structural immunology and crystallography help immunologists see the immune system in action: how t...
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Critical Review
Structural Immunology and Crystallography HelpImmunologists See the Immune System in Action:How T and NK Cells Touch Their Ligands
Yong Chen1,2, Yi Shi1,2, Hao Cheng1,2, Yun-Qing An3 and George F. Gao1,2,3
1CAS Key Laboratory of Pathogenic Microbiology and Immunology (CASPMI), Institute of Microbiology,Chinese Academy of Sciences, Beijing, People’s Republic of China2College of Life Sciences, Graduate University, Chinese Academy of Sciences, Beijing, People’s Republic of China3Department of Immunology, Capital Medical University, You-an-men, Beijing, People’s Republic of China
Summary
Host immune system is an important and sophisticated sys-tem, maintaining the balance of host response to ‘‘foreign’’ anti-gens and ignorance to the normal-self. To fulfill this achieve-ment the system manipulates a cell–cell interaction throughappropriate interactions between cell-surface receptors and cell-surface ligands, or cell-secreted soluble effector molecules totheir ligands/receptors/counter-receptors on the cell surface,triggering further downstream signaling for response effects. Tcells and NK cells are important components of the immunesystem for defending the infections and malignancies and main-taining the proper response against over-reaction to the host.Receptors on the surface of T cells and NK cells include a num-ber of important protein molecules, for example, T cell receptor(TCR), co-receptor CD8 or CD4, co-stimulator CD28, CTLA4,KIR, CD94/NKG2, LILR (ILT/LIR/CD85), Ly49, and so forth.These receptor molecules interact with their ligands on the tar-get cells, including major histocompatibility complex (MHC)(or human leukocyte antigen, HLA), CD80, CD86, and so forth.Detailed understanding of these receptor–ligand pair interac-tions is crucial for our full knowledge of the immune system,ultimately for us to manipulate the T cell and NK cell functions.Accumulations of the receptor–ligand complex crystal struc-tures in the recent years have provided us a unique angel to seehow the immune cells interacting with their partner cells. Inthis review, we discussed binding specificity, plasticity, and flexi-bility of the T cell and NK cell receptor/ligand interaction, fit-ting the structural data with their functions. Structural immu-
nology indeed helps us see how T and NK cells ‘‘touch’’ theirtarget cells in our immune system. � 2009 IUBMB
IUBMB Life, 61(6): 579–590, 2009
Keywords T cell; NK cell; structural immunology; crystallography;
immune system; receptor; ligand; interaction; MHC;
HLA; KIR; LILR.
INTRODUCTION
Vertebrates have had not only innate immunity but also are
able to mount defense mechanisms that constitute adaptive im-
munity to protect the body from foreign or changed self-attack.
The vertebrate immune system has a unique ability to mount a
highly specific response against almost any foreign antigens,
even those never seen before in the course of evolution, and
more importantly antigens of self-malignancies (self-changed
antigens). Cells involved in the immune system cover a broad
range of different types. Among them the cytotoxic lympho-
cytes play a crucial role in destroying the foreign or changed
self-antigens. The cytotoxic lymphocytes include at least the
following three types: T cells, NK (natural killer) cells, and the
NKT cells (natural killer T cells). T cells belong to a group of
lymphocytes expressing CD3, CD4/CD8, playing important
roles in antigen-specific cell-mediated immunity. NK cells are
large granular cytotoxic lymphocytes expressing CD16 and
CD56, but not CD3, constituting a major component of the
innate immune system. NKT cells are a heterogeneous group of
T cells that share properties of both T cells and NK cells, rec-
ognizing foreign or self lipids and glycolipids presented by non-
polymorphic CD1d.
Address correspondence to: Prof. George F. Gao, DPhil, CAS Key
Laboratory of Pathogenic Microbiology and Immunology, Institute of
Microbiology, Chinese Academy of Sciences, Beijing 100101, People’s
Republic of China. Tel: 186-10-64807688. Fax: 186-10-64807882.
E-mail: [email protected]
Received 15 March 2009; accepted 15 March 2009
ISSN 1521-6543 print/ISSN 1521-6551 online
DOI: 10.1002/iub.208
IUBMB Life, 61(6): 579–590, June 2009
The recognition molecule on T cells is the membrane-anch-
ored T cell antigen receptor (TCR) which recognize a compos-
ite ligand consisted by small antigen fragments and major histo-
compatibility complex (MHC) (or human lymphocyte antigen,
HLA, in human) (1). T cell receptors are classified as two dif-
ferent heterodimers, abTCR, and cdTCR. Receptors on NK cell
surface are divergent with different members as discussed in the
text later and interact with several groups of ligands, including
peptide MHC (pMHC).
The T cell/NK cell system is quite sophisticated and coordi-
nated each other in the immune system. To escape from killing
of T cells, cells infected by viruses or other pathogens would
have a lowered level of, or that have lost the expression of,
MHC class I molecules. However, these ‘‘missing-self’’ cells
can be recognized by NK cells which can spare normal cells
expressing adequate amounts of MHC class I molecules (2).
Thus, the altered expression of class I antigens, a common
event in tumor transformation or following viral infection with
CTL escape, leads to NK-mediated target cell lysis.
