Activation of lymphocytes induced by bronchial epithelial cells with prolonged RSV infection

Respiratory syncytial virus (RSV) preferentially infects airway epithelial cells,which might be responsible for susceptibility to asthma; however, the underlying mechanism is not clear. This study determined the activation of lymphocytes and drift of helper T (Th) subsets induced by RSV-infected human bronchial epithelial cells (HBECs) in vitro. HBECs had prolonged infection with RSV, and lymphocytes isolated from human peripheral blood were co-cultured with RSV-infected HBECs. Four groups were established, as follows: lymphocytes (group L); lymphocytes infected with RSV (group RL); co-culture of lymphocytes with non-infected HBECs (group HL); and co-culture of lymphocytes with infected HBECs (group HRL). After co-culture with HBECs for 24 hours, lymphocytes were collected and the following were determined in the 4 groups: cell cycle status; apoptosis rate; and concentrations of IL-4, IFN-γ, and IL-17 in the supernatants. Cell cycle analysis for lymphocytes showed a significant increase in S phase cells, a decrease in G1 phase cells, and a higher apoptosis rate in group HRL compared with the other three groups. In group HRL, the levels of IL-4, IFN-γ, and IL-17 in supernatants were also higher than the other three groups. For further study, lymphocytes were individually treated with supernatants from non-infected and RSV-infected HBECs for 24 h. We showed that supernatants from RSV-infected HBECs induced the differentiation of Th2 and Th17 subsets, and suppressed the differentiation of Treg subsets. Our results showed that HBECs with prolonged RSV infection can induce lymphocyte proliferation and apoptosis, and enhance the release of cytokines by lymphocytes. Moreover, subset drift might be caused by RSV-infected HBECs.     

Activation of human T lymphocytes via integrin signaling induced by RGD-disintegrins

Adhesive interactions play important roles in coordinating T cell migration and activation, which are mediated by binding of integrins to RGD motif found on extracellular matrix proteins. Disintegrins, isolated from snake venoms, contain the RGD sequence that confers selectivity to integrin interaction. We have investigated the ability of three RGD-disintegrins, ligands of alpha(5)beta(1) and alpha(v)beta(3), Flavoridin (Fl), Kistrin (Kr) and Echistatin (Ech), in modulating the activation of human T lymphocyte. The disintegrins induced T cell proliferation and CD69 expression. This activation parallels with actin cytoskeleton reorganization and tyrosine phosphorylation. Furthermore, the peptides induced focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K) activation. Finally, RGD-disintegrins were capable of driving NF-kappaB nuclear translocation and c-Fos expression, in a PI3K and ERK1/2 activities dependent manner. This report is the first to show that RGD-disintegrins interact with integrins on human T lymphocyte surface, modulating cell proliferation and activation of specific pathways coupled to integrin receptor.     

Activation of human T lymphocytes via the CD2 antigen results in tyrosine phosphorylation of T cell antigen receptor zeta-chains.

The phosphorylation of the invariant chains associated with the human TCR has been investigated after the stimulation of T lymphocytes with CD2 mAb T11(2) and T11(3), PHA, or phorbol 12,13-dibutyrate. As described previously, stimulation of T cells with either CD2 mAb or phorbol 12,13-dibutyrate resulted in the phosphorylation of the CD3 gamma-chain. The combination of T11(2) and T11(3) mAb also induced phosphorylation of the TCR zeta-chain. The phosphorylated zeta-polypeptide of CD2-activated cells was immunoprecipitated with antiphosphotyrosine antibodies and migrated to a 21- to 23-kDa position during SDS/PAGE. These results indicate that stimulation of human T cells via the CD2 Ag with the T11(2) and T11(3) mAb activates not only protein kinase C but also tyrosine kinase(s), resulting in the phosphorylation of the CD3 gamma-chain and the tyrosine phosphorylation of the zeta-chain, respectively.     

Activation of cytotoxic function in T lymphocytes.

The requirements for activation of cytotoxic function in mouse T lymphocytes were investigated. Initial generation of cytotoxicity in normal lymphocytes was equal in magnitude with either Con A or specific alloantigen, and in either case required DNA synthesis. Cytotoxic function in MLC-primed cells could also be regenerated by Con A, the magnitude and target specificity of the cytotoxicity thus generated being indistinguishable from that recalled by specific alloantigen. Cytotoxicity could also be regenerated by third party-stimulating cells; however, the cytotoxicity evoked by third party cells was always specific only for target cells of the original stimulating cell H-2 genotype. The data presented suggest that there are a number of ways to activate cytotoxicity in effector T cells, and are most consistent with a model for T cell triggering that minimizes a strict informational function of antigen-receptor interactions.     

