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Lymph node and spleen cells from normal mice were cultured for 3 days with polyoma virus-induced tumour, Ehrlich's ascites turnout or leukaemia L 1210 cells. This resulted in in vitro immunization of the lymphocytes, which were then transferred to irradiated target cells labelled with 51Cr. Normal, i.e. non-immune thioglycollate-stimulated peritoneal macrophages were also added to some tubes. Non-immune macrophages mixed with immunized lymphocytes showed a significantly increased ability to destroy tumour ceils as compared with macrophages in the absence of immunized lymphocytes. The immunized lymphocytes were almost entirely inactive alone. When the number of macrophages was kept constant the cytotoxicity was dependent on the number of viable immunized lymphocytes placed on the target cells. Immunized lymphocytes, in the presence of macrophages, only exhibited strong killing of the target cells against which they had been immunized; some lysis of ‘bystander’ cells was, however, seen provided specific target cells were present. Macrophage monolayers exposed to immunized lymphocytes upon contact with specific antigen became ‘armed’ and showed a significant cytotoxicity for specific target cells. When immunized lymphocytes and normal macrophages were treated with actinomycin D and puromycin, cytotoxicity was inhibited in the immunized lymphocytes but not in the macrophages. The possible mechanism of normal macrophage cooperation with immunized lymphocytes in the cytotoxic killing reaction is discussed. Results presented in this paper favour the view that immunologically specific cytophilic factor (presumptive cytophilic antibody) is involved in the macrophage-mediated cytotoxicity in the system studied. [ABSTRACT FROM AUTHOR]

Summary: Natural killer (NK) cells provide a major defence against human cytomegalovirus (HCMV) infection through the interaction of their surface receptors, including the activating and inhibitory killer immunoglobulin‐like receptors (KIRs), and human leucocyte antigen (HLA) class I molecules. Also γ marker (GM) allotypes, able to influence the NK antibody‐dependent cell‐mediated cytotoxicity, appear to be involved in the immunological control of virus infections, including HCMV. In some cases, their contribution requires epistatic interaction with other genes of the immune system, such as HLA. In the present report, with the aim of gaining insight into the immune mechanisms controlling HCMV, we have studied the possible associations among humoral and NK responses, and HCMV infections. In a previous study we assessed whether the KIR and HLA repertoire might influence the risk of developing symptomatic (n = 60) or asymptomatic (n = 60) disease after primary HCMV infection in the immunocompetent host. In the present study, the immunocompetent patients with primary symptomatic HCMV infection were genotyped for GM3/17 and GM23 allotypes, along with the 60 participants with a previous asymptomatic infection as controls. Notwithstanding the presence of missing data record, advanced missing data recovery techniques were able to show that individuals carrying the GM23 allotypes, both homozygous and heterozygous, GM17/17, HLA‐C2 and Bw4T KIR‐ligand groups are associated with the risk of developing symptomatic infection. Our findings on the role of both cellular and humoral immunity in the control of HCMV infection should be of value in guiding efforts to reduce HCMV‐associated health complications in the elderly, including immunosenescence, and in transplantation. [ABSTRACT FROM AUTHOR]

Intravenous immunoglobulin (IVIg) is successfully used in the treatment of autoimmune diseases involving self-reactive CD8+ T cells. However, its direct influence on the cytotoxic response remains unknown. Using an antigen cross-presentation assay and a mouse model of ovalbumin (OVA) immunization, we showed that IVIg decreases the in vitro activation, proliferation and cytokine secretion of OVA-specific CD8+ T cells (OT-I), as well as the in vivo generation of OVA-specific CD8+ T cells. In addition, IVIg significantly decreases the proportion of perforin- and CD107a-expressing CD8+ T cells, and inhibits the cytotoxic activity of OVA-activated OT-I cells. The interference of IVIg with the CD8+ T-cell response is associated with T-cell receptor blockade, therefore reducing the interaction between effector and target cells. A similar blockade is observed on human CD8+ T cells, suggesting that the observations reported here could apply to the IVIg-mediated improvement of CD8+ T-cell-mediated autoimmune conditions in human patients. [ABSTRACT FROM AUTHOR]

Summary α1,3-Galactosyltransferase gene-knockout pigs transgenic for porcine cytotoxic T-lymphocyte antigen 4 immunoglobulin (pCTLA4-Ig) have been produced to reduce T-cell-mediated rejection following xenotransplantation. The level of soluble pCTLA4-Ig in their blood was greatly in excess of the therapeutic level in patients, rendering the pigs immune-incompetent. Soluble pCTLA4-Ig produced by these transgenic pigs was evaluated for binding to porcine and human (h) B7 molecules, and for its inhibitory effect on allogeneic and xenogeneic human T-cell responses. Porcine CTLA4-Ig-expressing peripheral blood mononuclear cells (PBMCs) and aortic endothelial cells (AECs) were evaluated for their direct inhibitory effect on hCD4+ T-cell responses. Soluble pCTLA4-Ig and purified hCTLA4-Ig showed similar binding to pB7 molecules, but pCTLA4-Ig showed significantly less binding to hB7 molecules. The pCTLA4-Ig and hCTLA4-Ig inhibited the response of hCD4+ T cells to pAECs equally, but pCTLA4-Ig was less successful in inhibiting the human allogeneic response. The hCD4+ T-cell response to PBMCs from pCTLA4-Ig pigs was significantly lower than that of non-pCTLA4-Ig pigs. Although pCTLA4-Ig was detected in the cytoplasm of pCTLA4-Ig-expressing pAECs, only a minimal level of soluble pCTLA4-Ig was detected in the supernatant during culture, and pCTLA4-Ig-expressing pAECs did not inhibit the xenogeneic direct human T-cell response. High-level tissue-specific production of pCTLA4-Ig may be required for sufficient immunosuppression for organ or cell (e.g. islets) transplantation. [ABSTRACT FROM AUTHOR]

The reason(s) why individual cytotoxic T lymphocytes (CTL) possess a fast-acting, perforin/granzyme-mediated, as well as a much slower, Fas ligand (FasL) -driven killing mechanism is not clear, nor is the basis for wide variations in killing activity exhibited by individual CTL, ranging from minutes to hours. We show that perforin expression among individual, conjugated CTL varies widely, which can account for the heterogeneity in killing speeds exhibited by individual CTL. Despite a 2-hr lag in FasL-based killing, CTL lytic action is enhanced when the two mechanisms operate in concert. This is explained by finding that the two pathways in fact are jump-started simultaneously with the lag in FasL lytic action reflecting pre-lytic caspase-8 activation and BH3-interacting domain (BID) cleavage. The complementary action of the two lytic pathways, co-expressed at varying levels among individual CTL, facilitates the lytic action of late-stage poor perforin-expressing CTL, ensuring optimal cytocidal action throughout the CTL response. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are important effectors of both innate and adaptive immune responses. Although human and mouse NK cells are extensively characterized, much less is known about the rat cells, partly because of the current lack of reliable isolation techniques. We aimed to develop a method for isolating highly pure 'untouched' rat NK cells by negative selection from splenocytes. Thereafter, we characterized them phenotypically and functionally in comparison with those isolated by positive selection targeting the NKR-P1 receptor. Our novel method isolated highly pure untouched NK cells reproducibly with 97 ± 0·7% ( n = 7), 96·6 ± 0·8% ( n = 3) and 88·3 ± 1·5% ( n = 9) in LEWIS, Fischer and athymic nude rats, respectively. The positively selected NK cells were less homogeneous and exhibited undesired method-related activation profiles. Resting negatively selected NK cells were less proliferative and less robust compared with positively selected NK cells. Although resting positively selected NK cells were more cytotoxic, interleukin-2 (IL-2) activation increased the cytotoxicity of negatively selected cells three-fold. The negatively selected NK cells responded to cross-linking of the NKR-P1 receptor by calcium mobilization from intracellular stores. However, combined IL-2 and IL-12 activation resulted in significantly more interferon-γ release from positively selected NK cells. This new NK-cell isolation method will allow a deeper insight into rat NK-cell phenotypes and the roles of their receptors in the biology of these cells. [ABSTRACT FROM AUTHOR]

