- Email: [email protected]
Rescue Therapy by Immunoadsorption in Combination With Tacrolimus and Mycophenolate Mofetil for C4d-Positive Acute Humoral Renal Allograft Rejection
Rescue Therapy by Immunoadsorption in Combination With Tacrolimus and Mycophenolate Mofetil for C4d-Positive Acute Humoral Renal Allograft Rejection S.-M. Ji, Z.-H. Liu, J.-S. Chen, G.-Z. Sha, D.-X. Ji, and L.-S. Li ABSTRACT The aim of this study was to investigate the efficacy of immunoadsorption (IA) in combination with tacrolimus (TAC; 0.14 to 0.16 mg/kg/d) and mycophenolate mofetil (MMF; 1.5 to 2.0 g/d) rescue therapy for C4d-positive acute humoral rejection in nine cadaveric renal allograft recipients. Initial Panel reactive antibody (PRA-I and PRA-II levels were as high as 28.8% ⫾ 16.2% and 15.3% ⫾ 8.9%, IA therapy significantly decreased PRA-I and PRA-II levels to 5.9% ⫾ 2.9% and 2.2% ⫾ 0.6%, respectively. Total serum immunoglobulin levels were markedly decreased. Repeated allograft renal biopsy in nine patients revealed remission of acute humoral rejection (AHR), and the deposition of C4d disappeared and reduced. With a mean follow-up of 29.4 ⫾ 5.4 months, patient and allograft survivals were 100%, and renal function remained stable with a mean serum creatinine of 1.1 ⫾ 0.3 mg/dL. Our findings suggested that a therapeutic approach combining IA and TAC and MMF rescue improved the outcomes of AHR.
D
ESPITE THE MORE sophisticated use of immunosuppressive drugs and the improved understanding of cellular and humoral host responses to transplanted organs during recent years, early allograft rejection remains a barrier to successful kidney transplantation.1 There is increasing evidence that besides cellular immunity, antibody-mediated immune mechanisms also may cause acute rejection. These events are associated with a high rate of graft loss.2 Currently, the diagnosis of acute humoral rejection (AHR) is predominantly based on the detection of donor-specific antibodies by posttransplant crossmatches.3 The presence of donor-specific antibodies has been shown to be associated with typical histopathologic findings, such as accumulation of neutrophils in peritubular capillaries (PTC). Recent studies have suggested that endothelial deposition of the stable complement-split product C4d may indicate donor-specific alloantibody-mediated AHR.4 Furthermore, C4d deposition has been correlated with poor graft survival.5 The traditional antirejection treatment, such as methylprednisolone pulse therapy or intravenous injection of anti-CD3, has usually been ineffective to treat AHR. Plasma exchange and immunoadsorption are newly developed approaches to reduce the concentration of anti-HLA antibody in renal transplant recipients.6 Moreover, tacrolimus (TAC), a widely used calcineurin inhibitor (CNI), has been reported to reduce rejection or mitigate CNI nephro-
toxicity compared with cyclosporine (CsA)-based immunosuppressive therapy.7 The aim of this study was to investigate the efficacy of immunoadsorption (IA) in combination with TAC and mycophenolate mofetil (MMF) rescue therapy for C4d-positive acute humoral rejection of renal transplants. MATERIALS AND METHODS Patients During the period from December 2001 to June 2003, 137 patients received renal allografts from non– heart-beating donors. Prior to transplant, all blood from recipients must have shown a ⬍10% level of complement-dependent cytotoxicity. In addition, we used flow cytometry to detect panel reactive antibody (PRA), defining patients with a level ⬎ 10% as sensitized. Twenty-one recipients had one or more biopsy-proven acute rejection episodes. Histological findings were classified according to the Banff 97 classification for acute rejection, as borderline, or grades I to III rejection.8 C4d was detected by an indirect immunofluorescence method in renal tissues obtained from allograft From the Research Institute of Nephrology (S.-M.J., J.-S.C., G.-Z.S., D.-X.J.), Jinling Hospital, Nanjing University School of Medicine (Z.-H.L., L.-S.L.), Nanjing, China. Address reprint requests to Dr Shu-Ming Ji, The Research Institute of Nephrology, Jinling Hospital, 305 East Zhong Shan Road, Nanjing 210002, China. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.10.151
Transplantation Proceedings, 38, 3459 –3463 (2006)
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JI, LIU, CHEN ET AL Table 1. Patient Demographics Parameter
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Age (y) Gender Previous renal allografts Previous pregnancies Cold ischemia time (h) Pre-IA PRA (%) HLA-I HLA-II CDC (%) AHR time (day post-RT) Initial therapy
36 Female 0 2 9
20 Male 0 — 8
48 Male 0 — 8.5
54 Male 1 — 12
37 Male 0 — 11
51 Male 0 — 10
43.0 2.9 6 4 MP/M/T
19.0 12.9 7 5 MP/M/T
32.8 5.7 6 4 MP/M/C
54.0 15.0 5 2 MP/M/T
18.1 17.9 5 5 MP/M/C
18.3 37.7 6 4 MP/M/T
Case 7
Case 8
Case 9
39 Male 0 — 13
32 Female 0 3 11
52 Female 0 3 8.9
21.4 18.3 4 3 MP/M/C
18.3 16.0 8 5 MP/M/T
34.2 11.0 4 3 MP/M/C
IA, immunoadsorption; PRA, panel-reactive antibody; CDC, complement-dependent cytotoxic test; AHR, Acute hemoral rejection; MP, methylprednisolone; M, mycophenolate mofetil; T, tacrolimus; C, cyclosporine.
