The Enigmatic Nature of the TCR-pMHC Interaction: Implications for CAR-T and TCR-T Engineering.

The interaction of the T-cell receptor (TCR) with a peptide in the major histocompatibility complex (pMHC) plays a central role in the adaptive immunity of higher chordates. Due to the high specificity and sensitivity of this process, the immune system quickly recognizes and efficiently responds to the appearance of foreign and altered self-antigens. This is important for ensuring anti-infectious and antitumor immunity, in addition to maintaining self-tolerance. The most common parameter used for assessing the specificity of TCR-pMHC interaction is affinity. This thermodynamic characteristic is widely used not only in various theoretical aspects, but also in practice, for example, in the engineering of various T-cell products with a chimeric (CAR-T) or artificial (TCR-engineered T-cell) antigen receptor. However, increasing data reveal the fact that, in addition to the thermodynamic component, the specificity of antigen recognition is based on the kinetics and mechanics of the process, having even greater influence on the selectivity of the process and T lymphocyte activation than affinity. Therefore, the kinetic and mechanical aspects of antigen recognition should be taken into account when designing artificial antigen receptors, especially those that recognize antigens in the MHC complex. This review describes the current understanding of the nature of the TCR-pMHC interaction, in addition to the thermodynamic, kinetic, and mechanical principles underlying the specificity and high sensitivity of this interaction.

The gene therapy of collagen-induced arthritis in rats by intramuscular administration of the plasmid encoding TNF-binding domain of variola virus CrmB protein.

Wistar rats with collagen-induced arthritis were intramuscularly injected with the recombinant plasmid pcDNA/sTNF-BD encoding the sequence of the TNF-binding protein domain of variola virus CrmB protein (VARV sTNF-BD) or the pcDNA3.1 vector. Quantitative analysis showed that the histopathological changes in the hind-limb joints of rats were most severe in the animals injected with pcDNA3.1 and much less severe in the group of rats injected with pcDNA/sTNF-BD, which indicates that gene therapy of rheumatoid arthritis is promising in the case of local administration of plasmids governing the synthesis of VARV immunomodulatory proteins.

Phenotypic and functional characteristics of dendritic cells and contents of suppressor cell populations in peripheral blood of breast cancer patients.

The progression or the appearance of distant metastases in breast cancer (BC) is influenced by a variety of antitumor immune response suppression mechanisms. In this paper we study circulating dendritic cells (DC) and the suppressor cell populations in peripheral blood of breast cancer patients. The study of phenotypic and functional properties of DCs was performed in the samples of intact and TLR-stimulated whole blood from breast cancer patients and healthy women by multicolor flow cytometry. To determine the suppressor cell population among lymphocytes multicolor panel comprising markers CD 4, CD 25, CD 127, FoxP3 was used. It is showed that the formation of secondary foci of tumor growth in patients was accompanied by disturbances of the functional activity plasmocytoid DC and accumulation of cells with immunosuppressive functions.

Dendritic Cells Transfected with a DNA Construct Encoding Tumour-associated Antigen Epitopes Induce a Cytotoxic Immune Response Against Autologous Tumour Cells in a Culture of Mononuclear Cells from Colorectal Cancer Patients.

Significant effort has been devoted to developing effective cancer vaccines based on dendritic cells (DCs) loaded with various tumour antigens, including DNA constructs that carry sequences of tumour-associated antigens (TAAs). Such vaccines efficiently and selectively activate the T cell immune response. In this study, we describe a method to induce an antitumour immune response in mononuclear cell (MNC) cultures from colorectal cancer patients using DNA-transfected DCs encoding TAA epitopes of carcinoembryonic antigen, epithelial cell adhesion molecule and mucin 4. DCs were obtained from peripheral blood monocytes of colorectal cancer patients. Magnetic-assisted transfection was used to deliver the genetic constructs to DCs. To assess the potency of the immune response, the antitumour cytotoxic response was assessed by lymphocyte intracellular perforin and the MNC cytotoxic activity against autologous tumour cells. We showed that polyepitope DNA-transfected DCs enhanced MNC antitumour activity, increasing tumour cell death and the percentage of perforin-positive lymphocytes. In addition, DNA-transfected DCs elicited a cytotoxic response that was as efficient as that of tumour lysate-loaded DCs. Taken together, the data suggest that it is feasible to induce an antitumour immune response in colorectal MNCs using transfected DCs. Thus, the DNA construct reported in this study may potentially be used in therapeutic and prophylactic DC-based vaccines.

