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Departments of
* Medicine/Rheumatology and Immunology,
Biochemistry and Molecular Biology, and
Biostatistics, Mayo Clinic, Rochester, MN 55905
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain sequences were identified in three patients
and analyzed for their KIR gene expression pattern. Based on sharing of
TCR sequences, the clones were grouped into five clone families. The
repertoire of KIRs was diverse, even within each clone family; however,
the gene expression was not random. Three particular receptors,
KIR2DS2, KIR2DL2, and KIR3DL2, had significant differences in gene
expression frequencies between the clone families. These data suggest
that KIRs are successively acquired after TCR rearrangement, with each
clone family developing a dominant expression pattern. The patterns did
not segregate with the individual from whom the clones were derived,
indicating that peripheral selection in the host environment was not a
major shaping force. Several models were examined using a computer
algorithm that was designed to simulate the expression of KIRs at
various times during T cell proliferation. The computer simulations
favored a model in which KIR gene expression is inducible for a limited
time during the initial stages of clonal
expansion. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- dimers is increased (4).
CD4+CD28null T cells are
functionally distinct from normal CD4 T cells in that they secrete
large amounts of IFN-
, are resistant to apoptosis, and express
perforin and granzyme B, which conveys cytotoxic capability
(5, 6, 7, 8). Additionally, they express a variety of receptors
belonging to the killer Ig-like receptor (KIR) family (9, 10). The regulation of KIR expression on CD4 T cells is unclear. KIR expression on NK cells is dependent on IL-15 and starts after lineage commitment (11). The murine genome does not contain a gene family homologous to the human KIR cluster, but a sequential expression model has been suggested for the functionally similar Ly49 family (12). Under the proper conditions, murine NK1.1+Ly49A+Ly49G2/C/F/I/- NK cells are capable of initiating expression of Ly49F, Ly49G2, and Ly49C/I, as well as maintaining expression of Ly49A. Under similar conditions, NK1.1+Ly49G2+Ly49A/C/F/I/- NK cells were able to induce expression of Ly49C/I, although they were unable to induce expression of either Ly49A or Ly49F (13).
The close resemblance of CD4+CD28null T cells to NK-T cells may suggest that they represent a separate lineage that has been expanded to compensate for thymic dysfunction. NK-T cells are a subset of T cells that express the NK receptor CD161 in addition to the TCR and that are reactive to microbial glycolipids presented by the nonpolymorphic CD1d molecule (14, 15, 16). The TCR repertoire of these cells is highly restricted, typically involving rearrangement of the AV24-AJ18 gene segments. In contrast to classic NK-T cells, CD4+CD28null T cells do not express the conserved TCR that is characteristic of CD1d-restricted NK-T cells (9, 17). In the alternative model, expression of KIR molecules may be acquired by normal T cells during replicative senescence. Indeed, all CD4+CD28null T cells express the memory phenotype, and TCR studies in RA have shown that this population of T cells is dominated by relatively few clonally expanded T cells (17).
The principle focuses of the current study were to understand how the
KIR repertoire of the
CD4+CD28null T cell
populations in RA is formed and whether KIR expression occurs early in
T cell development or during peripheral clonal expansion. To address
these, we obtained multiple members of CD4 clone families sharing
identical TCR -chain sequences, determined the diversity of KIR gene
expression, and compared the results with different models of KIR
acquisition.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMCs from patients with RA were isolated using Ficoll-Hypaque
(Amersham Pharmacia Biotech, Arlington Heights, IL) density
centrifugation.
