Visualization and functional characterization of CD4+ T cell stimulation in the ileal lamina propria

Multiple sclerosis (MS) is characterized is by immune cell infiltration into the central nervous system (CNS) causing inflammation and subsequent demyelination associated with neuronal degeneration. According to the leading paradigm of the etiology of MS, pre-existing autoreactive T cells are primed in the periphery before invading the CNS. Combined with the increasingly important role of microbiota as potential triggers of autoimmune diseases, the gut possibly constitutes the site of action. The main aim of this study was to investigate whether encephalitogenic T cells get stimulated in gut-associated lymphoid tissues prior to experimental autoimmune encephalomyelitis (EAE), a rodent model for MS. Given that intracellular calcium signaling is a prerequisite for effective activation of T cells, cytosolic calcium levels constitute a useful indicator of stimulation. By combining the Förster resonance energy transfer (FRET)-based calcium sensor Twitch-2B with intravital two-photon microscopy, this study demonstrates that T cells specific for the CNS antigen myelin oligodendrocyte glycoprotein (MOG) as well as T cells specific for the control antigen ovalbumin (OVA) but not polyclonal T cells get stimulated in the ileal lamina propria. To investigate whether the ileal stimulation lead to activation-induced gene expression, transcriptome analysis of CD4+ T cells from the efferent mesenteric lymph, i.e. downstream of the stimulation in the small intestine, was performed. The RNA sequencing of MOG-specific CD4+ T cells from mice housed under specific pathogen-free (SPF) conditions indicated that stimulation in the small intestine induces Th17 cells with a pathogenic phenotype. In contrast, neither in germ-free mice, i.e. in the absence of microbiota, nor in polyclonal T cells, i.e. in the absence of extended calcium signaling in the ileal lamina propria, the upregulation of Th17 markers was observed. Furthermore, the transfer of gut-stimulated antigen-specific T cells to immunized recipient mice and subsequent analysis of the T cells’ migration capacities demonstrated that the ileal stimulation does not only alter gene expression but also influences T cell behavior. CD4+ T cells that had previously been stimulated by microbiota migrated more efficiently to lymphoid organs than T cells devoid of this stimulation. The second part of this study focused on another crucial event in the pathogenesis of MS/EAE, i.e. the infiltration of encephalitogenic T cells into the CNS. The requirement of specific adhesion molecules for T cell extravasation was examined by intravital two-photon imaging of encephalitogenic T cells at the blood-brain barrier in a transfer EAE model in the Lewis rat. This enabled visualization of the mode of action of antibodies blocking the entry of encephalitogenic T cells into the CNS. Furthermore, this study confirmed that the blocking antibody against integrin α4, which is already in use as treatment for MS, has the most pronounced therapeutic effect. The findings of the present study add to the mechanistic understanding of two critical steps in the pathogenesis of EAE, i.e., the peripheral stimulation by microbiota and the subsequent infiltration of encephalitogenic T cells into the CNS. Especially the priming process of autoreactive T cells in the periphery preceding CNS inflammation remains one of the least deciphered processes in the pathogenesis of CNS autoimmunity affecting millions of people worldwide