Antithymocyte globulins (ATGs) are polyclonal IgG preparations derived from rabbits immunized with the T-cell leukemia line Jurkat (ATG-Fresenius [ATG-F]) or human thymocytes (Thymoglobulin [THG]). ATGs are potent immunosuppressive and immunomodulatory agents for the therapy of aplastic anemia and for treatment and prevention of graft versus host disease in allogeneic hematopoietic stem-cell transplantation and solid organ graft rejection (1). ATGs cause depletion of T lymphocytes and other leukocytes through various mechanisms including complement or antibody-dependent lysis and the induction of apoptosis (1–3). In addition, immunomodulatory effects might also contribute to the benefits of ATG administration, although this aspect of ATG function is currently incompletely understood (4). Furthermore, ATG treatment can also result in overimmunosuppression of treated patients associated with an increased risk of opportunistic infections and posttransplant lymphoproliferative disease (1). It is evident that both beneficial and adverse effects of ATG treatment result from the interaction of ATG antibodies with leukocytes and other host cells. In contrast to therapeutic monoclonal antibody preparations, such as anti CD3 or IL2 receptor antibodies, rabbit ATGs are a mixture of specific antibodies and antibodies not directed to human antigens. Consequently, the functional activity of ATGs could be affected by variations regarding the antibody concentrations directed to different leukocyte antigens.
ATG-F and THG are used for the treatment of different medical conditions. The heterogeneous condition of the patients makes the evaluation of a diverse outcome of an ATG therapy challenging. The clinical efficacy could be affected by the condition of each individual patient or by deviation of the amounts of ATG antibodies directed to human cell surface antigens originating from different batches. Although the manufacturers take extensive measures to minimize variations in ATGs, it is impossible to avoid them in polyclonal antibody preparations.
In this study, we have compared different batches of ATG-F and THG regarding their reactivity to Jurkat cells and in vitro activated peripheral blood mononuclear cells (PBMCs). Furthermore, the reactivity of these ATGs to a panel of cell lines engineered to express ATG antigens at high levels was analyzed. Finally, we used a flow cytometric assay to compare different ATG preparations regarding their capability to mediate complement-dependent cytotoxicity (CDC) of human monocytes and lymphocytes. Our results demonstrate that there is little variation in ATG lots of both manufacturers. Therefore, it can be expected that ATG batches should not differ in their clinical efficacy.
RESULTS
Interaction of Different Batches of ATG With Jurkat Cells and Activated PBMCs
Binding to cell surface antigens is a prerequisite for ATGs to exert their clinical effects. Consequently, we compared ATGs derived from different batches regarding their ability to interact with human surface antigens. Different concentrations of ATG preparations were probed with the Jurkat cell line or with in vitro activated human PBMCs. Bound rabbit antibodies were detected with fluorescence-labeled secondary antibodies using fluorescence-activated cell sorting (FACS) analysis. A dose-dependent increase in the fluorescence signal was obtained with all samples (Fig. 1). Although all THG batches had a higher binding capacity to activated PBMCs than the ATG-F samples, there was little variation in respect of the binding signal obtained with different batches of both drugs.
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FIGURE 1.:
Interaction of different batches of antithymocyte globulin (ATG) with Jurkat cells and activated peripheral blood mononuclear cells (PBMCs). Jurkat cells (A) and in vitro activated PBMCs (B) were incubated with four different batches of ATG-Fresenius (ATG-F) (upper panels) or Thymoglobulin (THG) (lower panels) at final concentrations of 0.1–16.7 μg/mL. Bound rabbit antibodies were detected by allophycocyanin-conjugated donkey-anti-rabbit IgG. All data points represent the mean fluorescence intensity (MFI) from five experiments. Error bars indicate the SD.