biopsies. AHR was diagnosed according to the addition to the Banff ’97 Classification of Renal Allograft Rejection.9 Nine renal allograft recipients fulfilled the following criteria: severe allograft dysfunction, granulocytes in PTC, and C4d deposition in PTC. These patients were enrolled in this study. Patient demographics and clinical data are listed in Table 1. Initial immunosuppressive protocol included methylprednisolone pulse therapy for 3 days posttransplant, then CsA (in four patients, dose adjusted to trough levels) or TAC (in five patients, dose adjusted to trough levels), MMF (initial dose, 1.5 g/d orally with temporary reductions in four patients because of leukopenia or diarrhea), and prednisone.
Immunoadsorption Therapy
Pathological and Immunopathological Studies
RESULTS Clinical Features
An urgent bed-side biopsy was performed immediately after an abnormal elevation of serum creatinine and/or reduction in urine output. Biopsies obtained for immunofluorescent processing were frozen, embedded, and sectioned using standard techniques. A repeat allograft kidney biopsy was performed in nine patients to evaluate the effects of therapy on rejection lesions. C4d was detected by an indirect immunofluorescence method in renal tissues obtained from allograft biopsies.10 The intensity of endothelial C4d staining was staged as weak (⫹), moderate (⫹⫹), or strong (⫹⫹⫹). Some biopsies with weak C4d staining showed an inhomogeneous distribution of C4d: only a few C4d-positive PTC. In biopsies with moderate or strong C4d staining, all capillaries stained positive. Processing of tissues for histology and immunohistochemistry11 yielded the number of infiltrating CD4⫹, CD8⫹, CD68⫹ cells, as well as the expression of proliferating cell nuclear antigen (PCNA), interleukin IL-2 receptor (IL-2R), and HLA-DR antigens.
Rescue Therapy The first episodes of rejection were treated with intravenous steroid pulse therapy (methylprednisolone and 500 mg/d for 3 consecutive days). After the diagnosis of C4d-positive acute rejection, rescue therapy included IA and TAC (0.15 mg · kg⫺1 · d⫺1 orally, dose adjusted to trough levels), MMF (1.5 to 2.0 g/d orally). CsA was discontinued 12 hours before starting TAC, after informed consent was obtained. Prednisone at a dosage of 10 to 15 mg/d was continued in all patients. Continuous blood purification was performed immediately after abnormal elevation of serum creatinine and/or reduction in urine output.12
As the main antibody-removing therapy, IA therapy was performed with a Citem 10 Immunoadsorption System (Fresenius Absorber Technology, Bad Homburg, Germany) and a staphylococcal protein A column (Fresenius).13 Plasma was separated with a traditional separator, passed through the column for adsorption, and then reinfused. During each session, two to three plasma volumes were processed. Initially, recipients received daily treatment sessions (2 to 7 days) and two to three sessions per week afterward. In all patients, pre- and post-IA PRA reactivity was assessed by a flow cytometry test.
