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

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

Fibrin formation inhibits the in vitro cytotoxic activity of human natural and lymphokine-activated killer cells

The biological significance of the interaction of the haemostatic factors with tumour cells remains unclear. It has been hypothesized that fibrin deposition around tumour cells could help those cells to escape destruction by cytotoxic effector cells. To obtain direct evidence in support of this possibility, the effect of fibrin formation on in vitro cytotoxicity of human natural killer (NK) or lymphokine-activated killer (LAK) cells was investigated by comparing their cytotoxic activity with various human tumour cell lines in the presence of human serum or plasma. The data demonstrate that pre-incubation of human tumour cells with plasma, but not serum, substantially diminished or completely abrogated the cytotoxic effects of these killer cells. This was shown to be due to fibrin formation. The degree of coagulation and the number of radioactive tumour cells trapped in the clot correlated with the extent of inhibition of NK or LAK cytotoxicity. Abrogation of LAK activity was also observed when the effector cells were pre-exposed to plasma or when effector and target cells were simultaneously mixed with plasma and trapped in a fibrin clot. Similar results were obtained when, instead of whole plasma, the cytotoxic effect of LAK and NK cells was studied in the presence of fibrinogen and thrombin. When heparin was added, fibrin formation was prevented and no inhibition of LAK/NK cell cytotoxicity was observed. In studies of the mechanisms of inhibition of LAK cell activity by fibrin, target - effector cell conjugate formation was found to be blocked. When plasma was added post-binding (15-30 min after mixing effector and target cells) although coagulation occurred, no effect on cytotoxicity was observed, supporting the conclusion that fibrin interfered with binding rather than the lytic phase of cytotoxic cell activity. Thus, the present data demonstrate that fibrin deposition around tumour and/or effector cells can protect tumour cells from immune destruction and diminish the efficiency of the cytotoxic LAK/NK cells.     

Functional and phenotypic characterization of human peripheral blood stem cell harvests: a comparative analysis of cells from consecutive collections

A considerable number of patients with malignancies who are treated with high-dose therapy and hematopoietic stem cell transplantation subsequently relapse. Analyses of peripheral blood stem cell (PBSC) harvests obtained from 49 cancer patients showed that the PBSC harvest contained precursors for antitumor effector cells. Ex vivo manipulation of these harvests to maximize the antitumor effector cell activity may provide a new therapeutic approach to decrease or eliminate any minimal residual disease that remains after high-dose therapy. Characterization of PBSC from consecutive collections determined the collections best suited for ex vivo augmentation of antitumor cytotoxic effector cells. We report the results of a functional and phenotypical characterization of PBSC obtained from six consecutive collections from 18 cancer patients receiving granulocyte-macrophage colony-stimulating factor (GM- CSF) for hematopoietic stem/progenitor cell mobilization. The PBSC were evaluated for their cytotoxicity using the 51Cr-release assay. The frequency and subsets of lymphocytes were determined using flow cytometry with appropriate specific marker antibodies and differential cell counts. The content of hematopoietic progenitor cells in each collection was determined using a colony-forming unit granulocyte- macrophage (CFU-GM) culture assay. The frequency of cytotoxic effector cells including lymphokine-activated killer (LAK) cell precursors and lymphocytes was significantly greater (P < .05) in the early collections, whereas the later collections contained significantly (P < .05) more CFU-GM progenitor cells and fewer cytotoxic effector cells. Thus, our results show that PBSC obtained from advanced cancer patients do contain considerable levels of precursor cells for the generation of LAK cell populations. These results suggest that cells from the earlier collections are best suited for ex vivo manipulation to augment the antitumor effects.     

