Polyclonal anti-thymocyte globulins have been in clinical use for more than 30 years (1–3). Rabbit anti-thymocyte globulin (Thymoglobulin®; Genzyme Transplant, Cambridge, MA) is currently used in the treatment of aplastic anemia as well as in the prevention and treatment of allograft rejection and graft-versus-host disease (GVHD) in solid-organ and stem cell transplant recipients (4–12).
Thymoglobulin is prepared by immunizing pathogen-free rabbits with a cell suspension of human thymic tissue (thymocytes). After immunization, serum is harvested from the rabbits, and immunoglobulins against human thymocytes are isolated and subjected to a number of purification processes (5, 13, 14). Samples from more than 2600 immunized rabbits are pooled to achieve a high level of batch-to-batch consistency (13, 15).
Thymoglobulin is able to induce a variety of depleting and nondepleting biologic effects as a result of its diverse spectrum of antibody specificities (Table 1). Although T-cell depletion constitutes a primary mechanism of the immunosuppressive effects of Thymoglobulin, other mechanisms, such as modulation of cell surface antigens, emerge as major effectors in the immunological milieu (16–20). In fact, of the many different targets of Thymoglobulin that have been identified, relatively few are T-cell-specific antigens (16–19). Current understanding of the mechanisms of action of Thymoglobulin is reviewed in this article.
Binding of Thymoglobulin
The primary immunosuppressive effect of Thymoglobulin is T-cell depletion (18, 21, 22). Binding of Thymoglobulin to cell surface antigens leads to the elimination of target cell lines from the blood. The extent of peripheral lymphocyte depletion in the blood is dose-dependent (18). Although Thymoglobulin preferentially binds to T cells, it may also bind to B cells, dendritic cells, and other nonlymphoid cell lines, especially when greater doses are administered (15, 18, 23).
Flow cytometry has been used to provide a quantitative estimation of the total amount of antibodies in Thymoglobulin directed against T-cell and nonlymphoid cell lines in the blood (15, 18). This testing has revealed that a linear relationship exists between Thymoglobulin concentrations and the amount of antibodies directed against lymphocytes, monocytes, neutrophils, red blood cells, and platelets (Fig. 1). Antibody binding to lymphocytes is greater than that to monocytes and neutrophils at all Thymoglobulin concentrations, and binding to red blood cells and platelets is only achieved at relatively high concentrations. At high doses of Thymoglobulin, nonspecific binding to neutrophils and platelets can lead to undesirable effects, such as transient neutropenias and thrombocytopenias, in the clinical setting (15, 24–26). However, because of the high absolute numbers and turnover rates of these cell lines, these effects are short-lived.
Mechanisms of T-Cell Depletion
Thymoglobulin induces lymphocyte depletion in the peripheral blood primarily by complement-dependent cell lysis (21). Complement-dependent cell lysis in the peripheral blood compartment is dependent on the dose of Thymoglobulin administered and the density of antibody coating the surfaces of lymphocytes. High concentrations of Thymoglobulin, in the range of 100 to 1000 μg/mL, activate the complement pathway and induce lysis of both resting and preactivated T cells (19, 21, 30). Although T cells are the primary target of complement-dependent cell lysis, some transient depletion of B cells and dendritic cells is also observed at high doses (18, 19, 23, 28, 29).
Emerging evidence from in vitro studies suggests that T cells in the peripheral blood are depleted not only by complement-dependent lysis, but also by antibody-dependent cell-mediated cytotoxicity and activation-induced cell death (21). Low concentrations of Thymoglobulin, in the range of 0.1 to 1 μg/mL, have been shown to induce lysis of preactivated T cells, but not resting T cells, through an antibody-dependent cell-mediated cytotoxicity mechanism. Greater Thymoglobulin concentrations in the range of 10 to 100 μg/mL have been shown to trigger CD178 (CD95-L) expression by resting T cells and induce apoptosis of preactivated T cells through pathways mostly involving Fas/FasL interactions (CD178/CD95) (19, 21, 31, 32).
