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Keywords:

  • HLA antibodies;
  • humoral allograft rejection;
  • rejection risk;
  • renal allograft pathology

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Conflict of Interest Statement
  10. References

This study describes clinical relevance of subclinical antibody-mediated rejection (SAMR) in a cohort of 54 DSA-positive kidney transplant recipients receiving a deceased donor. In 3 months screening biopsies, 31.1% of patients met the criteria of SAMR. A total of 48.9% had an incomplete form of SAMR (g+/ptc+/C4d-negative) whereas 20% had no humoral lesions. Patients with SAMR at 3 months had at 1 year: a higher C4d score, ptc score, and arteriosclerosis score, higher rate of IFTA (100% vs. 33.3%, p < 0.01) and a higher rate of transplant glomerulopathy (43% vs. 0%, p = 0.02) compared to patients without 3-month SAMR. Patients with SAMR at 3 months exhibited at 1 year a higher class II MFImax-DSA and a lower mGFR compared to patients without SAMR (39.2 ± 13.9 vs. 61.9 ± 19.2 mL/min/1.73 m2 respectively, p < 0.01). The group of patients with C4d-negative SAMR at 3 months developed more ptc and IFTA lesions, and lower GFR at 1 year in comparison to biopsies without humoral lesions. SAMR is a frequent entity in KTR with preexisting DSAs and promotes subsequent GFR impairment and development of chronic AMR. C4d-negative SAMR patients displayed an intermediate course between the no-SAMR group and the C4d+ SAMR group. Screening biopsies may be useful to recognize patients more likely to develop SAMR.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Conflict of Interest Statement
  10. References

Nowadays, more than 30% of all patients awaiting a deceased donor kidney transplant are sensitized to human leukocyte antigens (HLAs). Numerous efforts have been devoted to improving graft survival in such patients following transplantation. Different protocols have recently emerged using B lymphocyte-depleting molecules (anti-CD20), intravenous immunoglobulin (IVIg) and plasmapheresis (1–8). In addition, sensitive DSA-screening techniques have emerged leading to better stratification of immunological risk (1–3).

Although these advances have improved short-term graft survival in individuals with anti-HLA or anti-ABO antibodies, they fail to prevent antibody-mediated rejection (AMR), particularly frequent in patients with preformed DSA. AMR is now well characterized, presenting typically with acute loss of graft function associated with specific morphological diagnostic criteria and specific staining for C4d (a product of the cleavage of the C4 component of complement) (9). Importantly, even in the absence of frank AMR, graft survival of positive DSA patients remains lower than in those without DSA (2,9). It may be that the new profoundly depleting therapies suppress clinical expression of AMR but only incompletely mute the histological lesions of humoral rejection. This argument has been recently reinforced by Haas et al. and Gloor et al. who described a new entity named subclinical AMR (SAMR) found in 12% of positive DSA kidney transplant recipients undergoing desensitization, and likely contributing to chronic transplant nephropathy (10,11).

In this study, we assessed the histological lesions at 3 months and 1 year in patients receiving deceased donor grafts, comparing those with preformed DSA to those without. Second, we evaluated the presence and extent of SAMR, and correlated the lesions found with functional and immunological parameters, particularly DSA, using recently available techniques, to further define the natural history of SAMR and its impact on graft outcome.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Conflict of Interest Statement
  10. References

Study design

From January 2002 to March 2007, 806 kidney transplantations were performed at our institution, of which 54 (6.6%) were in patients with preformed DSAs assessed by ELISA high definition (HD) receiving a single ABO-compatible deceased donor kidney transplant. This population of DSA-positive patients (group A) was compared to a control group of patients without preformed DSA (n = 83, group B), all having screening biopsies and measured glomerular filtration rate (GFR) at 3 months and 1 year.

Clinical protocols

All patients received induction treatment. Group A received a 10-day course of ATG (rabbit ATG, Thymoglobulin, Genzyme, Lyon, France) initiated before reperfusion at a dose of 75 mg/d. Group B received 20 mg intravenous Basiliximab (Simulect, Novartis Pharma AG, Basel), administered on days 0 and 4.

