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Originally published online as doi:10.1189/jlb.0204096 on October 21, 2004

Published online before print October 21, 2004
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(Journal of Leukocyte Biology. 2005;77:67-70.)
© 2005 by Society for Leukocyte Biology

Human V{gamma}9/V{delta}2 effector memory T cells express the killer cell lectin-like receptor G1 (KLRG1)

Matthias Eberl*,1, Rosel Engel*, Silke Aberle{dagger}, Paul Fisch{dagger}, Hassan Jomaa* and Hanspeter Pircher{ddagger}

* Biochemisches Institut, Infektiologie, Justus-Liebig-Universität Giessen, Germany; and
{dagger} Pathologisches Institut, Molekulare Pathologie, and
{ddagger} Institut für Medizinische Mikrobiologie und Hygiene, Immunologie, Albert-Ludwigs-Universität Freiburg, Germany

1 Correspondence: Biochemisches Institut, Infektiologie, Justus-Liebig-Universität Giessen, Friedrichstrasse 24, 35392 Giessen, Germany. E-mail: matthias.eberl{at}biochemie.med.uni-giessen.de


   ABSTRACT
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The killer cell lectin-like receptor G1 (KLRG1) is expressed in natural killer (NK) cells and effector memory {alpha}ß T cells. {gamma}{delta} T cells represent an unconventional lymphocyte population that shares characteristics of NK cells and T cells and links innate and adaptive immunity. V{gamma}9/V{delta}2 T cells comprise the majority of peripheral human {gamma}{delta} T cells and respond to the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). Here, we demonstrate that KLRG1 is expressed in a significant proportion of V{gamma}9/V{delta}2 T cells in cord blood and in the majority of peripheral V{gamma}9/V{delta}2 T cells from adult donors. KLRG1+ V{gamma}9/V{delta}2 T cells displayed an effector memory phenotype, as KLRG1 was expressed mainly in V{gamma}9/V{delta}2 T cells lacking CD27, CD45RA, CD62L, and CC chemokine receptor 7 (CCR7). Unlike {alpha}ß T cells, where possession of KLRG1 identified effector memory cells with impaired proliferative capacity, KLRG1+ V{gamma}9/V{delta}2 T cells were able to proliferate vigorously upon stimulation with HMB-PP in the presence of interleukin-2. Moreover, KLRG1 ligation on V{gamma}9/V{delta}2 T cells by antibodies did not inhibit HMB-PP-induced proliferation and cytokine production nor cytolysis of Daudi cells.

Key Words: {gamma}{delta} T lymphocytes • memory • killer inhibitory receptors • senescence • CD94

Fine-tuning of lymphocyte activity is achieved by molecules that belong to two distinct classes of major histocompatibility complex (MHC) class I-specific inhibitory receptors. While the killer immunoglobulin (KIR)-like receptors are type I integral membrane proteins that interact with particular human leukocyte antigen (HLA)-A, HLA-B, or HLA-C alleles, the killer cell lectin-like receptors (KLR) represent type II integral membrane proteins of the C-type lectin family. These include murine Ly49 proteins and CD94/NKG2 receptors that bind to HLA-E and related nonclassical class I molecules. The killer cell lectin-like receptor G1 (KLRG1) molecule is a recently described KLR that is expressed in natural killer (NK) cells and in a subset of {alpha}ß T cells [1 2 3 ]. Infection of mice with cytomegalovirus induces up-regulation of KLRG1 on the surface of NK cells, and KLRG1 engagement has been shown to inhibit effector functions of a murine NK cell line [4 ]. Besides NK cells, KLRG1 is expressed in a large fraction of murine effector CD8+ T cells, which are capable of exerting effector functions [5 ], while their ability to proliferate upon antigenic stimulation is severely impaired [6 ]. In humans, KLRG1 mRNA has been detected in lymphoid organs and in NK and basophilic cell lines [7 ]. In peripheral blood, KLRG1 cell-surface expression was detected in ~40% of CD8+ and ~20% of CD4+ antigen-experienced T cells, preferentially in the CC chemokine receptor (CCR)7– effector T cell pool and in ~50% of NK cells belonging to the CD56dim subset [8 ]. Similar to the situation in mice, KLRG1 identified human effector {alpha}ß T cells that secreted cytokines but were impaired in their proliferative capacity [8 ].

