Clinical grade multiparametric cell sorting and gene-marking of regulatory T cells.

BACKGROUND AIMS: Regulatory T cells (Tregs) are the main mediators of peripheral tolerance. Treg-directed therapy has shown promising results in preclinical studies of diverse immunopathologies. At present, the clinical applicability of adoptive Treg transfer is limited by difficulties in generating Tregs at sufficient cell dose and purity. METHODS: We developed a Good Manufacturing Practice (GMP) compliant method based on closed-system multiparametric Fluorescence-Activated Cell Sorting (FACS) to purify Tregs, which are then expanded in vitro and gene-marked with a clinical grade retroviral vector to enable in vivo fate tracking. Following small-scale optimization, we conducted four clinical-scale processing runs. RESULTS: We showed that Tregs could be enriched to 87- 92% purity following FACS-sorting, and expanded and transduced to yield clinically relevant cell dose of 136-732×106 gene-marked cells, sufficient for a cell dose of at least 2 × 106 cells/kg. The expanded Tregs were highly demethylated in the FOXP3 Treg-specific demethylated region (TSDR), consistent with bona fide natural Tregs. They were suppressive in vitro, but a small percentage could secrete proinflammatory cytokines, including interferon-γ and interleukin-17A. CONCLUSIONS: This study demonstrated the feasibility of isolating, expanding and gene-marking Tregs in clinical scale, thus paving the way for future phase I trials that will advance knowledge about the in vivo fate of transferred Tregs and its relationship with concomitant Treg-directed pharmacotherapy and clinical response.

Safety and feasibility of apheresis to harvest and concentrate parasites from subjects with induced blood stage Plasmodium vivax infection.

BACKGROUND: In the absence of a method to culture Plasmodium vivax, the only way to source parasites is ex vivo. This hampers many aspects of P. vivax research. This study aimed to assess the safety of apheresis, a method for selective removal of specific components of blood as a means of extracting and concentrating P. vivax parasites. METHODS: An iterative approach was employed across four non-immune healthy human subjects in single subject cohorts. All four subjects were inoculated with ~ 564 blood stage P. vivax (HMP013-Pv) and subjected to apheresis 10 to 11 days later. Blood samples collected during apheresis (haematocrit layers 0.5% to 11%) were tested for the presence and concentration of P. vivax by microscopy, flow cytometry, 18S rDNA qPCR for total parasites, and pvs25 qRT-PCR for female gametocyte transcripts. Safety was determined by monitoring adverse events. Malaria transmission to mosquitoes was assessed by membrane feeding assays. RESULTS: There were no serious adverse events and no significant safety concerns. Apheresis concentrated asexual parasites by up to 4.9-fold (range: 0.9-4.9-fold) and gametocytes by up to 1.45-fold (range: 0.38-1.45-fold) compared to pre-apheresis densities. No single haematocrit layer contained > 40% of all the recovered P. vivax asexual parasites. Ex vivo concentration of parasites by Percoll gradient centrifugation of whole blood achieved greater concentration of gametocytes than apheresis. Mosquito transmission was enhanced by up to fivefold in a single apheresis sample compared to pre-apheresis. CONCLUSION: The modest level of parasite concentration suggests that the use of apheresis may not be an ideal method for harvesting P. vivax. Trial Registration Australia New Zealand Clinical Trials Registry (ANZCTR) Trial ID: ACTRN12617001502325 registered on 19th October 2017. https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=373812.

Peripheral vascular access for therapeutic plasma exchange: A practical approach to increased utilization and selecting the most appropriate vascular access.

BACKGROUND: Therapeutic plasma exchange (TPE) is used in the treatment of many diseases. At present, peripheral vascular access (PVA) is an underutilized method of vascular access in TPE. It should be considered more frequently due its relatively low risk for adverse events, particularly infections. METHODS: The Advancing Vascular Access in Apheresis Working Group met in December 2017 for an extensive review and discussion of vascular access for TPE and developed a "road map" providing detailed information regarding clinical situations in which PVA-based TPE would and would not be appropriate. RESULTS: The road map is consistent with current recommendations that PVA should be used in combination with TPE whenever possible. PVA should be considered for patients who do not have existing central lines and who are stable. The patient should have peripheral veins that will allow for adequate treatment and must be able to comply with the process of achieving and maintaining peripheral access. There should be expert clinical assessment of veins, and this evaluation may include ultrasound and/or near infrared evaluation. Conditions that would prompt a switch from PVA to an alternate method of venous access include loss of venous access, patient preference, or development of a requirement for very frequent treatment over a long period of time. CONCLUSIONS: While PVA is not suitable for all patients requiring TPE, it has significant safety advantages over other approaches and should be employed whenever possible.

