Persistent infection with mouse hepatitis virus of low virulence in nude mice.

A persisting type of infection with wasting syndrome was established in congenitally athymic nude mice after intraperitoneal inoculation with a mouse hepatitis virus which was not fully pathogenic for heterozygous haired littermates. From the liver, spleen, lymph nodes, and brain of most infected nude mice, the virus was detected at high titers during aperiod from 6 to 35 days postinfection, occurrence of degenerative and necrotic lesions being correlated with virus titers in these organs. The titer of serum neutralizing antibody remained undetectable or very low in most diseases nude mice, whereas some animals resisting the infection could produce antibody at a later stage. In heterozygous haired mice, some lesions were detectable at a very early stage of infection in the spleen and liver, but they seemed to disappear with a marked elevation of the neutralizing antibody titer. Nude mice were able to resist the virus infection when they had previously received transfer of thymocytes from weanling heterozygous littermates.     

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.     

An epizootic Staphylococcus infection in a nude mouse colony

An epizootic of Staphylococcus infection causing abscesses was encountered in a small-scale breeding colony of nude mice of BALB/c background. The incidence of abscess was sporadic and mostly nude (nu/nu) mice aged over 3 months were affected. Staphylococcus aureus was isolated from the face, oral cavity, and feces of almost all nu/nu and heterozygous (nu/+) mice in the colony. After a prolonged time period, up to 10 to 14 months of age, almost all the S. aureus-carrying nu/nu mice produced abscesses and eventually died. Athymicity of the host seemed to be a prerequisite for the abscess formation since nu/+ mice were spared from the lesions. Also, transfer of immunocompetent spleen cells cured the abscesses of the affected nude mice.     

Natural T-cell regulation of spontaneous immunoglobulin secretion.

Splenic lymphocytes from nude (nu/nu), heterozygous/nude (+/nu), or wild type (+/+) mice were examined for their capacity to secrete immunoglobulin (Ig) in the absence of exogenous antigenic stimulation. Using the reverse hemolytic plaque assay, which measures spontaneous Ig secretion in vitro, whole spleen populations from both heterozygous/nude (+/nu) and nude (nu/nu) mice were found to have significantly fewer numbers of plaque-forming cells when compared with spleen cells from +/+ mice. Analysis of highly purified populations of T and B lymphocytes showed that increased numbers of B cells from +/+ mice were stimulated to secrete Ig when as few as 10% syngeneic +/+ T cells were added in vitro. In contrast, the same number of thymocytes suppressed the identical B-cell function. A comparison of splenic T cells obtained from either +/+ or +/nu mice revealed that T cells from +/nu animals stimulated additional plaque-forming activity by B cells from wild type or nude mice. The cellular mechanism underlying enhanced help by T cells from +/nu mice is unclear but may reflect a functionally restricted population of T cells inherited by heterozygous/ nude mice.     

Immunization of the nude mouse against mouse hepatitis virus through the transfer of sensitized lymphocytes]

Nude (nu/nu) mice failed to resist to virulent mouse hepatitis virus (MHV) infection after vaccination with inactivated virus. Resistance was induced in nu/nu mice by the transfer of spleen cells from heterozygous haired (nu/ +) mice concomitantly with the vaccination, and the effect was more remarkable with spleen cells from immunized nu/ + mice. Antibody was demonstrable in nu/nu mice having received nu/ + cells and survived challenge infection.     

Essential Role of T Cells in the Postexposure Prophylaxis of Rabies in Mice

Athymic nude mice injected intramuscularly with a street strain of rabies virus were not protected against rabies by postexposure administration of beta-propiolactone-inactivated rabies vaccine. In contrast, their normal littermates were completely protected from death by the same vaccination regimens. Nude mice did not produce IgG antibody as a result of the vaccine during the test period of 15 days, whereas normal littermates produced IgG antibody from day 5 after vaccination. However, passive immunization with antirabies hyperimmune mouse ascites showed that antibody was completely ineffective in protecting either nude mice or their normal littermates against rabies when given later than 2 days after infection. No significant difference in the induction of circulating interferon by the vaccination was noted in these mice. Passive transfer of immune spleen cells to nude mice immediately after infection resulted in 30 to 37.5% protection of the mice. Passively transferred spleen cells did not produce detectable amounts of neutralizing antibody in the recipient mice except on day 2 after the transfer, when a low level of antibody was detected. These observations demonstrate the essential role of T cells in the postexposure prophylaxis of rabies in mice. The mechanisms of the failure of postexposure vaccination in nude mice are discussed.     

