mimmunМедицинская иммунологияMedical Immunology (Russia)1563-06252313-741XSPb RAACI10.15789/1563-0625-GSF-2066mimmun-2066Research ArticleИММУНОЛОГИЧЕСКИЕ МЕТОДЫIMMUNOLOGICAL METHODSСтратегия гейтирования плазмобластов на примере иммунизации вакциной против гепатита ВGating strategy for plasmablast enumeration after hepatitis B vaccinationhttps://orcid.org/0000-0002-9858-7596БязроваМ. Г.ByazrovaM. G.

Бязрова Мария Георгиевна – младший научный сотрудник лаборатории иммунохимии ФГБУ «ГНЦ „Институт иммунологии“» Федерального медико-биологического агентства России; аспирант кафедры иммунологии, ФГБОУ ВО «Московский государственный университет имени М.В. Ломоносова»

Москва

Byazrova Maria G., Junior Research Associate, Laboratory of Immunochemistry, National Research Center “Institute of Immunology”, Federal Medical-Biological Agency of Russia; Postgraduate Student, Lomonosov Moscow State University

Moscow

mbyazrova@list.ruТоптыгинаА. П.ToptyginaA. P.

Топтыгина Анна Павловна – доктор медицинских наук, профессор, ведущий научный сотрудник лаборатории цитокинов ФБУН «Московский научно-исследовательский институт эпидемиологии и микробиологии имени Г.Н. Габричевского» Роспотребнадзора; профессор кафедры иммунологии, ФГБОУ ВО «Московский государственный университет имени М.В. Ломоносова»

Москва

Toptygina Anna P., PhD, MD (Medicine), Professor, Leading Research Associate, Cytokine Laboratory, G. Gabrichevsky Research Institute for Epidemiology and Microbiology; Professor, Department of Immunology, Lomonosov Moscow State University

Moscow

toptyginaanna@rambler.ruhttps://orcid.org/0000-0001-7493-0030МитинаТ. А.MitinaT. A.

Митина Татьяна Алексеевна – доктор медицинских наук, руководитель отделения клинической гематологии и иммунотерапии

Москва

Mitina Tatiana A., PhD, MD (Medicine), Head, Department of Clinical Hematology and Immunotherapy

Moscow

mi_69@inbox.ruhttps://orcid.org/0000-0002-6460-9427ФилатовА. В.FilatovA. V.

Филатов Александр Васильевич – доктор биологических наук, профессор, заведующий лабораторией иммунохимии ФГБУ «ГНЦ „Институт иммунологии“» Федерального медико-биологического агентства России; профессор кафедры иммунологии, ФГБОУ ВО «Московский государственный университет имени М.В. Ломоносова»

115522, Москва, Каширское ш., 24

Filatov Alexander V., PhD, MD (Biology), Professor, Head, Laboratory of Immunoc hemistry, National Research Center “Institute of Immunology”, Federal Medical-Biological Agency of Russia; Professor, Department of Immunology, Lomonosov Moscow State University

115522, Moscow, Kashirskoye Highway, 24

avfilat@yandex.ruФГБУ «ГНЦ „Институт иммунологии“» Федерального медико-биологического агентства России; ФГБОУ ВО «Московский государственный университет имени М.В. Ломоносова»РоссияNational Research Center “Institute of Immunology”, Federal Medical-Biological Agency of Russia; Lomonosov Moscow State UniversityRussian FederationФБУН Московский научно-исследовательский институт эпидемиологии и микробиологии имени Г.Н.Габричевского Роспотребнадзора; кафедра иммунологии Биологического факультета Московского государственного университета им. М.В. Ломоносова, 119192, Россия, МоскваРоссияLomonosov Moscow State University; G. Gabrichevsky Research Institute for Epidemiology and MicrobiologyRussian FederationГБУЗ МО «Московский областной научно-исследовательский клинический институт имени М.Ф. Владимирского»РоссияM. Vladimirsky Moscow Regional Research and Clinical InstituteRussian FederationФГБУ «ГНЦ „Институт иммунологии“» Федерального медико-биологического агентства России; ФГБОУ ВО «Московский государственный университет имени М.В. Ломоносова» ФГБУ «ГНЦ „Институт иммунологии“» Федерального медико-биологического агентства России; ФГБОУ ВО «Московский государственный университет имени М.В. Ломоносова» ФГБУ «ГНЦ „Институт иммунологии“» Федерального медико-биологического агентства России; ФГБОУ ВО «Московский государственный университет имени М.В. Ломоносова» ФГБУ «ГНЦ „Институт иммунологии“» Федерального медико-биологического агентства России; ФГБОУ ВО «Московский государственный университет имени М.В. Ломоносова» ФГБУ «ГНЦ „Институт иммунологии“» Федерального медико-биологического агентства России; ФГБОУ ВО «Московский государственный университет имени М.В. Ломоносова»РоссияNational Research Center “Institute of Immunology”, Federal Medical-Biological Agency of Russia; Lomonosov Moscow State UniversityRussian Federation20203112202022611851194Copyright © Бязрова М.Г., Топтыгина А.П., Митина Т.А., Филатов А.В., 20212021Бязрова М.Г., Топтыгина А.П., Митина Т.А., Филатов А.В.Byazrova M.G., Toptygina A.P., Mitina T.A., Filatov A.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.mimmun.ru/mimmun/article/view/2066

