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1.
Int Immunol ; 9(5): 665-77, 1997 May.
Article in English | MEDLINE | ID: mdl-9184912

ABSTRACT

X-linked immune deficient (Xid) mice fail to produce anti-phosphocholine (PC) antibodies even after immunization with Streptococcus pneumoniae. Consequently, Xid mice are extremely susceptible to infection with S. pneumoniae, PC-specific B cells appear to undergo clonal deletion in Xid mice; however, a new thymus-dependent form of PC, 6-(O-phosphocholine)hydroxyhexanoate (EPC), can rescue PC-specific B cells from the bone marrow presumably by providing T cell help before clonal deletion. Analysis of PC-specific IgG hybridomas from Xid mice revealed utilization of several V-D junctional variants of the VH1 gene segment rearranged to different D and JH gene segments. The majority of Xid anti-PC antibodies exhibit an Asp-->Gly95H replacement at the V-D junction. These Gly95H VH1 variants associate with kappa 1C L chains to produce anti-PC antibodies that: (1) have low relative affinity for PC, (ii) are heteroclitic for nitrophenylphosphocholine and (iii) fall to bind to or provide protection against S. pneumoniae. Single prototypic V-D variants of the T15 idiotype (Asp95H), M603 idiotype (Asn95H) and M167 idiotype (Asp95H-Ala96H) were also induced in Xid mice. The M603-like and M167-like antibodies bound to and protected against S. pneumoniae even though they exhibited Kas for PC which were lower than T15 idiotype+ antibodies. These data demonstrate that small changes in the V-D junctional sequence of the T15 (VH1) heavy chain alter L chain usage and the structure of the PC binding site so that the PC expressed on S. pneumoniae is no longer recognized.


Subject(s)
Antibodies, Antiphospholipid/metabolism , Binding Sites, Antibody , Gene Rearrangement, B-Lymphocyte, Heavy Chain , Immunoglobulin Heavy Chains/metabolism , Immunoglobulin Joining Region/metabolism , Immunoglobulin Variable Region/metabolism , Phosphorylcholine/immunology , Pneumococcal Infections/prevention & control , Amino Acid Sequence , Animals , Antibodies, Antiphospholipid/genetics , Antibody Affinity , Bacterial Adhesion/immunology , Base Sequence , Caproates/immunology , Haptens/immunology , Hybridomas/chemistry , Immunization, Passive , Immunoglobulin Allotypes/analysis , Immunoglobulin Heavy Chains/analysis , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Isotypes/analysis , Immunoglobulin Joining Region/analysis , Immunoglobulin Joining Region/genetics , Immunoglobulin Variable Region/analysis , Immunoglobulin Variable Region/genetics , Mice , Mice, Inbred CBA , Mice, Mutant Strains , Molecular Sequence Data , Phosphorylcholine/analogs & derivatives , Pneumococcal Infections/microbiology , Streptococcus pneumoniae/immunology , Streptococcus pneumoniae/pathogenicity , Virulence
2.
Hybridoma ; 16(6): 503-11, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9455702

ABSTRACT

Four rat x mouse hybridomas secreting monoclonal anti-idiotypic (anti-Id) antibodies (MAb) specific for the transgene-encoded antibody of the 207-4 transgenic mouse line, which carries the VH1/V kappa 24 gene segments of the IgA, phosphocholine-(PC) specific MOPC167 myeloma, were developed from a fusion of Ag8-X63.653 mouse cells with spleen cells from a rat immunized with MOPC167 and HPCM27 anti-PC antibodies. The anti-Id MAb were shown by ELISA to be specific for PC-binding proteins of VH1/V kappa 24 H and L chains of various isotypes. They did not bind VH1/V kappa 22, VH1/V kappa 8, or VH1/V kappa 1 PC-binding proteins or other IgA or IgM myeloma proteins. Analysis by flow cytometry demonstrated that these MAb bind to the transgene-encoded membrane immunoglobulin (sIgM) as expressed on > 95% of the B220 positive 207-4 spleen cells. All four MAb were able to inhibit the binding of MOPC167 to PC conjugated to bovine serum albumin. Differences in fine specificity of binding were demonstrated by differential staining of spleen cells of the 216-7 mu kappa delta Mem MOPC167 transgenic mice. In these mice endogenous H chains associate with the transgene encoded L chain to form MOPC167 crossreactive idiotopes. Two of the MAb, 28-4-3 and 28-6-20, stained significant numbers of cells, while MAb 28-5-15 did not bind to 216-7 cells. Three of the MAb, 28-5-15, 28-6-20, and 28-4-3, when conjugated to Sepharose beads, were able to induce DNA synthesis in cultures of 207-4 transgenic spleen cells. None of the MAb were able to induce an antibody response in vivo. These MAb should prove useful in staining PC-transgenic B cells for flow cytometry studies and in defining early cellular events in the activation of idiotype positive B cells by anti-Id antibodies.