Tolerance induced by chronic inhaled antigen in a murine asthma model is not mediated by endotoxin

Ovalbumin (OVA)-sensitized wildtype (WT) and endotoxin-resistant (ER) mice developed similar degrees of airways eosinophilia and serum OVA-specific IgE levels after acute aerosolized OVA challenge. WT mice demonstrated methacholine hyperreactivity, whereas ER mice showed no change in responsiveness. With chronic aerosolized OVA challenge, both WT and ER mice developed local tolerance, with resolution of airway eosinophilia but persistence of anti-OVA IgE in serum. Thus, the development of local tolerance with chronic aerosol exposure to OVA is independent of any potential effects of endotoxin in the OVA aerosol solution.     

An antigen expressed by avian neuronal cells is also expressed by activated T lymphocytes.

A monoclonal antibody, anti-BEN, initially characterized by its reactivity with an epitope present on the surface of avian bursa epithelial cells and neurons, also reacts with membrane molecules on some hemopoietic cells. In this study we examine BEN expression on lymphoid cells in thymus, spleen, and blood. We demonstrate that BEN is an activation antigen on mature T lymphocytes. It is not expressed on peripheral blood or splenic lymphocytes, but following mitogenic or allogeneic stimulation of blood lymphocytes it appears rapidly on a T cell subpopulation in parallel with the appearance of IL-2 receptors. BEN is also expressed on III-C5 cells, an avian IL-2-dependent permanent T cell line, and on immature CD4+CD8+ thymocytes. BEN is not expressed by resting or actively proliferating B cells. Biochemical analyses of the BEN protein on T lymphoblasts shows that the molecule is similar in size to the BEN molecules on bursa epithelial cells and on neurons. The physicochemical properties of the BEN protein and its tissue distribution differs from other known avian and mammalian T cell activation markers, differentiation antigens, and integrins. Thus BEN is a novel marker of activated T cells in birds.     

Maintenance of hyporesponsiveness to antigen by a distinct subclass of T lymphocytes.

Immunization with increasing doses of SRBC, in excess of 10(8), results in a progressive decline in the anti-SRBC PFC response. This hyporesponsive state is antigen specific and is reflected in a decrease of both T helper and B antibody-forming activity. We asked whether the apparent defect of T helper activity reflected a) an absence of alphaSRBC helper T cell activity, or b) the presence of SRBC-specific suppressor T cells within the hyporesponsive population. Our results indicate that at least a portion of hyporesponsiveness noted after antigen exposure to large doses of antigen can be ascribed to specific suppressor T cell activation. Fractionation of the suppressive T cell population using Ly antiserum showed that specific suppressive activity was mediated by a subclass of T cells (Ly2+), distinct from that committed to express helper function (Ly1).     

Activation of human T lymphocytes by ganglioside-containing liposomes

We investigated the in vitro stimulatory effect of ganglioside (G_M3, G_D1a, G_D1b, G_T1b, or G_Q1b)-containing liposomes on human immune cells. The effect of ganglioside-containing liposomes on the concentration of cytoplasmic free calcium ions ([Ca²⁺]₁) in human immunocytes was examined using the confocal laser fluorescence microscopic method. The G_D1a- and G_T1b-containing liposomes significantly increased [Ca²⁺]₁ of human T lymphocytes compared with the G_M3-, G_D1b- and G_Q1b-containing ones. The response of CD8⁺ and CD4⁺ cells was significantly higher than that of CD20⁺ cells. Our results show that the increase in [Ca²⁺]_i may be caused by not the number of sialic acids contained in the gangliosides but the conformation of the sialic acid moiety to protrude exteriorly from the liposomal membrane surface, and that a sort of receptor recognizing the sialic acid moiety exists on human T lymphocytes (both CD8⁺ and CD4⁺ cells), which may be involved in the activation of the cells. The present results are almost the same as those obtained for the rat T lymphocyte system previously reported. This clearly confirms that a sort of ganglioside surely stimulates T lymphocytes directly, which is not species-specific but conserved in humans and rats among animal species.     

T cell activation of primed rabbit blood lymphocytes by antigen.

