Background: Innate immunity affects infectious and inflammatory diseases. Results: Using RNAi and proteomic data, we identified a novel evolutionarily conserved protein network that modulates innate immunity. Conclusion: Studies using mutant C. elegans and mice demonstrate the utility of this network for disease investigation. Significance: This innate immunity network provides a novel set of targets for future innate immunity disease studies.

Regulatory T cells (Tregs) are a subset of immune cells that suppress the immune response. Treg therapy for inflammatory diseases is being tested in the clinic, with moderate success. However, it is difficult to isolate and expand Tregs to sufficient numbers. Engineered Tregs (eTregs) can be generated in larger quantities by genetically manipulating conventional T cells to express FOXP3. These eTregs can suppress in vitro and in vivo but not as effectively as endogenous Tregs. We hypothesized that ectopic expression of the transcription factor Helios along with FOXP3 is required for optimal eTreg immunosuppression. To test this theory, we generated eTregs by retrovirally transducing total human T cells (CD4+ and CD8+) with FOXP3 alone or with each of the 2 predominant isoforms of Helios. Expression of both FOXP3 and the full-length isoform of Helios was required for eTreg-mediated disease delay in a xenogeneic graft-versus-host disease model. In vitro, this corresponded with superior suppressive function of FOXP3 and full-length Helios-expressing CD4+ and CD8+ eTregs. RNA sequencing showed that the addition of full-length Helios changed gene expression in cellular pathways and the Treg signature compared with FOXP3 alone or the other major Helios isoform. Together, these results show that functional human CD4+ and CD8+ eTregs can be generated from total human T cells by coexpressing FOXP3 and full-length Helios.

In human T cell development, the mechanisms that regulate cell fate decisions after TCRβ expression remain unclear. We defined the stages of T cell development that flank TCRβ expression and found distinct patterns of human T cell development. In half the subjects, T cell development progressed from the CD4−CD8− double negative (DN) stage to the CD4+CD8+ double positive (DP) stage through an immature single positive (ISP) CD4+ intermediate. However, in some patients, CD4 and CD8 were expressed simultaneously and the ISP population was small. In each group of patients, CD3− ISP and DP thymocytes were subdivided into ISP1, ISP2, DP1, DP2, DP3, DP4, and DP5 developmental stages according to their expression of CD28, CD44, CD1a, CD7, CD45RO, and CD38. The ISP2, DP2, and DP3 thymocyte populations proliferated more robustly than ISP1 and DP1 and expressed markers consistent with TCRβ expression. After the DP3 stage, proliferation returned to baseline levels. We then analyzed protein levels of Ikaros, Helios, and Aiolos, the three Ikaros family members most abundantly expressed in human thymocytes. Ikaros and Helios expression increased transiently at the ISP2, DP2, and DP3 populations. Aiolos expression also increased at the ISP2, DP2, and DP3 stages, but its expression remained elevated throughout the DP4 and DP5 stages. In summary, we propose a model of human T cell development that reflects the asynchronous nature of TCRβ expression and we define the subpopulations of thymocytes that are highly proliferative and express Ikaros family members.

The Ikaros family of transcription factors includes five highly homologous members that can homodimerize or heterodimerize in any combination. Dimerization is essential for their ability to bind DNA and function as transcription factors. Previous studies showed that eliminating the function of the entire family blocks lymphocyte development while deletion of individual family members has relatively minor defects. These data indicate that multiple family members function during T cell development, so we examined the changes in expression of each family member as thymocytes progressed from the CD4−CD8− double negative (DN) to the CD4+CD8+ double positive (DP) developmental stage. Further, we compared the expression of each family member in murine and human thymocytes. In both species, Ikaros and Aiolos mRNA levels increased as thymocytes progressed through the DN to DP transition, but the corresponding increases in protein levels were only observed in mice. Further, Ikaros and Aiolos underwent extensive alternative splicing in mice, whereas only Ikaros was extensively spliced in humans. Helios mRNA and protein levels decreased during murine T cell development, but increased during human T cell development. These differences in the expression and splicing of Ikaros family members between human and murine thymocytes strongly suggest that the Ikaros family of transcription factors regulates murine and human T cell development differently, although the similarities across Ikaros family members may allow different proteins to fulfill similar functions.