Resistance of nude mice to herpes simplex virus and correlation with in vitro production of interferon.

Homozygous nude mice and their phenotypically normal littermates were infected intraperitoneally with herpes simplex virus (HSV). Nude mice did not show increased mortality rates. In fact, at lower virus doses they were consistently less susceptible than the controls. Spleen cells from nude mice, when challenged in vitro with HSV, produced equal amounts of interferon as spleen cells from the normal littermates.     

Tumoricidal responses in vitro of peritoneal macrophages from conventionally housed and germ-free nude mice

Peritoneal macrophages from untreated nude mice were nonspecifically cytotoxic to tumor cells in vitro and were more responsive to chemotactic stimuli than macrophages from normal mice or from phenotypically normal littermates of nude mice. Tumoricidal and chemotactic responses of activated macrophages from nude mice were quantitatively comparable to responses of macrophages from BCG-infected normal mice. Peritoneal macrophages from germ-free nude mice, however, were not tumoricidal in vitro. These observations suggest that environmental stimuli, rather than thymic deficiency per se, induced activated macrophages in nude mice.     

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.     

Experimental mucormycosis in congenitally athymic (nude) mice

Athymic nude (nu/nu) mice and their phenotypically normal (nu/+) littermates displayed a similar susceptibility to acute lethal infection withAbsidia corymbifera. Although the clinical manifestations of acute infection were also similar in both groups, the nude mice tended to develop more extensive lesions and were less effective in eliminating viableA. corymbifera spores than their heterozygous littermates. The results suggested that thymus-dependent processes did not play an essential role in primary resistance to mucormycosis.     

The asymmetry in idiotype-isotype expression in the response to phosphocholine is due to divergence in the expressed repertoires of Lyb-5+ and Lyb-5- B cells

The X-linked CBA/N immune defect was used to investigate the roles of Lyb-5- and Lyb-5+ B cells in the memory response to PC-KLH (phosphocholine-conjugated keyhole limpet hemocyanin). (CBA/N X BALB/c)F1 (CB) male mice express the xid mutation and thereby lack the Lyb-5+ B cell subset, whereas their female littermates are normal and express both Lyb-5+ and Lyb-5- B cells. After priming with PC-conjugated hemocyanin (PC-Hy) in complete Freund's adjuvant, female B cells produce three phenotypic sets of PC-KLH-specific antibody. The first set (group I) is dominated by T15+, IgM, IgA, and IgG3, PC-specific antibodies. The second subset (group II) is specific for phenylphosphocholine (PPC), and is dominated by T15-, IgG1, and IgG2 antibodies. The third set (group III) recognizes an epitope(s) composed of both the PPC hapten and carrier determinants. PC-Hy-primed B cells from immune defective CB male mice produce the same number of IgG1 and IgG2 plaque-forming cells (PFC) as do PC-Hy-primed normal female cells, and these PFC are also predominantly T15- and PPC specific (group II). In addition, a significant amount of group III IgG1 and IgG2 antibody is observed in the immune defective male response. In contrast to female B cells, immune defective male B cells produce a low IgM, IgA, and IgG3 memory response that is not composed of PC-specific (group I) antibodies; in fact, most of these antibodies arise from group III precursors and are not inhibited by either PC or PPC. PC-specific antibodies usually represent less than 25% of the anti-PC-KLH response in immune defective mice; however, these PC-specific antibodies are predominantly T15-. These data suggest that the Lyb-5-B cells in both normal and immune defective mice produce the T15-, IgG1, and IgG2 antibodies that dominate the secondary immune response to PC-KLH, and that the Lyb-5+ B cells produce the T15+, IgM, IgA, and IgG3 portion of the secondary response in normal mice. This hypothesis was confirmed by priming normal mice with the R36a strain of Streptococcus pneumoniae or with PC-Hy in saline. These forms of PC-antigen prime only the Lyb-5+ B cell subset. The adoptive transfer of these two B cell sources results in an anti-PC-KLH response that is T15 dominant and totally PC inhibitable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mucosal immunity to influenza without IgA: an IgA knockout mouse model

