Lectin-dependent Cell-mediated Cytotoxicity Research Articles

Altered nonspecific lymphocyte cytotoxicity in bronchoalveolar lavage of lung transplant recipients: can it be useful in monitoring rejection or infection?

A reliable method for determining the adequacy of immunosuppression of the lung allograft and the absence of rejection or infection does not exist. The purpose of this study is to evaluate the potential role of bronchoalveolar lavage (BAL) in monitoring the adequacy of immunosuppression of lung allografts and in identifying the possible presence of acute rejection (AR) or infection. Thirty-one consecutive lung transplant recipients were subjected to bronchoscopy, transbronchial biopsy, and BAL either as routine surveillance or for decline in lung function (n=68 episodes: 27 normal, 17 infections, six grade IAR, 10 grade II-III AR, and eight obliterative bronchiolitis). Diagnosis was always confirmed histologically and by microbiological workup. Harvested cells underwent phenotypic analysis, and T lymphocytes were subjected to nonspecific lectin-dependent cell-mediated cytotoxicity (LDCMC) and natural killer cytotoxic assays. BAL from normal grafts predominantly contained macrophages (72.8+/-4.4%), with lower neutrophil (13.9+/-4.1%) and lymphocyte (13.2+/-2.2%) populations. Grade II-III AR and infection were associated with an increase in the percentage of neutrophils (43.3+/-8.3% and 33.2+/-3.6%, respectively, P=0.02). BAL of normal allografts contained T cells with low LDCMC (7.4+/-4.5%) and natural killer cytotoxicity (6.3+/-3.4%), whereas grade II-III AR was associated with a significant elevation in LDCMC (32.5+/-11.6%, P=0.019). Pulmonary infection, regardless of its type, was associated with significant elevation in BAL natural killer cytotoxicity (23.9+/-4.9%, P=0.033). Patients with obliterative bronchiolitis, on the other hand, had a mild elevation in the percentage of neutrophils and lymphocytes in BAL, which did not reach statistical significance. However, BAL T-cell LDCMC was significantly elevated (37.6+/-13.7%, P=0.019) compared with normal and infected allografts. We conclude that phenotypic and nonspecific cytotoxic T-cell analysis of BAL, when complimented with microbiological studies, may be useful in surveillance of lung transplant recipients and in determining whether allografts are likely to be quiescent, or possibly affected by acute/chronic rejection or infection, necessitating further definitive action.

Lectin-dependent cell-mediated cytotoxicity and natural killer function in rejecting and infected lung allografts.

Differentiation between rejection and infection of lung allografts remains difficult. The effects of these two pathologic entities on the cytolytic activity of bronchoalveolar lavage (BAL) and PBL were investigated. Left lung allotransplantation was performed on 16 mongrel dogs of which 12 were available for complete studies. All animals received CA, AZA, and PRED for 2 weeks. Four grafts developed left lower lobe Gram negative pneumonia. The eight remaining recipients progressed gradually to severe rejection after acute reduction of immunosuppression. Cytolytic activity of blood and left lung BAL lymphocytes was quantitated by the natural killer (NK) and lectin-dependent cell-mediated cytotoxicity (LDCMC) assays. Two additional groups serving as controls were either given a 10-day course of immunosuppressants or had right lower lobe pneumonia induced by transbronchial inoculation of gram negative bacteria. Immunosuppressed control animals showed significant depression of PBL and BAL lymphocyte LDCMC and NK activity. Similarly, BAL lymphocytes expressed very low LDCMC in normal allografts (2.8 +/- 0.8%). Once rejection developed and progressed, LDCMC became significantly higher (15.6 +/- 2.2 and 52.7 +/- 2.8% in mild and severe rejection, respectively). There was no detectable NK activity in rejecting lung allografts. BAL lymphocytes from infected allografts, on the other hand, showed an elevation of both NK and LDCMC activity (9.1 +/- 1.1 and 14.6 +/- 1.0%, respectively). Similarly, bacterial pneumonia in control animals manifested an increase in NK and LDCMC activity in lung and blood. PBL lymphocytes of lung allograft recipients, however, had increased NK and LDCMC activity in both rejection and infection. LDCMC/NK activity ratio (LM/NK index) of lung lymphocytes was significantly higher in rejecting allografts (11.2 +/- 1.0 and 12.4 +/- 1.6 for mild and severe rejection, respectively) than in infected ones (1.2 +/- 0.3, P < 0.0001). It appears, from this study, that rejection of the lung allograft results in alterations in BAL lymphocyte phenotypes and functions that differ from those associated with bacterial infection. Such differences may be useful in distinguishing episodes of acute allograft rejection from bacterial infection.

