Tuesday, March 25, 2014

Successful use of miniphotopheresis for the treatment of graft-versus-host disease

Background : Extracorporeal photopheresis (ECP) is an important cell-based therapy for graft-versus-host disease (GVHD); however, the blood volume required per treatment to achieve a clinical response is unknown.
Study Design and Methods : We developed a mini-ECP technique (mini-ECP) using only 100 to 200 mL of whole blood for patients with contraindications for apheresis or low body weight. Sixteen patients (n = 13 acute, n = 3 chronic GVHD) with a median body weight of 19 kg (range, 7-48 kg) received 460 mini-ECP treatments with a median duration of 115 days (range, 49-973 days).
Results : Mini-ECP was well tolerated, and acute GVHD resolved completely in nine of 13 patients and partially in two patients but not in two patients. Cutaneous chronic GVHD exhibited a mixed response (one complete, one partial, and one no response).
Conclusion : These results indicate mini-ECP as a novel and less invasive therapy for patients with GVHD and contraindications for apheresis (read more)

HLA-DRB1 associations in individuals with single and multiple clinically relevant red blood cell antibodies

Background : A minority of red blood cell (RBC) alloantigen–exposed persons form antibodies. Responders are at high risk of developing additional antibodies upon subsequent transfusions. Several studies showed an association between particular HLA-DRB1 phenotypes and the development of specific RBC antibodies. This study evaluates the presence of HLA-DRB1 antigens in individuals with single or multiple RBC antibody specificities to explore whether the response against RBC antigens is associated with a summation of particular HLA-DRB1 susceptibility antigens.
Study Design and Methods : Frequencies of HLA-DRB1 alleles in individuals with antibodies against clinically relevant antigens were compared to a large population cohort to calculate odds ratios (ORs) for alloimmunization to different RBC antigens.
Results : The study cohort consisted of 941 individuals (female-to-male ratio, 3.8) possessing 1462 antibody specificities elicited by transfusion, pregnancy, transplantation, or a combination of these. Besides confirmation of known associations, new associations were identified for anti-E with DRB1*09 and for anti-S with DRB1*07 (ORs, 3.7 and 8.7, respectively). Multiple antibody formation was in a minority of cases associated with the presence of multiple DRB1 susceptibility genes. In multiple responders DRB1*15 was present in almost 40% of cases compared to approximately 25% in single-antibody responders and in the control population.
Conclusion : This study suggests that HLA-DRB1 restriction plays an important role for a first RBC antibody response but multiple antibody formation seems less dependent on the presence of particular HLA restriction genes, while HLA-DRB1*15 may represent a susceptibility phenotype enhancing formation of multiple RBC antibody specificities (read more)

Saturday, March 22, 2014

Expansion of Highly Differentiated Cytotoxic Terminally Differentiated Effector Memory CD8+ T Cells in a Subset of Clinically Stable Kidney Transplant Recipients: A Potential Marker for Late Graft Dysfunction

Despite the effectiveness of immunosuppressive drugs, kidney transplant recipients still face late graft dysfunction. Thus, it is necessary to identify biomarkers to detect the first pathologic events and guide therapeutic target development. Previously, we identified differences in the T-cell receptor Vβ repertoire in patients with stable graft function. In this prospective study, we assessed the long-term effect of CD8+ T-cell differentiation and function in 131 patients who had stable graft function. In 45 of 131 patients, a restriction of TCR Vβ diversity was detected and associated with the expansion of terminally differentiated effector memory (TEMRA; CD45RA+CCR7-CD27-CD28-) CD8+ T cells expressing high levels of perforin, granzyme B, and T-bet. This phenotype positively correlated with the level of CD57 and the ability of CD8+ T cells to secrete TNF-α and IFN-γ. Finally, 47 of 131 patients experienced kidney dysfunction during the median 15-year follow-up period. Using a Cox regression model, we found a 2-fold higher risk (P=0.06) of long-term graft dysfunction in patients who had increased levels of differentiated TEMRA CD8+ T cells at inclusion. Collectively, these results suggest that monitoring the phenotype and function of circulating CD8+ T cells may improve the early identification of at-risk patients (read more)

Pancreatic autoantibodies after pancreas–kidney transplantation – do they matter?

