Development of T-cell function in relation to T-cell set diversification in nu/nu mice

The differentiation pattern of splenic T-cell populations in germ- and pathogen-free nu/nu mice, as compared to nu/+ littermates, is characterized by two abnormal features: the expression of TL determinants on peripheral T cells and the delayed onset of their differentiation from the predominant Lyt-123:TL+ set into TL- cells of Lyt-1+ and Lyt-123+ phenotype, which, in these mice, does not occur until 10 weeks of age. We report here that the delayed onset of mitogen- or alloantigen-induced interleukin-2 synthesis and T-cell proliferation as well as the development of cytotoxic T-lymphocyte activity of enriched T-cell populations is strictly correlated with the time point of T-cell subset diversification in nu/nu mice and depends in particular on the presence of the Lyt-1 (TL-:Lyt-2-) T-cell set which is lacking in splenic T-cell populations of germ-free young nu/nu mice.     

Effects of tobacco glycoprotein (TGP) on the immune system. I. TGP is a T-independent B cell mitogen for murine lymphoid cells

It has been shown that tobacco glycoprotein (TGP), a polyphenol-rich glycoprotein antigen purified from cured tobacco leaves, is mitogenic for lymphoid cells in the spleen, peripheral blood, and bone marrow, but not for thymus cells. The proliferative response is not reduced by treatment of spleen cells or peripheral blood lymphocytes with anti-Thy-1.2 and complement, and spleen cells from the congenitally athymic (nu/nu) CD-1 proliferate as vigorously in response to TGP as do spleen cells from their heterozygous nu/+ littermates. In addition, TGP induces differentiation of mouse spleen cells into antibody-secreting cells, the majority of which secrete IgM, and the remainder mainly IgG and a few IgA. The differentiation into antibody-secreting cells induced by TGP occurs with spleen cells from nu/nu mice. It is concluded that TGP is a T-independent B cell mitogen for mouse lymphoid cells. On the basis of the ability of spleen cells from the LPS-nonresponder C3H/HEJ mice to respond to TGP with proliferation and differentiation into antibody-secreting cells, it is concluded that the effects of TGP are distinct from those of LPS and cannot be due to contamination of the TGP preparation with LPS.     

Characterization of mononuclear phagocytes in schistosomal egg granulomas of athymic mice

Macrophages from schistosomal egg granulomas of athymic mice (nu/nu GM) and their euthymic littermates (nu/+ GM) were analyzed phenotypically for the expression of antigens encoded by the I-A subregion of the major histocompatibility complex and for their ability to perform as antigen-presenting cells. Only 11 to 15% of nu/nu GM expressed I-A antigens as compared to 61.5 to 75% of nu/+ GM. Although both populations of cells appeared to be equally effective as antigen-presenting cells appropriately sensitized lymphocytes in the presence of specific antigens--soluble schistosomal egg antigen (SEA) and human gamma-globulin (HGG)--only nu/nu GM, but not nu/+ GM, were found to stimulate I-A-restricted proliferation of schistosome-sensitized T cell populations in the absence of SEA added in vitro. Furthermore, nu/nu GM but not nu/+ GM were shown to exhibit significant proliferative capacity in vitro, but this phenomenon could not account for the observed difference in SEA-independent T cell stimulation. Finally, culture supernatants from nu/nu GM displayed significant thymocyte-stimulating activity, consistent with interleukin 1, which was not observed in nu/+ GM. These findings point to significant differences between nu/nu GM and nu/+ GM, which may be part of an adaptive mechanism of granulomatous reactivity in the absence of a competent T cell system.     

