Oxidative stress and aging are known to alter the satellite III repeat (1q12) (SatIII(1q)) and telomere repeat (TR) copy numbers (CNs) in DNA of human cells. Extreme conditions of Antarctica could potentially affect the CNs of the repeats in human blood cells, the effect being possibly associated with inhibition of the antioxidant system and activation of apoptosis. The ribosomal DNA (rDNA), SatIII(1q), and TR repeat CNs were studied in leukocytes of 11 men who participated in an expedition to the Vostok Station from 2019 to 2020. To observe the dynamic changes in repeat CNs and the degree of repeat oxidation, six blood samples were taken: before the arrival in Antarctica and after 27, 85, 160, 270, and 315 days of wintering. To analyze the adaptive changes, expression levels were measured for the genes coding for BAX, BCL2, NOX4, NRF2, SOD1, and HIF1. A decrease in SatIII(1q) CN and an increase in TR CN were detected, while the rDNA CN remained stable in human blood cells during wintering. The changes, along with a decrease in the oxidation marker 8-oxodG in DNA, were associated with an increase in NOX4 activity, a decrease in NRF2 activity, and an increase in expression of the proapoptotic protein BAX. Wintering in Antarctica was therefore assumed to stimulate the adaptive response in the human body, including higher elimination of “ballast” cells with a higher level of DNA oxidation, a higher SatIII(1q) content, and a lower TR content from the bloodstream. An increase in ROS due to chronic NOX4 activation and a NRF2 blockage might play a significant role in the response.

The paper presents data of the investigations of subclinical reactivation of 7 human herpes viruses and bacteria Mycoplasmahominis, Ureaplasmaurealiticum and Chlamydiatrachomatis from 6 participants in a 14-day isolation study conducted in a small chamber with controlled environment. Purpose of analysis was to establish correlations between DNAs of latent viral and bacterial pathogens in bio-liquids of organism and indices of the natural and adaptive immune system. It was determined that short-term isolation in a small chamber may provoke reactivation of latent pathogens both of the viral and bacterial nature. The events can be triggered by changes in the immune status and appear in consequence of depressed immunological control of latent infections. Dynamics of the latent infections lytic cycle in the course of the study demonstrated a high degree of individual variability.

The authors present the results of studying the processes involved in maturation of dendritic cells (DCs) derived from CD14+-monocytes and TCR-activation of peripheral blood lymphocytes from 9 ISS Russian crew members. It was demonstrated that long-term habitation on low Earth's orbits brings about disorders both in DCs and Т-lymphocytes evidenced by reductions in mature DCs expressing the В7 family of co-stimulating molecules and also Т-lymphocytes expressing the molecules of positive CD28 co-stimulation. The investigation found an increased number of tolerogenic DCs with the CD80+CD86- immunophenotype and T-cells expressing the negative co-stimulation molecules CD152. TCR-stimulation of lymphocytes in vitro enabled detection of changes in the processes of associated with T-lymphocyte differentiation and synthesis of cytokines. The immune system reaction to prolonged exposure to the aggregate of spaceflight factors was distinctively individual.

The complex of space flight (SF) factors can have a negative impact on the human organism, including innate and adaptive immunity. One of the negative effects of impaired functional activity of the immune system is a decrease of latent immunological control of infections. During short- and long-term SFs, reactivation of latent viruses was shown, while the virus was detected in body fluids for some time after returning to Earth. Despite the fact that in most cases reactivation was asymptomatic, there are fears that during long expeditions into deep space, reactivation of latent pathogens can lead to the development of diseases that are dangerous to the crewmembers’ health. There is a lot of data about the reactivation of viral pathogens during SF, but the reactivation of bacterial agents in cosmonauts has received almost no attention. Due to the complexity of conducting research in space, an important role is played by ground-based analog experiments that simulate space station conditions. One of the most significant analogs of SF is the long-term Antarctic expedition, which makes it possible to obtain unique information about the effect of isolation, increased mental, and physiological stress on the reactivation of latent pathogens. Currently, latency is increasingly considered as a symbiotic existence of a pathogen and host, during which the human body acquires additional resistance to certain infectious agents. Thus, an important task of the upcoming research is a comprehensive analysis of the personal immunological status during SF, establishing its relationship with the latent infections reactivation and developing systems for monitoring, preventing, and treating the negative consequences of reactivation.

