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Transplantation:
doi: 10.1097/TP.0b013e3182a72115
Editorials and Perspectives: Overview

Down-Regulating Humoral Immune Responses: Implications for Organ Transplantation

Stegall, Mark D.1,3; Moore, Natalie1; Taner, Timucin2; Li, Han2; Dean, Patrick G.2

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Author Information

1 Department of Surgery, Division of Transplant Surgery and Department of Immunology, Mayo Clinic, Rochester, MN.

2 Division of Transplant Surgery, Mayo Clinic, Rochester, MN.

3 Address correspondence to: Mark D. Stegall, M.D., Department of Surgery, Division of Transplant Surgery, Mayo Clinic, 200 First Street Southwest, Rochester, MN 55905.

M.D.S. has research contracts with Millennium and Alexion Pharmaceuticals.

The authors declare no conflicts of interest.

E-mail: stegall.mark@mayo.edu

All authors participated equally in the planning and writing of this article.

Received 4 February 2013. Revision Requested 20 March 2013.

Accepted 19 June 2013.

Accepted September 20, 2013

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Abstract

Alloantibody can be a major barrier to successful organ transplantation; however, therapy to control antibody production or to alter its impact on the allograft remains limited. The goal of this review is to examine the regulatory steps that are involved in the generation of alloreactive B cells, with a specific emphasis on how known mechanisms relate to clinical situations in transplant recipients. Thus, we will examine the process of activation of mature, naïve B cells and how this relates to de novo antibody production. The role of long-lived plasma cells in persistent antibody production and the factors regulating their longevity will be explored. The regulation of memory B cells and their possible roles in alloimmunity also will be assessed. Finally, we will review current therapeutic approaches aimed at controlling alloantibody and assess their efficacy. By examining the pathways to antibody production mechanistically, we hope to identify important gaps in our current knowledge and gain insight into possible new therapeutic approaches to overcoming antibody in transplant patients.

Antibody against donor antigens is recognized as a major problem in clinical transplantation (1–5). The ability to “down-regulate” humoral alloimmune responses has emerged as one of the major unmet needs in transplantation. Although B cells have multiple functions including antigen presentation, T-cell stimulation, and immunoregulation via cytokine production, we will confine our discussion here to their role in the production of antibodies. We define “down-regulation” broadly to include any process that controls or ameliorates humoral responses functionally—ranging from preventing the humoral response, abrogating an existing response, or blocking the terminal effects of the response. Our major focus will be on the ways in which humoral responses are controlled at the cellular level and examine possible new areas for therapeutic intervention in transplant patients.

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Early Steps in B-Cell Regulation: Bone Marrow

The pathway to antibody production begins when B-cell progenitors in the bone marrow rearrange the immunoglobulin gene to form a unique antibody-like molecule (6). When inserted into the B-cell membrane, this so-called B-cell receptor can both interact with antigens in the marrow and transmit a signal to the B cell. Gene rearrangement is apparently random and it is estimated that almost half of the new pre–B cells generated react against “self” antigens (7). There are at least two regulatory “checkpoints” in the bone marrow—one at the pre–B-cell stage and another at the immature B-cell stage (Fig. 1). One of the primary outcomes of these checkpoints is the elimination of autoreactive B cells either by deletion or switching to a different B-cell receptor via a process called “receptor editing.” Only 10% of the B cells generated in the marrow survive these early regulatory steps and emerge as mature, naïve B cells.

FIGURE 1
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No transplant studies have specifically examined the impact of immunosuppression on these early stages of regulation and how they might be exploited to affect later humoral events.

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Regulation of Mature, Naïve B Cells: Secondary Lymphoid Tissue

Most mature, naïve B cells that survive early selection in the marrow migrate to secondary lymphoid tissue such as lymph nodes and spleen (8). Much of the biology of these cells before activation including the regulation of their longevity, mobility, and even proclivity to reactivity remains unclear. The phenotype of mature, naïve B cells is CD20+, CD19+, CD27-, IgD+, and IgM+, and they do not secrete antibody. It is thought that most mature, naïve B cells remain quiescent until they encounter cognate antigen.

B cells are capable of recognizing antigen directly via their cell-surface B-cell receptor and this can lead to activation. However, the most potent process of B-cell activation involves antigen presentation and help from T cells. B-cell activation is commonly separated as being either dependent or independent of T cells.

