Study of Dengue Virus Infection in SCID Mice Engrafted with Human K562 Cells

Here we report that severe combined immunodeficient (SCID) mice engrafted with human K562 cells (K562-SCID mice) can be used as an animal model to study dengue virus (DEN) infection. After intratumor injection into K562 cell masses of PL046, a Taiwanese DEN-2 human isolate, the K562-SCID mice showed neurological signs of paralysis and died at approximately 2 weeks postinfection. In addition to being detected in the tumor masses, high virus titers were detected in the peripheral blood and the brain tissues, indicating that DEN had replicated in the infected K562-SCID mice. In contrast, the SCID mice were resistant to DEN infection and the mock-infected K562-SCID mice survived for over 3 months. These data illustrate that DEN infection contributed directly to the deaths of the infected K562-SCID mice. Other serotypes of DEN were also used to infect the K562-SCID mice, and the mortality rates of the infected mice varied with different challenge strains, suggesting the existence of diverse degrees of virulence among DENs. To determine whether a neutralizing antibody against DEN in vitro was also protective in vivo, the K562-SCID mice were challenged with DEN-2 and received antibody administration at the same time or 1 day earlier. Our results revealed that the antibody-treated mice exhibited a reduction in mortality and a delay of paralysis onset after DEN infection. In contrast to K562-SCID, the persistently DEN-infected K562 cells generated in vitro invariably failed to be implanted in the mice. It seems that in the early stage of implantation, a gamma interferon activated, nitric oxide-mediated anti-DEN effect might play a role in the innate immunity against DEN-infected cells. The system described herein offers an opportunity to explore DEN replication in vivo and to test various antiviral protocols in infected hosts.     

Induction of tetravalent protective immunity against four dengue serotypes by the tandem domain III of the envelope protein

In the present study, the domain IIIs of all four dengue virus (DENV) serotypes were connected sequentially to construct the tandem domain III. The resulting DNA fragment was then cloned into pBAD/Topo ThioFusion plasmid. After induction, the Escherichia coli expression protein was purified and used to immunize BALB/c mice by subcutaneous route. The sera from mice immunized with the purified protein were confirmed to contain specific high antibody titers against DEN1, DEN2, and DEN4, and moderate antibody titer against DEN3. In suckling mouse model, 70% of the mice challenged with DEN1, DEN2, and DEN4 in combination with sera from mice immunized with the purified protein were protected, and 18% of the mice challenged with DEN3 in combination with the same sera were protected. Our data suggest that the tandem domain III of the envelope protein can be used as a potential tetravalent dengue vaccine based on a single antigen.     

The action of interleukin-4 on antigen-specific IgG1 and IgE production by interaction of in vivo primed B cells and carrier-specific cloned Th2 cells

The production of anti-trinitrophenyl (TNP) antibodies of different isotypes from in vivo primed B cells was studied using the plaque-forming cell method. It was shown that these B cells secrete anti-trinitrophenyl antibodies of different isotypes only in the presence of Th2 cells specific for keyhole limpet hemocyanin (KLH) and the hapten-carrier conjugate TNP-KLH. Lipopolysaccharide-stimulated primed B cells without cells from the Th2 clone did not produce anti-TNP-specific IgG1 or IgE antibodies even in the presence of the hapten-carrier antigen TNP-KLH. Supernatants from these Th2 clones cultured with antigen-presenting cells and the complete antigen were unable to activate primed B cells for antibody secretion. Cognate interaction between primed B cells and carrier-specific Th2 cells is a prerequisite for hapten-specific IgG1 or IgE production. Anti-IL-4 antibody inhibited secretion of anti-hapten IgE antibody. Therefore, for production of anti-hapten antibody of the IgE isotype IL-4 is also necessary.     

