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Biomedical subjects

A B Kantor

Publications and source records attributed to A B Kantor.

At least 19 recordsLinked to original sources
PubMed1999

Predominant VH genes expressed in innate antibodies are associated with distinctive antigen-binding sites.

Antibodies to phosphatidylcholine (PtC), a common constituent of mammalian and bacterial cell membranes, represent a large proportion of the natural antibody repertoire in mice. Previous studies of several mouse strains (e.g., C57BL/6) have shown that anti-PtC antibodies are mainly encoded by the VH11 and VH12 immunoglobulin heavy chain variable region gene families. We show here, however, that VH11 and VH12 encode only a small proportion of the anti-PtC antibodies in BALB/c mice. Instead, VHQ52-encoded antibodies predominate in this strain. In addition, two-thirds of the cells expressing VHQ52 family genes use a single gene (which, interestingly, has been previously shown to predominate in the anti-oxazolone response). We also show here that in anti-PtC antibodies from all strains, the distinctive antigen-binding sites associated with VHQ52 differ substantially from those associated with VH11 and VH12. That is, VHQ52-containing transcripts preferentially use the joining region JH4 rather than JH1 and exhibit more diverse complementarity-determining region 3 (CDR3) junctions with more N-region nucleotide additions at the gene segment junctions. Thus, the VH gene family that predominates in the anti-PtC repertoire differs among mouse strains, whereas the distinctive VHDJH rearrangements (CDR3, JH) associated with each VH gene family are similar in all strains. We discuss these findings in the context of a recent hypothesis suggesting that CDR3 structure, independent of VH framework, is sufficient to define the specificity of an antibody.

Animals↗
PubMed1998

Presence of germline and full-length IgA RNA transcripts among peritoneal B-1 cells.

Next to conventional B cells (or B-2 cells), peritoneal B-1 cells have been shown to contribute significantly to the production of IgA-secreting plasma cells in the gut. Evidence for this was mainly based on studies comprising manipulated animals, including lethally X-irradiated and transgenic mice. To examine the ability of peritoneal B-1 cells from untreated mice to switch actively to IgA in vivo, we performed RT-PCR analysis on FACS-sorted peritoneal B-cell subsets from untreated BALB/c mice in order to examine the presence of germline C alpha mRNA and mature C alpha mRNA transcripts. Germline C alpha and mature C alpha transcripts were readily detectable in peritoneal B-1 cells (defined as IgMbright/IgDdull), but not, or very little, in peritoneal B-2 cells (defined as IgMdull/IgDbright). Moreover, by subdividing the B-1-cell population in CD5+ B-1a cells and CD5- B-1b cells, it was shown that in vivo expression of germline C alpha and mature C alpha transcripts was largely restricted to the B-1b-cell lineage. These results indicate that peritoneal B-1 cells indeed are capable to switch to IgA under normal physiological conditions and hereby further support the view that B-1 cells contribute significantly to the mucosal IgA response, albeit this function appears to be restricted to the B-1b-cell subset.

Animals↗
PubMed1997

An unbiased analysis of V(H)-D-J(H) sequences from B-1a, B-1b, and conventional B cells.

Previous studies conclude that the repertoire of B-1a (CD5+ B) cells is highly restricted. Studies here, which use FACS sorting and single-cell PCR methodology to develop an unbiased representation of the IgH repertoires of B-1a, B-1b, and conventional B cells from the peritoneal cavity, demonstrate that the B-1a cell repertoire is more diverse than previously thought. Furthermore, adult B-1a cells have significantly fewer noncoded nucleotide (N) insertions than conventional B cells. However, B-1a cells are not defined by the absence of these regions, since such insertions are present in two-thirds of B-1a cell transcripts. All three B cell populations use a wide spectrum of V(H), D, and J(H) elements and display considerable diversity in complementarity-determining region 3 (CDR3). However, characteristic differences in the repertoires of all three B cell populations also exist, suggesting different selective and/or developmental forces act to shape each repertoire.

Animals↗
PubMed1997

Frequent occurrence of identical heavy and light chain Ig rearrangements.

