НОВЫЕ ВОЗМОЖНОСТИ РЕГУЛЯЦИИ ПРОТИВООПУХОЛЕВОГО ИММУННОГО ОТВЕТА

The tumor has different mechanisms capable of destroying the immunological protection. Population of regulatory cells, along with other factors provide “escape” of the tumor from immune surveillance. In our laboratory, we studied the features of quantitative changes of some subpopulations of peripheral blood lymphocytes in primary operable breast cancer (BC) and melanoma at different stages of tumor growth and in the process of tumor therapy. In 94.5% of patients with breast cancer were found to increase compared to the control amount of NKT-cells with the phenotype CD45+CD3+ CD16+CD56+, 78% increase in number of CD8+CD28– T-cells, and 20.5% increase in the number of patients Regulatory CD4+CD25+FOXP3+ T cells. Was found to depend on changes in the number of these cells from the stage of the disease. Patients with stage I and II disease there was a statistically significant increase in the percentage of CD8+CD28– T–cells and CD45+CD3+ CD16+CD56+ NKT-cells compared to the donor. At the same time in patients with stage III the number of cells of both populations declined and did not differ from the norm. Such dynamics of quantitative changes were typical for the main populations of effector cells antitumor immunity. In the evaluation of patients with disseminated melanoma was found that no increase in the number of cytotoxic CD45+CD8+CD11b+ T cells in the treatment dendritic cell vaccine (DCV) appears to be an indication to stop vaccine therapy, initially increased amount of CD3+CD8+CD16+. NKT-cells may serve as grounds for refusal of DCV. A brief description of the major co-receptor inhibitor of T-cells, and monoclonal antibodies that block the inhibitory molecule on immune and tumor cells in order to increase the efficiency of anti-tumor immune response. Encouraging clinical results have been obtained by using anti-CTLA-4 (ipilimumab), and anti-PD-1 (nivolumab) monoclonal antibodies.Опухоль обладает различными механизмами, способными разрушать иммунологическую защиту. Популяции регуляторных клеток наряду с другими факторами обеспечивают «ускользание» опухоли от иммунологического надзора. В нашей лаборатории было проведено изучение особенностей количественных изменений некоторых субпопуляций лимфоцитов периферической крови у первично-операбельных больных раком молочной железы (РМЖ) и диссеминированной меланомой на разных этапах опухолевого роста и в процессе противоопухолевой терапии. У 94,5% больных РМЖ было обнаружено повышение по сравнению с контролем количества NKT-клеток с фенотипом CD45+CD3+CD16+CD56+, у 78% повышение количества CD8+CD28- Т-клеток, и у 20,5% больных повышение количества регуляторных CD4+CD25+FOXP3+ Т-клеток. Выявлена зависимость изменений количества этих клеток от стадии заболевания: у пациенток с I и II стадиями заболевания отмечалось статистически значимое повышение процента CD8+CD28- Т-клеток и CD45+CD3+CD16+CD56+ NKT-клеток по сравнению с донорами. В то же время у пациенток с III стадией количество клеток обеих популяций снижалось и практически не отличалось от нормы. Подобная динамика количественных изменений была характерна и для основных популяций клеток-эффекторов противоопухолевого иммунитета. При обследовании больных диссеминированной меланомой было обнаружено, что отсутствие повышения количества цитотоксических CD45+CD8+CD11b+ Т-клеток в процессе лечения дендритноклеточной вакциной (ДКВ), по-видимому, является показанием к прекращению вакцинотерапии, а исходно повышенное количество CD3+CD8+CD16+ NKT-клеток может служить основанием для отказа от ДКВ. Приводится краткое описание основных ко-ингибиторных рецепторов Т-клеток и моноклональных антител, блокирующих ингибиторные молекулы на иммунокомпетентных и опухолевых клетках, с целью повышения эффективности противоопухолевого иммунного ответа. Обнадеживающие клинические результаты были получены при применении анти-CTLA-4 (ипилимумаб) и анти-PD-1 (ниволумаб) моноклональных антител