All patients received calcineurin inhibitors: cyclosporine was used until 2003, after which tacrolimus was used in all patients. All groups A and B patients received mycophenolate mofetil (CellCept, Roche Pharmaceuticals, Basel), 2 g preoperatively, followed by 1 g b.i.d., and prednisone, 500 mg preoperatively, 125 mg on day 1, then 20 mg/d for 15 days, progressively tapered to 10 mg/d at day 30.

In addition, all group A patients received four courses of IVIg (Endobuline, Baxter, Maurepas, France) at a dose of 2 g/kg administered over 96 h, the first course started before reperfusion, subsequent courses being given on days 21, 42 and 63. From 2006 onward, the final 18 patients in group A received additional prophylactic anti-CD20 therapy (Rituximab Roche pharmaceuticals), at a dose of 375 mg/m2 at day 4 and repeated according to absolute and relative numbers of CD19+ cells, together with plasmapheresis performed immediately posttransplant then three times per week for 3 weeks. No pretransplant desensitization treatment was used.

All CMV− recipients of a kidney from a CMV+ donor and all CMV+ recipients were given CMV prophylaxis.

Treatment of acute rejection episodes

Acute cellular rejection episodes were treated with high-dose steroids. Patients with acute AMR underwent plasma exchange and received Rituximab (325 mg/m2) and high-dose steroids. Plasma exchanges were continued until DSA were no longer detectable.

Antibody detection, specificity analysis and cross-match techniques

All immunological analyses were performed in one laboratory (Hôpital Saint-Louis, Paris, France). HLA typing of transplant recipients was performed by molecular biology (Innolipa HLA typing kit, Innogenetics, Belgium). For all kidney transplant donors, HLA A, B, DR and DQ tissue-typing was performed using the microlymphocytotoxicity technique with One-Lambda INC tissue-typing trays. Blanks and difficult typing were controlled by molecular biology.

For all patients, crossmatches were performed by two techniques: direct complement-dependent cytotoxicity (CDC) on T and B donor lymphocytes and antiglobulin-enhanced CDC (AHG-CDC) on donor and spleen nodal lymphocytes, the CDC and AHG-CDC crossmatches being performed on peak and current sera. All patients were transplanted with a current negative IgG T-cell CDC crossmatch against their donors. No flow crossmatch was performed.

Pretransplant sera were screened in all patients by HLA-specific ELISA assays for DSA (LAT-M, One-Lambda). In patients with positive DSA screening, specificities for anti-HLA class I antibodies were assessed using an HD single-antigen, ELISA (LAT-1HD, One-Lambda). For anti-HLA class II antibodies we performed an ELISA (LAT 2–40, One-Lambda) test, identifying DR and DQ subtypes on a panel of purified HLA antigens. Antibodies were defined by a score of 4, 6 or 8 reactivities. Both ELISA tests were performed as recommended by the manufacturer.

In addition, to the ELISA, all group A peak, day-0 and posttransplant sera (3 months and 1 year after transplantation) were retrospectively tested for the presence of DSA by Luminex SA. Briefly, identification of class I DSAs by Luminex SA (Luminex LABScreen Single Antigen, One-Lambda Inc., CA) uses a set of 96 beads, each coated with different HLA class I glycoproteins. Identification of DSA class II antibodies by Luminex SA was performed identically using a set of 76 beads coated with HLA class II antigens. Presence of antibodies is detected using a goat anti-human IgG coupled with phycoerythrin. The fluorescence of each bead is detected by a reader (LABscan) and recorded as the mean intensity of fluorescence (MFI). Both tests were performed as recommended by the manufacturer. All beads showing a normalized MFI >300 were considered positive. For each patient, we recorded the MFI of all DSAs classes I and II detected, as well as the maximum MFI value (MFImax).

Histology and immunochemistry

All patients underwent screening graft biopsies at 3 months and 1 year posttransplant. A screening biopsy was defined as a biopsy performed in patients in a steady state, without any context of acute graft failure or recent immunological event.

