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The co-inhibitory receptor killer-cell lectin like receptor G1 (KLRG1) is expressed on NK cells and antigen-experienced T cells and has been postulated to be a marker of senescence. Whilst KLRG1 has frequently been used as a marker of cellular differentiation, data are emerging indicating that KLRG1 plays an inhibitory role. In this review we examine evidence highlighting this view of KLRG1 with emphasis on the functional defects that arise during T cell differentiation with age that may, in part, be actively maintained by inhibitory receptor signalling.
The immune system undergoes a dramatic restructuring with age, leading to a decline in immune responses and an increased vulnerability of old individuals. The incidence and severity of infectious diseases, such as pneumonia (LaCroix et al. 1989), meningitis (Gorse et al. 1984), sepsis (Chattopadhyay and Al-Zahawi 1983), urinary tract infections (Ackermann and Monroe 1996), infection with respiratory syncytial virus (Barker and Mullooly 1980) or influenza (Sprenger et al. 1993) all increase with age. Indeed the mortality rate of older adults suffering urinary tract infections or tuberculosis is ten-fold higher than that of young adults (Yoshikawa 1997). This waning immunity in old age results from defects in numerous different leukocyte populations with the dysfunction being most pronounced in T cells. This T cell immune decline is marked by a dramatic decline in the number of naïve T cells as a result of a thymic atrophy (Douek et al. 1998; Linton and Dorshkind 2004). This reduced thymic output leads to the peripheral expansion of naïve and memory T cells to regenerate the T cell pool, which in turn leads to the accumulation of oligoclonally expanded, functionally impaired T cells (Akbar and Fletcher 2005; Messaoudi et al. 2004). These age-associated changes contribute to the inability of the aged immune system to respond to new antigenic challenge and mount optimum responses to vaccination (Goronzy et al. 2001).
There are numerous reports cataloging the phenotypic and functional changes to human T cells that occur during ageing (Table 1). Old individuals show an increased proportion of T cells that are highly differentiated, with similar phenotypic changes occurring in both CD4+ and CD8+ T cells during differentiation. However, the rate at which these changes happen varies within each subset, with age-related changes being more pronounced on CD8+ T cells due to a greater homeostatic stability of CD4+ T cells (Effros et al. 1994; Czesnikiewicz-Guzik et al. 2008; Goronzy et al. 2007). These highly differentiated cells have functional defects that may explain the decreased efficiency of the immune system in older individuals (Fletcher et al. 2005). Highly differentiated T cells are characterised by the loss of the cell surface co-stimulatory molecules CD27 and CD28, CD8+ T cells losing CD28 first followed by CD27 with the converse being true for CD4+ T cells (Appay et al. 2002; Amyes et al. 2003; Fletcher et al. 2005; Plunkett et al. 2005). Initially, it was thought that the loss of CD28 was a major factor in the reduced activation and function of these cells (Champagne et al. 2001; Effros et al. 2005). However, subsequent studies have suggested a greater plasticity with regard to co-stimulatory receptor expression and usage among T cells. For example, co-stimulation through ICOS, a CD28 family member, and CD137 and CD134, members of the TNF family, have all been shown to enhance the proliferation (Bukczynski et al. 2003; Serghides et al. 2005; Plunkett et al. 2007; Waller et al. 2007) and telomerase activity in CD8+CD28- T cells (Plunkett et al. 2007). This redundancy in co-stimulatory receptor usage suggests that changes in addition to the loss of co-stimulatory receptors are involved in T cell dysfunction during ageing. One such change may be a rise in co-inhibitory receptors, in particular the co-inhibitory receptor killer-cell lectin like receptor G1 (KLRG1).
In both mice and humans, KLRG1 expression is found on NK cells and antigen-experienced T cells (Blaser et al. 1998; Hanke et al. 1998; Voehringer et al. 2002). Human KLRG1 is also found on a subset of γδ T cells (Eberl et al. 2005) and in a large proportion of CD4+ and CD8+ T cells found in cord blood (Marcolino et al. 2004). In young adults, the expression of KLRG1 is about 40% on CD8+ T cells and 20% on CD4+ T cells (Voehringer et al. 2002). The expression of KLRG1 rises dramatically with age, with greater than 90% expression of KLRG1 being seen on CD8+ T cells in individuals over 65 years of age (Ouyang et al. 2003; Ito et al. 2006; Henson et al. 2009). The expression of KLRG1 increases not only with age but also with differentiation, with the highest percentage of expression being seen on memory cells and highly differentiated end stage cells (Voehringer et al. 2002; Thimme et al. 2005). In mice, KLRG1 has been used to identify memory precursor cells from effector T cells. Through the use of acute viral infection models it has been shown that KLRG1 can be used to distinguish short-lived effector CD8+ T cells (KLRG1high) and memory precursor effector CD8+ T cells (KLRG1low) (Joshi et al. 2007; Mousavi et al. 2008).