Antibody-mediated enhancement of infection by dengue virus of the P815 murine mastocytoma cell line

Dengue type 2 virus (DEN 2) could replicate only to a limited extent in a murine mastocytoma cell line, P815. The viral multiplication was enhanced 10- to 100-fold by mouse anti-DEN 2 antiserum or anti-DEN 2 type-specific monoclonal antibody diluted beyond their neutralizing titers. Cells incubated with virus-antibody mixtures changed morphologically, developing a mature mast cell-like appearance, 4-5 days after infection. The indirect fluorescent antibody technique showed that the enhancement of infection was caused by an increase in the number of DEN 2-infected cells. This is the first report that cells of mast cell lineage support dengue virus multiplication, and that virus production is enhanced in the presence of anti-dengue antibodies.     

Reaginic antibody formation in the mouse. X. Possible role of suppressor T cells in transient IgE antibody responses.

The kinetics of IgE antibody response to alum-absorbed dinitrophenyl derivatives of ovalbumin (DNP-OA) was dependent on the dose of immunogen. A persistent IgE antibody response was obtained when high responder BDF1 mice were immunized with a minimum (0.05 microgram) dose. An increase of the immunogen to 10 microgram depressed IgE antibody responses but enhanced IgG antibody responses of both hapten and carrier specificities. Determination of T helper cell activity and B memory cells after immunization with different doses of antigen indicated that minimum immunogen was favorable for developing helper activity, whereas 1 to 10 microgram immunogen were more favorable than a 0.05-microgram dose for developing both IgE and IgG B memory cells. Nevertheless, neither helper T cells nor B memory cells in the spleen explains a transient IgE antibody response to a high (10 microgram) dose of DNP-OA. Evidence was obtained that immunization with 10 microgram OA induced generation of antigen-specific suppressor T cells, which were not detectable after immunization with 0.05 microgram OA. Transfer of suppressor T cells to DNP-OA-primed mice depressed both anti-hapten and anti-carrier IgE antibody responses. The results suggested strongly that suppressor T cells are involved in a transient IgE antibody response to a high-dose immunogen.     

Suppression of IgE antibody production in SJL mice. V. Effect of irradiation and adult thymectomy on the suppression of IgE antibody production in SJL mice

Anti-DNP IgE antibody production was low and transient in SJL mice which were immunized with 1 microgram DNP-Nb and 1 mg A1(OH)3. The immunized SJL mice were irradiated (60-540 R) 1 day after challenge. A dose higher than 180 R induced enhancement of anti-DNP IgE antibody production as compared to nonirradiated control mice, suggesting the existence of irradiation-sensitive suppressor cells. Anti-DNP IgE antibody production was suppressed when immunized and irradiated SJL mice were injected with spleen cells from adult-thymectomized SJL mice. The donors of the spleen cells were thymectomized 2 or 4 months previously, and this suggests that the suppressor cells from unprimed mice are long-living T cells.     

IgE and IgG are synergistic in antigen-mediated release of thromboxane from human lung macrophages.

The mechanism of IgE-mediated release of thromboxane A2 from human lung macrophages has been studied using a monoclonal chimeric human/mouse IgE antibody and its specific antigen. The cells could be sensitized at 37 degrees C but not at 4 degrees C by incubation with IgE, and released a significant amount of thromboxane A2 (TXA2), measured as the stable hydrolysis product TXB2, in response to an anti-chimeric IgE antibody. In contrast, stimulation of IgE-sensitized macrophages with the specific antigen produced less than 10% of this response. A similar time course for the release of TXB2 and the formation of inositol monophosphate in the presence of LiCl was observed. Cleavage of the Fc domain of the anti-chimeric IgE antibody substantially eliminated its capacity to stimulate IgE-sensitized cells. However, the weak or undetectable response to chimeric IgE plus specific antigen was substantially potentiated by an antigen-specific chimeric IgG antibody. IgG-sensitized macrophages did not respond to antigen challenge by the release of TXB2. Preincubation of the cells with a monoclonal antibody against the low affinity receptor for IgE (Fc epsilon RII/CD23) did not prevent IgE sensitization. We conclude that cell-bound IgE antibody cannot induce the release of TXB2 but has fixed antigen which then must interact with specific IgG antibody and IgG receptors to induce mediator release.     

Regulation of antibody response in different immunoglobulin classes. II. Induction of in vitro IgE antibody response in murine spleen cells and demonstration of a possible involvement of distinct T-helper cells in IgE and IgG antibody responses.

