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TCR Rearrangement Express Highly Diverse Killer Cell Ig-Like Receptor Patterns1
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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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T cells
less frequently expressed the inhibitory receptors CD94:NKG2A and
KIR2DL1 than either 
T cells or NK cells. In contrast to NK
cells, not all NKR+ T cells expressed an inhibitory
receptor for autologous HLA class I. This lack of specific inhibitory
NKR was especially apparent on T cells of one donor. Overall,
T cells exhibited a distinct pattern of NKR expression different
from that of T and NK cells, which expressed highly similar NKR
repertoires. In one donor, analysis of TCR rearrangement revealed a
dominant subset of NKR+ T cells sharing identical TCR -
and -chains. Remarkably, among 55 T cell clones sharing the same
TCR rearrangement 18 different KIR phenotypes were seen,
suggesting that KIR expression was initiated subsequently to TCR
rearrangement. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although first characterized on NK cells, the KIR and CD94:NKG2
families are also found on subpopulations of peripheral blood T
cells (21, 22, 23), T cells (24, 25), and
T cells in the liver (26). Both CD4 and CD8 T cells
can express NKR, although the latter are much more common
(27). The NKR expressed by T cells have been shown to be
functional; stimulatory signals coming from the TCR can be overridden
by inhibitory signals generated through a KIR or CD94:NKG2A receptor,
thereby inhibiting target cell cytolysis and cytokine release
(22, 23, 24, 27, 28, 29, 30, 31, 32, 33). A common feature of
NKR+ T cells is a cell surface phenotype that is
characteristic of memory T cells; they lack CD28 and CD45RA, mostly
express CD45RO, and have high levels of CD18, CD44, CD29, and CD57
(28, 34). This has suggested that induction of NKR
expression on T cells occurs only after Ag stimulation.
The overall question to be addressed here is how the repertoire of expression of the different NKR in T cells compares with that which has been described for NK cells. Previous investigations have shown that individual NK cells express a variable number of different NKR (35, 36, 37), which can be as many as nine (38). Through expression of different combinations of NKR, a substantial diversity of NKR phenotype is present within an individual’s NK cell population. This diversity may help NK cell populations respond to cells infected by diverse viruses and other pathogens. A critical factor determining an individual’s repertoire of NKR expression is the cohort of NKR genes they inherit. Whereas the CD94 and NKG2 genes appear conserved within human populations, that is not the situation for the KIR gene family. KIR haplotypes differ in the number of genes they contain, with most variation being in the number (between 1 and 5) of activating receptors (9, 39, 40). Adding to the variation is polymorphism at some of the KIR genes (41). The consequence of such genetic differences is that the repertoire of NKR expression by NK cells varies between individual donors. A second factor that influences NKR expression by NK cells is the apparent requirement for each NK cell to be inhibitable by healthy cells expressing the autologous HLA class I type. This requirement for self tolerance is met by each NK cell expressing one or more inhibitory NKR with specificity for one or more autologous HLA class I allotypes (38).
Analysis of the cell surface phenotype using Abs specific for different
types of NKR has shown that T cells, like NK cells, can express more
than one type of NKR (21, 23). Another form of analysis
has been to examine the heterogeneity of TCRs within cultured
lines of T cells that were selected for expression of a particular KIR.
Mingari et al. (34) found such lines to be restricted to
particular V families. A similar restriction was seen in the
NKR+ T cell populations in the peripheral blood.
However, different V gene families dominated the T cells that were
derived from different donors but selected for expression of the same
KIR. In contrast to the results of Mingari et al., Andrea and Lanier
(27) did not find as consistent a picture when they
compared V usage in peripheral blood T cells that did or did not
express KIR3DL1. Although in some donors they found a dominance of
certain V families in KIR3DL1+ T cells, in
others the differences were more subtle, and in no donor was there
evidence for monoclonality or oligoclonality of the KIR3DL1-expressing
T cells.
