Distinct energy requirements for human memory CD4 T-cell homeostatic functions.

Differentiation and activation of CD4 memory T cells (T(mem) cells) require energy from different sources, but little is known about energy sources for maintenance and surveillance activities of unactivated T(mem) cells. Mitochondrial fatty acid oxidation (FAO) in human unactivated CD4 T(mem) cells was significantly enhanced by inhibition of glycolysis, with respective means of 1.7- and 4.5-fold for subjects <45 yr and >65 yr, and by stimulation of AMP-activated protein kinase, with respective means of 1.3- and 5.2-fold. However, CCL19 and sphingosine 1-phosphate (S1P), which control homeostatic lymphoid trafficking of unactivated T(mem) cells, altered FAO and glycolysis only minimally or not at all. Inhibition of CD4 T(mem)-cell basal FAO, but not basal glycolysis, significantly suppressed CCL19- and S1P-mediated adherence to collagen by >50 and 20%, respectively, and chemotaxis by >20 and 50%. Apoptosis of unactivated T(mem) cells induced by IL-2 deprivation or CCL19 was increased significantly by >150 and 70%, respectively, with inhibition of FAO and by >110 and 30% with inhibition of glycolysis. Anti-TCR antibody activation of T(mem) cells increased their chemotaxis to CCL5, which was dependent predominantly on glycolysis rather than FAO. The sources supplying energy for diverse functions of unactivated T(mem) cells differ from that required for function after immune activation.

Distinctive immunoregulatory effects of adenosine on T cells of older humans.

A role for adenosine in immunosenescence was investigated in T cells from older (≥65 yr) and younger (24-45 yr) healthy humans. Adenosine concentrations in cultures of activated T cells were significantly higher (P<0.0001) for older (145±47 nM, mean±sd) than younger (58±5.5 nM) subjects. Expression of the activation coreceptor CD28 was suppressed significantly by 0.1 to 1 µM exogenous adenosine, with greater effects of 1 µM (P<0.01) on T cells of younger (mean suppression of 67 and 65% for CD4 and CD8 T cells, respectively) than older (means of 42 and 46%) subjects. T-cell chemotaxis to CCL21 was suppressed significantly by 0.3 and 1 µM exogenous adenosine, with mean maximum decreases of 39 and 49%, respectively, for younger subjects and 28 and 31% for older subjects. Generation of IL-2 and IFN-γ by T cells of younger and older subjects was suppressed substantially only at adenosine levels of 3 µM or higher. Lower baseline expression of CD28 and chemotaxis to CCL21 and S1P for T cells from older subjects attributable to endogenous adenosine were reversed completely by two different A(2A) adenosine receptor antagonists without affecting T cells of younger subjects. Adenosine is an endogenous T-cell immunosuppressor in older humans, and A(2A) antagonists reverse adenosine-induced T-cell deficiencies of aging.

Gender specificity of altered human immune cytokine profiles in aging.

Cytokine generation by T cells and monocytes was determined for 50 subjects aged 65 yr or older and concurrently studied young subjects individually matched to each old subject for sex, race, and national origin. Highly significant differences between cytokine levels of old and young subjects all were gender specific. For T cells stimulated with anti-CD3 plus anti-CD28 antibodies, mean ratios of IFN-gamma generation for healthy old to young subjects were 0.22 for men (P<0.001; n=15) and 3.35 for women (P<0.001; n=13), and those of IL-17 were 0.30 for men (P<0.001) and no difference for women. CD8 T cells were the source of high IFN-gamma in healthy old women. For old men with an inflammatory or immune disease (n=10), mean old to young ratios of T-cell-generated IFN-gamma and IL-17 increased with disease severity up to 5.78 and 2.97 (both P<0.01), respectively, without changes for old women with similar diseases (n=12). For differentiated LPS-stimulated monocytes, old to young ratios of TNF-alpha and IL-6 generation were high only in women with immune or inflammatory disease (2.38, P<0.05 and 1.62, P<0.01, respectively), whereas ratios of IFN-gamma-evoked IP-10 chemokine were low in all groups. Alterations in immune cytokine profiles with aging show significant gender specificity.

Age-associated changes in basal NF-κB function in human CD4+ T lymphocytes via dysregulation of PI3 kinase.

Immune impairment and high circulating level of pro-inflammatory cytokines are landmarks of human aging. However, the molecular basis of immune dys-regulation and the source of inflammatory markers remain unclear. Here we demonstrate that in the absence of overt cell stimulation gene expression mediated by the transcription factor NF-κB is higher in purified and rested human CD4+ T lymphocytes from older compared to younger individuals. This increase of NF-κB -associated transcription includes transcripts for pro-inflammatory cytokines such as IL-1 and chemokines such as CCL2 and CXCL10. We demonstrate that NF-κB up-regulation is cell-intrinsic and mediated in part by phosphatidylinositol 3-kinase (PI3K) activity induced in response to metabolic activity, which can be moderated by rapamycin treatment. Our observations provide direct evidence that dys-regulated basal NF-κB activity may contribute to the mild pro-inflammatory state of aging.

