Peripheral Blood Lymphocyte Populations Research Articles

The role of immune factors in the development of preterm birth

Preterm birth (PB) carries a high risk of developing neuropsychological development disorders, cognitive and somatic problems in the child. The maternal immune system plays a critical role in maintaining a physiological pregnancy. However, specific mechanisms and disorders of pregnancy development have been little studied. There is a lack of research on the role of immune competent cell (ICC) activation in preterm labor leading to uterine contractility. Understanding the importance of immune system factors in the formation of preterm birth may allow the development of strategies to prolong pregnancy and, thus, lead to improved perinatal outcomes. The purpose of the work is to study the role of immune factors in the development of preterm birth. Seventy pregnant women in the third trimester with recurrent miscarriage (RPL) and 25 healthy pregnant women (control group) were examined. Observed pregnant women with RPL were divided into two groups: Group I – patients who gave birth prematurely (n = 30); and Group II – patients who gave birth at term (n = 40). The population and subpopulation composition of peripheral blood lymphocytes was studied by cytofluorimetry using monoclonal antibodies to: CD3, CD4, CD8, CD16, CD19 (JSC "Sorbent", Russia) (FITC), CD11b (Beckman Coulter, USA); CD14, CD25, CD69, CD71, CD28, CD107a, HLA-DR (Beckman Coulter, USA) (PE). To create a database and conduct statistical research, the capabilities of an Excel spreadsheet and application packages (Megastat and Statistica 6.0) were used. When determining statistical significance between the study groups, the Mann-Whitney test was used for independent groups and the Wilcoxon test for dependent groups. Modulation of the number and functional activity of ICC in patients with RPL had a significant correlation with pregnancy outcome. Inadequate activation of the immune system is a factor that can lead to miscarriage. Termination of pregnancy before term is associated with subpopulation shifts, increased activation potencies of immunocompetent cells compared to full-term birth, which is an important feature of the inflammatory response. The identified immune changes will allow the development of early prevention methods and new approaches to the treatment of this pregnancy complication.

Unswitched memory B cell deficiency in children with sickle cell disease and response to pneumococcal polysaccharide vaccine.

Early mortality in sickle cell disease (SCD) is attributed to increased infections due to loss of splenic function. Marginal zone B cells are important for initial opsonization of pathogens and can be absent in spleen histopathology in SCD. The frequency of unswitched memory B cells (UMBC), the circulating correlate of marginal zone B cells, reflects the immunologic function of the spleen. We hypothesized that asplenia in SCD is associated with alterations in the peripheral blood lymphocyte population and explored whether UMBC deficiency was associated with a clinical phenotype. We analyzed B cell subsets and clinical history for 238 children with SCD and 63 controls. The median proportion of UMBCs was lower in children with SCD compared with controls (4.7% vs. 6.6%, p < .001). Naïve B cells were higher in SCD compared with controls (80.6 vs. 76.3%, respectively, p = .02). UMBC frequency declined by 3.4% per year increase in age in SCD (95% CI: 2%, 4.7%, p < .001), but not in controls. A majority of children in all cohorts had an IgM concentration in the normal range for age and there were no differences between groups (p = .13). Subjects developed titers adequate for long-term protection to fewer serotypes in the polysaccharide vaccine than controls (14.7 vs. 19.4, p < .001). In this cohort, bacteremia was rare and specific clinical complications were not associated with UMBC proportion. In summary, UMBC deficiency occurs in SCD and is associated with age. Future studies should investigate B cell subsets prospectively and identify the mechanism of B cell loss in the spleen.

Participation of the spleen in the neuroinflammation after pilocarpine-induced status epilepticus: implications for epileptogenesis and epilepsy.

Epilepsy, a chronic neurological disorder characterized by recurrent seizures, affects millions of individuals worldwide. Despite extensive research, the underlying mechanisms leading to epileptogenesis, the process by which a normal brain develops epilepsy, remain elusive. We, here, explored the immune system and spleen responses triggered by pilocarpine-induced status epilepticus (SE) focusing on their role in the epileptogenesis that follows SE. Initial examination of spleen histopathology revealed transient disorganization of white pulp, in animals subjected to SE. This disorganization, attributed to immune activation, peaked at 1-day post-SE (1DPSE) but returned to control levels at 3DPSE. Alterations in peripheral blood lymphocyte populations, demonstrated a decrease following SE, accompanied by a reduction in CD3+ T-lymphocytes. Further investigations uncovered an increased abundance of T-lymphocytes in the piriform cortex and choroid plexus at 3DPSE, suggesting a specific mobilization toward the Central Nervous System. Notably, splenectomy mitigated brain reactive astrogliosis, neuroinflammation, and macrophage infiltration post-SE, particularly in the hippocampus and piriform cortex. Additionally, splenectomized animals exhibited reduced lymphatic follicle size in the deep cervical lymph nodes. Most significantly, splenectomy correlated with improved neuronal survival, substantiated by decreased neuronal loss and reduced degenerating neurons in the piriform cortex and hippocampal CA2-3 post-SE. Overall, these findings underscore the pivotal role of the spleen in orchestrating immune responses and neuroinflammation following pilocarpine-induced SE, implicating the peripheral immune system as a potential therapeutic target for mitigating neuronal degeneration in epilepsy.