Upon receiving cognate T-cell help, B cells appear to have three potential fates: (a) differentiate into early recirculating memory B cells (9), (b) form extrafollicular foci of short-lived plasmablasts that secret low-affinity antibody (10), or (c) form a germinal center within the B-cell follicle with the final product being affinity matured, often class-switched, long-lived memory B cells and plasma cells.

The initial regulation B-cell activation is partially dictated by the affinity of the B cell for antigen (high affinity for antigen appears to favor activation, whereas low affinity favors apoptosis) and likely involves both antigen-presenting cells and helper T cells. CD4+ T cells affect the subsequent fate of responding B cells via cell-cell interactions (e.g., CD40-CD40L and B7-CD28) and cytokines (interleukin [IL]-4 and IL-21, for example) (8). In germinal centers, both follicular dendritic cells and T follicular helper cells appear to play critical roles in the selection of which B cells either undergo apoptosis or survive to under go further rounds of replication, somatic hypermutation, and/or differentiation into memory B cells and long-lived plasma cells (LLPCs). Although the mechanisms affecting B-cell differentiation into either memory B cells or LLPCs are not entirely clear, the process does not appear to be stochastic. For example, differentiation into LLPCs appears to be associated with high-affinity receptor binding, whereas memory B-cell differentiation appears less dependent on receptor affinity and more related with their proclivity for survival (9). Germinal centers are transient structures that typically reach maximum size 1 to 2 weeks after antigenic stimulation and resolve within 3 to 4 weeks. (For a complete discussion of germinal centers, see ref. (8).)

We will discuss both LLPCs and memory B cells in detail below. However, we first will examine two situations in which activation of mature, naïve B cells may be clinically important in transplantation.

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De Novo Donor-Specific Antibody Formation

In the setting of alloimmunity, the use of immunosuppression and the persistence of antigen in the form of the allograft may significantly alter both the process of mature, naïve B-cell activation and their regulation after activation. For example, the incidence de novo donor-specific antibody (DSA) appears remarkably low after kidney transplantation. In one recent study, only 15% of patients develop de novo DSA at a mean of 4.6 years after transplantation (10). Another study showed very similar results (11). In these and other studies, the development of de novo DSA was associated with decreased long-term graft survival.

The fact that many cases of de novo DSA are associated with prior nonadherence or history of a clinical acute cellular rejection episode suggests that immunosuppression is a potent inhibitor of the activation of mature, naïve B cells (10). However, the fact that some cases of de novo DSA formation appear in compliant patients suggests that either T cells capable of helping naïve B cells emerge despite immunosuppression or some alloreactive B cells may differentiate into antibody-secreting cells in the absence of T-cell help. In some cases, apparent de novo DSA may actually be preformed DSA that was not detected before transplantation but can be seen with more extensive testing (12). In this setting, it is possible that the antibody-producing cells originate from an existing population of memory B cells that may not require T-cell help for activation.

It is possible that different immunosuppressive regimens may have differential efficacy in the prevention of de novo DSA such as has been suggested in recent studies involving CTLA-4 blockade using belatacept (13). Depletion or blockade of mature B cells using anti-CD20 or other agents also might prevent de novo DSA. However, given the relatively low incidence of de novo DSA formation, the efficacy and the risk/benefit ration for an individual patient remains to be determined.

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Acute Humoral Rejection

In previously unsensitized patients, acute humoral rejection likely involves the activation of mature, naïve B cells (14, 15). Commonly seen in combination with cellular rejection, these rejection episodes are heterogeneous in that they vary widely in severity, time from transplantation, and the relative role that antibody plays in graft dysfunction. Endpoints of these studies are difficult to define because, in many instances, chronic, irreversible graft injury already has occurred and graft function remains poor even if the acute humoral rejection episode is “successfully” treated. No randomized trials have been performed and consensus on treatment is lacking. Several groups have touted the use of bortezomib for the control of DSA in these settings. However, none of these studies have included control groups. Plasma exchange is usually effective in controlling DSA levels in the short-term. In more severe cases, blockade of terminal complement with eculizumab (usually in combination with plasma exchange) has been used anecdotally with success (16).