Warm ischemia induces alteration in lung immune cell functions

Warm ischemia is an important factor in early allograft dysfunction. To elucidate cellular events involved in such lung injury, we examined the effects of warm ischemia on the cytotoxic function of lymphocytes retrieved by bronchoalveolar lavage as compared with peripheral blood lymphocytes. Warm ischemia of the lung was induced in eight dogs by crossclamping left hilar structures for 1 hour. Bronchoalveolar cells from ischemia left and unaffected right lungs, as well as blood lymphocytes, were isolated before operation and 2 hours, 72 hours, and 7 days after operation. Lung and blood lymphocytes were assayed for natural killer and lectin-dependent cell-mediated cytotoxicity. Warm ischemia resulted in a significant impairment of natural killer activity within 2 hours of reperfusion (49% of preoperative control cytolysis, p less than 0.01). There was a significant increase in natural killer activity in bronchoalveolar lavage mononuclear cells 72 hours after reperfusion injury (178.4% of preoperative value, p less than 0.01). Interestingly, these functional alterations were not paralleled with changes seen in the peripheral blood lymphocytes or the opposite nonaffected lungs, where the natural killer activity appeared significantly depressed at 72 hours. Similarly, lectin-dependent cell-mediated cytotoxicity was noted to be increased in the bronchoalveolar lavage from the ischemic lung (179.5%, p less than 0.01) but decreased in the bronchoalveolar lavage from the nonaffected lung and peripheral blood lymphocytes at 72 hours after injury. We conclude that warm ischemia is associated with a functional alteration of the local lung immune cells. Such alteration is not observed in cells from the opposite lung or peripheral blood. The observed increase in nonspecific cytotoxicity of bronchoalveolar lymphocytes can be causative in the early damage seen in poorly preserved lung allografts.

ENHANCED PRODUCTION OF INTERFERON-γ BY T LYMPHOCYTES CLONED FROM REJECTED KIDNEY GRAFTS

Seventy-seven T cell clones were generated from cell blasts infiltrating rejected kidney allografts. All clones, either CD4 or CD8, displayed cytolytic activity evaluated by lectin-dependent cell-mediated cytotoxicity (LDCC) and natural killer activities. Furthermore, both types of clones were able to produce IFN-gamma following PHA stimulation. These data suggest that the graft infiltrate is characterized by T cell clones with cytolytic potential responsible for the killing of graft cells. The production of IFN-gamma, enhancing the class II MHC expression, may amplify the recipient immune response.