Membrane (CD8⁺PD-1⁺ and CD4+PD-1⁺) and soluble (sPD-1 and sPD-L1) forms of immune checkpoints in melanoma, breast cancer, and oral mucosal cancer patients: A observational study

Background. The PD-1/PD-L1 pathway plays an important role in tumor evasion from immunological surveillance. In addition to the membrane forms of PD-1 and PD-L1, there are soluble variants: sPD-1 and sPD-L1. Both membrane and soluble forms have immunoregulatory properties and can affect the function and number of different immune cell populations.&#x0D; Aim. To study the relationship between the initial level of CD8⁺PD-1⁺ and CD4+PD-1⁺ lymphocytes and soluble forms of sPD-1 and sPD-L1 with the percentage of the main effector and regulatory populations of peripheral blood (PB) lymphocytes and tumor-infiltrating lymphocytes.&#x0D; Materials and methods. The study included melanoma, breast cancer and the oral mucosa cancer patients. The percentage of cell populations of PB lymphocytes and tumor-infiltrating lymphocytes was determined by flow cytometry before treatment. The concentrations of sPD-1 and sPD-L1 proteins were studied in blood serum using enzyme immunoassay.&#x0D; Results. The relationship of the level of CD8⁺PD-1⁺ cells with certain populations of CD8-lymphocytes in PB and tumor tissue was found. In the PB of melanoma patients with CD8⁺CD11b⁺CD28⁺ and CD8⁺CD11b⁻CD28⁻ T cells, in breast cancer patients with a population of CD8⁺CD11b⁺CD28⁺ lymphocytes. In the tumor tissue of all patients there was a positive correlation with a population of regulatory CD8⁺CD11b⁻CD28⁻ T cells. The immunoregulatory properties of sPD-1 and sPD-L1 were confirmed. Both sPD-1 and sPD-L1 levels were positively correlated with the number of suppressor CD8⁺CD11b⁻CD28⁻ T cells and negatively with the level of CD8 lymphocytes, CD8⁺CD11b⁺CD28⁺ cytotoxic/memory T cells, B cells and activated CD25 lymphocytes.&#x0D; Conclusion. The results of the study can make a certain contribution to the study of the prognostic significance of membrane and soluble forms of PD-1 and PD-L1, taking into account the peculiarities of their relationship with suppressor and effector populations of lymphocytes of systemic and local immunity.

PSX-9 Gonadectomy Is Not Correlated with Altered T-Lymphocyte Populations in Senior Labrador Retrievers

Abstract Retrospective studies have suggested that gonadectomies, such as the spays and neuters common in companion animal populations, may be correlated with the development of autoimmune conditions later in the life of an animale. This study investigated the potential influences of gonadectomy on peripheral blood lymphocyte populations through flow cytometry. Twenty senior Labrador Retrievers (10 males and 10 females) were randomly selected from Four Rivers Kennel’s existing research colony according to their intact or altered status. Groups were balanced by age and body weight (average 11.4 y/o and 64.5 kg) as both have been previously shown to alter lymphocyte populations. A single blood collection was performed and stained with canine cross-reactive conjugated antibodies CD3-FITC, CD4-Superbright 600, CD8-Superbright 702, and a fixable lime viability stain (Invitrogen, Waltham MA). Samples were processed through a whole blood based lyse no-wash method and analyzed on an Attune NXT Flow Cytometer equipped with blue and violet lasers (ThermoFisher Waltham, MA). Measurement of cell populations was completed on a defined volume of each sample, and all subsets were gated to live, singlet lymphocytes. Statistical significance was determined through one-way ANOVAs on JMP Pro 16 (Cary, NC). There were no significant differences in CD4+/CD8+ ratio between groups (P = 0.56), or total lymphocyte populations (P = 0.34). Additionally, there were no significant differences between groups regarding CD3+ MFI (P = 0.25), CD8+ MFI (P = 0.70), or CD4+ MFI (P = 0.43). Overall, in age and body weight balanced senior Labrador Retrievers, spay or neuter status did not significantly influence T-cell populations. This suggests that previous gonadectomy does not influence the adaptive immune system as an animal ages. Further research is needed to clarify the relationship between gonadectomy and the later development of autoimmune disorders.

