Regulatory T cells in kidney transplant recipients: the effect of induction immunosuppression therapy

Background. Regulatory T cells have been suggested to down-regulate the alloimmune response. The aim of this prospective open study was to evaluate the effects of different inductive agents on peripheral blood regulatory T cells in kidney transplant patients and to analyse their association with short-term graft outcome.
Methods. Regulatory and effector T cell numbers in peripheral blood were determined by flow cytometry in 71 prospectively followed kidney transplant recipients at postoperative day 0, 7, 14, 21, 28, 60 and 90. Patients were treated with a calcineurin inhibitor-based triple immunosuppression with polyclonal rabbit anti-thymocyte globulin (rATG, n = 28), basiliximab, the anti-CD25 monoclonal antibody (n = 18) or without induction (controls, n = 25). Flow cytometry data were correlated to rejection incidence.
Results. Compared to controls, CD4⁺CD25⁺FoxP3⁺ regulatory T-cell expansion among CD4⁺ T cells was noticed in the rATG group at all post-transplant time-points by Day 14 (P < 0.001). A significant decrease in Treg frequency (P < 0.001) and concurrently a transient increase of CD4⁺CD25^low/–FoxP3⁺ population were observed in basiliximab-treated patients 7–60 days post-transplantation. Biopsy-proven acute rejection occurred in 16.7% of controls, 10.7% of the rATG group and in 11.1% of the basiliximab group. Higher CD4⁺FoxP3⁺/CD8⁺CD45RA⁺CD62L^– ratios were observed repeatedly in those patients after basiliximab induction who were rejection free (P < 0.01).
Conclusions. In this study, the rATG induction therapy was associated with an expansion of regulatory cells. Sustained high CD4⁺FoxP3⁺/Teff ratios were associated with the absence of rejection after basiliximab induction (read more).

Trends in immune function assay (ImmuKnow; Cylex™) results in the first year post-transplant and relationship to BK virus infection

Background. The ImmuKnow assay is a functional T-cell assay (TCA) that may quantify cellular immune responsiveness following renal transplantation. Using a standard protocol of TCA sampling in the first year post-transplant, we examined changes in TCA values over time and tested for an association between TCA and BK virus (BKV) infection as a marker of over-immunosuppression.
Methods. We performed a single-center retrospective analysis of 897 TCA results in 414 renal transplant recipients obtained at 0 (N = 122), 1 (N = 316), 6 (N = 258) and 12 (N = 201) months post-transplant from May 2005 to July 2009 with concurrent urine and blood BKV polymerase chain reaction measurements.
Results. Nearly 40% of patients experienced a decrease in TCA of >150 ng/mL from 1 to 6 months (mean 466–356 ng/mL, P < 0.0001) and remained stable from 6 to 12 months (mean 357 versus 370 ng/mL, P = 0.33). Neither a change in TCA of >150 ng/mL nor a TCA value of ≤225 ng/mL were associated with a diagnosis of BKV infection at 1 or 6 months, while TCA ≤225 ng/mL was associated with BKV infection at 12 months (P = 0.005).
Conclusions. A reduction in TCA from 1 to 6 months post-transplant is common and is not associated with conditions of over-immunosuppression, rendering the interpretation of changes in TCA during this time period difficult. BKV infection is associated with low TCA values at 12 months, suggesting that patients with low TCA values after 6 months may benefit from potential tailoring of immunosuppression or more aggressive monitoring to prevent subsequent BKV infection (read more).

Localization of Mesenchymal Stromal Cells Dictates Their Immune or Proinflammatory Effects in Kidney Transplantation

Multipotent mesenchymal stromal cells (MSC) have recently emerged as promising candidates for cell-based immunotherapy in solid-organ transplantation. However, optimal conditions and settings for fully harnessing MSC tolerogenic properties need to be defined. We recently reported that autologous MSC given posttransplant in kidney transplant patients was associated with transient renal insufficiency associated with intragraft recruitment of neutrophils and complement C3 deposition. Here, we moved back to a murine kidney transplant model with the aim to define the best timing of MSC infusion capable of promoting immune tolerance without negative effects on early graft function. We also investigated the mechanisms of the immunomodulatory and/or proinflammatory activities of MSC according to whether cells were given before or after transplant. Posttransplant MSC infusion in mice caused premature graft dysfunction and failed to prolong graft survival. In this setting, infused MSC localized mainly into the graft and associated with neutrophils and complement C3 deposition. By contrast, pretransplant MSC infusion induced a significant prolongation of kidney graft survival by a Treg-dependent mechanism. MSC-infused pretransplant localized into lymphoid organs where they promoted early expansion of Tregs. Thus, pretransplant MSC infusion may be a useful approach to fully exploit their immunomodulatory properties in kidney transplantation (read more).

