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Keywords:

  • Acute rejection;
  • antibody-mediated rejection;
  • kidney;
  • plasma cells;
  • transplant

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Case History
  5. Discussion
  6. Conclusion
  7. References

Splenectomy has been reported to have a beneficial effect in treating Acute antibody-mediated rejection (ABMR). This reason for this often rapid and profound beneficial effect is not readily apparent from what is known about normal splenic immunoarchitecture. While the spleen is rich in mature B cells, it has not been noted to be a repository for direct antibody-secreting cells. We present a case of a Native American female who received a renal transplant and developed a severe episode of ABMR. The patient was initially refractory to both plasmapheresis and IVIG. The patient underwent an emergent splenectomy with almost immediate improvement in her renal function and a rapid drop in her DR51 antibodies. Immunohistochemical stains of the spleen demonstrated abundant clusters of CD138+ plasma cells (>10% CD138 cells as opposed to 1% CD138 cells as seen in traumatic controls). Though this is a single case, these findings offer a rationale for the rapid ameliorative effect of splenectomy in cases of antibody rejection. It is possible that the spleen during times of excessive antigenic stress may rapidly turn over B cells to active antibody-secreting cells or serve as a reservoir for these cells produced at other sites.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Case History
  5. Discussion
  6. Conclusion
  7. References

Acute antibody-mediated rejection (ABMR) is associated with a poor prognosis (1). Treatment of ABMR includes plasmapheresis (PP), IVIG, and in some cases, Rituximab as well as Bortezomib (2–5). In refractory cases, emergent splenectomy has been reported to bring down anti-HLA antibody levels and improve function (6,7). The mechanism by which splenectomy would have such an immediate and profound effect is not entirely clear. Typically, the spleen contains only a very small proportion of direct antibody-secreting cells (e.g. CD138+ plasma cells) and is abundantly rich in mature B cells, removal of which would not be expected to lower antibody levels acutely. We describe the case of a patient who developed ABMR 1 week posttransplantation, which was refractory to PP and IVIG. This patient had a rapid recovery postsplenectomy with an almost immediate drop in anti-HLA antibody. The spleen was assessed revealing a large burden of CD138+ cells along with several other unique histopathologic features.

Case History

  1. Top of page
  2. Abstract
  3. Introduction
  4. Case History
  5. Discussion
  6. Conclusion
  7. References

The patient is a 65-year-old Native American female who received a deceased donor kidney transplant. The patient had a previous renal transplant that lasted approximately 8 years and was lost due to unspecified injuries leading to chronic fibrosis and tubular loss. In addition, the patient had six full-term pregnancies and multiple previous blood transfusions. Pretransplant her most recent PRA was 60%, which was also her peak. On pretransplant screening, Luminex testing detected very low levels (MFI < 500) of both class 1 and class 2 antibodies (including donor-specific anti-DR51 antibodies at MFI < 300) but both T and B cell cross matches by flow cytometry were negative pretransplant. The MFI of all anti-HLA antibodies noted were not of sufficient intensity to cause us to categorize any of these as absolute contraindicators for transplant. The recipient's HLA typing was A2/blank B35/39, DR58, DR14. The previous donor was A1/blank, B15, B18, DR4 and DR 7. The current donor was A2/A31, B35, B39, DR51/blank. Pretransplant nondonor-specific anti-HLA antibodies to A23, A24, A25, A26 and A32 were detected as were antibodies to BW4, B7 and B60. The only class 2 antibody noted was a donor-specific anti-DR51. The transplant procedure went uneventfully and the patient had immediate function. The patient received induction therapy with Thymoglobulin 1.25 mg/kg (max 100 mg) with rapid steroid taper for 5 days and was maintained on Tacrolimus, Mycophenolate Mofetil and Prednisone. Her Cr fell from 6.8 mg/dL to 1.2 mg/dL by day 4 postop. The patient came to the emergency room on postop day 7 complaining of no urine output. Cr was 2.1 mg/dL that evening, 3.6 in am, and 4.2 the following day; ultrasonography in the emergency department revealed no obstruction or fluid collection.