Clinical and pathogenetic significance of immunity factors in congenital generalized infection caused by the herpes simplex virus

Intrauterine infections are serious diseases that largely determine the level of infant mortality. Newborns who have had intrauterine infections often have long-term consequences, leading to disability. One of the intrauterine infections is a congenital infection caused by the herpes simplex virus (neonatal herpes). Neonatal herpes occurs less frequently (1:2000 live births) than cytomegalovirus fection, but the clinical symptoms in this disease are characterized by multiorgan damage.Purpose. To determine the role of immune factors in the development of congenital generalized HSV infection.Material and methods. Twenty-two newborns with a severe form of congenital generalized infection caused by the herpes simplex virus infection were examined (group I). The control group consisted of 26 healthy newborns born to women with uncomplicated pregnancy and childbirth. Determination of the population and subpopulation composition of peripheral blood lymphocytes and monocytes, the level of expression of activation markers, T-regulatory cells (Treg) was carried out by laser fl w cytometry using reagents from Immunotex (France), Caltag (USA), HyCultbiotechnology (Netherlands): FITC (fl escein isothiocyanate) — labeled CD3+, CD4+, CD8+, CD 16+, CD19+, CD282+ and PE (phycoerythrin)-labeled CD95+, CD25+, CD14+. Determination of the number of lymphocytes that have entered apoptosis using a diagnostic kit including Annexin-V, labeled with FITC and propidium iodide (PI), (Caltag, USA). The concentration of IFN-γ, IFN-α, IL-12 in the blood serum of newborns was determined by ELISA using BenderMedsistems test systems.Results. The development of a congenital generalized infection caused by the herpes simplex virus is associated with a lack of IFN-α, IFN-γ, IL-12 production, a decrease in the number of monocytes expressing TLR-2, a decrease in the relative number of CD8+, CD16+ lymphocytes, CD25+ activation markers, on the surface of NK cells, in combination with an increase in CD16 + CD95 +, AnnexinV + PI +, the number of Tregs.Conclusion. The results of the work indicate suppression of the early stages of the innate immune response, impaired effector function of immunocompetent cells, apoptosis processes

Alterations in Natural Killer Cells in Colorectal Cancer Patients with Stroma AReactive Invasion Front Areas (SARIFA).

Recently, our group introduced Stroma AReactive Invasion Front Areas (SARIFA) as an independent prognostic predictor for a poorer outcome in colon cancer patients, which is probably based on immunologic alterations combined with a direct tumor-adipocyte interaction: the two together reflecting a distinct tumor biology. Considering it is already known that peripheral immune cells are altered in colorectal cancer (CRC) patients, this study aims to investigate the changes in lymphocyte subsets in SARIFA-positive cases and correlate these changes with the local immune response. Flow cytometry was performed to analyze B, T, and natural killer (NK) cells in the peripheral blood (PB) of 45 CRC patients. Consecutively, lymphocytes in PB, tumor-infiltrating lymphocytes (TILs), and CD56+ and CD57+ lymphocytes at the invasion front and the tumor center were compared between patients with SARIFA-positive and SARIFA-negative CRCs. Whereas no differences could be observed regarding most PB lymphocyte populations as well as TILs, NK cells were dramatically reduced in the PB of SARIFA-positive cases. Moreover, CD56 and CD57 immunohistochemistry suggested SARIFA-status-dependent changes regarding NK cells and NK-like lymphocytes in the tumor microenvironment. This study proves that our newly introduced biomarker, SARIFA, comes along with distinct immunologic alterations, especially regarding NK cells.

Clinical and immunological criteria for prediction of thechronic course of cytomegalovirus infection on the background of hypoxic-ischemic damage of the central nervous system in children in the first year of life

Objective. To develop prognostic criteria for the chronic course of cytomegalovirus infection by studying disorders of the regulation of the immune response in children of the first year of life against the background of hypoxic-ischemic CNS damage.Materials and methods. 108 newborns with cytomegalovirus infection occurring against the background of perinatal hypoxicischemic lesions of the central unequal system were examined. All observed patients at 1 and 3 months of life conducted an immunological examination, including the determination of T and B-lymphocytes. Determination of the population and subpopulation composition of peripheral blood lymphocytes, activation markers was carried out by the method of one- and twoparameter phenotyping using reagents from Immunotex (France), FITC (fluorescein isothiocynate) — labeled with CD3+, CD4+, CD8+, CD20+ and PE (phycoerythrin) — labeled CD28+, CD40+. The results were recorded on a BECKMAN COULTER EPICSXL-II flow cytometer (USA) using standard protocols. The observation groups consisted of 78 children (72.2%) with an acute course of the disease (Group 1) and 30 children (27.3%) with a chronic course (Group 2).Results. Of the totality of the studied parameters of the cellular and humoral parts of the immune system, statistically significant for the prognosis of the chronic course of cytomegalovirus infection in children of the first year of life against the background of hypoxic-ischemic CNS damage were found: CD8, CD40, CD3+CD28+, CD20+CD40+. Using the PolyAnalist 3.5 Pro CNS package, systems of inequalities were obtained and a formula for predicting the chronic course of cytomegalovirus infection in children in the first year against the background of perinatal hypoxic-ischemic CNS damage was calculated.Conclusion. A statistically significant relationship was found between the prognosis of the chronic course of cytomegalovirus infection against the background of perinatal hypoxic-ischemic CNS damage and the level of CD20, CD4, costimulatory molecules CD3+CD28–, CD20+CD40+. The proposed diagnostic rules can be considered screening markers for the prognosis of the chronic course of cytomegalovirus infection against the background of perinatal hypoxic-ischemic CNS damage in newborns, which makes it possible to start specific therapy in a timely manner.

