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A correction has been published: N Engl J Med 2006;354(8):884.
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Background Conditioning with total lymphoid irradiation plus antithymocyte serum protects mice against acute graft-versus-host disease (GVHD) after hematopoietic-cell transplantation. We tested this strategy in humans.
Methods Thirty-seven patients with lymphoid malignant diseases or acute leukemia underwent an experimental conditioning regimen with 10 doses of total lymphoid irradiation (80 cGy each) plus antithymocyte globulin, followed by an infusion of HLA-matched peripheral-blood mononuclear cells from related or unrelated donors who received granulocyte colony-stimulating factor.
Results Of the 37 transplant recipients, only 2 had acute GVHD after hematopoietic-cell transplantation. Potent antitumor effects in patients with lymphoid malignant diseases were shown by the change from partial to complete remission. In the transplant recipients who underwent conditioning with total lymphoid irradiation and antithymocyte globulin, the fraction of donor CD4+ T cells that produced interleukin-4 after in vitro stimulation increased by a factor of five, and the proliferative response to alloantigens in vitro was reduced, as compared with normal control subjects and control subjects who underwent conditioning with a single dose of total-body irradiation (200 cGy).
Conclusions A regimen of total lymphoid irradiation plus antithymocyte globulin decreases the incidence of acute GVHD and allows graft antitumor activity in patients with lymphoid malignant diseases or acute leukemia treated with hematopoietic-cell transplantation.
A new approach to the prevention of acute GVHD takes advantage of the immune system's regulatory T cells. Two types of regulatory T cell in mice, natural killer T cells and CD4+CD25+ T cells, can prevent acute GVHD.11,12,13,14 These regulatory T cells inhibit the proliferation of and cytokine secretion by CD4+ and CD8+ donor T cells that injure the intestines, liver, and skin in acute GVHD.11,14 Nevertheless, direct tumor-killing activity mediated by donor CD8+ T cells remains unaffected.12 Thus, regulatory T cells can separate GVHD from the antitumor activity of the graft. Regulatory natural killer T cells of either donor or host origin have the unique capacity to prevent acute GVHD by secreting interleukin-4.13,14,15 Natural killer T cells recognize a nonpolymorphic, major-histocompatibility-complex class I–like antigen-presenting molecule, CD1d, which is present on both the donor and host antigen-presenting cells.16
Natural killer T cells constitute only 1 to 3 percent of all T cells in the spleen in normal mice. After repeated treatment with low-dose irradiation targeted to the spleen, thymus, and lymph nodes, however, the proportion of these cells progressively increases until ultimately they constitute the majority of T cells in the spleen and bone marrow.14,15 This change probably results from the resistance of natural killer T cells, as compared with conventional T cells, to radiation-induced apoptosis due to increased expression of antiapoptotic genes by the natural killer T cells.17 In preclinical studies, murine recipients of allogeneic bone marrow that underwent conditioning with anti–T-cell antibodies and repeated low-dose irradiation targeted to the lymphoid tissue (total lymphoid irradiation) were fully protected from GVHD, whereas mice that underwent conditioning with anti–T-cell antibodies and a single dose of total-body irradiation were not protected.14,15 Studies of the pattern of cytokine secretion by donor T cells in protected hosts showed a polarization toward a pattern of type 2 helper T (Th2) cells, with increased secretion of interleukin-4.15 The Th2 cells assist B cells to produce antibodies and reduce the inflammation promoted by type 1 helper T cells.
In the present study, we adapted the conditioning regimen of total lymphoid irradiation that reduces the incidence of acute GVHD in rodents to the treatment of humans with lymphoid malignant diseases or acute leukemia.
