Hisashi Bashuda

Requirement for natural killer T (NKT) cells in the induction of allograft tolerance

In this study, we investigated the role of Vα14 natural killer T (NKT) cells in transplant immunity. The ability to reject allografts was not significantly different between wild-type (WT) and Vα14 NKT cell-deficient mice. However, in models in which tolerance was induced against cardiac allografts by blockade of lymphocyte function-associated antigen-1/intercellular adhesion molecule-1 or CD28/B7 interactions, long-term acceptance of the grafts was observed only in WT but not Vα14 NKT cell-deficient mice. Adoptive transfer with Vα14 NKT cells restored long-term acceptance of allografts in Vα14 NKT cell-deficient mice. The critical role of Vα14 NKT cells to mediate immunosuppression was also observed in vitro in mixed lymphocyte cultures in which lymphocyte function-associated antigen-1/intercellular adhesion molecule-1 or CD28/B7 interactions were blocked. Experiments using IL-4- or IFN-γ-deficient mice suggested a critical contribution of IFN-γ to the Vα14 NKT cell-mediated allograft acceptance in vivo. These results indicate a critical contribution of Vα14 NKT cells to the induction of allograft tolerance and provide a useful model to investigate the regulatory role of Vα14 NKT cells in various immune responses.

A pilot study of operational tolerance with a regulatory T‐cell‐based cell therapy in living donor liver transplantation

Potent immunosuppressive drugs have significantly improved early patient survival after liver transplantation (LT). However, long‐term results remain unsatisfactory because of adverse events that are largely associated with lifelong immunosuppression. To solve this problem, different strategies have been undertaken to induce operational tolerance, for example, maintenance of normal graft function and histology without immunosuppressive therapy, but have achieved limited success. In this pilot study, we aimed to induce tolerance using a novel regulatory T‐cell‐based cell therapy in living donor LT. Adoptive transfer of an ex vivo‐generated regulatory T‐cell‐enriched cell product was conducted in 10 consecutive adult patients early post‐LT. Cells were generated using a 2‐week coculture of recipient lymphocytes with irradiated donor cells in the presence of anti‐CD80/86 monoclonal antibodies. Immunosuppressive agents were tapered from 6 months, reduced every 3 months, and completely discontinued by 18 months. After the culture, the generated cells displayed cell‐number‐dependent donor‐specific inhibition in the mixed lymphocyte reaction. Infusion of these cells caused no significant adverse events. Currently, all patients are well with normal graft function and histology. Seven patients have completed successful weaning and cessation of immunosuppressive agents. At present, they have been drug free for 16‐33 months; 4 patients have been drug free for more than 24 months. The other 3 recipients with autoimmune liver diseases developed mild rejection during weaning and then resumed conventional low‐dose immunotherapy. Conclusions: A cell therapy using an ex vivo‐generated regulatory T‐cell‐enriched cell product is safe and effective for drug minimization and operational tolerance induction in living donor liver recipients with nonimmunological liver diseases. (Hepatology 2016;64:632‐643)

Renal allograft rejection is prevented by adoptive transfer of anergic T cells in nonhuman primates

Anergic T cells generated ex vivo are reported to have immunosuppressive effects in vitro and in vivo. Here, we tested this concept in nonhuman primates. Alloreactive T cells were rendered anergic ex vivo by coculture with donor alloantigen in the presence of anti-CD80/CD86 mAbs before adoptive transfer via renal allograft to rhesus monkey recipients. The recipients were briefly treated with cyclophosphamide and cyclosporine A during the preparation of the anergic cells. Thirteen days after renal transplantation, the anergic T cells were transferred to the recipient, after which no further immunosuppressive agents were administered. Rejection-free survival was prolonged in all treated recipients, and 3 of 6 animals survived long term (410-880 days at studys end). In the long-surviving recipients, proliferative responses against alloantigen were inhibited in a donor-specific manner, and donor-type, but not third-party, skin allografts were also accepted, which demonstrated that antigen-specific tolerance had been induced. We conclude that anergic T cells generated ex vivo by blocking CD28/B7 costimulation can suppress renal allograft rejection after adoptive transfer in nonhuman primates. This strategy may be applicable to the design of safe clinical trials in humans.

