Analysis of the B cell receptor repertoire in six immune-mediated diseases.

B cells are important in the pathogenesis of many, and perhaps all, immune-mediated diseases. Each B cell expresses a single B cell receptor (BCR)1, and the diverse range of BCRs expressed by the total B cell population of an individual is termed the 'BCR repertoire'. Our understanding of the BCR repertoire in the context of immune-mediated diseases is incomplete, and defining this could provide new insights into pathogenesis and therapy. Here, we compared the BCR repertoire in systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Crohn's disease, Behçet's disease, eosinophilic granulomatosis with polyangiitis, and immunoglobulin A (IgA) vasculitis by analysing BCR clonality, use of immunoglobulin heavy-chain variable region (IGHV) genes and-in particular-isotype use. An increase in clonality in systemic lupus erythematosus and Crohn's disease that was dominated by the IgA isotype, together with skewed use of the IGHV genes in these and other diseases, suggested a microbial contribution to pathogenesis. Different immunosuppressive treatments had specific and distinct effects on the repertoire; B cells that persisted after treatment with rituximab were predominately isotype-switched and clonally expanded, whereas the inverse was true for B cells that persisted after treatment with mycophenolate mofetil. Our comparative analysis of the BCR repertoire in immune-mediated disease reveals a complex B cell architecture, providing a platform for understanding pathological mechanisms and designing treatment strategies.

Exhaustive Search of the Receptor Ligands by the CyCLOPS (Cytometry Cell-Labeling Operable Phage Screening) Technique.

Effective and versatile screening of the peptide ligands capable of selectively binding to diverse receptors is in high demand for the state-of-the-art technologies in life sciences, including probing of specificity of the cell surface receptors and drug development. Complex microenvironment and structure of the surface receptors significantly reduce the possibility to determine their specificity, especially when in vitro conditions are utilized. Previously, we designed a publicly available platform for the ultra-high-throughput screening (uHTS) of the specificity of surface-exposed receptors of the living eukaryotic cells, which was done by consolidating the phage display and flow cytometry techniques. Here, we significantly improved this methodology and designed the fADL-1e-based phage vectors that do not require a helper hyperphage for the virion assembly. The enhanced screening procedure was tested on soluble human leukocyte antigen (HLA) class II molecules and transgenic antigen-specific B cells that express recombinant lymphoid B-cell receptor (BCR). Our data suggest that the improved vector system may be successfully used for the comprehensive search of the receptor ligands in either cell-based or surface-immobilized assays.

Single-Cell RNA Sequencing of Peripheral Blood Reveals Immune Cell Signatures in Alzheimer's Disease.

The peripheral immune system is thought to affect the pathology of the central nervous system in Alzheimer's disease (AD). However, current knowledge is inadequate for understanding the characteristics of peripheral immune cells in AD. This study aimed to explore the molecular basis of peripheral immune cells and the features of adaptive immune repertoire at a single cell level. We profiled 36,849 peripheral blood mononuclear cells from AD patients with amyloid-positive status and normal controls with amyloid-negative status by 5' single-cell transcriptome and immune repertoire sequencing using the cell ranger standard analysis procedure. We revealed five immune cell subsets: CD4+ T cells, CD8+ T cells, B cells, natural killer cells, and monocytes-macrophages cells, and disentangled the characteristic alterations of cell subset proportion and gene expression patterns in AD. Thirty-one cell type-specific key genes, comprising abundant human leukocyte antigen genes, and multiple immune-related pathways were identified by protein-protein interaction network and pathway enrichment analysis. We also found high-frequency amplification clonotypes in T and B cells and decreased diversity in T cells in AD. As clone amplification suggested the activation of an adaptive immune response against specific antigens, we speculated that the peripheral adaptive immune response, especially mediated by T cells, may have a role in the pathogenesis of AD. This finding may also contribute to further research regarding disease mechanism and the development of immune-related biomarkers or therapy.