Type 1 diabetes recurrence has been documented in simultaneous pancreas–kidney transplants (SPKT), but this diagnosis may be underestimated. Antibody monitoring is the most simple, noninvasive, screening test for pancreas autoimmune activity. However, the impact of the positive autoimmune markers on pancreas graft function remains controversial. In our cohort of 105 SPKT, we studied the cases with positive pancreatic autoantibodies. They were immunosuppressed with antithymocyte globulin, tacrolimus, mycophenolate, and steroids. The persistence or reappearance of these autoantibodies after SPKT and factors associated with their evolution and with graft outcome were analyzed. Pancreatic autoantibodies were prospectively monitored. Serum samples were collected before transplantation and at least once per year thereafter. At the end of the follow-up (maximum 138 months), 43.8% of patients were positive (from pre-transplant or after recurrence) for at least one autoantibody – the positive group. Antiglutamic acid decarboxylase was the most prevalent (31.4%), followed by anti-insulin (8.6%) and anti-islet cell autoantibodies (3.8%). Bivariate analysis showed that the positive group had higher fasting glucose, higher glycated hemoglobin (HbA1c), lower C-peptide levels, and a higher number of HLA-matches. Analyzing the sample divided into four groups according to pre-/post-transplant autoantibodies profile, the negative/positive group tended to present the higher HbA1c values. Multivariate analysis confirmed the significant association between pancreas autoimmunity and HbA1c and C-peptide levels. Positivity for these autoantibodies pre-transplantation did not influence pancreas survival. The unfavorable glycemic profile observed in the autoantibody-positive SPKT is a matter of concern, which deserves further attention (read more)

Thursday, March 20, 2014

Preformed Donor-Specific Antibodies and Risk of Antibody-Mediated Rejection in Repeat Renal Transplantation

Background : Allograft outcomes in patients undergoing repeat renal transplantation are inferior compared to first-time transplant recipient outcomes. Donor-specific antibodies detected by solid-phase assays (DSA-SPA) may contribute to the worse prognosis. The influence of DSA-SPA on repeat renal transplantation outcomes has not been previously studied in detail.
Design : This study reports the findings in 174 patients who underwent repeat renal transplantation between years 2007 and 2012. These included 62 patients with preformed DSA-SPA detected by Luminex at the time of transplantation. Patients received standard and consistent immunosuppression and were monitored closely for evidence of rejection. Recipients who underwent desensitization were excluded from this analysis. Endpoints included development of biopsy-proven acute rejection and analysis of graft survival and function.
Results : Patients in the DSA-SPA-positive and DSA-SPA-negative groups received similar immunosuppression, and a similar proportion of recipients had a peak panel reactive antibody greater than 20%; the two groups differed with respect to human leukocyte antigen mismatches (4.7±1.1 vs. 4.1±1.7, P=0.024). Recipients with preformed DSA-SPA had higher rejection rates (54.8% vs. 34.8%, P=0.01), including higher rates of antibody-mediated rejection (AMR) (32.3% vs. 7.1%, P<0.001). Recipients who were DSA-SPA-positive and flow cytometry crossmatch (FCXM)-positive had a higher incidence of both AMR (OR 4.6, P=0.009) and of acute rejection (OR 3.57, P=0.02) as compared to those who were DSA-SPA-positive and FCXM-negative. Overall allograft survival was similar in the DSA-SPA-positive and DSA-SPA-negative groups (log-rank test=0.63, P=0.428). Differences in allograft function were detectable after 2 years (32.8±13.1 vs. 47±20.2 mL/min/1.73 m2, P=0.023) and may be reflective of more AMR among DSA-SPA-positive patients.
Conclusions : This analysis suggests that DSA-SPA increases the overall risk of acute rejection but does not appear to adversely impact allograft survival during the early follow-up period. Close monitoring of renal function and early biopsy for AMR detection appear to allow for satisfactory short-term allograft outcomes in repeat transplant recipients (read more)

Antithymocyte Globulin Is Associated With a Lower Incidence of De Novo Donor-Specific Antibodies in Moderately Sensitized Renal Transplant Recipients

Background : Recent evidence suggests that de novo donor-specific antibodies (dnDSA) are associated with antibody-mediated rejection (ABMR) and graft failure after kidney transplantation. The effects of induction immunosuppression on dnDSA are unknown.
Methods : The study population comprised 114 consecutive moderately sensitized (positive DSA and negative flow crossmatch) recipients who received deceased donor renal transplants between December 2009 and November 2011. Patients were divided into two groups based on induction immunosuppression: antithymocyte globulin (ATG) (n=85) or basiliximab (n=29) and were followed up for 36 months.
Results : Patients in the ATG group received a mean dose of 4.98 mg/kg±7.9 mg/kg, had a significantly higher PRA, and received more plasmapheresis and IVIG at the time of transplant. The incidence of dnDSA (P=0.02, HR=0.33, 95% CI 0.09–1.24) and ABMR (P=0.001, HR=0.9, 95% CI 0.04–0.87) was significantly lower in the ATG group. In multivariate regression analyses, ATG induction was the single most important variable associated with both ABMR and dnDSA.
Conclusions : In moderately sensitized deceased donor renal transplant recipients, induction with ATG is associated with a reduction in the occurrence of dnDSA and ABMR when compared with basiliximab (read more)

