Tumor inflating lymphocytes. Purification, expanding and cytotoxicity analisys on primary tumor cultures
- Authors: Yusubalieva G.M.1, Petrichuk S.V.2, Krivoshapkin A.L.3, Kedrova A.G.1, Ivanov Y.V.1, Vinokurov A.G.1, Kalinkin A.A.1, Sandjarov A.E.1, Kim S.V.1, Ponomarev A.V.1, Kuptsova D.G.2, Ischenko R.1, Troitskiy A.1, Baklaushev V.P.1
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Affiliations:
- Federal Scientific and Clinical Center of Specialized Types of Medical Care and Medical Technologies of the Federal Medical and Biological Agency of Russia
- National Medical Research Center for Children›s Health
- Novosibirsk State Medical University
- Issue: Vol 11, No 1 (2020)
- Pages: 49-58
- Section: Basic Science
- URL: https://journals.rcsi.science/clinpractice/article/view/33974
- DOI: https://doi.org/10.17816/clinpract33974
- ID: 33974
Cite item
Abstract
Background. Tumor Infiltrating Lymphocytes (TILs) is one of the most promising sources of autologous cytotoxic T-cells for adoptive immunotherapy, which has already shown high efficiency in the treatment of metastatic melanoma. However, the isolation of TILs from solid tumors is technically difficult. A suppressive tumor microenvironment, in particular, a high level of expression of check-point inhibitors PD-1 CTLA4, tissue hypoxia and other factors cause that T cells isolated from the tumor do not proliferate well and do not exhibit cytotoxic properties.
Aims. In this study, we isolated TILs from surgical material obtained by resection of solid tumors (primary and metastatic adenocarcinomas of various localization, melanoma, glioblastoma), studied their population composition and developed protocols for the purification expanding, and activation of CD4+, CD8+ cytotoxic antitumor lymphocytes.
Methods. An urgent task is the activation of TILs, turning off immunosuppressive mechanisms and increasing their antitumor cytotoxic activity. Various approaches are used for this: activation by a cocktail of cytokines and antibodies, editing the lymphocyte genome by knocking out suppressor genes or, conversely, transduction of activating genes, coincubation with feeder cells, etc. Cells were obtained from samples of resected tumors in 16 patients; in each case we obtain an autologous pair: the primary tumor culture and the TILs culture.
Results. We could isolate viable lymphocytes in 100% of cases. Isolated TILs were successfully expanded in our specialized medium using various combinations of IL-2, IL-15, IL-21, IL-7, anti-CD3 and anti-CD28. Immunophenotyping showed that the obtained TILs are a heterogeneous mixture of CD4+, CD8+ cells containing populations of CD3+CD8+CD45+(CTL) CD3+CD4+CD45+ (T-helpers), CD4+CD25+CD127- (Т-regulatory cells), CD3-CD56+CD45+ (NK-cells), CD3+CD56+CD45+ (Т-NK-cells). The initial cultures of TILs were also characterized by a high level of PD1 expression, indicating their low antitumor cytotoxicity. Using different protocols of isolation, expansion, and activation, we obtained a cell preparation containing 80% of CD8+ PD-1- activated TILs in an amount sufficient for adoptive therapy (500×106 or more). An in vitro study of the cytotoxicity of obtained TILs in primary cultures of homologous tumors using RTCA Icelligence showed high cytotoxicity, providing almost 100% tumor cell death.
Conclusion. Our developed protocol for the production and activation of TILs can be recommended for the phase I–II clinical trials of adoptive immunotherapy of recurrent, highly metastatic solid tumors.
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##article.viewOnOriginalSite##About the authors
G. M. Yusubalieva
Federal Scientific and Clinical Center of Specialized Types of Medical Care and Medical Technologies of the Federal Medical and Biological Agency of Russia
Email: gaukhar@gaukhar.org
ORCID iD: 0000-0003-3056-4889
SPIN-code: 1559-5866
MD, Cand. Sci. (Med.)
