Dynamics of B Cell Recovery In Kidney/Bone Marrow Transplant Recipients.

BACKGROUND: Previous studies identified B cell gene signatures and predominance of specific B cell subsets as a marker of operational tolerance after kidney transplantation. These findings suggested a role for B cells in the establishment or maintenance of tolerance. Here we analyzed B cell recovery in 4 subjects, 3 of whom achieved tolerance after combined kidney/bone marrow transplantation. METHODS: Peripheral B cell subsets were examined longitudinally by flow cytometry. Immunoglobulin heavy chain repertoire analysis was performed using next-generation sequencing. Lastly, the patients' serum reactivity to HLA was assessed by Luminex. RESULTS: B cell counts recovered approximately 1 year posttransplant except for 1 subject who experienced delayed reconstitution. This subject resumed immunosuppression for acute rejection at 10 months posttransplant and underwent preemptive retransplantation at 3 years for chronic rejection. B cell recovery was accompanied by a high frequency of CD20 + CD24CD38 transitional B cells and a diversified clonal repertoire. However, all 4 subjects showed prevalence of CD20 + CD27+ memory B cells around 6 months posttransplant when B cell counts were still low and the clonal B cell repertoire very limited. The predominance of memory B cells was also associated with high levels of somatically mutated immunoglobulin heavy chain variable sequences and transient serum reactivity to HLA. CONCLUSIONS: Our observations reveal the presence of memory B cells early posttransplant that likely escaped the preparative regimen at a time consistent with the establishment of tolerance. Further studies are warranted to characterize the functional properties of these persisting memory cells and evaluate their potential contribution to tolerance induction.

Filgrastim-Induced Crescentic Transformation of Recurrent IgG2λ GN.

Proliferative GN with monoclonal IgG deposits is an increasingly recognized form of GN, but its relation to hematologic malignancy remains poorly understood. Filgrastim, an analog for granulocyte colony-stimulating factor produced by recombinant DNA technology, is frequently used to stimulate bone marrow release of hematopoietic progenitor cells in preparation for stem cell transplant. We report an exceptional case of proliferative GN with monoclonal IgG2λ deposits in a young man whose disease progressed slowly to CKD, which was followed by a preemptive kidney transplant. The patient developed recurrent GN in the allograft and clinically detectable plasma cell neoplasm 9 years after the first renal manifestations. Contemporaneous with filgrastim administration for stem cell mobilization, the patient's slowly progressive GN underwent severe crescentic transformation, leading to rapidly progressive and irreversible allograft failure. This report explores the spectrum of GN with monoclonal IgG deposits and the pathophysiologic role of granulocyte colony-stimulating factor in exacerbation of preexisting GN.

Evidence to Support a Contribution of Polyreactive Antibodies to HLA Serum Reactivity.

BACKGROUND: Assessing the serum reactivity to HLA is essential for the evaluation of transplant candidates and the follow-up of allograft recipients. In this study, we look for evidence at the clonal level that polyreactive antibodies cross-reactive to apoptotic cells and multiple autoantigens can also react to HLA and contribute to the overall serum reactivity. METHODS: We immortalized B cell clones from the blood of 2 kidney transplant recipients and characterized their reactivity to self-antigens, apoptotic cells as well as native, denatured, and cryptic HLA determinants using enzyme-linked immunosorbent assay (ELISA), immunofluorescence, flow cytometry and Luminex assays. We also assessed the reactivity of 300 pretransplant serum specimens to HLA and apoptotic cells. RESULTS: We report here 4 distinct B cell clones cross-reactive to self and HLA class I. All 4 clones reacted to numerous HLA class I alleles but did not appear to target canonical "shared" epitopes. In parallel experiments, we observed a strong correlation between IgG reactivity to HLA and apoptotic cells in pretransplant serum samples collected from 300 kidney transplant recipients. Further analysis revealed that samples with higher reactivity to apoptotic cells displayed significantly higher class I percent panel-reactive antibodies compared to samples with low reactivity to apoptotic cells. CONCLUSIONS: We provide here (1) proof of principle at the clonal level that human polyreactive antibodies can cross-react to HLA, multiple self-antigens and apoptotic cells and (2) supportive evidence that polyreactive antibodies contribute to overall HLA reactivity in the serum of patients awaiting kidney transplant.

Tracking donor-reactive T cells: Evidence for clonal deletion in tolerant kidney transplant patients.

