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.     

Prevention of meningo/encephalomyelitis due to Sarcocystis neurona infection in mice is mediated by CD8 cells

Immunodeficient CD8 knockout mice were infected with Sarcocystis neurona merozoites, in order to determine the role of CD8 cells in protective immunity. Using a direct agglutination test, all infected mice seroconverted by selected time points. Infected mice developed splenomegaly and bilateral lymphadenopathy. Histological changes included marked follicular development in the spleen, endothelitis and moderate perivascular inflammation in the liver, and meningoencephalitis in the brain. Infected brains were positive for S. neurona by polymerase chain reaction. Corresponding to histopathological changes, there were decreased numbers of B-cells in the spleen. The mice did not have significant memory (CD44hi/CD4) or effector (CD45RBhi/CD4) populations present at the time of euthanasia. Flow cytometry confirmed the lack of CD8 cells. Taken together, these data support previous studies suggesting a critical role for CD8 cells in the prevention of menigoencephalitis in S. neurona-infected mice.     

Natural killer (NK) cells are present in mice with severe combined immunodeficiency (scid)

Spleen cells from C.B- 17 scid mice with severe combined immunodeficiency disease exhibit natural killer cell (NK) activity against YAC lymphoma targets in a standard 4-hr 51Cr release assay. The cytolytic activity is demonstrable only at high effector to target ratios but can be augmented at least sevenfold by the interferon inducer poly I:C. The pattern of target lysis is specific, because splenocytes from poly I:C-primed C.B-17 scid mice lyse NK-sensitive YAC cells and not the insensitive P815 mastocytoma. The presence of several NK-associated antigens on C.B-17 scid splenocytes was tested by pretreating cells with the appropriate antiserum plus complement before testing for NK activity. The results indicate that a proportion of NK effectors in C.B-17 scid mice bear surface NK 2.1 and Asialo GM1 but are negative for Thy-1.     

Lack of dendritic Thy-1+ epidermal cells in mice with severe combined immunodeficiency disease

C.B-17 scid (severe combined immunodeficiency disease) mice were used to evaluate the relationship of dendritic Thy-1+ epidermal cells (EC) to T lymphocytes (deficient in scid) and to NK cells (replete in scid). Epidermis from scid mice was deficient in dendritic Thy-1+ cells as determined by immunofluorescent staining of epidermal whole mounts. Similarly, epidermal cell suspensions from scid mice failed to proliferate in response to Con A, as compared with epidermal cell suspensions from C.B-17 control mice. Transplantation of normal bone marrow into scid mice reconstituted morphologically identifiable dendritic Thy-1+ EC in whole mounts, as well as Con A responsiveness of EC suspensions, thus indicating that the deficiency in dendritic Thy-1+ EC in scid mice is at the precursor level. These studies demonstrate that Thy-1+ EC are more closely related to T lymphocytes than to NK cells.     

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.     

Humoral immunity is not critical for protection against experimental infection with Sarcocystis neurona in B-cell-deficient mice

Immunodeficient B-cell-deficient mice (mmuMT) were infected with Sarcocystis neurona merozoites to determine the role of B cells and the humoral immune response in protective immunity. As expected, the mice did not seroconvert based on a direct agglutination test. Infected mice did not have significant changes in gross pathology at the time points examined. Histologic changes included mild perivascular and peribronchial infiltrate in the lungs; perivascular infiltrate and mild inflammatory sinusoidal foci in the liver; prominent high endothelial venules in the lymph nodes; and moderate cellular expansion of the periarteriolar sheaths (PALS) in the spleen. Changes resolved by day 60 postinfection. Mice developed significant CD4 and CD8 responses in lymphoid organs, including significant effector (CD45RB(high)) and memory (CD44(high)) CD4 and CD8 responses. Flow cytometry confirmed the lack of B cells. Overall, these data suggest that B cells are not critical to the protective immune response to SN infection.     

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.     

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.     

