Hirotaka Haruta

Role of Flk-1 in mouse hematopoietic stem cells.

It was reported that human hematopoietic stem cells in bone marrow were restricted to the CD34+KDR+ cell fraction. We found that expression levels of Flk‐1, a mouse homologue of KDR, were low or undetectable in mouse Lin−c‐Kit+Sca‐1+CD34low/− cells as well as Hoechst33342− cells (side population), which have long‐term reconstitution capacity. Furthermore, neither Flk‐1+CD34low/− cells nor Flk‐1+CD34+ cells had long‐term reconstitution capacity in mouse. Taken together with other observations using Flk‐1‐deficient mice, these results indicate that Flk‐1 is essential for the development of hematopoietic stem cells in embryo but not for the function of hematopoietic stem cells in adult mouse bone marrow.

Class-switching of the IgM type antiadenocarcinoma human antibody HB4C5 into an IgG1 type resulted in the loss of the antigen binding ability

The heavy and light chain genes (mu and lambda) of the human anti-adenocarcinoma monoclonal antibody HB4C5 (MAb-C5) were cloned from the human-human hybridoma cell line HB4C5 and co-expressed in Chinese hamster ovary (CHO) and COS-7 cells. ELISA assay and the RI imaging of the cancer tissue xenografted into nude mice showed that the recombinant MAb-C5 retained the original antigen binding activity. We then replaced the IgM constant region of the MAb-C5 heavy chain gene with the human IgG1 constant region gene and co-expressed it with the light chain gene. This recombination was confirmed by a complete DNA sequencing and Western-blot analysis. Despite the fact that the DNA sequence, the expressed protein size, and the assembly of heavy and light chains were indicated to be normal, the IgG1 type MAb-C5 could not bind to the original antigen. This result suggest that this antibody alters its antigen binding property upon class-switching.

Class — Specific Regulation of Immunoglobulin Production by Rose Bengal in Human B Cell Lines and Peripheral Blood Lymphocytes

We have previously reported that Rose Bengal (RB) can dance IgE production in rat spleen lymphocytes without increasing the production of IgG and IgM. To examine the effect of RB in human B cell lines, U-266 (IgE producer), H\My-2 (IgG producer) and NAT-30 (IgM producer) cells were cultured with RB, and the medium immunoglobulin (Ig) level was measured by ELISA. Though RB enhanced IgE production in rat lymphocytes, it inhibited IgE production in U-266 and IgG productionin HMy-2 cells in dose dependent manner. In NAT-30 cells, RB slightly inhibited IgM production at 0.1 μM, while it enhanced production at 10 μM. These results suggest that RB directly modulates Ig production in B cells, and that the modulatory effect is different between rat lymphocytes andhuman cell lines. Therefore, we examined the effect of RB on Ig production in human peripheral blood lymphocytes. We found that RB did not enhance IgE production in lymphocytes as well as human cell lines.

LIGHT CHAIN SHIFTING - CHARACTERIZATION OF LIGHT CHAIN REPLACEMENT IN HUMAN PLASMA B CELLS

Light chain shifting (LCS) is a phenomenon that some human antibody producing plasma B cells lost their original immunoglobulin light chain and begin to express new light chains which are different from original one. Each cells after LCS produced only one type of light chain and cell clone producing more than one light chain protein was not ever found. We have demonstrated that LCS is carried out by a secondary light chain gene rearrangement event and LCS-inducible cells also expressed V(D)J rearrangement-relating factors such as recombination activating genes RAG-1, RAG-2 and terminal deoxynucleotidyl transferase, all of which are usually not expressed in the plasma B cells.

Serum starvation induced secondary VλJλ rearrangement in a human plasma B cell line

HB4C5 is a human antibody producing plasma B cell line that expresses the recombination activating gene-1 (RAG-1) and RAG-2 constitutively, but undergoes few secondary immunoglobulin gene rearrangements when cultured in fetal bovine serum-containing medium. Here, we found that depletion of serum from the culture media induces secondary VlambdaJlambda rearrangement in this cell line. To investigate the induction mechanism of secondary VlambdaJlambda rearrangement, we assessed the expression levels of RAG-1 and RAG-2 products, Vlambda germline transcription level and the amount of Vlambda signal broken ends (SBE) in HB4C5 cells cultured in serum-supplemented or serum-free medium. Western-blot analysis showed that the expression level for the RAG-1 and RAG-2 proteins was not affected by the serum depletion. Vlambda germline transcript was found to be constitutively expressed in HB4C5 cell line and this transcription level was not affected by the lack of serum. On the other hand, the amount of Vlambda SBE was shown to be increased in HB4C5 cells cultured in serum-free medium, suggesting that this increased formation of Vlambda SBE at least partly contributed to the enhanced occurrence of secondary VlambdaJlambda rearrangement in HB4C5 cells cultured in the serum-free condition. These results indicate that expression of RAG proteins and Vlambda germline transcription is not enough to undergo secondary VlambdaJlambda rearrangement in this cell line.

VJ Rearrangement at Previously Excluded Igλ Allele in Human Plasma Cell Line NAT-30

NAT-30 (μ, λ) is a human plasma cell line derived from Namalwa, a human Burkitt lymphoma cell line (1). It was previously demonstrated that concanavalin A (Con A) stimulation of the NAT-30 cells caused the loss of the original light chain of the NAT-30 cells and the expression of the new light chains in this cell line (2). This phenomenon was referred to as Light Chain Shifting (2). In this report, we showed that the new VJ rearrangement followed by the transcription spontaneously occurred in NAT-30 cells without Con A stimulation at low frequency and Con A stimulation may enhance this phenomenon in the magnitude of several hundreds fold.

Removal of DNA in Cell Cultured Fluid Using BMMTM

We showed that the large amount of cellular DNA contaminated in cell cultured medium. For removal and/or reduction of contaminated DNA in bio-drugs, we want to show the applicability of the cuprammonium regenerated cellulose hollow fiber (BMMTM) virus removal filter. In cell culture step, that is, upstream manufacturing process of bio-drugs, the removability of DNA and the permeability of protein produced by cell using BMM were checked. Cell cultured fluid by hybridomas was prepared. Cell cultured fluid was filtered using BMM with mean pore size of 15nm and 35nm (BMM15 and BMM35). The amount of monoclonal antibodies (MAbs) (i.e., IgG and IgM) produced by cell were determined by ELISA. The removability of DNA by filtration was about 2 logs rejection and decreased with the increase of cell culture day. It may be caused by the differences of both dispersion state of DNA and fragmentation of DNA during culture. The permeabilities of IgG and IgM kept higher level than that of DNA in cultured fluid. DNA in protein solution was easily reduced comparing with naked DNA using BMM.