T cell responses to allogeneic major histocompatibility complex antigens present a formidable barrier to organ transplantation, necessitating long-term immunosuppression to minimize rejection. Chronic rejection and drug-induced morbidities are major limitations that could be overcome by allograft tolerance induction. Tolerance was first intentionally induced in humans via combined kidney and bone marrow transplantation (CKBMT), but the mechanisms of tolerance in these patients are incompletely understood. We now establish an assay to identify donor-reactive T cells and test the role of deletion in tolerance after CKBMT. Using high-throughput sequencing of the T cell receptor B chain CDR3 region, we define a fingerprint of the donor-reactive T cell repertoire before transplantation and track those clones after transplant. We observed posttransplant reductions in donor-reactive T cell clones in three tolerant CKBMT patients; such reductions were not observed in a fourth, nontolerant, CKBMT patient or in two conventional kidney transplant recipients on standard immunosuppressive regimens. T cell repertoire turnover due to lymphocyte-depleting conditioning only partially accounted for the observed reductions in tolerant patients; in fact, conventional transplant recipients showed expansion of circulating donor-reactive clones, despite extensive repertoire turnover. Moreover, loss of donor-reactive T cell clones more closely associated with tolerance induction than in vitro functional assays. Our analysis supports clonal deletion as a mechanism of allograft tolerance in CKBMT patients. The results validate the contribution of donor-reactive T cell clones identified before transplant by our method, supporting further exploration as a potential biomarker of transplant outcomes.

Expansion and somatic hypermutation of B-cell clones in rejected human kidney grafts.

BACKGROUND: B-cell infiltrates are common in rejected kidney allografts, yet their composition is still unclear. The aim of our study was to characterize the clonal composition of B-cell infiltrates of rejected human kidney grafts. METHODS: We used a molecular approach to characterize the partial B-cell repertoires of 5 failed human kidney grafts with detectable B-cell infiltrates. A comparison between the intragraft and blood repertoire was also conducted for 1 case. RESULTS: Redundant sequences were observed in both blood and graft, although the level of clonal amplification was significantly higher for the graft. Somatic hypermutations (SHMs) were also more frequent in sequences found in the graft compared to the blood. The rate of nonsilent mutations was significantly higher in complementarity determining regions (CDRs) compared to framework regions in blood sequences as well as in graft sequences found at low frequency. In contrast, this preferential distribution was lost in sequences found at high frequency in the graft, suggesting a lack of affinity maturation in situ. Lastly, follicular dendritic cells were undetectable in CD20 infiltrates in all samples examined. CONCLUSIONS: We provide here evidence that B-cell clones expand and undergo SHMs in situ. However, the even distribution of nonsilent SHM in high-frequency graft sequences together with the absence of follicular dendritic cells do not support the view that infiltrating B cells are part of functional germinal centers.

Partial therapeutic response to Rituximab for the treatment of chronic alloantibody mediated rejection of kidney allografts.

BACKGROUND AND OBJECTIVES: Chronic rejection leads to kidney allograft failure and develops in many kidney transplant recipients. One cause of chronic rejection, chronic antibody mediated rejection (CAMR), is attributed to alloantibodies. Maintenance immunosuppression including prednisone, mycophenolate mofetil (MMF) and calcineurin inhibitors may limit alloantibody production in some patients, but many maintain or develop alloantibody production, leading to CAMR. Therefore, no efficacious therapy to treat CAMR is presently available to prevent the progression of CAMR to kidney allograft failure. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: We performed a retrospective review of 31 subjects with CAMR, of which 14 received Rituximab and 17 subjects did not. Response to Rituximab was defined as decline or stabilization of serum creatinine for at least one year. Data reviewed included demographic, clinical, allograft, post-transplant, and pathological variables. Pathological variables in the diagnostic allograft biopsy were scored according to Banff criteria. RESULTS: The median survival time (MST) for allografts in the control group was 439 days, and for the Rituximab treated group was 685 days. The Rituximab group was dichotomous with 8 subjects showing a medial survival time of 1180 days, and 6 subjects having a median survival time of 431 days. The MST for the responders was statistically significant from the non-responders and controls. No pathological parameter distinguished any subset of subjects. CONCLUSIONS: These data show that Rituximab followed by standard maintenance immunosuppression shows a therapeutic effect in the treatment of CAMR, which is confined to a subset of treated subjects, not identifiable a priori.

