CD45RA or Leu-8 in the lamina propria. HML-1-positive cells were almost exclusively CD45RA-negative, but were found in both the CD29-positive and the CD29-negative subpopulation of LPL. In vitro stimulation of PBL showed that the expression of HML-1 was inducible on T cells by mitogens, phorbolester, Ag, and rIL-2. Expression of HML-1 was induced with a different time course and with differences in the response to the investigated stimuli compared with CD25. Activated HML-1-positive PBL were also predominantly CD45RA-negative. The findings show that HML-1 is an Ag, which is expressed in vivo on a specific subset of previously activated T cells in the unique environment of the intestinal mucosa, and which can be induced in vitro by different activation signals on PBL.     

The role of protein kinase C in transmembrane signaling by the T cell antigen receptor complex. Effects of stimulation with soluble or immobilized CD3 antibodies

Phorbol esters, such as phorbol myristate acetate (PMA), are known to be potent co-stimulants with calcium ionophores for activation of T lymphocytes. The most extensively studied intracellular effect of PMA is its ability to activate the cytoplasmic enzyme protein kinase C (pkC). Herein, we examined the role of pkC activation during T cell activation. During physiologic activation, this enzyme is activated by diacylglycerol which is generated through the hydrolysis of polyphosphoinositides. Therefore, we studied the activation of T lymphocytes induced by a synthetic diacylglycerol, dioctanoylglycerol. In contrast to PMA, this compound can be metabolized in T cells and presumably more closely mimics physiologic activation of pkC. Dioctanoylglycerol together with reagents that induce increases in intracellular free Ca2+ concentration, Ca2+ ionophores, or anti-cluster designation (CD)3 monoclonal antibodies (mAb) were able to induce interleukin 2 receptor expression and proliferation of T lymphocytes. Previous studies have demonstrated that the stimulation of T cells via the CD3/T cell antigen receptor complex by mAb against CD3 leads to an increase in cytoplasmic free Ca2+ and to an activation of pkC. Paradoxically, however, soluble CD3 antibodies do not cause proliferation of resting purified T cells. Inasmuch as immobilization of CD3 mAb has been shown to influence the agonist properties of such antibodies, we compared the ability of soluble and immobilized CD3 mAb to activate pkC. We demonstrated herein that soluble CD3 mAb cause only a very transient activation of pkC in the T cell leukemic line Jurkat. This pkC activation is markedly prolonged when Jurkat cells are stimulated with immobilized rather than soluble CD3 antibodies. These studies suggest that activation of pkC plays a major role in T cell activation and that the activation of pkC is influenced by the form in which CD3 mAb is presented to T cells.     

Mouse vascular endothelium activates CD8+ T lymphocytes in a B7-dependent fashion

Despite several studies examining the contribution of allorecognition pathways to acute and chronic rejection of vascularized murine allografts, little data describing activation of alloreactive T cells by mouse vascular endothelium exist. We have used primary cultures of resting or IFN-gamma-activated C57BL/6 (H-2(b)) vascular endothelial cells as stimulators and CD8(+) T lymphocytes isolated from CBA/J (H-2(k)) mice as responders. Resting endothelium expressed low levels of MHC class I, which was markedly up-regulated after activation with IFN-gamma. It also expressed moderate levels of CD80 at a resting state and after activation. Both resting and activated endothelium were able to induce proliferation of unprimed CD8(+) T lymphocytes, with proliferation noted at earlier time points after coculture with activated endothelium. Activated endothelium was also able to induce proliferation of CD44(low) naive CD8(+) T lymphocytes. Activated CD8(+) T lymphocytes had the ability to produce IFN-gamma and IL-2, acquired an effector phenotype, and showed up-regulation of the antiapoptotic protein Bcl-x(L). Treatment with CTLA4-Ig led to marked reduction of T cell proliferation and a decrease in expression of Bcl-x(L). Moreover, we demonstrate that nonhemopoietic cells such as vascular endothelium induce proliferation of CD8(+) T lymphocytes in a B7-dependent fashion in vivo. These results suggest that vascular endothelium can act as an APC for CD8(+) direct allorecognition and may, therefore, play an important role in regulating immune processes of allograft rejection.     

