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*
Departments of Structural Biology and Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305; and
DNAX Research Institute for Molecular and Cellular Biology, Palo Alto, CA 94306
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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signaling subunit (9). ILT/LIR proteins differ
from KIR by being principally expressed on cells with a phagocytic and
Ag-presenting function such as monocytes, macrophages, dendritic cells,
and B lymphocytes, although a subset of these inhibitory receptors are
expressed on peripheral NK and T lymphocytes (4, 5). The functional activity of human NK cells and a subset of CTL is influenced by the interaction of KIR with epitopes of HLA class I molecules. Upon binding their specific ligands, KIR recruit SHP-1 phosphatase via ITIMs located in their cytoplasmic tail, inhibiting the signaling processes associated with cellular activation (1). In NK cells, KIR genotype and expression repertoire determine self tolerance (10) and allow these cells to detect "missing self," i.e., the loss of cell surface HLA class I expression resulting from infection with certain viruses or malignant transformation. Isoforms of the KIR molecules that have short cytoplasmic tails lacking ITIM motifs may trigger effector cell activation through association with the adaptor molecule DAP12 (11). The precise role of KIR expression in CTL is unclear, although these receptors can modulate signals transmitted through the TCR/CD3 complex (12, 13, 14). KIR expression in T lymphocytes appears to be restricted to cells with a differentiated "memory" phenotype (15).
As the KIR and ILT/LIR receptors are encoded within the same genomic cluster, it is relevant to assess the features of ILT/LIR expression in peripheral lymphocytes and the extent of any coincidence with KIR expression, particularly as inhibitory receptors such as ILT2/LIR1 appear to be expressed on both NK and T lymphocytes and recognize HLA class I ligands (16). Investigation of these features will provide information on cell-type specific expression of genes within the LRC and may have important implications for the functional repertoires of cells with cytotoxic capacity. We have thus analyzed characteristics of the ILT2/LIR1 inhibitory receptor on T lymphocytes in comparison with expression of the related KIR. Previous studies have demonstrated the expression of ILT2/LIR1 by a proportion of T cells but have not addressed the coordination of ILT2/LIR1 and KIR expression or defined the specific types of T cells that express ILT2/LIR1. Our results indicate a certain overlap in expression of the two types of receptors but also demonstrate a differential regulation of Ig-like receptor acquisition which correlates with the development of resistance to activation-induced cell death in memory CTL.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Buffy coats obtained by leukapheresis of normal blood donors were purchased from Stanford Medical Center Blood Bank. Cord blood samples were obtained from normal term deliveries at the Maternity Unit, Packard Children’s Hospital, Stanford. Mononuclear cells were obtained from these products by Ficoll-Hypaque gradient centrifugation. HLA class I typing was performed on genomic DNA derived from PBMC using PCR followed by direct sequencing and sequence-specific primer amplification.
Flow cytometry
Three-color flow cytometry was performed on mononuclear cells
stained in a two-stage procedure with the ILT2/LIR1-specific mAb HPF1
(16) followed by FITC- or PE-labeled goat anti-mouse
reagent. After blocking with normal mouse serum (Sigma, St. Louis, MO),
cells were counterstained with directly conjugated (FITC or CyChrome)
Abs specific for the T cell markers CD3, CD4, CD8, TCR
,
TCR
, CD45RO, CD45RA, CD57, CD28, CD27, CD38, and the NK cell
markers CD16 and CD56. A mixture of PE-coupled KIR-specific mAbs
consisting of the anti-KIR reagents EB6 (anti-KIR2DL1,
anti-KIR2DS1; Coulter, Hialeah, FL), DX27 (anti-KIR2DL2/L3,
anti-KIR2DS2), DX9 (anti-KIR3DL1), and DX31 (anti-KIR3DL2)
was also used. T cell clones as well as peripheral mononuclear cells
were analyzed by flow cytometry using FITC-coupled mAbs specific for
the TCRV region families AV2, AV12.1, BV3.1, BV5a, BV5b, BV5c, BV6.7,
BV8a, BV12, BV13, BV14, BV16, BV21, BV22, and BV23 (Coulter and
Serotec, Raleigh, NC). For analysis of intracellular Bcl-2 levels by
four color flow cytometry, PBMC were first stained with appropriate PE,
Cychrome or APC-labeled Abs specific for cell surface markers,
permeabilized, and stained with FITC-labeled anti-Bcl-2 or IgG1
isotype control (Becton Dickinson, San Jose CA).
Ag-specific T lymphocytes were identified using synthetic HLA-A*0201 tetrameric complexes labeled with PE fluorochrome. HLA-A2 tetramers were refolded with HCMV pp65 peptide NLVPMVATV or human EBV BZLF1 peptide GLCTLVAML. Tetramer staining was performed at room temperature to minimize low affinity binding.
Cells (2 x 105) of each T cell clone and 1 x 106 PBMC were incubated at 4°C for 45 min with the appropriate Abs, washed, and analyzed on a FACScan or FACSort flow cytometer using CellQuest (Becton Dickinson) and FlowJo (TreeStar, San Carlos, CA) analysis software.
