Interleukin-2-activated murine cell lines with macrophage- and B-lymphoblast-lytic activity.

Interleukin-2 (IL-2)-activated murine killer cell lines with macrophage- and B-lymphoblastic-lytic activity were established, and their target specificity, surface markers, recognition-related structures, and requirements for optimal cell growth were characterized. Sustained growth of IL-2-activated lymphocytes was supported by the combination of IL-2 and IL-4-enriched T cell conditioned medium (CM), but was not supported by IL-2 alone or the combination of IL-2 and IL-3-containing CM in the presence of macrophages (M phi). The established line required continuous contact with M phi to maintain anti-M phi cytolytic activity. Flow cytometric analysis showed that the original line isolated by the first cloning was Thyl+, CD4-, and weakly CD8+, FcR+. The majority of these cells were CD3+ and TCR-V beta 8+. From this line, the CD3+, TCR-V beta 8+ and CD3-, TCR-V beta 8- clones were isolated by subcloning. The former clone showed Thyl+, CD3+, CD4-, CD8-, TCR-V beta 8+, FcR(+)-phenotype, and the latter clone showed Thyl+, CD3-, CD4-, CD8-, TCR-V beta 8-, FcR- phenotype. The original line and subclones showed a similar target specificity and killed resident or thioglycollate (TG)-induced peritoneal M phi and B-lymphoblasts, but did not kill T-lymphoblasts. Allogeneic M phi, M phi-like cell line P388D1, and B cell hybridoma were sensitive, whereas fresh lymphocytes, T cell lymphoma BW5147, natural killer (NK)-sensitive YAC-1, and NK-resistant P815 tumor cells were resistant to lysis by these cytotoxic lines. The addition of anti-H-2 heteroserum, anti-MHC class 1, anti-MHC class II, anti-CD3, or anti-TCR-V beta 8 monoclonal antibody (mAb) to assay cultures did not inhibit the anti-M phi cytolysis by these killer cells. In addition, the CD3- TCR-V beta 8- clone killed M phi and B lymphoblasts better than the CD3+, TCR-V beta 8+ clone. These results suggest that cytotoxic lines established in this study do not use the T cell receptor (TCR) molecules to recognize target cells and the MHC molecules are not involved in recognition. Anti-LFA-1 mAb partially inhibited anti-M phi-lysis, suggesting that the cell contact between targets and effectors is important in cytolysis. Our present data suggest that the culture condition containing IL-2, IL-4, and M phi may support the continuous growth of non-MHC-restricted killer cells with relative target specificity against M phi and B-lymphoblasts.     

Induction of natural killer cell activity in mice by injection of indomethacin

The natural killer (NK) cell system of mice in the peritoneal cavity is of very low to undetectable activity, and testing peritoneal NK cells is a useful model to study the influence of activating substances upon local injection. Injection of indomethacin at doses of 100-400 micrograms/mouse caused a marked activation of NK cell activity which was maximal at 3 days and lasted for a total of 6 days. A similar albeit less marked effect was observed with other cyclooxygenase inhibitors such as aspirin. Prostaglandin E2 reversed the activation of NK cells induced by injection of indomethacin. The cellular count of the peritoneal population was 2-fold elevated after indomethacin injection but the percentage of macrophages in the washed-out cell population was decreased from 60% (controls) to around 20%. The NK cell nature of the effector cells activated by indomethacin was substantiated by the finding that previous injection of anti-asialo GM1 antibody prevented activation. Interferon could not be detected in the peritoneal wash fluid after injection of indomethacin, suggesting interferon-independent activation. However, the possibility of small interferon quantities being locally produced could not be excluded. In further experiments we found after intraperitoneal injection of indomethacin not only cells that killed YAC-1 targets in a 4-hour assay but also killer cells that were insensitive to anti-asialo GM1 and killed P815 cells in an 18-hour assay. We assumed that these were macrophages and have done further experiments with in vitro grown bone-marrow-derived macrophages. These could be activated for killing of P815 targets by the addition of indomethacin, but (to a lesser degree) also for killing of YAC-1 lymphoma cells.     