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Regulation of LLPC

One of the major products of germinal centers are LLPCs (17–20). Before we discuss LLPCs in detail, it is important to examine evidence supporting their existence and their contribution to overall antibody production. Several cell types contribute to serum antibody levels, including plasmablasts, recently stimulated memory B cells that have converted to plasma cells, and LLPCs. Thus, antibody levels against some antigens might be maintained at stable levels for years (e.g., antibodies against human leukocyte antigen [HLA] in sensitized transplant candidates on the waiting list), suggesting the presence of a stable, long-lived antibody secreting population (LLPCs). In contrast, other antibodies generated secondary to recent exposure (or reexposure) to antigen might fluctuate, suggesting the presence of shorter-lived population of plasma cells (17). In the transplant setting, the persistent presence of donor antigen might provide a mechanism by which the constant stimulation of short-lived antibody-secreting cells could contribute to overall antibody production.

Several studies involving rituximab therapy suggest the presence of a LLPC population that is CD20-. For example, one study examined serum antibody levels in patients receiving prolonged treatment (five cycles) with the anti-CD20 antibody rituximab (a therapy that depletes mature B cells and memory B cells) (21). Low serum levels of IgG levels developed in 22.2% of patients and low IgM levels in 38.8% of patients. However, most patients showed no change in serum antibody levels and all patients demonstrated persistent antibody production resistant to rituximab. Thus, in some patients, CD20+ cells contribute significantly to overall antibody production. However, in the majority of patients, the majority of the serum antibody production (both IgG and IgM) is likely due to CD20- LLPCs. In a similar study, rituximab only showed a minimal impact on alloantibody levels in sensitized renal allograft candidates (22). However, when assessing these studies, it is important to consider that some B cells may be resistant to rituximab therapy despite expressing CD20.

The fact that the specificities and amount of antibodies in the serum against some types of antigens (e.g., HLA and viral antigens after vaccination) are maintained with high fidelity for many years also argues for the existence of a LLPC population rather than constant turnover with replacement from a pool of precursors (23). The poor correlation between the number of antigen-specific memory B cells in peripheral blood and the levels of long-lived serum antibodies against that specific antigen suggest that circulating memory B cells may not be the major source of long-lived antibody (23). Taken together, we believe that these data strongly suggest the existence of a pool of LLPCs. However, it is still possible that a pool of relevant memory B cells might exist in secondary lymphoid tissue and contribute to ongoing antibody production.

Compared with other B-cell types, LLPCs, especially in humans, have been studied less frequently and much less is known about their biology. This is due in part to the fact that LLPCs are hard to access (they are almost nonexistent in peripheral blood), they are extremely rare even in the bone marrow and lymph nodes (e.g., only 0.5% of nucleated bone marrow cells are LLPCs), and they are notoriously difficult to culture in vitro (most die immediately after isolation).

Fortunately, several recent studies are beginning to clarify the biology of these important cells (17–20). Unlike other B cells, LLPCs are nondividing, terminally differentiated cells that no longer express cell surface immunoglobulin. They are specialized cells that are protein factories whose major function is to secrete large amounts of high-affinity antibody. They have a distinctive appearance with a markedly enlarged cytoplasm and a shrunken nucleus (Fig. 2). The phenotype of LLPCs is CD20-, CD19-, CD27-, CD38+, and CD138+ (17–20).

FIGURE 2
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Some LLPCs that develop in peripheral lymphoid organs remain there, whereas others migrate out of these areas and home to the bone marrow. LLPCs in lymph nodes and spleen may have a shorter life span than those in the bone marrow, and over time, LLPCs in the marrow may contribute more to serum antibody levels (20).

LLPCs may live for decades, yet their longevity does not appear to be an intrinsic property. As already noted, most LLPCs die immediately after isolation and almost all die within a few days even in the most supportive culture conditions. Instead, it appears that it is the “microenvironment” in which LLPCs reside that is responsible for their longevity (20) (Fig. 3).