Primed marrow for autologous and allogeneic transplantation: a review comparing primed marrow to mobilized blood and steady-state marrow

Mobilized peripheral blood collections, obtained following either chemotherapy (with or without granulocyte colony-stimulating factor (G-CSF)) or G-CSF administration alone, are rapidly replacing traditional bone marrow harvests as the source of cells for hematopoietic stem cell transplantation. According to the Autologous Blood and Marrow Transplant and the International Bone Marrow Transplant Registries, for the years 1998 through 2000, blood stem cell (BSC) transplants accounted for about 80% of autologous transplants in the pediatric age group and more than 90% of the autologous transplants among adults. In allogeneic transplantation, where the donor is a healthy family member or normal volunteer, G-CSF-mobilized BSC transplants are being used more and more frequently, accounting for about 20% of allogeneic transplants in the pediatric age range and more than 40% of allogeneic transplants among adults during the same time period. It is not, therefore, too great a stretch to imagine that BSC transplants will soon be, if not already, in the majority for allogeneic transplantation among adults. The principal reason why this is happening is the prevailing view that BSC engraft more rapidly than marrow stem cells (MSC). However, this view is based on comparisons between primed circulating blood cells (BSC) and unprimed resident marrow cells in the steady state (SS-MSC). If the reason why BSC engraft faster than SS-MSC were a consequence of G-CSF used for mobilization, then would priming of MSC by G-CSF (Prim-MSC) accelerate engraftment of marrow as well? We reviewed the literature of the last 10 years to see if there were enough data to answer this question.     

Cytotoxicity of interleukin 2-activated lymphocytes for leukemia and lymphoma cells

Studies were undertaken to determine whether leukemia and lymphoma cells would be lysed by autologous and allogeneic lymphokine-activated killer (LAK) cells. Peripheral blood mononuclear cells (PBMC) from patients and normal donors were cultured for five days, 2 weeks, and 4 weeks with medium containing 2,500 units of recombinant interleukin 2 (IL-2) per mL, and their cytotoxicity was assayed by a five-hour 51Cr-release test. Of primary tumors isolated from patients with acute nonlymphoblastic leukemia, acute lymphoblastic leukemia, and non-Hodgkin's lymphoma, tumors of 37 out of 40 patients tested were shown to be susceptible to normal donors' LAK, and tumors of 18 of 20 patients tested were shown to be susceptible to autologous LAK. LAK cultured for longer periods showed a tendency to have lower cytotoxicity. LAK had also low, but significant, levels of cytotoxicity for nonmalignant target cells. Because PBMC expanded in IL-2-containing medium consisted mainly of OKT3-positive pan T cells, OKT8-positive suppressor/cytotoxic cells, and Leu-11-positive natural killer (NK) cells, and treatment with OKT3 and Leu-11 monoclonal antibodies (mAb) reduced LAK activity for autologous and allogeneic tumor cells, both T and NK cells appeared to be effector cells for LAK activity. Mechanisms of target-cell recognition in the LAK system seem to be different from those in alloreactive cytotoxic T lymphocytes (CTL) based on the results that, while cytotoxicity of alloreactive CTL was inhibited by the treatment of effector cells with mAb, OKT3, and OKT8, and by the treatment of target cells with a mAb that reacts with HLA class I antigen, LAK activity was not inhibited by the above treatment. When chromosomes of IL-2-expanded PBMC in nine patients and two normal individuals were analyzed, PBMC from one patient showed chromosomes of clonal abnormalities, and PBMC from five donors showed those of nonclonal abnormalities.     

G-CSF downregulates natural killer cell-mediated cytotoxicity in donors for hematopoietic SCT

In G-CSF-mobilized hematopoietic SCT (HSCT), natural killer (NK) cells have a critical role in GVHD and GVL effects. However, regulation of NK cell response to G-CSF remains unclear. This study assayed G-CSF effects in both HSCT donors and NK-92MI cells. The donors who received G-CSF had significantly decreased NK cell cytotoxicity. Levels of phosphatidylinositol 3-kinase (PI3K) and phosphorylated (p)-Akt, but not mammalian target of rapamycin (mTOR), were downregulated in NK cells from G-CSF-injected donors. G-CSF also decreased cytotoxicity without affecting viability and NF-κB of NK-92MI cells. PI3K and p-ERK expression were also decreased in G-CSF-treated NK-92MI cells, and their inhibitors, wortmannin and PD98059, respectively, both enhanced the downregulation of cytotoxicity. These effects were accompanied by decreased expression of a cytotoxicity-related gene, triosephosphate isomerase (TPI). Wortmannin, but not PD98059, enhanced the downregulation of TPI in G-CSF-treated NK-92MI cells, indicating a correlation between PI3K and TPI. We conclude that G-CSF-impaired NK cell cytotoxicity may accompany PI3K/Akt signaling. The effect is transient and NK cells may recover after G-CSF clearance, suggesting that G-CSF-mobilized HSCT may benefit both acute GVHD prevention and late-phase GVL promotion in HSCT recipients.     

