A Clinical Trial Aiming at Tolerance Induction by Adoptive Transfer of Ex Vivo-Induced , Donor-Specific Treg-Like Cells in Clinical Kidney Transplantation

1Department of Transplant Surgery, Mita Hospital, International University of Health and Welfare, Tokyo, Japan 2Department of Surgery, Kidney Center, Tokyo Women’s Medical University, Tokyo, Japan 3Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan 4Atopy Research Center, Juntendo University Hospital, Juntendo University School of Medicine, Tokyo, Japan Received date: 15 Oct 2017; Accepted date: 11 Dec 2017; Published date: 18 Dec 2017.


Introduction
The induction of immunological tolerance is an ultimate goal in organ transplantation.Today, the outcome of organ transplantation has been remarkably improved by the recent progress in many fields, especially in the development of potent and selective immunosuppressants.Many adverse effects of immunosuppressants, however, such as various opportunistic infections which may be fatal sometimes, nephrotoxicity which may lead to renal failure, malignancies and post-transplant lymphoproliferative disorders which may cause the fatal outcome hamper not only graft survivals but also the quality of life.Additionally hypertension, dyslipidemia, impaired glucose tolerance, cataract, glaucoma, osteoporosis and aseptic necrosis influence unfavorably on the quality of life of transplant patients.One of the greatest problems which influence long-term graft survival is a chronic rejection that cannot be successfully treated even with modern potent, selective immunosuppressants.
To overcome aforementioned problems, namely adverse effects of immunosuppressants and chronic rejection, tremendous basic experiments and clinical trials on the induction of immunological tolerance have been reported [1][2][3][4][5][6][7][8][9][10][11][12].These trials were divided into two categories, the induction of chimerism by bone marrow transplantation or the infusion of hematopoietic stem cells and the peripheral deletion of donor-responsive clone using alemtuzumab with T cell depleting antibody or immunosuppressants.In case of bone marrow transplantation, capillary leak syndrome (engraftment syndrome) and graft versus host disease (GVHD) remain to be solved [5,6], while in case of hematopoietic stem cells, GVHD has not been observed although chimerism was introduced [8].
To induce the immunological tolerance by the less-invasive method, we tried to induce donor-specific regulatory T (Treg)-like cells by coculture of donor and recipient lymphocytes in the presence of anti-CD80/86 monoclonal antibodies.We reported that the successful induction of the immunological tolerance by infusing ex vivo-induced, donor-specific Treg-like cells in rhesus monkey kidney transplantation [13].We conducted a clinical trial aiming at tolerance induction in kidney transplantation based upon the new protocol revised for the clinical trial with respect to twice stimulation of donor-specific Treg-like cells by donor lymphocytes (Figure 1).Open Access

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The purposes of the study are to investigate, (1) whether Treg-like cells can be induced ex vivo also in human by the same methods, (2) whether tolerance can be induced also in clinical kidney transplantation by using ex vivo-induced, donor-specific Treg-like cells?

Materials and Methods
This clinical trial of tolerance induction based upon the protocol shown in figure 1 was approved by the Institutional Review Board (the Ethics Committee of Tokyo Women's Medical University).Nine patients undergoing dialysis therapy were enrolled in this study after the informed consent, who all applied spontaneously to undergo kidney transplantation by this protocol.All kidney donors voluntarily applied to donate the kidney for the clinical trial.Age, gender, original diseases, the duration of dialysis, the relationship to the donor, and the number of mismatched HLA-A, -B and -DR antigens are shown in table 1.
After total 14 day co-culture, viable cells were collected, washed and centrifuged several times, tested for bacterial contamination and endotoxin (Endotoxin Single test Wako, Wako Pure Chemical Industries Ltd., Osaka, Japan), and suspended in 100 ml of physiological saline for intravenous administration to the recipients.All these procedures were done in the Cell Processing Center of Tokyo Women's Medical University.A sampling of cultured cells was used to study for lymphocytes subsets by Flow cytometry and mixed lymphocyte reaction (MLR) with lymphocytes from donor and 3 rd party.