При вакцинации происходит стимуляция В-клеток, и в кровотоке на короткое время появляются активированные В-лимфоциты, которые относятся к плазмобластам. Плазмобласты также наблюдаются при некоторых вирусных инфекциях. Количество плазмобластов может являться показателем успешности вакцинации или диагностическим признаком продолжающейся инфекции. Как правило, плазмобласты представлены немногочисленной популяцией клеток, определение которой встречает некоторые трудности. В исследовании приняли участие 15 здоровых добровольцев, которые были однократно иммунизированы рекомбинантной вакциной против гепатита В. Для определения плазмобластов были использованы меченные антитела, ранее полученные в нашей лаборатории. Использованные реагенты показали свою применимость для подсчета плазмобластов. Было проведено сравнение различных стратегий гейтирования плазмобластов. При окрашивании лимфоцитов иммунизированных добровольцев набором антител CD19-PE, CD3/CD14/CD16-FITC, CD27-PC5.5 и CD38-PC7 наблюдался отчетливый кластер плазмобластов с фенотипом CD27++CD38++. Включение в панель антитела CD20-FITC приводило к увеличению доли CD27++CD38++ плазмобластов среди CD19+ лимфоцитов до 60% и более. При замене антитела CD38 на антитело CD71 также обнаруживался отчетливый кластер плазмобластов, содержавший около 5% В-лимфоцитов. Две стратегии гейтирования плазмобластов с использованием комбинаций CD27/CD38 и CD27/CD71 мы сравнили в динамике на лимфоцитах одного вакцинированного добровольца. При использовании сочетания CD27/CD38 на 7-й день после вакцинации регистрировался острый и выраженный пик количества плазмобластов. Применение комбинации CD27/CD71 приводило к растягиванию пика на период с 7-го по 14-й день после вакцинации. Таким образом, временная динамика популяции CD27+CD71+ отличалась от появления классических плазмобластов с фенотипом CD27++CD38++. Это наталкивает на мысль, что в популяцию CD27++CD71+ входят не только плазмобласты, но также и другие типы активированных В-клеток. Был получен препарат малого поверхностного антигена гепатита В, меченного фикоэритрином (HBsAg-PE), с помощью которого было определено количество антигенспецифических плазмобластов. Результаты определения антигенспецифических клеток с помощью комплекса HBsAg-PE согласовались c данными, полученными методом ELISpot. Отработанная стратегия гейтирования плазмобластов в настоящее время используется нами для определения активированных В-клеток при инфекции, вызываемой вирусом SARS-CoV-2. На следующем этапе исследования эта методика будет использоваться для сортировки антигенспецифических В-лимфоцитов, что позволит провести секвенирование генов Ig и приступить к созданию новых человеческих антител против вирусных антигенов.

