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Genetic Control of Human NK Cell
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Heather G. Shilling, Neil Young, Lisbeth A. Guethlein,
Nathalie W. Cheng, Clair M. Gardiner, Dolly Tyan and Peter
Parham
J Immunol 2002; 169:239-247; ;
doi: 10.4049/jimmunol.169.1.239
http://www.jimmunol.org/content/169/1/239
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References
The Journal of Immunology
Genetic Control of Human NK Cell Repertoire1
Heather G. Shilling,* Neil Young,2* Lisbeth A. Guethlein,* Nathalie W. Cheng,*
Clair M. Gardiner,3* Dolly Tyan,† and Peter Parham4*
Through differential killer cell Ig-like receptor (KIR) and CD94:NKG2 gene expression, human NK cells generate diverse repertoires, each cell having an inhibitory receptor for autologous HLA class I. Using a new method for measuring repertoire difference
that integrates multiple flow cytometry parameters, we found individual repertoire stability, but population variability. Correlating repertoire differences with KIR and HLA genotype for 85 sibling pairs reveals the dominant influence of KIR genotype; HLA
genotype having a subtle, modulating effect on relative KIR expression frequencies. HLA and/or KIR genotype also influences
CD94:NKG2A expression. After HLA-matched stem cell transplantation, KIR repertoires either recapitulated that of the donor
or were generally depressed for KIR expression. Human NK cell repertoires are defined by combinations of variable KIR and HLA
class I genes and conserved CD94:NKG2 genes. The Journal of Immunology, 2002, 169: 239 –247.
*Departments of Structural Biology, and Microbiology and Immunology, Stanford
University School of Medicine, Stanford, CA 94305; and †Department of Pediatrics,
Cedars-Sinai Medical Center, Los Angeles, CA 90048
Received for publication February 19, 2002. Accepted for publication April 19, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This research was supported by National Institutes of Health Grants AI22039,
AI45865, and PO1 CA 49605. H.G.S. and L.A.G. were supported by National Institutes of Health Training Grant AI07290 and C.M.G. by a Leukemia Research Foundation Fellowship. Sample collection at City of Hope was supported by National
Institutes of Health Grant PO1 CA 30206.
2
Current address: Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, U.K. OX3 9DU.
3
Current address: Department of Biochemistry, Trinity College Dublin, Dublin 2,
Ireland.
4
Address correspondence and reprint requests to Dr. Peter Parham, Department of
Structural Biology, Sherman Fairchild Building, 299 Campus Drive West, Stanford
University School of Medicine, Stanford, CA 94305-5126. E-mail address:
[email protected]
complexes of HLA-E with peptides derived from HLA class I
leader sequences (11–13). Contrasting with KIR, genes encoding
CD94 and NKG2 family members are conserved in the human
population (14); phylogenetically they are also more conserved,
being present in mouse and linked to Ly49 genes in the NK complex (15–17). The human NK complex on chromosome 12 contains
genes for CD94 and NKG2 family members, as well as a nonfunctional gene, Ly49L; the only human homolog of the mouse
Ly49 gene family (18 –22).
A general rule is that NK cells cannot kill cells expressing a full
complement of autologous MHC class I allotypes, but can kill cells
expressing some combinations of allogeneic MHC class I (23). In
humans, this tolerance to self has been correlated with the expression of at least one inhibitory KIR or CD94:NKG2A receptor having specificity for self-HLA class I (4). Such observations, derived
from functional assays, provided evidence that HLA class I type
influences the repertoire of inhibitory HLA class I receptors expressed by peripheral blood NK cells. In contrast, population analysis of HLA-B- or -C-specific KIR expression revealed no difference between individuals who did, or did not, express a cognate
HLA class I ligand (24, 25).
Conversely, one study reporting no HLA effect implicated undefined, non-HLA genes as factors affecting NK cell expression of
the HLA-B-specific KIR (KIR3DL1) (24). Subsequent discovery
of KIR population diversity made the KIR locus on chromosome
19 a candidate for the non-HLA genes (7, 26 –31). The investigation described here tested this hypothesis and defined the magnitude of the role of HLA class I in NK cell repertoire selection. In
this analysis, the confounding effects of KIR and HLA genetic diversity were controlled by comparing NK cell receptor repertoires
in sibling pairs of known HLA and KIR types. The role of the KIR
genotype in determining KIR repertoire was further assessed by
following KIR expression after HLA-matched allogeneic and autologous stem cell transplantation. The results demonstrate a role
for both KIR and HLA class I genes in determining human NK cell
repertoires and resolve the seemingly paradoxical results of previous functional and genetic analyses.
Materials and Methods
Healthy sibling donors
5
Abbreviations used in this paper: KIR, killer cell Ig-like receptor; CML, chronic
myelogenous leukemia; MUD, matched unrelated donor; GVHD, graft-vs-host
disease.
Copyright © 2002 by The American Association of Immunologists, Inc.
