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We describe a recipient of combined kidney and hematopoietic-cell transplants from an HLA-matched donor. A post-transplantation conditioning regimen of total lymphoid irradiation and antithymocyte globulin allowed engraftment of the donor's hematopoietic cells. The patient had persistent mixed chimerism, and the function of the kidney allograft has been normal for more than 28 months since discontinuation of all immunosuppressive drugs. Adverse events requiring hospitalization were limited to a 2-day episode of fever with neutropenia. The patient has had neither rejection episodes nor clinical manifestations of graft-versus-host disease.
We have attempted to achieve persistent mixed chimerism and tolerance in humans after transplantation of combined HLA-matched kidney and hematopoietic cells, using a low-intensity conditioning regimen of total lymphoid irradiation and antithymocyte globulin. This regimen can induce tolerance of organ allografts in laboratory animals.4,5,6 It also provides protection against graft-versus-host disease when used in patients with hematologic malignant conditions who are given HLA-matched hematopoietic-cell transplants.7 Total lymphoid irradiation can facilitate tolerance of kidney transplants in some patients without the administration of donor hematopoietic cells.8
We enrolled six patients in a study in which the conditioning regimen was given for 10 days after kidney transplantation, and cryopreserved donor cells were infused thereafter. The first patient is described here.
Case Report
The patient was a 47-year-old white man with end-stage renal disease of unknown origin at the time of preemptive kidney transplantation. The kidney donor was his 49-year-old brother, who shared the HLA-type A1,26;B38,51;BW4;DRB1*04,12 with the patient. On the day of kidney transplantation (day 0), the patient received the first of five daily injections of rabbit antithymocyte globulin (1.5 mg per kilogram of body weight), and on day 1 he received the first of 10 doses of 80 cGy each of total lymphoid irradiation. Treatment with cyclosporine was initiated on day 0, with a target whole-blood trough level of 350 to 400 ng per milliliter. Prednisone was administered as premedication for antithymocyte globulin injections and was discontinued on day 10. The patient was discharged from the hospital on day 6, and he completed total lymphoid irradiation in the outpatient clinic on day 14. An intravenous infusion of cryopreserved donor cells was administered in the clinic immediately thereafter. Donor mononuclear cells had been mobilized into the peripheral blood by the administration of granulocyte colony-stimulating factor. They were highly enriched for CD34+ hematopoietic progenitor cells with the use of an immunomagnetic-bead column. A dose of 1x106 CD3+ T cells from the column effluent was injected with 8x106 enriched CD34+ progenitor cells per kilogram.
Mycophenolate mofetil (1000 mg twice a day) was administered for 1 month after the intravenous injection of donor cells. The patient was hospitalized on day 21 because of fever with neutropenia, and was discharged on day 23. Blood cultures were negative, but the fever rapidly resolved after antibiotics were administered. The patient had no further hospitalizations. Two kidney-biopsy specimens were obtained according to an outpatient protocol at 3 and 12 months. Cyclosporine was gradually tapered and was discontinued 6 months after the transplantation. During those 6 months, there were neither rejection episodes nor clinical manifestations of acute or chronic graft-versus-host disease, and the chimerism was persistent. The patient returned to work about 3 months after the transplantation, and graft biopsy specimens showed normal kidney tissue; no cellular infiltrates were seen. Antihypertensive medications were withdrawn shortly after the discontinuation of cyclosporine. The patient remains in good health 34 months after transplantation.
Methods
Conditioning
Total lymphoid irradiation (irradiation of the supradiaphragmatic lymph nodes and thymus and the subdiaphragmatic lymph nodes and spleen) was performed as described previously.9 Rabbit antithymocyte globulin (Thymoglobulin, Genzyme) was given intravenously, starting with an intraoperative injection. The patient received prophylactic medications against fungal, bacterial, and viral infections. The protocol was approved by the institutional review board at Stanford University School of Medicine, and all the patients and donors provided written informed consent.
