Effector and precursor phenotypes of lymphokine-activated killer cells in mice with severe combined immunodeficiency (scid) and athymic (nude) mice

The lineage of lymphokine-activated killer (LAK) cells is poorly understood. To examine the relationship between LAK and natural killer (NK) cells we utilized two congenitally immunodeficient mice, namely severe combined immunodeficient (scid) and athymic (nude) mice that lack T cells but have normal NK cells. LAK activity was evaluated by the ability to lyze NK-resistant P815 cells. When cultured with human recombinant interleukin 2, splenocytes of scid and nude mice could generate LAK activity at levels comparable to or more than those of normal C.B-17 mice. LAK effector cells in these immunodeficient mice as well as normal mice had the phenotype resembling that of NK cells with asialo-GM1 (aGM1) expression. In vivo treatment with anti-aGM1 antiserum completely abolished the induction of LAK activity from splenocytes of normal mice. In contrast, LAK activity in splenocytes of scid and nude mice was still demonstrable even after this treatment, indicating that most LAK precursors in both mice were cells without aGM1 antigen. The aGM1- progenitors for LAK activity, probably in common with NK progenitors, appeared to be more expanded in scid and nude mice than in normal mice. The use of such congenitally immunodeficient mice should be helpful in studying the differentiation step of LAK as well as NK cells from their precursors.     

Natural cytotoxic T cells (NCTC) that differ from natural killer (NK) and natural cytotoxic (NC) cells are present in Peyer's patches of mice

We have examined noninduced cytotoxicity of mouse gut associated and peripheral lymphoid tissues for a wide variety of syngeneic as well as allogeneic cell lines and lymphoblasts. Lymphoid cells from Peyer's patches were found to lyse these targets in a 3-hr chromium release assay whereas lymphoid cells from intestinal mucosa, mesenteric or peripheral lymph nodes, spleen, and thymus did not. The variety of targets toward which Peyer's patch cells were cytotoxic established the latter as nonspecific and H-2 unrestricted. The cell responsible for the lytic event was identified as possessing Thy 1.2 and Ia surface antigens. This naturally cytotoxic T cell (NCTC) was found to be adherent to nylon-wool but not to plastic plates. Although both natural killer cell (NK) and non-NK targets served as targets for the NCTC, the latter were further differentiable from NK cells by lack of asialo GM1 surface marker, which is present on NK cells. In addition, NCTC remained fully functional in mice given either of the drugs cyclophosphamide or cortisone. Each of these drugs, in the doses used, markedly reduced poly(I:C)-induced NK activity. Thus, NCTC differs from NK on the basis of the spectrum of targets against which it is functional, phenotypic surface markers, insusceptibility to stimulation with poly(I:C), and insensitivity to diminution by the immunosuppressive drugs cyclophosphamide and hydrocortisone. Since NCTC is a Thy 1.2 antigen-bearing cell and is detectable in a 3-hr cytotoxic assay, it also differs from the natural cytotoxic (NC) cell. NC lacks the Thy 1.2 marker and becomes detectable only in an 18-hr cytotoxic assay. Thus, NCTC is neither an NK nor an NC cell. We have discussed the possibility that the three naturally occurring cells may be related by being dedifferentiated descendants of an antigen-specific cytotoxic T lymphocyte (CTL). Alternatively, since NCTC is confined to an anatomical site prone to ample antigenic exposure and is still identifiable as a T cell, it may be in linear transition from the CTL to the NK or NC stages.     

