(1) Takashi Nishimura
Home Institution: Department of Anatomy, Tohoku University School of Medicine, Seiryo-Machi 2-1, Sendai 980, Japan.
Sponsor and Host Institution:
Professor Steven J. Burakoff, Department of Pathology and Division of Pediatric Oncology, Harvard Medical School, and the Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115
Dates of Visit: March 28, 1986 - August 31, 1987
Summary of Activity:
Project 1. Characterization of lymphokine activated-cell associated antigen (LAA) defined by monoclonal KBA antibody.
As previously reported (Cell. Immunol. 94, 122-132, 1985), we succeeded to establish a monoclonal antibody (KBA), which was strongly reactive against lymphokine-activated killer (LAK) cells and could block the cytotoxicity of LAK cells. The LAA antigen recognized by KBA MAb consisted of two distinct peptides with M.W. of 180K and 95K Da. Although the characteristics of the LAA were similar to that of LFA-1, the biological characteristics of LAA did not coincide with those of LFA-1. Therefore, I tried to evaluate the difference between LAA and LFA-1 using KBA MAb and MAb against LFA-1 (M17/5.2) produced by T. Springer. As a result, I demonstrated that (1) LAA and LFA-1 showed the same distribution pattern on lymphoid cell populations; (2) KBA MAb recognized different epitopes of LFA-1 molecules from that defined by M17/5.2 MAb; (3) KBA MAb could precipitate all LFA-1 molecules from LAK cells, while M17/5.2 MAb could precipitate a part of LFA-1 molecules from LAK cells. Considering these results together, LAA molecules defined by our KBA MAb is one epitope of LFA-1 molecules, distinct from LFA-1 molecules defined by M 1715.2 MAb. Moreover, it was strongly suggested that LAA epitopes were highly expressed on IL 2-activated T lymphocytes, such as LAK cells. Therefore, in near future, I would like to evaluate whether the expression of LAA epitopes of LFA-1 molecules are associated with T cell activation or not.
Project 2: Evaluation of the mechanisms of cytotoxic T lymphocytes (CTL) triggering.
It has been demonstrated that CTL-mediated cytotoxicity consists of three distinct process (binding, lethal and destruction). However, it remains unclear how CTL are triggered by target cells. The absence of the method to measure CTL activation after binding with target cells is one of major causes of the delay of the study about CTL triggering mechanisms. However, recently, it became possible to determine CTL triggering by measuring serine esterase (SE) activity released in culture supernatants. This time, I applied this method for the evaluation of mechanisms of CTL triggering after binding with target cells. Initially, I tried to demonstrate the signal requirements for SE release from CTL. When CTL were incubated with phorbol myristate acetate (PMA) plus A23187, great amounts of SE were released from CTL. In contrast, neither PMA alone or A23187 alone had effect on the release of SE release from CTL. These results strongly suggest the important role of protein kinase C (PK-C) on CTL triggering. Moreover, I demonstrated that long term treatment of CTL with PMA caused the inhibition of SE release from CTL in parallel with the reduction of CTL activity. In addition, long term treatment of CTL with PMA resulted in the total disappearance of PK-C activity in CTL. Therefore, we concluded that PK-C-dependent SE release (equivalent to cytolytic granule release) from CTL was essential for the triggering of CTL after binding with target cells. We also demonstrated that PK-C-dependent signals were important in LAK cell-mediated cytotoxicity. I believe that these study will be valuable for studying the mechanisms of cell-mediated cytotoxicity in future.
Project 3: Characterization of lymphokine-activated killer (LAK) cells generated from nude mouse spleen cells.
As previously reported (Immunol. Letters, 12, 77-82, 1986), we demonstrated that recombinant interleukin 2 (r-IL 2) allowed the generation of LAK cells from nude mouse spleen cells (Nude-LAK). Nude-LAK cells are characteristic of Thy 1.2+, L3T4-, Ly2-, asialo GM1-, which have different characteristics from that of normal LAK cells and NK cells. Recently, it was suggested that T cell receptor (TCR)!!