Peripheral blood samples were obtained from 104 healthy individuals: 53
donors representing 17 families, of which 5 were multigenerational, from
0022-1767/02/$02.00
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N
atural killer cells are lymphocytes of innate immunity
that kill cells and produce cytokines (1). NK cells contribute to early defense against viral infection and have
been implicated in alloreactions following bone marrow transplantation (1–3). NK cell clonal diversity is due to the combinatorial
expression of different cell surface molecules, including several
receptors for polymorphic epitopes of MHC class I, each encoded
by a conventional, nonrearranging gene. NK cells can express receptors for allogeneic as well as autologous MHC molecules, and
class I specificity of individual clones is determined by the array of
receptors present at the cell surface, rather than any individual
element (4, 5). In humans, the family of killer cell Ig-like receptors
(KIR)5 provides inhibitory, and some activating, receptors for
HLA-A, -B, -C, and -G (5). KIR are not present in mice, where
analogous functions are performed by the Ly49 family of lectinlike receptors (6). KIR are encoded by a compact, rapidly evolving
family of genes in the leukocyte receptor complex on human chromosome 19, which exhibits extensive haplotypic variation in gene
number and content, and allelic polymorphism for individual genes
(7–10).
Complementing KIR, the CD94:NKG2 lectin-like molecules
provide inhibitory and activating NK cell receptors specific for
240
Cedars-Sinai Medical Center (Los Angeles, CA); 4 individuals representing 2 families from the City of Hope Histocompatibility Laboratory (Duarte, CA); and from 47 individuals representing 17 families from Stanford
Medical Center Histocompatibility Laboratory (Stanford, CA).
GENETIC CONTROL OF HUMAN NK CELL REPERTOIRE
were: initial denaturation at 95°C for 5 min; 5 cycles of 97°C for 20 s, 62°C
for 45 s, and 72°C for 90 s; followed by 26 –30 cycles of 95°C for 20 s,
60°C for 45 s, and 72°C for 90 s; and a 7-min extension at 72°C.
KIR genotyping
Stem cell patients and donors
KIR nomenclature
KIR2DL1, KIR2DL3, KIR3DL1, and KIR3DL2 alleles were named according to guidelines used in naming HLA alleles. Briefly, an asterisk separates
the accepted gene designations (37) from three digits which distinguish
alleles that differ by nonsynonymous substitutions; fourth and fifth digits
were assigned to alleles that differ only by synonymous substitutions. Numerical order was assigned based on date of submission to GenBank; partial sequences and splice variants were excluded, as were sequences of
single PCR-derived clones.
Genomic DNA and cDNA preparation
Genomic DNA was prepared from 2 ⫻ 106–1 ⫻ 107 PBMC using a
QIAamp Blood kit (Qiagen, Valencia, CA). Total cellular RNA was prepared from 2 ⫻ 106–1 ⫻ 107 PBMC using RNAzol (Tel-Test, Friendswood, TX). First-strand cDNA was synthesized from ⬃5.0 ␮g RNA using
oligo(dT) (PE Applied Biosystems, Foster City, CA) and avian myeloblastosis virus/reverse transcriptase (Promega, Madison, WI) at 42°C for
90 min.
HLA class I determination and KIR epitope-typing
HLA-A and HLA-B Ags, including Bw4 and Bw6, were determined serologically by the laboratories supplying the sample. HLA-C type was
determined serologically or by PCR-sequence-specific primer analysis of
genomic DNA using C locus Sequence-Specific Primer Unitray test kits
(Pel-Freez Biologicals, Rogers, AR).
The presence of the class I HLA-C KIR epitopes was determined by
RT-PCR amplification. In addition, the HLA-Bw4 and Bw6 serological
typing results were confirmed by RT-PCR. The group 1 HLA-C epitope
was detected using specific sense primer (5⬘-CGA GTG AGC CTG CGG
AAC-3⬘) plus an HLA-C locus-specific antisense primer (5⬘-AGG ACA
GCT AGG ACA RCC-3⬘); the group 2 HLA-C epitope was detected with
a specific sense primer (5⬘-CCG AGT GAA CCT GCG GAA A-3⬘) and the
HLA-C generic primer. HLA-Bw4 epitopes were detected with a mixture of
three sense primers (5⬘-CCT GCG CAC CGC GCT CC-3⬘, 5⬘-CCT GCG
GAT CGC GCT CC-3⬘, and 5⬘-CCT GCG GAC CCT GCT CC-3⬘) with an
HLA-B locus-specific antisense primer (5⬘-TCC GAT GAC CAC AAC
TGC T-3⬘). HLA-Bw6 epitopes were detected with a specific sense primer
(5⬘-CCT GCG GAA CCT GCG CG-3⬘) paired with the same generic antisense primer. Primers were used at 0.5 ␮M in 25-␮l reactions using 1–2
␮l cDNA. Internal control primers specific for ␤-actin (sense, 5⬘-CTT
CGA GCA AGA GAT GGC CAC-3⬘; antisense, 5⬘-TTG CTG ATC CAC
ATC TGC TGG AAG-3⬘) were included at 0.05 ␮M. PCR conditions
Typing of genomic DNA for 10 KIR was performed as described by Uhrberg et al. (7), with modification. KIR2DL1v (KIR2DL1*004) was detected
with the KIR2DL1 forward primer and KIR2DS1 reverse primer. Detection
of KIR2DS5 was as modified by Vilches et al. (38).