Tuesday, March 18, 2014

Recombinant blood group proteins facilitate the detection of alloantibodies to high-prevalence antigens and reveal underlying antibodies: results of an international study

Background : Alloantibodies to high-prevalence red blood cell (RBC) antigens are not easily identified by routine serologic techniques. This multicenter study was conducted to test the effectiveness of recombinant blood group proteins (rBGPs) at regional and international RBC reference laboratories.
Study Design and Methods : Single or mixed soluble rBGPs (Lu, Yt, Kn, JMH, Sc, Rg, Ch, Do, and Cr) were assessed for their ability to inhibit the reactivity of antibodies to specific antigens. Initially, the effect of rBGPs was validated by testing panels of well-characterized patient serum samples containing antibodies to high-prevalence antigens in the hemagglutination inhibition assay. Subsequently, the rBGPs were prospectively used for routine antibody identification and the results were compared to those obtained with RBC-based diagnostics.
Results : Panels of predefined antibodies to high-prevalence antigens were completely and specifically neutralized by the corresponding rBGP specificities. For prospective identification, antibodies to high-prevalence antigens (n = 62) were specifically inhibited by the corresponding rBGP specificities except for some Complement Receptor 1–related antibodies, which may be directed to epitopes not expressed on the truncated recombinant Kn. In 14 cases, additional clinically relevant alloantibodies were identified. In cross-matching, the rBGPs were successfully used to inhibit the reactivity of clinically irrelevant antibodies to high-prevalence antigens to determine compatibility between donor and recipient.
Conclusion : rBGPs enable the identification of antibodies to high-prevalence antigens without the need for rare RBC reagents, which are often unavailable. Underlying antibodies can be reliably detected and cross-matching results validated, resulting in a more efficient blood supply for immunized patients (read more)

DQ molecules are the principal stimulators of de novo donor specific antibodies in non sensitized pediatric recipients receiving a first kidney transplant

Data on the different HLA-antibody (Ab) categories in pediatric kidney recipients developing de novo donor-specific Abs (DSA) after transplantation are scarce. We retrospectively evaluated 82 consecutive non-sensitized pediatric recipients of a first kidney graft for de novo HLA Ab occurrence and antigen specificity. At a median follow-up of 6-years, 29% of patients developed de novo DSA while 45% had de novo non-DSA. DSA appeared at 25-month median time post-transplant and were mostly directed towards HLA-DQ antigens. Considering each HLA antigen, the estimated rate of DQ DSA (7.55 per 100 person-years) was much higher than the rates observed for non-DQ DSA. The HLA-DQ Ab recognized determinants of the DQβ chain in 70% of cases, α chain in 25% of cases and both chains in one patient. Non-DSA appeared earlier than DSA, were largely directed against HLA-class I specificities that belonged to HLA -A and -B related cross-reacting epitope groups (CREG) in 56% of cases. Our results indicate a need for evaluating HLA-DQ compatibilities in kidney allocation, in order to minimize post-transplant development of de novoDSA, known to be responsible for antibody mediated rejection and graft loss (read more)

Wednesday, March 12, 2014

Vessel rejection secondary to HLA antibodies directed against the arterial conduit following pancreas transplantation from a separate donor

Whole-organ pancreas transplantation is typically carried out using a Y-graft derived from the donor iliac vessels. We describe a case in which a 31 year old male underwent a simultaneous pancreas-kidney (SPK) transplant, but in which vessels from a different donor were used for the arterial anastomosis of the pancreas graft. Although initially there was good function, 18 months post-transplant the patient was admitted with diabetic ketoacidosis secondary to pancreas graft failure. Radiological investigations revealed complete occlusion of the vascular Y-graft and laboratory investigations demonstrated donor-specific HLA antibodies (DSA) directed against HLA mismatches of the vessel donor (read more)

Monday, March 10, 2014

Regulation of MHC Class I Expression by Foxp3 and Its Effect on Regulatory T Cell Function

