Polyclonal Activation of Human B Lymphocytes in Vitro by Pokeweed Mitogen: a Simple Technique for the Simultaneous Assessment of Cell Proliferation, Generation of Plasma Cells, Plaque-forming Cells and Immunoglobulin Production

To determine whether or not B lymphocytes are committed to the synthesis of a single immunoglobulin heavy chain isotype during their differentiation into plasma cells, rabbit lymph node and Peyer's patch cells were separated into populations with and without membrane IgM, using a fluorescence-activated cell sorter (FACS). The potential of the µ-bearing (µ+) and non-µ-bearing (µ-) cells to give rise to plasma cells both in vivo after transfer into irradiated recipients and in vitro in the presence of pokeweed mitogen was assessed by immunofluorescence techniques, and the relative proportions of the cytoplasmic Ig-stained cells (CSC) synthesizing each class of heavy chains were determined. Most of the CSC arising in vitro from µ-bearing lymph node and Peyer's patch cells contained IgM; all IgM CSC appeared to be derived from µ+ cells. Peyer's patch lymphocytes, however, did not generate IgM CSC after cell transfer and thus may be functionally different from lymph node µ+ cells. It was found also that nearly all of the many IgA CSC generated by Peyer's patch lymphocytes either in culture or after transfer were derived from µ- cells. Further fractionation of these µ- cells with the FACS after they had been membrane stained with anti-b locus allotype reagents revealed that the precursors of IgA CSC belong to a minor population of cells which do have b locus light chain determinants on their membranes, although they do not have detectable µ-chains. These cells are not found in lymph nodes. Although the majority of Peyer's patch and lymph node cells were found to be precommitted to the synthesis of a single heavy chain isotype, a small proportion of cells may not be similarly restricted. Some of the CSC with membrane IgM were found to contain cytoplasmic IgA or IgG. In addition, µ+ populations did give rise to low numbers of IgA and IgG CSC. The implications of these results, obtained under experimental conditions, on the normal differentiation of B lymphocytes in situ are discussed.

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Tumor cell heterogeneity in multiple myeloma: antigenic, morphologic, and functional studies of cells from blood and bone marrow

Blood ◽

10.1182/blood.v73.7.1925.bloodjournal7371925 ◽

1989 ◽

Vol 73 (7) ◽

pp. 1925-1935

Author(s):

MA King ◽

DS Nelson

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Cell ◽

Tumor Cells ◽

B Lymphocytes ◽

Plasma Cells ◽

Bone Marrow Cells ◽

Cell Types ◽

Pokeweed Mitogen

Tumor cells from six patients with immunoglobulin G (IgG) multiple myeloma were analyzed for surface antigens, cytoplasmic paraprotein, morphology, and response to various culture conditions. The tumor marker was the paraprotein idiotype. Low numbers of tumor cells were found in the blood of most of the patients. In some patients, the circulating tumor cells were solely B lymphocytes, whereas in other patients, they were lymphoid, lymphoplasmacytoid, and plasmacytoid. Dual surface antigen analysis of blood and bone marrow cells confirmed that the tumor may be composed of a spectrum of cell types. Thus, cells may range from surface-idiotype+,CD19+,CD20+, PCA-1-,cytoplasmic- idiotype- lymphocytes, to CD19-,PCA-1+,cytoplasmic-idiotype+ plasma cells that are surface-idiotype- or weakly surface-idiotype+. In one patient, some of the tumor cells co-expressed surface idiotype and CD10. The tumor B lymphocytes were activated in vitro to synthesize paraprotein by pokeweed mitogen (PWM), and by low molecular weight B cell growth factor (BCGF). In contrast, spontaneous synthesis of paraprotein by more mature tumor cells was inhibited by agents that also inhibit nonmyeloma plasma cells. These agents included PWM, gamma interferon, and phorbol ester. The results demonstrate that in multiple myeloma there exist different tumor cell types that are similar, by a variety of criteria, to normal B lineage cells at different stages of differentiation. Thus, further evidence is provided for the hypothesis of myeloma cell differentiation.

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RESTRICTION OF GENE EXPRESSION IN B LYMPHOCYTES AND THEIR PROGENY

Journal of Experimental Medicine ◽

10.1084/jem.139.3.581 ◽

1974 ◽

Vol 139 (3) ◽

pp. 581-599 ◽

Cited By ~ 28

Author(s):

Patricia P. Jones ◽

John J. Cebra ◽

Leonard A. Herzenberg

Keyword(s):