Chronic humoral rejection of human kidney allografts associates with broad autoantibody responses.

BACKGROUND: Chronic humoral rejection (CHR) is a major complication after kidney transplantation. The cause of CHR is currently unknown. Autoantibodies have often been reported in kidney transplant recipients alongside antidonor human leukocyte antigen antibodies. Yet, the lack of comprehensive studies has limited our understanding of this autoimmune component in the pathophysiology of CHR. METHODS: By using a series of ELISA and immunocytochemistry assays, we assessed the development of autoantibodies in 25 kidney transplant recipients with CHR and 25 patients with stable graft function. We also compared the reactivity of five CHR and five non-CHR patient sera with 8027 recombinant human proteins using protein microarrays. RESULTS: We observed that a majority of CHR patients, but not non-CHR control patients, had developed antibody responses to one or several autoantigens at the time of rejection. Protein microarray assays revealed a burst of autoimmunity at the time of CHR. Remarkably, microarray analysis showed minimal overlap between profiles, indicating that each CHR patient had developed autoantibodies to a unique set of antigenic targets. CONCLUSION: The breadth of autoantibody responses, together with the absence of consensual targets, suggests that these antibody responses result from systemic B-cell deregulation.

Bortezomib in kidney transplant recipients with antibody mediated rejection: three case reports.

Here, we report our experience on three patients with AMR who were treated with bortezomib after other therapeutic interventions had failed. Bortezomib was well tolerated by two of the three patients. The third patient developed worsening thrombocytopenia following the second dose. Despite a low adverse event profile, none of the three patients conclusively responded to the bortezomib treatment. As a result of the difference in our results from that of other centers we feel that a larger prospective study is needed to define appropriate guidelines for the use of bortezomib in cases of acute rejection.

HLA-mismatched renal transplantation without maintenance immunosuppression.

Five patients with end-stage renal disease received combined bone marrow and kidney transplants from HLA single-haplotype mismatched living related donors, with the use of a nonmyeloablative preparative regimen. Transient chimerism and reversible capillary leak syndrome developed in all recipients. Irreversible humoral rejection occurred in one patient. In the other four recipients, it was possible to discontinue all immunosuppressive therapy 9 to 14 months after the transplantation, and renal function has remained stable for 2.0 to 5.3 years since transplantation. The T cells from these four recipients, tested in vitro, showed donor-specific unresponsiveness and in specimens from allograft biopsies, obtained after withdrawal of immunosuppressive therapy, there were high levels of P3 (FOXP3) messenger RNA (mRNA) but not granzyme B mRNA.

Comparison of two dosages of thymoglobulin used as a short-course for induction in kidney transplantation.

Thymoglobulin is used effectively as an induction agent in kidney transplantation, but the optimal dose is not well established. We evaluated the degree and durability of T-cell clearances with two different thymoglobulin regimens in adult kidney transplant recipients (KTR). Seven KTR received a 3-day thymoglobulin-based induction of 1.0 mg/kg/day while nine received 1.5 mg/kg/day, in addition to maintenance immunosuppression. Lymphocyte subsets were monitored for 6 months. Renal function, infections and malignancies were monitored for 24 months. T-cell subsets were significantly lower by day 30 with the thymoglobulin 1.5 mg/kg/day regimen when compared with the 1.0 mg/kg/day regimen; this trend was sustained at 6-month (CD3(+): 438 +/- 254 vs. 1001 +/- 532 cells/mm(3), P = 0.016). Renal function between the two groups was not significantly different at 6- and 24-months post-transplant. One case of BK Virus viremia in the 1.5 mg/kg/day thymoglobulin group was detected. No acute rejection episodes, cytomegalovirus infections, or malignancies were noted in either group. Thymoglobulin induction was efficacious in both groups, but with a significantly sustained T-cell clearance in the 1.5 mg/kg/day regimen. A more profound T-cell clearance within the first 6 months postinduction therapy may translate into a decreased risk of immunological injury and improved long-term outcome after kidney transplantation.

Slowing the progression of chronic allograft nephropathy by conversion from cyclosporine to tacrolimus: a randomized controlled trial.

