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L W Arnold

Publications and source records attributed to L W Arnold.

At least 37 records · Page 2Linked to original sources
PubMed1991

CD3 expression, modulation and signalling in T-cell subpopulations from MRL/Mp-lpr/lpr mice.

The expanded T-cell population of MRL/Mp-lpr2lpr mice is abnormal from a variety of standpoints. We have already shown that T-cell receptor expression and modulation are aberrant in the predominant CD4- CD8 (DN) T cell population. To investigate these abnormalities further, we examined CD3 expression and modulation in subpopulations of +/+ and lpr T cells and measured mitogen-induced Ca++ mobilization in DN lpr T cells. We found that expression and modulation of CD3 in CD4hi and CD8hi lpr single positive (SP) T cells are similar to that in +/+ T cells. We have, however, identified additional lpr cell subsets that are CD4lo or CD8lo. Their expression and modulation of CD3 are intermediate, between that of SP and DN lpr T cells. These subpopulations may thus represent a transitional stage between the SP and DN populations. The rapid modulation of CD3 in the DN population does not appear to be merely related to the lack of expression of CD4 or CD8, and may in fact cause (rather than result from) low CD3 expression. In addition, we observed impairment of CA++ mobilization in DN lpr T cells in response to concanavalin A or anti-CD3 antibody. These findings further define the abnormalities of T cells from lpr mice.

Animals↗
PubMed1991

Ig isotype switching in B lymphocytes. The effect of T cell-derived interleukins, cytokines, cholera toxin, and antigen on isotype switch frequency of a cloned B cell lymphoma.

The murine B cell lymphoma CH12.LX, which bears cell surface IgM specific for the phosphatidyl choline epitope of sheep red blood cells, is capable of spontaneous isotype switching in vitro. Switching to IgG3, IgG1, IgG2b, and IgA has been observed and variants expressing those isotypes have been isolated and cloned. We have developed a procedure for precise numerical evaluation of the frequency of switching to the several isotypes to which CH12.LX can switch. We have used a modified Poisson method which can distinguish between treatments which change isotype switch frequency and those which affect, in an isotype-specific fashion, growth or secretion rates of cells which have already switched. In this report we examine the effect of several cytokines, cholera toxin, hydroxyurea, and antigen on the isotype switch frequency of CH12.LX. The strongest effect observed was that of transforming growth factor-beta, which increases switch frequency 40-fold to an absolute switch frequency of 0.04 switch events (from IgM to IgA expression) per cell division. Interleukin-4 (IL-4) and cholera toxin also increase the switch frequency of CH12.LX while IL-5, IL-6 (with or without antigen), antigen (SRBC) alone, interferon-gamma, or hydroxyurea have no effect. We have shown that none of the cytokines studied change the relative frequency of switching to the available isotypes, only the absolute frequency of switching. We infer from this that the factors tested do not 'instruct' CH12.LX to switch to a particular isotype, but rather they deliver a 'go' signal to cells committed to switching to IgA at high frequency, rarely to IgG3, IgG1, or IgG2b, and never to IgG2a or IgE.

Animals↗
PubMed1990

High frequency expression of S107 VH genes by peritoneal B cells of B10.H-2aH-4bP/WTS mice.

Our previous analyses of peritoneal Ly-1 B cells indicate that a high percentage express VH genes of the VH11 and VH12 families, and that this bias is due to clonal selection. The antibodies encoded by these genes bind the same hapten, phosphatidyl choline (PtC). Twenty-one of 73 hybridomas generated from fusions with peritoneal Ly-1 and Ly-1 sister population B cells of B10.H-2aH-4bp/Wts mice produce anti-PtC specific antibodies. We show here that 19 of these express VH11 and VH12 family genes and two express VH36-60 family genes. To assess whether there is a bias in VH gene use among non-PtC-specific hybridomas we analyzed the remaining 52 hybridomas for VH family expression by using VH family-specific probes in an RNA dot blot assay and by Ig mRNA sequencing. We find a seven-fold increase in the expression of the VHS107 family genes, and only slight differences in the expression of VH genes of other families relative to splenic B cells. We attribute the increase in VHS107 gene expression to clonal selection inasmuch as five of the seven VHS107+ hybridomas express the same VH gene (V11) and VL association is nonrandom. The bias in VH gene use among the entire panel of 73 peritoneal hybridomas is to the extent that approximately one-third express one of three genes: the V11 gene of the S107 family, the CH34 gene of the VH11 family, and the VH12 family gene.

