Antithymocyte globulin affects the occurrence of acute and... : Transplantation (original) (raw)
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been established as a curative procedure for a variety of hematologic malignancies and other disorders (1). However, several complications after allo-HSCT remain lethal, such as regimen-related toxicities (RRT) caused by high-dose chemoradiotherapy, graft-versus-host disease (GVHD), and infections. To decrease their incidence, particularly RRT, reduced-intensity stem cell transplantation (RIST) has recently been developed for those who are not eligible for conventional stem cell transplantation because of old age or organ dysfunction (2). The primary concept of RIST is to enhance engraftment using intense immunosuppressive agents rather than myeloablative agents, with the expectation that donor-derived T cells will subsequently eradicate host-derived malignant cells, which is called a graft-versus-leukemia or graft-versus-tumor effect (3). Several immunosuppressive agents, such as purine analogs (cladribine, fludarabine) and antithymocyte globulin (ATG), have been investigated in the RIST procedure to facilitate engraftment (2,4). Both agents, especially ATG, have been shown to reduce the incidence of allograft rejection in allo-HSCT and solid organ transplantation by suppressing the regeneration of host T cells (5). Therefore, we conducted an initial phase I and II study of RIST against hematologic malignancies and found good results with a preimmunosuppressive regimen consisting of cladribine and ATG (6). All of the engrafted patients achieved the successful engraftment of complete donor chimerism, defined as more than 90% donor cells. After confirming the success of the initial trial, we performed RIST using a reduced immunosuppression regimen that did not include ATG, and this also led to successful engraftment (7). Although it is widely known that RRT is reduced by the application of RIST (2,4,6), the incidence and clinical impact of GVHD and infections after RIST have not been well characterized.
Initially, RIST is expected to contribute to reduced GVHD by decreasing the intensity of the “cytokine storm” that prepares the stage for the development of GVHD. However, there is a fine balance between the risk of GVHD or relapse and a graft-versus-leukemia or graft-versus-tumor effect. Hence, fine tuning of GVHD has become an important topic after a RIST procedure.
After nonspecific priming with a cytokine storm, the development of GVHD is initiated by donor T cells that recognize host peptides not present in the donor (8). In this immunologic setting, antigen-presenting cells could play a key role in the immune response after allogeneic transplantation (9). Among antigen-presenting cells, dendritic cells (DC) are the most efficient stimulators of T cells (10). The peripheral blood contains two subsets of immature DC (i.e., DC1 and DC2) (9,11). Both DC1 and DC2 induce the proliferation of allogeneic naive CD4+ T cells and lead to their differentiation into type 1 helper T cells (Th1) (DC1) or type 2 helper T cells (Th2) (DC2) (12). Polarization toward Th1 cells, which secrete interferon-α to promote the generation of cytotoxic T cells, could contribute to the development of GVHD, whereas polarization toward Th2 cells, which secrete interleukin (IL)-4 and IL-10, could result in the suppression of GVHD (13). Hence, it would be important to investigate the correlation between the kinetics of DC and T cells and the occurrence of GVHD after transplantation. In the setting of conventional marrow-ablative stem cell transplantation (CST), host DC and lymphocytes in most tissues are completely depleted by high-dose chemoradiotherapy (14,15); this is followed by the rapid establishment of donor DC chimerism (16). However, little is known about the kinetics of DC1 and DC2 after transplantation, particularly in the RIST setting.
Hence, we analyzed the onset and incidence of GVHD after RIST and compared the results with those observed after CST, particularly focusing on their correlation with the kinetics of the induction of mixed chimerism. Additionally, using flow cytometry analysis, we investigated whether the kinetics of DC and T cells was associated with GVHD. The fact that the background incidence and severity of GVHD are lower in Japan than in other countries (17) should enable a more precise analysis.