IgA knockout mice (IgA-/-) were generated by gene targeting and were used to determine the role of IgA in protection against mucosal infection by influenza and the value of immunization for preferential induction of secretory IgA. Aerosol challenge of naive IgA-/- mice and their wild-type IgA+/+ littermates with sublethal and lethal doses of influenza virus resulted in similar levels of pulmonary virus infection and mortality. Intranasal and i.p. immunization with influenza vaccine plus cholera toxin/cholera toxin B induced significant mucosal and serum influenza hemagglutinin-specific IgA Abs in IgA+/+ (but not IgA-/-) mice as well as IgG and IgM Abs in both IgA-/- and IgA+/+ mice; both exhibited similar levels of pulmonary and nasal virus replication and mortality following a lethal influenza virus challenge. Monoclonal anti-hemagglutinin IgG1, IgG2a, IgM, and polymeric IgA Abs were equally effective in preventing influenza virus infection in IgA-/- mice. These results indicate that IgA is not required for prevention of influenza virus infection and disease. Indeed, while mucosal immunization for selective induction of IgA against influenza may constitute a useful approach for control of influenza and other respiratory viral infections, strategies that stimulate other Igs in addition may be more desirable.     

Differentiation of B lineage cells from liver of neonatal mice: generation of immunoglobulin isotype diversity in vitro

The development and differentiation of B cells expressing different immunoglobulin (Ig) isotypes was studied in cultures of murine neonatal liver cells. Before culture, 5 to 15% of the liver cells were mu + pre-B cells; 1 to 3% had surface IgM and less than 0.1% had slgG. During 4 days in culture the number of pre-B cells declined, whereas the number of IgM B cells increased greater than 20-fold; IgG B cells also increased in number. Of the four subclasses, IgG3+ and IgG2b+ cells predominated, each representing 3 to 10% of the total B cells at day 4. IgG1+ and IgG2a+ cells were present in lower numbers, representing 1 to 5% and 0.3 to 2.5% of B cells, respectively. Most IgG+ cells also expressed sIgM. Only a minority (less than 10%) of the sIgM+ cells were sIgD+, and most sIgG+ cells were sIgD-. Few T cells were present in these cultures (less than 0.5% in newborn liver), and sIgG+ cells were generated in normal frequencies in cultures of cells from nude mice. The numbers of B cells expressing each IgG subclass were similar in cultures from athymic nu/nu mice, nu/+ heterozygous littermates, and normal BALB/c mice. Plasmablasts and plasma cells appeared over a 14-day culture interval, and these expressed cytoplasmic IgM, IgG3, IgG1, IgG2b, IgG2a, and IgA. Measurable amounts of the first four isotypes were detected in the culture supernatants by radioimmunoassay. These results indicate that neonatal B cells can undergo isotype switching in the absence of T cell help, and that the expression of sIgD may not be a prerequisite for cells to switch Ig isotypes.     

In vivo immune stimulation of mice by anti-μ antibodies: Generation of high serum levels of a low molecular weight IgM product

These studies show that anti-μ antibodies first injected into BALB/c mice as young adults exhibit a marked in vivo stimulatory effect, manifested by the appearance in circulation of large quantities of an aberrant IgM product. This stimulatory property extends to both rabbit and goat anti-μ antisera which have been raised against either myeloma or normal IgM but is not demonstrable for normal sera or antisera against γ or α heavy chains. The kinetics of appearance of this IgM product provide support for active generation upon stimulation, as opposed to immediate release of a preformed substance. Production of this form of IgM is accompanied by slight elevations in serum levels of IgG1 and normal IgM but unaltered levels of IgG2 and IgA. A molecular weight similar to that of IgG together with the demonstrated presence of light chains suggest that the aberrant product is likely a monomer of IgM. This stimulatory process appears to be thymus dependent because it cannot be induced in congenitally athymic (nude) mice unless they have been thymus reconstituted. Several test protocols involving adult-initiated anti-μ treatment resulted in immune responses to two thymus-dependent complex antigens (rabbit serum and sheep red blood cells) as well as generation of the aberrant IgM product in normal control mice but failed to render nude mice responsive to either antigen. It is thus apparent that although anti-μ antibodies can generate a stimulus in adult mice which results in production of an otherwise undetectable IgM product, the stimulus is not generally interpreted biologically as an immune “signal” complementary to antigen stimulation.     

Role of T-cell function in recovery from murine influenza infection.