Methods
Patients
Beginning on December 28, 2001, 37 consecutive patients were enrolled in a treatment protocol that was reviewed and approved by the Stanford University administrative panel on human subjects in medical research. Observations were carried out through December 1, 2004. Patients were eligible for the study if they had received a diagnosis of lymphoid malignant diseases or acute leukemia, were 50 years of age or older, were younger than 50 years but had preexisting medical conditions, or had received prior therapy and were considered to be at too high a risk for conventional myeloablative transplantation. For patients with lymphoid malignant diseases, no exclusions were made on the basis of disease status, sensitivity to chemotherapy, or prior bacterial or fungal infection. For patients with acute leukemia to be eligible, bone marrow aspirates were required to show less than 5 percent of blasts within six weeks before the start of the conditioning regimen. No patient refused to undergo the experimental conditioning regimen. All patients provided written informed consent. Patients were to be excluded if they were pregnant or if they had decompensated liver disease, a corrected pulmonary-diffusing capacity of less than 35 percent, a cardiac ejection fraction of less than 30 percent, a Karnofsky performance status of less than 50 percent, or serologic evidence of infection with the human immunodeficiency virus.
The Regimen
Total lymphoid irradiation was administered from a 15-MeV linear accelerator (photon beam) at a dose of 80 cGy daily, starting 11 days before transplantation, until a total of 10 doses (800 cGy) had been delivered. The irradiation consisted of a supradiaphragmatic mantle field, a subdiaphragmatic field that included an inverted Y, and splenic ports encompassing all major lymphoid organs, including the thymus, spleen, and lymph nodes, as used in the treatment of Hodgkin's disease.18 Antithymocyte globulin (Thymoglobulin, SangStat), at a dose of 1.5 mg per kilogram of body weight per day, was given intravenously on days –11 through –7, with day 0 being the day of transplantation (Figure 1A). All patients received prophylactic medications against bacterial, fungal, and viral infections and were monitored for cytomegalovirus and Epstein–Barr virus with the use of a blood polymerase-chain-reaction assay, as previously described.4,7 Immunosuppressive therapy after transplantation included oral cyclosporine started on day –3, at a dose of 6.25 mg per kilogram twice per day, and mycophenolate mofetil, at a dose of 15 mg per kilogram twice a day, started on the first day after transplantation. Among recipients of matched grafts from related donors, cyclosporine was tapered to discontinuation from day 56 to day 180, and mycophenolate mofetil was stopped on day 28. Among recipients of matched grafts from unrelated donors, cyclosporine was tapered to discontinuation from day 100 to day 180, and mycophenolate mofetil was tapered to discontinuation from day 42 to day 96.
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Patients with sibling donors were serologically matched for HLA-A and B antigens and were matched by high-resolution DNA typing for HLA-DRB1 alleles. Patients with unrelated donors were matched for HLA-A, B, and DRB1 alleles with the use of high-resolution (DNA sequencing) molecular typing and for HLA-C and DQB1 alleles with the use of low-resolution molecular typing.
Donor Cells
Related and unrelated donors received a five-day course of subcutaneous granulocyte colony-stimulating factor (at a dose of 16 µg and 10 µg per kilogram of body weight per day, respectively), and mononuclear cells were harvested by leukapheresis. The CD34+ and CD3+ content of cells in the graft was determined according to the standard guidelines of the International Society of Hematotherapy and Graft Engineering.20,21
Assessment of Chimerism and of Antithymocyte Globulin in Serum
The status of hematopoietic chimerism was determined at intervals of one to six months after transplantation by DNA genotyping of simple sequence-length polymorphic markers that encode short tandem repeats, performed by the histocompatibility laboratory at the Stanford Medical School, as previously described in detail.22 The analysis of chimerism was performed on whole blood and on blood mononuclear cells separated into T cells, B cells, and granulocytes with the use of immunomagnetic beads (Dynal) coated with monoclonal antibodies against CD3, CD19, and CD15, respectively. Samples were analyzed at SangStat for the presence of rabbit IgG, with the use of an enzyme-linked immunosorbent assay, and for the fraction of antithymocyte globulin that retained the capacity to bind to human CD3+ T cells (active antithymocyte globulin).19
