NOD/LtSz-Rag1null mice: an immunodeficient and radioresistant model for engraftment of human hematolymphoid cells, HIV infection, and adoptive transfer of NOD mouse diabetogenic T cells.

Development of a small animal model for the in vivo study of human immunity and infectious disease remains an important goal, particularly for investigations of HIV vaccine development. NOD/Lt mice homozygous for the severe combined immunodeficiency (Prkdcscid) mutation readily support engraftment with high levels of human hematolymphoid cells. However, NOD/LtSz-scid mice are highly radiosensitive, have short life spans, and a small number develop functional lymphocytes with age. To overcome these limitations, we have backcrossed the null allele of the recombination-activating gene (Rag1) for 10 generations onto the NOD/LtSz strain background. Mice deficient in RAG1 activity are unable to initiate V(D)J recombination in Ig and TCR genes and lack functional T and B lymphocytes. NOD/LtSz-Rag1null mice have an increased mean life span compared with NOD/LtSz-scid mice due to a later onset of lymphoma development, are radioresistant, and lack serum Ig throughout life. NOD/LtSz-Rag1null mice were devoid of mature T or B cells. Cytotoxic assays demonstrated low NK cell activity. NOD/LtSz-Rag1null mice supported high levels of engraftment with human lymphoid cells and human hemopoietic stem cells. The engrafted human T cells were readily infected with HIV. Finally, NOD/LtSz-Rag1null recipients of adoptively transferred spleen cells from diabetic NOD/Lt+/+ mice rapidly developed diabetes. These data demonstrate the advantages of NOD/LtSz-Rag1null mice as a radiation and lymphoma-resistant model for long-term analyses of engrafted human hematolymphoid cells or diabetogenic NOD lymphoid cells.

Lymphadenopathy, elevated serum IgE levels, autoimmunity, and mast cell accumulation in flaky skin mutant mice.

The autosomal recessive mutation "flaky skin" (fsn) causes pleiotropic abnormalities in the immune and hematopoietic systems accompanied by pathologic changes in the skin. Homozygotes (fsn/fsn) showed increased size and histological alterations in the spleen and lymph nodes. Abnormalities in lymphoid architecture of the spleen in fsn/fsn mice were accompanied by marked increases in total numbers of B cells, macrophages, and immature erythroid cells. Splenic B cells displayed elevated MHC class II expression. Serum IgE levels were greater than 100 microg/ml by 10 weeks of age, representing > 7000-fold increase compared with normal littermates. This increased IgE level was associated with elevated IL-4 production by spleen cells and with increased amounts of serum IL-4. Serum IgM, IgG1, and IgG2b levels were also increased in fsn/fsn mice while IgG3 was decreased. Autoimmunity in fsn/fsn mice was evidenced by glomerulonephritis accompanied by immune complex deposition in the kidneys, increased serum blood urea nitrogen levels, and the presence of circulating anti-double-stranded DNA autoantibodies. Pathological changes in the skin of fsn/fsn mice were characterized by epidermal hyperplasia and mixed dermal inflammation. Increased numbers of mast cells were also observed in the dermis of the truncal skin as well as in the epithelial stomach. These marked immunological abnormalities suggest that the fsn locus encodes a major immunoregulatory molecule important in multiple immune and hematopoietic functions.

Enhanced human CD4+ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice.

Genetic crosses produced NOD/LtSz mice doubly homozygous for the severe combined immunodeficiency (scid) mutation and the beta2m (B2m) null allele. Both NOD/LtSz-scid/scid and NOD/LtSz-scid/scid B2m(null) mice lacked mature lymphocytes and serum Ig. However, homozygosity for the B2m(null) allele also resulted in the absence of MHC class I expression, loss of NK cell activity, accumulation of iron in the liver, and rapid clearance of human IgG1. NOD/LtSz-scid/scid B2m(null) mice supported markedly elevated levels of human T cell engraftment, compared with NOD/LtSz-scid/scid control animals, following injection with human PBMC. The increased engraftment was associated with a major increase in the number of human CD4+ T cells. Following injection with 20 million human PBMC, levels of human CD4+ T cells in the peripheral blood and spleen of NOD/ LtSz-scid/scid B2m(null) mice were 6- to 7-fold higher than those in NOD/LtSz-scid/scid mice and >50-fold higher than those in C.B-17-scid/scid mice. The resulting normalization of CD4+/CD8+ ratios in NOD/LtSz-scid/scid B2m(null) mice is in sharp contrast to that observed in NOD/LtSz-scid/scid mice or in C.B-17-scid/scid mice. Circulating human IgG was cleared 6-fold more rapidly in NOD/LtSz-scid/scid B2m(null) mice than in NOD/LtSz-scid/scid mice. This rapid IgG clearance suggested a failure of the engrafted human lymphoid cells to maintain high circulating levels of human IgG. The higher levels of human CD4+ T cells and the normalization of the CD4:CD8 ratio that are observed in human PBMC-engrafted NOD/LtSz-scid/scid B2m(null) mice suggest that this system may be an excellent model for studies of HIV pathogenesis.

