S M Fu

Stimulation of human B lymphocytes by antibodies to IgM and IgG: Functional evidence for the expression of IgG on B-lymphocyte surface membranes

Abstract Isolated human tonsillar and peripheral blood B lymphocytes were induced to proliferate in vitro after exposure to F(ab′) 2 fragments of affinity purified antibodies to IgM, IgG, Fab, κ, and λ chain determinants. Low levels of DNA synthesis were observed in cultures containing anti-IgA antibodies, whereas DNA synthesis was not detected in cultures stimulated with anti-IgD. Divalent antibodies were essential for the generation of blastogenesis. These proliferative responses were T-cell independent and susceptible to suppression by excess numbers of monocytes. In addition, they were elicitable in cultures not containing FCS or 2-mercaptoethanol. Highly specific antibodies to IgG induced marked proliferation and this was similar in degree to that induced by anti-IgM. Subfractionation studies demonstrated that the anti-IgG responsive cells were contained to a major extent within the surface IgM + B-cell population. None of the antibody preparations elicited B-cell differentiation to antibody producing cells. Moreover, anti-μ antibodies completely abrogated Ig synthesis by pokeweed mitogen-stimulated cultures of unseparated tonsillar mononuclear cells. Anti-IgG antibodies similarly suppressed PWM-induced antibody production, although this was most apparent on the IgG response. In contrast anti-IgD antibodies both failed to suppress Ig production and in certain instances resulted in an increased level of Ig synthesis. These functional studies suggest that IgG molecules are intimately associated with the surface membrane of some B cells and that coexpression of IgG with IgM occurs. In addition, the observations emphasize further the divergent functional roles which surface IgM and IgG vs surface IgD have in B-cell proliferation and differentiation.

B-Lymphoid cell lines derived fromHLA-D homozygous donors

Multiple lymphoid cell lines were derived from 35HLA-D homozygous donors by EB-viral transformation of B lymphocytes. The expression of Ia-like alloantigens (HLA-DR) was studied by microcytotoxicity and by absorption with alloantisera exchanged through the Seventh Histocompatibility Workshop. B-lymphoid lines expressed the same specificities as normal B lymphocytes. Workshop antisera representing DRw1, DRw2, DRw3, and DRw7 gave well-defined typing patterns with cell lines derived from donors of corresponding D-locus specificities. A more complex reaction pattern was seen for antisera representing DRw4, DRw5, and DRw6. The available reagents could not discriminate between lines from donors homozygous for Dw4, Dw10, or D-KH. All lines studied, except for those from one donor homozygous for a unique D-locus determinant (D-SPO), could be assigned one of the provisional DRw specificities.The advantage of obtaining multiple cell lines from a single donor was evident. One line could not be typed by microcytotoxicity because it was lysed in all human sera tested, and some other lines gave weak cytotoxic reactions. Absorption studies, however, did indicate similar expression of DRw antigens on these lines. The availability of multiple lines from the same donor circumvented these difficulties.

Induction of Human Antibody Responses In Vitro with Emphasis on Allogeneic Helper Factors

Systems have been developed for the reproducible production of antigen-specific plaque forming cells for both tonsillar and peripheral blood B cells. Allogeneic helper activity was an essential supplement and monocyte removal was important, especially in the peripheral blood situation. Highly active allogeneic helper factors could be obtained from undirectional MLC supernatants which aided the proeuction of antigen-driven plaques. These factors also caused a polyclonal activation of B lymphocytes. A number of clinical applications of the above system were described. A defect in the T cells of patients with chronic lymphocytic leukemia with respect to the generation of helper activities was delineated.

Regulatory role of circulating monocytes in the differentiative and proliferative responses of human B lymphocytes