For KIR subtyping, primers designed to discriminate allele-specific
polymorphisms were paired with KIR2DL1, KIR2DL3, KIR3DL1, or
KIR3DL2 locus-specific primers. KIR3DL1 and KIR3DL2 subtyping were
as described by Gardiner et al. (29) and Shilling et al. (39); KIR2DL1 and
KIR2DL3 subtyping were as described by Shilling et al. (39). To supplement this, KIR2DL1, KIR2DL3, and KIR3DL1 locus-specific PCR products
from some sibling pairs were purified using a QIAquick PCR Purification
kit (Qiagen) and partially sequenced with the original amplification primers
by a dye terminator automated sequencer (Applied Biosystems).
Abs and flow cytometric analysis
mAbs DX27 (anti-KIR2DL2/KIR2DL3/KIR2DS2), DX9 (anti-KIR3DL1),
DX31 (anti-KIR3DL2; generously provided by L. Lanier, Cancer Research
Center, University of California, San Francisco, CA), EB6 (anti-KIR2DL1/
2DS1), and Z199 (anti-NKG2A) (Beckman-Coulter-Immunotech, Brea,
CA) were each used in combination with anti-CD3 (SK7) and anti-CD56
(NCAM16.2; BD Biosciences, Mountain View, CA) in individual threecolor flow cytometry assays of PBMC from each donor. After gating on the
CD3⫺CD56⫹ lymphocyte (i.e., NK cell) subset, KIR- or CD94:NKG2Aexpressing populations were identified using irrelevant, isotype-matched
control Ab stains to set the lower limit of the KIR- or CD94:NKG2Apositive gate, thereby excluding background fluorescence and receptornegative cells. Frequency and median fluorescence intensity of NK cells
binding each specific Ab were calculated for these populations. For 9 of the
104 donors surveyed, CD94:NKG2A expression was determined by gating
on NK cells which stained brightly with a CD94-specific Ab (HP-3D9; BD
Biosciences) instead of Z199. Anti-CD3 and anti-CD56 Abs were labeled
with PerCP and FITC, respectively, anti-KIR, anti-NKG2A, and anti-CD94
Abs were PE conjugated. Flow cytometric analysis was performed on a
FACScan flow cytometer using CellQuest software (BD Biosciences). The
four KIR-specific Abs used here detect all KIR of known specificity,
thereby providing a measure of functional KIR expression. As KIR-specific
Abs bind subsets of KIR, rather than individual allotypes, fluorescence
intensity levels likely encompass various Ab-binding affinities as well as
cell surface expression levels, and therefore reflect physical properties of
KIR repertoire.
Calculation of differences in frequency and median fluorescence
intensity of KIR expression from flow cytometry data
The difference in frequency of KIR expression by NK cells for each KIRspecific Ab stain was calculated according to the formula: (frequency (sample 1) ⫺ frequency (sample 2))/(mean frequency (samples 1 and 2)) ⫽
difference in frequency.
The mean was incorporated as a denominator here to amplify differences
between KIR-negative and KIR-positive samples. The four frequency differences were added to determine the “sum of frequency differences.” Differences in median fluorescence intensity were calculated using the same
formula, then added to give the “sum of median fluorescence intensity
differences.”
Statistical calculations
Correlations coefficients (r) were calculated using the formula r ⫽
Cov(X,Y)/␴X␴Y, where Cov(X,Y) ⫽ ⌺(X ⫺ ␮X)(Y ⫺ ␮Y); significance was
evaluated by two-tailed t test with n ⫺ 2 df.
Results
NK cell KIR repertoire is stable within an individual but varies
between individuals
Human NK cell clones differ in number and combination of KIR
expressed (4, 40). This mode of gene expression generates a diverse repertoire of KIR expression in peripheral blood NK cells.
To assess the stability of these expression patterns, we studied
peripheral blood NK cells of five healthy donors over a period of
about 1 year, analyzing cell surface KIR expression with four
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Eighteen patients receiving allogeneic stem cell transplants at the Stanford
University Medical Center for treatment of chronic myelogenous leukemia
(CML; n ⫽ 12) or acute myelogenous leukemia (n ⫽ 6) were studied. Of
these, 12 received bone marrow (n ⫽ 8) or G-CSF-mobilized peripheral
blood stem cells (n ⫽ 4) from HLA-matched sibling donors. The remaining
six patients received bone marrow from unrelated donors (MUD). This
group included 6 males and 12 females, with a median age of 39 years
(range, 21–54 years). Pretransplant myeloablative regimens included a
combination of busulfan, cyclophosphamide, and etoposide, with or without fractionated total-body irradiation. Posttransplant graft-vs-host disease
(GVHD), antiviral, and antibacterial prophylaxis were comparable (32–
35). Blood was drawn from donors and recipients before transplant and
from patients upon clinical engraftment or by day 30 posttransplant. Subsequent sample collection targeted days 30, 60, 90, and/or 100, 120, 150,
180, 270, and 1 year posttransplant.
