Specific cytotoxicity of human lymphocyte subpopulations defined by bacterial adherence.

It has been shown that lymphocytes can be subdivided into subpopulations based on their binding of bacteria. Monolayers of immobilized and fixed bacteria were used here to separate T cells into BA-T1T2, adherent to Escherichia coli-2 (EC-2+) and BA-T3T4, non-adherent to this strain of bacteria (Ec-2-) (our denomination). The cells were activated in mixed lymphocyte cultures (MLC) and tested for cytotoxic activity. The BA-T1T2 cells developed the same cytotoxic activity as the sham-separated T cells whereas BA-T3T4 cells did not become cytotoxic. When the T cells were separated into BA-T2 cells, adherent to Bacillus globigii (Bg+), and BA-T1T3T4, non-adherent, (Bg- cells became cytotoxic. Since BA-T1 cells, which represent 10-20% of T cells, are common to the two populations they appear to contain all T cells needed to develop the specific cytotoxicity for allogeneic cells. When the cells were first activated in MLC for 6 days and then separated by adherence to E. coli-2 or B. globigii, all cytotoxic cells were in the non-adherent fraction. We concluded that the subpopulation of T cells which are Ec-2+Bg- (less than 20%) contain all the cells required for the development of cytoxic cell function and that after activation they become Ec-2-Bg-.     

Natural killer cell function of human neonatal lymphocytes.

Human natural killer cell (NK cell) activity against K-562 target cell line was evaluated in full term cord blood (n = 30) and adult peripheral blood (n = 20) using 51Cr release assay. The level of NK cell activity was lower in cord blood compared to adult controls (39.6 +/- 11.4% vs 27.4 +/- 11.8% at effector:target ratio 50:1). Adult males showed a significantly higher NK activity compared to females. No sex difference was observed in cord blood. Furthermore, partially purified human leucocyte interferon (IFN alpha) increased in vitro NK cell function of both adult and newborn lymphocytes. The present results indicate that the appearance and maturation of human NK cells occurs during the intrauterine life of the human fetus.     

Isolation of human T lymphocytes by a negative selection procedure.

A single step method to isolate human T lymphocytes was achieved using a rosette technique to deplete non-T lymphocytes. Erythrocytes coated simultaneously with IgG and complement were used to form rosettes with cells bearing such receptors during a short incubation period and the rosetting cells removed by gradient centrifugation. T lymphocytes were isolated (greater than 96% T cells) without exposing to much experimental manipulations and in good recovery (mean 60%, range 40-75%).     

Natural killer activity of human blood lymphocytes

Natural killer (NK) activity is an operational designation. It implies the in vitro cytotoxicities registered in short-term tests exerted by lymphocytes derived from donors with no known immunization history against the particular target. The strength of the effect exerted by unmanipulated blood lymphocytes shows an individual variation. Short-term in vitro treatment with interferon elevates the lytic potential of lymphocytes. Owing to the heterogeneity of the cytotoxic blood lymphocytes with regard of cell surface properties it is not possible to separate all active cells and inactive cells in clean populations. A considerable enrichment of active cells can be achieved if nylon wool non-adherent, large, granular Fc gamma receptor positive SRBC receptor negative--or low-avidity SRBC receptor positive--OKM1-reactive cells are separated. Negative cells are concentrated in the Fc receptor and OKM1-negative high-avidity SRBC receptor positive high cell density subset. The activity of lymphocytes in the former category is potentiated by interferon and the latter acquire the lytic function if PHA is added to the assay system. Freshly separated, non-cultured tumor cells are not or weakly sensitive to the effect of unmanipulated lymphocytes. However, when the lymphocytes are treated with interferon prior to the assay a lytic potential can be induced even against these in allogeneic effector target combinations. Cytotoxic cells which acquired the function after in vivo and/or in vitro immunization are designated as 'cytotoxic T-lymphocytes' (CTL), and were shown to act on the basis of antigen recognition. The expression of known T-markers on at least a fraction of the active cells and the recognition of alloantigens in NK systems suggest that the distinction between CTL and NK cells is not as sharp as initially suggested.     

