Tetsuya Moroda

Circadian rhythm of leucocytes and lymphocyte subsets and its possible correlation with the function of the autonomic nervous system

There are physiological variations in the levels of leucocytes. Among these, the circadian rhythm is very important in terms of the magnitude. Since newly identified lymphocyte subsets (i.e. extrathymic T cells) have recently been detected, a comprehensive study of the circadian rhythm was conducted. All leucocytes were found to vary in number or proportion with a circadian rhythm and were classified into two groups. One group—granulocytes, macrophages, natural killer (NK) cells, extrathymic T cells, γδ T cells, and CD8+ subset—showed an increase in the daytime (i.e. daytime rhythm). The other group—T cells, B cells, αβ T cells, and CD4+ subset—showed an increase at night. Humans are active and show sympathetic nerve dominance in the daytime. Interestingly, granulocytes and lymphocyte subsets with the daytime rhythm were found to carry a high density of adrenergic receptors. On the other hand, lymphocyte subsets with the night rhythm carried a high proportion of cholinergic receptors. Reflecting this situation, exercise prominently increased the number of cells with the daytime rhythm. These results suggest that the levels of leucocytes may be under the regulation of the autonomic nervous system.

Impact of ductal resection margin status on long‐term survival in patients undergoing resection for extrahepatic cholangiocarcinoma

The current study was performed to clarify whether the presence of residual carcinoma in situ at ductal resection margins differs prognostically from residual invasive ductal lesions in patients undergoing surgical resection for extrahepatic cholangiocarcinoma.

Origin of CD57+ T cells which increase at tumour sites in patients with colorectal cancer

Human T cells carrying natural killer (NK) markers, CD57 or CD56 antigens, appear to be distinguishable from other T cell subsets in terms of their granular lymphocyte morphology and their numerical increase in patients with AIDS and in recipients of bone marrow transplantation. At the begining of this study, we observed that CD57+ T cells as well as CD56+ T cells were abundant at tumour sites in many patients with colorectal cancer. Since all these findings for CD57+ T cells are quite similar to those of extrathymic T cells seen in mice, we investigated how CD57+ T cells are distributed to various immune organs in humans. They were found to be present mainly in the bone marrow and liver, but to be completely absent in the thymus. Similar to the case of extrathymic T cells in mice, they were observed to consist of double‐negative CD48+ subsets as well as single‐positive subsets (preponderance of CD8+ cells), and to contain a considerable proportion of γδ T cells. These features are striking when compared with those of CD57+ T cells, which are characterized by an abundance of CD4+ subsets and αβ T cells. Not only at tumour sites but also in the peripheral blood, some patients with colorectal cancer displayed elevated levels of CD57+ cells. These results suggest that CD57+ T cells may be of extrathymic origin, possibly originating in the bone marrow and liver, and may be associated with tumour immunity, similar to another extrathymic population of CD56+ T cells in humans.

Autologous killing by a population of intermediate T-cell receptor cells and its NK1.1+ and NK1.1− subsets, using Fas ligand/Fas molecules

Self‐reactive clones, estimated by anti‐Vβ monoclonal antibodies (mAb) in conjunction with the Mls system, are confined to a population of intermediate (int) T‐cell receptor (TCR) (or CD3) cells (i.e. TCRint cells), but are not found among TCRhigh cells. The next questions to be answered are whether autologous killing is confined to TCRint cells and how such killing is mediated. In this study, 51Cr‐labelled thymocytes of syngeneic or allogeneic origin were used as target cells (4‐hr assay). When liver and splenic mononuclear cells (MNC) obtained from B6 mice were used as effector cells, prominent autologous killing was seen in liver MNC, but not splenic MNC. Such killing was not seen when thymocytes from B6‐lpr/lpr mice (i.e. Fas−) were used as target cells, nor when liver MNC from MRL‐gld/gld mice (i.e. Fas ligand−) were used as effector cells (target thymocytes of MRL‐+/+ mice). Cell separation experiments using a cell sorter revealed that autologous killing was mediated for the most part by CD3int cells, while allogeneic killing was mediated entirely by natural killer (NK) cells, TCRint cells and TCRhigh cells. Among CD3int cells, the NK1.1+ subset (i.e. NK1.1+ T cells) manifested a higher level of autologous killing than did the NK1.1− subset. Consistent with the results of a functional assay, it was found by reverse‐transcription–polymerase chain reaction (RT‐PCR) assay that CD3int cells among liver MNC showed the expression of Fas ligand mRNA, while thymocytes expressed Fas mRNA. When class I major histocompatibility complex (MHC)− thymocytes (from β2‐microglobulin‐deficient mice) were used as target cells, NK cells, but not CD3int cells, showed potent cytotoxicity. These results suggest that autologous killing is a major function of TCRint cells with self‐reactivity, and that such killing is mediated by means of Fas ligand/Fas molecules.

