Takenori Kawamura

Chronic human skin graft rejection in severe combined immunodeficient mice engrafted with human PBL from an HLA-presensitized donor.

Mice with severe combined immunodeficiency (C.B-17 scid [SCID]) accepted xenografts of adult human peripheral blood leukocytes injected intraperitoneally as evidenced by production of human immunoglobulin (IgG and IgM), and circulation of human leukocytes in peripheral blood. SCID mice also accepted human split-thickness skin xenografts. Passenger leukocytes present in small numbers in such skin grafts could also recirculate in host peripheral blood and make detectable levels of human immunoglobulin. To test the immunocompetence of the transferred human PBL, SCID mice received a human skin xenograft from a second donor (HLA-mismatched with the PBL donor) either before (n = 6) or after (n = 23) xenografting of PBL. Skin was monitored daily for signs of rejection, and rejection was scored by histology 3-4 weeks after the second graft (PBL or skin) was placed. Of 19 SCID injected with PBL from an HLA presensitized patient (L.G.), 7/19 (37%) rejected a subsequent HLA-mismatched skin xenograft. Two of six SCID (33%) rejected a previously established skin xenograft when PBL were administered afterward. The rejection of the human skin was chronic, of relatively late onset (3-4 weeks), and was characterized grossly by contraction, glassy surface, and thickening. Histopathologic examination showed lymphocyte infiltration into the dermis with endothelial cell cuffing and destruction of capillaries, as well as lymphocyte tagging of the basal epidermis, hyperkeratosis, lymphocyte exocytosis and single epidermal cell necrosis. Immunostaining with monoclonal antibody to human CD2 or mouse CD3 revealed that human, but not mouse T lymphocytes were tagging the dermis/epidermis junction and infiltrating the epidermis of rejecting skin grafts. We conclude that a form of human skin graft rejection may be reproduced in an SCID mouse. The immune status of the transferred cells (sensitized vs. normal) and the lymphocytes ability to recirculate in SCID peripheral blood appear to be factors limiting the rejection process.

DONOR-SPECIFIC CYTOTOXIC T LYMPHOCYTE HYPORESPONSIVENESS FOLLOWING RENAL TRANSPLANTATION IN PATIENTS PRETREATED WITH DONOR-SPECIFIC TRANSFUSIONS

Our purpose was to investigate the mechanism of the continuing beneficial effect of donor-specific transfusions in the cyclosporine era. We describe the development of donor-specific cytotoxic T lymphocyte hyporesponsiveness in peripheral blood lymphocytes obtained up to 2 years posttransplant in patients preconditioned with 3 DST plus azathioprine. In a group of 12 such patients, hyporesponsiveness developed gradually, becoming detectable in some patients as early as 1 month posttransplant and becoming statistically significant for the entire group at 9-12 months posttransplant. A complete specificity for donor alloantigens was seen in the hyporesponsiveness of some patients; in others, partial suppression of the response to a third party HLA-mismatched control was also seen. Although slight suppression of the mixed lymphocyte culture response was seen in some patients, overall there were no statistically significant differences in MLC responses to control or donor stimulators at any time point posttransplant as compared with pretransplant, pre-DST. The mechanism of donor-specific CTL hyporesponsiveness 2 years posttransplant was explored in one patient (HLA A1, 2, B 57, 60; DR 3, 6) who had received a 2-HLA haplotype-mismatched kidney transplant from her husband (HLA A2,--; B5, 8; DR4,--) following DST plus AZA pretreatment. Bulk culture CTL analysis showed specific nonresponsiveness to donor stimulators; however in the presence of exogenous recombinant IL-2, the antidonor response was restored to the level of pretransplant PBL. Limiting dilution analysis using recombinant IL-2 revealed equivalent precursor frequency of antidonor CTL in pre- and posttransplant PBL. These data suggest that the hyporesponsive PBL contained donor-specific CTL precursors but were deficient in helper function necessary for CTL maturation.

