FLIM of NAD(P)H in Lymphatic Nodes Resolves T-Cell Immune Response to the Tumor

Abstract

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.

Keywords: FAD; FLIM; NAD(P)H; T-lymphocyte; autofluorescence; immune response; metabolic coenzyme; tumor.

Figure 1

Fluorescence intensity of NAD(P)H and FAD in LNs of intact and tumor-bearing mice. (A) Representative intensity-based images in NAD(P)H (blue, em = 450–490), FAD (azure, em = 500–550) and EGFP (green, em = 500–570) channels. Examples of the regulatory FoxP3+ CD4+ T-cells, stably expressing EGFP, are marked by the white squares in all channels. (B) Quantification of fluorescence intensity-based redox ratio FAD/NAD(P)H and fluorescence intensity of NAD(P)H. Scatter dot plot displays the measurements for individual animals (dots), the mean and SEM (horizontal lines).

Figure 2

FLIM of NAD(P)H in LNs of intact and tumor-bearing mice. (A) Representative FLIM images of the mean fluorescence lifetime τ_m and long lifetime component τ₂. (B) Quantification of fluorescence lifetime parameters. Scatter dot plot displays the measurements for individual animals (dots) and the mean and SEM (horizontal lines).

Figure 3

Relative contributions of NAD(P)H in LNs of intact and tumor-bearing mice. (A) Representative FLIM images of the relative amplitude of free NADH α₁, upon bi- (all groups) and three- (“Large tumor” group) exponential fitting. (B) Quantification of relative contributions of free and bound NADH (α₁, α₂) and NADPH (α₃). The data are presented as mean ± SEM. * p < 0.03, compared to “Control” group; ^# p < 0.0001, compared to the “Small tumor” group. Three-exponential fitting was applied to the part of cells (28–83%) having long lifetime τ₂ within each LN; other cells were processed with bi-exponential fitting.

Figure 4

Expression of CD69 and CD25 in live CD4+ Th and CD8+ T-cells. Scatter dot plots display the measurements for individual animals (dots) and the mean and SEM (horizontal lines).

Figure 5

Expression of IFNγ and Ki67 in fixed CD4+ Th and CD8+ T-cells. Scatter dot plots display the measurements for individual animals (dots) and the mean and SEM (horizontal lines).