Natural killer (NK) cell immunodeficiency in patients with chronic myelogenous leukemia

Summary Defective natural killer (NK) cell populations from patients with chronic myelogenous leukemia (CML), that reacted with both HNK-1⁺ and B73.1⁺ antibodies, were obtained by a flluorescence-activated cell sorter (FACS). These fractions, along with NK fractions from normal donors which reacted with both antibodies, were expanded as bulk cultures or clones by limiting dilution, for 4 weeks in the presence of 10% interleukin 2 (IL 2), human type AB plasma, and irradiated human allogeneic mononuclear cells. Successfully established clones from patients with CML, with lytic activity against autologous and more differentiated neoplastic granulocytes, were generated more efficiently from B73.1⁺ than from HNK-1⁺ subsets. However, there were no significant differences among the generations of B73.1⁺ and HNK-1⁺ clones for both patients and normal donors with lytic activity against NK susceptible K-562 targets. Fresh myeloblast preparations from a blast crisis were found to be more susceptible to lysis by IL 2-proliferative B73.1⁺ and HNK-1⁺ clones than were fresh myelocyte preparations from a chronic phase CML patient, which were lytically susceptible to only B73.1⁺ clones. B73.1⁺ and HNK-1⁺ subsets from CML patients demonstrated major histocompatibility complex nonrestricted killing, and showed the following predominant phenotypes: B73.1⁺T3⁺T8⁺ or B73.1⁺T3⁺T8^– from B73.1⁺ subsets; and HNK-1^–T3⁺T8⁺ (initially HNK-1⁺) from HNK-1⁺ subsets. In contrast, B73.1⁺ and HNK-1⁺ clones from normal donors showed the following predominant phenotypes: B73.1⁺T3^–T8^–; and HNK-1^–T3^–T8^– or HNK-1^–T3^–T8⁺ (initially all HNK-1⁺). Short-term in vitro IL 2 or interferon treatment of fresh NK defective subsets from CML patients resulted in minimal cytotoxic augmentation. In contrast, defective NK cells from CML patients, whether HNK-1⁺ or B73.1⁺ subsets, proliferated with complete regeneration of cytolytic activity after a 3–4 week exposure to IL 2, but differed in phenotypic profiles as compared to those of normal donors. These observations imply that not only fresh defective NK cells but also the cytotoxically restored clones from CML patients are derived from different NK subsets and may represent undifferentiated forms of NK cells that may be arrested at an early stage of development by yet unknown mechanism(s). In vitro substantiation of autologous leukemia cell killing by IL 2-proliferative NK cell clones is encouraging and may allow for new in vivo immunotherapeutic modalities in CML patients.     

Natural killer (NK)-resistant human lung cancer cells are lysed by recombinant interleukin-2-activated NK cells

Natural killer (NK) cells are active in host defence against tumors. In order to determine if NK cells have the capacity to lyse human lung cancer cells, we evaluated blood NK cell activity against human lung carcinoma lines representing each of the commonest histological types of lung cancer, NCI-H157 (large cell), LICM107 and NCI-H146 (small cell), NCI-H226 (squamous cell), and LICM26 (adeno), and compared the results to their activity against a standard NK-sensitive target, K562, using a 16-hr 51Cr-release assay. At effector to target (E:T) ratios up to 50:1, NK activity was very low against each of the lung cancer cell lines compared to the K562 cells (NCI-H157 10 +/- 2%, LICM107 12 +/- 2%, NCI-H146 14 +/- 5%, NCI-H226 8 +/- 5%, and LICM26 7 +/- 3%, compared to K562 60 +/- 3%, P less than 0.001, for each compared to K562 cells). Recombinant interleukin 2 (IL-2) produced a dose-dependent augmentation of NK activity against each of the lung cancer cell lines, with doses as low as 0.25 U/ml being effective. The highest level of boosting was seen against NCI-H157 cells where NK activity (E:T, 50:1, IL-2, 250 U/ml) increased from 9 +/- 2 to 56 +/- 7%, P less than 0.001). Only brief exposure to IL-2 was necessary for augmentation to occur, with as little as 5 min being required for activation, although increased exposure times produced increased levels of augmentation. NK cells appeared to be the IL-2-responsive lytic cell population in these experiments as Leu 11b-depleted lymphocytes expressed little IL-2-mediated augmentation of activity against these target cells, and most of this IL-2-mediated augmentation of activity was located in the large granular lymphocyte-enriched fraction of the lymphocyte population. We conclude that normal blood NK cell activity against human lung cancer cell lines is low but that this activity can be markedly augmented by brief exposure of NK cells to low doses of recombinant IL-2, suggesting a potential role for IL-2 in the immunotherapy of human lung cancer.     