Altered cellular immune function in the atelectatic lung

Pulmonary atelectasis is common and may predispose the lung to infection. We have previously shown that atelectasis impairs alveolar macrophage antibacterial function. This study examines the effect of atelectasis on the cytotoxic function of lymphocytes harvested from the bronchoalveolar space of atelectatic lung segments by bronchoalveolar lavage. Specifically, we studied natural killer and lectin-dependent cell-mediated cytotoxicity in peripheral blood and bronchoalveolar lavage lymphocytes from the atelectatic lower lobes and contralateral normal lobes in a group of 8 dogs. We observed a decline of natural killer and lectin-dependent cell-mediated cytotoxicity to 62.7% and 61.5%, respectively, of preatelectasis control values in the affected lung lobes ( p < 0.01). Simultaneous measurements of cytotoxic activity of bronchoalveolar lavage lymphocytes harvested from the unaffected contralateral normal lungs were comparable with control values. On the other hand, natural killer and lectin-dependent cell-mediated cytotoxicity activities in peripheral blood lymphocytes were significantly increased in animals having right lower lobe atelectasis (166.7% and 154.7% of pretreated normal control, respectively, p < 0.01). Atelectasis was also associated with an influx of polymorphonuclear leukocytes into the bronchoalveolar compartment. These findings confirm the presence of natural killer cells and cytotoxic lymphocytes in the bronchoalveolar compartment and demonstrate an atelectasis-induced impairment of local bronchoalveolar lymphocyte function. Such a dysfunction of local lung cellular host defenses may render the atelectatic lung susceptible to infection.

Forskolin and prostaglandin E2 regulate the generation of human cytolytic T lymphocytes

In this paper we examine the characteristics of human cytolytic T lymphocytes (CTL) generated in the presence of forskolin and PGE2. Forskolin and PGE2 suppressed the generation of class-I-specific CTL. The CTL generated in the presence of forskolin and PGE2 had different characteristics which included their ability to proliferate in response to the alloantigen and their lectin-mediated cytolytic activity. The CTL generated in the presence of forskolin had normal proliferative response to the alloantigen, whereas the CTL generated in the presence of PGE2 showed a suppressed proliferative ability to the alloantigen. The two groups of CTL were then tested for their activity in the process of lectin-dependent cell-mediated cytotoxicity. After the addition of PHA into the chromium release assay the CTL generated in the presence of forskolin normally lysed the nonspecific targets, whereas the CTL generated in the presence of PGE2 did not show the normal response in lysing the nonspecific targets. The results suggest that the cytolytic machinery was intact when the CTL were generated in the presence of forskolin but CTL were not able to either recognize or lyse the target cell. However, the CTL generated in the presence of PGE2 did not share the same characteristics as the CTL generated in the presence of forskolin because the CTL generated in the presence of PGE2 were unable to kill even in the presence of lectin. It appears that the inhibitory effects of forskolin were mediated by cAMP and not by its effects on the potassium channels because the 1,9-dideoxy derivative of forskolin which did not activate adenylate cyclase also did not suppress the generation of CTL. However, it was not established whether the diverse effects of PGE2 on the generation of CTL were mediated by cAMP-dependent, -independent or by both mechanisms.

Liver-derived T cell clones in autoimmune chronic active hepatitis: Accessory cell function of hepatocytes expressing class II major histocompatibility complex molecules

Thirty T cell clones were generated from T cell blasts, infiltrating the liver of autoimmune chronic active hepatitis (CAH) patients, stimulated with autologous hepatocytes expressing class II major histocompatibility complex (MHC) molecules and interleukin 2 (IL2). Sixteen clones were CD4+ and 14 were CD8+; all were CD25+ and WT31+, revealing that all cell lines expressed the α β chains of T cell receptor. Five CD4+ and 4 CD8+ T clones proliferated in response to hepatocytes expressing both class I and class II antigens. The hepatocyte recognition was MHC restricted because only class II MHC-matched hepatocytes were able to stimulate the CD4+ T clones, while only class I-matched hepatocytes stimulated CD8+ T clones, and because MoAbs to monomorphic determinants of class II antigens or to class I antigens appeared to block the response of the CD4+ and CD8+ T clones, respectively. These findings, together with the observation that autologous irradiated peripheral blood mononuclear cells (iPBMC) were unable to stimulate the clones, indicate that the response of these clones was directed to a liver membrane antigen in association with class II or class I MHC molecules on the surface of the hepatocytes. All the CD8+ T clones and 5 CD4+ T clones expressed high cytotoxic activity in a lectin-dependent cell-mediated cytotoxicity assay; 10 CD8+ and 3 CD4+ T clones also showed natural killer (NK)-like function. The cytolytic machinery was also present in those clones (both CD8 and CD4) recognizing the HLA-matched hepatocytes. All liver-derived T clones were able to produce high amounts of interferon (IFN)-γ, as well as being capable of secreting IL2, following PHA stimulation.

