Polyreactive antigen-binding B (PAB-) cells are widely distributed and the PAB population consists of both B-1+ and B-1- phenotypes

B cells that make polyreactive antibodies (PAB+ cells) express polyreactive Ig receptors on their surface and can bind a variety of different antigens. The present study shows that PAB+ cells are widely distributed, are present in varying numbers in different lymphoid organs and that their phenotype varies depending on the organs from which they are isolated. Up to 10 times more cells in PAB+ enriched populations bind antigens as compared to PAB- populations. Comparison of PAB+ with B-1+ cells showed that a high percentage of PAB+ cells are B-1+, but that many PAB+ cells do not express B-1 cell surface markers and, in fact, are B-1-. It is concluded that the B cell population consists of PAB+/B-1+, PAB+/B-1-, PAB-/B-1+, and PAB-/B-1- cells. The presence of PAB+ cells in the thymus points to the possibility that PAB+ cells may carry endogenous host antigens from peripheral tissues to the thymus where they may contribute to immunological tolerance.     

Antigen-binding B cells and polyreactive antibodies

The present experiments were initiated to see if cells capable of binding antigens could make polyreactive antibodies. Fluorescein isothiocyanate-labeled self and non-self antigens were incubated with B cells from normal individuals. Antigenbinding cells were separated from non-antigen-binding cells by flow cytometry, immortalized with Epstein-Barr virus and analyzed at the clonal level for their capacity to make polyreactive antibodies. Four to six times more cells making polyreactive antibodies were found in the B cell subset that bound antigens than in the B cell subset that did not bind antigens. The majority of the polyreactive antibodies were of the immunoglobulin (Ig)M isotype. Immunoflow cytometry revealed that cell lines making polyreactive antibodies bound a variety of antigens (e.g., insulin, IgGFc and β-galactosidase), whereas cell lines making monoreactive antibodies bound only a single antigen. The binding of antigens to B cell lines that made polyreactive antibodies could be inhibited (range, 28%–57%) by both homogeneous and heterogeneous antigens. Both CD5⁺ and CD5^? antigen-binding B cells made polyreactive antibodies, but the frequency was slightly higher in the CD5⁺ antigen-binding (85%) as compared to the CD5^? antigen-binding (50%) population. Comparison of CD5⁺ B cells that bound antigens with CD5⁺ B cells that did not bind antigens showed that approximately 86% of the former, but only 15% of the latter, made polyreactive antibodies. It is concluded that cells capable of binding a variety of different antigens can make polyreactive antibodies and that antigen binding is a good marker for identifying polyreactive antibody-producing cells.     

Age-related depression of FDC accessory functions and CD21 ligand-mediated repair of co-stimulation

Morphological and kinetic studies of immune complex (IC) trapping by follicular dendritic cells (FDC) show marked age-related deficits. We postulated that a reduction in trapped IC, which generate CD21 ligands (L) on FDC, would lead to inadequate FDC-Ag-B cell interactions resulting in depressed Ab responses. To determine whether the age-related defect was the result of the aging of FDC or changes in the in vivo microenvironment of FDC (i.e. aging B and T cells), FDC-B cell-T cell-Ag interactions were studied in in vitro germinal centers where various combinations of old and young cells could be compared. Since we reasoned that reduced IC on FDC would generate less CD21L needed to stimulate the B cell co-receptor via CD21, we also examined the role of complement (C'). The hypothesis that aging reduces the accessory activity of FDC was tested with increasing numbers of FDC from young (12 weeks) or old (20 months) mice in the presence of young (12 weeks) B and T lymphocytes. The Ag-specific stimulatory activity of FDC was studied using the OVA-specific Ab response which was reduced by 40-50% in the presence of old FDC. Antigen-independent FDC-mediated co-stimulation was studied by using LPS to stimulate B-lymphocytes to produce immunoglobulin (Ig). In the presence of old FDC, co-stimulation was decreased by 70-80% in the LPS system.Incubation of aged FDC with IC and C' to provide FDC with CD21L restored co-stimulatory activity to near normal levels. In marked contrast, no defects in old B and T cells were apparent. The data suggest that the Ag handling capacity and co-stimulatory activity of old FDC become defective with aging and this appears to be a consequence of reduced trapping and presentation FDC-Ag and CD21L to B cells.     