CD4+CD28null T cell
frequency was determined by flow cytometry using FITC-conjugated
anti-CD4 and PE-conjugated anti-CD28 mAb (BD Biosciences, San
Jose, CA). The protocol was approved by the Mayo Clinic Institutional
Review Board (Rochester, MN) and all people gave written, informed
consent. Three patients who displayed expanded
CD4+CD28null T cell
populations were selected. TCR--chain variable region (BV)
gene segment usage in the
CD4+CD28+ and
CD4+CD28null populations
was analyzed by three-color flow cytometry using FITC-conjugated
BV-specific mAbs (anti-BV1, anti-BV2, anti-BV3, anti-BV11, anti-BV13S1,
anti-BV13S6, anti-BV14, anti-BV16, anti-BV17, anti-BV18, anti-BV20,
anti-BV21S3, anti-BV22 (Beckman Coulter, Miami, FL), anti-BV5S1,
anti-BV5S2/3, anti-BV6S7, anti-BV8, anti-BV12 (Endogen, Cambridge,
MA), and anti-BV7 (kindly provided by A. W. Boylston,
St. James’ University Hospital, Leeds, U.K.)), PE-conjugated
anti-CD28 mAb, and PerCP-conjugated anti-CD4 mAb (BD
Biosciences). TCR-BV elements that were overrepresented in the
CD4+CD28null population
were identified.
HLA-C and KIR genotyping
Genomic DNA was isolated using the DNA Isolation Kit for Mammalian Blood (Roche Molecular Biochemicals/Boehringer Mannheim, Indianapolis, IN). KIR genotypes were analyzed by PCR using KIR-specific primers (18). HLA-C alleles were analyzed by PCR using the Dynal HLA-C "Low Resolution" SSP kit (Dynal, Lake Success, NY) according to the manufacturer’s instructions.
Cloning of CD4+CD28null T cells
CD4+CD28null T cells
were sorted from PBMCs by FACS using FITC-conjugated anti-CD4 and
PE-conjugated anti-CD28 mAb (BD Biosciences). cDNA was amplified
with the appropriate TCR-BV/-chain constant region-specific
primer pairs corresponding to the overrepresented populations
previously determined by flow cytometry. Amplified products were
radiolabeled and separated on 5% polyacrylamide gels. Dominant bands
were eluted and directly sequenced (4). In parallel, the
sorted cells were cloned using limiting dilution. The clones were
maintained on 1.5 x 105/ml irradiated
EBV-transformed lymphoblastoid cells (treated with galactose
oxidase/neuraminidase to enhance cellular interactions)
(19), 25 ng/ml anti-CD3 mAb (Orthoclone OKT-3; Ortho
Diagnostics, Raritan, NJ), and 50 U/ml recombinant human IL-2
(Proleukin; Chiron, Emeryville, CA).
TCR -chain sequence analysis and KIR phenotyping
CD4+CD28null T cell
clones were harvested and total RNA was extracted using TRIzol reagent
(Invitrogen, Carlsbad, CA). cDNA was synthesized using an oligo(dT)
primer. Clones were screened for expression of in vivo expanded TCR
sequences by PCR amplification with the appropriate BV and -chain
constant region gene segment primers and TCR sequencing. The clones
from each individual were grouped into clone families based on the
expression of identical TCR -chains. The KIR repertoire for each
clone was analyzed by RT-PCR using KIR-specific primers described by
Uhrberg et al. (18). PBMCs from other individuals
expressing the appropriate KIRs were used as the positive controls.
Cytokine stimulation
Resting CD4+CD28null T cell clones were stimulated with either 50 ng/ml OKT-3, 10 ng/ml IL-12 (R&D Systems, Minneapolis, MN), 50 ng/ml IL-15 (R&D Systems), 50 ng/ml IL-18 (PeproTech, Rocky Hill, NJ), or a combination of 10 ng/ml IL-12 and 50 ng/ml IL-18 over the course of seven days. At the indicated times, 1.0 x 106 cells were harvested for RNA isolation and analyzed for expression of individual KIR transcripts.
Statistical analyses
Descriptive statistics were used to determine whether the five clone families could be distinguished based on the probability of expression of KIR2DL2, KIR2DS2, and KIR3DL2. Due to the small sample size, formal comparisons using all five groups were underpowered. The five clone families were partitioned into two larger groups, and univariate associations with the KIR2DL2, KIR2DS2, and KIR3DL2 were performed using Fisher’s exact test.