Despite the extensive use of KLRG1 as a marker of differentiation, KLRG1 possesses an immune receptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain, suggesting that it may play a functional role in the immune system. An inhibitory role for KLRG1 has been demonstrated in mice, with antibody-mediated cross-linking of KLRG1 being shown to inhibit cytolytic activity and IFNγ production in NK cells (Robbins et al. 2002). However, this result is at odds with a number of reports that failed to observe an inhibitory cytolytic effect (Hanke et al. 1998; Grundemann et al. 2006); these differing outcomes may be the result of Robbins et al. using an NK clone over-expressing KLRG1. In murine T cells, the cross-linking of TCR and KLRG1 by plate-bound antibodies was shown to lower Ca2+ influx (Beyersdorf et al. 2001) and to decrease IL-2 production (Tessmer et al. 2007). The use of KLRG1-transgenic mice showed that antigen-stimulated T cells in the presence of KLRG1’s ligand, E-cadherin, inhibited the proliferative capacity of CD8+ T cells (Grundemann et al. 2006).
Data is now emerging suggesting that KLRG1 plays an inhibitory role in human NK cells and T cells. KLRG1-mediated inhibition of NK cell function revealed that KLRG1/ligand interactions inhibit the cytolytic activity of polyclonal human NK cells by interfering with both degranulation and IFNγ release (Schwartzkopff et al. 2007). Consistent with murine data, the authors also show the degree of inhibition to be modest, and to require high expression levels of KLRG1’s ligand, E-cadherin (Schwartzkopff et al. 2007).
A recent study has demonstrated a role for KLRG1 as an inhibitory receptor in T cells. The authors used a CD4+ T cell hybridoma transduced with KLRG1 and showed that KLRG1-ligation inhibited the NFAT-signaling pathway and down-regulated CD95 mediated lysis (Rosshart et al. 2008). Furthermore, they also demonstrated that both KLRG1 and CD3/TCR signals have to be provided in a spatially restricted manner in order to inhibit T-cell activation (Rosshart et al. 2008), suggesting that KLRG1 inhibits T cell function only when MHC/antigen and KLRG1 ligands are expressed on the same target cells. Data from our laboratory has also shown KLRG1 to have an inhibitory effect on primary CD8+ T cells; we have shown that blocking KLRG1 signaling during TCR activation using antibodies against its ligand, E-cadherin, enhanced proliferative activity that was linked directly to an Akt-mediated increase in synthesis of cyclin D and E and a decrease in the cyclin inhibitor p27 (Henson et al. 2009). Whist we observed a significant enhancement in proliferative capacity in CD8+ T cells isolated from young individuals the effect was not as great as that of CD8+ T cells isolated from old individuals, suggesting that other, as yet unidentified, age-related defects are contributing to the poor proliferative responses of CD8+ T cells from old donors and these remain to be clarified.
KLRG1 has been shown to be a cadherin receptor, recognising E-, N- and R-cadherin (Grundemann et al. 2006; Ito et al. 2006; Schwartzkopff et al. 2007). The cadherins comprise a family of transmembrane glycoproteins that mediate Ca2+ dependent cell-cell adhesion (Gumbiner 2005). Classically, E-cadherin is expressed on epithelial cells and Langerhans cells, whereas N- and R-cadherin are expressed by the nervous system. We have shown E-cadherin to be expressed on peripheral blood cells, notably on myeloid DCs, with no expression of N-cadherin being found (Henson et al. 2009). Demonstrating that E-cadherin is found not only in the epithelium but on a wide range of antigen-presenting cells suggesting a broader range of scenarios for immune control by KLRG1/cadherin interactions. A recent report using a KLRG1-reporter cell assay with domain-deleted E-cadherin mutants (Δ1–Δ5) have shown that the first and the second extracellular domains of E-cadherin to be critical for interaction with KLRG1 (Rosshart et al. 2008).