Initiation of the immune response of the T cells starts with
interaction of TCR with its ligand pMHC. Current set of struc-
tural data of abTCR–pMHC complex reveals that the abTCR–pMHC interaction adopts an ‘‘induced-fit’’ model (3). This means
that the abTCR undergoes large conformational change or rear-
rangement when it engages on the pMHC, adjusting their fit to
the interactive ligands. On the other hand, CD8/MHC and NK
receptors/MHC adopts a ‘‘lock and key’’ model. ‘‘Induced fit’’
represents the adaptive receptor–ligand interaction, whereas ‘‘lock
and key’’ represents the innate receptor–ligand interaction (4).
As NK cells are bipartite and their functions were regulated
by a fine tuning between opposite signals delivered by inhibi-
tory and/or activating receptors (2). Human NK cells express
multiple receptors specific for different alleles of HLA class I
molecules expressed on normal cells. This interaction leads to
an inhibitory signal which prevents normal cell lysis. These
receptors can also be cataloged into two distinct structural fami-
lies: members of the immunoglobulin (Ig) receptor superfamily,
which mainly include the killer-cell Ig-like receptors (KIRs)
and leukocyte Ig-like receptor (LILR/LIR or immunoglobulin-
like transcript, ILT, or CD85). The other receptors are C-lectin
type receptors mainly composed of CD94/NKG2 heterodimer
and Ly49 homodimer.
As the late Professor Don C. Wiley (Harvard, GFG’s postdoc
mentor during 1999-2001) once said, ‘‘I do not know the biol-
ogy until I see it.’’ How can we see the biology? Structural
biology indeed helps the immunologists understand the immune
recognition in a simpler way, ‘‘see’’ the action. Peptide presen-
tation by MHC is a very good example. Don Wiley, Pamela
Bjorkman, and Jack Strominger and their colleagues’ work
made the T cell recognition of the foreign antigen really visible,
as for a long time immunologists did not understand why for-
eign antigens were recognized by the immune system always
correlate with the MHC. The peptide comfortably sitting on the
top of MHC helices’ ‘‘bed’’ gave us a clear picture. We really
see the biology, the immunobiology! Functional data were inter-
preted perfectly by the structural data.
As discussed earlier, the receptor–ligand interactions between
the T and NK cells are quite complicated and a lot of receptors/
ligands are involved in the cell–cell interface. In this review,
our discussion mainly focuses on the structural binding modes
of the T cell receptors and NK receptors. These receptors are
the major interests in our laboratory, such as LILR family and
TCR. Other receptors are mentioned briefly.
SPECIFICITY DETERMINED BY INTERACTIONOF ab T CELL RECEPTORS WITH PEPTIDE-MHC
T cell receptor (TCR) engaging with peptide-major histo-
compatibility complex (pMHC) is a complicated molecule–mol-
ecule interaction which still presents one of the most interesting
and enduring structural puzzles in biology. Recent progress on
TCR-pMHC complex structure (Fig. 1) truly reflects the beauty
of structural biology which vividly reveals the molecular basis
of their interaction: seeing the immunobiology by crystallogra-
phy. Similar to antibody molecules, abTCRs are generated by
recombination of a family of gene segments that form the
diverse TCR repertoire (as high as to 1014 different sequences)
42 Va (5) and 46 Vb (6) genes are used to constitute the first
two variable loops (CDR1 and CDR2), while the third variable
loops (CDR3) are formed by the randomly joining of V and J
(in TCR a), or by V, D, and J (in TCR b). The joining process
itself generates further diversity both by removing nucleotides
and by introducing nongermline nucleotides at each junction.
The diversity of TCR repertoire facilitates the existence of spec-
ificity of T cell response which is determined by recognition of
an antigenic peptide bound to a class I or class II MHC mole-
cule by the antigen-specific TCR.
Up to 20 TCR-pMHC complex structures have been pub-
lished in the past 10 years, but there is still no uniform agree-
ment among structural biologists and immunologists about how
to interpret this structural information. Here, we only want to
discuss the original hypothesis that TCR and MHC molecules
coevolved a ‘‘preference or specificity’’ to interact with one
another (7). To analyze the specificity determined by the inter-
action of abTCRs with pMHC, the binding interface can be
functionally and structurally segregated into four distinct com-
ponents that collectively form composite surfaces (8). In abTCRmolecules, the germline-encoded components (CDR1s and
CDR2s) and the highly diverse components (CDR3s) form the
pMHC-binding site; while in MHC molecules, the germline-
encoded components (a1 and a2 helices) and the highly diverse
components (a array of peptides) form the combining site
‘‘visible’’ to the TCR. Thus, the TCR–pMHC interface can be
segregated into invariant and variant parts based on different
sources in which the most variable CDR3 loops of the TCR are
positioned over the centre of the binding site where they contact
the peptide, whereas the relatively conserved CDR1 and CDR2
loops of the TCR are located on the top region of MHC helices
580 CHEN ET AL.
Figure 1. Interaction surface between T cell and antigen presenting cell (APC) (representing the known complex crystal structures).