Computer modeling
The proliferation of CD4+CD28null T cells was simulated as a stochastic branching process under the following assumptions: 1) simple cell division without cell death; 2) within each generation, there is a probability p, which is the same for all clone families, that a cell gains individual KIR expression; 3) in each generation, the cells are stochastically independent regarding the likelihood of KIR expression; and 4) KIR expression, once acquired, is not lost with further cell divisions.
Under these assumptions, the theoretical, or expected, proportion p of cells in the population with KIR expression after k generations at risk for gaining KIR expression is given by P = 1- (1-p)k. This can be defined as the probability that a cell, drawn at random from the population of cells after the k generation, will demonstrate KIR expression.
Data to estimate P come from several small samples of cells
(see Tables I
and III). From these data, P is estimated as
the ratio of the number of cells for which KIRs are observed divided by
the total number of cells. This is defined as 
. Then,
= 1-(1–p)k,
so 
= 1-
(1-)1/k.
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. If this is smaller than , the KIR
is expressed. We considered essentially five classes of models: 1) the
probability p is a nonzero constant for the first few
generations (2–5, 3–6, and 2–6 for models 1, 2, and 3,
respectively), and then is zero for all other generations; 2) the
probability p is initially zero and then becomes a nonzero
constant in the last several generations (20–25, 23–27, and 18–28
for models 4, 5, and 6, respectively); 3) the probability p
is a constant number for all generations up to 25 (model 7); 4) the
probability p is a nonzero constant for the first few
generations, with the number of generations during which this is a
valid being different for each individual clone family; the probability
p is then zero for all other generations (model 8); 5) the
probability p is initially zero and then becomes a nonzero
constant in the last several generations (up to 30 generations), with
the number of generations during which this is valid being different
for each individual clone family (model 9). For each of these models, a population corresponding to the last generation was simulated. Then, 20,000 samples of the sizes obtained in the experimental work were drawn from each simulated population. Thus, probability distributions for each model and sample size combination were obtained. These distributions were then used to calculate the likelihood function for each observed data set and appropriate models were compared according to the logs of the corresponding likelihood functions (20).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three patients with RA, who possessed expanded frequencies of
CD4+CD28null T cells (4.9,
29.0, and 34.6% for donors A, B, and C, respectively) were chosen for
this study. To identify dominant clonal populations in the subset of
CD4+CD28null T cells, the
BV gene segment usage in
CD4+CD28+ T cells and
CD4+CD28null T cells were
compared. Results from flow cytometric analysis using 19 BV-specific
mAbs are shown in Fig. 1. In all three
patients, the population of
CD4+CD28+ cells displayed a
diverse repertoire of TCR-BV gene segment usage, with each BV gene
segment being represented in 1–10% of the total
CD4+ population. Compared with the
CD4+CD28+ population, T
cells expressing particular BV gene segments were expanded in the
CD4+CD28null population and
gained dominance. In donor A, T cells expressing BV14 and BV17 were
expanded; donor B showed a dominance of BV13S1 and, to a lesser degree,
BV18; donor C had an increased frequency of
CD4+CD28null T cells
expressing BV18 and BV22. Sequence analysis of TCR -chain gene
sequences of these expanded populations yielded dominant clonal
sequences (Table I
).
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-chain sequence were isolated. The BV17+ T
cell clone, which was also expanded in vivo, was not found. In donor B,
clones with two BV13S1+ TCR sequences were
isolated, while in donor C, both BV18+ and
BV22+ T cell clones were isolated. In memory T
cells, the majority of -chains only associates with a single
-chain, suggesting that the isolated clones which share identical
-chains have arisen from the same progenitor cell (21).
Five to 13 T cell clones from each clone family were available for
study (Table I).
All three patients were characterized for their genomic representation
of KIR genes. Results of the KIR and HLA-C genotyping are shown in
Table II. Donor B lacked KIR2DS1, donor C
was negative for KIR2DS3 and KIR3DS1, and donor A possessed the gene
for each of the different isotypes.
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, A and B. Each clone family was obviously diverse
for the transcription of different KIR isoforms. In the clone family B1
of donor B, a total of 6 of the 11 KIR isoforms tested were found, and
individual clones possessed two to five KIR isoforms in different
combinations. The gene expression in clones from the family C2 of donor
C was more restricted, with only one to three KIR isotypes found.
