Monoclonal antibodies against a rat leucocyte antigen block antigen-induced T-cell responses via an effect on accessory cells.

The MRC OX-45 and OX-46 mouse monoclonal antibodies recognize a rat cell surface glycoprotein of 45,000 MW that is present on a wide variety of haematopoietic cells and on endothelial cells. MRC OX-45 IgG or F(ab')2 blocked the primary mixed lymphocyte response (MLR) and the secondary response of T lymphocytes to the soluble antigen DNP-BGG. In contrast, the antibodies had no effect on the cytotoxic activity of specific (CTL) or non-specific (NK) killer cells or on proliferative responses stimulated by lectins or oxidative mitogenesis. The inhibitory effect was at the level of stimulator cells rather than responders since mouse anti-rat xenogeneic MLRs were inhibited but rat anti-mouse responses were unaffected. However, the effect was not a direct one because inhibition was seen when irradiated spleen cells were used as stimulators but not when cell populations highly enriched for dendritic cells were used. In the latter case, inhibition potentiated by antibody could be restored if a peritoneal cell population enriched for macrophages was added back to the cultures. The inhibitory effects of these monoclonal antibodies seem most likely to be due to potentiation of nonspecific suppression by macrophages.     

Obtention of a heterohybridoma for production of type IgM monoclonal antibodies against the D antigen of the Rh system

The objective was to obtain a heterohybridoma capable of producing a monoclonal antibody with IgM type anti-D specificity (Rh system), that could be used as a reactive for hemoclasification. Mononuclear cells (MNC) were extracted from a blood sample of a highly sensitized woman, five days after giving birth to an Rh positive child. These were then transformed with the culture supernatant (CSN) of B05.8 cells, rich in Epstein Barr virus (EBV). Once transformed and in exponential growth, they were fused with K6H6/B5 line cells using PEG 4.000 as a fusing agent in a 1:1 proportion. After fusion, they were seeded in culture plates in order to evaluate the formation of hybrids and the secretion of specific antibodies in the CSN of each well. The efficiency of the fusion was 1.8 x 10(-6), making it possible to obtain an anti-D IgM producing clone, which we named BMS-9. This clone could be maintained in constant culture for three months, producing antibodies in a concentration of 4 microg/mL in de CSN. It was also possible to obtain antibodies with an Artificial Capilar System (ACS) reaching a concentration of 24 microg/mL. Potency was determined using Ror cells. In CSN at immediate centrifugation (IC): 1 x 32, score 52; 15' from incubation at room temperature (RT): 1 x 1,024 score 105. With that ACS product at IC: 1 x 32 score 54; 15' from incubation at RT: 1 x 8.192 score 136; and a 37 degrees C: 1 x 8,192 score 136. Reactivity was detected with red cells D(IIIa), D(IV), D(Va), D(VI) type IV, D(VII), DFR, DNU, STEM+, DAR and DAU. There was no reactivity with red cells D(IIIc), DI(Va), D(V) type II, D(VI) types I, II y III, Ro(HAR), DOL and weak D type II. During field study, no false negative or false positive reactions were detected. A stable heterohybridoma was obtained, producer of IgM type anti-D, with enough qualities to be used in blood typing. Given the excellent qualities of the antibody, we are evaluating dilution media and the addition of type IgG antibodies in order to manufacture a reactive for use in hemoclassification.     

MRC OX-52: a rat T-cell antigen.

A mouse monoclonal antibody MRC OX-52 has been shown to label rat T lymphocytes and thymocytes. The molecule precipitated by this antibody from both thymocytes and T lymphocytes had a two-chain structure of 120,000 MW and 95,000 MW.     

Induction of rat secretory IgA antibodies against cholera toxin by a synthetic peptide.

There is accumulating evidence concerning the possible importance of secretory IgA antibodies in defence mechanisms against infections of the gastrointestinal tract, including cholera. Intestinal IgA antibodies are also thought to play a major role in protection against the diarrhoeogenic effects of cholera toxin. We therefore attempted to induce secretory IgA antibodies towards a reactive synthetic peptide from the cholera toxin B subunit sequence. We report that rat biliary secretory IgA antibodies against the CTP3 peptide (residues 50-64 of the B subunit) were obtained by three intra-Peyer's patch immunizations, at 2-week intervals, with CTP3 conjugated to tetanus toxoid in complete Freund's adjuvant. Purified secretory IgA fractions from bile of such immunized rats reacted with the carrier toxoid, but also with the CTP3 peptide, and with the native cholera toxin, they also partially neutralized its biological activity, as assayed by inhibition of in vitro cholera toxin-induced cAMP production in mouse thymocytes.     