The patient demographics are listed in Table 1 and their clinical courses in Table 2. PRA Levels and Serum Immunoglobulin Changes
Initial PRA-I and PRA-II levels were 28.8% ⫾ 16.2% and 15.3% ⫾ 8.9%. IA therapy significantly decreased PRA-I and PRA-II levels to 5.9% ⫾ 2.9% and 2.2% ⫾ 0.6%. Total serum immunoglobulin levels were markedly decreased (Fig 1C, D, E), in particular serum IgG level before IA (614 ⫾ 170 mg/dL) versus after an IA session (21 ⫾ 79.1 mg/dL). Pathological Findings
In nine patients, an urgent bedside biopsy was performed 4.5 ⫾ 0.75 days after renal transplantation. The earlier the biopsy was performed, the faster effective therapy could be initiated. So early diagnostic biopsy was advocated. Histopathologic results are shown in Table 3. The common histopathologic features of the nine patients included: the presence of granulocytes in PTC, granulocytes in glomeruli, and in three patients capillary fibrin thrombi was observed (case 3). According to Banff criteria, we observed that the entire acute lesion was partially reversed after treatment. Immunopathological Evaluation
All patients showed positive staining for C4d in PTC and granulocyte accumulation in PTC (Table 4). The number of
RESCUE THERAPY FOR ACUTE HUMORAL REJECTION
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Table 2. Clinical Course in Patients With AHR Parameter
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Case 7
Case 8
Case 9
DGF Graft function at diagnosis Rejection type Number of IA sessions Reversal of rejection SCr after IA Duration of treatment when Scr2 (d) SCr (mg/dL) on latest Time (mo) on latest
Yes CBP Mixed 6 Yes 1.2 16 1.1 57
Yes CBP Mixed 7 Yes 1.4 10 1.2 32
Yes CBP AVR 5 Yes 1.8 18 1.5 30
Yes CBP AVR 6 Yes 3.8 13 0.9 28
Yes CBP Mixed 4 Yes 1.3 15 1.5 25
Yes CBP AVR 6 Yes 4.7 12 1.0 24
Yes CBP Mixed 5 Yes 1.9 17 1.3 24
Yes CBP AVR 8 Yes 2.2 21 1.1 23
Yes CBP AVR 7 Yes 1.4 14 1.2 22
DGF, delayed graft function; CBP, continuous blood purification; IA, immunoadsorption; AVR, acute vascular rejection.
CD4⫹, PCNA, IL-2R, and HLA-DR antigen-positive cells was increased in four patients with mixed acute rejection above that in five patients with acute vascular rejection: 402 ⫾ 87 versus 87 ⫾ 23 /mm2; 201 ⫾ 47 versus 34 ⫾ 12 /mm2; 87 ⫾ 12 versus 21 ⫾ 8 /mm2; and 84% ⫾ 23% versus 20% ⫾ 4%, respectively. Repeat allograft biopsies demonstrated that after therapy, the deposition of C4d disappeared or reduced and the number of CD4⫹, PCNA, IL-2R, and HLA-DR antigen-positive cells decreased in nine patients.
DISCUSSION
Herein we have reported that nine renal allograft recipients with C4d-positive AHR undergoing IA in combination with TAC and MMF rescue therapy showed successful reversal of rejection at a mean follow-up of 29 months. The satisfactory graft function and graft survival suggested efficacy of our combination treatment. It is sometimes difficult to make a clinical diagnosis of humoral involvement. The present diagnostic criteria for AHR are based on detection of donor-specific anti-HLA antibody. The stable complement-split product C4d has been suggested to be a useful marker reflecting antibodymediated graft injury. Collins et al demonstrated that endothelial C4d deposition in PTC was a characteristic histopathologic change of AHR.14 On such a basis, we used C4d staining in combination with granulocyte accumulation in PTC for the diagnosis of AHR. Our approach of prospective C4d staining in all patients allowed an early diagnosis of AHR and thus enabled a timely start of antihumoral therapy. These C4d-positive biopsies must be thoroughly examined to exclude “focal, smouldering rejection,” in particular in the setting of tubular HLA-DR expression. Such cases require therapy. The remaining patients must be closely followed. A subsequent rise in serum creatinine may indicate rejection, often due to transplant endartaritis. Our series of graft biopsies revealed that humoral factors seemed to play an important role in acute rejection epi-
Renal Function
After IA for 6.3 ⫾ 1.03 sessions, graft function dramatically improved in all nine patients (Table 2); the mean duration of treatment when serum creatinine decreased was 15.1 ⫾ 2.9 days. Graft Survival
After a mean duration of 29.4 ⫾ 5.4 months follow-up, patient and allograft survival are 100%. Renal function remained stable with a mean serum creatinine of 1.1 ⫾ 0.3 mg/dL (Table 2). Side Effects
As far as side effects are concerned, except the one patient (case 5) who experienced transient bacterial pneumonia after 56 d, there was neither serious infection nor other complications.
Table 3. Histopathological Evaluation at the Time of Rejection and Post-IA Therapy Glomerulitis
Case Case Case Case Case Case Case Case Case
1 2 3 4 5 6 7 8 9
Interstitial Infiltrates
Tubulitis
Vasculitis
Banff Grade
Before
After
Before
After
Before
After
Before
After
Before
After
3 3 2 3 3 2 3 2 3
1 1 1 1 2 1 1 1 1
2 2 1 1 2 1 2 1 1
0 1 0 0 0 0 1 0 0
1 2 2 2 1 2 2 1 1
0 1 0 0 0 1 1 0 0
1 1 3 2 1 3 2 2 3
0 1 0 0 0 1 1 0 0
II II III II II III III II III
BC Normal BC Normal Normal Normal Normal Normal BC
Before, before IA; After, in rebiopsy after reversion of AHR; BC, borderline change.
