Posttransplant Prophylactic Intravenous Immunoglobulin in Kidney Transplant Patients at High Immunological Risk: A Pilot Study

American Journal of Transplantation 2007; 7: 1185 1192 Blackwell Munksgaard C 2007 The Authors Journal compilation C 2007 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/j.1600-6143.2007.01752.x Posttransplant Prophylactic Intravenous Immunoglobulin in Kidney Transplant Patients at High Immunological Risk: A Pilot Study D. Anglicheau a,, A. Loupy a, C. Suberbielle b, J. Zuber a, N. Patey c, L.-H. Noël c, R. Cavalcanti a, M. Le Quintrec a, F. Audat d, A. Méjean e, F. Martinez a, M.-F. Mamzer-Bruneel a, E. Thervet a and C. Legendre a a Service de Transplantation Rénale et de Soins Intensifs, Hôpital Necker, APHP, Paris, F-75015 France; Université René Descartes, Paris, F-75006 France b Laboratoire d Histocompatibilité, Hôpital Saint-Louis, APHP, Paris, F-75010 France; Université Denis Diderot, Paris, F-75005 France c Laboratoire d Anatomie Pathologique, Hôpital Necker, APHP, Paris, F-75015 France; Université René Descartes, Paris, F-75006 France d Unité d hémaphérèse, Hôpital Necker, APHP, Paris, F-75015 France; Université René Descartes, Paris, F-75006 France e Service d Urologie, Hôpital Necker, APHP, Paris, F-75015 France; Université René Descartes, Paris, F-75006 France Corresponding author: D. Anglicheau, dany.anglicheau@nck.aphp.fr The effects of posttransplant prophylactic intravenous immunoglobulin (IVIg) were investigated in renal transplant recipients at high immunological risk. Thirty-eight deceased-donor kidney transplant recipients with previous positive complement-dependent cytotoxicity crossmatch (n = 30), and/or donor-specific anti-hla antibodies (n = 14) were recruited. IVIg (2 g/kg) was administrated on days 0, 21, 42 and 63 with quadruple immunosuppression. Biopsy-proven acute cellular and humoral rejection rates at month 12 were 18% and 10%, respectively. Glomerulitis was observed in 31% and 60% of patients at months 3 and 12, respectively, while allograft glomerulopathy rose from 3% at month 3 to 28% at 12 months. Interstitial fibrosis/tubular atrophy increased from 18% at day 0 to 51% and 72% at months 3 and 12 (p < 0.0001). GFR was 50 ± 17 ml/min/1.73 m 2 and 48 ± 17 ml/min/1.73 m 2 at 3 and 12 months. PRA decreased significantly after IVIg (class I: from 18 ± 27% to 5 ± 12%, p < 0.01; class II: from 25 ± 30% to 7 ± 16%, p < 0.001). Patient and graft survival were 97% and 95%, respectively and no graft was lost due to rejection (mean follow-up 25 months). In conclusion, prophylactic IVIg in highimmunological risk patients is associated with good one-year outcomes, with adequate GFR and a profound decrease in PRA level, but a significant increase in allograft nephropathy. Key words: Desensitization, donor-specific antibodies, intravenous immunoglobulin, positive crossmatch, sensitized patients Received 13 October 2006, revised 20 December 2006 and accepted for publication 15 January 2007 Introduction More than half of all patients awaiting a deceased-donor kidney transplant are sensitized to human leukocyte antigen (HLA). Although kidney transplantation is the preferred treatment of end-stage renal disease, transplant rates are extremely low in highly-sensitized patients (i.e. with high levels of preformed anti-hla antibodies, or panel reactive antibodies [PRA]) due to the additional immunologic barrier (1). Sensitization is a significant obstacle to both access and success in renal transplantation, both for patients with high PRA awaiting a deceased-donor kidney and for those specifically sensitized to their intended livingdonor graft. If transplanted, these patients experience an increased risk of rejection and reduced graft survival (2). The highly sensitized patient is therefore destined to remain waitlisted for an extended period of time (3). Various strategies have been developed to lower the immunologic barrier in sensitized patients (1,3). Current approaches include desensitization protocols (4 9), specific allocation protocols which allocate kidneys to patients based on avoidance of antigen sensitization (10) and paired donor exchange programs (11). However, desensitization protocols based on plasmapheresis, immunoadsorption or pre-transplant intravenous immunoglobulin (IVIg) administration have been designed primarily for living donor donation. Moreover, donor exchange programs and mismatch criteria to avoid antigen sensitization are not developed in all countries. Some studies have undertaken desensitization in sensitized recipients using pretransplant IVIg, a product that is known to possess immunomodulatory, immunoregulatory, and anti-inflammatory properties (3,12). It has been suggested that IVIg therapy results in lower anti-hla antibodies titers, fewer acute rejection episodes and improved long-term allograft outcomes for cardiac and renal allograft recipients (5 7,13 18). However, while IVIg has already been used pretransplantation in desensitization protocols 1185

