Satoshi Kusaka

Microchimerism linked to cytotoxic T lymphocyte functional unresponsiveness (clonal anergy) in a tolerant renal transplant recipient.

A patient was found to be functionally tolerant of a maternal kidney allograft as evidenced by good graft function 5 years after cessation of all immunosuppressive drug therapy. Despite normal in vitro proliferative and IL-2 responses, patient anti-donor 1° MLR cultures yielded little donor-specific CTL activity in either bulk or limiting dilution analysis (LDA) cultures. Using polymerase chain reaction, the patients PBL and skin were found to contain donor-derived Bw6+ cells. Removal of Bw6+ donor cells from the patient PBL with mAb and immunomagnetic beads before stimulation with donor PBL on day 0 failed to restore donor-specific CTL in either bulk 1° MLR or LDA cultures. Restimulation of 1° cultures with donor stimulator cells plus exogenous IL-2, however, completely restored anti-donor HLA class I-specific CTL, indicating class I-specific CTL precursors were not clonally deleted. Fresh patient PBL, as well as donor cell-enriched fractions, when added at the initiation of 3° MLR cultures, inhibited the generation of anti-donor CTL, whereas donor cell-depleted fractions did not. The inhibition was cell dose-dependent, was specific for the anti-donor response, and was radioresistant (1200 rad). Thus, the clinical tolerance observed in patients with microchimerism may be due to the presence of veto cells within the circulating donor cell pool.

Tolerance to Noninherited Maternal MHC Antigens in Mice

The phenomenon of tolerance to noninherited maternal Ags (NIMA) is poorly understood. To analyze the NIMA effect C57BL/6 (H-2b/b) males were mated with B6D2F1 (H-2b/d) females, whereby 50% of the offspring are H-2b/b mice that have been exposed to maternal H-2d alloantigens. Controls were H-2b/b offspring of C57BL/6 mothers, either inbred C57BL/6 mice or F1 backcross mice from breedings with H-2b/d fathers. We found that 57% of the H-2b/b offspring of semiallogeneic (H-2b/d) mothers accepted fully allogeneic DBA/2 (H-2d/d) heart grafts for >180 days, while similar transplants were all rejected by day 11 in controls (p < 0.0004). Foster nursing studies showed that both oral and in utero exposure to NIMA are required for this tolerogenic effect. An effect of NIMA was also found to extend the survival of skin grafts from a semiallogeneic donor (p < 0.02). Pretransplant analysis of splenocytes showed a 40–90% reduction of IL-2-, IL-5-, and IFN-γ-producing T cells responding to H-2d-expressing APC in NIMAd-exposed vs control mice. Injection of pregnant BALB/c-dm2 (H-2Ld-negative) female mice i.v. with H-2Ld61–80 peptide profoundly suppressed the offspring’s indirect pathway alloreactive CD4+ T cell response to H-2Ld. These results suggest that the natural exposure of the fetus and newborn to maternal cells and/or soluble MHC Ags suppresses NIMA-allospecific T cells of the offspring, predisposing to organ transplant tolerance in adult mice.

Clonotype Analysis of Human Alloreactive T Cells: A Novel Approach to Studying Peripheral Tolerance in a Transplant Recipient

The recognition of allo-MHC and associated peptides on the surface of graft-derived APC by host T cells (direct pathway allorecognition) plays an important role in acute rejection after organ transplantation. However, the status of the direct pathway T cells in stable long term transplants remains unclear. To detect alloreactive T cell clones in PBL and the allograft during the transplant tolerance, we utilized RT-PCR instead of functional assays, which tend to underestimate their in vivo frequencies. We established alloreactive CD4+ and CD8+ T cell clones from peripheral blood sampled during the stable tolerance phase of a patient whose graft maintained good function for 9 years, 7 without immunosuppression. We analyzed the sequence of TCR Vβ and Vα genes and made clonotype-specific probes that allowed us to detect each clone in peripheral blood or biopsy specimens obtained during a 1-year period before and after the rapid onset of chronic rejection. We found an unexpectedly high level of donor HLA-specific T cell clonotype mRNA in peripheral blood during the late tolerance phase. Strong signals for two CD4+ clonotypes were detected in association with focal T cell infiltrates in the biopsy. Chronic rejection was associated with a reduction in direct pathway T cell clonotype mRNA in peripheral blood and the graft. Our data are inconsistent with the hypothesis that direct pathway T cells are involved only in early acute rejection events and suggest the possibility that some such T cells may contribute to the maintenance of peripheral tolerance to an allograft.

