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Clinical and Translational Research

B Cells and Plasma Cells in Coronaries of Chronically Rejected Cardiac Transplants

Wehner, Jennifer R.1,2,4; Fox-Talbot, Karen1; Halushka, Marc K.1; Ellis, Carla1; Zachary, Andrea A.3; Baldwin, William M. III1,2

Author Information
doi: 10.1097/TP.0b013e3181d3f271

Advances in immunosupression have decreased the incidence of acute cell-mediated rejection of organ transplants, but the survival of cardiac and kidney allografts continues to be limited by chronic rejection (1). In cardiac transplantation, chronic rejection is caused by the development of vasculopathy in coronary arteries that is characterized by a diffuse concentric intimal proliferation and adventitial sclerosis (2–4). Endomyocardial biopsies, which are the standard method for assessing acute cardiac transplant rejection (5), are not useful in evaluating the pathologic changes in epicardial coronary arteries. Chronic rejection is usually detected by surveillance angiographic or intravenous ultrasound studies and pathologically assessed on autopsy. Allografts examined at autopsy introduce a number of confounding variables such as comorbidities (cancer, infections, sepsis, etc.) and postmortem cell autolysis. In this study, we have circumvented this problem by studying allografts removed because of chronic rejection at the time of retransplantation.

T cells have been studied extensively in cardiac allograft rejection because they are a major component of most acute rejection episodes. Increasingly sophisticated immunosuppressive regimens directed at T-cell responses have decreased graft loss because of acute rejection. Unfortunately, the rate of chronic rejection has not been significantly abated. In contrast to T cells, which act locally within the graft, most B cells reside within lymphoid organs. Although B cells constitute a small portion of circulating lymphocytes, substantial numbers of B cells and plasma cells have been demonstrated within transplanted organs, often in the nodules or sometimes forming tertiary lymphoid structures with distinct T- and B-cell compartments (6–8).

B-cell infiltrates have been described in renal transplants as features of both acute and chronic rejection. However, the significance and function of B-cell infiltrates is not fully understood. Some groups have reported that CD20+ B cells in renal allograft biopsies (9, 10) and explanted renal grafts (11–13) are associated with more severe rejection, but other groups have found that B cells do not correlate with decreased graft survival (14–16). Although the presence of intragraft B cells has not been found to correlate with C4d deposition or donor-specific antibody (DSA) in clinical studies, experimental models have demonstrated that tertiary lymphoid nodules can support the generation of memory T cells in skin grafts (17) and the production of DSA in segmental aortic grafts (7).

Considerably, less data are available on B cells and plasma cells in cardiac transplants. The majority of reports on B cells in cardiac allografts are confined to endomyocardial mononuclear cell infiltrates, known as the Quilty effect, which have been described in as many as 50% to 70% of all cardiac transplant recipients (18–22). Quilty lesions are characterized by nodular infiltrates that may contain compartmentalized B- and T-cell populations surrounding high-endothelial venules on the endomyocardial surface.

Previously, we reported gene microarray profiles of coronary arteries dissected from 24 human heart explants recovered in the operating room at the time of transplantation including 6 hearts with dilated cardiomyopathy without coronary lesions, 6 hearts with native atherosclerosis, and 12 cardiac transplants that were replaced because of transplant vasculopathy (8). Genes for immunoglobulins (heavy and light chains) and receptors (CR2; CD21) that are expressed by B lymphocytes were upregulated in 11 of 12 coronaries with vasculopathy compared with native atherosclerosis or no lesions. In five of these samples, these probes were increased in the range of 5- to 25-fold. The presence of B and plasma cells was confirmed by preliminary immunohistology on eight of these hearts.

The almost universal expression of B-cell genes in coronary arteries with vasculopathy and the paucity of B cells in atherosclerosis led us to more thoroughly evaluate the prevalence, location, and clonality of the B and plasma cells in transplant vasculopathy.