A novel rat monoclonal antibody directed against a population of activated mouse peritoneal macrophages

A hybridoma clone secreting rat monoclonal antibody (MAB) designated as 3F3.5F and which reacted with a population of activated tumoricidal mouse peritoneal macrophage (M phi) was produced by the fusion of mouse myeloma cells with rat spleen cells immunized against adherent BCG-activated mouse peritoneal exudate cells (adherent BCG-PEC). The antibody was cytotoxic and of the rat IgM class. The specific reactivity of the antibody with mouse primary cells and cell lines was examined by complement-dependent cytotoxicity and indirect immunofluorescence flow cytometry analysis. The antibody was found to bind to about 40% of the adherent BCG-PEC activated in vivo and elicited peritoneal macrophages activated in vitro by lymphokine and lipopolysaccharide (LPS), to about 35% of polymorphonuclear neutrophils (PMN) 15 hr after intraperitoneal injection of BCG, to about 30% of bone marrow cells from BCG-infected mice, to about 10% of P815 mastocytoma cells and to thioglycollate-induced PEC to some degree. It did not bind to other cells tested including BCG-induced peritoneal lymphocytes, non-tumoricidal PEC, thymocytes, spleen cells, resting bone marrow cells from normal mice, lymphomas, myelomas, fibroblasts, or macrophage-cell lines. Pretreatment of adherent BCG-PEC with MAB 3F3.5F and rabbit complement caused a considerable decrease in tumor cytotoxicity toward P815 cells, but the same pretreatment of non-adherent BCG-PEC had no inhibitory effect on natural killer activity for YAC-1 cells.     

In vivo activity of lymphokine-activated macrophages in host defense against neoplasia

Purified splenic macrophage (M phi) from normal DBA/2J mice and mice bearing P815 tumors were examined for responsiveness to lymphokine (LK) preparations containing high concentrations of IFN-gamma. For both normal and tumor-bearing M phi, LK treatment induced morphologic changes and increased the percentage of Ia+ cells from 35 to 55%. Although neither population exhibited spontaneous cytotoxicity toward P815 targets, LK treatment induced considerable tumoricidal activity in tumor-bearing M phi (32 to 80% lysis) but only minimal activity in normal M phi (8 to 17% lysis). Subcutaneous injection of 1 X 10(6)P815 cells into DBA/2J led to progressive tumor growth and death of 100% of the recipients after 27 +/- 3 days. Injection of a 1:18 mixture of P815 with either LK-activated normal or tumor-bearing M phi caused tumor regression after 10 days, and prolonged life until 43 +/- 4 days with tumor-bearing M phi and 39 +/- 3 days with normal M phi. Untreated normal or tumor-bearing M phi were unable to cause the effect (30 +/- 2 days), and lymphocytes could not be substituted for M phi (25 +/- 3 days). In x-irradiated recipients, no effect of LK-activated M phi could be observed (control = 19 +/- 2 days; LK-activated tumor-bearing M phi = 21 +/- 3 days). In addition, administration of an admixture of LK-treated M phi and x-rayed tumor before challenge with viable P815 enabled the recipient to inhibit tumor growth and caused tumor necrosis with inflammatory cell infiltration of the tumor. These observations suggest that, in part, LK-activated M phi may interact in vivo with host-derived cellular components and enhance the immune reactivity of the host against the tumor.     

Exposure to paclitaxel or vinblastine down-regulates CD11a and CD54 expression by P815 mastocytoma cells and renders the tumor cells resistant to killing by nonspecific cytotoxic T lymphocytes induced with anti-CD3 antibody