Kidney transplantation and inoculation of cultured cells
Laparoscopic donor nephrectomy and kidney transplantation in the iliac fossa along with laparoscopic splenectomy of the recipient were done.Immunosuppressants regimen consists of 8 mg/kg/day of cyclosporine (CsA), 2,000 mg/body/day of mycophenolate mofetil (MMF) and 5 ~ 10  mg/kg/day of methylprednisolone (MP).Basiliximab was avoided not to deplete CD25+ T cell.The doses of CsA and MMF were gradually reduced, adjusting each trough level around 150 ~ 250 ng/ml and 1.0 ~ 4.0 µg/ml, respectively for the first three months.The dose of MP was rapidly reduced to 20 mg/day on day 7, and further tapered to 8 mg/day on day 14.
On day 5 ~ 7, 25 ~ 30 mg/kg/day of cyclophosphamide (CPA) was given intravenously to deplete circulating leukocytes.Circulating leukocyte count was reduced to around 1,500/mm 3 within a few days and returned to the previous value within 14 ~ 21days.On 12 POD, 0.8 ~ 1.5 × 10 9 cultured cells were given intravenously to the recipient (Table 2).In three patients, cultured cells (0.075 ~ 0.8 × 10 9 ) were given later after 12 POD (resulting in the elongated culture period), on 19, 33 and 21 POD because of ascites due to preoperative continuous ambulatory peritoneal dialysis (CAPD), delayed urination due to postoperative acute tubular necrosis (ATN), and the leakage of pancreatic juice after splenectomy, respectively.Latter two patients were given an insufficient number of cultured cells, 0.075 × 10 9 on day 33 and 0.3 × 10 9 on day 21, respectively, because obtained viable cultured cells tended to decrease in number when the culture period of time was extended for more than 14 days (Table 2).
Thereafter MP was tapered down gradually, and the doses of CsA and MMF were reduced based upon the value of MLR with lymphocytes from the donor and 3 rd party, especially the ratio of MLR with the donor to 3 rd party lymphocytes.When the doses of CsA and MMF were reduced to 25 mg/day and 250 mg/day, respectively, first MMF was discontinued followed by further reduction of CsA.

Mixed lymphocyte reaction
Peripheral blood was obtained from the donor and the recipient, and PBMCs were prepared by centrifugation over Separate-L (Muto Pure Chemicals Co., Ltd., Tokyo, Japan).Thereafter 2 × 10 5 lymphocytes from the recipient were co-cultured with the same number of 30 Gyirradiated donor lymphocytes in 96-well round-bottomed plates (catalog 3799; Corning Inc., New York, NY).The cells were cultured for 6 days and then pulsed with 10 µCi of [3H] thymidine for the last 18 hours.The incorporated radioactivity was measured on a 1450 MicroBeta counter (Perkin Elmer Japan Co., Ltd., Yokohama, Japan).
Samplings of cultured cells (1 × 10 5 ) were incubated with 1 µg each of the indicated monoclonal antibodies for 30 minutes at 4℃ in PBS, washed twice, and analyzed on a FAC Scan equipped with Cellquest software version 3.3 (BD).The expression of CTLA-4 and FoxP3 was determined by intracytoplasmic staining using CD152 PE and FoxP3 FITC or PE after the cells were fixed with 2% formaldehyde and the membrane was permeabilized with BD Biosciences FoxP3 Staining Buffer Set (eBioscience, San Diego, USA).
Peripheral blood samplings of recipients were treated with the same procedures and assayed for flow cytometry.

Immunohistochemical staining of the biopsied specimen of the kidney grafts
The biopsied specimen was fixed in 10% buffered formalin and embedded in paraffin.H&E and PAS stains were performed for histological examination.The immunohistochemical staining assay using anti-CD4, -CD8, -CD20, -CD25, -FoxP3 and -granzyme B antibody was performed with iView DAB Detection Kit (Roche Tissue Diagnostics Japan Ventana, Tokyo, Japan) on a VENTANA BenchMark GX automated staining system (Roche Diagnostics Japan Ventana, Tokyo, Japan) to investigate the characteristics of infiltrated cells into kidney grafts.
Briefly, the tissue sections were deparaffinized with EZ Prep (Roche Tissue Diagnostics Japan Ventana) at 75℃, heat pretreated in Cell Conditioning 1 (CC1; Roche Tissue Diagnostics Japan Ventana) using "standard cell conditioning" for antigen retrieval at 100℃, and then incubated with anti-CD4 and -CD8 rabbit monoclonal antibody (Roche Tissue Diagnostics Japan Ventana), anti-CD20 primary antibody (Roche Tissue Diagnostics Japan Ventana), anti-CD25 mouse monoclonal antibody (NICHIREI Bioscience Inc., Tokyo, Japan), anti-human Foxp3 antibody (BioLegend Japan, Tokyo, Japan) and anti-granzyme B polyclonal antibody (Roche Tissue Diagnostics Japan Ventana) as a primary antibody, respectively, for 32 min at 37℃ after inactivation of the endogenous peroxidase with hydrogen peroxide for 4 min.They were then blocked using Endogenous Biotin Blocking Kit (Roche Tissue Diagnostics Japan Ventana), incubated with a biotinylated Ig secondary antibody for 8 min, and incubated with a streptavidin-HRP conjugate for 8 min at 37℃.The immunolocalized CD4, CD8, CD20, CD25, FoxP3 and granzyme B were visualized using a copper-enhanced DAB reaction.The slides were counterstained with Hematoxylin II (Roche Tissue Diagnostics Japan Ventana) for 4 min and Bluing Reagent (Roche Tissue Diagnostics Japan Ventana) for 4 min and coverslips were applied by an automated coverslipper (Tissue-Tek Film Automated Coverslipper; Sakura Finetek Japan, Tokyo, Japan).