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The current model of the microenvironment suggests that “stromal cells” provide survival factors for LLPCs such as IL-6 (20). Stromal cell lines and long-term cultured nonlymphoid cells from either the bone marrow or secondary lymphoid tissue have been shown to increase plasma cell survival in vitro and to increase antibody production. It is unclear if there are different types of stromal cells—some necessary to support hematopoietic stem cells and others to support the longevity of plasma cells. It also is possible that the LLPC plays a role in the development and maintenance of the microenvironment. One recent study suggested that IL-6 production by bone marrow stromal cells was not constitutive but was induced when cocultured with plasma cells in vitro (24). Antibody levels in the serum are maintained within relatively narrow ranges and it has been hypothesized that this is achieved by having a finite number of niches that support a finite number of LLPCs. However, the data to support a finite number of niches are lacking.

Another dimension of complexity of the microenvironment is suggested by emerging data that these ancillary cells vary between different lymphoid compartments such asthe bone marrow (megakaryocytes, macrophages, and eosinophils) (25, 26), spleen (dendritic cells/myeloid cells and basophils) (27, 28), and lymph nodes (monocytes/macrophages and neutrophils) (29, 30). Several in vitro studies suggest that these “accessory” cells support plasma cell survival via the production of prosurvival cytokines, including APRIL (a proliferation-inducing ligand), BAFF (B-cell–activating factor, a tumor necrosis factor family cytokine, also known as B lymphocyte stimulator), and IL-6 (20). However, in vivo studies have not borne out these in vitro findings. For example, treating mice with the drug ABT-737, which blocks Bcl-2, and Bcl-xL (prosurvival factors induced by APRIL) did not affect persistent plasma cells but did block earlier steps in B-cell development (i.e., the development of antigen-specific memory B cells and plasma cells) (31). Similarly, a recent trial of anti-BAFF antibody insensitized transplant candidates also failed to decrease serum alloantibody levels (32). These data highlight the resiliency of LLPCs and the possible existence of redundant prosurvival mechanisms.

When viewed from the perspective of immunoregulation, it appears that the immune cells commonly involved in regulation (regulatory T and B cells and dendritic cells) do not regulate LLPCs. Instead, it appears that a critical function of the immune system—the persistence of antibody production by LLPCs—may be greatly influenced by nonimmune stromal cells. A critical unanswered question is what is the mechanism by which a LLPC finally dies and how frequent does this occur in the bone morrow? Much more research is needed to clarify the interactions between LLPCs and the microenvironment.

In sensitized patients, it is likely that the most important source of persistent antidonor antibody is LLPCs. However, few studies have directly studied LLPCs in transplantation. One study examined LLPCs in the spleens of renal transplant recipients who underwent splenectomy eitherbefore or after transplantation as part of their desensitization regimens (33). When quantified using immunohistochemistry, plasma cells in the spleen were not reduced by conventional immunosuppression (tacrolimus, mycophenolate, and prednisone), rituximab, or polyclonal rabbit antithymocyte globulin (known to contain antibodies against LLPCs antigens such as CD38 and CD138). These results suggested that the reagents commonly used at the time were ineffective at depleting LLPCs in vivo.

Another group of studies directly examined LLPCs isolated from the bone marrow of sensitized renal transplant patients (34) (Fig. 4). Similar to the results of the splenectomy study, in vitro treatment with either rituximab, high-dose immunoglobulin, or polyclonal rabbit antithymocyte globulin had no effect on the viability or antibody production by LLPCs (34). In contrast, the proteasome inhibitor bortezomib (Food and Drug Administration approved for the treatment of resistant multiple myeloma and mantle cell leukemia) caused rapid induction of apoptosis and abrogated antibody production by LLPCs in vitro (35).

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Based on these data, a clinical trial of bortezomib monotherapy in highly sensitized renal transplant candidates was performed. The primary endpoint of the study was the reduction of bone marrow–derived, antigen-specific LLPCs in sensitized renal allograft candidates before transplantation (36). Bortezomib (up to 16 doses) caused a significant reduction in both allospecific and tetanus-specific LLPCs. In addition, the depletion of LLPCs enhanced the ability of plasma exchange to reduce serum alloantibody levels. Unfortunately, the effect on DSA on serum antibody levels in these highly sensitized patients was mild, limiting its clinical applicability.

However, the use of serum alloantibody levels as an endpoint for studies such as this places a very high bar for success and may obscure partial successes (37, 38). Antibodies have half-lives of 6 to 8 weeks; thus, serum levels might take months to decrease even if all antigen-specific LLPCs were eradicated.

