Monitoring Early Efficacy of the Checkpoint Immunotherapy Based on Fluorescence Lifetime Imaging Microscopy of the Patient-Derived Lymphocyte Subsets

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Diana V. Yuzhakova^1,a*#, Anastasia I. Kononova^1,2#, Anna V. Izosimova¹, Margarita M. Sazhina¹, Ilya D. Shchechkin¹, Artem M. Mozherov^1,3, Pavel A. Bureev¹, Dmitry A. Kuzmin^1,4, Alena I. Gavrina¹, Irina S. Shumskaya^1,5, Sergey V. Gamayunov^1,5, and Marina V. Shirmanova¹

¹Privolzhsky Research Medical University, 603005 Nizhny Novgorod, Russia

²Lobachevsky State University of Nizhny Novgorod, 603022 Nizhny Novgorod, Russia

³I. M. Sechenov First Moscow State Medical University, 119435 Moscow, Russia

⁴Sirius University of Science and Technology, 354340 Sirius Federal Territory, Russia

⁵Nizhny Novgorod Regional Clinical Oncology Dispensary, 603093 Nizhny Novgorod, Russia

^* To whom correspondence should be addressed.

^# These authors contributed equally to the work.

Received: April 16, 2026; Revised: June 15, 2026; Accepted: July 7, 2026

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Despite the success of immune checkpoint inhibitor (ICI) therapy, the problem of resistance makes the search for early response biomarkers critical. This study investigates the potential of monitoring autofluorescence of the NAD(P)H coenzyme in the T-lymphocyte subsets to assess early treatment efficacy. The ability of NAD(P)H FLIM to reflect sensitivity to ICI therapy was tested on the B16F0 mouse melanoma model and validated in the pilot study of the samples from melanoma patients during treatment. The mean fluorescence lifetime of NAD(P)H, τ_m, showed the best correlation with subsequent clinical response and was identified as a sensitive marker of early lymphocyte activation following anti-PD-1 or combination therapy. In the patients who responded to treatment, an increase in τ_m was observed, associated with metabolic reprogramming toward enhanced biosynthetic and bioenergetic pathways, as confirmed by the standard molecular analysis. Thus, the proposed approach represents a promising solution for personalized monitoring of early immunotherapy efficacy.

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KEY WORDS: immunotherapy, immune checkpoint inhibitors, melanoma, lymphocyte, biomarker, FLIM, metabolism

DOI: 10.1134/S0006297926601206

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