Cloned human cytotoxic T lymphocytes develop anomalous killer cell function.

Mixed lymphocyte cultures were set up between blood mononuclear cells and irradiated autologous or allogeneic B lymphoblasts infected with Epstein-Barr virus. The resulting cytotoxic effector cells were cloned and tested for activity against the stimulating B lymphoblast, K562 and melanoma targets. Specific clones which killed only the stimulating B lymphoblasts (cytotoxic T lymphocytes; CTL) were re-cloned and the subclones tested for cytolysis of B lymphoblasts and melanoma cells. Of seven primary CTL clones generated in allogeneic culture, 308 subclones developed the ability to kill melanoma cells and none retained specific CTL function. In the autologous system, 180 subclones were derived from three specific primary clones: of these, 13 (7%) retained specific function, 29 (16%) were able to kill both B lymphoblasts and melanoma cells, and 93 (52%) killed only the melanoma target. Testing of random clones demonstrated that whereas both B lymphoblast killing (CTL function) and melanoma cell killing (anomalous killer; AK function) were blocked by a monoclonal antibody to LFA-1, only CTL function was blocked by anti-T3 or anti-T8 antibodies. The factor(s) causing the progression of CTL to AK cells are discussed. These data thus demonstrate that the majority of CTL are capable of mediating AK cell function and are thus potentially suitable for passive immunotherapy.     

Zinc and bacterial adherence.

Zinc significantly enhances the ability of piliated Gram-negative and Gram-positive bacteria to attach to HeLa cells. This effect is related to the concentration of zinc and degree of bacterial piliation, and is not present with unpiliated organisms. Bacterial viability is not necessary for this effect, and sulfhydryl blockers decrease the response. These data suggest that zinc can bind to bacterial pili and augment bacterial adherence; in this manner, zinc may act as a virulence factor.     

Natural killer cells and spontaneous cell-mediated cytotoxicity in the human intestine.

Spontaneous cell-mediated cytotoxicity (SCMC) has been investigated in mononuclear cells (MNC) isolated from intestinal mucosa and autologous peripheral blood from human subjects. The proportion of cells with the NK-K phenotype (Leu 7+) were substantially lower in intestinal MNC than in autologous peripheral blood. SCMC of K-562 target cells when tested at an effector to target (E:T) ratio equivalent to that used for peripheral blood MNC was markedly deficient in intestinal MNC. This was not due to the effect of EDTA and collagenase used in the isolation process. However, at high E:T, ratios, significant cytotoxicity was demonstrated for most intestines examined probably reflecting a low proportion of effector cells within the intestinal MNC population. SCMC in both intestinal and autologous peripheral blood MNC were similarly related to the Leu 7+:T ratios used in the assay indirectly suggesting that the Leu 7+ cell may be responsible for the observed cytotoxicity. It is concluded that the apparent functional difference between similar cells derived from different sites may be largely related to differing proportions of effector cells. The findings indicate the need for specific definition of the effector cell and suggest that intestinal SCMC in health and various disease states requires re-appraisal.     

Blastogenic response of human lymphocytes to oral bacterial antigens: characterization of bacterial sonicates.

Soluble sonicate supernatant preparations were made from Actinomyces viscosus (ATCC 19246), A. naeslundii (ATCC 12104), two strains of Veillonella alcalescens (strain HV-1 and a human oral isolate), Streptococcus sanguis (ATCC 10556), S. mutans (strain 6715-T2), Bacteroides melaninogenicus (strain K110), and Leptotrichia buccalis (isolated from human dental plaque). These supernatants were characterized with reference to their chemical and antigenic components and their biological activity determined by using in vitro lymphocyte blastogenesis as a measure of the host's cellular immune response. The sonicate supernatant of each bacterium contained protein, neutral sugars, methylpentose, and nucleic acids. Protein was the major component in all except L. buccalis, in which neutral sugars predominated. The antigenic components in each supernatant were detected by using rabbit antisera prepared against the whole bacteria and the sonicate supernatant. The supernatants showed a complex antigenic distribution on immunoelectrophoretic analysis. The supernatants were shown to be antigenic and not mitogenic in nature, since neither cord blood lymphocytes nor all adult lymphocytes were stimulated. The supernatant antigen preparations showed a reproducible, dose-dependent, and kinetic response in vitro, which was similar to that seen with the antigen preparation streptokinase-streptodornase.     

