Nobumichi Hozumi

The B-cell transmembrane protein CD72 binds to and is an in vivo substrate of the protein tyrosine phosphatase SHP-1

BACKGROUNDnSignals from the B-cell antigen receptor (BCR) help to determine B-cell fate, directing either proliferation, differentiation, or growth arrest/apoptosis. The protein tyrosine phosphatase SHP-1 is known to regulate the strength of BCR signaling. Although the B-cell co-receptor CD22 binds SHP-1, B cells in CD22-deficient mice are much less severely affected than those in SHP-1-deficient mice, suggesting that SHP-1 may also regulate B-cell signaling by affecting other signaling molecules. Moreover, direct substrates of SHP-1 have not been identified in any B-cell signaling pathway.nnnRESULTSnWe identified the B-cell transmembrane protein CD72 as a new SHP-1 binding protein and as an in vivo substrate of SHP-1 in B cells. We also defined the binding sites for SHP-1 and the adaptor protein Grb2 on CD72. Tyrosine phosphorylation of CD72 correlated strongly with BCR-induced growth arrest/apoptosis in B-cell lines and in primary B cells. Preligation of CD72 attenuated BCR-induced growth arrest/death signals in immature and mature B cells or B-cell lines, whereas preligation of CD22 enhanced BCR-induced growth arrest/apoptosis.nnnCONCLUSIONSnWe have identified CD72 as the first clear in vivo substrate of SHP-1 in B cells. Our results suggest that tyrosine-phosphorylated CD72 may transmit signals for BCR-induced apoptosis. By dephosphorylation CD72. SHP-1 may have a positive role in B-cell signaling. These results have potentially important implications for the involvement of CD72 and SHP-1 in B-cell development and autoimmunity.

Transposition of two different intracisternal A particle elements into an immunoglobulin kappa-chain gene.

Each of two severely defective mouse kappa-chain genes has acquired a different intracisternal A particle (IAP) element within one of its introns. One IAP element generated 6-base-pair direct repeats upon insertion. In contrast, the other IAP element was not flanked by direct repeats and was missing a single nucleotide from its 3 terminus. Sequence analysis of the latter IAP element demonstrated that its long terminal repeats were not identical. Nevertheless, the long terminal repeats were organized like proviral long terminal repeats, and this IAP element did contain two regions that were analogous to retroviral priming sites for RNA-directed DNA synthesis. The region that corresponded to a retroviral tRNA primer binding site was complementary to the 3 ends of all mammalian phenylalanine tRNAs. These findings are discussed in the context of the presumed mode of transposition of IAP elements involving the reverse transcription of IAP RNA.

Effects of glycyrrhizin on immune‐mediated cytotoxicity

Intravenous administration of glycyrrhizin is known to decrease elevated plasma transaminase levels in patients with chronic viral hepatitis, in which immune‐mediated cytotoxicity by cytotoxic T lymphocytes and tumour necrosis factor (TNF)‐α is considered to play an important pathogenic role. However, the immunological interpretation of the transaminase‐lowering action of glycyrrhizin is not known. Studies were performed to elucidate this action immunologically by assessing the effects of glycyrrhizin on immune‐mediated cytotoxicity using an antigen‐specific murine CD4+ T hybridoma line, which exhibits cytotoxicity against antigen‐presenting cells after stimulation with specific antigen, and a murine TNF‐α‐sensitive fibroblast line. Glycyrrhizin inhibited the cytotoxic activity of the T cells against antigen‐presenting cells and also suppressed TNF‐α‐induced cytotoxicity in the TNF‐α‐sensitive cell line in vitro. These results suggest that the decrease of elevated transaminase levels by glycyrrhizin in patients with chronic viral hepatitis is mediated in part by inhibition of immune‐mediated cytotoxicity against hepatocytes.

Production of murine V-human cr1 chimeric anti-tag72 antibody using V region cdna amplified by PCR

A mouse/human chimeric B72.3-1-3 antibody was produced by construction of a novel expression vector mpSV2neo-EP1-V-Cr1. This vector contains the neo gene as a selection marker, the murine immunoglobulin heavy chain promoter and enhancer, the murine V region cDNA containing mRNA splicing joint sequences, amplified and cloned by the PCR technique directly from the B72.3 hybridoma RNA, and the human genomic Cr1 region. The expression vector containing the murine/human chimeric immunoglobulin heavy chain gene was transfected into heavy chain loss mutant cell line, B72.3Ml. Chimeric B72.3-1-3 antibody was produced at 2 micrograms/ml and retained full binding reactivity to TAG72 compared to the murine B72.3 parental antibody. Using this method, chimeric immunoglobulin molecules can be produced rapidly in comparison with the cDNA and genomic cloning techniques.

Efficient generation of respiratory syncytial virus (RSV)-neutralizing human MoAbs via human peripheral blood lymphocyte (hu-PBL)-SCID mice and scFv phage display libraries

RSV is one of the major causes of pneumonia and bronchiolitis in infants and young children and is associated with high mortality. RSV neutralizing human antibody (hu‐Ab) is known to mediate resistance to viral infection as well as to be an effective treatment for severe lower respiratory tract RSV infection. We have previously demonstrated that human primary and secondary immune responses can be established in severe combined immunodeficient mice engrafted with human peripheral blood lymphocytes (hu‐PBL‐SCID). By combining this animal model with the single‐chain Fv antibody (scFv) phage display library technique, we were able to investigate further its clinical potential by generating a panel of human scFvs that exhibit both high F glycoprotein (RSV‐F) binding affinities (∼108u2003M−1) and strong neutralizing activities against RSV infection in vitro. Sequencing analysis of the randomly isolated anti‐RSV‐F scFv clones revealed that they were derived from different VH families with mutations in the complementarity‐determining region 1 (CDR1). The results suggest that: (i) RSV‐F‐specific human immune responses and affinity maturation can be induced in hu‐PBL‐SCID mice; and (ii) this approach can be applied to generate large numbers of human scFvs with therapeutic potential. Despite the fact that hu‐PBL‐SCID mouse and human scFv phage display library have individually been established, our approach contributes a simple and significant step toward the generalization of antigen‐specific human monoclonal antibody (hu‐MoAb) production and their clinical applications.