Polyreactive antigen-binding B cells in the peripheral circulation are IgD+ and B7−

Polyreactive antibodies are naturally occurring antibodies, primarily of the IgM isotype, that are capable of reacting with a wide variety of different self and non-self antigens. Previously, we reported that a B cell capable of making polyreactive antibody has Ig receptors on its surface that can bind different antigens. The present investigation was initiated to characterize these polyreactive antigen-binding B cells further. A panel of fluorescein isothiocyanate-labeled antigens (insulin, IgG Fc fragment or β-galactosidase) served as probes to select polyreactive antigen-binding B cells by cell sorting. Our experiment revealed that these polyreactive antigen-binding B cells were mainly of the IgD isotype. They expressed high levels of CD40 and major histocompatibility complex class II molecules, but little or no B7-1, B7-2, or Fas. In contrast to the binding of antigens to monoreactive receptors (usually high affinity), the binding of antigens to polyreactive receptors (usually moderate or low affinity) did not up-regulate the expression of B7-1 or B7-2. Antigens that bound to polyreactive receptors, however, were internalized and degraded, although not as efficiently as antigens that bound to monoreactive receptors. Despite the ability of these B7⁻ cells to process antigens, they were not able to activate T cells in a mixed leukocyte reaction. It is concluded that polyreactive antigen-binding B cells have properties that are consistent with the ability to induce immunological tolerance.     

A unique polypeptide on avian antigen-specific suppressor T-cell radioiodinated in situ by antigen-lactoperoxidase conjugates

Antigen-specific receptors of B- or T-cells were selectively radiolabeled among the spleen cells from either human gammaglobulin immunized normal or bursectomized agammaglobulinemic chickens. Selective in situ radioiodination was accomplished by employing lactoperoxidase (LPO) covalently linked to antigen (Ag). Ag-specific receptors on B- or T-cells were allowed to bind Ag-LPO conjugates via the Ag portion of the conjugates and then to be selectively catalyzed for iodination by the LPO portion of the bound Ag-LPO conjugates. Radioiodinated cells were either processed for autoradiography to detect Ag-binding cells directly under the microscope or solubilized with detergents for protein analysis with two-dimensional (2-D) gel electrophoresis. On a cellular level, Ag-binding B- and T-cells were selectively radiolabeled and clearly visualized via autoradiography. On a molecular level, selectively radiolabeled Ag-specific membrane immunoglobulin of B-cells was demonstrated on 2-D gel autoradiographs. Furthermore, a unique polypeptide of Ag-specific T-cells with a reduced apparent mol. wt of 27 K and an apparent pI of 5.5-5.7 was demonstrated on 2-D gel autoradiograms. The 27 K molecule appears to be a T-cell receptor component itself, or a closely associated molecule.     

Enhancement of antigen-specific activation of CD8+ memory cytotoxic T cells by B cell-derived factors.

Purified CD8+ T cells from influenza A/WSN-immune BALB/c (H-2d) mice respond with the generation of secondary A/WSN-specific Tc cells in vitro when stimulated with a synthetic peptide (NPP) with a sequence derived from influenza A virus nucleoprotein with high affinity for Kd class I MHC molecules. The process of the conversion of NPP-Kd-responding Tc cell precursors into effector Tc cells in a population of CD8+ T cells occurs with no demonstrable requirements for accessory cells or their lymphokine products. The addition of culture supernatants from several mouse and human B cell lymphomas and LPS-activated normal mouse B cells to the culture of NPP-stimulated immune CD8+ T cells enhanced the induction of secondary Ag-specific Tc cells. None of the tested supernatants in the absence of Ag (NPP) induced cytolytic Tc cells, indicating that B cell-derived secretory factors can exert their activity only on Ag-exposed CD8+ T cells. The augmentatory effect of these supernatants on Ag-specific activation of memory CD8+ T cells was attributed to the synergism between B cell-derived factors and IL-2 which is produced endogenously in cultures of NPP-stimulated D8+ T cells. The possible role of B cell-derived helper factors is discussed.     