Freshly isolated murine intestinal intraepithelial lymphocytes (IEL) express CD95 ligand (CD95L), as shown by reverse transcription–polymerase chain reaction (RT–PCR) and fluorescence-activated cell sorter (FACS) analysis. Between 15 and 25% of IEL could be stained with an antibody to CD95L. Therefore it was investigated whether the CD95L/CD95 pathway was effective in IEL cytotoxicity. Stimulation of IEL in vitro with concanavalin A (Con A) induced a strong cytotoxic response, which was much higher when using CD95-expressing target cells. This effect was most evident when comparing the specific lysis of CD95-transfected target cells of the leukaemia cell line L1210 with that of the untransfected parental cell line. In addition, an antibody to CD95 was able to dramatically reduce the specific lysis of CD95-expressing target cells. After stimulation with Con A, which is able to bind to CD95L, the effects were more obvious compared with the triggering of the T-cell receptor (TCR)-αβ or γδ. On the other hand, EGTA reduced the Con A-induced cytotoxicity. Together these findings support a role of the CD95L/CD95 pathway in IEL cytotoxicity, even though the reaction was Ca2+ sensitive. As a function, CD95L-expressing IEL should be able to contribute to the elimination of CD95-expressing target cells in the intestine. [ABSTRACT FROM AUTHOR]

We have investigated the characteristics of antigen-specific reductions in routine immune responses to rat collagen type I (R-CI), chick collagen type II (C-CII) or bovine collagen type II (B-CII). Intravenous pretreatment with the appropriate soluble collagen or collagen-coupled spleen cells led to the development of antigen-specific reduced immune responses, the former treatment being more effective than the latter. In the case of CII, pretreatment with R-CI or non-related antigens was ineffective. However, pretreatment with denatured bovine-CII, native bovine-CII or chick-CII led to immune hyporesponsiveness for either the homologous or heterologous CII molecule. A delayed development of the diminished immune responses was observed for the cell-mediated immune response (CMI), as measured by in vivo delayed-type hypersensitivity (DTH), in that no reduction was evident at Day 7 but a significantly decreased response was observed at Day 14. Collagen-specific IgG and IgM antibody responses were consistently reduced by the pretreatment and remained reduced daring the study period. The antigen-specific hyporesponsive state was not sensitive to cyclophosphamide treatment and was not transferable with hyporesponsive spleen cells. Additionally, we have induced unresponsiveness to CII by treating mice with an antibody directed to T helper cells (GK1.5). This treatment led to profound reductions in CII CMI responses as well as CII antibody levels. However, this unresponsive state is not permanent and not transferable with spleen cells from treated mice. Them two types of procedures, soluble B-CII i.v. or GK1.5 treatment, not only resulted in CII hyporesponsive states, but also produced delayed onset and decrease incidence of arthritis in the appropriate strains. [ABSTRACT FROM AUTHOR]

A subpopulation of human lymphoid cells called natural killers is able to lyse certain normal and neoplastic targets in an in vitro cytotoxicity assay. The molecules which enable them to recognize sensitive cells, or permit turnout cells to escape remain unknown. In the studies described here we have compared some of the plasma membrane characteristics of a NK-sensitive human leukaemia cell line (K562) with those of a partially resistant subclone derived from it (K562-Clone I). Gel electrophoresis of cell-surface proteins radiolabelled by lactoperoxidase-catalysed iodination, periodate-borohydride tritiation, or biosynthetically by incubation with [3H]fucose did not reveal any reproducible differences between the sensitive and resistant lines. However, analysis of glycolipids showed that Clone I incorporated significantly more fucose than did the parental line, and that it synthesized a minor population of complex structures not found in the original K562. A subclone of Clone I(Clone I-Con ArI), made resistant to the toxic effects of concanavalin A, became sensitive once again to NK, and showed the parental glycolipid profile. These results suggest that the Clone I line, selected for resistance to NK, may have altered one or more of its intermediate oligosaccharides or pathways of fucose incorporation into glycolipid, and points to one process by which a tumour cell might modulate its surface to escape recognition by natural killers. [ABSTRACT FROM AUTHOR]

The mechanism by which a Db-specific helper clone (clone 9) and a hybridoma-derived Db-especific helper factor, referred to as ASHF, induced cytotoxic T lymphocyte (CTL) responses to alloantigens was investigated. Clone 9 or ASHF helped CBA thymocytes to produce CTL against B6 (H-2b), but not D2 (H-2d), alloantigens. However, when BDF1 (H-b,d) spleen cells or an equal mixture of B6 or D2 spleen cells were used as stimulator cells, CTL responses to both B6 and D2 were induced. This suggested that clone 9 cells or ASHF could induce the production of long-ranged, non-antigen-specific helper factor(s) in B6-stimulated cultures. One of these long-ranged factors produced in B6-stimulated cultures was found to be interleukin-2 (IL-2). Thus, clone 9, which is a non-IL-2 producer, increased the production of IL-2 by irradiated B6 spleen cells and by CBA anti-B6 cultures by 4·7- and 5·7-fold, respectively. ASHF did not increase the amount of IL-2 produced by irradiated B6 spleen cells but increased the amount of IL-2 produced in CBA anti-B6 cultures by 3·8-fold. Clone 9 cells or ASHF did not increase IL-2 production in D2-stimulated cultures. Upon stimulation with concanavalin A or antigen, clone 9 cells also produced a non-antigen-specific helper factor. This factor (IL-X) synergized with excess human recombinant IL.2 (rIL-2) in the polyclonal activation of BDF1 or D2 CTL precursors. A model that involves the participation of ASHF, clone 9 cells, IL-2 and IL-X in the induction of a cytotoxic response is proposed. [ABSTRACT FROM AUTHOR]

