SUMMARY ThGM Cells in CNS Autoimmunity ThGM cells (ThGM-CSF) are a newly described Th subset characterized by production of GM-CSF, but lacking expression of signature cytokines and master transcription factors (TF) for established Th cell lineages. In our analysis, ThGM cells were notably more abundant than Th2 and Th17 cells in the blood of healthy subjects, and even more so in MS patients. Although ThGM cells have some features of a distinct lineage, further study is needed to clarify that distinction, as well as their roles in health and disease. ThGM cells were highly encephalitogenic in the adoptive EAE model, and they can be readily found in the CNS of mice with direct EAE. Importantly, ThGM cells were enriched in the cerebrospinal fluid of MS patients, who also have greater numbers of circulating ThGM cells than healthy individuals. These findings suggest that ThGM cells contribute to the pathogenesis of CNS autoimmunity, but this subject has been only minimally studied. Over all, our data and published findings led us to hypothesize that ThGM cells play a significant pathogenic role in autoimmune neuroinflammation, MS and EAE. We will test this hypothesis in the following specific aims: Aim 1. To determine the origin of ThGM cells, and characterize the relationship between their GM-CSF expression and phenotype. Both human and mouse ThGM cells can be readily differentiated directly from naïve CD4+ T cells in vitro; however, it is unclear if ThGM cells in vivo originate from naïve precursors, or a Th lineage that switched its phenotype. We hypothesize that ThGM cells in vivo develop directly from naïve CD4+ T cells. In addition, we will study whether GM-CSF is a key determinant of ThGM cell phenotype, or these cells have a unique overall phenotype irrespective of GM-CSF expression. Aim 2. To study the role of ThGM cells in autoimmune neuroinflammation. Recent findings have suggested that ThGM cells contribute to CNS autoimmunity, as they are overrepresented in MS patients’ PBMCs, and they induce EAE in mice by passive transfer. This led us to hypothesize that ThGM cells play a significant pathogenic role in MS and EAE. In mice, we will characterize CNS autoimmunity induced by ThGM cells, while in the human system we will test their pathogenicity in organotypic brain slices, and characterize ThGM cells in MS brain lesions. Aim 3. To determine the role of candidate TFs in shaping ThGM phenotype. ThGM cells do not express master TFs of established Th lineages, suggesting that the ThGM phenotype is shaped by TF(s) differentially expressed in ThGM cells. Our findings form the basis for the hypothesis that the ThGM phenotype is directed by a set of TFs naturally overexpressed in ThGM cells compared to other Th cells. We will test this hypothesis using mice lacking candidate TFs and by knocking down expression of candidate TFs in human CD4+ T cells. Knowledge gained from these studies may modify the current concept of CNS autoimmunity.