Differential contributions of B7-1 and B7-2 to the development of murine experimental allergic conjunctivitis

B7-1 and B7-2 are the co-stimulatory molecules that are involved in activation of T cells. We investigated whether B7-1 and B7-2 play a role in the development of T cell-mediated experimental allergic conjunctivitis (EC). EC was induced in Balb/c mice by active immunization with ragweed (RW) followed by RW challenge in eye drops. These mice were treated with neutralizing anti-B7-1 Ab, anti-B7-2 Ab, both Abs, anti-cytotoxic T lymphocyte-associated Ag-4 (CTLA-4) Ab or normal IgGs as controls either during the induction phase or the effector phase. With regard to the induction phase treatment, EC was significantly attenuated when both anti-B7-1 and anti-B7-2 Abs were injected. In contrast, anti-CTLA-4 Ab treatment significantly exacerbated EC. With regard to the effector phase treatment, anti-B7-2 Ab alone significantly attenuated EC, while anti-CTLA-4 Ab tended to exacerbate EC. Collectively, B7-1 and B7-2 differently contribute to the development of EC during the induction and effector phases.     

Effects of LT-K63 and CpG2006 on phenotype and function of murine neonatal lymphoid cells

The immature state of the immune system of neonates makes them vulnerable to infectious agents, including Streptococcus pneumoniae. The aim of our study was to analyse and compare the effects of Escherichia coli heat-labile enterototoxin (LT)-K63 and CpG2006 on cells and key molecules of the neonatal immune system, using a previously established immunization model with pneumococcal polysaccharide of serotype 1 conjugated to tetanus toxoid (TT) (Pnc1-TT). The cellular response was evaluated by measuring cytokine secretion and proliferation upon in vitro stimulation with TT, the protein moiety of Pnc1-TT, and antibody (Ab) to both the polysaccharide (PS) and protein parts of the vaccine were measured by enzyme-linked immunosorbent assay (ELISA). Antigen (Ag)-presenting and co-stimulatory capacity of neonatal B-cells was evaluated by staining for major histocompatibility complex (MHC)II, CD80, CD86 and CD40. The results showed that both LT-K63 and CpG2006 significantly enhanced the neonatal Ab response to Pnc1-TT. Spleen cells from mice receiving LT-K63 showed enhanced proliferation and interferon (IFN)-gamma, interleukin (IL)-4, IL-5 and IL-10 secretion upon TT stimulation, whereas cells from mice receiving CpG2006 could only enhance IL-10 secretion. LT-K63 and to a lesser extent CpG2006 enhanced the capacity of B-cells to up-regulate the expression of co-stimulatory and activation markers compared with those of mice receiving Pnc1-TT alone. Thus, we conclude that LT-K63 markedly improves T-cell activation whereas the direct adjuvant effect of CpG2006 on neonatal B-cells may partly compensate for lower T-cell help resulting in enhanced neonatal Ab responses to both the TT and PS parts of the vaccine by both adjuvants.     

Early Expansion of Secondary B Cells after Primary Immunization with Antigen Complexed with IgE

IgE antibodies have potent immunoregulatory effects in vivo , and mice immunized with IgE–antigen (IgE/Ag) complexes exhibit a several hundred-fold higher humoral Ag-specific response than mice immunized with non-complexed Ag. In vitro studies indicate that this is a result of efficient endocytosis of the IgE/Ag complexes via the low-affinity receptor for IgE (CD23) on B cells, leading to efficient antigen presentation to T cells. Previous studies of IgE-induced Ab responses in vivo have only measured serum responses. The authors have now studied the up-regulated response as the number of IgG-, IgA-, IgE- and IgM-secreting single B cells in spleen, lymph nodes and bone marrow of mice immunized with IgE-anti-TNP + BSA-TNP (2,4,6-trinitrophenylated bovine serum albumin). IgE and Ag induced a greater than 500-fold increase of specific IgG-secreting spleen cells with the peak of the response 6 days after primary immunization. The response of other Ab isotypes and the response in other lymphoid organs was marginal. The rapid increase in the number of IgG-secreting cells in the spleen suggests that IgE/Ag complexes induce a secondary type of antibody response without requirement for conventional priming.     

