Akihiko Yano

The Role of Cytotoxic T Lymphocytes in the Pathogenesis of Vogt-Koyanagi-Harada Disease

Peripheral blood lymphocytes (PBL) were isolated from blood of 6 patients with Vogt-Koyanagi-Harada disease (VKH), and their cytotoxic activity was assayed using 51Cr release from the labelled cells; the cytotoxicity was expressed as percentage release in reference to the complete release using saponin. The PBLs from the patients showed a significant cytotoxic activity against P-36 human melanoma cells, and the specificity of the reaction was confirmed by the cold target inhibition test and also by the use of HeLa-S3 human cervical carcinoma cells and B-16 mouse melanoma cells, since no significant cytotoxicity was seen against these cells. The cytotoxic activity was greatly reduced by pretreatment of the PBLs with monoclonal antihuman Leu-1 antibody plus the rabbit complement. It was, therefore, thought that the cytotoxic activity against the melanoma cells is mainly due to T cells. In addition, the cytotoxic activity was specifically blocked by monoclonal antihuman Leu-2a antibody, the specificity of which is distributed on cytotoxic/suppressor T cell subset of normal human peripheral lymphocytes and normal human thymocytes. A possibility was discussed as regards the involvement of the cytotoxic T lymphocytes in the pathogenesis of VKH.

PPD‐Specific Proliferative Response in Humans: I. Analysis of PPD‐Specific Proliferative Cells from Tuberculous Pleurisy Patients and Healthy Controls with Monoclonal Antibodies Specific for Human T Subsets

Peripheral mononuclear cells (PBL) from tuberculin reaction (TR)‐negative tuberculous pleurisy patients proliferated poorly with PPD, while the cells of pleural effusion from these patients showed a proliferative response to PPD as well as did the healthy control PBL. Surface antigens of peripheral blood and pleural effusion were examined by using monoclonal antibodies. The Leu 1‐positive cell population can be divided into four groups, namely (1) Leu 1+, Leu 2a+, Leu 3a+, (2) Leu 1+, Leu 2a+, Leu 3a−, (3) Leu 1+, Leu 2a−, Leu 3a+, and (4) Leu 1+, Leu 2a−, Leu 3a− cell populations. Results of analysis of surface antigens of PPD‐specific proliferative cells in peripheral blood and pleural effusion from tuberculous pleurisy patients as well as healthy controls indicate that the PPD‐specific proliferative response is mediated by Leu 1+, Leu 2a−, Leu 3a+ cells and Leu 1+, Leu 2a−, Leu 3a− cells.

Participation of antigen presenting cells in glomerulonephritis.

The participation of glomerular antigen presenting (la positive) cells in the development of glomerulonephritis was analyzed. Antigen‐presentation by kidney cells was evident from the assay of ovalbumin‐speciflc T cell proliferation responses by these cells. Experimental serum sickness nephritis was induced in preimmunized rats by the intraperitoneal sensitization of oval‐bumin, and the participation of antigen presenting cells (APCs) in the peritoneal cavity and glomeruli was studied. APCs in the peritoneal cavity progressively increased in number following the antigen stimulation, while those in glomeruli were shown to increase in correlation with the development of glomerular hypercellularity. These results imply that the role of APCs in the sensitized area is superior in this model of immune complex nephritis

Serological cross-reactivity of murine A.TH anti-A.TL antibody (anti-I-Ek antibody) with la-like molecules of human antigen-presenting cells

The purified protein derivative (PPD)-specific proliferative responses of T cells from human peripheral blood are shown to be dependent on antigen-presenting cells (APC) which bear HLA-DR antigen detected by the monoclonal anti-HLA-DR antibody. The serological cross-reactivity of murine A.TH anti-A.TL antibody was observed in human APC. By absorption experiments using H-2 congenic mice, the serological cross-reactivity of A.TH anti-A.TL antibody with human APC is mapped in the I-E subregion. Thus, anti-I-Ek antibody reacts with the Ia-like molecule(s) on human APC. Murine allo-anti-I-Ek antibody does not always react with determinants of Ia-like molecule(s) on human APC, since this antibody did not eliminate PPD-specific proliferative responses in one particular case. Thus, anti-I-Ek antibody seems to react some type of the polymorphic determinants but not of the shared determinants of human Ia-like molecule(s) on APC. The relationship between the cross-reactive molecule detected by murine allo-anti-I-Ek antibody and the HLA-DR antigen remains to be analyzed.

Cellular FITC-linked immunospecific assay (Cell-FLISA) for detection of monoclonal antibodies against cell-surface antigens

A cellular fluorescein isothiocyanate (FITC)-linked immunospecific assay (Cell-FLISA) has been established using the recently developed fluorophotometer for microplates. In the Cell-FLISA system, monoclonal antibodies specific for the surface antigens of live cells are detected by measuring the fluorescence intensity of an FITC-labeled second antibody: goat anti-mouse immunoglobulin antibody. It takes only 2 min to count 96 samples in microplate wells using the fluorophotometer for microplates. Moreover, by this system, the analysis is finished within 2 hr. Thus, the Cell-FLISA system has advantages in screening a large number of samples, such as hybridoma cell lines secreting monoclonal antibodies against cell-surface antigens.