Subject(s)
Antibodies, Monoclonal/immunology , Immunoglobulin Idiotypes/immunology , Immunoglobulin kappa-Chains/immunology , Myeloma Proteins/immunology , Phosphorylcholine/immunology , Animals , Antibodies, Monoclonal/metabolism , Antibody Specificity , B-Lymphocytes/immunology , Binding Sites , Cattle , Immunoglobulin A/metabolism , Immunoglobulin Idiotypes/metabolism , Immunoglobulin M/metabolism , Lymphocyte Activation , Mice , Mice, Transgenic , Rats , Rats, Sprague-Dawley
3.
Am J Trop Med Hyg ; 52(5): 468-76, 1995 May.
Article in English | MEDLINE | ID: mdl-7771614

ABSTRACT

Severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood lymphocytes (hu-PBL) were evaluated as an animal model for demonstrating dengue (DEN) viral infection. Reconstituted mice (hu-PBL-SCID) that demonstrated successful engraftment by the presence of serum titers of human immunoglobulin (Ig) were inoculated intraperitoneally with DEN virus serotype 1 (DEN-1). Serial blood samples were taken postinoculation and assayed for virus in C6/36 cells. The identity of all viral isolates was confirmed by an immunofluorescence antibody assay using DEN-1 monoclonal antibody. A total of six experiments were performed using different procedures of reconstitution and infection, and in three of these experiments, DEN-1 virus was recovered from the hu-PBL-SCID mice. In the first successful experiment, DEN-1 virus was recovered on postinoculation day (PID) 24 from blood, spleen, thymus, and lung tissues of one of eight hu-PBL-SCID mice. A second group of eight hu-PBL-SCID mice were inoculated with human monocytes infected in vitro with DEN-1 virus. Virus was recovered from the blood of mice between PID 15 and 23, and from lung tissue of one of these mice. In a third experiment, seven SCID mice were treated initially with anti-asialo GM1 antibody to eliminate natural killer cells, and then were injected simultaneously with a mixture of hu-PBL and DEN-1 virus. Virus was demonstrated in the blood of one mouse on PID 38, and in another mouse on PID 8, 12, 20, 24, and 36.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Dengue/immunology , Disease Models, Animal , Mice, SCID , Animals , Dengue Virus/immunology , Dengue Virus/isolation & purification , Evaluation Studies as Topic , Female , Flow Cytometry , Humans , Immunoglobulin G/blood , Killer Cells, Natural/immunology , Lymphocytes/immunology , Male , Mice , Monocytes/immunology , Viremia/immunology
4.
J Immunol ; 152(10): 4873-83, 1994 May 15.
Article in English | MEDLINE | ID: mdl-8176209

ABSTRACT

The transgenic (TG) mouse strain 207-4, carries mu a + kappa transgenes ligated to the anti-phosphocholine (PC) VH1 and V kappa 24 V region genes from the MOPC-167 myeloma. Although B cells from mice carrying these transgenes respond both in vivo and in vitro to thymus-dependent Ags, they failed to proliferate in response to soluble goat anti-mu Ab or other soluble anti-Ig reagents. On the other hand, B cells from the Sp6 mu kappa anti-trinitrophenyl TG mouse line proliferated normally after stimulation with soluble anti-mu. However, the 207-4 anti-PC transgene positive (TG+) splenic B cells proliferated when stimulated with anti-mu, anti-idiotype, anti-allotype, or PC-conjugated to Sepharose beads. TG+ B cells were also induced to proliferate when stimulated with anti-Lyb-2; thus, their defect may be restricted to signaling through sigM. The lack of response to soluble anti-mu could not be reversed by addition of IL-4, by removal of T cells, by addition of anti-FcR Ab, or by stimulation with F(ab')2 anti-mu. Thus, the failure to proliferate was not caused by active T cell suppression or FcR-mediated inhibition. In mixed cultures of TG+ and transgene negative (TG-) spleen cells, the TG- cells were able to proliferate normally to soluble anti-mu, indicating that suppressive factors were not involved in the unresponsiveness of the TG+ anti-PC-specific B cells. These studies suggest that B cells in the 207-4 anti-PC TG mice exhibit a defect in activation through their sIgM receptors, and this unresponsiveness may reflect a form of Ag-induced tolerance.