Isolation of human splenic macrophages and lymphocytes by countercurrent centrifugal elutriation

Certain tissues, such as the spleen, are rich sources of mononuclear phagocytes (MP); however, separating the phagocytes from tissues and removing the contaminating cells have been difficult. We report here a method for the extraction and purification of human splenic MP that employs gentle homogenization of splenic fragments with a Tenbroeck tissue homogenizer, controlled digestion with purified collagenase to free MP from splenic stroma, incubation with DNase to dissociate cell clumps and purification by countercurrent centrifugal elutriation (CCE). With homogenization and enzymatic digestion most of the splenic nonspecific-esterase-positive cells are freed into suspension as determined by morphometric analysis of 2 micron sections from plastic embedded spleen stained for alpha-naphthyl butyrate esterase (ANB). Overall cell recovery after homogenization and enzyme treatment is 56 +/- 7%; no selective cell loss occurs as determined by differential cell counts at each purification step. CCE of up to 3 X 10(9) treated spleen cells results in recovery of 63 +/- 6% of the elutriated cells and separates nearly 50% of the recovered MP into enriched fractions. These MP are morphologically intact as determined by light and electron microscopy and are actively phagocytic. Highly purified (96%) autologous splenic lymphocytes are a useful by-product of this separation technique.     

Enhancement of spontaneous killer cytotoxicity by soluble factor

Two-stage stimulation of SK activity of normal human peripheral blood lymphocytes by soluble factors was demonstrated. The first stage was initiated by factors present in supernatant derived from normal B-LCL cultures. Only cell lines which could induce SK activity in culture in an MLR-type reaction had the capacity to produce the active factor. Supernatant factor required adherent cells to cause SK augmentation. The interaction of adherent cells plus supernatant factor resulted in the production of a second soluble factor which stimulated an increase in SK activity in responding lymphocyte populations. This second stage involved a different soluble factor which acted directly on the non-SK, Fc-negative lymphocyte population, and within 3 hr. Data obtained using antisera to interferon (IF) indicated that IF is a component of the second soluble factor, and not of the supernatant factor derived from the B-LCL.     

Comparative stimulation of granulocyte adherence and chemotaxis by bacterial products.

Many inflammatory stimuli increase both in vitro granulocyte adhesiveness and motility. The present investigation compared the effects of bacterial products on in vitro granulocyte adherence to nylon fiber and granulocyte motility. Escherichia coli endotoxin and Staphylococcus aureus and E. coli culture filtrates enhanced both motility and granulocyte adherence to nylon. Column chromatography of S. aureus culture filtrate demonstrated that similar molecular weight components stimulated both motility and granulocyte adherence. In contrast, endotoxin stimulated granulocyte adherence but not chemotaxis in plasma-free medium. In addition, a group D streptococcal culture filtrate enhanced motility but not adhesiveness of plasma-free granulocytes. These studies indicate that inflammatory stimuli may interact with granulocytes in concert to enhance both adhesiveness and motility, or they may interact independently to stimulate one in vitro function preferentially.     

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.     

Direct activity of human T lymphocytes and natural killer cells against Cryptococcus neoformans.