Immune Response to Toxoplasma gondii

Peripheral blood leukocytes (PBL) from patients with toxoplasmosis were shown to be highly responsive to in vitro stimulation with Toxoplasma gondii extract as measured by incorporation of [3H]methylated thymidine. Analysis of Toxoplasma‐specific proliferative cells in PBL by using monoclonal antibodies specific for human T cell subsets revealed that the Toxoplasma‐specific proliferation response of PBL from the patients was mediated by Leu 1, Leu 3a positive cells, that is, helper/inducer T cells. Tests for the Toxoplasma‐specific proliferation response may provide a readily available method for the diagnosis of congenital toxoplasmosis, especially during the newborn period.

Serological cross-reactivity of murine allo-anti-Ias antibody with the molecule on human antigen-presenting cells

Murine allo-anti-IaS (A.TL anti-A.TH) antibody cross-reacted with human B-cells and antigen-presenting cells, while the antibody did not cross-react with human T-cells, to be precise, PPD-specific proliferative T-cells. Furthermore, the murine allo-anti-IaS antibody could inhibit the PPD-specific responses of human PBL. Thus, the murine allo-anti-IaS antibody affixes on the Ia-like molecule of human antigen-presenting cells in PPD-specific responses. A genetic mapping study of the serological cross-reactive antibody, by absorbing with the spleen cells from B10.S(9R), A.TH, A.TL and SJL mice in order to determine the specificities for the I subregions of the H-2, revealed that the serological cross-reactivity with human PBL was caused by the anti-I-AS antibody.

Serological and biological cross-reactivity of class II antigens between mice and humans in antigen-specific T-cell proliferative responses

Many studies have already been reported with regard to the serological cross-reactivities between the polymorphic determinants of murine Ia antigens and human HLA-DR antigens. In this paper, we examined the biological cross-reactivity of the polymorphism of Class II antigens in the xenogeneic antigen-presenting cell (APC)-T-cell interaction. The data indicate that purified protein derivative (PPD)-specific human T cells were not stimulated by PPD-pulsed murine APC from B10.S(9R) which possess I-As and I-Ek molecules serologically cross-reacting with human Class II antigens. On the contrary, B10.S(9R) T cells primed to PPD were stimulated by PPD-pulsed human APC. The failure of the murine APC-human T-cell interaction was not caused by the suppressive effect in culture with ongoing xenogeneic mixed lymphocyte reactions (MLR) or other cell culture conditions. Thus, a hierarchy of antigen-presenting ability in the xenogeneic APC-T-cell interaction was shown to exist.

Activation of immunoglobulin allotype-specific T cells by B cells

To investigate the role of Ia and immunoglobulin (Ig) molecules of B cells in alloantigen-specific and nominal antigen-specific T-cell activations, the ability of B cells to stimulate Ig allotype-specific T cells was examined. T15-primed B10.BR T cells responded to MOPC 315 (IgA myeloma protein derived from BALB/c) as well as T15 but not to MOPC31c (IgG1 myeloma protein). These T cells were stimulated by papain-digested Fc fragment of T15. Thus, T15-primed B10.BR T cells were shown to be specific for Ig allotype of T15, that is, Igh-2a. T15-specific B10.BR T cells were selected by 10-day cultures with T15 in vitro. They responded to BALB.K spleen cells without addition of soluble T15 antigen to the assay culture. Stimulator cells in this mixed lymphocyte reaction (MLR)-like response between T15-specific B10.BR T cells and BALB.K spleen cells were Thy-1-, Ia+ cells and these responses were blocked by anti-Iak antibodies. Furthermore, Sephadex G-10-passed BALB.K B cells stimulated the proliferation of T15-specific B10.BR T cells, while they failed to stimulate allogeneic BALB/c spleen cells. The stimulating ability of B cells in this MLR-like response of T15-specific B10.BR T cells was shown to be genetically restricted, namely, both H-2 and non-H-2 genes are involved in the manifestation of the stimulating ability. This system will provide a useful model for studying the role of B-cell surface Ig and Ia molecules in the activation of antigen-specific T cells and alloreactive T cells.

Serological Cross‐Reactivities of Murine and Human Class II Antigen Determined by Murine Xeno Anti‐Human Class II Antibody

Murine anti‐human class II antibodies were shown to cross‐react with polymorphic determinants of murine class II antigens. The cross‐reacting antibodies were raised in B10.S(9R) mice by immunizing with human nylon wool adherent cells (Ad cells) from peripheral blood leukocytes. The B10.S(9R) anti‐human Ad cell antiserum bound to the molecules consisting of two chains with molecular weights of 35K and 28K dimers which were purified with a lentil‐lectin column. The B10.S (9R) anti‐human class II antiserum was also revealed to contain two distinct cross‐reacting antibodies with polymorphic determinants of murine class II antigens coded for by the I‐A subregion of the H‐2. One is specific for a determinant of class II molecules coded for by I‐Ab,d,q, and the other seems to be specific for class II molecules coded for by I‐Aa,k,r.