Antithymocyte globulins (AThG) are one class of polyclonal IgG antilymphocyte sera made by immunizing rabbits or horses against different lymphocyte preparations (Table 1). The cell type used as an immunogen determines the spectrum of antibodies generated, which in turn has important implications for specificity and activity. Various abbreviations have been used in the literature to refer to antilymphocyte and antithymocyte globulins, some of which are overlapping. For the sake of precision and clarity, we will distinguish between the available antihuman lymphocyte polyclonal antibody preparations, only some of which are made using unfractionated human thymocytes, by the following abbreviations: rabbit anti-thymocyte globulin (rAThG), equine anti-thymocyte globulin (eAThG), rabbit anti-T cell globulin (rATcG), and equine antilymphocyte globulin (eALG).
Polyclonal AThG have been used in solid organ transplantation for over 40 years (1–4), and yet our understanding of their mechanisms of action is still modest. In this review, we discuss new insights into the activity of AThG against human B lymphocytes. Antithymocyte globulins were initially prized in transplantation for their ability to kill or modulate T cells, successfully treat corticosteroid-resistant allograft cellular rejection, and for use as transplant induction therapy (1, 2, 5, 6). Human thymus was specifically used as the immunogen in these preparations to maximize the concentration of CD3+ T lymphocytes (7–9). Thus, mechanism of action of the resulting antisera was thought to be direct binding of antibodies to T-cell surface markers leading to complement mediated lysis and antibody-dependent cytotoxicity of CD4- and CD8-positive T cells. However, one of the first descriptions of the morphological changes in lymphocytes exposed to AThG in vitro (10, 11) reports what we now know to be classical apoptosis (12, 13). Over the last 20 years, however, it has become apparent that AThG preparations have multiple effects on many cell types involved in vascularized allograft rejection (14). It is now clear that AThGs contain antibodies specific for B-cell and plasma cell markers (15–19), can induce apoptosis and inhibit proliferation in B cells (15, 18–21), and can be used to effectively treat “steroid resistant” (22–24) and alloantibody-mediated rejection (25).
The B-cell immunomodulatory activity of AThG preparations is almost certainly a consequence of the unfractionated human thymocytes used as the immunogen in their manufacture (15, 18). Other antilymphocyte preparations use different immunogens, including activated T-cell lymphoblasts and the Jurkat T-cell line (Table 1). To make AThG, rabbits or horses are immunized against unfractionated, Ficoll density gradient isolated, human thymocytes. Although thymocyte preparations are composed of predominantly of T lymphocytes, they also contain B cells, plasma cells, and dendritic cells (18, 26–30). While antibodies against CD3, CD4, CD8, and the T-cell receptors (TCRs) appear in AThG at high titers, we also find antibodies specifically directed at B cell and plasma cell restricted surface antigens, as well as antibodies against antigens common to both B and T cells (i.e., major histocompatibility complex [MHC] Class I, CD95, etc.) (18, 19). This plurality of surface targets also gives rise to multiple mechanisms of action affecting the B-cell mediated alloresponse, including AThG-mediated B-cell apoptosis, FcR binding, complement mediated lysis, blockade of costimulatory molecules and cytokine receptors, and blocking the activation of T-helper cells necessary to support T-dependent B-cell activation.
Spectrum of B Cells in Thymic Inoculum
To understand why AThG preparations can contain B-cell specific antibodies, it is useful to consider the process of manufacture (31). Pediatric human thymi are routinely removed during cardiac surgical procedures on children as the large size of the thymus impairs surgical access to the heart. To prepare the immunogen for AThG, the thymus is macerated and collagenase digested, and the liberated thymocytes are then isolated by Ficoll density gradient centrifugation. As one might expect, the predominant cell population is that of CD3+ T cells in various stages of maturation, including CD4+, CD8+, and CD4+CD8+ immature cells. Antibodies generated against these cells are responsible for the T-cell activity of AThG. However, Ficoll density gradient isolates from thymus also contain B cells, dendritic cells, and likely some thymic endothelia. CD138+ plasma cells and CD20+ B cells are found at modest numbers in the thymus (Fig. 1A). CD20+ cells are found mostly in areas surrounding Hassall's corpuscles, whereas CD138+ plasma cells also localize primarily to the medullary cord areas (18, 19, 26–28, 32). These cells constitute approximately 1–2% of thymic lymphocytes, and express CD2, CD5, CD19, CD20, CD22, CD37, CD72, CD76, human leukocyte antigen (HLA)-DR, IgG, IgM and IgD, among other B-cell surface markers (27, 33). Although the function of these cells is not known, it is interesting that Ig transcripts produced by thymic B cells are biased towards the VH4 gene family, which is also associated with autoimmunity (30, 34, 35). Some investigators have postulated that these thymic B cells are involved in T-cell tolerance to self-B cell antigens and prevention of autoimmunity. Our group has recently described the presence of CD136 expressing plasma cells in the thymus as well (18, 19), although their function is unclear.
