Christina M. Celluzzi

Regulation of activation‐induced receptor activator of NF‐κB ligand (RANKL) expression in T cells

Receptor activator of NF‐κB ligand (RANKL) is a type II membrane protein of the TNF family and plays a critical role in the regulation of osteoclastogenesis. RANKL expressed on osteoblastic stromal cells has been shown to support osteoclast differentiation originated from hematopoietic precursors. Interestingly, RANKL is also expressed on cells of the immune system including T cells and dendritic cells. We have shown that anti‐CD3 could induce RANKL expression in T cell hybridoma A1.1 cells and splenic T cells. RANKL expressed on T cells could effectively induce osteoclast formation from the whole population of murine splenocytes. Furthermore, we have found that the induction of RANKL expression is solely dependent on TCR activation‐induced Ca2+ mobilization since its expression can be blocked by cyclosporine A and TMB‐8, a Ca2+ mobilization inhibitor. Additionally, treatment of A1.1 cells with ionomycin alone also strongly induces RANKL expression, while phorbol myristate acetate by itself does not. Moreover, although inhibition of c‐myc has significant effects on anti‐CD3‐induced Fas ligand (FasL) expression, we have found that the anti‐CD3‐induced RANKL expression is independent of c‐myc. Surprisingly, in contrast to its inhibitory effect on FasL expression, TGF‐β dramatically increased the expression of anti‐CD3‐induced RANKL expression. In addition to its potential role in immune responses, RANKL expressed on activated T lymphocytes may provide a mechanism for the communication between the immune and skeletal systems during immune responses and disease states such as rheumatoid arthritis.

A simple cryopreservation method for dendritic cells and cells used in their derivation and functional assessment

BACKGROUND: Cryopreservation and storage permitting multiple treatments with single donations is of practical importance to cellular therapies. HES and DMSO, used successfully in simple clinical procedures for freezing marrow and peripheral blood progenitor cells at –80°C, was tested on antigen‐presenting dendritic cells (DCs) and cells used in their derivation.

Dendritic cell culture : a simple closed culture system using ficoll, monocytes, and a table-top centrifuge

Dendritic cells (DCs) are potent antigen-presenting cells involved in the induction of T cell-mediated immune responses and as such have emerged as important candidates for cellular-based therapies. Critical to safe clinical use is the easy manipulation of DCs and their precursors in a closed system. We have developed a serum-free, closed culture system applying a simple wash-Ficoll centrifugation method to reduce platelet and red blood cell (RBC) contamination. This procedure optimized adherence of monocytes (44 +/- 10.9% recovery, >85% expressed CD14(+)/CD163(+)) for the generation of DCs from mononuclear cell (MNC) apheresis units. Most RBCs and up to 98% of platelets were removed. Following density sedimentation, cell viability remained high (98 +/- 2%) with only minimal loss of monocytes (3 +/- 3%). Importantly, Ficoll-treated monocytes retained their ability to differentiate to mature DCs demonstrated by morphology, phenotype (MHC class II(+), CD1a(+), CD80(+), CD86(+), and CD83(+)), ability to stimulate mixed lymphocyte responses (MLR), present antigen, and produce interleukin-12 (IL-12). Nonadherent CD3(+) (80 +/- 4%) were also isolated for functional assays. Ficoll can be easily incorporated into a simple adherence-based closed system for collection of lymphocytes and adherent monocytes for DC culture. The procedure is relatively fast (effective working time 5-6 h), does not impair monocyte function or induce substantial cell activation, and can be performed economically using equipment found in a typical blood banking environment.

Training practices of hematopoietic progenitor cell–apheresis and –cord blood collection staff: analysis of a survey by the Alliance for Harmonisation of Cellular Therapy Accreditation

As hematopoietic stem cell transplantation expands globally, identification of the key elements that make up high‐quality training programs will become more important to optimizing collection practices and quality of the products collected.

Training practices of cell processing laboratory staff: analysis of a survey by the Alliance for Harmonization of Cellular Therapy Accreditation

BACKGROUND AIMS Methods for processing products used for hematopoietic progenitor cell (HPC) transplantation must ensure their safety and efficacy. Personnel training and ongoing competency assessment is critical to this goal. Here we present results from a global survey of methods used by a diverse array of cell processing facilities for the initial training and ongoing competency assessment of key personnel. METHODS The Alliance for Harmonisation of Cellular Therapy Accreditation (AHCTA) created a survey to identify facility type, location, activity, personnel, and methods used for training and competency. A survey link was disseminated through organizations represented in AHCTA to processing facilities worldwide. Responses were tabulated and analyzed as a percentage of total responses and as a percentage of response by region group. RESULTS Most facilities were based at academic medical centers or hospitals. Facilities with a broad range of activity, product sources and processing procedures were represented. Facilities reported using a combination of training and competency methods. However, some methods predominated. Cellular sources for training differed for training versus competency and also differed based on frequency of procedures performed. Most facilities had responsibilities for procedures in addition to processing for which training and competency methods differed. Although regional variation was observed, training and competency requirements were generally consistent. CONCLUSIONS Survey data showed the use of a variety of training and competency methods but some methods predominated, suggesting their utility. These results could help new and established facilities in making decisions for their own training and competency programs.

Monocyte enrichment of mononuclear apheresis preparations with a multistep back-flush procedure on a cord blood filter.

INTRODUCTION: Monocytes or mononuclear cells have been investigated for the treatment of chronic wounds and spinal cord injuries, as well as serve as a source for dendritic or endothelial cell culture. Because these cells may have clinical benefit yet no rapid and inexpensive closed system for monocyte purification is commercially available, a method was investigated to enrich monocytes from mononuclear apheresis units using a cord blood filter.

Erratum: Training practices of hematopoietic progenitor cell-apheresis and -cord blood collection staff: analysis of a survey by the Alliance for Harmonisation of Cellular Therapy Accreditation (Trans (2014) 50, (3138-3144) 10.1111/trf.13173)

Celluzzi CM, Keever-Taylor C, Alurf M, Koh MBC, Rabe F, Rebulla P, Sacchi N, Sanders J, McGrath E, Loper K, on behalf of the Alliance for Harmonisation of Cellular Therapy Accreditation (AHCTA). Training practices of hematopoietic progenitor cell–apheresis and –cord blood collection staff: analysis of a survey by the Alliance for Harmonisation of Cellular Therapy Accreditation. Trans 2014;54:3138-44. The authors regret the misspelling of Mahmoud Aljurf, the third author of this manuscript.

Training practices of hematopoietic progenitor cell-apheresis and -cord blood collection staff

As hematopoietic stem cell transplantation expands globally, identification of the key elements that make up high‐quality training programs will become more important to optimizing collection practices and quality of the products collected.

Training practices of hematopoietic progenitor cell-apheresis and -cord blood collection staff: analysis of a survey by the Alliance for Harmonisation of Cellular Therapy Accreditation: HPC Collection Staff Training Practices

As hematopoietic stem cell transplantation expands globally, identification of the key elements that make up high‐quality training programs will become more important to optimizing collection practices and quality of the products collected.