Anglicheau et al. and posttransplant as a treatment of antibody-mediated rejections (16), its use as a posttransplant prophylactic agent has been poorly studied. We examined the utility of posttransplant IVIg in kidney transplant patients at high immunological risk as an alternative approach to reducing the alloimmune response. We hypothesized that IVIg would decrease anti-hla antibodies and limit the alloimmune response, thereby improving transplant outcome. We report the outcome of high immunological risk kidney transplant recipients treated with prophylactic IVIg initiated immediately before transplantation and then repeated every three weeks for a 3-month period, administered in combination with quadruple immunosuppressive therapy. Materials and Methods Study population This was a single-arm, pilot study conducted at a single transplant center in France. All patients were required to fulfil the following immunological criteria: previous positive T-cell anti-human globulin-enhanced (AHG)- complement-dependent cytotoxicity (CDC) crossmatch, and/or previous or current positive donor specific antibodies (DSA). All patients had a current negative T-cell IgG AHG-CDC crossmatch at the day of transplantation and were ABO compatible. Immunosuppression and concomitant medication Four courses of IVIg (Endobuline R, Baxter) at a dose of 2 g/kg were administered over a 96-h period. The first course was started before reperfusion, with subsequent courses being given on days 21, 42 and 63. Other than the first course, IVIg was administered in association with prophylactic anticoagulation (intravenous heparin 100 IU/kg/day). The immunosuppressive regimen consisted of a biological induction agent, either polyclonal antibodies (rabbit ATG, Thymoglobulin R, Genzyme, Lyon, France) or basiliximab (Simulect R, Novartis Pharma AG, Basel), a calcineurin inhibitor (tacrolimus, Prograf R, Astellas,Tokyo; or cyclosporine, Neoral R,Novartis Pharma AG), mycophenolate mofetil (CellCept R, Roche Pharmaceuticals, Basel), and steroids. A 10-day course of ATG was initiated before reperfusion at a dose of 75 mg/day, adjusted thereafter to maintain a lymphocyte count <200/mm 3. If an allergic reaction to ATG had been documented, or if ATG had been used following a previous transplant, basiliximab 20 mg was administered intravenously on days 0 and 4. Cyclosporine was used in all patients transplanted during 2002 and 2003, after which time tacrolimus was used in all highly sensitized transplants. During the first three months post-transplant, cyclosporine dose was adjusted to target a whole-blood concentration of 1000 1200 ng/ml 2 h after cyclosporine intake, whereas tacrolimus was adjusted to achieve trough levels of 8 12 ng/ml. All patients received mycophenolate mofetil (2 g preoperatively, followed by 1 g b.i. d. unless clinical or biological intolerance necessitated dose adjustment), and prednisone (500 mg preoperatively, 125 mg on day 1, then 20 mg/day for 15 days, progressively tapered to 10 mg/day at day 30 and continued to month 12). All cytomegalovirus (CMV)-positive recipients and CMV-negative recipients of a kidney from a CMV-positive donor, were given CMV prophylaxis with valacyclovir for the first 4 months posttransplant. Patient evaluation Mean follow-up was 25 months (12 54 months). All patients underwent protocol kidney biopsy at time of transplantation, and at months 3 and 12 posttransplant. All biopsies were evaluated by routine light microscopy and scored using the Banff 97 classification (19). The scoring of interstitial fibrosis and tubular atrophy (IF/TA) was identical to the Banff grading for these changes. C4d complement staining was performed by immunofluorescence staining with antihuman C4d monoclonal antibody (Quidel, Heidelberg, Germany) on a snap frozen portion of the allograft