Five non-Hodgkin’s lymphoma patients undergoing autologous peripheral blood stem cell transplantation at Stanford University Medical Center
were studied. This group included three males and two females, with a
mean age of 54 years (range, 39 –70 years). Patients underwent standard
G-CSF stem cell mobilization and harvest and comparable myeloablative
conditioning (36). Samples were collected before transplant and on the first
day that each patient showed clinical engraftment or by day 14 posttransplant. Samples were collected weekly for the first month following transplant and on days 60, 90, and 180 posttransplant.
PBMC were isolated by Ficoll-Hypaque gradient separation. All samples were collected with approval of the appropriate Institutional Review
Board.
The Journal of Immunology
mAbs of different KIR specificity. Flow cytometry analysis
showed that the proportion of NK cells binding each Ab and their
median level of binding were stable over time for all five donors
(Fig. 1, A–E). These parameters appeared not to be perturbed by
infection or other environmental stress, including running of a
marathon by the donor in Fig. 1A. The results showed that donors
have stable and characteristic patterns of KIR expression, or KIR
repertoires, which can be described in terms of eight flow cytometry measurements: two parameters for each of four Abs.
To compare KIR repertoires, paired sets of flow cytometry data
of the type shown in Fig. 1, A–E, were used to calculate summed
differences for the proportion of NK cells binding each Ab and for
the median level of Ab binding. In this way, a comparison between
two data sets was reduced to a single point on a two-dimensional
plot (Fig. 1F; see Materials and Methods for further details). Control comparisons confirmed the validity of this method; when frozen aliquots of the same sample of PBMC were thawed and analyzed in duplicate, and, on different days, the total differences were
small (data not shown). As expected from Fig. 1, A–E, comparison
of the repertoires measured from PBMC obtained from the same
donor on different occasions produced small differences. In contrast, pairwise comparison of unrelated individuals revealed a wide
range of differences (Fig. 1F). In sum, these results show that an
individual’s NK cell KIR repertoire is defined and stable over time,
but that repertoires are highly diversified within human
populations.
Differences in KIR repertoire are principally determined by KIR
genotype
Functional and genetic studies have given contradictory results
regarding the role of HLA class I polymorphism in determining
NK cell KIR repertoires, while the effects of KIR gene variation on
NK cell KIR repertoire remain unexplored. To define the contributions of these two gene families, we compared the KIR repertoires of 85 healthy sibling pairs and correlated differences in KIR
expression with identity or disparity at the HLA class I and
KIR loci.
A panel of 104 individuals from 36 families was studied (Fig.
2). Of 85 sibling pairs, 21 (25%) were HLA class I identical and 64
(75%) were disparate at one or both HLA class I haplotypes, as
determined by serology (HLA-A and -B) and/or PCR typing
(HLA-C). KIR identity of sibling pairs was determined through a
combination of low- and high-resolution PCR typing and selected
DNA sequencing; results were confirmed by segregation for 43 of
the 85 sibling pairs. Twenty-seven sibling pairs (32%) were KIR
identical and 58 (68%) were disparate at one or both KIR haplotypes. These numbers demonstrate random segregation of parental
HLA and KIR haplotypes. That the number of KIR-identical sibling
pairs exceeds 25% is because some families segregate more than
one copy of a common KIR haplotype. HLA class I and KIR polymorphisms independently segregated in the 36 families, as expected from location of HLA and KIR genes to different chromosomes (chromosomes 6 and 19, respectively). Seven pairs (8%)
were KIR and HLA identical; 20 (24%) were KIR identical, HLA
disparate; 14 (16%) were KIR disparate, HLA identical; and 44
(52%) were KIR and HLA disparate (Fig. 2).
Peripheral blood NK cells were analyzed for KIR expression by
flow cytometry (as in Fig. 1, A–E). For each sibling pair, the difference in NK cell KIR repertoire was calculated (as in Fig. 1F),
the results being displayed in four two-dimensional dot plots in
Fig. 3 according to whether the sibling pairs were KIR identical,
HLA identical (Fig. 3A); KIR identical, HLA disparate (Fig. 3B);
KIR disparate, HLA identical (Fig. 3C); or KIR disparate, HLA
disparate (Fig. 3D). Each dot plot has a distinct distribution, demonstrating the influence of KIR and HLA genes on the NK cell KIR
repertoire. However, KIR type is by far the dominant factor. In the
context of incompatibility at the other genetic complex, KIR identity gave greater similarity in repertoire than did HLA class I identity. Thus, siblings who are KIR identical, HLA disparate have a
much tighter distribution with lower summed difference (Fig. 3B)
than do siblings who are KIR disparate, HLA identical (Fig. 3C).
