Human Vγ9Vδ2 T cell expansion and their cytotoxic responses against cholangiocarcinoma.

Human Vγ9Vδ2 T lymphocytes are regarded as promising effector cells for cancer immunotherapy since they have the ability to eliminate several tumor cells through non-peptide antigen recognition. However, the cytotoxic function and the mechanism of Vγ9Vδ2 T cells leading to specific killing of cholangiocarcinoma cells are yet to be confirmed. In this study, we established a protocol for ex vivo expansion of Vγ9Vδ2 T cells from healthy donors' peripheral blood mononuclear cells by culture with zoledronate and addition of IL-2, and IL-15 or IL-18 or neither. Testing the cytotoxic capacity of cultured Vγ9Vδ2 T cells against cholangiocarcinoma cell lines showed higher reactivity than against control cells. Surface expression of CD107 was detected on the Vγ9Vδ2 T cells, suggesting that these cells limit in vitro growth of cholangiocarcinoma cells via degranulation of the perforin and granzyme pathway. Analysis of molecular signaling was used to demonstrate expression of pro- and anti-survival genes and a panel of cytokine genes in Vγ9Vδ2 T cells. We found that in the presence of either IL-15 or IL-18, levels of caspase 3 were significantly reduced. Also, IL-15 and IL-18 stimulated cells contained cytotoxicity against cholangiocarcinoma cells, suggesting that stimulated Vγ9Vδ2 T cells may provide a feasible therapy for cholangiocarcinoma.

Comparison of the efficacy of second and third generation lentiviral vector transduced CAR CD19 T cells for use in the treatment of acute lymphoblastic leukemia both in vitro and in vivo models.

T cells genetically engineered to express a chimeric antigen receptor (CAR) specifically binding to a CD19 antigen has become the frontline of hematological malignancies immunotherapy. Their remarkable antitumor effect has exerted complete remission in treating B-cell malignancies. Although successful patient treatment has been shown, improvement to the structure of CAR to enhance its safety and efficacy profile is warranted. Transduction with a lentiviral vector (LVV) leading to the expression of CARs is also a critical step in redirecting T cells to target specific tumor antigens. To improve the efficacy of CD19 CARs in this study, the transduction ability of second and third generations LVV were compared. Ex vivo expansion of CD19 CARs T cells from healthy donors' peripheral blood mononuclear cells was performed after transduction of T cells with second and third generations LVV. Transduction efficacy of transduced T cells was determined to show a higher percentage in the third generations LVV transduced cells, with no changes in viability and identity of cells characterized by immunophenotyping. Testing the cytotoxic capacity of third generations LVV-transduced T cells against target cells showed higher reactivity against control cells. Cytokine expression was detected on the CD19 CARs T cells, suggesting that these cells limit in vitro growth of B-cell leukemia via secretion of the pro-inflammatory cytokine IFN γ. To investigate whether the third generation LVV transduced T cells can limit CD19 lymphoma growth in vivo, an analysis of tumor burden in a mouse model assessed by bioluminescence imaging was performed. We found that, in the presence of CD19 CARs T cells, the level of tumor burden was markedly reduced. In addition, an increase in the length of survival in mice receiving CAR-CD19 T cells was also observed. This suggests that transduction with third generations LVV generate a functional CAR-CD19 T cells, which may provide a safer and effective therapy for B-cell malignancies.

Strategies to Improve Chimeric Antigen Receptor Therapies for Neuroblastoma.

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.

Antigen-Presenting Cell Characteristics of Human γδ T Lymphocytes in Chronic Myeloid Leukemia.

