Antibodies Of Various Isotypes Research Articles

Identification of Follicular T Cells in the Gut.

Humoral adaptive immune responses trigger the establishment of plasma B cells secreting antibodies of various isotypes that bind antigen specifically and with high affinity. Moreover, memory B cells will be generated. To accomplish this, B cells need assistance from a special subset of CD4 T cells, the so called follicular T cells that differentiate from naïve T cells in the course of the immune response. Therefore, the study of follicular T cells is of primordial interest when investigating the molecular and cellular determinants of adaptive immune responses. This is done by direct analysis of the cells isolated from mice following an immunological challenge but in many instances such analyses must involve follow-up studies in cell culture requiring living cells. Especially, in vitro experimentation necessitates isolation and sorting of follicular T cells. However, follicular T cells are generally difficult to handle because they are prone to apoptosis and cell death. This is particularly evident when dealing with follicular T cells residing in the gut since we observed that isolation and processing from murine gut notoriously results in very high loss rates when compared for example to cells obtained from immunized peripheral lymph nodes. To bypass these limitations, we developed a protocol that allows for efficient isolation of intact follicular T cells. The protocol introduced here illustrates isolation and handling of follicular T cells using murine Peyer's Patches as an example because they constantly harbor significant amounts of these cells.

FEATURES OF IMMUNE RESPONSE DURING VARIOUS SCHEMES OF USE OF BACTERIAL THERAPEUTIC VACCINE IMMUNOVAC VP-4

Aim. Study the dynamics of immunologic parameters in patients with chronic bacterial infections during various schemes of administration of Immunovac VP-4 vaccine. Materials and methods. Parameters of systemic immunity and levels of specific antibodies of various isotypes in blood sera and saliva against vaccine antigens of Staphylococcus aureus and Klebsiella pneumoniae were evaluated in patients (20 individuals, 18 - 50 years of age) distributed into 2 groups by vaccine administration type twice (before and 0,5 - 1,5 months after vaccine therapy course). Results. Local vaccination resulted in an increase of only IgA levels in saliva and both bacterial antigens, whereas parameters of systemic immunity before and after vaccination did not differ. Subcutaneous vaccination increased the level of sera antibodies of A- and G- isotypes against both bacterial antigens, normalized the decreased level of CD8" lymphocytes and an increased value of the immune regulating index; a tendency of increase of the percentage of CD3+ T-cells and reduction of the percentage of CD4+ T-helpers was observed. Conclusion. An optimal scheme of a combined vaccine therapy should be developed to obtain a complex effect, that would allow to simultaneously reach long-term local and systemic antibacterial immunity, as well as show immune modulating effect regarding cell compartment.

Antigenic modulation limits the effector cell mechanisms employed by type I anti-CD20 monoclonal antibodies

AbstractFollowing the success of rituximab, 2 other anti-CD20 monoclonal antibodies (mAbs), ofatumumab and obinutuzumab, have entered clinical use. Ofatumumab has enhanced capacity for complement-dependent cytotoxicity, whereas obinutuzumab, a type II mAb, lacks the ability to redistribute into lipid rafts and is glycoengineered for augmented antibody-dependent cellular cytotoxicity (ADCC). We previously showed that type I mAbs such as rituximab have a propensity to undergo enhanced antigenic modulation compared with type II. Here we assessed the key effector mechanisms affected, comparing type I and II antibodies of various isotypes in ADCC and antibody-dependent cellular-phagocytosis (ADCP) assays. Rituximab and ofatumumab depleted both normal and leukemic human CD20-expressing B cells in the mouse less effectively than glycoengineered and wild-type forms of obinutuzumab, particularly when human immunoglobulin G1 (hIgG1) mAbs were compared. In contrast to mouse IgG2a, hIgG1 mAbs were ineffective in ADCC assays with murine natural killer cells as effectors, whereas ADCP was equivalent for mouse IgG2a and hIgG1. However, rituximab's ability to elicit both ADCC and ADCP was reduced by antigenic modulation, whereas type II antibodies remained unaffected. These data demonstrate that ADCP and ADCC are impaired by antigenic modulation and that ADCP is the main effector function employed in vivo.

Fanca deficiency reduces A/T transitions in somatic hypermutation and alters class switch recombination junctions in mouse B cells.