In vitro immunogenicity prediction: bridging between innate and adaptive immunity.

Development of antidrug antibodies (ADAs) is an undesirable potential outcome of administration of biotherapeutics and involves the innate and adaptive immune systems. ADAs can have detrimental clinical consequences: they can reduce biotherapeutic efficacy or produce adverse events. Because animal models are considered poor predictors of immunogenicity in humans, in vitro assays with human innate and adaptive immune cells are commonly used alternatives that can reveal cell-mediated unwanted immune responses. Multiple methods have been developed to assess the immune cell response following exposure to biotherapeutics and estimate the potential immunogenicity of biotherapeutics. This review highlights the role of innate and adaptive immune cells as the drivers of immunogenicity and summarizes the use of these cells in assays to predict clinical ADA.

A new score including CD43 and CD180: Increased diagnostic value for atypical chronic lymphocytic leukemia.

Moreau score has been used to differentiate chronic lymphocytic leukemia (CLL) from other mature B-cell neoplasms. However, it showed limitations in Asian patients. Therefore, we conducted a new score system replacing CD5 and CD23 with CD43 and CD180 to evaluate its diagnostic value of CLL. 237 untreated samples diagnosed with mature B-cell neoplasms were collected and were randomly divided into an exploratory and a validation cohort by a 2:1 ratio. The expression of CD5, CD19, CD20, CD23, CD43, CD79b, CD180, CD200, FMC7, and surface immunoglobulin (SmIg) were analyzed among all the samples. A proposed score was developed based on the logistic regression model. The sensitivity and specificity of the proposed score were calculated by ROC curves. CD43/CD180, CD200, FMC7, and CD79b were included in our new CLL score, which showed a sensitivity of 91.8% and a specificity of 83.1%. These results were confirmed in a validation cohort with a sensitivity of 90.5% (p = 0.808) and a specificity of 79.5% (p = 0.639). In CD5 negative or CD23 negative CLL group, the new CLL score displayed improved sensitivity of 79.4% compared to Moreau score and CLLflow score (41.2% and 47.1%, respectively). In atypical CLL group, the new CLL score showed improved sensitivity of 84.2% compared to Moreau score and CLLflow score (61.4% and 64.9%, respectively). This proposed atypical CLL score helped to offer an accurate differentiation of CLL from non-CLL together with morphological and molecular methods, particularly in Chinese patients with atypical immunophenotype.

Surface antigen expression on Plasmodium falciparum-infected erythrocytes is modified in alpha- and beta-thalassemia.

In an attempt to determine the mechanism whereby thalassemia in its milder forms may protect against malaria, we have examined the expression of neoantigen at the surface of Plasmodium falciparum-parasitized thalassemic red cells. Neoantigen expression was estimated by measurement of antibody bound after incubation in serum from adults living in a malaria-endemic area, using a quantitative radiometric antiglobulin assay. We found that P. falciparum-parasitized alpha- and beta-thalassemic red cells bind greater levels of antibody from endemic serum than controls: mean binding ratios (+/- SE), respectively, for alpha- and beta-thalassemia compared with controls were 1.69 +/- 0.12 and 1.23 +/- 0.06 on a cell for cell basis, and 1.97 +/- 0.11 and 1.47 +/- 0.08 after a correction for surface area differences. Binding of antibody increased exponentially during parasite maturation. In addition, we found a small but significant degree of binding of naturally occurring antibody to parasitized red cells, the extent of which was also greater in thalassemia. The apparent protective effect of thalassemia against malaria may be related to enhanced immune recognition and hence clearance of parasitized erythrocytes.

Induction of human immunoglobulin synthesis and secretion in somatic cell hybrids of mouse myeloma and human B lymphocytes from patients with agammaglobulinemia.