KLRG1 contains one ITIM motif in its cytoplasmic domain, which mediates its effects through the recruitment of SHIP-1 and SHP-2 phosphatases and a tyrosine residue at position 7 in the ITIM (Xu et al. 2001; Tessmer et al. 2007). Murine KLRG1 also contains a PxxP motif in its cytoplasmic domain that could potentially interact with proteins containing SH3 domains (Abramson et al. 2002). KLRG1 forms both monomers and dimers, with a substantial fraction of KLRG1 being found on the cell surface as disulfide-linked trimeric and tetrameric complexes (Rosshart et al. 2008). The cysteine residues in human KLRG1 that are responsible for disulfide-linked multimer formation have not yet been defined; however, murine KLRG1 contains four cysteines proximal to the membrane in the extracellular domain that are probably not involved in intramolecular disulfide-bonding (Voehringer et al. 2001). It has been demonstrated that, in contrast to KLRG1-tetramers (Grundemann et al. 2006; Rosshart et al. 2008), monomeric KLRG1 shows little discernable binding to E-cadherin-expressing cells, suggesting that KLRG1 binds to E-cadherin with relatively low affinity. Multimerisation of KLRG1 increases the avidity and may thereby enhance the sensitivity for inhibition (Rosshart et al. 2008).
The effectors SHIP-1 and SHP-2 degrade PIP3 to PIP2, thus regulating the function of PI3K (Tessmer et al. 2007). PI3K plays a crucial role in a broad range of cellular functions in response to extracellular signals. A key downstream effector of PI3K is the serine-threonine kinase Akt, which, in response to PI3K activation, phosphorylates and regulates the activity of a number of targets, including kinases, transcription factors and other regulatory molecules (Donahue and Fruman 2004). The activation of Akt requires the binding of its pleckstrin homology (PH) domain to the phosphoinositide products of PI3K resulting in its recruitment to the plasma membrane. Once there, Akt activation is controlled by phosphorylation at two different sites, Thr308 and Ser473 (Alessi et al. 1996; Jacinto et al. 2006). Highly differentiated CD8+CD28-CD27- T cells are unable to phosphorylate Akt(ser473), with the Thr308 phosphorylation site being unaffected (Plunkett et al. 2007). We assessed whether signalling via KLRG1 contributes to any of the attenuated differentiation-related functional changes in CD8+ T cells. By blocking KLRG1 signalling during TCR activation using antibodies against its major ligand, E-cadherin, we showed a reversal of the defective Akt(ser473) phosphorylation in highly differentiated CD8+ T cells isolated from both young and old donors to the levels that are found after activation in the less differentiated CD8+ subsets (Fig. 1). This indicates that the defect in Akt phosphorylation is not a passive consequence of antigenic-driven differentiation of CD8+ T cells but that it is instead actively maintained by KLRG1 signalling (Henson et al. 2009).
Highly differentiated T cells accumulate with age; these cells have numerous defects including a decreased capacity for proliferation, an inability to produce IL-2, defective Akt(ser473) phosphorylation after activation, short telomeres and low telomerase activity, indicating that they are close to replicative senescence. In addition, these cells express increased levels of the inhibitory receptor KLRG1. Despite the extensive use of KLRG1 as a marker of differentiation, KLRG1 possesses an ITIM in its cytoplasmic domain, suggesting that it may play a functional role in the immune system. KLRG1 signalling has been shown to inhibit the cytolytic activity of polyclonal human NK cells and T cell hybridomas, as well as interfering with proliferation via Akt-mediated changes in cyclins and cyclin inhibitors. Therefore, signalling through KLRG1 may be responsible in part for the defects observed in highly differentiated T cells. It is well recognised that older humans have decreased responses to vaccination (Hayward et al. 1994; Stepanova et al. 2002; Wick et al. 2000) and it is possible that modulating certain inhibitory receptors like KLRG1 that are preferentially expressed in highly differentiated T cells, which expand during ageing, could potentially boost immunotherapeutic regimes such as vaccination for the aged.
This work was supported by a Research into Ageing Fellowship to S.M.H. and funding from the British Biotechnology and Biological Research Council to A.N.A.