A factor limiting previous studies to assess NKR expression by T cells was the reliance on Abs for detection and discrimination of NKR. The available Abs detect only a subset of KIR; some are cross-reactive with different types of KIR, and others have polymorphic specificity, so that only the allotypes expressed by certain donors are detected. A second limiting factor was incomplete knowledge of the KIR genotypes of the donors studied. This hinders assessment of whether the observed differences between donors were a consequence of KIR genetics or of functional interactions, either with the products of other host genes or with environmental factors such as pathogens. To address these issues we have taken the following two approaches: first, to focus analysis on T cells from blood donors who have been well characterized for KIR type, HLA type, and NK cell repertoire (38); and second, to supplement Ab-based assessment of NKR expression by T cells with RT-PCR typing, which can be both more specific and more comprehensive (39). Using this overall strategy we have studied the repertoire of NKR expression by T cell clones and peripheral blood T cells from two donors with distinctive KIR genotypes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three-color flow cytometry was performed on PBMC, stained with
FITC-coupled anti-CD16 mAb, Cy-Chrome-labeled anti-CD3 mAb
(both from Becton Dickinson, Mountain View, CA), and a mixture of
PE-coupled NKR-specific mAbs consisting of the anti-KIR reagents
EB6 (anti-KIR2DL1, anti-KIR2DS1; Coulter, Hialeah, FL), DX27
(anti-KIR2DL2/3, anti-KIR2DS2), DX9 (anti-KIR3DL1), DX31
(anti-KIR3DL2), and the CD94-specific mAb DX22. No specific Abs
were available for KIR2DL4, KIR2DS3–5, and KIR3DS1. Expression of the
CD94:NKG2A heterodimer was determined by calculating the percentage of
cells that stained brightly with the DX22 Ab. T cell clones and PBMC
were analyzed by flow cytometry using FITC-coupled mAbs specific for
the TCRV families 3, 5, 6, 8, 11, 12, 13, 14, 16, 17, 20, 21, and 22
(Coulter). mAbs specific for the T cell markers CD3, CD4, CD8,
TCR, and TCR; the NK cell markers CD16 and CD56; the T cell
memory markers CD45RO and CD57; as well as CD45RA Ag were used in
different fluorochrome combinations (Becton Dickinson). Cells (2
x 105) of each T cell clone and PBMC (1 x
106) were incubated for 45 min with the
appropriate Abs, washed, and analyzed with a FACScan flow cytometer
using CellQuest software (Becton Dickinson).
T cell cloning
PBMC were isolated from whole blood by Ficoll-Hypaque gradient separation. CD3+CD16-NKR+ T cells as well as CD3+CD16-NKR- control T cells were single-cell sorted using a FACStar cell sorter and cyt-clone software and hardware (Becton Dickinson). Sorted T cells were established in culture and maintained as previously described (42). Briefly, T cell clones were cultured in IMDM (Life Technologies, Gaithersburg, MD) containing 200 U/ml rIL-2 (provided by C. Reynolds, National Cancer Institute/Biological Response Modifier Program, Frederick, MD) and 0.1 µg/ml of PHA. Mixed allogeneic PBMC (1 x 106/ml; three donors) and cells of the JY B cell line (1 x 105/ml) 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 provide a control for background proliferation.
RNA preparation and RT
Total cellular RNA was extracted from 1 x 106 cultured T cells with feeders or with feeder cells alone using RNAzol according to manufacturer’s instructions (Tel-Test, Friendswood, TX). First-strand cDNA was synthesized from 1 µg of RNA by RT using oligo(dT) (Perkin-Elmer, Norwalk, CT) and Moloney murine leukemia virus reverse transcriptase (Life Technologies) in a volume of 25 µl at 42°C for 1 h.
NKR typing
PCR analysis of KIR and NKG2A expression was performed as
described previously (39). In brief, a panel of PCR primer
pairs was used to perform specific amplification of six groups of
inhibitory KIR (KIR2DL1–4 and KIR3DL1–2) and five groups of
noninhibitory KIR (KIR2DS1–4 and KIR3DS1) as well as the NKG2A gene.
Amplifications were performed with NKR-specific primers at a
concentration of 0.5 µM in 25-µl reactions for 30 cycles using 1
µl of cDNA. Internal control primers specific for -actin were
included in each PCR at a concentration of 0.05 µM. Feeders-only
cultures were used as controls for RT-PCR that monitored the presence
of residual NKR expression from irradiated feeder cells.
Typing for TCR V and V families
TCRV family-specific PCR was performed as described
previously (43). For each of 24 V families a specific
sense-primer and a common antisense primer matching the TCR constant
region were used. Similarly, V family-specific PCR was performed as
previously described (44) using sense primers for the
specific amplification of 27 V families in combination with a common
C-specific antisense primer. T cell clones were amplified for 30
cycles, and PBMC were amplified for 35 cycles using 1 µl of cDNA/25
µl reaction.
