Unsuprvised computational analysis of flow cytometry for the immunophenotyping of lymphiod and myeloid subsets across tissues in C57bl6 and Per2KO mice

San Miguel de Tucuman

Congreso; LXVII Reunión Anual de la Sociedad Argentina de Inmunología; 2019

Sociedad Argentina de Inmunología

Circadian rhythms are 24-h variations in physiological processes synchronizedby ambient cues and regulated by intracellular clocks. Immune functionscontrolled by circadian clocks allow organisms to mount more efficient responseswhile rhythm disruption interferes with cell biology and impairs immuneresponses. We performed an analysis of myeloid and lymphoid subsets in thelight (7 am) and dark (7 pm) phases in spleen, inguinal (ILN) and mesentericlymph (MLN) nodes of C57BL6 (WT) and clock gene deficient (Per2ko) mice. Weused multiparametric flow cytometry for the simultaneous enumeration of liveCD45+ cells, dendritic cells (CD11c+;MHCIIhi), macrophages (CD11b+;F4/80+),neutrophils (CD11b+;Ly6G+), T-lymphocytes (CD3+), CD4+ and CD8+ T-lymphocytes, B-lymphocytes (CD19+), NKT cells (NK1.1+;CD3+),, and NK cells(NK1.1+), (BD LSRFortessa) and t-Distributed Stochastic Neighbor Embedding(tSNE) for unsupervised analysis of dataset generated from different tissues ofWT and Per2ko mice. The frequency of NK and NKT cells was reduced at 7pmin ILN of WT and Per2ko mice (p<0.05). Macrophages, neutrophils and dendriticcells showed minor variations in ILN of WT and Per2ko mice. The T cellcompartment was higher at the dark phase in both strains, mainly in spleen (WTand Per2ko) and ILN (Per2ko) (p<0.05); for B cells a reduced frequency wasobserved only in Per2ko at 7 pm. We have defined frequency of myeloid andlymphoid subsets across strains and tissues at critical windows of the circadianrhythm. Describing these major players in one panel may give us a broader viewof the host competence and the possibility to predict immune outcomes.