In vitro induction of anti-DNP IgE as well as IgG1, IgG2a antibody responses was shown in murine spleen cell culture. Spleen cells primed three times with 1 mug of DNP-OA or DNP-Asc produced significant amounts of anti-DNP IgE as well as IgG antibodies by the in vitro stimulation with DNP-OA or DNP-Asc, respectively. Collaboration between DNP-primed B cells and carrier-primed T cells was required for the induction of both IgE and IgG antibodies with DNP-coupled T-dependent antigen. Carrier-specific T cells induced with a low dose of Asc (0.01 mug) showed helper function only on IgE antibody response, whereas T cells primed with a higher dose of Asc (10 mug) cooperated only with IgG-B cells. T cells primed with Asc in CFA showed helper function mainly on IgG antibody response but not on IgE antibody response. The result indicated the presence of a distinct population of T helper cells for IgE and IgG antibody responses. T-independent antigen (DNP-Ficoll) induced both anti-DNP IgE and IgG antibody responses in DNP-primed spleen cell population without the requirement of the collaboration of helper T cells.     

Reaginic antibody formation in the mouse. VII. Depression of the ongoing IgE antibody formation by suppressor T cells.

The ongoing IgE antibody formation against ovalbumin (OA) in high responder mice was depressed by i.v. injections of either native or urea-denatured ovalbumin (UD-OA). Adoptive transfer experiments to determine the helper function of spleen cells from the treated animals showed that helper function for both IgE and IgG antibody responses diminished after treatment. Evidence was obtained that treatment suppressed the expansion of IgE-G memory cells. When the same treatment with OA or UD-OA was given to OA-primed mice before the appearance of IgE antibody in their serum, OA-specific splenic suppressor T cells were demonstrable. Thus, the transfer of splenic T cells from treated mice into normal mice suppressed the primary IgE and IgG antibody responses of the recipeints to DNP-OA. It was also found that the transfer of the splenic T cells from UD-OA-treated mice into OA-primed mice depressed ongoing IgE antibody formation in the recipients. The results suggested strongly that the decrease of helper function and the depression of ongoing IgE antibody formation by repeated injections of UD-OA was caused by generation of antigen (OA)-specific suppressor T cells.     

Recombinant, live-attenuated tetravalent dengue virus vaccine formulations induce a balanced, broad, and protective neutralizing antibody response against each of the four serotypes in rhesus monkeys

Three tetravalent vaccine (TV) formulations of previously described monovalent dengue (DEN) virus vaccine candidates were compared to a tetravalent formulation of wild-type DEN viruses (T-wt) for replication in SCID mice transplanted with human liver cells (SCID-HuH-7) or for replication and immunogenicity in rhesus monkeys. TV-1 consists of recombinant DEN1, -2, -3, and -4, each with a 30-nucleotide deletion in the 3' untranslated region (Delta30). TV-2 consists of rDEN1Delta30, rDEN4Delta30, and two antigenic chimeric viruses, rDEN2/4Delta30 and rDEN3/4Delta30, both also bearing the Delta30 mutation. TV-3 consists of rDEN1Delta30, rDEN2Delta30, rDEN4Delta30, and a 10-fold higher dose of rDEN3/4Delta30. TV-1 and TV-2 were attenuated in SCID-HuH-7 mice with minimal interference in replication among the virus components. TV-1, -2, and -3 were attenuated in rhesus monkeys as measured by duration and peak of viremia. Each monkey immunized with TV-1 and TV-3 seroconverted to the four DEN components by day 28 with neutralization titers ranging from 1:52 to 1:273 and 1:59 to 1:144 for TV-1 and TV-3, respectively. TV-2 induced low antibody titers to DEN2 and DEN3, but a booster immunization after 4 months increased the neutralizing antibody titers to greater than 1:100 against each serotype and elicited broad neutralizing activity against 19 of 20 DEN subtypes. A single dose of TV-2 induced protection against wild-type DEN1, DEN3, and DEN4 challenge, but not DEN2. However, two doses of TV-2 or TV-3 induced protection against DEN2 challenge. Two tetravalent formulations, TV-2 and TV-3, possess properties of a successful DEN vaccine and can be considered for evaluation in clinical trials.     

