Mixed chimerism and immunosuppressive drug withdrawal after ... : Transplantation (original) (raw)
INTRODUCTION
The development of immune tolerance to organ allografts associated with mixed chimerism has been achieved in small inbred and large outbred laboratory animals (1,2). In most studies, the organ transplant recipients were conditioned with nonmyeloablative, total-body or total-lymphoid irradiation (TLI) and infused with donor bone marrow cells (1,2). In major histocompatability complex (MHC)-mismatched monkeys, long-term kidney graft acceptance was observed without immunosuppressive drugs when multilineage macrochimerism (>1.5% donor-type cells) developed among blood lymphocytes within the first month after organ and bone marrow transplantation (3). Graft-versus-host disease (GVHD) was not reported in the latter recipients, and macrochimerism was uniformly lost by 2 to 3 months, despite the continued acceptance of the kidney grafts (3).
Combined human leukocyte antigen (HLA)-matched donor organ and bone marrow transplantation using nonmyeloablative radiation host conditioning has been used in a clinical protocol for the treatment of multiple myeloma and end-stage renal failure (4). Multilineage macrochimerism was achieved transiently, and long-term kidney graft acceptance was observed thereafter in the absence of immunosuppressive drugs (4). Clinical studies of HLA-mismatched combined cadaver-kidney and bone-marrow transplantation have been reported in which recipients were given conventional immunosuppressive drugs (5,6) and developed transient macrochimerism without GVHD, but immunosuppressive drug withdrawal was not attempted (5,6).
Nonmyeloablative total-body irradiation conditioning followed by HLA-matched bone marrow or granulocyte colony-stimulating factor (G-CSF) “mobilized” hematopoietic progenitor cell transplantation has been used extensively to treat patients with hematological and lymphoid malignancies (7–10). Nonmyeloablative conditioning was used in recipients with advanced lymphomas who received HLA-mismatched bone marrow transplants, and severe GVHD associated with death was observed in some patients (11).
In the present report, four HLA-mismatched recipients of combined kidney and hematopoietic progenitor transplants were studied to determine whether posttransplant nonmyeloablative conditioning with TLI and antithymocyte globulin (ATG) can be used to achieve macrochimerism without severe GVHD and kidney allograft acceptance during and after immunosuppressive drug withdrawal. TLI has been shown to protect against GVHD in small and large animal studies (12–14). Three of four patients in our study achieved macrochimerism without GVHD, using the TLI-based conditioning regimen. Immunosuppressive drug dosages were gradually tapered according to protocol guidelines, and were completely withdrawn from the first enrolled patient at the end of 1 year.
MATERIAL AND METHODS
Methods
Patients.
Four patients were enrolled at the Stanford University Hospital in a trial of combined kidney and hematopoietic progenitor transplantation from non-HLA-matched living donors. The study protocol was approved by the Stanford University Medical Center Committee for Medical Human Subjects. Eligibility criteria included candidates who would receive living donor kidney transplants, who are at least 21 years old, and who agree to participate in and sign a consent form for a study designed to discontinue steroids and cyclosporine after transplantation. Patients were excluded if there were contraindications to rabbit ATG, radiation, serological evidence of HIV infection, presence of HepBsAg, HepC Ab or viral RNA, or HLA-screening panel reactivity of more than 20%. Patients who were pregnant, nursing, or seronegative for Epstein-Barr virus also were excluded. HLA typing of donors and recipients was performed by standard serological and molecular (DNA) techniques. The latter tests were used to distinguish HLA-A, HLA-B, or HLA-DR subtypes, when appropriate.
Donor progenitor cell collection.
Three to 6 weeks before kidney transplantation, donors were given a 5-day course of daily subcutaneous injections of recombinant human G-CSF. After the course was completed, a four–blood-volume leukapheresis was performed, and blood mononuclear cells were harvested. Mononuclear cells were separated on Isalex columns (Nexell Inc.) using immunomagnetic beads coated with anti-CD34 mAb. The CD34-selected hematopoietic progenitor cells were assayed for the percentage and absolute number of CD34+ and CD3+ cells by flow cytometry, and were then cryopreserved for subsequent infusion.