Specific Antigen Binding of ATGs Derived From Different Batches
In the next set of experiments, we addressed whether ATG preparations differ in regard of their antibody composition. For this purpose, we engineered the murine thymoma cell line Bw to retrovirally express different human leukocyte surface antigens that were previously identified to serve as antigens for ATG-F, THG, or for both preparations (1, 5). The high expression of the selected molecules (CD2, CD4, CD5, CD7, CD38, CD45, CD50, CD82, CD98, CD102, CD147, and HLA-DR) on the resultant Bw cells was confirmed by FACS analysis using mAb to these antigens (Fig. 2A). Specific reactivity to all Bw transductants selected for analysis of ATG-F binding was detected in all batches. There was little variation in the binding signals, indicating that similar amounts of antibodies to these antigens were present in all batches of ATG-F (Fig. 2B). Similar results were obtained with the Bw transductants that were selected for the analysis of THG-binding (Fig. 2C). In a previous study, we have demonstrated that ATG-F but not THG strongly interacts with CD102, whereas THG but not ATG-F contains significant amounts of antibodies to CD4 and major histocompatibility complex class II (HLA-DR) (5). These results could be confirmed when analyzing different batches of both antithymocyte preparations (Fig. 2B, C, data not shown).
FIGURE 2.:
Reactivity to selected human leukocyte antigens of different batches of antithymocyte globulin-Fresenius (ATG-F) and Thymoglobulin (THG). (A) Bw cell lines engineered to express the indicated human surface molecules were probed with specific mAbs and analyzed by flow cytometry (black histograms). Reactivity of these mAb to the parental Bw cell line is also shown (open histograms). Control Bw cells (open bars) or cells expressing the indicated antigens (black bars) were incubated with four different batches of ATG-F (B) or THG (C) at a final concentration of 16.7 μg/mL. All data points represent the mean fluorescence intensity (MFI) from five experiments. Error bars indicate the SD.
Competitive Binding of ATGs to Activated PBMCs and Jurkat Cells
ATGs contain antibodies to numerous human leukocyte surface antigens, including molecules that are not identified to date. Although, the experiments summarized in Figures 1 and 2 demonstrate similar amounts of antibodies directed to human PBMCs and to selected antigens in different ATG batches, we wanted to evaluate whether there are significant variations in the specificity profile of ATGs derived from different lots. We reasoned that compared with ATG batches with similar or identical antibody compositions, ATGs that vary in their antibody composition would have reduced capacity to compete for binding. Therefore, we generated labeled ATGs (ATG no. 1 and THG no. 2) using the fluorescence label DyLight 649. Jurkat cells and activated PBMCs were preincubated with varying concentrations of unlabeled ATGs derived from different batches and subsequently probed with fluorescence-labeled ATGs. The unlabeled competitor ATGs reduced the binding of fluorescence-labeled ATGs in a dose-dependent manner. Importantly, ATGs derived from the same batch as the labeled ATGs did not show a significantly higher capacity to reduce the fluorescence signal compared with competitor ATGs derived from different batches (Fig. 3). This indicates that also in respect of their overall antibody composition, there is little variation in ATGs derived from different batches. Furthermore, we analyzed the ability of ATG-F to inhibit binding of DyLight 649-labeled THG and the capacity of THG to compete with the binding of ATG-F-DyLight 649. ATG-F inhibition of the binding of THG to Jurkat cells was weaker than all batches of THG and vice versa. This is most likely due to differences in the composition of Jurkat reactive antibodies in these two ATG preparations. THG had a much stronger reactivity to activated PBMCs than ATG-F. Thus, THG blocked ATG-F binding more effectively than all batches of ATG-F. As expected, the ability of ATG-F to compete with THG for binding to activated PBMCs was much weaker (Fig. 3). Taken together, the results of our competitive binding experiments indicate that the antibody composition of ATGs do not significantly vary in different batches derived from one manufacturer, whereas preparations from different sources have a distinct antibody profile.