MATERIALS AND METHODS
Patients
This study was approved by the Institutional Review Board of the National Cancer Center Hospital. Patients with hematologic malignancies who were not eligible for CST because of their age or organ dysfunction and those with metastatic solid tumors were enrolled in the RIST protocol, while the other patients with hematologic malignancies underwent CST. A total of 72 patients underwent allogeneic peripheral blood stem cell (PBSC) transplantation from an HLA-matched related donor between June 1999 and September 2001. Overall, 39 patients underwent RIST and 33 underwent CST. The patient characteristics are shown in Table 1. We classified the patients into two populations based on preceding chemotherapy and the nature of the underlying disease: heavily pretreated (acute leukemias and non-Hodgkin’s lymphoma) and less heavily pretreated (chronic leukemia, myelodysplastic syndrome, severe aplastic anemia, and paroxysmal nocturnal hemoglobinuria).
Patient characteristics
Blood Stem Cell Collection
Donors were injected with granulocyte colony-stimulating factor at 5 μg/kg subcutaneously twice daily starting 3 days before the first collection of PBSC until the end of collection. Leukapheresis was performed daily using conventional techniques, and the target value of CD34+ cells was set at 3×106/kg of the recipient’s body weight. Collected donor PBSC were then cryopreserved without T-cell depletion using standard methods for subsequent thawing and infusion.
Conditioning Regimens
In the RIST group (Fig. 1), 20 patients received the cladribine or Flu/Bu/ATG regimen (“RIST with ATG”), which consisted of cladribine (Leustatin, Ortho Biotech, Raritan, NJ; 0.11 mg/kg for 6 days) or Flu (Fludara, Schering AG, Berlin, Germany; 30 mg/m2 for 6 days), Bu (busulfan; 4 mg/kg for 2 days), and rabbit ATG (Thymoglobulin, IMTIX-SANGSTAT, Lyons, France; 2.5 mg/kg for 4 or 2 days), whereas 19 received the Flu/Bu regimen (“RIST without ATG”), which consisted of Flu (30 mg/m2 for 6 days) and Bu (4 mg/kg for 2 days). In the RIST with ATG group, the dosage of ATG was decreased after confirming stable engraftment with a 4-day administration of ATG because of a previous observation of severely delayed recovery of CD4+ T cells with the addition of ATG. In the CST group, 17 patients received the Bu/Cy regimen, which consisted of Bu (4 mg/kg for 4 days) and Cy (cyclophosphamide; 60 mg/kg for 2 days), whereas 16 received the Cy/total body irradiation regimen, which consisted of Cy (60 mg/kg for 2 days) and fractionated total body irradiation (2 Gy twice a day for 3 consecutive days).
RIST protocol. In the RIST with ATG protocol, ATG was administered 2 days instead of 4 days (see text). PBSCT, peripheral blood stem cell transplantation.
Supportive Therapies
All patients received 300 μg/m2 granulocyte colony-stimulating factor (filgrastim) from day 6 after transplantation until they achieved an absolute neutrophil count greater than 1.0×109/L. Hemoglobin was maintained at above 7.0 g/dL and the platelet count was maintained at above 20×109/L with filtered and irradiated blood products. Antibacterial and antifungal prophylaxis consisted of 600 mg per day ciprofloxacin and 200 mg per day fluconazole. Prophylaxis against herpes simplex virus was performed with acyclovir at a dose of 1,000 mg per day orally or 750 mg per day intravenously (IV) from day −7 to day 35, followed by low-dose (400 mg/day) oral administration until the end of immunosuppressive therapy. Pneumocystis carinii prophylaxis included trimethoprim/sulfamethoxazole for 14 days before transplantation and twice weekly after engraftment.
Chimerism Analysis
T-cell donor-host chimerism analysis was performed with CD3+ cells by the polymerase chain reaction-based amplification of polymorphic short tandem repeat regions, as previously described (7). Peripheral blood mononuclear cells were separated by the Ficoll-Hypaque method and then by magnetic cell sorting, using anti-CD3 monoclonal antibody combined with immunomagnetic beads (Miltenyi Biotec, Germany), to give CD3-positive and -negative cells as final products.
