ITK signalling via the Ras/IRF4 pathway regulates the development and function of Tr1 cells

Abstract

Type 1 regulatory T (Tr1) cells differentiate in response to signals engaging the T cell receptor (TCR), express high levels of the immunosuppressive cytokine IL-10, but not Foxp3, and can suppress inflammation and promote immune tolerance. Here we show that ITK, an important modulator of TCR signalling, is required for the TCR-induced development of Tr1 cells in various organs, and in the mucosal system during parasitic and viral infections. ITK kinase activity is required for mouse and human Tr1 cell differentiation. Tr1 cell development and suppressive function of Itk deficient cells can be restored by the expression of the transcription factor interferon regulatory factor 4 (IRF4). Downstream of ITK, Ras activity is responsible for Tr1 cell induction, as expression of constitutively active HRas rescues IRF4 expression and Tr1 cell differentiation in Itk−/− cells. We conclude that TCR/ITK signalling through the Ras/IRF4 pathway is required for functional development of Tr1 cells.

Beyond type 1 regulatory T cells: co-expression of LAG3 and CD49b in IL-10-producing T cell lineages

“…LAG3 and CD49b co-expression was previously reported to be a cell surface signature for both mouse and human IL-10-producing CD4 + T cells that lack the expression of Foxp3 (also known as type 1 regulatory T cells, Tr1 cells) [15]. We and others have previously reported that co-culturing murine naïve CD4 + T cells with antigen presenting cells (APCs) in the presence of anti-CD3, anti-CD28, anti-IFN-γ, anti-IL-12, and IL-27 can efficiently induce the differentiation of Tr1 cells [40; 43; 44], which express high levels of LAG3 and CD49b. Our recent data also demonstrated that this protocol can induce IL-10 production in bulk T cell populations that include both CD4 + and CD8 + T cells (Fig.…”

“…IL-10 production can be significantly elevated in the pulmonary mucosa during the late stages of parasitic infection by Nippostrongylus brasiliensis ( Nb ), predominantly by CD4 + T cells that are LAG3/CD49b double positive [15; 45]. We recently observed that both Foxp3 − and Foxp3 + T cells are capable of producing IL-10 in the pulmonary tissues post Nb infection [40]. To determine whether co-expression of LAG3 and CD49b is exclusive to Foxp3 − Tr1 cell subset, we infected IL-10 GFP /Foxp3 RFP dual reporter mice with Nb , and analyzed the IL-10-producing T cells.…”

“…2–3). To determine whether this feature is applicable to IL-10-producing cells in other organs, we injected IL-10 GFP /Foxp3 RFP dual reporter mice with an anti-CD3ε antibody that has been shown to stimulate pronounced IL-10 production by T cells through TCR activation in vivo [23; 40]. We analyzed IL-10-producing T cells in blood, lymph nodes (LN), lung, fat and small intestine, found that co-expression of LAG3 and CD49b marked a portion of the IL-10-producing T cells following TCR activation in vivo , which again included Foxp3 + CD4 + , Foxp3 − CD4 + and CD8 + T cell subsets (Fig.…”

“…Cells from various organs were isolated as we recently described [40]. Briefly: blood cells were collected through cardiac puncture, and red blood cells were lysed before analysis; lungs were minced and digested in 0.2 mg/ml Liberase TL (Sigma, St. Luis, MO) in 37ºC for 15-30 mins, then filtered and red blood cells were lysed before analysis; intestines were flushed, opened longitudinally, and inner contents removed with the blunt end of scissors, then cut into 0.5-cm fragments, followed by digestion in 100 U/ml collagenase VIII (Sigma) in 37ºC for 1 hour, filtered, and lymphocytes isolated using gradient separation by 40% and 80% Percoll (GE Healthcare, Wilkes-Barre, PA) solutions; perigonadal adipose tissues were minced and digested in 500 U/ml collagenase I (Worthington Biochemical Corp., Lakewood, NJ) in 37ºC for 30 mins, filtered and red blood cells were lysed before analysis.…”

TCR/ITK signaling via mTOR tunes CD8+ T cell homeostatic proliferation, metabolism, and anti-tumor effector function

“…Triggering the TCR using anti-CD3 antibodies, has been shown to stimulate T cell activation and development in vivo (34), and we recently demonstrated that anti-CD3 antibody activates TCR and signals through ITK to drive type 1 regulatory T cell development in multiple organs (35). Given the observation that the lack of ITK, a TCR downstream mediator, is a negative tuner of CD8 + T cell HP, we examined the effect of TCR activation in regulating IL-7 mediated CD8 + T cell metabolism ( Figure 5C and Figure S3G&F).…”