Fluorescence Lifetime Imaging of NAD(P)H T Cells Autofluorescence in the Lymphatic Nodes to Assess the Effectiveness of Anti-CTLA-4 Immunotherapy.

Abstract

The main problem in the field of tumor immunotherapy is the lack of reliable biomarkers that allow pre-determining the susceptibility of individual patients to treatment, as well as insufficient knowledge about the resistance mechanisms. Biomarkers based on the autofluorescence of metabolic coenzymes in immune cells can become a powerful new predictor of early tumor response to treatment, whereas the optical FLIM method can be a tool to predict the effectiveness of immunotherapy, which allows preserving the spatial structure of the sample and obtaining results on the metabolic status of immune cells in real time. The aim of the study is to conduct a metabolic autofluorescence imaging study of the NAD(P)H metabolic coenzyme in immune cells of freshly isolated lymph nodes as a potential marker for assessing the effectiveness of an early response to immunotherapy.

Materials and methods: The study was carried out on C57Bl/6 FoxP3-EGFP mice with B16F0 melanoma implanted near the inguinal lymph node. The mice were injected with antibodies to CTLA-4 (Bio X Cell, USA) (250 μg per mouse, intraperitoneally on days 7, 8, 11, and 12 of the tumor growth). FLIM images in the nicotinamide adenine dinucleotide (phosphate) coenzyme (NAD(P)H) channel (excitation - 375 nm, reception - 435-485 nm) were received using an LSM 880 fluorescent confocal laser scanning microscope (Carl Zeiss, Germany) equipped with a FLIM Simple-Tau module 152 TCSPC (Becker & Hickl GmbH, Germany). Flow cytometry was conducted using a BD FACSAria III cell sorter (BD Biosciences, USA).

Results: Immunotherapy with checkpoint inhibitors resulted in marked metabolic rearrangements in T cells of freshly isolated lymph nodes in responder mice, with inhibition of the tumor growth. Fluorescence lifetime imaging data on NAD(P)H indicated an increase in the free fraction of NADH α₁, a form associated with glycolysis to meet high demands of the activated T cells and pro-inflammatory cytokine synthesis. In contrast, non-responder mice with advanced tumors showed low values of the ratio of free fraction to bound α₁/α₂, which may be related to mechanisms of resistance to therapy.The response to immunotherapy was verified by data on the expression of activation and proliferation markers by means of flow cytometry. The authors observed an increase in the production of the pro-inflammatory cytokine IFN-γ in effector T cells in responder mice compared to untreated controls and non-responders. In addition, an increase in the expression of the surface activation markers CD25 and CD69 was registered compared to untreated controls.

Conclusion: Use of the FLIM method allowed to demonstrate that autofluorescence of the NAD(P)H coenzyme is sensitive to the response to checkpoint immunotherapy and can be used as a reliable marker of the effectiveness of response to treatment.