Thymus dependent immunity against Tyzzer's disease in the mouse]

Nude (nu/nu) mice fail to resist to challenge infection of Tyzzer's disease after pretreatment with formalin-killed organisms that was effective for protecting heterozygous haired (nu/?) mice from challenge. Resistance was induced nu/nu mice after the transfer of spleen cells from immunized nu/? and concomitant formalin vaccine treatment.     

Recovery from a viral respiratory tract infection. IV. Specificity of protection by cytotoxic T lymphocytes

Immune spleen cells enhanced for influenza-specific cytotoxic activity after exposure to virus-infected stimulator cells in vitro effect recovery when transferred to nude and immunocompetent mice with influenza pneumonia (5). This protective effect correlated with the virus-specific cytotoxic activity of the transferred lymphocytes and is removed by treatment with anti-0 serum and complement. The experiments presented here indicate that spleen cells taken directly from mice undergoing a primary or secondary infection are less protective than immune spleen cells that are restimulated in vitro before transfer. This decreased ability to clear pulmonary virus and effect survival correlated with their relatively lower levels of influenza-specific cytotoxicity. Protection did not correlate with the level of natural killer cell activity of transferred cells. The results also indicate the immune spleen cells that are protective are influenza A subtype cross-reactive and are H-2-restricted; H-2d immune spleen cells effected recovery of H-2d but not H-2k challenged mice.     

T cells subsets responsible for clearance of Sendai virus from infected mouse lungs

T cell subsets responsible for clearance of Sendai virus from mouse lungs determined by adoptive transfer of immune spleen cell fractions to infected nude mice. T cells with antiviral activity developed in spleens by 7 days after intranasal infection. Spleen cell fractions depleted of Lyt-2+, Lyt-1+, or L3T4+ cells showed antiviral activity in vivo, although the degree of the activity was lower than that of control whole spleen cells. The antiviral activity of the Lyt-2+ cell-depleted fraction was consistently higher than that of L3T4+ (Lyt-1+)-depleted cells. In vitro cytotoxic activity against Sendai virus-associated, syngeneic lipopolysaccharide-blast cells was detected in stimulated cells from intraperitoneally immunized mice but was lost after depletion of Lyt-2+ cells. Multiple injection of anti-Sendai virus antibody into infected nude mice had no effect on lung virus titer. These results indicate that L3T4+ (Lyt-1+) and Lyt-2+ subsets are cooperatively responsible for efficient clearance of Sendai virus from the mouse lung.     

Regulation of murine IgE production in SJA/9 and nude mice. Potentiation of IgE production by recombinant interleukin 4

Serum IgE levels were determined in different strains of mice with enzyme-linked immunosorbent assay by using rat monoclonal anti-murine IgE antibodies in normal and in Nippostrongylus brasiliensis-infected mice. After infection, serum IgE levels were high in BALB/c and CB-20, low in SJL/J and SJA/20 mice, and not detected at all in SJA/9 and nude mice. Surface IgE-positive cells were greatly increased in BALB/c and SJL/J mice after infection, but not in SJA/9 and nude mice. Most surface IgE-positive spleen cells were also surface IgM- and surface IgD-positive. When spleen cells from SJA/9 or nude mice were stimulated in vitro with lipopolysaccharide and recombinant interleukin 4 (formerly B cell-stimulating factor 1), IgE was produced and detected in the supernatants of these cultures. In addition, surface IgE-positive cells could be detected in these cultures. Most of the surface IgE-positive cells were surface IgM- and surface IgD-negative, unlike those seen in the spleens of Nippostrongylus-infected BALB/c and SJL/J mice. These observations show that SJA/9 and nude mice have IgE-producing precursor B cells, and after appropriate stimulation interleukin 4 can induce them to secrete IgE.     

