One trick to solve the target discovery in the treatment of TILs

Cellular immunotherapy, also known as adoptive cellular immunotherapy (AIT), has a promising application prospect that has become the fourth pillar

At present, in addition to the chimeric antigen receptor-T cell (CAR-T) therapy, which has been widely used in clinical practice, in recent years, new therapies such as gene editing and modification of TCR-T, CAR-NK, CAR-M, and tumor-infiltrating lymphocyte (TIL) therapy have also gradually entered the clinic

In the field of solid tumor therapy, TILs therapy is considered one

01

TIL therapy in solid tumors

Application in treatment

Tumors are heterogeneous, are not static during development and are often able to evade immunotherapy such as immune checkpoint inhibitors, TCR-T, and CAR-T through antigenic epitope mutations or loss of expression

TILs are highly heterogeneous subsets of lymphocytes, with different cell subsets playing different roles

▲ Classification of tumor-infiltrating lymphocytes, in recent years, people have carried out a large number of clinical studies on TILs to observe and evaluate the therapeutic effect

02

Quick and precise "find"

TILs cells

With the rapid development of technology, flow cytometry can already be a good response to TILs cell recognition and analysis, the use of multi-parameter measurement, flow cytometry can be the cell markers fluorescence signal detection and strength determination, reflecting the proportion of screening cells or analyzing the distribution and content of substances on the cell surface and inside

 

Based on these advantages, flow cytometry analyzers can quickly find the 30% TILs that can react with tumor cells – in fact, TME contains not only tumor-associated antigen-reactive T cells (TAA-T), but also bystander T cells that recognize non-tumor (mainly viral) epitopes, and only about 30% of TILs can react with tumor cells.

03

Flow cytometry analyzer on

TILs target discovery

"Putting your fists to the punch"

Traditional TIL treatment is limited by the toxicity of high-dose IL-2, and its in vivo survival and function are not ideal, and in order to solve this problem, a new generation of TIL products

There are studies ^[8] using single-cell RNA sequencing in mouse models of non-small cell lung cancer to fully understand tumor-infiltrating T cells, with a focus on ti-Treg subpopulations, which was verified by flow cytometry analysis, and the results identified that Ccr8 in the two ti-Treg populations had unique expressions bound to the Treg activation marker; Another study[9] By flow cytometry and other analytical methods, it was found that the cytokines IL-10 and IL-35 (Ebi3-IL-12α heterodimer) were expressed differently by Treg cell subsets in the tumor microenvironment (TME), and that the transcriptome characteristics of T cell depletion in tumors were promoted collaboratively by modulating the expression of several inhibitory receptors
, CD8+ and failure-related transcriptome features.

The above examples illustrate how flow cytometry analyzers can "play a big role"
in the selection of targets.

▲ Mutual expression of IL-10 and IL-35 on mouse and human Treg cells Source Reference 9

04

Flow cytometry

Applications in other areas

In order to cope with the problem of how to match and transfuse blood in patients with autoimmune hemolytic anemia (AIHA), the advantages of high sensitivity of flow cytometry are used to analyze the individual differences and stability of blood group antigens, which can more effectively detect
alloantibodies mixed in autoantibodies.

Flow cytometry can also be used to identify and quantify different cell types in the preclinical stage, and there have been studies ^[10] using flow cytometry to assess levels of relevant inhibition receptors and cytokine-stimulated levels in patients with glioblastoma (GBM), and T cell receptor Vβ chain expansion
has also been evaluated in TILs and PBLs.

The results showed that GBM caused particularly severe characteristics of T cell failure in infiltrative T cells; In addition, the use of flow cytometry multiparameter measurements allows in-depth immunophenotyping analysis of TME to improve patient outcomes and treatment management
.

▲ In addition to TIL exhibiting molecular signatures and epigenetic atlases consistent with T cell failure10, flow cytometry analyzers can also be used in the field of hematology for the diagnosis, therapeutic evaluation and recurrence monitoring
of hematologic disorders.

It is worth mentioning that the use of CD45 antibody labeling gate method for multi-color immunolabeling has greatly improved the accuracy of immunotyping, and has now become an indispensable tool for diagnosing leukemia; It can also be used in immunology to detect lymphocyte subsets to assess the body's immune status and assist in disease diagnosis
.

05

Flow cytometry

Application scenarios

A typical monochromatic flow cytometer enables multiparametric analysis
of individual cells or particles at a rate of 10,000 or more cells or particles per second.

Combined with the development of an automated sample collection system, it is possible to measure multiple parameters
simultaneously.

Multiparametric analysis capabilities are particularly attractive in supporting the shift of focus from target centers to target-independent or mechanically informed phenotypic drug discovery
.

Flow cytometry has been applied at every stage of the drug discovery cycle, including target identification and validation, hit identification, lead and candidate drug selection, and safety studies
.

However, it is usually only possible to use it in
relatively low sample volumes.

This reflects the application scenario of the monochromatic flow cytometer "swallow-spit" sample processing method, which takes an hour or more for automated systems to read 96-well plates, so another solution is needed in the scale of detection running in many drug screening laboratories – multicolor flow cytometry
.