Bortezomib monotherapy also has been shown to be at least partially effective in multiple myeloma that is refractory to other therapy. Bortezomib monotherapy led to a partial response/complete response in 39% of patients (39). This combined endpoint increased to 78% when dexamethasone was added to bortezomib (40). Combination therapy such as lenalidomide, dexamethasone, and bortezomib resulted in 100% of patients experiencing at least a partial response and 74% showing a “very good partial response” (41). Thus, combination therapies are now standard of care when bortezomib is used for the treatment of multiple myeloma.

It is unclear what comparisons can be made between normal LLPCs and myeloma cells. However, because there are hundreds of published studies in myeloma and only a few studies of normal human plasma cells, the myeloma literature likely will provide important insights into possible targets for therapy. For example, recent studies have suggested that stromal cells convey resistance to bortezomib to myeloma cells (42). Examining similar interactions in LLPCs might be very enlightening.

Finally, although most antibody production in transplant patients appears to be the product of conventional immune responses, the concept of tertiary lymphoid tissue deserves mention. This consists of organized collection of lymphocytes in nonlymphoid peripheral organs where such immune aggregates are not normally found. These have been demonstrated in several clinical conditions including autoimmunity (43) and chronically rejecting renal allografts (44). B-cell clusters and even plasma cells have been demonstrated in kidneys undergoing rejection, but no studies have definitively shown that an allograft can be a major source of alloantibody. In fact, antibody levels tend to rise when an allograft is removed, suggesting that the graft is absorbing antibody rather than producing it in most cases, although the possibility of B-cell activation by inflammatory cytokines released after surgery also may play a role (45). It also has been suggested that the B-cell clusters in allografts may be participating in cellular immune responses (46).

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Animal Models and Down-Regulation of Humoral Responses

Animal models of antibody-mediated injury also might provide help in designing therapeutic trials aimed at down-regulating humoral responses. However, there may be significant differences between animal models and human humoral immune responses. For example, in contrast to human studies, bortezomib appears to be extremely effective in mice—even a single dose has been shown to lead to near-complete depletion of bone marrow–derived LLPCs (47). However, this dose (0.75 mg/kg) is much greater than that given to humans and the comparative effectiveness of bortezomib in mice and humans remains unclear. Another unusual aspect of mice is that the number of LLPCs tends to increase in the marrow of some mice as they age increasing to approximately 1% to 5% of nucleated bone marrow cells by 2 years of age (24). Although this may beviewed as a positive feature of mice that enhances the study of LLPCs, we view this as another instance in which human and mouse LLPCs may show significant differences in biology.

However, animal models also show other differences that might be instructive. For example, a recent study suggested that the spleen is the major source of antidonor antibody-secreting cells in murine heart allograft recipients early after transplantation but decreases over time (48). When B6 mice were sensitized with BALB/c splenocytes, the number of total IgG plasma cells were significantly higher in the spleen than in the bone marrow at days 7, 14, and 21 after transplantation. However, by 42 days, there were comparable numbers of IgG-secreting cells between the two compartments. Interestingly, between days 21 and 42, there was a decrease in splenic plasma cells and an increase in bone marrow plasma cells, suggesting that, in the long-term, the number of LLPCs in the marrow might overtake that of the spleen.

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Memory B Cells

Memory B cells are the other major product of germinal centers. Memory B cell encompasses a fairly broad range of cell types, the biology of which is only partially known. By definition, memory B cells are distinguishable from naïve B cells in that they show mutations of the immunoglobulin gene and respond to antigen more rapidly (49). In contrast to LLPCs, memory B cells are not terminally differentiated and can undergo further cycles of proliferation and mutation when they encounter antigen. Unlike LLPCs, memory B cells do not secrete antibody constitutively but convert to antibody-secreting cells soon after reexposure to antigen. The longevity of these newly converted antibody-secreting cells is unclear and whether or not they can become prototypical LLPCs is controversial.

Memory B cells can be isolated from lymph nodes, spleen, bone marrow, and peripheral blood. In fact, approximately 25% of all peripheral blood B cells appear to be memory B cells, suggesting that these cells are especially peripatetic roaming the body in search of their cognate antigen (50). The phenotype of human memory B cells is heterogeneous; however, they are commonly identified as CD27+, CD20+, CD38-, and CD138-. Memory B cells can be either IgM+, IgA+, or IgG+, with the latter being the most common.