Somatic cell hybrid clones were isolated from the fusion of RPC 5,4 mouse myeloma cells and B lymphocytes from three patients with agammaglobulinemia. One patient had X-linked agammaglobulinemia; the remaining two patients had common varied agammaglobulinemia. All three patients had B lymphocytes which fail to secrete immunoglobulin. The hybrid nature of the clones was established by examination of metaphase chromosome spreads. Most of the clones from all three patients expressed surface immunoglobulin of mouse and human parental origin. Clones from two of the patients had fewer cells with surface Ig than hybrids from normal persons, while clones from the third patient had large numbers of surface Ig fluorescent cells. Most of the clones from all three patients synthesized and secreted human and mouse immunoglobulin. As determined by sodium dodecyl sulfate acrylamide gel electrophoresis of radioactively labeled proteins, clones from each of the patients produced human gamma, alpha, and mu-heavy chains. These studies demonstrate the presence of functional structural genes coding for human immunoglobulin heavy chains in B lymphocytes of patients with agammaglobulinemia. Further, they represent induction in the somatic cell hybrids of a gene product not expressed in the parental B lymphocytes.

Gangliosides as markers for murine lymphocyte subpopulations.

Antibodies to GM1 ganglioside were used to study murine lymphocyte populations. In A, AKR, and BALB/c mice, anti-GM1 reacts with thymocytes and peripheral T cells. This reactivity of anti-GM1, studied by immunofluorescence, is independent of Thy-1 type and appears to be related to the reactivity of cross-reacting antibodies to asialo GM1 and GD1b, rather than GM1 itself. In addition, a subpopulation of lymphocytes reacting with anti-GM1 and anti-immunoglobulin has been found in approximately 26% of the peripheral lymphocytes of C3H mice, nude mice, and nude heterozygotes. This subpopulation is found in small numbers in A, AKR, and BALB/c mice. These studies demonstrate that antibodies to a chemically defined antigen can be used to identify T cells in many strains of mice and may delineate previously unrecognized lymphocyte subpopulations.

Inhibition of antibody-dependent cellular cytotoxicity and immunoglobulin synthesis by an antiserum prepared against a human B-cell Ia-like molecule.

Rabbit antisera to the human B-cell-specific antigen complex, p23,30, was used to define further the functional heterogeneity of isolated human lymphocyte subpopulations. Specific depletion of p23,30-bearing cells from Ig-negative cell populations and Ig-negative, E rosette-negative (Null) populations by either complement-mediated lysis or by physical separation on goat antirabbit Fab immunoabsorbent columns, eliminates the antibody-dependent cellular cytotoxic (ADCC) function. Furthermore, binding of anti-p23,30 serum to the effector cell surface inhibits ADCC but does not interfere with EA rosette formation. Apparently p23,30 represents a cell surface site which is distinct from the Fc receptor but which is important in the triggering of ADCC. In addition, depletion of p23,30-bearing cells from unfractionated cell populations, Ig-positive B-cell populations and Ig-negative, E rosette-negative (Null) populations eliminates the capacity of these populations to secrete immunoglobulin during subsequent culturing. Thus both the Ig-secreting cells and the ADCC effector cells within the Ig-negative, E rosette-negative (Null) population reside in the same population of cells which bears the p23,30 antigen.

A quantitative assay for transformation of bone marrow cells by Abelson murine leukemia virus.

A quantitative Abelson murine leukemia virus (A-MuLV) lymphoid cell transformation assay has been developed using a semisolid agarose culture system. Under these conditions lymphoid cell transformation was shown to vary linearly with the dose of A-MuLV used. The susceptibility of bone marrow cells from different strains of mice to A-MuLV-induced transformation can be estimated using the agarose assay. Strains with bone marrow cells of high, medium, and low susceptibility to A-MuLV can be identified. The assay has been used to study the susceptibility of cells from lymphoid organs of fetal and adult mice to A-MuLV. Cell suspensions from fetal liver, adult bone marrow, and adult spleen are susceptible to A-MuLV, while thymocytes are resistant to A-MuLV-induced transformation. Bovine serum albumin gradient fractionation of bone marrow cells before infection with A-MuLV demonstrates that the majority of A-MuLV-sensitive cells are recovered in a broad band partially overlapping the majority of the nucleated cells. The agarose assay system allows study of A-MuLV-lymphoid cell interaction at the level of single cell-single virus particle interaction.