T cell cloning
Single CD3+ HPF1+ T cells were sorted using a FACStar or FACSVantage cell sorter and Cyt-clone software (Becton Dickinson). Single-cell sorted T cell clones were established and maintained as described (17). Briefly, T cell clones were cultured in IMDM (Life Technologies, Gaithersburg, MD) containing 200 U/ml recombinant IL-2 (provided by C. Reynolds, National Cancer Institute/Biological Response Modifier Program, Frederick, MD) and 5% human T-STIM (Becton Dickinson). Mixed, allogeneic PBMC (1 x 106/ml; three donors) and 1 x 105/ml JY cells (EBV-transformed B lymphoblastoid cell line) were irradiated and used as feeder cells at the start of the culture and subsequently at weekly intervals. Irradiated feeder cells without T cell clones ("feeders-only" culture) were cultured in parallel to control for background proliferation.
RNA preparation and reverse transcription
Total cellular RNA was extracted from 3–5 x 106 T cell clones and the "feeders-only" culture using RNAzol according to the manufacturer’s instructions (Tel-Test, Friendswood, TX).
First strand cDNA was synthesized from 1 µg RNA by reverse transcription using oligo(dT) (Perkin-Elmer, Norwalk, CT) and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, MD) in a volume of 50 µl at 42°C for 1.5 h.
PCR amplification
RT-PCR analysis of KIR expression on T cell clones was performed
as described (18) using a panel of PCR primer pairs to
enable the specific amplification of six groups of inhibitory KIR
(KIR2DL1–4 and KIR3DL1–2) and six groups of noninhibitory KIR
(KIR2DS1–5 and KIR3DS1). Amplifications were performed with specific
primers at a concentration of 0.5 µM in 25-µl reactions for 30
cycles using 2 µl of cDNA per reaction. Internal control primers
specific for -actin were included in each PCR at a concentration of
0.05 µM. Feeders-only cultures were used as RT-PCR controls to
monitor the presence of residual transcripts from irradiated feeder
cells. The use of this RT-PCR analysis allowed us to detect expression
of all KIR genes, including those receptors that cannot be detected by
specific Abs.
TCRBV family-specific PCR was performed as previously described (19). For each of 25 TCRBV families a specific sense primer and a common antisense primer matching the TCRB constant region were used. T cell clones were amplified for 25 cycles, PBMC for 35 cycles using 1 µl of cDNA per 25-µl reaction.
TCRB gene rearrangements of T cell clones were determined by direct sequencing of products obtained by TCR family-specific PCR. Due to the monoclonality of the established T cell clones, no subcloning step was necessary to obtain unambiguous complementarity-determining region 3 (CDR3) sequences. PCR products were purified using a QIAquick PCR Purification Kit (Qiagen, Chatsworth, CA) and cycle-sequenced using dye-labeled deoxynucleotide terminators and a 373A automated sequencer (Applied Biosystems, Foster City, CA). TCRB rearrangements were determined in both directions using the family-specific and constant region-specific primers used for PCR amplification.
Functional analysis
T cell clones (2 x 104) reactive with tetrameric HLA complexes were incubated at 37°C in 96-well plates with irradiated peptide-pulsed stimulator cells at a ratio of 5:1 for the indicated time periods in IL-2 medium. Stimulator cells comprised the 721.221 HLA class I-negative B lymphoblastoid cell line transfected with cDNA for HLA-A*0201 and incubated in the presence of relevant nonamer peptides at 37°C overnight before washing and irradiation. Cultures for each time point were established in duplicate. Peptides were synthesized by F-moc chemistry and purified to greater than 90% purity by reverse phase HPLC (Sigma Genosys, Austin TX). Cells were analyzed by flow cytometry with fluorochrome-labeled anti-CD8 and anti-CD3 Abs. Cell viability was assessed by forward and side scatter characteristics and staining with FITC-labeled annexin V (Becton Dickinson). For anti-CD3 stimulation, T cell clones (2 x 105) were incubated in the presence of 1 µg/ml anti-CD3 mAb (clone UCHT1; Coulter) and 2 µg/ml recombinant protein G (Sigma) in IL-2 medium at 37°C for 6 h, washed, and assayed for Annexin V binding and propidium iodide exclusion by flow cytometry. Control incubations consisted of T cell clones incubated in the presence of protein G or medium alone. For analysis of PBLs, CD3+ CD8+ T cells were sorted into ILT2/LIR1+ KIR- and ILT2/LIR1+ KIR+ populations by flow cytometry before anti-CD3 stimulation and flow cytometric analysis as described above.
Statistical analysis
Analysis of differences in Ig-like receptor expression between flow cytometry-defined subsets was accomplished using a two-tailed Student t test and ANOVA.