Aberrant contraction of antigen-specific CD4 T cells after infection in the absence of gamma interferon or its receptor

Several lines of evidence from different model systems suggest that gamma interferon (IFN-gamma) is an important regulator of T-cell contraction after antigen (Ag)-driven expansion. To specifically investigate the role of IFN-gamma in regulating the contraction of Ag-specific CD4 T cells, we infected IFN-gamma-/- and IFN-gammaR1-/- mice with attenuated Listeria monocytogenes and monitored the numbers of Ag-specific CD4 T cells during the expansion, contraction, and memory phases of the immune response to infection. In the absence of IFN-gamma or the ligand-binding portion of its receptor, Ag-specific CD4 T cells exhibited normal expansion in numbers, but in both strains of deficient mice there was very little decrease in the number of Ag-specific CD4 T cells even at time points later than day 90 after infection. This significant delay in contraction was not due to prolonged infection, since mice treated with antibiotics to conclusively eliminate infection exhibited the same defect in contraction. In addition to altering the number of Ag-specific CD4 T cells, the absence of IFN-gamma signaling also changed the phenotype of cells generated after infection. IFN-gammaR1-/- Ag-specific CD4 T cells reacquired expression of CD127 more quickly than wild-type cells, and more IFN-gammaR1-/- CD4 T cells were capable of producing both IFN-gamma and interleukin 2 following Ag stimulation. From these data we conclude that IFN-gamma regulates the contraction, phenotype, and function of Ag-specific CD4 T cells generated after infection.     

Antigen dose-dependent predominance of either direct or sequential switch in IgE antibody responses.

Priming of CBA/J mice with minute doses of protein antigens (Ag) leads to high IgE antibody (Ab) titres in the immune sera of these animals. In contrast priming with large doses elicits only a marginal production of IgE Ab. In vitro restimulation of spleen cells from animals primed with large doses and lacking in vivo IgE Ab leads to a burst of IgE Ab-forming cells. This in vitro anamnestic response is lacking in mice primed with minute doses of Ag. In order to trace the cellular basis of the in vitro IgE memory response we have extended the analysis of the distribution of Ab isotypes to Ag-primed IgG1-deficient delta 5'S gamma 1 mice. The data presented here must be interpreted as followed. Priming of mice with minute doses of Ag leads to a direct switch from IgM to IgE Ab expression in both strains. These animals have high IgE Ab titres without establishing an IgE memory. The direct switch was verified by polymerase chain reaction and Southern blot analysis of switch circle DNA isolated from Ag-specific B cells of CBA/J mice primed with minute doses of Ag. In contrast to immunization with minute doses, priming with large doses of Ag fails to induce in vivo IgE Ab production in CBA/J and delta 5'S gamma 1 mice but establishes a B epsilon memory in CBA/J mice which involves IgG1-bearing intermediate B cells. In vivo these B epsilon memory cells do not enter the status of IgE Ab-producing cells. In vitro they can be released from this anergy and presumed suppression and develop in an anamnestic response into a large population of IgE Ab-forming B cells. This increase in the number of IgE Ab-producing cells after restimulation in vitro is lacking in delta 5'S gamma 1 mice, apparently because of their inability to generate IgG1-expressing precursor cells. The notion of a sequential switch and an IgG1 intermediate B epsilon memory status is also supported by depletion and inhibition experiments. Elimination of IgG1-expressing B cells in CBA/J mice primed with high doses of Ag prevents the IgE Ab burst after in vitro challenge with Ag. The data further suggest that the two switch pathways are not mutually exclusive and that the Ag dose can decide which pathway is preferentially used.     

Long-term anergy in orally tolerized mice is linked to decreased B7.2 expression on B cells

Durable antigen (Ag)-specific T- and B-cell anergy induced by oral tolerance is an attractive strategy for immunotherapy of allergic diseases. Here, we address the lasting effect of oral tolerance induction in na?ve or primed mice to ovalbumin (OVA) on antibody production. Single feeding with OVA prior to immunization or double feeding, before and after Ag priming, in A/Sn mice, induced a long-lasting suppression of IgE, IgG1 and IgG2a responses up to 8 months after immunization. In contrast, primed-fed mice had transient IgE inhibition. Naive and double-treated mice showed marked Ag-specific unresponsiveness and scarce cytokines production. Inhibition of IL-2 and IFN-gamma secretion in na?ve-fed mice were restored in the presence of anti-CD28 mAb plus Ag stimulation. The durable inhibition of Ab production in OVA-fed mice was related to the persistent decrease of B7.2 expression on B cells. Ag feeding in naive and primed status may be a prophylactic measure to avoid later Ag sensitization.     