The mouse gut T lymphocyte, a novel type of T cell. Nature, origin, and traffic in mice in normal and graft-versus-host conditions.

Lymphocytes of the mouse intestinal mucosa, identified in tissue sections or purified suspensions of intraepithelial lymphocytes as T cells (gut T lymphocytes [GTL]), were studied in normal mice or in beige mice (the equivalent of the Chediak-Higashi syndrome in man, characterized by giant granules in various cell types, including mast cells). Mice were studied in normal or in germ-free conditions, or during a graft versus host (GVH) reaction resulting from the injection of parental thymocytes into lethally irradiated F1 mice, a condition leading to massive accumulation of T lymphocytes of donor origin in the host gut mucosa. In normal as well as in GVH conditions, a high percentage of the gut IE lymphocytes contain granules (up to 80% in the beige mouse). These granules have ultrastructural, hostochemical and other features resembling those of mast cell granules; in beige mice, up to 50% of them can be shown to contain histamine. Granulated T cells are also found in the lamina propria. It appears that the GTL may progressively lose their surface T antigens when the granules become more developed. Kinetics of [3H]TdR labeling of the GTL, transfer experiments with T cells of various origins, selective [3H]TdR labeling and selective irradiation of the Peyer's patches (PP), and effect of thoraic duct (TD) drainage led to the conclusion that GTL are the progeny of T cells stimulated to divide in the PP microenvironment, which endows them with a gut-homing tendency. From the PP, these cells follow a cycle, migrating to the TD and to the blood to colonize the whole intestinal mucosa, the majority of them as dividing cells undergoing a single round of traffic, with some probably able to recirculate and becoming a more long-lived variety. Antigenic stimulation within the PP is necessary for the emergence of GTL progenitors, but their gut-homing property is unrelated to the antigen as shown with fetal gut grafts, notably in GVH where grafts syngeneic to the host or donor become similarly infiltrated by GTL. On the basis of their properties and of further evidence to be reported elsewhere, it is proposed that GTL belong to a special class of T lymphocytes, related to the immune defenses of the mucosal systems in general, and capable of acting as progenitors of mucosal mast cells.

The sequence of the mu transmembrane segment determines the tissue specificity of the transport of immunoglobulin M to the cell surface.

Membrane IgM is expressed on the surface of B lymphocytes. It is not transported to the surface of transfected plasmacytoma or COS cells. Here, we show that mutation of four hydrophilic amino acids in the microm transmembrane is sufficient to overcome the intracellular retention of membrane IgM in non-B cells. This suggests that the B cell-specific IgM-associated proteins that have been postulated to assist the transport of membrane IgM to the cell surface (3) act either by forming a hydrophobic sheath that surrounds the microm transmembrane segment or by displacing an interaction with this segment that would otherwise cause retention. Experiments with a CD8/mu hybrid H chain indicate that the proteins that assist the transport of membrane IgM to the B cell surface at most need the mu CH4 and transmembrane/cytoplasmic portion for interaction.

Detection of functional class II-associated antigen: role of a low density endosomal compartment in antigen processing.