B Lymphocytes ◽

Plasma Cells ◽

Relative Proportion ◽

Pokeweed Mitogen ◽

Allelic Exclusion ◽

Vitro Assay ◽

Membrane Immunoglobulin ◽

Cell Clones

Lymphocytes from b5/b9 rabbits were stained in suspension with fluorescent antiallotype antibody reagents to selectively label with fluorescent molecules those cells bearing membrane immunoglobulin (Ig) of the b5 or b9 allotype. After staining, the cells were separated by the fluorescence-activated cell sorter into populations markedly enriched in cells bearing b5 or b9 membrane Ig or totally depleted of cells with detectable membrane Ig. The potential of these separated cells to give rise to Ig-synthesizing plasma cells either in vivo after transfer into irradiated recipients or in vitro during culture in the presence of phytohemagglutinin or pokeweed mitogen was assessed by immunofluorescence. The relative proportion of b5 and b9 cytoplasmic Ig-stained cells (CSC) arising from the separated cells was determined to test directly whether B lymphocytes and their progeny are committed to the synthesis of Ig of one allotype. It was found that b5- and b9-bearing cells gave rise almost exclusively to b5- and b9-producing plasma cells, respectively, in both the in vivo and in vitro assay systems. Most of these CSC were probably not derived from previously existing CSC but arose as the result of the differentiation of lymphocytes with membrane Ig. When cell populations totally depleted of Ig-bearing lymphocytes were cultured, very few CSC were found, indicating that the majority of immediate precursors of CSC have membrane Ig. These results suggest that individual B cell clones are phenotypically restricted to the expression of immunoglobulin genes on one chromosome; the significance of this clonal allelic exclusion is discussed.

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Tumor cell heterogeneity in multiple myeloma: antigenic, morphologic, and functional studies of cells from blood and bone marrow

Blood ◽

10.1182/blood.v73.7.1925.1925 ◽

1989 ◽

Vol 73 (7) ◽

pp. 1925-1935 ◽

Cited By ~ 32

Author(s):

MA King ◽

DS Nelson

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Cell ◽

Tumor Cells ◽

B Lymphocytes ◽

Plasma Cells ◽

Bone Marrow Cells ◽

Cell Types ◽

Pokeweed Mitogen

Abstract Tumor cells from six patients with immunoglobulin G (IgG) multiple myeloma were analyzed for surface antigens, cytoplasmic paraprotein, morphology, and response to various culture conditions. The tumor marker was the paraprotein idiotype. Low numbers of tumor cells were found in the blood of most of the patients. In some patients, the circulating tumor cells were solely B lymphocytes, whereas in other patients, they were lymphoid, lymphoplasmacytoid, and plasmacytoid. Dual surface antigen analysis of blood and bone marrow cells confirmed that the tumor may be composed of a spectrum of cell types. Thus, cells may range from surface-idiotype+,CD19+,CD20+, PCA-1-,cytoplasmic- idiotype- lymphocytes, to CD19-,PCA-1+,cytoplasmic-idiotype+ plasma cells that are surface-idiotype- or weakly surface-idiotype+. In one patient, some of the tumor cells co-expressed surface idiotype and CD10. The tumor B lymphocytes were activated in vitro to synthesize paraprotein by pokeweed mitogen (PWM), and by low molecular weight B cell growth factor (BCGF). In contrast, spontaneous synthesis of paraprotein by more mature tumor cells was inhibited by agents that also inhibit nonmyeloma plasma cells. These agents included PWM, gamma interferon, and phorbol ester. The results demonstrate that in multiple myeloma there exist different tumor cell types that are similar, by a variety of criteria, to normal B lineage cells at different stages of differentiation. Thus, further evidence is provided for the hypothesis of myeloma cell differentiation.

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SELECTIVE TRIGGERING OF HUMAN T AND B LYMPHOCYTES IN VITRO BY POLYCLONAL MITOGENS

Journal of Experimental Medicine ◽

10.1084/jem.140.1.1 ◽

1974 ◽

Vol 140 (1) ◽

pp. 1-18 ◽

Cited By ~ 175

Author(s):

Melvyn Greaves ◽

George Janossy ◽

Michael Doenhoff

Keyword(s):

B Cells ◽

B Lymphocytes ◽

Human Lymphocytes ◽

Culture Conditions ◽

Pokeweed Mitogen ◽

Cell Populations ◽

T And B Cells ◽

Activated T Cells ◽

Lymphocyte Responses

Human lymphocytes from spleen and tonsils have been cultured with a variety of polyclonal mitogens. Cultures consisted of either unseparated T and B cells or alternatively purified T or B lymphocytes. The purity of the starting cell populations and the origin of activated lymphoblasts was analyzed with a panel of seven markers which discriminate between T and B cells. The selectivity of the lymphocyte responses was influenced by cell populations in a given culture, the mitogen used, and to a limited extent on culture conditions. Purified T lymphocytes from tonsil and spleen responded to phytohemagglutinin (PHA), pokeweed mitogen (PWM), and staphylococcal enterotoxin B (SEB). Purified B cells from spleen responded well to PWM, weakly to SEB and lipopolysaccharide, but not at all to PHA. Tonsil B cells responded weakly to PWM and SEB but not to PHA. Some B lymphocytes do respond to PHA in the presence of activated T cells. These results are discussed in relation to previously reported selective responses of human cells and parallel studies in animal species.

