Cancer Immunity 12:2 (2012)

Although Lloyd J. Old was involved in various studies of the interactions between cancer and the immune system, it seems to us that his ideas often originated from the studies of serological identification of mouse lymphocyte antigens. The findings from these studies allow us to distinguish cells of different lineages and differentiation stages, and also to distinguish leukemia cells from normal lymphocytes (1). It is amazing that essentially a single serological technique, i.e., the Trypan blue exclusion test (presumably introduced in Peter Gorer’s laboratory by Edward A. Boyse), was used to define a series of these antigens. This technique was used in conjunction with absorption analysis to elucidate the specificity of antisera, based on a vast knowledge of mouse immunogenetics. In the laboratory, Elisabeth Stockert (who was a technical assistant at that time) was the great master of these serological techniques, carrying out her own projects of cell surface antigens, as will be described below, while managing the day-to-day business of the laboratory. Similarly, Elizabeth A. Carswell and Gayla Geering investigated tumor necrosis factor (TNF) and retrovirus-associated intracellular antigens, respectively, while working as technical assistants. When Toshitada Takahashi left Nagoya, Japan, for New York in 1968, the TL and Lyt (Ly) series of antigens of thymocytes and peripheral T cells had already been identified by Drs. Old, Boyse, and Stockert (1-3), and congenic strains of mice for each antigen system had been established. Contemporaneously, Katsuaki Itakura was preparing to initiate genetic linkage studies of these differentiation antigens. In the late 1960s, the concept of T cells and B cells was becoming more and more widespread, and after the First International Immunology Congress in Washington, D.C., in 1971, this concept became widely accepted all over the world. The Thy-1 (Θ, CD90) and Lyt series of antigens are considered to be markers for thymocytes and peripheral T cells, whereas TL is a marker for thymocytes. Thus, Takahashi’s project was focused on B cells. When he started his study on cell surface antigen analysis of plasma cell leukemias (PCL), Yoshiyuki Hashimoto kindly taught him how to carry out day-to-day experiments. At that time, Dr. Hashimoto was a visiting investigator from the Biochemistry Institute in Tokyo, Japan, working on cell-mediated immunity against TL-positive leukemias. It is noteworthy that, as the founder of the Japanese Association of Cancer Immunology, Dr. Hashimoto greatly contributed to the progress in tumor immunology in Japan. Dr. Takahashi defined a novel PC.1 alloantigen that is expressed on PCL cells and mature antibody-forming cells (IgG type hemolytic plaque-forming cell, IgG-PFC), but not on peripheral B cells (4). Furthermore, he demonstrated reciprocal expression of surface immunoglobulin, which is expressed on peripheral B cells, but on neither IgG-PFC nor PCL cells. We had to wait until 1973 to see the first report of a B cell alloantigen by Hidetoshi Sato (5), although it is named Lyb-2 (CD72). This protein was later shown to be a ligand for Lyt-1 (CD5) and appears to be present on all B lymphocytes except for plasma cells. In 1973, Hiroshi Shiku, also from Nagoya, went to the SloanKettering Institute for Cancer Research (SKI) and started working on cell-mediated immunity with Herbert F. Oettgen and Michael A. Bean. Although the chromium release assay had already been established by Cerottini et al. and was widely used to target suspension cells, this assay was not always suitable for monolayer cell cultures derived from solid tumors. In order to detect killing activity against attached target cells, Dr. Bean developed a new microassay using [3H]proline for labeling, instead of chromium 51; this assay was used by Dr. Shiku for his phenotype analysis of T cell subpopulations. A very exciting result was obtained in 1975, namely, that the killer T cell population is relatively rich in Lyt-2/3 (CD8) surface antigens, but relatively poor in Lyt-1 antigen, suggesting that T cells with different functions could be distinguished on the basis of their Lyt phenotypes (6). Subsequently, Eiichi Nakayama clearly demonstrated selective blocking of cytotoxic cells by Lyt-2 and Lyt-3 antisera in vitro in the absence of complement, suggesting that Lyt-2/3 determinants on the surface of cytotoxic T cells have a close spatial relationship to the T cell receptor (7, 8). Furthermore, it was shown later by Drs. Nakayama and Akiko Uenaka in Japan that in vivo administration of Lyt-2/3 antibodies is able to block the cytotoxicity of killer T cells responsible for tumor rejection (9). Thus, Lyt-2/3 was demonstrated to be not merely the marker of killer T cells, but a molecule that is important to their key function. As for the phenotype of effector cells (Th1) and regulatory T cells (Treg), Shimon Sakaguchi and his colleagues demonstrated in 1982 that both populations express Lyt-1, but not Lyt-2/3, i.e., the CD4 phenotype (10, 11); they accomplished this by analysis of an organ-localized autoimmune disease model that develops in mice thymectomized on day 3 after birth. It is now well known that CD8 and CD4 molecules are involved, respectively, in the interactions between T cell receptor and target antigen peptides presented on major histocompatibility complex (MHC) class I and class II molecules. This research laid the groundwork for identifying the cell surface markers that allow cells to be experimentally and diagnostically separated and distinguished,