Effect of oestradiol dipropionate on the immune system of the pigeon,

Influence of female sex hormone, oestradiol dipropionate on the humoral and cell-mediated immune (CMI) responses of pigeon, Columba livia of different age groups and of both sexes were studied. The pigeons were administered with the hormone, immunized with sheep red blood cells (SRBC) and rosette forming cell (RFC), haemagglutinin (HA) and migration inhibition (MI) assays carried out on different days post-immunization. The hormone showed a differential effect: while the RFC and HA were enhanced in 1-month old hormone administered female pigeons, they were normal in 3 or 4 month old birds. The MI response was however depressed in both age groups, and the thymus and bursa were involuted. The hormonal influence was marginal in males: the hormone administered 1-month old male showed an elevated RFC level. The three month old males showed a depressed MI. Among the lymphoid organs, the bursal weight was enhanced. It is thus evident that the influence of female sex hormone oestradiol dipropionate on the immune system depends on the age and sex of the pigeons, persumably because of the endogenous hormonal levels.     

Human monoclonal IgM antibodies from foetal B-cell hybridomas directed against a surface antigen on human tumour cells.

In order to assess the existence of B lymphocytes capable of producing anti-tumour antibodies in non-tumour-bearing individuals, human lymphocytes derived from foetuses and adults were fused with the heteromyeloma cell line CB-F7. By indirect immunofluorescence, 29 out of 4,472 IgM-producing hybridomas (from 8 foetuses and 8 adults) were shown to produce antibodies which bind to colon carcinoma lines Colo205 and SW620, Raji lymphoma cells and small cell carcinoma of the lung. In vitro growth of tumour cells recognized by these antibodies was inhibited. The antibodies also mediated complement-dependent cytotoxicity. All antibodies tested recognized a cell surface molecule of 55 kDa. Southern blot hybridization analysis of hybridoma DNA with a human JH probe showed that the hybridomas were derived from clonally unrelated B cells. These results demonstrate that human foetal and adult B cells from non-tumour-bearing individuals are able to produce IgM antibodies recognizing defined cell surface molecules expressed on some tumour cells.     

T-cell enforced invariance of the antibody repertoire in the immune response against a bacterial carbohydrate antigen

The humoral response against the bacterial polysaccharide antigen alpha(1-->3) dextran (Dex) is controlled by J558 idiotype-(Id) specific T cells. These T cells of which the cell clone 178-4 Ts is a representative by all relevant criteria, recognize J558 Id-bearing B cells in an I-Ed-restricted manner. Costimulation via CD28/B7-1 but not via CD40/CD40L leads to T-cell activation. These T cells do not only suppress B cells producing the immunoglobulin (Ig)G3 isotype but also support the survival and clonal expansion of J558 Id positive B cells both in vivo and in vitro. This T-cell mediated dominance of the J558 idiotype limits the appearance of antibodies carrying other more diverse idiotypes which appear in immunized BALB/c nu/nu mice where no regulatory T cells occur. This T-cell mediated antibody invariance could be a strategy of the immune system responding to highly conserved antigens like polysaccharides, different from those against protein antigens, where diversity is assumed to be the basis for a successful response.     

AIDS: an immune response against the immune system. Role of a precise tridimensional molecular mimicry

This study describes some of the expected autoimmune consequences of a recently described virus-host molecular mimicry in patients infected with human immunodeficiency virus (HIV).Some physical and structural analogies have been recently identified between some precise antigenic sites of the trimeric ectodomain of the transmembrane envelope protein of AIDS-associated lentiviruses (HIV, Simian immunodeficiency virus and feline immunodeficiency virus) and some precise and crucial interleukin 2 (IL-2) antigenic sites of the corresponding infected species (man, monkey, cat). As expected, HIV-positive sera recognize human IL-2, and a cross-reactivity was found between the structurally and physically analogous antigenic sites of GP41 (HIV1) and human IL-2. The possible impact on AIDS-associated disorders of this tridimensional GP41 (HIV1)/human IL-2 molecular mimicry is suggested.     