A total of 266 screening biopsies were studied: 108 from group A patients (52 at 3 months, 47 at 1 year and 9 at 3 years) and 158 from group B patients (83 at 3 months and 75 at 1 year). Renal biopsies were fixed in alcoholic Bouin's fixative and stained by standard methods for routine microscopy. The biopsies were reviewed by two renal pathologists (DN and GH) and a nephrologist (AL), blinded to clinical information. Histological changes were graded according to the Banff 97 classification (12). In group A patients, additional immunohistochemical C4d staining was performed on paraffin sections using human polyclonal antibody (Biomedica). All group A patients with C4d-negative biopsies by peroxidase were also analyzed by a two-step indirect immunofluorescence (IF) method with a monoclonal antibody specific for C4d on frozen tissue (Quidel, Santa Clara, CA). This procedure confirmed C4d-negativity in group A patients with negative peroxidase C4d staining. The C4d score varied from 0 to 3 (negative, minimal, focal, diffuse), depending on the percentage of peritubular capillaries (PTC) having a linear staining pattern (13). A diagnosis of acute cellular or humoral rejection was based on published criteria in conjunction with allograft dysfunction (14). Humoral rejection definition was based on the Banff criteria classification (12). The IFTA score was the sum of ci+ct.

Subclinical AMR definition

Subclinical AMR was defined as the presence on a screening biopsy of glomerulitis (g 1–3), peritubular capillary leukocytic infiltration (ptc 1–3), and positive C4d (1–3) with either diffuse or focal C4d staining of peritubular capillaries, and the presence of positive class I or II DSA by Luminex SA (>300 MFI) in serum taken at the time of biopsy.

Glomerular filtration rate measurement

The glomerular filtration rate (GFR) was estimated by serum creatinine (SCr) value. In addition, all 137 patients underwent routine true GFR measurement at 3 months and 1 year posttransplant using 51Cr-EDTA clearance measurements. This method has been compared already to inulin clearance, the gold standard for GFR measurement and to MDRD calculation (15).

Data collection

For all patients, data was collected regarding: sex, recipient and donor age, cold ischemia time, donor/recipient CMV status, time to recovery of graft function, immunosuppressive drugs administered, SCr, GFR and level of proteinuria at 3 months and 1 year. Death with a functioning graft was considered as graft loss.

Statistical analysis

Results are expressed as mean ± SD for continuous variables, with the exception of MFIs where mean ± SEM is used. Comparisons were based on the χ2-test for categorical data and the Student's t-test for normally distributed continuous data. For parameters without Gaussian distribution, the Mann–Whitney U-test was used, and the Wilcoxon test was used to compare two paired groups.

Graft survival was assessed using Kaplan–Meier analysis, with survivals compared using the log-rank test. Probability values <0.05 were regarded as statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Conflict of Interest Statement
  10. References

Clinical and serological characteristics of our population

Among the 137 kidney transplantations in this study, 54 had DSA detected by ELISA HD (group A) whereas 83 did not have preformed DSA detected by ELISA (group B). In group A patients, the peak class I or II MFImax DSA value (Luminex) was 8557 ± 705. Twenty-two patients out of 54 (43%) had positive historical T- and/or B-cell crossmatch, whereas all group B patients had negative historical CDC crossmatch. Demographic data and baseline characteristics of patients are detailed in Table 1. Donor and recipient ages were similar in both groups. There was no difference in CMV donor/recipient status between groups A and B.

Table 1.  Patient characteristics and outcome
ParametersGroup A positive DSA (n = 54)Group B Negative DSA (n = 83)p-Value
  1. 1Values expressed in mean ± SEM.

Recipient characteristics
 Recipient age (years, mean ± SD)46 ± 1248 ± 14NS
 Donor age (years, mean ± SD)47 ± 1451 ± 14NS
 Retransplantation (%)71%9%<0.001
Immunology
 CDC positive crossmatch (n, %)22 (43%)0 (0%)<0.001
 Class I or II peak DSA pretransplant (ELISA HD)
   Score 4 DSA (n, %)14 (26%) 
   Score 6 DSA (n, %)18 (33%) 
   Score 8 DSA (n, %)22 (41%) 
 Class I or II peak DSA pretransplant MFImax18557 ± 705  
 Class I or II day-0 DSA MFImax15075 ± 599  
 HLA A + B mismatch (mean ± SD)2.1 ± 1.12.0 ± 1.1NS
 HLA DR mismatch (mean ± SD)1.2 ± 0.70.9 ± 0.70.1
Transplant characteristics
 Donor age (mean ± SD)47 ± 1451 ± 14NS
 Deceased donor (%)100%100%NS
Transplant outcome
 GFR at 3 months (mL/min/1.73 m2)52.7 ± 17.052.3 ± 19.0NS
 GFR at 1 year (mL/min/1.73 m2)47.3 ± 17.050.6 ± 16.0NS
 Proteinuria at 3 months (g/24h)0.24 ± 0.270.30 ± 0.48NS
 Proteinuria at 1 year (g/24h)0.35 ± 1.000.33 ± 0.60NS
 Acute T-cell-mediated rejection79NS
 Acute humoral rejection100<0.001
 Follow-up (months, mean ± SD)30.2 ± 16 54 ± 14<0.05 
 Graft loss (n, %)5 (9.2%)4 (4.8%)
 Patient death (n, %)2 (3.7%)1 (1.2%)