Natural killer cells in the thymus. Studies in mice with severe combined immune deficiency

The relationship between NK cell and T cell progenitors was investigated by using mice with severe combined immune deficiency (scid). Scid mice are devoid of mature T and B cells because they cannot rearrange their Ig and TCR genes. However, they have normal splenic NK cells. Thymus of scid mice, although markedly hypocellular, contains cells that lyse YAC-1, an NK-sensitive tumor cell. By flow cytometry, two populations of cells were identified in the scid thymus. Eighty percent of the cells were Thy-1+, IL-2R(7D4)+, J11d+, CD3-, CD4-, CD8- whereas the remaining were IL-2R-, J11d-, CD3-, CD4-, and CD8-. By cell sorting, all NK activity was found in the latter population, which is phenotypically similar to splenic NK cells. To determine if the thymus contains a bipotential NK/T progenitor cell, J11d+, IL-2R+ cells were cultured and analyzed for the generation of NK cells in vitro. These cells were used because they resemble 15-day fetal and adult CD4- CD8- thymocytes that are capable of giving rise to mature T cells. Cultured J11d+ thymocytes acquired non-MHC-restricted cytotoxicity, but in contrast to mature NK cells, the resulting cells contained mRNA for the gamma, delta, and epsilon-chains of CD3. This suggests that J11d+ cells are early T cells that can acquire the ability to kill in a non-MHC-restricted manner, but which do not give rise to NK cells in vitro. The differentiative potential of scid thymocytes was also tested in vivo. Unlike bone marrow cells, scid thymocytes containing 80% J11d+ cells failed to give rise to NK cells when transferred into irradiated recipients. Together these results suggest that mature NK cells reside in the thymus of scid mice but are not derived from a common NK/T progenitor.     

Human Taenia in severe combined immunodeficiency (SCID) mice

A rodent model for the development of the larval stages of human taeniid tapeworms would help advance immunodiagnosis in human and domestic animals and vaccine development for cysticercosis cellulosae or bovis in domestic animals. Here, Akira Ito and Mamoru Ito review recent results demonstrating the potential of the severe combined immunodeficiency (SCID) mouse for supporting development of the larval stages of Taenia saginata asiatica, T. saginata and T. solium.     

The function of antigen-presenting cells in mice with severe combined immunodeficiency

We have examined the antigen-presenting function of spleen cells in the C.B-17 scid mouse, a mutation that severely impairs the development of T and B lymphocytes. We show that antigen-presenting cells (APC) of SCID mice function normally in antigen-specific proliferative responses of primed T cells and in the antigen-specific activation of IL 2-producing T cell hybridomas. In both quantitative and qualitative terms, APC of SCID mice are equivalent to those of normal mice. These results indicate that the development and differentiation of APC function in vivo is independent of signals from mature, functional T or B lymphocytes.     

Natural killer (NK) activity of cultured S100 beta-positive T-leukemia cells

In order to clarify the function of human S100 beta-positive T-cells, S100 beta-positive T-leukemia cells (S100 beta TLC) were examined in vitro. S100 beta TLC were obtained from the peripheral blood of a patient with S100 beta-positive T-cell leukemia and enriched by an E-rosetting method. Two dimensional flow cytometric analysis indicated that the vast majority of the E-positive fraction were S100 beta TLC expressing CD3 and CD8 antigens. Although S100 beta TLC expressed CD3 antigen, they were negative for the alpha/beta and gamma/delta T-cell antigen receptor (TCR) defined by monoclonal antibodies (mabs) WT-31 and delta TCS-1, respectively. It was speculated that S100 beta TLC initially expressed alpha/beta TCR but lost it during malignant transformation. When S100 beta TLC were cultured for 24 h, they acquired cytotoxic activity towards various NK-sensitive cell lines including K-562, Molt-3 and CEM-CCLF, but did not exhibit lysing activity towards NK-resistant cell lines including Raji, Daudi and MT-1. Despite the NK-activity of cultured S100 beta TLC, they lacked the morphological features of large granular lymphocytes (LGL). S100 beta TLC did not exhibit lymphokine-activated killer (LAK) activity. When S100 beta TLC were cocultivated with NK-sensitive cells or NK-resistant cells, they selectively bound to NK-sensitive cells, indicating that they lysed target cells by cell-to-cell contact. The finding that S100 beta TLC lacked TCR molecules and their NK activity was not inhibited by mabs reactive with the CD3-TCR complex indicated that the CD3-TCR complex was not involved in their target recognition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin 3 enhanced interleukin 2-dependent maturation of NK progenitor cells in bone marrow from mice with severe combined immunodeficiency

