Effect of gamma radiation on resting B lymphocytes. II. Functional characterization of the antigen-presentation defect

The effect of radiation on three discrete Ag-presentation functions in resting B cells was examined: 1) Ag uptake and processing, 2) expression of processed Ag in the context of functional class II molecules, and 3) provision of necessary co-stimulatory, or "second," signals. Analysis of radiation's effect on B cell presentation of intact vs fragmented Ag or its effect on presentation by Ag-pulsed B cells indicated that damage to Ag uptake and processing could not account for the bulk of the radiation-induced Ag-presentation defect. Experiments with phosphatidylinositol hydrolysis as an indirect measure of TCR occupancy suggested that irradiation caused a fairly rapid (within 1 to 2 h) decrease in the ability of the B cell APC to display a stimulatory combination of Ag and class II molecule. Ag dose-response analyses demonstrated that when presenting a fragment of the Ag pigeon cytochrome c to a T cell clone, 3000 rad-treated B cell APC were able to stimulate approximately 50% as much phosphatidylinositol turnover as unirradiated B cells. It was also found that, in contrast to their inability to initiate T cell proliferation, and similarly to chemically cross-linked splenocytes, heavily irradiated resting B cells plus Ag induced a state of Ag hyporesponsiveness in T cell clones. This effect on T cells had the same Ag- and MHC-specificity as did receptor occupancy required for proliferation, indicating that heavily irradiated resting B cells bear functional class II molecules. Co-culture of T cells with allogeneic B cells and syngeneic heavily irradiated B cells or chemically cross-linked splenic APC plus Ag resulted in T cell proliferation and interfered with the induction of the hyporesponsive state. This co-stimulatory function was radiosensitive in resting allogeneic B cells. Together, these data support the hypothesis that the major functional consequences of radiation to resting B cell APC are a reduction in the effective display of Ag plus class II molecules and, probably what is more important, a loss in the ability to provide APC-derived co-stimulatory signals.     

Non-histone chromatin proteins of B lymphocytes stimulated by lipopolysaccharide. II. Phosphorylation.

Stimulation of mouse lymphocytes with the B lymphocyte specific mitogen lipopolysaccharide results in an increased rate of phosphorylation of non-histone chromatin proteins. An initial small increase in phosphorylation occurs during the first 2 h and a much larger increase after 24 h of culture with mitogen. The phosphorylated nuclear and cytoplasmic proteins were analysed by polyacrylamide gel electrophoresis and the stimulation index of each prominent peak measured. It was inferred that selective stimulation of the phosphorylation of individual proteins had occurred from: (1) the range of stimulation indices for different proteins, and (2) the appearance, after 8 h stimulation of an apparently newly phosphorylated non-histone chromatin protein of molecular weight 115 000. The pool size of ATP was monitored and showed only small changes during the first 24 h of exposure to lipopolysaccharide. Phosphatase activity was found to be associated with lymphocyte chromatin and nucleoplasm and may help to regulate the level of phosphorylation of non-histone chromatin proteins in vivo. To preserve phosphorylated proteins during their isolation phosphatase activity was inhibited by Na2MoO4. The selective changes in phosphorylation of nuclear proteins precede, and continue during, the stimulation of immunoglobulin and DNA synthesis. Our results are thus consistent with the hypothesis that phosphorylation of non-histone chromatin proteins plays a role in the regulation of gene expression in B lymphocytes.     

Regulation of IgM and IgD synthesis in B lymphocytes. II. Translational and post-translational events

Studies investigating the relative rates of biosynthesis of mu- and delta-polypeptide chains in normal resting B lymphocytes have shown that the translation rate of mu m is about sevenfold higher than that of delta m, thus reflecting the relative abundance of mRNA encoding these two chains. The turnover rate of cell surface IgM is faster, however, than that of cell surface IgD, resulting in higher expression of cell surface IgD relative to IgM under steady state conditions. LPS stimulation of B lymphocytes induces the complete cessation of synthesis of the delta-chain, thus accounting for the gradual disappearance of IgD from the cell surface of activated cells.     

Cloning and expression in Escherichia coli of three fragments of diphtheria toxin truncated within fragment B.

We have constructed three different truncated versions of diphtheria toxin (a 535-amino-acid polypeptide) which correspond to the N-terminal 290, 377, and 485 amino acids of the toxin. These lengths include one, three, and all four of the putative membrane-spanning sequences of the toxin which are thought to play a role in the translocation of fragment A into cells. Each of these three genes has been modified at its 3' end to code for a C-terminal cysteine (to allow for disulfide linkage of a targeting ligand) or a gene fusion with alpha-melanocyte-stimulating hormone. We have also substituted the native diphtheria tox promoter (ptox) with the lambda pR promoter in an effort to overexpress these proteins. The truncated genes are expressed in Escherichia coli from both the tox promoter in a constitutive fashion and from the pR promoter by using the heat-inducible cI857 repressor. The clones produce proteins which react with anti-diphtheria toxin serum, which migrate at the anticipated Mr on Western blots, and which have ADP-ribosyltransferase activity. Constitutive synthesis from ptox leads to severe proteolytic degradation even in a protease-deficient strain. High-level expression from the pR promoter in the same lon htpR strain allows the full-length polypeptides to accumulate but also stops the growth of the cells. It appears that removal of as few as 50 amino acids from the C-terminus of diphtheria toxin alters its conformation, making it a target for proteases and causing overexpression lethality in the host cells.     