We have developed a functional assay to identify processed antigen in subcellular fractions from antigen-presenting cells; stimulatory activity in this assay may be caused by either free peptide fragments or by complexes of peptide fragments and class II molecules present on organellar membrane sheets and vesicles. In addition, we have developed a functional assay to identify proteolytic activity in subcellular fractions capable of generating antigenic peptides from intact proteins. These techniques permit the direct identification of intracellular sites of antigen processing and class II association. Using a murine B cell line stably transfected with a phosphorylcholine (PC)-specific membrane-bound immunoglobulin (Ig), we show that PC-conjugated antigens are rapidly internalized and efficiently degraded to generate processed antigen within an early low density compartment. Proteolytic activity capable of generating antigenic peptide fragments from intact proteins is found within low density endosomes and a dense compartment consistent with lysosomes. However, neither processed peptide nor peptide-class II complexes are detected in lysosomes from antigen-pulsed cells. Furthermore, blocking the intracellular transport of internalized antigen from the low density endosome to lysosomes does not inhibit the generation of processed antigen. Therefore, antigens internalized in association with membrane Ig on B cells can be efficiently processed in low density endosomal compartments without the contribution of proteases present within denser organelles.

One synchronous wave of B cell development in mouse fetal liver changes at day 16 of gestation from dependence to independence of a stromal cell environment.

Precursor cells of the B lineage can be enriched from mouse fetal liver by FACS with the aid of the pre-B cell-specific mAb G-5-2. The cells are concomitantly enriched for cells expressing the pre-B cell-specific gene lambda 5, and for cells developing to LPS-reactive mature B cells. The enriched purified precursors are not influenced by rIL-2 through -7, alone or in combination, to develop to mitogen-reactive, sIg+ cells. Marginal proliferation of the precursors is observed in response to IL-3 plus -4, and IL-6 plus -7, and this does not change in the presence of stromal cells. Development to mitogen-reactive, sIg+ cells is dependent on interactions with embryonic stromal cells from fetal liver. Two mAbs raised against the stromal cells inhibit this development. Two phases of precursor cell development can be distinguished in fetal liver. Between days 13 and 15 of gestation, it is dependent on stromal cell interactions, thereafter, from days 16 to 19, it is independent. A sudden increase in the number of mitogen-reactive, sIg+ B lineage cells occurs within 24 h between days 16 and 17. All these results indicate that B cell development occurs in one wave with synchronous steps of changes from a mitogen-insensitive, sIg-, stromal cell dependent to a mitogen-reactive, sIg+, stromal cell-independent B lineage line.

The export of immunoglobulin D by human neoplastic B lymphocytes.

An investigation has been made into the ability of human neoplastic B lymphocytes expressing surface IgM and IgD to export IgD in culture. Cells that expressed surface Ig of the lambda light chain type frequently exported IgD (10/12 patients), whereas cells expressing surface Ig of the kappa light chain type exported no IgD, although most (8/11 patients) were able to export IgM. It appears, therefore, that in most of the 23 cases studied, cells synthesizing IgD with lambda light chains can both express and export IgD, whereas those synthesizing IgD kappa can only insert it into the surface membrane. This finding and the known preponderance of lambda in plasma IgD imply that the possession of a lambda chain facilitates the IgD secretory pathway, a conclusion that implicates a control mechanism subsequent to the surface/secretory dichotomy arising from different splicings of heavy chain messenger RNA.

Feedback suppression of the immune response in vitro. I. Activity of antigen-stimulated B cells.

Feedback regulation of the primary humoral immune response to sheep erythrocytes (SRBC) was studied in vitro. Whole spleen cells or spleen cell subpopulations were incubated with antigen for 4 d under Mishell-Dutton conditions (education) and the surviving cells tested for regulatory activity in fresh anti-SRBC spleen cell cultures assayed by measuring plaque-forming cells on day 4. The data indicate that (a) whole spleen cells educated with SRBC exert potent antigen-specific suppression in the assay culture, (b) surface Ig- (sIg-) cells (T cells) prepared by either nylon-wool separation or fractionation on rabbit anti-mouse-Ig-coated polystyrene Petri dishes failed to generate suppressive activity when educated alone, in 2-mercaptoethanol, or in the presence of additional macrophages, (c) surface Ig (sIg+) (B) cells educated alone also failed to generate suppressor cells, and (d) mixing sIg- (T) and sIg+, Lyt 123- (B) cells reconstituted the ability to induce suppressor cells under these conditions. The antigen-primed cell actually required to transfer suppression was also characterized by separating cells using anti-Ig coated dishes, by fluorescence-activated cell sorting and by anti-Lyt treatment. All these methods clearly identified sIg+ (B) and not sIg+ (T) cells as the important educated cells. It is concluded that under our conditions, T cell-dependent B cells triggered by antigen during primary in vitro cultures cause potent specific feedback suppression of humoral responses. Possible mechanisms for this suppression, including antigen blockade or anti-idiotypic responses, are discussed.