Control Patients
We studied two groups of control patients. One group of four patients was treated for the myelodysplastic syndrome with hematopoietic-cell transplantation after undergoing conditioning with three doses of fludarabine, at a dose of 30 mg per kilogram, followed by a single dose of total-body irradiation (200 cGy), and the same GVHD and antimicrobial prophylaxis as that received by patients who underwent the experimental conditioning regimen. The other group of five control patients received an allogeneic hematopoietic-cell transplant for the treatment of myeloma after undergoing conditioning with only total-body irradiation (200 cGy), as previously described.7,23
Cytokine Secretion by CD4+ and CD8+ T Cells
Peripheral-blood mononuclear cells from normal control subjects, control patients receiving total-body irradiation, and patients receiving total lymphoid irradiation were stimulated in vitro with phorbol myristate acetate and ionomycin for six hours, with brefeldin A (Sigma-Aldrich) added after two hours.12 The cells were stained with allophycocyanin anti-CD4 monoclonal antibody and with phycoerythrin or fluorescein isothiocyanate anti-CD8 monoclonal antibody and were subsequently permeabilized with the use of a saponin-based reagent (Cytofix-Cytoperm kit, BD Bioscience). After permeabilization, the cells were stained for intracellular cytokines with the use of fluorescein isothiocyanate anti–interleukin-2 monoclonal antibody, phycoerythrin anti–interleukin-4 monoclonal antibody, fluorescein isothiocyanate anti–interferon-
monoclonal antibody, and fluorescein isothiocyanate-anti–tumor necrosis factor 
(TNF-) monoclonal antibody, in accordance with the manufacturer's instructions. The cells were gated on CD4+ or CD8+ cells and analyzed for the percentage of gated cells that were positive on staining for each cytokine with the use of multicolor flow-cytometric analysis.12
Mixed-Lymphocyte–Reaction Assay
Mononuclear cells from normal control subjects and from patients who had undergone total lymphoid irradiation or total-body irradiation were enriched for CD4+ T cells by incubation with phycoerythrin–anti-CD4 monoclonal antibody, with the use of antiphycoerythrin-conjugated immunomagnetic beads, and the cells were then purified on columns (MACS system, Miltenyi Biotech). The CD4+ cells were more than 90 percent pure, as judged by flow cytometry. These cells were tested for proliferative immune responses to alloantigens in culture for five days in complete medium with irradiated (5000 cGy) stimulator cells made from a pool of mononuclear cells obtained from three normal subjects. The cultures were labeled with 3H-thymidine during the last 20 hours and harvested, and the supernatants were assayed in a scintillation counter.24
Statistical Analysis
Comparisons of the values of the results of laboratory tests of samples obtained from patients in different groups and from normal control subjects were calculated with the use of the Wilcoxon two-sample rank test. Actuarial overall survival and progression-free survival were calculated with the use of a Kaplan–Meier analysis.
Results
Patients' Characteristics
Table 1 summarizes the clinical features of the 37 patients enrolled in the study. Of these, 24 had lymphoid malignant diseases and 13 had acute leukemia. Twenty-eight patients had advanced disease, and 13 of the 24 patients with a lymphoid malignant disease had a relapse after undergoing autologous transplantation.
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The total lymphoid irradiation regimen, consisting of 10 doses of lymphoid irradiation of 80 cGy each, was administered over a period of 11 days, and antithymocyte globulin was given on each of the first 5 days (Figure 1A). Patients were admitted to the hospital for the administration of antithymocyte globulin. On completion of the regimen of total lymphoid irradiation, the patients were given an intravenous infusion of granulocyte colony-stimulating factor–mobilized mononuclear cells from HLA-matched related donors (23 patients, 62 percent) or unrelated donors (14 patients, 38 percent). The mean (±SD) absolute numbers of CD34+ hematopoietic progenitor cells and CD3+ T cells in these infusions were 5.8±2.4x106 per kilogram of body weight and 2.4±0.9x108 per kilogram, respectively.