Abstract Depletion of monocytes from peripheral blood mononuclear cells had a profound influence on mitogen- and antigen-induced B-cell proliferation and differentiation to antibody-secreting cells. Such depletion enhanced the generation of plasma cells identifiable by immunofluorescence and specific plaque-forming cells (PFC) against sheep erythrocytes in cultures containing pokeweed mitogen, staphylococcal protein A (SPA), concanavalin A (Con A), and phytohemagglutinin (PHA). This enhancement was especially marked in the cases of SPA and Con A. Without monocyte depletion only 0.1 to 0.4% of the cultured cells were shown to be plasma cells and as much as a 100-fold increase was seen with monocyte removal. Similar results were also obtained in the PFC assay. These studies suggest that Con A, SPA, and PHA may be considered as inducers of B-cell differentiation to plasma cells under appropriate conditions. In two B-cell differentiation systems initiated by antigen involving allogeneic helper cells and autologous helper factors, the monocyte-dependent inhibition was also demonstrated. In addition, monocyte depletion enhanced proliferation of B cells in the presence of irradiated autologous T cells and pokeweed mitogen. This enhancement was also seen when B cells were stimulated to divide by purified anti-μ antibodies. The addition of adherent cells to monocyte-depleted cultures confirmed the suppressive effect of excessive monocytes but also demonstrated that the presence of a certain number of monocytes was necessary for optimal responses in at least some of the systems studied. The striking effect of monocytes in these different systems emphasizes the importance of their consideration in B-cell stimulation studies, especially those involving human peripheral blood.

Phosphorylation of α,ß Subunits of 180/100-Kd Polypeptides (LFA-1) and Related Antigens

Human leukocyte function-associated antigens (LFA) have been defined by their association with human T lymphocyte-mediated cytolysis (1). One of these, LFA-1, is present on lymphocyte, thymocytes, monocytes, and granulocytes. Antibodies to LFA-1 have been shown to interfere with T lymphocyte-mediated cytotoxicity, NK cell-mediated cytolysis, and T cell proliferation to soluble antigens, alloantigens, and mitogens as well as various myeloid cell functions (1–4). OKM1 is a biomolecular structure and it has been identified to be the C3bi receptor of human monocytes and macrophages (5). Recently, LFA-1 and OKM1 were found to have a common s subunit and they belong to a human leukocyte differentiation antigen family (6).

Rapid generation of human T cell hybridomas.

Using the concept of a selectable surface marker we have generated screenable human T cell hybridomas within 4-7 days of fusion. OKT4+ T cell blasts were fused with MOLT4, an OKT4- T cell line, and OKT4+ fusion products were obtained by indirect rosetting technique. Supernatants of this heterogeneous population of hybrids were screened and those with activities of interest were subjected to cloning on soft agar and re-screening to dissect out various activities. Fusion efficiency, by staining, ranges from 6 to 80% depending on the state of activation of the T blasts and the growth phase of the MOLT4 line. This method allows for immortalization of T cell subpopulations, clones and malignant cells to study factor production and potentially messenger RNA for these factors. Additionally selectable surface markers may be used in human B cell and rodent:human fusions, avoiding the innate toxicity of various selection media.

Detection and Functional Studies of IL-2 Receptors on Activated Human B Cells

Initial description of the IL-2 receptor identifiable by monoclonal antibody anti-Tac suggested that such receptors were specific for activated T cells (1). However, using monoclonal antibody AT1 developed in our laboratory, and which exhibits similar specificity to anti-Tac, we have been able to demonstrate the presence of this antigen on activated B cells. Furthermore, after appropriate activation, B cells have been found to respond to IL-2 by proliferation.

IgA SPECIFIC T-CELL FACTOR PRODUCED BY A HUMAN T-T HYBRIDOMA*

A human T-T hybridoma was produced, which was a fusion product between and HGPRT-deficient T-cell line, Jurkat 3, and an OKT4+ activated peripheral blood T-cell. The hybrid expressed a receptor for IgM Fc and was negative for IgA Fc. It was shown to produce a factor capable of specifically enhancing IgA production and secretion by isolated human B-cells. The factor exerts its effect directly on B-cells and appears to be different from T-cell-replacing factors previously described.

RECOGNITION BY PREGNANCY SERUMS OF SEVERAL DISTINCT NON-HL-A ALLOANTIGENS EXPRESSED ON B CELLS AND THEIR RELATIONSHIP TO LD DETERMINANTS

Publisher Summary This chapter provides an overview of recognition by the pregnancy serums of several distinct non-HL-A alloantigens expressed on B cells and their relationship to LD determinants. Certain pregnancy sera detect a polymorphic system of alloantigens expressed selectively on B lymphocytes but not T lymphocytes. These antigens are distinct from HL-A determinants and for convenience have been termed HL-B determinants. The selected HL-B alloantisera detect antigens that closely parallel the distribution of specific LD determinants detected by MLC typing. In addition, there are a small number of individuals that bear the HL-B antigens associated with LD7a or LD8a but do not give the expected typing reaction in mixed lymphocyte cultures. Certain other HL-B sera, of different specificity, detect antigens without apparent relation to any of the present LD types.