Comparison of the fetal and adult functional B cell repertoires by analysis of VH gene family expression.

The functional B cell repertoire in BALB/c mice was assessed at various stages in ontogeny. This was done by analyzing VH gene family expression using the sensitive technique of in situ hybridization. The B cell repertoire was probed with the mitogen, LPS, and the antigen DNP. DNP was chosen because B cells responsive to this hapten appear very early in ontogeny. The APCs that developed after stimulation with LPS or DNP were analyzed for VH gene expression by in situ hybridization of individual cells using radiolabeled VH gene family probes. The results indicated that VH gene expression in fetal B cells after stimulation was distinct from adult B cells in that there was a biased expression of D proximal families. The results indicated that this bias was associated with developmental age and not a given differentiation stage in the B cell lineage. In addition, stimulation of fetal B cells with DNP resulted in a large increase in expression of member(s) of VH 36-60, suggesting that the early appearance of DNP-responsive B cells is not strictly correlated with preferential rearrangement of D proximal families, VH 7183 and VH Q52. However, the results suggested that a large proportion of pre-B cells that preferentially rearrange D proximal families early in ontogeny become part of the functional developing repertoire.

Restricted kappa chain expression in early ontogeny: biased utilization of V kappa exons and preferential V kappa-J kappa recombinations.

To determine the extent of kappa chain diversity in the preimmune repertoire early in development, kappa cDNA libraries were analyzed from 15-d old fetal omentum, 18-d-old fetal liver, and 3-wk old bone marrow. An anchored polymerase chain reaction approach was used to avoid bias for particular V kappa families. From the sequence analysis of 27 bone marrow clones, 10 different families and 20 unique V kappa genes were identified. In contrast, the V kappa expression in the fetus is highly restricted and clearly differs from the broader distribution see in 3-wk-old bone marrow. Although several V kappa families were represented in the fetal library including V kappa 9, V kappa 10, V kappa 4,5, V kappa 8, and V kappa 1, one or two members of individual families were observed repeatedly. The fetal liver and omentum libraries were found to be largely overlapping. Given the V kappa families/exons identified in the fetal sequences, the mechanism of kappa rearrangements in the early repertoire appears to occur predominantly by inversion. Importantly, the fetal repertoire was further restricted by dominant V kappa-J kappa combinations such as V kappa 4,5-J kappa 5, V kappa 9-J kappa 4, and V kappa 10-J kappa 1. Since in some cases independent rearrangements could be established, the results indicate a bias for particular V kappa-J kappa joins. The results also suggest that clonal expansion/selection in the fetal repertoire takes place after light chain rearrangement as opposed to at the pre-B cell level in the bone marrow. The restriction observed in kappa light chain expression together with known restrictions in gene usage and junctional diversity at the heavy chain level indicate a remarkably conserved fetal repertoire.

Ontogenic development of B-lymphocyte function and tolerance susceptibility in vivo and in an in vitro organ culture system.

The maturation of B-lymphocyte function during fetal development was studied in vivo and in an in vitro organ culture system. The results indicated that the progenitors for 2,4-dinitrophenol (DNP)-specific B cells are present as early as 14 d of gestation in liver and possibly as early as 15 d in spleen. In addition, it was found that the organ culture system supports the development of B lymphocytes as measured by an increase in both the percentage of surface immunoglobulin-positive cells and the frequency of clonable DNP-specific B cells after culturing. The majority of anti-DNP-secreting clones resulting from the antigenic stimulation of fetal B cells produced only the IgM isotype, and the ability to secrete the IgG isotypes increased as a function of gestational age. Because fetal DNP precursors from spleens and livers that had been incubated in organ culture resulted in a greater proportion of clones secreting IgG compared with age-matched uncultured controls, it was concluded that the maturation with regard to the ability to secrete IgG can occur in vitro. In studies relating to the ontogenetic development of tolerance susceptibility, it was found that up to one-half of the DNP-specific B-cell precursors from livers and spleens less than 18 or 19 d of gestation were resistant to tolerogen treatment for 24 h as if in a pretolerant phase. However, if tolerogen were present for 3--5 d during organ culture there was near total elimination of potential DNP clones. This finding suggested that the 24-h induction period was insufficient for affecting the DNP-specific precursors in livers and spleens from the earlier gestational ages, and that a proportion of precursors could subsequently form DNP clones in the splenic focus assay after the removal of tolerogen.

In vitro model for natural tolerance to self-antigens. Inhibition of the development of surface-immunoglobulin-negative lymphocytes into T-dependent responsive B cells by antigen.