Russian Federation, MoscowS. V. Petrichuk
National Medical Research Center for Children›s Health
Email: cito@list.ru
SPIN-code: 7026-6160
MD, Dr. Sci. (Med.), professor
Russian Federation, MoscowA. L. Krivoshapkin
Novosibirsk State Medical University
Email: alkr01@yandex.ru
MD, Dr. Sci. (Med.), professor
Russian Federation, NovosibirskA. G. Kedrova
Federal Scientific and Clinical Center of Specialized Types of Medical Care and Medical Technologies of the Federal Medical and Biological Agency of Russia
Email: kedrova.anna@gmail.com
SPIN-code: 3184-9760
MD, Dr. Sci. (Med.)
Russian Federation, МоскваYu. V. Ivanov
Federal Scientific and Clinical Center of Specialized Types of Medical Care and Medical Technologies of the Federal Medical and Biological Agency of Russia
Email: ivanovkb83@yandex.ru
ORCID iD: 0000-0001-6209-4194
SPIN-code: 3240-4335
MD, Dr. Sci. (Med.)
Russian Federation, MoscowA. G. Vinokurov
Federal Scientific and Clinical Center of Specialized Types of Medical Care and Medical Technologies of the Federal Medical and Biological Agency of Russia
Email: avinok@mail.ru
MD, Cand. Sci. (Med.)
Russian Federation, MoscowA. A. Kalinkin
Federal Scientific and Clinical Center of Specialized Types of Medical Care and Medical Technologies of the Federal Medical and Biological Agency of Russia
Email: akalinkin@mail.ru
MD, Cand. Sci. (Med.)
Russian Federation, MoscowA. E. Sandjarov
Federal Scientific and Clinical Center of Specialized Types of Medical Care and Medical Technologies of the Federal Medical and Biological Agency of Russia
Email: sanzh@mail.ru
ORCID iD: 0000-0003-1056-3053
SPIN-code: 5713-5791
врач-уролог, врач высшей категории, заведующий отделением урологии
Russian Federation, MoscowS. V. Kim
Federal Scientific and Clinical Center of Specialized Types of Medical Care and Medical Technologies of the Federal Medical and Biological Agency of Russia
Email: mrkims@mail.ru
врач анестезиолог-реаниматолог
Russian Federation, MoscowA. V. Ponomarev
Federal Scientific and Clinical Center of Specialized Types of Medical Care and Medical Technologies of the Federal Medical and Biological Agency of Russia
Email: limbt@mail.ru
research associate
Russian Federation, MoscowD. G. Kuptsova
National Medical Research Center for Children›s Health
Email: dg.kuptsova@gmail.com
ORCID iD: 0000-0001-7771-3314
SPIN-code: 7476-1524
junior research associate
Russian Federation, MoscowRoman Ischenko
Federal Scientific and Clinical Center of Specialized Types of Medical Care and Medical Technologies of the Federal Medical and Biological Agency of Russia
Email: ishenko@mail.ru
ORCID iD: 0000-0002-7999-8955
MD, Dr. Sci. (Med.)
Russian Federation, MoscowAlexander Troitskiy
Federal Scientific and Clinical Center of Specialized Types of Medical Care and Medical Technologies of the Federal Medical and Biological Agency of Russia
Email: dr.troitskiy@gmail.com
ORCID iD: 0000-0003-2143-8696
MD, Dr. Sci. (Med.)
Russian Federation, MoscowV. P. Baklaushev
Federal Scientific and Clinical Center of Specialized Types of Medical Care and Medical Technologies of the Federal Medical and Biological Agency of Russia
Author for correspondence.
Email: baklaushev.vp@fnkc-fmba.ru
ORCID iD: 0000-0003-1039-4245
https://fnkc-fmba.ru/about/komanda-upravleniya/
MD, Dr. Sci. (Med.)
Russian Federation, MoscowReferences
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