Nicole M. Chapman

mTOR Links Environmental Signals to T Cell Fate Decisions

T cell fate decisions play an integral role in maintaining the health of organisms under homeostatic and inflammatory conditions. The localized microenvironment in which developing and mature T cells reside provides signals that serve essential functions in shaping these fate decisions. These signals are derived from the immune compartment, including antigens, co-stimulation, and cytokines, and other factors, including growth factors and nutrients. The mechanistic target of rapamycin (mTOR), a vital sensor of signals within the immune microenvironment, is a central regulator of T cell biology. In this review, we discuss how various environmental cues tune mTOR activity in T cells, and summarize how mTOR integrates these signals to influence multiple aspects of T cell biology.

The T cell receptor-mediated phosphorylation of Pyk2 tyrosines 402 and 580 occurs via a distinct mechanism than other receptor systems

The tyrosine kinase Pyk2 is vital for integrating receptor‐mediated signals controlling adhesion and motility in neuronal, epithelial, and hematopoietic cell types. In T cells, the stimulation of the TCR and costimulatory, chemokine, cytokine, and integrin receptors leads to the phosphorylation of Pyk2 and the induction of its catalytic activity. However, our understanding of the mechanism of the TCR‐induced, site‐specific phosphorylation of this kinase is incomplete and contradictory. To address this issue, the role of individual signaling pathways in the phosphorylation of Pyk2 tyrosines 402 and 580 upon TCR activation was assessed in human T cells. In contrast to other receptor systems, the TCR‐induced phosphorylation of Pyk2 tyrosines 402 and 580 was dependent on the Src family kinases, Fyn or Lck. Interestingly, the TCR‐mediated phosphorylation of Pyk2 tyrosines 402 and 580 did not require Ca2+ influx, ZAP‐70 activation, actin cytoskeleton rearrangement, or PI3K function. These observations are different than other receptor systems, which require the induction of one or more of these pathways. Together, these data have defined more fully the mechanism for the TCR‐induced phosphorylation of specific sites on Pyk2, suggesting that the TCR has a distinct pathway for the activation of Pyk2 compared with other receptor systems.

mTOR signaling, Tregs and immune modulation.

Foxp3(+) Tregs are central regulators of immune tolerance. As dysregulated Treg responses contribute to disease pathogenesis, novel approaches to target the immunomodulatory functions of Tregs are currently under investigation. mTORC1 and mTORC2 are therapeutic targets of interest. Recent studies revealed that mTOR signaling impacts conventional T-cell homeostasis, activation and differentiation. Moreover, mTOR controls the differentiation and functions of Tregs, suggesting that its activity could be targeted to modulate Treg responses. Here, we summarize how Tregs suppress immune responses, their roles in disease development and methods used to alter their functions therapeutically. We also discuss the diverse effects exerted by mTOR inhibition on the development, homeostasis, and functions of conventional T cells and Tregs. We conclude with a discussion of how modulation of mTOR activity in Tregs may be therapeutically beneficial or detrimental in different disease settings.

Focal Adhesion Kinase Negatively Regulates Lck Function Downstream of the T Cell Antigen Receptor

Focal adhesion kinase (FAK) is a critical regulator of signal transduction in multiple cell types. Although this protein is activated upon TCR engagement, the cellular function that FAK plays in mature human T cells is unknown. By suppressing the function of FAK, we revealed that FAK inhibits TCR-mediated signaling by recruiting C-terminal Src kinase to the membrane and/or receptor complex following TCR activation. Thus, in the absence of FAK, the inhibitory phosphorylation of Lck and/or Fyn is impaired. Together, these data highlight a novel role for FAK as a negative regulator TCR function in human T cells. These results also suggest that changes in FAK expression could modulate sensitivity to TCR stimulation and contribute to the progression of T cell malignancies and autoimmune diseases.

mTOR and metabolic regulation of conventional and regulatory T cells

mTOR signaling links bioenergetic and biosynthetic metabolism to immune responses. mTOR is activated by diverse upstream stimuli, including immune signals, growth factors, and nutrients. Recent studies highlight crucial roles of mTOR signaling in immune functions mediated by conventional T cells and Tregs. In this review, we discuss the regulation of mTOR signaling in T cells and the functional impacts of mTOR and metabolic pathways on T cell‐mediated immune responses, with a particular focus on the differentiation and function of Tregs.

Distinct signaling pathways regulate TLR2 co-stimulatory function in human T cells.

Toll-like receptor 2 (TLR2) serves as a co-stimulatory receptor for human T cells by enhancing T cell receptor (TCR)-induced cytokine production and proliferation. However, it is unknown where signals from the TCR and TLR2 converge to enhance T cell activation. To address this gap, we examined changes in TCR-induced signaling following concurrent TLR2 activation in human T cells. Both proximal TCR-mediated signaling and early NFκB activation were not enhanced by TCR and TLR2 co-activation, potentially due to the association of TLR2 with TLR10. Instead, TLR2 co-induction did augment Akt and Erk1/Erk2 activation in human T cells. These findings demonstrate that TLR2 activates distinct signaling pathways in human T cells and suggest that alterations in expression of TLR2 co-receptors may contribute to aberrant T cell responses.