Human recombinant interleukin 2 (IL 2) and highly purified murine interleukin 3 (IL 3) were tested for their ability to generate NK activity in bone marrow cells from mice with severe combined immunodeficiency. IL 2 alone could dose dependently induce NK activity in marrow cells as determined by cytotoxicity against YAC-1 target cells. It was demonstrated that IL 3 had dual effects on the generation of NK activity in this culture system. The addition of IL 3 resulted in inhibition of NK cell activity seen at high concentrations of IL 2. In contrast, when IL 3 was added together with low concentrations of IL 2, the generation of NK cells as judged by cytotoxicity assay as well as the appearance of cells with NK phenotypes was markedly augmented. In some experiments, mice were treated with 5-fluorouracil (5-FU) to eliminate relatively differentiated NK precursors from bone marrow cells. It was noted that the residual immature marrow cells from 5-FU-treated mice showed little NK activity even after the culture with high concentrations of IL 2. Importantly, IL 3 could induce the generation of NK activity from 5-FU-treated marrow cells in the presence of IL 2. Kinetic studies indicated that NK activity was appreciably generated from 5-FU-treated marrow cells when preincubated with IL 3 at least for 12 hr and subsequently cultured with IL 2. The cells bearing IL 2 receptors appeared in 5-FU-treated marrow cells, even though cultured only with IL 3, which implied that IL 3 could support the development of very primitive NK cells from IL 2-unresponsive to IL 2-responsive states. These results suggested that IL 3 might play a crucial role for the IL 2-induced generation of NK cells in bone marrow through promoting the expression of IL 2R on NK progenitor cells.     

Retroviral producer cells cause sarcoma in severe combined immunodeficiency mice

Direct in situ introduction of retroviral producer cells might provide a form of treatment for localized tumors. A possible undesirable consequence of this treatment could be uncontrolled proliferation of the injected producer cells. To test this possibility, severe combined immunodeficiency (SCID) mice were reconstituted with human peripheral blood lymphocytes which were marked with a retroviral vector using a coculture method. Although specific measures were taken to remove the possible contaminating producer cells, a high percentage of mice developed fibrosarcoma 2–6 weeks after reconstitution. We hypothesized that tumors arose from a small number of contaminating producer cells in the inoculum. Tumor cells were consistently DNA tetraploid, a characteristic of the producer cell line. DNA extracted from tumor tissue was found to contain the gene (neomycin phosphotransferase) used to mark the producer cell line. Furthermore, SCID mice injected with 1 × 10⁴ producer cells developed tumors with analogous characteristics. This report indicates that the retroviral producer cell line is tumorigenic in immune-deficient animals.     

Model of angiogenesis in mice with severe combined immunodeficiency (SCID) and xenoengrafted with Epstein-Barr virus-transformed B cells