{gamma}{delta} T cells represent unconventional lymphocytes that share characteristics of NK cells and T cells and often express regulatory NK receptors, thereby linking innate and adaptive immunity [9 , 10 ]. Upon cross-linking CD94/NKG2A, for instance, antigenic activation of {gamma}{delta} T cells is down-modulated by inhibiting cell proliferation, cytokine production, and cytotoxicity [11 , 12 ]. Consequently, {gamma}{delta} T cells can detect target cells with patterns of MHC class I expression altered as a result of tumor transformation or viral infection, thus endowing them with a role in immunosurveillance originally attributed to NK cells only [13 ]. V{gamma}9/V{delta}2 T cells comprise the majority of peripheral human {gamma}{delta} T cells and are capable of responding to the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) [14 ]. In the present study, we have examined whether similar to NK cells and effector {alpha}ß T cells, HMB-PP-specific V{gamma}9/V{delta}2 T cells also express KLRG1.

Peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometry using anti-V{gamma}9-PC5 (Immu360) and anti-CD3-ECD (UCHT1) monoclonal antibody (mAb; Beckman Coulter, Krefeld, Germany) [15 ], while KLRG1 expression was monitored using the mAb 13A2-phycoerythrin (PE), described previously [8 ]. KLRG1 was expressed in ~40% of cord blood V{gamma}9/V{delta}2 T cells, and in the majority (80–90%) of peripheral V{gamma}9/V{delta}2 T cells from adult donors (Fig. 1 ). KLRG1 expression levels in the V{gamma}9– T cell subset, comprising mainly CD4+ and CD8+ {alpha}ß T cells, were lower in accordance with our earlier observations [8 ]. Coexpression analysis with a panel of memory cell markers revealed that KLRG1 expression was observed mainly in V{gamma}9/V{delta}2 T cells lacking CD27, CD45RA, CD62L, and CCR7, suggesting that KLRG1+ V{gamma}9+ cells exhibited an effector memory phenotype (Fig. 2 ). The fact that KLRG1 was predominantly found in peripheral CD45RA– V{gamma}9/V{delta}2 T cells and only to a lesser extent in cord blood cells is in line with previous data showing that the majority of adult V{gamma}9/V{delta}2 T cells displays a CD45RA– effector memory phenotype, whereas most neonatal V{gamma}9/V{delta}2 T cells are naive CD45RA+ cells [16 ]. No significant restriction was observed when costaining for the tetraspanin CD81, the C-type lectin CD94, or the activatory CD2 family member CD244 (2B4), suggesting that these markers can be expressed independently from each other.



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  		Figure 1. Human {gamma}{delta} T cells express KLRG1. (A) Typical dot-plot derived from adult PBMC labeled for CD3, V{gamma}9, and KLRG1, gated on CD3+ lymphocytes. (B) KLRG1 expression in V{gamma}9– and V{gamma}9+ CD3+ T cells from cord blood (hatched bars, n=7) and adult peripheral blood (solid bars, n=6). Data shown are means ± SD from individually analyzed donors.

 


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  		Figure 2. KLRG1+ {gamma}{delta} T cells exhibit an effector memory phenotype. PBMC were labeled with mAb specific for KLRG1, V{gamma}9, CD3, and the markers indicated. Data shown are expressed as percentage of positive cells among KLRG1+ V{gamma}9+ CD3+ lymphocytes from individually analyzed blood donors (n=4–9). Antibodies used were CCR7-fluorescein isothiocyanate (FITC) from R&D Systems (Wiesbaden, Germany) and CD27-PE, CD45RA-PE, CD62L-FITC, CD81-PE, CD94-PE, and CD244-PE from Beckman Coulter.