biopsy. A diagnosis of acute cellular or humoral rejection was based on light and immunofluorescence microscopic findings using published criteria in conjunction with allograft dysfunction (20). Acute cellular rejection episodes were treated with high-dose steroids. Patients with acute humoral rejection underwent plasma exchange therapy and received rituximab (375 mg/m 2 ), and high-dose steroids. Plasmapheresis was discontinued when DSA was no longer detectable. The number of rituximab infusions was adjusted to maintain CD19 + lymphocyte count <5/mm 3. Graft function was assessed by calculated creatinine clearance (Cockcroft- Gault formula (21)), and glomerular filtration rate (GFR) using 51 Cr-EDTA clearance measurements at months 3 and 12 posttransplant. Antibody detection, specificity analysis and crossmatch techniques All immunological analyses were performed in the same laboratory (Hôpital Saint-Louis, Paris, France). All pretransplant sera were screened by enzymelinked immunosorbent assay (ELISA, LAT-M R, Canoga Park, CA) to determine the presence or absence of anti-hla class I or class II antibodies of the IgG isotype. In all positive sera, PRA were measured using microlymphocytotoxicity in a panel of 30 selected lymphocytes and 26 Epstein-Barr virus transformed cell lines, and their peak values were noted. Class I and II PRA were also measured at day 0 and one month after completion of IVIg administration. All crossmatches were performed by two techniques: direct complement dependent cytotoxicity (CDC) on T and B donor lymphocytes and antiglobulin-enhanced CDC (AHG-CDC) on nodal lymphocytes of the donor. For all patients, the CDC and AHG-CDC crossmatch were performed on peak, when available, and on current sera. The patients were transplanted with current negative IgG T-cell CDC crossmatch against their specific donors. No flow crossmatches were performed. Statistical analysis Results are expressed as numerical values and percentages for categorical variables and as mean ± SD for continuous variables. Comparisons were based on the v 2 test for categorical data and the Student-t test for normally distributed continuous data. Since semiquantitative lesion scoring using the Banff classification is not normally distributed, the Friedman test was used to detect differences between repeated evaluations of Banff scores during the first year post-transplantation on protocol biopsies, and the Wilcoxon test was used to compare two paired groups. Statistical analyses were performed using Statview 5.0.1 R software (Abacus Concepts, Inc. Berkeley, CA). Probability values <0.05 were regarded as statistically significant. Results Patients characteristics Thirty-eight patients were recruited to the study, all of whom underwent transplantation during the period 2002 2005. Demographic data and baseline characteristics are summarized in Table 1. All patients received a graft from a deceased donor. The most frequent cause of end-stage renal disease was glomerulopathies (n = 10). Twenty-five 1186 American Journal of Transplantation 2007; 7: 1185 1192

IVIg in High-Immunological Risk Patients Table 1: Patient demographics and baseline characteristics Parameter n = 38 Recipient age (mean ± SD, years) 44.3 ± 11.7 Donor age (mean ± SD, years) 46.7 ± 16.2 Female recipient (%) 21 (55%) Deceased donor (%) 38 (100%) Cold ischemia time (mean ± SD, hours) 27.1 ± 7.8 Delayed graft function (%) 9 (23%) Number of transplantations (first/second/third) 13/20/5 HLA A + B mismatch (mean ± SD) 2.2 ± 1.0 HLA DR mismatch (mean ± SD) 1.1 ± 0.7 Sensitization Peak class I PRA number (mean ± SD, %) 50 ± 36% Peak class II PRA number (mean ± SD, %) 48 ± 35% Peak class I or II PRA number 80% (n, %) 17 (45%) Previous DSA (n, %) 14 (37%) CDC crossmatch Previous T-cell positive CDC crossmatch (n,%) 23 (60%) Previous B-cell positive CDC crossmatch (n,%) 16 (47%) DSA = donor specific antibodies; CDC = complement-dependent cytotoxicity. patients (66%) had had a previous transplant, for which the most common cause of failure was chronic rejection. Specific antibodies directed against the donor s HLA antigens were detected in fourteen patients. Seventeen patients (45%) had a previous PRA level >80%, due to blood transfusion in 11 cases and pregnancy in six cases. IVIg courses were stopped prematurely in nine patients due to acute humoral rejection (n = 3), primary nonfunction (n = 1), infection (n = 3) or