Again, the repertoire of receptor combinations on the individual T cell
clones was diverse and showed four of a possible eight combinations.
Similar diversity was seen for T cell clones belonging to the remaining
families, the data from which is summarized in Fig. 3. To exclude the possibility that this
diversity reflected biclonal populations, selected T cell clones were
sorted with BV-specific mAbs, recloned, and analyzed. In all of these
experiments, the original KIR transcription pattern was reconfirmed in
the subcloned populations.
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The repertoire analysis of KIR gene expression was done on
established T cell clones; therefore, it was important to consider
whether the observed diversity was generated during in vitro culture.
Several clones were randomly chosen, and the KIR transcription pattern
was analyzed at several time points during culture. At each time point,
the clones displayed identical patterns of gene expression (data not
shown). To analyze whether cytokines or TCR-mediated activation could
regulate the transcription of the KIRs in
CD4+CD28null T cells, a T
cell clone that transcribed mRNA for KIR2DL4, KIR2DL3, and KIR3DL2 was
selected and cultured with selected cytokines. Previous results have
shown that the expression of C-type lectin receptors on NK cells and
CD8 T cells can be regulated in vitro by IL-15 (22). IL-15
also induces KIR expression on progenitor cells during in vitro NK cell
differentiation (11). Moreover, it has been shown that KIR
expression can be modulated by TCR-mediated activation
(23). IL-12 was chosen because preliminary data suggested
that CD4+CD28null T cells
expressed high cell surface levels of the IL-12 receptor and that IL-12
could modify gene expression in
CD4+CD28null T cells
(24). IL-12 is also known to function synergistically with
IL-18 (25, 26). The clone was stimulated with
anti-CD3, IL-12, IL-15, IL-18, or a combination of IL-12 and IL-18
over the course of a week. During the in vitro culture, all clones were
also maintained in IL-2. On days 3 and 7, the cells were harvested, and
the KIR gene expression pattern was analyzed. Representative results
are shown for KIR2DL3, KIR3DL2, KIR2DL1, and KIR3DS1 (Fig. 4). After in vitro culture of this clone
with the various cytokines, as well as anti-CD3 mAb, there was no
change in the gene expression pattern of these particular receptors.
There was no down-regulation of KIR2DL3 and KIR3DL2, which were
previously transcribed by the clone, nor did the cytokines induce
transcription of KIR2DL1 or KIR3DS1. Therefore, these data would
suggest that under in vitro culture conditions, the gene expression
pattern in CD4+CD28null T
cells is not altered, and that the significant degree of diversity in
the gene expression of KIRs is acquired during in vivo clonal
expansion.
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Results in Fig. 2 document the enormous diversification in KIR
gene expression for each clone family. To address the issue of whether
this diversification was random or biased, the KIR expression pattern
within each CD4+CD28null T
cell clone family was compared. These results are summarized in Fig. 3.
It is apparent that certain KIRs are transcribed more frequently than
others. KIR2DL3 and KIR2DL4 are transcribed by a majority of T cell
clones irrespective of the TCR family grouping. In contrast, there are
other receptors, specifically KIR2DL1, KIR3DL1, KIR2DS1, KIR2DS3,
KIR2DS4, and KIR3DS1, that are not frequently transcribed by clones in
any of these TCR families. Therefore, we concentrated on the
transcription pattern of the three remaining receptors, KIR2DL2,
KIR2DS2, and KIR3DL2, which are found with wide variation among T cell
clone families. The frequency distribution of these receptors allowed
for the distinction of three patterns. The association of these
patterns with the different clone families was statistically
significant (Table III). The clone
families A1 and B1 were characterized by frequent transcription of each
of these three receptors. Transcription of these genes was infrequent
or totally absent in the B2 family. In contrast, in the remaining clone
families, C1 and C2, transcription of KIR3DL2 was detected in 
50%
of the clones, while transcription of KIR2DL2 and KIR2DS2 was again
infrequent. Taken together, despite the enormous diversification in KIR
transcription, three KIR isoforms, KIR2DL2, KIR2DS2, and KIR3DL2, can
be used to define a consensus pattern for a particular clone family. It
is of interest to note that these dominant patterns were clone-specific
and not necessarily donor-specific. Although clone families C1 and
C2 (both derived from donor C) had a similar pattern of
transcription, the clone families B1 and B2 from donor B were clearly
distinct. In the clone family B1, KIR3DL2 was most frequently
transcribed (80% of all clones tested), followed by KIR2DL2 (67%),
and KIR2DS2 (67%). In contrast, KIR2DL2 and KIR3DL2 were completely
absent in the clone family B2, and only KIR2DS2 was found at a low
frequency of 20%.