Donor Cells
Six weeks before transplantation, the donor received a 5-day course of subcutaneous injections of granulocyte colony-stimulating factor at a dose of 16 mg per kilogram per day, and mononuclear cells were harvested by means of leukopheresis. CD34+ cells were enriched with the use of an Isolex column (Baxter) and cryopreserved until they were infused.
Assessment of Chimerism
Chimerism was determined by means of DNA genotyping of simple sequence-length polymorphic markers that encode short tandem repeats, as described previously.10 Chimerism was assessed by analysis of T cells, B cells, natural killer cells, and granulocytes after enrichment of blood mononuclear cells on immunomagnetic beads (Dynabeads, Dynal) coated with monoclonal antibodies against CD3, CD19, CD56, and CD15, respectively.
Immunofluorescence Staining and Analysis of T-Cell Subgroups
Blood mononuclear cells were stained with fluorochrome-conjugated monoclonal antibodies against CD3, CD4, CD8, CD62L, CD45RA, CD45RO (BD Pharmingen), and V
24,Vβ11 (Beckman Coulter).11 Four-color analyses were performed by means of flow cytometry with the use of standard techniques and equipment (LSR and FACS Vantage cytometers, BD Biosciences).
T-Cell–Receptor Excision Circle Analysis
Sorted subgroups of T cells obtained by means of flow cytometry were cryopreserved and sent to the Human Vaccine Institute Immune Reconstitution Core Facility at Duke University for T-cell–receptor excision-circle (TREC) analysis with the use of a polymerase-chain-reaction–based assay.12
T-Cell Responses to Antigens
The mixed leukocyte reaction was performed by culturing peripheral-blood mononuclear cells as responder cells with irradiated allogeneic mononuclear cells or purified dendritic cells as stimulator cells and measuring 3H-thymidine incorporation.13,14 Dendritic cells were purified with the use of metrizamide gradients.15 Stimulation of mononuclear cells with tetanus toxoid and influenza virus antigens has been described previously.13,14 Concentrations of interleukin-2 and interferon-
in culture supernatants were measured with the use of commercial kits (BD Pharmingen).
Results
Figure 1A shows that mixed chimerism developed during the first month after transplantation and persisted until the last analysis, according to testing of short tandem repeats of DNA from purified white-cell subgroups. B cells and natural killer cells in blood contained the highest percentages of donor cells (70 to 85%). Granulocytes ranged between 30 and 70%. The lowest levels of chimerism were detected among T cells, and these levels remained in the range of 5 to 20% for more than 2 years after the transplantation. The level of serum creatinine decreased from about 6 mg per deciliter (530 µmol per liter) before transplantation to a stable level that has been below 2 mg per deciliter (177 µmol per liter) for more than 2 years after the transplantation (Figure 1B). Immunosuppressive drugs were discontinued 6 months after the transplantation.
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The culture of mononuclear cells obtained from the patient before the transplantation and 24 months after the transplantation with irradiated allogeneic mononuclear cells from two healthy subjects resulted in vigorous cellular proliferation as measured by means of 3H-thymidine incorporation (approximately 12,000 to 42,000 cpm) stimulated by cells from both healthy subjects (Figure 3A). Background incorporation without stimulator cells was less than 1000 cpm. A comparison of pretransplantation and post-transplantation responses of the recipient's T cells to dendritic cells from the donor showed that there was a significant reduction in the post-transplantation response after subtraction of background counts per minute (P=0.04, by Student's t-test) (Figure 3B). Figure 3C through 3F show that the patient's post-transplantation mononuclear cells were capable of responses to graded concentrations of influenza and tetanus toxoid antigens, as assessed by the secretion of the cytokines interferon- and interleukin-2, respectively, into the culture supernatants. The pattern of responses to alloantigens, influenza, and tetanus antigens was similar 18 months after transplantation (data not shown).
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In our patient, the persistence of mixed chimerism after combined kidney and hematopoietic-cell transplantation, the withdrawal of all immunosuppressive therapy, and the uninterrupted normal functioning of the kidney graft are all consistent with the phenomenon of induced immune tolerance in laboratory animals.5,6 We used the same conditioning regimen of total lymphoid irradiation and antithymocyte globulin that has been used in patients with hematolymphoid malignant conditions who received hematopoietic grafts,7 but we reduced the number of donor CD3+ T cells administered from about 200x106 to 300x106 cells per kilogram to 1x106 cells per kilogram in order to reduce the risk of graft-versus-host disease and increase the likelihood of achieving persistent mixed rather than complete chimerism.