The induction of hapten-specific immunological tolerance and immunity in B lymphocytes. II. Temporal and dose response relationships of tolerance induction in B lymphocytes of various differentiation states.

The induction of TNP-specific B lymphocyte tolerance by TNBS in sources representing various differentiation states was studied in an adoptive cell transfer system. An adoptive assay was utilized in which the delay of immunization with the T-independent antigen TNP-LPS resulted in an enhanced PFC response. TNBS induced tolerance in spleen cells which was independent of T cell activity, was dose dependent, and could be adoptively transferred. While bone marrow and spleen cells were susceptible to tolerogenesis after cell transfer, TNBS treatment of the donor induced unresponsiveness in splenocytes but not marrow cells. The tolerance dose response relationship and the effect of the temporal relationship between cell transfer and tolerogenesis were studied in B lymphocytes from various sources. Adult spleen cells were resistant to tolerance induction late in the adoptive response, and the tolerance induced by TNBS administration 1 hr after cell transfer was dose dependent. Athymic nude spleen cells and adult bone marrow cells displayed similar characteristics while fetal liver cells were somewhat more susceptible to the induction of unresponsiveness. Neonatal spleen cells were rendered tolerant at much lower doses and at any stage of the adoptive response. The hierarchy obtained in these studies in the order of decreasing resistance to tolerance induction is: adult normal and athymic nude spleen and adult bone marrow, fetal liver, and neonatal spleen. This variation in tolerogenesis appears to be due to the maturity of the cell types which may reflect differences in B lymphocyte sub-populations.     

Ig repertoire of human polyspecific antibodies and B cell ontogeny.

A total of 463 EBV Ig-secreting clones were derived from embryonic tissues, cord blood, and adult peripheral blood. Subcloning and analysis of the H and K loci (germline vs rearranged DNA status) of 44 primary clones insured clonality in at least 92% of cases. Whatever the cell origin, a somewhat constant proportion of clones (i.e., 11 to 16%) expressed polyspecific antibodies when tested on a panel of nine Ag, including self-Ag. The VH and VK repertoires have been studied using VH1-VH6 and VK1-VK4 family-specific probes. For all EBV clones the VH and VK utilization was similar to that of the normal untransformed population. A correlation was observed between the level of expression and the gene number for VH, whereas a clear distortion appeared for VK. Moreover, the usage pattern of VH and VK families of the polyspecific clones did not significantly differ from that of clones of unknown specificity, suggesting that polyspecificity was not linked to a restricted repertoire.     

Human B lymphocytes capable of spontaneous Ig production in short-term cultures. II. Relationship with the spontaneous DNA-synthesizing B cells

It has been previously demonstrated that normal nonimmunized individuals possess circulating and tissular B cells, which are capable of spontaneous immunoglobulin (Ig) production in short-term (3 days) cultures. We have also observed the occurrence of low levels of [3H]thymidine uptake early in such cultures. This work analyzes the relationship between both spontaneous B-cell functions in vitro: Kinetics studies revealed that both activities were temporarily related, as spontaneous DNA synthesis was maximal from 8 to 12 hr, and declined thereafter, when spontaneous Ig secretion was first detected in the culture supernatant: The abrogation of DNA synthesis at the culture initiation or during the period of early proliferation, but not after 24 hr, inhibited subsequent IgG secretion. The B cells responsible for spontaneous DNA synthesis and IgG secretion exhibited similar low densities, since both were recovered in the 42.5-45% Percoll fractions, and identical large size as determined by 1g sedimentation procedure, and in tonsil, were equally reactive with the BA-2 mouse monoclonal antibody. Finally, limiting dilution analysis showed that the precursor frequencies of both cells under study were similar. These results suggest that spontaneous DNA synthesis and IgG production are carried out by the same subset of in vivo-preactivated lymphoblastoid B cells.     