Innate immunity was investigated in six volunteers aged 28 to 44 years participating in the experiment with 120-day isolation in a controlled environment. Peripheral blood samples collected during isolation were analyzed to determine the content of monocytes and granulocytes expressing intracellular (TLR3, TLR8, TLR9) and surface (TLR1, TLR2, TLR4, TLR5, TLR6) TLRs, as well as the content of natural killer lymphocytes (CD3–CD16+CD56+). The system of pattern-recognition receptors of innate immunity cells exhibited heterogenic reaction to isolation with predominant activation throughout the entire experimental exposure. The revealed changes seem to reflect a complex adaptive process that occurs in the human immune system during the establishment of a dynamic balance with extreme environmental factors, including response to endogenous and exogenous immune stimuli.

Here, we report the changes in the expression of TLR-associated genes, genes of NF-kB, AP-1, and TLR4 pathways, genes coding for apoptotic proteins, cytokines, and chemokines, after rotation on the short-arm centrifuge. The study involved 10 almost healthy male volunteers aged 25-40. CD14+ monocytes were isolated from the testers’ blood before and after the exposure to the artificial gravity. The cells were incubated with TLR1-9 ligands for 24 hours to assess the monocytes’ reserve potential. The gene expression was evaluated by real-time PCR. In sum, no negative effect of rotation on the short-arm centrifuge on the functioning of TLR-associated branch of innate immunity was detected.

The results of studying the correlations between biochemical and immunological parameters in volunteers under conditions of 21-day “dry” immersion are presented. It is shown that, in comparison with the baseline period, at the final stage of the experiment, the number of statistically significant correlations (p ≤ 0.05, respectively, 27 and 51) between the parameters assessing the metabolic and immunological reactions of the body increased. The increase in the number of correlations was due mainly to an increase in the number of correlations between the indices of protein, carbohydrate, and lipid metabolism and indices characterizing the state of innate immunity. At the final stage of the experiment, the influence of protein and lipid metabolism on the cellular component of adaptive immunity increased. The revealed dynamics and the nature of correlations between biochemical and immunological indices indicate the metabolic regulation of the immune response under conditions of hypodynamic support unloading.

Показатели клеточного иммунитета человека в условиях создания искусственной гравитации с помощью центрифуги короткого радиуса 1 Федеральное государственное бюджетное учреждение науки Государственный научный центр Российской Федерации Институт медико-биологических проблем Российской академии наук, Министерство науки и высшего образования Российской Федерации

During space missions cosmonauts are exposed to a myriad of distinct stressors such as radiation, overloads, weightlessness, radiation, isolation in artificial environmental conditions, which causes changes in immune system. During space flights it is very difficult to determine the particular factor associated with the observed immunological responses. This makes ground-based experiments examining the effect of each space flight associated factor along of particular value. Determining mechanisms causing alterations in cosmonauts' immunity can lead to potential targets for different countermeasures. In the current article we present the study of the early period of adaptation of human innate immunity of 6 healthy test-subjects, 4 males and 2 females aged 25 through 40, to isolation factors (hypodynamia, psychological stress, artificial environment). We measured multiple parameters characterizing innate immunity status in blood samples at chosen time points before, during and after the mission. In the experiment, highly enhanced cytokine responses were observed upon ex vivo antigen stimulations in comparison to baseline values. For cellular parameters we found multidirectional dynamics with a persistent prevalence of increasing TLRs+ monocytes as well as TLRs expression. Our study provides evidence that even a short-term confinement leads to immune changes in healthy humans that may trigger aberrant immune response.