Subject(s)
Antibodies, Anti-Idiotypic/immunology , B-Lymphocytes/immunology , Immune Tolerance , Immunoglobulin M/immunology , Lymphocyte Activation , Animals , Antigens, Ly/analysis , Antigens, Ly/physiology , Cells, Cultured , Immunoglobulin M/genetics , Interleukin-4/pharmacology , Macrophage-1 Antigen/analysis , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Receptors, Fc/physiology
5.
J Clin Microbiol ; 31(5): 1394-6, 1993 May.
Article in English | MEDLINE | ID: mdl-8501252

ABSTRACT

A Western blot (immunoblot) assay was used to detect Campylobacter-specific immunoglobulin A in urine. Acute-phase urine samples from six children with Campylobacter diarrhea had titers ranging from 2 to 8. The highest titer was detected 4 days postonset. Campylobacter-specific immunoglobulin A was undetectable in the paired convalescent-phase specimens and urine samples from three control children.


Subject(s)
Blotting, Western/methods , Campylobacter Infections/immunology , Diarrhea/immunology , Immunoglobulin A/urine , Adolescent , Adult , Blotting, Western/statistics & numerical data , Campylobacter/classification , Campylobacter/isolation & purification , Campylobacter Infections/microbiology , Child , Child, Preschool , Diarrhea/microbiology , Evaluation Studies as Topic , Female , Humans , Luminescent Measurements , Male , Sensitivity and Specificity , Serotyping
7.
J Exp Med ; 174(5): 1189-201, 1991 Nov 01.
Article in English | MEDLINE | ID: mdl-1940797

ABSTRACT

Flow cytometric analysis of antigen-specific, idiotype-positive (id+), B cell development in transgenic mice expressing a rearranged M167-mu gene shows that large numbers of phosphocholine (PC)-specific, M167-id+ B cells develop in the spleen and bone marrow of these mice. Random rearrangement of endogenous V kappa genes, in the absence of a subsequent receptor-driven selection, should give rise to equal numbers of T15- and M167-id+ B cells. The observed 100-500-fold amplification of M167-id+ B cells expressing an endogenous encoded V kappa 24]kappa 5 light chain in association with the M167 VH1-id transgene product appears to be an antigen driven, receptor-mediated process, since no amplification of non-PC-binding M167 VH1/V kappa 22, T15-id+ B cells occurs in these mu-only transgenic mice. The selection and amplification of antigen-specific, M167-id+ B cells requires surface expression of the mu transgene product; thus, no enhancement of M167-id+ B cells occurs in the M167 mu delta mem-transgenic mice, which cannot insert the mu transgene product into the B cell membrane. Surprisingly, no selection of PC-specific B cells occurs in M167-kappa-transgenic mice although large numbers of B cells expressing a crossreactive M167-id are present in the spleen and bone marrow of these mice. The failure to develop detectable numbers of M167-id+, PC-specific B cells in M167-kappa-transgenic mice may be due to a very low frequency of M167-VH-region formation during endogenous rearrangement of VH1 to D-JH segments. The somatic generation of the M167 version of a rearranged VH1 gene may occur in less than one of every 10(5) bone marrow B cells, and a 500-fold amplification of this M167-Id+ B cell would not be detectable by flow cytometry even though the anti-PC antibody produced by these B cells is detectable in the serum of M167-kappa-transgenic mice after immunization with PC.


