Nrn1 expression and function in anergic T cells.
(A) Experimental scheme identifying Nrn1 in anergic T cells and qRT-PCR confirmation of Nrn1 expression in HA-specific CD4 cells recovered from HA-expressing host vs WT host activated with Vac_HA virus. (B) qRT-PCR and western blot detecting Nrn1 expression in naïve CD4+CD62LhiCD44lo Tn cell, CD4 effector CD4+Foxp3-CD44hiCD73-FR- Te cells and CD4 anergic CD4+Foxp3-CD44hiCD73+FR+ Ta cells. (C) Nrn1 expression was measured by qRT-PCR and western blot among naïve CD4+ T cells, CD4+Foxp3+ nTreg, and in vitro generated iTregs. (D) Nrn1 expression was detected by qRT-PCR and flow cytometry among WT naïve CD4+ cells and activated CD4+ cells on days 1, 2, and 3 after activation. Nrn1-/- CD4 cells were also stained for Nrn1 three days after activation. qPCR Data are presented as average ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Triplicates were used. Ordinary one-way ANOVA was performed for multi-comparison.
(E-J). Anergy induction in vivo. (E) Experimental outline evaluating anergy development in vivo: 2x106 Thy1.1+ Nrn1-/- or ctrl CD4 OTII T cells were co-transferred with 5x105 Thy1.2+Thy1.1- WT Treg cells into TCRα-/-mice. Cells were recovered on day 13 post-transfer. (F) Proportions and numbers of OTII cells recovered from recipient spleen; (G) IL2 secretion from OTII cells upon ex vivo stimulation with OVA peptide. (H) Foxp3+ cell proportion among Thy1.1+ Nrn1-/- or ctrl CD4 cells. (I & J) Nrn1-/- vs ctrl OTII cells recovered from the peptide-induced anergy model were subjected to bulk RNASeq analysis. GSEA comparing the expression of signature genes for anergy (I) and Treg (J) among ctrl and Nrn1-/- OTII cells.
Data are presented as mean ±SEM and representative of 3 independent experiments (N≥4 mice per group). *p<0.05, **p<0.01, ***p<0.001. Unpaired Student’s t-tests were performed.