Norikazu Nagata

Functional Analyses of B Cells in (NZW x BXSB) F1 Mice

Functions of B cells from (NZW x BXSB)F1 (W/BF1) mice are investigated. The W/BF1 mouse, which is an animal model for systemic lupus erythematosus (SLE) and immune thrombocytopenic purpura (ITP), produces anti-DNA and anti-platelet antibodies; W/BF1 mice show hypergammaglobulinemia (particularly increases in IgG2a and IgG2b). The ratio of small resting B cells to large activated B cells in W/BF1 mice is low compared to normal mice, suggesting that B cells in W/BF1 mice are already activated in vivo. Furthermore, small resting B cells separated by a Percoll density gradient technique show hyper-responsiveness to lipopolysaccharide (LPS) or anti-mu plus IL-4. This suggests that B cells in W/BF1 mice are genetically programmed to be easily activated, resulting in the overproduction of autoantibodies. A significant number of CD5+ B cells are found in the lymph nodes of old W/BF1 mice. These findings indicate that all cells in the B cell lineage of W/BF1 mice are already activated in vivo.

Attenuation of Ipr-graft-versus-host disease (GVHD) in MRL./lpr spleen cell-injected SCID mice by in vivo treatment with anti-Vβ8.1,2 monoclonal antibody

When MKL/lpr (H‐2k) spleen cells were intraperitoneally injected into C.B‐17‐scid/scid (severe combined immunodeficient (SCID)) (H‐2d) mice, the SCID (SCID‐MRL/lpr) mice manifested a severe wasting syndrome with weight loss, splenic atrophy, and lymphoid cell infiltration in the liver and lung, as seen in lpr‐GVHD. In contrast. MRL/+ spleen cell‐injected SCID (SCID‐MRL/+) mice did not show Ipr‐GVHD. The spleens of SCID‐MRL/lpr mice showed progressive increases in donor CD4+ and CD8+ T cells from 4 to 12 weeks after injection and a decrease in B cells at 12 weeks. SCID‐MRL/+ mice showed a stable engraftment of CD4+ and CD8+ T cells and a progressive increase in B cells. Analyses of T cell receptor (TCR) repertoires (Vβ6, Vβ8.1,2 and Vβ11) revealed that the Vβ8.1,2+ T cells were found more frequently in SCID‐MRL/lpr mice than in SCID‐MRL/+ mice. When SCID‐MRL/lpr mice were treated with intraperitoneal injection of an anti‐Vβ8.1,2+ (KJ16) MoAb, Vβ8.1,2+ T cells were markedly depleted, and the severity of/pr‐GVHD was attenuated at 4 and 8 weeks after treatment, in contrast to normal rat IgG‐injected SCID+MRL/lpr mice. However, the KJI6 MoAb‐treated SCID‐MRL/lpr mice suffered from severe lpr‐GVHD 12 weeks after treatment, although Vβ8.1,2+ T cells were still maintained at a low level. These findings suggest that Vβ8.1,2+ T cells are a major T cell population (hat mediates lpr‐GVHD in the early stage of lpr‐GVHD. but that in the later stage, the other T cell populations may proliferate naturally or in accordance with the depletion of Vβ8.1,2+ T cells, and contribute to the development of lpr‐GVHD.

Analyses of lpr-gvhd by adoptive transfer experiments using mrl/lpr-thy-1.1 congenic mice

When MRL/Mp- +/+ (MRL/+) mice are lethally irradiated and then reconstituted with MRL/Mp-lpr/lpr (MRL/lpr) spleen and/or bone marrow cells (BMCs), the mice develop a graft-versus-host disease (GVHD)-like syndrome which is known as lpr-GVHD. We analyzed lpr-GVHD by adoptive transfer experiments using congenic MRL/lpr-Thy-1.1 mice to distinguish the donor and recipient cells. MRL/+ mice were lethally (9.5 Gy) irradiated and then reconstituted with BMCs of MRL/lpr-Thy-1.1 mice treated with anti-Thy-1.1 monoclonal antibody (mAb) plus complement (C). The mice were sacrificed 5 to 6 weeks after bone marrow transplantation (BMT), and the spleen cells were transferred to second recipients. The second recipients (MRL/+ or MRL/lpr mice) were non-irradiated, sublethally (6 Gy) irradiated or lethally (9.5 Gy) irradiated. The lethally irradiated mice were also injected with syngeneic BMCs treated with anti-Thy-1.2 mAb plus C. When whole spleen cells (1 x 10(8) were injected into lethally irradiated MRL/+ mice, the mice showed short survival (1.2-1.5 months) and severe histological changes in the spleen (atrophy and fibrosis), liver (lymphoid infiltration in the Glissons sheath) and lung (lymphoid infiltration around the bronchus and vessel). The sublethally irradiated MRL/+ mice at 2 months after transfer showed histological changes similar to the lethally irradiated MRL/+ recipients, although the former survived more than 3 months, suggesting that histological changes do not reflect on mortality. These GVH-like diseases were not transferable to MRL/lpr mice; they developed autoimmune diseases.(ABSTRACT TRUNCATED AT 250 WORDS)

