Gonzalo G. Garcia

Age-Dependent Alterations in the Assembly of Signal Transduction Complexes at the Site of T Cell/APC Interaction

TCR interaction with peptide-MHC complexes triggers migration of protein kinases, actin-binding proteins, and other accessory molecules to the T cell/APC synapse. We used confocal immunofluorescence methods to show that the adapter protein LAT (linker for activation of T cells) and the guanine nucleotide exchange factor Vav also move to the APC interface in mouse CD4 T cells conjugated to anti-CD3 hybridoma cells, and in TCR-transgenic CD4 cells conjugated to APC bearing agonist (but not closely related nonagonist) peptides. The proportion of CD4+ T cells able to relocalize LAT or Vav, or to relocate cytoplasmic NT-AT (NF-ATc) from cytoplasm to nucleus, declines about 2-fold in aged mice. The decline in LAT relocalization is accompanied by a similar decline in tyrosine phosphorylation of LAT in CD4 cells stimulated by CD3/CD4 cross-linking. Two-color experiments show that LAT redistribution is strongly associated with relocalization of both NF-ATc and protein kinase C-θ among individual cells. LAT migration to the immunological synapse depends on actin polymerization as well as on activity of Src family kinases, but aging leads to only a small change in the percentage of CD4 cells that redistribute F-actin to the site of APC contact. These results suggest that defects in the ability of T cells from aged donors to move kinase substrates and coupling factors, including LAT and Vav, into the T cell/APC contact region may contribute to the decline with age in NF-ATc-dependent gene expression, and thus to defects in T cell clonal expansion.

Single-Cell Analyses Reveal Two Defects in Peptide-Specific Activation of Naive T Cells from Aged Mice

Confocal fluorescent microscopy was used to study redistribution of membrane-associated proteins in naive T cells from young and old mice from a transgenic stock whose T cells express a TCR specific for a peptide derived from pigeon cytochrome C. About 50% of the T cells from young mice that formed conjugates with peptide-pulsed APC were found to form complexes, at the site of binding to the APC, containing CD3ε, linker for activation of T cells (LAT), and Zap-70 in a central area and c-Cbl, p95vav, Grb-2, PLCγ, Fyn, and Lck distributed more uniformly across the interface area. Two-color staining showed that those cells that were able to relocalize c-Cbl, LAT, CD3ε, or PLCγ typically relocalized all four of these components of the activation complex. About 75% of conjugates that rearranged LAT, c-Cbl, or PLCγ also exhibited cytoplasmic NF-AT migration to the T cell nucleus. Aging had two effects. First, it led to a diminution of ∼2-fold in the proportion of T cell/APC conjugates that could relocalize any of the nine tested proteins to the immune synapse. Second, aging diminished by ∼2-fold the frequency of cytoplasmic NF-AT migration among cells that could generate immune synapses containing LAT, c-Cbl, or PLCγ. Thus naive CD4 T cells from old mice exhibit at least two separable defects in the earliest stages of activation induced by peptide/MHC complexes.

Age-Dependent Defects in TCR-Triggered Cytoskeletal Rearrangement in CD4+ T Cells

Previous research has shown that many of the CD4 T cells from older mice do not form functional immune synapses after conjugation with peptide-pulsed APC. We now show that the defect lies at a very early stage in the cytoskeletal reorganization that precedes movement of protein kinases and their substrates to the TCR/APC interface. Antagonist peptides presented to T cells from young mice induce migration of talin (but not paxillin, vinculin, or F-actin) to the APC contact zone, but CD4 T cells from older donors typically fail to show the talin polarization response. A spreading assay in which contact with anti-CD3-coated slides induces CD4 T cells to assume a conical shape and develop lammelopodia also shows a decline with age in the proportion of T cells that can initiate cytoskeletal changes in response to this simplified stimulus. Finally, the transition from detergent-soluble to cytoskeletal forms of the p16, p21, and p23 isoforms of CD3ζ in response to CD3/CD4/CD28 cross-linking is much stronger in young than in old T cells. Thus, defects in cytoskeletal reorganization triggered by initial contact between TCR and peptide-bearing APC precede, and presumably contribute to, defective activation of protein kinase-mediated signals in the first few minutes of the activation cascade in T cells from aged mice.