Fanconi anemia is a rare genetic disorder that can lead to bone marrow failure, congenital abnormalities, and increased risk for leukemia and cancer. Cells with loss-of-function mutations in the FANC pathway are characterized by chromosome fragility, altered mutability, and abnormal regulation of the nonhomologous end-joining (NHEJ) pathway. Somatic hypermutation (SHM) and immunoglobulin (Ig) class switch recombination (CSR) enable B cells to produce high-affinity antibodies of various isotypes. Both processes are initiated after the generation of dG:dU mismatches by activation-induced cytidine deaminase. Whereas SHM involves an error-prone repair process that introduces novel point mutations into the Ig gene, the mismatches generated during CSR are processed to create double-stranded breaks (DSBs) in DNA, which are then repaired by the NHEJ pathway. As several lines of evidence suggest a possible role for the FANC pathway in SHM and CSR, we analyzed both processes in B cells derived from Fanca(-/-) mice. Here we show that Fanca is required for the induction of transition mutations at A/T residues during SHM and that despite globally normal CSR function in splenic B cells, Fanca is required during CSR to stabilize duplexes between pairs of short microhomology regions, thereby impeding short-range recombination downstream of DSB formation.

Comparative Evaluation of HIV-1 Neutralization in External Secretions and Sera of HIV-1-Infected Women

Objectives:Although human immunodeficiency virus type 1 (HIV-1)-specific antibodies are detectable in external secretions by ELISA and western blot (WB), the presence of HIV-1 neutralizing antibodies is difficult to evaluate due to the low levels of immunoglobulins (Ig) and the presence of humoral factors of innate immunity. The objective of this study was to determine virus neutralization activity and the relative contribution of HIV-1-specific antibodies of various isotypes to virus neutralization in serum/plasma samples, cervicovaginal lavages (CVL), and rectal lavages (RL).Design:Serum/plasma, CVL, and RL samples were examined by ELISA, WB and HIV-1 neutralization assays. Selected samples were Ig depleted and analyzed for virus neutralization.Results:IgG specific for three HIV-1 ENV antigens was detected in all serum/plasma samples, while IgA to at least one ENV glycoprotein was found at the low levels in 95% samples. Serum/plasma samples had the ability to neutralize at least one of three clade B and two clade C viruses. The neutralizing titers were reduced significantly or became undetectable after IgG removal. In corresponding CVL and RL, HIV-1 ENV-specific IgG antibodies were readily detected compared to IgA. Furthermore, IgG in CVL had greater ability than IgA to reduce virus infectivity. The difference in HIV-1 neutralization before and after Ig depletion was not observed in RL, implying that innate humoral factors were involved in anti-HIV-1 activity.Conclusions:Results demonstrate that HIV-1-specific neutralizing antibodies are almost exclusively of the IgG isotype in serum/plasma and CVL samples. HIV-1-specific binding antibodies detected in RL are not responsible for neutralization activity, suggesting that the antibody-mediated virus neutralization in external secretions should be verified by means of a selective depletion of Ig.

The Human IgE-encoding Transcriptome to Assess Antibody Repertoires and Repertoire Evolution

Upon encounter with antigen, the B lymphocyte population responds by producing a diverse set of antigen-specific antibodies of various isotypes. The vast size of the responding populations makes it very difficult to study clonal evolution and repertoire composition occurring during these processes in humans. Here, we have explored an approach utilizing the H-EPSILON-encoding transcriptome to investigate aspects of repertoire diversity during the season of antigen exposure. We show through sequencing of randomly picked transcripts that the sizes of patients' repertoires are relatively small. This specific aspect of the transcriptome allows us to construct evolutionary trees pinpointing features of somatic hypermutation as it occurs in humans. Despite the small size of the repertoires, they are highly diverse with respect to VDJ gene usage, suggesting that the H-EPSILON-encoding transcriptome is a faithful mimic of other class-switched isotypes. Importantly, it is possible to use antibody library and selection technologies to define the specificity of clonotypes identified by random sequencing. The small size of the H-EPSILON-encoding transcriptome of peripheral blood B cells, the simple identification of clonally related sets of genes in this population, and the power of library and selection technologies ensure that this approach will allow us to investigate antibody evolution in human B lymphocytes of known specificity. As H-EPSILON repertoires show many of the hallmarks of repertoires encoding other isotypes, we suggest that studies of this type will have an impact on our understanding of human antibody evolution even beyond that occurring in the IgE-producing B cell population.

