Wednesday, September 30, 2015

Discarded Human Thymus Is a Novel Source of Stable and Long-Lived Therapeutic Regulatory T Cells

Regulatory T cell (Treg)–based therapy is a promising approach to treat many immune-mediated disorders such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease (GVHD). Challenges to successful clinical implementation of adoptive Treg therapy include difficulties isolating homogeneous cell populations and developing expansion protocols that result in adequate numbers of cells that remain stable, even under inflammatory conditions. We investigated the potential of discarded human thymuses, routinely removed during pediatric cardiac surgery, to be used as a novel source of therapeutic Tregs. Here, we show that large numbers of FOXP3+ Tregs can be isolated and expanded from a single thymus. Expanded thymic Tregs had stable FOXP3 expression and long telomeres, and suppressed proliferation and cytokine production of activated allogeneic T cells in vitro. Moreover, expanded thymic Tregs delayed development of xenogeneic GVHD in vivo more effectively than expanded Tregs isolated based on CD25 expression from peripheral blood. Importantly, in contrast to expanded blood Tregs, expanded thymic Tregs remained stable under inflammatory conditions. Our results demonstrate that discarded pediatric thymuses are an excellent source of therapeutic Tregs, having the potential to overcome limitations currently hindering the use of Tregs derived from peripheral or cord blood (read more)

Evaluation of the iBeads assay as a tool for identifying class I HLA antibodies.

In addition to antibodies targeting native class I human leukocyte antigens (HLA), the single antigen flow beads assay (SAFB) detects antibodies recognizing denatured forms (anti-dHLA). Acid treated SAFB and the modified SAFB reagent named iBeads are expected to distinguish anti-native (anti-nHLA) from anti-dHLA. Sera from 280 class I HLA-sensitized SAFB-positive kidney transplant candidates were retested with acid-treated SAFB and iBeads. Concordance between SAFB and iBeads, taking into account acid-treatment results, was described at global and locus levels. T-lymphocyte flow cytometry crossmatches (FCXM) were performed to identify an accurate iBeads MFI threshold allowing predicting FCXM results. Concordance between acid-treatment and iBeads assays was observed for 86.9% of alleles. The iBeads MFI were lower than for classical SAFB, especially for HLA-B and C alleles. Anti-dHLA identified with acid-treated SAFB were more frequently negative with iBeads for HLA-B and -C alleles. An iBeads MFI threshold of 1000 allowed predicting positive FCXM with 95.6% sensitivity, 91.6% negative predictive value and 0.08 negative likelihood ratio. The iBeads assay still has limitations, but might represent an invaluable alternative to SAFB for virtual crossmatch strategies in organ transplant allocation programs (read more)

Tuesday, September 22, 2015

Donor-specific antibody to trans-encoded donor HLA-DQ heterodimer

The majority of de novo donor specific HLA antibodies (DSAs) in transplant patients are directed to HLA-DQ antigens, which consist of a heterodimer of alpha and beta chains. Although a heterodimer can theoretically be cis- or trans-encoded, the sensitizing forms appear to be generally cis-forms. DSA to DQ trans-heterodimer has never been reported. We reviewed 360 post-kidney transplant recipients (transplant: 2002-2013; follow-up: 5.6±3.3years). DQ DSA was detected in 46 of 57 patients who developed DSA. DSA specificity was consistent with donor mismatched DQ trans-heterodimers in three patients: DQ2.5 (DQB1*02, DQA1*05), DQ2.3 (DQB1*02, DQA1*03), and DQ4.3 (DQB1*04, DQA1*03). Two of them eventually lost grafts (2 and 5years later) with allograft nephropathy. In conclusion, post-transplant patients may develop DSA to donor DQ trans-heterodimers. Further studies are warranted to determine the clinical significance of such DSAs (read more)