Epstein-Barr virus-transformed human B lymphocytes endocytose autologous and heterologous red blood cells

Epstein-Barr virus (EBV)-transformed B lymphocytes, either isolated from a patient with EBV-induced infectious mononucleosis or obtained by in vitro infection of B lymphocytes of donors in different clinical conditions, have been tested for the ability to endocytose particulate forms of antigens, such as human, sheep or mouse red blood cells. By light and electron microscopy, it has been found that EBV-transformed B cells are able to bind and internalize human autologous and allogeneic erythrocytes, as well as sheep and mouse erythrocytes, independent of the specificity of the secreted immunoglobulins. The ability of EBV-transformed B lymphocytes to endocytose and process particulate forms of autoantigens during infectious mononucleosis might have a role both in the production of heterophile antibodies and in EBV-induced autoimmunity.     

The blastogenic response of rabbit lymphocytes stimulated with autologous cells.

Rabbit WBC and spleen cells respond with marked blastogenesis and mitosis when cultured with mitomycin-C treated autologous sacculus rotundus, appendix and Peyer's patches cells in the one-way rabbit mixed leucocyte reaction (MLR). A lesser response is observed using stimulating cells of the other lymphoid organs. The blastogenic response in the one-way 'autologous MLR' cannot be attributed to mitogenic factors released from the mitomycin-C treated stimulator cells since it is also observed in the 'two-way autologous MLR', using untreated stimulator cells. Several explanations are proposed to account for the 'autologous MLR'.     

Novel rosette formation of murine spleen cells with autologous red blood cells

Spleen cells of normal BALB/c mice formed rosettes with autologous red blood cells, and the formation was calcium ion dependent. Peritoneal exudate cells, bone marrow cells and thymocytes did not form such rosettes. Spleen cells were passed over a Sephadex G-10 column or incubated on a plastic surface in order to eliminate adherent cells from them. Cells obtained by both these methods were unable to form rosettes. B cell-, T cell- and natural killer cell-enriched fractions in spleen cells were unable to form rosettes either. Some of the mouse IgG subclasses suppressed rosette formation when added to its site. These are monoclonal antibodies whose specificities are directed against Aspergillus niger glucose oxidase. Moreover, Aspergillus niger glucose oxidase suppressed the rosette formation when spleen cells had been treated with it as well as when it had been added to the site of rosette formation. These findings suggest that some murine spleen cells have receptors to a structure on autologous red blood cells, which is recognized by an anti-Aspergillus niger glucose oxidase monoclonal antibody.     

NK cells can recognize asialylated autologous lymphocytes and ABO-mismatched lymphocytes.

Peripheral blood lymphocytes (PBLs) are not normally susceptible to killing by autologous natural killer (NK) cells. Treatment of human PBLs with neuraminidase removes terminal sialic acid residues and renders them susceptible to cytotoxicity by autologous NK cells. This neuraminidase-induced natural killing can be inhibited with cold targets such as K-562, and also by human red blood cells of blood group A. In addition NK cells will kill certain allogeneic PBLs even when they are fully sialylated, and this killing appears to be directed against the carbohydrate ABO blood group of the foreign target. For example group O NK cells will kill untreated group A PBLs, and this cytotoxicity can be inhibited by red blood cells of group A, but not of group O. The implications of this finding for NK cell recognition of targets are discussed.     

Recognition of B-CLL cells experimentally infected with EBV by autologous T lymphocytes

We compared 5-day-old cultures of two B-CLL clones experimentally infected with EBV for their interaction with autologous T lymphocytes. The clone which was strongly activated by the virus stimulated autologous T cells. It was also damaged by the cytotoxic T cells which were generated in mixed cultures with autologous lymphoblastoid cell lines (LCL). Cultured, non-infected CLL cells were not lysed by these effectors. The other B-CLL clone, which was activated to considerably lesser extent by the virus, did not stimulate the autologous T lymphocytes. While, also in this case cytotoxic function was generated in the mixed T cell—LCL culture, the effectors did not damage the EBV-infected CLL cells. The results with B-CLL cells can be regarded as a model for the EBV genome carrier normal B lymphocytes. They substantiate the current concept that such cells persist in seropositive healthy individuals undisturbed by the specific immune response as long as they maintain the phenotype of resting cells. However, after activation they can be recognized and eliminated by T cells.     