GVHD Prophylaxis
Prophylaxis against acute GVHD was performed with cyclosporine (CsA) alone in RIST and with CsA and short-term methotrexate in CST. One patient who received RIST with ATG, four who received RIST without ATG, and three who received CST underwent rapid tapering of CsA in an attempt to induce a graft-versus-leukemia or graft-versus-tumor effect, because of chemotherapy-resistant acute leukemia, non-Hodgkin’s lymphoma, or relapsing disease after their first allogeneic transplantation. Rapid tapering was defined as a tapering rate of greater than 25% per week after engraftment or cessation of CsA by day 40. In the rest of the patients, except for severe aplastic anemia and paroxysmal nocturnal hemoglobinuria, CsA was tapered during 5 to 7 weeks with discontinuation by 16 weeks if no GVHD developed. If the patient developed grades II to IV acute GVHD, CsA was resumed to achieve the therapeutic level and methylprednisolone therapy was added at a dose of 1 to 2 mg/kg per day IV.
Acute and Chronic GVHD
Acute GVHD was diagnosed both clinically and histologically in all patients and was classified as grade I to IV according to the criteria of the consensus conference on acute GVHD, as previously described (18). Liver involvement was diagnosed with a biopsy specimen whenever feasible. We inspected and evaluated all organs commonly involved in chronic GVHD, regardless of whether symptoms existed. Chronic GVHD was diagnosed clinically and was classified as either limited or extensive (19). A biopsy specimen of suspected organ was taken if possible.
Infectious Episodes and Cytomegalovirus (CMV) Antigenemia Assay
Clinically definitive infection was defined as an illness that was associated with symptoms and signs consistent with an infection and microbiologic documentation of a pathogen. Microbiologic documentation consisted of isolation of the pathogen by culture from sterile or from nonsterile sites or histologic or immunohistologic evidence.
An antigenemia assay was performed at least once a week after engraftment using the monoclonal antibody HRP-C7 (Teijin, Tokyo, Japan) raised against CMV immediate-early antigen, and preemptive therapy guided by antigenemia against CMV disease was conducted, as previously described (20). To evaluate the risk factors for CMV infection, we used the same definition as Nichols et al. (21) for rising antigenemia (i.e., an increase greater than or equal to twice the initial antigenemia).
Immune Phenotypic Assays
To investigate the recovery time course of DC and T cells after allo-HSCT, we monitored their surface markers as follows. Peripheral blood was collected in tubes containing sodium heparin at 30, 60, 90, 120, 180, 240, 300, and 360 days after transplantation. Three-color immunofluorescent staining was performed using the whole-blood lysis technique, and cells were analyzed on a fluorescence-activated cell sorter (FACSCalibur; Becton Dickinson Immunocytometry Systems [BDIS], San Jose, CA). Briefly, heparinized peripheral blood was divided into 100-μl aliquots and stained with the three appropriate monoclonal antibodies for 30 min at 4°C in the dark. This was followed by the simultaneous lysis of erythrocytes and fixation of leukocytes using BD FACS Lysing Solution (BDIS) for 10 min at room temperature in the dark. Cells were then washed twice with CellWASH (BDIS). Finally, the cells were fixed with CellFIX (BDIS) for flow cytometry analysis. Data acquisition was performed with CellQuest software using a fluorescent or forward scatter threshold. Lymphocytes were identified by forward scatter and side scatter analysis based on 30,000 events, if possible. The following monoclonal antibodies were used in this study: mouse IgG1 (fluorescein isothiocyanate; FITC), mouse IgG1 (phycoerythrin; PE), IgG2b (PE), IgG1 (peridinin chlorophyll protein; PerCP), CD3 (PerCP), CD4 (PerCP), CD4/CD8 cocktail (FITC/PE), CD45 RA (FITC), CD45RO (PE), lineage cocktail 1 (FITC), HLA-DR (PerCP), CD11c (PE), and CD123 (PE). CD4/CD8 cocktail, CD45RA, and CD45RO were purchased from Immunotech (A Coulter Company, Marseilles, France), and the others were from BDIS. Lineage cocktail 1 consisted of CD3, CD14, CD16, CD19, CD20, and CD56 in one vial. CD4 cells were defined as CD4+CD3+, and CD8 cells were considered CD8+CD3+. Proportions of CD45RA and CD45RO were determined in CD4+ cells only. DC were defined as lineage− and HLA-DR+ lesions, and then separated into CD11c+ (DC1) or CD123+ (DC2) subsets.