Modeling of KIR gene expression by computer simulation
The diverse repertoire of KIR transcription in each T cell clone
family clearly indicated that KIR transcription occurred subsequent to
TCR expression. It was also obvious from our data that the likelihood
of transcription for each KIR isoform was not identical. Certain KIR
isoforms, such as KIR2DL4, were frequently transcribed, while others,
such as KIR2DL1, KIR3DL1, or KIR2DS1, were only infrequently found in
any of the clone families. In a similar pattern, KIR2DL4 was expressed
by essentially all KIR+ NK cells (27) and CD8
T cells (28). In addition, other receptors, including
KIR3DL1, KIR2DS1, and KIR2DL1, were only infrequently found on
KIR+ NK cells (27) and CD8 T cells
(28). Although minor influences of HLA type cannot be
excluded, peripheral selection does not appear a likely reason for
these irregular expressions because the patients included in this study
expressed different HLA-C variants. The KIR patterns, however, were not
completely random, and each clone family possessed a consensus pattern.
This observation raised the issue of whether KIR expression occurs
during the early stages of T cell expansion and the dominant pattern
represents a founder effect, or whether the clone-specific patterns
could also be explained by a cumulative expression during the late
stages of clonal expansion. Based on the frequencies of
CD4+CD28null T cells
expressing particular BV elements in the peripheral blood, the size of
each clone was on the order of 1% of all T cells. Therefore, the
clonal size could be estimated to be 1 x
109 cells, suggesting that a clone underwent
proliferation through a minimum of 30 generations.
As described in Materials and Methods, a computer model was
developed to simulate the stochastic branching process defined by
repeated cell divisions. We simulated the clonal proliferation of a
single T cell clone over the course of up to 30 cell divisions.
Although previous data suggest that expression of Ly49 receptors on NK
cells occur in a sequential manner (12), our data for KIR
gene expression was more consistent with a stochastic model. Therefore,
at various times during the proliferation, KIR2DS2, KIR2DL2, KIR3DL2,
KIR2DL3, and KIR3DL1 expression was initiated with a given fractional
probability over a defined number of cell divisions (Table IV). We initially based the models on
four basic assumptions. First, the probability of KIR expression was
gene-specific, but identical for the different clone families and
constant for a given number of cell cycles. Second, all clone families
had the same number of generations during which de novo KIR expression
could be induced. Third, KIR expression was not lost after it had been
expressed. Fourth, selective forces that may favor or disfavor the
proliferation of KIR-expressing cells were ignored. Several models were
tested using these assumptions (models 1–7, Table IV). In models 1, 2,
and 3, KIR expression occurred only in the early stages of clonal
expansion, between the second and fifth, third and sixth, or second and
sixth cell divisions, respectively. In contrast, models 4, 5, and 6
induced KIR expression at the later stages of proliferation, between
generations 20 and 25, 23 and 27, or 18 and 27, respectively. In model
7, KIR expression occurred over the entire course of clonal expansion,
from generations 1–25. Based on the simulations, a probability was
calculated that reflected the ability of each model to generate KIR
expression patterns identical to those found in the experimental data.