Expression of MHC class I molecules, which provide immune surveillance against intracellular pathogens, is higher on lymphoid cells than on any other cell types. In T cells, this is a result of activation of class I transcription by the T cell enhanceosome consisting of Runx1, CBFβ, and LEF1. We now report that MHC class I transcription in T cells also is enhanced by Foxp3, resulting in higher levels of class I in CD4+CD25+ T regulatory cells than in conventional CD4+CD25– T cells. Interestingly, the effect of Foxp3 regulation of MHC class I transcription is cell type specific: Foxp3 increases MHC class I expression in T cells but represses it in epithelial tumor cells. In both cell types, Foxp3 targets the upstream IFN response element and downstream core promoter of the class I gene. Importantly, expression of MHC class I contributes to the function of CD4+CD25+ T regulatory cells by enhancing immune suppression, both in in vitro and in vivo. These findings identify MHC class I genes as direct targets of Foxp3 whose expression augments regulatory T cell function (read more)

Wednesday, March 5, 2014

Red Blood Cell Transfusions and the Risk of Allosensitization in Patients Awaiting Primary Kidney Transplantation

Background : Most studies of HLA sensitization after red blood cell transfusion in transplant candidates were done before widespread use of leukoreduced blood and based on relatively insensitive, nonspecific antibody assays. We evaluated the effect of transfusion on the breadth and magnitude of HLA antibody formation using current, sensitive, HLA-specific immunoassays. 
Methods : Serial HLA antibody data were merged with transfusion data from the US Renal Data System for 1324 patients on the kidney transplant waitlist (2004–2010). Two study groups were identified: a matched cohort consisting of 89 patients who received transfusion and 251 patients who did not receive transfusion and a crossover cohort consisting of 69 patients. Changes in antibody levels and calculated panel-reactive antibody (CPRA) were compared using χ2 and Sign tests, respectively. Logistic regression was used to estimate the relative risk of antibody responses. 
Results : Among the matched cohort, 20% of those who received transfusion compared to 3% of those who did not receive transfusion exhibited an antibody response (P=0.001), whereas in the crossover cohort, 19% exhibited a response in those who received transfusion compared to 1% of those who did not receive transfusion (P=0.0001). Moreover, 26.3% of those who received transfusion had increased CPRA compared to 5.8% of those who did not receive transfusion . These effects were greater in women and blacks compared to men and whites, respectively. Importantly, patients who received transfusion were at an increased risk of a potentially crossmatch positive response (odds ratio=9.6, 95% confidence interval=3.0–30.7). 
Conclusions : Sensitization from transfusion can occur in up to 20% of transplant candidates, resulting in higher antibody levels and CPRA values that adversely impact access to transplantation. These results support transfusion avoidance whenever possible (read more)

Increased Negative Impact of Donor HLA-Specific Together With Non-HLA–Specific Antibodies on Graft Outcome

Background : De novo donor HLA-specific (dnDSA) and non-HLA antibodies including antiangiotensin type 1 receptor antibodies (AT1R-abs) have been associated with antibody-mediated rejection (AMR) and decreased graft survival as well as cellular-mediated rejection (CMR) and early onset of microvasculopathy in heart transplantation. The aim of our study was to determine the impact of anti–AT1R-ab and anti–donor HLA–specific antibody (DSA) on clinical outcomes. 
Methods : Pretransplant and posttransplant sera from 200 recipients transplanted between May 2007 and August 2011 were tested for DSA (Luminex-based single antigen bead assay) and AT1R-ab (enzyme-linked immunosorbent assay). Two cutoff levels (≥17 and ≥12 units) were used to define high and intermediate binding of AT1R-ab. Clinical parameters examined were 5-year AMR/CMR (≥grade 2), coronary artery vasculopathy, and survival. 
Results : At 2 years after transplant, freedom from AMR and/or CMR was 95.4% for those with no DSA (n=175), 66.9% for those with dnDSA (n=19), and 25% for those with DSA at transplant (n=6) (P<0.0001). Neither ≥17 nor ≥12 units of pretransplant levels indicated a significant difference in freedom from AMR and/or CMR. When both dnDSA and AT1R-ab ≥17 or ≥12 units were considered, freedom from AMR and/or CMR decreased to 50% and 45% (P<0.0001), respectively. Coronary artery vasculopathy and survival were not significantly impacted. 
Conclusions : These results show the increased negative impact of dnDSA and AT1R-ab on freedom from AMR and/or CMR and an increased hazard ratio when both parameters are considered. Both HLA- and non-HLA–specific antibodies seem to impact graft outcome in heart transplantation (read more)