Results
The number of viable cells obtained by co-culture of the recipient and irradiated donor lymphocytes in the presence of anti-CD80/86 monoclonal antibodies was 0.8 ~ 1.5 × 10 10 in case that the culture period was within 14 days, while the number of viable cells was decreased as the period of culture was extended to more than 14 days (Table 2).Cultured cells expressed CD4, CD25, CTLA-4, and FoxP3 (Figure 2).The mean incidence of CD4+CD25+CTLA-4+FoxP3+ cells in cultured cells was 13.03 ± 6.89%.Cultured cells depressed MLR with donor lymphocytes in a dose-dependent manner, while did not depress MLR with 3 rd party lymphocytes (Figure 3).Cultured cells (0.8 ~ 1.5 × 10 9 ) were given intravenously to the recipient on 12 2).
All patients well tolerated the administration of CPA, while leukocytopenia and the loss of hair were observed after the administration of CPA.Any adverse effects attributable to the intravenous infusion of cultured cells, such as fever, hypotension, and rash have not been observed when cultured cells were infused intravenously in all patients.
The phenotype of circulating lymphocytes of the recipients after infusing Treg-like cells is shown in figure 4. The incidence of CD4+CD25+CTLA-4+FoxP3+ cells in circulating lymphocytes was increased with time and reached the peak about 4 weeks after infusing cultured cells.
The ratio of with the donor to 3 rd lymphocytes was depressed with time after the intravenous infusion of cultured cells to 0.006 ~ 0.22 in 8 patients, whereas not depressed to 1.0 or less in remaining one patient (patient No.9, table 3) from1 month to12 months after the transplantation.Thereafter in three patients (patient No.1, 4 and 7), the ratio of MLR with the donor to 3 rd party lymphocytes was increased to 0.49-0.720(Table 3).
Table 3 shows the number of mismatched antigens, the dose of CsA, MMF and MP, the Banff grade, the type and the time of rejection episode and its treatment in addition to the ratio MLR with the donor to 3 rd party lymphocytes.Six out of 9 patients developed rejection episode between 220 and 378 POD, cellular rejection (IA) in 4 patients (patient No. 3, 4, 6 and 8), and mixed type (IIB) in two patients (patient No.1 and 5).
Figure 5 shows the postoperative course of patient No.2, in which a dose of CsA and MMF was reduced to 25 mg/day and 250 mg/day following discontinuing steroid, respectively.In case of patient No.3, after reducing CsA dose to 25 mg/day following discontinuing steroid and MMF, a cellular rejection occurred in 330 POD.After the recovery by the treatment, we started again low-dose CsA and MMF (Figure 6). Figure 7 shows the postoperative course of patient No.8.After reducing CsA dose to 10 mg/day following discontinuing steroid and MMF, a cellular rejection occurred in 307 POD.After it was relieved by the treatment, we started again low-dose triple regimen.Other two patients (patient No.1 and 5) developed rejection episode on 302 POD and 378POD, respectively, during reducing the doses of CsA and MMF following the discontinuation of steroid.All rejection episodes were relieved by steroid pulse with or without muromonab CD3 and low-dose triple regimen was started.
The average daily doses of CsA and MMF in the study group during the postoperative time course are compared to those in the control group (Figure 8).The control group consists of living-related ABO-compatible kidney transplantation performed during the same period in which basiliximab was given at the transplantation.The average daily dose of CsA and MMF in the study group was 40.3% and 45.6% of that in the study group at 12 months, and 51.2% and 51.0% of that in the study group at 18 months after the transplantation, respectively.
Figure 9 shows the immuno-histochemical findings of a biopsy specimen of kidney graft (case 2, 100 POD), showing CD4+, CD25+ and FoxP3+ cell infiltration.Despite the infiltration of CD8+ cells, the granzyme+ cell was not observed.The number of HLA mismatch antigens, the ratio of MLR with donor to 3 rd party lymphocytes, anti-HLA antibody, immunosuppressants, Banff grade, the type and the time of the rejection, and its treatment.*: donor-specific antibody