Space flight factors are known to cause a malfunction in the human immune system and lead to damage to blood vessels. The hemostatic function of endothelium during space missions and its interaction with human immunity has not been determined so far. In this work, we investigated the markers of endothelial activation and damage (plasma concentrations of soluble thrombomodulin fraction (sTM), von Willebrand factor (vWF), highly sensitive C-reactive protein (hs-CRP)), as well as the level of D-dimer and compared them to the immunological parameters characterizing the state of human humoral and cellular immunity. The immune status of long-duration ISS crewmembers was assessed by whole-blood testing, and comprehensive postflight immune assessment included the analysis of leukocyte distribution. Flow cytometry was applied to determine the absolute counts and the percentage of lymphocyte subsets: B cells (CD19 + ), T cells (CD3 + , CD3 + CD4 + , CD3 + CD8 + ), NK cells (CD3 − CD16 + CD56 + , CD11b + CD56 + ), and activated subsets (CD3 + CD25 + and CD3 + HLA-DR + ). The in vitro basal cytokine production was investigated in whole blood cell culture. The cytokines IFN-gamma, IL-1-beta, IL-4, IL-6, IL-10, IL-18, and TNF-alpha were measured in plasma and the 24-h supernatants by a sensitive enzyme-linked immunosorbent assay. A significant increase in the plasma levels of vWF and hs-CRP and a decrease in the concentration of sTM after spaceflights were detected. Divergent changes in the parameters characterizing the state of the immune system were observed. We propose that the changes revealed may lead to an increase in the procoagulant activity of blood plasma, suppression of protein C activation and thrombin inhibition, as well as to an increase in the adhesive-aggregate potential of platelets, especially in case of changes in the rheological characteristics of blood flow during re-adaptation to ground conditions. We also speculate that the immune system might play an important role in vessel damage during long-duration missions.

Expression of inborn immunity genes was studied in 10 essentially healthy male subjects aged 24 to 32 years in a 21-day dry immersion experiment without countermeasures. To evaluate the humoral immunity function, genes encoding Toll-like receptors, proteins mediating NF-kB, AP-1, Akt, TLR4; apoptosis proteins, and CLEC4E, HSPD1, PTGS2 were selected. Fenol-cloroform extraction was used to obtain total RNA from peripheral blood monocytes CD14+. Subsequently, total RNA was reverse transcribed to generate cDNA for real-time PCR. The results showed substantial changes in expression of these genes suggesting that changes in the TLRs function on the transcriptional level were at the very start of the immune response to 21-d DI.

Влияние дыхательных гипоксических газовых смесей (кислородно-азотной и кислородно-азотно-аргоновой) в условиях барокамеры на показатели клеточного иммунитета человекаФедеральное государственное бюджетное учреждение науки Государственный научный центр Российской Федерации -Институт медико-биологических проблем Российской академии наук

Cytomegalovirus (CMV)-specific T-cells from healthy donor are used as a treatment of CMV infection in patients receiving immunosuppressive therapy following transplantation. To choose the optimal donor and to select the best isolation method (MHC-tetramer or IFNy secretion assay) CMV-specific T-cells should be characterized by specificity and functionality. We compared the antigen specificity, diversity and functionality of T-cell responding to CMV pp65 and to three pp65-derived immunodominant epitopes: NLV restricted by HLA-A*02, TPR and RPH restricted by HLA-B*07. 87 healthy donors were divided into 4 groups according to the presence of HLA-A*02 and/or HLA-B*07. CMV-specific response was analyzed by flow cytometry. For 6 donors antigen-specific cells were isolated by MHC-tetramer staining and pp65 IFNγ secretion assay followed by FACS. cDNA libraries of T-cell receptor β-chains were prepared and sequenced by NGS. We found that pp65-specific T-cell response (IFNy-producing T-cell after stimulation with pp65 CMV) was strongly focused on pp65-derived epitopes NLV, TPR and RPH in fixed hierarchy. NLV-specific clones constituted most pp65-specific cells in presence of HLA-A*02 but not when HLA-B*07 was also present. NLV-specific fraction consisted of few large clones, TPR− and RPH-specific cells were more diverse. Many MHC-tetramer+ clones were non-functional as they were not detected in IFNy+ fraction after pp65 stimulation. In summary, HLA-genotype significantly affects magnitude and structure of pp65-specific T-cell response. It should be taken into consideration for choosing a donor of CMV-specific T cells. IFNy secretion assay produces more diverse and more functional cell product.