As noted in Table 1, other polyclonal antilymphocyte sera have been made against activated lymphocytes and cell lines, notably the rATcG product Fresenius-ATG, which is made against the Jurkat cell line. Unfortunately, no direct comparisons of rAThG (Genzyme) and rATcG (Fresenius) regarding B-cell directed antibodies have been published in the literature. There are only two reports suggesting in vitro activity of rATcG (Fresenius) against malignant plasma cells (20) and dendritic cells (36), but whether or not these have in vivo efficacy has yet to be determined. Several comparisons of other antilymphocyte sera such as eAThG (Upjohn) and eALG (MALG; University of Minnesota) have focused largely on T-cell specific antigens (37). However, work by Bonnefory-Berard and colleagues using rabbit antilymphocyte sera generated against a variety of lymphocyte preparations shows moderate to extensive apoptosis for all preparations when used against B cell lines in vitro (15). This finding likely reflects the presence of B cell specific antibodies, as well as a large amount of cross-reactivity against MHC-Class I, CD45, CD38, and other similarly shared antigens between B and T cells (15).
Evidence for Activity of AThG Against B Cells
Although AThG is likely to suppress B cell activation by eliminating CD4+ T cell help, there is strong in vitro and in vivo evidence that AThG preparations directly inhibit B cell proliferation and induce B cell death by apoptosis (Fig. 1B) and complement mediated lysis. In vitro (Fig. 2), AThG preparations have been shown to induce apoptosis in naïve, memory, and activated human B cells (18, 21), plasma cells (18, 19), human B cell lines (15, 18–20), and primary myeloma cells (19, 20). Studies of lymphocyte subset counts after rAThG administration in nonhuman primates and human subjects have demonstrated rAThG reduces CD19+ peripheral blood lymphocyte counts by 30–90% (38–41). In a nonhuman primate model, rAThG induced CD20+ B-cell depletion in multiple compartments including spleen, lymph node, and peripheral blood. The reduction is dose dependent, and B cell counts can be suppressed for two to six months after a course of therapy followed by chronic triple-agent immunosuppression (39, 40, 42). The effective serum concentrations of rATG that have been measured (1–250 μg/mL) are similar to those shown to have in vitro B cell activity.
What Are the Mechanisms of AThG B-Cell Activity?
The multiplicity of antigens that AThG preparations can bind to on human B cells gives rise to a multiplicity of mechanisms through which AThG modulates B and plasma cell function (Fig. 3). The most obvious mechanism is the induction of complement-mediated antibody dependent lysis, where the AThG antibodies bind to a spectrum of surface targets on the B cells (CD19, CD20, CD27, CD30, CD32, CD38, CD40, CD126, CD138, HLA-DR), bind complement, and lyse the target cells. Both rabbit and equine AThG are capable of binding human complement and inducing lymphocyte lysis (43). Other usual mechanisms of AThG-coated B-cell depletion are likely opsonization by macrophages and antibody-dependent cell-mediated cytotoxicity, which depend on Fc receptor mediated clearance of AThG coated cells.
Induction of complement independent apoptosis is another mechanism of B-cell depletion by AThG. rAThG has been shown to induce in vitro apoptosis of B-cell lines (15, 19), human memory and naïve B cells (18), normal (18) and malignant (18, 20) human plasma cells. The pathways activated by rAThG include caspase and cathepsin B dependent apoptotic cascades and direct loss of mitochondrial membrane potential (18, 19, 44). The surface proteins responsible for B and plasma cell apoptosis by AThG are likely to depend on the state of differentiation. CD95 specific antibodies are present in AThG preparations, and this caspase dependent pathway likely contributes to apoptosis of activated B cells, plasma cells and myeloma cells (18, 43, 44). Similarly, HLA-DR and CD30 ligation of naïve, activated, and memory cells, results in apoptosis via activation of cathepsin induced pathways and loss of the mitochondrial membrane potential (18, 44, 45). Cross-linking of the FcRγII (CD32) by either direct antibody binding or nonspecific Fc binding of AThG likely plays a role in augmenting caspase dependent apoptosis of naïve and memory B cells and possibly plasma cells (19, 46).
