Eva Gronowicz

Differentiation of B Cells: Sequential Appearance of Responsiveness to Polyclonal Activators

The responses to the polyclonal B‐cell activators dextran sulphate (DxS), lipo polysaccharide (LPS), and purified protein derivative from tuberculin (PPD) were followed along the differentiation process of fetal liver cells to mature B cells in irradiated hosts It was shown that these cells sequentially gained responsiveness to DxS. LPS, and PPD, in that order. It was also shown that the result of activation depends only on the differentiation degree of the target cells at the turn they are activated. Thus, more primitive cells can only divide, and most of the DxS‐. LPS‐, or PPD‐induced blasts are lg‐negative soon after the cells become responsive to each ligand. The response of more differentiated cells is characterized also by high‐rate antibody synthesis. These results provide a unique possibility of using polyclonal B‐cell activators as well‐defined functional markers for sub‐populations of B cells and shed a new light on the problem of immune B‐cell triggering

Heterogeneity of B cells: direct evidence of selective triggering of distinct subpopulations by polyclonal activators.

This investigation was undertaken to assess directly the previous postulate that distinct subsets of B cells in the adult mouse spleen are selectively triggered by different polyclonal B‐cell activators (PBA). Several strains of mice and lipopolysaccharide (LPS), purified protein derivative of tuberculin (PPD), and dextran sulphate (DS) were used in this study. Two experimental approaches were used: stimulation after addition of two PBAs simultaneously to cell cultures and eliminating the responding population to one PBA, by a hot pulse of radioactive thymidine, on a later response to another PBA. The results of these experiments indicated that DS stimulated a cell population completely different from that stimulated by the LPS‐ and PPD‐sensitive cells. When LPS and PPD Stimulations were compared, it was found that the cells responding to these PBAs were largely distinct, although some cells were sensitive to both these PBAs. The extension of the overlaps in these subsets (the number of cells that could be activated by either PBA) was found to vary from one strain to another. These experiments gave direct evidence of the existence of subsets that cm Inactivated by different PBAs. The present results also provided indications of the functional performance of distinct subsets of B cells on activation.

Responsiveness of Lymphoid Precursors to Polyclonal B‐Cell Activators

We have shown previously that in the differentiation of fetal liver cells to mature B cells in irradiated hosts, these cells sequentially gain responsiveness to the polyclonal B‐cell activators dextran‐sulphate (DxS), lipopolysaccharide (LPS)and purified protein derivative from tuberculin (PPD). in that order. In this paper we show that both fetal liver cells and adult bone marrow cells responded with proliferation to DxS. but not to LPS or PPD. However, neither fetal liver nor bone marrow cells gave rise to detectable numbers of high‐rate antibody‐secreting cells on short‐term stimulation by polyclonal B‐cell activators The lack of LPS and PPD responses of fetal liver and bone marrow cells could not be ascribed to the presence of inhibitory cells, and the DxS‐induced response in these cell populations was not dependent on adherent cells. However, LPS could inhibit the DxS response of fetal liver cells, possibly indicating that DxS‐responsive cells are precursors to B cells. Direct evidence was provided that DxS activated B‐Cell precursors in bone marrow. Thus, this cell population became responsive to LPS after DxS prestimulation. as measured by DNA synthesis. Bone marrow cells, sequentially stimulated with DxS and LPS. contained increased numbers of cells with surface immunoglobulin, although no significant increase in numbers of antibody‐secreting cells was obtained. These data indicate that DxS had the capacity to increase the rate of differentiation of B‐cell precursors. Finally, we show that the sequential appearance of responsiveness in B‐cell differentiation to polyclonal B‐cell activators is not due to lack of accessory cells during early stages in maturation

Mechanism of B-cell activation and paralysis by thymus-independent antigens. Additive effects between NNP-LPS and LPS in the specific response to the hapten.

Normal spleen cells showed a bell‐shaped dose response profile when stimulated in vitro with die thymus independent antigen (4 ‐hydroxy‐3,5 dinitiophenyl)acetyl (NNP)‐lipopolysaccharide (LPS) with regard to the development of high‐avidity plaque‐forming cells to NNP The addition of suboptimal concentrations of LPS to cultures stimulated by suboptimal concentrations of NNP LPS resulted in optimal induction of B cells in that affinity fraction Addition of LPS to cultures optimally stimulated by NNP‐LPS resulted in paralysis of the specific cells These results are interpreted in terms of the additive effects between the mitogenicity LPS and the mitogenicity of NNP LPS the latter being selectively focused on the specific cells, thus providing further evidence for the ‘one nonspecific signal’ hypothesis for immune activation of B cells

Quantity and Quality of Anti-Hapten Antibodies in Normal and in T Cell-Deprived Mice Studied at the Cellular Level

The Immune response of normal mice as well as of thymus‐deprived (TxB) mice against 2 different hapten‐protein conjugates (NNP‐BSA and NNP‐CG) was tested at various times after immunization at the cellular level. Normal mice developed both direct and indirect plaque‐forming cells against the hapten, as determined by a modified local haemolysis in gel assay for detection of anti‐hapten antibody‐secreting cells. However, TxB mice produced direct plaque‐forming cells to the same extent as normal mice, whereas the indirect plaque‐forming cell response was greatly impaired. In fact, significantly elevated levels of indirect plaque‐forming cells were not detected in such mice. Studies on the affinity of secreted antibodies by means of hapten inhibition revealed that the indirect plaque‐forming cells derived from normal immunized mice could be inhibited by decreasing concentrations of free hapten with time after immunization, indicating a gradual increase in the number of cells secreting antibodies of high affinity. No such change was detected in normal or TxB mice with regard to antibodies giving rise to direct lysis, presumably IgM antibodies.

The Polyclonal B Cell Activator Dextran‐Sulphate Induces Formation of Colony Stimulating Activity

The effect of dextran‐sulphate (DS), a polyclonal activator known to stimulate immature murine B‐cells, was assayed in a culture system allowing the growth of myeloid cells. It was known that DS induced the production of a myeloid colony stimulating factor (CSF) by cells from both spleen and bone marrow. Nylonwool purified mouse spleen cells, enriched for T cells, showed a dimished CSF production in response to DS, while CSF production in response to Con A was increased. Furthermore, DS induced CSF in both spleen and bone marrow cells from nude mice. Removal of macrophages did not affect CSF production. The CSF induced was non‐dialysable and no small molecular weight or lipoprotein inhibitors could be demonstrated. The results suggest that DS activates cells other than T cells or macrophages (possibly B cells or null cells) to produce a myeloid stem cell stimulating substance. These results indicate that interactions between lymphoid and myeloid cells can take place during differentiation.