Polyclonal antithymocyte globulin (ATG) belongs to a group of immunosuppressive drugs, currently used to prevent and treat acute steroid resistant graft rejection, as conditioning regimens for bone marrow transplantation and to treat hematological disorders such as aplastic anemia or graft-versus-host disease (1–3). ATG is a mixture of purified immunoglobulins M and G of sera from rabbits, horses, or goats immunized with human thymocytes or T-cell lines.
ATG contains antibodies against selectin and integrin family members and immunoglobulin superfamily molecules expressed on the surface of T lymphocytes. Other cell types such as endothelial or B-cells are also recognized by ATG due to shared epitopes between these cells and T-cells (4, 5).
Induction therapy based on continuous or discontinuous administration of ATG is associated with profound depletion of T-, B-, and NK-cells. It has been shown that CD3+ cell counts are lower up for years in patients treated with ATG (6–8). The key mechanisms of ATG action are T-cell depletion and functional alterations of the remaining cells (5, 9, 10). Potential mechanisms for prolonged CD3+ T-cell depletion and dysfunction after ATG treatment include sustained interference with leukocyte responses to chemotactic signals, and inhibition of integrin-mediated cellular adhesion. Furthermore, Fas-mediated activation-induced apoptosis along with potential cellular destruction of lymphoid precursor cells in the bone marrow might also affect peripheral T-cells (5, 9, 11).
Binding of ATG to distinct T-cell surface molecules and modulation of their expression as determined by flow cytometry occurs at low concentrations within 2 hr and may last for as long as 4 weeks (9, 12, 13). Plasma concentration of ATG in vivo depends of application regimens and ranges from 50 and 300 μg/mL (12, 14).
Particularly, a block and/or downregulation of adhesion molecules on leukocytes could play a role in reduced organ infiltration as well as diminished migration to the lymph system by preventing leukocyte adhesion to the endothelium (5, 15). Indeed, experiments with ATG monotherapy in cynomolgus monkeys demonstrated that ATG-coated lymphocytes were inhibited in their migration to the spleen or lymph nodes (9, 16). Upon contact with an antigen-presenting cell (APC), T-cell adhesion and signaling molecules including leukocytes function-associated antigen-1 (LFA-1), the T-cell receptor (TCR)/CD3 complex, and protein kinase C (PKCθ) are relocalized to the T-cell/APC interface. This spatial concentration of molecules, named the immunological synapse (IS) facilitates stable and prolonged interactions of T-cells with APCs and thereby sustained intracellular signaling, which is a prerequisite for full T-cell activation including cytokine production and proliferation as well as further lymphocyte differentiation (17–19). This congregation of specific adhesion and signaling molecules to the IS is an active process, initiated by TCR-mediated dynamic remodeling of the actin cytoskeleton (20). Incomplete IS formation results in an immature IS type that is determined by defective relocalization of the TCR/CD3 complex and is linked with T-cell hypo-responsiveness (17, 21, 22). Accordingly, interference with IS formation has previously been shown to be a mode of action of anti-inflammatory and antirheumatic agents (23–26), suggesting that prevention of T-cell activation by interference with IS formation represents a therapeutic strategy for controlling T-cell responsiveness such as in transplantation rejection. Here we investigated whether ATG impairment of T-cell adhesion properties may lead to disturbed T-cell/APC interactions.
Two preparations of ATG were used in this study: ATG Fresenius (batch SU 01 A-1, Fresenius Biotech, Munich, Germany) and Thymoglobulin (CH-B:TH118-H05, IMTIX, SangStat, Lyon, France), which have been produced by immunization of rabbits with a Jurkat T-cell line and human thymocytes and are referred to as ATG-1 and ATG-2, respectively.
Peripheral blood T-cells (purity >95%) were obtained from healthy volunteers (buffy coats, Red Cross, Vienna, Austria) using Ficoll-Paque (Amersham, Uppsala, Sweden) density gradient centrifugation followed by magnetic depletion of non-T-cells as described (27). The viability of T-cells was >90% as assessed by propidium iodide and trypan blue staining (data not shown).