Influence of gender and age of patients with oral mucosa cancer on the phenotype of systemic and local immunity

Background. The incidence of oral mucosa cancer (OMC) is higher in people over 50 years of age, and the aggressiveness of the course of the disease is higher in people under 50 years of age. In this context, it is of interest to clarify the mechanisms of immune disorders characteristic of patients of different age groups.Aim. To research systemic and local immunity in OMC patients and the relationship of peripheral blood lymphocyte population (PBLs) and tumor infiltrating lymphocytes (TILs) with the patient’s sex and age.Materials and methods. PBLs and TILs effector and suppressor populations were studied by flow cytometry in OMC patients aged 29 to 84 years.Results. The percentage of CD3-, CD3+CD4+ and CD3+CD8+T cells, regulatory CD4+CD25+CD127low/ –(CD4Treg) and CD8+CD11b–CD28–(CD8Тreg) T lymphocytes, CD4+PD-1+ and CD8+PD-1+ T cells was increased in TILs compared to PBLs. The levels of cytotoxic CD8+CD11b+CD28– T lymphocytes, NK, CD8+Perforin+ and CD16+Perforin+ cells in TILs were lower than in PBLs. The relationship between the level of CD4Treg and other TILs and PBLs depended on the patient’s sex. Age-related changes in the levels of NK and CD8 T-cells were observed in men, and CD4Treg – in women.Conclusion. Local immunity in OMC patients is highly immunosuppressive. The sex of patients influences the relationship between CD4Treg and other populations of PBLs and TILs, as well as age-related changes in the OMC patients’ immune system. This investigation results can make a certain contribution to personalized treatment of patients with OMC, taking into account differences in systemic and local immunity and in the immune response to the tumor in patients of different sex and age.

“Lymphocyte population in peripheral blood in children and adolescents with graves disease. Potential predictive tool for severity of the disease”

PurposeTo date, few data are available on the prognostic role of lymphocyte subsets in pediatric Graves’ Disease (GD). The aim of this retrospective study is to analyze the role of lymphocyte subtypes in predicting the severity of GD.MethodsData of 10 pediatric subjects aged <18 years with GD onset in the period November 2017–April 2021 were collected. The lymphocyte population was assessed at the onset of GD as well as hormonal and clinical data. The follow-up period was 2.4 ± 0.8 years.ResultsPearson correlation coefficient between CD4+ /CD8+ ratio and fT3 levels and thyroid volume at diagnosis was 0.72 (p = 0.04) and 0.81 (p = 0.004) respectively; that between CD4+ /CD8+ ratio and the TRAb titer at diagnosis and after 6, 12 and 24 months was 0.89, 0.89, 0.73 and 0.77 respectively (p = 0.02, p = 0.01, p = 0.03 and 0.04). The correlation coefficient of anti-thyroid drug (ATD) dose after 6 and 12 months with CD4+ /CD8 ratio was 0.88 and 0.78 (p = 0.001 and p = 0.02 respectively). Patients with a higher CD4+ /CD8+ ratio at diagnosis displayed higher fT3 levels (28.73 ± 2.18 vs 13.48 ± 2.19 pmol/L, p = 0.03) and higher TRAb titers (28.9 ± 11.2 vs 4.88 ± 0.97, p = 0.01).ConclusionCD4+/CD8+ ratio appears as a promising predictive tool to be considered together with other prognostic factors to better manage pediatric GD. These preliminary data need to be confirmed over a longer follow-up period and in larger cohorts.

Dynamics of Cytokines Concentration in the Blood Plasma of Patients after Multipotent Mesenchymal Stromal Cells Administration for Graft Versus Host Disease Prevention