Screening biopsies results

Results of the 3-month and 1-year screening biopsies compared by group are summarized in Table 2. The biopsies from DSA-positive recipients exhibited a higher rate of glomerulitis (g) and peritubular capillaritis (ptc) both at 3 months and 1 year. They also showed greater chronic lesions of transplant glomerulopathy and interstitial fibrosis/tubular atrophy (IF/TA), both at 3 months and 1 year.

Table 2.  3-month and 1-year screening biopsies results according to the presence or absence of DSA
 3-Month screening biopsy1-Year screening biopsy
Group A positive DSA (n = 52)Group B negative DSA (n = 83)p-ValueGroup A positive DSA (n = 47)Group B negative DSA (n = 75)p-Value
  1. g = glomerulitis; ptc = peritubular capillaritis; cg = transplant glomerulopathy; t = tubulitis; i = interstitial inflammation; IF/TA = interstitial fibrosis/tubular atrophy; v = vasculitis; cv = chronic vascular lesions (arteriosclerosis).

Sclerotic glomeruli (%)7 ± 9 8 ± 14NS 8.3 ± 13.810 ± 12NS
g0.8 ± 0.80.1 ± 0.4<0.0011.0 ± 0.80.2 ± 0.6<0.001
ptc1.1 ± 0.90.1 ± 0.4<0.0011.4 ± 0.90.1 ± 0.4<0.001
% g or ptc >077.5%10.8%<0.0188.3%15.0%<0.01
cg0.13 ± 0.380.02 ± 0.150.020.33 ± 0.660.09 ± 0.410.01
% cg >013.5%2.4%0.0225.5%6.8%0.03
C4d staining (0–3)0.8 ± 0.2 1.0 ± 0.2
C4d staining
 Negative (%)68.9% 49.0% 
 Focal (%)20.0% 42.5% 
 Diffuse (%)11.1% 8.5% 
Glomerular TMA (%)3.8%0%0%0% 
t0.2 ± 0.50.4 ± 0.8NS0.2 ± 0.5 0.2 ± 0.56NS
i0.5 ± 0.70.5 ± 0.8NS0.6 ± 0.70.4 ± 0.70.05
v0.0 ± 0.00.05 ± 0.3 NS0.02 ± 0.1 0.01 ± 0.10NS
IF/TA0.9 ± 0.90.5 ± 0.7<0.011.5 ± 1.01.0 ± 1.10.07
cv0.7 ± 0.80.7 ± 0.9NS 1.0 ± 0.950.9 ± 0.8NS
Borderline changes (%)21.1%6.0%<0.0114.9%6.7%NS
Cellular rejection (%)7.7%8.4%NS8.5%4.0%NS

C4d staining was studied in group A screening biopsies. Diffuse positive C4d PTC staining was only observed in 11.1% and 8.5% of 3 months and 1 year biopsies respectively, whereas focal positive C4d was observed in 20.0% and 42.5% of 3 months and 1 year biopsies. Finally, despite a high rate of subclinical humoral lesions, negative C4d staining was observed in 68.9% and 49.0% of 3 months and 1 year biopsies, respectively. Three-year screening biospsies performed in nine DSA positive patients revealed persisting acute humoral lesions: glomerulitis, ptc and C4d score of 0.72 ± 0.22, 1.33 ± 0.25 and 1.00 ± 0.30, respectively. These lesions correlated with a dramatic increase of chronic lesion at 3 years (transplant glomerulopathy score, arteriosclerosis score and IFTA score of 0.65 ± 0.22, 1.83 ± 0.34 and 2.12 ± 0.27, respectively). The evolution of acute and chronic lesions in group A patients (DSA positive) is presented in Figure 1. As can be seen, the acute lesions persist, largely unabated, from 3 months to 3 years, whereas the chronic lesions increase steadily and significantly over this period.