B7/CTLA4 pathway is essential for generating regulatory cells after intratracheal delivery of alloantigen in mice.

Background. The mechanism of hyporesponsiveness induced by intratracheal (IT) delivery of alloantigen was examined and its effect on cardiac graft survival was assessed in studies in mice. Methods. In CBA (H2k) mice, donor splenocytes were given by IT delivery 7 days before transplantation of a C57BL/10 (H2b) heart. To determine whether regulatory cells were involved in hyporesponsiveness, splenocytes from mice given IT delivery of alloantigen and antibodies for B7–1, B7–2, or CTLA4 were adoptively transferred to naïve secondary recipients 7 days after delivery; those recipients underwent heart transplantation the same day. Effects on cell proliferation and cytokine production of splenocytes from mice given IT delivery of alloantigen were examined in mixed leukocyte cultures (MLC). Results. Cardiac graft survival was significantly prolonged in mice given IT delivery of alloantigen (median survival time [MST], 81 days); those given syngeneic splenocytes rejected grafts acutely (MST, 7 days;P <0.05). Adoptive transfer of splenocytes also significantly prolonged survival of cardiac grafts in secondary recipients (MST, 62 days). When B7–1, B7–2, or CTLA4 antibody was combined with IT delivery of alloantigen in the first recipient, all grafts were rejected within 14 days in second recipients after adoptive transfer. In mixed leukocyte cultures, splenocytes from these mice did not respond to alloantigen and production of interleukin-4 and interleukin-10 was increased. Conclusions. Donor splenocytes delivered IT induced hyporesponsiveness and regulatory cells in our animal model, and such induction was dependent on B7–1, B7–2, and CTLA4 signals.

A crucial role of host CD80 and CD86 in rat cardiac xenograft rejection in mice.

BACKGROUND Graft rejection can be initiated by two primary pathways of antigen presentation: (a) direct activation of host T cells by donor-derived antigen presenting cells (APC) and (b) indirect presentation of processed graft antigens by host APC. METHODS We investigated the differential roles for direct and indirect antigen presentation by preventing the CD28 costimulatory pathway with monoclonal antibodies to rat or mouse CD80 and CD86 in a rat-to-mouse cardiac transplantation model. RESULTS Although the mouse anti-rat monoclonal antibodies to CD80 and CD86 did not significantly prolong the survival of rat cardiac xenografts in mice, the rat anti-mouse monoclonal antibodies to CD80 and CD86 did prolong the survival. Development of the anti-donor antibodies was inhibited, and the deposition of C3, IgM, and IgG on endothelium in the xenografts was mild in the anti-mouse CD80/CD86-treated mice. Infiltration of macrophages, neutrophils, and lymphocytes expressing perforin and interferon-gamma was decreased by the anti-mouse CD80/CD86 treatment. CONCLUSIONS These findings suggest that the indirect antigen presentation, which is mediated by CD80 and CD86 pathway on host APC, plays a crucial role in concordant cardiac xenograft rejection.

Induction of hyporesponsiveness to fully allogeneic cardiac grafts by intratracheal delivery of alloantigen.

Background. Soluble protein delivered through the mucosal surface can induce immunological unresponsiveness. The purpose of this study was to determine if prior exposure to alloantigen via the trachea could modulate the immune response to subsequent cardiac allografts. Methods. Hearts from C57BL/10(H2b) mice were transplanted into CBA(H2k) recipients. Recipient mice were given donor 1×107 splenocytes into the trachea with or without antibody specific for mouse CD80 (1G10) and/or CD86 (GL1) (100 &mgr;g each) 7 days before transplantation. Results. All grafts survived in recipients treated with intratracheal delivery of alloantigen for over 35 days (mean survival time [MST], 56 days), whereas naive control mice and mice treated with syngeneic antigen rejected grafts acutely (MST, 8 and 7 days, respectively). Interestingly, when 1G10, GL1, or both of them were combined with the protocol, the majority of grafts were rejected within 21 days after grafting (MST, 7, 15, and 17 days, respectively). Conclusion. Intratracheal delivery of alloantigen induced significantly prolonged survival of fully mismatched cardiac allografts and the effect was abrogated by the blockade CD80 and/or CD86 pathway.