The models were then compared using a likelihood ratio test. Results of
one representative comparison (model 3 vs 5) is given in Table V. Likelihood ratios that are
significantly >1 (or log likelihood ratios >0) indicate that the
model being analyzed is less likely to approximate the experimental
data than the model with which it is being compared. The simulations
suggested that the model in which KIR expression occurred during the
first several generations of clonal expansion was significantly more
likely to result in the experimentally observed expression patterns of
KIR2DL2 and KIR3DL1. A similar trend was seen for KIR2DS2. For KIR3DL2,
both models described the experimental results equally well. KIR2DL2,
KIR2DS2, and KIR3DL2 allowed for the distinction of the different clone
families (Table III). In contrast, the simulations favored the late
expression model for KIR2DL3, a receptor that was equally expressed in
all clone families.
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). In model 9, KIR
expression was induced during the 18th generation for each clone
family, and the algorithm then proceeded for varying numbers of
generations. To optimize the fit of the model with the experimental
data, it was assumed that clone families with a broader expression
pattern had a longer replicative history. One possible implication of
this model is that cells continue to acquire expression of KIRs until
sufficient affinity for self-MHC molecules is attained. In contrast,
model 8 allowed for a variable number of cycles during the initial
rounds of cell division (Table IV). The probabilities for these models
were determined, as with the previous models. By varying the numbers of
generations, the likelihood ratios comparing model 9 with 8 for the
different KIR receptors were calculated (Table V). In these
simulations, the probabilities were higher than in models 1–7,
reflecting that the number of generations was optimized to fit the
experimental data. The results of the early and late expression models
were no longer strikingly different for most receptors. However, even
under these conditions, the early expression model, model 8, was
significantly better at explaining the KIR2DL2 expression pattern
(Table V). In summary, the results from the simulations were consistent
with a stochastic, cumulative expression of KIR genes during clonal
replication. The distinct clone family-specific patterns of KIR
expression may indicate a founder effect during early replication
and/or differences in the number of cell cycles during which the cells
could be induced to express KIR genes. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and
-chains and do not show any TCR preference, a characteristic for
CD1d-restricted NK-T cells (17). Moreover, typical
CD4+ NK-T cells may express several NK
cell-related receptors, such as CD161, but generally not KIRs
(15). This suggests that
CD4+CD28null T cells are
distinct from the CD1d-restricted NK-T cell.
CD4+CD28null T cells
express markers of previous activation, including CD45RO, CD44, and
CD57 (4). Therefore, it could be argued that the
expression of KIRs represents a late developmental stage in T cells
undergoing chronic stimulation, as has been argued for the subset of
CD8+KIR+ T cells
(31). In vitro conditions that induce KIR expression have
not been found, neither for CD8 nor CD4 T cells. Our studies provide
clear evidence that KIR transcription in
CD4+CD28null T cells occurs
after termination of TCR rearrangement. T cell clones expressing
identical TCR -chain transcripts showed a diverse repertoire of KIR
expression. These results parallel previously published data on CD8 T
cells that share identical TCR - and -chains and also express
varying repertoires of KIRs (32). In vitro culture of
CD4+CD28null T cells was
not associated with further diversification, suggesting that KIR
transcription occurred during clonal expansion in vivo. Surprisingly,
transcription of particular KIRs in individual clone families,
including KIR2DL2, KIR2DS2, and KIR3DL2 was not random, and each family
possessed a preferred family-specific pattern. Based on computer
simulations, such patterns can emerge if KIR expression is limited to
the first generation cycles of an expanding T cell clone family. Such a
founder effect would indicate that commitment for KIR expression occurs
at an early, and not a late, developmental stage of selected CD4 T
cells. Alternatively, such patterns may emerge if clone families differ
largely in the number of generation cycles during which induction of
KIR expression is possible. The association of CD4+CD28null T cells with certain disease states has given rise to the hypothesis that these cells directly contribute to disease manifestations. In RA, frequencies of CD4+CD28null T cells correlate with extraarticular manifestations. Patients with nodular RA, and in particular patients with vasculitic complications of RA, have grossly expanded CD4+CD28null T cells (29). Also, in patients with early RA, the frequency of CD4+CD28null T cells is a prognostic marker that correlates with the risk of subsequent joint erosions (J. J. Goronzy and C. M. Weyand, unpublished observations). In patients with coronary artery disease, the frequency of CD4+CD28null T cells correlated with the risk of having acute coronary syndromes (30). Acute coronary syndromes develop if the atherosclerotic plaque is inflamed and develops a fissure or ulceration with subsequent thrombosis. Indeed, clonal expansion of CD4+CD28null T cells was found in the inflamed plaque of such patients (33). These findings raised the question of whether the involvement of these cells in tissue injury in certain diseases reflects unique regulatory or effector functions.