B cell stimulation develops upon vaccination, thus causing occurrence of activated B cells (plasmoblasts) in bloodstream. Similar cells are also observed in some viral infections. The contents of plasmablasts may be a marker of successful vaccination, or a diagnostic feature of ongoing infection. The plasmablasts are normally represented by a small cell subpopulation which is not easy to detect. A study was performed with 15 healthy volunteers who were subjected to a single immunization with a recombinant vaccine against hepatitis B virus. To identify the plasmablasts, we have used labeled antibodies prepared in our laboratory. These reagents were previously validated for counting the plasmablasts. Different gating strategies for plasmablast gating have been compared. Upon staining of lymphocytes from immunized volunteers, we observed a distinct cluster of plasmablasts with CD27++CD38++ phenotype using the following antibody set: CD19-PE, CD3/CD14/CD16-FITC, CD27-PC5.5 and CD38-PC7. Inclusion of a CD20-FITC antibody into the panel caused an increase of CD27++CD38++ plasmablast ratio among CD19+ lymphocytes to > 60%. Upon substitution of CD38 antibody by anti-CD71, a distinct plasmablast cluster was again revealed, which contained ca. 5 per cent В cells. Two strategies for the plasmablast gating using the CD27/ CD38 and CD27/CD71 combinations were compared in dynamics with lymphocyte samples from a single vaccinated volunteer. When applying the CD27/CD38 combination, a sharp and pronounced plasmablast peak was registered on day 7 post-vaccination. With CD27/CD71 combination, the peak was extended between day 7 and day 14 following immunization. Hence, time kinetics of the CD27+CD71+ population proved to be different from occurrence of classic plasmablasts with CD27++CD38++ phenotype. This finding suggests that the CD27++CD71+population contains both plasmablasts and other types of activated B cells. A minor HBV surface antigen was prepared and labeled with phycoerythrin (HBsAg-PE), thus allowing to quantify the antigen-specific plasmablasts. The results of HBsAg-PE-based detection of antigen-specific cells were in compliance with the data obtained by ELISpot technique. At the present time, we use the original plasmablast gating technique for detection of activated B cells in SARS-CoV-2 infection. At the next step, this technique will be applied to sorting of antigen-specific B cells, thus permitting sequencing of Ig genes and design of novel human antibodies against viral antigens.

B cell stimulation develops upon vaccination, thus causing occurrence of activated B cells (plasmoblasts) in bloodstream. Similar cells are also observed in some viral infections. The contents of plasmablasts may be a marker of successful vaccination, or a diagnostic feature of ongoing infection. The plasmablasts are normally represented by a small cell subpopulation which is not easy to detect. A study was performed with 15 healthy volunteers who were subjected to a single immunization with a recombinant vaccine against hepatitis B virus. To identify the plasmablasts, we have used labeled antibodies prepared in our laboratory. These reagents were previously validated for counting the plasmablasts. Different gating strategies for plasmablast gating have been compared. Upon staining of lymphocytes from immunized volunteers, we observed a distinct cluster of plasmablasts with CD27++CD38++ phenotype using the following antibody set: CD19-PE, CD3/CD14/CD16-FITC, CD27-PC5.5 and CD38-PC7. Inclusion of a CD20-FITC antibody into the panel caused an increase of CD27++CD38++ plasmablast ratio among CD19+ lymphocytes to > 60%. Upon substitution of CD38 antibody by anti-CD71, a distinct plasmablast cluster was again revealed, which contained ca. 5 per cent В cells. Two strategies for the plasmablast gating using the CD27/ CD38 and CD27/CD71 combinations were compared in dynamics with lymphocyte samples from a single vaccinated volunteer. When applying the CD27/CD38 combination, a sharp and pronounced plasmablast peak was registered on day 7 post-vaccination. With CD27/CD71 combination, the peak was extended between day 7 and day 14 following immunization. Hence, time kinetics of the CD27+CD71+ population proved to be different from occurrence of classic plasmablasts with CD27++CD38++ phenotype. This finding suggests that the CD27++CD71+ population contains both plasmablasts and other types of activated B cells. A minor HBV surface antigen was prepared and labeled with phycoerythrin (HBsAg-PE), thus allowing to quantify the antigen-specific plasmablasts. The results of HBsAg-PE-based detection of antigen-specific cells were in compliance with the data obtained by ELISpot technique. At the present time, we use the original plasmablast gating technique for detection of activated B cells in SARS-CoV-2 infection. At the next step, this technique will be applied to sorting of antigen-specific B cells, thus permitting sequencing of Ig genes and design of novel human antibodies against viral antigens.

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The authors declare that there are no conflicts of interest present.