MATERIALS AND METHODS

Tissue Samples

Between 1989 and 2008, a total of 26 cardiac transplants with chronic rejection were explanted during a second transplant procedure at The Johns Hopkins Hospital. Tissue samples from 16 of these explants contained intact coronary arteries and were used in this study. This retrospective study was approved by the Johns Hopkins Medical Institutional Review Board.

All tissues were operating room specimens and were immediately placed in formalin and processed in a timely matter for diagnostic purposes. The number of tissue blocks and vessels available for analysis from each patient was variable with an average of three tissue blocks containing coronary vessels per patient. Approximately three vessels per block were present in each patient.

Immunohistology

Five-micron sections were deparaffinized, and antigen retrieval was performed by immersing slides in two changes of Trilogy EDTA (pH 8.0; Cell Marque, Hot Springs, AR) in a steamer for a total of 60 min. The slides were rinsed and cooled in H2O for 5 min. Endogenous peroxidase activity was blocked by incubation in 0.3% H2O2 in methanol. Nonspecific protein activity was blocked with a serum-free protein block (DAKO Corporation, Carpinteria, CA). For IgG1 staining, the slides were incubated with an anti-human IgG1 (AbD Serotec, Oxford, UK) for 60 min. Secondary labeling and visualization of these slides were performed using the SuperPicture horseradish peroxidase-conjugated polymer DAB system from Zymed Laboratories. (San Francisco, CA). Additional primary antibodies used in staining were CD3 and syndecan-1 (Abcam, Cambridge, MA), CD20cy (Dako, Carpinteria, CA), IgG2, IgG3, and IgG4 (Zymed, San Francisco, CA), Kappa and Lambda (Diagnostic Biosystems, Pleasanton, CA), C4d (American Research Products, Belmont, MA), CXCL13 (R&D Systems, Minneapolis, MN). The sections were incubated with biotinylated anti-rabbit IgG or anti-mouse IgG (Jackson Labs, West Grove, PA) depending on the primary antibody, for 30 min at room temperature. Immunoperoxidase staining was performed using Vectastain ABC Elite (Vector Labs, Burlingame, CA). The avidin-biotin complex was visualized using a 3, 3′ diaminobenzidine peroxidase substrate (Vector Labs). The sections were counterstained in hematoxylin (Richard-Allen, Kalamazoo, MI).

Histopathologic Scoring

B-cell and plasma cell infiltrates were evaluated in a blinded fashion on tissue samples that contained vessels with allograft vasculopathy (AV). B-cell nodules were considered to be associated with a vessel if they were within one 20× power field of the smooth muscle cell layer of the affected vessel. Nodular B-cell infiltrates were categorized into groups of zero nodules, one to three nodules, or greater than three nodules. Adventitial plasma cells clustering around B-cell nodules were evaluated on the percent of the nodule encircled by plasma cells: 3+=more than 75% of the nodule; 2+=more than 50% to 75% of the nodule; 1+=25% to 50% of the B-cell nodule; 0=less than 25% of the nodule. T- and B-cell compartmentalization was noted when there was a clear separation of T and B cells. Histologic scoring of diffuse B-cell and plasma infiltrates and neointimal infiltrates was based on the degree of the vessel encircled by B-cell infiltrate; 3+=more than 75% of vessel; 2+=more than 50% to 75% of vessel; 1+=25% to 50% of vessel; and 0=less than 25% of vessel.

Assays for Antibodies to HLA

Lymphocyte cross-match tests were performed by complement-dependent cytotoxicity (anti-human globulin [AHG]-enhanced for T cells and one-wash for B cells) and by flow cytometry. Tests of human leukocyte antigen (HLA)-specific antibodies were by solid-phase immunoassay on the ELISA (Quik-ID class I and class II, GTI, Brookfield, WI) and Luminex platforms (Lifematch ID and LifeScreen kits; Tepnel LifeCodes, Stamford, CT; Single Antigen Bead kits; One Lambda, Canoga Park, CA).

Statistics

Statistical differences in the presence or absence of B cells between AV and native atherosclerosis were evaluated using a Chi-square test.