Graft invariant natural killer T-cell dose predicts risk of acute graft-versus-host disease in allogeneic hematopoietic stem cell transplantation

Invariant natural killer T (iNKT) cells are powerful immunomodulatory cells that in mice regulate a variety of immune responses, including acute GVHD (aGVHD). However, their clinical relevance and in particular their role in clinical aGVHD are not known. We studied whether peripheral blood stem cell (PBSC) graft iNKT-cell dose affects on the occurrence of clinically significant grade II-IV aGVHD in patients (n = 57) undergoing sibling, HLA-identical allogeneic HSCT. In multivariate analysis, CD4^– iNKT-cell dose was the only graft parameter to predict clinically significant aGVHD. The cumulative incidence of grade II-IV aGVHD in patients receiving CD4^– iNKT-cell doses above and below the median were 24.2% and 71.4%, respectively (P = .0008); low CD4^– iNKT-cell dose was associated with a relative risk of grade II-IV aGVHD of 4.27 (P = .0023; 95% CI, 1.68-10.85). Consistent with a role of iNKT cells in regulating aGVHD, in mixed lymphocyte reaction assays, CD4^– iNKT cells effectively suppressed T-cell proliferation and IFN- secretion in a contact-dependent manner. In conclusion, higher doses of CD4^– iNKT cells in PBSC grafts are associated with protection from aGVHD. This effect could be harnessed for prevention of aGVHD (read more).

Proteinuria after kidney transplantation

The prevalence of proteinuria at 1 year after renal transplantation ranges between 11% and 45% and is even higher in patients treated with inhibitors of the mammalian target of rapamycin (mTOR). Two main mechanisms can lead to proteinuria: an inadequate reabsorption of small proteins from proximal tubular cells damaged by ischemia-reperfusion injury, rejection, or toxic agents (tubular proteinuria) or an increased passage of albumin and/or protein with higher molecular weight (MW) because of a disruption of glomerular barrier caused by recurrent or de novo glomerulonephritis, transplant glomerulopathy, chronic rejection, or CNI toxicity (glomerular proteinuria). Proteinuric patients have worse patient and graft survival rates in comparison to non proteinuric patients. The amount of proteinuria is a reliable predictor of the allograft outcome. However, even microalbuminuria may be associated with a poor outcome. Treatment of proteinuria mainly rests on the management of the etiologic cause. Inhibitors of renin-angiotensin system (RAS) are useful in reversing microalbuminuria and can reduce proteinuria, but their efficacy in interfering with patient or graft survival is not demonstrated (read more).

HHV-6B is frequently found in the gastrointestinal tract in kidney transplantation patients

In immunosuppressed patients human herpesvirus 6 (HHV-6) reactivations are common. The aim of the study was to determine to which extent HHV-6 can be found in the gastrointestinal tract in kidney transplant recipients and in patients on chronic dialysis. The HHV-6 and cytomegalovirus (CMV) examinations were performed on gastro duodenal and colon biopsy specimens obtained from 81 kidney transplant recipients and on 46 chronic dialysis patients. The HHV-6 and CMV were demonstrated by immunohistochemistry detecting both HHV-6A and HHV-6B, and CMV-specific antigens. The HHV-6B-positive cells, were found in gastroduodenal biopsy specimens from 34% of the transplant recipients and 28% of the patients on chronic dialysis, CMV-positive cells were found in specimens from 53% of the transplant recipients and 28% of the patients on chronic dialysis. The HHV-6B positive cells were found in the colonic mucosa specimens from 36% of the transplant recipients and 22% of the patients on chronic dialysis, CMV-positive cells were found in specimens from 36% of the transplant recipients and 17% of the patients on chronic dialysis. The HHV-6B positive cells were found equally often in the gastroduodenal as in the colorectal mucosa. The HHV-6B positive cells as well as CMV positive cells were simultaneously found in every fifth of transplant recipients (read more).