Neonatal and adult splenic cell suspensions were labeled with fluorescein isothiocynate-anti-Ig and fractionated into surface-immunoglobulin- (s-Ig) positive and s-Ig-negative subpopulations by the fluorescence-activated cell sorter. The subpopulations were then tested by splenic focus assay for both frequency and tolerance susceptibility of clonable 2,4,-dinitrophenol (DNP) precursors. It was shown that both adult, and neonatal, s-Ig-negative subsets contained clonable DNP-specific B-cell precursors. However, because these precursors result in fewer clones secreting IgG, they appeared to be less mature than the s-Ig-positive precursors. In the absence of helper T cells, it was found that exposure of s-Ig-negative lymphocytes to tolerogen during the process in which they were acquiring surface receptors resulted in nearly total abrogation of potential DNP clones. This finding provides compelling evidence for clonal abortion.

A clonal analysis of the IgE response and its implications with regard to isotope commitment.

In a clonal analysis of the IgE response, it was found that a small proportion of primary or nonimmune B cells in spleen and mesenteric lymph nodes can be stimulated by antigen to produce IgE-secreting clones. In addition, there appears to be no substantial difference in the frequency of such cells between the classical low and high IgE responder strains. An analysis of immune, or memory, B cells revealed substantial increases in the frequency of B cells secreting IgE as compared with primary B cells, although the actual proportion of B cells secreting IgE remained relatively low. When the IgE-secreting clones derived from either primary or secondary B cells were reanalyzed for the presence of other isotypes, it was found that all clones secreting IgE were secreting at least one other isotype, with the majority secreting two or three other isotypes in addition to IgE. This demonstrates that there is no distinct subpopulation of B cells committed to IgE expression per se.

Diversity of the T-cell receptor BV repertoire in HIV-1-infected patients reflects the biphasic CD4+ T-cell repopulation kinetics during highly active antiretroviral therapy.

OBJECTIVES: Highly active antiretroviral therapy (HAART) induces a decline in viral load and a biphasic increase in peripheral blood CD4+ T-cell counts in HIV-infected patients. To evaluate the effect of HAART on T-cell receptor (TCR) diversity of repopulating naive and memory CD4+ T cells, complementarity determining region 3 (CDR3) spectratyping was performed. DESIGN: For four patients treated with HAART, CD45RO+ (memory) and CD45RA+ (naive) CD4+ T cells were isolated from peripheral blood leukocyte samples obtained 1 week before, 1-2 months after, and 9-11 months after start of treatment. METHODS: CDR3 regions were amplified by TCR-BV-specific nested PCR from CD4+ T-cell subsets. CDR3 size distributions and single-strand conformation polymorphism profiles were compared as an indication for TCR diversity. RESULTS: Increasing blood CD4+ T-cell counts during the first 2 months of treatment coincided with increased perturbation of CDR3 patterns in CD4+ T-cell subsets, suggesting an early oligoclonal repopulation. At later timepoints, CDR3 size diversity increased when T-cell counts did not substantially decrease. Memory and naive CD4+ T cells generally showed comparable levels of perturbation. CONCLUSION: Diversity of the TCR repertoire reflected biphasic T-cell repopulation during HAART, compatible with initial redistribution and later CD4+ T-cell production. Sustained elevation of T-cell counts will in principle result in restoration of TCR diversity.

T cell expansions in lymph nodes and peripheral blood in HIV-1-infected individuals: effect of antiretroviral therapy.

OBJECTIVE: To evaluate dynamics in CD8 T cell expansions during highly active antiretroviral therapy (HAART). DESIGN: Various T cell subsets were isolated from blood and lymph nodes and analysed for T cell receptor (TCR) diversity. METHODS: TCR complementarity determining region 3 (CDR3) spectratyping and single-strand conformation polymorphism (SSCP) analyses were performed in combination with sequencing to assess clonality of the subsets. RESULTS: Strongly skewed CDR3 patterns in total CD8 cells and the CD8 subsets CD45RO+CD27+ and CD45RO-CD27+ showed substantial dynamics in dominant CDR3 sizes, resulting in relative improvement of CDR3 size diversity in the first months of therapy. During sustained treatment, TCR diversity changed only moderately. SSCP profiles confirmed oligoclonality of TCR CDR3 perturbations. Various dominant CDR3 sizes for CD4 and CD8 T cells present in lymph nodes, but not in peripheral blood mononuclear cells, before the start of therapy emerged in peripheral blood early during therapy. CONCLUSIONS: HAART induces substantial changes in CD8 TCR diversity, eventually resulting in improvement of the repertoire. Clonal expansions observed in lymph nodes before therapy were observed in peripheral blood after therapy, suggesting that recirculation of CD4 and CD8 T cells from lymph nodes contributes to the early T cell repopulation. Decreased immune activation and possibly naive T cell regeneration subsequently decreased clonal expansions and perturbations in the CD8 TCR repertoire.

