Certain notable changes in critical cellular functions were observed in alloantigen-specific cultures that reached replicative senescence. Similar to senescent fibroblasts (Wang, Lee, and Pandey, 1994), CD8+ T cell cultures that reach senescence became resistant to apoptosis and expressed increased levels of bcl2 (Spaulding et al., 1999). Stimuli such as antibody to Fas, IL-2 withdrawal, mild heat shock, galectin-1 and staurosporine, all of which caused robust apoptosis in the early passage cells, caused only minimal cell death in the senescent cultures. A second major change involved the reduced ability of senescent cultures to respond to stress by upregulation of the major mammalian stress protein, hsp70 (Effros, Zhu, and Walford, 1994b). This defect is reminiscent of the overall reduction in responsiveness to physical and oxidative stress that is believed to play a major role in organismic aging (Golden et al., 2002).
One of the main advantages of studying T cells in long-term culture is the ability to monitor changes in expression over time in the large spectrum of known T cell surface markers reflecting lineage, activation status, adhesion, and memory. Using flow cytometry, we demonstrated that for nearly all such markers, there was no change in expression between early passage and senescent cultures (Perillo et al., 1993). The single exception was CD28, a key T cell-specific signaling molecule that plays diverse roles in T cell biology, including delivering the costimulatory second signal that is essential for activation/proliferation, stabilizing cyto-kine mRNAs, modulating cell trafficking, and regulating glucose metabolism. As T cells progress to senescence in culture, the proportion of cells that are CD28~ increases, and senescent cultures are >99% CD28~ (Effros et al., 1994a). The loss of CD28 expression is associated with complete suppression of gene expression of this key T cell signaling molecule.
Whereas characterization of senescence-associated changes using long-term primary T cell cultures has the advantage of allowing longitudinal analysis of the same population of cells over time, the model admittedly suffers from the absence of the in vivo milieu, which undoubtedly plays a role in marker expression patterns changes that occur in the context of the whole organism. Indeed, although loss of CD28 expression has been presumed to be the ultimate senescence-associated change based on cell culture analysis, it is possible that other cell surface antigens identified on ex vivo samples may delineate a more precise phenotype for the true end-stage senescent CD8+ T cell. One such marker change is the acquisition of CD57, which has been documented for virus-specific CD8+ T cells from HIV-infected persons that have undergone multiple rounds of proliferation (Brenchley et al., 2003). Interestingly, many features are shared by CD57+ and CD28~ T cells isolated ex vivo, reinforcing the notion that both the loss of CD28 expression and the acquisition of CD57 can be considered markers of senescent T cells (Demarest et al., 2001; Mach et al., 1997; Posnett et al., 1999; Weekes et al., 1999). Additional markers that have been identified on putatively senescent
CD28~ T cells isolated ex vivo are the KLRG1 NK inhibitory lectin-like receptor and CD56, an NK marker bearing the HNK-1 epitope (Ouyang et al., 2003a; Tarazona et al., 2000), findings that may explain earlier reports of MHC-unrestricted NK function in senescent cultures (Pawelec et al., 1986).
As CD8+ T cells undergo increasing numbers of cell divisions in long-term culture, they show a variety of alterations that would be predicted to dramatically affect in vivo immune function. For example, alloantigen-specific cultures from healthy donors produce increasing amounts of two pro-inflammatory cytokines, IL-6 and TNFa, as they approach replicative senescence (Effros et al., 2005), and virus-specific CD8+ T cells show decreased antigen-specific production of IFNg (Dagarag et al., 2004). HIV-specific CD8+ T cell cultures initiated from T cells of HIV-infected persons show a progressive decline in the ability to perform antigen-specific lysis of target cells pulsed with HIV peptides, a change that is accompanied by reduced expression of perforin (Dagarag et al., 2004; Yang et al., 2005). Moreover, one of the key antiviral activities believed to function protectively in vivo— inhibition of HIV replication—is markedly reduced in senescent HIV-specific CD8+ T cells (Dagarag et al., 2003). One can envision that such functional changes might exert pleiotropic influences on a variety of physiological processes in vivo, both in the context of aging and in chronic HIV infection.
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