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Results |
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Flow cytometric analysis of ILT2/LIR1 expression in PBMC of 20
normal donors using the specific mAb HPF1 demonstrated that the
principal cell types expressing this inhibitory receptor were monocytes
and B lymphocytes (Fig. 1
A),
as previously reported (16). In all individuals analyzed,
ILT2/LIR1 was expressed on close to 100% of
CD3- CD19+ B lymphocytes
and CD14+ CD16+ or
CD13+ CD16+ monocytes. In
contrast to the ubiquitous expression by B lymphocytes and monocytes,
ILT2/LIR1 was expressed on a subset of peripheral adult
CD3+ T lymphocytes and
CD3- CD56+ NK lymphocytes
(Fig. 1A) and a very small proportion of cord blood T
lymphocytes (mean = 1.7% SD = 1.2, n = 5).
ILT2/LIR1 was expressed on a mean of 48% (SD = 21.4) of T
lymphocytes and a mean of 23.5% (SD = 14.9) of
T-lymphocytes in the 20 normal adults. Expression of both ILT2/LIR1 and
KIR was restricted to CD8+ T lymphocytes, with
<1% of CD4+ T lymphocytes expressing Ig
superfamily inhibitory receptors (Fig. 1B). Although the
values for CD3+ cells appear quantitatively
similar to that for CD8+ cells, this is an
artifact of presenting the results as mean values. Analysis of
individual donors demonstrates that these values are not correlated and
are apparently influenced by variations in numbers of
CD8+ T cells between donors (data not shown).
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In view of the low numbers of ILT2/LIR1+ T
lymphocytes observed in cord blood samples and the previously reported
restriction of KIR expression to memory phenotype adult T lymphocytes,
we performed a subset analysis of T cells in the panel of 20 normal
individuals using the HPF1 Ab and a pool of Abs recognizing KIR2D and
KIR3D molecules. This analysis demonstrated that both
ILT2/LIR1+ and KIR+ T cells
are predominantly contained within the CTL memory/effector population
i.e., CD8+ CD56+
CD57+ CD27-
CD28- T cells (Fig. 2A-D).
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, E and F).
ILT2/LIR1+ T cells were of a higher proportion in
the CD45RA+ population, which may correlate with
the reported effector function of this subset of cells
(20). By comparison with KIR expression in the populations
defined by CD8, CD56, CD57, CD27, or CD28, ILT2/LIR1 was expressed by a
significantly higher number of T cells within each subset
(p < 0.0005). Coexpression of ILT2/LIR1 and KIR on CTLs
Our analysis of inhibitory receptor expression in T lymphocyte
subsets revealed a consistently greater number of cells positive with
the HPF1 Ab than the mixture of KIR-specific Abs within all subsets
tested. This raised the possibility that ILT2/LIR1 and KIR expression
may not be concomitant in these cells. To analyze this further, we
tested coexpression of ILT2/LIR1 and KIR in the peripheral lymphocytes
of twenty individuals. Although a consistent proportion of
KIR+ NK (gated on CD56+ or
CD16+ lymphocytes) or T lymphocytes (gated on
CD3+ lymphocytes) coexpressed ILT2/LIR1 (Fig. 3A), this feature was not
apparent when ILT2/LIR1+
CD3+ T-lymphocytes were analyzed for coexpression
of KIR (Fig. 3B). Although equivalent levels of KIR
expression were observed on ILT2/LIR1+ NK
lymphocytes, the majority of ILT2/LIR1+ T cells
expressed only this receptor and the minority coexpressed KIR. A
comparison of the relative proportions of ILT2/LIR1 and KIR
coexpression in the CD3+ population is shown in
Fig. 3, C and D.
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). In both
CD3+ CD8+ (Fig. 4A) and TCR+ (Fig. 4B) populations, 19 of 20 individuals expressed ILT2/LIR1 on
a higher percentage of T cells than KIR molecules. However, the
majority of these individuals expressed ILT2/LIR1 on a lower percentage
of their NK (CD3- CD56+)
cells (Fig. 4C) than KIR molecules with T cells
demonstrating an intermediate phenotype (Fig. 4D).
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To precisely investigate the characteristics of ILT2/LIR1 expression in T lymphocytes, we derived T cell clones by single cell sorting of CD3+ ILT2/LIR1+ lymphocytes from the peripheral blood of two donors who were previously analyzed in detail for the features of KIR expression in both NK (10) and T lymphocytes (21). The phenotype and TCR clonotype of the clones were analyzed in detail using flow cytometric and molecular techniques. Because the pool of KIR-specific Abs we used in the flow cytometric analysis detect all but four of the 12 known KIR molecules, a precise molecular analysis of T cell clones was required to unambiguously determine the full extent of overlap in Ig-like receptor expression.
ILT2/LIR1+ T cell clones are representative of peripheral blood T lymphocytes
The panels of ILT2/LIR1+ T cell clones
derived from donors NV and PP have similarities to the populations of
KIR+ T cell clones we have previously analyzed
(21) but also have significant differences (Table I). In short, although these cells
typically displayed the phenotypes of activated memory cells, the
panels comprised not only CD8+ T cells but also
included CD4- CD8-
TCR cells and rare CD4+ cells. Similar
proportions of TCR and TCR cells were derived from cloning
ILT2/LIR1+ T cells as were found in
KIR+ cloning.