The role of N-linked carbohydrates in the antigenicity of Taenia solium metacestode glycoproteins of 12, 16 and 18 kD.

The glycoproteins of 12-28 kD from Taenia solium metacestodes provide a high specificity and sensitivity for the serological diagnosis of the central nervous system infection, neurocysticercosis. Their widespread use as antigens for routine serological assays will require their production in large and reproducible amounts. Prior to determining the ideal strategy to produce these antigens at a large scale, it is important to determine the contribution of the carbohydrates to the antigenicity of these molecules, given the uncertainty of reproducing saccharidic epitopes in recombinant expression systems. In this study we examined this issue. The chemical oxidation of the carbohydrates of the 12-28 kD glycoproteins with sodium metaperiodate, reduced the antigenicity of the molecules to variable extents, with the more notable changes being detected for the 18 and 28 kD antigens. This approach was complemented by purification of the 12, 16 and 18 kD antigens, followed by the enzymatic deglycosylation of their abundant N-linked oligosaccharides. Silver-stained SDS-PAGE analysis indicated that the three deglycosylated antigens now migrated as 7 kD products, suggesting a protein backbone with a similar size, but different extents of glycosylation. By Western blot, the antigenicity of these antigens was diminished. This was more notable for the 18 kD antigen, which is more heavily glycosylated than the 12 or 16 kD glycoproteins. These data suggest that the antigenicity of the glycoproteins of T. solium is due to a combination of carbohydrate and protein epitopes.

Analysis of immune lesions in neurocysticercosis patients: central nervous system response to helminth appears Th1-like instead of Th2.

Neurocysticercosis (NCC) caused by the helminth Taenia solium is the most common parasitic infection of the human central nervous system (CNS) worldwide. Because clinical symptoms are associated with localized immunological responses in the brain, characterization of these responses are pivotal for understanding the pathogenesis of cysticercosis. Immunohistochemical analysis of brain specimens from several patients with cysticercosis revealed at least four types of immune responses, including: (i) an antibody response (IgM + plasma cells), (ii) a predominant NK response, (iii) an infiltrate with abundant macrophages and granulocytes, and (iv) an intense infiltrate with a predominance of macrophages and T cells. The intensity and type of immunity appeared to be associated somewhat with the parasite's viability and anatomical location. In most of the lesions, cell mediated responses were evident and proinflammatory cytokines including IL12 predominated. Moreover, IL4 was undetectable in the immune infiltrates. Thus, the CNS response to this helminth, unlike the systemic response, is predominately Th1-like.

The human nervous tissue in proximity to granulomatous lesions induced by Taenia solium metacestodes displays an active response.

In neurocysticercosis, the nervous tissue surrounding the brain lesion is affected as a consequence of the local immune response induced by a Taenia solium metacestode. In this study, a histological and immunohistochemical analysis of five brain specimens from patients with neurocysticercosis revealed a proinflammatory activity reflected by an apparently altered blood-brain barrier permeability, secretion of pro-inflammatory cytokines, and up-regulation of molecules associated with antigen presentation. There were also anti-inflammatory cytokines, as well as an active wound-healing process reflected by angiogenesis, collagen deposition and glial scar formation. This immune response displayed by the nervous tissue adjacent to chronic neurocysticercosis lesions appeared to be contributing to the local tissue damage, and hence, may be fundamental in the pathology of NCC.

Comparative expression of adult and fetal V gene repertoires.

A hallmark of the immune system is the extraordinary diversity associated with antibodies. This is made possible by a series of genetic rearrangements involving variable region gene segments. Considerable detail is known about these genetic mechanisms except for the enzymatic machinery involved. An important question in studies of the generation of diversity is whether V genes are selected for rearrangement mainly in a random manner or selected by particular developmental rules. Past studies have indicated that the acquisition of fetal and neonatal specificity repertoires is a nonrandom process. In this report, we review our studies that directly compare the adult and fetal/neonatal V gene repertoires. The evidence suggests that the adult repertoire is more diverse with indications of a random use of VH gene families. However, whether V genes are indeed randomly used in the adult remains to be clarified at the VH gene member level. The fetal repertoire, on the other hand, appears nonrandom in V gene usage. In addition, the fetal repertoire is mostly germline encoded with little evidence of junctional diversity. Taken together, the results indicate different rules for generation of the adult and fetal repertoires, findings most likely explain by distinct B cell subsets and B cell progenitors at early stages in ontogeny.