A kidney allograft biopsy was performed emergently on postop day 8, which revealed marked acute capillaritis and interstitial hemorrhage, diffuse peritubular capillary C4D deposition by immunofluorescense and glomerular endothelial cell injury (Figure 1). Upon admission, the patient also underwent screening by Luminex for any donor-specific antibodies. The patient underwent immediate PP with IVIG at 500 mg/kg along with 250 mg of Solumedrol. PP/IVIG were performed on postop day 8 and twice on postop day 9 where Cr continued to rise, and the patient remained anuric. Due to the severe histopathologic damage and anuria, the patient underwent a splenectomy the evening of postop day 9. At admission, Luminex cytoscreen detected specific anti-DR51 antibody at an MFI of 12 000. Subsequent cytoscreens after three PP sessions showed no decrease in antibody MFI. One day after splenectomy the patient had increasing urine output and the Cr started to drop. PP and IVIG were continued postsplenectomy daily for four more sessions and then every other day for five more sessions. By day 20 postsplenectomy Cr was now back down to 1.4 mg/dL and anti-DR51 MFI was markedly decreased (Figure 2). Three months posttransplant, the patient continues to have excellent renal function and anti-DR51 MFI remains under 500.

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Figure 1. (A–D). Renal transplant biopsy. (A) and (B). Diffuse congestion of peritubular capillaries with neutrophils is seen, with interstitial edema and interstitial hemorrhage (Hematoxylin and eosin, 100× and 200× magnification). (C) Diffuse staining of peritubular capillaries and glomerular capillary loops for C4d (Immunofluorescence, 200× magnification). (D) Ultrastructural examination of glomerular capillary loops revealed extensive endothelial cell swelling and vacuolization of cell cytoplasm, with focal subendothelial widening and early duplication of the glomerular basement membrane (8800× magnification). Biopsy of the renal allograft demonstrated diffuse congestion of the peritubular capillaries with neutrophils (acute capillaritis, Banff score ptc3) and infiltration of neutrophils into the interstitium. The inflammation was associated with diffuse interstitial edema, interstitial hemorrhage and acute tubular injury (Figures 1A, B). Glomeruli demonstrated acute glomerulitis (Banff score g1). There was no significant mononuclear interstitial inflammatory infiltrate and no tubulitis was identified. No viral inclusions were identified within tubular epithelium. Immunofluorescence staining for C4d performed on frozen tissue demonstrated diffuse, 3+ staining of peritubular capillaries (Figure 1C), and 2–3+ semilinear staining of the glomerular capillary loops. Electron microscopy revealed swelling and vacuolization of glomerular endothelial cell cytoplasm, with focal subendothelial widening and early duplication of the glomerular basement membrane (Figure 1D). Ultrastructural examination of the peritubular capillaries revealed swelling of endothelial cell cytoplasm with focal lamellation of the basement membrane.

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Figure 2. Course of serum creatinine and donor-specific anti-DR51 antibody.

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The spleen demonstrated the following immunoarchitectural features (Figure 3): Germinal centers composed of CD20+ B cells (upper left) with surrounding CD3+ T cells (upper right). They are seen as collections of lymphocytes surrounding a central artery in the routine H&E section (lower left). All sections are at 40× magnification. At higher magnification (400×, lower right), an increased number of plasma cells is demonstrated surrounding a blood vessel. They are confirmed as polyclonal plasma cells by CD138+, kappa and lambda reactivity.

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Figure 3. Immunohistology and immunohistochemistry of spleen.

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The spleen weighed a total of 75 g. It had a smooth capsule and was without masses or lesions. The microscopic evaluation was notable for a decrease in the white pulp, which was otherwise normal. The red pulp splenic cords were expanded by different cell types, including neutrophils, plasma cells and iron-containing macrophages. The sinusoids also contained a smaller number of the same cells, although not macrophages.