The prediction of autologous red cell survival

Nine donors predicted from earlier studies to have increased autologous red cell survival and four predicted to have lesser survival were selected from a pool of previously studied donors. Blood was drawn from these donors, and stored in Neutracel (R-Cutter Lab) preservative for eight hours at room temperature. The platelet rich plasma was removed, the additive solution was introduced, and the red cells were stored for 42 days in an approved refrigerator. At the end of the storage period, a 24 hour Cr-51 viability analysis was performed on each donor. Predicted red cell survival scores correlated reasonably well with the observed percent of red cell viability (r = 0.648). This study suggests that it may be possible to predict increased viability of autologous red cells in certain donors.     

The interaction of adenosinetriphosphate and inorganic phosphate with the sodium pump in red cells.

1. An increase in the intracellular concentration of inorganic phosphate (Pi) reduces the rate of the Na:K exchange catalysed by the Na pump in red cells. The inhibitory effect of Pi is exerted on the maximum rate of flux, Pi having no appreciable effect on the apparent affinity of the Na pump for either internal Na or external K. The effect of Pi is exerted along a rectangular hyperbola which tends to zero as Pi tends to infinity and is half-maximal at about 17 mM internal Pi. 2. Pi does not modify the rate of Na:Na exchange catalysed by the Na pump. 3. A reduction in the intracellular concentration of ATP reduces the maximum rate of Na:K exchange having no effect on the apparent affinity for either internal Na or external K. 4. The effects of ATP and Pi are mutually independent. 5 The lack of effect of ATP and Pi on the apparent affinity for internal Na is compatible with the idea that the affinity of the inner sites of the Na pump remains constant during a pump cycle. 6. The lack of effect of ATP on the apparent affinity for external K and the independence between the effects of ATP and Pi are difficult to explain if the only effect of ATP were its combination at a phosphorylating site. 7. The apparent affinities for K and phosphate become independent of the concentration of ATP, if it is assumed that in our experimental range the phosphorylating site is fully saturated with ATP, the rate of pumping being controlled by the state of occupation of a second non-phosphorylating site whose affinity for ATP is much lower. 8. The lack of effect of Pi on the apparent affinity for external K seems to indicate that during Na:K exchange the conformations of the pump that predominate are endowed with a reactivity towards inorganic phosphate and have the same high affinity for K in both their phospho and their dephospho states. 9. The kinetic behaviour of the Na pump in regard to its interactions with inner and outer cations, ATP and Pi seems to indicate that, in contrast with what happens with soluble allosteric proteins, in the active transport system ligand-induced changes in the reactivity are more important than ligand-induced changes in affinity. In this respect therefore the Na pump behaves as an allosteric 'V system'.     

The interaction of intestinal epithelial cells and intraepithelial lymphocytes in host defense

Intestinal intraepithelial lymphocytes (i-IEL) are located at the basolateral surfaces of intestinal epithelial cells (i-EC) and play important roles in the homeostasis of intestinal microenvironment. i-IEL comprise unique T cell populations including CD4^-CD8αα⁺ T cells expressing T cell receptor (TCR)αΒ or TCRγδ and CD4⁺ CD8αα⁺ T cells expressing TCR αΒ. We show here that CD4⁺ CD8αα⁺ i-IEL belongs to Th1 type T cells capable of responding to self-MHC class I on i-EC and that a significant fraction of i-IEL expressed Fas ligand (Fas-L) and induced apoptosis in the i-EC via Fas-dependent pathway. i-IEL may recognize and eliminate the effete i-EC for homeostatic regulation of intestinal epithelia. The interaction of i-EC and i-IEL through E-cadherin/α_EΒ₇ integrin is important for homing and maintenance of i-IEL in intestine.Listeria monocytogenes are also known to interact with E-cadherin on i-EC and invade into the epithelial cells. Invasion ofL. monocytogenes into i-EC activated NFk-B and subsequently up-regulated the expression of IL-15 gene, which has a NFk-B binding site at the promoter region. i-IEL, especially γδ T cells, were significantly activated to produce Th1 type cytokines at the early stage after oral infection withL. monocytogenes in mice and rats. The activation of i-IEL coincided with a peak response of IL-15 production by i-EC after infection. Taken together, mutual interaction of i-IEL and i-EC may be important not only for homeostatic regulation but also host defense against microbial infection in intestine.     

