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G Haughton

Publications and source records attributed to G Haughton.

At least 37 records · Page 2Linked to original sources
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

Mixed phenotype lymphomas of B10.H-2a-H-4bp/Wts mice: characterization of component cell populations.

Three murine lymphomas of H-2a origin were investigated. Each of these lymphomas was derived in B10.H-2aH-4bp/Wts (2a4b) mice after a hyperimmunization protocol involving sheep red blood cells. Analysis of cell suspensions derived from the splenic tumors revealed a mixed lymphoid phenotype of the total cell population. Both the T-cell and B-cell populations remained after multiple passages of the tumors in syngeneic mice. A multiparameter approach combining immunologic and molecular genetic techniques was taken to determine the malignant cell population within these lymphomas. This analysis included depletion experiments utilizing histocompatible (B10.A X 21M)F1 mice to distinguish normal host-derived cells from the malignant tumor cells, fluorescence-activated cell sorting of the two cell populations followed by in vivo growth of the sorted cells, in vitro and in vivo cloning attempts, and molecular analysis of genomic DNA derived from the splenic tumors for clonal rearrangements of the immunoglobulin heavy-chain and T-cell receptor loci. Each of the three lymphomas was shown to be a malignant B-cell tumor that has an associated nonneoplastic T-cell component. The nature and functional significance of the numerous T-cells present in the B-cell lymphomas remain unknown and are being investigated.

Animals↗
PubMed1988

Signaling to a B-cell clone by Ek, but not Ak, does not reflect alteration of Ak genes.

The mouse B-cell clone, CH12.LX (Iak, Ly-1+, mu+, delta+), can be induced to differentiate and secrete antibody in an antigen-specific, H-2-restricted manner. Induction requires two signals. One must be provided by the binding of specific antigen to the membrane IgM; the other is delivered by the binding of Ek-specific T-cell hybridomas to the Ek molecules of CH12.LX (Bishop and Haughton 1986). Previous studies demonstrated that Ek-specific monoclonal antibodies (mAbs) could substitute for T cells in delivering the second differentiative signal (Bishop and Haughton 1986). Although CH12.LX cells present Ak to Ak-restricted or alloreactive T-helper cells, neither T cells nor mAbs specific for Ak induce differentiation (Bishop and Haughton 1986). However, since the Akspecific mAbs tested previously were beta-chain-specific and the Ia epitope specificity of the T cells used was unknown, it is possible that the differentiative signal delivered to the CH12.LX class II molecule is chain-specific. Here we report the effects of ten additional Iak-specific mAbs upon the differentiation of CH12.LX. In addition, a cDNA library was prepared from CH12.LX cells, clones corresponding to the alpha and beta chains of the Ak molecule were isolated, and their nucleotide sequences were determined. Finally, the Ak and Ek molecules of CH12.LX and H-2k spleen cells were compared by two-dimensional gel electrophoresis to examine possible post-translational differences in the Iak molecules of CH12.LX.

Amino Acid Sequence↗
PubMed1988

Analysis of a murine B cell lymphoma, CH44, with an associated non-neoplastic T cell population. I. Proliferation of normal T lymphocytes is induced by a secreted product of the malignant B cells.

A non-neoplastic T cell population associated with a murine monoclonal B cell malignancy, CH44, was analyzed. Immunofluorescence on cell suspensions and immunoperoxidase staining on tissue sections using monoclonal antibodies to the antigens Thy1.2, Ly-1, L3T4, and Lyt-2 confirmed the presence of both TH (Ly-1/L3T4+, Lyt-2-) and Tc/s (Ly-1/L3T4-, Lyt-2+) T cell subpopulations. The non-neoplastic T cells were present in both a 0.6 and 2.1 g CH44-bearing spleen. T cells, not normally in liver in significant numbers, were found in liver tissue when the CH44 tumor cells were present. These data implied an active proliferation of the T cell populations within tissues containing the malignant B cells. Supernatant from an in-vitro-adapted cell line of CH44 (CH44.LX) was tested for its ability to induce proliferation of normal murine splenocytes and thymocytes. As assayed by tritiated thymidine incorporation, both spleen and thymus cells proliferated in the presence of CH44.LX supernatant. Although supernatant from two of nine other B cell lines was able to stimulate the proliferation of spleen cells, only CH44.LX could induce proliferation of thymus cells. Supernatant from the seven other B cell lines and three hybridomas had no measurable effect on either splenocytes or thymocytes in this assay. It is hypothesized that the presence of a non-neoplastic proliferating T cell population associated with a neoplastic B cell lymphoma during in vivo passaging of the tumor is the result of effects derived from a secreted product of the malignant B cells. Whether the T cells have any effect on the growth of the malignant B cells is not known.

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↗
PubMed1987

Role of the interleukin 2 receptor in differentiation of a clone of Ly-1+ B cells.