Animals↗
PubMed1989

Restricted immunoglobulin variable region gene usage by normal Ly-1 (CD5+) B cells that recognize phosphatidyl choline.

5-15% of lymphocytes in the peritoneums of normal adult B10.H-2aH-4bp/Wts (2a4b) mice are CD5+ (Ly-1) B cells that recognize phosphatidyl choline (PtC), a phospholipid component of all mammalian cells. We produced a set of IgM-secreting hybridomas from the peritoneal cells of normal, adult 2a4b mice. We found that this set of hybridomas shows a similarly high frequency of antibodies specific for PtC (21 of 86) that also react with bromelain-treated mouse erythrocytes. Restriction fragment analysis of Ig gene rearrangements and analysis of expressed Ig idiotypes reveal that these cells use a restricted set of variable region genes to generate the PtC-specific antibodies. The Ig genes used by the PtC-specific hybridomas appear to be the same as those found in the PtC-specific Ly-1 B cell lymphomas, CH27 and CH34.

Animals↗
PubMed1989

Early commitment to neoplasia in murine B- and T-cell lymphomas arising late in life.

Lymphomas arise spontaneously in C57BL/10.H-2aH-4b (2a4b) mice greater than 1 year old. The purpose of this study was to determine when cells become committed to neoplasia and whether genes linked to H-2a or H-4b are a factor in predisposition to genesis of lymphoma. Following transplantation of a pool of spleen cells in large groups of mice, lymphoma incidence increased 1.8- to 2.1-fold and mean latent periods decreased, compared with those in controls. Lymphomas arising in mice that received the same pool of spleen cells often displayed identical rearrangements of genes for immunoglobulin and T-cell receptor. This finding indicates that the lymphomas were derived from expanding clones of cells present in the donor mice prior to transfer. The age of the donor mice determined the latency of lymphomas arising after spleen cell transfer. When donors were 3-4 months old at the time of spleen cell transfer, lymphomas were detected in the recipients 1 year later. When mice 1.5-2 years old were used as donors, tumors were observed 3 months after transfer. Almost all of these tumors were clonally related B-cell lymphomas. We conclude that the lymphomas seen late in life arise from premalignant clones of cells that have become committed to neoplasia in relatively young animals. These clones normally follow an indolent course and are manifested clinically only in old age. Neither H-2a nor H-4b alone appeared to alter susceptibility to the genesis of lymphoma in similar experiments with H-2a and H-4b congenic mice.

Animals↗
PubMed1989

Biased immunoglobulin variable region gene expression by Ly-1 B cells due to clonal selection.

Most, if not all, autoantibodies specific for bromelain-treated mouse erythrocytes recognize the common membrane phospholipid, phosphatidyl choline (PtC). Anti-PtC antibodies are produced by 5%-15% of CD5+ Ly-1 B cells of normal unimmunized mice, but not by detectable numbers of conventional CD5- B cells. At 1 week of age PtC-specific B cells are undetectable but then increase dramatically over the next 3 to 4 weeks to reach adult numbers. We report here that PtC-specific Ly-1 B cells in B10.H-2aH-4bp/Wts mice predominantly express either of two heavy and kappa chain variable (V) region gene combinations. In addition, the sequence and length of DH genes are conserved among cells expressing the same V gene combination, and the V kappa-J kappa junctions of one group involve unusual splice sites. Preferential V gene rearrangement models are insufficient to explain the DH and V kappa-J kappa junctional sequences or the delayed appearance of this specificity, and so they cannot solely account for the high frequency of PtC-specific cells. These characteristics are more consistent with antigen selection. We therefore attribute the frequent use of the two V region gene combinations to selection for cells that express them and conclude that the expressed V gene repertoire of Ly-1 B cells in adult mice is influenced by antigen selection. Apparently, there is no selection for mutant anti-PtC antibodies of higher affinity during the formation of the Ly-1 B repertoire because the V region genes expressed by PtC-specific cells are unmutated. Our findings are consistent with an important, germ line-encoded function for the immunoglobulin products of these gene combinations.