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JI, LIU, CHEN ET AL Table 4. Immunopathological Evaluation at the Time of Rejection and Post-IA Therapy
Parameter
C4d in PTC Before After CD4⫹ cell (/mm2) Before After CD8⫹ cell (/mm2) Before After CD68⫹ cell (/mm2) Before After PCNA (/mm2) Before After IL-2R (/mm2) Before After HLA-DR (%) Before After
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Case 7
Case 8
Case 9
⫹⫹⫹ ⫺
⫹⫹⫹ ⫹
⫹⫹ ⫺
⫹⫹⫹ ⫹
⫹⫹⫹ ⫺
⫹⫹⫹ ⫹
⫹⫹ ⫺
⫹⫹⫹ ⫹
⫹⫹⫹ ⫺
479 89
399 104
110 98
154 54
369 43
168 78
563 77
107 86
224 32
254 111
293 98
307 210
317 102
477 111
218 132
187 198
257 166
283 102
372 93
380 108
511 104
595 87
621 110
428 68
139 17
401 98
382 102
168 57
201 58
32 10
40 21
50 8
31 10
301 12
47 14
50 19
99 12
187 0
43 10
31 8
53 12
23 10
97 20
29 21
46 8
97 8
92 10
42 12
23 10
84 4
43 2
99 4
28 8
79 4
PTC, peritubular capillaries.
sodes. Transplantation of a renal allograft in the presence of high-titer antibodies almost invariably resulted in a high graft failure rate and poor long-term survival. In nine recipients, adequate IA alleviated exposure of graft to antibodies and was effective to remove immunoglobins. However, transplant survival was markedly decreased amony C4d-positive patients. High PRA concentrations remain a high risk according to previous reports. Therefore, the combination of IA and other antirejection therapy seems vital for reversion of AHR. It may be speculated that early removal of pathogenetic alloantibodies prevents severe, probably irreversible, graft injury. Support for the efficacy of IA also comes from histopathologic evaluation of follow-up biopsies. In patients who responded to IA, we observed complete disappearance of granulocytes in PTC. Other histological signs of AHR were reduced or disappeared upon therapy as well. Repeat allograft biopsies demonstrated that after effective immunosuppressive therapy, the number of CD4⫹, PCNA, IL-2R, and HLA-DR antigen-positive cells was decreased in nine patients. Other than histopathologic signs of AHR, deposition of C4d along peritubular or glomerular endothelium was either not or only slightly altered by IA, which might be explained by stable long-term covalent binding of C4d to target structures. Theruvath et al demonstrated that rescue therapy with ATC and MMF was associated with a sustained decrease in antidonor antibodies, which was evident as soon as 2 months after starting therapy and continued progressively over a 12-month period.15 TAC-MMF treatment may limit both T-cell and B-cell responses. Previous studies have reported that MMF inhibits in vitro antibody production
and in vivo humoral responses and, when used in combination with TAC, limits B-cell responses in renal allograft recipients with acute humoral rejection.16 Interestingly, CsA appears to interfere with the metabolism of MMF, which may decrease the biological effects of this drug on alloantibody production.17 So, in such a combination protocol, effective control of antidonor antibody production may be better achieved with TAC plus MMF. In addition, it should be emphasized that prevention of chronic rejection remains a priority in organ transplantation. Recently, two randomized clinical trials in renal transplantation showed acute rejection rates lowered to approximately 8% to 15%.18 Immunosuppressive regimens that will specifically control both T-cell and B-cell responses may improve long-term graft survival. In conclusion, our data suggested that a therapeutic approach combining IA and TAC-MMF rescue showed high efficacy to reverse C4d-positive AHR, inhibit both the humoral and cellular arms of the rejection-cascade, and acheive increased graft survival for this group of high-risk AHR patients.