The interaction of intestinal epithelial cells and intraepithelial lymphocytes in host defense

Intestinal intraepithelial lymphocytes (i-IEL) are located at the basolateral surfaces of intestinal epithelial cells (i-EC) and play important roles in the homeostasis of intestinal microenvironment. i-IEL comprise unique T cell populations including CD4-CD8alphaalpha+ T cells expressing T cell receptor (TCR)alphabeta or TCRgammadelta and CD4+ CD8alphaalpha+ T cells expressing TCR alphabeta. We show here that CD4+ CD8alphaalpha+ i-IEL belongs to Th1 type T cells capable of responding to self-MHC class I on i-EC and that a significant fraction of i-IEL expressed Fas ligand (Fas-L) and induced apoptosis in the i-EC via Fas-dependent pathway. i-IEL may recognize and eliminate the effete i-EC for homeostatic regulation of intestinal epithelia. The interaction of i-EC and i-IEL through E-cadherin/alphaEbeta7 integrin is important for homing and maintenance of i-IEL in intestine. Listeria monocytogenes are also known to interact with E-cadherin on i-EC and invade into the epithelial cells. Invasion of L. monocytogenes into i-EC activated NFkappa-B and subsequently up-regulated the expression of IL-15 gene, which has a NFkappa-B binding site at the promoter region. i-IEL, especially gammadelta T cells, were significantly activated to produce Th1 type cytokines at the early stage after oral infection with L. monocytogenes in mice and rats. The activation of i-IEL coincided with a peak response of IL-15 production by i-EC after infection. Taken together, mutual interaction of i-IEL and i-EC may be important not only for homeostatic regulation but also host defense against microbial infection in intestine.     

Adenovirus-receptor interaction with human lymphocytes

Lymphocytes play a key role in cell-mediated immunity and are host cells for several viral and bacterial pathogens. Their importance in adenovirus (Ad) infections is not yet fully understood. The initial event, the attachment of Ad to lymphocytes and their subsets, was examined using flow cytometry. The study included analysis of stimulated T cells in binding assays with FITC-labeled Ad fiber. The results confirm that native peripheral lymphocytes express very small amounts of Ad receptors. Stimulation with PHA and interleukin 2 induced the expression. The presence of Ad DNA as a sign of internalization in stimulated cells was demonstrated using the polymerase chain reaction. The findings suggest that lymphocytes after stimulation can turn into target cells for Ad. This is particularly important if there are indications for persistence of Ad, and in the case of immunocompromised patients severe, life-threatening diseases can develop. J. Med. Virol. 51:252–257, 1997. © 1997 Wiley-Liss, Inc.     

Prolonged growth of activated B lymphocytes requires interaction with helper T cells

Resting B lymphocytes acquire upon activation responsiveness to soluble factors that support their growth and differentiation. We have studied the growth requirements of anti-mu pre-activated B lymphocytes in vitro. We found that activated B lymphocytes did respond to soluble factors from helper T cells by entering the cell cycle but cell-cell contact with specific helper T cells was required for prolonged growth of activated B lymphocytes. Thus, activated B lymphocytes must receive activation signals from helper T cells in order to stay in the proliferative phase of the cell cycle.     

The influence of autologous erythrocytes on active T-cells rosetting with sheep red blood cells and on DNA synthesis

The addition of autologous erythrocytes to a mixture of human peripheral blood lymphocytes and sheep red blood cells (SRBC) alters the spontaneous rosetting reaction in the test detecting the active fraction of T lymphocytes (ARFC). Increase in rosetting is more often observed in mixtures containing smaller human red blood cell: lymphocyte ratios, while inhibition occurred in those containing a higher ratio. Autologous erythrocytes also affected DNA synthesis when added to three-day lymphocyte cultures, unstimulated or stimulated with BCG. These effects were more strongly pronounced when phagocytic cells were removed from lymphocytes tested.     

Rosette formation of pig peripheral blood lymphocytes with sheep red blood cells.

