(J-368) Autofluorescence lifetime imaging resolves metabolic abnormalities in T cells from SLE patients

Introduction:: Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by aberrant immune response and chronic multiorgan systemic inflammation and is a leading causing of morbidity and mortality in young women.  Metabolic abnormalities in T cells play an important role in SLE pathogenesis. Optical metabolic imaging (OMI) is a label-free imaging technique that leverages the autofluorescence of NAD(P)H and FAD, two metabolic co-enzymes, to investigate cellular metabolism. Our objective is to 1) use OMI to investigate baseline metabolic differences between CD3+ T cells from healthy donors and SLE patients and 2) explore potential correlations between T cell metabolism and the SLE Disease Activity Index (SLEDAI), a standard of care measure used to assess disease activity in patients with SLE based on their clinical presentations and serological markers (0-105, 0=No activity, 5+ = active disease).

Materials and Methods:: Whole blood samples were obtained from SLE patients (n=4) and healthy donors (n=1). CD3+ T cells were then isolated and cultured in Human Plasma-Like Medium. After 24 hours, CD3+ T-Cells were imaged using a 40x/1.15 NA objective on a custom inverted 2P FLIM microscope with sequential acquisition of NAD(P)H [750nm ex. (440/80nm em.)] and FAD [890nm ex. (550/100nm em.)]. Single cells were automatically segmented through Cellpose to obtain single cell metabolism data. Figure 1 provides an example of a FLIM image with single cell information. Independent t-tests and Pearson’s correlation coefficient were used to calculate statistical significance and the correlation coefficient.

Results, Conclusions, and Discussions:: We observed significant differences in OMI measurements between an age matched healthy donor and an SLE patient with a SLEDAI score of 12. CD3+ T cells from the SLE patient displayed significantly lower NAD(P)H mean lifetime. Lower NAD(P)H mean lifetime is associated with cell activation, suggesting SLE patients have more active CD3+ T cells. The percentage of bound FAD in CD3+ T-cells was also found to have a slightly positive correlation with the SLEDAI. A higher SLEDAI score indicates more severe disease activity, thus our data suggest that bound FAD proportion increases in patients with more active SLE.

Our study provides novel insights into the metabolic characteristics of CD3+ T-cells in SLE patients using OMI. We observed significant differences in baseline metabolism between an SLE patient and an age-matched healthy donor. We also demonstrated a slight correlation between CD3+ T-cell metabolism and SLE disease activity, highlighting the potential of FLIM as a tool for evaluating immune dysregulation in patients with SLE. Furthermore, understanding the role cellular metabolism plays in SLE pathogenesis and progression could uncover new therapeutic targets and improve disease management strategies for SLE patients.


Acknowledgements (Optional): : This material is based
upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No.
DGE-1747503. Any opinions, findings, and conclusions or recommendations expressed in this material are those of
the author(s) and do not necessarily reflect the views of the National Science Foundation.

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Walsh AJ, Mueller KP, Tweed K, Jones I, Walsh CM, Piscopo NJ, Niemi NM, Pagliarini DJ, Saha K, Skala MC. Classification of T-cell activation via autofluorescence lifetime imaging. Nat Biomed Eng. 2021 Jan;5(1):77-88. doi: 10.1038/s41551-020-0592-z. Epub 2020 Jul 27. PMID: 32719514; PMCID: PMC7854821.