Functional analysis of Ras in Colletotrichum trifolii

Ras is a small monomeric GTP binding protein that transduces signals for growth and differentiation of eukaryotic organisms. Previously, a unique ras gene, designated Ct-ras, was cloned from the alfalfa fungal phytopathogen, Colletotrichum trifolii. Expression of Ct-Ras in mouse fibroblast cells (NIH3T3) demonstrated that Ct-ras is functionally similar to the mammalian ras genes since activating mutations of Ct-ras caused oncogenic phenotypes in nu/nu mice, including tumors. In C. trifolii, activated 'oncogenic' Ras (Val2) induced abnormal hyphal proliferation, defects in polarized growth and significantly reduced differentiation such as conidiation and appressorium formation in a nutrient dependent manner. Gene disruption of ct-ras was lethal. To further evaluate the function of Ct-Ras in C. trifolii, three different approaches were used: overexpression of cytosolic Ras by CAAX box deletion; expression of dominant negative Ct-RasT22N; and antisense ct-ras expression. Results showed that suppression of Ct-Ras activity significantly decreases fungal germination frequencies and hyphal growth rates. Taken together, these data suggest involvement of Ct-Ras in regulation of fungal cell growth and differentiation.     

Reconstitution of nude mouse T cell function in vivo: IL 2-independent effect of human T cells

The i.p. injection of 1 to 5 X 10(6) heavily irradiated human T lymphocytes resulted in the lasting reconstitution of T cell functions in young mice bearing the nu/nu mutation. IgM and IgG responses to immunization with sheep red cells or ovalbumin, splenic lectin responses, and the expression of easily detectable Thy-1 determinants on up to 20% of spleen cells could be documented for several months after the injection of human cells. Only a narrow cell dose range was effective. Injection of larger cell numbers not only failed to induce immune reconstitution but also resulted in the development of resistance to subsequent treatments. Only mature T cells, but not thymocytes, could induce nude mouse T cell development. Lymphoblasts from one patient with acute T cell leukemia consistently immune-reconstituted nude recipients. These cells were completely unable to produce IL 2 in vitro. In contrast, the IL 2-producing T cell line Jurkat was ineffective, indicating that the abilities to produce IL 2 and to induce nude mouse T cell development are independent. In an extension of earlier models of the nude mouse immune defect, two distinct T precursor cell pools are proposed as the major components of an extrathymic differentiation pathway. As an adequate trigger of differentiation, interaction with thymus-processed T cells guides the development of precursors in the first cell pool towards populating the second IL 2-responsive pool of T precursor cells.     

The immunotherapeutic effects in tumor-bearing mice of Ly-6 monoclonal antibodies

In vivo administration of Ly-6 mAb which recognize lymphoid differentiation Ag encoded for by the Ly-6 gene complex were found to have significant beneficial immunotherapeutic effects in tumor-bearing mice. The effectiveness of the mAb treatment in mice bearing sarcomas, leukemias, or melanomas was dependent on the host and not the tumor Ly-6 phenotype. The treatment was effective in nu/nu mice, although a more pronounced inhibition of tumor growth occurred in immunocompetent mice. The effectiveness of the therapy in immunocompetent mice was dependent on the dose of mAb and was influenced by the immunogenicity of the tumor. It ranged from significant growth inhibition of weakly immunogenic tumors to complete rejection of strongly immunogenic tumors. The results of cell-mediated cytotoxic assays of splenocytes from mAb-treated mice indicated that Ly-6 mAb treatment induced and/or augmented tumor-specific CTL as well as NK cell activity in these mice. Ly-6 mAb treatment represents a novel method for tumor immunotherapy using mAb recognizing lymphoid differentiation Ag with functional activities.     

Thymus differentiation and T-cell specificity in nu/nu +/+ mouse aggregation chimaeras