[Features of functional activity of dendritic cells in tumor growth].

During recent years much data, accumulated on biology, function and role of dendritic cells (DC) in cancer development, in a new way allow assessing their role in disease process. Identification of features of DC functional state as well as their interaction and influence on the immune cells in tumor growth can be used as a basis for a new approach to cancer therapy enhancing standard therapy efficacy. The review analyzes different mechanisms of escaping of tumor cell from immune surveillance involving DC as one of the main participants of antitumor immune response. Also the prospects of using DC for vaccination are discussed. DC can be promising target for therapeutic strategies and also can be used for formation of antitumor response and cell therapy.

Biological effects of individually synthesized TNF-binding domain of variola virus CrmB protein.

The biological characteristics of a 17-kDa protein synthesized in bacterial cells, a TNF-binding domain (VARV-TNF-BP) of a 47-kDa variola virus CrmB protein (VARV-CrmB) consisting of TNF-binding and chemokine-binding domains, were studied. Removal of the C-terminal chemokine-binding domain from VARV-CrmB protein was inessential for the efficiency of its inhibition of TNF cytotoxicity towards L929 mouse fibroblast culture and for TNF-induced oxidative metabolic activity of mouse blood leukocytes. The results of this study could form the basis for further studies of VARV-TNF-BP mechanisms of activity for prospective use in practical medicine.

Analysis of the levels of tumour necrosis factor (TNF), autoantibodies to TNF, and soluble TNF receptors in patients with rheumatoid arthritis.

OBJECTIVES: To evaluate the potential contribution made by autoantibodies against tumour necrosis factor (TNF) to the pathogenesis of rheumatoid arthritis (RA). METHOD: We used affinity chromatography methods and a magnetic separation procedure to purify human autoantibodies specific to TNF. The autoantibodies were used as calibration material to determine the absolute content of autoantibodies to TNF using an enzyme-linked immunosorbent assay (ELISA). TNF content and the levels of soluble TNF receptors types I and II (sTNF-RI and sTNF-RII) were determined using commercial ELISA test kits. RESULTS: We demonstrated significant increases in the levels of TNF, sTNF-RI, and sTNF-RII in the sera of patients with acute RA and in patients with RA who had responded positively to therapy compared with healthy controls. Levels of autoantibodies of the immunoglobulin (Ig)G2, IgG3, and IgG4 subclasses were significantly higher in sera from patients with acute RA than in sera from healthy controls. Level of autoantibodies of the IgG2 subclass were significantly higher in sera from patients with acute RA than in RA patients who had responded positively to therapy. CONCLUSIONS: Acute RA is associated with changes in levels of TNF and soluble receptors for TNF and also in levels of autoantibodies to TNF. Given the magnitude of the changes in levels of different subclasses of autoantibodies to TNF, we propose that these autoantibodies might contribute to the pathogenesis of RA.

Effect of mature dendritic cells primed with autologous tumor antigens from patients with epithelial ovarian cancer on stimulation of the cytotoxic immune response in culture of mononuclear cells.

For modulation of antitumor cytotoxic activity of mononuclear cells in vitro, autologous dendritic cells loaded with tumor lysate antigens were cultured with peripheral blood mononuclear cells from patients with epithelial ovarian cancer in the presence or absence of IL-12 and IL-18. The efficiency of modulation was evaluated by cytotoxic activity of mononuclear cells against autologous tumor cells, by the production of IFN-γ, IL-4, and by the count of perforin-containing lymphocytes. It was demonstrated that dendritic cells stimulated cytotoxic immune response in mononuclear cell culture. Maximum induction of cytotoxic activity of mononuclear cells was attained in case of dendritic cells combination with IL-12 and IL-18 (increased death of autologous tumor cells, accumulation of perforin-positive lymphocytes, enhanced production of IFN-γ).