Cytotoxic activity against tumour cells mediated by intermediate TCR cells in the liver and spleen

Morphological and phenotypic characterization in previous studies has indicated that intermediate (int) T‐cell receptor (TCR) cells or T natural killer (TNK) cells may stand at an intermediate position between NK cells and high TCR cells of thymic origin in phylogenetic development. In this study, a functional study on cytotoxic activity against various tumour targets was performed in each purified subset. When a negative selection method entailing in vivo injection of anti‐asialo GM1 antibody or anti‐interleukin (IL)‐2Rβ monoclonal antibody (mAb) was applied, IL‐2Rβ+ CD3− NK cells were found to have the highest NK activity while IL‐2Rβ+ int CD3 (or TCR) cells had a lower level of the NK activity. High CD3 cells (freshly isolated) did not have any such activity. Sorting experiments further revealed that the NK function mediated by int CD3 cells was augmented when they were exposed to anti‐CD3 mAb, anti‐TCRαβ, or anti‐TCRγδ mAb. This phenomenon was not observed in NK cells and high CD3 cells. More importantly, when anti‐CD3 mAb (or anti‐TCR mAb) was added to the assay culture, int CD3 cells became cytotoxic against even NK‐resistant tumour (FcγR−, Fas+) targets. Liver mononuclear cells or int CD3 cells exposed to anti‐CD3 mAb for 6 hr showed an elevated level of perforin in their cytoplasms. The present results suggest that int CD3 cells are usually non‐cytotoxic against various tumours but become functional after being stimulated via the TCR–CD3 complex.

Self-reactive forbidden clones are confined to pathways of intermediate T-cell receptor cell differentiation even under immunosuppressive conditions

It is believed that self‐reactive forbidden T‐cell clones are generated by ‘failure’ of the pathway of T‐cell differentiation in the thymus, if it is disturbed. We examined how such forbidden clones are generated under immunosuppressive conditions. Mice were treated with an injection of deoxyspergualin, FK506, or cycloporin A. From day 3, the number of cells yielded by various organs decreased. Because of the resistance of intermediate (int) T‐cell receptor (TCR) cells (i.e. TCRint cells), they became more prominent in proportion than TCRhigh cells. TCRhigh cells are conventional T cells generated through the mainstream in the thymus, whereas TCRint cells are primordial T cells generated by the extrathymic pathway or an alternative intrathymic pathway. Similar to untreated mice, forbidden Vβ3+ and Vβ11+ clones in C3H/He (Mls‐1b2a) mice were confined to TCRint cells after treatment; there was no leakage of forbidden clones into TCRhigh cells in the thymus and periphery. In parallel with the increase in the proportion of TCRint cells, the proportion of forbidden clones also increased under immunosuppressive states, especially in the liver. Liver mononuclear cells isolated from treated mice still had the potential to mediate autologous killing. The present results suggest that the generation of self‐reactive clones is highly restricted to the pathways of TCRint cell differentiation even under immunosuppressive conditions.

Participation of NK1.1+ T cells in the rejection of lpr αβT cells when bone marrow cells of lpr mice are transplanted into B6 mice

When C57BL/6 (B6) mice were irradiated (9 Gy) and received bone marrow (BM) cells of B6‐lpr/lpr mouse origin (i.e., lpr→B6), all mice died within 6 days. In the irradiated B6 mice, radioresistant CD3− IL‐2Rβ+ NK cells and IL‐2Rβ CD3int cells (i.e., CD3int cells of extrathymic origin) remained, especially in the liver. There were two subsets, NK1.1+ and NK1.1−, among the IL‐2Rβ+ CD3int cells. However, the NK1.1+ subset (i.e., NK1.1+ T cells) was much more radioresistant, and the majority of CD3int cells belonged to this subset in irradiated mice. The expansion of lymphocytes from injected BM cells did not occur in the irradiated B6 mice. However, such expansion did take place in irradiated B6‐lpr/lpr mice injected with both BM cells of B6‐lpr/lpr and B6 origin. As a result, the mice subjected to BM cells survived. Irradiated B6 mice were treated in vivo with anti‐NK1.1 mAb or anti‐asialoGM1 antibody to eliminate NK cells alone or both NK cells and NK1.1+ T cells. When irradiated B6 mice were pretreated with anti‐NK1.1 mAb, the mice could survive. These results suggest that intact NK1.1+ T cells of extrathymic origin may recognize abnormal BM cells with the lpr gene and inhibit the expansion of lymphocytes, including abnormal double‐negative CD4−8− cells, in B6‐lpr/lpr mice. To inhibit the expansion of lymphocytes, mechanisms other than Fas ligand/Fas molecules on extrathymic T cells may be responsible.

An allogeneic microenvironment influences the phenotype of intermediate T-cell receptor cells expanding in MRL-lpr/lpr mice.

MRL‐lpr/lpr (lpr) mice fall victim to autoimmune disease owing to a lymphoproliferative disorder mainly of double‐negative (DN) CD4− CD8−αβT cells expressing a low density of interleukin‐2 receptor β‐chain (IL‐2Rβ). It was previously revealed that the lpr gene is a defective Fas gene, into which an early transposon (ETn) of retrovirus is transfected. As a result of the failure of apoptosis, intermediate T‐cell receptor (TCR) cells (i.e. TCRint cells) with DN phenotype abnormally accumulate in the periphery of lpr mice. We investigated herein how these TCRint cells are selected in terms of CD4, CD8 and TCR in lpr mice. When a whole fraction of mononuclear cells (MNC) in various immune organs of lpr mice was injected into scid mice (allogeneic circumstance), CD8+ TCRint cells mainly expanded. They had a high density of IL‐2Rβ. This was true when bone marrow cells of lpr mice were injected into scid mice. On the other hand, when MNC of the spleen and bone marrow in lpr mice were injected into irradiated (9 Gy) lpr mice (syngeneic circumstance), the major expanding cells were DN TCRint cells expressing a low density of IL‐2Rβ. A cell‐sorting experiment for purified fractions demonstrated that only CD8+ cells reconstituted TCRint cells in scid mice. Namely, DN CD4− CD8− cells as well as CD4+ cells which once acquired the mature phenotype, no longer switched their phenotype. These results suggest that the phenotype of TCRint cells is influenced by the surrounding microenvironment.