Memory B cells appear to be very long-lived, but it is unclear if this is due to persistence of the original cells or constant turnover. The factors regulating the number, migration, and longevity of memory B cells remain to be delineated.

In certain disease states, such as autoimmune rheumatoid arthritis, ongoing conversion of memory B cells to antibody-secreting cells may be a major source of pathologic antibody (51, 52). Indeed, there are some data to support the concept that class-switched memory B cells are more likely to be autoreactive than LLPCs (53). In sensitized transplant patients, the role of memory B cells in ongoing antibody production is unclear. After transplantation, memory B cells may be responsible for the early increase in DSA serum levels observed in some sensitized patients and this likely contributes to the development early antibody-mediated rejection (AMR) (54). Similarly, memory B cells may also be responsible for the posttransplantation production of DSA in patients with a “historically positive crossmatch,” that is, transplant recipients who demonstrated no DSA at the time of transplantation but who had evidence of serum DSA at earlier time points. Assays for the isolation and study of memory B cells from peripheral blood in transplant patients have been described, but their correlation with clinical outcomes is unclear (55–58).

Because memory B cells usually express CD20, rituximab therapy is generally effective at depleting these cells. Thus, rituximab has been suggested to decrease early AMR rates after positive crossmatch kidney transplantation when combined with high-dose intravenous immunoglobulin (IVIG), further supporting the concept that memory B cells may play a role in early AMR (59).

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“Down-Regulating” The Impact of DSA Clinically
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Kidney Transplant Studies

In the absence of effective therapy to remove antibody-producing cells, many clinical protocols have focused on directly removing antibody or blocking its impact on the allograft. These protocols typically involve either high-dose IVIG or a combination of plasma exchange and low-dose IVIG both before and after kidney transplantation (59–61). Using these approaches, hyperacute rejection has been a rare event, but early AMR is seen in up to 40% of recipients. In addition, many of these recipients developed evidence of chronic antibody-mediated allograft injury, most commonly manifested by transplant glomerulopathy (62). Despite these immunologic events, however, actual 5-year graft survival rates of 70% have been achieved (2). One must remember that, for most of these highly sensitized patients, the only other option for renal replacement therapy is maintenance dialysis, which has an estimated 5-year survival of approximately 50% (1).

Early AMR episodes commonly demonstrate complement deposition in the allograft including C4d deposition in the peritubular capillaries (54). Therefore, blocking complement activation might ameliorate complement-mediated damage in some settings (63) (Fig. 5). One recent study tested the ability of terminal complement blockade with the anti-C5 antibody eculizumab in the prevention of AMR in the first 3 months after positive crossmatch living-donor kidney transplantation (64). In this group of 26 highly sensitized, positive crossmatch kidney transplant recipients, the incidence of AMR in the first 3 months was 7.7%, despite high levels of circulating DSA after the transplantation. This was significantly lower than the 41.2% rate of early AMR seen in a historical control group of sensitized patients receiving a similar plasma exchange-based desensitization protocol without eculizumab. These results are encouraging and are being further evaluated in an ongoing randomized, multicenter trial (65).

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In these studies, terminal complement blockade appeared to have no impact on the DSA production after transplantation. For example, a similar percentage of patients developed high levels of DSA with treatment as in the control group. The clinical effectiveness of eculizumab appeared to be due to its ability to ameliorate the impact of these high levels of DSA. In addition, the incidence of chronic antibody-mediated injury appeared unchanged either by the prevention of early AMR or by prolonged complement blockade.

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Liver Transplant Studies

In contrast to kidneys and hearts, the liver appears to be remarkably resistant to antibody-mediated injury (66). The liver also appears to protect other organs such as kidneys from antibody-mediated injury if they are from the same donor and transplanted at the same time (67, 68). Animal models have confirmed this effect. For example, donor-specific alloantibodies were cleared from the circulation in only 30 min, when serum was perfused through an extracorporeal liver of the donor origin (69).