other reasons (n = 2). Immunosuppressive drugs daily doses and blood concentrations are shown in Table 2. The mean blood concentration of cyclosporine and tacrolimus was within target range at all timepoints. Efficacy At a mean follow-up of 25 months, patient and graft survival were 97% and 95%, respectively (Table 3). Two patients experienced primary nonfunction. One was attributed to an antiphospholipid syndrome which was also responsible for the death of the patient secondary to intracerebral bleeding and refractory epilepsy. In another case, the patient experienced renal artery thrombosis. No graft loss was related to rejection. Eleven biopsy-proven acute rejection episodes occurred in 10 patients, five within the first 3 months post- Table 3: Efficacy parameters at months 3 and 12 (n = 38) Month 3 Month 12 Patient survival (%) 38 (100%) 37 (97%) Graft survival (%) 36 (95%) 36 (95%) Primary nonfunction 2 (5%) Renal function GFR (mean ± SD, ml/min/1.73 m 2 ) 50 ± 17 48 ± 17 Serum creatinine (mean ± SD, 147 ± 49 142 ± 53 lmol/l) Proteinuria (mean ± SD, g/day) 0.27 ± 0.27 0.49 ± 1.3 Acute rejection 5 (13%) 11 (29%) Acute cellular rejection (%) 3 (8%) 7 (18%) Acute humoral rejection (%) 2 (5%) 4 (10%) transplantation (Table 3). Seven acute cellular rejections occurred, of Grade IA in three patients, Grade IB in one patient, and Grade IIA in three recipients; all episodes were steroid-sensitive. Four acute humoral rejection episodes occurred, all of which were C4d-positive, and were each treated by additional anti-cd20 therapy and plasma exchanges with a favorable outcome. Mean serum creatinine for patients who experienced an acute humoral or cellular rejection was 187 ± 80 lmol/l and 135 ± 39 lmol/l, respectively, at a mean follow-up of 25 months. Borderline lesions were observed in three patients on protocol biopsy during the first year posttransplantation. Mean serum creatinine was 147 ± 49 lmol/l and 142 ± 53 lmol/l at months 3 and 12, respectively (p = ns). Measured GFR remained stable during the first year posttransplantation (mean 50 ± 17 ml/min/1.73 m 2 at month 3 and 48 ± 17 ml/min/1.73 m 2 at month 12, p = ns). Mean proteinuria increased from 0.27 ± 0.27 g/day at month 3 to 0.49 ± 1.3 g/day at month 12 (p = ns). Pathological changes in protocol biopsies Chronic renal damage increased over the first year posttransplant (Table 4). According to the Banff classification, specimen adequacy was obtained in 28 biopsies (74%) at day 0, 32 biopsies at 3 months (89%), and 26 biopsies at 12 months (72%). IF/TA increased from 18% at day 0 to 51% and 72% at months 3 and 12, respectively. IF/TA scores higher than Grade I rose from 4% at day 0 to 27% at month 3 and 40% at month 12. Glomerulis was frequently observed in 31% and 60% of protocol biopsies at months 3 and 12, respectively. Chronic allograft glomerulopathy was also frequently observed in 3% and 28% of protocol biopsies at months 3 and 12, respectively. C4d deposition was Table 2: Dose and blood concentration of immunosuppression agents (n = 38) Treatments Day 10 Month 3 Month 6 Month 12 Steroid dose (mean ± SD, g/day) 21 ± 5.7 11.4 ± 3.9 10 ± 3.1 9.6 ± 4.6 Mycophenolate mofetil dose (mean ± SD, g/day) 1.9 ± 0.2 1.8 ± 0.54 1.7 ± 0.7 1.7 ± 0.6 Cyclosporine C 2 (mean ± SD, ng/ml) 1246 ± 390 1228 ± 168 1062 ± 276 1134 ± 211 Tacrolimus C 0 (mean ± SD, ng/ml) 10.6 ± 2.8 11.9 ± 2.3 8.2 ± 2.8 8.2 ± 1.7 C = target whole-blood concentration 2 (C 2 ) or 12 hours (C 0 ) after drug intake. American Journal of Transplantation 2007; 7: 1185 1192 1187

Anglicheau et al. Table 4: Banff scores (mean ± SD) and percent of patients with histology scores >0 on protocol biospies Day 0 (n = 28) Month 3 (n = 32) Month 12 (n = 25) p c Glomerulitis (g) % with g score >0 0 31% 60% <0.0001 Mean g score 0 0.62 ± 1.04 a 1.04 ± 1.14 a <0.01 Interstitial infiltrate (i) % with i score >0 0 41% 48% 0.0001 Mean i score 0 0.53 ± 0.76 a 0.80 ± 1.04 a <0.05 Tubulitis (t) % with t score >0 0 31% 44% <0.001 Mean t score 0 0.50 ± 0.92 0.60 ± 0.82 ns Vasculitis (v) % with v score >0 0 12% 4% ns Mean v score 0 0.13 ± 0.34 0.04 ± 0.20 ns Acute rejection 6% 4% Allograft glomerulopathy (cg) % with cg score >0 5% 3% 28% <0.01 Mean cg score 0.05 ± 0.22 0.03 ± 0.18 0.44 ± 0.87 ns Interstitial fibrosis (ci) % with ci score >0 14% 59% 76% <0.0001 Mean ci score 0.18 ± 0.48 0.94 ± 0.98 a 1.48 ± 1.16 a,b 0.001 Tubular atrophy (ct) % with ct score >0 18% 