Activation of primed rabbit PBL by homologous antigen in the early proliferative phase (on days 3--5) mainly involves lymphocytes which neither secrete specific antibody nor contain immunoglobulin in their cell membrane. This stimulation is antigen-specific, and evidence is given that the major proportion of these cells are T lymphocytes. The B cells forming Av-CHO-specific PFC were studied by autoradiography on days 6 and 12 of culture. Since incorporation of radioactive thymidine was found in the majority of PFC, these cells are also in proliferation.     

Activation of CFTR chloride current by nitric oxide in human T lymphocytes.

Nitric oxide, which is produced by cytokine-activated mononuclear cells, is thought to play an important role in inflammation and immunity. While the function of nitric oxide as a direct cytotoxic effector molecule is well established, its function as a transducer molecule in immune cells is not. By use of whole-cell patch clamp recordings, we show that nitric oxide activates cystic fibrosis transmembrane conductance regulator CI- currents in normal human cloned T cells by a cGMP-dependent mechanism. This pathway is defective in cystic fibrosis-derived human cloned T cells. These findings not only delineate a novel transduction mechanism for nitric oxide but also support the hypothesis that an intrinsic immune defect may exist in cystic fibrosis.     

Activated and memory T lymphocytes in human milk.

Since activated macrophages and cytokines are found in human milk (HM), a flow cytometry study was conducted to determine whether T cells in HM display phenotypic markers of recent or previous activation. HM was collected during the first 3 d of lactation. The Paint-a-Gate program was used to optimize gating on the lymphocyte population. A mean +/- 1 SD of 4 +/- 3% of total HM leukocytes were lymphocytes and 96 +/- 3% were macrophages and granulocytes (N = 33 subjects). HM lymphocyte populations were further analyzed in five subjects. T cells (CD3+) represented 83 +/- 11% and B cells (CD19+) were 6 +/- 4% of HM lymphocytes. The mean CD4/CD8 ratio of T cells in HM was 0.88 (range 0.40-1.25). This ratio was significantly decreased compared to the peripheral blood (PB) of control adults (P less than 0.02) and postpartum women (P less than 0.02), due mostly to a significant increase in CD8+ CD3+ cells in HM compared to the PB of control adults (P less than 0.002) and postpartum women (P less than 0.05). T cells bearing markers of recent activation were significantly increased in HM compared to the PB of control adults: 85 +/- 7% of CD3+ cells in HM were HLA-DR+ (controls, 10 +/- 4%; P less than 0.001), and 15 +/- 6% of CD3+ cells in HM were IL-2R+ (controls, 6 +/- 2%; P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of a tyrosine protein kinase is an early event in the stimulation of T lymphocytes by interleukin-2

The ability of the T lymphocyte growth factor interleukin 2 (IL-2) to activate a tyrosine protein kinase in vivo was assessed by using antibodies to phosphotyrosine in conjunction with immunoblots. Treatment of the murine IL-2-dependent cytotoxic T cell line CTLL-2 with IL-2 resulted in an increase in tyrosine phosphorylation of several proteins of molecular weights ranging from 38,000 to 120,000. The tyrosine phosphorylation in the various proteins increased in a concomitant fashion and reached a maximum level within 15 min. The concentration of IL-2 required for inducing this phosphorylation was similar to that required for stimulating [3H]thymidine uptake, indicating that the increase in tyrosine phosphorylation correlated with the ability of IL-2 to stimulate the proliferation of the CTLL-2 cells. IL-2 was also found to induce the phosphorylation of proteins on tyrosine residues in short term cultures of human T lymphocytes. These results suggest that IL-2, like other polypeptide growth factors, acts by stimulating the activity of a tyrosine protein kinase.     

An induced fit hypothesis for antigen recognition by T lymphocytes: a role for specific antigen retention structures on antigen-presenting cells