Production of TNF-α and IL-1ß by peripheral blood mononuclear cells in carriers of different allele variants of the gene.

Associations of cytokine production by mononuclear cells and the TNF-α genetic polymorphism in positions -238, -308, -376, -857, -1031, and of IL-1ß in positions -31 and +3954 were studied. The data on distribution of allele and genotype incidence and on the level of spontaneous and mitogen-induced production of these cytokines by donor mononuclear cells demonstrated a statistically significant association of TNF-α production by mononuclear cells with polymorphic variants of the gene promoter regions -238>A (rs361525) and -857C>T (rs1799724). Carriers of -238GG/-308GG/-857CC/-1031NC genotype were characterized by low production of TNF-α, while carriers of -31TT/+3954CT genotype were characterized by low IL-1ß stimulation index in response to mitogen in comparison with carriers of other genotype combinations.

Tissue-specific expression of splice variants of human IL-4 and IL-6 gene mRNA.

The spectrum of alternatively spliced IL-4 and IL-6 gene mRNA was studied in peripheral blood mononuclears from healthy donors and in human fetal tissues. It was found that the expression of alternatively spliced IL-4 and IL-6 gene mRNA in fetal tissues is tissue specific and that hemopoiesis- and immunopoiesis-related tissues differ by the amount of IL-4 and IL-4δ2 mRNA. An mRNA variant IL-4alt3 carrying partial exon 3 deletion was for the first time identified in human mononuclear cells.

Evaluation of the expression of isoforms of stem cell factor mRNA in fetal tissues and mononuclear cells at different stages of human development.

We studied the expression of isoforms of stem cells factor mRNA forming as a result of alternative splicing. Both isoforms of stem cell factor mRNA forming as a result of alternative splicing were found in different fetal tissues. Changes in the expression of alternative isoforms of stem cell factor in peripheral blood mononuclear cells were demonstrated from the prenatal and neonatal periods to adult organism.

Ontogenetic features of the expression of mRNA isoforms for leukemia-inhibitory factor in human fetal tissues and mononuclear cells.

The expression of leukemia-inhibitory factor mRNA in human fetal tissues and mononuclear cells was studied during ontogeny. The expression of mRNA isoforms for leukemia-inhibitory factor was tissue-specific at the stage of prenatal development. The transition from antenatal and neonatal development to the postnatal period was accompanied by a decrease in the expression of mRNA isoforms for leukemia-inhibitory factor in mononuclear cells.

[Genetic and clinical and pathological characteristics of breast cancer in premenopausal and postmenopausal women].

This study involved 525 breast cancer (BC) patients of T2-4N0-2M0 stages at the age of 35 years and older. Significant differences in clinical and pathological characteristics between premenopausal and postmenopausal BC patients were found. Mostly marked differences were shown for positive lymph node correlation with distant metastasis, multicentric growth and local recurrence depending on menopause status. The prevalence of various morphological structures in primary tumors was appeared to be associated with different forms of tumor progression in pre- and postmenopausal women. We have studied polymorphisms in 15 genes involved in major cancer related pathways (apoptosis, interleukins, folate metabolism enzymes genes). We found that variant genotypes of MTHFR and DHFR genes were associated with an increased BC risk among premenopausal women while polymorphism in IL-18, p53 genes were associated with BC among postmenopausal women. These results demonstrate novel biological information, which points the different mechanisms contributed to breast cancer progression in premenopausal and postmenopausal women.

[Modulation of an antituberculous immune response in vitro, by using antigen-activated dendritic cells].