CH12.LX, an in vitro subclone of a murine B cell lymphoma that makes IgM reactive with sheep erythrocytes (SRBC), has cell surface receptors for the lymphokine interleukin 2 (IL 2). The binding of recombinant murine IL 2 to these receptors did not stimulate CH12.LX cells to differentiate and secrete antibody. However, the binding of either of two monoclonal antibodies (Mab) specific for the IL 2 receptor increased the proportion of CH12.LX cells that secrete hemolytic IgM. The effect did not require the presence of antigen. One of the Mab, 3C7, is known to block the binding of IL2 to its receptor on T cells, whereas the other, 7D4, which also reacts with the IL 2 receptor, does not block the binding of IL 2. The differentiation of CH12.LX induced by 3C7, but not that induced by 7D4, was inhibited by recombinant IL 2. Neither IL 2 (up to 200 U/ml) nor 3C7 (up to 10 micrograms/ml) had any significant influence on incorporation of [3H]thymidine; 7D4 at 10 micrograms/ml decreased thymidine incorporation by about 60%. Mitomycin C and hydroxyurea, which both inhibit the incorporation of [3H]thymidine into CH12.LX cells, also both induce antibody secretion. In both cases, the concentration necessary to cause differentiation is substantially lower than that needed to cause detectable inhibition of thymidine uptake. We conclude that the IL 2 receptor on CH12.LX cells is a functional signal transducing molecule, and we discuss the possible inverse relationship between proliferation and differentiation.

Animals↗
PubMed1987

Cyclosporine inhibition of CH series murine B-cell lymphomas.

To investigate the mechanisms by which cyclosporine (CsA) inhibits B-cell function, the effect of this agent on murine B-lymphoma cell lines of the CH series was tested. These lymphomas appear to be derived from a restricted B-cell population on the basis of their common expression of the Ly-1 cell surface marker and autoantibody products. Proliferation of each of the six cell lines tested was inhibited by CsA at doses without effect on the nonlymphoid HeLa cell line. The cell lines, however, differed from each other in their sensitivity to this agent. To correlate this sensitivity to other functional B-cell properties, the effect of lipopolysaccharide (LPS) on the proliferation of the CH cell lines was tested. Three of the lines showed enhanced proliferation to LPS; two were inhibited while one was unaffected. The cell lines that responded with increased proliferation to LPS were the most sensitive to CsA. These results indicate that sensitivity to CsA may be a common property of B cells of certain lineages, although the degree of sensitivity may be influenced by the activation properties of these cells.

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

Induced differentiation of a transformed clone of Ly-1+ B cells by clonal T cells and antigen.

The present study used cocultures of clonally derived B and T cells, together with an antigen reactive with the membrane Ig of the clonal B cells, to address the issue of B-cell differentiation requirements. The B cells were CH12.LX, an in vitro grown subclone of a murine B-cell lymphoma, which bears surface IgM reactive with sheep erythrocytes. The T cells were alloreactive T-helper-cell hybridomas. Very small numbers of T-helper cells could induce differentiation of cloned B cells without the presence of accessory cells, but such induction was dependent upon the presence of the antigen recognized by the B cell. Induced differentiation of the B cells did not depend on metabolic activity of the T cells, and metabolically active T cells could be replaced by fixed cells or by monoclonal antibody reactive with the class II molecule of the B cell to deliver an Ia-specific differentiative signal. T cells, or alloantibody that reacted with the I-E molecule, induced differentiation of the B cells; those that reacted with the I-A molecule did not. These results define the minimal requirements for major histocompatibility complex-restricted, T-cell-mediated induction of B-cell differentiation.

Animals↗
PubMed1986

Cyclosporine inhibition of a murine B cell lymphoma.

The effect of cyclosporine (CsA) on the CH12 murine B cell lymphoma was investigated to determine whether sensitivity to this agent is retained by malignant B cells. This tumour produces an antibody to bromelain-treated red blood cells and may represent transformation of a B cell with certain activation properties associated with early resting B cells. In in vitro cultures, the growth and proliferation of CH12 were inhibited by CsA in concentrations of 0.1-1.0 microgram/ml; these levels were ineffective against non-lymphoid tumours, although some non-specific cell toxicity was noted at higher levels. IgM antibody production, as measured by enzyme-linked immunosorbent assay (ELISA), was inhibited over the same range. CH12 cells stimulated by lipopolysaccharide, however, were less sensitive to CsA than untreated cells. These studies indicate that malignant B cells may be sensitive to CsA, perhaps reflecting their derivation from a functionally distinct B cell population with enhanced drug sensitivity.

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

Antigen-nonspecific T cell-derived factors in B cell activation: differences in the requirements for interleukin 2 in responses of unprimed and primed B cells.

The requirements for interleukin 2 (IL2) and other T cell-derived helper factors in the responses of unprimed and antigen-primed B cells to sheep erythrocytes were investigated. Unprimed B cells required both IL2 and additional factor(s), hereafter referred to as T cell-replacing factor (TRF), in addition to specific antigen, for antibody production. IL2 was required only during the early stages (approximately equal to 24 h) of culture while TRF was necessary only after this time and was then required throughout the remaining culture period. IL2 stimulated the appearance of Thy-1+ cells in unprimed "B cell" populations which could substitute for the function of IL2, implicating an indirect role, at least in part, for IL2 in the TRF assay. Furthermore, in contrast to the results with unprimed B cells, primed B cells required only late-acting TRF for optimal antibody responses. We suggest that IL2 activates residual T cells or precursors of T cells in B cell populations which then function, in the presence of specific antigen, to render B cells receptive to T cell-derived factors which promote B cell growth and differentiation.

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↗