Antibodies to a number of cell surface antigens expressed on mouse spleen cells were tested for their ability to inhibit antibody-dependent cell-mediated cytotoxicity (ADCC) of antibody-coated sheep erythrocytes (Ab-SE) in the absence of complement. Of the antibodies tested, only the antisera to Ly- 5 and H-2 antigens significantly inhibited ADCC by spleen cells reactive with those antisera. Inhibition by Ly-5 antisera was shown to be allotype-specific by inhibition experiments using C57BL/ 6-Ly-5.1 (B6-Ly-5.1) and congenic C57BL/6-Ly-5.2 (86-Ly-5.2) mice. Inhibition by Ly-5.1 antiserum appeared not to be due to competition for the Fc receptor (FcR), since in mixing experiments 'third-party' thymus cells treated with Ly-5 antiserum did not inhibit the cytotoxic activity of untreated cells. In comparing inhibition induced by antisera to H-2 and Ly-5 antigens, Ly- 5.1 antiserum was more inhibitory at nearly every dilution tested. In addition, F(ab')2 fragments of Protein A-purified Ly-5.1 antibody were inhibitory to RALR/c spleen effector cells in ADCC of Ab- SE, whereas F(ab')2 fragments of H-2 antibodies had no effect. Because ADCC of tumour cells may share a common lytic mechanism with ADCC of erythrocytes, several antisera to cell surface antigens found on spleen cells were tested for inhibition of ADCC to antibody-coated P815 tumour cells (Ab-P815). As seen in ADCC to Ab-SE, anti-Ly-5.1 was a more potent inhibitor than antibody against either H-2k or H-2d antigens. The results are discussed in the light of the ability of Ly-5 antisera to inhibit various types of cell-mediated killing. [ABSTRACT FROM AUTHOR]

The present study was undertaken to investigate the role of 'antigen processing' in influenza virus- specific cytotoxic T (Tc)-cell recognition. H-2 Db-restricted Tc-cell clones specific for the 1934 influenza nucleoprotein (NP) were tested in an in vitro proliferation assay for the recognition of intact virus, purified protein or peptide antigen. Inactivated virus required further 'processing', which was inhibited by either NH4Cl treatment or paraformaldehyde fixation of antigen-presenting cells (APC). Purified NP, by contrast, was readily presented by both normal peritoneal exudate cells and H-2 Db gene 'transfected' L cells. The response was not inhibited by either NH4Cl or prior paraformaladehyde treatment of APC. Peptone-induced peritoneal exudate cells presented ineffectively unless treated with NH4Cl or prefixed with paraformaldehyde. Comparison of the responses to either purified protein or a synthetic peptide implies that the epitope recognized by the three NP specific clones is not 'cryptic' and, therefore, that the purified protein, in this case, does not require 'processing'. [ABSTRACT FROM AUTHOR]

Multiple minor antigen incompatibilities produce unexpectedly strong DTH reactions in mice. These experiments showed that this is probably not due to the effect of a single minor incompatibility, and that the response to the MUC is relatively weak compared with that against multiple minor antigens. Assessment of the in vivo response to multiple minor antigens in delayed-type hypersensitivity responses and in priming for subsequent mixed lymphocyte culture generation of lymphocyte- mediated cytotoxicity revealed a major degree of cross-reactivity for antigens not present on the initial in vivo stimulus. Recruitment of cross-reactive cells in vivo might play a role in these strong responses to multiple minor antigens. [ABSTRACT FROM AUTHOR]

In order to explore the role of glutathione in cell-mediated cytotoxicity, we have examined the effect of the sulphydryl-reactive and glutathione-depleting agent 2-cyclohexene- 1 -one on antibody- dependent cellular cytotoxicity, spontaneous cell-mediated cytotoxicity, and cell-mediated lympho- lysis by human peripheral blood mononuclear cells. 2-Cyclohexene- 1 -one significantly inhibited (P < 0.001) both antibody-dependent and spontaneous cell-mediated cytotoxicity using three different cell-line targets, at three different killer: target cell ratios (10: 1,25: 1 and 50: 1). Using K-562 cell-line targets, spontaneous cell-mediated cytotoxicity was inhibited by 2-cyclohexene-1-one with an ID50 of 0.71 × 10-4 M-3.98 × 10- M, while antibody-dependent cellular cytotoxicity was less sensitive to inhibition, and required slightly higher concentrations of 1.48 × 10-4 M-3.98 × 10-4 M to achieve 50% inhibition. Similar results were seen with human colon tumour cell-line and Chang liver cell-line cells as targets. Maximal inhibition occurred when 2-cyclohexene-1-one was added to the cytotoxicity assay 60 mm prior to, at the start of, or within the first 60 mm of a 4-hr assay; inhibition of cytotoxicity occurred with pretreatment of effector cells; and no inhibition of cytotoxicity was observed with pretreatment of target cells. Both the allogeneic mixed leucocyte reaction and cell- mediated lympholysis were also significantly inhibited (P < 0.001) by 2-cyclohexene-1-one. These studies demonstrate that 2-cyclohexene-1-one is an effective inhibitor of cell-mediated cytotoxicity and suggest that glutathione, specific glutathione-protein interactions, or protein-bound sulphydryl groups are involved in allowing cells to carry out cytolysis. [ABSTRACT FROM AUTHOR]

Antibody-dependent cell-mediated cytotoxicity (ADCC) carried out by fresh and precultured peripheral blood mononuclear cells (PBMC) was compared. The results indicate that the cytotoxic capability was decreased in precultured PBMC, probably due to the effect of suppressor cells. The depletion of adherent cells before preincubation eliminated inhibitory effects, suggesting that the suppressor activity resided in this cell fraction. In addition, supernatants from adherent cell cultures were also able to suppress ADCC of non-adherent PBMC. Normal ADCC values were restored by supplementing the reaction medium with cycloheximide (Cy) 1 × 10-3 M. The enhancing effect of Cy was exerted upon whole and adherent PBMC, while the opposite effect was observed when ADCC was carried out by non-adherent cells. The fact that Cy increased ADCC activity by precultured PBMC might rule out the possibility that suppression could be ascribed to trivial factors such as cell death, overcrowding or steric hindrance. The results presented in this report support the premise that ADCC is under active immunoregulation. [ABSTRACT FROM AUTHOR]

An increase in macrophage tumouricidal activity in the spleen was demonstrated following intravenous infection of genetically resistant C57BL/10 mice with Listeria monocytogenes, but not after infection of BALB/c mice. However, tumouricidal macrophages appeared in the peritoneal cavity of both strains after infection, while NK cell activity was generally higher in BALB/c mice. The activation of tumouricidal macrophages and NK cells in the C57BL/B10 mice was T independent and, at least in the peritoneal cavity, radioresistant (600 rads). The relevance of these results to the genetic control of resistance to Listeria is discussed. [ABSTRACT FROM AUTHOR]