(A) Models of TCR/pMHCI/CD8aa/CD3ed/CD3ec complex, (B) TCR/pMHCII/CD4/CD3ed/CD3ec complex, and (C) co-stimulatory
molecules of CTLA-4/CD80 and CTLA-4/CD86 complexes. The TCR/pMHCI/CD8aa is modeled by superimposing two structures,
HLA-A2/CD8aa complex (PDB 1AKJ) and the TCR A6/HLA-A2/TaxP6A complex (PDB 1QRN); while the TCR/pMHCII/CD4 is
modeled by a series of superimpositions using three structures, CD4/I-Ak/peptide complex (PDB 1JL4), TCR HA1.7/HLA-DR1/
peptide complex (PDB 1FYT) and four-domain CD4 (PDB 1WIO). The positions of CD3ed (PDB 1XIW) and CD3ec (PDB 1SY6)
relative to TCR on the T cell are based on the proposal by Arnett et al. (88). Costimulatory system is modeled by two complex
structures, CTLA-4/CD80 complex (PDB 1I8L) and CTLA-4/CD86 complex (PDB 1I85). In the models, peptides are colored in or-
ange, MHCI heavy chain and MHCII b chain in yellow, MHCI b2m and MHCII a chain in green, CD8aa in red and blue, CD4 in
blue, TCR a chain in purple and TCR b chain in cyan, CD3e in marine, CD3d in magenta, CD3c in olive. The oval drawing in or-
ange color represents the structure-unknown D2 domain of CD86. These images were prepared using the program PYMOL.
Figure 2. Different interaction modes for ab TCRs and cd TCRs binding to their ligands. (A) Overview structure of 2C ab TCR
bound to its ligand H2-Kb-dEV8. MHCI heavy chain is colored in limon, MHCI b2m in marine, peptide in red, TCR a chain in
cyan, TCR b chain in pink. (B) Footprints of ab TCR CDR loops on the pMHC interface. It shows a stereotyped polarity of the
Va and Vb CDR loops on pMHC, in which the Va domain lies mainly over the amino-terminal end of the peptide and the a2 helix
(MHCI) or b1 helix (MHCII), while the Vb domain lies mainly over the carboxy-terminal region of the peptide and the respective
a1 helices. The whole pMHC interface is colored in limon, while the peptide in red. CDR1s and CDR2s marked with semitranspar-
ent cyan oval drawings, which constitute the relatively conserved components of the TCR. CDR3s marked with a semitransparent
magenta oval drawing constitute the high diversity components of the TCR. (C) Overview structure of G8 cd TCR bound to its
ligand MHC Ib T22. MHC Ib heavy chain is colored in yellow, MHC Ib b2m in magenta, TCR c chain in pink, TCR d chain in
blue. G8 interacts with T22 at a titled angle which differs from the ab TCRs. (D) Footprints of cd TCR CDR loops on the MHC
Ib interface. The surface of whole MHC interface is colored in limon. cd TCR G8 predominantly uses its CDR3d loop (marked
with semitransparent red oval drawing) to interact with its ligand T22, and the remaining CDRd and CDR3c (marked with semi-
transparent magenta oval drawing) bind to T22 with degenerate contacts.
581STRUCTURAL IMMUNOLOGY AND CRYSTALLOGRAPHY HELP IMMUNOLOGISTS
surrounding the central CDR3-peptide region (9, 10) (Figs. 2A
and 2B). This interaction mode suggests that the specificity of
TCR binding to pMHC is determined by two parts: contacts
between the germline-encoded TCR V-gene products and MHC
gene products, and also contacts between the diverse TCR
CDR3 regions and peptides in the MHC groove. Logically, we
can predict that invariant parts of TCR (CDR1 and CDR2) have
evolved to interact with MHC molecule in specific and con-
served way, leaving the CDR3-peptide region aside.
Nevertheless, it is not quite simple as that. Except the speci-
ficity for one MHC, the TCR also has cross-reactivity that it is
cross-reactive with many other MHC molecules (11). In princi-
ple, each abTCR can recognize any MHC allele, as there does
not seem to be class or allotype specificity by particular TCR
germline segments. This cross-reactivity is essential to enable
the TCR to dock and ‘‘scan’’ the peptide contents of many dif-
ferent MHC molecules. Although the usage preferences of the
TCR chains do exist in the T cell response, but it doesn’t mean
that each abTCR is specific to one MHC molecule.
Recent study focusing on the interaction of one particular
Vb segment (Vb8.2) with different I-A MHC alleles has dem-
onstrated the conservation of specific TCR-MHC interfacial
contacts (12, 13). A ‘‘codon hypothesis’’ which suggests that
each TCR variable-region gene product engages each type of
MHC through a ‘‘menu’’ of structurally coded recognition
motifs that have arisen through coevolution has been proposed
to clarify the existence of ‘‘TCR-MHC bias’’ or ‘‘germline-
encoded recognition’’ of MHC. Here, ‘‘bias’’ is simply a euphe-
mism for specificity, and cases of true cross-reactivity are more
correctly called ‘‘polyspecificity’’ (14). ‘‘Induce-fit’’ interaction
mode facilitates polyspecificity to occur which is usually seen
in the immune system of specific recognition by one protein of
a spectrum of diverse ligands.