72% 72% <0.0001 Mean ct score 0.21 ± 0.50 1.09 ± 0.96 a 1.44 ± 1.19 a <0.01 Chronic vascular changes (cv) % with cv score >0 32% 41% 71% 0.01 Mean cv score 0.39 ± 0.68 0.78 ± 1.10 1.00 ± 0.86 ns Arteriolar hyalinosis (ah) % with ah score >0 32% 56% 36% ns Mean ah score 0.46 ± 0.74 0.75 ± 0.84 0.40 ± 0.58 ns Interstitial fibrosis/tubular atrophy 18% 51% 72% <0.0001 C4d staining d 1/26 0/20 a Month 3 or 12 biopsy versus Day 0 biopsy. b Month 12 biopsy versus Month 3 biopsy. c Friedman test for quantitative variables and v 2 test for trend for categorical data. d Not performed in all cases due to insufficient material. detected in peritubular capillaries in 1/26 protocol biopsies at month 3 and 0/20 at month 12. PRA changes Before transplantation, all patients had anti-hla antibodies, with a mean class I and II PRA of (50 ± 36)% and (48 ± 35)% respectively. At time of transplantation, whereas 11 patients had no detectable anti-hla antibodies by ELISA, the remaining 27 patients (71%) had a mean class I and II PRA of 33 ± 28% and 47 ± 27%, respectively. In these patients, class I and II PRA decreased to (5 ± 12)% (p < 0.01) and 7 ± 16% (p < 0.001) respectively after the four IVIg courses (Figure 1). Among the 11 patients without anti-hla antibodies at time of transplantation, two developed positive PRA after IVIg infusions without any immunological complication. Among the 15 patients with class I PRA at transplantation, complete removal of class I antibodies was obtained in 12 patients, two patients had a decrease of 60% and 40%, respectively, and one remained stable (Figure 1). Two patients without class I antibodies at transplantation Class I Class II 100 100 75 P=0.01 75 P=0.001 PRA (%) 50 25 0 At Transplant Post-IVIg PRA (%) 50 25 0 At Transplant Post-IVIg Figure 1: Class I and class II anti- HLA antibodies titers before and after IVIg treatment. 1188 American Journal of Transplantation 2007; 7: 1185 1192

IVIg in High-Immunological Risk Patients developed antibodies. Ten out of 17 patients with class II PRA at transplantation had a complete antibody removal, whereas five patients had a partial removal of 52 ± 33%, and two patients increased their class II PRA (Figure 1). Two humoral rejections (10%) and one grade IIA acute cellular rejection (5%) occurred among the 21 patients without anti-hla antibodies after the 4 IVIg courses. Conversely, two humoral (25%) and two grade IIA acute cellular rejection (25%) occurred among the eight patients who did not achieve complete removal of class I and/or II anti-hla antibodies. Adverse events One patient developed severe headaches during each IVIg infusion, which responded to analgesic treatment. Two thrombotic episodes were observed during IVIg treatment. One patient experienced multiple venous thrombosis during the initial posttransplant period which was related to a severe antiphospholipid syndrome recurrence that resulted in the patient s death. One arterio-venous fistula thrombosis occurred immediately after the fourth course of IVIg in one patient. During the first year posttransplant, 16 infectious complications were observed. Three patients experienced viral complications including one CMV disease, one BK virus nephropathy responding to low-dose cidofovir and immunosuppression tapering, and one BK viremia without related nephropathy. Thirteen bacterial complications occurred (seven nonsevere pyelonephritis, two pneumonitis, two septicemia of unknown origin, one peritonitis and one parietal infection). Both cases of BK virus replication, and the cases of peritonitis and parietal infection occurred in patients who had been previously treated for acute humoral rejection prior to infection using steroid pulses, plasmapheresis and rituximab. No malignancy was observed during follow-up. Neither renal dysfunction nor acute renal failure related to IVIg infusion was reported. However, protocol biopsies unexpectedly showed vacuolisations of the proximal tubules consistent with osmotic injury in 15/38 (39%) patients which were potentially related to IVIg treatment. Discussion The benefit of IVIg is now recognized in kidney transplant recipients as an immunomodulatory agent of the antibodymediated alloimmune response (1). Prior to transplantation, administration of IVIg therapy to highly sensitized patients reduces allosensitization and anti-hla antibodies (4,6), while posttransplant IVIg has been used successfully