Paclitaxel and vinblastine are drugs with anti-microtubule activity that are commonly used in the treatment of numerous types of cancer. In this study, we investigated the effect of prior exposure to submaximal cytotoxic concentrations (EC(25) and EC(50)) of paclitaxel or vinblastine on the subsequent susceptibility of surviving P815 murine mastocytoma cells to cytolysis by major histocompatibility complex (MHC)-unrestricted mouse cytotoxic T lymphocytes that had been induced with anti-CD3 antibody. P815 cells that had survived culture for 24 h in the presence of paclitaxel (5 or 50 micro g/ml) or vinblastine (1.5 or 15 micro g/ml) were rendered resistant to anti-CD3-activated killer-T (AK-T) cell-mediated cytolysis in a standard (51)Cr-release assay. Resistance to killing was associated with a reduced ability of AK-T cells to form conjugates with drug-treated P815 target cells, suggesting a possible effect on adhesion molecules. Flow cytometric analysis of paclitaxel- or vinblastine-treated P815 cells revealed reduced cell-surface expression of the adhesion molecules LFA-1 (CD11a /CD18) and ICAM-1 (CD54). Similar results were obtained following paclitaxel or vinblastine treatment of Yac-1 lymphoma cells. RT-PCR analysis revealed reduced levels of mRNAs coding for CD11a and CD54 in paclitaxel- or vinblastine-pretreated P815 cells. Collectively, these data lead us to conclude that paclitaxel and vinblastine render P815 mastocytoma cells resistant to T cell-mediated cytotoxicity by interfering with CD11a and CD54 expression by the tumor cells. A similar effect by these drugs on tumor cells and/or leukocytes in cancer patients might compromise tumor-specific cell-mediated immune responses.     

In vivo and in vitro induction of natural killer cells by cloned human tumor necrosis factor

Summary The natural killer (NK) cell activity of mice in the peritoneal cavity is very low or undetectable and testing peritoneal NK cells is a useful model for studying the influence of activating substances upon local injection. Injection of tumor necrosis factor (TNF) at doses of 10–200 ng caused a marked activation of NK cell activity which was maximal after 24 h and declined rapidly on day 2. A similar effect was observed when interferons alpha and beta were injected, and there were additive results when interferon was injected together with TNF. The NK cell nature of the effector cells activated by TNF was substantiated by the finding that previous injection with anti-asialo GM 1 antibody prevented activation. Interferon could not be detected in the peritoneal wash fluid after injection of TNF suggesting interferon-independent activation. In further experiments after i.p. injection of TNF peritoneal exudate cells (PECs) only killed YAC-1 targets in a 4-h assay. There was no additional killing in an 18-h assay towards neither YAC-1 cells or P815 cells, suggesting that macrophages were not involved. Furthermore TNF was also active in vitro by activating NK cells in isolated human peripheral blood cells. However in the PECs stimulated in vitro no significant induction of cytotoxic capacities by TNF was measured. Our data suggest that the action of TNF is not restricted to the lysis of tumor cells but can also induce immunological properties in the host defense against virus infections and neoplasms.     

Cells of the J774 macrophage cell line are primed for antibody-dependent cell-mediated cytotoxicity following exposure to gamma-irradiation.

Activation of macrophages (M phi) for host defense against tumor cells follows a sequence of priming events followed by an initiating stimulus that results in production and release of cytotoxic molecules that mediate target cell killing. We have developed a model to study specific macrophage cytotoxicity in vitro utilizing a cultured murine M phi cell line, J774. Specific cytotoxicity of cultured human gastrointestinal tumor cells is achieved in the presence of murine IgG2a monoclonal antibody (mAb) 17-1-A. The ability of these cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) is greatly enhanced following gamma-irradiation. ADCC can be demonstrated at mAb 17-1-A concentrations greater than or equal to 1 microgram/ml and effector/target cell ratios greater than or equal to 2. Exposure to doses greater than or equal to 10 Gy of gamma-irradiation increases ADCC threefold. Varying the duration from J774 M phi exposure to gamma-irradiation until addition of antibody-coated target cells showed that the primed state for ADCC is stable for at least 8 days but approximately 24 hr is required for complete development of the primed state. mAb-dependent target cell death begins 8 hr after addition of mAb and labeled target cells to primed effector cells and is complete by 24 hr. Incubation of unirradiated J774 M phi effector cells with recombinant murine interferon-gamma (rmIFN-gamma) also results in enhanced ADCC, but the extent of target cell killing achieved is less than that following priming by gamma-irradiation. Concomitant priming of gamma-irradiated J774 M phi with rmIFN-gamma increases the extent of ADCC. Further study of irradiated J774 cells may elucidate the molecular pathways utilized by M phi for achieving and maintaining the primed state for ADCC. Irradiated J774 cells will also provide a homogenous, stably primed cell type in which to examine the mechanism(s) of cytotoxicity employed by tumoricidal M phi.     