Blockade by AThG of costimulatory signals from CD4+ T cells and accessory cytokines is another means by which AThG can inhibit B-cell responses. AThG contains antibodies to CD40 and CD28, both of which are costimulatory signaling molecules triggered by their cognate ligands on CD4 T cells. Blockade of these signals can lead to apoptosis during B-cell activation. In addition, AThG contains antibodies to the interleukin (IL)-6 receptor CD128, a key trophic factor in B cell and plasmablast maturation (19).
Which Antibodies in AThG Are Biologically Relevant for B Cells?
Of the mechanisms of AThG activity, the most intriguing is signaling through B-cell surface proteins leading to inactivation or apoptosis of B cells. Our group has used competitive binding assays to identify anti-B cell and plasma cell antibodies present in rAThG (Fig. 1B). In addition, others have noted the presence of antibodies to T-cell surface markers also expressed on B cells such as MHC-Class I, CD38, CD45, and CD95. Here we review some of the specific B-cell surface markers and biologically relevant pathways through which AThG binding to B cells surface proteins may modulate B-cell activity.
The MHC class II molecule HLA-DR is present on the surface of resting B cells and upregulated on activated B cells. rAThG contains anti-HLA DR antibodies, and these appear to contribute significantly to B cell apoptosis activity via mitochondrial membrane depolarization in naïve and activated B cells (18). Cross-linking of HLA-DR causes apoptosis on CD20+ B cells via mitochondrial membrane potential loss (45, 47–49). HLA-DR ligation also sensitizes B cells to apoptosis via the Fas/CD95 pathway (50). It is interesting to note that HLA-DR mediated apoptosis and signaling plays an important role in regulation of bone marrow hematopoeisis, and is a hypothetical mechanism of bone marrow suppression after AThG administration (51). As HLA-DR is downregulated on mature, bone-marrow resident plasma cells, this mechanism is not likely to be active in AThG-induced apoptosis of antibody secreting plasma cells (18, 19).
Although downregulated on mature plasma cells, CD20 is expressed on naïve, memory, and activated B cells. Ligation of CD20 can mediate B-cell apoptosis through a mechanism that utilizes caspase (52) and mitochondrial induced cell death pathways (53), a third nonclassical cell death pathway (54, 55), complement-mediated cell lysis (56), and antibody dependent cellular cytotoxicity (54, 57). Efficient activity of anti-CD20 antibodies appears to require crosslinking via FcRγIIa, and receptor mutations that alter Fc affinity for CD32 reduce the efficacy of murine monoclonal anti-CD20 antibodies (58).
CD30 is a member of the tumor necrosis factor superfamily, and is expressed on activated B and T cells, including germinal center B cells and CD3+ thymocytes. Expression of CD30 on CD3+ thymocytes likely accounts for the presence of anti-CD30 antibodies in AThG preparations. CD30 signaling leads to NF-κB activation, as well as the MAPK associated kinases ERK, JNK, and p38 (59–61). It is interesting that naïve IgD+ IgM+ B cells express CD30, and that activated B cells are capable of expressing CD153 (CD30L) (62). CD30 signaling by CD153 in IgD+ IgM+ germinal center B cells inhibits IL-4 induced class switching, induces growth inhibition in large cell lymphomas (63), and induces apoptosis in eosinophils (64). It is unclear which of these mechanisms may be at work in AThG ligation of CD30.
AThG preparations have been found contain high concentrations of FcR antibodies (65). Indeed, the high activity of AThG preparations against platelet expressed CD32 appears to be the major mechanism responsible for AThG induced thrombocytopenia (66, 67). On human B cells, the Fc receptor subtype expressed is the FcRγIIb inhibitory receptor, which binds IgG1 and IgG3 subtypes at high affinity, and IgG2 and IgG4 with a very low affinity (68). Interestingly, rabbit IgG has a higher binding affinity for human FcR than horse sera, and this may account for the increased efficacy of rAThG compared with equimolar concentrations of eAThG (46, 69, 70). FcRγII clustering on the surface of B germinal center B cells without B cell receptor engagement leads to inhibition of calcium influx, aborts B cell activation, and induces apoptosis (71–73). F(ab)2 fragments of rAThG are less effective in inducing B cell apoptosis, suggesting at least a partial role for such binding (18). Indeed, Fc binding of rAThG may enhance cross-linking of other B antibodies present in AThG such as HLA-DR, CD95, or CD30, leading to augmented apoptosis.
