ABSTRACT
OBJECTIVE: There is interest in using cytotoxic T lymphocyte antigen-4 (CTLA-4) immunotherapy to treat blood cancers. Unfortunately, patients with acute lymphoblastic leukaemia (ALL) frequently exhibit resistance to treatment and natural killer (NK) cell exhaustion. This study aims to increase the cytotoxic potency of natural killer cells by using CTLA-4 to block the Nalm-6 leukaemia cell line. MATERIALS AND METHODS: In this experimental study, NK cells were purified from the peripheral blood mononuclear cells (PBMCs) of 10 healthy people and assessed by flow cytometry for purity and viability. The purified cells were activated overnight at 37°C and 5% CO2 with interleukin-15 (IL-15, 10 ng/ml) followed by evaluation of expressions of CTLA-4, activating and inhibitory receptors, and the release of interferon gamma (IFN-γ) and granzyme B (GZM B). CTLA-4 expression on NK cells from recurrent ALL patients was also evaluated. Finally, the cytotoxic activity of NK cells was assessed after the CTLA-4 blockade. RESULTS: The purity of the isolated cells was 96.58 ± 2.57%. Isolated NK cells activated with IL-15 resulted in significantly higher CTLA-4 expression (8.75%, P<0.05). Similarly, CTLA-4 expression on the surface of NK cells from patients with ALL was higher (7.46%) compared to healthy individuals (1.46%, P<0.05). IL-15 reduced NKG2A expression (P<0.01), and increased expressions of NKP30 (P<0.05) and NKP46 (P<0.01). The activated NK cells released more IFN-γ (P<0.5) and GZM B (P<0.01) compared to unactivated NK cells. Blockade of CTLA-4 enhanced the NK cell killing potential against Nalm-6 cells (56.3%, P<0.05); however, IFN-γ and GZM B levels were not statistically different between the blocked and non-blocked groups. CONCLUSION: Our findings suggest that CTLA-4 blockage of Nalm-6 cells causes an increase in antitumour activity of NK cells against these cells. Our study also provides evidence for the potential of cancer immunotherapy treatment using blocking anti-CTLA-4 mAbs.
ABSTRACT
OBJECTIVE: Natural killer (NK) cells are critical immune cells for acute myeloid leukemia (AML) targeting. However, little is known about the relationship between using checkpoint inhibitors and heat shock protein 70 (Hsp70) as NK cell activators to control AML. Therefore, the study aims to find the best formulation of Hsp70, human PD-1 (Programmed cell death protein 1) blocker, and interleukin 15 (IL-15) to activate NK cells against AML. MATERIALS AND METHODS: In this experimental study, the NK cells were isolated from mononuclear cells (MNCs) by using magnetic activation cell sorting (MACS) and were activated using the different combinations of Hsp70, PD-1 blocker, and IL-15 and then followed by immunophenotyping, functional assays to estimate their killing potential, and evaluation of expression pattern of PRF1, PIK3CB, PD-1, AKT-1, FAS-L, TRAIL, and GER A and B. RESULTS: The expression of PD-1 was significantly (P<0.05) reduced after NK cell activation by the different formulas of IL-15, Hsp70, and PD-1 blocker. The expression of NKG2A in the treated NK cells was reduced particularly in the IL-15 (P<0.01) and IL-15+PD-1 blocker (P<0.05) groups. The addition of Hsp70 increased its expression. The cytotoxic effect of NK cells increased in all groups, especially in IL-15+PD-1 blocker besides increasing interferon-gamma (IFN-γ), Granzymes, and perforin expression (P<0.05). All IL-15+PD-1 blocker group changes were associated with the upregulation of PIK3CB and AKT-1 as key factors of NK cell activation. The presence of Hsp70 reduced IFN-γ releasing, and down-regulation of PIK3CB, AKT-1, Granzymes, and Perforin (P<0.05). CONCLUSION: We suggested the combination of IL-15 and PD-1 blocker could enhance the killing potential of AMLNK cells. Moreover, Hsp70 in combination with IL-15 and PD-1 blocker interferes activation of AML-NK cells through unknown mechanisms.
ABSTRACT
Coronavirus disease 2019 (COVID-19) is a newly emerging human infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Early diagnosis is essential to reducing the transmission rate and mortality of COVID-19. PCR-based tests are the gold standard for the confirmation of COVID-19, but immunological tests for SARS-CoV-2 detection are widely available and play an increasingly important role in the diagnosis of COVID-19. Nanomechanical sensors are biosensors that work based on a change in the mechanical response of the system when a foreign object is added. In this paper, a graphene-based nanoresonator sensor for SARS-CoV-2 detection was introduced and analyzed by using the finite element method (FEM). The sensor was simulated by coating a single-layer graphene sheet (SLGS) with a specific antibody against SARS-CoV-2 Spike S1 antigen. In the following, the SARS-CoV-2 viruses were randomly distributed on the SLGSs, and essential design parameters of the nanoresonator, including frequency shift and relative frequency shift, were evaluated. The effect of the SLGS size, aspect ratio and boundary conditions, antibody concentration, and the number of viruses variation on the frequency shift and relative frequency shift were investigated. The results revealed that, by proper selection of the nanoresonator design variables, a good sensitivity index is achievable for identifying the SARS-CoV-2 virus even when the number of the viruses are less than 10 per test. Eventually, according to the simulation results, by using SLGS geometry determination, an analytical relationship is presented to predict the limit of detection (LOD) of the sensor with the required sensitivity index. The results can be applied in designing and fabricating specific graphene-based nanoresonator sensors for SARS-CoV-2.
