Toshihito Hirai

Chronic Antibody-Mediated Rejection Is Reduced by Targeting B-Cell Immunity During an Introductory Period

Transplantation across blood group antigen and human leukocyte antigen (HLA) barriers are immunologically high risk. Both splenectomy and rituximab injection were developed to overcome those immunological barriers. The idea behind these treatments is to control B‐cell immunity before and after renal transplantation and antibody production. Between January 2001 and December 2004, recipients underwent pretransplant double‐filtration plasmapheresis (DFPP) and splenectomy at the time of transplantation in the ABO‐incompatible group (ABO‐I‐SPX; n= 45). From January 2005 to June 2009, a low dose of rituximab was given as an alternative to splenectomy (ABO‐I‐RIT; n = 57). As a control group, we selected 83 cases of ABO‐C living‐donor kidney transplantation between January 2001 and December 2007 (ABO‐C). We compared the graft survival rate and chronic antibody‐mediated rejection (C‐AMR) rate between ABO‐C and ABO‐I kidney transplantation with induction treatment. C‐AMR rates 2 years after the operation were 8.8, 3.5 and 28.9%, and de novo donor‐specific anti‐HLA antibody (DSHA) positive rates were 2.2, 1.7 and 18.1% in the ABO‐I‐SPX, ABO‐I‐RIT and ABO‐C groups, respectively. The ABO‐C group showed the highest rate of C‐AMR and de novo DSHA. B‐cell depletion protocols, such as splenectomy or rituximab administration, reduced C‐AMR after kidney transplantation.

Analysis of predictive and preventive factors for de novo DSA in kidney transplant recipients.

Background Development of de novo donor-specific anti-HLA antibodies (dnDSA) has been associated with poor graft outcome, although the preventive factor for its production is still elusive. We analyzed the incidence of dnDSA within 5 years posttransplant in 562 living-kidney transplant recipients to evaluate predicting and preventive factors for dnDSA development. Materials and Methods All patients were considered to be non-HLA sensitized, as determined by the preoperative single-antigen bead assay (SABA), although they included various ABO blood type compatibilities. Preoperative administration of rituximab was indicated for 48% of patients, mainly for ABO incompatible transplantation. We retrospectively compared the patients with dnDSA and those without. Results Development of dnDSA was observed in 27 of the total 562 patients (5%). Chronic rejection was more frequently observed in patients with dnDSA than in those without (41% vs. 6%, P<0.001). The dnDSA-positive patients showed decreased graft function and poorer graft survival rates than those who tested negative. In multivariate analysis, higher likelihood of dnDSA production was observed in male recipients (odds ratio 6.57, P=0.012) and patients with a higher number of HLA-DR mismatches (odds ratio 2.41, P=0.008), whereas lower likelihood was observed in patients treated with rituximab induction (odds ratio 0.33, P=0.040). Conclusion Results suggest that rituximab induction as a standard immunosuppression protocol may have a preventive effect for dnDSA production in the non-HLA sensitized low immunologic risk patients.

Significance of low‐level DSA detected by solid‐phase assay in association with acute and chronic antibody‐mediated rejection

We sought to clarify the controversial issue of whether detecting low‐level anti‐donor‐specific HLA antibody (HLA‐DSA) by single‐antigen flow‐bead assay (SAFB) may have a potential role in reducing acute and chronic antibody‐mediated rejection (AMR). We retrospectively studied the preoperative serum of ABO‐compatible living kidney transplantation recipients transplanted between 2001 and 2004 by SAFB using a Luminex platform. HLA‐DSA was detected only by SAFB in 24 patients, although all of them showed negative T‐cell and B‐cell complement‐dependent cytotoxicity (CDC) crossmatches. The HLA‐DSA patients went on to have surprisingly high levels of acute and chronic AMR despite being only weakly sensitized (acute AMR, 33.3%; chronic AMR, 41.7%). After 2005, we implemented SAFB routinely and any patient having a positive HLA‐DSA was considered to be a desensitization candidate. The 52 patients found to have HLA‐DSA underwent kidney transplantation after prior treatment with a single dose of rituximab (RIT) and three or four sessions of double‐filtration plasmapheresis (DFPP) in addition to regimens commonly used between 2001 and 2004. After 2005, there was a significant reduction in the occurrence of acute and chronic AMR (acute AMR, 4.7%, P < 0.001; chronic AMR, 4.7%, P < 0.001). The 5‐year graft survival rate also improved after implementing SAFB (83.3–98.1%, P = 0.032). The RIT/DFPP‐induction protocol may improve graft survival even in patients with low‐level DSA.