Xenoengraftment of human cells in mice with severe combined immunodeficiency (SCID) has been used as a model system to study the mechanisms of B-cell lymphomagenesis. In the study reported here, we determined that SCID mice can also be used as a model to study angiogenesis in B-cell lymphomas. The C.B-17 scid/scid mice were xenotransplanted with Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL), and we determined whether CD31, a marker found on endothelial cells, was detected in the human B-cell lymphomas that developed in these mice. Microvessel formation was identified by use of immunohistochemical staining for CD31. To assess possible mechanisms of angiogenic stimulus, we analyzed the expression of interleukin 8 (IL-8), a chemokine documented to promote angiogenesis, in non-small-cell lung cancer and bronchogenic carcinomas. We observed that a panel of LCL and LCL-lymphomas expressed IL-8 mRNA and protein. Neutralization of IL-8, however, did not inhibit lymphomagenesis, suggesting that IL-8 is not essential for angiogenesis in this model. To examine other parameters of angiogenesis, we identified expression of vascular endothelial growth factor in the lymphomas. These data suggest that angiogenesis accompanies EBV-associated B-cell lymphoma development, but IL-8 is not essential for this process. Thus, the SCID mouse model is amenable to testing of anti-angiogenic factors.     

Depletion of natural killer cells does not result in neurologic disease due to Sarcocystis neurona in mice with severe combined immunodeficiency

Sarcocystis neurona is an apicomplexan parasite that is the primary etiologic agent of equine protozoal myeloencephalitis in horses. Protective immune responses in horses have not been determined, but interferon-gamma (IFN-gamma) is considered critical for protection from neurologic disease in mice. The role of adaptive and innate immune responses in control of parasites was explored by infecting BALB/c, IFN-gamma knockout (GKO), and severe combined immune deficient (SCID) mice with S. neurona (10(4) sporocysts/mouse). Immune competent BALB/c mice eliminated parasites within 30 days, with no sign of neurologic disease, whereas GKO mice developed fulminant neurologic disease. In contrast, SCID mice remained healthy throughout the experimental period despite the persistence of parasite at low levels in some mice. Treatment with anti-IFN-gamma antibody resulted in neurologic disease in infected SCID mice. Although SCID mice lack adaptive immune responses, they have natural killer (NK) cells capable of producing significant quantities of IFN-gamma. Therefore, SCID mice were infected with sporocysts of S. neurona and treated with anti-asialo GM1. Depletion of NK cells, confirmed by flow cytometry, did not result in neurologic disease in SCID mice. These results indicate that IFN-gamma mediates protection from neurologic disease in SCID mice. Protective levels of IFN-gamma may originate from a low number of nondepleted NK cells or from a non-T cell, non-NK cell population.     

Adherent lymphokine-activated natural killer cells in normal and severe combined immunodeficiency mice: large granular lymphocytes with natural killer cell phenotype and high cytolytic activity

Plastic-adherent lymphokine-activated natural killer (LANK) cells were generated from nylon wool-nonadherent murine splenocytes cultured in recombinant interleukin-2 (IL-2). Under such conditions, adherent lymphokine-activated killer cells capable of killing natural killer (NK)-resistant targets were not generated. Adherent LANK cells proliferated rapidly and closely resembled NK cells in their morphology, cytotoxic reactivity, and surface marker expression. Mice with severe combined immunodeficiency (scid) were used to generate adherent LANK cells to define the role of T cells in LANK cell development. Scid lymphocytes responded to IL-2 by becoming adherent LANK cells with potent NK-like activity, suggesting that soluble lymphokines other than IL-2 that may have been produced by T cells were not required for the generation of LANK cell activity in mice.     

Natural killer (NK) cell-mediated cytolysis of Plasmodium falciparum-infected human red blood cells in vitro

The ability of human NK cells to inhibit the growth in vitro of the asexual blood stages of Plasmodium falciparum was tested. Purified NK cells from donors with no prior exposure to malaria significantly inhibited parasite growth after 48 hours of co-culture in the presence of human immune serum. This inhibition was completely abrogated by pre-treatment of the NK cells with an anti-CD95 (anti-Fas) monoclonal antibody and human Fas-Fc soluble protein. The level of growth inhibition was also substantially reduced by pre-treatment with an anti-CD56 antibody. These two antibodies caused reductions, to varying levels, of the amounts of NK cell-derived granzyme B (GrB) and pro-inflammatory cytokines, but only the anti-CD95 antibody affected the production of soluble Fas ligand (sFasL). Direct destruction of parasite-infected red cells by NK cells, in the absence of serum, was also observed in a standard 51Cr cytotoxicity test, during which N-carboxybenzoxy-L-lysine thiobenzil ester (BLT esterase) activity, which catalyzes serine protease granule release, was detected. The results obtained are indicative of a novel mechanism of NK cell-mediated cytotoxic activity against Plasmodium falciparum-infected red cells, which is mediated in part by both Fas and by GrB.     