DC Activation Assay
To investigate the immune function of DC, we analyzed the intracellular production of tumor necrosis factor (TNF)-α only for DC1, because we confirmed that few DC2 exist after allo-HSCT, as previously described (22). Briefly, heparinized whole blood was stimulated with 0.1 μg/mL lipopolysaccharide (Sigma Chemical Co, St. Louis, MO). Brefeldin (Sigma) was then added to inhibit cytokine secretion at a final concentration of 10 μg/mL, and the blood was incubated for 2 hours at 37° and 5% CO2 in polypropylene tubes. Next, 2 mM EDTA was added to arrest activation and remove adherent cells, the blood was separated into FACS tubes (200 μl each), and appropriate anti-surface markers (lineage cocktail 1 [FITC], HLA-DR [PerCP], and CD11c allophycocyanin [BDIS]) were added. After incubation for 30 min at room temperature in the dark, the blood was lysed for 10 min by adding 2 mL of FACS lysing solution (BDIS). Cells were then washed and permeabilized using FACS permeabilizing solution 2 (BDIS) for 10 min. After an additional wash, anti-cytokine antibody (TNF-α; PE) was added and the cells were incubated for 30 min at room temperature in the dark. Finally, the cells were washed and fixed for flow cytometry analysis.
Clinical Endpoints
The primary endpoints of this study were (1) the onset and incidence of acute and chronic GVHD in RIST with or without ATG, in comparison with those in CST; (2) the correlation between the onset and incidence of acute GVHD and chimera status within RIST; and (3) the correlation between GVHD and the recovery kinetics of DC and T cells. The secondary endpoints were the incidence of infection in the three regimens and the correlation between infectious episodes and GVHD or GVHD-related steroid therapy.
Statistical Methods
Differences among the three regimens were evaluated using the chi-square test. Continuous data were compared using the Kruskal-Wallis test. The times to the onset of acute and chronic GVHD, clinically definitive infection, and positive CMV antigenemia were estimated by the Kaplan-Meier method and compared using the log-rank test. To evaluate predictive factors, we performed univariate and multivariate analyses using a Cox proportional hazard model to adjust the hazard ratio for patients who did not have covariates. To determine the risk factors related to rising antigenemia, we first analyzed the probability of event against variables in a univariate analysis and then in a backward stepwise logistic regression analysis. The time course of DC recovery and the intracellular cytokine of DC1 and T cells were evaluated using a two-way analysis of variance.
RESULTS
Patient Characteristics
Both of the conditioning regimens in RIST were tolerated by all of the respective patients. There were nine patients with solid tumors in the RIST with ATG group. Thus, more patients in this group were not in remission and they were less heavily pretreated, compared with those who were treated with RIST without ATG or CST (P =0.047, 0.0008, respectively;Table 1).
The median numbers of infused CD3+, CD4+, and CD8+ cells were comparable among the three regimens (Table 1). Although the number of infused CD34+ cells in RIST without ATG was less than in the other two regimens (P =0.0054), all of the former patients showed prompt hematopoietic recovery. Collectively, all 72 patients successfully achieved engraftment. The median number of days in the neutropenic period was 11 in the RIST regimens (either with or without ATG) and 14 in the CST regimen (P <0.0001).