BACKGROUND: Chronic allograft nephropathy (CAN) is a multifactorial process with immunologic and nonimmunologic factors. Because tacrolimus (Tac) has been ascribed a beneficial effect on some of these factors when compared to cyclosporine A (CyA), a randomized controlled trial was conducted to investigate whether conversion from CyA to Tac can ameliorate the progression of renal dysfunction in kidney transplant recipients (KTR) with CAN. METHODS: Of the 46 patients with biopsy-proven CAN enrolled, 24 were converted from CyA to Tac, whereas 22 patients were maintained on CyA. Serum creatinine (SCrea), lipid profiles and an antihypertensive score (AHS) were determined after 3, 6 and 12 months. AHS is based on the total number and dosages of antihypertensive medications used. SCrea and AHS were additionally evaluated at 36 months. RESULTS: SCrea was decreased in the Tac group (Tac(baseline): 297 +/- 67 micromol/L; Tac(6): 261+/- 70 micromol/L, P < 0.001; Tac(12): 254 +/- 55 micromol/L, P < 0.001; Tac(36): 255 +/- 78 micromol/L, P = 0.235), whereas a significant increase of SCrea was detected in the CyA group (CyA(baseline): 279 +/- 77 micromol/L, CyA(12): 333 +/- 98 micromol/L, P < 0.001; CyA(36): 317 +/- 89 micromol/L, P < 0.001). Compared to CyA therapy, SCrea in the Tac group declined after 12 and 36 months (P = 0.011 and 0.048, respectively) as well as AHS (Tac(12): 59 +/- 13, CyA(12): 83 +/- 14, P < 0.001; Tac(36): 60 +/- 12, CyA(36): 84 +/- 14, P < 0.001). LDL cholesterol was lower in the Tac group after 12 months (Tac(12): 2.5 +/- 0.5 mmol/L, CyA(12): 3.5 +/- 0.6 mmol/L, P < 0.001). CONCLUSION: Conversion from CyA to Tac in KTR with CAN improves allograft function, lowers blood pressure, and reduces LDL cholesterol. This superior profile may translate into improved long-term graft survival.

Massachusetts General Hospital participation in Operation Unified Assistance for tsunami relief in Banda Aceh, Indonesia.

When Project HOPE invited Massachusetts General Hospital (MGH) to participate in a tsunami relief mission aboard the USNS Mercy hospital ship in January 2005, MGH responded swiftly and enthusiastically. Overcoming differences in medical styles and standards, as well as a variety of cultural and institutional differences, MGH volunteers collaborated successfully with Navy personnel and volunteers from other hospitals to deliver excellent care to a large number of tsunami victims. Lessons were learned regarding the need for better initial team orientation, more-open communication, and more-transparent decision-making; still, a rapid response of tertiary medicine, joining public and private resources, proved to be a powerful effective model.

2005 immunosuppressive strategies in kidney transplantation: which role for the calcineurin inhibitors?

The calcineurin inhibitors (CNIs) cyclosporine and tacrolimus have been the cornerstones of immunosuppressive strategies in clinical transplantation. Currently, regimens that are most widely used for induction and maintenance therapy include CNIs. However, many clinical trials aiming at reducing or eliminating CNIs have been performed in recent years. Here, we review and discuss current and future immunosuppressive strategies with a special emphasis on the role of CNIs, in the light of recent studies in the field of kidney transplantation. In the current era, CNIs still play an important role.

Prevalence and significance of anti-HLA and donor-specific antibodies long-term after renal transplantation.

Post-transplant circulating anti-human leukocyte antigens (HLA)-antibodies and C4d in allograft biopsies may be important in chronic rejection in renal transplant recipients (RTR). We determined the prevalence and significance of anti-HLA-antibodies and donor-specific antibodies (DSA). Sera were collected from 251 RTR >6 months post-transplant. Sera were tested using enzyme-linked immunosorbent assay (ELISA) screening for anti-HLA antibodies. Positive sera were retested with ELISA-specific panel for antibody specificity. A 11.2% of patients had anti-HLA antibodies and 4.4% had DSA. Anti-HLA antibodies were significantly associated with pretransplant sensitization, acute rejection and in multivariate analysis, higher serum creatinine (2.15 +/- 0.98 vs. 1.57 +/- 0.69 mg/dl in negative anti-HLA antibodies group). Allograft biopsies performed in a subset of patients with anti-HLA antibodies revealed that 66% had C4d in peritubular capillaries (0% in patients without antibodies). Anti-HLA antibodies were associated with a worse allograft function and in situ evidence of anti-donor humoral alloreactivity. Long-term RTR with an increase in creatinine could be screened for anti-HLA antibodies and C4d in biopsy.

