BCG-activation of leukocytes is sufficient for the generation of donor-independent innate anti-tumor NK and γδ T-cells that can be further expanded in vitro

ABSTRACT

Bacillus Calmette-Guérin (BCG), the nonpathogenic Mycobacterium bovis strain used as tuberculosis vaccine, has been successfully used as treatment for non-muscle invasive bladder cancer for decades, and suggested to potentiate cellular and humoral immune responses. However, the exact mechanism of action is not fully understood. We previously described that BCG mainly activated anti-tumor cytotoxic NK cells with upregulation of CD56 and a CD16⁺ phenotype. Now, we show that stimulation of human peripheral blood mononuclear cells with iBCG, a preparation based on BCG-Moreau, expands oligoclonal γδ T-cells, with a cytotoxic phenotype, together with anti-tumor CD56^high CD16⁺ NK cells. We have used scRNA-seq, flow cytometry, and functional assays to characterize these BCG-activated γδ T-cells in detail. They had a high IFNγ secretion signature with expression of CD27⁺ and formed conjugates with bladder cancer cells. BCG-activated γδ T-cells proliferated strongly in response to minimal doses of cytokines and had anti-tumor functions, although not fully based on degranulation. BCG was sufficient to stimulate proliferation of γδ T-cells when cultured with other PBMC; however, BCG alone did not stimulate expansion of purified γδ T-cells. The characterization of these non-donor restricted lymphocyte populations, which can be expanded in vitro, could provide a new approach to prepare cell-based immunotherapy tools.

KEYWORDS:

• Cancer immunology
• BCG
• NK activation
• gamma delta T lymphocytes
• bladder cancer
• immunotherapy

Acknowledgments

The authors would like to thank M. Muñoz (Crick Institute, UK) for a critical review of the manuscript; L. Martinez-Piñeiro (H. La Paz, Madrid) for BCG; A. Dopazo and the Genomics Unit of the National Centre for Cardiovascular Research (CNIC, Madrid, Spain) for their advice on scRNAseq analysis. The group of MVG belongs to the research network “Conexion cancer”-CSIC. MJF and AFGJ are registered PhD students at the Molecular Biosciences doctoral program of the Universidad Autónoma de Madrid (UAM).

Disclosure statement

EP, ER, and IM are employees of Biofabri, S.L.

Data availability statement

R code related to the main scRNA-seq figures can be found at https://github.com/algarji/BCG_scRNA-seq.

Raw data of the scRNA-seq analysis will be found at GEO (https://www.ncbi.nlm.nih.gov/geo/)

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/2162402X.2022.2160094

Author contributions

GE, MJF, AFGJ, CRV, HTR acquired, analyzed, and interpreted data

EP, NA, ER, IM provided mycobacterial reagents

NA, CM, ER, IM, and MVG provided material support

GE, MJF, and MVG wrote the manuscript

MVG supervised the study

All authors participated in critical revisions of the article

Additional information

Funding

This work was supported by the Spanish Ministry of Science and Innovation under Grants (RTC-2017-6379-1, RTI2018-093569-B-I00, PID2021-123795OB-I00 [Ministerio de Ciencia, Innovación y Universidades (MCIU)/Agencia Estatal de Investigación (AEI)/European Regional Development Fund (FEDER, EU)], SAF2017-83265-R (HTR); and the regional government of Madrid [COMUNIDAD DE MADRID S2017/BMD-3733-2 (MVG)]; MJF is a fellow of the INPhINIT Doctoral Programme from La Caixa Foundation (LCF/BQ/DI19/11730039). AFGJ is a recipient of a fellowship (FPU18/01698) from the Spanish Ministry of Science and Education.