Returning the issue of specificity determined by interaction
of abTCR with pMHC, it is reasonable that each TCR engages
on each pMHC molecule using distinct interaction codon which
is derived from the coordination of the germline-encoded recog-
nition bias and peptide-mediated editing of germline-encoded
recognition. And this distinct interaction codon determines the
specificity of TCR binding to pMHC.
HELP OF CO-RECEPTORS CD8/CD4 TO THEPEPTIDE-MHC/TCR BINDING
The interaction of the abTCR with pMHC is enhanced by
the recognition of the co-receptor CD8 or CD4 to the same
pMHC in the immune response when T cells ‘‘touch’’ the anti-
gen presenting cells (APC). Binding dynamics and complex
structures have shed light on this complex interactions for a sin-
gle ligand (pMHC) recognized simultaneously by two receptors,
both abTCR and co-receptor CD8 or CD4 (15–21).
CD8 (two forms of aa homodimer and ab heterodimer) acts
as a co-receptor in the function of cytotoxic T lymphocytes
(CTLs), whereas CD4 acts as a co-receptor in T helper (Th)
cells. Thus, CD8 interacts with class I pMHC (pMHCI), while
CD4 interacts with class II pMHC (pMHCII). In the current
model, both CD8 and CD4 bind to the same pMHC (class I or
class II, respectively) as the ab TCR and are thus called T cell
co-receptor [reviewed in (22)]. Crystal structures of both human
and murine CD8aa-pMHCI complexes (15, 17), muring
CD8ab-pMHC (23), and TL antigen-CD8aa complex (24) have
shown that the Ig-like domain is involved in the binding to
pMHC in a similar way to antibody-antigen binding as pre-
dicted in the earlier functional studies (Fig. 1A). The binding of
CD8aa and CD8ab to pMHC mainly involves the a3 domain
with some hydrogen bonds in the a2 and b2m domains away
from the peptide interface. CD4 molecule consisted of four Ig-
like domains and the complex structure involving human CD4
(the two N-terminal domains) and murine pMHC (I-Ak)
revealed that the N-terminal Ig-like V domain is the only one
of the four Ig-like domains directly binding to the two mem-
brane-proximal domains (a2 and b2 domains) (Fig. 1B) (18).
Although the binding modes of CD8aa-pMHCI and CD4-
pMHCII are completely different, but in both cases the binding
conformations ensure that the specificity for pMHC is deter-
mined by the antigen-specific TCR, whereas CD8 or CD4 act as
a universal co-receptor for any polymorphic pMHCs, confirming
their true ‘‘help’’ function as co-receptors.
Biophysically both CD8 and CD4 bind pMHC with a lower
affinity, faster kinetics and independently from the abTCRensuring that binding is dominated by the TCR rather than the
‘‘assistant’’ co-receptors. Furthermore, the BIAcore binding
experiments involving soluble reagents of pMHC, TCR and
CD8 or CD4 do not indicate any direct binding of the TCR
with CD8 or CD4, or enhancement by CD4 or CD8 to the bind-
ing of TCR-pMHC (19–21). However, one early study demon-
strated some enhancement of TCR-pMHC binding in the pres-
ence of soluble CD8 using BIAcore analysis (16). In the future,
a triple complex crystal structure involving the TCR-pMHCI-
CD8 or the TCR-pMHCII-CD4 complex will help us to under-
stand the nature of these crucial interactions that lie at the heart
of antigen recognition and T-cell activation. In both human and
mouse, the functions of CD8 involved in immune responses
have been extensively studied (25, 26).
Simian immunodeficiency virus (SIV) infection of rhesus
macaque (macaca mulatta) is widely used as an animal model
for human immunodeficiency virus (HIV) infection (27–29) as
well as other human diseases. However, for macaque monkeys,
little is known on the structures of the CTL-related molecules
(e.g., TCR, pMHC and CD8). Recently, we presented the crys-
tal structures of Mamu-A*01 complexed with two immunodom-
inant SIV epitopes: the nonamer CM9 of group specific antigen
(Gag, 181-189; CTPYDINQM) and the octamer TL8 of tran-
scription activator (Tat, 28-35; TTPESANL) for the rational
design of Mamu-A*01-restricted CTL epitopes with improved
binding, as a step towards development of AIDS vaccines (30).
Furthermore, recently we also present the crystal structure of
rhesus macaque CD8aa (rCD8aa) homodimer and found that
582 CHEN ET AL.
with two Thr43 residues in C-C’ loop, rCD8aa shows a unique
extra hydrogen bond in the homodimeric interface indicating a
tighter homodimeric interaction (31).
SECONDARY SIGNALS OF T CELL ACTIVATION
Specific immune responses are strictly regulated by cell–cell
interactions. When pMHC-peptide complex on APC cells is rec-
ognized by, TCR, it is called the first signal, the fully activation
of the T cell needs the secondary signal which occurs after the
co-receptor interactions (32).