Athymic (nude) mice and their normal littermates were intranasally inoculated with graded doses of A/WSN influenza virus. At a dose of 10³ EID₅₀, all mice survived the infection. In contrast, at a dose of 5 × 10⁴ EID₅₀, all mice died by 7 days. At intermediate doses of 5 × 10³ and 10⁴ EID₅₀, the nude mice were less resistant to the infection than their normal littermates, so that a higher proportion always died. Given a dose of 5 × 10³ EID₅₀, lung virus levels in both groups reached similar high levels by Day 5. Thereafter, virus levels in the normal mice rapidly fell so that no infectious virus could be detected by Day 18. In nude mice, the levels fell very slowly so that relatively high levels were still present at Day 18 in the surviving mice. At the height of the infection, high levels of cytotoxic T-cell activity was detected in the lungs of normal but not nude mice. Transfer to the nude mice of specific immune T cells raised from infected normal littermates enhanced survival of the nude mice and reduced the lung virus levels. Nude mice consistently showed a greater degree of lung consolidation than their normal littermates. Microscopically, the nude mouse lungs showed greater respiratory epithelial hyperplasia with minimal inflammatory cell infiltration in the foci of consolidation compared with their infected normal littermates. Under the conditions of these experiments, influenza-immune T cells seemed to inhibit rather than contribute to the generation of virus-mediated pulmonary pathology. The findings strongly suggest that T cells play an important positive role in the process of recovery from murine influenza infection.     

Cellular mechanisms in the protection against infection by Listeria monocytogenes in mice.

Listeria monocytogenes, in doses of 2-0 X 10(3) to 3-0 X 10(3) viable organisms, was injected into athymic nude mice, irradiated mice and mice treated with reticuloendothelial system-blocking agents. Viable counts on liver and spleen homogenates were made at intervals after infection. In both nude mice (nu/nu) and normal littermates (nu/+) of BALB/c background, the bacteria grew rapidly for 24 h but increased only slowly thereafter, to reach a plateau of about 10(5) per organ at 72 h. In nu/+ mice, the number of viable bacteria began to decrease after 6 to 9 days, with complete elimination by day 12. In nude mice, the number of Listeria remained at a stable level of approximately 10(5) per organ during the observation period of 21 days. In lethally irradiated nu/+ mice, bacteria grew progressively and extensively to reach 10(7) per spleen and 10(9) per liver by 72 h. Bacterial growth during the first 72 h was markedly enhanced by treatment with carbon particles, dextran sulphate 500 or silica. These enhancing effects were also observed in nude mice and in AKR, C3H/He and C57BL/6 animals. We conclude that both non-immune phagocytes and T cell-dependent mechanisms contribute to the resistance of mice to Listeria infection.     

Proliferation of hemolysin-plaque-forming cells in nude mice and normal littermates subjected to antigenic competition.

The increase of PFC per spleen and the development of hemolytic foci were examined to clarify the patterns of clonal expansion of B-lymphocytes in athymic nude mice (nu/nu) and normal littermates (nu/+) subjected to the procedure for antigenic competition between horse erythrocytes (HRBC) and sheep erythrocytes (SRBC). In normal littermates without pretreatment with HRBC, a small number of PFC and hemolytic foci of small size were detected 2-days after the challenge with SRBC. The number of PFC increased progressively from day 2 to day 4, and hemolytic foci increased in the number and size during the period. In nude mice, a small number of PVFC were detected on day 2 and the number increased only slightly from day 2 to day 4. No large hemolytic foci were detected during the period. In normal littermates subjected to the procedure for antigenic competition, the patterns of increase of PFC and development of hemolytic foci were similar to those in nude mice. In nude mice, the procedure for antigenic competition exerted almost no effect on the patterns.     

Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding

Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.     

IFN-gamma secreted by CD103+ dendritic cells leads to IgG generation in the mesenteric lymph node in the absence of vitamin A

Although the induction mechanism of secretory IgA has been well studied, that of IgG in the mucosal compartments is not well understood. In this study, vitamin A deficiency was convincingly shown to be associated with increased IgG in serum and intestinal fluid. We found increased numbers of IgG-secreting B cells in the lamina propria of the small intestine and mesenteric lymph node (MLN) of vitamin A-deficient (VAD) mice. Of note, IFN-γ secreted by MLN dendritic cells (DCs) was significantly augmented in VAD mice, unlike control mice, and CD103(+) DCs were the main subsets to secrete IFN-γ. The aberrant increase of IgG in VAD mice can be ascribable to IFN-γ, because IFN-γ(-/-) VAD mice have normal IgG levels and the addition of rIFN-γ increased IgG production by B cells cocultured with MLN DCs from IFN-γ(-/-) VAD mice. Oral feeding of antibiotics resulted in significant reduction of IgG in VAD mice, indicating a critical role for altered commensal bacteria for IgG class-switching recombination in the absence of vitamin A. Collectively, vitamin A deficiency provokes the generation of IFN-γ-secreting CD103(+) DCs, which may be a critical regulator for IgG generation in the MLN.     