Protective immune response to experimental infection with Sarcocystis neurona in 57BL/6 mice

Immunocompetent C57BL/6 mice were infected with Sarcocystis neurona merozoites to assess the protective immune response to active infection. Using a direct agglutination test, all infected mice seroconverted to S. neurona merozoite antigens by day 14 postinfection (PI). Further, mice developed splenomegaly and bilateral symmetrical lymphadenopathy by day 14 PI, which appeared to be resolving by day 28 PI. Histologic analysis revealed a marked increase in germinal center formation in the spleen and lymph nodes by day 14 PI. Corresponding to gross and histopathological changes, the percentage of B-cells decreased significantly by day 14 PI but then increased significantly and persisted at day 28 PI in the blood, spleen, and multiple lymph nodes. There was a sharp nonspecific significant decrease in CD4 percentages by day 14 PI in the blood, spleen, and lymph nodes. Early-activation CD8 lymphocytes (CD62/CD8) were significantly down-regulated coinciding with a significant compensatory up-regulation of memory (CD44/CD8) lymphocytes in multiple organs. We propose that the protective cell-mediated immune response to S. neurona involves both CD4 and CD8 cells, with CD8 lymphocytes appearing to play a more critical role.     

Human choriocarcinoma cell resistance to natural killer lysis due to defective triggering of natural killer cells

The trophoblast, the outermost layer of the human placenta, lacks expression of the classical human leukocyte antigen (HLA) class I molecules. This prevents allorecognition by T cells but raises the question of what protects the trophoblast from natural killer (NK) cells. In a previous study, we have shown that choriocarcinoma cell (CC) resistance to NK lysis was mainly independent of HLA class I molecules. In the present study, we postulated that CC may prevent activation of NK cells by failing to stimulate their triggering receptors (TR). To test this hypothesis, we evaluated the lysis of JAR and JEG-3 CC after effective cross-linking and activation of NK cells by means of lectins or antibodies. Our results show that NK-resistant CC were sensitive to lysis by unstimulated peripheral blood lymphocytes in the presence of phytohemagglutin (PHA), to antibody-dependent cell cytotoxicity in presence of anti-Tja antibodies, and to monoclonal antibody redirected killing using anti-TR antibodies anti-CD16 and anti-CD244/2B4. Finally, CC fail to express CD48, the ligand for CD244/2B4. These results indicate that the resistance of CC to lysis results primarily from defective NK cell activation, at least partially due to the lack of expression of ligands, such as CD48, involved in the triggering of NK cells.     

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.     

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.     

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.     

Renewal of natural killer cells in mice having elevated natural killer cell activity

The present study was designed to examined the dynamics of splenic natural killer (NK) cells under two conditions of enhanced NK cell activity: (1) CBA/J mice given polyinosinic-polycytidylic acid (poly-I:C), an NK-cell-enhancing agent, and 62) untreated athymic nude (nu/nu) mice. The 'total NK cell activity' of the spleen (percentage specific lysis corrected for changes in organ cellularity) increased 5-fold and 2.7-fold after poly-I:C treatment for 1 day and 4 days, respectively. An injection of hydroxyurea (HU), a cell-cycle-toxic drug, given together with either poly-I:C or saline to CBA/J mice resulted in both cases in a 25% reduction in total NK cell activity 1 day later. This suggests that the renewal rate of nondividing NK cells is similar in poly-I:C-treated and saline-injected mice, and that the NK-enhancing effect of poly-I:C is not due to a stimulation of proliferation among NK cell precursors. HU administered simultaneously with poly-I:C or saline for 4 days eliminated NK cell activity in both cases, indicating that spleen NK cell activity is mediated almost entirely by newly formed (less than or equal to 4 days) cells. In nude mice, NK cell activity was assayed at various intervals after an HU depletion period of 2 days. NK depletion was initially more rapid in nu/nu mice than in control (nu/+) mice, although equally profound, and the subsequent recovery of NK cell activity after cessation of HU was also more rapid than in control (nu/+) mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutated HLA-G3 localizes to the cell surface but does not inhibit cytotoxicity of natural killer cells

HLA-G plays an important role in the induction of immune tolerance. Various attempts to produce good manufacturing practice levels of HLA-G as a therapeutic molecule have failed to date partly due to the complicated structure of full-length HLA-G1. Truncated HLA-G3 is simpler and easier to produce than HLA-G1 and contains the expected functional epitope in its only α1 monomorphic domain. In this study, we engineered the ER retrieval and retention signal on HLA-G3’s cytoplasmic tail by replacing its RKKSSD motif with RAASSD. We observed that mutated HLA-G3 was highly expressed on the cell surface of transduced K562 cells but did not inhibit cytotoxicity of natural killer cells.     