Granulocyte colony-stimulating factor-mobilized allogeneic stem cell transplantation maintains graft-versus-leukemia effects through a perforin-dependent pathway while preventing graft-versus-host disease.

Minimization of graft-versus-host disease (GVHD) with preservation of the graft-versus-leukemia (GVL) effect is a crucial step to improve the overall survival of allogeneic bone marrow transplantation (BMT) for patients with hematological malignancies. We and other investigators have shown that granulocyte colony-stimulating factor (G-CSF)-mobilized allogeneic peripheral stem cell transplantation (PBSCT) reduces the severity of acute GVHD in murine models. In this study, we investigated whether G-CSF-mobilized PBSC maintain their GVL effect in a murine allogeneic transplant model (B6 --> B6D2F1). B6 mice (H-2(b)) were injected subcutaneously with human G-CSF (100 micrograms/kg/d) for 6 days and their splenocytes were harvested on day 7 as a source of PBSC. G-CSF mobilization dramatically improved transplant survival compared with nonmobilized controls (95% v 0%, P <.001). Systemic levels of lipopolysaccharide and tumor necrosis factor-alpha were markedly reduced in recipients of allogeneic G-CSF-mobilized donors, but cytolytic T lymphocyte (CTL) activity against host tumor target cells p815 was retained in those recipients. When leukemia was induced in recipients by coinjection of p815 tumor cells (H-2(d)) at the time of transplantation, all surviving recipients of G-CSF-mobilized B6 donors were leukemia-free at day 70 after transplant, whereas all mice who received T-cell-depleted (TCD) splenocytes from G-CSF-mobilized B6 donors died of leukemia. When splenocytes from G-CSF-mobilized perforin-deficient (pfp-/-) mice were used for transplantation, 90% of recipients died of leukemia, demonstrating that perforin is a crucial pathway mediating GVL effects after G-CSF-mobilized PBSCT. These data illustrate that G-CSF-mobilized allogeneic PBSCT separate GVL from GVHD by preserving perforin-dependent donor CTL activity while reducing systemic inflammation.     

Lymphokine-activated killer cell and natural killer cell activities in patients with systemic sclerosis.

OBJECTIVE. To determine the ability of T lymphocytes and natural killer (NK) cells from patients with systemic sclerosis (SSc) to respond to cytokines and to generate immune effector cells. METHODS. The numbers and percentages of peripheral blood T and NK cells were examined by 2-color flow cytometry, and NK and lymphokine-activated killer (LAK) cell function were measured in 4-hour 51Cr-release assays, in 34 patients with SSc. The patients were categorized into 3 subgroups: 10 had diffuse cutaneous disease of less than or equal to 3 years disease duration, 11 had diffuse cutaneous SSc of greater than 3 years duration, and 13 had limited cutaneous disease. RESULTS. Baseline and activated NK and T cell numbers and NK activity were normal in SSc patients. However, mean LAK activity was significantly depressed in all SSc subgroups. CONCLUSION. Decreased LAK cell function, despite normal numbers of circulating T and NK cells, indicates that SSc patients have poor ability to produce effector cells in response to interleukin-2.     