Interactions between B lymphocyte subpopulations. Augmentation of the responses of resting B lymphocytes by activated B lymphocytes

Mouse B lymphocytes were fractionated from normal T lymphocyte-depleted spleen cell populations using discontinuous percoll gradients and were stimulated with rabbit F(ab')2 anti-mouse mu-specific antibodies (anti-mu) plus the supernatant of Con A-stimulated rat spleen cells (SN) as a source of lymphokines. The responses of small (mean volume 120 mu 3), dense (greater than 1.087 specific gravity), resting (least spontaneous thymidine incorporation) B lymphocytes were augmented by irradiated (4000 rad), larger (mean volume greater than 170 mu 3), less dense (less than 1.081 specific gravity), activated (greater spontaneous thymidine incorporation) B lymphocytes. Proliferation was augmented 2- to 4-fold and polyclonal antibody-forming cell responses three- to sixfold. Maximal augmentation of the responses of 5 X 10(4) resting B cells was obtained with 10(4) activated B cells. Augmenting activity was specific for activated B lymphocytes in that responses were not augmented by irradiated thymocytes, T lymphoblasts, macrophages, or additional supernatant. B lymphocytes activated in vitro by LPS or anti-mu also had augmenting activity. Augmentation of responses was maximal only when activated B lymphocytes were added simultaneously with anti-mu. The interaction between activated and resting B lymphocytes did not appear to be genetically restricted. Interestingly, the augmenting activity of activated B cells could be reconstituted by a combination of supernatant and cell membranes from these cells but not by either alone, suggesting that two components are required, one soluble and the other membrane-bound. Thus, a functional interaction has been demonstrated between B lymphocyte subpopulations which differ in their state of activation, and this interaction appears to involve a novel mechanism of action.     

A caspase-independent pathway of MHC class II antigen-mediated apoptosis of human B lymphocytes.

MHC class II molecules have a crucial role in thymic selection and in generating Ag-specific T cell responses. There is extensive evidence for second messenger generation via MHC class II molecules, which can lead to apoptosis of B lymphocytes. We have examined HLA class II-mediated apoptosis in both normal and tumoral human B lymphocytes. Phosphatidylserine exposure and DNA fragmentation were observed in B cells within 24 h of stimulation via HLA class II. In marked comparison with Fas, the cell-permeable and irreversible caspase inhibitors zVAD-fmk and DEVD-fmk failed to inhibit HLA-DR-mediated apoptosis. No direct activation of caspase 3 was detected, and cleavage of pro-caspase 3 was not observed. Cleavage of poly(ADP-ribose) polymerase was detected via Fas but not via HLA class II. Although phosphatidylinositol-3-kinase has been implicated in HLA class I-mediated apoptosis, neither wortmannin nor LY294002 affected HLA class II-mediated apoptosis. CD95-sensitive cells were used to reveal that death occurred independently of CD95-CD95 ligand interactions. Overall, these data reveal a pathway of HLA-DR-mediated apoptosis that neither requires nor involves caspases. Moreover, it is phosphatidylinositol-3-kinase independent and Fas/CD95 independent. This pathway of HLA class II-mediated apoptosis could have an important role in the regulation of APC populations or in the control of malignant B lymphocyte proliferations.     

Immunopotentiating effects of amphotericin B. II. Enhanced in vitro proliferative responses of murine lymphocytes.

Enhanced in vitro proliferative responses to DNBSO3 were seen in lymph node cells and spleen cells after in vivo sensitization of mice with DNFB plus AmB compared with mice primed with DNFB alone. The T cell proliferation in the nylon column nonadherent fraction for both groups was highly similar, and the enhanced lymph node cell proliferation with AmB was demonstrated to be in the nylon adherent population consisting of both T and B cells. These and earlier studies of immunopotentiation by AmB are consistent with a mechanism that depends on selective interaction of the polyene with a subset of T cells and a resultant impairment of the normally induced suppressor regulation that limits the magnitude and duration of immune responses.     

Paratope and epitope mapping of the antithrombotic antibody 6B4 in complex with platelet glycoprotein Ibalpha

The monoclonal antibody 6B4 has a potent antithrombotic effect in nonhuman primates by binding to the flexible loop, also known as the beta-switch region (amino acids 230-242), of glycoprotein Ibalpha (GPIbalpha). This interaction blocks, in high shear stress conditions, the specific interaction between GPIbalpha and von Willebrand factor suppressing platelet deposition to the damaged vessel wall, a key event in the pathogenesis of arterial thrombosis. To understand the interactions between this antibody and its antigen at the amino acid level, we here report the identification of the paratope and epitope in 6B4 and GPIbalpha, respectively, by using computer modeling and site-directed mutagenesis. The docking programs ZDOCK (rigid body docking) and HADDOCK (flexible docking) were used to model the interaction of 6B4 with GPIbalpha and to delineate the respective paratope and epitope. 6B4 and GPIbalpha mutants were constructed and assayed for their capacity to bind GPIbalpha and 6B4, respectively. From these data, it is found that the paratope of 6B4 is mainly formed by five residues: Tyr(27D), Lys(27E), Asp(28), and Glu(93) located in light chain CDR1 and -3, respectively, and Tyr(100C) of the heavy chain CDR3. These residues form a valley, where the GPIbalpha flexible loop can bind via residues Asp(235) and Lys(237). The experimental results were finally used to build a more accurate docking model. Taken together, this information provides guidelines for the design of new derivatized lead compounds with antithrombotic properties.