Background: Cytomegalovirus (HCMV) infection is one of the major complications and causes of mortality in patients receiving immunosuppressive therapy following hematopoietic stem cell transplantation (HSCT). Antiviral drugs have limited efficacy and high toxicity while using CMV-specific T cells from healthy individuals as a cellular vaccine had proven to be safe and efficient. Conventional protocols rely on expanding virus-specific cells ex vivo while more recently it was proposed to transfuse relatively small numbers of minimally manipulated cells and allow them to expand in vivo. To isolate virus-specific cell one could use either the stimulation with overlapping pools of peptides covering the whole immunogenic viral protein followed by isolation of INFγ-producing cells, or direct isolations of cells bound to MHC-multimer loaded with known immunodominant peptides. Both methods have their advantages. The former approach isolates all activated clones irrespective of their MHC-restriction and supplements CD8+ cells with CD4+ fraction, while the latter method is faster and simpler, besides it captures cells irrespective of their ability to produce IFNγ. The efficiency of the two methods was not directly compared and it is currently unknown to what extent T-cell populations isolated by these two approaches are different in terms of absolute quantity and clonal composition. Aims: to compare the quantity and T-cell receptor repertoire composition of T cells specific to pp65 CMV and to pp65-derived immunodominant epitopes NLV restricted by HLA-A*02, TPR and RPH restricted both by HLA-B*07. Methods: PBMC of healthy donors with different combination of HLA-A*02 and HLA-B*07 alleles were stimulated with peptides of immunodominant epitopes or overlapping peptide pool covering the entire pp65 CMV (Miltenyi Biotec, Cat.-130-093-435). Antigen-specific T cells were detected by the intracellular staining for IFNγ and flow cytometry. We obtained fractions of virus-specific T cells from PBMC either by MHC-tetramer staining or IFNγ secretion assay followed by fluorescent activated cell sorting. Then we prepared cDNA libraries of TCR α- and β-chains and sequenced them using NGS. Characterization of TCR repertories was performed by proprietary bioinformatic pipeline. Results: T-cell response to pp65 CMV is strongly focused on immunodominant pp65-derived epitopes. In donors having HLA-A*02 and/or HLA-B*07 absolute majority of pp65-reactive cells are specific to one of the three immunodominant epitopes - NLV, TPR and RPH. Immunodominant antigens are structured in a fixed hierarchy. TPR and RPH are superior to NLV. When HLA-B*07 is present response to the NLV epitope is drastically reduced, most of the pp65-specific cells recognize either TPR or RPH epitopes. TCR repertoire of the NLV-specific T cells is highly skewed and consists of few large dominant clones. TPR and RPH-specific T cells are more clonally diverse. Substantial share of the cells belonging to the large clones specific to the immunodominant antigens do not secrete IFNγ upon antigenic stimulation. Nevertheless, they may be therapeutically valuable, as they might secrete other cytokines. Conclusion: In summary, CMV-specific response in donors positive for HLA-A*02 and/or HLA-B*07 is focused to the combination of three immunodominant epitopes - NLV, TPR and RPH. Substantial share of the cells are not IFNγ-producers. For the patients having these alleles it might be beneficial to use MHC-multimers for isolation of the therapeutic lymphocytes. Figure 1: A - Healthy donors with different combination of HLA-A*02 and HLA-B*07 B - Intracellular staining for IFNγ of CD8+ T cells after stimulation with pp65-derived peptides C - NGS to identify clonally diverse of pp65-sprcific T cells on combination of HLA-A*02 and HLA-B*07 Figure 1 Disclosures No relevant conflicts of interest to declare.