Thymus development and T cell differentiation were studied in mouse chimaeras produced by aggregating pre-implantation embryos of thymus-deficient nude BALB/c (nu/nu) and wild-type C57BL/6 (+/+) mice and vice versa. Chimaeras showed mosaic distribution of skin and coat pigmentation, of hair follicles, of glucosephosphate isomerase within all tested organs and of lymphocytes expressing the different major transplantation antigens (H-2). When tested for their capacity to generate vaccinia virus-specific and self-H-2 specific cytotoxic T cells, all chimaeras of BALB/c (nu/nu) H-2d in equilibrium C57BL/6 (+/+) H-2b type generated T cells of one or both parental origins that were specific for virus and for self-H-2 of the +/+ (H-2b) type only. In contrast, some BALB/c (+/+) H-2d in equilibrium C57BL/6 (nu/nu) H-2b chimaeras generated vaccinia virus-specific cytotoxic T cells specific for either H-2d (+/+) type or for H-2b (nu/nu) type. These asymmetrical results can be interpreted to indicate the following: (i) The +/+ thymus part alone is functional, but because of asymmetrical cross-reactivities of anti-self-H-2 specificities, the observed T cell restriction phenotypes differ. (ii) Both nu/nu and +/+ thymus parts are functional but immune response defects may be exaggerated in such chimaeras producing unexpected non-responsiveness to vaccinia virus linked to H-2d in H-2b (+/+) in equilibrium H-2d (nu/nu).     

Effect of immune heterozygous spleen cell transfer on resistance to mouse hepatitis virus infection in nude mice

The role of cell mediated immune response to mouse hepatitis virus (MHV) infection in mice was studied by transferring spleen cells from immune heterozygous littermates (nu/+). A suppressive effect on viral growth was seen in infected nude (nu/nu) mice, whereas immune nu/+ serum transfer had no effect. The protective effect of immune nu/+ spleen cells was significantly reduced by treatment with anti-theta serum plus complement but not with anti-Ig serum. In infected nu/nu mice which received transfers of immune nu/+ cells, neutralizing antibody appeared although the titer was not high enough to protect nu/nu mice from fatal infection. Histopathologically, lymphocyte infiltration in hepatic lesions was evident in infected nu/nu mice with nu/+ cell transfer, while it was slight without nu/+ cell transfer.     

Reactivation of the nuclei of reticulocytes and erythrocytes in heterokaryons]

The reactivation of the nuclei of erythrocytes, reticulocytes and bone marrow cells has been studied by means of hybridization of the pigeon erythroid cells with the human embryonic cells A1. The process of reactivation of the erythroid nucleus was shown to depend on the stage of erythroid cell differentiation. The nuclei of cells at the earlier stages of differentiation give a higher percentage of heterocaryons (40%, 70%) than those of more mature cells (9%). The activated nuclei of immatur cells formed nucleoli already 24 hrs, whereas those of mature erythrocytes only 3-5 days after the cell fusion.     

Local effects of granulomatous inflammation on functional activation of T cells in athymic mice

We investigated the effect of granulomatous inflammation in skin on lymphocyte maturation in athymic (nu/nu) mice. Hepatic egg granulomas developed in euthymic (nu/+) mice with schistosomiasis were transplanted into skin of nu/nu mice. During skin granuloma development the rate of DNA synthesis and interleukin 2 activity of lymphocytes from lymph nodes, with and without concanavalin A stimulation, showed that the nu/nu cells were activated to levels of untreated nu/+ lymph node cells. Activation of splenic lymphocytes was not detected in the grafted nu/nu mice. Also, immunohistochemical staining demonstrated an increase in cells expressing Thy 1.2, Lyt-1 or L3T4 surface markers in the skin and lymph nodes, but not in spleen. The findings indicate that a granulomatous reaction in nu/nu mouse skin induces local, but not systemic, proliferation and differentiation of lymphocytes, to a low degree compatible with resting nu/+ mice.     

Defective differentiation of natural killer cells in SJL mice. Role of the thymus