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The most striking finding of our previous study of
KIR+ T cell clones was the almost complete
restriction of TCRBV region gene usage in TCR clones from donor
NV. All such clones were TCRBV16 and expressed a highly restricted CDR3
sequence (21). We used PCR amplification and nucleotide
sequencing of TCRBV gene transcripts from
ILT2/LIR1+ T cell clones from donors NV and PP to
assess the extent of TCR diversity in these cells. A proportion of the
ILT2/LIR1+ TCR clones from donor NV
represented the population cloned previously as they expressed the same
TCRBV16 clonotype as the KIR+ T cell clones.
However, a further population of ILT2/LIR1+ T
cell clones expressed TCRB chain genes of diverse families and
clonotypes including TCRBV2, BV6, BV17, BV21, and BV23 (Table II). No motifs common to all represented
TCRBV families were found when the CDR3-encoding regions of these
transcripts were sequenced, suggesting that these clones originally
proliferated in response to unique peptide Ags. Similar results were
found in clones derived from donor PP, where clonal TCRBV transcripts
included TCRBV1, BV2, BV3, BV6, BV10, BV13, BV14, BV16, BV17, and BV21
(Table II).
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Clonal analysis of ILT2/LIR1 and KIR coexpression
Typing of KIR gene expression in each of the T cell clones by
RT-PCR and flow cytometry revealed a surprising finding. Although all
clones were ILT2/LIR1+, only a very small number
displayed the Ig-like receptor phenotype of the cells that predominated
in peripheral blood, i.e., ILT2/LIR1+
KIR-. From a total of 104 clones (including 20
from a third donor), only 19 did not react with KIR-specific mAbs and
the majority of these expressed KIR2DL4, KIR2DS4, or KIR3DS1, which are
not detectable by currently available Abs but can be detected at the
cDNA level (Fig. 5). Thus, only 3 of the
104 clones displayed a truly KIR- phenotype. The
remaining 101 clones displayed KIR phenotypes, as determined by RT-PCR
typing, similar to those described in our previous study
(21). These results indicate that our use of the pooled
KIR-specific Abs in the analysis of PBLs detected the majority
(
80%) of the KIR-expressing T cells.
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). Using an HLA-A*0201-HCMVpp65
tetramer, it is evident that the majority of these peptide-specific
CD8+ T cells express ILT2/LIR1 (Fig. 6B) but only a very small proportion express KIR or CD94
receptors (Fig. 6C). Similar results were detected in
another five donors using tetramers containing either HCMV- or
EBV-specific peptides (data not shown) confirming the validity of the
LRC receptor phenotype found in our analysis of peripheral blood
CD8+ lymphocytes (Fig. 4).
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In view of the discrepancy observed between the phenotype of peripheral blood ILT2/LIR1+ T cells and that of the in vitro expanded panel of T cell clones, we reasoned that the majority of ILT2/LIR1+ T cells did not survive the cloning procedure and only those clones, which were also KIR+, exhibited long-term in vitro growth characteristics. To investigate whether possible differences in the survival ability of T cell clones correlated with their expression of particular Ig-like receptors we performed another two experiments. After isolation and limited expansion of HCMVpp65 peptide-specific T cell clones using tetramers and single cell sorting, we tested the response of these ILT2/LIR1+ KIR- clones to in vitro stimulation with their cognate peptide/HLA class I restriction element in the presence of IL-2.
As shown in Fig. 7A,
incubation of T cell clones with their cognate peptide resulted in the
death of 80–90% of these cells by 48 h of culture, with a
slightly more rapid rate of cell death induced by stimulator cells
pulsed with higher peptide concentrations. In contrast, incubation with
stimulators pulsed with an equivalent concentration of irrelevant
peptide resulted in a decrease of viable CD8+
cells of only 5–10%. To compensate for any potential differences in
the affinity of clonally distributed TCRs for peptide-HLA complexes, we
also stimulated ILT2/LIR1+
KIR- and ILT2/LIR1+
KIR+ T cell clones with soluble anti-CD3 Ab
in the presence of protein G and IL-2. After 6 h of incubation
under these conditions, 50% of cells of the
KIR- clone bound annexin V, indicating the
translocation of plasma membrane phospholipids characteristic of the
early stages of apoptosis (22). In contrast,
KIR2DL4+ T cell clones displayed only a
background level of such annexin V binding (Fig. 7B).
