Kazuo Ohtsuka

Details of an isolation method for hepatic lymphocytes in mice

The liver comprises a unique lymphocyte population, i.e., extrathymic alpha beta T cells with TcR of intermediate intensity. In the present study, we attempted to determine what pretreatments were appropriate to isolate hepatic mononuclear cells (MNC) containing such intermediate alpha beta TcR cells in mice. Hepatic MNC were isolated from untreated mice and mice subjected to either bleeding or liver perfusion, and the intermediate alpha beta TcR cells in each preparation were identified. For reasons of simplicity, cell purity and cell yields, hepatic lymphocytes should be obtained from mice subjected to total bleeding. Additional information on extrathymic alpha beta T cells obtained by using the recommended method is also presented.

Radioresistance of Intermediate TCR Cells and Their Localization in the Body of Mice Revealed by Irradiation

Extrathymic generation of T cells in the liver and in the intestine was recently demonstrated. We investigated herein whether such T cells, especially those in the liver, are present in other organs of mice. This investigation is possible employing our recently introduced method with which even a minor proportion of extrathymic, intermediate TCR cells in organs other than the liver can be identified. Intermediate TCR cells expressed higher levels of IL‐2Rβ and LFA‐1 than bright TCR cells (i.e., T cells of thymic origin) as revealed by two‐color staining. Although intermediate TCR cells were present at a small proportion in the spleen and thymus, they predominated in these organs after irradiation (9 Gy) and bone marrow reconstitution, or after low dose irradiation (6 Gy). This was due to that intermediate TCR cells were relatively radioresistant, whereas bright TCR cells were radiosensitive. Microscopic observation and immunochemical staining showed that intermediate TCR cells in the spleen localized in the red pulp and those in the thymus localized in the medulla. These intermediate TCR cells displayed a large light scatter, similar to such cells in the liver. The present results suggest that intermediate TCR cells may proliferate at multiple sites in the body.

A Similar Expression Pattern of Adhesion Molecules between Intermediate TCR Cells in the Liver and Intraepithelial Lymphocytes in the Intestine

Two major populations of extrathymically differentiated T cells exist in the liver and intestine. Such T cells in the liver have TCR of intermediate intensity (i.e., intermediate TCR cells) and constitutively express IL‐2 receptor β‐chain (IL‐2Rβ), whereas those in the intestine, especially intraepithelial lymphocytes, have TCR of bright intensity, consisting of a mixture of IL‐2Rβ+ and IL‐2Rβ–. All mature thymocytes and thymus‐derived T cells seen in the peripheral immune organs are TCR‐bright+IL‐2Rβ– under resting conditions. When the expression pattern of adhesion molecules, including CD44, L‐selectin, LFA‐1 and ICAM‐1, was compared among these T‐cell populations, they displayed quite unique patterns of expression. All extrathymic T cells in the liver, intestine, and even other organs were CD44+L‐selectin– LFA‐1++ICAM‐1+, whereas thymocytes and thymus‐derived T cells were CD44– L‐selectin+LFA‐1+ICAM‐1–. This inverted expression of adhesion molecules between extrathymic T cells and thymus‐derived T cells might be associated with their unique tissue‐localization.

Intraepithelial lymphocytes in colon have similar properties to intraepithelial lymphocytes in small intestine and hepatic intermediate TCR cells.

Recently, properties of intraepithelial lymphocytes (IEL) in the colon (C-IEL) have been analyzed in comparison with those of IEL in the small intestine (SI-IEL). We compared the properties of C-IEL with those of SI-IEL and hepatic intermediate TCR cells, two other types of extrathymic T cells. C-IEL and intermediate TCR cells contain many NK1+T cells, although SI-IEL contain few. Vγ and Vδ usage of C-IEL was the same as that of SI-IEL, and that of intermediate TCR cells was different. C-IEL responded to Con A while SI-IEL did not. As to adhesion molecules, C-IEL include both extrathymic and thymus-originated type T cells. With age, TCR-αβ+CD4+CD8+ cells do not increase among C-IEL but do increase among SI-IEL. IL-2Rβ+ or CD4−CD8− C-IEL increase as observed in the liver. These results indicate that these organ-specific T cells have different roles at their respective sites and that they may be of different lineages.

Gut-associated lymphoid tissues in ulcerative colitis.

BACKGROUND The main feature of ulcerative colitis (UC) is numerous infiltration of not only lymphocytes and plasma cells but also neutrophils and macrophages, indicating acute on chronic inflammation. Recent studies show that apoptosis may play an important role in the regulation of gut-associated lymphoid tissues (GALT). Therefore, this study was performed to clarify apoptosis of lymphocytes in the peripheral blood and colonic mucosa of UC. METHODS Three-color flow cytometry was used to clarify apoptosis of lymphocytes in the peripheral blood and colonic mucosa of patients with active and inactive UC compared with controls using fluorescence-labeled monoclonal and polyclonal antibodies such as Fas (CD95), Fas ligand, CD4, CD8, CD45RO, etc. RESULTS The ratio of Fas and CD45RO double-positive cells in the peripheral blood of UC patients was significantly increased in CD8 but not CD4 T cells when compared with controls. The ratio of Fas-positive and CD45RO-negative cells was significantly increased in CD4 and CD8 T cells of UC when compared with controls. There was unbalanced immunoregulation between CD4 and CD8 T cells in the colonic mucosa of UC probably due to apoptosis through Fas-Fas ligand system. CONCLUSIONS Abnormal GALT system was found in UC probably due to dysregulation of T cells through Fas-Fas ligand system.

Increase in the proportion of CD56+T cells in patients with chronic type C hepatitis after treatment with α-interferon

Abstract Recently CD56 + T cells, which are unique T cells that have one of the NK markers and are few in peripheral blood mononuclear cells (PB MNC), have been found to be predominant in the liver. We investigated the phenotypical changes of PB MNC in 18 patients with chronic type C hepatitis before and during α-interferon (IFN-α) treatment. In the treatment response group ( n = 11 ), CD56 + T cells increased significantly ( P ). Conversely, no increase of CD56 + T cells was observed in the non-response group ( n = 7 ). Phenotypically studied, CD56 + T cells were mostly CD8 + , about half comprised γδ-TCR and all expressed high levels of LFA-1. These results suggest that CD56 + T cells might play an important role in the pathogenesis of chronic type C hepatitis.

In Vivo Activation of Extrathymic T Cells in Mice by Staphylococcal Enterotoxin B

Extrathymic T cell differentiation was recently known to occur in the sinusoids of the liver,1,2 and in the epithelial region of the intestine.3 Such extrathymic T cells in the liver and intraepithelial lymphocytes (IEL) in the intestine both have common and distinct properties with each other. Both extrathymic T cells become predominant in corresponding organs of aged humans and animals with the involved thymus, and comprise a considerable proportion of self-reactive forbidden clones, possibly due to the lack of negative selection systems for such clones. Staphylococcal enterotoxin B (SEB) that activates T cells is known as one of the super-antigens.4,5 We investigated herein how extrathymic T cells in the liver and intestine were activated when SEB was administered in mice.