Induction tolerance with donor hematopoietic stem cell infusion in kidney transplantation: a single-center experience in China with 10-year follow-up

Background. We performed the first clinical trial in China in which kidney transplantation was combined with donor hematopoietic stem cell (DHSC) infusion for tolerance induction. This study summarizes the 10-year follow-up results. Methods. From 2009 to 2017, 11 cases of living-related kidney transplantation combined with DHSC infusion were performed. Two of them were HLA-matched, and nine were HLA-mismatched. The DHSCs were mobilized using granulocyte colony-stimulating factor and harvested one day before transplantation. The recipients received consecutive total lymphoid irradiation for 3 days before kidney transplantation. The induction drug was anti-thymocyte globulin. DHSCs were infused on the 2 nd , 4 th and 6 th postoperative days. Results. One HLA-matched recipient induced 30-50% chimerism, and the others only induced less than 1% chimerism. Recipients had a low immune response to their donors while sustaining normal reactivity to non-donors in mixed lymphocyte reactions. All recipients were followed up for 717~3,918 days. One recipient lost allograft function, and 10 recipients had stable renal function. None of the 11 recipients had myelosuppression or graft-versus-host disease post transplantation. Our protocol did not increase the risk of infection. Allograft biopsy confirmed that one patient had mild acute rejection, and the other 10 recipients did not develop rejection. Five patients reduced the dose of immunosuppression. Conclusions. This study shows that long-term stable kidney allograft survival may be achieved with low-dose immunosuppression maintenance using our protocol.


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Although immunosuppression (IS) and antibodies have been developing progressively, the long-term survival of renal allografts is still unsatisfactory [1]. The main reason is rejection, either acute or chronic. In addition, many side effects, such as cardiovascular diseases and metabolic disorders caused by IS, also threaten the lives of recipients.
Therefore, tolerance induction is always the ultimate goal of organ transplantation. Some recipients underwent IS withdrawal post transplantation. The results are encouraging, but adverse events cannot be ignored [2]. we reported a proteomics study from 4 patients [3]. Here, for the first time, we report the long-term follow-up results of a kidney transplantation clinical tolerance study in China.
We attempted to induce transit chimerism through combined kidney transplantation with the infusion of donor CD34 + selected hematopoietic stem cells.

Patients
From April 2009 to December 2019, 11 pairs of subjects were enrolled in this study. The average age of recipients was 33.2 ± 5.1 years, ranging from 22 to 42 years. The age of donors ranged from 31 to 62 years, with an average of 50.5 ± 11.1 years. Ten recipients received primary and one received secondary renal transplantation. The flow crossmatches were all negative. Initially, the preoperative panel reactive antibodies (PRAs) were set at < 10%. Seven pairs of donors and recipients were parent-child relationships, and four pairs were siblings. Two pairs were full HLA-matched at six HLA loci, and the others were 1 to 3 HLA-mismatched. The study was registered in the Chinese Clinical Trial Registry (http://www.chictr.org.cn, ChiCTR-TNC-09000399) with written informed consent. The general information of donors and recipients is detailed in Table 1.

Clinical Protocol
Before collecting DHSCs, the donor received a 5-day course of subcutaneous injections of granulocyte colony-stimulating factor (G-CSF) at a dose of 7.5 mg/kg/day. G-CSF mobilized donor mononuclear cells into the peripheral blood. These cells were harvested by COM.
TEC Blood Cell Separator (Fresenius AG, Bad Homburg, Germany). CD34 + and CD3 + cells were assessed by flow cytometric analysis. One day before transplantation, DHSCs were harvested and stored at − 70 °C.