Chimerism Analysis
Chimerism was assessed with regard to T-cell lineage in patients who received RIST with or without ATG (Fig. 2A). Complete donor-type chimera (>90%) was achieved within 30 days in all patients who received RIST with ATG, whereas this took much longer (up to 90 days) in RIST without ATG, particularly for the T-cell lineage (P =0.038). Complete donor-type chimera was seen in RIST without ATG after 90 days.
(A) Incidence of acute GVHD (grades II-IV) and chimerism analysis of T cells in RIST with ATG or without ATG. (B) Incidence of chronic GVHD.
Acute GVHD
Grades II to IV acute GVHD were diagnosed in 10% (2/20) of RIST with ATG, 63% (12/19) of RIST without ATG, and 33% (11/33) of CST (P =0.0068, log-rank test;Table 2, Fig. 2A). Furthermore, a proportional hazard model showed that only ATG influenced the occurrence of grades II to IV acute GVHD (hazard ratio [HR]=0.16, P =0.014). These findings suggest that ATG is associated with a significantly lower incidence of acute GVHD. The numbers of patients who underwent rapid tapering of CsA were as follows: one in RIST with ATG, four in RIST without ATG, and three in CST. Among these, two in the RIST without ATG group developed grades II and IV acute GVHD, respectively, whereas one in the CST group developed grade II acute GVHD. The median number of days until the onset of acute GVHD in the three regimens was 53 in RIST with ATG, 74 in RIST without ATG, and 27 in CST (P =0.064;Table 2). Interestingly, the onset in RIST without ATG was delayed compared with that in CST. Moreover, there was a close correlation between the onset of acute GVHD and the timing of chimerism induction (Fig. 2A), suggesting that conversion to complete donor-type chimerism prepares the stage for the development of acute GVHD. The patients who received RIST without ATG also tended to be more likely to receive steroid therapy against GVHD compared with those with the other two regimens (P =0.048;Table 2). Overall, steroid therapy was effective and could be tapered immediately except in two patients in RIST with ATG, two in RIST without ATG, and two in CST. Among these six patients, five had received rapid tapering of CsA. Consequently, four patients died of GVHD or GVHD-related complications.
GVHD prophylaxis, incidence of GVHD, and steroid use
Chronic GVHD
Chronic GVHD was diagnosed in 30% (6/20) of RIST with ATG, 63% (12/19) of RIST without ATG, and 64% (21/33) of CST (P =0.017, log-rank test) (Table 2, Fig. 2B). Thus, the incidence of chronic GVHD was significantly lower in RIST with ATG than in the other two regimens. Progressive and extensive chronic GVHD was more frequent in RIST without ATG than in the other two regimens (Table 2), possibly because of the delayed onset of acute GVHD in RIST without ATG in a multivariate analysis (HR=2.63, P =0.0035). The therapeutic response against chronic GVHD with CsA, steroid, or both was satisfactory in all patients.
Immune Phenotype Analysis
In the analysis of T cells, there were fewer CD3+ and CD3+/CD8+ cells in the RIST regimens than in the CST regimen at day 30, but these numbers quickly recovered to within the normal range after day 60 (Fig. 3A,B). The recovery kinetics of CD3+/CD4+ cells were in the following order: CST > RIST without ATG > RIST with ATG (P <0.0001;Fig. 3C). RIST without ATG and CST showed normal ranges within 360 days after transplantation, whereas RIST with ATG did not show normal ranges. Notably, there were significantly fewer CD4+/CD45RA+ cells, a marker of naive CD4+ T cells, in RIST with ATG than in RIST without ATG (P <0.0001) or in CST (P <0.0001;Fig. 3D). Moreover, the recovery profiles in CST and RIST without ATG were similar throughout the initial year. On the other hand, CD4+/CD45RO+ cells, a marker of memory CD4+ T cells, showed a profile similar to CD3+/CD4+ (P <0.0001;Fig. 3E), whereas all three regimens recovered to within the normal range by 240 days after transplantation. These findings suggested that ATG contributed to the delayed reconstitution of CD4+ T cells after transplantation.
