Tumor inflating lymphocytes. Purification, expanding and cytotoxicity analisys on primary tumor cultures

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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.

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, Moscow

S. 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, Moscow

A. L. Krivoshapkin

Novosibirsk State Medical University

Email: alkr01@yandex.ru

MD, Dr. Sci. (Med.), professor

Russian Federation, Novosibirsk

A. 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, Москва

Yu. 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, Moscow

A. 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, Moscow

A. 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, Moscow

A. 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, Moscow

S. 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, Moscow

A. 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, Moscow

D. 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, Moscow

Roman 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, Moscow

Alexander 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, Moscow

V. 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, Moscow

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Supplementary files

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2. Fig. 1. Light microscopy of primary cultures of tumor cells (A – B) and primary cultures of cytotoxic tumors of filtering lymphocytes (D – E), × 200, phase contrast

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3. Fig. 2. Immunophenotyping of cytotoxic CD45 + CD3 + CD8 + TILs

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4. Fig. 3. The content of T-regulatory cells and the expression level of the marker of depletion of cytotoxic lymphocytes PD-1 in the final culture of TILs

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5. Fig. 4. Assessment of the cytotoxicity of the obtained preparations of autologous and allogeneic TILs in relation to cultures of primary tumor cells

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6. Fig. 1. Light microscopy of primary cultures of tumor cells (A – B) and primary cultures of cytotoxic tumor-infiltrating lymphocytes (D – E), × 200, phase contrast

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Indexing metadata
7. Fig. 2. Immunophenotyping of cytotoxic CD45 + CD3 + CD8 + TILs

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8. Fig. 3. The content of T-regulatory cells and the level of expression of the marker of depletion of cytotoxic lymphocytes PD-1 in the final culture of TILs

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9. Fig. 4. Evaluation of the cytotoxicity of the obtained preparations of autologous and allogeneic TILs in relation to cultures of primary tumor cells

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Copyright (c) 2020 Yusubalieva G.M., Petrichuk S.V., Krivoshapkin A.L., Kedrova A.G., Ivanov Y.V., Vinokurov A.G., Kalinkin A.A., Sandjarov A.E., Kim S.V., Ponomarev A.V., Kuptsova D.G., Ischenko R., Troitskiy A., Baklaushev V.P.

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