Novel GMP-Compatible Protocol Employing an Allogeneic B Cell Bank for Clonal Expansion of Allospecific Natural Regulatory T Cell

The adoptive transfer of natural regulatory T cells (nTreg) is a new option to reshape undesired immune reactivity in autoimmunity and transplantation toward “tolerance.” The first clinical trials using adoptive transfer of polyclonal nTreg demonstrated safety and hints of efficacy. However, the low frequencies of antigen-specific cells among the pool of polyclonal nTreg and their broad antigen nonspecific suppression are limitations of this approach regarding efficacy and safety. Recently, the isolation and expansion of (allo)antigen-specific nTreg have successfully been achieved by using Treg-specific activation markers but the yield is relatively low. Here, we describe a novel good manufacturing practice (GMP)-compatible expansion protocol of alloantigen-specific nTreg based on the stimulation of nTreg by allogeneic activated B cells. Their functionality and specificity are superior compared to polyclonal nTreg both in vitro and in vivo. Employing an allogeneic B cell bank, designed to cover the majority of HLA types, allows fast GMP-compliant manufacturing for donor-specific nTreg for clinical application in organ and stem cell transplantation. TCR repertoire analyses by next generation sequencing revealed impressive expansion by several log-steps of even very low-abundance alloantigen-specific nTreg clones. This novel method offers a simple approach for expanding antigen-specific nTreg and is characterized by high replicability and easy transferability to full GMP standards (read more)

Influence of preoperative anti-HLA antibodies on short- and long-term graft survival in recipients with or without rituximab treatment

We investigated the relationship between preoperative anti-HLA antibodies (DSA) and the graft survival rate in recipients who had or had not received rituximab (Rit) treatment. The subjects were categorized into four groups as follows: DSA+Rit-, n = 39; DSA-Rit-, n = 121; DSA+Rit+, n = 74; and DSA-Rit+, n = 47. We examined the influence of preoperative DSA on the incidence of graftrejection and the survival rate of recipients who had or who had not received rituximab before transplantation. The 6-month acute rejection rates based on graft biopsies were 39%, 19%, 15%, and 0% for the DSA+Rit-, DSA-Rit-, DSA+Rit+, and DSA-Rit+ groups. The rates of chronic antibody-mediated rejection after more than 6 months were 50%, 22%, 18%, and 0%. The 5-yeargraft survival rate was significantly lower in the DSA+Rit- group (84%) than in the other groups (95% for DSA-Rit-, 98% for DSA+Rit+, and 91% for DSA-Rit+). The rate of the appearance of de novoanti-HLA antibodies was higher in the groups that did not receive rituximab treatment. The rate ofgraft loss associated with chronic antibody-mediated rejection was also higher in the DSA+Rit- group than in the other groups (P=0.01). The presence of DSA and the administration of rituximabhad strong impacts on not only short-term graft rejection, but also long-term graft rejection and its association with the graft survival time (read more)

Translating in vitro prediction of cytotoxic T cell alloreactivity to hematopoietic stem cell transplantation outcome

INTRODUCTION: Previously we developed a weighted amino acid (AA) mismatch score predictive for cytotoxic T cell (CTL) alloreactivity (in vitro CTLp assay) based on the structure of the HLA class I molecule. The aim of this study is to confirm the clinical relevance of the CTLp assay and to validate the AA mismatch score as an alternative and easy to use tool to predict permissible mismatches in hematopoietic stem cell transplantation (HSCT).
METHODS: We selected patients transplanted with a 9/10 single HLA class I mismatched graft (n=171) at three Dutch HSCT centers. A CTLp assay was performed in 73 donor-recipient pairs. As a control we selected 168 10/10 HLA matched pairs that were matched to the 9/10 single HLA class I mismatched pairs for HSCT year, donor type, patient age and diagnosis.
RESULTS: We observed that pairs with negative a CTLp assay had statistically significant decreased incidence of mortality after HSCT comparable to that of 10/10 HLA matched pairs. However, the weighted AA mismatch score did not significantly predict any HSCT end point of interest.
CONCLUSION: Further investigation is needed to unravel the mechanisms involved in causing the beneficial effect of a negative CTLp assay, before other alternative tools to predict HSCT outcome may be developed (read more)

ABO antibody and complement depletion by immunoadsorption combined with membrane filtration--a randomized, controlled, cross-over trial

BACKGROUND: Potent antibody depletion techniques have paved the way to successful ABO-incompatible transplantation. Considering its efficiency regarding IgG removal, the use of non-antigen-specific semi-selective immunoadsorption (IA) has been advocated. One attractive strategy to overcome the caveat of incomplete IgM depletion and to interfere with complement activation could be the adjunctive use of membrane filtration (MF) to enhance the removal of macromolecules.
METHODS: To investigate the depletion efficiency of semi-selective IA plus MF, we conducted a randomized, controlled, cross-over trial including patients on regular IA treatment for indications outside recipient desensitization. According to the results of sample size calculation, 14 subjects were enrolled. Two treatment sequences, a single session of IA plus MF followed by IA alone after ≥7 days (and vice versa), were analysed.
RESULTS: IA plus MF markedly enhanced the median per cent reduction of ABO-specific IgM determined by flow cytometry (primary end point; 59 versus 23%, P < 0.001) and haemagglutination (2 versus 1 titre steps, P < 0.001), respectively. Combined treatment also substantially lowered C1q concentrations (86 versus 58% reduction, P < 0.001) and the functionality of classical complement as reflected by impaired in vitro C3 activation capability. IgG was strongly reduced without any additional effect of MF.
CONCLUSIONS: We demonstrate that the innovative strategy of combining MF with semi-selective IA may substantially increase IgM elimination and affect classical complement activation. Our findings suggest that this new treatment concept could be an efficient strategy for recipient desensitization in ABO- and HLA-incompatible transplantation (read more)

ABO-incompatible (ABO-i) kidney transplantation (KT) has emerged for overcoming the shortage of organ donors. Although this technique initially achieved only low graft survival due to isoagglutinin, recently developed desensitization protocols have improved survival to levels that are comparable to ABO-compatible KT. However, isoagglutinin is still regarded as a major obstacle to ABO-i KT. In this study, we evaluate the impact of isoagglutinin titer on clinical outcomes as well as factors that may influence isoagglutinin titers. In total, data from 95 patients who underwent ABO-i KT were analyzed. Preoperatively, rituximab administration and plasmapheresis were performed until the titer was reduced to ≤1:4. Retrospective analysis included blood group; timing and dosage of rituximab; isoagglutinin titer; number of plasmapheresis; and clinical outcomes including graft survival and serum creatinine. Graft survival was 95.8% (n = 91) and average serum creatinine at 1- and 1.5-year post-ABOi-KT was 1.3. Three patients died of sepsis. The identified predictors of titer-rebound after transplant were short interval (<7 days) between rituximab and first plasmapheresis (P = 0.004); high initial titer (≥256) (P = 0.023); low titer-reduction rate (P < 0.001); and blood group O (P < 0.001). One patient who experienced a rebound developed antibody-mediated rejection. With low-dose (200 mg) rituximab, the change in isoagglutinin titer-rebound was not significant and the infection rate was significantly decreased (P = 0.001). In conclusion, isoagglutinin titer-rebound within the first 2 weeks after KT may be a risk factor for rejection. The factors identified as affecting titer-rebound after KT were high initial isoagglutinin titer, low titer-reduction rate, short interval, and blood group O (read more)