Cytotoxic cell activity directed against paternal alloantigens was investigated in primiparous C57BL/10 and CBA/Ca mice using a microcytotoxicity assay. Most allogeneically or syngeneically mated females lacked effector cells in their spleens or paraaortic lymph nodes both during pregnancy and immediately postpartum. However, spleen, cells from 33% of C57BL/10 females mated to CBA/Ca males exhibited low levels of paternal target cell killing (P<0·05–0·01). Alloimmunization of virgin mice prior to mating resulted in only allogeneically mated females producing cytotoxic cells and alloantibody. These responses were not detectable during pregnancy bat appeared immediately postpartum. The ability of pregnancy to induce memory cell formation was tested by allowing females one successful pregnancy before challenging them postpartum with allogeneic spleen cells. Kinetic studies of cytotoxic cell production showed that C57BL/10 females that had borne (C57DL/10 × CBA/Ca)F1 litters responded earlier than their syngeneically meted sisters giving a peak response at 4 days compared to 7 days after immunization. This indicates that a single allogeneic pregnancy can prime the mother against paternal alloantigens suggesting that the conceptus is weakly immunogenic. [ABSTRACT FROM AUTHOR]

Groups of 6-week-old female C57B1/6 mice were fed a normal diet with recommended levels of all vitamins Dr a vitamin-deficient (VD) diet containing half of the recommended level of each vitamin. At different time periods (1-11 weeks) after the initiation of diets, basal natural killer (NK) activity, interleukin-2 (IL-2) and concanavalin A (Con A)-induced cytotoxic activity, Con A-induced IL-2 production and levels of allospecific cytotoxic T cell activity generated in a mixed lymphocyte culture (MLC), were studied in spleen cells derived from control and VD mice. Results indicated that: (i) spleen NK activity remained normal until 2 weeks after the initiation of VD diet, fell steeply to tow levels at the 4 and 5 week time points and remained depressed thereafter; (ii) IL-2- and Con A-induced levels of cytotoxic activity in spleen cells derived from VD mice declined at 4 weeks alter the institution of VD diet, and then remained low throughout the study; (iii) the capacity of spleen cells from VD mice to generate IL-2 in response to Con A and cytotoxic T cells in response to allogeneic spleen cells, was normal at 1 and 4 weeks alter initiation of the VD diet and was markedly depressed at the 6 and 9 week time points. These results suggest that partial combined deficiencies of dietary vitamins strongly influence assays of immune function. [ABSTRACT FROM AUTHOR]

The effect of neonatal thymectomy at various times after birth (Tx-1, Tx-7) on effector and suppressor I cells responsible for cell-mediated cytotoxicity (CMC) for allogeneic antigens was deter- mined. Following in-vitro primary mixed lymphocyte cultures, in the absence of T-cell growth factor (TCGF), alloreactive CMC was not detected in spleen cells of Tx-1 mice, but was detected in spleen cells of Tx-7 mice at as high levels as in those of sham-operated mice. However, in the presence of TCGF, as much alloreactive CMC was detected in spleen cells of Tx-1 mice as in those of Tx-7 mice. Furthermore, TCGF production was not detected in spleen cells of Tx-1 mice but was detected in those of Tx-7 mice. In in-vivo experiments, inhibition of allogeneic tumour growth and CMC in spleen cells showed the same pattern as in in-vitro experiments. These results sup- port the concept that the reduction of CMC in Tx-I mice might be due to a defect in helper function (TCGF-producing capacity) rather than to a defect in cytotoxic T lymphocytes and/or cytotoxic T lymphocyte precursors. Alloreactive suppressor T cells could not be induced in spleen cells of Tx-1 mice but were induced in spleen cells of Tx-7 mice. Therefore, it was suggested that alloreactive suppressor T cells require the presence of the thymus for 7 days after birth in their development. [ABSTRACT FROM AUTHOR]

A system is described which involves an in-vivo and an in-vitro step for the generation of strong primary cytotoxic T lymphocyte (CTL) responses to multiple minor alloantigens. The protocol consists of immunizing mice in the footpad with irradiated, minor alloantigen-different spleen cells. Four days later, cells from the lymph nodes draining the site of injection are cultured for 3 days in the absence of antigen to allow the development of cytotoxic activity. The analysis of the effector phase of the response indicates that the killer cells are H-2 restricted cytotoxic cells specific for minor alloantigens. The in-vitro phase of these cytotoxic responses is I cell-dependent and the responder cells proliferate strongly during the culture period. Both cytotoxic and proliferative responses are abolished by passing responder cells over Sephadex G-10 prior to culture. The results suggest that the maturation process of CTL-precursor (CTL-P) cells into effector CTL in vitro depends upon proliferating T cells and accessory cells. [ABSTRACT FROM AUTHOR]

The secreted IgMs of two human lymphoblastoid cell lines, RPMI-6410 and RPMI-8392, were purified. Antisera against these two IgMs were raised in rabbits and made idiotypically specific to the respective antigens through various absorption procedures. By immunofluorescence and radioimmunoassay techniques, the purified anti-idiotype antibodies were found to react also with the membrane Igs of the respective cell lines, but not with those of other cell lines. The purified anti-idiotype antibodies were then coupled with Chlorambucil to form antibody-drug conjugates, whose effectiveness in the in-vitro killing of target cells was evaluated by a chromium-release cytotoxicity assay. The results showed that these anti-idiotype antibody-Chiorambucil conjugates were specifically cytotoxic to lymphoblastoid cells that bore membrane Igs carrying the respective idiotypic determinant(s). Furthermore, the conjugates were far more effective in causing cytolysis to the target cells than either Chlorambucil or the anti-idiotype antibodies alone. [ABSTRACT FROM AUTHOR]

Spontaneous natural killer (NK) activity and antibody-dependent cellular cytotoxicity (ADCC) of blood lymphocytes against five human tumour cell lines (K562, Molt-4, HL-60, Chang, Daudi) and three mouse tumour lines (YAC, P815, RBL-5) were ten- to 100-fold lower than normal in six patients with Chediak-Higashi (CH) disease. NK and ADCC were defective at 4 hr, and less so at 18 hr. The NK activity in normals and CH patients was mediated in part by FcR+, E- effector cells. ADCC against human erythrocytes was normal in CH patients, as were lectin-dependent cytolysis and mixed lymphocyte proliferative responses. Phagocytosis of antibody-coated ox erythrocytes was normal in CH patients as well, These observations confirm that the CH syndrome is associated with a profound and selective defect in NK and ADCC activity against tumour cells, whereas other mononuclear cell-mediated functions are normal. [ABSTRACT FROM AUTHOR]

We have examined the development of specific cytotoxic T-cell activity in the lungs and the epithelium and lamina propria of the small intestine following tumour cell inoculation by subcutaneous or intraperitoneal routes. After an intraperitoneal injection of tumour cells, large amounts of cytotoxic activity are detectable in the lungs and lamina propria. In comparison, the epithelial lymphocytes of the small intestine display low cytotoxio activity. After a subcutaneous injection, little cytotoxicity is detectable except in the lungs and the development of such cytotoxicity has a much shorter time course compared with that induced by an intraperitoneal inoculation of tumour cells. The data indicate a marked difference in the functional capacity of lymphocytes from the epithelium and lamina propria of the small intestine. [ABSTRACT FROM AUTHOR]