Single-cell PCR analyses of expressed Ig H and L chain sequences presented here show that certain rearrangements occur repeatedly and account for a major segment of the well-studied repertoire of B-1 cell autoantibodies that mediate the lysis of bromelain-treated mouse erythrocytes, i.e. antibodies reactive with phosphatldyicholine (PtC). We repeatedly isolated at least 10 different types of VH region rearrangements, involving three distinct germline genes, among FACS-sorted PtC-binding B-1 cells from three strains of mice (C57BL/6J, BALB/c and C.B-17). The predominant rearrangement, VH11-DSP-JH1 (VH11 type 1), has been previously found in anti-PtC hybridomas in several studies. We show that within each of six mice from two strains (C57BL/6J and BALB/c), unique instances of IgH/IgL pairing arose either from different B cell progenitors prior to IgH rearrangement or from pre-B cells which expanded after IgH rearrangement but prior to IgL rearrangement. Together with other recurrent rearrangements described here, our findings demonstrate that clonal expansion of mature B cells cannot account for all repeated rearrangements. As suggested by initial studies of dominant idiotype expression, these findings confirm that clonal expansion is only one of the mechanisms contributing to the establishment of recurrent rearrangements.

Amino Acid Sequence↗
PubMed1996

Cy7PE and Cy7APC: bright new probes for immunofluorescence.

We demonstrate the utility of indotricarbocyanine (Cy7) conjugates of the phycobiliproteins phycoerythrin (PE) and allophycocyanin (APC) in flow cytometry. This is the first demonstration of the use of an APC tandem dye for fluorescence measurements. These resonance energy transfer tandem dyes can be excited by the phycobiliprotein-specific excitation wavelengths and fluoresce at wavelengths above 780 nm. The tandem dyes, when conjugated to antibodies, are suitable for flow cytometry and other immunofluorescence applications. These conjugates are easily detectable above the very low autofluorescence in this part of the spectrum. Indeed, the Cy7-conjugated PE tandem (Cy7PE) has a "brightness" (fluorescence signal over cellular autofluorescence) comparable to that of fluorescein, and the Cy7APC tandem has a "brightness" comparable to that of APC. These tandems are also easily distinguished from other commonly used fluorophores, making them suitable for high-order multiparametric analysis. We show an example of six-color immunofluorescence analysis by flow cytometry, simultaneously measuring fluorescences from fluorescein, PE, Cy5PE, Texas red, APC, and Cy7APC.

Antigens, CD↗
PubMed1996

V-gene usage and N-region insertions in B-1a, B-1b and conventional B cells.

The adult B-1a cell repertoire shows both substantial diversity and characteristic features. Recent studies using PCR to amplify variable-region transcripts from single, FACS-sorted B cells illuminate this apparent dichotomy. Although there is substantial diversity in the number of different rearrangements, characteristic patterns of VH-gene usage, combined with low use of N-region insertions when compared with conventional B cells, distinguish the B-1a repertoire. B-1b cells also have characteristic features of their own. These results are discussed in the context of key developmental and regulatory events which shape the B-1a and B-1b repertoires.

Animals↗
PubMed1995

Identification and kinetics of two recently bone marrow-derived B cell populations in peripheral lymphoid tissues.

In rats, the glycoprotein Thy-1 is expressed on recently bone marrow (BM)-generated B cells but not on mature recirculating follicular (RF) B cells. Here we demonstrate that Thy-1+ B cells consist of two phenotypically distinct, but developmentally related, populations: a population of newly formed (NF) B cells (IgMbr-IgDdu) that give rise to the second, less immature, Thy-1+ population of so-called early recirculating follicular (ERF) B cells (Thy-1+IgMduIgDbr) cells. These cells ultimately develop to RF-B cells (Thy-1-IgMbrIgDdu). Kinetic studies reveal that in absolute numbers per day most cells die at the transition of NF-B cells in the BM and those in the periphery: less cells die at later stages of B cell differentiation. Given the notion that this cell loss is not random, we speculate that NF-B cells and ERF-B cells may represent crucial steps during peripheral B cell development and their selection. Identification of their unique phenotype makes it possible to evaluate their roles in development of the antibody repertoire.