Analysis of proteolytic processing during specific antigen presentation

In this report we have studied the effect of protease inhibitors on B-cell-antigen processing. As a source of antigen-presenting B cells we have utilized transformants transfected with a vector carrying immunoglobulin (Ig) genes specific for the hapten trinitrophenyl (TNP). B-cell-specific (TNP-proteins) and nonspecific antigen-presentation activities were blocked to the same extent upon addition of inhibitors for protease and endosomal function. Interestingly, the effect of leupeptin, a thiol protease inhibitor, varied depending on the antigen and helper T cells utilized. These results suggest that specific groups of proteases may be required for antigen processing so that discrete antigenic epitopes in association with major histocompatibility complex molecules can be recognized by interacting T cells.

Deletion analysis of the c-Ha-ras oncogene promoter

Using transient assays of chloramphenicol acetyltransferase expression in CV‐1 cells, we have localized the human c‐Ha‐ras oncogene promoter to a 550 bp fragment located about 1 kb upstream from the ras coding sequence. Deletion analysis has revealed that a 100 nucleotide region 200 base pairs upstream of the putative initiation sites for transcription is essential for high levels of expression. Within this sequence lie two Spl binding sites and a consensus CCAAT box.

Parameters that govern the regulation of immunoglobulin delta heavy-chain gene expression.

The mu and delta immunoglobulin heavy-chain genes comprise a complex transcriptional unit in which a single mRNA precursor gives rise to mu- and delta-specific transcripts. During the immature B-cell stage, posttranscriptional processing events involving alternate splicing and cleavage-polyadenylation site selection give rise to mu- but not delta-encoding transcripts. In terminally differentiated B cells, delta mRNA is not synthesized because of a transcription termination event occurring upstream of the delta-gene locus. In an attempt to gain insight into the respective contributions of alternate splicing and cleavage-polyadenylation in the control of delta mRNA synthesis, we have constructed a set of plasmids in which membrane mu (mu m)-delta intergenic sequences containing the mu m poly(A) site but differing in splicing capacity were inserted in between a VH and delta gene. The mu m-delta insertion vectors were transfected into a B lymphoma line representative of an immature stage, and proximal mu m poly(A) site usage and delta mRNA synthesis were assessed. To determine unequivocally whether the mu m-delta intergenic region can regulate termination, the insertion vectors were also transfected into a B myeloma line, and transcription through the region was measured. In immature B-cell transfectants, splicing site selection was found to have a key role in determining poly(A) site utilization and concomitant delta mRNA expression. Mature delta mRNA synthesis was blocked by an upstream cleavage-polyadenylation event only when the proximal poly(A) site was associated with appropriate splicing signals. Furthermore, in vitro transcription assays revealed that the mu m-delta intergenic region is sufficient to regulate transcription termination within a 1,2430-base-pair region containing the mu m poly(A) site in myeloma transfectants. The mu m-delta insertion vectors provide an excellent model system for studying the regulatory aspects of this transcription termination event.

Cytotoxic function of a cloned helper T cell line

The cytotoxic function of a well-characterized helper T hybridoma was investigated utilizing 2,4,6-trinitrophenyl (Tnp) specific B cells. The T hybridoma which is ovalbumin (Ova) specific recognizes the Ova synthetic peptide 323-339 Tyr. A number of experiments using several different antigens, Ova, Tnp-Ova and 323-339 Tyr clearly demonstrated that the cytotoxic function of the T hybridoma is strictly correlated with antigen presentation.

Egr-1 mRNA expression is independent of regulatory proliferative responses in the immature B cell line WEHI-231

We have recently reported cellular growth arrest induced following crosslinking of surface IgM (sIgM) but not surface IgD (sIgD) in the WEHI-231 cell line, representative of the immature B cell stage, and its delta heavy chain (delta) transfectant. An initial report has indicated WEHI-231.7, a subclone of WEHI-231, failed to express Egr-1 mRNA following sIgM crosslinking, in contrast to significant up-regulation found in mature B lymphocytes. The implication for linkage between selective surface immunoglobulin (sIg) signal transduction, expression of immediate/early genes and control of cellular growth imposes an attractive model for induction of immature B cell tolerance. Our investigations examined the relationships between Egr-1 mRNA expression and growth regulation in WEHI-231, WEHI-231.7 and their respective delta-transfectants (WEHI-delta, WEHI-delta 7). We report sIgM and sIgD crosslinking leads to a rapid increase of Egr-1 mRNA expression in WEHI-231 and WEHI-delta but not in the subclone WEHI-231.7 and WEHI-delta 7. Nevertheless, both WEHI-231, WEHI-231.7 and their delta-transfectants demonstrate the ability to induce growth arrest following sIgM but not sIgD crosslinking. Furthermore, we found Egr-1 expression could be achieved by direct activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) circumventing the classical sIg activated phosphatidylinositol signal transduction pathway. Our results suggest Egr-1 expression does not directly participate in growth regulation of immature B cell clones but rather is a consequence of signal transduction through sIg.