Immunologic and physicochemical properties of enhancing soluble factors for IgG and IgE antibody responses.

Rabbits were immunized with dinitrophenyl-coupled Ascaris antigen (DNP-Asc) or ragweed antigen (DNP-Rag) included in aluminum hydroxide gel and their mesenteric lymph node cells were cultured for 24 hr in vitro in the presence of free homologous carrier. The cell-free supernatant thus obtained enhanced both IgG and IgE antihapten antibody responses of DNP-primed cells to DNP-heterologous carrier conjugate (DNP-keyhole limpet hemocyanin). Since the cell-free supernatant obtained from Rag-specific cells enhanced antibody response of hapten-primed cells raised by immunization with DNP-Asc, no carrier specificity was involved in the enhancement. It was found that treatment of primed cells with 10-5 M pactamycin suppressed the formation of the enhancing soluble factor, whereas the factor was readily formed in the presence of 2 mug/mol of cytosine arabinoside in the culture. The results indicated that cell proliferation was not required but de novo synthesis of protein was essential for the formation of soluble factor(s). The enhancing factor was not absorbed by either carrier-coated or anti-carrier antibody-coated immunosorbent. It was also found that the enhancing factor was formed by incubating primed cells with carrier-coated Sepharose. The cell-free supernatant containing no free carrier enhanced both IgG and IgE anti-hapten antibody responses. The activities of the cell-free supernatant to enhance IgG and IgE antibody responses were not absorbed by anti-Fab, anti-gamma-or anti-mu-chain antibody immunosorbent, indicating that the nonspecific enhancing factor did not possess immunoglobulin determinant. The cell-free supernatant was fractionated by sucrose density gradient ultracentrifugation and by gel filtration with three radiolabeled proteins, i.e., IgG, ovalbumin, and cytochrome C as markers. Enhancing activity for IgG antibody response was recovered in a fraction between ovalbumin peak (40,000 m.w.) and cytochrome C peak (20,000 m.w.). The activity for IgE antibody response was recovered in a fraction containing IgG marker (150,000 m.w.). By block electrophoresis, both activities were detected in beta globulin fraction. The results suggested that different T cell factors are involved in the IgG and IgE antibody responses.     

Monoclonal antibody specific for T cell-derived human IgE binding factors

A B cell hybridoma secreting monoclonal antibody against human IgE binding factors was obtained by immunization of BALB/c mice with partially purified IgE binding factors, and fusion of their spleen cells with SP-2/0-AG14 cells. The monoclonal antibody bound all of the 60,000, 30,000, and 15,000 dalton IgE binding factors from two T cell hybridomas and those from activated T cells of a normal individual. The antibody bound both IgE-potentiating factors, which had affinity for lentil lectin, and IgE-suppressive factors, which had affinity for peanut agglutinin. However, the monoclonal anti-IgE-binding factor bound neither Fc epsilon R on RPMI 8866 cells nor IgE binding factors from the B lymphoblastoid cells. A monoclonal antibody against Fc epsilon R on B cells (H107) bound the 60,000 and 30,000 dalton IgE binding factors from both T cell hybridomas and RPMI 8866 cells but did not bind the 15,000 dalton IgE binding factors from either T cells or B cells. The results indicate that T cell-derived IgE binding factors have a unique antigenic determinant that is lacking in both Fc epsilon R on B cells and B cell-derived IgE binding factors. The anti-IgE binding factor and anti-Fc epsilon R monoclonal antibodies both failed to stain cell surface components of IgE binding factor-producing T cell hybridomas. However, both antibodies induced the T cell hybridoma to form IgE binding factors. The results suggest that the T cell hybridomas bear low numbers of Fc epsilon R that share antigenic determinants with IgE binding factors secreted from the cells.     