FIGURE 3.:
Competitive binding of antithymocyte globulins (ATGs) to activated peripheral blood mononuclear cells (PBMCs) and Jurkat cells. Jurkat cells (A) or in vitro activated PBMCs (B) were preincubated with ATG-Fresenius (ATG-F) or Thymoglobulin (THG) derived from different batches at the indicated final concentrations. ATG-F- DyLight 649 (upper panels) or THG- DyLight 649 (lower panels) was added at a final concentration of 4.2 μg/mL and bound fluorescence-labeled antibodies were measured by fluorescence-activated cell sorting (FACS). All data points represent the mean fluorescence intensity (MFI) from five experiments. Error bars indicate the SD.
Cytotoxic Activity of Different Batches of ATG-F and THG
The clinical benefit of ATG treatment is largely ascribed to a rapid and profound depletion of T cells and other leukocytes by CDC (1, 2, 6). Consequently, we established a FACS-based cytotoxicity assay to compare different batches of ATGs regarding their ability to mediate complement dependent lysis of human lymphocytes and monocytes (Fig. 4A). As shown in previous in vitro studies, the ability of ATGs to induce cytotoxic effects on human cells in the presence of rabbit complement was analyzed (7, 8). Moreover, we also wanted to assess the cytotoxic effects of ATGs in a more physiological setting. Therefore, we performed additional experiments, where human serum instead of rabbit complement was added.
FIGURE 4.:
Cytotoxic activity of different batches of antithymocyte globulin-Fresenius (ATG-F) and Thymoglobulin (THG). Human peripheral blood mononuclear cells (PBMCs) were left untreated or incubated with ATG-F or THG derived from different batches at the indicated final concentrations. After incubation with human serum or rabbit complement, cells were analyzed by flow cytometry. Dead cells were identified by propidium iodide staining and excluded from the analysis. (A) Gating strategy to assess the number of viable lymphocytes and monocytes. Percent specific lysis induced by ATGs in lymphocytes (B) or monocytes (C) in the presence of human serum (upper diagrams) or rabbit complement (lower diagrams) is shown. Anti-major histocompatibility complex (MHC) class I mAb and purified rabbit IgG (rIgG) were used as positive and negative control for cell lysis. (Bars) Median cell lysis determined from five experiments. Error bars indicate the SD.
When PBMCs were incubated with ATG-F at a final concentration of 20 μg/mL, approximately 50% to 60% of lymphocytes and monocytes were lysed on addition of human serum. Under these conditions, cell lysis induced by THG was considerably higher (Fig. 4B, C). On addition of rabbit complement, the cytotoxic effects of the ATG preparations was much stronger, despite the fact that lower antibody concentrations were used (4 μg/mL; Fig. 4B, C). Our results demonstrate that different batches of ATG-F and THG mediated the complement dependent lysis of lymphocytes and monocytes with comparable efficiency. Furthermore, the cytotoxic effects exerted by THG were generally stronger than those mediated by ATG-F.
Interaction of Different ATG Batches With PBMCs From Kidney Transplant Recipients
In a therapeutic setting, ATGs are used for treatment and prevention of graft rejection episodes. Therefore, we wanted to evaluate the interaction of different batches of ATG-F and THG with PBMCs of transplant recipients.
First, the binding of different ATG batches to PBMCs of stable kidney transplant recipients or recipients with biopsy-proven acute rejection was analyzed (Fig. 5A, B). Patients' PBMCs were incubated with four batches of ATG-F and THG at different concentrations and analyzed by flow cytometry. Different batches of ATG-F or THG bound to the patients' PBMCs with comparable efficiency. In addition, the obtained fluorescence signal of the dose-dependent binding curves for all batches of THG was higher than for ATG-F.