ATG is mixture of antibodies against distinct adhesion and costimulatory molecules involved in IS formation and T-cell/APC interaction including CD11a (LFA-1 α chain), CD49d (VLA-4), CD50 (ICAM-3), and CD28 (5). In accordance with previous findings (5, 28), treatment of peripheral human T-cells with different concentrations of ATG-1 and ATG-2 (5 to 100 μg/mL) in medium devoid of complement (heat-inactivated serum) at 37°C led to a time- and concentration-dependent downregulation of the adhesion molecules CD11a (LFA-1 α chain), CD50 (ICAM-3) and CD49d (VLA-4; Fig. 1 and data not shown). The same adhesion molecules were similarly downregulated on Jurkat T-cells as assessed by flow cytometry using fluorescein isothiocyanate- or phycoerythrin-conjugated monoclonal antibodies (mAb; all BD Biosciences, San Jose, CA; Fig. 1 and data not shown).
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FIGURE 1.:
Expression of surface molecules on T-cells incubated with ATG. Peripheral blood T-cells (PBTL) and Jurkat T-cells (Jurkat) were incubated with indicated doses of ATG-1 or ATG-2. Surface molecules were measured by flow cytometry at indicated time points. Surface expression of CD11a (A), CD50 (B), and CD28 (C) on T-cells is shown. The diagrams depicts mean immunofluorescence intensities (MFI) of peripheral T-cells of three independent donors or JE6-1 Jurkat T-cells in three different experiments expressed in percent of untreated control±SEM. *P≤0.05; **P<0.01; ***P<0.001 compared to untreated samples (unpaired two-tail Student's t test).
Moreover, we found that the expression of the costimulatory molecule CD28 was significantly reduced by ATG treatment (Fig. 1C). To exclude that ATG prevents surface binding of the fluorescence-activated cell sorting (FACS) antibodies, T-cells where incubated on ice for 30 min in the presence or absence of 100 μg/mL ATG-1 or ATG-2. ATG did not influence binding of FACS antibodies corroborating that ATG indeed triggers downregulation of important adhesion molecules on the surface of T-cells (data not shown).
To analyze whether treatment of T-cells with ATG interferes with IS formation as characterized by relocalization of cytoskeletal, adhesion, and signaling molecules toward the T-cell/APC interface (Fig. 2A), we used a system using Jurkat T-cells and superantigen-pulsed Raji B-cells, which spontaneously and independently of stimulation, form conjugates and after 15 min of coincubation lead to a mature IS in a superantigen-dependent manner as earlier described (24–26). ATG-treated (3 hr) T-cells exhibited significantly reduced relocalization of CD3 and CD11a to the IS even at low ATG concentrations (10 and 20 μg/mL; Fig. 2B). Relocalization of F-actin and PKCθ to the IS was significantly inhibited at ATG concentrations of 50 μg/mL or higher, with the exception of PKCθ at 20 μg/ml ATG-2 (Fig. 2B). In general, ATG-1 and ATG-2 treatment resulted in similar effects (Fig. 2B). These findings show that ATG, partly independent of its effect on surface expression of the molecules, suppresses very early stages of T-cell activation by preventing relocalization of critical molecules to the central (CD3/PKCθ) and the peripheral region (LFA-1/F-actin) of the IS.
FIGURE 2.:
Influence of ATG on the formation of the immunological synapse. Jurkat T-cells were treated for 3 hours with different doses of ATG-1 or ATG-2 and stimulated for 15 minutes with superantigen-pulsed APCs (Raji B cells) or left unstimulated with unpulsed APCs. CD3, CD11a, F-actin, and PKCθ were visualized by indirect immunofluorescence (green). Relocalization of respective molecules was determined by counting at least 100 T-cell/APC conjugates per sample by two individuals in a blinded manner. (A) Typical examples of conjugates, negative and positive for relocalization of indicated molecules are shown. (B) The diagrams show the percentage of conjugates positive for molecule relocalization as mean±SEM of at least four independent experiments. Open bars, unpulsed APCs; black bars, superantigen-pulsed APCs as stimulator cells. *P≤0.05; **P<0.01; ***P<0.001 compared to superantigen-stimulated untreated controls.