The protocol for preparation of mature antigen-activated dendritic cells (DC) from peripheral blood monocytes from patients with pulmonary tuberculosis was optimized. The obtained DCs were concurrently cultured with mononuclear cells (MNC) to activate antigen recognition, by and without adding IL-18 for the formation of directed differentiation of naive T cells to T helper cells type 1 (Th1). Joint cultivation of specific autologous DCs and MNCs from patients with pulmonary tuberculosis is an effective way of activation of the latter, which appeared as an increase in the proliferative potential, stimulation of IFN-gamma production and formation of cytotoxic cells expressing perforin in response to the specific Mycobacterium tuberculosis antigen ESAT-6. The use of recombinant IL-18 to enhance the induction of a Th1 response statistically significantly increases all the study parameters of MNC activation.

Enhanced expression of TNF-α type-1 receptors by immune cells in active pulmonary tuberculosis.

BACKGROUND: Tumour necrosis factor-alpha (TNF-α) and its inhibitors are involved in both defence against tuberculosis (TB) and damage to the host by TB. Notably, the change in receptor expression on cell density is a key mechanism in regulation of the biological properties of cytokines. OBJECTIVE: To study the differences in TNF-α receptor (TNFR) expression in patients with active pulmonary tuberculosis (aPTB) in correlation with the parameters of disease severity. METHODS: TNFR1/2 levels on peripheral blood mononuclear cells (PBMCs) from 45 patients with aPTB and 150 healthy controls were analysed by flow cytometry using monoclonal antibodies and QuantiBRITE beads. Soluble TNFR1/2 and TNF-α in serum were measured using an enzyme-linked immunosorbent assay. RESULTS: TNFR1 expression in aPTB patients was increased in the main populations of immune cells. Patients who were Mycobacterium tuberculosis culture-positive on bronchoscopy had higher levels of the soluble forms of TNFR1 (sTNFR1) than M. tuberculosis-negative patients. CONCLUSION: Active TB was shown to cause activation of different immune cell types by increasing TNFR1 expression on cells and reducing sTNFR1 expression compared with healthy controls. M. tuberculosis-positive patients with disseminated infection had the highest sTNFR1 serum level compared with other patients, but did not differ in receptor expression on PBMCs.

Relationship between interleukin-1 type 1 and 2 receptor gene polymorphisms and the expression level of membrane-bound receptors.

The biological activity of the multifunctional cytokine interleukin-1 (IL-1) is mediated by its receptors. The aim of this study was to determine if an association exists between single nucleotide polymorphisms (SNPs) in the IL-1 type 1 and 2 receptor genes (IL1R1 and IL1R2) and the expression level of membrane-bound IL1Rs on subpopulations of mononuclear cells or serum levels of soluble IL-1 receptors. It was observed that healthy individuals with the genotype TT in SNP rs2234650:C>T had a lower percentage of intact CD14(+) monocytes expressing IL1R1 on their surface. The SNP rs4141134:T>C in IL1R2 has also been associated with the percentage of intact CD3(+) T cells expressing IL1R2. Furthermore, individuals carrying the CC allele of SNP rs4141134:T>C and the TT allele of SNP rs2071008:T>G in IL1R2 had a lower density of IL1R2s on the surface of CD14(+) monocytes in lipopolysaccharide (LPS)-stimulated PBMC cultures. In summary, this study demonstrated that IL-1 receptor gene polymorphisms could be one of the factors influencing the expression of membrane-bound IL-1 receptors (IL1R) on immunocompetent cells.

Characterization of erythroid cell-derived natural suppressor activity.