HLA-Haploidentical Donor Lymphocyte Infusions for Patients with Relapsed Hematologic Malignancies after Related HLA-Haploidentical Bone Marrow Transplantation

Treatment of relapse after related HLA-haploidentical T cell–replete bone marrow transplantation (haploBMT) with post-transplantation cyclophosphamide (PTCy) using haploidentical donor lymphocyte infusion (haploDLI) is not documented. All patients who received haploDLI after haploBMT with PTCy between June 2003 and October 2012 were identified and assessed for graft-versus-host disease (GVHD) and outcomes. Forty patients received 52 haploDLI doses. Sixteen patients had acute myeloid leukemia, 11 had lymphomas, and 34 had nonmyeloablative conditioning before haploBMT. The median time from haploBMT to relapse was 183 (range, 0 to 1399) days. The median age at haploDLI was 48 (range, 3 to 70) years. The first haploDLI doses were 1 × 105 CD3+ cells/kg with subsequent escalation. The most commonly used first haploDLI dose was 1 × 106 CD3+ cells/kg. The median follow-up after haploDLI was 7 (mean, 15.4; range, .5 to 96) months for the entire cohort, and 17.5 (mean, 28; range, 2.4 to 96) months for the responders. Acute GVHD developed in 10 patients (25%), 6 patients had grade 3 to 4, and 3 developed chronic GVHD. Twelve (30%) patients achieved a complete response (CR) with a median duration of 11.8 (mean, 22.5; range, .4 to 94) months. At last follow-up, 8 responders were alive in CR; 6 for over a year. HaploDLI for relapse after haploBMT is associated with acceptable toxicities and can result in durable responses (read more)

Monday, March 3, 2014

Prevalence, Incidence and Risk Factors for Donor-Specific Anti-HLA Antibodies in Maintenance Liver Transplant Patients

Although large retrospective studies have identified the presence of donor-specific antibodies (DSAs) to be a risk factor for rejection and impaired survival after liver transplantation, the long-term predicted pathogenic potential of individual DSAs after liver transplantation remains unclear. We investigated the incidence, prevalence and consequences of DSAs in maintenance liver transplant (LT) recipients. Two hundred sixty-seven LT recipients, who had undergone transplantation at least 6 months previously and had been screened for DSAs at least twice using single-antigen bead technology, were included and tested annually for the presence of DSAs. At a median of 51 months (min–max: 6–220) after an LT, 13% of patients had DSAs. At a median of 36.5 months (min–max: 2–45) after the first screening, 9% of patients have developed de novo DSAs. The sole predictive factor for the emergence of de novo DSAs was retransplantation (OR 3.75; 95% CI 1.28–11.05, p = 0.025). Five out of 21 patients with de novo DSAs (23.8%) developed an antibody-mediated rejection. Fibrosis score was higher among patients with DSAs. In conclusion, monitoring for the development of DSAs in maintenance LT patients is useful in case of graft dysfunction and to identify patients with a high risk of developing liver fibrosis (read more)

Everolimus Inhibits Anti-HLA I Antibody-Mediated Endothelial Cell Signaling, Migration and Proliferation More Potently Than Sirolimus

Antibody (Ab) crosslinking of HLA I molecules on the surface of endothelial cells triggers proliferative and pro-survival intracellular signaling, which is implicated in the process of chronic allograft rejection, also known as transplant vasculopathy (TV). The purpose of this study was to investigate the role of mammalian target of rapamycin (mTOR) in HLA I Ab-induced signaling cascades. Everolimus provides a tool to establish how the mTOR signal network regulates HLA I–mediated migration, proliferation and survival. We found that everolimus inhibits mTOR complex 1 (mTORC1) by disassociating Raptor from mTOR, thereby preventing class I–induced phosphorylation of mTOR, p70S6K, S6RP and 4E-BP1, and resultant class I–stimulated cell migration and proliferation. Furthermore, we found that everolimus inhibits class I–mediated mTORC2 activation (1) by disassociating Rictor and Sin1 from mTOR; (2) by preventing class I–stimulated Akt phosphorylation and (3) by preventing class I–mediated ERK phosphorylation. These results suggest that everolimus is more effective than sirolimus at antagonizing both mTORC1 and mTORC2, the latter of which is critical in endothelial cell functional changes leading to TV in solid organ transplantation after HLA I crosslinking. Our findings point to a potential therapeutic effect of everolimus in prevention of chronic Ab-mediated rejection (read more)