The B7 family is such one kind of co-stimulatory molecules
including CD28, CTLA4 (CD152) and their ligands B7-1
(CD80), B7-2 (CD86), ICOS and its ligands ICOSL, PD-1 and
its ligands PD-L1, PD-L2, and so on, which are playing a cru-
cial role in regulating the activation, differentiation, and toler-
ance of T lymphocytes. All of these costimulatory molecules
belong to the Ig (immunoglobulin-like) superfamily, as stimula-
tory or inhibitory functions. Stimulatory receptor CD28 is con-
stitutively expressed on naıve T cells, when engaged by B7-1
and B7-2 on APCs, positive signal will provide to initiate and
sustain T cell responses (33, 34). ICOS is not expressed consti-
tutively on naive T cells but can be induced rapidly on T cells
after TCR engagement (35), taking the T-cell proliferation and
differentiation as its major function (36). On the other hand,
CTLA4 is induced and expressed after T cell activation, which
can competitively binds B7-1 and B7-2 to down regulate or
even terminate the responses by providing negative signal (37).
Recently, PD-1 was also found to generates negative signal
expressed on T cell after binding PD-L1 and PD-L2 (38, 39).
Until now, the extracellular region structure of CTLA4-B7-1
complex (40) and CTLA4-B7-2 (only IgV domain) (41) have
been resolved. As CD28 may be hard for crystallization alone,
we only know the structure of CD28 extracellular region com-
plexed with the Fab fragment of a mitogenic antibody (42). PD-
1 and its ligands, another hot spot of B7 family, are still in pro-
gress. The structure of murine PD-1 (43), murine PD-1 and
human PD-L1 hybrid complex (44), murine PD-1 and murine
PD-L2 (45) complex have been reported. However, human PD-
1 and its complex still remains a mystery. Other important B7
family members as well as other T cell co-receptor molecules
are waiting for us to investigate.
INTERACTION OF cd T CELL RECEPTORSWITH THEIR LIGANDS
cd T cell receptors (TCRs) are one of the three lineages of
somatically recombined antigen receptors which also include
abTCRs and antibodies. Previous studies demonstrated that aband cd T cells have different functional roles in the immune
system (46), yet the identity of endogenous ligands for cd T
cells is still unclear. Compared with antibodies and abTCRs,cdTCRs have the highest potential CDR3 diversity (1018) gener-
ated by VDJ recombination, but limited diversity derived from
pairing of germline-encoded V regions, due to small numbers of
Vc and Vd segments (8). CDR3 length distribution in cdTCRsis more similar to antibodies than to abTCRs, as the CDR3dloops are long and variable, while the CDR3c loops are short
and constrained (47). However, cd T cell reactivity seems to
correlate with V gene usage, suggesting that CDR3 diversity
has little role on the recognition of specific ligands (48).
The molecular basis of the recognition of ligand by cdTCRsis not well understood. As the first co-crystal structure of
cdTCR bound to its ligand was reported (49), we can have a
glimpse of the molecular basis of the recognition of ligand by
cdTCRs. The structure shows that G8 cdTCR predominantly
use its CDR3d loop to bind the MHC class Ib molecule T22 at
a tilted angle with degenerate contacts for the remaining CDRdand CDR3c loops, differing from the straight-on approach
which abTCRs adopt to interact with pMHC (Figs. 2A, 2C, and
2D). Furthermore, the residues involved in the interface are
derived mainly from the germline-encoded Dd segments, and it
shows that G8 cdTCR has a germline-encoded basis for T22
recognition. Recent study has revealed that an autonomous
CDR3d is sufficient for the recognition of the ligands T10 and
T22 (50). The interaction mode of G8 cdTCR binding to T10/
T22 shows that the conserved amino acid motif in the CDR3dloop can have its ligand affinity ‘‘tuned’’ by different surround-
ing contexts of CDR3d sequence. All the data suggest that the
G8 cdTCR may function in the immune system with a hybrid
of innate and adaptive recognition strategies.
INTERACTION OF KIR WITH PEPTIDE-HLA
In contrast to TCR-MHC ‘‘induce-fit’’ mode, many of NK
cell receptors interact with MHC class I molecules as ‘‘lock and
key’’ and modulate cells activity. As shown in Fig. 3, KIR and
CD94/NKG2 dock on the a1 helix and a2 helix including pep-
tide (51–53) while Ly49 and LILR interacts with pMHC a3 do-
main and b2m (54–57).
The human KIR gene family contains closely linked 15
genes and two pseudogenes in the chromosome 19q13.4.
Human KIRs are type I transmembrane proteins containing ei-
ther two (KIR2D) or three (KIR3D) immunoglobulin-like
domains that recognize polymorphic HLA-C and HLA-B mole-
cules, respectively (4). The Ig-like domains have been desig-
nated D0, D1, and D2, or D1 and D2, from N to C-terminal in
KIR3D/KIR2D proteins. A short stalk region separates the Ig-
like domains from the transmembrane segment, and the cyto-
plasmic domains are variable in length. Some receptors possess
long (L) cytoplasmic domains with one or two ITIM sequences,
and other receptors have short (S) cytoplasmic domains without
signaling motifs.