A high vitamin E diet altered cell-mediated immune responses of maturing BALB/c mice. Serum corticosterone concentrations in these animals were also determined because of the pharmacological effect the glucocorticoid hormone has on immune systems. Mice receiving high vitamin E diet (twenty times higher than the vitamin E concentration in control diet) showed significant increase in antibody-dependent cellular cytotoxicity (ADCC) activity within a week after initiation of the diet. Adult levels of spleen and intestinal ADCC activities were reached at a much younger age in these high vitamin E-treated animals. In addition, there was a good correlation between increase in spleen and intestinal ADCC activities and the significantly lower than control concentration of serum corticosterone in these mice. However, after prolonged consumption of high vitamin E diet, decrease in ADCC activity was observed in these mice. No significant differences in PHA-induced spleen lymphocytes [3H]-thymidine incorporation, along with a decrease in serum corticosterone levels, were observed in mice given either high vitamin E or the control diets throughout most study periods. [ABSTRACT FROM AUTHOR]

Protective immunity towards a lethal dose of 104 living ADJ-PC-5 cells can be induced in 56% of BALB/c mice by treating them with 107 irradiated cells. This protection can be modulated by pretreatment of animals with lower numbers of irradiated turnout cells. Repeated injections of 10¹ cells before immunization and challenge with turnout cells lead to tumours in only 16% of the animals, whereas repeated injections of 105 cells shift the tumour incidence to 78%. The enhanced tumour incidence is paralleled by suppression of T-cell cytotoxicity which can be transferred by spleen cells into 100 r-irradiated BALB/c mice. The possible significance of induction of specific immunosuppression during the initial stages of turnout growth is discussed. It is stressed that this suppression becomes effective at a dose 10³ times lower than that which could induce protective immunity. [ABSTRACT FROM AUTHOR]

The nature of immunoglobulin and effector cells involved in the antibody-dependent cell-mediated adhesion and cytotoxicity to Wuchereria bancrofti larvae has been investigated. Human neutrophils and eosinophils purified from peripheral blood by metrizamide gradients readily adhered to the parasites in presence of IgG fraction of sera from majority of elephantiasis cases with amicrofilaraemia and many of the endemic normals. The cells from normal, microfilariae carriers and elephantiasis cases were equally effective in the adhesion reaction. While the adhered neutrophils killed the larvae, eosinophils were ineffective in this respect. DEC treatment of elephantiasis cases results in a significant reduction in the ability of their sera to promote cellular adhesion. [ABSTRACT FROM AUTHOR]

The complement-mediated lysis of human lymphocytes by three monoclonal anti-human T-cell antibodies OKT3.PAN, OKT4.IND and OKT8.SUP was studied. The percentages of Ficoll-Hypaque-isolated mononuclear cells lysed by these antibodies were respectively: 65% for OKT3.PAN, 39% for OKT4.IND and 20% for OKT8.SUP. Optimal lymphocytotoxic reactions were noticed when unabsorbed rabbit serum was used as the source of complement (C). Addition of heat-inactivated human, mouse and newborn calf sera but not of foetal calf serum inhibited the lyric activity of the antibodies. Treatment of peri- pheral mononuclear blood cells with OKT3.PAN and C abrogated their mitotic response to PHA and Con-A. Sheep erythrocyte rosetting lymphocytes (E+ cells) treated with OKT4.IND or OKT8.SUP and C exhibited no marked changes in responsiveness to PHA, Con-A or allogeneic non-T cells.However, only E+ cells enriched with OKT4.IND-reactive cells responded to purified protein derivative, proliferated in the autologous mixed lymphocyte reaction and were highly sensitive to hydrocortisone suppression when stimulated by PHA. Our data indicate that these monoclonal antibodies can be regarded as invaluable tools for enumeration, characterization and functional assessment of human T cells and their subclasses. [ABSTRACT FROM AUTHOR]

The combined use of an improved technique for inactivating cytotoxic T cells during the lytic reaction, with time lapse cinematography and isotope release assay, have shown that the initiation of the morphological phase of zeiosis corresponds to the time when the target cell is irreversibly programmed to lyse. It is suggested that rubidium release occurs during the phase of zeiosis. The rate of release of chromium is the result of two phases of variable length. The reversible phase (before programming for lysis) and the irreversible phase from the initiation of zeiosis to the final lytic event. The time required for programming for lysis to occur depends on the number of T cells reacting with the target cell. Thus at high ratios in tubes, where multiple interactions are possible, most target cells are programmed to lyse within 10 min. However, under conditions when T-cell:target-cell conjugates are kept in suspension to prevent multiple interactions, programming for lysis can take several hours. This provides an explanation for the apparent difference in timing of zeiosis and programming for lysis in previous publications. It is also shown that further T-cell interactions with the target cell after programming for lysis (i.e. during the irreversible phase), markedly influence the rate of chromium release. This provides an explanation for the fact that chromium release takes at least 3 h t6 reach plateau levels after inactivation of the T cells, whereas at high effector cell ratios, maximum levels of chromium release can occur within 1 h. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cell activity was found in the various lymphoid compartments of the normal guinea-pig, prominent in the spleen and absent in the thymus. Oestrogen treatment. which increased the Kurloff cell population in blood, spleen and thymus, did not alter NK cell activity in blood and spleen but markedly augmented the lytic capacity of the thymus. Resetting reactions and selective depletion studies in normal and oestrogen-treated animals revealed the NK cells to belong to a small population of E+ Kurloff cells, some of which were Fe+ and others apparently Fe+. Some of these natural killer cells in the spleen also had receptors for C3 and carried Ig (probably cytophilic). ln the lymph nodes, however, the NK cells were found to he E + lymphocytes, again some of which were Fe+ and others Fe+. [ABSTRACT FROM AUTHOR]

The experiments reported here were designed to investigate the role of major histocompatibility antigens alone in primary allogeneic responds in vitro. Mixed lymphocyte cultures were established using small numbers of responder spleen cells and semi-allogeneic or fully allogeneic stimulator spleen cells. The cytotoxicity generated in individual cultures was then detected by a sensitive 51Cr-release assay and the frequencies of responding cytotoxic T lymphocyte precursors (CTL-P) determined by limit dilution analysis. To investigate the responses of spleen cells to major histocompatibility (H-2) antigens alone, strains of BALB/c mice congenic at the H-2 locus were used as responder and stimulator cells. The frequencies of CTL-P responding in such congenic combinations were compared with those obtained using mouse strain combinations differing at minor histocompatibility (minor-H) loci in addition to H-2. A similar fraction of spleen cells responded regardless of whether the stimulator cells differed at the H-2 locus alone or at multiple minor-H loci in addition to H-2. It was concluded that minor-H antigenic differences do not contribute significantly to allogeneic responses. [ABSTRACT FROM AUTHOR]

