Transcriptional Program Regulating the Resistance of Memory T Cells to Tolerance in EAE

Background: Myelin-reactive CD4⁺ T cells in multiple sclerosis (MS) patients belong to the long-lived memory T cell pool. Immune suppression by CD4⁺Foxp3⁺ regulatory T cells (Tregs) is mediated through various mechanisms including inhibitory cytokines [e.g. transforming growth factor (TGF)-beta1 and interleukin- (IL)-10] and surface ligands. The possibility that Tregs might be used for the treatment of autoimmune diseases has recently gained momentum. Current therapeutic strategies in MS are focused on developing agents or manipulating pathways that stimulate Treg expansion to induce immunological tolerance. However, recent findings suggest that memory CD4⁺T cells resist Treg-mediated suppression.

Objectives: To identify the cellular and molecular mechanisms controlling the resistance of memory T cells to Treg-mediated suppression in EAE.

Methods: Microarray analysis was used to identify novel gene that discriminate memory and conventional effector CD4⁺T cells. Retrovirus transduction system was used to overexpress ATF3 in T cells and ATF3-/- mice were used to analyze the effects of ATF3 deficient on T cells function. Whole genome gene profiling was generated in peripheral blood mononuclear cells (PBMCs) from relapsing-remitting MS treated and untreated patients.

Results: Previously, we have demonstrated that unlike conventional effector CD4⁺ T cells, long-lived memory CD4⁺ T cells are less susceptible to CTLA4Ig-mediated tolerance in EAE. We analyzed the molecular basis underlying the resistance of memory T cells to immune regulation using gene microarray profiling of myelin-specific murine effector and memory CD4⁺ T cells. We found that activating transcription factor 3 (ATF3), a transcriptional repressor, is down-regulated in memory CD4⁺ T cells and this was confirmed in human memory CD4⁺ T cells. Using gain (Overexpression) and loss of function (Knockout mice) approaches, we demonstrated that ATF3 expression is required for the TGF-beta1-mediated T cell suppression by physically binding and stabilizing Smad3 protein expression, the transcriptional effector of TGF-beta1 signaling. In EAE, adoptive transfer of myelin-specific CD4⁺T cells overexpressing ATF3 failed to induce clinical disease in naïve syngeneic mice.  In humans, we found that ATF3 gene expression is upregulated in PBMCs isolated from MS patients that received interferon-beta (n=128) compared to untreated subjects (p = 0.0025). Altogether, these findings suggest that ATF3 maybe involved in the induction of tolerance in MS.

Conclusions: We identified for the first time a transcriptional pathway that regulates the resistance of memory CD4⁺ T cells to tolerance. We predict that ATF3 maybe a therapeutic target for T cell pathogenicity in multiple sclerosis and other autoimmune diseases.