To date, the crystal structures of three free-state KIRs
(KIR2DL1, 2DL2 and 2DL3) and two KIR/pMHC complexes
(KIR2DL2/HLA-Cw3, KIR2DL1/HLA-Cw4) have been
reported (2), while no crystal structures available for KIR3D
receptors. As shown in Fig. 4, for example, KIR2DL2 bind
583STRUCTURAL IMMUNOLOGY AND CRYSTALLOGRAPHY HELP IMMUNOLOGISTS
HLA-Cw3 ligands through the a1 and a2 helices and the C-ter-
minal portion of the peptide (52). The KIR D1D2 axis is
approximately orthogonal to the axis of the peptide with D1
mainly binds to the HLA a1 helix and D2 to a2. The interface
is highly charged, and shows electron complementary that are
critical for binding (Fig. 3B). A comparison of the KIR2DL2/
HLA-Cw3 and KIR2DL1/HLA-Cw4 complexes revealed that
due to side chain rearrangements, many conserved residues in
KIR2D and HLA-C mediate different interactions in the two
structures, explaining the allelic specificity of KIR2DLs/HLA
complexes (58). For example, different alleles of HLA-C have
either Ser 77/Asn80 or Asn77/Lys80, and these are recognized
by different isoforms of KIR2DL.
Furthermore, peptide preferences have been documented in
KIR2D binding to HLA-C molecules (59), although the function
of peptide selectivity is not very clear in NK cell receptors. As
shown in Fig. 4B, the KIR/HLA interaction results in the sensi-
tivity to substitutions at peptide positions 7 and 8. Gln71 of
KIR2DL2 is hydrogen bonded to the main-chain nitrogen atom
of P8 Ala (60). This hydrogen bond may restrict the size of the
side chain accommodated at this position, consistent with the
observed preference for Ala or Ser at the P8 position. By com-
parison, TCRs, which exhibit much greater peptide selectivity
than KIRs, generally focus on the central portion of the MHC-
bound peptide, at and around the P5 position (Fig. 2) (61).
Although the specificity of the inhibitory KIR have been
extensively characterized, very little is known about the ligands
for the KIR2DS and KIR3DS molecules. The similarity between
several pairs of the activating and inhibitory KIR suggests they
arose by gene duplication. However, in all cases studied, the
activating KIR either does not bind HLA class I, or binds with
an affinity much weaker than that of the paired inhibitory KIR.
INTERACTION OF LILR (ILT/LIR/CD85)WITH PEPTIDE-HLA
Leukocyte immunoglobulin-like receptors (LILRs/LIRs), also
called Immunoglobulin-like transcripts (ILTs) (62, 63) or CD85
(64), including 13 members (with two of them as pseudogenes)
of either activating receptors (LILRA) or inhibitory receptors
(LILRB), regulating a broad range of cells in the immune
responses. Since LILR was first discovered by David Cosman
and Marco Colonna in 1997 (62, 63, 65), lots of studies have
been performed on its ligands and structures.
According to the amino acid similarity responsible for the
interaction between LILRB1 (ILT2/LIR-1/CD85j) and HLA-A2/
b2m, LILR can be divided into two groups (56): Group 1 mem-
bers include the inhibitory receptors LILRB1 and LILRB2
(ILT4/LIR-2/CD85d), the activating receptors LILRA1 (LIR6/
CD85i) and LILRA2 (ILT1/LIR-7/CD85h), and the soluble re-
ceptor LILRA3 (ILT6/LIR-4/CD85e). Group 2 members mainly
comprise of LILR A4/A5/A6 and B3/B4/B5 (ILT 7/11/8 and
ILT 5/3/LIR8), showing less than 60% sequence identity with
Group 1 members.
Most of the LILR family members contain four Ig-like
domains in their extracellular domains (designated D1, D2, D3,
and D4 from N to C terminus). Current available crystal struc-
tures of LILR B1/B2/A2/A5 only include their D1 and D2
domains (D1D2) (55, 56, 66–69). Though the instability and
precipitation of wild-type LILRA2 (ILT1/LIR-7/CD85h) D1D2
has been documented (70), a cysteine-introduced mutation
(R142C) was developed to form a disulfide bond with the spare
Cys132, which stabilizes the protein and enhances the recombi-
nant production of the LILRA2 D1D2 protein (71).
As inhibitory receptors with ITIM-motifs in their cytoplas-
mic region, LILRB1 and LILRB2 have been reported to bind a
broad range of classical or nonclassical pMHCIs and MHC-like
molecules (UL18) and differentiate or impair many lymphocyte
cells (72–74). The complex crystal structures of the LILRB1-
Figure 3. Overview structure of NK receptors binding to a
pMHCI molecule (representing the known complex crystal
structures). NK receptors were marked by different circles with
corresponding colors as following: NKG2A (PDB 3CDG) in
light blue; CD94 (PDB 3CDG) in pink; KIR (PDB 1EFX) in
yellow; Ly49 (PDB 1P4L) in limon; LILRB1/B2 (PDB 1P7Q/
2DYP) in brown. MHC molecule (green and blue) (HLA-Cw3
as an example, PDB 1EFX) with peptide (red, GAVDPLLAL)
was located at the center.
584 CHEN ET AL.
HLA-A2 (56) (Fig. 5A), the LILRB2-HLA-G (55), and
LILRB1-UL18 (69) were also reported.
One of the most important questions we concerned about is
the binding specificity of LILR-pMHC. LILRB1/B2 D1D2
binds pMHCIs through a3 domain and b2m, respectively (Fig.