Nucleotide sequencing of TCR rearrangements
TCR and TCR rearrangements in T cell clones were
characterized by direct sequencing of template 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). Subsequently, products were cycle-sequenced employing
dye-labeled deoxynucleotide terminators and a 373A automated sequencer
(Applied Biosystems, Foster City, CA). The nucleotide sequences of TCR
- and -chain rearrangements were determined in both directions
using as sequencing primers the family-specific and constant
region-specific primers used for amplification.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and T cells express a
diverse repertoire of NKR combinations
T cell clones from two healthy donors were cultured from PBMC that
had been isolated by single-cell sorting using CD3-specific mAbs in
combination with a cocktail of KIR-specific and CD94:NKG2A-specific
mAbs. Eighty-two NKR+ T cell clones from donor NV
and 71 clones from donor PP were obtained. As controls, 20 T cell
clones expressing no NKR were also established from
NKR- T cells of each donor using the same
protocol. Flow cytometric analysis showed that 68 (83%) of the
NKR+ T cell clones from donor NV expressed
T cell receptors, and 14 (17%) expressed TCRs. In contrast, 30
(42%) of the NKR+ T cell clones from donor PP
expressed TCRs, and 41 (58%) expressed TCRs. The numbers
of vs T cell clones obtained from each donor reflected
the relative abundance of and cells in the
NKR+ T cell population in their peripheral blood
as determined by flow cytometry. For donor NV the
NKR+ population of peripheral blood T cells
comprised 76% T cells and 24% T cells; for donor PP it
was 45% cells and 55% T cells. For both donors, the
T cell clones obtained were all
CD4-CD8+, while the
T cell clones were mostly
CD4-CD8-, with a few
being CD4-CD8+. All
NKR+ T cell clones from both donors had the T
cell memory phenotype,
CD28-CD45RA-CD45RO+.
RNA was isolated from each T cell clone and typed for KIR and NKG2A by
RT-PCR-based methods we described previously (39). Clones
derived from NKR+ T cells expressed NKR, whereas
those derived from NKR- T cells did not,
indicating that the culture conditions we used did not induce NKR
expression in T cells. The expression of NKR by T cells (Fig. 1
, A and B) was
compared with that previously determined for NK cell clones obtained
from the same two donors (38). For each donor all of his
KIR genes are used by T cells as well as NK cells. None of the KIR was
restricted to one or the other cell type. As for NK cells, the
differential expression of KIR and CD94:NKG2A on T cells leads to a
diverse repertoire of 31 and 24 different phenotypes in the 82 and 71
clones studied from donors NV and PP, respectively. The frequency of
expression for most KIR genes was similar on NKR+
T cells and NK cells. The KIR2DL4 gene was expressed on every
NKR+ T cell and NK cell. The HLA-Bw4-specific
KIR3DL1 was infrequently expressed on the NK and
NKR+ T cells of donor NV, but was abundant on
both kinds of lymphocytes from PP. The five noninhibitory KIR of donor
NV (missing tyrosine-based inhibitory motifs in the cytoplasmic chain)
were expressed at comparable frequencies on NK cells and
NKR+ T cells, as was the only noninhibitory KIR
of donor PP, KIR2DS4.
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The expression of NKR by the and subsets of
NKR+ T cell clones obtained from the same
individual showed specific differences (Fig. 1
, C and
D). Particularly impressive was the high frequency of
expression of NKG2A by T cell clones compared with T cell
clones; this difference was seen for both donors, but was more
pronounced in NV. In expression of CD94:NKG2A, the T cells
resemble NK cells, and it is the T cells that are different.
Similarly, the higher frequency of KIR2DL2 expression by NV’s T cell
clones and that of KIR3DL1 by PP’s T cell clones are due to T
cells, while the expression of these receptors by +
cells resembles that of each individual’s NK cell clones. By contrast,
the low frequency of KIR2DL1 expression by T cell clones from both
donors is a feature that distinguishes both and T cell
clones from NK cell clones.