FIGURE 5.:
Interaction of different antithymocyte globulin (ATG) batches with patients' peripheral blood mononuclear cells (PBMCs). The interaction of four different batches of ATG-Fresenius (ATG-F) and Thymoglobulin (THG) with PBMCs from a stable kidney transplant recipient (left panels) and a recipient with acute rejection (right panels) is shown. Binding of ATG-F and THG antibodies at indicated concentrations was assessed by flow cytometry (A+B). To assess the cytotoxic activity, PBMCs were left untreated or incubated with four different batches of ATG-F or THG (20 μg/mL). After incubation with human serum, cells were analyzed by flow cytometry. Percent specific lysis induced by ATGs in lymphocytes or monocytes is shown (C+D). All data points represent the mean fluorescence intensity (MFI) from four measurements. Error bars indicate the SD.
Furthermore, we assessed the ability of different batches of ATG-F and THG to mediate complement dependent lysis of patients' lymphocytes and monocytes (Fig. 5C, D). After incubation with ATG at a final concentration of 20 μg/mL and subsequent addition of human serum, the percentage of lysed monocytes and lymphocytes did not significantly differ from batch to batch. As observed with PBMCs from healthy donors, compared with ATG-F preparations, the complement dependent lysis mediated by all batches of THG was substantially higher.
DISCUSSION
The first report describing the successful use of ATG in human kidney transplantation was published almost 50 years ago (9). Despite the enormous expansion of the armamentarium of immunosuppressive drugs, ATGs are still used in the field of solid organ transplantation, stem-cell transplantation and for the treatment of patients suffering from aplastic anemia. Importantly, the introduction of therapeutic monoclonal antibodies to CD3 or CD25 (muromonab, basiliximab, and daclizumab) has not resulted in a replacement of ATGs. In several studies, ATGs were found to be superior to these monoclonal antibodies (10–12). Furthermore, the clinical use of ATGs has continuously been extended regarding the therapeutic regimen and the medical indications, such as the treatment of various conditions including type I diabetes and B-cell-mediated autoimmune diseases such as systemic lupus erythematodes (13–15).
Initially, ATGs were often home made preparations produced by each individual clinic. Such preparations showed considerable variations in their clinical efficacy. For example, based on clinical data Wakabayashi et al. (16) identified poor and good preparations, which differed significantly in respect of their binding capacity to human T cells. During the last decades such preparations have been replaced by commercial products with THG and ATG-F being the most widely used ATGs. Although, manufacturers have considerably more possibilities to standardize antibody preparations, differences between batches that could potentially affect their efficacy remain a concern in the use of ATGs. Clinical effects observed with the administration of ATG can substantially vary. Therefore, it is difficult to predict whether such differences are mainly due to intraindividual differences and the conditions of the treated patients, or whether variations in the ATG preparations significantly contribute to such differences. Despite this, to our knowledge, there are no studies where commercial antithymocyte globulin preparations have been extensively analyzed in this respect.
In this study, we have tested four different batches of THG and ATG-F regarding their capacity to interact with human cells. Both preparations showed little variation in respect of their binding to Jurkat cells and to in vitro activated human PBMCs and to PBMCs from kidney transplant recipients. Previous studies have largely relied on blocking studies with mAbs to assess the interaction of ATGs with human distinct leukocyte surface molecules (15, 17–19). Although such studies have been invaluable for the identification of ATG antigens, this method can only detect rabbit antibodies that have identical or overlapping epitopes with the mAbs used. In addition, this approach does not allow obtaining reliable information about the amount of ATG antibodies directed to the antigens of interest. We have recently demonstrated that cells engineered to express high levels of ATG antigens are excellent tools to assess the interaction of ATG with human molecules (5). In the current study, we have also used this method to directly assess binding of different ATG batches to human leukocyte antigens from various molecule families, which were expressed at high levels on a murine cell line (Fig. 2). This novel approach allows for standardized measurement and comparison of ATG antibody amounts originating from different lots or sources to specific leukocyte antigens. We found that the amounts of antibodies to the tested leukocyte antigens were similar in the analyzed batches. Many ATG antigens have not been identified to date. Therefore, by demonstrating that the reactivity to known ATG antigens is similar in different batches, it cannot ruled out that there are batch to batch variations in the reactivity to unknown ATG antigens. However, we could show that different batches of ATGs had a similar capacity to reduce the binding of fluorescence-labeled ATGs to human cells (Fig. 3). This strongly suggests that the overall antibody composition of ATGs does not significantly vary from batch to batch. In contrast, the antibody composition seems to differ between ATG-F and THG. This is not only reflected by our binding studies to Bw-transductants expressing different leukocyte antigens but also by the fact that the obtained binding interaction to PBMCs and Jurkat cells was higher for THG.