HETEROGENEITY OF NK AND NKT LYMPHOCYTE POPULATIONS IN HEALTHY DONORS

Natural killer(NK) and  NKT lymphocytes are important components of innate immunity, and compose a first-line defense against cancer. These populations are characterized by high heterogeneity and are divided into several subpopulations, by differences in functional activity as well as CD56  and CD16  expression. Studying  heterogeneity for these  lymphocyte populations in healthy  donors  is important, due  to imbalance between  different  lymphocyte subsets in cancer patients. Changes in the ratio of these subpopulations may be of prognostic and clinical  significance in malignant diseases. The present  study was conducted with peripheral blood  lymphocytes in 50 healthy  donors. When  analysing  population of NK  lymphocytes we have identified 18.0±11.3% of antigen-positive cells, their fluctuations ranged  from 7.6 to 29.2%, whereas average number of cells with  CD3-CD56+ and  CD3-CD16+   phenotypes was equal  to 16,2±8.1%, and  11,0±6.7%, respectively. The  subpopulation analysis  showed  that  the  primary  pool  of NK  cells  was presented by CD56dimCD16dim cells  by  52.3±19.9 percent.  We  detected  minor   subpopulations,  e.g.,  CD56dimCD16bright,  CD56-CD16+, CD56brightCD16- (0.3±0.2%, 1.7±0.9%, and  1.3±0.6%,  respectively). Search  for  intracellular perforin has revealed  that the number of CD56+Perf+ cells comprized 25.1±14.8%, CD16+Perf+, 23.8±16.0%. Cytometric analysis showed that perforin is found, predominantly, in CD56dimCD16dim NK lymphocytes, whereas the cells with CD56dimCD16bright, CD56-CD16+, CD56brightCD16- immunophenotypes did not produce perforin. For the first time, we have discovered a subpopulation of NK cells with the СD56dimCD16dim immunophenotype that did not contain intracellular perforin (2.0%).  The NKT cell population with СD3+CD16/СD56+  phenotype was detected in 7.1% (25% – 3.45; 75% – 8.75) antigen-positive cells, within a range of 2.5 to 11.9%. Analysis with a combination of monoclonal antibodies CD3/CD56/CD16 has shown that the number of CD3+ CD56+ cells was 4.33% (25% – 2.25; 75% – 7.3), whereas the number of CD3+CD16+ was 3.087% (25% – 0.9; 75% – 6.2). These  data  demonstrate that  the differences in results  between  the CD3/CD16/CD56, and  CD3/CD16 test systems are statistically significant  (p < 0.05). It was shown that the primary-pool NKT-cells are CD56+CD16- cells, whose number is about 5.45% (25% – 2.95; 75% – 7.3) among  total CD3+ lymphocyte population. Two minor subpopulations were also detected which differed in expression  of CD56 and CD16 antigens. Hence, the level of CD56-CD16+ cells was 3% (25% – 0.25; 75% – 3.05),  and the number of CD56+CD16+ was equal to 0.67% (25% – 0.25; 75% – 0.9). Hence, the observed wide phenotypic diversity of NK and NKT-cells reflects their  ability  to exert  various  functional activities.  This  study, showing  high  heterogeneity of NK  and  NKT lymphocytes, may serve as a basis for the study of imbalances between  different  subpopulations of these cells in cancer patients

ASSOCIATION OF NKT- AND ACTIVATED CD25⁺ PERIPHERAL BLOOD LYMPHOCYTES WITH DISEASE FREE AND OVERALL SURVIVAL OF TRIPLE NEGATIVE BREAST CANCER PATIENTS

Background. We previously found that a decrease in the number of NKT cells and activated CD 25+ peripheral blood lymphocytes (PBLs) before neoadjuvant chemotherapy was associated with an increased likelihood of disease progression in patients with locally advanced triple-negative breast cancer (TN BC).The purpose of this study was to determine the relationship between the initial number of NKT-and CD 25+ PBLs and relapsefree survival (RFS)/overall survival (OS ) in patients with TN BC who received neoadjuvant chemotherapy with cisplatin and paclitaxel followed by surgery.Material and Methods. The study included patients with stage II and III TN BC. The follow-up time was 36 and 66.9 months. Immediately before chemotherapy, the percentage of CD 3+CD 16+CD 56+ (NKT) -, CD 25+- and CD 8+ PBLs was determined by flow cytometry. Statistical analysis of the data was carried out using the Statistics 7 software package. The Kaplan-Meier method was used to determine the relationship between immunological parameters and RFS/ OS .Results. The decreased level of NKT cells before treatment was associated with a decrease in the 3-year RFS [Me: 20.1 (0.533 and 39.7) months] compared to that observed in patients with higher percentage of these cells than in the control (Me was not achieved). There were no statistically significant differences in the 3-year OS between the groups. The initially reduced number of CD 25+ lymphocytes in comparison with the control was associated with decreased rates of both RFS and OS . The difference in DFS and OS was more significant between the groups of patients who simultaneously had an increased initial number of both NKT and CD 25+ cells and patients in whom both cell populations were below normal levels.Conclusion. The initial (prior to chemotherapy) number of NKT and activated CD 25+ PBLs can apparently be a predictive factor in TN BC patients, who received neoadjuvant chemotherapy with cisplatin and paclitaxel

MEMBRANE LEc EXPRESSION IN BREAST CANCER CELLS

Affine chromatography was used to isolate Lec antibodies from the sera of a healthy female donor with the high titers of these anti- bodies, which were labeled with biotin. The study enrolled 51 patients with primary breast cancer (BC). Antigen expression was found by immunohistochemistry and flow cytometry. With these two techniques being used, the detection rate of Lec expression in BC cells was 65% (33/51); the antigen was most frequently found by flow cytometry as compared with immunohistochemistry: 72 and 58% of cases, respectively