Amino Acid Sequence↗
PubMed1989

Ig isotype switching in B lymphocytes. A method for estimating isotype switch frequency in cloned B cell lymphomas.

We have developed a method for enumerating the frequency of Ig isotype switching in clones of B cells. The method adapts Poisson statistics to analyze the distribution of amounts of switched isotype produced by multiple subclones of cells and thus enables one to estimate the probability that a single cell will switch isotype in one cell generation. We have applied this method to determine the spontaneous switch frequency of two Ly-1+ B cell lymphomas of B10-H-2aH-4bp/Wts mice. Both CH12.LX and CH27.LX switch from IgM to IgA at very high frequencies (1 - 5 x 10(-3) switch events per cell division) and from IgM to IgG at low but detectable frequencies (10(-4) - 10(-5) switch events per cell division). Cloned IgG variants of CH12.LX switch to IgA at the same frequency as the IgM-producing cells. Bacterial lipopolysaccharide has a strong inhibitory effect on isotype switching by CH12.LX. Possible explanations for the observed preference for switching to IgA are discussed.

Animals↗
PubMed1988

Restricted Ig variable region gene expression among Ly-1+ B cell lymphomas.

The majority of the characterized Ly-1+ B cell lymphomas of B10.H-2aH-4bp/Wts origin (the CH series) bear surface Ig related by Ag specificity or idiotype or both. To determine the genetic basis for these structural similarities, we have sequenced the VH and VL region genes expressed by 10 CH lymphomas, and have compared their VH and V kappa gene rearrangements by Southern blot analysis to one another and to those of four other CH lymphomas. Sequence analysis identified only five different VH, and seven different VL genes, and indicated that these V genes are essentially unmutated. CH lymphomas which express the identical VH gene share at least one idiotope. Thus, the basis for shared idiotype and specificity is due in most cases to the use of the same V gene. This restriction in V gene expression is not due to the preferential use of V genes of any particular VH family or VL group, as the expressed V genes belong to four different VH families and four V kappa groups, and include V lambda 1 and V lambda 2. We hypothesize that Ag selection accounts for the restriction in V gene usage among CH lymphomas.

Animals↗
PubMed1988

Normal mouse peritoneum contains a large population of Ly-1+ (CD5) B cells that recognize phosphatidyl choline. Relationship to cells that secrete hemolytic antibody specific for autologous erythrocytes.

We have found that, in the peritoneums of normal adult mice, 5-15% of lymphocytes bind a fluorescent liposome probe. In ontogeny, cells with this specificity were shown to appear by 8 d after birth, and increase to the adult frequency by 2-3 wk. Some older mice contain an expanded population of these cells. We have shown that liposome binding occurs by cell surface IgM recognizing the common membrane phospholipid, phosphatidyl choline (PtC). Virtually all of these PtC-specific cells bear the cell surface marker Ly-1. Our results indicate that roughly 1 in 10 peritoneal Ly-1+ B cells has this single specificity. We have found that the precursors to all the cells that form plaques on protease-treated autologous erythrocytes (BrMRBC) are included in the PtC-specific population and can be isolated by FACS. We believe this is the first report of sorting large numbers of B cells with a single antigen specificity from normal, unimmunized animals. This method will allow for in vitro and in vivo studies of differentiative and proliferative properties of Ly-1+ B cells, which may help define their role in development and disease.

Aging↗
PubMed1988

Ig isotype switching in B lymphocytes. Isolation and characterization of clonal variants of the murine Ly-1+ B cell lymphoma, CH12, expressing isotypes other than IgM.