Persistent rotavirus infection in mice with severe combined immunodeficiency.

Rotaviruses are important pathogens of human infants and the infants of many animal species. The disease produced by these viruses can be described as an acute, self-limiting diarrheal disease, with virus replication localized to the differentiated epithelial enterocytes of the small intestine. Immunologically normal infants shed virus for approximately 5 to 12 days after the onset of infection. Recently, it has been shown that rotavirus can produce a chronic infection in severely immunocompromised children, with virus shedding and intermittent diarrhea lasting from 6 weeks to 2 years (G. A. Losonsky, J. P. Johnson, J. A. Winkelstein, and R. H. Yolken, J. Clin. Invest. 76:2362-2367, 1985; F. T. Saulsbury, J. A. Winkelstein, and R. H. Yolken, J. Pediatr. 97:61-65, 1980). These findings point to an important role for the immune system in recovery from the disease. The study described here examined the outcome of murine rotavirus infection in mice with severe combined B- and T-cell immunodeficiency (SCID) and in immunologically normal seronegative BALB/c mice. Persistent rotavirus infection was established in all mice with SCID which had been inoculated orally as pups. Low levels of virus replication and constant fecal virus shedding characterized the chronic infection. This is the first report of a persistent rotavirus infection in an animal model.     

Inhibition of tumor growth in mice with severe combined immunodeficiency is mediated by heat shock protein 70 (Hsp70)-peptide-activated,CD94 positive natural killer cells

Previously, we reported that the major stress-inducible heat shock protein 70 (Hsp70) acts as a recognition structure for natural killer (NK) cells, if localized on the cell surface of tumor cells. Incubation of purified NK cells with low-dose interleukin (IL)-2 (100 IU/mL) plus recombinant Hsp70-protein or the immunogenic 14-mer Hsp70-peptide TKDNNLLGRFELSG450-463, termed TKD (2 microg/mL), enhances the cytolytic activity against Hsp70 membrane-positive (CX+) but not against Hsp70-negative (CX-) tumor cells. Here, we show that the cytolytic activity against Hsp70-positive tumor cells is inducible by incubation of unseparated peripheral blood mononuclear cells (PBMNC) with low-dose IL-2 plus TKD. Cell sorting experiments revealed that within the PBMNC population CD94(+)/CD3(-) NK cells, and not CD94(-)/CD3(+) T cells, mediate the cytotoxic activity against Hsp70-positive tumor cells. The antitumoral effect of PBMNC stimulated either with IL-2 plus TKD or with IL-2 alone was assessed in tumor-bearing severe combined immunodeficiency/beige mice. A single intravenous (iv) injection of 40 x 10(6) IL-2 plus TKD-stimulated PBMNC (containing 5.2 x 10(6) NK cells) on day 4 results in a 60% reduction in tumor size, from 3.89 g to 1.56 g. In contrast, the adoptive transfer of the identical amount PBMNC stimulated with low-dose IL-2 only (containing 4.4 x 10(8) NK cells) reduces the tumor size only less than 10% (3.64 g). A phenotypic characterization of the excised tumors revealed that predominantly Hsp70-positive tumor cells were eliminated by TKD-activated PBMNC. Kinetic studies demonstrate that the in vivo cytolytic capacity of TKD-stimulated PBMNC is dependent on the effector to target cell ratio. An iv injection of effector cells on day 1 or 2 after tumor cell inoculation results in significantly smaller tumors (0.77 g or 0.89 g) on day 21 as compared with mice that were immunoreconstituted on day 4 or 8 (1.39 g or 2.23 g). The tumor size of nonimmunoreconstituted control animals was 3.55 g.