Human leukocyte antigen epitope analysis to assess complement- and non-complement-binding donor-specific antibody repertoire in a pediatric heart transplant recipient.

This case report summarizes the spectrum of anti-human leukocyte antigen (HLA) antibody reactivity determined by single-allele Luminex immunoglobulin G and C1q binding assays before transplant, during an episode of antibody-mediated rejection (AMR), and following treatment in a sensitized pediatric heart transplant (Tx) recipient. We were able to discriminate between complement- and non-complement-binding epitope-specific antibodies present against a single donor antigen (HLA-A2) during the progression of AMR and its resolution. Our findings illustrate the usefulness of determining antibody specificities against epitopes using various Luminex-based assays (read more)

Adsorption of chain type–specific ABO antibodies on Sepharose-linked A and B tetrasaccharides

BACKGROUND: Antigen-specific removal of anti-A and anti-B on immunoadsorption columns carrying the blood group A and B trisaccharides is one important component of some protocols used in ABO-incompatible organ transplantation. Because ABO antibodies exist requiring parts of the core saccharide chain for binding, the anti-A and -B–binding capacity of individual and combined, Sepharose-linked Types 1 through 4 A and B tetrasaccharides with that of the A and B trisaccharides was compared.

STUDY DESIGN AND METHODS: Sepharose-linked A and B tri- and tetrasaccharides were used to adsorb anti-A and -B from pooled blood group O serum. Remaining chain type–specific anti-A and -B were detected and quantified in enzyme-linked immunosorbent assays using wells coated with neoglycoproteins or recombinant mucins carrying A and B determinants on defined core saccharide chains.

RESULTS: Significantly more anti-A Type 3- and 4-specific immunoglobulin (Ig)G remained after adsorption on the A trisaccharide and the A Type 1 and A Type 2 tetrasaccharide than after adsorption on the A Types 3 and 4 tetrasaccharides. Selective adsorption of chain type–specific IgG anti-B was detected on Sepharose-linked B tetrasaccharides. In contrast, there were no chain type–specific IgM anti-A or -B. A combination of the A or B tetrasaccharides adsorbed a larger fraction of the IgG anti-A and -B repertoires than the corresponding trisaccharides.

CONCLUSION: There are chain type–specific anti-A and anti-B IgG, and an adsorber based on a combination of Types 1 through 4 A or B tetrasaccharides will be a more efficient adsorber than an adsorber based on the A or B trisaccharides (read more).

Interaction of the humoral immune system with peptides presented by class I major histocompatibility (MHC) complexes [B Lymphocyte Signaling and Transcription]

Antibodies usually bind to unprocessed antigens, while cytotoxic T cells react with peptides derived from intracellular antigens when presented by class I major histocompatibility (MHC) complexes. We screened human sera for antibodies reacting specifically with the influenza matrix protein (IMP) derived peptide (58-66) displayed by HLA-A*0201 complexes by ELISA. Among blood donors, high-titered HLA-A*0201/IMP (58-66) complex-specific IgG antibodies were detected in HLA-A*0201- females with a history of pregnancies. Extended analyses of specificity indicated that these antibodies interacted peptide-specific with the MHC complex. No antibodies were detected in HLA-A*0201+ female or male blood donors. In another cohort of 218 females on delivery, only HLA-A*0201- mothers had HLA-A*0201/IMP (58-66) antibodies, which were also detected in the cord blood of the newborns, demonstrating that HLA-A*0201/ IMP58-66 antibodies are produced in HLA-A*0201- mothers and enter the fetal blood system. Refolding the peptide IMP (58-66) with all HLA-A, -B and -Cw alleles of the mothers and newborns revealed, that these antibodies are allo-reactive and are binding in some cases to the peptide displayed by a MHC allele of an offspring. Therefore allo-MHC/IMP (58-66) antibodies might provide immunity in HLA-disparate pregnancies. These antibody responses specific to a peptide displayed by a class I MHC complex opens a new dimension of interactions between the cellular and humoral immune system (read more).