image

Figure 1. Evolution of acute (A) and chronic lesions (B) in DSA positive patients (group A) according to time of screening biopsy (mean ± SEM, data including nine additional screening biopsies performed at 3 years).

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DSA-level evolution in group A patients

Class I or II MFImax values declined from 8557 ± 705 in peak serum to 5075 ± 599 in day-0 serum (p < 0.01), decreasing to 2320 ± 590 at 3 months (p < 0.01 compared to day-0) and was 2575 ± 627 at 1 year (p = NS compared to 3 months). The ΔMFImaxpeak to day-0 was −31 ± 6%, the ΔMFImaxday-0 to 3 months was −63 ± 6% and finally, the ΔMFImax3 months to 1 year was +3.5 ± 1%. The pre- and posttransplant course of classes I and II DSA MFImax in group A patients is presented in Figure 2.

image

Figure 2. Class I or II DSA MFImax DSA course in group A.

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Outcome

Graft and patient survival:  One-year graft survival was 96.3% and 98.7% in groups A and B, respectively (p = NS), Finally, Kaplan–Meier analysis revealed that 4 years graft survival was 86.2% and 96.2% respectively, significantly lower in group A as compared to group B patients (log-rank test, p = 0.01, Figure 3). In group A, graft losses were due to: chronic rejection (n = 2), artery thrombosis (n = 1), autoimmune nephropathy recurrence (n = 1) and bacterial sepsis (n = 1). Two patients died during follow-up: the above patient with sepsis and one patient due to cerebral bleeding after losing his graft. In group B, four graft losses were observed, with one patient dying 3 years after transplantation. The posttransplant events and outcome parameters are detailed in Table 1.

image

Figure 3. Kaplan–Meier analysis of graft survival in groups A and B.

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Graft function:  There was no significant difference in GFR between groups A and B, either at 3 months or at 1 year, nor was there any significant difference in proteinuria between the two groups at either time period (Table 1).

Evolution of histological, immunological and functional parameters

Fifty-two of 54 patients in group A had adequate screening biopsies at 3 months; however, seven patients did not have adequate material for C4d staining. Of the remaining 45 biopsies: nine (20%) showed no lesions suggestive of SAMR (C4d−, g−, ptc−), whereas 14 biopsies (31.1%) showed lesions deemed SAMR at 3 months. In addition, 22 patients (48.9%) were C4d-negative, but showed lesions of glomerulitis and ptc. Morphological, immunological and functional parameters are compared between these groups at 3 months and 1 year in Table 3.