As previously shown, three distinct phenotypes exist in murine natural killer (NK) cell activity when it is evaluated by the endogenous levels of activity and the susceptibility to augmentation by interferon (IFN) and IFN inducers. The "low" phenotype has low levels of activity which can be poorly augmented by IFN, as in mice of SJL strain. The "inducible" phenotype exhibits low endogenous levels but can vigorously respond to IFN-mediated augmentation, as in A.SW strain. The "high" NK phenotype shows high levels of endogenous activity which can be augmented to still higher levels by IFN, as in B10.S mice. Since SJL mice with congenital absence of the thymus (nude) were of the inducible type, the effect of neonatal thymectomy was examined in the present study. Neonatal thymectomy was found to convert the low phenotype of SJL mice to the inducible, mimicking the effect of nu/nu genotype. Thymectomy as late as 25 days after birth was effective, but retransplantation of a syngeneic newborn or adult thymus, or thymocytes, failed to reverse the effect of thymectomy. The poor responsiveness of NK activity to IFN in SJL, therefore, is extrinsic to the NK cell lineage and is attributable to suppression or maturational block of NK cell differentiation by the thymus during the first few weeks of neonatal life. A series of experiments with bone marrow chimeras showed that the SJL recipients did not allow the expression of inducible or high phenotype by bone marrow progenitors from allogeneic donors with either phenotype. Therefore, the SJL recipients provide an environment which suppresses not only the development of IFN-sensitive NK cell precursors, but also the levels of endogenous NK cell activity. SJL bone marrow cells gave rise to NK activity of inducible phenotype in B10.S recipients, confirming the crucial role of the environment in which NK cell differentiation takes place.     

Cells with natural killer activity in the cerebrospinal fluid of normal mice and athymic nude mice with acute Sindbis virus encephalitis

Sindbis virus causes an acute, nonfatal inflammatory encephalitis in weanling BALB/c mice. Mononuclear inflammatory cells are present in the cerebrospinal fluid (CSF) as well as in the parenchyma of the brain. Both aspects of this inflammatory response were eliminated by treatment with cyclophosphamide. Athymic nude (nu/nu) mice developed no inflammation in the brain, but did develop a CSF pleocytosis that peaked on day 2 after infection. The time course of the appearance of cells in the CSF was earlier in nu/nu mice than their heterozygote (nu/+) littermates. The pleocytosis in nu/nu mice reached a peak on day 2, whereas in nu/+ mice the peak was on day 4, as it is in normal BALB/c mice. To determine whether some of the CSF cells in nu/nu mice may be natural killer (NK) cells, NK activity was measured in a 4-hr assay by using a YAC-1 target cell. NK cell activity in the spleen and peripheral blood was induced by infection with Sindbis virus in nu/nu mice with a similar time course to that of nu/+ mice (peak 1 day after infection). CSF from nu/nu mice had NK activity present 2 days after infection that was greater than that present in either the peripheral blood or spleen. BALB/c and nu/+ mice had insufficient cells present for assay at day 2, but BALB/c mice had NK activity present in the CSF 3 and 5 days after infection that exceeded that in the peripheral blood or spleen. Brain interferon was detectable on day 1 in nu/nu mice, but not until day 2 in nu/+ mice even though the amounts of brain virus were the same in the two groups at all time points. It is concluded that cells with NK activity contribute to the CSF pleocytosis induced by acute Sindbis virus encephalitis.     

Neoplasms in NIH type II athymic (nude) mice

Necropsy and histopathology were performed over an 18-month period on 173 NIH type II athymic (nude) mice and 53 NIH type II mice heterozygous at the nu locus. A total of 149 mice were used in studies of tumor transplantation while 77 mice were screened as part of the quality assurance program for the colony. Twenty-nine neoplasms were found in 173 nu/nu mice. Only one neoplasm, an ovarian granulosa cell tumor, was found in 53 nu/+ mice. In nu/nu mice, there were nineteen lymphosarcomas, nine ovarian granulosa cell tumors, and one transitional cell carcinoma of the urinary bladder. A greater number of lymphosarcomas occurred in mice greater than 6 months old. A greater number of tumors, particularly lymphosarcomas, were found in nu/nu mice than in nu/+ mice.     