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Defective in vitro immunoglobulin production in response to pokeweed mitogen in patients with Hodgkin's disease pretreatment and in remission

Clinical Immunology and Immunopathology ◽

10.1016/0090-1229(86)90112-1 ◽

1986 ◽

Vol 41 (2) ◽

pp. 281-289 ◽

Cited By ~ 3

Author(s):

Anne M. Dickinson ◽

Sandra M. McLanchlan ◽

Elizabeth A. Jacobs ◽

B. Fail ◽

S.J. Proctor

Keyword(s):

Hodgkin's Disease ◽

Hodgkin’S Disease ◽

Pokeweed Mitogen ◽

Immunoglobulin Production

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Delayed in Vitro Immunoglobulin Production by Cord Lymphocytes

PEDIATRICS ◽

10.1542/peds.65.3.497 ◽

1980 ◽

Vol 65 (3) ◽

pp. 497-500

Author(s):

Yukiaki Miyagawa ◽

Kenichi Sugita ◽

Atsushi Komiyama ◽

Taro Akabane

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Mononuclear Cells ◽

Pokeweed Mitogen ◽

Immunoglobulin Production ◽

Mitogen Stimulation ◽

Functional Immaturity ◽

Adult Cells

Pokeweed mitogen-induced immunoglobulin (Ig) production by cord lymphocytes was studied in vitro by Ig-secreting plaque-forming cell (Ig-PFC) assay. Although adult mononuclear cells generated all of IgM-, IgG-, and IgA-PFC, cord mononuclear cells generated only IgM-PFC when cultured for seven days. The number of cord IgM-PFC was 102 ± 26/104 mononuclear cells, being about one fourth of that of adult IgM-PFC. When cultured for 14 days, cord mononuclear cells formed increased numbers of IgM-PFC in contrast to adult cells, and yielded IgG-PFC as well, indicating delayed Ig production. Cord T cells were much less effective at helping adult B cells to differentiate into Ig-PFC as compared with adult T cells. Substitution of adult T cells for cord T cell markedly improved the response of cord B cells. The present study demonstrates Ig secretion by cord lymphocytes in response to pokeweed mitogen stimulation. The results further indicate that the delayed Ig production by cord lymphocytes is largely due to functional immaturity of the T cells.

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Effect of levamisole on pokeweed mitogen stimulation of immunoglobulin production in vitro

Immunopharmacology ◽

10.1016/0162-3109(81)90021-7 ◽

1981 ◽

Vol 3 (4) ◽

pp. 289-298 ◽

Cited By ~ 2

Author(s):

Jean-Louis Pasquali ◽

Constantine D. Tsoukas ◽

Sherman Fong ◽

Dennis A. Carson ◽

John H. Vaughan

Keyword(s):

Pokeweed Mitogen ◽

Immunoglobulin Production ◽

Mitogen Stimulation ◽

Stimulation Of

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Induction of in vitro differentiation and immunoglobulin synthesis of human leukemic B lymphocytes.

Journal of Experimental Medicine ◽

10.1084/jem.148.6.1570 ◽

1978 ◽

Vol 148 (6) ◽

pp. 1570-1578 ◽

Cited By ~ 112

Author(s):

S M Fu ◽

N Chiorazzi ◽

H G Kunkel ◽

J P Halper ◽

S R Harris

Keyword(s):

T Cells ◽

B Cells ◽

Plasma Cells ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Pokeweed Mitogen ◽

In Vitro Differentiation ◽

Immunoglobulin Synthesis

Successful induction of in vitro differentiation and immunoglobulin synthesis of the leukemic lymphocytes was carried out in two cases of chronic lymphocytic leukemia. Few plasma cells and little specific Ig secretion were detected in the cultures of isolated leukemic B cells in either the presence or the absence of autologous T cells. Up to 30% of the leukemic B cells matured to plasma cells, and a 32-fold increase in specific Ig synthesis was observed when T cells from normal individuals were added to the cultures of these leukemic B cells. In one of the two cases, autologous T cells were able to induce greater than 50% of the leukemic B cells to differentiate further to plasma cells in the presence of pokeweed mitogen. This markedly accelerated in vitro differentiation was only achieved with leukemic cells from cases in which there was evidence of slight differentiation in vivo. No evidence could be obtained for excessive suppressor T cells in these patients. However, a T-cell defect in the generation of allogeneic effect helper factors was identified. This defect may be responsible for the reduced rate of leukemic maturation in vivo.

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