Impaired Antibody Responses but Normal Proliferation of Specific CD4+ T Cells in Mice Lacking Complement Receptors 1 and 2

Severely impaired Ab responses are seen in animals lacking C (complement) factors C2, C3 or C4 as well as CR1/2 (C receptors 1 and 2). The molecular mechanism behind this phenomenon is not understood. One possibility is that C-containing immune complexes are endocytosed via CR2 on B cells and presented to specific CD4⁺ T cells, which would then proliferate and provide efficient help to specific B cells. In vitro , B cells can endocytose immune complexes via CR1/2 and present the Ag to T cells. Whether absence of this Ag presenting function in Cr2^−/− mice (mice lacking CR1/2) explains their low Ab response is unclear. To address this question, Cr2^−/− and wild type mice were transferred with OVA-specific T cells, obtained from the DO11.10 strain which has a transgenic TCR recognizing an OVA peptide. The animals were subsequently immunized with sheep red blood cells (SRBC) conjugated to OVA. Interestingly, proliferation of the OVA-specific T cells was normal in Cr2^−/− mice, although their Ab response to both SRBC and OVA was severely impaired. These observations suggest that the impaired Ab response in Cr2^−/− mice cannot be explained by a lack of appropriate induction of T cell help.     

Gamma delta T cells of the murine vagina: T cell response in vivo in the absence of the expression of CD2 and CD28 molecules

While little is known about their activation requirements and function, the intraepithelial T cells of the murine vagina express TCR complexes in which the antigen recognition components and the signaling components have unusual features. These vaginal T cells express an invariant V gamma 4/V delta 1 TCR and appear to be the only intraepithelial gamma delta T cells that exclusively use FcR gamma chains in their TCR complex. To further characterize the vaginal gamma delta T cells we isolated them from normal mice and from mice injected systemically with an activation-inducing dose of anti-TCR mAb. The isolated gamma delta T cells were examined by flow cytometry for their surface expression of a panel of adhesion, proteins, activation antigens and cellular interaction molecules (CD44, CD62L, CD45RB, LFA- 1, CD2 and CD28). The patterns of expression observed indicate that the vaginal gamma delta T cells of normal mice show the phenotype of effector T cells. The adhesion/co-stimulatory molecules CD28 and CD2 were not detected on vaginal gamma delta T cells, an interesting finding since the absence of CD2 from other T cells has been suggested to result in anergy. However, vaginal gamma delta T cells are responsive to TCR-mediated signals since injection of normal mice with pan-anti-TCR antibody or stimulating anti-gamma delta TCR antibody resulted in an increase in cell number and increased expression of transferrin and IL-2 receptors. These results indicate that vaginal gamma delta T cells might utilize other co-stimulatory molecules, if any, in connection with TCR-induced activation and differentiation. While the physiological function of vaginal gamma delta T cells remains unknown, the expression of an invariant V gamma 4/V delta 1 TCR, their exclusive use of gamma chain homodimers in their TCR, and the absence of CD2 and CD28 co-stimulatory molecules are a novel combination of properties that suggests specialized functional properties. Although vaginal gamma delta T cells share some features in common with gamma delta T cells that reside in other epithelial tissues, such as skin and intestine, the present studies provide additional evidence that vaginal gamma delta T cells are a highly specialized and distinct T cell population.      

Low responsiveness of hepatitis B virus-transgenic mice in antibody response to T-cell-dependent antigen: defect in antigen-presenting activity of dendritic cells.

The experiments presented here were performed to evaluate immune responsiveness of hepatitis B virus (HBV)-transgenic mice (transgenic mice), as a model of HBV-carrier state to a T-cell-dependent antigen, keyhole limpet haemocyanin (KLH). The transgenic mice which were completely unresponsive to hepatitis B surface antigen (HBsAg), responded poorly to KLH. The levels of anti-KLH antibodies (Ab) produced in vivo were significantly lower in transgenic mice compared with the normal control mice at respective immunizing doses of KLH. In addition, a little or no anti-KLH Ab production was detected in culture supernatants of KLH-primed transgenic mice spleen cells. KLH-primed T cells from normal and transgenic mice induced anti-KLH Ab production from transgenic B cells in the presence of antigen-presenting spleen adherent cells (SAC) from normal mice, but not those from transgenic mice. Depletion of dendritic cells from normal mice-derived SAC completely abrogated the anti-KLH Ab response in transgenic spleen cell culture and their addition to the culture restored the response. Low efficiency of transgenic dendritic cells was demonstrated in sodium periodate (NaIO4)-induced non-specific and allogenic antigen-induced T-cell proliferation. Finally, cytofluorometric analyses showed a reduced Ia antigen expression on transgenic dendritic cells. These results indicate that low responsiveness of transgenic mice in specific-antibody response is not due to functional defects in T cells or B cells but rather to a defect of antigen-presenting activity of dendritic cells.     