A role for Mlh3 in somatic hypermutation

Somatic hypermutation (SHM) and class switch recombination (CSR) allow B cells to make high affinity antibodies of various isotypes. Both processes are initiated by activation-induced cytidine deaminase (AID) to generate dG:dU mismatches in the immunoglobulin genes that are resolved differently in SHM and CSR to introduce point mutations and recombination, respectively. The MutL homolog MLH3 has been implicated in meiosis and DNA mismatch repair (MMR). Since it interacts with MLH1, which plays a role in SHM and CSR, we examined these processes in Mlh3-deficient mice. Although deficiencies in other MMR proteins result in defects in SHM, Mlh3 −/− mice exhibited an increased frequency of mutations in their immunoglobulin variable regions, compared to wild type littermates. Alterations of mutation spectra were observed in the Jh4 flanking region in Mlh3 −/− mice. Nevertheless, Mlh3 −/− mice were able to switch to IgG3 or IgG1 with similar frequencies to control mice. This is the first instance where a loss of a DNA repair protein has a positive impact on the rate of SHM, suggesting that Mlh3 normally inhibits the accumulation of mutations in SHM.

Host genetic and adjuvant factors influence epitope specificity to a major recombinant grass allergen.

The role of host genetic and adjuvant factors in the induction of immune responses to a major recombinant Kentucky bluegrass allergen was examined utilizing five strains of mice and two different adjuvants. Analysis of the recombinant allergen-specific antibodies induced in these strains indicated that the antibodies of various isotypes were differentially regulated. In terms of IgE antibody response, BDF1 and DBA/2 were characterized as high responder, whereas BALB/C, CBA/J and C57BL/6 were intermediate and SJL was a low responder. In different strains, both dextran sulfate (DS) and complete Freund's adjuvant (CFA), as adjuvants, induced recombinant allergen-specific IgE antibodies of similar titer, however, CFA induced higher IgG2a and lower IgM antibodies compared to DS. Further, analysis of T cell proliferative responses of the splenocytes of different strains demonstrated that these strains varied also in their capacity to respond to synthetic peptides. Furthermore, utilizing a panel of synthetic peptides corresponding to the recombinant allergen, we demonstrated that the antibodies induced by the recombinant allergen with CFA in different strains vary with respect to their epitope specificity. In the BDF1 strain, compared to DS, CFA as adjuvant induced recombinant allergen-specific antibodies of additional peptide specificity. Taken together, these results suggest that both host genetic background and adjuvants govern the fine specificity of antibodies produced against this recombinant Kentucky bluegrass allergen.

Approaches for the Study of T-Cell Influences on B1-Cell Function

B1 cells are a subset of B lymphocytes, found in many species, that differ from conventional B cells (B2) in phenotype and function. B1 cells produce large amounts of low-affinity IgM, appear to be self-renewing, respond primarily to T-independent antigens, and, in the peritoneal cavity, do not generate lymphoid follicles. While B2 lymphocyte-T lymphocyte interactions result in proliferation, differentiation, and production of high-affinity antibodies of various isotypes, the precise role of T cells in regulating B1-cell activity remains largely unresolved. In this review, we focus on describing in detail two approaches that we have used for studying B1-cell-T-cell interactions-reconstitution of allotype-congenic, severe combined immunodeficient (SCID) mice and treatment with regulatory cytokines.

Immune response to a single peptide containing an immunodominant region of hepatitis C virus core protein: the isotypes and the recognition site.