Gene expression profiling showed that these cells, in contrast to
normal CD4+ T cells, express perforin and
granzyme B (6).
CD4+CD28null T cells
isolated from patients with coronary artery disease exhibited cytotoxic
activity toward endothelial cells in a perforin-dependent manner
(34). In addition to their cytotoxic abilities, they
express a set of regulatory molecules that are usually found on NK
cells. CD4+CD28null T cells
express CD8 -chain dimers and not the CD8 -chain, reminiscent of
T cells that have undergone extrathymic T cell maturation
(4). In addition, MHC class I-recognizing receptors of the
KIR family are frequently found, particularly those recognized by the
mAb GL183 (9). KIRs comprise a multigenic, multifunctional
family and, depending upon their cytoplasmic domains, are subdivided
into inhibitory and activating isoforms (18). GL183
recognizes the inhibitory isoforms, KIR2DL2 and KIR2DL3, and the
stimulatory isoform, KIR2DS2. Analysis of KIR isoforms in CD4 T cell
clones from patients with RA demonstrated a preferential expression of
the stimulatory receptor KIR2DS2, often in the absence of the
inhibitory KIR2DL2 and KIR2DL3 isoforms. This prompted the hypothesis
that a preponderance of stimulatory receptors on
CD4+CD28null T cells may
predispose a person to autoimmune manifestations (9).
Indeed, the KIR2DS2 gene was found to be genetic risk factor of
vasculitic manifestations in patients with RA (35).
The mechanism through which KIR expression is regulated is currently unclear. In contrast to CD94/NKG2 gene transcription, KIR expression cannot be induced on mature T cells. In our experiments, KIR transcription was stable in vitro and could not be changed by the addition of cytokines that are known to influence NKG2 expression or have been shown to be important for KIR and NKG2 expression during NK cell maturation (11, 36).
In the absence of an experimental system that allows for the induction
of KIR expression on T cells, it remains debatable at which stage of
development KIR expression is induced. The multitude of different
markers on CD4+CD28null T
cells that are usually not expressed on normal CD4 T cells may favor
the interpretation that
CD4+CD28null T cells are a
special lineage and that KIR expression may occur early during
development. However, the loss of CD28 expression, which is one of the
hallmarks of CD4+CD28null T
cells, has been shown to occur with replicative senescence
(37). The transcriptional mechanism that leads to the
down-regulation of CD28 during replication is identical to the
transcriptional defect in
CD4+CD28null T cells,
raising the possibility that KIR expression is part of a senescence
program. Accordingly, it has been postulated for CD8 T cells that KIR
expression is a late event during clinical expansion. Vely et al.
(32) and Uhrberg et al. (28) isolated CD8 T
cell clone families with identical TCR-/TCR-
rearrangement. KIR expression typing in one clone family allowed for
the definition of 18 different KIR phenotypes, clearly demonstrating
that KIR expression was initiated subsequent to TCR rearrangement
(32). Vely et al. (32) sorted
CD158b+CD8+ and
CD158b-CD8+ T cells and
found identical productive and nonproductive TCR-/TCR-
transcripts in both subsets. Young et al. (31) have
provided evidence supporting a sequential program of gene expression
encoded in the human leukocyte receptor complex on chromosome 19q13.4.
In their model, genes belonging to the leukocyte Ig-like receptors were
expressed on CD8 T cells after activation at the stage of effector
cells, while KIR gene transcription was activated in the minor
proportion of activated T cells that survived clonal downsizing and
became long-term memory T cells.
