Gammadelta (γδ) T cells are found in both blood and tissues and have antiviral and antitumor properties. The frequency of γδ T cells in umbilical cord blood (UCB) is low, and the majority express δ1, in contrast to blood, whereas the main subset is δ2γ9 T cells. UCB γδ T cells are functionally immature, which together with their scarcity complicates the development of UCB γδ T cell therapies. We aimed to develop an effective expansion protocol for UCB γδ T cells based on zoledronate and IL-2. We found that culture with 5 μM zoledronate and 200 IU IL-2/ml medium for 14 days promoted extensive proliferation. The majority of the cultured cells were γ9δ2 T cells. The fold expansion of this, originally infrequent, subset was impressive (median and maximum fold change 253 and 1085, resp.). After culture, the cells had a polyclonal γδ T cell repertoire and the main memory subset was central memory (CD45RO+ CD27+). The cells produced cytokines such as IL-1B, IL-2, and IL-8 and displayed significant tumor-killing capacity. These results show that development of in vitro expanded UCB γδ T cell therapies is feasible. It could prove a valuable treatment modality for patients after umbilical cord blood transplantation.

BackgroundRare nucleated CD45 negative cells in peripheral blood may be malignant such as circulating tumor cells. Untouched isolation thereof by depletion of normal is favored yet still technological challenging. We optimized and evaluated a novel magnetic bead-based negative selection approach for enhanced enrichment of rare peripheral blood nucleated CD45 negative cells and investigated the problem of rare cell contamination during phlebotomy.MethodsFirstly, the performance of the magnetic cell separation system was assessed using leukocytes and cultivated fibroblast cells in regard to depletion efficiency and the loss of cells of interest. Secondly, a negative selection assay was optimized for high performance, simplicity and cost efficiency. The negative selection assay consisted of; a RBC lysis step, two depletion cycles comprising direct magnetically labelling of leukocytes using anti-CD45 magnetic beads followed by magnetic capture of leukocytes using a duopole permanent magnet. Thirdly, assay evaluation was aligned to conditions of rare cell frequencies and comprised cell spike recovery, cell viability and proliferation, and CD45 negative cell detection. Additionally, the problem of CD45 negative cell contamination during phlebotomy was investigated.ResultsThe depletion factor and recovery of the negative selection assay measured at most 1600-fold and 96%, respectively, leaving at best 1.5 × 104 leukocytes unseparated and took 35 min. The cell viability was negatively affected by chemical RBC lysis. Proliferation of 100 spiked ovarian cancer cells in culture measured 37% against a positive control. Healthy donor testing revealed findings of nucleated CD45 negative cells ranging from 1 to 22 cells /2.5 × 107 leukocytes or 3.5 mL whole blood in 89% (23/26) of the samples.ConclusionOur assay facilitates high performance at shortest assay time. The enrichment assay itself causes minor harm to cells and allows proliferation. Our findings suggest that rare cell contamination is unavoidable. An unexpected high variety of CD45 negative cells have been detected. It is hypothesized that a rare cell profile may translate into tumor marker independent screening.

Chimeric antigen receptors (CARs) are among the curative immunotherapeutic approaches that exploit the antigen specificity and cytotoxicity function of potent immune cells against cancers. Neuroblastomas, the most common extracranial pediatric solid tumors with diverse characteristics, could be a promising candidate for using CAR therapies. Several methods harness CAR-modified cells in neuroblastoma to increase therapeutic efficiency, although the assessment has been less successful. Regarding the improvement of CARs, various trials have been launched to overcome insufficient capacity. However, the reasons behind the inadequate response against neuroblastoma of CAR-modified cells are still not well understood. It is essential to update the present state of comprehension of CARs to improve the efficiency of CAR therapies. This review summarizes the crucial features of CARs and their design for neuroblastoma, discusses challenges that impact the outcomes of the immunotherapeutic competence, and focuses on devising strategies currently being investigated to improve the efficacy of CARs for neuroblastoma immunotherapy.