IntroductionDespite the large number of clinical studies on the use of multipotent mesenchymal stromal cells (MSCs) for the treatment and prevention of graft-versus-host disease (GVHD), the mechanism of their action in the organism is not well understood. The known data refer either to clinical effects or obtained in vitro. Due to the immunomodulatory effect of MSCs in the body, subpopulations of T cells and the concentration of cytokines involved in the immune response can change. The role of T cells and certain cytokines (TNF alpha, IL6, IL8, etc.) in the pathophysiology of GVHD is described. The aim of this investigation was to study the composition of T cells subpopulations and the concentration of cytokines in the peripheral blood of patients who received MSCs for GVHD prophylaxis.MethodsThe study included 21 patients who received hematopoietic stem cells donor's derived MSCs for the prevention of GVHD as part of the ClinicalTrials.gov Identifier NCT01941394 trial. The control group included 16 patients who did not receive MSCs. After signing informed consent, blood samples were taken from all patients during routine examinations on the day of restoration of the number of leukocytes to 1000 in μl (day 0), after 3 and after 30 days. MSCs were injected on day 0. None of the patients developed GVHD during this time. To analyze plasma cytokines and chemokines, the Bio-Plex Pro Human Cytokine Panel kit, 27-Plex (BioRad) was used, to determine the concentrations of IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL- 6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, bFGF, Eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1 (MCAF), MIP-1α, MIP-1β, PDGF-bb, RANTES, TNF-α, VEGF according to manufacturer's recommendations. Forward and side scattering parameters determined the peripheral blood lymphocytes population and then CD4+ or CD8+ lymphocytes were gated. For each of this population the composition of memory cells subset were determined by flow cytometry.ResultsSignificant differences were found between the 2 groups only on day 30 in the concentration of IL8 (17.0±3.2 pg/ml in the control group versus 32.8±4.1 pg/ml in the MSC group, p<0.0001). It has been shown that 30 days after MSCs, the number of CD4+ T cells in the peripheral blood of patients significantly increases compared to the group without MSCs (CD4 CM 38.9±9.0 vs 22.4±7.8, CD4 TM 97.0±32.5 vs 91.0±43.2, CD4 TE 5.0±1.9 vs 1.4±1.0, CD4 EM 34.6±20.1 vs 19.9±7.9, CD4CD25+ 27.6±6.6 vs 12.9±5.0). These cells produce IL8, which play an important role in immune cell homeostasis by activating antimicrobial neutrophils. Without the introduction of MSCs, the concentration of this protective against GVHD cytokine practically did not change within a month, whereas after the introduction of MSCs, it gradually increased almost 2 times. However, the dynamics of changes in the levels of the studied cytokines differed greatly between the 2 groups (Table). The concentration of IP10, which is involved in the development of GVHD, increased significantly faster and stronger in the group without MSCs. An increase in the concentration of other investigated cytokines associated with the activation of macrophages (MCP-1, MIP-1a, MIP-1b) did not depend on the MSCs administration. At the same time, in the MSC group, the concentration of the growth factor PDGF-bb necessary for the HSC proliferation increased significantly more actively. The IL9 concentration on day 0 was comparable to the level in healthy donors, and then gradually increased in both groups and after 30 days it was significantly higher than on day 0 in the MSC administration group. The concentration of G-CSF changed in a similar way.ConclusionChanges in the dynamics of T cells subpopulations and the concentration of cytokines in the blood after MSCs administration contribute to a faster recovery of patients after allogeneic bone marrow transplantation.The work were supported by the Russian Foundation for Basic Research, Project No. 19-29-04023. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Metabolic therapy of predicted complications in immunocompromised recipients before repeated corneal transplantation

One of the topical problems of modern ophthalmotransplantology is the graft engraftment after repeated keratoplasty. During repeated corneal transplantation, the frequency of graft rejection increases significantly. The study included 121 patients aged 19 to 89 years with corneal graft failure, who were scheduled for repeated corneal transplantation. Immunophenotyping of major and small populations of peripheral blood lymphocytes was performed by flow cytometry (CytoFlex BC, USA). The intensity of energy metabolism in lymphocyte populations was determined by the activity of succinate dehydrogenase and NADH dehydrogenase by immunocytochemical method using flow cytometry. An increase in the content of B lymphocytes (p = 0.004) and a decrease in Th17 lymphocytes (p = 0.013) were revealed after the use of a course of metabolic therapy. Against the background of therapy, the activity of SDH in the T lymphocyte population significantly increases (p = 0.034). In addition, in the studied populations of lymphocytes in the recipient group, against the background of metabolic therapy, the normalization of SDH activity is observed: the number of recipients with low and high enzyme activity decreases. After a course of metabolic therapy, a significant decrease in NADHDH activity was revealed (p = 0.034). Indicators of lymphocyte populations and mitochondrial enzyme activity in recipients after a course of metabolic therapy indicated a more favorable prognosis for repeated corneal transplantation. Evaluation of the results of repeated keratoplasty a year after surgery showed that 59 recipients received transparent graft engraftment, and in 62 patients the graft became cloudy in the period from 1 to 8 months after surgery. In the group of patients with transparent graft engraftment, the percentage of recipients receiving metabolic therapy was significantly higher than in the group of recipients with graft opacity (41%±2.05% vs 21%±2.91%, p 0.001). Conducting metabolic therapy before surgery reduces the number of realized unfavorable prognoses of the result of rekeratoplasty, and monitoring the activity of dehydrogenases and the content of lymphocyte populations allows us to evaluate the effectiveness of therapeutic and preventive measures in immunocompromised recipients.

Flow cytometry evaluation of the rhesus monkey cellular immunity following the Zaire ebolavirus (Filoviridae; Ebolavirus: Zaire ebolavirus) experimental infection

The outbreaks of the Zaire ebolavirus (ZE) disease (ZED) that have arisen in the last decade determine the need to study the infection pathogenesis, the formation of specific immunity forming as well as the development of effective preventive and therapeutic means. All stages of fight against the ZED spread require the experimental infection in sensitive laboratory animals, which are rhesus monkeys in case of this disease .The aim of the study is to evaluate the rhesus monkey cellular immunity following the ZE experimental infection by the means of flow cytometry (cytofluorimetry). Male rhesus monkeys were intramuscularly infected by the dose of 15 LD50 (dose of the pathogen that causes 50% mortality of infected animals) of the ZE, the Zaire strain (ZEBOV). Levels of 18 peripheral blood lymphocyte populations of the animals before the ZE experimental infection and at the terminal stage of the disease were assessed using flow cytometry. The certain changes in the levels of the lymphocyte populations were observed following infection, indicating simultaneous activation and suppression of the immune system during ZED. The increase in content was observed for T-lymphocytes, T-helper and cytotoxic T-lymphocytes expressing the corresponding markers of early activation. The decrease was recorded for T-lymphocytes and double-positive T-lymphocytes expressing corresponding markers of late activation, as well as natural killer cells expressing CD8 (p &lt; 0.05). For the first time in the Russian Federation, the rhesus monkey cellular immunity before and after the ZE experimental infection was assessed using flow cytometry.