An investigation was made of the proxies of cytotoxic T cells induced by Con A and exposure to LCM virus-infected cells. As a basis for such studies, the optimal conditions for in vitro Con A stimulation of in vivo LCM virus-primed C3H mouse splenocytes were determined. The most potent cytoxicity was obtained when responder cells were cultured in the presence of Con A in a concentration of 2 μg/ml for 3 days, but strong cytoxicity was also measured on days 2 and 4 When stimulation was performed by the homologous antigen maximal response was seen on day 4 although marked cytotoxicity was also noted on day 3. Effector cells produced by the two different procedures showed equally high degrees of cytotoxicity against LCM virus-infected target cells, whereas they did not appear cytotoxic against non-infected targets. If LCM virus-immune mice were treated intravenously with 280 μg of Con A per animal, moderate cytotoxicity was demonstrable in splenocytes from these mice 1, 2 and 3 days after treatment. The in vitro generation of secondary cytotoxicity by Con A as well as by the homologous antigen was found to be totally dependent on DNA synthesis. The reactivated cells were investigated for in vivo anti-viral effect by measuring their ability to protect intracerebrally LCM virus-infected mice from a fatal outcome of this infection. LCM virus-primed splenocytes stimulated by the homologous antigen caused complete protection, while Con A-reactivated cells did not protect at all. Secondary cytotoxic cells stimulated by Con A and by LCM virus showed fairly similar in vitro characteristics, but fundamentally different in vivo qualities. [ABSTRACT FROM AUTHOR]

Isolated P815 karyoplasts are up to four times more susceptible to lysis by T cells than intact cells, suggesting that the target cell nucleus or a nuclear associated structure may be particularly sensitive to cell-mediated cytotoxicity. Cytoplasts showed variable susceptibility. The morphological changes seen by time-lapse cinematography show that the first change seen with intact cells is a burst of zeiosis (membrane blebbing), whereas neither cytoplasts nor karyoplasts exhibit zeiosis. These observations suggest that zeiosis results from changes in the cytoskeletal system. This is discussed in relation to the possibility that T cells kill target cells by causing physical damage to a critical organelle inside the cell rather than to the membrane itself. [ABSTRACT FROM AUTHOR]

Electron micrographs of material fixed during the first 10 mm of a T-cell cytotoxic system showed T-cell projections and T-cell burrowing into target cells. These observations were made possible by using a system with a very high rate of killing. The projections vary in shape and size, and can push deeply into the target cell, distorting organelles in their path, including the nucleus. The projections contain fine fibrillar material, to the exclusion of organelles. They push the target cell membrane in front of them to form pockets approximating to the shape of the projection. Areas of close contact occur between the projections and the target cell membrane, particularly at the leading edges. The likelihood that these projections develop as a result of Contact with specific antigen, and are involved in the Cytotoxic mechanism is discussed. [ABSTRACT FROM AUTHOR]

Antibody dependent cytotoxicity of chicken erythrocytes by purified rat eosinophils, neutrophils, macrophages and K cells has been compared by 51Cr release and time lapse microcinematography. Techniques have been developed for purifying these effector cell types. Both eosinophils and neutrophils cause rapid release of 51Cr from erythrocytes. Time lapse observations indicated that this was the result of phagocytosis. Eosinophils show rapid membrane movement and repeatedly engulf and regurgitate the erythrocytes. On the other hand, neutrophuls become quiescent after phagocytosing erythrocytes, and remain quiescent until the remains of the cell are expelled. Neutrophils presumably have a mechanism for the release of soluble material, as 51Cr is released rapidly. Macrophages show a similar quiescence after phagocytosis, but in these cells there is apparently no rapid mechanism to expel material, as there is no significant 51Cr release over 20 h. K cells appear to damage chicken erythrocytes more slowly than they destroy tumour cells. Mast cells cause antibody-Independent cytotoxicity which can be attributed to the release of toxic materials. None of these effector cells produced the type of lysis seen with antibody and complement. [ABSTRACT FROM AUTHOR]

Secondary cytotoxic T cells were generated in vitro by culturing WE3-LCM virus-specific memory CBA/H spleen cells with WE3-LCM virus-infected syngeneic peritoneal cells at 37° for 5 days, and were found to be highly potent in reducing virus titres in the visceral organs of syngeneic recipients when transferred 24 h before intravenous virus challenge (e.g. 3.1 × 106 cells transferred gave approximately 3 logs reduction of virus titre). Protection was measured by titrating spleens for virus in a plaque assay, usually 2 days post virus-challenge. Intravenously administered secondary effectors did not, however, elicit a reduction in brain virus titres by 3 days after intracerebral inoculation of virus. Cells mediating protection were sensitive to treatment with anti-θ ascitic fluid plus complement. Protection occurred only when donors of secondary cells, infected stimulator cells and recipients shared H-2K or H-2D, and there was a similar genetic restriction for these effectors to efficiently lyse virus-infected targets in vitro. Spleen cells from ectromelia virus-memory mice were cultured with ectromelia virus-infected syngeneic peritoneal ‘stimulator’ cells and generated secondary effector cells which protected syngeneic recipients from subsequent challenge with ectromelia but not LCM virus. However, secondary effector cells derived from in vitro stimulation of spleen cells from WE3-LCM virus-memory mice with WE3-LCM virus-infected syngeneic stimulator cells caused a significant reduction in spleen virus titres in syngeneic recipients challenged with either ectromelia or LCM virus. If WE3-LCM virus-infected stimulator cells were fixed and responder spleen ceils were either from very old (6–12 months post-priming) WE3-LCM virus-memory mice, or from ARM-LCM virus-memory mice, this unidirectional cross-protection of ectromelia virus-challenged mice was less pronounced. One explanation for the unidirectional cross-protection was that the transferred secondary effectors contained and released infectious LCM virus (or defective virions) which adsorbed to recipients' spleen cells; these latter cells displayed both LCM and ectromelia virus-specific antigenic patterns after ectromelia virus challenge, rendering them susceptible to specific T-cell mediated lysis. Clear specificity of effector cells was demonstrated at the effector:target level in vitro between LCM virus and ectromelia virus. (Experiments to compare the in vivo activity of secondary effectors with primary effector T cells raised in vivo or in vitro were hampered because of the large amounts of infectious virus in the latter two populations.) Secondary effectors reduced spleen, lung and liver virus titres when transferred 24 h after virus challenge, but were less efficient in protecting recipients than when given before virus. [ABSTRACT FROM AUTHOR]

Immune responses were examined after immunization with chicken erythrocytes (CRBC) in mice, Cytotoxicity of spleen cells was assessed by the release of 51Cr from labelled target cells. (1) At early stages (day 4–7) after primary intraperitoneal immunization, direct cytotoxicity of spleen cells was raised efficiently in C57BL/6 and AKR mice, but not in C3H,/He, SL and DDD mice. Delayed hypersensitivity and antibody production were raised to almost the same extent in all the strains at such periods. (2) Effector cells in direct cytotoxicity were θ-positive and IgG-positive, and glass-nonadherent and Nylon wool column-adherent. Effector cells in antibody-dependent cell-mediated cytotoxicity in the presence of antibody to CRBC were eliminated by treatment with anti-IgG serum, but not by treatment with anti-θ serum. (3) Cytotoxicity and antibody production were raised efficiently after intraperitoneal or intravenous immunization, but not after footpad immunization. On the other hand, delayed hypersensitivity developed most efficiently after footpad immunization. [ABSTRACT FROM AUTHOR]