Conditioning of the recipients and transplantation.
On day 0, all recipients received a kidney transplant from the progenitor cell donor, and received an IV infusion of 1.5 mg/kg rabbit ATG (Sangstat Inc., Fremont, CA). Three of four patients were given five more doses of ATG on days 1, 3, 5, 9, and 14. Total lymphoid irradiation, targeted to the lymph nodes, spleen, and thymus, was administered to each patient using a 6-million electron volt (MeV) linear accelerator, at a dose of 80 cGy starting on day 1, until a total of 10 doses (800 cGy) were delivered to all patients (15). Three patients received four TLI treatments on days 1 through 4, and six treatments on days 7 through 11, including 1 day with two treatments. The CD34+ donor cells were infused intravenously immediately after the last dose of TLI on day 11. Patient 3, with cardiac abnormalities, received only two doses of ATG on days 0 and 1. Ten doses of TLI were administered over 18 days, and CD34+ donor cells were given on day 18.
Steroids were begun in all patients on day 0 (before ATG administration) at the equivalent of 1 mg/kg per day of prednisone; and cyclosporine (Neoral; Novartis Inc., East Hanover, NJ) was started at conventional doses 3 days before the donor cell infusion in all patients. Patients were given daily ganciclovir for 6 months, and daily trimethoprim/sulfamethoxasole for 12 months as routine prophylaxis for cytomegalovirus and pneumocystis infections, respectively.
Monitoring of chimerism.
Chimerism in patients was monitored on serial posttransplant samples of recipient blood cells by immunofluorescent staining, when appropriate mAbs were available, and by analysis of DNA segments containing variable-length short tandem repeats (STRs), using a modification of a technique by Scharf et al. (16). Chimerism was determined among mononuclear cells from Ficoll/Hypaque gradients (Sigma, St. Louis, MO) and from lymphocyte subsets by flow cytometry, or by STR analysis after separation of mononuclear cells with immunomagnetic beads (Dynal Inc., Lake Success, NY) and specific mAbs (Becton Dickinson, Mountain View, CA). Limited cell numbers restricted serial subset analysis to T and B, or T and natural killer (NK) cells. After polymerase chain reaction (PCR) amplification of DNA segments from informative STR loci, PCR products with fluorochrome tags were analyzed on an ALF or ALF Express automated sequencing apparatus, using ALF Fragment Manager Software (Amersham Pharmacia Biotech, Piscataway, NJ). The sensitivity of this assay is 1% of donor-type cells in reference donor–host cell mixtures. Microchimerism was determined by nested PCR amplification of DNA, using donor-type HLA-DR-specific primers, as described elsewhere (17). The sensitivity of the latter assay is at the level of 0.01% of donor-type cells.
Monitoring of blood lymphocyte subsets.
Peripheral blood mononuclear cells were stained with fluorochrome conjugated mouse anti-human CD3, TCRαβ, CD4, CD8, CD56, CD19, and CD161 mAbs (purchased from Becton Dickinson Inc.) and analyzed by two-color flow cytometry (18). (Anti-HLA-2 mAb was a gift of Dr. L. A. Herzenberg, Department of Genetics, Stanford University, Stanford, CA.) Cells were gated by light scatter to include lymphocytes, and propidium iodide staining was used to exclude dead cells (18,19). To determine the absolute number of T-cell subsets, the percentage of positively staining cells within the lymphocyte light scatter gate was multiplied by the absolute number of mononuclear cells per mm3 harvested from the Ficoll/Hypaque gradients on the same day.