Hematopoietic stem cell mobilization with G-CSF induces innate inflammation yet suppresses adaptive immune gene expression as revealed by microarray analysis

OBJECTIVE: Granulocyte colony-stimulating factor (G-CSF) is used to boost granulocyte counts in immunocompromised patients, but its effects on the immune system may be counterproductive. We tested the hypothesis that G-CSF-mobilized peripheral blood stem cell (PBSC) products are immunologically downregulated based on gene microarray analysis. METHODS: Ten peripheral blood samples from normal donors for allogeneic PBSC transplantation were obtained before and after administration of G-CSF and tested on Affymetrix Human U133 Plus 2.0 GeneChip microarrays and by flow cytometry. Significant changes in gene expression after G-CSF were reported by controlling the false discovery rate at 5%. The quantitative real-time polymerase chain reaction method was used to validate expression of representative genes. RESULTS: All immune cells measured, including neutrophils, monocytes, lymphocytes, and dendritic cells, were significantly increased after G-CSF. In terms of gene expression, inflammatory and neutrophil activation pathways were upregulated after G-CSF. However, adaptive immune-related gene expression, such as antigen presentation, co-stimulation, T-cell activation and cytolytic effector responses, were downregulated. CONCLUSION: Despite significant increases in lymphocytes and antigen-presenting cells, G-CSF-mobilized PBSC allografts exhibit a suppressive adaptive immune-related gene-expression profile. However, innate and inflammatory responses are elevated. Our data provides an explanation for the potentially immunosuppressive effects observed after G-CSF administration.     

Lymphokine-activated killer cell and natural killer cell activities in patients with systemic sclerosis

Objective. To determine the ability of T lymphocytes and natural killer (NK) cells from patients with systemic sclerosis (SSc) to respond to cytokines and to generate immune effector cells. Methods. The numbers and percentages of peripheral blood T and NK cells were examined by 2-color flow cytometry, and NK and lymphokine-activated killer (LAK) cell function were measured in 4-hour ⁵¹Cr-release assays, in 34 patients with SSc. The patients were categorized into 3 subgroups: 10 had diffuse cutaneous disease of ≤3 years disease duration, 11 had diffuse cutaneous SSc of >3 years duration, and 13 had limited cutaneous disease. Results. Baseline and activated NK and T cell numbers and NK activity were normal in SSc patients. However, mean LAK activity was significantly depressed in all SSc subgroups. Conclusion. Decreased LAK cell function, despite normal numbers of circulating T and NK cells, indicates that SSc patients have poor ability to produce effector cells in response to interleukin-2.     

Effects of recombinant interleukin 2 on immunological effector cells of the peripheral blood in patients with HBe antigen-positive chronic hepatitis

Changes in peripheral blood immunological effector cells after systemic administration of recombinant interleukin 2 (rIL2) were studied in 14 patients with HBe antigen-positive chronic hepatitis. The patients were intravenously injected with rIL2 for 4 weeks, and the experiments were performed using mononuclear cells isolated from the peripheral blood during and after rIL2 administration. No significant changes in OKT4-positive cell population, OKT8-positive cell population, OKT4/OKT8 ratio and Leu7-positive cell population were detected during and after intravenous injection of rIL2. However, the natural killer (NK) cell and lymphokine-activated killer (LAK) cell activities significantly increased in the fourth week of rIL2 administration, while the K cell activity in the antibody-dependent cell-mediated cytotoxicity reaction, antibody response, lectin-induced lymphocyte transformation and IL1 production of LPS-stimulated monocytes were not affected. These results suggest that IL2 increases the NK cell and LAK cell activities in vivo.     

Lysis of human alveolar macrophages by lymphokine-activated killer cells

In order to determine if human LAK cells were cytotoxic against autologous AM phi, we studied the ability of human peripheral blood MNCs, stimulated in vitro with recombinant human IL-2, to lyse AM phi in a four-hour 51Cr-release assay. These cells showed significant cytotoxicity against autologous AM phi. The AM phi which had been cultured for four days served as better targets than freshly isolated AM phi. Kinetic study showed that the lysis of AM phi was proportional to the incubation time of MNCs with IL-2 and that LAK cells against AM phi required two days of in vitro culture with IL-2 for their induction. Freshly isolated MNCs did not lyse AM phi but did lyse K562 target cells, indicating that AM phi are natural killer-resistant. The phenotypes of effector cells against AM phi were found to be CD8+ or CD16+ (or both). These studies indicate that IL-2 can generate LAK cells against autologous AM phi, and this cytolytic activity must be taken into account when IL-2 or LAK cells are used for immunomodulation in patients with cancer.     