Lymphocytes constitute a critical component of host defenses against cryptococcosis. Previously, we demonstrated that human lymphocytes cultured with interleukin-2 formed conjugates with, and directly inhibited the growth of, Cryptococcus neoformans. Here, we explore the anticryptococcal activity of freshly isolated, highly purified populations of human peripheral blood lymphocytes. Lymphocytes were incubated with encapsulated C. neoformans for 24 h, after which the lymphocytes were lysed, dilutions and spread plates were made, and CFU were counted. Fungistasis was determined by comparing growth in wells with and without lymphocytes. Nylon wool-nonadherent peripheral blood mononuclear cells (NWNA PBMC) were highly fungistatic, even if either T cells or natural killer (NK) cells were depleted by panning. A mixed population of T cells and NK cells, obtained by rosetting NWNA PBMC with sheep erythrocytes, completely inhibited cryptococcal growth, whereas the nonrosetting cells had little fungistatic activity. CD4+, CD8+, and CD16/56+ lymphocytes, isolated by positive immunoselection, had potent growth-inhibitory activity. In contrast, purified B cells had no activity. Fungistasis was seen even in the absence of opsonins. Antifungal activity was markedly diminished when surface receptors on NWNA PBMC were cleaved by treatment with trypsin or bromelain. Supernatants from stimulated lymphocytes or concentrated lymphocyte sonicates were not active. Lymphocyte-mediated fungistasis was seen with two different strains of C. neoformans. CD4+, CD8+, and CD16/56+ lymphocytes formed conjugates with C. neoformans, as observed under Nomarski differential interference contrast microscopy and videomicroscopy. These data demonstrate that freshly isolated peripheral blood T cells and NK cells have the capacity to bind and directly inhibit the growth of C. neoformans.     

Activated lymphocytes reduce adherence of Aspergillus fumigatus.

Lymphocytes comprise up to 30% of the cells present in human bronchoalveolar lavage fluid and thus could participate in host response to infectious Aspergillus fumigatus conidia. We have examined the possibility that lymphocytes might play a role during early infection by either damaging the fungus or interfering with adherence. When incubated with A. fumigatus conidia for 20 h, highly purified 5-day-old lymphocytes activated with either IL-2 or phytohaemagglutinin, but not untreated lymphocytes, were consistently able to reduce residual fungal biomass as estimated by a metabolic assay. T lymphocytes, but not NK cells, appeared to be responsible for this activity. Lymphocytes bound both A. fumigatus conidia and hyphae, and the antifungal activity of the lymphocytes required direct lymphocyte fungus contact. In a separate set of experiments using release of 51Cr from 51Cr-loaded fungi as an estimate of fungal damage, lymphocyte-induced loss of fungal biomass was found to be due to loss of fungal adherence rather than to direct fungal damage. The detached hyphae were also found to be metabolically intact and to have normal morphology by electron microscopy. These data demonstrate that IL-2- and phytohaemagglutinin-activated lymphocytes exhibit a contact-dependent ability to reduce adherence of germinating conidia of A. fumigatus to a surface.     

Isolation from human serum of an inactivator of bacterial lipopolysaccharide.

By a series of chromatographic procedures involving precipitation by salt, gel filtration, anionic exchange, and hydroxyapatite elution, a protein--termed the lipopolysaccharide inactivator (LPS-I)--has been isolated from normal human serum. As a result of treatment of bacterial lipopolysaccharide (LPS) by LPS-I, the treated LPS loses its toxicity for mice and reactivity in the Limulus assay and appears to be irreversibly disaggregated. The inactivation of the LPS by the purified LPS-I is temperature and time dependent and is not blocked by the addition of irreversible inhibitors of serine esterases. The LPS inactivator migrates as an alpha-globulin in whole serum and has a sedimentation velocity of approximately 4.5S. Characteristics of the inactivated LPS are briefly described using internally labeled LPS.     

Factors influencing bacterial adherence to biomaterials.

The adherence of bacteria to implanted medical devices is believed to be important in the development of implant associated infections. Measures which reduce bacterial adherence should reduce the incidence of these infections. However, in order to assess the importance of adherence, the effectiveness of methods to reduce adherence, and compare data from different laboratories, the conditions of the in vitro studies on adherence need to be specified. There are currently no correct and incorrect methods, however, methods used need to be carefully described. The studies reported here indicate that the definition of adherence needs to be established, with the use of polystyrene as the reference material recommended. Since the adherent organisms lose adherence traits with culture, cultures must be selected for adherence regularly. It is important to control the number of organisms/ml but the volume used is not important. The medium used to grow the organisms and the use of stationary, rocking or flow conditions will alter adherence and need to be specified and be consistent within a set of experiments. Culture conditions, methods of rinsing the material, methods of elution and counting, or direct counting of organisms on the material need to be specified. Finally, as much information as possible on the bulk and surface properties of the material should be provided. The handling of the material for the experiments should be careful and defined. Fingerprints, contact with protein, wet surfaces vs dry surfaces, etc., will all affect the subsequent adherence. The materials should not be re-used since the removal of the adherent proteins or the biofilm is very difficult. Progress can be made in this important area if the details of procedures are specified.     