Table 3.  Group A (positive DSA) patients stratified according to histological status at 3 months
ParametersC4d+g+ptc+ (SAMR) 14 Patients[LEFTWARDS ARROW]p-Value[RIGHTWARDS ARROW]C4d−g+ptc+ 22 Patients[LEFTWARDS ARROW]p-Value[RIGHTWARDS ARROW]C4d−g−ptc− 9 Patientsp vs. SAMR
Parameters at 3 months
 C4d (0–3)2.21 ± 0.8 <0.0010 ± 0NS0 ± 0<0.001
 g (0–3)1.32 ± 0.97<0.050.77 ± 0.46<0.0010 ± 0<0.01
 cg (0–3)0.29 ± 0.610.080.04 ± 0.14NS0 ± 0NS
 ptc (0–3)1.68 ± 0.720.061.15 ± 0.82<0.0010 ± 0<0.001
 IF/TA (0–3)0.86 ± 0.95NS1.04 ± 0.900.0470.37 ± 0.51NS
 Arteriosclerosis0.75 ± 0.61NS0.82 ± 0.89NS0.37 ± 0.74NS
Parameters at 1 year
 C4d (0–3)1.60 ± 0.990.0330.50 ± 0.87NS0.29 ± 0.490.012
 g (0–3)1.23 ± 1.00NS1.05 ± 0.690.0760.50 ± 0.650.09
 cg (0–3)0.31 ± 0.63NS0.14 ± 0.31NS0.09 ± 0.20NS
 %cg > 06/14 = 43%0.074/22 = 18%NS0/9 = 0%0.02
 ptc (0–3)1.90 ± 0.980.061.35 ± 0.690.0370.64 ± 0.85<0.01
 IF/TA (0–3)1.60 ± 0.83NS1.46 ± 1.18NS1.00 ± 1.410.10
 % IF/TA > 014/14 = 100%0.05617/22 = 77.2%0.0183/9 = 33.3%<0.001
 Arteriosclerosis1.37 ± 0.91NS1.10 ± 1.02NS0.50 ± 0.580.03
Graft function
 GFR at 3 months (mL/min/1.73 m2)48.8 ± 12.7NS55.8 ± 19.2NS60.2 ± 18.60.069
 GFR at 1 year (mL/min/1.73 m2)39.2 ± 13.9NS45.9 ± 17.70.02461.9 ± 19.2<0.01
 Last SCr (μmol/L)163 ± 55 NS161 ± 117NS117 ± 42 0.046
DSA level
 Month 3 class I MFImax960 ± 257NS493 ± 135NS1891 ± 1432NS
 Month 3 class II MFImax3291 ± 972 0.061214 ± 417 NS176 ± 1050.04
 Year 1 class I MFImax383 ± 139NS683 ± 404NS1600 ± 1251NS
 Year 1 class II MFImax2904 ± 10010.07711 ± 220NS0 ± 00.05

As seen in Table 3, a clear pattern emerges. The 14 SAMR (Cd4+, g+, ptc+) patients at 3 months had worse morphological lesions (higher C4d, ptc, arteriosclerosis score, and higher rate of transplant glomerulopathy and IFTA lesions) and worse functional parameters at 1 year than the 22 patients who were C4d-negative but g+, ptc+. These latter patients in turn exhibited higher ptc, IFTA lesions, and lower GFR at 1 year than the 9 C4d−, g−, ptc− patients.

Importantly, although the C4d+g+ptc+ and C4d−g+ptc− groups differed from one another mainly in severity of lesions, both groups differed dramatically from the C4d−g−ptc− group. For example, at 12 months, transplant glomerulopathy (cg) was absent from the C4d−g−ptc− group but present in both the C4d+g+ptc+ group (43%, p = 0.02) and the C4d−g+ptc+ group (18%, p = NS). Similarly, GFR in the C4d−g−ptc− group was significantly better than in either the C4d+g+ptc+ group (p < 0.01) or the C4d−g+ptc+ group (p = 0.024). Differences between the three groups were not always significant because of small sample size, but the relative constancy of the trends strongly suggests that the general pattern is valid.

Distinctions between groups at 3 months were not fixed over time. Indeed, as Figure 4 shows, there was a great deal of variation with 20 patients changing group between successive biopsies, in both directions. Five patients changed from C4d+ to C4d−, although retaining glomerular and PTC lesions. In 14 patients, by contrast, there was a shift from C4d-negativity to C4d-positivity: 12 had prior glomerular and PTC lesions, with no lesions in 2.

image

Figure 4. Changes in screening biopsy status between 3 months and 1 year (cases which did not change groups between biopsies are not included).

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Further demonstrating these changes, eight patients went from C4d−g+ptc+ at 3 months to C4d+g+ptc+ at 1 year. Comparing these eight cases with 11 C4d−g+ptc+ cases who remained C4d-negative, we found that lesions were generally worse, but reached significance only in the greater severity of overall glomerular lesions among those who became C4d-positive (1.75 ± 0.93 vs. 0.95 ± 0.47, p = 0.02).

There were no significant differences between class I MFImax levels between the three groups at either 3 months or 1 year. By contrast, class II MFImax levels showed the same declining severity between groups as the morphological lesions and GFR at both periods, though results were only marginally significant (Table 3).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Conflict of Interest Statement
  10. References

This study assesses for the first time the histopathological course in kidney transplant recipients with positive DSA without pretransplant desensitization treatment. We demonstrated a monomorphic association of persistent subclinical humoral lesions, occurring at some point in 48/52 (92.3%) of patients. The most important finding is that SAMR at 3 months was associated with worse glomerular and tubulointerstitial scarring, higher SCr and worse GFR at 1 year.