Analysis of the cardiovascular risk profile in stable kidney transplant recipients after 50% cyclosporine reduction.

BACKGROUND: Long-term use of cyclosporine (CsA) contributes to post-transplant cardiovascular disease (CVD). Hence, a reduction in CsA dosage in kidney transplant recipients (KTR) may improve long-term outcomes. We analyzed the effects of 50% CsA dose reduction on the CVD risk profile in stable KTR. METHOD: Thirty-one KTR on a regimen of CsA, prednisone and mycophenolate mofetil (MMF) were studied. Patients were randomized to either a) continue their previously determined CsA dose (control group, n = 15) or b) lower their CsA dose by 50% (CsA reduction group, n = 16). Renal function, blood pressure, lipid profile, plasma homocysteine (HCY), C-reactive protein (CRP), fibrinogen, and uric acid were compared at baseline and at 6 months. RESULTS: At 6 months, there was a significant improvement in allograft function, systolic blood pressure, number of anti-hypertensive medications and serum uric acid levels in the CsA reduction group. No significant decrease in plasma HCY, CRP, fibrinogen or improvement in lipid profile was found. In contrast, in the Control group, there was a significant increase in HCY, uric acid, and triglycerides. No acute rejection occurred in either group. CONCLUSIONS: A greater reduction in CsA dose could further improve CVD risk profiles, although this may increase the risk of acute or subclinical rejection.

Interferon-alpha therapy in liver transplant recipients: lack of association with increased production of anti-HLA antibodies.

Interferon-alpha (IFN) is a useful treatment for active HCV infection. In kidney transplantation, IFN has been shown to trigger acute rejection with de novo anti-HLA antibodies. Interferon-alpha has not been reported to enhance the risk of acute rejection in HCV-positive liver transplant recipients (LTRs). Sera were collected from 44 LTRs greater than 6 months post-transplant. Sera were tested with ELISA for the presence and the specificity of anti-HLA antibodies. The prevalence of anti-HLA antibodies was 11% and was not significantly different in 13 HCV-positive recipients who received IFN, compared with 10 who did not receive IFN (8% vs. 20%), or with 21 HCV-negative recipients (10%). None of the patients had an acute rejection after starting IFN. In this study, LTRs receiving IFN did not have an increased frequency of anti-HLA antibodies. This may partially explain the safety of IFN previously reported in LTRs requiring antiviral therapy.

Vasopeptidase inhibition affords greater renoprotection than angiotensin-converting enzyme inhibition alone.

The renoprotective efficacy of the vasopeptidase inhibitor omapatrilat (OMA) was compared with that of enalapril (ENA) in male Munich-Wistar rats subjected to 5/6 nephrectomy. ENA and OMA administered beginning on day 2 after surgery were equally effective in normalizing systolic BP (SBP) and preventing glomerulosclerosis (GS) for 12 wk. Micropuncture studies of rats performed using a similar treatment protocol demonstrated greater reduction of glomerular capillary hydraulic pressure with OMA than with ENA, at similar mean arterial pressures. To investigate whether these glomerular hemodynamic differences might be associated with differences in chronic renoprotective efficacy, additional rats were included in a protocol in which treatment was delayed until 4 wk after surgery (after the onset of hypertension and proteinuria) and continued for a longer period. Both treatments normalized SBP, but OMA resulted in more sustained reduction of proteinuria than did ENA. At week 20, OMA- and ENA-treated rats exhibited less GS than did untreated (control) rats at week 12, but only the difference in control versus OMA values was statistically significant [GS scores: control (12 wk), 36 +/- 4%; ENA (20 wk), 22 +/- 6%; OMA (20 wk), 14 +/- 2%]. The remaining ENA-treated rats were euthanized at 32 wk because of rapidly increasing proteinuria, whereas the remaining OMA-treated rats demonstrated a substantially slower increase in proteinuria until euthanasia at 50 wk. At this extremely late time point, OMA-treated rats exhibited GS scores similar to those of ENA-treated rats at 32 wk and control rats at 12 wk [GS scores: ENA (32 wk), 34 +/- 5%; OMA (50 wk), 38 +/- 8%]. It is concluded that, in this model, OMA affords greater long-term renoprotection than ENA when doses are adjusted to yield equivalent control of SBP.