RESULTS
Clinical Outcome
The age and gender of the four patients enrolled in the study are shown in Table 1. Renal failure was the result of polycystic kidney disease (patient 1), glomerulonephritis (patients 2 and 4), and lupus nephritis (patient 3). Patients received kidney transplants on day 0 from living donors who were either unrelated (patients 1 and 4) or related (parents of patients 2 and 3). In all cases there was at least one HLA-A, HLA-B, or HLA-DR mismatched antigen present in the donor (Table 1). G-CSF “mobilized” blood mononuclear cells were harvested by apheresis from the donors 3 to 6 weeks before transplantation surgery. The mononuclear cells were selected for CD34+ hematopoietic progenitors by immunomagnetic beads (≥75% purity), and cryopreserved. On day 0, recipients were given the first of up to six intravenous injections of ATG (1.5 mg/kg), as described in Materials and Methods. Starting on day 1, 10 doses (80 cGy each) of TLI were given. An infusion of the donor CD34+ cells (3–5×106/kg) was given immediately after the completion of TLI (day 11 in patients 1, 2, and 4, and day 18 in patient 3). Contamination of the infused donor cells with CD3+ T cells was 3 to 4×104/kg.
Clinical characteristics of combined transplant recipients
Patients received maintenance steroids starting on day 0, and maintenance cyclosporine (Neoral) beginning 3 days before the donor cell infusion. Conventional maintenance doses of both drugs were given for the first 6 months. Maintenance doses were tapered to discontinuation by 9 and 12 months, respectively, if recipients developed macrochimerism within 3 months, failed to show serological and histological evidence of rejection, and failed to show reactivity to donor cells in the MLR during drug withdrawal. The daily doses of prednisone (0–10 mg/day) and the daily doses of cyclosporine (0–300 mg/day) for the four patients at the last observation point (days 116 to 374 posttransplant) are shown in Table 1.
None of the patients developed systemic viral or bacterial infections or any clinical or laboratory abnormalities indicative of GVHD during the observation period. However, patient 1 developed herpes zoster, which was confined to a single dermatome, at 6 months. Patient 2 experienced an early-rejection episode (Table 1), and a graft biopsy at day 13 showed a histological pattern consistent with early humoral rejection (clotting in the peritubular capillaries). The rejection episode was resolved with antirejection drugs and multiple plasmaphereses. The current serum creatine levels are between 1.0 and 1.7 mg/dL (Table 1). Kidney biopsies performed on patient 1 at 6 months (just before tapering of prednisone of to less than 5 mg/day) and at 10 months (just before gradual withdrawal of cyclosporine) showed no evidence of acute or chronic rejection and no cellular infiltrate. A biopsy of performed on patient 3 just before a similar withdrawal of prednisone showed no rejection.
Studies of Chimerism
To determine whether the infused donor CD34+ hematopoietic progenitor cells engrafted with subsequent development of macrochimerism, recipient blood samples were obtained at serial intervals, and the percentage of donor-type cells among blood mononuclear cells was determined by STR analysis of DNA,or by immunofluorescent staining, or both.
Figure 1 shows the changes in the percentage of donor-type cells among all mononuclear cells and among the CD3+ T cells and CD56+ NK cells purified by immunomagnetic bead separation in blood samples obtained from patient 1. Chimerism was not detected at 7 days after the donor cell infusion, and the peak levels of chimerism occurred at 17 days. At the latter time points, mononuclear, NK, and T cells contained 5, 5, and 2% of donor-type cells, respectively. Numbers of available CD19+ B cells were insufficient to perform donor-type cell analysis. At 27 days after the cell injection, donor-type cells were between 1 and 2% of the mononuclear, NK, and T cells, and by 32 days chimerism was detected by STR analysis only among T cells (Fig. 1). At 87 days, mononuclear cells showed only microchimerism by HLA-DR PCR analysis, and by 143 days, microchimerism was not detected (<0.001%) among mononuclear and T cells (Fig. 1). During the period that macrochimerism was detected by STR, similar percentages of donor-type, T, B, and NK cells were measured after staining with anti-HLA-A2 mAb (data not shown).