Nematospiroides dubius: susceptibility to infection and the development of resistance in hypothymic (nude) BALB/c mice.

BALB/c-nu/nu mice and their intact nu/+ littermates are equally susceptible to infection with third-stage larvae of Nematospiroides dubius. Unlike their heterozygous littermates, however, the nu/nu mice are unable to form ganulomata in the intestinal wall and become only partially resistant to rechallenge. Following two or more infections, nu/nu mice maintain a high burden of adult intestinal worms, whereas worms are lost from immune nu/+ mice. Studies in T cell-injected nu/nu mice suggest that a full complement of T cells is needed to develop maximum resistance against the infective third-stage larvae and to expel adult worms. Measurement of serum immunoglobulin levels indicate that infected nu/+ mice have very high levels of IgG1 whereas the levels of IgG2a are reduced. In infected T cell-injected nu/nu mice, IgG1 levels increase with the number of T cells injected, whereas IgG2a levels are variable but always higher than in infected nu/+ mice.     

Taenia crassiceps: Serum and surface immunoglobulins in metacestode infections of mice

Serum levels of IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 were measured weekly for 8 weeks by radial immunodiffusion in pooled sera from female BALB/c and BDF1 mice with primary and secondary Taenia crassiceps infections and age-matched normal control mice of each strain. Although increases in levels of all immunoglobulin classes occurred during primary and secondary infections in both strains of mice, the only consistent changes common to both strains of mice were higher levels of IgG1 and IgG3 in early weeks of secondary infections as compared to primary infections, and high levels of IgG1 late in primary infections. High levels of IgG3 occurred late in primary infections in BDF1 mice but not in BALB/c mice. It was not possible to correlate increased levels of any one immunoglobulin class either with cytotoxic activity of early immune serum or with the onset of the cellular encapsulation response in secondary infections. IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 could be demonstrated on the surface of washed fixed larvae from long-term infected donor mice by the indirect fluorescent antibody method. Living T. crassiceps larvae were capable of shedding fluorescent label within 1 hr at room temperature, but not at 4 C after staining with either rabbit anti-T. crassiceps serum or rabbit anti-mouse immunoglobulin serum and fluorescein-conjugated goat anti-rabbit globulin.     

Echinostoma caproni: kinetics of IgM, IgA and IgG subclasses in the serum and intestine of experimentally infected rats and mice

The kinetics of specific immunoglobulin M, A and IgG subclasses against Echinostoma caproni (Trematoda: Echinostomatidae) were analyzed in serum and intestinal fluid of two host species (Wistar rats and ICR mice) in which the course of the infection markedly differs. In rats, the worms were rapidly expelled, whereas E. caproni evokes in mice long-lasting infection. The pattern of antibody responses in both serum and intestinal samples was different in each host species. Serum responses in mice were characterized by significant increases of IgM, IgA, total IgG, IgG1 and IgG3, but not IgG2a. In contrast, serum responses in rats showed elevated levels of IgM, probably in relation to thymus-independent antigens, and slight increases of total IgG, IgG1 and IgG2a. At the intestinal level, increases of IgM and IgA levels were observed in mice. In regard to IgG subclasses, increases in both IgG1 and IgG2a were detected. Later decreases to normal values in IgG2a were also detected. In rats, only increases in total IgG and IgG2a were found. According to our results the development of long-lasting E. caproni infections in mice appears to be associated with a dominance of Th2 responses at the systemic level and balanced Th1/Th2 responses at the local level, characterized by initial increases in IgG1 and IgG2a levels. In contrast, the worm expulsion appears to be related to increases in local IgG2a levels.     