Increased G-CSF responsiveness of bone marrow cells from hematopoietic cell phosphatase deficient viable motheaten mice.

The mouse mutation viable motheaten (me(v)) results in defects in the expression and catalytic activity of the cytoplasmic protein tyrosine phosphatase known as hematopoietic cell phosphatase (HCP). This reduction in HCP activity leads to the aberrant regulation of several myeloid and lymphoid cell lineages, including substantial increases in numbers of granulocytes. The differentiation, proliferation, and survival of cells in this lineage are normally supported by granulocyte-colony stimulating factor (G-CSF). In this study we have determined the consequences of the loss of HCP activity in me(v)/me(v) mice on the response of bone marrow cells to G-CSF. Bone marrow from these mice exhibited substantial increases in clonogenic and proliferative responses to G-CSF. These enhanced activities of G-CSF correlated with an increase in the level of immature granulocytic, G-CSF receptor positive cells in the bone marrow. These results suggested the possibility that HCP may regulate the G-CSF receptor by a direct interaction. However, under conditions where the previously described interaction between the erythropoietin receptor and HCP was readily observed, HCP did not detectably associate with the G-CSF receptor.

Role of natural killer cells on engraftment of human lymphoid cells and on metastasis of human T-lymphoblastoid leukemia cells in C57BL/6J-scid mice and in C57BL/6J-scid bg mice.

The severe combined immunodeficiency (scid) mutation was backcrossed onto the C57BL/6J strain background in order to study the role of natural killer (NK) cells in rejection of normal and malignant human lymphohematopoietic cells. C57BL/6J-scid/scid mice showed severe loss of mature T and B cells accompanied by increased percentages of NK1.1+ cells and myeloid cells. Although little or no serum immunoglobulin was detectable prior to 2 months of age, all mice tested had circulating immunoglobulin by 7.5 months of age. C57BL/6J-scid/scid mice had markedly elevated levels of both hemolytic complement activity and NK cell activity compared with C57BL/6J - (+/+) controls. Weekly injections with anti-NK1.1 antibody resulted in elimination of NK cell activity in C57BL/6J-scid/scid mice throughout 8 weeks of treatment. Although human CEM-C7 T lymphoblastoid tumor cells grew slowly in unmanipulated C57BL/6J-scid/scid mice, anti-NK1.1 treatment resulted in increased growth accompanied by metastasis of human lymphoma cells to the brain, liver, and kidney. In contrast to T lymphoblastoid tumor cells, nonmalignant human peripheral blood mononuclear cells engrafted at low levels in anti-NK1.1-treated as well as in unmanipulated C57BL/6-scid/scid mice. Backcrossing of the beige (bgJ) mutation onto the C57BL/6-scid/scid genetic stock caused decreased NK cell activity accompanied by granulocyte defects. C57BL/6-scid/scid bgJ)/bgJ) mice showed metastasis of human CEM-C7 cells to the brain and other organs but supported only low levels of engraftment with human peripheral blood mononuclear cells. These results demonstrate that NK cells, in the absence of an adaptive immune system, function in resistance to metastasis of human lymphomas and suggest that innate immune factors in addition to NK cell function mediate resistance to engraftment of normal human peripheral blood leukocytes.

HIV-1 infectivity of human T cells in a human/murine chimeric fetal thymic organ culture system.

Human cord blood (HuCB) can colonize a murine fetal thymus organ culture (FTOC) and generate phenotypically immature (CD4+ CD8+) and mature (CD4+ CD8-; CD4- CD8+) T cells. We have used this model system to demonstrate that the human T cells that develop in this culture system can be infected with HIV-1. A cytopathic and non-cytopathic patient isolate of HIV-1 were used to infect FTOC established using C.B-17 or NOD/LtSz.scid/scid strain fetal thymic lobes colonized with HuCB. At 13-15 days after infection, FTOC were placed in co-culture with human PHA-blasts. These co-cultures demonstrated the presence of replicating HIV-1. Few human CD45+ cells were detectable in the thymic lobes that were infected with HIV-1, while high numbers of human CD45+ T cells were present in the uninfected cultures. These results demonstrate the cytopathicity of HIV-1 on human T lymphocytes that have developed in a HuCB colonized FTOC system.