We have investigated the ability of male-mouse spleen cells before and after culture in the absence of deliberate antigenic stimulation to show specific cytotoxicity to syngeneic embryo-fibroblast cells. The data suggest that cytotoxicity which develops spontaneously in such spleen cell cultures is directed primarily against embryo-associated antigens. Syngeneic Con-A-stimulated spleen cells, which, unlike fresh normal spleen cells, are also lysed by rabbit anti-mouse embryo antisera, are also a suitable target to demonstrate spontaneously developing cytotoxicity. [ABSTRACT FROM AUTHOR]

Cultured female spleen cells develop less cytotoxicity to embryo-associated antigens than do cultured male spleen cells. This difference is less pronounced if the cells are treated before culture with a specific hetero-anti-B cell antiserum. Female non-T cells (especially from primiparous animals) can decrease the spontaneously developing T-cell cytotoxicity in male splenic T lymphocytes upon culture. By artificially manipulating the T cell: non-T cell ratio in the spleen population prior to culture, it seems one can alter the type of cytotoxic mechanism (to embryo-associated antigens) which develops in the cultures. [ABSTRACT FROM AUTHOR]

The mechanisms whereby neutrophils become cytotoxic to chicken erythrocyte (CRBC) target cells were investigated in a system of lectin-dependent neutrophil-mediated cytotoxicity (LDNMC). Through the use of drugs and specific metabolic inhibitors, LDNMC was found to be dependent on energy supplied by anaerobic glycolysis and on other active metabolic functions of the neutrophil. 2-Iodoacetamide, 2-deoxy-D-glucose, diisopropyl-fluorophosphate, colchicine, cytochalasin B, and dibutyryl cyclic AMP all caused dose-dependent inhibition of cytotoxicity, while inhibitors of protein and nucleic acid synthesis were without effect. Cell surface membrane-active agents, such as chloroquine, hydrocortisone and chlorpromazine inhibited cytotoxicity, while vitamin A caused enhancement. Lectins which agglutinated neutrophils, but not necessarily CRBC, such as phytohaemagglutinin (PHA-P), concanavalin A (Con A), soybean agglutinin (SBA), and Ricinus communis agglutinin (RCA), mediated cytotoxicity while lectins which did not cause agglutination, such as pokeweed mitogen (PWM), did not mediate cytotoxicity. Preincubation of neutrophils, but not CRBC with PHA-P, resulted in time-dependent enhancement of cytotoxicity, while pre-incubation with Con A yielded progressive inhibition of cytotoxicity. These studies suggest that lectin binding to the cell surface causes alterations of the membrane, that LDNMC requires cell to cell surface contact, and that cytotoxicity depends on active metabolic processes. [ABSTRACT FROM AUTHOR]

Isolated normal human peripheral neutrophils became cytotoxic to chicken red blood cells (CRBC) in the presence of phytohaemagglutinin (PHA) and concanavalin A (Con A), a phenomenon which we have termed lectin-dependent neutrophil-mediated cytotoxicity (LDNMC). Substantial cytotoxicity could be demonstrated by I h of incubation at 37°. Isolated human peripheral lymphocytes were not cytotoxic to CRBC in the presence of these lectins, even after 18 h of incubation. Both PHA and Con A exhibited dose responses over a wide concentration range and displayed progressive, time-dependent cytotoxicity. Cytotoxicity for both PHA and Con A was greater at 37° than at 22°, and was undetectable at 4°. CRBC as target cells were much more readily lysed than either sheep or human erythrocytes. Erythrophagocytosis did not appear to play a role. [ABSTRACT FROM AUTHOR]

[75Se]L-selenomethionine can be employed as a label for following lymphocytes in vivo. Its properties are broadly similar to those of 51Cr with regard to uptake, release of the isotope on cell death and reutilization; thus relatively high levels of 75Se in lymphoid tissue indicate the presence of viable labelled cells. It differs from 51Cr in that active release of selenium occurs from the labelled cells when the ambient cell concentration is low. No significant reutilization of leaked label occurs. Leakage of 75Se label occurs in vivo; this differs in the spleen and lymph nodes. Evidence is presented to suggest that 75Se losses reflect the transit times of cells through lymphoid organs. [ABSTRACT FROM AUTHOR]

Cell-mediated cytotoxicity in virulent rodent malaria has been demonstrated in vitro, whereby splenic cells effected specific lysis of 51Cr-labelled erythrocytes from parasitized animals. More than one cellular cytolytic effector system appeared to be operative in the mouse. One effector system involved splenic macrophages, from normal or immune animals, which were increasingly cytotoxic to target cells in the presence of antibody. A second effector system involved nylonpurified immune spleen cells which were significantly more cytotoxic than similarly prepared normal spleen cells in the presence of immune serum. Although antibody alone was not cytolytic, the data strengthen the concept that immune spleen cells and antibody can interact in a co-operative fashion to mediate cytotoxic reactions in malaria. [ABSTRACT FROM AUTHOR]

The experiments described demonstrate that the in vitro lysis of chicken erythrocytes (CRC) by allergized mouse spleen cells (ASC) is an antibody-dependent phenomenon. Supernatants obtained from cultures of ASC were shown to contain antibody which could effect antibody-dependent cell-mediated cytotoxicity. The cytolysis observed with ASC was inhibited by both an IgG fraction and a F(ab')2 portion of rabbit anti-mouse IgG. No inhibition was obtained by pretreating the ASC with AKR anti-C3Hθ serum and guinea-pig complement. [ABSTRACT FROM AUTHOR]

Fourteen days after injection with Detroit-6 cells (a human cell line) rats have high titres of antibody capable of inducing cytotoxicity towards Detroit-6 cells in normal spleen cells. At the same time, the spleen cells from the injected rats are cytotoxic for Detroit-6 cells in vitro. However, killing by allergized spleen cells is apparently not entirely antibody induced, since heterologous anti-thymocyte sera (absorbed with rat bone marrow cells) inhibit target cell killing by spleen cells from injected rats but do not appreciably inhibit killing of antibody-coated target cells by normal spleen cells. Wistar spleen cells 'activated' by transfer into irradiated August rats are cytotoxic for August hepatoma cells. This cytotoxicity is also inhibited by the absorbed anti-thymocyte sera. It is suggested that the cytotoxicity of spleen cells in vitro, in both these allogeneic and xenogeneic systems, is mediated by thymus-derived (T) cells. [ABSTRACT FROM AUTHOR]