Background:Cytomegalovirus (CMV) is the opportunistic infection that persists in healthy individuals asymptomatically and reactivates in immunodeficient host, e.g. after allo‐HSCT. The main cause of viral reactivation is the lack of CMV‐specific T cells. The adoptive cellular therapy with CMV‐specific T cells seems very attractive clinical option to accelerate virus‐specific T‐cell reconstitution after allo‐HSCT. Here we report the preliminary data about CMV‐specific T‐cell reconstitution in allo‐HSCT patients.Aims:To identify a group of patients after allo‐HSCT who are in high‐risk of CMV reactivation and could be eligible for prophylaxis with CMV‐specific T cells.Methods:To detect CMV‐specific CD8+ cells we utilized MHC I tetramers loaded with the two immunodominant epitopes of viral pp65 protein: NLVPMVATV (NLV) and TPRVTGGGAM (TPR) presented in HLA‐A∗02 and ‐B∗07 respectively.We analyzed CD3+CD8+ cytotoxic T‐lymphocytes (CTLs) in peripheral blood and bone marrow of 7 patients on day +30 after allo‐HSCT. All patients were CMV‐seropositive. 5 patients were HLA‐A∗02 positive and 2 HLA‐B∗07 positive.Leukocyte cell suspension (5∗106 white blood cells per test) after preliminary red blood cells lysing with Lysing Buffer (BD Pharm Lyse™) were incubated with the protein tyrosine kinase inhibitor dasatinib for 1 hr at 37°С to increase surface expression of both TCR and CD8. NLV/TPR‐ specific cytotoxic T‐lymphocytes (CTLs) were identified by flow cytometry using PE‐Cy7 labeled anti‐CD45 antibody (Ab), Alexa Fluor 700 labeled anti‐CD3 Ab and PerCP‐Cy5.5 labeled anti‐CD8 Ab (Sony Biotechnology Inc.) and in house produced MHC class I tetramers conjugated with Phycoerythrin and loaded with NLV or TPR peptides. Alexa Fluor™ 750 NHS Ester (Succinimidyl Ester) was used (Invitrogen™) to exclude dead cells. Flow cytometry was performed on a BD FACSCanto™ II Flow Cytometer using BD FACSDiva™ software. Two‐platform method was used to calculate absolute CMV‐specific CD3+CD8+ count in peripheral blood.Results:According to our data, the lowest rates of CMV‐specific CTLs are presented in patients who underwent allo‐HSCT with post‐transplant high‐dose cyclophosphamide (PT‐Cy, 50 mg/kg on +3, +4 day) as GVHD prophylaxis.Results are presented in Table 1.Summary/Conclusion:Clinical option to accelerate virus‐specific T‐cell reconstitution using adoptive cellular therapy with CMV‐specific T cells seems very attractive. Modern technologies make this possible but first we should identify the patients after allo‐HSCT who benefit from this effective but very expensive therapy. Patients with using PT‐Cy as GVHD prophylaxis are the possible candidates for this treatment. Further studies CMV‐specific T‐cell reconstitution will help to expand the group of patients in high risk.image

Interleukin-8 (IL-8, CXCL8) belongs to major chemokines to stimulate migration of neutrophils and monocytes/macrophages (Mc/Mphs) into the inflammation sites. We studied the direct effects of IL-8 on the functionality of human Mc/Mphs in vitro. CD14-positive cells were isolated from human peripheral blood mononuclear cells (PBMCs) by positive magnetic separation and were further cultured with or without lipopolysaccharide (LPS, 1.0 μg/ml) for 24 h. We showed that upon LPS activation of Mc/Mphs, IL-8 reduced markedly both the percentages and median fluorescence intensity (MFI) of CD16 (FcγRIII)-positive cells among CD14high cells, as well as in cells that reduced the expression of СD14 during their culturing. IL-8 was also found to be capable of reducing the expression of СD124 (IL-4 receptor subunit alpha, IL-4RA), with concomitant enhancement of the expression of both CD119 (interferon-gamma receptor 1) and CD197 (CCR7) in Mph cells. In addition, IL-8 up-regulated production of IL-6 and IL-1β [but not tumor necrosis factor-α (TNF-α) and IL-10] by activated Mc/Mphs. Our results suggest the ability for IL-8 to directly favor pro-inflammatory M1-type Mph activity.