The absence of resistance in congenitally athymic nude mice toward infection with the intestinal nematode, Trichuris muris: resistance restored by lymphoid cell transfer

Congenitally athymic (nude) mice maintained infections of Trichuris muris for at least 40 days post-infection, whereas phenotypically normal mice expelled the worms by 18 days post-infection. Complete worm expulsion occurred in nude mice which had been given spleen cells. On the other hand, a partial resistance to the infection was observed in nude mice which received thymus or mesenteric lymph node cells. No significant worm reduction was seen by injection of immune serum.     

Evidence for extrathymic development of TNK cells. NK1+ CD3+ cells responsible for acute marrow graft rejection are present in thymus-deficient mice.

The predominant mechanism responsible for acute specific rejection of allogeneic and parental bone marrow by irradiated mice is due to a cell (TNK) that expresses the NK cell surface markers NK1 and ASGM1 as well as TCR. Here we analyze the question as to whether TNK cells require a functional thymus for their development. Using adoptive cell transfer assays, evidence is presented that, as is the case in normal mice, NK1+ CD3+ effector cells are responsible for rejection in thymus-deficient nude mice and that the specificity of rejection is indistinguishable from that of normal mice. To reveal the presence of TNK cells in the spleen of nude mice, double staining for NK1 and CD3 followed by FACS analysis was done. It is shown that NK1+ CD3+ cells are present in the spleens of nude but not euthymic mice, suggesting that the lack of a functional thymus stimulates either Ag expression or the number of TNK cells. In support of this finding, the treatment of irradiated marrow reconstituted mice with cyclosporin A leads to the appearance of TNK cells in the spleen. The relative efficiency of spleen cells from nude and cyclosporin A-treated mice to transfer resistance in adoptive cell transfers was assessed and found to be higher than that of normal spleen, consistent with the higher frequency of these cells in thymus-defective mice. The fate of NK1+ CD3+ cells subsequent to stimulation with an allogeneic marrow graft indicates that these cells proliferate in nude mice without gaining cytolytic activity. In euthymic mice, however, NK1+ CD3+ cells appear transiently but disappear in favor of CD4+ and CD8+ cells that proliferate in response to an allogeneic marrow graft. The CD8+ cells express cytolytic activity with specificity similar to that of the acute rejection mechanism, consistent with the suggestion that TNK cells differentiate into CD8+ killer cells. The reason why TNK cells in nude mice fail to differentiate into CD8+ CTL is explained by the lack of Th cells.     

Fluctuation of antiviral resistance in the intestinal tracts of nude mice infected with a mouse adenovirus

Oral administration of adenovirus strain K87 to BALB/c nude mice resulted in viral proliferation in the intestinal tract up to around week 6 at which point replication was suppressed. In other words, the host acquired resistance. However, this resistance was temporary and the viral infection persisted over a long period with repeated periods of proliferation and resistance. That the appearance of this resistance is the result of infecting mice with the virus and is not due to age difference per se was made clear through experimentation with nude mice of different age groups. However, it was indicated that increase in age is involved in the decreased rate of reproliferation following initial suppression. No evidence of the virus was obtained from any other organ throughout the infection. Furthermore, throughout the persistent infection, even during the aforementioned periods of resistance, no neutralizing antibody was detected from sera, intestinal wall or intestinal content. When spleen cells from BALB/c heterozygous littermate mice was transferred to the nude mice, an earlier onset of antiviral resistance was seen than in nude mice without the transfer, and this was accompanied by a rise in neutralizing antibody titer. From these results, it is believed that the resistance characteristic of nude mice infected by mouse adenovirus is dependent on some factor other than the neutralizing antibody invoked resistance exhibited by euthymic mice.     

Augmentation of transfer of experimental autoimmune thyroiditis (EAT) in mice by irradiation of recipients

Experimental autoimmune thyroiditis (EAT) can be adoptively transferred to normal syngeneic recipients using spleen cells from susceptible strains of mice primed in vivo with mouse thyroglobulin (MTg) and lipopolysaccharide (LPS) following in vitro activation of spleen cells by culture with MTg. Irradiation of recipient animals markedly augments the severity of thyroiditis induced in this system. Irradiation of recipients does not alter the time course of the development of thyroiditis, nor does it alter the requirement for both in vivo priming and in vitro activation of spleen cells for the development of EAT. Spleen cells from EAT-resistant strains of mice (e.g., Balb/c) do not induce EAT in irradiated recipients. Irradiated recipients develop significant levels of anti-MTg antibodies while unirradiated recipients have little detectable antibody response. The augmenting effect of irradiation can be substantially reversed by transferring naive spleen cells to recipients prior to the transfer of MTg/LPS-primed in vitro-activated spleen cells. In addition athymic CBA/Tufts nude mice develop more severe EAT than CBA/Tufts nude/+ littermates following transfer of activated CBA/J spleen cells. These data suggest that natural suppressor cells may regulate the development of EAT at the effector cell level.     