Retroperitoneoscopic Living Donor Nephrectomy: Experience of 425 Cases at a Single Center

BACKGROUND AND PURPOSE Laparoscopic living donor nephrectomy (LLDN) is a standard method of donor nephrectomy. Most cases of LLDN are transperitoneal. Retroperitoneal access, however, implies a direct approach to the retroperitoneal organs without interfering with any of them. Since 2001, we have been trying to establish the technique of retroperitoneoscopic live donor nephrectomy (RPLDN). To assess the safety, feasibility, and usefulness of RPLDN, we reviewed the experience with this technique at our institution. PATIENTS AND METHODS From July 2001 to March 2009, 425 patients underwent live donor renal transplantation at our institution with allografts procured by RPLDN. Study variables included operative time, time to retrieval of the kidney, blood loss, warm ischemia time, length of hospital stay, number and length of renal vessels, graft function, and complications. RESULTS Mean follow-up was 53 months. Donor nephrectomy was performed successfully in all patients. The complication rate was 4.9%. In one case, the procedure was changed to open donor nephrectomy because of severe adhesion in the renal hilum from previous surgery. Ureteral complications occurred in four patients, who were successfully treated with retrograde ureteral stent placement. None of the donors needed readmission. Mean warm ischemia time was 4.8 minutes. Creatinine levels returned to normal in all patients, and long-term allograft function was good. Serum creatinine levels at postoperative days 1, 7, and 14 were 3.7 mg/dL, 1.4 mg/dL, and 1.4 mg/dL on average, respectively. Slow graft function was noted in four (1.1%) cases but returned to the normal level within 2 weeks after surgery. One-year donor survival was 99%, and 1-year graft survival was 98.2%. CONCLUSIONS Excellent donor safety and allograft function were obtained with RPLDN. These results suggest that RPLDN could be an option for LLDN.

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.

A Novel Approach Inducing Transplant Tolerance by Activated Invariant Natural Killer T Cells With Costimulatory Blockade

Invariant natural killer T (iNKT) cells are one of the innate lymphocytes that regulate immunity, although it is still elusive how iNKT cells should be manipulated for transplant tolerance. Here, we describe the potential of a novel approach using a ligand for iNKT cells and suboptimal dosage of antibody for CD40–CD40 ligand (L) blockade as a powerful method for mixed chimerism establishment after allogenic bone marrow transplantation in sublethally irradiated fully allo recipients. Mixed‐chimera mice accepted subsequent cardiac allografts in a donor‐specific manner. High amounts of type 2 helper T cytokines were detected right after iNKT cell activation, while subsequent interferon‐gamma production by NK cells was effectively inhibited by CD40/CD40L blockade. Tolerogenic components, such as CD11clowmPDCA1+ plasmacytoid dendritic cells and activated regulatory T cells (Tregs) expressing CD103, KLRG‐1 and PD‐1, were subsequently augmented. Those activating Tregs seem to be required for the establishment of chimerism because depletion of the Tregs 1 day before allogenic cell transfer resulted in a chimerism brake. These results collectively suggest that our new protocol makes it possible to induce donor‐specific tolerance by enhancement of the innate ability for immune tolerance in place of the conventional immunosuppression.

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 (H2k) underwent transplantation of C57BL/6 male (H2b) hearts and received a single dose of danazol (0.4, 1.2 or 4 mg/kg/day) by intraperitoneal injection on the day of transplantation and for 6 days 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 4 mg/kg/day) mice was 28 and 63 days, respectively, compared with 7 days in untreated mice. Moreover, secondary CBA recipients given whole splenocytes or CD4+ cells from primary danazol‐treated (4 mg/kg/day) CBA recipients 30 days after transplantation had prolonged allograft survival (MSTs, 29 and 60 days, 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.