Mesenchymal Stromal Cells: A New Tool against Graft-versus-Host Disease?

Mesenchymal stromal cells (MSCs) represent a heterogeneous subset of multipotent cells that can be isolated from several tissues including bone marrow and fat. MSCs exhibit immunomodulatory and anti-inflammatory properties that prompted their clinical use as prevention and/or treatment for severe graft-versus-host disease (GVHD). Although a number of phase I-II studies have suggested that MSC infusion was safe and might be effective for preventing or treating acute GVHD, definitive proof of their efficacy remains lacking thus far. Multicenter randomized studies are ongoing to more precisely assess the impact of MSC infusion on GVHD prevention/treatment, whereas further research is performed in vitro and in animal models with the aims of determining the best way to expand MSCs ex vivo as well as the most efficient dose and schedule of MSCs administration. After introducing GVHD, MSC biology, and results of MSC infusion in animal models of allogeneic hematopoietic cell transplantation, this article reviews the results of the first clinical trials investigating the use of MSC infusion as prevention or treatment of GVHD (read more).

Antibody-Mediated Rejection of Single Class I MHC-Disparate Cardiac Allografts

Murine CCR5^−/− recipients produce high titers of antibody to complete MHC-mismatched heart and renal allografts. To study mechanisms of class I MHC antibody-mediated allograft injury, we tested the rejection of heart allografts transgenically expressing a single class I MHC disparity in wild-type C57BL/6 (H-2^b) and B6.CCR5^−/− recipients. Donor-specific antibody titers in CCR5^−/− recipients were 30-fold higher than in wild-type recipients. B6.K^d allografts survived longer than 60 days in wild-type recipients whereas CCR5^−/− recipients rejected all allografts within 14 days. Rejection was accompanied by infiltration of CD8 T cells, neutrophils and macrophages, and C4d deposition in the graft capillaries. B6.K^d allografts were rejected by CD8^−/−/CCR5^−/−, but not μMT^−/−/CCR5^−/−, recipients indicating the need for antibody but not CD8 T cells. Grafts recovered at day 10 from CCR5^−/− and CD8^−/−/CCR5^−/− recipients and from RAG-1^−/− allograft recipients injected with anti-K^d antibodies expressed high levels of perforin, myeloperoxidase and CCL5 mRNA. These studies indicate that the continual production of antidonor class I MHC antibody can mediate allograft rejection, that donor-reactive CD8 T cells synergize with the antibody to contribute to rejection, and that expression of three biomarkers during rejection can occur in the absence of this CD8 T cell activity (read more).

Clinically significant anti-A1 in a presumed ABO-identical hematopoietic stem cell transplant recipient: a case report

BACKGROUND: Subgroups of the blood group A (ABO) are generally not considered ABO incompatible for hematopoietic progenitor cell (HPC) transplant.

CASE REPORT: A 54-year-old female presented for HPC transplantation for acute leukemia. No HLA-matched donor was identified, so she received a peripheral blood stem cell graft from an HLA-mismatched unrelated donor. On pretransplant testing, both the donor and the recipient typed as blood group A. On Day +67 after transplant, the recipient had a transfusion reaction consisting of an increase in temperature, rigors, and shaking chills during infusion of a unit of group A red blood cells (RBCs). A transfusion reaction workup revealed an ABO discrepancy with both anti-A (1+) and anti-B (3+) identified in the patient's serum as well as a positive direct antiglobulin test with monoclonal anti-IgG antisera. Anti-A₁ were identified serologically and in an eluate. Hemolysis was clinically significant, requiring blood transfusion. No ABO typing discrepancies were found on pretransplant testing in either the recipient or the donor. DNA sequencing for blood group A subgroups performed after the transfusion reaction on blood collected before the transplant showed the donor to be type A₁ and the recipient as A₂. Unfortunately, the patient experienced graft failure requiring reconditioning and reinfusion of additional cells from the original HPC donor. On Day +94 after the second transplant, the patient died with severe acute gastrointestinal graft-versus-host disease.

CONCLUSION: This report describes a blood group A₂ patient who developed an anti-A₁ causing clinically significant hemolysis after HPC transplant from an A₁ donor (read more).