RESULTS

Overall Prevalence of B-Cell and Plasma Cell Infiltrates Associated With AV

In agreement with our microarray data reported previously, B and plasma cells were a consistent feature of AV. In fact, all 16 of this expanded group of patients had infiltrates of B cells (14 of 16), plasma cells (15 of 16), or both (13 of 16).

Patterns of B-Cell Infiltrates Associated With AV Lesions

B-cell infiltrates associated with AV lesions displayed three distinct patterns: adventitial nodules, diffuse adventitial infiltration, and neointimal infiltration (Table 1). To compensate for the variable number of blocks and vessels per patient, we averaged the scores in all vessels for each patient for each of the infiltrate patterns. The most common finding was B-cell nodules in the adventitia of arteries with AV (Fig. 1A–C). Thirteen of 16 patients (81%) analyzed demonstrated B-cell nodules associated with affected arteries. Nodular adventitial infiltrates were compartmentalized into B- and T-cell regions as shown in Figure 2 in 7 of 13 patients (44%). The second most common pattern of B-cell infiltrates was diffuse cellular infiltrates in adventitial tissues (Fig. 1B), which occurred in 10 of the 16 patients (63%) and were accompanied by T-cell infiltrates. Of note was the finding that these B-cell infiltrates were frequently surrounded by dense fibrosis in the adventitia. Within the vessel, B cells infiltrated the neointima in 7 of the 16 patients evaluated (44%; Fig. 1A, inset).

TABLE 1
TABLE 1:
Clinical and histologic characteristics of explanted hearts from patients with AV
FIGURE 1.
FIGURE 1.:
Epicardial coronary arteries with allograft vasculopathy showing distinct patterns of B-cell and plasma cell infiltrates. (A–C) Immunohistochemistry staining with CD20 showing B-cell infiltrates as adventitial nodules (A–C), diffuse adventitial infiltrates (B), or neointimal infiltrates (A). (D) Immunohistochemistry staining with syndecan-1 showing plasma cell infiltrates in the sclerotic adventitia. (A–D) 2×. Insets; 20×.
FIGURE 2.
FIGURE 2.:
Epicardial coronary artery with allograft vasculopathy and adventitial nodular mononuclear cell infiltrates showing compartmentalization of B and T lymphocytes and plasma cells. Serial sections are stained by immunoperoxidase for B lymphocytes (CD20; A and B), T lymphocytes (CD3; C) and plasma cells (syndecan-1; D). Magnification: (A) 2× and (B–D) 20×. *Arteriole.

Many patients had more than one pattern of B-cell infiltrate. All but one of the patients with diffuse infiltrates also had adventitial nodules, but 4 of 13 patients with B-cell nodules had only nodular infiltrate. Seven patients had vessels sampled at multiple sites ranging from proximal, middle, and distal regions. B cells were located in all regions, although the pattern of distribution and the extent of infiltrating B cells throughout the length of each vessel was variable (data not shown).

Quilty Lesions Are Not Associated With Patterns of B-Cell Infiltrates

We reviewed all patient endomyocardial biopsies taken before explantation to look for a possible association of endomyocardial Quilty lesions and epicardial B-cell infiltrates in patients with chronic rejection. Half of the patients (8 of 16) had a Quilty lesion present in at least one of their previous endomyocardial biopsies. Of the eight patients with Quilty lesions, six patients had one to three adventitial B-cell nodules, whereas the remaining two patients had greater than three adventitial B-cell nodules. Diffuse B-cell infiltrates were present in five of the eight patients with Quilty lesions. Only four of the eight patients with Quilty lesions had infiltrating B cells in the neointima. Therefore, although all patients with Quilty lesions had epicardial B-cell infiltrates, the presence of Quilty lesions on the endocardium was not predictive of the extent or pattern of B-cell infiltrate in the epicardium surrounding coronary arteries with AV.