We have selected and cloned variant cells from the murine B cell lymphoma, CH12, that produce a variety of other Ig isotypes in addition to or in place of the original IgM and IgD. Variants were selected by flow cytometry and automated cloning and isotype production was analyzed by membrane immunofluorescence and ELISA of culture fluids. Variants have been isolated that produce the single isotypes IgA, IgG2b, and IgG3, as well as variants that produce more than one isotype simultaneously, i.e., IgM, IgD, and IgA; IgG2b and IgA; IgG3 and IgA. All isotypes have been seen as cell surface proteins and all except IgD have been found in culture supernatants. All isotypes display the same idiotype and Ag-binding specificity for phosphatidyl choline as the original IgM and all are translated from the same VDJH and VJ kappa gene assemblies. Production of more than one isotype by a variant clone is due to simultaneous production of all the isotypes by each cell within the clone. The finding that the variants producing more than one isotype are all tetraploid suggests the interesting possibility that each isotype is derived from an independently switching chromosome. All isotype variants can be stimulated by LPS to secrete the appropriate Ig isotype at an increased rate similar to the IgM expressing parent. The variants differ in stability; some have remained stable for more than 9 months in culture, whereas other have undergone further isotype switching. The facts that some isotypes have not been seen, that multistep switching has occurred, and that many variants produce IgA in addition to another isotype are discussed in relation to current notions of isotype switching mechanisms.

Animals↗
PubMed1988

A monoclonal antibody to the insect prothoracicotropic hormone.

The prothoracicotropic hormone (PTTH) is an insect cerebral peptide that stimulates the prothoracic glands to produce the steroid hormone ecdysone thus initiating molting and metamorphosis. "Big" PTTH, one of several molecular forms of the neurohormone, was isolated from brains of the tobacco hornworm Manduca sexta, and fractionated by high-pressure liquid chromatography (HPLC) for use in antibody production. A murine polyclonal antiserum and a monoclonal antibody (MAb) have been generated using this highly purified preparation of big PTTH. Antisera and hybridoma supernatants were screened with an indirect, brain whole-mount immunocytological assay, and antibody specificity was confirmed by immunocytological, ELISA, and functional criteria. In brain whole-mount preparations, the MAb (A2H5) and antiserum specifically immunostained the lateral protocerebral neurosecretory cells (L-NSC III), the prothoracicotropes, which produce PTTH. This immunostaining was blocked by preadsorbing the antibodies with big PTTH. Analysis of the elution of HPLC-fractionated big PTTH with an in vitro bioassay for the neurohormone and an ELISA employing the A2H5 MAb resulted in peaks of activity that were superimposable. Finally, the antiserum and A2H5 MAb inhibited big PTTH activation of the prothoracic glands to synthesize ecdysone in the in vitro bioassay for the neurohormone. With these specific antibodies, the organization of the PTTH neuroendocrine axis has been defined. It is now evident that both of the peptidergic neurons that comprise the L-NSC III are prothoracicotropes, and that the corpora allata are the neurohemal organs for the release of big PTTH into the hemolymph. This study indicates that these specific antibodies will be useful in investigations of numerous aspects of the biology of this cerebral neuroendocrine axis.

Animals↗
PubMed1986

Phosphatidyl choline is recognized by a series of Ly-1+ murine B cell lymphomas specific for erythrocyte membranes.

Cells from 6 of 14 different Ly-1+ murine B cell lymphomas bound to synthetic liposomes encapsulating fluorescein. The liposomes were made from distearoyl phosphatidyl choline (DSPC), distearoyl phosphatidyl glycerol (DSPG), and cholesterol. In all cases, liposome binding was due to recognition of phosphatidyl choline by the surface IgM on the tumor cells. Liposome binding could be inhibited by DSPC but not by DSPG, and the number of liposomes bound per cell was directly related to the cell surface concentration of IgM. The IgM secreted by a hybridoma derived from one of the lymphomas, CH12, was shown to agglutinate liposomes, and was used in a solid-phase immunoassay to study inhibition of liposome binding by pure phospholipids; DSPC and sphingomyelin both inhibited, whereas DSPG did not. The Ig borne by the six lymphomas that bind phosphatidylcholine also bind to both SRBC and bromelain-treated mouse erythrocytes. The idiotypic of CH12 IgM is similar to that expressed by Ly-1+ normal splenic B cells of the same specificity. The significance of these data in relation to other commonly studied autoantigens, and to the restricted specificity of normal Ly-1+ B cells is discussed.