Estimating Baseline Kidney Function in Hospitalized Patients with Impaired Kidney Function

Background and objectives : Inaccurate determination of baseline kidney function can misclassify acute kidney injury (AKI) and affect the study of AKI-related outcomes. No consensus exists on how to optimally determine baseline kidney function when multiple preadmission creatinine measurements are available.
Design, setting, participants, & measurements : The accuracy of commonly used methods for estimating baseline serum creatinine was compared with that of a reference standard adjudicated by a panel of board-certified nephrologists in 379 patients with AKI or CKD admitted to a tertiary referral center.
Results : Agreement between estimating methods and the reference standard was highest when using creatinine values measured 7–365 days before admission. During this interval, the intraclass correlation coefficient (ICC) for the mean outpatient serum creatinine level (0.91 [95% confidence interval (CI), 0.88–0.92]) was higher than the most recent outpatient (ICC, 0.84 [95% CI, 0.80–0.88]; P<0.001) and the nadir outpatient (ICC, 0.83 [95% CI, 0.76–0.87; P<0.001) serum creatinine. Using the final creatinine value from a prior inpatient admission increased the ICC of the most recent outpatient creatinine method (0.88 [95% CI, 0.85–0.91]). Performance of all methods declined or was unchanged when the time interval was broadened to 2 years or included serum creatinine measured within a week of admission.
Conclusions : The mean outpatient serum creatinine measured within a year of hospitalization most closely approximates nephrologist-adjudicated serum creatinine values (read more).

Transfusion-associated graft-versus-host disease in a liver transplant recipient: an unusual presentation and review of the literature

BACKGROUND: Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare, nearly universally fatal complication from transfusion of nonirradiated cellular blood components, occurring when a recipient's immune system is unable to recognize and destroy transfused T lymphocytes. Irradiation of cellular components eliminates this risk. We present an unusual case of a liver transplant recipient developing TA-GVHD 13 weeks after transfusion of a random unit of nonirradiated red blood cells (RBCs) that happened to be from a donor homozygous for an HLA haplotype shared by the patient.

STUDY DESIGN AND METHODS: This study was a single case review of a liver transplant recipient who developed skin GVHD and marrow aplasia. Clinical course and the chimerism studies involving the patient, the liver donor, and the blood donor are detailed.

RESULTS: The patient presented 3 months posttransplant with GVHD of his skin and marrow aplasia. In addition to standard antigraft immunosuppression, this patient had started the interleukin-1 receptor antagonist anakinra on Posttransplant Day 13 for an acute gout flare. Chimerism studies on the patient's peripheral blood identified a population of CD3 cells that did not originate with either the patient or his liver donor. HLA studies and microsatellite profiling of the unknown CD3 population identified the source of the patient's TA-GVHD, a unit of nonirradiated, nonleukoreduced apheresis RBCs.

CONCLUSION: Use of an immunomodulating agent may have contributed to the development of TA-GVHD in a liver transplant patient who received a random unit of nonirradiated RBCs by chance from an unrelated haploidentical donor (read more).

Antibodies to K-α 1 Tubulin and Collagen V Are Associated With Chronic Rejection After Lung Transplantation

Bronchiolitis obliterans syndrome (BOS), the clinical correlate of chronic rejection after lung transplantation, is the leading obstacle to better long-term outcomes. We previously instituted a clinical protocol to screen for donor-specific human leukocyte antigen (HLA) antibodies (DSA) and a preemptive antibody-directed therapy protocol consisting of rituximab and/or intravenous immune globulin. In this study, we retrospectively analyzed serum samples from lung transplant recipients (n = 108) for antibodies to self-antigens (K-α 1 tubulin and collagen V) before and after antibody-directed therapy and correlated the results with the subsequent development of BOS. Seventy-two of the 108 recipients developed antibodies to self-antigens. There was a correlation between the development of antibodies to self-antigens and DSA. Sixteen of the 54 patients who had antibodies to self-antigens and were treated with antibody-directed therapy cleared the antibodies, and they were significantly less likely to develop BOS than those who had persistent antibodies. Furthermore, those who cleared DSA after treatment but had persistent antibodies to self-antigens were significantly more likely to develop BOS than those who cleared these antibodies. We conclude that antibodies to self-antigens are an important risk factor for the development of BOS (read more).