Macrophage – T cell interaction in experimental mycobacterial infection. Selective regulation of co-stimulatory molecules on Mycobacterium- infected macrophages and its implication in the suppression of cell-mediated immune response

The most important immunopathological consequence of experimental mycobacterial infection is the suppression of T cell-mediated immune response to both mitogens and mycobacterial antigens. We registered that there was decreased concanavalin A-induced spleen cell proliferation in infected susceptible BALB/c mice as compared to normal mice. In resistant (C3H/HeJ) mice, infection with the bacteria did not induce any suppression in the mitogen-induced lymphoproliferation. Likewise, delayed-type hypersensitivity (DTH) responses, to keyhole limpet hemocyanin and mycobacterial crude soluble antigen were suppressed in infected BALB/c mice but not in C3H/HeJ mice. This depressed T helper cell function may either be due to defective T cell-receptor occupancy by antigen-Ia complex or altered co-stimulatory signals provided by antigen-presenting cells. In the present study, we have investigated the status of certain co-stimulatory molecules on the infected macrophages from both susceptible and resistant mice. Our results demonstrate that upon mycobacterial infection, the macrophages are rendered incapable of delivering the co-stimulatory signals to T helper cells, possibly due to the involvement of prostaglandin, as inhibition of its biosynthesis by indomethacin reversed the defect. Furthermore, the selective regulation was bacteria-induced as killing of the bacteria by rifampicin abrogated the derangements in the expression of co-stimulatory molecules on the Mycobacterium-infected macrophages. Our observations revealed that upon infection with Mycobacterium tuberculosis, B7 was down-regulated while ICAM-1 was increased only in BALB/c but not in C3H/HeJ mice. Expression of VCAM-1 did not change during the infection in either strain of mice. We found that these changes in ICAM-1 and B7 expression on the surface of infected macrophages resulted in inhibition of DTH-mediating functions of T helper cells from BALB/c mice. The results obtained in this study describe not only a novel immune evasion strategy adopted by Mycobacterium, but also open up the possibility of immunotherapy of mycobacterial infection by selective manipulation of co-stimulatory molecules.     

The effects of DNA containing CpG motif on dendritic cells

Dendritic cells (DC) are specialized antigen-presenting cells. DC can acquire and process antigens in the periphery before maturing and migrating to secondary lymphoid tissues where they present the antigens and deliver co-stimulatory signals to T cells. We describe an immunostimulatory oligonucleotide containing a CpG motif that stimulated murine DC to up-regulate co-stimulatory molecules, induce T-cell proliferative responses and secrete interleukin-12 in vitro. Administration of this oligonucleotide, but not of a control oligonucleotide lacking this motif, to mice led to the disappearance of DC from the marginal zone and T-cell areas of spleen, but not from heart or kidney. The same CpG did not cause maturation of monocyte-derived human DC in vitro, but lipopolysaccharide-treated monocyte-derived DC showed enhanced functional activity and up-regulated co-stimulatory molecules.     

Selective suppression of antigen-specific Th2 cells by continuous micro-dose oral tolerance

The effect on antigen (Ag)-specific Th2 response as well as IgE production of continuous oral administration of micro-doses of Ag was investigated. Transgenic (Tg) mice carrying the α β-T cell receptor (TCR) genes specific for ovalbumin (OVA) peptide fragment 323 – 339 were continuously fed with micro-doses of OVA (100 μg/day) for 14 days. Mice were first immunized by OVA in alum and pertussis toxin 7 days before the oral feeding and given a second immunization 1 day after the oral treatment. This feeding regimen tolerized Th2 but not Th1 responses as shown by decrease of Ag-driven cell proliferation and cytokine secretion of IL- 4 but not of IL-2 or IFN-γ as well as by the absence of Ag-specific antibody production of IgE and IgG1, but not of IgG2a or total IgG. Numbers of clonotype-specific TCR-high CD4-positive T cells in peripheral lymphoid tissues markedly decreased in the orally treated group but not in the control group. However, total numbers of CD4-positive T cells in thymus, spleen and lymph nodes were not affected by the oral treatment, indicating that tolerance induction in Th2 cells was mainly due to the down-regulation of TCR and not clonal deletion. The population of antigen-presenting cells expressing B7-2 (CD86) Ag on the surface was decreased in the spleen of the mice which underwent the feeding regimen. The present results suggest that Ag-specific low responsiveness in Th2 cells, which resulted in suppres sion of the Ag-specific IgE production, can be achieved by continuous feeding with microdoses of Ag.     