Auditory stimulation of opera music induced prolongation of murine cardiac allograft survival and maintained generation of regulatory CD4+CD25+ cells

BackgroundInteractions between the immune response and brain functions such as olfactory, auditory, and visual sensations are likely. This study investigated the effect of sounds on alloimmune responses in a murine model of cardiac allograft transplantation.MethodsNaïve CBA mice (H2k) underwent transplantation of a C57BL/6 (B6, H2b) heart and were exposed to one of three types of music--opera (La Traviata), classical (Mozart), and New Age (Enya)--or one of six different single sound frequencies, for 7 days. Additionally, we prepared two groups of CBA recipients with tympanic membrane perforation exposed to opera for 7 days and CBA recipients exposed to opera for 7 days before transplantation (pre-treatment). An adoptive transfer study was performed to determine whether regulatory cells were generated in allograft recipients. Immunohistochemical, cell-proliferation, cytokine, and flow cytometry assessments were also performed.ResultsCBA recipients of a B6 cardiac graft that were exposed to opera music and Mozart had significantly prolonged allograft survival (median survival times [MSTs], 26.5 and 20 days, respectively), whereas those exposed to a single sound frequency (100, 500, 1000, 5000, 10,000, or 20,000 Hz) or Enya did not (MSTs, 7.5, 8, 9, 8, 7.5, 8.5 and 11 days, respectively). Untreated, CBA mice with tympanic membrane perforations and CBA recipients exposed to opera for 7 days before transplantation (pre-treatment) rejected B6 cardiac grafts acutely (MSTs, 7, 8 and 8 days, respectively). Adoptive transfer of whole splenocytes, CD4+ cells, or CD4+CD25+ cells from opera-exposed primary allograft recipients resulted in significantly prolonged allograft survival in naive secondary recipients (MSTs, 36, 68, and > 100 days, respectively). Proliferation of splenocytes, interleukin (IL)-2 and interferon (IFN)-γ production was suppressed in opera-exposed mice, and production of IL-4 and IL-10 from opera-exposed transplant recipients increased compared to that from splenocytes of untreated recipients. Flow cytometry studies showed an increased CD4+CD25+ Forkhead box P3 (Foxp3)+ cell population in splenocytes from those mice.ConclusionOur findings indicate that exposure to opera music, such as La traviata, could affect such aspects of the peripheral immune response as generation of regulatory CD4+CD25+ cells and up-regulation of anti-inflammatory cytokines, resulting in prolonged allograft survival.

An agonistic anti-BTLA mAb (3C10) induced generation of IL-10-dependent regulatory CD4+ T cells and prolongation of murine cardiac allograft.

Background The co-inhibitory receptor B and T lymphocyte attenuator (BTLA) has been implicated in the regulation of autoimmunity and may potentially play an important role in allograft tolerance. This study investigated the effect of an agonistic anti-BTLA mAb (3C10) in the fully major histocompatibility complex–mismatched murine cardiac transplantation. Methods CBA mice underwent transplantation of C57BL/6 hearts and received one dose of 3C10 on the day of transplantation (day 0) or four doses of 3C10 on day 0, 3, 6, and 9. Adoptive transfer studies were performed to determine whether regulatory cells were generated. Moreover, to confirm the requirement for regulatory T cell and Th-2 cytokines, anti-interleukin (IL)-2 receptor alpha antibody (PC-61) or anti-IL-10 antibody (JES-2A5) was administered to a 3C10-treated CBA recipient. Results CBA mice treated with one and four doses of 3C10 prolonged allograft survival (median survival times [MSTs], 43 and >100 days, respectively). Secondary CBA recipients given whole splenocytes or CD4+ cells from primary 3C10-treated CBA recipients had significantly prolonged survival of C57BL/6 hearts (MSTs, >100 in both). Also, flow cytometry studies showed an increased CD4+CD25+Foxp3+ cell population in 3C10-treated mice. Additionally, IL-2 and interferon-&ggr; production were suppressed in 3C10-treated mice, and IL-4 and IL-10 from 3C10-treated CBA mice increased. Moreover, 3C10 directly suppressed alloproliferation in a mixed leukocyte culture. However, administration of PC-61 or JES-2A5 clearly attenuated prolonged survival of 3C10-treated mice (MSTs, 15.5 and 13.5 days, respectively). Conclusion 3C10 could control acute rejection by its suppressive effect on alloreactive T cells and induction of IL-10-dependent regulatory CD4+ T cells.