The aim of the experiments was to find a T cell-mediated cytotoxic reaction where genetically determined low responsiveness is under the control of recessive genes. The T cell-mediated immune response to DBA/2 mastocytoma cells P 815-X2 in different inbred strains, F1 hybrids and congenic resistant strains of mice was determined using the 51Crk-release method. Two types of low responsiveness were found. The first already well documented type is characterized by dominance of low responsiveness in genetical analysis. It is due to tolerance because of the identity of the H-2 linked specificities of the recipient and of DBA/2 (H-2d) mice from which the turnout is derived. The second and new type of low responsiveness in T cell-mediated immunity was found in DBA/1 mice. In this strain low responsiveness is inherited as a recessive trait. The cytotoxic response of DBA/1J mice against the C57B1/6 lymphoma EL 4 is normal. Thus, their low responsiveness is restricted to a limited number of membrane determinants. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells can kill target cells by either necrotic or apoptotic mechanisms. Using the 51Cr-release assay to measure necrotic death of target cells, neonatal NK cells had low NK activity (K562 targets) and high lymphokine-activated killer (LAK) activity (Daudi targets) compared with adult cells, as has been previously reported. Using a 125I-deoxyuridine (125I-UdR) release assay, cord cells were shown to also have higher apoptotic LAK activity against YAC-1 target cells. Interleukin-4 (IL-4) inhibited interleukin-2 (IL-2)-induced necrotic killing of target cells by adult effectors but had no such inhibitory effect on cord cells. In contrast, IL-4 inhibited both adult and cord LAK cytotoxicity of YAC-1 target cells by apoptotic mechanisms with higher suppression observed in cord cell preparations. Using a colorimetric substrate conversion assay, IL-2 induced higher, and IL-4 had a more significant suppressive effect on, cord cell granzyme B enzyme activity compared with adult cells, paralleling apoptosis cytotoxicity data. Co-culture of either adult or cord LAK cells with IL-4 had a similar inhibitory effect on granzyme B protein expression, as detected by Western blotting. In contrast, IL-4 did not inhibit perforin expression, thereby defining IL-4 as a cytokine that can differentially regulate the NK cell-mediated cytotoxicity processes of apoptosis and necrosis. The differential sensitivity of cord cells to cytokine regulation of cytotoxicity may also have implications for cord blood transplantations, as NK cells are known to function as an effector cell in both graft-versus-host disease and in the graft-versus-leukaemia phenomena. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a chronic autoimmune disease of unknown aetiology. Recently, superantigens have been implied in the pathogenesis of RA. Superantigens activate a large fraction of T cells leading to the production of cytokines and proliferation. In addition, superantigens direct cellular cytotoxicity towards major histocompatibility complex (MHC) class II-expressing cells. There is now increasing evidence that cytotoxic T cells may be involved in the pathogenesis of RA. In the inflamed synovia class II-positive synovial fibroblasts (SFC) are found. In the present study it was tested whether MHC class II-positive SFC serve as target cells for superantigen-induced cellular cytotoxicity. SFC were stimulated with interferon-γ to express class II antigens, then they were cultivated in the presence of CD4-positive T cells with or without staphylococcal enterotoxins (SE). Cytotoxicity of T cells was measured as release of lactate dehydrogenase from SFC. Specific cytotoxicity was only found in the presence of class II-positive SFC depending on the dose of SE. Maximum lysis was seen after 20 hr. T-cell cytotoxicity was inhibited by antibodies to MHC class II antigens. The data suggest that class II-positive SFC not only function as accessory cells for SE-mediated T-cell proliferation and interleukin-2 production but may also be the targets of superantigen-mediated cellular cytotoxicity. [ABSTRACT FROM AUTHOR]

We have previously reported the identification by the murine monoclonal antibody (mAb) 1C4 of the first leucocyte receptor which is involved in natural cytotoxicity (NC) against WEHI-164; the NC-1.1 receptor. We report herein the identification and characterization of a second leucocyte receptor which is involved in NC, NC-2 (MW 50 000), identified by a rat anti-mouse mAb D9 (immunoglobulin G2a; IgG2a). Flow cytometric analysis showed that NC-2 was expressed on<6% of splenic leucocytes of different inbred mouse strains and 96% of the cells of a mast-cell line which has high NC activity. In vitro treatment of splenic leucocytes with the D9 mAb blocked effector cell–WEHI-164 target cell conjugation and NC by ≈ 50% without affecting natural killing (NK). Western blot analysis of affinity purified NC-2 and NC-1.1 using the D9 and 1C4 mAbs showed specific reactivity of the proteins with D9 and 1C4, respectively. Pretreatment of splenic leucocytes with both mAbs blocked NC 84%, a result which almost doubled that caused by either mAb alone. Flow cytometric screening of 16 different mouse cell lines showed that 19% of the cell lines expressed both receptors, 6% expressed only NC-2, 44% expressed mainly or only NC-1.1 and the remaining cells expressed neither receptor. These data indicate that D9 identifies a xeno-antigen, NC-2, which is expressed on cells mediating NC and not NK, and that it is not the previously described NC-1.1 allo-antigen. We conclude that NC-2 is likely to be one of a number of receptor molecules on cells mediating NC against tumour cells. [ABSTRACT FROM AUTHOR]

Chlorpromazine (CPZ) has been previously shown to protect against endotoxin [lipopolysacchar- ide (LPS)] lethality and inhibit the release of tumour necrosis factor in vivo. We investigated at the cellular level whether this was due to direct inhibition of tumour necrosis factor-α (TNF-α) synthesis, using LPS-stimulated THP-1 human monocytic leukemia cells. We also studied the effect of CPZ on human TNF-α action by assessing TNF-α cytotoxicity on mouse fibrosarcoma L929 cells. CPZ (1–100 μM) inhibited TNF-α production in THP-1 cells in a dose dependent manner by a maximum of 80%. This effect was comparable to that of two well-known inhibitory drugs, dexamethasone and cyclicAMP. Inhibition was also evident at the mRNA level. On the other hand CPZ (10–25 μM) also inhibited TNF-α activity: in fact it reduced the cytotoxicity of TNF-α on L929 cells (EC50 was increased four times) and could provide protection even as a post- treatment. CPZ inhibited TNF-induced apoptosis in L929 cells, as detected by analysis of nuclear morphology. However, since we showed that apoptosis was very limited, and was not the main mode of cell death in our conditions, this could not explain the overall protection. Since CPZ did not interfere with either the oligomerization state of TNF-α or its receptor binding, our data suggest that it reduced cytotoxicity by inhibiting some steps in the TNF-α signalling pathways. [ABSTRACT FROM AUTHOR]

Treatment of highly purified natural killer (NK) cells with the protein-tyrosine kinase (PTK) inhibitors, genistein and herbimycin A, diminished their ability to lyse K562 target cells by as much as 100%. The ability of NK cells to bind to K562 cells was not affected by PTK inhibition. However, activation of phospholipase C (PLC) in response to K562 cell binding (as measured by inositol phosphate turnover) was decreased by as much as 75% when PTK activity was inhibited. Furthermore, there was an increase in tyrosine phosphorylation of NK cell PLCγ2 after exposure to K562 target cells. These data indicate that a PTK is involved in the activation of NK PLC by tumour target cells in the cytotoxic response. [ABSTRACT FROM AUTHOR]