Animals↗
PubMed1995

Defective B cell development and function in Btk-deficient mice.

Mutations in the Bruton's tyrosine kinase (Btk) gene have been linked to severe early B cell developmental blocks in human X-linked agammaglobulinemia (XLA), and to milder B cell activation deficiencies in murine X-linked immune deficiency (Xid). To elucidate unequivocally potential Btk functions in mice, we generated mutations in embryonic stem cells, which eliminated the ability to encode Btk pleckstrin homology or kinase domains, and assayed their effects by RAG2-deficient blastocyst complementation or introduction into the germline. Both mutations block expression of Btk protein and lead to reduced numbers of mature conventional B cells, severe B1 cell deficiency, serum IgM and IgG3 deficiency, and defective responses in vitro to various B cell activators and in vivo to immunization with thymus-independent type II antigens. These results prove that lack of Btk function results in an Xid phenotype and further suggest a differential requirement for Btk during the early stages of murine versus human B lymphocyte development.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1995

De novo development and self-replenishment of B cells.

Previous studies distinguished two murine B cell lineages: the conventional lineage, which comprises the majority of B cells, and the Ly-1 B lineage (B-1a), which represents a small percentage of total adult B cells. A third subset, B-1b cells, shares many properties with B-1a cells, including the characteristic ability to self-replenish, but does not express Ly-1 (CD5). Reconstitution studies presented here show that (i) although the B220- population in adult spleen and bone marrow contains very little progenitor activity for B-1a cells, it can reconstitute roughly half the normal number of B-1b cells; (ii) B-1 progenitors present in adult bone marrow and spleen function at low levels in adult animals; (iii) peritoneal B-1 cells (principally B-1b) that develop following bone marrow transfer, like B-1 cells from normal animals, are capable of substantial self-replenishment; and (iv) conventional B cells do not expand (self-replenish) in adoptive recipients, although they can persist for long periods. Collectively, these progenitor and self-replenishment characteristics provide a developmental base for distinguishing B-1a, B-1b and conventional B cells.

Age Factors↗
PubMed1994

CD43 (S7) expression identifies peripheral B cell subsets.

CD43 (leukosialin) expression has previously been demonstrated on the surface of developing B cells in mouse bone marrow and on plasma cells induced in vitro, but not on peripheral B cells in spleen. Here we show that CD43, as recognized by mAb S7, is indeed expressed on a small population of splenic B cells. Flow cytometric phenotyping of normal mice and radiation chimeras reveals that CD43/S7 is expressed on virtually all (> 90 to 95%) splenic B-1 cells and the majority of peritoneal B-1 cells, but not on conventional B cells. The expression of CD43/S7, in conjunction with other cell surface markers, clearly distinguishes B-1 cells from follicular, marginal zone, and immature B cells in the unstimulated adult spleen and permits further phenotyping of these subsets. The phenotype of splenic and peritoneal B-1 cells in normal BALB/c and BAB/25 mice is essentially identical with the exception that all peritoneal B-1 cells express CD11b (Mac-1) and some lack CD43/S7 and heat stable Ag (as detected by the mAb 53-10) expression. Although splenic B-1, marginal zone, and immature B cells share many phenotypic characteristics, these studies show that, in addition to CD43, they differ with respect to the expression levels of a variety of Ags including heat stable Ag, B220, and the B cell activation Ag B7.

Aging↗
PubMed1993

Aggregation of hapten-bearing liposomes mediated by specific antibodies.

We studied specific membrane-membrane interactions mediated by ligand-receptor binding in a model system, which consisted of (a) FG3P, the fluorescein hapten attached to a phospholipid by a peptidyl spacer as described previously (Petrossian, A., A.B. Kantor, and J.C. Owicki. 1985. J. Lipid Res. 26:767-773), (b) antifluorescein monoclonal antibodies (MAbs), and (c) phospholipid vesicles (liposomes) into which the FG3P was incorporated. The aggregation of the hapten-bearing liposomes by four MAbs was studied by differential centrifugation. The ability of the MAbs to induce vesicle aggregation varied considerably and correlated inversely with affinity. Aggregation by one of the MAbs was studied in more detail by turbidimetry and freeze-fracture electron microscopy of samples frozen throughout the course of the aggregation. Rapid freezing was achieved with a double propane-jet apparatus. The aggregate morphologies and the time evolution of the aggregate size distribution were obtained from the two-dimensional fracture views with a stereological correction. The aggregation kinetics were simulated by considering dynamical aggregation according to a mass-action model with two parameters, the rate constants for antibody-mediated vesicle aggregation and disaggregation. Both rate constants were orders of magnitude lower than the rate constants for the corresponding interactions of antibodies with haptens either in solution or on vesicles under nonaggregating conditions.

