Demonstration of a splenic cytotoxic effector cell in mice of genotype SCID/SCID.BG/BG

Splenocytes from mice of genotype scid/scid.bg/bg were tested in vitro to characterize the nature of the immunological deficit in these doubly mutant animals. The cells were unresponsive to the mitogens LPS and Con A and to alloantigens, as predicted for scid/scid genotype. Splenocytes from scid/scid.bg/bg lysed the NK cell-sensitive target cell line YAC at levels approximately 50% lower than those observed for scid/scid splenocytes. Splenocytes from SCID-beige mice failed to lyse the NK-resistant, LAK-sensitive cell line P815 but showed high levels of activity against the murine placental cell line Be6. Lytic activity was found in both nonadherent and plastic adherent cells and was eliminated by pretreatment of the effectors with anti-asialo-GM1 and complement. Incubation of 1 x 10(5) splenocytes with hrIL-2 failed to induce blastogenesis in scid/scid.bg/bg cells but produced a response in cultures of scid/scid or bg/bg spleen cells. However, blastogenesis and elevated levels of LAK-type killing were observed following incubation of higher numbers of scid/scid.bg/bg splenocytes in hrIL-2. Thus, doubly mutant scid/scid.bg/bg mice have reduced NK cell activity, in comparison to scid/scid mice, and appear to possess LAK-like effector cells and LAK cell precursors.     

Cardiovascular adaptations of pregnancy in T and B cell-deficient mice

The pathophysiology of gestational hypertensive disorders is incompletely defined. T lymphocytes are implicated. Both T and natural killer (NK) cells express RAS and, in implantation sites, NK cells are highly enriched. We hypothesized that T cells and/or NK cells contribute to circulatory control during pregnancy. Using radiotelemetry of arterial pressure, heart rate, and activity, mice without T and B cells (genotypes BALB/c-Rag2(-/-) and NOD.scid) were examined at baseline and across pregnancy. These strains differ in NK cell competency, with Rag2(-/-) being normal and NOD.scid impaired. Circulatory features differed between these inbred strains. Rag2(-/-); had blood pressure responses to pregnancy that did not differ from congenic normal mice. NOD.scid had higher midgestational blood pressure compared with normoglycemic NOD mice (3-5 mm Hg greater than NOD; P < 0.004). In comparison to controls, both T and B strains had much higher heart rates after first trimester that did not remit until parturition (>30 bpm greater than control; P < 0.0001). NOD.scid had additional anomalies, including 90% depletion of circulating NK cells and elevated (57%) proliferation of uterine NK cells within implantation sites. These data demonstrate immune control of midgestational heart rate and suggest NK cells contribute to midpregnancy regulation of mean arterial pressure.     

Defective repair of radiation-induced chromosomal damage in scid/scid mice.

The murine severe combined immunodeficiency (scid) mutation interferes with normal recombination of immunoglobulin and T-cell receptor genes. This immunologic defect results in a lack of fully differentiated B and T cells in scid/scid mice. Animals homozygous for the scid mutation also display increased sensitivity to the damaging effects of ionizing radiation. We report here our observations of high frequencies of radiation-induced chromatid interchanges and intrachanges in bone marrow cells and fibroblasts from scid/scid mice. The presence of these aberrant chromosome structures suggests that a delay in strand rejoining underlies the increased sensitivity of scid/scid mice to ionizing radiation. The scid mutation may provide important clues for understanding the relationship between mitotic recombination and DNA repair in higher eukaryotic cells.     

Characterization of murine decidual natural killer (NK) cells and their relevance to the success of pregnancy

The identification of lytic cells in 6.5-day to 9.5-day murine decidua as NK cells has been extended. The cells with natural killer (NK) activity in early decidua were nonphagocytic and heterogeneous in size as assessed by velocity sedimentation at unit gravity. The numbers of lytic cells were reduced by treatment with anti-asialo GM1 in vivo and they were absent from the decidua of bg/bg mice. Thus, decidual NK cells were not distinct from NK cells in other tissues. The decline in the levels of decidual NK activity as pregnancy progressed was attributed to their regulation by other cells present in decidua by midgestation. The development of NK activity in decidua was dependent upon the presence of an embryo, however, decidual NK cells were not essential for successful pregnancy because viable offspring were obtained from mice lacking decidual NK activity. It was shown that NK cells from either spleen or decidua were unlikely to cause damage to embryos during the first half of pregnancy as freshly dissociated 9.5- and 11.5-day embryonic cells resisted NK lysis. Furthermore, blastocysts were not damaged by coincubation with splenic or decidual NK cells and were viable upon subsequent embryo transfer. These studies indicate that decidual NK cells are not essential for successful pregnancy and are not necessarily detrimental to early embryos. It is suggested that decidual NK cells may play other nonimmunological roles during embryonic development.     