Responses of mouse lymphocytes to extracellular ATP. II. Extracellular ATP causes cell type-dependent lysis and DNA fragmentation

Extracellular ATP (ATPo) caused dose-dependent lysis of YAC-1 and P-815 mouse tumor cells. This event, assessed by 51Cr release, was accompanied by sustained depolarization of the plasma membrane potential and Ca2+ influx. Plasma membrane depolarization and Ca2+ influx occurred within a few seconds of ATPo addition to both cell types, whereas 51Cr was released without apparent lag in YAC-1 cells and after 2 h in P-815 cells. Furthermore, a rise in [Ca2+]i was required for ATPo-dependent lysis of YAC-1 but not P-815 cells. In P-815 cells, ATPo caused an early and [Ca2+]i-independent DNA fragmentation that occurred at lower nucleotide concentrations than those required to trigger 51Cr release. Instead in YAC-1 cells very low concentrations of ATPo caused early lysis (ED50 for lysis about 200 microM) accompanied by only barely detectable DNA fragmentation. Previous studies disclosed that lymphokine-activated killer cells are fully resistant to the membrane-perturbing effects of ATPo. We show that lymphokine-activated killer cells also do not undergo DNA fragmentation even in the presence of high ATPo concentrations. This study complements previous observations on the lytic effects of ATPo and shows that this nucleotide can also cause DNA fragmentation, one of the earliest target cell alterations observed during CTL-mediated lysis.     

Influence of dietary restriction and aging on natural killer cell activity in mice

Natural killer cells (NK) are believed to defend against tumor growth. Because rodents subjected to dietary restriction without malnutrition live longer and develop spontaneous tumors less often or later in life than unrestricted controls, we measured NK activity in restricted and in unrestricted mice. An age-related decline in NK responses to YAC-1 tumor target cells was detected in both groups. NK responses for control mice were highest in 2- to 3-mo-old mice, sharply reduced in middle-age mice (14 to 15 mo), and slightly reduced further in old mice (30 to 33 mo). At all ages the response of restricted mice was less than that of controls. However, after injection with Poly I:C (which increases NK activity), old restricted mice showed NK cytolysis not different from young mice on either diet, and substantially higher responses than old unrestricted mice. In addition, restricted mice showed increased in vitro generation of cytotoxic T lymphocytes (CTL) to YAC-1 and P815 compared with age-matched controls. Restricted mice may better resist cancer via an NK system very responsive to induction signals coupled with a CTL system more effective than that of unrestricted controls.     

Phenotypic and functional heterogeneity of thymic and splenic lymphokine activated killer cells relative to ornithine sensitivity

We have previously reported the selective inhibition of cytotoxic T lymphocytes (CTL) by 10 mM ornithine (ORN) relative to natural killer (NK) cell-derived lymphokine activated killer cells (LAK). To determine if this were due to differences in the progenitor cells or the type of stimulus, we used cortisone-resistant thymocytes (CRT) as a source of mature T cells for induction of LAK and CTL, and compared the results with spleen. Thymic and splenic CTL precursors (CTLp) from C57B1/6 (B6) mice were CD8+, ASGM1-, ORN sensitive. Splenic LAK precursors (LAKp) were CD8-, ASGM1+, ORN resistant when assayed against both YAC-1 and P815 tumor targets. In contrast, CRT-derived LAKp were CD8-, ASGM1+, ORN resistant against YAC-1, whereas LAKp against P815 were CD8+, ASGM1+, ORN sensitive. ORN sensitivity was also observed among CTL and LAK in DBA/2 mice and was associated with CD8+ phenotype. Therefore, our initial observation of differential ORN sensitivity in CTL vs LAK was a function of the progenitor cells; furthermore, CD8+ cytolytic cells are ORN sensitive whether activated by antigen (CTL) or IL-2 (T-LAK).     