Elicitation of antigen-induced primary and secondary murine IgE antibody responses in vitro

Described herein are methods for eliciting and quantitating primary and secondary murine IgE antibody responses in vitro, and the important role of antigen concentration in determining the level of IgE produced during an immune response. The methods for quantitating IgE antibody levels in culture supernatant fluids and in serum by ELISA are presented in detail. The specificity of such methods was confirmed in that (1) no other isotype of antibody registered in the IgE-ELISA, and (2) parallel determinations of IgE antibody concentrations could be obtained by independent analysis using Fc epsilon RI-dependent basophil degranulation. We examined various parameters of cell donor immunization and lymphoid cell culture which allow for optimal in vitro primary and secondary IgE responses. High relative antigen doses result in diminished IgE antibody responses in experimental animals, a finding confirmed herein. High antigen concentrations in vitro also result in relative suppression of IgE antibody synthesis. This was also true for in vitro production of IgG1 and IgA antibodies. Conversely, IgM and IgG2a responses were elicited at both low and high antigen concentrations; IgG2b and IgG3 were not produced under the conditions of priming and culture used herein. Finally, production of IgE in vitro depended on the presence of carrier-primed CD4+ T cells and hapten-specific B cells. Generation of maximal IgE antibody secretion, and hence elicitation of an allergic reaction, is thus dependent on the amount of antigen acting as stimulant for the immune response.     

IgE formation in the rat following infection with Nippostrongylus brasiliensis: I. Proliferation and differentiation of IgE-bearing cells

Sprague-Dawley rats were infected with Nippostrongylus brasiliensis larvae, and IgE formation was studied. Before infection, the serum IgE level was less than 0.4 μg/ml. The IgE level began to increase from the 10th day of infection, reached its maximum (50–100 μg/ml) at the 14th day and gradually declined. Reinfection of the rats resulted in an increase of the serum IgE level within 7 days. The IgE antibody response to N. brasiliensis antigens did not parallel the increase of IgE synthesis. In most animals, the antibody became detectable in the serum at the 21st day when the total IgE level already began to decrease. The animals showed a secondary IgE antibody response upon reinfection. Both mesenteric lymph nodes and spleen cell suspensions were examined for the presence of IgE-bearing cells (IgE-B cells) and IgE-forming cells by fluorescent antibody technique. The IgE-bearing lymphocytes became detectable in the mesenteric lymph nodes and spleen at the 8th day of infection. The proportion of the IgE-B cells in nonadherent cell population gradually increased and reached maximum at the 14th day; about 20% of immunoglobulin (Ig)-bearing cells in the mesenteric lymph nodes and 10% of Ig-bearing cells in spleen bore IgE on their surface. Evidence was obtained that these lymphocytes synthesized IgE. The IgE-forming cells were detected in both mesenteric lymph nodes and spleen of the infected animals. The number of IgE-forming cells was greater in the mesenteric lymph nodes than in spleen, indicating that the regional lymph nodes are the major source of serum IgE in the N. brasiliensis-infected animals.     

Demonstration of phosphorylcholine-specific IgE B cells in CBA/N mice.

CBA/N mice, which did not make anti-PC IgM or IgG antibody against PC-conjugated T-dependent or T-independent antigens, produced IgE antibody to PC-determinant when they were immunized with PC-KLH. PC-specificity of IgE antibody produced in CBA/N mice was determined by inhibition of PCA reaction with free PC-hapten or C-polysaccharide or by absorption of reaginic activity in the serum with C-polysaccharide. The presence of T15 idiotype on anti-PC IgE antibody produced in CBA/N x BALB/c F1 males also showed that anti-PC IgE antibody in defective mice was PC-specific. The results suggest that PC-specific B epsilon cells may belong to a subpopulation distinct from PC-specific precursors for IgM and IgG responses.     