Peripheral blood lymphocytes of normal young pigs formed spontaneous and EAC rosettes with sheep red blood cells. Pretreatment of the SRBC with a sulphydryl reagent increased the percentage of spontaneous rosettes and the number of SRBC bound by the lymphocytes. The influence of an incubation period prior to centrifugation and the duration and temperature of the following incubation were tested for spontaneous and EAC rosettes. The importance is stressed to define the number of bound SRBC to call a lymphocyte a "rosette-forming cell". Rosette formation is interpreted as a quantitative rather than a qualitative marker.     

The migration of blood-borne lymphocytes into rat popliteal lymph nodes stimulated with xenogeneic red blood cells.

The recruitment of radiolabelled blood-borne lymphocytes into rat popliteal lymph nodes (PLN) was investigated following the injection of varying doses of chicken (CRBC) or sheep red blood cells (SRBC) into the hind foot pad. Within the dose range tested (10(5)-10(8) RBC) an increase in the dose of injected antigen resulted in elevated levels of lymphocyte recruitment into the draining popliteal lymph node. The kinetics of lymphocyte recruitment with the same dose of CRBC or SRBC was similar even though these red cells differ in size and are antigenically non-cross-reactive. While little is known of the mechanisms which control the rate of entry of blood-borne lymphocytes into antigen-stimulated lymph nodes, the extent of lymphocyte recruitment was shown to be directly related to the quantity of antigen injected.     

Recognition of autologous and allogeneic lymphocytes and tumor cells by human natural killer cells

The shedding of the mobile Fc receptor (FcR1) and the depletion of the immobile Fc receptor (FcR11) bearing human lymphocytes revealed that human natural killer cells belong to the FcR11-bearing population. Anti-beta-2-microglobulin treatment of the effector cells decreased natural cytotoxicity against some target cells and the detectability of HLA antigens, indicating that histocompatibility antigens or related structures may be involved in natural cytotoxicity. Using a panel of 29 autologous and allogenic PHA-stimulated target cells and peripheral lymphocytes from the same donors as the effector cells, distinct cytotoxic responses against allogeneic and autologous target cells were observed. A computer analysis of selective natural cytotoxicity distinguished seven different groups of target cells that may represent common structures for NK recognition.     

Lysis of nonnucleated red blood cells by cytotoxic T lymphocytes

To establish the role of direct membrane damage in cytotoxic T lymphocytes (CTL)-mediated lysis we investigated whether CTL would be capable of lysing nonnucleated target cells. By antibody-mediated targeting we show that CTL are indeed capable of lysing sheep and human red blood cells.     

Human B lymphocytes produce leukocyte interferon after interaction with foreign cells.

Culture filtrate antigens of Micropolyspora faeni grown in a synthetic medium in a stirred fermentor were characterized. The culture filtrate antigens were fractionated by preparative isoelectric focusing with a pH gradient of 3.5 to 5.5. The fractions were pooled according to their reaction with rabbit anti-M. faeni sera. A pool containing two major antigens which were resolved by analytical isoelectric focusing and polyacrylamide disc gel electrophoresis was obtained. One antigen was stainable with Coomassie blue and periodic acid-Schiff stain and was determined to have a mass of 51,000 daltons. The other antigen was stainable only with Coomassie blue and was determined to have a mass of 29,000 daltons. When used at 1 mg/ml, this pool reacted with the sera from all patients with farmer's lung disease by immunodiffusion but failed to react with control sera.     

Enhanced adhesion of autologous lymphocytes to gamma-interferon-treated human endothelial cells in vitro

An adhesion assay was developed using human umbilical vein endothelial cell cultures and autologous and allogeneic lymphocytes separated from cord blood. Endothelial monolayers cultured in plain or gamma-interferon (IFN gamma)-supplemented medium were cocultured with lymphocytes for 1 h and non-adherent lymphocytes removed by washing. Autologous and allogeneic lymphocytes exhibited significantly increased adhesion with IFN gamma-treated cells compared with untreated controls. The increased adhesion to IFN gamma-treated endothelial cells was significantly inhibited when autologous or allogeneic lymphocytes were pre-treated with saturating amounts of an anti-CD4 monoclonal antibody. The results indicate that IFN gamma enhances lymphocyte binding to endothelium and that the CD4 molecule may be involved in this process. This could be an important mechanism in targetting the migrating of T-helper (Th) cells to areas of chronic inflammation and in antigen presentation by endothelial cells.