A novel negative regulator molecule, Cho-1, is involved in the cytotoxicity by human natural killer cells but not in cytotoxic T lymphocytes.

We previously reported the cytotoxic negative regulatory molecule, Cho-1, that was expressed on the cell surface of rat fetal fibroblast cells in the cytotoxicity by natural killer (NK) cells. This molecule was IFN-gamma-inducible, but appeared to be different from MHC class I. It was expressed on NK-resistant cells but not on NK-sensitive murine target cells such as YAC-1. In this paper, first we determined whether Cho-1 could also act as the negative regulatory molecule in a human NK-resistant HEPM line. Our data strongly suggested that Cho-1 could act as such a negative regulatory molecule in human NK cytotoxicity. The immunoprecipitates made with HEPM cell lysate and anti-MHC class I monoclonal antibody (mAb) did not react against anti-Cho-1 mAb, indicating that Cho-I was different from MHC class I. Second, an assessment was made as to whether or not this molecule is involved in the cytotoxicity of CD8 (+) cytotoxic T lymphocytes (CTL) against human autologous tumor cells. The data indicated that although this cell surface molecule was expressed on certain tumor lines, it was not involved in the cytotoxic mechanism of CTL. Thus, Cho-1 appeared to be the novel regulatory molecule in the NK cytotoxic mechanism.     

Characterization by monoclonal antibodies of a target cell antigen complex recognized by nonspecific cytotoxic cells

Fish nonspecific cytotoxic cells (NCC)3 recognize and lyse a large variety of human and mouse transformed cells. In an effort to determine the Ag recognized by NCC on these targets, mAb were raised against NC-37 target cells. Four anti-NC-37 mAb were chosen for further characterization based on their effects on NCC lysis of target cells. Purified mAb 18C2 and 1E7 (IgM isotype) inhibited NCC killing of the following targets: U937, MOLT-4, K562, HL-60, DAUDI, NC-37, P815, and YAC-1. The dose-dependent inhibitory activity occurred at the target cell level and ranged from 50 to 70% at a concentration of 50 micrograms/well when compared to noninhibitory mAb 7C6 and 1D4 (IgG isotype). Similarly, mAb 18C2 protected the fish parasite Tetrahymena pyriformis from lysis by NCC when compared to mAb 7C6. Adsorption experiments demonstrated that the inhibitory effect on NC-37 lysis by NCC could be removed in a titratable fashion by incubation of mAb 1E7 with any one of the other target cell lines, but it could not be removed by incubation with effector cells. The inhibitory activity of mAb 1E7 and 18C2 was shown to be caused by the inhibition of conjugate formation between effector and NC-37 target cells. The relative membrane concentration of the antigenic determinants recognized by these mAb on the target cells was studied by flow cytometry using FITC-labeled mAb. These experiments showed that all four mAb bound to the surface of the cells tested. Biochemical analysis with Western blots and immunoprecipitation showed that mAb 18C2 and 1E7 recognize two Ag in NC-37 lysates: a larger protein of around 80 kDa and a smaller one of 42 kDa.     

Tumour-infiltrating lymphocytes mediate lysis of autologous squamous cell carcinomas of the head and neck

