To identify a Tr1 cell DEG signature in an experimental malaria model that would allow functional testing of candidate signature molecules in vivo, we next infected triple reporter (Il10gfp x Ifngyfp x foxp3rfp) C57BL/6 mice with P. berghei ANKA (PbA) and then isolated Th0 (CD4+ GFP- YFP- RFP-), Th1 (CD4+ GFP- YFP+ RFP-) and Tr1 (CD4+ GFP+ YFP+ RFP-) cells from the spleens by cell sorting at day 4 p.i.. RNA was isolated from Th0, Th1 and Tr1 cells and RNAseq employed to identify differentially expressed genes between the CD4+ T cell subsets

Control of the intracellular parasites causing malaria requires IFN?+ Tbet+ CD4+ T (Th1) cells generated by infected hosts. IL-10 produced by Th1 cells mitigates subsequent inflammation and related pathology. However, IL-10-producing Th1 (Tr1) cells can also promote parasite persistence, as well as impair immunity following re-infection or vaccination. Here, we identify a transcriptional signature that distinguished Th1 cells from Tr1 cells in experimental malaria caused by Plasmodium berghei (PbA) ANKA infection of C57BL/6 mice. This was achieved by infecting triple reporter (Il10gfp x Ifngyfp x foxp3rfp) C57BL/6 mice with PbA and then isolating Th0 (CD4+ GFP- YFP- RFP-), Th1 (CD4+ GFP- YFP+ RFP-) and Tr1 (CD4+ GFP+ YFP+ RFP-) cells from the spleens by cell sorting at day 4 p.i.. RNA was isolated from Th0, Th1 and Tr1 cells and RNAseq employed to identify differentially expressed genes (DEGs) between the CD4+ T cell subsets. We focused our attention on differences between Tr1 and Th1 cells and found 2031 DEGs in Tr1 cells compared to Th1 cells, with 1025 and 1006 of these significantly up- and down-regulated, respectively. Up-regulated DEGs included the co-inhibitory receptors Ctla4, Havcr2 (encoding TIM3) and Lag3; chemokine receptors, including Ccr2, Ccr5, and Cxcr6, and transcription factors Maf, Ahr and Prdm1. Together, this work provides new insights into CD4+ T cell development during malaria that offer opportunities for strategies to modulate their functions to improve anti-parasitic immunity