Subject(s)
B-Lymphocytes/immunology , Gene Rearrangement , Genes, Immunoglobulin , Immunoglobulin Idiotypes/analysis , Immunoglobulin mu-Chains/genetics , Phosphorylcholine/pharmacology , Animals , Antibody Affinity , Base Sequence , Bone Marrow/immunology , Female , Immunoglobulin Variable Region/genetics , Immunoglobulin kappa-Chains/genetics , Immunoglobulin kappa-Chains/immunology , Immunoglobulin mu-Chains/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Molecular Sequence Data , RNA, Messenger/analysis
8.
J Immunol ; 146(8): 2568-77, 1991 Apr 15.
Article in English | MEDLINE | ID: mdl-1901880

ABSTRACT

The combined expression of the M167 mu/kappa anstiphosphocholine (PC) transgenes with the x-linked immunodeficiency gene, xid, results in an almost total failure to develop B cells in the peripheral lymphoid organs of such mice. Although there is no significant difference between the normal transgene positive (TG+) female offspring and the immunodeficient TG+ xid males with respect to the number of B220+ pre-B cells and IgM+B220+B cells that develop in their bone marrow, the hemizygous xid males have 85% fewer B cells in their spleens than the phenotypically normal heterozygous F1 females. In xid M167-mu-transgenic mice, PC-specific B cells also fail to develop in the spleen; however, numerous B cells bearing the mua+VH1(+)-transgene product associated with endogenous kappa L chains that do not give rise PC-specific antibodies are present. In the phenotypically normal TG+ (B6.CBA/N x mu 243-4)F1 female mice, PC-specific B cells represent almost 10% of the total B cell population, and these B cells express an M167-Id that has been produced by association of the VH1 transgene product with an endogenous V kappa 24L chain. B cells expressing the normally dominant T15-Id are not detectable in the spleens of these M167 mu-transgenic mice. Furthermore, M167-Id+ B cells are present at a fivefold lower level in the bone marrow of mu-TG+ normal mice than in their spleens. These data suggest that the PC-specific B cells that develop in TG+ xid mice are either clonally deleted via some "IgR-directed" mechanism or they fail to receive the appropriate signals to exit the bone marrow or to enter the peripheral lymphoid tissues. This hypothesis is supported by the finding that TNP-specific B cells develop normally and do not undergo clonal deletion in xid mice carrying the Sp6 mu/kappa anti-TNP transgenes.


Subject(s)
B-Lymphocytes/immunology , Immune Tolerance/genetics , Phosphorylcholine/immunology , X Chromosome , Animals , Bone Marrow Cells , Flow Cytometry , Genetic Linkage , Immunoglobulin M/genetics , Immunoglobulin kappa-Chains/genetics , Mice , Mice, Transgenic , Spleen/immunology
9.
Hybridoma ; 10(1): 121-35, 1991 Feb.
Article in English | MEDLINE | ID: mdl-1709612

ABSTRACT

An IgG1 mouse monoclonal antibody (MAb) specific for a mouse IgM allotypic determinant in the a, c, f, g, h, and j haplotypes was derived from a fusion of SP2/O-Ag14 mouse myeloma cells with C57BL/6 mouse spleen cells (Igh-Cb) immune to TC31, a MAb of the IgMa allotype. MAb from one hybridoma derived from this fusion (designated DS1) was demonstrated to bind in an ELISA to immunoglobulin bearing the IgMa allotype (TC31, MOPC104E), but not to immunoglobulin bearing the IgMb allotype (C.BPC112). Fluorescein-conjugated DS1 was shown to bind to the surface of BALB/cByJ splenic B cells, but was shown to have negligible binding on C57BL/6J cells. Similarly, DS1-conjugated Sepharose beads were able to stimulate in vitro proliferation of BALB/c, but not C57BL/6 splenic B cells. DS1 was unable to bind to spleen cells from BALB/c allotype congenic strains, BAB/14 (Igh-Cb) and C.AL-20 (Igh-Co), demonstrating that DS1 recognizes a determinant under the control of a gene linked to the Igh-C gene complex. Using sera from recombinant inbred lines, the determinant defined by DS1 was shown to be linked to the Igh-1 locus. Furthermore, the determinant was localized to the CH1 domain of the mu heavy chain. Sera from BALB/cByJ, NMRI, CBA/J, SEA/GnJ, RIIIs/J, and CE/J mouse strains were shown to bind to DS1 in an ELISA, while sera from A/J, SJL/J, NZB/B1NJ, AKR/J, C57BL/6J, and C57BL/10SnJ mouse strains did not bind to DS1. From these data we propose that DS1 is reactive with specificity Igh-6.1, which was originally defined by an allotypic antiserum developed by Black et al. (Immunogenetics 7:213, 1978).


