Assessment of T-cell response to the tumor is important for diagnosis of the disease and monitoring of therapeutic efficacy. For this, new non-destructive label-free methods are required. Fluorescence lifetime imaging (FLIM) of metabolic coenzymes is a promising innovative technology for the assessment of the functional status of cells. The purpose of this work was to test whether FLIM can resolve metabolic alterations that accompany T-cell reactivation to the tumors. The study was carried out on C57Bl/6 FoxP3-EGFP mice bearing B16F0 melanoma. Autofluorescence of the immune cells in fresh lymphatic nodes (LNs) was investigated. It was found that fluorescence lifetime parameters of nicotinamide adenine dinucleotide (phosphate) NAD(P)H are sensitive to tumor development. Effector T-cells in the LNs displayed higher contribution of free NADH, the form associated with glycolysis, in all tumors and the presence of protein-bound NADPH, associated with biosynthetic processes, in the tumors of large size. Flow cytometry showed that the changes in the NADH fraction of the effector T-cells correlated with their activation, while changes in NADPH correlated with cell proliferation. In conclusion, FLIM of NAD(P)H in fresh lymphoid tissue is a powerful tool for assessing the immune response to tumor development.

Personalized strategies in glioblastoma treatment are highly necessary. One of the possible approaches is drug screening using patient-derived tumor cells. However, this requires reliable methods for assessment of the response of tumor cells to treatment. Fluorescence lifetime imaging microscopy (FLIM) is a promising instrument to detect early cellular response to chemotherapy using the autofluorescence of metabolic cofactors. Here, we explored FLIM of NAD(P)H to evaluate the sensitivity of patient-derived glioma cells to temozolomide (TMZ) in vitro. Our results demonstrate that the more-responsive cell cultures displayed the longest mean fluorescence lifetime τm after TMZ treatment due to an increase in the protein-bound NAD(P)H fraction α₂ associated with a shift to oxidative phosphorylation. The cell cultures that responded poorly to TMZ had generally shorter τm, i.e., were more glycolytic, and showed no or insignificant changes after treatment. The FLIM data correlate well with standard measurements of cellular drug response—cell viability and proliferation index and clinical response in patients. Therefore, FLIM of NAD(P)H provides a highly sensitive, label-free assay of treatment response directly on patient-derived glioblastoma cells and can become an innovative platform for individual drug screening for patients.

Background: The wide variability in clinical responses to anti-tumor immunotherapy drives the search for personalized strategies. One of the promising approaches is drug screening using patient-derived models composed of tumor and immune cells. In this regard, the selection of an appropriate in vitro model and the choice of cellular response assay are critical for reliable predictions. Fluorescence lifetime imaging microscopy (FLIM) is a powerful, non-destructive tool that enables direct monitoring of cellular metabolism on a label-free basis with a potential to resolve metabolic rearrangements in immune cells associated with their reactivity. Objective: The aim of the study was to develop a patient-derived glioma explant model enriched by autologous peripheral lymphocytes and explore FLIM of the redox-cofactor NAD(P)H in living lymphocytes to measure the responses of the model to immune checkpoint inhibitors. Methods: The light microscopy, FLIM of NAD(P)H and flow cytometry were used. Results: The results demonstrate that the responsive models displayed a significant increase in the free NAD(P)H fraction α₁ after treatment, associated with a shift towards glycolysis due to lymphocyte activation. The non-responsive models exhibited no alterations or a decrease in the NAD(P)H α₁ after treatment. The FLIM data correlated well with the standard assays of immunotherapy drug response in vitro, including morphological changes, the T-cells activation marker CD69, and the tumor cell proliferation index Ki67. Conclusions: The proposed platform that includes tumor explants co-cultured with lymphocytes and the NAD(P)H FLIM assay represents a promising solution for the patient-specific immunotherapeutic drug screening.

Recent advances in cancer immunotherapy have great promise for the treatment of solid tumors. One of the key limiting factors that hamper the decoding of physiological responses to these therapies is the inability to distinguish between specific and nonspecific responses. The identification of tumor-specific lymphocytes is also the most challenging step in cancer cell therapies such as adoptive cell transfer and T cell receptor (TCR) cloning. Here, we have elaborated a protocol for the identification of tumor-specific T lymphocytes and the deciphering of their repertoires. B16 melanoma engraftment following anti-PD1 checkpoint therapy provides better antitumor immunity compared to repetitive immunization with heat-shocked tumor cells. We have also revealed that the most error-prone part of dendritic cell (DC) generation, i.e., their maturation step, can be omitted if DCs are cultured at a sufficiently high density. Using this optimized protocol, we have achieved a robust IFNγ response to B16F0 antigens, but only within CD4+ T helper cells. A comparison of the repertoires of IFNγ-positive and -negative cells shows a prominent enrichment of certain clones with putative tumor specificity among the IFNγ+ fraction. In summary, our optimized protocol and the data provided here will aid in the acquisition of broad statistical data and the creation of a meaningful database of B16-specific TCRs.

In humans and mice, the T-cell receptor (TCR) of each effector/memory T-cell clone recognizes one to several cognate peptide–MHC complexes (pMHC). Limited knowledge of TCR repertoire specificities restricts our capacity to rationally interpret this information, both diagnostically and in preclinical research. In this study, we (i) developed and validated a cost-efficient wet lab and computational pipeline to identify mouse TCRs specific to particular peptides, (ii) produced a dataset of helper T cell (Th) TCR beta chain CDR3s specific to B16 melanoma neoantigens in the I-Ab pMHCII context, available in VDJdb, and (iii) applied this dataset to track tumor-specific T-cell responses to the CTLA4 blocking immunotherapy in the orthotopic B16 melanoma model. We showed that B16-specific TCR motifs expanded in both Th and regulatory T cell (Treg) repertoires of tumor-bearing mice, with stronger expansion in the Th subset of mice treated with the CTLA4 blocking antibody. The response was stochastic across individual mice with respect to the specific TCR motifs and target peptides involved—each mouse displayed a unique response pattern, likely reflecting the natural diversity of molecular pathways underlying antitumor immunity. We also showed that CTLA4 blockade promotes prominent Th clonal expansion and induces general, nontumor-specific Th-to-Treg plasticity. Altogether, we provide a universal pipeline for the investigation of mouse T-cell responses at the antigen-specific level, facilitating the development and validation of immunotherapeutic and vaccination approaches.

Distribution of identified B16F10-specific TCRβ CDR3 clusters with 1 amino acid mismatch allowed