We have used one single peptide covering the 17 N-terminal amino acids of the hepatitis C virus (HCV) core protein (c) to analyse the human immune response against B-cell epitope(s) within this region. The sequence MSTNPKPQRKTKRNTNR was obtained from two sequenced HCV genomes, and the peptide was synthesized by a newly developed method. The peptide was assayed with 144 human sera which had all been assayed for antibodies to HCV (anti-HCV) using commercial assays. Forty-nine sera were found to be positive for anti-HCV using these assays; 40 of these were found to be positive with our anti-HCV IgG peptide assay. The class (IgM, IgG) and subclass (IgA1, IgG1-4) specific reactions were determined using the polyclonal and monoclonal anti-HCV peptide enzyme immunoassays. Isotypes of mainly IgG1 and IgG3, but also IgG4, IgM and IgG2, gave specific reactions with this region. Using omission peptide analogues of the region 1-18, the sequence RKTKRNTN within residues 9-16 was common to 34 out of 37 sera of which the IgG antibody binding site could be mapped. It is unusual for a single peptide assay to have such high sensitivity since B cell epitopes within a protein are often discontinuous. It seems that at least 80% of HCV infected individuals develop antibodies of various isotypes to the antigenic site RKTKRNTN, located in the N-terminal portion of the HCV core. Thus, the immune response to this peptide should be further investigated with regard to the reactive Ig isotypes developing during HCV infection.

Epitope analysis of the oncofetal antigen alphafetoprotein using monoclonal antibodies

Alphafetoprotein (AFP), an oncofetal antigen, plays very important roles in the early embryonic life and oncogenesis. Under various physiological and pathological conditions AFP exhibits microheterogeneity, probably as a result of differential expression of its epitopes. To analyse the epitopes we have developed a panel of monoclonal antibodies against human AFP purified by a new and efficient method using an immunoadsorbent consisting of polyclonal antibodies immobilized on cyanogen bromide activated Sepharose. Clones producing antibodies of various isotypes, e.g. IgG 1, IgG 2a, IgG 2b. IgA and IgM have been subcloned and characterized. The antibodies showed high avidity for AFP (with half-maximal binding concentrations between 0.012 and 3.87 nM). Mutual inhibition efficiencies of a panel of 14 monoclonal antibodies were determined by RIA. Based on these inhibition data a computer program was used to group these antibodies with respect to their “epitope specificity distance”. As a result of this grouping, clones have been identified which can recognize at least five different epitopes on AFP. This panel of antibodies may be very useful for analysis of the epitoplc variation of AFP under various physiological and pathological conditions.

Humoral immune response to mammalian allergens.

Solid-phase enzyme immunoassay and passive hemagglutination were used to study the occurrence of antibodies of various isotypes against feline and bovine epithelial extracts in RAST-positive patients, exposed subjects (cat fanciers and senior veterinary students) and healthy controls. A high proportion of the patients had positive results, whereas the findings in exposed subjects varied, depending on the antigen and test technique. These observations caution against making far-reaching conclusions on the basis of narrow studies.

Switch region content of hybridomas: the two spleen cell Igh loci tend to rearrange to the same isotype.

We have examined the switch region content of 25 hybridomas that secret antibodies of various isotypes with specificity for phosphocholine or glycoproteins of herpes simplex virus. These Southern hybridization experiments included probes for the murine JH region as well as probes for the mu, gamma 3, gamma 1, gamma 2b, gamma 2a, and alpha switch regions. For 22 of the hybridomas, the deletion model of the heavy chain switch fits the data well--all switch regions upstream of the rearranged (and expressed) switch regions are deleted and all switch regions downstream remain in the germline configuration. As exceptions to a simple deletion model of the switch recombination, we have observed two, and perhaps three, examples of switch region rearrangements downstream of an expressed heavy chain gene. The 25 hybridoma DNA samples include 28 rearranged gamma switch regions; the sizes of at least 25 of these rearranged fragments are consistent with recombination in the tandemly repeated sequences associated with gamma genes. For those hybridomas with two spleen cell-derived Igh loci, including three mu-expressers, three gamma 3-expressers, four gamma 1-expressers, and one gamma 2b-expresser, the two loci tend to be rearranged to the same switch region, suggesting that the heavy chain switch rearrangement is an isotype-specific event. The exceptions within this group include three hybridomas in which the switch seems to be incomplete--on one chromosome the JH complex is rearranged to the S gamma 3 region, while on the other it remains associated with the S mu region. A second group of hybridomas, which includes four gamma 3-expressers, have both gamma 3 and gamma 1 switch rearrangements. Each of these four hybridomas includes three rearranged JH segments, suggesting that they may be the result of an unusual differentiative pathway or a technical artifact. These experiments suggest that the heavy chain switch rearrangement in normal spleen cells is a deletion event that occurs within tandemly repeated elements. The rearrangement is mediated by factors with partial, or perhaps complete, isotype specificity.