REFERENCES 1. Colvin RB: The renal allograft biopsy. Kidney Int 50:1069, 1996 2. McKenna RM, Takemoto SK, Terasaki PI: Anti-HLA antibodies after solid organ transplantation. Transplantation 69:319, 2000 3. Nickeleit V, Zeiler M, Gudat F, et al: Detection of the complement degradation product C4d in renal allografts: diagnostic and therapeutic implications. J Am Soc Nephrol 13:242, 2002
RESCUE THERAPY FOR ACUTE HUMORAL REJECTION 4. Watschinger B, Pascual M: Capillary C4d deposition as a marker of humoral immunity in renal allograft rejection. J Am Soc Nephrol 13:2420, 2002 5. Herzenberg AM, Gill JS, Djurdjew O, et al: C4d deposition in acute rejection: an independent long-term prognostic factor. J Am Soc Nephrol 13:234, 2002 6. Hickstein H, Korten G, Bast R, et al: Immunoadsorption of sensitized kidney transplant candidates immediately prior to surgery. Clin Transplant 16:97, 2002 7. Kliem V, Radermacher J, Hiss M, et al: Conversion to tacrolimus for acute corticosteroid- and antibody-resistant rejection following kidney transplantation. Transplant Proc 31:37S, 1999 8. Racusen LC, Solez K, Colvin RB, et al: The BANFF 97 working classification of renal allograft pathology. Kidney Int 55:713, 1999 9. Racusen LC, Colvin RB, Solez K, et al: Antibody-mediated rejection criteria—an addition to the Banff’97 Classification of Renal Allograft Rejection. Am J Transplant 3:708, 2003 10. Liu Z-H, Chen S-F, Chen Z-H, et al: Peritubular capillary C4d deposition in acute renal allograft rejection. Nephrol Dial Transplant 12:415, 2003 11. Yang J-W, Zhou H, Liu Z-H, et al: Relationship between various immunological markers expressed in renal allograft tissue and acute cellular rejection. Nephrol Dial Transplant 13:232, 1997 12. Tao J, Jl D-X, Gong D-H, et al: Effects of continuous high volume hemofiltration in renal transplant recipients with severe
3463 pulmonary infection and acute respiratory distress syndrome. Nephrol Dial Transplant 12:240, 2003 13. Jl D-X, Gong D-H, Ren B, et al: Clinical application and effects of the protein A based immunoadsorption. Nephrol Dial Transplant 13:408, 2004 14. Collins AB, Schneeberger EE, Pascual MA, et al: Complement activation in acute humoral renal allograft rejection: diagnostic significance of C4d deposits in peritubular capillaries. J Am Soc Nephrol 10:2208, 1999 15. Theruvath TP, Saidman SL, Mauiyyedi S, et al: Control of antidonor antibody production with tacrolimus and mycophenolate mofetil in renal allograft recipients with chronic rejection. Transplantation 72:77, 2001 16. Gelder TV: Mycophenolate mofetil: How to furthure improve using an already successful drug? Am J Transplant 5:199, 2005 17. Miller J, Mendez R, Pirsch JD, et al: Safety and efficacy of tacrolimus in combination with mycophenolate mofetil (MMF) in cadaveric renal transplant recipients. TAC/MMF Dose-Ranging Kidney Transplant Study Group. Transplantation 69:875, 2000 18. Johnson C, Ahsan N, Gonwa T, et al: Randomized trial of tacrolimus (Prograf) in combination with azathioprine or mycophenolate mofetil vs. cyclosporine (Neoral) with mycophenolate mofetil after cadaveric kidney transplantation. Transplantation 69:834, 2000
D
ESPITE THE MORE sophisticated use of immunosuppressive drugs and the improved understanding of cellular and humoral host responses to transplanted organs during recent years, early allograft rejection remains a barrier to successful kidney transplantation.1 There is increasing evidence that besides cellular immunity, antibody-mediated immune mechanisms also may cause acute rejection. These events are associated with a high rate of graft loss.2 Currently, the diagnosis of acute humoral rejection (AHR) is predominantly based on the detection of donor-specific antibodies by posttransplant crossmatches.3 The presence of donor-specific antibodies has been shown to be associated with typical histopathologic findings, such as accumulation of neutrophils in peritubular capillaries (PTC). Recent studies have suggested that endothelial deposition of the stable complement-split product C4d may indicate donor-specific alloantibody-mediated AHR.4 Furthermore, C4d deposition has been correlated with poor graft survival.5 The traditional antirejection treatment, such as methylprednisolone pulse therapy or intravenous injection of anti-CD3, has usually been ineffective to treat AHR. Plasma exchange and immunoadsorption are newly developed approaches to reduce the concentration of anti-HLA antibody in renal transplant recipients.6 Moreover, tacrolimus (TAC), a widely used calcineurin inhibitor (CNI), has been reported to reduce rejection or mitigate CNI nephro-