Lymphocyte depletion in the peripheral lymphoid tissues, such as the spleen and axillary lymph nodes, occurs mainly by T-cell apoptosis (Fig. 2) (18). This T-cell apoptosis in lymphoid tissues differs from that mediated by the classic activation-induced pathway (33). It does not require prior exposure to interleukin-2, nor does it involve CD178/CD95 receptor interactions. Rather, it appears to be dependent on activation-induced proliferation of T cells and associated with the release of active cathepsin B from lysosomes into the T-cell cytosol. In vitro experiments have shown that activation-associated apoptosis is dose-dependent, occurs rapidly within 6 to 12 hr after the binding of Thymoglobulin to T cells, and is completed within 48 hr.
Time Course of T-Cell Depletion
Lymphocyte depletion occurs rapidly after the administration of Thymoglobulin (4, 5). A substantial reduction in peripheral T-cell counts is immediately observed, and monitoring of circulating CD3+ T cells shows that absolute counts <50 cells/μL are reached within 2 to 3 hr. Recovery of peripheral T-cell counts occurs gradually after the cessation of Thymoglobulin treatment. After a single dose of Thymoglobulin, T-cell counts begin to return toward baseline after about 10 days (34). Typically about 40% of patients treated with a course of Thymoglobulin (mean of 6 doses at 1.5 mg/kg/day) recover more than 50% of the initial lymphocyte count at 3 months (4). However, changes in the lymphocyte subsets and counts, particularly of CD4+ T cells, may be long-lasting (35).
Peripheral and Central T-Cell Depletion
Substantial T-cell depletion in the peripheral blood compartment after the administration of Thymoglobulin has been well documented (4, 5). More recently, studies have been conducted to determine the extent of T-cell depletion in lymphoid tissues during Thymoglobulin treatment (18, 19).
In a series of experiments using cynomolgus monkeys, administration of Thymoglobulin at doses ranging from 1 to 20 mg/kg was shown to induce transient dose-dependent depletion of CD2+, CD3+, CD4+, CD8+, CD20+, and CD56+ lymphocyte subsets in the peripheral blood (18). Of interest, Thymoglobulin treatment also induced dose-dependent depletion of these lymphocyte subsets in the spleen and axillary lymph nodes (Fig. 2). Thus, Thymoglobulin depletes T cells not only in the periphery but also in these secondary lymphoid tissues where the vast majority of T cells reside and antigen presentation occurs. Notably, no lymphocyte depletion was observed in the thymus at any dosing level, indicating that Thymoglobulin has limited access to this organ (18).
Modulation is a major functional mechanism of Thymoglobulin. Modulation refers to the reversible decrease in the expression of cell surface molecules that occurs due to cross-linking between antibody and cell surface antigen (36). Once the antigen-antibody complex is internalized and no longer expressed, its related pathway is inhibited for as long as the antibody is present. Modulation is an important mechanism of Thymoglobulin as this mechanism affects molecules that regulate T-cell activation and molecules that are involved in leukocyte-endothelial interactions. Thymoglobulin treatment down-modulates the expression of several molecules that control T-cell activation, including the T-cell receptor (TCR)/CD3 complex, CD2, CD4, CD5, CD6, and CD8 (18). Down-modulation of these cell surface antigens has been observed both in the peripheral blood and peripheral lymphoid tissues. This process of binding and modulating multiple surface molecules occurs independently of de novo protein synthesis and is unique to polyclonal biological agents. This effect is observed within 2 hr of Thymoglobulin administration and may last for as long as 4 weeks. This rapid and extended modulation likely accounts for much of the prolonged hyporesponsiveness of T cells after Thymoglobulin therapy (13, 18, 37).
Effects of Thymoglobulin on Adhesion Molecules and Chemokines
Functional antibodies in Thymoglobulin down-regulate the cell surface expression of a number of integrins and intercellular adhesion molecules (ICAMs) that facilitate leukocyte adhesion to the endothelium (19, 38). Thymoglobulin contains antibodies to lymphocyte Peyer's patch adhesion molecule-1 (LPAM-1), very late antigen 4 (VLA-4), leukocyte function-associated antigen (LFA-1), ICAM-1, ICAM-2, and ICAM-3 (17–19, 27, 39, 40). Thymoglobulin also contains antibodies that bind several chemokine receptors involved in regulating leukocyte-endothelium adhesion and leukocyte migration, including CXCR4, CCR5, and CCR7 (19).