5A). This orientation differs largely from the docking mode of
KIR onto pMHC as we have shown earlier. The size of the
LILRB1-HLA-A2 interface (�1,700 A2) is larger than those of
KIR-pMHC interfaces (�1,500 A2) and similar to those of TCR-
pMHC complexes (1,700�1,900 A2) (9). About 70% of the
LILRB1-HLA-A2 binding surface is involved in contacts with
14 b2m residues. This relative dominance of b2m is unprece-
dented among HLA-binding receptors, and is partly responsible
for the broad recognition to both classical and nonclassical
pMHCIs of LILRB1.
Comparison of the binding pattern of LILRB1-HLA-A2 with
CD8aa-HLA-A2 demonstrated that steric hindrance exists
between CD8aa and LILRB1 consistent with functional data
(72), even though the footprints of these two molecules onto
HLA-A2 show little overlapping (Fig. 5B).
However, the effects, mechanisms, and structures of many
LILR activating receptors remain unknown. Some LILR activat-
ing receptors with short cytoplasmic tail were proved to recruit
the c-chain of FceRI with charged Arg residue in transmem-
brane region to activate the immune system via ITAM motifs of
FceRIc (75–77). Shiroishi et al. demonstrated the crystal struc-
ture of LILRA5 (ILT11/LIR-9/CD85f) in 2006 and found its
nonbinding with several classical and non-classical pMHCI
molecules (66). As a member of ‘‘Group 1’’ activating recep-
tors, LILRA2, shows more than 80% sequence identity to LILR
B1/B2, but also proved to be a nonbinder of pMHCIs (77). The
exact mechanism for this nonbinding is yet elusive.
Recently, we report LILRA2 extracellular D1D2 domain
crystal structure at 2.6 A and the structure reveals structural
shifts of the corresponding pMHC-binding amino acid residues
in comparison with LILR B1/B2, explaining its non-binding
property to pMHCI molecules (78). We also identify the transi-
tion of 310 in LILRB1/B2 to b-strand in LILRA2 with great
influences on the local structure which only exist in the pMHC-
binding receptors. Moreover, in the structure, LILRA2 forms a
domain swapped dimer. Further mutational work of these key
swapping residues yields a monomeric form, confirming that the
domain-swapping is amino acid sequence-specific. The structure
described by our group not only supports the dimer conforma-
tion in solution observed earlier but also implies a stress
induced regulation by dimerization, consistent with its particular
heat shock promoter.
INTERACTION OF CD94/NKG2 WITH HLA
CD94 and NKG2 encode type II transmembrane proteins of
the C-type lectin-like family. CD94 can be expressed on the
cell surface as a disulfide-linked homodimer or as a disulfide-
linked heterodimer with NKG2A or NKG2C (4). NKG2A has
an ITIM in its cytoplasmic domain and CD94/NKG2A hetero-
dimers function as inhibitory receptors while CD94/NKG2C
heterodimers serve as activating receptors.
In contrast to the promiscuity of most NK receptors, the
human CD94/NKG2 family solely recognizes a nonclassical
MHC molecule, HLA-E (79). The members of the NKG2 fam-
ily, including NKG2A, -2B, -2C, and -2E, dimerize with CD94
in vitro (4) with different binding affinity to HLA-E, such as
the CD94/NKG2A binds with HLA-E more tightly than CD94/
Figure 4. Detailed binding region between KIR2DL2 and HLA-Cw3. (A) Zoom-in of the interface of KIR2DL2 D1D2 and HLA-
Cw3 (PDB 1EFX). (B) Down-view of HLA-Cw3 a1/a2/peptide binding with the molecular surface of KIR2DL2 colored by electro-
static potential (positive: blue, negative: red).
585STRUCTURAL IMMUNOLOGY AND CRYSTALLOGRAPHY HELP IMMUNOLOGISTS
NKG2C. To fully understand the mode of CD94/NKG2A inter-
action with HLA-E, the structural investigation is essential (79).
The crystal structure of CD94/NKG2A-HLA-E was shown in
Fig. 6. CD94 mainly binds to HLA-E a2 helix while NKG2A
binds to a1 helix with this orientation similar to the docking
mode of TCRs/KIRs onto pMHC, but is completely distinct
from those of LILR or Ly49 NK receptors. Analysis of the elec-
trostatic surfaces of HLA-E and CD94/NKG2A highlighted a
role for charge complementarity at the CD94/NKG2A-HLA-E
interface (Fig. 6B). The buried surface area (BSA) of the inter-
face was about 2,100 A2, which is marginally larger than TCR/
pMHC complex, with CD94 and NKG2A contributing �69 and
�31%, respectively. The peptide contributed 23% of the BSA
at the HLA-E interface, in which CD94 subunit played a much
more marked role (80%) in interacting with the peptide com-
pared with the NKG2A chain (20%).
As we discussed earlier, classical and nonclassical pMHCIs
molecules can be recognized by a diverse set of NK receptors.
However, NK cell recognition of MHC-like ligands, (i.e.,
MICA/MICB and ULBP1-4 in humans and H60, RAE-1 pro-
teins, and MULT1 in mice) is conserved across species and
mediated predominantly through the homodimeric NKG2D
receptors (80).