NKR+ T cell clones from NV and PP expressed
multiple NKR (two to eight receptors per cell for NV and two to seven
receptors per cell for PP). For both donors a majority of the T cell
clones expressed either three or four NKR (Fig. 2, A and B). In
comparison with the autologous NK cell clones, the T cell clones
expressed slightly fewer NKR, on the average. This can be seen in the
reduction by one of the range in receptor number and in the reduction
of the mean number of receptors expressed per cell (3.9 for NV’s T
cell clones compared with 4.2 for his NK cell clones, 3.7 for PP’s T
cell clones compared with 4.5 for his NK cell clones). These
differences between NKR+ T cells and NK cells are
principally due to differences in the expression of inhibitory NKR
(Fig. 2, C and D) rather than noninhibitory NKR
(Fig. 2, E and F). The two donors differ in their
KIR types, with NV having genes for several noninhibitory KIR that are
not possessed by PP. This disparity results in NV’s
NKR+ T cells expressing up to five noninhibitory
receptors, whereas PP’s cells express only one (Fig. 2, E
and F).
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). For donor NV, every
NKR+ T cell clone expressed an inhibitory
receptor specific for an autologous HLA class I allotype. By contrast,
this criterion was met by only 41 of the 71 NKR+
T cell clones from donor PP. Of the 30 clones that did not express a
receptor for autologous HLA class I, 24 were T cells, and six
were cells. Whereas 80% of PP’s T cell clones had an
inhibitory receptor, this was only true for 27% of the T cell
clones. This difference between the two types of T cell clone can
largely be attributed to the more frequent expression of CD94:NKG2A
(Fig. 1, C and D). Similar differences in
CD94:NKG2A expression between peripheral blood and cells
were seen using flow cytometry; CD94:NKG2A was expressed by 25% (NV)
and 17% (PP) of NKR+ T cells, and by 58%
(NV) and 62% (PP) of NKR+ T cells (data
not shown).
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T cells with identical TCR can
be highly diversified for KIR expression
To examine the clonal diversity of NKR+ T
cells, their expression of TCR V segments was analyzed by
family-specific PCR. Each of the
NKR++ T cell clones
from donor NV expressed the V16 gene segment. None of the control
NKR- T cells expressed V16; instead, they
used various other V segments (data not shown).
NKR+ T cell clones from donor PP expressed
one of three different V families: V11 (eight clones), V14
(eight clones), and V21 (six clones). Cell surface expression of the
V-chains assigned by PCR typing was confirmed by flow cytometric
analysis of the T cell clones using V family-specific mAbs (data not
shown).
To determine the extent to which the restricted use of V segments
found in the NKR+ T cell clones reflects
the in vivo situation, PBMC from the two donors were analyzed by flow
cytometry (Fig. 4A). For donor NV,
35% of NKR+ T cells expressed V16 (Fig. 4D), and 85% of the peripheral T cells expressing V16
were NKR+. V16+ T cells
were also predominantly of CD8+ type (Fig. 4C). The subset of peripheral T cells expressing both V16
and NKR (Fig. 4D) was estimated to comprise approximately
4% of NVs PBMC. No other V family was found on peripheral blood
NKR+ T cells of donor NV at such high frequency,
although other V families were clearly represented. Flow cytometry
was also used to assess the expression of different KIR within the
subset of peripheral blood
NKR+V16+ T cells, and
the results were similar to those obtained by KIR typing of the
cultured V16+ T cell clones by RT-PCR typing
(Fig. 5). Thus, in pattern of NKR and TCR
expression, the panel of NKR+ T cell clones from
donor NV represents the dominant V family expressed by
NKR+ T cells in the peripheral blood.
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families expressed by the NKR+ T cell clones,
only V21 was expressed by substantial numbers (15%) of
NKR+ T cells in the peripheral blood. By
contrast, the V11 and V14 families, which were the two V
families most commonly expressed by PP’s NKR+ T
cell clones, were expressed by relatively few
NKR+ T cells in the peripheral blood (Fig. 4A).
Since NKR++ T cell
clones from donor NV were strongly biased toward usage of the V16
gene segment, we were interested to determine the heterogeneity of
their TCR -chain gene rearrangements. Five different V
rearrangements were represented in the panel of 68 T cell clones
(Fig. 6A). A predominant
rearrangement (II) was common to 55 clones, while four less frequent
rearrangements (IV, V, I, and III) were represented by one, two, three,
and seven clones, respectively. All five gene rearrangements led to
shared characteristic features in the CDR3 region of the TCR
V-chain. Aspartic acid followed by arginine was found in all five
rearrangements at positions 97–98 encoded by the D gene-segment and
its flanking N nucleotides. This motif is determined either by the D1
germline segment (probably rearrangements II, III, and IV) or by N
nucleotide addition (rearrangements I and V). Moreover, three of the
five rearrangements share the same J element, and they differ from each
other by no more than two amino acids in the CDR3 region. In the clones
expressing rearrangement II, the V19 gene segment was also
rearranged and expressed at the mRNA level (data not shown). However,
its nucleotide sequence showed this to be an unproductive
rearrangement, resulting in a stop codon in the CDR3 region, consistent
with the assignment of V19 as a pseudogene (45). Thus,
in clones with rearrangement II, V16 is the only functionally
rearranged TCR-V gene segment.