Radiation Sensitivity of Resting and Activated Nonspecific Cytotoxic Cells of T Lineage and NK Lineage

Natural killer (NK) cell-mediated killing of tumor cells is a radiation-sensitive function that in most subjects is completely abrogated by treatment of the effector cells with 3,000 cGy. The radiation sensitivity of LAK (lymphokine-activated killer) cells and their precursors, the bulk of which are NK cells, is undetermined. In this study, functional cytotoxicity assays and electron microscopy were used to determine the effect of radiation on the cytotoxic function of NK cells, LAK cells (generated by three-day culture of peripheral blood lymphocytes with IL-2), and LAK cell precursors (lymphocytes irradiated prior to culture with IL-2). For comparison, we analyzed the radiation sensitivity of lectin-dependent cell-mediated cytotoxicity (LDCC), which is primarily a function of CD3+ CD8+ granular lymphocytes. We also analyzed the radiation sensitivity of nonspecific cytotoxicity mediated by mitogen-activated T cells (AK activity). Following 3,000 cGy irradiation, NK cells retained their ability to bind to tumor cell targets but, as shown by both morphologic and functional analyses, they did not undergo activation after conjugate formation, and were unable to release the content of their granules. In order to evaluate LDCC, lymphocytes were depleted of CD16+ cells and tested in a cytotoxicity assay in the presence of Con A. The radiation sensitivity curve was comparable to that of NK cell-mediated cytotoxicity. IL-2-treated lymphocytes (LAK cells) were relatively radioresistant as compared with untreated NK cells, and their cytotoxic function was not abrogated until treatment with greater than 10,000 cGy. Cells receiving such radiation doses displayed cytoplasmic blebbing and damage of their cytoskeletal structures, with disruption of centrioles and microtubules, and disarray of the intermediate filaments. As was shown with NK cells, irradiated LAK cells formed conjugates with tumor targets but failed to degranulate. The radiation sensitivity of nonspecific cytotoxicity mediated by mitogen-activated T cells was identical to that of LAK effector cells. Doses up to 2,000 cGy did not prevent generation of LAK cells from blood lymphocytes, but 3,000 cGy did so. Blast transformation similar to that observed in IL-2-stimulated controls occurred when lymphocytes irradiated with 3,000 cGy were cultured with IL-2. These transformed cells were not cytotoxic and displayed a normal cytoskeletal apparatus but did not bear electron-dense granules.(ABSTRACT TRUNCATED AT 400 WORDS)

Radiation sensitivity of resting and activated nonspecific cytotoxic cells of T lineage and NK lineage