Immunology of pregnancy: renewed interest

The long-standing question of pregnancy immunological paradox has been generating renewed interest. Recent insights have emerged from studies in pregnant mice and humans demonstrating a number of mechanisms that prevent potentially harmful effects of maternal anti-paternal allo-antibodies (complement inhibition, partial deletion of maternal B cells specific of paternal antigens), cytotoxic CD8+ T cells (lack of HLA-A and HLA-B expression on trophoblast, local immunosuppressive molecules, transient tolerance of paternal allo-antigens specific T cells) and uterine NK cells directed against fetal-derived trophoblast cells (limited NK cytotoxic potential, trophoblast resistance to NK killing). Interestingly, it appears that not only decidual NK cell/trophoblast interactions are not harmful for the fetus but are beneficial for the placental vascularization and its subsequent development. A recent report has indeed demonstrated that during pregnancy most of the combinations of uterine KIR (killer cell immunoglobulin-like receptor) NK cell receptors and fetal HLA-C molecules expressed by trophoblast led to normal pregnancies, whereas mothers lacking activating KIR of the AA genotype when the fetus possessed HLA-C of the C2 group were at a greatly increased risk of severe preeclampsia pathology.     

Lack of B cell leakiness in BALB/cA-nu, scid double mutant mice.

BALB/cA mice homozygous for both nu and scid mutations (BALB/cA-nu/nu, scid/scid) were developed by mating between BALB/cA-scid and BALB/cA-nu. These mice have greater longevity than C.B-17-scid because no thymic lymphoma occurs in them unlike in the latter. C.B-17-scid is known to show the leaky phenomenon in which a few clones of functional T and B cells develop in aged C.B-17-scid. Unexpectedly, the leaky B cells and T cells were absent or suppressed in BALB/cA-nu, scid mice when cytokine expressions were determined by RT-PCR, lymphocyte phenotypes by flow cytometry and serum immunoglobulin levels by ELISA. These results indicate that B cell leakiness may be induced by leaked T cells. BALB/cA-nu, scid mice may be useful as a recipient in allo- and xeno-transplantation experiments because of the absence of both thymic lymphomas and leakiness, in addition to lack of hair.     

Effects of probiotic bacteria on humoral immunity to Candida albicans in immunodeficient bg/bg-nu/nu and bg/bg-nu/+ mice

Germfree beige-nude ( bg/bg-nu/nu) and beige-heterozygous ( bg/bg-nu/+) mice were colonized with a pure culture of Candida albicans or with a probiotic bacterium (Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus casei, or Bifidobacterium infantis). Probiotic-colonized mice were subsequently challenged orally with C. albicans. The effect of prior colonization with probiotic bacteria on the antibody responses of the immunodeficient mice to alimentary tract colonization with C. albicans was compared to the antibody responses of the gnotobiotic mice colonized only with C. albicans. This study demonstrated that, although the probiotic bacteria did not induce a vigorous antibody response to their own antigens, they altered the antibody responses of mice to C. albicans. In T cell competent bg/bg-nu/+mice, B. infantis enhanced and focused IgG1, IgG2A, and IgA responses to C. albicans antigens. Some of the probiotic bacteria also enhanced the IgG1 and IgG2A antibody responses of bg/bg-nu/nu mice to C. albicans antigens. This study not only shows the value of gnotobiotic animal models in demonstrating that probiotic bacteria can affect the capacity of mice to form antibodies to C. albicans, but it also points out their usefulness in comparing the capacity of different probiotic bacteria to produce beneficial health effects in mice.     