Functional character and augmentation of lymphocytes in regional lymph nodes of patients with lung cancer

It appears that lymph node metastases are more frequent in lung cancer than in other cancers because of impaired defensive mechanisms in the regional lymph nodes. However, little is known about the immunologic function of regional lymph node lymphocytes (RLNL) in patients with lung cancer. We have studied the immunologic properties of RLNL in comparison with peripheral blood lymphocytes (PBL). We measured the natural killer (NK) cell activity of RLNL and PBL in patients with lung cancer and found that the NK activity was significantly more depressed in the RLNL than in the PBL. In contrast, interleukin-2 (IL-2) production was markedly higher in the RLNL than in the PBL. The cytotoxic effect of RLNL in nonmetastatic lymph nodes on target cells (such as K562 cells) or PC-3 and PC-10 cells (NK-resistant, human lung cancer of adenocarcinoma and epidermoid carcinoma, respectively) was significantly enhanced by in vitro incubation with recombinant IL-2 (rIL-2). Furthermore, we clarified that both rIL-2 and OK-432, which is a biologic response modifier and IL-2 inducer as well, augmented the cytotoxicity of RLNL and that these effector cells were lymphokine-activated killer (LAK) cells. The depletion of lymphocyte subsets by pretreatment with specific monoclonal antibody showed that the LAK activity in RLNL was mediated by CD3+ and CD8+ cells, whereas the lymphocyte subsets contributing the LAK activity in PBL were CD3+ and CD16+ cells. It was concluded that a majority of the effector cells in RLNL were LAK cells of the cytotoxic T cell population.     

Daratumumab-mediated lysis of primary multiple myeloma cells is enhanced in combination with the human anti-KIR antibody IPH2102 and lenalidomide

Despite recent treatment improvements, multiple myeloma remains an incurable disease. Since antibody-dependent cell-mediated cytotoxicity is an important effector mechanism of daratumumab, we explored the possibility of improving daratumumab-mediated cell-mediated cytotoxicity by blocking natural killer cell inhibitory receptors with the human monoclonal anti-KIR antibody IPH2102, next to activation of natural killer cells with the immune modulatory drug lenalidomide. In 4-hour antibody-dependent cell-mediated cytotoxicity assays, IPH2102 did not induce lysis of multiple myeloma cell lines, but it did significantly augment daratumumab-induced myeloma cell lysis. Also in an ex vivo setting, IPH2102 synergistically improved daratumumab-dependent lysis of primary myeloma cells in bone marrow mononuclear cells (n=21), especially in patients carrying the FcγRIIIa-158F allele or the FcγRIIa-131R allele, who bind IgG1 with lower affinity than patients carrying the FcγRIIIa-158V allele or the FcγRIIa-131H allele. Finally, a further synergistically improved myeloma cell lysis with the daratumumab-IPH2102 combination was observed by adding lenalidomide, which suggests that more effective treatment strategies can be designed for multiple myeloma by combining daratumumab with agents that independently modulate natural killer cell function.     

Enhancement of MHC-unrestricted cytotoxic activity of human CD56+ CD3− natural killer (NK) cells and CD3+ T cells by rhamnogalacturonan: Target cell specificity and activity against NK-insensitive targets