Subject(s)
Antibodies, Monoclonal , Immunoglobulin M , Alleles , Animals , Antibodies, Anti-Idiotypic , Antibody Specificity , B-Lymphocytes/immunology , Epitopes/genetics , Genes, Immunoglobulin , Hybridomas/immunology , Immunoglobulin Allotypes/genetics , Immunoglobulin Heavy Chains/genetics , Immunoglobulin M/genetics , Lymphocyte Activation , Mice , Mice, Inbred Strains
10.
Lab Invest ; 59(2): 181-91, 1988 Aug.
Article in English | MEDLINE | ID: mdl-2457132

ABSTRACT

In pursuing the mechanism of endotoxin action, we examined the effect of lipopolysaccharide (LPS) and its chemically defined components, lipid A and lipid X on cultured bovine endothelial cells. We report that LPS and lipid A caused detachment and altered morphology of endothelial cells while lipid X did not. Phorbol myristate acetate, a compound known to activate protein kinase C, also caused endothelial cell detachment. Morphologic changes were readily apparent in the endothelial cells after 6 hours of exposure to lipopolysaccharide (1 microgram/ml); at that time many of the cells had contracted and formed bleblike structures on the surface. Large vacuoles, dense bodies, and pyknotic nuclei were found in the detaching cells, indicating necrosis or cell death. Preceding the morphologic changes and actual detachment, endothelial cell DNA and RNA synthesis was impaired by LPS. The changes in DNA and RNA synthesis occurred within 4 hours of exposure to 1 microgram/ml of LPS when the cells were still able to maintain normal levels of ATP. In addition to the inhibition of nucleic acid synthesis, protein synthesis was inhibited after 6 and 8 hours of LPS exposure. DNA, RNA, and protein synthesis returned to control levels after 24 hours of exposure. Investigation on the cultured bovine endothelial cells as a model for LPS action was useful in that these cells are sensitive to relatively low levels of LPS and the endothelium may be an important target in sepsis.


Subject(s)
Endothelium, Vascular/cytology , Glycolipids/pharmacology , Lipid A/pharmacology , Lipopolysaccharides/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Animals , Cattle , Cell Adhesion/drug effects , Cell Line , Cell Survival , DNA/biosynthesis , Dose-Response Relationship, Drug , Endothelium, Vascular/drug effects , Microscopy, Electron , Microscopy, Electron, Scanning , Protein Biosynthesis , RNA/biosynthesis
11.
Proc Natl Acad Sci U S A ; 85(10): 3546-50, 1988 May.
Article in English | MEDLINE | ID: mdl-3130629

ABSTRACT

Transgenic mice carrying immunoglobulin genes coding for mu heavy chain and kappa light chain have been used to study the mechanisms involved in allelic and isotypic exclusion. We report here that individual cells from transgenic mice carrying a functionally rearranged mu heavy chain gene (capable of generating both membrane and secreted forms of IgM) can rearrange an endogenous mu heavy chain gene and simultaneously produce both transgenic and endogenous IgM. These "double-producing" cells express both endogenous and transgenic IgM in the cytoplasm (detected by immunohistology) and on the cell surface (detected by multiparameter fluorescence-activated cell sorter analysis). In addition, they secrete mixed IgM molecules containing both transgenic and endogenous mu heavy chains (detected in serum by radioimmune assay). The transgenic mice studied also have relatively large numbers of cells that produce endogenous immunoglobulin in the absence of detectable transgenic immunoglobulin ("endogenous-only cells"). The mechanisms that generate double-producing cells and endogenous-only cells appear to be under genetic control because the frequencies of these B-cell populations are characteristic for a given transgenic line. Thus, our findings indicate that more is involved in triggering allelic exclusion than the simple presence or absence of membrane mu heavy chains (as has been previously postulated).