Natural killer (NK) cell-mediated killing of tumor cells is a radiation- sensitive function that in most subjects is completely abrogated by treatment of the effector cells with 3,000 cGy. The radiation sensitivity of LAK (lymphokine-activated killer) cells and their precursors, the bulk of which are NK cells, is undetermined. In this study, functional cytotoxicity assays and electron microscopy were used to determine the effect of radiation on the cytotoxic function of NK cells, LAK cells (generated by three-day culture of peripheral blood lymphocytes with IL-2), and LAK cell precursors (lymphocytes irradiated prior to culture with IL-2). For comparison, we analyzed the radiation sensitivity of lectin-dependent cell-mediated cytotoxicity (LDCC), which is primarily a function of CD3+ CD8+ granular lymphocytes. We also analyzed the radiation sensitivity of nonspecific cytotoxicity mediated by mitogen-activated T cells (AK activity). Following 3,000 cGy irradiation, NK cells retained their ability to bind to tumor cell targets but, as shown by both morphologic and functional analyses, they did not undergo activation after conjugate formation, and were unable to release the content of their granules. In order to evaluate LDCC, lymphocytes were depleted of CD16+ cells and tested in a cytotoxicity assay in the presence of Con A. The radiation sensitivity curve was comparable to that of NK cell-mediated cytotoxicity. IL-2-treated lymphocytes (LAK cells) were relatively radioresistant as compared with untreated NK cells, and their cytotoxic function was not abrogated until treatment with greater than 10,000 cGy. Cells receiving such radiation doses displayed cytoplasmic blebbing and damage of their cytoskeletal structures, with disruption of centrioles and microtubules, and disarray of the intermediate filaments. As was shown with NK cells, irradiated LAK cells formed conjugates with tumor targets but failed to degranulate. The radiation sensitivity of nonspecific cytotoxicity mediated by mitogen-activated T cells was identical to that of LAK effector cells. Doses up to 2,000 cGy did not prevent generation of LAK cells from blood lymphocytes, but 3,000 cGy did so. Blast transformation similar to that observed in IL-2- stimulated controls occurred when lymphocytes irradiated with 3,000 cGy were cultured with IL-2. These transformed cells were not cytotoxic and displayed a normal cytoskeletal apparatus but did not bear electron- dense granules.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of T Lymphocytes Cloned from Rejected Kidney Allograft: High Frequency of Cytotoxic T Cell Precursor

A high proportion of CD8 positive cells and inverted CD4/CD8 ratio were found in peripheral blood mononuclear cells and in freshly isolated kidney-graft infiltrating cells in two patients who underwent irreversible acute rejection. Seventy seven T cell clones were generated from the T cell blasts infiltrating rejected kidney allografts. The majority of T cell clones obtained showed CD8 phenotype in accordance to uncloned graft infiltrating cells. All clones (both CD8 and CD4) displayed cytolytic activity evaluated by lectin-dependent cell-mediated cytotoxicity and natural killer (NK) activities. None of the clones presented lymphokine activated killer phenomenon. These data suggest that the graft infiltrate is characterized by T cell clones with cytolytic potential and that these T cell clones may be responsible for the killing of graft cells by a CTL or NK type mechanism.

Effects of lentinan on cytotoxic functions of human lymphocytes.

The in vitro effects of lentinan on natural killer (NK), antibody-dependent cell-mediated cytotoxicity (ADCC), lectin-dependent cell-mediated cytotoxicity (LDCC) and mitogen-induced blast transformation were studied in patients with solid tumors and chronic lymphocytic leukemia (CLL). NK activity was measured against 51Cr-labelled K-562 targets, ADCC against antibody-coated chicken red cells. LDCC and natural cell-mediated cytotoxicity (NCMC) was assessed using 3H-thymidine prelabelled HEp-2 targets. Mitogen (PHA- and Con A-) induced blast transformation was measured by thymidine incorporation. Blastogenesis and LDCC was not influenced by lentinan. 1 microgram/ml lentinan increased NCMC of tumor-bearing subjects. The most prominent enhancement of NK and ADCC activity was seen in CLL patients, where a dose-related increase was seen (from 0.01 to 1 microgram/ml).

The low affinity 40,000 Fc gamma receptor and the transferrin receptor can be alternative or simultaneous target structures on cells sensitive for natural killing.