We found that the nonmyeloablative regimen of total lymphoid irradiation and antithymocyte globulin caused only minor neutropenias (Figure 1B). All the patients had persistent lymphopenia, with a marked reduction in CD4+ helper T cells, after transplantation (Figure 1C). Despite the lymphopenia, antithymocyte globulin that bound to lymphocytes (as judged with the use of immunofluorescent staining) was not detected in the recipients' serum after day 7 (Figure 1D). There were too few circulating host T cells at the time of transplantation to determine whether an increase in host natural killer T cells had occurred, as observed in the rodent spleen and bone marrow.14,15
Multilineage donor hematopoietic-cell engraftment, including T cells, B cells, and granulocytes, was uniformly achieved within 56 days after transplantation (Figure 2A). However, the donor T cells subsequently declined in number and were markedly reduced or undetectable within 75 to 200 days after transplantation in 6 of the 37 patients (Figure 2B). The decline in the number of donor B cells and granulocytes was similar to that for the donor T cells (data not shown). In four of these six patients, the loss of donor cells occurred during tumor progression or relapse.
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All patients were monitored after transplantation for the principal manifestations of acute GVHD — diarrhea, rash, and abnormal results on liver-function tests. Standard scores for GVHD on a scale from grade 0 through grade IV were used during the first 100 days after transplantation to diagnose acute GVHD,25 and thereafter, patients were evaluated for chronic GVHD, which was classified as absent, limited, or extensive.26,27
Among the 24 patients with lymphoid malignant diseases (of whom 13 received a transplant from a sibling and 11 from an unrelated donor), acute GVHD was scored as grade 0 in 22 patients, grade I in 1 patient, and grade III in 1 patient (Table 2). GVHD in this last patient responded to corticosteroids. Among the 13 patients with acute leukemia (of whom 10 received a graft from a sibling and 3 from an unrelated donor), acute GVHD was scored as grade 0 in all patients (Table 3).
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Survival and Tumor Response
Among transplant recipients with lymphoid malignant diseases, the follow-up for the first enrolled patient was 1069 days, and for the last enrolled patient it was 222 days; the median follow-up among the surviving patients was 482 days (Table 2). For the 13 patients with acute leukemia, the period of follow-up for the first patient enrolled and the last patient enrolled was 215 and 1041 days, respectively, and the median follow-up of surviving patients was 446 days (Table 3).
Of the 24 patients with lymphoid malignant diseases, 17 (71 percent) survived, all with a Karnofsky performance status score of 100 percent (indicating normal daily functioning) (Table 2). 28 Of the 13 patients with acute leukemia, 10 (77 percent) survived, all of them with a performance status of 90 percent or more (Table 3). For patients who died, the causes of death are given in the footnotes to Table 2 and Table 3.
Only 4 of the 24 patients who received a transplant as treatment for lymphoid malignant diseases were in complete remission when the regimen of total lymphoid irradiation and antithymocyte globulin was started. Eighteen of these 24 patients had evidence of residual disease (partial remission) on clinical examination or on computed tomography, positron-emission tomography, or both, and two patients had progressive disease (Table 2). Of the four patients in complete remission at the time of transplantation, three were still in complete remission at the last follow-up visit and the fourth had a relapse. Of the 18 patients with measurable disease at the start of the conditioning regimen, 12 had complete remission without detectable acute GVHD. All patients whose condition changed from partial to complete remission were in complete remission at the last follow-up visit. Four of 18 patients who had partial remission at the start of the conditioning regimen of total lymphoid irradiation and antithymocyte globulin still had evidence of disease at the last observation; of these 4 patients, 2 had disease progression. The two remaining patients who had partial remission at the time of transplantation died from causes other than relapse within six months after transplantation, and for that reason their disease status could not be ascertained by tumor imaging. However, autopsy revealed microscopical tumor foci in one patient (Patient 7).
