Obtaining tumour-specific T cells in a mouse melanoma model

Abstract

Abstract 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. 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.