Rapid blood clearance of mouse IgG2a and human IgG1 in many nude and nu/+ mouse strains is due to low IgG2a serum concentrations

 We reported previously that the blood clearance of injected mouse IgG2a was extremely rapid in many strains of nude and nu/+ mice. In an attempt to determine the cause of this phenomenon, the levels of endogenous IgG2a in the blood of these mice was assayed. It was found that the serum level of IgG2a was extremely low in many of these mice, below 50 μg/ml, which is 20–100 times lower than the expected normal value. Great heterogeneity between individual mice was observed in their blood level of IgG2a, and there was an excellent correlation between low blood IgG2a levels and rapid clearance of injected IgG2a. Thus, the blood IgG2a levels are so low that a novel, previously undescribed effect occurs, namely the rapid clearance of small amounts of injected IgG2a. The clearance is due primarily to binding sites in the spleen and liver. The low level of endogenous IgG2a is not due to the lack of a thymus, since it occurs in nu/+ as well as nude mice, but can probably be attributed to the very clean environment in which these mice are raised. In assays of sera from approximately 50 mouse strains, low IgG2a levels were found in all nude colonies and also in some normal mouse strains. Some nude mice displayed relatively normal IgG2a clearance rates despite having low levels of endogenous IgG2a. In repeated bleedings of individual mice, IgG2a levels were found to fluctuate greatly. A similar clearance effect was observed with a human IgG1 Ab injected into mice. This rapid clearance of injected IgG, of certain subclasses, represents a practical problem for many experiments in which antibodies are used for diagnosis or therapy, and several methods of circumventing the problem are discussed. Received: 15 August 1977 / Accepted: 14 October 1997     

A comparison of antibody titres in mouse uterine fluid after immunization by several routes, and the effect of the uterus on antibody titres in vaginal fluid

Measurements of specific antibody titres in uterine fluid of mice immunized by different routes indicated that two immunizations in the pelvic presacral space using aluminium hydroxide as adjuvant was a simple and effective way to elicit a significant IgA and IgG response. Higher IgA and IgG titres were produced in uterine fluid by subcutaneous immunization with antigen in Freund's complete adjuvant followed by intravaginal boosting without adjuvant, but this immunization involved both a toxic adjuvant and repeated applications of large doses of antigen in the vagina. Intragastric immunization produced an IgA response in the uterus but no IgG. Local intravaginal priming and boosting with large doses of antigen without adjuvant produced an IgA response in uterine fluid, but was less effective for IgG and was inefficient in terms of time and the amount of antigen used. Hysterectomy reduced the concentration of specific IgA in vaginal fluid of immunized mice to no more than 5% of normal, indicating that most of the IgA in vaginal fluid originates in the uterus. In contrast, IgG titres were not significantly different in hysterectomized and intact mice. IgA titres in vaginal fluid were at least partly restored to normal levels in sham-hysterectomized mice.     

Neonatally induced tolerance to soluble protein antigens. II. A role for the thymus in prolonging tolerance

Mice were rendered tolerant to bovine serum albumin (BSA) or fowl γ-globulin (FGG) by neonatal injection. Spleen and thymus cells from tolerant mice were able to suppress responsiveness of normal adult spleen cells, but only if tolerant donor mice were between the ages of 6 weeks and the age at which mice were no longer tolerant (10 weeks for BSA tolerance and 20 weeks for FGG tolerance). To determine whether T-cell-dependent suppression was obligatory for the maintenance of tolerance, neonatal nude and euthymic littermate mice were injected with tolerizing doses of FGG. FGG-specific B-cell tolerance in nude mice lasted until the mice were 8 weeks of age. In sharp contrast, B-cell tolerance in euthymic littermates lasted until 22 weeks of age. These results are consistent with a “fail-safe” role of T-cell-dependent immune suppression in the maintenance of tolerance.     

Serum steroid levels in intact and endocrine ablated BALB/c nude mice and their intact littermates

An investigation was made of the serum steroid levels found in intact and endocrine ablated nude mice of both sexes and in their intact homozygous littermates. The results showed that nude mice have a normal steroidogenesis, but with decreased levels of circulating steroids compared to those of the littermates. The efficacy of the endocrine ablations was confirmed by the reduction in serum oestrone following oophorectomy, and by the reduction in serum testosterone and progesterone following orchiectomy. The normal steroidogenesis in nude mice, and the similarities between mouse and man with regard to changes in serum steroids following oophorectomy and orchiectomy, support the usefulness of human tumor xenograft models for the study of hormone-tumor interactions.