to treat steroid-resistant acute rejection episodes (16,22,23). However, desensitization protocols using IVIg seem particularly suitable to living donor donation and less appropriate for recipients of a deceased-donor kidney. In the absence of an intended living donor, highly sensitized patients may wait many years for a compatible graft from a deceased donor. Moreover, in a randomized study evaluating IVIg as an agent to lower allosensitization, Jordan et al. observed that despite an initial reduction of anti-hla antibody levels by IVIg, PRA level returned to near-baseline six months after IVIg infusion (6). In the context of deceased donors, this would lead to repeated infusions to obtain a sustained desensitization effect, and desensitization protocols to remove circulating alloantibodies with IVIg are both technically demanding and expensive. In this pilot study, we evaluated whether administering IVIg after transplantation could reduce the alloimmune response in patients at high immunological risk, defined by the presence of DSA and/or positive CDC crossmatch. Our results showed that at 12 months posttransplant, patients treated with posttransplant IVIg experience good graft outcome, adequate renal function and a profound decrease in PRA, but that chronic allograft lesions worsened. Prophylactic use of IVIg in which administration begins only after transplantation has not been well-studied. In previous trials that have reported the benefit of IVIg as a desensitization agent, patients received initial infusions pretransplant with additional infusions subsequently. Glotz et al. (5) studied the clinical outcome of 13 highly sensitized patients desensitized by three IVIg courses who received a further three courses of IVIg posttransplant. Graft survival at 1 year was 84% with 7.6% immune-mediated graft loss. One-year creatinine was not available and protocol biopsies were not performed. Similarly, Jordan et al. (6) also reported findings from a series of 16 patients who underwent transplantation after successful desensitization, and who received additional IVIg infusions monthly for 4 months. At 2 years, graft survival was 80% and serum creatinine was numerically higher in the IVIg group versus a placebo group (mean ± SEM, 1.68 ± 0.28 vs. 1.28 ± 0.13 mg/dl, p = 0.29). No data were available concerning histological progression. Surprisingly, although the authors claimed the desensitization protocol was effective, and that reduction of the anti-hla antibodies titer prior to transplantation would improve graft outcome, Jordan et al. reported no differences in graft survival and graft function, but more rejection episodes in the IVIg-treated group compared to placebo (6). Noteworthy, whereas our patients did not receive IVIg before transplantation as a desenzitization agent, the acute rejection rate we report in the present study is quite similar to those previously reported in protocols involving IVIg before transplantation for sensitized patients (1). One month after completion of the IVIg administration, we observed a complete disappearance of PRA in the majority of the patients with alloantibodies at transplantation. This result is not typical of those reported in pretransplantation desensitization studies (6), that showed only partial decrease of PRA after IVIg, and must be interpreted American Journal of Transplantation 2007; 7: 1185 1192 1189

Anglicheau et al. cautiously because of the lack of long-term follow-up. One may also consider the consequences of immunosuppressive drugs, mainly antithymocyte glogulins on PRA levels. It was previously suggested that ATG may have anti-b cell and plasma cell activity. For example, Zand et al. reported that rabbit ATG strongly induced apoptosis in vitro against naive, activated B cells and bone marrow resident plasma cells at clinically relevant concentrations (24). The current study offers more complete efficacy and safety data on post-transplant desensitization with IVIg by providing both 1-year graft outcome data and systematic histological evidence and GFR values, in a very similar but larger cohort of patients. Although we observed a high rate of biopsy-proven acute rejection, findings were encouraging in that the high risk of antibody-mediated hyperacute rejection and early graft loss was avoided; furthermore, when humoral rejection occurred it was reversible and did not have a significant deleterious effect