Rapid depletion of T cells and other leukocytes is generally regarded as a major mechanism for the potent immunosuppressive effects of ATG treatment. Therefore, we also compared the ability of ATGs derived from different lots to mediate CDC to lymphocytes and monocytes in vitro. In line with the results obtained in our binding studies, we found that cytotoxic effects mediated by the analyzed batches of ATGs were similar. Furthermore, we could demonstrate that all batches of both ATG preparations also mediated complement-dependent lysis of patients' lymphocytes and monocytes. Therefore, it can be expected that under clinical conditions, the functional interaction with PBMCs of transplant recipients will not differ from batch to batch. In one ATG-F preparation (ATG-F no. 2), the reactivity to cells expressing human antigens seemed to be slightly lower. However, this ATG-F preparation strongly reacted with all human antigens tested. Furthermore, the ability of this ATG preparation to bind to human PBMCs and to mediate complement-dependent lysis of human leukocytes was affected to an even lesser extent.
Overall, the high conformity in ATG preparations of both manufacturers found in our study makes variations of different batches of ATGs in respect of their clinical efficacy unlikely. Moreover, the methods described in this study allow for a comprehensive evaluation of an ATG preparation in clinical use regarding their antibody composition and their ability to mediate lysis of human leukocytes. ATG-F and THG are polyclonal antibody preparations from different sources and our data demonstrate that these two preparations differ in their antibody composition and in their functional activity. Thus, it is not surprising that there are profound differences in the regimen of these two preparations.
Our findings also have implications on contrasting results regarding the immunomodulatory effects exerted by THG in vitro. Although several studies have reported on the ability of THG to induce regulatory T cells, Broady et al. (20) did not find evidence for such an effect. In preliminary experiments, we observed that THG and ATG-F treatment induced upregulation of CD25 but not Treg function in human T cells (unpublished results). From this study, it seems that the contradictory findings regarding the ability of ATGs to induce Tregs cannot be explained by different functional effects of the lots used by the investigators.
MATERIALS AND METHODS
Human Subjects
Human peripheral blood samples were obtained from healthy volunteers or kidney transplant recipients from the Department of Internal Medicine III, Clinical Division of Nephrology and Dialysis, Medical University of Vienna. Informed consent was obtained from all donors and the study was approved by the ethical review board of the General Hospital and Medical University of Vienna. Renal transplant recipients aged between 22 and 72 years were categorized into stable transplant recipients (n=7) and patients with biopsy-proven acute rejection (n=2). Stable transplant recipients were defined to have serum creatinine levels less than 3 mg/dL, whereas acute rejection episodes underwent histologic diagnosis and were graded according to the Banff criteria.
Cell Culture
PBMCs and serum were derived from human peripheral blood samples and cultured as described (21). For activation of PBMCs, cells (2Ă—106 cells/mL) were cultured for 72 hr in the presence of phorbol myristate acetate and ionomycin (both purchased from Sigma-Aldrich, Deisenhofen, Germany, final concentration of 100 nM).
Bw 5147, a mouse thymoma cell line (designated within this work as Bw cells) and the Jurkat cell clone 41-19 (designated as Jurkat cells) were cultured as described (22).