Effects of spleen cells and serum on transfer of immunity to Strongyloides venezuelensis infection in hypothymic (nude) mice

The course of Strongyloides venezuelensis infection in congenitally hypothymic (nu/nu) mice and their heterozygous thymus-bearing littermates (nu/+) was followed. Unlike the infected nu/+ mice, the nu/nu mice were unable to expel the worms until the end of the observation period (98 days post-infection). In addition, about three times as many eggs were counted at the peak level of infection in faeces of the infected nu/nu mice in comparison with the nu/+ mice. No acquired resistance to rechallenge was observed among the nu/nu mice. Auto-reinfection within the infected nu/nu mice could not be supposed in the present study. The worm expulsion mechanism was generated by nu/nu mice which had been given syngeneic spleen cells from intact +/+ mice. The expulsion of adult worms, as well as the protection against migrating larvae, occurred anamnestically when spleen cells from immune +/+ mice were transferred. The serum transfer, however, only caused a retardation of larval migration. The results support the hypothesis that direct worm immunity and worm expulsion are a T cell-dependent phenomenon.     

Brugia pahangi in nude mice: protective immunity to infective larvae is Thy 1.2+ cell dependent and cyclosporin A resistant

Mechanisms of protective immunity to larvae of Brugia pahangi were studied in congenitally athymic nude C3H/HeN mice and their syngeneic heterozygous littermates. An average 11% of subcutaneous larval inocula was recovered from control nudes 28 days after inoculation. No worms were recovered from nude recipients of viable splenic Thy 1.2+ T lymphocytes from heterozygotes which had killed a priming dose of B. pahangi larvae. Primed T lymphocytes, depleted of either Lyt 1.1+ or Lyt 2.1+ cells or incubated with anti-Thy 1.2 monoclonal antibody and complement, failed to protect nude mice against a larval challenge. Nor were primed B lymphocytes depleted by Thy 1.2+ T cell contaminants protective. Treatment with cyclosporin A (CsA) did not increase the numbers of worms recovered from heterozygotes nor did CsA treatment of heterozygous cell donors abolish the ability of primed Thy 1.2+ T lymphocytes to transfer protection to nude mice. IgG but not IgM antibody titres to B. pahangi antigens were depressed in all CsA-treated mice. CsA treatment of nude mice had no direct effect upon development of B. pahangi larvae. These results show that protective immunity to larvae of B. pahangi in mice depends upon small numbers of Thy 1.2+ T cells which are CsA-resistant.     

Histopathological studies on experimental Cryptococcosis in nude mice

Cryptococcosis in nude (nu/nu) mice was examined histopathologically. In addition, effects of carrageenan and lymph node cell transfer againstCryptococcus infection were investigated. As controls, heterozygous littermates (nu/+mice) and mice of strain ddy (ddy mice) were employed.Each mouse was inoculated intravenously with 10⁵ yeast cells ofCryptococcus neoformans suspended in 0.2 ml phosphate-buffered saline. Two mice out of each group were sacrificed at appropriate intervals up to 25 days after inoculation and histopathological sections were prepared from them. They were stained with H & E and by PAS method. Histopathological characterristic in the brain was cyst formation with no cellular response. The brain was more severely in the nu/nu mice than in either the nu/+ or ddy mice. In the liver, there was a major difference in histopathological findings between the nu/nu and either of the other groups of mouse. In the nu/nu mice, cyst formation with no cellular response was induced, and on the contrary granuloma formation in the nu/+ and ddy mice. However, the granuloma formation was inhibited in the livers of the nu/+ and ddy mice by administration of carrageenan, and induced in the nu/nu mice by cell transfer. In the spleen and lymph nodes, lesions were severer in nu/nu mice than in either the nu/+ or ddy mice.These results suggested that the fungus' invasiveness of mice was strongly influenced by T-cell dependent mechanism.     