Calcium Homeostasis Change in CD4<Superscript>+</Superscript> T Lymphocytes from Human Peripheral Blood during Differentiation <Emphasis Type="Italic">in vivo</Emphasis>

Resting naive CD4⁺CD45R0^?CD45RA⁺ T cells are sensitive to ionomycin. In contrast, resting CD4⁺CD45RA^?CD45R0⁺ memory T cells show resistance to this Ca²⁺ ionophore. In the present study, the ability of activated T lymphocytes to respond to ionomycin during the transition from naive precursors into memory T cells has been analyzed. Activated CD4⁺CD45RA⁺CD45R0⁺ T cells are always present both in human peripheral blood (HPB) and in the ionomycin-resistant (IR) fraction. Therefore, some activated T cells are resistant toward the Ca²⁺ ionophore. CD69 molecules are markers of the very early stage of T cell activation. However, CD4⁺CD69⁺ T cells have never been found in the IR fraction. Thus, the majority of CD4⁺ T lymphocytes at the early stage of activation are ionomycin-sensitive cells. The proportion of CD4⁺CD25⁺ T cells did not differ significantly in HPB and in the IR fraction. The presence of CD4⁺CD25⁺ T lymphocytes in the IR fraction reflects changes in the Ca²⁺-signaling pathway at this differentiation step of activated cells. Depending on the expression level of CD25 molecules, the population of CD4⁺CD25⁺ cells is divided in T-regulatory (CD25^high) and proliferating (CD25^low) subpopulations. The action of ionomycin results in a decrease in the portion of the CD4⁺CD25^low T-cells, but it leads to an increase in the proportion of the CD4⁺CD25^high T lymphocytes. Consequently, greater portion of CD4⁺CD25^high T lymphocytes and smaller portion of CD4⁺CD25^low T cells are IR cells. Expression of HLA-DR molecules can be used as the marker for the late activation step. The IR fraction is significantly rich in CD4⁺HLA-DR⁺ T lymphocytes in comparison to the blood of the same donor. The link between different differentiation steps of CD4⁺ T-lymphocytes and alterations in calcium ion homeostasis is discussed.     

Differential dynamics of CD4(+) and CD8(+) T-lymphocyte proliferation and activation in acute simian immunodeficiency virus infection

Although lymphocyte turnover in chronic human immunodeficiency virus and simian immunodeficiency virus (SIV) infection has been extensively studied, there is little information on turnover in acute infection. We carried out a prospective kinetic analysis of lymphocyte proliferation in 13 rhesus macaques inoculated with pathogenic SIV. A short-lived dramatic increase in circulating Ki-67(+) lymphocytes observed at 1 to 4 weeks was temporally related to the onset of SIV replication. A 5- to 10-fold increase in Ki-67(+) CD8(+) T lymphocytes and a 2- to 3-fold increase in Ki-67(+) CD3(-) CD8(+) natural killer cells accounted for >85% of proliferating lymphocytes at peak proliferation. In contrast, there was little change in the percentage of Ki-67(+) CD4(+) T lymphocytes during acute infection, although transient increases in Ki-67(-) and Ki-67(+) CD4(+) T lymphocytes expressing CD69, Fas, and HLA-DR were observed. A two- to fourfold decline in CD4(+) T lymphocytes expressing CD25 and CD69 was seen later in SIV infection. The majority of Ki-67(+) CD8(+) T lymphocytes were phenotypically CD45RA(-) CD49d(hi) Fas(hi) CD25(-) CD69(-) CD28(-) HLA-DR(-) and persisted at levels twofold above baseline 6 months after SIV infection. Increased CD8(+) T-lymphocyte proliferation was associated with cell expansion, paralleled the onset of SIV-specific cytotoxic T-lymphocyte activity, and had an oligoclonal component. Thus, divergent patterns of proliferation and activation are exhibited by CD4(+) and CD8(+) T lymphocytes in early SIV infection and may determine how these cells are differentially affected in AIDS.     

Phenotypic comparison of the three populations of human lymphocytes defined by CD45RO and CD45RA expression.