Subject(s)
B-Lymphocytes/immunology , Genes, Immunoglobulin , Immunoglobulin M/genetics , Receptors, Antigen, B-Cell/genetics , Animals , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Immunoglobulin kappa-Chains/genetics , Immunoglobulin mu-Chains/genetics , Mice , Mice, Inbred Strains , Mice, Transgenic
12.
Cell Immunol ; 85(1): 1-14, 1984 Apr 15.
Article in English | MEDLINE | ID: mdl-6424947

ABSTRACT

Mouse spleen cells were stimulated to proliferate in vitro by soluble affinity-purified heterologous antibodies to mouse delta. Antibodies from goat or rabbit antisera to TEPC 1017, a mouse IgD myeloma protein, were purified on an affinity column of TEPC 1033, a second mouse IgD myeloma protein. Maximum uptake of [3H]thymidine in the range of 60,000 cpm was obtained after 48 hr of culture with anti-delta at concentrations of 50 micrograms/ml. In contrast, the hybridoma 10-4.22 anti-delta was nonmitogenic at similar concentrations. The proliferative response was not impaired upon removal of T cells by treatment with an anti-thymocyte serum (ATS), nor by removal of adherent cells by passage of spleen cells over Sephadex G-10 columns and counter-flow centrifugation. Splenic lymphocytes isolated on the fluorescence activated cell sorter (FACS) with intermediate-to-high amounts of surface IgD (sIgD) were responsive to soluble anti-delta, while IgD-negative cells, or cells with low amounts of sIgD, were unresponsive. Spleen cells from mice less than 4 weeks of age, or from mice carrying the X-linked B cell defect (xid), were unresponsive to anti-delta. These results indicate that anti-delta acts similarly to anti-mu in stimulating a proliferative response by later maturing B cells, which are characterized by a high density of sIgD.


Subject(s)
Antibodies, Anti-Idiotypic/immunology , Immunoglobulin Heavy Chains/immunology , Immunoglobulin delta-Chains/immunology , Lymphocyte Activation , Animals , Antibodies, Monoclonal/immunology , B-Lymphocytes/immunology , Cell Division , Goats/immunology , Immunoglobulin Fab Fragments/immunology , Immunoglobulin mu-Chains/immunology , Kinetics , Macrophages/immunology , Male , Mice , Rabbits/immunology , Solubility , Spleen/cytology
13.
J Immunol ; 132(3): 1544-9, 1984 Mar.
Article in English | MEDLINE | ID: mdl-6420465

ABSTRACT

Mice expressing the xid gene exhibit an altered immune response to phosphocholine (PC)-conjugated keyhole limpet hemocyanin (KLH). Less than 25% of their anti-PC-KLH response is PC specific, and most of these antibodies lack the normally predominant T15 idiotype. These findings suggested that immune defective mice might employ different variable region genes than normal mice in their anti-PC response. To examine this possibility, we characterized by Southern blot analysis the gene family encoding PC-VH regions and determined the amino acid sequence and fine specificity of binding of a T15-, IgG2, PC-specific hybridoma (1B8E5) produced by fusion of the SP2/O cell line and PC-KLH immune CBA/N spleen cells. Southern blot analysis of DNA from CBA/N mice by using a PC-VH probe (S107 VH) revealed a hybridization pattern virtually identical to that of DNA from normal CBA/J mice, indicating that CBA/N mice do not suffer from a gross deletion of PC-VH genes. Analysis of the 1B8E5 antibody reveals that both the binding specificity and relative affinity of this antibody are different from the anti-PC antibodies of the T15, M167-M511, and M603 families. The complete amino acid sequence of the heavy (H) chain variable region shows that 1B8E5 uses a VH segment identical to the allelic form of T15 (C3) but has a unique D region of three amino acids and use the JH1 joining segment. Both the DH and JH regions are unusual when compared to PC-specific antibodies from normal mice, which have a D region composed of five to eight amino acids and use the JH1 joining segment. The amino terminal sequence of the 1B8E5 light (L) chain demonstrates that this anti-PC antibody carries a Vk3 subgroup L chain. Chains from this subgroup have not previously been found in association with PC-binding antibodies. Thus, the Vk, DH, and JH segments expressed in 1B8E5 make this hybridoma unique in terms of the anti-PC antibodies studied to date, and suggests that additional PC-specific antibodies exist in inbred mice that employ "unusual" V gene segments.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Binding Sites, Antibody , Choline/analogs & derivatives , Immunoglobulin Idiotypes/genetics , Immunoglobulin Variable Region/genetics , Phosphorylcholine/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/genetics , Antibody Specificity , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Hybridomas/immunology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Immunoglobulin kappa-Chains/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred CBA , Mitochondria, Liver/metabolism , Phosphorylcholine/immunology
14.
J Immunol Methods ; 63(3): 299-307, 1983 Oct 28.
Article in English | MEDLINE | ID: mdl-6138381

