Next Generation Sequencing of T regulatory cells (GFP+, dTomato+) and ex-T regulatory cells (GFP-, dTomato+) harvested from lymph nodes of bone marrow chimeras (50% Foxp3GFP-tdT  or Foxp3GFP-Fth/-tdT and 50% C57BL/6 bone marrow into Rag2-/- recipients)
Purpose: To address whether FTH regulates gene expression in TREG cells, irrespective of bystander inflammation. For this, we compared the gene expression profile of TREG and Ex-TREG cells sorted from the lymph nodes of mixed BM chimeras Results: Using an optimized data analysis workflow, we mapped an average 30.14 million sequence reads per sample to the mouse genome (build mm39) and identified 34,321 transcripts in the mRNA of Treg and Ex-Treg cells from mixed bone marrow chimeras. Differential gene expression was performed using the DESeq2 R package (v.1.32) and gene expression was modeled by genotype and condition. Differentially expressed genes were considered for genes with an adjusted p-value<0.05 and an absolute log2 fold change>0. Conclusions: Analysis of RNAseq data showed that Fth deletion in TREG cells (CD45.2+GFP+tdT+) developing from the BM of Foxp3GFP-Fth?/?-tdT mice, up-regulated 149 genes and down-regulation of 96 genes, compared to TREG cells from control Foxp3GFP-tdT mice. Ex-TREG cells (CD45.2+GFP-tdT+) up-regulated 90 genes and down-regulated 36 genes. The genes regulated in TREG and ex-TREG cells originating from the BM of Foxp3GFP-Fth?/?-tdT vs. Foxp3GFP-tdT mice were associated with the oxidative stress response regulated by NRF2 . This suggests that FTH exerts cell-autonomous control of TREG cell redox homeostasis, irrespective of bystander inflammation. Overall design: Methods: T-reg and Ex-T-reg cells were collected from the lymph nodes of mixed bone marrow chimeras, 6 weeks after reconstitution. Mixed bone chimeras were generated by non-lethally irradiating Rag2-/- mice and reconstituting with a mix of 50% Foxp3GFP-tdT  or Foxp3GFP-Fth/-tdT and 50% C57BL/6 bone marrow (n=3-4 per combination). RNA was extracted and libraries prepared. mRNA expression profiles were generated by next generation sequencing,  using Illumina High output kit v2.5 and NexSeq500 sequencer.