Characterisation of the carbohydrate components of Taenia solium metacestode glycoprotein antigens.

Human neurocysticercosis is caused by Taenia solium metacestodes. It usually affects the central nervous system of humans and can be confused with other brain pathologies. The Lens culinaris-binding glycoproteins from this parasite have been shown to be ideal targets for the development of a highly specific immunoassay for the diagnosis of neurocysticercosis. In the present study we characterised the carbohydrates associated with five antigenic glycoproteins of T. solium metacestodes in the range of 12-28 kilodaltons. Lectin-affinities and enzymatic deglycosylations suggested that each of the five antigens contain various glycoforms of asparagine-linked carbohydrates of the hybrid, complex and probably high mannose type. These carbohydrates accounted for at least 30-66% of the apparent molecular mass of the glycoconjugates. In contrast, there was no evidence for the presence of O-linked carbohydrates. Lectin affinity patterns suggested that the sugars are short and truncated in their biosynthetic route, and that some contain terminal galactose moieties. Elucidating the precise structure of the carbohydrates and establishing their role in antigenicity will be essential to design strategies to produce them in large and reproducible amounts for the development of improved immunoassays.

Ig heavy chain CDR3 size diversities are similar after conventional peripheral blood and ex vivo expanded hematopoietic cell transplants.

It is largely unknown whether the immune repertoire can be reconstituted successfully after high-dose chemotherapy and transplantation using ex vivo expanded hematopoietic stem cell (HSC) grafts. It is critically important for the transplant outcome that immune repertoire reconstitution progresses after ex vivo expanded HSC graft transplants at least as efficiently as that seen after conventional HSC transplants. Previously, we showed that the T cell receptor V beta (TCRVB) third complementarity determining region (CDR3) diversification after ex vivo expanded bone marrow (BM) HSC graft transplants was similar to that seen after conventional peripheral blood stem cell transplants (PBSCTs). In the present study, the CDR3 diversity of the six immunoglobulin (Ig) heavy chain variable region gene (V(H)) families was examined in five breast cancer patients who were transplanted with ex vivo expanded BM HSCs as the only source of stem cells. For comparison, 12 healthy adults and four conventional PBSCT recipients were also studied. Using both CDR3 fingerprinting and single strand conformation polymorphism (SSCP) methodologies, it is shown that the contribution of the V(H) families to the overall repertoire among healthy adults is highly variable and not always proportional to V(H) family member size. After both ex vivo expanded HSC transplants and conventional PBSCTs, the V(H) CDR3 repertoires were limited in size diversity at 6 weeks post transplant. By 6 months, however, V(H) families displayed a repertoire diversity that was as complex as that seen in healthy adults. No difference was seen between ex vivo expanded HSC graft transplant recipients and conventional PBSCT recipients in V(H) repertoire diversity. In one patient there was a follow-up analysis 12 months after ex vivo expanded graft transplant, and the diversity of the V(H) families was maintained. In all patients, the amino acid size of the CDR3 regions fell within adult limits at all time points post transplant. These results indicate that B cell repertoire regeneration after ex vivo expanded hematopoietic cell graft transplants is similar to that seen after conventional PBSCT.

Analysis of the peripheral immune response in patients with neurocysticercosis: evidence for T cell reactivity to parasite glycoprotein and vesicular fluid antigens.

In neurocysticercosis (NCC), it is thought that the long-term survival of the parasite within the human brain is due in part to the ability of the cestode to suppress the local immune response. When the parasite dies, the immunosuppression is apparently lost and a strong local inflammatory response then develops. In contrast, little is known about the immunologic response that may occur in the peripheral immune system of these patients. In this study, the status of the peripheral (extracerebral) cellular and humoral response was evaluated in patients with a history of NCC. The in vitro proliferation of peripheral blood mononuclear cells to mitogens and foreign antigens was similar in patients and controls. Importantly, a substantive response was elicited by two Taenia solium metacestode antigens. In addition, 8 of 10 patients had a detectable humoral response to the antigenic glycoproteins of the cestode. Considering both the cellular and humoral response, all of the patients with NCC presented an active peripheral immunity.