Extracellular idiotypic immunoglobulin arising from human leukemic B lymphocytes.

The peripheral blood lymphocytes of nine out of nine patients with typical surface Ig-positive chronic lymphocytic leukemia but no paraprotein visible on serum electrophoresis have been shown by radioimmunoassay to export small amounts of pentameric IgM during culture (in the range of 2.4-7.2 ng/10(7) cells per h); three out of nine also exported monomeric IgD (0.7-1.4 ng/10(7) cells per h). Immunoglobulin turned over on the cell surface did not appear to contribute to material in the culture fluid, except possibly as vesicle-bound Ig. In three cases, which included two of the IgD producers, anti-idiotypic antibody raised against the cell surface Fab mu was used to demonstrate the idiotypic nature of the exported Ig. Anti-idiotypic antibody was also used to measure levels of idiotypic Ig in the sera of these three patients as a proportion of the total Ig. Total serum IgM was depressed in all three patients, and the idiotypic IgM represented 43%, 65%, and 96% of the IgM. The findings suggest that in typical chronic lymphocytic leukemia involving B lymphocytes, the export of a small amount of idiotypic Ig by the neoplastic cells in a common or even usual occurrence.

Lymphocyte membrane IgG and secreted IgG are structurally and allotypically distinct.

We have demonstrated that there are structurally distinct membrane and secreted IgG2a immunoglobulin molecules. The membrane heavy chain is both larger and more acidic than the secreted molecule. This difference is not a result of different N-glycosidic-linked oligosaccharide chains. The membrane heavy chain also is antigenically different from its secreted homologue. This is based on the fact that secreted IgG2a molecules express an allotypic determinant absent on membrane molecules. We discussed the genetic control and gene organization of membrane and secreted immunoglobulin heavy chain sequences and suggest mechanisms controlling the expression of the simian virus 40 genome as models for alternate gene expression of membrane and secreted heavy chain polypeptide chains from the same DNA sequence. The possible biological significance of the membrane immunoglobulin acting as a recognition site for regulatory T cells also is discussed. The difference between membrane and secreted immunoglobulin is proposed as a possible explanation for the manner in which T cells interact with IgG on memory B cells in the presence of a large excess of IgG present in body fluids.

B-cell differentiation in the CBA/N mouse. I. Slower maturation of mitogen and antigen-responsive B cells in mice expressing an X-linked defect.

The effect of age on the mitogenic and antigenic responsiveness of B cells is examined in spleen cell cultures of CBA/N and (CBA/N X DBA/2) F1 mice. Spleen cells from young male F1 mice (4- to 6-wk old) show lower mitogenic responses to lipopolysaccharide, a lower frequency of sheep erythrocytes (SRBC)-reactive B-cell precursors, and a lower percentage of Ig-bearing cells than age-matched female F1 mice. The expression of all three functions were found to increase with the age of the F1 male mice. Whereas male F1 mice at 60 wk of age showed an equivalent percentage of Ig-bearing spleen cells and a similar mitogenic responsiveness to LPS when compared to adult female F1 mice, the frequency of SRBC-reactive B-cell precursors remained threefold lower. These findings reveal that there is a slower maturation of B cells in mice expressing the X-linked defect and suggests that the defect has differential effects on the mechanisms of antigen and mitogen activation of B cells.