Immunohistochemistry was performed on paraffin sections of routinely formalin-fixed tissue, using the Ventana XLT or Benchmark instruments (Ventana Medical Systems, Tucson, AZ) with their standard protocol. A battery of primary antibodies was used including those for B cells (CD20, CD79a), T cells (CD3, CD4 and CD8), dendritic cells (CD21), NK cells (CD56), macrophages (CD68), neutrophils (CD15) and plasma cells (CD138+). Additionally, in situ hybridization was used to determine kappa and lambda reactive cells after assuring an adequate amount of RNA. All antibodies were from Ventana except for CD56, which was supplied by Cell Marque (Rocklin, CA). Antibody diluent was used as the negative control. Cytochemical staining for reticulin using Snook's method and iron using Gomori's method was also performed. Results of staining confirmed the expected immunoarchitectural features of the spleen, although the T-cell periarteriolar lymphoid sheath was moderately diminished (8,9). Plasma cells were noted throughout the red pulp and were polyclonal. The kappa: lambda ratio was approximately 2–3:1. The iron stain highlighted a large number of iron-containing macrophages throughout the red pulp. Reticulin stain did not show an increase in reticulin-positive fibers.

Six spleens were utilized as controls read by the same pathologist and immunohistochemistry protocol was performed in the exact same manner. These six ‘control’ spleens were from otherwise healthy individuals who had splenectomy performed due to trauma. The case had a cellular component of 10% CD138 bearing cells, some in clear aggregates. The six control spleens had no more than 1% CD138 bearing cells. None of the control splenic samples showed aggregation of CD138 cells or splenitis. The other cellular components and architecture were not remarkably different to the case, except for a relative paucity of CD20 cells as compared to controls; however, the cellular components of CD20 cells differed enough between control samples that differences to the case were difficult to definitely assess.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Case History
  5. Discussion
  6. Conclusion
  7. References

This patient had a particularly severe form of early ABMR, presumably due to the donor-specific anti-DR51 antibody present. The patient had an initial negative clinical response to the first three sessions of PP and IVIG and had markedly elevated levels of DR51 antibody. The decision to proceed to splenectomy was based on the severity of the histopathology, anuria and high antibody levels. While splenectomy has been reported to have immediate beneficial effects in ABMR, its mechanism of action is not entirely clear. We decided to carefully look at the immunophenotype of cells in the spleen along with its histopathology. The major findings were a 10-fold increase in CD138+ cells along with marked inflammation of the spleen when compared to control spleens. To our knowledge, this degree of CD138+ cells has not been described before, and was clearly different than our control samples. These findings raise the possibility that in some cases of severe ABMR the spleen either captures or sequesters CD138 cells, or quick differentiation of B cells into antibody-secreting CD138+ cells occurs during this high antigenic stress. This may help explain the salutary effect of splenectomy in cases of severe ABMR. It is quite possible that these splenic findings are also time dependent and time sensitive. This degree of antibody response and the contribution of the spleen to total CD138 burden may be quite different early posttransplant or postantigenic stimulation than at later time points.

Due to the long life span of anti-HLA antibodies, it is possible that both removal and rapid decrease of antibody production are both needed when antibody production is high. In these cases, our findings would indicate that splenectomy may be an effective way to decrease antibody production (and/or total body burden) under these specific circumstances and timing. However, as a single case, any conclusions must be couched with caution. Thus, we cannot be sure that the patient would not have responded without splenectomy nor that every case of ABMR has similar splenic findings.

Nonetheless, this case illustrates that under conditions of ABMR, the spleen may produce or ‘house’ abundant numbers of antibody-secreting cells and this offers one explanation for the observation of the efficacy of this intervention in cases of ABMR (possibly cases where production is high and simple removal is insufficient).

In addition, we, of course, cannot with certainty know whether the CD138+ cells in the spleen were secreting the anti-DR51 antibodies either. However, given these caveats, we feel this case offers sufficient novel insights to warrant further study of mechanisms and sites of antibody production under the stress of ABMR.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Case History
  5. Discussion
  6. Conclusion
  7. References

Though this is a single case, these findings offer a rationale for the rapid ameliorative effect of splenectomy in cases of antibody rejection. It is possible that the spleen during times of excessive antigenic stress may rapidly turn over B cells to active antibody-secreting cells or serve as a reservoir for these cells produced at other sites.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Case History
  5. Discussion
  6. Conclusion
  7. References