AThG preparations contain anti-CD38 antibodies in relatively high concentrations. Ligation of CD38 by AThG may induce modest apoptosis in activated B cells, while preventing migration of activated B cells from the blood by blocking adhesion to CD31 in high endothelial venules (18, 74). CD38 is upregulated on human B cells after activation, and is one of the defining markers of B-cell activation and the plasma cell phenotype (including CD20− CD138+ sIg− HLA-DR−). The exact role of CD38 in mature human B cell activation and development is currently unclear. CD38 has several functions including enzymatic production of calcium mobilizing compounds during cell activation and signal transduction in human leukocytes (75). Agonistic CD38 ligation on immature B cells suppresses human B cell lymphopoiesis and induces apoptosis (76, 77), similar to the putative function of CD38 signaling on immature T cells in the thymus (78). In contrast, CD38 signaling rescues activated CD27− B cells from apoptosis within the germinal center by upregulation of bcl-2 (79). In human B cells activated by CD40L and IL-4 stimulation, ligation of CD38 induces moderate levels of apoptosis (18). Finally, binding of CD38 to its ligand CD31 on human vascular endothelial cells during the rolling phase of B cell adhesion promotes migration of B cells from the blood into lymphoid and nonlymphoid tissues. Thus, activity of AThG antibodies with respect to CD38 is likely to involve inhibition of early B lymphopoiesis, apoptosis of activated B cells, complement mediated lysis of activated B and plasma cells, and blockade of B cell and plasma cell migration from the blood.
Although CD95 is not a B-cell specific molecule, crosslinking of surface expressed HLA-DR on naïve and memory B cells increases their sensitivity to CD95 triggered apoptosis (50). The CD95 apoptotic pathway is caspase dependent, and can be blocked by pan-caspase inhibitors such as z-VAD-fmk (18, 43). As noted above, HLA-DR crosslinking also activates a potent mitochondrial death program (18, 45). Thus, the combination of anti-HLA-DR and anti-CD95 antibodies described above may synergize the efficacy of AThG preparations that contain antibodies to both proteins (18, 43, 50, 51).
Plasmablasts and early plasma cells all express high levels of the IL-6 receptor protein CD126 (80). IL-6 is essential for Ig class switching and differentiation of human plasmablasts (81). When combined with IL-2 or IL-10, IL-6 enhances B cell differentiation into antibody secreting plasma cells (82). Antibodies directed against CD126 are present in moderate amounts in AThG, and CD126 blockade may prevent plasma cell differentiation in previously activated B cells and plasmablasts (19). IL-6 receptor blockade has also been reported to induce apoptosis of human myeloma cells and cell lines, suggesting another possible mechanism for the activity of AThG against myeloma (83).
Syndecan-1 (CD138) is expressed on human bone-marrow resident plasma cells at a high level. Its putative function is to improve plasma cell adhesion to extracellular matrix components, including chondroitin/heparin sulfates via 5 glycosaminoglycan binding sites. Syndican-1 has been hypothesized to mediate adhesion of plasma cells toglycosaminoglycan binding sites. Syndican-1 has been bone-marrow stromal cells, and promote plasma cell survival by binding and “capturing” hepatocyte and fibroblast growth factors. rAThG contains moderate levels of anti-CD138. Given that CD138 has not been reported to have a direct association with proapoptotic pathways, it is likely that anti-CD138 antibodies in AThG preparations function by two mechanisms: 1) inducing complement-mediated lysis of human CD138+ fully differentiated plasma cells; and 2) preventing stromal cell and growth factor interactions. CD138 does associated with fibroblast and hepatocyte growth factors, which appear to be required for plasma cell survival and differentiation.
Variability of AThG Preparations
Variability of efficacy and binding of AThG preparations has been a theoretical concern given the polyclonal nature of these preparations (84, 85). There are several potential sources for variability of AThG preparations: lot-to-lot variability and species variability. Lot-to-lot variability in the manufacture of AThG might result from variability in the cell types present in pooled human thymus immunogen preparations, or genetic variability in the rabbit repertoire and immune response to the immunogens. The former can be minimized by pooling sera from many animals, while the latter can be minimized by using genetically identical animal strains for immunization. Pooling serum is a necessity for rabbit preparations, and rAThG lots generally contain purified IgG from at least 50–75 individual rabbits of a similar genetic background. For eAThG, sera is pooled from a number of animals as well, although the larger volumes of sera obtained from horses likely leads to pooling of sera from a smaller number of animals.