The Outcomes of Nivolumab Fixed Dose 40 Mg Therapy in Patients with Relapsed or Refractory Classical Hodgkin Lymphoma

Background Currently, the recommended dose of nivolumab for patients with relapsed or refractory classical Hodgkin lymphoma (r/r сHL) is 3 mg/kg. Nevertheless, published clinical cases indicate the possible efficacy of lower doses of nivolumab. Moreover, experimental studies provided the rationale for possible reduction of nivolumab dose in patients with solid tumors (Agrawal et al. 2016). The presented data creates prerequisites for studying the lower nivolumab doses efficacy and safety in the r/r cHL therapy. Patients and Methods This study included 42 patients (14 male/28 female) with r/r cHL who were treated with nivolumab 40 mg every 2 weeks. The median age of patients was 36 (22-53) years. The median number of prior therapy lines was 4 (2-7). Prior treatment contained high dose chemotherapy with ASCT in 9 pts (21%), brentuximab vedotin in 14 pts (33%) and allo-HSCT in 1 pt (2%). Four pts (9,5%) had the partial response (PR) and the remaining 38 pts (90,5%) had the disease progression (PD) at the moment of nivolumab initiation. B-symptoms were present in 23 pts (55%), ECOG status was grade 0-I in 25 pts (59,5%), grade II in 12 pts (29%), grade III in 4 pts (9,5%) and grade IV in 1 pt (2%). The primary endpoint was the overall response rate (ORR) determined by positron-emission tomography/computed tomography (PET/CT) using LYRIC criteria every 3 months. Key secondary endpoints included progression-free survival (PFS) and overall survival (OS). Adverse events (AE) were evaluated according to CTCAE 4.03. The patient group characteristics were evaluated using descriptive statistics methods, the survival analysis was performed using Kaplan-Meyer method (SPSS Statistics v.17). Results The median number of nivolumab cycles was 24 (2-38). The response was evaluated in 41 out of 42 pts. The ORR was 66%. The best response included complete response (CR) in 39%, PR in 27%, stable disease in 5%, PD in 2%, indeterminate response (IR) in 27% of pts. With a median follow-up of 27,5 mo (11,3-34,5) 41 pts (97,6%) were alive, the median OS was not reached. The 2-year PFS was 44,5% (95% CI, 28,2-59,6) The nivolumab therapy was discontinued in 39 pts (93%) due to scheduled discontinuation in 14 pts (33%), PD in 13 pts (31%), grade 3-4 AE in 2 pts (5%), change of therapy because of insufficient response in 6 pts (14%) and other reasons in 4 pts (10%). The progression of disease during nivolumab therapy was present in 14 (33%) pts and after nivolumab discontinuation in 6 (14%) pts. After disease progression 30 pts (71%) were retreated with nivolumab monotherapy or in combination with chemotherapy. The median time to additional therapy was 14,5 mo (4,2 -32,9). The adverse events of any severity were observed in 30 pts (71%). Grade 3 or higher AE were present in 4 pts (9,5%), including grade 3 arthralgia, grade 3 anemia, grade 4 pneumonia and pneumonitis, grade 4 increased level of alanine aminotransferase and grade 5 MDS in 1 pt. A significant reduction of PD1+CD3+ cell population of peripheral blood lymphocytes was observed after first nivolumab cycle (median 0.7% (0-1.7) versus 33% (15.7-80.1) before therapy initiation, p = 0.02, Wilcoxon signed-rank test). Conclusion Our study demonstrated the efficacy and safety of nivolumab 40 mg therapy. The presented results are comparable to previously published data of nivolumab 3 mg/kg therapy in patients with r/r cHL. Thus, this creates a basis for further direct comparative study of nivolumab efficacy in different doses Disclosures No relevant conflicts of interest to declare.

Catfish lymphocytes expressing CC41-reactive leukocyte immune-type receptors (LITRs) proliferate in response to Edwardsiella ictaluri infection in vitro

Monoclonal antibodies (mAbs) CC34 and CC41 recognize overlapping subsets of leukocyte immune-type receptors (LITRs). The mAb CC34 was raised against the clonal TS32.15 cytotoxic T cell line and the mAb CC41 was raised against the clonal NK cell line TS10.1. In this study, an in vitro model was developed to monitor CC34- and CC41-reactive cells in response to Edwardsiella ictaluri infection. Briefly, head kidney leukocytes and peripheral blood lymphocytes (PBL) were isolated from individual catfish and labeled with CellTrace Violet and CellTrace FarRed dye, respectively. Head kidney-derived macrophages were infected with E. ictaluri and then cocultured with autologous PBL. The combined cell cultures were then analyzed using flow cytometry. A significant increase in CC41 staining was observed in the PBL population at 2, 5 and 7 days after culture, which suggest that LITRs are involved in cell-mediated immunity to E. ictaluri.