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A). This population also
contained significantly (p < 0.005) lower
intracellular levels of the survival molecule Bcl-2 (Fig. 8B). The results of these two experiments indicate that
expression of KIR by T cells correlates with the acquisition of
resistance to activation-induced cell death by apoptosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The phenotypic assessment of peripheral blood T cell subsets definitively shows that ILT2/LIR1 and KIR genes are expressed in the same types of T cells, i.e., differentiated, Ag-experienced cytotoxic cells which may have lost the requirement for costimulation. However, this overlap in expression of the related genes is not necessarily coordinate as the proportions of ILT2/LIR1+ cells always exceeded that of KIR+ cells within each subset. The more widespread expression of ILT2/LIR1 is also supported by phenotypic analysis of T cell clones and Ag-specific T cells. ILT2/LIR1+ clones and peripheral T cells displayed a diverse assortment of TCRV region gene usage in contrast to KIR+ cells. Also, ILT2/LIR1+ clones included CD4-CD8- and rare CD4+ T cells in addition to the CD8+ cells characteristically expressing KIR.
Analysis of coexpression of the two receptor types demonstrated the predominance of ILT2/LIR1+ KIR- T cells in peripheral blood and the use of tetrameric HLA class I constructs confirmed this pattern of receptor expression within a population of CD8+ T cells specific for distinct viral peptides.
The derivation of ILT2/LIR1+ T cell clones allowed us to define a major functional difference between cells expressing ILT2/LIR1 alone or in concert with KIR. The use of established in vitro cloning procedures failed to produce clones representative of the major phenotype observed ex vivo, as the only cells that displayed significant in vitro proliferation were KIR+ and not ILT2/LIR1+ KIR-. Further functional analysis of these clones demonstrated that ILT2/LIR1+ KIR- clones were susceptible to apoptosis when activated through the TCR/CD3 complex, whereas KIR+ clones were not. This result was confirmed by analysis of these T cell populations derived from peripheral blood, which additionally demonstrated a lower level of the cell survival molecule Bcl-2 in ILT2/LIR1+ KIR- CD8+ T cells.
It could be argued that without an Ab to specifically identify KIR2DL4,
we do not currently know the true extent of KIR2DL4 expression in the
serologically KIR- population. However, if all
of the ILT2/LIR1+ KIR-
cells expressed KIR2DL4 we would expect a far higher proportion of the
in vitro expanded clones to display this phenotype than was actually
detected (18% of ILT2/LIR1+ clones compared
with 60% of peripheral blood ILT2/LIR1+ T
cells). This suggests that the data on KIR expression we obtained by
flow cytometry are not a gross underestimate of peripheral
KIR+ T cells.
These phenotypic and functional results allow us to propose a scheme for the sequential acquisition of LRC-encoded receptors by activated CTLs. In our previous study of KIR+ T cell clones (21), we suggested that the diverse KIR phenotypes displayed by a dramatically expanded clone of T lymphocytes represented the end result of a program of KIR gene activation which began with the expression of KIR2DL4, a receptor which has been demonstrated to be expressed in all KIR+ NK or T cells (23).
Our present study supports such a serial mechanism of LRC receptor
expression and allows this to be extended to a stage before the
expression of KIR2DL4. The dramatically increased proportion of
activated T cells that are ILT2/LIR1+
KIR-, as exemplified by the tetramer binding
population, can be regarded as effector CTL because they comprise the
greater number of Ag-specific cells, are CD56+
(24) and are prone to apoptosis when stimulated
(25). This association of ILT2/LIR1 gene expression with T
cell activation may be related to our finding of an NCAM
(CD56)-associated transcription element in the 5'-untranslated region
of ILT2/LIR1 cDNA clones (N.Y., unpublished data). However, the precise
signals determining Ig-like receptor gene expression are currently
unknown. Although certain cytokines, such as IL-15 (26),
TGF- (27), and IL-10 (28), have been
reported to induce or modulate expression of the lectin-like receptors
CD94 and NKG2A on T cells, no effects on LRC-encoded Ig-like receptor
expression have been observed. The relevance of KIR2DL4 expression at
the initiation of KIR gene expression in T cells is unclear. Although
this receptor has been detected at the mRNA level in all
KIR+ cells, its expression on the cell surface is
currently controversial. One study reports KIR2DL4 expression on all
peripheral NK cells (23), whereas another suggests that
KIR2DL4 is only expressed on the surface of decidual NK cells in
pregnancy (29). The significance of the apparent
specificity of KIR2DL4 for the nonclassical HLA-G class I molecule
(23, 29) is also unclear because HLA-G expression is
highly tissue restricted (30). Specific reagents to
reliably detect KIR2DL4 are required before these matters can be fully
resolved.
The relatively small population of KIR+ cells represent clones that have made the transition to a state of long-lived memory T cells in a scheme that is consistent with the recently demonstrated linear differentiation of specific memory CTL in transgenic mice (31). The proportions of KIR+ and KIR- cells within the ILT2/LIR1+ T cell population are consistent with the numbers of effector and memory cells reported in the anti-HY response of these mice. This situation is likely to be observed in chronic viral infections because the pool of responsive peptide-specific T cells will continually be exposed to Ag (32), becoming activated and expanding in number before maintaining dynamic homeostasis through apoptosis, with a small percentage of cells remaining as KIR+ memory cells. Such a predominance of effector cells may not be observed where viral infections are cleared and only the relatively scarce Ag-specific memory cells will be detectable (33).