Published reports indicate that CD45RO-CD45RAbright T cells are native T cells, CD45RObrightCD45RA- T cells are memory T cells, and that concomitant loss of CD45RA expression and gain of CD45RO expression occurs during transition from naive to memory status. Thus, following in vitro activation of CD45RO- CD45RAbright T cells, a subset of transitional CD45ROdimCD45RAdim T cells is observed before conversion to a CD45RObrightCD45RA- phenotype is completed. Interestingly, all three of these phenotypic subsets are represented in the circulating human lymphocyte pool. We thus used dual-color flow cytometry to phenotypically characterize CD45RObrightCD45RA-, CD45ROdimCD45RAdim, and CD45RO- CD45RAbright lymphocytes. Both the CD45RObrightCD45RA- and CD45ROdimCD45RAdim subsets consisted almost entirely of T cells, whereas the CD45RO-CD45RAbright subset contained T cells plus essentially all of the B and natural killer cells. Additional studies used three-color flow cytometry to assess activation markers on T cells within the three subsets defined by CD45RO/CD45RA expression. CD25 expression increased with conversion from naive to memory status (5% of CD45RO-CD45RAbright, 24% of CD45ROdimCD45RAdim, and 42% of CD45RObrightCD45RA- T cells), whereas CD38 expression decreased during conversion (76, 53, and 27%, respectively). We also assessed the fluorescent intensities of CD11a, CD2, and CD44, shown by others to be increased on memory, compared to naive T cells. Visual inspection of fluorescence cytograms confirmed these findings, and further showed that transitional T cells express these markers at levels indistinguishable from those for naive T cells. These findings suggest that acquisition of CD25 and loss of CD38 occur relatively early in the naive-to-memory transition process, being evident in the transitional cell subset. In contrast, increased expression of CD11a, CD2, and CD44 appear to represent late events, occurring after loss of CD45RA and gain of CD45RO has been completed.     

SC5 mAb represents a unique tool for the detection of extracellular vimentin as a specific marker of Sezary cells

Circulating malignant Sézary lymphocytes result from a clonal proliferation of memory/activated CD4(+)CD45RO(+) T lymphocytes primarily involving the skin. Recently, the CD158k/KIR3DL2 cell surface receptor has been identified to phenotypically characterize these cells. We previously described a mAb termed SC5 that identifies an unknown early activation cell membrane molecule. It is expressed selectively by T lymphocytes isolated from healthy individuals upon activation, and by circulating Sézary syndrome lymphocytes. In addition, we found that SC5 mAb was reactive with all resting T lymphocytes once permeabilized, indicating that SC5 mAb-reactive molecule might present distinct cellular localization according to the T cell activation status. In this study, we show for the first time that SC5 mAb recognizes the intermediate filament protein vimentin when exported to the extracellular side of the plasma membrane of viable Sézary malignant cells. We demonstrate that SC5 mAb is unique as it reacts with both viable malignant lymphocytes and apoptotic T cells. As vimentin is also detected rapidly at the cell membrane surface after normal T lymphocyte activation, it suggests that its extracellular detection on Sézary cells could be a consequence of their constitutive activation status. Finally, as a probable outcome of vimentin cell surface expression, autoantibodies against vimentin were found in the sera of Sézary syndrome patients.     

Lymphocyte subpopulations of regional lymph nodes in human colon and gastric adenocarcinomas

 In order to study the host immune response to tumours, previous knowledge of the cellular composition of regional draining lymph nodes is necessary. Enlarged regional lymph nodes are a common finding in colon and gastric adenocarcinomas. We have studied the cellular composition of normal non-reactive and of regional draining lymph nodes of colon and gastric adenocarcinomas. In normal non-reactive lymph nodes, T lymphocytes (CD2⁺, CD7⁺) constituted the largest fraction of the lymphoreticular cells. These lymphocytes were mainly CD4⁺, and there were more cells expressing the CD45RA isoform of the CD45 antigen than CD45RO. Reactive lymph nodes presented a decreased proportion of CD4⁺ CD45RA⁺ cells and an increased number of B cells. Although most of the T cells in the reactive nodes were CD4⁺ CD45RO⁺, their proportion was similar to that found in normal non-reactive nodes. We studied the presence of the molecules CD28 and CD80 involved in the processes of interaction and activation of T and B lymphocytes. The CD28 molecule was found in all the T lymphocytes, while the CD80 molecule was weakly expressed on the B lymphocyte membrane. Received: 4 January 1996 / Accepted: 28 May 1996     

Vdelta1 T lymphocytes expressing a Th1 phenotype are the major gammadelta T cell subset infiltrating the liver of HCV-infected persons