Tissue responses against Cladosporium trichoides and its parasitic forms in congenitally athymic nude mice and their heterozygous littermates

The tissue responses against Cladosporium trichoides and its parasitic forms were studied using nude (nu/nu) mice and their heterozygous (nu/+) littermates of BALB/c background.1.0,0.1 and 0.01% cell suspensions were prepared from a culture broth which had been inoculated with the C. trichoides and cultured with reciprocal shaking at 27 ° C for 7 days. Sixty nu/nu or 60 nu/+ mice were divided into three groups consisting of 20 each which was allotted to one of the three cell suspensions. Each mouse was inoculated intravenously with 0.1 ml of either the cell suspensions. Two mice from each of the six groups were sacrificed at adequate intervals until 30 days after inoculation and histopathologic sections stained with H & E or by PAS were prepared from their visceral organs.There were no characteristic findings in the nu/nu and nu/+ mice inoculated with the 0.01% cell suspension. When inoculated with the 1.0% cell suspension, the brain was the favorite target organ in both groups of mice and the kidney was the second. When inoculated with the 0.1% cell suspension, brain lesions were observed only in the nu/nu mice. The susceptibility of the nu/nu mice was higher than that of the nu/+ mice.The parasitic forms in the brain of the nu/nu and nu/+ mice were slender septate true hyphae with or without polymorphonuclear leucocyte infiltrate, while in the liver, spleen and lung of both groups of mice the parasitic forms were short thick hyphae, moniliform hyphae, chlamydospores or round cells (sclerotic cells). Many giant cells containing fungal elements appeared in the liver of the nu/nu mice.     

A "lymphokine-like" soluble product that induces proliferation and maturation of B cells appears in the serum-free supernatant of a T cell hybridoma as a consequence of mycoplasmal contamination

The serum-free supernatant of a cloned murine T cell hybridoma supports the proliferation and maturation to Ig secretion of purified B cells (mu+ cells) from BALB/c nu/nu mice, but has no effect on the proliferation of nylon wool-selected BALB/c nu/+ splenic T cells. Although the supernatant activates B cells without co-stimulation, it synergizes with anti-mu for the proliferative response. The induction of B cell proliferation and maturation to Ig secretion is directly related to contamination of the hybridoma by Mycoplasma hyorhinis. Hybridoma cells freed of mycoplasma by detergent treatment fail to produce active supernatant, and reinfection of the treated cells reconstitutes the activity. Furthermore, deliberate infection of a mycoplasma-free unrelated T cell hybridoma, as well as the monocytic cell line P388D1, results in the production of supernatants with B cell proliferating activity. Mycoplasma organisms isolated from the supernatant induce B cell proliferation without subsequent maturation to Ig secretion. Gel filtration chromatography of the supernatant from mycoplasma-contaminated hybridoma cells yields two peaks of activity. The first peak, found at the exclusion limit of the gel, results in B cell proliferation without maturation and may be attributed to mycoplasma organisms. The second peak (average m.w. 90,000) results in B cell proliferation as well as differentiation to Ig secretion. A "lymphokine-like" soluble product released by Mycoplasma hyorhinis is most likely responsible for this B cell activation, because fractionation of the supernatant from deliberately contaminated P388D1 cells gives essentially the same results, and gel filtration of mycoplasma-free supernatants does not generate any active fractions. The possibility should be considered that mycoplasma-derived soluble products may be among the many factors controlling in vitro B cell growth and maturation.     