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clonotype for each
NKR+ T cell clone in the NV panel, their V
gene rearrangements were determined. Among the 66
NKR+ T cell clones having V rearrangement I,
II, III, or IV, different V gene rearrangements were found in
combination with each V rearrangement, and all clones with identical
V rearrangements had identical V rearrangements. Clones with the
type I, II, or III V16 rearrangement expressed two different
-chains. Thus, a total of seven different rearrangements were
expressed by the 66 T cell clones, and these involved five different
V segments and five different J segments (Fig. 6B).
The seven V rearrangements had no shared characteristics,
contrasting with their V counterparts. Even in cases where one V
segment was held in common among cells having the same V
rearrangement, they differed substantially in their V CDR3 region
and were also joined to different J segments. All seven
rearrangements are in-frame and could give rise to full-length
-chains. Not known is whether any of the T cell clones of clonotype
I, II, or III expresses both the -chains at their cell surfaces,
since their simultaneous cell surface expression could not be analyzed
with the Abs available.
Finding that only four different TCR clonotypes were represented
in 66 NKR+ T cell clones provided an
unprecedented opportunity to assess the extent to which NKR expression
distinguished clones of identical clonotype. Typing was performed by
both RT-PCR and flow cytometry, and both techniques gave concordant
results (Fig. 7). Whereas only the single
T cell of clonotype IV was distinguished by expression of
NKG2A:CD94, there were many differences in the expression of KIR (Fig. 8). Most informative was the analysis of
clonotype II that accounted for 55 NKR+ T
cell clones from donor NV. Among these cells with identical TCR were 18
different KIR phenotypes that varied in frequency and
number of KIR expressed. All the clones expressed KIR2DL2 and KIR2DL4,
and diversity was mediated by the differential expression and
combination of seven other KIR. The frequency of expression of these
KIR was in the order KIR3DL2>2DS4>2DS2>3DS1>3DL1>2DL1
2DS1.
KIR-mediated diversification of T cell clones with clonotypes I, II,
and V was also seen (Fig. 8), although to a lesser extent because of
the fewer numbers of clones analyzed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR clonotype, but different KIR
phenotypes. Particularly striking are the 55 clones of clonotype II
that divide into 18 distinct groups on the basis of differential
expression of five KIR genes. These data reveal the considerable
potential for NKR expression to modulate the functional activities of T
cells. We previously showed that mature NK cells express at least one inhibitory KIR or CD94:NKG2A receptor with specificity for self class I (38). This restriction in the NKR repertoire is in accord with the missing self model and ensures self tolerance of the peripheral NK cell compartment (46). A similar restriction was not apparent in our panel of NKR+ T cell clones. Whereas NV’s T cell clones all express an inhibitory receptor for autologous HLA class I determinants, this is only true for some 60% of PP’s clones. This suggests either that there is no requirement for T cells to express an inhibitory NKR for autologous class I or that additional receptors that we have not considered here are fulfilling that role.
ILT2, also called LIR1, is another type of inhibitory receptor with Ig-like domains that is distinct from KIR (19, 20). ILT2/LIR1 is specific for the viral homologue UL18, but exhibits also weak binding to relatively nonpolymorphic sites on HLA class I molecules (47). In peripheral blood of both donors NV and PP, expression of ILT2/LIR1 and KIR was found on distinct but overlapping T cell subsets. Similarly, ILT2/LIR1 expression was found on a substantial fraction of NKR+ T cell clones by flow cytometry. However, in studies with a soluble LIR1 fusion protein, LIR1 was found to bind to one of NV’s HLA-B allotypes, but none of PP’s class I allotypes (data not shown). It therefore seems unlikely that those NKR+ T cells in PP that lack KIR and CD94:NKG2A receptors for autologous class I allotypes are functionally inhibited by ILT2/LIR1. On the other hand, the existence of mature NKR+ T cells without self-specific inhibitory NKR should not pose a principal problem to the host. In contrast to NK cells that rely on engagement of their inhibitory receptors by self class I epitopes to be self tolerant, T cells are already selected for self tolerance by TCR-driven processes in the thymus.