Human γδ T lymphocytes play a role in the immune system defense against cancer. Their broad anti-cancer activity against different types of cancers makes them outstanding candidates for cancer immunotherapy. An issue of recent interest is whether their antigen presentation features are similar to mature dendritic cells. The antigen-presenting cell (APC)-like phenotype and function of γδ T lymphocytes have been confirmed in many clinical trials. In this study, to support the strong role played by Vγ9Vδ2 T cells against cancer, we provide evidence that Vγ9Vδ2 T cells activated with chronic myeloid leukemia (CML) cell lysate antigens can efficiently express an APC phenotype and function. Vγ9Vδ2 T cells derived from normal peripheral blood mononuclear cells were activated with tumor cell lysate, and the tumor-activated Vγ9Vδ2 T cells could recognize and kill CML through their cytotoxic activity. In conclusion, the Vγ9Vδ2 T cells activated by cancer cell lysate showed APC characteristics, and this may greatly increase interest in investigating their therapeutic potential in hematologic malignancies. Abbreviations: CML: chronic myeloid leukemia; APC: antigen-presenting cell; TCR: T cell receptor; MHC: major histocompatibility complex; N-BPs: nitrogen-containing bisphosphonates; IPP: isopentenyl pyrophosphate; PBMC: peripheral blood mononuclear cells; NKG2D: natural killer receptor group 2, member D; TRAIL: tumor necrosis factor-related apoptosis-inducing ligand.

Expansion of Gammadelta T Cells from Cord Blood: A Therapeutical Possibility.

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.

Enrichment of human Vγ9Vδ2 T lymphocytes by magnetic poly(divinylbenzene-co-glycidyl methacrylate) colloidal particles conjugated with specific antibody.

γδ T cells play a significant role in protection against cancer. Purification of γδ T cells is needed for insight when studying their anti-cancer functionality and their utilization in adoptive cell therapy. To improve the purification of γδ T cells, in this work, a composite material based on magnetic nanoparticles was developed for purification of Vγ9Vδ2 T cells, the predominant subset of γδ T lymphocytes in human peripheral blood. The epoxy-functionalized magnetic poly(divinylbenzene-co-glycidyl methacrylate) particles (mPDGs) were bio-conjugated with anti-human Vδ2 antibody to provide specific recognition sites for T cell receptors of Vγ9Vδ2 T cells. Using fluorescence-activated cell sorting (FACS) analysis, separation of Vγ9Vδ2 T cells from peripheral blood mononuclear cells of healthy donors was confirmed with high purity [89.77% (range 87.00-91.80, n = 3)]. More interestingly, the immobilized particles did not affect the viability of purified cells as high cell viability was indicated (>90%). By combining the properties of magnetic nanoparticles with specific antibodies, these immobilized particles were shown to be used as a cell-friendly purification tool of Vγ9Vδ2 T lymphocytes without any limits for the further use of cells. The purified Vγ9Vδ2 T cells using the antibody-immobilized epoxy-functionalized mPDGs could be used directly without a detachment step for further cultivation and expansion. This highlights the advantages of this method in allowing the study of cell function and further investigation of such rare T cell populations in immunotherapy.

Enhanced Sensitivity for Detection of Plasmodium falciparum gametocytes by magnetic nanoparticles combined with enzyme substrate system.

The highly sensitive and specific detection of Pfg377 gene of Plasmodium falciparum gametocyte using Magnetic Nanoparticles PCR Enzyme-Linked Gene Assay (MELGA) was successfully developed. The MELGA included amplification of the Pfg377 gene by polymerase chain reaction (PCR) using magnetic nanoparticles (MNPs)-conjugated forward primer and biotinylated reverse primer, followed by post-analytical process using horseradish peroxidase (HRP)-conjugated streptavidin (SA). The complexes of MELGA product were incubated with the peroxidase substrate and hydrogen peroxide to produce the signal for colorimetric measurement. Altogether, the MELGA technique provided a highly sensitive and specific detection at 1 P. falciparum gametocyte/µL, which was more efficient than that of microscopic examination and rapid diagnostic tests (RDTs). Additionally, the MELGA could detect target gene at femtogram level, which was greater sensitive than the conventional PCR, nested PCR and loop-mediated isothermal amplification (LAMP). The MELGA technique could become a novel and practical method that overcome limitation of traditional gametocyte detection.

Advances in rare cell isolation: an optimization and evaluation study.

BACKGROUND: Rare 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. METHODS: Firstly, 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. RESULTS: The 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. CONCLUSION: Our 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.