T-cell Responses To Antigens
The mixed lymphocyte reaction was performed by culturing peripheral-blood mononuclear cells as responder cells with irradiated donor or irrelated third part allogeneic mononuclear cells as stimulator cells and measuring 3 H -thymidine incorporation. Roundbottom culture plates were stimulated with plated-coated anti-CD3 antibody and soluble anti-CD28 antibody (both from eBioscience) in RPMI 1640 medium and then were incubated for 5 to 7 days in a humidified 37 °C, 5% CO 2 incubator. Next, 3 H -thymidine was added 18 h before the end of the culture period. The cells were then harvested onto glass fiber mats for the measurement of 3 H -thymidine incorporation.

Statistics
Data are presented as the mean ± standard deviation. Statistical analysis (SPSS 18.0 software, SPSS, Inc., Armonk, NY, USA) was performed using the two-tailed independent Student's t test (paired or unpaired) after a demonstration of homogeneity of variance with the F test. Statistical significance was set as p < 0.05.

Results
Donor bone marrow mobilization of hematopoietic stem cells Before stem cell harvest, G-CSF (7.5 µg/kg/d) was used for 5 consecutive days in donors.

Change Of Blood Cells And Lymphocyte Subsets After Tli
After TLI, a dramatic decrease in lymphocyte counts occurred in all patients immediately, reaching 0 after ATG by day 0. Before TLI (D-4), the lymphocyte count was (1.70 ± 0.61) × 10 9 /L with a rapid decrease after 3 days (D-1). However, the number of white blood cells and neutrophils remained stable. After TLI at D-1, the proportions of CD19 + B cells (3.23 ± 1.10 vs. 12.80 ± 5.58) significantly decreased and CD56 + CD16 + NK cells (10.9 ± 2.84 vs. 15.36 ± 4.70) increased compared to those before TLI (D-1). The percentage of CD8 + T cells increased, while CD3 + T cells and CD4 + T cells had no significant change (Table 3).
Each individual data point is shown in Fig. 3. All patients had persistent lymphopenia, and then the lymphocytes gradually recovered after one month. The lymphocyte count increased and returned to the normal level approximately 6 months post transplantation.

Adverse Events
During TLI, 1 patient experienced fatigue, and 5 patients had mild diarrhea. During DHSC infusion, 1 patient exhibited oxygen saturation decline, chest tightness and discomfort.
After stopping the infusion, the symptoms improved, while the rest of the recipients did not show any adverse events. Previously, suspected graft-versus-host disease (GVHD) was reported. After one month of follow-up, skin symptoms disappeared, and GVHD did not occur in the other 10 recipients. The infection risk did not increase compared with that of the routine protocol. No bone marrow suppression was observed in 11 recipients.

T Cell Reactivity To Donor And Non-donor Allo-antigens
At 6 months after renal transplantation, we detected the T cell reactivity of recipients to donor and non-donor allo-antigens. Compared to pretreatment, T cell proliferation in recipients upon donor antigens was significantly reduced post transplantation but remained similar to that upon non-donor antigens. We also set a positive internal control using anti-CD3 and anti-CD28 antibody stimulation. For the external control, we tested T cell reactivity from a healthy volunteer (Fig. 5). These results demonstrated that our protocol induced a donor-specific low response of T cells in recipients.