We found that the entity of SAMR, defined by Haas et al. and Gloor et al. (10,11) with positive C4d staining, g+ and ptc+ and positive DSAs (with a probable variant being described by Lerut et al. (16)), was actually extremely common in our patients with preexisting DSA. It occurred in 34.4% of biopsies evaluated for C4d at 3 months, 12 months or both 3 and 12 months. Apart from these C4d-positive cases, there was another group of biopsies (39.4% of those evaluated for C4d) that were C4d-negative, but g+ and ptc+ at 3 months, 12 months or both. Finally, 26.2% of patients had biopsies that were negative for all three elements (C4d−, g−, ptc−) on at least one biopsy, although only one patient was persistently negative at both 3- and 12-month biopsy.

More impressive was the number of cases that evolved from one category to another. Overall, eight cases were persistently C4d+g+ptc+ and nine patients were persistently C4d−g+ptc+ at both 3 and 12 months. However, as illustrated in Figure 4, 20 cases changed from C4d-positivity to C4d-negativity and vice versa. At any one time, C4d+ cases had worse associated light microscopic lesions than C4d-negativity cases. For example, at 12 months: glomerulitis (1.43 ± 0.86 vs. 0.77 ± 0.53, p = 0.008) and ptc (1.81 ± 0.77 vs. 1.29 ± 0.84, p = 0.05). However, once established at 3 or 12 months, the glomerular and PTC lesions tended to persist, although fluctuating according to C4d status. The reasons for these changes in C4d-positivity are not obvious, but a few remarks should be made. First, all biopsies were protocol biopsies; none was performed during an episode of active rejection, so that clinical rejection does not account for C4d status. C4d can disappear within days after beginning antirejection treatment (17), but this explanation could be invoked in only 3/31 patients and clearly cannot explain the majority of C4d-negative cases, nor the cases changing from C4d-negativity to C4d-positivity on later biopsy.

C4d deposition is a footprint of antibody-triggered classical complement activation (18,19). Cases of normal biopsies with positive PTC C4d deposition have been reported, speculated to be related to the mechanisms underlying ‘accommodation’. Consequently, the term ‘C4d deposition without morphological evidence of active rejection’ has been added to the Banff 2007 classification (13). (Indeed, we had two such cases, and neither subsequently developed major lesions.)

One of the novel findings of this study is that microcirculation inflammation and DSA but without C4d predicts later events. The entity C4d−g+ptc+ observed in 22 patients at 3 months might be a truncated form of SAMR probably due to antibodies of too low avidity or inability to fix complement (20,21). This argument is reinforced by the fact that the group with C4d−g+ptc+ had lower DSA titers as compared to patients having the complete criteria of subclinical AMR. Nevertheless, this hypothesis could not be validated because the antibody affinity has not been studied in the present paper. In addition, only C4d has been tested, which cannot formally rule out complement activation.

In this study, we found marked differences in behavior between the DSA classes: class I did not vary significantly between the SAMR, C4d−g+ptc+, and no SAMR groups at either 3 months or 1 year. By contrast class II values in the two g+, ptc+ groups were strikingly higher at both time intervals, and mirrored the differences in the histological lesions profile. Other authors have also demonstrated the relationship of class II Ab to subsequent transplant glomerulopathy and IFTA occurrence (22,23).

Table 3 shows the differences between the three different categories of lesions at 3 months, most of the differences between C4d+g+ptc+ and C4d−g−ptc− cases being highly significant. Furthermore, these differences persisted to a lesser degree at 1 year, despite numerous changes between categories. There is a gradation of decreasing severity of lesions between the three categories for almost all of the morphological and clinical parameters examined, although these differences do not always reach significance. Attention is particularly called to the differences in function between the three categories, especially in light of a recent study by Bartel et al. (24). Our study confirmed their observation that initially, DSA may occur without impaired graft function, but went further in demonstrating that in the absence of specific treatment, SAMR may promote subsequent chronic AMR, resulting in significantly higher SCr values at last follow-up (illustrated in Figure 5).