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.

Macrophage colony-stimulating factor inhibits tumor necrosis factor production and prolongs skin graft survival

Background. Despite the availability of a variety of immunosuppressive agents, acute rejection and infection after organ transplantation remain serious problems. Methods and Results. We examined the effect of macrophage colony-stimulating factor (M-CSF) on the production of tumor necrosis factor (TNF) in a Bacille de Calmette Guérin-lipopolysaccharide–challenged mouse model. Both serial and repeated injections of M-CSF inhibited TNF production in a dose-dependent manner. Electrophoretic mobility shift assay showed that M-CSF–induced inhibition of TNF production was a result of suppression of nuclear factor-kappaB. High-dose M-CSF significantly prolonged skin graft survival in mice with orthotopic transplantation compared with the control and low-dose M-CSF groups. The combined administration of low-dose M-CSF and cyclosporine also significantly prolonged graft survival compared with the control and low-dose single agent-treated groups. Conclusions. Our results indicate that M-CSF at a high dose is a potent inhibitor of cytokine production and can potentially be used as an immunosuppressive agent for allograft rejection.

Effects of interleukin 2 receptor b chain (P75)-specific monoclonal antibody on the generation of cytotoxic T lymphocytes and suppressor T cells in mixid lymphocyte culture

The interaction of interleukin 2 (IL-2) with its receptor (IL-2R) plays an essential role in the proliferation and differentiation of T cells. The IL-2R beta-chain is considered to function directly in the intracellular signal transduction. In this study, we investigated using a newly established IL-2R beta-chain-specific monoclonal antibody (MAb) (TU-25) and an IL-2R alpha-chain-specific MAb (H-31). The IL-2-induced proliferation of concanavalin blasts and the mixed lymphocyte reaction (MLR) were suppressed by TU-25 in combination with H-31. This combination had a greater suppressive effect than each of them alone. The generation of cytotoxic T lymphocytes (CTL) using a cell-mediated lympholysis (CML) assay, was not inhibited by TU-25 alone. TU-25 in combination with H-31 suppressed the generation of CTL completely in this assay even if recombinant IL-2 (rIL-2) was added. Although the CTL generation was inhibited, cells that suppressed a fresh MLR were preserved. Our study suggests that the combination of TU-25 with H-31 completely blocks the functional high-affinity binding site of IL-2 but does not inhibit the generation of suppressor cells. This may lead to immunosuppressive therapy using an IL-2R beta-chain-specific MAb in combination with an IL-2R alpha-chain-specific MAb in clinical organ transplantation.

Analysis of suppressor T cells induced by donor-specific transfusion (DST): establishment of a human T cell hybridoma producing an antigen-nonspecific suppressor factor.

Formation of suppressor T cells (Ts) induced by donor-specific transfusion (DST) is one of the most commonly suggested mechanisms for the beneficial effect of DST. In this study, we established a human T cell hybridoma derived from the peripheral blood lymphocytes (PBL) of a DST-treated patient, which produced an antigen-nonspecific suppressor factor. Post-DST PBL were fused with an azaguanine-resistant mutant of a human T cell leukemia cell line, CCRF-CEM(AG). After selection and cloning, we established one clone producing the mixed lymphocyte reaction (MLR) inhibitory factor (C524: 18%-43% suppression). Suppressive activity of the supernatant obtained from C524 after activation by PHA was highly augmented (64%-88% MLR suppression). This factor inhibited MLR dose-dependently in an antigen-nonspecific and HLA non-restricted manner. These results indicated that Ts clones could be generated in patients receiving DST and that the immunoregulatory factors produced by activated clones may play a role in the prolongation of renal allograft survival.