Age-associated alterations in inducible gene transcription in human CD4+ T lymphocytes.

Age associated immune dysregulation results in a pro-inflammatory state and increased susceptibility to infections and autoimmune diseases. Studies show that signaling initiated at the T cell antigen receptor (TCR) is impaired in CD4+ T cells from old compared to young mice. Here we examined TCR-inducible gene expression changes in CD4+ T cells during human aging. We reveal a dichotomy in gene expression mediated by the inducible transcription factor NF-κB. Most NF-κB target genes are not induced in a sustained manner in cells derived from older compared to younger individuals. However, a subset of NF-κB target genes including genes associated with chronic pro-inflammatory state in the elderly, such as interleukin 1 and 6, continue to be up-regulated even in the absence of NF-κB induction. In addition, we identify other widespread changes in gene expression between cells derived from older and younger individuals. Surprisingly, many of the most noteworthy age-associated changes in human CD4+ T cells differ from those seen in murine models. Our studies provide the first view of age-associated alteration of TCR-inducible gene expression in human CD4+ T cells.

CXCL12-induced partitioning of flotillin-1 with lipid rafts plays a role in CXCR4 function.

Lipid rafts play an important role in signal integration and in the cellular activation of a number of cytokine and growth factor receptors. It has recently been demonstrated that flotillin proteins are recruited to lipid raft microdomains upon cellular activation and play a role in neural cell regeneration, receptor signaling and lymphocyte activation. However, little is known about the relevance of the flotillin proteins during T cell responses to chemoattractant stimulation. To this end, cytoplasmic and lipid raft fractions from human T cells were analyzed for flotillin protein redistribution prior to and after CXCL12 stimulation. Flotillin-1, but not flotillin-2, redistributes to lipid rafts upon CXCR4 ligation. Moreover, in CXCL12-treated T cells, flotillin-1 also associates with several raft proteins including LAT, CD48 and CD11a but not Lck. In addition, an increase in CXCR4 association with flotillin-1 in lipid rafts was observed after chemokine treatment. RNAi technology was also utilized to inhibit the expression of flotillin-1, resulting in an inhibition of CXCL12-mediated signaling, function and CXCR4 recruitment into lipid rafts. Together, these data suggest that the increased association of cellular flotillin-1 with lipid raft microdomains during chemokine exposure may play an important role in chemokine receptor signaling and receptor partitioning with lipid rafts.

Histone acetylation is associated with differential gene expression in the rapid and robust memory CD8(+) T-cell response.

To understand the molecular basis for the rapid and robust memory T-cell responses, we examined gene expression and chromatin modification by histone H3 lysine 9 (H3K9) acetylation in resting and activated human naive and memory CD8(+) T cells. We found that, although overall gene expression patterns were similar, a number of genes are differentially expressed in either memory or naive cells in their resting and activated states. To further elucidate the basis for differential gene expression, we assessed the role of histone H3K9 acetylation in differential gene expression. Strikingly, higher H3K9 acetylation levels were detected in resting memory cells, prior to their activation, for those genes that were differentially expressed following activation, indicating that hyperacetylation of histone H3K9 may play a role in selective and rapid gene expression of memory CD8(+) T cells. Consistent with this model, we showed that inducing high levels of H3K9 acetylation resulted in an increased expression in naive cells of those genes that are normally expressed differentially in memory cells. Together, these findings suggest that differential gene expression mediated at least in part by histone H3K9 hyperacetylation may be responsible for the rapid and robust memory CD8(+) T-cell response.

Interleukin-12-induced interferon-gamma production by human peripheral blood T cells is regulated by mammalian target of rapamycin (mTOR).

Depending on the type of external signals, T cells can initiate multiple intracellular signaling pathways that can be broadly classified into two groups based on their sensitivity to the immunosuppressive drug cyclosporin A (CsA). Interleukin (IL)-12-mediated interferon (IFN)-gamma production by activated T cells has been shown to be CsA-insensitive. In this report, we demonstrate that the IL-12-induced CsA-resistant pathway of IFN-gamma production is sensitive to rapamycin. Rapamycin treatment resulted in the aberrant recruitment of Stat3, Stat4, and phospho-c-Jun to the genomic promoter region resulting in decreased IFN-gamma transcription. IL-12-induced phosphorylation of Stat3 on Ser-727 was affected by rapamycin, which may be due to the effect of rapamycin on the IL-12-induced interaction between mammalian target of rapamycin (mTOR) and Stat3. In accordance with this, reduction in the mTOR protein level by small interfering RNA resulted in suppression of Stat3 phosphorylation and decreased production of IFN-gamma after IL-12 stimulation. These results suggest that mTOR may play a major role in IL-12-induced IFN-gamma production by activated T cells.