Compartmentalized Lymphoid Nodules Exhibit Evidence of Functional Antigen-Presenting Units

The formation of compartmentalized tertiary lymphoid nodules suggested a functional role for these cells. Critical for the formation of a functional tertiary lymphoid nodule is a central follicular dendritic cell (FDC) network. FDCs are known to secrete CXCL13, a B-cell chemoattractant, and also have complement receptors that facilitate in antigen presentation. Immunohistochemical staining using CD21 for FDCs revealed that compartmentalized lymphoid aggregates, but not diffuse infiltrates, contained a FDC network (data not shown). C4d staining was seen on FDCs in these nodules (Fig. 3C). In addition, CXCL13 staining was more extensive in nodules than in diffuse infiltrates (Fig. 3D).

FIGURE 3.
FIGURE 3.:
Immunohistochemistry stains of C4d deposition and CXCL13 in patients with allograft vasculopathy (AV). C4d deposits are shown on the endothelium of a coronary artery with AV (A) and capillaries of adjacent myocardium (B). Lymphoid nodules stained for C4d (C), CXCL13 (D), B cells (CD20; E), and T cells (CD3; F). Magnification: (A) 40×; (B–D) 20×; Insets 60×.

Plasma Cell Infiltrates Are Associated With AV Lesions

Similar to B-cell infiltrates, plasma cell infiltrates were seen in three patterns: clusters around B-cell nodules, diffuse adventitial infiltrates, and neointimal infiltrates (Table 1). Almost 70% of the patients (12 of 16) analyzed had plasma cells in clusters around B-cell nodules (Fig. 2D), whereas more than 80% of patients (13 of 16) had diffuse plasma cell infiltrates in the adventitia (Fig. 1D). Plasma cell infiltrates were also present in the neointima of seven patients (44%) with AV lesions. All but one patient had plasma cell infiltrates. Moreover, the extent of plasma cell infiltrate was not dependent on the extent or location of B-cell infiltration.

Plasma Cells in Coronaries With AV Are Heterogeneous

Compact collections of plasma cells in the neointima, adventitia, and epicardium raised the question of the clonality of plasma cells in these regions. Patients with sufficient tissue and plasma cells were evaluated for cytoplasmic content of all four IgG subclasses by immunohistochemistry. In 10 of 12 patients (83%) with sufficient tissue, all four IgG subclasses were present within plasma cell infiltrates. The remaining two patients showed the presence of only IgG2 and IgG3. However, in these samples, the quality of IgG1 staining was inadequate. In addition, plasma cells in nodules were heterogeneous for kappa and lambda light chains, indicating that the plasma cell infiltrates were polyclonal (see Figure, Supplemental Digital Content 1,http://links.lww.com/TP/A174).

B-Cell and Plasma Cell Infiltrates Encompass all Patient Groups

To explore the relationship between B-cell and plasma cell infiltrates with length of graft survival, we divided the patient population into short (<3 years), intermediate (3–12 years), and long (>12 years) graft survival groups (Table 2). In both the short and long graft survival groups, three of four grafts had one to three adventitial B-cell nodules, and one heart had no B-cell nodules. The two patients with greater than three adventitial B-cell nodules were in the intermediate graft survival group. Diffuse adventitial B-cell infiltrates were only present in one of four hearts in the short graft survival group, and no neointimal B-cell infiltrates were seen in this group. However, the long graft survival group had diffuse B-cell infiltrates in all the hearts and neointimal B cell infiltrates in three of four of the hearts.

TABLE 2
TABLE 2:
B-cell and plasma cell infiltrates by length of graft survival

Similarly, we examined the presence of plasma cells in relation to length of graft survival. The presence of plasma cells surrounding B-cell nodular infiltrates was slightly higher (3 of 4) in the long graft survival group than the short survival group (2 of 4). Likewise, the presence of diffuse adventitial plasma cells were slightly higher in the long graft survival group (4 of 4) compared with the short survival group (2 of 4). The presence of neointimal plasma cells between these two groups was the same (1 of 4). Overall, there was only a slight increase in B-cell or plasma cell infiltrates with length of graft survival. In addition, we saw no correlation between the genders nor age of patient with the presence or pattern of B-cell or plasma cell infiltrates (Table 1).