The conditioning regimen was not associated with notable adverse events. The patient was discharged from the hospital 6 days after transplantation, and the donor-cell infusion was given in the outpatient clinic. Evidence of adequate immune reconstitution was the absence of opportunistic infections, normal in vitro T-cell responses to tetanus toxoid and influenza antigen, and a vigorous mixed leukocyte reaction to third-party stimulator cells. By contrast, there was a weak response of post-transplantation T cells from the recipient to dendritic cells from the donor.
We examined changes in T-cell subgroups after transplantation to elucidate the source, extent, and kinetics of naive and memory T-cell reconstitution after the initial severe lymphopenia. TREC analysis showed that naive CD8+ T cells returned more rapidly than naive CD4+ T cells, probably because of expansion in the periphery in response to T-cell depletion rather than generation of new cells in the thymus.
The second patient enrolled in the study had a biopsy-confirmed relapse of focal segmental glomerulosclerosis, and severe proteinuria developed during the first week after transplantation. Treatment included a prolonged course of plasmapheresis, antithymocyte globulin, and rituximab. Chimerism was not detected. The patient is receiving cyclosporine and mycophenolate mofetil, and there have been no rejection episodes in 26 months; the current serum creatinine level is 1.2 mg per deciliter (106.1 µmol per liter).
Transient chimerism developed in the third patient, with donor-type cells accounting for a peak of 35 to 65% of natural killer cells, granulocytes, and B cells but only 2% of T cells. The chimeric state was gradually lost in all lineages by the fifth month. A mild rejection episode that developed while the cyclosporine dose was being tapered was reversed by a brief course of corticosteroids, and the current serum creatinine level is 1.1 mg per deciliter (97.2 µmol per liter) 20 months after transplantation while the patient is receiving cyclosporine therapy alone.
The fourth, fifth, and sixth patients were enrolled in the past 6 months, after the protocol was changed to deliver 10 doses of 120 cGy each, instead of 80 cGy each, of total lymphoid irradiation to enhance chimerism. Levels of early peak chimerism were as high as the level in the first patient, with donor-type cells accounting for 39 to 86% of T cells and 31 to 93% of natural killer cells, B cells, and granulocytes.
In a series of seven patients with multiple myeloma given HLA-matched combined kidney and bone marrow transplants, five had transient rather than persistent chimerism, yet all immunosuppressive drugs were discontinued in the five patients.18 These drugs could not be discontinued in two patients, both of whom had persistent complete chimerism, because of the development of graft-versus-host disease.18 Our study shows that it is feasible to achieve persistent mixed chimerism and organ-transplantation tolerance in humans, without the development of graft-versus-host disease.
Supported by a grant (1PO1HL-075462) from the National Heart, Lung, and Blood Institute.
Dr. Scandling reports receiving lecture fees from Astellas and research support from Astellas, Novartis, and Roche; and Dr. Busque, research support from Astellas, Isotechnika, Pfizer, and Novartis. No other potential conflict of interest relevant to this article was reported.
We thank Anne DeLuca for coordination of laboratory and clinic studies; Aditi Mukhopadhyay for technical assistance; Paula Colmenero, Ph.D., for help with performance of in vitro functional studies; and Mary Hansen for preparation of an earlier version of the manuscript.
Source Information
From the Departments of Medicine (J.D.S., S.D.-J., J.A.S., R.L., S.S.), Surgery (S.B., M.T.M.), Pathology (C.B., E.G.E.), and Radiation Oncology (R.T.H.), Stanford University School of Medicine, Stanford, CA.
Address reprint requests to Dr. Strober at the Department of Medicine, Stanford University School of Medicine, Center for Clinical Science Research Bldg., Suite 2215, 269 W. Campus Dr., Stanford, CA 94305, or at sstrober{at}stanford.edu.
References
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