Discussion
Antigen recognition without co-stimulatory signals is known to lead to T cell anergy.Anti-CD80/86 antibodies inhibit CD28 and promote CTLA-4 ligation of CD80/86, leading to express Treg-specific transcription factor, forkhead box P3 (FoxP3), which suppresses IL-2 transcription via interacting with AML-1/Runx1 (leucine zipper domain), histone de-acetylases (amino-terminal repressor domain), and NF-AT (forkhead domain) [14,15].We reported that anergic T cells induced by the co-culture of recipient lymphocytes with irradiated donor lymphocytes in the presence of anti-CD80/86 antibodies, expressed CD4, CD25, CTLA-4 and FoxP3 and demonstrated donor-specific hypo-responsiveness in MLR in a dosedependent manner, named ex vivo-induced, donor-specific Treg-like cells, and that tolerance induction was successfully achieved in rhesus monkey kidney transplantation by infusing ex vivo-induced Treg-like cells intravenously to the recipient [13].
We conducted the clinical trial of tolerance induction in the kidney transplantation to investigate, (1) whether Treg-like cells can be produced also in human by the same (2) whether tolerance can be induced also in clinical kidney transplantation by using ex vivo-induced, donorspecific Treg-like cells?
Viable cultured cells obtained by the co-culture of recipient lymphocytes with irradiated donor lymphocytes in the presence of anti-human CD80/86 antibodies expressed CD4, CD25, CTLA-4 and FoxP3 and depressed MLR with donor lymphocytes in a dose-dependent manner, while did not depressed MLR with 3 rd party lymphocytes, suggesting that donor-specific Treg-like cells can be introduced also in human subjects (Figure 2,3).
The ratio of MLR of the recipient with donor lymphocytes to that with 3 rd party lymphocytes is considered to indicate the specificity of the suppressive effect of Treg-like cells.On conducting the clinical trial, we considered the decrease in the ratio of MLR with donor lymphocytes to 3 rd party lymphocytes would be a key indicator of the immunological responsiveness of the recipient to donor antigens, and therefore a possible indicator of reducing immunosuppressants.
In the process of reducing immunosuppressants referring to the ratio of MLR with the donor to 3 rd party lymphocytes, a cellular rejection (IA) occurred on 330 POD after reducing CsA dose to 25 mg/day following discontinuing steroid and MMF in case 3 (Figure 6), although the ratio of MLR with the donor to the 3 rd party was suppressed to 0.02.Also in case of patient No.8, after reducing CsA dose to 10 mg/day following discontinuing steroid and MMF, a cellular rejection (IA) occurred on 307 POD although the ratio of MLR with the donor to the 3 rd party was suppressed to 0.06 (Figure 6).Additionally, in case of patient No.5, a mixed type rejection occurred on 378 POD after reducing CsA and MMF to 25 mg/day and 250 mg/day, respectively following discontinuing steroid, even though the ratio of MLR with the donor to the 3 rd party was depressed to 0.006.
Finally in 6 out of 9 patients, the rejection occurred on 220 ~ 378 POD in the process of reducing CsA and/or MMF following reducing and/or discontinuing steroid and MMF, even though the ratio of MLR with the donor to 3 rd party lymphocytes were depressed to 0.006 ~ 0.22.While in case 4, although triple regimen had been maintained (CsA: 125 mg/day, MMF: 500 mg/day, MPS: 2 mg), a cellular rejection (IA) occurred on 220 POD (Table 3).
From above-mentioned facts several problems to be solved arise.Can the ratio of MLR with the donor to 3 rd party lymphocytes be a key indicator of immunological responsiveness of the recipient to donor antigens?And can it be an indicator of reducing immunosuppressants?Can infuse donorspecific Treg-like cells suppress in vivo the immunological responsiveness of the recipient to donor antigens enough to reduce and/or withdraw immunosuppressants in human subjects, same as rhesus monkey?[13] Can ex vivo-induced, donor-specific Treg-like cells continue to function in vivo and expand in the recipient for long period?
Regarding the point at issue, "To what extent the dose of immunosuppressants can be reduced?"would depend upon "To what extent MLR with donor can be depressed compared to that with 3 rd party?", it could not be settled in this study.The intravenous infusion of ex vivoinduced, donor-specific Treg-like cells suppressed the immunological responsiveness of the recipient to donor antigens enough to reduce immunosuppressants to some extent, but not enough to withdraw them completely in the clinical kidney transplantation.
The suppressive activity of CD4+CD25+Treg cells was reported to be transferred to CD4+CD25-T cells via direct cell-to-cell contact (infectious tolerance [16]), and the co-culture of human CD25+Treg cells with CD25-CD4+T cells was also reported to lead to the development of an additional CD4+T suppressor cell population (CD4+Treg cells), which emerge from the CD4+CD25-T cell population and suppress the proliferation of freshly isolated conventional CD4+T cells [17].
Finally, the occurrence of the rejection might be related to memory cells probably generated by encountering donor antigens before the infusion of Treg-like cells.It is likely that initial immunosuppression, the triple regimen consisting of CsA, MMF, and MP, would not be enough to inhibit producing memory T and/or B cells against donor antigens.We are reconsidering a new protocol including the administration of a small dose of thymoglobulin and rituximab on 0 POD to inhibit the production of T and B memory cells, based upon outcomes of the pilot study adding thymoglobulin to the previous protocol, and of the in vitro culture of Treglike cells with thymoglobulin and human plasma containing the compliment.