toxicity compared with cyclosporine (CsA)-based immunosuppressive therapy.7 The aim of this study was to investigate the efficacy of immunoadsorption (IA) in combination with TAC and mycophenolate mofetil (MMF) rescue therapy for C4d-positive acute humoral rejection of renal transplants. MATERIALS AND METHODS Patients During the period from December 2001 to June 2003, 137 patients received renal allografts from non– heart-beating donors. Prior to transplant, all blood from recipients must have shown a ⬍10% level of complement-dependent cytotoxicity. In addition, we used flow cytometry to detect panel reactive antibody (PRA), defining patients with a level ⬎ 10% as sensitized. Twenty-one recipients had one or more biopsy-proven acute rejection episodes. Histological findings were classified according to the Banff 97 classification for acute rejection, as borderline, or grades I to III rejection.8 C4d was detected by an indirect immunofluorescence method in renal tissues obtained from allograft From the Research Institute of Nephrology (S.-M.J., J.-S.C., G.-Z.S., D.-X.J.), Jinling Hospital, Nanjing University School of Medicine (Z.-H.L., L.-S.L.), Nanjing, China. Address reprint requests to Dr Shu-Ming Ji, The Research Institute of Nephrology, Jinling Hospital, 305 East Zhong Shan Road, Nanjing 210002, China. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.10.151
Transplantation Proceedings, 38, 3459 –3463 (2006)
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JI, LIU, CHEN ET AL Table 1. Patient Demographics Parameter
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Age (y) Gender Previous renal allografts Previous pregnancies Cold ischemia time (h) Pre-IA PRA (%) HLA-I HLA-II CDC (%) AHR time (day post-RT) Initial therapy
36 Female 0 2 9
20 Male 0 — 8
48 Male 0 — 8.5
54 Male 1 — 12
37 Male 0 — 11
51 Male 0 — 10
43.0 2.9 6 4 MP/M/T
19.0 12.9 7 5 MP/M/T
32.8 5.7 6 4 MP/M/C
54.0 15.0 5 2 MP/M/T
18.1 17.9 5 5 MP/M/C
18.3 37.7 6 4 MP/M/T
Case 7
Case 8
Case 9
39 Male 0 — 13
32 Female 0 3 11
52 Female 0 3 8.9
21.4 18.3 4 3 MP/M/C
18.3 16.0 8 5 MP/M/T
34.2 11.0 4 3 MP/M/C
IA, immunoadsorption; PRA, panel-reactive antibody; CDC, complement-dependent cytotoxic test; AHR, Acute hemoral rejection; MP, methylprednisolone; M, mycophenolate mofetil; T, tacrolimus; C, cyclosporine.
biopsies. AHR was diagnosed according to the addition to the Banff ’97 Classification of Renal Allograft Rejection.9 Nine renal allograft recipients fulfilled the following criteria: severe allograft dysfunction, granulocytes in PTC, and C4d deposition in PTC. These patients were enrolled in this study. Patient demographics and clinical data are listed in Table 1. Initial immunosuppressive protocol included methylprednisolone pulse therapy for 3 days posttransplant, then CsA (in four patients, dose adjusted to trough levels) or TAC (in five patients, dose adjusted to trough levels), MMF (initial dose, 1.5 g/d orally with temporary reductions in four patients because of leukopenia or diarrhea), and prednisone.
Immunoadsorption Therapy
Pathological and Immunopathological Studies
RESULTS Clinical Features
An urgent bed-side biopsy was performed immediately after an abnormal elevation of serum creatinine and/or reduction in urine output. Biopsies obtained for immunofluorescent processing were frozen, embedded, and sectioned using standard techniques. A repeat allograft kidney biopsy was performed in nine patients to evaluate the effects of therapy on rejection lesions. C4d was detected by an indirect immunofluorescence method in renal tissues obtained from allograft biopsies.10 The intensity of endothelial C4d staining was staged as weak (⫹), moderate (⫹⫹), or strong (⫹⫹⫹). Some biopsies with weak C4d staining showed an inhomogeneous distribution of C4d: only a few C4d-positive PTC. In biopsies with moderate or strong C4d staining, all capillaries stained positive. Processing of tissues for histology and immunohistochemistry11 yielded the number of infiltrating CD4⫹, CD8⫹, CD68⫹ cells, as well as the expression of proliferating cell nuclear antigen (PCNA), interleukin IL-2 receptor (IL-2R), and HLA-DR antigens.
Rescue Therapy The first episodes of rejection were treated with intravenous steroid pulse therapy (methylprednisolone and 500 mg/d for 3 consecutive days). After the diagnosis of C4d-positive acute rejection, rescue therapy included IA and TAC (0.15 mg · kg⫺1 · d⫺1 orally, dose adjusted to trough levels), MMF (1.5 to 2.0 g/d orally). CsA was discontinued 12 hours before starting TAC, after informed consent was obtained. Prednisone at a dosage of 10 to 15 mg/d was continued in all patients. Continuous blood purification was performed immediately after abnormal elevation of serum creatinine and/or reduction in urine output.12
As the main antibody-removing therapy, IA therapy was performed with a Citem 10 Immunoadsorption System (Fresenius Absorber Technology, Bad Homburg, Germany) and a staphylococcal protein A column (Fresenius).13 Plasma was separated with a traditional separator, passed through the column for adsorption, and then reinfused. During each session, two to three plasma volumes were processed. Initially, recipients received daily treatment sessions (2 to 7 days) and two to three sessions per week afterward. In all patients, pre- and post-IA PRA reactivity was assessed by a flow cytometry test.