Monoclonal antibodies against Neisseria gonorrhoeae: production of antibodies directed against a strain-specific cell surface antigen.

Hybridomas secreting monoclonal antibodies against an apparent strain-specific cell surface antigen of Neisseria gonorrhoeae were produced. Spleen cells from BALB/c mice immunized with whole gonococci were fused with mouse myeloma cell line Sp2/0, and hybrid cells were selected in culture. One hybridoma that secreted antibodies reactive with the immunizing strain was cloned by limiting dilution to obtain cell lines secreting monoclonal antibodies. These antibodies reacted with purified outer membranes from the immunizing strain as well as with whole gonococci. Binding of antibodies to whole gonococci was highly strain specific, with most gonococcal strains showing less than 1% of the binding with the immunizing strain. Antibodies did not bind to the other Neisseria species tested. Binding of monoclonal antibodies to whole gonococci of the immunizing strain was not dependent on state of piliation. The extent of antibody binding did vary in different colonial variants of the immunizing strain. Antibody bound to cells from colonies that were transparent or of intermediate opacity, but did not bind to cells from deeply opaque colony variants.     

Characterization of immune complexes containing cytomegalovirus-specific IgM antibodies following a kidney graft.

In order to demonstrate the viral specificity of IgM-containing immune complexes (IgM-CIC) detected by a C1q assay in renal allograft recipients developing a CMV infection, a technique is described allowing: 1) the dissociation of IgM-CIC by action of an acid buffer, and 2) the characterization of the viral specificity of IgM antibodies released by this treatment. This step was performed by ELISA and Western Blot. When technique was applied to the follow-up of a renal allograft recipient developing a recurrent CMV infection within 2 months post-graft, it was found that the IgM-CIC detected on the day of the graft were not CMV-specific, whereas the IgM-CIC detected during the second month after transplantation contained CMV-specific IgM antibodies. These CMV-specific IgM-CIC were detected as early as the urinary viral excretion. It was shown by Western Blot analysis that these IgM antibodies reacted with a 45-47 kDa viral polypeptide which is a viral target for specific humoral response at the early phase of CMV infection.     

Hormonal influence on the secretory immune system of the eye: androgen control of secretory component production by the rat exorbital gland

Androgens are known to regulate the level of secretory component (SC) in tears of male rats. The purpose of the present study was to explore the underlying mechanism of this hormone action by (i) identifying the ocular tissue(s) involved in SC production; and (ii) determining whether androgens increase SC production by this tissue. We also examined whether androgen administration influenced the concentration of SC in tears of female rats. Ocular tissues from adult Sprague-Dawley rats were cultured in the presence or absence of cycloheximide in the incubation medium. Secretory component in the culture media was measured by an RIA which detects primarily free SC. Analysis of media obtained after incubation of exorbital (lacrimal) glands, 'lid' tissues, globes, and Harderian glands revealed that only exorbital glands released substantial amounts of SC. This exorbital gland production of SC, which was significantly greater in tissues from male rats, as compared to those of female rats, was reduced by approximately 50% when cycloheximide was present in the culture medium. To determine whether SC production by exorbital glands was influenced by androgens, orchiectomized glands was influenced by androgens, orchiectomized rats were administered either saline or testosterone (2.0 mg/day for 4 days), and exorbital glands were cultured 24 hr after the last injection. Testosterone treatment in vivo induced a significant, cycloheximide-sensitive increase in SC production in vitro, compared to the glandular SC output of saline-injected controls. It is interesting that similar androgen treatment of ovariectomized females also resulted in elevated tear SC concentrations and enhanced output of SC by their exorbital glands in vitro. These findings indicate that the exorbital gland is primarily responsible for SC production in the rat eye and that androgens may modulate the synthesis of SC in this gland.     

Evidence for long-term memory of the mucosal immune system: milk secretory immunoglobulin A against Shigella lipopolysaccharides.

