Our previous research showed that Akt phosphorylation of the RNA-binding protein, (RBP) heterogeneous nuclear ribonucleoprotein (hnRNP) A1, is dependent on TCR signal strength, and occurs under conditions of induced T regulatory (Treg) cell differentiation. We have shown hnRNPA1 is required for optimal Treg differentiation by performing knockdown experiments, and our present research is focused on identifying a role for Akt phosphorylation in hnRNPA1 function. HnRNPA1 is phosphorylated by Akt at S199 and our lab has generated a new mutant mouse model, hnRNPA1-S199A (mA1), in which hnRNPA1 will no longer be Akt-phosphhorylated. Our initial characterization of mA1 mouse model failed to reveal major abnormalities in immune cells populations at steady state except for changes in the Treg proportion in the mesenteric lymph nodes. To investigate the effect of the mA1 on the development of oral tolerance we used an adoptive transfer model in which OT-II mA1 or OT-II WT naïve T cells were transferred into congenic strains and then the mice were given OVA food. Results indicate that mA1 OT-II T cells have a proliferation defect in vivo and we also observed a significant reduction in the induction of OVA-specific Treg in the mA1 T cells. Similar results were obtained when adoptively transferred OT-II mA1 T cells were challenged with limiting doses of specific peptide, given iv. RNASeq analysis of the transferred mA1 and WT T cells is being performed. These results suggest that Akt phosphorylation of hnRNPA1 is necessary for T cell response to low dose antigen and Treg induction in vivo. Our studies identify a new mechanism by which RBP influence T cell proliferation and Treg differentiation which has implications for tolerance to food antigens in the gut. Supported by NIH (F31-AI152320-02) NIH (R01-AI125513-03)

The activation and differentiation of CD4+ T cells is a complex process that is controlled by many factors. A critical component of the signaling pathway triggered following T-cell receptor (TCR) engagement is the serine threonine kinase Akt. Akt is involved in the control of many cellular processes including proliferation, metabolism, and differentiation of specific TH-cell subsets. Recent work has shown that, depending on the nature or strength of the TCR activation, Akt may activate different sets of substrates which then lead to differential cellular outcomes. Akt plays an important role in controlling the strength of the TCR signal and several recent studies have identified novel mechanisms including control of the expression of negative regulators of TCR signaling, and the influence on regulatory T cells (Treg) and TH17 differentiation. Many of these functions are mediated via control of the FoxO family of transcription factors, that play an important role in metabolism and Th cell differentiation. A theme that is emerging is that Akt does not function in the same way in all T-cell types. We highlight differences between CD4 and CD8 T cells as well as between Treg, TH17, and TFH cells. While Akt activity has been implicated in the control of alternative splicing in tumor cells, recent studies are emerging that indicate that similar functions may exist in CD4 T cells. In this mini review, we highlight some of the recent advances in these areas of Akt function that demonstrate the varied role that Akt plays in the function of CD4 T cells.

The interaction between T-cell receptor (TCR) and a specific peptide-bound major histocompatibility complex (MHC) class II molecule on an antigen-presenting cell stimulates naive CD4+ T cells to differentiate into either effector T (Teff) or regulatory T cells (Treg). Previous studies have shown that CD4+ T cell fate decision is controlled by TCR signal strength. High dose of antigen promotes the differentiation into Teff cells, while low dose of antigen favors the Treg fate. We previously reported that the Akt/mTOR signaling pathway is involved in this process of alternate fate decision. High or low dose of antigen results in differential Akt phosphorylation and subsequent Akt substrate specificity. Upon high dose of antigen, dually phosphorylated Akt (T308 and S473) phosphorylates FoxO1, which excludes it from the nucleus; while under low dose of antigen, phosphorylated Akt (T308) activates RNA processing factors hnRNP L and hnRNP A1. Our lab has shown that both hnRNP L and A1 are necessary for the induction of Tregs in vitro. Other studies reported the identification of S199 as the phosphorylation site of hnRNP A1 by Akt. However, the relationship between Akt phosphorylation on S199 of hnRNP A1 and Treg induction is unclear. In order to investigate this, we are performing RIP-Seq on RNAs bound by hnRNP A1 under low or high stimulatory condition. We have generated a mutant mouse in which the hnRNP A1 S199 site has been mutated to Alanine. We are currently examining the impact of this mutation on the nature of RNAs bound by hnRNP A1 in T cells stimulated by low or high dose of antigen. In conclusion, our goal is to determine the mechanism by which Akt phosphorylation of hnRNP A1 is implicated in the CD4+ T cell differentiation programs.

Regulatory T cells (Treg) play a critical role in preventing autoimmunity diseases and maintaining tolerance in various tissues, particularly the gut. Peripheral derived Tregs (pTregs) are especially crucial in gut homeostasis and tolerance to commensal and food antigens. We have previously shown that knockdown of the RNA-binding protein, heterogeneous nuclear ribonucleoprotein (hnRNP) A1, reduces in vitro pTreg induction upon low TCR stimulation. In addition, we have shown that, under conditions of low TCR stimulation, in which pTregs are induced, hnRNP A1 is phosphorylated by Akt. In order to explore the impact of hnRNP A1 phosphorylation by Akt on pTregs induction we have generated a mutant mouse model, hnRNPA1-S199 in which the known Akt phosphorylation site on hnRNP A1 is mutated to Alanine. We have examined the induction of pTregs in vivo using an adoptive transfer model of OT-II TCR transgenic mutant (hnRNPA1-S199A) and wild-type naïve CD4 T cells followed by oral OVA administration. In addition, we are examining the induction of pTregs in vitro using OVA peptide presented by wild-type and mutant (antigen presenting cells) APC from the spleen. Preliminary data showed a decrease in pTreg induction and proliferation when naïve mutant T cells are stimulated either in vitro or in vivo with OVA. Furthermore, we observe that mutant APC have a reduced ability to induce proliferation and pTreg induction in both mutant and WT T cells. These data suggest that hnRNP A1 has important functions in both T cells induction and antigen presentation.

T regulatory cells (Treg) play a significant role in maintaining self-tolerance and preventing autoimmune diseases. We and others have shown that low dose favors Treg and T helper (Th) 2 cell differentiation, while high Ag dose stimulation activates the PI3K/Akt/mTOR pathway, favoring inflammatory Th1 and Th17 cell differentiation (Tconv). Differences in PI3K/Akt/mTOR signaling not only affects T cell fate but our research shows that Akt phosphorylation of the RNA-binding protein, (RBP) heterogeneous nuclear ribonucleoprotein (hnRNP) A1, is dependent on TCR signal strength. RBPs such as hnRNPA1 are emerging as regulators of RNA processing and stability in immune cells, and the effect of RBP on T cell differentiation is a growing subject of interest. We have shown hnRNPA1 is required for optimal Treg differentiation by performing knockdown experiments, and our present research is focused on identifying a role for Akt phosphorylation in hnRNPA1 function. HnRNPA1 is known to have a single Akt phosphorylation site at S199 and our lab has generated a new mutant mouse model, hnRNPA1-S199A (mA1). This mutation affects the ability of Akt to phosphorylate hnRNPA1 in all immune cells. Using Cytek’s Aurora flow cytometer we characterized the immune cell populations of mA1 at steady state. Preliminary data do not indicate any major changes in innate immune and B cells populations at steady state. We observed modest changes in CD4 T cell population frequencies at steady state. Current work using In vitro skewing suggests that Akt phosphorylation of hnRNPA1 influences Treg fate. This project suggests a novel mechanism by which Akt modulates T cell fate.