Kazunori Onoé

Regulation of experimental autoimmune uveitis in rats--separation of MHC and non-MHC gene effects.

Experimental autoimmune uveitis (EAU) is an organ‐specific autoimmune disease and has served as a model of certain ocular inflammatory conditions in man. The present study was aimed at separating the effects of MHC and non‐MHC genes on the development of EAU in the rat. EAU‐susceptible LEW (RT1l) EAU‐resistant WKAH (RTIk), and WKAH.1L (RTF) MHC congenic strain of WKAH background rats were immunized with retinal soluble antigen (S‐Ag) in Freunds complete adjuvant (FCA). LEW rats showed typical EAU, while neither WKAH nor WKAH. 1L congenic rats developed EAU. However, when an additional i.v. injection of Bordetella pertussis was given, all rat strains developed EAU. Furthermore, when immunized with peptide M, an 18‐mer synthetic peptide, which corresponds to amino acid positions 303–320 of bovine S‐Ag, and given an additional i.v. injection of B. pertussis, LEW and WKAH. IL rats developed EAU, whereas WKAH did not. When ACI (RTIavl), BUF (RTIb), LEJ (RTIl), W(RTIk), F344(RTI1v1), BN (RTIq), NIG‐III (RTIq), TO (RT1I), and SDJ (RT1u) rats were immunized with peptide M or S‐Ag and then given B. pertussis, all strains developed EAU by immunization with S‐Ag plus B. pertussis, but only F344 and NIG‐III developed EAU by immunization with peptide M. These findings suggest that susceptibility to EAU in rats is controlled by both MHC and non‐MHC genes; and that in the absence of B. pertussis adjuvant, the form of disease induced by native S‐Ag in FCA is governed by non‐MHC gene(s). However, this effect of non‐MHC gene(s) could no longer be observed when the rats were also injected with B. pertussis adjuvant at sensitization.

Interleukin-1-dependent mitogenic responses induced by protoscoleces of Echinococcus multilocularis in murine lymphocytes.

Mitogenic effects of protoscoleces (PSCs) of Echinococcus multilocularis on murine lymphocytes were studied. Spleen cells from normal BALB/c mice showed significant proliferative responses when cocultured with PSCs. Proliferative responses were observed in both the T and B cell populations. The PSCs also stimulated cells of the macrophage/monocyte lineage to secrete inter‐leukin‐1 (IL‐1). Depletion of plastic‐ and Sephadex G‐10‐adherent cells from the spleen cell population significant‐ly reduced the proliferative responses to PSCs and the low responsiveness was restored by addition of plastic‐ad‐herent cells to these cultures. Furthermore, addition of anti‐lL‐l serum to the spleen cell cultures stimulated with PSCs completely suppressed the proliferative responses. These findings demonstrate that the mitogenic effect of PSC on lymphocytes depends on EL‐1 secreted by cells of macrophage/monocyte lineage.

Distribution of MEL-14+ Cells in Various Lymphoid Tissues

The distribution of MEL-14+ lymphocytes was investigated by both fluorocytometric analysis and complement-dependent-cellular-cytotoxicity (CDCC) tests in which rabbit anti-rat Ig was added with complement at a secondary step. When CDCC was employed to detect MEL-14+ cells, almost half of the thymocytes were found to be MEL-14+ in various strains of mice. This high proportion of MEL-14+ cells stands in striking contrast to prior reports. Furthermore, when determined by fluorocytometric analysis, MEL-14+ cells were found to comprise more than 80% of the cells in the thymus. The MEL-14+ thymocytes comprised both immature subsets (CD4-8-, CD4+8+) and mature subsets (CD+8-, CD4-8+). MEL-14 brightly positive (MEL-14high) cells, however, were located mainly in mature T cell subpopulations within the thymus. The MEL-14high thymocytes appeared to be susceptible to the CDCC method. Most of MEL-14+ cells present in spleens and lymph nodes were shown to be included in the MEL-14high population. The MEL-14+ cells susceptible to treatment with MEL-14, rabbit anti-rat Ig plus complement in the spleen and lymph node were restricted to cells of the T-lineage. These data suggest that T cells may change from cells with low expression of the MEL-14 antigens at their surface to cells with high MEL-14 antigens in the process of differentiation. Furthermore, these findings indicate that MEL-14 molecules may be used as a surface marker to characterize an important T cell subpopulation.