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Figure 5. Histological changes in renal screening biopsies in the same patient at 3 months (A), 1 year (B) and 3 years after transplantation (C). Glomerulitis (g 1–3), ptc (ptc 1–3, arrows) are observed in all three biopsies. (B and C) IFTA lesions. In addition, (C) shows transplant glomerulopathy (arrow) with segmental duplication of glomerular basement membrane associated with persisting glomerulitis and capillaritis (arrow). C4d score is 0, 1 and 2 in A, B and C respectively, and serum creatinine value is 66, 108 and 155 μmol/L, respectively.

Importantly, our results confirmed that the humoral lesion profile was almost entirely confined to the high immunological risk group A. By contrast, other lesions at 1 year tended to be modestly, but not significantly, worse in group A. These results accord with those of Gloor et al. (11). Comparing histological findings in a cohort of ABO-incompatible positive crossmatch (receiving conditioning treatment) with conventional living donor transplants, they found no significant differences between the groups with regard to IFTA and chronic vasculopathy scores, although a higher rate of transplant glomerulopathy (22% at 1 year) was demonstrated in HLA-incompatible patients. One difference between groups A and B was the use of ATG in group A vs. basiliximab in group B, based on results published by other groups showing superiority of ATG over basiliximab in terms of acute rejection incidence (25). Consequently, it is conceivable that the use of ATG in high immunological risk patients may have contributed to improve the success rate of transplantation in this group.

In this study, we did not find strong correlations between the degree of initial DSA or class and ptc or C4d. This lack of correlation may be related to the different initial protocols used for group A patients, particularly the addition of Rituxan and plasmapheresis in the last 18 of 54 group A patients. Importantly, our strategy has changed over time, leading to the sequential addition of prophylactic anti-CD20 and plasmapheresis in group A patients since 2006. In fact we found obvious differences between these 2 protocols, an issue which will be addressed in a subsequent communication. The aim of the present study was to define an immunopathological entity, and the fluctuations within that entity. The succeeding communication will deal with how different therapeutic strategies influence the course of that entity.

Several limitations must be acknowledged in our study including its small sample size and a mean follow-up of 30.2 ± 16 months in group A. Consequently, because only five graft losses were observed in group A, we were forced to use GFR and histological lesions as a proxy for graft survival, a reasonable stance because both low GFR and histological scarring lesions (cg, IF/TA) at 1 year have been correlated with worse long-term graft outcome (26,27). Finally, group B was defined on the basis of DSA-negativity by ELISA, not by the more sensitive Luminex SA, possibly missing some patients with low DSA-positivity and accounting for the low level of humoral lesions seen in group B. However, ELISA alone was able to distinguish the great majority of cases showing the humoral profile.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Conflict of Interest Statement
  10. References

This study is the first one to assess SAMR in a cohort of DSA-positive kidney transplant recipients without pretransplant desensitization treatment. We demonstrated that SAMR is a frequent finding in patients with preformed DSA. Beyond the group with defined SAMR is a group exhibiting glomerulitis and ptc, although C4d staining is negative. Furthermore, there are frequent transformations from C4d-positivity to negativity and vice versa over time, with accompanying fluctuations in glomerulitis and ptc. Both C4d+g+ptc+ and C4d−g+ptc+ cases are associated with worse histological parameters, renal function and higher class II DSA MFImax levels than C4d−g−ptc− cases. Finally, the entity of humoral lesions without C4d deposition may represent a milder but progressive form of AMR.

The subclinical nature of these processes emphasizes the usefulness of screening biopsies in this population because recognition of subclinical humoral lesions identifies those patients more likely to develop subsequent chronic AMR, so that the immunosuppressive regimen may be modified. These results merit further prospective randomized studies concerning the most appropriate therapeutic approach to SAMR.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Conflict of Interest Statement
  10. References

The Necker Hospital authors are members of the Centaure group together with Nantes and Lyon. The Necker Hospital is a member of the DIVAT database group.

Funding source: This study received no external funding.

Conflict of Interest Statement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Conflict of Interest Statement
  10. References

The authors have no conflict of interest related to this study.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Conflict of Interest Statement
  10. References