To evaluate the effects of inhibited IS formation, which is necessary for stable conjugate formation (17, 20), we analyzed the impact of ATG on conjugate formation using an allogeneic cell system (25). This system monitors the initiation of an anti-T-cell response by detecting conjugate formation between freshly isolated T-cells and allogeneic monocyte-derived dendritic cells (DCs) as APCs. As shown in Figure 3, stable conjugate formation was inhibited by treatment of T-cells with 50 μg/mL ATG, but not at concentrations of 10 μg/mL (data not shown). The higher ATG concentrations needed for inhibition of conjugate formation correlated well with the effect on IS formation (Fig. 2). These findings indicate a profound block of ATG-treated T-cells on the formation of IS and stable conjugates with APCs.
FIGURE 3.:
Effects of ATG on T-cell conjugate formation with allogeneic DCs. Mature monocyte-derived DCs were generated by incubation of CD14 positive cells isolated from buffy coat mononuclear cells, with granulocyte macrophage colony stimulating factor and interleukin-4 for 5 days and subesequent stimulation with 100 ng/ml lipopolysaccharide for 2 days (
25). T-cells and DCs were stained for 30 min at 37°C with 10 μM Snarf-1 carboxylic acid (Molecular Probes; green emission detected in channel FL-1) and 0.5 μM chloromethylfluorescein diacetate Cell Tracker (Molecular Probes; red emission detected in channel FL-3), respectively. T-cells were incubated for 3 hours with different concentrations of ATG-1 and ATG-2. (A) Representative fluorescence microscopic photographs of peripheral blood T-cells (green cells) treated without or with 50 μg/mL ATG-1 or ATG-2 for 3 hr and incubated for 2 hr with allogeneic DCs. (B) Representative FACS analysis plots of T-cells treated without or with 50 μg/mL ATG-1 or ATG-2 for 3 hr and incubated for 2 hr with allogeneic DCs. Conjugates are detected in the upper right quadrant. (C) The diagram shows mean conjugate formation efficiency expressed in percent of untreated control±SEM of four different T-cell/DC combinations using cells obtained from four different donors. ***
P<0.001 compared to untreated conjugates (Student's
t test).
Polyclonal antibodies have been used for more than 40 years in solid organ transplantation (29, 30) but our understanding of their mechanisms of action besides cell depletion is still rudimentary. ATG depletes peripheral blood T-cells through induction of cell death by both complement-dependent lysis and activation-associated apoptosis (10, 31, 32). Interestingly, T-cells that escape depletion in vivo display hyporesponsiveness in mixed leukocyte reactions (11) as do in vitro ATG-treated T-cells (11, 31).
The initial step of T-cell-driven immune responses is the formation of the IS between APCs and T-cells (22). We show here that ATG treatment blocks IS formation, as revealed by inhibition of the translocation of F-actin, the TCR/CD3 complex and CD11a into the IS. Since CD11a is necessary for the formation of high-affinity T-cell/APC interactions via the IS (33, 34), reduced amounts of CD11a and other integrins at the IS due to blocked relocalization processes could impair conjugate formation of ATG-treated T-cells with DCs. Moreover, a direct block of several surface molecules and their subsequent down-regulation by ATG could contribute to the inhibited efficiency of T-cells to form conjugates with APCs.
These data demonstrate that ATG profoundly blocks the earliest stages of T-cell/APC interactions indicating that, apart from its lymphocyte-depleting capacity, a discrete IS disturbing potential may become operative and underlie the observed T-cell dysfunction in patients treated with ATG.
ACKNOWLEDGMENTS
We thank Bianca Weissenhorn for excellent technical assistance.
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