Quantitative analysis of MEL-14 expression on various lymphocyte subpopulations

Amounts of gp90MEL-14 molecules in various lymphocyte subpopulations were analyzed. The expression of gp90MEL-14 was rapidly up-regulated in the course of usual procedures for preparing single cell suspensions. On the other hand, the gp90MEL-14 expression was down-modulated after short term culture in the presence of PMA. Addition of NaN3 in the medium for preparing cell suspension was shown to stabilize the expression profile of the gp90MEL-14. By this simple improvement, we could successfully evaluate the precise amounts of gp90MEL-14 expressed on various lymphoid cells which appeared to reflect the expression pattern of gp90MEL-14 in vivo. The proportion of MEL-14+ cells and amounts of gp90MEL-14 on the cells were clearly associated with the anatomical and functional distance from endothelial cells of HEV in peripheral lymph nodes. The present findings, thus, support the role of gp90MEL-14 molecules as homing receptors for HEV that was originally proposed by Weissman and his colleagues.

Functional studies on MEL-14+ and MEL-14− T cells in peripheral lymphoid tissues

Functions of MEL-14+ T cells and MEL-14- T cells in peripheral lymphoid tissues were analyzed and compared. The MEL-14- T cells, representing a minor subpopulation of spleen and lymph node T cells, generated considerably higher mixed lymphocyte reaction and mitogen responses than the MEL-14+ T cells in any lymphoid tissues studied. Furthermore, upon stimulation with ConA the MEL-14- CD8+ T cells produced significantly larger amounts of IL-2 and IFN-gamma than MEL-14+ CD8+ T cells did. A similar but less marked observation was obtained with the CD4+ T cell population. Furthermore, when B10.BR mice were immunized with AKR (Mls-1a) spleen cells, the proportion of the Mls-1a reactive V beta 6+ T cells from draining lymph nodes increased and a substantial proportion of the increasing V beta 6+ T cells was shown to be MEL-14-. The present findings on the whole indicate that MEL-14- T cells in the peripheral lymphoid tissues are at functionally high levels and may represent memory cells which have been previously stimulated in vivo.

Influence of a Small Number of Mature T Cells in Donor Bone Marrow Inocula on Reconstitution of Lymphoid Tissues and Negative Selection of a T Cell Repertoire in the Recipient

Allo‐chimerism and clonal elimination of self antigen (Ag) (Ia + Mls‐1a) reactive Vβ6+ T cells were analyzed and compared between allogeneic bone marrow (BM) chimeras reconstituted with BM cells which had been treated with anti‐Thy‐1 monoclonal antibody (mAb) plus complement (C) (T– chimeras) and BM chimeras which had been reconstituted with BM cells pretreated with anti‐Thy‐1 mAb alone (T+ chimeras). When lethally irradiated AKR (Mls‐1a) mice were reconstituted with BM cells from B10 or B10 H‐2 congenic mice, both T+ and T– chimeras were entirely free of signs of graft‐versus‐host reaction (GVHR). However, complete replacement of the AKR lymphoid tissues by donor BM cells was accomplished at an early stage in T+ chimeras but not in T– chimeras. On the other hand, clonal elimination of Vβ6+ T cells reactive to the recipient Ag (Mls‐1a) was abolished in T+ chimeras but successfully induced in T– chimeras. The Vβ6+ T cells not eliminated in T+ chimeras showed depressed responses against Mls‐1a antigens. The findings herein demonstrate that T cells which contaminate a BM inoculum survive in recipient mice after treatment with anti‐Thy‐1 mAb without C in vitro followed by BMT. The surviving T cells have been estimated to represent fewer than 0.5% of the BM cells inoculated. These cells appear to accelerate the full replacement of recipient lymphoid tissues by donor cells. Furthermore, the T cells which survive in the marrow inoculum influence eventually the development of a tolerant state in the T cell repertoire of the donor.

FINE ANALYSIS OF RECOGNITION SITES OF MONOCLONAL ANTIBODIES, 1B7 AND 1E4, USING L CELL TRANSFECTANTS OF RECOMBINANT MOUSE MHC CLASS II GENES

Recognition sites of two monoclonal antibodies (mAbs), 1B7 and 1E4, specific for murine MHC class II were determined, using eleven L cell transfectants which express recombinant class II (I‐A) molecules. 1B7 positive cells possessed a haplotype k in the α‐helix of the β‐chain, whereas 1E4 positive cells had haplotype b at the α‐helix of the β‐chain. The α‐helix of the β‐chains is regarded as an important site for antigen binding. Thus, the 1B7 and 1E4 mAbs may be useful for analysis of functional sites on class II molecules involved in antigen recognition.