Long-term Follow-up Results
By December 31, 2019, the median follow-up period was 3,671 days. Nine recipients underwent protocol biopsy 1-3 years after transplantation. Only one recipient had mild rejection (Banff IA grade, Banff 07 criteria), and the remaining 10 had no rejection (Table 4). The first recipient presented renal allograft dysfunction at 4 years post transplantation. Proteinuria and poor control of hypertension occurred in the fourth year.
The biopsy indicated glomerulonephritis relapse without rejection. Therefore, this patient returned to immunosuppressive drug treatment. Unfortunately, he had to receive hemodialysis at 4 years after kidney transplantation due to allograft loss. Ten patients had stable allograft function. Among them, 5 patients reduced the dosage of immunosuppressive agents, 4 patients used tacrolimus 1.0 to 3.0 mg/d, and 1 patient used rapamycin 1 mg/d, mofetil mycophenolate 0.5 to 1 g/d and prednisone 5 mg/d.  Hospital, operational tolerance was induced in 7 of 10 patients, 4 of whom remained off IS [4,7]. The Stanford University protocol found that 24/29 HLA-identical living donor kidney transplant recipients withdrew IS. However, this protocol has not been successful in HLAmismatched donor/recipient pairs [8,9]. A total of 26/37 HLA-mismatched, unrelated and related living donor kidney transplant recipients were removed from IS at Northwestern University [6,10].
Our protocol was similar to that of Stanford University, but the donor hematopoietic cell product did not control for the dose of CD34 + and CD3 + cells. We used TLI 3 days before kidney transplantation. Each dose was 160 ~ 170 cGy, and the total dose in 3 days was 480 ~ 510 cGy. However, Stanford University used TLI after kidney transplantation (80 ~ 120 cGy × 10 times, a total of 800 ~ 1,200 cGy) [8,9]. As we were the first to carry out clinical studies on kidney transplantation immune tolerance induction in China, we integrated irradiation protocols of Harvard and Stanford and developed our protocol for safety and efficacy. Learning from the theory of re-education of autologous or allogeneic hematopoietic stem cell transplantation in autoimmune diseases and leukemia [11][12][13][14][15], we infused a small dose of hematopoietic stem cells three times to prevent GVHD. Our longterm follow-up results showed that none of the patients had GVHD.
It is widely believed that successful induction of chimerism is essential for the induction of immune tolerance [3,9,16,17], and the three major centers in the United States also induce different degrees of chimerism. Harvard University induces transient chimerism [18], Stanford University induces mixed chimerism, and Northwestern University induces complete chimerism [19][20][21]. The Stanford University program successfully achieved continuous mixed chimerism in HLA-matched living kidney transplantation and completely withdrew immunosuppressive drugs. This protocol has also successfully achieved continuous mixed chimerism in HLA-mismatched living kidney transplantation; however, complete discontinuation of immunosuppressive drugs has not been achieved. Although immune tolerance was not induced in HLA-mismatch recipients, they found that persistent mixed chimerism prevented both acute and chronic rejection [8,9]. In our study, 2 donors were HLA-matched, 9 were HLA haplotype matched, and 1 recipient induced 30-50% chimerism. Unfortunately, chimerism disappeared after half a year. In addition, the chimeric rate of the other recipients was low. This may be the reason complete immune tolerance was not induced.
The ATG dose in our protocol was relatively low compared with the corresponding doses used at the three US centers, and they also used cyclophosphamide, anti-CD2 antibody and fludarabine; second, we used noncontrolling hematopoietic stem cells. The number of CD34 + cells infused was lower, but the number of CD3 + cells was higher. Stanford University controlled hematopoietic stem cell transplantation with CD34 + cells (10 × 10 6 /kg) and CD3 + cells (1 × 10 6 /kg). Our long-term follow-up results showed that 11 patients did not have GVHD or severe infection, and two patients had slight leukopenia for long-term follow-up, suggesting that our TLI protocol is safe. Therefore, we can increase the strength of pretreatment in the future, including increasing the TLI and/or ATG dose. recipients had nephritis relapse [20], which also suggested that we should be strictly screened when enrolling recipients and that caution should be taken when reducing or discontinuing immunosuppressive agents. Although the remaining 10 recipients were followed up for stable allograft function, we did not monitor the DSA. In the future, we should test DSA in all recipients.

Conclusion
Induction of immune tolerance in kidney transplantation remains very difficult, especially in recipients with HLA mismatches. This study shows that, in China, our protocol is safe and feasible without increasing the risk of infection.    Mixed lymphocyte reaction. This figure represents the T cell reactivity of patient #2. Before kidney transplantation and pretreatment, recipient T cell proliferation significantly increased upon donor or third-part alloantigen stimulation, but after transplantation, T cell proliferation upon donor antigen stimulation significantly decreased compared to that of the third-part alloantigen, suggesting a low