Krüppel-like factor 2: a key transcription factor mediates CD4+ T cells migration in experimental autoimmune encephalomyelitis

Autoreactive CD4+ T cells migrating into the central nervous system (CNS) has been established as a key driver for the multiple sclerosis (MS) disease onset. Once inside the CNS, these cells are activated by CNS-specific antigens, triggering neuroinflammation, and further neurodegeneration. In recent times, the inhibition of the autoreactive CD4+ T cell migration became a promising therapeutic strategy. However, like other treatments, it cannot fully prevent the MS disease progression and comes with the risk of severe infections. To develop better therapies, a deeper understanding of the molecular mechanisms guiding CD4+ T cell migration is essential. Our study aims to reveal how autoreactive CD4+ T cells migrate through key stages—from the bloodstream to secondary lymphoid organs (SLOs), back into circulation, and ultimately into the CNS. Our observations are based on the experimental autoimmune encephalomyelitis (EAE) model, a well-established model for MS. We focused on the two transcription factors, krüpple-like factor 2 (Klf2) and forkhead box protein O1 (Foxo1), identified from our previously published genome-wide CRISPR/Cas9 screening data for their role in regulating autoreactive CD4+ T cell migration. Using CRISPR/Cas9 single-gene knockout technology, we examined the individual functions of Klf2 and Foxo1 in T cell migration in an adoptive transfer EAE model, along with the distinct functions of their downstream effector molecules. Our study highlights Klf2 as a key transcription factor controlling T cell migration in EAE. Knocking out Klf2 caused T cells to accumulate in the parathymic lymph nodes (ptLNs) and spleen. Based on our findings, this is due to an increase in C-C chemokine receptor 7 (Ccr7) expression and the absence of sphingosine-1-phosphate receptor 1 (S1pr1). Additionally, Klf2 deficiency reduced Itga4 and Itgb1 expression, disrupting very late antigen 4 (VLA4) and preventing T cells from adhering to CNS blood vessels. As a result, Klf2-KO cells cannot infiltrate the CNS to induce EAE. Interestingly, knocking out Foxo1, an upstream regulator of Klf2, led T cells to a higher accumulation in ptLNs but did not affect their migration to the CNS. This is likely because Foxo1 promotes Ccr7 expression, but its absence does not impact Itga4. Beyond migration, our findings reveal Klf2 as a crucial switch between the activated and migratory states of T cells. Without Klf2, autoreactive T cells remain in an activated state, showing increased CD25 expression and elevated pro-inflammatory cytokines (TNF-α, IFN-γ, and IL-17A). This abnormal activation, combined with their failure to infiltrate the CNS, suggests that Klf2 is essential for shifting T cells into a migratory phenotype in SLOs. By uncovering these molecular pathways, our study offers new insights into potential therapeutic targets that could block autoreactive T cell migration into the CNS without broadly suppressing the immune system.