Tumour-infiltrating lymphocytes (TIL) and tumours from six patients with squamous cell carcinomas of the head and neck (SCCHN) were investigated. The six tumours all expressed major histocompatibility complex (MHC) class I antigens both in vivo and as tumor cell lines grown in vitro. In addition, the cancer cells either overexpressed the tumour-suppressor gene product p53 or harboured human papilloma virus 16/18 (HPV). The TIL were expanded in vitro in the presence of interleukin-2, immobilised anti-CD3 mAb and soluble anti-CD28 mAb. Expanded TIL cultures contained both CD4+and CD8+T cells, but generally contained few CD56+CD3-cells of the natural killer (NK) phenotype. CD8+T cells dominated the individual TIL cultures from five of the six patients and showed significant autologous tumour cell lysis. In TIL cultures derived from four of these tumour-reactive TIL cultures, killing could be partially blocked by an anti-MHC class I mAb. TIL cultures reacting with autologous tumour cells also showed strong TCR/CD3-redirected cytotoxicity when assayed against hybridoma cells expressing anti-TCR/CD3 mAb as well as natural-killer(NK)-like activity. A number of TIL cultures devoid of autologous tumour cell lysis were capable of lysing the natural-killer(NK)-sensitive K562 cell line suggesting that the SCCHN cells themselves are resistant to NK-like lysis. In conclusion, TIL cultures from head and neck carcinomas contain T cells which, upon expansion in vitro, can lyse autologous tumour cells in a MHC-class-I-restricted fashion. Thus, the results of the present study document that carcinomas of the head and neck in some patients are infiltrated by cytotoxic T cell precursors potentially capable of rejecting the autologous tumour.     

A Human Anti-M2 Antibody Mediates Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Cytokine Secretion by Resting and Cytokine-Preactivated Natural Killer (NK) Cells

The highly conserved matrix protein 2 (M2) is a good candidate for the development of a broadly protective influenza vaccine that induces long-lasting immunity. In animal models, natural killer (NK) cells have been proposed to play an important role in the protection provided by M2-based vaccines through a mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC). We investigated the ability of the human anti-M2 Ab1-10 monoclonal antibody (mAb) to activate human NK cells. They mediated ADCC against M2-expressing cells in the presence of Ab1-10 mAb. Furthermore, NK cell pro-inflammatory cytokine and chemokine secretion is also enhanced when Ab1-10 mAb is present. We also generated cytokine-preactivated NK cells and showed that they still displayed increased effector functions in the presence of Ab1-10 mAb. Thus, our study has demonstrated that human resting and cytokine-preactivated NK cells may have a very important role in the protection provided by anti-M2 Abs.     

Target cell susceptibility to immune lysis and expression of MHC antigens are independent of position in the cell cycle

We have investigated the effects of progression through the mitotic cell cycle upon the tumor (P815) cell line's expression of H-2 antigens and susceptibility to immune cytolysis. By using a combination of DNA quantitation by Hoechst 33342 staining and fluorescence-activated cell sorter analysis we were able to obtain pure populations of G1, S, and G2+M cells for study. Furthermore, by employing a fluorescence quenching technique utilizing bromodeoxyuridine substitution we were able to monitor the times required for the P815 cells to transit each of the various phases. By using mAb and cloned CTL populations with well-defined specificities we observed that neither H-2 antigen expression nor lytic susceptibility to mAb plus complement or CTL fluctuated with cell cycle progression. Each of the three major MHC loci (H-2Kd, -Dd, and -Ld) was analyzed. These results demonstrated that expression of H-2 antigens and susceptibility to immune lysis were independent of a cell's position in the cell cycle.     

Large-scale expansion of CD3<Superscript>+</Superscript>CD56<Superscript>+</Superscript> lymphocytes capable of lysing autologous tumor cells with cytokine-rich supernatants

We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMC) by the timed addition of cytokine-rich supernatants collected from allogeneic PBMC cultures stimulated with anti-CD3 monoclonal antibody (mAb) (allogeneic CD3 supernatants; ACD3S). These cytotoxic effectors belonged primarily to CD56(+) natural killer (NK) cells, and the cell subset with the greatest cytotoxic activity was an otherwise rare population of CD3(+)CD56(+) cells (NKT cells) that expand dramatically under these conditions. CD3(+)CD56(+) cytotoxic effectors were generated from the PBMC of 16 patients with several types of cancer. The CD3(+)CD56(+) cell subset expanded significantly and efficiently lysed NK- as well as lymphokine-activated killer (LAK)-sensitive targets. More importantly, ACD3S-activated CD3(+)CD56(+) cells were capable of efficiently lysing autologous tumor cells including metastatic colorectal, ovarian, breast, lung and pancreatic tumor cells as well as melanoma cells. ACD3S-expanded CD3(+)CD56(+) cells exhibited increased levels of cytoplasmic interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), gamma-interferon (IFN-gamma) and perforin. CD3(+)CD56(+) cell-mediated cytotoxicity was not restricted by major histocompatibility complex (MHC) gene products, since it was not blocked by anti-MHC class I mAb but was highly inhibited in the presence of CD2- and CD18-specific mAb. These data suggest that CD3(+)CD56(+) cells expanded under the presence of ACD3S may be utilized in clinical protocols for cancer immunotherapy.     

