Metabolism, migration and memory in cytotoxic T cells

Abstract

The transcriptional and metabolic programmes that control CD8(+) T cells are regulated by a diverse network of serine/threonine kinases. The view has been that the kinases AKT and mammalian target of rapamycin (mTOR) control T cell metabolism. Here, we challenge this paradigm and discuss an alternative role for these kinases in CD8(+) T cells, namely to control cell migration. Another emerging concept is that AMP-activated protein kinase (AMPK) family members control T cell metabolism and determine the effector versus memory fate of CD8(+) T cells. We speculate that one link between metabolism and immunological memory is provided by kinases that originally evolved to control T cell metabolism and have subsequently acquired the ability to control the expression of key transcription factors that regulate CD8(+) T cell effector function and migratory capacity.

Figure 1. Mechanism of activation of AKT

When there are low levels of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P₃) in the plasma membrane, AKT is in an inactive conformation and cannot be phosphorylated by it upstream activating kinase 3-phosphoinositide-dependent protein kinase 1 (PDK1) (not shown). When PtdIns(3,4,5)P₃ levels increase in the plasma membrane, for example following the activation of phosphoinositide 3-kinase (PI3K), AKT binds PtdIns(3,4,5)P₃ through its phecktrin homology (PH) domain. Binding of PtdIns(3,4,5)P₃ the PH domain induces a conformational change within the kinase domain of AKT allowing PDK1 to phosphorylate the critical residue required for AKT kinase activity, threonine 308 (Thr308). Mammalian target of rapamycin complex 2 (mTORC2) also phosphorylates AKT at the C terminal serine 473 (Ser473) site to fully activate its kinase activity. PDK1 has a PH domain that can bind PtdIns(3,4,5)P₃ but the binding of PtdIns(3,4,5)P₃ to PDK1 is not essential for PDK1 catalytic activity.

Figure 2. AKT control of adhesion and chemokine receptor expression

When AKT is strongly activated (for example in cytotoxic T lymphocytes (CTLs) that are cultured with interleukin-2 (IL-2)) FOXO transcription factors, which regulate the transcription of target genes such as the transcription factor Kruppel-like factor 2 (KLF2), are sequestered in the cytoplasm where they bind 14-3-3 proteins thus preventing FOXO transcriptional activity. When AKT is inactive or activated suboptimally (for example in quiescent naïve T cells or CTLs cultured with IL-15) FOXO transcription factors can gain access to the nucleus and drive the expression of KLF2. KLF2 in turn induces the expression of multiple adhesion and chemokine receptors, such as CD62L, and sphingosine 1-phosphate receptor 1 (S1P₁), which control migration of T cells.

Figure 3. Regulation of mTORC1 activity

mTOR activity is regulated by the balance of multiple signalling pathways. phosphoinositide 3-kinase (PI3K)–AKT signalling activates mammalian target of rapamycin complex 1 (mTORC1) through the phosphorylation of the GTPase-activating protein TSC2 (tuberous sclerosis complex 1) at position Ser1462. This phosphorylation event results in the dissociation of the TSC1–TSC2 complex and inhibits the intrinsic GTPase activity of the small G protein RHEB, resulting in the accumulation of GTP-bound RHEB, which is a positive regulator of mTORC1. AKT also phosphorylates 40kDa proline-rich AKT1 substrate (PRAS40) at position Thr246, resulting in its dissociation from mammalian target of rapamycin (mTOR) thus blocking PRAS40-mediated mTORC1 inhibition. AMP-activated protein kinase (AMPK) activity inhibits mTORC1 by phosphorylating both TSC2 and the mTORC1 component raptor. Phosphorylation of TSC2 (at Thr1227 and Ser1345) by AMPK positively regulates its GAP activity, leading to RHEB-mediated GTP hydrolysis, thereby inhibiting mTORC1 activity. Phosphorylation of raptor (at Ser722 and Ser792) facilitates its binding to 14-3-3 proteins (not shown) and the subsequent inhibition of mTORC1 function.

Figure 4. mTORC1 regulation of effector versus memory CD8⁺ T cell fate

Mammalian target of rapamycin complex 1 (mTORC1) controls CD8⁺ T cell migration by regulating the expression of Kruppel-like factor 2 (KLF2), which controls the expression of chemokine and adhesion molecules that are crucial for lymph node homing. mTORC1 also controls the expression of T-bet, which controls the expression of the CXC-chemokine receptor 3 (CXCR3) and P-selectin ligands, both of which are crucial for the trafficking of effector CD8⁺ T cells to sites of inflammation. In addition, mTORC1 regulates the balance of T-bet versus eomesodermin expression, transcription factors that are known regulators of effector versus memory CD8⁺ T cell function. T-bet is required for cytotoxicity and the production of effector cytokines whereas eomesodermin expression is required for the acquisition of the memory phenotype. AMPK, AMP-activated protein kinase; CCR7, CC-chemokine receptor 7; IL-7R, interleukin-7 receptor; S1P₁, sphingosine-1-phosphate receptor 1.

Figure 5. Model for how differential AKT and mTOR signalling controls effector versus memory CD8⁺ T cell formation

A | Naïve CD8⁺ T cells (T_N) circulate between the blood, lymph nodes and efferent lymphatics. Crucial to this trafficking cycle is the expression of CD62L (which mediates adhesion to high endothelial venules (HEVs)), CC-chemokine receptor 7 (CCR7; directs transendothelial migration into lymph nodes) and sphingosine 1-phosphate receptor 1 (S1P₁; directs T cell migration into the efferent lymphatics). B | During an infection, CD8⁺ T cells, which have been activated by their cognate antigen presented to them on antigen presenting cells, within the lymph node cease trafficking, proliferate and differentiate to produce CTL. After a period, CTLs exit the lymph node and any cells that no longer express CD62L or CC-chemokine receptor 7 (CCR7) will be unable to re-enter secondary lymphoid tissue. Moreover, such strongly activated CTL will upregulate the expression of pro-inflammatory chemokine receptors such as CXC-chemokine recpetor 3 (CXCR3) and P-selectin and E-selectin ligands allowing them to traffic across inflamed endothelium to sites of infection and inflammation. As the infection resolves, cytokines become limiting at the site of inflammation and in the absence of these survival signals effector CTL will undergo apoptosis (not shown). The loss of cytokine signalling will also cause loss of AKT/mTOR signalling and could allow re-expression of CD62L and CCR7 on some CTL and hence allow these cells to return to secondary lymphoid tissues. This would bring them into proximity of the stromal cells that make the homeostatic cytokines IL-7 and IL-15. Such cells will have the potential to generate memory T cells. An alternate possibility is that during the initial immune activation any cells that only weakly activate AKT/mTOR signalling within the lymph nodes could retain the expression of CD62L and CCR7 and hence immediately resume a pattern of homing to secondary lymphoid organs which would favour their development into memory T cells (T_M).