EXPRESSION PATTERNS AND ROLES OF THE IKAROS FAMILY OF TRANSCRIPTION FACTORS IN HUMAN REGULATORY T CELL DEVELOPMENT AND FUNCTION.

Abstract

Regulatory T cells (Tregs) are a small subset of immune cells that are responsible for downregulating the immune response and maintaining immune tolerance. Tregs are generally defined by a high expression of the transcription factor FOXP3. Dysfunction of Treg activity or number is the basis of many inflammatory diseases. Thus, there has been much research on understanding Treg development in the thymus and activity in the periphery and Tregs are being studied as a potential cellular therapy. One form of Treg therapy is generating engineered Tregs (eTregs), which involves expressing Treg genes in conventional T cells through retroviral or lentiviral transduction. The work described here investigates the role of the Ikaros family of transcription factors in eTreg function and thymic Treg development. The Ikaros family members, namely Helios and Eos, have been implicated as critical mediators of Treg induction and function. In the first study, we hypothesized that ectopic expression of Helios with FOXP3 is required for optimal engineered Treg immunosuppression. In the second study, we hypothesized that Ikaros family member expression correlates with Treg marker expression and defines points of Treg lineage commitment within CD4+ mature single positive (MSP) Tregs in the thymus. In the first study, we generated eTregs by retrovirally transducing total human T cells with combinations of FOXP3, Helios (Hel-FL) and Δ3B Helios (Hel-Δ3B), a relevant splice variant of Helios. FOXP3+Hel-FL eTregs were the only eTregs able to delay disease in a xenogenic Graft versus Host Disease model. In vitro, FOXP3+Hel-FL CD4+ eTregs suppressed T cell proliferation more effectively than FOXP3 and FOXP3+Hel-Δ3B CD4+ eTregs. However, both FOXP3+Hel-FL CD8+ eTregs and FOXP3+Hel-Δ3B CD8+ eTregs were more effective than FOXP3 alone. RNA Sequencing of the CD4+ and CD8+ eTregs demonstrated that the addition of Hel-FL to FOXP3 in eTregs changed gene expression in cellular pathways and the Treg signature compared to FOXP3 alone or FOXP3+Hel-Δ3B. Thus, overexpression of Hel-FL with FOXP3 in eTregs changed gene expression in Tconvs and mediated immunosuppression in vivo and in vitro. Additionally, there is a functional difference between the endogenous splice variants of Helios in mediating CD4+ and CD8+ T cell immunosuppression. In the second study, we used novel CD4+ mature single positive (MSP) thymocyte populations that our laboratory previously defined to track CD4+ CD25+ FOXP3+ human Treg development from human thymus samples. We then characterized protein expression of Ikaros family members and Treg markers of Tregs from each of these populations. We found that a majority of Tregs can be found in the distinct MSP6 population and these Tregs have heterogenous expression of Helios and CD39 and CD127. Within the MSP1-MSP5 populations expression of Ikaros family members transiently changed and Helios and Eos correlated with the percent of Tregs within each population. Thus, using Ikaros family members and Treg markers within subsets of CD4+ thymocytes, we were able to more precisely determine where thymic Tregs originate from and critical points of Treg development. Overall, the results of this research provide further insight into the role of the Ikaros family members in Treg function and development and can be used to improve current Treg therapy.