Recently, it has been suggested that memory T cells can be distinguished into two types (effector and central) based on the expression of CCR7 chemokine receptors, correlating with their in vivo trafficking (34). Due to the unavailability of the Abs defining the CCR7 chemokine receptor we have been unable to directly assess how this correlates with the expression of specific Ig-like receptors. We would suggest that the major population of ILT2/LIR1+ KIR- cells represent the effector cells characteristic of an initial immune expansion, while future studies should allow us to define whether the ILT2/LIR1+ KIR+ cells we have identified are comparable with the effector memory subset defined by Sallusto et al. (34).
Our results also indicate a possible role for KIR in the maintenance of
memory T cell survival and resistance to activation-induced cell death.
Effector CD8+ cells have lower levels of the cell
protective molecule Bcl-2 (35), which results in
sensitivity to apoptosis induced by fratricide (36) or
TNF- (37). Our findings support these reports and
suggest that KIR expression in T cells is associated with resistance to
activation-induced cell death mediated through stimulation of the TCR.
At present, we cannot assign this anti-apoptotic role directly, as
it is possible that KIR expression in memory cells is simply coordinate
with that of another molecule(s) that confers this survival feature
(38). However, KIR have been suggested to bind the p85
subunit of PI3-kinase leading to activation of the anti-apoptotic
AKT kinase (39), and the functionally equivalent Ly49
receptors in mice may be involved in the selection and survival of
individual NK clones (40).
The differential expression of ILT2/LIR1 and KIR in activated T
lymphocytes is consistent with differences in the structure and ligand
binding specificities of these molecules. Whereas KIR bind defined
epitopes in the 1 domain of HLA-C or HLA-B molecules, the larger
size of the four Ig-domain ILT2/LIR1 molecule is compatible with its
binding to the common 3 domain of class I in an extended
conformation, similar to that of CD8 (41). If such a model
is correct, the molecular dimensions of ILT2/LIR1, in addition to the
nature of its ligand binding site, would allow concomitant binding of
both TCR and ILT2/LIR1 to the same HLA class I ligand molecule. This
would enable the recruitment of phosphatases directly to the site of
TCR signaling and the subsequent diminution of cellular activation.
In conclusion, our study indicates a program of sequential expression of Ig-superfamily receptors encoded within the LRC, with ILT2/LIR1 expression as an initial event in early stage, activated cytotoxic effector T cells followed by the acquisition of KIR at the stage of transition to differentiated memory cells. The expression of KIR appears to correlate with a resistance to activation-induced cell death in these cell populations, although the requirement for KIR interaction with self-HLA class I in this process is currently unknown.
In view of the close developmental and functional relationships between NK and CD8+ T lymphocytes (42), further investigation of the events occurring in activated CD8+ T cells should enable the use of such cells as an appropriate working model for analysis of LRC gene transcription and expression. Future studies will allow us to determine the potential role of these Ig-like receptors in the long-term survival of memory T cells.
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Acknowledgments |
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Footnotes |
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2 Current address: Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, U.K.
3 Address correspondence and reprint requests to Dr. Neil Young at his current address: Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Headington Oxford, OX3 9DU U.K.
4 Current address: Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich Heine University Medical Center, Düsseldorf, Germany.
5 Current address: University of California San Francisco, Department of Microbiology and Immunology, San Francisco, CA 94143.
6 Abbreviations used in this paper: KIR, killer cell Ig-like receptor; ILT, Ig-like transcript(s); LRC, leukocyte receptor complex; LIR, leukocyte Ig-like receptor(s); CDR3, complementarity-determining region 3.