The role of the low avidity 40,000 dalton receptor for IgG (Fc gamma R) present on K562 and U937 cells in sensitivity to natural killing (NK) was studied by using a murine monoclonal antibody (mAb) specific for the 40,000 dalton Fc gamma R (alpha Fc gamma R mAb). Pretreatment of K562 target cells with intact alpha Fc gamma R mAb or its Fab fragment or anti-transferrin receptor (alpha TFR) mAb partially blocked in a dose-dependent manner, NK activity to K562 cells. However, combined pretreatment with alpha Fc gamma R and alpha TFR mAb completely blocked NK activity against K562 targets. As compared with K562 cells, lower levels of NK were elicited against Molt-4, U937, HL-60, and Daudi targets. Although NK activity to Molt-4 targets was not affected by alpha Fc gamma R mAb, it was fully prevented by pretreatment with alpha TFR mAb. In contrast, NK to U937 cells was not influenced by alpha TFR mAb, but it was strongly inhibited by alpha Fc gamma R mAb. The resistance of 3H-TdR-prelabeled adherent HEp-2 cells to natural cell-mediated cytotoxicity was not affected by either mAb. Lectin-dependent cell-mediated cytotoxicity (LDCC) against HEp-2 cells due to the presence of concanavalin A, and was completely abrogated by pretreatment of the targets with alpha TFR mAb, but was unaffected by alpha Fc gamma R mAb. By use of the flow cytometer, a significant correlation was detected between the relative expression of 40,000 dalton Fc gamma R and the susceptibility to NK, whereas the expression of TFR was discordant from NK sensitivity. As determined in the single cell cytotoxicity assay alpha Fc gamma R mAb reduced the frequency of target binding effector cells without affecting the number of dead bound targets. This pattern of inhibition was found against both K562 and U937 targets. Alternatively, alpha TFR mAb inhibited both binding and killing of K562 and Molt-4 targets. Because pretreatment of HEp-2 cells with alpha TFR mAb did not influence conjugate formation, the blocking of LDCC to HEp-2 cells by alpha TFR mAb can be related to post-binding events. These data show that although both the 40,000 dalton Fc gamma R and the TFR can be target structures for NK cell recognition, the TFR may also play an important role in the post-binding events.

Concanavalin A Dependent Cell-mediated Cytotoxicity (CDCMC) in Bronchoalveolar Lavage (BAL) Fluid of Patients with Interstitial Lung Diseases (ILD): Evidence of Cytolytic T-Lymphocyte (CTL) Activity

Previous investigations have demonstrated alterations in the composition and function of immune cells in BAL fluid of patients with certain ILD. However, the activity of CTL in these disorders has not been examined, although CTLs are one of the major effector cell components of immune responses. A problem in measuring the function of CTL in idiopathic ILD has been that the target antigen is unknown. To circumvent this difficulty, we used the CDCMC assay to measure CTL activity in patients with ILD. In this assay multivalent lectin binds to glycoproteins on both target cells and effector lymphocytes, causing their attachment in the absence of specific recognition of cell surface molecules. 1 Bonavida B Bradley TP Studies on the induction and expression of T cell-mediated immunity. V. Lectin-induced nonspecific Cell-mediated cytotoxicity by alloimmune lymphocytes. Transplantation. 1976; 21: 94 Crossref PubMed Scopus (73) Google Scholar , 2 Emeson EE Norin AJ Veith FJ Lectin-dependent cell-mediated cytotoxicity. A new and simple method to quantitate cytotoxic T cell activity in dogs. Transplantation. 1982; 33: 365 Crossref PubMed Scopus (22) Google Scholar

Indomethacin abrogates the suppression by cyclosporin A of lectin-dependent cell-mediated cytotoxicity to HEp-2 cells