on 1-year graft survival. Nevertheless, one of the most important findings of the protocol biopsies is the high incidence of transplant glomerulopathy at 12 months, that may suggest the relatively poor performance of post transplant IVIg to prevent antibody-mediated chronic glomerular endothelial damage. These findings are consistent with those of Gloor et al. who reported histological findings 1 year after positive crossmatch living-donor kidney transplantation (25). In this study, according to the DSA level, recipients of positive crossmatch underwent pretransplant conditioning using plasmapheresis followed by low-dose IVIg after each plasmapheresis or received only a single course of IVIg 2 g/kg immediately prior to transplantation. With this approach, Gloor et al. reported a 1-year acute rejection rate of 38%, with 17 antibody-mediated rejections observed among 37 patients. Allograft glomerulopathy was observed in 22% of patients with a positive crossmatch compared to 8% in a control group of patients. However, these strategies based on plasmapheresis are only conceivable for living donor donation. With a rather similar high immunological risk population, we report very similar results, but with a strategy available in case of deceased donor donation without pretransplant conditioning. In our series, allograft glomerulopathy was found in 28% of patients at 1 year, which would suggest persistence of an antibody-mediated alloimmune injury (26), although only a few patients had C4d in peritubular capillaries at the 1- year biopsy. Isolated glomerulitis, without either DSA or C4d staining, does not completely fulfill the diagnostic criteria for typical humoral rejection, and its significance remains a source of debate. Isolated transplant glomerulitis/glomerulopathy might be a truncated form of humoral rejection, due to more intensive immunosuppression. The apparent absence of DSA may result from low levels of alloantibodies undetectable in the blood but bound to the graft endothelium. Using highly sensitive techniques (flow cytometric crossmatch assay and single-antigen Flowbead analysis of alloantibodies) Gloor et al. reported that low levels of donor-specific alloantibody persisted in the majority of patients despite desensitization protocols (15). Thus, as described by Gloor et al., in most patients desensitization and subsequent immunosuppression does not result in absolute removal of all donor-specific alloantibodies but rather a decrease in the number of antibodies and reduced antibody activity (15). It is also likely that antibodies against non-hla molecules, which are closely associated with PRA (27), contribute to chronic glomerular endothelial damage that in turn promotes duplication of glomerular basement membrane (28). In our study, unfortunately, antibody detection and specificity analysis were limited to anti-hla molecules and only assessed by microlymphocytotoxicity and ELISA assays that may limit the ability to detect low levels of alloantibodies. Protocol biopsies demonstrated that despite good GFR at 3 months and 1 year posttransplant, there was an increasingly high rate of IF/TA lesions. Similar findings were reported by Gloor et al. (25), who showed that the distribution of interstitial fibrosis, tubular atrophy, vasculopathy and arteriolar hyalinosis did not differ between conventional and positive crossmatch living-donor kidney transplant recipients at time of transplant and at 12 months posttransplant (25). It is noteworthy that in the Gloor study all kidneys were from living donors and experienced only minimal chronic lesions on biopsies taken at the time of transplantation (Banff ci score >0 was reported in only 9% of cases). In the present study, all patients received a graft from a deceased donor, and chronic lesions were more pronounced at transplantation (Banff ci score >0 in 14% of high immunological risk patients). The potential nephrotoxicity of IVIg should also be considered, since renal dysfunction has been associated with administration of IVIg (29 31). Patients who are highly HLA-sensitized require high doses of IVIg, which may result in higher rates of infusion-related complications. These remain poorly understood, particularly in kidney transplant recipients, who may be more vulnerable to adverse effects related to the osmolarity of IVIg. Consistent with this hypothesis, a sizeable proportion of biopsies demonstrated