ABSTRACT

A method for the rapid separation of murine spleen cells into subpopulations on the basis of their size has been developed using counterflow centrifugation. Upon separation of normal spleen cells with a mean cell volume of 125.5 +/- 6.0, 5 fractions of cells were obtained with mean cell volumes which ranged from 107.8 +/- 3.2 microns3 in fraction 1 to 152.7 +/- 4.9 microns3 in fraction 5. The cells in these 5 fractions were characterized by analysis on a fluorescence activated cell sorter (FACS) after staining with fluorescein conjugated anti-mu, delta, Ia, or Thy 1.2 antibodies, and by assaying for the presence of non-specific esterase activity. Surface Ig+, Ia+ B lymphocytes and Thy 1.2 T lymphocytes were present in all 5 fractions. However, while these T and B lymphocytes accounted for virtually all of the cells in the first 3 fractions, non-T, non-B cells were found in fractions 4 and 5, and represented 30% of the total population in the 5th fraction. Comparison of the intensity of anti-mu, delta or Ia staining of the B cells in fractions 1-5 revealed differences which suggested that B cell size correlated with different activation states of these cells. Increases in the intensity of the staining of T lymphocytes by anti-theta antibodies were also noted in the various fractions. The capacity of the B and T cells in each fraction to proliferate to B or T cell mitogens, respectively, was proportional to their frequency within the fractions. By contrast, the fractions containing larger cells were enriched in cells which proliferated in vitro in the absence of added mitogen. Furthermore, only fractions containing larger cells had the capacity to stimulate allogeneic T cell proliferation in a mixed lymphocyte reaction. Our data suggest that this technique provides a useful method for separating splenocytes on the basis of cell size. Use of this methodology provides a way to correlate cell size with phenotypic surface markers and functional abilities.


Subject(s)
Cell Separation/methods , Centrifugation/methods , Lymphocytes/cytology , Macrophages/cytology , Spleen/cytology , Animals , Antigens, Surface/analysis , Histocompatibility Antigens Class II/analysis , Lymphocyte Activation , Lymphocyte Culture Test, Mixed , Lymphocytes/immunology , Mice , Receptors, Antigen, B-Cell/analysis , Thy-1 Antigens
16.
J Exp Med ; 154(5): 1500-16, 1981 Nov 01.
Article in English | MEDLINE | ID: mdl-6975349

ABSTRACT

B lymphocyte-enriched cell populations cultured with mitogens in initial suspension cultures formed colonies in soft agar when the same mitogenic agent was present in the lower layer of a two-layer soft agar system. Colony formation depended upon the presence of T cells in the initial culture, and was optimal after an initial 72-h culture with phytohemagglutinin (PHA; 12.5 microliters/ml), pokeweed mitogen (PWM; 2.5 micrograms/ml), or protein A (10 micrograms/ml). The colonies could be picked from the agar and propagated by feeding every 3 d with medium supplemented with a growth factor-containing tissue culture supernate. The growth factor-containing supernate was prepared by stimulating pools of human peripheral blood mononuclear cells for 72 h with PHA or PWM. The lines propagated in this manner were membrane Ig+, lacked sheep erythrocyte rosette-forming ability, and did not ingest latex. They lacked the Epstein-Barr nuclear antigen (EBNA) and had 46 chromosomes. Such lines have been propagated for over 1 yr. One line (BL1) was subjected to limiting dilution cloning and a line, BL1.1, was prepared that contained 96% lambda-bearing cells and no kappa-bearing cells. This line was also EBNA negative. This procedure can thus be used to prepare and clone long-term lines of nontransformed human B lymphocytes.