Although the common mucosal immune system has generally been considered to have only short-term memory, recent data suggest that long-term memory exists for Shigella virulence plasmid antigens. Because such antigens might cross-react with environmental antigens, we investigated milk for the persistence of antibodies to the specific Shigella lipopolysaccharide (LPS) antigens. Enzyme-linked immunosorbent assays to detect secretory immunoglobulin A (sIgA) against Shigella flexneri and Shigella sonnei LPS in milk samples were developed; 15 random milk samples tested on different days correlated from one day to the next (P = 0.0001). Of 18 Mexican mothers, 18 (100%) had one or more milk samples positive for anti-S. flexneri LPS, 14 (78%) had one or more milk samples positive for anti-S. sonnei LPS, and 14 (78%) had one or more milk samples positive for both. Of 27 Houston mothers, 16 (59%) had one or more milk samples positive for anti-S. flexneri LPS, 7 (26%) had one or more milk samples positive for anti-S. sonnei LPS, and 5 (19%) had one or more milk samples positive for both. Mexican mothers were significantly more likely than Houston mothers to have at least one sample with a positive titer for anti-S. flexneri LPS (P less than 0.02) and at least one sample with a positive titer for anti-S. sonnei LPS (P less than 0.002). Although the Houston women had a lower rate of titer positivity for both Shigella species, the rate was too high to be consistent with short-lived mucosal immunity. It is unlikely that 18 of the 27 Houston women had shigellosis during or just prior to lactation. The data suggest that there exists a long-term hormonally driven memory in the secretory immune system for Shigella spp.     

Role of secretory antibodies in the defence against infections

Adaptive immunity mediated by secretory antibodies is important in the defence against mucosal infections. Specific secretory immunoglobulin A (SIgA) can inhibit initial pathogen colonization by performing immune exclusion both on the mucosal surface and within virus-infected secretory epithelial cells without causing tissue damage. Resistance against toxin-producing bacteria such as Vibrio cholerae appears to be particularly dependent on SIgA antibodies. Like natural infections, live topical vaccines or adequate combinations of inactivated vaccines and mucosal adjuvants give rise not only to SIgA antibodies, but also to long-standing serum IgG and IgA responses. The intranasal route of vaccine application could be particularly attractive to achieve this result, but only if successful stimulation is obtained without the use of toxic adjuvants. The degree of protection after vaccination may range from complete inhibition of reinfection to reduction of symptoms. In this scenario it is generally difficult to determine unequivocally the relative importance of SIgA versus serum antibodies. However, infection models in knockout mice strongly support the notion that SIgA exerts a decisive role in protection and cross-protection against a variety of infectious agents.     

Anti-idiotypic immune responses against adjuvant-free isologous IgM monoclonal antibodies and their augmentation by complex formation between IgM and albumin in bovine serum.

In previous studies we demonstrated that the hypermutated isologous myeloma protein MOPC 315 (isotype IgA; λ2) is recognized by T helper cells like an ordinary foreign protein antigen. To what extent can an immune system recognize and respond to V domains from the primary (pre-immune) repertoire? To study this question we made 21 BALB/c hybridoma anti-2, 4, 6 trinitrophenyl monoclonal antibodies (mAb) of IgM; λ isotype. All mAb purified from supernatants containing fetal bovine serum had formed spontaneous complexes with bovine serum albumin possibly by way of disulfide interchange. Twenty of twenty-one mAb from this source elicited IgG₁ anti-idiotypic (Id) Ab when given as a single adjuvant-free dose of 200 μg. For 12 of them even 10 μg was sufficient. This indicated that BSA augmented the anti-Id responses by a carrier effect. Three of the mAb were therefore purified from ascites fluid and from serum-free medium. Only one of them then induced humoral anti-Id responses in BALB/c mice when given as a single adjuvant-free dose of 100 μg. The other two became immunogenic when emulsified in Freund's complete adjuvant. The results indicate that some IgM mAb exist whose Id determinants alone can elicit substantial anti-Id responses because they are recognized like ordinary foreign protein antigens. Since albumin in fetal bovine serum forms complexes with IgM and greatly augments its immunogenicity, serum-free medium should be used for production of human or humanized therapeutic IgM mAb. A possible role for Id of IgM Ab as cardinal autoantigens is discussed.     

Effect of adsorbents on IgM and IgG measles antibodies.