Age-associated changes in glycosylation of CD43 and CD45 on mouse CD4 T cells

We have recently shown that treatment of T cells from aged mice with an endopeptidase specific for O‐linked glycoproteins can restore synapse formation and early activation markers to CD4 cells from aged mice. New data show that the sialidase from Clostridium perfringens, but not from Vibrio cholerae, can increase activation of CD4 cells from both old and young mice as measured by calcium signals, expression of CD25 and CD69, and secretion of IL‐2. Lectin binding assays showed alterations with age in the levels, accessibility or conformation of multiple glycoproteins on the surface of CD4 cells. While some alterations were due to the accumulation of memory cells with age, others were age sensitive and found exclusively in the naive subset or both naive and memory subsets. Furthermore, analysis of the sialic acid links α(2,3)Gal/GalNAc and α(2,6)Gal/GalNAc in immunoprecipitated CD43 and CD45 molecules confirm that age alters the glycosylation of specific proteins that regulate TCR interaction with antigen presenting cells. These data support the idea that changes in T cell surface glycosylation could play an important role in immune senescence.

T cells in aging mice: genetic, developmental, and biochemical analyses

Summary:  A combination of approaches – gene mapping, biomarker analysis, and studies of signal transduction – has helped to clarify the mechanisms of age‐related change in mouse immune status and the implications of immune aging for late‐life disease. Mapping studies have documented multiple quantitative trait loci (QTL) that influence the levels of age‐sensitive T‐cell subsets. Some of these QTL have effects that are demonstrable in young‐adult mice (8 months of age) and others demonstrable only in middle‐aged mice (18 months). Biomarker studies show that T‐cell subset levels measured at 8 or 18 months are significant predictors of lifespan for mice dying of lymphoma, fibrosarcoma, mammary adenocarcinoma, or all causes combined. Mice whose immune systems resemble that of young animals, i.e. with low levels of CD4+ and CD8+ memory T cells and relatively high levels of CD4+ T cells, tend to outlive their siblings with the opposite subset pattern. Biochemical analyses show that T cells from aged mice show defects in the activation process within a few minutes of encountering a stimulus and that the defects precede the recognition by the T‐cell receptor of agonist peptides on the antigen‐presenting cell. Defective assembly of cytoskeletal fibers and hyperglycosylation of T‐cell surface glycoproteins contribute to the immunodeficiency state, and indeed treatment with a sialylglycoprotein endopeptidase can restore full function to CD4+ T cells from aged donors in vitro.

Age-related defects in CD4+ T cell activation reversed by glycoprotein endopeptidase

CD4+ T cells from old mice show defects in the activation process including deficiency in the formation of immunosynapses with antigen‐presenting cells. We show that CD4+ T cells from old mice express unusually high levels of glycosylated forms of the bulky T cell glycoprotein CD43, particularly on a subset of functionally anergic cells expressing P‐glycoprotein. T cells from old donors also show a decline in the association of CD43 with cytoskeletal matrix and in the proportion of T cells that can exclude CD43 from the synapse. O‐sialoglycoprotein endopeptidase, which removes the external domain of CD43 and other O‐sialoglycoproteins from the aged naive CD4+ T cells of TCR‐transgenic mice, restores early agonist‐independent stages and later agonist‐dependent stages of synapse formation as well as expression of the activation markers CD69 and CD25 to the levels found in the young mice. These data support a model in which O‐glycosylated forms of T cell surface molecules, including CD43, are largely responsible for age‐related defects in TCR signaling and function.