Received for publication November 1, 2000. Accepted for publication January 12, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 E. Fourmentraux-Neves, A. Jalil, S. Da Rocha, C. Pichon, S. Chouaib, G. Bismuth, and A. Caignard Two opposite signaling outputs are driven by the KIR2DL1 receptor in human CD4+ T cells Blood, September 15, 2008; 112(6): 2381 - 2389. [Abstract] [Full Text] [PDF]
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E. Morel and T. Bellon HLA Class I Molecules Regulate IFN-{gamma} Production Induced in NK Cells by Target Cells, Viral Products, or Immature Dendritic Cells through the Inhibitory Receptor ILT2/CD85j J. Immunol., August 15, 2008; 181(4): 2368 - 2381. [Abstract] [Full Text] [PDF]
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A. Iannello, O. Debbeche, S. Samarani, and A. Ahmad Antiviral NK cell responses in HIV infection: I. NK cell receptor genes as determinants of HIV resistance and progression to AIDS J. Leukoc. Biol., July 1, 2008; 84(1): 1 - 26. [Abstract] [Full Text] [PDF]
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A. Iannello, O. Debbeche, S. Samarani, and A. Ahmad Antiviral NK cell responses in HIV infection: II. viral strategies for evasion and lessons for immunotherapy and vaccination J. Leukoc. Biol., July 1, 2008; 84(1): 27 - 49. [Abstract] [Full Text] [PDF]
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M. Maffei, F. Ghiotto, M. Occhino, M. Bono, A. De Santanna, L. Battini, G. L. Gusella, F. Fais, S. Bruno, and E. Ciccone Human Cytomegalovirus Regulates Surface Expression of the Viral Protein UL18 by Means of Two Motifs Present in the Cytoplasmic Tail J. Immunol., January 15, 2008; 180(2): 969 - 979. [Abstract] [Full Text] [PDF]
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C. S. Wagner, G. C. Riise, T. Bergstrom, K. Karre, E. Carbone, and L. Berg Increased Expression of Leukocyte Ig-Like Receptor-1 and Activating Role of UL18 in the Response to Cytomegalovirus Infection J. Immunol., March 15, 2007; 178(6): 3536 - 3543. [Abstract] [Full Text] [PDF]
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 C. Griffin, E. C. Y. Wang, B. P. McSharry, C. Rickards, H. Browne, G. W. G. Wilkinson, and P. Tomasec Characterization of a highly glycosylated form of the human cytomegalovirus HLA class I homologue gpUL18 J. Gen. Virol., November 1, 2005; 86(11): 2999 - 3008. [Abstract] [Full Text] [PDF]
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S. E. Kirwan and D. N. Burshtyn Killer Cell Ig-Like Receptor-Dependent Signaling by Ig-Like Transcript 2 (ILT2/CD85j/LILRB1/LIR-1) J. Immunol., October 15, 2005; 175(8): 5006 - 5015. [Abstract] [Full Text] [PDF]
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 R. Thimme, V. Appay, M. Koschella, E. Panther, E. Roth, A. D. Hislop, A. B. Rickinson, S. L. Rowland-Jones, H. E. Blum, and H. Pircher Increased Expression of the NK Cell Receptor KLRG1 by Virus-Specific CD8 T Cells during Persistent Antigen Stimulation J. Virol., September 15, 2005; 79(18): 12112 - 12116. [Abstract] [Full Text] [PDF]
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 J. Xu, A. N. Vallejo, Y. Jiang, C. M. Weyand, and J. J. Goronzy Distinct Transcriptional Control Mechanisms of Killer Immunoglobulin-like Receptors in Natural Killer (NK) and in T Cells J. Biol. Chem., June 24, 2005; 280(25): 24277 - 24285. [Abstract] [Full Text] [PDF]
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N. Anfossi, J.-M. Doisne, M.-A. Peyrat, S. Ugolini, O. Bonnaud, D. Bossy, V. Pitard, P. Merville, J.-F. Moreau, J.-F. Delfraissy, et al. Coordinated Expression of Ig-Like Inhibitory MHC Class I Receptors and Acquisition of Cytotoxic Function in Human CD8+ T Cells J. Immunol., December 15, 2004; 173(12): 7223 - 7229. [Abstract] [Full Text] [PDF]
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J. van Bergen, A. Thompson, A. van der Slik, T. H. M. Ottenhoff, J. Gussekloo, and F. Koning Phenotypic and Functional Characterization of CD4 T Cells Expressing Killer Ig-Like Receptors J. Immunol., December 1, 2004; 173(11): 6719 - 6726. [Abstract] [Full Text] [PDF]
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M. R. Snyder, T. Nakajima, P. J. Leibson, C. M. Weyand, and J. J. Goronzy Stimulatory Killer Ig-Like Receptors Modulate T Cell Activation through DAP12-Dependent and DAP12-Independent Mechanisms J. Immunol., September 15, 2004; 173(6): 3725 - 3731. [Abstract] [Full Text] [PDF]
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N. Anfossi, S. H. Robbins, S. Ugolini, P. Georgel, K. Hoebe, C. Bouneaud, C. Ronet, A. Kaser, C. B. DiCioccio, E. Tomasello, et al. Expansion and Function of CD8+ T Cells Expressing Ly49 Inhibitory Receptors Specific for MHC Class I Molecules J. Immunol., September 15, 2004; 173(6): 3773 - 3782. [Abstract] [Full Text] [PDF]
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M. Urosevic, J. Kamarashev, G. Burg, and R. Dummer Primary cutaneous CD8+ and CD56+ T-cell lymphomas express HLA-G and killer-cell inhibitory ligand, ILT2 Blood, March 1, 2004; 103(5): 1796 - 1798. [Abstract] [Full Text] [PDF]