Emv30null NOD-scid mice. An improved host for adoptive transfer of autoimmune diabetes and growth of human lymphohematopoietic cells.

When used as hosts in passive transfer experiments, a stock of NOD/Lt mice congenic for the severe combined immunodeficiency (scid) mutation have provided great insight to the contributions of various T-cell populations in the pathogenesis of autoimmune insulin-dependent diabetes mellitus (IDDM). Moreover, NOD-scid mice support higher levels of human lymphohematopoietic cell growth than the C.B-17-scid strain in which the mutation originated. However, the ability to perform long-term lymphohematopoietic repopulation studies in the NOD-scid stock has been limited by the fact that most of these mice develop lethal thymic lymphomas beginning at 20 weeks of age. These thymic lymphomas are characterized by activation and subsequent genomic reintegrations of Emv30, an endogenous murine ecotropic retrovirus unique to the NOD genome. To test the role of this endogenous retrovirus in thymomagenesis, we produced a stock of Emv30null NOD-scid mice by congenic replacement of the proximal end of chromosome 11 with genetic material derived from the closely related NOR/Lt strain. Thymic lymphomas still initiate in Emv30null NOD-scid females, but their rate of progression is significantly retarded since the frequency of tumors weighing between 170 and 910 mg at 25 weeks of age was reduced to 20.8% vs. 76.2% in Emv30% segregants. The thymic lymphomas that did develop in Emv30null NOD-scid mice were not characterized by a compensatory increase in mink cell focus-forming proviral integrations, which initiate thymomagenesis in other susceptible mouse strains. Significantly, the ability of standard NOD T-cells to transfer IDDM to the Emv30null NOD-scid stock was not impaired.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice.

The scid mutation was backcrossed ten generations onto the NOD/Lt strain background, resulting in an immunodeficient stock (NOD/LtSz-scid/scid) with multiple defects in adaptive as well as nonadaptive immunologic function. NOD/LtSz-scid/scid mice lack functional lymphoid cells and show little or no serum Ig with age. Although NOD/(Lt-)+/+ mice develop T cell-mediated autoimmune, insulin-dependent diabetes mellitus, NOD/LtSz-scid/scid mice are both insulitis- and diabetes-free throughout life. However, because of a high incidence of thymic lymphomas, the mean lifespan of this congenic stock is only 8.5 mo under specific pathogen-free conditions. After i.v. injection of human CEM T-lymphoblastoid cells, splenic engraftment of these cells was fourfold greater in NOD/LtSz-scid/scid mice than in C.B17/Sz-scid/scid mice. Although C.B-17Sz-scid/scid mice exhibit robust NK cell activity, this activity is markedly reduced in both NOD/(Lt-)+/+ and NOD/LtSz-scid/scid mice. Presence of a functionally less mature macrophage population in NOD/LtSz-scid/scid vs C.B-17Sz-scid/scid mice is indicated by persistence in the former of the NOD/Lt strain-specific defect in LPS-stimulated IL-1 secretion by marrow-derived macrophages. Although C.B-17Sz-scid/scid and C57BL/6Sz-scid/scid mice have elevated serum hemolytic complement activity compared with their respective +/+ controls, both NOD/(LtSz-)+/+ and NOD/LtSz-scid/scid mice lack this activity. Age-dependent increases in serum Ig levels (> 1 micrograms/ml) were observed in only 2 of 30 NOD/LtSz-scid/scid mice vs 21 of 29 C.B-17/Sz-scid/scid animals. The multiple defects in innate and adaptive immunity unique to the NOD/LtSz-scid/scid mouse provide an excellent in vivo environment for reconstitution with human hematopoietic cells.

Increased B lymphopoiesis in genetically sex steroid-deficient hypogonadal (hpg) mice.

Interleukin 7 (IL-7) responsive B lineage precursors were greatly expanded in genetically hypogonadal female (HPG/Bm-hpg/hpg) mice that have a secondary deficiency in gonadal steroidogenesis. Estrogen replacement in these mice resulted in a dose-dependent reduction in B cell precursors. More modest increases were documented in genetically normal mice that were surgically castrated. These findings complement other recent observations that B lymphopoiesis selectively declines in pregnant or estrogen-treated animals. Sex steroids have long been known to influence such disparate processes as bone physiology and tumor growth, in addition to their importance for reproductive function. We now show that these hormones are important negative regulators of B lymphopoiesis.