Genetically manipulated bacterial toxin as a new generation mucosal adjuvant

Cholera toxin (CT) and heat-labile toxin (LT) of Escherichia coli act as adjuvants for the enhancement of mucosal and serum antibody (Ab) responses to mucosally co-administered protein antigen (Ag). Both LT and CT induce B7-2 expression on antigen-presenting cells (APCs) for subsequent co-stimulatory signalling to CD4+ T cells. CT directly affects CD4+ T cells activated via the TCR-CD3 complex with selective inhibition of Th1 responses whereas LT maintains Th1 cytokine responses with inhibition of interleukin (IL)-4 production. Interestingly, while CT failed to induce mucosal adjuvant activity in the absence of IL-4, LT did so. Nontoxic mutant (m)CTs (S61F and E112K) retain adjuvant properties by inducing CD4+ Th2 cells, which provided effective help for the Ag-specific mucosal immunoglobulin (Ig)A, as well as serum IgG1, IgE and IgA Ab responses. The mCT E112K has been shown to exhibit two distinct mechanisms for its adjuvanticity. Firstly, mCT enhanced the B7-2 expression of APCs. Secondly, this nontoxic CT derivative directly affected CD4+ T cells and selectively inhibited Th1 cytokine responses. Thus, several lines of evidence indicate that enzyme activity can be separated from adjuvant properties of CT and this offers promise for the development of safe delivery of vaccines for mucosal IgA responses.     

Structural aspects of human IgM antibodies expressed in chronic B lymphocytic leukemia.

BACKGROUND: Malignant B cells from patients with chronic B lymphocytic leukemia (B CLL) generally express both surface IgM and the pan T cell antigen CD5, a characteristic of the B1 population of B lymphocytes. The IgM on the surface of these B CLL cells is frequently polyreactive with respect to its capacity to recognize multiple structurally dissimilar antigens (Ag). OBJECTIVES: To understand the structural characteristics of the polyreactive binding sites of human IgM molecules expressed on B CLL cells by: (1) analyzing the nucleotide and protein sequences of the variable (V) domains of five IgM molecules expressed in cases of B CLL and; (2) utilizing these sequences to generate three-dimensional (3D) models of Fv (VL - VH) molecules. STUDY DESIGN: Peripheral blood leukocytes obtained from five cases of B CLL were tested for polyreactive binding properties by assessing their capacity to bind mouse IgG by indirect immunofluorescence. The V region genes of light and heavy chains were amplified using the polymerase chain reaction, subsequently cloned and their nucleotide sequences obtained. Translated amino acid sequences of the V domains were used to generate homology models of the Fv molecules. RESULTS: Low affinity binding of mouse IgG was demonstrated for all B CLL samples examined, confirming the polyreactive nature of the IgM expressed on these cells. There was an absence or minimal mutation within V region genes when compared to germline Ig genes. Junctional diversity was not observed for VL regions, although truncations and insertions were frequent in D minigenes of VH regions. The binding sites were predicted to form either relatively flat surfaces with occasional protrusions or cavities at the VL - VH domain interface. Aromatic side chains covered a large proportion of the potential binding surfaces in the models of B CLL Fv components. DISCUSSION: Primary DNA sequences can be categorized as germline, suggesting that the B cells involved in B CLL are germline or naive in origin. The medium to large HCDR3s provide the majority of probable contact residues for antigens. While prominent aromatic residues are likely to engage in binding patterns which are conserved (e.g. mouse Ig reactivity), the diverse binding sites predicted for B CLL-derived IgMs also have properties which are conducive to polyreactive antigen binding.