Rhamnogalacturonan-mediated enhancement of MHC-unrestricted cytotoxicity was studied with freshly isolated CD56⁺CD3^– natural killer (NK) cells, interleukin-2 (IL-2)-activated CD56⁺ lymphokine-activated killer (LAK) cells und IL-2/anti-CD3-activated T cells as effector cells using NK-sensitive and NK-insensitive tumor cells as targets. The rhamnogalacturonan fractions IM, IP, and IQ were prepared from commercially available extracts ofViscum album. The dose/response relation of IM, IP, and IQ demonstrated the presence of various concentrations of cytotoxicity-enhancing compounds in all three fractions that were identified as rhamnogalacturonans by degradation studies with poly--d-galacturonidase (EC 3.2.1.15) and -1,6-rhamnosidase (EC 3.2.1.40). Specific cytotoxicity of all three effector cell populations as well as the respective rhamnogalacturonan-mediated cytotoxicity enhancement was readily inhibited in a dose-dependent manner by 60%-deacetylated mannose pentaacetate. Rhamnogalacturonan-mediated enhancement of cytotoxicity of fresh CD56⁺ NK cells was also observed with four of five NK-insensitive tumor cells as targets, indicating that the effector-cell/tumor-cell bridging activity of rhamnogalacturonans renders NK-insensitive targets susceptible to NK-mediated lysis. Moreover, the rhamnogalacturonan-mediated cytotoxicity enhancement became even more prominent when lymphokine-activated CD56⁺ LAK and CD3⁺ T cells were assayed with the NK-insensitive tumor cell targets.Abbreviations E/T effector cell/tumor cell ratio - IL interleukin - LAK lymphokinje-activated killer - LDH lactic acid dehydrogenase - MHC major histocompatibility complex - NK natural killer - PBMC peripheral blood mononuclear cells - PNAC peripheral non-adherent cells     

Resistance of some leukemic blasts to lysis by lymphokine activated killer (LAK) cells

Peripheral blood mononuclear cells (PBMC) from healthy donors and AML patients in remission were stimulated with phytohemagglutinin (PHA) and recombinant interleukin-2 (IL-2). These stimulated cells (lymphokine activated killer (LAK) cells) showed increased DNA synthesis as measured by 3H-Thymidine uptake. A synergistic effect of PHA and IL-2 was found. LAK cells' ability to kill acute myeloid leukemia (AML) blasts was investigated by the 51Cr release assay. LAK cells showed a cytotoxicity (over 10% specific 51Cr release) against 9/12 leukemic blasts, even at effector/target (E/T) ratios as low as 5:1. However, on average only 22.2% (SD 11.8) and 36.5% (SD 12.5) 51Cr release were obtained in 4- and 18-hour cytotoxicity assays, respectively, at an E/T ratio of 20:1. Leukemic blasts in 3/12 AML cases and normal PBMC were entirely resistant to lysis, even at an E/T ratio of 80:1. Susceptibility to lysis was not correlated to peanut-agglutinin receptor expression. LAK cells were more cytotoxic towards the K-562 cell line (natural killer activity) than unstimulated PBMC.     

Antibody-dependent cell-mediated immune reactions--fundamentals and clinico-diagnostic relevance (author's transl)

Antibody-dependent cell-mediated cytotoxicity is realized by means of humoral antibodies and unsensitized effector cells which interact with appropriate target structures inducing target cell destruction. Specificity of this immunological effector mechanism is mediated by immunoglobulins. Autologous, allogenic or xenogenic effector cells serve as efficient inductors of cell lysis. The in vivo relevance is still unclear. The present state of our knowledge has been surveyed and several samples are given showing the potential clinical importance of this immune mechanism in control of intracellular virus infections, bacterial and parasitological diseases, tumor and graft rejection, and autoimmune diseases. It has been attempted to interpret several apparently different immune phenomena (antibody-dependent cell-mediated cytotoxicity, "spontaneous" or "natural" cell-mediated cytotoxicity, mitogen-induced cell-mediated cytotoxicity and cytotoxic reactions of activated macrophages) under a common aspect.     

Blocking of influenza virus-induced cell-mediated cytotoxicity by hemagglutinin-specific monoclonal antibody

The relative importance of three major proteins of influenza virus in the mechanism of induction of cell-mediated cytotoxicity (natural killer cell activity) was assessed by an overnight chromium-51-release assay using radiolabeled K-562 cells as target cells and Ficoll-Hypaque-purified peripheral-blood lymphocytes as effector cells. Incubation of peripheral-blood lymphocytes with influenza virus (whether type-A or type-B) showed that intact and formalin-inactivated influenza virus enhanced cell-mediated cytotoxicity equally. The stimulation by intact or inactivated virions was comparable to that induced by the two major internal mediators of positive natural killer cell regulation, namely interferon and interleukin-2. This virus-induced cell-mediated cytotoxicity, which was mediated by human natural-killer 1+ cells, could be blocked only with monoclonal antibodies to the hemagglutinin and not with antinucleoprotein or antimatrix protein monoclonal antibodies, results indicating that the hemagglutinin of influenza virus is a potent mediator of natural killer cell stimulation in vitro.     