PHENOTYPIC COMPOSITION OF PERIPHERAL BLOOD LYMPHOCYTES AND THEIR COOPERATION IN PATIENTS WITH CHRONIC MERCURY INTOXICATION IN A POST-CONTACT PERIOD

Introduction. Based on the current understanding of the progression of professional chronic mercury intoxication, it is extremely important to study the regulatory activity of immunocompetent cells after the cessation contact with mercury to develop effective therapeutic measures. The purpose of the study was to study the population and subpopulation spectrum of peripheral blood lymphocytes and their cooperation in patients with chronic mercury intoxication in a distant postexposure period. Material and methods. Phenotyping of lymphocytes in the blood of the subjects was carried out by the method of indirect immunofluorescence using monoclonal antibodies to the molecules CD3+, CD4+, CD8+, CD9+, CD16+, CD20+, CD21+, CD23+, CD25+, CD95+. Results. In patients with chronic mercury intoxication in the long-term postexposure period, there were revealed features of the immune system functioning, indicating hyperactivation of both T- and B-components of the immune system. An increase in the total population of T-lymphocytes was established due to an increase in cells with receptors CD4+ (T-lymphocyte-helper cells) and CD16+ (killer cells), as well as an increase in the number of mature B-lymphocytes (CD20+) and pre-B-immature-lymphocytes (CD9+). Changes in the system of lymphocyte apoptosis, characterized by an increase in the number of cells expressing receptors for readiness for Fas-dependent apoptosis (CD95+), have been recorded. The established relationships between populations and subpopulations of lymphocytes indicate their importance in the implementation of the immune response, high activity and contingency between the components of the immune system in persons with chronic mercury intoxication after the termination of contact with the toxicant. Conclusion. The results obtained are the basis for long-term monitoring of the health status and improvement of the tactics of treating patients with neurointoxication with mercury in the postexposure period.

1280P - A burst of highly differentiated CD4 TL identifies a subset of fast progressors, and correlates with hyperprogressive disease in NSCLC patients treated with ICI

BackgroundHyperprogressive disease (HPD) is a new pattern of tumor response described with immune checkpoint inhibitors (ICI), characterized by a sharp acceleration of tumor growth after the administration of the treatment. Several definitions based on clinical and/or radiological criteria have been proposed, although different factors limit their applicability. The mechanistic rationale has still not been elucidated. Our group has monitored lymphocyte populations in peripheral blood from non-small cell lung cancer patients (NSCLC) under ICI treatment with flow cytometry, and correlated the findings with HPD. Methods41 NSCLC patients treated with ICI after progression to≥1 lines of chemotherapy for advanced disease have been included. We analyzed lymphocyte subpopulations from peripheral blood samples obtained immediately before the administration of the 1st and 2nd cycle of ICI. The expression of the following markers has been considered: CD3, CD4, CD8, CD27, CD28, CD45RA, CD62L, PD1. We correlated the findings with HPD as defined by TGR (Champiat S, 2017) and TGKR (Sâada-Bouzid E, 2017). ResultsOverall response rate (ORR) was 23.8%, and disease control rate (DCR) was 31%. 10 patients (23.8%) presented HPD by TGKR, and 7 patients (16.7%) by TGR. 9 patients (21.4%) could not be evaluated for HPD, mainly due to progression by non-measurable disease or death before further radiological. Median progression free survival (mPFS) was 6 weeks for HPD by TGR and 6.3 weeks by TGKR. We found that the mean proportion of post/pre-treatment LT CD4+ CD27- CD28- was significantly higher for HPD patients (1.86; CI95% 1.14 to 2.58 than non-HPD (1.09; CI95% 0.89 to 1.30) (p=0.0033). An increase > 30% in LT CD4+/CD27-/CD28- after the first cycle of immunotherapy was associated with fast progression (ORR 0%, mPFS 6 weeks [CI95% 5.8 to 6.2], 8-weeks PFS 0%). Cohen’s kappa between HPD by TGR and by LT was 0.503 (p=0.004). ConclusionsHPD as defined by LT CD4+ CD27- CD28- burst > 30% might complement the limitations of radiological criteria as it does not require the evaluation of the tumor kynetics previous to the beggining of immunotherapy, nor it is influenced by non-measurable disease or tumor burden. Legal entity responsible for the studyThe authors. FundingAECC, ISCIII. DisclosureAll authors have declared no conflicts of interest.