Regulation of mTOR Activity in Snell Dwarf and GH Receptor Gene-Disrupted Mice

The involvement of mammalian target of rapamycin (mTOR) in lifespan control in invertebrates, calorie-restricted rodents, and extension of mouse lifespan by rapamycin have prompted speculation that diminished mTOR function may contribute to mammalian longevity in several settings. We show here that mTOR complex-1 (mTORC1) activity is indeed lower in liver, muscle, heart, and kidney tissue of Snell dwarf and global GH receptor (GHR) gene-disrupted mice (GHR-/-), consistent with previous studies. Surprisingly, activity of mTORC2 is higher in fasted Snell and GHR-/- than in littermate controls in all 4 tissues tested. Resupply of food enhanced mTORC1 activity in both controls and long-lived mutant mice but diminished mTORC2 activity only in the long-lived mice. Mice in which GHR has been disrupted only in the liver do not show extended lifespan and also fail to show the decline in mTORC1 and increase in mTORC2 seen in mice with global loss of GHR. The data suggest that the antiaging effects in the Snell dwarf and GHR-/- mice are accompanied by both a decline in mTORC1 in multiple organs and an increase in fasting levels of mTORC2. Neither the lifespan nor mTOR effects appear to be mediated by direct GH effects on liver or by the decline in plasma IGF-I, a shared trait in both global and liver-specific GHR-/- mice. Our data suggest that a more complex pattern of hormonal effects and intertissue interactions may be responsible for regulating both lifespan and mTORC2 function in these mouse models of delayed aging.

Age-related Defects in Moesin/Ezrin Cytoskeletal Signals in Mouse CD4 T cells

Cytoskeletal proteins of the ezrin-radixin-moesin (ERM) family contribute to T cell activation in response to Ag, and also to T cell polarization in response to connective tissue matrix proteins and chemokine gradients. Previous work has shown that T cells from aged mice are defective in their ability to develop molecular linkages between surface macromolecules and the underlying cytoskeletal framework, both for proteins that move to the synapse and those that are excluded from the site of T cell-APC interaction. T cells from aged mice also show defective cytoskeletal rearrangements and lamellipodia formation when placed in contact with slides coated with Abs to the TCR/CD3 complex. In this study, we show that old CD4 T cells differ from young CD4 T cells in several aspects of ERM biochemistry, including ERM phosphorylation and ERM associations with CD44, CD43, and EBP50. In addition, CD4 T cells from aged mice show defects in the Rho GTPase activities known to control ERM function.

Age-Related Defects in the Cytoskeleton Signaling Pathways of CD4 T Cells

It has been postulated that the cytoskeleton controls many aspects of T cell function, including activation, proliferation and apoptosis. Recent advances in our understanding of F-actin polymerization and the Ezrin-Radixin-Moesin (ERM) family of cytoskeleton signal proteins have provided new insights into immunological synapse formation during T cell activation. During aging there is a significant decline of T cell function largely attributable to declines in activation of CD4 T cells and defects in the formation of the immunological synapse. Here we discuss recent progress in the understanding of how aging alters F-actin and ERM proteins in mouse CD4 T cells, and the implications of these changes for the T cell activation process.

CD43‐independent augmentation of mouse T‐cell function by glycoprotein cleaving enzymes

Previous work has shown that the function of mouse CD4+ T cells can be augmented by an enzyme, O‐sialoglycoprotein endopeptidase (OSGE), which cleaves surface CD43, suggesting the idea that the high levels of glycosylated CD43 found on T cells from aged mice may contribute to immune senescence. New results now show that OSGE improves T‐cell function even in mice lacking CD43, showing that other glycoproteins must contribute to the OSGE effect on function. Evaluation of other enzymes found two whose ability to stimulate CD4 activation was higher in aged than in young T cells. One of these, PNGase F, is a glycosidase specific for N‐linked glycans, and the other, ST‐Siase(2,3) from Salmonella typhimurium, is specific for α2,3‐linked terminal sialic acid residues. Parallel lectin‐binding experiments showed that removal of α2,3‐linked sialic acid residues vulnerable to PNGase F and ST‐Siase(2,3) was also greater in old than in young T cells. The preferential ability of PNGase F and ST‐Siase(2,3) to improve the function of T cells from aged mice may involve cleavage of glycoproteins containing α2,3‐linked sialic acid residues on N‐linked or O‐linked glycans or both.