NK cells expansion <i>in vitro</i> is followed by loss of inhibitory KIR expression

NK cells are innate lymphocytes that are able to eliminate altered cells, which makes them promising for the immunotherapy of viral diseases and tumors. The NK cell population is characterized by high phenotypic and functional diversity. In particular, in the pool of highly differentiated NK cells in the presence of cytomegalovirus (HCMV), a population of adaptive cells can be formed, characterized by a high lifespan and high cytotoxicity. However, in order to carry out a cytotoxic reaction, a NK cell must undergo a licensing process, during which it acquires the expression of NKG2A and KIRs. Currently, there are many effective methods of NK cell accumulation for subsequent use in therapy, one of them is the stimulation with IL-2 and K562-mbIL21 feeder cells. Highly differentiated adaptive-like NK cells are able to expand in respond to such stimulation. However, the phenotype of actively expanding NK cells dynamically changes. Loss of inhibitory KIR expression during intense proliferation of NK cells may adversely affect their cytotoxic potential. This work shows that highly differentiated CD56dimNKG2C+ NK cells from HCMV-seropositive individuals have a high proportion of KIR2DL2/3+ cells. This may indicate a high stability of KIR receptor expression in this population. We have shown that CD56dimNKG2C+ clonal cultures obtained by stimulation with IL-2 and K562- mbIL21 are characterized by high stability of KIR2DL2/3 expression compared to NKG2C-negative and less differentiated CD56brightNKG2C+. Also, in heterogeneous cultures of adaptive NK cells precursors CD57- CD56dimNKG2C+, a higher expression level of KIR2DL2/3 was observed in comparison with NKG2C-negative cultures of CD57-CD56dimNKG2C-. Thus, the accumulation of NK cells upon stimulation with IL-2 and K562- mbIL2 feeder cells can lead to loss of expression of KIR receptors and a decrease in their functional activity. However, cultures of highly differentiated NK cells of HCMV-seropositive individuals CD56dimNKG2C+, as well as cultures of precursors of adaptive NK cells CD57-CD56dimNKG2C+, are characterized by a greater stability of KIR2DL2/3 expression. As a result, stimulation with IL-2 and K562-mbIL21 feeder cells can be used to accumulate adaptive-like cells and their progenitors with stable inhibitory KIR expression and high cytotoxic potential

DISTRIBUTION OF <i>MICA</i> ALLELES IN THE RUSSIAN POPULATION

Stress factors, infections, tumor transformation of the cells of organism induce the expression of MICA protein, which is a ligand for the NKG2D receptor of NK and T cells. The interaction of the NKG2D receptor on the surface of the cells of the immune system with MICA results in activation of lymphocytes and elimination of the ligand carrier. The MICA gene has a high level of polymorphism. To date, 87 alleles have been described; their products differ in ability to activate cytotoxic lymphocytes, that can affect the progression of a number of diseases, such as cancer, viral infections, autoimmune diseases. The distribution of MICA alleles in different ethnic groups varies considerably. The analysis of MICA polymorphism in a current ethnos is necessary for revealing the relationships between certain MICA alleles and different diseases. Goal. This work is aimed at studying of the distribution of MICA alleles in Russian population. Materials and methods. Polymorphism of MICA was analyzed according to the procedure proposed by Yizhou Zoe and Peter Stastny. The procedure included: 1) isolation of genomic DNA from whole blood; 2) PCR for amplification of a fragment of the MICA gene; 3) sequencing of the resulting PCR fragments. Analysis of the results of sequencing was carried out using the programs Vector NTI and Chromas Lite. Results. The genotype of the MICA alleles of 119 donors has been determined. Of the 87 MICA alleles described in the literature, 15 were found among the samples studied. The frequencies of MICA alleles were the following: *002 – 19.3%, *004 – 6.7%, *007 – 3.0%, *008 – 35.7%, *009 – 10.1%, *010 – 5.0%, *011 – 3.8%, *012 – 2.1%, *016 – 2.5%, *017 – 3.4% *018 – 5.5%, *019 – 0.4%, *027 – 1.3%, *053 – 0.8%, *068 – 0.4%. The distribution of MICA alleles in Russia was found to be similar to that of European countries. When comparing literary data for different countries of the world, it was found that the differences in the distribution of MICA alleles are expressed mainly between races, and not nations. Conclusions. In this paper, the distribution of MICA alleles in Russian population has been analyzed. It turned out to be very similar to those of other European countries and has a number of significant differences from the ethnoses of the Mongoloid race (Japan, China, Korea). The analysis of the distribution of MICA alleles in the Russian population may be useful for identifying the predisposition of individuals to certain diseases

Analysis of the association of the polymorphism of the CLIC1, MSH5, C6orf26, C6orf25 genes with the expression level of the HSPA1B gene