Induction of persistent allograft tolerance in the rat by combined treatment with anti-leukocyte function-associated antigen-1 and anti-intercellular adhesion molecule-1 monoclonal antibodies, donor-specific transfusion, and FK506

We previously reported that a short course of treatment with anti-LFA-1 and anti-ICAM-1 monoclonal antibodies (mAbs) led to a persistent acceptance of mouse cardiac allografts, which resulted from the induction of allospecific tolerance. In the present study, we tested the effect of anti-LFA-1 and anti-ICAM-1 mAbs on rat allograft rejection and analyzed the mechanisms underlying allograft tolerance. In sharp contrast to the mouse case, a short course of treatment with anti-LFA-1 and anti-ICAM-1 mAbs led to a persistent acceptance in only half of the treated rats when MHC was compatible but mismatched for minor antigens, and was virtually ineffective when MHC was fully incompatible. However, treatment with these mAbs combined with donor-specific transfusion and FK506 consistently led to a persistent acceptance, even when the MHC was fully incompatible. Donor-specific tolerance was induced by this treatment, as estimated by skin challenging. In the tolerant rats, proliferative response and CTL generation against donor-type alloantigen were severely impaired but partially restored by exogenous interleukin-2. Limiting dilution analysis demonstrated that the precursor frequency of CTL was decreased in the tolerant rats, as compared with the naive rats. These results suggest that donor-reactive T cells were partially deleted and rendered anergic in the periphery.

Prevention of fetal bowel allograft rejection by combined treatment with anti-ICAM-1 and anti-LFA-1 antibodies.

Prevention and treatment of allograft rejection remain the major issues in clinical small bowel transplantation. New strategies for manipulating immune responses using more powerful immunosuppressive agents continue to be evaluated. The fetal small bowel from BALB/c (H-2d) or C3H/He (H-2k) mice was transplanted into the space between the peritoneum and rectus abdominis of adult C3H/He (H-2k) recipient mice. Syngeneic (n = 6) and allogeneic transplant groups were made. In the allogeneic group, the recipient mice were subdivided into three groups, depending on the duration of combined treatment with anti-LFA-1 and anti-ICAM-1 monoclonal antibodies (MAbs): untreated (n = 10), 7-day course (n = 10), and 4-week course (n = 14). A dose of 50 micrograms/mouse/d each of both MAbs was given intraperitoneally, immediately after transplantation and on the consecutive days. All mice were killed 4 weeks after transplantation, and the graft as well as the recipient spleen were taken for histological examination, graft survival ratio, mixed lymphocyte reaction (MLR) assay and cytotoxic T lymphocyte (CTL) assay. All grafts in the syngeneic group survived with normal villi, whereas all grafts in the allogeneic group without treatment disappeared within 4 weeks. All grafts in the allogeneic group with a 7-day course of MAb treatment showed marked disruption of the mucosa with massive cellular infiltration. However, in the allogeneic group treated for 4 weeks, all allografts had adequate growth and demonstrated normal villi with minimal cellular infiltration. Splenocytes from allografted recipient mice without MAb treatment showed markedly increased MLR and CTL activity, compared with the activity seen in the syngeneic MLR and CTL.(ABSTRACT TRUNCATED AT 250 WORDS)