All 13 patients with acute leukemia were in the first or a subsequent complete remission before starting the conditioning regimen. Remission status was assigned on the basis of histologic analysis of samples of bone marrow obtained six weeks before the start of the regimen. Of these 13 patients, 9 had a first complete remission and 4 had two or four remissions. Of the nine who had a first complete remission, seven continued to be in complete remission and two had a relapse. Of the four who underwent transplantation while in a second or fourth complete remission, one continued to have a complete remission and three had a relapse. Among the patients with lymphoid malignant diseases, the actuarial overall survival and progression-free survival rates were 62 percent and 55 percent, respectively; among the patients with acute leukemia, the actuarial overall survival and progression-free survival rates were 73 percent and 69 percent, respectively.
Severe Infections
Four patients were hospitalized because of viral infection, fungal infection, or both: one patient had a gastrointestinal cytomegalovirus infection, one had a fungal pneumonitis, and two had multidermatomal herpes zoster or multifocal herpes simplex virus type 1 infection. These four patients and an additional five patients also had bacterial infections. All viral, bacterial, and fungal infections were controlled with the use of antimicrobial therapy, with the exception of one fatal case of bacteremia. In total, 28 of the 37 patients had no evidence of any clinically significant infection.
Function of CD4+ T Cells
We obtained purified mononuclear cells from the blood of the transplant recipients within one to seven months after transplantation and stimulated these cells in vitro with phorbol myristate acetate and ionomycin. The stimulated cells were analyzed for the accumulation in the CD4+ and CD8+ T cells of interferon-, interleukin-4, interleukin-2, and TNF- with the use of immunofluorescent staining and flow cytometry. As shown in Figure 3A, the mean percentages of CD4+ and CD8+ cells that expressed intracellular interferon-, interleukin-2, or TNF- did not differ significantly among the patients who had undergone total lymphoid irradiation, the normal controls, and the control patients who had received transplants and had undergone conditioning with low-dose total-body irradiation (P=0.13 to 0.94 for interferon-, P=0.06 to 0.90 for interleukin-2, and P=0.14 to 0.90 for TNF-, as calculated with the Wilcoxon rank test). Control patients who had received transplants were those without acute GVHD. In contrast, among patients who had undergone total lymphoid irradiation, the mean percentage of CD4+ T cells that expressed interleukin-4 was significantly higher than among the normal controls (P=0.04 by the Wilcoxon rank test) or among control subjects who had undergone total-body irradiation with or without fludarabine (P=0.05 by the Wilcoxon rank test); the mean percentage of interleukin-4–positive CD4+ cells in the total-lymphoid-irradiation group was about five times that in the control groups.
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Discussion
In our study, the incidence of acute GVHD, grades II through IV, among recipients of hematopoietic-cell transplants who had undergone a conditioning regimen of total lymphoid irradiation plus antithymocyte globulin (1 of 37 patients, 3 percent) was markedly lower than that reported in studies of nonmyeloablative conditioning with total-body irradiation, chemotherapeutic agents, or both, conducted by researchers at our institution or elsewhere.1,2,3,4,5,6,7,8,9,10 This reduction in the incidence of acute GVHD is especially noteworthy because 14 of the 37 patients received transplants from unrelated donors. In previous studies of recipients of hematopoietic-cell transplants from unrelated donors, the incidence of acute GVHD grades II through IV was more than 50 percent.5,6,7,9,10
In addition to the low incidence of acute GVHD among the transplant patients, 12 of 16 patients with lymphoid malignant diseases who entered the study while in partial remission and who were evaluated for tumor status after transplantation were in complete remission. The contributions of the conditioning regimen of total lymphoid irradiation and of the antitumor activity of the graft to these changes are difficult to distinguish from each other, except in seven patients who had clearing of the tumor outside the field of the total lymphoid irradiation. Of the 17 patients who entered the study in complete remission, 12 continued to have complete remission, with a median follow-up of 425 days.