 
To examine KLRG1 expression in proliferating {gamma}{delta} T cells, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE)-stained PBMC were incubated with HMB-PP and interleukin (IL)-2 or with IL-2 alone. In the absence of HMB-PP, V{gamma}9/V{delta}2 T cells did not proliferate, whereas addition of HMB-PP led to a significant proliferation of KLRG1+ V{gamma}9+ CD3+ cells that lost the CFSE label almost completely (Fig. 3A ). In contrast to KLRG1, proliferating V{gamma}9/V{delta}2 T cells remained negative for CD27 throughout the entire culture period of 6 days. As KLRG1+ {alpha}ß T cells have an impaired proliferative capacity upon stimulation [8 ], we next sought to examine the proliferative potential of purified KLRG1+ {gamma}{delta} T cells. To generate these cells, V{gamma}9/V{delta}2 T cells were first expanded from PBMC in the presence of HMB-PP and IL-2. After 7 days, KLRG1+ cells were purified by positive selection on a VarioMACS magnetic cell sorter (Miltenyi Biotec, Bergisch Gladbach, Germany), using biotinylated mAb 13A2 and antibiotin microbeads (Miltenyi Biotec); purities of KLRG1+ cells were >92.5%, as assessed with streptavidin-PE (Beckman Coulter). In the presence of {gamma}{delta} T cell-depleted, autologous PBMC as feeder cells (<0.05% {gamma}{delta}+), prepared by using the T cell receptor {gamma}/{delta} MicroBead kit (Miltenyi Biotec), restimulation of CFSE-stained KLRG1+ V{gamma}9/V{delta}2 T cells with HMB-PP and IL-2 resulted in vigorous cell division (Fig. 3B) . Thus, these data show that KLRG1+ V{gamma}9/V{delta}2 T cells were able to proliferate upon stimulation in vitro.



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  		Figure 3. Proliferative potential of KLRG1+ {gamma}{delta} T cells. (A) CFSE-stained PBMC (2x105) were incubated in IL-2 (100 U/ml) containing RPMI-1640 medium with or without 1 nM HMB-PP. After 6 days, cultures were labeled with mAb specific for CD3, V{gamma}9, and KLRG1 or CD27. Representative dot-plots gated on V{gamma}9+ CD3+ lymphocytes are shown. (B) V{gamma}9/V{delta}2 T cells were expanded from PBMC in the presence of 1 nM HMB-PP and 100 U/ml IL-2. After 7 days, KLRG1+ cells were purified, stained with CFSE, and cultured (104) with {gamma}{delta}-depleted PBMC (2x105), 1 nM HMB-PP, and no or 100 U/ml IL-2. After 6 more days, cultures were labeled with mAb specific for CD3 and V{gamma}9. The histograms show CFSE staining of gated V{gamma}9+ CD3+ lymphocytes.

 
Cross-linking of KLRG1 on NK cells has been reported to inhibit cytokine production and cell-mediated cytotoxicity [4 ]. To test the effect of KLRG1 ligation on the proliferative response of V{gamma}9/V{delta}2 T cells, CSFE-stained PBMC were labeled with biotinylated anti-KLRG1 mAb 13A2 and stimulated in streptavidin-coated 96-well plates (Pierce, Perbio Science, Bonn, Germany) with HMB-PP and IL-2. After 5 days, cell proliferation was assessed by determining the dilution of the CFSE signal in V{gamma}9+ CD3+ lymphocytes. Figure 4A shows that antibody-mediated cross-linking of KLRG1 did not affect the proliferative response of V{gamma}9/V{delta}2 T cells, whereas ligation of CD94 with biotinylated anti-CD94 mAb (clone HP-3D9; Alexis Biochemicals, Grünberg, Germany) significantly impaired the division of these cells. We also failed to observe an inhibitory effect of KLRG1 ligation when production of tumor necrosis factor {alpha} and interferon-{gamma} was examined by intracellular cytokine staining (data not shown). Likewise, anti-KLRG1 mAb did not inhibit the cytolytic activity of KLRG1+ V{gamma}9/V{delta}2 T cell clones toward the human Burkitt’s lymphoma cell line Daudi [13 ], in contrast to anti-CD94 mAb (Fig. 4B) .