LY49 AND OTHER NK CELL RECEPTORS
In humans, classical pMHC molecules are recognized by the
Ig superfamily of NK receptors, such as KIR and LILR,
whereas in mice they are recognized by structurally divergent
NK receptors of the C-type lectin superfamily, termed Ly49
(81). Ly49 receptors are transmembrane glycoproteins expressed
on murine NK cells and on a subset of cytotoxic T-cells.
The best characterized Ly49/MHCIs interaction is that
between the inhibitory Ly49A receptor and its H-2Dd ligand
(57). Ly49A is a homodimer on the NK cell surface, with an
interchain disulfide in the stalk region. In the crystal structure
of the Ly49A/H-2Dd complex, the Ly49A homodimer contacts
H-2Dd through two distinct interfaces, however, the site-directed
mutants of both H-2Dd and Ly49A designed on the basis of the
crystal structure of the complex identified that the primary bind-
ing site for Ly49A on MHCIs formed by the a2 and a3 domains
of H-2Dd and b2-microglobulin (b2m) (Fig. 3) (82). It is one
side of the pMHCI binding platform, away from the peptide
antigen. This concave region partially overlaps the binding site
for CD8 (15) with the total buried surface of approximately
3400A2.
Nevertheless, there are other groups of NK cell receptors,
such as NKR-P1, LAIR-1, Siglec-7/9, NCR, and so forth. Natu-
ral cytotoxicity receptors (NCR) is a set of Ig superfamily
receptors mainly including NKp46, NKp30, and NKp44
involved in the triggering of natural cytotoxicity (83). Recent
studies have unveiled the structures of NCR and identified the
ligands of NKp46 and NKp44 as the hemagglutinin (HA) of
influenza virus and the hemagglutinin-neuraminidase (HN) of
parainfluenza virus (84, 85). These findings indicate how NCR-
expressing NK cells recognize target cells infected by influenza
or parainfluenza without the decreased expression of target-cell
MHCI protein.
CONCLUSIVE REMARKS AND PERSPECTIVES
Immunologists always want to know what and how were the
related cells involved in immune responses. Although more and
more functional data presented for the cell–cell interaction, the
Figure 5. Detailed binding region between LILRB1 and HLA-A2, in comparison with CD8 foot print. (A) Zoom-in the interface
of LILRB1 D1D2 and HLA-A2 (PDB 1P7Q). (B) Footprints of LILRB1 and CD8aa binding to the molecular surface of HLA-A2
(Side-view) (CD8/HLA-A2 PDB 1AKJ).
586 CHEN ET AL.
molecular basis under these processes is in the spotlight for us
to further understand the immune system in action. In this arti-
cle, we reviewed the crystal structures of some MHC-centered
molecules and protein complexes as well as recent progress on
the T and NK cell recognition, especially the ligand-receptor
binding (or touch) modes. We also try to fit the functional data
with the complex structures, explaining the touch of the
immune cell (T and NK cells) of the APCs.
So far, from the comparatively limited number of TCR/
pMHC structures, we can conclude that the TCR binding to
pMHC with an interaction codon which is determined by the
germline-encoded recognition bias and the peptide-mediated
editing of germline-encoded bias. However, there are still some
cases of TCR-pMHC complexes that have minimal contacts
with germline-encoded residues yet still maintain the stereotypi-
cal polarity (86, 87). More structures of TCR-pMHC complexes
are in need to refine the codon concept. Despite the TCR-
pMHC interaction, the structure of TCR-pMHC-CD4 (CD8)-
CD3 four-molecule complex which represent the intact immu-
nological synapse should be determined in the future, if we want
to know how the signaling process work at molecular level.
Many NK cell receptors bind to MHC molecules, some of
which follow the similar docking mode as TCR-pMHC. Emerg-
ing evidence suggests that NK cells, previously considered an
‘‘ancient’’ immune effector cell type, have more likely
coevolved with T cells, given that both of these lymphocytes
are focused on recognition of classical and non-classical MHC
molecules. Nevertheless, while the term ‘‘NK receptor’’ has
been used to describe molecules that were first discovered on
NK cells, the majority of these NK receptors are expressed on
at least a subset of T lymphocytes, in particular on cd TCR1 T
cells and on activated CD81 T cells, and more importantly on
the surface of other cell types, for example, dendritic cells or
monocytes or other myeloid cells.
As we know, NK cell function depends on the delicate bal-
ance of triggering and inhibitory signals. Previous extensive
functional and structural studies on inhibitory receptors on NK
cells provide the fundamental analysis for the ‘‘suspension’’ of
immune responses. However, more and more evidence shows
up indicating that NK cells can be activated to lyse target even
though the inhibitory signal exists or the amount of MHC mole-
cules on the target cells remains normal. On the other hand, for
example, although human red blood cells do not express
MHCIs, NK cells do not attack them; therefore, these cells may
lack ligands capable of engaging the activating NK cell recep-
tors. Until now, biological rationale for many NK activating
receptors has remained an enigma, including both function and
structure. That should be one of our major tasks in the future.
To ‘‘see’’ the immune system in action needs both functional
and structural approaches to answer the ‘‘what and how’’
questions.
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