BACKGROUND: Hepatitis C infection induces an acute and chronic liver inflammation that may lead to cirrhosis, liver failure, or hepatocarcinoma. Since the role of alphabeta T lymphocytes in hepatitis C virus (HCV) immunopathology has been analyzed extensively, we investigated the distribution and functional activation of gammadelta T cell subsets in chronically HCV-infected patients. MATERIALS AND METHODS: Blood samples and liver biopsies from 35 patients with compensated chronic HCV infection were compared in terms of T cell subset distribution, expression of activation markers, gammadelta T cell receptor (TCR) repertoire, and pattern of cytokine production. Moreover, we analyzed whether these immunological parameters were associated with other clinical observations (plasma viremia, ALT levels, Ishak index). RESULTS: Differing from peripheral blood distribution, a specific compartmentalization of Vdelta1 T cells (p < 0.001) was observed in the liver of HCV patients. These cells represented a relevant fraction of intrahepatic T lymphocytes (1.8-8.7%) and expressed the memory/effector phenotype (CD62-L- CD45-RO+CD95+). This phenotype was consistent with selective homing upon antigen recognition. Mitogenic stimulation of Vdelta1 + T lymphocytes recruited in the liver revealed the T helper cell type 1 (Th1) pattern of cytokine secretion. Interestingly, the frequency of interferon-gamma (IFN-gamma)-producing Vdelta1 T cells was associated with an higher degree of liver necroinflammation, measured by the Ishak index. Finally, the T-cell repertoire analysis revealed the absence of Vgamma selection in the TCR repertoire of intrahepatic Vdelta1 T cells. CONCLUSIONS: gammadelta T cell distribution in the peripheral blood differs from the Vdelta1 T cell subset because it is policlonally activated and recruited in the liver of chronic HCV-infected patients. During HCV-infection, this T cell subset may release Th1 cytokines and contribute to the necroinflammatory liver disease.     

Activation of Wnt signaling arrests effector differentiation in human peripheral and cord blood-derived T lymphocytes

The canonical Wnt/β-catenin signaling pathway plays an important role in thymocyte development and T cell migration, but little is known about its role in naive-to-effector differentiation in human peripheral T cells. We show that activation of Wnt/β-catenin signaling arrests human peripheral blood and cord blood T lymphocytes in the naive stage and blocks their transition into functional T effector cells. Wnt signaling was induced in polyclonally activated human T cells by treatment either with the glycogen synthase kinase 3β inhibitor TWS119 or the physiological Wnt agonist Wnt-3a, and these T cells preserved a naive CD45RA(+)CD62L(+) phenotype compared with control-activated T cells that progressed to a CD45RO(+)CD62L(-) effector phenotype, and this occurred in a TWS119 dose-dependent manner. TWS119-induced Wnt signaling reduced T cell expansion, as a result of a block in cell division, and impaired acquisition of T cell effector function, measured by degranulation and IFN-γ production in response to T cell activation. The block in T cell division may be attributed to the reduced IL-2Rα expression in TWS119-treated T cells that lowers their capacity to use autocrine IL-2 for expansion. Collectively, our data suggest that Wnt/β-catenin signaling is a negative regulator of naive-to-effector T cell differentiation in human T lymphocytes. The arrest in T cell differentiation induced by Wnt signaling might have relevant clinical applications such as to preserve the naive T cell compartment in Ag-specific T cells generated ex vivo for adoptive T cell immunotherapy.     

Decreased expression of CD69 in chronic fatigue syndrome in relation to inflammatory markers: evidence for a severe disorder in the early activation of T lymphocytes and natural killer cells