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  		Figure 4. KLRG1 ligation by mAb does not affect the proliferative and cytotoxic response of {gamma}{delta} T cells. (A) CFSE-stained PBMC, unlabeled (upper right) or prelabeled with 50 µg/ml biotinylated anti-KLRG1 (lower left) or anti-CD94 mAb (lower right), were incubated in streptavidin-coated 96-well plates in the presence of 10 nM HMB-PP and 100 U/ml IL-2 or in medium only (upper left). Proliferation was determined as dilution of the CFSE signal in the V{gamma}9+ CD3+ lymphocyte gate on day 5. Plots are typical from five independent experiments. (B) Cytolytic activity of the KLRG1+ V{gamma}9/V{delta}2 T cell clones GD40, PF16, PF60, and PF2 against Daudi target cells in the absence of mAb (med) or in the presence of anti-KLRG1, anti-CD94, or IgG1{kappa} isotype control mAb (30 µg/ml each). The cytolytic activtiy was determined in a standard 51Cr release assay [13] using effector:target ratios of 3:1 (hatched bars) and 10:1 (solid bars). Data shown are mean values of percent specific lysis from experiments performed in triplicates. Standard deviations were <3%.

 
Expression of KLRG1 on human V{gamma}9/V{delta}2 T cells, as demonstrated in the present report, appears to contradict earlier investigations that failed to detect KLRG1 expression in intraepithelial T cell subsets, including CD8{alpha}{alpha}+ {gamma}{delta}+ lymphocytes, in mice [17 ]. However, it is important to note that KLRG1 expression values on {alpha}ß T cells are considerably higher in humans than in mice [8 ]. As KLRG1 expression is induced dramatically in {alpha}ß T cells during viral and parasitic infection [4 , 6 ], the discrepancy between humans and mice is most likely due to the multiple infections experienced by humans during their longer lifetime compared with mice housed in clean facilities. This interpretation fits in well with the concept that V{gamma}9/V{delta}2 effector memory T cell levels rise from childhood onward, as a result of constant exposure to microbial pathogens, so that in adults, they are mainly characterized by expression of CD45RO and lack of CCR7 [15 , 16 , 18 ].

In contrast to the well-established inhibition by anti-CD94 mAb [11 , 12 ], we failed to observe a significant inhibitory effect in human V{gamma}9/V{delta}2 T cells by anti-KLRG1 mAb. This result is in apparent contrast to the reported inhibitory function of KLRG1 in NK cells [4 ]. However, it is important to stress that those experiments had been performed with a murine NK cell line derived from p53–/– mice, which overexpressed KLRG1 as a result of retroviral transduction [4 ]. In our study, human {gamma}{delta} T cells expressing physiological levels of KLRG1 were analyzed. The impaired proliferative capacity of KLRG1+ {alpha}ß T cells in peripheral blood led to the notion that KLRG1 may represent a marker for senescent lymphocytes [6 , 8 ]. The present report demonstrates that the proliferative capacity of KLRG1+ cells differs considerably between the {alpha}ß and {gamma}{delta} T cell subsets in human adult peripheral blood and that KLRG1 expression and impaired proliferation are not necessarily correlated in all lymphocytes.


   ACKNOWLEDGEMENTS
 
This work was supported in part by the Else Kröner-Fresenius-Stiftung, the Fonds der Chemischen Industrie, and the Deutsche Forschungsgemeinschaft (SFB620, Teilprojekte B2 and C4). Blood samples from healthy donors were kindly provided by the Institut für Klinische Immunologie und Transfusionsmedizin, Universitätsklinikum Giessen. Cord blood samples were kindly provided by Dr. Reinhard Berner, Universitätskinderklinik Freiburg. We thank Stephen Batsford for comments on the manuscript.

Received February 18, 2004; revised June 8, 2004; accepted September 16, 2004.


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