Sera from rabbits immunized with L-16 measles virus absorded with monkey blood cells; kaolin and blood cells; and MnCl2 and heparin were examined in haemagglutination inhibition (HI) and neutralization tests. Kaolin and MnCl2 adsorbed primarily HI IgM antibodies from the early immunization period. The adsorbents used had no influence on HI and neutralization IgG antibodies. Human convalescent serum gave similar results, i.e. only IgG antibodies were found and they were not affected by kaolin and MnCl2 with heparin.     

CD8(+) T-cell priming against a nonsecreted Listeria monocytogenes antigen is independent of the antimicrobial activities of gamma interferon

Sublethal infection of mice with recombinant Listeria monocytogenes expressing a model epitope in either secreted or nonsecreted form results in similar CD8(+) T-cell priming. Since nonsecreted bacterial proteins have no obvious access to the endogenous major histocompatibility complex (MHC) class I presentation pathway, presentation of these antigens requires destruction of the bacterium to reveal the nonsecreted molecules to an exogenous MHC class I presentation pathway. Gamma interferon (IFN-gamma), a cytokine made by multiple cell types in response to L. monocytogenes infection, could be required for exogenous presentation of nonsecreted bacterial antigens via its capacity to upregulate the expression of molecules involved in antigen presentation, its capacity to activate macrophages to kill bacteria to expose nonsecreted molecules or both. IFN-gamma knockout (KO) mice were used to address the requirement for IFN-gamma in CD8(+) T-cell priming against (i) a model exogenous antigen and (ii) secreted and nonsecreted L. monocytogenes antigens. We demonstrate that IFN-gamma KO mice are capable of cross-presenting the model exogenous antigen ovalbumin to prime CD8(+) T-cell responses that are only slightly weaker than that in wild-type (WT) mice. Despite their extreme susceptibility to primary L. monocytogenes infection, previously immunized and naive IFN-gamma KO mice were able to generate CD8(+) T-cell responses against both secreted and nonsecreted L. monocytogenes antigens which were similar to responses of WT mice. Interestingly, IFN-gamma KO mice were as capable as WT mice in mediating the characteristic drop in bacterial load in the liver at 4 h postinfection, although the IFN-gamma KO mice have exacerbated bacterial loads as early as 24 h postinfection. These results demonstrate that the regulatory functions of IFN-gamma are not required for priming of CD8(+) T cells by cross-presentation of a model exogenous antigen or in response to a nonsecreted L. monocytogenes antigen. In addition, the capacity of IFN-gamma to activate the microbicidal activities of macrophages is not required for the very early innate immune response to L. monocytogenes or priming of CD8(+) T cells against a nonsecreted bacterial antigen.     

The effect of aging on the secretory immune system of the eye.

The objective of the present study was to examine the influence of aging on the ocular secretory immune system of the eye. Levels of IgA and free secretory component (FSC) were measured in lacrimal glands and/or tears of 0.6, 1.3, 3, 8 and 17-month-old male and female rats. In addition, the FSC output of lacrimal tissue cultured in vitro was evaluated. During the period from 0.6 to 1.3 months of age, the content of tear IgA increased nine- and 13-fold in females and males, respectively. This rise was paralleled by changes in the concentration of tear FSC. Prior to the onset of puberty, FSC could be detected in only 7% of tear samples, whereas after pubertal maturation, tear FSC levels had attained adult concentrations. This tear FSC profile was similar to the age-related pattern of FSC output by lacrimal tissue incubated in vitro. Following puberty, tear IgA content continued to increase in both sexes until adulthood (3 months of age) and then plateaued in females from 8 to 17 months of age. In contrast, tear IgA in males appeared to stabilize from 3 to 8 months and then rose significantly to the highest levels at 17 months of age. This increase in males was also reflected in their lacrimal tissue: IgA content underwent a six-fold elevation from 3 to 17 months. Of interest is that the differential kinetics involved in tear IgA and FSC expression resulted in an age-associated decline in the FSC/IgA ratio from post-puberty to senescence. A striking finding in these studies was the persistence of a sexual dimorphism in the secretory immune system of the eye. After pubertal development, IgA and FSC levels were significantly higher in tears of males, compared to those of females, at all ages tested up to 17 months. These gender- and age-related variations in tear IgA and FSC amounts could not be accounted for by changes in either the volume of, or total protein content in, tears.