We investigated the direct effects of human granulocyte colony-stimulating factor (G-CSF) on functionality of human T-cell subsets. CD3(+) T-lymphocytes were isolated from blood of healthy donors by positive magnetic separation. T cell activation with particles conjugated with antibodies (Abs) to human CD3, CD28 and CD2 molecules increased the proportion of cells expressing G-CSF receptor (G-CSFR, CD114) in all T cell sub-populations studied (CD45RA(+)/CD197(+) naive T cells, CD45RA(-)/CD197(+) central memory T cells, CD45RA(-)/CD197(-) effector memory T cells and CD45RA(+)/CD197(-) terminally differentiated effector T cells). Upon T-cell activation in vitro, G-CSF (10.0 ng/ml) significantly and specifically enhanced the proportion of CD114(+) T cells in central memory CD4(+) T cell compartment. A dilution series of G-CSF (range, 0.1-10.0 ng/ml) was tested, with no effect on the expression of CD25 (interleukin-2 receptor a-chain) on activated T cells. Meanwhile, G-CSF treatment enhanced the proportion of CD38(+) T cells in CD4(+) naive T cell, effector memory T cell and terminally differentiated effector T cell subsets, as well as in CD4-central memory T cells and terminally differentiated effector T cells. G-CSF did not affect IL-2 production by T cells; relatively low concentrations of G-CSF downregulated INF-gamma production, while high concentrations of this cytokine up-regulated IL-4 production in activated T cells. The data obtained suggests that G-CSF could play a significant role both in preventing the development of excessive and potentially damaging inflammatory reactivity, and in constraining the expansion of potentially cytodestructive T cells.

Interleukin-8 (IL-8, CXCL8) is one of the main chemokines that stimulates the migration of neutrophils, monocytes and lymphocytes into the inflammatory focus. The aim of this study was to investigate the direct influence of IL-8 on the functionality of human monocytes/macrophages (Mc (Mph)) upon their activation by lipopolysaccharide (LPS). CD14+ cells were isolated from blood mononuclear cells (MNCs) by positive magnetic separation. Surface markers (CD16, CD119, CD124, CD197) in Mc (Mph) cultures were determined by flow cytometry, while IL-10, IL-6, IL-1 ß and tumor necrosis factor-а (TNF-а) concentrations in cell supernatants by solid-phase enzyme-linked immunosorbent assay. IL-8 was found to be capable of significantly reducing the number of CD16+ (FcyRIII) cells among activated Mc (Mph). At the same time, IL-8 detectably increased the number of cells expressing CD1 1 9 (receptor to interferon-y) and CD197 (CCR7), reducing the number of cells carrying CD124 (receptor to IL-4). In addition, IL-8 was able to enhance the secretion of IL-6 and IL-1 ß by activated Mph cells, without significantly affecting the production of TNF-а and IL-10. The data obtained indicate the ability of IL-8 to directly favor the pro-inflammatory activity of Mph cells.

CD3+T lymphocytes were isolated from normal donors by positive magnetic separation. Activation of the T cells with particles conjugated with antibodies to CD3, СD28 and СD2 molecules led to substantial increase in T cell production of interleukin-8 (IL-8). An interleukin-8 receptor (CXCR1, CD181) was initially expressed in 13.3% of T lymphocytes. Activation of T lymphocytes resulted into a detectable increase of CD181+ cell number among CD4+ naïve cells and CD4+ terminally-differentiated effector cells, and, conversely, into decrease of their number among CD4+ effector memory cells. Activation of T lymphocytes was assessed by membrane expression of CD25 molecule (receptor for IL-2). IL-8 (0.01-10.0 ng/ml) was shown to markedly reduce activation of both CD4- and CD4+ effector memory T cells, as well as terminallydifferentiated T effectors, without significantly affecting activation of naive T lymphocytes and central memory T cells. IL-8 noticeably increased IL-2 production by activated Т cells, caused a reduced IL-10 production, and did not significantly affect the secretion of IFNγ and IL-4. The data obtained suggest a significance of IL-8 for direct regulation of adaptive T cell responses.