Development of tumor cell resistance to syngeneic cell-mediated cytotoxicity during growth of ascitic mastocytoma P815Y.

The immune reactivity to tumor cells within a progressively growing tumor mass in the syngeneic host has been analyzed by studying the cell-mediated cytolytic response of DBA/2 mice to the ascitic mastocytoma P815Y. Peritoneal cells from P815Y tumor-bearing hosts were fractionated by velocity sedimentation at unit gravity. Cell-mediated cytotoxicity of fractionated and unfractionated cells was measured by 51Cr-release from tumor target cells. The cell separation procedure revealed significant levels of specific cell-mediated cytotoxicity to P815Y within peritoneal cell populations at 8-16 days after tumor cell inoculation. Tumor cells purified from the peritoneal cell populations of mice injected with 10(3) tumor cells 10 days previously were as susceptible to syngeneic and allogeneic cell-mediated cytotoxicity as P815Y grown in vitro. However, tumor cells obtained from mice 16 days after tumor inoculation were resistant to cytolysis by syngeneic, but not allogeneic, effector cells. In addition, day 16 tumor cells did not inhibit syngeneic cell-mediated cytotoxicity against P815Y grown in vitro. Immunoglobulin was not detected on day 16 tumor cells and no circulating antibody to P815Y was found in the ascitic fluid of day 16 tumor-bearing mice. These results indicate that tumor cells may escape immune attack by loss of expression of cell surface tumor-associated antigens in the absence of circulating antibody against tumor.     

Phenotypic analysis of nylon-wool-adherent suppressor cells that inhibit the effector process of tumor cell lysis by lymphokine-activated killer cells in patients with advanced gastric carcinoma

The causes of down-regulation of cytotoxic immune responses in cancer patients have not been fully evaluated. We previously demonstrated that T-cell-growth-factor-activated peripheral blood lymphocytes (PBL) with the surface phenotype CD8⁺ CD11b^–, from patients with widespread metastasis of gastric carcinoma, inhibited the effector process of lymphokine-activated-killer(LAK)-cell-mediated cytolysis. In this study, we examined suppressor cell activity in freshly prepared PBL from 18 patients with advanced gastric carcinoma, and 10 normal healthy individuals. The suppressor cell activity was assayed by recording whether or not PBL inhibited directly the effector process of LAK cell cytotoxicity. Most of the PBL suspensions from cancer patients showed that they contained a population of cells that can directly inhibit the effector phase of tumor cell lysis of the cytotoxic cells. To analyze further the PBL responsible for the suppression, the cells were passed over a nylon-wool column. Nylon-wool-adherent cells significantly augmented the suppression, while the cells passing through abrogated the suppressive effect. Most nylon-wool-adherent cells from 10 normal healthy controls did not inhibit the cytotoxic reaction. To determine further the suppressor-effector population in nylon-wool-adherent cells, negative-selection studies using CD8-, CD4- or CD11b-coated magnetic beads, and positive-selection studies using CD8- or CD4-coated magnetic beads were performed. Finally the results suggest that the suppressor-effector cells comprise at least two different surface phenotypes: CD8⁺ T and CD8^–CD11b⁺ cells. The possible role of CD4⁺ T cells and HLA-DR⁺ LeuM3⁺ macrophages as suppressor cells was ruled out in nylon-wood-adherent cells. CD8⁺ T and possibly CD8^–CD11b⁺ cells apparently suppressed the efferent limb of the antitumor immunity. The selective immune suppression mediated by these cells may partly be concerned with escape mechanisms of gastric carcinoma from the host immune surveillance system.Abbreviations TCGF T cell growth factor - PBL peripheral blood leucocytes - LAK lymphokine-activated killer - NK natural killer - CTL cytotoxic T lymphocytes