Effect of plasmapheresis on ATG (Thymoglobulin) clearance prior to adoptive T cell transfer.

The effect of anti-thymocyte globulin (ATG) on the outcome of hematopoietic stem cell transplantation (SCT) is dependent on formulation, dose and exposure. However, ATG levels are not routinely measured and therapeutic levels are not well defined. In ex vivo T cell-deplete SCT, the potential effect of residual ATG has important implications on the timing of adoptive T cell transfer. Here we measured active rabbit ATG concentration using a flow cytometry-based method that can be implemented in any laboratory. Three adult patients received 6 mg/kg Thymoglobulin over 4 days, leading to peak plasma active ATG concentration of 20.8 ± 1.4 µg/mL, suggesting volume of distribution of 16-19 L. The half-life of active ATG was 6.1 ± 0.7 days and plasmapheresis at Day 25 ± 1 post-transplant reduced mean plasma concentration from 1.25 to 0.61 µg/mL. Total ATG and active ATG do not have a constant relationship because of differences in volumes of distribution and half-lives. Thymoglobulin can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro at concentrations as low as 0.03 µg/mL, with a log-linear relationship between ATG concentration and ADCC. Plasmapheresis can remove ATG but likely has modest biological impact when performed 4 weeks after 6 mg/kg ATG.

Outcomes after major or bidirectional ABO-mismatched allogeneic hematopoietic progenitor cell transplantation after pretransplant isoagglutinin reduction with donor-type secretor plasma with or without plasma exchange.

BACKGROUND: Major ABO mismatch in hematopoietic progenitor cell transplantation (HPCT) is associated with a range of immunohematologic consequences including progenitor cell infusion (PCI)-related hemolysis, delayed red blood cell engraftment, and pure red cell aplasia (PRCA). Although pretransplant (recipient) isoagglutinin reduction may be associated with decreased immunohematologic complications in this setting, there is no consensus with respect to strategies for isoagglutinin reduction. STUDY DESIGN AND METHODS: This observational study assessed the efficacy of a standardized pretransplant isoagglutinin reduction strategy incorporating donor-type secretor plasma infusions with or without plasma exchange to prevent PCI-associated hemolysis and PRCA in major or bidirectional ABO-mismatched peripheral blood HPCT. All major or bidirectional ABO-mismatched HPCTs performed between 1999 and 2010 were identified from an institutional database. Immunohematologic outcomes were determined retrospectively by review of individual medical records. RESULTS: In total 110 major or bidirectional ABO-mismatched HPCTs had been performed. No patient developed hemolysis after PCI. With respect to PRCA incidence, 16 patients (15%) were excluded due to early mortality and three (3%) due to incomplete data; of the remaining 91 patients, five (5%) developed PRCA. Patients with PRCA had significantly higher pretransplant isoagglutinin titers (p = 0.0001) compared to those who did not develop PRCA. CONCLUSIONS: Use of a standardized pretransplant isoagglutinin reduction strategy including donor-type secretor plasma infusions is both safe and efficient in preventing PCI-associated hemolysis and is associated with low rates of posttransplant PRCA.

Posttransplant thrombotic microangiopathy: sensitivity of proposed new diagnostic criteria.

BACKGROUND: Objective diagnosis of transplantation-associated thrombotic microangiopathy (TA-TMA) has traditionally been difficult due to the multiple potential etiologies of thrombocytopenia and red blood cell fragmentation occurring after allogeneic hematopoietic stem cell transplantation (SCT). To attempt to address this issue of diagnostic uncertainty, two new diagnostic criteria for TA-TMA have recently been proposed: the Bone Marrow Transplant Clinical Trials Network (BMT-CTN) and the International Working Group (IWG) criteria. However, both newly proposed criteria are yet to be clinically validated. STUDY DESIGN AND METHODS: All 15 cases of TA-TMA previously diagnosed at the authors' institution between December 2001 and March 2008 were retrospectively reclassified under the newly proposed BMT-CTN and IWG criteria. RESULTS: Potential diagnostic pitfalls were identified in both the BMT-CTN and the IWG TA-TMA criteria. The main limitation of the BMT-CTN criteria appeared to be need for concurrent renal and/or neurologic dysfunction to be manifest at TA-TMA diagnosis, which was present in only 73% of our patient cohort. For the IWG criteria, the main limitation to TA-TMA diagnosis appeared to be the requirement for schistocytosis of more than 4%, which was present in only 27% of these patients. CONCLUSION: Our experience suggests that potentially significant diagnostic pitfalls remain with both recently proposed TA-TMA diagnostic criteria, pitfalls that are likely to limit the diagnostic sensitivity of both. It is recommended that further clinical correlation of both the BMT-CTN and the IWG criteria be undertaken before either is routinely adapted into SCT practice.