The dominance of KIR over HLA is well illustrated when the
data points for all sibling pairs are placed on the same dot plot and
differentiated according to either KIR (Fig. 3E) or HLA identity
(Fig. 3F). The data points for all KIR-identical pairs cluster close
to the origin, irrespective of their HLA status (Fig. 3E), whereas the
data points for HLA-identical pairs distribute throughout all but the
outer fringe of the distribution (Fig. 3F). The distributions for KIRdisparate sibling pairs split into two subpopulations (Fig. 3, C–F),
where the subpopulation with higher difference consists of pairs in
which one sibling (but not the other) lacks reactivity with a KIR
Ab (DX9 or EB6), due either to the absence of a KIR gene
(KIR3DL1 or KIR2DL1/2DS1) or presence of an allele giving a
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FIGURE 1. Patterns of KIR expression by peripheral blood NK cells
can be used to define and compare NK cell KIR repertoires. A–E, Flow
cytometry data for five donors whose peripheral blood NK cells were assayed at intervals over a period of 13 mo for binding for KIR-specific Abs.
Each panel shows the data from one donor. The Abs encompass the
HLA-A, -B, and -C-specific KIR and were EB6 (E; KIR2DL1/S1), DX27
(u; KIR2DL2–3/S2), DX9 (⽧; KIR3DL1), and DX31 (Œ; KIR3DL2).
Each identical symbol in a panel represents a different time point. F, The
data from A–E is used to compare KIR repertoires. Each symbol on the
two-dimensional plot derives from two sets of flow cytometry data and
provides a measure of the overall differences in frequency of cells binding
each Ab and the overall differences in the level of binding. Comparisons
are for pairs of unrelated individuals (⽧) and, as controls, for data sets
obtained from the same person, but from blood samples drawn on different
occasions (‚). The range of possible value for each sum is 0 – 8.
241
242
GENETIC CONTROL OF HUMAN NK CELL REPERTOIRE
null phenotype (KIR3DL1*004) with DX9 (29). Thus, KIR genes
dominate over HLA class I genes in determining the NK cell repertoire of KIR expression in peripheral blood NK cells.
Apparent from the distribution of repertoire differences between
sibling pairs is that the two summed differences are not completely
independent variables. Thus, siblings who have large differences in
Ab-binding frequencies tend to have large differences in Ab-binding levels (Fig. 3E). This correlation between the sum of differences in frequency and median levels of Ab binding is statistically
significant (r ⫽ 0.72, p ⬍ 0.01; Fig. 3E). It demonstrates that
polymorphisms in the KIR gene family influence the frequency of
NK cells that express a particular KIR as well as other characteristics assessed by Ab binding, including the level of cell surface
expression and Ab-binding affinity.
HLA genotype influences the frequencies of KIR-expressing NK
cells
To determine the contribution of HLA class I to the NK cell KIR
repertoire, we considered just the subset of KIR-identical sibling
pairs. For these 27 pairs the differences in repertoire between siblings in HLA-identical pairs were compared with the differences
between the siblings in HLA-disparate pairs. As seen in Fig. 4, the
7 HLA-identical pairs have similarly low values for the sum of
differences for the frequency of Ab-binding cells, whereas the 20
HLA-disparate KIR-identical siblings exhibited a much wider
range of values. This difference has statistical significance in a t
test ( p ⬍ 0.0001). In contrast, the HLA-identical and HLA-disparate pairs exhibited a similar range of values for the summed
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FIGURE 2. KIR genotyping of 85 HLA-typed sibling pairs. The sibling pairs are organized into four groups according to genotype: KIR-identical,
HLA-identical (A), KIR-identical, HLA-disparate (B), KIR-disparate, HLA-identical (C) and KIR-disparate, HLA-disparate (D). Siblings having matching
HLA-A, -B, and -C allotypes by serology (HLA-A and -B) and/or PCR typing (HLA-C) were considered to be HLA identical. Siblings with matching KIR
typing and allelic subtyping results were considered KIR identical, with the exception of one sibling pair (no. 48), found to be KIR disparate by partial
sequencing of KIR locus-specific PCR products. The presence of KIR genes is indicated with white boxes, with allelic subtypes shown only for those sibling
pairs having matching combinations of KIR genes. Dark gray boxes indicate KIR genes that are absent. In heterozygous individuals, allele names are
separated with a slash. Alleles which are not discriminated by subtyping are listed together, e.g., as KIR2DL3*002 or 6; KIR3DL1*002 refers here to a set
of similar alleles which includes KIR3DL1*002, *003, and *006 – 8.
The Journal of Immunology
243
FIGURE 4. HLA class I influences the relative frequencies of cells expressing KIR but not their levels of surface expression. Shown are KIR
repertoire comparisons for the 27 KIR-identical sibling pairs. They are
distinguished as HLA identical (f), HLA different but having identical
HLA-B and -C KIR epitopes (u) or HLA and KIR epitope different (䡺).
FIGURE 3. KIR genes dominate HLA class I genes in determining human peripheral blood NK cell KIR repertoires. Sibling NK cell KIR repertoires were compared for 85 HLA- and KIR-typed sibling pairs. Sibling
pairs are organized into four groups: KIR identical, HLA identical (A), KIR
identical, HLA disparate (B), KIR disparate, HLA identical (C) and KIR
disparate, HLA disparate (D). Each symbol (F) represents the repertoire
difference between two siblings. E and F, Both contain all of the data points
from A–D. E, Sibling pairs are distinguished according to KIR identity (f)
or difference (䡺); F, they are distinguished according to HLA identity (⽧)
or difference (〫). Included in E is a linear regression trend line, calculated
using all data, which shows the correlation between summed differences in
frequency and surface level of KIR expression.
differences in the median level of Ab binding. Thus, the effect of
the HLA class I genotype on the NK cell KIR repertoire is to
modify the relative frequencies of cells expressing particular KIR,
but not the surface levels of KIR expression.