Heat  shock proteins (HSP, heat  shock proteins) form one of the cellular  molecular systems with chaperone activity, aimed  at stabilizing  the structure of intracellular proteins, ensuring  the resistance of cells to stress, renaturation of incorrectly folded  and  elimination of denatured intracellular proteins.Our task was to look for an association between  the presence of single nucleotide polymorphisms in selected  regions of the genome  and the basal level of transcriptional activity of the HSPA group genes, namely  HSPA1A/B, HSPA1A, HSPA1B, HSPA6  and  HSPA8  in mononuclear leukocytes  (PBMC), analyzed  in our  experiments volunteers from the population of the middle  part of Russia in order  to analyze  the universality of the biological  effects of these SNPs.The study was performed on DNA  and RNA isolated  from peripheral blood lymphocytes of 16 donors. Genotyping was performed by the polymerase chain reaction (PCR) followed by sequencing of the PCR product. To assess the level of gene expression  of the HSPA group, cDNA was synthesized on an RNA template isolated  from  PBMC cell fraction samples, followed  by real-time polymerase chain  reaction.The  following types of polymorphisms were genotyped: rs400547 (A/G), rs1150793 (G/A), rs707936 (A/G), rs707915 (A/T), rs376510 (T/C) located in the CLIC1,  MSH5, C6orf26, MSH5, C6orf25genes,  respectively. We determined the basal level of transcription of genes of constitutively expressed and inducible proteins of the HSP70 family and searched for the association of their  expression  with polymorphisms.It was found  that  in PBMC cells, DNA that has the following genotype: AG/AA (SNP rs400547), AG/GG (rs1150793), AG (rs707936), TArs707915, TC (rs376510), in the regions we studied, is associated with a decrease in the transcription of the HSPA1B  gene (p = 0.02) compared with homozygotes: GG (SNP rs400547), AA (rs1150793), GG (rs707936), TT (rs707915), CC  (rs376510).Associations of these  polymorphisms with  gene  expression  of HSPA1A, HSPA6  and  HSPA8 have  not  been  identified.The CLIC1,  MSH5, C6orf26, C6orf25 genes,  in which  the  polymorphisms studied by us are present, are located in the same locus near  the HSPA1B  gene on the 6th chromosome. We found  a decrease in HSPA1B  gene expression  in the presence of single nucleotide polymorphisms in nearby genes may indicate spatial interactions of this locus and the HSPA1B gene locus, and that a change in the genotype  CLIC1, MSH5, C6orf26, C6orf25 may entail a change in the expression  of closely arranged genes which are functionally significant  for the cell

Targeted Delivery of HSP70 to Tumor Cells via Supramolecular Complex Based on HER2-Specific DARPin9_29 and the Barnase:Barstar Pair

(1) Background: We have previously shown that the use of an artificial supramolecular two-component system based on chimeric recombinant proteins 4D5scFv-barnase and barstar-heat shock protein 70 KDa (HSP70) allows targeted delivery of HSP70 to the surface of tumor cells bearing HER2/neu antigen. In this work, we studied the possibility to using DARPin9_29-barnase as the first targeting module recognizing HER2/neu-antigen in the HSP70 delivery system. (2) Methods: The effect of the developed systems for HSP70 delivery to human carcinomas SK-BR-3 and BT474 cells hyperexpressing HER2/neu on the activation of cytotoxic effectors of the immune cells was studied in vitro. (3) Results: The results obtained by confocal microscopy and cytofluorimetric analysis confirmed the binding of HSP70 or its fragment HSP70-16 on the surface of the treated cells. In response to the delivery of HSP70 to tumor cells, we observed an increase in the cytolytic activity of different cytotoxic effector immune cells from human peripheral blood. (4) Conclusions: Targeted modification of the tumor cell surface with molecular structures recognized by cytotoxic effectors of the immune system is among new promising approaches to antitumor immunotherapy

Reduced Immunosenescence of Peripheral Blood T Cells in Parkinson&rsquo;s Disease with CMV Infection Background

Immunosenescence is a process of remodeling the immune system under the influence of chronic inflammation during aging. Parkinson&rsquo;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&minus; T cells and CD57+CD56+ T cells was significantly reduced in PD patients compared with CMV-seropositive age-matched healthy individuals. The CD57+CD56&minus; 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

Increased Susceptibility of the CD57⁻ NK Cells Expressing KIR2DL2/3 and NKG2C to iCasp9 Gene Retroviral Transduction and the Relationships with Proliferative Potential, Activation Degree, and Death Induction Response

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

Alterations in Proteostasis System Components in Peripheral Blood Mononuclear Cells in Parkinson Disease: Focusing on the HSP70 and p62 Levels

Parkinson disease (PD) is attributed to a proteostasis disorder mediated by &alpha;-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&rsquo; 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