Cell surface adhesion molecules and leukocyte infiltration play a key role in ischemia-reperfusion injury and rejection processes in organ transplantation (19). Homing of leukocytes requires both leukocyte adhesion to the endothelium and chemokine signaling (38). The main steps involved in leukocyte homing include leukocyte tethering (sticking) and rolling, followed by leukocyte exposure to a chemotactic stimulus (activation) and arrest, as shown in Figure 3.
Leukocyte tethering and rolling is, in part, mediated by integrins on the surface of leukocytes, such as LPAM-1 and VLA-4 (38). Arrest of rolling leukocytes is mediated by activated integrins on the surface of leukocytes, including LPAM-1, VLA-4, and LFA-1, as well as ICAMs on the surface of the endothelium. Chemokines are secreted polypeptides that bind to receptors on the surface of leukocytes, including CXCR4, CCR5, and CCR7. Chemokines bound to these surface receptors transmit signals through G proteins that are important for activating the integrins involved in leukocyte adhesion and for directing the migration of leukocytes into and within the extravascular space.
Effects of Thymoglobulin on Leukocyte Homing
Thymoglobulin down-modulates of the cell surface expression of LPAM-1, VLA-4, LFA-1, ICAM-1, ICAM-2, and ICAM-3, thereby inhibiting leukocyte tethering, rolling, and arrest and subsequently inhibiting the firm adhesion of leukocytes to the endothelium (19). Binding of Thymoglobulin to the chemokine receptors, CXCR4, CCR5, and CCR7 inhibits exposure to chemotactic stimulus, resulting in further down-regulation of integrins and decreased leukocyte responses to chemotactic signals (19).
The effects of Thymoglobulin on leukocyte-endothelial interactions in ischemia-reperfusion injury were examined in cynomolgus monkeys (41, 42). In one of these studies, animals reperfused without Thymoglobulin showed rolling of leukocytes within 5 min, with most leukocytes adhering to the endothelium within 30 min. Reperfusion in the presence of Thymoglobulin, however, markedly reduced the rolling of leukocytes and almost completely inhibited the adhesion of leukocytes to the endothelium (41). The therapeutic implications of these antichemotactic and antiadhesion effects are broad and are particularly relevant in transplantation. Therefore, the administration of Thymoglobulin before reperfusion may be necessary to prevent leukocyte invasion into the allograft tissue and thereby, minimize ischemia-reperfusion injury.
Homeostasis-Driven Proliferation of T Cells
In addition to the canonical antigen-driven proliferation of T cells, the repopulation of T cells after depletion with polyclonal antibodies, such as Thymoglobulin, also results from homeostasis-driven proliferation. The empty space in the lymphocyte compartment created by T-cell depletion induces the proliferation of residual naïve and memory T cells to fill the space and regain the previously stationary distribution in cell population densities (43, 44). In contrast to antigen-driven proliferation, homeostasis-driven proliferation is not dependent on foreign antigen stimulus (45–47). Instead, it requires the interaction of the TCR with major histocompatibility complex (MHC) molecules loaded with self-peptides. Unlike antigen-driven proliferation, homeostasis-driven proliferation shows a slow rate of expansion, no significant increase in the size of the proliferating cells, and no acquirement of effector T-cell functions (48–50).
Homeostasis-driven proliferation after T-cell depletion is associated with changes in the ratios of CD4+ to CD8+ T cells and naïve to memory T cells. After T-cell depletion with Thymoglobulin, there is a pronounced and persistent inversion in the ratio of CD4+ to CD8+ T cells (35). The low ratio of CD4+ to CD8+ T cells may reflect differences in the regeneration pathways of the 2 T-cell subsets (51–54). Regeneration of CD4+ T cells is dependent on the thymus, whereas regeneration of CD8+ T cells occurs extrathymically (55, 56). Because of thymic involution, adults show persistent low levels of CD4+ T cells and a relative overregeneration of CD8+ T cells (35, 51).