06

High throughput, multi-color

Aid in drug discovery

One step ahead

Bó Lè ZE5 flow cytometry analyzer has the advantages of high speed, automatic, flexible and different from monochrome flow cytometer, ZE5 a 96-well plate running time of less than or equal to 12 minutes, a 384-well plate running time of less than or equal to 50 minutes, greatly shortening the time
required for analysis.

Faced with application scenarios such as the analysis of TILs cells, ZE5 can quickly complete the analysis and detection of different cells by taking advantage of its high throughput and multi-staining
.

It is worth mentioning that the high-throughput detection supported by ZE5 can analyze up to 100,000 events per second with no data loss; Flow tubes or multi-well plates, low or high speed, low or high throughput, 1 color or 25 colors, flexible sample handling methods and automated program settings enable efficient analysis and response to complex application scenarios
.

▲ Bó Lè ZE5 flow cytometry analyzer

Bó Lè offers a wide selection of fluorescent dyes, from StarBright, a highly compatible high-brightness dye, to other common dyes of different wavelengths
.

These dyes meet a wide range of targets from Bó Lè's carefully selected people and mice, giving your multicolor flow cytometry design a wide range of options, ensuring outstanding cell resolution and high-quality flow cytometry data
.

The combination of adoptive therapy of TILs with traditional and emerging therapies is the development trend
of future clinical treatment.

Further research and identification of TILs subsets and functions in different tumors is the basis for the development of new methods of
tumor immunotherapy.

Flow cytometry can play its own unique role in these problems and challenges and influence the process of pre-drug discovery – the Böhler's ZE5 Flow Cytometry Analyzer greatly simplifies the research process
.

Resources:

Yang Han,Chen Chao,Wang Chong.
Research Progress on Immunotherapy of Tumor Invasive Lymphocytes[J].
Shanghai Medicine,2022,43(11):17-21.
[2] Quan Jiale, Kang Yanliang, Zhang Wanli, Tian Xun, Gao Xiangdong.
Research Progress on Adoptive Cell Therapy in Tumor Immunotherapy[J].
Pharmaceutical Advances,2021,45(10):725-734.
Zhang Qingqing,Xu Lianrong.
Research Progress on CAR-T and TCR-T Therapy in Tumor Adoptive Cell Immunotherapy[J].
New Medicine,2021,52(03):165-169.
Chen Yuanyuan,Cai Jing,Jiang Chuyi.
Research Progress of Tumor Invasive Lymphocyte Adoptive T Cell Therapy in Cervical Cancer[J].
Henan Medical Research,2021,30(01):189-192.
[5] Recent advances in high-throughput flow cytometry for drug discovery[J].
Expert Opinion on Drug Discovery,2020(prepublish).
[6] Bai Suhang, Yang Xiaoyue, Zhang Nan, Zhang Fuhan, Shen Zongyi, Yang Na, Zhang Wensai, Yu Yuan, Yang Zhao.
Research Progress on the Role of Tumor Invasive Lymphocytes in Solid Tumor[J].
Chinese Journal of Bioengineering,2019,35(12):2308-2325.
DOI:10.
13345/j.
cjb.
190300.
[7] Phenotyping of tumor infiltrating immune cells using mass-cytometry (CyTOF).
Methods Enzymol.
2020; 632:339-368.
doi: 10.
1016/bs.
mie.
2019.
07.
025.
Epub 2019 Oct 18.
PMID: 32000904.
[8] Therapeutic depletion of CCR8+ tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy.
J Immunother Cancer.
2021 Feb; 9(2):e001749.
doi: 10.
1136/jitc-2020-001749.
PMID: 33589525; PMCID: PMC7887378.
[9] Adaptive plasticity of IL-10+ and IL-35+ Treg cells cooperatively promotes tumor T cell exhaustion.
Nat Immunol.
2019 Jun; 20(6):724-735.
doi: 10.
1038/s41590-019-0346-9.
Epub 2019 Apr 1.
PMID: 30936494; PMCID: PMC6531353.
[10] T-Cell Exhaustion Signatures Vary with Tumor Type and Are Severe in Glioblastoma.
Clin Cancer Res.
2018 Sep 1; 24(17):4175-4186.
doi: 10.
1158/1078-0432.
CCR-17-1846.
Epub 2018 Feb 7.
PMID: 29437767; PMCID: PMC6081269.
[11] Zhao Fengyong, Zhang Yuyu, Wang Zhongying, Xu Ting, Li Qin, Zhang Jiamin, Xiang Dong, Zhu Ziyan.
Study on the detection of blood group alloantibodies in patients with autoimmune hemolytic anemia under autoantibody mask by flow cytometry[J].
Clinical Blood Transfusion and Testing,2022,24(01):6-10.
[12] Effect of cryopreservation on delineation of immune cell subpopulations in tumor specimens as determinated by multiparametric single cell mass cytometry analysis.
BMC Immunol.
2017 Feb 2; 18(1):6.
doi: 10.
1186/s12865-017-0192-1.
PMID: 28148223; PMCID: PMC5288879.