Analysis of therapeutic potential of monocytic myeloid-derived suppressor cells in cardiac allotransplantation.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) are attractive immune cells to induce immune tolerance. To explore a strategy for improving the efficacy of MDSC therapies, we examined the impact of adoptive transfer of several types of MDSCs on graft rejection in a murine heart transplantation model. METHODS: We analyzed the effects of induced syngeneic and allogeneic bone marrow-derived MDSCs (BM-MDSCs) on graft survival and suppressive capacity. We also compared the ability of syngeneic monocytic MDSCs (Mo-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs) to inhibit graft rejection and investigated the suppression mechanisms. RESULTS: Both syngeneic and allogeneic donor- or allogeneic third-party-derived BM-MDSCs prolonged graft survival, although syngeneic BM-MDSCs inhibited anti-donor immune responses most effectively in vitro. Syngeneic Mo-MDSCs, rather than PMN-MDSCs, were responsible for immune suppression through downregulating inducible nitric oxide synthase (iNOS) and expanded naturally occurring thymic originated Treg (nTreg) in vitro. Adoptive transfer of Mo-MDSCs, but not PMN-MDSCs, prolonged graft survival and increased Treg infiltration into the graft heart. CONCLUSION: Recipient-derived Mo-MDSCs are most effective in prolonging graft survival via inhibiting T cell response and nTreg infiltration.

Anti-CD321 antibody immunotherapy protects liver against ischemia and reperfusion-induced injury.

The prognosis of the liver transplant patients was frequently deteriorated by ischemia and reperfusion injury (IRI) in the liver. Infiltration of inflammatory cells is reported to play critical roles in the pathogenesis of hepatic IRI. Although T lymphocytes, neutrophils and monocytes infiltrated into the liver underwent IRI, we found that neutrophil depletion significantly attenuated the injury and serum liver enzyme levels in a murine model. Interestingly, the expression of CD321/JAM-A/F11R, one of essential molecules for transmigration of circulating leukocytes into inflammatory tissues, was significantly augmented on hepatic sinusoid endothelium at 1 h after ischemia and maintained until 45 min after reperfusion. The intraportal administration of anti-CD321 monoclonal antibody (90G4) significantly inhibited the leukocytes infiltration after reperfusion and diminished the damage responses by hepatic IRI (serum liver enzymes, inflammatory cytokines and hepatocyte cell death). Taken together, presented results demonstrated that blockade of CD321 by 90G4 antibody significantly attenuated hepatic IRI accompanied with substantial inhibition of leukocytes infiltration, particularly inhibition of neutrophil infiltration in the early phase of reperfusion. Thus, our work offers a potent therapeutic target, CD321, for preventing liver IRI.

A Clinical Trial With Adoptive Transfer of Ex Vivo-induced, Donor-specific Immune-regulatory Cells in Kidney Transplantation-A Second Report.

BACKGROUND: Although the outcome of kidney transplantation (KTx) has improved, various adverse effects of immunosuppressants and chronic rejection aggravate the long-term prognosis of patients. Therefore, the induction of immune tolerance may be an effective therapeutic strategy. METHODS: A clinical trial aiming at immune tolerance induction was conducted in kidney transplant recipients from HLA mismatched living donors by infusing autologous donor-specific regulatory T cells (Treg). To obtain Treg, recipient's peripheral blood mononuclear cells were cocultured with irradiated donor cells in the presence of anti-CD80/CD86 monoclonal antibody for 2 weeks. For preconditioning, splenectomy + cyclophosphamide (CP) was employed in the first series (group A; n = 9). In group B, splenectomy was substituted by preadministration of rituximab (group B; n = 3). In the latest cases, rituximab + rabbit antithymocyte globulin was administered instead of cyclophosphamide (group C; n = 4). Twelve days after KTx, the cultured cells were intravenously infused, and immunosuppressants were gradually tapered thereafter. RESULTS: Although mixed lymphocyte reaction was remarkably suppressed in a donor-specific fashion, 6 out of 9 patients from group A, 1 out of 3 from group B, and 1 out of 4 from group C developed acute rejection within 1 year after KTx. Complete cessation of immunosuppression was not achieved, and a small dose of immunosuppressants was continued. CONCLUSIONS: The adoptive transfer of autologous ex vivo-expanded Treg is 1 of the options to possibly induce alloimmune hyporesponsiveness. However, in the present study, further regimen optimization is still required and should be the focus of future investigations.

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

UNLABELLED: 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).

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 CD4CD25Foxp3 cell population in 3C10-treated mice. Additionally, IL-2 and interferon-γ 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.

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

BACKGROUND: Interactions 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. METHODS: Naïve 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. RESULTS: CBA 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. CONCLUSION: Our 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.

Danazol induces prolonged survival of fully allogeneic cardiac grafts and maintains the generation of regulatory CD4(+) cells in mice.

