In this study, we address the role of glucose uptake in T cell development, homeostasis, and activation as well as explore the mechanism by which the CD28 costimulatory signal regulates glucose uptake. Limiting glucose uptake resulted in decreased T cell proliferation and IFN-γ production, while increasing Glut1 protein levels through transgenic expression did not appear to affect T cell development or homeostasis, but did result in increased cell size and cytokine production upon activation. In addition, aged Glut1 transgenic mice showed increased Ig levels in the serum and deposition in the kidney. We implicate the involvement of the PI3K/Akt pathway (5
), and demonstrate that other CD28-signaling pathways are also required to augment TCR-mediated induction of Glut1. In particular, constitutive activation of Akt was sufficient to increase glucose uptake in resting T cells, but was insufficient toachieve levels of glucose uptake seen during costimulation unless Glut1 protein was up-regulated independently by strong TCR signals. This suggested that Akt activation downstream of CD28 signaling may regulate Glut1 surface localization or activity, but a separate pathway was necessary to up-regulate Glut1 protein. Together, these data indicate that regulation of glucose uptake is a critical aspect of T cell activation, and that Akt-independent regulation of Glut1 expression and Akt-dependent regulation of Glut1 trafficking and glucose uptake activity may account for the central role of CD28 in glucose metabolism of activated T cells.
This work delineates a vital role for glucose and glucose uptake in immunity. Despite availability of other nutrients, glucose was required for increased growth, IL-2 production, and proliferation upon T cell stimulation. Conversely, increased T cell activation that occurred when Glut1 was over-expressed suggested that glucose uptake is normally limiting in T cell stimulation. It should be noted, however, that in addition to decreased glucose metabolism, glucose limitation can lead to ER stress and activation of 5′AMP-activated protein kinase that may complicate these analyses (33
). Nevertheless, increased activation of Glut1 transgenic T cells demonstrates that glucose uptake is normally insufficient to allow maximal immune response. The extensive increase (>10-fold) in glucose uptake that occurs after 1 day of stimulation () relative to the increase glucose uptake provided by the Glut1 transgene (3-4-fold; ) suggests that glucose may be particularly limiting in the early phases of T cell activation. Consistent with glucose limitation early in T cell activation, Tamás et al. (34
) recently showed an important role for 5′AMP-activated protein kinase activation following TCR stimulation, which may promote more efficient ATP generation or use of alternative fuels while Glut1 protein is synthesized and glucose uptake increases to meet cellular demands. Although increases in energy availability are necessary for early T cell activation, maintaining the appropriate balance of nutrient uptake and energy production is also critical for T cell homeostasis as increased glucose uptake led to possible immune pathology with age.
Blockade of costimulation with CTLA4-Ig or anti-ICAM on CD3-stimulated T cells cultured on LPS-stimulated macrophages prevented the maximal increase of glucose uptake in T cells. Despite the wide variety of other cell-cell interactions and soluble factors that LPS-activated macrophages may provide, these costimulation pathways provide a necessary combination of signals for the regulation of T cell glucose uptake upon activation. Common features of costimulation include enhancement of TCR signals and activation of PI3K/Akt. The combination of these signaling pathways may be critical for up-regulation of glucose uptake. Conversely, receptors that decrease these signaling pathways, such as CTLA4, may decrease glucose uptake. Although signals down-stream of CD28 that regulate glucose metabolism are not entirely clear, the PI3K/Akt pathway appears to play a critical role. The Akt pathway is well known to control trafficking of the insulinsensitive glucose transporter Glut4 (35
) and we have directly shown Akt regulation of Glut1 cell surface trafficking in a lymphoid cell line in response to cytokine signaling (17
). Akt has also been shown to regulate cell surface trafficking of Glut1 in the mammary gland and was required to meet the increased metabolic demand associated with lactation (36
). In addition to regulation of cell surface levels, Akt may also control Glut1 transporter activity through activation of the mammalian target of rapamycin (17
). Glut1 glycosylation also appeared to be PI3K-dependent, although Akt activation was not sufficient to substantially alter this Glut1 modification. Therefore, Akt may play a critical role in response to costimulation to promote trafficking of Glut1 protein to the cell surface and to stimulate Glut1 activity.
The capacity of Akt signaling to control Glut1 localization or activity may combine with pathways that up-regulate or modify Glut1 to jointly mediate CD28-induced glucose uptake. In addition to activation of the PI3K/Akt pathway, CD28 also augments CD3 signals that may play a critical and underappreciated role to upregulate and modify Glut1 protein. Other likely signaling pathways that may regulate Glut1 protein levels downstream of CD28 include activation of the TEC kinases, TEC and ITK, which can play a role in positive regulation of PLC-γ (37
). Another signaling pathway that may link CD28 to increased Glut1 protein levels may be the small GTPase GEF, Vav1 (38
). Vav1 associates with the membrane in a PI3K-independent mechanism (39
) and is known to potently induce activation of NFAT, NF-κB, and AP1 (40
). Each of these transcription factors could be responsible for induction of Glut1 protein expression downstream of CD28 and explain how CD3 signals alone, when strong enough, are capable of inducing increased Glut1 expression.
Increased glucose uptake may benefit T cell activation through a number of mechanisms. T cell stimulation increases the intracellular need for ATP and other high energy molecules (41
). This need is met primarily by an increase in glycolysis, resulting in substantial lactate production (3
). However, glycolysis is not the only cellular metabolic pathway that may benefit from increased intracellular glucose levels. The pentose phosphate pathway, which produces pentose sugars required for nucleic acid synthesis and NADPH for reducing power and lipid synthesis, is also upregulated in response to T cell activation (42
). In addition, increased glucose metabolism may alter signaling pathways to enhance activation and survival. In both hematopoietic cell lines and Glut1 transgenic T cells, we have shown elevated levels of glucose metabolism to lead to increased levels of phospho-GSK3αβ (25
). This inhibitory phosphorylation of GSK3 may augment cellular activation by reducing nuclear export of transcription factors such as NFAT (43
) as well as protect against apoptosis by stabilizing the anti-apoptotic Bcl-2 family member Mcl-1 (25
). Together, these pathways establish signaling pathway coordination between nutrient sensing, proliferation, and cell survival.
The evidence presented in this study implicates regulation of glucose uptake and Glut1 as critical features of immune function. In diabetes research, it has become clear that pathways that regulate glucose transporter translocation are a central aspect of insulin-dependent promotion of metabolism and growth of insulin-responsive tissues. T cells do not typically respond directly to insulin, yet comparable to insulin regulation of Glut4, CD28 provides a signal to T cell metabolism and growth that is mediated by induction of Glut1 and regulation of Glut1 trafficking. Similar to T cell activation, cancer cells also often show elevated Glut1 expression and glycolytic metabolism (45
). Understanding the regulatory pathways underlying this metabolic phenotype may therefore provide useful insight into immunological and neoplastic diseases, as well as illustrate the critical role that nutrition may have on immune function.