Several recent studies have examined the impact of the liver on alloantibody in more detail. In a prospective study of 90 liver transplant recipients with detectable DSA before transplantation, DSA was undetectable at 1 year after transplantation (3). In another study, 23 highly presensitized kidney transplant candidates were transplanted with auxiliary liver grafts from the same donor (70, 71). After transplantation, the crossmatch turned negative in most, but not all, of these patients, all of whom maintained normal renal allograftfunction.

The mechanisms underlying this phenomenon remain to be clarified. In the rat model, antibody and complement split products accumulate on sinusoidal endothelial cells, lowering the circulating antibody levels (69). Hence, it is possible that the abrogation of the DSA is merely secondary to immunoadsorption of the antibody by the liver, with its vast vascular space. In humans, DSA against HLA class I are cleared from the circulation more effectively than class II DSA in liver transplant recipients possibly because of the higher levels of class I expression on the liver sinusoidal endothelial cells or due to the production of soluble antigens, of which the liver is the largest source (72,73). Likewise, in the rat model, the risk of AMR increases as the size of the liver allograft is reduced (74).

In short, accumulating evidence supports the notion that the liver allograft may be a potential biological filter for preexisting DSA. Whether it can prevent de novo or ongoing humoral responses remains to be elucidated.

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Tolerance and B Cells

Immunologic tolerance offers the promise of freedom from both the risk of immunologic graft loss and avoidance of the long-term complications of immunosuppression (75, 76). Certainly, any tolerance protocol will require durable B-cell unresponsiveness. Unfortunately, several protocols aimed at achieving allograft tolerance in humans have been complicated by the development of de novo DSA (77, 78). This suggests that, even when evidence of T-cell tolerance exists, naïve B cells can become activated and undergo transformation into LLPCs. It is not known if this process is T independent or some T cells are not affected by the tolerance protocols. However, it is clear that the development of de novo DSA is higher in some of these protocols than it is with conventional immunosuppression and may limit their implementation.

Others have suggested that B-cell tolerance might be achieved by the deletion of all naïve B cells at the time of transplantation and that the subsequent repopulation of this pool would not include alloreactive B cells (79, 80). However, it is unclear if this event would actually occur.

The fact that new naïve B cells are generated continuously and that donor antigen persists indefinitely presents a daunting task for the development of durable tolerance therapy. New naïve B cells likely will recognize different donor antigens from the naïve B cells that were present at the time of transplantation. Even the way that we think of HLA as a single antigen oversimplifies the true plethora of donor antigens that the recipient’s immune system may be able to recognize. Studies from vaccine biology suggest that the process of antigen presentation can result in several distinct peptides stimulating different immune cells even when a relatively simple viral protein is the antigen (80). Thus, down-regulating all of the possible antidonor naïve B cells in an antigen-specific way appears difficult.

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Future Directions

The mechanisms regulating humoral immune responses involve many different phases of B-cell development. New concepts such as the impact of the bone marrow microenvironment on LLPC longevity have broadened our view of immunoregulation. However, our understanding of how later stages of B-cell development such as the longevity of memory B cells and how LLPCs finally die remain unclear. These basic science questions have important implications for transplant patients.

Clearly, our understanding of the role of antidonor antibody in organ transplantation has improved significantly over the past decade. However, the mechanisms of antibody formation in different clinical situations—acute AMR, de novo formation, and ongoing production in sensitized patients—are still unclear. The role of LLPCs is becoming clearer and understanding their biology, although difficult to elucidate, is an important area for future study. In addition, defining the contribution of memory B cells and naïve B cells to both acute and chronic allograft injury isimportant.

Existing therapy to prevent antibody-mediated damage is only partially effective (Table 1) (63). New therapies are sorely needed to better control humoral immunity and improve the outcomes of many types of organ transplants. Studies from other disease conditions such as multiple myeloma and autoimmunity will likely provide important insights into possible new approaches. We suggest that protocols aimed more specifically at depleting LLPCs are required and these will need to be much more aggressive than those currently used. Understanding the risk-benefit ratio for an individual patient will be an important component of deciding what therapies to employ.

TABLE 1
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Keywords

Humoral immunity; Alloantibody; Plasma cells; Complement; Memory B cells; Immunoregulation; Tolerance; Kidney transplantation; Antibody-mediated rejection

Copyright © 2013 by Lippincott Williams & Wilkins

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