(A to E) Phenotypic recovery of T cells after RIST with ATG, RIST without ATG, and CST. CD3+, CD3+/CD8+, CD3+/CD4+, CD4+/CD45RA+, and CD4+/CD45RO+, respectively. The mean value and standard error are shown by dot and whisker plots. The shaded area shows the reference range (25th to 75th percentile in 12 healthy adult donors).
In infused grafts, there were significantly more DC2 than DC1 (P =0.008;Fig. 4A). After engraftment, the recovery kinetics of blood DC and the intracytoplasmic production of TNF-α in DC1 cells approached normal ranges, with no essential difference among the three regimens (P =0.40 and 0.61, respectively;Fig. 4B,C). Interestingly, the DC1/DC2 ratio was greater than 1.0 in all three regimens by day 30 after transplantation, despite the predominance of DC2 in infused cells (Fig. 4D).
(A) DC1 and DC2 counts in infused graft. DC counts in peripheral blood (B), intracytoplasmic TNF-α of DC as a percentage (C), and the DC1/DC2 ratio (D) were measured after RIST with ATG, RIST without ATG, and CST. The mean value and standard error are shown by dot and whisker plots. The shaded area shows the reference range (25th to 75th percentile in 12 healthy adult donors).
Infections
The incidence of clinically definitive infection was not different among the three regimens (Table 3). Although the relative incidence of fungal and viral infection could not be meaningfully analyzed because of the small number of patients, the incidence of viral infection tended to be higher in CST than in the other two regimens. CMV diseases consisted of three cases of gastroenteritis and one case of cystitis, whereas adenovirus infection was manifested as hemorrhagic cystitis in all cases. Although there was no difference in the incidence of CMV antigenemia itself, the numbers of episodes of rising antigenemia were marginally different among the three regimens (P =0.08) (Table 3). In a univariate analysis using the Cox regression hazard model, factors identified as marginally significant were steroid therapy for clinically definitive infection [HR=1.8, 95% confidence interval (CI)=0.96–3.3, P =0.066] and grades II to IV acute GVHD and steroid therapy for CMV antigenemia (HR=1.8 and 1.8, 95% CI=0.9–3.3 and 0.95–3.3, P =0.079 and 0.073, respectively). Multivariate analysis did not show any independent significant factors. Grades II to IV acute GVHD and steroid therapy were identified as risk factors for rising antigenemia in a univariate analysis (odds ratio=17.2 and 26.7, 95%CI=3.4–86 and 3.2–219, P =0.0005 and 0.0022, respectively). A backward stepwise logistic regression analysis showed that only steroid therapy independently influenced the incidence of rising antigenemia.
Numbers of clinically definite infections and CMV antigenemia among the three regimens
DISCUSSION
Although RIST has been the subject of recent intense clinical research, the benefit of ATG, with regard to the occurrence of GVHD, immune kinetics, and infectious complications, has not been established. The effect of rabbit ATG in the prevention of GVHD was previously evaluated in two randomized studies on bone marrow transplantation from an HLA-matched unrelated donor after conditioning with a conventional Cy/total body irradiation regimen (23). As a result, the overall incidence of extensive chronic GVHD was lower in patients who received ATG than in those who did not (39% vs. 62%). In this study, we analyzed the contribution of ATG by focusing on the occurrence of acute and chronic GVHD and the recovery kinetics of T cells and DC. We also examined the incidence and clinical characteristics of infectious episodes after RIST with ATG. Consequently, we found that the incidence of acute and chronic GVHD in RIST was notably lower with ATG. Although it has been controversial whether the incidence of GVHD is lower after RIST or CST, it is noteworthy that in our study a lower dose of ATG in the RIST procedure suppressed the development of acute and chronic GVHD after allo-HSCT.