The patient demographics are listed in Table 1 and their clinical courses in Table 2. PRA Levels and Serum Immunoglobulin Changes
Initial PRA-I and PRA-II levels were 28.8% ⫾ 16.2% and 15.3% ⫾ 8.9%. IA therapy significantly decreased PRA-I and PRA-II levels to 5.9% ⫾ 2.9% and 2.2% ⫾ 0.6%. Total serum immunoglobulin levels were markedly decreased (Fig 1C, D, E), in particular serum IgG level before IA (614 ⫾ 170 mg/dL) versus after an IA session (21 ⫾ 79.1 mg/dL). Pathological Findings
In nine patients, an urgent bedside biopsy was performed 4.5 ⫾ 0.75 days after renal transplantation. The earlier the biopsy was performed, the faster effective therapy could be initiated. So early diagnostic biopsy was advocated. Histopathologic results are shown in Table 3. The common histopathologic features of the nine patients included: the presence of granulocytes in PTC, granulocytes in glomeruli, and in three patients capillary fibrin thrombi was observed (case 3). According to Banff criteria, we observed that the entire acute lesion was partially reversed after treatment. Immunopathological Evaluation
All patients showed positive staining for C4d in PTC and granulocyte accumulation in PTC (Table 4). The number of
RESCUE THERAPY FOR ACUTE HUMORAL REJECTION
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Table 2. Clinical Course in Patients With AHR Parameter
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Case 7
Case 8
Case 9
DGF Graft function at diagnosis Rejection type Number of IA sessions Reversal of rejection SCr after IA Duration of treatment when Scr2 (d) SCr (mg/dL) on latest Time (mo) on latest
Yes CBP Mixed 6 Yes 1.2 16 1.1 57
Yes CBP Mixed 7 Yes 1.4 10 1.2 32
Yes CBP AVR 5 Yes 1.8 18 1.5 30
Yes CBP AVR 6 Yes 3.8 13 0.9 28
Yes CBP Mixed 4 Yes 1.3 15 1.5 25
Yes CBP AVR 6 Yes 4.7 12 1.0 24
Yes CBP Mixed 5 Yes 1.9 17 1.3 24
Yes CBP AVR 8 Yes 2.2 21 1.1 23
Yes CBP AVR 7 Yes 1.4 14 1.2 22
DGF, delayed graft function; CBP, continuous blood purification; IA, immunoadsorption; AVR, acute vascular rejection.
CD4⫹, PCNA, IL-2R, and HLA-DR antigen-positive cells was increased in four patients with mixed acute rejection above that in five patients with acute vascular rejection: 402 ⫾ 87 versus 87 ⫾ 23 /mm2; 201 ⫾ 47 versus 34 ⫾ 12 /mm2; 87 ⫾ 12 versus 21 ⫾ 8 /mm2; and 84% ⫾ 23% versus 20% ⫾ 4%, respectively. Repeat allograft biopsies demonstrated that after therapy, the deposition of C4d disappeared or reduced and the number of CD4⫹, PCNA, IL-2R, and HLA-DR antigen-positive cells decreased in nine patients.
DISCUSSION
Herein we have reported that nine renal allograft recipients with C4d-positive AHR undergoing IA in combination with TAC and MMF rescue therapy showed successful reversal of rejection at a mean follow-up of 29 months. The satisfactory graft function and graft survival suggested efficacy of our combination treatment. It is sometimes difficult to make a clinical diagnosis of humoral involvement. The present diagnostic criteria for AHR are based on detection of donor-specific anti-HLA antibody. The stable complement-split product C4d has been suggested to be a useful marker reflecting antibodymediated graft injury. Collins et al demonstrated that endothelial C4d deposition in PTC was a characteristic histopathologic change of AHR.14 On such a basis, we used C4d staining in combination with granulocyte accumulation in PTC for the diagnosis of AHR. Our approach of prospective C4d staining in all patients allowed an early diagnosis of AHR and thus enabled a timely start of antihumoral therapy. These C4d-positive biopsies must be thoroughly examined to exclude “focal, smouldering rejection,” in particular in the setting of tubular HLA-DR expression. Such cases require therapy. The remaining patients must be closely followed. A subsequent rise in serum creatinine may indicate rejection, often due to transplant endartaritis. Our series of graft biopsies revealed that humoral factors seemed to play an important role in acute rejection epi-
Renal Function
After IA for 6.3 ⫾ 1.03 sessions, graft function dramatically improved in all nine patients (Table 2); the mean duration of treatment when serum creatinine decreased was 15.1 ⫾ 2.9 days. Graft Survival
After a mean duration of 29.4 ⫾ 5.4 months follow-up, patient and allograft survival are 100%. Renal function remained stable with a mean serum creatinine of 1.1 ⫾ 0.3 mg/dL (Table 2). Side Effects
As far as side effects are concerned, except the one patient (case 5) who experienced transient bacterial pneumonia after 56 d, there was neither serious infection nor other complications.