Derivation and characterization of a dengue type 1 host range-restricted mutant virus that is attenuated and highly immunogenic in monkeys

We recently described the derivation of a dengue serotype 2 virus (DEN2mutF) that exhibited a host range-restricted phenotype; it was severely impaired for replication in cultured mosquito cells (C6/36 cells). DEN2mutF virus had selected mutations in genomic sequences predicted to form a 3' stem-loop structure (3'-SL) that is conserved among all flavivirus species. The 3'-SL constitutes the downstream terminal similar95 nucleotides of the 3' noncoding region in flavivirus RNA. Here we report the introduction of these same mutational changes into the analogous region of an infectious DNA derived from the genome of a human-virulent dengue serotype 1 virus (DEN1), strain Western Pacific (DEN1WP). The resulting DEN1 mutant (DEN1mutF) exhibited a host range-restricted phenotype similar to that of DEN2mutF virus. DEN1mutF virus was attenuated in a monkey model for dengue infection in which viremia is taken as a correlate of human virulence. In spite of the markedly reduced levels of viremia that it induced in monkeys compared to DEN1WP, DEN1mutF was highly immunogenic. In addition, DEN1mutF-immunized monkeys retained high levels of neutralizing antibodies in serum and were protected from challenge with high doses of the DEN1WP parent for as long as 17 months after the single immunizing dose. Phenotypic revertants of DEN1mutF and DEN2mutF were each detected after a total of 24 days in C6/36 cell cultures. Complete nucleotide sequence analysis of DEN1mutF RNA and that of a revertant virus, DEN1mutFRev, revealed that (i) the DEN1mutF genome contained no additional mutations upstream from the 3'-SL compared to the DEN1WP parent genome and (ii) the DEN1mutFRev genome contained de novo mutations, consistent with our previous hypothesis that the defect in DEN2mutF replication in C6/36 cells was at the level of RNA replication. A strategy for the development of a tetravalent dengue vaccine is discussed.     

登革2型病毒结合血管内皮细胞分子机制的初步研究

以ECV304细胞为对象分析登革病毒感染血管内皮细胞的机制。2型登革病毒(DEN2)吸附后微量蚀斑法测定ECV304细胞上清释放的病毒滴度,证实该细胞对DEN2感染有一定的敏感性。机械刮取或胰蛋白酶消化法收集ECV304细胞分离膜蛋白,SDS—PAGE见胰酶处理样品缺失一43 kDa的膜蛋白。将ECV304细胞膜蛋白与^35S—Met标记的DEN2进行病毒重叠蛋白结合试验(VOPBA),有29、34和43kDa的3种膜蛋白可与DV结合,其中29 kDa的蛋白对胰酶耐受。培养的ECV304细胞中加入重组E蛋白(rEgp)对DEN2吸附进行阻断试验,微量蚀斑法与间接免疫荧光表明rEgp抑制DEN2感染该细胞。VOPBA中rEgp可阻断病毒与细胞膜蛋白的结合。结果表明ECV304细胞表面可能存在29、34、43 kDa的3种与DEN2结合的相关蛋白,DEN2E蛋白可直接介导DV感染血管内皮细胞。     

Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells

Trastuzumab (Herceptin), a humanized IgG1 antibody raised against the human epidermal growth factor receptor 2 (HER2/neu), is the main antibody in clinical use against breast cancer. Pre-clinical evidence and clinical studies indicate that trastuzumab employs several anti-tumour mechanisms that most likely contribute to enhanced survival of patients with HER2/neu-positive breast carcinomas. New strategies are aimed at improving antibody-based therapeutics like trastuzumab, e.g. by enhancing antibody-mediated effector function mechanisms. Based on our previous findings that a chimaeric ovarian tumour antigen-specific IgE antibody showed greater efficacy in tumour cell killing, compared to the corresponding IgG1 antibody, we have produced an IgE homologue of trastuzumab. Trastuzumab IgE was engineered with the same light- and heavy-chain variable-regions as trastuzumab, but with an epsilon in place of the gamma-1 heavy-chain constant region. We describe the physical characterisation and ligand binding properties of the trastuzumab IgE and elucidate its potential anti-tumour activities in functional assays. Both trastuzumab and trastuzumab IgE can activate monocytic cells to kill tumour cells, but they operate by different mechanisms: trastuzumab functions in antibody-dependent cell-mediated phagocytosis (ADCP), whereas trastuzumab IgE functions in antibody-dependent cell-mediated cytotoxicity (ADCC). Trastuzumab IgE, incubated with mast cells and HER2/neu-expressing tumour cells, triggers mast cell degranulation, recruiting against cancer cells a potent immune response, characteristic of allergic reactions. Finally, in viability assays both antibodies mediate comparable levels of tumour cell growth arrest. These functional characteristics of trastuzumab IgE, some distinct from those of trastuzumab, indicate its potential to complement or improve upon the existing clinical benefits of trastuzumab.     