Induction of antiidiotypic antibodies by donor-specific blood transfusions. Establishment of a human-mouse hybridoma secreting the MLR-inhibiting factor

We describe a patient transfused with 200 ml of donor fresh whole blood three times at 2-week intervals. Three weeks after the last transfusion, transplantation and splenectomy were done at the same time. Splenic cells from this DST pretreated patient were fused with murine myeloma cells (X63-Ag8, 653). With DST pretreatment, various clones were developed in vivo, and finally 69 human immunoglobulin-secreting clones were obtained. Modulation of the alloantigen-specific MLR by supernatants from 69 clones showed various degrees of suppression or augmentation. The hybridoma clone 7 and clone 2, which had been secreting IgG antibody for more than 6 months, showed some degree of suppression in the alloantigen-specific MLR (mean suppression = 63%, 46% respectively). According to the result of MLR, clone 7 antibody was directed against recipient lymphocytes and clone 2 antibody was against donor lymphocytes. Immunoprecipitation was carried out by clone 7-IgG and clone 2-IgG. Clone 7-IgG specifically precipitated 1 molecule from the recipient lymphocyte with a molecular weight of 120 KD, similar to the molecular weight range reported for T cell receptors. Clone 2-IgG precipitated a 20 KD molecule from the donor lymphocyte. The data suggest that DST induces antibodies directed against the blood donor alloantigen-specific receptors on the recipients T lymphocytes--and, at the same time, induces antibodies against donor lymphocyte antigens. These antibodies may be essential to prolongation of kidney allograft survival following DST.

Expression of the interleukin 2 receptor beta chain (p75) in renal transplantation--applicability of anti-interleukin-2 receptor beta chain monoclonal antibody.

The interaction of interleukin 2 with specific cellular receptors plays an essential role in the allostimulated proliferation and differentiation of T cells. Recent chemical linking studies have demonstrated that the human high-affinity IL-2 receptor is a membrane complex composed of at least two distinct subunits, which are the p55 (alpha-chain) and p75 (beta-chain) subunits. The IL-2R beta chain is supposed to play a role in the signal transduction of IL-2, but the exact mechanism is still unknown. In this study, we investigated the effects of a newly established anti-IL-2R beta chain monoclonal antibody (MoAb, TU-27) on the induction of cytotoxic T lymphocytes (CTLs) using the cell-mediated lympholysis (CML) assay. TU-27 in combination with H-31, a MoAb directed against the IL-2R alpha chain, produced inhibition of cytotoxicity, while TU-27 alone could not inhibit cytotoxicity, while TU-27 alone could not inhibit cytotoxicity at any concentration. TU-27 plus H-31 prevented the expansion of CD4+ cells and CD8++ cells in mixed lymphocyte culture (MLC). Furthermore, we examined the serial changes in the expression of the IL-2R beta chain on peripheral blood lymphocytes from renal transplant recipients using two-color immunofluorescence flow cytometry, so as to investigate correlations between IL-2R beta chain expression and the occurrence of allograft rejection. Here, we report that the IL-2R beta chain is expressed on CD4-positive (CD4+) cells and strongly CD8-positive (CD8(+)+) cells in association with acute rejection, indicating that IL-2R beta chain expression appears to increase on alloreactive T cells.

Immunosuppressive mechanism of 15-deoxyspergualin on sinusoidal lining cells in swine liver transplantation: Suppression of MHC class II antigens and interleukin-1 production

To elucidate the precise mechanism of action of 15-deoxyspergualin (DSG) in swine liver transplantation, the expression of MHC class II antigens (Ia) on hepatic sinusoidal lining cells (SLC) and their production of interleukin-1 (IL-1) were examined. In our previous study, we isolated sinusoidal endothelial cells (SEC) and Kupffer cells (KC) by enzymatic digestion and centrifugal elutriation, and demonstrated that both SEC and KC present alloantigens effectively and generated IL-1 in response to allogenic or lipopolysaccharide stimulation. Animals were divided into three groups: group 1, nontransplanted normal controls (n = 3); group 2, no immunosuppressive treatment following liver transplantation (n = 5); group 3, DSG (0.8 mg/kg/day) intravenously for 7 days following liver transplantation (n = 5). At 1 week after transplantation, the three liver grafts in groups 2 and 3 were processed for the study of Ia expression and IL-1 production on SEC and KC. The expression of Ia was detected in 21.5 +/- 4.7% of SEC and 24.3 +/- 11.1% of KC in group 1. In group 3, Ia expression was suppressed compared with group 2, being 3.6 +/- 2.8% versus 22.0 +/- 2.8% for SEC (P less than 0.02) and 15.5 +/- 11.3% versus 24.3 +/- 7.1% for KC. IL-1 production by SEC and KC was respectively 11,483 +/- 3311 cpm and 9077 +/- 2161 cpm in group 1. In group 3, IL-1 production was inhibited compared with that in group 2, being 7190 +/- 883 cpm versus 19,297 +/- 5182 cpm for SEC (P less than 0.05) and 16,130 +/- 3769 cpm versus 25,857 +/- 3963 cpm for KC.(ABSTRACT TRUNCATED AT 250 WORDS)