The effects of cyclosporin A, prostaglandin E1 and indomethacin were studied on lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 human epipharynx carcinoma target cells. LDCC activity by human peripheral blood lymphocytes was evaluated by detachment from the monolayer of [3H]thymidine-prelabelled HEp-2 cells in a 24-h assay at 50:1 effector:target cell ratio in the presence of 25 μg/ml concanavalin A. Under these conditions, but without concanavalin A, considerable natural cell-mediated cytotoxicity was not elicited although LDCC was significantly augmented in the presence of concanavalin A. Addition of both cyclosporin A (0.1, 1.0 or 10 μg/ml) and prostaglandin E1 (10−8, 10−7 or 10−6 M) dose-dependently suppressed LDCC activity. Indomethacin (0.1, 1.0 or 10 μg/ml) did not in itself influence LDCC although suppression of LDCC by cyclosporin A, but not prostaglandin E1, was abrogated in the presence of indomethacin. Similar to indomethacin, acetyl salicylic acid also reversed the inhibition of LDCC by cyclosporin A. In parallel experiments, cyclosporin A elicited a more than two-fold increase of prostaglandin E production under LDCC assay conditions as measured by radioimmunoassay. Contrary to LDCC, depression of concanavalin A induced blastogenesis by cyclosporin A was not influenced by indomethacin, suggesting that the inhibition by cyclosporin A of LDCC and concanavalin A-induced blastogenesis proceed via different mechanisms.

Heterogeneity of Human Natural Killer Cells

The cell surface antigens and specificity of cultures of human natural killer (NK) cells, propagated in the presence of interleukin 2, were analyzed at the single-cell level. With the use of a micropipette isolation procedure, clones were initiated from cells of defined phenotype. The cell surface markers on the resultant clones were not stable and often diverged from those on the cells selected for initiation. Clones manifesting NK activity had low expression of T4 and T8 and most had some expression of T10, whereas clones without detectable cytotoxic activity had a considerable proportion of cells expressing T4 and/or T8 antigens. Within a cytotoxic clone, single cells shown to have killer activity were shown to express often T10 and infrequently T8; T4 was not detected on any of the reactive cells examined. The NK, reactive cells were found to express also antibody-dependent cell-mediated cytotoxicity and lectin-dependent cell-mediated cytotoxicity (LDCC), and some of the individual NK cells were shown to have the ability to lyse more than one type of target cell. However, there appeared to be considerable heterogeneity among the cells in the clones, with only 50-60% of cells displaying lytic activity against K562 cells at a given time. In addition, one cytotoxic clone appeared to have specific immune alloreactivity as well as NK and LDCC activities. The results of this study indicated that the cell surface phenotype and specificity of cytotoxic reactivity of cultured human NK cells were heterogeneous and suggested that such heterogeneity may be due to variations in the levels of differentiation and/or activation.

Effects of interleukin-2, OK-432 and interferon-gamma on in vitro induction of nonspecific killer cells by concanavalin A in mice.

Human interleukin-2 (IL-2), OK-432 and mouse interferon-gamma (IFN-gamma) were evaluated for their beneficial effects on in vitro induction of nonspecific killer cells by concanavalin A (Con A). Nonspecific killer cells could be induced by culturing C57BL/6 spleen cells for 4 days with Con A. Con A activated killer cells showed direct cell-mediated cytotoxicity (DCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) against natural killer (NK) sensitive YAC-1 cells, and LDCC against NK-resistant EL-4 cells in a 4 hr-51Cr release assay. Higher DCMC and LDCC reactivity against YAC-1 cells, and higher LDCC reactivity against EL-4 cells were obtained with spleen cells cultured with Con A plus IL-2, or Con A plus OK-432 than with those cultured with Con A alone. No beneficial effect of IFN-gamma and no additive effects of IL-2 plus OK-432, or IL-2 plus IFN-gamma were observed in the induction of nonspecific killer cells by Con A.

Cytolytic T cell clones derived from liver tissue of patients with chronic hepatitis B.