Best characterized of the HLA class I-specific inhibitory KIR
are those specific for epitopes of HLA-B or -C, which correlate
with polymorphisms in the carboxyl terminal part of the ␣1 helix.
The HLA-Bw4 epitope, formed by some sequence motifs at positions 77– 83 in HLA-B, is recognized by KIR3DL1. HLA-C
epitopes, defined by alternative motifs at positions 77 and 80, are
bound by KIR2DL2/2DL3/2DS2 and 2DL1/2DS1, respectively
(41– 44). Because different HLA types can have the same KIR
epitopes, we distinguished KIR-identical, HLA-disparate sibling
pairs according to whether they were identical or different for KIR
epitopes (Fig. 4). Repertoire differences were similar, there being
no statistically significant difference between the two HLA-disparate groups. When the expression of individual KIR was analyzed
for these sibling pairs, neither KIR frequency nor fluorescence
intensity differences between siblings were consistently correlated
with the presence or absence of a relevant KIR epitope. However,
CD94:NKG2A expression is affected by HLA and KIR genotypes
The stability of CD94:NKG2A expression was studied using the
same five donors and samples used for KIR (Fig. 1). For each
individual, the frequency of NK cells expressing CD94:NKG2A
and their median level of surface expression were stable over time.
However, these parameters varied among the five donors (data not
shown). The stability of the individual CD94:NKG2A phenotype
parallels that seen for KIR.
CD94:NKG2 expression by NK cells from the 85 sibling pairs
was measured. A range of difference between siblings, in both
frequency and level of CD94:NKG2A expression, was observed
(Fig. 5). The differences were least for KIR- and HLA-identical
siblings (Fig. 5A), showing that the combination of KIR and HLA
influences the repertoire of CD94:NKG2A expression. The differences were lower for KIR-disparate, HLA-identical siblings than
for either KIR-identical, HLA-disparate siblings or siblings disparate at both KIR and HLA. Thus, HLA type appears to override KIR
type in affecting CD94:NKG2 expression. However, the effect is
weaker than the influences of HLA and KIR on KIR expression and
for this sample size did not reach statistical significance.
We also compared expression of CD94:NKG2A and KIR in the
donor panel, independently of familial relationships. This revealed
a statistically significant inverse correlation between the total frequency of cells expressing KIR and the frequency of cells expressing CD94:NKG2. This was seen either when the total frequency of
KIR-expressing cells was roughly estimated by summation of the
frequencies of KIR expression for the four Abs (Fig. 5E, r ⫽
⫺0.39 ( p ⬍ 0.01)) or when it was calculated from individual KIR
frequencies, taking account of knowledge that KIR are expressed
in random combinations (4) (Fig. 5F, r ⫽ ⫺0.40 ( p ⬍ 0.01),
respectively). This result demonstrates some coupling between expression of the CD94:NKG2A receptor and KIR receptors.
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KIR- and HLA-identical sibling pairs and KIR-identical, HLA-disparate, KIR epitope-matched pairs were significantly different
( p ⬍ 0.01). Likewise, KIR- and HLA-identical pairs were different
from KIR-identical, HLA-disparate, KIR epitope-disparate pairs
( p ⬍ 0.001). Together, these results imply that HLA modification
of the NK cell KIR repertoire cannot be explained simply in terms
of the HLA-B and -C sequence motifs.
244
Reconstitution of NK cell receptor repertoire following stem cell
transplantation
Reconstitution of NK cell receptor expression was followed in 18
patients undergoing stem cell transplantation for treatment of CML
or acute myelogenous leukemia. Twelve of these transplants involved HLA-matched sibling donors, five involved HLA-matched
unrelated donors (MUD), and one an unrelated donor that was
HLA-C disparate, but otherwise HLA matched. Consistent with
random segregation of parental KIR and HLA haplotypes, 3 (25%)
of the 12 (25%) sibling donor/recipient pairs were KIR identical.
All of the HLA-matched unrelated donor/recipient pairs were KIR
disparate.
The three donor/patient pairs with identical KIR genotypes had
similar KIR repertoires (Fig. 6A), exhibiting summed differences
comparable to those of healthy KIR- and HLA-identical sibling
pairs (Fig. 3A). Thus, the hematologic malignancy suffered by the
patients in these pairs had little effect on their KIR expression,
indicating that, in general, the KIR repertoires measured in patients
before transplant will reflect their healthy repertoires before the
onset of malignant disease. The KIR-disparate donor/recipient
pairs gave a wide range of summed differences, as did the MUD
pairs (Fig. 6A), similar to those observed for healthy HLA-identical, KIR-disparate sibling pairs (Fig. 3C).