Non-Catalytic Functions of Pyk2 and Fyn Regulate Late Stage Adhesion in Human T Cells

T cell activation drives the protective immune response against pathogens, but is also critical for the development of pathological diseases in humans. Cytoskeletal changes are required for downstream functions in T cells, including proliferation, cytokine production, migration, spreading, and adhesion. Therefore, investigating the molecular mechanism of cytoskeletal changes is crucial for understanding the induction of T cell-driven immune responses and for developing therapies to treat immune disorders related to aberrant T cell activation. In this study, we used a plate-bound adhesion assay that incorporated near-infrared imaging technology to address how TCR signaling drives human T cell adhesion. Interestingly, we observed that T cells have weak adhesion early after TCR activation and that binding to the plate was significantly enhanced 30–60 minutes after receptor activation. This late stage of adhesion was mediated by actin polymerization but was surprisingly not dependent upon Src family kinase activity. By contrast, the non-catalytic functions of the kinases Fyn and Pyk2 were required for late stage human T cell adhesion. These data reveal a novel TCR-induced signaling pathway that controls cellular adhesion independent of the canonical TCR signaling cascade driven by tyrosine kinase activity.

Functions of the FAK family kinases in T cells: beyond actin cytoskeletal rearrangement

T cells control the focus and extent of adaptive immunity in infectious and pathological diseases. The activation of T cells occurs when the T cell antigen receptor (TCR) and costimulatory and/or adhesion receptors are engaged by their ligands. This process drives signaling that promotes cytoskeletal rearrangement and transcription factor activation, both of which regulate the quality and magnitude of the T cell response. However, it is not fully understood how different receptor-induced signals combine to alter T cell activation. The related non-receptor tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are phosphorylated downstream of the TCR and several costimulatory and adhesion receptors. FAK family proteins integrate receptor-mediated signals that influence actin cytoskeletal rearrangement and effector T cell responses. In this review, we summarize the receptor-specific roles that FAK and Pyk2 control to influence T cell development and activation.

Proline-rich tyrosine kinase 2 controls PI3-kinase activation downstream of the T cell antigen receptor in human T cells

TCR‐induced signaling controls T cell activation that drives adaptive immunity against infections, but it can also induce dysfunctional T cell responses that promote pathologic disease. The PI3K pathway regulates many downstream effector responses after TCR stimulation. However, the molecular mechanisms that induce PI3K function downstream of the TCR are not fully understood. We have previously shown that Pyk2 is activated downstream of the TCR in a PI3K‐independent manner. Although Pyk2 controls adhesion, proliferation, and cytokine production in T cells, the mechanisms by which it controls these processes are not known. In this study, we generated Pyk2‐deficient human T cells to elucidate further the role that this kinase plays in TCR‐induced effector functions and signaling. We observed that Pyk2 localized with the p85 regulatory subunit of PI3K at the LAT complex and that PI3K‐dependent signaling was impaired in Pyk2‐deficient T cells. Likewise, functions downstream of PI3K, including IFN‐γ production and proliferation, were also suppressed in human T cells deficient in Pyk2. Collectively, these data demonstrate that Pyk2 is a critical regulator of PI3K function downstream of the TCR.

Exposure of Human CD4 T Cells to IL-12 Results in Enhanced TCR-Induced Cytokine Production, Altered TCR Signaling, and Increased Oxidative Metabolism.

Human CD4 T cells are constantly exposed to IL-12 during infections and certain autoimmune disorders. The current paradigm is that IL-12 promotes the differentiation of naïve CD4 T cells into Th1 cells, but recent studies suggest IL-12 may play a more complex role in T cell biology. We examined if exposure to IL-12 alters human CD4 T cell responses to subsequent TCR stimulation. We found that IL-12 pretreatment increased TCR-induced IFN-γ, TNF-α, IL-13, IL-4 and IL-10 production. This suggests that prior exposure to IL-12 potentiates the TCR-induced release of a range of cytokines. We observed that IL-12 mediated its effects through both transcriptional and post-transcriptional mechanisms. IL-12 pretreatment increased the phosphorylation of AKT, p38 and LCK following TCR stimulation without altering other TCR signaling molecules, potentially mediating the increase in transcription of cytokines. In addition, the IL-12-mediated enhancement of cytokines that are not transcriptionally regulated was partially driven by increased oxidative metabolism. Our data uncover a novel function of IL-12 in human CD4 T cells; specifically, it enhances the release of a range of cytokines potentially by altering TCR signaling pathways and by enhancing oxidative metabolism.