Given the multiple mechanisms of action of rabbit AThG, one wonders if there are structural differences between rabbit, horse and human IgG that might alter their efficacy for use in human immunomodulation. Both rabbit and horse antithymocyte globulin preparations are made from purified IgG fractions of immunized sera. However, rabbits express only one IgG heavy chain (86), compared to seven equine IgG heavy chains (87) and four human (IgG1, IgG2, IgG3, IgG4). Rabbit IgG is known to bind human complement and interact with the human FcRγII (CD32) and FcRγIII (CD16) to aid in antibody dependent cellular cytotoxicity (ADCC) (70). We were not able to find any published literature describing the equine IgG isotypes present in eAThG (ATGAM) or eALG (MALG), nor their relative concentrations. This may be of some importance, as only two of the seven equine IgG isotypes (IgGa, IgGb) mediate complement binding (88), and high concentrations of the IgG(T) subtype can bind and compete out complement and ADCC activity of other equine IgG isotypes leading to diminished cytotoxicity (89). Variations between batches of eALG in IgG subtype proportions may explain the greater lot-to-lot variation in efficacy observed by some investigators compared with rAThG (17).
There is no current consensus on standard assays for assessing the potential variability of AThG preparations. Presumably the manufacturers monitor biological activity of each lot by several different assays, although this information does not appear on package inserts. Several methods for comparing AThG preparations have been proposed including flow cytometry measures of lymphocyte surface binding, apoptotic efficacy and lytic activity (17, 84, 85, 90). There is general agreement in the literature that AThG preparations differ between manufacturers (17, 84, 90), likely due to differences in immunogen and the species immunized described above. Some investigators have reported differences between lots of AThG produced by different species and manufacturers, although these studies have used one to three lots with a limited number of assays (84), while others have found no differences in B and myeloma cell apoptotic activity between five lots of rAThG (18, 85). It is unclear if any putative in vitro differences in AThG preparations, either between different products or between different lots, portend clinically significant differences in patient responses to AThG in vivo.
Clinical Use of AThG in Treatment of Antibody-Mediated Renal Allograft Rejection
Given the multiplicity of antibodies in AThG preparations with the potential to modulate B cell function, AThG can be used to treat acute alloantibody mediated renal allograft rejection. Antibody-mediated rejection (AMR) most often occurs in graft recipients who have been sensitized against donor HLA antigens by previous transfusion, pregnancy, or solid organ transplants. Such “alloimmunization” results in two populations of allospecific IgG class-switched cells: actively secreting plasma cells and quiescent memory B cells. In programs that perform flow-cytometric cell or HLA-coated bead cross-matching, antibody mediated rejection is sometimes detected in patients who initially had undetectable donor-specific antibody levels. In these cases, quiescent donor-specific memory B cells become activated upon B-cell receptor (BCR) crosslinking by donor HLA. Such activation leads to rapid B cell proliferation and plasmablast secretion of complement binding donor-HLA directed IgG isotypes. The hallmark of such rejections is the triad of C4d staining in peritubular capillaries of the renal biopsy, the new presence of donor-specific IgG antibody, and histologic evidence of renal tubular or glomerular damage (91).
Effective treatment of AMR involves: 1) removal of circulating donor-specific antigen by plasmapheresis, 2) interruption of T cell help by adequate immunosuppression, 3) inducing the apoptosis or lysis of activated B cells and plasmablasts, and 4) preventing the development of long-lived CD138+ allospecific plasma cells. A number of approaches have been reported in the literature, all of which combine plasmapheresis with the administration of either intravenous immunoglobulin (92–95), AThG (25), or rituximab (96–98). AThG preparations appear to have some advantages over IVIG or rituximab in treatment of AMR. In contrast to rituximab, rAThG induces apoptosis and complement mediated lysis in plasmablasts and plasma cells in vitro (18, 85). This is likely due to the rapid downregulation of CD20 expression in activated B cells and plasma cells. In addition, rabbit IgG has a higher affinity than human IgG for the FcγIIRb inhibitory receptor, which may improve efficacy in plasma cell and B cell binding over IVIG. AThG preparations also combine anti-T cell activity to block costimulation of activated B cells. Some reports, however, have noted the presence of increased alloantibody titers after AThG administration (99, 100), although highly sensitized patients given rAThG induction therapy in a large prospective study had better graft and patient survival than those without induction therapy (101). Better delineation of these issues will require prospective randomized trials comparing AThG-based therapies for AMR to intravenous immunoglobulin and/or rituximab, and large cross-sectional studies of alloantibody appearance and specificity in renal transplant recipients who have received a variety of induction and maintenance immunosuppression therapies.
The author would like to thank Didier Mandelbrodt, Millie Samaniego, and Ignacio Sanz for stimulating conversations that helped improve this work, and Alicia Henn for her proofreading of the manuscript. This paper is dedicated to the memory of Peter Rowley, a close collaborator and colleague, who recently died.
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