Significance of qualitative and quantitative evaluations of anti-HLA antibodies in kidney transplantation

In this study, we retrospectively investigated the relationship between the presence/titers of donor‐specific (DSA)/nondonor‐specific antibody (NDSA) and the rate of graft rejection after transplantation. The subjects comprised 34 recipients who tested positive by FlowPRA® Screening. The recipients were divided into two groups; 22 recipients with DSA and 12 recipients with NDSA, as detected using FlowPRA® Single Antigen I and II beads. The antibodies were also quantitatively examined using the molecules of equivalent soluble fluorochrome (MESF) method. Nine of the 22 recipients with DSA (9/22, 40%) developed antibody‐mediated rejection (AMR), while none of the 12 recipients with NDSA (0/12, 0%) developed AMR (P < 0.01). In a quantitative analysis of the MESF data, patients with DSA with MESF values of over 3000 frequently showed AMR (8/11, 73%). In contrast, one of the patients with DSA with MESF values of <3000 showed AMR (1/11, 9%). One of the 12 patients (1/12, 8%) with NDSA showed cellular rejection (T‐cell‐mediated rejection), regardless of the MESF values. In patients with DSA, an MESF value of 3000 may be a useful cutoff value for identifying patients at a high risk for AMR.

Comparison of the acute rejection incidence rate in spousal donor transplantation before and after anti-CD20 antibody (rituximab) protocol as desensitization therapy.

Recipients of spousal donor transplantation (SDT) have poorer histocompatibility and higher human leukocyte antigen (HLA) sensitization due to pregnancy than those receiving related donor transplantation (RDT). Thus, SDT carries a higher risk of acute rejection (AR). In our department, patients at a high immunological risk, such as those with ABO incompatibility and HLA sensitization, were considered for desensitization by double filtration plasmapheresis and preoperative administration of rituximab. In this study we compared the AR incidence rates between SDT and RDT according to their immunological risk. We performed RDT in 279 and SDT in 100 patients, a total of 379 cases, between 2000 and 2008; 48.7% of RDT and 67.0% of SDT cases were considered to be at a high immunological risk and underwent preoperative desensitization (P = 0.002). Even though the AR incident rate of SDT was higher than RDT in the low immunological risk group, in which the patients had undergone transplantation without desensitization (RDT 24.4%, SDT 37.0%, P = 0.012), there was no significant difference between the two donor type groups in the high immunological risk group, in which transplantation with desensitization occurred (RDT 21.3%, SDT 31.3%, P > 0.05). Preoperative administration of rituximab significantly reduced AR from 37.4% to 10.6% (P < 0.001), especially T‐cell mediated rejection (36.4% to 20.2%, P = 0.01). SDT no longer carries a high risk when appropriate desensitization, including the use of rituximab, is performed. Overall, the five‐year graft survival rates were similar between RDT and SDT.

Clonal Deletion Established via Invariant NKT Cell Activation and Costimulatory Blockade Requires In Vivo Expansion of Regulatory T Cells

Recently, the immune‐regulating potential of invariant natural killer T (iNKT) cells has attracted considerable attention. We previously reported that a combination treatment with a liposomal ligand for iNKT cells and an anti‐CD154 antibody in a sublethally irradiated murine bone marrow transplant (BMT) model resulted in the establishment of mixed hematopoietic chimerism through in vivo expansion of regulatory T cells (Tregs). Herein, we show the lack of alloreactivity of CD8+T cells in chimeras and an early expansion of donor‐derived dendritic cells (DCs) in the recipient thymi accompanied by a sequential reduction in the donor‐reactive Vβ‐T cell receptor repertoire, suggesting a contribution of clonal deletion in this model. Since thymic expansion of donor DCs and the reduction in the donor‐reactive T cell repertoire were precluded with Treg depletion, we presumed that Tregs should preform before the establishment of clonal deletion. In contrast, the mice thymectomized before BMT failed to increase the number of Tregs and to establish CD8+T cell tolerance, suggesting the presence of mutual dependence between the thymic donor–DCs and Tregs. These results provide new insights into the regulatory mechanisms that actively promote clonal deletion.