Changes in the percentage of donor-type cells among blood mononuclear cells and lymphocyte subsets after combined kidney and hematopoietic progenitor transplantation. Percentage of donor-type cells was determined by short tandem repeat (STR) analysis of all mononuclear cells (Ficoll-Hypaque) or CD3+, CD56+, or CD19+ mononuclear cells separated by immunomagnetic beads. * Microchimerism was detected by PCR analysis using donor-specific HLA-DR primers but not by STR analysis (patient 1, days 32 and 87). ** Microchimerism was not detected (patient 1, day 143).
Serial blood samples from patient 2, obtained at weekly intervals for the first month after the donor cell infusion, failed to detect macrochimerism, but microchimerism was detected at the level of about 1 in 5×103 cells. During the end of the second month after the donor cell infusion, even microchimerism was no longer detected. Macrochimerism among the mononuclear, T, and B cells of patient 3 is shown in Figure 1. Chimerism was first detected 11 days after donor cell infusion and was present at peak levels of 10 to 15% at 18 days. There was a threefold reduction of chimerism at 67 and 81 days, and at 95 days chimerism among mononuclear, T, and B cells was undetectable by STR analysis. Microchimerism analysis was not performed thereafter due to donor matching of HLA-DR. Patient 4 had a pattern of macrochimerism that was similar to patients 1 and 3, as judged by staining for donor-type cells with anti-HLA-A2 mAb. Macrochimerism peaked at 2 months, with 2 to 16% of donor-type T, B, or NK cells and became undetectable at the end of the third month (data not shown). Sorted dendritic cells from patient 1 (MHC class IIhi CD19−CD3−CD14−CD56−) and CD14+ monocytes from patient 3 tested during the first month also had 10 to 15% of donor-type cells (data not shown).
Changes in White Blood Cell Counts and T-cell Subsets
Figure 2 shows the changes in the absolute white blood cell count patients 1, 2, and 3 starting from day 0 (day of kidney transplantation). Within the first 28 days, the nadir white blood cell counts were higher than 3×103/mm3 in the patients; however, the counts were intermittently reduced to approximately 2×103/mm3 during the first 90 days after transplantation and were subsequently increased when the daily dose of ganciclovir was decreased. The absolute number of CD3+ T cells was less than 50 cells/mm3 for the three patients during the first month (Fig. 2). At approximately 3 to 4 months, the CD3+ T cell count rose to at least 100 cells/mm3, and by 6 months levels of 200 to 400 cells/mm3 were observed. Although the levels of CD8+ T cells were slightly less than normal range (350–600 cells/mm3) by 9 months, in patient 1 the levels of CD4+ T cells remained fivefold less than the normal range (500–1200 cells/mm3) at that time. Patient 4 had a similar pattern of T-cell recovery after transplantation (data not shown).
Changes in the white blood cell (WBC) counts and absolute number of CD4, CD8, and total T cells after combined kidney and hematopoietic progenitor transplantation. Serial blood samples were obtained at indicated time points for each patient. T cells show all CD3+ cells, and CD4 and CD8 T cells show CD4+TCRαβ+ and CD8+TCRαβ+ cells, respectively.
Immunosuppressive Drug Withdrawal
Patient 1 met the monitoring criteria for immunosuppressive drug withdrawal, including donor-specific unresponsiveness in the MLR at monthly intervals after 7 months (stimulation index ≤2 to donor and ≥10 to third-party stimulator cells), and two graft biopsies without evidence of rejection. Prednisone was completely withdrawn at 9 months and cyclosporine at the end of 12 months (Fig. 3).
Complete withdrawal of immunosuppressive drugs without rejection episodes in patient 1. The daily dose of prednisone, concentrations of creatinine in the serum, and trough levels of cyclosporine in whole blood at serial time points are shown.