Although the differences in NKR expression by the two donors are many,
within each individual the expression of NKR by NK cell clones and that
by T cell clones from each individual are largely similar, but with a
few potentially important differences. The biggest difference is the
reduced frequency with which NKR+ T cells express
CD94:NKG2A compared with NK cells, which approaches zero for
NKR+ T cells. It is this difference that
largely accounts for the absence of an inhibitory receptor for
autologous HLA class I on 40% of PP’s NKR+ T
cells. Also common to both donors was a reduced frequency of KIR2DL1,
the inhibitory receptor for HLA-C allotypes with the N77, K80 amino
acid sequence motif. Although for NV the frequency of KIR2DL2
expression by T cells was higher than that for either NK cells
or T cells, this may not be a general property because of the
limited number of T cell clonotypes represented in the clones and their
ubiquitous expression of KIR2DL2. Overall, the similarities in
frequency with which KIR are expressed by NK cells and
NKR+ T cells indicate that the mechanisms that
diversify the expression of KIR in populations of these cells are very
similar.
Much of the analysis we report was made upon NKR+ T cell clones that were stimulated and cultured in vitro, and in this regard T cells expressing NKRs are more difficult to isolate than NK cells because of their lack of a generic marker. The extent to which the clones reflect the in vivo populations is therefore of importance; to address this question we have, wherever possible, compared the clones with the NKR+ T cells in peripheral blood. Such comparison shows that clones we have isolated and analyzed do not provide complete representation of the peripheral blood NKR+ T cells of either donor. One possible source of bias is that cells expressing certain combinations of NKR were not captured by the Ab cocktail used in isolating NKR+ T cells. This cocktail did not include Abs that bind to either KIR2DL4 or the recently described KIR2DL5 (9, 48), and the weak affinity of the CD94-specific Ab for the CD94:NKG2C heterodimer may have meant that this Ab was inefficient in capturing cells via interaction with CD94:NKG2C. A second potential source of bias is that the in vitro cloning procedure did not equally promote the growth of all cells isolated with the Ab cocktail.
For donor NV, one-third of the peripheral blood
NKR+ T cells and all 68 of the T cell
clones express V16. Clearly, the in vitro culture was strongly
biased toward furthering the growth and survival of V16-expressing
cells; however, this selection involved the dominant V family of the
peripheral blood NKR+ T cells, and the data
collected from these cells are therefore likely to have physiological
relevance to this particular donor’s history of immune response. The
dominance of V16 NKR+ T cells of the type seen
in donor NV is a phenomenon that may not have generality, being seen in
just a fraction of the human population, or may even be unique to this
donor. Indeed, the high diversity and polymorphism of KIR genotype and
the low frequency of unrelated individuals with identical KIR genes
provide a genetic context in which individuality in the patterns of NKR
expression by human T cells could actually be the common theme
(39, 40).
In donor NV, five TCR clonotypes account for the 68 clones, and
for all four clonotypes that were represented by more than one clone,
there is diversification due to differential KIR expression. That
different combinations of KIR are expressed by T cells with the same
TCR strongly indicates that KIR expression was turned on in mature T
cells that had already undergone TCR gene rearrangement and thymic
selection. This model is consistent with the memory phenotype of
V16+NKR+ T cells in
peripheral blood. The alternative model, that KIR expression preceded
TCR gene rearrangement, is not consistent with the memory phenotype and
is inherently unlikely because it requires the occurrence of 18
independent, but identical, sets of TCR and gene rearrangements
in immature T cells with different KIR phenotypes.
It is possible that KIR-mediated diversification of T cells with identical clonotype occurred during in vitro culture. It was recently reported that murine CD8 T cells acquire Ly-49 receptors upon in vitro culture with IL-2, IL-4, or IL-15 (49). For several reasons we consider an analogous induction of KIR genes during the cloning procedure unlikely. First none of the control clones cultured from NKR- T cells expressed KIR at any stage during culture. Second stimulation of CMV-specific NKR- T cells with stimulatory cytokines and cognate peptide did not result in acquisition of KIR. Many other deliberate attempts to induce KIR expression in T cells in vitro have failed, although Mingari et al. have reported the induction of CD94:NKG2A (50). Recently, down-regulation of KIR was reported on NKR+ T cell clones that were deprived of specific Ag (51). Since we do not know the Ag specificity of our NKR+ T cell clones, we were unable to perform a similar experiment with our T cell clones. However, analysis of KIR expression levels over time did not reveal any signs of KIR gene down-regulation during culture. Therefore, it is likely that the diverse KIR phenotypes seen on single-cell sorted T cells with identical TCR are not a consequence of in vitro changes, but represent changes in the NKR expression status that occurred in vivo.