Following transplantation, patients’ peripheral blood was sampled at intervals over the course of 1 year. Posttransplant KIR
repertoires were compared with those of the donor and patient
before transplant. The patients could be divided into three groups
based on reconstitution of KIR and CD94:NKG2A expression. For
the eight patients of group 1 (five MUD, three sibling donors), the
reconstituted repertoire of KIR expression was like that of the
donor and distinct from that of the patient before transplant (Fig.
6, B and C). Upon initial engraftment, few peripheral blood NK
cells expressed KIR, while the majority were CD94:NKG2A positive. The frequency of KIR expression gradually increased during
the subsequent 6 –9 mo, reaching levels comparable to those of the
donor. Concomitant with increasing KIR expression, the proportion of NK cells expressing CD94:NKG2A gradually lessened. Patients in this group (group 1) had good recovery from transplantation with no major clinical complications.
Reconstitution in the five patients of group 2 (all sibling donors)
was characterized by reduced frequencies of KIR-positive NK
cells that persisted throughout the first year posttransplant, while
CD94:NKG2A expression frequencies remained high (Fig. 6D).
Although fewer NK cells expressed KIR, the hierarchy of KIR
expression within the KIR-positive population resembled that of
the donor. All but one of the group 2 patients experienced no major
clinical complications during the first year posttransplant. A similar pattern of reconstitution was observed in patients receiving
autologous stem cell transplants (Fig. 6E). Five patients (group 3)
exhibited idiosyncratic patterns of NK cell receptor reconstitution
(data not shown). They all suffered serious complications within
the first year after transplant, including chronic GVHD, grade IV
acute GVHD, and CML relapse.
Discussion
We developed a simple, robust, and quantitative method for comparing NK cell repertoires of KIR and CD94:NKG2A expression.
Study of five healthy donors over a 1-year period showed that each
person’s repertoire was stable and unaffected by infection or other
environmental stress. This raises our previous observation of
KIR3DL1 expression stability (24) to a general principal. The validity of this conclusion of individual repertoire stability is underscored by the fact that the same method of comparison showed that
each donor has a different repertoire. Thus, each donor’s repertoire
is genetically predetermined, a characteristic of innate defense
mechanisms. Moreover, the variation from one donor to another
shows that no one repertoire is optimal.
From a practical standpoint, repertoire stability meant that reliable assessment of a person’s repertoire could be based on a single
blood donation. This made feasible the analysis of ⬎100 individuals from whom correlations of NK cell repertoire difference with
KIR and HLA genetics were made. By studying 85 sibling pairs
from 36 different families, we were able to combine a fair sampling of the human population with valuable simplification of a
complex genetics. Comparison of sibling pairs demonstrated that
the KIR genotype is the dominant factor determining the repertoire
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FIGURE 5. CD94:NKG2A expression by NK cells is influenced by
HLA class I and KIR type. Differences in frequency of cells expressing
CD94:NKG2A and in level of expression are shown for the 85 sibling
pairs. As for KIR repertoire comparisons, CD94:NKG2A expression differences were calculated by dividing the absolute difference between two
measurements by the mean of the two values. Sibling pairs are organized
into four groups: KIR identical, HLA identical (A); KIR identical, HLA
disparate (B); KIR disparate, HLA-identical (C); and KIR disparate, HLA
disparate (D). E and F compare relative frequency of CD94:NKG2A and
KIR expression by NK cells of the 104 individual donors. E, The frequencies of the four measured KIR-positive subpopulations were simply
summed (as subpopulations overlap, the total frequency exceeds 100%). F,
The actual percentage of NK cells expressing any KIR or KIR combination
was estimated from the individual frequencies and knowledge of their random association. The formula (100 ⫺ (100 ⫺ percent EB6⫹NKs)(100 ⫺
percent DX27⫹NKs)(100 ⫺ percent DX9⫹NKs)(100 ⫺ percent
DX31⫹NKs)) calculates the percentage of NK cells expected to express
one or more KIR. The linear regression trend lines show the inverse
correlation.
GENETIC CONTROL OF HUMAN NK CELL REPERTOIRE
The Journal of Immunology
of KIR expression on NK cells, as is vividly seen from comparison
of KIR-identical pairs with KIR-disparate pairs in the absence of
any knowledge of HLA class I type (Fig. 3, E and F). Now evident
is that the KIR genes themselves are the undefined, non-HLA genes
previously implicated in controlling KIR expression (24).
In healthy individuals, the influence of the HLA genotype on NK
cell KIR expression is subordinate to that of KIR, only being detectable when analysis was restricted to the subset of KIR-identical
sibling pairs. Then it became clear that the KIR repertoires of
KIR-identical, HLA-identical siblings were more similar than those
of KIR-identical, HLA-disparate siblings. Importantly, the impact
of HLA is to change the frequencies of KIR-expressing cells, not
the surface levels of KIR expression. This provides good evidence
that the HLA class I genotype imposes selection during development of the NK cell receptor repertoire and is consistent with functional observations showing that human NK cells express an inhibitory receptor for autologous HLA class I, though not
necessarily for allogeneic HLA class I (4). Formally, it is possible
that the HLA effect we observed is not due to HLA class I but to
other linked genes of the HLA region. However, we consider this
an unlikely possibility, given the well-established role of HLA
class I polymorphisms in human NK cell receptor biology (23).
