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Lunch & Poster Display session

38P - Obtaining tumour-specific T cells in a mouse melanoma model

Date

12 Dec 2019

Session

Lunch & Poster Display session

Presenters

Diana Yuzhakova

Citation

Annals of Oncology (2019) 30 (suppl_11): xi12-xi15. 10.1093/annonc/mdz448

Authors

D.V. Yuzhakova1, A. Izosimova1, L. Barbashova1, E. Zagaynova1, G. Sharonov1, D. Chudakov2

Author affiliations

  • 1 Institute Of Experimental Oncology And Biomedical Technologies, Privolzhsky Research Medical University, 603005 - Nizhny Novgorod/RU
  • 2 Laboratory Of Immunosequencing Methods, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 - Moscow/RU
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Resources

Abstract 38P

Background

One of most promising strategies for cancer immunotherapy is adoptive T cell therapy (ACT). In the frame of this approach, tumor-specific lymphocytes (TSL) are infused into patients to recognize and destroy tumor cells. The key step of ACT is identification of TSL within the tumor-infiltrating lymphocytes (TILs). However, current procedures of obtaining TSL are very labor-intensive and expensive or have not demonstrated the sufficient clinical benefits. The purpose of this study was to develop an effective and simple method for TSL identification in a mouse melanoma model.

Methods

The experiments were carried out on C57BL/6 mice bearing B16F0 mouse melanoma model. TILs were obtained by disaggregation of tumor nodules, and mouse spleen T-cells were harvested by magnetic separation. Tumor reactivity of T-lymphocytes was assessed by IFNγ secretion assay or intracellular staining. IFNγ+ T-cells were collected by BD FACSAria III cell sorter (BD Biosciences, USA).

Results

To obtain melanoma specific T-cells we isolated TILs from tumor nodules and sorted live activated IFNγ+ T-cells. We identified 1-6% CD4+ and 3-8% CD8+ IFNγ+ activated T-lymphocytes. With bioinformatic analysis of T-cell receptor repertoires we detected the clusters of cells that recognize the same antigens and are enriched in IFNγ+ cell fraction compared to IFNγ- cells. This fact is an additional evidence of tumor specify of these cells. To check tumor specificity of bioinformatically identified T-cells we have used one of the common approaches for TSL identification – in vitro stimulation of T-cells by APCs loaded with tumor antigen. First, we established mice with stable immunity against B16F0 and harvested T-cells from spleen. Then T-cells were cocultured with B16F0-loaded APCs. To improve the efficiency, we have optimized several parameters, such as additional immunization of the mice and co-cultivation conditions. As a result, we registered 1% of B16F0-specific CD4+ T-lymphocytes against the background of non specifically activated cells.

Conclusion

We have developed a new method to obtain potential TSL from tumor nodules. Future experiments will be directed to compare their repertoires with B16F0-specific T-cells.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

Ministry of Education and Science of the Russian Federation (grant No 14.W03.31.0005).

Disclosure

All authors have declared no conflicts of interest.

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