Eradication of a large MOPC-315 tumor in athymic nude mice by chemoimmunotherapy with Lyt2+ splenic T cells from melphalan-treated BALB/c mice bearing a large MOPC-315 tumor

Summary We have previously shown that spleen cells from BALB/c mice that are in the process of eradicating a large MOPC-315 tumor following low-dose (2.5 mg/kg) melphalan (l-phenylalanine mustard) therapy are effective in preventing tumor progression upon adoptive transfer into BALB/c mice bearing a barely palpable tumor that had been treated with a subcurative dose of melphalan [Mokyr et al. (1989) Cancer Res 49: 4597]. Here we show that such spleen cells in conjunction with a subcurative dose of drug (adoptive chemoimmunotherapy, ACIT) can cause the complete regression of a large (15–20 mm) s.c. MOPC-315 tumor in a large percentage of T-cell-deficient (athymic nude) tumor-bearing mice. Spleen cells that were effective in ACIT of athymic nude mice displayed in vitro a substantial direct lytic activity against MOPC-315 tumor cells, and the lytic activity was greatly enhanced when the spleen cells were cultured for 5 days with or without mitomycin-C-treated MOPC-315 stimulator tumor cells. The cells responsible for the therapeutic effectiveness of the spleen cells in ACIT of athymic nude mice, as well as the cells responsible for the direct in vitro anti-MOPC-315 lytic activity of the spleen cells, were of the Lyt 2 and not the L3T4 phenotype. Most of the athymic nude mice that completely eradicated a large MOPC-315 tumor as a consequence of ACIT were capable of rejecting a challenge with 30–100 times the minimal lethal tumor dose for 100% of normal BALB/c mice administered more than 1 month after the ACIT. The ability of these athymic nude mice to resist the tumor challenge was associated with the presence of a greatly elevated percentage of cells expressing T cell surface markers in their spleens. Thus, it is conceivable that splenic Lyt 2⁺ T cells from melphalan-treated BALB/c mice bearing a large MOPC-315 tumor mediate their therapeutic effectiveness in ACIT of athymic nude mice bearing a large MOPC-315 tumor, at least in part, through direct cytotoxicity for MOPC-315 tumor cells. In addition, eradication of a large MOPC-315 tumor through cooperation between antitumor immunity and melphalan toxicity endues the athymic nude mice with an elevated percentage of T cells in their secondary lymphoid organs, and these T cells are probably responsible for the long-lasting protective antitumor immunity exhibited by these mice.Supported by research grant IM-435A from the American Cancer Society and research grant B-8806 from the Bane EstateIn partial fulfillment of the requirements for the Doctor of Philosophy DegreeRecipient of Career Development Award CA-01350 from the National Cancer Institute     

Cytotoxicity Induced by Monoclonal Antibody to α-Fetoprotein Coadministered with Spleen Cells of Nude Mice

This paper describes an attempt to effectively induce antibody-dependent cell-mediated cytotoxicity (ADCC) in nude mice. A monoclonal antibody against α-fetoprotein, 80G, coadministered with spleen cells from other nude mice bearing HuH-7N (xenograft of human hepatoma cell line, HuH-7) significantly suppressed the growth of HuH-7N as compared to treatment with 80G alone. 80G with spleen cells from normal nude mice also had some suppressive effect. In contrast, no effect was observed with each spleen cells alone as well as 80G alone. These results suggest that further supply of effector cells could enhance ADCC activity in nude mice.     

Activation of murine B lymphocytes by suramin

Suramin stimulated DNA synthesis in spleen cell cultures of all inbred strains of mice tested, including, for example, CBA, DBA/2, C57BL/6, and the lipopolysaccharide (LPS)-nonresponsive strain C3H/HeJ. The cells responding to the drugs were removed by passage through nylon wool columns, but they were not eliminated by in vivo treatment of the mice with anti-Thy 1.2 antibody. Spleen cells of homozygous nude mice (C57BL/6 or BALB/c background) were as reactive as those of their heterozygous littermates. Collectively the data show that suramin is a B-cell mitogen in the mouse.