vacuolization of the proximal tubules, consistent with osmotic injury. IVIG is an expensive therapy as well as antithymocyte globulin. Is it cost-effective to use as an adjunctive agent to improve outcome of kidney transplantation in highly sensitized patients? Although this point needs a specific pharmaco-economic study, as emphasized by Jordan et al., the costs savings to Medicare (the sole provider for ESRD services in the United States) to remove half the highly sensitized patients from dialysis by performing a successful transplantation would be $1.4 billion over a 3-year period (6). Nevertheless, this pilot study was of course not designed to access a pharmaco-economic evaluation. We used a 10-day course of rabbit antithymocyte globulin, a protocol that has been already applied to highimmunological risk patients (5,15). Obviously our objective 1190 American Journal of Transplantation 2007; 7: 1185 1192

IVIg in High-Immunological Risk Patients was not to test a minimization of the induction therapy. However, due to the expensive cost of this treatment and to the fact that short-courses of thymoglobulin have been reported as very effective as well, a shortened protocol might be evaluated (32 34). One of the weaknesses of the current investigation is that the induction and maintenance immunosuppression should have been kept constant. With regard to induction therapy, the use of basiliximab was restricted to patients with an allergic reaction to ATG, or if ATG had been used previously. With regard to the calcineurin inhibitor, the allocation of cyclosporin/tacroliumus was sequential. The decision to replace cyclosporin by tacrolimus was justified by the results published with a tacrolimus-based therapy in high immunological risk patients (5,6). However, we failed to find any differences on acute rejection rate, 1-year graft function and histological changes among cyclosporinand tacrolimus-treated patients. Another weakness of the study is related to the technologies of antibodies detection and histocompatibility testing. If all pretransplant sera were screened by ELISA to determine the presence or absence of anti-hla class I or class II antibodies of the IgG isotype, one of the limitations of this study is that antigen specific technologies were not available at our laboratory at the time of transplant of all the patients. Particularly anti-hla class II antibodies were not identified for all of them. B cells constitutively display both class I and class II antigens, whereas T cells generally express only class I. Moreover, B cells are a more sensitive indicator of class I antibodies than T cells because they express higher cell surface densities of class I molecules. Therefore, a positive B-cell crossmatch result may indicate anti-class II or weak anti-class I reactivities (33). It would have been important to have complete determination of both class I and class II specificities of DSA in these patients to further analyze the efficacy of IVIg according to the class of DSA, as it has been previously reported by Akalin et al. (33). Moreover, we did not have the possibility of perform flow crossmatches. Our clinical strategy is to transplant with current negative IgG T-cell CDC crossmatch. Flow crossmatches would have been particularly useful in the case of patients with a high PRA and negative AHG-CDC crossmatches. A flow crossmatch negative and no detected DSA would predict a satisfactory outcome without evidence of antibody mediated disease unless a new antibody emerged following transplantation. However, a positive flow crossmatch might predict the presence of non-hla directed DSA. The patients in whom the worst outcome would be expected are those with donor specific antibodies. In conclusion, four courses of IVIg administered posttransplant in high immunological risk kidney transplant recipients induce a profound decrease in PRA levels, associated with good graft outcome without immune-mediated graft loss after a mean follow-up of 25 months. However, protocol biopsies show a high rate of chronic lesions clearly suggestive of antibody-mediated chronic rejection, contrasting with the good renal function observed. The reasons for this mismatch between clinical and physiological evaluation and histological findings are unclear, and longer follow-up of these lesions is therefore of utmost importance. 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