Abstract 1557: Characterization of AB154, a humanized anti-TIGIT antibody, for use in combination studies

Abstract BACKGROUND: AB154 is a humanized antibody that blocks human TIGIT (T-cell immunoreceptor with Ig and ITIM domains), an inhibitory receptor expressed on natural killer (NK) cells, CD8+ T cells, CD4+ T cells and regulatory T cells (Treg). DNAM-1 (DNAX Accessory Molecule-1) is an activating receptor found on NK cells, monocytes and a subset of T cells that competes with TIGIT for shared ligands CD155 (PVR) and CD112 (Nectin-2), expressed by cancer and antigen-presenting cells. TIGIT blockade prevents binding to its ligands and shifts the immune balance towards a more favorable DNAM-1 interaction. AB154 has the potential to promote sustained immune activation and tumor clearance, particularly in combination with other immunotherapies such as AB122 (α-PD1). METHODS: Binding affinity of AB154 was determined in CHO cells over-expressing human TIGIT and in human T cells. TIGIT blockade was quantified using a TIGIT-expressing reporter gene cell line. TIGIT and PD-1 expression in cancer patient PBMCs and dissociated tumor cells (DTCs) were assessed by flow cytometry. Gene expression of these markers were also derived from TCGA (The Cancer Genome Atlas), GTEX (Genotype-Tissue Expression Project), RNAseq, and by immunohistochemistry in various tumor types and normal tissues. A receptor occupancy (RO) assay was developed using a competing α-TIGIT antibody and validated ex vivo in whole blood leukocytes from healthy donors and cancer patients. RESULTS: AB154 binds to and blocks human TIGIT with sub-nanomolar affinity. Data assembled from TCGA identified tumor types in which expression of TIGIT is greater than PD-1, equivalent to PD-1, or less than PD-1. Expression of TIGIT and CD155 at the protein level was confirmed by IHC. Immunophenotyping performed on dissociated human tumor cells demonstrated strong correlation between TIGIT and PD-1 expression on immune cells. The intensity of TIGIT staining was lowest on conventional CD4+ T cells while its intensity in Treg and CD8+ T cells was 1.5 to 3-fold higher on average. Using flow cytometry, we profiled lymphocyte populations in peripheral whole blood and demonstrated target engagement by AB154 on T cells and NK cells in the low nanomolar range in both healthy and cancer patients (ex vivo). This receptor occupancy assay is being used to monitor target engagement in AB154 dosed patient samples in an ongoing Phase 1 dose escalation study in oncology patients. CONCLUSION: Blockade of multiple immune checkpoint proteins can confer effective and durable responses in the treatment of cancer. The data presented will provide: 1) the basis for selection of tumor types by TIGIT RNA and protein expression profiles, 2) rationale for combining AB154 with AB122 (α-PD-1) in upcoming clinical trials, 3) methodology to evaluate TIGIT receptor occupancy and 4) preliminary PK/PD data from the AB154 Phase 1 dose escalation study in oncology subjects. Citation Format: Amy E. Anderson, Daniel DiRenzo, Susan Lee, Akshata Udyavar, Kimberline Gerrick, Hema Singh, Xiaoning Zhao, Lixia Jin, Lisa Seitz, Nigel P. Walker, Matthew J. Walters, Joanne B. Tan. Characterization of AB154, a humanized anti-TIGIT antibody, for use in combination studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1557.

Percentage of CD19+ Cells in Peripheral Blood Lymphocytes After Rituximab-Based Desensitization as a Predictor of Acute Antibody-Mediated Rejection in ABO-Incompatible Kidney Transplantation

BackgroundRituximab (RIT) is effective as a part of the desensitization therapy before ABO-incompatible kidney transplantation (ABOi-KTx), and a single dose of RIT at 375 mg/m2 or less is recommended. However, adequate RIT dose recommendations have not yet been established for individual recipients. Therefore, we evaluated the relationship between the proportion of B cells in peripheral blood and acute antibody-mediated rejection (AAMR). MethodsForty-four consecutive ABOi-KTx recipients were enrolled in this retrospective study. Before transplantation, subjects were treated with RIT at various doses, ranging from 65 to 400 mg/body (46–263 mg/m2), followed by plasmapheresis and intravenous immunoglobulin as a desensitization therapy. The percentage of CD19+ cells in the total peripheral blood lymphocytes population (%CD19) was determined the day before transplantation. Transplant recipients were divided into 2 groups according to pretransplant %CD19, as follows: low %CD19 group, ≤ 1.2% (n = 35) and high %CD19 group, > 1.2% (n = 9). The relationship between %CD19 and incidence of AAMR was evaluated, and the predicting factors for AAMR incidence were determined by univariate and multivariate analyses. ResultsThe incidence of AAMR was significantly higher in the high %CD19 group than in the low %CD19 group (44.4% vs 5.7%, P = .006). Furthermore, multivariate analysis showed that %CD19 > 1.2% was the only independent factor to predict AAMR, with an odds ratio of 14.31 (P = .038). ConclusionHigh %CD19 values after rituximab administration in ABOi-KTx recipients implies insufficient depletion of B cells, which can lead to AAMR.

Dynamics of nasopharyngeal colonisation & correlations with lymphocyte populations in the adenoid & peripheral blood of chronic otitis media prone children

Dynamics of nasopharyngeal colonisation & correlations with lymphocyte populations in the adenoid & peripheral blood of chronic otitis media prone children