Interactions between natural killer cells and antibody Fc result in enhanced antibody neutralization of human immunodeficiency virus type 1

Antibodies can prevent lentivirus infections in animals and may play a role in controlling viral burden in established infection. In preventing and particularly in controlling infection, antibodies likely function in the presence of large quantities of virus. In this study, we explored the mechanisms by which antibodies neutralize large inocula of human immunodeficiency virus type 1 (HIV-1) on different target cells. Immunoglobulin G (IgG) from HIV-infected patients was tested for neutralizing activity against primary R5 strains of HIV-1 at inocula ranging from 100 to 20,000 50% tissue culture infective doses. At all virus inocula, inhibition by antibody was enhanced when target cells for virus growth were monocyte-depleted, peripheral blood mononuclear cells (PBMCs) rather than CD4(+) lymphocytes. However, enhanced inhibition on PBMCs was greatest with larger amounts of virus. Depleting PBMCs of natural killer (NK) cells, which express Fc receptors for IgG (FcgammaRs), abrogated the enhanced antibody inhibition, whereas adding NK cells to CD4(+) lymphocytes restored inhibition. There was no enhanced inhibition on PBMCs when F(ab')(2) was used. Further experiments demonstrated that the release of beta-chemokines, most likely through FcgammaR triggering of NK cells, contributed modestly to the antiviral activity of antibody on PBMCs and that antibody-coated virus adsorbed to uninfected cells provided a target for NK cell-mediated inhibition of HIV-1. These results indicate that Fc-FcgammaR interactions enhance the ability of antibody to neutralize HIV-1. Since FcgammaR-bearing cells are always present in vivo, FcgammaR-mediated antibody function may play a role in the ability of antibody to control lentivirus infection.     

Depletion of resident alveolar macrophages does not prevent Fas-mediated lung injury in mice

Activation of the Fas/Fas ligand (FasL) system in the lungs results in a form of injury characterized by alveolar epithelial apoptosis and neutrophilic inflammation. Studies in vitro show that Fas activation induces apoptosis in alveolar epithelial cells and cytokine production in alveolar macrophages. The main goal of this study was to determine the contribution of alveolar macrophages to Fas-induced lung inflammation in mice, by depleting alveolar macrophages using clodronate-containing liposomes. Liposomes containing clodronate or PBS were instilled by intratracheal instillation. After 24 h, the mice received intratracheal instillations of the Fas-activating monoclonal antibody Jo2 or an isotype control antibody and were studied 18 h later. The Jo2 MAb induced increases in bronchoalveolar lavage fluid (BALF) total neutrophils, lung caspase-3 activity, and BALF total protein and worsened histological lung injury in the macrophage-depleted mice. Studies in vitro showed that Fas activation induced the release of the cytokine KC in a mouse lung epithelial cell line, MLE-12. These results suggest that the lung inflammatory response to Fas activation is not primarily dependent on resident alveolar macrophages and may instead depend on cytokine release by alveolar epithelial cells.     

A severe form of human combined immunodeficiency due to mutations in DNA ligase IV

DNA ligase IV (LigIV) deficiency was identified as the molecular basis for a severe form of combined immunodeficiency in two microcephalic siblings with cellular radiosensitivity. In one patient the diagnosis was made directly after birth, allowing analysis of the role of LigIV in the development of specific immune cells. Absolute numbers of B cells were reduced 100-fold and alphabeta T cells 10-fold, whereas gammadelta T cells were normal. Spectratyping of all three cell populations showed a diverse repertoire, but sequencing of IgH V(D)J junctions revealed shorter CDR3 regions due to more extensive nucleotide deletions among D and J elements and fewer N nucleotide insertions. Clonal restriction of IgG-expressing, but not IgM-expressing, B cells and the lack of primary and secondary lymph node follicles indicated impaired class switch recombination. Observations in the older sibling showed that this rudimentary immune system was able to mount specific responses to infection. However, partial Ab responses and extensive amplification of gammadelta T cells could not prevent a life-threatening course of viral and bacterial infections, the development of an EBV-induced lymphoma, and immune dysregulation reflected by severe autoimmune cytopenia. Impaired generation of immune diversity under conditions of limited LigIV activity can cause a human SCID variant with a characteristic immunological phenotype.