Salivary and serum antibodies in patients with Yersinia enterocolitica infection

The systemic and secretory antibody response in patients with yersiniosis was studied by measuring Yersinia antibodies of various isotypes (IgG, IgA and IgM) and the total concentrations of the corresponding Ig classes in serum and saliva. Specific antibody activities of IgG (IgG antibody concentration divided by IgG concentration) were almost identical in serum and saliva of all patients although the pair of values varied from patient to patient. Almost all salivary IgG of these patients was therefore probably a transudate from the blood. Specific antibody activities of IgA and of IgM, on the other hand, varied independently in serum and saliva. Occasional great differences between serum and saliva values indicate that most of the salivary IgA and IgM (more than 90%) is produced locally at least in some individuals. The local anti-Yersinia response was restricted to IgA in some individuals, to IgM in others, whilst yet other patients produced salivary antibodies of both isotypes.

Effects of a Crystalline Silica on Antibody Production to T-Dependent and T-Independent Antigens in Balb/c Mice

A crystalline silica (standard quartz DQ12 with particle size less than 5 micrometers) is able to stimulate in Balb/c mice the production of antibodies of various isotypes to the T-dependent antigen trinitrophenylated keyhole limpet hemocyanin. Under the same experimental conditions silica was not able to stimulate antibody response to the T-independent antigen trinitrophenylated Ficoll. These results support the possibility that adjuvanticity of silica is correlated to its effects on one or more of the various functions carried out by macrophages on immune responses.

A new function for platelets: IgE-dependent killing of schistosomes.

Several killing mechanisms against schistosomes have been described in vitro, involving cellular and humoral factors. Neutrophils, eosinophils--with an accessory role for mast cells--monocytes and macrophages have been shown to exhibit cytotoxic properties against Schistosoma mansoni larvae, in association with antibodies of various isotypes or with complement (reviewed in ref. 1). Lymphocyte participation in effector functions is mediated mainly through lymphokines inducing cytotoxic macrophages, and, in certain cases, directly by T cells. The experiments reported here show that platelets, taken from rats after specific periods of infection with S. mansoni, were able to kill schistosomula, and that normal human or rat platelets acquired toxic properties towards the same target in the presence of serum from infected individuals. The humoral factor involved in this process was shown to be IgE, and evidence was obtained of a Fc receptor for IgE on human and rat platelets. The passive transfer of immune platelets to normal rats conferred a high degree of protection towards a challenge infection by the parasite.

Regulation of the antibody response against hapten-coupled erythrocytes by monoclonal antihapten antibodies of various isotypes

The plaque-forming cell (PFC) response of C57BL/6 mice to NP-coupled sheep red blood cells can be inhibited by administration of microgram doses of a variety of monoclonal anti-NP antibodies of various isotypes prior to immunization. NP-specific PFC are most strongly affected but a drastic reduction of the SRC-specific response also occurs. The latter depends on physical association of the immunizing red cells with NP groups. Inhibition was obtained with antibodies of all IgG subclasses, but was marginal or absent when up to 100 μg IgM or IgD antibodies or F(ab′) 2 fragments were administered. Our experiments lead us to conclude that antibody feedback inhibition is not the function of a particular IgG subclass and that the role of the variable region in feedback inhibition is sufficiently explained by its affinity for the antigen. It was noted that small doses of a monoclonal NP-specific IgM antibody failed to enhance the response to NP-SRC.

Subclass restriction of murine antibodies: V. The IgG plaque-forming cell response to thymus-independent and thymus-dependent antigens in athymic and euthymic mice

Antigens differ in their abilities to stimulate antibodies of various isotypes. Many thymus-independent (TI) polysaccharide antigens stimulate largely IgG3 and IgM antibodies while thymus-dependent (TD) protein antigens stimulate predominantly IgG1 and smaller amounts of other isotypes. Here we determine whether thymus dependence or independence is a property of antigens which is expressed equally by all isotypes. To do this nu/+ and nu/nu mice were immunized with several TI and TD antigens and antibody responses of IgM and the four IgG subclasses measured. We found that, within the conditions of these experiments, all IgG isotypes were influenced equally by the presence or absence of T lymphocytes. Second, in agreement with J. L. Press ( J. Immunol. 126, 1234, 1981), we propose a division of TD antigens into two types based upon the ability to stimulate responses in the CBA/N mouse.