Danazol, a derivative of testosterone, is useful for treatment of endometriosis as well as pretreatment for in vitro fertilization and embryo transfer, although its mechanisms of action are unclear. The aim of this study was to investigate the effect of danazol on alloimmune responses in murine heart transplantation. CBA male mice (H2(k) ) underwent transplantation of C57BL/6 male (H2(b) ) hearts and received a single dose of danazol (0.4, 1.2 or 4mg/kg/day) by intraperitoneal injection on the day of transplantation and for 6days thereafter. An adoptive transfer study was performed to determine whether regulatory cells were generated. The median survival time (MST) of allografts in danazol-treated (1.2 and 4mg/kg/day) mice was 28 and 63days, respectively, compared with 7days in untreated mice. Moreover, secondary CBA recipients given whole splenocytes or CD4(+) cells from primary danazol-treated (4mg/kg/day) CBA recipients 30days after transplantation had prolonged allograft survival (MSTs, 29 and 60days, respectively). Cell proliferation, interleukin (IL)-2 and interferon-γ were suppressed in danazol-treated mice, whereas IL-4 and IL-10 were up-regulated. Moreover, danazol directly suppressed allo-proliferation in a mixed leukocyte culture. Flow cytometry showed an increased CD4(+) CD25(+) Foxp3(+) cell population in splenocytes from danazol-treated mice. Danazol prolongs cardiac allograft survival and generates regulatory CD4(+) cells.

Prolongation of renal allograft survival by anergic cells: advantages and limitations.

Even in the era of pharmacological calcineurin inhibitors, a current major challenge in organ transplantation remains the development of immunosuppressive regimens that protect against rejection. One potentially effective procedure is the use of donor-specific anergic T cells generated ex vivo and adoptively transferred back into the recipient after transplantation. In our own work, we first investigated the effect of anergic cells on the prolongation of graft survival in non-human primates. In six animals, half of the recipients survived for over one yr (all animals died or were killed within eight yr). The cause of death was acute renal failure because of cellular rejection (one), uncontrolled bleeding after renal biopsy (two), hydronephrosis probably because of ureteral stenosis (one), and chronic rejection (one). The remaining animal was killed at the end of the study. No infection, malignancy, or signs of graft versus host disease (GVHD) was observed in any of these monkeys. Except for the one animal that died of acute cellular rejection, there was no evidence of tubular infiltration by mononuclear cells, glomerular damage, or parenchymal necrosis. In all animals surviving for more than one yr, a mild grade of interstitial fibrosis, an increase in mesangial matrix, or glomerulopathy was noted. In two of three monkeys, no vascular narrowing of the luminal area caused by fibrointimal thickening of arteries was noted, and arteriosclerotic change was dominant. In this chapter, we summarize the efficacy and limitations of our strategy.

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 study's 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.

Role of the CTLA4 pathway in hyporesponsiveness induced by intratracheal delivery of alloantigen.

BACKGROUND: The authors previously reported that intratracheal delivery (ITD) of donor alloantigen induced donor-specific hyporesponsiveness to C57BL/10 cardiac allografts in CBA recipients and that blockade of the B7 pathways abrogated that hyporesponsiveness. In this study, the authors used a CD28-deficient model to evaluate which signal, either through CD28 or cytotoxic T-lymphocyte-associated antigen (CTLA4), is involved in the induction of hyporesponsiveness. METHODS: Seven days before transplantation of hearts from C3H/HeJ (H2k) mice into C57BL/6 (H2b) or CD28-deficient (C57BL/6 background) mice, the transplant recipients were given ITD of donor splenocytes (1 x 10(7)), alone or in combination with human CTLA4-immunoglobulin (Ig) (200 microg). RESULTS: ITD of C3H splenocytes induced donor-specific hyporesponsiveness to C3H cardiac grafts in C57BL/6 recipients (graft median survival time [MST], 40 days). Administration of CTLA4-Ig concurrently with ITD abrogated the prolonged allograft survival (MST, 12 days). Interestingly, ITD of C3H splenocytes induced prolonged survival of C3H allografts in CD28-deficient recipients (MST, 55 days). Furthermore, administration of CTLA4-Ig combined with ITD of C3H splenocytes abrogated the prolonged survival of C3H allografts in CD28-deficient recipients (MST, 7 days), whereas recipients given isotype-control antibody in combination with ITD of splenocytes had prolonged survival of C3H allografts (MST, 58 days). CONCLUSIONS: Taken together, the authors' findings indicate that a signal through CTLA4, rather than through CD28, plays an important role in the induction of hyporesponsiveness by ITD of alloantigen in this model.

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 (H2 ) mice, donor splenocytes were given by IT delivery 7 days before transplantation of a C57BL/10 (H2 ) 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.