!complex might be important for the recognition of target cells by double negative killer cells. Therefore, it was anticipated that TCR!!!might be also involved in Nude-LAK cell-mediated cytotoxicity. To clarify this problem, we investigated the TCR expression on Nude-LAK cells using both flow cytometry and gene technology. As a result, it was demonstrated that Nude-LAK cells did not express either TCR!!
!defined by F23.1 MAb or T3 molecules defined by 2C11 on their cell surface. Moreover, Nude-LAK cells had no any message for TCR!!
,!,.Therefore, I concluded that TCR were not involved in Nude-LAK cell-mediated cytotoxicity. Thus, it was strongly suggested that TCR were not always necessary for the recognition mechanisms of killer cells. It remains unclear what molecules are important for the recognition mechanisms of Nude LAK cells. However, I demonstrated that LFA-l molecules are at least essential for Nude-LAK cell-mediated cytotoxicity. These findings suggest the existence of another recognition molecules besides TCR on Nude-LAK cells and would be available for understanding of the recognition mechanisms of natural immunity systems. ACKNOWLEDGEMENTS:
I greatly thank Drs. Steven J. Burakoffand Steven H. Herrmann (Dana-Farber Cancer Institute) for their kind help during my staying in Boston. I also would like to thank Dr. Kakefuda (NIH), who always help may family out of several troubles about medical insurance and gave me great encouragement.
Publication:
Takashi Nishimura, Steven J. Burakoff, and Steven H. Herrmann, Protein kinase C required for cytotoxic T lymphocyte triggering. J. Immunol. 139, 2888-2891, 1987.
(2) Masao Kimoto
Home Institution: Saga Medical School
Sponsor and Host Institution:
Alfred Singer, M.D.
Immunology Branch
National Cancer Institute
National Institute of Health
Dates of Visit: from January 31, 1988 to February 13, 1988
Summary of Activities:
The title of my research subject is “Analysis of the regulation of immune response gene expression. “The objectives of my visit is to exchange information with scientists concerning my research subject and to promote my research activities. The achievements obtained through my visit this time will be described in the following paragraphs. I obtained many up-to-date information concerning my research subject. I also obtained several monoclonal antibodies which will be useful for my study. I discussed on the possibility of collaboration of experiments concerning my current research projects with several scientists.
l. Attendance and poster presentation at the UCLA Symposium “B cell development” held at Taos, New Mexico from January 31 to February 6.
The major topics presented at this symposium were rearrangement and expression of immunoglobulin genes, interrelationships of B cell subsets, diversification of B cell repertoire, development of B cells, B cell growth and differentiation factors, antigen presentation by B cells, autoimmunity and immunodeficiency, oncogenes and B cell transformation, mechanisms of B cell activation and transgenic mouse models for immune regulation. About four hundred scientists from all over the world attended the meeting and discussed on these topics. We presented our data entitled “The B lymphocyte lineage related gene mb-1: cDNA structure and RNA expression in different cell lineages” by S. Kashiwamura, N. Sakaguchi, M. Kimoto, P. Thalmann and F. Melchers. This is a completely new method which is able to analyse the function of B cell specific gene function. Many scientists joined the discussions concerning our presentation.
2. Visit and seminar presentation at Mayo Clinic, Rochester, Minnesota from February 10 to 12.
I presented a seminar entitled “Immunologic analysis of E a gene transgenic mouse” following the introduction by Dr. David McKean, Professor at the Department of Immunology, Mayo Clinic. My talk presented some new data which suggest the possibility of the existence of new la specificities present in E!!
