A pattern of natural killer cell (NK cell) heterogeneity determines proliferative and functional responses to activating stimuli in individuals. Obtaining the progeny of a single cell by cloning the original population is one of the ways to study NK cell heterogeneity. In this work, we sorted single cells into a plate and stimulated them via interleukin (IL)-2 and gene-modified K562 feeder cells that expressed membrane-bound IL-21 (K562-mbIL21), which led to a generation of phenotypically confirmed and functionally active NK cell clones. Next, we applied two models of clone cultivation, which differently affected their phenotype, lifespan, and functional activity. The first model, which included weekly restimulation of clones with K562-mbIL21 and IL-2, resulted in the generation of relatively short-lived (5–7 weeks) clones of highly activated NK cells. Levels of human leukocyte antigen class II molecule—DR isotype (HLA-DR) expression in the expanded NK cells correlated strongly with interferon-γ (IFN-γ) production. The second model, in which NK cells were restimulated weekly with IL-2 alone and once on the sixth week with K562-mbIL21 and IL-2, produced long-lived clones (8–14 weeks) that expanded up to 10⁷ cells with a lower ability to produce IFN-γ. Our method is applicable for studying variability in phenotype, proliferative, and functional activity of certain NK cell progeny in response to the stimulation, which may help in selecting NK cells best suited for clinical use.

Immunosenescence is a process of remodeling the immune system under the influence of chronic inflammation during aging. Parkinson’s disease (PD) is a common age-associated neurodegenerative disorder and is frequently accompanied by neuroinflammation. On the other hand, cytomegalovirus (CMV), one of the most spread infections in humans, may induce chronic inflammation which contributes to immunosenescence, differentiation and the inflation of T cells and NK cells. Currently, there is no clear understanding of immunosenescence severity in PD patients infected with CMV. In this study, we analyzed differentiation stages and immunosenescence characteristics of T cells and NK cells in 31 patients with mild and moderate PD severity, 33 age-matched and 30 young healthy donors. The PD patients were 100% CMV-seropositive compared to 76% age-matched and 73% young CMV-infected healthy donors. The proportion of effector memory T cells re-expressing CD45RA, CD57⁺CD56⁻ T cells and CD57⁺CD56⁺ T cells was significantly reduced in PD patients compared with CMV-seropositive age-matched healthy individuals. The CD57⁺CD56⁻ T cell proportion in PD patients was similar to that of CMV-seropositive young healthy donors. Thus, PD is characterized by reduced peripheral blood T cell immunosenescence, even against the background of CMV infection.

Parkinson disease (PD) is attributed to a proteostasis disorder mediated by α-synuclein accumulating in a specific brain region. PD manifestation is often related to extraneuronal alterations, some of which could be used as diagnostic or prognostic PD biomarkers. In this work, we studied the shifts in the expression of proteostasis-associated chaperones of the HSP70 family and autophagy-dependent p62 protein values in the peripheral blood mononuclear cells (PBMC) of mild to moderate PD patients. Although we did not detect any changes in the intracellular HSP70 protein pool in PD patients compared to non-PD controls, an increase in the transcriptional activity of the stress-associated HSPA1A/B and HSPA6 genes was observed in these cells. Basal p62 content was found to be increased in PD patients’ PBMC, similarly to the p62 level in substantia nigra neural cells in PD. Moreover, the spontaneous apoptosis level was increased among PBMC and positively correlated with the p62 intracellular level in the PD group. A combined HSPA6- and p62-based analysis among 26 PD patients and 36 age-matched non-PD controls pointed out the diagnostic significance of these markers, with intermediate sensitivity and high specificity of this combination when observing patients diagnosed with PD.

Background/objectives: The functional activity of a certain tumor determines the effectiveness of primary NK cells and NK-92 cell line-based cancer therapy; their therapeutic effectiveness against different tumors can vary. This work provides a direct simultaneous comparison of the cytotoxic effects of in vitro-activated peripheral NK (pNK) cells and NK-92 cells in spheroid models of BT-474, MCF7 and SKOV-3 carcinomas and uncovers the reasons for the differential effectiveness of NK cells against tumors. Methods: Tumor spheroids of similar size and shape, obtained from agarose molds, were incubated with NK-92 or pNK cells for 24 h. Tumor cell death was detected using flow cytometry or confocal microscopy. Cytokine production, granzyme B levels and NK cell degranulation analyses were performed, along with pNK and target-cell phenotypic characterization. Results: While NK-92 and pNK cells lysed BT-474 spheroids with comparably low efficiency, pNK cells were more capable of eliminating MCF7 and SKOV-3 spheroids than NK-92 cells were. The results of the functional and phenotypic analyses strongly support the participation of the NKG2D-NKG2DL pathway in pNK cell activation induced by the most sensitive cytotoxic attack on SKOV-3 spheroids, whereas the CX3CR1-CX3CL1 axis appears to be involved in the pNK reaction against MCF-7 spheroids. Conclusions: We provide a new approach for the preliminary identification of the most promising NK cell receptors that can alter the effectiveness of cancer therapy depending on the specific tumor type. Using this approach, NK-92 cells or pNK subsets can be selected for further accumulation and/or genetic modification to improve specificity and reactivity.

Nowadays, the use of genetically modified NK cells is a promising strategy for cancer immunotherapy. The additional insertion of genes capable of inducing cell suicide allows for the timely elimination of the modified NK cells. Different subsets of the heterogenic NK cell population may differ in proliferative potential, in susceptibility to genetic viral transduction, and to the subsequent induction of cell death. The CD57⁻NKG2C⁺ NK cells are of special interest as potential candidates for therapeutic usage due to their high proliferative potential and certain features of adaptive NK cells. In this study, CD57⁻ NK cell subsets differing in KIR2DL2/3 and NKG2C expression were transduced with the iCasp9 suicide gene. The highest transduction efficacy was observed in the KIR2DL2/3⁺NKG2C⁺ NK cell subset, which demonstrated an increased proliferative potential with prolonged cultivation. The increased transduction efficiency of the cell cultures was associated with the higher expression level of the HLA-DR activation marker. Among the iCasp9-transduced subsets, KIR2DL2/3⁺ cells had the weakest response to the apoptosis induction by the chemical inductor of dimerization (CID). Thus, KIR2DL2/3⁺NKG2C⁺ NK cells showed an increased susceptibility to the iCasp9 retroviral transduction, which was associated with higher proliferative potential and activation status. However, the complete elimination of these cells with CID is impeded.

Anti-cancer targeted therapy is a promising approach. However, the identification of target molecules over-expressed in a wide range of tumors remains a significant challenge. The aim of this study was to analyze the expression of cell membrane-exposed heat shock protein 70 kDa (mHSP70) on different tumor cells and to develop a nanoscale delivery system based on a monoclonal antibody (mAb) that recognizes mHSP70 and uses chitosan core–shell nanoparticles (NPs). Several types of tumor cells (breast, pancreas, colon, prostate cancers, and some lymphomas) expressed mHSP70 as was determined by flow cytometry and confocal microscopy both in 2D and 3D cultures. Core NPs were formed by chitosan (C) conjugated to allocolchicinoid, which was used as a model drug (D). mAbs (A) targeting mHSP70 were complexed with succinylchitosan and used as NP shells forming final CAD-NPs. These NPs were characterized by size, charge, and functional activity. CAD-NPs were shown to have additional toxicity in comparison with CD-NPs in mHSP7-positive cells. Taken collectively, this study shows that mAb to mHSP70 can be used as a targeting vector in antitumor therapy.