Isolation of spontaneous and interferon inducible natural killer like cells from human colonic mucosa: lysis of lymphoid and autologous epithelial target cells.

Viable mononuclear and epithelial cells were dispersed from human colonic tissue by treatment with collagenase and ethylene diamino-tetra acetate (EDTA) and separated by centrifugal elutriation. Using a single cell cytotoxic assay, functional endogenous and interferon responsive mononuclear cytotoxic cells were detected. Compared to peripheral blood lymphocyte associated killer cells that had been exposed to similar treatment, these colonic killer cells demonstrated lower efficiency cytotoxicity of Molt-4 target cells. Furthermore, inefficient, but interferon responsive cytotoxic cells were present which bound and lysed freshly isolated autologous epithelial cells. The cytotoxicity of these colonic mononuclear natural killer (NK) like cells appeared specific in that cells bound but did not lyse NK resistant Raji cells, even after interferon activation.     

Anchorage and lymphocyte function. Acquisition of spontaneous motile behaviour by human blood lymphocytes and its modulation by concanavalin A.

The majority of splenic lymphocytes were motile, showing lamellipodial activity almost immediately after purification. In contrast, fresh blood lymphocytes were non-motile and maintained their spherical suspension morphology. The number of motile blood lymphocytes increased markedly during a 2-day in vitro culture period. This increase was enhanced by high cell density and required a metabolically active cell with protein synthesis but not exogenous mitogens. The spontaneous development of motility in different subpopulations of blood lymphocytes was analysed by means of monoclonal antibodies. The results indicated that cells which were motile immediately after purification were almost exclusively non-T lymphocytes. Lymphocytes which became motile during in vitro culture included both T and non-T cells. Substrate adhesion mediated by concanavalin A (Con A) changed the morphology of motile T lymphocytes and instead of being polar, the cells flattened over the substratum and acquired a non-polar shape. Furthermore, the morphogenetic response induced by Con A-mediated substrate adhesion appeared to distinguish T and non-T lymphocytes. Thus, the length of the cell perimeter showing lamellar activity was greater in T than in non-T lymphocytes, and the degree of polarity was greater in non-T (with and without B-cell markers) than in T lymphocytes.     

Isolation of human small bowel intraepithelial lymphocytes by annexin V-coated magnetic beads

Human intestinal intraepithelial lymphocytes (IEL) are important effector cells of the mucosal immune system and their study is hampered by the difficulty of their isolation. The molecular study of enriched samples of IEL is mandatory in the diagnosis of enteropathy-associated T-cell lymphoma and refractory celiac sprue. In order to isolate human small bowel IEL, we took advantage of the stress that intestinal epithelial cells (IEC) suffer during the conventional initial steps of IEL isolation, which induces their apoptosis but not that of IEL. After cell individualization by dithiothreitol and ethylenediamine tetraacetic acid, two-thirds of human IEC can be stained with Annexin-V due to their surface exposure of phosphatidyl serine, a sign of apoptosis. This percentage increases to 95% after performing a density gradient to enrich for IEL. This allows for the use of Annexin-V-coated magnetic beads, originally designed for the removal of dead cells from cell cultures, to obtain >95% pure, 99% viable and untouched IEL after two rounds of depletion. This simple procedure has proven useful for the isolation of human IEL for functional and molecular studies and can conceivably facilitate the diagnosis of intestinal lymphoid malignancies that rely upon the study of pure IEL preparations.