!transgenic lymphoid cells. About thirty scientists attended the seminar and discussed on my presentation. After my seminar, I discussed individually with Drs. D. Mckean, P. Leibson, B. Abraham, E. Hay, K. Donovan, V. Lennon, C. David, C. Krco, B Wei, M. Robinson, R. Little, S. Savarirayan, S. Banerjee, K. Hillman, T. Haqqi, J, Martin, C. Nickerson, L. Kerr, B. Beck. These scientists work on the functions of Ir genes. I obtained a lot of information concerning my current research projects. Dr. David McKean gave me monoclonal anti-Ia antibodies which will be very useful for my research. 3. Visit and seminar presentation at the National Institute of Health (NIH), Bethesda, Maryland.
On February 11th, I presented seminar titled “Immunological analysis of E!!
!gene transgenic mouse.” This was chaired by Dr. Alfred Singer, Senior investigator, Immunology Branch, NCI, NIH. About 30 scientists attended the seminar and discussed concerning my presentation. I also talked individually with Drs. E. Shevach, G. Shearer, S. Sharrow, D. Margulies, J. Berzofsky, A. Singer and R. Germain. They talked their own works and also gave suggestions on my research projects. Dr. R. Germain is working on the association of!!!and!!!chain of Ia molecule using L cell transfection system. His project is intimately related to my current research and we have discussed on the possibility of collaboration with each other. (3) Hiromi Fujiwara
Home Institution: Biomedical Center, Osaka University Medical School
Sponsors and Host Institutions:
Prof. Mark I. Greene, Pennsylvania Univ. School of Medicine and Dr. Gene M. Shearer, NCI, NIH
Dates of Visit: September 14, 1987 - October 4, 1987
Summary of Activities:
l) With Prof. Mark I. Greene at Pennsylvania University
The purpose of my stay at Pennsylvania University is a) to initiate collaborative studies concerning “Functional properties and physiological effects of TSTGF”, b) to learn technique of in situ hybridization and c) to exchange the most recent informations about T cell differentiation and/or proliferation in the thymus. I set up two, but mutually related collaborative studies. Our experiments performed in Prof. Greene’s laboratory demonstrated that TSTGF which was produced and purified in our laboratory (Osaka Univ. Med. Sch.) was capable of supporting the growth of thymic leukemia cell line and CTL line, both of which express phenotypes of immature T cells in the thymus. Based on these results, we discussed our future collaborative experiments to investigate the effect of the TSTGF on the differentiation of the above immature type of T cell lines along with the induction of their proliferation.
I learned the technique of in situ hybridization from Dr. Kokai in Prof. Greene’s laboratory.
I made a seminar entitled “Purification and Physiological Significance of TSTGF”, discussed with members in Prof. Greene’s laboratory in the field of T cell differentiation and proliferation in the thymus.
2) With Drs. Gene M. Shearer, Alfred Singer, Gilbert Jay and William E. Paul at NCI, NIH.
I discussed and exchanged the most recent informations in the following aspects:
a) Immunosuppression which is common to tumor-bearing state, AIDS and GVH.
b) Roles of each subset of T cells in mediating anti-allograft rejection and anti-tumor immunity.
c) T cell differentiation/proliferation in the thymus.
I obtained several suggestions from Dr. W.E. Paul about how to analyze the effect of TSTGF on the proliferation and/or differentiation of immature thymocytes and to further purify TSTGF.
3) With Dr. Philip D. Greenberg at Washington University
I made a seminar entitled “TSTGF; its biochemical and functional characterization” and exchanged informations about T cell differentiation and proliferation.
Through 3 week-stay, I was able to initiate two collaborative studies and obtain a lot of new informations concerning the title of the present study.
(4) Kenichi Matsubara
Home Institution: Institute for Molecular and Cellular Biology, Osaka University, 1-3, Yamada-oka, Suita, Osaka 565, Japan
Sponsor and Host Institution: (See below)
Dates of Visit: January 30, 1988-February 14, 1988 (16 days)
Summary of Activities:
To study the results of intensive discussions on human gene project in USA, six major administrative organizations and many individual scientists who took part in the discussions were selected for interviews. The report has been orally presented in the meeting sponsored by Mombusho “Bio-gann project”, and in a preparatory workshop for “Human gene projects” sponsored by Human Frontier Science Programme.