Full reconstitution of the immune deficiency in scid mice with normal stem cells requires low-dose irradiation of the recipients

Mice homozygous for an autosomal recessive mutation for the scid gene exhibit a defect that specifically impairs lymphoid differentiation but not myelopoiesis. Such mice can be cured of their lymphoid deficiency by grafts with normal bone marrow, although full reconstitution of lymphoid function is seldom obtained. Long-term bone marrow cultures (LTBMC) are devoid of all mature B and pre-B cells but contain lymphoid stem cells. We therefore reconstituted scid mice with LTBMC cells to study the kinetics of B lymphocyte reconstitution in normal and irradiated (4 Gy) scid recipients and in irradiated (9.5 Gy) co-isogenic C.B-17 mice. Detectable colony-forming B cells rapidly increased in the spleen and bone marrow of irradiated C.B-17 and irradiated scid recipients, reaching normal levels between 4 and 6 wk post-grafting. Unirradiated scid recipients showed limited reconstitution in spleen and very poor reconstitution in bone marrow. Unirradiated scid recipients also had relatively few surface Ig+ cells in spleen or bone marrow, whereas both groups of irradiated recipients had normal numbers between 4 and 6 wk post-reconstitution. Normal levels of cytotoxic T cell activity by 8 wk after reconstitution were observed only in the irradiated C.B-17 and irradiated scid recipients. Analysis of mice reconstituted with cells from LTBMC indicates that these cultures contain lymphoid stem cells with significant proliferative and self-renewal potential, and that full reconstitution of lymphoid function requires prior irradiation of the scid recipient.     

Early B-cell precursors in scid mice: normal numbers of cells transformable with Abelson murine leukemia virus (A-MuLV)

scid mice lack detectable B and T lymphocytes; there are no typical pre-B cells as defined by c mu and surface markers in their bone marrow and their thymus contains only 1% of the normal number of cells. In these characters scid mice seem to lack lymphoid stem cells. However, some mice have detectable serum immunoglobulin and others develop thymomas; both observations indicate that the block in lymphoid development is not absolute. To determine whether scid mice have any B-cell precursors, we looked for pre-B cells by their ability to be transformed by Abelson murine leukemia virus (A-MuLV). Surprisingly, scid mice contain as many B-cell precursors transformable with A-MuLV as normal control mice. Cell-surface markers specific for pre-B and B cells were detected on the A-MuLV-transformed bone marrow cells of both scid and normal mice, indicating that the A-MuLV-transformed cells belong to the B lineage. Interestingly, the same surface markers were undetectable on nontransformed scid bone marrow cells. We conclude from these results that scid mice have normal numbers of early B-cell precursors but that their differentiation into functional B cells is severely impaired.     

Adoptive transfer of BALb/c mouse splenocytes reduces lesion severity and induces intestinal pathophysiologic changes in the Mycobacterium avium Subspecies paratuberculosis beige/scid mouse model

Successful immune reconstitution would enhance resistance of beige/scid mice to chronic infection with Mycobacterium avium subspecies paratuberculosis, but may cause damage to intestinal tissue. Therefore, we investigated the effect of adoptive transfer of BALB/c mouse splenocytes on lesion severity and intestinal physiology in beige/scid mice infected with M. paratuberculosis. Mice were inoculated intraperitoneally (i.p.) with M. paratuberculosis, and two weeks later were inoculated i.p. with viable spleen cells from immune-competent BALB/c mice. Mice were necropsied 12 weeks after infection when engraftment of lymphocytes, clinical disease, pathologic lesions, and intestinal electrophysiologic parameters were evaluated. Lymphocytes were rare in control beige/scid mice not inoculated with spleen cells. In contrast, high numbers of CD4+, CD8+, and B220+ lymphocytes were detected in the spleen of all beige/scid mice (n = 24) inoculated with spleen cells, indicating that adoptive transfer resulted in successful engraftment of donor lymphocytes (immune reconstitution). Immune reconstitution of M. paratuberculosis-infected beige/ scid mice significantly reduced the severity of clinical disease and pathologic lesions, and numbers of bacteria in the liver. However, intestinal electrophysiologic parameters studied in vitro indicated that intestinal tissues from reconstituted beige/scid mice had reduced short-circuit current responses (due to reduced ion secretion) following electrical, glucose, and forskolin stimulation. These abnormal responses suggested that neural or epithelial cells in the intestine were damaged. We conclude that successful immune reconstitution of beige/scid mice enhance their resistance to M. paratuberculosis infection, but may cause pathophysiologic changes associated with intestinal inflammation.     

