Stem-like T cells as bioindicators and orchestrators of the disease process in MS and animal models - There is an urgent need for a better understanding of the propagation of encephalitogenic autoimmune responses and the identification of relevant immune cells and their specific characteristics, which could serve as biomarkers and therapeutical targets. CD4+ T cells show critical pathogenic roles in multiple sclerosis (MS) in preclinical models and human disease. Within the central nervous system (CNS), the encephalitogenic autoimmune CD4+ T cells infiltrate the MS brain lesions, and these T cells recirculate to the peripheral immune system, where they can be maintained and activated. The mechanisms that support long-term maintenance of the pro-encephalitogenic autoimmune CD4+ T cells remain a mystery. The long-lived stem-like CD4+ T cells (CD4+ TSTEMS) with enhanced capacities for effector differentiation were proposed to contribute to the onset and exacerbation of MS disease in humans and in the corresponding animal models. Unlike the well-characterized CD8+ TSTEMS involved in anti-tumor and anti-viral immune responses, these MS-related autoimmune CD4+ TSTEMS are still understudied. Moreover, the current markers of CD4+ TSTEMS are contextual, e.g., T cell factor 1 (Tcf1, encoded by Tcf7) is expressed in non-stem cells, including naive T cells. A lack of precision analytical tools and clearly defined markers hinders further progress in understanding the stem-like paradigm. Our published results uncovered that pro-encephalitogenic CD4+ pre-effectors de novo induced from naive T cells become programmed for diverse autoimmune TEFFS differentiation in an MS model, lending support to the stem-like paradigm. We developed a strategy to overcome the lack of biomarkers issue by identifying a consensus signature from the transcriptomes of various bona fide stem cells. We used this consensus signature and identified a conserved stemness transcriptomic profile specifically in mouse pre-effectors and human T cells from the peripheral blood of MS patients but not in MS-free controls. Furthermore, we have developed a new single- cell sequencing analytical approach enabled by an advanced scoring method, which retrieves biologically relevant information based on the cumulative expression of multiple genes involved in specific biological processes. We extend these exciting research endeavors in this proposal and dedicate Aims 1 and 2 to rigorously test the hypothesis that a continuing stemness and encephalitogenic TEFFS differentiation results in a functional and transcriptomic diversification of CD4+ TSTEM lineages under evolving autoimmune conditions. Furthermore, we have also found increased expression of the homeodomain-only protein (Hopx), in pro-encephalitogenic pre- effectors. Mouse Hopx and human HOPX are well-established orchestrators of stemness in progenitor populations. Intriguingly, we found that Hopx also critically enhanced the survival of Foxp3+ TREGS, suppressing disease in MS models. Therefore, in Aim 3, we will test the hypothesis that Hopx orchestrates independent mechanisms in pro-encephalitogenic TSTEMS and anti-encephalitogenic TREGS, resulting in functionally opposing outcomes impacting the autoimmune response.