Transplantability and MHC antigen expression of tumor mast cells

A cloned cell line was established from tumor cells spontaneously developed in a coculture of an autoreactive T cell line (1/+ T1) and 30 Gy-irradiated MRL/+ spleen cells with Con A supernatants. Morphological studies and studies of histamine content and modes of histamine release after stimulation with compound 48/80 revealed that the cell line (MRL-MC3) had mast cell characteristics. MRL-MC3 was transplantable not only to MRL/+, MRL/lpr and AKR/J (H-2k) mice but also to BALB/c and (BALB/c x DBA/2) F1 (H-2d) mice, although the allogeneic mice survived twice as long as syngeneic mice after i.v. injection. In addition, after i.v. injection, the mast cells infiltrated the livers and spleens of syngeneic (MRL/+) mice, however the lymph nodes around the mesenterium to the parapylorus in allogeneic (BALB/c) mice. A mast cell line (BALB-MC) was also established from a lymph node of MRL-MC3-injected BALB/c mice. Cell surface marker analyses revealed clear differences between the BALB-MC and the original MRL-MC3, which was positive for the expression of MHC class I antigens (K, D), I-E antigen and c-abl-encoded (anti-pEX-2 antibody-reactive) proteins, but not for I-A on the cell surface. In contrast, BALB-MC showed positive only for the MHC class I antigens (K, D) on the surface, and also positive for anti-pEX-2 antibody-reactive cytoplasmic proteins, as seen in MRL-MC3. Mast cells obtained from MRL-MC3-injected MRL/+ mice showed the same staining pattern as MRL-MC3. BALB-MC induced shorter survival times (approximately half) in both MRL/+ and BALB/c mice than MRL-MC3.(ABSTRACT TRUNCATED AT 250 WORDS)

Auto-MHC Class II-reactive T cell line obtained from MRL/+ Mice suffering from lpr-GVHD. II. Analyses of functional characteristics of T cell line by in vivo administration

Functional characteristics of an autoreactive (I-Ek-restricted) T cell line (l/+ T1), previously established from MRL/M(p-)+/+(MRL/+) mice with lpr-GVHD, were analyzed in vivo. Intravenous injection of l/+ T1 cells to non-irradiated H-2k (MRL/+ or AKR) mice (but not H-2d mice) induced enhanced spontaneous proliferation of recipient spleen cells; this was also I-Ek self-restricted. This augmented self-reactivity seemed to be mediated by recipient L3T4+ T cells, since few l/+ T1 cells were detected in the spleen cells of l/+ T1-injected AKR mice by cell surface marker analyses, and the treatment of the spleen cells with anti-Thy-1.1 antibody (Ab) or anti-L3T4 Ab plus complement abolished this enhanced spontaneous proliferation. The production of IgM rheumatoid factor (RF) in AKR mice and IgG RF in MRL/+ mice increased, although no enhancement of anti-ssDNA Ab production was observed. Judging from both spleen B cell proportion and serum Ig levels, autoantibody induction by the injection of l/+ T1 cells was not associated with polyclonal B cell activation. When lethally irradiated B10 congenic mice were used as recipients, B10. BR mice showed elevated levels of IgM anti-ssDNA and IgM RF 1 wk after l/+ T1 cell injection; it is likely that lethal irradiation causes autoantigens, particularly DNA, to be exposed. These findings suggest that the autoreactivity of l/+ T1 cells can be transferred to recipient L3T4+ T cells via T-T interaction or the immunological network, and that increased autoreactivity induces autoantibody production in the presence of autoantigen stimulation. In contrast to the stimulatory effects observed in AKR and MRL/+ mice, MRL/Mp-lpr/lpr(MRL/lpr) mice showed a different response to the injection of l/+ T1 cells; spontaneous proliferation of spleen cells and autoantibody production were not enhanced, and suppression of the mitogen responses was observed. It is discussed that lpr-GVHD may be due to these unusual features of MRL/lpr mice.