Human NK cell infusions prolong survival of metastatic human neuroblastoma-bearing NOD/scid mice

Aim Several lines of evidence suggest that NK cell immunotherapy may represent a successful approach in neuroblastoma (NB) patients refractory to conventional therapy. However, homing properties, safety and therapeutic efficacy of NK cell infusions need to be evaluated in a suitable preclinical murine NB model. Materials and methods Here, the therapeutic efficacy of NK cell infusions in the presence or absence of NK-activating cytokines have been evaluated in a NB metastatic model set up in NOD/scid mice, that display reduced functional activity of endogenous NK cells. Results In NOD/scid mice the injected NB cells rapidly reached all the typical sites of metastatization, including bone marrow. Infusion of polyclonal IL2-activated NK cells was followed by dissemination of these cells into various tissues including those colonized by metastatic NB cells. The early repeated injection of IL2-activated NK cells in NB-bearing NOD/scid mice significantly increased the mean survival time, which was associated with a reduced bone marrow infiltration. The therapeutic effect was further enhanced by low doses of human recombinant IL2 or IL15. Conclusion Our results indicate that NK-based adoptive immunotherapy can represent a valuable adjuvant in the treatment of properly selected NB patients presenting with metastatic disease, if performed in a minimal residual disease setting. Roberta Castriconi and Alessandra Dondero equally contributed to the work.     

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.     

Occurrence of mature B (IgM+, B220+) and T (CD3+) lymphocytes in scid mice

Scid mice with and without detectable serum Ig (scid Ig+ and scid Ig- mice, respectively) were examined for the presence of mature "leaky" lymphocytes by flow microfluorimetry with the use of antibodies to B (IgM, B220) and T (CD3, CD4, CD8) lymphocyte surface Ag. The data showed that leaky scid mice are more frequent than is evident from serum Ig analysis and that the incidence of detectable B and T cells increases with age. IgM+ B220+ cells were not detectable in young adult mice (3 mo old), but in old mice (greater than or equal to 1 yr old) they were routinely present in the peritoneal cavity though not in the spleen. Striking differences in the representation of T cell subsets were seen in the thymus of these two age groups. Most young adult mice contained CD3- populations of CD4/CD8 double positive cells, and in some cases, CD4 or CD8 single positive cells as well. By contrast, identifiable T lineage cells in old mice were all CD3+ and predominantly single positive for CD4 or CD8. Detectable peripheral T cell populations numbered less than 10(5) cells, and the representation of T subset markers (CD4, CD8) varied widely among individual mice; further, Southern blot analysis of TCR gene rearrangements in the DNA of polyclonally stimulated lymphoid cultures from these mice showed very restricted heterogeneity relative to that of cultures from normal mice. We conclude that most leaky mice contain very few T cell clones.     

Tumor necrosis factor is involved in the T cell-independent pathway of macrophage activation in scid mice

We analyzed the T cell-independent production of IFN-gamma in the severe combined immunodeficiency (scid) mutant mouse. Spleen cells from scid mice secreted high levels of IFN-gamma in response to heat-killed Listeria monocytogenes (HKLM), but not to the T cell stimulus ConA. This response was ablated by prior removal of adherent macrophages. IFN-gamma secretion in vitro was preceded by the rapid production of TNF and was inhibited by addition of neutralizing mAb to TNF. Moreover, injection of scid mice with anti-TNF mAb increased the severity of infection with live Listeria and inhibited macrophage activation for class II-MHC expression. Finally, IFN-gamma secretion and class II-MHC expression were also inhibited by an antibody to asialoGM1, a reagent known to impair host NK cell function. These results suggest that TNF is a critical cytokine in the T cell-independent pathway of macrophage activation in scid mice.     

