Background Adoptive T cell therapy is a promising treatment strategy for tumor patients. However, when entering the tumor microenvironment (TME), T cells lose their effector function showing reduced degranulation and cytokine secretion. Besides T cell inhibition through checkpoint pathways (i.e. PD-1/L1, CTLA–4), suppressor cells (i.e. TAM, Treg) and cytokines (i.e. IL–10, TGF, VEGF), various metabolites of the TME also counteract antitumoral activities. Among the latter, lactate and extracellular acidosis are byproducts of the cancer metabolism and commonly observed in high concentrations in solid tumors. Previous experiments showed that tumor lactic acidosis selectively targets the signaling pathway including JNK/c-Jun and p38, resulting in inhibition of IFN-γ production. In contrast, granule exocytosis, which is regulated via the MEK1/ERK pathway, was moderately affected. Based on the contrasting effects on these two essential T cell effector activities, we investigated in more detail the effects of lactic acidosis on the killing process conducted by T cells.
Material and Methods Tumor cells and cytotoxic T cells were co-cultured in lactic acid or regular culture medium and analyzed for effector function by flow cytometry and cell-mediated cytotoxicity assays. Additionally, ‘in-channel micropatterning’ in combination with artificial intelligence (AI) aided image analysis was used to visualize and analyze T cell cytotoxicity and mobility on a single cell level. Usage of collagen-matrices allowed the observation of T cell activity in a physiological three-dimensional environment. Cell metabolism was analyzed by Seahorse technology.
Results In the presence of lactic acid, IFN-γ production was strongly inhibited, while degranulation was only moderately reduced. Detailed analysis of the different processes involved in T cell cytotoxicity revealed that T cell recognition of tumor cells resulted in less secretion of cytotoxins (perforin, granzyme B and granzyme A). Lytic activity against tumor cells was strongly reduced at low T cell to tumor cell ratio (1:2). This deficiency could be compensated by increasing the T cell to tumor cell ratio (10:1). Using live cell imaging we investigated underlying mechanisms that might explain how higher T cell to target cell ratios might overcome lactic acid inhibition. T cells in lactic acid covered less distance, they moved for longer time periods and made less contacts with tumor cells in comparison to T cells cultured in regular culture medium.
Conclusions Micropatterning and AI based image analysis allows for detailed assessment of the processes involved in T cell-mediated killing such as mobility, speed, directionality and attachment on target cells. Lactic acidosis is hampering T cell killing activity by reducing the T cell’s capacity to find its target cell and attach to it. Repeated addition of T cells or neutralization of lactic acidosis in the TME are means to overcome these deficits and hold promise to improve the outcome of T cell-based immunotherapies.
Disclosure Information A.J. Fischbeck: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Modest; IBIDI GmbH. A.N. Mendler: None. M. Balles: A. Employment (full or part-time); Significant; IBIDI GmbH. J. Schwarz: A. Employment (full or part-time); Significant; IBIDI GmbH. R. Zantl: A. Employment (full or part-time); Significant; IBIDI GmbH. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; IBIDI GmbH. E. Noessner: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Modest; IBIDI GmbH.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.