The role of L3T4+ and Lyt-2+ T cells in the IgE response and immunity to Nippostrongylus brasiliensis

The role of L3T4+ and Lyt-2+ T cells in protective immunity to Nippostrongylus brasiliensis (Nb) was studied in BALB/c mice that were depleted of either the L3T4+ or Lyt-2+ T cell population by injection with rat mAb specific for the appropriate determinant. Host responses to Nb infection including spontaneous elimination of adult worms, development of intestinal mucosal mast cell hyperplasia and the generation of a polyclonal IgE response were all completely blocked by 0.5 mg anti-L3T4 antibody administered simultaneously with Nb inoculation. However, administration of 0.5 mg of anti-Lyt-2 antibody at the same time and 7 days after inoculation with Nb had no effect on any of these responses. Injection of anti-L3T4 antibody as late as 9 days after Nb inoculation interfered with spontaneous cure of Nb infection and anti-L3T4 antibody injection 11 days after Nb inoculation inhibited serum IgE levels measured on day 13 by 50%. In addition, administration of anti-L3T4 antibody at the time of the peak serum IgE response, 13 days after Nb inoculation, accelerated the decline in serum IgE levels. Injection of previously Nb-infected mice with anti-L3T4 antibody at the time of a second Nb inoculation prevented the development of a secondary IgE response but did not affect immunity to Nb infection based on finding no adult worms in the intestines of these mice. These data indicate that 1) L3T4+ T cells are required for spontaneous cure of Nb infection, development of intestinal mucosal mast cell hyperplasia, and the generation and persistence of an IgE response during primary infection with Nb and 2) L3T4+ T cells are required for a considerable time after inoculation for optimal development of these responses. However, L3T4+ T cells are not required for all protective responses in immune mice. In contrast, our data indicate that considerable depletion of the Lyt-2+ T cell population has no significant effect on either worm expulsion or the generation of serum IgE responses.     

Monoclonal antibody (H107) inhibiting IgE binding to Fc epsilon R(+) human lymphocytes

A hybridoma-producing monoclonal antibody blocking the binding of human IgE to lymphocytes Fc receptor (Fc epsilon R) was established by the fusion of murine myeloma cells. P3X63.653.Ag8, with BALB/c spleen cells immunized with Fc epsilon R(+) human B lymphoblastoid cell line cells, RPMI1788. A clone of the hybridoma (H107) produced a monoclonal IgG2b antibody that inhibited the rosette formation of Fc epsilon R(+) human B lymphoblastoid cell line cells (RPMI1788, RPMI8866, CESS, Dakiki, and IM9) with fixed ox red blood cells (ORBC) conjugated with human IgE (IgE-ORBC). In contrast, the rosette formation with IgG-conjugated ORBC (IgG-ORBC) on Fc gamma R(+), Fc epsilon R(-) Daudi cells were not affected by the H107 antibodies. A close association of Fc epsilon R and the antigenic determinant recognized by H107 antibody was suggested by the following results. First, the bindings of 125I-labeled IgE (125I-IgE) or 125I-labeled H107 IgG2b antibody (125I-H107) to RPMI8866 cells were inhibited by cold human IgE and H107 IgG2b but not by other classes of human Ig (IgA and IgG), MPC11 IgG2b, or unrelated monoclonal antibodies. Second, H107 antibody reacted with Fc epsilon R(+) B cell lines but not with Fc epsilon R(-) B cell lines as determined by an indirect immunofluorescence. Third, Fc epsilon R(+) cells were depleted by the incubation in the dish coated with H107 antibody or IgE but not in the dish coated with unrelated antibodies. Finally, there was a correlation between the increase of Fc epsilon R(+) cells and that of H107(+) cells in the peripheral blood lymphocytes of the patients with atopic dermatitis. The surface antigens on Fc epsilon R(+) RPMI8866 cells recognized by H107 antibodies had the molecular size of 45,000 as determined by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.