The distribution of MHC class I and MHC class II molecules also may influence the novel homeostatic CD8+/CD4+ ratio. Interaction of TCR with MHC class I molecules leads to the proliferation of CD8+ T cells, whereas interaction of TCR with MHC class II molecules leads to the proliferation of CD4+ T cells (49, 50, 57). The higher quantity of MHC class I expression in the periphery may contribute to the over-generation of CD8+ T cells during repopulation.
Homeostasis-driven proliferation also has been associated with changes in the ratio of naïve to memory T cells. During homeostasis-driven proliferation, naïve T cells have been shown to acquire the cell surface markers and functional properties of antigen-induced memory T cells, resulting an overrepresentation of memory phenotype T cells (49, 58, 59). Because the regeneration of naïve T cells is dependent on thymopoiesis, the overrepresentation of memory phenotype T cells is especially apparent in the CD4+ T-cell compartment, which also depends on thymic capacity for CD4+ T-cell regeneration (51, 54).
Expansion of Specific Subset Population
Homeostasis-driven proliferation appears to be associated with the expansion of specific T-cell subsets that may have a regulatory function. After lymphocyte depletion with Thymoglobulin, increased numbers of CD8+ T cells coexpressing CD57, a molecule primarily expressed on natural killer cells, is observed (35, 51). About 70% to 80% of regenerated CD8+ T cells coexpress the marker CD57 (35). This marker has been associated with regulatory suppressor functions and is reciprocally expressed with the co-stimulatory molecule CD28 (51, 60, 61). CD8+CD28− alloantigen-specific T-suppressor cells have been shown to induce up-regulation of ILT3 and ILT4—two members of a family of inhibitory receptors expressed by monocytes and dendritic cells—rendering these antigen-presenting cells tolerogenic (62, 63). Thus, the expansion of CD8+CD57+CD28− T cells during homeostasis-driven proliferation may play an important role in regulating the immune response after lymphocyte depletion.
Perhaps the best-characterized regulatory T-cell subset is the CD4+CD25+ lymphocytes expressing CTLA-4 and GITR cell surface molecules and the Foxp3 transcription factor (64). Emerging evidence in both rodents and humans demonstrates that these CD4+CD25+ T cells play an important role in maintaining self-tolerance to autoantigens and tolerance toward alloantigens (65, 66). In a recent studies, CD4+CD25+ T cells were shown to be relatively spared, compared with CD8+ T cells, CD4+ naïve T cells, CD45RA+ memory T cells, and central memory T cells, after lymphocyte depletion with Thymoglobulin (67, 68).
Homeostasis-driven proliferation after lymphocyte depletion with Thymoglobulin is associated with the expansion of specific subpopulations of highly differentiated cells that appear to have a regulatory function. Additional phenotypic and functional studies are needed to fully characterize the generation and role of these cells in transplantation.
Polyclonal antibodies are biological agents with unique mechanisms of action in comparison to small molecule immunosuppressants and monoclonal antibody preparations. Thymoglobulin acts as a rapid T-cell-depleting agent in both the blood and peripheral lymphoid tissues. The major pathways for T-cell depletion are complement-dependent cell lysis in the blood compartment and apoptotic cell death in the lymphoid tissues. Thymoglobulin also modulates cell surface molecules that regulate T-cell activation as well as adhesion molecules and chemokine receptors involved in leukocyte-endothelial interactions. Thymoglobulin treatment is associated with both short- and long-term changes in T-cell populations. After lymphocyte depletion, T-cell repopulation leads to an altered homeostasis characterized by age-dependent changes in the ratios of CD4+ to CD8+ T cells and naïve to memory T cells. This new homeostasis is also characterized by the expansion of specific T-cell subsets that have been shown to exhibit regulatory-suppressor functions, such as CD8+CD57+CD28− T cells. In addition, CD4+CD25+Foxp3+ regulatory T cells are more resistant to depletion than other cells types and therefore are relatively enriched. Further investigation of the mechanisms of action of Thymoglobulin will be important to guiding therapy with this agent in the transplant setting as well as in autoimmune disorders.
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