UNSUPERVISED COMPUTATIONAL ANALYSIS OF FLOW CYTOMETRY FOR THE IMMUNOPHENOTYPING OF LYMPHOID AND MYELOID SUBSETS ACROSS TISSUES IN C57BL6 AND PER2KO MICE.

Tucuman

Congreso; LXVII Reunión Científica Annual de la Sociedad Argentina de Inmunología.; 2019

Sociedad Argentina de Inmunología

Circadian rhythms are endogenous 24-h variations in different physiological processes synchronized by ambiental cues and regulated by intracellular clocks. Immune functions controlled by circadian clocks allow organisms to mount most efficient responses while rhythm disruption interferes with cell biology and impairs immune responses. We performed a throughout analysis of myeloid and lymphoid subsets in the light (7 am) and dark (7 pm) phases in spleen, inguinal (ILN) and mesenteric lymph (MLN) nodes of C56BL6 (WT) and clock gene deficient (Per2ko) mice. We used multiparameter flow cytometry for the simultaneous enumeration of live CD45+ cells, dendritic cells, macrophages, neutrophils, T lymphocytes, CD4+ and CD8+ lymphocytes, B lymphocytes, NKT cells, and NK cells (BD LSRFortessa) and t-distributed Stochastic Neighbor Imbedding (tSNE) for unsupervised analysis of dataset generated from different tissues of WT and Per2ko mice. The frequency of NK and NKT cells was reduced at 7pm in ILN of WT and Per2ko mice (p<0.05). Macrophages, neutrophils and dendritic cells showed minor variations in ILN of WT and Per2ko mice. The T cell compartment (CD3+) was higher at the dark phase in both strains, mainly in spleen (WT and Per2ko) and ILN (Per2ko) (p<0.05); for B cells a reduced frequency was observed only in Per2ko at 7 pm. We have defined frequency of myeloid and lymphoid subsets across strains and tissues at critical windows of the circadian rhythm. Describing these major players in one panel may give us a broader view of the host competence and the possibility to predict immune outcomes.