The memory phenotype of all NKR+ T cells in
peripheral blood implies that induction of NKR expression occurs only
after T cells have been stimulated by Ag, a model supported by the
absence of KIR on T cells in fetal thymus and cord blood
(27). That all T cell clones from donor NV comprise
five clonotypes using V16 chains with common residues in their CDR3
loops is also evidence for clonal expansion driven by some form of Ag.
V16 was shown to dominate the clonal expansions of
CD8+CD57+ T cells that
occurred in four patients following transplantation of allogeneic bone
marrow from an HLA-identical sibling (52). Steinle et al.
showed that an HLA-B35-specific alloreactive T cell expressed a similar
type of V16 chain (53). Such associations raise the
possibility that T cells bearing such V16-containing receptors could
have specificity for alloantigens. In healthy donors expansions of such
cells might arise as a result of blood transfusion or pregnancy.
Alternatively, some types of superantigen or specific Ag could be
responsible for the expansion of V16-expressing cells, and these
might be associated with infections that either occur or are
reactivated following transplantation. In this context it is important
to note that the culture system we used to generate T cell clones could
have favored the growth of NKR+ T cells with
allospecificity, because the feeder cells were pooled PBMC from
allogeneic donors. Selection by alloantigens could also explain why a
majority of the T cell clones from donor PP expressed V
families that were represented at relatively low frequency in the
NKR+T cell population of the peripheral
blood.
The results of this study demonstrate that individual
NKR+ and T cells express variable
combinations of receptors that collectively have a complexity
approaching that of NK cells. This complexity can also introduce
considerable heterogeneity within the populations of memory-type T
cells that constitute a clone as defined by the TCR. The functional
consequences of differential KIR expression on clones of
NKR+ T cells in the course of an immune response
are largely unknown. Redirected lysis experiments show that both types
of inhibitory NKR, KIR and CD94:NKG2A, are functional in the analyzed T
cell clones (data not shown). Other studies have shown that inhibitory
signaling through NKR can affect TCR-mediated functions, and in the
case of a melanoma-specific response this property has the deleterious
effect of preventing T cell-mediated killing of tumor cells
(31). Very little is known about the role of noninhibitory
NKR on T cells. It was recently shown that the adapter molecule DAP12,
also known as KARAP, is involved in the activation of NK cells through
noninhibitory KIR (54, 55). RT-PCR analyses show that a
fraction of approximately 20% of donor NVs NKR+
T cells expresses DAP12. Differential expression of DAP12 was also seen
within T cells with identical KIR phenotype and TCR (data not shown).
If DAP12 is essential for signal transduction through noninhibitory
KIR, its differential expression would introduce another level of
heterogeneity into clones of NKR+ T cells. In
summary, the present study shows that expression of inhibitory and
noninhibitory KIR in a clone of T cells produces 18 different KIR
phenotypes, each of which involves two to seven different receptors
that individually or in combination have the potential to modify T cell
responses to Ag.
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Footnotes |
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2 Current address: Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich Heine University Medical Center, Dusseldorf, Germany.
3 Current address: Immunobiological Research Institute of Siena Research Center, Chiron S.p.A., Siena, Italy.
4 Current address: Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, U.K.
5 Current address: University of California San Francisco, Department of Microbiology and Immunology, San Francisco, CA 94143.
6 Address correspondence and reprint requests to Dr. Peter Parham, Departments of Structural Biology and Microbiology and Immunology, Stanford University School of Medicine, Sherman Fairchild Building, Stanford, CA 94305.
7 Abbreviations used in this paper: NKR, NK cell receptors; KIR, killer-cell Ig-like receptor; CDR, complementarity-determining region.
Received for publication September 6, 2000. Accepted for publication January 3, 2001.
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) and NK cell clones (
) expressing each receptor are
given. In C (donor NV) and D (donor PP)
the percentage of NKR+ 
), and the percentage of NKR+ 
) are shown. For donor NV, the data are from 82 T cell clones (68
).