Circulating Donor-Specific Antibodies Were Not Found at the Time of Explantation

The presence of B and plasma cells along with compartmentalized infiltrate nodules suggested a local immune response to the transplant. Therefore, we looked for DSAs in patient serum at the time of explantation. Serum samples were available for the 10 patients who were retransplanted after 1998. DSAs were not detected in the serum from any of these 10 patients. Two patients had low levels of antibodies to major histocompatibility complex class II antigens that were not present on the donor heart.

C4d Deposition in Coronary Arteries and Capillaries

The tissue sections with sufficient myocardium adjacent to coronaries were stained with C4d to assess complement deposition. Two of 14 patients showed strong diffuse capillary C4d staining, and one patient demonstrated a weak diffuse capillary C4d staining in the myocardium (Fig. 3B). In these three patients, the endothelium of main coronary arteries were not well enough preserved for definitive evaluation of C4d; however, medium-sized branching arteries showed C4d deposits (Fig. 3A). There was no correlation between C4d positivity and the length of graft survival or B-cell and plasma cell infiltrates (Table 2).

Studies of B-Cell and Plasma Cell Infiltrates in Control Patients With Open Heart Surgery and Native Atherosclerosis

To eliminate the possibility that B-cell and plasma cell infiltrates were a nonspecific inflammatory response to open heart surgery, we examined native hearts from six patients who had undergone open heart surgery 7 to 10 years before explantation. The indication for open heart surgery included coronary artery bypass grafts, stent placement, and valve replacement. To maximize the possibility of finding surgically related inflammatory responses, we examined the surgical site as indicated by suture sites. Control coronary arteries revealed minimal inflammatory infiltrates, and specifically, no epicardial lymphoid nodules were seen surrounding the repaired coronary arteries.

Hearts with native atherosclerosis that were removed from nine patients who received primary cardiac transplants were analyzed for the presence of B-cell, plasma cell and T-cell infiltrates (Table 3). Two patients had one small mixed B- and T-cell nodule adjacent to the affected coronary artery, and there were no diffuse or neointimal B-cell infiltrates (see Figure, Supplemental Digital Content 2,http://links.lww.com/TP/A175). Only one patient demonstrated minimal diffuse plasma cell infiltrates. T cells were scattered diffusely within the atherosclerotic lesions.

TABLE 3
TABLE 3:
Clinical and histologic characteristics of hearts from patients with native atherosclerosis

DISCUSSION

This study demonstrates that B or plasma cells are a consistent component of AV. In fact, B or plasma cells were found in the coronary arteries from all 16 patients. In contrast, only a minority of vessels with native atherosclerosis (2 of 9 patients) had clusters of B cells, which is consistent with the existing concepts of a prominent role for T cells and macrophages in the pathogenesis of atheroscerosis (23, 24). However, prominent B-cell infiltrates have been described in atherosclerotic lesions of patients with rheumatoid arthritis (25). These data substantiate our previously reported data from microarrays, in which transcripts from immunoglobulin genes were consistently elevated in 12 coronary arteries with AV, but not in 6 coronary arteries with native atherosclerosis (8).

We have characterized three main patterns of B-cell and plasma cell infiltration. Most frequently, B and plasma cells formed nodules in the adventitia of coronary arteries with AV; 13 of the 16 hearts had nodules of B cells, 12 of which contained nodules of both B and plasma cells. Nodules of B cells have been reported in renal transplants, but this finding is confined to only a subset of patients. In endomyocardial biopsies from cardiac transplants, B cells can be a prominent feature of the Quilty effect. In the current diagnostic formulation (26), the Quilty effect is defined as nodular endocardial infiltrates that are distinguished from acute rejection by the presence of B lymphocytes and plasma cells in a background of fibrosis and prominent vascularity. Although several recent publications have emphasized the B-cell content of these lesions and have reported correlations with acute rejection (20, 21, 27), the relationship of Quilty lesions and chronic rejection remains unclear. We reviewed all endomyocardial biopsies from the hearts in our study before explantation and identified the Quilty effect in one or more biopsies from only 8 of the 16 patients. Therefore, nodular infiltrates of B and plasma cells were more frequently found in the epicardium adjacent to coronaries with AV than in the endocardium. Although some characteristics of Quilty lesions are present in the nodules described here, the epicardial location of the nodules excludes them from being conventional Quilty lesions. In addition, the presence of Quilty lesions was not indicative of the extent or pattern of epicardial B-cell infiltrates.