Effect of fetal exposure to ultrasound on the development of functional, antigen-specific B lymphocytes in fetal and neonatal BALB/c mice.

In this study, the influence of fetal exposure to ultrasound on the development of immunocompetence is addressed. Pregnant BALB/c mice were exposed to various intensities of ultrasound ranging from 0.1-3.0 W/cm2. B cells from 19 day old fetal livers or 5 and 10 day old neonatal spleens were assessed for the following: (a) differentiation into plasma cells after mitogenic stimulation with lipopolysaccharide (LPS); (b) development and frequency of 2,4-dinitrophenyl (DNP)-specific B cells, and (c) the ability to produce antibody in response to DNP. Comparison between ultrasound-exposed and sham-treated groups did not reveal any evident differences in the above tests. The results suggest that ultrasound neither hinders nor augments the development of immunocompetent B cells.

Effect of fetal exposure to ultrasound on B lymphocyte function and antibody class production.

The purpose of this study was to determine if ultrasound radiation would affect the ability of antigen-stimulated B cells and their clonal progeny to undergo the isotype switch and produce diverse antibody classes. Pregnant BALB/c mice were exposed to various intensities of ultrasound (0.1-3.0 W/cm2) at different ages of gestation. Spleens from the resulting offspring were removed five or ten days after birth. The splenocytes were analyzed for the frequency of B cells capable of responding to the hapten 2,4 dinitrophenyl (DNP) using the splenic fragment assay, a B cell cloning assay. The DNP-responsive B cell clones were then classified on the basis of isotype expression. It was found that ultrasonic radiation during gestation did not alter the frequency of B cells responding to DNP. Furthermore, ultrasound had no apparent effect on the B lymphocyte's capacity to switch to different isotypes after antigenic stimulation. Thus, the results indicate that prenatal exposure to ultrasound does not appreciably affect the genetic and cellular processes necessary for the isotype switch and antibody class production.

Effect of fetal exposure to ultrasound on B cell development in BALB/c mice.

Ultrasound is a major tool for clinical diagnosis, especially during prenatal life. Therefore, it is important that the potential bioeffects of ultrasound be determined. In this report, the effects of ultrasound on the development of B lymphocytes is studied. Pregnant BALB/c mice were exposed to intensities of ultrasound ranging from 0.1-3.0 W/cm2, receiving either a single exposure on day 11, or multiple exposures on days 14, 15, and 18 or 19 of gestation. Fetal livers were removed on day 19 of gestation whereas spleen cells were obtained from 5 and 10 day old neonates. These cell populations were analyzed for: (a) the frequency of B220+ B lineage cells; (b) the frequency of immunoglobulin-positive (Ig+) cells, and (c) the ability to proliferate in response to the B cell mitogen lipopolysaccharide (LPS). None of the tests performed revealed any substantial differences between ultrasound-exposed versus sham-treated control animals. In addition, the development of blood cell types other than B lineage cells remained unaffected by exposure to ultrasound. Therefore, exposure to ultrasound at the intensities used does not appear to hinder hemopoiesis or the normal development of B lymphocytes during fetal and early neonatal life.

Aged mice display an altered pulmonary host response to Francisella tularensis live vaccine strain (LVS) infections.

Aging is a complex phenomenon that has been shown to affect many organ systems including the innate and adaptive immune systems. The current study was designed to examine the potential effect of immunosenescence on the pulmonary immune response using a Francisella tularensis live vaccine strain (LVS) inhalation infection model. F. tularensis is a Gram-negative intracellular pathogen that can cause a severe pneumonia. In this study both young (8-12 week old) and aged (20-24 month old) mice were infected intranasally with LVS. Lung tissues from young and aged mice were used to assess pathology, recruitment of immune cell types and cytokine expression levels at various times post infection. Bacterial burdens were also assessed. Interestingly, the lungs of aged animals harbored fewer organisms at early time points of infection (day 1, day 3) compared with their younger counterparts. In addition, only aged animals displayed small perivascular aggregates at these early time points that appeared mostly mononuclear in nature. However, the kinetics of infiltrating polymorphonuclear neutrophils (PMNs) and increased cytokine levels measured in the bronchial alveolar lavage fluid (BALF) were delayed in infected aged animals relative to young infected animals with neutrophils appearing at day 5 post infection (PI) in the aged animals as opposed to day 3 PI in the young infected animals. Also evident were alterations in the ratios of mononuclear to PMNs at distinct post infection times. The above evidence indicates that aged mice elicit an altered immune response in the lung to respiratory F. tularensis LVS infections compared to their younger counterparts.