There is some evidence that patients with chronic fatigue syndrome (CFS) suffer from immune abnormalities, such as immune activation and decreased immune cell responsivity upon polyclonal stimili. This study was designed to evaluate lymphocyte activation in CFS by using a CD69 expression assay. CD69 acts as a costimulatory molecule for T- and natural killer (NK) cell activation. We collected whole blood from CFS patients, who met CDC criteria, and healthy volunteers. The blood samples were stimulated with mitogens during 18 h and the levels of activated T and NK cells expressing CD69 were measured on a Coulter Epics flow cytometer using a three color immunofluorescence staining protocol. The expression of the CD69 activation marker on T cells (CD3+, CD3+CD4+, and CD3+CD8+) and on NK cells (CD45+CD56+) was significantly lower in CFS patients than in healthy subjects. These differences were significant to the extent that a significant diagnostic performance was obtained, i.e. the area under the ROC curve was around 89%. No differences either in the number of leukocytes or in the number or percentage of lymphocytes, i.e. CD3, CD4, CD8 and CD19, could be found between CFS patients and the controls. Patients with CFS show defects in T- and NK cell activation. Since induction of CD69 surface expression is dependent on the activation of the protein kinase C (PKC) activation pathway, it is suggested that in CFS there is a disorder in the early activation of the immune system involving PKC.     

In vitro and in vivo activated T cells display increased sensitivity to PGE2.

In this study we report that in vitro activation of T cells increased the cyclic AMP response to subsequent prostaglandin E2 (PGE2) stimulation severalfold per cell. This sensitization of T cells to PGE2-induced cyclic AMP generation was observed when the T cells had been stimulated in vitro for 5 days with either the CD3 monoclonal antibody OKT3, phytohemagglutinin, or the combination phytohemagglutinin plus the phorbol ester PMA. Enhanced cyclic AMP generation following mitogenic activation was seen in response to both PGE2 and forskolin, direct activator of the adenylate cyclase, indicating that the amount of adenylate cyclase had increased during the in vitro activation course. In order to investigate whether various T cell subsets in general and in vivo activated T cells in particular would differ in their susceptibility to PGE2, we isolated CD4+, CD8+, CD4-CD8-, CD4+CD45RO+ ("memory"), and CD4+CD45RA+ ("virgin") T cells and studied PGE2-mediated inhibition of CD3-induced proliferation, as well as cyclic AMP generation in response to PGE2, respectively. We found that CD8+ T cells are more susceptible to PGE2 inhibition and produce more cyclic AMP than CD4+ T cells. Double-negative T cells (enriched for gamma delta T cell receptor positive cells) were found to be sensitive to PGE2 as well. Within the CD4+ T cell population, CD45RO+ ("memory") T cells were significantly more sensitive to PGE2-mediated suppression than CD45RA+ ("virgin") T cells. CD45RO+ cells required a 10-fold lower dose of PGE2 for half-maximum suppression of proliferation. However, no difference in cyclic AMP production could be demonstrated between these two subsets. We propose that substantial heterogeneity exists among peripheral blood T lymphocyte subsets regarding their sensitivity to the immunosuppressive action of PGE2 and that the sensitivity of individual cells changes in the course of an immune response.     

Accumulation of CD45RO<Superscript>+</Superscript> cells in peritoneal carcinomatous fluid favours survival of ovarian carcinoma patients

In 44 patients with advanced ovarian carcinoma (OC) a fraction of CD45RO(+) lymphocytes in the blood and peritoneal carcinomatous fluid (PCF) was investigated. Thirty-one patients received cisplatinum with cyclophosphamide +/- doxorubicin. This group was followed from 2.2 to 9 years (mean: 45 months). In 23 out of 31 patients, the percentage of CD45RO(+) lymphocytes was higher in the PCF than in the blood samples. Patients with these higher lymphocyte levels experienced longer survival than those who did not show any excess of CD45RO(+) lymphocytes in PCF ( P=0.02). This was further verified by the use multivariate Cox analysis which included an assessment of the percentage of CD45RO(+) lymphocytes in PCF, age, FIGO status, histology, treatment (CAP or CP) and residual disease (RD) post-surgery. This analysis revealed that two factors had an independent power of prediction: RD ( P=0.02) and the percentage of CD45RO(+) cells in PCF ( P=0.04). Therefore, CD45RO(+) lymphocytes were studied in further detail in a group of 20 patients. This study revealed that PCF CD45RO(+) lymphocytes were characterized by: (1) a higher proportion of cells co-expressing activation markers (HLA-DR, CD28) and higher levels of mRNA for CXC chemokines (IP-10, IL-8) and for IL-10, but with lower levels for IL-2; (2) higher levels of Ki67, bcl-2 and p53 mRNA as compared to those in blood. In conclusion, in the present study it was found that an accumulation of activated CD45RO(+) cells in PCF had a beneficial effect on the survival of patients receiving platinum-based chemotherapy.