In this study, five different T cell clone families from three different patients with RA were included. In these five clone families, KIR gene transcription was diverse, which is clearly consistent with the model that KIR expression occurred after completion of TCR rearrangement. However, KIR gene transcription was not random. Certain KIR molecules were found more frequently than others. KIR2DL4 was expressed by virtually all T cell clones. Transcripts for KIR2DL3 were also consistently found in all five clone families, although at a lower frequency. The receptors KIR2DL2, KIR2DS2, and KIR3DL2 were variably transcribed in the different clone families. In some clone families, most clones were positive for each of these receptors. This was in contrast to other clone families, in which fewer of the T cell clones transcribed these receptors. The remaining receptors, KIR2DL1, KIR3DL1, KIR2DS1, KIR2DS3, KIR2DS4, and KIR3DS1, were infrequently transcribed. In general, the hierarchy of gene expression was similar to that previously reported for CD8+ T cells (28) and NK cells (27). Further evidence that the expression pattern was not random came from statistical analyses of gene expression in the different clone families. These analyses suggested that at least three different dominant patterns could be distinguished between the five clone families. We considered several models to explain this bias. It is possible that the repertoire of KIR expression was shaped by selective forces, depending on the MHC class I polymorphisms expressed by the original host. We have previously shown that stimulatory KIRs were able to promote proliferative responses of T cell clones, suggesting that the recognition of self-MHC class I molecules may lead to clonal expansion (9). Ugolini et al. (38) have used a KIR2DL3-HLACw3 transgenic mouse system and have found that KIR2DL3 is involved in T cell survival. In contrast, several studies have not found any evidence that KIR cell surface expression is directly affected by the nature or expression levels of HLA class I molecules (11, 39). In the study described here, two T cell clone families that showed a clear difference in KIR expression patterns were derived from the same patient, suggesting that peripheral selection is not a dominant force imposing KIR expression patterns.
Alternatively, the dominant pattern may reflect a founder effect and de novo KIR expression may be limited to early T cell expansion. We simulated KIR expression during early or late stages of clonal expansion for varying numbers of cell cycles. There was no indication in our experimental data that expression occurs in an ordered, sequential process. Therefore, the models were built on the assumption that KIR expression occurs as a stochastic process with a given probability for each KIR gene. The simulations indicated that the experimental data were consistent with a model in which KIR expression occurred after TCR rearrangement but during the early generation cycles of T cell clones undergoing expansion. If this model is correct, CD4+CD28null T cells would likely be a separate lineage that acquires KIRs during early clonal expansion after Ag encounter. Alternatively, clone families may have grossly differed in the number of generation cycles during which they were amenable to KIR expression. In these simulations, we could not clearly distinguish between models of early and late gene expression to fit the experimental data.
In summary, our data demonstrate that
CD4+CD28null T cells
acquire a diverse repertoire of KIR expression. Diversification within
different clone families sharing the same TCR -chain is consistent
with a model of accumulative KIR expression after completion of TCR
rearrangement. The profile of KIR expression on individual T cell clone
families is not random. If KIR expression patterns reflect a founder
effect, then stochastic events in KIR expression during the first
generations of T cell clonal expansion may have significant downstream
effects and determine the behavior of this clone during pathological
immune responses.
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Footnotes |
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2 Address reprint requests and correspondence to Dr. Jörg J. Goronzy, Department of Medicine and Immunology, Mayo Clinic and Foundation, 401 Guggenheim Building, 200 First Street Southwest, Rochester, MN 55905. E-mail address: goronzy.jorg{at}mayo.edu
3 Abbreviations used in this paper: RA, rheumatoid arthritis; BV, -chain variable region; KIR, killer Ig-like receptor.
Received for publication December 5, 2001. Accepted for publication February 13, 2002.
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T cell receptor diversity. Science 286:958. TCR rearrangement express highly diverse killer cell Ig-like receptor patterns. J. Immunol. 166:3923.This article has been cited by other articles:
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) and the
CD4+CD28null (
) T cell populations.
Several BV gene segments were found to be overrepresented on the
CD4+CD28null T cells. TCR 