The subtlety of HLA class I selection on NK cell repertoires
arises because NK cells express multiple KIR, so that selection for
cells expressing inhibitory receptors specific for autologous HLA
class I causes only small reductions in the relative frequencies of
cells expressing other KIR. This subtlety may have contributed to
the failure of previous studies to see any influence of HLA class I
on the expression of HLA-B- or HLA-C-specific KIR (24, 25). In
those studies, comparison of the effect of the HLA class I difference was not made in the context of KIR identity and any effect due
to HLA would have been obscured by the greater effects of the KIR
genotype difference. Also critical was that the earlier studies used
KIR epitope motifs of HLA-B and -C molecules as measures of
HLA identity; these are simplified measures of KIR ligands which,
in this study, did not reveal the HLA effect in selection of the NK
cell KIR repertoire. Thus, the results obtained here are compatible
with and resolve the seemingly paradoxical results obtained in previous investigations. Moreover, our data suggest that the HLA effect on the KIR repertoire may be governed by complex interactions between KIR and HLA molecules; these could include allelic
fine specificities for the human KIR and as yet undiscovered KIR
specificities.
Clinical stem cell transplantation provides a system for examining the reconstitution of NK cell repertoires under conditions of
HLA and/or KIR genetic difference. The patients we studied
formed three groups, with those experiencing no major clinical
complications following transplantation making up the first two. In
the majority of allogeneic HLA-matched transplants, the patterns
of KIR expression became like that of the donor, confirming the
importance of the KIR genotype revealed in the comparison of
healthy sibling pairs. In some allogeneic transplants and autologous transplants, the relative expression of the different KIR genes
was like the donor, but the overall percentage of NK cells expressing KIR was reduced compared with the donor. One possibility
was that CD56dim KIR⫹ cells were replaced by CD56bright KIR⫺
cells (45, 46). This did not seem to be the case, because lack of
KIR expression was seen in both the CD56bright and CD56dim populations. In contrast, allogeneic transplants between HLA-matched
unrelated individuals tended to reconstitute NK cell populations
with frequencies of KIR expression comparable to those of the
donor. Thus, in the transplant situation, some degree of genetic
incompatibility may facilitate induction of KIR expression. A potentially related phenomenon is that in vitro culture of human NK
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FIGURE 6. Distinct patterns of NK cell receptor repertoire reconstitution after stem cell transplantation. A, Comparisons between the pretransplant NK cell KIR repertoires of 18 allogeneic transplant patients and their
MUD or sibling donors, identified as KIR-identical, HLA-identical sibling
donor/patient pairs (f), KIR-disparate, HLA-identical sibling donor/patient
pairs (䡺), KIR-disparate, HLA-identical MUD/patient pairs (〫), and KIRdisparate, and HLA-disparate MUD/patient pairs (E). The HLA-disparate
patient is mismatched for one HLA-C allele, but shares the same KIR
epitopes as the donor. The same pretransplant NK cell KIR repertoires
were compared with the 1 year posttransplant repertoires for the patients in
group 1 (B); each symbol represents a different patient. Empty symbols
show comparisons between posttransplant repertoire and recipient pretransplant repertoire; filled symbols represent comparisons between posttransplant repertoires and the donor repertoires. These results indicate that
posttransplant KIR repertoires, in these patients, become like those of the
donors. Posttransplant KIR and CD94:NKG2A NK cell expression frequencies of representative patients in group 1 (C) and group 2 (D) are
compared with donor and recipient pretransplant expression; group 1 patients are characterized by a return to donor-like KIR expression, whereas
for group 2, KIR expression remains depressed. E, Posttransplant KIR
expression in a representative autologous transplant patient. Each receptorspecific Ab is shown with a different symbol and are EB6 (䡺; KIR2DL1/
S1), DX27 (f; KIR2DL2–3/S2), DX9 (‚; KIR3DL1), DX31 (Œ;
KIR3DL2), and Z199 (E; CD94: NKG2A).
245
246
than KIR genotype on CD94:NKG2A expression is that HLA genotypes vary in the number of KIR ligands they provide, whereas
the great majority of KIR genotypes provide all of the different
types of inhibitory KIR with specificity for HLA class I. Consequently, the HLA class I genotype will usually dictate the number
of KIR that can serve as inhibitory receptors for autologous HLA
class I and thus the proportion of NK cells needing CD94:NKG2A
expression to be tolerant of self. A second, and not necessarily
mutually exclusive, possibility is that the HLA effect is due to
polymorphism affecting either the expression or function of
HLA-E, the ligand for CD94:NKG2A.
Acknowledgments
We thank Dr. Grumet and the Stanford Medical Center Histocompatibility
Laboratory staff; Dr. Rodriguez and the City of Hope Bone Marrow Transplant Program; Dr. Khakoo for help in collecting samples from healthy
individuals; Drs. Shizuru, Blume, and Negrin and Kathryn Tierney for
collection of transplant patient samples and helpful advice; Dr. Uhrberg for
providing KIR epitope-typing primers; and Dr. Brown for help and advice
with statistical analysis.
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