Conclusion
Ex vivo-induced Treg-like cells by co-culture of donor and recipient lymphocytes in the presence of anti-CD80/86 monoclonal antibody showed donor-specific hyporesponsiveness in MLR in a dose-dependent manner.The infusion of Treg-like cells to recipients induced hyporesponsiveness of the recipient so as to reduce immunosuppressants approximately by 50% in clinical kidney transplantation, but did not realize immunological tolerance.To induce immunological tolerance, further study was required.
n H U B f o r S c i e n t i f i c R e s e a r c h Citation: Teraoka S, Koyama I, Bashuda H, Uchida K, Tonsho M, et al. (2017) A Clinical Trial Aiming at Tolerance Induction by Adoptive Transfer of Ex Vivo-Induced, Donor-Specific Treg-Like Cells in Clinical Kidney Transplantation.J Transplant Res 2(1): doi http://dx.doi.org/10.16966/2473-1730.115

Figure 3 :
Figure 3: Mixed lymphocytes reaction (MLR) with donor and third party lymphocytes in the presence of cultured cells, which depressed MLR with donor lymphocytes in dose-dependent manner, while did not with 3 rd party lymphocytes.

Figure 4 :
Figure 4: Phenotype of circulating lymphocytes of the recipients after infusing Treg-like cells.

Figure 5 :
Figure 5: Clinical course of patient No.2 (53 y.o., male, 1 mismatched against donor HLA-antigens).The minimum ratio of MLR with the donor to the 3 rd party was 0.07.Immunosuppressants were reduced and maintained with 25 mg/day of CsA and 250 mg/day of MMF without any rejection episode.

Figure 6 :
Figure 6: Clinical course of patient No.3 (41 y.o., female, 2 mismatched against donor HLA-antigens).The minimum ratio of MLR with the donor to the 3 rd party was 0.02.When reducing CsA to 25 mg/day following the discontinuation of MP and MMF, cellular rejection (IA) occurred on 330 POD, which was resolved by steroid pulse therapy and maintained with a reduced dose of CsA and MMF.

Figure 7 :
Figure 7: Clinical course of patient No.8 (34 y.o., male, 2 mismatched against donor HLA-antigens).The minimum ratio of MLR with the donor to the 3 rd party was 0.06.When reducing CsA to 10 mg/day following the discontinuation of MP and MMF, cellular rejection (IA) occurred on 307 POD, which was resolved by steroid pulse therapy and maintained with a small dose triple regimen.

Figure 8 :
Figure 8: Average daily dose of CsA (A) and MMF (B) at given time (postoperative months) in study group as compared to control group (living-related ABO-compatible kidney transplantation performed during the same period in which basiliximab was given at the induction).

Table 1 :
Demography of patients enrolled in the clinical trial.

Table 2 :
POD following 2-3 days-consecutive intravenous administration of Cyclophosphamide dosage and the number of infused cells (TReg-like cells).