Defense mechanisms of mice against Fonsecaea pedrosoi infection

Defense mechanisms of a host against Fonsecaea pedrosoi infection were studied histopathologically using athymic nude (nu/nu) mice of BALB/c background and their heterozygous (nu/+) littermates. Thirty male nu/nu and 30 nu/+ mice, weighing 16–19 g, were employed in this experiment. The nu/nu or nu/+ mice were divided into 3 groups consisting of 10 each. Furthermore, 4 nu/nu mice were supplemented to investigate effects of lymph node cell transfer. Subglobose cells of F. pedrosoi Tsuchiya strain were obtained from a culture in brain heart infusion glucose (1%) broth with reciprocal shaking at 37 °C for 17 days, and then 0.02, 0.1 and 0.5% cells suspensions were prepared. Each cell suspension was allotted to one group of the nu/nu or nu/+ mice. 0.1 ml of the cell suspension was inoculated into a tail vein, then one mouse from each group was sacrificed 1, 2, 4, 6, 8, 10, 14, 18, 21 and 25 days after inoculation. In both the nu/nu and nu/+ mice, the brain, kidneys and heart were affected severely with the strain in that order. Histopathologically, the defense mechanisms of the nu/+ mice against the fungus infection consisted chiefly of 2 steps: first, of non-immune phagocytosis by polymorphonuclear leucocytes (PMNs), and second, of granuloma formation induced by cell-mediated immunity. Those of the nu/nu mice consisted only of one step: phagocytosis by PMNs. A difference in susceptibility to the strain between the nu/nu and nu/+ mice changed according to the amount of the fungal cells inoculated. When inoculated with the 0.02% cell suspension, the resistance of the nu/nu mice was stronger than that of the nu/+ mice. In contrast, when inoculated with the 0.5% cell suspension, the former was affected more severely than the latter. There were little differences in the susceptibility to the strain between the nu/nu and nu/+ mice inoculated with the 0.1% cell suspension. These data seem to indicate that the phagocytic function of PMNs of the nu/nu mice was more active than that of the nu/+ mice, and the nu/nu mice inoculated with the 0.5% cells suspension (beyond the phagocytic capacity) lost resistance against the fungus infection. When the nu/nu mice were transferred with lymph node cells before inoculation of the strain, granulomata were formed to prevent hyphae from growing freely in the tissue.     

Distribution of terminal deoxynucleotidyl transferase in bovine serum albumin gradient-fractionated thymocytes and bone marrow cells of normal and leukemic mice.

The distribution of terminal deoxynucleotidyl transferase (TdT) peaks I and II, in single cell suspensions of thymuses, bone marrow, and peripheral lymphoid organs fractionated in discontinuous bovine serum albumin gradients, was examined in a variety of mouse strains and Fischer 344 rats to relate the normal patterns of thymocyte differentiation to the leukemic process. TdT peaks I and II were found in fractions A (10 to 23%), B (23 to 26%), and C (26 to 29%) of the thymus of both normal and leukemic C57BL/6 mice, whereas only peak I was found in the same fractions of AKR mice. TdT in bone marrow was found mainly in fraction A in both normal and leukemic mice. The specific activity of TdT in this fraction, which comprises only 1 to 5% of the total bone marrow cell population, was similar to that of the thymus. The cell population of fraction A of the bone marrow was found to increase (10 to 15-fold) in leukemic mice. Only low levels of TdT activity were found in either whole or fractionated bone marrow of athymic NIH Swiss (nu/nu) mice.     

5'-Nucleotidase in Dictyostelium: protein purification, cloning, and developmental expression

5'-Nucleotidase (5NU) in Dictyostelium discoideum is an enzyme that shows high substrate specificity to 5'-AMP. The enzyme has received considerable attention in the past because of the critical role played by cyclic AMP in cell differentiation in this organism. Degradation of cAMP by cAMP phosphodiesterase (PDE) produces 5'-AMP, the substrate of 5NU. During the time course of development, the enzyme activity of 5NU increases and becomes restricted to a narrow band of cells that form the interface between the prestalk/prespore zones. We have purified a polypeptide associated with 5NU enzyme activity. Protein sequence of this peptide was obtained from mass spectrometry and Edman degradation. Polymerase chain reaction PCR amplification of genomic DNA using degenerate oligonucleotides and a search of sequences of a cDNA project yielded DNA fragments with sequence corresponding to the peptide sequence of 5NU. In addition, a clone was found that corresponded to the classical 'alkaline phosphatase' (AP) as described in several organisms. The sequences of the 5NU and AP cDNAs were not similar, indicating they are the products of separate genes and that both genes exist in Dictyostelium. Analysis of the expression of 5nu during Dictyostelium development by Northern blotting determined that the gene is developmentally regulated. Southern blot analysis showed a single form of the 5nu gene. Targeted gene disruption and knockout mutagenesis using the 5nu sequences suggested that a 5nu mutation may be lethal.