Natural cellular resistance of beige mice against Cryptococcus neoformans

Previous reports have demonstrated that natural killer (NK) cells are capable of inhibiting the growth of Cryptococcus neoformans in vitro, and recent studies indicate that adoptively transferred NK cell-enriched spleen cell populations enhance clearance of cryptococci from the tissues of cyclophosphamide-pretreated recipients. The primary objective of these studies was to confirm that NK cells participate in early clearance of C. neoformans in vivo. Secondarily, the anti-cryptococcal activities of polymorphonuclear leukocytes and macrophages were examined. Seven-week-old C57BL/6 bg/+ mice, which have normal levels of NK cell activity, were compared with their bg/bg littermates, which have impaired NK cell function. One and 3 days after injecting both groups of mice i.v. with 2 X 10(4) cryptococci, we assessed the NK cell activities in spleens, lungs, and livers and clearance of the organism from corresponding tissues as determined by the mean log10 numbers of cryptococcal colony-forming units (CFU) per organ. Three days postinfection, the mean numbers of cryptococcal CFU in lungs and spleens of bg/+ mice were significantly lower than in the corresponding organs of bg/bg mice. NK cell activities in spleens and lungs of bg/+ mice were significantly higher than were the NK cell activities in similar cell populations from bg/bg mice. In contrast, the mean numbers of cryptococcal CFU in livers of the two groups of animals were nearly equivalent, a situation not unexpected, since liver NK cell activities were extremely low and similar in both groups of animals. Although these data indicated a correlation between early clearance of cryptococci from tissues and levels of NK cell activities in the corresponding tissues, it was also possible that differences in phagocytic cell function between the bg/+ and bg/bg animals could account for the observed differences in clearance of cryptococci from the tissues. Therefore, phagocytic cells from the two groups of animals were compared with respect to their abilities to phagocytize and inhibit the growth of cryptococci and to their abilities to respond to chemotactic stimuli in vivo. Peritoneal PMNL from bg/+ and bg/bg mice were similar in their abilities to phagocytize and inhibit the growth of cryptococci, as well as in their chemotactic responses to viable cryptococci or sodium caseinate. In addition, there were no differences in splenic macrophage functions between the two groups of mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Immunohistochemical and ultrastructural evidence for myelopoiesis in the scid/scid mouse thymus

The ultrastructure of scid mouse thymus (a small encapsulated epithelial mass within the precardial fat pad) is described. The epithelium did not form cortex or medulla and hence remained relatively undifferentiated. Small unmyelinated nerves innervated the capsule, the major blood vessels and were distributed between the epithelial cells. Fenestrated blood vessels were common. Thymocytes were not identified but numeous granulocytes, mast cells and some fibroblasts, macrophages and interdigitating cells were present. All stages of granulopoiesis were observed in scid thymus. A very small number of immunoreactive ER-MP58 cells indicated bone marrow derived myeloid precursor cells, and low numbers of ER-MP12⁺ and ER-MP20⁺ mononuclear cells indicated stages of myeloid cells committed to the granulocyte/macrophage lineage. Cells containing proliferating nuclear cell antigen (cells in G1, S and G2-M stage) were present throughout the thymic mass. BALB/c thymuses contained cortical foci of p53⁺ cells whereas in scid mice, p53 positive cells were scattered singly throughout the thymus. This study indicates that the presence of moderately extensive myelopoiesis within the scid mouse thymus has potential for the study of extramedullary hematopoiesis, and also is important to bear this function in mind when using the scid mouse as an immunological model for thymus reconstitution and for creating organoid cultures.     

Establishment of a set of combined immunodeficient DA/Slc-Foxn1(rnu) Lyst(bg) congenic rat strains.

The congenitally athymic nude rat is used for studying cancer and transplantation owing to its hairlessness and T-cell defective function caused by the Foxn1(rnu) gene. However, NK cell activity of the nude rat is markedly increased. It is known that NK cells play a major role in rejection of xenografts and in cytotoxicity against tumor cells. Thus, the athymic nude rat with impaired NK cell activity should be a useful model for extensive studies. The DA-Lyst(bg)/Lyst(bg) rat, a model for human Chediak-Higashi syndrome (CHS) is characterized by diluted-coat color and impairment of NK cell activity. We planned to establish a combined immunodeficient double mutant rat introgressed with the Foxn1(rnu) and Lyst(bg) genes and a set of congenic strains having an identical genetic backgrounds simultaneously. Based on the phenotypic and genetic characteristics of the parental rat strains, the new strains were produced using continuous backcross and diagnosis with molecular genetic techniques. Each disease gene was diagnosed with PCR-RFLP or the long-nested PCR method. Furthermore, we used a marker-assisted congenic strategy based on scanning the genetic backgrounds of the parental rats with 461 rat microsatellite markers. We think that the newly established DA/Slc-Foxn1(rnu)/Foxn1(rnu) Lyst(bg)/Lyst(bg) double mutant will be useful as a severe disease model for human CHS, and the set of DA/Slc-Foxn1(rnu) Lyst(bg) congenic strains which have impaired NK cell activity and/or defective T cell function should be useful for studying in cancer research, xenotransplantation, immune function and other wide-ranging studies.