The nature of T lymphocyte recognition of foreign antigens is not known, despite recent advances in elucidating the cellular structures that may be involved in the specific interactions. The central difficulty in this process is that T cells respond to foreign antigen only in the context of major histocompatibility complex (MHC) antigens expressed by another antigen-presenting cell. In addition, T cells that interact with class II MHC antigens do not bind foreign protein antigens in their native form, but seem to recognize only proteolytic peptide fragments as the relevant antigen. The simplest explanation for these observations is that the class II MHC antigens themselves bind antigenic peptides to form the appropriate determinant that interacts with the antigen-specific T cell receptor. However, to date no such antigenic complex has been found with MHC antigens despite rigorous attempts at their demonstration. One alternative explanation described here is that there is no preexisting foreign antigen-MHC antigen complex prior to interaction with T cells, and it is the T cells that cause the two moieties to become associated for recognition by a single antigen-specific T cell receptor. Central to this mechanism is that foreign antigenic peptides must be associated with specific antigen retention structures (SARS) expressed by antigen-presenting cells which retain and protect the peptide on the cell surface. These SARS, upon interaction with T cell membrane moieties, would subsequently associate with MHC antigens. A hypothesis to describe this mechanism is developed to account for published observations of antigen processing by antigen-presenting cells and T cell antigen recognition, and makes several predictions that are experimentally testable. This mechanism is also generally applicable to other cellular interactions in which soluble peptide mediators may become associated with surface components of one cell type, and this newly formed complex is in turn recognized by a receptor on a second cell type to deliver functional signals.     

A voltage-gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes.

Activation of T lymphocytes results in an increase in intracellular Ca2+ due in large part to influx of extracellular Ca2+. Using the patch clamp technique, an inward current in Jurkat T lymphocytes was observed upon depolarization from a holding potential of -90 mV but not from -60 mV. This whole-cell current was insensitive to tetrodotoxin, carried by Ba2+, and blocked by Ni2+. Occupancy of the T lymphocyte antigen receptor increased the current's magnitude. These data suggest that antigen receptor-induced Ca2+ entry in T lymphocytes may be mediated by a voltage-regulated Ca channel.     

Differences in target cell DNA fragmentation induced by mouse cytotoxic T lymphocytes and natural killer cells

Fragmentation of YAC-1 target cell DNA during cytolysis mediated by mouse natural killer (NK) cells and cytotoxic T lymphocytes (CTL) was compared. Cleavage of nuclear chromatin was always an extensive and early event in CTL-mediated cytolysis, whereas with NK cell-mediated killing the degree of DNA fragmentation showed an unexpected relationship to the effector:target (E:T) ratio. At low NK:YAC-1 ratios, DNA fragmentation and 51Cr release were equivalent and increased proportionately until a ratio of about 50:1 was reached; at higher ratios, 51Cr release increased as expected but DNA fragmentation decreased dramatically. Comparison of time course data at E:T ratios producing similar rates of 51Cr release showed that the target cell DNA fragmentation observed in NK killing was not nearly as rapid nor as extensive as that observed with CTL effectors. These results suggest that NK cells induce target cell injury via two different mechanisms. One mechanism would involve lysis mediated by cell-to-cell contact, while the other may induce DNA fragmentation via a soluble mediator. In support of this notion, cell-free culture supernatants containing NK cytotoxic factor (NKCF) induced DNA fragmentation in YAC-1 cells. The DNA fragments induced by NK cells and NKCF-containing supernatants consisted of oligonucleosomes indistinguishable from those induced by CTL. The results presented here show distinct differences in target cell DNA fragmentation induced by CTL and NK cells, and suggest that these two effectors use different mechanisms to achieve the same end. CTL seem to induce DNA fragmentation in their targets by direct signaling, whereas NK cells may do so by means of a soluble factor.     

Activation of T lymphocytes by the Fc portion of immunoglobulin

T lymphocytes are stimulated to release T-cell-replacing factors in response to Fc fragments of human IgG. Lyt 1⁺23^? T cells are directly triggered to factor production by Fc subfragments, derived from intact Fc fragments by macrophage-dependent enzymatic cleavage. These factor(s) replace T cell function in two Fc-mediated immune responses; induction of polyclonal antibody synthesis, and potentiation of anti-SRBC responses.     

The proliferative response of human lymphocytes to antigen is suppressed preferentially by lymphocytes precultured with the same antigen.

Human blood lymphocytes activated in vitro with antigen to which the donor is reactive are capable of suppressing the secondary proliferative response of autochthonous fresh cells to antigen. Both antigen-specific and antigen-nonspecific suppression can be detected in each experiment. These suppressor cells act by decreasing the number of lymphocytes entering the proliferative response rather than by slowing or otherwise inhibiting ongoing proliferation. The suppressor cells must be added soon after fresh cells are stimulated with antigen to be effective, but the suppressor cells themselves need not proliferate to exert their effect. Suppressor cells are optimally effective when added in numbers equal to those of the responding population, but still exert a significant effect at one-eighth that number.