短双歧杆菌和嗜酸性乳杆菌对小鼠抗肿瘤活性的激活作用

用活的短双歧杆菌,嗜酸性乳杆菌和BCG给昆明小鼠腹腔注射,在原位激活后,由肝脏分离的非实质性细胞(NPC)对YAC-1,P815和L929的细胞毒性作用,以及NPC和Kupffer(KC)培养上清中所显示出对三株瘤细胞的肿瘤坏死因子(TNF)活性都比对照组明显增强。其次,胸腺细胞和脾细胞对ConA刺激的增殖反应也明显增强。实验结果表明双歧杆菌和乳杆菌在小鼠体内诱发抗肿瘤活性的启动能力与BCG几乎相等。     

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

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

A triggering structure recognized by G7 monoclonal antibody on porcine lymphocytes and granulocytes.

Monoclonal antibody (mAb) G7 has been developed and appears to recognize a triggering structure on porcine natural killer (NK) cells and granulocytes. G7 mAb binds to approximately 13% of lymphocytes, 70% of monocytes, and greater than 95% of granulocytes. G7 mAb does not react with B cells. G7 mAb immunoprecipitates a heterodispersed molecule of approximately 40 kDa. Functionally, whole but not F(ab')2 fragments of G7 mAb enhance NK killing of Fc receptor positive K562, U937, and MOLT-4 targets but not Fc receptor negative CEM, WEHI-164, or YAC-1 targets. Both whole and F(ab')2 fragments of G7 mAb inhibit lymphocyte-mediated antibody-dependent cellular cytotoxicity. Interestingly, G7 mAb induces dramatic levels of granulocyte killing against nucleated K562 targets. These results suggest that G7 mAb recognizes a trigger molecule involved in porcine cellular cytotoxicity.     

Modulation of macrophage activation and resistance by suppressor T lymphocytes in chronic murine Schistosoma mansoni infection

Activated macrophages (M phi) appear responsible for at least part of the concomitant resistance in mice infected with Schistosoma mansoni. We found that as murine S. mansoni progressed from acute (8 to 12 wk of infection) to chronic (16 to 24 wk) stages, acquired resistance decreased (57% resistance to challenge with cercariae at 8 wk vs 28% by 24 wk, p less than 0.05), as did macrophage activation (21% +/- 2 killing of schistosomula by 8 wk M phi vs 8% +/- 2 for 24 wk M phi, p less than 0.01). T cells from the spleens of 8 wk-infected mice were capable of activating M phi from naive animals when stimulated with worm antigens (24% +/- 2 killing vs 8% +/- 2 induced by normal T cells, p less than 0.01); T cells obtained from 24 wk-infected mice did not activate M phi (13% +/- 2 killing). Furthermore, T cells from 24 wk-infected animals suppressed activation of M phi by 8 wk T cells. The addition of 10(5) 24 wk T cells to 3 X 10(5) antigen-stimulated 8 wk T cells reduced subsequent M phi killing from 27% +/- 4 to 13% +/- 2 (p less than 0.05). Week 24 T cells (3 X 10(5] reduced this additionally to 9% +/- 1 (p less than 0.01), a value no greater than that of unstimulated M phi. The subpopulation of T cells responsible for suppression of M phi activation was Lyt-2+1- nonadherent T cells. Cyclophosphamide treatment of chronically infected mice resulted in enhanced resistance (41%), M phi activation (18% +/- 1 killing), and T cell activation of naive M phi (10% +/- 1 killing). Thus, during chronic S. mansoni infection, resistance to reinfection wanes in parallel to and perhaps because of development of suppressor T cells that interfere with T-dependent M phi activation.