Table 3. Histopathological Evaluation at the Time of Rejection and Post-IA Therapy Glomerulitis
Case Case Case Case Case Case Case Case Case
1 2 3 4 5 6 7 8 9
Interstitial Infiltrates
Tubulitis
Vasculitis
Banff Grade
Before
After
Before
After
Before
After
Before
After
Before
After
3 3 2 3 3 2 3 2 3
1 1 1 1 2 1 1 1 1
2 2 1 1 2 1 2 1 1
0 1 0 0 0 0 1 0 0
1 2 2 2 1 2 2 1 1
0 1 0 0 0 1 1 0 0
1 1 3 2 1 3 2 2 3
0 1 0 0 0 1 1 0 0
II II III II II III III II III
BC Normal BC Normal Normal Normal Normal Normal BC
Before, before IA; After, in rebiopsy after reversion of AHR; BC, borderline change.
3462
JI, LIU, CHEN ET AL Table 4. Immunopathological Evaluation at the Time of Rejection and Post-IA Therapy
Parameter
C4d in PTC Before After CD4⫹ cell (/mm2) Before After CD8⫹ cell (/mm2) Before After CD68⫹ cell (/mm2) Before After PCNA (/mm2) Before After IL-2R (/mm2) Before After HLA-DR (%) Before After
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Case 7
Case 8
Case 9
⫹⫹⫹ ⫺
⫹⫹⫹ ⫹
⫹⫹ ⫺
⫹⫹⫹ ⫹
⫹⫹⫹ ⫺
⫹⫹⫹ ⫹
⫹⫹ ⫺
⫹⫹⫹ ⫹
⫹⫹⫹ ⫺
479 89
399 104
110 98
154 54
369 43
168 78
563 77
107 86
224 32
254 111
293 98
307 210
317 102
477 111
218 132
187 198
257 166
283 102
372 93
380 108
511 104
595 87
621 110
428 68
139 17
401 98
382 102
168 57
201 58
32 10
40 21
50 8
31 10
301 12
47 14
50 19
99 12
187 0
43 10
31 8
53 12
23 10
97 20
29 21
46 8
97 8
92 10
42 12
23 10
84 4
43 2
99 4
28 8
79 4
PTC, peritubular capillaries.
sodes. Transplantation of a renal allograft in the presence of high-titer antibodies almost invariably resulted in a high graft failure rate and poor long-term survival. In nine recipients, adequate IA alleviated exposure of graft to antibodies and was effective to remove immunoglobins. However, transplant survival was markedly decreased amony C4d-positive patients. High PRA concentrations remain a high risk according to previous reports. Therefore, the combination of IA and other antirejection therapy seems vital for reversion of AHR. It may be speculated that early removal of pathogenetic alloantibodies prevents severe, probably irreversible, graft injury. Support for the efficacy of IA also comes from histopathologic evaluation of follow-up biopsies. In patients who responded to IA, we observed complete disappearance of granulocytes in PTC. Other histological signs of AHR were reduced or disappeared upon therapy as well. Repeat allograft biopsies demonstrated that after effective immunosuppressive therapy, the number of CD4⫹, PCNA, IL-2R, and HLA-DR antigen-positive cells was decreased in nine patients. Other than histopathologic signs of AHR, deposition of C4d along peritubular or glomerular endothelium was either not or only slightly altered by IA, which might be explained by stable long-term covalent binding of C4d to target structures. Theruvath et al demonstrated that rescue therapy with ATC and MMF was associated with a sustained decrease in antidonor antibodies, which was evident as soon as 2 months after starting therapy and continued progressively over a 12-month period.15 TAC-MMF treatment may limit both T-cell and B-cell responses. Previous studies have reported that MMF inhibits in vitro antibody production
and in vivo humoral responses and, when used in combination with TAC, limits B-cell responses in renal allograft recipients with acute humoral rejection.16 Interestingly, CsA appears to interfere with the metabolism of MMF, which may decrease the biological effects of this drug on alloantibody production.17 So, in such a combination protocol, effective control of antidonor antibody production may be better achieved with TAC plus MMF. In addition, it should be emphasized that prevention of chronic rejection remains a priority in organ transplantation. Recently, two randomized clinical trials in renal transplantation showed acute rejection rates lowered to approximately 8% to 15%.18 Immunosuppressive regimens that will specifically control both T-cell and B-cell responses may improve long-term graft survival. In conclusion, our data suggested that a therapeutic approach combining IA and TAC-MMF rescue showed high efficacy to reverse C4d-positive AHR, inhibit both the humoral and cellular arms of the rejection-cascade, and acheive increased graft survival for this group of high-risk AHR patients.
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