Animals↗
PubMed1986

Antigen-specific B cell tumors of mice.

The topics covered in this review include: the origin and availability of murine B cell lymphomas bearing immunoglobulins of known antigen reactivity; their etiology and the role of antigen in lymphomagenesis; the extent to which such tumors are true analogs of normal B cells; and the knowledge of B cell differentiation which has been derived from the study of these cells. There is little or no evidence that B lymphomas with preselected antigen specificity can be induced at will. However, there is evidence that genetic factors influencing idiotype specific regulation can predispose particular subsets of B cells to neoplastic transformation, and thereby indirectly influence the antigen specificity of the resultant lymphomas. The bulk of evidence suggests that, except for growth control characteristics, recently derived B cell lymphomas retain many of the features of normal lymphocytes. Thus, mechanisms elucidated by study of lymphomas are probably reflective of normal B cell biology.

Animals↗
PubMed1985

Three classes of signalling molecules on B-cell membranes.

The question of whether surface immunoglobulin and Ia molecules have a signalling function in helper T cell-dependent activation of B cells has been evaluated. Two sources of B cells have been used, one a purified population of hapten-binding B cells, the other a B-cell lymphoma, CH12, with known antigen specificity. Evidence is presented that both immunoglobulin and Ia molecules are receptors actively involved in the initial activation of resting B cells. Nevertheless, the requirements for ligand binding to either receptor can be bypassed under appropriate conditions, and the implications of this result for the function of these molecules is discussed. With respect to B-cell Ia, the authors present data that demonstrate two distinct functions of this molecule, one as a restricting element for T-cell activation, the second as a signalling receptor for B-cell excitation. On the CH12 surface, the I-A molecule fulfills the former function, but T-cell interactions with I-A fail to result in B-cell stimulation, suggesting that B-cell Ia may limit helper T cell-B cell interactions. We suggest that the binding of antigen surface immunoglobulin and binding of helper T-cell receptors to the appropriate Ia molecule(s) results in the activation of genes that encode for a third class of membrane B-cell receptors, those that bind B-cell stimulating factors.

Animals↗
PubMed1985

Cross-reactive idiotypes and common antigen binding specificities expressed by a series of murine B-cell lymphomas: etiological implications.

A series of 27 B-cell lymphomas (designated the CH series), induced in B10.H-2aH-4b p/Wts mice by intense adoptive immunization with sheep erythrocytes, was found to represent a subset of the total B-cell repertoire. This subset was characterized by expression of a limited number of Ig heavy chain variable regions, as evidenced by the presence of cross-reactive idiotypes and common antigen binding specificities. Twenty-one of the 27 CH lymphomas studied were classified into five groups, defined by a particular cross-reactive idiotype; four of these groups were linked in a single network. Seven of 16 idiotypes defined by absorption analysis were present on lymphomas bearing either kappa or lambda light chains and so were localized to the heavy chain variable region. The surface Ig on 14 CH lymphomas was found to be specific for epitopes on certain erythrocytes (bromelain-treated autologous erythrocytes, sheep, and chicken erythrocytes) or E. coli. We propose that the CH lymphomas represent the malignant counterparts of a subset of idiotypically related, normal B cells in B10.H-2aH-4b p/Wts mice. Perturbation of this idiotype network, by hyperimmunization with an antigen for which some of the members are specific (sheep erythrocytes), increases the risk for neoplasia. Possible mechanisms for this are discussed.

Animals↗