Animals↗
PubMed1993

Evidence that intestinal IgA plasma cells in mu, kappa transgenic mice are derived from B-1 (Ly-1 B) cells.

B6-Sp6 transgenic mice carry fully rearranged (BALB/c-derived, Igh-Ca allotype) mu heavy chain and kappa light chain transgenes, specific for trinitrophenyl, on a C57BL background (Igh-Cb allotype). FACS analyses show that the majority of B cells in peripheral lymphoid organs and bone marrow (BM) express transgenic IgM exclusively. A small proportion of the B cells, however, express endogenous IgM, usually concomitant with transgenic IgM. Three criteria establish that the endogenous IgM expressing B cells belong to the B-1 cell lineage. (i) Endogenous IgM expressing B cells in B6-Sp6 mice have the same localization pattern as B-1 cells from normal animals: they are enriched in the peritoneal cavity. (ii) The endogenous IgM+ B cells have the phenotype of B-1 cells: the endogenous IgM+ peritoneal B cells express Mac-1 (CD11b) and low levels of IgD, and most also express CD5 (Ly-1). (iii) B6-Sp6 BM poorly reconstitutes endogenous IgM+ B cells, just as adult BM from normal mice poorly reconstitutes B-1 cells. In contrast, B cells which only express the transgene are readily reconstituted by B6-Sp6 BM. The few endogenous IgM+ cells in the B6-Sp6 BM recipients are located in the peritoneal cavity and have the phenotype of B-1b cells (previously the Ly-1 B sister population), which are known to be reconstituted by adult BM. Two-color immunofluorescence staining of tissue sections from the gut and from isolated gut lamina propria cells shows the presence of many IgA containing cells, about one-third of which simultaneously express cytoplasmic (transgenic) IgM. The C-region of this IgA is produced by endogenous C alpha genes, because the transgene encodes only for C mu. Furthermore, the majority of gut IgA containing cells do not express the idiotype of the transgene, indicating that most of the gut IgA cells are encoded by endogenous VH genes and thus the result of an isotype switch from endogenous IgM expressing B cells. Since the endogenous IgM+ cells are B-1 cells (both B-1a and B-1b), the data strongly indicate that the intestinal IgA plasma cells also belong to the B-1 cell lineage.

Animals↗
PubMed1993

Origin of murine B cell lineages.

Until recently, the hematopoietic stem cells (HSC) that appear early in ontogeny were thought to constitute a homogeneous, self-replenishing population whose developmental potential remains constant throughout the life of the animal. Studies reviewed here, however, demonstrated clear differences in the developmental potential of fetal and adult progenitor populations (including FACS-sorted HSC). These studies, which chart the ability of various progenitor sources to reconstitute functionally distinct B cell populations, define three B cell lineages: B-1a cells (CD5 B cells), derived from progenitors that are present in fetal omentum and fetal liver but are largely absent from adult bone marrow; B-1b cells ("sister" population), derived from progenitors that are present in fetal omentum, fetal liver, and also in adult bone marrow; and conventional B cells, whose progenitors are missing from fetal omentum but are found in fetal liver and adult bone marrow. B-1a and B-1b cells share many properties, including self-replenishment and feedback regulation of development. These B cell studies, in conjunction with evidence for a similar developmental switch for T cells and erythrocytes, suggest that evolution has created a "layered" immune system in which successive progenitors (HSC) reach predominance during development and give rise to differentiated cells (B, T, etc) responsible for progressively more complex immune functions.

Animals↗