Bw transductants were generated as described previously (22). Briefly, expression plasmids encoding for the indicated molecules and green fluorescent protein (GFP) were generated by cloning polymerase chain reaction-amplified cDNAs encoding the molecules of interest into the retroviral expression vector pCJK2 as previously described (23). Bw cell lines expressing high levels of human surface molecules or GFP were generated by retroviral transduction as previously described (24).
For cytotoxicity assay, Bw transductants expressing GFP (designated as GFP+ Bw cells) were fixed with fixation medium (An Der Grub Bio Research, Kaumberg, Austria) according to the manufacturer's brochure.
ATG Preparations
Four different batches of ATG-F (Fresenius Biotech, Gräfelfing, Germany) and THG (Genzyme, Cambridge, MA) were tested in the described experiments: ATG-F no. 1 (T12N-2), ATG-F no. 2 (U01A-2), ATG-F no. 3 (Z07E-2), and ATG-F no. 4 (A03C-2); THG no. 1 (C9062H04), THG no. 2 (C9074H27), THG no. 3 (C9078H17), and THG no. 4 (C0074H14).
To generate fluorescence-labeled ATGs, ATG-F no. 1 and THG no. 2 were conjugated with the amine-reactive fluorescent dye DyLight 649 (Pierce Biotechnology, Meridian Rd, Rockford) according to the manufacturer's protocol.
Antibodies and Flow Cytometric Analysis
Flow cytometric measurements were performed using a FACScalibur flow cytometer supported by CELLQUEST software (Becton Dickinson, San Jose, CA). For the detection of bound ATGs, APC-conjugated donkey-anti- rabbit-IgG(H+L) antibodies (Jackson ImmunoResearch, West Grove, PA) were used. Competitive binding studies were performed by probing Jurkat cells and in vitro activated PBMCs with different batches of ATG-F or THG at the indicated concentrations. After preincubation for 10 min, ATG-F-DyLight 649 or THG-DyLight 649 was added at a final concentration of 4.2 μg/mL for 20 min. Samples were washed and bound fluorescence-labeled ATGs were measured by FACS.
Antigen expression of Bw transductants was confirmed by incubation of mAbs directed to the indicated molecules and PE-labeled goat-anti-mouse-IgG (H+L) antibodies (Jackson ImmunoResearch). CD2, CD4, CD5, CD7, CD38, CD45, CD50, CD98, CD102, CD147, and HLA-DR mAbs were produced at our institute. CD82 mAb was purchased from BioLegend (San Diego, CA).
Activation of human PBMCs was controlled after 72 hr by staining for CD25 and CD69 with APC-labeled mAbs (BD Bioscience Pharmingen, San Diego, CA) directed to these activation markers.
Cytotoxicity Assay
Tubes (1.4 mL, round bottom, Thermo Fisher Scientific, Meridian Rd, Rockford) containing freshly isolated PBMCs (1.3Ă—105 cells at 6Ă—106/mL in culture medium) were left untreated or incubated with ATG-F, THG, ChromPure Rabbit IgG (Jackson ImmunoResearch) at the indicated final concentrations for 60 min at 4°C. Cells treated with an anti-major histocompatibility complex class I mAb (clone W6/32; final concentration 6.7 μg/mL) were also analyzed. Afterwards, samples were treated with 20 μL of medium, human serum or rabbit complement-MA (Cedarlane Laboratories, Ontario, Canada). After an incubation period of 60 min at 37°C, fixed GFP expressing Bw cells (5Ă—103) and propidium iodide (Sigma-Aldrich) were added to each sample. Subsequently, samples were immediately subjected to flow cytometric analysis. Fixed GFP+ Bw cells were used to standardize the acquired volume for each sample and the analysis was stopped when 1Ă—103 GFP+ Bw cells had been acquired. The number of viable monocytes and lymphocytes was determined for each sample by gating propidium iodide-negative cells according to their size and granularity. The percentage of specific lysis was calculated using the formula:
ACKNOWLEDGMENTS
The authors appreciate the excellent technical assistance of Petra Cejka, Claus Wenhart, and Johannes Popow.
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