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L. D. Lieto, F. Borrego, C.-h. You, and J. E. Coligan Human CD94 Gene Expression: Dual Promoters Differing in Responsiveness to IL-2 or IL-15 J. Immunol., November 15, 2003; 171(10): 5277 - 5286. [Abstract] [Full Text] [PDF]
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 S. O. Schonland, C. Lopez, T. Widmann, J. Zimmer, E. Bryl, J. J. Goronzy, and C. M. Weyand Premature telomeric loss in rheumatoid arthritis is genetically determined and involves both myeloid and lymphoid cell lineages PNAS, November 11, 2003; 100(23): 13471 - 13476. [Abstract] [Full Text] [PDF]
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 A. Gati, N. Guerra, C. Gaudin, S. Da Rocha, B. Escudier, Y. Lecluse, A. Bettaieb, S. Chouaib, and A. Caignard CD158 Receptor Controls Cytotoxic T-Lymphocyte Susceptibility to Tumor-Mediated Activation-Induced Cell Death by Interfering with Fas Signaling Cancer Res., November 1, 2003; 63(21): 7475 - 7482. [Abstract] [Full Text] [PDF]
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D. Belkin, M. Torkar, C. Chang, R. Barten, M. Tolaini, A. Haude, R. Allen, M. J. Wilson, D. Kioussis, and J. Trowsdale Killer Cell Ig-Like Receptor and Leukocyte Ig-Like Receptor Transgenic Mice Exhibit Tissue- and Cell-Specific Transgene Expression J. Immunol., September 15, 2003; 171(6): 3056 - 3063. [Abstract] [Full Text] [PDF]
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M. Shiroishi, K. Tsumoto, K. Amano, Y. Shirakihara, M. Colonna, V. M. Braud, D. S. J. Allan, A. Makadzange, S. Rowland-Jones, B. Willcox, et al. Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G PNAS, July 22, 2003; 100(15): 8856 - 8861. [Abstract] [Full Text] [PDF]
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C. A. Stewart, J. van Bergen, and J. Trowsdale Different and Divergent Regulation of the KIR2DL4 and KIR3DL1 Promoters J. Immunol., June 15, 2003; 170(12): 6073 - 6081. [Abstract] [Full Text] [PDF]
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X. Gu, A. Laouar, J. Wan, M. Daheshia, J. Lieberman, W. M. Yokoyama, H. R. Katz, and N. Manjunath The gp49B1 Inhibitory Receptor Regulates the IFN-{gamma} Responses of T Cells and NK Cells J. Immunol., April 15, 2003; 170(8): 4095 - 4101. [Abstract] [Full Text] [PDF]
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Y.-J. Chwae, M. J. Chang, S. M. Park, H. Yoon, H.-J. Park, S. J. Kim, and J. Kim Molecular Mechanism of the Activation-Induced Cell Death Inhibition Mediated by a p70 Inhibitory Killer Cell Ig-Like Receptor in Jurkat T Cells J. Immunol., October 1, 2002; 169(7): 3726 - 3735. [Abstract] [Full Text] [PDF]
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N. Guerra, F. Michel, A. Gati, C. Gaudin, Z. Mishal, B. Escudier, O. Acuto, S. Chouaib, and A. Caignard Engagement of the inhibitory receptor CD158a interrupts TCR signaling, preventing dynamic membrane reorganization in CTL/tumor cell interaction Blood, September 26, 2002; 100(8): 2874 - 2881. [Abstract] [Full Text] [PDF]
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M. Nikolova, P. Musette, M. Bagot, L. Boumsell, and A. Bensussan Engagement of ILT2/CD85j in Sezary syndrome cells inhibits their CD3/TCR signaling Blood, July 18, 2002; 100(3): 1019 - 1025. [Abstract] [Full Text] [PDF]
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M. R. Snyder, L.-O. Muegge, C. Offord, W. M. O'Fallon, Z. Bajzer, C. M. Weyand, and J. J. Goronzy Formation of the Killer Ig-Like Receptor Repertoire on CD4+CD28null T Cells J. Immunol., April 15, 2002; 168(8): 3839 - 3846. [Abstract] [Full Text] [PDF]
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T. Bellon, F. Kitzig, J. Sayos, and M. Lopez-Botet Mutational Analysis of Immunoreceptor Tyrosine-Based Inhibition Motifs of the Ig-Like Transcript 2 (CD85j) Leukocyte Receptor J. Immunol., April 1, 2002; 168(7): 3351 - 3359. [Abstract] [Full Text] [PDF]
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R. L. Allen, T. Raine, A. Haude, J. Trowsdale, and M. J. Wilson Cutting Edge: Leukocyte Receptor Complex-Encoded Immunomodulatory Receptors Show Differing Specificity for Alternative HLA-B27 Structures J. Immunol., November 15, 2001; 167(10): 5543 - 5547. [Abstract] [Full Text] [PDF]
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J. Dunne, S. Lynch, C. O'Farrelly, S. Todryk, J. E. Hegarty, C. Feighery, and D. G. Doherty Selective Expansion and Partial Activation of Human NK Cells and NK Receptor-Positive T Cells by IL-2 and IL-15 J. Immunol., September 15, 2001; 167(6): 3129 - 3138. [Abstract] [Full Text] [PDF]
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), 1 µM HCMVpp65 peptide (
), or 10 µM EBV
BZLF1 peptide (
), and the viability of CD8+ cells was
determined by flow cytometry at indicated time points.
B, ILT2/LIR1+ KIR2DL4- and
ILT2/LIR1+ KIR2DL4+ T cell clones were
incubated with 1 µg/ml soluble anti-CD3 in the presence of 2
µg/ml protein G for 6 h and analyzed for binding of annexin V by
flow cytometry.