Adoptive transfer of diabetes into immunodeficient NOD-scid/scid mice. Relative contributions of CD4+ and CD8+ T-cells from diabetic versus prediabetic NOD.NON-Thy-1a donors.

Precise definition of the role of both CD4 and CD8 T-cell subsets from NOD mice in the adoptive transfer of diabetes has been complicated by the possibility that endogenous T-cells may be recruited. Two newly created NOD congenic stocks, NOD.NON-Thy-1a and NOD/LtSz-scid, have been used as T-cell donors and recipients, respectively, to eliminate contributions from endogenous T-cells and thus to define the requirement for transferred T-cell subsets as a function of underlying diabetes development in the NOD donor. Total T-cells and T-cell subsets prepared from either prediabetic or diabetic NOD.NON-Thy-1a donors were adoptively transferred into 6-wk-old NOD-scid/scid recipients that were monitored for diabetes development. Both flow cytometric and histological analysis of recipient spleen and pancreas after adoptive transfer showed lymphocytes of donor (Thy1.1+) origin exclusively. Total T-cell and enriched CD4+ T-cell preparations from both diabetic and young prediabetic donors transferred diabetes to NOD-scid/scid recipients. However, the mean time to diabetes onset was doubled when CD4+ lymphocytes were isolated from prediabetic versus diabetic donors, and these transfers were complicated by the generation of small but significant numbers of CD8+ cells over time. Enriched CD8+ populations alone were unable to transfer disease. More rigorous exclusion of CD8+ cells by means of anti-CD8 MoAb treatment in vivo of the recipients of enriched CD4+ cells demonstrated a significant difference in the diabetogenic potency of CD4+ lymphocytes from diabetic versus nondiabetic donors. Diabetes was adoptively transferred to 58% of the recipients of enriched CD4+ lymphocytes from diabetic donors. In contrast, none of the recipients of enriched CD4+ lymphocytes from young prediabetic donors developed diabetes after MoAb treatment in vivo. The ability of a T-cell population to produce severe insulitis and sialitis in NOD-scid/scid recipients of T-cells closely paralleled its ability to induce diabetes. In an effort to suppress insulitis by suppression of macrophage migration to the islets, NOD-scid/scid mice were treated with silica in conjunction with adoptive transfer of T-cells from diabetic donors. Chronic silica treatment failed to deplete tissue macrophages and did not prevent diabetes development after transfer of unfractionated T-cells. Evidence is discussed indicating that the age-associated differences in ability of CD4+ T-cells to adoptively transfer diabetes in the absence of the CD8+ T-cells subset is a function of prior, chronic exposure of the CD4+ lymphocytes to beta-cell antigens in the donor.(ABSTRACT TRUNCATED AT 400 WORDS)

Intrathymic islet cell transplantation reduces beta-cell autoimmunity and prevents diabetes in NOD/Lt mice.

Intrathymic transplantation of syngeneic islets into adolescent NOD/Lt mice was performed to establish whether the thymus would serve as an immunoprivileged site for beta-cell engraftment, and whether this treatment would prevent the development of diabetes by eliciting tolerance to islet antigens. Intrathymic injection of cells from 200 NOD islets into 4-wk-old female NOD/Lt mice produced a significant reduction in the severity of insulitis at 24 wk of age. Furthermore, diabetes development was strongly suppressed (11% incidence) compared with controls (100% incidence). Both thymus histology and thymic insulin content revealed a rapid loss of the implanted beta-cells with < 1% remaining 1 wk posttransplantation. Despite the rapid loss of thymus-implanted islet cells, evidence for tolerance induction to islet cell antigens was obtained by adoptive transfer of splenic leukocytes from these mice into NOD-scid/scid recipients. After adoptive transfer of splenic leukocytes from 24-wk-old untreated prediabetic donors, 4 of 5 NOD-scid/scid recipients developed diabetes within 4 wk, and none of the recipients became diabetic after transfer of splenocytes from intrathymic islet-implanted donors. Intrathymic islet transplantation did not lead to reduction of sialitis in females with reduced severity of insulitis, indicating that the protective effect was tissue specific. This also was reflected in adoptive transfer experiments, because equal severity of sialitis was observed in NOD-scid/scid recipients of spleen cells from either islet transplanted or control NOD/Lt mice. In conclusion, the data suggest that intrathymic injection of islet cells prevents diabetes by stimulating immunological tolerance to beta-cells.