In 7 of 13 patients, adventitial nodules contained compartmentalized B- and T-cell zones characteristic of tertiary lymphoid neogenesis. This phenomenon has been studied extensively in chronic inflammatory diseases such as rheumatoid arthritis. In patients with rheumatoid arthritis, B-cell nodules have been described in coronary arteries and synovium (25, 28). There are reports (6, 29) of tertiary lymphoid follicles in renal transplants and a few reports in experimental (7, 30) and clinical heart transplants (7, 8). The organization of lymphoid tissue at the site of immune response allows a continued reaction to the immune stimulus. The fact that these nodules contained FDCs that stained positively for C4d provides evidence that they are functional. Complement split products are known to localize antigen to complement receptors on FDCs (31, 32). Dendritic cells in these nodules also expressed CXCL13 in a similar pattern to the staining reported for lymphoid neogenesis in rheumatoid arthritis (28), suggesting a mechanism for the recruitment of B cells.

In addition to B-cell and plasma cell nodules, diffuse infiltrates were found in the adventitia surrounding vessels with AV. All but two of the hearts had diffuse infiltrates of B or plasma cells in the adventia, and half of the hearts had diffuse infiltrates of both B and plasma cells in the adventia. More than half (9 of 16) of the hearts had infiltrates of B or plasma cells in the neointimal, whereas almost a third of the hearts (5 of 16) had B-cell and plasma cell infiltrates in the neointima of vessels with AV. These neointimal and advential infiltrates were often embedded in fibrosis. Mengel et al. (33) have reported that the transcripts associated with B and plasma cells are a signature of scarring in renal transplants. Recent data from experimental models indicate that B cells can promote fibrosis (34, 35). Adventitial fibrosis has functional consequence because it prevents compensatory outward remodeling of coronary arteries in response to AV.

The finding that nodules of plasma cell infiltrates stained heterogeneously for heavy and light chains indicated that the nodules did not result from expansions of single clones of B cells. This heterogeneity also decreases the likelihood that the nodules are related to posttransplant lymphoproliferative disorder.

The availability of multiple segments from several individual coronary arteries allowed us to determine whether infiltrates were continuous or focal. We found that B and plasma cells were prevalent throughout the vessels and were not confined to one region or single coronary vessel. However, the composition and degree of infiltration varied along the length of the vessels.

With the pervasiveness of B cells and especially plasma cells with demonstrable cytoplasmic immunoglobulins in this group of patients, we tested the possibility that there may be a high incidence of DSA in these patients. However, we found no evidence of DSA at the time of explantation. The absence of antibodies to HLA in the circulation does not exclude the possibility that antibodies to HLA were absorbed to the transplant or that the plasma cells produced tissue-specific antibodies or autoantibodies, both of which have been reported in clinical and experimental cardiac transplants (34). In fact, 3 of 14 transplants had diffuse C4d deposits in the capillaries in myocardium adjacent to the coronary arteries, which are consistent with complement activation by donor reactive antibodies.

In summary, B and plasma cells are consistent findings in and around coronary arteries with AV. These cells were found most frequently in the nodules, some of which had distinct compartmentalization that typifies tertiary lymphoid nodules. In addition, diffuse infiltrates of B and plasma cells were found in fibrotic areas of the neointima and adventitia. The frequency of B and plasma cells within and surrounding vessels with AV was significantly higher than in coronaries with native atherosclerosis or surgical procedures.

ACKNOWLEDGMENTS

We would like to thank Rene Rodriguez and Carmela Tan for their advice in selecting a reagent for human C4d.

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Keywords:

Chronic allograft vasculopathy; B cells; Plasma cells

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