Using the same type of RIST regimen, Slavin et al. (2) reported that the incidences of grades I to IV and grades III and IV acute GVHD was 46% (12/26) and 25% (6/26), respectively, whereas the incidence of chronic GVHD was 35% (9/26); these are comparable to values previously published for the CST procedure (24). We think that the lower incidence of GVHD in our study reflects the more homogeneous distribution of HLA in Japan. Bornhauser et al. (25) used a Flu/Bu without ATG regimen, and the incidences of grades II to IV acute and chronic GVHD was 15% (3/20) and 25% (5/20), respectively, which were even lower than our findings. Moreover, the median number of days to the onset of acute GVHD was 17, which was shorter than in our analysis. However, it is not possible to directly compare their results with either our present findings or those of Slavin and coworkers because they used methotrexate or mycophenolate mofetil in addition to CsA as prophylaxis against GVHD, whereas we did not. Likewise, additional intensive immunosuppression with CAMPATH-1H resulted in the reduction of acute and chronic GVHD (26). Furthermore, we still have to consider the possibility that the difference in the patients’ background, including the disease status and pretransplantation treatment history, affected the occurrence of acute GVHD, although this was discounted by the results of a multivariate analysis.
An additional important finding in this study is the close chronologic relationship between the onset of acute GVHD and the induction of mixed T-cell chimerism in RIST. Whenever GVHD occurred, the onset was significantly delayed in RIST without ATG compared with the conventional regimen, despite the fact that the overall incidence of acute GVHD in RIST without ATG was high compared with that in CST. Other recent reports have noted that the achievement of T cell complete chimera preceded the onset of acute GVHD (4,27) (i.e., the incidence of GVHD might become low under mixed T-cell chimerism, although controversial data have been published; (28). In any event, the results suggest that the delayed achievement of donor T-cell chimerism may contribute to the delayed onset of acute GVHD.
There is a concern that the use of ATG may increase the rate of serious infections, including CMV infection (29). However, in the present study, our analysis of risk factors for CMV infection, especially rising antigenemia, showed that both acute GVHD and steroid therapy were significant factors, whereas ATG was not. Moreover, the observed incidence of clinically definitive infection, which included CMV, was comparable among the three regimens. It is likely that the higher risk of CMV infection with the use of ATG more than offsets the risk of more frequent use of steroids to control the higher incidence of GVHD in those who were not treated with ATG.
In our immunophenotype analysis, the reconstitution of CD3+/CD4+ cells could be clearly divided into three characteristic patterns. The number of CD4+/CD45+RA cells (naive CD4+ T cell) in RIST with ATG remained significantly lower than in the other two regimens. Because ATG has a prolonged half-life in humans (30), the combination of CsA and ATG could intensively deplete T lymphocytes in donor PBSC graft to suppress the regeneration of T lymphopoiesis after engraftment (31). Therefore, it is likely that this delayed reconstitution of naive CD4+ T cells resulted in the lower incidence of GVHD in RIST with ATG, because interaction between naive CD4+ T cells and DC is critical for mediating the immune response (32). DC2 induce naive CD4+ T cells toward Th2 cells, which secrete immunosuppressive cytokines, IL-4 and IL-10, to suppress GVHD. Hence, the kinetics of naive CD4+ T cells may play a crucial role in the development of GVHD. In our study, the DC1/DC2 ratio was greater than 1.0 after day 30, with no difference among the three regimens. Because the function of DC1 seemed to be the same in the three cohorts, the low number of naive CD4+ T cells may have partly contributed to the development of GVHD.
CONCLUSION
We examined the effect of adding ATG to a RIST conditioning regimen. We found that the development kinetics of acute and chronic GVHD after RIST are complex, because they are sensitively modulated by the use of immunosuppressive therapy before and after transplantation and the variable use of steroids. Nonetheless, our results suggest that naive CD4+ T cells may affect the development of GVHD and that the delayed achievement of complete T-cell chimerism in a regimen without ATG may be responsible for the delayed onset of acute GVHD. The control of GVHD with ATG may still outweigh the suggested risk of infections, which is increased with the use of steroid. Considering these findings together, the conditioning regimen and immunosuppressive strategy should be carefully designed after RIST and should depend on the urgent risk of disease recurrence and GVHD.
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