Patient 2’s drug regimen was not tapered because of the early rejection episode, lack of macrochimerism, and an anti-donor response in the MLR (data not shown). Prednisone was withdrawn from patient 3 at 8 months after a normal graft biopsy, and withdrawal of cyclosporine is dependent on continuing monitoring results. Patient 4 has not reached the point of drug withdrawal.
DISCUSSION
Four patients were given combined kidney and G-CSF “mobilized” hematopoietic progenitor cell transplants after nonmyeloablative posttransplant conditioning with TLI and ATG. The posttransplant TLI and ATG conditioning regimen followed by the infusion of donor bone marrow after 10 doses of TLI has been successful in achieving mixed chimerism and long-term acceptance of heart transplants without GVHD in rodents (20,21). In addition, the feasibility of inducing immune tolerance to cadaver kidney allografts using pretransplant TLI without donor cell infusions in humans has been reported previously, and graft survival after immunosuppressive drug withdrawal was followed for up to 12 years (17,22).
Three patients without rejection episodes developed mixed chimerism, with up to 16% donor-type cells present among T, B, and NK cells, during the first 3 months after transplantation. Similar levels of chimerism were observed in sorted dendritic cells and monocytes, but macrochimerism was not detected among unfractionated white blood cells that contained more than 90% granulocytes cells during the first 3 months (data not shown). Macrochimerism in the mononuclear cell subsets was transient and undetectable after 2 months in patient 1, and was lost during the third month in patients 3 and 4. Long-term acceptance of kidney allografts without immunosuppressive drugs has been reported after transient chimerism in both monkeys and humans given donor bone marrow cell infusions (3,4). Although patients 1, 3, and 4 developed macrochimerism, patient 2 experienced an early humoral rejection episode on day 8 and failed to develop macrochimerism. The latter episode in patient 2 may have been due to the presence of pretransplant serum anti-donor antibodies that were not detected by the usual cross-match procedures, including antibody-mediated cytolysis and flow cytometry.
None of the four patients developed GVHD or systemic viral or bacterial infections. The absence of GVHD is likely due to the use of purified CD34+ cells because the levels of contaminating CD3+ T cells (3–4×104 cells/kg) has been reported to be below the threshold for the induction of GVHD in myeloablated recipients given HLA-mismatched progenitor transplants (23). The TLI and ATG conditioning protects against GVHD in rodents because of the predominance of NK T cells in the host lymphoid tissues (14). Interestingly, a five- to 10-fold increase in the percentage of NK T cells (CD161+CD3+) among CD3+ T cells in the blood of all recip-ients in our study was observed during the first 3 months (data not shown). A recent report showed that CD161+CD3+ T cells in human bone marrow markedly suppress the MLR (24). A marked reduction in the absolute number of CD3+ T cells, especially among CD4+ T cells, was observed in the first 6 months in all patients, which was similar to that observed in a previous study of TLI and ATG conditioning for kidney transplantation (25).
The lack of systemic bacterial infection was not surprising because the nadir white blood cell counts did not fall lower than 2×103 cells/mm3 in all four patients shortly after conditioning with TLI and ATG. Although all four patients developed a marked lymphopenia, the risk of systemic viral or pneumocystis infection was reduced by the administration of ganciclovir and trimethoprim/sulfamethoxazole during the posttransplant observation period. Immunosuppressive drugs were completely withdrawn in patient 1 over a 12-month period, and prednisone was withdrawn from patient 3 over an 8-month period, according to protocol monitoring criteria.
In conclusion, macrochimerism without GVHD, severe infection, or myeloablation can be achieved after combined HLA-mismatched kidney and hematopoietic progenitor transplantation using a posttransplant conditioning regimen of TLI and ATG that can be applied to cadaver organ transplantation. Further observations are required to determine whether complete immunosuppressive drug withdrawal can be achieved in additional patients, and whether lack of rejection episodes after withdrawal is durable.
Acknowledgments.
The authors thank Mary Hansen for preparation of the manuscript and Mojgan Haririfar for monitoring of patients.
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