To study the role of cell-mediated immunity in chronic hepatitis B (cHB) we have cloned T cells from liver biopsies of 14 patients with cHB. As a first step, T cell lines were established from lymphocytes infiltrating the liver by culturing the biopsied specimens with autologous feeder cells and interleukin 2 (IL2). Fifty-eight clones obtained by limiting dilution showed phenotypic stability over periods of 2-26 weeks. Of the 58 clones 50 were of the "cytotoxic/suppressor" T cell subset (CD8) as defined by the monoclonal antibody T811. Only 8 of 58 clones were of the "helper/inducer" phenotype (CD4) as defined by the monoclonal antibody T151. Functional studies on 9 clones (7 of CD8+ phenotype, 2 of CD4+ phenotype) revealed high cytotoxic activity of all of these clones in a lectin-dependent cell-mediated cytotoxicity assay reflecting the killing capacity of cytotoxic T lymphocytes. All 9 clones lacked significant natural killer activity, while two additional CD8+ clones that also expressed the VEP13 antigen showed significant natural killer activity. For none of the clones tested could killer cell activity (ADCC) be demonstrated. The studies demonstrate that the clonal expansion of T lymphocytes from liver biopsies of patients with cHB in the absence of detectable antigen is possible. The propagation of in vivo activated cytolytic T lymphocytes points to an active role of these cells in the pathogenesis of this disease.

Contrasting effects of RNA and protein synthesis blocking on natural and lectin-dependent cell-mediated cytotoxicity against adherent HEp-2 cells

In this study, earlier observations concerning the independence of both natural (NCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) from DNA synthesis have been confirmed. In addition, blocking of RNA synthesis by actinomycin D and of protein synthesis, reversibly by puromycin (PM) and irreversibly by emetine (EM) had different effects on NCMC and LDCC against 3H-thymidine-prelabeled HEp-2 target cells. Similarly to the Con A-induced proliferation of lymphocytes, LDCC activity was also inhibited by blocking of RNA and protein synthesis. NCMC to HEp-2 target cells was not affected by blocking of RNA synthesis, while both PM and EM strongly enhanced NCMC activity.

Heterogeneity of Human Natural Killer Cells&lt;xref ref-type="fn" rid="FN1"&gt;2&lt;/xref&gt;

The cell surface antigens and specificity of cultures of human natural killer (NK) cells, propagated in the presence of interleukin 2, were analyzed at the single-cell level. With the use of a micropipette isolation procedure, clones were initiated from cells of defined phenotype. The cell surface markers on the resultant clones were not stable and often diverged from those on the cells selected for initiation. Clones manifesting NK activity had low expression of T4 and T8 and most had some expression of T10, whereas clones without detectable cytotoxic activity had a considerable proportion of cells expressing T4 and/or T8 antigens. Within a cytotoxic clone, single cells shown to have killer activity were shown to express often T10 and infrequently T8; T4 was not detected on any of the reactive cells examined. The NK, reactive cells were found to express also antibody-dependent cell-mediated cytotoxicity and lectin-dependent cell-mediated cytotoxicity (LDCC), and some of the individual NK cells were shown to have the ability to lyse more than one type of target cell. However, there appeared to be considerable heterogeneity among the cells in the clones, with only 50-60% of cells displaying lytic activity against K562 cells at a given time. In addition, one cytotoxic clone appeared to have specific immune alloreactivity as well as NK and LDCC activities. The results of this study indicated that the cell surface phenotype and specificity of cytotoxic reactivity of cultured human NK cells were heterogeneous and suggested that such heterogeneity may be due to variations in the levels of differentiation and/or activation.

The Appearance of Cytotoxic Lymphocytes in Unstimulated and Concanavalin A‐Activated Human Peripheral Mononuclear Cells

The presence of a mitogenic lectin during the cytotoxicity assay enables the detection of non-specific cytotoxicity (lectin-dependent cell-mediated cytotoxicity (LDCC]. Human mononuclear cells cultured at 37 degrees C in vitro became cytotoxic after 1 day, as detected by the LDCC. In some individuals, the presence of adherent cells during culture inhibited the development of cytotoxicity. Activation by concanavalin A (Con A) resulted in enhanced cytotoxicity after a few days in culture. There was no clear relationship between the lectin doses required for optimal induction of cytotoxicity and [3H]thymidine uptake. High Con A concentrations that induced a proliferative response often inhibited cytotoxicity in mononuclear cells depleted of adherent cells.