Subject(s)
B-Lymphocytes/cytology , Lymphocyte Activation , B-Lymphocytes/immunology , Cell Line , Cells, Cultured , Clone Cells/cytology , Clone Cells/immunology , Growth Substances/pharmacology , Humans , Phytohemagglutinins/pharmacology , Pokeweed Mitogens/pharmacology , Receptors, Antigen, B-Cell , Staphylococcal Protein A/pharmacology , T-Lymphocytes/cytology , Time Factors
17.
J Immunol ; 127(3): 873-6, 1981 Sep.
Article in English | MEDLINE | ID: mdl-6167630

ABSTRACT

Two monoclonal rat anti-mouse IgM antibodies, Bet 1 and Bet 2, are described in this paper. Bet 1 defines a new allotypic determinant, Igh-6.5, expressed on IgM molecules in serum and on B lymphocytes, whereas Bet 2 recognizes a determinant on IgM molecules of all mouse strains tested. Both reagents bind to the IgM myeloma protein MOPC104E, but not to IgG myeloma proteins, including FLOPC21, MOPC21, and UPC10. Using serum from various mouse strains to inhibit the binding of Bet 1 to MOPC104E, 3 distinct inhibition patterns were found. BALB/c, DBA/2, and CBA sera inhibited strongly, C56BL/6 (B6), SJL, AKR, and NZB sera inhibited weakly, and A and AL sera showed no inhibition of binding of BET 1 to MOPC104E. All sera tested were equivalent in their inhibition of the binding of Bet 2 to MOPC104E. When spleen cells from different mouse strains were reacted with fluorescein-conjugated Bet 1 (F-Bet 1) and subjected to flow microfluorometry analysis, 3 types of staining patterns, corresponding to those obtained with the serum inhibition assay, were also found. The determinant recognized by Bet 1 is controlled by a gene linked to the Igh-C gene complex. C.AL20 behaved like AL, and C.B20 and BAB/14 behaved like B6, both in the serum inhibition assay and in flow microfluorometry analysis of spleen cells stained with F-Bet 1. In addition, the capacity of serum from individual (BALB/c X B6) X B6 backcross progeny to inhibit the binding of Bet 1 to MOPC104E was linked to the expression of the Ig-1a marker.


Subject(s)
Antibodies , Epitopes , Immunoglobulin Allotypes , Immunoglobulin M , Animals , Antibodies, Anti-Idiotypic , Antibodies, Monoclonal , Binding, Competitive , Fluorescent Antibody Technique , Genetic Linkage , Mice , Mice, Inbred A , Mice, Inbred AKR , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Inbred DBA , Mice, Inbred NZB , Myeloma Proteins/immunology , Rats
19.
J Immunol ; 124(4): 1721-6, 1980 Apr.
Article in English | MEDLINE | ID: mdl-6965958

ABSTRACT

The ability of various cell populations to bind and present the thymus-independent antigen TNP-Ficoll to a responding cell population was assessed. The in vitro antibody response to TNP-Ficoll depends upon the presence of B lymphocytes and plastic-adherent accessory cells, but does not require T lymphocytes. Purified B cells were the most effective population in binding and presenting TNP-Ficoll, and adherent cells did not perform this function. Antigen binding and presentation was antigen specific and could be blocked with anti-mu antibody, but not by antibodies directed against other immunoglobulin classes. Spleen cells from mice genetically unresponsive to TNP-Ficoll (CBA/N X BALB/c F1 males) were equally effective as normal spleen cells in antigen binding and presentation. We conclude that the initial events in the induction of the antibody response involves antigen binding by B cells, and that subsequent activation of the subset of B cells that can respond to TNP-Ficoll proceeds either via B cell-B cell interaction or B cell-dependent transfer of antigen to macrophage-like cells.


Subject(s)
Antibody Formation , Antigens , Immunity, Cellular , Thymus Gland/immunology , Animals , B-Lymphocytes/immunology , Cell Adhesion , Cell Survival , Female , Ficoll/immunology , Immunoglobulin M , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Inbred DBA , Rabbits , T-Lymphocytes/immunology , Trinitrobenzenes/immunology
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