In preclinical studies, the protection afforded against GVHD by the conditioning regimen of total lymphoid irradiation plus antithymocyte globulin in wild-type host mice was dependent on host natural killer T cells, since protection was lost in mice in which the CD1d gene was inactivated, causing developmental failure of natural killer T cells in the thymus and other lymphoid tissue.15 After the wild-type mice were treated with total lymphoid irradiation, the natural killer T cells were the main source of host interleukin-4, which subsequently increased the production of interleukin-4 by donor T cells.14,15 In the current study, we were unable to measure directly the number and function of host natural killer T cells, because assays were limited to the blood samples obtained immediately after the patients underwent the conditioning regimen, and these samples contained too few host T cells for testing. Subsequently, we enrolled seven additional patients, and by increasing the volume of the blood samples obtained from each of the patients, we were able to measure the percentage of natural killer T cells in five of these patients. The median percentage of natural killer T cells among all T cells was increased by a factor of 10 after the patients underwent the experimental conditioning regimen, owing to a more profound decrease in the absolute number of non–natural killer T cells than of natural killer T cells (data not shown).
After the patients underwent transplantation, we found a marked increase in the production of interleukin-4 by donor CD4+ T cells in the transplant recipients, as compared with interleukin-4 production by CD4+ T cells in the normal control subjects, as was observed in mice.15 These donor CD4+ T cells also showed a marked reduction in their proliferative response to alloantigenic stimulation in the mixed-lymphocyte reaction. Donor CD4+ T cells from patients who underwent conditioning with total-body irradiation, rather than total lymphoid irradiation plus antithymocyte globulin, showed neither an increased production of interleukin-4 nor a reduced proliferative response.
Although we could not distinguish the separate contributions of total lymphoid irradiation and antithymocyte globulin to protection against GVHD, preclinical studies have shown that the combination of total-body irradiation and antithymocyte globulin fails to provide protection against GVHD.14,15 It is likely that the changes in the function of donor T cells in the patients who underwent total lymphoid irradiation reduced the risk of acute GVHD, since donor T cells with a profile of increased Th2 cytokine secretion have a decreased capacity to induce GVHD in rodents.29,30,31
In conclusion, a regimen of nonmyeloablative conditioning of total lymphoid irradiation plus antithymocyte globulin given before hematopoietic-cell transplantation for lymphoid malignant diseases or acute leukemia can markedly decrease the incidence of acute GVHD while retaining the antitumor effect of the graft.
Supported in part by the National Institutes of Health (grants P01-CA49605, P01-HL-075462, P01-HL-57443, RO1-HL-58250, and RO1-AI-37683) and by a Faculty Scholar Award from the American Society of Hematology (to Dr. Lowsky).
We are indebted to Dr. Kartoosh Heydari for data on natural killer T cells in blood samples obtained from seven patients outside this study; to Dr. Tyson Holmes for the graphic display of data on T-cell chimerism for all patients; and to Aditi Mukhopadhyay for assistance in the acquisition and processing of blood samples.
Source Information
From the Departments of Medicine (R.L., T.T., Y.P.L., S.D.-J., J.A.S., G.G.L., K.E.S.-G., L.J.J., K.G.B., R.S.N., S.S.), Pathology (F.C.G.), Radiation Oncology (R.T.H.), and Health Research and Policy (D.A.B.), Stanford University School of Medicine, Stanford, Calif.
Address reprint requests to Dr. Strober at the Center for Clinical Sciences Research Building, Rm. 2215, 269 W. Campus Dr., Stanford, CA 94305-5290.
References
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Related Letters:
Protective Conditioning for Acute Graft-versus-Host Disease
Ayala E., Kharfan-Dabaja M., Lowsky R., Negrin R. S., Strober S.
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N Engl J Med 2005;
353:2718, Dec 22, 2005.
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cpm). The mean values in five consecutive patients with sufficient yields of CD4+ T cells in each group are shown. The asterisks indicate a statistically significant difference (P
0.05) between the TLI group and the other groups.