Nucleated erythroid cells (NEC) have been previously reported to the capable of suppressing antibody-mediated primary (IgM) and secondary (IgG) immune responses to thymus-dependent antigens. In the present study we indicated that NEC, separated from the spleens of mice following phenylhydrazine treatment were able to suppress directly the proliferative response of preactivated B cells to lipopolysaccharide (LPS) in vitro. While being active in suppressing B cell blastogenesis, NEC, however, failed to reduce both cell proliferation and cytotoxic T lymphocyte (CTL) generation in an allogeneic mixed lymphocyte culture (MLC). NEC also lacked a significant effect on interleukin (IL)-2 production and utilization by concanavalin A (Con A)-activated T lymphocytes. The NEC-derived suppression of B cell proliferation was, at least in part, mediated by soluble molecules. The specific blockade of transforming growth factor (TGF)-beta synthesis with antisense oligodeoxynucleotides (OD) binding TGF-beta mRNA, as well as the neutralization of TGF-beta activity with anti-TGF-beta antibodies (Ab), resulted in a detectable diminished ability of the NEC-conditioned medium (CM) to suppress B cell blastogenesis. Taken together, the results suggest that: 1) NEC may suppress directly B cell responses, while not affecting T cell ones; 2) NEC may mediate their natural suppressor (NS) activity partially through releasing TGF-beta.

Production of cytokines by immature erythroid cells derived from human embryonic liver.

It is well known that regulatory interactions between hematopoietic and lymphoid cells are mediated by different mediators. The cells of erythroid lineage are not an exception and have a regulatory effect on hemato- and immunopoiesis that can be mediated through the production of cytokines i.e. by soluble factors - a universal mechanism for cell regulation in hematopoietic and immune systems. It has been previously shown that erythroid progenitor cells from mice express mRNA of cytokines such as IL-1 alpha and beta, IL-4, IL-6, IFN-gamma, GM-CSF and TGF-beta. In this report we present the results of the production of the main immunoregulatory cytokines by erythroid cells derived from human embryonic liver. It was revealed that the cell population enriched with erythroid progenitors, isolated from human fetal liver, can produce IL-1 beta, IL-2, IL-4, IL-6. The levels of production of cytokines by immature erythroid progenitor cells is compared to the levels of corresponding cytokines produced by mitogen-stimulated peripheral blood mononuclear cells. The production of these cytokines changed quantitatively under the effect of erythropoietin, and are correlated with the expression of differentiation markers of erythroid cells such as AG-EB and Glycophorin A. The role of cytokine production by erythroid cells in hemato- and immunopoiesis and the mechanisms of self-regulation of proliferation and differentiation of erythroid progenitor cells is discussed.

Cytokine gene expression in erythroid cells.

Erythroid nuclear cells have been shown to exert regulatory effects on immunopoiesis. We have reported that some of these influences might be mediated via soluble factors secreted by nuclear erythroid cells. In this report we describe our estimate of the cytokine gene expression in cells isolated from individual erythroid colonies by Reverse transcription-Polymerase chain reaction. We found in erythroid cells, originated from the bone marrow precursors obtained from phenylhydrazine-treated mouse, the expression of the following cytokine genes: IL-1 alpha and IL-1 beta, IL-4, IL-6, GM-CSF, gamma-IFN and TGF-beta. In contrast, the erythroid cells derived from newborn mouse spleen precursor cells expressed IL-1 alpha, IL-1 beta, IL-4, IL-6 and GM-CSF mRNAs but not gamma-IFN and TGF-beta mRNAs. No detectable levels of IL-2, IL-3 and IL-5 mRNAs were expressed in nuclear erythroid cells. These data provide evidence of the expression of mRNAs coding in the set of immunoregulatory cytokines in immature erythroid progenitor cells in mouse.

[Immunotropic properties of the causative agent of viral hepatitis C]. / Immunotropnye svoistva vozbuditelia virusnogo gepatita C.

In this review the immunomodulating properties of the causative agent of viral hepatitis C are characterized on the basis of experimental data, obtained by Russian and foreign researchers during recent 3-5 years. The short characterization of the causative agent is presented and a number of adaptation mechanisms making it possible for hepatitis C virus to resist the action of the immune protective system of the host are considered. The role of individual protein products of the virus in the immunopathogenesis of the disease and the mechanisms of their action on the molecular level are discussed in detail on the basis of the results of mouse and in vitro experiments.